Vascular Smooth Muscle Cell Phenotype Research Articles

Abstract 1128: Smooth Muscle Myosin Ii Dynamics And Specificity To Muscle Type

Smooth muscle myosin II (SMII), the main motor protein in vascular smooth muscle cells (VSMC), generates the contractile forces crucial to VSMC function. SMII filaments assemble into bundles that bind and pull on the actin cytoskeleton. VSMCs exist on a functional spectrum between the contractile and synthetic phenotypes. Our previous work established a discernible difference in EGFP-SMII filaments between phenotypes. The synthetic phenotype is highly dynamic with SMII filaments freely assembling into bundles and dissociating into monomers. The contractile phenotype features stabilized SMII bundles leading to organized contractile machinery. While known that VSMC function changes with artery size, differences in SMII dynamics remain understudied. We will investigate these differences in SMII between VSMCs of large conducting, small conducting, and resistive arteries to determine SMII functional properties unique to each VSMC type. We will extend this to looking at smooth muscle cells (SMC) from other tissue types. We aim to identify tissue-specific SMII properties to discover mechanisms that predispose specific tissues to pathologies and potential tissue-specific targets for therapeutics. We recently engineered a novel endogenously-tagged EGFP-SMII mouse, which allowed us to observe changes in VSMC contractile machinery directly and to better understand SMII dynamics. We isolated SMCs from the EGFP-SMII mouse from the vasculature, gastrointestinal, and genitourinary tracts. We measured SMII filament assembly, SMII fluorescent recovery after photobleaching (FRAP), and whole-cell and organoid contractility using traction force microscopy. With the isolation of single cells, we observed a very rapid transition in aortic VSMC phenotype, from contractile to synthetic, with the contractile machinery demonstrating synthetic changes accordingly. The physiologic, native whole-tissue state had stable SMII dynamics, while isolated cells (in a pathologic, cultured state) exhibited rapid filament turnover. We also observed greater stability in aortic tissue ex vivo compared to other vessels and tissues containing SMII. These results suggest aortic SMII has unique properties and pathways specific to the type and state of the tissue.

Abstract 3142: Pik3c3 Safeguards Smooth Muscle Identity By Promoting Lysosome-mediated Yap1 Degradation

Background: Mature vascular smooth muscle cells (VSMCs) are quiescent and geared for maintaining vascular homeostasis by expressing a unique repertoire of contractile genes. However, key factors in controlling the contractile phenotype of VSMCs are poorly defined. Class III PI3K, PIK3C3 (Phosphatidylinositol 3-Kinase Catalytic Subunit Type 3) is a critical regulator of vesicular trafficking. Although PIK3C3 is known to play an essential role in cardiac homeostasis, its role in VSMCs remains elusive. Methods: To elucidate the role of Pik3c3 in regulating SMC fate and plasticity, we generated smooth muscle-specific Pik3c3 inducible knock out (KO) mice with SMC-lineage tracing. Proteomic mass spectrometry, next-generation RNA sequencing, SMC fate mapping coupled with single-cell RNA sequencing were applied to determine the phenotype and trajectories of SMCs after Pik3c3 ablation. Results: Specific ablation of Pik3c3 in SMCs results in severe gastrointestinal pseudo-obstruction and premature death. Analysis of aorta from the KO mice revealed dramatic vascular remodeling including dilatation, thickening of the vascular wall, spontaneous neointimal formation that was largely contributed by cells of SMC origin, and complete loss of contractility. Proteomics, bulk RNA-seq, and scRNA-seq studies of SMC-restricted Pik3c3 KO mouse aorta showed near complete loss of smooth muscle identity and the acquisition of an intermediate smooth muscle progenitor-like cell phenotype that underwent re-programming into multiple cell types including macrophages, T cells and osteoblasts. Mechanistically, we found that Pik3c3 deletion in VSMCs induces the accumulation of YAP1 (Yes associated protein 1) and its downstream target genes; this elevated YAP1 program appears to result from a blockade of autophagic lysosome-mediated degradation of YAP1, leading to phenotypic modulation of SMCs. Importantly, pharmacological inhibition of YAP by Verteporfin or genetic deletion of Yap1 rescues the vascular phenotype of Pik3c3 SM-specific KO mice. Conclusion: Our study reveals PIK3C3 as a master regulator safeguarding VSMC identity, and perturbation of the PIK3C3-YAP axis may be a novel mechanism underlying VSMC-driven vascular diseases in humans.

