Search

Ziyu Liu,1,2 Leilei Huang,3,4 Liwei Sun,1 Hui Nie,3,4 Yuqi Liang,1,2 Jinwen Huang,3,4 Fanhong Wu,3,4 Xin Hu1 1The Laboratory of Cancer Biology, China-Japan Union Hospital of Jilin University, Jilin University, Changchun, Jilin, People’s Republic of China; 2Department of Biochemistry and Molecular Biology, School of Life Science, Jilin University, Changchun, Jilin, People’s Republic of China; 3Shanghai Engineering Research Center of Green Fluoropharmaceutical Technology, Shanghai, People’s Republic of China; 4Department of Pharmaceutical Engineering, School of Chemical and Environmental Engineering, Shanghai Institute of Technology, Shanghai, People’s Republic of ChinaCorrespondence: Xin Hu; Fanhong Wu Email huxin@jlu.edu.cn; wfh@sit.edu.cnPurpose: Erianin is a small chemical compound extracted from Dendrobium chrysotoxum and has excellent antineoplastic effects against a variety of cancers. Combretastatin A-4 (CA4) is the most effective member of natural phenolic stilbene compounds isolated from the African willow tree Combretum caffrum. Ecust004 (Chemical Formula: C18H21NO7S) is a drug candidate optimized from structure–activity relationship studies of the sulfamate derivatives of Erianin and CA4, which has better bioavailability and pharmacokinetic profiles than Erianin and CA4.Methods: To investigate the antitumor activity of Ecust004 in different types of breast cancer cells, MDA-MB-231 and MCF7 cells were treated with Ecust004. MTT and CCK8 were used to determine the effects of Ecust004 on cell proliferation. Wound-healing and Transwell assays were used to evaluate the migration and invasion level of cells treated with Ecust004. The expression of genes and proteins associated with epithelial–mesenchymal transition was detected by RT-PCR and Western blotting. In vivo studies further clarified the functional effects of Ecust004.Results: Ecust004 treatment decreased the growth and proliferation of MDA-MB-231 and MCF7 cells at a lower dosage than Erianin. In addition, compared to Erianin and CA4, Ecust004 can better inhibit the invasion and migration of MDA-MB-231 and MCF7 cells. Accordingly, the expression of genes associated with epithelial–mesenchymal transition, such as E-cadherin and vinculin, was increased. Finally, compared with Erianin and CA4, Ecust004 exhibited a better anti-tumor activity in vivo.Conclusion: Ecust004 inhibits the proliferation, invasion, and migration of breast cancer cells, and therefore represents a potential agent for development as an antitumor drug.Keywords: Ecust004, Erianin, breast cancer, EMT

In this study, we fabricated poly(3-hydroxybutyrate-3-hydroxyvalerate) (PHBV) coatings doped with Gd3+ (1, 5, and 10 × 10−4 mol/L) on Ti6Al4V alloy for the first time to promote soft tissue sealing around dental implants. The corrosion resistance of Gd3+-modified PHBV-coated Ti6Al4V was studied by electrochemical and immersion tests, respectively, whereas CCK-8 and RT-PCR evaluated the biocompatibility to human gingival fibroblasts (HGFs) and human umbilical vein endothelial cells (HUVECs). It was found that the Gd3+-modified PHBV coating could enhance the corrosion resistance of Ti6Al4V. In vitro cell tests showed that PHBV coatings with and without Gd3+ addition could promote adhesion and proliferation of HGFs and HUVECs, showing a Gd3+ content-dependent manner. Moreover, it was found that the PDA-PHBV@1Gd showed the best proliferation to HGFs by up-regulating gene expressions of VINCULIN, ITGB1, and ITGA3, whereas the best response to HUVECs with the highest gene expression of eNOS and HIF-1α genes was found in the PDA-PHBV@5Gd-coated group.

Acute liver failure (ALF) is a rare and critical medical condition. This study was designed to investigate the protective effects and underlying mechanism of ACY1215 in ALF mice. Our findings suggested that ACY1215 treatment ameliorates the pathological hepatic damage of ALF and decreases the serum levels of ALT and AST. Furthermore, ACY1215 pretreatment increased the level of ATM, γ-H2AX, Chk2, p53, p21, F-actin and vinculin in ALF. Moreover, ACY1215 inhibited the level of NLRP3, ASC, caspase-1, IL-1β and IL-18 in ALF. The ATM inhibitor KU55933 could decrease the level of ATM, γ-H2AX, Chk2, p53, p21, F-actin and vinculin in ALF with ACY1215 pretreatment. The F-actin inhibitor cytochalasin B decreased the level of F-actin and vinculin in ALF with ACY1215 pretreatment. However, cytochalasin B had no effect on protein levels of ATM, Chk2, p53 and p21 in ALF with ACY1215 pretreatment. Cytochalasin B could dramatically increase the level of NLRP3, ASC, caspase-1, IL-1β and IL-18 in ALF with ACY1215 pretreatment. These results indicated that ACY1215 exhibited hepatoprotective properties, which was associated with the inhibition of NLRP3 inflammasome, and this effect of ACY1215 was connected with upregulation of the ATM/F-actin mediated signalling pathways.

The actin-binding protein vinculin is a major constituent of focal adhesion, but its role in neuronal development is poorly understood. We found that vinculin deletion in mouse neocortical neurons attenuated axon growth both in vitro and in vivo. Using functional mutants, we found that expression of a constitutively active vinculin significantly enhanced axon growth while the head-neck domain had an inhibitory effect. Interestingly, we found that vinculin-talin interaction was dispensable for axon growth and neuronal migration. Strikingly, expression of the tail domain delayed migration, increased branching, and stunted axon. Inhibition of the Arp2/3 complex or abolishing the tail domain interaction with actin completely reversed the branching phenotype caused by tail domain expression without affecting axon length. Super-resolution microscopy showed increased mobility of actin in tail domain expressing neurons. Our results provide novel insights into the role of vinculin and its functional domains in regulating neuronal migration and axon growth.

