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Angiopoietin-1 requires oxidant signaling through p47phox to promote endothelial barrier defense.

Ghosh CC, Mukherjee A, David S, Milam KE, Hunter JT, Parikh SM - PLoS ONE (2015)

Bottom Line: Using primary human microvascular endothelial cells (HMVECs), we found that Angpt-1 stimulation induces phosphorylation of p47phox and a brief oxidative burst that is lost when chemical inhibitors of NOX activity or siRNA against the NOX component p47phox were applied.All of these changes were associated with weakened barrier function.These results suggest an essential role for NOX signaling in Angpt-1-mediated endothelial barrier defense against mediators of systemic inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center for Vascular Biology Research and Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, United States of America.

ABSTRACT

Background: Reactive oxygen species (ROS) are largely considered to be pathogenic to normal endothelial function in disease states such as sepsis. We hypothesized that Angiopoietin-1 (Angpt-1), an endogenous agonist of the endothelial-specific receptor, Tie-2, promotes barrier defense by activating NADPH oxidase (NOX) signaling.

Methods and findings: Using primary human microvascular endothelial cells (HMVECs), we found that Angpt-1 stimulation induces phosphorylation of p47phox and a brief oxidative burst that is lost when chemical inhibitors of NOX activity or siRNA against the NOX component p47phox were applied. As a result, there was attenuated ROS activity, disrupted junctional contacts, enhanced actin stress fiber accumulation, and induced gap formation between confluent HMVECs. All of these changes were associated with weakened barrier function. The ability of Angpt-1 to prevent identical changes induced by inflammatory permeability mediators, thrombin and lipopolysaccharides (LPS), was abrogated by p47phox knockdown. P47phox was required for Angpt-1 to activate Rac1 and inhibit mediator-induced activation of the small GTPase RhoA. Finally, Angpt-1 gene transfer prevented vascular leakage in wildtype mice exposed to systemically administered LPS, but not in p47phox knock out (p47-/-) littermates.

Conclusions: These results suggest an essential role for NOX signaling in Angpt-1-mediated endothelial barrier defense against mediators of systemic inflammation. More broadly, oxidants generated for signal transduction may have a barrier-promoting role in vascular endothelium.

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p47phox is required for Angpt-1-mediated barrier defense against thrombin.(A) Transendothelial resistance assay (TER) of confluent HMVECs treated with thrombin (1 U/ml), with p47phox or control siRNA, and with and without Angpt-1 (300 ng/ml). n = 4 experiments per condition. To enable comparisons between conditions, the baseline absolute resistance of each well was used to normalize subsequent readings for the respective well. (B) Data from (A) quantified as the change in normalized resistance 30 minutes after thrombin addition. ***p<0.001. (C) Transendothelial resistance assay (TER) of control- vs. p47phox-siRNA-treated HMVECs to which LPS (10 ng/ml) and Angpt-1 (300 ng/ml) were applied. The change was recorded 1 hour after LPS and Angpt-1 addition (n = 3 experiments per condition). ***p< 0.001, n = 4 experiments per condition.
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pone.0119577.g004: p47phox is required for Angpt-1-mediated barrier defense against thrombin.(A) Transendothelial resistance assay (TER) of confluent HMVECs treated with thrombin (1 U/ml), with p47phox or control siRNA, and with and without Angpt-1 (300 ng/ml). n = 4 experiments per condition. To enable comparisons between conditions, the baseline absolute resistance of each well was used to normalize subsequent readings for the respective well. (B) Data from (A) quantified as the change in normalized resistance 30 minutes after thrombin addition. ***p<0.001. (C) Transendothelial resistance assay (TER) of control- vs. p47phox-siRNA-treated HMVECs to which LPS (10 ng/ml) and Angpt-1 (300 ng/ml) were applied. The change was recorded 1 hour after LPS and Angpt-1 addition (n = 3 experiments per condition). ***p< 0.001, n = 4 experiments per condition.

Mentions: Based on the ability of Angpt-1 to counteract thrombin-induced RhoA activation in a p47phox-dependent fashion (Fig. 3), we asked whether Angpt-1 could counteract thrombin-induced barrier dysfunction, and if so, whether this form of barrier defense required p47phox. Compared to control siRNA, p47phox silencing had no effect on basal barrier function without the presence of thrombin (Fig. 4A, left of thrombin arrow). Upon addition of thrombin, p47phox-siRNA HMVECs responded with a greater fall in resistance than control-siRNA HMVECs. Angpt-1 substantially attenuated the thrombin-induced fall in resistance of control siRNA-treated HMVECs. This protective effect was lost in p47phox-siRNA HMVECs (Fig. 4A,B). These results show that reduction of p47phox exacerbates barrier dysfunction induced by the canonical RhoA activator, thrombin, and that Angpt-1’s barrier-protective effect against thrombin relies on intact p47phox expression.


