Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

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Chapter 10

THE ROLE OF PROTEIN KINASE C

IN DIABETIC RETINAL

VASCULAR ABNORMALITIES

Jennifer K. Sun1 and George L. King2

1Beetham Eye Institute and Eye Research Section, Joslin Diabetes Center, and the Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts; and 2Section on Vascular Cell Biology and Complications, Joslin Diabetes Center, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts

1.INTRODUCTION

Diabetes mellitus affects more than 16 million people in the United States,1 and over 171 million individuals worldwide.2 Its manifestations are both macroand microvascular in nature; they include peripheral vascular disease, coronary artery disease, and atherosclerosis as well as retinopathy, nephropathy, and autonomic neuropathy. Diabetic retinopathy develops in almost all people with type 1 diabetes and in more than 60% of those with type 2 diabetes within the first 20 years of disease.3 It is the leading cause of new blindness in working age adults in the western world.4

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J.S. Penn (ed.), Retinal and Choroidal Angiogenesis, 187–202.

© Springer Science+Business Media B.V. 2008

2.PROGRESSION AND TREATMENT OF DIABETIC RETINOPATHY

Diabetes-associated changes are present in the retinal vasculature even before the first clinically recognized signs of diabetic retinopathy. These changes include decreases in retinal blood flow,5 the loss of retinal pericytes,6 and thickening of capillary basement membranes.7

The first standardized grading system for clinically recognizable lesions was created in 1968 at the Airlie House Convention in Alexandria, Virginia.8 The Airlie House Classification of Diabetic Retinopathy was subsequently modified by both the Diabetic Retinopathy Study (DRS)9 and the Early Treatment of Diabetic Retinopathy Study (ETDRS).10 Early signs of clinically apparent diabetic retinopathy include microaneurysms, intraretinal hemorrhages, and cotton wool spots. Retinal ischemia occurs later in the disease and is often accompanied by changes in venous caliber and intraretinal microvascular abnormalities (IRMAs). In some patients, advanced retinal ischemia leads to the formation of neovascular vessels on the optic disc or elsewhere in the retina. This stage is known as proliferative diabetic retinopathy (PDR). Development of neovascularization can lead to further complications such as vitreous hemorrhage or traction retinal detachments. Macular edema due to increased vascular permeability can present at any stage of the disease and is one of the most common causes for vision loss in diabetes.11

Large-scale studies, including the Diabetes Control and Complications Trial (DCCT)12 and the United Kingdom Prospective Diabetes Study (UKPDS),13 established a strong relationship between intensive glycemic control and a decreased risk of progression of diabetic retinopathy. Other studies, such as the DRS14 and the ETDRS,15 also proved the efficacy of laser-based treatment methods such as scatter (panretinal) photocoagulation and focal macular laser in managing high-risk PDR and clinically significant macular edema, respectively. Over the last half-century, treatment of diabetes and its attendant complications has improved significantly. However, a need still exists for more effective treatments. As understanding grows of the molecular mechanisms behind microvascular pathology, new treatments have been proposed that specifically target these mechanisms. This paper reviews the literature surrounding one signaling molecule of recent interest, protein kinase C (PKC). It discusses PKC’s role in the mechanisms underlying diabetic retinopathy as well as the development of PKC inhibitors to prevent retinopathy and its complications.

3.MOLECULAR MECHANISMS UNDERLYING DIABETIC RETINOPATHY

Hyperglycemia appears to be the unifying etiologic factor that underlies the diverse vascular complications of diabetes. Multiple clinical studies have

demonstrated a significant correlation between levels of glycosylated hemoglobin and the incidence and progression of diabetic retinopathy.13,16,17

The mechanisms by which hyperglycemia leads to retinal vascular endothelial damage have not yet been fully clarified. However, several molecular pathways involved in glucose metabolism have been elucidated.

Elevations in blood glucose lead to an increased flux of glucose through glycolysis and affect the ratio of NAD to NADPH. Non-enzymatic reactions of glucose also result in the generation and accumulation of advanced glycation endproducts (AGE) and reactive oxygen species.18,19 In turn, these agents may activate the signal transduction pathway of diacylglycerol (DAG)-PKC (Figure 1). The DAG-PKC pathway acts upon functional enzymes, signaling proteins, cytokine expression, and cell cycle factors and transcription factors. Through these actions, it affects multiple facets of vascular function, including retinal hemodynamics, leukocyte adhesion, cell growth, extracellular matrix regulation, endothelial cell permeability, and angiogenesis.20 Through its effects on the microvasculature, PKC may play an important role in the two major causes of diabetic ocular morbidity: macular edema and proliferative retinopathy.

4.CLASSIFICATION AND STRUCTURE OF PKC

The family of PKC is composed of at least 12 serine/threonine isoforms.21 These related enzymes serve as intracellular signaling systems for a number of growth factors, hormones, and cytokines. Each PKC molecule comprises a single polypeptide chain with an N-terminal regulatory domain and a C- terminal catalytic domain. Whereas the regulatory domain binds phospholipid cofactors and calcium, the catalytic domain is responsible for the enzyme’s kinase activity.22 The PKCs fall into three categories (classic, novel, and atypical) based on distinctive factors in their catalytic and regulatory domains.23 Classic PKCs are calcium dependent and are activated by both phosphatidylserine (PS) and DAG. Novel PKCs are calciumindependent, but are also regulated by PS and DAG. In contrast, atypical

PKCs are calcium-independent and not regulated by DAG, but are sensitive to PS.24

Figure 10-1. The role of PKCβ in diabetic retinopathy.

