蛋白激酶D在血管生成与重构过程中的作用及分子机制

The protein kinase D (PKD) is a subfamily of serine/threonine kinases, which was identified within 20 years and has been implicated in regulating cell proliferation, differentiation, polarization, vesicle transportation and secretion. In mammals, PKD has three distinct isoforms encoded by three different genes. It has been shown that PKD could play a role in proliferation, permeability, and tube formation in cultured endothelial cells. However, it remains to be determined whether PKD could regulate vascular endothelial homeostasis, angiogenesis and vascular remodeling in vivo. The specific phosphorylation substrates for PKD in endothelial cells also remain unclear. To address these questions, we generated all three different flox mice of PKD genes, and further demonstrated that endothelial cell specific deletion of all three subtypes of PKD by Tie2-Cre results in embryonic lethality, and abnormal vascular development characterized by aberrant vessel patterning, decreased vessel density, and dilated vessel diameter. We also found that PKD triple deletion decreases VEGF-induced tube formation in cultured endothelial cells. All these in vivo and in vitro data suggested that PKD really plays an important role in endothelial cells. Based on these preliminary results, we plan to characterize the function and the molecular mechanisms of PKD in mouse embryonic vascular development, the substrates of PKD in endothelial cells, the signaling networks mediated by PKD. We also plan to examine the role of PKD in tumor angiogenesis and vascular remodeling after injury.

蛋白激酶D是最近二十年来才发现的一类丝氨酸/苏氨酸激酶，在调控细胞增殖、分化、极性、囊泡转运和分泌等方面有重要作用。PKD在哺乳动物中有三种不同的亚型，不同亚型之间存在功能共冗。体外实验表明PKD可能调节内皮细胞的增殖、通透性与管状结构形成，但PKD如何调控在体血管内皮细胞稳态、血管生成及血管重构的作用尚不清楚，PKD在内皮细胞中的特异性的磷酸化底物尚不清楚。为了解答这些问题，我们构建了所有三种PKD亚型的flox小鼠。我们发现内皮细胞特异性地PKD三敲除会导致胚胎致死，并伴随着明显的血管发育异常；我们进一步发现在培养的内皮细胞中PKD三敲除会降低VEGF刺激诱导的管状结构的形成。在这些发现的基础上，我们拟进一步确定PKD在小鼠胚胎血管发育中的作用与分子机制，研究PKD在内皮细胞中的磷酸化底物与分子调控网络，并探讨PKD在成年小鼠肿瘤血管生成与血管损伤重构过程中的作用。

蛋白激酶D（PKD）是一类丝氨酸/苏氨酸激酶，在调控细胞增殖、分化、极性、囊泡转运和分泌等方面有重要作用。PKD在哺乳动物中有三种不同的亚型，不同亚型之间存在功能共冗。到目前为止，内皮细胞PKD在调节血管生成、血管功能与血管重构等方面的作用尚不十分清楚，对于PKD在内皮细胞中的磷酸化底物也不清楚。为了回答这些问题，我们引进了所有三种PKD亚型的flox小鼠及内皮细胞特异性的Tie2-Cre小鼠与可诱导的iPdgfb-Cre小鼠，并进一步建立了内皮细胞特异性的PKD三敲除小鼠。我们发现Tie2-Cre介导的、内皮细胞特异性的PKD三敲除会导致小鼠胚胎致死，并伴随着明显的血管与肝脏发育异常，主要表现为腹部血管密度下降、肝脏体积减小、肝脏静脉窦内腔变大以及肝脏中内皮细胞与肝细胞凋亡增加；体外培养体系也证明了PKD基因敲除会减少VEGF诱导的管状结构的形成以及eNOS活性的上升，但是不影响肝脏细胞与内皮细胞的生长，也不影响成年小鼠肝脏的再生过程；在新生小鼠内皮细胞中敲除所有三种PKD亚型会导致视网膜血管发育异常，主要表现为视网膜血管通透性增加、血管面积减少与密度增加；在成年小鼠血管内皮细胞中敲除PKD基因会降低ACh引起的血管舒张，但是不影响导丝拉伤与颈动脉结扎诱导的血管重构过程。我们的这些研究结果为进一步了解PKD在血管生理与病理过程中的作用提供了重要的依据。

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