Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

pathology in the retina. This study showed that although leukocyte/monocyte adhesion in the retina is related to oxidative stress rather than directly to hyperglycemia, it can be prevented using either a non-specific PKC

inhibitor, d-α-tocopherol, or the PKCβ-specific inhibitor ruboxistaurin (RBX).38

6.2Changes in basement membrane and ECM

Another change observed early in the course of diabetes is the thickening of capillary basement membranes.7 This thickening results from an increased deposition of ECM and leads to alterations in vascular permeability as well as cellular adhesion, proliferation, differentiation, and gene expression.22 Documented changes in diabetic basement membranes include increases in type IV and VI collagen and increases in fibronectin and laminin.48-51 PKC inhibitors, such as staurosporine and calphostin, act to prevent glucosestimulated transcription of collagen IV in cultured mesangial cells.52 Phorbol ester and other PKC agonists stimulate type IV collagen and fibronectin expression.52,53

PKC may mediate the glucose-induced overexpression of ECM components through its effects on transforming growth factor β (TGFβ) and angiotensin-II.54 It has been shown that the glucose-induced activation of PKC is a key component of the process by which TGFβ stimulates the production of type IV collagen, fibronectin, and laminin in cultured mesangial cells.55 A possible mechanism by which hyperglycemia increases

TGFβ is via the regulatory action of PKC on the transcription factors c-fos and c-jun.56,57 These factors are proto-oncogenes that regulate gene

transcription through the AP-1 binding site.58 Several studies have established that the AP-1 binding sequence is common to the promoter regions of TGF β1,59 fibronectin,60 and laminin.61

6.3Vascular permeability and angiogenesis

Diabetic retinal and renal vessels are markedly more permeable than their nondiabetic counterparts to macromolecules such as albumin.62 Research suggests that PKC isoforms may play an important role in the mechanisms by which hyperglycemia leads to vascular permeability. It has been shown that phorbol esters increase cultured endothelial cell permeability through the activation of PKC.63 Increasing levels of the PKCβ1 isoform in dermal endothelial cells enhance the effect of phorbol esters on vascular permeability.64 PKCα is activated by hyperglycemia in porcine aortic endothelial cells and also serves to increase cell permeability.65 Furthermore, the permeability effects of high glucose and phorbol esters can be reduced in

rat skin tissue by PKC inhibition.66 One hypothesis is that PKC activation increases vascular permeability through its effects on phosphorylation of tight junction proteins, thereby affecting endothelial cell contractility. Specifically, PKC is known to regulate the phosphorylation of cytoskeletal proteins such as caldesmon, vimentin, talin, and vinculin.67-69 Phorbol esters also cause redistribution of cytoskeleton elements such as actin and vimentin.70

PKC isoforms also appear to play a role in the development of diabetesassociated neovascularization through their influence on the activity of growth factors such as vascular endothelial growth factor (VEGF).22 VEGF has mitogenic and pro-permeability effects on endothelial cells. A number of experimental and clinical studies have examined the role of VEGF in the

pathogenesis of PDR. VEGF levels are significantly increased in the vitreous fluid and aqueous humor of patients with PDR.71,72 In human vascular

smooth muscle cells, increased expression of VEGF due to hyperglycemia can be prevented by administration of PKC inhibitors.73 These properties are mediated via activation of PKCβ, through tyrosine phosphorylation of phospholipase Cγ.74 It has been found that inhibition of PKCβ by the selective inhibitor RBX blocks VEGF’s proliferative, angiogenic, and propermeability effects.40,75 Using a model of ischemia-induced proliferative retinopathy, there is a significant increase in VEGF-mediated retinal neovascularization in transgenic mice overexpressing the PKCβ2 isoform, and a corresponding decrease in angiogenic activity in PKCβ-null mice.76

7.INHIBITION OF PKC

A number of studies have examined the effect of PKC inhibitors such as staurosporine, H-7, GF109203X, and chelerythrine.20 Their in vitro utilization has been effective in blocking PKC effects, thereby demonstrating an association between PKC activation and decreased retinal blood flow, thickening of basement membranes, and increased vascular permeability and angiogenesis. However, in vivo use of nonspecific inhibitors may be limited by their effects on PKC isoforms that perform vital, non-pathogenic functions throughout the body. Indeed, a recent trial of a non-selective PKC inhibitor, PKC412, as a therapeutic agent for diabetic macular edema resulted in approximately 10% of the patient population being withdrawn from the study due to systemic toxic effects, including gastrointestinal side effects and hepatotoxicity.77

Currently, interest in agents that may be used successfully in vivo has focused primarily on the PKCβ-specific agent, RBX. This PKC inhibitor has

a fifty-fold higher affinity for PKCβ1/2 than for other PKC isoforms (α, γ, δ, ε, η, and ζ).40 Perhaps because of this high selectivity for PKCβ, its safety profile appears to be better than that of previously tested, non-selective PKC inhibitors. Recent clinical studies have examined the efficacy of RBX in improving outcomes related to diabetic nephropathy78 and retinopathy.79

