Quercetin Inhibits IL-1β-Induced Inflammation, Hyaluronan Production and Adipogenesis in Orbital Fibroblasts from Graves' Orbitopathy

Management of Graves' orbitopathy (GO) is challenging, as no reliable, specific, and safe medical therapeutic agents have yet been developed. We investigated the effect of quercetin in primary cultured orbital fibroblasts from GO, targeting pathways of inflammation, aberrant accumulation of extracellular matrix macromolecules, and adipose tissue expansion. Quercetin significantly attenuated intercellular adhesion molecule-1 (ICAM-1), interleukin (IL) -6, IL-8, and cyclooxygenase (COX) -2 mRNA expression, and inhibited IL-1β-induced increases in ICAM-1, IL-6, and IL-8 mRNA. Increased hyaluronan production induced by IL-1β or tumor necrosis factor-α was suppressed by quercetin in a dose- and time-dependent manner. Treatment with noncytotoxic doses of quercetin inhibited accumulation of intracytoplasmic lipid droplets and resulted in a dose-dependent decrease in expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer-binding protein (C/EBP) α, and C/EBPβ proteins. In conclusion, inhibition of inflammation, hyaluronan production, and adipogenesis by the natural plant product quercetin in vitro provides the basis for further study of its potential use in the treatment of GO

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Cyclic ADP-Ribose (cADPR) Mediate Ca2+ Signaling in Cardiac Hypertrophy Induced by β-Adrenergic Stimulation.

Ca2+ signaling plays a fundamental role in cardiac hypertrophic remodeling, but the underlying mechanisms remain poorly understood. We investigated the role of Ca2+-mobilizing second messengers, NAADP and cADPR, in the cardiac hypertrophy induced by β-adrenergic stimulation by isoproterenol. Isoproterenol induced an initial Ca2+ transients followed by sustained Ca2+ rises. Inhibition of the cADPR pathway with 8-Br-cADPR abolished only the sustained Ca2+ increase, whereas inhibition of the NAADP pathway with bafilomycin-A1 abolished both rapid and sustained phases of the isoproterenol-mediated signal, indicating that the Ca2+ signal is mediated by a sequential action of NAADP and cADPR. The sequential production of NAADP and cADPR was confirmed biochemically. The isoproterenol-mediated Ca2+ increase and cADPR production, but not NAADP production, were markedly reduced in cardiomyocytes obtained from CD38 knockout mice. CD38 knockout mice were rescued from chronic isoproterenol infusion-induced myocardial hypertrophy, interstitial fibrosis, and decrease in fractional shortening and ejection fraction. Thus, our findings indicate that β-adrenergic stimulation contributes to the development of maladaptive cardiac hypertrophy via Ca2+ signaling mediated by NAADP-synthesizing enzyme and CD38 that produce NAADP and cADPR, respectively

A schematic model showing signaling pathways underlying β-AR-mediated NAADP-synthesizing enzyme and CD38 activation.

<p>Binding of ISO to β-AR stimulates AC, thus activating PKA. PKA induces Ca²⁺ influx that leads to activation of an NAADP-synthesizing enzyme (NSE) to produce NAADP. NAADP-mediated Ca²⁺ mobilization from the acidic Ca²⁺ stores results in activation of CD38. cADPR produced by CD38 in the endocytic vesicles induces Ca²⁺ release from SR Ca²⁺ stores. cADPR-mediated Ca²⁺ release induces SOCE, resulting in a sustained Ca²⁺ signal. NE: norepinephrine; SR: sarcoplasmic reticulum; NSE: NAADP-synthesizing enzyme; AC, adenylyl cyclase; PKA, protein kinase A; SOCE, store-operated Ca²⁺ entry.</p

CD38 KO mice are protected from the ISO-mediated cardiac hypertrophic response.

<p><b>(A)</b> Histological analysis of hematoxylin/eosin stained heart sections from WT and CD38 KO mice following vehicle/ISO infusion. <b>(B)</b> The mean cross-sectional areas and heart weight/body weight (HW/BW) ratios of WT and CD38 KO mice were calculated following vehicle/ISO infusion. <b>(C and D)</b> Masson’s trichrome staining reveals substantial ventricular interstitial fibrosis in WT hearts compared to CD38 KO hearts. <b>(E and F)</b> Echocardiography assessment of fractional shortening (FS) and ejection fraction (EF) following vehicle/ISO infusion in WT and CD38 KO mice. <b>(G-I)</b> ADPR cyclase activity and cADPR and NAADP formation in WT and CD38 KO mice following vehicle/ISO infusion. *, P<0.05 versus WT. #, P<0.05 versus CD38 KO. §, P<0.05 versus WT + ISO. Values are the mean ± SEM of three independent experiments.</p

ISO-stimulated cADPR and NAADP production in cardiomyocytes.