Background Prostate cancer (PCa) is a leading cause of death in men, and effective treatment of PCa requires further development. Our study aimed to investigate the potential role of vinculin (VCL) in PCa progression in vitro and in vivo. Methods We investigated the methylation level of the VCL promoter based on the TCGA database. The knockdown efficacy of VCL gene expression was confirmed by quantitative polymerase chain reaction, Western blot analysis, and immunofluorescence. Furthermore, morphological changes in PCa cells were detected using phalloidin staining. The mobility of PCa cells was measured using transwell assays and high-content analysis. Moreover, cell growth and viability were determined using the colony formation and cell counting kit-8 assays. The role of VCL in tumor growth in vivo was investigated using a subcutaneous xenograft model generated by injecting tumor cells into the right flank of BALB/c nude mice. Results The methylation level of the VCL promoter in PCa was significantly downregulated concomitant with age and the progression of nodal metastasis. VCL expression was markedly decreased by shRNA. Importantly, VCL knockdown significantly changed the cell morphology; inhibited the migration, invasion, and movement; and repressed colony formation and viability of PCa cells in vitro. Furthermore, downregulation of VCL suppressed tumor growth in vivo. Conclusions Our study comprehensively evaluated the role of VCL in PCa progression in vivo and in vitro. The findings of the present study suggest that VCL can be a potential target for PCa prognosis and treatment.

In the process of epithelial-mesenchymal transition (EMT), epithelial cancer cells transdifferentiate into mesenchymal-like cells with high motility and aggressiveness, resulting in the spread of tumor cells. Immune cells and inflammation in the tumor microenvironment are the driving factors of EMT, but few studies have explored the core targets of the interaction between EMT and tumor immune cells. We analyzed thousands of cases of gastric cancer and gastric tissue specimens of TCGA, CPTAC, GTEx and analyzing QPCR and IHC data of 56 gastric cancer patients in SYSU Gastric Cancer Research Center. It was known that EMT has an important connection with the infiltration of NK cells, and that the expression of vinculin may be the target of the phenomenon. The increased expression of vinculin is closely related to the aggressiveness and distant metastasis of cancer, which affects the survival prognosis of the patient. Moreover, through in vitro experiments under 3D conditions, we found that vinculin, cell invasion and metastasis are clearly linked. VCL can affect EMT and tumor immunity by regulating EPCAM gene expression. The role and mechanism of action of vinculin have been controversial, but this molecule may downregulate EpCAM (epithelial cellular adhesion molecule) and its own role in gastric cancer through DNA methylation, causing NK cells to enrich into tumor cells and kill tumor cells. At the same time, it promotes the occurrence of EMT, which in turn causes tumor metastasis and thus poorer prognosis.

Integrin-dependent adhesions mediate reciprocal exchange of force and information between the cell and the extracellular matrix. These effects are attributed to the "focal adhesion clutch," in which moving actin filaments transmit force to integrins via dynamic protein interactions. To elucidate these processes, we measured force on talin together with actin flow speed. While force on talin in small lamellipodial adhesions correlated with actin flow, talin tension in large adhesions further from the cell edge was mainly flow-independent. Stiff substrates shifted force transfer toward the flow-independent mechanism. Flow-dependent force transfer required talin's C-terminal actin binding site, ABS3, but not vinculin. Flow-independent force transfer initially required vinculin and at later times the central actin binding site, ABS2. Force transfer through integrins thus occurs not through a continuous clutch but through a series of discrete states mediated by distinct protein interactions, with their ratio modulated by substrate stiffness.

Fractures have a great impact on health all around the world and with fracture healing optimization; this problem could be resolved partially. To make a practical contribution to this issue, the knowledge of bone tissue, cellularity, and metabolism is essential, especially cytoskeletal architecture and its transformations according to external pressures. Special physical and chemical characteristics of the extracellular matrix (ECM) allow the transmission of mechanical stimuli from outside the cell to the plasmatic membrane. The osteocyte cytoskeleton is conformed by a complex network of actin and microtubules combined with crosslinker proteins like vinculin and fimbrin, connecting and transmitting outside stimuli through EMC to cytoplasm. Herein, critical signaling pathways like Cx43-depending ones, MAPK/ERK, Wnt, YAP/TAZ, Rho-ROCK, and others are activated due to mechanical stimuli, resulting in osteocyte cytoskeletal changes and ECM remodeling, altering the tissue and, therefore, the bone. In recent years, the osteocyte has gained more interest and value in relation to bone homeostasis as a great coordinator of other cell populations, thanks to its unique functions. By integrating the latest advances in relation to intracellular signaling pathways, mechanotransmission system of the osteocyte and bone tissue engineering, there are promising experimental strategies, while some are ready for clinical trials. This work aims to show clearly and precisely the integration between cytoskeleton and main molecular pathways in relation to mechanotransmission mechanism in osteocytes, and the use of this theoretical knowledge in therapeutic tools for bone fracture healing.

Diamond-like carbon (DLC) is a biocompatible material that has many potential biomedical applications, including in orthopaedics. DLC layers doped with Cr at atomic percent (at.%) of 0, 0.9, 1.8, 7.3, and 7.7 at.% were evaluated with reference to their osteoinductivity with human bone marrow mesenchymal stromal cells (hMSCs), immune activation potential with RAW 264.7 macrophage-like cells, and their effect on apoptosis in Saos-2 human osteoblast-like cells and neonatal human dermal fibroblasts (NHDFs). At mRNA level, hMSCs on DLC doped with 0.9 and 7.7 at.% of Cr reached higher maximum values of both RUNX2 and alkaline phosphatase. An earlier onset of mRNA production of type I collagen and osteocalcin was also observed on these samples; they also supported the production of both type I collagen and osteocalcin. RAW 264.7 macrophages were screened using a RayBio™ Human Cytokine Array for cytokine production. 10 cytokines were at a concentration more than 2 × as high as the concentration of a positive control, but the values for the DLC samples were only moderately higher than the values on glass. NHDF cells, but not Saos-2 cells, had a higher expression of pro-apoptotic markers Bax and Bim and a lower expression of anti-apoptotic factor BCL-XL in proportion to the Cr content. Increased apoptosis was also proven by annexin V staining. These results show that a Cr-doped DLC layer with a lower Cr content can act as an osteoinductive material with relatively low immunogenicity, but that a higher Cr content can induce cell apoptosis.

In recent years, protein decomposition has become of increasing interest for the use in forensic estimation of the postmortem interval (PMI). Especially skeletal muscle tissue has proven to be a prime target tissue, among other reasons, due to its large abundance in the human body. In this regard, it is important to know whether there are any intra- and intermuscular differences in the behavior of protein degradation. Thus, samples from different locations within several skeletal muscles as well as from cardiac and smooth muscle tissue samples were collected from three autopsy cases with varying degree of decomposition. Samples were analyzed by SDS-PAGE and Western blotting and compared for protein degradation patterns. Intramuscular variations turned out to be minimal and without major influence for the use of the method. Observed intermuscular differences provide possibilities for future improvement of the precision and temporal application range. The results of this study show the strengths and current limitations of protein degradation-based PMI estimation and provide a deeper understanding of intraindividual postmortem protein degradation processes.

