The Role Of Nicotinamide Phosphoribosyl-Transferase In Cerebral Endothelial Cell Function And Ischemic Injury

BackgroundsNicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme of the salvage biosynthesis of nicotinamide adenine dinucleotide (NAD), which converts nicotinamide into nicotinamide mononucleotide (NMN). By synthesizing NAD, NAMPT participates in various important physiological and pathophysiological processes. NAMPT is expressed in many types of cells, which can also be secreted through paracrine and endocrine actions by some of these cells. In the braina NAMPT is expressed in neurons and endothelial cells but not in astrocytes and microglial cells. Intracellular nicotinamide phosphoribosyltransferase (iNAMPT) is an essential enzyme in the NAD biosynthetic pathway. Extracellular form of this protein (eNAMPT) has been reported to act as a cytokine, involved in immunity, metabolism, aging, inflammation, and responses to stress.Ischemic stroke is an age-related disease and happens more frequently in elderly population, causes severe neuronal injury and consequently high mortality and morbidity. Both neurons and non-neuronal cells such as cerebral microvasculature and glial cells are involved in ischemic stroke. Immediately after ischemic stroke, the death and dysfunction of cerebral microvascular endothelial cells cause brain-blood barrier (BBB) breakdown and lead to vasogenic edema, this in turn causes vasospasm and aggravates ischemic injury. During recovery, endothelial cells play an essential role in angiogenesis, this is important for the recovery of neuronal functions. As such, it is necessary to assess the effect of neuroprotective agents on cerebral microvascular endothelial cells during ischemic injury and recovery.It has been reported that the level of NAMPT in serum increased after ischemic stroke. Knocking down or inhibiting the activity of NAMPT decreased NAD level and aggravated ischemic injury in the mouse and rat, over-expression of NAMPT or addition of nicotinamide mononucleotide (NMN), a direct production of NAMPT, reduced ischemia-induced cerebral injury in the rat. NAMPT has a neuroprotective effect on ischemia through its enzymatic activity for NAD production that can ameliorate mitochondrial dysfunction. In addition, NAD has been identified as a neuroprotective agent based on the evidence that it reduced ischemia-induced neuronal death and axonal degeneration. These data suggest that NAMPT may be an attractive therapeutic target for both the prevention and treatment of ischemic stroke.NAMPT promotes endothelial cell proliferation, migration and capillary-like tube formation, suggesting that NAMPT plays an important role in angiogenesis. NAMPT was found to be a critical protective protein in endothelial cells, by preventing endothelial cells from apoptosis, injury, and aging. The findings demonstrate the NAMPT as a protector of endothelial cells against various stresses. In addition, both in vitro and in vivo experiments suggest that NAMPT may also be related to inflammatory reactions in endothelial cells.We have found that the level NAMPT increased in serum while decreased in brain in aged mice in previous study. The change of NAMPT in serum has direct effect to cerebral vascular endothelial cells. Besides, many clinical studies have found that the level of NAMPT also increased in patients with metabolic disease such as diabetes and obesity, which, as we all know, with greater chance to ischemic stroke and often have more serious consequences.So, we want to know whether the changes of iNAMPT and eNAMPT have an effect in cerebral vascular endothelial cells, if there are, what impact do they have on cerebral vascular endothelial cells after ischemic injury, whether they aggravate the injury or play a protective role, and the possible mechanism.AimsIn the present study, we aimed to investigate the effects of iNAMPT and eNAMPT on physiological function of celebral vascular endothelial cells. Then we aimed to clarify the effects of iNAMPT on elebral vascular endothelial cells after ischemic injury and to reveal the possible mechanism.1. To investigate the effect of NAMPT on physiological function of celebral vascular endothelial cells.2. To clarify the effect of iNAMPT on OGD-induced acute injury and subsequent recovery in cerebral vascular endothelial cells.MethodsbEnd.3cells were derived from mouse cerebral microvascular endothelial cells. We used FK866(a specific inhibitor of NAMPT) to inhibit enzymatic activity of NAMPT and to mimic NAD depletion, and used nicotinamide mononucleotide (NMN, the direct product of NAMPT) to replenish intracellular NAD. Recombination human NAMPT (wild type) and human NAMPT/H247A (mutation) were used to act as eNAMPT. Ischemic injury was induced by OGD for0.5or4h; Western blotting analysis and cellular immunofluorescence assay were performed to detect the expression of NAMPT and LC3; MTT reduction assay was done to detect cell viability; PI-Hoechst dyeing was used to detect apoptosis and necrosis; cell scratching was performed as a model of cell migration; Angiogenesis was detected using rat aortic ring of angiogenesis model; LDH release was used to detect the damage of cells after OGD/recovery (OGD/R); NAD/NADH Quantification Kit was used to detect the level of NAD in cells.ResultsPart I Effect of NAMPT on physiological function of celebral vascular endothelial cells.Western-blotting and cellular immunofluorescence assay showed that NAMPT was expressed in bEnd.3cells. We found that the intracellular NAD level was reduced by FK866, an inhibitor of NAMPT, and was replenished by NMN, the direct production of NAMPT. MTT reduction assay and cell healing test showed that viability and migration of bEnd.3cells decreased upon application of FK866(10-100nM), in a concentration-and time-dependent manner, NMN0.1-1mM concentration-dependently abolished cell viability and migration reduction caused by FK866, while NMN alone had no effect. FK866100nM induced cell apoptosis. Rat aortic ring model of angiogenesis showed that FK866100nM inhibited microvessel growth while10nM inhibited migration of endothelial cells. These results indicated that NAMPT is essential for the survival of cerebral vascular endothelial cells through synthesizing NAD. In addition, we found that both NAMPT and NAMPT/H247A0.1-1.0μg/ml promoted bEnd.3cell proliferation and migration. This result showed that eNAMPT promotes proliferation and migration of cerebral endothelial cells, possibly through the control of non-enzymatic activity.Part II Effect of iNAMPT on OGD-induced acute injury and subsequent recovery in cerebral vascular endothelial cells After0.5h OGD, we found that cell viability decreased by about20%, but LDH release and cell counting did not change. Cell viability returned to normal after recovery for24-48h. This indicated that OGD0.5h induced a transient injury to cerebral vascular endothelial cells. During24-48h recovery, the level of NAD decreased. Application with FK86624h before OGD further reduced the level of NAD which could be replenished by NMN. Reduction of NAD before OGD with FK866significantly aggravated OGD-induced decreasing of cell viability, and replenishment of NAD with NMN abolished such an aggravation by FK866, but had no effect on OGD-induced injury. Application with FK866and NMN after OGD had no effect on cell viability. These results indicated that during mild ischemic injury, preventing NAD from being depleted could protect cerebral endothelial cells from acute ischemic injury, but NAD was not involved in the recovery process.After OGD for4h, cell viability decreased by about50%. LDH release increased and cell number decreased. These results showed that4h OGD obviously induced injury to cerebral vascular endothelial cells. The level of NAD gradually increased during24-48h recovery. Application with FK866aggravated OGD-induced decreasing of NAD level, but NMN had no effect on NAD level after OGD and recovery. FK8661-10nM had no effect on cell viability after OGD and recovery. Interestingly, we found that NMN1mM alleviated4h OGD-induced cell viability decrease, promoted cell recovery, and decreased LDH release. These results showed that during serious ischemic injury, NMN had a protective effect on cerebral vascular endothelial cells.At last, we explored the possible mechanism of iNAMPT to cerebral vascular endothelial cells after ischemia. We found that both application with FK866and4h OGD can induced cell autophagy, NMN and the autophagy inhibitor LY294002abolished cell autophagy induced by FK866and4h OGD. These results showed that cell autophagy might be involved in the effect of iNAMPT on cerebral vascular endothelial cells after ischemic injury. Conclusions:1. Through synthesizing NAD, iNAMPT is involved in important physiological functions of cerebral vascular endothelial cells, iNAMPT is essential for the survival of cerebral vascular endothelial cells; eNAMPT promotes proliferation and migration of cerebral endothelial cells, possibly through the control of non-enzymatic activity.2. During mild ischemic injury, preventing NAD depletion can protect cerebral endothelial cells from acute ischemic injury; during servious ischemic injury, replenishing of NAD has a protective effect on OGD-induced injury in cerebral vascular endothelial cells; Cell autophagy may participate in this process.