Gut microbial co-metabolite 2-methylbutyrylcarnitine exacerbates thrombosis via binding to and activating integrin α2ß1.

Thrombosis represents the leading cause of death and disability upon major adverse cardiovascular events (MACEs). Numerous pathological conditions such as COVID-19 and metabolic disorders can lead to a heightened thrombotic risk; however, the underlying mechanisms remain poorly understood. Our study illustrates that 2-methylbutyrylcarnitine (2MBC), a branched-chain acylcarnitine, is accumulated in patients with COVID-19 and in patients with MACEs. 2MBC enhances platelet hyperreactivity and thrombus formation in mice. Mechanistically, 2MBC binds to integrin α2ß1 in platelets, potentiating cytosolic phospholipase A2 (cPLA2) activation and platelet hyperresponsiveness. Genetic depletion or pharmacological inhibition of integrin α2ß1 largely reverses the pro-thrombotic effects of 2MBC. Notably, 2MBC can be generated in a gut-microbiota-dependent manner, whereas the accumulation of plasma 2MBC and its thrombosis-aggravating effect are largely ameliorated following antibiotic-induced microbial depletion. Our study implicates 2MBC as a metabolite that links gut microbiota dysbiosis to elevated thrombotic risk, providing mechanistic insight and a potential therapeutic strategy for thrombosis.

Restraining of glycoprotein VI- and integrin α2ß1-dependent thrombus formation by platelet PECAM1.

The platelet receptors, glycoprotein VI (GPVI) and integrin α2ß1 jointly control collagen-dependent thrombus formation via protein tyrosine kinases. It is unresolved to which extent the ITIM (immunoreceptor tyrosine-based inhibitory motif) receptor PECAM1 and its downstream acting protein tyrosine phosphatase PTPN11 interfere in this process. Here, we hypothesized that integrin α2ß1 has a co-regulatory role in the PECAM1- and PTPN11-dependent restraint of thrombus formation. We investigated platelet activation under flow on collagens with a different GPVI dependency and using integrin α2ß1 blockage. Blood was obtained from healthy subjects and from patients with Noonan syndrome with a gain-of-function mutation of PTPN11 and variable bleeding phenotype. On collagens with decreasing GPVI activity (types I, III, IV), the surface-dependent inhibition of PECAM1 did not alter thrombus parameters using control blood. Blockage of α2ß1 generally reduced thrombus parameters, most effectively on collagen IV. Strikingly, simultaneous inhibition of PECAM1 and α2ß1 led to a restoration of thrombus formation, indicating that the suppressing signaling effect of PECAM1 is masked by the platelet-adhesive receptor α2ß1. Blood from 4 out of 6 Noonan patients showed subnormal thrombus formation on collagen IV. In these patients, effects of α2ß1 blockage were counterbalanced by PECAM1 inhibition to a normal phenotype. In summary, we conclude that the suppression of GPVI-dependent thrombus formation by either PECAM1 or a gain-of-function of PTPN11 can be overruled by α2ß1 engagement.

CC16 drives VLA-2-dependent SPLUNC1 expression.

