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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 induces a p47phox-dependent oxidative burst in endothelium.(A) Titration and validation of p47phox siRNA delivery into HMVECs. (B) Example epifluorescence (excitation 495 nm: emission 520 nm) and corresponding bright field images of individual HMVECs loaded with CM-H2DCFDA, pre-treated with Apo (650 μM), control siRNA, or p47phox siRNA, and then treated with Angpt-1 (300 ng/ml). Images shown were taken 5 minutes after Angpt-1 addition. Scale bar 10 μm. (C) Planimetric quantification of CM-H2DCFDA fluorescence from above conditions (n = 3–5 experiments per condition). ***p<0.001 compared to p47phox siRNA+Angpt-1. (D-G) Cells transfected with Hyper-3 for 48 hours, underwent serum starvation for 2 hours, were treated with 10 μM H2O2 or Angpt-1 (300 ng/ml), and changes in fluorescence intensity were measured by live cell imaging microscopy. Representative images after 2 minutes of H2O2(D-E) and 5 minutes with Angpt-1 (F-G) are shown. (H) Hyper-3 transfected HMVECs were, treated with chemicals (Apo and VAS 2870), siRNA p47phox or Tie2-Fc (500 ng/ml) before addition of Angpt-1 (300 ng/ml). Results were analyzed by one-way ANOVA followed by post-hoc corrections for multiple comparisons. ***p<0.001,***p<0.01 relative to Angpt-1 alone.
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pone.0119577.g001: Angpt-1 induces a p47phox-dependent oxidative burst in endothelium.(A) Titration and validation of p47phox siRNA delivery into HMVECs. (B) Example epifluorescence (excitation 495 nm: emission 520 nm) and corresponding bright field images of individual HMVECs loaded with CM-H2DCFDA, pre-treated with Apo (650 μM), control siRNA, or p47phox siRNA, and then treated with Angpt-1 (300 ng/ml). Images shown were taken 5 minutes after Angpt-1 addition. Scale bar 10 μm. (C) Planimetric quantification of CM-H2DCFDA fluorescence from above conditions (n = 3–5 experiments per condition). ***p<0.001 compared to p47phox siRNA+Angpt-1. (D-G) Cells transfected with Hyper-3 for 48 hours, underwent serum starvation for 2 hours, were treated with 10 μM H2O2 or Angpt-1 (300 ng/ml), and changes in fluorescence intensity were measured by live cell imaging microscopy. Representative images after 2 minutes of H2O2(D-E) and 5 minutes with Angpt-1 (F-G) are shown. (H) Hyper-3 transfected HMVECs were, treated with chemicals (Apo and VAS 2870), siRNA p47phox or Tie2-Fc (500 ng/ml) before addition of Angpt-1 (300 ng/ml). Results were analyzed by one-way ANOVA followed by post-hoc corrections for multiple comparisons. ***p<0.001,***p<0.01 relative to Angpt-1 alone.

Mentions: Given the role of NOXs in regulating GTPases and our previous findings in support of a GTPase mechanism of Angpt-1 barrier defense [8,9], we hypothesized that NOXs may be involved in Angpt-1-mediated barrier defense. We focused on p47phox based upon a large body of literature describing its functions in the endothelium (summarized in [30]. We first validated a siRNA against p47phox in HMVECs (Fig. 1A). We then used the cell-permeable reactive oxygen species detector dye, DCFDA, to follow the oxidative burst induced by Angpt-1 as previously reported [11,13,14]. We found that the chemical NOX inhibitor, Apo, or p47phox siRNA was sufficient to abrogate this burst (Fig. 1B,C). To overcome the non-specificity of the chemical redox probe, we used genetically defined redox probe-plasmid, Hyper-3[16,19].Using time-lapse imaging, we observed that H2O2 as well as Angpt-1 generate intracellular ROS (Fig. 1D-G).The effect of Angpt-1 was significantly reducedwhen Hyper-3 transfected cellswere pretreated with VAS2870, Apo, or p47phox siRNA (Fig. 1H).We also explored whether Angpt-1 phosphorylates p47phox and may alter the localization of p47phox from cytosol to membrane to augment ROS production. We found that Angpt-1 induces phosphorylation of p47phox at serine-304, an important regulatory site for p47phox (S1 Fig.).


