12 Diabetic Retinopathy

Contact: Available from American Diabetes Association. 1701 North Beauregard Street, Alexandria, VA 22311. (800) 232-3472. Website: www.diabetes.org.

Summary: This study describes the incidence and progression of diabetic retinopathy (diabetes associated eye disease) in relation to medical risk indicators as well as visual acuity outcome after a continuous follow up period of 10 years in patients with type 1 diabetes treated under routine care. The incidence and progression of retinopathy and their association to HbA1c (glycosylated hemoglobin, a measurement of blood glucose levels over time), blood pressure, urinary albumin (protein in the urine), serum creatinine levels (a measure of kidney function), and insulin dosage were studied prospectively in 452 patients with type 1 diabetes. In patients still alive at follow up (n = 344), 61 percent (69 patients) developed any retinopathy, 45 percent (51 patients) developed background retinopathy, and 16 percent (18 patients) developed sight threatening retinopathy. Progression from background to sight threatening retinopathy occurred in 56 percent (73 patients out of 131 patients). In 2 percent (6 patients of 335), visual acuity dropped to less than 0.5 and in less than 1 percent (3 patients of 340) to less than 0.1. Patients who developed any retinopathy and patients who progressed to sight threatening retinopathy had higher mean HbA1c levels over time compared to those who remained stable. Patients who developed any retinopathy had higher levels of mean diastolic blood pressure, whereas no differences were seen in systolic blood pressure levels between the groups. Analysis showed mean HbA1c to be an independent risk indicator for both development and progression of retinopathy, whereas mean diastolic blood pressure was only a risk indicator for the incidence of retinopathy. The authors stress that metabolic control is an important risk indicator for both development and progression of retinopathy, whereas diastolic blood pressure is important for the development of retinopathy in type 1 diabetes. However, overall the number of patients who became blind during 10 years of follow up was low. 3 figures. 3 tables. 40 references.

Federally Funded Research on Diabetic Retinopathy

The U.S. Government supports a variety of research studies relating to diabetic retinopathy. These studies are tracked by the Office of Extramural Research at the National Institutes of Health.2 CRISP (Computerized Retrieval of Information on Scientific Projects) is a searchable database of federally funded biomedical research projects conducted at universities, hospitals, and other institutions.

Search the CRISP Web site at http://crisp.cit.nih.gov/crisp/crisp_query.generate_screen. You will have the option to perform targeted searches by various criteria, including geography, date, and topics related to diabetic retinopathy.

For most of the studies, the agencies reporting into CRISP provide summaries or abstracts. As opposed to clinical trial research using patients, many federally funded studies use animals or simulated models to explore diabetic retinopathy. The following is typical of the type of information found when searching the CRISP database for diabetic retinopathy:

2 Healthcare projects are funded by the National Institutes of Health (NIH), Substance Abuse and Mental Health Services (SAMHSA), Health Resources and Services Administration (HRSA), Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDCP), Agency for Healthcare Research and Quality (AHRQ), and Office of Assistant Secretary of Health (OASH).

Studies 13

•Project Title: A NEW THERAPY FOR DIABETIC MACULAR EDEMA

Principal Investigator & Institution: Ma, Jian-Xing; Laureate Professor; Medicine; University of Oklahoma Hlth Sciences Ctr Health Sciences Center Oklahoma City, Ok 73126

Timing: Fiscal Year 2003; Project Start 30-SEP-2003; Project End 31-AUG-2005

Summary: (provided by applicant): This is a R21/R33 phase-combined proposal aiming to develop a new treatment for diabetic macular edema using peptide angiogenic inhibitors. Vascular leakage is an early feature of diabetic retinopathy and can result in diabetic macular edema. Over-expression of VEGF is a major causative factor leading to vascular leakage in diabetic retinopathy. Currently, there is no satisfactory treatment for macular edema which remains a major cause of vision loss in diabetic patients. Plasminogen kringle 5 (K5) is a potent angiogenic inhibitor. Our recent studies have shown that K5 significantly decreases vascular leakage in the retina in the experimental diabetes, laser-induced choroid neovascularization and oxygen-induced retinopathy rat models. The K5induced reduction of vascular leakage requires only less than one-tenth of the dose needed for the inhibition of neovascularization. Furthermore, our preliminary data suggest that the K5-induced reduction of vascular leakage may be through blocking hypoxia-induced VEGF over-expression in the retina, primarily in Muller cells. We hypothesize that a sustained ocular delivery of K5 may induce a longterm reduction of vascular leakage in diabetic retina and thus, may have therapeutic effect on cyctoid macular edema (CME) secondary to cataract surgery and diabetic macular edema. In the R21 phase, we propose to first reveal the mechanisms for the K5induced down-regulation of VEGF expression and identify the receptor or binding protein on the cell surface which mediates the K.5-induced reduction of permeability. As diabetic macular edema is a chronic complication of diabetes and requires a longterm treatment, we propose to develop a KS-polymer pellet to achieve a sustained release of K5. The ocular delivery routes of the K5 pellet will be optimized and the pharmacokinetics will be studied in rats. The long-term effect of the K5 pellet on vascular leakage will be determined in a diabetic rat model. The R21 phase will achieve the following goals: 1),to reveal the mechanism and identify the receptor mediating the K5 action, 2) to develop a sustained delivery system for K5 and 3) to prove the concept that a sustained delivery of K5 can induce a prolonged reduction of vascular leakage, The R21 phase will provide essential tools and information for starting the R33 phase. In the R33 phase, we will study the pharmacokinetics of K5 in ocular tissues and optimize the delivery route in normal dogs, With the optimized delivery route, the efficacy of K5 on reduction of vascular leakage will be confirmed in a dog model of vascular leakage induced by intravitreal injection of IGF-1. The possible toxicity of K5 to the retinal vasculature and retinal structure will be examined in both rats and dogs by histochemistry. The retinal function will be examined by ERG recoding. Although this project does not reach clinical trials, the proposed studies will obtain pre-clinical data such as pharmacokinetics, delivery route, efficacy and toxicity from more than one species, which are essential and useful for starting clinical trials. These studies will contribute to the development of a new treatment for CME and for diabetic macular edema. This new treatment will use natural human peptides and will be less invasive. This new therapy, if successful, can prevent vision loss from macular edema in diabetic patients.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

14 Diabetic Retinopathy

•Project Title: ALDOSE REDUCTASE AND DIABETIC EYE DISEASE

Principal Investigator & Institution: Petrash, J Mark.; Professor; Ophthalmology and Visual Sci; Washington University Lindell and Skinker Blvd St. Louis, Mo 63130

Timing: Fiscal Year 2002; Project Start 16-JUL-1988; Project End 31-MAR-2005

Summary: (Adapted from applicant's abstract): Diabetes mellitus is a major cause of blindness due to irreversible structural and functional changes to the lens and retinal vasculature. Mounting evidence from work with experimental animals suggests that aldose reductase, the first enzyme of the polyol pathway of glucose metabolism, plays a key role in the pathogenesis of diabetic eye disease. Inhibition of aldose reductase provides a therapeutically attractive means to delay the onset and/or progression of diabetic complications in the eye. However, effective drug therapy will depend on inhibitors with a high degree of binding specificity - a criterion not met by the currently available inhibitors. The aldo-keto reductase superfamily contains enzymes with structural and kinetic properties similar to aldose reductase. Many of these aldo-keto reductases are high affinity receptors for the same aldose reductase inhibitors previously evaluated but withdrawn from clinical trials. The long-range objective or our application is to identify structural features of aldose reductase and related enzymes that explain their functional differences and perhaps provide distinguishing features that can be exploited in drug design. A corollary goal is to establish the physiological role of aldose reductase, as long-term inhibitor therapy is likely to be required for effective prevention of diabetic eye disease. Three specific aims are proposed to address these goals: (1) We will characterize the functional properties and expression pattern in the normal and diabetic eye of a newly discovered human enzyme that appears to have functional properties strikingly similar to aldose reductase; (2) Using a combination of mutagenesis and x-ray crystallography, we will test the hypothesis that the C-terminal domain is a structural feature that distinguishes the function of aldose reductase from other closely-related enzymes; (3) We will test the hypothesis that the physiological role of aldose reductase has been conserved in the budding yeast S. cerevisiae and that strains containing deletions of yeast aldose reductase can be marker rescued by the human aldose reductase gene.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: ANGIOSTATIN--MOLECULAR MECHANISMS AND INTERACTIONS

Principal Investigator & Institution: Sane, David C.; Associate Professor; Internal Medicine; Wake Forest University Health Sciences Winston-Salem, Nc 27157

Timing: Fiscal Year 2002; Project Start 30-SEP-1998; Project End 31-OCT-2003

Summary: (Adapted from Investigator's Abstract): Abnormalities in angiogenesis contribute to the pathogenesis of a variety of diseases including tumor growth and metastases, diabetic retinopathy, arthritis, psoriasis, and atherosclerosis. Angiostatin is a recently-discovered inhibitor of angiogenesis that has been shown to prevent the growth and metastasis of experimental tumors. The goal of this proposal is to understand the molecular mechanisms by which angiostatin inhibits cellular growth and migration. Based on its striking homology with hepatocyte growth factor (HGF), a known inducer of angiogenesis, it is hypothesized that angiostatin may act as a competitive inhibitor of HGF, perhaps by binding, but not activating the c-met receptor. This hypothesis will be tested in cell proliferation and migration assays and with competitive cellular binding studies. Another potential mechanism of action of angiostatin involves its ability to displace plasminogen and thereby inhibit plasmin-

Studies 15

mediated pericellular proteolytic activities, including the activation of procollagenases and the conversion of latent TGF-B to the active form. A final mechanism that to be examined is the potential ability of angiostatin to inhibit vitronectin-mediated haptotaxis, by disrupting urokinase receptor (UPAR) or integrin-mediated adherence of cells to this extracellular matrix protein. Recombinant vitronectin (wild and mutant forms) will be used to evaluate angiostatin's effect on VN-supported haptotaxis. Preliminary studies demonstrate that HGF (like angiostatin) binds to vitronectin. Domain deletion mutants of VN will be used to determine the VN binding site of HGF. The ability of angiostatin and HGF to compete for the same binding site on VN will also be examined using ELISA-based assays. By virtue of its ability to mediate haptotaxis and also approximate either pro or anti-angiogenic factors close to the cell, VN may modulate angiogenesis. These studies will lead to a better understanding of the mechanisms of angiogenesis inhibition by angiostatin.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: ANTI-ANGIOGENIC SIGNALING MOLECULES IN RETINAL CELLS

Principal Investigator & Institution: Rahimi, Nader; Ophthalmology; Boston University Medical Campus 715 Albany St, 560 Boston, Ma 02118

Timing: Fiscal Year 2002; Project Start 01-FEB-2002; Project End 31-JAN-2005

Summary: Angiogenesis, formation of new blood vessels from pre-existing vessels is a hallmark of many eye diseases such as age-related macular degeneration, proliferative diabetic retinopathy, retinopathy of prematurity and vascular glaucoma, which are among leading cause of visual loss in the USA and throughout the world. Vascular endothelial growth factor (VEGF), the major stimulator of angiogenesis, elicits its effect by binding to and activating two endothelial receptors namely VEGFR-1/FLK-1 and VEGFR-2/FLT-1. Although activation of VEGFR-2 has been demonstrated to be an essential requirement for induction of angiogenesis, the role of VEGFR-1 in angiogenesis is largely unknown. We will investigate the molecular mechanisms responsible for action of VEGFR-1, such as activation of signaling molecules or induction of immediate early genes (IEG) that might drive its anti-angiogenesis effects in endothelial cells. The significance of the results obtained from the proposed study lies in their potential to provide fundamental information on how VEGFR-1 communicates to control/restrain angiogenesis in endothelial cells. The importance of angiogenesis in ocular diseases is well recognized. our long-term goal is to begin to apply the information obtained from this project to the design of strategies to regulate angiogenesis in clinical settings.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: CELL COMMUNICATION IN THE VERTEBRATE RETINA

Principal Investigator & Institution: Miller, Robert F.; Professor; Neuroscience; University of Minnesota Twin Cities 200 Oak Street Se Minneapolis, Mn 554552070

Timing: Fiscal Year 2002; Project Start 30-SEP-1978; Project End 31-MAR-2004

Summary: (Verbatim from applicant's abstract): This research application represents a broad investigation into the mechanisms of cell communication in the vertebrate retina. Our objectives are centered on three main issues. These include (1) the mechanisms in the inner retina which are subserved by the release of glutamate and the different types of glutamate receptors, both ionotropic and metabotropic, which interact to regulate the excitability of ganglion cells; (2) the identification and biophysical characterization of voltage-gated Ca2+ channels in ganglion cells with special emphasis on T-type Ca2+ channels, their pharmacological properties and cellular distribution in the dendrites and

16 Diabetic Retinopathy

soma. We plan to evaluate how T-type Ca2+ channels contribute to nerve impulse generation and whether these ion channels play a role in amplifying synaptic currents which are generated on the dendrites. The third broad mission of this application is dedicated to a better understanding of dendritic physiology by recording from dendrites and isolated dendrosomes to further define the physiological properties of AMPA, NMDA and KA receptors. The methods used in this study will include electrophysiology, Ca2+ imaging and the use of photolysis to introduce chemical agents quickly and apply them locally at visually targeted dendritic regions. These experiments will be carried out in the amphibian retina and are designed to enhance our understanding about the mechanisms by which cells interact with one another and contribute to the excitability of retinal ganglion cells. The health-related implications of this research are extensive and relate to the mechanisms which control cellular functions of ganglion cells and how these mechanisms may help or hinder the stability of these cells when confronted with the stress of several different disease states, including glaucoma and diabetic retinopathy.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: COHORT REGISTRY OF TYPE 1 DIABETES

Principal Investigator & Institution: D'alessio, Donn J.; Preventive Medicine; University of Wisconsin Madison 750 University Ave Madison, Wi 53706

Timing: Fiscal Year 2001; Project Start 01-MAY-1987; Project End 31-AUG-2005

Summary: (Adapted from Investigator's Abstract) The decade between 1987 and 1997, spanning the Diabetes Control and Complications Trial (DCCT) and other clinical trials, saw confirmation of the importance of intensive therapy to control glycemic levels in Type 1 diabetes. Accompanying this advance in diabetes treatment, molecular studies emerged and provided clues to possible pathways for long term vascular complications perhaps most notably in the area of advanced glycation end products. The investigators' point out that their geographically based cohort presents an excellent opportunity to examine how these recent advances in clinical diabetes and basic science apply to prevention of complications in the community. Detailed information from this cohort followed from diabetes diagnosis during 1987-1992 includes a valuable longitudinal plasma bank. The cohort provides a unique baseline to compare diabetes management, glycemic control and outcomes in the post-DCCT era. As the investigators enrolled incident cases, they have information from diagnosis on children and adolescents. They propose to expand their existing data base, to use their existing resources including the plasma bank, and to incorporate promising new pathogenic testing by: (a) recruiting over 5 years a new post-DCCT cohort, (b) completing a 9 year follow-up for microvascular outcomes on the existing cohort, and performing antibody studies for methylglyoxal modified protein (an advanced glycation end product, or AGE) and sex hormone testing on our stored plasma. These proposed activities will allow the investigators to pursue the following specific aims: (1) compare trends in diabetes management and outcomes for individuals diagnosed before and after the announcements and dissemination of the DCCT results; (2) firmly establish retinopathy incidence and change in urinary albumin excretion rates through the first 9 years of Type 1 diabetes and their relationship to early risk factors; and (3) validate the role of a new plasma marker for intracellular AGE in glycemic control, retinopathy and change in urinary albumin excretion rates.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

Studies 17

•Project Title: COLOR DOPPLER IMAGING OF THE RETINA AND CHOROID

Principal Investigator & Institution: Glucksberg, Matthew R.; Professor and Chairman; Biomedical Engineering; Northwestern University 633 Clark Street Evanston, Il 60208

Timing: Fiscal Year 2002; Project Start 01-JUN-2000; Project End 31-MAY-2004

Summary: Defects in the regulation of retinal and choroidal flow are part of the etiology of diabetic retinopathy, glaucoma, and other vision-threatening disorders, yet the physics and physiology controlling of blood flow to the retina is poorly understood, at least in part because of the limitations of current methods of measuring blood flow. The goal of this research is to quantitatively study the control of flow and the hemodynamics in the choroidal and retinal circulations and their relationships to retinal disease. The hypothesis is that blood flow in the retinal and choroidal circulations is not homogeneous and that increased heterogeneity in blood flow may be an early indicator of dysfunction of the retinal and choroidal circulations. As part of this work simultaneous, continuous and quantitative measurements of tissue perfusion in the choroidal and retinal circulations will be made to allow study of how the retina and choroidal circulations interact in response to physiological conditions. Previous investigations of the role of the vasculature in health and disease have been hampered by the limits of technology. In this project Color Doppler Optical Coherence Tomography (CDOCT), a novel non-invasive imaging technology, will be adapted to measure hemodynamic parameters in the circulations that serve the retina. The specific aims will first address the instrumentation and quantification of blood flow and then validate the results using in-vivo comparison to Laser Doppler Flowmetery, the most commonly used current method of assessing perfusion. The method will then be used in animal experiments to determine the effects of perfusion pressure and blood gasses on the regional distribution of blood flow and local hematocrit in the retinal and choroidal circulations, with and without ganglionic blockade and other maneuvers which act differently on the two circulations.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: DIABETES AND INCONTINENCE

Principal Investigator & Institution: Brown, Jeanette S.; Professor and Director; Obstetrics, Gynecology & Reproductive Scis; University of California San Francisco 500 Parnassus Ave San Francisco, Ca 941222747

Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-AUG-2006

Summary: Jeanette S. Brown, M.D. is Professor of Obstetrics Gynecology and of Epidemiology & Biostatistics at the University of California, San Francisco (UCSF). She is Co-Director of the newly established UCSF Women?s Health Clinical Research Center and Director of the UCSF Women?s Urologic Research Group. Dr. Brown also developed and now is Director of UCSF Women?s Health Research Center Fellowship in Clinical Research and Associate Director of the UCSF/ San Francisco VAMC (SFVAMC) Women?s Health Clinical Research Fellowship. Additionally, Dr. Brown is Director of the UCSF Women?s Continence Center. Formal training in clinical research methods and support from a 4-year Mentored Clinical Scientist Award provided the experience and skills to become an independent investigator and build a multidisciplinary research team in patient-oriented research. The proposed research is designed to determine among women with diabetes: prevalence and incidence of urinary incontinence by type (urge, stress, and mixed) and severity, both overall and by race; risk factors associated with incontinence, especially aspects of diabetes severity (duration, treatment, glycemic control, presence of microvascular complications

18 Diabetic Retinopathy

including retinopathy, nephropathy and neuropathy) that are associated with greater risk or severity of urinary incontinence; and whether interventions including glycemic control or weight reduction prevent or reduce severity of urinary incontinence among women with diabetes. Questionnaires, laboratory, and outcome data from a triad of diabetes studies including women with impaired glucose tolerance, type 2 and type 1 diabetes and 2 population-based cohort studies that include women with and without diabetes will be analyzed. Dr. Brown has the enthusiastic support of her department UCSF investigators and other internationally recognized clinical researchers to pursue her research goals. She is a senior mentor to young clinical investigators and will develop a unique joint Urogynecology and Urology 3-year clinical research fellowship.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: DIABETIC MACULAR EDEMA CLINICAL RESEARCH NETWORK

Principal Investigator & Institution: Beck, Roy W.; Director; Jaeb Center for Health Research, Inc. Suite 350 Tampa, Fl 33647

Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-JAN-2009

Summary: (provided by applicant): This application is submitted in response to the RFA for a Coordinating Center (CC) to serve the needs of the Diabetic Macular Edema Clinical Research Network. The network is expected to include 25 clinics, a Fundus Photograph Reading Center (FPRC), Study Chairman's Office, and a CC. The main objective of this network is to evaluate promising new treatment approaches for diabetic macular edema (DME). The role of the CC is critical for the success of a multi-center study. The importance to the network of having an experienced CC, particularly one with experience in eye disease trials, is sufficiently self-evident in the RFA that it needs no justification statements here. While the core principles followed by a CC in study protocol design, quality control, study conduct, and statistical analyses have changed little in the last 20 years, through technologic advances the approach to carrying out many CC functions has changed considerably. Advances in computing and in access to the Internet have provided new opportunities for a CC to enhance quality control measures and at the same time increase efficiency. This evolution in CC trial management will continue as technology continues to advance. For essentially all aspects of the project, we will draw upon our experience in our past and current studies. We have coordinated numerous multi-center eye disease investigator groups and have experience with all of the CC functions and responsibilities that will be part of this project. We have considerable experience with study groups conducting multiple, concurrent and consecutive protocols, a likely aspect of this project. We have been innovative in our approach to clinical trial conduct and management and have relied extensively on using the Internet to not only increase efficiency but also in many ways to enhance quality control measures. For a number of years we have worked to develop a data management system that is as generic as possible so a new study can be readily added to an existing data management structure. This extends to website development in which the web-based applications we have developed for our prior and current studies will serve as a template for this project. This proposal will detail the capabilities of the Jaeb Center to serve as the CC for the network and our plans to carry out the objectives of the project.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

Studies 19

•Project Title: DIABETIC MACULAR EDEMA CLINICAL RESEARCH NETWORK - FUND*

Principal Investigator & Institution: Davis, Matthew D.; Professor; Ophthalmology and Visual Sci; University of Wisconsin Madison 750 University Ave Madison, Wi 53706

Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-JAN-2009

Summary: (provided by applicant): The University of Wisconsin Fundus Photograph Reading Center (UW FPRC) proposes to serve as photograph reading center for the DME Clinical Research Network. To accomplish this, we propose to: (I) Collaborate with other network investigators to develop ideas for clinical trials of promising new DME treatments, construct study protocols, carry out approved studies, analyze the resultant data, and prepare manuscripts. (2) Adapt or develop procedures for documenting and grading DME. We can provide protocols for DME imaging (color stereoscopic fundus photographs, fluorescein angiograms, and optical coherence tomograms), and are experienced at orienting, certifying, and giving feedback to clinic photographers. By extending the Early Treatment Diabetic Retinopathy Study classification, we have protocols for evaluation/grading of DME from color photographs and angiograms. We are developing a protocol for assessment of OCT scans. We are prepared to modify these protocols further, if necessary, to define the eligibility and outcome criteria appropriate to network goals. The UW FPRC principal investigator and staff are experienced at participation in collaborative multi-center clinical trials (e.g., Diabetic Retinopathy Study, Early Treatment of Diabetic Retinopathy Study, Diabetes Control and Complications Trial, and several drug trials) and in networks to conduct them (e.g., the Study of the Ocular Complications of AIDS), and are strongly committed to the study of DME and its potential treatments as a major public health priority.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: DIABETIC RETINOPATHY: AR1 AS A NOVEL THERAPEUTIC TARGET

