Caveolin-1 scaffolding domain promotes leukocyte adhesion by reduced basal endothelial nitric oxide-mediated ICAM-1 phosphorylation in rat mesenteric venules

Abstract

Exogenously applied caveolin-1 scaffolding domain (CAV) has been shown to inhibit inflammatory mediator-induced nitric oxide (NO) production and NO-mediated increases in microvessel permeability. However, the effect of CAV on endothelial basal NO that prevents leukocyte adhesion remains unknown. This study aims to investigate the roles of exogenously applied CAV in endothelial basal NO production, leukocyte adhesion, and adhesion-induced changes in microvessel permeability. Experiments were conducted in individually perfused rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). NO was quantified with fluorescence imaging in DAF-2-loaded vessels. Perfusing venules with CAV inhibited basal NO production without affecting basal Lp. Resuming blood flow in CAV-perfused vessels significantly increased leukocyte adhesion. The firmly adherent leukocytes altered neither basal Lp nor adherens junction integrity. Increases in Lp occurred only upon formyl-Met-Leu-Phe application that induces release of reactive oxygen species from the adherent leukocytes. The application of NO synthase inhibitor showed similar results to CAV, and NO donor abolished CAV-mediated leukocyte adhesion. Immunofluorescence staining showed increases in binding of ICAM-1 to an adhesion-blocking antibody concurrent with a Src-dependent ICAM-1 phosphorylation following CAV perfusion. Pre-perfusing vessels with anti-ICAM-1 blocking antibody or a Src kinase inhibitor attenuated CAV-induced leukocyte adhesion. These results indicate that the application of CAV, in addition to preventing excessive NO-mediated permeability increases, also causes reduction of basal NO and promotes ICAM-1-mediated leukocyte adhesion through Src activation-mediated ICAM-1 phosphorylation. CAV-induced leukocyte adhesion was uncoupled from leukocyte oxidative burst and microvessel barrier function, unless in the presence of a secondary stimulation.

Keywords: ICAM-1 phorsphorylation; caveolin-1; leukocyte adhesion; microvessel permeability; reactive oxygen species.

Fig. 1.

The application of Antennapedia homeodomain (AP)-caveolin-1 (CAV) inhibits basal nitric oxide (NO). A: the time-dependent changes in cumulative DAF-2 fluorescence intensity (FI_DAF) from one representative experiment before (○) and after (●) treatment with 10 μM AP-CAV, followed by 10 μM SNP (△) perfusion for 40 min. B: time-dependent changes in cumulative FI_DAF from two representative experiments in the absence (●) or presence of (○) of 500 μM l-NMMA. C: summary of NO production rate derived from cumulative FI_DAF in each group of vessels. The dashed arrow indicates the time when DAF-2 fluorescence reached steady state. The arrow indicates the time when testing solution was added. †Significant decreases from control (P < 0.05).

Fig. 2.

Perfusion of AP-CAV for 30 min has no effect on basal hydraulic conductivity (Lp). A: a representative experiment shows the time course of Lp changes when the vessel was perfused with AP-CAV (10 μM). B: summary results of four experiments.

Fig. 3.

Reduction of basal NO by AP-CAV induced significant leukocyte adhesion without increasing Lp in the absence of a secondary stimulation. A: paired video images from two rat venules. The two images on the left show the same vessel under control conditions and after AP-CAV (10 μM)-induced leukocyte adhesion. The two images on the right show that the application of sodium nitroprusside (SNP) abolished AP-CAV-induced leukocyte adhesion. B: the time course of Lp changes from a representative experiment showing that AP-CAV (10 μM)-induced leukocyte adhesion (25/100 μm of vessel length) did not increase Lp, unless formyl-Met-Leu-Phe-OH (fMLP) (10 μM) was added to the perfusate. C: summary graph showing the correlation between the number of adherent leukocytes (per 100 μm vessel length) and the changes in Lp in four group of studies. Perfusion vessels with AP-CAV at 1 μM (n = 3) and 10 μM (n = 5) show AP-CAV dose-dependent increases in leukocyte adhesion and fMLP-induced increases in Lp. The application of SNP attenuated AP-CAV-induced leukocyte adhesion (n = 4), and replacing AP-CAV with scrambled AP-CAV (AP-CAV-X) showed no effect on leukocyte adhesion (n = 3). The blank bars represent the control values. The arrows indicate the procedures of 30 min of AP-CAV or AP-CAV-X perfusion followed by 10 min of resumed blood flow. The dashed line bars represent values measured after resumed blood flow in AP-CAV or AP-CAV-X perfused vessels. *Significant increases from the control values (P < 0.05). †Significant decreases from AP-CAV group (P < 0.05).

Fig. 4.

l-NMMA, a NO synthase (NOS) inhibitor, showed similar effects on leukocyte adhesion and microvessel permeability to those of CAV. A: time course of Lp changes from a representative experiment. B: summary of the number of adherent leukocytes (per 100-μm vessel length) and the corresponding changes in microvessel Lp (n = 3). *Significant increases from control (P < 0.05).

Fig. 5.

Confocal images of the co-staining of VE-cadherin and adherent leukocytes illustrating that CAV-induced leukocyte adhesion did not change VE-cadherin distribution. A: VE-cadherin staining under control conditions and after CAV-induced leukocyte adhesion. The third image shows the double staining of VE-cadherin and adherent leukocytes (indicated by arrows). B: magnified images from three different vessels showing no changes in VE-cadherin at the adhesion sites.

Fig. 6.

AP-CAV-induced increase in ICAM-1 adhesive capacity causes leukocyte adhesion. A: representative confocal images of anti-ICAM-1 blocking antibody mAb1A29 and vascular cell nuclei co-staining from five groups of studies and the secondary antibody control (ICAM-1 is shown in green, and nuclei are red). B: quantification of total fluorescence intensity (FI) of ICAM-1 per unit area of vessel wall under control conditions (n = 4), after AP-CAV (1 μM, n = 3; and 10 μM, n = 4) perfusion in the absence and presence of SNP (n = 3), and after AP-CAV-X perfusion (n = 3). C: perfusion of vessels with mAb1A29 significantly attenuated AP-CAV (10 μM)-induced leukocyte adhesion (n = 5 per group). *Significant increase from control (P < 0.05). †Significant decrease from AP-CAV group (P < 0.05).

Fig. 7.

AP-CAV-induced leukocyte adhesion involves Src activation-mediated ICAM-1 phosphorylation at tyrosine 526 (Y526). A: representative confocal images demonstrate increases in ICAM-1 phosphorylation at Y526 following AP-CAV perfusion relative to that of the control. Such increased ICAM-1 phosphorylation was blocked by the application of a Src kinase inhibitor, PP1, and was absent in vessels perfused with AP-CAV-X or secondary antibody alone. Phosphorylated ICAM-1 at Y526 is shown in green, and nuclei are red. B: summary of changes in FI of phospho-Y526-ICAM-1 relative to control in each experimental group (n = 3 per group). C: perfusion of vessels with PP1 that prevented ICAM-1 phosphorylation also significantly attenuated AP-CAV induced leukocyte adhesion (n = 3). *Significant increase from control (P < 0.05). †Significant decrease from AP-CAV group (P < 0.05).