Smooth Muscle Cell Phenotypic Modulation Research Articles

SREBP1 regulates Lgals3 activation in response to cholesterol loading

Aberrant smooth muscle cell (SMC) plasticity is etiological to vascular diseases. Cholesterol induces SMC phenotypic transition featuring high LGALS3 (galectin-3) expression. This proatherogenic process is poorly understood for its molecular underpinnings, in particular, the mechanistic role of sterol regulatory-element binding protein-1 (SREBP1), a master regulator of lipid metabolism. Herein we show that cholesterol loading stimulated SREBP1 expression in mouse, rat, and human SMCs. SREBP1 positively regulated LGALS3 expression (and vice versa), whereas Krüppel-like factor-15 (KLF15) acted as a negative regulator. Both bound to the Lgals3 promoter, yet at discrete sites, as revealed by chromatin immunoprecipitation-qPCR and electrophoretic mobility shift assays. SREBP1 and LGALS3 each abated KLF15 protein, and blocking the bromo/extraterminal domain-containing proteins (BETs) family of acetyl-histone readers abolished cholesterol-stimulated SREBP1/LGALS3 protein production. Furthermore, silencing bromodomain protein 2 (BRD2; but not other BETs) reduced SREBP1; endogenous BRD2 co-immunoprecipitated with SREBP1’s transcription-active domain, its own promoter DNA, and that of Lgals3. Thus, results identify a previously uncharacterized cholesterol-responsive dyad—SREBP1 and LGALS3, constituting a feedforward circuit that can be blocked by BETs inhibition. This study provides new insights into SMC phenotypic transition and potential interventional targets.

The Atypical Cadherin FAT1 Limits Mitochondrial Respiration and Proliferation of Vascular Smooth Muscle Cells.

Smooth muscle cells contribute to cardiovascular disease, the leading cause of death worldwide. The capacity of these cells to undergo phenotypic switching in mature arteries of the systemic circulation underlies their pathogenic role in atherosclerosis and restenosis, among other vascular diseases. Growth factors and cytokines, extracellular matrix components, regulation of gene expression, neuronal influences, and mechanical forces contribute to smooth muscle cell phenotypic switching. Comparatively little is known about cell metabolism in this process. Studies of cancer and endothelial cell biology have highlighted the importance of cellular metabolic processes for phenotypic transitions that accompany tumor growth and angiogenesis. However, the understanding of cell metabolism during smooth muscle cell phenotypic modulation is incipient. Studies of the atypical cadherin FAT1, which is strongly upregulated in smooth muscle cells in response to arterial injury, suggest that it has important and distinctive functions in this context, mediating control of both smooth muscle cell mitochondrial metabolism and cell proliferation. Here we review the progress made in understanding how FAT1 affects the smooth muscle cell phenotype, highlighting the significance of FAT1 as a processed protein and unexpected regulator of mitochondrial respiration. These mechanisms suggest how a transmembrane protein may relay signals from the extracellular milieu to mitochondria to control metabolic activity during smooth muscle cell phenotypic switching.

Abstract 360: Epigenomic Changes Govern Smooth Muscle Cell Phenotype Shift In Marfan Syndrome Aortic Root Aneurysm

Background: Vascular smooth muscle cell (SMC) phenotype modulation produces a mixed proteolytic/collagen synthetic state in Marfan syndrome (MFS) aortic root aneurysm. While the transcriptomic changes associated with this process are established, upstream regulators governing this plasticity are poorly characterized. Methods/Results: We performed concurrent single-cell RNA sequencing (scRNAseq) and assay for transposase-accessible chromatin using sequencing (scATACseq) on aortic root aneurysm tissue from adult Fbn1 C1041G/+ (MFS) mice and littermate controls. MFS/control scRNAseq data analysis identified four SMC subtypes including MFS-specific ‘modulated’ cells (modSMCs) which enriched for extracellular matrix organization and collagen synthesis pathway activation. These SMC clusters were projected onto the MFS/control scATAC dataset via integrative label transfer to study dynamic chromatin accessibility during SMC phenotype modulation. We compared DNA accessibility in modSMCs versus mature SMCs, finding 336 enriched and 29 suppressed peaks, suggesting increased open chromatin during modulation. Using chromVAR, we identified 242 enriched transcription factor motifs overrepresented in modSMCs. Motifs representing central but nonspecific transcription factor families including numerous AP-1 (FOS/JUN/ATF) heterodimers and TEAD family members showed highest enrichment, while enriched TWIST1, HAND2, and SMAD2:SMAD3:SMAD4 complex motifs suggested more specific functions. To functionally validate these findings, we examined TWIST1 as a potential regulator of SMC modulation in vitro via lentiviral overexpression in MFS patient-derived aortic SMCs.In SMCs with forced TWIST1 overexpression, we found heightened promoter region DNA accessibility (via bulk ATACseq) and increased mRNA expression (via RT-PCR) for specific modSMC markers (e.g., COL1A1 and LUM confirming TWIST1 promotes collagen synthesis. Conclusions: Integrated single-cell transcriptomic/epigenomic analysis permits identification of critical upstream regulatory signals promoting disease-specific cell phenotype changes. TWIST1 is a potential driver of SMC modulation and a target for therapeutic agent design in MFS aortic aneurysm.

Single-Cell Analysis Uncovers Osteoblast Factor Growth Differentiation Factor 10 as Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation Associated with Plaque Rupture in Human Carotid Artery Disease.

(1) Background: Vascular smooth muscle cells (VSMCs) undergo a complex phenotypic switch in response to atherosclerosis environmental triggers, contributing to atherosclerosis disease progression. However, the complex heterogeneity of VSMCs and how VSMC dedifferentiation affects human carotid artery disease (CAD) risk has not been clearly established. (2) Method: A single-cell RNA sequencing analysis of CD45− cells derived from the atherosclerotic aorta of Apolipoprotein E-deficient (Apoe−/−) mice on a normal cholesterol diet (NCD) or a high cholesterol diet (HCD), respecting the site-specific predisposition to atherosclerosis was performed. Growth Differentiation Factor 10 (GDF10) role in VSMCs phenotypic switch was investigated via flow cytometry, immunofluorescence in human atherosclerotic plaques. (3) Results: scRNAseq analysis revealed the transcriptomic profile of seven clusters, five of which showed disease-relevant gene signature of VSMC macrophagic calcific phenotype, VSMC mesenchymal chondrogenic phenotype, VSMC inflammatory and fibro-phenotype and VSMC inflammatory phenotype. Osteoblast factor GDF10 involved in ossification and osteoblast differentiation emerged as a hallmark of VSMCs undergoing phenotypic switch. Under hypercholesteremia, GDF10 triggered VSMC osteogenic switch in vitro. The abundance of GDF10 expressing osteogenic-like VSMCs cells was linked to the occurrence of carotid artery disease (CAD) events. (4) Conclusions: Taken together, these results provide evidence about GDF10-mediated VSMC osteogenic switch, with a likely detrimental role in atherosclerotic plaque stability.

RIP2 Knockdown Attenuates Vascular Smooth Muscle Cells Activation via Negative Regulating Myocardin Expression.

RIP2 is an adaptor protein contributing to the activation of nuclear factor-κB induced by TNF receptor-associated factor (TRAF) and nucleotide oligomerization domain (NOD)-dependent signaling implicated in innate and adaptive immune response. Beyond regulation of immunity, we aimed to elucidate the role of RIP2 in vascular smooth muscle cell (VSMC) phenotypic modulation. In the current study, we observed that RIP2 showed an increased expression in VSMCs with PDGF-BB stimulation in a dose-dependent manner. Knockdown of RIP2 expression mediated by adenovirus dramatically accelerated the expression of VSMC-specific differentiation genes induced by PDGF-BB. Silencing of RIP2 inhibited proliferative and migratory ability of VSMCs. Additionally, we demonstrated that RIP2 knockdown can promoted myocardin expression. Furthermore, RIP2 inhibition also can attenuate the formation of intimal hyperplasia. These findings suggested that RIP2 played an important role in regulation of VSMCs differentiation, migration, and proliferation that may due to affect myocardin expression. Our results indicated that RIP2 may be a novel therapeutic target for intimal hyperplasia.