Principal Investigator & Institution: Smith, Sylvia B.; Professor; Cellular Biology and Anatomy; Medical College of Georgia 1120 15Th St Augusta, Ga 30912

Timing: Fiscal Year 2004; Project Start 01-JAN-2004; Project End 31-DEC-2007

Summary: (provided by applicant): The goal of this project is to determine the pathogenesis of and neuroprotection against retinal ganglion cell (RGC) death in diabetic retinopathy. Many RGCs die within the first 2 years of disease onset. The RGC death is thought to be due to overstimulation of the N-methyl-D-aspartate (NMDA) receptor that leads to excessive levels of intracellular calcium, which triggers the cell death cascade. Glutamate, which is elevated in the vitreous body and retina of diabetic patients, is the primary excitotoxin that activates the NMDA receptor. Homocysteine, which accumulates in the plasma of diabetic patients, induces RGC death when injected intravitreally. NMDA receptor activation requires co-activation of its glycine binding site and D-serine is the endogenous physiologic ligand for this site. Serine racemase is the enzyme responsible for the endogenous generation of D-serine. One of the goals of the project is to elucidate the molecular events involved in the extracellular accumulation of the MDA receptor agonists, glutamate and homocysteine, and the coagonist D-serine. D-serine and serine racemase are expressed in retina, but their involvement in diabetes has not been investigated. AIM 1 will test the hypothesis that diabetes is associated with increased levels of D-serine and serine racemase leading to enhanced activation of the NMDA receptor by glutamate and homocysteine. AIM 2 will test the hypothesis that diabetes is associated with altered function of transport systems

20 Diabetic Retinopathy

for glutamate (EAATs, x[c-]), homocysteine and D-serine (ATB0,+) and that their altered function may provide the molecular basis for the diabetes-associated increase in extracellular levels of glutamate, homocysteine and D-serine. Therapeutic intervention strategies targeted at blocking NMDA receptor stimulation could prevent RGC death and may delay other manifestations of diabetic retinopathy. Type 1 sigma receptor (sigmaR1) is a nonopiate, nonphencyclidine binding site that demonstrates robust neuroprotective properties including inhibition of ischemia-induced glutamate release and depressed neuronal responsivity to NMDA receptor stimulation. SigmaR1 is expressed abundantly in RGCs and continues to be expressed under hyperglycemic conditions. Agonists specific for sigmaR1 may have potential as therapeutic agents in providing neuroprotection in the early stages of diabetic retinopathy. Our preliminary data show that (+)-pentazocine, a sigmaR1 agonist, prevents RGC death in vitro induced by glutamate and homocysteine and in vivo induced by diabetes. AIM 3 will test the hypothesis that sigmaR1 agonists will be protective against RGC death characteristic of diabetic retinopathy.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: DYSLIPIDEMIA AND RETINAL ENDOTHELIAL CELL DYSFUNCTION

Principal Investigator & Institution: Busik, Julia V.; Physiology; Michigan State University 301 Administration Bldg East Lansing, Mi 48824

Timing: Fiscal Year 2003; Project Start 15-JUN-2003; Project End 31-MAY-2005

Summary: (provided by applicant): Diabetic retinopathy represents the leading cause of blindness in adults. Diabetic retinopathy is a disease of the retinal microvessels characterized by capillary occlusions, microaneurysms, selective loss of pericytes, acellular capillaries, hypertrophy of the basement membrane, angiogenesis and neovascularization. While the initial determinants of retinal microvascular damage are not well understood, recent studies suggest that diabetic retinopathy is a low-grade chronic inflammatory disease. As such, recent studies document increased leukocyte attachment and transmigration into the vascular intima. The increased adherence of leukocytes to endothelial cells likely involves induction of specific adhesion molecules, such as ICAM-1. The factors elevating cellular adhesion molecules are not well defined, but likely involve hyperglycemia and dyslipidemia associated with diabetes mellitus. Our preliminary studies show that treating human retinal vascular endothelial (hRVE) cells with specific fatty acids leads to the induction of ICAM-1, as well as alterations in several signaling pathways. We hypothesize that exposure of hRVE cells to specific fatty acids leads to the formation of specific bioactive lipids that cause alterations in cell signaling and gene expression and lead to increased expression of adhesion molecules and leukocyte attachment. To test this hypothesis, the following aims are proposed: 1) To evaluate the effects of fatty acids and glucose on adhesion molecule expression and cell signaling in hRVE cells. 2) To correlate changes in adhesion molecule expression and cell signaling to the production of specific bioactive lipids. The outcome of this work will lead to a better understanding of how diabetic dyslipidemia contributes to altered hRVE cell phenotype and the onset and progression of diabetic retinopathy.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: EFFECT OF ISOPROSTANES ON RETINAL TRANSMITTER RELEASE

Principal Investigator & Institution: Opere, Catherine A.; Pharmacy Sciences; Creighton University 2500 California Plaza Omaha, Ne 68178

Studies 21

Timing: Fiscal Year 2003; Project Start 15-APR-2003; Project End 31-MAR-2005

Summary: (provided by applicant): Isoprostanes (IsoP's) are a series of prostaglandinlike compounds that are formed in abundance in vivo by a non-enzymatic, free radical catalyzed peroxidation of arachidonic acid independent of cyclooxygenase. In addition to providing a reliable measure of in vivo and in vitro oxidative stress, IsoP's can exert pharmacological effects in some tissues. A review of literature revealed that very few studies have addressed the effects of IsoP's on neurotransmitter release from central or peripheral tissues. Although IsoP's have been reported to modulate sympathetic neurotransmission from anterior uveal tissues, no studies have investigated the potential regulatory effects of these compounds on neurotransmission in the retina. In the present study, we will test the hypothesis that IsoP's can modulate glutamate, gamma-aminobutyric acid (GABA) and dopaminergic transmission in the retina both in vitro and in vivo. The overall objective of the present study is to examine the effect of different series of IsoP's (A1, E1, E2, F1, F2 and F3) on glutamate, GABA and dopamine release from retinae both in vitro and in vivo. Experiments in this project have, therefore, been designed to answer the following questions: (i) do different IsoP's alter the release and/or availability of glutamate, GABA and dopamine in vitro and in vivo? (ii) are the effects produced by IsoP's comparable to those of other arachidonic acid metabolites (prostaglandins and thromboxanes)? (iii) what is the role of presynaptic IsoP heteroreceptors in the effects caused by these compounds on glutamate, GABA and dopamine in vitro and in vivo? We anticipate that the results of the present study will improve our understanding of the basic mechanisms involved in the effects of IsoP's on retinal glutaminergic, GABAergic and dopaminergic transmission. Furthermore, these studies may reveal pharmacologically/toxicologically accessible sites for the action of IsoP's in the retina. We hope that observations made in this project will be applicable to diseases of the retina associated with the generation of free radicals and oxidative damage such as ischemia, glaucoma, diabetic retinopathy and age-related macular degeneration.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: EPIDEMIOLOGY OF AGE-RELATED OCULAR DISEASE

Principal Investigator & Institution: Klein, Ronald; Professor; Ophthalmology and Visual Sci; University of Wisconsin Madison 750 University Ave Madison, Wi 53706

Timing: Fiscal Year 2002; Project Start 16-JUN-1987; Project End 31-MAY-2007

Summary: (Applicant's Abstract) This proposal describes the follow-up of a populationbased cohort study aimed at determining the long-term (15-year) impairments of aging. In addition we will determine the incidence and associated risk factors for other agerelated ocular conditions such as branch retinal vein occlusion, retinal arteriolar ernboli, and epiretinal membranes. We will examine age-related hyperopic shift in refraction. The population was 43-86 years of age at the census prior to the first survey in 1987-88. Standardized protocols for interviews, examinations, ocular photography, and grading have been employed during the baseline (n--4,926), the 5-year (n7-3,816) and 10-year (n7-2764) examinations. Refusal rates have been low. Because this cohort initially included a substantial number of middle-aged adults, the study provides a unique opportunity to follow the course of these eye conditions and document their natural history as this population enters the age of marked increase in disease incidence. The study in its initial prevalence survey and at the 5 and I 0-year follow-ups obtained information about cardiovascular disease, hypertension, diabetes and other medical conditions, cigarette smoking, nutritional supplements, light exposure, drug use history, and blood factors (e.g. glycosylated hemoglobin, total and HDL cholesterol). The 15-year

22 Diabetic Retinopathy

follow-up is essential because the cohort is maturing and the number of cases of disease will be great enough to test many of the hypotheses that could not be precisely tested when the population was younger. In addition, at the 5-year examination, additional questions were added regarding subjective assessment of visual ability and history of falls and fractures, while continuing to monitor other risk factors and ocular variables that were measured at baseline. We will examine the relationship of impaired vision from specific age related eye conditions to self-reported visual function, falls, and fractures and nursing home placement. Findings regarding age-related maculopathy, cataract other retinal diseases will be of great public health importance in helping to predict the requirement for visual care and rehabilitative services as the population ages, and in directing further efforts at preventing these conditions.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: EVALUATING ALTERNATE RETINOPATHY SCREENING INTERVENTIONS

Principal Investigator & Institution: Walker, Elizabeth A.; Associate Professor; Epidemiology & Population Health; Yeshiva University 500 W 185Th St New York, Ny 10033

Timing: Fiscal Year 2002; Project Start 07-SEP-2000; Project End 31-AUG-2005

Summary: Diabetic retinopathy is serious, costly and prevalent, especially among minority populations. Annual ophthalmic examinations and appropriate follow-up care would result in substantial savings in preventable vision loss, health care costs, and lost productivity. However, most people do not receive these services. In our previous study, we doubled the rate of ophthalmic examinations in a low-income AfricanAmerican population using a multi-component intervention. We now propose to evaluate the incremental effects and costs of components of that intervention in a broader diabetes population, including Spanish-speaking patients The specific aims of the proposed study are to: 1) test the hypothesis that a tailored telephone intervention will result in a higher rate of ophthalmic examinations than a standard print intervention; 2) improve understanding about reasons why people obtain an ophthalmic examination and assess differences in subgroups (gender and Spanish language preference); 3) improve understanding about reasons why people who are diagnosed with diabetic eye disease do or do not receive recommended follow-up treatment; and 4) conduct cost-effectiveness analyses. The study design is a randomized, controlled intervention trial with masking; the individual is the unit of sampling, assignment, and analyses. A total of 800 patients with type 1 or type 2 diabetes mellitus who have not had a dilated eye examination in the last year will be sampled from the databases of two large urban health systems. After eligibility is ascertained and written informed consent is obtained, patients will be randomized within site by gender and preferred language (Spanish or English) to either the tailored telephone group or the standard print group. Sample size determinations are based on having sufficient power to detect differences between groups at the p<0.05 level of significance. The main study outcome will be receipt of a dilated eye exam at 6 months post-randomization as ascertained by medical record abstraction, with a second outcome assessment conducted at 18 months post randomization. Receipt of follow-up care for diagnosed eye disease will also be assessed. Preand post-intervention telephone interviews will provide data on changes in knowledge, beliefs, risk perceptions and behavior regarding ophthalmic exams. Cost data will be collected using standardized methods. Study results will inform implementation and dissemination of practical, low-cost interventions to increase

Studies 23

ophthalmic examinations and follow-up care, and thereby contribute to a decrease in vision loss and health care costs in diabetes populations.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: EXPRESSION AND REGULATION OF RETINAL ANGIOTENSIN II

Principal Investigator & Institution: Senanayake, Preenie E.; Cleveland Clinic Foundation 9500 Euclid Ave Cleveland, Oh 44195

Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2006

Summary: (provided by applicant): The long-term goal of my research is to understand the role of hypertension in the pathogenesis of diabetic retinopathy. In humans and other species with vascularized retinas, the rates of glucose and oxygen utilization by the retina are 3-fold higher than in any other tissue. The retinal circulation is highly sensitive to local tissue metabolic needs and susceptible to damage from circulatory dysfunction. In diabetic patients, capillary non-perfusion has been shown to precede neovascularization. Capillary ischemia could be due at least in part to obstruction caused by abnormal or accelerated growth of vascular smooth muscle cells. The increases in angiotensin (Ang) II and insulin levels that occur in hypertension and noninsulin dependent diabetes (type 2) may contribute to this process within the small ophthalmic arteries. Thus, insulin and Ang II could act in a synergistic manner. Inhibiting the renin-angiotensin system (RAS) confers a therapeutic benefit in the treatment of diabetic retinopathy. Although the pathogenesis of diabetic retinal complications is not fully understood, emerging evidence implicates the RAS, with its mitogenic and trophic actions and its influence on angiogenesis. Ang II may be produced locally in the retina, and could play a role in the development and/or maintenance of proliferative diabetic retinopathy. Our hypothesis is that the retina is a target tissue for Ang II in rats with hypertension and/or diabetes. We will test this hypothesis in the stroke-prone spontaneously hypertensive rat (SHRSP) with streptozocin-induced diabetes, now shown to develop signs of diabetic retinopathy that are reversed by an Ang receptor antagonist. Specific aims are: 1) To determine the tissue-specific expression of Ang receptor subtype transcripts and proteins, 2) To examine tissue-specific expression of angiotensinogen, renin, angiotensin converting enzyme and Ang II in the retina. Molecular, biochemical and immunohistochemical approaches will be combined. 3) To assess the mechanism of regulation of Ang II in the retina under physiological and genetic perturbations of the RAS. Electroretinography will be used to evaluate retinal function, which will be correlated with blood pressure measured by tail cuff. These studies will not only expand our understanding of the function of the RAS in a novel tissue, but may also have important therapeutic implications. Ultimately retinal Ang receptors could be specifically targeted with selective therapeutic agents to prevent the development of proliferative diabetic retinopathy.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: FEMTOSECOND LASER VITREOUS PHOTODISRUPTION

Principal Investigator & Institution: Mcdonnell, Peter J.; Director; Ophthalmology; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218

Timing: Fiscal Year 2003; Project Start 30-SEP-2003; Project End 31-AUG-2006

Summary: (provided by applicant): The National Eye Institute has reported that Diabetic Retinopathy is a major cause of blindness. Due to the prevalence of this disease, NEI's program objective for the next 5 years includes the development of better

24 Diabetic Retinopathy

methods of prevention and therapy of diabetic retinopathy. It is this project's long term objective to develop a non-invasive, trans-pupillary photodisruption laser system to perform safe and effective vitreolysis for the prevention and treatment of diabetic retinopathy. The specific objectives include: 1. Develop and optimize a trans-pupillary femtosecond laser delivery system to perform vitreolysis in rabbit eyes. 2. Measure development, growth and complications in rabbit eyes after core vitreolysis and lysis of cortical vitreous adjacent to the retina. 3. Determine the efficacy of laser vitreolysisinduced PVD in inhibiting hemorrhage and tractional retinal detachment in a VEGFBFGF growth factor-induced model of vitreoretinal angiogenesis. The laser delivery system and laser parameter optimization will be tested on animal cadaver eyes. Paired sample live-animal experiments will be performed to assess the safety and efficacy of the developed procedure.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: GENE EXPRESSION CHANGES IN RETINAL NEOVASCULARIZATION

Principal Investigator & Institution: Duh, Elia J.; Professor; Ophthalmology; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218

Timing: Fiscal Year 2002; Project Start 01-AUG-1999; Project End 31-JUL-2004 Summary: This abstract is not available.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: GENE THERAPY FOR RETINAL DETACHMENT

Principal Investigator & Institution: Tomasek, James J.; Professor & Vice Chair for Research; Cell Biology; University of Oklahoma Hlth Sciences Ctr Health Sciences Center Oklahoma City, Ok 73126

Timing: Fiscal Year 2003; Project Start 16-JAN-2003; Project End 31-DEC-2005

Summary: (provided by applicant): Retinal detachment with subsequent loss of vision is a major clinical problem associated with a number of ocular diseases, including proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Surgery, the only treatment currently available, has a high rate of recurrence of fibrous epiretinal membrane formation and retinal detachment. Smooth muscle (SM) alphaactin containing myofibroblasts are a major cell type present in the epiretinal membranes that form in PVR and PDR and it is these cells that are responsible for generation of force leading to retinal detachment. The long-term goal of this research is to develop a gene therapy-based approach that targets and blocks the contraction of the myofibroblast. The first objective of this project is to develop a myofibroblast-specific promoter. Recent studies have suggested that the regulation of SM alpha-actin expression is different in myofibroblasts and smooth muscle cells and these studies have begun to identify regulatory elements in the promoter bf SM alpha-actin that might be responsible for these differences. Therefore, the first specific aim is to identify regulatory elements within the SM alpha-actin promoter that are specific to myofibroblasts in epiretinal membranes. To address this aim we will use transgenic mice containing a SM alpha-actin promoter/LacZ transgene in which specific regulatory elements have been deleted or mutated. These mice will be mated with a transgenic mouse model in which an epiretinal membrane containing myofibroblasts forms and contracts, with subsequent retinal detachment. The second part of our long-term goal is to be able to block the contraction of myofibroblasts in the epiretinal membranes. The expression of SM alpha-actin in myofibroblasts is functionally related to the ability of these cells to

Studies 25

generate large amounts of contractile force. Therefore, the second specific aim is to determine whether knocking out the SM alpha-actin gene will decrease or inhibit retinal detachment. Specifically we will determine the length of time it takes for retinal detachment to occur in control and SM alpha-actin-null mice. These studies will provide the basis for developing a gene therapy-based approach that can specifically target myofibroblasts in epiretinal membranes and block their generation of contractile force thereby blocking epiretinal membrane contraction and retinal detachment.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: GENETIC CONTROL OF VASCULAR REMODELING

Principal Investigator & Institution: Dumont, Daniel J.; Associate Professor; Sunnybrook & Women's Coll Hlth Scis Ctr S132 Toronto, on M4n 3M5

Timing: Fiscal Year 2002; Project Start 14-AUG-2001; Project End 31-JUL-2005

Summary: Angiogenesis, the growth of blood vessels from preexisting vessels, which is known to play a pivotal role in the progression of several different diseases, including diabetic retinopathy, arteriosclerosis and cancer. To-date, virtually all genes that have been shown to play a role in early vascular development of the embryo, are also expressed in the endothelium of these diseased tissues. Thus, implies that the disregulation of gene expression in the endothelium in these diseased tissues recapitulates the genes expressed in the activated endothelium of the developing embryo. The genetic control of this vessel growth is currently poorly understood, however several studies using gene-targeting in mice have revealed that numerous diverse signaling pathways produce overlapping defects in vascular remodeling. The working hypothesis for this proposal is that the defects observed in these different mouse mutants are the consequence of misregulation of a common subset of genes whose expression control vascular remodeling. This research will use gene expression profiling by gene chip and SAGE analysis to examine the genes expressed in four different mouse mutants that affect very different signaling pathways and result in overlapping vascular remodeling defects. The differential expression of genes in the heart and yolk sacs of the Tek/Tie2, Endoglin, VEGFR3, and Notch1 mouse mutants will be compared. This work will provide considerable insight into the role of these disparate signaling modalities in the angiogenic response.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: GENETICS OF DIABETIC RETINOPATHY

Principal Investigator & Institution: Hanis, Craig L.; Professor; Human Genetics Center; University of Texas Hlth Sci Ctr Houston Box 20036 Houston, Tx 77225

Timing: Fiscal Year 2002; Project Start 01-JAN-1999; Project End 31-DEC-2003

Summary: (Applicant's Abstract) Diabetic retinopathy substantially contributes to the morbidity of type 2 diabetes mellitus and is a strong predictor of subsequent, often early, mortality in those with diabetes. Susceptibility to type 2 diabetes has long been known to have a substantial genetic component. Not only does diabetes aggregate in families, but so do its complications. Preliminary results demonstrate an 8.3 fold increased risk for retinopathy in diabetic siblings of a diabetic with no retinopathy. It is likely that diabetes susceptibility alleles impact the clinical courses of the disease and development of retinopathy. It is also plausible that other genes influence susceptibility to retinopathy, but exert their influence only after the development of diabetes. To determine the contribution of genetic factors to diabetic retinopathy, 1,000 Mexican Americans with type 2 diabetes distributed in 750 sibling pairs will undergo detailed

26 Diabetic Retinopathy

eye examinations on 2 occasions (2.5 years apart). Examinations will include stereoscopic fundus photography and scoring according to standard protocols. Except for the retinal examinations, these individuals have been and are being characterized in ongoing studies in Starr County, Texas. These characterization includes genotypes at markers spanning the entire genome at an average distance of 8 to 10 centi-Morgans. All marker data will be available prior to the completion of the first round of retinal examinations. Documentation of the presence and severity of diabetic retinopathy in this sibling pair genotype resource and an additional confirmatory set of 200 individuals with diabetes will permit: 1) Determining the sibling pair concordance for retinopathy, 2) Localizing retinopathy susceptibility loci based on two-point and multipoint sibling pair linkage analysis, and 3) Identification of variation by DNA sequence scanning of genes in linked regions impacting on the presence and development of retinopathy. The end result will be improved understanding of mechanisms and exploitable pathways for moving retinopathy treatment from palliative to preventive.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: GLIAL-NEURONAL INTERACTIONS IN THE RETINA

Principal Investigator & Institution: Newman, Eric A.; Professor; Neuroscience; University of Minnesota Twin Cities 200 Oak Street Se Minneapolis, Mn 554552070

