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Caveolin-1-eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability.

Siddiqui MR, Komarova YA, Vogel SM, Gao X, Bonini MG, Rajasingh J, Zhao YY, Brovkovych V, Malik AB - J. Cell Biol. (2011)

Bottom Line: We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation.Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A.Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.

ABSTRACT
Endothelial barrier function is regulated by adherens junctions (AJs) and caveolae-mediated transcellular pathways. The opening of AJs that is observed in caveolin-1(-/-) (Cav-1(-/-)) endothelium suggests that Cav-1 is necessary for AJ assembly or maintenance. Here, using endothelial cells isolated from Cav-1(-/-) mice, we show that Cav-1 deficiency induced the activation of endothelial nitric oxide synthase (eNOS) and the generation of nitric oxide (NO) and peroxynitrite. We assessed S-nitrosylation and nitration of AJ-associated proteins to identify downstream NO redox signaling targets. We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation. Inhibition of eNOS or RhoA restored AJ integrity and diminished endothelial hyperpermeability in Cav-1(-/-) mice. Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A. Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

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Restoration of normal paracellular permeability in Cav-1−/− endothelial monolayers and vessels by inhibition of either RhoA or eNOS. (a) Immunofluorescent staining of MLVECs isolated from Cav-1−/− and Cav-1/eNOS double knockout (DKO) mice for β-catenin (green), F-actin (red), and nuclei (blue). Cav-1−/− cells were treated with Rho inhibitor C3 transferase and AP-CSD peptide. Bar, 10 µm. (b) β-catenin accumulation at AJs as in Fig. 1 e; *, P < 0.01 as compared with Wt control; **, P < 0.01 as compared with Cav-1−/−; n = 10. (c) Endothelial permeability to EBA; mean and SEM are as in Fig. 1 e; *, P < 0.01 as compared with Wt control; **, P < 0.05 as compared with Cav-1−/−; n = 4. (d) Lung weight changes after a step increase in transvascular oncotic pressure gradient. Recordings were smoothed by averaging successive groups of five points. Lungs isolated from Wt and Cav-1−/− mice were perfused with 0% BSA for 10 min, with 10% BSA for 30 min, and with 0% BSA for 10 min; an additional Cav-1−/− group received AP-CSD peptide starting at 10 min of BSA profusion. AP-CSD peptide reversed lung weight loss during high albumin perfusion in Cav-1−/− lungs and largely restored the transvascular fluid filtration rate between 40 and 50 min. (e) The filtration rate was calculated from the initial slope of slow exponential component of lung weight gain; mean and SEM are as in Fig. 1 e; *, P < 0.05 as compared with Wt control; n = 5–9.
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fig4: Restoration of normal paracellular permeability in Cav-1−/− endothelial monolayers and vessels by inhibition of either RhoA or eNOS. (a) Immunofluorescent staining of MLVECs isolated from Cav-1−/− and Cav-1/eNOS double knockout (DKO) mice for β-catenin (green), F-actin (red), and nuclei (blue). Cav-1−/− cells were treated with Rho inhibitor C3 transferase and AP-CSD peptide. Bar, 10 µm. (b) β-catenin accumulation at AJs as in Fig. 1 e; *, P < 0.01 as compared with Wt control; **, P < 0.01 as compared with Cav-1−/−; n = 10. (c) Endothelial permeability to EBA; mean and SEM are as in Fig. 1 e; *, P < 0.01 as compared with Wt control; **, P < 0.05 as compared with Cav-1−/−; n = 4. (d) Lung weight changes after a step increase in transvascular oncotic pressure gradient. Recordings were smoothed by averaging successive groups of five points. Lungs isolated from Wt and Cav-1−/− mice were perfused with 0% BSA for 10 min, with 10% BSA for 30 min, and with 0% BSA for 10 min; an additional Cav-1−/− group received AP-CSD peptide starting at 10 min of BSA profusion. AP-CSD peptide reversed lung weight loss during high albumin perfusion in Cav-1−/− lungs and largely restored the transvascular fluid filtration rate between 40 and 50 min. (e) The filtration rate was calculated from the initial slope of slow exponential component of lung weight gain; mean and SEM are as in Fig. 1 e; *, P < 0.05 as compared with Wt control; n = 5–9.

Mentions: To demonstrate the causal link between RhoA activity and destabilization of AJs, we attempted to restore integrity of AJs in Cav-1−/− MLVECs by inhibiting RhoA. Treatment of Cav-1−/− endothelium with the Rho inhibitor C3-transferase significantly reduced phosphorylation of myosin light chain (MLC), a downstream effector of RhoA/ROCK, in Cav-1−/− cells (Fig. S2). It also rescued β-catenin accumulation at AJs and restored permeability of Cav-1−/− monolayers to the Evans blue albumin (EBA) tracer (Fig. 4, a–c). Treatment of Cav-1−/− endothelium with Cav-1 scaffold domain (CSD)–antennapedia (AP) fusion peptide, which binds to and suppresses eNOS catalytic activity (Bucci et al., 2000), superoxide scavenger TEMPOL, and l-NNA as well as Cav-1/eNOS deficiency (DKO) also restored the integrity of AJs and the permeability of Cav-1−/− monolayers (Fig. 4, a–c). We concluded therefore that nitration of p190A provides a reversible switch mechanism regulating RhoA activity and thereby endothelial barrier integrity.


Caveolin-1-eNOS signaling promotes p190RhoGAP-A nitration and endothelial permeability.

