Yasukazu Ohmoto

Plasma Concentrations of a Novel, Adipose-Specific Protein, Adiponectin, in Type 2 Diabetic Patients

Adiponectin is a novel, adipose-specific protein abundantly present in the circulation, and it has antiatherogenic properties. We analyzed the plasma adiponectin concentrations in age- and body mass index (BMI)-matched nondiabetic and type 2 diabetic subjects with and without coronary artery disease (CAD). Plasma levels of adiponectin in the diabetic subjects without CAD were lower than those in nondiabetic subjects (6.6+/-0.4 versus 7.9+/-0.5 microg/mL in men, 7.6+/-0.7 versus 11.7+/-1.0 microg/mL in women; P<0.001). The plasma adiponectin concentrations of diabetic patients with CAD were lower than those of diabetic patients without CAD (4.0+/-0.4 versus 6.6+/-0.4 microg/mL, P<0.001 in men; 6.3+/-0.8 versus 7.6+/-0. 7 microg/mL in women). In contrast, plasma levels of leptin did not differ between diabetic patients with and without CAD. The presence of microangiopathy did not affect the plasma adiponectin levels in diabetic patients. Significant, univariate, inverse correlations were observed between adiponectin levels and fasting plasma insulin (r=-0.18, P<0.01) and glucose (r=-0.26, P<0.001) levels. In multivariate analysis, plasma insulin did not independently affect the plasma adiponectin levels. BMI, serum triglyceride concentration, and the presence of diabetes or CAD remained significantly related to plasma adiponectin concentrations. Weight reduction significantly elevated plasma adiponectin levels in the diabetic subjects as well as the nondiabetic subjects. These results suggest that the decreased plasma adiponectin concentrations in diabetes may be an indicator of macroangiopathy.

Adiponectin, an Adipocyte-Derived Plasma Protein, Inhibits Endothelial NF-κB Signaling Through a cAMP-Dependent Pathway

BackgroundAmong the many adipocyte-derived endocrine factors, we found an adipocyte-derived plasma protein, adiponectin, that was decreased in obesity. We recently demonstrated that adiponectin inhibited tumor necrosis factor-&agr; (TNF-&agr;)–induced expression of endothelial adhesion molecules and that plasma adiponectin level was reduced in patients with coronary artery disease (Circulation. 1999;100:2473–2476). However, the intracellular signal by which adiponectin suppressed adhesion molecule expression was not elucidated. The present study investigated the mechanism of modulation for endothelial function by adiponectin. Methods and ResultsThe interaction between adiponectin and human aortic endothelial cells (HAECs) was estimated by cell ELISA using biotinylated adiponectin. HAECs were preincubated for 18 hours with 50 &mgr;g/mL of adiponectin, then exposed to TNF-&agr; (10 U/mL) or vehicle for the times indicated. NF-&kgr;B–DNA binding activity was determined by electrophoretic mobility shift assays. TNF-&agr;–inducible phosphorylation signals were detected by immunoblotting. Adiponectin specifically bound to HAECs in a saturable manner and inhibited TNF-&agr;–induced mRNA expression of monocyte adhesion molecules without affecting the interaction between TNF-&agr; and its receptors. Adiponectin suppressed TNF-&agr;–induced I&kgr;B-&agr; phosphorylation and subsequent NF-&kgr;B activation without affecting other TNF-&agr;–mediated phosphorylation signals, including Jun N-terminal kinase, p38 kinase, and Akt kinase. This inhibitory effect of adiponectin is accompanied by cAMP accumulation and is blocked by either adenylate cyclase inhibitor or protein kinase A (PKA) inhibitor. ConclusionsThese observations raise the possibility that adiponectin, which is naturally present in the blood stream, modulates the inflammatory response of endothelial cells through cross talk between cAMP-PKA and NF-&kgr;B signaling pathways.

