Inhibition of smooth muscle cell proliferation in vitro by leflunomide, a new immunosuppressant, is antagonized by uridine

Immunology Letters, 47 (1995) 171-174 0165.2478/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved IMLET 2411

Inhibition of smooth muscle cell proliferation in vitro by leflunomide, a new immunosuppressant, is antagonized by uridine Rajan

V. Nair

a.b, Wei Cao a, Randall

E. Morris

a**

a Laboratory for Transplantation Immunology, Dept. of Cardiorhoracic Surgery, Stanford Uniuersity School of Medicine, Stanford. CA: USA h Dept. of General Surgery. University of Arizona College of Medicine. Tucson, AZ: USA (Received Key words: Leflunomide

17 April 1995; revised 12 May 1995; accepted 5 June 1995)

(HWA 486, LFM); Chronic rejection; Graft vascular disease (GVD); Smooth muscle cell proliferation: (uridine) biosynthesis

1. Summary Chronic rejection in the form of graft vascular disease (GVD) continues to plague clinical transplantation of vascularized organs. The histopathology of this lesion is characterized by neointimal hyperplasia, smooth muscle cell proliferation, and obliterative arteriopathy. Due to the lack of effective medical therapy for preventing or reversing these chronic vascular changes, retransplantation remains the final resort in treatment. Some of the newer immunosuppressive agents, including the new isoxazole derivative leflunomide (LFM), have shown efficacy in preventing chronic rejection in animal models of transplantation. Although its mechanism of action remains incompletely elucidated, previous work using lymphocytes in vitro suggests that the drug might act as a tyrosine kinase inhibitor, an inhibitor of de novo pyrimidine biosynthesis, or both. In order to elucidate whether the efficacy of LFM in vivo is attributable not only to anti-proliferative effects on the recipient immune system but also to direct effects on mesenchymal cells in the donor organ, we examined the effects of LFM on a transformed 9EllG murine smooth muscle cell (M-SMC) line in vitro. We demonstrate here that the active metabolite of LFM, A77 1726, dose-dependently inhibits the constitutive and growth-factor stimulated proliferation of M-SMC in vitro. Furthermore, the anti-proliferative effect of the drug can be reversed by the addition of uridine to the culture medium. These results suggest that inhibition of

* Corresponding author: Randall E. Morris, MD Director, Laboratory for Transplantation Immunology Dept. of Cardiothoracic Surgery Falk Cardiovascular Research Building, Rm CV-116A Stanford University School of Medicine 300 Pasteur Drive Stanford, CA 943055247 U.S.A. SSDI 0165-2478(95)00074-7

Pyrimidine

uridine biosythesis appears to be a mechanism by which LFM exerts anti-proliferative effects on both lymphocytes and smooth muscle cells, and this dual action may be responsible for its efficacy in preventing GVD in vivo.

2. Introduction Leflunomide (LFM) is a novel isoxazole immunosuppressive prodrug which, following in vivo administration, is converted to its active, water-soluble metabolite, A77 1726. LFM has been shown in Phase II clinical trials to be a safe and effective treatment for rheumatoid arthritis [l] and has been shown to prevent acute kidney, small bowel, and heart allograft rejection in canine, rodent, and monkey models of organ transplantation [2,3,4]. Despite recent advances in the prevention and reversal of acute rejection in various animal models, few immunosuppressive agents have been able to prevent chronic rejection in the form of graft vascular disease (GVD), a common cause of long-term vascularized graft failure. Recent work by MacDonald [5] and our group [6] showed that LFM inhibits intimal thickening in rat heart and arterial allograft models of chronic rejection and halts progression of pre-existing GVD. The pathogenesis of GVD in all models of chronic rejection involves two distinctly separate but undoubtedly interrelated mechanisms: (1) activation of the immune cells of the recipient and (2) proliferation and migration of the mesenchymal cells in the vessels of the donor allograft induced by a variety of factors, including growth factors. Rapamycin (RPM) [7] and mycophenolic acid (MF’A) [8] effectively inhibit GVD in several animal models. It has also been shown that these drugs directly inhibit the proliferation of rat vas171

cular smooth muscle cells (R-VSMC) in vitro 19,101. These findings suggest that control of GVD by RPM and MPA may result from suppression of both the recipient immune system as well as the growth factorstimulated proliferation of smooth muscle cells in donor vessels [ 111. Although the exact mechanism of action of LFM remains elusive, experiments in different laboratories, including ours, have demonstrated that LFM may interfere with one or two distinct targets in activated lymphocytes: inhibition of cytokine receptor-associated tyrosine kinases [ 12,131 or inhibition of pyrimidine nucleotide biosynthesis [14,15,16]. Given leflunomide’s success in controlling chronic rejection in vivo, we evaluated the antiproliferative effect of the active metabolite, A77 1726, on a transformed mm-me smooth muscle cell (M-SMC) line 9EllG in vitro. We now report that LFM inhibits M-SMC proliferation in cell culture, and that this effect can be antagonized by addition of uridine to the culture medium. Our current results extend our preliminary observation that leflunomide antagonizes growth-factor mediated smooth muscle cell proliferation in vitro [17].

