RÉSUMÉ
This study explored the effect of Tianma Gouteng Decoction on oxidative stress induced by angiotensin â ¡(Angâ ¡) in vascular smooth muscle cell(VSMC) and its molecular mechanism. Primary rat VSMC were cultured using tissue block method, and VSMC were identified by α-actin immunofluorescence staining. Angâ ¡ at a concentration of 1×10~(-6) mol·L~(-1) was used as the stimulating factor, and Sprague Dawley(SD) rats were orally administered with Tianma Gouteng Decoction to prepare drug serum. Rat VSMC were divided into normal group, model group, Chinese medicine group, and inhibitor(3-methyladenine, 3-MA) group. Cell counting kit-8(CCK-8) assay was used to detect cell proliferation activity. Bromodeoxyuridine(BrdU) flow cytometry was used to detect cell cycle. Transwell assay was used to detect cell migration ability. Enzyme-linked immunosorbent assay(ELISA) was used to detect the activity of superoxide dismutase(SOD), catalase(CAT), and malondialdehyde(MDA) in VSMC. The intracellular reactive oxygen species(ROS) fluorescence intensity was detected using DCFH-DA fluorescent probe. Western blot was used to detect the expression of PTEN-induced putative kinase 1(PINK1), Parkin, p62, and microtubule-associated protein 1A/1B-light chain 3(LC3-â ¡) proteins in VSMC. The results showed that Tianma Gouteng Decoction-containing serum at a concentration of 8% could significantly inhibit VSMC growth after 48 hours of intervention. Compared with the normal group, the model group showed significantly increased cell proliferation activity and migration, significantly decreased levels of SOD and CAT, significantly increased levels of MDA, significantly enhanced ROS fluorescence intensity, significantly decreased expression of PINK1, Parkin, and LC3-â ¡ proteins, and significantly increased expression of p62 protein. Compared with the model group, the Chinese medicine group showed significantly reduced cell proliferation activity and migration, significantly increased levels of SOD and CAT, significantly decreased levels of MDA, significantly weakened ROS fluorescence intensity, significantly increased expression of PINK1, Parkin, and LC3-â ¡ proteins, and significantly decreased expression of p62 protein. Compared with the Chinese medicine group, the addition of the mitochondrial autophagy inhibitor 3-MA could block the intervention of Tianma Gouteng Decoction-containing serum on VSMC proliferation, migration, mitochondrial autophagy, and oxidative stress levels, with statistically significant differences. In summary, Tianma Gouteng Decoction has good antioxidant activity and can inhibit cell proliferation and migration. Its mechanism of action may be related to the activation of the mitochondrial autophagy PINK1/Parkin signaling pathway.
Sujet(s)
Angiotensine-II , Prolifération cellulaire , Médicaments issus de plantes chinoises , Muscles lisses vasculaires , Stress oxydatif , Protein kinases , Rat Sprague-Dawley , Ubiquitin-protein ligases , Animaux , Médicaments issus de plantes chinoises/pharmacologie , Stress oxydatif/effets des médicaments et des substances chimiques , Rats , Muscles lisses vasculaires/effets des médicaments et des substances chimiques , Muscles lisses vasculaires/cytologie , Muscles lisses vasculaires/métabolisme , Mâle , Prolifération cellulaire/effets des médicaments et des substances chimiques , Ubiquitin-protein ligases/métabolisme , Ubiquitin-protein ligases/génétique , Protein kinases/métabolisme , Protein kinases/génétique , Myocytes du muscle lisse/effets des médicaments et des substances chimiques , Myocytes du muscle lisse/métabolisme , Espèces réactives de l'oxygène/métabolisme , Mouvement cellulaire/effets des médicaments et des substances chimiques , Transduction du signal/effets des médicaments et des substances chimiques , Cellules cultivées , Superoxide dismutase/métabolismeRÉSUMÉ
Glycolysis is a major determinant of pulmonary artery smooth muscle cell (PASMC) proliferation in pulmonary hypertension (PH). Circular RNAs (circRNAs) are powerful regulators of glycolysis in multiple diseases; however, the role of circRNAs in glycolysis in PH has been poorly characterized. The aim of this study was to uncover the regulatory mechanism of a new circRNA, circNAP1L4, in human pulmonary artery smooth muscle cell (HPASMC) proliferation through the host protein NAP1L4 to regulate the super-enhancer-driven glycolysis gene hexokinase II (HK II). CircNAP1L4 was downregulated in hypoxic HPASMCs and plasma of PH patients. Functionally, circNAP1L4 overexpression inhibited glycolysis and proliferation in hypoxic HPASMCs. Mechanistically, circNAP1L4 directly bound to its host protein NAP1L4 and affected the ability of NAP1L4 to move into the nucleus to regulate the epigenomic signals of the super-enhancer of HK II. Intriguingly, circNAP1L4 overexpression inhibited the proliferation but not the migration of human pulmonary arterial endothelial cells (HPAECs) cocultured with HPASMCs. Furthermore, pre-mRNA-processing-splicing Factor 8 (PRP8) was found to regulate the production ratio of circNAP1L4 and linear NAP1L4. In vivo, targeting circNAP1L4 alleviates SU5416 combined with hypoxia (SuHx)-induced PH. Overall, these findings reveal a new circRNA that inhibits PASMC proliferation and serves as a therapeutic target for PH.
