Brighton and Sussex Medical School Department of Clinical and experimental investigation, University of Sussex, Falmer East Sussex, Brighton, BN1 9PS, UK.
Brighton and Sussex Medical School Department of Medical Education, Mayfield House, Falmer East Sussex, Brighton, BN1 9PH, UK.
Mol Med. 2018 May 16;24(1):22. doi: 10.1186/s10020-018-0026-5.
Endothelial injury is an early and enduring feature of cardiovascular disease. Inflammation and hypoxia may be responsible for this, and are often associated with the up-regulation of several transcriptional factors that include Hypoxia Inducible Factor-1 (HIF-1). Although it has been reported that HIF-1α is detectable in plasma, it is known to be unstable. Our aim was to optimize an assay for HIF-1α to be applied to in vitro and in vivo applications, and to use this assay to assess the release kinetics of HIF-1α following endothelial injury.
An ELISA for the measurement of HIF-1α in cell-culture medium and plasma was optimized, and the assay was used to determine the best conditions for sample collection and storage. The results of the ELISA were validated using Western blotting and immunohistochemistry (IHC). In vitro, a standardized injury was produced in a monolayer of rat aortic endothelial cells (RAECs) and intracellular HIF-1α was measured at intervals over 24 h. In vivo, a rat angioplasty model was used. The right carotid artery was injured using a 2F Fogarty balloon catheter. HIF-1α was measured in the plasma and in the arterial tissue (0, 1, 2, 3 and 5 days post injury).
The HIF-1α ELISA had a limit of detection of 2.7 pg/mL and was linear up to 1000 pg/ mL. Between and within-assay, the coefficient of variation values were less than 15%. HIF-1α was unstable in cell lysates and plasma, and it was necessary to add a protease inhibitor immediately after collection, and to store samples at -80 °C prior to analysis. The dynamics of HIF-1α release were different for the in vitro and in vivo models. In vitro, HIF-1α reached maximum concentrations approximately 2 h post injury, whereas peak values in plasma and tissues occurred approximately 2 days post injury, in the balloon injury model.
HIF-1α can be measured in plasma, but this requires careful sample collection and storage. The carotid artery balloon injury model is associated with the transient release of HIF-1α into the circulation that probably reflects the hypoxia induced in the artery wall.
内皮损伤是心血管疾病的早期和持续特征。炎症和缺氧可能是造成这种情况的原因，并且通常与几种转录因子的上调有关，包括缺氧诱导因子-1（HIF-1）。尽管已经报道 HIF-1α可在血浆中检测到，但它是不稳定的。我们的目的是优化 HIF-1α 的测定方法，以应用于体外和体内应用，并使用该测定方法评估内皮损伤后 HIF-1α 的释放动力学。
优化了用于测量细胞培养介质和血浆中 HIF-1α 的 ELISA，并使用该测定方法确定了最佳的样本收集和储存条件。使用 Western 印迹和免疫组织化学（IHC）验证 ELISA 的结果。在体外，在单层大鼠主动脉内皮细胞（RAEC）上产生标准化损伤，并在 24 小时内的不同时间点测量细胞内 HIF-1α。在体内，使用大鼠血管成形术模型。使用 2F Fogarty 球囊导管损伤右侧颈总动脉。在血浆和动脉组织（损伤后 0、1、2、3 和 5 天）中测量 HIF-1α。
HIF-1α ELISA 的检测限为 2.7pg/mL，线性范围高达 1000pg/mL。批内和批间变异系数值小于 15%。HIF-1α在细胞裂解物和血浆中不稳定，有必要在收集后立即添加蛋白酶抑制剂，并在分析前将样品储存在-80°C。体外和体内模型中 HIF-1α 释放的动力学不同。在体外，HIF-1α 在损伤后约 2 小时达到最大浓度，而在球囊损伤模型中，血浆和组织中的峰值出现在损伤后约 2 天。
可以在血浆中测量 HIF-1α，但这需要仔细收集和储存样本。颈动脉球囊损伤模型与 HIF-1α 短暂释放到循环中有关，这可能反映了动脉壁缺氧。
