Trapidil Inhibits Human Mesangiai Cell Proliferation: Effect on PDGF Β

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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. After rinsing with [0.01 to 10% FBS, 20 ng/ml PDGF-BB (Pepro Tech Inc., Rocky1 ml binding buffer (RPM! 1640, pIus 25 mt Hepes, pH 7.4, and Hill, New Jersey, USA), 10 ng/ml bFGF (Pepro Tech Inc.) or 202 mg/ml BSA), cells were incubated with 5 ng/ml '251-PDGF-BB 1004 Gesualdo ci a!: Trapidil inhibits MC proliferation

A B

1200 600 1000 0 * 0 0 0 * 800 >< x400 ** -oci) 0a) E E

** 0a) 0cis 200

0 0 0 48 96 0 96

Time, hours Time, hours C

600

o * 0 X 400 .0a) E C a) o200

Fig. 2. Effect of Trapidil on NIH 3T3 fibroblasts proliferation. Cells were serum- and insulin-starved for 48 hours. Thereafter, they were challenged with either 10% FBS (A), 20 ng/ml PDGF-BB (B) or 10 ng/ml bFGF (C), 0 in the presence or absence of different concentrations of Trapidil (0: 0 0 48 96 isg/ml, •: 100 rg/ml; 0: 200 /LgIml; •: 400 jsglml). Data are presented as mean SCM of 4experiments,each performed in quadruplicate. °P < Time, hours 0.05, < 0.01 versus cell proliferation induced by mitogen alone.

(S.A.: 1000 Ci/mmol, Amity S.r.l., Milan, Italy) for two hours at Sequential assay. HMC were preincubated with various concen- 4°C, with constant gentle rotatory agitation. trations of Trapidil for two hours at 37°C. Thereafter, cultures At the end of the incubation period, cells were washed thricewere extensively washed in ice-cold binding buffer and standard with ice-cold PBS containing 1 mr't CaCI2 and 2 mg/mi BSA andbinding assay was then performed. In some experiments, cells then solubilized by adding 1,0 ml of 20 mrvi Hepes, pH 7.4, 1%were exposed to 400 jrg/ml Trapidil for 48 hours at 37°C. HMC Triton X-i00, 10% (vol/vol) glycerol, and 0.1 mg/mI BSA. Cell-were subsequently rinsed four times in 4 ml of ice-cold binding bound radioactivity was counted in a gamma counter. Nonspecificbuffer, before performing binding experiments. binding was determined in the presence of a 100-fold excess of Acid washing assay. These experiments were aimed to deter- purified PDGF-BB. Specific binding was calculated by subtractingmine whether the competing activity of Trapidil on PDGF-BB non specific binding from the total counts bound per well. binding could be removed by acid washing of HMC. To this aim, Coincubation assay. HMC were incubated for two hours at 4°Ccells were preincubated with Trapidil for two hours at either 4° or with 1251-PDGF-BB and various concentrations of Trapidil. Then,37°C, then washed with binding medium and subsequently ex- cultures were washed thrice in ice-cold binding buffer. Cellposed to medium containing 0.1% acetic acid (pH 3.52) with 150 associated 1251 radioactivity was extracted as described above. mM NaCI and 2 mg/ml BSA for eight minutes at 4°C {2, 33, 4I. Gesualdo et al: Trapidil inhibits MC proliferation 1005

A B 7. 6

4 C

0 3 3C .00 2 U- n0

0 0 100 200 400 800

Trapidil, pg/mi Trapidil, pg/mi C

Fig. 3. Effect ofTrapidil on '251-PDGF-i3BbindingtolIARand ,VlH 3T3 fibroblasts.A. Coincuhation assay. Human MC were incubated with S nglmt '2-I-PDGF-BB and (Ho 8(X) sg/ml Trapidil for 2 hours at 4 C. Then. ccli associated radu,aeti ity was determined. 8. Sequential assay. Human MC were preincubated with the indicated concentrations at Trapidil for 2 hours at 3TC. Thereafter, cells were extensively washed with binding 0 400 800 medium and specific PDGF 138 binding wu measured. C. Sequential assay performed on Nil-i 3T3 libroblasts. Data are given as the means t il) of three separate experiments. each perlhrmed in quadruplicate. */) < 01)5, Trapidil, pg/mi "P C 0.01 versus control.

