Key Words: Protein kinase C, diacylglycerol, diabetes, diabetic retinopathy, retinal blood flow, vascular permeability, angiogenesis, ruboxistaurin

INTRODUCTION

Numerous studies, including the Diabetes Control and Complications Trial (DCCT) and the United Kingdom Prospective Diabetes Study (UKPDS), have demonstrated a close correlation between increased blood glucose levels and diabetic complications affecting multiple organ systems, including the brain, cardiovascular system, kidneys, and eyes. Complications from this disease may be divided into macrovascular, including coronary artery disease, atherosclerosis, and peripheral vascular disease; and microvascular, including retinopathy, nephropathy, and neurovascular defects. Intensive control of blood glucose can successfully delay the onset, and slow the progression, of diabetic retinopathy, nephropathy, neuropathy, and cardiac abnormalities (1).

In addition to systemic factors such as hyperglycemia, local tissue responses are equally important in the development of pathologies both in the type and the severity of complications. For example, some vascularized organs such as the pulmonary system do not manifest significant pathological functions even though they are exposed to the same levels of hyperglycemia and other systemic metabolites that exist in the diabetic condition. Another clear example of the importance of tissue response is in the area of angiogenic response. In the retina, excessive proliferation of endothelial cells is the hallmark of diabetic proliferative retinopathy. In contrast, myocardium and peripheral limbs exhibit too little neovascularization in response to hypoxia even though all the tissues are exposed to hyperglycemia, oxidative stress, or inflammatory cytokines. Thus, a complete understanding requires the clarification of changes in systemic and local factors.

Hyperglycemia has been shown to affect several signal transduction pathways including the elevation of diacylglycerol (DAG) levels and the activation of protein kinase C (PKC) and MAP kinases (2,3). We have focused on the effects of PKC activation since this pathway is involved in regulating vascular function in a variety of ways including endothelial permeability regulation, vasoconstriction, extracellular matrix synthesis and turnover, cell growth, angiogenesis, activating cytokines, and adherence of leukocytes. This chapter will review the role of PKC in diabetes and its complications, particularly in respect to PKC’s actions affecting retinal blood flow, basement membrane and extracellular matrix changes, and vascular permeability and angiogenesis.

PKC

Protein kinase C (PKC) is part of a serine/threonine kinase family that phosphorylates key proteins involved in cardiovascular and endothelial function. It catalyzes the phosphate group transfer from ATP to other substrate proteins. PKC has individual isozymes, which are selectively activated in vascular tissues by diacylglycerol (DAG). Synthesis of DAG is in turn upregulated in vascular tissues by hyperglycemia. Differences in structure and substrate requirements have yielded approximately 12 different PKC isoforms. These differences allow classification of PKC into three different groups: Conventional (cPKC), novel (nPKC), and atypical PKCs (aPKC) (Table 1) (4, 5–9).

PS phosphatidylserine

Each PKC molecule contains a single polypeptide chain with an N-terminal regulatory region and a C-terminal catalytic region. The different isoforms are products of separate genes (except for PKCβ1 and β2 which are same gene alternate splice variants).

The three PKC groups are characterized by differences in the four conserved domains, referred to as C1–C4. All PKC isozymes contain C3 and C4, which are located in the C-terminal catalytic portion of the molecule. The C3 site is involved in binding ATP, whereas the C4 site recognizes substrates to be phosphorylated by PKC (5–9). The cPKC polypeptide structure contains all four conserved domains and five variable regions. The components required for interaction with DAG, phorbol esters, phosphatidylserine, and calcium are located in the C1 and C2 domains in the N-terminal portion of the molecule. In contrast to the cPKC subclass, both nPKC and aPKC possess C2-like regions, that do not bind calcium, so that only cPKC is calcium dependent (Table 1).

DAG-PKC PATHWAY

In cells, DAG is the physiologic activator of PKC. DAG, in turn, is derived from multiple pathways, including the hydrolysis of phosphatidylinositol (PI) by phospholipase C (PLC), and by synthesis from dihydroxyacetone phosphate and glycerol 3-phosphate. However, several studies have shown that PI hydrolysis does not lead to hyperglycemia-induced increases in DAG in vascular cells. De novo synthesis involving the metabolism of dihydroxyacetone phosphate into lysophosphatidic acid and then phosphatidic acid (PA) has been shown to lead to glucose-induced DAG formation

(10–12).

Hyperglycemia mediated upregulation of DAG causing corresponding increases in PKC activity has been demonstrated in the retina, aorta, heart, monocytes, and glomeruli from animals and humans with diabetes (10–12). PKC isoforms are diffusely located in mammalian tissue and vary widely in regard to their tissue localization. These isoforms are differentially activated within these tissues (Table 2). Of all the isoforms, the largest fraction is that of PKCβ, which belongs to the cPKC family. This isoform family, as mentioned earlier, requires DAG for activation. Studies have shown that the PKCβ isoform demonstrates the most significant increase in a variety of vascularized tissues in hyperglycemic states. It is also one of the isoforms activated in hyperglycemic states in monocytes and leukocytes (13–17). However, PKCα and δ isoforms have also been reported to be activated in several tissues including the retina, heart, kidney, and the monocytes.