Rationale: CC16 (Club Cell Secretory Protein) is a protein produced by club cells and other non-ciliated epithelial cells within the lungs. CC16 has been shown to protect against the development of obstructive lung diseases and attenuate pulmonary pathogen burden. Despite recent advances in understanding CC16 effects in circulation, the biological mechanisms of CC16 in pulmonary epithelial responses have not been elucidated. Objectives: We sought to determine if CC16 deficiency impairs epithelial-driven host responses and identify novel receptors expressed within the pulmonary epithelium through which CC16 imparts activity. Methods: We utilized mass spectrometry and quantitative proteomics to investigate how CC16 deficiency impacts apically secreted pulmonary epithelial proteins. Mouse tracheal epithelial cells (MTECS), human nasal epithelial cells (HNECs) and mice were studied in naïve conditions and after Mp challenge. Measurements and main results: We identified 8 antimicrobial proteins significantly decreased by CC16-/- MTECS, 6 of which were validated by mRNA expression in Severe Asthma Research Program (SARP) cohorts. Short Palate Lung and Nasal Epithelial Clone 1 (SPLUNC1) was the most differentially expressed protein (66-fold) and was the focus of this study. Using a combination of MTECs and HNECs, we found that CC16 enhances pulmonary epithelial-driven SPLUNC1 expression via signaling through the receptor complex Very Late Antigen-2 (VLA-2) and that rCC16 given to mice enhances pulmonary SPLUNC1 production and decreases Mycoplasma pneumoniae (Mp) burden. Likewise, rSPLUNC1 results in decreased Mp burden in mice lacking CC16 mice. The VLA-2 integrin binding site within rCC16 is necessary for induction of SPLUNC1 and the reduction in Mp burden. Conclusion: Our findings demonstrate a novel role for CC16 in epithelial-driven host defense by up-regulating antimicrobials and define a novel epithelial receptor for CC16, VLA-2, through which signaling is necessary for enhanced SPLUNC1 production.

Carcinoma-associated fibroblast-derived lysyl oxidase-rich extracellular vesicles mediate collagen crosslinking and promote epithelial-mesenchymal transition via p-FAK/p-paxillin/YAP signaling.

Carcinoma-associated fibroblasts (CAFs) are the main cellular components of the tumor microenvironment and promote cancer progression by modifying the extracellular matrix (ECM). The tumor-associated ECM is characterized by collagen crosslinking catalyzed by lysyl oxidase (LOX). Small extracellular vesicles (sEVs) mediate cell-cell communication. However, the interactions between sEVs and the ECM remain unclear. Here, we demonstrated that sEVs released from oral squamous cell carcinoma (OSCC)-derived CAFs induce collagen crosslinking, thereby promoting epithelial-mesenchymal transition (EMT). CAF sEVs preferably bound to the ECM rather than being taken up by fibroblasts and induced collagen crosslinking, and a LOX inhibitor or blocking antibody suppressed this effect. Active LOX (αLOX), but not the LOX precursor, was enriched in CAF sEVs and interacted with periostin, fibronectin, and bone morphogenetic protein-1 on the surface of sEVs. CAF sEV-associated integrin α2ß1 mediated the binding of CAF sEVs to collagen I, and blocking integrin α2ß1 inhibited collagen crosslinking by interfering with CAF sEV binding to collagen I. CAF sEV-induced collagen crosslinking promoted the EMT of OSCC through FAK/paxillin/YAP pathway. Taken together, these findings reveal a novel role of CAF sEVs in tumor ECM remodeling, suggesting a critical mechanism for CAF-induced EMT of cancer cells.

Dynamic-stiffening collagen-coated substrate enhances osteogenic differentiation of mesenchymal stem cells through integrin α2ß1.

Matrix stiffness dynamically increases during the bone formation process. Enhancement of the osteogenic differentiation of mesenchymal stem cells (MSCs) by the dynamic stiffening of the substrate has been reported in previous research. However, the mechanism by which the dynamic stiffening of the matrix effects the osteogenic differentiation of MSCs remains quite unknown. A previously reported dynamic hydrogel system with dynamic matrix stiffening was used in this study to investigate the mechanical transduction mechanism of MSCs. The integrin α2ß1 and phosphorylation focal adhesion kinase levels were evaluated. The results indicated that dynamic stiffening of the matrix mediated the activation of integrin α2ß1, and further influenced the focal adhesion kinase (FAK) phosphorylation level of MSCs. In addition, integrin α2 is a probable integrin subunit that causes integrin ß1 activation during the matrix dynamic stiffening process. The integrin ß1 is the main integrin subunit regulating the osteogenic differentiation of MSCs induced by FAK phosphorylation. Overall, the results suggested that the dynamic stiffness facilitated the osteogenic differentiation process of the MSCs by regulating the integrin-α2ß1-mediated mechanical transduction pathway, which implied that integrin α2ß1 played a crucial role in the physical biological coupling in the dynamic matrix microenvironment.

