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Dental Materials Journal Jan 2023After periodontal tissue injury, reconstruct soft tissue sealing around the tooth surface is of fundamental importance to treat periodontitis. Among multiple cell types,...
After periodontal tissue injury, reconstruct soft tissue sealing around the tooth surface is of fundamental importance to treat periodontitis. Among multiple cell types, fibroblast plays a central role in reestablishing functional periodontium. To enhance fibroblast activity, a novel metal-organic framework-based nanoplatform is fabricated using mesoporous Prussian blue (MPB) nanoparticles to load baicalein (BA), named MPB-BA. Drug release test displayed sustained BA release of MPB-BA. Cell proliferation, transwell migration and wound healing tests revealed accelerated fibroblast proliferation and migration for the established MPB-BA nanoplatform. Moreover, vinculin immunofluorescence staining, western blot and quantitative real-time PCR analysis showed up-regulated vinculin protein and integrin α5 and integrin β1 gene expressions for MPB-BA, suggesting improved cell adhesion. In addition, hematoxylin and eosin (H&E) and Masson trichromatic staining suggested superior anti-inflammatory and collagen fiber reconstruction effects for MPB-BA in a rat experimental periodontitis model in vivo. Our study may provide a promising strategy for the treatment of periodontitis.
Topics: Rats; Animals; Vinculin; Metal-Organic Frameworks; Wound Healing; Fibroblasts; Periodontitis
PubMed: 36244739
DOI: 10.4012/dmj.2022-096 -
American Journal of Respiratory Cell... Oct 2016Increased vascular endothelial cell (EC) permeability is a result of intercellular gap formation that may be induced by contraction-dependent and contraction-independent...
Increased vascular endothelial cell (EC) permeability is a result of intercellular gap formation that may be induced by contraction-dependent and contraction-independent mechanisms. This study investigated a role of the adaptor protein vinculin in EC permeability induced by contractile (thrombin) and noncontractile (IL-6) agonists. Although thrombin and IL-6 caused a similar permeability increase in human pulmonary ECs and disrupted the association between vinculin and vascular endothelial-cadherin, they induced different patterns of focal adhesion (FA) arrangement. Thrombin, but not IL-6, caused formation of large, vinculin-positive FAs, phosphorylation of FA proteins, FA kinase and Crk-associated substrate, and increased vinculin-talin association. Thrombin-induced formation of talin-positive FA and intercellular gaps were suppressed in ECs with small interfering RNA-induced vinculin knockdown. Vinculin knockdown and inhibitors of Rho kinase and myosin-II motor activity also attenuated thrombin-induced EC permeability. Importantly, ectopic expression of the vinculin mutant lacking the F-actin-binding domain decreased thrombin-induced Rho pathway activation and EC permeability. In contrast, IL-6-induced EC permeability did not involve RhoA- or myosin-dependent mechanisms but engaged Janus kinase/signal transducer and activator of transcription-mediated phosphorylation and internalization of vascular endothelial-cadherin. This process was vinculin independent but Janus kinase/tyrosine kinase Src-dependent. These data suggest that vinculin participates in a contractile-dependent mechanism of permeability by integrating FA with stress fibers, leading to maximal RhoA activation and EC permeability response. Vinculin inhibition does not affect contractile-independent mechanisms of EC barrier failure. This study provides, for the first time, a comparative analysis of two alternative mechanisms of vascular endothelial barrier dysfunction and defines a specific role for vinculin in the contractile type of permeability response.
PubMed: 27115795
DOI: 10.1165/rcmb.2015-0328OC -
Shigella IpaA mediates actin bundling through diffusible vinculin oligomers with activation imprint.Cell Reports Apr 2023Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin...
Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin head and tail domains that bind to actin filaments. Here, we show that Shigella IpaA triggers major allosteric changes in the head domain, leading to vinculin homo-oligomerization. Through the cooperative binding of its three vinculin-binding sites (VBSs), IpaA induces a striking reorientation of the D1 and D2 head subdomains associated with vinculin oligomerization. IpaA thus acts as a catalyst producing vinculin clusters that bundle actin at a distance from the activation site and trigger the formation of highly stable adhesions resisting the action of actin relaxing drugs. Unlike canonical activation, vinculin homo-oligomers induced by IpaA appear to keep a persistent imprint of the activated state in addition to their bundling activity, accounting for stable cell adhesion independent of force transduction and relevant to bacterial invasion.
