Genes In Vascular Smooth Muscle Cells Research Articles

Role of EZH2-mediated epigenetic modification on vascular smooth muscle in cardiovascular diseases: A mini-review.

Vascular smooth muscle cells (VSMCs) are integral to the pathophysiology of cardiovascular diseases (CVDs). Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, plays a crucial role in epigenetic regulation of VSMCs gene expression. Emerging researches suggest that EZH2 has a dual role in VSMCs, contingent on the pathological context of specific CVDs. This mini-review synthesizes the current knowledge on the mechanisms by which EZH2 regulates VSMC proliferation, migration and survival in the context of CVDs. The goal is to underscore the potential of EZH2 as a therapeutic target for CVDs treatment. Modulating EZH2 and its associated epigenetic pathways in VSMCs could potentially ameliorate vascular remodeling, a key factor in the progression of many CVDs. Despite the promising outlook, further investigation is warranted to elucidate the epigenetic mechanisms mediated by EZH2 in VSMCs, which may pave the way for novel epigenetic therapies for conditions such as atherosclerosis and hypertension.

HRD1 reduction promotes cholesterol-induced vascular smooth muscle cell phenotypic change via endoplasmic reticulum stress.

Phenotypic change of vascular smooth muscle cells (VSMCs) is the main contributor of vascular pathological remodeling in atherosclerosis. The endoplasmic reticulum (ER) is critical for maintaining VSMC function through elimination of misfolded proteins that impair VSMC cellular function. ER-associated degradation (ERAD) is an ER-mediated process that controls protein quality by clearing misfolded proteins. One of the critical regulators of ERAD is HRD1, which also plays a vital role in lipid metabolism. However, the function of HRD1 in VSMCs of atherosclerotic vessels remains poorly understood. The level of HRD1 expression was analyzed in aortic tissues of mice fed with a high-fat diet (HFD). The H&E and EVG (VERHOEFF'S VAN GIESON) staining were used to demonstrate pathological vascular changes. IF (immunofluorescence) and WB (western blot) were used to explore the signaling pathways in vivo and in vitro. The wound closure and transwell assays were also used to test the migration rate of VSMCs. CRISPR gene editing and transcriptomic analysis were applied in vitro to explore the cellular mechanism. Our data showed significant reduction of HRD1 in aortic tissues of mice under HFD feeding. VSMC phenotypic change and HRD1 downregulation were detected by cholesterol supplement. Transcriptomic and further analysis of HRD1-KO VSMCs showed that HRD1 deficiency induced the expression of genes related to ER stress response, proliferation and migration, but reduced the contractile-related genes in VSMCs. HRD1 deficiency also exacerbated the proliferation, migration and ROS production of VSMCs induced by cholesterol, which promoted the VSMC dedifferentiation. Our results showed that HRD1 played an essential role in the contractile homeostasis of VSMCs by negatively regulating ER stress response. Thus, HRD1 in VSMCs could serve as a potential therapeutic target in metabolic disorder-induced vascular remodeling.

Higher matrix stiffness promotes VSMC senescence by affecting mitochondria-ER contact sites and mitochondria/ER dysfunction.

Abdominal aortic aneurysm (AAA) is a prevalent condition characterized by the weakening and bulging of the abdominal aorta. This study aimed to investigate the impact of a stiff matrix on vascular smooth muscle cells (VSMCs) in AAA development. Bioinformatics analysis revealed that differentially expressed genes (DEGs) in VSMCs of an AAA mouse model were enriched in cellular senescence and related pathways. To simulate aging-related changes, VSMCs were cultured on stiff matrices, and compared to those on soft matrices, the VSMCs cultured on stiff matrices exhibited cellular senescence. Furthermore, the mutual distance between mitochondria and endoplasmic reticulum (ER) in VSMCs was increased, indicating altered mitochondria-endoplasmic reticulum contacts (MERCs). The observed upregulation of reactive oxygen species (ROS) levels, antioxidant gene expression, and decreased mitochondrial membrane potential suggested the presence of mitochondrial dysfunction in VSMCs cultured on a stiff matrix. Additionally, the induction of ER stress-related genes indicated ER dysfunction. These findings collectively indicated impaired functionality of both mitochondria and ER in VSMCs cultured on a stiff matrix. Moreover, our data revealed that high lipid levels exacerbated the effects of high matrix stiffness on VSMCs senescence, MERC sites, and mitochondria/ER dysfunction. Importantly, treatment with the antilipemic agent CI-981 effectively reversed these detrimental effects. These findings provide insights into the role of matrix stiffness, mitochondrial dysfunction, ER stress, and lipid metabolism in AAA development, suggesting potential therapeutic targets for intervention.

LncRNA JPX-Enriched Chromatin Microenvironment Mediates Vascular Smooth Muscle Cell Senescence and Promotes Atherosclerosis.

