Šárka Lhoták

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.

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.

Mouse models of cystathionine β-synthase deficiency reveal significant threshold effects of hyperhomocysteinemia

Untreated cystathionine β‐synthase (CBS) deficiency in humans is characterized by extremely elevated plasma total homocysteine (tHcy>200 µΜ), with thrombosis as the major cause of morbidity. Treatment with vitamins and diet leads to a dramatic reduction in thrombotic events, even though patients often still have severe elevations in tHcy (>80 µΜ). To understand the difference between extreme and severe hyperhomocysteinemia, we have examined two mouse models of CBS deficiency: Tg‐hCBS Cbs–/– mice, with a mean serum tHcy of 169 µΜ, and Tg‐I278T Cbs–/– mice, with a mean tHcy of 296 µΜ. Only Tg‐I278T Cbs–/– animals exhibited strong biological phenotypes, including facial alopecia, osteoporosis, endoplasmic reticulum (ER) stress in the liver and kidney, and a 20% reduction in mean survival time. Metabolic profiling of serum and liver reveals that Tg‐I278T Cbs–/– mice have significantly elevated levels of free oxidized homocysteine but not protein‐bound homocysteine in serum and elevation of all forms of homocysteine and S‐adenosyl‐homocysteine in the liver compared to Tg‐hCBS Cbs–/– mice. RNA profiling of livers indicate that Tg‐I278T Cbs–/– and Tg‐hCBS Cbs–/– mice have unique gene signatures, with minimal overlap. Our results indicate that there is a clear pathogenic threshold effect for tHcy and bring into question the idea that mild elevations in tHcy are directly pathogenic.— Gupta, S., Kuhnisch, J., Mustafa, A., Lhotak, S., Schlachterman, A., Slifker, M. J., Klein‐Szanto, A., High, K. A., Austin, R. C., Kruger, W. D. Mouse models of cystathionine β‐synthase deficiency reveal significant threshold effects of hyperhomocysteinemia. FASEB J. 23, 883–893 (2009)

Electrical stimulation of the vagus nerve modulates the histamine content of mast cells in the rat jejunal mucosa.

Mast cells are best known for their participation in allergic reactions. However, a number of recent studies suggest that mast cells are subject to nervous control. In the gut mucosa, mast cells are intimately associated with nerves, and the psychologically conditioned release of RMCP II (a mucosal mast cell-derived mediator) has been reported. These data suggest the potential for CNS regulation of intestinal mucosal mast cells. In this study, we stimulated the cervical vagi and found an increased histamine content in mucosal mast cells, without apparent degranulation. Furthermore, these changes could be prevented by subdiaphragmatic vagotomy. These data support the potential for intestinal mucosal mast cell regulation by the central nervous system and suggest modulation of mast cells without degranulation.

Prothrombotic Effects of Hyperhomocysteinemia and Hypercholesterolemia in ApoE-Deficient Mice

Objective—We tested the hypothesis that hyperhomocysteinemia and hypercholesterolemia promote arterial thrombosis in mice. Methods and Results—Male apolipoprotein E (Apoe)-deficient mice were fed one of four diets: control, hyperhomocysteinemic (HH), high fat (HF), or high fat/hyperhomocysteinemic (HF/HH). Total cholesterol was elevated 2-fold with the HF or HF/HH diets compared with the control or HH diets (P<0.001). Plasma total homocysteine (tHcy) was elevated (12 to 15 &mgr;mol/L) with the HH or HF/HH diets compared with the control or HF diets (4 to 6 &mgr;mol/L; P<0.001). Aortic sinus lesion area correlated strongly with total cholesterol (P<0.001) but was independent of tHcy. At 12 weeks of age, the time to thrombotic occlusion of the carotid artery after photochemical injury was >50% shorter in mice fed the HF diets, with or without hyperhomocysteinemia, compared with the control diet (P<0.05). At 24 weeks of age, carotid artery thrombosis was also accelerated in mice fed the HH diet (P<0.05). Endothelium-dependent nitric oxide–mediated relaxation of carotid artery rings was impaired in mice fed the HF, HH, or HF/HH diets compared with the control diet (P<0.05). Conclusions—Hyperhomocysteinemia and hypercholesterolemia, alone or in combination, produce endothelial dysfunction and increased susceptibility to thrombosis in Apoe-deficient mice.

