A laminin 511 matrix is regulated by TAZ and functions as the ligand for the α6Bß1 integrin to sustain breast cancer stem cells.

Understanding how the extracellular matrix impacts the function of cancer stem cells (CSCs) is a significant but poorly understood problem. We report that breast CSCs produce a laminin (LM) 511 matrix that promotes self-renewal and tumor initiation by engaging the α6Bß1 integrin and activating the Hippo transducer TAZ. Although TAZ is important for the function of breast CSCs, the mechanism is unknown. We observed that TAZ regulates the transcription of the α5 subunit of LM511 and the formation of a LM511 matrix. These data establish a positive feedback loop involving TAZ and LM511 that contributes to stemness in breast cancer.

Dual inhibition of αvß6 and αvß1 reduces fibrogenesis in lung tissue explants from patients with IPF.

RATIONALE: αv integrins, key regulators of transforming growth factor-ß activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvß6) and fibroblasts (αvß1) in fibrotic lungs. OBJECTIVES: We evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis. METHODS: Selective αvß6 and αvß1, dual αvß6/αvß1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-ß cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-ß signaling. Bleomycin-challenged mice treated with dual αvß6/αvß1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation. MEASUREMENTS AND MAIN RESULTS: Inhibition of integrins αvß6 and αvß1 was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional αv integrins. Antifibrotic efficacy of dual αvß6/αvß1 integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone. CONCLUSIONS: In the fibrotic lung, dual inhibition of integrins αvß6 and αvß1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-ß.

Tumour exosome integrins determine organotropic metastasis.

Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6ß4 and α6ß1 were associated with lung metastasis, while exosomal integrin αvß5 was linked to liver metastasis. Targeting the integrins α6ß4 and αvß5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

Association of VLA4, 5, 6 and PSGL1 expression levels with engraftment in autologous HPSC transplantation in multiple myeloma patients.

The most promising therapy for leukemia is hematopoietic stem cell transplantation. Engraftment of HPSCs mainly depends on some factors such as adhesion molecules, including VLAs. This study tried to delineate the relationship between HPSCs engraftment and expression level of PSGL1 and VLA4, 5, and 6 genes in candidate MM patients for autologous bone marrow transplantation. Firstly, the CD 34+ HPSCs were collected from multiple myeloma (MM) patients after five days of G-CSF therapy through apheresis processes. Then, the patients were categorized into two groups of good and bad prognosis depending on engraftment time (Less or more than 18 days). Followingly, the expression of PSGL1 and VLA4, VLA5, and VLA6 genes were assessed by the qRT-PCR technique in each patient. Finally, the correlation between the genes and engraftment time was investigated to determine the prognostic role of each gene on HPSCs transplantation. Our findings demonstrated that there is a significant correlation between VLA4 (P=< 0.0001) and 5 (P = 0.005) levels and HPSCs engraftment time. As the higher levels of VLA4 and 5, the shorter time HPSCs engraftment occurs. In contrast, there was no significant correlation between VLA6 (P = 0.2) and PSGL1 (P = 0.3) genes levels and engraftment time. So that, the patients with a good prognosis had a higher level of VLA4 and VLA5, but no relation was found between VLA6 and PSGL1. It is concluded that VLA4 and VLA5 expression could be considered a significant prognostic factor for HPSC transplantation.

Shear-Induced CCN1 Promotes Atheroprone Endothelial Phenotypes and Atherosclerosis.

