Adhesion Of Vascular Smooth Muscle Cells Research Articles

Targeting deubiquitinase OTUB1 protects vascular smooth muscle cells in atherosclerosis by modulating PDGFRβ.

Atherosclerosis is a chronic artery disease that causes various types of cardiovascular dysfunction. Vascular smooth muscle cells (VSMCs), the main components of atherosclerotic plaque, switch from contractile to synthetic phenotypes during atherogenesis. Ubiquitylation is crucial in regulating VSMC phenotypes in atherosclerosis, and it can be reversely regulated by deubiquitinases. However, the specific effects of deubiquitinases on atherosclerosis have not been thoroughly elucidated. In this study, RNAi screening in human aortic smooth muscle cells was performed to explore the effects of OTU family deubiquitinases, which revealed that silencing OTUB1 inhibited PDGF-BB-stimulated VSMC phenotype switch. Further in vivo studies using Apoe-/- mice revealed that knockdown of OTUB1 in VSMCs alleviated atherosclerosis plaque burden in the advanced stage and led to a stable plaque phenotype. Moreover, VSMC proliferation and migration upon PDGF-BB stimulation could be inhibited by silencing OTUB1 in vitro. Unbiased RNA-sequencing data indicated that knocking down OTUB1 influenced VSMC differentiation, adhesion, and proliferation. Mass spectrometry of ubiquitinated protein confirmed that proteins related to cell growth and migration were differentially ubiquitylated. Mechanistically, we found that OTUB1 recognized the K707 residue ubiquitylation of PDGFRβ with its catalytic triad, thereby reducing the K48-linked ubiquitylation of PDGFRβ. Inhibiting OTUB1 in VSMCs could promote PDGFRβ degradation via the ubiquitin-proteasome pathway, so it was beneficial in preventing VSMCs' phenotype switch. These findings revealed that knocking down OTUB1 ameliorated VSMCs' phenotype switch and atherosclerosis progression, indicating that OTUB1 could be a valuable translational therapeutic target in the future.

Shizukaol C alleviates trimethylamine oxide-induced inflammation through activating Keap1-Nrf2-GSTpi pathway in vascular smooth muscle cell

BackgroundCardiovascular disease is one of the main causes of global mortality, and there is an urgent need for effective treatment strategies. Gut microbiota-dependent metabolite trimethylamine-N-oxide (TMAO) promotes the development of cardiovascular diseases, and shizukaol C, a natural sesquiterpene isolated from Chloranthus multistachys with various biological activities, might exhibit beneficial role in preventing TMAO-induced vascular inflammation. PurposeThe purpose of this study was to investigate the anti-inflammatory effects and the underlying mechanisms of shizukaol C on TMAO-induced vascular inflammation. MethodsThe effect and underlying mechanism of shizukaol C on TMAO-induced adhesion molecules expression, bone marrow-derived macrophages (BMDM) adhesion to VSMC were evaluated by western blot, cell adhesion assay, co-immunoprecipitation, immunofluorescence assay, and quantitative Real-Time PCR, respectively. To verify the role of shizukaol C in vivo, TMAO-induced vascular inflammation model were established using guidewire-induced injury on mice carotid artery. Changes in the intima area and the expression of GSTpi, VCAM-1, CD68 were examined using haematoxylin-eosin staining, and immunofluorescence assay. ResultsOur data demonstrated that shizukaol C significantly suppressed TMAO-induced adhesion molecule expression and the bone marrow-derived macrophages (BMDM) adhesion in vascular smooth muscle cells (VSMC). Mechanically, shizukaol C inhibited TMAO-induced c-Jun N-terminal kinase (JNK)-nuclear factor-kappa B (NF-κB)/p65 activation, and the JNK inhibition was dependent on the shizukaol C-mediated glutathione-S-transferase pi (GSTpi) expression. By further molecular docking and protein-binding analysis, we demonstrated that shizukaol C directly binds to Keap1 to induce Nrf2 nuclear translocation and upregulated GSTpi expression. Consistently, our in vivo experiment showed that shizukaol C elevated the expression level of GSTpi in carotid arteries and alleviates TMAO-induced vascular inflammation. Conclusion: Shizukaol C exerts anti-inflammatory effects in TMAO-treated VSMC by targeting Keap1 and activating Nrf2-GSTpi signaling and resultantly inhibits the downstream JNK-NF-κB/p65 activation and VSMC adhesion, and alleviates TMAO-induced vascular inflammation in vivo, suggesting that shizukaol C may be a potential drug for treating TMAO-induced vascular diseases.

