Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

20 to 25% fewer cells in the ganglion cell layer compared with age-matched control mice. There was a modest, but significant, decrease in the thickness of the whole retina and the INLs and outer nuclear layers (ONLs) in mice that had been diabetic for 10 wk. TUNEL analysis and detection of active caspase-3 revealed that cells of the ganglion cell layer were dying by apoptosis. Electron microscopic analysis detected morphologic features characteristic of apoptosis, including margination of chromatin and crenated nuclei of cells in the ganglion cell layer. These data suggest that as with the diabetic rat, neurons in the ganglion cell layer of diabetic mouse retinas die and this death occurs through an apoptotic pathway. The observation is an important one given the abundance of transgenic mice available that can permit analysis of various genetic contributions to diabetic retinopathy. It is probably not surprising that ganglion cells are affected in diabetic mice given the findings of Kowluru (53) that metabolic changes in retinas of diabetic mice were quite similar to those of diabetic rats. Both species showed increased oxidative stress, protein kinase C (PKC) activity, and NO levels in the retina. To determine whether other factors associated with diabetes can exaggerate the neuronal cell death, we have exploited a mouse model with a defect in the cystathionine-β-synthase gene, which has an elevation of plasma homocysteine. Elevated levels of homocysteine have been reported in patients with diabetic retinopathy (54,55). Preliminary studies suggest that inducing diabetes in these mice leads to increased death of cells in the ganglion cell layer and alterations of the nerve fiber layer (56).

Ganglion cell loss has been reported in other mouse models of diabetes. One of the most promising models to be characterized recently is the spontaneously diabetic Ins2Akita mouse (57). This mouse carries a spontaneous mutation of one insulin gene and heterozygous mice develop diabetes within 4 to 5 wk after birth. The mice have been studied through 28 wk morphometrically and by TUNEL analysis. The morphometric analysis revealed approx 23% fewer cells in the ganglion cell layer of affected mice, in addition to significant decreases in the thickness of the IPL and the INL. There is increased apoptosis and accelerated loss of neurons in the retina of Ins2Akita mice. The investigators contend that these changes parallel and extend observations of neuronal degeneration in streptozotocin-diabetic rats and postmortem tissue from humans with diabetes and may explain the loss of vision in diabetic retinopathy.

Apoptosis of cells in the ganglion cell layer has been reported also in a mouse model for type 2 diabetes. Ning and colleagues (58) investigated neuroretinal apoptosis in the KKAY mouse and reported significantly more TUNEL positive cells in the ganglion cell layer than in control mice. Many apoptotic cells were observed in the inner portion of the INL as well. The investigators also reported microangiopathy in early stage of diabetes.

A number of proteins, thought to be involved in the pathogenesis of diabetic retinopathy, especially the angiogenic component, have been investigated and several of these proteins have been localized to the retinal ganglion cell layer. Included among this group is IGF-1, a growth-promoting polypeptide that can act as an angiogenic agent in the eye. IGF-1 is thought to play a central role in the pathogenesis of proliferative diabetic retinopathy. Interestingly messenger RNA (mRNA) encoding IGF-1 and its receptor is expressed throughout the neural retina, including retinal ganglion cells, as well as vascular, Müller and retinal pigment epithelial (RPE) cells (59). The angiopoietin (Ang)/Tie-2 system, also thought to play a role in vascular integrity and