Timing: Fiscal Year 2002; Project Start 01-JUL-1990; Project End 31-MAR-2005

Summary: (Verbatim from applicant's abstract): The long-term objective of this project is to determine the functions of glial cells (Muller cells and astrocytes) in the mammalian retina. It is widely recognized that glial cells have important support functions in the retina, including uptake of neurotransmitters and regulation of extracellular potassium and pH. The role of glial cells in direct modulation of neuronal activity is not yet understood, however. In the preceding project period, we demonstrated that intercellular Ca2+ waves can be propagated through glial cells in the rat retina and that these glial Ca2+ waves modulate spike activity in neighboring neurons. In the proposed project period, we will extend our studies of glial modulation of neuronal activity and explore additional aspects of glial Ca2+ signaling with the goal of determining the significance of these interactions in vivo. The specific aims for the project period are: (1) to identify natural stimuli that elicit Ca2+ signals in retinal glial cells; the hypotheses to be tested are that: (a) chemicals released under normal or pathological conditions evoke glial Ca2+ increases, and (b) light stimulation evokes glial Ca2+ increases; (2) to test the hypothesis that spontaneous Ca2+ oscillations in glial cells modulate the activity of neighboring neurons, using regression analysis to correlate neuronal spike activity and membrane potential with Ca2+ levels in adjacent glial cells displaying spontaneous Ca2+ oscillations; (3) to characterize mechanisms of glial cell modulation of neuronal activity; the hypotheses to be tested are that: (a) excitatory neuronal modulation is mediated by release of glutamate from glial cells onto neurons, and (b) inhibitory modulation is mediated indirectly by glial activation of inhibitory amacrine cells; (4) to elucidate the mechanism by which Ca2+ waves are propagated in retinal glial cells; the hypothesis to be tested is that wave propagation is mediated by the release of ATP, which functions as an extracellular messenger; and (5) to characterize physiological changes in retinal glial cells elicited by propagation of Ca2+ waves; the hypotheses to be tested are that: (a) Ca2+ increases modulate inward rectifier potassium and Ca2+- dependent potassium conductances, and (b) Ca2+ increases generate intracellular pH variations in retinal glial cells. Glial cells have been implicated in many types of retinal pathology, including diabetic retinopathy, glaucoma, and macular degeneration. Knowledge of the basic physiological properties of retinal glial cells and their

Studies 27

interactions with retinal neurons will add to our understanding of how these cells contribute to retinal pathology. The research outlined in this application will provide significant progress towards this goal.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: HIF-1 AS A THERAPEUTIC TARGET IN DIABETIC RETINOPATHY

Principal Investigator & Institution: Ihnat, Michael A.; University of Oklahoma Hlth Sciences Ctr Health Sciences Center Oklahoma City, Ok 73126

Timing: Fiscal Year 2002; Project Start 16-SEP-2002; Project End 31-AUG-2007

Summary: Diabetic retinopathy is a major complication of diabetes and a leading cause of blindness. In its later stages this disease is characterized by a hyperproliferation of retinal blood vessels, or angiogenesis. Hypoxia inducible factor -1 (HIF-1) is a transcription factor that is rapidly induced in response to hypoxia and regulates the expression of several genes critical to angiogenesis and glucose regulation. HIF-1 has been shown to be significantly increased in the retina in response to hypoxia and to advanced glycation products (AGEs), altered macromolecules associated with diabetes. The overall hypothesis driving these studies is that inhibition of angiogenesis through the specific disruption of HIF-1 is a novel, early target for potentially controlling proliferative retinopathy. The goal of specific aim one is to target HIF-lalpha, a critical subunit of HIF-1, at the messenger RNA level through the use of ribozymes, or catalytic pieces of RNA. The goal of specific aim two is to find specific peptide inhibitors of the interaction between the HIF-1alpha and the HIF-1beta (ARNT) subunit using phage peptide display. Both the ribozymes and the peptides will be introduced locally into the retina using adenovirus. The effect of these agents on angiogenesis and on downstream targets of HIF-1 (VEGF, PAl-l) will be examined using an oxygen-induced mouse ischemia model. Using these molecular approaches we seek to define a role for HIF-1 in proliferative diabetic retinopathy and to determine whether this transcription factor represents a valid target for therapy in this disease state.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: HIGHER ORDER VISUAL PERCEPTION: LOW VISION IN ELDERLY

Principal Investigator & Institution: Odom, James V.; Medicine; West Virginia University P. O. Box 6845 Morgantown, Wv 265066845

Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2006

Summary: (provided by applicant): Low vision is a major health problem, especially among the elderly; reduced vision impairs their ability to navigate in their environment, impairs their ability to perform activities of daily living independently, and places them at greater risk for accidents and death. Relatively little research has been directed to understanding the visual control of mobility and daily behavior in older Americans with reduced vision. Almost none of the research that has been performed has examined higher order perceptual functions in the performance of tasks of daily living including mobility. Our long-range objective is to examine the impact of reduced vision on higher order perceptual abilities and the relationship of differences in higher order perception on the performance of simulated and real tasks of everyday living. In order to accomplish this long-range goal we have brought together a team of vision researchers, clinicians, and computer scientists. For the purposes of this small, pilot study grant application, our aims are more specific and restricted. One set of higher order variables that have been identified in the literature as particularly important in mobility are those related to the detection of optic flow and its components. However, we have been

28 Diabetic Retinopathy

unable to find research related to low vision patients' abilities use optic flow for detecting heading or navigating in their environment. Our specific aim for this grant is to conduct a series of six experiments to provide data on the ability of patients with low vision to detect optic flow information.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: HIGH-RESOLUTION IMAGING OF OCULAR MELANOMA AND RETINA

Principal Investigator & Institution: Bartsch, Dirk-Uwe G.; Associate Professor; Ophthalmology; University of California San Diego La Jolla, Ca 920930934

Timing: Fiscal Year 2002; Project Start 01-FEB-2001; Project End 31-JAN-2004

Summary: (Applicant's Description): High-resolution imaging allows improved understanding of retinal disease and pathological conditions, particularly in prognosis of malignant choroidal melanoma, age-related macular degeneration, diabetic retinopathy, ocular complications of AIDS and glaucoma. The eye can only be regarded as a diffraction-limited optical system up to a pupil diameter of 3 mm. Standard retinal imaging technology uses a 3 mm imaging aperture to avoid the higher order aberrations of the outer cornea. However, if these higher order aberrations can be compensated, diffraction-limited imaging at 7 or 8 mm pupil diameter can be achieved. The numerical aperture of the diffraction-limited eye at 7 or 8 mm pupil will allow to visualize retinal detail that was previously not achievable in ophthalmology. To achieve this goal we plan to develop a wavefront sensor that will allow us to measure the existing aberrations of the measured eye. We will test it in an eye model, in animal eyes and in human eyes. The wavefront sensor will be attached to a fundus camera and scanning laser ophthalmoscopes. The second aim is to use an adaptive wavefront compensator to correct the aberrations in a feed-back loop setup. We will test the complete system of wavefront sensor and wavefront compensator in an eye model, in animal eyes and in human eyes. The wavefront compensator will be attached to a fundus camera and scanning laser ophthalmoscopes. The third aim is to used digital image processing to correct for the portion of the residual aberrations that could not be compensated with the adaptive wavefront compensator due to mechanical considerations. Our group has previous developed a wavefront sensor and wavefront compensator based on a micromachined membrane deformable mirror. Due to the limited number of electrodes, slow speed of our computer system and the mechanical stiffness of the membrane we were not able to completely correct all measured aberrations. Our preliminary work shows that the system is capable of allowing wavefront correction. In this study we plan to improve the acquisition speed of our wavefront sensor and wavefront compensator to allow rapid wavefront compensation. Our preliminary results have shown that even the best aberration compensation still suffers from residual wave aberrations. Since we can measure and characterize these aberrations, we can develop digital inverse filter based on our experience in image reconstruction to correct the acquired images.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: HUMAN ALKALINE PHYTOCERAMIDASE CONTROL OF ANGIOGENESIS

Principal Investigator & Institution: Mao, Cungui; Medical University of South Carolina P O Box 250854 Charleston, Sc 29425

Timing: Fiscal Year 2002; Project Start 26-SEP-2002; Project End 31-AUG-2007

Studies 29

Summary: (provided by applicant): Angiogenesis, the process of formation of new capillaries from preexisting blood vessels, is essential for the proper organ development and tissue repair (1). However, uncontrollable angiogenesis may lead to pathologies such as chronic inflammation (2), diabetic retinopathy (3), rheumatoid arthritis (4), and growth of solid tumors (5). Our long-term goals are to define the role of human alkaline phytoceramidase (haPHC) in regulating angiogenesis and to develop this concept into a new strategy to treat angiogenesis-related diseases by targeting this enzyme, haPHC, a novel enzyme which the PI identified recently, cleaves phytoceramide to generate phytosphingosine (PHS), which is in turn phosphorylated to generate phytosphingosine-1-P (PHS-1-P) through the action of sphingoid base kinases (6). Our additional preliminary data demonstrate that 1) PHS-1-P protects human umbilical vein endothelial cells (HUVEC) from apoptosis induced by serum starvation and induces migration of HUVEC; 2) the levels of PHS-1-P are regulated by haPHC through controlling the generation of its precursor, PHS from phytoceramide; 3) haPHC mRNA is highly expressed in placenta in which angiogenesis occurs actively; and 4) downregulation of haPHC by antisense oligodeoxynucleotide (ODN) suppresses growth of HUVEC. These results suggest the hypothesis that haPHC regulates angiogenesis by regulating the levels of the potential angiogenic stimulator, PHS-1-P. Specific aim 1: to determine the role of the human alkaline phytoceramidase in growth and survival of human umbilical vein endothelial cells. Our hypothesis is that haPHC regulates growth and survival of endothelial cells by controlling the generation of PHS-1-P. To test this hypothesis, we will determine 1) whether up-regulation of haPHC, by adenovirusmediated transfection of sense cDNA, elevates the levels of PHS-1-P in HUVEC and results in cell proliferation whereas down-regulation of haPHC by antisense cDNA transfection reduces the levels of PHS-1-P and results in apoptosis and/or growth suppression of HUVEC; and 2) whether apoptosis and/or growth inhibition induced by the downregulation of haPHC is alleviated or suppressed by exogenous PHS-1-P. We expect that downregulation of haPHC will lower the level of PHS- 1-P and induce growth suppression and apoptosis of HUVEC, while exogenous PHS-1-P will suppress these effects of haPHC down-regulation. Specific aim 2: to determine mechanism of the human alkaline phytoceramidase action. In our preliminary studies, we demonstrate that haPHC 1) is highly expressed in placenta and heart and localized to the Golgi apparatus and endoplasmic reticulum; 2) hydrolyzes NBD-Clz-phytoceramide preferentially in vitro, and 3) is activated by Ca2+,but inhibited by sphingosine (6). These results support the hypothesis that haPHC has very restricted substrate specificity; its action is regulated by second messengers; and its expression is compartment and tissue specific. To test this hypothesis, we will purify haPHC and determine its substrate specificity using different phytoceramide analogs, effects of cations and lipids on its activity, its cellular localization, and tissue specific expression. We expect that the purified haPHC 1) prefers phytoceramide with a specific acyl chain as its endogenous substrate, 2) is activated by a physiologic concentration of Ca2+, but inhibited by a low concentration of sphingosine, 3) is localized to the Golgi apparatus and ER, 4) is highly expressed in tissues where angiogenesis actively occurs. Specific aim 3: to determine the role of the alkaline phytoceramidase in angiogenesis. Our hypothesis is that haPHC regulates developmental angiogenesis and vascularogenesis by regulating the generation of PHS-1-P. To test this hypothesis, we will disrupt the mouse alkaline phytoceramidase (maPHC) gene and analyze developmental angiogenesis and vascularogenesis in maPHC null versus wild type mice. We expect that the mice lacking maPHC will have insufficient angiogenesis, which may lead to abnormalities in embryonic development. These studies should validate our hypothesis that haPHC has an important role in angiogenesis in a definitive way.

30 Diabetic Retinopathy

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: HYPERSPECTRAL IMAGING OF OXYGEN SATURATION IN THE ONH

Principal Investigator & Institution: Khoobehi, Bahram; Professor; Ophthalmology; Louisiana State Univ Hsc New Orleans New Orleans, La 70112

Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2006

Summary: (provided by applicant): The development of a non-invasive means of measuring oxygen saturation in the fundus of the human eye would be useful in the diagnosis and monitoring of numerous disorders, including diabetic retinopathy, arterial venous occlusion disease, and glaucoma. In these studies, a practical system to evaluate oxygen saturation in the retina and optic nerve head using a recent innovation, hyperspectral imaging, will be developed. The hyperspectral technique measures spectral changes within the visible and infrared spectra and provides information on the molecular state of hemoglobin. The hyperspectral imaging device will allow measurement - non-invasively and in real time - of reduction and/or elevation in tissue oxygenation. The distinct optical signature of biological materials such as oxyhemoglobin and deoxy-hemoglobin as a function of their reflectance spectra will enable determination of their relative concentrations. In recent years, reflectance oximetry has been developed for the non-invasive measurement of oxygen saturation changes in the vessels of the fundus using double, triple, and multiple wavelength reflectance imaging. The hyperspectral reflectance oximetry that will be employed in these studies will permit the first non-invasive measurement for oxygen saturation in the optic nerve head tissue, and the hyperpectral data to be collected will intrinsically include all of the multiple wavelength spectra obtained in earlier approaches. The new system will be tested in two specific aims: 1) hyperspectral imaging will be used to non-invasively evaluate the stimulus-response relationship between perturbations in intraocular pressure (lOP) (10-50 mm Hg) and oxygen saturation in optic nerve head tissues and in retinal artery/vein pairs for a graded series of hypoxic states, and 2) the same studies will be performed in eyes with early stage experimental glaucoma. With this new approach, it will be possible to determine how acute changes in lOP alone or in combination with chronic lOP elevation (glaucoma) affect the three distinct microcirculations of the optic nerve head (surface nerve fiber layer; prelaminar region; lamina cribrosa) independently and/or collectively. The proposed studies are motivated by the potential for clinical application of this innovative technology in the early diagnosis of and monitoring of therapy for ocular vascular diseases in which the associated hypoxia may eventually lead to loss of vision.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: IMAGING FOCAL HYPOXIA IN DIABETIC RETINOPATHY

Principal Investigator & Institution: Maman, Anne-Marie; Oxygen Enterprises, Ltd 207 St. Mark's Sq Philadelphia, Pa 19104

Timing: Fiscal Year 2003; Project Start 01-JUN-2003; Project End 31-MAY-2004

Summary: (provided by applicant): Diabetic retinopathy is the leading cause of blindness in working age Americans. In these patients there is pathologic growth of new blood vessels in the inner retina. These poorly formed blood vessels burst, bleeding into the retina and obscuring vision. Current treatment for these patients is to detect the neovascular formations and ablate tissue around the vessels, intending to reduce or eliminate further angiogenic response. It is proposed that new blood vessels are formed

Studies 31

in response to local tissue hypoxia, but it is not possible at this time to identify focal areas of hypoxia in the retina. Oxygen Enterprises has developed a safe, accurate and robust method that is capable of detecting focal hypoxia in tissues. Through this Phase I SBIR Grant, the company proposes to apply their technology to the understanding and measuring oxygen in the retina of the eye. Initial application will be in biomedical research. Longer term goals will be to apply this technology into clinical practice, allowing clinicians to diagnose and treat patients with diabetic retinopathy earlier and more effectively. The company proposes to develop a phosphorescence lifetime imaging system that will provide an opticalmethod for the clinician that yields a topographic map, clearly identifying focal areas of hypoxia and differentiating them from areas of normoxia. During Phase 1 the company proposes to synthesize gram quantities of a new Oxyphors (phosphor) for suitable for pre-clinical testing and to construct a minimally invasive imaging system for use in ophthalmological research. The imaging system will be assembled and adapted for measuring oxygen in the retina of two animal models, piglets and rats, and the existing imaging software will be revised into a technically competent and user-friendly operating system. Oxygen Enterprises' imaging system is expected to offer sensitive and specific detection and quantification of focal hypoxia in retinal tissue - which is anticipated to allow earlier and more accurate diagnosis of retinopathy - and a diagnostic system with high stability and reliability, providing a clear and unambiguous display of retinal pathology. Emphasis will be on making the system user friendly and on providing it at a reasonable cost.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: IMPROVING THE QUALITY OF DIABETES EYE CARE

Principal Investigator & Institution: Lee, Paul P.; Professor; Ophthalmology; Duke University Durham, Nc 27710

Timing: Fiscal Year 2003; Project Start 30-SEP-2003; Project End 31-AUG-2007

Summary: (provided by applicant): Up to 95 percent of the more than 6, 000 to 24, 000 new cases of blindness and the ten thousands of cases of visual impairment due to diabetes each year in the United States can be prevented. Most efforts to reduce the visual loss have focused upon raising the 50 percent rate of annual eye exams on the assumption that patients entering the eye care system will subsequently do well. However, new qualitative and quantitative data indicate that significant deficiencies exist in the competency (diagnostic accuracy and treatment patterns) and technical process quality of care (history taking and examination elements) of eye care providers. Community-Based optometrists and general ophthalmologists correctly staged the severity of retinopathy in our pilot study about a third of the time, while roughly 40 percent of both groups reported performing at least 60 percent of the recommended history and examination elements on patient visits. Our proposed study is a randomized controlled trial that seeks to compare to a control group the efficacy of two levels of structured interventions that directly address both competency and technical process quality. One intervention will be "low technology" with paper-based aids (posters with standard reference photos and structured visit forms). The Second "high technology" intervention uses similarly structured aids in a digital tool (PDA). The control group will be exposed to usual and customary continuing professional education and educational feedback. The proposed measures are readily implemented and are accepted by providers. Because the study encompasses both general (comprehensive) ophthalmologists and optometrists, it includes the major community providers of eye care for patients. Retina specialist will be excluded, since they provide the level of care that is the goal of improvement to be attained with these interventions. The insights

32 Diabetic Retinopathy

from the study results should be applicable to other areas of diabetes care and health care in general.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: INDUCED APOPTOSIS IN AGE RELATED MACULAR DEGENERATION

Principal Investigator & Institution: Ferguson, Thomas A.; Associate Professor; Ophthalmology and Visual Sci; Washington University Lindell and Skinker Blvd St. Louis, Mo 63130

Timing: Fiscal Year 2002; Project Start 01-AUG-1999; Project End 31-JUL-2005

Summary: (Applicant's Description) Neovascularization is the major cause of vision loss in patients with agerelated macular degeneration (AMD), diabetic retinopathy, and retinopathy of prematurity. AMD is the leading cause of blindness in the Western world in individuals over 60 years of age. Since a large proportion of the population is living well beyond this age, this is a significant threat to the quality of life in elderly people. In patients with AMD new vessel growth (angiogenesis) beneath the retina from the underlying choroid (choroidal neovascularization or CNV) is the major or cause of severe visual loss in these patients. We recently examined the role of apoptosis in controlling new vessel growth in the eye by examining the function of two molecules, Fas (CD95) and FasL (CD95L). Our studies revealed that FasL plays a significant role in controlling CNV, where FasL+ retinal pigment epithelial cells (RPE) prohibit the growth and development of new Fas+ subretinal vessels that damage vision. Studies described in this proposal are designed to thoroughly understand the role of Fas/FasL and apoptosis in the pathogenesis of AMD. We propose 5 aims. Aim 1 we will more completely evaluate the role of Fas/FasL in CNV in a mouse model using normal, Fas, and FasL defective mice. In Aim 2 we will study cell endothelial cells derived from the choroid and compare these to endothelial cells derived from other areas. We will examnine cell death, proliferation, and differentiation using in vitro models and characterize the role of the Fas antigen in these processes. Aim 3 will contains experiments to explore the function of FasL on RPE cells and determine how growth factors and MMP inhibitors can affect FasL function in these cells that are crucial in controlling CNV. Aim 4 will explore potential treatment modalities in CNV applying the knowledge we have gained concerning the regulation of FasL expression to the animal model. Finally, studies in Aim 5 will evaluate clinical specimens from patients AMD for Fas/FasL expression. Our studies should provide important insights into one of the leading causes of blindness in the western world.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: INTRACELLULAR GLYCATION AND DIABETIC COMPLICATIONS

Principal Investigator & Institution: Brownlee, Michael A.; Anita and Jack Saltz Professor; Medicine; Yeshiva University 500 W 185Th St New York, Ny 10033

Timing: Fiscal Year 2002; Project Start 01-APR-1984; Project End 31-JUL-2006

Summary: The long-term objectives of the proposed work are to elucidate the mechanisms by which hyperglycemia-induced intracellular reactive oxygen species (ROS) produce diabetic retinopathy. The specific research proposed in this application will elucidate the role played by ROS-induced glyoxalase I substrates, which are precursors of intracellular advance glycation endproducts and mediators of hyperglycemia-induced angiopoietin-2 gene expression in retinal Muller cells. Specific Aim 1 will evaluate the effect of hyperglycemia-induced intracellular reactive oxygen

Studies 33

species (ROS) on glyoxalase I substrates, advanced glycation endproducts derived from these substrates, and diabetic retinopathy in uncoupling protein-2 (UCP-2) knockout and glyoxalase I transgenic mice. Streptozotocin diabetes will be induced in UCP-2 knockout, glyoxalase I transgenic, and wild type mice. Retinal concentrations of intracellular oxidative stress products, ROS-induced glyoxalase I substrates, and glyoxalase-I substrate-derived AGEs will be determined in diabetic and non-diabetic mice. Retinopathy will be assessed by semi-quantitative RT-PCR and in situ hybridization at early time-points, and by quantitative morphology at late time-points. Specific Aim 2 will evaluate the effect of inhibition of hyperglycemiainduced ROS on glyoxalase I substrates, advanced glycation endproducts derived from these substrates, and diabetic retinopathy in uncoupling protein-2 (UCP-2) knockout, glyoxalase I transgenic, and wild type mice. Streptozotocin diabetes will be induced in UCP-2 knockout, glyoxalase I transgenic, and wild type mice. In selected groups of these animals, hyperglycemia-induced intracellular ROS will be inhibited by treatment with either MnTBAP or EUK8, structurally distinct SOD/catalase mimetic compounds. Retinal endpoints will be assessed as described in Specific Aim 1. Specific Aim 3 will characterize the mechanism of Angiopoietin 2 transcriptional control by glyoxalase I substrates in cultured primary retinal Muller cells. A hyperglycemia-responsive mouse angiopoietin-2-luciferase reporter vector has been constructed which contains 2.5kb 5'- flanking sequence. Progressive deletions from the 5' end of the Ang-2 promoter will be constructed using restriction endonucleases or PCR methods. The glyoxalase-I substrate responsive region will be identified, and known transcription factor binding sequences altered by sitedirected mutagenesis. EMSA will be performed with indicated consensus oligonucleotides from the glyoxalase-I responsive region. Supershift experiments will use commercially available antibodies. IP-westerns of cell extracts will be immunoblotted with antibodies to glyoxalase I-substrate- derived advanced glycation endproducts.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: LIVE MICROSCOPY AND CYTOMETRY IN VASCULAR BIOLOGY

Principal Investigator & Institution: Lin, Charles P.; Massachusetts General Hospital 55 Fruit St Boston, Ma 02114

