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SURVEY OF OPHTHALMOLOGY VOLUME 47 • SUPPLEMENT 2 • DECEMBER 2002

The Potential Role of PKC  in Diabetic Retinopathy and Macular Edema Lloyd Paul Aiello, MD, PhD Harvard Medical School and Joslin Diabetes Center, Boston, Massachussetts, USA Abstract. Diabetic retinopathy is one of the most debilitating complications of diabetes mellitus. Despite major advances in understanding the pathogenesis of this disease and the efficacy of current therapies, diabetic retinopathy remains the leading cause of new-onset blindness among working-age people. The mainstay of current therapy, laser photocoagulation, is useful in preventing blindness and severe vision loss but is not often effective in restoring lost visual acuity. In addition, troublesome side effects and potentially serious complications may occur. Diabetic retinopathy is characterized by a progression of abnormalities. Nonproliferative retinopathy results from a series of biochemical and cellular changes that ultimately cause progressive retinal ischemia. The subsequent elaboration of growth factors in response to ischemia leads to the development of proliferative retinopathy, which is characterized by aberrant neovacularization of the retina—potentially leading to severe, irreversible visual loss. Increased retinal vascular leakage may also occur at any stage in this process, resulting in macular edema and possible progressive visual impairment. Although numerous biochemical factors are thought to play a role in the development of retinopathy, activation of protein kinase C (PKC), specifically the beta isoform of PKC (PKC ), is implicated for both the early and late-stage manifestations of retinopathy. Studies suggest that orally administered LY333531, a -isoform specific PKC inhibitor, may be effective in ameliorating retinopathy progression, proliferation, and retinal vascular leakage. The status of ongoing clinical trials aimed at addressing the efficacy of PKC  with regard to diabetesinduced retinal complications and perspectives on the role of PKC  are presented. (Surv Ophthalmol 47(Suppl 2):S263–S269, 2002. © 2002 Elsevier Science Inc. All rights reserved.) Key words. diabetes • diabetic retinopathy • macular edema • protein kinase C

One of the most debilitating and costly complications of diabetes mellitus is diabetic retinopathy. In the United States, DR remains the leading cause of blindness in the adult working-age population,4 despite ongoing advances in our understanding of the pathophysiology of the disease and the availability of effective sight-saving treatments such as laser photocoagulation. Diabetic macular edema is a frequent, sight-threatening complication of retinopathy that may occur at any stage of the disease.15,35 When treatment is provided in a timely and appropriate manner, up to 90% of severe

visual loss from proliferative diabetic retinopathy, and 50% of moderate visual loss from diabetic macular edema, can be prevented.4,16,17 Nonetheless, this leaves many patients at risk of blindness and severe visual loss, in addition to the large numbers of asymptomatic patients who fail to seek treatment until some loss of vision has already occurred. As we strive to preserve vision in these individuals and treat diabetic retinopathy earliest stages, interventions that target the initial subclinical changes in the microvasculature of the retina are gaining ever increasing importance.

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Treatment of Diabetic Retinopathy and Macular Edema Laser photocoagulation is the current mainstay of therapy for diabetic retinopathy and diabetic macular edema, and it is indicated for essentially all patients when retinopathy progresses to the more advanced, proliferative stage of diabetic retinopathy or to clinically significant macular edema.4,16 With laser photocoagulation, visual function is preserved to a greater extent than if left untreated, although at the expense of destruction of portions of the retina by the laser burns themselves. Photocoagulation therapy can be associated with side effects such as decreased peripheral and night vision and changes in color perception. In some instances, retinopathy continues to progress despite timely and appropriate laser photocoagulation. The side effects associated with laser therapy may become particularly troublesome as patients enter their second and third decades after treatment, presumably due to the continued expansion of the retinal scars.31,37,39 Under rare but potentially serious circumstances, complications of laser therapy may occur, including panretinal photocoagulation through the macular region, laser-induced rupture of Bruch’s membrane, laser burns to the fovea, and laser-induced lenticular burns. Although laser photocoagulation is used to treat both proliferative diabetic retinopathy and clinically significant macular edema, its effectiveness in preventing visual loss from clinically significant macular edema is less than that for proliferative diabetic retinopathy. Laser photocoagulation for macular edema prevents further moderate visual loss by only about 50% and does not usually restore vision loss that has occurred prior to treatment. Although the visual loss associated with macular edema may be less severe than that associated with proliferative diabetic retinopathy, it affects larger numbers of individuals.15,35 The drawbacks inherent to the use of laser therapy in patients with retinopathy and macular edema have fueled the search for interventions that target the disease at earlier stages, before substantive retinal damage and/or vision loss has occurred.

