Filippi Irene, Saltarella Ilaria, Aldinucci Carlo, Carraro Fabio, Ria Roberto, Vacca Angelo, Naldini Antonella
Cellular and Molecular Physiology Unit, Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy.
Istituto Toscano Tumori, Firenze, Italy.
Cell Physiol Biochem. 2018;46(1):203-212. doi: 10.1159/000488423. Epub 2018 Mar 21.
BACKGROUND/AIMS: Hypoxia is a powerful stimulator of angiogenesis under physiological as well as pathological conditions. Normal endothelial cells (EC), such as human umbilical vein EC (HUVEC), are relatively affected by hypoxic insult in terms of cell survival. In contrast, EC from tumors are particularly resistant to hypoxia-induced cell death. Previous reports have shown that EC in bone marrow from multiple myeloma (MM) patients had a hypoxic phenotype, even under normoxic conditions. The aim of this study was to evaluate whether HUVEC and MMEC adapt differently to hypoxia.
Cell proliferation was assessed by the CyQUANT assay. Cdc25A, p21, Bax, Bcl-xl, BNIP3, glucose transporter (GLUT)-1, monocarboxylate transporter (MCT)-4 and carbonic anhydrase (CA)IX mRNA expression was determined by qRT-PCR. HIF-1α, BNIP3, Beclin-1, LC3B, livin, Bax, Bcl-xl, p21, p62 and β-actin protein expression was analyzed by western blot. Apoptosis was determined by TUNEL assay. Silencing of BNIP3 was achieved by stealth RNA system technology.
While HUVEC survival was reduced after prolonged hypoxic exposure, MMEC were completely unaffected. This difference was also significant in terms of livin, cdc25A and p21 expression. Hypoxia induced apoptosis and inhibited autophagy in HUVEC, but not in MMEC, where hypoxic treatment resulted in a more sustained adaptive response. In fact, MMEC showed a more significant increase in the expression of genes regulated transcriptionally by hypoxia-inducible factor (HIF)-1α. Interestingly, they showed higher expression of BNIP3 than did HUVEC, indicating a more pronounced autophagic (and pro-survival) phenotype. The potential role of BNIP3 in EC survival was confirmed by BNIP3 siRNA experiments in HUVEC, where BNIP3 inhibition resulted in reduced cell survival and increased apoptosis.
These findings provide further information on how hypoxia may affect EC survival and could be important for a better understanding of EC physiology under normal and pathological conditions, such as in multiple myeloma.
背景/目的：缺氧是生理和病理条件下血管生成的强大刺激因素。正常内皮细胞（EC），如人脐静脉内皮细胞（HUVEC），在细胞存活方面相对受缺氧损伤的影响。相比之下，肿瘤来源的内皮细胞对缺氧诱导的细胞死亡具有特别的抗性。先前的报道表明，即使在常氧条件下，多发性骨髓瘤（MM）患者骨髓中的内皮细胞也具有缺氧表型。本研究的目的是评估HUVEC和MMEC对缺氧的适应是否不同。
通过CyQUANT检测评估细胞增殖。通过qRT-PCR测定Cdc25A、p21、Bax、Bcl-xl、BNIP3、葡萄糖转运蛋白（GLUT）-1、单羧酸转运蛋白（MCT）-4和碳酸酐酶（CA）IX mRNA表达。通过蛋白质印迹分析HIF-1α、BNIP3、Beclin-1、LC3B、livin、Bax、Bcl-xl、p21、p62和β-肌动蛋白的蛋白表达。通过TUNEL检测确定细胞凋亡。通过隐形RNA系统技术实现BNIP3的沉默。
长时间缺氧暴露后HUVEC的存活率降低，而MMEC完全不受影响。在livin、cdc25A和p21表达方面，这种差异也很显著。缺氧诱导HUVEC细胞凋亡并抑制自噬，但在MMEC中不发生，在MMEC中缺氧处理导致更持久的适应性反应。事实上，MMEC中缺氧诱导因子（HIF）-1α转录调控的基因表达增加更为显著。有趣的是，它们的BNIP3表达高于HUVEC，表明具有更明显的自噬（和促存活）表型。HUVEC中的BNIP3 siRNA实验证实了BNIP3在内皮细胞存活中的潜在作用，其中BNIP3抑制导致细胞存活率降低和细胞凋亡增加。
这些发现提供了关于缺氧如何影响内皮细胞存活的进一步信息，对于更好地理解正常和病理条件下（如多发性骨髓瘤）的内皮细胞生理学可能很重要。
