Smooth Muscle Cell Contractile Phenotype Research Articles

Piezo1 mediated calcium-calcineurin signaling pathway regulates gastrointestinal smooth muscle cell phenotype in a stiffness dependent manner

Background: Gastrointestinal (GI) disorders like chronic constipation are more prevalent with aging. Aging GI tract becomes stiffer and exhibits delayed GI transit. Smooth muscle cells (SMCs) are involved in controlling GI transit and show reduced contractility with age. Piezo1 is a mechanically activated ion channel that responds to membrane tension by opening its pore to allow flow of ions such as calcium (Ca2+) and regulates various cellular processes through Ca2+-calcineurin signaling pathway. We wondered GI stiffness changes might control SMC contractile to non-contractile phenotype through Piezo1. Objective: To determine how substrate stiffness regulates GI SMC contractile phenotype through Piezo1. Hypothesis: SMC Piezo1 senses GI tissue stiffness changes and alters SMC phenotype. Methods: Pathway analysis on human colon single cell sequencing data was performed using Gene Set Enrichment Analysis (GSEA) pre-ranked module with publicly available gene-sets from Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Immortalized human jejunal SMCs (HuSMCs) and freshly dissociated SMCs from control (CTRL, Myh11-creERT2) and SMC specific Piezo1 knockout [Myh11-creERT2xPiezo1 Flox (Piezo1-KO)] mouse colons (primary SMCs) were cultured on soft (1kPa) and stiff (50kPa) substrates to model age-dependent shifts in intestinal stiffness for 48 hours either in the presence/absence of Piezo1 siRNA, Piezo1 agonist (Yoda1, 1μM), and calcineurin blocker (Cyclosporin A, CsA, 1μM) and used for Ca2+ imaging, RT-qPCR and immunostaining. Results: GSEA showed Piezo1+ve SMCs were significantly enriched in pathways involved in the regulation of Ca2+ signaling compared to Piezo1−ve SMCs (p<0.05). HuSMCs and primary SMCs showed increased Piezo1 expression (HuSMCs:1.1±0.2-fold, n=8, p<0.05; primary SMCs:2.1±0.1-fold, n=3, p<0.05) and cytoplasmic Ca2+ (3.6-fold, n=10, p<0.05) in response to Yoda1 on stiff substrates compared to soft. HuSMCs on stiff substrate showed decreased expression of contractile SMC markers smoothelin ( SMTN:0.7±0.2-fold, n=7, p<0.05) and transgelin ( TAGLN:0.6±0.1-fold, n=7, p<0.05) which was rescued by Piezo1 siRNA treatment ( SMTN:1.2±0.1-fold, n=2-7, p<0.05; TAGLN:1.15±0.02-fold, n=2-7, p<0.05) (Piezo1 is necessary). Stimulation by Yoda1 of HuSMCs on the soft substrate resulted in decreased expression of SMTN (0.7±0.3-fold, n=2-7, p<0.05) and TAGLN (0.7±0.1-fold, n=2-7, p<0.05) (Piezo1 is suffcient). CTRL primary SMCs on stiff substrate showed decreased expression of Smtn (0.4±0.1-fold, n=3, p<0.05) and Tagln (0.3±0.2-fold, n=3, p<0.05) compared to SMCs on soft substrate which was reversed when SMCs from Piezo1-KO mice were cultured on these substrates ( Smtn:1.8±0.5-fold, n=3, p<0.05; Tagln:2.4±0.07-fold, n=3, p<0.05). Treatment of CsA in HuSMCs cultured on stiff substrate also rescued from loss of SMTN (1.3±0.1-fold, n=3, p<0.05) and TAGLN (1.7±0.3-fold, n=3, p<0.05) expression and prevented nuclear factor of activated T cells (NFAT) nuclear translocation. Summary and Conclusion: GI SMC Piezo1 senses stiffness changes and regulates SMC phenotype through Ca2+-calcineurin signaling pathway. Targeting Piezo1 might be protective from age induced reduced GI SMC contractility. NIH DK052766, DK123549. 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.

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

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

Orphan Nuclear Receptor NR4A3 Promotes Vascular Calcification via Histone Lactylation.