Table 2

Differential Expression and Activation of PKC Isoforms in Tissues and Cultured Cells

Under Normal and Hyperglycemic Conditions

This preferential activation of different PKC isoforms in different tissues in hyperglycemic states is not fully understood. Several theories have been proposed. First, calcium levels may change the overall affinity of PKC isoforms to DAG. It has been suggested that the PKCβ isoform may be more sensitive to DAG than the nPKC or aPKC isoforms when low calcium levels are present (though the latter are overall calcium independent). Another possibility is that because of the specific subcellular loca- tionofsomeisoforms,theymaybedifferentiallyactivatedincasesofhyperglycemia-induced increases in DAG. For example, because PKCβ is present intracellularly, rather than being membrane-bound, the mitochondrial-Golgi apparatus could preferentially activate it. Third, varying ratios of synthesis and degradation could contribute to different concentrations of isoform reported in different tissues (29–31). Fourth, it is also possible that the activation of PKCβ isoform appears to be greater than other isoforms due to our ability to measure the changes between control and diabetic states.

DIABETES AND RETINAL BLOOD FLOW

Capillary pericyte loss is among the earliest and most specific features of clinical diabetic retinopathy. Retinal pericytes serve a key role in maintaining capillary integrity and function. Their loss can lead to vessel dysfunction, vascular permeability, increase in vessel diameter and loss of regulatory tone, endothelial cell proliferation, and formation of microaneurysms. All these changes may in turn alter retinal blood flow dynamics and result in retinal ischemia. This ischemia can cause release of growth factors that stimulate new, unhealthy blood vessel formation and proliferation. This stage of proliferative

retinopathy can lead to hemorrhages and fibrosis, and eventually either vitreous hemorrhage or retinal detachment with poor visual outcome. The increased loss of pericytes is likely due to apoptosis as a response to diabetes-induced decreases in PDGF-B receptor’s actions. We have shown that PDGF-β levels are actually increased in the retina of diabetic animals, suggesting that diabetes and possibly PKC activation may cause PDGF-B resistance in retinal pericytes, affecting permeability and blood flow (30).

Some hemodynamic abnormalities are evident and can be recognized before any clinical signs of retinopathy are evident. Circulatory abnormalities have been reported in Type 1 diabetic patients with no clinical signs of retinopathy. Retinal blood flow is significantly decreased in these patients as shown by video fluorescein angiography (VFA) (31). Laser Doppler methods have also shown decreased arterial blood velocity in diabetic patients with no-to-minimal retinopathy (32). With longer duration of disease and progression to proliferative retinopathy, retinal blood flow will actually increase (33–36). Proposed mechanisms to explain initial decreases in retinal blood flow include diabetes-related changes in vasoactive factors in concordance with the PKC signal transduction pathway resulting in increased resistance to blood flow in early stages of diabetes.

It has been reported that injection of the PKC activator, phorbol dibutyrate, into the vitreous of healthy rats resulted in a significant increase in arteriovenous passage time. This paralleled the increase noted in 2–4 week diabetic rats in the same study. In addition, intravitreal administration of R59949, a DAG kinase inhibitor, into healthy rats demonstrated increased levels of total retinal DAG and showed dose-dependent decreases in retinal blood flow (37).

PKC could exert its effects on retinal vascular reactivity by changing the expression of certain endothelium-derived growth factors. One such factor is endothelin-1 (ET-1), a potent vasoconstrictor which has been identified in capillary endothelial cells, pericytes, and other retinal cells (38, 39). Retina samples from diabetic rats showed increased mRNA levels of ET-1. If ET-1 is given as an intravitreal injection to nondiabetic rats, there is increased retinal vasoconstriction and resultant decreases in retinal blood flow. Taking another approach, injecting the ET-1 receptor antagonist, BQ-123, dose dependently increases retinal blood flow in diabetic rats. In addition, when bovine retinal capillary endothelial cells and pericytes are exposed to high glucose concentrations, both membranous PKC activity and ET-1 expression are increased (17). Following the administration of two compounds, PKC inhibitor (GF 109203X) and mitogen-acti- vated protein kinase inhibitor (PD 98059), the noted increase in ET-1 is inhibited. Another endothelium-derived growth factor altering vasoreactivity is nitric oxide (NO), which, in contrast to ET-1, is a vasodilator. In response to increased glucose levels, both expression and production of NO are decreased in cultured retinal endothelial cells (40). This decrease can be partially restored by inhibiting PKC. The combined effects of an increase in ET-1 (a vasoconstrictor) and a decrease in NO (a vasodilator) could contribute to overall vasoconstriction and resulting increased resistance to blood flow. Therefore, these endothelium-derived growth factors may exert significant vasoactive effects to promote the initial decrease in retinal blood flow observed in early diabetes.

Leukostasis may also play a significant role in diabetic pathology, though it likely does not entirely explain the observed decrease in retinal blood flow. Leukocytes and monocytes show increased adhesion to retinal endothelial cells in response to the PKC activation seen in early diabetes (41). This increased adhesion is more directly related to oxidative stress