Ординатура / Офтальмология / Английские материалы / Retinal Vascular Disease_Joussen, Gardner, Kirchhof_2007

7. Early Treatment Diabetic Retinopathy Study Research

fundus photographic screening population. Invest Ophthalmol Vis Sci 44:767 – 771

11.van Hecke MV, Dekker JM, Stehouwer CD, Polak BC, Fuller JH, Sjolie AK, Kofinis A, Rottiers R, Porta M, Chaturvedi N (2005) EURODIAB prospective complications study: Diabetic retinopathy is associated with mortality and cardiovascular disease incidence: the EURODIAB prospective complications study. Diabetes Care 28(6):1383 – 1389

12.Wilkinson CP, Ferris FL 3rd, Klein RE, Lee PP, Agardh CD, Davis M, Dills D, Kampik A, Pararajasegaram R, Verdaguer JT (2003) Global Diabetic Retinopathy Project Group: Proposed international clinical diabetic retinopathy and diabetic macular edema disease severity scales. Ophthalmology 110(9):1677 – 82

13.Zoega GM, Gunnarsdottir T, Bjornsdottir S, Hreietharsson AB, Viggosson G, Stefansson E (2005) Screening compliance and visual outcome in diabetes. Acta Ophthalmol Scand 83(6):687 – 90

Core Messages

Diabetes-induced degeneration of retinal capillaries during the early (nonproliferative) stages of diabetic retinopathy appears to contribute to the later progression of the retinopathy. Thus, inhibiting the degeneration of retinal capillaries is likely to be a meaningful therapeutic target to inhibit visual loss in diabetes

All laboratory animal species tested to date have been found to develop at least the early microvascular stages of diabetic retinopathy, but progression to the more advanced lesions has been less common

Multiple diverse therapies have been found to inhibit the early stages of diabetic retinopathy in animals, and anti-inflammatory effects seem to be common to a number of the therapies

Diabetic retinopathy is today the leading cause of acquired blindness among young adults throughout the developed world. It classically has been regarded as a disease of the microvasculature of the retina, and the natural history of the disease has been divided into an early, nonproliferative (or background) stage, and a later, proliferative stage. Nonproliferative diabetic retinopathy currently is diagnosed ophthalmoscopically based on the presence of retinal vascular abnormalities, including dilation of retinal veins, retinal microaneurysms, intraretinal microvascular abnormalities (which include intraretinal new vessels), areas of capillary nonperfusion, retinal hemorrhages, cotton wool spots (infarctions within the nerve fiber layer), edema, and exudates. Proliferative diabetic retinopathy is diagnosed based on the presence of new vessels on the surface of the retina. The resulting preretinal new vessels are the major cause of vitreous hemorrhage and consequent visual loss. Retinal edema is the other major contributor to visual impairment in diabetes.

Available evidence suggests that abnormalities that occur in the early stages of the retinopathy underlie the progression of the ocular disease, ultimately leading to neovascularization. Molecular mechanisms involved in the neovascular response, and development of therapies to inhibit this process, are major areas of research interest at present, but are beyond the scope of this chapter. In this chapter, we will focus on the use of animal models to investigate the pathogenesis of the early, nonproliferative stages of diabetic retinopathy and therapeutic approaches

to the inhibition of the retinopathy that have come out of such research. This review will be restricted to in vivo studies.

19.1.1.1Early Stages of Diabetic Retinopathy: Histopathology

Histologically, the early stages of diabetic retinopathy in man and animals are characterized by the presence of saccular capillary microaneurysms, pericyte-deficient capillaries, and obliterated and acellular capillaries [41]. Pericyte loss is evident as an excessive number of pericyte “ghosts” on viable capillaries, the “ghost” referring to a pocket in basement membrane that formally was occupied by the pericyte. Acellular capillaries apparently were functional capillaries that degenerated until only a basement membrane tube remains. Acellular, degenerate capillaries are not perfused, and are regarded as histologic markers of nonperfused capillaries. Although devoid of nuclei, these degenerate vessels sometimes are not truly acellular, and may be filled with cytoplasmic processes of glial cells [48]. Whether the invasion of retinal capillaries by glia in diabetes is secondary to capillary degeneration, or whether it initiates vessel occlusion and degeneration, is not known.