Medial arterial calcification is a chronic systemic vascular disorder distinct from atherosclerosis and is commonly observed in patients with chronic kidney disease, diabetes, and aging individuals. We previously showed that NR4A3 (nuclear receptor subfamily 4 group A member 3), an orphan nuclear receptor, is a key regulator in apo (apolipoprotein) A-IV-induced atherosclerosis progression; however, its role in vascular calcification is poorly understood. We generated NR4A3-/- mice and 2 different types of medial arterial calcification models to investigate the biological roles of NR4A3 in vascular calcification. RNA-seq was performed to determine the transcriptional profile of NR4A3-/- vascular smooth muscle cells under β-glycerophosphate treatment. We integrated Cleavage Under Targets and Tagmentation analysis and RNA-seq data to further investigate the gene regulatory mechanisms of NR4A3 in arterial calcification and target genes regulated by histone lactylation. NR4A3 expression was upregulated in calcified aortic tissues from chronic kidney disease mice, 1,25(OH)2VitD3 overload-induced mice, and human calcified aorta. NR4A3 deficiency preserved the vascular smooth muscle cell contractile phenotype, inhibited osteoblast differentiation-related gene expression, and reduced calcium deposition in the vasculature. Further, NR4A3 deficiency lowered the glycolytic rate and lactate production during the calcification process and decreased histone lactylation. Mechanistic studies further showed that NR4A3 enhanced glycolysis activity by directly binding to the promoter regions of the 2 glycolysis genes ALDOA and PFKL and driving their transcriptional initiation. Furthermore, histone lactylation promoted medial calcification both in vivo and in vitro. NR4A3 deficiency inhibited the transcription activation and expression of Phospho1 (phosphatase orphan 1). Consistently, pharmacological inhibition of Phospho1 attenuated calcium deposition in NR4A3-overexpressed vascular smooth muscle cells, whereas overexpression of Phospho1 reversed the anticalcific effect of NR4A3 deficiency in vascular smooth muscle cells. Taken together, our findings reveal that NR4A3-mediated histone lactylation is a novel metabolome-epigenome signaling cascade mechanism that participates in the pathogenesis of medial arterial calcification.

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

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

Pancreatic Tumor Organoid-Derived Factors from Cachectic Patients Disrupt Contractile Smooth Muscle Cells

Patients with pancreatic cancer often suffer from cachexia and experience gastrointestinal symptoms that may be related to intestinal smooth muscle cell (SMC) dysfunction. We hypothesized that pancreatic tumor organoids from cachectic patients release factors that perturb the SMC’s contractile characteristics. Human visceral SMCs were exposed to conditioned medium (CM) from the pancreatic tumor organoid cultures of cachectic (n = 2) and non-cachectic (n = 2) patients. Contractile proteins and markers of inflammation, muscle atrophy, and proliferation were evaluated by qPCR and Western blot. SMC proliferation and migration were monitored by live cell imaging. The Ki-67-positive cell fraction was determined in the intestinal smooth musculature of pancreatic cancer patients. CM from the pancreatic tumor organoids of cachectic patients did not affect IL-1β, IL-6, IL-8, MCP-1, or Atrogin-1 expression. However, CM reduced the α-SMA, γ-SMA, and SM22-α levels, which was accompanied by a reduced SMC doubling time and increased expression of S100A4, a Ca2+-binding protein associated with the synthetic SMC phenotype. In line with this, Ki-67-positive nuclei were increased in the intestinal smooth musculature of patients with a low versus high L3-SMI. In conclusion, patient-derived pancreatic tumor organoids release factors that compromise the contractile SMC phenotype and increase SMC proliferation. This may contribute to the frequently observed gastrointestinal motility problems in these patients.

