SC COBRE: HUMAN ALKALINE PHYTOCERAMIDASE REGULATION OF ANGIOGENESIS

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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.