Asprosin aggravates atherosclerosis via regulating the phenotype transformation of vascular smooth muscle cells

Phenotype transformation of vascular smooth muscle cells (VSMCs) plays an important role in the development of atherosclerosis. Asprosin is a newly discovered adipokine, which is critical in regulating metabolism. However, the relationship between asprosin and phenotype transformation of VSMCs in atherosclerosis remains unclear. The aim of this study is to investigate whether asprosin affects the progression of atherosclerosis by inducing phenotype transformation of VSMCs. We established an atherosclerosis model in ApoE−/− mice and administered asprosin recombinant protein and asprosin antibody to mice. Knocking down asprosin was also as an intervention. Interestingly, we found a correlation between asprosin levels and atherosclerosis. Asprosin promoted plaque formation and phenotype transformation of VSMCs. While, AspKD or asprosin antibody reduced the plaque lesion and suppressed vascular stiffness in ApoE−/− mice. Mechanistically, asprosin induced phenotype transformation of MOVAs by binding to GPR54, leading to Gαq/11 recruitment and activation of the PLC-PKC-ERK1/2-STAT3 signaling pathway. Si GPR54 or GPR54 antagonist partially inhibited the action of asprosin in MOVAs. Mutant GPR54-(267, 307) residue cancelled the binding of asprosin and GPR54. In summary, this study confirmed asprosin activated GPR54/Gαq/11-dependent ERK1/2-STAT3 signaling pathway, thereby promoting VSMCs phenotype transformation and aggravating atherosclerosis, thus providing a new target for the treatment of atherosclerosis.

Divergent and Compensatory Effects of BMP2 and BMP4 on the VSMC Phenotype and BMP4's Role in Thoracic Aortic Aneurysm Development.

Vascular smooth muscle cells (VSMCs) play a key role in aortic aneurysm formation. Bone morphogenetic proteins (BMPs) have been implicated as important regulators of VSMC phenotype, and dysregulation of the BMP pathway has been shown to be associated with vascular diseases. The aim of this study was to investigate for the first time the effects of BMP-4 on the VSMC phenotype and to understand its role in the development of thoracic aortic aneurysms (TAAs). Using the angiotensin II (AngII) osmotic pump model in mice, aortas from mice with VSMC-specific BMP-4 deficiency showed changes similar to AngII-infused aortas, characterised by a loss of contractile markers, increased fibrosis, and activation of matrix metalloproteinase 9. When BMP-4 deficiency was combined with AngII infusion, there was a significantly higher rate of apoptosis and aortic dilatation. In vitro, VSMCs with mRNA silencing of BMP-4 displayed a dedifferentiated phenotype with activated canonical BMP signalling. In contrast, BMP-2-deficient VSMCs exhibited the opposite phenotype. The compensatory regulation between BMP-2 and BMP-4, with BMP-4 promoting the contractile phenotype, appeared to be independent of the canonical signalling pathway. Taken together, these results demonstrate the impact of VSMC-specific BMP-4 deficiency on TAA development.

LncRNA RP11-301G19.1 is required for the maintenance of vascular smooth muscle cell contractile phenotype via sponging miR-17-5P/ATOH8 axis.

Long noncoding RNAs (LncRNAs) play essential roles in regulating gene expression in various biological processes. However, the function of lncRNAs in vascular smooth muscle cell (VSMC) transformation remains to be explained. In this work, we discover that a new bone marrow protein (BMP) signaling target, lncRNA RP11-301G19.1, is significantly induced in BMP7-treated VSMCs through lncRNA microarray analysis. Addition of BMP signaling inhibitor LDN-193189 attenuates the expression of ACTA2 and SM-22α, as well as the mRNA level of RP11-301G19.1. Furthermore, lncRNA RP11-301G19.1 is critical to the VSMC differentiation and is directly activated by SMAD1/9. Mechanistically, knocking down of RP11-301G19.1 leads to the decrease of ATOH8, another BMP target, while the forced expression of RP11-301G19.1 reactivates ATOH8. In addition, miR-17-5p, a miRNA negatively regulated by BMP-7, contains predicted binding sites for lncRNA RP11-301G19.1 and ATOH8 3'UTR. Accordingly, overexpression of miR-17-5p decreases the levels of them. Together, our results revealed the role of lncRNA RP11-301G19.1 as a miRNA sponge to upregulate ATOH8 in VSMC phenotype transformation.

Targeting deubiquitinase OTUB1 protects vascular smooth muscle cells in atherosclerosis by modulating PDGFRβ.