Pharmacological Inhibition of Glycoprotein VI- and Integrin α2ß1-Induced Thrombus Formation Modulated by the Collagen Type.

BACKGROUND: In secondary cardiovascular disease prevention, treatments blocking platelet-derived secondary mediators pose a risk of bleeding. Pharmacological interference of the interaction of platelets with exposed vascular collagens is an attractive alternative, with clinical trials ongoing. Antagonists of the collagen receptors, glycoprotein VI (GPVI), and integrin α2ß1, include recombinant GPVI-Fc dimer construct Revacept, 9O12 mAb based on the GPVI-blocking reagent Glenzocimab, Syk tyrosine-kinase inhibitor PRT-060318, and anti-α2ß1 mAb 6F1. No direct comparison has been made of the antithrombic potential of these drugs. METHODS: Using a multiparameter whole-blood microfluidic assay, we compared the effects of Revacept, 9O12-Fab, PRT-060318, or 6F1 mAb intervention with vascular collagens and collagen-related substrates with varying dependencies on GPVI and α2ß1. To inform on Revacept binding to collagen, we used fluorescent-labelled anti-GPVI nanobody-28. RESULTS AND CONCLUSION: In this first comparison of four inhibitors of platelet-collagen interactions with antithrombotic potential, we find that at arterial shear rate: (1) the thrombus-inhibiting effect of Revacept was restricted to highly GPVI-activating surfaces; (2) 9O12-Fab consistently but partly inhibited thrombus size on all surfaces; (3) effects of GPVI-directed interventions were surpassed by Syk inhibition; and (4) α2ß1-directed intervention with 6F1 mAb was strongest for collagens where Revacept and 9O12-Fab were limitedly effective. Our data hence reveal a distinct pharmacological profile for GPVI-binding competition (Revacept), GPVI receptor blockage (9O12-Fab), GPVI signaling (PRT-060318), and α2ß1 blockage (6F1 mAb) in flow-dependent thrombus formation, depending on the platelet-activating potential of the collagen substrate. This work thus points to additive antithrombotic action mechanisms of the investigated drugs.

Stiff substrates inhibit collagen accumulation via integrin α2ß1, FAK and Src kinases in human atrial fibroblast and myofibroblast cultures derived from patients with aortal stenosis.

The aim of the study was to confirm whether cell substrate stiffness may participate in the regulation of fibrosis. The involvement of integrin α2ß1, focal adhesion kinase (FAK) and Src kinase in signal transmission was investigated. Human atrial fibroblasts and myofibroblasts were cultured in both soft (2.23 ± 0.8 kPa) and stiff (8.28 ± 1.06 kPa) polyacrylamide gels. The cells were derived from the right atrium of patients with aortal stenosis undergoing surgery. The isolated cells, identified as fibroblasts or myofibroblasts, were stained positively with α smooth muscle actin, vimentin and desmin. The cultures settled on stiff gel demonstrated lower intracellular collagen and collagen type I telopeptide (PICP) levels; however, no changes in α1 chain of procollagen type I and III expression were noted. Inhibition of α2ß1 integrin by TC-I 15 (10-7 and 10-8 M) or α2 integrin subunit silencing augmented intracellular collagen level. Moreover, FAK or Src kinase inhibitors increased collagen content within the culture. Lower TIMP4 secretion was reported within the stiff gel cultures but neither MMP 2 nor TIMP-1, 2 or 3 release was altered. The stiff substrate cultures also demonstrated lower interleukin-6 release. Substrate stiffness modified collagen deposition within the atrial fibroblast and myofibroblast cultures. The elasticity of the cellular environment exerts a regulatory influence on both synthesis and breakdown of collagen. Integrin α2ß1, FAK and Src kinase activity participates in signal transmission, which may influence fibrosis in the atria of the human heart.

