Yasumi Shintani

Glucocorticoid Excess Induces Superoxide Production in Vascular Endothelial Cells and Elicits Vascular Endothelial Dysfunction

Abstract— Glucocorticoid (GC) excess often elicits serious adverse effects on the vascular system, such as hypertension and atherosclerosis, and effective prophylaxis for these complications is limited. We sought to reveal the mechanism underlying GC-induced vascular complications. Responses in forearm blood flow to reactive hyperemia in 20 GC-treated patients were significantly decreased to 43±8.9% (mean±SEM) from the values obtained before GC therapy (130±14%). An administration of vitamin C almost normalized blood flow responses. In human umbilical vein endothelial cells (HUVECs), production of hydrogen peroxide was increased up to 166.5±3.3% of control values by 10−7 mol/L dexamethasone (DEX) treatment (P <0.01). Concomitant with DEX-induced hydrogen peroxide production, intracellular amounts of peroxynitrite significantly increased and those of nitric oxide (NO) decreased, respectively (P <0.01). Immunoblotting analysis using anti-nitrotyrosine antibody showed that peroxynitrite formation was increased in DEX-treated HUVECs. Using inhibitors against metabolic pathways for generation of reactive oxygen species (ROS), we identified that the major production sources of ROS by DEX treatment were mitochondrial electron transport chain, NAD(P)H oxidase, and xanthine oxidase. These findings suggest that GC excess causes overproduction of ROS and thereby perturbs NO availability in the vascular endothelium, leading to vascular complications in patients with GC excess.

Potent induction of activin A secretion from monocytes and bone marrow stromal fibroblasts by cognate interaction with activated T cells.

Activin A is a multifunctional cytokine essential for cell differentiation and apoptosis including erythroid cell differentiation in the bone marrow. In addition, activin A is induced by inflammation and exerts anti‐inflammatory effects. However, the mechanism of activin A induction is still unclear, especially by inflammatory processes. Here we show that activin A secretion from monocytes and bone marrow stromal fibroblasts, its major sources in the bone marrow, is markedly enhanced by cognate interaction with activated T cells. This process is mediated by CD40/CD40 ligand interaction as well as concomitantly secreted T cell‐derived cytokines, granulocyte macrophage‐colony stimulating factor, and interferon‐γ. Furthermore, stromal fibroblasts as well as monocytes provide a costimulatory signal to anti‐CD3‐treated T cells via CD80 and CD86 to maintain the enhanced activin A production. These findings suggest that activin A is potently induced in the bone marrow and may play a role in the suppression of inflammatory or immune processes.

Interleukin-1 β enhances and interferon-γ suppresses activin A actions by reciprocally regulating activin A and follistatin secretion from bone marrow stromal fibroblasts

Activin A is a multi‐functional cytokine with a potent stimulation on erythroid cell differentiation in the bone marrow. The actions of activin A are determined by a balance of the levels of activin A and its inhibitor, follistatin (FS). However, the regulation of its actions in the bone marrow has been unclear. Here we show that bone marrow‐derived stromal fibroblasts are the major source of activin A and FS in the bone marrow, and that the production of activin A is enhanced by interleukin‐1β (IL‐1β) and lipopolysaccharide (LPS), whereas interferon‐γ (IFN‐γ) inhibits the secretion of activin A by stromal fibroblasts. Concomitantly, IL‐1β as well as LPS inhibits and IFN‐γ stimulates FS secretion from stromal fibroblasts. Thus, these cytokines potently regulate activin A actions by reciprocal modulation of activin A and FS secretion from stromal fibroblasts. Because activin A exhibits anti‐inflammatory effects in various tissues, up‐regulation of activin A actions by IL‐1β and endotoxin in the bone marrow may play a protective role against inflammatory processes as well as anaemia. The present results also suggest that the inhibitory effect of IFN‐γ on erythropoiesis is mediated at least in part by a suppression of activin A actions in bone marrow.

Serum follistatin concentrations are increased in patients with septicaemia

Follistatin (FS) is the specific binding protein of activin, a growth and differentiation factor of many cell types. Both factors have almost ubiquitous tissue distributions. In vitro, FS is secreted by vascular endothelial cells and this can be stimulated by bacterial compounds. For this reason, serum FS levels were examined in patients with septicaemia.

Radioimmunoassay for activin A/EDF: Method and measurement of immunoreactive activin A/EDF levels in various biological materials