Rationale: ZO-1 (Zonula occludens-1), a plasma membrane-associated scaffolding protein regulates signal transduction, transcription, and cellular communication. Global deletion of ZO-1 in the mouse is lethal by embryonic day 11.5. The function of ZO-1 in cardiac myocytes (CM) is largely unknown. Objective: To determine the function of CM ZO-1 in the intact heart, given its binding to other CM proteins that have been shown instrumental in normal cardiac conduction and function. Methods and Results: We generated ZO-1 CM-specific knockout (KO) mice using α-Myosin Heavy Chain-nuclear Cre (ZO-1cKO) and investigated physiological and electrophysiological function by echocardiography, surface ECG and conscious telemetry, intracardiac electrograms and pacing, and optical mapping studies. ZO-1cKO mice were viable, had normal Mendelian ratios, and had a normal lifespan. Ventricular morphometry and function were not significantly different between the ZO-1cKO versus control (CTL) mice, basally in young or aged mice, or even when hearts were subjected to hemodynamic loading. Atrial mass was increased in ZO-1cKO. Electrophysiological and optical mapping studies indicated high-grade atrioventricular (A-V) block in ZO-1cKO comparing to CTL hearts. While ZO-1-associated proteins such as vinculin, connexin 43, N-cadherin, and α-catenin showed no significant change with the loss of ZO-1, Connexin-45 and Coxsackie-adenovirus (CAR) proteins were reduced in atria of ZO-1cKO. Further, with loss of ZO-1, ZO-2 protein was increased significantly in ventricular CM in a presumed compensatory manner but was still not detected in the AV nodal myocytes. Importantly, the expression of the sodium channel protein NaV1.5 was altered in AV nodal cells of the ZO-1cKO versus CTL. Conclusions: ZO-1 protein has a unique physiological role in cardiac nodal tissue. This is in alignment with its known interaction with CAR and Cx45, and a new function in regulating the expression of NaV1.5 in AV node. Uniquely, ZO-1 is dispensable for function of the working myocardium.

The ACMG/AMP variant classification framework was intended for highly penetrant Mendelian conditions. While it is appreciated that clinically relevant variants exhibit a wide spectrum of penetrance, accurately assessing and expressing the pathogenicity of variants with lower penetrance can be challenging. The vinculin (VCL) gene illustrates these challenges. Model organism data provide evidence that loss of function of VCL may play a role in cardiomyopathy and aggregate case-control studies suggest low penetrance. VCL loss of function variants, however, are rarely identified in affected probands and therefore there is a paucity of family studies clarifying the clinical significance of individual variants. This study, which aggregated data from >18,000 individuals who underwent gene panel or exome testing for inherited cardiomyopathies, identified 32 probands with VCL loss-of-function variants and confirmed enrichment in probands with dilated cardiomyopathy (odds ratio [OR] = 9.01; confidence interval [CI] = 4.93–16.45). Our data revealed that the majority of these individuals (89.5%) had pediatric onset of disease. Family studies demonstrated that heterozygous loss of function of VCL alone is insufficient to cause cardiomyopathy but that these variants do contribute to disease risk. In conclusion, VCL loss-of-function variants should be reported in a diagnostic setting but need to be clearly distinguished as having lower penetrance.

Exosomes are cell-secreted microvesicles that play important roles in epithelial ovarian cancer (EOC) progression, as they are constantly secreted into ascites fluids. While cells spontaneously release exosomes, alterations in intracellular calcium or extracellular pH can release additional exosomes. Yet, little is known about how these exosomes compare to those that are continuously released without stimulation and how they mediate cellular activities important in cancer progression. Here, we demonstrate that chelation of extracellular calcium leads to release of chelation-induced exosomes (CI-exosomes) from OVCAR-3 EOC cells. CI-exosomes display a unique miRNA profile compared to naturally secreted exosomes (SEC-exosomes). Furthermore, treatment with CI- and SEC-exosomes leads to differential biophysical and functional changes including, adhesion and migration in EOC-derived fibroblasts that suggest the development of a malignant tumor microenvironment. This result highlights how tumor environmental factors contribute to heterogeneity in exosome populations and how different exosome populations mediate diversity in stromal cell behavior.

Purpose: To use label-free proteomic method to investigate the mechanism of action of nuanggong zhitong decoction (NZD) on primary dysmenorrhea (PD). Methods: A mouse model of PD was established through oxytocin administration. The mice were divided into control group (normal mice), model group (PD mice administered normal saline), and treatment group (mice given NZD). The serum levels of PGE2 and PGF2α in the mice were measured by ELISA. The differentially expressed proteins (DEPs) among the three groups were revealed by identifying the proteins that were up-regulated (or down-regulated) in model group and down-regulated (or up-regulated) in the treatment group. The DEPs in the three groups were identified using Nano- HPLC-MS/MS, and their functions were investigated using bioinformatics analyses. The accuracy of proteomics was verified with western blot analysis. Results: Thirty-eight up-regulated and 66 down-regulated DEPs were identified. Bioinformatics analysis revealed that the DEPs were related to immune response, signal conduction, protein binding, and metabolism. STRING analysis indicated a total of 53 DEPs have direct or indirect functional links. Western blot results revealed that levels of Stat1, Rock1, vinculin and vaveolin-1 were consistent with the results of proteomic analysis. Conclusion: These findings provide further insights into the mechanism underlying the protective effects of NZD. Keywords: Primary dysmenorrhea, Uterus, Nuangong zhitong decoction, Vinculin, Caveolin, Differentially expressed proteins (DEPs), Bioinformatics

Matrix stiffness regulates the physiology of the cells and plays an important role in maintaining its homeostasis. It has been reported to regulate cell division, proliferation, migration, extracellular uptake and various other physiological processes. The alteration in matrix stiffness has also been well reported in various disease pathologies. However, in ocular system, Keratoconus (KC) is an ideal model to study the effect of matrix stiffness on endocytosis since the progression of the disease is controlled by increasing the stromal elasticity. Our study using corneal epithelial and retinal pigment epithelial cell lines showed that ocular cells do respond to matrix stiffness by altering their morphology and endocytic uptake of FITC-Dextran 20 kDa. Further, by using KC epithelium as a clinical model, we hypothesize that change in stromal elasticity may also affect the endocytosis of KC epithelium. Our results clearly showed alteration in the expression of actin binding proteins such as Phosphorylated Cofilin, Profilin, Focal adhesion kinase, and Vinculin. Apart from cytoskeletal rearrangement proteins, we also observed endocytic proteins such as Clathrin, Caveolin1 and Rab 11 to be affected by matrix stiffness. Our study thus establishes connecting role between endocytosis and matrix stiffness which could be used to understand the pathophysiology of keratoconus that it is influenced by both mechanical and biochemical factors.

