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CHAPTER 1. STUDIES ON MACULAR DEGENERATION

Overview

In this chapter, we will show you how to locate peer-reviewed references and studies on macular degeneration.

The Combined Health Information Database

The Combined Health Information Database summarizes studies across numerous federal agencies. To limit your investigation to research studies and macular degeneration, you will need to use the advanced search options. First, go to http://chid.nih.gov/index.html. From there, select the “Detailed Search” option (or go directly to that page with the following hyperlink: http://chid.nih.gov/detail/detail.html). The trick in extracting studies is found in the drop boxes at the bottom of the search page where “You may refine your search by.” Select the dates and language you prefer, and the format option “Journal Article.” At the top of the search form, select the number of records you would like to see (we recommend 100) and check the box to display “whole records.” We recommend that you type “macular degeneration” (or synonyms) into the “For these words:” box. Consider using the option “anywhere in record” to make your search as broad as possible. If you want to limit the search to only a particular field, such as the title of the journal, then select this option in the “Search in these fields” drop box. The following is what you can expect from this type of search:

•Age-Related Macular Degeneration

Source: Access. 17(7): 30-34. August 2003.

Contact: Available from American Dental Hygienists' Association (ADHA). 444 North Michigan Avenue, Chicago, IL 60611. (312) 440-8900. E-mail: mail@adha.net. Website: www.adha.org.

Summary: Age-related macular degeneration (AMD) is the most common cause of vision loss and blindness in people over age 65, and it can affect people at younger ages as well. The risk of developing AMD steadily increases with age. This article reviews the anatomy and function of the normal retina. The author also identifies the risk factors for AMD and its causes, as well as its diagnosis and treatment. A diagnosis of AMD means

4Macular Degeneration

a significant decrease in vision, and, in some cases, complete blindness. The author discusses the issues of AMD patients' everyday activities, and reviews the communication skills that dental hygienists can use with low-vision individuals. The author notes that the ability to cope with AMD depends on how much vision is lost, how sudden or gradual the change is, the patient's pre-illness coping style, and, especially, the patient's support system. One side bar lists assistive devices for people with low vision; another provides the contact information for four resource organizations. 6 figures. 12 references.

Federally Funded Research on Macular Degeneration

The U.S. Government supports a variety of research studies relating to macular degeneration. 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 macular degeneration.

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 macular degeneration. The following is typical of the type of information found when searching the CRISP database for macular degeneration:

•Project Title: ABCR, MACULAR DYSTROPHIES AND DEGENERATION

Principal Investigator & Institution: Lupski, James; Professor; Molecular and Human Genetics; Baylor College of Medicine 1 Baylor Plaza Houston, Tx 77030

Timing: Fiscal Year 2002; Project Start 05-SEP-2002; Project End 31-AUG-2006

Summary: (provided by applicant): We identified the causative role of either compound heterozygous or homozygous mutations of ABCR in classic autosomal recessive Stargardt's macular dystrophy. The ABCR gene (also known as ABCA4) encodes a photoreceptor-specific ATP-binding cassette transporter, which has been shown recently to transport retinal or a derivative thereof. ABCR mutations also have a role in some forms of retinitis pigmentosa and a large fraction of cone-rod dystrophy. Thus, ABCR is likely to be responsible for a substantial portion of diverse retinal pathologies. That some heterozygous ABCR mutations are associated with the multifactorial disorder Age-Related Macular Degeneration suggests an even more prominent role for ABCR in visual impairment. We and others have proposed a model, the "ABCR activity vs. retinal disease phenotype hypothesis," in which the severity of retinal dystrophic phenotype is inversely proportional to the residual fraction of ABCR wild-type activity. That is, an RP phenotype results from essentially zero ABCR activity associated with two null

mutations, while a late-onset Stargardt's macular dystrophy results from a selected combination of mutations that retain substantial ABCR activity. In this model,

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 5

heterozygous ABCR mutations represent a dominant susceptibility locus for AMD. The common final pathophysiological pathway is influenced by how much A2E (a conjugate of retinaldehyde and phosphatidylethanolamine that appears to be transported by the "ABCR flippase" and is the major toxic constituent of lipofuscin that accumulates in the flecks of STGD patients and drusen of those with AMD) accumulates in the retinal pigment epithelium. Using an assay to assess protein expression and ATP-binding activity we will perform functional analyses of disease-associated ABCR mutations. Further, we will systematically investigate and classify synergistic effects and the functional consequences of complex alleles. We will also develop a novel transporter assay to directly evaluate ABCR transporter/flippase function and the effects of diseasecausing mutation. Functional analyses of ABCR and mutations in retinopathy patients will expand greatly our knowledge of normal ABCR function and the dysfunction resulting in retinal pathology. Understanding the molecular pathway involving ABCR opens new avenues for the predictive diagnosis of selected retinal diseases and may lead to novel prophylactic and interventional therapies for these devastating disorders.

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

•Project Title: AGE-INDUCED PHENOTYPE OF THE RETINAL PIGMENT EPITHELIUM

Principal Investigator & Institution: Handa, James T.; Associate Professor; Ophthalmology; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218

Timing: Fiscal Year 2001; Project Start 01-JUL-2001; Project End 31-MAY-2006

Summary: (provided by applicant): While a number of genes have been postulated to be involved in age-related macular degeneration (AMD), the leading cause of blindness among the elderly in the US, a comprehensive mRNA phenotype of the retinal pigment epithelium (RPE) in vivo is at present unknown. Determination of an RPE mRNA phenotype could identify the genes critical to the development of AMD, identify markers of disease, and lead to new preventative and treatment strategies. Basal deposits in Bruch's membrane are an early recognizable change that occur prior to degeneration of the RPE and are a histopathological marker for AMD. Recently, our laboratory identified an age-dependent accumulation of advanced glycation end products (AGEs) in Bruch's membrane and basal deposits. AGEs are structures formed during the series of nonenzymatic reactions between sugars or other precursors, and long-lived proteins that alter the phenotype of a variety of cell types. Our laboratory has also recently shown that AGEs alter the expression of genes involved in matrix regulation, cell polarity, and apoptosis in RPE cells. Our long term goal is to define an extensive mRNA phenotype of the RPE in health and AMD which will in turn, yield insights into the pathogenesis of AMD. Specifically, we want to define the component of that phenotype which is regulated by AGEs. We hypothesize that the presence of AGEs in basal deposits is responsible for a switch in the phenotype of RPE cells that is consistent with alterations in matrix regulation, RPE cell polarity, and apoptosis. To test this hypothesis we ask 3 questions: 1. Are the mRNA phenotypes of macular RPE cells overlying basal deposits and normal Bruch's membrane different? 2. Does AGE modified matrix induce an RPE mRNA phenotype in vitro that is a subset of the phenotype expressed by RPE cells overlying basal deposits? 3. Is a subset of the AGE induced mRNA phenotype of RPE cells mediated by the Receptor for Advanced Glycation End products? To answer these questions we will use laser capture microdissection to obtain pure samples of RPE cells from tissue specimens for mRNA phenotyping by microarray analysis. We will also utilize an in vitro AGE-matrix system to determine what subset of genes is regulated by AGEs. This project hopes to establish

6Macular Degeneration

an extensive mRNA phenotype of the RPE in both health and AMD, and define a subset of genes induced by AGEs.

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

•Project Title: AGE-RELATED MACULOPATHY: COMPLEMENT-MEDIATED EVENTS

Principal Investigator & Institution: Johnson, Lincoln V.; None; University of California Santa Barbara 3227 Cheadle Hall Santa Barbara, Ca 93106

Timing: Fiscal Year 2002; Project Start 01-AUG-1996; Project End 31-MAR-2006

Summary: (provided by applicant): The objective of the proposed studies is to test the hypothesis that the process of drusen formation is stimulated by complement-mediated inflammatory events involving retinal pigmented epithelial (RPE) cells. Drusen are extracellular deposits that form between the RPE and Bruch's membrane, and are a significant risk factor for age-related macular degeneration (AMD). In AMD, functional compromise and ultimately death of RPE cells as a consequence of drusen formation is thought to lead to secondary degeneration of retinal photoreceptor cells and visual loss. Studies of age-related human diseases that, like AMD, involve cellular degeneration and the formation of insoluble deposits (e.g., Alzheimer's disease, atherosclerosis and kidney glomerulonephritis) now implicate complement activation and inflammatory events as key elements in disease processes. Data that we generated during the prior application period strongly suggest that complement activation also plays an important role in the formation of ocular drusen. We observed that terminal complement complexes and complement regulatory molecules are present in drusen, as are molecules with potential complement-activating properties. Furthermore, our observations show that RPE cells overlying drusen exhibit a compromised molecular phenotype that is consistent with a well-characterized cellular response to complement attack. Thus, these observations suggest an AMD disease process that is consistent with that of other age-related human diseases. A process that involves a primary pathogenic or age-related stimulus, the effects of which are exacerbated by localized, self-perpetuating, complement-mediated tissue destruction and inflammatory sequelae that persist over decades. The studies proposed here will examine the hypothesis that complement-mediated events contribute both to the process of drusen formation, and to the functional compromise of drusenassociated RPE cells. The studies will target the following specific aims: (1) To determine if RPE cell compromise is the result of complement-mediated attack, (2) To characterize the nature of the complement-mediated processes involved in drusen formation, and (3) To examine RPE cell responses to complement attack utilizing an in vitro model system.

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

•Project Title: ANALYSIS OF HUMAN VMD2: A MODEL FOR RPE GENE REGULATION

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

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

Summary: (Adapted from Applicant's abstract): The long-term objective of this project is to better understand the molecular mechanisms that underlie retinal pigment epithelium (RPE)-specific gene expression. The RPE is important for the function and survival of retinal photoreceptors. Abnormalities in the RPE have been implicated in the pathogenesis of photoreceptor degeneration both in the RCS rat and in human agerelated macular degeneration (AMD), the leading cause of irreversible blindness in

Studies 7

elderly Americans. In addition, mutations in RPD65, RLBP1, TIMP3, and VMD2, four genes that are specifically or preferentially expressed in the RPE, have been shown to cause Leber's Congenital Amaurosis (LCA), retinitis pigmentosa (RP), Sorsby's fundus dystrophy (SFD), and vitelliform macular dystrophy (Best's disease), respectively. However, despite the importance of the RPD, and the importance of genes that are specifically or preferentially expressed in the RPE, little is known about the molecular mechanisms that regulate RPE-specific gene expression. Therefore, the goal of this proposal is to begin elucidating such mechanisms using human VMD2 (Best's disease gene) as a model system. More specifically, the following questions will be addressed: What are the cis-acting DNA elements that regulate VMD2 expression? Do different RPE-specific genes share common regulatory elements? What are the trans-acting factors that bind to the DNA regulatory elements? Are mutations in RPE promoters and/or RPE transcription factors involved in human disease? For answering these questions, DNA elements in the promoter region of VMD2 will be identified by sequence comparison with the other RPE-specific promoters and by biochemical analysis using DNase I footprint and electrophoretic mobility shift (EMSA) assays. The promoter region of VMD2 will be functionally analyzed using a combination of transient transfection and transgenic mouse approaches. Finally, to clone and characterize cDNAs for the trans-acting that bind to the DNA regulatory elements in the VMD2 promoter, we will use the yeast one-hybrid approach with a human RPE cDNA library. Increased understanding of RPE gene regulation should help provide new insights into diseases involving abnormalities of RPE gene expression. In addition, the knowledge will be useful in the development of RPE-targeted gene therapy and the generation of transgenic mice with targeted expression of exogenous proteins to the RPD. Furthermore, since Best's disease shares some clinical and histological features with AMD, studies of human VMD2 expression may have important implications for understanding macular degeneration.

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

8 Macular Degeneration

•Project Title: ANTIOXIDANT METABOLISM IN CHLAMYDOMONAS

Principal Investigator & Institution: Niyogi, Krishna K.; Plant and Microbial Biology; University of California Berkeley Berkeley, Ca 94720

Timing: Fiscal Year 2001; Project Start 01-JAN-1999; Project End 31-DEC-2003

Summary: Reactive oxygen species (ROS) are generated inevitably in cells of all aerobic organisms, from bacteria plants and animals. Coping with the potentially damaging consequences of ROS generation is a problem of broad biological significance. The involvement of excited and highly reactive intermediates in oxygenic photosynthesis poses unique problems for algae and plants with respect to the generation of ROS. The proposed research plan is focused on the question of how photosynthetic organisms prevent or minimize photo-oxidative damage caused by ROS. To dissect antioxidant defenses in the chloroplast, mutants of the unicellular green alga Chlamydomonas reinhardtii, have been isolated. Several mutants affect the metabolism of xanthophyll pigments, which have critical roles in photoprotection in all algae and plants. An npq1 lor1 double mutant, which is deficient in lutein and zeaxanthin, undergoes photooxidative bleaching in high light, presumably due to increased production and/or decreased quenching of ROS, especially singlet oxygen. The proposed research will examine the types of photooxidative damage and the responses of various antioxidant systems in npq1 lor1 cells. Isolation of the genes affected in the xanthophyll mutants will enable determination of the molecular basis for the mutant phenotypes and will provide a strong foundation for further genetic analysis, including isolation of suppressors of npq1 lor1. The generation and/or scavenging of other ROS is perturbed in another group of mutants, which will be characterized using a combination of molecular genetic, biochemical, and physiological approaches. Understanding the metabolism and function of xanthophylls and other antioxidants in algae and plants will provide insights into the important roles of these molecules in preventing age-related diseases such as macular degeneration in humans and will enable engineering of antioxidant metabolism in plants to benefit human nutrition and health.

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

•Project Title: ANTIOXIDANT SYSTEMS AND AGE RELATED MACULAR DEGENERATION

Principal Investigator & Institution: Sternberg, Paul W.; Thomas Aaberg Professor; Ophthalmology; Emory University 1784 North Decatur Road Atlanta, Ga 30322

Timing: Fiscal Year 2001; Project Start 01-AUG-1989; Project End 31-JUL-2004

Summary: (Adapted from the applicant's abstract): This research program focuses on the hypotheses that age-related macular degeneration (ARMD) can result from oxidative injury to the retinal pigment epithelium (RPE) and that glutathione (GSH) may protect the retina and RPE cells from ARMD-associated with oxidative injury. Previous work has shown that there is a shift in blood plasma redox status to a more oxidized state with aging, placing all tissues at risk for age-related diseases; there also appears to be a more oxidized redox state in patients' with more advanced ARMD. In vitro studies suggest that oxidative injury-induced apoptotic cell death in RPE cells may involve mitochondrial damage. Further, dietary inducers can stimulate increased GSH synthesis leading to elevated intracellular GSH, with concomitant increased protection against oxidative injury. In this project, the investigators propose to answer the following questions which will test their hypotheses: (1) how redox status is associated with ARMD; (2) what mechanism is involved in shifting GSH redox status to a more oxidized state that affects RPE cell function; (3) whether one can modulate GSH synthetic

Studies 9

capacity in human RPE cells by controlling the rate-limiting enzyme for GSH synthesis; and (4) what mechanisms are involved in the redox regulation of apoptosis in cultured human RPE cells. Biochemical studies, including HPLC, viability studies, measures of RPE function, TUNEL assay and other assays for apoptosis, and assays for mitochondrial function, will evaluate human blood plasma samples, cultured human RPE cells, and experimental animals. These studies should increase our understanding of the pathogenesis of ARMD, while directly suggesting new treatment strategies.

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

•Project Title: APOPTOSIS IN RETINAL DETACHMENTS

Principal Investigator & Institution: Zacks, David N.; 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 30-SEP-2002; Project End 31-AUG-2007

Summary: (provided by applicant): Retinal diseases such as retinal detachments and macular degeneration are a leading cause of vision loss and blindness in this country and around the world. Despite available surgical and medical treatments for these diseases, patients still experience a large amount of vision loss secondary to the disruption in nutritional support provided to the retina by the underlying tissues. This disruption causes death of the retina, and of the photoreceptor cells in particular. Apoptosis, or programmed cell death, has been implicated as the mechanism by which photoreceptor cells die, but the molecular mechanisms of activation, transduction and execution of the apoptotic cascade in retinal diseases are poorly understood. The main hypothesis of this research proposal is that photoreceptor cell death during retinal detachments and macular degeneration occurs via apoptosis. The research outlined in this application will use a newly developed rodent model of retinal detachments and molecular biologic and genetic techniques to gain new insight into apoptotic photoreceptor cell death. This will allow us to develop and test new neuroprotective therapies for improving visual outcomes in retinal disease.