3. Materials and Methods 3.1. Reagents For in vitro studies, A77 1726 [a gift from Dr. R. Bartlett at Hoechst AG (Wiesbaden, Germany)] was dissolved in dimethyl sulfoxide [DMSO; Sigma (St. Louis, MO)] to make a 50 mM stock solution. Uridine [Sigma (St. Louis, MO)] was prepared as a 10 mM stock solution in phosphate buffered saline [PBS; Oxoid (Hampshire, England)]. Working solutions of A77 1726 and uridine, as well as the stock and working solutions of human recombinant basic fibroblast growth factor [bFGF; Life Technologies/Gibco BRL (Gaithersburg, MD)] ,were prepared using serum-free medium @FM> [Ex-Cell 320; JRH Biosciences (Lenexa, KS)]. Radiolabelled 3H-thymidine 13H-TdR, specific activity = 84 Ci/mmol; New England Nuclear (Boston, MA)] was diluted into alpha-MEM buffer [Bio Whittaker Inc. (Walkersville, MD)] to make a stock solution of 0.1 mCi/ ml. 3.2. Cells Murine 9EllG cells (generously donated by Dr. G.K. Owen, University of Virginia) were used for all experiments. This cell line was derived from the P19 mouse teratocarcinoma cell line [ATCC (Rockville, MD)] which constitutively expresses alpha-smooth muscle actin (a SM-actin). The P19 cell line was treated 172

with retinoic acid to stimulate differentiation, selected on the basis of inducible smooth muscle myosin heavy chain (SM-myosin-HC) expression, then dilutionally cloned to yield a homogeneous population (the 9EllG subclone). Based on experience in Dr. Owen’s laboratory, the 9EllG cell line demonstrates a stable phenotype through multiple passages. The stock cultures were maintained in T75 culture flasks with complete alphaMEM growth medium containing 10% fetal calf serum [Tissue Culture Biologicals (Tulare, CA)], L-glutamine (2 mM1, penicillin (100 U/ml), streptomycin (100 ug/ml), and HEPES buffer (10 mM). Growth medium was changed every 3-4 days; when the cells reached approximately 70% confluence in the T75 flasks, cultures were either split 1:12 for passage of maintenance cultures or were seeded into experimental culture microplate wells. 3.3. Proliferation

assays

When experimental wells were prepared, stock cultures of 9EllG cells were trypsinized and plated into sterile Linbro 96-well flat-bottom tissue culture plates [Flow Laboratories (McLean, VA)] at a final concentration of 2 X lo4 cells/ml (approximately 4,000 cells/ well). Cells were incubated at 37°C in 5% humidified CO, for 24 hours to allow the cells to adhere. The culture medium was decanted, replaced with serum-free medium @FM), and the cells were again incubated under the same conditions for 48 hours. After this period of serum deprivation, the medium overlying the adherent cells was again decanted, replaced with fresh SFM, and different concentrations (O-100 uM) of A 77 1726 were added either alone or in combination with uridine (O-50 uM). In some experiments, mitogenic stimulus with bFGF (IO ng/ml) was added 0.5 hours after addition of the drug, while in other experiments, no stimulus was used. The plates were then re-incubated using the same conditions for 48 hours, at which time 1.O uCi of 3H-TdR was added. After incubation with 3H-TdR for 24 hours, cells were trypsinized and harvested onto glass fiber filters using either a Filtermateharvester [Packard Instrument Co. (Meriden, CT)] or a PHD harvester [Cambridge Technologies Inc. (Watertown, MA)]. Beta emissions from each filter disk were quantitated as counts per minute (cpm) using either a TOPCOUNT or a TRI-CARB liquid scintillation counter [Packard Instrument Co. (Meriden, CT or Downers Grove, IL, respectively)]. 3.4. Statistics Results of scintillation counting were expressed as a mean percentage of cpm of either stimulated control

(for experiments using bFGF as stimulus) or unstimulated control (for experiments using no added growth factor). Statistical analysis was performed using a twotailed Student’s T-test of two samples with unequal variances. Values of p I 0.05 were considered statistically significant.