Progression of Diabetic Retinopathy and Macular Edema Retinopathy generally proceeds through several stages,4,6 beginning with nonclinically evident biochemical and cellular alterations, and continuing through early and late-stage complications of the disease. The preclinical stages of diabetic retinopathy include changes in cellular biochemistry, leukocyte adhesion retinal blood flow, and the loss of retinal pericytes.4,6a,8,10 Subsequently, disease becomes clinically evident through the formation of microaneurysms, retinal dot/blot hemorrhages, cotton-wool

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spots, venous beading, and vascular loops and/or reduplications.4 Morphological alterations such as these mark the nonproliferative stage of diabetic retinopathy. The ensuing progressive ischemia of the retina results in numerous cellular alterations including the release of a variety of growth factors, many of which are potent stimulators of angiogenesis. Reduction in endogenous angiogenic inhibitors may also occur.40a The balance of these factors are thought to play a major role in the onset of aberrant neovascularization, which characterizes the proliferative stage of diabetic retinopathy. The newly formed vessels, which are prone to bleeding, may result in vitreous hemorrhage. Fibrovascular contraction of the new vessels can occur and cause retinal detachment and irreversible vision loss. In these advanced stages of retinopathy, patients are also prone to develop vision loss or impairment from clinically significant macular edema, although as noted above, this complication may develop at any stage of retinopathy.

Mechanisms Underlying Diabetic Retinopathy Complications: Early and Late-Stage Abnormalities The hyperglycemia associated with diabetes mellitus is thought to be the underlying factor in the development of diabetic complications. Indeed, the incidence and progression of retinopathy has been closely correlated with glycated hemoglobin levels across numerous major epidemiologic studies.25 The early stages of retinopathy are thus attributed to the adverse effects of hyperglycemia in microvascular tissues. Elevated glucose levels lead to increased flux through the polyol pathway, the generation of advanced glycation endproducts (AGE), and the generation of reactive oxygen species.18,19,45 In turn, these processes have been reported to result in enhanced generation of diacylglycerol, a physiologic activator of the protein kinase C (PKC) pathway (Fig. 1).7,28,45 For example, chronic exposure of retinal pericytes to AGE (3 uM methylglyoxal-modified bovine serum albumin) leads to a 2-fold increase of cellular diacylglycerol.14 Increased oxidative stress can also activate PKC and, indeed, the various pathways are likely interrelated.26 PKC is a family of related enzymes which function as signaling components for a variety of growth factors, hormones, neurotransmitters, and cytokines.33 Among the different PKC isoforms, the beta isoform (PKC ) is the predominant isozyme activated in vascular tissues during hyperglycemia.20,28,40 The involvement of PKC  in these very early diabetes-induced vascular complications has raised interest in this molecule as a potential target for therapeutic inhibition. In addition, with advancing retinopathy, progressive retinal hypoxia may contribute to the elaboration of a wide array of growth modulators, resulting

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important pathogenic factor across many stages of diabetic retinopathy.

