Study On Visfatin/Nampt As A Novel Vasoactive Substance And As A Novel Therapeutic Target For Stroke

Visfatin/Nampt is a protein of interest. Visfatin/Nampt is the rate-limiting enzyme in mammalian NAD biosynthesis. Visfatin/Nampt converts nicotinamide into NMN, with NMN being subsequently converted to NAD by Nmnat. Identification of this molecule took more than 40 years, since its enzymatic activity was first described in 1957 and its molecular identity was found in 2000s. It was originally characterized as a presumptive cytokine named pre-B cell colony-enhancing factor (PBEF). This protein drew much more interest after reported as a visceral fat-derived adipokine renamed visfatin. Visfatin/Nampt gene knockout is lethal in mice, indicating the importance of visfatin/Nampt for the life. The yeast nicotinamidase Pnc1, a functional equivalent of mammalian visfatin/Nampt, is a stress- and calorie-responsive longevity protein. Pnc1 and visfatin/Nampt both positively regulate the activity of another longevity protein called Sir2 in yeast and sirtuin-1 in mammals. Furthermore, visfatin/Nampt exerts anti-apoptotic action and protects cell from death in many cell types. However, the dunction of visfatin/Nampt in cardiovascular system is largely unknown. We investigated the role of visfatin/Nampt in cardiovascular system in this thesis.In the first section, we determined whether visfatin/Nampt acts as a relaxing factor and/or a growth factor secreted from PVAT and involved in the regulation of vascular function, and if so, to explore the underlying mechanisms. Visfatin/Nampt was preferentially expressed in Sprague-Dawley rat and monkey aortic PVAT, compared with subcutaneous and visceral adipose tissues. The PVAT-derived visfatin/Nampt was found to be a VSMC growth factor rather than a VSMC relaxing factor, which were proved by visfatin/Nampt specific antibody/inhibitor and direct observation of recombinant visfatin/Nampt. Exogenous visfatin/Nampt stimulated VSMC proliferation in a dose- and time-dependent manner via extracellular signal-regulated kinase (ERK 1/2) and p38 signaling pathways. This proliferative effect was further confirmed by enhancement of DNA synthesis and upregulation of proliferative marker Ki-67. Visfatin/Nampt had no anti-apoptotic effect on normal cultured VSMCs, and it exerted anti-apoptotic effect only under cell apoptosis induced by H2O2, excluding a role of anti-apoptotsis in the visfatin/Nampt-induced VSMC proliferation. Insulin receptor knockdown did not show any action on visfatin/Nampt effect. However, visfatin/Nampt acted as nicotinamide phosphoribosyltransferase to biosynthesize nicotinamide mononucleotide (NMN), which mediated proliferative signaling pathways and cell proliferation, mimetic to visfatin/Nampt effect. The present study provides a molecular link of visfatin/Nampt for the paracrine action of PVAT, demonstrates a novel function of visfatin/Nampt in promoting VSMC proliferation, and reveals NMN as a novel signal molecule to trigger the proliferative process.In the second section, we investigated the involvement of visfatin/Nampt in varied metabolic status and explored the change of visfatin/Nampt level in serum and local adipose in eight kinds of animal models (LH, SHR, SHR-SP, CR, AS, GK, nicotine injection and old rats). Firstly, we examined detailed characteristics of subcutaneous, visceral and PVAT in these eight animal models. Using enzyme immunoassay, we found that visfatin/Nampt serum concentrations increased only in LH compared with control (89.3±9.2 ng/ml vs 57.8±5.6 ng/ml; P<0.05). We determined expression of visfatin/Nampt in subcutaneous, visceral and PVAT using western blot analysis. Different from serum visfatin/Nampt level, expression of visfatin/Nampt in local adipose tissues were changed diversely. In LH, expression of visfatin/Nampt in visceral adipose tissue significantly increased (+88%; P<0.01). In SHR, visfatin/Nampt in PVAT decreased (-33%; P<0.01). In SHR-SP, visfatin/Nampt expression decreased