Dedifferentiation Of Smooth Muscle Cells Research Articles

#899 The effect of exercise training on microRNAs in patients undergoing hemodialysis

Abstract Background and Aims Physical inactivity is a risk factor for mortality and morbidity in patients undergoing hemodialysis (HD). Therefore, physical exercise (PE) is recommended as part of the management of HD patients. Although the benefits of PE for HD patients have been well documented in the literature, the pathophysiological mechanism still needs to be fully understood. Recently, microRNAs (miRNAs) have come to light as potential mediators of PE's therapeutic effects in healthy individuals. Currently, no information is available about how PE affects the expression of miRNAs in HD patients, mainly for osteogenic differentiation, a critical process in forming vascular calcification. Therefore, this observational longitudinal study aims to assess the expression of miR-9 and miR-30b, two regulators of the osteogenic commitment from endothelial progenitor cells, in HD patients engaged in an interdialytic exercise program. Method Male HD patients aged between 50 and 80 could walk for at least six meters were enrolled. Exclusion criteria were uncorrected anemia, acute infectious disease, uncontrolled hyperparathyroidism, active oncologic disease, and severe cardio-respiratory, musculoskeletal, or neurological conditions inhibiting exercise training. Patients were engaged in a structured interdialytic program supervised by an exercise facilitator. Exercise training was performed at home or the dialysis centre based on the patient's needs and preferences. It consisted of a 10-minute session/day of low-intensity walking and/or resistance exercises. The speed was increased weekly and controlled with a metronome application on the patient's smartphone. The maximum distance covered evaluates the patients' physical performance, as measured by the 6-minute walking test (6MWT). It was assessed at baseline and the 6-month follow-up. Blood samples for miRNA analysis were collected during the short interdialytic period at baseline and at the 6-month follow-up. MiRNAs will be extracted from serum samples using the miRNeasy Serum/Plasma Advanced Kit. Statistical analysis will be performed using SPSS 21.0. The difference between the two groups was assessed using a t-test for paired data. P-values<0.05 were considered statistically significant. Results Patients’ characteristics at baseline are shown in Table 1. Five patients were enrolled in the study, three undergoing exercise training at the dialysis centre and two at home. At six months, miR-30b expression appeared significantly upregulated in all patients (p<0.03) (Fig. 1). At the same time, the upregulation of miR-9 did not reach significance (p = 0.07). In one patient, miR-9 was not detected (Fig. 2). The mean distance covered during the 6MWT did not improve at six months (255 ± 129 meters) compared to baseline (235 ± 118 meters) (p = 0.49). Conclusion Preliminary results of this longitudinal observational study show that miR-30b was upregulated in patients engaged in the exercise training period at the 6-month follow-up despite no improvement in physical performance. The upregulation of miR-30b levels can potentially reduce the dedifferentiation of vascular smooth muscle cells into an osteoblastic-like phenotype. At the end of our study, novel evidence concerning the potential effect of exercise training on miRNA expressions correlated to the vascular calcification process in HD patients will be available.

A pathway to precision medicine: coronary and cerebral arterial transcriptomic analysis reveals distinct pathophysiological adaptations in two clinically relevant porcine models of heart failure

