Osteoprotegerin regulates cancer cell migration through SDF-1/CXCR4 axis and promotes tumour development by increasing neovascularization

We previously reported that OPG is involved in ischemic tissue neovascularization through the secretion of SDF-1 by pretreated-OPG endothelial colony-forming cells (ECFCs). As the vascularization is one of the key factor influencing the tumour growth and cancer cell dissemination, we investigated whether OPG was able to modulate the invasion of human MNNG-HOS osteosarcoma and DU145 prostate cancer cell lines in vitro and in vivo. Cell motility was analysed in vitro by using Boyden chambers. Human GFP-labelled MMNG-HOS cells were inoculated in immunodeficient mice and the tumour nodules formed were then injected with OPG and/or FGF-2, AMD3100 or 0.9% NaCl (control group). Tumour growth was manually followed and angiogenesis was assessed by immunohistochemistry. In vitro, SDF-1 released by OPG-pretreated ECFCs markedly attracted both MNNG-HOS and DU145 cells and induced spontaneous migration of cancer cells. In vivo, tumour volumes were significantly increased in OPG-treated group compared to the control group and OPG potentiated the effect of FGF-2. Concomitantly, OPG alone or combined with FGF-2 increased the number of new vasculature compared to the control group. Interestingly AMD3100, an inhibitor of SDF-1, prevented the in vivo effects of OPG induced by SDF-1 This study provides experimental evidence that OPG promotes tumour development trough SDF-1/CXCR4 axis

Expression of Protease-Activated Receptor 1 and 2 and Anti-Tubulogenic Activity of Protease-Activated Receptor 1 in Human Endothelial Colony-Forming Cells

Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50–100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25–100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade

Mechanistic study of the proangiogenic effect of osteoprotegerin

Osteoprotegerin (OPG), a soluble tumour necrosis factor receptor superfamily member, inhibits RANKL-mediated osteoclastogenesis. We have previously reported that OPG enhances the proangiogenic properties of endothelial colony-forming cells (ECFCs) in vitro, and promotes vasculogenesis in vivo. Here we investigated how OPG promotes neovascularisation. Proteomic experiments showed that OPG pretreatment affected ECFCs protein expression in two ways, 23 spots being down-regulated and 6 upregulated. These spots corresponded to proteins involved in cell motility, adhesion, signal transduction and apoptosis. In keeping with these proteomic results, we found that OPG induced ECFCs adhesion to activated endothelium in shear stress conditions, promoting intermediate but not focal adhesion to fibronectin and collagen. Treatment with OPG induced a reorganization of the ECFCs cytoskeleton, with the emergence of cell protrusions characteristic of a migratory phenotype. These effects correlated with decreased FAK phosphorylation and enhanced integrin αVβ3 expression. OPG drastically reduced caspase-3/7 activities and maintained ECFCs viability after 48 h of treatment. All these effects were significantly attenuated by ECFCs incubation with the CXCR4 antagonist AMD-3100, and by prior heparan sulphate proteoglycan disruption. The proangiogenic properties of OPG appeared to be mediated by the proteoglycan syndecan-1, although OPG 1-194 lacking its heparin-binding domain still had pro-vasculogenic effects in vitro and in vivo. These results suggest that OPG may interact with ECFCs by binding to HSPGs/syndecan-1, thereby induce an anti-adhesive effect and promoting ECFCs migration through a SDF-1/CXCR4 dependent pathway

Acidic preconditioning improves the proangiogenic responses of endothelial colony forming cells

Objective: Acidosis is present in several pathological conditions where vasculogenesis takes place including ischemia, tumor growth and wound healing. We have previously demonstrated that acidosis induces human CD34+ cell apoptosis. Considering that endothelial colony-forming cells (ECFC) are a subpopulation of CD34+ cells and key players in vasculogenesis, in the present study we investigated the effect of acidosis on the survival and functionality of ECFC. Approach and results: Endothelial colony-forming cells obtained by differentiation of human cord blood CD34+ cells in endothelial growth medium-2 for 14–21 days were exposed at pH 7.4, 7.0 or 6.6. We found that acidosis failed to induce ECFC apoptosis and, although an early reduction in proliferation, chemotaxis, wound healing and capillary-like tubule formation was observed, once the medium pH was restored to 7.4, ECFC proliferation and tubulogenesis were augmented. Stromal cell derived factor-1 (SDF1)-driven migration and chemokine receptor type 4 surface expression were also increased. The maximal proangiogenic effect exerted by acidic preconditioning was observed after 6 h at pH 6.6. Furthermore, preconditioned ECFC showed an increased ability to promote tissue revascularization in a murine model of hind limb ischemia. Immunoblotting assays showed that acidosis activated AKT and ERK1/2 and inhibited p38 pathways. Proliferation rises triggered by acidic preconditioning were no longer observed after AKT or ERK1/2 inhibition, whereas p38 suppression not only mimicked but also potentiated the effect of acidosis on ECFC tubule formation abilities. Conclusions: These results demonstrate that acidic preconditioning greatly increases ECFC-mediated angiogenesis in vitro including ECFC proliferation, tubulogenesis and SDF1-driven chemotaxis and is a positive regulator of microvessel formation in vivo.Fil: Mena, Hebe Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Lokajczyk, Anna. Université Paris Descartes; Francia. Inserm; FranciaFil: Dizier, Blandine. Inserm; FranciaFil: Strier, Sergio E.. Ciudad Autónoma de Buenos Aires. Hospital "Bernardino Rivadavia"; ArgentinaFil: Voto, Liliana S.. Ciudad Autónoma de Buenos Aires. Hospital "Juan A. Fernández"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Boisson Vidal, Catherine. Université Paris Descartes; Francia. Inserm; FranciaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Negrotto, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin