Jayasinghe Caren, Simiantonaki Nektaria, Michel-Schmidt Romi, Kirkpatrick Charles James
Institute of Pathology, Johannes Gutenberg University, 55131 Mainz, Germany.
Int J Mol Med. 2009 Jan;23(1):49-55.
The functionality of large-vessel endothelial cells, such as human umbilical vein endothelial cells (HUVEC), may differ significantly from that in the microvasculature. We established a method for the isolation of human colonic microvascular endothelial cells (HCMEC). Since colonic diseases are often accompanied by hypoxia we examined its effects on HCMEC of five individuals in comparison with HUVEC, with respect to the secretion of the soluble form of the two important vascular endothelial growth factor (VEGF) receptors, VEGFR-1 and 2. After dissociation by dispase/collagenase of mucosal and submucosal tissue obtained from normal adult colon, HCMEC were isolated using CD31-coated magnetic beads and cultivated as monolayers. Subsequent characterization studies demonstrated the endothelial phenotype, including VEGFR-1 and 2 mRNA and protein expression. sVEGFR expression analyses were performed using ELISA. Under hypoxic conditions significantly enhanced levels of sVEGFR-1 on HUVEC were observed (p<0.001), while in HCMEC there was a markedly variable reaction to hypoxia, with cases of enhanced, unchanged and reduced expression. sVEGFR-2 was significantly decreased in HCMEC under hypoxia (p<0.001). In contrast, the responses of sVEGFR-2 levels to hypoxia in HUVEC were variable, that is, either unchanged or up-regulated. The different secretion profiles of sVEGFR-1 and 2 between HUVEC and HCMEC under normoxia and hypoxia underline the importance of using a functionally adequate and relevant microvasculature for in vitro studies of colonic diseases. The homogeneously reduced sVEGFR-2 levels in hypoxic HCMEC provide evidence for a novel microvascular endothelium-specific biomarker in hypoxia-response processes.
大血管内皮细胞（如人脐静脉内皮细胞，HUVEC）的功能可能与微血管中的功能有显著差异。我们建立了一种分离人结肠微血管内皮细胞（HCMEC）的方法。由于结肠疾病常伴有缺氧，我们比较了五名个体的HCMEC与HUVEC在缺氧条件下两种重要血管内皮生长因子（VEGF）受体VEGFR-1和2的可溶性形式分泌情况的影响。从正常成年结肠获取的黏膜和黏膜下组织经dispase/胶原酶解离后，使用包被CD31的磁珠分离HCMEC，并培养成单层。随后的特性研究证实了内皮细胞表型，包括VEGFR-1和2的mRNA及蛋白表达。使用ELISA进行sVEGFR表达分析。在缺氧条件下，观察到HUVEC上sVEGFR-1水平显著升高（p<0.001），而HCMEC对缺氧的反应明显不同，有表达增强、不变和降低的情况。缺氧条件下HCMEC中的sVEGFR-2显著降低（p<0.001）。相比之下，HUVEC中sVEGFR-2水平对缺氧的反应各不相同，即不变或上调。常氧和缺氧条件下HUVEC与HCMEC之间sVEGFR-1和2的不同分泌模式强调了在结肠疾病体外研究中使用功能合适且相关的微血管的重要性。缺氧HCMEC中sVEGFR-2水平均匀降低为缺氧反应过程中一种新的微血管内皮特异性生物标志物提供了证据。
