Sanjana Dayal

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.

Protein Phosphatase 2A Methyltransferase Links Homocysteine Metabolism with Tau and Amyloid Precursor Protein Regulation

Alzheimers disease (AD) neuropathology is characterized by the accumulation of phosphorylated tau and amyloid-β peptides derived from the amyloid precursor protein (APP). Elevated blood levels of homocysteine are a significant risk factor for many age-related diseases, including AD. Impaired homocysteine metabolism favors the formation of S-adenosylhomocysteine, leading to inhibition of methyltransferase-dependent reactions. Here, we show that incubation of neuroblastoma cells with S-adenosylhomocysteine results in reduced methylation of protein phosphatase 2A (PP2A), a major brain Ser/Thr phosphatase, most likely by inhibiting PP2A methyltransferase (PPMT). PP2A methylation levels are also decreased after ectopic expression of PP2A methylesterase in Neuro-2a (N2a) cells. Reduced PP2A methylation promotes the downregulation of Bα-containing holoenzymes, thereby affecting PP2A substrate specificity. It is associated with the accumulation of both phosphorylated tau and APP isoforms and increased secretion of β-secretase-cleaved APP fragments and amyloid-β peptides. Conversely, incubation of N2a cells with S-adenosylmethionine and expression of PPMT enhance PP2A methylation. This leads to the accumulation of dephosphorylated tau and APP species and increased secretion of neuroprotective α-secretase-cleaved APP fragments. Remarkably, hyperhomocysteinemia induced in wild-type and cystathionine-β-synthase +/− mice by feeding a high-methionine, low-folate diet is associated with increased brain S-adenosylhomocysteine levels, PPMT downregulation, reduced PP2A methylation levels, and tau and APP phosphorylation. We reported previously that downregulation of neuronal PPMT and PP2A methylation occur in affected brain regions from AD patients. The link between homocysteine, PPMT, PP2A methylation, and key CNS proteins involved in AD pathogenesis provides new mechanistic insights into this disorder.

Cerebral Vascular Dysfunction Mediated by Superoxide in Hyperhomocysteinemic Mice

Background and Purpose— Hyperhomocysteinemia is an emerging risk factor for stroke, but little is known about effects of hyperhomocysteinemia on cerebral vascular function. We tested the hypothesis that chronic hyperhomocysteinemia in mice causes endothelial dysfunction in cerebral arterioles through a mechanism that involves superoxide. Methods— Mice heterozygous for a targeted disruption of the cystathionine β-synthase gene (Cbs +/−) and their wild type littermates (Cbs +/+) were fed either a control diet or a high-methionine diet for 10 to 12 months. Results— Plasma total homocysteine was elevated with the high-methionine diet compared with the control diet for both Cbs +/+ (7.9±1.0 versus 5.0±0.5 μmol/L; P <0.05) and Cbs +/− (20.5±3.1 versus 8.2±0.9 μmol/L; P <0.001) mice. Dilatation of cerebral arterioles (≈30 μm baseline diameter) was measured in vivo in response to the endothelium-dependent dilator acetylcholine or the endothelium-independent dilator nitroprusside. Vasodilatation to acetylcholine was impaired with the high-methionine diet compared with the control diet for both Cbs +/+ and Cbs +/− mice (P <0.01). Dilatation of arterioles to acetylcholine was restored toward normal by the superoxide scavenger tiron (P <0.05). Vasodilatation to nitroprusside was not influenced by Cbs genotype or diet. Dihydroethidium (DHE) staining for vascular superoxide was elevated in Cbs +/− mice fed the high-methionine diet and was inhibited by apocynin or Nω-nitro-l-arginine methyl ester, implicating NAD(P)H oxidase and nitric oxide synthase as potential sources of superoxide. Conclusions— Superoxide is a key mediator of endothelial dysfunction in the cerebral circulation during diet-induced hyperhomocysteinemia.

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.

