Trapidil Inhibits Human Mesangiai Cell Proliferation: Effect on PDGF Β
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Kidney International, Vol. 46 (1994), pp. 1002—1009 Trapidil inhibits human mesangial cell proliferation: Effect on PDGF a-receptor binding and expression LORETO GESUALDO, SALVATORE Di PAOLO, ELENA RANIERI, and FRANCESCO PAOLO SCHENA, with the technical assistance of ANNALISA BRUNACCINI Chair and Division of Nephrology, University of Bad, Ban, Italy Trapidil inhibits human mesangial cell proliferation: Effect on PDCF mediate, at least in part, the biological effect of other growth a-receptor binding and expression. Mesangial cell (MC) proliferation, a factors, as demonstrated for EGF, and elects PDGF as a potential histopathologic feature common to many human glomerular diseases, is regulated by several growth factors through their binding to specific cell autocrine regulator of MC proliferation [14]. surface receptors. Platelet-derived growth factor (PDGF) is a peptide MC play a pivotal role in the course of glomerular injury: they exerting a potent mitogenic activity on MC. Recently, an increasedfirst act as target cells for several mediators, which are released expression of both PDGF protein and its receptor has been localized in locally or are delivered through the systemic circulation [4, 15, 16]. the mesangial areas of several experimental as well as human proliferative Thereafter, they may drive the evolution of pathologic lesions glomerulonephritides (GN). Thus, it may be postulated that the inhibition of PDGF action could prevent MC proliferation during mesangial prolif- towards resolution or progression of the glomerular disease. MC erative GN. Trapidil, an antiplatelet drug, has been shown to inhibit the proliferation, a histopatologic feature common to different forms growth of several cell types both in vitro and in vivo. The present study was of glomerulonephritides [17], is likely to influence the regulation aimed at evaluating the effect of Trapidil on human MC in vitro. The of glomerular microvascular system and extracellular matrix pro- addition of 100 to 400 jsg/ml Trapidil significantly reduced cell prolifera- duction. tion induced by different growth factors (FCS, PDGF-BB, bFGF, EGF), the highest inhibitory effect being on PDGF-BB stimulated MC growth. Several experimental as well as human proliferative glomeru- The effect of the drug was dose-dependent and seemingly specific: aspirin lonephritides were shown to display an increased expression of was devoid of any anti-proliferative action, while dypiridamole proved to both PDGF protein and its receptor in mesangial cells [18—24]. be toxic. Receptor binding experiments showed that Trapidil competitively These data indicate PDGF as a growth factor primarily implicated inhibited 251-PDGF-BB binding to its cell surface receptors, without inducing receptor internalization, at least after short-term (2 hr) incuba- in the pathogenesis of mesangial proliferative glomerulonephrit- tion. In contrast, long-term (48 hr) exposure to 400 pg/mI Trapidil caused ides. Consequently, it may be postulated that the inhibition of a sharp increase of PDGF-BB binding. Similar effects on cell proliferation PDGF action could prevent MC proliferation and the activation and 25I-PDGF-BB binding were observed when NIH-3T3 fibroblasts were exposed to the test substance. Finally, we evaluated the influence ofof other growth factor pathways during mesangial proliferative Trapidil on PDGF 13-receptor gene expression and found that 24 hour glomerulonephritides. exposure to 400 pg/mI Trapidil moderately decreased steady-state mRNA Trapidil is an anti-platelet drug active against various aggregat- levels, whereas 48 hours of incubation increased PDGF 13-receptor ing agents, such as collagen, ADP, arachidonic acid, PAF and transcript levels by approximately threefold. We conclude that Trapidil is calcium ionophore [25, 26]. It exerts its action by blocking the able to strongly inhibit human MC proliferation, a process in whichbiosynthesis of thromboxane A2 and antagonizing its effect at the inhibition of PDGF-BB binding to its receptors and modulation of PDGF J3-receptor gene expression might be involved. receptor level, and by stimulating the synthesis and release of prostacyclin [25, 26]. Moreover, Trapidil has been shown to inhibit the growth of several cell types both in vitro and in vivo [27—31]. In the present study, we have explored the possibility that Platelet-derived growth factor (PDGF) exerts a striking mitogenicTrapidil exerts an antiproliferative effect on human MC in vitro. activity on mesangial cells (MC), along with a broad range ofWe show that the drug is able to strongly inhibit cell growth and biological effects, including chemoattraction and regulation ofthat it interferes with the binding of PDGF-BB to its receptor and matrix metabolism [1—4]. All these biological activities are medi-with the regulation of PDGF 13-receptor transcript levels. ated through the binding of the growth factor to cell surface receptors, which transduce extracellular events into intracellular Methods