Amyloid Aggregates of Smooth-Muscle Titin Impair Cell Adhesion.
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ABSTRACT:
SUBMITTER: Bobylev AG
PROVIDER: S-EPMC8123791 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing β-sandwiches, as well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500-1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMTHMW, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-β structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-β structure-unlike other amyloidogenic proteins-formed without changes in the SMTHMW secondary structure. SMTHMW aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMTHMW amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis.
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Project description:Actin-myosin II filament-based contractile structures in striated muscle, smooth muscle, and nonmuscle cells contain the actin filament-cross-linking protein alpha-actinin. In striated muscle Z-disks, alpha-actinin interacts with N-terminal domains of titin to provide a structural linkage crucial for the integrity of the sarcomere. We previously discovered a long titin isoform, originally smitin, hereafter sm-titin, in smooth muscle and demonstrated that native sm-titin interacts with C-terminal EF hand region and central rod R2-R3 spectrin-like repeat region sites in alpha-actinin. Reverse transcription-PCR analysis of RNA from human adult smooth muscles and cultured rat smooth muscle cells and Western blot analysis with a domain-specific antibody presented here revealed that sm-titin contains the titin gene-encoded Zq domain that may bind to the alpha-actinin R2-R3 central rod domain as well as Z-repeat domains that bind to the EF hand region. We investigated whether the sm-titin Zq domain binds to alpha-actinin R2 and R3 spectrin repeat-like domain loops that lie in proximity with two-fold symmetry on the surface of the central rod. Mutations in alpha-actinin R2 and R3 domain loop residues decreased interaction with expressed sm-titin Zq domain in glutathione S-transferase pull-down and solid phase binding assays. Alanine mutation of a region of the Zq domain with high propensity for alpha-helix formation decreased apparent Zq domain dimer formation and decreased Zq interaction with the alpha-actinin R2-R3 region in surface plasmon resonance assays. We present a model in which two sm-titin Zq domains interact with each other and with the two R2-R3 sites in the alpha-actinin central rod.
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Project description:While the development of second- and third-generation drug-eluting stents (DES) have significantly improved patient outcomes by reducing smooth muscle cell (SMC) proliferation, DES have also been associated with an increased risk of late-stent thrombosis due to delayed re-endothelialization and hypersensitivity reactions from the drug-polymer coating. Furthermore, DES anti-proliferative agents do not counteract the upstream oxidative stress that triggers the SMC proliferation cascade. In this study, we investigate biocompatible amphiphilic macromolecules (AMs) that address high oxidative lipoprotein microenvironments by competitively binding oxidized lipid receptors and suppressing SMC proliferation with minimal cytotoxicity. To determine the influence of nanoscale assembly on proliferation, micelles and nanoparticles were fabricated from AM unimers containing a phosphonate or carboxylate end-group, a sugar-based hydrophobic domain, and a hydrophilic poly(ethylene glycol) domain. The results indicate that when SMCs are exposed to high levels of oxidized lipid stimuli, nanotherapeutics inhibit lipid uptake, downregulate scavenger receptor expression, and attenuate scavenger receptor gene transcription in SMCs, and thus significantly suppress proliferation. Although both functional end-groups were similarly efficacious, nanoparticles suppressed oxidized lipid uptake and scavenger receptor expression more effectively compared to micelles, indicating the relative importance of formulation characteristics (e.g., higher localized AM concentrations and nanotherapeutic stability) in scavenger receptor binding as compared to AM end-group functionality. Furthermore, AM coatings significantly prevented platelet adhesion to metal, demonstrating its potential as an anti-platelet therapy to treat thrombosis. Thus, AM micelles and NPs can effectively repress early stage SMC proliferation and thrombosis through non-cytotoxic mechanisms, highlighting the promise of nanomedicine for next-generation cardiovascular therapeutics.
