Richard C. Austin

Homocysteine-induced endoplasmic reticulum stress causes dysregulation of the cholesterol and triglyceride biosynthetic pathways

Hepatic steatosis is common in patients having severe hyperhomocysteinemia due to deficiency for cystathionine beta-synthase. However, the mechanism by which homocysteine promotes the development and progression of hepatic steatosis is unknown. We report here that homocysteine-induced endoplasmic reticulum (ER) stress activates both the unfolded protein response and the sterol regulatory element-binding proteins (SREBPs) in cultured human hepatocytes as well as vascular endothelial and aortic smooth muscle cells. Activation of the SREBPs is associated with increased expression of genes responsible for cholesterol/triglyceride biosynthesis and uptake and with intracellular accumulation of cholesterol. Homocysteine-induced gene expression was inhibited by overexpression of the ER chaperone, GRP78/BiP, thus demonstrating a direct role of ER stress in the activation of cholesterol/triglyceride biosynthesis. Consistent with these in vitro findings, cholesterol and triglycerides were significantly elevated in the livers, but not plasmas, of mice having diet-induced hyperhomocysteinemia. This effect was not due to impaired hepatic export of lipids because secretion of VLDL-triglyceride was increased in hyperhomocysteinemic mice. These findings suggest a mechanism by which homocysteine-induced ER stress causes dysregulation of the endogenous sterol response pathway, leading to increased hepatic biosynthesis and uptake of cholesterol and triglycerides. Furthermore, this mechanism likely explains the development and progression of hepatic steatosis and possibly atherosclerotic lesions observed in hyperhomocysteinemia.

Role of hyperhomocysteinemia in endothelial dysfunction and atherothrombotic disease

AbstractHyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease, including ischemic heart disease, stroke, and peripheral vascular disease. Mutations in the enzymes responsible for homocysteine metabolism, particularly cystathionine β-synthase (CBS) or 5,10-methylenetetrahydrofolate reductase (MTHFR), result in severe forms of HHcy. Additionally, nutritional deficiencies in B vitamin cofactors required for homocysteine metabolism, including folic acid, vitamin B6 (pyridoxal phosphate), and/or B12 (methylcobalamin), can induce HHcy. Studies using animal models of genetic- and diet-induced HHcy have recently demonstrated a causal relationship between HHcy, endothelial dysfunction, and accelerated atherosclerosis. Dietary enrichment in B vitamins attenuates these adverse effects of HHcy. Although oxidative stress and activation of proinflammatory factors have been proposed to explain the atherogenic effects of HHcy, recent in vitro and in vivo studies demonstrate that HHcy induces endoplasmic reticulum (ER) stress, leading to activation of the unfolded protein response (UPR). This review summarizes the current role of HHcy in endothelial dysfunction and explores the cellular mechanisms, including ER stress, that contribute to atherothrombosis.

Activation of the Unfolded Protein Response Occurs at All Stages of Atherosclerotic Lesion Development in Apolipoprotein E–Deficient Mice

Background—Apoptotic cell death contributes to atherosclerotic lesion instability, rupture, and thrombogenicity. Recent findings suggest that free cholesterol (FC) accumulation in macrophages induces endoplasmic reticulum (ER) stress/unfolded protein response (UPR) and apoptotic cell death; however, it is not known at what stage of lesion development the UPR is induced in macrophages or whether a correlation exists between UPR activation, FC accumulation, and apoptotic cell death. Methods and Results—Aortic root sections from apolipoprotein E–deficient (apoE−/−) mice at 9 weeks of age (early-lesion group) or 23 weeks of age (advanced-lesion group) fed a standard chow diet were examined for markers of UPR activation (GRP78, phospho-PERK, CHOP, and TDAG51), apoptotic cell death (TUNEL and cleaved caspase-3), and lipid accumulation (filipin and oil red O). UPR markers were dramatically increased in very early intimal macrophages and in macrophage foam cells from fatty streaks and advanced atherosclerotic lesions. Although accumulation of FC was observed in early-lesion–resident macrophage foam cells, no evidence of apoptotic cell death was observed; however, UPR activation, FC accumulation, and apoptotic cell death were observed in a small percentage of advanced-lesion–resident macrophage foam cells. Conclusions—UPR activation occurs at all stages of atherosclerotic lesion development. The additional finding that macrophage apoptosis did not correlate with UPR activation and FC accumulation in early-lesion–resident macrophages suggests that activation of other cellular mediators and/or pathways are required for apoptotic cell death.

Hyperhomocysteinemia and its role in the development of atherosclerosis

Numerous epidemiological studies have demonstrated that hyperhomocysteinemia (HHcy) is a strong and independent risk factor for cardiovascular disease. HHcy can result from a deficiency in the enzymes or vitamin cofactors required for homocysteine metabolism. Several hypotheses have been proposed to explain the cellular mechanisms by which HHcy promotes cardiovascular disease, including oxidative stress, endoplasmic reticulum (ER) stress and the activation of pro-inflammatory factors. Studies using genetic- and diet-induced animal models of HHcy have now demonstrated a direct causal relationship between HHcy, endothelial dysfunction and accelerated atherosclerosis. These recently established animal models of HHcy provide investigators with important in vivo tools to (i) further understand the cellular mechanisms by which HHcy contributes to endothelial dysfunction and atherosclerosis, and (ii) develop therapeutic agents useful in the treatment of cardiovascular disease.

