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.

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.

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.

Biphasic α2ß1 Integrin Expression in Breast Cancer Metastasis to Bone.

Integrins participate in the pathogenesis and progression of tumors at many stages during the metastatic cascade. However, current evidence for the role of integrins in breast cancer progression is contradictory and seems to be dependent on tumor stage, differentiation status, and microenvironmental influences. While some studies suggest that loss of α2ß1 enhances cancer metastasis, other studies suggest that this integrin is pro-tumorigenic. However, few studies have looked at α2ß1 in the context of bone metastasis. In this study, we aimed to understand the role of α2ß1 integrin in breast cancer metastasis to bone. To address this, we utilized in vivo models of breast cancer metastasis to bone using MDA-MB-231 cells transfected with an α2 expression plasmid (MDA-OEα2). MDA cells overexpressing the α2 integrin subunit had increased primary tumor growth and dissemination to bone but had no change in tumor establishment and bone destruction. Further in vitro analysis revealed that tumors in the bone have decreased α2ß1 expression and increased osteolytic signaling compared to primary tumors. Taken together, these data suggest an inverse correlation between α2ß1 expression and bone-metastatic potential. Inhibiting α2ß1 expression may be beneficial to limit the expansion of primary tumors but could be harmful once tumors have established in bone.

Implication of integrin α2ß1 in senescence of SK-Mel-147 human melanoma cells.

This investigation addressed the impact of integrin-initiated signaling pathways on senescence of tumor cells. In a model of human SK-Mel-147 melanoma cells, the silencing of integrin α2ß1 strongly reduced cell proliferation and enhanced the percentage of SA-ß-Gal-positive cells, a phenotypic feature of cellular senescence. These changes were accompanied by a significant increase in the activity of Akt and mTOR protein kinases and also in the expression of p53 and p21 oncosuppressors. Pharmacological inhibition of Akt and mTORC1 and genetic inhibition of p53 and p21 reduced the senescence of α2ß1-depleted SK-Mel-147 cells to the level of control cells. Based on our earlier data on the non-canonical functions of Akt isomers in the invasion and anoikis of SK-Mel-147 cells, we investigated the role of Akt isomers in senescence induced by α2ß1 suppression. The inhibition of Akt1 strongly reduced the percentage of SA-ß-Gal-positive cells in the α2ß1-depleted cell population, while the inhibition of Akt2 did not have a noticeable effect. Our data demonstrated for the first time that α2ß1 is involved in the protection of tumor cells against senescence and that senescence, which is induced by the downregulation of α2ß, is based on a signaling mechanism in which Akt1 performs a non-canonical function.

Integrin α2ß1 as a negative regulator of the laminin receptors α6ß1 and α6ß4.

Integrin α2ß1 is a widely expressed collagen I receptor which also mediates laminin-111 binding in some cell types, but the functional relevance of collagen versus laminin binding for different cell types is poorly understood. Here we use AFM-based singe-cell force spectroscopy (SCFS) to compare α2ß1-mediated adhesion strength to collagen and laminin in different cell types. Chinese Hamster Ovary (CHO) cells stably expressing integrin α2ß1 (CHO-A2) displayed enhanced adhesion to collagen, but weak adhesion to laminin, consistent with a role of α2ß1 as a receptor only for collagen in these cells. Inversely, the α2ß1-deficient CHO wildtype cells (CHO-WT) showed weak adhesion to collagen, but strong adhesion to laminin-111, in turn suggesting that integrin α2ß1 expression suppresses laminin binding. Analogous results were obtained in a pair of SAOS-2 human osteosarcoma cell lines. Again, wildtype cells (SAOS-WT) adhered strongly to laminin and poorly to collagen, while expression of integrin α2ß1 (SAOS-A2) induced strong adhesion to collagen, but reduced adhesion to laminin. Expression of α2ß1 also shifted cell spreading preference from laminin to collagen and suppressed laminin-dependent transmigration. In agreement with reduced laminin adhesion, α2ß1 expression downregulated transcription and expression of integrin subunits α6 and ß4, components of the main laminin-111 binding receptors integrin α6ß1 and α6ß4 in these cells. Integrin α6 and ß4 expression was also reduced when α2 expression was chemically induced using tetradecanoyl-phorbol-acetate (TPA). Our results thus show that integrin α2ß1 expression negatively regulates integrin α6ß1 and α6ß4-mediated adhesion, spreading and invasion on laminin in different cancer cell types. In contrast to SAOS-WT, but similar to SAOS-A2 osteosarcoma cells, primary Human osteoblasts (HOB) cells express α2 but only low levels of ß4 integrin, preferentially adhere to and spread on collagen over laminin and show suppressed laminin-dependent transmigration. By enhancing collagen binding directly and suppressing laminin binding indirectly through laminin receptor downregulation, α2ß1 expression may thus re-direct migrating cancer cells from laminin-rich to collagenous tissues and partially revert osteosarcoma cells towards an untransformed phenotype.

