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The reaction of halothane, 2-bromo-2-chloro-1,1,1-trifluoroethane (1), with aldehydes and ketones in the presence of bases was found to give 1-alkyl- or 1-aryl-2-bromo-2-chloro-3,3,3-trifluoropropanols (2) or 2-chloro-3,3-difluoro-2-propenols (3) selectively in good to moderate yields depending on the bases and reaction conditions.

OBJECTIVE In white adipose tissue, adipocytes and adipocyte precursor cells are enmeshed in a dense network of type I collagen fibrils. The fate of this pericellular collagenous web in diet-induced obesity, however, is unknown. This study seeks to identify the genetic underpinnings of proteolytic collagen turnover and their association with obesity progression in mice and humans. RESEARCH DESIGN AND METHODS The hydrolysis and degradation of type I collagen at early stages of high-fat diet feeding was assessed in wild-type or MMP14 (MT1-MMP)-haploinsufficient mice using immunofluorescent staining and scanning electron microscopy. The impact of MMP14-dependent collagenolysis on adipose tissue function was interrogated by transcriptome profiling with cDNA microarrays. Genetic associations between MMP14 gene common variants and obesity or diabetes traits were examined in a Japanese cohort (n = 3,653). RESULTS In adult mice, type I collagen fibers were cleaved rapidly in situ during a high-fat diet challenge. By contrast, in MMP14 haploinsufficient mice, animals placed on a high-fat diet were unable to remodel fat pad collagen architecture and display blunted weight gain. Moreover, transcriptional programs linking type I collagen turnover with adipogenesis or lipogenesis were disrupted by the associated decrease in collagen turnover. Consistent with a key role played by MMP14 in regulating high-fat diet–induced metabolic programs, human MMP14 gene polymorphisms located in proximity to the enzyme's catalytic domain were closely associated with human obesity and diabetes traits. CONCLUSIONS Together, these findings demonstrate that the MMP14 gene, encoding the dominant pericellular collagenase operative in vivo, directs obesogenic collagen turnover and is linked to human obesity traits.

2,3-Didehydro-1,2-bis(methoxycarbonyl)-6-methoxypiperidine ( 4 ), prepared from l -lysine by using electrochemical oxidation, was cyclopropanated with high diastereoselectivity (96.6% de), and the cyclopropanated product was transformed to optically active 2,3-methanopipecolic acid ( 1 ). In this transformation, the 6-methoxy group of 4 was found to be an effective chiral auxiliary.

Japan's climate, with several months of high temperatures, and high humidity, is well suited to the growth of various kinds of fungi. They grow not only on food, wood, and textiles, but also on paintings. We have examined a variety of instances of deterioration caused by the growth of fungi on paintings and have isolated and identified more than a hundred of these fungi. Of these, the typical ones are Alternaria, Cladosporium, Fusarium, Aspergillus, Trichoderma and Penicillium . Since 1982 we have studied a fungicide-containing varnish to test its ability to inhibit fungal growth. The varnish, made from synthetic resin containing Vinyzene, a fungicide, was sprayed on about 100 paint samples. Annual observations have confirmed that it has a fungicidal effect. This paper reports on the effect of the Vinyzene-containing varnish on paint and its utility as a fungicide.

Urine type IV collagen concentrations in type 2 diabetic patients were measured by enzyme immunoassay which has crossreactivity with only intact type IV collagen, and the clinical usefulness for estimating the early phase of diabetic nephropathy was evaluated. Precision of the measurement system was satisfactory for clinical use and the value did not influenced by the presence of sediments in urine. In whole type 2 diabetic patients (N=132), urine type IV collagen concentration (microg/g of creatinine) increased with development of nephropathy and showed significantly increase even in normoalbuminuria when compared with that in normal control subjects (N=117). In type 2 diabetic patients (N=100) with mild microalbuminuria (less than 100 mg/g of creatinine), multiple regression analysis revealed that HbA1C was extracted as a significant valuable for urine type IV collagen, while body mass index was extracted as a significant valuable for urine albumin. In these subjects, urine type IV collagen was significantly lower in the patients with good metabolic control (HbA1C8.0%) than those with poor control (HbA1or =8.0%), while the urine albumin was not significantly different between those two groups. These results suggest that measurement of urine type IV collagen in type 2 diabetic patients is useful for detection of early phase of diabetic nephropathy.