Abstract 454: The Effect Of High-fat Diet On Vascular Smooth Muscle Cell Mechanics And Migration In Apoe -/- Mice

Atherosclerosis remains a leading cause of cardiovascular diseases, which cause causes about 30% of global death. Vascular smooth muscle cells (VSMCs) are the major cellular components of arterial wall. VMSCs play critical role in the atherosclerosis development by phenotypic switching from a contractile phenotype to a synthetic phenotype, migration from media to intima of arterial wall, and deposition as foam cells in atherosclerotic plaque. In this study, we tested the effect of high-fat diet on the VSMC mechanics by comparing the mechanics and migration of VSMCs isolated from apolipoprotein-E knockout (ApoE -/- ) and wild type (WT) mice. Atomic force microscope (AFM) was employed to test cell adhesion and stiffness of VSMCs. AFM was also used to examine live VSMC submembranous cytoskeleton organization. A data-driven mathematical models-based image and signal processing software was used to analyze AFM data. Two-way ANOVA was used to test statistical significance for all experiments. The results demonstrated significant differences in the actin cytoskeletal organization and cell mechanics between ApoE -/- VSMCs and WT cells upon high-fat diet. However, no significant difference in cell stiffness was observed between normal diet-fed ApoE -/- and WT VSMCs. These interesting findings on the effect of high-fat diet on VSMC mechanics will improve our knowledge of the mechanism underlying the contribution of VSMC in the development of atherosclerosis.

Effects of ROS-Sensitive Nanoparticles Loaded with Interstitial Cell Derived Factors on Vascular Smooth Cell Adhesion, Proliferation, and Endothelialization

Local high concentrations of stromal cell-derived factor 1 (SDF-1) attract and trap stem cells to involve in the vascular repair when the skin is damaged. In this research, mercaptan ketone polymer (PRGY) was selected as a nano-carrier to prepare ROS-sensitive nanoparticles (NPs) loaded with SDF-1 by means of volatilization of a complex emulsion solvent. The NPs were characterized by a transmission electron microscope (TEM) and a particle size (PS) analyzer, the protein content of the NPs was evaluated by a BCA method, the cytotoxicity was evaluated by CCK8, and the NPs were subjected to the in vitro release (IVR) test. An acute mouse wound model was established, the pathological effect of NP solution on major organs of mice was observed by HE staining after intravenous injection, and the effect of targeted release of NP on animal model was evaluated. To analyze the effect of NPs on wound healing, the mouse models were rolled into different groups. They were sacrificed two weeks after the wound healing was completed, the blood vessels were fixed with perfusion solution, and the common artery where the wound was located was taken for follow-up study. The results suggested that the NPs exhibited a PS of 122.57±18.33 nm, a loading rate of 1.7%, and spherical and uniform surfaces. The IVR tests showed that the NPs could release rapidly under high ROS conditions. The wound model of mice was established, and the injection of NP solution revealed that it could target the wound area. The healing speed in the NP group was the fastest (P <0.05). Smooth muscle cells (SMCs) and endothelial cells (ECs) were isolated from common artery, and proliferation of them in the NP group was found to be obvious (P <0.05), and the adhesion of vascular SMCs could be accelerated at the same time.

Macrophages regulate vascular smooth muscle cell function during atherosclerosis progression through IL-1β/STAT3 signaling

Vascular smooth muscle cells (VSMCs) play a central role in atherosclerosis progression, but the functional changes in VSMCs and the associated cellular crosstalk during atherosclerosis progression remain unknown. Here we show that scRNA-seq analysis of proximal adjacent (PA) and atherosclerotic core (AC) regions of human carotid artery plaques identifies functional alterations in macrophage-like VSMCs, elucidating the main state differences between PA and AC VSMCs. And, IL-1β mediates macrophage-macrophage-like VSMC crosstalk through regulating key transcription factors involved in macrophage-like VSMCs functional alterations during atherosclerosis progression. In vitro assays reveal VSMCs trans-differentiated into a macrophage-like phenotype and then functional alterations in response to macrophage-derived stimuli. IL-1β promots the adhesion, inflammation, and apoptosis of macrophage-like VSMCs in a STAT3 dependent manner. The current findings provide interesting insight into the macrophages-macrophage-like VSMC crosstalk, which would drive functional alterations in the latter cell type through IL-1β/STAT3 axis during atherosclerosis progression.