both in visceral adipose (-23%; P<0.05) and PVAT (-47%; P<0.01). Calorie restriction upregulated visfatin/Nampt expression in visceral adipose (+132%; P<0.01) and in PVAT (+78%; P<0.01). In AS rats, visfatin/Nampt was downregulated in PVAT (-40%; P<0.01). In old rats, visfatin/Nampt was downregulated in visceral adipose and PVAT significantly. The discrepancy between levels of visfatin in serum and local adipose indicates that local adipose visfatin is more sensitive to systemic metabolism homeostasis and may contribute to local adipose adaptation to metabolic status without accompanying alteration of serum level in metabolic relating diseases.In the third section, we demonstrate that visfatin/Nampt is a neuroprotector under ischemic stress. We detected visfatin/Nampt expression in brain and neurons, in line with previous reports. Remarkably, visfatin/Nampt was significantly upregulated in peri-infarct area and infarct core of MCAO model. Inhibition of visfatin/Nampt accelerated the death of SHR-SP rats and augmented the infarction in MCAO rats, whereas overexpression of visfatin/Nampt in local brain protected against stroke injury in MCAO rats, indicating visfatin/Nampt is a critical protective factor in ischemic stroke. Furthermore, the neuroprotection of visfatin/Nampt was confirmed in cultured neurons of OGD model. It was found that inhibition or knockdown of visfatin/Nampt aggravated neuronal injury, evidenced by reduction of neuronal cells and cell variability, enhancement of LDH release, upregulation of pro-apoptotic proteins, downregulation of anti-apoptotic proteins, collapse of mitochondrial membrane potential, augmentation of mitochondrial cytochrome c release, and increase of neuronal apoptosis and death. In contrast, overexpression of visfatin/Nampt reduced neuronal injury. All of these in vivo and in vitro results identify visfatin/Nampt as a chief dictator for neuron survival in ischemia and implicate visfatin/Nampt as a crucial factor involved in energy balance regulation. Thus, our results provide new insights into endogenous defense mechanism in ischemic stroke, and suggest that visfatin/Nampt is a new therapeutic target for stroke prevention and treatment.In the forth section, we studied the effect of PTU on AS in rodents. This study was to characterize vascular function in rat atherosclerosis induced by high cholesterol plus antithyroid drug propylthiouracil (HC+PTU) and provide novel insights into hypothyroidism-associated atherosclerosis. Mechanisms for causal relationship between hypothyroidism and atherosclerosis are largely unknown. Rats were treated with HC+PTU for long-term (2 to 10 weeks) and short-term (3 days to 1 week), or HC and PTU alone for short-term to find which factor involved in early changes of HC+PTU treatment. In vivo T4 intervention and in vitro vascular smooth muscle cell (VSMC) culture were used to know direct and indirect effect of PTU. This hypothyroidism-associated atherosclerosis was characterized by aortic contraction loss for several kinds of contracting agents in long-term treatment and rapid onset of vasoconstriction reduction in short-term treatment. Accordingly, aortic VSMC apoptosis appeared as early as 3 days, developed within 2 weeks, and was very severe at 6 and 10 weeks after HC+PTU treatment. However, endothelial anticontractile effect was preserved and endothelial nitric oxide synthase upregulated in short-term treatment. Detailed experiments revealed PTU rather than HC induced rapid onset of vasoconstriction reduction and VSMC apoptosis, which was prevented by T4 supplement. VSMC culture study clarified cell apoptosis was not due to direct effect of PTU at serum concentration, but it was caused by reduced anti-apoptotic action due to reduced T3 and T4. The jun N-terminal kinases and caspase-3 were involved in hypothyroidism-induced cell apoptosis. These data delineate a novel mechanism for hypothyroidism-associated atherosclerosis initiated by VSMC apoptosis rather than endothelial dysfunction.