Heat Shock Protein 70 Prevents Hyperoxia-Induced Disruption of Lung Endothelial Barrier via Caspase-Dependent and AIF-Dependent Pathways

Abstract

Exposure of pulmonary artery endothelial cells (PAECs) to hyperoxia results in a compromise in endothelial monolayer integrity, an increase in caspase-3 activity, and nuclear translocation of apoptosis-inducing factor (AIF), a marker of caspase-independent apoptosis. In an endeavor to identify proteins involved in hyperoxic endothelial injury, we found that the protein expression of heat-shock protein 70 (Hsp70) was increased in hyperoxic PAECs. The hyperoxia-induced Hsp70 protein expression is from hspA1B gene. Neither inhibition nor overexpression of Hsp70 affected the first phase barrier disruption of endothelial monolayer. Nevertheless, inhibition of Hsp70 by using the Hsp70 inhibitor KNK437 or knock down Hsp70 using siRNA exaggerated and overexpression of Hsp70 prevented the second phase disruption of lung endothelial integrity. Moreover, inhibition of Hsp70 exacerbated and overexpression of Hsp70 prevented hyperoxia-induced apoptosis, caspase-3 activation, and increase in nuclear AIF protein level in PAECs. Furthermore, we found that Hsp70 interacted with AIF in the cytosol in hyperoxic PAECs. Inhibition of Hsp70/AIF association by KNK437 correlated with increased nuclear AIF level and apoptosis in KNK437-treated PAECs. Finally, the ROS scavenger NAC prevented the hyperoxia-induced increase in Hsp70 expression and reduced the interaction of Hsp70 with AIF in hyperoxic PAECs. Together, these data indicate that increased expression of Hsp70 plays a protective role against hyperoxia-induced lung endothelial barrier disruption through caspase-dependent and AIF-dependent apoptotic pathways. Association of Hsp70 with AIF prevents AIF nuclear translocation, contributing to the protective effect of Hsp70 on hyperoxia-induced endothelial apoptosis. The hyperoxia-induced increase in Hsp70 expression and Hsp70/AIF interaction is contributed to ROS formation.

Proteomic analysis of mitochondrial proteins in the guinea pig heart following long-term normobaric hyperoxia

“…It is involved in protein import into organelles, such as mitochondria and endoplasmic reticulum [47]. Recently, it has been reported that hyperoxia can induce an increase in HSP70 expression due to formation of ROS [48]. Similar with these findings, NBO induced upregulation of HSP70 in our study.…”

Melatonin receptor agonist protects against acute lung injury induced by ventilator through up-regulation of IL-10 production

“…Another study demonstrated that enhanced Hsp70 expression attenuates apoptosis in ALI [30]. Moreover, HSP70 overexpression inhibits caspase-3 activation and PARP cleavage [31,32].…”

Targeted heat shock protein 72 for pulmonary cytoprotection

“…Damage occurs to the lung endothelial cells, leading to barrier disruption between the lung surface and the vasculature . In studies investigating the connection between hyperoxia and lung barrier disruptions, researchers demonstrated increased HSP72 protein expression in pulmonary artery endothelial cells exposed to 95% oxygen and specific inhibition of the ATP‐dependent, caspase‐directed apoptosis pathway and the ATP‐independent AIF pathway by HSP72. Disruption of HSP72 activity, either with a general heat shock protein inhibitor (KNK437) or specific disruption using siRNA, increased endothelial cell apoptosis as measured by TUNEL assay, caspase‐3 activity, and AIF translocation from the cytoplasm into the nucleus.…”

“…This long induction period may have evolved in order to deliver a chronic cytoprotectant to the cells following an acute injury in which secondary effects, such as apoptosis, can occur hours later. In the case of hyperoxic exposure of the lungs, HSP72 induction reaches its maximum 24 h after exposure . Hyperoxia causes a biphasic barrier disruption of the endothelial monolayer, with the first phase occurring 1–3 h after exposure and the second phase, largely attributable to apoptosis, occurring at 24 hours .…”

“…13 Damage occurs to the lung endothelial cells, leading to barrier disruption between the lung surface and the vasculature. 14 In studies investigating the connection between hyperoxia and lung barrier disruptions, researchers demonstrated increased HSP72 protein expression in pulmonary artery endothelial cells exposed to 95% oxygen 15 and specific inhibition of the ATP-dependent, caspase-directed apoptosis pathway and the ATP-independent AIF pathway 15…”