Abstract P126: FXR1 Regulates Vascular Smooth Muscle Cell Cytoskeletal Dynamics By Multiple Mechanisms

Introduction: Hypertension is a major risk factor for cardiovascular disease and stroke. Optimally treated hypertensive patients have a 50% greater cardiovascular risk than untreated normotensive subjects, presenting a need for additional targets. Vascular smooth muscle cells (VSMC) play a critical role in vascular contractility and regulation of blood pressure. Fragile X-related protein (FXR1) is a muscle-enhanced RNA binding protein. In addition to containing mRNA binding domains, FXR1 has an agenet-like domain for protein-protein interactions and four WH2 motif domains to mediate actin dynamics. The structure of FXR1 supports its role in post-transcriptional regulation and though largely ignored, cytoskeletal dynamics via potential protein-protein interactions. This study will test the hypothesis that FXR1 regulates vascular contractility by RNA stability and protein interactions. Results: RNA immuno-precipitation sequencing (RIPseq) analysis in human VSMC identified that FXR1 binds to mRNA that participate in VSMC contractility and cytoskeletal reorganization, and FXR1 depletion decreases mRNA abundance and stability of these transcripts. Mass-spectrometry and co-immunoprecipitation identified that FXR1 interacts with members of the WAVE complex, a five-subunit protein complex involved in the formation of the actin cytoskeleton, including CYFIP1 (Cytoplasmic FMR1-interacting protein 1) and Actin Related Protein 2/3 complex (ARP2). The WAVE complex is activated by an ARP2-mediated interaction with Rac1, promoting actin remodeling and cytoskeletal reorganization in a GTP-dependent manner. FXR1 depletion decreases small GTPases RAC1 and CDC42 activation in VSMC. Additionally, FXR1 depletion decreases VSMC lamellipodia formation, adhesion, migration and collagen gel contraction. Novel, VSMC-specific FXR1 conditional knock out mice show decreased diastolic (P < 0.05) blood pressure at baseline compared to controls. Conclusion: These data are the first to suggest that FXR1 regulates mRNA stability of contractile proteins and interacts with members of the WAVE complex. Deletion of FXR1 abrogates VSMC cytoskeletal reorganization, resulting in a hypotensive phenotype in VSMC-specific conditional knockout mice.

FXR1 Decreases Blood Pressure by Regulating Vascular Contractility

IntroductionHypertension is a major risk factor for cardiovascular disease and stroke. Optimally treated hypertensive patients still have a 50% greater cardiovascular risk than untreated normotensive subjects. Despite current medication, half a million deaths in the United States include hypertension as a primary contributing cause in 2018 presenting a need for additional targets. Vascular smooth muscle cells (VSMCs) are the fulcrum of vascular disease, particularly hypertension. VSMCs play a critical role in vascular contractility and the regulation of blood pressure. Fragile X‐related protein (FXR1) is a muscle‐enhanced RNA binding protein and we previously found siRNA knock down of FXR1 increases inflammatory mRNA stability. Overexpression of FXR1 decreases inflammatory mRNA stability in VSMC. Little is known concerning FXR1 protein binding partners and its role in vascular disease. The specific aim of this study is to test the hypothesis that FXR1 regulates vascular contractility by RNA stability and protein interactions.ResultsWe generated a novel, VSMC‐specific FXR1 conditional knock out mouse (FXR1VSMC/VSMC) in order to establish an in vivo role of FXR1 in vascular disease. Preliminary data indicates that these mice show decreased diastolic (P < 0.05) blood pressure at baseline compared to controls. Gene ontology of RNA immuno‐precipitation sequencing analysis in human VSMCs identified that FXR1 binds to mRNA that participate in VSMC contractility and regulation of blood pressure‐ related pathways. Although considered an RNA stability protein, mass‐spectrometry identified that FXR1 interacts with proteins related to contractile processes such as cell migration, adhesion and stress fiber formation. FXR1 depletion in human VSMCs decreases mRNA abundance of contractile machinery. Future studies will determine the effect of FXR1 depletion on mRNA stability of these transcripts. Additionally, siRNA knock down of FXR1 decreased VSMC adhesion, migration and collagen gel contraction corroborating in vivo observations.ConclusionThese data are the first to suggest FXR1 regulates blood pressure and vascular contractility potentially by two mechanisms: mRNA stability and functional activity by protein‐protein interactions. The findings support FXR1 activity may represent a target for therapeutic invention to regulate blood pressure.