BACKGROUND: Atherosclerosis occurs preferentially at the blood vessels encountering blood flow turbulence. The matricellular protein CCN1 is induced in endothelial cells by disturbed flow, and is expressed in advanced atherosclerotic lesions in patients and in the Apoe-/- mouse model. The role of CCN1 in atherosclerosis remains undefined. METHODS: To assess the function of CCN1 in vivo, knock-in mice carrying the integrin α6ß1-binding-defective mutant allele Ccn1-dm on the Apoe-/- background were tested in an atherosclerosis model generated by carotid artery ligation. Additionally, CCN1-regulated functional phenotypes of human umbilical vein endothelial cells, or primary mouse aortic endothelial cells isolated from wild-type and Ccn1 dm/dm mice, were investigated in the in vitro shear stress experiments under unidirectional laminar shear stress (12 dyn/cm2) versus oscillatory shear stress (±5 dyn/cm2) conditions. RESULTS: We found that Ccn1 expression was upregulated in the arterial endothelium 3 days after ligation before any detectable structural changes, and intensified with the progression of atherosclerotic lesions. Compared with Apoe-/- controls, Ccn1 dm/dm/ Apoe-/- mice were remarkably resistant to ligation-induced plaque formation (n=6). These mice exhibited lower oxidative stress, expression of endothelin-1 and monocyte chemoattractant protein-1, and monocyte homing. CCN1/α6ß1 critically mediated flow-induced activation of the pleiotropic transcription factor nuclear factor-κB and therefore the induction of atheroprone gene expression in the mouse arterial endothelium after ligation (n=6), or in cultured human umbilical vein endothelial cells or primary mouse aortic endothelial cells exposed to oscillatory shear stress (n=3 in triplicate). Interestingly, the activation of nuclear factor-κB by CCN1/α6ß1 signaling prompted more production of CCN1 and α6ß1. Blocking CCN1-α6ß1 binding by the Ccn1-dm mutation or by T1 peptide (derived from an α6ß1-binding sequence of CCN1) disrupted the positive-feedback regulation between CCN1/α6ß1 and nuclear factor-κB, and prevented flow-induced atheroprone phenotypic alterations in endothelial cells or atherosclerosis in mice. CONCLUSIONS: These data demonstrate a causative role of CCN1 in atherosclerosis via modulating endothelial phenotypes. CCN1 binds to its receptor integrin α6ß1 to activate nuclear factor-κB, thereby instigating a vicious circle to persistently promote atherogenesis. T1, a peptide antagonist selectively targeting CCN1-α6ß1, can be further optimized for developing T1-mimetics to treat atherosclerosis.

The N-terminal domain of the R28 protein promotes emm28 group A Streptococcus adhesion to host cells via direct binding to three integrins.

Group A Streptococcus (GAS) is a human-specific pathogen responsible for a wide range of diseases, ranging from superficial to life-threatening invasive infections, including endometritis, and autoimmune sequelae. GAS strains express a vast repertoire of virulence factors that varies depending on the strain genotype, and many adhesin proteins that enable GAS to adhere to host cells are restricted to some genotypes. GAS emm28 is the third most prevalent genotype in invasive infections in France and is associated with gyneco-obstetrical infections. emm28 strains harbor R28, a cell wall-anchored surface protein that has previously been reported to promote adhesion to cervical epithelial cells. Here, using cellular and biochemical approaches, we sought to determine whether R28 supports adhesion also to other cells and to characterize its cognate receptor. We show that through its N-terminal domain, R28Nt, R28 promotes bacterial adhesion to both endometrial-epithelial and endometrial-stromal cells. R28Nt was further subdivided into two domains, and we found that both are involved in cell binding. R28Nt and both subdomains interacted directly with the laminin-binding α3ß1, α6ß1, and α6ß4 integrins; interestingly, these bindings events did not require divalent cations. R28 is the first GAS adhesin reported to bind directly to integrins that are expressed in most epithelial cells. Finally, R28Nt also promoted binding to keratinocytes and pulmonary epithelial cells, suggesting that it may be involved in supporting the prevalence in invasive infections of the emm28 genotype.

CCN2 promotes drug resistance in osteosarcoma by enhancing ABCG2 expression.

In recent years, osteosarcoma survival rates have failed to improve significantly with conventional treatment modalities because of the development of chemotherapeutic resistance. The human breast cancer resistance protein/ATP binding cassette subfamily G member 2 (BCRP/ABCG2), a member of the ATP-binding cassette family, uses ATP hydrolysis to expel xenobiotics and chemotherapeutics from cells. CCN family member 2 (CCN2) is a secreted protein that modulates the biological function of cancer cells, enhanced ABCG2 protein expression and activation in this study via the α6ß1 integrin receptor and increased osteosarcoma cell viability. CCN2 treatment downregulated miR-519d expression, which promoted ABCG2 expression. In a mouse xenograft model, knockdown of CCN2 expression increased the therapeutic effect of doxorubicin, which was reversed by ABCG2 overexpression. Our data show that CCN2 increases ABCG2 expression and promotes drug resistance through the α6ß1 integrin receptor, whereas CCN2 downregulates miR-519d. CCN2 inhibition may represent a new therapeutic concept in osteosarcoma.