PKC Inhibition: A Potential Therapy for Diabetic Retinopathy and Macular Edema

Fig. 1. Diabetes-induced activation of protein kinase C (PKC).

in aberrant neovascularization and retinal vascular leakage. Although numerous growth factors, including insulin-like growth factors I and II and basic fibroblast growth factor, have been implicated in retinal neovascularization, experimental and clinical evidence suggests that vascular endothelial growth factor (VEGF) plays a dominant role in this process.30 Studies have demonstrated that the blockade of VEGF action is sufficient to inhibit retinal neovascularization in several experimental models of ischemic retinopathy.1,5,34,36 Experimental studies have further demonstrated that while the cellular signaling process for VEGF is complex, one critical component, particularly in terms of its mitogenic and permeability-inducing effects, is the activation of PKC .3,46 PKC has also been implicated in the stimulation of VEGF expression, although this is not observed in all studies. Thus, through increases in diacylglycerol hyperglycemia may activate PKC and possibly contribute the early stages of diabetic retinopathy. In advanced disease where VEGF levels are elevated, PKC may play an important role in mediating this growth factor’s action. If PKC also mediates VEGF expression, then a reinforcing cycle could arise. These findings therefore suggest that activation of PKC  may be an

There are at least 13 different isoforms of the PKC family, which are broadly grouped into three subcategories, classical, novel, and atypical, depending upon the organization of their catalytic and regulatory domains.33,43 PKC  falls into the classical category, in that its activation is dependent upon the release of intracellular calcium.28, 33 The activation of PKC may result in a number of effects on the vasculature that are characteristic of those seen in retinopathy. These include changes in smooth muscle contractility and increases in basement membrane protein synthesis, endothelial permeability, and angiogenesis.7,22,28 PKC activation may also result in the elaboration of cytokines,7,28 including vasoactive factors such as endothelin and angiogenic factors such as transforming growth factor-beta and VEGF. Interest in suppressing these processes in diseases such as retinopathy has resulted in the study of a wide array of PKC-inhibitory compounds, with varying degrees of isoform selectivity (Table 1).22,43 Rottlerin, for example, inhibits the delta PKC isoform with some selectivity; however, it is also an inhibitor of other important enzymes, including protein kinase A and calmodulin. Indolocarbazoles also inhibit multiple PKC isoforms as well as non-PKC enzymes. PKC412 is a potent inhibitor of VEGF and inhibits , , and  PKC isoforms as well as the kinase domain-containing receptor component of the VEGF receptor and the platelet derived growth factor receptor beta chain.38 LY333531 is a highly selective inhibitor for the PKC  isoform and has remarkably little effect on unrelated enzymes (Table 2).23 The selectivity of the LY333531 compound might theoretically reduce the potential for side effects when used systemically in patients.

TABLE 1

Protein Kinase C Inhibitors Inhibitor Rottlerin Indolocarbazoles PKC412 Bisindolylmaleimides LY333531

Characteristics Natural product; some protein kinase C (PKC)  selectively, also inhibits PKA, calmodulin, and casein kinase II General PKC inhibitor, also inhibits phosphorylase kinase, PKA, myosin light chain kinase, endothelial growth factor (EGF), and p60src (UCN01, CGP412521, Go6976) N-benzoylstaurosporine, partially selective kinase inhibitor, inhibits vascular EGF, plateletderived growth factor, and c-kit receptor phosphorylation (phase I trials) Less potent than indolocarbazoles, greater selectivity for PKC, some with PKC , , or  isoform selectivity (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9: 16,21-dimetheno-1H, 13Hdebenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione, PKC  selective inhibitor (phase III trials)

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Surv Ophthalmol 47 (Suppl 2) December 2002 TABLE 2

Kinase Selectivity of LY333531 Kinase PKC  PKC I PKC II PKC  PKC  PKC  PKC  PKC PKA Ca calmodulin Casein kinase src-tyrosine kinase Rat brain PKC

IC50 (nM) 360 4.7 5.9 300 250 600 100,000 52 100,000 6,200 100,000 100,000 3,200

PKC protein kinase C. Smaller numbers represent greater selectivity. Adapted from Jirousek.23

