Dedifferentiation Of Smooth Muscle Cells Research Articles

The Effect of Retinoids in Vascular Smooth Muscle Cells: From Phenotyping Switching to Proliferation and Migration.

Atherosclerosis, a term derived from the Greek "athero" (atheroma) and "sclerosis" (hardening), is a long-standing process that leads to the formation of atheromatous plaques in the arterial wall, contributing to the development of atherosclerotic cardiovascular disease. The proliferation and migration of vascular smooth muscle cells (VSMCs) and the switching of their phenotype play a crucial role in the whole process. Retinoic acid (RA), a natural derivative of vitamin A, has been used in the treatment of various inflammatory diseases and cell proliferation disorders. Numerous studies have demonstrated that RA has an important inhibitory effect on the proliferation, migration, and dedifferentiation of vascular smooth muscle cells, leading to a significant reduction in atherosclerotic lesions. In this review article, we explore the effects of RA on the pathogenesis of atherosclerosis, focusing on its regulatory action in VSMCs and its role in the phenotypic switching, proliferation, and migration of VSMCs. Despite the potential impact that RA may have on the process of atherosclerosis, further studies are required to examine its safety and efficacy in clinical practice.

Dedifferentiation- and aging-induced loss of mechanical contractility and polarity in vascular smooth muscle cells: Heterogeneous changes in macroscopic and microscopic behavior of cells in serial passage culture

Dedifferentiation and aging of vascular smooth muscle cells (VSMCs) are associated with serious vascular diseases, such as arteriosclerosis and aneurysm. However, how cell dedifferentiation and aging affect cellular mechanical behaviors at the single-cell and intracellular structure levels remains unclear. An in-depth understanding of these interactions is extremely important for understanding the mechanism underlying VSMC mechanical integrity and homeostatic regulation of vascular walls. Herein, we systematically investigated changes in VSMC morphology, structure, contractility, and motility during dedifferentiation and aging induced by serial passage culture using traction force microscopy with elastic micropillar substrates, laser nanodissection of cytoskeletons, confocal fluorescence microscopy, and atomic force microscopy. We found that VSMC dedifferentiation started in the middle stage of serial passage culture, accompanied by a transient cell spreading in the cell width and decrease in contractile protein expression. Dedifferentiated VSMCs showed a significant decrease in the contraction and stiffness of individual actin stress fibers; however, their overall cell traction forces were maintained. Simultaneously, a significant increase in cell motility and the number of actin fibers was observed in dedifferentiated VSMCs, which may be associated with the enhancement of cell migration and disruption of cell/tissue integrity during the early stage of vascular diseases. As cell senescence progressed in the later stage of serial passage culture, VSMCs displayed reduced cell spreading and migration with decrease in the overall cell traction forces and drastic reduction in mechanical polarity of cell structures and forces. These results suggested that cell senescence causes loss of mechanical contractility and polarity in VSMCs, which may be an important factor in vascular disease progression. The experimental systems established in this study can be powerful tools for understanding the mechanisms underlying cellular dedifferentiation and aging from a biomechanical perspective.

Continuous infusion of resolvin D2 in combination with Angiotensin-II show contrary effects on blood pressure and intracardiac artery remodeling

Specialized pro-resolving mediators (SPMs) are key effectors of resolution of inflammation. This is highly relevant for cardiac and vessel remodeling, where the net inflammatory response contributes to determine disease outcome. Herein, we used a mice model of angiotensin (Ang)–II–induced hypertension to study the effect of the SPM Resolvin D2 (RvD2), on hypertension and cardiac remodeling. By using subcutaneous osmotic minipumps, mice were treated with PBS or Ang-II in combination with or without RvD2 for two weeks. Mice receiving RvD2 gained less blood pressure increase compared to Ang-II alone. Surprisingly, however, examination of intracardiac arteries revealed that RvD2 treatment in combination with Ang-II exacerbated Ang–II–induced fibrosis. Measures of vascular smooth muscle cell dedifferentiation correlated with the level of vascular remodeling, indicating that this dedifferentiation, including increased proliferation and migration, is a contributing factor. RNA sequencing of left ventricle cardiac tissue supported these findings as pathways related to cell proliferation and cell differentiation were upregulated in mice treated with Ang-II in combination with RvD2. Additionally, the RNA sequencing also showed upregulation of pathways related to SPM metabolism. In line with this, Mass spectrometry analysis of lipid mediators showed reduced cardiac levels of the arachidonic acid derived metabolite leukotriene E4 in RvD2 treated mice. Our study suggests that continuous infusion through osmotic minipumps should not be the recommended route of RvD2 administration in future studies.

