Neuropeptide Y in Revascularizing Ischemic Tissues

描述（来自申请的逐字描述）：心脏和外周 血管疾病，血管生成可以改善血液流动，促进长期 缺血组织的存活。这个复杂的过程涉及内皮细胞 (ECs)与血管平滑肌细胞（VSMC）粘附、迁移、增殖 和差异化。体液因子，如碱性成纤维细胞生长因子 血管内皮生长因子（VEGF）和碱性成纤维细胞生长因子（bFGF）已被认为是 这个过程的调解人。然而，血管也有丰富的神经支配， 已知含有营养因子的交感神经。最近我们 发现神经肽Y（NPY），一种交感神经递质， 血管生成在体外和体内，与效力和疗效相当， bFGF或VEGF。此外，人内皮细胞具有完整的NPY自分泌功能， 系统，其合成NPY及其受体（Rs; Y1-Y 5）以及 “转化酶”，二肽基肽酶IV（DPPIV）。在过去的两年里， 在体内大鼠血管生成模型中， 闭塞和因此肢体缺血导致大量NPY从 缺血性腿，但没有检测到NPY受体，从而防止NPY发挥其 血管生成活性。在后续研究中，我们证明了局部NPY 给药，既上调NPY Rs，又恢复毛细血管密度， 骨骼肌中观察到的水平在非缺血腿。基于这些 令人兴奋的发现，我们现在建议建立，如果NPY构成一个新的 缺血刺激的内源性血管生成系统，并确定 血管生成受体亚型和NPY作用机制。为此，两名 提出了缺血模型：后肢（大鼠和小鼠）和心脏（大鼠）。 具体目的是确定：1）缺血/缺氧是否上调NPY释放 其Rs的表达与血管生成的时空关系 在缺血组织和离体EC中的作用; 2）NPY的作用机制 血管生成作用及其对DPPIV的依赖性，以及与其他 生长因子：（bFGF，VEGF，一氧化氮）; 3）5个NPY R中的哪一个是 血管生成;和4）如果特异性NPY R激动剂使缺血性血管再生， 心脏和后肢我们将重点关注Y2和Y 5亚型，它们是 我们最近的研究结果表明，但主要的血管NPY的作用， R，Yl，也将被调查。这些研究将在大鼠中进行 用缓释小丸中的NPY（或其他因子）治疗或过表达 NPY基因，以及在NPY、Y1、Y2和Y 5敲除（KO）小鼠中。血管生成 A（s）将通过使用KO研究其mRNA和蛋白质表达来确定 小鼠和/或特异性NPY R激动剂或拮抗剂在上述缺血性 模型和体外血管生成测定中。NPY的血管生成活性将 在结构上进行评估（例如，通过血管造影、组织病理学、毛细血管/纤维 比率和有丝分裂指数）和功能（通过局部血流，心脏 输出和运动评分量表）。这项研究将首次建立 NPY在缺血驱动的血管生成中的作用，并确定特异性 NPY Rs介导该作用。除了提供对 神经肽Y作为神经源性血管生成介质的作用，这些发现将打开 治疗缺血性疾病的新途径。

DESCRIPTION (Verbatim from the application): In the heart and peripheral vascular diseases, angiogenesis can improve blood flow and facilitate long-term survival of ischemic tissues. This complex process involves endothelial cell (ECs) and vascular smooth muscle cell (VSMC) adhesion, migration, proliferation and differentiation. Humoral agents such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) have been suggested as mediators of this process. However, vessels are also richly innervated with sympathetic nerves, which are known to contain trophic factors. Recently, we discovered that neuropeptide Y (NPY), a sympathetic transmitter stimulates angiogenesis in vitro and in vivo, with potency and efficacy comparable to those of bFGF or VEGF. In addition, human ECs possess a complete NPY autocrine system, which synthesizes NPY and its receptors (Rs; Y1-Y5)as well as the "converting enzyme," dipeptidyl peptidase IV (DPPIV). In the last two years we made a major discovery that in the in vivo rat angiogenic model, femoral artery occlusion and hence limb ischemia results in substantial NPY release from the ischemic leg yet no NPY Rs are detected, thus preventing NPY from exerting its angiogenic activity. In a follow up study, we demonstrated that local NPY administration, both up-regulates NPY Rs and restores capillary density in the skeletal muscles to the level observed in the non-ischemic leg. Based on these exciting discoveries, we now propose to establish if NPY constitutes a new endogenous angiogenic system stimulated by ischemia, and to determine the subtypes of angiogenic Rs and mechanisms of NPY action. To this end, two ischemic models are proposed: the hindlimb (rat and mouse) and the heart (rat). Specific aims are to determine: 1) if ischemia/hypoxia up-regulates NPY release and the expression of its Rs in spatial and temporal relation to angiogenesis in the ischemic tissues, and in vitro in ECs; 2) the mechanisms of NPY's angiogenic effects and its dependence on DPPIV, and interactions with other growth factors: (bFGF, VEGF, nitric oxide); 3) which of the five NPY Rs are angiogenic; and 4) if the specific NPY R agonist revascularizes the ischemic heart and hindlimb. We will focus on the Y2 and Y5 subtypes, which are suggested by our recent findings, but the role of the predominant vascular NPY R, the Yl, will also be investigated. These studies will be performed in rats treated with NPY (or other factors) in slow release pellets or over-expressing the NPY gene, and in NPY, Yl, Y2, and Y5 knockout (KO) mice. The angiogenic A(s) will be determined by studying their mRNA and protein expression using KO mice and/or specific NPY R agonists or antagonists in the above ischemic models, and in the in vitro angiogenic assays. NPY's angiogenic activity will be assessed structurally (e.g. by angiograms, tissue pathology, capillary/fiber ratio, and mitotic index) and functionally (by regional blood flow, cardiac output and motor scoring scale). This research will be the first to establish the role of NPY in ischemia-driven angiogenesis, and to determine the specific NPY Rs mediating this action. In addition to providing an understanding of the NPY's role as a mediator of neurogenic angiogenesis, these findings will open new avenues for treatment of ischemic diseases.