HSP70 FUNCTIONS AND DYNAMICS IN HEAT-SHOCKED AND HYPOXIA HUMAN ENDOTHELIAL CELLS

The 70 and 90-kDa heat shock proteins, HSP70 and HSP90, exert cardiovascular cytoprotective function. However, ATP-dependent chaperone dynamic in the vascular pathophysiological environment is unknown. We hypothesized that endothelial cells (EC) adapt to stress by modulating chaperome oligomerization. We showed that bed vascular-independent HSC70 and HSP70 multimeric complexes accumulate after heat shock (HS) and are decreased by hypoxia in primary human veins and coronary artery endothelial cells by native gel. HSP90 dimer, inducible HSP70, and HSP40 macromolecular complexes increase after HS recovery (HSR) while HSC70 returns to baseline. To understand the function of HSP70, we evaluated HSP70 inhibition using VER-155008 and YM-1, which resulted in decreased basal EC proliferation, impaired migration, and angiogenesis at HSR. Interestingly, HSP70 is essential to physiological cell alignment induced by physiological laminar shear stress in vein but not arteries. HSP70 inhibition leads to the disassembly of cell-surface junctional proteins platelet/endothelial cell adhesion molecule-1 (PECAM-1)/ CD31 and VE-Cadherin, downregulated steady state PECAM-1 and endothelial nitric oxide synthase (eNOS) expression and did not affect VE-cadherin levels. We discovered that PECAM-1 and VE-cadherin were immunoprecipitated with HSP70. Finally, the physiological readout uncovered that cytosolic HSP70 preserves vein/artery human endothelial monolayer permeability. These results shed light that HSR and hypoxia regulate HSPs by a new layer of complexity: oligomerization, in addition to classical co-chaperone/ NEF interactions. We demonstrated that HSP70 performs quality control of intercellular EC junctions, mediates mechanotransduction, and maintains monolayer integrity. Our work mechanistically advances our understanding of human HSP70 function in maintaining quiescent endothelium and possible contribution to EC-dysfunction related to cardiovascular diseases. FAPESP 2018/13739-8