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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. Cardiac mesoderm enhancer-associated noncoding RNA (CARMN) is a lncRNA located immediately upstream of the microRNAs-143 and -145 (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 a potential 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 proatherogenic vSMC phenotype and accelerates the development of the plaques in vivo. Method and Results: Expression of CARMN lncRNA was silenced using locked nucleic acids antisense oligonucleotides (GapmeRs) in human coronary arterial smooth muscle cells, revealing that GapmeR-mediated loss of CARMN negatively affects miR-143 and miR-145 microRNA expression. RNA sequencing of CARMN-depleted human coronary arterial smooth muscle cells 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 human coronary arterial smooth muscle cell proliferation in a microRNA-independent manner. In humans and mice, CARMN and associated microRNAs were downregulated in advanced versus early atherosclerotic lesions. Using a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) knockout approach, we explored the implications of CARMN depletion during atherosclerosis in vivo. Consistent with in vitro results, the knockout of CARMN impaired the expression of miR-143 and miR-145 under homeostatic conditions. Importantly, when atherosclerosis was induced in these mice, CARMN knockout increased the volume, size, proinflammatory Lgals3 (galectin 3)-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 proatherogenic phenotype in vitro and accelerates the development of atherosclerosis in vivo.

Supplemental Digital Content is available in the text., Rationale: Despite increasing understanding of the prognostic importance of vascular stiffening linked to perivascular fibrosis in hypertension, the molecular and cellular regulation of this process is poorly understood. Objectives: To study the functional role of microRNA-214 (miR-214) in the induction of perivascular fibrosis and endothelial dysfunction driving vascular stiffening. Methods and Results: Out of 381 miRs screened in the perivascular tissues in response to Ang II (angiotensin II)-mediated hypertension, miR-214 showed the highest induction (8-fold, P=0.0001). MiR-214 induction was pronounced in perivascular and circulating T cells, but not in perivascular adipose tissue adipocytes. Global deletion of miR-214−/− prevented Ang II-induced periaortic fibrosis, Col1a1, Col3a1, Col5a1, and Tgfb1 expression, hydroxyproline accumulation, and vascular stiffening, without difference in blood pressure. Mechanistic studies revealed that miR-214−/− mice were protected against endothelial dysfunction, oxidative stress, and increased Nox2, all of which were induced by Ang II in WT mice. Ang II-induced recruitment of T cells into perivascular adipose tissue was abolished in miR-214−/− mice. Adoptive transfer of miR-214−/− T cells into RAG1−/− mice resulted in reduced perivascular fibrosis compared with the effect of WT T cells. Ang II induced hypertension caused significant change in the expression of 1380 T cell genes in WT, but only 51 in miR-214−/−. T cell activation, proliferation and chemotaxis pathways were differentially affected. MiR-214−/− prevented Ang II-induction of profibrotic T cell cytokines (IL-17, TNF-α, IL-9, and IFN-γ) and chemokine receptors (CCR1, CCR2, CCR4, CCR5, CCR6, and CXCR3). This manifested in reduced in vitro and in vivo T cell chemotaxis resulting in attenuation of profibrotic perivascular inflammation. Translationally, we show that miR-214 is increased in plasma of patients with hypertension and is directly correlated to pulse wave velocity as a measure of vascular stiffness. Conclusions: T-cell–derived miR-214 controls pathological perivascular fibrosis in hypertension mediated by T cell recruitment and local profibrotic cytokine release.

Rationale: The pathogenesis of pulmonary arterial hypertension (PAH) remains unclear. The 4 microRNAs representing the miR-143 and miR-145 stem loops are genomically clustered. Objective: To elucidate the transcriptional regulation of the miR-143/145 cluster and the role of miR-143 in PAH. Methods and Results: We identified the promoter region that regulates miR-143/145 microRNA expression in pulmonary artery smooth muscle cells (PASMCs). We mapped PAH-related signaling pathways, including estrogen receptor, liver X factor/retinoic X receptor, transforming growth factor-β (Smads), and hypoxia (hypoxia response element), that regulated levels of all pri-miR stem loop transcription and resulting microRNA expression. We observed that miR-143-3p is selectively upregulated compared with miR-143-5p during PASMC migration. Modulation of miR-143 in PASMCs significantly altered cell migration and apoptosis. In addition, we found high abundance of miR-143-3p in PASMC-derived exosomes. Using assays with pulmonary arterial endothelial cells, we demonstrated a paracrine promigratory and proangiogenic effect of miR-143-3p–enriched exosomes from PASMC. Quantitative polymerase chain reaction and in situ hybridization showed elevated expression of miR-143 in calf models of PAH and in samples from PAH patients. Moreover, in contrast to our previous findings that had not supported a therapeutic role in vivo, we now demonstrate a protective role of miR-143 in experimental pulmonary hypertension in vivo in miR-143−/− and anti-miR-143-3p–treated mice exposed to chronic hypoxia in both preventative and reversal settings. Conclusions: MiR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, whereas inhibition of miR-143-3p blocked experimental pulmonary hypertension. Taken together, these findings confirm an important role for the miR-143/145 cluster in PAH pathobiology.

Rationale: Despite improved understanding of the underlying genetics, pulmonary arterial hypertension (PAH) remains a severe disease. Extensive remodeling of small pulmonary arteries, including proliferation of pulmonary artery smooth muscle cells (PASMCs), characterizes PAH. MicroRNAs (miRNAs) are noncoding RNAs that have been shown to play a role in vascular remodeling. Objective: We assessed the role of miR-145 in PAH. Methods and Results: We localized miR-145 in mouse lung to smooth muscle. Using quantitative PCR, we demonstrated increased expression of miR-145 in wild-type mice exposed to hypoxia. PAH was evaluated in miR-145 knockout and mice treated with anti-miRs via measurement of systolic right ventricular pressure, right ventricular hypertrophy, and percentage of remodeled pulmonary arteries. miR-145 deficiency and anti-miR–mediated reduction resulted in significant protection from the development of PAH. In contrast, miR-143 anti-miR had no effect. Furthermore, we observed upregulation of miR-145 in lung tissue of patients with idiopathic and heritable PAH compared with unaffected control subjects and demonstrated expression of miR-145 in SMC of remodeled vessels from such patients. Finally, we show elevated levels of miR-145 expression in primary PASMCs cultured from patients with BMPR2 mutations and also in the lungs of BMPR2 -deficient mice. Conclusions: miR-145 is dysregulated in mouse models of PAH. Downregulation of miR-145 protects against the development of PAH. In patient samples of heritable PAH and idiopathic PAH, miR-145 is expressed in remodeled vessels and mutations in BMPR2 lead to upregulation of miR-145 in mice and PAH patients. Manipulation of miR-145 may represent a novel strategy in PAH treatment.