Timing: Fiscal Year 2002; Project Start 01-SEP-2002; Project End 31-AUG-2007

Summary: (provided by applicant): This is a multidisciplinary, collaborative research program bringing together investigators from several institutions to work across technology and disciplinary boundaries. The group shares a common interest in vascular biology, particularly in the eye, and specifically in the application of modern optical technology to answer critical questions related to vascular biology. The technology platform will be based on the scanning laser ophthalmoscope and the realtime in vivo confocal microscope previously developed at Schepens Eye Research Institute and at the Wellman Laboratories of Photomedicine. The existing technology will be enhanced with new development to improve image resolution, contrast, sensitivity, methods for quantification, and flexibility of imaging in living animals. Specific questions to be addressed include: 1. What are the cellular processes governing normal vascular development and stabilization? 2. What are the factors governing angiogenesis, Iymphangiogenesis, and immune cell trafficking? 3. What are the cellular mechanisms for the development of sickle cell and diabetic retinopathy? 4. Can we visualize early changes in the retinal pigment epithelium noninvasively in vivo? 5. Can we detect circulating cells in vivo without drawing blood? Is the number of circulating tumor cells a good predictor for tumor burden and response to therapy? Imaging at the

34 Diabetic Retinopathy

cellular level will enable biologists to study problems in living animals over time, gaining physiological insights beyond what can be obtained by classic static measurement (histology, immunocytochemistry, etc.), substantially reducing the number of animals required to answer these critical questions.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: LONGITUDINAL STATISTICAL METHODS FOR OPHTHALMOLOGY

Principal Investigator & Institution: Davidow, Amy L.; Prev Med and Community Health; Univ of Med/Dent Nj Newark Newark, Nj 07107

Timing: Fiscal Year 2002; Project Start 01-FEB-2001; Project End 31-JAN-2005

Summary: (Applicant's Abstract) The proposed project is a two year study to develop inferential and study design aspects of a linear statistical model appropriate to the analysis of longitudinal ophthalmologic data and to apply the improved model in a reanalysis of a data set obtained from the Early Treatment Diabetic Retinopathy Study (ETDRS). The relevant statistical model is the repeated-measures random effects/AR(l) model for continuous data due to Heitjan and Sharma. This model incorporates crosscorrelation between eyes and longitudinal correlation among measurements obtained from a single eye. The re-analysis of the ETDRS data set will consider the effects of aspirin on visual acuity measured continuously. A previously published analysis (ETDRS Report Number 8) analyzed a categorical version of visual acuity, ignoring longitudinal correlation and handling cross-correlation between eyes by analyzing eyes separately. The repeated measures random effects /AR(l) model has already been utilized by Heitjan and Sharma in a published study of intraocular pressure, using the expected information to perform statistical inference. Simulation studies have shown that it performs well in the balanced data case. Our first objective is to develop better inferential methods so as to 1) handle unbalanced (i.e., missing) data such as that arising when only one eye of two has the disease of interest and/or when the number of followup visits varies with subject, 2) control for the use of a possibly mis-specified variance structure, and 3) compensate for the use of estimated variance parameters in the standard error of the fixed effects, a procedure that may result in an underestimation of the standard error. This objective will be accomplished by the following: 1) replacing the expected information with the observed information, the observed information being a more reliable method of performing statistical inference in the presence of missing data,

2)using the robust (sandwich) variance estimator, a method that can control for a possibly mis-specified variance structure, and 3) approximating the degrees of freedom so as to control for the possibility of underestimated standard errors. Methodological advances will be incorporated into a computer package to be made available on the World Wide Web. Our second objective is to carry out power and sample size calculations for several different longitudinal ophthalmologic study designs, under various assumptions about the prevalence of bi-ocular versus uni-ocular disease, differential treatment allocation, loss-to-follow-up, and expected adherence to study treatment(s). The implications of treatments applied at the eye-level (e.g., photocoagulation) as well as systemically applied treatments (e.g., aspirin) on sample size, frequency of evaluation, and power will be considered.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: MATRIX METALLOPROTEINASES AND DIABETIC NEPHROPATHY

Principal Investigator & Institution: Thrailkill, Kathryn M.; Arkansas Children's Hospital Res Inst Research Institute Little Rock, Ar 72202

Studies 35

Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-JUL-2006

Summary: (provided by applicant): Matrix metalloproteinases (MMPs) are a family of zinc-dependent proteinases that are involved in the breakdown and remodeling of extracellular matrix (ECM). Dysregulation of MMP activity has been implicated in many pathologic processes characterized by degradation of connective tissue matrices, including rheumatoid arthritis, periodontal disease and metastatic cancer. Recent studies both in vitro and in animal models of diabetes suggest that hyperglycemiamediated alterations in MMP secretion, activation or action may also contribute to the development of diabetes-related complications including diabetic retinopathy and nephropathy. Based on these findings one can hypothesize that the mesangial accumulation and renal hypertrophy characteristic of diabetic nephropathy may result from reduced matrix degradation caused by a hyperglycemia-mediated suppression of renal MMP activity. In fact, preliminary clinical data from our laboratory confirm that in children with type 1 DM, serum MMP-2 concentrations are suppressed in the face of uncontrolled hyperglycemia, yet normalize with near-normalization of blood glucose levels. In the present study, we propose to investigate the hypothesis that MMPs are involved in the pathogenesis of diabetic nephropathy by measuring concentrations of specific MMPs (MMP-2, -8 and -9), concentrations of the naturally occurring inhibitors of MMPs (Tissue Inhibitor of Matrix Metalloproteinases, TIMP-1 and -2), and concentrations of the MMP-activated growth factor, insulin-like growth factor-I (IGF-I) in the serum and urine of patients with type 1 DM. We will examine levels of MMPs, TIMPs, and IGF-I in these biologic fluids among diabetic patient subgroups, ages 14-40 years, chosen to represent various time points in the natural history of d abet c nephropathy. Moreover, we will examine the correlation between observed differences in MMPFl'lMP/IGFconcentrations and differences in glycemic control at the time of study, as indicated by HbA1 c measurements and concurrent (72 hour) Continuous Subcutaneous Glucose Monitoring (CGMS). We anticipate that this study will provide preliminary evidence to establish a link between dysregulation of MMP activity and the pathogenesis of nephropathy in type 1 DM.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: MECHANISM OF MUTANT TIMP-3 INDUCED NEOVASCULARIZATION

Principal Investigator & Institution: Tolentino, Michael J.; Ophthalmology; University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Timing: Fiscal Year 2002; Project Start 01-APR-2001; Project End 31-MAR-2006

Summary: (Applicant's Abstract) The main objective of this proposal is to provide Dr. Tolentino a mechanism for rigorous training both technically and conceptually in gene therapy and molecular biology. Dr. Tolentino's long term goal is to become an independent investigator focusing on understanding the molecular mechanisms and developing treatments for pathologic ocular neovascularization seen in blinding conditions such as macular degeneration and diabetic retinopathy. The training plan proposed is essential for Dr. Tolentino to successfully pursue these long-term goals. The broad long-term research objective of this grant proposal is to understand the biochemical mechanisms that lead to choroidal neovascularization (CNV). The development of CNV in age related macular degeneration (AMD) is the leading cause of blindness in the United States. Patients with Sorsby's fundus dystrophy, an autosomal dominant form of macular degeneration, have similar clinical findings to AMD including CNV. These patients have been found to have a mutation in the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene. The specific aims of this study are:

36 Diabetic Retinopathy

Specific aim 1: To test the hypothesis that CNV induced by mutant TIMP-3 (mTIMP-3) is caused by loss of functionality or direct stimulation of angiogenesis by mTIMP-3. To accomplish this aim, mTIMP-3 transfected endothelial cell will be assayed for their ability to proliferate, form tubes and migrate to angiogenic stimuli in vitro. Sub retinal injections into normal and transgenic mice of adenovirus expressing mTIMP-3 will be performed to determine mTIMP-3's ability to cause sub retinal neovascularization in vivo. Specific aim 2: To test the hypothesis that upregulation of vascular endothelial growth factor (VEGF) and/or down regulation of pigment epithelial derived growth factor (PEDF) is involved in mTlW-3 induced CNV. Both mTIMP-3 transfected retinal pigment epithelial (RPE) cells and a transgenic mouse model with a human TIMP-3 mutation that develops CNV will be assayed for protein and mRNA upregulation using ELISA, northern blot, immunohistochemical and in situ hybridization techniques. Specific aim 3: To develop and test gene vectors expressing inhibitors of angiogenesis (angiostatin, endostatin, PEDF) and test their ability to inhibit subretinal neovascularization in the mutant transgenic mouse.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: MECHANISMS OF EPITHELIAL ALTERATIONS IN DIABETIC CORNEA

Principal Investigator & Institution: Ljubimov, Alexander V.; Director; Cedars-Sinai Medical Center Box 48750, 8700 Beverly Blvd Los Angeles, Ca 900481804

Timing: Fiscal Year 2002; Project Start 01-AUG-2001; Project End 31-JUL-2005

Summary: (provided by applicant): Diabetic retinopathy has been the leading cause of blindness in the United States since 1974. It is manifest by progressive changes in the microvasculature of the diabetic eye, leading to intravitreal hemorrhages, retinal edema, neovascularization, and detachments. Along with the retina, cornea, lens and iris are also affected by diabetes. Many diabetics suffer from diabetic keratopathy that includes recurrent erosions, epithelial fragility, abnormal wound healing and increased susceptibility to injury. Altered epithelial-stromal interactions and epithelial basement membrane (BM) defects likely contribute to diabetic keratopathy. Despite clinical importance of diabetic corneal disease, the molecular mechanisms of this complication are not understood. In our preliminary studies, the expression of many BM components and proteinases has been analyzed in normal and diabetic human corneas. We show that: 1. Diabetic retinopathy (DR) corneas have severely decreased epithelial BM immunostaining for laminin-1, laminin-10, nidogen-1/entactin, and for epithelial integrin alpha3 beta1; 2. Gene expression of BM proteins and integrin alpha3 beta1 is not changed in diabetic and DR corneal epithelium; 3. Gene and protein expression of matrix metalloproteinase (MMP)-10 increases in diabetic and DR corneal epithelium and stroma, and MMP-3 expression increases in diabetic and DR corneal stroma. The data suggest that major components of corneal epithelial BM are altered in diabetes and especially DR due to elevated activity of specific proteinases, e.g., of MMP-1O that is expressed in the epithelium. Our hypothesis is that corneal epithelial BM in diabetes and DR undergoes degradation by elevated proteinases, notably by MMP-10. Proteinase expression and activity may be stimulated by specific growth factors activated by diabetic microenvironment. These alterations may constitute the molecular mechanism of corneal epithelial abnormalities in diabetes. Specific Aim 1.To characterize the effect of MMP-10 on the integrity of corneal epithelial BM and integrin alpha3 beta1 and on wound healing in organ-cultured human corneas. Specific Aim 2. To identify by gene array analysis growth factors and cytokines abnormally expressed in diabetic and DR corneas and examine their effects on MMP-10 and wound healing in normal organ-

Studies 37

cultured corneas. Specific Aim 3. To assess by gene array analysis the expression levels of various proteinases in diabetic, DR and normal human corneas. Identify and analyze additional proteinases with elevated expression in diabetic corneas. Specific Aim 4. To attempt blocking BM and integrin degradation in human diabetic and DR organ-culture corneas. Neutralizing antibodies to specific growth factors and proteinases (primarily, MMP-10), and various MMP inhibitors including clinically approved tetracyclines will be tested in organ-cultured corneas. These studies could lead to the development of novel therapeutics that would block the progression of diabetic keratopathy.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: MECHANISMS OF PAI-1 INDUCED ANTI-ANGIOGENESIS

Principal Investigator & Institution: Mulligan-Kehoe, Mary J.; Surgery; Dartmouth College 11 Rope Ferry Rd. #6210 Hanover, Nh 03755

Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2007

Summary: (provided by applicant): Endothelial cells lining the vascular wall are normally maintained in a differentiated, quiescent state by anti-angiogenic molecules. Pro-angiogneic molecules destabilize the quiescent endothelium into migratory, proliferative endothelial cells that form new capillary blood vessels. The steady turn over rate of the vascular endothelium is maintained by a tightly controlled balance of proand anti-angiogenic molecules that have cell survival and death functions, respectively. A shift in the balance can alter the turn over rate of the endothetium and disrupt vascular homeostasis. It has been shown that vascular endothelial growth factor (VEGF) has a role in atherosclerotic plaque development. This is supported by studies which show that angiogenesis is associated with increases in plasmin and metalloproteinase activity which increases plaque susceptibility to acute rupture or plaque destabilization. Several animal studies have shown a reduction in atherosclerotic plaque neovascularization and plaque growth after treatment with angiogenesis inhibitors. Plasminogen activator inhibitor-1 (PAl-1) is over expressed in the intimal layer in the atherosclerotic vessel wall in human coronary artery disease. This has led to one hypothesis that PAl-1 expression is a means of controlling localized plasminogen activators that contribute to extracellular matrix degradation and smooth muscle cell migration. We have constructed recombinant truncated PAl-1 (rPAI-1) proteins to examine the anti-angiogenic activity of PAl-1 in "inactive" conformations (absence of reactive center loop). One rPAI-1 protein, rPAI-123, has potent anti-angiogenic activity. The activity of rPAI-123 induces cleavage of plasmin into angiostatin and inhibits the bioavailability of VEGF. In this study, we will utilize rPAI-123 to: Aim I: Clarify the rPAI-123 interactions that induce cleavage of plasmin into angiostatin; Aim II: Determine if rPAI-123 inhibits VEGF release from heparan sulfate; and Aim Ill: Define the anti-angiogenic/pro-apoptotic signaling pathways in rPAI-123 treated aortic endothelial cells. Due to the potent anti-angiogenic activity of rPAI-123 protein, it may ultimately play a useful role in the treatment of diiseases characterized by excessive angiogenesis, such as progression and stabilization of atherosclerotic plaques, diabetic retinopathy, and certain types of cancer.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: MECHANISMS OF PHOTORECEPTOR DEATH IN PHOTIC INJURY

Principal Investigator & Institution: Dunaief, Joshua L.; Assistant Professor; Ophthalmology; University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Timing: Fiscal Year 2002; Project Start 01-JUL-2000; Project End 31-MAY-2005

38 Diabetic Retinopathy

Summary: Age related macular degeneration, the most common cause of blindness among people aged 50 and older in the United States, results in photoreceptor degeneration. Similarly, retinitis pigmentosa, retinal detachment and ischemic diabetic retinopathy all lead to photoreceptor death. The loss of photoreceptors is the ultimate cause of significant visual loss. The mechanism of photoreceptor degeneration in these diseases is poorly understood, but is known to occur through apoptosis. This programmed cell death is a highly ordered and regulated cellular suicide pathway that has been well defined in lymphocytes. This application proposes to draw from the rich knowledge of apoptosis in lymphocytes to elucidate mechanisms of photoreceptor cell death in the photic injury animal model of retinal degeneration. This model has been studied extensively at the cellular but not yet at the molecular level. Good evidence suggests that photoreceptor degeneration in this model occurs through apoptosis. The ability of anti-apoptotic genes expressed in transgenic mice to inhibit photic injury induced cell death will be tested. Specifically, the ability of anti-oxidant genes and antiapoptosis genes that act upstream or downstream in apoptosis pathways to inhibit photoreceptor degeneration will be evaluated. Further, the intracellular localization of cytochrome c, a mediator of apoptosis, and the role of caspase activation will be probed. Caspases are proteases involved in a number of apoptotic pathways. These studies will define critical apoptotic pathways and suggest therapeutic interventions for the blinding disorders that result from photoreceptor degeneration. The proposed study is well within the realm of feasibility. The principal investigator has experience with molecular biology, transgenic mice and retinal histology. The mentor is an international leader in the molecular mechanisms of apoptosis, and the comentor has extensive experience with transgenic mouse models of retinal disease and gene therapy. This proposal should serve as a good launching pad for the PI's career devoted to understanding the molecular basis of photoreceptor degeneration.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: MICROENVIRONMENT OF THE RETINA

Principal Investigator & Institution: Linsenmeier, Robert A.; Professor; Biomedical Engineering; Northwestern University 633 Clark Street Evanston, Il 60208

Timing: Fiscal Year 2002; Project Start 01-JUL-1983; Project End 31-JUL-2007

Summary: (provided by applicant): The long-term objectives of this work are to understand aspects of the retinal microenvironment related to oxygen and pH, and how these relate to energy metabolism and function of the mammalian retina in vivo. While this work will be done in animals, it is particularly relevant to blinding diseases that affect the relationships between the circulation and retinal neurons in humans. During the next project period, our main interests are in diabetic retinopathy, retinal detachment, and retinal arterial occlusive disease. However, the results will also provide fundamental information that may be relevant to other types of retinal dysfunction. The proposed work will be done primarily on intact anesthetized cats, since their retina provides a good model for much of the human retina. The techniques are primarily to use oxygen and pH sensitive microelectrodes to map out retinal oxygen levels, pH and electrical activity (the electroretinogram) with high spatial and temporal resolution, as we have done previously under other experimental conditions. Following the measurements, mathematical modeling of diffusion will be used to extract metabolic parameters that are not apparent from the measurements alone, and to perform simulations of situations that may not be amenable to experimentation. Some measurements of retinal histology will also be made. The project has 5 specific aims. 1) We will study intraretinal oxygenation following photocoagulation, because the

Studies 39

mechanism by which photocoagulation blocks neovascularization is still unclear. 2) We will use information about oxygenation after photocoagulation from specific aim 1 to create an appropriate two-dimensional diffusion model of this situation, with the hope of providing a better rationale for the density and size of lesions designed to treat retinopathy. 3) We will study retinal oxygenation in the detached retina in order to understand the basis for the protective effect of hyperoxia in retinal detachment, which has been shown recently in cats. 4) We will study pH after retinal arterial occlusion, to understand the potential role of acidosis in damaging the retina. 5) We will investigate the influence of anesthesia on the metabolic measurements we make, and will study metabolic differences between the cat and primate retinas.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: MOLECULAR AND CELLULAR MECHANISMS OF VASCULAR ANOMALIES

Principal Investigator & Institution: Olsen, Bjorn R.; Hersey Professor of Cell Biology; Oral and Developmental Biology; Harvard University (Medical School) Medical School Campus Boston, Ma 02115

Timing: Fiscal Year 2003; Project Start 15-SEP-2003; Project End 31-MAR-2008

Summary: (provided by applicant): This Program Project, entitled '"Molecular and Cellular Mechanisms of Vascular Anomalies," represents the concerted and collaborative efforts of three research groups, two in Boston and one in Brussels, Belgium, to elucidate the causes and abnormal mechanisms that are responsible for vascular anomalies in the skin. Commonly called birthmarks, these anomalies include infantile hemangioma, a vascular tumor found in 5-10% of Caucasian children at 1 year of age, and vascular malformations. Hemangiomas usually appear a few days after birth, grow rapidly for a few weeks to months, and then slowly regress over a 5-10 year period. In most cases, no treatment is needed, but sometimes vital, structures can be obstructed or distorted causing serious problems. In contrast to hemangiomas, malformations do not regress, but grow with the child, and can become life-threatening. The investigators have recently found that hemangiomas contain clonal expansions of abnormal endothelial cells, and they have discovered two types of mutations that cause localized abnormalities in the skin of patients with venous malformations and glomuvenous malformations. In three research Projects, supported by three Cores, the Program investigators propose to examine and test the hypotheses that hemangiomas result from somatic mutations in genes that control endothelial cell proliferation and/or maturation from precursor cells, causing rapid growth of abnormal capillaries. In addition to identifying such genes and mutations, it is also proposed to identify genes responsible for rare cases of inherited hemangiomas and to establish mouse models allowing further studies of detailed pathological mechanisms. Finally, they now propose to generate mouse models for venous and glomuvenous malformations, characterize the cellular and molecular consequences of the causative mutations, and search for mutations in additional families. The proposed studies should lead to a better understanding of the pathogenesis of hemangiomas and malformations and therefore a basis for development of effective therapies. In addition, a better understanding of the causes and mechanisms of vascular anomalies will provide novel insights into blood vessel formation and growth. This will add significantly to the efforts to develop novel antiangiogenic therapies for cancer, diabetic retinopathy, and rheumatoid arthritis.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

40 Diabetic Retinopathy

•Project Title: MOLECULAR MECHANISMS OF MEKK3-SIGNALING IN ANGIOGENESIS

Principal Investigator & Institution: Su, Bing; Associate Professor; Immunology; University of Texas Md Anderson Can Ctr Cancer Center Houston, Tx 77030

Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2007

Summary: (provided by applicant): Angiogenesis is essential for normal physiological processes such as organ development and wound healing. It is also critical to pathological processes such as tumor growth, atherosclerosis, rheumatoid arthritis, and diabetic retinopathy. Therefore, blocking angiogenesis could be a powerful therapeutic intervention for treating diseases requiring formation of new blood vessels. However, the underlying molecular mechanism of intracellular signal transduction in this process remains largely unexplored. We recently generated Mekk3-knockout mice through homologous recombination and demonstrated that MEKK3, a Ser/Thr protein kinase belonging to the mitogen-activated protein kinase (MAPK) kinase gene family, is essential for angiogenesis. The long-term goals of this study are to elucidate the molecular mechanisms of angiogenesis regulated by MEKK3 signaling. Our SPECIFIC AIM 1 is to determine the function of MEKK3 in endothelial cells (ECs) during embryonic development by examining the morphology, proliferation and apoptosis of ECs in the E8.5 to E10 wild-type, Mekk3+/- and Mekk3-/- fetuses. We will also isolate embryonic ECs from the E9-E9.5 fetuses and establish EC lines by using polyoma middle T antigen to transform the primary cultures. In SPECIFIC AIM 2, we will determine whether MEKK3 is a specific upstream activator of JNK1/2, ERK1/2, p38 and ERK5 MAPKs in ECs by using in vitro kinase assays. We will determine whether the induction of these MAPKs is defective in Mekk3-/- embryos, purified ECs and EC lines. In addition, we will determine whether MEKK3 is specifically activated by angiogenic stimulation by using in vitro kinase, in-gel kinase and MEKK3 phosphorylation assays. Furthermore, we will isolate and clone MEKK3-associated proteins by coprecipitation, chromatography, and yeast two-hybrid screening. In Specific Aim 4, we plan to generate Mekk3 floxed ES cells and mice, and use these mice to create EC-specific Mekk3 knock out (Mekk3 (EC-null)) mice and Mekk3-deficient ECs with Cre recombinase. Function of MEKK3-signaling will be studied specifically in ECs using these mice. In addition to revealing the MEKK3-signaling in angiogenesis, the outcome from this study will provide conceptual and material resources for studying MEKK3 and its homologues in many other physiological and pathological processes. Most importantly, this study may discover novel molecular targets for therapeutic intervention for treating diseases requiring formation of new blood vessels like cancer.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: MONOCYTE VEGF & PKC, MARKERS FOR DIABETIC COMPLICATIONS

