Endothelial nitric oxide synthase affects both early and late collateral arterial adaptation and blood flow recovery after induction of hind limb ischemia in mice

ObjectiveThe goals of this study were to determine if endothelial nitric oxide synthase (eNOS) affects both early and late collateral arterial adaptation and blood flow recovery after severe limb ischemia in a mouse model and to determine if eNOS-derived NO is necessary for recruitment of chemokine (C-X-C motif) receptor 4 (CXCR4)+ vascular endothelial growth factor receptor-1 (VEGFR1)+ hemangiocytes to the site of ischemia.MethodsTwo studies were completed. In the first, hind limb ischemia was induced by unilateral femoral artery excision in three groups: C57Bl6 (wild-type), eNOS−/−, and C57Bl/6 mice treated with NG-nitro-l-arginine methyl ester (L-NAME) from 1 day before excision through day 3 after excision (early L-NAME group). These groups were studied on day 3 after induction of ischemia. In the second study, hind limb ischemia was induced in C57Bl/6 mice (wild-type) and C57Bl/6 mice treated with L-NAME from days 3 through 28 after induction of ischemia. These groups were studied day 28 after ischemia induction. Dependent variables included hind limb perfusion, collateral artery diameter, and the number and location of hemangiocytes within the ischemic hind limb.ResultsIn the first study, toe gangrene developed in the eNOS−/− and early L-NAME treatment groups by day 2. These groups demonstrated less blood flow recovery and smaller collateral artery diameter than the wild-type group. Hemangiocytes were present within the adventitia of collateral arteries in the wild-type group but were only sparsely present, in a random pattern, in the eNOS−/− and early L-NAME treatment groups. In the second study, the late L-NAME group showed less blood flow recovery and smaller collateral artery diameter on day 28 of ischemia than the wild-type group. Hemangiocytes were present in a pericapillary distribution in the wild-type group, but were present only sparsely in the late L-NAME treatment group.ConclusionEarly (day 3) and late (day 28) adaptive responses to hind limb ischemia both require eNOS–derived NO. NO is necessary for normal hemangiocyte recruitment to the ischemic tissue.Clinical RelevanceThis study demonstrates that endothelial nitric oxide synthase (eNOS)-derived NO is requisite for both the early and late vascular recovery phases in response to hind limb ischemia. Moreover, it demonstrates that recruitment of hemangiocytes, a specific subset of vascular progenitor cells that display vascular endothelial growth factor receptor 1 (VEGFR1) and chemokine (C-X-C motif) receptor 4 (CXCR4) surface antigens, is dependent on the presence of NO. These findings enhance understanding of the basic biologic mechanisms in the recovery from ischemia. The need for eNOS-derived NO suggests that clinical strategies to enhance postocclusive flow recovery in peripheral artery disease might be more successful if eNOS or NO, or both, are applied as a part of the treatment paradigm. Hemangiocytes are an important part of the cellular response to ischemia. The present findings underscore their importance and suggest that preservation or enhancement of eNOS within the ischemic limb might improve the recruitment of these critical reparative cells to the site of ischemic injury