Differentiation and Growth-Arrest-Related lncRNA (DAGAR): Initial Characterization in Human Smooth Muscle and Fibroblast Cells.

Vascular smooth muscle cells (SMCs) can transition between a quiescent contractile or "differentiated" phenotype and a "proliferative-dedifferentiated" phenotype in response to environmental cues, similar to what in occurs in the wound healing process observed in fibroblasts. When dysregulated, these processes contribute to the development of various lung and cardiovascular diseases such as Chronic Obstructive Pulmonary Disease (COPD). Long non-coding RNAs (lncRNAs) have emerged as key modulators of SMC differentiation and phenotypic changes. In this study, we examined the expression of lncRNAs in primary human pulmonary artery SMCs (hPASMCs) during cell-to-cell contact-induced SMC differentiation. We discovered a novel lncRNA, which we named Differentiation And Growth Arrest-Related lncRNA (DAGAR) that was significantly upregulated in the quiescent phenotype with respect to proliferative SMCs and in cell-cycle-arrested MRC5 lung fibroblasts. We demonstrated that DAGAR expression is essential for SMC quiescence and its knockdown hinders SMC differentiation. The treatment of quiescent SMCs with the pro-inflammatory cytokine Tumor Necrosis Factor (TNF), a known inducer of SMC dedifferentiation and proliferation, elicited DAGAR downregulation. Consistent with this, we observed diminished DAGAR expression in pulmonary arteries from COPD patients compared to non-smoker controls. Through pulldown experiments followed by mass spectrometry analysis, we identified several proteins that interact with DAGAR that are related to cell differentiation, the cell cycle, cytoskeleton organization, iron metabolism, and the N-6-Methyladenosine (m6A) machinery. In conclusion, our findings highlight DAGAR as a novel lncRNA that plays a crucial role in the regulation of cell proliferation and SMC differentiation. This paper underscores the potential significance of DAGAR in SMC and fibroblast physiology in health and disease.

Smooth muscle-specific deletion of Ccn2 causes severe aorta malformation and atherosclerosis.

Cellular Communication Network Factor 2 (CCN2) is a matricellular protein implicated in fibrotic diseases, with ongoing clinical trials evaluating anti-CCN2-based therapies. By uncovering CCN2 as abundantly expressed in non-diseased artery tissue, this study aimed to investigate the hypothesis that CCN2 plays a pivotal role in maintaining smooth muscle cell (SMC) phenotype and protection against atherosclerosis. Global- and SMC-specific Ccn2 knockout mouse models were employed to demonstrate that Ccn2 deficiency leads to SMC de-differentiation, medial thickening, and aorta elongation under normolipidemic conditions. Inducing hyperlipidemia in both models resulted in severe aorta malformation and a 17-fold increase in atherosclerosis formation. Lipid-rich lesions developed at sites of the vasculature typically protected from atherosclerosis-development by laminar blood flow, covering 90% of aortas, and extending to other vessels, including coronary arteries. Evaluation at earlier time points revealed medial lipid accumulation as a lesion-initiating event. Fluorescently labelled LDL injection followed by confocal microscopy showed increased LDL retention in the medial layer of Ccn2 knockout aortas, likely attributed to marked proteoglycan enrichment of the medial extracellular matrix. Analyses leveraging data from the Athero-Express study cohort indicated relevance of CCN2 in established human lesions, as CCN2 correlated with SMC marker transcripts across 654 transcriptomically profiled carotid plaques. These findings were substantiated through in situ hybridization showing CCN2 expression predominantly in the fibrous cap. This study identifies CCN2 as a major constituent of the normal artery wall, critical in regulating SMC differentiation and aorta integrity, and possessing a protective role against atherosclerosis development. These findings underscore the need for further investigation into the potential effects of anti-CCN2-based therapies on the vasculature.

