Abstract 008: Perivascular Cell-specific Knockout of the Stem Cell Pluripotency Gene Oct4 Inhibits Angiogenesis in Part by Attenuating Perivascular and Endothelial Cell Migration

Abstract

Objective: Angiogenesis requires coordinated migration of endothelial cells (EC) and perivascular cells, including smooth muscle cells and pericytes (SMC-P). Perivascular cell-specific mechanisms by which SMC-P migrate and invest EC remain largely unknown. Herein, we used Myh11-CreER T2 SMC-P lineage tracing, combined with SMC-P specific knockout of the stem cell pluripotency gene Oct4, to test the hypothesis that SMC-P derived Oct4 regulates perivascular cell migration and recruitment necessary for angiogenesis. Methods and Results: Myh11-CreER T2 ROSA floxed STOP eYFP Oct4 WT/WT and Myh11-CreER T2 ROSA floxed STOP eYFP Oct4 FL/FL littermate mice were injected with tamoxifen from 6-8 weeks of age to permanently label Myh11-expressing cells with eYFP, without or with Oct4 KO, respectively. Mice were subjected to either corneal alkali burn or hindlimb ischemia (HLI), monitored by live confocal imaging and laser doppler perfusion respectively, and sacrificed for tissue analysis. SMC-P Oct4 KO resulted in markedly impaired eYFP+ (SMC-P derived) migration and increased vascular leak following corneal alkali burn. EC neovascular area and migration distance were also significantly decreased in SMC-P specific Oct4 KO mice. Following HLI, SMC-P Oct4 KO mice had impaired perfusion recovery and decreased capillary density. Slit3 was expressed in eYFP+ cells of the microvasculature in both cornea and muscle and was downregulated following Oct4 KO. RNA-seq and qRT-PCR analysis of cultured SMC revealed dysregulation of Slit3 as well as additional members of the Slit-Robo pathway of guidance genes, including downregulation of the receptors Robo1 and Robo2 and upregulation of Slit2 following loss of Oct4. Conclusions: Taken together, we demonstrate that SMC-P derived Oct4 is essential for angiogenesis in both corneal alkali burn and HLI models. These effects are at least partially due to Oct4-dependent regulation of the Slit-Robo pathway in SMC-P, with Oct4 KO resulting in dysregulated migration of both EC and SMC-P. To our knowledge, this is the first direct evidence that loss of a single gene exclusively in SMC-P impacts angiogenesis following injury.