Senescence is a series of degenerative changes in the structure and physiological function of an organism. Whether JPX (just proximal to XIST)-a newly identified age-related noncoding RNA by us-is associated with atherosclerosis is still unknown. Our study was to investigate the role of JPX and provide insights into potential therapies targeting atherosclerosis. We analyzed clinical data from multiple tissues including meniscus tissue, leukemia cells, and peripheral blood monocytes to identify age-related noncoding RNAs in senescent vascular smooth muscle cells (VSMCs). The molecular mechanism of JPX was investigated by capture hybridization analysis of RNA targets and chromatin immunoprecipitation. IGVTools and real-time quantitative polymerase chain reaction were used to evaluate the JPX expression during phenotype regulation in age-related disease models. The therapeutic potential of JPX was evaluated after establishing an atherosclerosis model in smooth muscle-specific Jpx knockout mice. JPX expression was upregulated in activated ras allele (H-rasV12)-induced senescent VSMCs and atherosclerotic arteries. JPX knockdown substantially reduced the elevation of senescence-associated secretory phenotype (SASP) genes in senescent VSMCs. Cytoplasmic DNA leaked from mitochondria via mitochondrial permeability transition pore formed by VDAC1 (voltage-dependent anion channel 1) oligomer activates the STING (stimulator of interferon gene) pathway. JPX could act as an enhancer for the SASP genes and functions as a scaffold molecule through interacting with phosphorylated p65/RelA and BRD4 (bromodomain-containing protein 4) in chromatin remodeling complex, promoting the transcription of SASP genes via epigenetic regulation. Smooth muscle knockout of Jpx in ApoeKO mice resulted in a decrease in plaque area, a reduction in SASP gene expression, and a decrease in senescence compared with controls. As an enhancer RNA, JPX can integrate p65 and BRD4 to form a chromatin remodeling complex, activating SASP gene transcription and promoting cellular senescence. These findings suggest that JPX is a potential therapeutic target for the treatment of age-related atherosclerosis.

Silencing of PKG1 Gene Mimics Effect of Aging and Sensitizes Rat Vascular Smooth Muscle Cells to Cardiotonic Steroids: Impact on Fibrosis and Salt Sensitivity.

Background Marinobufagenin, NKA (Na/K-ATPase) inhibitor, causes vasoconstriction and induces fibrosis via inhibition of Fli1 (Friend leukemia integration-1), a negative regulator of collagen synthesis. In vascular smooth muscle cells (VSMC), ANP (atrial natriuretic peptide), via a cGMP/PKG1 (protein kinase G1)-dependent mechanism, reduces NKA sensitivity to marinobufagenin. We hypothesized that VSMCfrom old rats, due to downregulation of ANP/cGMP/PKG-dependent signaling, would exhibit heightened sensitivity to the profibrotic effect of marinobufagenin. Methods and Results Cultured VSMCfrom the young (3-month-old) and old (24-month-old) male Sprague-Dawley rats and young VSMCwith silenced PKG1 gene were treated with 1 nmol/L ANP, or with 1 nmol/L marinobufagenin, or with a combination of ANP and marinobufagenin. Collagen-1, Fli1, and PKG1 levels were assessed by Western blotting analyses. Vascular PKG1 and Fli1 levels in the old rats were reduced compared with their young counterparts. ANP prevented inhibition of vascular NKA by marinobufagenin in young VSMC but not in old VSMC. In VSMC from the young rats, marinobufagenin induced downregulation of Fli1 and an increase in collagen-1 level, whereas ANP blocked this effect. Silencing of the PKG1 gene in young VSMC resulted in a reduction in levels of PKG1 and Fli1; marinobufagenin additionally reduced Fli1 and increased collagen-1 level, and ANP failed to oppose these marinobufagenin effects, similar to VSMC from the old rats with the age-associated reduction in PKG1. Conclusions Age-associated reduction in vascular PKG1 and the resultant decline in cGMP signaling lead to the loss of the ability of ANP to oppose marinobufagenin-induced inhibition of NKA and fibrosis development. Silencing of the PKG1 gene mimicked these effects of aging.

A novel role for the histone modifier PRDM6 and an opportunity to understand hypertension

