Vascular Smooth Muscle Cell Phenotype Research Articles

Characterisation of Lipoma-Preferred Partner as a Novel Mechanotransducer in Vascular Smooth Muscle Cells.

In arteries and arterioles, a chronic increase in blood pressure raises wall tension. This continuous biomechanical strain causes a change in gene expression in vascular smooth muscle cells (VSMCs) that may lead to pathological changes. Here we have characterised the functional properties of lipoma-preferred partner (LPP), a Lin11-Isl1-Mec3 (LIM)-domain protein, which is most closely related to the mechanotransducer zyxin but selectively expressed by smooth muscle cells, including VSMCs in adult mice. VSMCs isolated from the aorta of LPP knockout (LPP-KO) mice displayed a higher rate of proliferation than their wildtype (WT) counterparts, and when cultured as three-dimensional spheroids, they revealed a higher expression of the proliferation marker Ki 67 and showed greater invasion into a collagen gel. Accordingly, the gelatinase activity was increased in LPP-KO but not WT spheroids. The LPP-KO spheroids adhering to the collagen gel responded with decreased contraction to potassium chloride. The relaxation response to caffeine and norepinephrine was also smaller in the LPP-KO spheroids than in their WT counterparts. The overexpression of zyxin in LPP-KO VSMCs resulted in a reversal to a more quiescent differentiated phenotype. In native VSMCs, i.e., in isolated perfused segments of the mesenteric artery (MA), the contractile responses of LPP-KO segments to potassium chloride, phenylephrine or endothelin-1 did not vary from those in isolated perfused WT segments. In contrast, the myogenic response of LPP-KO MA segments was significantly attenuated while zyxin-deficient MA segments displayed a normal myogenic response. We propose that LPP, which we found to be expressed solely in the medial layer of different arteries from adult mice, may play an important role in controlling the quiescent contractile phenotype of VSMCs.

Reprogramming endothelial and vascular smooth muscle cells to prevent and treat hypertension

The major pathophysiological characteristic of hypertension is the occurrence of small artery remodeling and endothelial dysfunction. There is also solid evidence showing that microcirculation abnormalities occur prior to the onset of hypertension. However, the mechanism(s) that trigger these changes prior to the elevation of blood pressure are unknown, and this may limit our ability to identify the cause of this disease and effectively treat it. In hypertension, as with aging, the vasculature becomes less susceptible to repair. One of the reasons is because endothelial cells start to deteriorate and present with exacerbated endothelial-to-mesenchymal transition (EndMT). Likewise, vascular smooth muscle cells (VSMC) also dedifferentiate into a synthetic phenotype, whereby they start to produce and secrete extracellular vesicles with a high migration and proliferation capacity for repairing vascular injury. Uncontrolled EndMT and/or VSMC phenotype switching contributes to vascular diseases, but the initial trigger for these conditions is unidentified. Importantly, EndMT and synthetic VSMC exhibit plasticity and can return to adopt an endothelial cell-like fate and present contractile phenotype again, respectively. Therefore, in this hypothesis we will take advantage of this plasticity, and we propose to manipulate this fate by inducing partial cellular reprogramming without passing through the pluripotent state. Specifically, we suggest that activation of the three master transcription factors, Oct-4, Sox-2, and Klf-4 (collectively termed OSK) will reprogram endothelial cells and prevent and reduce EndMT and VSMC synthetic phenotype. It was recently shown that activation of OSK was able to restore lost vision in old mice, and cancer risk was reduced by excluding c-Myc. Therefore, OSK treatment could provide new possibilities for vascular rejuvenation and treatment of hypertension.

MiR-590-3p Promotes the Phenotypic Switching of Vascular Smooth Muscle Cells by Targeting Lysyl Oxidase.