Atherosclerosis is a chronic artery disease that causes various types of cardiovascular dysfunction. Vascular smooth muscle cells (VSMCs), the main components of atherosclerotic plaque, switch from contractile to synthetic phenotypes during atherogenesis. Ubiquitylation is crucial in regulating VSMC phenotypes in atherosclerosis, and it can be reversely regulated by deubiquitinases. However, the specific effects of deubiquitinases on atherosclerosis have not been thoroughly elucidated. In this study, RNAi screening in human aortic smooth muscle cells was performed to explore the effects of OTU family deubiquitinases, which revealed that silencing OTUB1 inhibited PDGF-BB-stimulated VSMC phenotype switch. Further in vivo studies using Apoe-/- mice revealed that knockdown of OTUB1 in VSMCs alleviated atherosclerosis plaque burden in the advanced stage and led to a stable plaque phenotype. Moreover, VSMC proliferation and migration upon PDGF-BB stimulation could be inhibited by silencing OTUB1 in vitro. Unbiased RNA-sequencing data indicated that knocking down OTUB1 influenced VSMC differentiation, adhesion, and proliferation. Mass spectrometry of ubiquitinated protein confirmed that proteins related to cell growth and migration were differentially ubiquitylated. Mechanistically, we found that OTUB1 recognized the K707 residue ubiquitylation of PDGFRβ with its catalytic triad, thereby reducing the K48-linked ubiquitylation of PDGFRβ. Inhibiting OTUB1 in VSMCs could promote PDGFRβ degradation via the ubiquitin-proteasome pathway, so it was beneficial in preventing VSMCs' phenotype switch. These findings revealed that knocking down OTUB1 ameliorated VSMCs' phenotype switch and atherosclerosis progression, indicating that OTUB1 could be a valuable translational therapeutic target in the future.

Carrageenan maintains the contractile phenotype of vascular smooth muscle cells by increasing macromolecular crowding in vitro

BackgroundThe contractile phenotype of vascular smooth muscle cells (VSMCs) results in good diastolic and contractile capacities, and its altered function is the main pathophysiological basis for diseases such as hypertension. VSMCs exist as a synthetic phenotype in vitro, making it challenging to maintain a contractile phenotype for research. It is widely recognized that the common medium in vitro is significantly less crowded than in the in vivo environment. Additionally, VSMCs have a heightened sense for detecting changes in medium crowding. However, it is unclear whether macromolecular crowding (MMC) helps maintain the VSMCs contractile phenotype.PurposeThis study aimed to explore the phenotypic, behavioral and gene expression changes of VSMCs after increasing the crowding degree by adding carrageenan (CR).MethodsThe degree of medium crowding was examined by a dynamic light scattering assay; VSMCs survival and activity were examined by calcein/PI cell activity and toxicity and CCK-8 assays; VSMCs phenotypes and migration were examined by WB and wound healing assays; and gene expression was examined by transcriptomic analysis and RT-qPCR.ResultsNotably, 225 μg/mL CR significantly increased the crowding degree of the medium and did not affect cell survival. Simultaneously, CR significantly promoted the contraction phenotypic marker expression in VSMCs, shortened cell length, decreased cell proliferation, and inhibited cell migration. CR significantly altered gene expression in VSMCs. Specifically, 856 genes were upregulated and 1207 genes were downregulated. These alterations primarily affect the cellular ion channel transport, microtubule movement, respiratory metabolism, amino acid transport, and extracellular matrix synthesis. The upregulated genes were primarily involved in the cytoskeleton and contraction processes of VSMCs, whereas the downregulated genes were mainly involved in extracellular matrix synthesis.ConclusionsThe in vitro study showed that VSMCs can maintain the contractile phenotype by sensing changes in the crowding of the culture environment, which can be maintained by adding CR.Graphical

Single-Nucleus Multiomic Analyses Identifies Gene Regulatory Dynamics of Phenotypic Modulation in Human Aneurysmal Aortic Root.

Aortic root aneurysm is a potentially life-threatening condition that may lead to aortic rupture and is often associated with genetic syndromes, such as Marfan syndrome (MFS). Although studies with MFS animal models have provided valuable insights into the pathogenesis of aortic root aneurysms, this understanding of the transcriptomic and epigenomic landscape in human aortic root tissue remains incomplete. This knowledge gap has impeded the development of effective targeted therapies. Here, this study performs the first integrative analysis of single-nucleus multiomic (gene expression and chromatin accessibility) and spatial transcriptomic sequencing data of human aortic root tissue under healthy and MFS conditions. Cell-type-specific transcriptomic and cis-regulatory profiles in the human aortic root are identified. Regulatory and spatial dynamics during phenotypic modulation of vascular smooth muscle cells (VSMCs), the cardinal cell type, are delineated. Moreover, candidate key regulators driving the phenotypic modulation of VSMC, such as FOXN3, TEAD1, BACH2, and BACH1, are identified. In vitro experiments demonstrate that FOXN3 functions as a novel key regulator for maintaining the contractile phenotype of human aortic VSMCs through targeting ACTA2. These findings provide novel insights into the regulatory and spatial dynamics during phenotypic modulation in the aneurysmal aortic root of humans.