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

•Project Title: ATHEROSCLEROSIS AND RISK OF AGE-RELATED MACULOPATHY

Principal Investigator & Institution: Cruickshanks, Karen J.; Professor; Ophthalmology and Visual Sci; University of Wisconsin Madison 750 University Ave Madison, Wi 53706

Timing: Fiscal Year 2001; Project Start 01-FEB-2001; Project End 31-JAN-2003

Summary: (Applicant's Abstract) The purpose of this epidemiologic study is to evaluate the association of markers of generalized atherosclerosis with the incidence of early and late stage age-related maculopathy in a population-based, nested incident case-control study. This proposal builds on the population-based studies of aging in Beaver Dam, WI: The Beaver Dam Eye Study (EY06594) and the Epidemiology of Hearing Loss Study (AG11099). Participants with incident early age-related maculopathy or incident latestage age-related maculopathy as determined from grading of standardized photographs taken at two examinations five years apart will be eligible as cases for this study. Participants without age-related maculopathy at the time of the 1 0-yr follow-up eye examination will be eligible as controls. Incident early cases will be frequencymatched by age and gender to controls using a two-to-one ratio of controls to cases. Incident late age-related maculopathy cases will be frequency-matched to controls by age and gender using a three-to-one ratio of controls to cases. Based on expected 5-yr

10Macular Degeneration

incidence rates and participation rates in both studies, we estimate that a total of 189 cases with incident early age-related maculopathy and 378 controls and 34 cases with incident late age-related maculopathy and 102 controls will be included in this study. Generalized atherosclerosis will be measured by intima-medial thickness and the presence of plaque in the carotid arteries. Stored videotapes of B mode ultrasound scans of the carotid arteries were obtained as of the Epidemiology of Hearing Loss Study, using a modification of the Atherosclerosis Risk in Communities Study protocol. These scans will be read, and measurements of the intima-medial thickness made, using the Atherosclerosis Risk in Communities ultrasound reading protocol. Age-related maculopathy is well-recognized to be the leading cause of blindness among older adults and a significant public health problem for older adults. As yet there is no way to prevent or effectively treat this disorder. This study will provide important epidemiologic information about the role of atherosclerosis in the etiology of age-related maculopathy. It takes advantage of an unique opportunity to evaluate this association by utilizing an existing population-based cohort with standardized assessments of the incidence of age-related maculopathy and standardized carotid artery ultrasound scans.

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

•Project Title: BIOCHEMISTRY AND PHARMACOLOGY OF THE MACULAR CAROTENOIDS

Principal Investigator & Institution: Bernstein, Paul S.; Assistant Professor; Ophthalmology and Visual Scis; University of Utah Salt Lake City, Ut 84102

Timing: Fiscal Year 2001; Project Start 01-AUG-1997; Project End 31-JUL-2003

Summary: The human macula, the specialized region of the retina responsible for high resolution visual acuity, selectively accumulates two xanthophy11 carotenoids derived from the diet, lutein and zeaxanthin. Several recent epidemiological studies have demonstrated a strong inverse correlation between dietary intake of lutein and zeaxanthin and the risk of progression Age-Related Macular Degeneration (AMD) the leading cause of blindness among the elderly in the United States. It is thought that the macular carotenoids protect against least-induced damage to the retina by filtering out damaging wavelengths of light and by acting as antioxidants. The biochemical mechanisms that mediate the selective uptake, concentration, and stabilization of the macular carotenoids are unknown. In lower animals, such as lobsters and cyanobacteria, specialized carotenoid-binding proteins perform these tasks. It is hypothesized that comparable carotenoid-binding proteins may have a similar role in the human macula. A major goal of this project is to understand the biochemical processes responsible for the specific deposition of lutein and zeaxanthin the macula, with special emphasis on the search for potential carotenoid-binding proteins. This project will also investigate the properties of carotenoidprotein and carotenoid-lipid interactions through quantitative binding studies and various spectroscopic methods, including resonance Raman spectroscopy, in model systems and in intact retinal tissue. The experiments of this proposal may provide new insights into the biochemical basis of the specific uptake lutein and zeaxanthin into the macula. Derangements of the mechanisms of uptake and stabilization of the macular carotenoids could have profound impact on the progression of AMD and inherited retinal dystrophies. Anticipated interventional clinical studies may be able to take advantage of the specific uptake systems to increase the level of macular carotenoid pigment and perhaps retard or prevent the progressive blindness produced by these diseases.

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

Studies 11

•Project Title: BIOCHEMISTRY OF THE RETINA SPECIFIC TRANSPORTER ABCR/RIM

Principal Investigator & Institution: Nathans, Jeremy H.; Professor; Molecular Biology and Genetics; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218

Timing: Fiscal Year 2001; Project Start 01-JAN-1999; Project End 31-DEC-2003

Summary: The goal of this research program is to develop an in vitro assay for the functional characterization of the retina-specific ABC transporter, ABCR/RIM. Mutations in the gene encoding ABCR/RIM are responsible for Stargardt disease (STGD), an autosomal recessive early onset form of macular degeneration, and have also been reported in a subset of patients with age related macular degeneration (AMD). Unpublished data implicate other mutations in this gene in some cases of autosomal recessive retinitis pigmentosa (RP). We aim to (1) identify the transported substrate and (2) develop methods for producing recombinant human ABCR/RIM that can be used to test the effect of mutations on the structure, function, and stability of this protein. Identifying the transported molecule(s) may reveal new compounds or new roles for known compounds. Determining whether the transported molecule is transported in conjunction with specific binding proteins, and studying its synthesis, degradation, and processing may lead both to the identification of additional candidate genes for retinal diseases, and to the identification of additional targets for drug development. Production of a large number of different ABCR/RIM proteins, each carrying a mutation found in humans with STGD, AMD, or RP will allow us to determine the biochemical defect in each case, and will allow a correlation to be drawn between the clinical characteristics of the affected subjects and the biochemical defects resulting from the mutations they carry.

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

•Project Title: BONE MARROW-DERIVED STEM CELL TRANSPLANTATION TO RETINA

Principal Investigator & Institution: Mcloon, Steven C.; Professor; Neuroscience; University of Minnesota Twin Cities 200 Oak Street Se Minneapolis, Mn 554552070

Timing: Fiscal Year 2003; Project Start 01-JUN-2003; Project End 31-MAY-2006

Summary: (provided by applicant): Degeneration of retinal photoreceptor neurons, such as that seen in age-related macular degeneration (AMD), is the most common cause of blindness in the United States. There are compelling reasons to believe that subretinal cell transplantation could be used to replace missing photoreceptor neurons. No effective and practical source of cells for transplantation is currently available. The goal of this project is to develop cells to be used for transplantation to replace photoreceptor neurons in AMD and related diseases. Bone marrow-derived stem cells offer numerous advantages over other cell types as a possible source of donor cells. These cells can differentiate into neurons. They are readily available, and if used for autologous transplantation to the retina, they would not have the same immunological consequences inherent in the use of other cell types. To our knowledge, no other laboratories are investigating bone marrow-derived stem cells for transplantation to the retina. At this time, there is no evidence that bone marrow-derived stem cells can differentiate into retinal neurons. The specific aim of this preliminary investigation is to determine conditions that would allow these cells to differentiate as photoreceptor neurons or other retinal cell types. The study has three sequential steps. First, treat GFPlabeled, bone marrow-derived stem cells in ways likely to induce the photoreceptor phenotype. This includes culturing cells in factors such as FGF-2, EGF, retinoic acid,

12Macular Degeneration

sonic hedgehog and taurine, and/or transfecting the cells with a gene for the photoreceptor cell specific transcription factor, Crx. Second, co-culture treated bone marrow-derived stem cells with embryonic retina or transplant the cells to the subretinal space in animals depleted of photoreceptor cells. Third, assess histologically the differentiation of bone marrow-derived stem cells in the retinal co-cultures or after transplantation to the retina by determining their laminar distribution in the host retina and by immunohistochemistry with antibodies specific to retinal cell types.

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

•Project Title: CHARACTERIZATION OF RPE MONOLAYER FOR TRANSPLANT

Principal Investigator & Institution: Mckay, Brian S.; Ophthalmology; University of Arizona P O Box 3308 Tucson, Az 857223308

Timing: Fiscal Year 2003; Project Start 13-JAN-2003; Project End 31-DEC-2005

Summary: (provided by applicant): Age-related macular degeneration (AMD) is a disease that affects many in our aging population. The end result of this disease is blindness and there is no cure. Current treatments center on re-establishing contact between the neural sensory retina and its supporting tissue, the retinal pigment epithelium (RPE). There are two current surgical methods to accomplish this task, either macular translocation or RPE transplantation. Macular translocation involves detaching the retina, cutting it circumferentially, and mechanically rotating the retina such that the macula is placed onto a healthy bed of RPE. This is a very technical and difficult surgery that requires significant follow up and may not be appropriate for many patients. RPE transplantation, can in animal models of disease, rescue retinal degeneration suggesting that this may be a valuable treatment option. However, RPE grafts in AMD patients suffer a high rate of graft rejection, limiting the usefulness of this treatment. This study is focused on preparing cultures of RPE cells from adult humans that are candidates for transplantation. The proposal is based on the novel observation that coordinated cadherin adhesion causes differentiation of human RPE cells. This observation suggests that it may be possible to produce RPE monolayers in culture that function as the tissue in vivo, and may yield beneficial results for transplantation. Significantly, our data suggests that the appropriate monolayers of RPE can be produced from passaged RPE that have expanded and proliferated extensively. This new method opens the opportunity to harvest a small number of RPE from a patient, expand the cells to produce enough for transplantation, stimulate differentiation, then transplant the patient's own RPE cells back under the retina. This procedure would avoid the graft rejection problems encountered using fetal RPE cells, RPE cell lines, or cultured RPE from generic donor tissue. The goals of this pilot project are to determine whether the methods developed are broadly applicable to RPE cultures from a spectrum of donors at different ages, to determine whether the 'tissue-type' monolayers produced express the functional characteristics necessary for function after transplantation, and to determine whether the monolayers can be produced on transplantable substrates. The results from this project will significantly impact both the treatment of the disease and the basic studies that seek a cure.

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

•Project Title: CHOROIDAL CIRCULATION & AGE RELATED MACULAR DEGENERATION

Principal Investigator & Institution: Grunwald, Juan E.; Professor; Ophthalmology; University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Studies 13

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

Summary: (Applicant's Description): Age-related macular degeneration (AMD) is the leading cause of blindness in people over the age of 65 in the United States. About 10 percent of the U.S. population will develop AMD and 1 percent may suffer from the more advanced, exudative form of the disease that leads to severe visual impairment. The basic mechanism involved in the development of this disease is not clearly understood. One of the most common manifestations of this disease, however, is the deposition of materials that are not adequately removed from the retinal pigment epithelium-Bruch's Membrane interface. Several recent reports and some preliminary data from our laboratory have suggested abnormalities of the choroidal circulation in AMD. These reports are of great interest because this circulation supplies all metabolites and removes all waste products from the outer retina. An abnormal choroidal circulation could have a role in the process that leads to the accumulation of these abnormal substances. In addition, a decreased choroidal vascular supply could also lead to ischemia and hypoxia of the outer retina and this could be associated with the development of choroidal neovascularization, a blinding complication of AMD. The main aim of this application is to use the state-of-the-art non-invasive technique of laser Doppler flowmetry to investigate whether choroidal blood flow and its regulation are abnormal in AMD, and whether a more abnormal choroidal circulation is associated with a higher risk of developing choroidal neovascularization and the more advanced and blinding stages of AMD. A better understanding of the mechanisms associated with the development of AMD could, in the long run, help us devise new treatment strategies for this devastating disease. In addition, the knowledge gained through this investigation could also help in the identification of patients at risk of developing choroidal neovascularization, thus enabling earlier treatment.

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

•Project Title: CONFERENCE--RETINAL DEGENERATION

Principal Investigator & Institution: Anderson, Robert E.; Professor; Ophthalmology; University of Oklahoma Hlth Sciences Ctr Health Sciences Center Oklahoma City, Ok 73126

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

Summary: (Applicant?s Abstract) The primary goal of the forthcoming X International Symposium on Retinal Degenerations, a satellite meeting of the XV International Congress of Eye Research, is to promote the exchange of current information and ideas amongst basic and clinical scientists in the interest of fostering new advances in our understanding of basic mechanisms and the development of therapeutic interventions in acquired and inherited retinal degenerations. Special effort has been made to introduce new investigators into the field and to include established investigators with clinical and basic science backgrounds. We have reached a point in the history of scientific endeavor at which there is common interest among basic and clinical scientists in understanding the fundamental mechanisms underlying retinal diseases. This meeting, which has been held biennially since 1984, has been responsible for fostering many of the collaborative research projects that have provided vital new information. As with previous meetings, the 2002 conference will feature a rich mixture of investigators, ranging from cell and developmental biologists to clinician-scientists from major research/clinical centers throughout the world. The 2002 meeting will be held in Antalya, Turkey on Sept. 29 - Oct. 5, 2002 as a satellite meeting of the XV International Congress of Eye Research to be held in Jerusalem from Oct. 6 - 11. The meeting will have eight sessions of paper presentations and dedicated time for poster discussion. The program will be finalized 3-

14Macular Degeneration

4 months before the meeting and will include the very latest information. Topics covered in previous meetings include new genes and loci, gene therapy, macular degeneration, animal models, medical therapies, mechanisms of cell death, transplantation, and retinal prostheses. A highly favorable aspect of the nine meetings held to date is the intimate environments that have provided for formal and informal discussions among established scientists with diverse backgrounds. This relaxed atmosphere has allowed clinicians and researchers from all parts of the to meet each other and learn of each other?s work. From the first meeting held in 1984, we have encouraged young persons to attend. Beginning in 2000, we supported the attendance of eleven students, postdoctoral fellows, and young scientists new to the field, who had the opportunity to meet and interact with established scientists. This is a key feature that distinguishes the International Symposium on Retinal Degenerations from other scientific meetings.

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

•Project Title: DEVELOPMENT OF A NEW ANTIANGIOGENIC TUMOR BLOCKER SBD 1

Principal Investigator & Institution: Bojanowski, Krzysztof; Ceo/ Scientist; Sunny Biodiscovery, Inc. 675 Hegenberger Rd, 2Nd Fl Oakland, Ca 946011919

Timing: Fiscal Year 2002; Project Start 01-JUN-2002; Project End 31-OCT-2003

Summary: Inhibition of tumor angiogenesis-the growth of new blood vessels towards the tumor mass-is a new and promising approach to anti-cancer therapy. The results of Phase I/II clinical trials completed so far with several angiogenesis inhibitors validate the concept of tumor angiogenesis as effective target for anti-cancer therapy. However, the same studies stress the need for novel, more potent angiogenesis inhibitors. We addressed this need by isolating the human urine a new protein (SBD.1), which specifically blocks the proliferation of capillary endothelial cells in vitro (ID50=15NG/ML), angiogenesis in chorioallentoic membrane assay, and two growth in vivo (Lewis Lung Carcinoma, T/C=0.04 at 20umum/kg/day), placing SBD.1 among the most potent known angiogenesis inhibitors. Sequencing of SBD/1 showed it is a novel protein and its mild proteolysis resulted in the generation of smaller peptides without losing the inhibitory activity. Here, we propose to a) clone and express SBD.1 in a recombinant system; b) isolate and sequence the activity SBD.1 fragments; c) test the purified SBD.1 on prostate, lung and breast carcinoma human xenografts in nude mice to further assess its anti-tumor activity This project will result in the cloning of a novel therapeutically-active protein, which may be an important endogenous regulator of angioand tumorigenesis in humans. PROPOSED COMMERCIAL APPLICATIONS: The development of SBD.1 addresses a pressing need in the pharmaceutical industry for a new, better angiogenesis inhibitor. Currently targeting anti-cancer therapy, the use of SBD.1 might be later extended to the treatment of vascular pathologies, such as macular degeneration and certain cardiovascular diseases. Meanwhile, SBD.1 can also be commercialized as reagent for laboratory research on angiogenesis. Taken together, these applications represent a substantial potential market for SBD.1

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

•Project Title: DIABETES AND CARDIOVASCULAR DISEASE IN FILIPINA WOMEN

Principal Investigator & Institution: Araneta, Maria R.; Family and Preventive Medicine; University of California San Diego 9500 Gilman Dr, Dept. 0934 La Jolla, Ca 92093

Studies 15

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

Summary: (provided by applicant): This proposal is designed to assess the relationship of noninsulin dependent diabetes (NIDDM) and its covariates to macrovascular and microvascular disease in a previously unstudied ethnic cohort by: 1. Providing support to an "under-represented investigator" Maria Rosario G. Araneta, Ph.D., an epidemiologist of Filipina descent. The present proposal will enable Dr. Araneta the opporunity to gain research experience in diabetes epidemiology, through the mentorship of Dr. Elizabeth Barrett-Connor, and collect and analyze data on which to base a subsequent research grant application on diabetes research. 2. Continuation of the UCSD Filipina Womens Health Study, a cross-sectional study of Filipina women aged 40 and above in San Diego County, through: a) a follow-up telephone interview to determine changes in morbidity, mortality, and medication use since their initial clinical evaluation in 1995-1999, and b) record review validation of reported cardiovascular disease. 3. Conducting statistical analyses of newly obtained data and data from the clinical evaluation to explore the relationship between NIDDM and its covariates, and the metabolic syndrome and its components to macrovascular (cardiovascular and lower extremity arterial disease) and microvascular disease (microalbuminuria, nephropathy, diabetes retinopathy, visual impairment and macular degeneration) among Filipina women.