<p><b>(A)</b> Time course of cADPR and NAADP production following ISO treatment. <b>(B and C)</b> Differential effects of Ca²⁺ second messenger inhibitors on ISO-induced cADPR and NAADP formation. *, P<0.05 versus control (Con) cADPR level. #, P<0.05 versus control (Con) NAADP level. §, P<0.05 versus ISO-induced cADPR level. ¶, P<0.05 versus ISO-induced NAADP level. Values are the mean ± SEM of three independent experiments.</p

Echocardiographic assessment of CD38 WT and CD38KO mice at baseline.

<p>Echocardiographic assessment of CD38 WT and CD38KO mice at baseline.</p

ISO-stimulated Ca²⁺ increase and cADPR and NAADP production in wild-type (WT) and CD38 knock-out (KO) mice.

<p><b>(A)</b> Representative tracings of the Ca²⁺ response to ISO in cardiomyocytes obtained from WT and CD38 KO mice. (right panel) A direct comparison of the mean [Ca²⁺]_i during sustained increases in [Ca²⁺]_i. The data shown are analyzed at 150 s. <b>(B)</b> ISO-stimulated cADPR production in WT and CD38 KO mice. <b>(C)</b> ISO-stimulated NAADP production in WT and CD38 KO mice. *, <i>P</i>< 0.05 versus WT controls. #, <i>P</i> < 0.05 versus CD38 KO controls. §, P<0.05 versus WT + ISO. Values are the mean ± SEM of three independent experiments.</p

ISO-stimulated Ca²⁺ increase in cardiomyocytes.

<p><b>(A)</b> Representative tracings of the [Ca²⁺]_i response to 2 μM ISO in the presence of an adenylate cyclase inhibitor and a cAMP antagonist. <b>(B, C)</b> Representative tracings of the [Ca²⁺]_i response to 2 μM ISO in the presence of thapsigargin (Thap), 8-Br-cADPR, xestospongine C (XesC), or bafilomycin A1 (Baf). <b>(D)</b> Representative tracings of the [Ca²⁺]_i response to 2 μM ISO or 50 nM NAADP-AM in the presence or absence of extracellular Ca²⁺. <b>(E)</b> Representative tracings of the [Ca²⁺]_i response to NAADP-AM after pretreatment with or without 8-Br-cADPR. <b>(F)</b> Representative tracings of the [Ca²⁺]_i response to 100 nM deaza-cADPR in the absence and presence of extracellular Ca²⁺. <b>(G)</b> Representative tracings of the [Ca²⁺]_i response to 2 μM ISO in cardiomyocytes after transfection with scrambled or Stim1 siRNA. Values are the mean ± SEM of three independent experiments.</p

Outcome of adult severe or very severe aplastic anemia treated with immunosuppressive therapy compared with bone marrow transplantation: Multicenter trial

To compare survival rates and long-term complications after bone marrow transplantation (BMT) or treatment with immunosuppressive agents (ISA) in the management of adult aplastic anemia (AA) and to identify prognostic factors associated with improved survival, we evaluated 229 adult AA patients treated with ISA from 1990 to 2001 and compared the results with those for 64 BMT recipients. Of 156 patients with severe aplastic anemia (SAA) or very severe AA treated with ISA (antithymocyte globulin [ATG]or ATG plus cyclosporine), 46.8% showed complete or partial response and 7.1% had relapses. After long-term follow-up, 1 case each of acute leukemia, myelodysplastic syndrome, and paroxysmal nocturnal hemoglobinuria developed. The 6-year survival rate was 69%. Response to ISA, disease severity, and low absolute neutrophil count (ANC) (less than or equal to200/mm(3)) were associated with poor survival. Patient age, sex, initial platelet count, etiology, or treatment regimen did not significantly affect survival. Cox regression analysis showed low ANC to be the only pretreatment variable significantly associated with poor survival (P = .000). Of 64 BMT recipients, 82.8% had sustained engraftment, and 12.5% experienced graft failure. Twenty (31.3%) of the patients developed grade II to IV acute graft-versus-host disease (GVHD), and 12 (18.8%) of the patients developed chronic GVHD. The 6-year survival rate was 79%. Patient age and sex, disease severity, etiology, ANC, initial platelet count, and treatment regimen did not affect survival. Survival of 83 AA patients, aged 14 to 40 years, treated with ISA was not statistically significant from that of 61 adult AA patients who underwent BMT (6-year survival rate, 65% and 79%, respectively). However, BMT in adult AA achieved long-term engraftment and a lower relapse rate than ISA. These results suggest that ISA can achieve a high response rate and long-term survival among patients with adult AA, regardless of disease severity. Further studies with larger numbers of patients and long-term follow-up are needed. (C) 2003 The Japanese Society of Hematology