Renin-secreting cells are critical for survival. These cells control blood pressure (BP), fluid-electrolyte homeostasis, tissue perfusion, kidney development, and repair of damaged glomeruli 1 Sequeira-Lopez M.L.S. Gomez R.A. Renin cells, the kidney, and hypertension. Circulation Research. 2021; 128: 887-907 Crossref PubMed Scopus (12) Google Scholar . Renin-secreting cells descend from mesodermal-derived precursors that differentiate into the mural cells of the kidney arterial tree 1 Sequeira-Lopez M.L.S. Gomez R.A. Renin cells, the kidney, and hypertension. Circulation Research. 2021; 128: 887-907 Crossref PubMed Scopus (12) Google Scholar . Mature renin-secreting cells are confined to the tips of the renal arterioles near the glomeruli, thus their name juxtaglomerular (JG) cells 1 Sequeira-Lopez M.L.S. Gomez R.A. Renin cells, the kidney, and hypertension. Circulation Research. 2021; 128: 887-907 Crossref PubMed Scopus (12) Google Scholar (Figure 1a). Other renin-lineage cells along the afferent arterioles can re-express renin in response to decreased kidney perfusion or fluid losses, resulting in an increased number of renin-secreting cells to restore homeostasis 1 Sequeira-Lopez M.L.S. Gomez R.A. Renin cells, the kidney, and hypertension. Circulation Research. 2021; 128: 887-907 Crossref PubMed Scopus (12) Google Scholar . Understanding how these cells differentiate and regulate renin expression and release may improve the management of hypertension and cardiovascular and kidney diseases. In a recent article published in the Journal of Clinical Investigation, Gunawardhana and colleagues present a novel approach to study the underlying mechanisms of hypertension and hypothesize a possible role for the vascular smooth muscle cell gene Prdm6 in determining the abundance of renin-secreting cells. 2 Gunawardhana K.L. Hong L. Rugira T. Uebbing S. Kucharczak J. Mehta S. Karunamuni D.R. Cabera-Mendoza B. Gandotra N. Scharfe C. Polimanti R. Noonan J.P. Mani A. A systems biology approach identifies the role of dysregulated PRDM6 in the development of hypertension. The Journal of Clinical Investigation. 2023; : 133 Google Scholar

Genetic Deletion of FXR1 Reduces Intimal Hyperplasia and Induces Senescence in Vascular Smooth Muscle Cells

Vascular smooth muscle cells (VSMC) play a critical role in the development and pathogenesis of intimal hyperplasia indicative of restenosis and other vascular diseases. Fragile-X related protein-1 (FXR1) is a muscle-enhanced RNA binding protein whose expression is increased in injured arteries. Previous studies suggest that FXR1 negatively regulates inflammation, but its causality in vascular disease is unknown. In the current study, RNA-sequencing of FXR1-depleted VSMC identified many transcripts with decreased abundance, most of which were associated with proliferation and cell division. mRNA abundance and stability of a number of these transcripts were decreased in FXR1-depleted hVSMC, as was proliferation (P<0.05); however, increases in beta-galactosidase (P<0.05) and γH2AX (P<0.01), indicative of senescence, were noted. Further analysis showed increased abundance of senescence-associated genes with FXR1 depletion. A novel SMC-specific conditional knockout mouse (FXR1SMC/SMC) was developed for further analysis. In a carotid artery ligation model of intimal hyperplasia, FXR1SMC/SMC mice had significantly reduced neointima formation (P<0.001) after ligation, as well as increases in senescence drivers p16, p21, and p53 compared with several controls. These results suggest that in addition to destabilization of inflammatory transcripts, FXR1 stabilized cell cycle-related genes in VSMC, and absence of FXR1 led to induction of a senescent phenotype, supporting the hypothesis that FXR1 may mediate vascular disease by regulating stability of proliferative mRNA in VSMC.

Identification of Novel Gene Regulatory Networks for Dystrophin Protein in Vascular Smooth Muscle Cells by Single-Nuclear Transcriptome Analysis.

Duchenne muscular dystrophy is an X-linked recessive disease caused by mutations in dystrophin proteins that lead to heart failure and respiratory failure. Dystrophin (DMD) is not only expressed in cardiomyocytes and skeletal muscle cells, but also in vascular smooth muscle cells (VSMCs). Patients with DMD have been reported to have hypotension. Single nuclear RNA sequencing (snRNA-seq) is a state-of-the-art technology capable of identifying niche-specific gene programs of tissue-specific cell subpopulations. To determine whether DMD mutation alters blood pressure, we compared systolic, diastolic, and mean blood pressure levels in mdx mice (a mouse model of DMD carrying a nonsense mutation in DMD gene) and the wide-type control mice. We found that mdx mice showed significantly lower systolic, diastolic, and mean blood pressure than control mice. To understand how DMD mutation changes gene expression profiles from VSMCs, we analyzed an snRNA-seq dataset from the muscle nucleus of DMD mutant (DMDmut) mice and control (Ctrl) mice. Gene Ontology (GO) enrichment analysis revealed that the most significantly activated pathways in DMDmut-VSMCs are involved in ion channel function (potassium channel activity, cation channel complex, and cation channel activity). Notably, we discovered that the DMDmut-VSMCs showed significantly upregulated expression of KCNQ5 and RYR2, whereas the most suppressed pathways were transmembrane transporter activity (such as anion transmembrane transporter activity, inorganic anion transmembrane transporter activity, import into cell, and import across plasma membrane). Moreover, we analyzed metabolic pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) using "scMetabolism" R package. DMDmut-VSMCs exhibited dysregulation of pyruvate metabolism and nuclear acid metabolism. In conclusion, via the application of snRNA-seq, we (for the first time) identify the potential molecular regulation by DMD in the upregulation of the expression of KCNQ5 genes in VSMCs, which helps us to understand the mechanism of hypotension in DMD patients. Our study potentially offers new possibilities for therapeutic interventions in systemic hypotension in DMD patients with pharmacological inhibition of KCNQ5.