Cells were extensively washed with binding medium and 125J using random primer extension and added at 1 X 106 cpm/ml. PDGFbinding was then determined as described above. Electrophoresis of 20 tg of total RNA was carried out in 1% agarose gel with 2.2 M formaldehyde. The RNA was then trans- RNAisolationand Northern analysis ferred overnight to a nylon membrane (Schleicher & Sehuell, Human MC were rested for 48 hours in serum-free medium andDassel, Germany). Prehybridization and hybridization were per- then incubated for 0 to 48 hours with 20 ng/ml PDGF BB in theformed for 18 hours at 42°C in a buffer containing 50% form- presence or absence of 400 g/ml Trapidil. Cells were lysed withamide, 5xSSC, 5x Denhart's solution, 0.1% SDS, 100 g/ml 4 M guanidium isothiocyanate containing 25 m sodium citrate,denatured salmon sperm DNA. The blots were then washed once pH 7.0, 0.5% sarcosyl and 0.1 ms 2-3-mercaptoethanol. Totalin 2x SSC, 0.1% SDS at 22°C for five minutes, and once in the RNA was isolated by the single-step method, using phenol andsame buffer at 55°C for 30 minutes, Finally, the membranes were chioroform/isoamylalcohol, as previously described [35]. Levels of washed in lx SSC, 0.1% SDS at 55°C for a further 30 minutes. PDGF /3-receptor mRNA were determined by Northern analysis,After drying, membranes were exposed to a Kodak X-OMAT film using a 751 base pair fragment of the human PDGF 13-receptorwith intensifying screens at —70°C. Membranes were subsequently eDNA [36], isolated from pGEM-1 plasmid with Pst I. The eDNAstripped and rehybridized with a (32P)-labeled glyceraldehyde-3- was labeled with (32P) dCTP (3,000 Ci/mmol, Amersham, UK)phosphate dehydrogenase (GAPDH) eDNA probe, added at 2 X 1006 Gesualdoet a!: Trapidil inhibits MC proliferation

-4 01 were challenged with FBS, PDGF or bFGF and the effect of Trapidil was then tested. Figure 2 demonstrates that Trapidil also affected fibroblast growth, with a dose-dependent fashion. Note

cells that, at comparable concentrations, the drug exhibited its highest inhibitory effect towards PDGF-BB-induced cell growth. Previous investigations focused on PDGF antagonistic effect of 00,000 0 Trapidil and postulated an inhibition of PDGF interaction with target cells. Therefore, we wondered whether Trapidil might

ImoVi interfere with the first step of the chain of events leading to cell activation by PDGF, namely the binding of the growth factor to specific cell surface receptors. Figure 3A shows that Trapidil bound, inhibited specific binding of '251-PDGF-BB to human MC, when 01 BB both ligands were added simultaneously (coincubation assay). This assay does not allow distinction between inhibition resulting from an interaction between Trapidil and PDGF receptor and inhibition resulting from a direct interaction of the test substance

