Cell-surface HSP70 associates with thrombomodulin in endothelial cells
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.
KeywordsEndothelial cells Atherosclerosis HSP70 Thrombomodulin Cell stress Shear stress
70 kDa heat shock protein family
Proximity ligation assay
Protein C activated
Vascular endothelial growth factor
Low laminar shear stress
- PDI, PDIA1
Protein disulfide isomerase A1
We thank Ana L. Garippo and Laura Ventura for technical support. We are grateful to Prof. Marcelo L. Santoro, from Instituto Butantan, for advice and reagents.
T.L.S.A. conceived the project, designed and performed most experiments, analyzed data, and wrote the manuscript; G.V. and A.C.P. designed, performed, and discussed shear stress-associated proteomic analysis; L.Y.T. performed en face immunofluorescence experiments; A.I.S.M. performed DUOLINK and immunoprecipitation experiments; and F.R.M.L. conceived the project, analysis results, discussed experiments, and wrote the manuscript.
Work was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (Process number 15/06210-2), Centro de Pesquisa, Inovação e Difusão FAPESP (CEPID “Processos Redox em Biomedicina—Redoxoma,” Grant 13/07937-8), and Fundação Zerbini.
Compliance with ethical standards
Animal studies were performed in male C57BL/6 6-week old following approval from to Ethics Committee of the Heart Institute and School of Medicine from University of São Paulo, Brazil.
Conflict of interest
The authors declare that they have no conflict of interest.
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