Funding: This work was supported by Action Medical Research/ The Chief Scientist (CSO) Office, Scotland [GN 2614], Royal Society [RG160373], Carnegie Trust [50341], Wellcome Trust ISSF grant 105621/Z/14/Z, and RS Macdonald Charitable Trust grants to SP and Engineering and Physical Sciences Research Council [EP/P030017/1], Biotechnology and Biological Sciences Research Council [BB/P027148/1], and the European Research Council Starting Grant ABLASE [640012] grants to MCG. SP is a Royal Society University Research Fellow. Following its association with dyslexia in multiple genetic studies, the KIAA0319 gene has been extensively investigated in different animal models but its function in neurodevelopment remains poorly understood. We developed the first human cellular knockout model for KIAA0319 in RPE1 retinal pigment epithelia cells via CRISPR-Cas9n to investigate its role in processes suggested but not confirmed in previous studies, including cilia formation and cell migration. We observed in the KIAA0319 knockout increased cilia length and accelerated cell migration. Using Elastic Resonator Interference Stress Microscopy (ERISM), we detected an increase in cellular force for the knockout cells that was restored by a rescue experiment. Combining ERISM and immunostaining we show that RPE1 cells exert highly dynamic, piconewton vertical pushing forces through actin-rich protrusions that are surrounded by vinculin-rich pulling sites. This protein arrangement and force pattern has previously been associated to podosomes in other cells. KIAA0319 depletion reduces the fraction of cells forming these actin-rich protrusions. Our results suggest an involvement of KIAA0319 in cilia biology and cell–substrate force regulation. Publisher PDF

Peri-implantitis is an unsolved but critical problem with dental implants. It is postulated that creating a seal of gingival soft tissue around the implant neck is key to preventing peri-implantitis. The objective of this study was to determine the effect of UV surface treatment of titanium disks on the adhesion strength and retention time of oral connective tissues as well as on the adherence of mucosal fibroblasts. Titanium disks with a smooth machined surface were prepared and treated with UV light for 15 min. Keratinized mucosal tissue sections (3 × 3 mm) from rat palates were incubated for 24 h on the titanium disks. The adhered tissue sections were then mechanically detached by agitating the culture dishes. The tissue sections remained adherent for significantly longer (15.5 h) on the UV-treated disks than on the untreated control disks (7.5 h). A total of 94% of the tissue sections were adherent for 5 h or longer on the UV-treated disks, whereas only 50% of the sections remained on the control disks for 5 h. The adhesion strength of the tissue sections to the titanium disks, as measured by tensile testing, was six times greater after UV treatment. In the culture studies, mucosal fibroblasts extracted from rat palates were attached to titanium disks by incubating for 24, 48, or 96 h. The number of attached cells was consistently 15–30% greater on the UV-treated disks than on the control disks. The cells were then subjected to mechanical or chemical (trypsinization) detachment. After mechanical detachment, the residual cell rates on the UV-treated surfaces after 24 and 48 h of incubation were 35% and 25% higher, respectively, than those on the control surfaces. The remaining rate after chemical detachment was 74% on the control surface and 88% on the UV-treated surface for the cells cultured for 48 h. These trends were also confirmed in mouse embryonic fibroblasts, with an intense expression of vinculin, a focal adhesion protein, on the UV-treated disks even after detachment. The UV-treated titanium was superhydrophilic, whereas the control titanium was hydrophobic. X-ray photoelectron spectroscopy (XPS) chemical analysis revealed that the amount of carbon at the surface was significantly reduced after UV treatment, while the amount of TiOH molecules was increased. These ex vivo and in vitro results indicate that the UV treatment of titanium increases the adhesion and retention of oral mucosa connective tissue as a result of increased resistance of constituent fibroblasts against exogenous detachment, both mechanically and chemically, as well as UV-induced physicochemical changes of the titanium surface.

Herein, we propose an array of gold (Au)-coated SU-8 cantilevers with microgrooves for improved maturation of cardiomyocytes and describe its applications to drug-induced cardiac toxicity tests. Firstly, we evaluated the effect of cell culture substrates such as polydimethylsiloxane (PDMS), polyimide (PI), and SU-8 on the cardiomyocyte's maturation. Among these, the SU-8 with microgroove structures exhibits improved cardiomyocyte maturation. Further, thin layers of graphene and Au are coated on SU-8 substrates and the effects of these materials on cardiomyocyte maturation are evaluated by analyzing the expression of proteins such as alpha-actinin, Connexin 43 (Cx43), and Vinculin. While both conductive materials enhanced protein expression when compared to bare SU-8, the Au-coated SU-8 substrates demonstrated superior cardiomyocyte maturation. The cantilever structure is constructed using microgroove patterned SU-8 with and without an Au coating. The Au-coated SU-8 cantilever showed maximum displacement of 17.6 ± 0.3 μm on day 21 compared to bare SU-8 (14.2 ± 0.4 μm) owing to improved cardiomyocytes maturation. Verapamil and quinidine are used to characterize drug-induced changes in the contraction characteristics of cardiomyocytes on bare and Au-coated SU-8 cantilevers. The relative contraction forces and beat rates changed according to the calcium and sodium channel related drugs. Matured cardiomyocytes are less influenced by the drugs compared to immature cardiomyocytes and showed reliable IC50 values. These results indicate that the proposed Au-coated SU-8 cantilever array could help improve the accuracy of toxicity screening results by allowing for the use of cardiomyocytes that have been matured on the drug screening platform.