Topics: Bacterial Proteins; Antigens, Bacterial; Actins; Vinculin; Shigella; Protein Binding
PubMed: 37071535
DOI: 10.1016/j.celrep.2023.112405 -
Cellular Signalling Jun 2016Endothelial cell (EC) barrier disruption induced by edemagenic agonists such as thrombin is a result of increased actomyosin contraction and enforcement of focal...
Endothelial cell (EC) barrier disruption induced by edemagenic agonists such as thrombin is a result of increased actomyosin contraction and enforcement of focal adhesions (FA) anchoring contracting stress fibers, which leads to cell retraction and force-induced disruption of cell junctions. In turn, EC barrier enhancement by oxidized phospholipids (OxPAPC) and other agonists is a result of increased tethering forces due to enforcement of the peripheral actin rim and enhancement of cell-cell adherens junction (AJ) complexes promoting EC barrier integrity. This study tested participation of the mechanosensitive adaptor, vinculin, which couples FA and AJ to actin cytoskeleton, in control of the EC permeability response to barrier disruptive (thrombin) and barrier enhancing (OxPAPC) stimulation. OxPAPC and thrombin induced different patterns of FA remodeling. Knockdown of vinculin attenuated both, OxPAPC-induced decrease and thrombin-induced increase in EC permeability. Thrombin stimulated the vinculin association with FA protein talin and suppressed the interaction with AJ protein, VE-cadherin. In contrast, OxPAPC stimulated the vinculin association with VE-cadherin. Thrombin and OxPAPC induced different levels of myosin light chain (MLC) phosphorylation and caused different patterns of intracellular phospho-MLC distribution. Thrombin-induced talin-vinculin and OxPAPC-induced VE-cadherin-vinculin association were abolished by myosin inhibitor blebbistatin. Expression of the vinculin mutant unable to interact with actin attenuated EC permeability changes and MLC phosphorylation caused by both, thrombin and OxPAPC. These data suggest that the specific vinculin interaction with FA or AJ in different contexts of agonist stimulation is defined by development of regional actyomyosin-based tension and participates in both, the barrier-disruptive and barrier-enhancing endothelial responses.
Topics: Cadherins; Cell Adhesion; Cell Membrane Permeability; Cells, Cultured; Cytoskeleton; Endothelial Cells; Endothelium, Vascular; Focal Adhesions; Humans; Myosin Light Chains; Phospholipids; Talin; Thrombin; Vinculin
PubMed: 26923917
DOI: 10.1016/j.cellsig.2016.02.015 -
PloS One 2019Vinculin (Vcn) is a ubiquitously expressed cytoskeletal protein that links transmembrane receptors to actin filaments, and plays a key role in regulating cell adhesion,...
Vinculin (Vcn) is a ubiquitously expressed cytoskeletal protein that links transmembrane receptors to actin filaments, and plays a key role in regulating cell adhesion, motility, and force transmission. Metavinculin (MVcn) is a Vcn splice isoform that contains an additional exon encoding a 68-residue insert within the actin binding tail domain. MVcn is selectively expressed at sub-stoichiometic amounts relative to Vcn in smooth and cardiac muscle cells. Mutations in the MVcn insert are linked to various cardiomyopathies. In vitro analysis has previously shown that while both proteins can engage filamentous (F)-actin, only Vcn can promote F-actin bundling. Moreover, we and others have shown that MVcn can negatively regulate Vcn-mediated F-actin bundling in vitro. To investigate functional differences between MVcn and Vcn, we stably expressed either Vcn or MVcn in Vcn-null mouse embryonic fibroblasts. While both MVcn and Vcn were observed at FAs, MVcn-expressing cells had larger but fewer focal adhesions per cell compared to Vcn-expressing cells. MVcn-expressing cells migrated faster and exhibited greater persistence compared to Vcn-expressing cells, even though Vcn-containing FAs assembled and disassembled faster. Magnetic tweezer measurements on Vcn-expressing cells show a typical cell stiffening phenotype in response to externally applied force; however, this was absent in Vcn-null and MVcn-expressing cells. Our findings that MVcn expression leads to larger but fewer FAs per cell, in conjunction with the inability of MVcn to bundle F-actin in vitro and rescue the cell stiffening response, are consistent with our previous findings of actin bundling deficient Vcn variants, suggesting that deficient actin-bundling may account for some of the differences between Vcn and MVcn.