The substrate stiffness at physiological range significantly modulates vascular cell behavior

Changes in the stiffness of the cellular microenvironment are involved in many pathological processes of blood vessels. Substrate stiffness has been shown to have extensive effects on vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs). However, the material stiffness of most previously reported in-vitro models is ranging from ~100 kPa to the magnitude of MPa, which does not match the mechanical properties of natural vascular tissue (10–100 kPa). Herein, we constructed hydrogel substrates with the stiffness of 18–86 kPa to explore the effect of physiological stiffness on vascular cells. Our findings show that, with the increase of stiffness at the physiological range, the cell adhesion and proliferation behaviors of VECs and VSMCs are significantly enhanced. On the soft substrate, VECs express more nitric oxide (NO), and VSMCs tend to maintain a healthy contraction phenotype. More importantly, we find that the number of differentially expressed genes in cells cultured between 18 kPa and 86 kPa substrates (560 in VECs, 243 in VSMCs) is significantly higher than that between 86 kPa and 333 kPa (137 in VECs, 172 in VSMCs), indicating that a small increase in stiffness within the physiological range have a higher impact on vascular cell behaviors. Overall, our results expanded the exploration of how stiffness affects the behavior of vascular cells at the physiological range.

Systemic deficiency of vitronectin is associated with aortic inflammation and plaque progression in ApoE-Knockout mice.

Optimal cell spreading and interplay of vascular smooth muscle cells (VSMC), inflammatory cells, and cell adhesion molecules (CAM) are critical for progressive atherosclerosis and cardiovascular complications. The role of vitronectin (VTN), a major cell attachment glycoprotein, in the pathogenesis of atherosclerosis remains elusive. In this study, we attempt to examine the pathological role of VTN in arterial plaque progression and inflammation. We found that, relative expression analysis of VTN from the liver of Apolipoprotein E (ApoE) Knockout mice revealed that atherosclerotic progression induced by feeding mice with high cholesterol diet (HCD) causes a significant downregulation of VTN mRNA as well as protein after 60 days. Promoter assay confirmed that cholesterol modulates the expression of VTN by influencing its promoter. Mimicking VTN reduction with siRNA in HCD fed ApoE Knockout mice, accelerated athero‐inflammation with an increase in NF‐kB, ICAM‐1, and VCAM‐1 at the site of the plaque along with upregulation of inflammatory proteins like MCP‐1 and IL‐1β in the plasma. Also, matrix metalloprotease (MMP)‐9 and MMP‐12 expression were increased and collagen content was decreased in the plaque, in VTN deficient condition. This might pose a challenge to plaque integrity. Human subjects with acute coronary syndrome or having risk factors of atherosclerosis have lower levels of VTN compared to healthy controls suggesting a clinical significance of plasma VTN in the pathophysiology of coronary artery disease. We establish that, VTN plays a pivotal role in cholesterol‐driven atherosclerosis and aortic inflammation and might be a useful indicator for atherosclerotic plaque burden and stability.

Alternative C3 Complement System: Lipids and Atherosclerosis.