Background Integrins play a prominent role in osteogenic differentiation by transmitting both mechanical and chemical signals. Integrin expression is closely associated with tensile stress, which has a positive effect on osteogenic differentiation. We investigated the relationship between integrin αVβ3 and tensile stress. Methods Human fibroblasts were treated with c (RGDyk) and lentivirus transduction to inhibit function of integrin αVβ3. Y-15, cytochalasin D and verteporfin were used to inhibit phosphorylation of FAK, polymerization of microfilament and function of nuclear YAP, respectively. Fibroblasts were exposed to a cyclic tensile stress of 10% at 0.5 Hz, once a day for 2 h each application. Fibroblasts were harvested on day 4 and 7 post-treatment. The expression of ALP, RUNX2, integrin αVβ3, β-actin, talin-1, FAK, vinculin, and nuclear YAP was detected by Western blot or qRT-PCR. The expression and distribution of integrin αVβ3, vinculin, microfilament and nuclear YAP. Results Cyclic tensile stress was found to promote expression of ALP and RUNX2. Inhibition of integrin αVβ3 activation downregulated the rearrangement of microfilament and the expression of ALP, RUNX2 and nuclear YAP. When the polymerization of microfilament was inhibited the expression of ALP, RUNX2 and nuclear YAP were decreased. The phosphorylation of FAK induced by cyclic tensile stress reduced by the inhibition of integrin αVβ3. The expression of ALP and RUNX2 was decreased by inhibition of phosphorylation of FAK and inhibition of nuclear YAP. Conclusions Cyclic tensile stress promotes osteogenesis of human fibroblasts via integrin αVβ3-microfilament axis. Phosphorylation of FAK and nuclear YAP participates in this process.

It has been hypothesized that the host, viral factors, and secreted cytokines (especially TNF-α) play roles in the pathogenesis of secondary dengue infections. Mass spectrometry-based proteomic screening of cytoskeleton fractions isolated from human endothelial (EA.hy926) cells upon dengue virus (DENV) infection and TNF-α treatment identified 450 differentially altered proteins. Among them, decreased levels of moesin, actin stress fiber rearrangements, and dot-like formations of vinculin were observed with western blot analyses and/or immunofluorescence staining (IFA). In vitro vascular permeability assays using EA.hy926 cells, seeded on collagen-coated transwell inserts, showed low levels of transendothelial electrical resistance in treated cells. The synergistic effects of DENV infection and TNF-α treatment caused cellular permeability changes in EA.hy926 cells, which coincided with decreasing moesin levels and the production of abnormal organizations of actin stress fibers and vinculin. Functional studies demonstrated moesin overexpression restored transendothelial permeability in DENV/TNF-α-treated EA.hy926 cells. The present study improves the understanding of the disruption mechanisms of cytoskeleton proteins in enhancing vascular permeability during DENV infection and TNF-α treatment. The study also suggests that these disruption mechanisms are major factors contributing to vascular leakage in severe dengue patients.

Gastrointestinal (GI) disease is common in systemic sclerosis, and novel biomarkers are needed to identify and monitor these patients. Dr. Suliman and colleagues identify anti-vinculin autoantibodies in a subset of systemic sclerosis patients which associate with GI symptom severity, although more work is needed to determine their clinical utility.

Understanding the biological and morphological reactions of human cells towards different dentinal derivate grafting materials is fundamental for choosing the type of dentin for specific clinical situations. This study aimed to evaluate human periodontal ligament fibroblasts (hPLF) cells exposed to different dentinal derivates particles. The study design included the in vitro evaluation of mineralized dentine (SG), deproteinized and demineralized dentine (DDP), and demineralized dentine (TT) as test materials and of deproteinized bovine bone (BIOS) as the positive control material. The materials were kept with the hPLF cell line, and the evaluations were made after 24 h, 72 h, and 7 days of in vitro culture. The evaluated outcomes were proliferation by using XTT assays, the morphological characteristics by light microscopy (LM) and by the use of scanning electron microscopy (SEM), and adhesion by using confocal microscopy (CLSM). Overall, the experimental materials induced a positive response of the hPLFs in terms of proliferation and adhesion. The XTT assay showed the TT, and the SG induced significant growth compared to the negative control at 7 days follow-up. The morphological data supported the XTT assay: the LM observations showed the presence of densely packed cells with a modified shape, the SEM observations allowed the assessment of how fibroblasts exposed to DDP and TT presented cytoplasmatic extensions, and SG and BIOS also presented the thickening of the cellular membrane. The CLMS observations showed the expression of the proliferative marker, as well as and the expression of cytoskeletal elements involved in the adhesion process. In particular, the vinculin and integrin signals were stronger at 72 h, while the actin signal remained constantly expressed in all the follow-up of the sample exposed to SG material. The integrin signal was stronger at 72 h, and the vinculin and actin signals were stronger at 7 days follow-up in the sample exposed to DDP material. The vinculin and integrin signals were stronger at 72 h follow-up in the sample exposed to TT material, vinculin and integrin signals appear stronger at 24 h follow-up in the sample exposed to BIOS material. These data confirmed how dentinal derivates present satisfying biocompatibility and high conductivity and inductivity properties fundamental in the regenerative processes. Furthermore, the knowledge of the effects of the dentin’s degree of mineralization on cellular behavior will help clinicians choose the type of dentine derivates material according to the required clinical situation.

Capacitive-resistive electric transfer (CRET) therapies have been proposed as strategies for regeneration of cutaneous tissue lesions. Previous studies by our group have shown that intermittent stimulation with 448 kHz CRET currents at subthermal densities promotes in vitro proliferation of human stem cells involved in tissue regeneration. The present study investigates the effects of the in vitro exposure to these radiofrequency (RF) currents on the proliferation and migration of keratinocytes and fibroblasts, the main cell types involved in skin regeneration. The effects of the electric stimulation on cell proliferation and migration were studied through XTT and wound closure assays, respectively. The CRET effects on the expression and location of proteins involved in proliferation and migration were assessed by immunoblot and immunofluorescence. The obtained results reveal that electrostimulation promotes proliferation and/or migration in keratinocytes and fibroblasts. These effects would be mediated by changes observed in the expression and location of intercellular adhesion proteins such as β-catenin and E-cadherin, of proteins involved in cell-to-substrate adhesion such as vinculin, p-FAK and the metalloproteinase MMP-9, and of other proteins that control both processes: MAP kinases p-p38, p-JUNK and p-ERK1/2. These responses could represent a mechanism underlying the promotion of normotrophic wound regeneration induced by CRET. Indeed, electric stimulation would favor completion of granulation tissue formation prior to the closure of the outer tissue layers, thus preventing abnormal wound cicatrization or chronification.

