Temple University School of Medicine

Temple University School of Medicine

Harinder Singh and Victor RizzoCardiovascular Research Center and the Dept of Anatomy and Cell Biology

Temple University School of Medicine Philadelphia, PA 19140

Results

Acknowledgements This work is supported by grants from NIH and AHA.

Abstract

• Cell Culture: Primary Bovine Aortic Endothelial Cells (BAEC) and grown in MCDB-131 culture media.• Lipid-raft/caveoale disassembly: Methyl-β-Cyclodextrin (MβCD) (10mM)• Caveolin-1siRNA: BAEC’s transfected with caveolin-1siRNA(100nM), ScrambledsiRNA using DharmaFECT-1 (Dharmacon, Inc., Lafayette, CO). • Inhibition of NADPH oxidase and eNOS enzyme: 50μM gp91-ds-tat peptide (Biosynthesis Inc.), L-NAME (Sigma Aldrich) for 45’ prior to stimulation, 100uM Uric acid (Sigma Aldrich)•Western Blotting: SDS-PAGE in 5-20% gradient gels, nitrocellulose membranes, indicated primary and secondary antibodies conjugated with HRP. Chemiluminiscence substrate for detection.• Immunoprecipitation: BAEC lysate immunoprecipitaed with anti-nitrotyrosine conjugated Dynabeads. • Colloidal silica technology: MLECs apical (luminal) plasma membranes were coated with silica and subfractionated to purify their subtending caveolae. Proteins (5 µg) from the indicated fractions (P: plasma membranes; V: caveolae;P-V: membranes stripped of caveolae) (Yang B et al., 2007)• In vivo: C57Blk WT and Cav1KO mice (8wk) were injected with either saline or TNFα and aortic tissue was processed for IHC using nitrotyrosine and VCAM-1 antibodies.

Methods

Inflammatory cytokine induced protein tyrosine nitration in WT and Cav1KO mouse aorta’s

Figure 2: IHC for nitrotyrosine staining in Aorta’s derived from C57BL/6J WT and Cav1KO mice injected with saline or TNFα(3μg/25g) i.v. for 3 hours

WT-Saline

Cav1KO-TNF

WT-TNF

Cav1KO-Saline

Inflammatory cytokine induced adhesion molecule expression in WT and Cav1KO mouse aorta’s

Figure 3: IF staining for VCAM-1 in aortic sections derived from C57BL/6J WT and Cav1KO mice injected with saline or TNFα(3μg/25g) i.v. for 3 hours

Caveolae regulate nitroxidative signaling via localized nitration of Src-family kinase in endothelial cells

Peroxynitrite (ONOO-) mediated tyrosine nitration of proteins has been implicated in vascular pathologies. The regulatory mechanism of cross-reaction between O2

- and NO for ONOO generation and subsequent nitroxidative signaling is poorly understood. Endothelial membrane caveolae, localize NAPDH oxidase enzyme complex (O2

-) and eNOS (NO), suggesting caveolae may act as compartments for radical generation and oxidative/nitrative signaling. Endothelial cells were challenged with TNFα and tyrosine nitration of proteins was measured (ONOO- generation).The blockade of NADPH oxidase (gp91ds-tat) and/or eNOS (L-NAME), as well as scavenging of ONOO- (Uric acid), inhibited protein nitration. Similarly, cells depleted of caveolae (Cav1siRNA), showed attenuation in protein nitration.The functional significance of ONOO generation was evaluated by studying proinflammatory signaling and markers of endothelial dysfunction. TNFα induced phosphorylation of Src family kinases (SFKs), IkB and NFkB as well as ICAM-1/VCAM-1 expression, whereas inhibition of radicals and loss of caveolae attenuated signaling. Given that SFKs localize to caveolae and IkB/NFkB are redox signaling mediators, targets of ONOO were probed. The SFK (not IkB/NFkB) was found to be nitrated in a Caveolae dependent manner. Also, inhibition of SFKs (PP2) showed decreased activation of IkB and NFkB, suggesting Src nitration regulates NFkB pathway. Similarly, In vivo studies on Cav1KO mice showed loss of Src nitration and adhesion molecule expression, when compared to WT mice injected with TNFα. Our results indicate that caveolae act as reaction centers for ONOO generation and nitroxidative signaling regulating endothelial dysfunction.  

TNFα induced protein tyrosine nitration in cultured endothelial cells

Figure 4: BAEC’s were pretreated with DPI, L-NAME, Uric Acid, Cav1siRNA and stimulated with TNFα. Cell lysates were collected and Western-blotted with nitrotyrosine antibody.

Figure 1: BAEC’s were processed using colloidal silica technology to Isolate apical (luminal) Membrane caveolae. The indicated fractions (P: plasma membranes; V: caveolae; P-V: membranes stripped of caveolae) were resolved by SDS-PAGE and immunoblotted with gp91(Nox2) p22phox, p47phox, Nox4 and eNOS (Yang B et al., 2007)

NADPH oxidase components and eNOS are localized in caveolae

Blockade of SFK attenuates TNFα induced NFkB activation and adhesion molecule expression

Targets of Protein tyrosine nitration

Figure 7: TNFα stimulated BAEC lysates were incubated with anti-nitrotyrosine antibody conjugated Dynabeads. Immunoprecipitates were Western-blotted with indicated primary antibodies.

Figure 8: BAEC lysates from scrambled and Cav1siRNA pre-treatments were collected and immunoprecipitaed using dynabeads counjugated with nitrotyrosine antibodies and western blotted for total SFK antibody

TNFα induced SFK nitration is dependent on caveolin-1

Figure 9: BAEC lysates pre-treated with SFK inhibitor, PP2, and stimulate with TNFα were probe for indicated signaling molecules and adhesion molecule antibodies

Inhibition of O2.-, NO and disruption of caveolae, attenuates adhesion molecule expression

Figure 5: BAEC’s pretreated with gp91ds-tat peptide, L-NAME, Cav1siRNA, and MβCD, were stimulated with TNFα. and western-blotted with indicated primary antibodies

Conclusions

Inflammatory cytokine induced nitroxidative signaling and NFkB activation

Figure 6: TNFα stimulated BAEC lysates pretreated with inhibitors and caveolae disrupting agents were western-blotted with above mentioned signaling molecules