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Objective— The pathogenic role of macrophage apoptosis in atherosclerosis is still debatable, but it is considered to be a suppressor of plaque progression in early stages but a promoter of plaque necrosis in advanced stages. Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that plays a pivotal role in stress-induced apoptosis. In the current study, we investigated the functions of ASK1 in hyperlipidemia-induced atherosclerosis. Methods and Results— We generated ASK1 and apolipoprotein E (apoE) double-knockout mice (ASK1 −/− /apoE −/− ) and analyzed atherosclerosis in ASK1 −/− /apoE −/− mice fed a high-cholesterol diet for 12 weeks. ASK1 −/− /apoE −/− mice had accelerated hyperlipidemia-induced atherosclerosis, which was characterized by less apoptosis of macrophages and fewer necrotic areas, and more macrophages and elastolysis compared with apoE −/− mice. Bone marrow transplantation from ASK1 −/− or wild-type to apoE −/− mice confirmed the above observation that the recipient mice of ASK1 −/− donors had more pronounced hyperlipidemia-induced atherosclerosis than recipient mice of wild-type donors. Conclusion— These findings suggest that ASK1 suppresses hyperlipidemia-induced atherosclerosis via increased macrophage apoptosis and that ASK1 may cause pronounced plaque vulnerability via necrotic core development.

Introduction: CCL22 (macrophage-derived chemokine) is a member of the CC-family of chemokines. The functional role of CCL22 as a chemokine involves migration/ recruitment of monocytes and the stimulation of platelet activity. In this study, we investigated the relationship between CCL22 and atherosclerosis in monocytes/ macrophages of mice and humans with respect to atherosclerosis. Hypothesis: We assessed the hypothesis that CCL22 is involved in the process of atherosclerosis and can be a biological marker for progression of atherosclerosis and atherosclerotic disease. Methods: Apolipoprotein E deficient (apoE-/-) mice as atherosclerotic models were used for this study. Male apoE-/- mice were weaned at 8 weeks of age onto a high-cholesterol diet consisting of 1.25% cholesterol and were maintained on this diet for 12 weeks or 24 weeks. Another group of apoE-/- mice was maintained for 30 weeks on a normal chow diet. Serum CCL22 concentrations were measured by ELISA and expression of CCL22 mRNA in the atheroma were evaluated by real-time PCR. Furthermore immunohistochemical staining was performed. In vitro, the expressions of CCL22 in the mouse monocytes and macrophages were examined and discussed. The expression of CCL22 human monocytes/ macrophages and human atherosclerotic lesions were similarly investigated. Results: ApoE-/- mice had predominately high concentrations of CCL22 compared with wild-type (C57BL/6J) mice. ApoE-/- mice developed atherosclerotic lesions with aging and this was influenced by a high-cholesterol diet. CCL22 concentrations increased with aging and a high-cholesterol diet. Their atherosclerotic lesions, especially in macrophages contained abundant levels of CCL22. When the mouse monocyte differentiated into macrophage, the cells showed similar expression of CCL22. Similarly, human macrophages also expressed abundant levels of CCL22 more than monocytes. In addition, immunohistochemical analysis of human atherosclerotic lesions and coronary stenotic lesion revealed that macrophages express CCL22. Conclusion: In conclusion, these results suggested that this molecule is involved in the atherogenic processes and CCL22 may represent an attractive molecular marker for progression of atherosclerosis.

Objective— To study the effect of granulocyte macrophage–colony-stimulating factor (GM-CSF) on histamine metabolism in arteriosclerosis, the expression of histidine decarboxylase (HDC; histamine-producing enzyme), histamine receptors 1 and 2 (HH1R and HH2R), and GM-CSF was investigated in human and mouse arteriosclerotic carotid arteries. Furthermore, the molecular mechanisms of GM-CSF–induced HDC and HH1R expression in monocytic U937 cells were investigated. Methods and Results— Immunohistochemistry showed that atherosclerotic human coronary and mouse ligated carotid arteries contained HDC-expressing macrophages. Gene expression of HDC, HH1R, HH2R, and GM-CSF was also detected in the lesions. In U937 cells, GM-CSF enhanced histamine secretion and gene expression of HDC and HH1R. A promoter assay showed that GM-CSF enhanced gene transcription of HDC and HH1R but not HH2R. Conclusion— The present results indicate that HDC and HHR are expressed in arteriosclerotic lesion, and that GM-CSF induces HDC and HH1R expression in monocytes. Locally produced histamine might participate in atherogenesis by affecting the expression of atherosclerosis-related genes in monocytes and smooth muscle cells.

Objective— Histamine and histamine receptors are found in atherosclerotic lesions, and their signaling and subsequent proatherogenic or proinflammatory gene expression are involved in atherogenesis. In the present study, we generated apolipoprotein E (apoE) and histamine synthesizing histidine decarboxylase double knockout (DKO) mice on a C57BL/6J (wild-type mice) background to clarify the roles of histamine in atherosclerosis. Methods and Results— Wild-type, apoE knockout (KO), and DKO mice were fed a high-cholesterol diet to analyze hyperlipidemia-induced atherosclerosis. Compared with wild-type mice, apoE-KO mice showed increased expression of histamine and its receptors, corresponding to increased atherosclerotic lesion areas and expression of inflammatory regulators, such as nuclear factor-κB, scavenger receptors, inflammatory cytokines, and matrix metalloproteinases. Histamine deficiency after deletion of histidine decarboxylase reduced atherosclerotic areas and expression of a range of the inflammation regulatory genes, but serum cholesterol levels of DKO mice were higher than those of apoE-KO mice. Conclusion— These results indicate that histamine is involved in the development of atherosclerosis in apoE-KO mice by regulating gene expression of inflammatory modulators, an action that appears to be independent of serum cholesterol levels. In addition to acute inflammatory response, histamine participates in chronic inflammation, such as hyperlipidemia-induced atherosclerosis, and might be a novel therapeutic target for the treatment of atherosclerosis.

Objective— Histamine increases endothelial nitric oxide (NO) production as an endothelium-dependent vasodilator, which acts as a vasoconstrictor in atherosclerotic coronary arteries. To investigate the relation between histamine and NO production in intimal smooth muscle cells (SMCs), we studied the effect of histamine on inducible NO synthase (iNOS) expression in the SMCs. Methods and Results— In cultured human intimal SMCs, histamine increased NO production, iNOS expression, and NF-κB nuclear translocation, which were inhibited by histamine H1 blocker and NF-κB inhibitor. Luciferase assay using −8.3 kb upstream of human iNOS promoter region and electrophoretic mobility shift assay suggested that a NF-κB motif located at −3922 to −3914 would be necessary for histamine-inducible promoter activity. In addition, H1 blocker, NF-κB inhibitor, and dominant negative IκBα or IκB kinase β downregulated the histamine-induced iNOS promoter activity. In the human aorta, histamine content was estimated to be 310±66 pmol/mg protein in the atherosclerotic intima, while that was to be 43±22 pmol/mg protein in the media ( P Conclusions— Histamine stimulates intimal SMCs to increase iNOS expression via H1 receptors and NF-κB signaling pathway. Histamine could be one of NO-regulating factors, by inducing iNOS expression in intimal SMCs, and may be related to atherogenesis.