We investigated the clinical characteristics of patients with acute aortic dissection (AAD) and miR-590-3p levels in serum, tissue, and vascular smooth muscle cells. The effect of miR-590-3p on the vascular smooth muscle cell phenotype was assessed, and the regulation of lysyl oxidase by miR-5903p was determined. C57BL/6 mice were used to investigate the incidence of AAD and effects of miR-5903p on AAD. The miR-590-3p levels were measured in the aortae of mice, and hematoxylin and eosin staining and Masson staining were performed to identify the morphological features of the aorta. Comparative analysis revealed significant differences in clinical characteristics between patients with AAD and healthy control subjects, with most patients with AAD exhibiting concomitant hypertension and nearly 50% having atherosclerosis. Lysyl oxidase was a direct target of miR-590-3p. Lysyl oxidase overexpression inhibited switching of the vascular smooth muscle cell phenotype from contractile to synthetic, but miR-590-3p overexpression significantly reversed this change. In the mouse model, miR-590-3p upregulation increased the incidence of AAD to 93.3%, and its incidence decreased to 13.3% after miR-590-3p inhibition. Hematoxylin and eosin and Masson staining revealed that the miR-590-3p agomiR group had a greater loss of the contractile phenotype in the dissected aortic wall and an increased number of muscle fibers in the aortic wall, which contributed to thickening of the aortic wall and the formation of a false lumen in aortic dissection. miR-590-3p might be pivotal in the pathogenesis of AAD. Thus, targeting miR-590-3p or its downstream pathways could represent a therapeutic approach for AAD.

Vascular smooth muscle cells in intracranial aneurysms

Intracranial aneurysm (IA) is a severe cerebrovascular disease characterized by abnormal bulging of cerebral vessels that may rupture and cause a stroke. The expansion of the aneurysm accompanies by the remodeling of vascular matrix. It is well-known that vascular remodeling is a process of synthesis and degradation of extracellular matrix (ECM), which is highly dependent on the phenotype of vascular smooth muscle cells (VSMCs). The phenotypic switching of VSMC is considered to be bidirectional, including the physiological contractile phenotype and alternative synthetic phenotype in response to injury. There is increasing evidence indicating that VSMCs have the ability to switch to various phenotypes, including pro-inflammatory, macrophagic, osteogenic, foamy and mesenchymal phenotypes. Although the mechanisms of VSMC phenotype switching are still being explored, it is becoming clear that phenotype switching of VSMCs plays an essential role in IA formation, progression, and rupture. This review summarized the various phenotypes and functions of VSMCs associated with IA pathology. The possible influencing factors and potential molecular mechanisms of the VSMC phenotype switching were further discussed. Understanding how phenotype switching of VSMC contributed to the pathogenesis of unruptured IAs can bring new preventative and therapeutic strategies for IA.

Deficiency of protein inhibitor of activated STAT3 exacerbates atherosclerosis by modulating VSMC phenotypic switching

Background and aimsPhenotypic switching of vascular smooth muscle cells (VSMCs) plays an essential role in the development of atherosclerosis. Protein inhibitor of activated STAT (Pias) regulates VSMCs phenotype via acting as sumo E3 ligase to promote protein sumoylation. Our previous study indicated that Pias3 expression decreased in atherosclerotic lesions. Therefore, this study aimed to explore the role of Pias3 on VSMCs phenotype switching during atherosclerosis. MethodsApoE−/− and ApoE−/−Pias3−/− double-deficient mice were fed with high-fat/high-cholesterol diet to induce atherosclerosis. Aorta tissues and primary VSMCs were collected to assess plaque formation and VSMCs phenotype. In vitro, Pias3 was overexpressed in A7r5, a VSMCs cell line, by transfection with Pias3 plasmid. Real-time quantitative PCR, immunoblotting, immunoprecipitation, were used to analyze the effect of Pias3 on VSMCs phenotypic switching. ResultsPias3 deficiency significantly exacerbated atherosclerotic plaque formation and promoted VSMCs phenotypic switching to a synthetic state within lesion. In vitro, overexpressing Pias3 in VSMCs increased the expression of contractile markers (myosin heavy chain 11, calponin 1), while it decreased the level of synthetic marker (vimentin). Additionally, Pias3 overexpression blocked PDGF-BB-induced VSMCs proliferation and migration. Immunoprecipitation and mass spectrometry results showed that Pias3 enhanced sumoylation and ubiquitination of vimentin, and shortened its half-life. Moreover, the ubiquitination level of vimentin was impaired by 2-D08, a sumoylation inhibitor. This suggests that Pias3 might accelerate the ubiquitination-degradation of vimentin by promoting its sumoylation. ConclusionsThese results indicate that Pias3 might ameliorate atherosclerosis progression by suppressing VSMCs phenotypic switching and reducing vimentin protein stability.