Abstract 11990: Insulin-Like Growth Factor Binding Protein 2 is a Novel Marker of Early Smooth Muscle Cell Phenotype Modulation in Thoracic Aortic Aneurysm

Introduction: Single cell RNA sequencing (scRNAseq) enables high resolution analysis of vascular smooth muscle cell (SMC) phenotype modulation in thoracic aortic aneurysm. Insulin-like growth factor binding protein-2 ( Igfbp2 ) expression by SMCs is activated early in a murine Marfan syndrome (MFS) aortic aneurysm model. IGFBP2 modulates IGF1 bioavailability and interacts directly with SMCs via integrins. We hypothesize that IGFBP2 is a marker of early SMC modulation in human aortopathy. Methods: Surgically resected aortic aneurysm tissue was collected from patients spanning the clinical spectrum of monogenic aortopathies ( ACTA2, FBN1, TGFBR1 (n=2), TGFB2, or SMAD3 variants). Tissues were digested and processed for scRNAseq. SMC clusters from all patients were jointly analyzed with pseudotemporal trajectory analysis to reconstruct the SMC phenotypic continuum. Primary MFS human (n=5) aortic root SMCs underwent IGFBP2 silencing via siRNA followed by bulk RNAseq. Results: scRNAseq identified two main SMC clusters, comprising 39% of all cells in the dataset, including a quiescent contractile SMCs and a modulated, synthetic subset. Pseudotemporal ordering generated a continuous ‘pseudotime’ scale inversely correlated with contractile gene (e.g., CNN1, MYH11) expression, modeling gradual de-differentiation in vivo . We identified similar markers of late SMC modulation (e.g., TNFRSF11B , MMP2, CYTL1, COL1A1 ) in patients of all genotypes. IGFBP2 expression showed comparatively earlier activation and peak with reduced expression late in pseudotime. Silencing IGFBP2 in human MFS SMCs in vitro significantly depressed contractile gene expression. Conclusions: SMC modulation occurs in consistent pattern across hereditary aortopathy subtypes. IGFBP2 activation occurs early in this trajectory, marking cells in phenotypic transition. IGFBP2 may promote contractile phenotype maintenance, presenting a novel pathway for potential therapies.

Abstract 12453: Smooth Muscle-Specific Latexin Deficiency Prevents Platelet-Derived Growth Factor-Mediated Vascular Smooth Muscle Cell Phenotypic Switch and Neointimal Formation

Introduction: Vascular smooth muscle cell (SMC) phenotypic switch from a contractile to a synthetic phenotype plays a significant role in vascular pathologies, including atherosclerosis, restenosis, and aneurysm. Latexin (LXN) is a novel inflammatory protein that is highly expressed in SMCs; the role of LXN in SMCs biology, however, remains largely unknown. Methods: Both global and SMC-specific LXN-knockout mice were generated to determine the in vivo function of LXN in SMC remolding. Neointimal formation induced by mouse carotid artery ligation was assessed by HE staining. Mouse SMCs were isolated from thoracic aortas and used for biological and functional assays. Western blot (WB), immunohistochemistry (IHC), RNA-seq, and quantitative polymerase chain reaction (qPCR) were used to assess protein and gene expression. BrdU incorporation assay and Boyden’s chamber assay were used to assess SMC proliferation and migration. Results: The expression of contractile SMC markers were down-regulated, while synthetic SMC markers were up-regulated in the mouse model of carotid artery ligation. LXN expression was significantly increased in mouse carotid arteries after ligation-induced injury. Furthermore, we found that both global and SMC-specific deletion of LXN significantly attenuated neointimal formation after injury, implicating an essential role of LXN in the modulation of SMC phenotype. Our ex vivo and in vitro functional assays showed that LXN deficiency attenuates SMC proliferation and migration, which is consistent with our RNA-seq data showing that the expression of cell proliferation-related genes was down-regulated in LXN deficient mouse SMCs. Mechanistically, we identified platelet-derived growth factor receptor alpha as a critical mediator involved in regulating SMC phenotypic switch by LXN. Conclusions: Our results for the first time identify LXN as a critical mediator in regulating SMC phenotypic switch and suggest that inhibition of LXN may represent a novel therapeutic strategy for the treatment of proliferative vascular diseases such as atherosclerosis and restenosis.

Abstract 12006: Smooth Muscle Cell Embryologic Origin Does Not Define Propensity for Phenotype Modulation in Murine Marfan Syndrome Aortic Root Aneurysm