Familial hypercholesterolemia (FH) is increasingly associated with inflammation, a phenotype that persists despite treatment with lipid lowering therapies. The alternative C3 complement system (C3), as a key inflammatory mediator, seems to be involved in the atherosclerotic process; however, the relationship between C3 and lipids during plaque progression remains unknown. The aim of the study was to investigate by a systems biology approach the role of C3 in relation to lipoprotein levels during atherosclerosis (AT) progression and to gain a better understanding on the effects of C3 products on the phenotype and function of human lipid-loaded vascular smooth muscle cells (VSMCs). By mass spectrometry and differential proteomics, we found the extracellular matrix (ECM) of human aortas to be enriched in active components of the C3 complement system, with a significantly different proteomic signature in AT segments. Thus, C3 products were more abundant in AT-ECM than in macroscopically normal segments. Furthermore, circulating C3 levels were significantly elevated in FH patients with subclinical coronary AT, evidenced by computed tomographic angiography. However, no correlation was identified between circulating C3 levels and the increase in plaque burden, indicating a local regulation of the C3 in AT arteries. In cell culture studies of human VSMCs, we evidenced the expression of C3, C3aR (anaphylatoxin receptor) and the integrin αMβ2 receptor for C3b/iC3b (RT-PCR and Western blot). C3mRNA was up-regulated in lipid-loaded human VSMCs, and C3 protein significantly increased in cell culture supernatants, indicating that the C3 products in the AT-ECM have a local vessel-wall niche. Interestingly, C3a and iC3b (C3 active fragments) have functional effects on VSMCs, significantly reversing the inhibition of VSMC migration induced by aggregated LDL and stimulating cell spreading, organization of F-actin stress fibers and attachment during the adhesion of lipid-loaded human VSMCs. This study, by using a systems biology approach, identified molecular processes involving the C3 complement system in vascular remodeling and in the progression of advanced human atherosclerotic lesions.

Patterned Electrospinning: A Method of Generating Defined Fibrous Constructs Influencing Cell Adhesion and Retention.

A critical component of tissue engineering is the ability to functionally replace native tissue stroma. Electrospinning is a technique capable of forming fibrous constructs with a high surface area for increased cell-material interaction and enhanced biocompatibility. However, physical and biological properties of electrospun scaffolds are limited by design controllability on a macroscale. We developed a methodology for generating electrospun scaffolds with defined patterns and topographic features to influence physical properties and biological interactions. Five unique design electrospinning target collectors were fabricated to allow for generation of defined polymeric scaffold patterns including lines, sinusoids, squares, zigzags, and solid. Poly(lactic-co-glycolic) acid was electrospun under identical conditions utilizing these varied targets, and constructs generated were examined as to their physical configuration, mechanical and chemical properties, and their ability to foster vascular smooth muscle cell adhesion and retention at 24 h. Modifying collector designs led to significant differences in fiber target coverage ranging from 300 mm2 for solid (100% of the target area) to 217.8 mm2 for lines (72.6% of the target area). Measured fiber excess, residual open area, and contact angle (hydrophobicity) followed the same trend as fiber target coverage with respect to the collector pattern: lines > sinusoids > squares > zigzags > solid. Similarly, the line design allowed for the greatest cell adhesion and retention (258 ± 31 cells), whereas solid exhibited the lowest (150 ± 15 cells); p < 0.05. There was a strong direct correlation of cell adhesion to construct residual open area (R2 = 0.94), normalized fiber excess (R2 = 0.99), and fiber grammage (R2 = 0.72), with an inverse relationship to fiber target coverage (R2 = 0.94). Our results demonstrate the ability to utilize patterned collectors for modifying macroscopic and microscopic electrospun scaffold features, which directly impact cell adhesion and retention, offering translational utility for designing specific tissue constructs.

Circular RNA UVRAG Mediated by Alternative Splicing Factor NOVA1 Regulates Adhesion and Migration of Vascular Smooth Muscle Cells.

The movement of abnormal vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia in vein graft disease. Circular RNAs (circRNAs) are single stranded RNAs with 3’ and 5’ ends covalently joined together. They have been shown to regulate cell function in many diseases. NOVA1 is considered to be a brain-specific splicing factor that plays an important role in the nervous system and cancer. The role of NOVA1 in VSMCs remains unclear. In the present study, transcriptome sequencing was used to identify differentially expressed circRNAs in the rat vein graft model. A novel circRNA, circUVRAG, was decreased in the grafted vein and stably located in the cytoplasm. Knockdown of circUVRAG suppressed VSMC adhesion and migration. In addition, we demonstrated that the alternative splicing factor NOVA1 co-located with UVRAG pre-mRNA in the nucleus and modulated the production of circUVRAG. These new discoveries may serve as a potential means to treat intimal hyperplasia after vein grafts.