The stiffness-controlled release of interleukin-6 by cardiac fibroblasts is dependent on integrin α2ß1.

Cardiac fibroblasts are able to sense the rigidity of their environment. The present study examines whether the stiffness of the substrate in cardiac fibroblast culture can influence the release of interleukin-6 (IL-6), interleukin-11 (IL-11) and soluble receptor of IL-6 (sIL-6R). It also examines the roles of integrin α2ß1 activation and intracellular signalling in these processes. Cardiac fibroblasts were cultured on polyacrylamide gels and grafted to collagen, with an elasticity of E = 2.23 ± 0.8 kPa (soft gel) and E = 8.28 ± 1.06 kPa (stiff gel, measured by Atomic Force Microscope). Flow cytometry and ELISA demonstrated that the fibroblasts cultured on the soft gel demonstrated higher expression of the α2 integrin subunit and increased α2ß1 integrin count and released higher levels of IL-6 and sIL-6R than those on the stiff gel. Substrate elasticity did not modify fibroblast IL-11 content. The silencing of the α2 integrin subunit decreased the release of IL-6. Similar effects were induced by TC-I 15 (an α2ß1 integrin inhibitor). The IL-6 levels in the serum and heart were markedly lower in α2 integrin-deficient mice B6.Cg-Itga2tm1.1Tkun/tm1.1Tkun than wild type. Inhibition of Src kinase by AZM 475271 modifies the IL-6 level. sIL-6R secretion is not dependent on α2ß1 integrin. Conclusion: The elastic properties of the substrate influence the release of IL-6 by cardiac fibroblasts, and this effect is dependent on α2ß1 integrin and kinase Src activation.

Perlecan, a modular instructive proteoglycan with diverse functional properties.

This study reviewed some new aspects of the modular proteoglycan perlecan, a colossal proteoglycan with a 467 kDa core protein and five distinct functional domains. Perlecan is a heparan sulphate proteoglycan that transiently displays native CS sulphation motifs 4-C-3 and 7-D-4 during tissue morphogenesis these are expressed by progenitor cell populations during tissue development. Perlecan is susceptible to fragmentation by proteases during tissue development and in pathological tissues particularly in domains IV and V. The fragmentation pattern of domain IV has been suggested as a means of grading prostate cancer. Domain V of perlecan is of interest due to its interactive properties with integrin α5ß1 that promotes pericyte migration enhancing PDGF-BB-induced phosphorylation of PDGFRß, Src homology region 2 domain-containing phosphatase-2, and focal adhesion kinase supporting the repair of the blood brain barrier following ischaemic stroke. Fragments of domain V can also interact with α2ß1 integrin disrupting tube formation by endothelial cells. LG1-LG2, LG3 fragments can antagonise VEGFR2, and α2ß1 integrin interactions preventing angiogenesis by endothelial cells. These domain V fragments are of interest as potential anti-tumour agents. Perlecan attached to the luminal surfaces of endothelial cells in blood vessels acts as a flow sensor that signals back to endothelial and smooth muscle cells to regulate vascular tone and blood pressure. Perlecan also acts as a flow sensor in the lacuno-canalicular space regulating osteocytes and bone homeostasis. Along with its biomechanical regulatory properties in cartilaginous tissues this further extends the functional repertoire of this amazingly diverse functional proteoglycan.