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

•Project Title: DIRECTED GENE TRANSFER INTO RPE CELLS

Principal Investigator & Institution: Chaum, Edward; Ophthalmology; University of Tennessee Health Sci Ctr Memphis, Tn 38163

Timing: Fiscal Year 2001; Project Start 01-FEB-1998; Project End 31-JAN-2003

Summary: The retinal pigment epithelium (RPE) plays a critical role in maintaining the health and function of the neurosensory retina. Inherited and acquired RPE dysfunction is responsible for a number of important blinding diseases including certain forms of retinitis pigmentosa, retinal dystrophy and age-related macular degeneration. We believe that the greatest promise in the potential treatment of inherited and acquired retinal degeneration lies in the development and refinement of directed gene therapy to the host retinal pigment epithelium. Our working hypothesis is that directed liposomemediated gene transfer can transfect RPE cells in vivo with high efficiency and can deliver functional genes which are capable of sustained expression in host RPE. The conceptual basis for this hypothesis is that the intrinsic phagocytic function of the RPE makes this cell an ideal host recipient for liposome-mediated gene transfer. Liposomemediated gene transfer allows transfection of large molecular weight DNA s, potentially translatable genes capable of replacing the defective genes which result in retinal degeneration. Specific Aims and Methods 1: Develop and optimize an efficient liposomemediated gene transfer protocol for mammalian retinal pigment epithelial cells in vitro: i) assess and optimize conditions of in vitro endocytosis of liposomes in human and rabbit RPE cells, ii) determine relative delivery efficiencies of different liposome preparations in humans and rabbit RPE cells, and iii) assess relative toxicity of different liposome preparations on cells in vitro. 2: Assess exogenous DNA uptake and expression frequencies in RPE cells by liposome-mediated gene transfer utilizing the reporter gene beta-galactosidase or the selective marker gene neomycin resistance: i) quantitate uptake of exogenous DNA sequences, ii) determine in vitro culture and transfection conditions which maximize DNA uptake in the human and rabbit RPE cell lines, iii) quantitate transcription and translation of exogenous DNA sequences. 3: Optimize this liposome-mediated gene transfer system for mammalian retinal pigment

16Macular Degeneration

epithelial cells in vivo: I) assess toxicity of liposome/DNA complexes in the living retina, ii) assess transfection efficiencies in the retinal pigment epithelium, iii) optimize conditions for gene transfer to RPE cells in vivo and assess duration of expression of transfected genes in the living retina.

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

•Project Title: DRUSEN AND AMD: SUB-TYPE ISOLATION AND CHARACTERIZATION

Principal Investigator & Institution: Hollyfield, Joe G.; Director of Research; Cleveland Clinic Foundation 9500 Euclid Ave Cleveland, Oh 44195

Timing: Fiscal Year 2002; Project Start 02-SEP-2002; Project End 31-AUG-2006

Summary: (provided by applicant): The broad, long-term objective of this application is to define in molecular terms the linkage between the accumulation of soft drusen below the retinal pigment epithelium (RPE) in the macula and the increased risk of developing age-related macular degeneration (AMD). The presence of soft drusen in the macula is the hallmark risk factor for developing AMD. Surprisingly little is known of the composition or origin of drusen. To this end a novel method for drusen isolation has been developed that allows the collection of microgram quantities of drusen from donor eye tissue. At the time of isolation, different drusen sub-types can be identified and separated for use in studies that will characterize their molecular composition. The diagnostic utility of drusen in AMD can be likened to that of blood levels of cholesterol in atherosclerosis. The presence and abundance of drusen, like the level of cholesterol in the blood, indicates the degree to which a patient is at risk for developing the disease. Because of the relationship of drusen and AMD, understanding the composition of different drusen sub-types will provide important information on possible pathways that are causally involved in drusen development. Novel proteins or common modifications of proteins present in drusen, should provide insight as to potential drug targets of therapeutic agents to treat AMD. The current application is focused on exploiting this drusen isolation procedure to define the molecular composition, distribution and cellular origin of drusen sub-types in normal and AMD tissues. The three specific aims are: (1) To test the hypothesis that different sub-populations of drusen can be isolated from donor eye tissue. (hard vs. soft, foveal vs. peripheral, old vs. older, spherical vs. flat, amber vs. clear, opaque vs. granular, etc.). (2) To test the hypothesis that different structural features of drusen sub-populations reflect different molecular composition (light microscopy, histochemistry, electron microscopy, SDS/PAGE, Western blotting, mass spectrometry). (3) To test the hypothesis that some of the molecules present in drusen are novel and are not found in Bruch's membrane.

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

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 17

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: ELASTIC FIBER DEFECTS IN PSEUDOXANTHOMA ELASTICUM

Principal Investigator & Institution: Boyd, Charles D.; Professor of Cell Biology & Program Dire; None; University of Hawaii at Manoa Honolulu, Hi 96822

Timing: Fiscal Year 2001; Project Start 01-FEB-2000; Project End 31-JAN-2003

Summary: The aim of this proposal is to understand the biological and pathogenetic basis of elastic fiber defects in a heritable disorder of cutaneous, vascular and ocular tissue, pseudoxanthoma elasticum (PXE). PXE is a heritable disease characterized by the accumulation of abnormal elastic fibers in several elastic tissues, particularly the skin, arteries and the elastic Bruch's membrane of the retina. The aberrant deposition of calcified elastic fibers in these tissues is responsible for the development of the clinical symptoms characteristic of PXE and these include inelastic skin lesions, retinal hemorrhage with partial loss of vision and vascular defects such as gastrointestinal bleeding and myocardial infarction. The pattern of inheritance of PXE is complex and both autosomal dominant and recessive forms of the disease have been reported. Over the last few years, several groups of investigators have attempted to identify the gene mutation(s) responsible for PXE but it has only been in the last two years that three separate laboratories, including our own, have located a major locus for this disease on the short arm of chromosome 16. We have now successfully identified a region at 16p13.1 of 820kb that contains 6 candidate genes, at least one of which will contain the mutations responsible for PXE. With this new information, we hope to identify the 'PXE gene', the mutations in this gene in a cohort of PXE patients and determine a possible function for the product of the PXE gene. These goals are focused on elucidating the biology of PXE and using this information to provide a better understanding of the role of elastic fibers in more common elastic tissue diseases, particularly those skin, vascular and eye disorders characterized by tissue calcification such as the many dystrophic

18Macular Degeneration

calcification disorders of skin, aneurysms, atherosclerosis and age-related macular degeneration.

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

•Project Title: ENDOTHELIAL TRANSMIGRATION ACROSS THE RPE BARRIER

Principal Investigator & Institution: Hartnett, Mary E.; Associate Professor of Ophthalmology; Ophthalmology; University of North Carolina Chapel Hill Office of Sponsored Research Chapel Hill, Nc 27599

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

Summary: (provided by applicant): Choroidal neovascularization (CNV) is the primary cause of severe vision loss in age-related macular degeneration (AMD), but little is known about the complex mechanisms leading to CNV growth into the neurosensory retina. Human histopathology of occult CNV in AMD has shown that contact between the endothelial cells (EC) of the choriocapillaris and the retinal pigment epithelium (RPE) leads to compromise of the RPE barrier, which precedes the growth of CNV into the neurosensory retina. We will test the hypothesis that RPE-EC contact causes a functional change in the RPE monolayer that permits the transmigration of EC across the RPE. Specifically, we will determine whether 1) EC contact with RPE causes compromised RPE barrier function and structure; 2) EC contact with RPE causes a major change in the ratio of angiogenic stimulators to inhibitors (i.e., VEGF: PEDF) in the RPE; and 3) RPE-EC contact specifically activates cell-associated RPE VEGF isoforms that enable transmigration of EC across the RPE monolayer. Methods will include measurement of transepithelial electrical resistance (TER), cell counts, viability tests, and staining for ZO-1, n-cadherin, and actin in human cell co-cultures of RPE and EC to evaluate RPE barrier structure and function; ELISA, Western blot, and Northern blot to quantitate VEGF:PEDF ratios under various culture conditions; staining for actin and barrier proteins, TER, permeability, and RT-PCR to quantitate soluble and cellassociated VEGF isoforms; and fluorescent labeling of RPE and EC to follow transmigration of EC. Approximately 90% of the legal blindness that occurs in patients with AMD is the result of CNV that originates from the choriocapillaris and grows beyond the natural boundaries of Bruch's membrane and the outer blood-retinal barrier of the RPE into the neurosensory retina. Increased knowledge of the mechanisms that cause this destructive phenomenon may lead to future means to prevent CNV in the neurosensory retina, and thus, reduce vision loss in AMD.

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 2001; Project Start 16-JUN-1987; Project End 31-MAY-2002

Summary: (Applicant's Abstract) This proposal describes a population-based cohort study aimed at determining the long-term (10-year) incidence and progression of the most common vision-threatening conditions of adult Americans, age-related cataract and maculopthy, and their risk factors. 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) and the 5-year follow-up (n=3,816) 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

Studies 19

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-year follow-up 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 10-year 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. Frequencies of agerelated maculopathy differ between whites and ethnic minority groups. We will determine the relation of risk factors (cardiovascular disease, hypertension, diabetes, other characteristics as described above) for age-related maculopathy in nonHispanic whites, nonHispanic blacks and Mexican Americans participating in the National Health and Nutrition Examination Survey III (NHANES III) and whites and blacks participating in the Atherosclerosis Risk in Communities (ARIC) study. Existing graded fundus photographs will be used. Findings regarding cataract and age-related maculopathy 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: ETIOLOGIC STUDIES OF AGE-RELATED MACULAR DEGENERATION

Principal Investigator & Institution: Seddon, Johanna M.; Associate Professor; Massachusetts Eye and Ear Infirmary 243 Charles St Boston, Ma 02114

Timing: Fiscal Year 2003; Project Start 01-MAR-1996; Project End 31-MAR-2007

Summary: (provided by applicant): We hypothesize that underlying susceptibility gene

(s) are critical to the development of age-related macular degeneration (AMD) and that they most likely interact with environmental factors to trigger the development and progression of this disease. We propose a multi-pronged approach to the identification of susceptibility gene(s) for age-related macular degeneration (AMD). During the initial grant period we developed a large genetic AMD database including large families and multiplex families with affected sib-pairs, and have identified several areas of genetic linkage to pursue. During the next grant period, we propose a multipronged approach to build upon this database, with the goal of finding the gene or genes associated with AMD susceptibility. We will use family-based and population-based case-control association studies, combined with an expanded genome-wide sib-pair study for this project. Single nucleotide polymorphisms in high priority positional or functional AMD candidate loci will be genotyped for the association studies and linkage disequilibrium and transmission disequilibrium tests will be performed. Sib-pair linkage analysis using both quantitative and qualitative traits on the expanded genome-wide scan will be completed. We will collect family history and risk factor data and conduct studies examining these risk factors for AMD. These data will provide important information to analyze the influence of environmental factors as information about specific genetic factors involved in the disease is identified. This study should provide important new information about the etiology of AMD which is the leading cause of irreversible

20Macular Degeneration

blindness among elderly individuals worldwide, so that preventative or treatment measures can be developed.

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

•Project Title: FUNCTIONAL ANALYSIS OF THE RETINAL REGULATORY PROTEIN RB

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

Timing: Fiscal Year 2001; Project Start 01-JAN-1998; Project End 31-DEC-2002

Summary: The candidate's long-term career goal is to combine his clinical interests in retinal diseases with his research interests in molecular biology. Washington University in general, and the sponsor's laboratory in particular, provides an exceptional setting for the candidate to pursue this goal. The sponsor's laboratory is a vigorous intellectual environment where many important contributions have been made in understanding the structure and function of the retinoblastoma protein (Rb) in health and disease. Further, the sponsor has an outstanding record of training successful clinician-scientists. The candidate's research will focus on the normal structure and function of Rb. While Rb was initially discovered through its role in the development of retinal tumors, it is now clear that Rb plays an important role in normal growth and differentiation of the retina. Understanding the normal function of Rb is likely to have significant implications not only for retinoblastoma, but also for macular and retinal degenerations, photoreceptor regeneration, and retinal transplantation. The candidate proposes a series of experiments to further elucidate the interaction between Rb and the E2F family of transcription factors. This interaction appears to be critical for cell cycle regulation and cellular differentiation. When Rb binds E2Fs, it blocks transcription from cell cycle genes that have E2F sites. In this proposal, the candidate will use a wide range of techniques, including transfection assays, and in vitro and in vivo binding studies to address the following specific aims: (1) determine regions in domains A and B of Rb that are responsible for transcriptional repression, (2) determine regions in domains A and B that mediate interdomain binding to form the active repressor motif, and (3) identify retinaspecific transcription factors that are inhibited by Rb.

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

•Project Title: FUNCTIONS OF UBIQUITINYLATION IN THE MAMMALIAN RETINA

Principal Investigator & Institution: Obin, Martin S.; None; Tufts University Boston Boston, Ma 02111

Timing: Fiscal Year 2001; Project Start 01-APR-1997; Project End 31-MAR-2004

Summary: (Adapted from applicant's abstract): Selective (nonlysosomal) proteolysis serves two vital functions in cells : (1) it controls steady-state levels of proteins whose concentrations and activities must be precisely regulated to maintain homeostasis and differentiation pathways; and (2) it protects cells from insults such as oxidative stress, viral infection and mutations by degrading potentially cytotoxic damaged or aberrant proteins. The importance of selective proteolysis to cell regulation and to stress and disease resistance is underscored by the fact that eukaryotic cells possess a highly conserved and exquisitely selective proteolytic pathway that recognizes and degrades both native regulatory proteins as well as aberrant proteins resulting from stress and disease. This pathway requires ubiquitin, a protein which is abundant in all cells; hence, the pathway is called the ubiquitin-dependent pathway (UDP). Studies from the

Studies 21

applicant's laboratory indicate that the UDP functions in mammalian ROS and suggest that the visual transduction GTP-binding protein (G protein), transducin, is a UDP substrate. These observations suggest a novel mechanism by which photoreceptor proteins may be selectively regulated as well as the potential for modulation of the visual transduction cascade by the UDP. One long-term objective of the applicant's research program is to determine the function of the UDP in photoreceptors. In pursuit of this goal, the proposed research will (1) confirm the presence of the UDP protease in rat ROS using immunoelectron microscopy and (2) demonstrate UDP proteolytic function in cell-free preparations of gradient-purified bovine ROS (specific aim 1). The applicant will subsequently determine (3) if purified transducin is degraded by the UDP in ROS preparations and (4) the extent to which light-induced transducin dissociation alters its degradation by the ROS UDP (specific aim 2). In humans, protein damage in the normal retina resulting from (photo)oxidation is implicated in age-related macular degeneration (AMD), and expression of aberrant proteins in the diseased retina is causally associated with the pathogenesis of progressive retinal degenerations (retinitis pigmentosa). Thus, an additional long-term objective of the applicant's research is to determine how damaged and mutant proteins are removed from cells in the intact and diseased retina. In pursuit of this goal, experiments described under specific aim 3 will

(1) use immunohistochemistry and western blotting to confirm a previous report of increased UDP-modified protein in retinas of Long-Evans rats exposed to photic stress (prolonged bright light) and to determine where within retinal cells these increases are manifest; (2) determine if increased protein flux through the UDP in response to photic stress reflects increased UDP efficacy and/or increased substrate availability; and (3) initiate studies designed to elucidate the molecular and biochemical mechanisms by which UDP efficacy is altered in response to photic stress.

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

•Project Title: GENE EXPRESSION ANALYSIS IN MICROCAPTURED RETINAL CELLS

Principal Investigator & Institution: Adler, Ruben; Arnall Patz Distinguished Professor; Ophthalmology; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218

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

Summary: (provided by applicant): Retinal degenerative diseases are a major cause of visual disability and blindness worldwide. Age-related macular degeneration (AMD), for example, is the leading cause of blindness in the elderly in the Western world. Current treatments do little to alter the inexorable loss of vision due to retinal degenerations. Several studies have shown that intraocular injection of factors such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), or basic fibroblast growth factor-2 (FGF2), slows photoreceptor cell death caused by specific mutations or exposure to constant light. However, the clinical usefulness of these findings may be limited, because rescue effects are partial and transient, and some factors appear to have unwanted side effects. Elucidation of the mechanism by which survival factors delay retinal degenerations appears necessary in order to maximize benefits and minimize side effects. Recent studies from our laboratories have suggested that CNTF, BDNF and FGF2 do not act directly on photoreceptors; rather, they appear to act indirectly through other cells, most likely M ller cells. Based on these observations, we propose to investigate the molecular changes triggered by neurotrophic factors in M ller cells. The studies involve the combined use of two complementary and demanding state-of-the-art techniques: the generation of cDNA from individual cells, and their analysis using custom designed retinal cDNA microarrays. We will then

22Macular Degeneration

establish which of these changes are important for photoreceptor survival. The potential impact of the identification of these molecules is clear, since they could offer new avenues for the treatment of these devastating diseases.

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

•Project Title: GENE EXPRESSION PROFILES OF RETINAL DEGENERATION

Principal Investigator & Institution: Cepko, Constance L.; Professor; Genetics; Harvard University (Medical School) Medical School Campus Boston, Ma 02115

Timing: Fiscal Year 2003; Project Start 01-JAN-2003; Project End 31-DEC-2007

Summary: (provided by applicant): Many diseases that ultimately lead to blindness are caused by the degeneration of photoreceptor (PR) cells. Cones typically die after rods, with kinetics somewhat dependent upon the particular disease. Rod loss followed by cone loss is also seen in cases where a genetic etiology has not been established, as in some forms of macular degeneration. While humans are able to function quite well without rods, the loss of cone-mediated vision is devastating. The reason(s) that cones die in these cases is unknown. However, since cone loss can be initiated by events that are not intrinsic to cones, these events must include some type of cell-cell interaction, perhaps including the action of a secreted molecule(s). Such a process may be susceptible to interruption through the application of a pharmacological or a cell based therapy. In addition to progressive diseases such as retinitis pigmentosa, there is a mouse model, the cyclin D1 knock-out (KO) mouse, in which degeneration is arrested. The cause of PR death, as well the cause of the arrest, are unknown. This model may provide some insight into how degeneration can be arrested in progressive diseases. We are seeking to use retinal microarrays to define the gene expression changes that accompany PR death in mice, with an emphasis on the events that lead to cone death. In addition, we will characterize the gene expression changes that accompany the arrest of PR degeneration in the cyclin D1 mutant. We further plan to characterize the expression patterns of such genes in normal and pathological tissue. Finally, we will explore the function of some of these genes using genetic approaches in mice.

