INHIBITION OF HUMAN ENDOTHELIAL HSP70 INDUCES ENDOTHELIAL DYSFUNCTION

Background: Heat shock protein (HSP) chemical inhibition is being considered for its therapeutic effects in oncological and neurodegenerative diseases, with the 70 kDa HSP family as a relevant target. However, the global profile of its inhibition in vascular cells is unknown. Methods: Here, we uncover the effects of HSP70 inhibition in primary human umbilical vein endothelial cells (HUVECs) using label-free quantitative proteomics, confocal microscopy to follow platelet and monocyte adhesion, inhibitory antibody biochemical competition assay and endothelial permeability quantification using the Electric Cell-Substrate Impedance Sensing (ECIS) system. Central findings: We discovered extensive proteome remodeling in HSP70-inhibited cells in the protein-folding machinery of the endoplasmic reticulum, adhesion proteins, endothelial barrier and cytoskeletal remodeling and other processes. HUVECs pre-treated with VER-155008, an ATP-competitive inhibitor, increased monocyte adhesion. VER-155008 and YM-1, an allosteric inhibitor, stimulated human platelet adhesion to HUVECs. We confirmed stimulation of vWF secretion with confocal microscopy and identified GPIbα as the receptor responsible for the observed platelet-HUVECs interaction for cells treated with VER-155008. A label-free biosensor, ECIS, demonstrated that HSP70 regulates endothelial barrier permeability. Conclusions: Together, our data reinforce that cytosolic HSP70 is an essential molecular chaperone functioning as a central hub that maintains quiescent and unstressed primary human endothelial cells. HSP70 is a relevant hemostatic regulator. Future studies are warranted to carefully assess vessel responses to HSP70-targeted therapies.