P91	Bone-marrow mononuclear cells induce NO-dependent vasodilation: involvement in their pro-angiogenic potential.
D.You, L.Waeckel, T.Ebrahimian, O.Blanc-Brude, S.Le Ricousse-Roussanne, G.Tobelem, J.Vilar, M.Duriez, B.I.Lévy, J-S.Silvestre
INSERM U689, Paris, FR.

Considerable efforts have been focused on the development of therapeutic strategies designed to increase vessel growth after ischemia. Therapeutic delivery of bone-marrow mononuclear cells (BMC) has been shown to stimulate neovascularization after ischemic injury. However, mechanisms of BMC beneficial effects remain unclear. We hypothesized that the pro-angiogenic potential of BMC is mediated through nitric oxide (NO)-dependent vasodilation. Intraluminal administration of BMC induced a rapid (5 min) dose-dependent vasodilation in isolated femoral arteries (246micrometer basal diameter to 272micrometer for 0.5.106 BMC/ml, p<0.01). Interestingly, Sca-1+ BMC induced vasodilation by 3.9-fold over Sca-1- BMC (p<0.001). Ex vivo expanded human EPC also induced vasodilation (246micrometer basal diameter to 292micrometer for 0.5.106 EPC/ml, p<0.01). eNOS, but not iNOS nor nNOS, protein was detected in BMC and EPC, as assessed by western-blotting. BMC-induced vasodilation was reduced by 1.8-fold with NOS inhibitor pretreatment (L-NAME, 10-5 M , 1h p<0.01) or by 2.7-fold using BMC isolated from eNOS-deficient mice (p<0.001). BMC-induced vasodilation was abrogated when BMC were pretreated with neutralizing CXCR4 antibodies. The absence of endothelium or treatment of isolated vessels with neutralizing SDF-1 antibodies also hampered BMC-related effects. This suggested that BMC-induced vasodilation required adhesion to endothelium through SDF-1/CXCR4 interactions. BMC-induced vasodilation may participate to their pro-angiogenic effects. Using a model of surgically-induced hindlimb ischemia in mice, we evidenced that BMC induced a marked vasodilation in ischemic femoral artery. Pretreatment of BMC with L-NAME reduced angiographic score by 2.4-fold and blood foot perfusion by 1.5-fold compared to animals receiving untreated BMC (n=6, p<0.05). BMC isolated from eNOS-deficient mice displayed an impairement in their pro-angiogenic potential which was partially restored by cotreatment with NO donnor (SNP, 10-5 M , 1h). Similarly, EPC pro-angiogenic effect was abrogated by pretreatment with L-NAME (n=5, p<0.05 versus untreated EPC). In conclusion, BMC-induced NO-dependent vasodilation can contribute, at least in part, to post-ischemic neovascularization.

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