Introduction: Vascular smooth muscle cells (SMC) undergo phenotype modulation (silenced contractile genes and activated proteolytic/synthetic genes) in Marfan syndrome (MFS) aortic aneurysm. Aortic root SMCs develop from the second heart field (SHF) proximally and neural crest (NC) distally. We hypothesize that both developmental lineage and hemodynamic factors dictate SMC vulnerability to phenotype change, promoting focal aortic root aneurysm. Methods/Results: Aortic roots from adult MFS and littermate control mice (n=4 each sex) with a knockin fluorescent SHF tracing construct ( Nkx2-5 cre ) were sorted for lineage-resolved single cell RNA sequencing (scRNAseq). Modulated SMCs (modSMC) were enriched in MFS vs. controls (18.1% of all SMCs vs. 2.2%) and originated from both SHF (15.5%) and NC (20.2%) lineages, ruling out complete lineage specificity. NC-derived modSMCs expressed more aggrecan ( Acan ) and sclerostin ( Sost ) while small proteoglycans decorin ( Dcn ) and lumican ( Lum ) were enriched in SHF modSMCs. Insulin-like growth factor binding protein-2 ( Igfbp2 ) was a lineage-independent modSMC marker. RNA in situ hybridization localized Igfbp2 + SMCs within SHF/NC overlap in the aortic root. To study regional SMC propensity for modulation due to hemodynamic stresses, we generated aortic pressure overload via 27-gauge transverse aortic arch constriction (TAC) or sham surgery on healthy Myh11 cre / Rosa tdT (SMC-traced) mice. TAC induced mild root dilation vs . sham (1.93±0.07 mm vs. 1.69±0.09 mm, n=4, p<0.01). scRNAseq showed SMC phenotype modulation comparable to MFS in TAC mice and spatially concentrated in the aortic root. Conclusions: Embryologic origin does not dictate aortic SMC propensity for pathologic phenotype change in MFS but does promote distinct ECM proteoglycan gene activation. Aortic pressure overload incites root SMC modulation, suggesting hemodynamic effects may promote regional susceptibility to SMC-mediated aortic remodeling.

Abstract 12444: Smooth Muscle-Specific Deletion of Perk Prevents Atherosclerotic Plaque Formation in Mice

Introduction: Lineage tracing and single cell RNA sequencing (scRNA-Seq) studies have identified a subset of phenotypically modulated vascular smooth muscle cells (SMCs) that appear with atherosclerotic plaque formation in hyperlipidemic mice. These modulated SMCs (mSMCs) are de-differentiated, and increase expression of macrophage, fibroblast and stem cell markers. We previously showed that SMCs exposed to free cholesterol in vitro activate an endoplasmic reticulum unfolded protein response (UPR) and assume an mSMC phenotype, and blocking Perk specifically abolishes this SMC modulation. Methods: Here, we sought to define the role of Perk in atherosclerotic plaque formation using SMC-specific Perk knockout in C57BL6 mice ( Perk SMC-KO ) and inducing hyperlipidemia by PSCK9 DY overexpression and a high fat diet for 12 weeks. Results: Aortic Oil Red O staining and en face aortic analysis revealed no plaque formation in the Perk SMC-KO aorta, with the exception of some small plaques in the root, whereas the wild-type (WT) aortas had extensive plaque, especially in the aortic arch (N = 10, p<0.0001, Fig. 1A ). scRNA-Seq analysis of aortic tissue from the root to the arch from these mice showed no major differences in cell clusters between Perk SMC-KO and WT mice at baseline ( Fig. 1B ). In hyperlipidemic WT mice, SMC-derived cluster 8 expands and a new cluster 7 appears (circled in red, Fig. 1B ). In Perk SMC-KO mice, cluster 8 is similarly expanded with hyperlipidemia, while cluster 7 is completely absent ( Fig. 1B , bottom right). Cluster 7 in the WT atherosclerotic aortas is the only cluster with Perk expression and has significantly increased expression of macrophage markers and a subset of fibroblast-specific markers when compared to cluster 8. Conclusions: These data indicate that SMC phenotypic modulation plays a major role in atherosclerotic plaque formation and Perk is a key regulator of the SMC modulation that specifically contributes to atherosclerotic plaque burden.

PI 3-kinase isoform PI3Kalpha controls smooth muscle cell functionality and protects against aortic aneurysm formation