Abstract 13229: Coiled-Coil Domain-Containing Protein 80 Protects Against Aortic Dissection by Maintaining the Contractile Smooth Muscle Cell Phenotype

Background: Aortic dissection (AD) is a life-threatening medical emergency characterized by adverse vascular remodeling. Effective prevention and treatment options are lacking for AD. Coiled-coil domain-containing protein 80 (CCDC80) plays an essential role in regulating vascular remodeling. In the present study, we aimed to define the role of CCDC80 in the formation and development of AD. Methods: The expression and subcellular localization of CCDC80 were determined in human and mouse AD. CCDC80 knockout mice (CCDC80 –/– ) and vascular smooth muscle cell (VSMC)-specific CCDC80 knockout mice (CCDC80 fl/fl SM22α Cre + ) were treated with angiotensin II (Ang II) or Ang II combined with β-aminopropionitrile monofumarate (BAPN) to induce AD models. RNA-Seq analysis combined with immunoprecipitation and blockage of the JAK2/STAT3 signaling pathway in CCDC80 –/– mice were used to evaluate the effect of CCDC80 deficiency on this pathway. Results: CCDC80 was downregulated in VSMCs in human and mouse AD. CCDC80 – / – mice challenged with Ang II and BAPN developed AD with higher frequency and severity compared with their littermate controls (66.7% vs. 100%, p < 0.05). Remarkably, compared with littermate controls treated with Ang II, CCDC80 –/– mice (8.3% vs. 87.5%, p < 0.05) and CCDC80 fl/fl SM22α Cre + mice (12.5% vs. 80%, p < 0.05) frequently developed AD with severe elastin fragmentation and collagen deposition. CCDC80 interacted with JAK2; CCDC80 deficiency could promote VSMC phenotype switching, proliferation, and migration as well as matrix metalloproteinase production by activating the JAK2/STAT3 signaling pathway. The JAK2/STAT3 pathway-specific inhibitor ameliorated adverse vascular remodeling and reduced AD formation (90% vs. 55%, p < 0.05) in CCDC80 knockout mice. Conclusion: CCDC80 deficiency exacerbates the progression of AD by activating the JAK2/STAT3 pathway involved in regulating the phenotype switching and function of VSMCs. Our findings suggest that CCDC80 is a potential target for the prevention and treatment of AD.

Anti-fouling plus: Engineered stent coating with inflammation-regulation capability enables enhanced tissue healing

Coagulation, inflammation, and hyperplasia frequently restrict the effectiveness of cardiovascular stents. It is critical to modify stent surfaces to allow for in situ tissue healing. Interrelated coagulation and inflammation can impede endothelialization and are critical to the long-term performance of cardiovascular implants. This study proposes a relatively bio-inert “Anti-fouling plus” coating that is effective in anti-coagulation and anti-inflammation at the early stage of implantation and provides mild inflammatory responses during tissue remodeling process. The phosphorylcholine (PC)-based polymer PM(PCLA) is reacted onto a polydopamine-treated polylactic acid (PLA) surface. PC-based polymers that mimic cell membranes present remarkable antiinflammation and anticoagulation properties. The composite of epigallocatechin gallate (EGCG) and tempol is loaded on the PM(PCLA) coating to achieve “anti-fouling plus” which can mediate inflammatory responses by scavenging reactive oxygen species. In vitro hemocompatibility and cell tests demonstrate the effectiveness of coatings in inhibiting thrombus and inflammation. Interestingly, this study originally reveals a novel strategy that the anti-fouling plus coating can direct vascular tissue healing on stent via promoted endothelialization and mediated contractile smooth muscle cell phenotype by switching inflammatory cells to a pro-repair phenotype during tissue healing process, even if the coating is relatively bio-inert.

Abstract P1018: Peptidyl-prolyl Cis-trans Isomerase-like 4 Is A Novel Regulator Of Smooth Muscle Cell Remodeling