Peroxynitrite Causes Endoplasmic Reticulum Stress and Apoptosis in Human Vascular Endothelium: Implications in Atherogenesis

Objective—Peroxynitrite, a potent oxidant generated by the reaction of NO with superoxide, has been implicated in the promotion of atherosclerosis. We designed this study to determine whether peroxynitrite induces its proatherogenic effects through induction of endoplasmic reticulum (ER) stress. Methods and Results—Human vascular endothelial cells treated with Sin-1, a peroxynitrite generator, induced the expression of the ER chaperones GRP78 and GRP94 and increased eIF2&agr; phosphorylation. These effects were inhibited by the peroxynitrite scavenger uric acid. Sin-1 caused the depletion of ER–Ca2+, an effect known to induce ER stress, resulting in the elevation of cytosolic Ca2+ and programmed cell death (PCD). Sin-1 treatment was also found, via 3-nitrotyrosine and GRP78 colocalization, to act directly on the ER. Adenoviral-mediated overexpression of GRP78 in endothelial cells prevented Sin-1–induced PCD. Consistent with these in vitro findings, 3-nitrotyrosine was observed and colocalized with GRP78 in endothelial cells of early atherosclerotic lesions from apolipoprotein E–deficient mice. Conclusions—Peroxynitrite is an ER stress-inducing agent. Its effects include the depletion of ER–Ca2+, a known mechanism of ER stress induction. The observation that 3-nitrotyrosine–containing proteins colocalize with markers of ER stress within early atherosclerotic lesions suggests that peroxynitrite contributes to atherogenesis through a mechanism involving ER stress.

Decreased Endogenous Production of Hydrogen Sulfide Accelerates Atherosclerosis

Background— Cystathionine &ggr;-lyase (CSE) produces hydrogen sulfide (H2S) in the cardiovascular system. The deficiency of CSE in mice leads to a decreased endogenous H2S level, an age-dependent increase in blood pressure, and impaired endothelium-dependent vasorelaxation. To date, there is no direct evidence for a causative role of altered metabolism of endogenous H2S in atherosclerosis development. Methods and Results— Six-week-old CSE gene knockout and wild-type mice were fed with either a control chow or atherogenic paigen-type diet for 12 weeks. Plasma lipid profile and homocysteine levels, blood pressure, oxidative stress, atherosclerotic lesion size in the aortic roots, cell proliferation, and adhesion molecule expression were then analyzed. CSE-knockout mice fed with atherogenic diet developed early fatty streak lesions in the aortic root, elevated plasma levels of cholesterol and low-density lipoprotein cholesterol, hyperhomocysteinemia, increased lesional oxidative stress and adhesion molecule expression, and enhanced aortic intimal proliferation. Treatment of CSE-knockout mice with NaHS, but not N-acetylcysteine or ezetimibe, inhibited the accelerated atherosclerosis development. Double knockout of CSE and apolipoprotein E gene expression in mice exacerbated atherosclerosis development more than that in the mice with only apolipoprotein E or CSE knockout. Conclusions— Endogenously synthesized H2S protects vascular tissues from atherogenic damage by reducing vessel intimal proliferation and inhibiting adhesion molecule expression. Decreased endogenous H2S production predisposes the animals to vascular remodeling and early development of atherosclerosis. The CSE/H2S pathway is an important therapeutic target for protection against atherosclerosis.

The chemical chaperone 4-phenylbutyrate inhibits adipogenesis by modulating the unfolded protein response

Recent studies have shown a link between obesity and endoplasmic reticulum (ER) stress. Perturbations in ER homeostasis cause ER stress and activation of the unfolded protein response (UPR). Adipocyte differentiation contributes to weight gain, and we have shown that markers of ER stress/UPR activation, including GRP78, phospho-eIF2α, and spliced XBP1, are upregulated during adipogenesis. Given these findings, the objective of this study was to determine whether attenuation of UPR activation by the chemical chaperone 4-phenylbutyrate (4-PBA) inhibits adipogenesis. Exposure of 3T3-L1 preadipocytes to 4-PBA in the presence of differentiation media decreased expression of ER stress markers. Concomitant with the suppression of UPR activation, 4-PBA resulted in attenuation of adipogenesis as measured by lipid accumulation and adiponectin secretion. Consistent with these in vitro findings, female C57BL/6 mice fed a high-fat diet supplemented with 4-PBA showed a significant reduction in weight gain and had reduced fat pad mass, as compared with the high-fat diet alone group. Furthermore, 4-PBA supplementation decreased GRP78 expression in the adipose tissue and lowered plasma triglyceride, glucose, leptin, and adiponectin levels without altering food intake. Taken together, these results suggest that UPR activation contributes to adipogenesis and that blocking its activation with 4-PBA prevents adipocyte differentiation and weight gain in mice.

