SC COBRE: HUMAN ALKALINE PHYTOCERAMIDASE REGULATION OF ANGIOGENESIS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。血管生成，即从原有血管中形成新的毛细血管的过程，对器官发育和组织修复至关重要。然而，不可控的血管生成可能导致诸如慢性炎症、类风湿关节炎和实体瘤生长等疾病。我们的长期目标是明确人类碱性植物神经酰胺酶（haPHC）在调节血管生成中的作用，并将这一概念发展为针对该酶治疗血管生成相关疾病的新策略。haPHC是PI最近发现的一种新型酶，它可以裂解一种神经酰胺羟神经酰胺生成鞘氨醇，而鞘氨醇在鞘氨醇激酶的作用下被磷酸化生成鞘氨醇-1-磷酸（S1P）。S1P介导血管生成和血管发生。我们的研究表明：1)haPHC mRNA在胎盘中高表达，在胎盘中血管生成和血管发生活跃；2) haPHC及其同源神经酰胺酶共同调节S1P水平。这些结果支持haPHC通过调节S1P水平来调节血管生成的假设。为了验证这一假设，我们提出了三个具体目标：目的1：确定haPHC在人脐静脉内皮细胞（HUVEC）生长和存活中的作用。我们将确定haPHC上调是否会提高HUVEC中S1P的水平并导致细胞增殖，而haPHC下调是否会产生相反的效果；以及外源S1P是否减轻或抑制haPHC下调诱导的生长抑制。目的2：确定人碱性植物神经酰胺酶的作用机制。我们将在HaCaT细胞中表达haPHC，并确定其底物特异性、阳离子和脂质对其活性、细胞定位和组织特异性表达的影响。目的3：确定碱性植物神经酰胺酶在血管生成中的作用。我们将产生碱性植物神经酰胺酶（maPHC）敲除小鼠，并分析maPHC缺失与野生型小鼠的发育性血管生成和血管发生。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Angiogenesis, the process of formation of new capillaries from preexisting blood vessels, is essential for the proper organ development and tissue repair. However, uncontrollable angiogenesis may lead to pathologies such as chronic inflammation, rheumatoid arthritis, and solid-tumor growth. Our long-term goals are to define the role of human alkaline phytoceramidase (haPHC) in regulating angiogenesis and to develop this concept into a new strategy to treat angiogenesis-related diseases by targeting this enzyme. haPHC, a novel enzyme which the PI identified recently, cleaves hydroxylceramide, one type of ceramides, to generate sphingosine, which is in turn phosphorylated to generate sphingosine-1-phosphate (S1P) through the action of sphingosine kinases. S1P mediates angiogenesis and vascular genesis. Our studies demonstrate that 1) haPHC mRNA is highly expressed in placenta in which angiogenesis and vascular genesis occur actively; and 2) haPHC and its homologous ceramidases co-regulate the levels of S1P. These results support the hypothesis that haPHC regulates angiogenesis by regulating the levels of S1P. To test this hypothesis, we proposed three specific aims: Aim 1: to determine the role of haPHC in growth and survival of human umbilical vein endothelial cells (HUVEC). We will determine whether haPHC up-regulation elevates the levels of S1P in HUVEC and results in cell proliferation whereas haPHC down-regulation has the opposite effects; and whether growth inhibition induced by haPHC down-regulation is alleviated or suppressed by exogenous S1P. Aim 2: to determine mechanism of the human alkaline phytoceramidase action. We will express haPHC in HaCaT cells and determine its substrate specificity, effects of cations and lipids on its activity, its cellular localization, and tissue specific expression. Aim 3: to determine the role of the alkaline phytoceramidase in angiogenesis. We will generate alkaline phytoceramidase (maPHC) knockout mice and analyze developmental angiogenesis and vascular genesis in maPHC null versus wild type mice.