A 52-year-old woman was admitted to Yame General Hospital because of persistent cough, wheeze, and shortness of breath at age 48. Chest X-ray and computed tomography (CT) showed bilateral centrilobular shadows. Pulmonary function test revealed obstructive dysfunction. She also had chronic sinusitis. Initially, diffuse panbronchiolitis was diagnosed and she was given macrolides, but no improvement was observed. Thus video-assisted thoracoscopic lung biopsy (VATS) was performed in order to establish a definitive diagnosis. Histopathological findings were compatible with a diagnosis of follicular bronchiolitis. Treatment with corticosteroid (oral prednisolone, 50 mg/day) improved her condition. However, on reducing the steroid doze, her symptoms and chest X-ray film/CT findings became exacerbated. In addition, polyarthritis appeared. Further investigations revealed a diagnosis of rheumatoid arthritis. Only 2 cases of follicular bronchiolitis preceding rheumatoid arthritis have been reported in Japan.

The exocyst is an octameric molecular complex that drives vesicle trafficking in adipocytes, a rate-limiting step in insulin-dependent glucose uptake. This study assessed the role of the exocyst complex in regulating free fatty acid (FFA) uptake by adipocytes. Upon differentiating into adipocytes, 3T3-L1 cells acquire the ability to incorporate extracellular FFAs in an insulin-dependent manner. A kinetic assay using fluoresceinated FFA (C12 dodecanoic acid) uptake allows the real-time monitoring of FFA internalization by adipocytes. The insulin-dependent uptake of C12 dodecanoic acid by 3T3-L1 adipocytes is mediated by Akt and phosphatidylinositol 3 (PI3)-kinase. Gene silencing of the exocyst components Exo70 and Sec8 significantly reduced insulin-dependent FFA uptake by adipocytes. Consistent with the roles played by Exo70 and Sec8 in FFA uptake, mCherry-tagged Exo70 and HA-tagged Sec8 partially colocalize with lipid droplets within adipocytes, suggesting their active roles in the development of lipid droplets. Tubulin polymerization was also found to regulate FFA uptake in collaboration with the exocyst complex. This study demonstrates a novel role played by the exocyst complex in the regulation of FFA uptake by adipocytes.

Human peptide transporter 1 (PEPT1) mediates the cellular uptake of di- and tripeptides and peptide-like drugs in the small intestine. In the present study, we examined the regulation of PEPT1 by anticancer drugs in the gastric cancer cell line MKN45. PEPT1 was expressed and functioned in MKN45 cells. The transport activity and mRNA expression of the facilitative glucose transporter 1 (GLUT1) were significantly decreased by 5-fluorouracil treatment, but those of PEPT1 were slightly increased. Cisplatin treatment affected neither PEPT1 nor GLUT1 activity. In conclusion, PEPT1 expressed in MKN45 cells are resistant against the cellular injury induced by 5-fluorouracil and cisplatin.

Thiazolidinediones, a new class of antidiabetic drugs that increase insulin sensitivity, have been shown to be ligands for peroxisome proliferator-activated receptor gamma (PPARgamma). Recent studies demonstrating that PPARgamma occurs in macrophages have focused attention on its role in macrophage functions. In this study, we investigated the effect of thiazolidinediones on monocyte proliferation and migration in vitro and the mechanisms involved. In addition, we examined the therapeutic potentials of thiazolidinediones for injured atherosclerotic lesions. Troglitazone and pioglitazone, the two thiazolidinediones, as well as 15-deoxy-delta12,14-prostaglandin J2 inhibited in a dose-dependent manner the serum-induced proliferation of THP-1 (human monocytic leukemia cells) and of U937 (human monoblastic leukemia cells), which permanently express PPARgamma. These ligands for PPARgamma also significantly inhibited migration of THP-1 induced by monocyte chemoattractant protein-1 (MCP-1). Troglitazone and 15-deoxy-delta12,14-prostaglandin J2 significantly suppressed the mRNA expression of the MCP family-specific receptor CCR2 (chemokine CCR2 receptor) in THP-1 at the transcriptional level. Furthermore, troglitazone significantly inhibited MCP-1 binding to THP-1. Oral administration of troglitazone to Watanabe heritable hyperlipidemic (WHHL) rabbits after balloon injury suppressed acute recruitment of monocytes/macrophages and accelerated re-endothelialization. These results suggest that thiazolidinediones have therapeutic potential for the treatment of diabetic vascular complications.

