Martins-Green Manuela, Petreaca Melissa, Yao Min
Department of Cell Biology and Neuroscience, University of California, Riverside, California, USA.
Methods Enzymol. 2008;443:137-53. doi: 10.1016/S0076-6879(08)02008-9.
The molecular mechanisms by which endothelial permeability occurs are often studied more readily in vitro, underscoring the importance of the use of systems that mimic human endothelium in vivo. We present an assay that accurately models human endothelium by use of primary human microvascular endothelial cells (hMVEC), because permeability primarily occurs at the microvascular level, and transwell filter units coated with Matrigel, extracellular matrix that mimics basal lamina, the matrix that is tightly associated with endothelium and is critical for its proper function. As a tracer molecule, we used 3-kDa dextran-FITC to detect leakage through the small gaps present in the early stages of permeability induction. The permeability-inducing agents IL-8 and VEGF were added to the lower chamber of the transwell units to mimic inflammatory conditions in vivo. After optimization, we were able to minimize basal permeability and to detect rapid changes in permeability stimulated by IL-8 and VEGF, similar to that observed in vivo. Furthermore, we have used this system to delineate the importance of the transactivation of VEGFR2 in IL-8-induced permeability and have confirmed the relevance of this signaling in vivo, suggesting that our permeability assay system adequately mimics the in vivo situation. Therefore, this system can be used to better understand the molecular mechanisms of human vascular permeability in a more in vivo-like setting and, thus, may be used to test effective therapeutics to prevent and treat diseases involving persistent permeability.
内皮通透性发生的分子机制通常在体外更容易研究，这突出了使用在体内模拟人内皮的系统的重要性。我们提出了一种检测方法，通过使用原代人微血管内皮细胞（hMVEC）来精确模拟人内皮，因为通透性主要发生在微血管水平，并且使用涂有基质胶的Transwell滤器单元，基质胶是模仿基膜的细胞外基质，基膜是与内皮紧密相关且对其正常功能至关重要的基质。作为示踪分子，我们使用3 kDa葡聚糖 - FITC来检测在通透性诱导早期出现的小间隙中的渗漏。将通透性诱导剂IL - 8和VEGF添加到Transwell单元的下室以模拟体内炎症状态。经过优化，我们能够将基础通透性降至最低，并检测到IL - 8和VEGF刺激引起的通透性快速变化，类似于在体内观察到的情况。此外，我们已使用该系统来阐明VEGFR2的反式激活在IL - 8诱导的通透性中的重要性，并已在体内证实了该信号传导的相关性，这表明我们的通透性检测系统充分模拟了体内情况。因此，该系统可用于在更类似体内的环境中更好地理解人血管通透性的分子机制，从而可用于测试预防和治疗涉及持续性通透性疾病的有效疗法。