Spaceflight effects on human vascular smooth muscle cell phenotype and function

The cardiovascular system is strongly impacted by the hazards of spaceflight. Astronauts spending steadily increasing lengths of time in microgravity are subject to cardiovascular deconditioning resulting in loss of vascular tone, reduced total blood volume, and diminished cardiac output. Appreciating the mechanisms by which the cells of the vasculature are altered during spaceflight will be integral to understanding and combating these deleterious effects as the human presence in space advances. In this study, we performed RNA-Seq analysis coupled with review by QIAGEN Ingenuity Pathway Analysis software on human aortic smooth muscle cells (HASMCs) cultured for 3 days in microgravity and aboard the International Space Station to assess the transcriptomic changes that occur during spaceflight. The results of our RNA-Seq analysis show that SMCs undergo a wide range of transcriptional alteration while in space, significantly affecting 4422 genes. SMCs largely down-regulate markers of the contractile, synthetic, and osteogenic phenotypes including smooth muscle alpha actin (αSMA), matrix metalloproteinases (MMPs), and bone morphogenic proteins (BMPs). Additionally, components of several cellular signaling pathways were strongly impacted including the STAT3, NFκB, PI3K/AKT, HIF1α, and Endothelin pathways. This study highlights the significant changes in transcriptional behavior SMCs exhibit during spaceflight and puts these changes in context to better understand vascular function in space.

Small nucleolar RNA host gene 18 controls vascular smooth muscle cell contractile phenotype and neointimal hyperplasia.

Long non-coding RNA (LncRNA) small nucleolar RNA host gene 18 (SNHG18) has been widely implicated in cancers. However, little is known about its functional involvement in vascular diseases. Herein, we attempted to explore a role for SNHG18 in modulating vascular smooth muscle cell (VSMC) contractile phenotype and injury-induced neointima formation. Analysis of single-cell RNA sequencing and transcriptomic datasets showed decreased levels of SNHG18 in injured and atherosclerotic murine and human arteries, which is positively associated with VSMC contractile genes. SNHG18 was upregulated in VSMCs by TGFβ1 through transcription factors Sp1 and SMAD3. SNHG18 gene gain/loss-of-function studies revealed that VSMC contractile phenotype was positively regulated by SNHG18. Mechanistic studies showed that SNHG18 promotes a contractile VSMC phenotype by up-regulating miR-22-3p. SNHG18 up-regulates miR-22 biogenesis and miR-22-3p production by competitive binding with the A-to-I RNA editing enzyme, adenosine deaminase acting on RNA-2 (ADAR2). Surprisingly, we observed that ADAR2 inhibited miR-22 biogenesis not through increasing A-to-I editing within primary miR-22, but by interfering with the binding of microprocessor complex subunit DGCR8 to primary miR-22. Importantly, perivascular SNHG18 overexpression in the injured vessels dramatically up-regulated the expression levels of miR-22-3p and VSMC contractile genes, and prevented injury-induced neointimal hyperplasia. Such modulatory effects were reverted by miR-22-3p inhibition in the injured arteries. Finally, we observed a similar regulator role for SNHG18 in human VSMCs and a decreased expression level of both SNHG18 and miR-22-3p in diseased human arteries; and we found that the expression level of SNHG18 was positively associated with that of miR-22-3p in both healthy and diseased human arteries. We demonstrate that SNHG18 is a novel regulator in governing VSMC contractile phenotype and preventing injury-induced neointimal hyperplasia. Our findings have important implications for therapeutic targeting snhg18/miR-22-3p signalling in vascular diseases.

CNPY2 governs PDGF‑BB‑treated vascular smooth muscle cell proliferation, migration and phenotypic transformation via the Akt/mTOR/GSK‑3β signaling pathway.