125IPDGF with the growth factor. Preincubation of cells with Trapidil alone, followed by extensive washing and then exposure to labeled ligand 0 (sequential assay) enables discrimination between these two pos- a 0 0 sibilities and definitely demonstrated that Trapidil exerts its inhibitory effect by interacting with PDGF receptor (Fig. 3B). Fig. 4. Effect of chronic exposure to Trapidi! on '251-PDGF-BB binding. Sequential assay performed on NIH 3T3 fibroblasts yielded Human MC were incubated with 400 jLg/ml Trapidil for 48 hours at 37°C. similar results (Fig. 3C). Thereafter, cells were rinsed 4timesin 4 ml of ice-cold binding medium, However, the above findings still raise two possibilities: Trapidil before performing binding experiments. °P < 0.01 versus control. might inhibit PDGF binding to its cell surface receptor and/or induce a reduction of receptor number (that is, surface receptor translocation). To further clarify this issue, human MC were I 0 cpm/ml, to account for small differences in RNA loading andpreincubated with 400 xg/ml Trapidil at either 4 or 37°C; transfer. thereafter the cells were washed at pH 3.52 before measurement of '251-labeled PDGF binding, to dissociate the drug from cell Statistical analysis binding. Washing the cells with an acidic medium almost com- Dose- and time-response curves of cell counts were analyzed bypletely reversed the inhibition of PDGF binding, regardless of the two-way analysis of variance on raw data. Receptor bindingtemperature of preincubation (control: 3.450.43 fmol/105 cells; experiments were analyzed by unpaired Student's t-test. Trapidil-treated cells, 4°C: 3.030.250 fmol/105 cells; 37°C: 2.71 0.380 fmol/105 cells; N =3).Thus, experiments performed with Results cells maintained at 37°C yielded a conclusion identical to that Several growth factors (FBS, PDGF-BB, bFGF and EGF) wereobtained in experiments performed at 4°C, when PDGF receptor able to stimulate human MC proliferation, with PDGF resultingcannot be internalized: Trapidil exerts a fainty effect, if any, on as the most potent mitogen after FBS (Fig. 1). The addition ofreceptor disappearance from cell surface [2, 33, 34J. Finally, Trapidil caused a growth inhibition that was dose-dependent (Fig.long-term (48 hr) exposure to 400 jig/mi Trapidil caused a 150% I). After 96 hours of incubation with 100 xWml Trapidil, averageincrease of 1251-PDGF binding to human MC (Fig. 4). inhibition of cell proliferation induced by either FBS, PDGF-BB, The results of receptor binding experiments prompted us to bFGF or EGF was 41.8, 65.3, 38.7 and 56%, respectively. It isinvestigate the kinetics of Trapidil effect on PDGF-f3 receptor therefore apparent that Trapidil exerted its maximum inhibitorymRNA. Northern blot analysis of PDGF-f3 receptor transcript effect on PDGF-stimulated cells (Fig. IB). revealed a 30% decrease of PDGF-/3 receptor mRNA levels after Aspirin, at the same doses of Trapidil, was devoid of any24 hour exposure of PDGF-stimulated human MC to 400 jxg/ml significant effect: 100 jxg/ml aspirin inhibited PDGF-BB-inducedTrapidil, whereas at 48 hours 3-receptor transcript levels dis- cell proliferation by 6.8 3.2%after96 hours. Dypiridamoleplayed an almost threefold increase over control cells (Fig. 5). resulted in being toxic for human MC: at doses of 10 to 100 iM (5 to 50 agJml), comparable to those used in other cell culture Discussion systems, it induced cell lysis, as confirmed by LDH assay. Cell proliferation assays showed that Trapidil exerts a strong Trypan blue dye exclusion test, LDH assay of cell supernatantsinhibition of human MC growth induced by different mitogens, as well as the rapid increase of cell proliferation following thethe strongest effect being on PDGF-stimulated cell proliferation. challenge with fresh 10% FBS-supplemented medium demon-Similar results were obtained when NIH-3T3 fibroblasts were strated that the drug was devoid of any cytotoxic effect on humananalyzed. MC in culture (data not shown). Noteworthy, Trapidil did not Previous reports described a suppression of PDGF-induced induce any significant variation in cell detachment at the dosesproliferation in different cell culture systems pretreated with tested, compared to FBS-stimulated cells. Trapidil [27—31]. Onishi et a! [27] postulated that Trapidil might We next analyzed whether the anti-proliferative effect of Tra-function as a competitive inhibitor of PDGF to its receptor, thus pidil was specific for human MC. To this aim, NIH 3T3 fibroblasts preventing PDGF from inducing its mitogenic effect. On the other Gesualdo et at: Trapidil inhibits MC proliferation 1007