LY333531—Experimental Studies One of the earliest changes of retinopathy to occur, even before the onset of clinical symptoms, is a change in retinal blood flow.4,8 In an experimental rat model of diabetes where retinal mean circulation time is increased, the change was evident when untreated control diabetic animals were compared to their nondiabetic counterparts (Fig. 2). In contrast, in animals fed LY333531 for a 2-week period, the effect of diabetes on retinal circulation time was abrogated (Fig. 2).21 Similarly, the effects of growth factors on retinal permeability may also be ameliorated by the inhibition of PKC .46 As shown in Fig. 3, intravitreal treatment of rats with concentrations of VEGF observed in the human eye during active diabetic retinopathy increases retinal permeability, as assessed by vitreous fluorescein leakage.46 In rats orally administered LY333531, however, the ability of VEGF to increase permeability was effectively blocked after

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1 week of treatment with the inhibitor (Fig. 3).46 Similar studies have shown that LY333531 can prevent the diabetes-induced retinal vascular leakage observed after as little as 2 weeks of diabetes in rats. These experimental findings suggest that the inhibition of PKC  may be an effective means of blocking some of the earliest pathologic changes in the retina (i.e., changes in retinal blood flow and permeability). In a sophisticated series of experiments, Suzuma and colleagues have begun to assess the role of PKC  in a model of ischemia-induced retinopathy that effectively mimics the vascular changes characteristic of proliferative diabetic retinopathy. Fig. 4 shows retinal cross sections of transgenic mice which were engineered to overexpress PKC 2 in the endothelium of their vasculature.41 Ischemia-induced retinopathy in control animals causes a neovascularization of the retina that is characteristic of that observed with this model (Fig. 4A). In contrast, transgenic, PKC  overexpressing animals display a markedly increased neovascularization response to the ischemia (Fig. 4B). These results suggest that activation of this PKC isoform has an important role in neovascularization of the retina. This is also suggested by the fact that animals with a targeted disruption (‘knock-out’) of the PKC 1 and 2 genes29 have a markedly diminished neovascularization response to the same ischemic insult (Figs. 4C and 4D).41 In each case, the changes between the control and experimental groups were statistically significant.41 Inhibition of PKC  using LY333531 produces similar results in a pig model of neovascularization using branch retinal vein occlusion as the ischemic insult.11 Treatment of these animals with LY333531 over 3 months suppressed the neovascularization induced by the retinal vein occlusion.11 These studies suggest that inhibition of PKC  using LY333531 may also be an effective means of blocking the new vessel formation characteristic of the later, proliferative stages of diabetic retinopathy. The encouraging results in these and other experimental studies have led to an evaluation of LY333531 in the clinical setting as a potential preventive therapy for retinopathy and macular edema in diabetic patients.

LY333531—Clinical Studies

Fig. 2. LY333531 and retinal mean circulation time. (Reprinted with permission from Ishii et al.21 Copyright 1996 American Association for the Advancement of Science.)

A number of phase I studies of LY333531 have been conducted using single and multiple doses in a variety of patient populations, including the elderly and patients with diabetes.12,13 Results of these studies have shown the compound to be readily bioavailable. Reporting of adverse events has been generally similar between placebo and LY333531 groups, and no clinically relevant changes in EKG or other parameters have been noted.12,13 Preliminary pharmacodynamic data have also been obtained in some of

PKC  AND DIABETIC RETINOPATHY AND MACULAR EDEMA

S267 Fig. 3. Protein kinase C  inhibition and retinal permeability. VEGF vascular endothelial growth factor. (Reprinted with permission from Aiello et al.3 Copyright 1997 American Diabetes Association.)

these studies. In a randomized, double-masked, placebo-controlled, parallel study, patients with type 1 or type 2 diabetes ( 10 years duration) and no or very mild retinopathy were treated with either LY333531 or placebo over the course of 1 month.2 At this stage of their disease, these patients exhibit decreased retinal blood flow. In the placebo-treated patients, retinal mean circulation time, an inverse indicator of retinal blood flow, was abnormal, as is usually observed in patients with diabetes of this duration. In contrast, these changes were normalized in patients treated with LY333531 in a dose-dependent fashion, reaching statistical significance at the 32 mg daily dose.2 Similarly, the changes in retinal blood flow observed in these patients were also normalized by treatment with LY333531,2 although the trend did not reach statistical significance in this small study.