Phenotype switching of vascular smooth muscle cells (VSMCs) is a pathological process in various vascular diseases. Canopy FGF signaling regulator 2 (CNPY2) has previously been found to be abnormally expressed in ApoE-/- mice and aortic endothelial cells, indicating it may have an important role in vascular diseases. The present study aimed to determine the role and mechanism of CNPY2 in VSMC phenotype switching. Following stimulation with platelet-derived growth factor type BB (PDGF-BB), the expression of CNPY2 in VSMCs was detected using reverse transcription-quantitative PCR and western blot analysis. Subsequently, to explore the regulatory effects of CNPY2 on VSMCs, CNPY2 expression was knocked down by transfection with short hairpin RNA and cell viability, proliferation, migration and phenotypic transformation indicators were detected. Western blot analysis was also used to detect the phosphorylation of Akt/mTOR/GSK-3β pathway-associated proteins downstream of CNPY2. In addition, pretreatment with the Akt pathway activator SC79 was performed to further explore the regulatory mechanisms of CNPY2. The results revealed that CNPY2 expression was upregulated in PDGF-BB-stimulated VSMCs. In addition, the knockdown of CNPY2 inhibited PDGF-BB-induced VSMC hyperproliferation, cell cycle arrest, migration and phenotypic transformation, as well the activation of Akt/mTOR/GSK-3β pathway-associated proteins. Pretreatment with SC79 significantly reversed the inhibitory effects of CNPY2 knockdown on the proliferation, cell cycle arrest, migration and phenotypic transformation of the model cells. In summary, the present study indicates that CNPY2 regulates the abnormal proliferation, migration and phenotypic transformation of PDGF-BB-stimulated VSMCs via activation of the Akt/mTOR/GSK-3β signaling pathway.

Ercc1 DNA repair deficiency results in vascular aging characterized by VSMC phenotype switching, ECM remodeling, and an increased stress response.

Cardiovascular diseases are the number one cause of death globally. The most important determinant of cardiovascular health is a person's age. Aging results in structural changes and functional decline of the cardiovascular system. DNA damage is an important contributor to the aging process, and mice with a DNA repair defect caused by Ercc1 deficiency display hypertension, vascular stiffening, and loss of vasomotor control. To determine the underlying cause, we compared important hallmarks of vascular aging in aortas of both Ercc1Δ/- and age-matched wildtype mice. Additionally, we investigated vascular aging in 104 week old wildtype mice. Ercc1Δ/- aortas displayed arterial thickening, a loss of cells, and a discontinuous endothelial layer. Aortas of 24 week old Ercc1Δ/- mice showed phenotypical switching of vascular smooth muscle cells (VSMCs), characterized by a decrease in contractile markers and a decrease in synthetic markers at the RNA level. As well as an increase in osteogenic markers, microcalcification, and an increase in markers for damage induced stress response. This suggests that Ercc1Δ/- VSMCs undergo a stress-induced contractile-to-osteogenic phenotype switch. Ercc1Δ/- aortas showed increased MMP activity, elastin fragmentation, and proteoglycan deposition, characteristic of vascular aging and indicative of age-related extracellular matrix remodeling. The 104 week old WT mice showed loss of cells, VSMC dedifferentiation, and senescence. In conclusion, Ercc1Δ/- aortas rapidly display many characteristics of vascular aging, and thus the Ercc1Δ/- mouse is an excellent model to evaluate drugs that prevent vascular aging in a short time span at the functional, histological, and cellular level.

CircHAT1 regulates the proliferation and phenotype switch of vascular smooth muscle cells in lower extremity arteriosclerosis obliterans through targeting SFRS1.

This study aimed to decipher the mechanism of circular ribonucleic acids (circRNAs) in lower extremity arteriosclerosis obliterans (LEASO). First, bioinformatics analysis was performed for screening significantly down-regulated cardiac specific circRNA-circHAT1 in LEASO. The expression of circHAT1 in LEASO clinical samples was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The protein expression of splicing factor arginine/serine-rich 1 (SFRS1), α-smooth muscle actin (α-SMA), Calponin (CNN1), cyclin D1 (CNND1) and smooth muscle myosin heavy chain 11 (SMHC) in vascular smooth muscle cells (VSMCs) was detected by Western blotting. Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU) and Transwell assays were used to evaluate cell proliferation and migration, respectively. RNA immunoprecipitation (RNA-IP) and RNA pulldown verified the interaction between SFRS1 and circHAT1. By reanalyzing the dataset GSE77278, circHAT1 related to VSMC phenotype conversion was screened, and circHAT1 was found to be significantly reduced in peripheral blood mononuclear cells (PBMCs) of LEASO patients compared with healthy controls. Knockdown of circHAT1 significantly promoted the proliferation and migration of VSMC cells and decreased the expression levels of contractile markers. However, overexpression of circHAT1 induced the opposite cell phenotype and promoted the transformation of VSMCs from synthetic to contractile. Besides, overexpression of circHAT1 inhibited platelet-derived growth factor-BB (PDGF-BB)-induced phenotype switch of VSMC cells. Mechanistically, SFRS1 is a direct target of circHAT1 to mediate phenotype switch, proliferation and migration of VSMCs. Overall, circHAT1 regulates SFRS1 to inhibit the cell proliferation, migration and phenotype switch of VSMCs, suggesting that it may be a potential therapeutic target for LEASO.

