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-IkappaB-alpha) were assessed by immunohistochemical staining of these atherosclerotic lesions. GRP78/94, HSP70, and phospho-IkappaB-alpha 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-IkappaB-alpha were observed in the lipid-rich necrotic core. Increased HSP70 and phospho-IkappaB-alpha 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.