Traditional treatments for heart failure (HF) with reduced ejection fraction (HFrEF) are ineffective at treating HF with preserved ejection fraction (HFpEF), establishing a need for more targeted therapeutic approaches. To date, the comparative vascular adaptions induced by these two distinct forms of HF have been largely unexplored. Therefore, the objective of this study was to compare HFrEF and HFpEF pathophysiological adaptions in both coronary and cerebral microvasculature in two clinically relevant swine models of HF. We hypothesized that HFrEF and HFpEF will induce differing pathophysiological adaptations within the microvasculature of the heart as well as peripheral tissues, such as the brain. Intact female Ossabaw swine (2 months old) were fed a Western Diet for 4 months to develop metabolic syndrome (HOMA-IR: HFrEF 2.94 ± 0.34; HFpEF 2.95 ± 0.59). At 6 months of age, animals were either subjected to 90 minutes ischemia followed by reperfusion (I/R) to induce a myocardial infarction (MI) recapitulating cardiometabolic HFrEF (38.0 ± 3.9% EF) or were aortic banded to induce pressure-overload with relevance to cardiometabolic HFpEF (61.5 ± 1.7% EF). Following humane euthanasia, coronary arterioles (139.5 ± 4.9 μm inner diameter) and middle cerebral artery second order pial arterioles (281.1 ± 16.7 μm) from each model were dissected and either cannulated and pressurized for myography experiments or flash frozen for RNA isolation, sequencing, and predictive pathway analysis. Smooth muscle dependent U46619 (thromboxane A2 agonist)-induced constriction of cerebral arterioles was not different between groups (HFrEF 46.9 ± 10.3%; HFpEF 50.7 ± 4.8%), however, smooth muscle dependent dilation to sodium nitroprusside (SNP, nitric oxide mimetic) was significantly reduced in HFrEF (37.1 ± 14.0%) compared to HFpEF (83.6 ± 4.6%), suggesting the dilatory capacity of peripheral vessels is impaired when EF% is reduced. Furthermore, RNA-sequencing comparison between HFrEF and HFpEF uncovered 35 differentially expressed genes in the coronary (n=3) and 23 differentially expressed genes in cerebral (n=3) arterioles (q ≤ 0.1). Ingenuity pathway analysis predicted upstream regulators that code for angiotensinogen, STAT3, and interferon-Ƴ were inhibited (z-score > -2.0) in the HFrEF model, which was associated with decreased proliferation of smooth muscle cells and vasculogenesis with increased organismal death and inflammation in the coronary arterioles. Furthermore, analysis of the cerebral arterioles predicted inhibition of angiotensinogen (z-score > -2.0) in the HFrEF model which was associated with increased fibroblast cell death, dedifferentiation of smooth muscle cells, and recruitment of macrophages. Together, these data reveal distinct pathophysiological functional and transcriptomic adaptions between HFrEF and HFpEF in the coronary and cerebral arterioles. Additionally, these data provide three candidate genes that regulate these pathophysiological processes and could be targeted for potential therapeutic intervention. Funding: REGENCOR and Department of Defense (DOD) W81XWH-18-1-0179. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

KLF13 promotes VSMCs phenotypic dedifferentiation by directly binding to the SM22α promoter.

Krüppel-like factor 13 (KLF13), a zinc finger transcription factor, is considered as a potential regulator of cardiomyocyte differentiation and proliferation during heart morphogenesis. However, its precise role in the dedifferentiation of vascular smooth muscle cells (VSMCs) during atherosclerosis and neointimal formation after injury remains poorly understood. In this study, we investigated the relationship between KLF13 and SM22α expression in normal and atherosclerotic plaques by bioanalysis, and observed a significant increase in KLF13 levels in the atherosclerotic plaques of both human patients and ApoE-/- mice. Knockdown of KLF13 was found to ameliorate intimal hyperplasia following carotid artery injury. Furthermore, we discovered that KLF13 directly binds to the SM22α promoter, leading to the phenotypic dedifferentiation of VSMCs. Remarkably, we observed a significant inhibition of platelet-derived growth factor BB-induced VSMCs dedifferentiation, proliferation, and migration when knocked down KLF13 in VSMCs. This inhibitory effect of KLF13 knockdown on VCMC function was, at least in part, mediated by the inactivation of p-AKT signaling in VSMCs. Overall, our findings shed light on a potential therapeutic target for treating atherosclerotic lesions and restenosis after vascular injury.

Murine double minute 2-mediated estrogen receptor 1 degradation activates macrophage migration inhibitory factor to promote vascular smooth muscle cell dedifferentiation and oxidative stress during thoracic aortic aneurysm progression

Estrogen receptor 1 (ESR1) has been recently demonstrated as a potential diagnostic biomarker for thoracic aortic aneurysm (TAA). However, its precise role in the progression of TAA remains unclear. In this study, TAA models were established in ApoE-knockout mice and primary mouse vascular smooth muscle cells (VSMCs) through treatment with angiotensin (Ang) II. Our findings revealed a downregulation of ESR1 in Ang II-induced TAA mice and VSMCs. Upregulation of ESR1 mitigated expansion and cell apoptosis in the mouse aorta, reduced pathogenetic transformation of VSMCs, and reduced inflammatory infiltration and oxidative stress both in vitro and in vivo. Furthermore, we identified macrophage migration inhibitory factor (MIF) as a biological target of ESR1. ESR1 bound to the MIF promoter to suppress its transcription. Artificial MIF restoration negated the mitigating effects of ESR1 on TAA. Additionally, we discovered that murine double minute 2 (MDM2) was highly expressed in TAA models and mediated protein degradation of ESR1 through ubiquitination modification. Silencing of MDM2 reduced VSMC dedifferentiation and suppressed oxidative stress. However, these effects were reversed upon further silencing of ESR1. In conclusion, this study demonstrates that MDM2 activates MIF by mediating ESR1 degradation, thus promoting VSMC dedifferentiation and oxidative stress during TAA progression.