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

•Project Title: GENETIC MODEL OF RETINAL PIGMENT EPITHELIUM DEGENERATION

Principal Investigator & Institution: Vollrath, Douglas E.; Associate Professor; Genetics; Stanford University Stanford, Ca 94305

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

Summary: (provided by applicant): Animal genetic models have been essential to the understanding and treatment of human retinal degenerative disease. The importance of the retinal pigment epithelium (RPE) to photoreceptor function is widely recognized. Primary degeneration of RPE cells is thought to be central to the etiology of several significant human retinal disorders including age-related macular degeneration (AMD) and pigmentary retinopathies associated with mitochondrial dysfunction, yet no animal genetic model of a primary RPE cell degeneration exists. This proposal describes a strategy to create such a model using mouse genetics. A mouse strain will be generated in which RPE cells gradually and postnatally degenerate and die due to RPE specific loss of mitochondrial function. RPE cell degeneration should induce secondary photoreceptor cell degeneration and choroidal atrophy. A detailed structural and functional analysis of the effects of RPE cell loss on the RPE and adjacent tissues will be performed at various ages. At an appropriate stage in the degeneration, the model will

Studies 23

be perturbed by modulating light exposure. A model of primary RPE cell degeneration will be useful for understanding the interdependence of RPE and photoreceptor cells and of the RPE and choroid, for understanding pathogenic processes secondary to RPE cell death, and for investigating potential therapies in a setting in which RPE cell function is progressively compromised.

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

•Project Title: GENETIC STUDIES OF OCULAR ANGIOGENESIS

Principal Investigator & Institution: D'amato, Robert J.; Associate Professor; Children's Hospital (Boston) Boston, Ma 021155737

Timing: Fiscal Year 2001; Project Start 01-AUG-1999; Project End 31-JUL-2004

Summary: Angiogenesis, the formation of new blood vessels, is a tightly regulated function determined by the local balance of endogenous angiogenesis stimulators versus inhibitors. The central hypothesis for this proposal is that the angiogenic balance varies between individuals and that this variation is in large part genetically determined. Indeed, epidemiological data suggests that different racial populations have varied susceptibility to ocular neovascularization. We have surveyed inbred mouse strains to see if mice have a range of angiogenic diversity that models that of humans. Surprisingly, we found a large range of angiogenic responses to pellets of basic fibroblast growth factor (bFGF) implanted in the corneas of different strains. The difference between the lowest response and the highest response was 1200 percent. To date, the most sensitive strain is an albino mouse known as 129. In this strain, corneal bFGF pellets induce very aggressive corneal and iris neovascularization as compared to C57 black mice which have moderate corneal angiogenesis and no iris neovascularization. Interestingly, tyrosinase positive substrains of 129 mice (which are pigmented), retain the overly aggressive corneal neovascular response but do not have iris neovascularization. To evaluate the aggressive corneal angiogenic response of the 129 mice we propose to breed them with other inbred strains with lower responses. We will then characterize the angiogenic phenotype of the offspring of the crosses and will use genetic mapping to identify the chromosomal locus that segregates with this phenotype. If there are candidate genes in the region, these will be screened for alterations in DNA sequence. The goal of this proposal is to characterize the angiogenic phenotypes of different murine inbred strains and to identify the genetic contributions to these phenotypes. Identifying the genes controlling ocular angiogenesis in mice will help find similar genes in humans, the characterization of which may suggest new therapies for pathologic neovascularization seen in neovascular glaucoma and macular degeneration.

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

•Project Title: GENETICS OF AGE RELATED MACULAR DEGENERATION

Principal Investigator & Institution: Klein, Michael L.; Director; Ophthalmology; Oregon Health & Science University Portland, or 972393098

Timing: Fiscal Year 2001; Project Start 01-AUG-1998; Project End 31-JUL-2003

Summary: Age-related macular degeneration (AMD) is recognized as the leading cause of blindness in the United States. It affects nearly 1.5 million older Americans and causes loss of vision in more than 7 percent of individuals over 75 years of age. Currently, there are no established means of preventing AMD. The only proven effective treatment, laser photocoagulation, is successful in only a small proportion of cases. While the etiology of AMD is unknown, there is considerable evidence implicating a strong genetic

24Macular Degeneration

component for the disease. Advances in genomic screening and analysis methodologies make a direct genetic approach to the etiology, pathophysiology, and ultimate therapy of AMD viable. Recent successes with other complex traits, which share genetic and epidemiological similarities with AMD, support the idea that identification of genetic loci responsible for AMD is an achievable goal. The long-term objectives of this project are to identify genes responsible for AMD, develop diagnostic tools to identify patients at risk of developing the disease, and to understand its molecular pathophysiology. This understanding will allow the development of preventive measures and improved methods of treatment. The immediate goal of this research proposal is to map genetic loci cosegregating with AMD in a number of affected families. We will employ both parametric and several nonparametric linkage analysis methods. To achieve these goals, we propose to: 1) Continue to collect additional kindreds containing multiple affected living members; 2) Complete genome-wide screening of AMD families, beginning with a set of candidate loci; 3) Fine-map loci suggestive of linkage and conduct detailed multipoint parametric and nonparametric linkage analysis; and 4) Refine identified loci and begin studies to identify the specific genetic defects responsible for AMD in these families.

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

•Project Title: GENETICS OF AGE RELATED MACULOPATHY

Principal Investigator & Institution: Gorin, Michael B.; Associate Professor; Ophthalmology; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, Pa 15260

Timing: Fiscal Year 2001; Project Start 01-SEP-1993; Project End 31-JUL-2002

Summary: Age Related Maculopathy (ARM) is the leading cause of vision loss in the elderly population in the United States and Western world and is a major public health issue. Epidemiologic studies have indicated that heredity is a significant risk factor and family studies have further substantiated that ARM can be inherited as a dominant disease with late age of onset and variable expressivity. ARM is not well suited for traditional genetic investigations due to difficulties of clinical ascertainment and the small pedigrees because of its late onset. However, nonparametric linkage methods including Affected Pedigree Member method and simIBD provide a means of determining genetic loci that contribute to ARM susceptibility using small and intermediate-sized families. In our previous project we ascertained over 200 ARM families and are in the process of completing a candidate locus screening as well as a genome-wide scan of the first 120 families with 161 autosomal markers (average spacing of 20 cM). We have established a classification system that allows us to evaluate a stringently defined ARM population as well as larger sets of patients with less severe and/or ambiguous phenotypes. Several markers used in the initial candidate gene screening and chromosome-wide panels have provided results that suggest linkage with ARM. These will be investigated during the next grant period. We are proposing to expand our recruitment of ARM families to 1000 families and pursue a combination of genome-wide scans (200 and 350 families) with 10 cM resolution and focused genotyping based upon the tentative positively linked loci determined during the first grant period. The large number of families is necessary to confirm and further resolve potential ARM loci so that we can undertake candidate gene screening of our ARM population. The families that are not used in the genome-wide scans will be used for the focused genotyping effort, in the candidate gene screening program, and for disequilibrium linkage studies.

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

Studies 25

•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 2001; 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 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: 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

26Macular Degeneration

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 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 9500 Gilman Dr, Dept. 0934 La Jolla, Ca 92093

Timing: Fiscal Year 2001; 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

Studies 27

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: HOMEOSTATIC ABILITY OF THE RETINAL PIGMENT EPITHELIUM

Principal Investigator & Institution: Miller, Sheldon S.; Scientific Director; Optometry; University of California Berkeley Berkeley, Ca 94720

Timing: Fiscal Year 2001; Project Start 01-FEB-1978; Project End 31-AUG-2005

Summary: (provided by applicant): The retinal pigment epithelium (RPE) lies in the back of the eye between the neural retina and its choroidal blood supply. This layer of epithelial cells serves to protect the health and integrity of the outer retina. In the choroid, abnormal blood vessel growth or choroidal neovascularization (CNV) occurs in diseases such as age related macular degeneration (AMD) - the major cause of blindness for people over the age of 60. Vascular endothelial growth factor (VEGF) and other proor anti-angiogenic factors are constitutively secreted by the RPE into the extracellular space on both sides of the epithelium. The effects of these molecules on RPE physiology are not known. Evidence from human pathologic specimens and from rodent models suggests that increased VEGF production by the RPE is the source of CNV. Research in this area is severely hampered by the lack of a small animal model in which VEGF secretion by the RPE induces CNV. In preliminary experiments, we have used gene transfer techniques to develop a rat model for stimulating blood vessel growth in the back of the eye. This model will be used to test the hypothesis that secretion of VEGF by RPE is critically important in generating choroidal neovascularization (CNV). Gene transfer techniques will be also used to inhibit or reduce this VEGF-induced abnormal blood vessel growth. These experiments should provide a deeper understanding of CNV and the basis for a potential therapy for diseases like AMD. Normally, there is a very close anatomical relationship between the RPE and the retina. Separation of these two tissues occurs in a whole host of pathologies that lead to the abnormal accumulation of fluid in the extracellular or subretinal space. This separation or retinal detachment can lead to a loss of vision. One of our goals is to develop a rat model of retinal detachment. This model will allow us evaluate putatively therapeutic molecules that work directly on the RPE to remove fluid from the subretinal space. This removal will restore the normal anatomical relationship between RPE and retina and should improve vision.

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

•Project Title: HYPERSPECTRAL MICROSCOPE FOR DRUSEN PHENOTYPING

Principal Investigator & Institution: Truitt, Paul W.; 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): The demonstration of a Fourier-transform hyperspectral microscope for high resolution spectral ex vivo characterization of human retinal tissue is the aim of this project. The spectral reflectance and intrinsic fluorescence nature of normal and pathological retinal tissue must be established without the interfering effects of the intervening ocular media. This motivates the immediate goal of integrating the Fourier-transform imaging spectrometer (FTIS) to a microscope. A hyperspectral microscope may be used in many areas of biological and medical research; tissue classification (normal vs. pathological) and characterization, detection of

28Macular Degeneration

bacteria, gene identification, chromatin distribution, drug safety and efficacy, molecular imaging, and countless other applications. The Fourier-transform technology provides for a large number of highly resolved spectral bands simultaneously spatial at each pixel in the scene. Since fluorescence intensities are several orders of magnitude less than the reflectance signal, it is necessary to use a spectrometer with the highest possible sensitivity and wavelength selectivity. The FTIS has the advantage over traditional dispersive-based spectrometers in that entrance aperture and spectral resolution are decoupled. Thus, the slit can be increased to allow more light into the system without reducing spectral resolution. Further, because the FTIS is a constant wave number device, it offers improved spectral resolution in the visible to short wave infrared bands for a given detector size over conventional dispersive spectral imagers. Finally, the spatially modulated device collects all spectral data at once, avoiding any issues with spectral registration that otherwise occur. The six-month Phase I project will demonstrate the sensitivity of the hyper-spectral imaging methodology for spectrally phenotyping drusen in age-related macular degeneration (ARMD) eyes. Our approach builds on Kestrel's existing hyperspectral instruments and software, developed through Department of Defense. High spectral (2-4 nm) and spatial resolution images will be collected of histological specimens from two normal donor eyes and two eyes with clinical evidence or pre-mortem diagnosis of ARMD. The integrated system will be calibrated and tested. Illumination requirements for recording fluorescence will be established. A detailed, multi-source data set will be produced consisting of measurements of reflectance and transmission spectra, and intrinsic fluorescence spectra.

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

•Project Title: IMMUNE RESPONSES IN MACULAR DEGENERATION

Principal Investigator & Institution: Cousins, Scott W.; Professor; Ophthalmology; University of Miami-Medical Box 248293 Coral Gables, Fl 33124

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

Summary: (Applicant's Description): Age-related macular degeneration (ARMD) is a progressive disorder of the retina occurring in people older than age 50 associated with vision loss coupled with a spectrum of specific clinical, physiological and histopathological features. The pathogenic mechanisms for these specific changes are unknown, but circumstantial evidence suggests a role for a macrophage-mediated component in ARMD. We propose the general hypothesis that choroidal macrophages observed in ARMD are blood-derived monocytes recruited to the choriocapillaris to scavenge debris and deposits trapped in Bruch's membrane. In this project, we propose two specific aims to explore macrophage activation under conditions relevant to ARMD. In aim 1, we will use freshly isolated blood monocytes from normal subjects or ARMD patients, to establish the existence of high and low activity monocytes and to determine if high activity is a prognostic marker for ARMD progression. In aim 2, using a mouse model for age-related subRPE extracellular deposit formation recently developed in our laboratory, we will induce high activity monocytes in mice and establish that these macrophages will increase the severity of RPE degeneration or rate of progression of deposit formation.

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

Studies 29

•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 2001; Project Start 01-AUG-1999; Project End 31-JUL-2003

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: INHIBITION OF CNV USING A TRANSGENIC PEDF MODEL

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-2002; Project End 31-JUL-2005

Summary: (provided by applicant): Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in the United States. Choroidal neovascularization (CNV) is largely responsible for severe vision loss from AMD. As new and promising treatments emerge for the treatment of choroidal neovascularization, in vivo approaches will be needed not only to evaluate efficacy, but also to address issues important for clinical trials (e.g., dosing, toxicity). We thereby propose to use an inducible transgenic mouse model as an innovative approach to the study of inhibition of CNV. Specifically, we propose to study the effects of pigment epithelium-derived factor (PEDF), which has emerged as a potent inhibitor of angiogenesis, with dramatic inhibitory effects on corneal, retinal, and choroidal neovascularization. Our long-term oal is to develop PEDF as a ther~eutic modality for

30Macular Degeneration

the prevention as well as treatment of choroidal neovascularization. We hyaothesize that PEDF can cause regression of existing choroidal neovascularization, in addition to inhibiting CNV formation. Accordingly, we propose the following Specific Aims: (1) Develop a transgenic PEDF mouse model with retina-specific, inducible expression of PEDF. Aim 1 will include the characterization of spatial, temporal, and quantitative expression of induced PEDF in the mouse model. (2) Define the potential of PEDF as a therapeutic agent for choroidal neovascularization using the laser-induced CNV model. Aim 2 will determine if transgenic PEDF expression can prevent choroidal neovascularization as well as cause regression of existing CNV. In addition, Aim 2 will address the PEDF dosage requirements required for prevention of CNV, as well as the duration of PEDF presence necessary for prevention. Finally, Aim 2 will determine if transgenic PEDF expression causes choroidal endothelial cell apoptosis in vivo. In our studies, we will adapt the tetracycline-inducible gene expression system to induce photoreceptor-specific expression of PEDF. We will study the effects of transgenic PEDF expression in mice on laser-induced choroidal neovascularization. It is anticipated that our proposal will provide a strong foundation for further studies regarding the mechanism of PEDF action. In addition, our studies will provide a new experimental approach for the study of candidate inhibitors of CNV, providing important data regarding efficacy, therapeutic dose, frequency of administration, and toxicity.

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

•Project Title: INVESTIGATIONS OF HUMAN CONE DIRECTIONALITY

Principal Investigator & Institution: Burns, Stephen A.; Senior Scientist; Schepens Eye Research Institute Boston, Ma 02114

Timing: Fiscal Year 2001; Project Start 01-NOV-1987; Project End 31-AUG-2006

Summary: (provided by applicant): The eye is a highly specialized optical system. Its proper function requires tradeoffs between image quality at a single wavelength and the need to operate over a wide range of conditions., Retinal image quality is determined by both the refractive elements of the eye (cornea and lens) and the cone photoreceptors. The cone photoreceptors play a critical role, since they preferentially capture light from a particular location in properties of the pupil, and because they discretely sample the retinal image. The interaction of cones and the refractive properties of the eye is not well understood, and is highly variable across individuals. Understanding of this interaction has high clinical relevance, since over 1,000,000 patients a year are undergoing some form of refractive surgery. Refractive surgery does not yet take into account the large intra-subject variations. If future plans for enhanced refractive strategies are to improve vision then they must be designed with an understanding of the tradeoffs and optimizations involved in image formation. This proposal will make both basic measurements of all aspects of image formation, and ask questions as to whether the aberrations of the eye arise from random errors, or are actually in some sense beneficial, providing increased uniformity of image quality with changes in the spectral content of the retinal image. We will measure the optical quality of the eye over the visible spectrum using modern wavefront sensing techniques. Using modern optical techniques we will obtain measurements of cone directional sensitivity and cone packing density. We will validate new optical techniques using psychophysical measurements. We will combine information on the wave aberrations of the eye with the measurements of directional selectivity and packing density of the cone photoreceptors to obtain individualized estimate of retinal image quality for both monochromatic and polychromatic light. Retinal image quality will be examined as a function of age and refractive error. We will use these data to test whether the incorporation of cone

Studies 31

information into the estimates of retinal image quality generates a better description of patient visual performance. Finally, we will test whether the cones can actively respond to changes in the eye by re-orienting. Our long-term goal is to understand the factors involved in the initial stages of image formation in the human eye, including the contributions of the cornea, lens, and cone photoreceptors, as well as their interactions.

