Three of the major biochemical pathways implicated in the pathogenesis of hyperglycemia induced vascular damage (the hexosamine pathway, the advanced glycation end product (AGE) formation pathway and the diacylglycerol (DAG)-protein kinase C (PKC) pathway) are activated by increased availability of the glycolytic metabolites glyceraldehyde-3-phosphate and fructose-6-phosphate. We have discovered that the lipid-soluble thiamine derivative benfotiamine can inhibit these three pathways, as well as hyperglycemia-associated NF-kappaB activation, by activating the pentose phosphate pathway enzyme transketolase, which converts glyceraldehyde-3-phosphate and fructose-6-phosphate into pentose-5-phosphates and other sugars. In retinas of diabetic animals, benfotiamine treatment inhibited these three pathways and NF-kappaB activation by activating transketolase, and also prevented experimental diabetic retinopathy. The ability of benfotiamine to inhibit three major pathways simultaneously might be clinically useful in preventing the development and progression of diabetic complications.

Retinal capillary closure induced by hyperglycemia is the principal pathophysiologic abnormality underlying diabetic retinopathy, but the mechaniss by which this induction occurs are not clear. Treatment of diabetic rats for 26 weeks with aminouanidine, an inhibitor of advanced glycosylation product formation, prevented a 2.6-fold accumulation of these products at branching sites of precapillary arterioles where abnormal periodic acid/Schiff reagentpositive deposits also occurred. A dine treatment completely prevented abnormal endothelial cell proliferation and signiflcantiy diminshed pericyte dropout. After 75 weeks, untreated diabetic animals developed an 18.6-fold increase in the number of acellular capillaries and formed capillary microaneurysms, characteristic pathologic features of background diabetic retinopathy. In contrast, aminoguanidinetreated diabetic animals had only a 3.6-fold increase in acellular capillaries and no microaneurysms. These findings indicate that advanced glycosylation product accumulation contributes to the development of diabetic retinopathy and suggest that a anqidine may have future therapeutic use in this disorder.Pathologic retinal changes are found in virtually 100%o of patients having type I diabetes for 15 yr or longer (1). Underlying the complex clinical manifestations of diabetic retinopathy are two fundamental abnormalities: increased retinal vascular permeability and progressive retinal vessel closure (2, 3). Digested retinal vasculature preparations from diabetic patients and animals characteristically show early pericyte loss which is followed by acellular capillary development and microaneurysm formation (4,5). The severity of these changes is associated with the degree of chronic hyperglycemia to which the diabetic retina has been exposed, but the mechanisms by which elevated glucose levels cause retinal damage are currently not known (6, 7). Potentially important mechanisms include increased polyol pathway activity, activation of protein kinase C by de novo diacylglycerol synthesis, and altered cell/matrix functions induced by accumulated advanced glycosylation end products (8-11).Recently the nucleophilic hydrazine compound aminoguanidine has been shown to inhibit the formation of advanced glycosylation products on collagen and basement membrane both in vitro and in vivo (12,13 pepsin/0.2% HCL. After 10 min in a water bath at room temperature, the retina was subjected to digestion by 3% trypsin/0.2 M Tris for 3.5 hr at 370C to remove the inner limiting membrane. The sample was then placed on a glass slide, and further digestion with trypsin was monitored under a dissecting microscope. The intact isolated retinal vessel preparation was washed with distilled H20 and air-dried. Retinal vessel preparations were stained with periodic acid/ Schiff reagent/hematoxylin (15) and photographed using a

No abstract

Pericyte loss is an early pathologic feature of diabetic retinopathy, consistently present in retinae of diabetic humans and animals. Because pericyte recruitment and endothelial cell survival are controlled, in part, by the angiopoietin/Tie2 ligand/receptor system, we studied the expression of angiopoietin-2 and -1 in relation to the evolution of pericyte loss in diabetic rat retinae, using quantitative retinal morphometry, and in retinae from mice with heterozygous angiopoietin deficiency (Ang-2 LacZ knock-in mice). Finally, recombinant angiopoietin-2 was injected into eyes of nondiabetic rats, and pericyte numbers were quantitated in retinal capillaries. Angiopoietin-1 protein was present in the normal maturing retina and was upregulated 2.5-fold in diabetic retinae over 3 months of diabetes. In contrast, angiopoietin-2 protein was consistently upregulated more than 30-fold in the retinae of diabetic rats, preceding the onset of pericyte loss. Heterozygous angiopoietin-2 deficiency completely prevented diabetes-induced pericyte loss and reduced the number of acellular capillary segments. Injection of angiopoietin-2 into the eyes of normal rats induced a dose-dependent pericyte loss. These data show that upregulation of angiopoietin-2 plays a critical role in the loss of pericytes in the diabetic retina. Diabetes 53

