Elucidation of role of vascular local hormones on endothelial differentiation by developmental cell bilology and its application to vascular regeneration therapy.

内皮功能障碍在增殖性血管疾病中起着关键作用。对血管内皮细胞功能的分子理解有助于血管再生治疗。本研究主要从发育细胞生物学的角度阐明血管局部激素，尤其是利钠肽的病理生理学意义。我们证实了血管内皮生长因子(VEGF)调节血管局部激素的内皮分泌，包括C型利钠肽(CNP)，并报道了血管内皮生长因子在人类冠状动脉粥样硬化病变中的表达增加。血管内皮生长因子及其受体(Flk-1、Flt-1)在活化的内皮细胞、去分化的血管平滑肌细胞和巨噬细胞中表达。结论1.氧化低密度脂蛋白上调培养的人冠状动脉内皮细胞中血管内皮生长因子的表达。采用细胞分选技术分离Flk-1阳性的小鼠ES细胞，将其培养在IV型胶原包被的血管内皮生长因子包被的培养皿上，诱导其分化为内皮细胞。在胶原凝胶3D培养中，我们还成功地与Flk-1 ES细胞形成了管状结构。阐明了胚胎干细胞向类胚体分化过程中CNP及其受体鸟苷环化酶B的基因表达。我们报道了CNP在大鼠胚胎和新生大鼠肺组织中的表达。我们构建了CNP重组腺病毒，并利用该重组腺病毒证实，在兔股动脉球囊损伤后，CNP的过度表达导致新生内膜形成的抑制、血管平滑肌细胞的再分化和再内皮化的加速。我们还成功地克隆了人cGMP依赖的蛋白激酶I型α，并建立了cGK转基因小鼠(cGK。Tg)。在CGK中。TG、CGK在脑、心脏、血管和骨骼肌中有较强的表达。使用CGK。Tg，我们证实了CGK在光化学损伤模型中具有血管保护作用。

Endothelial dysfunction plays the pivotal role in proliferative vascular diseases. Molecular understanding of endothelial cell function leads to vascular regeneration therapy. This research project aims at elucidation of the pathophygiological significance of vascular local hormones, especially natriuretic peptides, mainly by the approach of developmental cell biology.1. We demonstrated that VEGF (vascular endothelial growth factor) modulates endothelial secretion of vascular local hormones, including CNP (C-type natriuretic peptide) and reported that VEGF expression is augmented in human coronary atherosclerotic lesions. VEGF and its receptor (Flk-1,flt-1) expression were detected in activated endothelial cells, de-differentiated vascular smooth muscle cells and macrophages. Oxidized LDL up-regulated VEGF expression in cultured human coronary endothelial cells.2. Using cell sorting technique, we isolated Flk-1 positive mouse ES cells, cultured them on collagen IV coated dishes with VEGF and made them differentiated into endothelial cells. In collagen gel 3D culture, we also succeeded in tube formation with Flk-1 ES cells. We also clarified the gene expression of CNP and its receptor, Guanylate Cyclase B during the course of differentiation of ES cells to embryoid body. We reported CNP expression in rat embryo and neonatal lung tissue.3. We constructed CNP cDNA recombinant adenovirus and using the CNP adenovirus demonstrated that over-expression of CNP in balloon-injured rabbit femoral artery resulted in suppression of neointimal formation, re-differentiation of vascular smooth muscle cells and acceleration of re-endothelialization. We also succeeded in cloning human cGMP-dependent protein kinase(cGK)type Iα and developed cGK transgenic mice(cGK. Tg). In cGK. Tg, strong cGK expression was detected in the brain, heart, blood vessels and skeletal muscle. Using the cGK. Tg, we demonstrated that cGK can act for vascular protection in photo-chemical injury model.