Protein phosphatase inhibitors and heat preconditioning prevent Hsp27 dephosphorylation, F‐actin disruption and deterioration of morphology in ATP‐depleted endothelial cells

Abstract

The vascular endothelium response to ischemic depletion of ATP was studied in vitro. Endothelial cells (EC) cultured from human aorta or umbilical vein were incubated in a glucose-free medium containing CCCP or rotenone. Such blockade of energy metabolism caused a drop in ATP, destruction of actin filaments, morphological changes, and eventually disintegration of EC monolayer within 2-2.5 h. While ATP fell and F-actin collapsed, the 27-kDa heat shock protein (Hsp27) lost basal phosphorylation and became Triton X-100-insoluble forming granules inside the cell nuclei. Protein phosphatase (PP) inhibitors (okadaic acid, cantharidin, sodium orthovanadate) did not delay the ATP decrease in energydeprived EC but arrested both the alterations in the Hsp27 status and the changes for the worse in F-actin and cell morphology. Similarly, the Hsp27 dephosphorylation/insolubilization/granulation and the cytoskeletal and morphological disturbances resulting from lack of ATP were suppressed in heat-preconditioned (thermotolerant) cultures, this effect being sensitive to quercetin, a blocker of Hsp induction. The longer preservation of the cytosolic pool of phosphorylated Hsp27 during ATP depletion in the PP inhibitor-treated or thermotolerant EC correlated with the acquired resistance of F-actin and morphology. These data suggest that PP inhibitors as well as heatinducible Hsp(s) can protect ischemia-stressed cells by preventing the ATP loss-provoked protein dephosphorylation and breakdown of the actin cytoskeleton.

Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion

“…Previously, quercetin was shown to abolish the heat preconditioning-induced stabilization of F-actin in ATP-depleted endothelial cells, which suggested the protective role for stressinduced HSPs (21,22). In the present study, quercetin, when added to H9c2 cells, did not affect normal morphology (not shown) and restoration of the ATP level following the metabolic preconditioning (Fig.…”

“…Recovery periods following either priming stress continued for 16-18 h before the challenging ATP depletion. Quercetin was added at a 30 M concentration to some samples of the cells before the stressful preconditioning (21,22); this drug was present in the incubation medium during the priming stress and the next 10 h, and then the drug-containing medium was replaced by the usual growth medium. To perform the challenging ATP depletion, we placed the cells in the CCCPcontaining medium supplemented with 10 mM 2-deoxyglucose and incubated them at 37°C in a CO2 incubator (21,22).…”

S-Thiolation of HSP27 Regulates Its Multimeric Aggregate Size Independently of Phosphorylation

“…For example, swelling-induced damage is a pivotal mechanism of injury during ischemia and reperfusion (60,61); thus, HSP27-mediated cell strengthening in ischemia/reperfusion would be a potential mechanism of endogenous cardioprotection. This would be consistent with a number of cellular studies showing that small HSPs provide protection against simulated ischemia involving a hypotonic stress that causes swelling-induced damage (57,(62)(63)(64)(65)(66).…”

Cofilin mediates ATP depletion-induced endothelial cell actin alterations

“…Dynamic functions of the actin cytoskeleton, meditated through shifts in monomeric (G-actin) and filamentous (Factin) actin levels, play an important role in cellular morphology and permeability of the vascular endothelium (22). Progressive disassembly and shortening of the microfilaments of endothelial cells were observed during ATP depletion, and rapid reassembly was seen during cell recovery (24,25,33). Previous in vivo studies have evaluated actin cytoskeletal changes during ischemic injury (31,48).…”