Principal Investigator & Institution: King, George L.; Professor and Research Director; Joslin Diabetes Center Boston, Ma 02215

Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-JUL-2004

Summary: (provided by applicant): Hyperglycemia is the major cause of diabetic microvascular complications including retinopathy, nephropathy and neuropathy. Early diagnosis of microvascular complications in these tissues has been difficult due to the long duration of disease required for the onset of clinical symptoms and the general inaccessibility of vascular and neurological tissues for analysis. Animal studies have clearly demonstrated that microvascular pathologies in the retina and renal glomeruli

Studies 41

can manifest years before the onset of clinical symptoms. Activation of protein kinase C (PKC), specifically the B isoforms and increases in VEGF levels in the retina and renal glomeruli have been shown to correlate with severity of hyperglycemia induced vascular pathologies in diabetic animals. However, due to tissue access problems, almost all of the data regarding the increases in PKC activities and VEGF levels have been measured in vascular tissues from animal models of diabetes. Plasma levels of PKC do not exist and plasma VEGF levels do not consistently correlate with corresponding levels in the microvascular tissues. Recently, we have measured PKC activities in the circulating mononuclear cells and they correlated with the severity of diabetic retinopathy and nephropathy in type 1 and 2 diabetic patients. In addition, the expression of VEGF levels in the circulating monocytes from humans and diabetic rats can be increased by PKC activation. Similarly, increases in PKC activation and, potentially, VEGF expression in the circulating monocytes responded in parallel to diabetes in the vascular tissues of retina, renal glomeruli and arteries. In addition, monocyte activation has been noted by multiple studies in the cardiovascular and microvascular circulations including the retina and renal glomeruli from diabetic animals. Thus, we are proposing to test the hypothesis that increases in PKC activation and VEGF levels in the circulating monocyte are similar to those found in the retina and renal glomeruli in response to hyperglycemia or diabetes. Therefore, the ability to measure VEGF levels and PKC activities in circulating monocytes, which can be easily accessed, could be used as surrogate markers of diabetic microvascular disease especially diabetic retinopathy and nephropathy. We are proposing to test three specific aims in diabetic and control patients 1) To correlate VEGF levels with PKC activity in circulating monocyte with severity of retinopathy and nephropathy. 2) To determine whether glycemic control can alter the activation of PKC and VEGF levels in circulating monocytes. 3) To correlate PKC activity and VEGF levels in circulating monocytes with VEGF level in the ocular fluids obtained during surgery. These data can provide definitive evidence to determine whether further studies are needed to establish these two parameters in the circulating monocyte as surrogate markers of diabetic microvascular diseases.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: NEUROTRANSMITTER TRANSPORTERS IN THE RETINA

Principal Investigator & Institution: Sarthy, Vijay P.; Magerstadt Professor; Ophthalmology; Northwestern University Office of Sponsored Research Chicago, Il 60611

Timing: Fiscal Year 2002; Project Start 01-AUG-2000; Project End 31-JUL-2004

Summary: (Adapted from applicant's abstract): As the premier excitatory synaptic transmitter, glutamate has the potential to influence the function of most neuronal circuits in the retina. In addition, glutamate can also act as a potent neurotoxin when present at a high level in the retinal microenvironment. Therefore, extracellular glutamate needs to be maintained at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and to protect neurons from excitoxic damage and cell death. The long-term objective of the proposed project is to identify mechanisms that operate to maintain glutamate levels in the retina. Extracellular glutamate level is normally kept low by the action of potent uptake systems present in retinal neurons and Muller (glial) cells, and inadequate clearance of glutamate may result in excitotoxic neuronal loss. Although glial glutamate transporters are known to play a major role in glutamate uptake, their specific contribution to glutamate homeostasis has not been directly examined because glial-specific, glutamate uptake inhibitors are not available.

42 Diabetic Retinopathy

Mice with targeted disruption of glial glutamate transporter genes, GLT-1- and GLAST, provide an alternative means to investigate the function of glial glutamate transporters. The present proposal is concerned with neurochemical and immunolocalization studies in GLAST-knockout mice, and tests the hypothesis that GLAST plays a crucial role in regulating retinal glutamate levels, and in metabolic trafficking in the retina. The specific goals of the proposal are to determine whether normal metabolic signaling, and metabolite transfer from Muller cells to photoreceptors is disrupted by loss of GLAST; to examine whether GLAST-deficiency provokes compensatory changes in other glutamate transporters; to determine whether intracellular and extracellular glutamate levels are altered in GLAST-knockout mice; to determine whether NMDA and AMPA glutamate receptor expression is modified in GLAST-null mice; and finally to examine whether loss of GLAST leads to major changes in GABAergic neurons in the retina. Because extracellular glutamate levels have been reported to be elevated in glaucoma and diabetic retinopathy, the proposed studies are crucial for elucidating the cellular mechanisms responsible for elevation in glutamate levels in the retina, and for considering glutamate transporters as potential therapeutic targets.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: NEW STATISTICAL METHODS FOR DEMOGRAPHIC DATA ANALYSIS

Principal Investigator & Institution: Wahba, Grace; Bascom and I.J Schoenburg Professor of s; Biostatistics/Med Informatics; University of Wisconsin Madison 750 University Ave Madison, Wi 53706

Timing: Fiscal Year 2002; Project Start 01-DEC-1992; Project End 31-DEC-2002

Summary: (Adapted from the Applicant's Abstract): This research is for the further development of a new class of multi variate semi-parametric model building methods' known collectively as Smoothing Spline Analysis of Variance, (SS-ANOVA) which are suitable for the analysis of data from large cohort studies, either epidemiologic or clinical trials, with many qualitatively different variables observed over several time points. These methods represent an attempt to obtain flexible empirical relationships between multiple complex responses and predictors. If such models can be fitted to the data, then estimated sensitivities of the responses to various predictors can be obtained and the existence of associations between various variables of interest can be tested. The models reduce to standard parametric models if the data suggest that nonparametric terms in the model are not present. SS-ANOVA models have been built and tested for the prediction of multi variate and multi categorical responses and methods developed which allow the analysis of large complex data sets. The investigators will extend this work in several directions: Development of methods to prescreen large, complex data sets for patterns of relationships that warrant further examination; more sophisticated model selection methods, extension to nonparametric multi variate density estimation for the purpose of uncovering conditional and time dependent relationships among the variables, and the development of threshold models. Data from the Wisconsin Epidemiological Study of Diabetic Retinopathy and the Beaver Dam Eye Study will be used to examine the models under study for their reasonableness and for their ability to answer questions meaningful to the study scientists. The results will have broad applicability to other large epidemiological studies as well as to clinical trials. The research software will be developed into a user friendly form, documented, and made publicly available.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

Studies 43

•Project Title: NON-COX ARACHIDONIC ACID METABOLITES AND ANGIOGENESIS

Principal Investigator & Institution: Rao, Gadiparthi N.; Associate Professor; Medicine; University of Tennessee Health Sci Ctr Memphis, Tn 38163

Timing: Fiscal Year 2004; Project Start 01-APR-2004; Project End 31-MAR-2008

Summary: (provided by applicant): Inflammation that follows tissue injury is believed to be important in the initiation and progression of various diseases including, atherosclerosis, cancer, and retinopathy. Phospholipase A2s (PLA2s), a group of enzymes that breakdown phospholipids generating arachidonic acid and lysophospholipids have been implicated in inflammation. One of the major events underlying the progression of atherosclerosis is angiogenesis. Endothelial cell (EC) migration and proliferation are critical events in angiogenesis. Emerging evidence suggests that PLA2, arachidonic acid and its eicosanoid metabolites play a role in the regulation of cell migration, proliferation, and apoptosis. In addition, recent investigations using nonsteroidal anti-inflammatory drugs reveal a potential role for eicosanoids in angiogenesis. Based on this knowledge, we hypothesize that eicosanoids, particularly the lipoxygenase-monooxygenase metabolites of arachidonic acid, play an important role in angiogenesis and thereby influence the pathogenesis of atherosclerosis. To test the role of eicosanoids in angiogenesis we will address the following four specific aims: 1. To identify eicosanoids produced in human microvascular endothelial cells (HMVEC) and determine their effects on angiogenesis using in vitro and in vivo models. 2. To determine the effects of angiogenic eicosanoids on HMVEC migration and proliferation. 3. To test the role of the Jak/STAT and PI3K/Akt pathways in angiogenic eicosanoid-induced HMVEC migration and proliferation. 4. To identify the effector molecules of eicosanoid-induced angiogenesis and study the mechanisms underlying their regulation of expression in HMVEC and vascular smooth muscle cells. The results of this proposal will provide novel information on the identification of specific angiogenic eicosanoids and on elucidation of the underlying mechanisms by which these lipid molecules stimulate angiogenesis. Such knowledge, in turn, could be useful in developing therapeutics in the prevention of progression of diseases such as atherosclerosis.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: NONPEPTIDE SOMATOSTATIN AGONISTS FOR RETINOPATHY

Principal Investigator & Institution: Shapiro, Gideon; Somatocor Pharmaceuticals, Inc. Ste 125 Alachua, Fl 32615

Timing: Fiscal Year 2003; Project Start 01-FEB-2003; Project End 31-JAN-2004

Summary: (provided by applicant): Diabetic retinopathy (DR) is the leading cause of blindness in the United States. The disorder is characterized by aberrant neovascularization ultimately leading to blindness. Currently, there is no drug treatment for DR patients and pannretinal laser coagulation surgery is the only option to delay blindness. Somatostinergic drugs are growth factor inhibitors that offer the potential to treat a probable cause of diabetic retinopathy by blocking key mediating steps in disease progression. Clinical trials with somatostatin peptide drugs in DR patients indicate that somatostatinergic drug therapy can stop neovascularization and improve visual acuity. However, the clinical results have been highly variable and have been best for patients receiving high dosage regimens or continuous parenteral treatment. These results are consistent with an inadequate blood-retinal barrier penetration of somatostatin peptide drugs to reach target retinal tissues. New non-

44 Diabetic Retinopathy

peptide lipophilic somatostatin receptor agonists described herein have the potential to effectively penetrate the BRB. Using pharmacological in vitro and animal testing models of DR this study aims to establish structure activity relationships for this innovative class of compounds. The final goal is to identify a small potent somatostinergic molecule that readily accesses target retinal tissue for clinical testing in DR patients.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: NOVEL DIAGNOSTICS WITH OPTICAL COHERENCE TOMOGRAPHY

Principal Investigator & Institution: Fujimoto, James G.; Professor of Pharmacology; Center for Cancer Research; Massachusetts Institute of Technology Room E19-750 Cambridge, Ma 02139

Timing: Fiscal Year 2002; Project Start 01-SEP-1985; Project End 31-MAY-2005

Summary: (Adapted from applicant's abstract): This is a collaborative multi-disciplinary program involving investigators at the Massachusetts Institute of Technology and the New England Eye Center. Optical Coherence Tomography (OCT) is an emerging technology for ophthalmic performance that can perform non-contact, non-invasive real time cross sectional imaging of the retina and the anterior eye. Standard ophthalmic OCT has a 10 micron axial resolution, ten times higher than conventional ultrasound. Our objective is to develop new OCT technology, which achieves a quantum leap in performance over current ophthalmic OCT and to demonstrate this technology for the early diagnosis and monitoring of disease of the retina and anterior eye. We will increase OCT axial image resolution by 5 to 10 times from 10 microns to about 1-2 microns. And also develop multi-wavelength, spectroscopic OCT techniques to improve differentiation of morphology and permit micron scale functional imaging. Our hypothesis is that these advances will dramatically enhance ability to image structural morphology such as intraretinal features, as well as improve the accuracy in reproducibility of morphometric measurements such as retinal thickness or nerve fiber layer thickness. We propose to: 1. Develop ultra high resolution OCT technology for imaging the retina and anterior eye. 2. Image transgenic retinal mouse models and cross sectional and longitudinal studies. 3. Perform cross sectional patient studies to investigate the capabilities of ultra-high resolution OCT for identifying micro-structural changes associated with posterior segment diseases. 4. Investigate ultra high resolution OCT for improving the accuracy of morphometric imaging of retinal thickness and nerve fiber layer thickness. 5. Investigate imaging structures in the anterior eye at the cellular level. 6. Perform preliminary clinical studies to investigate the ability of ultra high resolution OCT to identify ocular pathologies in the anterior eye segment. 7. Develop and demonstrate spectroscopic OCT techniques enabling spectrally resolved tomographic imaging on the micron scale. 8. Demonstrate spectroscopic OCT imaging of oxygen saturation in retinal and choroidal vasculature. 9. Perform preliminary clinical studies to assess the effectiveness of ultra high resolution structural, morphometric, or spectroscopic OCT imaging to diagnosis and monitor the progression of retinal disease. This proposal involves the development of new instrumentation which will have wide spread research and clinical applications as well as the development and validation of methods for the early diagnosis and monitoring of diseases including AMD, glaucoma, and diabetic retinopathy.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

Studies 45

•Project Title: OPTIMIZED RETINAL CAMERA

Principal Investigator & Institution: Truitt, Paul W.; Kestrel Corporation 3815 Osuna Ne Albuquerque, Nm 87109

Timing: Fiscal Year 2003; Project Start 01-APR-2000; Project End 31-MAY-2005

Summary: (provided by applicant): A low-cost, high-resolution, high-contrast color digital camera optimized for ophthalmology will be demonstrated. This Optimized Retinal Camera will be specifically tested for its effectiveness in meeting the image quality requirements for the screening and assessment of pre-proliferative and proliferative diabetic retinopathy in both traditional clinical settings and in telemedicine. The proposed device exploits recent technological advances in high sensitivity charge coupled device (CCD) cameras and digital signal processing electronics. Today's CCD cameras do not have the dynamic range to image the human retina. The human retina is characterized by regions of high reflectivity (20-40 percent), such as the optic disc, and very low reflectivity (<2 percent), such as the macula and fovea. Further, these existing digital cameras treat each of the color channels in the same manner and do not consider the special, red-saturated characteristics of the retina. The approach builds on existing fundus imaging technology developed by Kestrel for the National Eye Institute. The proposed Optimized Retinal Camera will be shown to offer significant improvement over existing digital color cameras by addressing each of the deficiencies mentioned above. Joslin Diabetes Center, the University of Iowa Department of Opthalmology, and the University of New Mexico Health Sciences Center will provide independent, "masked" evaluation of the optimized digital retinal images.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: PARACRINE REGULATION OF ANGIOGENESIS BY MURAL CELLS

Principal Investigator & Institution: Nicosia, Roberto F.; Professor; Pathology; University of Washington Grant & Contract Services Seattle, Wa 98105

Timing: Fiscal Year 2002; Project Start 01-APR-1995; Project End 31-MAR-2006

Summary: Angiogenesis plays a critical role in the revascularization of ischemic organs and in the progression of cancer, atherosclerosis, rheumatoid arthritis, and diabetic retinopathy. The outcome of angiogenesis depends on neovessel survival. Developing vessels uses the angiopoietin/Tie2 system to acquire a stabilizing layer of mural cells (smooth muscle cells/pericytes). The mechanisms regulating this process are, however, poorly understood because the Tie2 receptor is reportedly expressed in endothelial cells but not in mural cells. Using the rat aorta model of angiogenesis we found that Tie2 is transiently expressed in intimalderived mesenchymal cells that have the capacity to differently into mural cells. Tie2+ mural precursor cells migrate and secrete matrix metalloproteinases in response to Ang-1 which they produce, and Ang-2, which is produced by endothelial cells. Based on the additional observation that the intimal/subintimal layers of the rat aorta contain Flk- 1+ and Tie2+ non-endothelial mesenchymal cells and have angioinformative properties we postulate that Tie2+ muralprecursor cells arise from vascular progenitor cells capable of both endothelial and mural cell differentiation. We linked mural cell recruitment to the p38 MAPK signaling pathway by demonstrating that pharmacologic inhibition of p28, which is activated upon Tie2 stimulation, abrogates mural cell development resulting in naked neovessels. Based on these observations the specific aims of this grant focus on the following hypotheses. 1) The aortic wall. contains vascular progenitor cells capable of both endothelial and mural cell differentiation. 2. The muscular wall of blood vessels

46 Diabetic Retinopathy

originates from Tie2+ mural precursor cells. 3. Mural cell recruitment during angiogenesis is selectively mediated by the p38 MAPK pathway. Vascular progenitor cells of neonatal, young and old rat aortas will be identified by immunohistochemistry and confocal microscopy and isolated by surface marker-based magnetic beads technology. Their angioformative and mural cell differentiation properties will be studied in models of vascular organ culture, microvessel assembly and chemotaxis. The role of Tie2 and p38 MAPK pathway in mural cell recruitment will be studied by gene transfer technology using adeno-associated viral vectors carrying wild type or dominant-negative genes Mural cell recruitment will be analyzed by Immunohistochemistry, confocal microscopy and image analysis. Gene/protein expression and function will be evaluated by Northern and Western analysis, RT-PCR, kinase assays, gel zymography, in situ hybridization, and immunohistochemistry,. These studies will define key cellular and molecular mechanisms of vessel wall development. This knowledge may lead to novel approaches for the stabilization of neovessels in ischemic conditions, the induction of vascular regression in angiogenesisdependent disorders, and the bioengineering of blood vessels for therapeutic applications.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: PATHOGENIC MECHANISMS UNDERLYING DIABETIC RETINOPATHY

Principal Investigator & Institution: Medof, M Edward.; Pathology; Case Western Reserve University 10900 Euclid Ave Cleveland, Oh 44106

Timing: Fiscal Year 2004; Project Start 15-JUN-2004; Project End 31-MAY-2008

Summary: (provided by applicant): The pathogenic processes that give rise to microangiopathic retinopathy and other vascular complications in diabetes are poorly understood. Several new lines of evidence have pointed toward inflammation as playing a heretofore unrecognized role. Retinal cells and vasculature and other self cells are protected from attack by autologous complement proteins by a set of intrinsic membrane regulatory proteins. These regulators are the decay accelerating factor (DAF or CD55), the membrane cofactor protein (MCP or CD46), and the membrane inhibitor of reactive lysis (CD59). Previous studies from our lab have shown that all three surface proteins are highly expressed in the retina and its associated vasculature in levels similar to those on glomerular cells and on systemic vasculature, sites where intrinsic regulatory activity is critical. In recent in vitro work, we have shown that DAF's regulatory function is 1) >90% inactivated by incubation with glucose or ribose, 2) rapidly abrogated by methylglyoxal incubation, and 3) lost due to modification of one or more active site lysine and arginine residues, the latter to argprymidine. In in vivo work, we have shown that endogenous DAF protein isolated from diabetic retinas is modified by several sugar adducts including argpyrimidine. The proposed studies are directed toward 1) determining the functional effects of chemical modifications in DAF, MCP, (and CD59) that are induced by hyperglycemia and other metabolic abnormalities that pertain in the diabetic state, 2) structurally characterizing the types and sites of the modifications on the regulators, 3) analyzing the functions and structures of endogenous DAF, MCP, (and CD59) proteins isolated from retinas and other tissues of diabetics, and 4) examining whether the pathological changes that are associated with retinopathy and other diabetic complications develop more rapidly in Daf1-/- (murine DAF homolog), Crry-/- (murine MCP surrogate), CD59a-/- (murine CD59 homolog) and double Daf1-/- / -/- Curry-/- or Daf1-/- / CD59a-/- mice experimentally made diabetic with streptozotocln. Since vasculopathy and retinopathy are debilitating

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complications that eventually affect most patients with diabetes, fully understanding the mechanisms involved in their development is important in designing effective therapeutic interventions.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: PHYSIOLOGY OF RETINAL PERICYTES

Principal Investigator & Institution: Puro, Donald G.; Ophthalmology and Visual Sciences; University of Michigan at Ann Arbor 3003 South State, Room 1040 Ann Arbor, Mi 481091274

Timing: Fiscal Year 2002; Project Start 01-APR-1999; Project End 31-MAR-2007

Summary: (provided by applicant): Our long-range objective is to elucidate the pathophysiological mechanisms that disrupt microvascular function early in the course of diabetic retinopathy. A vital function lost soon after the onset of diabetes is the autoregulatory control of the retinal microcirculation. The resulting inefficiency in the distribution of blood flow compromises the function, and eventually, the viability of retinal neurons, glia and vascular cells. At present, a critical gap in our understanding of how diabetes alters microvascular function is the limited knowledge of the mechanisms by which local vasoactive signals regulate capillary perfusion in the retina. Our proposed studies are based on a new working hypothesis. Namely, voltage changes induced by vasoactive signals that act at distal capillary sites must be transmitted electrotonicly via gap junction pathways to proximal pericytes. This intercellular transmission is necessary because proximal, but not distal, pericytes contain the contractile apparatus necessary to regulate the diameter of the microvascular lumen. Using dual perforated-patch recordings and other techniques, we will compare intercellular communication within pericyte-containing microvessels freshly isolated from retinas of control and diabetic rats. To address the mechanisms by which diabetes disrupts distal-to-proximal communication within the retinal microvasculature, the specific aims of our proposed studies will test the hypotheses that (1) in diabetes a disruption of electrotonic transmission between retinal pericytes compromises the transduction mechanism by which a vasoactive signal acting at a distal capillary site elicits a contraction or relaxation of the proximal portion of a pericyte-containing microvessel, (2) PKC-beta isoforms play a critical role in the mechanism by which gap junction pathways are disrupted in microvessels of the diabetic retina and (3) ET/A endothelin receptors mediate disruption of intercellular communication within diabetic microvessels. Over the long-term, elucidating the mechanisms by which diabetes disrupts the ability of local vasoactive signals to regulate capillary perfusion will facilitate the development of new strategies to ameliorate, and hopefully, prevent sightthreatening complications of this disease.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: PILOT--CHEMICAL PROFILING OF ORPHAN NUCLEAR RECEPTORS

Principal Investigator & Institution: Downes, Michael R.; Baylor College of Medicine 1 Baylor Plaza Houston, Tx 77030

Timing: Fiscal Year 2002; Project Start 15-AUG-2002; Project End 31-JUL-2007

Summary: A solid clinical rationale exists for the discovery of novel orphan nuclear receptor (ONR) therapeutic ligands. The intimate associate, for example, between PPARgamma and carbohydrate and lipid metabolism, for example, finds clear expression in a variety of metabolic and aging disorders from atherosclerosis and lipid

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metabolism, for example, finds clear expression in a variety of metabolic and aging disorders, from atherosclerosis and diabetes, to Alzheimer's disease, decreased skin elasticity, male erectile dysfunction, pulmonary fibrosis, and atherosclerosis, and ocular diseases such as diabetic retinopathy, glaucoma, cataract formation, and age-related macular degeneration (AMD). This proposal will focus on the discovery of novel chemical tools for the purpose of advancing ONR research. A technology platform will be assembled that will facilitate the screening of chemical compound libraries for molecules able to modulate ONR-mediated transcription. Chemical screens that will be established to achieve this objective will consist of both in vivo and in vitro bas assays developed specifically for a high throughput (HTS) 384 well format. The assays developed exploit the agonist induced association of receptor ligand binding domains (LBDs) with nuclear receptor co-regulators and their derivative peptides. Currently we have diverse 18000 chemical compound library of mostly synthetic compounds that we have demonstrated to be viable for screening against ONRs. A pilot a screen of these compounds against FXR and identified novel compounds that robustly activate transcription of this ONR. The major goal of this project is to identify potent, specific compounds and make them freely available to the academic community to contribute high quality and unrestricted research on ONR function.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: POST-DPP FOLLOW-UP STUDY

Principal Investigator & Institution: Crandall, Jill P.; Medicine; Yeshiva University 500 W 185Th St New York, Ny 10033

Timing: Fiscal Year 2003; Project Start 15-AUG-1994; Project End 31-JAN-2008

Summary: (provided by applicant): The Diabetes Prevention Program is a multicenter controlled clinical trial examining the efficacy of an intensive life-style intervention or metformin to prevent or delay the development of diabetes in a population selected to be at high risk due to the presence of impaired glucose tolerance (IGT). Development of diabetes, defined by 1997 ADA criteria, is the primary outcome while cardiovascular disease and its risk factors are important secondary outcomes. The DPP began recruitment in mid-1996. At the time of this application, total study exposure is a mean of approximately 3 years (range 2 to 5) with a total of approximately 10,000 patient years in the 3,234 volunteers in the 3-arm study. On the basis of a statistically significant and clinically compelling decrease in the development of diabetes in the life-style intervention and metformin-treated groups (58% and 31% reductions, respectively) compared with the placebo treated group, the DPP Data Monitoring Board and NIDDK ended the masked treatment phase of the study in May, 2001, one year earlier than originally planned. This application is designed to take further advantage of the scientifically and clinically valuable cohort of DPP volunteers and the large volume of data collected during the study. The highly compliant DPP cohort, including 45% minorities, is the largest IGT population ever studied. Moreover, the subcohort that has developed diabetes (n approximately 700) has been followed from near the exact time of diabetes onset. Clinically important research questions remain in the wake of the DPP. The carefully collected, centrally measured and graded data in this cohort should help to answer, definitively, a number ofvolunteers with impaired glucose tolerance and volunteers whose diabetes developed during the DPP to determine the natural history of diabetic complications. This analysis will be epidemiologic in nature with all groups being pooled with prior treatment being used as a co-variate. Finally, the same data will be used to examine the effects of gender, age, and race/ethnicity on diabetes and its vascular complication.