Ursolic acid inhibits NLRP3 inflammasome activation and alleviates vascular smooth muscle injury in Kawasaki disease

Kawasaki disease (KD) is a kind of autoimmune disease with systemic vasculitis as the main pathological change. It is critical to explore potential new therapeutic agents to address KD disease and its complications. Ursolic acid (UA) is a pentacyclic triterpene (PT) carboxylic acid that has a number of important pharmacological activities, however, the possible effects of UA on the progression of KD and the mechanism are still unclear. Here we investigated the effects of UA on KD. We revealed that UA improved arterial injury in KD mice. UA improved vascular inflammation in KD mice. In addition, Ursolic suppressed NLR Family Pyrin Domain Containing 3 (NLRP3) inflammasome activation. We further found UA restrained vascular smooth muscle cell (VSMC) dedifferentiation, therefore suppressing KD progression. In summary, UA suppressed NLRP3 inflammasome activation as well as alleviated vascular smooth muscle injury in KD. We thought UA could act as a drug of KD.

Colchicine Blocks Abdominal Aortic Aneurysm Development by Maintaining Vascular Smooth Muscle Cell Homeostasis.

Development of non-surgical treatment of human abdominal aortic aneurysm (AAA) has clinical significance. Colchicine emerges as an effective therapeutic regimen in cardiovascular diseases. Yet, whether colchicine slows AAA growth remain controversy. Here, we demonstrated that daily intragastric administration of low-dose colchicine blocked AAA formation, prevented vascular smooth muscle cell (SMC) phenotype switching and apoptosis, and vascular inflammation in both peri-aortic CaPO4 injury and subcutaneous angiotensin-II infusion induced experimental AAA mice models. Mechanistically, colchicine increased global mRNA stability by inhibiting the METTL14/YTHDC1-mediated m6A modification, resulting in increased sclerostin (SOST) expression and consequent inactivation of the WNT/β-catenin signaling pathway in vascular SMCs from mouse AAA lesions and in cultured human aortic SMCs. Moreover, human and mouse AAA lesions all showed increased m6A methylation, decreased SOST expression, and skewed synthetic SMC de-differentiation phenotype, compared to those without AAA. This study uncovers a novel mechanism of colchicine in slowing AAA development by using the METTL14/SOST/WNT/β-catenin axis to control vascular SMC homeostasis in mouse aortic vessels and in human aortic SMCs. Therefore, use of colchicine may benefit AAA patients in clinical practice.

Vav2 promotes ductus arteriosus anatomic closure via the remodeling of smooth muscle cells by Rac1 activation.

The ductus arteriosus (DA), bridging the aorta and pulmonary artery, immediately starts closing after birth. Remodeling of DA leads to anatomic obstruction to prevent repatency. Several histological changes, especially extracellular matrices (ECMs) deposition and smooth muscle cells (SMCs) migration bring to anatomic closure. The genetic etiology and mechanism of DA closure remain elusive. We have previously reported a novel copy number variant containing Vav2 in patent ductus arteriosus (PDA) patients, but its specific role in DA closure remains unknown. The present study revealed that the expression of Vav2 was reduced in human patent DA, and it was less enrichment in the adjacent aorta. Matrigel experiments demonstrated that Vav2 could promote SMC migration from PDA patient explants. Smooth muscle cells with Vav2 overexpression also presented an increased capacity in migration and downregulated contractile-related proteins. Meanwhile, SMCs with Vav2 overexpression exhibited higher expression of collagen III and lessened protein abundance of lysyl oxidase, and both changes are beneficial to DA remodeling. Overexpression of Vav2 resulted in increased activity of Rac1, Cdc42, and RhoA in SMCs. Further investigation noteworthily found that the above alterations caused by Vav2 overexpression were particularly reversed by Rac1 inhibitor. A heterozygous, rare Vav2 variant was identified in PDA patients. Compared with the wild type, this variant attenuated Vav2 protein expression and weakened the activation of downstream Rac1, further impairing its functions in SMCs. In conclusion, Vav2 functions as an activator for Rac1 in SMCs to promote SMCs migration, dedifferentiation, and ECMs production. Deleterious variant potentially induces Vav2 loss of function, further providing possible molecular mechanisms about Vav2 in PDA pathogenesis. These findings enriched the current genetic etiology of PDA, which may provide a novel target for prenatal diagnosis and treatment. KEY MESSAGES: Although we have proposed the potential association between Vav2 and PDA incidence through whole exome sequencing, the molecular mechanisms underlying Vav2 in PDA have never been reported.This work, for the first time, demonstrated that Vav2 was exclusively expressed in closed DAs. Moreover, we found that Vav2 participated in the process of anatomic closure by mediating SMCs migration, dedifferentiation, and ECMs deposition through Rac1 activation. Our findings first identified a deleterious Vav2 c.701C>T variant that affected its function in SMCs by impairing Rac1 activation, which may lead to PDA defect. Vav2 may become an early diagnosis and an effective intervention target for PDA clinical therapy.