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Project description:OBJECTIVE:The investment of newly formed endothelial cell tubes with differentiated smooth muscle cells (SMC) is critical for appropriate vessel formation, but the underlying mechanisms remain unknown. We previously showed that depletion of focal adhesion kinase (FAK) in the nkx2.5 expression domain led to aberrant outflow tract (OFT) morphogenesis and strove herein to determine the cell types and mechanisms involved. METHODS AND RESULTS:We crossed fak(loxp) targeted mice with available Cre drivers to deplete FAK in OFT SMC (FAK(wnt) and FAK(nk)) or coronary SMC (FAK(cSMC)). In each case, depletion of FAK led to defective vasculogenesis that was incompatible with postnatal life. Immunohistochemical analysis of the mutant vascular structures revealed that FAK was not required for progenitor cell proliferation, survival, or differentiation into SMC but was necessary for subsequent SMC recruitment to developing vasculature. Using a novel FAK-null SMC culture model, we found that depletion of FAK did not influence SMC growth or survival, but blocked directional SMC motility and invasion toward the potent endothelial-derived chemokine, platelet-derived growth factor PDGFBB. FAK depletion resulted in unstable lamellipodial protrusions due to defective spatial-temporal activation of the small GTPase, Rac-1, and lack of Rac1-dependent recruitment of cortactin (an actin stabilizing protein) to the leading edge. Moreover, FAK null SMC exhibited a significant reduction in stimulated extracellular matrix degradation. CONCLUSIONS:FAK drives PDGFBB-stimulated SMC chemotaxis/invasion and is essential for SMC to appropriately populate the aorticopulmonary septum and the coronary vascular plexus.
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Project description:Integrins regulate mechanotransduction between smooth muscle cells (SMCs) and the extracellular matrix (ECM). SMCs resident in the walls of airways or blood vessels are continuously exposed to dynamic mechanical forces due to breathing or pulsatile blood flow. However, the resulting effects of these forces on integrin dynamics and associated cell-matrix adhesion are not well understood. Here we present experimental results from atomic force microscopy (AFM) experiments, designed to study the integrin response to external oscillatory loading of varying amplitudes applied to live aortic SMCs, together with theoretical results from a mathematical model. In the AFM experiments, a fibronectin-coated probe was used cyclically to indent and retract from the surface of the cell. We observed a transition between states of firm adhesion and of complete detachment as the amplitude of oscillatory loading increased, revealed by qualitative changes in the force timecourses. Interestingly, for some of the SMCs in the experiments, switching behaviour between the two adhesion states is observed during single timecourses at intermediate amplitudes. We obtain two qualitatively similar adhesion states in the mathematical model, where we simulate the cell, integrins and ECM as an evolving system of springs, incorporating local integrin binding dynamics. In the mathematical model, we observe a region of bistability where both the firm adhesion and detachment states can occur depending on the initial adhesion state. The differences are seen to be a result of mechanical cooperativity of integrins and cell deformation. Switching behaviour is a phenomenon associated with bistability in a stochastic system, and bistability in our deterministic mathematical model provides a potential physical explanation for the experimental results. Physiologically, bistability provides a means for transient mechanical stimuli to induce long-term changes in adhesion dynamics-and thereby the cells' ability to transmit force-and we propose further experiments for testing this hypothesis.
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Project description:Vascular smooth muscle cell (VSMC) migration play a key role in the development of intimal hyperplasia and atherosclerosis. Galectin-1 (Gal-1) is a redox-sensitive ?-galactoside-binding lectin expressed in VSMCs with intracellular and extracellular localizations. Here we show that VSMCs deficient in Gal-1 (Gal-1-KO) exhibited greater motility than wild type (WT) cells. Likewise, Gal-1-KO-VSMC migration was inhibited by a redox-insensitive but activity-preserved Gal-1 (CSGal-1) in a glycan-dependent manner. Gal-1-KO-VSMCs adhered slower than WT cells on fibronectin. Cell spreading and focal adhesion (FA) formation examined by phalloidin and vinculin staining were less in Gal-1-KO-VSMCs. Concomitantly, FA kinase (FAK) phosphorylation was induced to a lower extent in Gal-1-KO cells. Analysis of FA dynamics by nocodazole washout assay demonstrated that FA disassembly, correlated with FAK de-phosphorylation, was faster in Gal-1-KO-VSMCs. Surface plasmon resonance assay demonstrated that CSGal-1 interacted with ?5?1integrin and fibronectin in a glycan-dependent manner. Chemical crosslinking experiment and atomic force microscopy further revealed the involvement of extracellular Gal-1 in strengthening VSMC-fibronectin interaction. In vivo experiment showed that carotid ligation-induced neointimal hyperplasia was more severe in Gal-1-KO mice than WT counterparts. Collectively, these data disclose that Gal-1 restricts VSMC migration by modulating cell-matrix interaction and focal adhesion turnover, which limits neointimal formation post vascular injury.