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

•Project Title: ISOPRENOID METABOLISM IN THE RETINA

Principal Investigator & Institution: Fliesler, Steven J.; Professor; Ophthalmology; St. Louis University St. Louis, Mo 63110

Timing: Fiscal Year 2001; Project Start 01-MAR-1988; Project End 31-MAR-2005

Summary: (provided by applicant): The long-range goal of this project is to determine the biological roles of cholesterol (CHOL) and related molecules in the retina in both normal and pathological states. Oxygenated derivatives of CHOL and other sterols occur naturally in cells and tissues, being formed both by autoxidation as well as enzymatically. Such "oxysterols" regulate normal cellular physiology, but also are potent cytotoxins that have been implicated in diseases such as atherosclerosis, diabetes, and cancer. The involvement of oxysterols in retinal diseases is unknown and has yet to be investigated. However, given the association between hypercholesterolemia and atherosclerosis as risk factors in prevalent retinal diseases such as age-related macular degeneration (AMD), research in this area seems warranted. Herein, we evaluate the formation and biological activity of oxysterols in the retina of normal rats in comparison with those that have been treated with a drug (AY9944) that causes accumulation of 7- dehydrocholesterol (7DHC) in the retina and other tissues. AY9944-treated rats are an animal model for the Smith-Lemli-Opitz syndrome (SLOS), a common, autosomal recessive disease with associated ophthalmic defects, including retinal dysfunction. New results presented herein show that AY9944-treated rats develop retinal dysfunction prior to obvious histological damage, yet when exposed to intense green light for only 24 h, a massive, rapid retinal degeneration ensues that is much more severe and extensive than occurs in normal rats under the same conditions. We will examine the time course of retinal degeneration in AY9944-treated rats relative to controls, in both normal, dim cyclic lighting and with the "light damage" paradigm, correlating retinal structure and function with the formation, amounts, and types of oxysterols in the retina. We will compare the effects of intravitreally-injected oxysterols on the structure and function of the retina in normal rats, with and without pretreatment with dimethylthiourea (DMTU), a potent antioxidant. We also will evaluate the ability of DMTU pretreatment to reduce or prevent both oxysterol formation and the retinal damage observed in AY9944-treated and normal rats following exposure to intense green light.

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

•Project Title: MACULAR DEGENERATION: GENETICS OF 4 DISTRINCT PHENOTYPES

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

Timing: Fiscal Year 2001; Project Start 02-JUL-2001; Project End 31-MAY-2006

Summary: The focus of this proposal is to study the biological basis of macular degeneration as will be seen through four large independent pedigrees with distinct

32Macular Degeneration

forms of macular degeneration segregating in Mendelian fashion. We are in the process of positional cloning and candidate gene analysis of these pedigrees, with current emphasis on early onset atrophic macular degeneration. Currently no treatment is available for these debilitating diseases. Cloning of these genes will provide an opportunity to look at the process of degeneration of macula from four biological genetic perspectives. The four forms of macular degeneration we are studying are: (1). Early onset autosomal dominant atrophic macular degeneration (adMD), (2). X-linked cone-rod dystrophy (COD1), (3). Late onset atrophic macular degeneration (adMD) and

(4). Hemorrhagic macular atrophy. We mapped the disease locus for early onset adMD to a 4.9 cM interval on chromosome 6q (6q-adMD) and for COD1 to about 1 cM at Xpll. We have excluded most of the known macular degeneration loci for late onset adMD and hemorrhagic macular degeneration, and a genome wide scan to localize the disease genes is in progress. We have constructed physical and transcript maps of the 6q-adMD interval. Characterization of genes corresponding to candidate ESTs in the critical region is currently in progress. We will work toward positional cloning by: (a) localizing the disease gene to a small interval by ascertaining additional members of the pedigrees and analyzing new markers, (b) Characterizing candidate genes, and (c) Screening candidate genes for mutations. Once the gene(s) for above macular degeneration(s) (MD) is cloned, we will study the possible association between the macular degeneration gene and other phenotypic forms of macular diseases including AMD. Because we can not work on all four pedigrees simultaneously, some of the work is planned for sequential analysis. This study will result in identification of gene mutations causing selective degeneration of macula. These genes will help in understanding the mechanism underlying the variable age of onset and variable rate of progression of the above degenerations and will assist in developing effective treatments either to slow the rate of progression or to delay the age of onset of the disease.

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 2001; 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: 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

Studies 33

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: MECHANISM OF ORGANELLE DEGRADATION IN THE LENS

Principal Investigator & Institution: Duvoisin, Robert M.; Associate Scientist; Ophthalmology; Weill Medical College of Cornell Univ New York, Ny 10021

Timing: Fiscal Year 2001; Project Start 01-JAN-1999; Project End 31-DEC-2001

Summary: The nucleus and organelles of lens fiber cells are degraded during cell differentiation. Because light would be scattered by these membranous particles, this process is necessary for clear vision. Very little is known about the biochemical and cellular mechanisms of this degradation and their regulation. A similar phenomenon occurs in reticulocytes, precursors of red blood cells. Following nucleus expulsion, organelles, including mitochondria and endoplasmic reticulum, are degraded. It has been proposed for a number of years that this process involves lipoxygenase, an enzyme that dioxygenases arachidonic acid and other polyenoic fatty acids. Several mechanisms have been suggested, all based on lipoxygenase modifying organelle membrane lipids or membrane-associated proteins. We have accumulated data that support a totally new mechanism: we propose that the soluble enzyme lipoxygenase assembles into a multimeric structure that forms pores in the membranes of organelles. Such pores would allow the cytoplasmic protein degradation machinery to gain access to the lumenal compartment and initiate the degradation of the organelle. We also found that lipoxygenase is expressed in the lens, most strongly in the peripheral fiber cells where nucleus and organelle degradation occur. We hypothesize that organelle degradation in the lens uses a similar mechanism as in reticulocytes. This proposal will test this hypothesis by identifying the lipoxygenase isozyme expressed in lens, analyzing its regulation of expression, gaining a better understanding of how lipoxygenase permeates membranes, and studying the expression of lipoxygenase in animal model and human cataractous lenses.

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; Ophthalmology; University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Timing: Fiscal Year 2001; Project Start 01-JUL-2000; Project End 31-MAY-2005

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

34Macular Degeneration

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: MICROCONTACT PRINTER FOR OPHTHALMIC TISSUE ENGINEERING

Principal Investigator & Institution: Bloom, David M.; Alces Technology, Inc. 4265 Willowbrook Ln Wilson, Wy 83014

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

Summary: (provided by applicant): This project will demonstrate a prototype microcontact printing aligner for use in ophthalmic tissue engineering. The performance of the microcontact printing aligner will be refined and validated by experiments conducted in collaboration with the Stanford Ophthalmic Tissue Engineering Laboratory. Functioning much like traditional rubber-stamping methods, microcontact printing is widely used to print molecules, such as growth factors or extracellular matrix proteins, in well-defined locations at the nanoscale. This project focuses on microcontact printing applied to a tissue engineering solution for age-related macular degeneration. Using microcontact printing on autologous lens capsule, the Stanford Ophthalmic Tissue Engineering Laboratory is developing a replacement retinal pigment epithelium for treating age-related macular degeneration. Current microcontact printing techniques depend strongly on the skill and ability of the researcher. A prototype tool will be built to improve the reproducibility of microcontact printing. This will require investigation into and enhancement of the stamping materials and techniques. Additionally, this tool will allow for printing of multiple layers, with alignment between the layers. A tool for aligned microcontact printing is a significant enhancement over the current techniques, and something that is neither commercially available, nor easily developed in most biological research facilities.

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

Studies 35

•Project Title: MOLECULAR BASIS OF SYNDROMIC RETINITIS PIGMENTOSA

Principal Investigator & Institution: Hayflick, Susan J.; Associate Professor; Molecular and Medical Genetics; Oregon Health & Science University Portland, or 972393098

Timing: Fiscal Year 2001; Project Start 01-JAN-1999; Project End 31-DEC-2003

Summary: The goal of this project is to isolate and characterize the gene for a form of syndromic retinitis pigmentosa (RP), called Hallervorden-Spatz syndrome (HSS) and characterized by abnormal electroretinogram, lipofuscin accumulation in the retinal pigment epithelium, and early, rapidly progressive pigmentary retinopathy. This autosomal recessive disorder of childhood includes extrapyramidal dysfunction with iron accumulation in the basal ganglia. Though lipid peroxidation is an hypothesized mechanism leading to the HSS phenotype, no knowledge exists of the molecular or biochemical defect. We propose a molecular genetic approach to understanding this syndromic form of RP. Our specific aims are to 1) identify the gene for HSS, designated NBIA1 (Neurodegeneration with Brain Iron Accumulation, type 1) by completing the physical map of the critical region, identifying and screening candidate genes, and demonstrating deleterious mutations; 2) develop the molecular diagnosis of HSS using mutation studies and genotype-phenotype correlation; 3) characterize the HSS gene and its protein product at the tissue, cellular, subcellular and molecular levels using homology to model organisms, sequence analysis, histopathology, immunohistochemistry and studies of tissue expression patterns; and 4) isolate the murine homolog of the HSS gene and develop a mouse model for HSS in order to study its pathophysiology. Knowledge about the HSS gene will allow molecular diagnosis in individuals suspected to have this disease. As well, prenatal diagnosis of this fatal condition will be feasible. By delineating the pathophysiologic process in HSS, we may begin to develop rational therapies, which may be of benefit in treating other forms of RP, as well. Rare diseases often illuminate the mechanisms at work in common, related disorders. An advantage to studying syndromic RP is that the pleiotropic manifestations provide a context to help delineate the mechanism of retinopathy. The HSS gene is not retina-specific, and a defect in it must account for rod photoreceptor degeneration as well as regional brain iron accumulation. Furthermore, since defects in this non-retina- specific process may cause other forms of syndromic and isolated RP and may be integral in disorders of lipofuscin accumulation, including aging macular degeneration, identification of the HSS gene may lead to greater understanding of RP as well as the macular dystrophies associated with senescence.

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

•Project Title: MOLECULAR MECHANISMS UNDERLYING OPTIC NERVE REGENERATION

Principal Investigator & Institution: Chen, Dong F.; Assistant Professor; Schepens Eye Research Institute Boston, Ma 02114

Timing: Fiscal Year 2001; Project Start 01-APR-2000; Project End 31-MAR-2005

Summary: (Verbatim from applicant's abstract): The long-term objective of this research plan is to elucidate the molecular mechanisms underlying nerve growth and regeneration. It is generally believed that neurons in the brain and retina of mammals are unable to regenerate nerve fibers, which presents a great challenge for the treatment of retinal degenerative disorders and damage. Recent data have established that retinal ganglion cells (RGCs) are intrinsically regulated in their ability to extend nerve fibers, and that the proto-oncogene Bcl-2 is a key regulator supporting the mechanism for nerve elongation. This growth-stimulating activity of Bcl-2 does not seem to be a

36Macular Degeneration

consequence of its well-known anti-apoptotic function. The central question here is the mechanisms by which Bcl-2 promotes the regeneration of retinal nerve fibers. It has long been recognized that neural differentiation impacts nerve growth. It is now evident that overexpression of Bcl-2 suppresses the function of a differentiation signal, p53-mediated transcriptional activation, and expression of terminal differentiation markers in neurons. Therefore, it is hypothesized that Bcl-2 may function as a repressor of the p53-p21WAF1 pathway inhibiting neural terminal differentiation and thus, maintaining the ability of RGCs to grow nerve fibers. Using genetically-engineered mouse models as well as novel retinotectal co-cultures, this research plan is aimed at elucidating functional pathway(s) through which Bcl-2 may support optic nerve regeneration. Our goals are: (1) to investigate whether Bcl-2 inhibits the terminal differentiation of RGCs by determining levels and timing of RGC markers and differentiation-related protein expression in retinas of wild type and Bcl-2 mutant mice; (2) to test whether Bcl-2 supports nerve regeneration via suppression of the differentiation signal, p53-p21WAF1 pathway. Here, functional interactions between Bcl-2 and p53 will be assessed by comparing transcriptional activity, subcellular localization, and phosphorylation of p53 in wild type, Bcl-2 mutant, and Bcl-2/p53 double mutant mice; and (3) to determine whether Bcl-2 acts on a common cellular pathway to support nerve regeneration and neuronal survival by comparing the ability of RGCs from Bcl-x, and Bcl-2 transgenic mice to survive and regenerate nerve fibers. Given the functional importance of Bcl-2 in neuronal survival and nerve regeneration, these studies should provide valuable information for the development of neuroprotective drugs and therapeutic strategies for numerous diseases mediated by dysregulated cell death and nerve growth, including glaucoma and macular degeneration.

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

•Project Title: MOLECULAR RISK FACTORS FOR AGE-RELATED MACULOPATHY

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

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

Summary: (provided by applicant): This is a revised application by a new investigator to assess the role of inflammation in age-related maculopathy (ARM). ARM comprises the leading cause of incurable blindness among older adults in the US and other developed countries. However, the basic molecular pathways involved in the pathogenesis of ARM remain unknown, few potentially modifiable risk factors have been identified, and treatment remains inadequate. We hypothesize that the pathologic changes occurring in both the early and late stages of ARM are mediated by cells and molecules associated with inflammation and that the pro-inflammatory state that gives rise to these changes is at least in part a systemic rather than merely local phenomenon. The proposed studies will build upon a broadly based and growing body of research that supports a key role for inflammatory/immune-mediated processes in ARM pathogenesis. This research suggests several pathways through which inflammation could mediate the development of ARM, including RPE damage and repair, drusen formation, degeneration of Bruch's membrane, endothelial dysfunction in choroidal vessels, increased oxidative stress, decreased bioavailability of antioxidants, as well as the direct or indirect promotion of angiogenesis. Through its use of archived blood specimens from the Physicians' Health Study, Women's Health Study, women's Antioxidant Cardiovascular Disease Study, Nurses' Health Study, and Health Professionals Follow-up Study, this proposal represents an exceptionally cost-effective and efficient means to investigate the proposed hypothesis using a prospective nested case-control study design. The Specific

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Aims are to investigate 1) the relationship of systemic markers /mediators of inflammation (IL-6, C-reactive protein, fibrinogen, haptoglobin, circulating adhesion molecules, and tumor necrosis factor-alpha receptors) with incident ARM, 2) the separate relationships of these inflammatory molecules with dry and neovascular ARM lesions, 3) whether the relationship of inflammation with ARM is independent of other risk factors such as cigarette smoking, and 4) the interrelationships among the biomarkers and which independendy predict incident ARM. These aims will be accomplished through measurement using highly sensitive assays of inflammatory biomarkers in blood specimens collected at baseline (i.e. prior to the development of ARM) and stored since that time below -80xC. Biomarker levels will be compared among subjects who eventually developed ARM and control subjects who remained free of ARM, and the analysis will be extended to control for other risk factors. The longterm objective and clinical relevance of this research is to shed light on potential underlying biological mechanisms of ARM pathogenesis and suggest avenues for new preventive or therapeutic approaches, as well as to identify clinically useful biomarkers for identification of individuals at increased risk of ARM.

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

•Project Title: MOLECULAR STUDIES OF RETINAL DEGENERATION IN DROSOPHILA

Principal Investigator & Institution: Colley, Nansi J.; Associate Professor; Ophthalmology and Visual Sci; University of Wisconsin Madison 750 University Ave Madison, Wi 53706

Timing: Fiscal Year 2001; Project Start 01-AUG-1990; Project End 31-JUL-2005

Summary: (provided by applicant): The objective of the proposed research is to utilize Drosophila as a model for studying hereditary human diseases that cause retinal degeneration and eventual blindness [retinitis pigmentosa (RP) and age-related macular degeneration (AMD)]. The complexity and variations of human RP and AMD suggest that there are multiple subtypes of the diseases, each with distinct genetic and biochemical bases. This complexity, the infrequent availability of ocular tissues from RP and AMD patients, and the broad base of knowledge of Drosophila molecular genetics, combine to make Drosophila a powerful animal model for studying inherited retinal degeneration disorders. We propose to use an integrated strategy of biochemical, cell biological, electrophysiological, genetic, and molecular approaches to identify and characterize mutations that cause defects in protein transport and targeting. We have identified three mutant lines of flies that display secretory pathway defects and retinal pathology. We will identify the corresponding genes and subject them to a detailed genetic and molecular analysis. In addition, we will continue to screen 12,000 individual mutant lines for retinal degeneration. The screen is based on a simple morphological phenotype that may be screened in live flies under the dissecting microscope. Mutants that define constituents of the secretory pathway and protein targeting will be subjected to a detailed characterization. Our findings will be utilized to screen a highly defined set of human AMD and RP patients for similar defects. Genetic analysis in Drosophila remains a powerful means of rapidly identifying genes that are essential for protein trafficking and normal photoreceptor function. It is anticipated that genes identified in this study will provide insights for the genetics of AMD and RP in humans.

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

38 Macular Degeneration

•Project Title: MRI PROBES FOR MAPPING RETINAL ACTIVITY IN VIVO

Principal Investigator & Institution: Louie, Angelique Y.; Biomedical Engineering Div; University of California Davis Sponsored Programs, 118 Everson Hall Davis, Ca 95616

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

Summary: (Applicant?s Abstract) This proposal describes the development of probes for three-dimensional imaging of retinal activity. These probes are magnetic resonance imaging (MRI) contrast agents that are sensitive to membrane potentials. It is the signal from water protons that provides the image in typical MRI images and contrast agents interact with water to enhance that signal. In our previous work we have shown that by blocking the interaction of water with the contrast agent we can "turn off? enhancement, and then selectively "turn on" enhancement by removing the block. In these previous experiments we added a blocking group to the contrast agent that is removable by enzyme cleavage, so that signal enhancement was dependent upon enzyme cleavage. Based on these results we hypothesize that we can modulate the ability of an agent to enhance MRI signal by adding a group that is sensitive to membrane potentials, thus making signal enhancement dependent on membrane activity. In our research design we propose to: 1) Design and synthesize an agent based on a known optical indicator of membrane potentials 2) Characterize the ability of the new agent to enhance contrast in cell-free and cell culture systems. 3) Perform studies leading to the application of the new agents in eyes in vivo. These studies include determining the optimal route of delivery for the agent, and its biodistribution in model animal studies. These investigations will ultimately be extended to rodent models of retinal degeneration to map the loss of retinal activity over time and correlate the results with those observed by traditional methods. Results of the proposed studies will provide a noninvasive method to image membrane activity in deep tissues; this may aid in the early diagnosis of retinal disease. While our ultimate goal is to apply these agents in studies of macular degeneration, these agents would be useful for observations of any system in which membrane signaling activity plays a role.