Deficiency of Glutathione Peroxidase-1 Sensitizes Hyperhomocysteinemic Mice to Endothelial Dysfunction

Objective—We tested the hypothesis that deficiency of cellular glutathione peroxidase (GPx-1) enhances susceptibility to endothelial dysfunction in mice with moderate hyperhomocysteinemia. Methods and Results—Mice that were wild type (Gpx1+/+), heterozygous (Gpx1+/−), or homozygous (Gpx1−/−) for the mutated Gpx1 allele were fed a control diet or a high-methionine diet for 17 weeks. Plasma total homocysteine was elevated in mice on the high-methionine diet compared with mice on the control diet (23±3 versus 6±0.3 &mgr;mol/L, respectively;P <0.001) and was not influenced by Gpx1 genotype. In mice fed the control diet, maximal relaxation of the aorta in response to the endothelium-dependent dilator acetylcholine (10−5 mol/L) was similar in Gpx1+/+, Gpx1+/−, and Gpx1−/− mice, but relaxation to lower concentrations of acetylcholine was selectively impaired in Gpx1−/− mice (P <0.05 versus Gpx1+/+ mice). In mice fed the high-methionine diet, relaxation to low and high concentrations of acetylcholine was impaired in Gpx1−/− mice (maximal relaxation 73±6% in Gpx1−/− mice versus 90±2% in Gpx1+/+ mice, P <0.05). No differences in vasorelaxation to nitroprusside or papaverine were observed between Gpx1+/+ and Gpx1−/− mice fed either diet. Dihydroethidium fluorescence, a marker of superoxide, was elevated in Gpx1−/− mice fed the high-methionine diet (P <0.05 versus Gpx1+/+ mice fed the control diet). Conclusions—These findings demonstrate that deficiency of GPx-1 exacerbates endothelial dysfunction in hyperhomocysteinemic mice and provide support for the hypothesis that hyperhomocysteinemia contributes to endothelial dysfunction through a peroxide-dependent oxidative mechanism.

Hyperhomocysteinemia, endothelial dysfunction, and cardiovascular risk: the potential role of ADMA.

Hyperhomocysteinemia is an emerging risk factor for cardiovascular disease and stroke. The mechanisms underlying the pathophysiology of hyperhomocysteinemia are not completely defined, but endothelial dysfunction resulting from impaired bioavailability of nitric oxide is a consistent finding in experimental models. One potential mechanism for decreased nitric oxide bioavailability is inhibition of endothelial nitric oxide synthase by its endogenous inhibitor, asymmetric dimethylarginine (ADMA). Elevated plasma levels of ADMA have been found in association with hyperhomocysteinemia and endothelial dysfunction in both animals and humans. Additional studies are required to determine the mechanisms by which ADMA accumulates in hyperhomocysteinemia and to define the importance of ADMA in the endothelial dysfunction of hyperhomocysteinemia in vivo.

ADMA and hyperhomocysteinemia

Hyperhomocysteinemia is a risk factor for cardiovascular disease and stroke. Like many other cardiovascular risk factors, hyperhomocysteinemia produces endothelial dysfunction due to impaired bioavailability of endothelium-derived nitric oxide (NO). The molecular mechanisms responsible for decreased NO bioavailability in hyperhomocysteinemia are incompletely understood, but emerging evidence suggests that asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, may be a key mediator. Homocysteine is produced during the synthesis of ADMA and can alter ADMA metabolism by inhibiting dimethylarginine dimethylaminohydrolase (DDAH). Several animal and clinical studies have demonstrated a strong association between plasma total homocysteine, plasma ADMA, and endothelial dysfunction. These observations suggest a model in which elevation of ADMA may be a unifying mechanism for endothelial dysfunction during hyper-homocysteinemia. The recent development of transgenic mice with altered ADMA metabolism should provide further mechanistic insights into the role of ADMA in hyperhomocysteinemia.