RBX is a bisindoylmaleimide compound that preferentially inhibits PKCβ1 and PKCβ2 over other PKC isoforms. When orally administered to diabetic rats, RBX successfully increases retinal blood flow as measured by mean circulation time and improves glomerular filtration rates and albumin excretion.40 Oral administration in diabetic rats also reduces microvasculature flow disturbances caused by leukocyte entrapment.80 Intravitreal injection of the compound in the same animal model has been found to decrease PKC activation and increase retinal blood flow.39 Consistent with the theoretical effects of inhibition of PKCβ, RBX has additional antipermeability and anti-angiogenic effects. It suppresses VEGFmediated vascular permeability in vivo75 and prevents the development of retinal neovascularization in a pig model of ischemic retinal disease.81

Clinical phase I and II trials demonstrated that oral RBX (Eli Lilly Co., Indianapolis, IN) is well tolerated for periods of up to a month in doses up to 32 mg a day. In these studies, a significant amelioration of retinal blood flow

and mean circulation time was found in patients with no or minimal retinopathy and diabetes of duration less than 10 years.82,83 These

improvements occurred despite a lack of change in either fasting blood glucose levels or hemoglobin A1c (HbA1c). Subsequent studies have revealed a favorable safety profile in patients taking 32 mg/day for up to 3 years.79

RBX also appears to have some efficacy in reducing the non-ophthalmic microvascular complications of diabetes. Although it did not improve sensory symptom scores in patients with peripheral diabetic neuropathy in phase III trials,84 a recent pilot study of RBX use in type 2 diabetic patients with proteinuria suggests that its ameliorating effects on renal function are additive to intensive glycemic control and blood pressure regulation by angiotensin inhibition.78

Phase III trials on the ophthalmic effects of RBX have focused on endpoints related to decreases in diabetic macular edema and neovascularization. One trial investigating RBX’s effect on diabetic macular edema found that it did not prevent progression of macular edema or decrease the need for macular grid/focal laser. However, subgroup analysis that excluded patients with poor glycemic control (HbA1c > 10) found a 31% risk reduction in progression of diabetic macular edema.85 Further analysis suggested that patients taking 32 mg/day of RBX were less likely to develop edema involving the central macula, and that when they did, their visual acuities were better than their placebo-taking counterparts.86 Additional trials

investigating the ability of RBX to ameliorate diabetic macular edema are ongoing.

The Protein Kinase C (beta) Inhibitor Diabetic Retinopathy Study Group (PKC-DRS) reported initial results from its phase II/III clinical trial utilizing RBX in July of 2005.79 This study enrolled 252 patients with type 1 or type 2 diabetes and moderately severe to very severe nonproliferative diabetic retinopathy in at least 1 eye. Subjects were randomized to one of three dose levels of RBX, from 8 to 32 mg/day administered orally. The drug was found to have a favorable safety profile, with no clinically significant differences between treatment and placebo groups. Although the primary end point for the study (photographically determined progression of diabetic retinopathy or the use of laser photocoagulation) was not met after three years, there was a significant benefit in terms of decreased rates of moderate visual loss for patients treated with 32 mg/day of RBX.79 Based on the results of a second phase III trial that also demonstrated that RBX reduces sustained moderate vision loss in patients with diabetic retinopathy, FDA approval is currently being sought for the compound.84

8.CONCLUSIONS

There is an increasing preponderance of literature that suggests an important role for PKC in the development of diabetic retinopathy. Hyperglycemia activates the DAG-PKC pathway, which in turn regulates a number of vascular functions. Studies show that PKC has a direct effect on retinal blood flow and leukostasis, ECM deposition and basement membrane thickening, and vascular permeability and angiogenesis. Recent investigations have examined the potential role of PKC inhibitors in the treatment of diabetic retinopathy. RBX, an oral PKCβ-selective inhibitor, significantly decreases the extent of visual loss in diabetic patients with no to minimal retinopathy over the course of three years. This drug will likely undergo evaluation for FDA approval for the treatment of diabetic retinopathy and possibly diabetic nephropathy in the near future. It is hoped that future clinical and experimental investigations will more clearly elucidate the potential of PKC inhibitors in the treatment of diabetic retinopathy.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health Grants K12 EY-16335 (J.K.S.) and DK-59725 (G.L.K.) and by the American Diabetes Association Grant 1-05-RA-61 (G.L.K.).

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86.Eli Lilly and Co. Data indicate that Lilly’s ruboxistaurin may have a potentially beneficial effect on diabetes-induced eye disease. Corporate news, Oct. 26, 2004 (accessed at http://www.prnewswire.com/cgi-bin/micro_stories.pl?ACCT=916306&TICK=LLY&STORY=/ www/story/10-26-2004/0002310518&EDATE=Oct+26,+2004 on August 23, 2006).