Enhancer of zeste homolog 2 facilitates phenotypic transition of vascular smooth muscle cells leading to aortic aneurysm/dissection.

Thoracic aortic aneurysms (TAAs) are a major cause of death owing to weaker blood vessel walls and higher rupture rates in affected individuals. Vascular smooth muscle cells (VSMCs) are the predominant cell type within the aortic wall and their dysregulation may contribute to TAA progression. Enhancer of zeste homolog 2 (EZH2), a histone methyltransferase, is involved in several pathological processes; however, the biological functions and mechanisms underlying VSMC phenotype transition and vascular intimal hyperplasia remain unclear. The present study aimed to determine the involvement of EZH2 in mediating VSMC function in the development of TAAs. The expression of EZH2 was revealed to be elevated in patients with thoracic aortic dissection and TAA mouse model through western blotting and reverse transcription-quantitative PCR experiments. Subsequently, a mouse model was established using β-aminopropionitrile. In vitro, EdU labeling, Transwell assay, wound healing assay and hematoxylin-eosin staining revealed that knocking down the Ezh2 gene could reduce the proliferation, invasion, migration, and calcification of mouse primary aortic smooth muscle cells. Flow cytometry analysis found that EZH2 deficiency increased cell apoptosis. Depletion of Ezh2 in mouse primary aortic VSMCs promoted the transformation of VSMCs from a synthetic to a contractile phenotype. Using RNA-sequencing analysis, it was demonstrated that Ezh2 regulated a group of genes, including integrin β3 (Itgb3), which are critically involved in the extracellular matrix signaling pathway. qChIP found Ezh2 occupies the Itgb3 promoter, thereby suppressing the expression of Itgb3. Ezh2 promotes the invasion and calcification of VSMCs, and this promoting effect is partially reversed by co-knocking down Itgb3. In conclusion, the present study identified a previously unrecognized EZH2-ITGB3 regulatory axis and thus provides novel mechanistic insights into the pathophysiological function of EZH2. EZH2 may thus serve as a potential target for the management of TAAs.

Nupr1-mediated vascular smooth muscle cell phenotype transformation involved in methamphetamine induces pulmonary hypertension

AimsNuclear protein 1 (Nupr1) is a multifunctional stress-induced protein involved in the regulation of tumorigenesis, apoptosis, and autophagy. However, its role in pulmonary hypertension (PH) after METH exposure remains unexplored. In this study, we aimed to investigate whether METH can induce PH and describe the role and mechanism of Nupr1 in the development of PH.Methods and resultsMice were made to induce pulmonary hypertension (PH) upon chronic intermittent treatment with METH. Their right ventricular systolic pressure (RVSP) was measured to assess pulmonary artery pressure. Pulmonary artery morphometry was determined by H&E staining and Masson staining. Nupr1 expression and function were detected in human lungs, mice lungs exposed to METH, and cultured pulmonary arterial smooth muscle cells (PASMCs) with METH treatment. Our results showed that chronic intermittent METH treatment successfully induced PH in mice. Nupr1 expression was increased in the cultured PASMCs, pulmonary arterial media from METH-exposed mice, and METH-ingested human specimens compared with control. Elevated Nupr1 expression promoted PASMC phenotype change from contractile to synthetic, which triggered pulmonary artery remodeling and resulted in PH formation. Mechanistically, Nupr1 mediated the opening of store-operated calcium entry (SOCE) by activating the expression of STIM1, thereby promoting Ca2+ influx and inducing phenotypic conversion of PASMCs.ConclusionsNupr1 activation could promote Ca2+ influx through STIM1-mediated SOCE opening, which promoted METH-induced pulmonary artery remodeling and led to PH formation. These results suggested that Nupr1 played an important role in METH-induced PH and might be a potential target for METH-related PH therapy.Graphical 1. Chronic intermittent methamphetamine exposure can induce the development of pulmonary arterial hypertension.2. Nupr1 plays a key role in the development of methamphetamine-related pulmonary arterial hypertension.3. Nupr1 mediates PASMCs phenotypic transformation via STIM1signal axis, which results in the development of methamphetamine-related pulmonary arterial hypertension.