Capillary occlusion initially occurs in single, isolated capillaries, and in the early stages has no clinical consequences. As more and more capillaries become occluded, however, the retina presumably becomes ischemic, causing elaboration of one or

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Fig. 19.1.1.1. Retinal microvasculature of diabetic patient (isolated by the trypsin digest technique). Ma microaneurysm, narrow arrow pericyte ghost, wide arrow acellular capillary

more growth factors, such as vascular endothelial growth factor (VEGF). Thus, capillary vaso-oblitera- tion and the resulting increase in acellular capillaries appears to represent a discrete event that progressively contributes to the development of retinal ischemia, and ultimately neovascularization. Mechanisms believed to contribute to the capillary occlusion and/or obliteration in diabetes include: (1) occlusion of the vascular lumen by white blood cells or platelets [18, 75, 78], and (2) death of cells of the vessel wall as a result of exogenous (cytokines, etc.) or endogenous abnormalities within the vascular cells themselves.

Retinal capillary endothelial cells and pericytes have been found to die by an apoptotic-like (TUNEL-positive) process in diabetic humans, and in diabetic or experimentally galactosemic rats [122]. The rate of death of these cells in hyperglycemic rats becomes significantly greater than normal at about 6 – 8 months of disease, but is not demonstrable sooner. Although statistically greater than normal, the number of TUNEL-positive capillary cells detected in the retina of diabetic rats is quite small at any given duration of diabetes. For example, diabetes of 8 months duration resulted in only 9±6 TUNEL-positive capillary cells in diabetic rats [122]. This is not unexpected, since apoptotic cells are known to be rapidly cleared by phagocytosis [37]. Although the duration that dying cells are able to be stained using the TUNEL technique is known to be short (less than 1 day), it is still reasonable to ask whether or not the cumulative effect of this small number of cells undergoing apoptosis over a period of months (in rodents) or years (in humans) is sufficient to account for the capillary degeneration that is characteristic of diabetic retinopathy. The ability of a therapy to inhibit diabetes-induced apoptosis of capillary cells has been found to pre-

dict the ability of the therapy to inhibit the degeneration of retinal capillaries [95], thus suggesting that the apoptotic process is an important component in the pathogenesis of the retinopathy.

Damage to nonvascular cells of the retina (including ganglion cells) in diabetic humans also has been detected histologically [17], and functionally [22], and a possible role of the neural disease in the pathogenesis of diabetic retinopathy has been postulated [20]. Consistent with a possible role of apoptosis in the death of retinal neurons, numerous initiator and effector caspases have been found to become activated in the retina of diabetic animals or in retinal cells incubated in elevated glucose concentration [123]. Compared to capillary cells, more neurons become TUNEL-positive or caspase 3-positive in retinas of diabetic rats, and do so at a faster rate [14]. As with capillary cells, however, the rate of neuronal death also is very small compared to the total number of neurons.

Apoptosis might not be the only form of cell death occurring in diabetic retinopathy. Joussen and coworkers [75] have reported that only 9 days of diabetes caused an increase in intracellular accumulation of propidium iodide (commonly used as a marker of necrosis) in the retinal vasculature. Thus, focusing solely on apoptotic cell death might underestimate the total number of cells dying at any time. Nevertheless, propidium iodide likely overestimates necrosis, since it has been found to enter even viable cells having impaired membrane integrity [151].

The basement membrane that surrounds retinal capillaries thickens in diabetes, and had been postulated to play a role in development of the retinopathy in previous decades. This view had become less popular in recent years, but that now needs to be reexamined again in light of recent findings that the degeneration of retinal endothelial cells and pericytes in galactose-fed rats can be significantly inhibited by inhibiting synthesis of fibronectin, a component of the basement membrane [145].

Although all vascular cells should be exposed to the same concentration of blood glucose in a given individual, there is unexplained regional variability in susceptibility to diabetes-induced microvascular disease between embryologically similar tissues, and even within the same retina. Microvessels isolated from cerebral cortical of dogs with either diabetes or experimental galactosemia of 5 years duration possessed none of the microaneurysms, acellular capillaries, and pericyte ghosts that occurred in retinal vessels of the same animals [91]. Similar conclusions have been reached in diabetic humans [42]. In addition, microaneurysms and acellular capillaries have been found to develop in a nonuniform distri-

bution even within the same retina in diabetic patients [33] and in experimentally diabetic or galactosemic dogs, lesions being more common in the superior and temporal portions of the retina [89]. Likewise, neovascularization in diabetic patients has been noted to be more common in the superior and temporal portions of the retina than in other regions [155]. Currently available hypotheses regarding the pathogenesis of diabetic retinopathy do not account adequately for the geographically unequal distribution of the microvascular disease, and suggest that local factors are important. The only biochemical abnormality identified to date that shows a similar nonuniform distribution across the retina in patients (and thus might contribute to the development of the histopathology) is a diabetes-induced increase in activity of the proinflammatory caspase, caspase 1 [154].