Increased Collagen Turnover Impairs Tendon Microstructure and Stability in Integrin α2ß1-Deficient Mice.

Integrins are a family of transmembrane proteins, involved in substrate recognition and cell adhesion in cross-talk with the extra cellular matrix. In this study, we investigated the influence of integrin α2ß1 on tendons, another collagen type I-rich tissue of the musculoskeletal system. Morphological, as well as functional, parameters were analyzed in vivo and in vitro, comparing wild-type against integrin α2ß1 deficiency. Tenocytes lacking integrin α2ß1 produced more collagen in vitro, which is similar to the situation in osseous tissue. Fibril morphology and biomechanical strength proved to be altered, as integrin α2ß1 deficiency led to significantly smaller fibrils as well as changes in dynamic E-modulus in vivo. This discrepancy can be explained by a higher collagen turnover: integrin α2ß1-deficient cells produced more matrix, and tendons contained more residual C-terminal fragments of type I collagen, as well as an increased matrix metalloproteinase-2 activity. A greatly decreased percentage of non-collagenous proteins may be the cause of changes in fibril diameter regulation and increased the proteolytic degradation of collagen in the integrin-deficient tendons. The results reveal a significant impact of integrin α2ß1 on collagen modifications in tendons. Its role in tendon pathologies, like chronic degradation, will be the subject of future investigations.

Exosomes with low miR-34c-3p expression promote invasion and migration of non-small cell lung cancer by upregulating integrin α2ß1.

Exosomes play critical roles in regulating various physiological and pathological processes, including immune stimulation, immune suppression, cardiovascular diseases, and cancers. Recent studies show that exosomes that transport specific microRNAs (miRNAs) are involved in tumor development. However, the molecular mechanism by which tumor invasion and migration are regulated by exosomes from non-small cell lung cancer (NSCLC) is not well understood. Here, we show that exosomes shuttling low levels of miR-34c-3p are involved in NSCLC progression. Our results showed that exosomes derived from NSCLC cells carrying low levels of miR-34c-3p could be transported into the cytoplasm of NSCLC cells and accelerate NSCLC invasion and migration by upregulating integrin α2ß1. A luciferase assay revealed that integrin α2ß1 was the direct target of miR-34c-3p, and overexpression of integrin α2ß1 could promote the invasion and migration of NSCLC cells. The analysis of exosomes derived from clinical serum samples indicated that the expression of miR-34c-3p was significantly downregulated in exosomes from NSCLC patients compared with that of normal controls. A549-derived exosomes promoted NSCLC cells lung metastases in vivo. Exosomes shuttling low levels of miR-34c-3p were associated with the progression of NSCLC in vitro and in vivo. Our data demonstrate that exosomes shuttling low levels of miR-34c-3p can accelerate the invasion and migration of NSCLC by upregulating integrin α2ß1. MiR-34c-3p can be a diagnostic and prognostic marker for NSCLC. High expression of integrin α2ß1 is positively related to the migration and metastasis of NSCLC cells.

Integrin-specific hydrogels modulate transplanted human bone marrow-derived mesenchymal stem cell survival, engraftment, and reparative activities.

Stem cell therapies are limited by poor cell survival and engraftment. A hurdle to the use of materials for cell delivery is the lack of understanding of material properties that govern transplanted stem cell functionality. Here, we show that synthetic hydrogels presenting integrin-specific peptides enhance the survival, persistence, and osteo-reparative functions of human bone marrow-derived mesenchymal stem cells (hMSCs) transplanted in murine bone defects. Integrin-specific hydrogels regulate hMSC adhesion, paracrine signaling, and osteoblastic differentiation in vitro. Hydrogels presenting GFOGER, a peptide targeting α2ß1 integrin, prolong hMSC survival and engraftment in a segmental bone defect and result in improved bone repair compared to other peptides. Integrin-specific hydrogels have diverse pleiotropic effects on hMSC reparative activities, modulating in vitro cytokine secretion and in vivo gene expression for effectors associated with inflammation, vascularization, and bone formation. These results demonstrate that integrin-specific hydrogels improve tissue healing by directing hMSC survival, engraftment, and reparative activities.