5.THE DAG-PKC PATHWAY

DAG can be derived from either the hydrolysis of phosphatidylinositides

(PI) or from the metabolism of phosphatidylcholine (PC) by phospholipase C (PLC) or D (PLD).24,26 However, studies of glomerular mesangial cells

and aortic smooth muscle cells reveal that hyperglycemia does not increase PI hydrolysis.27,28 It is more likely that increases in DAG result from de novo

synthesis involving the metabolism of dihydroxyacetone phosphate into lysophosphatic acid and then phosphatidic acid (PA).

Upregulation of DAG occurs both acutely and chronically in the hyperglycemic state. An increase in glucose levels from 5.5 mM to 22 mM caused increased levels of DAG and higher specific activity for PKC in rat retinal endothelial cells within 3-5 days.29 More persistent upregulation of DAG is seen in the aortic tissue of diabetic dogs up to five years after the onset of hyperglycemia.27 Work by Inoguchi et al. suggests that subsequent euglycemic control may not necessarily correct elevations in DAG levels. High DAG levels were sustained in the aortas but not the hearts of diabetic rats after islet cell transplantation.30

PKC isoforms are differentially localized and activated within mammalian tissues. Whereas PKCθ is found in skeletal muscle and haematopoietic tissue and PKCγ is seen primarily in the central nervous system, PKCβ is present in multiple vascularized organs, including the retina, kidney, and heart.20 Specific PKC isoforms that have been shown to be active in the diabetic rat retina include PKCα, β1, β2, and ε.29 Of all of these isoforms, the largest fraction is that of PKCβ2. PKCβ is a classic PKC isoform that requires DAG for activation. Additional immunoblotting studies suggest that in vascularized tissues in general, it is primarily PKCβ that is activated in states of hyperglycemia.30,31 Furthermore, hyperglycemia also activates PKCβ in monocytes and leukocytes.32

The preferential activation of certain isoforms of PKC in different cell types is incompletely understood. Three potential mechanisms for this activation specificity have been proposed. First, interactions between levels of DAG and calcium may allow specific activation of PKCβ over other PKC isoforms. It is possible that PKCβ is more sensitive to DAG elevations, especially in the presence of lower concentrations of calcium, than other classic or novel isoforms of PKC.33 Second, increasing DAG levels associated with glucose metabolism in the mitochondria and Golgi complex could cause preferential activation of PKCβ because of its intracellular rather than membrane-bound location. Finally, differential rates of synthesis and degradation of the PKC isoforms could also explain differing rates of isoform activity between different tissue types.22

6.VASCULAR ALTERATIONS RELATED TO PKC ACTIVATION

Within the spectrum of changes due to diabetes, multiple vascular alterations on a cellular and functional level have been ascribed to the activation of PKC. The following section will review the literature regarding PKC activity and its effects on retinal hemodynamics and leukostasis; extracellular matrix (ECM) and basement membranes; and vascular permeability and angiogenesis, with special attention to mechanisms potentially related to diabetic retinopathy.

6.1Retinal hemodynamics and leukostasis

It is well established that retinal circulatory changes are a hallmark of early diabetes in the eye, and that they can appear even before the onset of clinically recognized retinopathy. Decreases in retinal blood flow have been documented by video fluorescein angiography34 and laser Doppler35 in

patients with relatively short disease duration. Later in the disease, particularly after the development of PDR, retinal blood flow increases.36,37

Increased adherence of leukocytes and monocytes to the retinal endothelium is also present in subclinical diabetic retinopathy, but can occur in the presence of insulin resistance alone, without diabetes or overt hyperglycemia.38 Several studies suggest a connection between the DAG-PKC pathway and changes in retinal blood flow. The early impairment in retinal circulation can be mimicked by injection of a PKC activator, phorbol dibutyrate, into the vitreous cavity of normal rats. This injection causes a decrease in blood flow that is similar to that seen in 2-4 week diabetic rats.29 Intravitreal injection of a DAG kinase inhibitor (R59949) both increases retinal DAG levels and decreases retinal blood flow in a dose-dependent fashion.39 Furthermore, oral PKC inhibition can normalize retinal blood flow changes as well as changes in glomerular filtration rate and albumin excretion rate in

diabetic rats.40

A possible mechanism by which PKC activity could alter retinal vasoreactivity is through affecting expression of vasoactive factors such as endothelin-1 (ET-1), a potent vasoconstrictor, or nitrous oxide (NO), which acts as a vasodilator. ET-1 is present in both retinal capillary endothelial cells and retinal pericytes,20 and increased levels of ET-1 mRNA have been found in retinal tissue from diabetic rats.41 Studies have shown that intravenous administration of ET-1 results in decreased retinal blood flow secondary to vasoconstriction in nondiabetic rats.42,43 Futhermore, treatment with either phosphoramidon, an endothelin-converting enzyme inhibitor, or the endothelin type A receptor antagonist BQ-123 inhibits the effects of ET- 1 and increases retinal blood flow.42 Clear links have been demonstrated between elevations in glucose levels, increased PKC activity, and higher levels of both endothelin-converting enzyme and ET-1 expression.44,45 The increase in ET-1 levels seen with increased glucose levels is reduced with a general PKC inhibitor, GF109203X.44 Recent studies suggest that PKC regulates ET-1 expression through increasing levels of platelet-derived growth factor (PDGF)-BB.46 High glucose concentrations also lead to overexpression of endothelial nitric oxide synthase (NOS) and subsequent decreased production of NO in cultured retinal endothelial cells. In one study, PKC inhibition partially reversed the effect of hyperglycemia on NO production.47

PKC activation also plays a role in the increased leukocyte and monocyte adhesion to retinal endothelial cells seen in early diabetes. Abiko et al. recently demonstrated that increased leukostasis alone probably does not suffice to explain diabetic decreases in retinal blood flow. However, it is still possible that leukostasis plays a contributing role in worsening diabetic