Two-Dimensional Peptide Assembly via Arene-Perfluoroarene Interactions for Proliferation and Differentiation of Myoblasts.

Supramolecular assembly based on aromatic interactions can provide well-defined nanostructures with an understanding of intermolecular interactions at the molecular level. The peptide assembly via a supramolecular approach can overcome the inherent limitations of bioactive peptides, such as proteolytic degradations and rapid internalizations into the cytosol. Although extensive research has been carried out on supramolecular peptide materials with a two-dimensional (2D) structure, more needs to be reported on biological activity studies using well-defined 2D peptide materials. Physical and chemical properties of the 2D peptide assembly attributed to their large surface area and flexibility can show low cytotoxicity, enhanced molecular loading, and higher bioconjugation efficiency in biological applications. Here, we report supramolecular 2D materials based on the pyrene-grafted amphiphilic peptide, which contains a peptide sequence (Asp-Gly-Glu-Ala; DGEA) that is reported to bind to the integrin α2ß1 receptor in 2D cell membranes. The addition of octafluoronaphthalene (OFN) to the pyrene-grafted peptide could induce a well-ordered 2D assembly by face-centered arene-perfluoroarene stacking. The DGEA-peptide 2D assembly with a flat structure, structural stability against enzymatic degradations, and a larger size can enhance the proliferation and differentiation of muscle cells via continuous interactions with cell membrane receptors integrin α2ß1 showing a low intracellular uptake (15%) compared to that (62%) of the vesicular peptide assembly. These supramolecular approaches via the arene-perfluoroarene interaction provide a strategy to fabricate well-defined 2D peptide materials with an understanding of assembly at the molecular level for the next-generation peptide materials.

The effects of inhibition and siRNA knockdown of collagen-binding integrins on human umbilical vein endothelial cell migration and tube formation.

Blood vessels in the body are lined with endothelial cells which have vital roles in numerous physiological and pathological processes. Collagens are major constituents of the extracellular matrix, and many adherent cells express several collagen-binding adhesion receptors. Here, we study the endothelium-collagen interactions mediated by the collagen-binding integrins, α1ß1, α2ß1, α10ß1 and α11ß1 expressed in human umbilical vein endothelial cells (HUVECs). Using qPCR, we found expression of the α10 transcript of the chondrocyte integrin, α10ß1, along with the more abundant α2, and low-level expression of α1. The α11 transcript was not detected. Inhibition or siRNA knockdown of the α2-subunit resulted in impaired HUVEC adhesion, spreading and migration on collagen-coated surfaces, whereas inhibition or siRNA knockdown of α1 had no effect on these processes. In tube formation assays, inhibition of either α1 or α2 subunits impaired the network complexity, whereas siRNA knockdown of these integrins had no such effect. Knockdown of α10 had no effect on cell spreading, migration or tube formation in these conditions. Overall, our results indicate that the collagen-binding integrins, α1ß1 and α2ß1 play a central role in endothelial cell motility and self-organisation.

Collagen Type XI Inhibits Lung Cancer-Associated Fibroblast Functions and Restrains the Integrin Binding Site Availability on Collagen Type I Matrix.

The tumor microenvironment, including cancer-associated fibroblast (CAF), plays an active role in non-small cell lung cancer (NSCLC) development and progression. We previously reported that collagen type XI and integrin α11, a collagen receptor, were upregulated in NSCLC; the latter promotes tumor growth and metastasis. We here explored the role of collagen type XI in NSCLC stroma. We showed that the presence of collagen type XI in collagen type I matrices inhibits CAF-mediated collagen remodeling and cell migration. This resulted in the inhibition of CAF-dependent lung-tumor cell invasion. Among the collagen receptors expressed on CAF, we determined that DDR2 and integrin α2ß1, but not integrin α11ß1, mediated the high-affinity binding to collagen type XI. We further demonstrated that collagen type XI restrained the integrin binding site availability on collagen type I matrices, thus limiting cell interaction with collagen type I. As a consequence, CAFs failed to activate FAK, p38 and Akt one hour after they interacted with collagen type I/XI. We concluded that collagen type XI may have a competitive negative feedback role on the binding of collagen type I to its receptors.