19.1.1.2Animal Models of the Early Stages of Diabetic Retinopathy

Many animal species have been studied as possible models of diabetic retinopathy, and lesions that have been found to develop in various models are summarized in Table 19.1.1.1. The early stages of diabetic retinopathy have been found to develop in all mammalian species studied who have had diabetes for relatively long durations. Each of the different species have been found to have their own advantages and disadvantages, so their use will depend on desired endpoints and experimental design. Most of the studies of diabetic retinopathy in animals to date have focused on insulin-deficient models (type 1 diabetes), as opposed to models of insulin resistance (type 2 diabetes).

Studies in humans have shown considerable similarities in the retinopathy that develops in the different types of diabetes [99]; and in animals, retinal lesions have been found to be the same whether due to experimental induction of diabetes (alloxan, streptozotocin, growth hormone, pancreatectomy) or spontaneous diabetes (unpublished). Likewise, experimental elevation of blood hexose level by feeding galactose has resulted in development of a dia- betic-like retinopathy in dogs [52, 53, 80, 81, 152], rats [59, 87, 142, 143, 164], and mice [90].

The morphologic similarity of retinal lesions in diabetes and the experimental galactosemia has suggested that the two retinopathies share a common final pathway leading from elevated blood hexose level to the retinal vascular disease [53]. Consistent with this, both diabetesand galactose-induced retinopathies have been inhibited in rodents by antioxidants [108] or deletion of ICAM [76], but the retina in galactosemia differed from diabetes with respect

Table 19.1.1.1. Diabetes-induced retinal histopathology in various species

to activation of proapoptotic caspases [123] and in the ability of aminoguanidine to inhibit capillary cell death and degeneration [95].

19.1.1.2.1 Dogs and Cats

Diabetic dogs have been shown repeatedly to develop morphologic lesions of the retinopathy that are indistinguishable from those of background retinopathy seen in diabetic patients, including capillary microaneurysms, acellular (and nonperfused) capillaries, pericyte ghosts, varicose and dilated capillaries [or intraretinal microvascular abnormalities (IRMAs)] [48]. The lesions in diabetic dogs can be inhibited by strict regulation of glycemia with exogenous insulin [50]. Microaneurysms, leukocyte and platelet plugging of vessels, and degenerating endothelial cells likewise were observed in cats after several years of diabetes [71]. Capillary aneurysms, degeneration of capillary cells, and vaso-obliteration usually do not begin to appear in these animals until about 2 – 3 years after induction of diabetes. The cost, slow development of lesions, and lack of availability of antibodies or molecular biology techniques have made dog and cat models less suitable for current studies to understand molecular mechanisms involved in the development of diabetic retinopathy. Neovascularization has been observed to develop within the retina in long-term diabetic dogs, but pre-retinal neovascularization has not been detected.

19.1.1.2.2 Rats

The streptozotocin-diabetic rat has been the primary model for research into the pathogenesis of the vascular lesions of diabetic retinopathy [87]. A signifi-

18 months of diabetes) [58]. Diabetes also has been reported to cause loss of retinal ganglion cells in Ins2Akita and C57Bl/6J mice [13]. In contrast to the reported loss of retinal ganglion cells from C57Bl/6 mice after only 14 weeks of diabetes [116], however, diabetic C57Bl/6 mice in our hands did not show detectable loss of ganglion cells by any of three independent methods (number of cells in ganglion cell layer of retinal cross-sections, retrograde labeling of

retinal ganglion cells with fluorescent dye, or TUNEL staining) during up to 1 year duration of diabetes [58].

Genetically modified mice now are being used to explore the pathogenesis of the retinopathy in diabetic mice. Mice deficient in the genes encoding for the leukocyte adhesion molecules CD18, ICAM-1, and lipid regulation have been used to study the pathogenesis of diabetic retinopathy [76], and studies involving numerous other genes (including iNOS, PARP, COX2) are ongoing (Kern, unpublished). The principal advantages of mice are cost, availability of reagents, and ability to generate genetically modified animals for study. The principal disadvantages of mice for studies of the retinopathy are the small size of the retina (and consequently the quantitatively small number of lesions that can be detected per retina), and the difficulty in unambiguously identifying and quantifying pericytes and pericyte “ghosts.” Diabetes-induced retinal neovascularization has not been detected in any purely diabetic mouse model to date.