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

•Project Title: MULTI-AGENT COLLABORATION FOR AMD SUBTYPE CLASSIFICATION

Principal Investigator & Institution: Williams, Andrew B.; Electrical and Computer Engineering; University of Iowa Iowa City, Ia 52242

Timing: Fiscal Year 2003; Project Start 01-FEB-2003; Project End 31-JAN-2006

Summary: (provided by applicant): Age-related macular degeneration (AMD) is the most common cause of blindness in developed countries and as such represents a very significant public health problem a number of specific genes, and the discovery, characterization, and eventual therapeutic control of these genes represent major goals of the vision research community. Although the strategies for gene discovery have become very powerful in recent years, there remains a major obstacle to the discovery of genes that underlie common, late-onset diseases like AMD. That obstacle is that clinicians cannot reliably sort patients with different molecular subtypes of late-onset disease into sufficiently homogeneous groups. The purpose of this project is to use the power of multi-agent systems computer technology in a novel way to aid clinicians in the collaborative development of a robust classification system based upon the ophthalmoscopic features of AMD. The result of this project will contribute to an NIH's Innovations in Biomedical Information and Science and Technology Program goal of speeding the progress of biomedical research through the development tools for

Studies 39

electronic collaboration that will have impact on broader areas of biomedical research. We hypothesize that a multi-agent approach to this problem will result in a classification system with greater reproducibility and discriminative power than a system developed by clinicians without such computer assistance. The availability of populations of AMD patients with lower molecular complexity will significantly increase the power of statistical techniques for AMD gene discovery. In addition to this immediate and specific benefit, the strategies we will develop during this project for objectively interfacing medical experts with each other as well as with computers will have applications in the search for other late-onset disease genes as well as in the development of multi-center and multidisciplinary clinical trials of new therapeutic approaches. The proposed system, the Intelligent Distributed Ontology Consensus system (IDOCS) goes beyond conventional groupware by addressing drawbacks to direct, synchronous interaction by providing an autonomously coordinated, asynchronous interaction and collaboration platform among clinicians through their representative intelligent agents. IDOCS will provide a generic meta-data infrastructure using XMLJRDF to make it easily configurable for other diseases.

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

•Project Title: OPHTHALMIC IMAGING USING ADAPTIVE OPTICS AND OCT

Principal Investigator & Institution: Werner, John S.; Professor; Ophthalmology; University of California Davis Sponsored Programs, 118 Everson Hall Davis, Ca 95616

Timing: Fiscal Year 2003; Project Start 30-SEP-2003; Project End 31-AUG-2008

Summary: (provided by applicant): The purpose of this BRP is to develop and evaluate new instrumentation that will permit unprecedented three dimensional imaging of single cells in the human retina, specifically rod and cone photoreceptors and ganglion cells. An interdisciplinary team will combine adaptive optics (AO), enabling the best lateral resolution for retinal imaging, with optical coherence tomography (OCT), providing the best axial resolution for retinal imaging. Two instruments will be developed using complementary OCT imaging modalities, flood illumination and enface scanning. These instruments will be used to study cellular morphology associated with normal aging, age-related macular degeneration (AMD) and glaucoma. The instruments will be compared quantitatively with each other and with existing retinal imaging devices. The project will be led by UC-Davis COCD), where a highperformance AO system has been developed in collaboration with the Lawrence Livermore National Laboratory (LLNL). This collaboration will be expanded to include a team of OCT experts at LLNL and Indiana University (IU). The IU team has previously collaborated with LLNL through the Center for Adaptive Optics and has already developed a working prototype AO-OCT system for retinal imaging. In this BRP project, LLNL will construct one AO-OCT instrument at the UCD site to be tested clinically in years 3-5, while the second AO-OCT instrument will be developed at IU in collaboration with LLNL and tested in the laboratory in years 4-5. Comparisons of AO-OCT and functional measures will be obtained at UCD and IU. Both instruments will be made available for use by scientists and clinicians who are not part of the BRP, and will be refined through the course of the project period. UCD has expertise in vision science, aging, and evaluation of AMD and glaucoma progression and treatment LLNL has a long history of research on AO for astronomy and has transferred some of its AO technology to vision science at UCD. LLNL also has expertise in OCT, and has pioneered its application to in vivo imaging of oral and vascular structures. The IU team has experience in AO-OCT and vision science with specific expertise in visual optics and retinal electrophysiology. This BRP is buttressed by consultants who have developed

40Macular Degeneration

ophthalmic OCT technology at the University of Texas and Carl Zeiss Meditec. The Zeiss group has already transferred OCT technology to the clinic via commercial development and will facilitate incorporation of user friendly interfaces for our AOOCT systems. This BRP thus combines the unique expertise of engineers, vision scientists and clinicians who have experience working together to effect a smooth transition from the laboratory to applications. This synergistic team will develop a new generation of instruments to advance vision science, permit retinal dysfunction to be studied in vivo in a way that will offer new insights into normal aging, the pathogenesis of glaucoma and macular degeneration, and a reliable method to monitor novel treatments for retinal disease.

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

•Project Title: OPSINS, G PROTEIN PATHWAYS AND REGULATION IN RPE CELLS

Principal Investigator & Institution: Fong, Henry K.; Doheny Eye Institute 1450 San Pablo St Los Angeles, Ca 90033

Timing: Fiscal Year 2001; Project Start 01-AUG-1990; Project End 31-JUL-2002

Summary: (Adapted from applicant's abstract): Vertebrate RPE and Muller cells contain a cytoplasmic visual pigment homolog termed RGR (for RPE retinal G protein-coupled receptor) which binds retinoid and shares sequence similarity with retinochrome, a photoisomerase in squid photoreceptors. This application hypothesizes that RGR may function as a retinal isomerase in the vertebrate visual cycle, be involved in a primitive form of phototransduction, play a role in circadian rhythm, or act as a sensor of free retinaldehyde. To test these hypotheses, research will investigate RGR ligandand protein-binding properties and analyze the phenotype resulting from mutation of the RGR gene in a RGR-less mouse model system. The endogenous chromophore of bovine RGR will be identified and photoisomerization of retinal bound to natural and recombinant RGR will be investigated. A splice variant of the human RGR gene termed RGR-d has been identified in which the predicted sixth transmembrane domain is deleted. To investigate the possibility that RGR-d is involved in diseases of the retina such as age-related macular degeneration (AMD), studies will characterize human RGR-d retinaldehyde-binding properties, subcellular localization and other possible altered properties.

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

•Project Title: P27KIP1 AND RPE CELL CYCLE

Principal Investigator & Institution: Defoe, Dennis M.; Anatomy and Cell Biology; East Tennessee State University Box 70565 Johnson City, Tn 37601

Timing: Fiscal Year 2003; Project Start 04-APR-2003; Project End 31-MAR-2005

Summary: (provided by applicant): Abnormal growth or turnover of the retinal pigment epithelium (RPE) is associated with an alarming number of ocular disorders (i.e., proliferative vitreo-retinopathy (PVR), age-related macular degeneration, developmental hamartomas, congenital hypertrophy of the RPE), accounting for significant health costs. An understanding of the basic mechanisms for establishing and maintaining appropriate epithelial cell number and size is crucial to finding ways to alleviate disease consequences and to facilitate their repair. The aim of the proposed research is to define the mechanisms that regulate growth, through cell multiplication and enlargement of cell size, during RPE development in situ. While many exogenous influences can regulate cell proliferation, they do so by ultimately impinging on

Studies 41

machinery of the cell division cycle. Consequently, the proposed studies focus on mechanisms operating in the RPE cells as they withdraw from the cell cycle during differentiation of the epithelial layer in vivo. Experiments will make use of a mutant mouse with targeted disruption of the gene coding for the cell cycle regulatory protein p27Kip1, in which there is an abnormal expansion of the RPE rnonolayer. The overall hypothesis is that loss of p27Kip1 leads to an increase in the number or density of epithelial cells as a result of an enhancement in the rate of cell proliferation, or of a delay in the timing of cell cycle exit prior to differentiation. This hypothesis will be tested using immunohistochemical techniques to determine the magnitude of cell division, as well as cell death, at various developmental ages. The alternative hypothesis that the thickening of the RPE layer is due to an increase in cell size will be examined by morphometric analysis of images obtained by confocal microscopy.

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

•Project Title: PEDF AND PHOTORECEPTOR PROTECTION: A NAKED DNA APPROACH

Principal Investigator & Institution: Cao, Wei; Ophthalmology; University of Oklahoma Hlth Sciences Ctr Health Sciences Center Oklahoma City, Ok 73126

Timing: Fiscal Year 2003; Project Start 01-JAN-2003; Project End 31-DEC-2005

Summary: (provided by applicant): PEDF and photoreceotor protection: a naked DNA approach. Photoreceptor cell death is an irreversible event in many blinding diseases including retinitis pigmentosa, age-related macular degeneration and retinal detachment. Pigment epithelium derived factor (PEDF), a secreted protein with both neurotrophic and anti-angiogenic properties, is made in vivo by a variety of cells in both fetal and adult animals. Within the retina, it is secreted by retinal pigment epithelial (RPE) cells into the interphotoreceptor matrix, placing it in a prime physical location to affect the underlying photoreceptor cells. We recently reported that the intravitreal injection of PEDF protected photoreceptor cells against constant light induced damage in a rat model. An alternative method for delivering a protein factor to cells and tissues is to use only the DNA which codes for the protein. Our preliminary data show that primary cell cultures of rat retinal neurons can be transfected with a naked DNA coding for enhanced green fluorescent protein under the control of the photoreceptor cellspecific promoter, mouse interphotoreceptor retinol binding protein. We have performed similar experiments expressing other reporter genes under control of other photoreceptor cell specific promoters. These experiments suggest that we are able to transfect PEDF cDNA into retinal neurons which will then express the PEDF protein. I am seeking three years of funding in this proposal to develop a novel approach using plasmid DNA cassettes expressing PEDF and apply it to the retina to prolong useful vision by delaying or preventing retinal degeneration. This novel method is actually being employed in phase I clinical trials with the injection of a naked DNA encoding the angiogenesis factor, VEGF, into the hearts of cardiac patients. In several phase II trials, direct injection of DNA has also demonstrated significant therapeutic effects in cancer patients. The specific aims of this proposal are: I) To demonstrate the expression and protection efficiency of transfected naked plasmid DNA, encoding PEDF, in cultured rodent retinal cells. 2) To maximize the in vivo expression of naked plasmid PEDF in the eye, through the optimization of the route of administration, such as topical application, anterior chamber/intravitreal/subretinal injection. 3) To evaluate the protective efficacy of this protocol to prevent the retinal degeneration induced by light or by heredity. The proposed studies are innovative and could have a significant clinical impact as a novel

42Macular Degeneration

therapeutic approach to delay or prevent retinal degeneration and to prolong useful vision.

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

•Project Title: PHOTORECEPTOR CELL DEVELOPMENT AND SIGNAL TRANSDUCTION

Principal Investigator & Institution: Carthew, Richard W.; Professor; Biochem/Molecular & Cell Biol; Northwestern University 633 Clark St Evanston, Il 60208

Timing: Fiscal Year 2003; Project Start 01-MAR-1993; Project End 31-JUL-2006

Summary: (provided by applicant): Growth factors are important signaling proteins that control cell growth, survival, and differentiation in the developing and adult retina. The ultimate objective of the proposed research is to understand the molecular mechanisms that growth factors use to regulate cell behavior in the retina. We address this question by studying the differentiation of photoreceptor and cone cells in the Drosophila eye that are regulated by growth factor ligands for the Sevenless and EGF Receptor Tyrosine Kinases. This could provide important insights into how retinal cell differentiation is regulated, and how errors in growth factor signal transduction can lead to retinal diseases such as macular degeneration and retinitis pigmentosa. A paradox of growth factor signaling is how cells respond so specifically to a signal that appears to carry little informational content. For example, the Pros gene becomes transcriptionally activated as an immediate-early target of EGFR in a subset of cells (R7 and cone) that are activated by EGFR. A combinatorial mechanism restricts Pros enhancer responsiveness to EGFR activation. Other required inputs include two transcription factors (Lola, Lozenge) and a cell-cell signal mediated by the Notch Receptor. Pros transcription increases specifically in the R7 photoreceptor where it is required for its proper differentiation. The mouse homolog of Pros, Prox-1, is also required for proper development of the eye. The R7specific transcriptional response requires activation of the Sevenless receptor, which induces expression of another immediate-early gene, Phyllopod. Phyllopod associates with a transcriptional repressor called Tramtrack, and this results in ubiquitination and degradation of Tramtrack protein. Tramtrack destruction may be the sole essential action of Sevenless to activate Pros. Our approach has been to characterize molecular interactions between proteins in the GFR/Sevenless pathways and a target gene, Pros. As such, Pros serves as an experimental model gene. We use a combination of genetic and biochemical experiments. The goal of the proposed research is to further characterize EGFR and Sevenless signaling by: 1) determining how other signals like Notch integrate with the EGFR signal to regulate Pros, 2) determining how EGFR and Sevenless exert different effects on Pros transcription, 3) characterizing a second model target gene, Phyllopod, as a way to compare and contrast with Pros regulation.

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; 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

Studies 43

expression in a variety of metabolic and aging disorders from atherosclerosis and lipid 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: PREVENTING DEPRESSION IN MACULAR DEGENERATION

Principal Investigator & Institution: Rovner, Barry W.; Psychiatry and Human Behavior; Thomas Jefferson University Office of Research Administration Philadelphia, Pa 191075587

Timing: Fiscal Year 2001; Project Start 06-FEB-2001; Project End 31-JAN-2006

Summary: Applicant's This application describes a randomized controlled clinical trail to evaluate the efficacy of a brief, standardized cognitive psychotherapy, Problem Solving Therapy (PST), to prevent incident depressive disorder (DSM-IV diagnoses) in elderly patients with age-related macular degeneration (AMD). AMD is the most common cause of blindness in older adults and limits the ability to read, see familiar faces, and walk independently. Almost 2 million persons (about 5 percent of the U.S. population over age 65) are now affected and their number will triple by the year 2020. We will target patients with neovascular AMD (NV-AMD), a form of AMD that can lead to sudden vision loss, substantial disability, and depression. Because depression is itself disabling and not likely to be recognized nor treated by ophthalmologists, preventing depression is clearly important. We will recruit 230 non-depressed AMD patients from the retinovascular clinic of Wills Eye Hospital with newly diagnosed NV-AMD one eye, who already have AMD in the fellow eye. Because they have recently developed bilateral vision loss they will be at high risk for depression. We will randomize subjects to PST or a usual care control condition in this 6-month clinical trial. The primary outcome measure will be a DSM-IV diagnosis of depression diagnosed by a geriatric psychiatrist masked to treatment assignment. We will evaluate subjects at baseline, month 2 (immediately post-intervention), month 6 (for the primary efficacy analysis) and month 12 (to evaluate sustained effects). Although depressive disorder is the primary outcome, we will also assess the impact of PST on levels of disability and vision-related quality of life.

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

44 Macular Degeneration

•Project Title: PROSPECTIVE STUDY OF RISK FACTORS FOR EYE DISEASE

Principal Investigator & Institution: Hankinson, Susan E.; Associate Professor; Brigham and Women's Hospital 75 Francis Street Boston, Ma 02115

Timing: Fiscal Year 2001; Project Start 01-JAN-1995; Project End 31-DEC-2004

Summary: We propose to investigate several lifestyle and genetic factors in relation to age-related macular degeneration (AMD) and primary open angle glaucoma (POAG) in two prospectively followed cohorts of women and men. Specifically, we will evaluate dietary intake of antioxidants and fat (including specific types of fat), postmenopausal hormone use and variants in the ATP-binding cassette-transporter retina (ABCR) gene in relation to both wet and dry AMD, and antioxidant intake, smoking, and systemic blood pressure in relation to POAG. The Nurses' Health Study (NHS) began in 1976 among 121,700 women ages 30-55 at that time. About 89,000 participants completed an extensively validated semiquantitative food frequency questionnaire (FFQ) in 1980 and every 2-4 years since. The Health Professionals Follow-up Study (HPFS) began in 1986 among 52,000 men ages 45-75, all of whom completed a FFQ at baseline and every four years since. Both groups have been sent a questionnaire biennially to update exposure information and reports of major illnesses, including AMD and POAG. Information has been collected repeatedly on specific vitamin supplement use, smoking, diagnosis of hypertension, reported blood pressure, and postmenopausal hormone use among other factors. Over 32,000 blood samples were collected in the NHS in 1989-90 and over 18,000 in the HPFS in 1993. In the proposed study we will confirm reports of AMD and POAG by contacting the participant's ophthalmologist, and obtaining detailed information from the optical record, including fundus photographs for those with AMD. A case will be considered to have AMD if it is judged to be sufficient to result in a visual acuity loss of at least 20/30 and is confirmed by a standardized review of the fundus photograph; wet and dry types will be carefully delineated by photographic review. A case will be considered to have POAG if confirmed by medical record review and is documented to have visual field loss. We anticipate 554 cases of exudative and 833 cases of dry AMD, and 1049 cases of POAG. Stratified and multivariate techniques will be used to quantify the risk of AMD and POAG according to the level of exposure after controlling for potentially important confounders; analyses will be conducted among participants who reported having a recent eye exam. Overall, the prospective design, large size of the cohorts, the high follow-up rates, repeated exposure measures, and carefully confirmed disease definitions provide a unique opportunity to evaluate several hypotheses of public health importance.

