Nampt/Visfatin Promotes Post-Ischemic Neovasculairzation Via Enhancing Endothelial Progenitor Cell Function

Accumulating experimental and clinical evidence indicated that the level ofcirculating EPCs predicts the occurrence and outcome of cerebro-cardiovascular eventsand diabetes. Recent small-scale clinical studies also underscored the transplantation ofEPCs in therapy of ischemic heart disease. However, it has been shown that someendogenous regulatory factors contribute to EPCs dysfunction for impairedneovascularization and subsequent poor wound healing in certain diseases such asdiabetes and hypertension. NAD+is well-established cofactor regulating electron transportchain and cellular energy production, and has been involved in various signalingtransduction as an energy sensor. As the rate-limiting enzyme of NAD+biosynthesis,NAMPT has been reported to localize in both intra-and extracellular compartments, and isa pivotal regulator in cell survival, immune responses, circadian control and metabolicabnormalities. We previously demonstrated the neuroprotective effect of NAMPT incerebral ischemia. We hypothesized there might be an association between NAD+andEPCs biological function in ischemia.To investigate whether NAD+level is influenced by the ischemia, we surveyed theblood NAD+level in mice subjected hindlimb ischemia We also isolated bone marrow(BM) from HLI-performed mice, and quantified the NAD+level in BM. Because NAMPTis the limiting-enzyme for NAD+biosynthesis, we measured NAMPT expression in BM.To investigate whether NAD+level is influenced by the ischemia, we surveyed the bloodNAD+level in mice subjected hindlimb ischemia (HLI), and found that the blood NAD+level increased markedly at3and7days after HLI and returned to baseline at14dayspost-HLI. We also isolated bone marrow (BM) from HLI-performed mice, and quantifiedthe NAD+level in BM. Interestingly, HLI induced enhancement of BM NAD+levelssimilarly. Because NAMPT is the limiting-enzyme for NAD+biosynthesis, we measuredNAMPT expression in BM. Accordingly, the BM NAMPT expression increased after HLI.We used a well-established NAMPT inhibitor FK866to inhibit NAMPT enzymaticactivity and depleted NAD+, and then studied the influence of NAD+depletion on EPCsmotivation after ischemia. Using fluorescence-activated cell sorting (FACS), wedetermined the relative frequency of EPCs in blood and BM. The HLI-induced increase ofblood EPCs (Sca-1/VEGFR2) frequency was markedly abolished by FK866treatment (i.p.,1.5mg/kg/day).We isolated EPCs from BM using magnetic-activated cell sorting and cultured themin vitro. With the endothelial culture medium, EPCs can proliferate, migrate and formtubes. NAD+depletion by FK866reduced EPCs proliferation, attenuated EPCs migration,and disrupted EPCs tube formation. We therefore conclude that intracellular NAD+levelregulates EPCs biology.To investigate the potential role of NAMPT in the regulation of NAD+level on EPCsbiology, we generated transgenic mice overexpressing NAMPT (NAMPT-Tg mice) anddominant-negative NAMPT (DN-NAMPT-Tg mice, mutant H247A) driven by theubiquitin promoter. Compared with NAMPT protein, the DN-NAMPT protein has noNAD+-biosynthesis enzymatic activity in vitro. The NAD+level in BM from NAMPT-Tgmice was higher than that from wild-type mice, whereas DN-NAMPT-Tg mice BMexhibited lower NAD+level, suggesting the overexpression of exogenous NAMPT indeedinfluenced the in vivo NAD+level. Robust expression of his-tagged NAMPT orDN-NAMPT in BM-derived EPCs from transgenic mice was readily detectable. Weperformed functional analysis on these EPCs. Compared with wild-type EPCs, NAMPT-TgEPCs displayed enhanced proliferation, migration ability and tube formation. All thesephenotypes were not observed in DN-NAMPT-Tg EPCs.Next, we analysed whether modulation of NAD+level influences post-ischemicneovascularization in vivo. In wild-type mice, the enhancement of blood/BM EPCsfrequency induced by HLI was further augmented in NAMPT-Tg mice but not inDN-NAMPT-Tg mice. We also found that NAMPT-Tg mice, but not DN-NAMPT-Tg mice,exhibited increased capillary density of ischemic gastrocnemius muscle at2weeks afterHLI. Additionally, the number of proliferating endothelial cells (CD31+/Ki-67+) was higherin NAMPT-Tg mice but not in DN-NAMPT-Tg mice. Transgene of NAMPT markedlyenhanced blood perfusion in the ischemic hind limb. We further studied neovascularizationpost-ischemic stroke in mice. NAMPT-Tg mice exhibited enhancement ofneovascularization in injured brain area.All these results indicate that NAD+derived from NAMPT is essential for EPCsfunctions in post-ischemic vascular repair.