Sox9 Accelerates Vascular Aging by Regulating Extracellular Matrix Composition and Stiffness.

Vascular calcification and increased extracellular matrix (ECM) stiffness are hallmarks of vascular aging. Sox9 (SRY-box transcription factor 9) has been implicated in vascular smooth muscle cell (VSMC) osteo/chondrogenic conversion; however, its relationship with aging and calcification has not been studied. Immunohistochemistry was performed on human aortic samples from young and aged patients. Young and senescent primary human VSMCs were induced to produce ECM, and Sox9 expression was manipulated using adenoviral overexpression and depletion. ECM properties were characterized using atomic force microscopy and proteomics, and VSMC phenotype on hydrogels and the ECM were examined using confocal microscopy. In vivo, Sox9 was not spatially associated with vascular calcification but correlated with the senescence marker p16 (cyclin-dependent kinase inhibitor 2A). In vitro Sox9 showed mechanosensitive responses with increased expression and nuclear translocation in senescent cells and on stiff matrices. Sox9 was found to regulate ECM stiffness and organization by orchestrating changes in collagen (Col) expression and reducing VSMC contractility, leading to the formation of an ECM that mirrored that of senescent cells. These ECM changes promoted phenotypic modulation of VSMCs, whereby senescent cells plated on ECM synthesized from cells depleted of Sox9 returned to a proliferative state, while proliferating cells on a matrix produced by Sox9 expressing cells showed reduced proliferation and increased DNA damage, reiterating features of senescent cells. LH3 (procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3) was identified as an Sox9 target and key regulator of ECM stiffness. LH3 is packaged into extracellular vesicles and Sox9 promotes extracellular vesicle secretion, leading to increased LH3 deposition within the ECM. These findings highlight the crucial role of ECM structure and composition in regulating VSMC phenotype. We identify a positive feedback cycle, whereby cellular senescence and increased ECM stiffening promote Sox9 expression, which, in turn, drives further ECM modifications to further accelerate stiffening and senescence.

Phenotypic switching of vascular smooth muscle cells in atherosclerosis, hypertension, and aortic dissection.

Vascular smooth muscle cells (VSMCs) play a critical role in regulating vasotone, and their phenotypic plasticity is a key contributor to the pathogenesis of various vascular diseases. Two main VSMC phenotypes have been well described: contractile and synthetic. Contractile VSMCs are typically found in the tunica media of the vessel wall, and are responsible for regulating vascular tone and diameter. Synthetic VSMCs, on the other hand, are typically found in the tunica intima and adventitia, and are involved in vascular repair and remodeling. Switching between contractile and synthetic phenotypes occurs in response to various insults and stimuli, such as injury or inflammation, and this allows VSMCs to adapt to changing environmental cues and regulate vascular tone, growth, and repair. Furthermore, VSMCs can also switch to osteoblast-like and chondrocyte-like cell phenotypes, which may contribute to vascular calcification and other pathological processes like the formation of atherosclerotic plaques. This provides discusses the mechanisms that regulate VSMC phenotypic switching and its role in the development of vascular diseases. A better understanding of these processes is essential for the development of effective diagnostic and therapeutic strategies.

Piezo1-mediated regulation of smooth muscle cell volume in response to enhanced extracellular matrix rigidity.

Decreased aortic compliance is a precursor to numerous cardiovascular diseases. Compliance is regulated by the rigidity of the aortic wall and the vascular smooth muscle cells (VSMCs). Extracellular matrix stiffening, observed during ageing, reduces compliance. In response to increased rigidity, VSMCs generate enhanced contractile forces that result in VSMC stiffening and a further reduction in compliance. Mechanisms driving VSMC response to matrix rigidity remain poorly defined. Human aortic-VSMCs were seeded onto polyacrylamide hydrogels whose rigidity mimicked either healthy (12 kPa) or aged/diseased (72 kPa) aortae. VSMCs were treated with pharmacological agents prior to agonist stimulation to identify regulators of VSMC volume regulation. On pliable matrices, VSMCs contracted and decreased in cell area. Meanwhile, on rigid matrices VSMCs displayed a hypertrophic-like response, increasing in area and volume. Piezo1 activation stimulated increased VSMC volume by promoting calcium ion influx and subsequent activation of PKC and aquaporin-1. Pharmacological blockade of this pathway prevented the enhanced VSMC volume response on rigid matrices whilst maintaining contractility on pliable matrices. Importantly, both piezo1 and aquaporin-1 gene expression were up-regulated during VSMC phenotypic modulation in atherosclerosis and after carotid ligation. In response to extracellular matrix rigidity, VSMC volume is increased by a piezo1/PKC/aquaporin-1 mediated pathway. Pharmacological targeting of this pathway specifically blocks the matrix rigidity enhanced VSMC volume response, leaving VSMC contractility on healthy mimicking matrices intact. Importantly, upregulation of both piezo1 and aquaporin-1 gene expression is observed in disease relevant VSMC phenotypes.