Hours 0 24 48

— Trapidil — + +

PDGF n-receptor

GAPDH

7 PDGF+TRAP

Fig.5. A. Total RNAwasisolated from human MC cultured in the presence of 20 nglml PDGF-BB and with or without 400 p.gIml Trapidil for up to 48 hours; 20 p.g of RNAforeach time point was separated in 1% denaturing agarose gel and PDGF transferred to nitrocellulose. The filter was hybridized with a fragment from PDGF p-receptor cDNA, then stripped and rehybridized with a glyceraldehyde-phosphate dehydrogenase (GAPDH) probe, as internal standard, and subjected to autoradiography. Represented is one of three separate experiments. B. the hybridization signals obtained in A were quantified by scanning of autoradiograms and expressed 0 24 48 relative to the level of zero time, after adjusting to the GAPDH signal.Points are the means of three separate Time, hours experiments. Symbols are: (0) PDGF +TRAP;(L) PDGF.

hand,both EGF and bFGF are able to induce PDGF genedemonstrated that a polyclonal anti-PDGF antibody was able to expression and protein synthesis in MC and would exert theirinhibit EGF-stimulated mesangial cell growth with a dose-depen- mitogenic effect, at least in part, via the PDGF loop [31•dent effect. In preliminary experiments, we found that 48-hour Therefore, a unifying hypothesis which postulates that Trapidilincubation with 2 sg/ml polyclonal anti-PDGF-AB antibody in- exerts its growth inhibitory effect on different mitogens throughhibited bFGF-induced human MC proliferation by 31%, a value the inhibition of PDGF interaction with target cells appearsroughly corresponding to the inhibition exerted by 100 sg/ml intriguing. Indeed, Silver, Jaft'er and Abboud [141 previouslyTrapidil. 1008 Gesualdoet at: Trapidil inhibits MC proliferation

