CARMN Loss Regulates Smooth Muscle Cells and Accelerates Atherosclerosis in Mice

Abstract

Rationale: In the microenvironment of atherosclerotic lesions, vascular smooth muscle cells (vSMCs) switch to a dedifferentiated state but the underlying molecular mechanisms driving this switch are not fully understood. Long noncoding RNAs (lncRNAs) are dysregulated during vascular pathology, but relatively little is known about their involvement in controlling vSMCs function. CARMN is a lncRNA located immediately upstream of the microRNAs (miRNAs) miR-143 and miR-145, both involved in vSMCs function. Objective: We investigated the role of the lncRNA CARMN, independent from miR-143 and miR-145, as potential a regulator of vSMC phenotypes in vitro and the consequences of its loss during the development of atherosclerosis in vivo. We hypothesized that loss of CARMN is a primary event controlling the functional switch towards pro-atherogenic vSMC phenotype and accelerates the development of the plaques in vivo. Methods and Results: Expression of CARMN lncRNA was silenced using GapmeRs in human coronary arterial smooth muscle cells (hCASMCs), revealing that GapmeR-mediated loss of CARMN negatively affects miR-143 and miR-145 miRNA expression. RNA sequencing of CARMN-depleted hCASMCs revealed large transcriptomic changes, associated with vSMC proliferation, migration, inflammation, lipid metabolism and dedifferentiation. The use of miR-143 and miR-145 mimics revealed that CARMN regulates hCASMC proliferation in a miRNA-independent manner. In human and mouse, CARMN and associated miRNAs were downregulated in advanced versus early atherosclerotic lesions. Using a CRISPR-Cas9 knock-out approach, we explored the implications of CARMN depletion during atherosclerosis in vivo. Consistent with in vitro results, the knock-out of CARMN impaired the expression of miR-143 and miR-145 under homeostatic conditions. Importantly, when atherosclerosis was induced in these mice, CARMN knock-out increased the volume, size, pro-inflammatory LGALS3-expressing cells content and altered plaque composition, yielding an advanced phenotype. Conclusions: We identified the early loss of CARMN lncRNA as critical event which primes vSMCs towards a pro-atherogenic phenotype in vitro and accelerates the development of atherosclerosis in vivo.

Noncoding RNAs: biology and applications—a Keystone Symposia report

“…Lalwani focused on the impact of CARMN . Baker's group previously showed that loss of CARMN can enhance vascular smooth muscle cell proliferation, migration, inflammation, and plasticity, and accelerate atherosclerosis in mice 162 . In addition, differential expression of CARMN isoforms in cardiac progenitor cells affects differentiation at different stages of development.…”

WNT5B promotes vascular smooth muscle cell dedifferentiation via mitochondrial dynamics regulation in chronic thromboembolic pulmonary hypertension

“…Activation of VSMC proliferation and migration is associated with VSMC phenotype switching to synthetic and dedifferentiated states (Basatemur et al, 2019). Despite progress in understanding the VSMC phenotype among various cardiovascular diseases (Fernandes et al, 2021; Furmanik et al, 2020; He et al, 2020; Vacante et al, 2021), the underlying mechanisms are not clearly understood in CTEPH. Previously studies revealed that DNA methylation changes in VSMCs are associated with the pathogenesis of CTEPH (Barst et al, 2004; Philibert et al, 2012; Y. Wang et al, 2018).…”

Non-coding RNA-Associated Therapeutic Strategies in Atherosclerosis

“…On silencing CARMN by GapmeR strategy and overexpressing miR-143 and−145 by mimics at the same time, hCASMCs migration and VSMC identifying markers were observed to restore, whereas hCASMCs proliferation remained increased, revealing the independent role of the lncRNA in regulating VSMC proliferation. Further in vivo studies showed CARMN was a protective non-coding RNA in atherogenesis consistently ( 54 ).…”