Rhesus rotavirus receptor-binding site affects high mobility group box 1 release, altering the pathogenesis of experimental biliary atresia.

Biliary atresia (BA) is a neonatal inflammatory cholangiopathy that requires surgical intervention by Kasai portoenterostomy to restore biliary drainage. Even with successful portoenterostomy, most patients diagnosed with BA progress to end-stage liver disease, necessitating a liver transplantation for survival. In the murine model of BA, rhesus rotavirus (RRV) infection of neonatal mice induces an inflammatory obstructive cholangiopathy that parallels human BA. The model is triggered by RRV viral protein (VP)4 binding to cholangiocyte cell-surface proteins. High mobility group box 1 (HMGB1) protein is a danger-associated molecular pattern that when released extracellularly moderates innate and adaptive immune response. In this study, we investigated how mutations in three RRV VP4-binding sites, RRVVP4-K187R (sialic acid-binding site), RRVVP4-D308A (integrin α2ß1-binding site), and RRVVP4-R446G (heat shock cognate 70 [Hsc70]-binding site), affects infection, HMGB1 release, and the murine model of BA. Newborn pups injected with RRVVP4-K187R and RRVVP4-D308A developed an obstruction within the extrahepatic bile duct similar to wild-type RRV, while those infected with RRVVP4-R446G remained patent. Infection with RRVVP4-R446G induced a lower level of HMGB1 release from cholangiocytes and in the serum of infected pups. RRV infection of HeLa cells lacking Hsc70 resulted in no HMGB1 release, while transfection with wild-type Hsc70 into HeLa Hsc70-deficient cells reestablished HMGB1 release, indicating a mechanistic role for Hsc70 in its release. Conclusion: Binding to Hsc70 contributes to HMGB1 release; therefore, Hsc70 potentially serves as a therapeutic target for BA.

Anti-GPVI nanobody blocks collagen- and atherosclerotic plaque-induced GPVI clustering, signaling, and thrombus formation.

BACKGROUND: The collagen receptor glycoprotein VI (GPVI) is an attractive antiplatelet target due to its critical role in thrombosis but minor involvement in hemostasis. OBJECTIVE: To investigate GPVI receptor involvement in platelet activation by collagen-I and atherosclerotic plaque using novel blocking and non-blocking anti-GPVI nanobodies (Nbs). METHODS: Nb effects on GPVI-mediated signaling and function were assessed by western blot and whole blood thrombus formation under flow. GPVI clustering was visualized in thrombi using fluorescently labeled Nb28. RESULTS: Under arterial shear, inhibitory Nb2 blocks thrombus formation and platelet activation on collagen and plaque, but only reduces adhesion on plaque. In contrast, adhesion on collagen, but not plaque, is decreased by blocking integrin α2ß1. Adhesion on plaque is maintained despite inhibition of integrins αvß3, α5ß1, α6ß1, and αIIbß3. Only combined αIIbß3 and α2ß1 blockade inhibits adhesion and thrombus formation to the same extent as Nb2 alone. Nb2 prevents GPVI signaling, with loss of Syk, Lat, and PLCÉ£2 phosphorylation, especially to plaque stimulation. Non-blocking fluorescently labeled Nb28 reveals distinct GPVI distribution patterns on collagen and plaque, with GPVI clustering clearly apparent on collagen fibers and less frequent on plaque. Clustering on collagen fibers is lost in the presence of Nb2. CONCLUSIONS: This work emphasizes the critical difference in GPVI-mediated platelet activation by plaque and collagen; it highlights the importance of GPVI clustering for downstream signaling and thrombus formation. Labeled Nb28 is a novel tool for providing mechanistic insight into this process and the data suggest Nb2 warrants further investigation as a potential anti-thrombotic agent.

Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin α2ß1.

In Alport mice, activation of the endothelin A receptor (ETA R) in mesangial cells results in sub-endothelial invasion of glomerular capillaries by mesangial filopodia. Filopodia deposit mesangial matrix in the glomerular basement membrane (GBM), including laminin 211 which activates NF-κB, resulting in induction of inflammatory cytokines. Herein we show that collagen α1(III) is also deposited in the GBM. Collagen α1(III) localized to the mesangium in wild-type mice and was found in both the mesangium and the GBM in Alport mice. We show that collagen α1(III) activates discoidin domain receptor family, member 1 (DDR1) receptors both in vitro and in vivo. To elucidate whether collagen α1(III) might cause podocyte injury, cultured murine Alport podocytes were overlaid with recombinant collagen α1(III), or not, for 24 h and RNA was analyzed by RNA sequencing (RNA-seq). These same cells were subjected to siRNA knockdown for integrin α2 or DDR1 and the RNA was analyzed by RNA-seq. Results were validated in vivo using RNA-seq from RNA isolated from wild-type and Alport mouse glomeruli. Numerous genes associated with podocyte injury were up- or down-regulated in both Alport glomeruli and cultured podocytes treated with collagen α1(III), 18 of which have been associated previously with podocyte injury or glomerulonephritis. The data indicate α2ß1 integrin/DDR1 co-receptor signaling as the dominant regulatory mechanism. This may explain earlier studies where deletion of either DDR1 or α2ß1 integrin in Alport mice ameliorates renal pathology. © 2022 Boys Town National Research Hospital. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Melatonin suppresses the metastatic potential of osteoblastic prostate cancers by inhibiting integrin α2 ß1 expression.

Advanced prostate cancer often develops into bone metastasis, which is characterized by aberrant bone formation with chronic pain and lower chances of survival. No treatment exists as yet for osteoblastic bone metastasis in prostate cancer. The indolamine melatonin (N-acetyl-5-methoxytryptamine) is a major regulator of the circadian rhythm. Melatonin has shown antiproliferative and antimetastatic activities but has not yet been shown to be active in osteoblastic bone lesions of prostate cancer. Our study investigations reveal that melatonin concentration-dependently decreases the migratory and invasive abilities of two osteoblastic prostate cancer cell lines by inhibiting FAK, c-Src, and NF-κB transcriptional activity via the melatonin MT1 receptor, which effectively inhibits integrin α2 ß1 expression. Melatonin therapy appears to offer therapeutic possibilities for reducing osteoblastic bone lesions in prostate cancer.

Alternagin-C, an alpha2beta1 integrin ligand, attenuates collagen-based adhesion, stimulating the metastasis suppressor 1 expression in triple-negative breast tumor cells.

Triple-negative breast cancer has an aggressive clinical course and its treatment has been challenging due to high metastatic risk. Molecular targets have been sought to provide better strategies for this type of cancer. Integrins are cell adhesion receptors involved in tumor progression and α2ß1 integrin, a collagen receptor, has a key role in breast metastasis. Disintegrins, a family of proteins from snake venoms, selectively block the function of integrin receptors. Alternagin-C (ALT-C), a disintegrin-like protein purified from Bothrops alternatus venom, binds to α2ß1 integrin, attenuating inflammation and angiogenesis, and decreasing metalloprotease levels in the tumor microenvironment, which suggests anti-metastatic effects. However, its mechanisms of action in metastatic tumor cells have not been fully explored. Here, we investigated ALT-C effects in a triple-negative breast cancer cell line (MDA-MB-231) to elucidate how α2ß1 integrin affects cellular adhesion, migration and gene expression related to metastasis. We observed that ALT-C attenuated cell adhesion of MDA-MB-231 cells to collagen I. α2 integrin subunit silencing in MDA-MB-231 cells did not inhibit cell adhesion and migration to collagen I, indicating that other integrins play a crucial role in cell motility for this cell line. ALT-C also stimulated the metastasis suppressor 1 (MTSS1) expression and decreased metalloproteases MMP9 and MMP2. Therefore, we suggest that ALT-C contributes to impair metastasis, preventing extracellular matrix degradation and tumor attachment to collagen I, increasing MTSS1. This study is the first to elucidate the anti-metastatic mechanism involving a disintegrin-like protein from snake venom targeting α2ß1 integrin and stimulating a metastasis suppressor.