19.1.1.2.4 Primates

Microaneurysms and retinal hemorrhages were detected in primates after 10 – 15 years of chronic hyperglycemia [19, 47, 110], as were retinal ischemia, other lesions, alterations in the blood-retinal barrier, and maculopathy [73, 74, 97, 98, 160]. The main advantages of primates in the study of diabetic retinopathy are: (1) the presence of a macula, thus allowing investigation of macular edema, a major cause of visual impairment in diabetes, and (2) the likelihood that many antibodies and molecular probes generated against human samples will work also on the primates. The major disadvantages of this model are the cost and slow development of lesions. Pre-retinal neovascularization has not been detected in diabetic primates.

19.1.1.3Insulin Therapy to Inhibit Development of Retinopathy

Intensive insulin therapy has been shown to inhibit development of vascular lesions of diabetic retinopathy in patients [43], dogs [50], and rats (transplanted with exogenous islets [64]). Therapeutically, improved glycemic control is less effective at inhibiting the development of retinopathy if initiation of the therapy is delayed; lesions of the retinopathy were observed to continue to develop for at least some interval after elimination of elevated blood hexose level in diabetic patients [43], diabetic dogs [55] and rats [150], and in some, but not all, studies of galactosemic animals [57, 82, 138]. A possible biochemical basis of this continued progression of retinopathy

even after elimination of hyperglycemia has been suggested in studies of diabetic rats and galactosemic rats where oxidative stress and nitric oxide production remained significantly greater than normal even months after elimination of hyperglycemia [104].

19.1.1.4Is Diabetic Retinopathy a Chronic Inflammatory Disease?

Retinas from diabetic animals exhibit biochemical and physiological abnormalities which, in composite, have features that include inflammatory processes. In retinas of diabetic animals, induction of iNOS and COX-2, as well as increase in nitric oxide and prostaglandins [24, 25, 44, 45, 125], have been reported, presumably as a consequence of a diabetesinduced translocation of NF-κB to the nucleus of retinal cells [176]. Inhibition of COX2 has been reported to inhibit the diabetes-induced upregulation of retinal VEGF [12] and increase in retinal vessel permeability [78], and to inhibit death of retinal endothelial cells cultured in diabetic-like concentrations of glucose [44]. Less selective cyclo-oxygenase inhibitors have inhibited development of the retinopathy in diabetic dogs and rodents [92], as well as the increase in vascular permeability in diabetic rodents [78]. Blocking FasL in vivo has been shown to prevent endothelial cell damage, vascular leakage, and platelet accumulation in diabetes, indicating that the Fas/ FasL system can contribute to the diabetes-induced damage that leads to development of the retinopathy [77]. Increases in TNF- and IL-1 levels have been shown in the vitreous of diabetic patients and in retinas of STZ-induced diabetic rodents [78]. Diabetic mice genetically deficient in TNF- have been reported in an abstract to be protected from galac- tose-induced retinopathy [111]. Activity of caspase- 1, the enzyme responsible for interleukin-1b production, is increased in retinas of diabetic mice and galactose-fed mice, and diabetic humans, and retinal Müller cells incubated in elevated glucose concentration [123].

Leukostasis is an important component of the inflammatory process, and adhesion of leukocytes to the vascular wall has been found to be significantly increased in retinas of diabetic animals [75]. Investigators have reported that diabetes increased expression of ICAM-1 (intracellular adhesion molecule-1) in retina [78] and interaction of this protein with the CD18 adhesion molecule on monocytes and neutrophils contributed to the diabetes-induced increase in leukostasis within retinal vessels [120]. Leukostasis has been postulated to be an important factor in death of retinal endothelial cells in diabetes. Consistent with this postulate, mice that are genetically deficient in either ICAM or CD18 developed signifi-

kinase C inhibitors [6], tyrosine kinase inhibitors [124], aspirin [78], a cyclooxygenase 2 inhibitor [78], steroids [153], VEGF receptor antagonist, and TNF- receptor antagonists [78].

Taken together, these numerous defects in diabetes are consistent with a diabetes-induced inflammatory response in the retina. Interestingly, the stimulus for these inflammatory-like changes appears to be glucose itself, since many of these changes can be detected in retinal cells incubated in vitro in diabetic-like concentrations of glucose. Thus, as long as the blood sugar remains elevated, this inflammatory response is inappropriately maintained, and apparently contributes to the development of tissue damage.