CircACTA2 inhibits NLRP3 inflammasome-mediated inflammation via interacting with NF-κB in vascular smooth muscle cells

circACTA2 derived from the smooth muscle α-actin gene plays an important role in the regulation of vascular smooth muscle cell (VSMC) phenotype. The activation of NLRP3 inflammasome is involved in VSMC phenotypic switching. However, the mechanistic relationship between circACTA2 and NLRP3 inflammasome during vascular remodeling remains poorly understood. Here, we showed that circACTA2 was down-regulated in human intimal hyperplasia. circACTA2 overexpression in circACTA2 transgenic mice significantly decreased the neointimal hyperplasia induced by vascular injury, which is concomitant with a decrease in IL-18, IL-1β, TNF-α, and IL-6 levels. Gain- and loss-of-function studies revealed that circACTA2 alleviated VSMC inflammation by suppressing the activation of NLRP3 inflammasome. Mechanistically, circACTA2 inhibited the expression of NF-κB p65 and p50 subunits and interacted with p50, which impedes the formation of the p50/p65 heterodimer and nuclear translocation induced by TNF-α, thus resulting in the suppression of NLRP3 gene transcription and inflammasome activation. Furthermore, circACTA2 overexpression mitigated inflammation via repressing NLRP3 inflammasome-mediated VSMC pyroptosis. Importantly, employing a decoy oligonucleotide to compete with circACTA2 for binding to p50 could attenuate the expression of NLRP3, ASC, and caspase-1. These findings provide a novel insight into the functional roles of circACTA2 in VSMCs, and targeting the circACTA2–NF-κB–NLRP3 axis represents a promising therapeutic strategy for vascular remodeling.

Smooth Muscle Heterogeneity and Plasticity in Health and Aortic Aneurysmal Disease.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the medial layer of the aorta, which plays a critical role in the maintenance of aortic wall integrity. VSMCs have been suggested to have contractile and synthetic phenotypes and undergo phenotypic switching to contribute to the deteriorating aortic wall structure. Recently, the unprecedented heterogeneity and diversity of VSMCs and their complex relationship to aortic aneurysms (AAs) have been revealed by high-resolution research methods, such as lineage tracing and single-cell RNA sequencing. The aortic wall consists of VSMCs from different embryonic origins that respond unevenly to genetic defects that directly or indirectly regulate VSMC contractile phenotype. This difference predisposes to hereditary AAs in the aortic root and ascending aorta. Several VSMC phenotypes with different functions, for example, secreting VSMCs, proliferative VSMCs, mesenchymal stem cell-like VSMCs, immune-related VSMCs, proinflammatory VSMCs, senescent VSMCs, and stressed VSMCs are identified in non-hereditary AAs. The transformation of VSMCs into different phenotypes is an adaptive response to deleterious stimuli but can also trigger pathological remodeling that exacerbates the pathogenesis and development of AAs. This review is intended to contribute to the understanding of VSMC diversity in health and aneurysmal diseases. Papers that give an update on VSMC phenotype diversity in health and aneurysmal disease are summarized and recent insights on the role of VSMCs in AAs are discussed.

Apolipoprotein E (ApoE) Rescues the Contractile Smooth Muscle Cell Phenotype in Popliteal Artery Aneurysm Disease.

Popliteal artery aneurysm (PAA) is the most frequent peripheral aneurysm, primarily seen in male smokers with a prevalence below 1%. This exploratory study aims to shed light on cellular mechanisms involved in PAA progression. Sixteen human PAA and eight non-aneurysmatic popliteal artery samples, partially from the same patients, were analyzed by immunohistochemistry, fluorescence imaging, Affymetrix mRNA expression profiling, qPCR and OLink proteomics, and compared to atherosclerotic (n = 6) and abdominal aortic aneurysm (AAA) tissue (n = 19). Additionally, primary cell culture of PAA-derived vascular smooth muscle cells (VSMC) was established for modulation and growth analysis. Compared to non-aneurysmatic popliteal arteries, VSMCs lose the contractile phenotype and the cell proliferation rate increases significantly in PAA. Array analysis identified APOE higher expressed in PAA samples, co-localizing with VSMCs. APOE stimulation of primary human PAA VSMCs significantly reduced cell proliferation. Accordingly, contractile VSMC markers were significantly upregulated. A single case of osseous mechanically induced PAA with a non-diseased VSMC profile emphasizes these findings. Carefully concluded, PAA pathogenesis shows similar features to AAA, yet the mechanisms involved might differ. APOE is specifically higher expressed in PAA tissue and could be involved in VSMC phenotype rescue.

