Yoshitaka Murata

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, β-cell apoptosis was also repressed, and reversely, β-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 β-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.

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 (&agr;-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.

Overexpression of Peroxiredoxin 4 Attenuates Atherosclerosis in Apolipoprotein E Knockout Mice

AIMnA 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.nnnRESULTSnTo 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.nnnINNOVATIONnIn this study, we demonstrated for the first time that PRDX4 suppressed the development of atherosclerosis in apoE(-/-) mice fed a high-cholesterol diet.nnnCONCLUSIONnThese 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.

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.

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.

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. 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.

Histamine increases the expression of LOX-1 via H2 receptor in human monocytic THP-1 cells

Lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1) is a member of the scavenger receptor family, and is known to be expressed in monocytes/macrophages. We investigated the effect of histamine on the expression of LOX‐1 in cells of the human monocytic leukemia cell line THP‐1. Histamine as well as forskolin and dibutyryl cyclic AMP (Bt2‐cAMP) stimulated the THP‐1 monocytes to express the LOX‐1 gene at the transcription level. This histamine effect on LOX‐1 gene expression, via the histamine H2 receptor‐mediated cAMP signal transduction pathway, was reduced after differentiation of the cells into macrophages, even though forskolin and Bt2‐cAMP still enhanced the gene expression. The alteration of the responsiveness of LOX‐1 expression to histamine was related to suppressed expression of the H2 receptor in THP‐1 macrophages. The switch of the predominant class of histamine receptors between H1 and H2 would modulate the effects of histamine on LOX‐1 gene expression in monocytes and macrophages, and therefore, would play a certain role in the inflammatory aspects of atherogenesis.

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-&kgr;B, 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.

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. 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.

Histamine Upregulates the Expression of Inducible Nitric Oxide Synthase in Human Intimal Smooth Muscle Cells via Histamine H1 Receptor and NF-κB 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-&kgr;B nuclear translocation, which were inhibited by histamine H1 blocker and NF-&kgr;B inhibitor. Luciferase assay using −8.3 kb upstream of human iNOS promoter region and electrophoretic mobility shift assay suggested that a NF-&kgr;B motif located at −3922 to −3914 would be necessary for histamine-inducible promoter activity. In addition, H1 blocker, NF-&kgr;B inhibitor, and dominant negative I&kgr;Bα or I&kgr;B 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<0.001). Conclusions—Histamine stimulates intimal SMCs to increase iNOS expression via H1 receptors and NF-&kgr;B signaling pathway. Histamine could be one of NO-regulating factors, by inducing iNOS expression in intimal SMCs, and may be related to atherogenesis.