Capillary Degeneration in Diabetic Retinopathy

Fig. 1. (A) Low-power view of retinal histopathology in a patient having nonproliferative diabetic retinopathy. There is a large area of capillary degeneration in the photo, indicated by the absence of dark nuclear stain in most vessels. Numerous microaneurysms are along the top and bottom of the micrograph. (B) Close-up of vascular histopathology in a diabetic patient. Degenerate capillaries are indicated by arrows, and saccular capillary microaneurysm is indicated by asterisk (*).

growth factor (VEGF). VEGF is known to be a key molecule leading to retinal permeability and neovascularization in diabetes and other retinal diseases [14–16].

VASCULAR NONPERFUSION IN DIABETES: MECHANISMS

Capillary nonperfusion can be due either to temporary or permanent occlusion/degeneration. Degenerate capillaries that are detected via histologic preparations of the isolated vasculature (trypsin digest or elastase methods) apparently once were functional capillaries that degenerated until only a basement membrane tube remains. These degenerate capillaries are no longer perfused, and have been used as histologic markers of nonperfused capillaries [10]. Although devoid of nuclei, these degenerate vessels sometimes are not truly acellular, and may be filled with cytoplasmic processes of glial cells [17].

Nonperfusion of capillaries also might be temporary. Temporary occlusions do not always cause damage to the capillary or nearby tissue, but repeated ischemic insults in a chronic disease like diabetes likely could cause progressive injury. Moreover, the neural retina of diabetic animals has been shown to be more sensitive to ischemia [18]. Small nonperfused areas observed in some retinas of diabetic patients later were found to be reperfused, and even the entire fundus became reperfused in a small number of other diabetic patients [19]. It is not clear if the reperfusion occurred in vessels that originally were occluded, or if other patent vessels took their place to supply blood to the ischemic region.

Mechanisms believed to contribute to the nonperfusion and degeneration of retinal capillaries in diabetes include occlusion of the vascular lumen by white blood cells, platelets, or other cells (notably glial cell processes), or altered hemodynamics. These mechanisms are not mutually exclusive.

1. Vasoocclusion by white blood cells. Using either ex vivo or in vivo techniques, diabetes increases adhesion of leukocytes to the vascular wall in diabetic animals [20–34]. Moreover, instances have been reported where the circulation of fluorescent dye injected into

the blood or using in situ (whole mount) perfusion methods is blocked by an immobile leukocyte, suggesting that the leukostasis is contributing to the capillary nonperfusion in diabetic retinopathy [27, 35]. Although individual instances of temporary capillary occlusion by a blood cell might be short-lived, cumulative effects of such repeated ischemia/reperfusion injuries over a prolonged interval are not known. Leukocyte stiffness has been reported to be increased in diabetes, thus making the cells less filterable and more likely to occlude retinal vessels [21, 36]. Abnormal leukocyte adherence to retinal vessels in diabetes occurs via expression of ICAM-1 and other adhesion molecules on the endothelial surface. Diabetes increases expression of ICAM-1 and other adhesion molecules in retinas of animals and humans [24, 28, 37–39], and interaction of this adhesion molecule with the CD18 adhesion molecule on leukocytes contributes to the diabetes-induced increase in adherence of white blood cells to the vascular wall in retinal vessels [24]. Diabetic mice lacking ICAM-1 and CD18 do not develop either the diabetes-induced increase in leukostasis, vascular permeability, or degeneration of retinal capillaries [33], providing strong evidence that white blood cells likely contribute to the eventual capillary damage and degeneration that is characteristic of diabetic retinopathy. Leukocytes have been found to be associated with capillary closure in retinas of spontaneously diabetic monkeys [40].

Although evidence suggesting a role for white blood cells in the development of the retinopathy is accumulating [33, 41, 42], whether or not leukostasis [23, 24, 26, 27, 33, 39, 43, 44] per se is a good parameter of the process of leading to capillary degeneration or diabetic retinopathy is less clear. A disconnect between leukostasis per se and the degeneration of retinal capillaries in diabetes was suggested by evidence that 12-lipoxygenase−/− diabetic mice did not develop the diabetes-induced increase in leukostasis, but nevertheless developed the capillary degeneration of diabetic retinopathy [45].

