P694The induction of homeobox genes by disturbed flow limits inflammation at atherosusceptible sites

Abstract

Atherosclerosis develops at branches and bends of arteries that are exposed to disturbed blood, whereas regions exposed to uniform flow are protected. Disturbed flow generates low, oscillatory wall shear stress (WSS) which promotes atherosclerosis by inducing EC expression of inflammatory molecules. Conversely, high unidirectional WSS is protective. We studied the transcriptome at low and high WSS regions in the porcine aorta using micro arrays and observed differential expression of multiple Homeobox (Hox) genes. These transcription factors regulate embryonic development and morphogenesis. We hypothesize that Hox genes also influence EC responses to WSS. This project aims to: (1) assess the influence of WSS on Hox gene expression, (2) assess the effects of Hox genes on inflammatory activation in sheared EC. EC were isolated from high and low WSS regions of the porcine aorta, prior to measurement of Hox gene expression by qPCR. The expression of HoxB7 and HoxB9 in the murine aorta was assessed at the protein level by en face fluorescent staining. Porcine aortic EC and human umbilical vein EC were exposed to shear stress for 72 hours using an in vitro orbital shaking system. Alternatively, cells were exposed to oscillatory or unidirectional WSS using a commercial pump system. The expression of Hox genes at these regions was assessed by qPCR using gene specific primers. Gene silencing technologies were used to attenuate Hox expression in sheared EC, and subsequent effects on inflammatory activation was assessed by qPCR and immunofluorescent staining. qPCR revealed that EC expression of multiple Hox genes was increased at the inner curvature (low WSS) compared to the outer curvature (high WSS) of the porcine aorta. Similarly, en face staining demonstrated that expression of HoxB7 and HoxB9 proteins in EC were higher at the inner compared to the outer curvature of the murine aortic arch. Consistent with these observations, low oscillatory WSS induced the expression of the above Hox genes in cultured HUVEC and PAEC, while high unidirectional WSS did not. Silencing of HoxB9 and HoxB7 significantly enhanced the expression of E-selectin, MCP1 and VCAM-1 in EC exposed to low oscillatory WSS, indicating that Hox genes exert anti-inflammatory effects. We conclude that disturbed flow induces multiple Hox genes that limit inflammatory activation of EC. These findings shed light on the complex effects of WSS on inflammatory signaling pathways and indicate that vascular inflammation at atheroprone sites is governed by a balance between the activities of pro- and anti-inflammatory molecules.