Histamine deficiency decreases atherosclerosis and inflammatory response in apolipoprotein E knockout mice independently of serum cholesterol level.

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. CONCLUSIONS: 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.

Apoptosis signal-regulating kinase 1 deficiency accelerates hyperlipidemia-induced atheromatous plaques via suppression of macrophage apoptosis.

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. CONCLUSIONS: 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.

Unsymmetrically substituted disilyne Dsi(2)(i)PrSi-Si≡Si-SiNpDsi(2) (Np = CH(2)(t)Bu): synthesis and characterization.

The unsymmetrically substituted disilyne, Dsi(2)(i)PrSi-Si≡Si-SiNpDsi(2) (Np = CH(2)(t)Bu) 2, was synthesized and characterized by X-ray crystallography to show a trans-bent structure with a silicon-silicon triple bond length of 2.0569(12) Å. The (29)Si chemical shifts of the triply bonded silicon atoms of 2 are quite different, being observed at 62.6 ppm for the Dsi(2)(i)PrSi side and 106.3 ppm for the Dsi(2)NpSi side, indicating different hybridizations on the triply bonded silicon atoms at each site.

Histamine upregulates the expression of inducible nitric oxide synthase in human intimal smooth muscle cells via histamine H1 receptor and NF-kappaB signaling pathway.

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-kappaB nuclear translocation, which were inhibited by histamine H1 blocker and NF-kappaB inhibitor. Luciferase assay using -8.3 kb upstream of human iNOS promoter region and electrophoretic mobility shift assay suggested that a NF-kappaB motif located at -3922 to -3914 would be necessary for histamine-inducible promoter activity. In addition, H1 blocker, NF-kappaB inhibitor, and dominant negative IkappaB alpha or IkappaB kinase beta 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<0.001). CONCLUSIONS: Histamine stimulates intimal SMCs to increase iNOS expression via H1 receptors and NF-kappaB signaling pathway. Histamine could be one of NO-regulating factors, by inducing iNOS expression in intimal SMCs, and may be related to atherogenesis.

Role of histamine produced by bone marrow-derived vascular cells in pathogenesis of atherosclerosis.

To clarify the role of histamine-producing cells and its origin in atherosclerosis, we investigated histidine decarboxylase (HDC; histamine-producing enzyme) expression in murine arteries with vascular injuries after the animal had received transplanted bone marrow (BM) from green fluorescent protein (GFP)-transgenic mice. The neointima in the ligated carotid arteries contained BM-derived HDC+ cells that expressed macrophage (Mac-3) or smooth muscle cell antigen (alpha-SMA). In contrast, the HDC+ BM-derived cells, which were positive for Mac-3, were mainly located in the adventitia in the cuff replacement model. In apolipoprotein E-knockout mice on a high cholesterol diet, BM-derived cells expressing Mac-3 in the atheromatous plaques were also positive for HDC. In comparison with wild-type mice, HDC-/- mice showed reduced neointimal thickening and a decreased intima-to-media ratio after ligation and cuff replacement. These results indicate that histamine produced from BM-derived progenitor cells, which could transdifferentiate into SMC- or macrophage-like cells, are important for the formation of neointima and atheromatous plaques.

Granulocyte macrophage-colony stimulating factor increases the expression of histamine and histamine receptors in monocytes/macrophages in relation to arteriosclerosis.

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. CONCLUSIONS: 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. The presence of histamine-producing macrophages and gene expression of histamine receptors and GM-CSF was demonstrated in arteriosclerotic lesions. In monocytic U937 cells, GM-CSF upregulated the expression of histamine and HH1R. Coordinated expression of histamine and its receptors by GM-CSF would participate in atherogenesis by affecting monocytic and SMC gene expression.

Difference in responsiveness of human esophageal squamous cell carcinoma lines to epidermal growth factor for MMP-7 expression.