Oxidative stress is known to be involved in growth control of vascular smooth muscle cells (VSMCs). We and others have demonstrated that angiotensin II (Ang II) has an important role in vascular remodeling. Several reports suggested that VSMC growth induced by Ang II was elicited by oxidative stress. Gax, growth arrest-specific homeobox is a homeobox gene expressed in the cardiovascular system. Over expression of Gax is demonstrated to inhibit VSMC growth. We previously reported that Ang II down-regulated Gax expression. To address the regulatory mechanism of Gax, we investigated the significance of oxidative stress in Ang II-induced suppression of Gax expression. We further examined the involvement of mitogen-activated protein kinases (MAPKs), which is crucial for cell growth and has shown to be activated by oxidative stress, on the regulation of Gax expression by Ang II. Ang II markedly augmented intracellular H2O2 production which was decreased by pretreatment with N-acetylcystein (NAC), an anti-oxidant. Ang II and H2O2 decreased Gax expression dose-dependently and these effects were blocked by administration of both NAC and pyrrolidine dithiocarbamate (PDTC), another anti-oxidant. Ang II and H2O2 induced marked activation of extracellular signal-responsive kinase1/2 (ERK1/2), which was blocked by NAC. Ang II and H2O2 also activated p38MAPK, and they were blocked by pre-treatment with NAC. However, the level of activated p38MAPK was quite low in comparison with ERK1/2. Ang II- or H2O2 -induced Gax down-regulation was significantly inhibited by PD98059, an ERK1/2 inhibitor but not SB203580, a p38MAPK inhibitor. The present results demonstrated the significance of regulation of Gax expression by redox-sensitive ERK1/2 activation.

Insulin stimulates glucose transport by promoting exocytosis of the glucose transporter Glut4 (refs 1, 2). The dynamic processes involved in the trafficking of Glut4-containing vesicles, and in their targeting, docking and fusion at the plasma membrane, as well as the signalling processes that govern these events, are not well understood. We recently described tyrosine-phosphorylation events restricted to subdomains of the plasma membrane that result in activation of the G protein TC10 (refs 3, 4). Here we show that TC10 interacts with one of the components of the exocyst complex, Exo70. Exo70 translocates to the plasma membrane in response to insulin through the activation of TC10, where it assembles a multiprotein complex that includes Sec6 and Sec8. Overexpression of an Exo70 mutant blocked insulin-stimulated glucose uptake, but not the trafficking of Glut4 to the plasma membrane. However, this mutant did block the extracellular exposure of the Glut4 protein. So, the exocyst might have a crucial role in the targeting of the Glut4 vesicle to the plasma membrane, perhaps directing the vesicle to the precise site of fusion.