Dual role of pericyte α6ß1-integrin in tumour blood vessels.

The α6ß1-integrin is a major laminin receptor, and formation of a laminin-rich basement membrane is a key feature in tumour blood vessel stabilisation and pericyte recruitment, processes that are important in the growth and maturation of tumour blood vessels. However, the role of pericyte α6ß1-integrin in angiogenesis is largely unknown. We developed mice where the α6-integrin subunit is deleted in pericytes and examined tumour angiogenesis and growth. These mice had: (1) reduced pericyte coverage of tumour blood vessels; (2) reduced tumour blood vessel stability; (3) increased blood vessel diameter; (4) enhanced blood vessel leakiness, and (5) abnormal blood vessel basement membrane architecture. Surprisingly, tumour growth, blood vessel density and metastasis were not altered. Analysis of retinas revealed that deletion of pericyte α6-integrin did not affect physiological angiogenesis. At the molecular level, we provide evidence that pericyte α6-integrin controls PDGFRß expression and AKT-mTOR signalling. Taken together, we show that pericyte α6ß1-integrin regulates tumour blood vessels by both controlling PDGFRß and basement membrane architecture. These data establish a novel dual role for pericyte α6-integrin as modulating the blood vessel phenotype during pathological angiogenesis.

Role of Extracellular Vesicle Surface Proteins in the Pharmacokinetics of Extracellular Vesicles.

Extracellular vesicles (EVs) are small membrane vesicles secreted from cells and have great potential as drug delivery carriers. Surface proteins on EV membranes might play roles in pharmacokinetics. One method which can be used to study the role of surface membrane of EV is to modify the inner space of EV. In the present study, we constructed a plasmid DNA expressing a fusion protein of Gag protein derived from Moloney murine leukemia virus (Gag) and Gaussia luciferase (gLuc) (Gag-gLuc) to modify the inner space of EVs. EVs were collected from B16BL6 melanoma cells, transfected with the plasmid, and isolated by a differential ultracentrifugation method. Gag-gLuc EVs were negatively charged globular vesicles with a diameter of approximately 100 nm. gLuc labeling of the Gag-gLuc EVs was stable in serum. gLuc activity of Gag-gLuc EVs was minimally decreased by proteinase K (ProK) treatment, indicating that gLuc was modified in the inner space of EV. Then, to evaluate the effect of the surface proteins of EVs on their pharmacokinetics, Gag-gLuc EVs treated with ProK were intravenously administered to mice. Volume of distribution (Vd) was significantly smaller for treated EVs than untreated EVs. Moreover, integrin α6ß1, an integrin known to be involved in lung targeting, was degraded after ProK treatment. The ProK treatment significantly reduced the lung distribution of EVs after intravenous injection. These results indicate that the surface proteins of EVs such as integrin α6ß1 play some roles in pharmacokinetics in terms of reducing Vd and their distribution to the lung.

Probing the acidic residue within the integrin binding site of laminin-511 that interacts with the metal ion-dependent adhesion site of α6ß1 integrin.

Laminins are major cell-adhesive proteins of basement membranes that interact with integrins in a divalent cation-dependent manner. Laminin-511 consists of α5, ß1, and γ1 chains, of which three laminin globular domains of the α5 chain (α5/LG1-3) and a Glu residue in the C-terminal tail of chain γ1 (γ1-Glu1607) are required for binding to integrins. However, it remains unsettled whether the Glu residue in the γ1 tail is involved in integrin binding by coordinating the metal ion in the metal ion-dependent adhesion site of ß1 integrin (ß1-MIDAS), or by stabilizing the conformation of α5/LG1-3. To address this issue, we examined whether α5/LG1-3 contain an acidic residue required for integrin binding that is as critical as the Glu residue in the γ1 tail; to achieve this, we undertook exhaustive alanine substitutions of the 54 acidic residues present in α5/LG1-3 of the E8 fragment of laminin-511 (LM511E8). Most of the alanine mutants possessed α6ß1 integrin binding activities comparable with wild-type LM511E8. Alanine substitution for α5-Asp3198 and Asp3219 caused mild reduction in integrin binding activity, and that for α5-Asp3218 caused severe reduction, possibly resulting from conformational perturbation of α5/LG1-3. When α5-Asp3218 was substituted with asparagine, the resulting mutant possessed significant binding activity to α6ß1 integrin, indicating that α5-Asp3218 is not directly involved in integrin binding through coordination with the metal ion in ß1-MIDAS. Given that substitution of γ1-Glu1607 with glutamine nullified the binding activity to α6ß1 integrin, these results, taken together, support the possibility that the critical acidic residue coordinating the metal ion in ß1-MIDAS is Glu1607 in the γ1 tail, but no such residue is present in α5/LG1-3.