Abstract Background Class I PI 3-kinase isoform PI3Kα is a lipid kinase and signals downstream of receptor tyrosine kinases. Smooth muscle cells (SMCs) lacking PI3Kα are characterized by impaired proliferation, migration and survival. Mice, harbouring a smooth muscle specific PI3Kα deficiency (SM-PI3Kα−/−), display reduced vascular wall thickness and impaired vascular remodeling in response to vessel injury. We hypothesize that SM-PI3Kα−/− mice are prone to aortic aneurysm (AA) formation due to impaired SMC functions. Herein, we investigated, how PI3Kα-dependent signaling in SMCs affect aortic aneurysm (AA) formation, aortic wall structure, and expression of extracellular matrix (ECM) components. Methods and results AA formation in SM-PI3Kα−/− mice and wild-type littermates was examined by means of the “porcine pancreatic elastase” (PPE) AA model. PPE was infused into the infrarenal aorta to induce AA formation. Ultrasound examination revealed a significantly increased aortic diameter in SM-PI3Kα−/− mice (1.22±0.12 mm) compared to wild-type animals (0.96±0.02 mm, p=0.014). These data indicate a protective function of SM-PI3Kα in AA formation. In addition, the media thickness in the abdominal aorta was significantly reduced in SM-PI3Kα−/− mice (29.0±3.1 vs. 42.5±4.1 μm). Ultrastructural analysis of aortic wall morphology in SM-PI3Kα−/−mice using transmission electron microscopy (TEM) showed a deranged tunica media with detached SMCs and increased apoptotic cell death. Consequently, SM-PI3Kα deficiency significantly diminished responsiveness of aortic rings to vasodilator acetylcholine and NO-donor nitroglycerin, further indicating impaired aortic wall structure. Western blots demonstrated a reduced elastin and fibrillin expression in SMCs from SM-PI3Kα−/− mice. Furthermore, immunofluorescence stainings of PI3Kα−/− and wild-type SMCs, cultured for seven days under 10% fetal calf serum containing DMEM medium, showed significantly disturbed structures of elastin-, fibrillin-1- and collagen-1-fibers. These data indicate that PI3Kα signaling contributes to elastic fiber homeostasis thus affecting SMC phenotypic modulation. Immunoblots demonstrated that PDGF and insulin induced phosphorylation and inactivation of key regulators of SMC differentiation and dedifferentiation including FoxO1, FoxO3a, Foxo4, and GSK3b, respectively, were reduced or even abrogated in PI3Kα−/− SMCs. Conclusion These data show that deficiency of PI3Kα in SMCs promotes the formation and progression of AA. Causative is a deranged aortic structure of SM-PI3Kα−/− aortae which can likely be attributed to an impaired production of elastic fiber components by PI3Kα−/− SMCs. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Abstract MP13: Small RNA Profiling Identifies Mir-200b As A Repressor Of Vascular Smooth Muscle Cell Migration And Proliferation

Background and Objectives: A large body of research has identified miRNA as pivotal regulators of smooth muscle cell (SMC) fate in various vascular diseases. However, global changes of miRNA expression profile during SMC phenotypic modulation have not been well characterized. As the targetome of a given miRNA is highly dependent on the cell type and context, comparative analysis of mRNA and miRNA expression profiles can provide decisive information for assessing the versatility in miRNA functions and discovering novel miRNA modulating SMC phenotype. Our goal is to define the miRNA signatures in differentiated and de-differentiated SMC, the miRNA overall contribution to the transcriptome, and novel functionally relevant miRNA. Methods: By performing RNAseq and small RNAseq, we defined the mRNA/miRNA expression profiles in aortic SMC cultured in differentiation or de-differentiation media with Platelet Derived Growth Factor-BB (PDGF-BB) and performed functional studies on differentially expressed miRNAs (DEmiR). Results: We found that PDGF-BB-induced SMC de-differentiation generated 48 significant DEmiRs, including 15 downregulated and 33 upregulated DEmiRs. Integrative analysis predicted that nearly 40% (1335 of 3523) of differentially expressed transcripts were direct targets of DEmiRs. The miR-200 cluster (miR-200a, miR-200b and miR-429) was found potently downregulated after PDGF-BB treatment, as well as Tet2 and Myocardin knockdown, while its expression increased in rapamycin treated SMC. We found that miR-200b overexpression prevented PDGF-BB-induced SMC migration and proliferation, while miR-200b inhibition had an opposite effect. Finally, we identified Quaking, a potent inducer of SMC phenotypic switching, as a primary target of miR-200b. Conclusion: Our study predicted a marked contribution of miRNA in regulating SMC transcriptomic profile. miR-200b was identified as a novel pro-differentiation miRNA in SMC through inhibition of phenotypic switching.

Single-cell analysis uncovers osteoblast factor GDF10 as mediator of vascular smooth muscle cell phenotypic modulation associated with plaque rupture in human carotid artery disease

Background and Aims: Vascular smooth muscle cells (VSMC) are not terminally differentiated and undergo complex phenotypic changes during atherosclerosis. However, the complex heterogeneity of VSMC and how VSMC de-differentiation affects human carotid artery disease (CAD) risk has not been clearly established. The aim of the present study is to comprehensively characterized the transcriptomic profile of phenotypically modulated VSMC and to identify mediators of VSMC transition to osteogenic like cells with likely detrimental role for atherosclerosis plaque stability.

