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M Bartoli, T Lemtalsi, DH Platt, MB Marrero, RB Caldwell; High Glucose-Induced ROS Production Inhibits Shp-1 Activity and Enhances STAT3 Activation in Retinal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1320.
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Purpose:Diabetic retinopathy (DR) is characterized by increased VEGF production and neo-angiogenesis. High glucose and reactive oxygen species (ROS) are important etiologic agents in DR. It has been shown that the signal transducer and activator of transcription 3 (STAT3) plays an important role in angiogenesis by modulating VEGF expression. We have shown that in retinal endothelial cells (REC) VEGF induces STAT3 activation and that high glucose exaggerates this effect. The purpose of this study was to determine how high glucose affects STAT3 activation in REC. Methods:Bovine REC were cultured in the presence of 25mM glucose (HG) or 5mM glucose (NG). REC cultured either in HG or NG conditions were also treated with the ROS scavengers SOD and uric acid or with the NOS inhibitor L-NAME. Immunoprecipitation and western blotting analysis were carried-out to analyze the phosphorylation pattern of STAT3 and Shp1 (PTP1C). Results:In REC cultured in NG, VEGF treatment stimulated a transient STAT3-tyrosine phosphorylation that starts at 5min and ends at 60min after VEGF induction. After 48h exposure to HG, STAT3 activation was sustained and did not decrease at 60 or 90 min post-treatment. After 5 days of HG treatment STAT3 tyrosine phosphorylation was also evident in the basal condition. Analysis of the phosphorylation pattern of the STAT3 negative modulator Shp-1 indicated that HG completely abolishes Shp-1 phosphorylation, thus inhibiting its activity. Treatments with uric acid and L-NAME completely restored Shp-1 tyrosine phosphorylation. SOD partially restored Shp-1 phosphorylation. Conclusion:Our data indicate that high glucose induces constitutive activation of STAT3 in REC. This effect is due to the inhibition of the specific phosphatase Shp-1. ROS generated by high glucose treatment appear to abolish Shp-1 activity resulting in STAT3 constitutive activation. Furthermore, the effect of uric acid, SOD and L-NAME in restoring Shp-1 activity implicates the ROS peroxynitrite that is known to be an important inducer of vascular dysfunction in diabetes
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