Topics: Animals; Cell Line; Cell Movement; Focal Adhesions; Gene Expression Regulation; Mechanotransduction, Cellular; Mice; Models, Molecular; Protein Domains; Vinculin
PubMed: 31483833
DOI: 10.1371/journal.pone.0221962 -
Calpain inhibition decreases myocardial fibrosis in chronically ischemic hypercholesterolemic swine.The Journal of Thoracic and... Jan 2022Calpain activation during ischemia is known to play critical roles in myocardial remodeling. We hypothesize that calpain inhibition (CI) may serve to reverse and/or...
OBJECTIVES
Calpain activation during ischemia is known to play critical roles in myocardial remodeling. We hypothesize that calpain inhibition (CI) may serve to reverse and/or prevent fibrosis in chronically ischemic myocardium.
METHODS
Yorkshire swine were fed a high-cholesterol diet for 4 weeks followed by placement of an ameroid constrictor on the left circumflex artery to induce myocardial ischemia. 3 weeks later, animals received either: no drug; high-cholesterol control group (CON; n = 8); low-dose CI (0.12 mg/kg; LCI, n = 9); or high-dose CI (0.25 mg/kg; HCI, n = 8). The high-cholesterol diet and CI were continued for 5 weeks, after which myocardial tissue was harvested. Tissue samples were analyzed by western blot for changes in protein content.
RESULTS
In the setting of hypercholesterolemia and chronic myocardial ischemia, CI decreased the expression of collagen in ischemic and nonischemic myocardial tissue. This reduced collagen content was associated with a corresponding decrease in Jak/STAT/MCP-1 signaling pathway, suggesting a role for Jak 2 signaling in calpain activity. CI also decreases the expression of focal adhesion proteins (vinculin) and stabilizes the expression of cytoskeletal and structural proteins (N-cadherin, α-fodrin, desmin, vimentin, filamin, troponin-I). CI had no significant effect on metabolic and hemodynamic parameters.
CONCLUSIONS
Calpain inhibition may be a beneficial medical therapy to decrease collagen formation in patients with coronary artery disease and associated comorbidities.
Topics: Animals; Calpain; Chemokine CCL2; Collagen; Coronary Artery Disease; Disease Models, Animal; Fibrosis; Glycoproteins; Hypercholesterolemia; Janus Kinase 2; Myocardial Ischemia; Myocardium; STAT Transcription Factors; Signal Transduction; Swine; Ventricular Remodeling
PubMed: 32359903
DOI: 10.1016/j.jtcvs.2019.11.150 -
International Journal of Molecular... May 2023Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn's disease (CD). However, these drugs are not without adverse events,...
Therapy with anti-tumor necrosis factor (TNF) has dramatically changed the natural history of Crohn's disease (CD). However, these drugs are not without adverse events, and up to 40% of patients could lose efficacy in the long term. We aimed to identify reliable markers of response to anti-TNF drugs in patients with CD. A consecutive cohort of 113 anti-TNF naive patients with CD was stratified according to clinical response as short-term remission (STR) or non-STR (NSTR) at 12 weeks of treatment. We compared the protein expression profiles of plasma samples in a subset of patients from both groups prior to anti-TNF therapy by SWATH proteomics. We identified 18 differentially expressed proteins ( ≤ 0.01, fold change ≥ 2.4) involved in the organization of the cytoskeleton and cell junction, hemostasis/platelet function, carbohydrate metabolism, and immune response as candidate biomarkers of STR. Among them, vinculin was one of the most deregulated proteins ( < 0.001), whose differential expression was confirmed by ELISA ( = 0.054). In the multivariate analysis, plasma vinculin levels along with basal CD Activity Index, corticosteroids induction, and bowel resection were factors predicting NSTR.
Topics: Humans; Crohn Disease; Tumor Necrosis Factor Inhibitors; Vinculin; Tumor Necrosis Factor-alpha; Antineoplastic Agents; Remission Induction; Infliximab
PubMed: 37240037
DOI: 10.3390/ijms24108695 -
Nature Cell Biology Jul 2015Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved...
Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin's interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function.
Topics: Actins; Blotting, Western; Cell Line; Cell Line, Tumor; Fluorescence Resonance Energy Transfer; Focal Adhesions; Humans; Integrins; Luminescent Proteins; Microscopy, Fluorescence; Models, Molecular; Mutation; Nanostructures; Nanotechnology; Paxillin; Protein Binding; Protein Structure, Tertiary; RNA Interference; Talin; Vinculin
PubMed: 26053221
DOI: 10.1038/ncb3180 -
The Korean Journal of Physiology &... Mar 2020Interstitial cells of Cajal (ICC) are known as the pacemaker cells of gastrointestinal tract, and it has been reported that acute gastroenteritis induces intestinal...
Interstitial cells of Cajal (ICC) are known as the pacemaker cells of gastrointestinal tract, and it has been reported that acute gastroenteritis induces intestinal dysmotility through antibody to vinculin, a cytoskeletal protein in gut, resulting in small intestinal bacterial overgrowth, so that anti-vinculin antibody can be used as a biomarker for irritable bowel syndrome. This study aimed to determine correlation between serum anti-vinculin antibody and ICC density in human stomach. Gastric specimens from 45 patients with gastric cancer who received gastric surgery at Kangwon National University Hospital from 2013 to 2017 were used. ICC in inner circular muscle, and myenteric plexus were counted. Corresponding patient's blood samples were used to determine the amount of anti-vinculin antibody by enzyme-linked immunosorbent assay. Analysis was done to determine correlation between anti-vinculin antibody and ICC numbers. Patients with elevated anti-vinculin antibody titer (above median value) had significantly lower number of ICC in inner circular muscle (71.0 240.5, p = 0.047), and myenteric plexus (12.0 68.5, p < 0.01) compared to patients with lower anti-vinculin antibody titer. Level of serum anti-vinculin antibody correlated significantly with density of ICC in myenteric plexus (r = -0.379, p = 0.01; Spearman correlation). Increased level of circulating anti-vinculin antibody was significantly correlated with decreased density of ICC in myenteric plexus of human stomach.
PubMed: 32140042
DOI: 10.4196/kjpp.2020.24.2.185 -
Journal of Molecular Biology Apr 2019Debilitating heart conditions, notably dilated and hypertrophic cardiomyopathies (CMs), are associated with point mutations in metavinculin, a larger isoform of the...
Debilitating heart conditions, notably dilated and hypertrophic cardiomyopathies (CMs), are associated with point mutations in metavinculin, a larger isoform of the essential cytoskeletal protein vinculin. Metavinculin is co-expressed with vinculin at sub-stoichiometric ratios in cardiac tissues. CM mutations in the metavinculin tail domain (MVt) occur within the extra 68-residue insert that differentiates it from the vinculin tail domain (Vt). Vt binds actin filaments (F-actin) and promotes vinculin dimerization to bundle F-actin into thick fibers. While MVt binds to F-actin in a similar manner to Vt, MVt is incapable of F-actin bundling and inhibits Vt-mediated F-actin bundling. We performed F-actin co-sedimentation and negative-stain EM experiments to dissect the coordinated roles of metavinculin and vinculin in actin fiber assembly and the effects of three known metavinculin CM mutations. These CM mutants were found to weakly induce the formation of disordered F-actin assemblies. Notably, they fail to inhibit Vt-mediated F-actin bundling and instead promote formation of large assemblies embedded with linear bundles. Computational models of MVt bound to F-actin suggest that MVt undergoes a conformational change licensing the formation of a protruding sub-domain incorporating the insert, which sterically prevents dimerization and bundling of F-actin by Vt. Sub-domain formation is destabilized by CM mutations, disrupting this inhibitory mechanism. These findings provide new mechanistic insights into the ability of metavinculin to tune actin organization by vinculin and suggest that dysregulation of this process by CM mutants could underlie their malfunction in disease.
Topics: Actins; Animals; Cardiomyopathies; Chickens; Humans; Models, Molecular; Point Mutation; Protein Binding; Protein Domains; Protein Interaction Maps; Vinculin
PubMed: 30844403
DOI: 10.1016/j.jmb.2019.02.024