2. Vasoocclusion by platelets. Platelet microthrombi have been detected in the retinas of diabetic rats and humans, and have been spatially associated with apoptotic endothelial cells [46, 47]. Nevertheless, the selective antiplatelet drug (clopidogrel) did not prevent neuronal apoptosis, glial reactivity, capillary cell apoptosis, or degeneration of retinal capillaries in diabetic rats [48], thus providing no support for a postulated role of platelet aggregation in the development of capillary occlusion in diabetes. Moreover, aspirin (delivered at low doses that should have inhibited platelet aggregation) did not [49] or only modestly [50] inhibited the progression of diabetic retinopathy in clinical trials.

3. Hemodynamics. Many studies of diabetes indicate that there are alterations in blood flow to the retina [51–54]. Reduction in flow might be due to diabetes-induced increase in vascular resistance or viscosity, or to a reduction in metabolic activity in the retina which thus reduces the metabolic demand for flow. Whatever the cause, subsequent impairments to flow, even if slight, have been speculated to allow temporary stasis until backpressure increases.

4. Invasion of the vascular lumen by other cell types. Cellular processes from retinal glial cells have been found inside of occasional degenerate capillaries (identified from the basement membrane tube that surrounds vessels) [17, 55, 56]. It is not clear whether this glial invasion precedes and causes the capillary to degenerate or is a result of the capillary cells dying (thus opening spaces for the glial cell to expand into).

5. Growth factor withdrawal. Intravitreal administration of VEGF antagonists has been reported to cause apparent nonperfusion or regression of neovascular tufts in diabetic retinopathy [57, 58]. The later reappearance of the neovascular tufts in the same area of retina in some patients [57], however, suggests that the treatment had reduced perfusion of the vessels, but apparently had not caused regression.

MOLECULAR CAUSES OF CAPILLARY DEGENERATION

The molecular mechanisms by which capillary degeneration occurs in diabetes have not been studied in humans, human studies instead focusing on the retinopathy as a whole. Thus, the primary focus of the present discussion on molecular causes of dia- betes-induced degeneration of retinal capillaries will focus largely on animal studies. Factors or pathways involved in the capillary degeneration in early stages of diabetic retinopathy have been identified primarily using pharmacologic inhibitors or genetically modified animals.

Metabolic control. Intensive insulin therapy, blood pressure medications, and lipid-low- ering therapy all have been shown to inhibit the development of diabetic retinopathy in patients [59–63]. Consistent with this, animal studies have demonstrated that these therapies likewise inhibited degeneration of the retinal vasculature in diabetes [64–67], and they demonstrate that the therapies did inhibit degeneration of the retinal vasculature. Likewise, lipid levels have been shown to influence the development or progression of the retinopathy in diabetic animals [68, 69].

Pathways secondary to poor metabolic control of diabetes. Metabolic sequelae of hyperglycemia have been extensively studied to identify potential causes responsible for the development of diabetic retinopathy and its associated vascular abnormalities. A variety of therapies have reduced the number of TUNEL-positive capillary cells or degenerate capillaries compared to control [27, 33, 39, 44, 48, 67, 70–77], suggesting that related metabolic abnormalities also contribute to the capillary cell death. Tables 1 and 2 summarize a number of therapies or genetic modifications that have been reported to inhibit degeneration of retinal capillaries in diabetic animals. TUNEL-positive retinal capillary cells are a much less reproducible finding in diabetic mice than in diabetic rats (Kern, unpublished).

UNEXPLAINED ASPECTS OF DIABETES-INDUCED DEGENERATION OF RETINAL CAPILLARIES

Nonuniform degeneration of capillaries within the same retina. Despite the evidence indicating that hyperglycemia is a (or the) major determinant of capillary degeneration in diabetic retinopathy, capillary degeneration (like other lesions of the retinopathy) does not develop uniformly across even the same retina of diabetic dogs or patients [78, 79]. The superior temporal portion of retina develops significantly more pathology than, for example, inferior nasal retina. Likewise, midperipheral retina is more prone to undergo capillary nonperfusion in diabetic retinopathy than is the posterior or anterior retina [13].

Why does it take so long for capillary degeneration to become apparent in diabetic retinopathy? As mentioned earlier, vascular remodeling is a normal process, and so all