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Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: PROTEIN PRODUCTION OF PEDF-POTENT ANTIANGIOGENIC AGENT

Principal Investigator & Institution: Wei, Lisa L.; Genvec, Inc. 65 W Watkins Mill Rd Gaithersburg, Md 20878

Timing: Fiscal Year 2003; Project Start 01-MAY-2003; Project End 31-OCT-2003

Summary: (provided by applicant): Exudative age-related macular degeneration (AMD) and proliferative diabetic retinopathy (DR), due to aberrant choroidal and retinal neovascularization respectively, are two of the leading major causes of blindness in the US. PEDF (Pigment Epithelium-Derived Factor) is a potent endogenous antiangiogenic/neurotrophic factor and is purported to be the key natural regulator of vascularity in the eye. In experimental disease models, PEDF blocks choroidal and retinal neovascularization. Based on these compelling data, we hypothesize that administration of PEDF protein may treat blinding ocular neovascular diseases such as wet AMD. It will be the overall goal of the SBIR Phase II to generate preclinical data enabling potential clinical testing of PEDF protein. In this Phase I application, we will test the feasibility of generating PEDF for preclinical studies. We propose: 1) to generate reproducible stocks of high quality PEDF protein and 2) to demonstrate the biological activity/potency of the purified protein product. This bioactive recombinant human PEDF will be used for future PEDF pharmacokinetic studies, to test efficacy of purified PEDF protein in ocular disease models, to test various delivery approaches, and to determine whether PEDF-based product should be advanced to clinical testing. Furthermore, the methods and procedures generated in the Phase I SBIR grant may lead to future viable manufacturing processes and assays that could lead to clinical grade material.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: RACE AND LONG-TERM DIABETES SELF-MANAGEMENT IN AN HMO

Principal Investigator & Institution: Ross-Degnan, Dennis; Harvard Pilgrim Health Care, Inc. 93 Worcester St Wellesley, Ma 02481

Timing: Fiscal Year 2002; Project Start 15-JAN-2002; Project End 31-DEC-2004

Summary: (provided by applicant): This project will examine the complex relationships between race, diabetes self-management (including self-monitoring of blood glucose and diabetes drug therapy), glycemic control, and diabetes complications in a managed care setting over a nine-year period. African Americans with diabetes are less likely to be in glycemic control, a major risk factor for development of complications, including nephropathy, retinopathy, and peripheral vascular disease. Randomized controlled trials suggest that diabetes self-management including patient education, drug therapy, changes in diet, and regular exercise can improve glycemic control in the African American population. However, there is little epidemiological evidence regarding the role of race/ethnicity as a determinant of adherence to recommended diabetes selfmanagement practices, or regarding the relationship between self-management, glycemic control, and subsequent clinical outcomes. Further, previous studies of race and diabetes self-management have been limited by short study periods, inadequate sample size, and reliance on self-reported measures of self-monitoring of blood glucose. The clinical setting for this study is Harvard Vanguard Medical Associates (HVMA), a large multi-site, multi-specialty group affiliated with Harvard Pilgrim Health Care.

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HVMA consists of 14 health centers serving over 300,000 people in the Boston area. We will use an open cohort design to enroll all adult (l8 years) patients between 1991 and 1999 who have 24 months or more of uninterrupted enrollment in HVMA following ascertainment of non-gestational diabetes, defined by (1) hospital discharge diagnosis of diabetes mellitus, (2) outpatient diagnoses of diabetes mellitus, HbAlc lab test result 8.0, or use of a diabetes drug (insulin, sulfonylurea, or metformin). We estimate that the cohort will include approximately 1,800 adults identified as African American and 5,000 identified as Caucasian. Access to HVMA computerized medical records, hospital emergency room and inpatient claims, lab, and pharmacy data will allow us to create reliable, objective measures of self-monitoring (home glucose monitor test strip use), drug therapy. glycemic control (HbAlc lab results), and diabetes complications (as measured in outpatient visits, emergency room visits, and hospitalizations). Stratifying by type of drug therapy (insulin/combined therapy vs. oral therapy), we will use descriptive analyses, generalized linear mixed models, and proportional hazards models to (1) identify racial differences in self-management practices and diabetes-related health outcomes over time; (2) assess whether African American race is an independent predictor of self-monitoring practice or adherence to drug regimen; and (3) whether there are racial/ethnic differences in the association between self-management and specific clinical endpoints, including glycemic control (HbAlc<8.0) and the incidence of diabetes-related complications over the nine-year study period.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: REGULATION OF EXPRESSION OF SECRETED FLT-1

Principal Investigator & Institution: Huckle, William R.; Biomedical Sciences and Pathobiology; Virginia Polytechnic Inst and St Univ 460 Turner Street, Suite 306 Blacksburg, Va 24060

Timing: Fiscal Year 2004; Project Start 01-APR-2004; Project End 31-MAR-2006

Summary: (provided by applicant): Vascular endothelial growth factor (VEGF) is a major mediator of neovascularization in embryonic development and adult vascular homeostasis. VEGF contributes to adaptive angiogenesis in ischemic tissue as well as excessive vessel formation in proliferative vascular pathologies. VEGF and its signaling pathways thus may provide a means of either promoting angiogenesis in vascular insufficiency or inhibiting angiogenesis in diabetic retinopathy or in tumors. Responses to VEGF are mediated by two endothelial cell-surface receptors, Fit-1 and KDR. An RNA processing variant of Fit-1 mRNA produces a secreted form of Fit-1 ("sFIt-1 ") that binds VEGF with high affinity and can inhibit biological responses to VEGF. Previous studies indicate that the ratio of sFIt-l:FIt-1 can vary under physiological conditions, suggesting that molecular mechanisms exist to specifically control the mRNA processing events leading to sFIt-l. We postulate that the relative expression of sFIt-1 and Fit-1 is a significant biological determinant of vascular responsiveness to VEGF. The goals of the proposed research are to identify new experimental systems and biochemical assays to discover the regulatory mechanisms governing sFIt-1 expression. Our specific aims are 1) to identify biological models appropriate for the study of regulated sFIt-1 expression, 2) to identify nuclear factors involved in Fit-1 pre-mRNA processing, and 3) to determine the post-transcriptional mechanism by which protein kinase C activation alters sFIt-1 expression. In Aim 1, we will quantify sFIt-1:Fit-1 expression in cellor animal-based models of vascular development and adult angiogenesis. Under Aim 2, we will probe the interaction of specific and unknown processing factors with Fit-1 pre-mRNA using cell-free systems. In Aim 3, we will assess the role of altered mRNA stability and pre-mRNA 3rocessing in the induction of sFIt-1

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expression by phorbol ester. These studies will increase our understanding of the mechanisms of both adaptive and therapeutic angiogenesis, and may provide rationale for developing novel pharmacological interventions to modulate sFlt-1 expression and, thereby, VEGF responsiveness.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: REGULATION OF HEMANGIOGENESIS BY ADENOVIRAL E4 GENES

Principal Investigator & Institution: Rafii, Shahin; Professor of Medicine; Weill Medical College of Cornell Univ New York, Ny 10021

Timing: Fiscal Year 2002; Project Start 01-SEP-2002; Project End 31-AUG-2007

Summary: (provided by applicant): Endothelial and hematopoietic cells are ideal targets for gene therapy because are readily accessible to gene therapy vectors via the circulation and play a critical role in the progression of disease processes including inflammation and tumor angiogenesis. Adenoviral (Ad) vectors which could infect quiescent vascular cells provide ideal vectors to introduce genes into vascular endothelium as well as hematopoietic stem and progenitor cells with high efficiency and low toxicity. However, expression of genes by Ad vectors has been hampered by infiltration of inflammatory cells and intravascular activation of neo-intimal cells. Despite numerous studies defining the role of immune response to Ad vectors the exact mechanism whereby Ad vectors modulate activation status of ECs and hematopoietic cells and subsequent inflammatory response is not know. We have shown that introduction of E1-E4+, but not E1-E4-Ad vectors into primary ECs results in profound alteration in the proliferation and inflammatory status of the ECs. Infection of ECs with E4+ Ad vectors result in the generation of unique state where ECs do not proliferate or undergo apoptosis. This state is also associated with the upregulation of inflammatory adhesion molecules including ICAM, VCAM, and CD34. Moreover, E4 mediated activation of ECs enhance transendothelial migration of hematopoietic precursor cells and proinflammatory cells. These data clearly demonstrate that one or a combination of E4 gene products encoded by seven distinct E4 open reading frames (ORFs) regions may play a critical role in modulation of angiogenic and inflammatory potential of ECs. Based on these data, we hypothesize that gene products encoded by the E4 region of Ad vectors pirate the molecular machinery of ECs resulting in transformation of ECs into a unique pro-angiogenic and proinflammatory state. Identification of E4ORF genes that modulate angiogenic and inflammatory potential of ECs hematopoietic cells will facilitate designing strategies to attenuate vascular toxicity associated with Ad vector gene therapy. The mechanism by which E4ORFs gene products modulate angiogenic and inflammatory potential will be investigated through studying the following experiments: 1) Identifying specific E4ORF genes that alone or in combination modulate angiogenic and inflammatory potential of ECs in in vitro and in vivo models. 2) Assess the effect of E4ORFs gene products in the modulation of angiogenic potential of circulating VEGFR2+AC133+endothelial precursor cells (EPCs) and hematopoietic stem and progenitor cells. 3) Define the mechanism(s) whereby E4ORFs modulate the motility and mitogenic potential of ECs and hematopoietic progenitor and stem cells. 4) Define the role of E4ORFs in physiological models of angiogenesis and inflammation. These experiments may culminate in identification of E4ORFs that promote or inhibit angiogenesis, and diminish E4 mediated vascular toxicity. Incorporation of proangiogenic E4ORFs in conjunction with transgenes expressing VEGF, will facilitate developing clinical strategies to enhance collateral formation in ischemic myocardium or limbs. Conversely, E4ORFs with anti-angiogeni properties may be used in clinical

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strategies that are targeted at inhibiting tumor angiogenesis or blocking aberrant angiogenesis in clinical scenarios, such as diabetic retinopathy.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: REGULATION OF VASCULAR DEVELOPMENT IN THE MOUSE RETINA

Principal Investigator & Institution: Ash, John D.; University of Oklahoma Hlth Sciences Ctr Health Sciences Center Oklahoma City, Ok 73126

Timing: Fiscal Year 2002; Project Start 16-SEP-2002; Project End 31-AUG-2007

Summary: Proliferative retinopathies (PRs) are a classification of diseases that include sickle cell retinopathy, diabetic retinopathy, branch vein occlusion retinopathy, and retinopathy of prematurity. These diseases all are characterized by the loss of normal blood vessels in the retina and subsequent uncontrolled growth of abnormal blood vessels in the vitreous of the eye. As in humans, retinal ischemia in mice induces growth of abnormal vessels in the vitreous. In contrast to humans, mice are only transiently affected by the growth of abnormal blood vessels in the vitreous. Within a short period, normal blood vessels will re-grow within the mouse retina and the abnormal vessels in the vitreous will spontaneously resolve. The regrowth of normal retinal vessels provide mice with an endogenous mechanism for long-term protection from proliferative retinopathy. We hypothesize that the rodent retina is competent to promote retinal vascular regrowth through the expression of genes that are essential regulators and effectors of blood vessel growth and stability. Our goal is to identify the molecular regulators and effectors of vascular growth in the mouse retina. We have previously demonstrated that transgenic expression of either activated transforming growth factor 131(TGF-131) or leukemia inhibitory factor (LIF) in the lens alters the environment within the retina so that it is no longer competent for vascular development. Our strategy is to identify the genes that are expressed in wild type retinas which can promote vascular development, but that are not expressed in retinas which have lost the ability to support vascular development. In the current proposal, we will use transgenic mice in which we can induce the expression of TGF-131 (aim 1), or LIF (aim 2) in retinal photoreceptors, so that we can turn off retinal competence for vascular development at ages corresponding to the beginning, to the middle, and to completion of normal retinal vascular development (aims 1 and 2). With these mice, we will test the hypothesis that cytokines reduce growth of vascular endothelial cells or increase vascular degeneration. We will also test the hypothesis that cytokines down-regulate the expression of specific genes that are necessary for retinal vascularization (aim 3). This will allow us to identify genes that are regulators and effectors of retinal vascular development in mice.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: REGULATION OF VEGF MRNA STABILITY BY HYPOXIA

Principal Investigator & Institution: Furneaux, Henry M.; Associate Professor; Cell Biology; University of Connecticut Sch of Med/Dnt Bb20, Mc 2806 Farmington, Ct 060302806

Timing: Fiscal Year 2002; Project Start 01-JAN-1999; Project End 31-DEC-2003

Summary: (Adapted from Investigator's Abstract): Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis in diabetic retinopathy, cancer and coronary heart disease. The expression of VEGF is tightly controlled to the cellular oxygen tension. Previous studies have shown that VEGF mRNA is markedly stabilized under hypoxic conditions. The molecular mechanisms underlying the regulation of

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VEGF mRNA stability are not well understood. In the preliminary results the applicant showed that the stability of VEGF mRNA is controlled by a cis acting element in its 3'UTR. They have also identified a trans acting factor (HuR) that binds to this element and increases the expression of VEGF. In addition, they show that HuR itself is regulated by hypoxia. This proposal will focus on the role of HuR in hypoxic regulation of VEGF mRNA stability. The proposed studies may be important since they could lead to the generation of new drugs that will regulate VEGF expression at the post transcriptional level. The specific aims of the proposal are the following: (1) To determine the fine structure of the HuR/VEGF mRNA complex. To generate mutations in both the mRNA element and HuR and determine their effect on VEGF expression; (2) To design selective inhibitors of HuR/VEGF mRNA complexes; (3) To identify and clone HuR associate proteins that modify its activity; (4) To determine how hypoxia regulates HuR expression; and (5) To determine how HuR stabilizes VEGF mRNA.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: REMOTE EXPERT SCREENING FOR DIABETIC RETINOPATHY

Principal Investigator & Institution: Meeder, Torre; Eyetel Corporation 15078 Stillfield Pl Centreville, Va 20120

Timing: Fiscal Year 2002; Project Start 30-SEP-1999; Project End 31-MAR-2004

Summary: (provided by applicant): Half of diabetic patients do not visit an ophthalmologist as recommended, resulting in frequent and unnecessary vision loss. The NEI has set as an Healthy People 2010 objective to increase the screening rate for diabetic retinopathy. EyeTel-lmaging and the Wilmer Eye Institute are developing technology to screen diabetic patients during their visits to their primary care physician (PCP). The DigiScope, a low cost, quasi-automatic computerized fundus camera acquires digital stereoscopic fundus images and assesses visual acuity. The data are automatically transferred, via the Internet, to a reading center where expert readers, supervised by retinal specialists, identify patients with retinopathy and refer them to an ophthalmologist. The DigiScope will be provided to PCPs without capital investment on their part and a service fee will be collected from health care providers. The goals of Phase I were exceeded with the successful development of the DigiScope and the results of an independent study. In comparison with the 'gold standard', the DigiScope had a sensitivity of 98.5 percent and a specificity of 92 percent. The goals of phase II are to meet all the specifications for implementation in PCPs offices, develop the reading center and assess the impact on screening rate in different health care environments.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: RENIN ANGIOTENSIN SYSTEM BLOCKAGE-DN (RASS)

Principal Investigator & Institution: Mauer, S Michael.; Professor; Pediatrics; University of Minnesota Twin Cities 200 Oak Street Se Minneapolis, Mn 554552070

Timing: Fiscal Year 2003; Project Start 16-MAR-1997; Project End 31-MAY-2008

Summary: (provided by applicant): Diabetes nephropathy (DN) is the most important cause of kidney failure. Patients (pts) with Type 1 diabetes mellitus (DM) who develop DN have a markedly increased death rate from kidney failure, coronary artery disease and stroke. Glycemia only partly explains why some pts develop these DM complications. Further, since tight blood sugar control is extremely difficult to maintain, other efforts need to be made to reduce risks of DM complications. Renin-angiotensin system (RAS) inhibitors slow the progress of established DN. The specific aim of this study is to determine whether treatment at the early stages of DM can slow or stop DN

54 Diabetic Retinopathy

structural changes. The long-term objective is to prevent DN from developing. Two hundred eight five pts ages 16-59 with 2-29 yrs of Type 1 DM and no renal functional abnormalities have been randomized into a parallel, double-blind, placebo-controlled study involving 3 groups (95 pts/group). Each group receives an angiotensinconverting enzyme inhibitor (ACEI) (enalapril), or an angiotensin II receptor blocker (losartan), or placebo. All pts have their usual DM management. Baseline studies included measures of glomerular filtration rate (GFR), urinary albumin excretion rate (UAE), blood pressure (BP), and a percutaneous renal biopsy. Pts are followed by quarterly measures of BP, HbA1C, UAE, and drug compliance. There are annual measures of GFR and a repeat renal biopsy after 5 yrs in the study. The main endpoint is kidney structural changes over time, especially mesangial fractional volume [Vv(Mes/glom)]. Secondary endpoints will be other DN structural measures and measures of kidney function (UAE, GFR). These studies will determine whether RAS blockage in the early stages of DN can prevent the early kidney structural changes in this important disorder. Ancillary studies will evaluate the effects of treatment group on the development and progression of diabetic retinopathy and will develop predictors of study participants' compliance.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: RETINAL BLOOD FLOW AND MICROTHROMBI IN TYPE 1 DIABETES

Principal Investigator & Institution: Lorenzi, Mara; Associate Professor; Schepens Eye Research Institute Boston, Ma 02114

Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-AUG-2005

Summary: (provided by applicant): The greatest likelihood of lessening the threat of diabetic retinopathy is through prevention. Preventative strategies must target pathogenic events that occur early in diabetes, and incorporate methods for monitoring outcomes longitudinally and noninvasively. Previous studies suggest that the formation of platelet-fibrin microthrombi could be an early pathogenic event in the closure and obliteration of diabetic retinal capillaries, and do show reduced retinal blood flow in patients with short diabetes duration and no or minimal retinopathy. The hypothesis to be tested in this project is that the reduced retinal blood flow reflects increased vascular resistance due to microthrombosis, and, operationally, that antiplatelet agents normalize the reduced retinal blood flow. The project aims to (1) confirm that, under basal conditions, retinal blood flow measured with the laser Doppler method in a large group of type 1 diabetic patients with no or minimal retinopathy differs from the flow measured in ageand sex-matched nondiabetic control subjects; (2) determine whether the response of retinal blood flow to low-dose aspirin--expected to be mostly or solely antithrombotic--administered for two months versus placebo, differs between the diabetic and the control group; and (3) determine whether the response of retinal blood flow to low-dose aspirin in type 1 diabetic patients differs from the response to another antiplatelet agent, which is highly selective and acts downstream of the site of aspirin action. The study is designed as a two-arm (diabetic patients and controls), parallelgroup, double-masked, randomized, placebo-controlled trial, involving individuals 1840 years of age with diabetes duration of one to ten years and no or minimal retinopathy. The diabetic subjects will be recruited from the Diabetes Center of the Massachusetts General Hospital. The retinal blood flow measurements will be performed with the Canon Laser Blood Flowmeter, an instrument approved by the FDA and usable with ease in the clinical setting. A finding that early reduction of blood flow reflects the occurrence of microthrombosis in the retina of short-term diabetic patients

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will have identified a pathogenic process for diabetic retinopathy, a surrogate endpoint and a probe for its early detection, and a target for early intervention using benign and clinically available drugs.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: RETINAL IMAGE QUALITY IN RETINAL-DISEASED EYES