biochemical signals [4, 5—13]. Interestingly, multiple peptide growth factors, and PDGF itself, induce PDGF-A and -B chain Cell culture mRNA in MC [14]. This suggests that endogenous PDGF may Human MC lines were established employing a modification of a previously published technique [32]. Glomeruli were isolated from histologically normal portions of human kidneys, obtained at Received for publication February 26, 1993 the time of nephrectomy for renal carcinoma, by passage through and in revised form May 5, 1994 serially graded sieves (425 and 180 jrm-mesh brass filters) and Accepted for publication May 5, 1994 collection on 75 rm-mesh nylon filters. The glomeruli were © 1994 by the International Society of Nephrology decapsulated by forced flow through a 18-gauge needle, washed 1002 Gesualdo et al: Trapidil inhibits MC proliferation 1003 80000 A B 60000 a) .0 60000 * 50000 E * E C * 40000 a) 40000 0 0 30000 UUU) 0 48 96 0 48 96 Time,hours Time, hours 60000 60000D Fig. 1. Effect of Trapidil on human MC growth. Cells were serum- and insulin-starved for 48 a) 50000 a)50000 .0 .0 hours. Thereafter, they were challenged with E E either 10% FBS (A), 20 ng/ml PDGF-BB (B), 10 C 40000 C40000 ng/ml bFGF (C) or 20 ng/ml EGF (D), in the a) a) presence or absence of different concentrations of 0 30000 030000 Trapidil (0: 0 p.g/ml, •: 100 Lg/ml; El: 200 rg/ ml; •: 400 jig/mI). The hatched lines represent 20000 20000________________ ______ humanMC growth in the presence of 0.01% FBS (basal). Data are presented as means SEM of 7 0 48 96 48 °6 experiments, each performed in quadruplicate. *p < 0.01, 'P < 0.05 versus cell proliferation Time, hours Time,hours inducedby mitogen alone. twice, incubated for 30 minutes with 750 U/mI of bacterialng/ml EGF (Collaborative Research, Inc., Bedford, Massachu- collagenase type IV (Sigma Chemical Co., St. Louis, Missouri,setts, USA)] in the presence or absence of Trapidil (Master USA) and finally resuspended in RPM! 1640 medium (GibcoPharma, Parma, Italy), acetylsalicylic acid (ASA, Sigma Chemical Laboratories, Grand Island, New York, USA) supplemented withCo.) or dypiridamole (Sigma Chemical Co.). At the selected times 17% heat-inactivated fetal bovine serum (FBS, Hyclone Labora-(0 to 96 hours), cell growth was evaluated by cell counting. Briefly, tories, Inc., Logan, Utah, USA), 100 U/mI penicillin, 100 jtg/mlcells were trypsinized for I to 2 minutes at 37°C. The enzymatic streptomycin, 2 mi L-glutamine, 2 m sodium pyruvate, 1%activity was blocked by the addition of I mI/well of 17% FBS- (vol/vol) non-essential amino acids, 5 j.tg/ml insulin, 5 jig/mIsupplemented medium. Cell detachment was helped by gentle transferrin and 5 ng/mI selenium. In the present study four humanabrasion with a plastic scraper and verified by phase-contrast MC lines between passage 4 and 12 were used. NIH 3T3microscopy. Cell suspension obtained was centrifuged at 600 Xg fibroblasts were provided by Dr. HE. Abboud, and were grown inand resuspended in Isoton III solution (Coulter Electronics LTD, the same medium used for HMC. Luton, Beds, UK). Finally, cells were counted in a Coulter Counter (Coulter Electronics LTD). Since PBS and other mito- Morphological and immunohistochemical characterization of gens could occasionally induce cell detachment, cell counting was HMC also performed on cell supernatants after exposure to mitogens. After passage 3, MC looked like elongated spindle-shaped cells. No glomerular, endothelial, or epithelial cells were observed, as Cell viability judged by phase contrast microscopy. Moreover, the purity of the culture was assessed by immunoperoxidase staining for interme- Cell viability was determined by Trypan blue dye exclusion test diate filaments and surface antigens. The cells showed an intenseand LDH assay. Supernatants, collected from cells seeded in positivity for desmin, laminin and vimentin and were negative forserum-free medium and exposed to the different mitogens and keratin, factor Vill-related antigens, common leukocyte antigendrugs tested, were centrifuged and LDH concentration deter- (CD 45) and macrophage antigen (CD 68). mined. Supernatants obtained from sonicated cells were used as a positive control. Furthermore, to definitely exclude a cytotoxic Cell counts effect of Trapidil on human MC, cells incubated for four days with Cells (10 cells/well), grown in complete RPMI, were renderedand without the drug were challenged with fresh medium contain- quiescent by removal of FBS for 72 hours. Serum deprivation diding 10% FBS, and cell proliferation evaluated. not affect cell viability as assessed by morphology, Trypan blue vital dye exclusion test and lactate dehydrogenase (LDH) assay. PDGF-binding experiments After 72 hours of incubation in serum-free medium, cells were washed once with Hanks' Balanced Salt Solution (HBSS, Gibco Standard binding assay. Binding experiments were performed Laboratories) without calcium and magnesium and re-fed withon confluent MC cells (about i0 cells/well), made quiescent by serum-free medium. Cells were stimulated with several substancesincubation in serum-free medium for 48 hours.