Myeloid-derived growth factor suppresses VSMC dedifferentiation and attenuates postinjury neointimal formation in rats by activating S1PR2 and its downstream signaling.

Restenosis after angioplasty is caused usually by neointima formation characterized by aberrant vascular smooth muscle cell (VSMC) dedifferentiation. Myeloid-derived growth factor (MYDGF), secreted from bone marrow-derived monocytes and macrophages, has been found to have cardioprotective effects. In this study we investigated the effect of MYDGF to postinjury neointimal formation and the underlying mechanisms. Rat carotid arteries balloon-injured model was established. We found that plasma MYDGF content and the level of MYDGF in injured arteries were significantly decreased after balloon injury. Local application of exogenous MYDGF (50 μg/mL) around the injured vessel during balloon injury markedly ameliorated the development of neointimal formation evidenced by relieving the narrow endovascular diameter, improving hemodynamics, and reducing collagen deposition. In addition, local application of MYDGF inhibited VSMC dedifferentiation, which was proved by reversing the elevated levels of osteopontin (OPN) protein and decreased levels of α-smooth muscle actin (α-SMA) in the left carotid arteries. We showed that PDGF-BB (30 ng/mL) stimulated VSMC proliferation, migration and dedifferentiation in vitro; pretreatment with MYDGF (50-200 ng/mL) concentration-dependently eliminated PDGF-BB-induced cell proliferation, migration and dedifferentiation. Molecular docking revealed that MYDGF had the potential to bind with sphingosine-1-phosphate receptor 2 (S1PR2), which was confirmed by SPR assay and Co-IP analysis. Pretreatment with CCG-1423 (Rho signaling inhibitor), JTE-013 (S1PR2 antagonist) or Ripasudil (ROCK inhibitor) circumvented the inhibitory effects of MYDGF on VSMC phenotypic switching through inhibiting S1PR2 or its downstream RhoA-actin monomers (G-actin) /actin filaments (F-actin)-MRTF-A signaling. In summary, this study proves that MYDGF relieves neointimal formation of carotid arteries in response to balloon injury in rats, and suppresses VSMC dedifferentiation induced by PDGF-BB via S1PR2-RhoA-G/F-actin-MRTF-A signaling pathway. In addition, our results provide evidence for cross talk between bone marrow and vasculature.

Hsa_circ_0031891 targets miR-579-3p to enhance HMGB1 expression and regulate PDGF-BB-induced human aortic vascular smooth muscle cell proliferation, migration, and dedifferentiation.

Atherosclerosis (AS) is an underlying cause of the majority of coronary artery disease (CAD), in which proliferation, migration, and dedifferentiation of vascular smooth muscle cells (VSMCs) exert vital roles. It has been reported that circular RNAs (circRNAs) are associated with the VSMCs function. Here, we undertook to explore the biological function and mechanism of hsa_circ_0031891 in a platelet-derived growth factor-BB (PDGF-BB)-induced AS cell model. Hsa_circ_0031891 and microRNA-579-3p (miR-579-3p) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation and migration were detected using Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), wound healing, and transwell assay. Protein levels of alpha-smooth muscle actin (α-SMA), smooth muscle protein 22-α (SM22-α), Osteopontin, and High mobility group box-1 (HMGB1) were determined using western blot assay. After predicting via a variety of bioinformatics software, the binding between miR-579-3p and hsa_circ_0031891 or HMGB1 was validated using dual-luciferase reporter and RNA pull-down assays. Increased hsa_circ_0031891 and HMGB1 and reduced miR-579-3p were found in CAD patients and PDGF-BB-induced human aortic vascular smooth muscle cells (HA-VSMCs). Moreover, hsa_circ_0031891 deficiency relieved PDGF-BB-mediated HA-VSMC proliferation, migration, and dedifferentiation. Mechanically, hsa_circ_0031891 modulated HMGB1 expression via sponging miR-579-3p. Hsa_circ_0031891 boosted PDGF-BB-induced proliferation, migration, and dedifferentiation partly by regulating the miR-579-3p/HMGB1 axis, hinting at a feasible therapeutic strategy for AS.