Sujet(s)
Prolifération cellulaire , Glycolyse , Hexokinase , Hypertension pulmonaire , Myocytes du muscle lisse , Artère pulmonaire , ARN circulaire , Humains , Hexokinase/métabolisme , Hexokinase/génétique , Artère pulmonaire/métabolisme , Artère pulmonaire/anatomopathologie , Hypertension pulmonaire/métabolisme , Hypertension pulmonaire/génétique , Myocytes du muscle lisse/métabolisme , ARN circulaire/génétique , ARN circulaire/métabolisme , Animaux , Souris , Mâle , Cellules cultivées , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/cytologieRÉSUMÉ
Pulmonary hypertension (PH) arises from increased pulmonary vascular resistance due to contraction and remodeling of the pulmonary arteries. The structural changes include thickening of the smooth muscle layer from increased proliferation and resistance to apoptosis. The mechanisms underlying apoptosis resistance in PH are not fully understood. In cancer cells, high expression of aquaporin 1 (AQP1), a water channel, is associated with apoptosis resistance. We showed AQP1 protein was expressed in pulmonary arterial smooth muscle cells (PASMCs) and upregulated in preclinical PH models. In this study, we used PASMCs isolated from control male rats and the SU5416 plus hypoxia (SuHx) model to test the role of AQP1 in modulating susceptibility to apoptosis. We found the elevated level of AQP1 in PASMCs from SuHx rats was necessary for resistance to apoptosis and that apoptosis resistance could be conferred by increasing AQP1 in control PASMCs. In exploring the downstream pathways involved, we found AQP1 levels influence the expression of Bcl-2, with enhanced AQP1 levels corresponding to increased Bcl-2 expression, reducing the ratio of BAX to Bcl-2, consistent with apoptosis resistance. These results provide a mechanism by which AQP1 can regulate PASMC fate.
Sujet(s)
Apoptose , Aquaporine-1 , Hypoxie , Indoles , Muscles lisses vasculaires , Myocytes du muscle lisse , Artère pulmonaire , Pyrroles , Animaux , Aquaporine-1/métabolisme , Aquaporine-1/génétique , Mâle , Artère pulmonaire/métabolisme , Artère pulmonaire/anatomopathologie , Artère pulmonaire/cytologie , Rats , Myocytes du muscle lisse/métabolisme , Myocytes du muscle lisse/anatomopathologie , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/anatomopathologie , Muscles lisses vasculaires/cytologie , Pyrroles/pharmacologie , Indoles/pharmacologie , Hypoxie/métabolisme , Rat Sprague-Dawley , Hypertension pulmonaire/métabolisme , Hypertension pulmonaire/anatomopathologie , Cellules cultivées , Protéines proto-oncogènes c-bcl-2/métabolisme , Protéines proto-oncogènes c-bcl-2/génétique , Modèles animaux de maladie humaineRÉSUMÉ
The regulatory factor X7 (RFX7) is a vital mediator in atherosclerosis. This study aims to discuss the effect and underlying mechanism of RFX7 on the regulation of oxidized low-density lipoprotein (ox-LDL) -induced proliferation and migration of vascular smooth muscle cells (VSMCs).Ox-LDL was used to construct atherosclerosis in vitro model. The mRNA and protein levels of RFX7 and Sirtuin 4 (SIRT4) were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot assays. The cellular functions were measured via 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT), EdU, flow cytometry, and wound healing assay assays. The interaction between RFX7 and SIRT4 promoter was validated using chromatin immunoprecipitation and dual-luciferase reporter assays.The stimulation with ox-LDL elevated the viability of VSMCs and decreased the mRNA and protein levels of RFX7 and SIRT4 in VSMCs in a dose-dependent manner. Functionally, RFX7 overexpression restrained the VSMC viability, proliferation, and migration induced by ox-LDL, but facilitated VSMC apoptosis. RFX7 elevated SIRT4 expression via binding to its promoter. Furthermore, overexpressing either SIRT4 or RFX7 inactivated JAK2/STAT3 signaling, causing a decrease in VSMC proliferation and migration and an increase in VSMC apoptosis when exposed to ox-LDL. The impact of RFX7 overexpression on JAK2/STAT3 signaling and cellular function following ox-LDL exposure was abrogated by SIRT4 silencing.The heightened RFX7 expression restrained the proliferation and migration of ox-LDL-stimulated VSMCs via SIRT4-mediated inactivation of JAK2/STAT3 pathway.
