Thrombin receptor PAR-1 activation on endothelial progenitor cells enhances chemotaxis-associated genes expression and leukocyte recruitment by a COX-2-dependent mechanism.

BACKGROUND: Endothelial colony forming cells (ECFC) represent a subpopulation of endothelial progenitor cells involved in endothelial repair. The activation of procoagulant mechanisms associated with the vascular wall's inflammatory responses to injury plays a crucial role in the induction and progression of atherosclerosis. However, little is known about ECFC proinflammatory potential. AIMS: To explore the role of the thrombin receptor PAR-1 proinflammatory effects on ECFC chemotaxis/recruitment capacity. METHODS AND RESULTS: The expression of 30 genes known to be associated with inflammation and chemotaxis was quantified in ECFC by real-time qPCR. PAR-1 activation with the SFLLRN peptide (PAR-1-ap) resulted in a significant increase in nine chemotaxis-associated genes expression, including CCL2 and CCL3 whose receptors are present on ECFC. Furthermore, COX-2 expression was found to be dramatically up-regulated consequently to PAR-1 activation. COX-2 silencing with the specific COX-2-siRNA also triggered down-regulation of the nine target genes. Conditioned media (c.m.) from control-siRNA- and COX-2-siRNA-transfected ECFC, stimulated or not with PAR-1-ap, were produced and tested on ECFC capacity to recruit leukocytes in vitro as well in the muscle of ischemic hindlimb in a preclinical model. The capacity of the c.m. from ECFC stimulated with PAR-1-ap to recruit leukocytes was abrogated when COX-2 gene expression was silenced in vitro (in terms of U937 cells migration and adhesion to endothelial cells) as well as in vivo. Finally, the postnatal vasculogenic stem cell derived from infantile hemangioma tumor (HemSC) incubated with PAR-1-ap increased leukocyte recruitment in Matrigel(®) implant. CONCLUSIONS: PAR-1 activation in ECFC increases chemotactic gene expression and leukocyte recruitment at ischemic sites through a COX-2-dependent mechanism.

Thrombospondin-1 is a plasmatic marker of peripheral arterial disease that modulates endothelial progenitor cell angiogenic properties.

OBJECTIVE: We examined whether plasma levels of angiogenic factors are altered in plasma of patients with peripheral arterial disease (PAD) and whether these factors affect endothelial progenitor cell-induced angiogenesis. METHODS AND RESULTS: Plasma was collected from 184 patients with PAD and 330 age-matched healthy controls. Vascular endothelial growth factor and placental growth factor concentrations did not differ between the groups, whereas we found a linear correlation between PAD disease and thrombospondin (TSP)-1 plasma level. TSP-1 was expressed in newly formed vessels in PAD patients having received local injections of bone marrow mononuclear cells. To analyze the functional role of TSP-1 during neoangiogenesis, we used a Matrigel-plug assay and showed that vascularization of implanted Matrigel-plugs was increased in TSP-1(-/-) mice. Moreover, injections of TSP-1 in C57Bl6/J mice after hindlimb ischemia induced a significant decrease of blood flow recovery. To investigate the effects of TSP-1 on human endothelial colony-forming cell (ECFC) angiogenic potential, recombinant human TSP-1 and a small interfering RNA were used. In vitro, TSP-1 N-terminal part significantly enhanced ECFC adhesion, whereas recombinant human TSP-1 had a negative effect on ECFC angiogenic potential. This effect, mediated by CD47 binding, modulated stromal cell-derived factor 1/CXC chemokine receptor 4 pathway. CONCLUSIONS: TSP-1 is a potential biomarker of PAD and ECFC-induced angiogenesis, suggesting that TSP-1 modulation might improve local tissue ischemia in this setting. ( CLINICAL TRIAL REGISTRATION: NCT00377897.).