Timothy S. Kern

Amelioration of Vascular Dysfunctions in Diabetic Rats by an Oral PKC β Inhibitor

The vascular complications of diabetes mellitus have been correlated with enhanced activation of protein kinase C (PKC). LY333531, a specific inhibitor of the β isoform of PKC, was synthesized and was shown to be a competitive reversible inhibitor of PKC β1 and β2, with a half-maximal inhibitory constant of ∼5 nM; this value was one-fiftieth of that for other PKC isoenzymes and one-thousandth of that for non-PKC kinases. When administered orally, LY333531 ameliorated the glomerular filtration rate, albumin excretion rate, and retinal circulation in diabetic rats in a dose-responsive manner, in parallel with its inhibition of PKC activities.

A central role for inflammation in the pathogenesis of diabetic retinopathy

Diabetic retinopathy is a leading cause of adult vision loss and blindness. Much of the retinal damage that characterizes the disease results from retinal vascular leakage and nonperfusion. Diabetic retinal vascular leakage, capillary nonperfusion, and endothelial cell damage are temporary and spatially associated with retinal leukocyte stasis in early experimental diabetes. Retinal leukostasis increases within days of developing diabetes and correlates with the increased expression of retinal intercellular adhesion molecule‐1 (ICAM‐1) and CD18. Mice deficient in the genes encoding for the leukocyte adhesion molecules CD18 and ICAM‐1 were studied in two models of diabetic retinopathy with respect to the long‐term development of retinal vascular lesions. CD18−/− and ICAM‐1−/− mice demonstrate significantly fewer adherent leukocytes in the retinal vasculature at 11 and 15 months after induction of diabetes with STZ. This condition is associated with fewer damaged endothelial cells and lesser vascular leakage. Galactosemia of up to 24 months causes pericyte and endothelial cell loss and formation of acellular capillaries. These changes are significantly reduced in CD18‐ and ICAM‐1‐deficient mice. Basement membrane thickening of the retinal vessels is increased in long‐term galactosemic animals independent of the genetic strain. Here we show that chronic, low‐grade subclinical inflammation is responsible for many of the signature vascular lesions of diabetic retinopathy. These data highlight the central and causal role of adherent leukocytes in the pathogenesis of diabetic retinopathy. They also underscore the potential utility of anti‐inflammatory treatment in diabetic retinopathy.

Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy.

To reconstruct the mechanisms for the vasoobliteration that transforms diabetic retinopathy into an ischemic retinopathy, we compared the occurrence of cell death in situ in retinal microvessels of diabetic and nondiabetic individuals. Trypsin digests and sections prepared from the retinas of seven patients (age 67 +/- 7 yr) with .9 +/- 4 yr of diabetes and eight age- and sex-matched nondiabetic controls were studied with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) reaction which detects preferentially apoptotic DNA fragmentation. The count of total TUNEL+ nuclei was significantly greater in the microvessels of diabetic (13 +/- 12 per one-sixth of retina) than control subjects (1.3 +/- 1.4, P = 0.0016), as were the counts of TUNEL+ pericytes and endothelial cells (P < 0.006). The neural retinas from both diabetic and nondiabetic subjects were uniformly TUNEL-. Retinal microvessels of rats with short duration of experimental diabetes or galactosemia and absent or minimal morphological changes of retinopathy, showed TUNEL+ pericytes and endothelial cells, which were absent in control rats. These findings indicate that (a) diabetes and galactosemia lead to accelerated death in situ of both retinal pericytes and endothelial cells; (b) the event is specific for vascular cells; (c) it precedes histological evidence of retinopathy; and (d) it can be induced by isolated hyperhexosemia. A cycle of accelerated death and renewal of endothelial cells may contribute to vascular architectural changes and, upon exhaustion of replicative life span, to capillary obliteration.

Contributions of inflammatory processes to the development of the early stages of diabetic retinopathy.

