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, 112 (7), 1049-57

Inhibition of GAPDH Activity by poly(ADP-ribose) Polymerase Activates Three Major Pathways of Hyperglycemic Damage in Endothelial Cells

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Inhibition of GAPDH Activity by poly(ADP-ribose) Polymerase Activates Three Major Pathways of Hyperglycemic Damage in Endothelial Cells

Xueliang Du et al. J Clin Invest.

Abstract

In this report, we show that hyperglycemia-induced overproduction of superoxide by the mitochondrial electron transport chain activates the three major pathways of hyperglycemic damage found in aortic endothelial cells by inhibiting GAPDH activity. In bovine aortic endothelial cells, GAPDH antisense oligonucleotides activated each of the pathways of hyperglycemic vascular damage in cells cultured in 5 mM glucose to the same extent as that induced by culturing cells in 30 mM glucose. Hyperglycemia-induced GAPDH inhibition was found to be a consequence of poly(ADP-ribosyl)ation of GAPDH by poly(ADP-ribose) polymerase (PARP), which was activated by DNA strand breaks produced by mitochondrial superoxide overproduction. Both the hyperglycemia-induced decrease in activity of GAPDH and its poly(ADP-ribosyl)ation were prevented by overexpression of either uncoupling protein-1 (UCP-1) or manganese superoxide dismutase (MnSOD), which decrease hyperglycemia-induced superoxide. Overexpression of UCP-1 or MnSOD also prevented hyperglycemia-induced DNA strand breaks and activation of PARP. Hyperglycemia-induced activation of each of the pathways of vascular damage was abolished by blocking PARP activity with the competitive PARP inhibitors PJ34 or INO-1001. Elevated glucose increased poly(ADP-ribosyl)ation of GAPDH in WT aortae, but not in the aortae from PARP-1-deficient mice. Thus, inhibition of PARP blocks hyperglycemia-induced activation of multiple pathways of vascular damage.

Figures

Figure 1
Figure 1
Effect of GAPDH antisense ODNs on GAPDH activity in BAECs. Each bar represents the mean ± SEM of four separate experiments. *P < 0.01 compared with cells incubated in 5 mM glucose alone.
Figure 2
Figure 2
Effect of GAPDH antisense ODNs on pathways of hyperglycemic damage in BAECs. (a) PKC activation; (b) hexosamine pathway activation; (c) intracellular AGE formation; (d) NF-κB activation. *P < 0.01 compared with cells incubated in 5 mM glucose. AU, arbitrary units. For a through c, each bar represents the mean ± SEM of four separate experiments. For d, each bar represents the mean ± SEM of fluorescence from 40 cells measured in an in situ DNA–protein binding assay.
Figure 3
Figure 3
Effect of genes that alter mitochondrial superoxide production and of PARP inhibition on poly(ADP-ribosyl)ation of GAPDH (a), and on GAPDH activity (b), in BAECs. Cells were incubated in 5 mM glucose or 30 mM glucose alone, in 30 mM glucose plus either control, UCP-1– or MnSOD-expressing adenoviral vectors, and in 30 mM glucose plus 3 μM PJ34. Each bar represents the mean ± SEM of four separate experiments. *P < 0.01 compared with cells incubated in 5 mM glucose alone.
Figure 4
Figure 4
Effect of hyperglycemia on poly(ADP-ribosyl)ation of GAPDH in aortas from WT and PARP-1 KO mice. Each bar represents the mean ± SEM of four separate experiments. *P < 0.01 compared with aortas incubated in 5 mM glucose.
Figure 5
Figure 5
Effect of hyperglycemia and genes that alter mitochondrial superoxide production on PARP activity in BAECs. Cells were incubated in 5 mM glucose or 30 mM glucose alone, in 30 mM glucose plus either control, UCP-1– or MnSOD-expressing adenoviral vectors, and in 30 mM glucose plus 3 μM PJ34. Each bar represents the mean ± SEM of four separate experiments. *P < 0.01 compared with cells incubated in 5 mM glucose alone.
Figure 6
Figure 6
Effect of hyperglycemia and genes that alter mitochondrial superoxide production on DNA strand breaks in BAECs. Cells were incubated in 5 mM glucose (a) or 30 mM glucose alone (b), or in 30 mM glucose plus either control (c), UCP-1–expressing (d) or MnSOD-expressing (e) adenoviral vectors. Fluorescent micrographs from single-cell electrophoresis assay. (f) Quantitation of DNA strand breaks from single-cell electrophoresis assay. Each bar represents the mean ± SEM of 40 cells for each incubation condition. *P < 0.01 compared with cells incubated in 5 mM glucose alone.
Figure 7
Figure 7
Effect of PARP inhibition on hyperglycemia-induced pathways of vascular damage in BAECs. (a) PKC activation; (b) hexosamine pathway activation; (c) intracellular AGE formation; (d) NF-κB activation. *P < 0.01 compared with cells incubated in 5 mM glucose. For a through c, each bar represents the mean ± SEM of four separate experiments. For d, each bar represents the mean ± SEM of fluorescence from 40 cells measured in an in situ DNA–protein binding assay.

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