Introduction: Smooth muscle cell (SMC) remodeling is a complex and multifaceted process that plays an essential role in the maintenance and function of various organ systems in the body. In response to physiological and pathological stimuli, smooth muscle cells exhibit remarkable plasticity, undergoing morphological and functional changes that can impact vascular tone. Dysregulation of SMC remodeling can contribute to the development and progression of numerous pathological conditions, such as hypertension and atherosclerosis. However, the role of SMC remodeling in intracranial aneurysm (IA) formation has yet to be explored. Objective-Method: In this study, we aimed to elucidate the role of PPIL4, an IA-implicated gene, in SMC remodeling using a targeted experimental approach. We performed immunostaining and bulk RNA sequencing (RNA-seq) in human primary brain vascular smooth muscle cells (HPBVSMC) treated with lentiviral shRNA and open-reading-frame (ORF) particles to individually downregulate and overexpress PPIL4, respectively. Results: We found that PPIL4 downregulation results in significant upregulation of the genes encoding contractile proteins, including ACTA2, CNN1, TNNT1, and TNNT2, in HPBVSMC and leads to a significantly increased cell size, favoring a contractile SMC phenotype. Conversely, overexpressing PPIL4 using ORF-lentiviral particles caused downregulation of contractile proteins, increased expression of MMP1 and MMP3, and promoted a synthetic phenotype in SMCs. Furthermore, PPIL4 overexpression caused cell elongation and spindle-like cell formation in HPBVSMC. Conclusion: In conclusion, our findings highlight the critical role of PPIL4 in modulating SMC remodeling, suggesting a potential mechanistic link between PPIL4 dysregulation, SMC remodeling, and IA formation, which warrants further investigation.

Stable hydrogel adhesion to polydimethylsiloxane enables cyclic mechanical stimulation of 3D-bioprinted smooth muscle constructs.

During normal urination, smooth muscle cells (SMCs) in the lower urinary tract (LUT) are exposed to mechanical signals that have a critical impact on tissue structure and function. Nevertheless, the mechanisms underlying the maintenance of the contractile phenotype of SMCs remain poorly understood. This is due, in part, to a lack of studies that have examined the effects of mechanical loading using three-dimensional (3D) models. In this study, surface modifications of polydimethylsiloxane (PDMS) membrane were evaluated to investigate the effects of cyclic mechanical stimulation on SMC maturation in 3D constructs. Commercially available cell stretching plates were modified with amino or methacrylate groups to promote adhesion of 3D constructs fabricated by bioprinting. After 6 days of stimulation, the effects of mechanical stimulation on the expression of contractile markers at the mRNA and protein levels were analyzed. Methacrylate-modified surfaces supported stable adhesion of the 3D constructs to the membrane and facilitated cyclic mechanical stimulation, which significantly increased the expression of contractile markers at the mRNA and protein levels. These effects were found to be mediated by activation of the p38 MAPK pathway, as inhibition of this pathway abolished the effects of stimulation in a dose-dependent manner. These results provide valuable insights into the role of mechanical signaling in maintaining the contractile phenotype of bladder SMCs, which has important implications for the development of future treatments for LUT diseases.

Glut10 restrains neointima formation by promoting SMCs mtDNA demethylation and improving mitochondrial function

Neointimal hyperplasia is a major clinical complication of coronary artery bypass graft and percutaneous coronary intervention. Smooth muscle cells (SMCs) play a vital roles in neointimal hyperplasia development and undergo complex phenotype switching. Previous studies have linked glucose transporter member 10(Glut10) to the phenotypic transformation of SMCs. In this research, we reported that Glut10 helps maintain the contractile phenotype of SMCs. The Glut10-TET2/3 signaling axis can arrest neointimal hyperplasia progression by improving mitochondrial function via promotion of mtDNA demethylation in SMCs. Glut10 is significantly downregulated in both human and mouse restenotic arteries. Global Glut10 deletion or SMC-specific Glut10 ablation in the carotid artery of mice accelerated neointimal hyperplasia, while Glut10 overexpression in the carotid artery triggered the opposite effects. All of these changes were accompanied by a significant increase in vascular SMCs migration and proliferation. Mechanistically, Glut10 is expressed primarily in the mitochondria after platelet-derived growth factor-BB (PDGF-BB) treatment. Glut10 ablation induced a reduction in ascorbic acid (VitC) concentrations in mitochondria and mitochondrial DNA (mtDNA) hypermethylation by decreasing the activity and expression of the Ten-eleven translocation (TET) protein family. We also observed that Glut10 deficiency aggravated mitochondrial dysfunction and decreased the adenosinetriphosphate (ATP) content and the oxygen consumption rate, which also caused SMCs to switch their phenotype from contractile to synthetic phenotype. Furthermore, mitochondria-specific TET family inhibition partially reversed these effects. These results suggested that Glut10 helps maintain the contractile phenotype of SMCs. The Glut10-TET2/3 signaling axis can arrest neointimal hyperplasia progression by improving mitochondrial function via the promotion of mtDNA demethylation in SMCs.