A radioimmunoassay (RIA) for the measurement of activin A, which is identical to erythroid differentiation factor (EDF), has been developed. A specific antiserum against activin A/EDF was raised in rabbits using a mixture of recombinant EDF and polyvinyl pyrrolidone. Of the compounds tested this polyclonal antibody cross-reacted only with bovine inhibin (3.2%) and human TGF-beta (4.2%). The least detectable value in this assay was 0.06 ng/tube. The within- and between-assay coefficients of variation at three different concentrations were 3.6-9.8% and 3.4-7.7%, respectively. Using this RIA, immunoreactive activin A/EDF levels in various biological fluids and tissues were examined. The dose-response curves of porcine follicular fluid and ovarian extract were parallel to the standard curve, and porcine follicular fluid contained high activin A/EDF immunoreactivity (1050 ng/ml). On gel chromatography of porcine follicular fluid, the major immunoreactivity was eluted in the same position as authentic activin A/EDF. Human placental extract and amniotic fluid had relatively high immunoreactive activin A/EDF levels (174 ng/g wet wt. and 63.9 ng/ml, respectively), but the dose-response curve of amniotic fluid was not parallel to the standard curve. Among rat tissues, the ovary showed the highest activin A/EDF immunoreactivity (163 ng/g wet wt.) much lower than that of porcine ovary (1020 ng/g wet wt.). Low immunoreactive activin A/EDF levels were detected in most parts of rat brain (8.7-14.2 ng/g wet wt.), except for the pituitary gland (70.0 ng/g wet wt.). The initial plasma half clearance time (t1/2) of exogenous activin A/EDF was 14 min in the rat and the plasma FSH concentration did not change significantly during this period. These results suggest that this RIA system has sufficient sensitivity and specificity to measure activin A/EDF concentrations in biological materials, and that the reproductive tissues are the main sources of activin A/EDF.

Lung cancer containing growth hormone-releasing hormone associated with hypertrophic osteoarthropathy. Case report.

In a 49-year-old man, large-cell carcinoma of the lung was accompanied by raised serum levels of growth hormone (GH) and growth hormone-releasing hormone (GHRH) and hypertrophic osteoarthropathy. Immunohistochemically the tumour contained GHRH but not GH. The osteoarthropathy disappeared after resection of the primary tumour and did not reappear after its recurrence. The high serum GH level presumably was due to ectopic GHRH production in the tumour. The hypertrophic osteoarthropathy was not clearly attributable to these hormones.

Inhibition of Fibrin Monomer Polymerization by Bence Jones Protein in a Patient with Primary Amyloidosis

The mechanism of inhibition of fibrin monomer polymerization was studied in a patient with primary amyloidosis. Thrombin and reptilase times of the patients purified fibrinogen (Fbg) were remarkably prolonged, and polymerization of the patients fibrin monomer was disturbed. Fbg-Bence Jones protein (BJP) complex was demonstrated by immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis on the patients purified Fbg. The patients BJP not only prolonged the thrombin time of normal Fbg but also inhibited the polymerization of normal fibrin monomer. These results suggested that in this patient fibrin monomer polymerization was inhibited by binding of BJP to Fbg.

Immunoassays for Activin and Follistatin: Results in Normal and Diseased Subjects

Activin (ACT) and inhibin (INH) are gonadal proteins purified on the basis of bioactivity to stimulate or inhibit follicle-stimulating hormone (FSH) secretion from the pituitary (1). Activin is a 25-kDa homodimeric protein composed of the two β-subunits of INH linked by interchain disulfide bonds, and it comprises three different forms (β A β A, β A β B, and β B β B). Follistatin (FS) is a monomeric glycosylated protein isolated from ovarian follicular fluid (FF), and is now known to neutralize the action of ACT by forming a stable complex with it (2–4). Molecular cloning has revealed the presence of two FS core proteins of 288 and 315 amino acids (FS-288 and FS-315, respectively) as a result of the alternative splicing of the precursor messenger ribonucleic acid (mRNA) (5). Further work by Sugino et al. identified six molecular forms of porcine FS caused by truncation and glycosylation (6).

Localization of activin A/EDF in the normal and diabetic rat pancreas: Immunohistochemical andIn Situ hybridization studies

We examined the immunohistochemical localization of activin A/erythroid differentiation factor (EDF) in the pancreases of normal and diabetic rats with insulin-dependent diabetes mellitus or noninsulin-dependent diabetes mellitus to elucidate how activin A/EDF modulates insulin secretion. In both the normal and the diabetic pancreas, all of the cells staining with antirecombinant human (rh) activin A/EDF antiserum were insulin-producing B-cells, and they gradually decreased in number with the development of diabetic changes in both types of diabetic rats. No rh activin A/EDF immunoreactivity was detected in A-, D-, or pancreatic polypeptide (PP) cells in the islets, or in the exocrine cells. Thein situ hybridization (ISH) method showed that activin A/EDF mRNA is localized in activin A/EDF-positive cells. These data indicated that activin A/EDF localizes in insulin-producing B-cells of normal and diabetic pancreases, and may act as an autocrine modulator of insulin secretion.

Deoxycorticosterone-secreting adrenocortical carcinoma

A case of DOC-secreting adrenocortical carcinoma in a 66-year-old man is reported. He had hypertension, hypokalemia, suppressed PRA, and excessive serum levels of DOC. His serum aldosterone level was normal. The resected adrenal mass weighed 230 g. Histologically, the tumor was mainly composed of compact cells associated with necrosis and atypical mitoses. Invasion of venous structure, sinusoids, and capsule was also present. Immunohistochemically, P450 C21 (21 -hydroxylase) was positive in many tumor cells, and P450 C17(17 α-hydroxylase) was intensely positive in a relatively small number of tumor cells. The patient died 9 months after operation due to rupture of metastatic liver tumor.Endocr Pathol 4:165–168, 1993.