Association of Multiple Cellular Stress Pathways With Accelerated Atherosclerosis in Hyperhomocysteinemic Apolipoprotein E-Deficient Mice

Background—A causal relation between hyperhomocysteinemia (HHcy) and accelerated atherosclerosis has been established in apolipoprotein E–deficient (apoE−/−) mice. Although several cellular stress mechanisms have been proposed to explain the atherogenic effects of HHcy, including oxidative stress, endoplasmic reticulum (ER) stress, and inflammation, their association with atherogenesis has not been completely elucidated. Methods and Results—ApoE−/− mice were fed a control or a high-methionine (HM) diet for 4 (early lesion group) or 18 (advanced lesion group) weeks to induce HHcy. Total plasma homocysteine levels and atherosclerotic lesion size were significantly increased in early and advanced lesion groups fed the HM diet compared with control groups. Markers of ER stress (GRP78/94, phospho-PERK), oxidative stress (HSP70), and inflammation (phospho-IκB-&agr;) were assessed by immunohistochemical staining of these atherosclerotic lesions. GRP78/94, HSP70, and phospho-IκB-&agr; immunostaining were significantly increased in the advanced lesion group fed the HM diet compared with the control group. HSP47, an ER-resident molecular chaperone involved in collagen folding and secretion, was also increased in advanced lesions of mice fed the HM diet. GRP78/94 and HSP47 were predominantly localized to the smooth muscle cell–rich fibrous cap, whereas HSP70 and phospho-IκB-&agr; were observed in the lipid-rich necrotic core. Increased HSP70 and phospho-IκB-&agr; immunostaining in advanced lesions of mice fed the HM diet are consistent with enhanced carotid artery dihydroethidium staining. Interestingly, GRP78/94 and phospho-PERK were markedly increased in macrophage foam cells from early lesions of mice fed the control or the HM diet. Conclusions—Multiple cellular stress pathways, including ER stress, are associated with atherosclerotic lesion development in apoE−/− mice.

Interrelationship Between Cardiac Hypertrophy, Heart Failure, and Chronic Kidney Disease: Endoplasmic Reticulum Stress As a Mediator of Pathogenesis

Synthesis of transmembrane and secretory proteins occurs within the endoplasmic reticulum (ER) and is extremely important in the normal functioning of both the heart and kidney. The dysregulation of protein synthesis/processing within the ER causes the accumulation of unfolded proteins, thereby leading to ER stress and the activation of the unfolded protein response. Sarcoplasmic reticulum/ER Ca2+ disequilibrium can lead to cardiac hypertrophy via cytosolic Ca2+ elevation and stimulation of the Ca2+/calmodulin, calcineurin, NF-AT3 pathway. Although cardiac hypertrophy may be initially adaptive, prolonged or severe ER stress resulting from the increased protein synthesis associated with cardiac hypertrophy can lead to apoptosis of cardiac myocytes and result in reduced cardiac output and chronic heart failure. The failing heart has a dramatic effect on renal function because of inadequate perfusion and stimulates the release of many neurohumoral factors that may lead to further ER stress within the heart, including angiotensin II and arginine-vasopressin. Renal failure attributable to proteinuria and uremia also induces ER stress within the kidney, which contributes to the transformation of tubular epithelial cells to a fibroblast-like phenotype, fibrosis, and tubular cell apoptosis, further diminishing renal function. As a consequence, cardiorenal syndrome may develop into a vicious circle with poor prognosis. New therapeutic modalities to alleviate ER stress through stimulation of the cytoprotective components of the unfolded protein response, including GRP78 upregulation and eukaryotic initiation factor 2α phosphorylation, may hold promise to reduce the high morbidity and mortality associated with cardiorenal syndrome.

Contributions of hyperhomocysteinemia to atherosclerosis: Causal relationship and potential mechanisms

Hyperhomocysteinemia (HHcy) is considered an independent risk factor for cardiovascular disease, including ischemic heart disease, stroke, and peripheral vascular disease. Mutations in the enzymes and/or nutritional deficiencies in B vitamins required for homocysteine metabolism can induce HHcy. Studies using genetic‐ or diet‐induced animal models of HHcy have demonstrated a causal relationship between HHcy and accelerated atherosclerosis. Oxidative stress and activation of proinflammatory factors have been proposed to explain the atherogenic effects of HHcy. Recently, HHcy‐induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been found to play a role in HHcy‐induced atherogenesis. This review will focus on the cellular mechanisms of HHcy in atherosclerosis from both in vivo and in vitro studies. The contributions of ER stress and the UPR in atherogenesis will be emphasized. Results from recent clinical trials assessing the cardiovascular risk of lowering total plasma homocysteine levels and new findings examining the atherogenic role of HHcy in wild‐type C57BL/6J mice will also be discussed.