MiR-33a Expression Attenuates ABCA1-Dependent Cholesterol Efflux and Promotes Macrophage-Like Cell Transdifferentiation in Cultured Vascular Smooth Muscle Cells.

Recent evidence suggests that the majority of cholesterol-laden cells found in atherosclerotic lesions are vascular smooth muscle cells (VSMC) that have transdifferentiated into macrophage-like cells (MLC). Furthermore, cholesterol-laden MLC of VSMC origin have demonstrated impaired ABCA1-dependent cholesterol efflux, but it is poorly understood why this occurs. A possible mechanism which may at least partially be attributed to cholesterol-laden MLC demonstrating attenuated ABCA1-dependent cholesterol efflux is a miR-33a expression, as a primary function of this microRNA is to silence ABCA1 expression, but this has yet to be rigorously investigated. Therefore, the VSMC line MOVAS cells were used to generate miR-33a knockout (KO) MOVAS cells, and we used KO and wild-type (WT) MOVAS cells to delineate any possible proatherogenic role of miR-33a expression in VSMC. When WT and KO MOVAS cells were cholesterol-loaded to convert into MLC, this resulted in the WT MOVAS cells to exhibit impaired ABCA1-dependent cholesterol efflux. In the cholesterol-loaded WT MOVAS MLC, we also observed a delayed restoration of the VSMC phenotype when these cells were exposed to the ABCA1 cholesterol acceptor, apoAI. These results imply that miR-33a expression in VSMC drives atherosclerosis by triggering MLC transdifferentiation via attenuated ABCA1-dependent cholesterol efflux.

The downstream network of STAT6 in promoting vascular smooth muscle cell phenotypic switch and neointimal formation.

Intimal thickening caused by the excessive multiplication of vascular smooth muscle cells (VSMCs) is the pathological process central to cardiovascular diseases, including restenosis. In response to vascular injury, VSMCs would undergo phenotypic switching from a fully differentiated, low proliferative rate phenotype to a more pro-proliferative, promigratory, and incompletely-differentiated state. The lack of a full understanding of the molecular pathways coupling the vascular injury stimuli to VSMCs phenotype switching largely limits the development of medical therapies for treating intima hyperplasia-related diseases. The role of signal transducers and activators of transcription 6 (STAT6) in modulating the proliferation and differentiation of various cell types, especially macrophage, has been well investigated, but little is known about its pathophysiological role and target genes in restenosis after vascular injury. In the present work, Stat6-/- mice were observed to exhibit less severe intimal hyperplasia compared with Stat6+/+ mice after carotid injury. The expression of STAT6 was upregulated in VSMCs located in the injured vascular walls. STAT6 deletion leads to decreased proliferation and migration of VSMCs while STAT6 overexpression enhances the proliferation and migration of VSMCs companies with reduced expression of VSMCs marker genes and organized stress fibers. The effect of STAT6 in mouse VSMCs was conserved in human aortic SMCs. RNA-deep-sequencing and experiments verification revealed LncRNA C7orf69/LOC100996318-miR-370-3p/FOXO1-ER stress signaling as the downstream network mediating the pro-dedifferentiation effect of STAT6 in VSMCs. These findings broaden our understanding of vascular pathological molecules and throw a beam of light on the therapy of a variety of proliferative vascular diseases.

ETV2 Enhances CXCL5 Secretion from Endothelial Cells, Leading to the Promotion of Vascular Smooth Muscle Cell Migration.

Abnormal communication between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) promotes vascular diseases, including atherogenesis. ETS variant transcription factor 2 (ETV2) plays a substantial role in pathological angiogenesis and the reprogramming of ECs; however, the role of ETV2 in the communication between ECs and VSMCs has not been revealed. To investigate the interactive role of ETV2 in the EC to VSMC phenotype, we first showed that treatment with a conditioned medium from ETV2-overexpressed ECs (Ad-ETV2 CM) significantly increased VSMC migration. The cytokine array showed altered levels of several cytokines in Ad-ETV2 CM compared with those in normal CM. We found that C-X-C motif chemokine 5 (CXCL5) promoted VSMC migration using the Boyden chamber and wound healing assays. In addition, an inhibitor of C-X-C motif chemokine receptor 2 (CXCR2) (the receptor for CXCL5) significantly inhibited this process. Gelatin zymography showed that the activities of matrix metalloproteinase (MMP)-2 and MMP-9 increased in the media of VSMCs treated with Ad-ETV2 CM. Western blotting revealed a positive correlation between Akt/p38/c-Jun phosphorylation and CXCL5 concentration. The inhibition of Akt and p38-c-Jun effectively blocked CXCL5-induced VSMC migration. In conclusion, CXCL5 from ECs induced by ETV2 promotes VSMC migration via MMP upregulation and the activation of Akt and p38/c-Jun.