Endothelial cells control vascular smooth muscle cell cholesterol levels by regulating 24-dehydrocholesterol reductase expression

Communication of vascular cells is essential for the control of organotypic functions of blood vessels. In this context, vascular endothelial cells (EC) act as potent regulators of vascular smooth muscle cell (VSMC) functions such as contraction and relaxation. However, the impact of ECs on the gene expression pattern of VSMCs is largely unknown. Here, we investigated changes of the VSMC transcriptome by utilizing 3D human vascular organoids organized as a core of VSMCs enclosed by a monolayer of ECs. Microarray-based analyses indicated that interaction with ECs for 48 h down-regulates expression of genes in VSMCs controlling rate-limiting steps of the cholesterol biosynthesis such as HMGCR, HMGCS1, DHCR24 and DHCR7. Protein analyses revealed a decrease in the abundance of DHCR24 (24-dehydrocholesterol reductase) and lower cholesterol levels in VSMCs co-cultured with ECs. On the functional level, the blockade of the DHCR24 activity impaired adhesion, migration and proliferation of VSMCs. Collectively, these findings indicate that ECs have the capacity to instruct VSMCs to shut down the expression of DHCR24 thereby limiting their cholesterol biosynthesis, which may support their functional steady state.

Stiffness of Vascular Smooth Muscle Cells from Monkeys Studied using Atomic Force Microscopy

Vascular smooth muscle cells (VSMC) are the main cellular components of blood vessel walls and bear external mechanical forces caused by blood flow and pressure. In this report, we have verified the following hypothesis through experiments: The increase in VSMC stiffness may be mainly due to changes in vascular stiffness due to aging. Although aging enhances the stiffness and adhesion of VSMC, there is no significant difference in apparent elastic modulus and adhesion between the VSMC obtained by male and female. The effect of aging through the ECM-integrin-cytoskeleton axis is related to increased VSMC stiffness and matrix adhesion rather than gender.

Behaviour of Vascular Smooth Muscle Cells on Amine Plasma-Coated Materials with Various Chemical Structures and Morphologies.

Amine-coated biodegradable materials based on synthetic polymers have a great potential for tissue remodeling and regeneration because of their excellent processability and bioactivity. In the present study, we have investigated the influence of various chemical compositions of amine plasma polymer (PP) coatings and the influence of the substrate morphology, represented by polystyrene culture dishes and polycaprolactone nanofibers (PCL NFs), on the behavior of vascular smooth muscle cells (VSMCs). Although all amine-PP coatings improved the initial adhesion of VSMCs, 7-day long cultivation revealed a clear preference for the coating containing about 15 at.% of nitrogen (CPA-33). The CPA-33 coating demonstrated the ideal combination of good water stability, a sufficient amine group content, and favorable surface wettability and morphology. The nanostructured morphology of amine-PP-coated PCL NFs successfully slowed the proliferation rate of VSMCs, which is essential in preventing restenosis of vascular replacements in vivo. At the same time, CPA-33-coated PCL NFs supported the continuous proliferation of VSMCs during 7-day long cultivation, with no significant increase in cytokine secretion by RAW 264.7 macrophages. The CPA-33 coating deposited on biodegradable PCL NFs therefore seems to be a promising material for manufacturing small-diameter vascular grafts, which are still lacking on the current market.

Statin-mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration.