19.1.1.5 Therapies

The concept that diabetic retinopathy includes aspects of an inflammatory disease suggests new therapeutic strategies and targets at which to inhibit the retinopathy. A variety of therapies reported are listed below, listing their effects on inflammatory changes in the retina where possible. Their postulated targets are summarized in Fig. 19.1.1.2 and Table 19.1.1.2.

Available diabetic animal models

Table 19.1.1.2. Inhibition of diabetes-induced retinal injury in animals

aControversial [56]

19.1.1.5.1Insulin and Other Glucose-Lowering Drugs

The best evaluated therapeutic agent that inhibits the development of diabetic retinopathy is insulin or other glucose-lowering drugs. This group of agents have been found to inhibit development and pro-

gression of retinopathy in type 1 diabetes, in type 2 diabetes, and in diabetic dogs [50]. Recent studies have demonstrated a pro-survival action of insulin on retinal cells independent of its effect on glucose concentrations [137].

19.1.1.5.2 Aspirin and Salicylates

In 5-year studies of diabetic dogs, aspirin significantly inhibited the formation of acellular capillaries and retinal hemorrhages but was less effective on microaneurysms and pericyte ghosts in those animals [92]. Prospective clinical trials to assess the possible effect of aspirin on diabetic retinopathy in patients have yielded contradictory results. Aspirin treatment resulted in a statistically significant (although weak) inhibition of the mean yearly increase in the number of microaneurysms in the DAMAD trial [36], whereas no beneficial effect was observed on any aspect of retinopathy in the ETDRS trial [46]. The lack of effect of aspirin in the ETDRS likely is attributable, in part, to the greater severity of retinopathy at the onset than in the DAMAD trial, especially since animal and clinical studies have shown that retinopathy tends to resist arrest once it is initiated [55]. Perhaps more importantly, both patient studies utilized doses of aspirin that were less than those used in the dog studies. The concentration of aspirin used in the dog study was moderate (22 mg/kg BW), consistent with doses used to treat arthritis in patients (equivalent to approximately 1.5 g of aspirin per day in a 70 kg patient), whereas doses used in the DAMAD trial and ETDRS were lower.

The ability of three different nonsteroidal anti-in- flammatory drugs (aspirin, sodium salicylate and sulfasalazine) to inhibit the development of the retinopathy recently was examined in experimentally diabetic rats [175]. Moderate doses of each of these salicylate derivatives significantly inhibited the dia- betes-induced increase in retinal capillary cell death and formation of acellular capillaries. Aspirin is known to inhibit production of prostaglandins, but salicylate has much less of this activity, suggesting that the common action of aspirin, sodium salicylate and sulfasalazine to inhibit retinopathy was not primarily mediated by inhibition of prostaglandins. Each of these salicylates can inhibit the NF-κB pathway (which is involved in the pathogenesis of the inflammatory response), and evidence indicates that the drugs did indeed inhibit activation of the NF-κB, as well as retinal expression of ICAM, iNOS, COX2 (gene products regulated by NF-κB dependent transcription). These data demonstrate that moderate doses of salicylates inhibit early lesions of diabetic retinopathy, seemingly via inhibition of NF-κB, and are consistent with the premise that diabetic retinopathy is a chronic inflammatory disease.

19.1.1.5.3 Cyclooxygenase Inhibitor

Nepafenac is a potent inhibitor of cyclooxygenases that can be applied in eye drops, and meloxicam is a selective inhibitor of COX-2. Both compounds have been found to inhibit diabetes-induced leukocyte adhesion in retinal vessels of diabetic rats [78]. Meloxicam also reduced eNOS levels, inhibited NF-κB activation in the diabetic retina, and modestly, but significantly, reduced TNF- levels in the retina; but its effect on histologic lesions of diabetic retinopathy was not studied. Long-term topical administration of nepafenac significantly inhibited the diabetesinduced increase in the number of TUNEL-positive capillary cells, acellular capillaries, and pericyte ghosts [94].

19.1.1.5.4 Eternacept

Eternacept is a soluble TNF- receptor that acts as competitive inhibitor to block effects of TNF- binding to cells. Eternacept reduced leukocyte adherence in all retinal blood vessel types of rats diabetic for 1 week compared to control [78]. Eternacept did not reduce retinal VEGF levels, but it inhibited bloodretinal barrier breakdown and NF-κB activation in the diabetic retina. No effects of the therapy on histologic lesions of the retinopathy were reported in diabetes, but mice genetically deficient in TNF- have been reported in an abstract to be protected from galactose-induced retinopathy [111].

