Early and delayed tolerance to simulated ischemia in heat-preconditioned endothelial cells: a role for HSP27

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Abstract
An ischemia-mimicking metabolic stress in cultured endothelial cells from the human aorta or umbilical vein caused ATP depletion, a rise in cytosolic free Ca2+, fragmentation and aggregation of actin microfilaments, retraction of the cytoplasm, and disintegration of cell monolayer. Simultaneously, the constitutive heat shock protein 27 (HSP27) underwent dephosphorylation and formed granules inside cell nuclei. Prior heat shock (45°C, 10 min) in confluent cultures conferred two phases (early and delayed) of tolerance to simulated ischemia. Although heat preconditioning did not retard the ATP drop and the free Ca2+overload within ischemia-stressed cells, each phase of the tolerance was manifested in longer preservation of normal cell morphology during the stress. Cells exhibiting the early tolerance within 3 h after heating altered the F-actin response to ischemic stress; no microfilament debris but, instead, translocation of F-actin to the tight submembranous layer was observed. In contrast, the delayed cytoprotection preserved the preexisting F-actin bundles under simulated ischemia; this happened only after 12- to 14-h post-heat shock recovery, elevating the intracellular HSP content, and was sensitive to blockers of HSP synthesis, cycloheximide and quercetin. The dephosphorylation and intranuclear granulation of HSP27 were markedly suppressed in both phases of the heat-induced tolerance. Without heat pretreatment, similar attenuation of the HSP27 dephosphorylation/granulation and the actin cytoskeleton stability during simulated ischemia were achieved by treating cells with the protein phosphatase inhibitors cantharidin or sodium orthovanadate. We suggest that prior heat shock ameliorates the F-actin response to ischemic stress by suppressing the HSP27 dephosphorylation/granulation; this prolongs a sojourn in the cytosol of phosphorylated HSP27, which protects microfilaments from the disruption and aggregation.