Adipocyte-Derived Plasma Protein, Adiponectin, Suppresses Lipid Accumulation and Class A Scavenger Receptor Expression in Human Monocyte-Derived Macrophages

Background —Excessive lipid accumulation in macrophages plays an important role in the development of atherosclerosis. Recently, we discovered an adipocyte-specific plasma protein, adiponectin, that is decreased in patients with coronary artery disease. We previously demonstrated that adiponectin acts as a modulator for proinflammatory stimuli and inhibits monocyte adhesion to endothelial cells. The present study investigated the effects of adiponectin on lipid accumulation in human monocyte-derived macrophages. Methods and Results —Human monocytes were differentiated into macrophages by incubation in human type AB serum for 7 days, and the effects of adiponectin were investigated at different time intervals. Treatment with physiological concentrations of adiponectin reduced intracellular cholesteryl ester content, as determined using the enzymatic, fluorometric method. The adiponectin-treated macrophages contained fewer lipid droplets stained by oil red O. Adiponectin suppressed the expression of the class A macrophage scavenger receptor (MSR) at both mRNA and protein levels by Northern and immunoblot analyses, respectively, without affecting the expression of CD36, which was quantified by flow cytometry. Adiponectin reduced the class A MSR promoter activity, as measured by luciferase reporter assay. Adiponectin treatment dose-dependently decreased class A MSR ligand binding and uptake activities. The mRNA level of lipoprotein lipase as a marker of macrophage differentiation was decreased by adiponectin treatment, but that of apolipoprotein E was not altered. Adiponectin was detected around macrophages in the human injured aorta by immunohistochemistry. Conclusions —The adipocyte-derived plasma protein adiponectin suppressed macrophage-to-foam cell transformation, suggesting that adiponectin may act as a modulator for macrophage-to-foam cell transformation.

Adipocyte-Derived Plasma Protein Adiponectin Acts as a Platelet-Derived Growth Factor-BB–Binding Protein and Regulates Growth Factor–Induced Common Postreceptor Signal in Vascular Smooth Muscle Cell

Background—Vascular smooth muscle cell proliferation plays an important role in the development of atherosclerosis. We previously reported that adiponectin, an adipocyte-specific plasma protein, accumulated in the human injured artery and suppressed endothelial inflammatory response as well as macrophage-to-foam cell transformation. The present study investigated the effects of adiponectin on proliferation and migration of human aortic smooth muscle cells (HASMCs). Methods and Results—HASMC proliferation was estimated by [3H] thymidine uptake and cell number. Cell migration assay was performed using a Boyden chamber. Physiological concentrations of adiponectin significantly suppressed both proliferation and migration of HASMCs stimulated with platelet-derived growth factor (PDGF)-BB. Adiponectin specifically bound to 125I-PDGF-BB and significantly inhibited the association of 125I-PDGF-BB with HASMCs, but no effects were observed on the binding of 125I-PDGF-AA or 125I-heparin–binding epidermal growth factor (EGF)–like growth factor (HB-EGF) to HASMCs. Adiponectin strongly and dose-dependently suppressed PDGF-BB–induced p42/44 extracellular signal–related kinase (ERK) phosphorylation and PDGF &bgr;-receptor autophosphorylation analyzed by immunoblot. Adiponectin also reduced PDGF-AA–stimulated or HB-EGF–stimulated ERK phosphorylation in a dose-dependent manner without affecting autophosphorylation of PDGF &agr;-receptor or EGF receptor. Conclusions—The adipocyte-derived plasma protein adiponectin strongly suppressed HASMC proliferation and migration through direct binding with PDGF-BB and generally inhibited growth factor–stimulated ERK signal in HASMCs, suggesting that adiponectin acts as a modulator for vascular remodeling.

Macrophage infiltration correlates with tumor stage and angiogenesis in human malignant melanoma: Possible involvement of TNFα and IL‐1α

We examined whether macrophage infiltration is associated with angiogenesis in cutaneous melanoma. The numbers of macrophages and microvessels increased significantly with increasing depth of tumor and with tumor angiogenesis. Macrophage infiltration thus appeared to provide a useful diagnostic marker for the progression of cutaneous melanoma. We further examined whether human melanoma cells produce angiogenic factors in response to macrophage‐derived cytokines, tumor necrosis factor alpha (TNFα) and interleukin‐1 alpha (IL‐1α). Treatment of melanoma cells with TNFα and IL‐1α in vitro enhanced the production of interleukin‐8 (IL‐8) and vascular endothelial growth factor (VEGF), and of basic fibroblast growth factor (bFGF) to a lesser degree, in human melanoma cells. Lipopolysaccharide (LPS)‐activated human monocytes enhanced production of IL‐8, VEGF, TNF α, as well as IL‐1α, but not bFGF. Co‐culture of human monocytes and human melanoma cells was also found to significantly enhance production of IL‐8 and VEGF in the absence and presence of LPS, compared with either monocytes or melanoma cells alone. The production of IL‐8 and VEGF from co‐cultured melanoma cells and LPS‐activated monocytes was blocked when anti‐TNF‐α antibody or anti‐IL‐1α antibody was co‐administrated. This is direct evidence that production of the potent angiogenic factors IL‐8 and VEGF from melanoma cells is up‐regulated through TNFα and/or IL‐1α secreted by activated monocytes/macrophages, influencing both tumor growth and angiogenesis in melanomas. Int. J. Cancer 85:182–188, 2000. ©2000 Wiley‐Liss, Inc.