Siddiqui MR, Komarova YA, Vogel SM, Gao X, Bonini MG, Rajasingh J, Zhao YY, Brovkovych V, Malik AB - J. Cell Biol. (2011)

Restoration of normal paracellular permeability in Cav-1−/− endothelial monolayers and vessels by inhibition of either RhoA or eNOS. (a) Immunofluorescent staining of MLVECs isolated from Cav-1−/− and Cav-1/eNOS double knockout (DKO) mice for β-catenin (green), F-actin (red), and nuclei (blue). Cav-1−/− cells were treated with Rho inhibitor C3 transferase and AP-CSD peptide. Bar, 10 µm. (b) β-catenin accumulation at AJs as in Fig. 1 e; *, P < 0.01 as compared with Wt control; **, P < 0.01 as compared with Cav-1−/−; n = 10. (c) Endothelial permeability to EBA; mean and SEM are as in Fig. 1 e; *, P < 0.01 as compared with Wt control; **, P < 0.05 as compared with Cav-1−/−; n = 4. (d) Lung weight changes after a step increase in transvascular oncotic pressure gradient. Recordings were smoothed by averaging successive groups of five points. Lungs isolated from Wt and Cav-1−/− mice were perfused with 0% BSA for 10 min, with 10% BSA for 30 min, and with 0% BSA for 10 min; an additional Cav-1−/− group received AP-CSD peptide starting at 10 min of BSA profusion. AP-CSD peptide reversed lung weight loss during high albumin perfusion in Cav-1−/− lungs and largely restored the transvascular fluid filtration rate between 40 and 50 min. (e) The filtration rate was calculated from the initial slope of slow exponential component of lung weight gain; mean and SEM are as in Fig. 1 e; *, P < 0.05 as compared with Wt control; n = 5–9.
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fig4: Restoration of normal paracellular permeability in Cav-1−/− endothelial monolayers and vessels by inhibition of either RhoA or eNOS. (a) Immunofluorescent staining of MLVECs isolated from Cav-1−/− and Cav-1/eNOS double knockout (DKO) mice for β-catenin (green), F-actin (red), and nuclei (blue). Cav-1−/− cells were treated with Rho inhibitor C3 transferase and AP-CSD peptide. Bar, 10 µm. (b) β-catenin accumulation at AJs as in Fig. 1 e; *, P < 0.01 as compared with Wt control; **, P < 0.01 as compared with Cav-1−/−; n = 10. (c) Endothelial permeability to EBA; mean and SEM are as in Fig. 1 e; *, P < 0.01 as compared with Wt control; **, P < 0.05 as compared with Cav-1−/−; n = 4. (d) Lung weight changes after a step increase in transvascular oncotic pressure gradient. Recordings were smoothed by averaging successive groups of five points. Lungs isolated from Wt and Cav-1−/− mice were perfused with 0% BSA for 10 min, with 10% BSA for 30 min, and with 0% BSA for 10 min; an additional Cav-1−/− group received AP-CSD peptide starting at 10 min of BSA profusion. AP-CSD peptide reversed lung weight loss during high albumin perfusion in Cav-1−/− lungs and largely restored the transvascular fluid filtration rate between 40 and 50 min. (e) The filtration rate was calculated from the initial slope of slow exponential component of lung weight gain; mean and SEM are as in Fig. 1 e; *, P < 0.05 as compared with Wt control; n = 5–9.
Mentions: To demonstrate the causal link between RhoA activity and destabilization of AJs, we attempted to restore integrity of AJs in Cav-1−/− MLVECs by inhibiting RhoA. Treatment of Cav-1−/− endothelium with the Rho inhibitor C3-transferase significantly reduced phosphorylation of myosin light chain (MLC), a downstream effector of RhoA/ROCK, in Cav-1−/− cells (Fig. S2). It also rescued β-catenin accumulation at AJs and restored permeability of Cav-1−/− monolayers to the Evans blue albumin (EBA) tracer (Fig. 4, a–c). Treatment of Cav-1−/− endothelium with Cav-1 scaffold domain (CSD)–antennapedia (AP) fusion peptide, which binds to and suppresses eNOS catalytic activity (Bucci et al., 2000), superoxide scavenger TEMPOL, and l-NNA as well as Cav-1/eNOS deficiency (DKO) also restored the integrity of AJs and the permeability of Cav-1−/− monolayers (Fig. 4, a–c). We concluded therefore that nitration of p190A provides a reversible switch mechanism regulating RhoA activity and thereby endothelial barrier integrity.

Bottom Line: We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation.Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A.Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.

ABSTRACT
Endothelial barrier function is regulated by adherens junctions (AJs) and caveolae-mediated transcellular pathways. The opening of AJs that is observed in caveolin-1(-/-) (Cav-1(-/-)) endothelium suggests that Cav-1 is necessary for AJ assembly or maintenance. Here, using endothelial cells isolated from Cav-1(-/-) mice, we show that Cav-1 deficiency induced the activation of endothelial nitric oxide synthase (eNOS) and the generation of nitric oxide (NO) and peroxynitrite. We assessed S-nitrosylation and nitration of AJ-associated proteins to identify downstream NO redox signaling targets. We found that the GTPase-activating protein (GAP) p190RhoGAP-A was selectively nitrated at Tyr1105, resulting in impaired GAP activity and RhoA activation. Inhibition of eNOS or RhoA restored AJ integrity and diminished endothelial hyperpermeability in Cav-1(-/-) mice. Thrombin, a mediator of increased endothelial permeability, also induced nitration of p120-catenin-associated p190RhoGAP-A. Thus, eNOS-dependent nitration of p190RhoGAP-A represents a crucial mechanism for AJ disassembly and resultant increased endothelial permeability.

Show MeSH
Related in: MedlinePlus