A novel co-culture assay to evaluate the effects of sympathetic innervation on vascular smooth muscle differentiation

Dedifferentiation of vascular smooth muscle cells (VSMCs) from a functional phenotype to an inverse synthetic phenotype is a symptom of cardiovascular disorders, such as atherosclerosis and hypertension. The sympathetic nervous system (SNS) is an essential regulator of the differentiation of vascular smooth muscle cells (VSMCs). In addition, numerous studies suggest that SNS also stimulates VSMCs to retain their contractile phenotype. However, the molecular mechanisms for this stimulation have not been thoroughly studied. In this study, we used a novel in vitro co-culture method to evaluate the effective cellular interactions and stimulatory effects of sympathetic neurons on the differentiation of VSMCs. We co-cultured rat neural-like pheochromocytoma cells (PC12) and rat aortic VSMCs with this method. Expression of VSMCs contractile genes, including smooth muscle actin (acta2), myosin heavy chain (myh11), elastin (eln), and smoothelin (smtn), were determined by quantitative real-time-PCR analysis as an indicator of VSMCs differentiation. Fold changes for specific contractile genes in VSMCs grown in vitro for seven days in the presence (innervated) and absence (non-innervated) of sympathetic neurons were 3.5 for acta2, 6.5 for myh11, 4.19 for eln, and 4 for smtn (normalized to Tata Binding Protein (TBP)). As a result, these data suggest that sympathetic innervation promotes VSMCs' contractile gene expression and also maintains VSMCs' functional phenotype.

Semicarbazide-Sensitive Amine Oxidase (SSAO) and Lysyl Oxidase (LOX) Association in Rat Aortic Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are the main stromal cells in the medial layer of the vascular wall. These cells produce the extracellular matrix (ECM) and are involved in many pathological changes in the vascular wall. Semicarbazide-sensitive amine oxidase (SSAO) and lysyl oxidase (LOX) are vascular enzymes associated with the development of atherosclerosis. In the vascular smooth muscle cells, increased SSAO activity elevates reactive oxygen species (ROS) and induces VSMCs death; increased LOX induces chemotaxis through hydrogen peroxide dependent mechanisms; and decreased LOX contributes to endothelial dysfunction. This study investigates the relationship between SSAO and LOX in VSMCs by studying their activity, protein, and mRNA levels during VSMCs passaging and after silencing the LOX gene, while using their respective substrates and inhibitors. At the basal level, LOX activity decreased with passage and its protein expression was maintained between passages. βAPN abolished LOX activity (** p < 0.01 for 8 vs. 3 and * p < 0.05 for 5 vs. 8) and had no effect on LOX protein and mRNA levels. MDL72527 reduced LOX activity at passage 3 and 5 (##&nbsp;p < 0.01) and had no effect on LOX protein, and mRNA expression. At the basal level, SSAO activity also decreased with passage, and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (****&nbsp;p < 0.0001 for 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 5 (** p < 0.01) and 8 (**** p < 0.0001), and Aoc3 mRNA levels at passage 8 (* p < 0.05). βAPN inhibited SSAO activity (**** p < 0.0001 for 5 vs. 3 and 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 3 (* p < 0.05), and Aoc3 mRNA levels at passage 3 (* p < 0.05). Knockdown of the LOX gene (**** p < 0.0001 for Si6 vs. Sictrl and *** p < 0.001 for Si8 vs. Sictrl) and LOX protein (** p < 0.01 for Si6 and Si8 vs. Sictrl) in VSMCs at passage 3 resulted in a reduction in Aoc3 mRNA (####&nbsp;p < 0.0001 for Si6 vs. Sictrl and ###&nbsp;p < 0.001 for Si8 vs. Sictrl) and VAP-1 protein (#&nbsp;p < 0.05 for Si8 vs. Sictrl). These novel findings demonstrate a passage dependent decrease in LOX activity and increase in SSAO activity in rat aortic VSMCs and show an association between both enzymes in early passage rat aortic VSMCs, where LOX was identified as a regulator of SSAO activity, protein, and mRNA expression.

Protection against angiotensin II-induced hypertension in mice lacking sphingosine kinase 1 in vascular smooth muscle cells