Sujet(s)
Mouvement cellulaire , Prolifération cellulaire , Kinase Janus-2 , Lipoprotéines LDL , Muscles lisses vasculaires , Facteur de transcription STAT-3 , Transduction du signal , Sirtuines , Facteur de transcription STAT-3/métabolisme , Lipoprotéines LDL/pharmacologie , Lipoprotéines LDL/métabolisme , Kinase Janus-2/métabolisme , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/cytologie , Humains , Sirtuines/métabolisme , Sirtuines/génétique , Athérosclérose/métabolisme , Cellules cultivées , Myocytes du muscle lisse/métabolisme , Myocytes du muscle lisse/effets des médicaments et des substances chimiques , Protéines mitochondrialesRÉSUMÉ
An intricate reciprocal relationship exists between adherent synthetic cells and their extracellular matrix (ECM). These cells deposit, organize, and degrade the ECM, which in turn influences cell phenotype via responses that include sensitivity to changes in the mechanical state that arises from changes in external loading. Collagen-based tissue equivalents are commonly used as simple but revealing model systems to study cell-matrix interactions. Nevertheless, few quantitative studies report changes in the forces that the cells establish and maintain in such gels under dynamic loading. Moreover, most prior studies have been limited to uniaxial experiments despite many soft tissues, including arteries, experiencing multiaxial loading in vivo. To begin to close this gap, we use a custom biaxial bioreactor to subject collagen gels seeded with primary aortic smooth muscle cells to different biaxial loading conditions. These conditions include cyclic loading with different amplitudes as well as different mechanical constraints at the boundaries of a cruciform sample. Irrespective of loading amplitude and boundary condition, similar mean steady-state biaxial forces emerged across all tests. Additionally, stiffness-force relationships assessed via intermittent equibiaxial force-extension tests showed remarkable similarity for ranges of forces to which the cells adapted during periods of cyclic loading. Taken together, these findings are consistent with a load-mediated homeostatic response by vascular smooth muscle cells.
Sujet(s)
Muscles lisses vasculaires , Myocytes du muscle lisse , Muscles lisses vasculaires/cytologie , Muscles lisses vasculaires/métabolisme , Animaux , Phénomènes biomécaniques , Myocytes du muscle lisse/cytologie , Myocytes du muscle lisse/métabolisme , Ingénierie tissulaire , Contrainte mécanique , Phénomènes mécaniques , Collagène/métabolisme , Bioréacteurs , Mise en charge , Matrice extracellulaire/métabolisme , Rats , Test de matériaux , Aorte/cytologieRÉSUMÉ
Yohimbine (YHB) has been reported to possess anti-inflammatory, anticancer, and cardiac function-enhancing properties. Additionally, it has been reported to inhibit the proliferation, migration, and neointimal formation of vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor (PDGF) stimulation by suppressing the phospholipase C-gamma 1 pathway. However, the transcriptional regulatory mechanism of YHB controlling the behavior of VSMCs is not fully understood. In this study, YHB downregulated the expression of cell cycle regulatory proteins, such as proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4), and cyclin E, by modulating the transcription factor FOXO3a in VSMCs induced by PDGF. Furthermore, YHB decreased p-38 and mTOR phosphorylation in a dose-dependent manner. Notably, YHB significantly reduced the phosphorylation at Y397 and Y925 sites of focal adhesion kinase (FAK), and this effect was greater at the Y925 site than Y397. In addition, the expression of paxillin, a FAK-associated protein known to bind to the Y925 site of FAK, was significantly reduced by YHB treatment in a dose-dependent manner. A pronounced reduction in the migration and proliferation of VSMCs was observed following co-treatment of YHB with mTOR or p38 inhibitors. In conclusion, this study shows that YHB inhibits the PDGF-induced proliferation and migration of VSMCs by regulating the transcription factor FOXO3a and the mTOR/p38/FAK signaling pathway. Therefore, YHB may be a potential therapeutic candidate for preventing and treating cardiovascular diseases such as atherosclerosis and vascular restenosis.
Sujet(s)
Mouvement cellulaire , Prolifération cellulaire , Protéine O3 à motif en tête de fourche , Muscles lisses vasculaires , Myocytes du muscle lisse , Facteur de croissance dérivé des plaquettes , Yohimbine , Muscles lisses vasculaires/cytologie , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/effets des médicaments et des substances chimiques , Protéine O3 à motif en tête de fourche/métabolisme , Prolifération cellulaire/effets des médicaments et des substances chimiques , Mouvement cellulaire/effets des médicaments et des substances chimiques , Animaux , Facteur de croissance dérivé des plaquettes/métabolisme , Facteur de croissance dérivé des plaquettes/pharmacologie , Myocytes du muscle lisse/métabolisme , Myocytes du muscle lisse/effets des médicaments et des substances chimiques , Phosphorylation/effets des médicaments et des substances chimiques , Yohimbine/pharmacologie , Rats , Transduction du signal/effets des médicaments et des substances chimiques , Sérine-thréonine kinases TOR/métabolisme , p38 Mitogen-Activated Protein Kinases/métabolisme , Focal adhesion kinase 1/métabolisme , Cellules cultivées , Paxilline/métabolisme , Rat Sprague-Dawley , MâleRÉSUMÉ
It has been reported that, in the spontaneously hypertensive rat (SHR) model of hypertension, different components of the G-protein/adenylate cyclase (AC)/Calcium-activated potassium channel of high conductance (BK) channel signaling pathway are altered differently. In the upstream part of the pathway (G-protein/AC), a comparatively low efficacy has been established, whereas downstream BK currents seem to be increased. Thus, the overall performance of this signaling pathway in SHR is elusive. For a better understanding, we focused on one aspect, the direct targeting of the BK channel by the G-protein/AC pathway and tested the hypothesis that the comparatively low AC pathway efficacy in SHR results in a reduced agonist-induced stimulation of BK currents. This hypothesis was investigated using freshly isolated smooth muscle cells from WKY and SHR rat tail artery and the patch-clamp technique. It was observed that: (1) single BK channels have similar current-voltage relationships, voltage-dependence and calcium sensitivity; (2) BK currents in cells with a strong buffering of the BK channel activator calcium have similar current-voltage relationships; (3) the iloprost-induced concentration-dependent increase of the BK current is larger in WKY compared to SHR; (4) the effects of activators of the PKA pathway, the catalytic subunit of PKA and the potent and selective cAMP-analogue Sp-5,6-DCl-cBIMPS on BK currents are similar. Thus, our data suggest that the lower iloprost-induced stimulation of the BK current in freshly isolated rat tail artery smooth muscle cells from SHR compared with WKY is due to the lower efficacy of upstream elements of the G-Protein/AC/BK channel pathway.
