Endoplasmic reticulum stress and glycogen synthase kinase-3ß activation in apolipoprotein E-deficient mouse models of accelerated atherosclerosis.

OBJECTIVE: The goal of this study was to examine the role of endoplasmic reticulum (ER) stress signaling and the contribution of glycogen synthase kinase (GSK)-3ß activation in hyperglycemic, hyperhomocysteinemic, and high-fat-fed apolipoprotein E-deficient (apoE(-/-)) mouse models of accelerated atherosclerosis. METHODS AND RESULTS: Female apoE(-/-) mice received multiple low-dose injections of streptozotocin (40 µg/kg) to induce hyperglycemia, methionine-supplemented drinking water (0.5% wt/vol) to induce hyperhomocysteinemia, or a high-fat (21% milk fat+0.2% cholesterol) diet to induce relative dyslipidemia. A subset of mice from each group was supplemented with sodium valproate (625 mg/kg), a compound with GSK3 inhibitory activity. At 15 and 24 weeks of age, markers of ER stress, lipid accumulation, GSK3ß phosphorylation, and GSK3ß activity were analyzed in liver and aorta. Atherosclerotic lesions were examined and quantified. Hyperglycemia, hyperhomocysteinemia, and high-fat diet significantly enhanced GSK3ß activity and also increased hepatic steatosis and atherosclerotic lesion volume compared with controls. Valproate supplementation blocked GSK3ß activation and attenuated the development of atherosclerosis and the accumulation of hepatic lipids in each of the models examined. The mechanism by which GSK3ß activity is regulated in these models likely involves alterations in phosphorylation at serine 9 and tyrosine 216. CONCLUSIONS: These findings support the existence of a common mechanism of accelerated atherosclerosis involving ER stress signaling through activation of GSK3ß. Furthermore, our results suggest that atherosclerosis can be attenuated by modulating GSK3ß phosphorylation.

Valproate attenuates accelerated atherosclerosis in hyperglycemic apoE-deficient mice: evidence in support of a role for endoplasmic reticulum stress and glycogen synthase kinase-3 in lesion development and hepatic steatosis.

We have previously shown that glucosamine promotes endoplasmic reticulum (ER) stress in vascular cells leading to both inflammation and lipid accumulation--the hallmark features of atherosclerosis. Pretreatment with glycogen synthase kinase (GSK)-3 inhibitors protects cultured cells from ER stress-induced dysfunction. Here we evaluate the potential role of GSK-3 on the pro-atherogenic effects of hyperglycemia and ER stress. We show that GSK-3-deficient mouse embryonic fibroblasts do not accumulate unesterified cholesterol under conditions of ER stress. Furthermore, GSK-3 inhibitors, including valproate, attenuate ER stress-induced unesterified cholesterol accumulation in wild-type mouse embryonic fibroblasts. In vivo we show that hyperglycemic apoE-deficient mice have accelerated atherogenesis at the aortic root compared with normoglycemic control mice. Mice fed a diet supplemented with 625 mg/kg valproate have significantly reduced lesion volume relative to nonsupplemented controls. Valproate supplementation has no apparent effect on the plasma levels of either glucose or lipids or on the expression of diagnostic markers of ER stress in the lesion. Significant reductions were observed in total hepatic lipids (>50.4%) and hepatic GSK-3beta activity (>55.8%) in mice fed the valproate diet. In conclusion, dietary supplementation with low levels of valproate significantly attenuates atherogenesis in hyperglycemic apoE-deficient mice. The in vivo anti-atherogenic effects of valproate are consistent with its ability to inhibit GSK-3 and interfere with pro-atherogenic ER stress signaling pathways in vitro.

Diabetes, hyperglycemia and accelerated atherosclerosis: evidence supporting a role for endoplasmic reticulum (ER) stress signaling.

Diabetes mellitus is associated with both micro- and macrovascular complications that can lead to significantly elevated incidence of retinopathy, nephropathy, neuropathy, myocardial infarction and stroke. The diabetic cardiovascular mortality rate exceeds 70% and individuals with diabetes are 2-3 times more likely to die from myocardial infarction and stroke than those with no history of diabetes even after controlling for other cardiovascular risk factors. Despite the profound clinical importance of vascular disease in patients with diabetes mellitus, our understanding of the molecular and cellular mechanisms by which diabetes promotes these vascular complications is incomplete. Endoplasmic reticulum (ER) stress and the unfolded protein response pathways have been previously associated with the development of several different diseases, including neurodegenerative disorders, cancer, and obesity. In addition, ER stress has been directly implicated in complications that are associated with diabetes, including pancreatic b cell dysfunction and insulin resistance. In this review we examine the potential role of endoplasmic reticulum stress in the initiation and progression of hyperglycemia-associated atherosclerosis.

Evidence supporting a role for endoplasmic reticulum stress in the development of atherosclerosis in a hyperglycaemic mouse model.

We previously observed a correlation between elevated levels of vascular endoplasmic reticulum (ER) stress and accelerated atherosclerotic plaque development in chronically hyperglycemic apolipoprotein-deficient (ApoE(-/-)) mice. We hypothesize that ER stress plays a causative role in diabetic atherogenesis. Here we examine the temporal relation between the onset of hyperglycemia, glucosamine accumulation in the vessel wall, ER stress, and the development of atherosclerosis. We demonstrate, by using streptozotocin-induced hyperglycemic ApoE(-/-) mice, that conditions of hyperglycemia increase intracellular glucosamine levels and endothelial ER stress levels in the endothelium before the onset of atherosclerosis. At 15 weeks of age, hyperglycemic mice have significantly larger atherosclerotic lesions (0.120 +/- 0.023 vs. 0.065 +/- 0.021 mm2; p = 0.001) relative to normoglycemic mice. Significantly, hyperglycemia-associated accelerated atherosclerosis is observed before the onset of dyslipidemias, suggesting that leveled glucose is sufficient to promote atherogenesis independently. Diagnostic markers of elevated ER-stress levels are increased in macrophage-derived foam cells in early and advanced atherosclerotic lesions. Dietary supplementation with valproate, a small branched-chain fatty acid that interferes with ER-stress signaling, significantly attenuates accelerated atherogenesis in this model. Together, these data are consistent with a causative role for hyperglycemia-associated ER stress in the development and progression of diabetic atherosclerosis.

Induction of GRP78 by valproic acid is dependent upon histone deacetylase inhibition.

Valproic (2-propylpentanoic) acid is a commonly used drug in the treatment of bipolar disorder and epilepsy. The molecular mechanism that underlies its clinical efficacy remains controversial and is complicated by the broad range of intracellular effects of valproic acid, including its ability to inhibit histone deacetylase (HDAC) and induce protein chaperone expression. Here we show that an established HDAC inhibitor, trichostatin A, promotes ER chaperone expression in HEK293 cells. Furthermore, we use chemical derivatives of valproic acid to show that the ability to promote GRP78 levels directly correlates with the induction of histone H4 hyperacetylation. These results suggest that exposure to valproic acid enhances chaperone expression by a mechanism that involves histone hyperacetylation.