Principal Investigator & Institution: Shahidi, Mahnaz; Associate Professor; Ophthalmology and Visual Scis; University of Illinois at Chicago 1737 West Polk Street Chicago, Il 60612

Timing: Fiscal Year 2002; Project Start 01-SEP-2002; Project End 31-AUG-2005

Summary: (provided by applicant): The optical properties of the eye and its imperfections limit visual performance, the ability for an individual to view the world, and retinal imaging, the ability for an ophthalmologist to view the retinal tissue. Recent advances in wavefront sensing and adaptive optics technologies have allowed measurement and correction of monochromatic wavefront aberrations in healthy human eyes. However, it is likewise important to investigate disease-related changes in the optics of the eye, since they can significantly contribute to degradation of both visual performance and resolution for retinal imaging. Particularly, there is a need to differentiate between vision loss that results from retinal disease and visual performance that is impaired due to imperfect optics, in order to anticipate optimal outcome for therapies applied to improve neural function of the retina in eyes with imperfect optics, or to foresee consequences of procedures that are targeted to improve the optical property of eyes with diseased retinas. Equally important is a need for high-resolution imaging of the retinal tissue that may be achieved by compensation for ocular aberrations with the use of adaptive optical components and image processing methodologies. Such imaging would allow visualization of fine retinal structures, thus providing for better understanding of retinal pathophysiology and enhanced diagnostic evaluation of retinal diseases. In the current research proposal, our novel technique for optical section retinal imaging will be coupled with wavefront sensing technology. Imaging will be performed in subjects diagnosed with diabetic retinopathy and agerelated macular degeneration and the optical performance of retinal-diseased and healthy eyes will be compared. The relation between ocular aberrations and retinal imaging resolution will be determined. High-resolution retinal imaging will be achieved by compensation for ocular aberrations. Findings from the research study will provide knowledge on the nature and extent of disease-related changes in the optical properties of the eye, that is of value for evaluation of optical factors that contribute to degradation of visuai performance and for achievement of high-resolution retinal imaging in subjects with retinal diseases that are considered the most prevalent causes of blindness.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: RETINAL OXIMETER FOR CLINICAL ASSESSMENTS

Principal Investigator & Institution: Beach, James M.; Photon Industries Bldg 9313, Rm 167 Stennis Space Center, Ms 39529

Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2004

Summary: (provided by applicant): In this SBIR we propose to develop a retinal vessel oxygen saturation monitoring system for investigational and clinical use. Retinal oximetry has been the subject of intense research and development since Hickam et al published the first photographic means in 1963. Studies of the retinal circulation and oxygen saturation have implicated oxygen status in diseases of the retina, including vein

56 Diabetic Retinopathy

occlusion, diabetic retinopathy, glaucoma and optic atrophy. Oximetry studies have shown impairment of retinal blood flow regulation in diabetes and improvement in retinal oxygenation by pan-retinal photocoagulation treatment for proliferative diabetic retinopathy. Recently, studies have used imaging oximetry technique to evaluate side effects of glaucoma medications on retinal oxygenation. Thus, there is growing interest in retinal oximetry for monitoring retinal pathology. We propose here to develop an instrument we call OXYCAM for determining changes in oxygenation from distinct regions of the retina. This ability to detect subtle changes will be important in preventing the blinding complications of retinovascular disease. The long-term objectives of this project are 1) to make available a standardized commercial system for oximetric evaluation of retinal disease in research institutions and clinics and 2) to determine the role of oximetry as a retinal evaluation tool, which will lead to a commercial market for oximetry products. The specific aims of Phase I include 1) development of the dual-wavelength OXYCAM Imaging system for oximetric assessment at different levels of the retinal circulation and 2) validation of the OXYCAM performance in normal subjects. Coverage of retinal vessel networks in single dualwavelength recordings will be achieved with pairs of large-format CCD sensors and a dichroic image splitter. Our longer term aims in Phase II include 1) development of automated vessel tracking and oximetry analysis software, 2) refinement of oximetry technique to improve accuracy and reproducibility in clinical work and 3) multi-site investigations into the role of oximetry in diagnosis, management and treatment of retinal disease.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: RETINAL OXYGENATION IN DIABETIC RETINOPATHY

Principal Investigator & Institution: Berkowitz, Bruce A.; Professor; Anatomy and Cell Biology; Wayne State University 656 W. Kirby Detroit, Mi 48202

Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-JUL-2005

Summary: (provided by applicant): Current treatments for diabetic retinopathy are not entirely successful. We reason that therapies could be more rapidly developed and evaluated than is currently possible if a patient's risk of developing retinopathy could be predicted early in the course of the disease. We have developed a unique functional magnetic resonance imaging (fMRI) technique that measures the change in partial pressure of oxygen (deltaPO2) in the vitreous humor while the subject breathes carbogen (95% O2: 5% CO2). Using this acute retinal "stress" test, we have shown that carbogen breathing in rats and mice at 3-4 months of diabetes (before the appearance of retinal lesions) produces a significantly reduced retinal deltaPO2 compared to normals. A subnormal deltaPO2 is consistent with the presence of hypoxia but it cannot yet be unambiguously interpreted as a measure of hypoxia. In addition, in 3 month diabetic rats, aminoguanidine (AMG) treatment prevented the decrease in retinal deltaPO2. AMG is known to prevent retinal lesion formation in diabetic rats and inhibit the activity of both the inducible form of nitric oxide synthase (iNOS) and protein kinase C (PKC) (among other actions). Hypothesis: Subnormal retinal deltaPO2 in experimental diabetes is correlated with retinal hypoxia (Aim 1), and increased iNOS and PKC activity (Aims 2 and 3) but precedes the appearance of histopathology. Specific Aim 1) To compare retinal PO2 and deltaPO2 before, and at 2 wks, and 3 and 9 months of diabetes in rats. fMRI and a carbogen challenge will be used to measure the retinal deltaPO2. To measure PO2, a perfluorocarbon droplet will be injected into the preretinal vitreous over superior or inferior retina and 19F magnetic resonance spectroscopy performed. Specific Aim 2) To compare the retinal deltaPO2 (at 3 months) and histology

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(at 3 and 15 months) in normal and diabetic rats with and without treatment with a selective iNOS inhibitor (L-NIL (L-N(6)-(1-iminoethyl)lysine)), and in normal, diabetic, and diabetic iNOS knockout mice. Specific Aim 3) To compare the retinal deltaPO2 (at 3 months) and histology (at 3 and 15 months) in normal and diabetic rats with and without treatment with a selective PKC inhibitor (LY333531). In addition, we will compare retinal deltaPO2 in normal mice, transgenic mice that overexpress PKC beta II, diabetic mice, and diabetic PKC beta II-knockout mice. The results of the proposed studies will prove whether or not changes in retinal deltaPO2 correlate with the development of retinal hypoxia or multiple biochemical abnormalities that are associated with diabetic retinopathy but cannot be measured in vivo by existing techniques. The results of these studies could lead to the development of a non-invasive real time method for the early evaluation of diabetic retinopathy and its treatment.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: RETINAL VESSEL MEASUREMENT AND CHARACTERIZATION SYSTEM

Principal Investigator & Institution: Wilson, Mark P.; Kestrel Corporation 3815 Osuna Ne Albuquerque, Nm 87109

Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2004

Summary: (provided by applicant): Retinal microvascular abnormalities are related to many ocular and systemic diseases including diabetic retinopathy, hypertension, stroke, and cerebral micro-vascular diseases. For example generalized arteriolar narrowing and arteriovenous nicking appear to be irreversible long-term markers of hypertension. Findings suggest that retinal photography may be useful for assessing risk stratification and screening for retinal disease in appropriate populations. Highly evolved imaging solutions and computer processing power have opened a door to quantify these abnormalities. A methodology is suggested that is efficient and comprehensive because of computer automation, that is repeatable, free of interand intra-reader variability, precise, and consistent with the human analysts' techniques. We propose to develop and validate a system that extracts retinal vessels, classifies their bifurcations and crossings, and quantifies a diameter measurement of the retinal vasculature system. The Auto-Cal system will use advanced morphological and Gaussian filtering techniques to segment the vessel network. Once segmented the crossings and bifurcations will be classified through morphological skeletonization path traversing and pruning algorithms. The vessel diameter is then calculated by fitting the gray level profile to Gaussian parameters that describe a typical vessel. The system will be tested on 50 digital images, which will have their 8 major vessels manually measured with a previously developed computer-aided tool. High statistical correlation with manually derived vessel diameters will determine the success of the process. The specific Phase I aims are to 1) Develop and validate a fully automatic retinal vessel extraction algorithm to segment the arteries and veins from a digital fundus photograph, 2) Develop and validate a vessel bifurcation and crossing recognition algorithm, and 3) Develop and validate a vessel diameter measurement algorithm. In Phase II the Auto-Cal tool will be used in a comparative study to examine 400 pre-proliferative diabetic retinopathy patients. The vessels have been manually measured. These data will be used to validate the system against a large "ground truth" population.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

58 Diabetic Retinopathy

•Project Title: RISK FACTORS FOR DIABETIC RETINOPATHY IN BLACK AMERICANS

Principal Investigator & Institution: Roy, Monique S.; Ophthalmology; Univ of Med/Dent Nj Newark Newark, Nj 07107

Timing: Fiscal Year 2002; Project Start 01-DEC-1992; Project End 31-JUL-2004

Summary: Diabetic retinopathy remains one of the leading causes of blindness in the United States. This disease, and its associated visual impairment, represent a major public health problem particularly for AfricanAmerican diabetic patients among whom the prevalence of diabetes is high with an increasing incidence. In a previous study (The New Jersey 725), we had assembled a large (n=725) cohort of AfricanAmericans with type 1 diabetes, identified from the New Jersey Hospital Discharge Data files. In that study, we delineated frequency of retinopathy and associated visual impairment (which are high, 64% and 11% respectively) as well as identified (in addition to duration of diabetes) three systemic modifiable risk factors, poor glycemic control, systemic hypertension and renal disease significantly associated with the presence and severity of diabetic retinopathy in this group of patients. The overall goal of the present prospective study is to determine the natural history of diabetic retinopathy in African-Americans with type 1 diabetes and examine the relationship between systemic risk factors listed above and progression of the retinopathy so that therapeutic interventions, targeted at these risk factors, can be appropriate designed. In order to achieve oral overall goal, we plan to obtain follow-up examinations of the patients from the previous study to ascertain () the 6-year incidence and progression of diabetic retinopathy (as graded from standard fundus photographs) and associated incidence of visual impairment and (b) how duration of diabetes and systemic risk factors (glycemic control, blood pressure, and renal disease), as measured at the baseline examination, relate to incidence and progression of diabetic retinopathy. Clinical evaluation of patients will include standardized protocols for ocular examination, fundus photography, structured clinical interview, and blood pressure measurement; laboratory evaluation will include blood assays to evaluate glycemic control, insulin insufficiency, and cholesterol levels, and urine assays to assess diabetic renal disease. Results obtained on incidence and progression of diabetic retinopathy and risk factors for disease progression this population group will provide the data to understand the natural history of the disease in African-Americans with type 1 diabetes. Such data are also essential for planning public health education measures, estimating needs for medical services, and designing community-based intervention studies for the prevention of diabetic complications in this group of Americans.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: ROLE OF ALK1 TGF-BETA SIGNALNG ON ANGIOGENESIS

Principal Investigator & Institution: Oh, S Paul.; Physiology and Functional Genomics; University of Florida Gainesville, Fl 32611

Timing: Fiscal Year 2002; Project Start 01-AUG-2000; Project End 31-JUL-2004

Summary: The establishment and maintenance of a vascular supply are essential for the survival of normal and neoplastic tissues. With the exception of early embryogenesis, new blood vessels are formed from pre-existing vessels by a process called angiogenesis. Angiogenesis occurs during embryonic development, ovarian follicular development and wound healing, and during pathogenesis including solid tumors, diabetic retinopathy and rheumatoid arthritis. The long term goal of my research program is to elucidate biological, molecular and genetic mechanisms underlying regulation of

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angiogenesis during development and post-natal life. Angiogenesis can be separated into two phases: activation and resolution phases. In activation phase the endothelial cells degrade perivascular matrices, migrate and proliferate, while in resolution phase, endothelial cells cease migration and proliferation. Mechanisms controlling these two distinct phases of angiogenesis remain to be understood. Working hypothesis is that activation-resolution phases are determined by a balance between positive and negative regulators: in activation phase, positive regulators predominate, whereas resolution phase is achieved and maintained by the dominance of negative regulators. While a great deal is known about positive regulators (or angiogenic factors), very little is known about the negative regulators involved in the resolution phase of angiogenesis. With the results from our recent molecular genetic studies about the activin receptorlike kinase-1 (ALK1) deficient mice, we hypothesize that ALK-1 may mediate a negative regulator for the angiogenic balance. The specific aims of this proposal is to test this hypothesis using in vivo and in vitro approaches. Results from this proposal would enhance our current understanding on the angiogenic balance. This knowledge can be readily applicable to various vascular pathogenesis including atherosclerosis, retinopathy and hereditary hemorrhagic telangiectasia.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: ROLE OF COMPLEMENT IN VASCULAR DIABETIC COMPLICATIONS

Principal Investigator & Institution: Halperin, Jose A.; None; Harvard University (Medical School) Medical School Campus Boston, Ma 02115

Timing: Fiscal Year 2002; Project Start 15-SEP-2002; Project End 31-JUL-2006

Summary: (provided by applicant): Epidemiological studies have established that hyperglycemia is responsible for the microand macrovascular disease that characteristically complicates human diabetes. However, the mechanism by which hyperglycemia causes vascular complications is poorly understood, in part because of the known absence of adequate animal models of diabetic complications. Indeed, no single animal model of diabetes reproduces the extensive vascular proliferative complications in the combination and intensity seen in humans. Thus, it appears that neither hyperglycemia itself nor the multiple signals activated by hyperglycemia are sufficient to induce in animals human-like vascular diabetic complications. This implies that there must be one or more genes and/or pathways necessary for the development of the diabetic vascular complications in humans that in animals differ substantially in their sensitivity to hyperglycemia and/or glycation. We have identified that the gene encoding for the complement regulatory membrane protein CD59, which inhibits formation of the membrane attack complex (MAC), is structurally different in humans and animals because hCD59 contains a glycation motif, formed by its H44 residue at approximately 5A angstroms from K41. We have shown that the K41-H44 motif makes hCD59 sensitive to inactivation by glycation, and that the H44 residue is not present in CD59 from other species. We postulate that glycation-inactivation of hCD59 due to its unique H44 residue could represent the elusive link between hyperglycemia and vascular proliferative complications of human diabetes. Consistently, high levels of glycated CD59 are found in diabetic urine, plasma and tissues; and glycated CD59 colocalizes with increased MAC deposition in kidneys, nerves and veins from diabetic subjects. Increased MAC deposition in the target tissues releases growth factors and cytokines that stimulate cell proliferation and induce synthesis of collagen type IV by glomerular mesangial cells. Increased MAC-induced mitogenic signals in diabetic tissues would act synergistically with other hyperglycemia-induced pathways causing

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vascular proliferative diabetic complications. In this application, we propose to use available mCd59KO and transgenic hCD59 mice to further investigate the causative role of glycation-inactivation of hCD59 in the pathogenesis of vascular proliferative complications of diabetes. We will phenotype diabetic mCd59KO, mCd59KO/hCD59WT, and mCd59KO/hCD59GIn44 variant transgenic mice for the development of human diabetic-like vascular disease, Mice will be made diabetic by injection of streptozotocin, and phenotyped by histopathology and parameters of retinal hemodynamic and renal function. We expect that in mCd59KO and in mCd59KO/hCD59WT mice, hyperglycemia will trigger a vascular proliferative response comparable to that seen in human diabetes. Instead, the hCD59GIn44 variant (resistant to glycation-inactivation) should protect mCd59KO from hyperglycemiainduced proliferative disease. We expect that these experiments will provide: 1) clear evidence for the role of glycation-inactivation of hCD59 in the pathogenesis of the proliferative vascular complications of diabetes; 2) needed animal models to study mechanism, therapy and prevention of diabetic complications; and 3) strong evidence to support future studies on complement and diabetes in humans.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: RUNX SPECIFIC ANGIOGENESIS INHIBITORS

Principal Investigator & Institution: Passaniti, Antonino; Pathology; University of Maryland Balt Prof School Baltimore, Md 21201

Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 31-MAR-2004

Summary: (provided by applicant): Recruitment of new blood vessels (angiogenesis) is required for tumor growth and metastasis. Therefore, the development of angiogenesis inhibitors represents a new approach that may increase the effectiveness of existing cancer treatments. Runt family genes (Runx1,2,3) are transcription factors that playa key role in vascular development including endothelial cell (EC) migration and stem cell recruitment to promote angiogenesis. Runx DNA binding is enhanced by association with the product of the Cbf gene which forms a trimeric DNA binding complex whose 3-dimensional (3D) structure has recently been determined. Computer-aided rational drug design (CADD) can be used to identify chemical compounds with the potential to become therapeutic agents. CADD database searching involves screening of a 3D chemical database to select small molecules that "fit" in the binding site of interest on the target biomolecule. The identified small molecules are then obtained and subjected to experimental assays to select those with the appropriate biological activity. Use of a database of commercially available compounds avoids the need for chemical synthesis, thereby facilitating the identification of active compounds. Our hypothesis is that specific inhibition of Runx-mediated transcriptional activation will inhibit EC migration and angiogenesis. Our goals are to use CADD, in combination with the available 3D structure of Runt to identify compounds with a high potential to bind selectively to Runt. Both the DNA and Cbf binding regions of Runt will be individually targeted. The selected compounds will be obtained and subjected to experimental testing using assays to identify compounds with the desired Runt-binding activities to verify that they are Runt-specific antagonists. An EC migration assay will then be used to screen candidate compounds for biological activity. These approaches are one of the first attempts to inhibit angiogenesis via transcriptional targeting. Since inhibiting Runx transcriptional activity should reduce angiogenesis, the growth of both hematopoietic and solid tumors that depend on a blood supply for survival and growth will be inhibited. The lead compounds developed from this application could also find utility in non-cancer

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situations, such as macular degeneration, atherosclerosis, or diabetic retinopathy, where uncontrolled angiogenesis is responsible for the pathology.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: STATISTICAL METHODS FOR OPHTHALMOLOGIC AND CLUSTER DATA

Principal Investigator & Institution: Rosner, Bernard A.; Professor; Brigham and Women's Hospital 75 Francis Street Boston, Ma 02115

Timing: Fiscal Year 2002; Project Start 01-AUG-1998; Project End 31-JUL-2005

Summary: (provided by applicant): Correlated data are the norm in ophthalmologic data, due to correlated response for paired eyes. An abundance of methods are now available that enhance standard models by incorporating clustering effects (Rosner, 1984; Liang and Zeger, 1986). These methods are limited usually to either normally distributed or binary outcomes. However, many scales used in ophthalmology are continuous, but non-normally distributed (e.g. visual field area). Other scales are ordinal and non-normally distributed, but are sometimes treated as normally distributed as well (e.g. diabetic retinopathy grade). Nonparametric methods are a natural approach for such scales. In this proposal, we propose a model for incorporating clustering effects for ranked data and use this model to extend tests such as the Mann-Whitney U test for the clustered data situation. In addition, we propose extensions to allow for tied values and to adjust for other covariate effects. Another issue in ophthalmologic data is that some endpoints are composite in nature (e.g. nuclear cataract, cortical cataract, PSC cataract, control). An interesting issue is that the risk factor profile for some risk factors may be different for different types of cataract, while for other risk factors it may be the same. (Marshall & Chisholm, 1985). We propose to use a flexible type of polytomous regression model in this setting and to enhance it by considering matched designs as well as outcome categories that are not mutually exclusive. Furthermore, we propose to develop a user-friendly software package to easily fit such models including use of stepwise regression strategies. A third area of interest is the use of correlated data methods in a small sample setting. Most previous methods developed have good asymptotic properties but depend on at least a moderately sized sample for their validity.We propose to extend existing methods of exact inference by incorporating clustering effects and making these methods available by exportable software. Finally, the area under the ROC curve is frequently used as a measure of goodness-of-fit for logistic regression models. However, its use is problematic if separate logistic models are fit for the right and left eyes of an individual in ophthalmologic studies. We propose to extend traditional ROC curve methods (Hanley and McNeil, 1982) to the clustered data situation where outcome on fellow eyes may be either concordant (bilateral cases) or discordant (unilateral cases).