Diabetes causes metabolic and physiologic abnormalities in the retina, and these changes suggest a role for inflammation in the development of diabetic retinopathy. These changes include upregulation of iNOS, COX-2, ICAM-1, caspase 1, VEGF, and NF-κB, increased production of nitric oxide, prostaglandin E2, IL-1β, and cytokines, as well as increased permeability and leukostasis. Using selective pharmacologic inhibitors or genetically modified animals, an increasing number of therapeutic approaches have been identified that significantly inhibit development of at least the early stages of diabetic retinopathy, especially occlusion and degeneration of retinal capillaries. A common feature of a number of these therapies is that they inhibit production of inflammatory mediators. The concept that localized inflammatory processes play a role in the development of diabetic retinopathy is relatively new, but evidence that supports the hypothesis is accumulating rapidly. This new hypothesis offers new insight into the pathogenesis of diabetic retinopathy, and offers novel targets to inhibit the ocular disease.

Characterization of the mechanism for the chronic activation of diacylglycerol-protein kinase C pathway in diabetes and hypergalactosemia

Similar vascular pathological conditions are observed in diabetic animals and those with diet-induced hypergalactosemia. Both diabetes and hypergalactosemia are believed to cause vascular dysfunction via a common biochemical mechanism. In this study, we have found that both diabetes and hypergalactosemia in the short term (2–4 months) can increase total diacylglycerol (DAG) levels by 52 ± 9 and 74 ± 13% in the retina and aorta, respectively, of diabetic dogs, and by 94 ± 9 and 78 ± 11% in the retina and aorta, respectively, in dogs with hypergalactosemia as compared with normal control animals (P < 0.01). The elevation of DAG levels was maintained for 5 years in the aortas of diabetic and hypergalactosemic dogs. To characterize the mechanism of the DAG increases, we have determined that total DAG levels were significantly increased in cultured macro- and microvascular cells exposed to elevated glucose (22 mM) and galactose (16.5 mM) levels. These increased levels were not prevented by sorbinil, an aldose reductase inhibitor. One of the sources of the increased DAG levels was probably derived from de novo synthesis from both hexoses as determined by radiolabeling studies. Intracellularly, the DAG elevation activated protein kinase C (PKC) activity with increases of 58 ± 12% (P < 0.05) and 66 ± 8% (P < 0.01) in the membrane fraction of cultured aortic smooth muscle cells exposed to elevated glucose and galactose levels, respectively. These findings have clearly demonstrated a possible common biochemical mechanism by which hyperglycemia and hypergalactosemia can chronically activate the DAG-PKC pathway in the vasculature and could be a possible explanation for the development of diabetic vascular complications.

Progression of Incipient Diabetic Retinopathy During Good Glycemic Control

To assess the extent to which the progression of diabetic retinopathy can be arrested by improved glycemic control, 35 normal dogs were randomly divided into a nondiabetic and three alloxan-induced diabetic groups prospectively identified according to glycemic control: poor control for 5 yr (PC), good control for 5 yr (GC), and poor control for 2.5 yr followed by good control for 2.5 yr (PGC). To achieve good control, insulin was given twice daily together with a measured diet so that hyperglycemia and glucosuria were mild and infrequent, and HbA, was comparable to normal. Retinal capillary aneurysms and other lesions developed during 60 mo of poor control (group PC) and were inhibited if good control was begun promptly within 2 mo (group GC). In group PGC, retinopathy was absent or equivocal at 2.5 yr of poor control and, surprisingly, was found to develop subsequently despite good glycemic control. Retinopathy in group PGC was greater at autopsy than at 2.5 yr and was greater than in group GC. The results indicate that retinopathy may be preventable but tends to resist arrest even in its incipient stages, before more than the first few aneurysms have appeared.

Activation of PKC-δ and SHP-1 by hyperglycemia causes vascular cell apoptosis and diabetic retinopathy