Platelets promote breast cancer cell MCF-7 metastasis by direct interaction: surface integrin α2ß1-contacting-mediated activation of Wnt-ß-catenin pathway.

BACKGROUND: Integrin-mediated platelet-tumor cell contacting plays an important role in promoting epithelial-mesenchymal transition (EMT) transformation of tumor cells and cancer metastasis, but whether it occurs in breast cancer cells is not completely clear. OBJECTIVE: The purpose of this study was to investigate the role of integrin α2ß1 in platelet contacting to human breast cancer cell line MCF-7 and its effect on the EMT and the invasion of MCF-7 cells. METHODS: Human platelets were activated by thrombin, and separated into pellets and releasates before the co-incubation with MCF-7 cells. Cell invasion was evaluated by transwell assay. The surface integrins on pellets and MCF-7 cells were inhibited by antibodies. The effect of integrin α2ß1 on Wnt-ß-catenin pathway was assessed by integrin α2ß1-silencing and Wnt-ß-catenin inhibitor XAV. The therapeutic effect of integrin α2ß1-silencing was confirmed in the xenograft mouse model. RESULTS: Pellets promote the invasion and EMT of MCF-7 cells via direct contacting of surface integrin α2ß1. The integrin α2ß1 contacting activates Wnt-ß-catenin pathway and promotes the expression of EMT proteins in MCF-7 cells. The activated Wnt-ß-catenin pathway also promotes the autocrine of TGF-ß1 in MCF-7 cells. Both Wnt-ß-catenin and TGF-ß1/pSmad3 pathways promote the expression of EMT proteins. Integrin α2ß1-silencing inhibits breast cancer metastasis in vivo. CONCLUSIONS: The direct interaction between platelets and tumor cells exerts its pro-metastatic function via surface integrin α2ß1 contacting and Wnt-ß-catenin activation. Integrin α2ß1-silencing has the potential effect of inhibiting breast cancer metastasis.

Integrin-EGFR interaction regulates anoikis resistance in colon cancer cells.

Anoikis resistance is an essential property of cancer cells that allow the extra-cellular matrix-detached cells to survive in a suspended state in body fluid in order to metastasize and invade to distant organs. It is known that integrins play an important role in anoikis resistance, but detailed mechanisms are not well understood. Here we report that highly metastatic colon cancer cells showed a higher degree of anoikis resistance than the normal intestinal epithelial cells. These anoikis-resistant cancer cells express high-levels of integrin-α2, ß1, and activated EGFR in the anchorage-independent state than the anchorage-dependent state. In contrast, normal intestinal epithelial cells failed to elevate these proteins. Interestingly, a higher co-association of EGFR with integrin-α2ß1/-α5ß1 was observed on the surface of anoikis-resistant cells. Thus, in the absence of extra-cellular matrix, integrins in association with EGFR activates downstream effectors ERK and AKT and suppress Caspase-3 activation to induce anoikis resistance as was confirmed from the gene-ablation and pharmacological inhibitor studies. Interestingly, these anoikis-resistant cancer cells express high-level of cancer stem cell signatures (CD24, CD44, CD133, EpCAM) and pluripotent stem cell markers (OCT-4, SOX-2, Nanog) as well as drug-resistant pumps (ABCG2, MDR1, MRP1). Altogether, our findings unravel the interplay between integrin-α2ß1/-α5ß1 and EGFR in anoikis resistance and suggest that the resistant cells are cancer initiating or cancer stem cells, which may serve as a promising target to combat metastasis of cancer.

Peptostreptococcus anaerobius promotes colorectal carcinogenesis and modulates tumour immunity.