CD8+ T Cells Drive Plaque Smooth Muscle Cell Dedifferentiation in Experimental Atherosclerosis.

Atherosclerosis is driven by the infiltration of the arterial intima by diverse immune cells and smooth muscle cells (SMCs). CD8+ T cells promote lesion growth during atherosclerotic lesion development, but their role in advanced atherosclerosis is less clear. Here, we studied the role of CD8+ T cells and their effects on SMCs in established atherosclerosis. CD8+ T cells were depleted in (SMC reporter) low-density lipoprotein receptor-deficient (Ldlr-/-) mice with established atherosclerotic lesions. Atherosclerotic lesion formation was examined, and single-cell RNA sequencing of aortic SMCs and their progeny was performed. Additionally, coculture experiments with primary aortic SMCs and CD8+ T cells were conducted. Although we could not detect differences in atherosclerotic lesion size, an increased plaque SMC content was noted in mice after CD8+ T-cell depletion. Single-cell RNA sequencing of aortic lineage-traced SMCs revealed contractile SMCs and a modulated SMC cluster, expressing macrophage- and osteoblast-related genes. CD8+ T-cell depletion was associated with an increased contractile but decreased macrophage and osteoblast-like gene signature in this modulated aortic SMC cluster. Conversely, exposure of isolated aortic SMCs to activated CD8+ T cells decreased the expression of genes indicative of a contractile SMC phenotype and induced a macrophage and osteoblast-like cell state. Notably, CD8+ T cells triggered calcium deposits in SMCs under osteogenic conditions. Mechanistically, we identified transcription factors highly expressed in modulated SMCs, including Runx1, to be induced by CD8+ T cells in cultured SMCs in an IFNγ (interferon-γ)-dependent manner. We here uncovered CD8+ T cells to control the SMC phenotype in atherosclerosis. CD8+ T cells promote SMC dedifferentiation and drive SMCs to adopt features of macrophage-like and osteoblast-like, procalcifying cell phenotypes. Given the critical role of SMCs in atherosclerotic plaque stability, CD8+ T cells could thus be explored as therapeutic target cells during lesion progression.

Ginger protects against vein graft remodeling by precisely modulating ferroptotic stress in vascular smooth muscle cell dedifferentiation

Vein graft failure (VGF) is associated with vein graft (VG) intimal hyperplasia, which is characterized by abnormal accumulation of vascular smooth muscle cells (VSMCs). Most neointimal VSMCs are derived from pre-existing VSMCs via a process of VSMC phenotypic transition, also known as dedifferentiation. There is increasing evidence to suggest that ginger or its bioactive ingredients may block VSMC dedifferentiation, exerting vasoprotective functions; however, the precise mechanisms have not been fully characterized. Therefore, we investigated the effect of ginger on VSMC phenotypic transition in VG remodeling after transplantation. Ginger significantly inhibited neointimal hyperplasia and promoted lumen opening in a 3-month VG, which was primarily achieved by reducing ferroptotic stress. Ferroptotic stress is a pro-ferroptotic state. Contractile VSMCs did not die but instead gained a proliferative capacity and switched to the secretory type, forming neointima after vein transplantation. Ginger and its two main vasoprotective ingredients (6-gingerol and 6-shogaol) inhibit VSMC dedifferentiation by reducing ferroptotic stress. Network pharmacology analysis revealed that 6-gingerol inhibits ferroptotic stress by targeting P53 while 6-shogaol inhibits ferroptotic stress by targeting 5-lipoxygenase (Alox5), both promoting ferroptosis. Furthermore, both ingredients co-target peroxisome proliferator-activated receptor gamma (PPARγ), decreasing PPARγ mediated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1 (Nox1) expression. Nox1 promotes intracellular reactive oxygen species (ROS) production and directly induces VSMC dedifferentiation. In addition, Nox1 is a ferroptosis-promoting gene that encourages ferroptotic stress production, indirectly leading to VSMC dedifferentiation. Ginger, a natural multi-targeted ferroptotic stress inhibitor, finely and effectively prevents VSMC phenotypic transition and protects against venous injury remodeling.