The Long Noncoding RNA Cardiac Mesoderm Enhancer-Associated Noncoding RNA (Carmn) Is a Critical Regulator of Gastrointestinal Smooth Muscle Contractile Function and Motility

Visceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract that regulate GI motility. SMC contraction is regulated by posttranslational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality, but the mechanisms regulating SMC-specific contractile gene expression, including the role of long noncoding RNAs (lncRNAs), remain largely unexplored. Herein, we reveal a critical role of Carmn (cardiac mesoderm enhancer-associated noncoding RNA), an SMC-specific lncRNA, in regulating visceral SMC phenotype and contractility of the GI tract. Genotype-Tissue Expression and publicly available single-cell RNA sequencing (scRNA-seq) data sets from embryonic, adult human, and mouse GI tissues were interrogated to identify SMC-specific lncRNAs. The functional role of Carmn was investigated using novel green fluorescent protein (GFP) knock-in (KI) reporter/knock-out (KO) mice. Bulk RNA-seq and single nucleus RNA sequencing (snRNA-seq) of colonic muscularis were used to investigate underlying mechanisms. Unbiased in silico analyses and GFP expression patterns in Carmn GFP KI mice revealed that Carmn is highly expressed in GI SMCs in humans and mice. Premature lethality was observed in global Carmn KO and inducible SMC-specific KO mice due to GI pseudo-obstruction and severe distension of the GI tract, with dysmotility in cecum and colon segments. Histology, GI transit, and muscle myography analysis revealed severe dilation, significantly delayed GI transit, and impaired GI contractility in Carmn KO vs control mice. Bulk RNA-seq of GI muscularis revealed that loss of Carmn promotes SMC phenotypic switching, as evidenced by up-regulation of extracellular matrix genes and down-regulation of SMC contractile genes, including Mylk, a key regulator of SMC contraction. snRNA-seq further revealed SMC Carmn KO not only compromised myogenic motility by reducing contractile gene expression but also impaired neurogenic motility by disrupting cell-cell connectivity in the colonic muscularis. These findings may have translational significance, because silencing CARMN in human colonic SMCs significantly attenuated contractile gene expression, including MYLK, and decreased SMC contractility. Luciferase reporter assays showed that CARMN enhances the transactivation activity of the master regulator of SMC contractile phenotype, myocardin, thereby maintaining the GI SMC myogenic program. Our data suggest that Carmn is indispensable for maintaining GI SMC contractile function in mice and that loss of function of CARMN may contribute to human visceral myopathy. To our knowledge this is the first study showing an essential role of lncRNA in the regulation of visceral SMC phenotype.

Trehalose attenuates abdominal aortic aneurysm formation by inducing autophagy in smooth muscle cells.

Trehalose is a naturally occurring disaccharide, which has been identified as an autophagy inducer and exhibits protective effect in cardiovascular diseases such as myocardial infraction and atherosclerosis. However, the functional role of trehalose in abdominal aortic aneurysm (AAA) remains undefined. To study the effect of trehalose in AAA, trehalose (1 g/kg per day) were given for 14 continuous days in a mouse model of elastase-induced abdominal aortic aneurysm. On day 14, ultrasound was performed to measure aortic diameter before the abdominal aortas were harvested and processed for further analysis. Verhoeff-Van Gieson staining and TUNEL staining were performed on paraffin sections to evaluate vascular histology and apoptosis, immunofluorescence staining and Western-blot were performed to evaluate expression of autophagy markers. Echocardiography and in situ pictures demonstrated that trehalose attenuated infrarenal aorta dilation. Verhoeff-Van Gieson staining showed elastin degradation was improved in trehalose-treated group. Compared with vehicle-treated mice, trehalose treatment restored smooth muscle cell contractile phenotype with increased α-SMA, Calponin and Myh11 expression. Furthermore, trehalose also attenuated cell apoptosis and leukocytes infiltration. Importantly, trehalose induced autophagy with decrease SQSTM1/p62 accumulation, increased lamp2 expression and LC3B conversion. Trehalose attenuated AAA progression with decreased inflammation and restored SMC contractile phenotype by inducing autophagy. These results demonstrated the therapeutic potential of trehalose in AAA.