Expression of MMP-7 in human esophageal squamous cell carcinoma lines (TE-9 and -10) was investigated. Under normal culture conditions, immunocytochemical staining and enzymography demonstrated the production of MMP-7 in their cytoplasm and its gelatinolytic activity in the medium of TE-9 and -10 cell cultures. When EGF was added to the cultures, Western blotting and RT-PCR analysis showed a dose-dependent increase in the amount of MMP-7 synthesized in the TE-9 cells, whereas in TE-10 cells, EGF failed to stimulate MMP-7 production. For luciferase reporter analysis of MMP-7 transcription, pMMP7LucWI (1132-bp) and pMMP7LucWII (334-bp) were cloned in the luciferase pGL3-basic vector. The promoter activity was enhanced from 1.5- to 2-fold by the addition of EGF to TE-9 cells transfected with pMMP7LucWI or WII, even though the response was low as compared with that of 12-O-tetra-decanoyl-phorbol-13-acetate (TPA); and in TE-10 cells, only TPA enhanced the promoter activity of MMP-7. Luciferase promoter analysis using a pMMP7WII mutant series revealed that the AP-1 site was essential for transcription of the MMP-7 gene in TE-9 cells, and Tcf-I was also an important site, and that to a lesser degree, Tcf-II and PEA3s participated in the transcription of the MMP-7 gene in these cells. Unlike the results with TE-9 cells, in TE-10 cells, EGF failed to stimulate transcription of the MMP-7 gene; and up-regulation of the promoter activity by TPA was dependent on the AP-1 site and to lesser degree, on Tcf-I and Tcf-II. These results suggest that EGF plays also an important role in MMP-7 production of TE-9 cells and that there is a difference not only in EGF-intracellular signaling system but also in regulation mechanisms of MMP-7 transcription by beta-catenin-Tcf and/or PEA3 system between these 2 carcinoma lines.

Overexpression of peroxiredoxin 4 attenuates atherosclerosis in apolipoprotein E knockout mice.

AIM: A growing body of evidence has shown that increased formation of oxidized molecules and reactive oxygen species within the vasculature (i.e., the extracellular space) plays a crucial role in the initiation and progression of atherosclerosis and in the formation of unstable plaques. Peroxiredoxin 4 (PRDX4) is the only known secretory member of the antioxidant PRDX family. However, the relationship between PRDX4 and susceptibility to atherosclerosis has remained unclear. RESULTS: To define the role of PRDX4 in hyperlipidemia-induced atherosclerosis, we generated hPRDX4 transgenic (Tg) and apolipoprotein E (apoE) knockout mice (hPRDX4(+/+)/apoE(-/-)). After feeding the mice a high-cholesterol diet, they showed fewer atheromatous plaques, less T-lymphocyte infiltration, lower levels of oxidative stress markers, less necrosis, a larger number of smooth muscle cells, and a larger amount of collagen, resulting in thickened fibrous cap formation and possible stable plaque phenotype as compared with apoE(-/-) mice. We also detected greater suppression of apoptosis and decreased Bax expression in hPRDX4(+/+)/apoE(-/-) mice than in apoE(-/-) mice. Bone marrow transplantation from hPRDX4(+/+) donors to apoE(-/-) mice confirmed the antiatherogenic aspects of PRDX4, revealing significantly suppressed atherosclerotic progression. INNOVATION: In this study, we demonstrated for the first time that PRDX4 suppressed the development of atherosclerosis in apoE(-/-) mice fed a high-cholesterol diet. CONCLUSION: These data indicate that PRDX4 is an antiatherogenic factor and, by suppressing oxidative damage and apoptosis, that it may protect against the formation of vulnerable (unstable) plaques.

Overexpression of peroxiredoxin 4 protects against high-dose streptozotocin-induced diabetes by suppressing oxidative stress and cytokines in transgenic mice.

Peroxiredoxin 4 (PRDX4) is one of a newly discovered family of antioxidative proteins. We generated human PRDX4 (hPRDX4) transgenic (Tg) mice, displaying a high level of hPRDX4 expression in the pancreatic islets, and then focused on the functions of PRDX4 in a type 1 diabetes mellitus (T1DM) model using a single high dose of streptozotocin (SHDS). After SHDS-injection, Tg mice showed significantly less hyperglycemia and hypoinsulinemia and a much faster response on glucose tolerance test than wild-type (WT) mice. Morphologic and immunohistochemical observation revealed that the pancreatic islet areas of Tg mice were larger along with less CD3-positive lymphocyte infiltration compared with WT mice. Upon comparison between these two mouse models, ß-cell apoptosis was also repressed, and reversely, ß-cell proliferation was enhanced in Tg mice. Real-time RT-PCR demonstrated that the expression of many inflammatory-related molecules and their receptors and transcription factors were significantly downregulated in Tg mice. These data indicate that PRDX4 can protect pancreatic islet ß-cells against injury caused by SHDS-induced insulitis, which strongly suggests that oxidative stress plays an essential role in SHDS-induced diabetes. This study, for the first time, implicates that PRDX4 has a pivotal protective function against diabetes progression in this T1DM model.