Inhibition of Focal Adhesion Kinase Signaling by Integrin α6ß1 Supports Human Pluripotent Stem Cell Self-Renewal.

Self-renewal of human embryonic stem cells and human induced pluripotent stem cells (hiPSCs)-known as pluripotent stem cells (PSC)-is influenced by culture conditions, including the substrate on which they are grown. However, details of the molecular mechanisms interconnecting the substrate and self-renewal of these cells remain unclear. We describe a signaling pathway in hPSCs linking self-renewal and expression of pluripotency transcription factors to integrin α6ß1 and inactivation of focal adhesion kinase (FAK). Disruption of this pathway results in hPSC differentiation. In hPSCs, α6ß1 is the dominant integrin and FAK is not phosphorylated at Y397, and thus, it is inactive. During differentiation, integrin α6 levels diminish and Y397 FAK is phosphorylated and activated. During reprogramming of fibroblasts into iPSCs, integrin α6 is upregulated and FAK is inactivated. Knockdown of integrin α6 and activation of ß1 integrin lead to FAK phosphorylation and reduction of Nanog, Oct4, and Sox2, suggesting that integrin α6 functions in inactivation of integrin ß1 and FAK signaling and prevention of hPSC differentiation. The N-terminal domain of FAK, where Y397 is localized, is in the nuclei of hPSCs interacting with Oct4 and Sox2, and this immunolocalization is regulated by Oct4. hPSCs remodel the extracellular microenvironment and deposit laminin α5, the primary ligand of integrin α6ß1. Knockdown of laminin α5 resulted in reduction of integrin α6 expression, phosphorylation of FAK and decreased Oct4. In conclusion, hPSCs promote the expression of integrin α6ß1, and nuclear localization and inactivation of FAK to supports stem cell self-renewal. Stem Cells 2016;34:1753-1764.

Integrin α6ß1 Expressed in ESCs Instructs the Differentiation to Endothelial Cells.

Adhesion of embryonic stem cells (ESCs) to the extracellular matrix may influence differentiation potential and cell fate decisions. Here, we investigated the inductive role of binding of integrin α6ß1 expressed in mouse (m)ESCs to laminin-1 (LN1) in mediating the differentiation of ESCs to endothelial cells (ECs). We observed that α6ß1 binding to LN1 was required for differentiation to ECs. α6ß1 functioned by recruiting the adaptor tetraspanin protein CD151, which activated FAK and Akt signaling and mediated the EC lineage-specifying transcription factor Er71. In contrast, association of the ESC-expressed α3ß1, another highly expressed LN1 binding integrin, with CD151, prevented α6ß1-mediated differentiation. CD151 thus functioned as a bifurcation router to direct ESCs toward ECs when α6ß1 associated with CD151, or prevented transition to ECs when α3ß1 associated with CD151. These observations were recapitulated in mice in which α6 integrin or CD151 knockdown reduced the expression of Er71-regulated angiogenesis genes and development of blood vessels. Thus, interaction of α6ß1 in ESCs with LN1 activates α6ß1/CD151 signaling which programs ESCs toward the EC lineage fate.

Growth hormone in the presence of laminin modulates interaction of human thymic epithelial cells and thymocytes in vitro.