angiogenesis, has been analyzed in diabetic retinas (60). Molecular methods were used to study the expression of Ang-1, Ang-2, and Tie-2 in the retinas of streptozotocininduced diabetic rats. Results of in situ hybridization analysis revealed Ang-1, Ang-2, and Tie-2 mRNA expression in the ganglion cell layer and the INL. In particular, Ang- 2 mRNA expression increased in the ganglion cell layer and was accompanied by a reduction of α smooth-muscle actin-positive perivascular cells. These changes may suggest a role for Ang-2 in the mechanism of pericyte loss in diabetic retinopathy. CTGF has been postulated to have prosclerotic and angiogenic properties and has been characterized also in the diabetic rat model (61). In comprehensive molecular studies, diabetes was associated with a greater than twofold increase in CTGF mRNA levels and the major site of CTGF gene and protein expression in the retina of diabetic rats was the ganglion cell layer. The authors theorized that ciliary neurotrophic factor (CNTF) plays a key role in mediating diabetes-associated retinal pathology. Platelet-derived growth factor (PDGF) has been studied in diabetic retina and found to be expressed in the ganglion cell layer (62). Until recently, the source of PDGF was not known, but studies from the Gardiner group have confirmed ganglion cells as the principal source of PDGF. In situ hybridization studies using streptozotocin diabetic mice demonstrated that PDGF-A and -B were predominantly expressed by the retinal ganglion cells/nerve fiber layer in both normal and diabetic mice, and this localization pattern did not alter in diabetes. Interestingly, there was a significant decrease of PDGF-B mRNA levels in diabetic retina when compared to nondiabetic controls, which may have significant implications for the vascular complications of diabetic retinopathy. Expression of vascular endothelial growth factor (VEGF), implicated in retinal neovascularization, is upregulated in ganglion cells, the IPL and cells of the INL in studies of retinas of human patients with diabetic retinopathy (63). Others have demonstrated that advanced glycation end products (AGEs) can increase VEGF mRNA levels in the ganglion, INLs and RPE cell layers of the rat retina (64). The data lend support to a role for AGEs in the pathogenesis of diabetic retinopathy through their ability to increase retinal VEGF gene expression.

In addition to proteins associated with angiogenesis, other groups have studied various proteins that might be linked with diabetes. Among these studies is work showing that glutamate receptors and calcium binding proteins are increased in diabetes (65). The vulnerability of neuronal cells to excessive levels of glutamate, coupled with the observation by several groups that glutamate levels may be increased in diabetes (7–9) prompted the study of expression of glutamate receptors and calcium-binding proteins in streptozotocin-induced diabetic rats. Upregulation of glutamate receptors (N-methyl- D-aspartate receptor [NMDAR]1 and GluR2/3) was observed in the ganglion, amacrine, and bipolar cells as well as in the IPL and outer plexiform layer at 1 mo of diabetes and was further enhanced at 4 mo. Immunoreactivity of calcium-binding proteins (calbindin and parvalbumin) was also increased. The data suggest changes on glutamate and calcium metabolism in the diabetic retina.

Owing to observations of ganglion cell death in diabetes, our group has been interested in neuroprotective proteins that are expressed in diabetes. Among these we have analyzed the expression of the type 1 sigma receptor (σR1) in retinas of diabetic mice (66). σR1 is a nonopiate and nonphencyclidine binding site that has numerous pharmacological and

physiological functions. In particular, agonists for σR1 have been shown to afford neuroprotection against overstimulation of the NMDAR. We analyzed whether ganglion cells cultured under hyperglycemic conditions and ganglion cells of diabetic mice continue to express σR1. Reverse-transcriptase (RT)-polymerase chain reaction (PCR), in situ hybridization, Western blot analysis, and immunolocalization studies confirmed that ganglion cells cultured under hyperglycemic conditions continue to express σR1 as do ganglion cells of diabetic mice. Studies are currently underway to determine whether ligands for σR1 may prove useful in inhibiting the apoptosis observed in diabetic retinopathy.

AMACRINE, BIPOLAR, AND HORIZONTAL CELLS

The cell type most studied within the INL of the retina during diabetes is the Müller cell. The three neuronal cell types of this layer, amacrine, bipolar, and horizontal have not been investigated extensively in diabetic retinopathy. This may be in part because of the fact that Müller cells have distinctive radial fibers and are also positive for GFAP and other markers allowing them to be analyzed more easily. There are known markers for bipolar, amacrine, and horizontal cells now available and as investigators use these it will be possible to determine how extensively these neuronal cell types are involved in diabetes. There certainly have been studies reporting that cells of the INL undergo apoptosis (34) and reporting that the thickness of this layer is reduced in diabetic retinopathy (34,52,57,67). Further, reports that the IPL, which is the synaptic layer between the neurons of the INL and the ganglion cell layer, is reduced in diabetic retinopathy (34), are highly suggestive of involvement of neurons in the INL during the disease.