ADAM15 (named for a disintegrin and metalloprotease 15, metargidin) is a membrane-anchored glycoprotein that has been implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. To characterize the potential roles of ADAM15 during development and in adult mice, we analyzed its expression pattern by mRNA in situ hybridization and generated mice carrying a targeted deletion of ADAM15 (adam15 ؊/؊ mice). A high level of expression of ADAM15 was found in vascular cells, the endocardium, hypertrophic cells in developing bone, and specific areas of the hippocampus and cerebellum. However, despite the pronounced expression of ADAM15 in these tissues, no major developmental defects or pathological phenotypes were evident in adam15 ؊/؊ mice. The elevated levels of ADAM15 in endothelial cells prompted an evaluation of its role in neovascularization. In a mouse model for retinopathy of prematurity, adam15 ؊/؊ mice had a major reduction in neovascularization compared to wild-type controls. Furthermore, the size of tumors resulting from implanted B16F0 mouse melanoma cells was significantly smaller in adam15 ؊/؊ mice than in wild-type controls. Since ADAM15 does not appear to be required for developmental angiogenesis or for adult homeostasis, it may represent a novel target for the design of inhibitors of pathological neovascularization.Metalloprotease-disintegrins (ADAMs) are a family of membrane-anchored glycoproteins that are related to snake venom metalloproteases and integrin ligands, termed disintegrins. ADAMs have been implicated in fertilization, myogenesis, neurogenesis, and protein ectodomain shedding (for recent reviews, see references 5, 55, 62, and 63). ADAMs are also thought to have roles in cell-cell adhesion, for example, through interactions with integrins (9, 18, 49) or syndecans (32, 71). Over 30 ADAMs have been identified to date, and yet the function of less than a third of these proteins during development and in adulthood has been characterized. ADAM1 and -2 (fertilin ␣ and ␤) and ADAM3 have critical roles in fertilization (12, 13, 50, 64). ADAM9 and -12 are not required for development or adult survival (39, 76), although ADAM12 reportedly has a role in processing the heparin-binding epidermal growth factor (EGF)-like growth factor HB-EGF (3), in adipogenesis and myogenesis (34, 39), and in the pathology of muscular dystrophy (38). ADAM13 has been implicated in cranial neural crest migration in Xenopus laevis (2). The tumor necrosis factor alpha-converting enzyme (TACE/ADAM17) has a role in releasing the ectodomain of a number of physiologically relevant proteins, including tumor necrosis factor alpha (4, 47); ligands of the EGF receptor, such as HB-EGF (45, 70) and transforming growth factor ␣ (52); and the amyloid precursor protein (8). ADAM17/TACE is essential for survival of newborn mice, presumably because transforming growth factor ␣ and other ligands of the EGF receptor require functional TACE for their proper activation (...

Methylglyoxal is a highly reactive dicarbonyl degradation

Pericytes provide vascular stability and control endothelial proliferation. Pericyte loss, microaneurysms, and acellular capillaries are characteristic for the diabetic retina. Platelet-derived growth factor (PDGF)-B is involved in pericyte recruitment, and brain capillaries of mice with a genetic ablation of PDGF-B show pericyte loss and microaneurysms. We investigated the role of capillary coverage with pericytes in early diabetic retinopathy and the contribution to proliferative retinopathy using mice with a single functional allele of PDGF-B (PDGF-B ؉/؊ mice). As assessed by quantitative morphometry of retinal digest preparations, pericyte numbers in nondiabetic PDGF-B ؉/؊ mice were reduced by 30% compared with wild-type mice, together with a small but significant increase in acellular capillaries. Pericyte numbers were reduced by 40% in diabetic wild-type mice compared with nondiabetic wild-type controls. Pericyte numbers were decreased by 50% in diabetic PDGF-B ؉/؊ mice compared with nondiabetic wild-type littermates, and the incidence of acellular capillaries was increased 3.5-fold when compared with nondiabetic PDGF-B ؉/؊ mice. To investigate the effect of pericyte loss in the context of ongoing angiogenesis, we subjected mice to hypoxia-induced proliferative retinopathy. As a result, PDGF-B ؉/؊ mice developed twice as many new blood vessels as their wild-type littermates. We conclude that retinal capillary coverage with pericytes is crucial for the survival of endothelial cells, particularly under stress conditions such as diabetes. At high vascular endothelial growth factor levels, such as those in the retinopathy of prematurity model, pericyte deficiency leads to reduced inhibition of endothelial proliferation in vivo. Diabetes 51:3107-3112, 2002

Vascular endothelial growth factor (VEGF) is a major contributor to retinal neovascularization. The possible participation of VEGF and its high-affinity tyrosine kinase receptors, flk-1 and flt-1, in early background diabetic retinopathy was studied in the streptozotocin-induced diabetic rat model of experimental retinopathy using in situ hybridization, blotting techniques, and immunohistochemistry. Diabetic retinopathy was assessed by quantitative morphometry of retinal digest preparations. The number of acellular capillaries increased 2.7-fold in diabetic animals with diabetes' duration of 6 months compared with nondiabetic controls. VEGF expression was not detectable by in situ hybridization in nondiabetic rats but was highly increased in the ganglion cell layer and in the inner and outer nuclear layers of retinas from diabetic animals. VEGF protein was extractable only from diabetic retinas, and a strong immunolabeling was detected in vascular and perivascular structures. Increased flk-1 and flt-1 mRNA levels were also found in the ganglion cell and both nuclear layers of diabetic samples only. Dot blot and Western blot analyses confirmed the increase in flk-1 mRNA and protein in diabetic retinas. Also, flk-1 immunoreactivity was associated with vascular and nonvascular structures of the inner retinas from diabetic animals. These data obtained from a rodent model in which retinal neovascularization does not occur support the concept that the VEGF/VEGF receptor system is upregulated in early diabetic retinopathy.