BACKGROUND: Several evidences indicate that hormones and neuropeptides function as immunomodulators. Among these, growth hormone (GH) is known to act on the thymic microenvironment, supporting its role in thymocyte differentiation. The aim of this study was to evaluate the effect of GH on human thymocytes and thymic epithelial cells (TEC) in the presence of laminin. RESULTS: GH increased thymocyte adhesion on BSA-coated and further on laminin-coated surfaces. The number of migrating cells in laminin-coated membrane was higher in GH-treated thymocyte group. In both results, VLA-6 expression on thymocytes was constant. Also, treatment with GH enhanced laminin production by TEC after 24 h in culture. However, VLA-6 integrin expression on TEC remained unchanged. Finally, TEC/thymocyte co-culture model demonstrated that GH elevated absolute number of double-negative (CD4(-)CD8(-)) and single-positive CD4(+) and CD8(+) thymocytes. A decrease in cell number was noted in double-positive (CD4(+)CD8(+)) thymocytes. CONCLUSIONS: The results of this study demonstrate that GH is capable of enhancing the migratory capacity of human thymocytes in the presence of laminin and promotes modulation of thymocyte subsets after co-culture with TEC.

Integrin-α5ß1 is not required for mural cell functions during development of blood vessels but is required for lymphatic-blood vessel separation and lymphovenous valve formation.

Integrin α5ß1 is essential for vascular development but it remains unclear precisely where and how it functions. Here, we report that deletion of the gene encoding the integrin-α5 subunit (Itga5) using the Pdgfrb-Cre transgenic mouse line, leads to oedema, haemorrhage and increased levels of embryonic lethality. Unexpectedly, these defects were not caused by loss of α5 from Pdgfrb-Cre expressing mural cells (pericytes and vascular smooth muscle cells), which wrap around the endothelium and stabilise blood vessels, nor by defects in the heart or great vessels, but were due to abnormal development of the lymphatic vasculature. Reminiscent of the pathologies seen in the human lymphatic malformation, fetal cystic hygroma, α5 mutants display defects both in the separation of their blood and lymphatic vasculature and in the formation of the lymphovenous valves. As a consequence, α5-deficient mice develop dilated, blood-filled lymphatic vessels and lymphatic capillaries that are ectopically covered with smooth muscle cells. Analysis of the expression of Pdgfrb during lymphatic development suggests that these defects probably arise from loss of α5ß1 integrin in subsets of specialised Prox1(+)Pdgfrb(+) venous endothelial cells that are essential for the separation of the jugular lymph sac from the cardinal vein and formation of the lymphovenous valve leaflets.

Matricellular protein Cyr61 bridges lysophosphatidic acid and integrin pathways leading to cell migration.

Lysophosphatidic acid (LPA), a potent bioactive lipid found in atherosclerotic lesions, markedly induces smooth muscle cell (SMC) migration, which is an important process in atherogenesis. Therefore, understanding the mechanism of LPA-induced SMC migration is important. Several microarray databases suggest that the matricellular protein Cyr61 is highly induced by LPA. We hypothesized that Cyr61 mediates LPA-induced cell migration. Our data show that LPA induced temporal and spatial expression of Cyr61, which promptly accumulated in the cellular Golgi apparatus and then translocated to the extracellular matrix. Cyr61 antibody blockade and siRNA inhibition both diminished LPA-induced SMC migration, indicating a novel regulatory role of Cyr61. SMCs derived from LPA receptor 1 (LPA1) knock-out mice lack the ability of Cyr61 induction and cell migration, supporting the concept that LPA1 is required for Cyr61 expression and migration. By contrast, PPARγ was not found to be involved in LPA-mediated effects. Furthermore, focal adhesion kinase (FAK), a nonreceptor tyrosine kinase important for regulating cell migration, was activated by LPA at a late time frame coinciding with Cyr61 accumulation. Interestingly, knockdown of Cyr61 blocked LPA-induced FAK activation, indicating that an LPA-Cyr61-FAK axis leads to SMC migration. Our results further demonstrate that plasma membrane integrins α6ß1 and ανß3 transduced the LPA-Cyr61 signal toward FAK activation and migration. Taken together, these data reveal that de novo Cyr61 in the extracellular matrix bridges LPA and integrin pathways, which in turn, activate FAK, leading to cell migration. The current study provides new insights into mechanisms underlying cell migration-related disorders, including atherosclerosis, restenosis, and cancers.

α6ß1- and αV-integrins are required for long-term self-renewal of murine embryonic stem cells in the absence of LIF.