Adropin inhibits the phenotypic modulation and proliferation of vascular smooth muscle cells during neointimal hyperplasia by activating the AMPK/ACC signaling pathway.

In-stent restenosis (ISR) remains an inevitable problem for some patients receiving drug-eluting stent (DES) implantation. Intimal hyperplasia is an important biological cause of ISR. It has been previously reported that adropin is a potentially protective factor in cardiovascular disease. Therefore, the present study investigated the function of adropin in inhibiting smooth muscle cell (SMC) phenotype modulation and proliferation, causing intimal hyperplasia. A total of 56 patients who visited the hospital consecutively (25 with ISR and 31 without ISR), who were followed up between April 2016 and March 2019, 1 year following DES, were analyzed to evaluate the association between in-stent neointimal volume and adropin serum levels. Rat aorta smooth muscle cells (RASMCs) were used to determine the effects of adropin on their phenotypic modulation and proliferation using western blot, MTT, PCR and immunofluorescence analyses. Adropin serum levels in the ISR group were significantly lower than those in the non-ISR group. Furthermore, linear regression analysis revealed that only adropin levels were negatively associated with neointimal volume in both groups. The overall adropin levels of the 56 patients and the percentages of neointimal volume revealed a strong negative association. In vitro, adropin suppressed angiotensin II (Ang II)-induced phenotypic modulation in RASMCs by restoring variations of osteopontin and α-smooth muscle actin. Furthermore, compared with the Ang II group, adropin markedly decreased the percentage of G2/M-phase cells. Finally, adropin negatively regulated the phenotypic modulation and proliferation of RASMCs via the AMP-activated protein kinase/acetyl-CoA carboxylase (AMPK/ACC) signaling pathway. In conclusion, an independent, negative association was revealed between adropin and intimal hyperplasia; specifically, adropin inhibited the phenotypic modulation and proliferation of RASMCs by activating the AMPK/ACC signaling pathway. Therefore, adropin may be used as a potential predictor and therapeutic target for intimal hyperplasia and ISR.

Inactivation of SERCA2 Cys674 accelerates aortic aneurysms by suppressing PPARγ

Inactivation of Cys674 (C674) in the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) causes intracellular Ca2+ accumulation, which activates calcineurin-mediated nuclear factor of activated T-lymphocytes (NFAT)/NF-κB pathways, results in the phenotypic modulation of smooth muscle cells (SMCs) to accelerate angiotensin II-induced aortic aneurysm. Our goal was to investigate the mechanism involved. We used heterozygous SERCA2 C674S knock-in (SKI) mice, where half of C674 was substituted by serine, to mimic partial irreversible oxidation of C674. The aortas of SKI mice and their littermate wild-type mice were collected for RNA sequencing, cell culture, protein expression, luciferase activity and aortic aneurysm analysis. Inactivation of C674 inhibited the promoter activity and protein expression of PPARγ, which could be reversed by inhibitors of calcineurin or NF-κB. In SKI SMCs, inhibition of NF-κB by pyrrolidinedithiocarbamic acid (PDTC) or overexpression of PPARγ2 reversed the protein expression of SMC phenotypic modulation markers, and inhibited cell proliferation, migration, and macrophage adhesion to SMCs. Pioglitazone, the activator of PPARγ, blocked the activation of NFAT/NF-κB, reversed the protein expression of SMC phenotypic modulation markers and inhibited cell proliferation, migration, and macrophage adhesion to SMCs in SKI SMCs. Furthermore, pioglitazone also ameliorated angiotensin II-induced aortic aneurysms in SKI mice. The inactivation of SERCA2 C674 promotes the development of aortic aneurysm by disrupting the balance between PPARγ and NFAT/NF-κB. Our study highlights the importance of C674 redox status in regulating PPARγ to maintain aortic homeostasis.

Role of NFAT in the Progression of Diabetic Atherosclerosis.