Sujet(s)
Calcium , Hypertension artérielle , Iloprost , Canaux potassiques calcium-dépendants de grande conductance , Muscles lisses vasculaires , Rats de lignée SHR , Rats de lignée WKY , Vasodilatateurs , Animaux , Canaux potassiques calcium-dépendants de grande conductance/métabolisme , Muscles lisses vasculaires/effets des médicaments et des substances chimiques , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/cytologie , Rats , Calcium/métabolisme , Iloprost/pharmacologie , Hypertension artérielle/métabolisme , Hypertension artérielle/traitement médicamenteux , Vasodilatateurs/pharmacologie , Myocytes du muscle lisse/métabolisme , Myocytes du muscle lisse/effets des médicaments et des substances chimiques , Mâle , Artères/effets des médicaments et des substances chimiques , Artères/métabolisme , Queue/vascularisation , Transduction du signal/effets des médicaments et des substances chimiquesRÉSUMÉ
Vascular calcification (VC) is a cardiovascular disease characterized by calcium salt deposition in vascular smooth muscle cells (VSMCs). Standard in vitro models used in VC investigations are based on VSMC monocultures under static conditions. Although these platforms are easy to use, the absence of interactions between different cell types and dynamic conditions makes these models insufficient to study key aspects of vascular pathophysiology. The present study aimed to develop a dynamic endothelial cell-VSMC co-culture that better mimics the in vivo vascular microenvironment. A double-flow bioreactor supported cellular interactions and reproduced the blood flow dynamic. VSMC calcification was stimulated with a DMEM high glucose calcification medium supplemented with 1.9 mM NaH2PO4/Na2HPO4 (1:1) for 7 days. Calcification, cell viability, inflammatory mediators, and molecular markers (SIRT-1, TGFß1) related to VSMC differentiation were evaluated. Our dynamic model was able to reproduce VSMC calcification and inflammation and evidenced differences in the modulation of effectors involved in the VSMC calcified phenotype compared with standard monocultures, highlighting the importance of the microenvironment in controlling cell behavior. Hence, our platform represents an advanced system to investigate the pathophysiologic mechanisms underlying VC, providing information not available with the standard cell monoculture.
Sujet(s)
Différenciation cellulaire , Techniques de coculture , Muscles lisses vasculaires , Myocytes du muscle lisse , Calcification vasculaire , Humains , Calcification vasculaire/métabolisme , Calcification vasculaire/anatomopathologie , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/anatomopathologie , Muscles lisses vasculaires/cytologie , Myocytes du muscle lisse/métabolisme , Myocytes du muscle lisse/anatomopathologie , Cellules cultivées , Survie cellulaire , Facteur de croissance transformant bêta-1/métabolisme , Sirtuine-1/métabolisme , Cellules endothéliales/métabolisme , Cellules endothéliales/anatomopathologie , BioréacteursRÉSUMÉ
Vascular smooth muscle cells (VSMCs) envelop vertebrate brain arteries and play a crucial role in regulating cerebral blood flow and neurovascular coupling. The dedifferentiation of VSMCs is implicated in cerebrovascular disease and neurodegeneration. Despite its importance, the process of VSMC differentiation on brain arteries during development remains inadequately characterized. Understanding this process could aid in reprogramming and regenerating dedifferentiated VSMCs in cerebrovascular diseases. In this study, we investigated VSMC differentiation on zebrafish circle of Willis (CoW), comprising major arteries that supply blood to the vertebrate brain. We observed that arterial specification of CoW endothelial cells (ECs) occurs after their migration from cranial venous plexus to form CoW arteries. Subsequently, acta2+ VSMCs differentiate from pdgfrb+ mural cell progenitors after they were recruited to CoW arteries. The progression of VSMC differentiation exhibits a spatiotemporal pattern, advancing from anterior to posterior CoW arteries. Analysis of blood flow suggests that earlier VSMC differentiation in anterior CoW arteries correlates with higher red blood cell velocity and wall shear stress. Furthermore, pulsatile flow induces differentiation of human brain PDGFRB+ mural cells into VSMCs, and blood flow is required for VSMC differentiation on zebrafish CoW arteries. Consistently, flow-responsive transcription factor klf2a is activated in ECs of CoW arteries prior to VSMC differentiation, and klf2a knockdown delays VSMC differentiation on anterior CoW arteries. In summary, our findings highlight blood flow activation of endothelial klf2a as a mechanism regulating initial VSMC differentiation on vertebrate brain arteries.