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 induces a p47phox-dependent oxidative burst in endothelium.(A) Titration and validation of p47phox siRNA delivery into HMVECs. (B) Example epifluorescence (excitation 495 nm: emission 520 nm) and corresponding bright field images of individual HMVECs loaded with CM-H2DCFDA, pre-treated with Apo (650 μM), control siRNA, or p47phox siRNA, and then treated with Angpt-1 (300 ng/ml). Images shown were taken 5 minutes after Angpt-1 addition. Scale bar 10 μm. (C) Planimetric quantification of CM-H2DCFDA fluorescence from above conditions (n = 3–5 experiments per condition). ***p<0.001 compared to p47phox siRNA+Angpt-1. (D-G) Cells transfected with Hyper-3 for 48 hours, underwent serum starvation for 2 hours, were treated with 10 μM H2O2 or Angpt-1 (300 ng/ml), and changes in fluorescence intensity were measured by live cell imaging microscopy. Representative images after 2 minutes of H2O2(D-E) and 5 minutes with Angpt-1 (F-G) are shown. (H) Hyper-3 transfected HMVECs were, treated with chemicals (Apo and VAS 2870), siRNA p47phox or Tie2-Fc (500 ng/ml) before addition of Angpt-1 (300 ng/ml). Results were analyzed by one-way ANOVA followed by post-hoc corrections for multiple comparisons. ***p<0.001,***p<0.01 relative to Angpt-1 alone.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4356555&req=5

pone.0119577.g001: Angpt-1 induces a p47phox-dependent oxidative burst in endothelium.(A) Titration and validation of p47phox siRNA delivery into HMVECs. (B) Example epifluorescence (excitation 495 nm: emission 520 nm) and corresponding bright field images of individual HMVECs loaded with CM-H2DCFDA, pre-treated with Apo (650 μM), control siRNA, or p47phox siRNA, and then treated with Angpt-1 (300 ng/ml). Images shown were taken 5 minutes after Angpt-1 addition. Scale bar 10 μm. (C) Planimetric quantification of CM-H2DCFDA fluorescence from above conditions (n = 3–5 experiments per condition). ***p<0.001 compared to p47phox siRNA+Angpt-1. (D-G) Cells transfected with Hyper-3 for 48 hours, underwent serum starvation for 2 hours, were treated with 10 μM H2O2 or Angpt-1 (300 ng/ml), and changes in fluorescence intensity were measured by live cell imaging microscopy. Representative images after 2 minutes of H2O2(D-E) and 5 minutes with Angpt-1 (F-G) are shown. (H) Hyper-3 transfected HMVECs were, treated with chemicals (Apo and VAS 2870), siRNA p47phox or Tie2-Fc (500 ng/ml) before addition of Angpt-1 (300 ng/ml). Results were analyzed by one-way ANOVA followed by post-hoc corrections for multiple comparisons. ***p<0.001,***p<0.01 relative to Angpt-1 alone.
Mentions: Given the role of NOXs in regulating GTPases and our previous findings in support of a GTPase mechanism of Angpt-1 barrier defense [8,9], we hypothesized that NOXs may be involved in Angpt-1-mediated barrier defense. We focused on p47phox based upon a large body of literature describing its functions in the endothelium (summarized in [30]. We first validated a siRNA against p47phox in HMVECs (Fig. 1A). We then used the cell-permeable reactive oxygen species detector dye, DCFDA, to follow the oxidative burst induced by Angpt-1 as previously reported [11,13,14]. We found that the chemical NOX inhibitor, Apo, or p47phox siRNA was sufficient to abrogate this burst (Fig. 1B,C). To overcome the non-specificity of the chemical redox probe, we used genetically defined redox probe-plasmid, Hyper-3[16,19].Using time-lapse imaging, we observed that H2O2 as well as Angpt-1 generate intracellular ROS (Fig. 1D-G).The effect of Angpt-1 was significantly reducedwhen Hyper-3 transfected cellswere pretreated with VAS2870, Apo, or p47phox siRNA (Fig. 1H).We also explored whether Angpt-1 phosphorylates p47phox and may alter the localization of p47phox from cytosol to membrane to augment ROS production. We found that Angpt-1 induces phosphorylation of p47phox at serine-304, an important regulatory site for p47phox (S1 Fig.).

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