Abstract Background Sphingosine-1-phosphate (S1P), a lipid mediator produced by sphingosine kinases (Sphks), significantly contributes to the maintenance of cardiovascular homeostasis. Mice lacking Sphk1 globally are protected against angiotensin II-induced hypertension, yet, due to pleiotropic action of S1P, it has been challenging to identify a specific cell type responsible for this protection. Purpose The aim of this study was to investigate the role of Sphk1 expressed in vascular smooth muscle cells (VSMCs) in the development of angiotensin II (AngII)-induced hypertension in mice. Methods Male, 12-week-old C57BL/6J wild type (WT) or VSMC-specific, Cre-mediated, Sphk1 knockout (Sphk1VSMC−/−) mice underwent either Sham or AngII (490 ng/min/kg s.c.) treatment for 2 weeks, using a surgically implanted osmotic minipump. Blood pressure was measured by the tail-cuff method. Vascular structure and function were examined ex vivo using wire (vasoconstrictor/vasorelaxant responses) and pressure myography (myogenic tone, structural and elasticity parameters). Gene set enrichment analysis (GSEA) was performed using transcriptomic profiles of the isolated mesenteric arteries (MA, RNASeq). Expression of proteins was analyzed by Western blot. Results Deletion of Sphk1 in VSMCs resulted in 5x and 40x lower expression of Sphk1 mRNA in MA and aorta compared to WT mice respectively. A 2-week infusion of AngII resulted in a development of higher systolic blood pressure (SBP) in WT mice compared to Sphk1VSMC−/− mice (mean SBP ± SEM: 168±4.3 mmHg vs. 132±4.4 mmHg respectively, p&amp;lt;0.05). Studies of ex vivo mesenteric artery (MA) function revealed diminished both, depolarization-induced (in response to KCl), as well as G protein-coupled receptor-elicited (in response to phenylephrine and thromboxane A2 analogue) contraction of MA derived from hypertensive Sphk1VSMC−/− mice compared to WT mice. In the same time, significantly impaired endothelial vasorelaxation was observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice. Moreover, MA from Sphk1VSMC−/− mice were characterized by the significantly reduced myogenic response evoked by intravascular pressure elevation (maximal myogenic response ± SEM: 19.7±1.8% for WT normotensive, 26.2±1.8% for WT AngII vs. 19.0±2.5% for Sphk1VSMC−/− AngII mice), and an increased stiffness compared to WT mice. GSEA found that the lack of Sphk1 gene in VSMCs significantly downregulated molecular pathways related to smooth muscle contraction. In line with transcriptomic studies, significantly reduced level of the Rho-associated protein kinase (Rock) has been observed in MA of hypertensive Sphk1VSMC−/− compared to hypertensive WT mice using Western blot. Conclusions This study revealed that mice lacking Sphk1 gene in VSMCs are protected against the development of AngII-induced hypertension likely due to the lowered myogenic tone in resistance arteries, which may be mediated by autocrine effects of Sphk1 on Rock proteins. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): The National Science Centre, Poland

Single-Cell RNA Sequencing Reveals the Pathogenic Relevance of Intracranial Atherosclerosis in Blood Blister-Like Aneurysms.

BackgroundIntracranial non-branching site blood blister-like aneurysms (BBA) are extremely rare and vicious. Their etiology remains elusive, and no molecular study has been carried out to reveal its pathogenic relevance to intracranial atherosclerosis. To investigate its transcriptomic landscape and underlying potential pathogenesis, we performed single-cell RNA sequencing with extensive pathological validation.MethodsIn total, 12,245 cells were recovered for single-cell RNA sequencing analysis from 1 BBA and 2 saccular intracranial aneurysms (IAs). Unbiased clustering using Seurat-based pipeline was used for cellular landscape profiling. Cellchat was used to understand intracellular communications. Furthermore, 10 BBAs and 30 IAs were retrospectively collected for pathological validations like scanning electron microscopy, H&E stain, Masson stain, Verhoeff Van Gielson stain, and immunofluorescence.ResultsSingle-cell transcriptome profiled 14 total subclusters in 6 major groups, namely, 6 monocyte/macrophage clusters, 2 T&NK clusters, 3 vascular smooth muscle cell (VSMC) clusters, 1 dendritic cell, 1 B cell, and 1 endothelial cell cluster. The only mural cell identified in BBAs was VSMC-2 cluster, while mural cells in IAs comprise most clusters of VSMCs and endothelial cells. Upregulated genes in BBA-derived VSMCs are related to arterial mineralization and atherosclerosis, such as PTX3, SPP1, LOX, etc., whereas vasodilation and physiological regulatory genes such as MGP, ACTA2, and MYL9 were conversely enriched in conventional IA-derived VSMCs. Immune cells in the BBA were predominantly macrophages, with a low fraction of T&NK cells, while conventional IAs had a higher percentage of T&NK. Gene enrichment analysis suggested that macrophages in BBA were highly enriched in lipid metabolism as well as atherosclerosis. Ligand–receptor interaction suggested that secretory phosphoprotein 1 (also known as osteopontin) played a major role in mediating the intracellular communication between VSMC and macrophages, especially in BBA. Pathological experiments corroborate with the bioinformatic findings and further characterized BBAs as a thin-walled thrombotic aneurysm with severe atherosclerotic lesions, where ApoE+ macrophages and OPN+ mural cells are intimately involved in the inflammation process.ConclusionsThe preexisting intracranial atherosclerosis might predispose the parent artery to the pathogenic occurrence of BBAs. These data shed light on the pathophysiology of intracranial aneurysms and might assist in the further resolution of the complexity in aneurysm pathogenesis.