In summary, phase I studies of LY333531 have shown the compound to be well tolerated in patients with type 1 or type 2 diabetes for a period of up to 30 days and at doses of up to 32 mg/day. Thus far, the drug does not appear to be associated with any undue adverse events, and early results suggest that it may ameliorate some of the retinal vascular dysfunctions characteristic of diabetes. A number of studies are being planned to investigate the ability of LY333531 to slow or halt the progression to proliferative diabetic retinopathy and slow or reverse the progression of macular edema. Two phase II/III studies evaluating these endpoints have just been completed and no concerns have been raised by the independent safety monitoring board to date. Results of these important trials are expected in mid 2003. Phase III trials have been initiated and planning for additional studies is under way.

Fig. 4. Neovascularization and protein kinase C (PKC) : retinal cross sections in genetically engineered mice. (Reprinted from Suzuma et al.41 Copyright 2002 National Academy of Sciences, USA.)

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Conclusions Diabetic retinopathy and diabetic macular edema continue to be major causes of visual impairment in patients with diabetes. Vascular changes as a result of diabetes-associated hyperglycemia are evident in the retina even before the onset of clinical findings or symptoms of either diabetic retinopathy or diabetic macular edema. Laser photocoagulation, although clearly beneficial, is not usually effective in restoring lost vision and is most useful only in the later stages of disease, after irreversible retinal pathology has already occurred. Side effects and potentially serious complications of laser photocoagulation, as well as the need for repeated treatments, underscore the need for therapies that can target these diseases at an earlier stage and in a nondestructive manner. The enzyme PKC, specifically the beta isoform, appears to play an important role in mediating the adverse effects of hyperglycemia in susceptible tissues. The targeted inhibition of PKC  is now possible using agents such as LY333531, which appear thus far to have minimal interference with normal metabolism. Experimental studies using LY333531 over the last 6 years indicate that the compound can effectively inhibit certain pathological changes associated with retinopathy, including retinal permeability and new vessel formation. Early phase I trials in humans have shown LY333531 to be well tolerated at therapeutic doses. Preliminary pharmacodynamic results suggest that the drug is indeed effective in ameliorating some of the vascular dysfunction characteristic of early retinopathy. These findings continue to support the possibility that PKC  inhibition may slow or reverse the progression of diabetic retinopathy and diabetic macular edema. In addition, preliminary studies suggest that inhibition of PKC  may benefit diabetes-induced neuropathy,9,24,44 nephropathy27 and cardiovascular disease.32,42 If proven effective for these indications in ongoing and future clinical trials, the availability of PKC  selective inhibitors such as LY333531 may add a powerful new therapeutic option to our present armamentarium against the debilitating retinal disease of diabetes, and perhaps benefit other complications of diabetes as well.

References 1. Adamis AP, Shima DT, Tolentino MJ, et al: Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate. Arch Ophthalmol 114:66–71, 1996 2. Aiello LP, Bursell S, Devries T: Protein kinase C beta selective inhibitor LY333531 ameliorates abnormal retinal hemodynamics in patients with diabetes. Diabetes 48:A19, 1999 3. Aiello LP, Bursell SE, Clermont A, et al: Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor. Diabetes 46:1473–80, 1997

