A study by Erdmann et al. in 2009, revealed a region on 3q22.3, encompassing the MRAS gene as a risk factor for coronary artery disease (CAD). MRAS encodes muscle Ras, a GTPase acting as a signal transducer in TNF signalling and other acute phase response pathways. eQTL data suggests that CAD risk variants in the MRAS gene primarily increase MRAS mRNA levels in the arterial tissue and vascular smooth muscle cells (SMCs). However, MRAS’s exact role in atherogenesis is still elusive. This study aims to investigate the function of MRAS on SMC behaviour to identify the signalling pathways that are differentially regulated by MRAS modulation in SMCs and how they influence cellular function and CAD disease progression. Human primary aortic SMCs transfected with MRAS-specific siRNA and murine SMCs derived from our Mras-/-ApoE-/- double knockout (dKO) mouse model were subjected to functional assays including proliferation, migration and apoptosis. Cell Migration was assessed using a wound-healing assay with an ibidi 4-well insert in a 12 well plate measuring the cellular confluence, as a parameter for migration ability. Proliferation was evaluated through Hoechst staining for nuclei and the bromodeoxyuridine (BrdU) assay, tracking DNA synthesis. Moreover, apoptosis was analysed using a caspase-3 colorimetric assay, measuring enzymatic activity at 405 nm. MRAS siRNA knockdown in human SMCs, as well as MRAS deficiency in mouse SMCs, resulted in increased migration and proliferation, along with reduced apoptotic activity at basal levels. Given that proinflammatory cytokines such as TNF and IL-6 promote atherosclerotic progression, SMCs were stimulated with these cytokines to evaluate changes in migration and proliferation. MRAS downregulation in human SMCs further enhanced migration and proliferation while decreasing apoptosis upon stimulation with TNF (10 ng/ml) and IL-6 (5 and 10 ng/ml), suggesting a functional interaction between MRAS signaling and cytokine-mediated pathways. Conversely, overexpression of MRAS conducted via the AMAXA nucleofection procedure resulted in reduced proliferation, and migration along with increased apoptosis, confirming the findings observed in MRAS downregulated cells and highlighting the impact of MRAS on these pro-atherogenic mechanisms. Bulk RNA sequencing of murine SMCs lacking Mras revealed differential expression of multiple genes associated with cGMP/cAMP signaling, suggesting that SMCs may enhance/upregulate this pathway to compensate for the loss of Mras. As MRAS is highly conserved between human and mice, an in vivo atherosclerosis study on plaque characteristic in mice was also performed. It revealed that the impact of Mras deficiency was sex-specific as significant differences in plaque size, collagen content and macrophage staining were only observed in male Mras-/-ApoE-/- dKO mice as compared to B6.ApoE-/- male mice. On the other hand, no significant difference was observed in female mice. Conclusively, this research study suggests a potential link between MRAS and SMC processes during atherosclerosis contributing to the pathophysiology of CAD. This may occur by regulating plaque stabilisation, probably by promoting increased SMC migration and proliferation, while reducing SMC apoptosis within the fibrous cap of the intima. These processes may contribute to a reduced risk of plaque rupture and consequently reduce hospitalizations in CAD patients.