That said, the literature in this area is sparse compared to that for Müller or ganglion cells. Studies of the diabetic rat models have revealed a 22 and 14% reduction in the thickness of the IPL and INL, respectively (34). This study did not examine which cell types of the INL might have been lost during the disease. Studies with the Ins2Akita mice revealed a reduction of the IPL of 17 and 27% in the central vs peripheral retinas, respectively (57). Additionally, the INL was reduced by 16% in these diabetic mice. In this study, a marker of starburst amacrine cells, choline acetyltransferase, revealed significantly fewer subtypes of this amacrine cell in the diabetic mice than controls. Studies from streptozotocin-induced diabetic mice showed a reduced thickness of the INL, though specific cell types that were lost were not reported (52). Seki and colleagues reported that dopaminergic amacrine cells degenerate in diabetic rat retinas, as revealed by reduction in tyrosine hydroxylase immunoreactivity (68). The levels of this protein in diabetic retinas were decreased to one-half of controls. Sophisticated electrophysiological studies of diabetic rat retinas by Kaneko and coworkers evaluated the scotopic threshold response and the oscillatory potential of the ERG (69). Their data showed a significantly prolonged mean latency of the oscillatory potential, which reflects altered function of dopaminergic amacrine cells. These data suggest disordered neurotransmission of the amacrine cells in the inner retinal layers of diabetic rats. In studies by Park and colleagues (67), postsynaptic processes of horizontal cells in the deep invaginations of the photoreceptors of streptozotocin diabetic rats showed degenerative changes within 1 wk of diabetes onset. Some amacrine cells and a few horizontal cells showed necrotic features at 12 wk postonset of diabetes. Agardh and co-workers

have observed a decrease in the numbers of horizontal cells and decreased branching of their terminals in diabetic rats (70). Studies of proteins whose expression might be altered in neurons of the INL in diabetic retinopathy have been quite limited, although Ng and colleagues showed that amacrine and bipolar cells alter their expression of glutamate receptors and calcium-binding proteins (71).

PHOTORECEPTOR CELLS

Alterations in photoreceptor cells have been reported in the diabetic retina. In a 2003 study using streptozotocin-induced diabetic rats, which were not maintained on insulin, Park et al. (67) reported a remarkable reduction in the thickness of the ONL 24 wk after the onset of diabetes. TUNEL analysis of these retinas showed some apoptotic nuclei at 4 wk postonset of diabetes and this number increased as diabetes progressed. The authors suggest that visual loss associated with diabetic retinopathy could be attributed to an early phase of substantial photoreceptor loss, in addition to later microangiopathy. Nork used histological methods to distinguish L/M cones from S-cones in humans with diabetic retinopathy and found “selective and widespread” loss of the S-cones was found in diabetic retinopathy (72). The investigator concluded that in diabetic retinopathy the acquired tritan-like color vision loss could be caused, or contributed to, by selective loss of the S-cones. Landenranta et al. (73) point out that PDR is rarely associated clinically with retinitis pigmentosa (RP). They note than humans with diabetic retinopathy demonstrate a spontaneous regression of retinal neovascularization associated with long-standing diabetes mellitus when RP becomes clinically evident. They test this theory using the rd mouse (rd1/rd1), which has a marked reduction of photoreceptor cells within the first 3 wk of postnatal life. When the rd mice are subjected to conditions of oxygen-induced proliferative retinopathy they fail to mount reactive retinal neovascularization. Their data would suggest that marked photoreceptor cell loss, characteristic of RP is incompatible with PDR.

RETINAL PIGMENT EPITHELIUM

The RPE is a remarkable cell type with numerous critical functions that maintain the health and integrity of the retina, particularly the adjacent photoreceptor cells. It not only plays a key role in retinoid metabolism and the regeneration of rhodopsin, it also phagocytoses shed outer segment photoreceptor disks, contains pigment granules thought to reduce light scattering, and mediates the transport of numerous nutrients. Owing to tight junctions between cells, the RPE contributes to the blood retinal barrier. Glucose transport across this barrier is mediated by the sodium-independent glucose transporter GLUT1. Alterations in the blood–retinal barrier may affect glucose delivery to the retina and may have major implications in the development of two major diabetic complications, namely insulin-induced hypoglycemia and diabetic retinopathy (74). Thus, it is not surprising that studies of the role of RPE, especially during proliferative stages have been reported. In addition to the glucose transporter, another transporter has been examined in cultured RPE and in retina/RPE of diabetic mice. This work, from our laboratory, explored the regulation of a transporter for the essential vitamin folate under diabetic conditions. Folate is essential for synthesis of RNA, DNA, and proteins

