Vascular Biology Laboratory, Heart Institute (InCor), University of São Paulo School of Medicine, Av. Enéas Carvalho Aguiar, 44, Annex II, 9th Floor, São Paulo, Brazil.
Laboratory of Genetics and Molecular Cardiology, Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo, Brazil.
Cell Stress Chaperones. 2019 Jan;24(1):273-282. doi: 10.1007/s12192-018-00964-y. Epub 2019 Jan 15.
Heat shock protein-70 (HSP70) is crucial for proteostasis and displays cell-protective effects. Meanwhile, enhanced levels of cell-surface (cs) and secreted HSP70 paradoxically associate with pathologic cardiovascular conditions. However, mechanisms regulating csHSP70 pool are unknown. We hypothesized that total and csHSP70 expressions are modulated by hemodynamic forces, major contributors to endothelial pathophysiology. We also investigated whether thrombomodulin, a crucial thromboresistance cell-surface protein, is a csHSP70 target. We used proteomic/western analysis, confocal microscopy, and cs-biotinylation to analyze the pattern and specific characteristics of intracellular and csHSP70. HSP70 interaction with thrombomodulin was investigated by confocal colocalization, en face immunofluorescence, proximity assay, and immunoprecipitation. Thrombomodulin activity was assessed by measured protein C activation two-step assay. Our results show that csHSP70 pool in endothelial cells (EC) exhibits a peculiar cluster-like pattern and undergoes enhanced expression by physiological arterial-level laminar shear stress. Conversely, total and csHSP70 expressions were diminished under low shear stress, a known proatherogenic hemodynamic pattern. Furthermore, total HSP70 levels were decreased in aortic arch (associated with proatherogenic turbulent flow) compared with thoracic aorta (associated with atheroprotective laminar flow). Importantly, csHSP70 co-localized with thrombomodulin in cultured EC and aorta endothelium; proximity ligation assays and immunoprecipitation confirmed their physical interaction in EC. Remarkably, immunoneutralization of csHSP70 enhanced thrombomodulin activity in EC and aorta ex vivo. Overall, proatherogenic hemodynamic forces promote reduced total HSP70 expression, which might implicate in disturbed proteostasis; meanwhile, the associated decrease in cs-HSP70 pool associates with thromboresistance signaling. Cell-surface HSP70 (csHSP70) expression regulation and csHSP70 targets in vascular cells are unknown. We showed that HSP70 levels are shear stress-modulated and decreased under proatherogenic conditions. Remarkably, csHSP70 binds thrombomodulin and inhibits its activity in endothelial cells. This mechanism can potentially explain some deleterious effects previously associated with high extracellular HSP70 levels, as csHSP70 potentially could restrict thromboresistance and support thrombosis/inflammation in stress situations.
热休克蛋白 70（HSP70）对蛋白质稳态至关重要，并具有细胞保护作用。同时，细胞表面（cs）和分泌的 HSP70 水平升高与病理心血管状况相反相关。然而，调节 csHSP70 池的机制尚不清楚。我们假设总 HSP70 和 csHSP70 的表达受血流动力学的调节，血流动力学是内皮生理病理的主要贡献者。我们还研究了血栓调节蛋白是否是 csHSP70 的靶点，血栓调节蛋白是一种至关重要的抗血栓细胞表面蛋白。我们使用蛋白质组学/蛋白质印迹分析、共聚焦显微镜和 cs 生物素化来分析细胞内和 csHSP70 的模式和特定特征。通过共聚焦共定位、正面免疫荧光、接近测定和免疫沉淀研究 HSP70 与血栓调节蛋白的相互作用。通过两步蛋白 C 激活测定评估血栓调节蛋白的活性。我们的结果表明，内皮细胞（EC）中的 csHSP70 池表现出独特的簇状模式，并通过生理动脉水平层流剪切力增强表达。相反，总 HSP70 和 csHSP70 的表达在低剪切力下减少，低剪切力是一种已知的促动脉粥样硬化血流动力学模式。此外，与胸主动脉（与抗动脉粥样硬化的层流相关）相比，主动脉弓（与促动脉粥样硬化的湍流相关）中的总 HSP70 水平降低。重要的是，csHSP70 在培养的 EC 和主动脉内皮中与血栓调节蛋白共定位；接近连接测定和免疫沉淀证实了它们在 EC 中的物理相互作用。值得注意的是，csHSP70 的免疫中和增强了 EC 和主动脉离体的血栓调节蛋白活性。总体而言，促动脉粥样硬化的血流动力学力促进总 HSP70 表达减少，这可能与蛋白质稳态紊乱有关；同时，csHSP70 池的相关减少与血栓抵抗信号相关。血管细胞中 csHSP70（csHSP70）表达的调节和 csHSP70 靶标尚不清楚。我们表明 HSP70 水平受剪切力调节，并在促动脉粥样硬化条件下减少。值得注意的是，csHSP70 与血栓调节蛋白结合并抑制其在内皮细胞中的活性。该机制可能可以解释先前与细胞外 HSP70 水平升高相关的一些有害影响，因为 csHSP70 可能限制血栓抵抗并在应激情况下支持血栓形成/炎症。