Basal ATP release signals through the P2Y2 receptor to maintain the differentiated phenotype of vascular smooth muscle cells

Background and aimsVascular smooth muscle cell (VSMC) dedifferentiation contributes substantively to vascular disease. VSMCs spontaneously release low levels of ATP that modulate vessel contractility, but it is unclear if autocrine ATP signaling in VSMCs is critical to the maintenance of the VSMC contractile phenotype. MethodsWe used pharmacological inhibitors to block ATP release in human aortic smooth muscle cells (HASMCs) for studying changes in VSMC differentiation marker gene expression. We employed RNA interference and generated mice with SMC-specific inducible deletion of the P2Y2 receptor (P2Y2R) gene to evaluate resulting phenotypic alterations. ResultsHASMCs constitutively release low levels of ATP that when blocked results in a significant decrease in VSMC differentiation marker gene expression, including smooth muscle actin (SMA), smooth muscle myosin heavy chain (SMMHC), SM-22α and calponin. Basal release of ATP represses transcriptional activation of the Krüppel-Like Factor 4 (KFL4) thereby preventing platelet-derived growth factor-BB (PDGF-BB) from inhibiting expression of SMC contractile phenotype markers. SMC-restricted conditional deletion of P2Y2R evoked dedifferentiation characterized by decreases in aortic contractility and contractile phenotype markers expression. This loss was accompanied by a transition to the synthetic phenotype with the acquisition of extracellular matrix (ECM) proteins characteristic of dedifferentiation, such as osteopontin and vimentin. ConclusionsOur data establish the first direct evidence that an autocrine ATP release mechanism maintains SMC cytoskeletal protein expression by inhibiting VSMCs from transitioning to a synthetic phenotype, and further demonstrate that activation of the P2Y2R by basally released ATP is required for maintenance of the differentiated VSMC phenotype.

#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.

Abstract 2150: PTEN Protects Against Vascular Smooth Muscle Cell Pro-inflammatory Dedifferentiation Through Metabolic Re-wiring

Vascular smooth muscle cells (SMCs) play a critical role in intimal hyperplasia in atherosclerosis and restenosis. SMCs in diseased arteries exhibit a modulated phenotype with reduced contractile gene expression, elevated proliferation, and a proinflammatory/profibrotic phenotype. Metabolic reprogramming accompanies and is essential for SMC phenotypic modulation. Our group previously demonstrated that the tumor suppressor gene, PTEN, contributes to the maintenance of the SMC contractile phenotype. Analysis of human GTEx project data established a negative correlation between expression of PTEN and inflammatory genes in human arterial tissues. As a metabolic regulator, the function of PTEN in SMC metabolism has not been examined. We subjected SMC lineage tracing mice with systemic (sPTEN) and inducible, SMC-specific (iPTEN) PTEN overexpression to disease models of atherosclerosis and carotid artery (CA) injury. Compared to control mice, sPTEN and iPTEN mice are protected from atherosclerotic lesion formation and ligation-induced neointima formation. The observed vascular protection effect of PTEN was associated with reduced inflammation and reduced expression of key regulators of glycolysis, including GLUT1, HIF1a and PKM2, which were up-regulated in dedifferentiated SMCs. Consistently, unbiased metabolomics analysis confirmed that CA injury induced a SMC metabolic phenotype, including accumulation of pyruvate, reduction of OXPHOS, and increased fatty acid synthesis, which was rescued in iPTEN mice. Through high throughput screening, we previously identified and validated 5-azacytidine (5-aza) as potent transcriptional activator of PTEN expression. 5-aza reversed injury-induced SMC dedifferentiation in WT, but not PTEN inducible KO (PTEN iKO) mice. Our new scRNA-seq data further suggest that 5-aza inhibited injury-induced SMC metabolic reprogramming and dedifferentiation in a PTEN-dependent manner. Altogether, the data support that PTEN antagonizes the metabolic changes in SMC dedifferentiation and thereby maintains a contractile SMC phenotype. Pharmacological induction of PTEN by 5-aza can serve as a viable approach to rescue SMC metabolic reprogramming and phenotypic modulation in vascular disease.

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.