Background Macrophages play a central role in the development of atherosclerosis. However, the signaling pathways that regulate their function are not well understood. The Rho-associated coiled-coil containing kinases (ROCK1 and ROCK2) are serine-threonine protein kinases that are involved in the regulation of the actin cytoskeleton. Recent studies suggest that ROCK1 in macrophages and bone marrow (BM)-derived cells mediates atherogenesis. However, a similar role for ROCK2 in the pathogenesis of atherosclerosis has not been determined. Methods and Results The BMs from wild-type (WT), ROCK2+/− and ROCK2−/− mice were transplanted into irradiated recipient LDLr−/− mice and atherosclerosis was induced with a 16-week high-cholesterol diet. Compared to WT BM transplanted (BMT) mice, ROCK2+/− BMT and ROCK2−/− BMT mice showed substantially less lipid accumulation in the aorta (8.46 ± 1.42% and 9.80 ± 2.34% vs. 15.64 ± 1.89%, p<0.01 for both) and decreased atherosclerotic lesions in the subaortic sinus (158.1 ± 44.4 and 330.1 ± 109.5 ×103μm2 vs. 520.2 ± 125.7 ×103μm2, p<0.01 for both). These findings correlated with decreased foam cell formation (2.27 ± 0.57 vs. 4.10 ± 0.3, p<0.01) and increased cholesterol efflux (17.65 ± 0.6 vs. 9.75 ± 0.8, p<0.05) in ROCK2-deficient mice that are mediated, in part, through the PPARγ-LXR-ABCA-1 pathway in macrophages. Conclusions ROCK2 contributes to atherosclerosis, in part, by inhibiting PPARγ-mediated reverse cholesterol transport in macrophages, which contributes to foam cell formation. These findings suggest that inhibition of ROCK2 in macrophages may have therapeutic benefits in preventing the development of atherosclerosis.

The bone morphogenetic protein (BMP) signaling pathway plays a central role during vasculature development. Mutations or dysregulation of the BMP pathway members have been linked to arteriovenous malformations. In the present study, we investigated the effect of the BMP modulators bone morphogenetic protein endothelial precursor-derived regulator (BMPER) and twisted gastrulation protein homolog 1 (TWSG1) on arteriovenous specification during zebrafish development and analyzed downstream Notch signaling pathway in human endothelial cells. Silencing of bmper and twsg1b in zebrafish embryos by morpholinos resulted in a pronounced enhancement of venous ephrinB4a marker expression and concomitant dysregulated arterial ephrinb2a marker expression detected by in situ hybridization. As arteriovenous specification was disturbed, we assessed the impact of BMPER and TWSG1 protein stimulation on the Notch signaling pathway on endothelial cells from different origin. Quantitative real-time PCR (qRT-PCR) and western blot analysis showed increased expression of Notch target gene hairy and enhancer of split, HEY1/2 and EPHRINB2. Consistently, silencing of BMPER in endothelial cells by siRNAs decreased Notch signaling and downstream effectors. BMP receptor antagonist DMH1 abolished BMPER and BMP4 induced Notch signaling pathway activation. In conclusion, we found that in endothelial cells, BMPER and TWSG1 are necessary for regular Notch signaling activity and in zebrafish embryos BMPER and TWSG1 preserve arteriovenous specification to prevent malformations.