AIELLO 4. Aiello LP, Gardner TW, King GL, et al: Diabetic retinopathy. Diabetes Care 21:143–56, 1998 5. Aiello LP, Pierce EA, Foley ED, et al: Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc Natl Acad Sci USA 92:10457–61, 1995 6. American Diabetes Association: Diabetic retinopathy. Diabetes Care 23(Suppl 1):S73–S76, 1993 6a. Barouch FC, Miyamoto K, Allport JR, et al. Integrin mediated neutrophil adhesion and retinal leukostasis in diabetes. Invest Ophthamol Vis Sci 4:1153–8, 2000. 7. Bursell S-E, King GL: Can protein kinase C inhibition and vitamin E prevent the development of diabetic vascular complications? Diab Res Clin Pract 45:169–82, 1999 8. Bursell SE, Clermont AC, Kinsley BT, et al: Retinal blood flow changes in patients with insulin-dependent diabetes mellitus and no diabetic retinopathy. Invest Ophthalmol Vis Sci 37:886–97, 1996 9. Cameron NE, Cotter MA: Effects of protein kinase Cbeta inhibition on neurovascular dysfunction in diabetic rats: interaction with oxidative stress and essential fatty acid dysmetabolism. Diabetes Metab Res Rev 18:315–23, 2002 10. Cogan DG, Toussaint D, Kubawara T: Retinal vascular patterns. IV. Diabetic retinopathy. Arch Ophthalmol 66:100–12, 1961 11. Danis RP, Bingaman DP, Jirousek M, Yang Y: Inhibition of intraocular neovascularization caused by retinal ischemia in pigs by PKCbeta inhibition with LY333531. Invest Ophthalmol Vis Sci 39:171–9, 1998 12. Demolle D, de Suray JM, Onkelinx C: Pharmacokinetics and safety of multiple oral doses of LY333531, a PKC beta inhibitor, in healthy subjects. Clin Pharmacol Ther 65:189, 1999 13. Demolle D, de Suray JM, Vandenhende F, Onkelinx C: LY333531 single escalating oral dose study in health volunteers. Diabetologia 41(Suppl 1):A354, 1998 14. Denis U, Lecomte M, Paget C, et al: Advanced glycation endproducts induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction. Free Radic Biol Med 33:236– 47, 2002 15. Ferris FL, Patz A: Macular edema. A complication of diabetic retinopathy. Surv Ophthalmol 28(Suppl):452–61, 1984 16. Ferris FL 3rd: How effective are treatments for diabetic retinopathy? JAMA 269:1290–1, 1993 17. Ferris FL 3rd, Davis MD, Aiello LM: Treatment of diabetic retinopathy. N Engl J Med 341:667–78, 1999 18. Friedman EA: Advanced glycosylated end products and hyperglycemia in the pathogenesis of diabetic complications. Diabetes Care 22(Suppl 2):B65–71, 1999 19. Giugliano D, Ceriello A, Paolisso G: Oxidative stress and diabetic vascular complications. Diabetes Care 19:257–67, 1996 20. Inoguchi T, Battan R, Handler E, et al: Preferential elevation of protein kinase C isoform beta II and diacylglycerol levels in the aorta and heart of diabetic rats: differential reversibility to glycemic control by islet cell transplantation. Proc Natl Acad Sci USA 89:11059–63, 1992 21. Ishii H, Jirousek MR, Koya D, et al: Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor. Science 272:728–31, 1996 22. Ishii H, Koya D, King GL: Protein kinase C activation and its role in the development of vascular complications in diabetes mellitus. J Mol Med 76:21–31, 1998 23. Jirousek MR, Gillig JR, Gonzalez CM, et al: (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-d ione (LY333531) and related analogues: isozyme selective inhibitors of protein kinase C beta. J Med Chem 39:2664–71, 1996 24. Kamei J, Mizoguchi H, Narita M, Tseng LF: Therapeutic potential of PKC inhibitors in painful diabetic neuropathy. Expert Opin Investig Drugs 10:1653–64, 2001 25. Klein R, Klein BE, Moss SE, et al: Glycosylated hemoglobin predicts the incidence and progression of diabetic retinopathy. JAMA 260:2864–71, 1988