and is thus critical for cell survival. Our lab characterized the polarized distribution of two folate transport proteins in RPE (75) and then studied the regulation of reducedfolate transporter (RFT-1), under hyperglycemic conditions (76). Exposure of RPE cells to 45 mM glucose for as short an incubation time as 6 h resulted in a 35% decrease in methyltetrahydrofolate (MTF) uptake. Kinetic analysis showed that the hyperglycemia-induced attenuation was associated with a decrease in the maximal velocity of the transporter with no significant change in the substrate affinity. Semiquantitative RT-PCR demonstrated that the mRNA encoding RFT-1 was significantly decreased in cells exposed to high glucose, and Western blot analysis showed a significant decrease in protein levels. The uptake of (3H)-MTF in RPE of diabetic mice was reduced by approx 20%, compared with that in nondiabetic, age-matched control animals. Semiquantitative RT-PCR demonstrated that the mRNA encoding RFT-1 was decreased significantly in RPE of diabetic mice. These findings demonstrated for the first time that hyperglycemic conditions reduce the expression and activity of RFT-1 and may have profound implications for the transport of folate by RPE in diabetes. Stevens and colleagues reported a similar finding of downregulation of the human taurine transporter by glucose in cultured RPE cells (77). The implications for decreased taurine are potentially serious owing to the apparent role of this amino acid as an osmolyte and as an antioxidant.

Other laboratories have examined various proteins in RPE during diabetes to attempt to understand the role of RPE in the pathogenesis of diabetic retinopathy. Rollin et al. (78) examined atrial natriuretic peptide (ANP), an endogenous inhibitor of the synthesis and angiogenic action of VEGF, in plasma and vitreous humor of human subjects with PDR. They found that vitreous ANP concentrations were significantly higher in patients with active PDR compared to patients with quiescent PDR, diabetes without PDR, or controls. ANP was detected in the fibrovascular epiretinal tissue of patients with PDR. They concluded that patients with diabetes with active neovascularization have significantly higher levels of ANP in the vitreous humor than those without active PDR. Patients with diabetes without PDR were also found to have significantly higher vitreous ANP levels than nondiabetic patients. In the fibrovascular epiretinal tissue of these patients, ANP was localized to vascular, glial, fibroblast-like, and RPE cells.

One of the treatments used commonly in PDR is thermal laser photocoagulation. Framme and Roider (79) used fundus autofluorescence imaging to study the RPE of nearly 190 human subjects who had undergone the laser photocoagulation. Initially, autofluorescence decreases in the area of laser lesions 1 h after laser treatment, but more than 1 mo posttreatment, it increases. The authors point out the usefulness of this method in evaluating RPE destruction and subsequent proliferation after continuous wave laser photocoagulation. A zone of atrophy noted as dark spots can signify denaturation of neurosensory retinal tissue. Further studies (80) suggest that autofluorescence imaging may replace invasive fluorescein angiography in many cases to verify therapeutic laser success and reveal extent of RPE damage.

Bensaoula and Ottlecz have evaluated the RPE of the diabetic rats using electron microscopic methods (81). Their ultrastructural studies revealed a significant deepening of basal infoldings in the RPE and a noticeable increase in the size of the extracellular

space between the basal infoldings of 5-mo streptozotocin-induced diabetic rats. They suggest that this increase extracellular space may contribute to increased alteration of the RPE barrier function in progressive diabetes.

ENDOTHELIAL CELLS

The retina receives blood from two sources: the choroid, which supplies the outer retina, and the central retinal artery, which ramifies on the inner region of the retina. Within the vasculature of the inner retina, the capillary network is distributed in a superficial network within the nerve fiber layer and an intraretinal network at the level of the INL. The capillaries of the INL tend to be more involved in diabetes. Two cell types are found in the capillaries. The endothelial cells, the cytoplasm of which makes up the lining or wall of the capillaries, and the pericytes, which are on the surface of the capillaries. The effects of diabetes on protein expression and function of retinal endothelial cells have been studied extensively. This review highlights some of the more recent reports (within the last 3 yr) of the effects of diabetes on retinal endothelial cells.