Dihydroartemisinin inhibits restenosis after balloon angioplasty via circHSPA4/miR-19a-5p axis.

Dihydroartemisinin (DHA) inhibits restenosis following balloon angioplasty. However, data on the mechanisms of DHA activity in restenosis remains scant. Here, we investigated the role of circRNAs in mediating the inhibitory activity of DHA in neointimal formation. We used total RNA sequencing data to profile the expression of mRNA, circRNA and small RNA in sham, vascular balloon injury (VBI) and DHA-treated groups. CCK8 and EdU assays were employed to analyze cell proliferation, while qRT-PCR and Western blot were used to analyze the RNA or protein expression. In addition, we used RNA immunoprecipitation and luciferase reporter assay to assess the binding of circHSPA4 with miR-19a-5p. RNA sequencing demonstrated that circHSPA4 was upregulated in VBI. Treatment with DHA effectively suppressed the upregulation of the circHSPA4. In addition, analysis of platelet-derived growth family factor bb (PDGFbb)-induced HA-VSMCs showed upregulation of circHSPA4, which was associated with cell proliferation and differentiation. CircHSPA4 was shown to induce dedifferentiation and proliferation of smooth muscle cells. PDGFBB-induced overexpression of CircHSPA4 in HA-VSMCs led to suppression of miR-19a-5p, a phenomenon that was reversed by DHA, in concentration-dependent fashion. In addition, miR-19a-5p reduced the dedifferentiation and proliferation of the smooth muscle cells. Our data demonstrated that CircHSPA4 regulates proliferation and differentiation of smooth muscle cells. DHA and miR-19a-5p modulates CircHSPA4 and can be used as coated drugs on balloon catheter to improve the success rate of vascular remodeling.

Abstract 101: Crosstalk Of Pluripotency And Innate Immunity In Smooth Muscle Cell Phenotypic Transitions

Objectives: Smooth muscle cell ( SMC ) phenotypic transitions play a critical role in the pathogenesis of cardiovascular diseases. However, the molecular mechanisms responsible for these transitions are not well understood. We previously found that the embryonic stem cell/pluripotency factor OCT4 is reactivated in SMC in human and mouse atherosclerotic plaques and plays an atheroprotective role by regulating SMC investment into the protective fibrous cap. Loss of OCT4 in SMC is associated with SMC de-differentiation toward a macrophage-like phenotypic state resulting in increased lipid accumulation and phagocytosis. However, the endogenous molecular mechanism responsible for atheroprotective SMC-OCT4-dependent effects remains unknown. Methods and Results: Herein, we performed combinatory bioinformatics analyses on the in vivo and in vitro wild-type and SMC- Oct4 knockout mouse samples and found that loss of Oct4 in SMC resulted in up-regulation of the innate immune receptor TLR4 in numerous experimental conditions. Notably, the TLR4 promoter does not have any putative OCT4 binding sites. First - Using publicly available OCT4 ChIPseq datasets and long non-coding ( lnc )RNA databases, we identified several lncRNAs, including IGF2AS, MALAT1, PLATR10, BACE1, and SRA based on the OCT4 occupancy at promoter and enhancer regions of lncRNA, which in turn, directly target TLR4. To confirm the OCT4-dependent activation of the identified lncRNAs in SMC, qRT-PCR was performed in mouse aortic SMC transfected with Oct4 -blocking siRNA. We found that the Oct4 knockdown in SMC led to significant decreases in all five selected lncRNAs and increases in TLR4 and its downstream gene, TRIF. Second - Intriguingly, we also demonstrated that TLR4 signaling, in turn, promotes hydroxymethylation of the OCT4 promoter and reactivates OCT4 in SMC. Third - Using a Tlr4 -reporter and SMC-lineage tracing mouse model, we also demonstrated that medial differentiated SMC from healthy arteries, but not phenotypically modulated lesion SMC, express TLR4. Conclusions: Together, these results indicate a role of TLR4-signaling in SMC both upstream and downstream of OCT4, suggesting that pluripotency/innate immunity crosstalk plays a critical role in SMC phenotypic transitions.