Nuclear factor of activated T cells (NFAT) is a transcription factor with a multidirectional regulatory function, that is widely expressed in immune cells, including cells in the cardiovascular system, and non-immune cells. A large number of studies have confirmed that calcineurin/NFAT signal transduction is very important in the development of vascular system and cardiovascular system during embryonic development, and plays some role in the occurrence of vascular diseases such as atherosclerosis, vascular calcification, and hypertension. Recent in vitro and in vivo studies have shown that NFAT proteins and their activation in the nucleus and binding to DNA-related sites can easily ɨnduce the expression of downstream target genes that participate in the proliferation, migration, angiogenesis, and vascular inflammation of vascular wall related cells in various pathophysiological states. NFAT expression is regulated by various signaling pathways, including CD137-CD137L, and OX40-OX40L pathways. As a functionally diverse transcription factor, NFAT interacts with a large number of signaling molecules to modulate intracellular and extracellular signaling pathways. These NFAT-centered signaling pathways play important regulatory roles in the progression of atherosclerosis, such as in vascular smooth muscle cell phenotypic transition and migration, endothelial cell injury, macrophage-derived foam cell formation, and plaque calcification. NFAT and related signaling pathways provide new therapeutic targets for vascular diseases such as atherosclerosis. Hence, further studies of the mechanism of NFAT in the occurrence and evolution of atherosclerosis remain crucial.

Plasma Small Extracellular Vesicle‐Carried miRNA‐501‐5p Promotes Vascular Smooth Muscle Cell Phenotypic Modulation‐Mediated In‐Stent Restenosis

Vascular smooth muscle cell (VSMC) phenotypic modulation plays an important role in the occurrence and development of in-stent restenosis (ISR), the underlying mechanism of which remains a key issue needing to be urgently addressed. This study is designed to investigate the role of plasma small extracellular vesicles (sEV) in VSMC phenotypic modulation. sEV were isolated from the plasma of patients with ISR (ISR-sEV) or not (Ctl-sEV) 1 year after coronary stent implantation using differential ultracentrifugation. Plasma sEV in ISR patients are elevated markedly and decrease the expression of VSMC contractile markers α-SMA and calponin and increase VSMC proliferation. miRNA sequencing and qRT-PCR validation identified that miRNA-501-5p was the highest expressed miRNA in the plasma ISR-sEV compared with Ctl-sEV. Then, we found that sEV-carried miRNA-501-5p level was significantly higher in ISR patients, and the level of plasma sEV-carried miRNA-501-5p linearly correlated with the degree of restenosis (R2 = 0.62). Moreover, miRNA-501-5p inhibition significantly increased the expression of VSMC contractile markers α-SMA and calponin and suppressed VSMC proliferation and migration; in vivo inhibition of miRNA-501-5p could also blunt carotid artery balloon injury induced VSMC phenotypic modulation in rats. Mechanically, miRNA-501-5p promoted plasma sEV-induced VSMC proliferation by targeting Smad3. Notably, endothelial cells might be the major origins of miRNA-501-5p. Collectively, these findings showed that plasma sEV-carried miRNA-501-5p promotes VSMC phenotypic modulation-mediated ISR through targeting Smad3.

TSP-1 (Thrombospondin-1) Deficiency Protects ApoE-/- Mice Against Leptin-Induced Atherosclerosis.

Hyperleptinemia, hallmark of obesity, is a putative pathophysiologic trigger for atherosclerosis. We previously reported a stimulatory effect of leptin on TSP-1 (thrombospondin-1) expression, a proatherogenic matricellular protein implicated in atherogenesis. However, a causal role of TSP-1 in leptin-driven atherosclerosis remains unknown. Approach and Results: Seventeen-weeks-old ApoE-/- and TSP-1-/-/ApoE-/- double knockout mice, on normocholesterolemic diet, were treated with or without murine recombinant leptin (5 µg/g bwt, IP) once daily for 3 weeks. Using aortic root morphometry and en face lesion assay, we found that TSP-1 deletion abrogated leptin-stimulated lipid-filled lesion burden, plaque area, and collagen accumulation in aortic roots of ApoE-/- mice, shown via Oil red O, hematoxylin and eosin, and Masson trichrome staining, respectively. Immunofluorescence microscopy of aortic roots showed that TSP-1 deficiency blocked leptin-induced inflammatory and smooth muscle cell abundance as well as cellular proliferation in ApoE-/- mice. Moreover, these effects were concomitant to changes in VLDL (very low-density lipoprotein)-triglyceride and HDL (high-density lipoprotein)-cholesterol levels. Immunoblotting further revealed reduced vimentin and pCREB (phospho-cyclic AMP response element-binding protein) accompanied with augmented smooth muscle-myosin heavy chain expression in aortic vessels of leptin-treated double knockout versus leptin-treated ApoE-/-; also confirmed in aortic smooth muscle cells from the mice genotypes, incubated ± leptin in vitro. Finally, TSP-1 deletion impeded plaque burden in leptin-treated ApoE-/- on western diet, independent of plasma lipid alterations. The present study provides evidence for a protective effect of TSP-1 deletion on leptin-stimulated atherogenesis. Our findings suggest a regulatory role of TSP-1 on leptin-induced vascular smooth muscle cell phenotypic transition and inflammatory lesion invasion. Collectively, these results underscore TSP-1 as a potential target of leptin-induced vasculopathy.