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Project description:BackgroundA new mechanism for intercellular communication has recently emerged that involves intercellular transfer of extracellular vesicles (EVs). Several studies have indicated that EVs may play a potential role in cell-to-cell communication between macrophage foam cells and vascular smooth muscle cells (VSMCs) in atherosclerotic lesion.Methods and resultsThis study involved the comparison of circulating EVs from atherosclerotic patients and control participants. The results showed that the circulation of the patients contained more leukocyte-derived EVs and that these EVs promoted more VSMC adhesion and migration than those of healthy participants. We then established a macrophage foam cell model and characterized the EVs from the macrophages. We used flow cytometric analyses and cell migration and adhesion assays and determined that the foam cells generated more EVs than the normal macrophages and that the foam cell-derived EVs were capable of promoting increased levels of VSMC migration and adhesion. Furthermore, we performed a proteomic analysis of the EVs. The data showed that the foam cell-derived EVs may promote VSMC adhesion and migration by regulating the actin cytoskeleton and focal adhesion pathways. In addition, Western blotting revealed that foam cell-derived EVs could promote the phosphorylation of ERK and Akt in VSMCs in a time-dependent manner. We also found that foam cell-derived EVs could enter the VSMCs and transfer integrins to the surface of these cells.ConclusionsThe data in our present study provide the first evidence that EVs from foam cells could promote VSMC migration and adhesion, which may be mediated by the integration of EVs into VSMCs and the subsequent downstream activation of ERK and Akt.
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Project description: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-?5 antibody coated AFM probe, we confirmed that cell stiffness was increased by AII, while we observed no change in adhesion to an ?5 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-?5 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.
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Project description:Molecular mechanisms responsible for abnormal endometrial vasculature in women receiving long-acting progestin-only contraceptives (LAPCs) are unknown. We hypothesize that LAPCs impair vascular smooth muscle cell (VSMC) and pericyte proliferation and migration producing thin-walled hyperdilated fragile microvessels prone to bleeding. Proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (αSMA) double-immunostaining assessed VSMC differentiation and proliferation in endometria from women before and after DepoProvera (Depo) treatment and from oophorectomized guinea pigs (OVX-GPs) treated with vehicle, estradiol (E2), medroxyprogesterone acetate (MPA), or E2+MPA. Whole-genome profiling, proliferation, and migration assays were performed on cultured VSMCs treated with MPA or etonogestrel (ETO). Endometrial vessels of Depo-administered women displayed reduced αSMA immunoreactivity and fewer PCNA (+) nuclei among αSMA (+) cells (P < 0.008). Microarray analysis of VSMCs identified several MPA- and ETO-altered transcripts regulated by STAT1 signaling (P < 2.22 × 10(-6)), including chemokine (C-C motif) ligand 2 (CCL2). Both MPA and ETO reduce VSMC proliferation and migration (P < 0.001). Recombinant CCL2 reversed this progestin-mediated inhibition, whereas a STAT1 inhibitor abolished the CCL2 effect. Similarly, the endometria of MPA treated OVX-GPs displayed decreased αSMA staining and fewer PCNA (+) nuclei in VSMC (P < 0.005). In conclusion, LAPCs promote abnormal endometrial vessel formation by inhibiting VSMC proliferation and migration.
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