Sujet(s)
Différenciation cellulaire , Cercle artériel du cerveau , Hémodynamique , Muscles lisses vasculaires , Danio zébré , Animaux , Cercle artériel du cerveau/embryologie , Muscles lisses vasculaires/physiologie , Muscles lisses vasculaires/cytologie , Muscles lisses vasculaires/métabolisme , Humains , Myocytes du muscle lisse/physiologie , Myocytes du muscle lisse/métabolisme , Cellules endothéliales/physiologie , Cellules endothéliales/métabolismeRÉSUMÉ
Plasmid transfection in cells is widely employed to express exogenous proteins, offering valuable mechanistic insight into their function(s). However, plasmid transfection efficiency in primary vascular endothelial cells (ECs) and smooth muscle cells (SMCs) is restricted with lipid-based transfection reagents such as Lipofectamine. The STING pathway, activated by foreign DNA in the cytosol, prevents foreign gene expression and induces DNA degradation. To address this, we explored the potential of STING inhibitors on the impact of plasmid expression in primary ECs and SMCs. Primary human aortic endothelial cells (HAECs) were transfected with a bicistronic plasmid expressing cytochrome b5 reductase 4 (CYB5R4) and enhanced green fluorescent protein (EGFP) using Lipofectamine 3000. Two STING inhibitors, MRT67307 and BX795, were added during transfection and overnight post-transfection. As a result, MRT67307 significantly enhanced CYB5R4 and EGFP expression, even 24 hours after its removal. In comparison, MRT67307 pretreatment did not affect transfection, suggesting the inhibitor's effect was readily reversible. The phosphorylation of endothelial nitric oxide synthase (eNOS) at Serine 1177 (S1177) by vascular endothelial growth factor is essential for endothelial proliferation, migration, and survival. Using the same protocol, we transfected wild-type and phosphorylation-incapable mutant (S1177A) eNOS in HAECs. Both forms of eNOS localized on the plasma membrane, but only the wild-type eNOS was phosphorylated by vascular endothelial growth factor treatment, indicating normal functionality of overexpressed proteins. MRT67307 and BX795 also improved plasmid expression in human and rat aortic SMCs. In conclusion, this study presents a modification enabling efficient plasmid transfection in primary vascular ECs and SMCs, offering a favorable approach to studying protein function(s) in these cell types, with potential implications for other primary cell types that are challenging to transfect.
Sujet(s)
Cellules endothéliales , Protéines membranaires , Plasmides , Transfection , Humains , Plasmides/génétique , Protéines membranaires/génétique , Protéines membranaires/métabolisme , Cellules endothéliales/métabolisme , Cellules endothéliales/cytologie , Myocytes du muscle lisse/métabolisme , Myocytes du muscle lisse/cytologie , Protéines à fluorescence verte/génétique , Protéines à fluorescence verte/métabolisme , Animaux , Nitric oxide synthase type III/génétique , Nitric oxide synthase type III/métabolisme , Cellules cultivées , Phosphorylation , Rats , Expression des gènes , Muscles lisses vasculaires/cytologie , Muscles lisses vasculaires/métabolismeRÉSUMÉ
Vascular smooth muscle cell (VSMCs) is one of the important cell types in artery. VSMCs stiffening may regulate vascular stiffness and contribute to the development of vulnerable plaques. Thrombin, an enzyme in coagulation system, is involved in pathological processes of atherosclerosis. Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4) plays an important role in regulating inflammation and may have cardiovascular protective effect. Therefore, the elucidation of the mechanisms underlying ITIH4-mediated VSMCs stiffening helps to provide new ideas and potential targets for the diagnosis and treatment of atherosclerosis. In this study, we used specific ITIH4 expression vector and siRNA methods to transfect VSMCs. Our results found that ITIH4 expression increased VSMCs stiffness, meanwhile, ITIH4 siRNA decreased VSMCs stiffness. ITIH4 increased acetylated α-tubulin and inhibited ERK1/2 and JNK, but not P38 MAPK. ERK inhibitor (PD98059) or JNK inhibitor (SP600125) treatment increased acetylated α-tubulin expression and cell stiffness in VSMCs. ITIH4 was downregulated by thrombin treatment, ITIH4 partly reversed the effect of thrombin on acetylated α-tubulin and VSMCs stiffness. These results indicated that ITIH4 regulated acetylated α-tubulin expression in VSMCs and was against the effects of thrombin on VSMCs stiffness. JNK and ERK signaling pathways were proved to participate in this process.