The decision of long-term institutionalization of patients with Alzheimer's disease (AD) is based on multiple patients' and caregivers' characteristics. It is very important to find out factors associated with institutionalization for the timely institutionalization, i.e. for the prevention of premature institutionalization. Among those factors, caregiver burden is reportedly one of the most important factors in Europe and North America. However, no studies have been carried out in Japan. In order to address this issue, we studied outcome of patients with AD.Subjects were 211 patients who fulfilled the criteria of the National Institute of Neurological Disease and Stroke/Alzheimer's Disease and Related Disorders Association for probable AD. Diagnosis of AD was made through extensive examinations including MRI and PET/SPECT of the brain. The patients included 149 women and 62 men; the mean age was 73.1 +/- 8.0 (SD) years. The Japanese version of Zarit Caregiver Burden Interview (ZBI), which is a validated tool for assessment of the burden of caregivers for dementia patients, was used to evaluate caregivers' burden through the interview of a principal caregiver by trained nurses. Cognitive, functional, and neuropsychiatric impairments were assessed with standardized instruments of the Mini-Mental State Examination (MMSE), Hyogo Activities of Daily Living Scale (HADLS), Neuropsychiatric Inventory (NPI), respectively. We annually sent a questionnaire to the caregivers to collect information on the patients' status. It included questions whether and when they died or were institutionalized and whether they had used formal social supports such as home care, day care, and respite care services.Time to death or institutionalization.One-hundred and fifty subjects were followed for at least one year and 51 of them met either end point. The Cox proportional hazard model demonstrated that the baseline caregiver burden was a significant predictor of the end point even after controlling possible confounders. Among these, the use of day care and home care services during the course were significant protective factors. The baseline cognitive and functional disturbances but not neuropsychiatric manifestations were significantly associated with the end point.The caregiver burden is a significant predictor for the death or institutionalization of the patients with AD. The caregiver education which decrease the caregiver burden and prompt the usage of the social care services are necessary to protect premature institutionalization.

Background —Vascular endothelial growth factor (VEGF) is an important angiogenic factor reported to induce migration and proliferation of endothelial cells, enhance vascular permeability, and modulate thrombogenicity. VEGF expression in cultured cells (smooth muscle cells, macrophages, endothelial cells) is controlled by growth factors and cytokines. Hence, the question arises of whether VEGF could play a role in atherogenesis. Methods and Results —Frozen sections from 38 coronary artery segments were studied. The specimens were characterized as normal with diffuse intimal thickening, early atherosclerosis with hypercellularity, and advanced atherosclerosis (atheromatous plaques, fibrous plaques, and totally occlusive lesions). VEGF expression as well as the expression of 2 VEGF receptors, flt-1 and Flk-1, were studied with immunohistochemical techniques in these samples at the different stages of human coronary atherosclerosis progression. The expression of VEGF mRNA was also studied with reverse transcription–polymerase chain reaction. Normal arterial segments showed no substantial VEGF expression. Hypercellular and atheromatous lesions showed distinct VEGF positivity of activated endothelial cells, macrophages, and partially differentiated smooth muscle cells. VEGF positivity was also detected in endothelial cells of intraplaque microvessels within advanced lesions. In totally occlusive lesions with extensive neovascularization, intense immunostaining for VEGF was observed in accumulated macrophages and endothelial cells of the microvessels. Furthermore, VEGF mRNA expression was detected in atherosclerotic coronary segments but not in normal coronary segments. The immunostainings for flt-1 and Flk-1 were detected in aggregating macrophages in atherosclerotic lesions and also in endothelial cells of the microvessels in totally occlusive lesions. Conclusions —These results demonstrate distinct expression of VEGF and its receptors (flt-1 and Flk-1) in atherosclerotic lesions in human coronary arteries. Considering the multipotent actions of VEGF documented experimentally in vivo and in vitro, our findings suggest that VEGF may have some role in the progression of human coronary atherosclerosis, as well as in recanalization processes in obstructive coronary diseases.

Shear stress dilates blood vessels and exerts an antiproliferative effect on vascular walls. These effects are ascribed to shear stress-induced, endothelium-derived vasoactive substances. Endothelin-converting enzymes (ECEs), the enzymes that convert big endothelin-1 (ET-1) to ET-1, have recently been isolated and the corresponding proteins have been termed ECE-1 and ECE-2. Furthermore, two isoforms of human ECE-1 have been demonstrated and termed ECE-1 alpha and ECE-1 beta. In this study, to elucidate the role of ECE-1 under shear stress we examined the effect of physiologic shear stress on the mRNA expression of ECE-1 and ET-1 in cultured bovine carotid artery endothelial cells (BAECs) and human umbilical veins (HUVECs), and also ECE-1 alpha mRNA expression in HUVECs. ECE-1 mRNA expression was significantly downregulated by shear stress in 24 h, both in BAECs and HUVECs, in a shear stress intensity-dependent manner. The expression of ECE-1 alpha mRNA was also attenuated by shear stress in HUVECs. ET-1 mRNA expression showed a concordant decrease with ECE-1 mRNA expression. These results suggest that shear stress-induced gene regulation of ET-1 and ECE-1 mRNA expression can contribute to the decrease of ET-1 peptide level by shear stress.