Heterogeneous expression of alternatively spliced lncRNA mediates vascular smooth cell plasticity

9p21.3 locus polymorphisms have the strongest correlation with coronary artery disease, but as a noncoding locus, disease connection is enigmatic. The lncRNA ANRIL found in 9p21.3 may regulate vascular smooth muscle cell (VSMC) phenotype to contribute to disease risk. We observed significant heterogeneity in induced pluripotent stem cell-derived VSMCs from patients homozygous for risk versus isogenic knockout or nonrisk haplotypes. Subpopulations of risk haplotype cells exhibited variable morphology, proliferation, contraction, and adhesion. When sorted by adhesion, risk VSMCs parsed into synthetic and contractile subpopulations, i.e., weakly adherent and strongly adherent, respectively. Of note, >90% of differentially expressed genes coregulated by haplotype and adhesion and were associated with Rho GTPases, i.e., contractility. Weakly adherent subpopulations expressed more short isoforms of ANRIL, and when overexpressed in knockout cells, ANRIL suppressed adhesion, contractility, and αSMA expression. These data suggest that variable lncRNA penetrance may drive mixed functional outcomes that confound pathology.

Taxifolin ameliorates abdominal aortic aneurysm by preventing inflammation and apoptosis and extracellular matrix degradation via inactivating TLR4/NF-κB axis.

Abdominal aortic aneurysm (AAA) is a serious aortic disease with high mortality. Vascular smooth muscle cells (VSMCs) loss is a prominent feature of AAA. Taxifolin (TXL) is a natural antioxidant polyphenol and possesses therapeutic functions in numerous human diseases. This study aimed to investigate TXL's impact on VSMC phenotype in AAA. In vitro and in vivo of VSMC injury model was induced by angiotensin II (Ang II). The potential function of TXL on AAA was determined using Cell Counting Kit-8, flow cytometry, Western blot, quantitative reverse transcription-PCR, and enzyme-linked immunosorbent assay. Meanwhile, TXL mechanism on AAA was checked by a series of molecular experiments. Also, TXL function on AAA in vivo was further evaluated using hematoxylin-eosin staining, TUNEL assay, Picric acid-Sirius red staining and immunofluorescence assay in C57BL/6 mice. TXL alleviated Ang II-induced VSMC injury mainly by enhancing VSMC proliferation and weakening cell apoptosis, alleviating VSMC inflammation, and reducing extracellular matrix (ECM) degradation of VSMCs. Furthermore, mechanistic studies corroborated that TXL reversed the high levels of Toll-like receptor 4 (TLR4) and p-p65/p65 induced by Ang II. Also, TXL facilitated VSMC proliferation and reduced cell apoptosis, repressed inflammation, and ECM degradation of VSMCs, while these effects were reversed by TLR4 overexpression. In vivo studies further confirmed that TXL owned the function of alleviating AAA, such as alleviating collagen fiber hyperplasia and inflammatory cell infiltration in AAA mice, and repressing inflammation and ECM degradation. TXL protected VSMCs against Ang II-induced injury through activating TLR4/noncanonical nuclear factor-kappaB(NF-κB).

SGC agonist BAY1021189 promotes thoracic aortic dissection formation by accelerating vascular smooth muscle cell phenotype switch

Thoracic aortic dissection (TAD) is common but lethal cardiovascular disease with high mortality. This study aimed to expound whether and how sGC-PRKG1 signaling pathway might promote the formation of TAD. Our work identified two modules with high relevance to TAD using WGCNA method. Combined with previous studies, we focused on the participation of endothelial NOS (eNOS) in the progression of TAD. Through immunohistochemistry, immunofluorescence and western blot we verified that eNOS expression was elevated in the tissues of patients and mice with aortic dissection, and the phosphorylation Ser1177 of eNOS was activated. In a BAPN-induced TAD mouse model, sGC-PRKG1 signaling pathway promotes TAD formation by inducing vascular smooth muscle cells (VSMCs) phenotype transition, which was demonstrated as a decrease in markers of the contractile phenotype of VSMCs such as αSMA, SM22α, and Calponin. These results were also verified by experiments in vitro. To explore the further mechanism, we conducted immunohistochemistry, western blot and quantitative RT-PCR (qPCR), the results of which indicated that sGC-PRKG1 signaling pathway was activated when TAD occurred. In conclusion, our current study revealed that sGC-PRKG1 signaling pathway could promote TAD formation by accelerating VSMCs phenotype switch.

