Introduction Atherosclerosis develops at branches and bends of arteries exposed to disturbed blood flow, whereas regions exposed to uniform flow are protected. Disturbed flow generates low, oscillatory wall shear stress (WSS) which promotes atherosclerosis by inducing endothelial cell (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 microarrays and observed differential expression of multiple Homeobox (Hox) genes, which regulate embryonic development and morphogenesis. We hypothesise that Hox genes influence EC responses to WSS. This project aimed to: (1) assess the effects of WSS on Hox gene expression, and (2) use gene silencing to determine the effects of Hox genes on inflammatory activation.
Methods EC were isolated from the outer (high WSS) and inner (low WSS) curvatures of porcine aortae prior to measurement of Hox gene expression by quantitative real-time PCR (qPCR). The expression of HOXA9 and HOXB9 proteins in the murine aortic arch was assessed at the protein level by en face fluorescence staining. Human umbilical vein EC (HUVEC) were exposed to flow for 72 h using an in vitro orbital shaking system, which generates high WSS at the periphery of a cell culture well and low WSS at the centre. Alternatively, cells were exposed to oscillatory or unidirectional WSS using an IBIDI pump system. The expression of Hox genes in sheared HUVEC was assessed by qPCR. Hox genes were silenced in sheared EC prior to the assessment of inflammatory activation by qPCR and immunofluorescence staining.
Results qPCR revealed that EC expression of multiple Hox genes (HOXA1, HOXA9, HOXA10, HOXB4, HOXB7, HOXB9, HOXD8, HOXD9) was increased at the low WSS region compared to the high WSS region of the porcine aorta (p < 0.05). Similarly, en face staining demonstrated that endothelial expression of HOXA9 and HOXB9 proteins was higher at a low WSS site compared to a high WSS region (p < 0.05). Consistent with these observations, low oscillatory WSS induced the expression of the above Hox genes in cultured HUVEC while high unidirectional WSS did not. Silencing of HOXB9 significantly enhanced the expression of E-selectin, MCP1 and VCAM-1 in EC exposed to low oscillatory WSS (p < 0.05), indicating that HOXB9 exerts anti-inflammatory effects. Conversely, silencing of other Hox genes had little or no effect on inflammatory molecule expression.
Conclusions We conclude that disturbed flow induces multiple Hox genes and that HOXB9 limits inflammatory activation of EC. Thus, although disturbed flow promotes inflammation, it also activates anti-inflammatory HOXB9 which promotes physiological homeostasis. These findings highlight the complex effects of WSS on vascular inflammation and indicate that inflammation at atheroprone sites is governed by a balance between the activities of pro- and anti-inflammatory molecules.
- hox genes