Abstract Shear stress is known to dilate blood vessels and exert antiproliferative effects on vascular walls; these effects have been ascribed to shear stress–induced upregulation of endothelium-derived vasoactive substances, mainly nitric oxide and prostacyclin. We have demonstrated the significance of C-type natriuretic peptide (CNP) as a novel endothelium-derived relaxing peptide (EDRP) that shares a cGMP pathway with nitric oxide. Adrenomedullin is a recently isolated EDRP that elevates intracellular cAMP as prostacyclin does. To elucidate the possible role of these EDRPs under shear stress, we examined the effect of physiological shear stress on CNP mRNA expression in endothelial cells derived from the human umbilical vein (HUVECs), bovine aorta (BAECs), and murine lymph nodes (MLECs) as well as adrenomedullin mRNA expression in HUVECs. CNP mRNA was stimulated prominently in HUVECs under shear stress of 15 dyne/cm 2 in a time-dependent manner (4 hours, sixfold increase compared with that in the static condition; 24 hours, 30-fold increase). Similar results were obtained in BAECs (4 hours, twofold increase; 24 hours, threefold increase) and MLECs (4 hours, threefold increase; 24 hours, 10-fold increase). Augmentation of CNP mRNA expression that was dependent on shear stress intensity was also observed (5 dyne/cm 2 , 2.5-fold increase of static; 15 dyne/cm 2 , 4.5-fold increase). Increased CNP secretion was also confirmed by the specific radioimmunoassay for CNP. Adrenomedullin mRNA expression in HUVECs increased under shear stress of 15 dyne/cm 2 in a time-dependent manner (4 hours, 1.2-fold increase of static; 24 hours, threefold increase) and shear stress intensity–dependent manner (15 dyne/cm 2 , threefold increase compared with that at 5 dyne/cm 2 ). These results suggest that the coordinated augmentation of mRNA expression of these novel EDRPs may constitute shear stress–dependent vasodilator and antiproliferative effects.

C-type natriuretic peptide (CNP) was originally isolated from porcine brain extracts (1). CNP has a primary structure similar to α-ANP and BNP consisting of 22 amino acids, and the ring portion consisting of 17 amino acids is highly homologous to α-ANP and BNP (Fig. 1). Different from ANP and BNP, CNP lacks the C-terminal tail, and has a Cys residue at the C-terminus. Another species of CNP is CNP-53, which has the N-terminal extension of 31 amino acids compared with CNP-22 (2). Molecular cloning of the CNP precursor in the pig, rat, human, and mouse has revealed that the primary structure of CNP-22 is identical in these species (1,3–5). However, two amino acid substitutions are noted in CNP-53 between human and porcine/rat/mouse precursors. ProCNP consists of 103 amino acids and a single arginine residue precedes CNP-53 and CNP-22. Using reverse-phase high-performance liquid chromatography (HPLC), we demonstrated that the major molecular forms in the human brain are CNP-22 and CNP-53 (4).

各種澱粉粒の酵素分解性とX線回折図,フォトペーストグラム,ヨード呈色度(“ 青価”)あるいはSEM観察による形態との関連について検討し,以下の知見を得た. I)一部の例外はあるが,分解性の高い澱粉粒のX線回折図はA図形,分解性の低いものはB図形もしくはC図形を示すことがわかった. II)フォトペーストグラフィーによる糊化開始温度と酵素分解性との間には相関はみられなかったが,フォトペーストグラムは,一部の例外を除き,分解性の高い澱粉粒は透光度の増加が二段の変化でおこり,分解の低いものは一段の変化でおこることがわかった. III)各種澱粉粒のSEM観察の結果,分解性の高い群に属する澱粉粒は,いずれも粒が角ばっていて,多面形のものが多いが,分解性の低い群に属する澱粉粒は,いずれも粒がなめらかで,卵形だとか,球形を示していることがわかった. なお,X線回折に関してこ便宜をはかってくださいました武庫川女子大学家政学部教授滝山一善先生に深謝いたします.

