Wang Li, Chen Qing-wei, Li Gui-qiong, Ke Da-zhi
Department of Geriatrics Cardiology, Chongqing Medical University, Chongqing, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2012 Jan;40(1):50-6.
To clarify whether ghrelin could promote in vitro rat cardiac microvascular endothelial cells (CMECs) angiogenesis and related mechanisms.
CMECs were isolated from myocardial tissue of adult male SD rats and characterized by the immunocytochemistry staining with Factor VIII and the capacity of in vitro capillary tube-like formation. The mRNAs and protein expressions of ghrelin and its receptor (growth hormone secretagogue receptor, GHS-R) of CMECs were determined by RT-PCR, Immunofluorescence, ELISA and Western blot. Proliferation, migration and in vitro angiogenesis as well as ERK2 phosphorylation of CMECs were tested in the presence of ghrelin (10(-9) - 10(-7) mol/L) with or without pretreatment with specific MAPK/ERK2 inhibitor PD98059.
Purity of CMECs characterized by immunocytochemistry staining with Factor VIII was about 95%, and the cells showed a high ability to form the capillary tube-like structures on Matrigel. Ghrelin and GHS-R were constitutively expressed in CMECs. Proliferation, migration and in vitro angiogenesis capacities of CMECs (72.20 ± 5.72 vs. 28.60 ± 5.13, P < 0.001; 71.00 ± 7.78 vs. 28.60 ± 5.13, P < 0.001) as well as ERK2 phosphorylation (0.92 ± 0.13 vs. 0.29 ± 0.04, P < 0.001; 1.15 ± 0.16 vs. 0.29 ± 0.04, P < 0.001) were significantly enhanced by exogenous ghrelin (10(-8) - 10(-7) mol/L). PD98059 abolished ghrelin-induced ERK2 phosphorylation and in vitro angiogenesis.
Ghrelin and its receptor are expressed in CMECs and ghrelin could stimulate CMECs in vitro angiogenesis through activation of MAPK/ERK2 signaling pathway.
明确胃饥饿素是否能促进体外培养的大鼠心脏微血管内皮细胞（CMECs）血管生成及其相关机制。
从成年雄性SD大鼠心肌组织中分离CMECs，通过VIII因子免疫细胞化学染色及体外形成毛细血管样结构的能力进行鉴定。采用RT-PCR、免疫荧光、ELISA和蛋白质免疫印迹法检测CMECs中胃饥饿素及其受体（生长激素促分泌素受体，GHS-R）的mRNA和蛋白表达。在存在胃饥饿素（10⁻⁹ - 10⁻⁷ mol/L）的情况下，检测CMECs的增殖、迁移和体外血管生成以及ERK2磷酸化，同时进行或不进行特异性MAPK/ERK2抑制剂PD98059预处理。
以VIII因子免疫细胞化学染色鉴定的CMECs纯度约为95%，细胞在基质胶上表现出较高的形成毛细血管样结构的能力。胃饥饿素和GHS-R在CMECs中组成性表达。外源性胃饥饿素（10⁻⁸ - 10⁻⁷ mol/L）显著增强了CMECs的增殖、迁移和体外血管生成能力（72.20 ± 5.72对28.60 ± 5.13，P < 0.001；71.00 ± 7.78对28.60 ± 5.13，P < 0.001）以及ERK2磷酸化（0.92 ± 0.13对0.29 ± 0.04，P < 0.001；1.15 ± 0.16对0.29 ± 0.04，P < 0.001）。PD98059消除了胃饥饿素诱导的ERK2磷酸化和体外血管生成。
胃饥饿素及其受体在CMECs中表达，胃饥饿素可通过激活MAPK/ERK2信号通路刺激CMECs体外血管生成。