Murine Models of Hyperhomocysteinemia and Their Vascular Phenotypes

Hyperhomocysteinemia is an established risk factor for arterial as well as venous thromboembolism. Individuals with severe hyperhomocysteinemia caused by inherited genetic defects in homocysteine metabolism have an extremely high incidence of vascular thrombosis unless they are treated aggressively with homocysteine-lowering therapy. The clinical value of homocysteine-lowering therapy in individuals with moderate hyperhomocysteinemia, which is very common in populations at risk for vascular disease, is more controversial. Considerable progress in our understanding of the molecular mechanisms underlying the association between hyperhomocysteinemia and vascular thrombotic events has been provided by the development of a variety of murine models. Because levels of homocysteine are regulated by both the methionine and folate cycles, hyperhomocysteinemia can be induced in mice through both genetic and dietary manipulations. Mice deficient in the cystathionine beta-synthase (CBS) gene have been exploited widely in many studies investigating the vascular pathophysiology of hyperhomocysteinemia. In this article, we review the established murine models, including the CBS-deficient mouse as well as several newer murine models available for the study of hyperhomocysteinemia. We also summarize the major vascular phenotypes observed in these murine models.

Glutathione Peroxidase-1 Plays a Major Role in Protecting Against Angiotensin II–Induced Vascular Dysfunction

Levels of reactive oxygen species, including hydrogen peroxide, increase in blood vessels during hypertension and in response to angiotensin II (Ang II). Although glutathione peroxidases are known to metabolize hydrogen peroxide, the role of glutathione peroxidase during hypertension is poorly defined. We tested the hypothesis that glutathione peroxidase-1 protects against Ang II–induced endothelial dysfunction. Responses of carotid arteries from Gpx1-deficient (Gpx1+/− and Gpx1−/−) and Gpx1 transgenic mice, and their respective littermate controls, were examined in vitro after overnight incubation with either vehicle or Ang II. Under control conditions, relaxation to acetylcholine (ACh; an endothelium-dependent agonist) was similar in control, Gpx1+/−, and Gpx1 transgenic mice, whereas in Gpx1−/− mice, responses to ACh were impaired. In control mice, ACh-induced vasorelaxation was not affected by 1 nmol/L of Ang II. In contrast, relaxation to ACh in arteries from Gpx1+/− mice was inhibited by ≈60% after treatment with 1 nmol/L of Ang II, indicating that Gpx1 haploinsufficiency markedly enhances Ang II–induced endothelial dysfunction. A higher concentration of Ang II (10 nmol/L) selectively impaired relaxation to ACh in arteries from control mice, and this effect was prevented in arteries from Gpx1 transgenic mice or in arteries from control mice treated with polyethylene glycol-catalase (which degrades hydrogen peroxide). Thus, genetic and pharmacological evidence suggests a major role for glutathione peroxidase-1 and hydrogen peroxide in Ang II–induced effects on vascular function.

Hydrogen Peroxide Promotes Aging-Related Platelet Hyperactivation and Thrombosis

Background— The incidence of thrombotic events increases during aging, but the mechanisms are not well understood. To investigate the prothrombotic role of oxidative stress during aging, we tested the hypothesis that aged mice overexpressing the antioxidant enzyme glutathione peroxidase-1 (Gpx1) are protected from experimental thrombosis. Methods and Results— Susceptibility to carotid artery thrombosis was first examined in wild-type C57BL/6J mice. After photochemical injury of the carotid artery, the time to stable occlusion was significantly shorter in 12- and 18-month-old mice compared with 4-month-old mice (P<0.01). Unlike wild-type mice, transgenic mice overexpressing Gpx1 (Gpx1 Tg) did not exhibit shortened times to occlusion of the carotid artery at 12 or 18 months of age. Wild-type mice also exhibited increased susceptibility to venous thrombosis after inferior vena cava ligation at 12 or 18 months of age (P<0.05 versus 4 months of age). Gpx1 Tg mice were protected from this aging-related enhanced susceptibility to venous thrombosis. Age-dependent platelet hyperactivation, evidenced by increased hydrogen peroxide, fibrinogen binding, and activation of fibrinogen receptor &agr;IIb&bgr;3, was observed in thrombin-activated platelets from wild-type but not Gpx1 Tg mice (P<0.05). Enhanced platelet activation responses in aged mice were also prevented by polyethylene glycol–catalase or apocynin, an inhibitor of NADPH oxidase. Aged mice displayed increased intraplatelet expression of p47phox and superoxide dismutase-1, suggesting a mechanistic pathway for increased hydrogen peroxide generation. Conclusions— Our findings demonstrate that hydrogen peroxide is a key mediator of platelet hyperactivity and enhanced thrombotic susceptibility in aged mice.