Emerging evidence implicates a role of the gut microbiota in colorectal cancer (CRC). Peptostreptococcus anaerobius (P. anaerobius) is an anaerobic bacterium selectively enriched in the faecal and mucosal microbiota from patients with CRC, but its causative role and molecular mechanism in promoting tumorigenesis remain unestablished. We demonstrate that P. anaerobius adheres to the CRC mucosa and accelerates CRC development in ApcMin/+ mice. In vitro assays and transmission electron microscopy revealed that P. anaerobius selectively adheres to CRC cell lines (HT-29 and Caco-2) compared to normal colonic epithelial cells (NCM460). We identified a P. anaerobius surface protein, putative cell wall binding repeat 2 (PCWBR2), which directly interacts with colonic cell lines via α2/ß1 integrin, a receptor frequently overexpressed in human CRC tumours and cell lines. Interaction between PCWBR2 and integrin α2/ß1 induces the activation of the PI3K-Akt pathway in CRC cells via phospho-focal adhesion kinase, leading to increased cell proliferation and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. NF-κB in turn triggers a pro-inflammatory response as indicated by increased levels of cytokines, such as interleukin-10 and interferon-γ in the tumours of P. anaerobius-treated ApcMin/+ mice. Analyses of tumour-infiltrating immune cell populations in P. anaerobius-treated ApcMin/+ mice revealed significant expansion of myeloid-derived suppressor cells, tumour-associated macrophages and granulocytic tumour-associated neutrophils, which are associated with chronic inflammation and tumour progression. Blockade of integrin α2/ß1 by RGDS peptide, small interfering RNA or antibodies all impair P. anaerobius attachment and abolish P. anaerobius-mediated oncogenic response in vitro and in vivo. Collectively, we show that P. anaerobius drives CRC via a PCWBR2-integrin α2/ß1-PI3K-Akt-NF-κB signalling axis and identify the PCWBR2-integrin α2/ß1 axis as a potential therapeutic target for CRC.

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers-Danlos syndrome-associated substitutions.

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2ß1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2ß1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2ß1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

Impact of Itga2-Gp6-double collagen receptor deficient mice for bone marrow megakaryocytes and platelets.

The two main collagen receptors on platelets, GPVI and integrin α2ß1, play an important role for the recognition of exposed collagen at sites of vessel injury, which leads to platelet activation and subsequently stable thrombus formation. Both receptors are already expressed on megakaryocytes, the platelet forming cells within the bone marrow. Megakaryocytes are in permanent contact with collagen filaments in the marrow cavity and at the basal lamina of sinusoids without obvious preactivation. The role of both collagen receptors for megakaryocyte maturation and thrombopoiesis is still poorly understood. To investigate the function of both collagen receptors, we generated mice that are double deficient for Gp6 and Itga2. Flow cytometric analyses revealed that the deficiency of both receptors had no impact on platelet number and led to the expected lack in GPVI responsiveness. Integrin activation and degranulation ability was comparable to wildtype mice. By immunofluorescence microscopy, we could demonstrate that both wildtype and double-deficient megakaryocytes were overall normally distributed within the bone marrow. We found megakaryocyte count and size to be normal, the localization within the bone marrow, the degree of maturation, as well as their association to sinusoids were also unaltered. However, the contact of megakaryocytes to collagen type I filaments was decreased at sinusoids compared to wildtype mice, while the interaction to type IV collagen was unaffected. Our results imply that GPVI and α2ß1 have no influence on the localization of megakaryocytes within the bone marrow, their association to the sinusoids or their maturation. The decreased contact of megakaryocytes to collagen type I might at least partially explain the unaltered platelet phenotype in these mice, since proplatelet formation is mediated by these receptors and their interaction to collagen. It is rather likely that other compensatory signaling pathways and receptors play a role that needs to be elucidated.

The Role of Genetic Variants (rs869109213 and rs2070744) Of the eNOS Gene and BglII in the α2 Subunit of the α2ß1 Integrin Gene in Diabetic Retinopathy in a Tunisian Population.