In an effort to understand the pathogenesis of diabetic retinopathy, especially the proliferative phase, a number of investigations have been performed to determine whether hyperglycemia induces potentially damaging molecules in retinal endothelial cells. Members of the Kern laboratory reported that hyperglycemia increases NO and PGE(2) in endothelial cells. They believe that this increase in these molecules contributes to the pathogenesis of diabetic retinopathy (15,82). Additional studies from this group examined the role of poly(ADP-ribose) polymerase (PARP) in the development of diabetic retinopathy (83). Activity of PARP increased in whole retina and in endothelial cells and pericytes of diabetic rats. They found that inhibition of PARP or nuclear factor (NF)-κB inhibited the hyperglycemia-induced cell death in retinal endothelial cells. Thus, PARP activation plays an important role in the diabetes-induced death of retinal capillary cells, at least in part via its regulation of NF-κB. Similar results were obtained by Kowluru and co workers (84), who demonstrated that high glucose activates NF-κB and elevates NO and lipid peroxides in both retinal endothelial cells and pericytes. They further reported that the effects could be inhibited by antioxidants. Additional studies from the Kowluru laboratory examined the role of advanced AGEs in accelerating retinal capillary cell death under in vitro conditions using bovine retinal endothelial cells and pericytes (85). Her studies incubating these cells with AGE-bovine serum albumin showed a 60% increase in oxidative stress and NO. She proposed that inhibiting AGEs in the retinal capillary cells could prevent their apoptosis, and perhaps ultimately the development of retinopathy in diabetes. Other studies from this laboratory have examined the cytokine IL-1β and have determined that it too accelerates apoptosis of retinal capillary cells via activation of NF-κB (86), and showed that the process is exacerbated in highglucose conditions.

Interesting studies published in 2005 by members of the Grant and Scott laboratories (87) have shown that stromal cell-derived factor (SDF)-1 increases as PDR progresses and it induces human retinal endothelial cells to increase expression of vascular cell adhesion molecule-1, a receptor for very late antigen-4 found on many hematopoietic progenitors. They found also that SDF-1 reduced tight cellular junctions by reducing occludin expression. The investigators used a mouse model of proliferative retinopathy and found that the

new vessels that developed originated from hematopoietic stem cell-derived endothelial progenitor cells. SDF-1, when injected intravitreally in mice can induce retinopathy. The authors theorized that SDF-1 plays a major role in proliferative retinopathy and may be an ideal target for the prevention of proliferative retinopathy.

Kondo and colleagues have also exploited mice to study the pathogenesis of neovascularization. They used mice in which the insulin receptor or the IGF-1 receptor was eliminated from endothelial cells and found decreased neovascularization in these experimental animals (88,89). They conclude that insulin and IGF-1 signaling in endothelium play a role in retinal neovascularization through the expression of vascular mediators, with the effect of insulin being most important in this process.

Other studies have focused on the expression and function of the sodium dependent glucose transporter, GLUT1 in endothelials cells. Fernandes et al. (90) studied GLUT1 in endothelial cells cultured under conditions of experimental diabetes and reported that levels were decreased in the in vivo models and in the retinal endothelial cells exposed to elevated glucose concentrations. The decreased abundance of GLUT1 may be associated with its increased degradation by an ubiquitin-dependent mechanism. Electron microscopic immunohistological methods have also been used to study GLUT1 transporter in retinas of diabetic rats (91), but no difference in immunoreactivity for GLUT1 was observed between control and diabetic retinas. In contrast, Zhang et al. (92) reported that high glucose actually increased GLUT1-mediated glucose transport in cultured retinal endothelial cells.

Interesting studies have been reported recently about the effects that leptin, an adipocytederived hormone, might have on endothelial proliferation and angiogenesis (93). Suganami and colleagues used a mouse model of ischemia and demonstrated more pronounced retinal neovascularization in 17-d-old transgenic mice overexpressing leptin than in agematched wild-type littermates. Interestingly, the ischemia-induced retinal neovascularization was markedly suppressed in 17-d-old leptin-deficient ob/ob mice. This suggests that leptin can stimulate ischemia-induced retinal neovasucularization possibly through the upregulation of endothelial VEGF, thereby suggesting that leptin antagonism may offer a novel therapeutic strategy to prevent or treat diabetic retinopathy. Chibber and coworkers have demonstrated that PKC β2-dependent phosphorylation of core 2 GlcNAc- T promotes leukocyte-endothelial cell adhesion (94). They suggest that this may represent a novel regulatory mechanism in mediating increased leukocyte-endothelial cell adhesion and capillary occlusion in diabetic retinopathy.