Sujet(s)
Système de signalisation des MAP kinases , Muscles lisses vasculaires , Thrombine , Thrombine/pharmacologie , Thrombine/métabolisme , Système de signalisation des MAP kinases/effets des médicaments et des substances chimiques , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/cytologie , Muscles lisses vasculaires/effets des médicaments et des substances chimiques , Animaux , Myocytes du muscle lisse/métabolisme , Myocytes du muscle lisse/effets des médicaments et des substances chimiques , Rigidité vasculaire/effets des médicaments et des substances chimiques , Cellules cultivées , Rats , Humains , Rat Sprague-Dawley , Hormones peptidiques/métabolisme , Hormones peptidiques/pharmacologie , Hormones peptidiques/génétiqueRÉSUMÉ
Microplastics (MPs) are recognized as a major environmental problem due to their ubiquitous presence in ecosystems and bioaccumulation in food chains. Not only humans are continuously exposed to these pollutants through ingestion and inhalation, but recent findings suggest they may trigger vascular inflammation and potentially worsen the clinical conditions of cardiovascular patients. Here we combine headspace analysis by needle trap microextraction-gas chromatography-mass spectrometry (HS-NTME-GC-MS) and biological assays to evaluate the effects of polystyrene, high- and low-density polyethylene MPs on phenotype, metabolic activity, and pro-inflammatory status of Vascular Smooth Muscle Cells (VSMCs) the most prominent cells in vascular walls. Virgin and artificially aged MPs (4 weeks at 40 °C and 750â¯W/m2 simulated solar irradiation) were comparatively tested at 1â¯mg/mL to simulate a realistic exposure scenario. Our results clearly show the activation of oxidative stress and inflammatory processes when VSMCs were cultured with aged polymers, with significant overexpression of IL-6 and TNF-α. In addition, volatile organic compounds (VOCs), including pentane, acrolein, propanal, and hexanal as the main components, were released by VSMCs into the headspace. Type-specific VOC response profiles were induced on vascular cells from different MPs.
Sujet(s)
Chromatographie gazeuse-spectrométrie de masse , Inflammation , Interleukine-6 , Microplastiques , Stress oxydatif , Microplastiques/toxicité , Inflammation/induit chimiquement , Stress oxydatif/effets des médicaments et des substances chimiques , Interleukine-6/métabolisme , Facteur de nécrose tumorale alpha/métabolisme , Composés organiques volatils/toxicité , Polystyrènes/toxicité , Muscles lisses vasculaires/effets des médicaments et des substances chimiques , Muscles lisses vasculaires/cytologie , Humains , Polyéthylène/toxicité , Cellules cultivées , Myocytes du muscle lisse/effets des médicaments et des substances chimiquesRÉSUMÉ
Vascular calcification, which is a major complication of diabetes mellitus, is an independent risk factor for cardiovascular disease. Osteogenic differentiation of vascular smooth muscle cells (VSMCs) is one of the key mechanisms underlying vascular calcification. Emerging evidence suggests that macrophage-derived extracellular vesicles (EVs) may be involved in calcification within atherosclerotic plaques in patients with diabetes mellitus. However, the role of macrophage-derived EVs in the progression of vascular calcification is largely unknown. In this study, we investigated whether macrophage-derived EVs contribute to the osteogenic differentiation of VSMCs under high glucose conditions. We isolated EVs that were secreted by murine peritoneal macrophages under normal glucose (EVs-NG) or high glucose (EVs-HG) conditions. miRNA array analysis in EVs from murine macrophages showed that miR-17-5p was significantly increased in EVs-HG compared with EVs-NG. Prediction analysis with miRbase identified transforming growth factor ß receptor type II (TGF-ß RII) as a potential target of miR-17-5p. EVs-HG as well as miR-17-5p overexpression with lipid nanoparticles inhibited the gene expression of Runx2, and TGF-ß RII. Furthermore, we demonstrated that VSMCs transfected with miR-17-5p mimic inhibited calcium deposition. Our findings reveal a novel role of macrophage-derived EVs in the negative regulation of osteogenic differentiation in VSMCs under high glucose conditions.
Sujet(s)
Différenciation cellulaire , Vésicules extracellulaires , Glucose , microARN , Muscles lisses vasculaires , Myocytes du muscle lisse , Ostéogenèse , Transduction du signal , Facteur de croissance transformant bêta , microARN/génétique , microARN/métabolisme , Animaux , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/cytologie , Glucose/pharmacologie , Glucose/métabolisme , Ostéogenèse/effets des médicaments et des substances chimiques , Ostéogenèse/génétique , Facteur de croissance transformant bêta/métabolisme , Souris , Myocytes du muscle lisse/métabolisme , Vésicules extracellulaires/métabolisme , Calcification vasculaire/métabolisme , Calcification vasculaire/génétique , Calcification vasculaire/anatomopathologie , Récepteur de type II du facteur de croissance transformant bêta/métabolisme , Récepteur de type II du facteur de croissance transformant bêta/génétique , Mâle , Souris de lignée C57BL , Sous-unité alpha 1 du facteur CBF/métabolisme , Sous-unité alpha 1 du facteur CBF/génétiqueRÉSUMÉ
Abnormal proliferation, migration, and foam cell formation of Vascular smooth muscle cells (VSMCs) each play a role in the development of atherosclerosis (AS). Schisandrin (Sch) is the active lignan ingredient with broad-spectrum pharmacological effects. However, the role of Sch in the AS process is not clear. Therefore, this study was proposed to explore the therapeutic effect and potential mechanism of Sch on VSMCs. Ox-LDL was selected to create an atherosclerosis injury environment for VSMCs and macrophages. The MTT assay, Oil red O staining, wound healing, transwell experiments and ELISA were used to investigate the phenotype effects of Sch. Network pharmacology, molecular docking, flow cytometry, and western blot were used to investigate the underlying mechanisms of Sch on AS progression. Our findings implied that Sch treatment inhibited the proliferation and migration of VSMCs, and suppressed the ROS production and inflammatory cytokines up-regulation of VSMCs and macrophages. Moreover, Sch reduced lipid uptake and foam cell formation through downregulating LOX-1. Mechanistically, we found that Sch can inhibit the activation of JAK2/STAT3 signaling by targeting JAK2, and arrest cell cycle in GO/G1 phase. In summary, Sch can inhibit VSMCs proliferation and migration by arresting cell cycle and targeting JAK2 to regulating the JAK2/STAT3 pathway. Sch may serve as a potential drug for patients with AS.