Abstract 478: Role Of FXR1 And Senescence In Intimal Hyperplasia And Vascular Biology

Introduction: Despite the advent of stents, intimal hyperplasia subsequent to vascular interventional procedures remains a major obstacle. Vascular smooth muscle cells (VSMC) play a critical role in the pathogenesis of intimal hyperplasia; therefore regulation of VSMC gene expression is a logical intervention point. FXR1 is a muscle-enhanced RNA binding protein and expression is increased in injured arteries. We have shown that modulation of FXR1 levels affects stability and abundance of inflammatory transcripts in VSMC, suggesting that FXR1 is a negative regulator of inflammation. This drives our hypothesis that FXR1 is involved in mitigating vascular disease and regulating inflammatory and proliferative mRNA in VSMC. Approach/Results: We developed a novel VSMC-specific conditional knockout mouse (FXR1 VSMC/VSMC ). In a carotid artery ligation model of intimal hyperplasia FXR1 VSMC/VSMC mice, have significantly reduced neointima formation (p&lt;0.001) post-ligation compared to controls. To determine the mechanism of these effects, we knocked down FXR1 in human VSMC and observed decreased proliferation (p&lt;0.05) as well as increased beta galactosidase (p&lt;0.05) and gamma H2AX (p&lt;0.01) staining, indicative of senescence. Senescent cells exhibit a senescence associated secretory phenotype (SASP) with characteristic gene expression leading to increased inflammation in the tissue microenvironment. RIP-sequencing demonstrated that FXR1 interacts with transcripts involved in cell cycle control, and stability of these transcripts is decreased with FXR1 KO. qPCR analysis from FXR1 KO mouse VSMC show increased transcripts associated with senescence (p21, p16, p53) as well as increased SASP-associated mRNA. Furthermore, wild-type human VSMC cultured in conditioned media from cells transfected with FXR1 siRNA show increased SASP mRNA and increased proliferation compared with cells cultured in conditioned media from scrambled control cells. Summary &amp; Conclusions: Our results are the first to suggest that in addition to destabilization of inflammatory transcripts, FXR1 may stabilize cell cycle related genes in VSMC, and absence of FXR1 leads to induction of a senescent phenotype, an increase in SASP genes, and reduction of intimal hyperplasia.

The Role of FXR1 and Senescence in Vascular Biology and Intimal Hyperplasia

IntroductionDespite the advent of stents, intimal hyperplasia subsequent to vascular interventional procedures remains a major obstacle to success. Vascular smooth muscle cells (VSMC) play a critical role in the development and pathogenesis of intimal hyperplasia indicative of restenosis; therefore regulation of gene expression in VSMC represents a logical intervention point to attenuate this syndrome. FXR1 is a muscle‐enhanced RNA binding protein and expression is increased in injured human and mouse arteries. We have shown that modulation of FXR1 levels affects abundance and stability of inflammatory transcripts in VSMC, suggesting that FXR1 is a negative regulator of inflammation. This drives our hypothesis that FXR1 is involved in mitigating vascular disease and acts as a crucial regulator of inflammatory and proliferative mRNA in VSMC.Approach/ResultsBecause FXR1 expression is increased in injured arteries, we have developed a novel VSMC‐specific conditional knockout mouse (FXR1VSMC/VSMC). In a carotid artery ligation model of intimal hyperplasia, FXR1VSMC/VSMC mice have significantly reduced neointima formation (p&lt;0.001) post‐ligation compared to several controls. To determine the mechanism of these effects, we knocked down FXR1 in human VSMC by siRNA and observed decreased proliferation (p&lt;0.05) as well as an increase in beta galactosidase (p&lt;0.05) and gamma H2AX (p&lt;0.01) staining compared with controls, indicative of senescence. Senescent cells exhibit phenotypic changes called the senescence associated secretory phenotype (SASP) with characteristic gene expression leading to increased inflammation in the tissue microenvironment. RIP‐sequencing demonstrated that FXR1 interacts with transcripts involved in cell cycle control, and RNA stability of these transcripts is decreased with FXR1 KO. qPCR analysis from FXR1 KO mouse VSMC show an increase in transcripts associated with senescence (p21, p16, p53) as well as an increase in SASP‐associated mRNA compared with controls. Furthermore, wild‐type human VSMC cultured in conditioned media from cells transfected with FXR1 siRNA show an increase in SASP mRNA and an increase in proliferation compared with cells cultured in conditioned media from cells transfected with a scrambled control.Summary &amp; ConclusionsOur results are the first to suggest that in addition to destabilization of inflammatory transcripts, FXR1 may stabilize cell cycle related genes in VSMC, and absence of FXR1 leads to induction of a senescent phenotype, an increase in SASP gene expression, and reduction of intimal hyperplasia.

The epigenetic enzyme DOT1L orchestrates vascular smooth muscle cell-monocyte crosstalk and protects against atherosclerosis via the NF-κB pathway.

