Thrombospondin-2 Expression During Retinal Vascular Development and Neovascularization

Abstract

Purpose: To determine thrombospondin-2 (TSP2) expression and its impact on postnatal retinal vascular development and retinal neovascularization. Methods: The TSP2-deficient (TSP2 -/ -) mice and a line of TSP2 reporter mice were used to assess the expression of TSP2 during postnatal retinal vascular development and neovascularization. The postnatal retinal vascularization was evaluated using immunostaining of wholemount retinas prepared at different postnatal days by collagen IV staining and/or TSP2 promoter driven green fluorescent protein (GFP) expression. The organization of astrocytes was evaluated by glial fibrillary acidic protein (GFAP) staining. Retinal vascular densities were determined using trypsin digestion preparation of wholemount retinas at 3-and 6-weeks of age. Retinal neovascularization was assessed during the oxygen-induced ischemic retinopathy (OIR). Choroidal neovascularization (CNV) was assessed using laser-induced CNV. Results: Using the TSP2-GFP reporter mice, we observed significant expression of TSP2 mRNA in retinas of postnatal day 5 (P5) mice, which increased by P7 and remained high up to P42. Similar results were observed in retinal wholemount preparations, and western blotting for GFP with the highest level of GFP was observed at P21. In contrast to high level of mRNA at P42, the GFP fluorescence or protein level was dramatically downregulated. The primary retinal vasculature developed at a faster rate in TSP2-/ -mice compared with TSP2 + / + mice up to P5. However, the developing retinal vasculature in TSP2 + / + mice caught up with that of TSP2 -/ -mice after P7. No significant differences in retinal vascular density were observed at 3-or 6-weeks of age. TSP2-/ -mice also exhibited a similar sensitivity to the hyperoxia-mediated vessel obliteration and similar level of neovascularization during OIR as TSP2 + / + mice. Lack of TSP2 expression minimally affected laserinduced CNV compared with TSP2 + / + mice. Conclusions: Lack of TSP2 expression was associated with enhanced retinal vascularization during early postnatal days but not at late postnatal times, and minimally affected retinal and CNV. However, the utility of TSP2 as a potential therapeutic target for inhibition of ocular neovascularization awaits further investigation.

Negative regulators of angiogenesis: important targets for treatment of exudative AMD

“…This was further confirmed by using wholemount staining and Western blot analysis of TSP2-GFP reporter mice. However, TSP2 protein levels (GFP-reporter) were undetectable at P42 [72]. This is consistent with limited expression of TSP2 protein observed in adult retina.…”

“…We showed that the primary retinal vasculature develops at a faster rate in Thbs2 −/− mice with a minimal impact on the rate of vascular cell proliferation and apoptosis. Thbs2 -deficiency also minimally affected the degree of retinal and choroidal neovascularization [72]. However, TSP2 level was significantly up-regulated in P17 mice subjected to OIR.…”

“…This could be attributed to increased oxidative stress at this stage and needs further verification. We also showed that although lack of TSP1 had no effect on expression of TSP2 level during normal development or OIR in the retina, the level of TSP1 was significantly lower in the absence of TSP2 [72]. Thus, lack of TSP2 expression in the retina does not cause a compensatory increase in TSP1 levels.…”

Negative regulators of angiogenesis, ocular vascular homeostasis, and pathogenesis and treatment of exudative AMD

“…[ 26 ] In the retinal vasculature, pericytes are the major source of TSP2 expression, and its expression is further enhanced under oxidative stress. [ 27 28 ] However, the cell autonomous role of TSP2 expression in retinal vascular cells and the characteristics of TSP2-null vascular cells compared with wild type cells have yet to be studied, as we have reported for TSP1. However, we recently demonstrated that the germline deletion of TSP2 minimally affects the postnatal retinal vascular development and neovascularization.…”

“…However, we recently demonstrated that the germline deletion of TSP2 minimally affects the postnatal retinal vascular development and neovascularization. [ 28 ]…”