PKC  AND DIABETIC RETINOPATHY AND MACULAR EDEMA 26. Kowluru RA: Diabetes-induced elevations in retinal oxidative stress, protein kinase C and nitric oxide are interrelated. Acta Diabetol 38:179–85, 2001 27. Koya D, Haneda M, Nakagawa H, et al: Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes. FASEB J 14:439–47, 2000 28. Koya D, King GL: Protein kinase C activation and the development of diabetic complications. Diabetes 47:859–66, 1998 29. Leitges M, Schmedt C, Guinamard R, et al: Immunodeficiency in protein kinase cbeta-deficient mice. Science 273: 788–91, 1996 30. Miller JW, Adamis AP, Aiello LP: Vascular endothelial growth factor in ocular neovascularization and proliferative diabetic retinopathy. Diabetes Metab Rev 13:37–50, 1997 31. Morgan CM, Schatz H: Atrophic creep of the retinal pigment epithelium after focal macular photocoagulation. Ophthalmology 96:96–103, 1989 32. Naruse K, King GL: Protein kinase C and myocardial biology and function. Circ Res 86:1104–6, 2000 33. Nishizuka Y: Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258: 607–14, 1992 34. Ozaki H, Seo MS, Ozaki K, et al: Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization. Am J Pathol 156:697–707, 2000 35. Patz A, Schatz H, Berkow JW, et al: Macular edema—an overlooked complication of diabetic retinopathy. Trans Am Acad Ophthalmol Otolaryngol 77:OP34–42, 1973 36. Robinson GS, Pierce EA, Rook SL, et al: Oligodeoxynucleotides inhibit retinal neovascularization in a murine model of proliferative retinopathy. Proc Natl Acad Sci USA 93: 4851–6, 1996 37. Rutledge BK, Wallow IH, Poulsen GL: Sub-pigment epithelial membranes after photocoagulation for diabetic macular edema. Arch Ophthalmol 111:608–13, 1993 38. Seo MS, Kwak N, Ozaki H, et al: Dramatic inhibition of retinal and choroidal neovascularization by oral administration of a kinase inhibitor. Am J Pathol 154:1743–53, 1999

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39. Shah SS, Schachat AP, Murphy RP, Fine SL: The evolution of argon laser photocoagulation scars in patients with the ocular histoplasmosis syndrome. Arch Ophthalmol 106:1533– 6, 1988 40. Shiba T, Inoguchi T, Sportsman JR, et al: Correlation of diacylglycerol level and protein kinase C activity in rat retina to retinal circulation. Am J Physiol 265:E783–93, 1993 40a. Spranger J, Osterhoff M, Reimann M, et al. Loss of the antiangiogenic pigment epithelial derived factor in patients with angiogenic eye disease. Diabetes 50:2641-5, 2001. 41. Suzuma K, Takahara N, Suzuma I, et al: Characterization of protein kinase C beta isoforms action on retinoblastoma protein phosphorylation, vascular endothelial growth factorinduced endothelial cell proliferation, and retinal neovascularization. Proc Natl Acad Sci USA 99:721–6, 2002 42. Wakasaki H, Koya D, Schoen FJ, et al: Targeted overexpression of protein kinase C beta2 isoform in myocardium causes cardiomyopathy. Proc Natl Acad Sci USA 94:9320–5, 1997 43. Way KJ, Chou E, King GL: Identification of PKC-isoform-specific biological actions using pharmacological approaches. Trends Pharmacol Sci 21:181–7, 2000 44. Way KJ, Katai N, King GL: Protein kinase C and the development of diabetic vascular complications. Diabet Med 18: 945–59, 2001 45. Ways DK, Sheetz MJ: The role of protein kinase C in the development of the complications of diabetes. Vitam Horm 60: 149–93, 2000 46. Xia P, Aiello LP, Ishii H, et al: Characterization of vascular endothelial growth factors effect on the activation of protein kinase C, its isoforms, and endothelial cell growth. J Clin Invest 98:2018–26, 1996

The author has received travel reimbursement from Eli Lilly & Company for presentation expenses. Reprint address: Lloyd Paul Aiello, MD, PhD, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215.