Histone H3 dimethylation at lysine 79 is a key epigenetic mark uniquely induced by methyltransferase disruptor of telomeric silencing 1-like (DOT1L). We aimed to determine whether DOT1L modulates vascular smooth muscle cell (VSMC) phenotype and how it might affect atherosclerosis in vitro and in vivo, unravelling the related mechanism. Gene expression screening of VSMCs stimulated with the BB isoform of platelet-derived growth factor led us to identify Dot1l as an early up-regulated epigenetic factor. Mouse and human atherosclerotic lesions were assessed for Dot1l expression, which resulted specifically localized in the VSMC compartment. The relevance of Dot1l to atherosclerosis pathogenesis was assessed through deletion of its gene in the VSMCs via an inducible, tissue-specific knock-out mouse model crossed with the ApoE-/- high-fat diet model of atherosclerosis. We found that the inactivation of Dot1l significantly reduced the progression of the disease. By combining RNA- and H3K79me2-chromatin immunoprecipitation-sequencing, we found that DOT1L and its induced H3K79me2 mark directly regulate the transcription of Nf-κB-1 and -2, master modulators of inflammation, which in turn induce the expression of CCL5 and CXCL10, cytokines fundamentally involved in atherosclerosis development. Finally, a correlation between coronary artery disease and genetic variations in the DOT1L gene was found because specific polymorphisms are associated with increased mRNA expression. DOT1L plays a key role in the epigenetic control of VSMC gene expression, leading to atherosclerosis development. Results identify DOT1L as a potential therapeutic target for vascular diseases.

Human umbilical cord mesenchymal stem cell-derived exosomal microRNA-148a-3p inhibits neointimal hyperplasia by targeting Serpine1

BackgroundRestenosis is inevitable when patients undergo percutaneous transluminal angioplasty due to neointimal hyperplasia (NIH). Human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Exos) have been studied in the field of cardiovascular diseases. However, the effects and mechanisms of hucMSC-Exos on NIH are unclear. We aimed to investigate whether MSC-Exos regulate vascular smooth muscle cell (VSMC) functions to inhibit NIH and explore the underlying mechanisms. MethodsHucMSCs and mouse VSMCs were isolated and characterized by flow cytometry and immunofluorescence. HucMSC-Exos were identified by transmission electron microscopy, nanoparticle tracking analysis and western blots. Exosomes (Exos) were intravenously injected into mice with left common carotid artery ligation, and their effects on NIH were assessed by haematoxylin and eosin (H&E) and immunohistochemistry staining. The effects of hucMSC-Exos on VSMCs were evaluated by Cell Counting Kit-8, scratch wound, Transwell and Western blot assays. MicroRNA sequencing data in the Gene Expression Omnibus and mRNA sequencing results were used to identify potential molecules in hucMSC-Exos and target genes in VSMCs, respectively. We tested the regulatory effect of microRNAs in Exos and target genes in VSMCs using overexpression and knockdown experiments. ResultsPrimary hucMSCs, VSMCs and hucMSC-Exos were isolated and characterized. Administration of hucMSC-Exos suppressed NIH after artery ligation. H&E and immunohistochemistry results showed that hucMSC-Exos decreased the intima and media area and intima/media ratio, increased the contractile phenotype protein SM22a in the media layer and downregulated Serpine1 expression in the carotid artery. Exos were ingested by VSMCs, which inhibited migration and upregulated SM22a expression by suppressing Serpine1 expression in vitro. MiR-148a-3p was enriched in hucMSC-Exos and repressed Serpine1 by targeting its 3’ untranslated region. Moreover, exosomal miR-148a-3p suppressed VSMC phenotypic switching and migration by targeting Serpine1. ConclusionsWe found that hucMSC-Exos inhibited NIH in a mouse carotid artery ligation model and that the inhibitory effects on VSMC phenotypic switching and migration were mediated by delivery of miR-148a-3p to VSMCs to target Serpine1.

MiR-222-5p promotes dysfunction of human vascular smooth muscle cells by targeting RB1.

Coronary atherosclerosis (AS) is characterized by the formation of plaque in the vessel wall. The structural and functional changes of vascular smooth muscle cells (VSMCs) can promote plaque formation and induce plaque instability. To investigate the functions and mechanism of miR-222-5p in VSMCs under the treatment of oxidized low-density lipoprotein (ox-LDL). miR-222-5p expression in ox-LDL-treated VSMCs and the serum of Apolipoprotein E (ApoE) knockout mice was detected by reverse transcription quantitative polymerase chain reaction. The viability and migration of VSMCs were detected by Cell Counting Kit-8 and Transwell assays. Protein levels of proliferation and migration-related factors were evaluated by western blotting. Luciferase reporter assays were performed to explore the binding between miR-222-5p and retinoblastoma susceptibility protein (RB1) gene in VSMCs. ApoE-knockout mice were infected with the lentivirus inhibiting miR-222-5p expression to explore the effect of miR-222-5p on pathological changes. Hematoxylin and eosin (H&E) staining, trichrome staining, and Oil Red O staining were conducted to determine the necrotic core area and atherosclerotic lesion size in the ascending aorta of ApoE-knockout mice. With the accumulation of ox-LDL concentration and treatment time, miR-222-5p expression was gradually upregulated in VSMCs. Similarly, miR-222-5p expression was increased in the serum of ApoE-knockout mice. miR-222-5p knockdown inhibited the proliferative and migratory abilities of ox-LDL-treated VSMCs, and the inhibitory effect on cellular behaviors was then significantly reversed by co-knockdown of RB1. RB1 is a downstream target gene of miR-222-5p, and miR-222-5p bound with 3'-untranslated region of RB1 in VSMCs. We further confirmed that miR-222-5p knockdown alleviated pathological changes and inhibited lipid deposition in the serum of ApoE-knockout mice in vivo. miR-222-5p accelerates the dysfunction of VSMCs and promotes pathological changes and lipid deposition in ApoE-knockout mice by targeting RB1. The study may provide novel therapeutic targets for AS.

