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

Kondrikov D, Fulton D, Dong Z, Su Y - PLoS ONE (2015)

Bottom Line: Furthermore, we found that Hsp70 interacted with AIF in the cytosol in hyperoxic 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.Association of Hsp70 with AIF prevents AIF nuclear translocation, contributing to the protective effect of Hsp70 on hyperoxia-induced endothelial apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Apoptosis contributes to hyperoxia-induced disruption of lung endothelial barrier.(A and B) PAECs were exposed to normoxia or to hyperoxia for 24 to 48 h after which caspase-3 activity (A) and nuclear AIF protein level (B) was assayed. (C) PAECs were treated with and without the caspase-3 inhibitor Z-VAD-FMK (50 μM) and exposed to normoxia and hyperoxia for 48 h. TEER was continuously monitored as described in Materials and Methods. Results are expressed as mean ± SE; n = 4. * P<0.05 vs normoxia; **P<0.05 vs. hyperoxia+vehicle; #P<0.05 vs. normoxia+Z-VAD.
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pone.0129343.g004: Apoptosis contributes to hyperoxia-induced disruption of lung endothelial barrier.(A and B) PAECs were exposed to normoxia or to hyperoxia for 24 to 48 h after which caspase-3 activity (A) and nuclear AIF protein level (B) was assayed. (C) PAECs were treated with and without the caspase-3 inhibitor Z-VAD-FMK (50 μM) and exposed to normoxia and hyperoxia for 48 h. TEER was continuously monitored as described in Materials and Methods. Results are expressed as mean ± SE; n = 4. * P<0.05 vs normoxia; **P<0.05 vs. hyperoxia+vehicle; #P<0.05 vs. normoxia+Z-VAD.

Mentions: Because the second phase of lung endothelial barrier disruption was accompanied with endothelial apoptosis, we investigate whether apoptotic pathways contribute to the disruption of lung endothelial integrity. As shown in Fig 4A and 4B, caspase-3 activity and apoptosis-inducing factor (AIF) in the nuclear fraction were significantly increased in lung endothelial cells exposed to hyperoxia for 48 h, indicating that both caspase-dependent and-independent apoptotic pathways participate hyperoxia-induced disruption of lung endothelial integrity. Moreover, the caspase-3 inhibitor Z-VAD-FMK partially prevented hyperoxia-induced lung endothelial barrier disruption (Fig 4C), suggesting that hyperoxia-induced disruption of endothelial monolayer integrity is due to increased endothelial apoptosis via caspase-3 pathway in part and that caspase-independent pathway, i.e., AIF, may also contribute to endothelial apoptosis of hyperoxic PAECs.


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

Kondrikov D, Fulton D, Dong Z, Su Y - PLoS ONE (2015)

Apoptosis contributes to hyperoxia-induced disruption of lung endothelial barrier.(A and B) PAECs were exposed to normoxia or to hyperoxia for 24 to 48 h after which caspase-3 activity (A) and nuclear AIF protein level (B) was assayed. (C) PAECs were treated with and without the caspase-3 inhibitor Z-VAD-FMK (50 μM) and exposed to normoxia and hyperoxia for 48 h. TEER was continuously monitored as described in Materials and Methods. Results are expressed as mean ± SE; n = 4. * P<0.05 vs normoxia; **P<0.05 vs. hyperoxia+vehicle; #P<0.05 vs. normoxia+Z-VAD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4465980&req=5

pone.0129343.g004: Apoptosis contributes to hyperoxia-induced disruption of lung endothelial barrier.(A and B) PAECs were exposed to normoxia or to hyperoxia for 24 to 48 h after which caspase-3 activity (A) and nuclear AIF protein level (B) was assayed. (C) PAECs were treated with and without the caspase-3 inhibitor Z-VAD-FMK (50 μM) and exposed to normoxia and hyperoxia for 48 h. TEER was continuously monitored as described in Materials and Methods. Results are expressed as mean ± SE; n = 4. * P<0.05 vs normoxia; **P<0.05 vs. hyperoxia+vehicle; #P<0.05 vs. normoxia+Z-VAD.
Mentions: Because the second phase of lung endothelial barrier disruption was accompanied with endothelial apoptosis, we investigate whether apoptotic pathways contribute to the disruption of lung endothelial integrity. As shown in Fig 4A and 4B, caspase-3 activity and apoptosis-inducing factor (AIF) in the nuclear fraction were significantly increased in lung endothelial cells exposed to hyperoxia for 48 h, indicating that both caspase-dependent and-independent apoptotic pathways participate hyperoxia-induced disruption of lung endothelial integrity. Moreover, the caspase-3 inhibitor Z-VAD-FMK partially prevented hyperoxia-induced lung endothelial barrier disruption (Fig 4C), suggesting that hyperoxia-induced disruption of endothelial monolayer integrity is due to increased endothelial apoptosis via caspase-3 pathway in part and that caspase-independent pathway, i.e., AIF, may also contribute to endothelial apoptosis of hyperoxic PAECs.

Bottom Line: Furthermore, we found that Hsp70 interacted with AIF in the cytosol in hyperoxic 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.Association of Hsp70 with AIF prevents AIF nuclear translocation, contributing to the protective effect of Hsp70 on hyperoxia-induced endothelial apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology & Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, GA 30912, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus