Contribution of distinct platelet integrins to binding, unfolding, and assembly of fibronectin.

Fibronectin (FN) fibrillogenesis depends on the binding of FN to cellular receptors and subsequent unfolding of bound FN. Integrins αIIbß3, αvß3, and α5ß1 are known to assemble FN fibrils on platelets. In our study, we examined the contribution of these integrins to FN binding, unfolding, and assembly on platelets in suspension and adherent platelets in the presence or absence of agonists. Phorbol 12-myristate 13-acetate (PMA), but not adenosine diphosphate (ADP), induced binding of FN to platelets in suspension. In contrast, adherent platelets were able to deposit FN on their surfaces in the absence of agonists. ß3 integrins had a major impact on the interaction of FN on platelets. αvß3 showed a similar contribution to the binding of FN as αIIbß3 on PMA-stimulated platelets in suspension but had a lesser contribution to unfolding and deposition of FN on adherent platelets. α5ß1 also participated in the interaction of FN with platelets by mediating the unfolding and assembly of FN, but to a lesser extent than ß3 integrins. None of the distinct antibodies directed against one of the three integrins caused a complete inhibition of binding, unfolding, and assembly of FN by platelets. Thus, it is likely that αIIbß3, αvß3, and α5ß1 or another still unknown receptor can be substituted.

The exosomal integrin α5ß1/AEP complex derived from epithelial ovarian cancer cells promotes peritoneal metastasis through regulating mesothelial cell proliferation and migration.

PURPOSE: Epithelial ovarian cancer (EOC) is one of the most malignant cancers in the gynecologic system. Many patients are diagnosed at an advanced stage with disseminated intra-peritoneal metastases. EOC spreads via both direct extension and trans-coelomic spread. However, the interplay between human peritoneal mesothelial cells (HPMCs) and EOC cells is still ambiguous. We hypothesize that integrins (ITG) in HPMCs may play important roles in EOC metastasis. METHODS: The expression of different integrin subtypes from HPMCs was assessed using Western blotting. The expression of integrin α5ß1 (ITGA5B1) and its co-localization with asparaginyl endopeptidase (AEP) in HPMCs derived from EOC patients (EOC-HPMCs) were assessed using immunofluorescence. The role and mechanism of the exosomal ITGA5B1/AEP complex in HPMCs was assessed using both in vitro and in vivo assays. A retrospective study involving 234 cases was carried out to assess ITGA5B1 and AEP levels in circulating sera and ascites of EOC patients, as well as associations between ITGA5B1/AEP expression and overall survival. RESULTS: We found that ITGA5B1was highly expressed and co-localized with AEP in EOC cells, and that the exosomal ITGA5B1/AEP complex secreted by EOC cells played an important role in the proliferation and migration of HPMCs. High levels of exosomal ITGA5B1/AEP were also found in circulating sera and ascites of EOC patients, and the expression of ITGA5B1/AEP in EOC tissues was found to be negatively associated with overall survival. CONCLUSIONS: Our data indicate that EOCs may regulate the function of HPMCs through exosomal ITGA5B1/AEP, which may be crucial for peritoneal metastasis.

Expression and function of homing-essential molecules and enhanced in vivo homing ability of human peripheral blood-derived hematopoietic progenitor cells after stimulation with stem cell factor.

Hematopoietic stem cell (HSC) homing from blood to bone marrow is a multistep process involving rolling, extravasation, migration, and finally adhesion in the correct microenvironment. With view to the hematopoietic recovery after clinical stem cell transplantation, we investigated the effect of stem cell factor (SCF) on the expression and the adhesive function of the alpha4beta1 and alpha5beta1 integrins very-late antigen (VLA)-4 and VLA-5 on peripheral blood-derived hematopoietic progenitor cells. After SCF stimulation, the expression of VLA-4 and VLA-5 on CD34+/c-kit+ cells obtained from healthy donors increased from 54% to 90% and from 3% to 82%, respectively. For patient-derived cells, the increase was 67% to 90% and 12% to 46%. The proportion of mononuclear cells adhering to the fibronectin fragment CH296 increased by stimulation with SCF from 14% to 23%. Accordingly, functional studies showed an approximate 30% increase of adherent long-term culture-initiating cell. The improvement of the homing abilities of SCF-stimulated HSC was confirmed by transplantation into sublethally irradiated nonobese diabetic-scid/scid mice. Six weeks after the transplantation, in eight of eight animals receiving human HSC with the addition of SCF, a profound multilineage hematopoietic engraftment was detected, whereas in the control group receiving only HSC, none of eight animals engrafted. Our data provide the first in vivo evidence that stimulation with cytokines improves the homing ability of transplanted human hematopoietic progenitor cells.

Lipopolysaccharide induces expression of fibronectin alpha 5 beta 1-integrin receptors in human monocytic cells in a protein kinase C-dependent fashion.

LPS is an outer-membrane glycolipid component of gram-negative bacteria known for its fervent ability to activate monocytic cells and for its potent proinflammatory capabilities. In addition, LPS triggers the release of cytokines and chemokines as well as cell-cell adhesion molecules. We postulate that LPS may also affect the expression of matrix-binding integrin receptors, thereby modulating cell-adhesive functions in monocytic cells. To test this hypothesis, we investigated the effects of LPS on the expression of the integrin alpha(5)beta(1), a fibronectin receptor, in a human monocytic cell line (U937) as well as in isolated human peripheral blood mononuclear cells (PBMCs). We found that LPS increased the expression of alpha(5)beta(1) receptors and enhanced the adherence of U937 cells and PBMCs to fibronectin-coated surfaces; this was blocked by anti-alpha(5)beta(1) antibodies. LPS increased alpha(5)-subunit mRNA accumulation in a dose- and time-dependent manner. The induction by LPS occurred, at least in part, at the level of gene transcription as indicated by experiments using alpha(5) intact and deletion promoter constructs. LPS-induced alpha(5) gene transcription was associated with rapid induction of conventional PKC-alpha protein and activity, was blocked by PKC inhibitors, and was mimicked by lipid A. Finally, we found that an anti-CD14 antibody was able to inhibit the LPS response. Overall, the data suggest that LPS stimulates alpha(5) gene transcription via CD14 and PKC-dependent signals to enhance the expression of functional alpha(5)beta(1) receptors in monocytic cells. This process may help stimulate monocytic cell activation and facilitate their migration into fibronectin-containing tissues during infection.