significant features of human diabetic retinopathy (36). Pancreatectomized or spontaneously diabetic cats have demonstrated local ischemia and tissue hypoxia in the retina (37), and an acidification of the inner retina that is associated with capillary dropout (38). Diabetic dogs have demonstrated degenerative capillaries, and other histopathology characteristic of retinopathy in diabetic patients (39, 40).

Rodent models (rats and mice), chemically made diabetic with streptozotocin or alloxan, are routinely employed to investigate the development of diabetic retinopathy. Mice present an advantage because they can be genetically manipulated to identify the genes responsible for capillary degeneration (40–43). Capillary dropout and other early histopathological signs of diabetic retinopathy can be observed in rodents at much shorter duration (8–12 months) of diabetes compared to dogs (3–5 years). In addition, the smaller size of the animal provides an advantage in that multiple pathways and therapies can be evaluated using a large number of rodents. Further details about animal models to investigate capillary dropout and other pathology are compiled by Dr. Kern in Chap. 5.

In vitro studies using isolated retinal capillary cells (endothelial cells and pericytes) have helped elucidate the mechanism of capillary dropout in diabetes. These models are useful because the specific gene of interest can be overexpressed or silenced, and the cells can be exposed to specific activators or inhibitors of the pathway of interest without much difficulty (44–47). However, the results should be used with caution because the culture conditions can greatly modify the cells and the in vivo model where multiple diabetes-induced biochemical, physiological, structural, and functional alterations that could work in synergy to contribute to capillary dropout cannot be duplicated.

POTENTIAL MECHANISMS FOR CAPILLARY DROPOUT

At least two mechanisms are thought to contribute to capillary occlusion and obliteration in diabetes: death/apoptosis of capillary cells and vascular occlusion by white blood cells or platelets. In addition, it is quite possible that there could be an interplay between these mechanisms, since capillary cell apoptosis could create an environment favorable for microthrombosis and leukocyte adhesion to endothelial cells, and vice versa.

Capillary Cell Apoptosis

Programmed cell death, commonly termed “apoptosis,” may be an important mechanism that contributes to capillary dropout (22). Retinal microvascular cells (pericytes and endothelial cells) and neuronal cells are lost selectively via apoptosis before other histopathology is detectable (48–50), and it is possible that this apoptosis over time contributes to capillary degeneration in DR. In diabetic patients, retinal capillary cells are reported to perish by accelerated cell death (22), and this phenomenon is replicated in animal models of diabetic retinopathy. Apoptosis of retinal microvascular cells in diabetic or galactosefed rodents can be detected as early as 5–6 months after induction of diabetes, and acellular capillaries and pericyte ghosts are seen after 10–12 months of diabetes (22,51–53).

The loss of retinal capillary pericytes is considered the earliest morphologic lesion observed in diabetic retinopathy (54, 55). Pericytes with almost no replicative capacity in the adult organism fail to renew (56). This loss of retinal pericytes leaves pockets in the basement membrane, commonly termed as “pericyte ghosts.” Apoptotic capillary

cells observed in the retina at any given duration of diabetes are very limited in number (22, 57, 58), but this may well be sufficient to result in significant pericyte loss (and appearance of pericyte ghosts) over time. The histopathology of diabetic retinopathy develops over decades in humans and more than 1 year in rats, but apoptosis is a rapidly consummated phenomenon, and apoptotic cells are detectable for only a few hours (59). Thus, accelerated apoptosis could account for the pericyte loss and formation of ghosts in diabetic retinopathy. Although pericyte loss is correlated with acellular capillaries during diabetic microangiopathy (60–62), the specific contribution of pericyte loss to capillary dropout in the pathogenesis of diabetic retinopathy remains unclear. However, studies demonstrating a co-survival relationship of pericytes and endothelial cells in developing and adult vasculature (63) suggest that pericytes might have a role in supporting endothelial cell survival. The mean ratio of perictye nuclei to endothelial cell nuclei is 1:1 in normal retinal capillaries, but this ratio decreases to 1:4 (and later 1:10) in diabetic retinopathy (63, 64). Since pericytes are endothelial-supporting cells and they regulate endothelial cells intricately, the loss of pericytes could have negative consequences on endothelial cell survival (for further discussion, see Chap. 10).