Tailoring Hydrogel Composition and Stiffness to Control Smooth Muscle Cell Differentiation in Bioprinted Constructs.

Reliable in vitro cellular models are needed to study the phenotypic modulation of smooth muscle cells (SMCs) in health and disease. The aim of this study was to optimize gelatin methacrylate (GelMA)/alginate hydrogels for bioprinting three-dimensional (3D) SMC constructs. Four different hydrogel groups were prepared by mixing different concentrations (% w/v) of GelMA and alginate: G1 (5/1.5), G2 (5/3), G3 (7.5/1.5), and G4 (7.5/3). GelMA 10% was used as control (G5). A circular structure containing human bladder SMCs was fabricated by using an extrusion-based bioprinter. The effects of the mixing ratios on printability, viability, proliferation, and differentiation of the cells were investigated. Rheological analysis showed that the addition of alginate significantly stabilized the change in mechanical properties with temperature variations. The group with the highest GelMA and alginate concentrations (G4) exhibited the highest viscosity, resulting in better stability of the 3D construct after crosslinking. Compared to other hydrogel compositions, cells in G4 maintained high viability (> 80%), exhibited spindle-shaped morphology, and showed a significantly higher proliferation rate within an 8-day period. More importantly, G4 provided an optimal environment for the induction of a SMC contractile phenotype, as evidenced by significant changes in the expression of marker proteins and morphological parameters. Adjusting the composition of GelMA/alginate hydrogels is an effective means of controlling the SMC phenotype. These hydrogels support bioprinting of 3D models to study phenotypic smooth muscle adaptation, with the prospect of using the constructs in the study of therapies for the treatment of urethral strictures.

Construction of a high-throughput aorta smooth muscle-on-a-chip for thoracic aortic aneurysm drug screening

Thoracic aortic aneurysm (TAA), in which arteries enlarge asymptomatically over time until dissection or rupture occurs, is a serious health risk. The mainstay of TAA treatment remains surgical repair due to the lack of effective drugs. The complex etiology and pathogenesis of TAA, including hemodynamic alterations and genetic factors, lead to inaccuracies in preclinical models for drug screening. Previously, our group designed an aorta smooth muscle-on-a-chip to emulate human aorta physiology and pathophysiology and screened three promising therapeutic drugs targeting mitochondrial dynamics in TAA. On this foundation, we updated the one-channel chip to an eighteen-well chip platform with four polydimethylsiloxane layers. Benefiting from this high-throughput chip, we rapidly screened multiple drugs simultaneously using distinct cell lines in vitro. In addition, we observed the abnormal activation of hypoxia-inducible factor 1-alpha (HIF-1alpha) in aortas from TAA patients by Western blot and bioinformatics analyses. Intriguingly, this phenomenon was replicated only when smooth muscle cells (SMCs) were strained on the chip. We then screened seven specific HIF-1alpha inhibitors and selected the two most effective drugs (2-methoxyestradiol and digoxin) by quantitative PCR and colorimetric methods. The results demonstrated that these two drugs can improve respiratory chain function and rescue the SMC contractile phenotype, showing applicability for the clinical treatment of TAA. This high-throughput aorta smooth muscle-on-a-chip will become a potential preclinical model for TAA drug screening.

Bves maintains vascular smooth muscle cell contractile phenotype and protects against transplant vasculopathy via Dusp1-dependent p38MAPK and ERK1/2 signaling