Abstract 650: Disrupting Neointimal Formation In Mouse And Human Vascular Tissues Through Targeting Connexin 43

Vascular damage caused by stent placement triggers smooth muscle cell (SMC) de-differentiation, leading to an aggressively proliferating phenotype that thickens the vessel wall and blocks the artery, which contributes to stent failure. We have identified the gap junction protein connexin 43 (Cx43) as a significant contributor to neointima progression. Phosphorylation of Cx43 by mitogen-activated protein kinase (MAPK) promotes its binding to the cell cycle regulator cyclin E and its kinase CDK2, promoting SMC proliferation. Beyond this, we do not know how these proteins regulate SMC proliferation in humans. We tested the hypothesis that disrupting Cx43-cyclin E binding could reduce neointima formation in human blood vessels. We used peptide arrays to identify cyclin E binding sites on Cx43 and to define critical Cx43 MAPK-phosphorylation residues that control their interactions. Based on this, we created a 15 amino acid, stearate-linked, cell-permeable, connexin mimetic peptide termed CycliCx that binds cyclin E. Experiments were controlled using Cx43 mimetic peptides designed against regions not bound by cyclin E. In cultured human coronary artery SMC, the CycliCx peptide reduces Cx43 MAPK-phosphorylation, disrupts Cx43-cyclin E interactions, and prevents PDGF-induced SMC proliferation. Application of the CycliCx peptide to mouse carotid arteries inhibits neointima formation in a 14-day ligation-induced neointima model (n=8, male and female). We developed an ex vivo model of PDGF-induced neointimal formation in excess human saphenous vein tissues recovered after CABG surgery. In the human saphenous vein preparations treated with PDGF, the CycliCx peptide disrupts neointima formation after 14 days (n=9). In conclusion, our data suggest that disrupting Cx43-cyclin E interactions provides protection against pathological SMC proliferation. These data highlight Cx43 as a potential drug target to prevent human neointima development.

Smooth Muscle Cell Klf4 Expression Is Not Required for Phenotype Modulation or Aneurysm Formation in Marfan Syndrome Mice-Brief Report.

Smooth muscle cell (SMC) phenotypic reprogramming toward a mixed synthetic-proteolytic state is a central feature of aortic root aneurysm in Marfan syndrome (MFS). Previous work identified Klf4 as a potential mediator of SMC plasticity in MFS. MFS (Fbn1C1041G/+) mouse strains with an inducible vascular SMC fluorescent reporter (MFSSMC) with or without SMC-specific deletion of Klf4 exons 2 to 3 (MFSSMC-Klf4Δ) were generated. Simultaneous SMC tracing and Klf4 loss-of-function (Klf4Δ mice) was induced at 6 weeks of age. Aneurysm growth was assessed via serial echocardiography (4-24 weeks). Twenty-four-week-old mice were assessed via histology, RNA in situ hybridization, and aortic single-cell RNA sequencing. MFS mice demonstrated progressive aortic root dilatation compared with control (WTSMC) mice regardless of Klf4 genotype (P<0.001), but there was no difference in aneurysm growth in MFSSMC-Klf4Δ versus MFSSMC (P=0.884). Efficient SMC Klf4 deletion was confirmed via lineage-stratified genotyping, RNA in situ hybridization, and immunohistochemistry. Single-cell RNA sequencing of traced SMCs revealed a highly similar pattern of phenotype modulation marked by loss of contractile markers (eg, Myh11, Cnn1) and heightened expression of matrix genes (eg, Col1a1, Fn1) between Klf4 genotypes. Pseudotemporal quantitation of SMC dedifferentiation confirmed that Klf4 deletion did not alter the global extent of phenotype modulation, but reduced expression of 23 genes during this phenotype transition in MFSSMC-Klf4Δmice, including multiple chondrogenic genes expressed by only the most severely dedifferentiated SMCs (eg, Cytl1, Tnfrsf11b). Klf4 is not required to initiate SMC phenotype modulation in MFS aneurysm but may exert regulatory control over chondrogenic genes expressed in highly dedifferentiated SMCs.

Conserved miR-370-3p/BMP-7 axis regulates the phenotypic change of human vascular smooth muscle cells

Endothelial dysfunction and inflammatory immune response trigger dedifferentiation of vascular smooth muscle cells (SMCs) from contractile to synthetic phenotype and initiate arterial occlusion. However, the complex vascular remodeling process playing roles in arterial occlusion initiation is largely unknown. We performed bulk sequencing of small and messenger RNAs in a rodent arterial injury model. Bioinformatic data analyses reveal that six miRNAs are overexpressed in injured rat carotids as well as synthetic-type human vascular SMCs. In vitro cell-based assays show that four miRNAs (miR-130b-5p, miR-132-3p, miR-370-3p, and miR-410-3p) distinctly regulate the proliferation of and monocyte adhesion to the vascular SMCs. Individual inhibition of the four selected miRNAs strongly prevents the neointimal hyperplasia in the injured rat carotid arteries. Mechanistically, miR-132-3p and miR-370-3p direct the cell cycle progression, triggering SMC proliferation. Gene ontology analysis of mRNA sequencing data consistently reveal that the miRNA targets include gene clusters that direct proliferation, differentiation, and inflammation. Notably, bone morphogenic protein (BMP)-7 is a prominent target gene of miR-370-3p, and it regulates vascular SMC proliferation in cellular and animal models. Overall, this study first reports that the miR-370-3p/BMP-7 axis determines the vascular SMC phenotype in both rodent and human systems.