S-propargyl-cysteine promotes the stability of atherosclerotic plaque via maintaining vascular muscle contractile phenotype.

Introduction: Plaque rupture in atherosclerosis contributes to various acute cardiovascular events. As a new sulfide-containing donor, S-propargyl-cysteine (SPRC) has been reported to play a beneficial role in cardioprotection, potentially through its anti-inflammatory, anti-oxidative and anti-atherogenic activities. Our previous study observed an increase in eNOS phosphorylation in endothelial cells. However, it remains unclear whether SPRC influences vascular smooth muscle cells (VSMCs) within the plaque and if this effect contributes to plaque stabilization. Methods: An atherosclerotic unstable plaque mouse model was established by subjecting ApoE-/- mice to tandem stenosis of the right carotid artery along with a Western diet. Daily SPRC administration was conducted for 13weeks. Plaque morphology and stability were assessed using MRI scanning and histopathological staining. In our in vitro studies, we stimulated human artery vascular smooth muscle cells (HAVSMCs) with platelet-derived growth factor-BB (PDGF-BB), both with and without 100μM SPRC treatment. Cell phenotype was assessed using both Western blot and Real-time PCR. Cell proliferation was assessed using the BrdU cell proliferation kit and immunofluorescence of Ki-67, while cell migration was measured using scratch wound healing and transwell assay. MiR-143-3p overexpression and knockdown experiments were used to investigate whether it mediates the effect of SPRC on VSMC phenotype. Results and Discussion: SPRC treatment reduced plasma lipid levels, increased collagen content and decreased cell apoptosis in atherosclerotic plaques, indicating improved plaque stability. Both in vivo and in vitro studies elucidated the role of SPRC in preserving the contractile phenotype of VSMCs through up-regulation of miR-143-3p expression. Furthermore, SPRC suppressed the pro-proliferation and pro-migration effects of PDGF-BB on HAVSMCs. Overall, these findings suggest that the inhibitory effect of SPRC on phenotype switch from contractile to synthetic VSMCs may contribute to its beneficial role in enhancing plaque stability.

WNTA5-mediated miR-374a-5p regulates vascular smooth muscle cell phenotype transformation and M1 macrophage polarization impacting intracranial aneurysm progression

miR-374a-5p expression and localization in intracranial aneurysm (IA) tissues were detected, and its correlation with vascular smooth muscle cells (VSMCs) and macrophage markers was analyzed. Using platelet-derived growth factor-BB (PDGF-BB) induced VSMC model, elastase-induced IA rat model. Subsequently, miR-374a-5p was knocked down or overexpressed. We investigated the effects of miR-374a-5p on phenotypic conversion, and in vivo experiments were also carried out to verify the findings. The targeted relationship between miR-374a-5p and WNTA5 was analyzed. The effect of WNT5A inhibition on VSMC phenotypic transformation and THP-1-derived macrophage polarization was explored. Clinical studies have shown that miR-374a-5p was upregulated in IA patients. miR-374a-5p was negatively correlated with SM22α, α-SMA, CD206, and positively correlated with CD86. In vitro experiments showed that knocking down miR-374a-5p reversed the promotion of SM22α and α-SMA expression by PDGF-BB, while overexpression of miR-374a-5p had the opposite effect. In addition, knocking down miR-374a-5p also reversed the decrease in Calponin, TIMP3, TIMP4, and IL-10 levels caused by PDGF-BB, and further reduced the levels of MMP1, MMP3, MMP9, IL-1β, IL-6, and TNF-α. These findings were further validated in vivo. In IA rats, there were notable increases in both systolic and diastolic blood pressure, along with an elevated M1/M2 ratio and the occurrence of vascular lesions. However, these symptoms were improved after knocking down miR-374a-5p. Furthermore, miR-374a-5p could target the WNT signals (WNT2B, WNT3, and WNT5A). miR-374a-5p regulated the VSMC phenotypic conversion and M1 macrophage polarization by targeting WNT5A, thereby impacting the progression of IA.