Overexpression of cellular glutathione peroxidase rescues homocyst(e)ine-induced endothelial dysfunction

Abstract

Homocyst(e)ine (Hcy) inhibits the expression of the antioxidant enzyme cellular glutathione peroxidase (GPx-1) in vitro and in vivo, which can lead to an increase in reactive oxygen species that inactivate NO and promote endothelial dysfunction. In this study, we tested the hypothesis that overexpression of GPx-1 can restore the normal endothelial phenotype in hyperhomocyst(e)inemic states. Heterozygous cystathionine beta-synthase-deficient (CBS((-/+))) mice and their wild-type littermates (CBS((+/+))) were crossbred with mice that overexpress GPx-1 [GPx-1((tg+)) mice]. GPx-1 activity was 28% lower in CBS((-/+))/GPx-1((tg-)) compared with CBS((+/+))/GPx-1((tg-)) mice (P < 0.05), and CBS((-/+)) and CBS((+/+)) mice overexpressing GPx-1 had 1.5-fold higher GPx-1 activity compared with GPx-1 nontransgenic mice (P < 0.05). Mesenteric arterioles of CBS((-/+))/GPx-1((tg-)) mice showed vasoconstriction to superfusion with beta-methacholine and bradykinin (P < 0.001 vs. all other groups), whereas nonhyperhomocyst(e)inemic mice [CBS((+/+))/GPx-1((tg-)) and CBS((+/+))/GPx-1((tg+)) mice] demonstrated dose-dependent vasodilation in response to both agonists. Overexpression of GPx-1 in hyperhomocyst(e)inemic mice restored the normal endothelium-dependent vasodilator response. Bovine aortic endothelial cells (BAEC) were transiently transfected with GPx-1 and incubated with dl-homocysteine (HcyH) or l-cysteine. HcyH incubation decreased GPx-1 activity in sham-transfected BAEC (P < 0.005) but not in GPx-1-transfected cells. Nitric oxide release from BAEC was significantly decreased by HcyH but not cysteine, and GPx-1 overexpression attenuated this decrease. These findings demonstrate that overexpression of GPx-1 can compensate for the adverse effects of Hcy on endothelial function and suggest that the adverse vascular effects of Hcy are at least partly mediated by oxidative inactivation of NO.

Figure 1

Tissue GPx-1 activity in wild-type mice [CBS^(+/+)/GPx-1^(tg−), n = 6], heterozygous CBS knockout mice [CBS^(−/+)/GPx-1^(tg−), n = 7], GPx-1 transgenic mice [CBS^(+/+)/GPx-1^(tg+), n = 5], and GPx-1 transgenic, heterozygous CBS knockout mice [CBS^(−/+)/GPx-1^(tg+), n = 5]. *, P < 0.05 vs. CBS^(+/+)/GPx-1^(tg−); $, P < 0.01 vs. CBS^(+/+)/GPx-1^(tg−); §, P < 0.01 vs. CBS^(−/+)/GPx-1t^g−.

Figure 2

Mesenteric microvascular response to superfusion with β-methacholine in CBS^(+/+)/GPx-1^(tg−) mice (●), CBS^(−/+)/GPx-1^(tg−) mice (○), CBS^(+/+)/GPx-1^(tg+) mice (▿), and CBS^(−/+)/GPx-1^(tg+) mice (▾). *, P < 0.001 vs. all other groups.

Figure 3

NO release from BAEC into the medium over 2 h. Cells were transfected with pCMV-GPx-1, pCMV-SelD, and pGEM3-tRNA^Sec or sham-transfected and preincubated with the indicated concentrations of dl-homocysteine or l-cysteine for 4 h. The buffer was then changed to a thiol-free buffer, and DAF-2 fluorescence in the supernatant was measured after stimulation with BK 10⁻⁵ mol/liter for 2 h as described in Materials and Methods. n = four experiments. *, P < 0.05 vs. control; **, P < 0.005 vs. control.

Figure 4

cGMP accumulation in BASMC after short-term coculture with BAEC. BAEC were grown in transwell inserts, transfected with pCMV-GPx-1, pCMV-SelD, and pGEM3-tRNA^Sec or sham-transfected and preincubated with the indicated concentrations of dl-homocysteine or l-cysteine for 4 h. BAEC were then transferred for coincubation with BASMC, incubated with 3-isobutyl-1-methylxanthine for 15 min, and then stimulated with BK 10⁻⁵ mol/liter for 60 s. cGMP was measured in the ethanol extract of BASMC by using an enzyme immunoassay. n = three experiments. *, P < 0.01 vs. BK-stimulated control; **, P < 0.001 vs. BK-stimulated control.