Development and preliminary evaluation of an integrin α2ß1-targeted PET probe as a supplement and alternative of PSMA imaging for prostate cancer.

An integrin α2ß1-targeted PET probe (68Ga-IABtP) was developed to serve as a supplement and alternative of PSMA imaging for prostate cancer. 68Ga-IABtP was synthesized by labeling the precursor peptide with 68Ga with 93% labeling yield and 4.14 MBq/µg specific radioactivity. 68Ga-IABtP showed no specific uptake in LNCaP prostate cancer cell with low integrin α2ß1 expression but significantly increased uptake in PC-3 prostate cancer cell with high integrin α2ß1 expression, which could be specifically blocked by the integrin α2ß1 monoclonal antibody. The efflux experiments demonstrated that 68Ga-IABtP could rapidly penetrate into PC-3 cell after cell binding, thereby prolonging the residence time in the tumor and allow enough time for probe clearance from the circulation and non-specific organs. The biodistribution study indicated that 68Ga-IABtP showed no specific accumulation in non-target organs and was quickly cleared from the kidney. The in vivo PET-CT imaging demonstrated that 68Ga-IABtP showed no specific uptake in LNCaP tumor but could specifically accumulate in the PC-3 tumor, and was rapidly cleared from spleen, intestine, kidney and liver, resulting in excellent contrast effect with low background signal and high target to non-target ratios.

The voltage-gated K+ channel Kv1.3 modulates platelet motility and α2ß1 integrin-dependent adhesion to collagen.

Kv1.3 is a voltage-gated K+-selective channel with roles in immunity, insulin-sensitivity, neuronal excitability and olfaction. Despite being one of the largest ionic conductances of the platelet surface membrane, its contribution to platelet function is poorly understood. Here we show that Kv1.3-deficient platelets display enhanced ADP-evoked platelet aggregation and secretion, and an increased surface expression of platelet integrin αIIb. In contrast, platelet adhesion and thrombus formation in vitro under arterial shear conditions on surfaces coated with collagen were reduced for samples from Kv1.3-/- compared to wild type mice. Use of collagen-mimetic peptides revealed a specific defect in the engagement with α2ß1. Kv1.3-/- platelets developed significantly fewer, and shorter, filopodia than wild type platelets during adhesion to collagen fibrils. Kv1.3-/- mice displayed no significant difference in thrombus formation within cremaster muscle arterioles using a laser-induced injury model, thus other pro-thrombotic pathways compensate in vivo for the adhesion defect observed in vitro. This may include the increased platelet counts of Kv1.3-/- mice, due in part to a prolonged lifespan. The ability of Kv1.3 to modulate integrin-dependent platelet adhesion has important implications for understanding its contribution to normal physiological platelet function in addition to its reported roles in auto-immune diseases and thromboinflammatory models of stroke.

The Stiffness of Cardiac Fibroblast Substrates Exerts a Regulatory Influence on Collagen Metabolism via α2ß1 Integrin, FAK and Src Kinases.

