Differential expression of angiogenic factors during postnatal retinal vascular development in Norrie disease mice.

Abstract

Abstract: : Purpose:To characterize defects of the retinal vasculature of Ndph knockout (Ko) mice and to identify the underlying molecular pathways affected by the Ndph knockout mutation during postnatal retinal development. Methods: For visualization of retinal vasculature whole mount retinal preparations of different time points (postnatal day 5 (P5) to 21) stained with lectin B4 (Bandeiraea simplicifolia) were used. To study the expression of factors involved in sprouting angiogenesis, retinae of Ndph Ko mice and age–matched controls (n = 4) were prepared from p5–p21, and DNaseI treated total RNA was used for quantitative RT–PCR by the ABI PRISM 7000 Sequence Detection System together with MGB–TaqMan probes for Agpt1;Agpt2; Efnb2; Ephb4; Fzd4; Igf1; Igf2; Itgav; Itgb3; Ndph; Pdgfb; Pdgfrb; Tie1; Tie2; Vegfr1 (Flt–1); Vegfr2 (Flk–1)and Vegfa (Assay by design; ABI, Rotkreuz, Switzerland). Results: A major delay in the outgrowth of the superficial retinal vessels towards the periphery and a complete absence of the deep capillary network was observed. The failure of sprouting angiogenesis into the deep retinal layers was represented by a cauliflower–like cell clump consisting of both endothelial cells and pericytes. Gene expression studies by real time PCR at P5 and P10 identified slightly but significantly decreased transcript levels for Pdgfb, Pdgfrb and Tie1 in knockout mice. From P10 onwards, an increasing Vegfa level was detected, whereas other factors, in particular angiopoietins, appeared unaffected. At P15 and P21, the same factors that were reduced at P5 and P10 showed increasing levels of expression. Of particular note, a 4 – 6 fold increase of Integrin ß₃ transcript occurred between P 10 and P21. Conclusions: These data suggest an important role of the Ndph protein for the outgrowth of retinal vascular sprouts during development of the deeper retinal capillaries. Compensatory upregulation of integrins involved in angiogenesis are observed. Upregulation of Vegfa likely reflects the hypoxic situation within the retinas of Ndph knockout mice.

Keywords: retinal development • gene/expression • vascular cells