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Pentoxifylline-augmented antiproliferative effects of adrenomedullin on vascular smooth muscle cells

2000; Elsevier BV; Volume: 2; Issue: 3 Linguagem: Inglês

10.1016/s1388-9842(00)00072-6

1879-0844

Stefan Dunzendorfer, Christian Meierhofer, Qingbo Xu, Christian J. Wiedermann,

Peptidase Inhibition and Analysis

Adrenomedullin (ADM) is an endogenous peptide with both vasodilating and smooth muscle cell proliferation-inhibiting properties. It was first isolated from phaeochromocytoma and adrenal gland but is also found in the heart, lungs and kidneys. In addition to its endocrine capacities paracrine properties have been discussed for ADM, because endothelial and smooth muscle cells (SMCs) synthesize ADM and express its receptors [1–3]. ADM has vasodilating and positive inotropic effects, enhances diuresis, and decreases mean arterial pressure, and pulmonary artery pressure. There are hard facts and evidence that ADM has beneficial hemodynamic effects in patients with congestive heart failure [4], where the plasma concentration of ADM increases in proportion to the severity of illness. The antiproliferative effect of ADM on SMCs depends on cyclic adenosine monophosphate (cAMP) elevation which is believed to be mediated by calcitonin gene-related peptide (CGRP) receptors and opposes vessel wall remodeling [5]. Pentoxifylline (PTX) improves left ventricular function in heart failure [6]. It may act by modulating endotoxin-triggered immune activation by virtue of its anti-tumor necrosis factor properties [7,8]. However, the phosphodiesterase blocker PTX increases cAMP and affects some tumor necrosis factor-independent pathways of inflammation [8], suggesting that additional mechanisms may be involved. It is conceivable that the dysregulated SMC growth (associated with heart failure) is due to the impaired ability of ADM to inhibit cell proliferation as a result of down regulation of ADM-receptor (CGRP-receptor 1)-coupled adenylate cyclase [9]. In that case, upregulation of cAMP by PTX can be expected to augment the antiproliferative ability of ADM. The aim of this study was to test in vitro whether the antiproliferative effect of ADM is augmented by PTX. If this is indeed the case, then it can be assumed that impaired antiproliferative effect of ADM on smooth muscle cells is a pathogenetic principle in heart failure and its correction by PTX might represent one possible mechanism of action of this methylxanthine. SMCs were obtained by enzymatic digestion from the aorta of Sprague–Dawley rats. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). SMCs were cultured in a humidified atmosphere of 95% air/5% CO2 in medium-199 (GIBCO BRL, Gaithersburg, MD, USA), supplemented with 10% fetal calf serum (FCS; Biological Industries, Kibbutz Beit Haemek, Israel), penicillin (100 U/ml), and streptomycin (100 μg/ml). These cultures in exponential growth were harvested every week using 0.05% trypsin/0.02% EDTA and passaged at a ratio of 1:3 into new culture flasks coated with gelatin 0.2% (Sigma Chemical Corp., St. Louis, MO, USA). Typical growth experiments were performed with SMCs at passage levels of 5–15. Cells were plated at a density of 5×103 cells/100 μl culture medium into 96-well culture plates. Cells were allowed to adhere for 24 h, then the medium was removed and cells were washed with phenol red-free Hanks’ balanced salt solution without Ca2+ and Mg2+ (HBSS; Gibco BRL Technologies, Vienna, Austria) and further incubated with medium-199/0.2% FCS for 48 h to establish quiescence. After washing, quiescent cells were treated with the following agents dissolved in medium-199/2.5% FCS for 48 h. Effects of PTX (Hoechst-Marion-Roussel Pharmaceuticals, Vienna, Austria) as a potent inhibitor of phosphodiesterases, the inhibitor of nitric oxide (NO) formation l-nitroarginine methylester (l-NAME), dibutyryl-cAMP, human recombinant platelet-derived growth factor AB (PDGF), angiotensin II (all Sigma Chemical Corp., St. Louis, MO, USA), vasoactive intestinal peptide (VIP), ADM and CGRP (all Neosystems, Strasbourg, France) on SMC growth were monitored using the colorimetric MTT assay [3-(4,5-dimethyldiazol-2yl)-2,5-diphenyl tetrazolium bromide; Sigma Chemical Corp., St. Louis, MO, USA]. This assay is based on the conversion of the tetrazolium salt MTT to formazan crystals. It detects living but not dead cells, and the absorbance generated is directly proportional to