Background Distinct fecal microbiota profiles are reported to be associated with various subtypes of IBS. Circulating antibodies to cytolethal-distending-toxin B (CdtB) and vinculin are proposed as biomarkers to identify post-infectious IBS. The aim of our study was to analyze serum levels of anti-CdtB and anti-vinculin antibodies in patients with different Functional Gastrointestinal Disorders (FGID) and their correlation with the composition of fecal microbiome. Methods The study cohort comprised 65 prospectively recruited individuals: 15 with Diarrhea-type-IBS (IBS-D), 13 with Constipation-type-IBS (IBS-C), 15 with functional dyspepsia (FD) and 22 healthy controls. FGID subgroups were defined according to Rome III criteria. Serum levels of anti-CdtB and anti-vinculin antibodies were measured by ELISA. Fecal microbiome composition analysis and assessment of dysbiosis were performed by GA-map®-Dysbiosis Test. Results Positivity rate either for anti-CdtB or anti-vinculin antibodies was higher in the IBS-C group (76.9%) compared to IBS-D (40.0%), FD (60%) and healthy (63.6%) groups. Dysbiosis was more frequent in subjects positive for anti-CdtB antibodies and in IBS-C patients, who showed an increased amount of opportunistic/pro-inflammatory bacteria and reduced gut protective bacteria. IBS-C patients showed a high inter-individual variation of bacterial communities compared to other FGID subgroups and healthy individuals, whereas microbial profiles of patients with IBS-D and FD were overlapping with those of healthy controls. No bacteria markers showed significant differences between FGID subgroups and healthy controls. Conclusion Neither anti-CdtB/anti-vinculin antibodies nor fecal microbial profiles allowed to discriminate between specific FGID subgroups. Dysbiosis was more frequent in patients presenting with anti-CdtB antibodies and in IBS-C patients.

The cytoplasmic tails of classical cadherins form a multiprotein cadherin-catenin complex (CCC) that constitutes the major structural unit of adherens junctions (AJs). The CCC in AJs forms junctional clusters, "E clusters," driven by cis and trans interactions in the cadherin ectodomain and stabilized by α-catenin-actin interactions. Additional proteins are known to bind to the cytoplasmic region of the CCC. Here, we analyze how these CCC-associated proteins (CAPs) integrate into cadherin clusters and how they affect the clustering process. Using a cross-linking approach coupled with mass spectrometry, we found that the majority of CAPs, including the force-sensing protein vinculin, interact with CCCs outside of AJs. Accordingly, structural modeling shows that there is not enough space for CAPs the size of vinculin to integrate into E clusters. Using two CAPs, scribble and erbin, as examples, we provide evidence that these proteins form separate clusters, which we term "C clusters." As proof of principle, we show, by using cadherin ectodomain monoclonal antibodies (mAbs), that mAb-bound E-cadherin forms separate clusters that undergo trans interactions. Taken together, our data suggest that, in addition to its role in cell-cell adhesion, CAP-driven CCC clustering serves to organize cytoplasmic proteins into distinct domains that may synchronize signaling networks of neighboring cells within tissues.

Background/aim Proteomics is an approach that can detect differentially expressed proteins between cancerous and non-cancerous tissue samples. Previously, we found that vinculin was predominantly expressed in pancreatic cancerous tissues compared to adjacent non-cancerous tissues by performing proteomic differential display analysis. However, the clinicopathological significance of vinculin in pancreatic cancer has not yet been documented. Materials and methods The GEPIA2 and the Human Protein Atlas databases were used to analyze vinculin expression levels in cancerous tissue samples and investigate whether its expression level is clinically associated with patient survival. Results Vinculin mRNA expression levels were solely increased in pancreatic cancer tissues, and increased expression was inversely related to patient survival. Higher levels of vinculin protein were found in pancreatic cancer tissues. In contrast, faint staining of vinculin was observed throughout the normal pancreatic tissues. Conclusion Vinculin may be an unfavorable prognostic indicator for patients with pancreatic cancer.

Transcriptomes associated with wooden breast (WB) were characterized in broilers at two different market ages. Breasts (Pectoralis major) were collected, 20-min postmortem, from male Ross 308 broilers slaughtered at 6 and 7 weeks of age. The breasts were classified as “non-WB” or “WB” based on palpation hardness scoring (non-WB = no abnormal hardness, WB = consistently hardened). Total RNA was isolated from 16 samples (n = 3 for 6 week non-WB, n = 3 for 6 week WB; n = 5 for 7 week non-WB, n = 5 for 7 week WB). Transcriptome was profiled using a chicken gene expression microarray with one-color hybridization technique, and compared between non-WB and WB samples of the same age. Among 6 week broilers, 910 transcripts were differentially expressed (DE) (false discovery rate, FDR < 0.05). Pathway analysis underlined metabolisms of glucose and lipids along with gap junctions, tight junction, and focal adhesion (FA) signaling as the top enriched pathways. For the 7 week broilers, 1,195 transcripts were identified (FDR < 0.05) with regulation of actin cytoskeleton, mitogen-activated protein kinase (MAPK) signaling, protein processing in endoplasmic reticulum and FA signaling highlighted as the enriched affected pathways. Absolute transcript levels of eight genes (actinin-1 – ACTN1, integrin-linked kinase – ILK, integrin subunit alpha 8 – ITGA8, integrin subunit beta 5 – ITGB5, protein tyrosine kinase 2 – PTK2, paxillin – PXN, talin 1 – TLN1, and vinculin – VCL) of FA signaling pathway were further elucidated using a droplet digital polymerase chain reaction. The results indicated that, in 6 week broilers, ITGA8 abundance in WB was greater than that of non-WB samples (p < 0.05). Concerning 7 week broilers, greater absolute levels of ACTN1, ILK, ITGA8, and TLN1, accompanied with a reduced ITGB5 were found in WB compared with non-WB (p < 0.05). Transcriptional modification of FA signaling underlined the potential of disrupted cell-cell communication that may incite aberrant molecular events in association with development of WB myopathy.