Abstract 13125: The Role of FXR1 and Cellular Senescence in Vascular Pathology

Introduction: Despite the advent of stents, intimal hyperplasia subsequent to vascular interventional procedures remains a major obstacle to success. Vascular smooth muscle cells (VSMC) play a critical role in the development and pathogenesis of intimal hyperplasia indicative of restenosis; therefore regulation of gene expression in VSMC represents a logical intervention point to attenuate this syndrome. FXR1 is a muscle-enhanced RNA binding protein and expression is increased in injured human and mouse arteries. We have shown that modulation of FXR1 levels affects abundance and stability of inflammatory transcripts in VSMC, suggesting that FXR1 is a negative regulator of inflammation. This drives our hypothesis that FXR1 is involved in mitigating vascular disease and acts as a crucial regulator of inflammatory and proliferative mRNA in VSMC. Approach/Results: Because FXR1 expression is increased in injured arteries, we have developed a novel VSMC-specific conditional knockout mouse (FXR1 VSMC/VSMC ). In a carotid artery ligation model of intimal hyperplasia, FXR1 VSMC/VSMC mice have significantly reduced neointima formation (p&lt;0.001) post-ligation compared to several controls. To determine the mechanism of these effects, we knocked down FXR1 in human VSMC by siRNA and observed decreased proliferation (p&lt;0.05) as well as an increase in beta galactosidase (p&lt;0.05) and gamma H2AX (p&lt;0.01) staining compared with controls, indicative of senescence. Senescent cells exhibit phenotypic changes called the senescence associated secretory phenotype (SASP) with characteristic gene expression leading to increased inflammation in the tissue microenvironment. RNA sequencing demonstrated a decrease in abundance of cell cycle control genes. This was validated by actinomycin D RNA stability assays, and further qPCR analysis showed an increase in abundance of SASP-related mRNA with FXR1 depletion. VSMC knockout of FXR1 via Cre adenovirus in mouse cells validated these results. Summary &amp; Conclusions: Our results are the first to suggest that in addition to destabilization of inflammatory transcripts, FXR1 may stabilize cell cycle related genes in VSMC, and absence of FXR1 leads to induction of a senescent phenotype and reduction of intimal hyperplasia.

Therapeutic MK2 inhibition blocks pathological vascular smooth muscle cell phenotype switch.

Vascular procedures, such as stenting, angioplasty, and bypass grafting, often fail due to intimal hyperplasia (IH), wherein contractile vascular smooth muscle cells (VSMCs) dedifferentiate to synthetic VSMCs, which are highly proliferative, migratory, and fibrotic. Previous studies suggest MAPK-activated protein kinase 2 (MK2) inhibition may limit VSMC proliferation and IH, although the molecular mechanism underlying the observation remains unclear. We demonstrated here that MK2 inhibition blocked the molecular program of contractile to synthetic dedifferentiation and mitigated IH development. Molecular markers of the VSMC contractile phenotype were sustained over time in culture in rat primary VSMCs treated with potent, long-lasting MK2 inhibitory peptide nanopolyplexes (MK2i-NPs), a result supported in human saphenous vein specimens cultured ex vivo. RNA-Seq of MK2i-NP–treated primary human VSMCs revealed programmatic switching toward a contractile VSMC gene expression profile, increasing expression of antiinflammatory and contractile-associated genes while lowering expression of proinflammatory, promigratory, and synthetic phenotype–associated genes. Finally, these results were confirmed using an in vivo rabbit vein graft model where brief, intraoperative treatment with MK2i-NPs decreased IH and synthetic phenotype markers while preserving contractile proteins. These results support further development of MK2i-NPs as a therapy for blocking VSMC phenotype switch and IH associated with cardiovascular procedures.

The Expanding Role of Alternative Splicing in Vascular Smooth Muscle Cell Plasticity.

Vascular smooth muscle cells (VSMCs) display extraordinary phenotypic plasticity. This allows them to differentiate or dedifferentiate, depending on environmental cues. The ability to ‘switch’ between a quiescent contractile phenotype to a highly proliferative synthetic state renders VSMCs as primary mediators of vascular repair and remodelling. When their plasticity is pathological, it can lead to cardiovascular diseases such as atherosclerosis and restenosis. Coinciding with significant technological and conceptual innovations in RNA biology, there has been a growing focus on the role of alternative splicing in VSMC gene expression regulation. Herein, we review how alternative splicing and its regulatory factors are involved in generating protein diversity and altering gene expression levels in VSMC plasticity. Moreover, we explore how recent advancements in the development of splicing-modulating therapies may be applied to VSMC-related pathologies.