Aluminum corrosion induced by microbial contamination of jet fuel has become one of the most important troubles. The object of the present work is to study the corrosion of aluminum caused by the growth of fungi isolated from jet fuel system. Of the fungi isolated, Cladosporium resinae appeared to be the most predominant. Many kinds of filamentous fungi were obtained from slime of jet fuel, water layer of storage tank, and sludges on stringer and lower panel of integral fuel tank of YS-11 jet planeResults obtained were as follows.(1) After 330 day incubation, exfoliation and corrosion occurred with small blisters on the surface of aluminum alloy, 6061 specimen.(2) The cross section of the 6061 specimen indicated that the corrosion pits by Cladosporium resinae were extensively evident thoughout the aluminum body.(3) The specimens of high purity aluminum (1099 and 1100), anodized aluminum, and aluminum alloy 5052 were severely damaged by the attack of the fungus.(4) An extruded bar of 1100 was also attacked by Cladosporium resinae. Corrosion pits began to develop after 30 day culture of the fungus.(5) Large amounts of sludge deposits were found on the stringer and lower skin panel of the integral tank of YS-11 jet plane. Widespread corrosions were found in the area covered by the growth of the fungus.(6) Discussions were also made over the development of tests for the effects of protective coatings.

In Japan Air Self-Defense Force, serious corrosion problems were encountered, regarding to the presence of the microorganisms in JP-4 fuel. Fuel was dripping from a tank in jet fuel aircraft. Microorganisms were cultured after being isolated from the JP-4 fuel underground storage tank, tank-lorry, water separator, and aircraft fuel tank. The effects of microbiological corrosion were examined on the specimens of 61-S aluminum alloy which was used in aircraft fuel tanks.The results were as follows:(1) Seventeen sorts of bacteria and three sorts of fungi were found to be bred by the utilization of JP-4 as nutritious element.(2) After culturing for 140 days, the surfaces of some aluminum specimens were covered with brown powdery blisters caused by the growth of microorganisms and other contaminations.(3) Severe corrosion pits were made on aluminum specimens, on which Micrococcus caseolyticus, Comamonas, and Vibrio faecalis were cultured.(4) It was observed that the surface of aluminum alloy specimens was covered with mycelium mats of Cladosporium resinae and tremendous corrosion existed under the mats.As the results, it is concluded that the preparation of filter-separator equipment such as ultrafine filter will be effective for the purpose of perfect removal of microorganisms from aircraft fuel.

Fungal contamination of electronic equipment has been recognized as a serious problem in the past few years. Many plastic materials such as epoxy, polyethylene and polypropylene are components of electronic equipment. The deterioration of these materials resulting from fungal growth is a very important problem. The predominant fungi isolated from deteriorated printed circuit, IC and silicon wafer plate were species of Trichoderma viride, Alternaria alternata, Cladosporium sphaeropermum, Fusaxim semitectum and Pesta- lotia adusta. The results of tests carried out are described and discussed

Growth arrest-specific homeobox (Gax) gene was isolated from rat aorta cDNA library and its expression was largely confined to the cardiovascular tissues. Gax gene was rapidly downregulated by platelet-derived growth factor in vascular smooth muscle cells (VSMCs) and overexpressed Gax was reported to reduce the neointimal thickening after balloon injury in vivo. We have demonstrated that angiotensin II (Ang II) stimulates vascular growth. In contrast, we also reported that C-type natriuretic peptide (CNP) is secreted from vascular endothelial cells to act as a novel endothelium-derived relaxing peptide and inhibits vascular growth via cGMP cascade. In the present study, we examined the effects of Ang II and CNP on Gax gene expression in VSMCs. In quiescent rat aortic VSMCs, Gax mRNA (2.3 kb) level became negligible 6 hours after the addition of Ang II (10 −6 mol/L). The inhibitory action of Ang II on Gax mRNA expression (ED 50 : 10 −11 mol/L) was almost completely blocked by an AT 1 R antagonist, CV11974. In contrast, CNP 10 −6 mol/L augmented Gax mRNA expression to exhibit 1.8-fold increase of the control 12 hours after the stimulation. This effect of CNP was mimicked by the addition of 8-bromoadenosine 3′:5′-cyclic monophosphate. The addition of C-ANF[4–23], an atrial natriuretic peptide-C receptor-specific agonist and devoid of stimulating cGMP production, exhibited no effect on Gax mRNA expression. Simultaneous administration of Ang II and CNP revealed that CNP (10 −6 mol/L) significantly attenuated the inhibitory action of Ang II (10 −10 mol/L) on Gax mRNA expression. These results suggest that Gax is a common transcription factor involved in the signaling pathway of vascular growth for Ang II and CNP and regulates the cell cycle and/or phenotype of VSMCs for vascular remodeling in hypertension and atherosclerosis.