Surfactant Protein A, a Novel Regulator for Smooth Muscle Phenotypic Modulation and Vascular Remodeling-Brief Report.

The objective of this study is to determine the role of SPA (surfactant protein A) in vascular smooth muscle cell (SMC) phenotypic modulation and vascular remodeling. Approach and Results: PDGF-BB (Platelet-derived growth factor-BB) and serum induced SPA expression while downregulating SMC marker gene expression in SMCs. SPA deficiency increased the contractile protein expression. Mechanistically, SPA deficiency enhanced the expression of myocardin and TGF (transforming growth factor)-β, the key regulators for contractile SMC phenotype. In vivo, SPA was induced in medial and neointimal SMCs following mechanical injury in both rat and mouse carotid arteries. SPA knockout in mice dramatically attenuated the wire injury-induced intimal hyperplasia while restoring SMC contractile protein expression in medial SMCs. These data indicate that SPA plays an important role in SMC phenotype modulation and vascular remodeling in vivo. SPA is a novel protein factor modulating SMC phenotype. Blocking the abnormal elevation of SPA may be a potential strategy to inhibit the development of proliferative vascular diseases.

YY1 directly interacts with myocardin to repress the triad myocardin/SRF/CArG box-mediated smooth muscle gene transcription during smooth muscle phenotypic modulation

Yin Yang 1 (YY1) regulates gene transcription in a variety of biological processes. In this study, we aim to determine the role of YY1 in vascular smooth muscle cell (VSMC) phenotypic modulation both in vivo and in vitro. Here we show that vascular injury in rodent carotid arteries induces YY1 expression along with reduced expression of smooth muscle differentiation markers in the carotids. Consistent with this finding, YY1 expression is induced in differentiated VSMCs in response to serum stimulation. To determine the underlying molecular mechanisms, we found that YY1 suppresses the transcription of CArG box-dependent SMC-specific genes including SM22α, SMα-actin and SMMHC. Interestingly, YY1 suppresses the transcriptional activity of the SM22α promoter by hindering the binding of serum response factor (SRF) to the proximal CArG box. YY1 also suppresses the transcription and the transactivation of myocardin (MYOCD), a master regulator for SMC-specific gene transcription by binding to SRF to form the MYOCD/SRF/CArG box triad (known as the ternary complex). Mechanistically, YY1 directly interacts with MYOCD to competitively displace MYOCD from SRF. This is the first evidence showing that YY1 inhibits SMC differentiation by directly targeting MYOCD. These findings provide new mechanistic insights into the regulatory mechanisms that govern SMC phenotypic modulation in the pathogenesis of vascular diseases.

Genetic and Epigenetic Mechanisms Underlying Vascular Smooth Muscle Cell Phenotypic Modulation in Abdominal Aortic Aneurysm.

Abdominal aortic aneurysm (AAA) refers to the localized dilatation of the infra-renal aorta, in which the diameter exceeds 3.0 cm. Loss of vascular smooth muscle cells, degradation of the extracellular matrix (ECM), vascular inflammation, and oxidative stress are hallmarks of AAA pathogenesis and contribute to the progressive thinning of the media and adventitia of the aortic wall. With increasing AAA diameter, and left untreated, aortic rupture ensues with high mortality. Collective evidence of recent genetic and epigenetic studies has shown that phenotypic modulation of smooth muscle cells (SMCs) towards dedifferentiation and proliferative state, which associate with the ECM remodeling of the vascular wall and accompanied with increased cell senescence and inflammation, is seen in in vitro and in vivo models of the disease. This review critically analyses existing publications on the genetic and epigenetic mechanisms implicated in the complex role of SMCs within the aortic wall in AAA formation and reflects the importance of SMCs plasticity in AAA formation. Although evidence from the wide variety of mouse models is convincing, how this knowledge is applied to human biology needs to be addressed urgently leveraging modern in vitro and in vivo experimental technology.