4. Results

4.1. A77 M-SMC

1726 Inhibits

Constitutive

Proliferation

of URIDINE(PM)

The transformed murine smooth muscle cell (MSMC) line 9EllG underwent constitutive replication in the presence of serum-free medium as assessed by 3H-TdR incorporation. This constitutive proliferation was effectively and dose-dependently inhibited by A77 1726 (Fig. 1). At a dose of 100 uM, 3H-TdR incorporation was maximally inhibited (13 f 1% of untreated control, p < 0.001); moreover, the IC,, for inhibition of constitutive proliferation was 5- 10 uM. 4.2. The anti-proliferative effect of A77 1726 on bFGF-stimulated M-SMC can be reversed by the addition of uridine Previous work [151, including our own [14,16], suggested that LFM might mediate its anti-proliferative effects in lymphocytes by depleting the available cellular pool of pyrimidine nucleosides, presumably through inhibition of one or more enzymes in the de novo pyrimidine biosynthetic pathway. To explore this possibility in non-immune cells, we evaluated the effect of I-T

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Fig. 1. A77 1726 dose-dependently inhibits ‘H-TdR incorporation in constitutively replicating M-SMC in serum-free medium. Adherent M-SMC were incubated in the presence of A77 1726 (O-100 uM) for 72 hours; ‘H-TdR was added 24 hours prior to harvest and scintillation counting. The values are plotted as a percentage of control (unstimulated) specific binding kpm). Data points are expressed as meanf SEM. [The mean incorporated radioactivity in the control wells is 2604+ 226 cpm].

Fig. 2. Increasing doses of uridine added to cell culture can completely reverse the inhibitory effect of A77 1726 on ’ H-TdRincorporation in bFGF-stimulated M-SMC. In this experiment done in quadruplicate, varying doses of A77 1726 (O-100 uM) and uridine (O-10 uM) were added to adherent M-SMC in the presence of 10 ng/ml bFGF and incubated for 72 hours; ‘H-TdR was added 24 hours prior to harvest and scintillation counting. The values are plotted as a percentage of control (bFGF-stimulated M-SMC in the absence of uridine or A77 1726) specific binding &pm). Data points are expressed as mean f SEM. [The average incorporated radioactivity in the control wells is 3039 f 301 cpm].

increasing concentrations of uridine on growth factor (bFGF)-stimulated M-SMC treated with A77 1726 at varying doses; these results are shown in Fig. 2. Uridine dose-dependently reversed the anti-proliferative effect of A77 1726; in the presence of 10 uM uridine, there was no statistically significant difference between the values for 3H-TdR incorporation in cells treated with 50 uM A77 1726 compared to untreated cells (p = 0.97).

5. Discussion Leflunomide (LFM) is an isoxazole prodrug which is structurally dissimilar to all of the immunosuppressive agents in current clinical use. Its open-ring form is the active metabolite, A77 1726. In T cells, A77 1726 has been shown to inhibit proliferation in response to IL-2, anti-CD3 [ 181, or the combination of anti-CD28 and PMA [19]. Furthermore, at lower doses between 1 and lOuM, the drug has been shown to inhibit B-cell proliferative responses to LPS [20]. More importantly, LFM has been shown to prolong comeal, cardiac, renal, and small bowel allografts in a variety of species, including rodents, dogs, and non-human primates [2,3,4]. In addition, it has also shown efficacy in both preventing and reversing acute rejection in concordant [21] and discordant [22] xenografts, presumably by inhibiting xenospecific antibody formation and blocking the isotype switch from IgM to IgG [23]. One of the current limiting factors in the long-term success of clinical organ transplantation is chronic re173

jection. In vascularized organs, it is characterized by neointimal hyperplasia and smooth muscle cell proliferation of the medium- and small-caliber arteries of the allograft, leading to obliterative arteriopathy. This disease is usually progressive, and no uniformly effective medical therapy short of re-transplantation is currently available. A few of the newer immunosuppressive agents, including RPM and MPA as well as LFM, have demonstrated varying degrees of success in preventing the histopathologic manifestations of chronic vascular rejection in animal models. The mechanism by which LFM acts has been a source of controversy. Initial studies showed that LFM inhibited autophosphorylation of both the EGF receptor as well as the PDGF receptor [13]. Recent work suggests another possible site of action, namely inhibition of the de novo pyrimidine biosynthetic pathway [ 14,15,16]. Using purified enzyme preparations, M. Loeffler and colleagues have recently shown that A77 1726 preferentially inhibits dihydro-orotate dehydrogenase (DHODH) [personal communication], the fourth enzyme in the de novo pyrimidine biosynthetic pathway that catalyzes the conversion of dihydro-orotate to orotate. Our results showed that the active metabolite of LFM, A77 1726, effectively and dose-dependently inhibited in vitro proliferation of the 9EllG murine smooth muscle cell line. More importantly, we have demonstrated that the anti-proliferative effects of this novel agent can be reversed by the addition of increasing concentrations of uridine to the culture medium. Considering the results of previous work with lymphocytes in vitro [ 14,15,16], the ability of LFM to interfere with pyrimidine metabolism appears to be a mechanism common to both immune and non-immune cells. Like RPM and MPA, LFM effectively prevents intima1 thickening in several animal models of GVD. From the currently available data, we speculate that LFM may derive its efficacy not only from its effects on recipient immune cells but also from its ability to inhibit proliferation of smooth muscle cells in the vessels of the donor organ. If our in vitro data using a mouse smooth muscle cell line are generalizable to the effects of LFM on smooth muscle proliferation in vivo in other species, our findings might explain the efficacy of LFM and its analogue for prevention and treatment of chronic rejection.

Acknowledgements

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