Y Feng

Oral Carnosine Supplementation Prevents Vascular Damage in Experimental Diabetic Retinopathy

Backgrounds/Aims: Pericyte loss, vasoregression and neuroglial activation are characteristic changes in incipient diabetic retinopathy. In this study, the effect of the antioxidant and antiglycating dipeptide carnosine was studied on the development of experimental diabetic retinopathy. Materials/Methods: STZ-induced diabetic Wistar rats were orally treated with carnosine (1g/kg body weight/day). Retinal vascular damage was assessed by quantitative morphometry. Retinal protein extracts were analyzed for markers of oxidative stress, AGE-formation, activation of the hexosamine pathway and changes in the expression of Ang-2, VEGF and heat shock proteins Hsp27 and HO-1. Glial cell activation was analyzed using Western blot analysis and immunofluorescence of GFAP expression and retinal neuronal damage was histologically examined. Results: Oral carnosine treatment prevented retinal vascular damage after 6 months of experimental hyperglycemia. The protection was not caused by ROS- or AGE-inhibition, but associated with a significant induction of Hsp27 in activated glial cells and normalization of increased Ang-2 levels in diabetic retinas. A significant reduction of photoreceptors in retinas of carnosine treated animals was noted. Conclusion: Oral carnosine treatment protects retinal capillary cells in experimental diabetic retinopathy, independent of its biochemical function. The vasoprotective effect of carnosine might be mediated by the induction of protective Hsp27 in activated glial cells and normalization of hyperglycemia-induced Ang-2.

Endothelial survival factors and spatial completion, but not pericyte coverage of retinal capillaries determine vessel plasticity

Pericyte loss and capillary regression are characteristic for incipient diabetic retinopathy. Pericyte recruitment is involved in vessel maturation, and ligand‐receptor systems contributing to pericyte recruitment are survival factors for endothelial cells in pericyte‐free in vitro systems. We studied pericyte recruitment in relation to the susceptibility toward hyperoxia‐induced vascular remodeling using the pericyte reporter X‐LacZ mouse and the mouse model of retinopathy of prematurity (ROP). Pericytes were found in close proximity to vessels, both during formation of the superficial and the deep capillary layers. When exposure of mice to the ROP was delayed by 24 h, i.e., after the deep retinal layer had formed [at postnatal (p) day 8], preretinal neovascularizations were substantially diminished at p18. Mice with a delayed ROP exposure had 50% reduced avascular zones. Formation of the deep capillary layers at p8 was associated with a combined up‐regulation of angiopoietin‐1 and PDGF‐B, while VEGF was almost unchanged during the transition from a susceptible to a resistant capillary network. Inhibition of Tie‐2 function either by soluble Tie‐2 or by a sulindac analog, an inhibitor of Tie‐2 phosphorylation, resensitized retinal vessels to neovascularizations due to a reduction of the deep capillary network. Inhibition of Tie‐2 function had no effect on pericyte recruitment. Our data indicate that the final maturation of the retinal vasculature and its resistance to regressive signals such as hyperoxia depend on the completion of the multilayer structure, in particular the deep capillary layers, and are independent of the coverage by pericytes.

Mediation of FoxO1 in Activated Neuroglia Deficient for Nucleoside Diphosphate Kinase B during Vascular Degeneration

The pathogenesis of diabetic retinopathy is closely associated with the breakdown of the neurovascular unit including the glial cells. Deficiency of nucleoside diphosphate kinase B (NDPK-B) results in retinal vasoregression mimicking diabetic retinopathy. Increased retinal expression of Angiopoietin-2 (Ang-2) initiates vasoregression. In this study, Muller cell activation, glial Ang-2 expression, and the underlying mechanisms were investigated in streptozotocin-induced diabetic NDPK-B deficient (KO) retinas and Muller cells isolated from the NDPK-B KO retinas. Muller cells were activated and Ang-2 expression was predominantly increased in Muller cells in normoglycemic NDPK-B KO retinas, similar to diabetic wild type (WT) retinas. Diabetes induction in the NDPK-B KO mice did not further increase its activation. Additionally, cultured NDPK-B KO Muller cells were more activated and showed higher Ang-2 expression than WT cells. Muller cell activation and Ang-2 elevation were observed upon high glucose treatment in WT, but not in NDPK-B KO cells. Moreover, increased levels of the transcription factor forkhead box protein O1 (FoxO1) were detected in non-diabetic NDPK-B KO Muller cells. The siRNA-mediated knockdown of FoxO1 in NDPK-B deficient cells interfered with Ang-2 upregulation. These data suggest that FoxO1 mediates Ang-2 upregulation induced by NDPK-B deficiency in the Muller cells and thus contributes to the onset of retinal vascular degeneration.

Identification of new genes and pathways involved in the vasoregression mimicking diabetic retinopathy

Aims: Diabetic retinopathy is the most common microangiopathy in diabetes. It is morphologically characterized by vasoregression including pericyte loss and increased formation of acellular capillaries. Recently, we characterized a transgenic rat with a universal overexpression of PKD-2, in which c- terminal polycystein-2 is truncated. The transgenic rats showed severe vasoregression in the retinas at the second month of life. To elucidate the mechanisms of vasoregression in the retina, we analysed transcriptional alterations in this transgenic rats. Materials and methods: Profiles of gene expression were evaluated in rats before and after vasoregression, at the first and the third month of life, respectively. Age-matched SD rats served as controls. RNA was isolated from individual retinas and microarray analysis was performed with affymetrix gene chips according to the protocols published (http://www.ma.uni-heidelberg.de/inst/zmf/affymetrix/). Results: Numerous genes were altered during retinal vasoregression. 3267 genes were upregulated while 2924 genes were significantly downregulated (cutoff p<0.001). Serine/cysteine peptidase inhibitor, lectin-galactose binding and CD74 antigen are the highest (approximately 6-fold) upregulated genes. A number of genes related to neuronal cells, glial cells and endothelial cells were regulated in 3-month transgenic retinas compared to control SD retinas. Analysis of signalling clusters showed many signalling pathways were significantly involved during vessel degeneration in the retina. The most significant pathways include fatty acid elongation in mitochondria, mRNA splicing, adipocytokine signaling pathways and antigen processing and presentation pathways. Conclusion: Our data from microarray analysis of a new transgenic rat, a model for vasoregression of diabetic retinopathy, suggests that we have identified a number of genes and pathways which are involved in the development of retinal vasoregression. Further analysis of such genes may provide new insights for discovering new mechanisms underlying vasoregression also in diabetic retinopathy.