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

Studies 45

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: PROTEOMIC STUDIES OF AGE RELATED MACULAR DEGENERATION

Principal Investigator & Institution: Crabb, John W.; Professor and Staff Manager; Cleveland Clinic Foundation 9500 Euclid Ave Cleveland, Oh 44195

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

Summary: (provided by applicant): The long-term goal of this research is to understand the molecular mechanisms of drusen formation and Bruch's membrane thickening in age-related macular degeneration (AMD). The unifying hypothesis is that protein modifications are causally involved in both processes. Drusen are extracellular deposits that form between the retinal pigment epithelium and Bruch's membrane and confluent drusen are the hallmark risk factor for developing AMD. The progression of AMO might be slowed or halted if drusen and Bruch's membrane changes can be modulated. The proposed research will identify proteins and lipids in drusen and Bruch's membrane from healthy and AMD donor tissues and characterize associated protein modifications and reactive lipid fragments. Four specific aims will test the following hypotheses: (i) that the drusen proteome differs among drusen sub-types; (ii) that the Bruch's membrane proteome varies with age and state of health; (iii) that lipid oxidation products in drusen and Bruch's membrane vary with age and state of health; (iv) that oxidative protein modifications contribute to drusen formation and Bruch's membrane thickening. Mass spectrometric methods will be used to identify and characterize proteins, phospholipids and lipid oxidation products. Immunocytochemistry will be used to confirm protein localization to drusen and Bruch's membrane. Western analyses, electrophoretic mobility shifts and bioinformatic tools will be used to detect protein modifications. The results will help establish a firm link between oxidative damage and the pathogenesis of AMD and provide new opportunities for developing strategies and therapies for preventing or limiting the complications of AMD.

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

•Project Title: QUANTIFICATION OF DRUSEN IN MACULAR DEGENERATION

Principal Investigator & Institution: Friberg, Thomas R.; Professor; Ophthalmology; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, Pa 15260

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

Summary: (Applicant's Abstract) Age-related macular degeneration (AMD) is the most common cause of blindness in the United Sates in Americans over the age of 50. Drusen are by-products of rod and cone metabolism and their presence in the ocular fundus is a

46Macular Degeneration

clinical sign of AMD. It is known that patients whose eyes harbor drusen are at special risk of visual loss from AMD. In a prospective, multi-centered trial, the Age-Related Eye Disease Study (AREDS), some 5000 patients have been enrolled and followed at sixmonth intervals to determine risk factors for AMD as well as to measure the influence of certain multivitamins and micronutrients on the progression of macular degeneration and cataract. In another prospective, randomized, controlled trial, the Prophylactic Treatment of AMD Trial (PTAMD), patients with multiple large drusen have been enrolled to determine whether a minimal laser treatment applied one time provides any benefit with respect to lowering rates of visual loss and rates of development of choroidal neovascularization (CNVM). During the PTAMD and AREDS trials, photographs of the fundi of each participant have been taken at least annually in the form of Ektachrome color slides. These slides are evaluated by human readers in a Reading Center and the extent of the drusen is categorized accordingly. The applicant proposes to perform a quantitative analysis on the color slides of the macula of all patients who have been enrolled into the PTAMD and AREDS trials at the Pittsburgh site, as well as other patients who have AMD and have been followed similarly for a minimum of three years. The original slides will be digitized onto CD disks for permanent archiving, and will be analyzed using a specially-written computer algorithm which semi-automatically detects drusen in digital images. Once detected, quantitative measurements of the area of drusen extent and size of each drusen are generated for each image. This method is more precise than characterizing drusen using traditional long and more subjective protocols, and may be valuable in determining the risks of a given patient developing choroidal neovascularization and visual loss before such events occur. That is, such continuous quantitative data may, when analyzed, provide a more accurate and sensitive assessment of risk compared to conventional measurement techniques.

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

•Project Title: REACTIVE OXYGEN-INDUCED DNA DAMAGE IN RETINAL CELLS

Principal Investigator & Institution: Godley, Bernard F.; Senior Scientist; Ophthalmology and Visual Scis; University of Texas Medical Br Galveston 301 University Blvd Galveston, Tx 77555

Timing: Fiscal Year 2001; Project Start 01-MAR-2000; Project End 31-JUL-2001

Summary: This project examines the hypothesis that reactive oxygen intermediates (ROI) overwhelm declining antioxidant defenses in aged RPE leading to mitochondrial DNA damage, cell dysfunction and death. This chain of events may be a primary mechanism of aging and of acquired retinal diseases such as AMD. A major objective of this study is to assess the basal levels, formation and repair of oxidative DNA damage in nuclear and mitochondrial DNA in primary RPE cells obtained from young versus aged human donors. We will test the hypothesis that mitochondrial DNA damage may accelerate the production of ROI that leads to further damage and functional decline. The following specific aims will be addressed to meet the objectives of this proposal: 1) To examine the formation and repair of oxidative DNA damage from rod outer segment-fed human retinal pigment epithelium (RPE) cell cultures; 2) To elucidate the temporal sequence and downstream cellular consequences in oxidantexposed cultured human RPE cells in relation to mitochondrial DNA damage, mitochondrial transcription and function, and cell death; 3) To evaluate the linkage between reactive oxygen intermediates (ROI), antioxidant enzyme systems, and oxidant-induced DNA damage; 4) To study the relevance of the glutathione S-transferase pathway in protecting RPE cells under oxidative stress: a study with mGSTA4-4 transfected cells; 5) To examine the

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formation of mitochondrial DNA damage and repair kinetics in RPE from aged human eyes, and eyes with AMD. Successful accomplishment of these specific aims will provide fundamental mechanistic insights regarding the molecular pathobiology of reactive oxygen in aging RPE cells. The ultimate goal of this project is to reveal and test novel clinical strategies to reduce morbidity from agerelated macular degeneration.

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

•Project Title: READING IN PERIPHERAL VISION

Principal Investigator & Institution: Chung, Susana T.; Associate Professor; None; Indiana University Bloomington P.O. Box 1847 Bloomington, in 47402

Timing: Fiscal Year 2001; Project Start 01-FEB-2000; Project End 31-JAN-2005

Summary: (Adapted From The Applicant's Abstract): This is a grant directed at the problem of reading in low vision patients. Specifically, the PI is interested in those cases where macular damage makes it necessary to read with peripheral retina. Reading is difficult under such conditions. Proposed research will investigate three hypotheses about the causes of that difficulty: Grouping/crowding effects are more damaging in the periphery. Consequently, Aim 1 seeks to minimize these effects. Peripheral representation of phase is inaccurate. Aim 2 will attempt to enhance reading speed by minimizing the need for phase information. Patients lack of practice with peripheral reading. Aim 3 will examine the effects of extensive practice with peripheral reading. Experiments will be carried out with normally-sighted subjects as well as with subjects having central scotomata.

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

•Project Title: RECOMBINANT H-VEGI AS AN ANTICANCER THERAPEUTIC

Principal Investigator & Institution: Medynski, Daniel C.; Proteomtech, Inc. 5980 Horton St, Ste 405 Emeryville, Ca 94608

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

Summary: (provided by applicant): Blocking or inhibiting blood vessel growth (angiogenesis) remains a critical focal point for anticancer drug development. Studies on Vascular Endothelial Growth Inhibitor (VEGI; TNFSF-15) secreted from human endothelial cells have demonstrated that it is a potent negative regulator of angiogenesis that induces growth arrest and apoptosis in endothelial cells. Unfortunately, because of a limitation in the quantity of VEGI obtained in this manner and the difficulty encountered in the production of the recombinant form of this protein, preclinical studies using systemically administered VEGI to assess its therapeutic potential as an anti-cancer or anti-angiogenesis agent have not been possible. In addition, despite a considerable effort, no commercial or academic laboratory has succeeded in producing large quantities of this valuable molecule. We have now developed a procedure to produce VEGI in E. coil. In this proposal, three specific objectives will be pursued: 1. In a small scale pilot study, a number of expression vector constructs encoding variants of the four isoforms of VEGI will be expressed in E. coil as inclusion bodies, purified, and refolded. They will then be tested in endothelial cell growth arrest assays to isolate refolded, biologically potent VEGI isoforms. 2. Expression, purification, and refolding of isoforms of VEGI already identified as biologically active in pilot studies will be pursued at a much larger scale. One hundred milligrams to gram quantities of highly purified biologically active VEGI isolated from E. coli inclusion bodies will be prepared for in vivo animal studies. 3. The active isoforms of VEGI will be utilized in rodent xenograft studies to determine if VEGI has a potent antiangiogenic or anticancer effect

48Macular Degeneration

when systemically administered. The long term objective of this study is to develop VEGI into an effective drug in diseases involving angiogenesis - particularly cancer, but also atherosclerosis and blindness (macular degeneration).

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

•Project Title: REMOVAL OF THE LIPOFUSCIN COMPONENT A2E FROM RPE CELLS

Principal Investigator & Institution: Rodriguez-Boulan, Enrique; Ophthalmology; Weill Medical College of Cornell Univ New York, Ny 10021

Timing: Fiscal Year 2003; Project Start 01-JAN-2003; Project End 31-DEC-2005

Summary: (provided by applicant): This project will develop methods to reduce the amounts of N-retinyledene-N-retinylethanolamine (A2E) in the retinal pigment epithelia (RPE). The excessive accumulation of this lipofuscin component contributes to the onset of age-related macular degeneration (ARMD). Thus, any methods that can remove A2E from the RPE will be useful in developing potential therapies to treat this disease. To test the efficiency of various methods to remove A2E from cultured RPE cells, we will use a fluorescence plate reader and fluorescence microscopy to measure both the intracellular concentration and the subcellular location of A2E. Initial results using an in vitro system have already identified several drugs which are capable of removing A2E from phospholipid membranes. Subsequent work will focus on delivering these drugs to the Iysosomes of RPE, the site of A2E subcellular accumulation. Recent evidence suggests that A2E impairs the ability of RPE to degrade phospholipids derived from phagocytosed outer segment. Each day, the RPE is presented with shed outer segment particles, and must complete the digestion of these particles before the next day. Any delay in the digestion of the OSphospholipids, which comprises more than half of the total OS mass, will result in the gradual accumulation of these phospholipids. Once the development of methods that can remove A2E is achieved, the effectiveness of these systems in restoring the ability of A2E-treated RPE to degrade OS phospholipids will be examined. A flourescence-based lipid degradation assay has already been developed in this lab.

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

•Project Title: RETINAL ANTIOXIDANT GENES INDUCED BY PHOTIC INJURY

Principal Investigator & Institution: Chen, Lin; Ophthalmology; University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2008

Summary: (provided by applicant): The candidate, Lin Chen, is determined to become a physician scientist and devote herself to both patient care and vision research. Her immediate career goal is to become an independent investigator. Although she has completed her graduate training in both basic science and clinical medicine, she will greatly benefit from the mentored clinical scientist development award. During the award period, she will acquire new research skills such as microarray analysis and mouse genetics, and obtain further training in the responsible conduct of research, and in communication and networking. The Scheie Eye Institute provides an ideal research environment for the candidate's career development. Dr. Chen's mentor, Dr. Jean Bennett, has a long track record of training scientists at all levels. She and the six other eye research faculty in the Kirby Center for Molecular Ophthalmology are eager to support Dr. Chen's career development. These faculty members have expertise in ophthalmic molecular biology, histology, gene transfer, animal models of retinal

Studies 49

degeneration, and animal ERG. The Kirby Center is a cohesive team of eye researchers with state-of-the-art equipment occupying an 11,000 sq. ft. floor in the heart of the Penn biomedical research community. The scientific objective of this proposal is to understand the role of oxidative stress in age-related macular degeneration, the leading cause of irreversible blindness in the elderly. Specifically, this proposal will investigate the role of natural antioxidant genes in the mouse photic injury model, a model of photo-oxidative photoreceptor injury, and in human AMD specimens. The research design will employ microarray analysis of differences in antioxidant gene expression in normal mouse retinas versus photic injury retinas and in AMD eyes versus normal agematched eyes. It will also use transgenic and knockout mice to investigate the role of ceruloplasmin, an antioxidant found in Dr. Chen's preliminary studies to be upregulated following photic injury. This information will help define the mechanisms by which the retina protects itself from oxidative stress, which is important in the pathogenesis of AMD, as demonstrated by the Age-Related Eye Disease Study. New information about the specific mechanisms of oxidative damage and natural antioxidant protection in the retina will suggest new agents for protection and treatment of patients with AMD, preserving their vision.

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

•Project Title: RETINAL DEGENERATION AND CHLORIDE CHANNELS.

Principal Investigator & Institution: Hartzell, H Criss.; Professor; Cell Biology; Emory University 1784 North Decatur Road Atlanta, Ga 30322

Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2007

Summary: (provided by applicant): The ability to read this page without magnification depends upon the integrity of the macula, a small region of the retina including the fovea. Macular degeneration is the leading cause of blindness in developed countries. Age-related macular degeneration (AMD) is a progressive degeneration of the macula that affects approximately 20% of individuals over the age of 65, but its causes remain unknown. The hypothesis driving this proposal is that CI currents play a role in phagocytosis of shed photoreceptor discs by the retinal pigment epithelium (RPE). Defects in this process can lead to macular degeneration as the result of accumulation of retinoids and lipofuscin pigment in the subretinal space. We propose that CI channels are important in normal phagocytosis because they are involved in the regulation of cell volume during ingestion of large quantities of outer segments. A variety of well-known CI channels including CFTR, CIC-2, CIC-3, and CIC-5 are expressed in RPE cells and recently it has been suggested that bestrophin, an RPE protein that causes Best macular dystrophy, is the founding member of a new family of CI channels. The goal of this project is to characterize the CI currents, especially bestrophin-mediated currents, that are expressed in RPE cells and to understand their function. There are three specific aims. (1) To determine the properties of bestrophin CI channels. We will test the hypothesis that bestrophins are subunits of a chloride channel by patch clamp analysis of heterologously expressed bestrophins. (2) To characterize chloride channels in RPE cells. This aim tests the hypothesis that several types of CI channels are functionally specialized for specific RPE functions. The strategy is to use whole-cell and patch clamp recording to characterize CI channels in RPE cells and to compare them to the properties of known CI channels, including bestrophin. (3) To determine the role of CI channels in photoreceptor disc phagocytosis. This aim will test the hypothesis that CI channels are important in phagocytosis of rod outer segments by RPE cells. This hypothesis will be tested by determining the effects of pharmacological inhibitors and antisense knockdown of CI currents on the phagocytosis of rod outer segments by RPE.

50Macular Degeneration

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

•Project Title: RETINAL GENE EXPRESSION IN LIGHT AND DARKNESS

Principal Investigator & Institution: Weitz, Charles J.; Associate Professor; Neurobiology; Harvard University (Medical School) Medical School Campus Boston, Ma 02115

Timing: Fiscal Year 2001; Project Start 01-AUG-2001; Project End 31-MAY-2004

Summary: (Applicant's abstract) Retinal sensitivity to light is controlled both by the level of illumination (light adaptation at the photoreceptor and network levels) and by an endogenous circadian clock. Considerable evidence suggests that broad and coordinated programs of gene expression underlie the changes in sensitivity driven by light and by the circadian clock, respectively, and that these programs overlap or converge in common pathways. Although there are sporadic examples of retinal transcripts regulated by light, a circadian clock, or both, there is as yet no comprehensive view of retinal gene expression in a natural light-dark cycle or in constant darkness. Microarray technology, developed for massively parallel analysis of gene expression, now permits detailed investigation of biological processes too complex for piecemeal approaches. Described here is a proposal to use cDNA microarrays to analyze gene expression in the mouse retina on a genome-wide scale. Two kinds of microarrays will be used for the analysis. The first contains 40,000 unique, fully sequenced mouse cDNAs, representing on the order of 50% of all mouse genes. The second is a custom microarray which will contain a few-thousand unique retinal cDNAs that encode secreted and transmembrane proteins, to be obtained in a large-scale "secretion trap" screen. This cDNA set will be highly enriched for molecules involved in intercellular communication, such as neuropeptides, receptors, and ion channels, known and unknown, likely to play a role in the control of retinal sensitivity. Retinal gene expression will be monitored at 4-hour intervals over two days, both from mice kept in a 24 hour light dark cycle and from mice kept in constant darkness. The incorporation of both conditions into a single experiment is a key-feature of the proposal. This experiment should provide comprehensive, parallel views of the dynamics of (i) diurnal gene expression over the course of a natural light-dark cycle and (ii) circadian clockregulated gene expression. The design will permit transcripts regulated by light to be distinguished from those regulated by a circadian clock, and it should provide insight into how the natural temporal pattern in a light-dark cycle arises from the interaction of a light-driven transcriptional program with a clock-driven one. Overlap between the two programs will likely highlight transcripts involved in changes in sensitivity of the retina to light. It is likely that a comprehensive view of natural retinal gene expression programs in light and darkness will provide deep insights into retinal physiology and open new avenues of inquiry regarding the coordinated responses of the retina to light. Aberrant transcriptional events are known to underlie light-induced photoreceptor degeneration, and knowledge of retinal gene expression programs will almost certainly stimulate progress in understanding retinal diseases such as age-related macular degeneration and retinitis pigmentosa.