Inhibition of Intimal Thickening By PRH (Proline-Rich Homeodomain) in Mice.

Late vein graft failure is caused by intimal thickening resulting from endothelial cell (EC) damage and inflammation which promotes vascular smooth muscle cell (VSMC) dedifferentiation, migration, and proliferation. Nonphosphorylatable PRH (proline-rich homeodomain) S163C:S177C offers enhanced stability and sustained antimitotic effect. Therefore, we investigated whether adenovirus-delivered PRH S163C:S177C protein attenuates intimal thickening via VSMC phenotype modification without detrimental effects on ECs. PRH S163C:S177C was expressed in vitro (human saphenous vein-VSMCs and human saphenous vein-ECs) and in vivo (ligated mouse carotid arteries) by adenoviruses. Proliferation, migration, and apoptosis were quantified and phenotype was assessed using Western blotting for contractile filament proteins and collagen gel contraction. EC inflammation was quantified using VCAM (vascular cell adhesion protein)-1, ICAM (intercellular adhesion molecule)-1, interleukin-6, and monocyte chemotactic factor-1 measurement and monocyte adhesion. Next Generation Sequencing was utilized to identify novel downstream mediators of PRH action and these and intimal thickening were investigated in vivo. PRH S163C:S177C inhibited proliferation, migration, and apoptosis and promoted contractile phenotype (enhanced contractile filament proteins and collagen gel contraction) compared with virus control in human saphenous vein-VSMCs. PRH S163C:S177C expression in human saphenous vein-ECs significantly reduced apoptosis, without affecting cell proliferation and migration, while reducing TNF (tumor necrosis factor)-α-induced VCAM-1 and ICAM-1 and monocyte adhesion and suppressing interleukin-6 and monocyte chemotactic factor-1 protein levels. PRH S163C:S177C expression in ligated murine carotid arteries significantly impaired carotid artery ligation-induced neointimal proliferation and thickening without reducing endothelial coverage. Next Generation Sequencing revealed STAT-1 (signal transducer and activator of transcription 1) and HDAC-9 (histone deacetylase 9) as mediators of PRH action and was supported by in vitro and in vivo analyses. We observed PRH S163C:S177C attenuated VSMC proliferation, and migration and enhanced VSMC differentiation at least in part via STAT-1 and HDAC-9 signaling while promoting endothelial repair and anti-inflammatory properties. These findings highlight the potential for PRH S163C:S177C to preserve endothelial function whilst suppressing intimal thickening, and reducing late vein graft failure.

Vascular smooth muscle cells in intimal hyperplasia, an update.

Arterial occlusive disease is the leading cause of death in Western countries. Core contemporary therapies for this disease include angioplasties, stents, endarterectomies and bypass surgery. However, these treatments suffer from high failure rates due to re-occlusive vascular wall adaptations and restenosis. Restenosis following vascular surgery is largely due to intimal hyperplasia. Intimal hyperplasia develops in response to vessel injury, leading to inflammation, vascular smooth muscle cells dedifferentiation, migration, proliferation and secretion of extra-cellular matrix into the vessel's innermost layer or intima. In this review, we describe the current state of knowledge on the origin and mechanisms underlying the dysregulated proliferation of vascular smooth muscle cells in intimal hyperplasia, and we present the new avenues of research targeting VSMC phenotype and proliferation.

Ferroptotic stress facilitates smooth muscle cell dedifferentiation in arterial remodelling by disrupting mitochondrial homeostasis.