Immunohistochemical detection of N-homocysteinylated proteins in humans and mice.

N-homocysteinylation of epsilon-amino group of protein lysine residues by homocysteine (Hcy) thiolactone has been implicated in vascular disease in humans. We have previously generated polyclonal rabbit anti-N-Hcy-protein IgG antibodies that specifically recognize the Nepsilon-Hcy-Lys epitope on N-homocysteinylated proteins. The present work was undertaken to examine the utility of these antibodies for the immunohistochemical detection of N-homocysteinylated proteins in biological samples. We found that the rabbit antibody specifically detected N-Hcy-protein in a dot-blot assay, that the signal resulting from the reaction of the antibody with N-Hcy-protein depended on the amount of the antigen, and that the sensitivity of the assay was protein-dependent. The rabbit anti-N-Hcy-protein IgG also specifically detected Nepsilon-Hcy-Lys epitopes in human tissues, as shown by positive immunohistochemical staining of myocardium and aorta samples from cardiac surgery patients, and a lack of staining when the antibody was pre-adsorbed with N-Hcy-albumin. We also observed increased immunohistochemical staining for N-Hcy-proteins in aortic lesions from ApoE-/- mice with hyperhomocysteinemia induced by a high methionine diet, relative to ApoE-/- mice fed a control chow diet. In conclusion, polyclonal rabbit anti-N-Hcy-protein antibody can detect and monitor N-homocysteinylated proteins in human and mouse tissues with good sensitivity and specificity.

Hyperhomocysteinemia induced by methionine supplementation does not independently cause atherosclerosis in C57BL/6J mice

A causal relationship between diet‐induced hyperhomocysteinemia (HHcy) and accelerated atherosclerosis has been established in apolipoprotein E‐deficient (apoE−/−) mice. However, it is not known whether the proatherogenic effect of HHcy in apoE−/− mice is independent of hyperlipidemia and/or deficiency of apoE. In this study, a comprehensive dietary approach using C57BL/6J mice was used to investigate whether HHcy is an independent risk factor for accelerated atherosclerosis or dependent on additional dietary factors that increase plasma lipids and/or inflam mation. C57BL/6J mice at 4 wk of age were divided into 6 dietary groups: chow diet (C), chow diet + methionine (C+M), western‐type diet (W), western‐type diet + methionine (W+M), atherogenic diet (A), or atherogenic diet + methionine (A+M). After 2, 10, 20, or 40 wk on the diets, mice were sacrificed, and the levels of total plasma homocysteine, cysteine, and glutathione, as well as total plasma cholesterol and triglycerides were analyzed. Aortic root sections were examined for atherosclerotic lesions. HHcy was induced in all groups supplemented with methionine, compared to diet‐matched control groups. Plasma total cholesterol was significantly increased in mice fed the W or A diet. However, the W diet increased LDL/IDL and HDL levels, while the A diet significantly elevated plasma VLDL and LDL/IDL levels without increasing HDL. No differences in plasma total cholesterol levels or lipid profiles were observed between methionine‐supplemented groups and the diet‐matched control groups. Early atherosclerotic lesions containing macrophage foam cells were only observed in mice fed the A or A + M diet. Furthermore, lesion size was significantly larger in the A + M group compared to the A group at 10 and 20 wk; however, mature lesions were never observed even after 40 wk on these diets. The presence of lymphocytes, increased hyaluronan staining, and the expression of endoplasmic reticulum (ER) stress markers were also increased in atherosclerotic lesions from the A + M group. Taken together, these results suggest that HHcy does not independently cause atherosclerosis in C57BL/6J mice even in the presence of increased total plasma lipids induced by the W diet. However, HHcy can accelerate atherosclerotic lesion development under dietary conditions that increase plasma VLDL levels and/or inflammation.—Zhou, J., Werstuck, G. H., Lhoták, Š., Shi, Y. Y., Tedesco, V., Trigatti, B., Dickhout, J., Majors, A. K., DiBello, P. M., Jacobsen, D. W., Austin, R. C. Hyperhomocysteinemia induced by methionine supplementation does not independently cause atherosclerosis in C57BL/6J mice. FASEB J. 22, 2569–2578 (2008)