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Aims Atherosclerosis develops near branches and bends of arteries that are exposed to disturbed blood flow which exerts low wall shear stress (WSS). These mechanical conditions alter endothelial cells (EC) by priming them for inflammation and by inducing turnover. Homeobox (Hox) genes are developmental genes involved in the patterning of embryos along their anterior–posterior and proximal–distal axes. Here we identified Hox genes that are regulated by WSS and investigated their functions in adult arteries. Methods and results EC were isolated from inner (low WSS) and outer (high WSS) regions of the porcine aorta and the expression of Hox genes was analysed by quantitative real-time PCR. Several Hox genes (HoxA10, HoxB4, HoxB7, HoxB9, HoxD8, HoxD9) were significantly enriched at the low WSS compared to the high WSS region. Similarly, studies of cultured human umbilical vein EC (HUVEC) or porcine aortic EC revealed that the expression of multiple Hox genes (HoxA10, HoxB9, HoxD8, HoxD9) was enhanced under low (4 dyn/cm2) compared to high (13 dyn/cm2) WSS conditions. Gene silencing studies identified Hox genes (HoxB9, HoxD8, HoxD9) that are positive regulators of inflammatory molecule expression in EC exposed to low WSS, and others (HoxB9, HoxB7, HoxB4) that regulated EC turnover. We subsequently focused on HoxB9 because it was strongly up-regulated by low WSS and, uniquely, was a driver of both inflammation and proliferation. At a mechanistic level, we demonstrate using cultured EC and murine models that bone morphogenic protein 4 (BMP4) is an upstream regulator of HoxB9 which elicits inflammation via induction of numerous inflammatory mediators including TNF and downstream NF-κB activation. Moreover, the BMP4-HoxB9-TNF pathway was potentiated by hypercholesterolaemic conditions. Conclusions Low WSS induces multiple Hox genes that control the activation state and turnover of EC. Notably, low WSS activates a BMP4-HoxB9-TNF signalling pathway to initiate focal arterial inflammation, thereby demonstrating integration of the BMP and Hox systems in vascular pathophysiology.

Introduction Atherosclerosis, a disease of arteries that can cause heart attack and stroke, is influenced by local blood flow patterns which exert wall shear stress (WSS) on endothelial cells (EC). Low, oscillatory WSS (LWSS) promotes atherosclerosis by inducing EC apoptosis and activation, while high, unidirectional WSS (HWSS) is athero-protective. We recently used microarray technology coupled to computational fluid dynamics to study the transcriptome of EC at regions of the porcine aorta exposed to LWSS or HWSS. The study revealed differential expression of multiple genes (GATA4, HAND2, TWIST1, FZD5, BMP2, SLIT2, PDGFRA, FBN2 and GJA5) that co-ordinate embryonic development. We hypothesised that this gene set includes regulators of EC survival in response to haemodynamic forces. Methods EC were isolated from inner (LWSS) and outer (HWSS) curvatures of the porcine aortic arch using collagenase and the expression of particular developmental genes was determined using quantitative RT-PCR (n = 6). The expression of GATA4 in the murine aorta was assessed at the protein level by en face fluorescent staining and confocal microscopy (n = 5). Porcine aortic EC (n = 7) or human umbilical vein EC (n = 7) were exposed to flow using an orbiting 6-well plate (210 rpm) which generates LWSS (centre) and HWSS (periphery). Alternatively, cells were exposed to LWSS or HWSS using an IBIDI™ pump system (n = 3). The expression of specific developmental genes was determined by quantitative RT-PCR. Developmental genes were silenced in sheared EC using two different gene-specific siRNA sequences prior to assessment of apoptosis using antibodies that recognise active caspase-3 (n = 3). Results Quantitative RT-PCR revealed that the expression of multiple developmental genes (GATA4, HAND2, TWIST1, FZD5, BMP2, SLIT2, PDGFRA, and FBN2) was elevated at the LWSS compared to the HWSS region of the porcine aorta (p < 0.05). Similarly, en face staining demonstrated that expression of GATA4 protein in EC was higher at a LWSS compared to a HWSS site of the murine aortic arch (p < 0.05). GATA4, HAND2, TWIST1, FZD5, BMP2, SLIT2, PDGFRA, and FBN2 were induced in HUVEC or PAEC exposed to LWSS for 72 h using either the orbital or IBIDI™ systems (p < 0.05). The rate of caspase-3 activation was significantly higher in EC exposed to LWSS compared to HWSS (3.1% vs 0.7%; p < 0.0001; n = 10). Silencing of GATA4, FZD5 and BMP2 significantly reduced apoptosis in EC exposed to LWSS (1.2%, 0.6% and 1.6%; p < 0.05). Conclusions We conclude that LWSS promotes EC apoptosis via a mechanism that involves the induction of GATA4, FZD5 and BMP2; molecules that have a well-defined role in embryonic development. Further work is required to define the molecular mechanisms that underly the induction of apoptosis by these molecules. Our observations illuminate the molecular mechanisms that regulate the focal nature of vascular injury and atherosclerosis.