Characterization of the role of integrin α5β1 in platelet function, hemostasis and experimental thrombosis

Objective: Integrins are key regulators of various platelet functions. The pathophysiological importance of most platelet integrins has been investigated, with the exception of α5β1, a receptor for fibronectin. The aim of this study was to characterize the role of α5β1 in megakaryopoiesis, platelet function, and to determine its importance in hemostasis and arterial thrombosis. Approach and results: We generated a mouse strain deficient for integrin α5β1 on megakaryocytes and platelets (PF4Cre-α5-/-). PF4Cre-α5-/- mice were viable, fertile and presented no apparent signs of abnormality. Megakaryopoiesis appears unaltered as evidence by a normal megakaryocytes morphology and development, which is in agreement with a normal platelet count. Expression of the main platelet receptors and the response of PF4Cre-α5-/- platelets to a series of agonists were all completely normal. Adhesion and aggregation of PF4Cre-α5-/- platelets under shear flow on fibrinogen, laminin or von Willebrand factor were unimpaired. In contrast, PF4Cre-α5-/- platelets displayed a marked decrease in adhesion, activation and aggregation on fibrillar cellular fibronectin and collagen. PF4Cre-α5-/- mice presented no defect in a tail-bleeding time assay and no increase in inflammatory bleeding in a reverse passive Arthus model and a lipopolysaccharide pulmonary inflammation model. Finally, no defects were observed in three distinct experimental models of arterial thrombosis based on ferric chloride-induced injury of the carotid artery, mechanical injury of the abdominal aorta or laser-induced injury of mesenteric vessels. Conclusion: In summary, this study shows that platelet integrin α5β1 is a key receptor for fibrillar cellular fibronectin but is dispensable in hemostasis and arterial thrombosis.

Integrin α5β1 nano-presentation regulates collective keratinocyte migration independent of substrate rigidity

Nanometer-scale properties of the extracellular matrix influence many biological processes, including cell motility. While much information is available for single-cell migration, to date, no knowledge exists on how the nanoscale presentation of extracellular matrix receptors influences collective cell migration. In wound healing, basal keratinocytes collectively migrate on a fibronectin-rich provisional basement membrane to re-epithelialize the injured skin. Among other receptors, the fibronectin receptor integrin α5β1 plays a pivotal role in this process. Using a highly specific integrin α5β1 peptidomimetic combined with nanopatterned hydrogels, we show that keratinocyte sheets regulate their migration ability at an optimal integrin α5β1 nanospacing. This efficiency relies on the effective propagation of stresses within the cell monolayer independent of substrate stiffness. For the first time, this work highlights the importance of extracellular matrix receptor nanoscale organization required for efficient tissue regeneration.

CDCA attenuates LUAD pathogenesis progression via integrin α5β1/FAK/p53 signalling pathway

BackgroundLung cancer is the leading cause of cancer-associated death and includes non-small-cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC comprises approximately 80–85% of all lung cancers. Chenodeoxycolic acid (CDCA), one of the primary bile acids, has been reported to inhibit proliferation in carcinoma cells. To date, the role of CDCA in the migration, invasion and apoptosis of LUAD remains unknown. MethodsWestern blotting and quantitative real-time PCR were used to test the protein and mRNA expression levels in LUAD cell lines. Cell Counting Kit-8 (CCK-8) and clone formation assays were performed to evaluate the proliferation ability of different kinds of cells in vitro. Transwell assay were utilized to assess the motility of tumor cells. RNA-seq was performed to identify the transcriptional profile in A549 cells after treatment with CDCA. A xenograft model was established to study the effect of CDCA on LUAD growth in vivo. GraphPad Prism 8 software was used for statistical analysis, and a P value < 0.05 was considered statistically significant.ResultsCDCA can significantly inhibited the migration and invasion capabilities of LUAD cells. Furthermore, CDCA promoted the progression of apoptosis in LUAD cells. Mechanistically, we revealed that CDCA blocked the integrin α5β1 signalling pathway by inhibiting the expression of integrin α5 and β1 subunits and p-FAK in LUAD cells. Furthermore, we found that CDCA increased the levels of p53, a downstream gene of the integrin α5β1/FAK pathway. ConclusionOur findings indicated that CDCA attenuates LUAD pathogenesis via the integrin α5β1/FAK/p53 axis in LUAD cells.

Cellular Integrin α5β1 and Exosomal ADAM17 Mediate the Binding and Uptake of Exosomes Produced by Colorectal Carcinoma Cells

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size−exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5β1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes.

