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Derick G. Holt, Leah Owen, Helen Huang, Subrata K. Das, Jacquelyn Simonas, Balamurali K. Ambati; Identification Of Novel Sflt-1 Regulators Through Genome Wide Expression Analysis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6409.
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Corneal neovascularization (KNV) is a central feature in the pathogenesis of many blinding corneal disorders. We have previously demonstrated that the soluble form of VEGF receptor 1 (sFlt-1) is the main preserver of corneal avascularity. In order to identify novel genes involved in the regulation of KNV, we have utilized genome-wide microarray analysis of three mice strains who share the same genetic background but have differing susceptibility to KNV. Statistical analysis was performed to identify groups of genes according to their expression patterns and functional properties. Further, we utilize knock-down techniques to investigate whether candidate genes of interest may act through regulation of sFlt-1.
Microarray data obtained from corneal samples derived from MRL/MpJ, C57/B6, and Pax6+/- mice were analyzed using genome-wide clustering and Sequential Bonferroni analyses. Reverse-transcriptase PCR was utilized to independently confirm the expression patterns of specific genes of interest. Knock-down experiments using target-specific siRNAs were carried out in a cell culture system.
Differentially expressed genes fell into several important functional classes including extracellular matrix regulators, angiogenesis factors, transcription factors, and regulators of alternative splicing. The expression pattern of several candidate genes was confirmed using quantitative RT-PCR. Raver2 and SamHD1 were identified as candidate positive and negative regulators of sFlt-1 expression, respectively. Knock down experiments using plasmids expressing shRNA suggest that two novel factors may act to regulate sFlt-1 levels.
Genome-scale microarray analysis across a spectrum of murine models has identified several promising candidate regulators of corneal avascularity, including two factors which may act by modulating the expression of the key regulator sFlt-1.
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