The impact of the pro-atherogenic MRAS gene in smooth muscle cells

Abstract

Cardiovascular disease (CVDs) including coronary artery disease (CAD), cardiomyopathy, myocardial infarction and stroke, are the leading cause of death globally. The preeminent cause of CVDs is atherosclerosis, an inflammatory thickening of the vessel wall. Numerous risk factors including smoking, alcohol consumption, obesity, hypertension, stress, age, gender and genetic predisposition, contribute to its development. Genome wide association studies (GWAS) have pinpointed more than 393 loci marked by single nucleotide variants significantly associated with CAD. However, the causal mechanisms behind these associations remain unclear for which functional follow-up studies are very essential to identify the causal genetic variation or gene, to better understand the link between these loci and the disease. A study by Erdmann et al. in 2009, revealed a region on 3q22.3, which encompasses the MRAS gene as a risk factor for CAD. In this doctoral dissertation, the CAD-risk gene MRAS was investigated. MRAS encodes muscle Ras, a small GTPase that acts as a signal transducer in tumour necrosis factor (TNF) signalling and other related acute phase response signalling pathways. According to eQTL data, MRAS risk variants for CAD increase MRAS mRNA levels primarily in the arterial tissue. Recently, it has been indicated that functional MRAS variants are specific to vascular smooth muscle cells (SMCs). The exact role of MRAS in atherogenesis, the underlying mechanism of CAD, and the therapeutic potential of targeting MRAS is still elusive. Therefore, we investigated the function of MRAS in vascular SMCs, one of the key cell types in the etiology of atherosclerosis and plaque stabilization. Human primary aortic SMCs transfected with MRAS-specific siRNA and murine aortic SMCs derived from our Mras-/-ApoE-/- double knockout (dKO) mouse model was subjected to functional assays including proliferation, migration and apoptosis. The siRNA mediated knockdown of MRAS in human SMCs increased migration and proliferation with and without stimulation of TNF, IL-6 and IL-1ß. In line with that, the absence of Mras in murine SMCs led to significant increase in proliferation, enhanced migration and reduced apoptotic activity compared to control B6.ApoE-/- SMCs. Stimulation with TNF, IL-6 and IL-1ß enhanced the proliferative effect of Mras deficiency, indicating an interplay of these cytokines and MRAS pathways. Moreover, bulk RNA sequencing revealed that Mras knockout in murine SMCs led to differential gene expression of numerous genes involved in cGMP and cAMP signalling, suggesting that SMCs upregulate cGMP/cAMP signalling to compensate for the loss of Mras. The in vivo atherosclerosis study on plaque characteristic 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 mice. Male Mras-/-ApoE-/- dKO mice showed significantly higher levels of collagen content, MoMa content and SMC content as compared to B6.ApoE-/- male mice. In conclusion, our data indicates that lower MRAS levels in the vessel wall, particularly in SMCs, provides protection against CAD by stabilizing atherosclerotic plaques, and thereby decreasing the risk of plaque rupture and subsequent hospitalization.