Single-cell RNA sequencing reveals the vascular smooth muscle cell phenotypic landscape in aortic aneurysm

Background and objectivesPhenotypic switching in vascular smooth muscle cells (VSMCs) has been linked to aortic aneurysm, but the phenotypic landscape in aortic aneurysm is poorly understood. The present study aimed to analyse the phenotypic landscape, phenotypic differentiation trajectory, and potential functions of various VSMCs phenotypes in aortic aneurysm.MethodsSingle-cell sequencing data of 12 aortic aneurysm samples and 5 normal aorta samples (obtained from GSE166676 and GSE155468) were integrated by the R package Harmony. VSMCs were identified according to the expression levels of ACTA2 and MYH11. VSMCs clustering was determined by the R package ‘Seurat’. Cell annotation was determined by the R package ‘singleR’ and background knowledge of VSMCs phenotypic switching. The secretion of collagen, proteinases, and chemokines by each VSMCs phenotype was assessed. Cell‒cell junctions and cell–matrix junctions were also scored by examining the expression of adhesion genes. Trajectory analysis was performed by the R package ‘Monocle2’. qPCR was used to quantify VSMCs markers. RNA fluorescence in situ hybridization (RNA FISH) was performed to determine the spatial localization of vital VSMCs phenotypes in aortic aneurysms.ResultsA total of 7150 VSMCs were categorize into 6 phenotypes: contractile VSMCs, fibroblast-like VSMCs, T-cell-like VSMCs, adipocyte-like VSMCs, macrophage-like VSMCs, and mesenchymal-like VSMCs. The proportions of T-cell-like VSMCs, adipocyte-like VSMCs, macrophage-like VSMCs, and mesenchymal-like VSMCs were significantly increased in aortic aneurysm. Fibroblast-like VSMCs secreted abundant amounts of collagens. T-cell-like VSMCs and macrophage-like VSMCs were characterized by high chemokine levels and proinflammatory effects. Adipocyte-like VSMCs and mesenchymal-like VSMCs were associated with high proteinase levels. RNA FISH validated the presence of T-cell-like VSMCs and macrophage-like VSMCs in the tunica media and the presence of mesenchymal-like VSMCs in the tunica media and tunica adventitia.ConclusionA variety of VSMCs phenotypes are involved in the formation of aortic aneurysm. T-cell-like VSMCs, macrophage-like VSMCs, and mesenchymal-like VSMCs play pivotal roles in this process.7meMEx4JhpJfRQ-F56EwAbVideo

Apelin Regulation of K-Cl Cotransport in Vascular Smooth Muscle Cells as a Potential Target for Cardiovascular Disease

