Cell-surface HSP70 associates with thrombomodulin in endothelial cells

Area of Science:

• Cardiovascular Biology
• Cellular Stress Response

Background:

• Heat shock protein-70 (HSP70) is vital for proteostasis and cell protection.
• Elevated cell-surface (cs) and secreted HSP70 are paradoxically linked to cardiovascular disease.
• Mechanisms governing csHSP70 levels in vascular cells remain unclear.

Purpose of the Study:

• To investigate if hemodynamic forces modulate total and csHSP70 expression in endothelial cells.
• To determine if thrombomodulin is a target of csHSP70.
• To elucidate the role of HSP70 in endothelial pathophysiology under varying flow conditions.

Main Methods:

• Proteomic and western blot analyses for HSP70 expression.
• Confocal microscopy and cell-surface biotinylation to characterize HSP70 localization.
• Co-localization, proximity ligation, and immunoprecipitation assays to assess HSP70-thrombomodulin interaction.
• Protein C activation assays to measure thrombomodulin activity.

Main Results:

• Physiological laminar shear stress enhanced csHSP70 expression in endothelial cells (EC) in a cluster pattern.
• Low shear stress, a pro-atherogenic condition, diminished total and csHSP70 levels.
• Total HSP70 was lower in the aortic arch (turbulent flow) than the thoracic aorta (laminar flow).
• csHSP70 co-localized and physically interacted with thrombomodulin in EC and aorta endothelium.
• Blocking csHSP70 increased thrombomodulin activity in EC and ex vivo aorta.

Conclusions:

• Pro-atherogenic hemodynamic forces reduce total HSP70, potentially disrupting proteostasis.
• Decreased csHSP70 under these conditions correlates with impaired thromboresistance signaling.
• csHSP70 binds to and inhibits thrombomodulin activity in endothelial cells.
• This interaction may explain adverse effects of high extracellular HSP70, promoting thrombosis and inflammation.