Growing evidence has demonstrated that biomimetic nanovesicles produced from specific cells show bioactive properties such as anti-tumor or anti-inflammatory activities. However, the properties of these nanovesicles are very diverse, depending on their cell sources. In this study, human tonsil-derived mesenchymal stem cells (TMSCs) were used in the production of functional biomimetic nanovesicles with anti-senescence. TMSCs were isolated from human tonsil tissue obtained by tonsillectomy. TMSC-derived nanovesicles (TMSC-NVs) were produced by serial extrusion using a mini-extruder. Western blotting and particle analysis were performed for characterization of TMSC-NVs. They were applied to both replicative and ultraviolet B-induced senescent human dermal fibroblasts in vitro. Following six days of treatment, analysis of the proliferation and senescence level of fibroblasts was performed using cell counting and senescence-associated β-galactosidase assay, respectively. Treatment with TMSC-NVs enhanced the cell proliferation and reduced the activity of senescence-associated β-galactosidase in both replicative and ultraviolet B-induced senescent cells. Treatment with TMSC-NVs resulted in increased expression of extracellular matrix and anti-oxidant genes. Treatment with TMSC-NVs resulted in reduced expression of vinculin in focal adhesion. These results show that TMSC-NVs have an effect on recovering from cellular senescence by oxidative stress and can be applied as useful materials for the development of skin rejuvenation.

Background The importance of an efficient seal created by soft (connective) tissues around newly inserted dental implants has become increasingly clear. This study was aimed to compare the compatibility of fibroblasts – the main cell type in connective tissues – with three dental materials commonly employed for the manufacture of implant abutments, i.e. zirconia (ZrO2), titanium oxide (TiO2) and polyether-ether-ketone (PEEK), as well as the effects of UVC photofunctionalization of the materials prior to the contact with cells. Materials and methods Human fibroblasts (MRC-5 cell line) were allowed to attach and grow up to 72 h on disks of each material, in absence or after UVC photofunctionalization. Cell viability as well as the expression of five genes related to extracellular matrix (ECM) homeostasis (fibronectin, collagen 1A1, vinculin, metalloproteases 2 and 9) were assessed at various times after cell seeding. Results The viability of fibroblasts was higher when cultured on zirconia disks, in the order ZrO2 > TiO2 > PEEK. The lower viability observed with untreated PEEK could be however recovered after UVC photofunctionalization. Early upregulation was observed of genes involved in cell adhesion and migration (FN1, VCL) as well as in ECM remodeling (MMP9). UVC photofunctionalization significantly enhanced these effects with all three materials tested. At variance, the expression of Col1A1 gene – coding for collagen type I-alpha 1 – was precociously downregulated, but UVC photofunctionalization could largely reverse this phenomenon. Conclusions Our observations suggest that fibroblasts cultured on UVC treated surfaces present an increased/accelerated activation of factors involved in the formation of new connective tissue. UVC photofunctionalization of dental implant abutments could thus enhance/accelerate the formation of soft tissue seals capable of preventing bacterial invasion.

Objective: Deficiency in vitamin B12/folate (methyl donor deficiency [MDD]) produces cardiovascular outcomes during aging and fetal programming effects in newborns of MDD mothers. Whether fetal programming provokes long-term effects on aorta remains largely unknown. Approach and Results: We investigated the impact of fetal programming on ascending aorta of aged rats born from mothers subjected to MDD during gestation/lactation. We performed morphological and molecular examinations of ascending aortas in 21 days- and 400 days-aged rats with initial MDD fetal programming (iMDD) compared with control matched rats. iMDD induces remodeling of the ascending aorta in aged rats, with collagen deposition ( P =0.0008), decreased thickness of elastin ( P P =0.0002). Proteomic analyses, Western blotting, and immunohistochemical examination revealed decreased expression of α-smooth muscle actin, vinculin, SM22α (smooth muscle 22α), and N-cadherin and increased expression of TGF (transforming growth factor) β1. Elastin breaks were correlated to increased neutrophil elastase ( P =0.0002), cathepsin-K ( P =0.0002), cathepsin-S ( P P P =0.02). Proximity Duolink ligation assay showed homocysteinylation of actin-associated and extracellular matrix proteins, including SM22α ( P =0.01), N-cadherin ( P =0.0008), and vinculin ( P =0.001), which was associated with elastin breaks ( P =0.002) and increased expression of MARS (methionyl-tRNA synthetase; involved in irreversible protein homocysteinylation). Furthermore, we observed an inverse relationship between elastin breaks and blood pressure (systolic, P =0.004 and diastolic, P =0.0007). Conclusions: MDD fetal programming produced altered integrity and remodeling of ascending aorta during aging and irreversible MARS-associated homocysteinylation of key proteins of extracellular matrix and elastin homeostasis. This contributes to understanding why homocysteine-lowering vitamin B supplementation fails to relieve vascular complications in adulthood.

For cellular adaptation in mechanical environments, it is important to consider transmission of forces from the outside to the inside of cells via a focal molecular complex. The focal molecular complex, which consists of integrin, talin, vinculin and actin, is known to form in response to a force applied via the extra-cellular matrix (ECM). In the early formation process of the complex, the complex–actin connection is reinforced. These structural changes of the nascent complex result in an increase in its mechanical integrity and overall stiffness, possibly leading to the maturation of the nascent complex by enhancing force transmission. In this study, we hypothesized that the complex component talin is a crucial factor in increasing the stiffness of the nascent complex. To test the hypothesis, we used atomic force microscopy (AFM) to measure the stiffness of the nascent complex using a probe coated with fibronectin. Stiffness measurements were conducted for intact and talin knocked-down cells. Our results demonstrated that talin was required to increase the stiffness of the nascent complex, which could be caused by the reinforced connection between the complex and actin filaments mediated by talin.

The adhesion morphology of a cell monolayer results in a mechanical force inside cells, between cells, or between cells and substrates. The mechanical force regulates the differentiation of stem cells, but its influence on cell fusion is seldom studied. The present study is focused on osteoclast precursors, RAW264.7 monocytes, which can fuse into multinucleated cells (MNCs) responsible for bone resorption. Cells were cultured on circular and ring-like patterned substrates. Then, cell fusion, cell-substrate traction force, and force-sensitive molecules in different regions were measured and analyzed. Results showed that MNCs mainly appeared in the interior of the ring-like pattern and the central zone of the circular pattern, where both cell-substrate traction force and in-plane maximal shear stress were smaller than that at the patterns' edge. The immunostaining results revealed that F-actin, vinculin, β-catenin, and E-cadherin were highly distributed at the edge of patterns. High seeding density of cells promoted mechanical force-dependent fusion. When calcium-dependent cell-cell connections were inhibited by E-cadherin antibody or low-calcium medium, the fusion into MNCs was greatly reduced. Thus, the morphology of cell monolayer decides the mechanical state of cell-substrate interaction and cell-cell connection, ultimately regulating the fusion of osteoclast precursors.