Smooth muscle cell (SMC) phenotypic switch from a quiescent 'contractile' phenotype to a dedifferentiated and proliferative state underlies the development of cardiovascular diseases (CVDs); however, our understanding of the mechanism is still incomplete. In the present study, we explored the potential role of ferroptosis, a novel nonapoptotic form of cell death, in SMC phenotypic switch and related neointimal formation. We found that ferroptotic stress was triggered in cultured dedifferentiated SMCs and arterial neointimal tissue of wire-injured mice. Moreover, pro-ferroptosis stress was activated in arterial neointimal tissue of clinical patients who underwent carotid endarterectomy. Blockade of ferroptotic stress via administration of a pharmacological inhibitor or by global genetic overexpression of glutathione peroxidase-4 (GPX4), a well-established anti-ferroptosis molecule, delayed SMC phenotype switch and arterial remodelling. Conditional SMC-specific gene delivery of GPX4 using adreno-associated virus in the carotid artery inhibited ferroptosis and prevented neointimal formation. Conversely, ferroptosis stress directly triggered dedifferentiation of SMCs. Transcriptomics analysis demonstrated that inhibition of ferroptotic stress mainly targets the mitochondrial respiratory chain and oxidative phosphorylation. Mechanistically, ferroptosis inhibition corrected the disrupted mitochondrial homeostasis in dedifferentiated SMCs, including enhanced mitochondrial ROS production, dysregulated mitochondrial dynamics, and mitochondrial hyperpolarization, and ultimately inhibited SMC phenotypic switch and growth. Copper-diacetyl-bisN4-methylthiosemicarbazone (CuATSM), an agent used for clinical molecular imaging and that potently inhibits ferroptosis, prevented SMC phenotypic switch, neointimal formation and arterial inflammation in mice. These results indicate that pro-ferroptosis stress is likely to promote SMC phenotypic switch during neointimal formation and imply that inhibition of ferroptotic stress may be a promising translational approach to treat CVDs with SMC phenotype switch.

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.

Phosphorylation of the smooth muscle master splicing regulator RBPMS regulates its splicing activity.

We previously identified RBPMS as a master regulator of alternative splicing in differentiated smooth muscle cells (SMCs). RBPMS is transcriptionally downregulated during SMC dedifferentiation, but we hypothesized that RBPMS protein activity might be acutely downregulated by post-translational modifications. Publicly available phosphoproteomic datasets reveal that Thr113 and Thr118 immediately adjacent to the RRM domain are commonly both phosphorylated. An RBPMS T113/118 phosphomimetic T/E mutant showed decreased splicing regulatory activity both in transfected cells and in a cell-free in vitro assay, while a non-phosphorylatable T/A mutant retained full activity. Loss of splicing activity was associated with a modest reduction in RNA affinity but significantly reduced RNA binding in nuclear extract. A lower degree of oligomerization of the T/E mutant might cause lower avidity of multivalent RNA binding. However, NMR analysis also revealed that the T113/118E peptide acts as an RNA mimic which can loop back and antagonize RNA-binding by the RRM domain. Finally, we identified ERK2 as the most likely kinase responsible for phosphorylation at Thr113 and Thr118. Collectively, our data identify a potential mechanism for rapid modulation of the SMC splicing program in response to external signals during the vascular injury response and atherogenesis.

Estrogen receptor α regulates phenotypic switching and proliferation of vascular smooth muscle cells through the NRF1-OMI-mitophagy signaling pathway under simulated microgravity.

Vascular remodeling during microgravity exposure results in postflight cardiovascular deconditioning and orthostatic intolerance in astronauts. To clarify the underlying mechanism, we investigated whether estrogen receptor α (ERα)-NRF1-OMI-mitophagy signaling was involved in the dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) under simulated microgravity. Phenotypic markers, mtDNA copy number and mitochondrial biogenesis, mitochondrial dynamics and mitophagy in rat thoracic artery smooth muscle cells were examined. Four-week hindlimb unweighting (HU) was used to simulate microgravity in rats and 10% serum was used to induce VSMCs dedifferentiation in vitro. The effects of ERα-NRF1-OMI signaling on mitophagy, phenotypic switching and proliferation of VSMCs, and cerebrovascular remodeling in HU rats were studied by genetic manipulation and chronic drug intervention. We found that ERα is positively associated with contractile phenotype switching but inversely correlated with synthetic phenotype switching and proliferation of VSMCs both in vivo and in vitro. During the dedifferentiation process of VSMCs, reduced mtDNA copy number, disturbed mitochondrial biogenesis and respiration, and perturbed fission-fusion-mitophagy signaling were detected, which were reversed by ERα overexpression. Mechanistically, the ERα downstream protein OMI preserved the mitochondrial Parkin level by increasing its protein stability, thereby protecting mitophagy. In line with this, we found that activating ERα signaling by propyl pyrazole triol (PPT) could alleviate the synthetic phenotype switching and proliferation of HU rat cerebral VSMCs by reestablishing fission-fusion-mitophagy hemostasis. The current study clarified a novel mechanism by which inhibited ERα-NRF1-OMI-mitophagy signaling resulted in synthetic phenotype switching and proliferation of VSMCs and cerebrovascular remodeling under simulated microgravity.