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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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Related in: MedlinePlus

Angpt-1 mediated barrier defense in acute systemic inflammation requires p47phox.(A) Spectrophotometric quantification at 620 nm of intravenously injected Evans blue dye extravasation into the lungs of wildtype littermates (p47+/+) and p47phox KO mice (p47−/−) 16 hours after LPS (15 mg/kg IP) with prior control adenovirus (control) or Angpt-1 adenovirus (1 x 109pfu/mouse) gene transfer. (B-D) Lung photomicrographs from above conditions (representative of n = 3–5 mice per condition). Scale bar 50 μm.
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pone.0119577.g005: Angpt-1 mediated barrier defense in acute systemic inflammation requires p47phox.(A) Spectrophotometric quantification at 620 nm of intravenously injected Evans blue dye extravasation into the lungs of wildtype littermates (p47+/+) and p47phox KO mice (p47−/−) 16 hours after LPS (15 mg/kg IP) with prior control adenovirus (control) or Angpt-1 adenovirus (1 x 109pfu/mouse) gene transfer. (B-D) Lung photomicrographs from above conditions (representative of n = 3–5 mice per condition). Scale bar 50 μm.

Mentions: We and others have previously shown that Angpt-1 (or mimetics thereof) counteract critical manifestations of sepsis in experimental rodent models [6,7,9,34–37]. LPS injection in mice induces systemic inflammation that mimics features of the early cytokine storm in sepsis, such as severe vascular leakage. To evaluate the significance of the proposed p47phox-dependent barrier defense mechanism of Angpt-1 in vivo, we administered LPS with or without Angpt-1 gene transfer by adenovirus in p47−/− mice and their wildtype littermates. LPS induced comparable vascular leakage in p47−/− and wildtype mice. Inwildtype mice, Angpt-1 gene transfer prevented the LPS-induced increase in lung permeability (Fig. 5A). However, the anti-permeability effect of Angpt-1 against LPS was lost in p47−/− mice. In addition, LPS functioned as a “double-edged sword” by decreasing the activation of pTie2 and reducing the level of total Tie2 protein without affecting the total protein level of Rac1 (S3 Fig.). As previously shown [9,35], LPS-induced cellular infiltration of the lungs was also attenuated by Angpt-1, but again, deletion of p47phox abrogated this protective effect (Fig. 5B-D).


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)

Angpt-1 mediated barrier defense in acute systemic inflammation requires p47phox.(A) Spectrophotometric quantification at 620 nm of intravenously injected Evans blue dye extravasation into the lungs of wildtype littermates (p47+/+) and p47phox KO mice (p47−/−) 16 hours after LPS (15 mg/kg IP) with prior control adenovirus (control) or Angpt-1 adenovirus (1 x 109pfu/mouse) gene transfer. (B-D) Lung photomicrographs from above conditions (representative of n = 3–5 mice per condition). Scale bar 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0119577.g005: Angpt-1 mediated barrier defense in acute systemic inflammation requires p47phox.(A) Spectrophotometric quantification at 620 nm of intravenously injected Evans blue dye extravasation into the lungs of wildtype littermates (p47+/+) and p47phox KO mice (p47−/−) 16 hours after LPS (15 mg/kg IP) with prior control adenovirus (control) or Angpt-1 adenovirus (1 x 109pfu/mouse) gene transfer. (B-D) Lung photomicrographs from above conditions (representative of n = 3–5 mice per condition). Scale bar 50 μm.
Mentions: We and others have previously shown that Angpt-1 (or mimetics thereof) counteract critical manifestations of sepsis in experimental rodent models [6,7,9,34–37]. LPS injection in mice induces systemic inflammation that mimics features of the early cytokine storm in sepsis, such as severe vascular leakage. To evaluate the significance of the proposed p47phox-dependent barrier defense mechanism of Angpt-1 in vivo, we administered LPS with or without Angpt-1 gene transfer by adenovirus in p47−/− mice and their wildtype littermates. LPS induced comparable vascular leakage in p47−/− and wildtype mice. Inwildtype mice, Angpt-1 gene transfer prevented the LPS-induced increase in lung permeability (Fig. 5A). However, the anti-permeability effect of Angpt-1 against LPS was lost in p47−/− mice. In addition, LPS functioned as a “double-edged sword” by decreasing the activation of pTie2 and reducing the level of total Tie2 protein without affecting the total protein level of Rac1 (S3 Fig.). As previously shown [9,35], LPS-induced cellular infiltration of the lungs was also attenuated by Angpt-1, but again, deletion of p47phox abrogated this protective effect (Fig. 5B-D).

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