Vinculin is a highly conserved protein involved in cell proliferation, migration, and adhesion. However, the effects of vinculin on gastric cancer (GC) remain unclear. Therefore, we aimed to explore the functional role of vinculin in GC, as well as its underlying mechanism. Expression of vinculin in patients with GC was analyzed by real-time polymerase chain reaction, Western blot analysis, and immunohistochemistry. Overall survival was evaluated by the Kaplan-Meier method with the log-rank test. The relationship between vinculin and clinicopathological characteristics of patients with GC was further identified. In addition, we assessed the expression of vinculin in GC cell lines. Besides, vinculin was suppressed or overexpressed by transfection with small interfering (si-vinculin) or pcDNA-vinculin and then cell viability, cell apoptosis, and/or migration was respectively examined by the 3-(4, 5-dimethylthiazole-2-yl)-2, 5-biphenyl tetrazolium bromide assay, flow cytometer, and scratch assay, respectively. Moreover, the cell cycle- and apoptosis-related proteins were detected by Western blot analysis. The expression of vinculin was significantly increased in the GC tissues and cells compared with the nontumor tissues or cells. Vinculin protein positive staining was mainly located in the cell membrane and cytoplasm. Moreover, vinculin was significantly associated with Tumor Node Metastasis (TNM) and poor differentiation. Patients with high vinculin levels had significantly worse overall survival than those with low levels. Suppression of vinculin significantly decreased cell viability and migration and promoted cell apoptosis. However, overexpression of vinculin statistically increased cell viability but had no effects on cell apoptosis. Vinculin promotes GC proliferation and migration and predicts poor prognosis in patients with GC.

Epithelial wound healing is essential for maintaining the function and clarity of the cornea. Successful repair after injury involves the coordinated movements of cell sheets over the wounded region. While collective migration has been the focus of studies, the effects that environmental changes have on this form of movement are poorly understood. To examine the role of substrate compliancy on multi-layered epithelial sheet migration, we performed traction force and confocal microscopy to determine differences in traction forces and to examine focal adhesions on synthetic and biological substrates. The leading edges of corneal epithelial sheets undergo retraction or contraction prior to migration, and alterations in the sheet's stiffness are affected by the amount of force exerted by cells at the leading edge. On substrates of 30 kPa, cells exhibited greater and more rapid movement than on substrates of 8 kPa, which are similar to that of the corneal basement membrane. Vinculin and its phosphorylated residue Y1065 were prominent along the basal surface of migrating cells, while Y822 was prominent between neighboring cells along the leading edge. Vinculin localization was diffuse on a substrate where the basement membrane was removed. Furthermore, when cells were cultured on fibronectin-coated acrylamide substrates of 8 and 50 kPa and then wounded, there was an injury-induced phosphorylation of Y1065 and substrate dependent changes in the number and size of vinculin containing focal adhesions. These results demonstrate that changes in substrate stiffness affected traction forces and vinculin dynamics, which potentially could contribute to the delayed healing response associated with certain corneal pathologies.

To improve initial osteoblast adhesion and subsequent osseointegration, TiO2 nanotubes layer was constructed on the titanium (Ti) surface by anodic oxidation (AO), with an additional hydroxyapatite (HA) coating to form the AO/HA surface. Tests on in vitro cellular activity displayed that the AO surface, especially the AO/HA surface, promoted initial adhesion, proliferation and differentiation of osteoblast cells. The modified AO and AO/HA surfaces further presented an up-regulated gene expression of osteogenic and adhesion markers collagen type 1 (COL), osteopontin (OPN), osteocalcin (OCN) and vinculin. In addition, in vivo experiments with a rat model demonstrated that the AO surface, particularly the AO/HA surface, achieved earlier osseointegration and a superior bone bonding ability compared with Ti. Our study shed light on a synergistic role played by nanotopography and HA in promoting osteoblast adhesion, proliferation, differentiation and osseointegration, thus suggesting a promising method for better modifying the implant surface.

Although titanium (Ti) alloys have been widely used as implant materials, the bioinertness of pristine Ti impairs their bioactivity and early osseointegration. In the present work, we prepared TiO2 nanotubes (TNT) layer on the titanium (Ti) surface by anodic oxidation. The anodized surface was functionalized with human bone morphogenetic protein-2 coating to form the hBMP-2/TNT surface. The release behavior of hBMP-2 on the hBMP-2/TNT surface displayed a controlled and sustained pattern, compared to that on the hBMP-2/Ti surface, which showed a rapid release. In vitro cellular activity tests demonstrated that both TNT and hBMP-2/Ti surfaces, particularly the hBMP-2/TNT surface, enhanced adhesion, proliferation, and differentiation of osteoblast cells. Increased cell adhesion, improved cytoskeleton organization, and immunofluorescence staining of vinculin were observed on the modified surfaces. The TNT, hBMP-2/Ti, and hBMP-2/TNT surfaces, especially the hBMP-2/TNT surface, further displayed an upregulated gene expression of adhesion and osteogenic markers vinculin, collagen type 1, osteopontin, and osteocalcin, compared to the pristine Ti surface. In vivo experiments using a rat model demonstrated that the TNT and hBMP-2/Ti surfaces, in particular the hBMP-2/TNT surface, improved osseointegration and showed a superior bone bonding ability compared to Ti. Our study revealed a synergistic role played by TiO2 nanotubes nanotopography and hBMP-2 in promoting initial osteoblast adhesion, proliferation, differentiation, and osseointegration, thus suggesting a promising method for better modifying the implant surface.

Vinculin is a key player in sensing and responding to external mechanical cues such as extracellular matrix stiffness. Increased matrix stiffness is often associated with certain pathological conditions including hypertension induced cellular cytoskeleton changes in vascular smooth muscle (VSM) cells. However, little is known on how stiffness affects cytoskeletal remodeling via vinculin in VSM cells. Thus, we utilized matrices with elastic moduli that simulate vascular stiffness in different stages of hypertension to investigate how matrix stiffness regulates cell cytoskeleton via vinculin in synthetic VSM cells. Through selecting a suitable reference gene, we found that an increase in physiologically relevant extracellular matrix stiffness (2–50 kPa) downregulates vinculin gene expression but upregulates vinculin protein expression. This discrepancy, which was not observed previously for non-muscle cells, suggests that the vinculin-mediated mecahnotransduction mechanism in synthetic VSM cells may be more complex than those proposed for non-muscle cells. Also adding to previous findings, we found that VSM cell growth may be impeded by substrates that are either too soft or too rigid.