A Glutathione Precursor Reduces Oxidative Injury to Cultured Embryonic Cardiomyocytes.

BACKGROUND: Newborn infants are highly vulnerable to oxidative stress. Following birth asphyxia, oxidative injury due to ischemia-reperfusion can result in significant brain and heart damage, leading to death or long-term disability. STUDY QUESTION: The study objective was to evaluate the effectiveness of antioxidant gamma-L-glutamyl-L-cysteine (γGlu-Cys) in inhibiting oxidative injury to cultured embryonic cardiomyocytes (H9c2 cells). STUDY DESIGN: Control and γGlu-Cys-treated (0.5 mM) H9c2 cells were incubated under 6-hour ischemic conditions followed by 2-hour simulated reperfusion. MEASURES AND OUTCOMES: To quantify oxidative stress-induced apoptosis sustained by cardiomyocytes, lactate dehydrogenase (LDH) release and the presence of cytosolic cytochrome c were measured, as well as the number of secondary lysosomes visualized under electron microscopy. RESULTS: Compared to controls, H9c2 cells coincubated with γGlu-Cys during ischemia-reperfusion exhibited a significant reduction in both LDH release into the incubation medium [23.88 ± 4.08 (SE) vs. 9.95 ± 1.86% of total; P = 0.02] and the number of secondary lysosomes [0.070 ± 0.009 (SD) vs. 0.043 ± 0.004 per µm; P = 0.01]. Inhibition of LDH release with γGlu-Cys was the same (P = 0.67) as that of a caspase inhibitor. The significant increase in cytosolic cytochrome c (P = 0.01) after ischemia-reperfusion simulation further supports γGlu-Cys's role in apoptosis prevention. CONCLUSIONS: It is concluded that the glutathione precursor γGlu-Cys protects cultured embryonic cardiomyocytes from apoptosis-associated oxidative injury.

Treatment with the matricellular protein CCN3 blocks and/or reverses fibrosis development in obesity with diabetic nephropathy.

Fibrosis is at the core of the high morbidity and mortality rates associated with the complications of diabetes and obesity, including diabetic nephropathy (DN), without any US Food and Drug Administration-approved drugs with this specific target. We recently provided the first evidence that the matricellular protein CCN3 (official symbol NOV) functions in a reciprocal manner, acting on the profibrotic family member CCN2 to inhibit fibrosis in a mesangial cell model of DN. Herein, we used the BT/BR ob/ob mouse as a best model of human obesity and DN progression to determine whether recombinant human CCN3 could be used therapeutically, and the mechanisms involved. Eight weeks of thrice-weekly i.p. injections (0.604 and 6.04 µg/kg of recombinant human CCN3) beginning in early-stage DN completely blocked and/or reversed the up-regulation of mRNA expression of kidney cortex fibrosis genes (CCN2, Col1a2, TGF-ß1, and PAI-1) seen in placebo-treated diabetic mice. The treatment completely blocked glomerular fibrosis, as determined by altered mesangial expansion and deposition of laminin. Furthermore, it protected against, or reversed, podocyte loss and kidney function reduction (rise in plasma creatinine concentration); albuminuria was also greatly reduced. This study demonstrates the potential efficacy of recombinant human CCN3 treatment in DN and points to mechanisms operating at multiple levels or pathways, upstream (eg, protecting against cell injury) and downstream (eg, regulating CCN2 activity and extracellular matrix metabolism).

Blood-brain barrier transport pathways for cytoprotective thiols.

The purpose of this study is to further define transport pathways for biological thiols by blood-brain barrier (BBB) endothelial cells, as a means of identifying endogenous cytoprotective mechanisms and potential therapeutic protocols for oxidative injury. Similar low-affininty, high-capacity passive carriers for glutathione (GSH) were observed at both the luminal (blood-facing) and abluminal (brain-facing) plasma membranes of BBB endothelial cells. These carriers are voltage dependent, favoring outward movement of intact peptide across both membrane domains, including efflux at the luminal plasmalemma where γ-glutamyl transpeptidase is located. Although present at both cell surfaces, the carriers are distributed unequally, with more appearing in the abluminal membrane. By contrast, high-affinity, low-capacity sodium-dependent GSH cotransport (Na-GSH) is observed only at the abluminal membrane, indicative of an inwardly directed active peptide carrier at the brain-facing plasma membrane. Treatment of cultured BBB endothelial cells with the GSH precursor γ-glutamyl-cysteine reduces cell damage under conditions simulating ischemia and reperfusion. These findings are consistent with the presence of (1) a typical γ-glutamyl cycle at the luminal membrane of BBB endothelial cells, (2) a significant efflux pathway at the abluminal membrane allowing passive movement of BBB GSH into brain extracellular fluid, (3) a Na-dependent, brain-to-blood pathway for transcellular transport of GSH, and (4) a mechanism for cytoprotection by γ-glutamyl cysteine, under conditions of ischemia and reperfusion.