Tamio Hagiwara

Human Urotensin II Accelerates Foam Cell Formation in Human Monocyte-Derived Macrophages

Human urotensin II (U-II), the most potent vasoconstrictor peptide identified to date, and its receptor (UT) are involved in hypertension and atherosclerosis. Acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) converts intracellular free cholesterol into cholesterol ester (CE) for storage in lipid droplets and plays an important role in the formation of macrophage-derived foam cells in atherosclerotic lesions. We examined the effects of U-II on ACAT-1 expression and CE accumulation in human monocyte-derived macrophages. U-II increased ACAT activity in a concentration-dependent manner after 7 days in monocyte primary culture. Immunoblotting analysis showed that U-II at 25 nmol/L increased ACAT-1 protein expression level by 2.5-fold, which was completely abolished by anti–U-II antibody, selective UT receptor antagonists (urantide and 4-aminoquinoline), a G-protein inactivator (GDP-&bgr;-S), a c-Src protein tyrosine kinase inhibitor (PP2), a protein kinase C (PKC) inhibitor (rottlerin), a mitogen-activated protein kinase kinase (MEK) inhibitor (PD98059), or a Rho kinase (ROCK) inhibitor (Y27632). Northern blotting analysis indicated that among the 4 ACAT-1 mRNA transcripts (2.8-, 3.6-, 4.3-, and 7.0-kb), the 2.8- and 3.6-kb transcript levels were selectively upregulated by ≈1.7-fold by U-II (25 nmol/L). Further, U-II (25 nmol/L) significantly increased acetylated LDL (acetyl-LDL)–induced CE accumulation in monocyte-derived macrophages but not scavenger receptor class A (SR-A) function as assessed by endocytic uptake of [125I]acetyl-LDL. Our results suggest that U-II may play a novel role in the formation of macrophage-derived foam cells by upregulating ACAT-1 expression via the UT receptor/G-protein/c-Src/PKC/MEK and ROCK pathways but not by SR-A, thus contributing to the relatively rapid development of atherosclerosis in hypertension.

Influence of SNPs in cytokine-related genes on the severity of food allergy and atopic eczema in children

Although many single nucleotide polymorphism (SNP) studies have reported an association of atopy, allergic diseases and total serum immunoglobulin E (IgE) levels, almost all of these studies sought risk factors for the onset of these allergic diseases. Furthermore, many studies have analyzed a single gene and hardly any have analyzed environmental factors. In these analyses, the results could be masked and the effects of other genes and environmental factors may be decreased. Here, we described the correlation between four genes [interleukin (IL)‐4 (C‐590T), IL‐4 receptor (A1652G), FCER1B (G6842A) and STAT6 (G2964A)] in connection with IgE production; the role of IL‐10 (C‐627A) as a regulatory cytokine of allergy; and the severity of food allergy (FA) and atopic eczema (AE) in 220 Japanese allergic children. In addition to these SNPs, environmental factors, i.e., patients attitude, indoor envirmonment, and so on, were also investigated in this study.

The requirement for and mobilization of calcium during induction by sodium ascorbate and by hydrogen peroxide of cell death

The requirement for and mobilization of Ca2+ ions during induction of cell death by sodium ascorbate were compared with those during induction of cell death by hydrogen peroxide. When HL-60 cells were incubated with sodium ascorbate, a rapid increase in the intracellular concentration of Ca2+ ions and subsequent apoptotic cell death, characterized by cell shrinkage, nuclear fragmentation and cleavage of internucleosomal DNA to yield fragments that were multiples of 180-200 base pairs, were induced. However, these effects of sodium ascorbate were significantly reduced in Ca2+-depleted medium. By contrast, hydrogen peroxide induced similar apoptosis associated phenomena in the presence and in the absence of extracellular Ca2+ ions. The intracellular concentration of the reduced form of glutathione was not significantly affected and glutathione disulfide was undetectable during the early stages of apoptosis. These data suggest that sodium ascorbate and hydrogen peroxide initiate cell death by different mechanisms.

Identification of the regulatory region required for ubiquitination of the cyclin kinase inhibitor, p21.

The expression of cyclin kinase inhibitor p21 is regulated by the ubiquitin-proteasome protein degradation system, as well as by transcriptional regulation. Generally, ubiquitination is regulated by the phosphorylation of the substrate. In this study, we identified the region of p21 responsible for the regulation of ubiquitination. Since the phosphorylation sites of p21 are distributed in the C-terminal region, we constructed sequential C-terminal truncated fragments and examined their ubiquitination in eukaryotic cells. The ubiquitination was observed in the 1-164 (full length) and 1-157 fragments with the same efficiency, but not in the 1-147 fragment. The lack of ubiquitination in the 1-147 fragment was unlikely due to the removal of a Lys residue at position 154, since the p21 K154R mutant was ubiquitinated as efficiently as the full-length p21. Furthermore, the 148-157 deleted form of p21 was not ubiquitinated, just like the 1-147 fragment. Thus, the C-terminal 148-157 region, not a ubiquitination site by itself, should contain an essential regulatory region for the efficient ubiquitination of p21.

Beneficial Role of Tetrahydrobiopterin on Various Cardiovascular Diseases and Regulation of its Levels under Pathological Conditions

Abstract 5,6,7,8-Tetrahydrobiopterin (BH4) is an csscntial cofactor for production of nitric oxide (NO) by N O synthase (NOS). Nitric oxide (NO) is an important signaling mo!ecu!e for the regulation o f vita! functions such as vascular tone, neurotransmission and immune surveiüance. Accumu!ated evidences show that although NOS mainly re!eases N O under nonnal conditions, NOS also produces Superoxide anion and hydrogen peroxide when BH4 is decreased, suggesting the possibility that NOS is a source of reactive oxygen species (ROS) under pathological conditions. In fact, supplementation of BH4 restores N O production and availability in various diseases including hyperlipidemia, diabetes, hypertension and ischemia-reperfusion. Moreover, recent studies show that administration o f BH4 protects tissues against ischemia-reperfusion injuiy in heart, stomach and liver. Oxidative stress is a common mechanism underlying the development of vascular diseases and reperfusion injury. Regulation of BH4 levels under oxidative stress is important to understand the role of BH4 on the development of vascular diseases and reperfusion injury. We recently described that oxidative stress transiently decreased BH4 levels, and then markedly increased its levels in vascular endothelial cells. Supplementation of BH4 appears to be important therapeutic strategies in vascular diseases and reperfusion injury, and induction of BH4 synthesis may endogenous defense system against oxidative stress-mediated cardiovascular diseases.