Angiopoietin-1 Requires Oxidant Signaling through p47phox to Promote Endothelial Barrier Defense

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

Obstructive Sleep Apnea: An Overview and Discussion of Inpatient Management

Obstructive sleep apnea (OSA) affects a predicted 22 million people in the United States with 80% of moderate to severe cases being undiagnosed.1 It has been shown that uncontrolled OSA can have an effect on cardiovascular health, specifically heart failure and recurrence of atrial fibrillation, as well as daytime alertness and quality of life.1,2 Continuous positive airway pressure (CPAP) is the primary treatment for OSA; CPAP is a nasally applied device that provides constant positive pressure that keeps the upper airway open while an individual is sleeping.3 Compliance with CPAP is a continuous battle medical providers fight, and many patients opt not to wear their CPAP complaining about comfort and convenience.4OSA is a national health epidemic; therefore, it is important to risk stratify patients while they are admitted in the hospital. Whether the hospital admission be for cardiovascular problems or not, if a person fits the demographics or voices a concern for having sleep apnea, health providers need to consult an inpatient sleep service or provide patient with an outpatient follow-up appointment. Addressing patients with sleep concerns who may be experiencing OSA while they are in the hospital helps to further treat a health issue that can affect the larger picture of a patient’s health. OSA control can help prevent exacerbations of further health issues and keep patients out of the hospital. Keywords: Obstructive sleep apnea, cardiovascular, continuous positive airway pressur

Angpt-1 mediated barrier defense in acute systemic inflammation requires p47phox.

<p>(<b>A</b>) 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>B-D</b>) Lung photomicrographs from above conditions (representative of n = 3–5 mice per condition). Scale bar 50 μm.</p

p47phox enables Angpt-1 to counteract cellular structural rearrangements induced by thrombin.

<p>(<b>A-H</b>) Confluent HMVECs were treated with control siRNA, thrombin (1 U/ml, <b>A-D</b>), or the combination of Angpt-1 (300 ng/ml) plus thrombin (<b>E-H</b>). After 15 minutes, cells were fixed, permeabilized, and stained for nuclei (blue, DAPI), VE-cadherin (green), or F-actin (red). White arrows indicate paracellular gaps. (<b>I-P</b>) Repeat of the above experiments, replacing control siRNA with p47phox siRNA. Representative of n = 3–4 experiments per condition. Scale bar 10 μm.</p

Angpt-1 induces a p47phox-dependent oxidative burst in endothelium.

<p>(<b>A</b>) Titration and validation of p47phox siRNA delivery into HMVECs. (<b>B</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. (<b>C</b>) Planimetric quantification of CM-H₂DCFDA fluorescence from above conditions (n = 3–5 experiments per condition). ***p<0.001 compared to p47phox siRNA+Angpt-1. <b>(D-G)</b> 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<b>(D-E)</b> and 5 minutes with Angpt-1 <b>(F-G)</b> are shown. <b>(H)</b> 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.</p

p47phox is required for Angpt-1-mediated barrier defense against thrombin.

<p>(<b>A</b>) 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>B</b>) Data from (<b>A</b>) quantified as the change in normalized resistance 30 minutes after thrombin addition. ***p<0.001. <b>(C)</b> 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.</p

Angpt-1 requires NOX activity to inhibit thrombin-induced RhoA activation.

<p>Confluent HMVECs were treated with vehicle (NT), Angpt-1 (300 ng/ml), Apo (650 μM), and/or thrombin (1 U/ml). Cells were lysed in the manufacturer-provided buffer 15 minutes after treatments for RhoA G-LISA, a pulldown method for RhoA-GTP detection. The results were then normalized to total RhoA, as determined by densitometry of Western blots (n = 4–6 experiments per condition). *p< 0.05.</p