Abstract
Glutathione (GSH) biosynthesis is essential for cellular redox homeostasis and antioxidant defence. The rate-limiting step requires glutathione cysteine ligase (GCL), which is composed of the catalytic (GCLc) and modulatory (GCLm) subunits. To evaluate the contribution of GCLc to endothelial function we generated endothelial-specific GCLc haploinsufficient (GCLc e/+ mice). In murine lung endothelial cells (MLEC) derived from these mice we corroborated a 50% reduction in GCLc levels compared to fibroblasts from the same mice. MLEC obtained from haploinsufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1176) and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT) mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in GCLc e/+ male mice, which was corrected by pre-incubation with ethyl-ester GSH and BH4. To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and GCLc e/+ mice. We observed that obstructed kidneys from GCLc e/+ mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is critical for endothelial function and anti-fibrotic response.
Highlights
Endothelial dysfunction constitutes a major challenge from both the biological and clinical standpoints as it is fundamentally associated to the vascular damage present in major pathological entities such as hypertension, diabetes, atherosclerosis, and aging [1]
In the present study we have studied the specific contribution of endothelial GSH to endothelial function
Using murine lung endothelial cells (MLEC) from endothelial-specific Gclc haplo-insufficient Gclc(e/ +) mice we have shown a reduction in GSH levels as well as increased
Summary
Endothelial dysfunction constitutes a major challenge from both the biological and clinical standpoints as it is fundamentally associated to the vascular damage present in major pathological entities such as hypertension, diabetes, atherosclerosis, and aging [1]. Antioxidant responses to injury are elicited through multiple pathways. Among these pathways, the endogenous nucleophile glutathione (GSH) is of paramount importance for redox homeostasis, due to its high intracellular concentrations (1–10 mM), its capacity to interact with peroxidases and its potential to act as an electron donor for free radicals [4,5,6]. GSH synthesis proceeds through two major steps regulated by the enzymes glutamate cysteine ligase (GCL) and glutathione synthetase (GS). The interaction between the two subunits and that of both of them together with their common substrates, cysteine and glutamate, dictates the final efficiency of GSH synthesis [10]
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