the number of cells [10]. Optical density was measured at 570 nm in an ELISA reader (Labsystems, Helsinki, Finland). Data are expressed as mean and S.E. Means were compared by Mann–Whitney U-test and Kruskal–Wallis non-parametric analysis of variance. P<0.05 was considered significant. N refers to the number of different cell cultures. Statistical analyses were performed using the StatView software package (Abacus Concepts, Berkley, CA, USA). ADM and CGRP each at 0.01 and 0.1 μmol/l inhibited FCS-stimulated increase in SMC growth; VIP (0.01 pmol/l–0.1 μmol/l) alone or in combination with each of the other peptides tested was inactive at any concentration (Fig. 1a). At 1 μmol/l the human beta-CGRP[8-37], a CGRP receptor antagonist, abolished antiproliferative effects of 0.01 μmol/l of ADM or CGRP (mean±S.E. optical densities as ratios of medium control: ADM vs. ADM/beta-CGRP[8-37], 0.83±0.032 vs. 0.96±0.023, n=4, P<0.05; CGRP vs. CGRP/beta-CGRP[8-37], 0.88±0.035 vs. 1.00±0.008, n=3, P<0.05); addition of PDGF (0.01 nmol/l) reversed the inhibitory effects of ADM or CGRP (CGRP vs. CGRP/PDGF, 0.78±0.046 vs. 1.08±0.041, n=6, P<0.01; ADM vs. ADM/PDGF, 0.80±0.034 vs. 1.02±0.060, n=6, P<0.01) indicating that growth inhibition is mediated by CGRP receptors and not due to toxic effects of the peptides on SMCs. PTX (1 mmol/l) mimicked the effects of ADM and CGRP by inhibiting cell proliferation (Fig. 1a). Concomidant administration of PTX (1 mmol/l) with ADM or CGRP (0.01 μmol/l) augmented the antiproliferative effects achieved by either of the substances alone (Fig. 1b), whereas combinations with l-NAME (0.01 μmol/l–1 mmol/l) showed no modifying effect (data not shown). As expected, elevation of intracellular cAMP with dibutyryl-cAMP (0.01 and 1 μmol/l) significantly augmented antiproliferative effects of 0.1 μmol/l CGRP on SMCs (CGRP vs. CGRP/dibutyryl-cAMP, 0.65±0.016 vs. 0.55±0.009 and 0.51±0.009, respectively, n=3, P<0.05). Angiotensin II (1 pmol/l–0.01 μmol/l) did not affect SMC growth (data not shown). Heart failure is characterized inter alia by an increase of the arterial vessel tone and vascular resistance. Since binding sites for ADM have also been found in the heart, it is supposed that ADM is an endogenous regulator of cardiac functions [11]. Cardiac myocytes and non-myocytic cells secrete almost equal amounts of ADM [12], which causes vasodilation via activation of receptor-dependent adenylate cyclase, thus increasing intracellular cAMP levels [9] and targeting vascular SMCs via activation of a cAMP–protein kinase A-sensitive signaling pathway [13]. Our results are in agreement with previous reports indicating that ADM inhibits FCS-stimulated proliferation of SMCs via CGRP receptors [3,5,9], because inhibitory effects of ADM and CGRP were abolished by the selective CGRP receptor antagonist beta-CGRP[8-37] in our experiments. NO production is inversely correlated with the progression of clinical severity of congestive heart failure as determined by the New York Heart Association class [14]. NO acts as the most potent vasodilating substance and inhibits SMC proliferation [15,16]. Its synthesis is modulated by ADM which activates NO synthase. In our experiments, the potent NO synthase inhibitor l-NAME failed to reverse the antiproliferative effects of ADM, suggesting a different main target of ADM in SMC growth. Vascular remodeling, which is characterized by dysfunction of endothelial cells and SMC proliferation, is thought to be augmented by angiotensin II involving a diversity of intracellular signal transduction cascades [17]. In our study angiotensin II failed to attenuate antiproliferative effects of ADM, indicating distinct cellular targets for ADM in affecting growth regulation of SMCs. In contrast to the above-mentioned mechanisms we found cAMP-dependent and, therefore, PTX-sensitive regulation of SMC proliferation. Since ADM induces cAMP elevation via CGRP-receptor 1 and PTX activates the cAMP-effector pathway [8], which augments the antiproliferative effects of ADM, we suggest that the beneficial effects of PTX in heart failure may be achieved by growth inhibition of SMCs synergistically with ADM. Thus, PTX may antagonize elevation of peripheral vascular resistance in heart failure. We, therefore, encourage further studies measuring parameters of vessel remodeling in heart failure after PTX treatment. We thank Mrs Rajam Csordas-Iyer for editorial assistance and critical reading of the manuscript.