Sujet(s)
Mouvement cellulaire , Prolifération cellulaire , Cyclooctanes , Kinase Janus-2 , Lignanes , Muscles lisses vasculaires , Composés polycycliques , Facteur de transcription STAT-3 , Transduction du signal , Kinase Janus-2/métabolisme , Facteur de transcription STAT-3/métabolisme , Mouvement cellulaire/effets des médicaments et des substances chimiques , Prolifération cellulaire/effets des médicaments et des substances chimiques , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/effets des médicaments et des substances chimiques , Muscles lisses vasculaires/cytologie , Lignanes/pharmacologie , Transduction du signal/effets des médicaments et des substances chimiques , Cyclooctanes/pharmacologie , Composés polycycliques/pharmacologie , Humains , Myocytes du muscle lisse/effets des médicaments et des substances chimiques , Myocytes du muscle lisse/métabolisme , Points de contrôle du cycle cellulaire/effets des médicaments et des substances chimiques , Animaux , Athérosclérose/anatomopathologie , Athérosclérose/métabolisme , Athérosclérose/traitement médicamenteuxRÉSUMÉ
When stimulated, vascular smooth muscle cells (VSMCs) change from a differentiated to a dedifferentiated phenotype. Dedifferentiated VSMCs have a key activity in cardiovascular diseases such as in-stent restenosis. MicroRNAs (miRNAs) have crucial functions in conversion of differentiated VSMCs to a dedifferentiated phenotype. We investigated the activity of miR-411-5p in the proliferation, migration, and phenotype switch of rat VSMCs.Based on a microRNA array assay, miR-411-5p expression was found to be significantly increased in cultured VSMCs stimulated by platelet-derived growth factor-BB (PDGF-BB). A CCK-8 assay, transwell assay, and scratch test were performed to measure the effect of miR-411-5p on the proliferation and migration of PDGF-BB-treated VSMCs. MiR-411-5p promoted expression of dedifferentiated phenotype markers such as osteopontin and tropomyosin 4 in PDGF-BB-treated VSMCs. Using mimics and inhibitors, we identified the target of miR-411-5p in PDGF-BB-treated VSMCs and found that calmodulin-regulated spectrin-associated protein-1 (CAMSAP1) was involved in the phenotypic switch mediated by PDGF-BB.By inhibiting expression of CAMSAP1, miR-411-5p enhanced the proliferation, migration, and phenotype switch of VSMCs.Blockade of miR-411-5p interaction with CAMSAP1 is a promising approach to treat in-stent restenosis.
Sujet(s)
Bécaplermine , Mouvement cellulaire , Prolifération cellulaire , microARN , Muscles lisses vasculaires , Myocytes du muscle lisse , Phénotype , Animaux , microARN/génétique , microARN/métabolisme , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/cytologie , Muscles lisses vasculaires/effets des médicaments et des substances chimiques , Rats , Bécaplermine/pharmacologie , Cellules cultivées , Myocytes du muscle lisse/métabolisme , Myocytes du muscle lisse/effets des médicaments et des substances chimiques , Rat Sprague-Dawley , Mâle , Ostéopontine/métabolisme , Ostéopontine/génétiqueRÉSUMÉ
Vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis play important roles in many physiological processes and pathological conditions. To identify genetic influences on VSMC behavior, we measured these traits and undertook genome-wide association studies in primary umbilical artery-derived VSMCs from >2000 individuals. Although there were no genome-wide significant associations for VSMC proliferation or migration, genetic variants at two genomic loci (7p15.3 and 7q32.3) showed highly significant associations with VSMC apoptosis (P = 1.95 × 10-13 and P = 7.47 × 10-9, respectively). The lead variant at the 7p51.3 locus was associated with increased expression of the GSDME and PALS2 genes in VSMCs. Knockdown of GSDME or PALS2 in VSMCs attenuated apoptotic cell death. A protein co-immunoprecipitation assay indicated that GSDME complexed with PALS2. PALS2 knockdown attenuated activated caspase-3 and GSDME fragmentation, whilst GSDME knockdown also reduced activated caspase-3. These findings provide new insights into the genetic regulation of VSMC apoptosis, with potential utility for therapeutic development.
