QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online December 22, 2004
doi: 10.1242/10.1242/jcs.01612

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by El-Remessy, A. B. Articles by Caldwell, R. B. Search for Related Content PubMed PubMed Citation Articles by El-Remessy, A. B. Articles by Caldwell, R. B.

Oxidative stress inactivates VEGF survival signaling in retinal endothelial cells via PI 3-kinase tyrosine nitration

¹ Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912, USA
² Department of Pharmacology, Medical College of Georgia, Augusta, GA 30912, USA
³ Department of Pathology, Medical College of Georgia, Augusta, GA 30912, USA
⁴ Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912, USA
⁵ Department of Ophthalmology, Medical College of Georgia, Augusta, GA 30912, USA

View larger version (12K): [in a new window]   Fig. 1. High glucose and peroxynitrite inactivate VEGF pro-survival and accelerate endothelial cell death. Statistical analysis of caspase-3 activity showing significant increases in apoptosis in cells treated with peroxynitrite (PN) or cultured in high glucose (HG) compared to normal glucose (NG). Exogenous VEGF (40 ng/ml) protected cells cultured in normal glucose from serum starvation-induced apoptosis but did not rescue cells cultured in high glucose or peroxynitrite. FeTPPS (2.5 µM) a specific peroxynitrite decomposition catalyst restored the pro-survival effect of VEGF in cells maintained in high glucose cultures. Similar results were obtained in another three experiments. (*P<0.05 compared to NG, P<0.05 compared to HG.)

View larger version (41K): [in a new window]   Fig. 2. Oxidative stress-induced apoptosis in high glucose, high osmolarity and peroxynitrite. (A) Representative images of Hoechst 33258 nuclear staining show apoptotic cells that were identified based on their shrunken, irregular or fragmented nuclei. Quantification of these data is shown in Table 2. (B) A representative immunoblot, using cell lysates (50 µg), for cleaved PARP and ß-actin. Below: statistical analysis of quantification of cleaved PARP showing significant increases in apoptosis in cells treated with high glucose (HG), 3-O-methyl glucose (3mG) or peroxynitrite in comparison to cells cultured in normal glucose (NG). (*P<0.05 vs NG.)

View larger version (28K): [in a new window]   Fig. 3. High glucose and peroxynitrite inactivate VEGF pro-survival via alteration of Akt-1/P38 MAP kinase activation. (A) High glucose (HG) and peroxynitrite (PN) significantly decreased phosphorylation of Akt-1 compared to normal glucose (NG) even in the presence of exogenous VEGF (40 mg/ml). (*P<0.05 compared to NG.) (B) High glucose (HG) and peroxynitrite (PN) significantly increased phosphorylation of p38 MAP kinase in basal or VEGF (40 mg/ml)-stimulated conditions. FeTPPS (2.5 µM) a specific peroxynitrite decomposition catalyst blocked the increases in p38 MAP kinase phosphorylation in both basal and VEGF stimulated conditions. Identical results were obtained in another two experiments. (*P<0.05 compared to NG, P<0.05 compared to HG.)

View larger version (31K): [in a new window]   Fig. 4. Tyrosine nitration inactivates PI 3-kinase/Akt-1 pro-survival pathway. (A) Immunoprecipitation with p85 subunit of PI 3-kinase and western blot analysis using anti-nitrotyrosine antibody showed that cells treated with high glucose (HG) or peroxynitrite (PN) had significantly more nitration on the regulatory p85 subunit compared with cells cultured in normal glucose (NG). (*P<0.05 compared to NG.) This effect was blocked by the specific peroxynitrite decomposition catalyst FeTPPS (2.5 µM) and the specific nitration inhibitor epicatechin (100 µM). (B) Immunoprecipitation with p110 subunit of PI 3-kinase and western blot analysis using p85 antibody showed that under high glucose (HG) or peroxynitrite (PN) treatments, the p85 subunit was hardly detected in the immunopreicipitate of the catalytic p110 subunit compared with cells cultured in normal glucose (NG). Cells were stimulated with VEGF (40 ng/ml) in the presence or absence of the peroxynitrite decomposition catalyst (FeTPPs). The association between p85 and p110 was restored by treatment with FeTPPs (2.5 µM). (C) High glucose (HG) and peroxynitrite (PN) decreased Akt-1 activity significantly compared to normal glucose (NG). Akt-1 kinase activity was restored by treatment of high glucose cultures with a specific peroxynitrite inhibitor (2.5 µM), NOS inhibitor (L-NAME, 0.5 mM) and superoxide dismutase (SOD, 100 U/ml). A western blot of phospho GSK-3, the substrate of Akt-1 kinase is shown at the top. (*P<0.05 compared to NG.) (D) Statistical analysis of caspase-3 activity showing significant increases in apoptosis in cells cultured in high glucose (HG) or treated with peroxynitrite (PN) compared to normal glucose (NG). These effects were blocked by the specific nitration inhibitor epicatechin (100 µM). Similar results were obtained in another three experiments. (*P<0.05 compared to NG.)

View larger version (20K): [in a new window]   Fig. 5. Transfection of active Akt-1 restores cell survival and inhibits p38 MAP kinase phosphorylation. (A) Statistical analysis of caspase-3 activity shows that transfection with myr-Akt-1 significantly blocks increases in caspase-3 activity in cells cultured in high glucose (HG) or peroxynitrite (PN) compared to cells transfected with ß-galactosidase (ß-gal). (*P<0.05 compared to ß-gal.) (B) Transfection with myr-Akt-1 decreases p38 MAP kinase phosphorylation in cells cultured in high glucose (HG) or peroxynitrite (PN) compared to cells transfected with ß-galactosidase. A representative image of immunoblot analysis of phospho p38 MAP kinase and ß-actin is shown at the top. Similar results were obtained in another two experiments. (*P<0.05 compared to NG, P<0.05 compared to HG and PN.)

View larger version (20K): [in a new window]   Fig. 6. Inhibition of p38 MAP kinase with SB203580 restores cell survival and Akt-1 phosphorylation. (A) Statistical analysis of caspase-3 activity shows that inhibition of p38 MAP kinase significantly reduced increases of caspase-3 activity in cells cultured in high glucose (HG) or peroxynitrite (PN) compared to cells cultured in normal glucose (NG). (*P<0.05 compared to NG, P<0.05 compared to PN.) (B) Inhibition of p38 MAP kinase restores Akt-1 phosphorylation in cells cultured in high glucose (HG) or peroxynitrite (PN) compared to cells cultured in normal glucose (NG). A representative image of immunoblot analysis, using cell lysates (50 µg), for phospho Akt-1 and ß-actin is shown at the top. Similar results were obtained in another two experiments. (*P<0.05 compared to NG.)

View larger version (13K): [in a new window]   Fig. 7. A schematic representation of the proposed mechanism by which high glucose, via its effect on peroxynitrite, inactivates the VEGF/PI 3-kinase/Akt-1 pro-survival pathway and stimulates cell death via activation of p38 MAP kinase pathway. Nitration of PI 3-kinase is proposed as a mechanism by which high glucose switches off the VEGF pro-survival pathway and triggers the pro-apoptotic pathway.