Endothelial cells are considered a little less susceptible to damage in diabetes than pericytes (65), and their ability to replicate and replace neighboring cells, though at a very slow rate, could contribute to this (56, 64). Although endothelial cells are capable of making up the deficit for a finite period of time, it is likely that with exposure to the diabetic milieu over time, the replicative capacity of the endothelial cells is exhausted, as occurs in all somatic cells which reach their Hayflick limit (66, 67). In response to cell death in DR, the endothelium maintains a higher than normal rate of cell division over time, and this would be predicted to overextend its replicative capacity. Indeed, it has been suggested that exposure to the diabetic environment could induce a senescent phenotype in endothelial cells so that the normal Hayflick limit is not attainable (63). The precise contribution of endothelial cell apoptosis to capillary dropout remains to be determined. However, it is noteworthy that the ability of a therapy to reduce retinal capillary cell apoptosis in diabetes was predictive of its ability to inhibit retinal capillary degeneration (50). This suggests that apoptosis is an important contributor to capillary dropout in DR. Finally, it is important to note that apoptosis might not be the sole form of cell death in DR, as necrosis has been suggested as well (68).

Proinflammatory Changes/Leukostasis

Diabetes precipitates several molecular and functional abnormalities in the retina suggesting that proinflammatory pathways are a critical contributor in the development of diabetic retinopathy (69). Abnormalities characteristic of inflammation are observed in diabetic retinas and retinal capillary cells cultured in high glucose conditions. These include elevations in proinflammatory cytokines, prostaglandins, nitric oxide (NO), the nuclear transcription factor (NF)-kB, and adhesion molecules coupled with up-regula- tion of leukostasis. These proinflammatory processes could contribute to capillary dropout in diabetic retinopathy by increasing leukostasis and/or via increasing apoptosis of capillary cells (68, 70–73).

The levels of several proinflammatory cytokines including interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and IL-8 are increased in the vitreous of patients with

274 Kowluru et al.

proliferative diabetic retinopathy and in retinas from diabetic rodents (70, 71, 74–76). Inflammation is one of the processes implicated in the apoptosis of retinal cells (68, 71, 77), and TNF-α is considered as an important mediator of apoptosis of retinal endothelial cells in diabetes (78).

The eicosanoid prostaglandin E2 (PGE2) together with the inducible enzyme that catalyzes its formation, cyclooxygenase-2 (COX-2), are elevated in the retina of diabetic rats (72,79–83). Besides regulating the levels of vascular endothelial growth factor (VEGF) and retinal vascular permeability during diabetes (82), COX-2 also mediates leukostasis and acellular capillary formation in the retinal microvasculature of diabetic rats (72,81).

Nitric oxide is an important mediator of inflammation. Its formation is catalyzed by nitric oxide synthases. Inducible nitric oxide synthase (iNOS), in particular, has been reported to contribute to cytotoxicity in some cell types. The expression of iNOS and the levels of NO and peroxynitrite (a product of the reaction between superoxide and NO) are elevated in the retina of diabetic patients and rats (50, 83–89). Diabetic mice that have their iNOS gene knocked out exhibited a significant decrease in retinal capillary degeneration (90).

NF-kB, a transcription factor that regulates many genes participating in the inflammatory process and apoptosis, is considered to be a major inducer of most of the proinflammatory proteins including IL-1β, TNF-α, COX-2, iNOS, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) (91). NF-kB is activated in the retina and retinal capillaries in diabetic conditions (92–95), and it has been implicated in promoting the apoptosis of retinal capillary cells and the formation of acellular capillaries (92). Inhibition of NF-kB activation is associated with a reduction in pericyte loss and degeneration of retinal capillaries in diabetic animals (85, 93, 96).

Adhesion molecules including ICAM-1 and VCAM-1 are also up-regulated in the vitreous and serum of patients with diabetic retinopathy (97–99). ICAM-1 and CD18 are considered particularly important in the development of diabetic retinopathy and are suggested to exert their effects via activation of leukostasis (100, 101).

Increased leukostasis has been demonstrated to play an important role in the pathogenesis of diabetic retinopathy (68, 69, 81, 95, 102–104). Leukocytes become less deformable and more activated in diabetes and may be involved in retinal capillary nonperfusion, vascular leakage and endothelial cell damage (105). Acridine orange leukocyte fluorography and fluorescein angiography studies have shown trapped leukocytes directly associated with areas of capillary occlusion and nonperfusion in the diabetic retinal microcirculation (105, 106). Interestingly, with subsequent disappearance of leukocytes, some capillaries remained nonperfused. A detailed discussion of leukostasis and its contribution to capillary dropout in diabetic retinopathy is provided in Chap. 13.

Microthrombosis/Platelet Aggregation

Accumulation of platelets has been observed in the retinal vasculature of diabetic subjects, and these platelet microthrombi were spatially associated with apoptotic endothelial cells (107). Studies have shown that advanced glycation end products (AGEs) inhibit prostacyclin production and induce plasminogen activator inhibitor-1 in microvascular endothelial cells suggesting that AGEs have the ability to cause platelet aggregation and fibrin stabilization, resulting in a predisposition to thrombogenesis