Sujet(s)
Apoptose , Muscles lisses vasculaires , Myocytes du muscle lisse , Apoptose/génétique , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/cytologie , Humains , Myocytes du muscle lisse/métabolisme , Étude d'association pangénomique , Caspase-3/métabolisme , Caspase-3/génétique , Prolifération cellulaire/génétique , Protéines membranaires/métabolisme , Protéines membranaires/génétique , Mouvement cellulaire/génétique , Cellules cultivéesSujet(s)
Athérosclérose , Myocytes du muscle lisse , Humains , Athérosclérose/anatomopathologie , Athérosclérose/métabolisme , Myocytes du muscle lisse/anatomopathologie , Myocytes du muscle lisse/métabolisme , Animaux , Tumeurs/anatomopathologie , Muscles lisses vasculaires/anatomopathologie , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/cytologieSujet(s)
Aorte , Sous-unité alpha du facteur-1 induit par l'hypoxie , Muscles lisses vasculaires , Myocytes du muscle lisse , Phénotype , Phosphatidylinositol 3-kinases , Protéines proto-oncogènes c-akt , Transduction du signal , Humains , Protéines proto-oncogènes c-akt/métabolisme , Muscles lisses vasculaires/métabolisme , Muscles lisses vasculaires/cytologie , Aorte/métabolisme , Aorte/cytologie , Sous-unité alpha du facteur-1 induit par l'hypoxie/métabolisme , Sous-unité alpha du facteur-1 induit par l'hypoxie/génétique , Phosphatidylinositol 3-kinases/métabolisme , Myocytes du muscle lisse/métabolismeRÉSUMÉ
Glutamine (Gln), known as the most abundant free amino acid, is widely spread in human body. In this study, we demonstrated the protective effects of glutamine against mouse abdominal aortic aneurysm (AAA) induced by both angiotensin II (AngII) and calcium phosphate (Ca3(PO4)2) in vivo, which was characterized with lower incidence of mouse AAA. Moreover, histomorphological staining visually presented more intact elastic fiber and less collagen deposition in abdominal aortas of mice treated by glutamine. Further, we found glutamine inhibited the excessive production of reactive oxide species (ROS), activity of matrix metalloproteinase (MMP), M1 macrophage activation, and apoptosis of vascular smooth muscle cells (VSMCs) in suprarenal abdominal aortas of mice, what's more, the high expressions of MMP-2 protein, MMP-9 protein, pro-apoptotic proteins, and IL-6 as well as TNF-α in protein and mRNA levels in cells treated by AngII were down-regulated by glutamine. Collectively, these results revealed that glutamine protected against mouse AAA through inhibiting apoptosis of VSMCs, M1 macrophage activation, oxidative stress, and extracellular matrix degradation.
Sujet(s)
Angiotensine-II , Anévrysme de l'aorte abdominale , Apoptose , Glutamine , Activation des macrophages , Muscles lisses vasculaires , Myocytes du muscle lisse , Stress oxydatif , Animaux , Anévrysme de l'aorte abdominale/anatomopathologie , Anévrysme de l'aorte abdominale/prévention et contrôle , Anévrysme de l'aorte abdominale/métabolisme , Apoptose/effets des médicaments et des substances chimiques , Souris , Glutamine/pharmacologie , Angiotensine-II/pharmacologie , Activation des macrophages/effets des médicaments et des substances chimiques , Muscles lisses vasculaires/effets des médicaments et des substances chimiques , Muscles lisses vasculaires/anatomopathologie , Muscles lisses vasculaires/cytologie , Humains , Myocytes du muscle lisse/effets des médicaments et des substances chimiques , Myocytes du muscle lisse/métabolisme , Stress oxydatif/effets des médicaments et des substances chimiques , Espèces réactives de l'oxygène/métabolisme , Modèles animaux de maladie humaine , Mâle , Macrophages/effets des médicaments et des substances chimiques , Macrophages/métabolisme , Macrophages/immunologie , Aorte abdominale/anatomopathologie , Aorte abdominale/effets des médicaments et des substances chimiques , Matrix metalloproteinase 9/métabolisme , Matrix metalloproteinase 2/métabolisme , Facteur de nécrose tumorale alpha/métabolisme , Interleukine-6/métabolisme , Phosphates de calciumRÉSUMÉ
(-)-Epicatechin gallate (ECG) is beneficial to the treatment of cardiovascular diseases (CVDs), especially atherosclerosis (AS) through antioxidant stress, but there is a lack of detailed mechanism research. In this study, the therapeutic target of ECG was determined by crossing the drug target and disease target of CVDs and AS. The combination ability of ECG with important targets was verified by Discovery Studio software. The abnormal proliferation of vascular smooth muscle cells (VSMCs) induced by Ang-II and the oxidative damage of AML 12 induced by H2O2 were established to verify the reliability of ECG intervention on the target protein. A total of 120 ECG targets for the treatment of CVDs-AS were predicted by network pharmacology. The results of molecular docking showed that ECG has strong binding force with VEGFA, MMP-9, CASP3 and MMP-2 domains. In vitro experiments confirmed that ECG significantly reduced the expression of VEGFA, MMP-9, CASP3 and MMP-2 in Ang-II-induced VSMCs, and also blocked the abnormal proliferation, oxidative stress and inflammatory reaction of VSMCs by inhibiting the phosphorylation of PI3K signaling pathway. At the same time, ECG also interfered with H2O2-induced oxidative damage of AML 12 cells, decreased the expression of ROS and MDA and cell foaming, and increased the activities of antioxidant enzymes such as SOD, thus playing a protective role.