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Wendy Dailey, Kenneth P Mitton, Kimberly Drenser, Kevin Roumayah, Michael Thomas Trese; Norrin induces expression of genes characteristic of the BRB in primary human Retinal Endothelial Cells.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1553.
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VEGF initiates break down of the BRB by increasing the expression of enzymes that degrade the cell-to-cell junction proteins and by inducing leukocyte adhesion proteins that allow monocytes to stick to the lining of the vessels. Norrin is a Wnt signaling ligand that can stabilize vessels, restoring the BRB in the OIR mouse model retina. Here we examined the expression of genes involved in the BRB to identify norrin induced changes at a cellular level.
mRNA was isolated from Human Retinal Microvascular Endothelial (HRMECs) that had been cultured for 24 hours with or without norrin (250 ng/ml). Two SuperArray, 84 gene, RT-PCR Arrays (Angiogenesis & Endothelial Cell Growth Factor) were then used to compare expression differences in control and norrin treated HRMECs. Additionally, HRMECs were treated with VEGFA165b (100ng/ml) with or without Norrin (250ng/ml) for 24hrs and Claudin-5 and VE-Cadherin Taqman assays were used to compare changes in the expression of these cell-to-cell junction proteins.
In the “Angiogenesis” panel seven genes were down-regulated by greater than 2X after norrin treatment; BMP2 (-31), TGFA (-9), HGF (-5), CXCL6 (-4),TYMP (-3), FOX04 (-2) and SPINK5 (-2) . In the “Endothelial Cell” panel fifteen genes were down-regulated greater than 4X however most of these had low expression in both treated & control samples. The exception was EDN1 which had a moderate level of expression in the control and was 5 fold lower in the norrin treated HRMECs. Other genes down regulated were PDGFRA (-67), TNFSF10 (-54), SELPLG (-31), PLAU (-31), ANGPT1 (-34) and COL18A (-3). No genes in the Angiogenesis panel were up-regulated. The expression of two genes in the Endothelial panel were increased by two fold; SERPINE1 (2) & THBD (2). VEGFA, itself was not dysregulated. Both Claudin-5 and VE-Cadherin expression was greater in cells treated with a [Norrin and VEGFA165b] versus those treated with VEGF165b alone.
Norrin appears to alter the expression of several genes involved in plasmin induced ECM degradation (SPINK5, PLAU, SERPINE1). A decrease in the expression of two genes (SELPLG & BMP2) involved in leukocyte adhesion was also seen. Importantly, the expression of Claudin-5 and VE-Cadherin genes was greater when norrin was added to VEGF induced HRMECs. Norrin may act in multiple ways to shore up the BRB.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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