This study demonstrates and evaluates the changes in rat vascular smooth muscle cell biomechanics following statin-mediated cholesterol depletion. Evidence is presented to show correlated changes in migration and adhesion of vascular smooth muscle cells to extracellular matrix proteins fibronectin and collagen. Concurrently, integrin α5 expression was enhanced but not integrin α2. Atomic force microscopy analysis provides compelling evidence of coordinated reduction in vascular smooth muscle cell stiffness and actin cytoskeletal orientation in response to statin-mediated cholesterol depletion. Proof is provided that statin-mediated cholesterol depletion remodels total vascular smooth muscle cell cytoskeletal orientation that may additionally participate in altering ex vivo aortic vessel function. It is concluded that statin-mediated cholesterol depletion may coordinate vascular smooth muscle cell migration and adhesion to different extracellular matrix proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell. Not only does cholesterol induce an inflammatory response and deposits in foam cells at the atherosclerotic plaque, it also regulates cellular mechanics, proliferation and migration in atherosclerosis progression. Statins are HMG-CoA reductase inhibitors that are known to inhibit cellular cholesterol biosynthesis and are clinically prescribed to patients with hypercholesterolemia or related cardiovascular conditions. Nonetheless, the effect of statin-mediated cholesterol management on cellular biomechanics is not fully understood. In this study, we aimed to assess the effect of fluvastatin-mediated cholesterol management on primary rat vascular smooth muscle cell (VSMC) biomechanics. Real-time measurement of cell adhesion, stiffness, and imaging were performed using atomic force microscopy (AFM). Cellular migration on extra cellular matrix (ECM) protein surfaces was studied by time-lapse imaging. The effect of changes in VSMC biomechanics on aortic function was assessed using an ex vivo myograph system. Fluvastatin-mediated cholesterol depletion (-27.8%) lowered VSMC migration distance on a fibronectin (FN)-coated surface (-14.8%) but not on a type 1 collagen (COL1)-coated surface. VSMC adhesion force to FN (+33%) and integrin α5 expression were enhanced but COL1 adhesion and integrin α2 expression were unchanged upon cholesterol depletion. In addition, VSMC stiffness (-46.6%) and ex vivo aortic ring contraction force (-40.1%) were lowered and VSMC actin cytoskeletal orientation was reduced (-24.5%) following statin-mediated cholesterol depletion. Altogether, it is concluded that statin-mediated cholesterol depletion may coordinate VSMC migration and adhesion to different ECM proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell and aortic function.

Salvia miltiorrhiza-derived miRNAs suppress vascular remodeling through regulating OTUD7B/KLF4/NMHC IIA axis.

Objective: Abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) are essential for vascular remodeling. Natural compounds with diterpene chinone or phenolic acid structure from Salvia miltiorrhiza, an eminent medicinal herb widely used to treat cardiovascular diseases in China, can effectively attenuate vascular remodeling induced by vascular injury. However, it remains unknown whether Salvia miltiorrhiza-derived miRNAs can protect VSMCs from injury by environmental stimuli. Here, we explored the role and underlying mechanisms of Salvia miltiorrhiza-derived Sal-miR-1 and 3 in the regulation of VSMC migration and monocyte adhesion to VSMCs induced by thrombin.Methods: A mouse model for intimal hyperplasia was established by the ligation of carotid artery and the injured carotid arteries were in situ-transfected with Sal-miR-1 and 3 using F-127 pluronic gel. The vascular protective effects of Sal-miR-1 and 3 were assessed via analysis of intimal hyperplasia with pathological morphology. VSMC migration and adhesion were analyzed by the wound healing, transwell membrane assays, and time-lapse imaging experiment. Using loss- and gain-of-function approaches, Sal-miR-1 and 3 regulation of OTUD7B/KLF4/NMHC IIA axis was investigated by using luciferase assay, co-immunoprecipitation, chromatin immunoprecipitation, western blotting, etc.Results: Salvia miltiorrhiza-derived Sal-miR-1 and 3 can enter the mouse body after intragastric administration, and significantly suppress intimal hyperplasia induced by carotid artery ligation. In cultured VSMCs, these two miRNAs inhibit thrombin-induced the migration of VSMCs and monocyte adhesion to VSMCs. Mechanistically, Sal-miR-1 and 3 abrogate OTUD7B upregulation by thrombin via binding to the different sites of the OTUD7B 3'UTR. Most importantly, OTUD7B downregulation by Sal-miR-1 and 3 attenuates KLF4 protein levels via decreasing its deubiquitylation, whereas decreased KLF4 relieves its repression of transcription of NMHC IIA gene and thus increases NMHC IIA expression levels. Further, increased NMHC IIA represses VSMC migration and monocyte adhesion to VSMCs via maintaining the contractile phenotype of VSMCs.Conclusions: Our studies not only found the novel bioactive components from Salvia miltiorrhiza but also clarified the molecular mechanism underlying Sal-miR-1 and 3 inhibition of VSMC migration and monocyte adhesion to VSMCs. These results add important knowledge to the pharmacological actions and bioactive components of Salvia miltiorrhiza. Sal-miR-1 and 3-regulated OTUD7B/KLF4/NMHC IIA axis may represent a therapeutic target for vascular remodeling.