Purpose: In this study, we investigated the association of two polymorphisms (rs869109213 and rs2070744) in the eNOS gene and one polymorphism BglII in the α2ß1 integrin gene (ITGA2) with the risk of diabetic retinopathy (DR) in a Tunisian population. Methods: The study investigated of 110 type 2 diabetes mellitus (T2DM) and 127 DR patients. The genotypes of the eNOS 4b/4a (rs869109213) and -786T/C (rs2070744) polymorphisms and of the BglII polymorphism of ITGA2 were studied using the PCR or PCR-RFLP method. Results: The genotype distributions of the two polymorphisms in eNOS 4b4a and eNOS (-786T/C) were significantly different between T2DM and DR patients (p < .004 and p = .033, respectively). These polymorphisms were associated with the risk of DR (OR = 2.65, 95%CI [1.45-4.84], p = .002) for the eNOS 4b4a genotype and (OR = 2.43, 95%CI [1.06 - 5.56], p = .036) for the CC genotype of the eNOS gene (-786T/C). Similarly, the genotype distribution of the BglII polymorphism was significantly different between the two groups studied (p = .037). This polymorphism was associated with an increased risk of DR (OR = 4.03, 95% CI [1.17 - 7.85], p = .022) for BglII(+/+). Conclusion: The present study suggests that the polymorphisms 4b4a and -786T/C in the eNOS gene might be associated with DR. In addition, the BglII polymorphism in the ITGA2 gene was a risk factor for DR.

Tumor-associated macrophages promote bladder tumor growth through PI3K/AKT signal induced by collagen.

The tumor microenvironment is associated with various tumor progressions, including cancer metastasis, immunosuppression, and tumor sustained growth. Tumor-associated macrophages (TAMs) are considered an indispensable component of the tumor microenvironment, participating in the progression of tumor microenvironment remodeling and creating various compounds to regulate tumor activities. This study aims to observe enriched TAMs in tumor tissues during bladder cancer development, which markedly facilitated the proliferation of bladder cancer cells and promoted tumor growth in vivo. We determined that TAMs regulate tumor sustained growth by secreting type I collagen, which can activate the prosurvival integrin α2ß1/PI3K/AKT signaling pathway. Furthermore, traditional chemotherapeutic drugs combined with integrin α2ß1 inhibitor showed intensive anticancer effects, revealing an innovative approach in clinical bladder cancer treatment.

Implication of Integrin α2ß1 in Proliferation and Invasion of Human Breast Carcinoma and Melanoma Cells: Noncanonical Function of Akt Protein Kinase.

Blocking the expression of integrin α2ß1, which was accomplished by transduction of α2-specific shRNA, resulted in significant inhibition of proliferation and clonal activity in human MCF-7 breast carcinoma and SK-Mel-147 melanoma cells. Along with these changes, deprivation of α2ß1 caused a sharp decrease in melanoma cell invasion in vitro. Analysis of integrin-mediating signal pathways that control cell behavior revealed a significant increase in activity of Akt protein kinase in response to depletion of α2ß1. The increase in Akt activity that accompanies a suppressive effect on cell invasion contradicts well-known Akt function aimed at stimulation of tumor progression. This contradiction could be explained by the "reversed" (noncanonical) role played by Akt in some cells that consists in suppression rather than promotion of invasive phenotype. To test this suggestion, the effects of Akt inhibitors on invasive activity of SK-Mel-147 cells were investigated. If the above suggestion is true, then inhibition of Akt in cells depleted of α2ß1 should result in the restoration of their invasive activity. It appeared that treatment with LY294002, which inhibits all Akt isoforms (Akt1, Akt2, Akt3), not only failed to restore the invasive phenotype of melanoma cells but further attenuated their invasive activity. However, treatment of the cells with an Akt1-specific inhibitor significantly increased their invasion. Thus, the stimulating effect of α2ß1 integrin on invasion of melanoma cells is realized through a mechanism based on inhibition of one of the Akt isoforms, which in these cells exhibits a noncanonical function consisting in suppression of invasion.