Functional characterization of HUVEC-CS:: Ca2+ signaling, ERK 1/2 activation, mitogenesis and vasodilator production
Functional characterization of HUVEC-CS:: Ca2+ signaling, ERK 1/2 activation, mitogenesis and vasodilator production
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DOI：
10.1677/joe.0.1820485
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发表时间：
2004-09-01
影响因子：
4
通讯作者：
Bird, IM
中科院分区：
文献类型：
--
作者：
Gifford, SM;Grummer, MA;Bird, IM
虽然存在许多内皮细胞系，但很少有人源的具有接近亲本内皮细胞的特征。我们衍生了永生化人脐静脉内皮细胞（HUVEC-C）的亚系（HUVEC-CS），其在标准生长培养基中增殖并表现出正乙酰化低密度脂蛋白（AcLDL）摄取，表达eNOS、CD 31和ve-cadherin，并且当在Matrigel上生长时自发形成毛细血管样结构。HUVEC-CS还在有丝分裂、激酶活化和血管扩张剂产生的水平上维持内皮细胞特性。与原代HUVEC细胞一样，HUVEC-CS表达血管扩张剂产生所必需的许多关键蛋白，包括上皮一氧化氮合酶（eNOS）、HSP 90、cav-1和cav-2、cPLA（2）和考克斯-1和cov-2。蛋白质印迹分析未检测到前列腺素I合酶（PGIS），这与PGI（2）产量最低的原代HUVEC一致。检测血管紧张素11（All）、缓激肽、ATP和生长因子受体。ATP诱导细胞内游离Ca 2+浓度（[Ca 2 +]（i））呈剂量和时间依赖性升高。最初，ATP刺激P2 Y受体而不是P2 X受体，如通过毒胡萝卜素排空内部Ca 2+储存后ATP不能启动Ca 2+反应所证明的。所有的缓激肽、表皮生长因子（EGF）和血管内皮生长因子（VEGF）也引起了细胞亚群中[Ca 2 +]（i）的升高。ATP、碱性成纤维细胞生长因子（bFGF）、EGF和VEGF诱导有丝分裂发生，并在10分钟内引起ERK 2激活增加。L-精氨酸至L-瓜氨酸转化试验表明，ATP、EGF和VEGF诱导eNOS活性显著增加，这与诱导Ca 2+动员和ERK 2激活的能力相关。总之，HUVEC-CS确实是内皮细胞，并且似乎在功能上与原代HUVEC非常相似。这些细胞将被证明是基础科学和治疗科学未来研究的宝贵工具。
While many endothelial cell lines exist, few are of human origin with characteristics close to the parent endothelial cell. We derived a subline (HUVEC-CS) of immortalized human umbilical vein endothelial cells (HUVEC-C) that proliferate in standard growth media and exhibit positive acetylated low-density lipoprotein (AcLDL) uptake, express eNOS, CD31 and ve-cadherin, and spontaneously form capillary-like structures when grown on Matrigel. HUVEC-CS also maintain endothelial cell characteristics at the level of mitogenesis, kinase activation and vasodilator production. Like primary HUVEC cells, HUVEC-CS express many of the key proteins necessary for vasodilator production, including epithelial nitric oxide synthase (eNOS), HSP 90, cav-1 and -2, cPLA(2), and COX-1 and -2. Prostaglandin I synthase (PGIS) was not detectable by Western blot analysis, consistent with primary HUVEC in which PGI(2) production is minimal. Receptors were detected for angiotensin 11 (All), bradykinin, ATP and growth factors. ATP induced a dose- and time-dependent rise in the intracellular free Ca2+ concentration ([Ca2+](i)). Initially, ATP stimulates P2Y receptors rather than P2X receptors, as demonstrated by the inability of ATP to initiate a Ca2+ response subsequent to emptying of the internal Ca2+ stores by thapsigargin. All, bradykinin, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) also caused a rise in [Ca2+](i) in a subset of the cells. ATP, basic fibroblastic growth factor (bFGF), EGF and VEGF induced mitogenesis and caused a rise in ERK 2 activation within 10 min. L-Arginine to L-citrulline conversion assays showed that ATP, EGF and VEGF induced a significant rise in eNOS activity, and this correlates with an ability to induce Ca2+ mobilization and ERK 2 activation. In conclusion, HUVEC-CS are indeed endothelial cells and appear to be functionally very similar to primary HUVEC. These cells will prove a valuable tool for future studies in both basic and therapeutic sciences.