Aging, Angiogenesis, and the Neuropeptide Y (NPY) System

血管生成的年龄依赖性障碍导致缺血性血管疾病进展加快，伤口愈合延迟，以及肿瘤生长和扩散速度减慢。血管生成减少的机制尚未完全阐明，但可能包括内皮反应性的改变、细胞与基质的相互作用以及血管生成因子及其受体的缺乏。神经肽Y(NPY)通常被认为是交感神经递质和血管收缩因子。最近，我们的实验室在体外和在大鼠后肢缺血模型中发现了其作为血管生成和有丝分裂因子的新的和有效的活性，对血管平滑肌和内皮细胞具有促进作用。这项研究的总体目标是确定NPY系统的变化是否与年龄相关的血管生成障碍有关。其具体目的是确定：1)NPY介导的血管生成是否随着年龄的增长而减少；2)老年动物在生理条件下和对血管生成刺激的反应中发生的NPY合成/释放减少；3)NPY血管生成受体(Y1、Y2、Y5)及其转换酶二肽基肽酶IV(DPPIV)的表达是否随着年龄的增加而改变。这些研究将在动物(幼鼠和老年鼠)和细胞模型上进行。在两个年龄组之间，将比较自发的以及NPY和bFGF诱导的小鼠主动脉萌发。采用小鼠后肢缺血模型，研究衰老对NPY诱导的缺血血管生成的影响。新血管将通过CD31免疫染色和乳胶灌流铸型来检测。将确定全身和局部NPY水平，以及血管生成刺激的表达。该细胞模型将包括NPY对年轻和老年小鼠主动脉内皮细胞的刺激，并在NPY受体表达的同时测量它们的有丝分裂反应。通过检测老龄和幼年动物缺血组织中生长因子和NPY受体的水平，以及NPY和NPY治疗后的缺血组织中生长因子和NPY受体的水平，将建立bFGF和NPY血管生成系统之间的相互作用及其与年龄相关的调节。此外，还将通过主动脉萌发实验，研究bFGF-和NPY中和抗体对Y2基因敲除小鼠新生血管的交叉抑制作用以及bFGF介导的血管生成。这项研究将有助于建立与年龄相关的血管生成变化的机制(S)，这可能对开发新的2W治疗策略具有重要意义-促进缺血性疾病的血管生成。

Age-dependent impairment of angiogenesis leads to increased progression of ischemic vascular diseases, delayed wound healing, as well as slower tumor growth and spread. The mechanisms of reduced angiogenesis have not been fully elucidated yet but may include alterations in endothelial responsiveness, cell-matrix interactions and lack of angiogenic factors and their receptors. Neuropeptide Y (NPY) is commonly known as a sympathetic transmitter and vasoconstrictor. Recent our laboratory has established its new and potent activities as an angiogenic and mitogenic factor for vascular smooth muscle and endothelial cells in vitro, and in vivo in the rat hindlimb ischemic model. The overall goal of this research is to establish if alternations in the NPY system are involved in the age-related impairment of angiogenesis. The specific aims are to determine if: 1) NPY-mediated angiogenesis is diminished with age; 2) NPY synthesis/release occurring under physiological condition and in response to angiogenic stimuli is reduced in aged animals; 3) expression of the NPY angiogenic receptors (Y1, Y2, Y5) and its converting enzyme dipeptidyl peptidase IV (DPPIV, which converts NPY to an angiogenically active fragment, NPY3-36) is altered with age 4) age-related changes in NPY angiogenic systems are due to decreased levels of basic fibroblast growth factor (bFGF) in aging. These studies will be perform4ed on both animal (young and aged mice) and cellular models. Spontaneous, as well as NPY- and bFGF-induced sprouting of mouse aortas will be compared between both age groups. The mouse hindlimb ischemic model will be employed to determine the effect of aging on NPY-induced angiogenesis in ischemia. New vessels will be detected by CD31 immunostaining and by their casting by perfusion with latex. Systemic and local NPY levels, as well as expression of angiogenic stimuli, will be determined. The cellular model will include NPY stimulation of aortic endothelial cells from young and aged mice and measuring their mitogenic response in parallel to the expression of NPY receptors. Interactions between bFGF and NPY angiogenic systems and their age-related modulations will be established by determining the levels of growth factors and NPY receptors in ischemic tissues of aged and young animals, also after NPY or bFGF treatment. Moreover, cross-inhibition with neutralizing antibody of bFGF- and NPY-induced neovascularization as well as bFGF-mediated angiogenesis in Y2 knockout mice will be investigated using aortic sprouting assay. This research will help to establish the mechanism(s) of age-related changes in angiogenesis, which may have important implications for the development of ne2w therapeutic strategies in older patients-an enhancement of angiogenesis in the ischemic disease.