Recent studies have reported that oncofetal fibronectin may play a role in the proliferation of endothelial cells during diabetic retinopathy. Khan et al. studied the expression of oncofetal fibronectin variants in human vitreous samples obtained from patients undergoing vitrectomy for PDR (95). They also used an animal model of diabetes and cultured endothelial cells in their work and showed that diabetes-induced upregulation of oncofetal fibronectin is, in part, dependent on hyperglycemia-induced transforming growth factor-β1 and endothelin-1. Their data may suggest that oncofetal fibronectin is involved in endothelial cell proliferation.

An interesting comparative study by Grammas and Riden (96) showed that retinal endothelial cells are more susceptible to oxidative stress and increased permeability than brain-derived endothelial cells. They reported that retinal endothelial cells release

higher levels of superoxide, have less glutathione peroxidase activity, and lower levels of superoxide dismutase and ZO-1 than brain-derived endothelial cells. Unlike brainderived endothelial cells in which ZO-1 levels increase in response to glucose, in retinal endothelial cells, ZO-1 levels are unaffected by glucose. These findings suggest that greater oxidative stress and lower-junctional protein levels in retinal endothelial cells may contribute to blood/retinal barrier dysfunction in diabetic retinopathy.

Hammes and colleagues (97) have studied the role of pericyte function in control of endothelial cell proliferation. They used PDGF-B (PDGF-B[+/–] mice) and studied pericyte numbers. They showed that retinal capillary coverage with pericytes is crucial for the survival of endothelial cells, particularly under stress conditions such as diabetes. When VEGF levels are high, as in diabetic retinopathy, pericyte deficiency may contribute to endothelial proliferation. Others have reported that the adhesive characteristics of pericytes are impaired when they are cultured with endothelial cells that have been exposed to high-hexose concentrations (98). These data suggest considerable interactions of endothelial cells and adjacent pericytes in maintaining capillary integrity during diabetes.

Thus, the studies of endothelial cells and their associated pericytes have focused primarily on various factors that induce their death or their proliferation during diabetes. The availability of the bovine retinal endothelial cell line has facilitated these studies in much the same way that availability of the Müller cell line and isolated primary Müller cells has made the dissection of the role of that cell type possible in diabetes.

SUMMARY

It is clear from the literature reviewed above that every cell type in the retina has been reported to be affected to some degree in diabetic retinopathy. Certain cell types, such as endothelial cells, are obvious cell types to study owing to the dramatic vascular changes that appear particularly later in diabetes. The availability of endothelial cells for in vitro studies has made the analysis of these cells very feasible. Similarly, the availability of a Müller cell line (18), a ganglion cell line (RGC-5) (99), and a RPE cell line (ARPE-19) (100) make studies of the effects of hyperglycemia on these cell types not only very doable, but likely to yield informative results. Other cell types such as amacrine, horizontal, bipolar have not been cultured to purity and no cell lines have yet been developed to ask similar questions about them. The recently available antibody markers for these cell types should yield more data about the consequences of diabetes on their function. A few studies of photoreceptor cells in diabetes have provided intriguing results that deserve repetition. The availability of the 661W photoreceptor cell line (101) will permit more thorough investigation of the susceptibility of these cells to hyperglycemia.

The importance of the availability of these cells for study and of various animal models for analysis is the information they may be able to provide regarding the pathogenesis of diabetes. Which cell type or types is/are the first to be affected by diabetes is not known. Many studies have focused on the long-term consequences of diabetes, but fewer studies have probed in models of diabetes precisely which cells are affected at the outset of diabetes. An alternative approach could be to determine which genes encoding which proteins are the first to be affected in diabetes. The field of diabetes research is already

benefiting from data generated heretofore about dysfunction of various cells and it is predicted that sophisticated molecular and cell biological methods will lead to important discoveries in the next few years.

ACKNOWLEDGMENT

I thank those present and former members of my laboratory, H. Naggar, P. Martin, B. Mysona, T. Van Ells, S. Ola, A. El-Sherbeny, and P. Roon, who worked diligently in trying to understand the consequences of diabetes on various functions of several retinal cell types. I also thank my colleague and collaborator, Dr. Vadivel Ganapathy, for the many useful discussions over the past several years concerning diabetic retinopathy. Studies from our laboratory described in this review were supported by National Institutes of Health, National Eye Institute: EY014560, EY12830, and EY13089.

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