Within this frame, inhibition of cellular binding of PDGF bypathologic manifestations of the disease should help clarify the Trapidil represents the first possible target. Binding inhibitionrole of inhibitory agents (such as Trapidil and other receptor experiments demonstrated that Trapidil does interfere withantagonists, anti-sense oligonucleotides, blocking antibodies, sol- PDGF binding to its cell surface receptors by competitive inhibi-uble receptors) able to reduce mesangial cell proliferation and the tion. This conclusion is supported by both sequential radiorecep-overall biological effects of growth factors in the treatment of tor assay and acid wash experiments. The latter studies alsomesangial proliferative glomerulonephritis. suggest that Trapidil does not induce receptors disappearance from cell surface, at least after short-term incubation. Acknowledgments Simultaneous competition assay showed that the contemporary This study was partly supported by the C.N.R. Target Project on addition of PDGF and Trapidil blunted the binding-inhibitoryBiotechnology and Bioinstrumentation (90.115.70, 91.1253.70, 92.1272.70, effect of the drug, and would imply that the test substance displays93.1102.PF7O), the C.N.R. Joint Program USA-Italy (90.1553.04, a lower affinity for PDGF receptor than the natural ligand. This91.466.04, 92.749.04), by grants (40%: 90.5 151, 91.1882 and 60%: 90.2422, 91.6986) from the Ministero dell'Universita' e della Ricerca Scientifica e conclusion is consistent with the finding of the lack of anTecnologica, Rome, Italy. We thank Dr. Trascinelli of Master-Pharma, anti-proliferative effect of Trapidil, when its addition is delayed upParma, Italy, who provided us with Trapidil. Portions of this work were to four hours after the exposure of cells to PDGF, and mostpresented at the European Dialysis Transplant Association meeting, Paris, available surface receptors are occupied by the natural ligandAugust 31 through September 2, 1992, and the American Society of Nephrology annual meeting, Baltimore, Maryland, November 14—18, (data not shown). 1992. Abstracts have been published in the EDTA abstract book, pg. 11, Binding experiments performed after 48 hours of exposure of1992 and JAm Soc Nephrol 3: 453, 1992. We thank Dr. V. Montinaro for HMC to Trapidil revealed a sharp up-regulation of PDGF-BB the critical review of the manuscript and Mrs. Rosa Filazzola for typing binding. Finally, Trapidil was shown to influence the level ofassistance. PDGF-/3 receptor transcript in human MC: 24 hours of exposure to 400 g/ml Trapidil caused a moderate decrease of target gene Reprint requests to F.P. Schena, M.D., Division of Nephrology, University expression, whereas at 48 hours Trapidil-treated cells exhibited aof Ban, Piazza G. Cesare, 11, 70124 Bari Italy. marked increase of specific mRNA transcript, seemingly support- ing the results of PDGF-BB binding studies. Presently we cannot References explain the latter finding. More specifically, we cannot distinguish 1. Ross R, RAINES EW, BOWEN-POPE DF: The biology of platelet between a direct effect of Trapidil, or its metabolites, on mRNA derived growth factor. Cell 46:155—169, 1986 transcript levels, or an adaptive response of cells to PDGF 2. SHULTZ PJ, DICORLETO PE, SILVER BJ, Ansoun HE: Mesangial cells receptor binding-inhibition. express PDGF mRNAs and proliferate in response to PDGF. Am J In conclusion, our results demonstrate that Trapidil: (i) exerts Physiol 255:F674—F684, 1988 3. BARNES JL, HEVEY KA: Glomerular mesangial cell migration in an inhibitory role on human MC and NIH 3T3 fibroblasts growth, response to platelet-derived growth factor. Lab Invest 62:379—382, as induced by several growth factors; (ii) competitively antago- 1990 nizes PDGF-BB binding to its specific surface receptors and 4. ABnOUD HE: Platelet-derived growth factor and mesangial cells. modulates f3-receptor transcript levels. Kidney mt 41:58 1—583, 1992 5. BISHAYEE S, MAJUNDAR S, KHIRE J, DAS M: Ligand-induced dimer- In our opinion the above considerations, while casting some izatjon of the PDGF-receptors. J Biol Chem 264:11699—11905, 1989 doubts on the complete mechanism of action of Trapidil, do not 6. MENE P, ABBOUD HE, DUBYAK GR, SCARPA A, Du MJ: Effects of diminish the potential relevance of the findings presented here. In PDGF on inositol phosphates, Ca ,andcontraction of mesangial fact, the available evidence from human and animal studies cells. Am J Physiol 253:F458—F463, 1987 supports the view that PDGF is a prime candidate to mediate or 7. HART CE, FORSTEOM JW, KELLY JD, SEIFERT RA, SMITH RA, Ross R, MURRAY MJ, BOWEN-POPE DF: Two classes of PDGF receptors modulate biologic responses of MC to glomerular injury, both recognize different isoforms of PDGF. Science 240:1529—1531, 1988 directly and through the activation of other growth factor path- 8. RHEE SG, SUM PG, RYu SH, LEE SY: Studies of inositol phosphates ways, such as TGF-f3. On the other hand, the PDGF effect is and specific phospholipase C activity. Science 244:546—550, 1989 mediated through the binding to its specific cell surface receptors. 9. SEIFERT RA, HART CE, PHILIPS PE, FORSTROM JW, Ross R, MURRAY M, BOwEN-POPE DF: Two different subunits associate to create Therefore, any substance able to interfere with the above binding, isoform specific PDGF-receptors. J Biol Chem 264:8771—8778, 1989 even in the presence of high levels of receptor expression, should10. 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HELDIN CH, OSTMAN A, ERIK550N A, SIEGBAHN A, CLAESSON-WELSH The present study suggests, but does not permit a definite L, WESTERMARK B: Platelet-derived growth factor: Isoform-specific conclusion, that the anti-proliferative effect of Trapidil is medi- signalling via heterodimeric or homo-dimeric receptor complexes. ated through the interference with PDGF loop. In any event, it Kidney mt 41:571—574, 1992 clearly demonstrates that Trapidil blocks PDGF-p receptor inter-14. SILVER BJ, JAFEER FE, ABBOUD HE: Platelet-derived growth factor synthesis in mesangial cells: induction by multiple peptide mitogens. action with the natural ligand on cell surface. Proc NatlAcad Sci USA 86:1056—1060, 1989 A more comprehensive understanding of the temporal relation-15. KA5HGARIAN M: Mesangium and glomerular disease. Kidney mt ship between the expression of different growth factors and the 17:141—154, 1980 Gesualdo et a!: Trapidil inhibits MC proliferation 1009

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