To address endothelial function on vascular walls exposed to oxidative stress, we investigated the effect of oxidative stress on the secretion of endothelin-l (ET-1) from cultured bovine carotid artery endothelial cells (BAECs). Concentrations of ET-I in the media were measured by a specific radioimmunoassay and ET-I mRNA expression was estimated by Northern blot analysis. Treatment of BAECs with 0.5-2.0 mM H 2 O 2 for 3 h suppressed both ET-I secretion and ET-1 mRNA expression in a dose-dependent manner compared to control. Attenuation of ET-1 mRNA expression by H 2 O 2 was revealed to take place at the transcriptional level. The addition of N G -nitro-L-arginine-methyl ester (L-NAME) 10 μM a specific nitric oxide synthase inhibitor. had no effect on H 2 O 2 -induced suppression of ET-I mRNA expression. Suppression of ET secretion under oxidative stress observed in the present study is proposed to be a compensatory mechanism of endothelial cells to inhibit vasoconstriction and proliferation during oxidative stress.

Abstract —Vascular endothelial growth factor (VEGF) has been recognized as an angiogenic factor that induces endothelial proliferation and vascular permeability. Recent studies have also suggested that VEGF can promote macrophage migration, which is critical for atherosclerosis. We have reported that VEGF is remarkably expressed in activated macrophages, endothelial cells, and smooth muscle cells within human coronary atherosclerotic lesions, and we have proposed the significance of VEGF in the progression of atherosclerosis. To clarify the mechanism of VEGF expression in atherosclerotic lesions, we examined the regulation of VEGF expression by oxidized low density lipoprotein (Ox-LDL), which is abundant in atherosclerotic arterial walls. A recent report has revealed that peroxisome proliferator–activated receptor-γ (PPARγ) is expressed not only in adipocytes but also in monocytes/macrophages and has suggested that PPARγ may have a role in the differentiation of monocytes/macrophages. Furthermore, 9- and 13-hydroxy-( S )-10,12-octadecadienoic acid (9- and 13-HODE, respectively), the components of Ox-LDL, may be PPARγ ligands. Therefore, we investigated the involvement of PPARγ in the regulation of VEGF by Ox-LDL. PPARγ expression was detected in human monocyte/macrophage cell lines, human acute monocytic leukemia (THP-1) cells, and human coronary artery endothelial cells (HCAECs). Ox-LDL (10 to 50 μg/mL) upregulated VEGF secretion from THP-1 dose-dependently. VEGF mRNA expression in HCAECs was also upregulated by Ox-LDL. The mRNA expression of VEGF in THP-1 cells and HCAECs was also augmented by PPARγ activators, troglitazone (TRO), and 15-deoxy-Δ 12,14 -prostaglandin J 2 (PGJ2). In contrast, VEGF expression in another monocyte/macrophage cell line, human histiocytic lymphoma cells (U937), which lacks PPARγ expression, was not augmented by TRO or PGJ2. We established the U937 cell line, which permanently expresses PPARγ (U937T). TRO and Ox-LDL augmented VEGF expression in U937T. In addition, VEGF production by THP-1 cells was significantly increased by exposure to 9-HODE and 13-HODE. In conclusion, Ox-LDL upregulates VEGF expression in macrophages and endothelial cells, at least in part, through the activation of PPARγ.