Monocyte chemoattractant protein-1 expression is enhanced by granulocyte-macrophage colony-stimulating factor via Jak2-Stat5 signaling and inhibited by atorvastatin in human monocytic U937 cells.

The proinflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is expressed in inflammatory and atherosclerotic lesions. GM-CSF is known to enhance monocytic expression of monocyte chemoattractant protein-1 (MCP-1). However, the molecular mechanism(s) by which GM-CSF up-regulates the MCP-1 expression remains to be clarified. Thus, in this study, we examined our hypothesis that GM-CSF up-regulates the MCP-1 expression via Jak2-Stat5 signaling pathway. In human monocytic cell line U937, GM-CSF increased MCP-1 expression in protein and mRNA levels. Furthermore, analysis of the GM-CSF promoter element revealed that the STAT5 (signal transducer and activator of transcription-5) transcription factor binding site, located between -152 and -144 upstream of the transcription start site, as well as Janus kinase-2-mediated Stat5 activation were necessary for the GM-CSF-induced transcriptional up-regulation of the MCP-1 gene. This GM-CSF-induced MCP-1 expression, measured as both protein and mRNA levels, was down-regulated by atorvastatin, a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor. However, this decrease in MCP-1 expression was not at the transcriptional level of MCP-1 gene but rather at the level of the stability of MCP-1 mRNA. These results indicate that GM-CSF regulates MCP-1 expression via Janus kinase-2-Stat5 pathway and by a novel regulatory mechanism of statins to reduce inflammatory reactions by down-regulating the expression of monocytic MCP-1, which promotes atherogenesis.

Acute inflammatory reactions caused by histamine via monocytes/macrophages chronically participate in the initiation and progression of atherosclerosis.

Previously we demonstrated that histidine decarboxylase (HDC), which produces histamine from l-histidine, was detected in monocytes/macrophages located in human atherosclerotic lesions. As monocytic migration is a key event of atherogenesis, we investigated whether histamine induces monocytic expression of monocyte chemoattractant protein (MCP)-1 and its receptors CCR2-A and -B, and also endothelial expression of ICAM-1 and VCAM-1. Furthermore, we studied the effect of interleukin (IL)-4, which inhibits the HDC expression, on the expression of MCP-1 and CCR2. Histamine stimulated monocytes, but not macrophages, to express MCP-1 and CCR2-A and -B. The expression of MCP-1 was inhibited by histamine H2 blocker. In contrast, IL-4 enhanced CCR2 expression but not MCP-1. Histamine stimulated endothelial cells to express ICAM-1 and VCAM-1. These results indicate that histamine and IL-4, which are both synthesized in the arterial intima, chronically participates in the pathogenesis of atherosclerosis via the enhanced expression of monocytic MCP-1, CCR2 and endothelial adhesion molecules.

Matrix metalloproteinase-12 gene expression in human vascular smooth muscle cells.

BACKGROUND: Matrix metalloproteinases (MMPs) play an important role in smooth muscle cell (SMC) migration and proliferation during vascular remodelling. To investigate the expression of MMP-12 by SMCs, we examined the protein secretion and mRNA expression of MMP-12 by cultured medial SMCs and intimal SMCs derived from human aortic atherosclerotic lesions. To further elucidate the molecular mechanism for MMP-12 expression in SMCs, we determined the sequence requirements for MMP-12 gene transcriptional activity. RESULTS: Cultured medial SMCs and intimal SMCs showed substantial MMP-12 expression at both the protein and mRNA levels. A series of 5'-deletion and site-directed mutants of the human MMP-12 promoter demonstrated that an AP-1 site spanning -81 to -75 bp was critical for the MMP-12 promoter activity in SMCs. An electrophoretic mobility shift assay confirmed the AP-1 binding activity in SMCs and showed that the protein bound to the AP-1 site consisted predominantly of c-Jun, JunD and Fra-1. Two structurally different inhibitors of phosphatidylinositol 3-kinase, wortmannin and LY294002, inhibited MMP-12 transcriptional activity and AP-1 binding. CONCLUSION: These results indicated the expression of MMP-12 in vascular SMCs and showed that the MMP-12 gene expression was dependent on the AP-1 binding activity. Phosphatidylinositol 3-kinase signalling may be involved in MMP-12 transcriptional activation through AP-1 binding activity.