Background/Aims: Apelin and its signaling through the G-protein coupled receptor (APJ, gene symbol APLNR) regulate cardiovascular function via two mechanisms: 1) By promoting nitric oxide (NO)-mediated vasodilation, impaired by oxidized low-density lipoproteins (oxLDL); and 2) By inducing cell proliferation via the phosphatidylinositol-3-kinase (PI3K)/protein kinase B/Akt pathway (PI3K/Akt) and mitogen activated protein kinase (MAPK) pathways. The potassium chloride cotransporter (KCC1,3,4; SLC12A4,6,7) controls cell volume, and regulates cardiovascular function through proliferation, migration, and blood pressure control. Importantly, KCC regulatory mechanisms and apelin/APJ signaling pathways overlap placing KCC as a potential target for apelin/APJ to elicit its cardioprotective effects. Therefore, apelin’s action on KCC activity was examined in contractile and synthetic rat aortic vascular smooth muscle cells (VSMCs). Methods: KCC activity was measured by atomic absorption spectrophotometry in chloride (Cl-) and Cl--free medium with sulfamate (Sf-) as Cl- replacement, and with rubidium (Rb+) as a potassium (K+) congener. The calculated difference between Rb+ transport in the presence of chloride (Cl-) and sulfamate (Sf-) is the Cl--dependent Rb+ influx (i.e., K-Cl cotransport activity). Apelin-13 (1 µM) was added either during flux (acute effect) and/or in the growth media (chronic effect) based on the experimental goals. KCC activity was characterized with respect to the VSMC phenotypes, in the presence or absence of apelin and corresponding inhibitors of the signaling pathways, oxLDL, and as a function of various physiological factors described below. Results: The APJ receptor was expressed in both contractile and synthetic VSMC phenotypes, the former also possessing the soluble guanylyl cyclase-coupled protein kinase G (PKG) receptor, critical for NO-mediated signaling. In general, KCC activity was higher in synthetic vs. contractile VSMCs, consistent with enhanced migration and proliferation in the former. In addition, apelin-mediated activation of KCC was modulated by extracellular sodium [Na+]o, osmolality, length of apelin treatment (acute or chronic) and VSMC phenotype (contractile vs synthetic). Based on selective inhibitors, apelin activated KCC through the (NO)/soluble guanylate cyclase (sGC)/protein kinase G (PKG) (NO/sGC/PKG)-, PI3K/Akt- and MAPK-dependent pathway(s). Furthermore, apelin rescued the inhibition of KCC by oxLDL. Altogether, results suggest apelin/APJ as an important modulator of KCC activity to sustain cell volume regulation and cardiovascular function. More recently, the apelinergic system has been proposed as a novel target for the treatment of Corona virus disease 2019 (COVID-19) and, given the significant overlap between the regulatory mechanisms of this system and KCC, and their role in cardiovascular disease (CVD), this study opens new avenues to identify potential targets for diverse implementation strategies. Conclusion: A better understanding of apelin effects on KCC will help design a novel therapeutic approach to treat atherosclerosis-linked cardiovascular diseases, including COVID-19-associated mortality.

MicroRNA regulation of phenotypic transformations in vascular smooth muscle: relevance to vascular remodeling.

Alterations in the vascular smooth muscle cells (VSMC) phenotype play a critical role in the pathogenesis of several cardiovascular diseases, including hypertension, atherosclerosis, and restenosis after angioplasty. MicroRNAs (miRNAs) are a class of endogenous noncoding RNAs (approximately 19-25 nucleotides in length) that function as regulators in various physiological and pathophysiological events. Recent studies have suggested that aberrant miRNAs' expression might underlie VSMC phenotypic transformation, appearing to regulate the phenotypic transformations of VSMCs by targeting specific genes that either participate in the maintenance of the contractile phenotype or contribute to the transformation to alternate phenotypes, and affecting atherosclerosis, hypertension, and coronary artery disease by altering VSMC proliferation, migration, differentiation, inflammation, calcification, oxidative stress, and apoptosis, suggesting an important regulatory role in vascular remodeling for maintaining vascular homeostasis. This review outlines recent progress in the discovery of miRNAs and elucidation of their mechanisms of action and functions in VSMC phenotypic regulation. Importantly, as the literature supports roles for miRNAs in modulating vascular remodeling and for maintaining vascular homeostasis, this area of research will likely provide new insights into clinical diagnosis and prognosis and ultimately facilitate the identification of novel therapeutic targets.

Calcium promotes vascular smooth muscle cell phenotypic switching in Marfan syndrome

Fibrillin 1 (Fbn1) mutations cause Marfan syndrome (MFS), with aortic root dilatation, dissection, and rupture. Few studies reported the blood calcium and lipid profile of MFS, and the effect of vascular smooth muscle cell (VSMC) phenotypic switching on MFS aortic aneurysm is unclear. Here, we aimed to investigate the role of calcium-related VSMC phenotypic switching in MFS. We retrospectively collected MFS patients' clinical data, performed bioinformatics analysis to screen the enriched biological process in MFS patients and mice, and detected markers of VSMC phenotypic switching on Fbn1C1039G/+ mice and primary aortic vascular smooth muscle cells. We found that patients with MFS have elevated blood calcium levels and dyslipidemia. Furthermore, the calcium concentration levels were increased with age in MFS mice, accompanied by the promoted VSMC phenotypic switching, and SERCA2 contributed to maintaining the contractile phenotype of VSMCs. This study provides the first evidence that the increased calcium is associated with the promoted VSMC phenotype switching in MFS. SERCA may become a novel therapeutic target for suppressing aneurysm progression in MFS.