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; 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

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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: ROLE OF PI3 KINASE AND ITS DOWNSTREAM TARGET BCL-XL IN RPE

Principal Investigator & Institution: Le, Yun Zheng; 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: My laboratory is interested in the roles of lipid second messengers generated in the phosphatidylinositol (PI) cycle in phototransduction, retinal degeneration, and protection of retinal cells from apoptosis. One messenger, PI-3,4,5-P3, generated by the PI-3 kinase (PI3K), plays an important role in cell protection, cell proliferation, glucose homeostasis, membrane trafficking and cytoskeletal rearrangement. In neuronal cells, activation of PI3K has been shown to protect the cells from stress-induced apoptosis. However, the functions of PI3K in visual systems are not well understood. Retinal pigment epithelial (RPE) cells are post-mitotic cells that provide the "blood-retinal barrier", and control the flow of nutrients and waste products between the retina and the blood. Death of these cells leads to death of rod and cone photoreceptor cells, and compromise in RPE function may be a contributing factor in age-related macular degeneration. Since Bcl-x, a downstream target of PI3K is highly expressed in RPE cells under cytotoxic stress, it is likely that PI3K plays an important role in RPE cell survival. We hypothesize that PI3K and BOI-xL are major survivor factors in RPE. To test this hypothesis, we will use a genetic approach to disrupt PI3K and Bcl-x (BCl-xL) in RPE,

52Macular Degeneration

and compare the functional, biochemical, and structural phenotypes of the mutant mice with those of wild type mice. Since the conventional PI3K(p85odp110o_) and Bcl-x knockout mice are neonatal or embryonic lethal, mouse models of PI3K and Bcl-x null mutation will be generated in a RPE specific fashion, using the Cre/Iox system. Therefore, I propose 1) to generate and characterize transgenic mice expressing RPEspecific Cre driven by RPE65 promoter; 2) to generate RPE-specific PI3K and/or Bcl-x null mice and test the hypothesis that PI3K and BCl-xL are involved in RPE cell survival,

3)to test the hypothesis that compromised RPE cells may exacerbate the death of photoreceptor cells with PI3K and/or Bcl-x null mutations using the RPEspecific PI3K, or Bcl-x null mice generated in this study and the ABCR null (RPE degeneration) mice. A major goal of this project in the context of the COBRE program is to establish mouse models that can be used in future studies to elucidate the mechanisms of inherent retinal degeneration.

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

•Project Title: ROS MEMBRANE PROTEINS AND RETINAL DEGENERATIVE DISEASES

Principal Investigator & Institution: Molday, Robert S.; Professor; University of British Columbia 2075 Wesbrook Pl Vancouver,

Timing: Fiscal Year 2001; Project Start 01-APR-1978; Project End 31-AUG-2005

Summary: (Adapted from applicant's abstract): Rod and cone outer segments are unique compartments of photoreceptor cells where the process of phototransduction occurs. The overall goal of this research program is to identify and characterize outer segment membrane proteins and elucidate their role in i) the visual process, ii) outer segment morphogenesis, structure, and renewal, and iii) inherited retinal degenerative diseases. The specific aims of the current grant period are as follows: 1) To study the structural, functional, and regulatory properties of ABCR, the photoreceptor ATP binding cassette (ABC) transporter of photoreceptors, and determine its cellular and subcellular distribution in rod and cone photoreceptors. 2) To develop a heterologous cell (COS-1) system to express wild-type and mutant forms of ABCR for structure-function analyses. This system will be used to determine how missense mutations in ABCR implicated in Stargardt disease alter the structure of ABCR and its possible function as a transporter. 3) To identify, clone, characterize and localize novel low abundant rod outer segment membrane proteins as a first step in determining their role in outer segment structure, function and renewal and their possible involvement in inherited retinal degenerative diseases. For these studies, a variety of current biochemical, molecular biology, cell biology and biophysical methods will be employed along with a unique panel of highly specific monoclonal antibodies and cDNAs to ABCR and novel, undefined outer segment proteins. The results of these studies should lead to new information about the morphogenesis, structure and function of rod and cone outer segments and provide insight into the role of ABCR in the visual cycle and retinal degenerative diseases including Stargardt macular dystrophy and age-related macular degeneration.

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

•Project Title: RPE65 IN RETINAL METABOLISM AND DEGENERATION

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

Timing: Fiscal Year 2001; Project Start 30-SEP-2000; Project End 31-AUG-2005

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Summary: (Adapted from the applicant's abstract): The retinal pigment epithelium (RPE) plays a critical role in the maintenance of normal photoreceptor functions and has been implicated in several visual disorders, including macular degenerations and dystrophies. The investigator has cloned and characterized the first known RPE-specific human gene, RPE65, and has shown that mutations in this gene are responsible for certain forms of autosomal recessive childhood-onset severe retinal dystrophy (arCSRD), a finding supported by reports of RPE65 defects in Leber's congenital amaurosis. A research program has been developed to study the function of RPE65 in the normal biology of the retina and in the disease state, based on the view that RPE65 is necessary for the isomerase activity involved in the conversion of vitamin A to 11-cis retinal. Four specific aims have been identified for the proposed funding period. (1) Recombinant protein-protein interactions and enzyme activity will be studied in cultured cells transfected with RPE65 expression constructs in order to distinguish between the two prevailing hypotheses about the specific role of RPE65 in RPE retinoid metabolism. (2) Site-directed mutagenesis will be used with assays of expression and protein function to elucidate the role of RPE65 mutations in the pathogenesis of arCSRD to test the hypothesis that disease-associated mutations in RPE65 result in functional null alleles that disrupt the 11-cis retinal biosynthetic pathway. This aim will include further characterization of mutations present in patient populations. (3) Because preliminary information indicates that RPE65 is down-regulated by a variety of factors that are known to be related to aging and disease processes, and because decreased levels of RPE65 are implicated in retinal degeneration, the mechanisms involved in this down-regulation will be investigated, as well as the effects of aging and other physiological conditions on RPE65 expression. (4) Effects of the RPE65 mutation in a large animal model of arCSRD will be characterized in assays of the biochemistry and enzymology of retinoid metabolism, to test the hypothesis that defects resulting from RPE65 mutations will be amenable to retinal replacement therapy. The long-term goals of this project are to elucidate the mechanisms by which RPE65 defects contribute to retinal degeneration, and to lay the groundwork for the development of therapeutic approaches to the disease.

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,

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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 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: SIBLING STUDY OF AGE-RELATED MACULAR DEGENERATION

Principal Investigator & Institution: Dryja, Thaddeus P.; Professor; Massachusetts Eye and Ear Infirmary 243 Charles St Boston, Ma 02114

Timing: Fiscal Year 2003; Project Start 30-SEP-2003; Project End 31-AUG-2008

Summary: (provided by applicant): We propose to recruit pairs of siblings for a molecular genetic search for genes having a role in the development of neovascular agerelated macular degeneration (AMD). Two types of sibling pairs will be recruited, extremely discordant sibpairs and extremely concordant sibpairs. Extremely discordant sibpairs will be composed of one member, the index sib, with neovascular AMD, and the second member with few or no aging signs of the macula at the age at which the index sibling developed neovascular AMD. Extremely concordant sibpairs will be composed of two siblings with neovascular AMD. Leukocyte DNA will be purified from blood samples collected from these siblings. The sibpairs will form the basis for a genome-wide linkage study to search for chromosomal regions where the extremely discordant pairs, on average, share fewer alleles than expected by chance alone, and where the extremely concordant sibpairs, on average, share more alleles than expected. Chromosomal regions having these properties are likely to harbor AMD genes. In addition, candidate genes that may have a role in susceptibility to AMD, such as ABCA4 and apoE, will be analyzed by evaluating DNA sequence variations in those genes and looking for a correlation between any alleles and neovascular AMD. To our knowledge, a molecular genetics approach to finding genes for neovascular AMD based on extremely discordant and extremely concordant sibpairs has not been previously carried out by any other group of investigators. If successful, our work should provide substantial progress toward identifying the genetic causes of neovascular AMD, one of the leading causes of legal blindness among the elderly.

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

•Project Title: SUBCONJUNCTIVAL ROUTE TO PROLONG CORTICOSTEROID DELIVERY

Principal Investigator & Institution: Kompella, Uday B.; Associate Professor; Pharmaceutical Sciences; University of Nebraska Medical Center Omaha, Ne 681987835

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

Studies 55

Summary: (provided by applicant): This study proposes subconjunctivally injectable biodegradable nanoand micro-particles to sustain the delivery of budesonide, a corticosteroid, to the posterior segment of the eye for a few months. The concept of continuous delivery of ultra-low amounts of budesonide to the posterior segment of the eye will significantly advance the therapy of disorders associated with difficult to reach tissues such as choroid, retina, and vitreous. Budesonide, a very potent corticosteroid with high local activity, low systemic activity, and vascular endothelial growth factor (VEGF)-inhibitory activity, is likely to find application in treating multiple inflammatory, proliferative, and neovascular disorders of the eye. The proposed study will enable the PI to begin establishing his research with this promising new therapeutic agent for ocular therapies. In this study, budesonide particles will be prepared using poly(lactic-co-glycolic acid) (PLGA), a biodegradable polymer that has been used in surgical sutures for over 30 years. The proposed research on subconjunctival budesonide-PLGA particles for prolonged budesonide delivery is likely to advance the delivery of other therapeutic agents targeted to the posterior segment. The objective of this study is to test the hypothesis that subconjunctival injection of budesonide-PLGA particles will sustain budesonide delivery to the posterior segment for up to 4 months. The specific aims of this study are: (1) To prepare and characterize biodegradable particles capable of releasing budesonide for about four months. (2) To determine whether the tissue budesonide levels increase with increasing subconjunctival dose of budesonide-PLGA(poly(lactic-co-glycolic acid) particles, without inducing lens opacities or ocular hypertension. This study entails fabrication of budesonide-PLGA particles and in vivo drug delivery studies. The proposed budesonide-delivery system is likely to benefit several disorders of the eye including proliferative vitreoretinopathy, cystoid macular edema, macular degeneration, uveitis, sarcoidosis, and scleritis. Based on this study, the PI will submit an RO-l proposal to assess subconjunctival budesonide-PLGA particles for the therapy of posterior segment disorders associated with inflammation and/or VEGF elevation.

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 2001; 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

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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: THERAPY FOR DOMINANTLY INHERITED RETINAL DEGENERATIONS

Principal Investigator & Institution: Duncan, Jacque L.; Ophthalmology; University of California San Francisco 500 Parnassus Ave San Francisco, Ca 94122

Timing: Fiscal Year 2001; Project Start 01-SEP-2000; Project End 31-AUG-2005

Summary: Retinitis pigmentosa (RP) is a heterogeneous group of hereditary retinal degenerations (RDs) that affects 1 in 3,500 people worldwide. Agerelated macular degeneration (AMD) affects as many as 1 in 4 people by the age of 75 and is the leading cause of blindness in people over age 50 in the US. There are currently no effective treatments to prevent photoreceptor degeneration and vision loss in patients with RP, or in most patients with AMD. Research efforts are being directed toward between understanding of the pathogenesis of these RDs and to develop therapies for them. The Sponsor's laboratory has recently demonstrated that subretinal injection of recombinant adeno-associated virus vectors for sustained injection of ribozymes in a rodent model of RP can delay photoreceptor loss and elevate a- and b-wave amplitudes in the ERG for at least 3 months. The relationship between rod and cone function with ribozymes, is largely unknown. The goals of the proposed experiments are to determine the duration of cone and rod photoreceptor survival and functional rescue by single and multiple administrations of ribozymes and to determine how late in the degenerative process ribozyme administration can rescue retinal function in S334ter and P23H rhodopsin mutant rat lines. These mutations in the rhodopsin gene are similar to those found in dominantly inherited human RP. We hypothesize that rescue of rod cell survival and function will also benefit cone cell survival and function. The proposed research and training plan provides enormous opportunities to help patients with both RP and AMD. The expertise the Candidate will gain using animal models of RD to develop and deliver therapies for RDs will be extremely valuable in her future career as an independent researcher.

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

•Project Title: UNIFYING GENETICS EPIDEMIOLOGY OF MACULAR DEGENERATION

Principal Investigator & Institution: Pericak-Vance, Margaret A.; Professor; Medicine; Duke University Durham, Nc 27706

Timing: Fiscal Year 2001; Project Start 15-JUN-2000; Project End 31-MAY-2005

Summary: Age-related macular degeneration (AMD) is the most common cause of severe vision loss among individuals over age 50 in the U.S. The socioeconomic impact is considerable, and is expected to become greater as the U.S. population ages. Unfortunately, treatment options remain limited because the etiology of this devastating disease remains unknown. Considerable evidence implicates a combination of genetic, environmental, and biological factors in the pathogenesis of AMD as such suggested genetic effect involves the ATP-binding transporter (ABCR) gene. We thus hypothesize that underlying susceptibility gene(s) are critical to the development of AMD, and likely

Studies 57

interact with environmental factors to trigger both the development and progression of the disease. The purpose of this study is to elucidate the genetic susceptibility to the development of AMD and to analyze the interaction of these genes with environmental influences. We will use both candidate gene and genomic screening approaches to identify these genes. Already identified genetic and environmental risk factors will be evaluated and current statistical methodologies will be adapted to examine the possible interactions. Multiplex (greater than 1 AMD affected/family) families will be recruited for the genetic analyses and detailed risk factor information will be collected on all study participants. Family-based association methods will be used to evaluate candidate genes, environmental risk factors and genegene and gene-environment interactions. The information derived from this study will further our understanding of this complex disease. The identification of specific susceptibility genes and evaluation of geneenvironment interaction will be crucial for future studies in unraveling the etiology of AMD and developing better treatments.

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

•Project Title: VISUAL CYCLE IN HUMAN PHOTORECEPTOR AND RPE DISEASE

Principal Investigator & Institution: Cideciyan, Artur V.; Ophthalmology; University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

Timing: Fiscal Year 2001; Project Start 05-AUG-2000; Project End 31-JUL-2004

Summary: (Adapted from applicant's abstract): Biochemical and physiological studies in vitro and in retina-specific ABC transporter (ABCR) -/- knockout mice suggest that ABCR accelerates recovery of rod photoreceptor resensitization after intense light exposure by transporting isomerized chromphore, all-trans-retinal, across the rod outer segment disk membrane. The current proposal is to test hypotheses about the role of ABCR in human disease as follows: (1) Study the visual cycle abnormalities in patients with retinopathy due to ABCR mutations with the goals of dissecting the contributions of primary rod effects vs. secondary disease consequences and learning the relationship between primary rod abnormalities and the genotype; (2) Investigate the basis of rod visual loss in these patients by testing the hypothesis that desensitization by equivalent light contributes to the visual loss, and determine if short term trial of unilateral light reduction can alter rod sensitivity and select mutations; (3) Test whether heterozygotes of ABCR mutations show visual cycle abnormalities and to approach from the visual function perspective the issue of ABCR sequence variance as risk factors in age related macular degeneration.

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

•Project Title: VISUAL DYSFUNCTION AND AGING--UNDERLYING MECHANISMS

Principal Investigator & Institution: Owsley, Cynthia; Professor; Ophthalmology; University of Alabama at Birmingham Uab Station Birmingham, Al 35294

Timing: Fiscal Year 2001; Project Start 01-APR-1983; Project End 31-DEC-2005

Summary: (Adapted from Applicant's abstract): A ubiquitous problem of growing old is difficulty seeing at night and under low illumination. Poor vision under reduced light levels in the elderly hinders their performance of daily activities, and has been linked to the occurrence of motor vehicle collisions and falls, which result in injury and elevated mortality risk. Even for those older adults who are free of significant eye disease there can be scotopic dysfunction, suggesting that these impairments are due to a biological aging of the visual system. Optical changes in the aged eye cannot account for the

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severity of night/low luminance vision problems, suggesting that they have a neural origin. Older adults with early age-related macular degeneration (AMD) exhibit similar night/low luminance vision problems, but theirs are typically more severe. Our longterm goal is to identify the causes of scoptic deficits in older adults and in those with early AMD so that treatment interventions to minimize or reverse these deficits can be developed thus enhancing older adults' quality of life. Studies will focus on retinal mechanisms that may underlie these impairments. The specific aims of this research plan are: (1) to identify mechanisms of scotopic sensitivity impairment and dark adaptation delays in aging and early AMD using flash electroretinography and dark adaptometry; (2) to develop a questionnaire instrument for measuring health-related quality of life that is targeted at nighttime/low luminance vision problems; and (3) to evaluate the impact of vitamin A in reversing or minimizing scotopic sensitivity and delays in dark adaptation and in enhancing vision-targeted health-related quality of life in aging and early AMD.

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 27706

Timing: Fiscal Year 2001; 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 measures. Our data are unique both in terms of the length of time over which patients are followed and in the range of health outcome monitored. (3) We will assess the impact of diabetes, glaucoma, and macular degeneration on total Medicare and Medicaid program cost and on costs privately incurred. Program cost will include cost of vision and non-vision services. In our framework, program cost and patient adherence to guidelines will be jointly determined. (4) We will replicate the analysis of the first 2 objectives for complications of diabetes other than for eyes. We will also specify and estimate a dynamic model of utilization of Medicare-covered services by persons with diabetes -- both vision and nonvision care. The main database will be the National Long-Term Care Survey (NLTCS) for 1982-2001, merged with Medicare claims data for 1982-2001. Eleven papers are planned.

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