19.1.1.5.5 Aminoguanidine

diabetes-induced polyol accumulation in retina [56]. Effects of aldose reductase inhibitors have been controversial in galactose-fed rats [142] and dogs [81]. A deficiency in studies of the polyol pathway to date is that extent to which the pathway was inhibited in the retina has been assessed only based on steady-state levels of polyol, not on in vivo measurement of flux

19 III through the pathway. Moreover, many studies have not made any effort to demonstrate the extent to which the pathway was inhibited in the retina in vivo, so that data from different studies are difficult to compare.

19.1.1.5.8Antisense Oligonucleotides Against Fibronectin

Thickening of retinal capillary basement membrane and overexpression of basement membrane components are closely associated with the development of diabetes-induced vascular disease. Intravitreal injection of antisense oligonucleotides targeted against fibronectin was found to significantly reduce fibronectin mRNA and protein level in retina of galactosefed rats, to partially inhibit thickening of the retinal capillary basement membrane, and to inhibit formation of acellular capillaries in this galactosemic model [145].

19.1.1.5.9 Benfotiamine

Administration of benfotiamine to the diabetic rats for 36 weeks significantly inhibited the development of acellular capillaries in retinas of diabetic rats [68]. This lipid-soluble thiamine derivative was used since it is known to activate transketolase. The authors postulated that benfotiamine should convert the accumulated fructose-6-phosphate and glyceralde- hyde-3-phosphate into pentose-5-phosphates, thereby diverting sugar metabolism away from the impairment in glycolysis. Consistent with this hypothesis, they demonstrated that benfotiamine significantly inhibited several hyperglycemia-induced abnormalities including activities of protein kinase C and the hexosamine pathway and formation of advanced glycation endproducts. Which of these (or other) pathways is the ultimate cause of the microvascular pathology in the retina of these diabetic rats remains to be clarified.

19.1.1.5.10 Blood Pressure Medications

Clinical studies have detected a beneficial effect of ACE inhibitors on diabetic retinopathy. The EUCLID Study Group investigated the effect of ACE inhibitor, lisinopril, on retinopathy in normotensive type 1 diabetic patients. In that 24-month study, retinopa-

thy was found to have progressed by at least one level in 23.4 % of control patients and in only 13.2 % of patients treated with lisinopril [30]. Thus, ACE inhibitor therapy resulted in a 50 % reduction in the progression of the retinopathy. The UKPDS clinical trial likewise revealed that the ACE inhibitor, captopril, inhibited a two-step progression of retinopathy by 34 % over a 9-year period [161]. The observed inhibition of retinopathy by ACE inhibitors remains unexplained. Corrosion cast and SEM studies of Otsuka Long-Evans Tokushima Fatty (OLETF) diabetic rats showed that cilazapril therapy inhibited diabetes-induced changes in capillary tortuosity, caliper and density in long-term studies [16]; and a recent abstract reported that captopril dramatically inhibited the early stages of diabetic retinopathy (capillary degeneration) [173].

19.1.1.5.11 Nerve Growth Factor

Administration of nerve growth factor significantly inhibited the diabetes-induced obliteration of retinal capillaries in rats [63] and mice (Kern, unpublished). The mechanism of this action remains under investigation, but one possibility under investigation is the known induction of antioxidant enzymes by nerve growth factor [62, 129, 130, 146, 177].

19.1.1.5.12 Protein Kinase Inhibitors

Inhibitors of protein kinase C have been demonstrated to inhibit a variety of diabetes-induced biochemical and physiological abnormalities in the retina [6], but they have not yet been reported to inhibit diabe- tes-induced histopathology of the retinal vasculature in animal models. LY333531 inhibited diabetesinduced increases in leukostasis and decreases in retinal blood flow in rats [1]. Clinical trials using this inhibitor have been evaluated in advanced stages of diabetic retinopathy (where the results did not achieve statistical significance) [133]. Less selective inhibitors of protein kinases have been found to inhibit diabetes-induced increases in retinal permeability [124].

19.1.1.5.13 Pyridoxamine

Pyridoxamine was found to significantly inhibit the diabetes-induced dropout of retinal capillaries at 29 weeks of diabetes in rats [149]. Simultaneously, it inhibited upregulation of laminin protein and mRNA for extracellular matrix proteins in the retina. Pyridoxamine is an inhibitor of the formation of advanced glycation endproducts and lipoxidation endproducts.

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