The CCN1-YAP regulatory pathway is a novel angiomodulatory circuit that controls vascular growth and expansion in the retina

Abstract

Purpose : CCN1 is a secreted extracellular matrix protein that localizes pericellularly and plays an essential role in local angiomodulation and barriergenesis in the retina. CCN1 is a bona fide target of the transcriptional coactivator Yes-Associated Protein (YAP), a core component of the Hippo pathway that controls cell differentiation and tissue expansion. In cultured endothelial cells (ECs), a positive feedforward loop between vascular endothelial growth factor (VEGF) and YAP promotes the mitogenic effects of VEGF. Conversely, CCN1 signals are associated with YAP phosphorylation and inactivation. This study is designed to determine the in vivo relevance of the CCN1-YAP crosstalk. Herein, we test the hypothesis that CCN1 is both an upstream regulator and downstream target of YAP and that the negative CCN1-YAP feedback circuit regulates angiogenic cell behavior and function in the retina.

Methods : Loss-of-function of CCN1 or YAP is carried out by crossing mice with floxed CCN1 or YAP alleles with tamoxifen-activated UBC-CreER^T2 mice. Retinal vascular alterations are characterized at postnatal day (P) 4 and P7 using immunohistochemical and biochemical techniques. CCN1 promoter enrichment with YAP is determined by chromatin immunoprecipitation (ChIP) assay. Gene and protein expression is quantified by quantitative real time PCR and immunoblotting. CCN1 and YAP functional interactions in human retinal ECs is examined by ChIP and phosphorylation assays. Two-tailed Student’s t-test is used for statistical analyses.

Results : YAP interacts with the myocardin-related transcription factor to activate the endogenous CCN1 promoter. Loss of YAP function in mice is associated with 71% reduction of CCN1 promoter enrichment with YAP, decreased CCN1 gene expression and reduced retinal EC migration and differentiation. Conversely, loss of function of CCN1 in mice is associated with accumulation of active non-phosphorylated YAP leading to EC hyperprolifeartion and formation of a thicker denser vasculature. Sustained expression of CCN1 in VEGF-stimulated ECs provides the cells with a soft compliant matrix putting them in in low contractility, YAP repressive quiescent states.

Conclusions : CCN1-YAP crosstalk promotes correct sprouting and branching patterns of developing blood vessels in the retina. It likely shapes gradients of VEGF activity in areas of uniform ligand expression.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.