Background and aimsVascular smooth muscle cell (VSMC) plasticity is tightly associated with the pathological process of vasculopathy. Blood vessel epicardial substance (Bves) has emerged as an important regulator of intracardiac vasculogenesis and organ homeostasis. However, the involvement and role of Bves in VSMC plasticity and neointimal lesion development remain unclear. MethodsWe used an in vivo rat model of graft arteriosclerosis and in vitro PDGF-treated VSMCs and identified the novel VSMC contractile phenotype-related gene Bves using a transcriptomic analysis and literature search. In vitro knockdown and overexpression approaches were used to investigate the mechanisms underlying VSMC phenotypic plasticity. In vivo, VSMC-specific Bves overexpression in rat aortic grafts was generated to assess the physiological function of Bves in neointimal lesion development. ResultsHere, we found that Bves expression was negatively regulated in aortic allografts in vivo and PDGF-treated VSMCs in vitro. The genetic knockdown of Bves dramatically inhibited, whereas Bves overexpression markedly promoted, the VSMC contractile phenotype. Furthermore, RNA sequencing unraveled a positive correlation between Bves and dual-specificity protein phosphatase 1 (Dusp1) expression in VSMCs. We found that Bves knockdown restrained Dusp1 expression, but enhanced p38MAPK and ERK1/2 activation, resulting in the loss of the VSMC contractile phenotype. In vivo, an analysis of a rat graft model confirmed that VSMC-specific Bves and Dusp1 overexpression in aortic allografts significantly attenuated neointimal lesion formation. ConclusionsBves maintains the VSMC contractile phenotype through Dusp1-dependent p38MAPK and ERK1/2 signaling, and protects against neointimal formation, underscoring the important role of Bves in preventing transplant vasculopathy.

Icariside II Restores Vascular Smooth Muscle Cell Contractile Phenotype by Enhancing the Focal Adhesion Signaling Pathway in the Rat Vascular Remodeling Model

Vascular smooth muscle cell (VSMC) phenotypic transition represents the fundamental pathophysiological alteration in the vascular remodeling process during the initiation and progression of cardiovascular diseases. Recent studies have revealed that Icariside II (ICS-II), a flavonol glycoside derived from the traditional Chinese medicine Herba Epimedii, exhibited therapeutic effects in various cardiovascular diseases. However, the therapeutic efficacy and underlying mechanisms of ICS-II regarding VSMC phenotypic transition were unknown. In this study, we investigated the therapeutic effects of ICS-Ⅱ on vascular remodeling with a rat’s balloon injury model in vivo. The label-free proteomic analysis was further implemented to identify the differentially expressed proteins (DEPs) after ICS-II intervention. Gene ontology and the pathway enrichment analysis were performed based on DEPs. Moreover, platelet-derived growth factor (PDGF-BB)-induced primary rat VSMC was implemented to verify the restoration effects of ICS-II on the VSMC contractile phenotype. Results showed that ICS-II could effectively attenuate the vascular remodeling process, promote SMA-α protein expression, and inhibit OPN expression in vivo. The proteomic analysis identified 145 differentially expressed proteins after ICS-II intervention. Further, the bioinformatics analysis indicated that the focal adhesion signaling pathway was enriched in the ICS-II group. In vitro studies showed that ICS-II suppressed VSMC proliferation and migration, and promoted VSMC contractile phenotype by modulating the focal adhesion signaling pathway. Taken together, our results suggest that ICS-II attenuates the vascular remodeling process and restores the VSMC contractile phenotype by promoting the focal adhesion pathway.

A Compressed Collagen Construct for Studying Endothelial-Smooth Muscle Cell Interaction Under High Shear Stress.

The coculture of vascular endothelial cells (ECs) on collagen gels containing smooth muscle cells (SMCs) has been carried out to investigate cellular interactions associated with blood vessel pathophysiology under wall shear stress (WSS) conditions. However, due to a lack of gel stiffness, the previous collagen gel coculture constructs are difficult to use for pathologic higher WSS conditions. Here, we newly constructed a coculture model with centrifugally compressed cell-collagen combined construct (C6), which withstands higher WSS conditions. The elastic modulus of C6 was approximately 6 times higher than that of the uncompressed collagen construct. The level of α-smooth muscle actin, a contractile SMC phenotype marker observed in healthy arteries, was elevated in C6 compared with that of the uncompressed construct, and further increased by exposure to a physiological level WSS of 2 Pa, but not by a pathological level of 20 Pa. WSS conditions of 2 and 20 Pa also induced different expression ratios of matrix metalloproteinases and their inhibitors in the C6 coculture model but did not in monocultured ECs and SMCs. The C6 coculture model will be a powerful tool to investigate interactions between ECs and SMCs under pathologically high WSS conditions.

Hexarelin attenuates abdominal aortic aneurysm formation by inhibiting SMC phenotype switch and inflammasome activation.