BACKGROUND: The growth properties and self-renewal capacity of embryonic stem (ES) cells are regulated by their immediate microenvironment such as the extracellular matrix (ECM). Integrins, a central family of cellular ECM receptors, have been implicated in these processes but their specific role in ES cell self-renewal remains unclear. RESULTS: Here we have studied the effects of different ECM substrates and integrins in mouse ES cells in the absence of Leukemia Inhibitory Factor (LIF) using short-term assays as well as long-term cultures. Removal of LIF from ES cell culture medium induced morphological differentiation of ES cells into polarized epistem cell-like cells. These cells maintained epithelial morphology and expression of key stemness markers for at least 10 passages in the absence of LIF when cultured on laminin, fibronectin or collagen IV substrates. The specific functional roles of α6-, αV- and ß1-integrin subunits were dissected using stable lentivirus-mediated RNAi methodology. ß1-integrins were required for ES cell survival in long-term cultures and for the maintenance of stem cell marker expression. Inhibition of α6-integrin expression compromised self-renewal on collagen while αV-integrins were required for robust ES cell adhesion on laminin. Analysis of the stemness marker expression revealed subtle differences between α6- and αV-depleted ES cells but the expression of both was required for optimal self-renewal in long-term ES cell cultures. CONCLUSIONS: In the absence of LIF, long-term ES cell cultures adapt an epistem cell-like epithelial phenotype and retain the expression of multiple stem cell markers. Long-term maintenance of such self-renewing cultures depends on the expression of ß1-, α6- and αV-integrins.

Effect of integrin combined laminin on peripheral blood vessel of cerebral infarction and endogenous nerve regeneration.

The third Chinese nationwide survey on causes of death states that cerebrovascular disease, accounting for 22.45% of total deaths, ranks as the first cause of death among rural and urban residents. It has become a serious public health problem since it has the highest disability and fatality rate among single diseases. Cerebral infarction is the most common cerebrovascular disease. In order to enhance the treatment response of cerebral infarction, this paper established male Sprague Dawley (SD) rat reperfusion model with 2 h of cerebral artery embolism by suture method. Neurological function deficit was scored according to rat improvement 24 h after model establishment, and 50 rats with scores between 10 and 13 were included in an ultimate experiment and were randomly divided into 5 groups: undisturbed control group, vascular endothelial growth factor (VEGF) up-regulated vessel group, endostatin down-regulated vessel group, ventricle injected Cxc Chemokin Receptor 4 (CXCR4) antibody group, ventricle injected α6ß1 antagonist (GoH3 antibody) group, respectively. The experiment was initiated after grouping and measurement of the relative data. The obtained results showed that the behavioral recovery of the VEGF group was more obvious compared with the control group, and the differences were statistically significant. The research was carried out using decreased modified neurological severity scores (mNSS), and the time a rat took to remove a pasted object. However, the behavioral recovery in the endostatin group, anti-CXCR4 group and GoH3 group was slow, and the difference was statistically significant, which showed as slowly decreased mNSS scoring and inconspicuous improved time of a rat removing a sticker. Compared with the control group, the number of peripheral BrdU+/ vWF+ cells of rat cerebral infarction in the VEGF group increased, and the peripheral VEGF expression quantity of cerebral infarction increased, thus the difference was statistically significant. However, cells in the endostatin group, anti-CXCR4 group, and GoH3 group were fewer and VEGF expression was reduced, and the difference was statistically significant. All these findings suggest that the promotion of angiogenesis after cerebral infarction can better provide the vascular niche for the proliferation, migration and differentiation of neural stem/progenitor cells (NSPCs), thereby further promoting endogenous nerve regeneration. NSPCs can always closely connect with vessels through the interaction of integrin α6ß1 and laminin; furthermore, under the support provided by the vascular niche and the chemotaxis of stromal cellderived factor (SDF-1), NSPCs can migrate from the subventricular zone (SVZ) to the periphery of infarction.

Strain-induced differentiation of fetal type II epithelial cells is mediated via the integrin α6ß1-ADAM17/tumor necrosis factor-α-converting enzyme (TACE) signaling pathway.

Mechanical forces are critical for normal fetal lung development. However, the mechanisms regulating this process are not well-characterized. We hypothesized that strain-induced release of HB-EGF and TGF-α is mediated via integrin-ADAM17/TACE interactions. Employing an in vitro system to simulate mechanical forces in fetal lung development, we showed that mechanical strain of fetal epithelial cells actives TACE, releases HB-EGF and TGF-α, and promotes differentiation. In contrast, in samples incubated with the TACE inhibitor IC-3 or in cells isolated from TACE knock-out mice, mechanical strain did not release ligands or promote cell differentiation, which were both rescued after transfection of ADAM17. Cell adhesion assay and co-immunoprecipitation experiments in wild-type and TACE knock-out cells using several TACE constructs demonstrated not only that integrins α6 and ß1 bind to TACE via the disintegrin domain but also that mechanical strain enhances these interactions. Furthermore, force applied to these integrin receptors by magnetic beads activated TACE and shed HB-EGF and TGF-α. The contribution of integrins α6 and ß1 to differentiation of fetal epithelial cells by strain was demonstrated by blocking their binding site with specific antibodies and by culturing the cells on membranes coated with anti-integrin α6 and ß1 antibodies. In conclusion, mechanical strain releases HB-EGF and TGF-α and promotes fetal type II cell differentiation via α6ß1 integrin-ADAM17/TACE signaling pathway. These investigations provide novel mechanistic information on how mechanical forces promote fetal lung development and specifically differentiation of epithelial cells. This information could be also relevant to other tissues exposed to mechanical forces.

Integrin α6ß1 is the main receptor for vascular laminins and plays a role in platelet adhesion, activation, and arterial thrombosis.

BACKGROUND: Laminins are major components of basement membranes, well located to interact with platelets upon vascular injury. Laminin-111 (α1ß1γ1) is known to support platelet adhesion but is absent from most blood vessels, which contain isoforms with the α2, α4, or α5 chain. Whether vascular laminins support platelet adhesion and activation and the significance of these interactions in hemostasis and thrombosis remain unknown. METHODS AND RESULTS: Using an in vitro flow assay, we show that laminin-411 (α4ß1γ1), laminin-511 (α5ß1γ1), and laminin-521 (α5ß2γ1), but not laminin-211 (α2ß1γ1), allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin α6ß1 and the glycoprotein Ib-IX complex, which binds to plasmatic von Willebrand factor adsorbed on laminins. Glycoprotein VI did not participate in the adhesive process but mediated platelet activation induced by α5-containing laminins. To address the significance of platelet/laminin interactions in vivo, we developed a platelet-specific knockout of integrin α6. Platelets from these mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. α6ß1-Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The tail bleeding time and blood loss remained unaltered, indicating normal hemostasis. CONCLUSIONS: This study reveals an unsuspected important contribution of laminins to thrombus formation in vivo and suggests that targeting their main receptor, integrin α6ß1, could represent an alternative antithrombotic strategy with a potentially low bleeding risk.

Neuropilin-2 regulates α6ß1 integrin in the formation of focal adhesions and signaling.

The neuropilins (NRPs) contribute to the function of cancer cells in their capacity as VEGF receptors. Given that NRP2 is induced in breast cancer and correlates with aggressive disease, we examined the role of NRP2 in regulating the interaction of breast cancer cells with the ECM. Using epithelial cells from breast tumors, we defined NRP2(high) and NRP2(low) populations that differed in integrin expression and adhesion to laminin. Specifically, the NRP2(high) population adhered more avidly to laminin and expressed high levels of the α6ß1 integrin than the NRP2(low) population. The NRP2(high) population formed numerous focal adhesions on laminin that were not seen in the NRP2(low) population. These results were substantiated using breast carcinoma cell lines that express NRP2 and α6ß1 integrin. Depletion experiments revealed that adhesive strength on laminin but not collagen is dependent on NRP2, and that VEGF is needed for adhesion on laminin. A specific interaction between NRP2 and α6ß1 integrin was detected by co-immunoprecipitation. NRP2 is necessary for focal adhesion formation on laminin and for the association of α6ß1 integrin with the cytoskeleton. NRP2 also facilitates α6ß1-integrin-mediated activation of FAK and Src. Unexpectedly, we discovered that NRP2 is located in focal adhesions on laminin. The mechanism by which NRP2 regulates the interaction of α6ß1 integrin with laminin to form focal adhesions involves PKC activation. Together, our data reveal a new VEGF-NRP2 signaling pathway that activates the α6ß1 integrin and enables it to form focal adhesions and signal. This pathway is important in the pathogenesis of breast cancer.