P04.18 Heterogeneity and plasticity of integrin α5β1 expression in glioblastoma stem cells

BACKGROUND Glioblastoma (GBM) is the most frequent and deadliest type of central nervous system tumors. Despite the treatment by the Stupp protocol, almost all patients relapse and new therapeutic protocols have been unsuccessful for ameliorating patient survival. Molecular heterogeneity of GBM and existence of glioma stem cells (GSC) may be linked to therapy resistance and recurrence. We demonstrated earlier that α5β1 integrin is a GBM therapeutic target which participate to therapy resistance; a high expression in patient tumors is linked to a worse prognosis. Expression of α5β1 integrin is heterogeneous inter- and intra-tumorally. We particularly addressed the role of glioma stem cell plasticity in the modulation of the integrin expression. Stem cells reside in specific niches (perivascular or hypoxic niches) in the tumor and are at the origin of the more differentiated tumor cell bulk. Metabolism is known to change between the different GSC states and may be affected by or may affect the integrin expression. The aim of our work is therefore to consider the expression of the integrin α5β1 in relationship with GSC differentiation or in hypoxic environment and with cell metabolism. MATERIAL AND METHODS Ten different patient-derived glioma stem cell lines were investigated. Cell culture in stem cell medium (neurospheres) or differentiation medium (adherent cell monolayer) was made in normoxia (21% O2) or hypoxia (1%O2). Alternatively, chemically-induced hypoxia (cobalt chloride/desferoxiamine) was used. Integrin expression was kinetically checked at the mRNA (RT-qPCR) or protein (Western blot) levels. Cell metabolism was investigated with the Seahorse Xfp technology and by HRMAS-NMR. RESULTS No GSC lines (neurospheres) expressed the α5β1 integrin. Interestingly, only half of them did after differentiation suggesting a first level of heterogeneity. A second level of heterogeneity was observed in hypoxic conditions provoking induction of integrin α5β1 expression in only some non-differentiated GSC. Three categories of GSC were thus characterized: one able to express the integrin in hypoxia and after differentiation, one never expressing it and the third one only after differentiation. Cell metabolism differed between GSC before and after differentiation and in presence of integrin α5β1 antagonists. Specific glioma regulator network analysis revealed new targets to be inhibited concomitantly with the integrin. CONCLUSION Data suggest that α5β1 integrin expression may be induced by different signaling pathways. Molecular switches may occur either when stem cells differentiate to tumor cells but also directly in stem cells in hypoxic niches. Characterization of α5β1 integrin expression drivers may help to find new therapeutic targets but also to delineate subpopulation of patients who would benefit from an anti-integrin strategy.

The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB

Vascular endothelial cells (EC) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, the hallmarks of the severe disease. However, the mechanisms by which EC are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin α5β1 signaling. Incubation of human umbilical vein EC with whole spike, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and enhanced the expression of leukocyte adhesion molecules VCAM1 and ICAM1, the adhesion of peripheral blood leukocytes, and the permeability of the monolayer. Inhibitors of integrin α5β1 activation prevented these effects. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin α5β1 in EC to activate Rho GTPases, eNOS pathways, and the NF-κB gene expression program responsible for vascular leakage and leukocyte infiltration, respectively. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin α5β1 as a promising target for treating vascular inflammation in COVID-19.

MO569MACROPHAGE PROMOTE VASCULAR CALCIFICATION VIA THE MIGRASOMES/ INTEGRIN Α5Β1 PATHWAY IN CKD

Background and Aims Patients with chronic kidney disease (CKD) have a predisposition to develop vascular calcification due to dysregulated homeostatic mechanisms. Macrophages can promote vascular calcification by releasing diverse extracellular vehicles including the newly found migrasomes (M-mig). Our previous research had found that M-mig provide nucleating foci for calcific mineral formation and initiating bone mineralization process. However, the specific mechanism by which M-mig influence the formation of vascular calcification remains incompletely understood. Method To study calcifying M-mig, we exposed M-mig to high Ca/P (Ca/P=3 mmol/L calcium/2 mmol/L phosphate) and/or with LPS for 1, 3, 5,7 days. The expression of M-mig surface integrin α5β1 was determined by fluorescence staining. To block the M-mig-integrin α5β1 mediated calcification, we modulated the expression of integrin α5 using siRNAs to produce M-migintegrin α5- or using 20 nM ATN-161 (small peptide antagonist of integrin α5β1) or integrin α5 antibody under high Ca/P stimulation. The stray mice artery co-cultivate with M-mig integrin α5- under high level Ca/P. Then the calcifying M-mig were assessed by TEM, Fluo-3 staining and calcium content assay. Results We discovered that Ca/P-stimulated macrophages released M-mig capable of mineralization. Amorphous calcium phosphate mineral deposit the surface or internal of M-mig. The M-mig exhibited increased Ca/P mineral content, implying aggregate larger calcifying M-mig that develop over time. Significantly, following a 7 days incubation with high level Ca/P, fiber tube and vesicle structure of M-mig showed rupture or fragmentation and the expression of M-mig surface integrin α5β1 increased. Pre-treatment with integrin α5β1 antagonist or block by integrin α5 antibody significantly reduced the calcifying M-mig formation. Further investigation showed that M-mig induced stray mice artery microcalcification while M-migintegrin α5- exhibited a reduce microcalcification. Conclusion Our finding revealed an association between microcalcification and integrin α5β1 signalling in the fiber tube and vesicle structure of M-mig and provide a new insight into vascular calcification in CKD.