Decreased food intake and body weight in pancreatic polypeptide-overexpressing mice

BACKGROUND & AIMS Pancreatic polypeptide (PP) is a 36-amino acid hormone produced by F cells within the pancreatic islets and the exocrine pancreas. The definitive function of PP in mammalian physiology remains to be determined. This study examined the effects of chronic overexpression of PP through the development of PP transgenic mice. METHODS PP transgenic mice were created by using mouse PP complementary DNA under the control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter (pCAGGS expression vector). RESULTS A unique line of transgenic mice was created that overexpresses PP in the pancreatic islets with low levels of expression in other tissues including the brain. Plasma PP concentrations were more than 20 times higher than those of control littermates. However, PP overproduction led to postnatal lethality in half of the pups because of markedly decreased milk intake. The remaining PP transgenic mice gained less weight with specifically reduced food intake and fat mass compared with controls, a result that was more evident in male than in female mice. The transgenic mice exhibited a reduced rate of gastric emptying of a solid meal but had normal oxygen consumption and fasting leptin levels. Immunoneutralization with anti-PP antiserum reversed the phenotypic changes of transgenic animals. CONCLUSIONS PP could be involved in feeding and body weight regulation partly through regulation of gastric emptying.

Transfection of interleukin-8 increases angiogenesis and tumorigenesis of human gastric carcinoma cells in nude mice

The growth and spread of tumour cells depends on adequate vasculature. We have previously reported that the expression of interleukin-8 (IL-8) directly correlates with the vascularity of human gastric carcinomas. To provide evidence for a causal role of IL-8 in angiogenesis and tumorigenicity of human gastric cancer, we used the lipofectin method to stably transfect the human TMK-1 gastric carcinoma cells (low endogenous IL-8) with an IL-8 expression vector or control vector. Transfection with IL-8 did not affect the proliferation of cultured cells, yet the culture supernatants of the transfected (but not control) cells stimulated proliferation of human umbilical vein endothelial cells. The IL-8-transfected and control cells were injected into the gastric wall of nude mice. IL-8-transfected cells produced rapidly growing, highly vascular neoplasms as compared to control cells. These results provide direct evidence for the role of IL-8 in the angiogenesis and tumorigenicity of human gastric carcinomas.

Expression of interleukin-12 in synovial tissue from patients with rheumatoid arthritis.

OBJECTIVE To examine the importance of interleukin-12 (IL-12) as a factor in the interferon-gamma (IFNgamma)-dominant T cell cytokine response in the synovial tissue of patients with rheumatoid arthritis (RA). METHODS The expression of IL-12 in synovial tissue samples from patients with chronic RA (> or = 2 years) was compared with that in samples from osteoarthritis (OA) patients by detection of IL-12 p40 messenger RNA (mRNA) using reverse transcriptase-polymerase chain reaction, measurement of IL-12 p70 protein in culture supernatants of tissue cells by immunoassay, and immunostaining of tissue sections with anti-IL-12 p70. The production of IFNgamma by RA synovial tissue cells cultured with or without IL-12 was determined. In addition, T cells were obtained 14 days after culturing RA synovial tissue cells with IL-2 alone or with IL-2 plus IL-12, neutralizing anti-IL-12, or IL-4, and cytokine patterns (i.e., IFNgamma and IL-4 levels) were determined by stimulating cells for 24 hours with anti-CD3 plus phorbol myristate acetate. RESULTS Synovial tissues from RA patients more strongly expressed IL-12 p40 mRNA than did OA tissues. Dissociated tissue cells from 21 of 37 RA patients spontaneously released detectable amounts of IL-12 p70 (> or = 12.5 pg/ml) in culture, whereas production of IL-12 by OA tissues was limited. By immunohistochemical analysis, IL-12-producing cells were localized mainly in the sublining layer of RA synovium, and mostly expressed the CD68 antigen. Levels of IFNgamma production by RA synovial tissue cells were potently and selectively enhanced by IL-12. The ability of IL-2-expanding synovial T cells to produce IFNgamma was augmented by costimulation with IL-12 and diminished by anti-IL-12, while it was not affected by IL-4. CONCLUSION These data suggest that IL-12, produced mainly by macrophage-lineage cells, may be involved in IFNgamma-dominant cytokine production by infiltrating T cells in joints with chronic RA.