Calcium in Vascular Smooth Muscle Cell Elasticity and Adhesion: Novel Insights Into the Mechanism of Action.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the arterial wall. These cells play a critical role in maintaining vascular homeostasis including vasoconstriction and vasodilatation through active contraction and relaxation. Dysregulation of VSMC function alters the response of blood vessels to mechanical stress, contributing to the pathogenesis of vascular diseases, particularly atherosclerosis and hypertension. The stiffness of VSMCs is a major regulator of vascular function. Previous studies suggest that intracellular Ca2+ controls the stiffness of VSMCs by a mechanism involving myosin contractile apparatus. More recent studies highlight important functions of cytoskeletal α-smooth muscle actin (α-SMA), α5β1 integrin, and integrin-mediated cell-extracellular matrix (ECM) interactions in Ca2+-dependent regulation of VSMC stiffness and adhesion to the ECM, providing novel insights into the mechanism of calcium action.

Regulation of Vascular Smooth Muscle Cell Stiffness and Adhesion by [Ca2+]i: An Atomic Force Microscopy-Based Study - CORRIGENDUM.

The intracellular concentration of calcium ion ([Ca2+]i) is a critical regulator of cell signaling and contractility of vascular smooth muscle cells (VSMCs). In this study, we employed an atomic force microscopy (AFM) nanoindentation-based approach to investigate the role of [Ca2+]i in regulating the cortical elasticity of rat cremaster VSMCs and the ability of rat VSMCs to adhere to fibronectin (Fn) matrix. Elevation of [Ca2+]i by ionomycin treatment increased rat VSMC stiffness and cell adhesion to Fn-biofunctionalized AFM probes, whereas attenuation of [Ca2+]i by 1,2-Bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) treatment decreased the mechanical and matrix adhesive properties of VSMCs. Furthermore, we found that ionomycin/BAPTA-AM treatments altered expression of α 5 integrin subunits and α smooth muscle actin in rat VSMCs. These data suggest that [Ca2+]i regulates VSMC elasticity and adhesion to the extracellular matrix by a potential mechanism involving changing dynamics of the integrin-actin cytoskeleton axis.

A Calcium Mediated Mechanism Coordinating Vascular Smooth Muscle Cell Adhesion During KCl Activation.

Efficient mechanotransduction in vascular smooth muscle cells (VSMCs) is intimately coupled to physical coupling of the cell to extracellular matrix proteins (ECM) by integrins. Integrin adhesion receptors are essential for normal vascular function and defective integrin signaling is associated with cardiovascular disease. However, less is known about the mechanism of integrin activation in VSMCs in relation to vasoregulation. Our laboratory previously demonstrated that the vasoconstrictor Angiotensin II increases VSMC stiffness in concert with enhanced adhesion to fibronectin (FN), indicating an important role for adhesion in contraction. However, the mechanism of this coordination remains to be clarified. In this study, intracellular Ca2+ ([Ca2+]i) was hypothesized to link integrin activation through inside-out signaling pathways leading to enhanced adhesion in response to AII. By using atomic force microscopy (AFM) with an anti-α5 antibody coated AFM probe, we confirmed that cell stiffness was increased by AII, while we observed no change in adhesion to an α5 integrin antibody. This indicated that increases in cell adhesion to FN induced by AII were occurring through an integrin activation process, as increased membrane integrin expression/receptor density would have been accompanied by increased adhesion to the anti-α5 antibody. Further studies were performed using either KCl or BAPTA-AM to modulate the level of [Ca2+]i. After KCl, VSMCs showed a rapid transient increase in cell stiffness as well as cell adhesion to FN, and these two events were synchronized with superimposed transient increases in the level of [Ca2+]i, which was measured using the Ca2+ indicator, fluo-4. These relationships were unaffected in VSMCs pretreated with the myosin light chain kinase inhibitor, ML-7. In contrast, unstimulated VSMCs incubated with an intracellular calcium chelator, BAPTA-AM, showed reduced cell adhesion to FN as well the expected decrease in [Ca2+]i. These data suggest that in VSMCs, integrin activation is linked to signaling events tied to levels of [Ca2+]i while being less dependent on events at the level of contractile protein activation. These findings provide additional evidence to support a role for adhesion in VSMC contraction and suggest that following cell contractile activation, that adhesion may be regulated in tandem with the contractile event.