Cellular apoptosis induced by hyperglycemia occurs in many vascular cells and is crucial for the initiation of diabetic pathologies. In the retina, pericyte apoptosis and the formation of acellular capillaries, the most specific vascular pathologies attributed to hyperglycemia, is linked to the loss of platelet-derived growth factor (PDGF)-mediated survival actions owing to unknown mechanisms. Here we show that hyperglycemia persistently activates protein kinase C-δ (PKC-δ, encoded by Prkcd) and p38α mitogen-activated protein kinase (MAPK) to increase the expression of a previously unknown target of PKC-δ signaling, Src homology-2 domain–containing phosphatase-1 (SHP-1), a protein tyrosine phosphatase. This signaling cascade leads to PDGF receptor-β dephosphorylation and a reduction in downstream signaling from this receptor, resulting in pericyte apoptosis independently of nuclear factor-κB (NF-κB) signaling. We observed increased PKC-δ activity and an increase in the number of acellular capillaries in diabetic mouse retinas, which were not reversible with insulin treatment that achieved normoglycemia. Unlike diabetic age-matched wild-type mice, diabetic Prkcd−/− mice did not show activation of p38α MAPK or SHP-1, inhibition of PDGF signaling in vascular cells or the presence of acellular capillaries. We also observed PKC-δ, p38α MAPK and SHP-1 activation in brain pericytes and in the renal cortex of diabetic mice. These findings elucidate a new signaling pathway by which hyperglycemia can induce PDGF resistance and increase vascular cell apoptosis to cause diabetic vascular complications.

Retinal ganglion cells in diabetes

Diabetic retinopathy has long been recognized as a vascular disease that develops in most patients, and it was believed that the visual dysfunction that develops in some diabetics was due to the vascular lesions used to characterize the disease. It is becoming increasingly clear that neuronal cells of the retina also are affected by diabetes, resulting in dysfunction and even degeneration of some neuronal cells. Retinal ganglion cells (RGCs) are the best studied of the retinal neurons with respect to the effect of diabetes. Although investigations are providing new information about RGCs in diabetes, including therapies to inhibit the neurodegeneration, critical information about the function, anatomy and response properties of these cells is yet needed to understand the relationship between RGC changes and visual dysfunction in diabetes.

Experimental Galactosemia Produces Diabetic-like Retinopathy

Six normal dogs were made galactosemic by feeding a 30% D-galactose diet, and were followed up to 5 yr. For comparison, 10 normal dogs and 10 alloxan-diabetic dogs were concurrently fed the diet less the galactose supplement. Retinopathy occurred in each of four dogs glactosemic 3 or more yr, and was absent at lesser durations of galactosemia, and from normal dogs not given the galactose supplement. The retinopathy was marked by saccular capillary aneurysms, hemorrhages, nonperfused or acellular vessels, tortuous hypertrophic capillaries, loss of capillary pericytes, and other lesions typical of diabetic patients and alloxan-diabetic dogs. In galactose-fed dogs, blood galactose varied between 0 (fasted) and 250 mg/ dl (postprandial), and gly cosy la ted hemoglobin levels became supranormal. In contrast to diabetic dogs, blood levels of glucose, free fatty acids, and branchedchain amino acids were not elevated in the galactosemic dogs, and their serum insulin seemed normal. The results suggest that the level of blood hexose is itself an important determinant of retinopathy.

Ischemic vascular damage can be repaired by healthy, but not diabetic, endothelial progenitor cells.

Endothelial precursor cells (EPCs) play a key role in vascular repair and maintenance, and their function is impeded in diabetes. We previously demonstrated that EPCs isolated from diabetic patients have a profound inability to migrate in vitro. We asked whether EPCs from normal individuals are better able to repopulate degenerate (acellular) retinal capillaries in chronic (diabetes) and acute (ischemia/reperfusion [I/R] injury and neonatal oxygen-induced retinopathy [OIR]) animal models of ocular vascular damage. Streptozotocin-induced diabetic mice, spontaneously diabetic BBZDR/Wor rats, adult mice with I/R injury, or neonatal mice with OIR were injected within the vitreous or the systemic circulation with fluorescently labeled CD34+ cells from either diabetic patients or age- and sex-matched healthy control subjects. At specific times after administering the cells, the degree of vascular repair of the acellular capillaries was evaluated immunohistologically and quantitated. In all four models, healthy human (hu)CD34+ cells attached and assimilated into vasculature, whereas cells from diabetic donors uniformly were unable to integrate into damaged vasculature. These studies demonstrate that healthy huCD34+ cells can effectively repair injured retina and that there is defective repair of vasculature in patients with diabetes. Defective EPCs may be amenable to pharmacological manipulation and restoration of the cells’ natural robust reparative function.