Interferon‐γ–inducing activity of interleukin‐18 in the joint with rheumatoid arthritis

Objective To examine the levels of interleukin-18 (IL-18) bioactivity within the rheumatoid arthritis (RA) joint, and the differential effects of IL-12 and IL-18 on interferon-γ (IFNγ) production by T cell infiltrates. Methods Expression of IL-18 protein and messenger RNA (mRNA) was determined by enzyme-linked immunosorbent assay and reverse transcriptase–polymerase chain reaction, respectively. The biologic activity of IL-18 was detected on the basis of IFNγ secretion from IL-18–responding human myelomonocytic KG-1 cells. To determine the extent of inhibitory activity on binding of IL-18 to its receptor, a [125I]–IL-18 binding inhibition assay was performed, using a Chinese hamster ovary cell line transfected with a murine IL-18 receptor. Results The amount of IL-18 protein detected in both the serum and synovial fluid of RA patients was markedly larger than that detected in the serum and synovial fluid of osteoarthritis (OA) patients, and serum IL-18 levels correlated with the levels of serum C-reactive protein. IFNγ production by KG-1 cells was more strongly stimulated in synovial fluid samples from RA patients than in samples from OA patients, and this activity was largely diminished in the presence of anti–IL-18 antibody. In contrast, the activity of IL-18 binding inhibition in the serum and synovial fluid of RA patients was not significantly elevated compared with that in OA patients. RA synovial tissues showed increased expression of IL-18 mRNA and increased IL-18 protein synthesis compared with that in OA tissues. Purified CD14+ macrophages, but not activated fibroblast cell lines, from RA synovium were able to release mature IL-18, although both cell types expressed its transcripts. IL-18 alone showed a negligible effect on IFNγ production by RA synovial tissue cells, in contrast to IL-12, which was directly stimulatory. However, IL-12–induced IFNγ production was synergistically enhanced by IL-18, and yet was >50% reduced by neutralization of endogenous IL-18 with anti–IL-18 antibody. Conclusion These results indicate that IL-18, produced predominantly by tissue macrophages, primarily potentiates IL-12–induced IFNγ production by T cell infiltrates in RA synovium. Detection of significant IL-18 bioactivity in the joints, despite the presence of IL-18 binding inhibitors, supports an integral role of this cytokine in perpetuating the IFNγ-dominant T cell cytokine response in RA.

Interleukin 8 and vascular endothelial growth factor — prognostic factors in human gastric carcinomas?

Gastric carcinoma cells express potent angiogenic factors including vascular endothelial growth factor (VEGF). We previously reported that interleukin-8 (IL-8) acts as an angiogenic factor for human gastric carcinomas. More recently, we found that IL-8 upregulates matrix metalloproteinase-9 (MMP-9) expression and increases invasive activity of gastric carcinoma cells. The purpose of this study was to determine whether the expression of IL-8 and VEGF correlates with clinicopathological parameters in human gastric carcinomas. IL-8 and VEGF expression levels were measured by an enzyme-linked immunosorbent assay (ELISA) in 56 gastric carcinomas and the surrounding normal mucosa. Macroscopic and histopathological tumour findings, presence of metastasis and prognosis were obtained from the patient records and endoscopic, surgical and pathological reports. IL-8 protein levels were higher in most neoplasms than in the corresponding normal mucosal tissue. In contrast, VEGF expression in the tumours was similar to that in normal mucosa. The IL-8 level in the neoplasms correlated significantly with the depth of invasion, venous invasion and lymphatic invasion. VEGF expression in the tumours correlated well with the depth of invasion and lymph node metastasis. No correlation between IL-8 and VEGF expression in the tumours was observed. The survival rates of patients with tumours displaying high IL-8 and VEGF expression levels were significantly lower (P<0.05) than those of patients with tumours displaying low IL-8 and VEGF expression. The results suggest that IL-8 and VEGF may be independent and important prognostic factors in human gastric carcinomas.