Department of Pharmacy, Center for Drug Research, Pharmaceutical Biology, University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany.
J Mol Cell Cardiol. 2012 Jan;52(1):196-205. doi: 10.1016/j.yjmcc.2011.10.020. Epub 2011 Oct 31.
Endothelial hyperpermeability followed by edema formation is a hallmark of many severe disorders. Effective drugs directly targeting endothelial barrier function are widely lacking. We hypothesized that the hawthorn (Crataegus spp.) extract WS® 1442, a proven multi-component drug against moderate forms of heart failure, would prevent vascular leakage by affecting endothelial barrier-regulating systems. In vivo, WS® 1442 inhibited the histamine-evoked extravasation of FITC-dextran from mouse cremaster muscle venules. In cultured human endothelial cells, WS® 1442 blocked the thrombin-induced FITC-dextran permeability. By applying biochemical and microscopic techniques, we revealed that WS® 1442 abrogates detrimental effects of thrombin on adherens junctions (vascular endothelial-cadherin), the F-actin cytoskeleton, and the contractile apparatus (myosin light chain). Mechanistically, WS® 1442 inhibited the thrombin-induced rise of intracellular calcium (ratiometric measurement), followed by an inactivation of PKC and RhoA (pulldown assay). Moreover, WS® 1442 increased endothelial cAMP levels (ELISA), which consequently activated PKA and Rap1 (pulldown assay). Utilizing pharmacological inhibitors or siRNA, we found that PKA is not involved in barrier protection, whereas Epac1, Rap1, and Rac1 play a crucial role in the WS® 1442-induced activation of cortactin, which triggers a strong cortical actin rearrangement. In summary, WS® 1442 effectively protects against endothelial barrier dysfunction in vitro and in vivo. It specifically interacts with endothelial permeability-regulating systems by blocking the Ca(2+)/PKC/RhoA and activating the cAMP/Epac1/Rap1 pathway. As a proven safe herbal drug, WS® 1442 opens a novel pharmacological approach to treat hyperpermeability-associated diseases. This in-depth mechanistic work contributes to a better acceptance of this herbal remedy.
内皮细胞通透性增加继而导致水肿形成是许多严重疾病的一个标志。目前广泛缺乏直接针对内皮屏障功能的有效药物。我们假设，经过验证的多成分药物山楂（Crataegus spp.）提取物 WS® 1442 可以通过影响内皮屏障调节系统来预防血管渗漏。在体内，WS® 1442 抑制了组胺诱导的从小鼠提睾肌静脉中 FITC-葡聚糖的漏出。在培养的人内皮细胞中，WS® 1442 阻断了凝血酶诱导的 FITC-葡聚糖通透性。通过应用生化和显微镜技术，我们揭示 WS® 1442 可消除凝血酶对黏附连接（血管内皮钙黏蛋白）、F-肌动蛋白细胞骨架和收缩装置（肌球蛋白轻链）的有害影响。在机制上，WS® 1442 抑制了凝血酶诱导的细胞内钙升高（比率测量），随后导致 PKC 和 RhoA 失活（下拉测定）。此外，WS® 1442 增加了内皮细胞中的 cAMP 水平（ELISA），这继而激活了 PKA 和 Rap1（下拉测定）。利用药理学抑制剂或 siRNA，我们发现 PKA 不参与屏障保护，而 Epac1、Rap1 和 Rac1 在 WS® 1442 诱导的 cortactin 激活中发挥关键作用，从而引发强烈的皮质肌动蛋白重排。总之，WS® 1442 可有效防止体外和体内的内皮屏障功能障碍。它通过阻断 Ca(2+)/PKC/RhoA 并激活 cAMP/Epac1/Rap1 途径来特异性地与内皮通透性调节系统相互作用。作为一种经过验证的安全草药药物，WS® 1442 为治疗通透性相关疾病开辟了新的药理学方法。这项深入的机制研究有助于更好地接受这种草药疗法。