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: STRUCTURE/FUNCTION OF CONNECTIVE TISSUE GROWTH FACTOR

Principal Investigator & Institution: Grotendorst, Gary R.; Professor; Cell Biology and Anatomy; University of Miami-Medical Box 248293 Coral Gables, Fl 33124

Timing: Fiscal Year 2002; Project Start 01-SEP-2001; Project End 31-AUG-2005

Summary: Fibrotic disorders represent the largest segment of human disease for the populations of North America, Europe and Japan. These disorders are characterized by an overproduction of connective tissue (primarily collagen) and are widely recognized

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to be initiated in response to some type of injury which leads to the chronic inflammation and subsequent overproduction of growth factors that encourage the formation of connective tissue. There are no current therapies short of transplant or grafts which are effective to control the progression of any fibrotic disorder. Intensive studies over the last 25 years now suggest that a common mechanism may underlie many of these disorders. Thus, an advancement in our understanding of the cellular and molecular control mechanisms which regulate this process could have a significant impact on a wide range of human diseases, including atherosclerosis, pulmonary fibrosis, glomerulosclerosis, arthritis, glaucoma, diabetic retinopathy and others. One common growth factor which has been demonstrated to be overproduced in all fibrotic disorders examined to date is TGFb, which is believed to be an initiator of the connective tissue formation in these disorders. During the course of our studies on mechanisms whereby TGFb stimulates connective tissue formation, we discovered another growth factor, Connective Tissue Growth Factor (CTG F). CTGF synthesis selectively induced in mesenchymal cells by TGFb. We have found that agents which block CTGF synthesis or action are effective inhibitors of TGFb induced fibroblastic cell proliferation and collagen synthesis. This suggests that CTGF may be an important therapeutic target for the control of connective tissue formation in fibrotic disorders. Recently, we have determined that the individual domains of CTGF are responsible for signaling either the mitogenic activity (C-terminal domain of CTGF) or matrigenic activity (N-terminal domain). Our data also indicate that CTGF has an absolute requirement for the presence of EGF for mitogenic and IGF-2 for matrigenic activity. The current project will determine in a more detailed manner the role of the CTGF, CTG F, lGF and EGF receptors and the roles of EGF and IGF factor for signaling the mitogenic and matrigenic actions induced by TGFb in fibroblastic cells.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: STUDY CHAIR CORE CENTER:DME CLINICAL RESEARCH NETWORK

Principal Investigator & Institution: Aiello, Lloyd P.; Associate Professor; Joslin Diabetes Center Boston, Ma 02215

Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-JAN-2006

Summary: (provided by applicant): Diabetic macular edema (DME) remains a leading cause of visual loss in patients with diabetes mellitus. Increased understanding of this condition has prompted investigations of novel therapies for the treatment of DME, several of which are nearing clinical trial evaluation. Although therapeutic methodology may vary widely, the basic components requiring careful consideration in the design, implementation and analyses of high quality clinical trials are often similar. Furthermore, study sites with particular retinopathy trial expertise and experience are an excellent resource for a variety of such trials. The goal of this proposal is to establish a clinical trial network to evaluate new treatments for diabetic macular edema that may be more effective, better tolerated, or more readily administered than the present standard of laser photocoagulation. This initiative will utilize a framework specifically designed to provide accelerated clinical research, standardized treatment protocols, leveraged recruitment power of multiple centers and optimal resource utilization. These benefits will be realized through the advantages of a dedicated disease approach, coordinated administration, knowledgeable leadership, experienced clinical centers and efficient (re)utilization of components common to multiple trials. This application for the Study Chair core of the Diabetic Macular Edema Clinical Research Network exploits the PI's unique qualifications to serve as Study Chair for this network. Over the past

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decade, the PI has achieved international recognition through participation in clinical and research evaluation of diabetic retinopathy and DME. The PI is currently the Study Chair of 3 ongoing, multicenter (100 sites), randomized, clinical trials evaluating new therapies for diabetic retinopathy and DME. These studies are run under the PI's guidance using a nearly identical administrative and clinical framework as that presented in this proposal. Thus, the PI has unique direct experience in protocol development and operational management required within such a Network. Additionally, the Joslin Diabetes Center provides exceptional support for trial networks with its close integration of all diabetes-associated health care professionals and its basic and clinical research and technology prowess. This proposal details the manner in which the specific goals of the network will be fostered under the guidance of the Study Chair.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: SUPPLEMENTAL OXYGEN FOR HYPOXIA-RELATED RETINAL DESEASES

Principal Investigator & Institution: Nguyen, Quan D.; Medicine; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218

Timing: Fiscal Year 2002; Project Start 01-SEP-2001; Project End 31-AUG-2006

Summary: (Candidate's Abstract) Hypoxia is thought to play a role in the pathogenesis of several ocular diseases. It is Clear that hypoxia is the driving force behind the development of retinal neovascularization (NV) in patients with ischemic retinopathies, including diabetic retinopathy and central and branch retinal vein occlusions. Macular edema is another major cause of decreased vision in ischemic retinopathies and it is suspected, but not yet proven that hypoxia plays a critical role. Likewise, it is suspected, but not yet proven, that hypoxia contributes to the development of choroidal neovascularization (CNV) in patients with age-related macular degeneration (AMD). Supplemental inspired oxygen provides a means to enhance oxygenation in the retina and choroid. We propose to use supplemental inspired oxygen to test several hypotheses related to the role of hypoxia in ocular NV and macular edema. The following experimental questions will be addressed. (1) Does supplemental inspired oxygen cause regression or slow progression of retinal NV that is not high risk in patients with diabetic retinopathy? (2) Does supplemental oxygen cause improvement in macular edema in patients with ischemic retinopathy? (3) Does supplemental oxygen cause stabilization or improvement in AMD patients with predominantly occult subfoveal CNV? (4) Does supplemental oxygen decrease recurrent CNV in AMD patients with predominantly classic CNV treated with photodynamic therapy? The answers to these questions will provide important insights into the pathogenesis of retinal diseases that could lead to the development of new treatments.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: TESTS OF MODELS OF RETINAL/OPTIC NERVE DISEASES&ADAPTION

Principal Investigator & Institution: Hood, Donald C.; Professor; Psychology; Columbia Univ New York Morningside 1210 Amsterdam Ave, Mc 2205 New York, Ny 10027

Timing: Fiscal Year 2002; Project Start 01-AUG-1977; Project End 31-JUL-2005

Summary: A major long-term objective is to develop techniques for localizing the sites, and identifying the mechanisms of diseases of the retina and optic nerve. The multifocal electroretinogram (mERG) and visual evoked potential (mVEP) techniques are relatively

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new ways for measuring the physiological activity of local retinal and cortical activity. By studying the mERGs from patients with a variety of retinal/optic nerve problems we aim to develop a conceptual framework for relating changes in the mERG to sites and mechanisms of damage (aim 1a). In addition, some retinal diseases affect retinal mechanisms of adaptation and studies are designed to understand these changes. The mVEP technique is less developed and studies are proposed to improve this technology. These include the development of a method for measuring and specifying the strength of the signal in the mVEP response (aim 1b). Specific studies are proposed to understand the contribution of cone pathways to the mVEP, to assess repeat reliability, to optimize recording techniques, and to develop norms. The second long-term objective is to improve our understanding of the sites and mechanisms of particular diseases of the retina/optic nerve through studies employing behavioral (e.g. visual fields), structural (e.g. nerve fiber layer analysis) and electrophysiological techniques (e.g. mERG and mVEP). As part of aim 2, the mERG and visual field measures will be employed to better understand retinal damage in patients with retinitis pigmentosa and diabetic retinopathy and to assess possible damage following retinal surgery for macular holes and macular pucker. In addition, the sites and mechanisms of glaucomatous damage will be studied. These studies include a comparison of structural and functional measures to better understand the mechanism(s) of damage, an attempt to detect early damage, an assessment of the effect of acute decreases in intraocular pressure, and an assessment of whether there are selective deficits in the cone pathways. Finally, patients with acute optic neuritis will be followed with behavioral and mVEP techniques. An attempt will be made to understand the recovery o vision following optic neuritis (ON).

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: THE ROLE OF ARNT IN VASCULAR AND CARDIAC DEVELOPNENT

Principal Investigator & Institution: Ramirez-Bergeron, Diana L.; Anatomy; University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Timing: Fiscal Year 2003; Project Start 09-JUN-2003; Project End 31-MAY-2008

Summary: (provided by applicant): The objective of this K01 award is to provide intensive mentoring and research training to the minority principal investigator that will allow for an effective transition into an independent academic researcher. The candidate will receive scientific and career mentorship from well established, highly motivational sponsors and advisory committee members. In the past, the investigator has focused on hematopoietic development. The proposed research will redirect her research to acquisiton of new skills in the areas of cardiovascular development and hypoxic biology. The proposal is based on work from Dr. Simon's laboratory encompassing work in hematopoiesis and vasculogenesis. Mutations in various subunits of the Hypoxia Inducible Factor (HIF) family, an important hypoxic transcriptional mediator, have contributed to the understanding the role 02 tension plays during early embryonic development. Abrogation of the Arnt subunit of the HIF heterodimer resulted in embryonic lethality by 10.5 days postcoitum (dpc) with yolk sac, placental, cardiac, and vascular defects. The goal of this proposal is to characterize the role of 02 in cardiovascular development, particularly on endothelial cells. The specific aims are to 1) determine how hypoxia influences endothelial behavior, 2) examine the hematopoietic and angiogenic potential of intraembryonic tissues of Amt -/- mice, and 3) determine the effect of hypoxia in heart development. Accomplishing these specific aims will provide an understanding of hypoxic signals in mediating biological responses required for the growth and differentiation of the cardiovascular system. Furthermore, the

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examination of the cellular and molecular responses to hypoxia will provide important insights into various disease states, including tumor growth, diabetic retinopathy, preeclampsia, wound healing, and ischemia. Additionally, the detailed studies of this proposal and the senior guidance of the faculty committee will assist in commanding the investigator into a leader in the field of cardiovascular biology.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: THERAPEUTIC POTENTIAL OF A NOVEL ANGIOGENIC INHIBITOR

Principal Investigator & Institution: Ryan, James C.; Genekeys, Llc 1067 Blue Marlin Dr Charleston, Sc 29412

Timing: Fiscal Year 2003; Project Start 01-MAY-2003; Project End 31-OCT-2003

Summary: (provided by applicant): Retinal neovascularization and vascular leakage are common causes leading to retinal damage in diabetic retinopathy. Currently, there is no satisfactory treatment to stop neovascularization or to reduce vascular permeability in diabetic retinopathy, which is a leading cause of blindness in the US and the industrialized countries. Accumulating evidence suggests that decreased levels of endogenous angiogenic inhibitor (e.g. pigment epithelium-derived factor, PEDF) in the retina and vitreous as well as increased angiogenic stimulators (e.g. vascular endothelial growth factor, VEGF) play a critical role in the development of retinal neovascularization. Therefore, delivery of angiogenic inhibitors into the eye is believed to be a promising therapy for retinal neovascularization. Kallikrein-binding protein (KBP, or kallistatin) is a specific inhibitor of tissue kallikrein. It belongs to the family of serine proteinase inhibitors (serpin) and has significant sequence homology with the potent angiogenic inhibitor, PEDF. Recently, we have found that KBP specifically inhibits the proliferation of primary endothelial cells and down-regulates VEGF expression. This vascular activity is independent of its interactions with tissue kallikrein. Moreover, KBP levels are decreased in the vitreous of patients with proliferative diabetic retinopathy and in the retina of a diabetic animal model. Our hypothesis is that KBP is a potent angiogenic inhibitor, which inhibits retinal neovascularization and reduces vascular leakage. The principal objective of this Phase I project is to determine the efficacy of KBP as an antiangiogenic agent in vivo. This Phase I project will determine if KBP can inhibit retinal neovascularization in an animal model of oxygeninduced retinopathy. Recombinant KBP will be injected intravitreally into newborn rats with oxygen-induced retinopathy. Its effect on retinal neovascularization will be evaluated by fluorescein angiography and quantification of pre-retinal vascular cells. This project will also determine if KBP can reverse vascular hyper-permeability in the retina of diabetic animals. VEGF is a major factor responsible for hyper-permeability in diabetic retinopathy. Given that KBP decreases VEGF levels, it may reverse hyperpermeability. This study will use oxygen-induced retinopathy and streptozotocin (STZ)- induced diabetic rats as models. The effect of KBP injection on vascular permeability will be evaluated by the Evans Blue leakage method.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: THICKENING OF BASEMENT MEMBRANE IN DIABETIC RETINOPATHY

Principal Investigator & Institution: Roy, Sayon; Ophthalmology; Boston University Medical Campus 715 Albany St, 560 Boston, Ma 02118

Timing: Fiscal Year 2004; Project Start 01-JAN-2004; Project End 31-DEC-2007

66 Diabetic Retinopathy

Summary: (provided by applicant): Vascular basement membrane thickening is a prominent and characteristic lesion of diabetic retinopathy. The goal of this project is to test the hypothesis that the thickened vascular basement membrane (BM) occurring in diabetes plays a role in the development and progression of serious structural and functional abnormalities of diabetic retinopathy. The hypothesis is based on recent findings that human retinal vessels manifest alterations that can be induced by the thickened BMs and, in turn, can compromise the integrity of the vessels' inner lining. In retinal capillary cells of diabetic individuals there is increased production of the BM proteins, fibronectin (FN), collagen IV (coil IV), and laminin (LM). Antisense oligonucleotides developed in our laboratory specifically down-regulate overexpression of the three BM components in microvascular endothelial cells grown in high glucose medium. Furthermore, inhibition of FN overexpression with the FN antisense oligonucleotide partially prevented the development of thickened vascular BM in retinal capillaries of galactose-fed rats, an animal model of diabetic retinopathy, with beneficial consequences to histological lesions. To completely prevent or reverse vascular BM thickening, and regulate functional abnormalities, the development of a more effective antisense strategy is necessary. Having identified antisense oligos that independently down-regulate FN, coil IV or LM expression in retinal vascular cells, in the proposed studies we plan to: (1) Establish whether combined antisense oligo approach prevents vascular BM thickening in rat retinas, and affects FN, coil IV, LM turnover in matrix. (2) Determine if downregulation of the specific BM genes reverses BM thickening, reduces vascular lesions, and affects cellular processes in retinas of diabetic rats. (3) Determine whether high glucose-induced or diabetes-induced altered expression of BM components plays a role in vascular permeability. FN, coil IV, and LM protein level will be monitored by Western blot analysis and immunohistochemistry; RNA level will be analyzed by RT-PCR. Retinal capillary BM width will be measured by morphometric analysis of electron micrographs. Vascular lesions will be assessed from retinal trypsin digests and image analysis. Findings from this project will establish or exclude a pathogenetic link between a discrete biosynthetic abnormality and the development of structural and functional lesions of diabetic retinopathy.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: THREE-DIMENSIONAL OCT FOR DETECTION OF MACULAR EDEMA

Principal Investigator & Institution: Zhou, Qienyuan; Laser Diagnostic Technologies, Inc. 10864 Thornmint Rd San Diego, Ca 92127

Timing: Fiscal Year 2002; Project Start 30-SEP-2002; Project End 31-MAR-2003

Summary: (provided by applicant): Macular edema(ME) is a common cause of significant visual loss in a wide variety of ocular condition, including age-related macular degeneration (AMD) and diabetic retinopathy (DR). The early detection and classification of ME is of special importance in DR, since the optimum treatment depends on position, type, and extension of the ME. Our long term goal is to develop a clinically useful tool to map macula thickness, to screen for clinically significant macular edema, and to provide high resolution images of retinal cross-sections at any arbitrary retinal locations. In the proposed technique, the rapid transverse imaging mode of the scanning laser ophthalmoscope (SLO) will be combined with high depth resolution and sectioning capability of the optical coherence tomography (OCT). This instrument will be able to image the retina in three dimensions, delineate ME boundaries, and quantify ME volume. During Phase l of this study, we will focus our effort on proof of feasibility. We will modify a breadboard system based on a Topographic Scanning System

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TopSS(Laser Diagnostic Technologies, Inc.) perform in vivo measurements on a few normal subjects and a few ME patients, develop 3-D image processing software, and identify potential problems and improvements for Phase II work.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: THROMBOSPONDIN-1 AND RETININAL VASCULAR HOMEOSTASIS

Principal Investigator & Institution: Sheibani, Nader; Assistant Professor; Ophthalmology and Visual Sci; University of Wisconsin Madison 750 University Ave Madison, Wi 53706

Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2006

Summary: (provided by applicant): Diabetes predominantly affects the microvascular circulation of the retina resulting in a range of structural changes that are unique to this tissue. These changes include an early persistent loss of pericytes from retinal microvessels, thickening of the basement membrane, followed by hyperproliferation of endothelial cells (ECs) and abnormal vascularization of the retina, which ultimately results in blindness. Retinal vascularization is normally restricted to the superficial and deep layers of the retina. Other ocular sites such as cornea, lens, and vitreous are normally vascular free. It has been hypothesized that a negative regulator of angiogenesis is responsible for these vascular restrictions. We have recently demonstrated that thrombospondin-1 (TSP1), a potent natural inhibitor of angiogenesis, is present at ocular avascular sites and TSPt expression is dramatically down regulated with diabetes. Our hypothesis is that TSP1 is an important modulator of retinal vascular homeostasis whose alterations under pathological conditions such as diabetes and/or ischemia results in retinal neovascularization. The studies proposed here will investigate the role of TSP1 in retinal vascular development and ischemia-induced neovascularization. We will examine the expression of TSP1 in the retina and retinal capillaries during development and determine whether its expression is altered during oxygen-induced ischemic retinopathy. We will compare development of retinal vasculature and their total area in the retina of normal, TSP1 deficient, and TSP1 overexpressing transgenic mice. We will determine whether lack or over-expression of TSP1 influences retinal vascular development and neovascularization in response to hypoxia. Identification of TSP1 as a modulator of ocular vascularization and the study of its mechanisms of action in retinal vascular cells will provide insight into the defects that contribute to retinal neovascularization. This knowledge will provide the rationale for development of new therapeutic approaches for the prevention and/or treatment of ocular diseases with a neovascular component.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: TRANSCLERAL DELIVERY OF DRUGS FROM A COLLAGEN SYSTEM

Principal Investigator & Institution: Devore, Dale P.; Xium, Llc 55 Beach St, #8 Westerly, Ri 02891

Timing: Fiscal Year 2004; Project Start 01-JUN-2004; Project End 31-MAY-2005

Summary: (provided by applicant) The overall objective of this project is to develop an injectable or implantable matrix system to provide sustained delivery of therapeutic drugs to the posterior chamber of the eye for treatment of macular degeneration, diabetic retinopathy and other ocular diseases. More effective methods of drug delivery to the posterior segment of the eye are needed for treatment of posterior segment

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disease. Current ocular drug delivery systems primarily include topical drops, subconjunctival and peribulbar injections, oral administration, intravitreal injections and implants. Each of these delivery routes has significant limitations for posterior drug delivery. Localized drug delivery would be a great advantage in treating posterior segment disease where systemic side effects can be avoided. The sclera of the eye has been found to be permeable to a wide variety of compounds and may be a possible route for local drug delivery. The objectives of the Phase I project are to formulate collagen-based matrices that can be injected or implanted beneath the conjunctiva and to measure the in vitro human scleral permeability of several drugs including dexamethasone, methotrexate, and vancomycin delivered in these systems. The specific aims are to formulate injectable in situ polymerizing collagen solutions and solid collagen matrices containing fluorescein-labeled drugs and to measure the transcleral diffusion of the target drugs using a specially designed perfusion chamber at Emory University. Injectable, in situ polymerizable collagen will be prepared from animal hide as described in U.S. Patent 5,492,135. Solid collagen will be prepared from chemically derivatized collagen by exposing the derivatized collagen to ultraviolet light. Both preparations will be dosed with fluorescein-labeled drugs. Diffusion through human sclera will be measured in the in vitro perfusion chamber (Emory University). Results will be analyzed to determine the most effective matrix system for sustained drug delivery. This system will then be used in Phase II studies in animal models.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: VEGF EXPRESSION AND ER STRESS IN DIABETIC RETINOPATHY

Principal Investigator & Institution: Roybal, Christopher N.; Biochem and Molecular Biology; University of New Mexico Albuquerque Controller's Office Albuquerque, Nm 87131

Timing: Fiscal Year 2002; Project Start 27-MAR-2003; Project End 31-AUG-2005

Summary: (provided by applicant): Our goal is to determine how VEGF translation and transcription is affected by ER-stress and if this mechanism plays a role in experimental retinophathy. Recent publications have shown a correlation between ER-stress and increased levels of VEGF mRNA and protein. Protein synthesis attenuation occurs during ER-stress and has prompted us to examine the role of a proposed internal ribosome entry site (IRES), located in the 5' UTR, in the translation initiation of VEGF synthesis. The role of ER-stress on angiogenesis will be further explored using an in vitro diabetic retinopathy model. The Streptozotocin (STZ) and retinopathy of prematurity (ROP) diabetic rat models are the identified models in which these studies will take place. By confirming or disproving the role of ER-stress on angiogenesis we will develop a greater understanding of vascular associated complications seen in diabetes.

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: VISION DISABILITIES IN LOW VISION

Principal Investigator & Institution: Massof, Robert W.; Professor; Ophthalmology; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218

Timing: Fiscal Year 2002; Project Start 01-AUG-1999; Project End 31-JUL-2003

Summary: (Adapted from the Investigator's Abstract) The proposed research has the goal of developing and validating interval psychometric scales of visual function limitations and vision disabilities. These interval scales will be developed using Rasch probablistic measurement models applied to ordinal patient rating responses to

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individual questions. Once developed and validated, these scales will be independent of the particular assessment used, as long as the instrument is calibrated to the scale. The significance of the proposed research is that it will provide a means of estimating measurements of latent functional ability variables for individual patients with visual impairments. In the future, these measurements can be used for parametric studies, epidemiological studies, and clinical outcome studies. The proposed research will identify the number and nature of functional ability scales. It will determine the dependence of those scales on the diagnosis of visual system disorder, the type of visual impairment, the existence of co-morbidities, and the patient's history of rehabilitation. Existing visual function instruments (NEI-VFQ, VF-14, ADVS, and VAQ) and two general function instrument's individual items will be evaluated with respect to scales. To estimate the scales, a large set of specific cognitive and motor activities (e.g., writing a check) will be classified according to functional domain (reading, fine and gross visual-motor, visual information processing [e.g., recognition, localization, orientation], or mobility). In telephone interviews, low vision patients will be asked to rate the difficulty of performing each activity. Rasch analysis will be used to test the hypothesis that there is a global functional ability scale and to test the validity of the a priori visual function domains. Principal component analysis of response residuals will be used to evaluate the dimensionality of visual function limitations. Patients also will be asked to rate the difficulty of achieving specific activity goals (e.g., cook a meal, manage personal finances) and Rasch analysis will be used to estimate a vision disability scale. Item ordering and item intervals on the scales and scale validity will be compared across diagnostic groups (AMD, glaucoma, diabetic retinopathy, RP, CVA, and anterior segment disorders) and for different types of visual impairments (e.g., acuity loss and contracted visual fields). Person measures of functional ability will be evaluated as a function of severity of visual impairments (visual acuity, contrast sensitivity, visual fields, dark adaptation, color vision). Determining if the NEI-VFQ, VF-14, ADVS, VAQ, SF-36, and SIP can be calibrated to common scales will test the hypothesis that there is a common functional ability variable(s).

Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

•Project Title: VISUAL IMPAIRMENT, TREATMENT, AND EFFECTS ON THE ELDERLY

Principal Investigator & Institution: Sloan, Frank A.; J. Alexander Mcmahon Professor; Ctr/Hlth Policy Law & Mgmt; Duke University Durham, Nc 27710

Timing: Fiscal Year 2002; Project Start 15-APR-2001; Project End 31-MAR-2005

Summary: This four-year study has four major objectives. (1) For elderly persons with diabetes mellitus diabetic retinopathy, glaucoma, and macular degeneration -- diseases that can cause serious impairments to vision and blindness -- we will analyze determinants of utilization of eye care services, and whether or not Medicare beneficiaries with diabetes/diabetic retinopathy and glaucoma receive care in accordance with minimal process of eye care standards. Such standards have been disseminated as guidelines by professional organizations. In this phase, we will address these issues. How do data on patient self-report of diagnosis compare with those obtained from physicians' diagnoses? What proportions of persons with the study diseases receive care at least at the minimum level of guidelines? Holding many factors constant, are there racial differences in treatments for these diseases? (2) We will study the longitudinal course of these diseases, measured in terms of visual and general functional status -- physical, cognitive -- and in terms of placement in a nursing home, and survival. We will analyze effects of care on changes in vision and the other outcome