Hexarelin, a synthetic growth hormone-releasing peptide, is shown to be protective in cardiovascular diseases such as myocardial infraction and atherosclerosis. However, the functional role of hexarelin in abdominal aortic aneurysm (AAA) remains undefined. The present study determined the effect of hexarelin administration (200μg/kg twice per day) in a mouse model of elastase-induced abdominal aortic aneurysm. Echocardiography and in situ pictures showed hexarelin decreased infrarenal aorta diameter. Histology staining showed elastin degradation was improved in hexarelin-treated group. Hexarelin rescued smooth muscle cell contractile phenotype with increased α-SMA and decreased MMP2. Furthermore, hexarelin inhibited inflammatory cell infiltration, NLRP3 inflammasome activation and IL-18 production. Particularly, hexarelin suppressed NF-κB signaling pathway which is a key initiator of inflammatory response. These results demonstrated that hexarelin attenuated AAA development by inhibiting SMC phenotype switch and NF-κB signaling mediated inflammatory response.

Engagement of the CXCL12-CXCR4 Axis in the Interaction of Endothelial Progenitor Cell and Smooth Muscle Cell to Promote Phenotype Control and Guard Vascular Homeostasis.

Endothelial progenitor cells (EPCs) are involved in vascular repair and modulate properties of smooth muscle cells (SMCs) relevant for their contribution to neointima formation following injury. Considering the relevant role of the CXCL12–CXCR4 axis in vascular homeostasis and the potential of EPCs and SMCs to release CXCL12 and express CXCR4, we analyzed the engagement of the CXCL12–CXCR4 axis in various modes of EPC–SMC interaction relevant for injury- and lipid-induced atherosclerosis. We now demonstrate that the expression and release of CXCL12 is synergistically increased in a CXCR4-dependent mechanism following EPC–SMC interaction during co-cultivation or in response to recombinant CXCL12, thus establishing an amplifying feedback loop Additionally, mechanical injury of SMCs induces increased release of CXCL12, resulting in enhanced CXCR4-dependent recruitment of EPCs to SMCs. The CXCL12–CXCR4 axis is crucially engaged in the EPC-triggered augmentation of SMC migration and the attenuation of SMC apoptosis but not in the EPC-mediated increase in SMC proliferation. Compared to EPCs alone, the alliance of EPC–SMC is superior in promoting the CXCR4-dependent proliferation and migration of endothelial cells. When direct cell–cell contact is established, EPCs protect the contractile phenotype of SMCs via CXCL12–CXCR4 and reverse cholesterol-induced transdifferentiation toward a synthetic, macrophage-like phenotype. In conclusion we show that the interaction of EPCs and SMCs unleashes a CXCL12–CXCR4-based autoregulatory feedback loop promoting regenerative processes and mediating SMC phenotype control to potentially guard vascular homeostasis.

Complex role of RhoA in regulating vascular smooth muscle cell phenotypes in type 2 diabetes

The incidence of type 2 diabetes mellitus (T2DM) is growing globally, and the major cause of morbidity and mortality in these patients is the premature development of cardiovascular disease. Consequently, medical interventions, such as coronary artery bypass graft surgery and widespread statin prescriptions, are common in this patient group. Smooth muscle cells are the major structural component of the vascular wall. They play a crucial role in post-bypass recovery to successfully revascularize the heart by switching between differentiated (contractile) and dedifferentiated (synthetic) phenotypes. However, in patients with T2DM, these cells have functional defects that may affect bypass integration. RhoA is a small GTPase that regulates many functions, such as motility and phenotypic regulation of smooth muscle cells. RhoA is dependent upon a stimulus, and it can drive the contractile smooth muscle cell phenotype present in the healthy condition or the (mal)adaptive phenotypes prevalent in disease or in response to injury. We hypothesize that RhoA deregulation plays a major role in vascular complications of T2DM. This protein is deregulated in T2DM smooth muscle cells, which may in part explain the functional defects of smooth muscle tissue and the subsequent failure rate of bypass in these patients. An important consideration in this circumstance is the use of statin therapies because these further inhibit RhoA activity. The effect of inhibition of RhoA activity in patients with T2DM who have a bypass is currently unknown.