Angiopoietin-1 requires oxidant signaling through p47phox to promote endothelial barrier defense.

Ghosh CC, Mukherjee A, David S, Milam KE, Hunter JT, Parikh SM - PLoS ONE (2015)

p47phox is required for Angpt-1-mediated barrier defense against thrombin.(A) Transendothelial resistance assay (TER) of confluent HMVECs treated with thrombin (1 U/ml), with p47phox or control siRNA, and with and without Angpt-1 (300 ng/ml). n = 4 experiments per condition. To enable comparisons between conditions, the baseline absolute resistance of each well was used to normalize subsequent readings for the respective well. (B) Data from (A) quantified as the change in normalized resistance 30 minutes after thrombin addition. ***p<0.001. (C) Transendothelial resistance assay (TER) of control- vs. p47phox-siRNA-treated HMVECs to which LPS (10 ng/ml) and Angpt-1 (300 ng/ml) were applied. The change was recorded 1 hour after LPS and Angpt-1 addition (n = 3 experiments per condition). ***p< 0.001, n = 4 experiments per condition.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356555&req=5

pone.0119577.g004: p47phox is required for Angpt-1-mediated barrier defense against thrombin.(A) Transendothelial resistance assay (TER) of confluent HMVECs treated with thrombin (1 U/ml), with p47phox or control siRNA, and with and without Angpt-1 (300 ng/ml). n = 4 experiments per condition. To enable comparisons between conditions, the baseline absolute resistance of each well was used to normalize subsequent readings for the respective well. (B) Data from (A) quantified as the change in normalized resistance 30 minutes after thrombin addition. ***p<0.001. (C) Transendothelial resistance assay (TER) of control- vs. p47phox-siRNA-treated HMVECs to which LPS (10 ng/ml) and Angpt-1 (300 ng/ml) were applied. The change was recorded 1 hour after LPS and Angpt-1 addition (n = 3 experiments per condition). ***p< 0.001, n = 4 experiments per condition.
Mentions: Based on the ability of Angpt-1 to counteract thrombin-induced RhoA activation in a p47phox-dependent fashion (Fig. 3), we asked whether Angpt-1 could counteract thrombin-induced barrier dysfunction, and if so, whether this form of barrier defense required p47phox. Compared to control siRNA, p47phox silencing had no effect on basal barrier function without the presence of thrombin (Fig. 4A, left of thrombin arrow). Upon addition of thrombin, p47phox-siRNA HMVECs responded with a greater fall in resistance than control-siRNA HMVECs. Angpt-1 substantially attenuated the thrombin-induced fall in resistance of control siRNA-treated HMVECs. This protective effect was lost in p47phox-siRNA HMVECs (Fig. 4A,B). These results show that reduction of p47phox exacerbates barrier dysfunction induced by the canonical RhoA activator, thrombin, and that Angpt-1’s barrier-protective effect against thrombin relies on intact p47phox expression.

Bottom Line: Using primary human microvascular endothelial cells (HMVECs), we found that Angpt-1 stimulation induces phosphorylation of p47phox and a brief oxidative burst that is lost when chemical inhibitors of NOX activity or siRNA against the NOX component p47phox were applied.All of these changes were associated with weakened barrier function.These results suggest an essential role for NOX signaling in Angpt-1-mediated endothelial barrier defense against mediators of systemic inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center for Vascular Biology Research and Division of Nephrology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, United States of America.

ABSTRACT

Background: Reactive oxygen species (ROS) are largely considered to be pathogenic to normal endothelial function in disease states such as sepsis. We hypothesized that Angiopoietin-1 (Angpt-1), an endogenous agonist of the endothelial-specific receptor, Tie-2, promotes barrier defense by activating NADPH oxidase (NOX) signaling.

Methods and findings: Using primary human microvascular endothelial cells (HMVECs), we found that Angpt-1 stimulation induces phosphorylation of p47phox and a brief oxidative burst that is lost when chemical inhibitors of NOX activity or siRNA against the NOX component p47phox were applied. As a result, there was attenuated ROS activity, disrupted junctional contacts, enhanced actin stress fiber accumulation, and induced gap formation between confluent HMVECs. All of these changes were associated with weakened barrier function. The ability of Angpt-1 to prevent identical changes induced by inflammatory permeability mediators, thrombin and lipopolysaccharides (LPS), was abrogated by p47phox knockdown. P47phox was required for Angpt-1 to activate Rac1 and inhibit mediator-induced activation of the small GTPase RhoA. Finally, Angpt-1 gene transfer prevented vascular leakage in wildtype mice exposed to systemically administered LPS, but not in p47phox knock out (p47-/-) littermates.

Conclusions: These results suggest an essential role for NOX signaling in Angpt-1-mediated endothelial barrier defense against mediators of systemic inflammation. More broadly, oxidants generated for signal transduction may have a barrier-promoting role in vascular endothelium.

Show MeSH
Related in: MedlinePlus