Abstract 528: Endothelial Estrogen Receptor Alpha Deletion Promotes Abdominal Aortic Aneurysm In Male Mice

Introduction: Although men have a greater incidence of abdominal aortic aneurysm (AAA) compared with women, the role of sex-hormones in mediating these differences is not well understood. Given the known effect of estrogen receptor alpha (ERα) signaling in maintaining vascular wall homeostasis, the present study examines its role in the pathogenesis of AAA. Specifically, we scrutinize whether endothelial cell (EC) ERα protects against AAA via increased lysyl oxidase (LOX) activation and regulation of vascular smooth muscle cell (VSMC) phenotype. Methods: AAA was induced in male and female ECERα knockout (ECERαKO) mice and their littermates (n=6-11) by administering β-Aminopropionitrile (BAPN) via drinking water (1 mg/ml; 0.15 g/kg/d) from 3 days prior to angiotensin II (Ang II; 1 μg/kg/day) infusion for 2 weeks. The development of AAA was assessed by ultrasound measurement of aortic luminal dilation and histology. Relevant molecular mechanisms were examined in abdominal aorta and in human aortic endothelial cells (HAECs). Results: BAPN Ang II resulted in an increased incidence of AAA in male ECERαKO mice (72%), whereas female ECERαKO mice did not develop aneurysms. No difference in AAA incidence was observed between male and female littermate control mice (12.5%). Elastin fragmentation was significantly higher only in male ECERαKO mice compared to littermates, while female ECERαKO and their littermates had comparable elastin fragmentation. Ang II receptor type 1a mRNA expression was higher in male ECERαKO, whereas female ECERαKO mice had higher Ang II receptor type 1b mRNA expression. Gene expression of smooth muscle alpha-actin and smooth muscle 22 (contractile VSMC markers) was significantly higher in female aortas compared to males irrespective of genotype, whereas, osteopontin, synthetic VSMC marker was only upregulated in male ECERαKO. HAECs treated with estradiol (10 nM, 24 h) significantly increased LOX activity. Conclusions: Our findings identify a critical role of endothelial ERα in protection against AAA in male mice likely through increased LOX activity and maintenance of VSMC phenotype. Further studies are needed to delineate the crosstalk between endothelial ERα and VSMCs, and their impact in the pathogenesis of AAA.

Abstract 496: Inhibition Of Tirna-gly-gcc Ameliorates Neointimal Formation Via Cbx3-mediated Vascular Smooth Muscle Cells Phenotypic Switching

Introduction: Vascular smooth muscle cells (VSMCs) are the main component of blood vessels and synthetic VSMCs play an essential role in vascular diseases. tRNA-derived fragments (tRFs) are a new class of non-coding RNAs involved in a variety of pathological processes, but their biological functions and mechanisms in VSMCs phenotype transition and vascular intimal hyperplasia are unclear. Hypothesis: We assessed the hypothesis that tRFs play important role in VSMCs phenotype transition, regulation of tRFs could ameliorates neointimal formation. Methods: Differential expression profiles of tRFs from different phenotype of VSMCs was acquired using small-RNA sequence. The expression of tiRNA-Gly-GCC was confirmed in VSMCs, vascular tissues and plasma from healthy and atherosclerosis using quantitative real-time PCR and RNA fluorescence in situ hybridization. Gain-and loss- of function were performed using mimics and inhibitors to investigate the role of tiRNA-Gly-GCC in VSMCs phenotypic transition and neointimal formation after vascular injury. Dual-luciferase reporter gene and rescue experiments were performed to demonstrate the relationship between tiRNA-Gly-GCC and CBX3. Results: tiRNA-Gly-GCC is upregulated in synthetic VSMCs, atherosclerotic arteries, plasma and the balloon injured carotid artery of rats. Functionally, the inhibition of tiRNA-Gly-GCC represses VSMCs proliferation, migration, and reversed dedifferentiation, whereas the overexpression of tiRNA-Gly- GCC have contrary effects. Mechanistically, tiRNA-Gly-GCC performs these functions on VSMCs via downregulating the expression of CBX3. Finally, the inhibition of tiRNA-Gly-GCC could ameliorate neointimal formation after vascular injury in vivo . Conclusions: In conclusion, tiRNA-Gly-GCC is a mediator of VSMCs phenotypic switching by targeting CBX3, inhibition of tiRNA-Gly-GCC suppresses neointimal formation.