Information about mechanical strain in the extracellular space is conducted along collagen fibers connected with integrins and then transmitted within cells. An aim of the study is to verify the hypothesis that the stiffness of cardiac human fibroblast substrates exerts a regulatory effect on collagen metabolism via integrin α2ß1 and downstream signaling. The experiments were performed on human cardiac fibroblasts cultured on stiff or soft polyacrylamide gels. Extracellular and intracellular collagen content, metalloproteinase-1 (MMP-1), metalloproteinase-9 (MMP-9) and expression of the α1 chain of the procollagen type I gene (Col1A1) were elevated in cultures settled on soft substrate. The substrate stiffness did not modify tissue inhibitors of matrix metalloproteinase capacity (TIMPs 1-4). Integrin α2ß1 inhibition (TC-I 15) or α2 subunit silencing resulted in augmentation of collagen content within the culture. Expression of Col1A1 and Col3A1 genes was increased in TC-I 15-treated fibroblasts. Total and phosphorylated levels of both FAK and Src kinases were elevated in fibroblasts cultured on stiff substrate. Inhibition of FAK (FAK kinase inhibitor 14) or Src kinase (AZM 47527) increased collagen content within the culture. The substrate stiffness exerted a regulatory influence on collagen metabolism via integrin α2ß1 and its downstream signaling (FAK and Src kinases) in cardiac fibroblasts.

Functional heterogeneity of IFN-γ-licensed mesenchymal stromal cell immunosuppressive capacity on biomaterials.

Mesenchymal stromal cells (MSCs) are increasingly combined with biomaterials to enhance their therapeutic properties, including their immunosuppressive function. However, clinical trials utilizing MSCs with or without biomaterials have shown limited success, potentially due to their functional heterogeneity across different donors and among different subpopulations of cells. Here, we evaluated the immunosuppressive capacity, as measured by the ability to reduce T-cell proliferation and activation, of interferon-gamma (IFN-γ)-licensed MSCs from multiple donors on fibrin and collagen hydrogels, the two most commonly utilized biomaterials in combination with MSCs in clinical trials worldwide according to ClinicalTrials.gov Variations in the immunosuppressive capacity between IFN-γ-licensed MSC donors on the biomaterials correlated with the magnitude of indoleamine-2,3-dioxygenase activity. Immunosuppressive capacity of the IFN-γ-licensed MSCs depended on the αV/α5 integrins when cultured on fibrin and on the α2/ß1 integrins when cultured on collagen. While all tested MSCs were nearly 100% positive for these integrins, sorted MSCs that expressed higher levels of αV/α5 integrins demonstrated greater immunosuppressive capacity with IFN-γ licensing than MSCs that expressed lower levels of these integrins on fibrin. These findings were equivalent for MSCs sorted based on the α2/ß1 integrins on collagen. These results demonstrate the importance of integrin engagement to IFN-γ licensed MSC immunosuppressive capacity and that IFN-γ-licensed MSC subpopulations of varying immunosuppressive capacity can be identified by the magnitude of integrin expression specific to each biomaterial.

Selectivity of the collagen-binding integrin inhibitors, TC-I-15 and obtustatin.

Integrins are a family of 24 adhesion receptors which are both widely-expressed and important in many pathophysiological cellular processes, from embryonic development to cancer metastasis. Hence, integrin inhibitors are valuable research tools which may have promising therapeutic uses. Here, we focus on the four collagen-binding integrins α1ß1, α2ß1, α10ß1 and α11ß1. TC-I-15 is a small molecule inhibitor of α2ß1 that inhibits platelet adhesion to collagen and thrombus deposition, and obtustatin is an α1ß1-specific disintegrin that inhibits angiogenesis. Both inhibitors were applied in cellular adhesion studies, using synthetic collagen peptide coatings with selective affinity for the different collagen-binding integrins and testing the adhesion of C2C12 cells transfected with each. Obtustatin was found to be specific for α1ß1, as described, whereas TC-I-15 is shown to be non-specific, since it inhibits both α1ß1 and α11ß1 as well as α2ß1. TC-I-15 was 100-fold more potent against α2ß1 binding to a lower-affinity collagen peptide, suggestive of a competitive mechanism. These results caution against the use of integrin inhibitors in a therapeutic or research setting without testing for cross-reactivity.