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(Circulation Research. 1995;77:1095-1106.)
© 1995 American Heart Association, Inc.

Articles

T-Cell Lymphokines, Interleukin-4 and Gamma Interferon, Modulate the Induction of Vascular Smooth Muscle Cell Tissue Plasminogen Activator and Migration by Serum and Platelet-Derived Growth Factor

Weizheng Wang, Hong Jun Chen, Kenneth N. Giedd, Allan Schwartz, Paul J. Cannon, LeRoy E. Rabbani

From the Division of Cardiology, Department of Medicine, Columbia University College of Physicians and Surgeons, New York, NY.

Correspondence to LeRoy E. Rabbani, MD, Division of Cardiology, Department of Medicine, Columbia University College of Physicians and Surgeons, 630 West 168th St, New York, NY 10032.

	Abstract

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Abstract Platelet-derived growth factor (PDGF)–induced smooth muscle cell (SMC) fibrinolysis is necessary for SMC migration. In order to determine whether the T-cell lymphokines interleukin-4 (IL-4) and gamma interferon (-IFN) affect SMC fibrinolysis and migration, we examined the effects of human recombinant IL-4 and -IFN on human aortic SMC tissue-type plasminogen activator (TPA), urokinase-type plasminogen activator (UPA), and plasminogen activator inhibitor type-1 (PAI-1) antigen production, as determined by enzyme-linked immunosorbent assays. Although IL-4 had no direct effect on SMC TPA antigen, IL-4 potentiated SMC TPA antigen levels and activity in conditioned media and cellular lysates in media containing 2% fetal bovine serum but did not change UPA or PAI-1 production. -IFN attenuated IL-4 augmentation of SMC TPA antigen production in conditioned media, although -IFN itself had no direct effects on SMC TPA and PAI-1 antigen production. IL-4 augmented PDGF induction of SMC TPA antigen. -IFN inhibited PDGF induction of SMC TPA antigen and IL-4 potentiation of this process. -IFN diminished the promigratory effects of both IL-4 and PDGF on in vitro SMC migration. Tranexamic acid, a plasmin inhibitor, abrogated the stimulation of SMC migration by IL-4. Therefore, IL-4 and -IFN modulate the induction of SMC TPA and SMC migration by 2% fetal bovine serum and PDGF.

Key Words: • interleukin-4 • fibrinolysis • gamma interferon • platelet-derived growth factor • smooth muscle cells

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The role of T cells in the pathogenesis of atherosclerosis and angioplasty restenosis remains poorly defined. T cells are present in all stages of atherosclerotic plaques, ranging from the early fatty streak to the mature fibrotic plaque.^{1 2} The majority of T cells in human atherosclerotic plaques are memory cells expressing surface antigens, such as HLA-DR, CD45RO, and the VLA-1 integrin, which denote a state of late or chronic activation.² In vitro activation of memory T cells effects transcription of the genes for -IFN, interleukin-3 to interleukin-6, and granulocyte/macrophage colony stimulating factor as well as secretion of these lymphokines into the culture medium.^{2 3} Indeed, it has been postulated that the CD45RO+ memory T cells extant in a state of late activation in the atherosclerotic plaque release lymphokines such as IL-4 and -IFN locally in the arterial wall during atherogenesis.³ Moreover, T cells may also play a role in influencing the growth of vascular SMCs in the aforementioned diseases. Cyclosporin A, an immunosuppressive drug that inhibits activation of T lymphocytes, inhibits SMC proliferation after balloon injury to the rat carotid artery.⁴ Furthermore, cyclosporin A or T-cell deficiency in athymic nude rats results in decreased SMC hyperplasia in venous grafts exposed to arterial pressure.⁵

Two lymphokines, IL-4 and -IFN, are secreted by different T-cell subsets and usually exhibit opposing effects on a variety of biological processes including fibrinolysis. IL-4 has been demonstrated to increase monocyte net fibrinolytic activity by stimulating human monocytes to produce TPA⁶ and to suppress formation of PAI-2, one of the endogenous inhibitors of TPA.⁷ Human IL-4 also stimulates the expression of UPA in cultured human microvascular endothelial cells.⁸ -IFN, which usually opposes the actions of IL-4, has been demonstrated to inhibit tumor necrosis factor–mediated induction of endothelial cell UPA proteolysis of ECM.⁹

The effects of T-cell lymphokines on vascular SMC fibrinolysis and consequently on SMC migration have been heretofore unknown. The intima of normal blood vessels usually is devoid of SMCs.¹⁰ However, SMC migration from the media into the intima of blood vessels is a major event underlying the pathogenesis of atherosclerosis, angioplasty restenosis, and vein graft intimal hyperplasia.¹¹ The factors controlling SMC migration have only recently been clarified. Platelets have been demonstrated to stimulate SMC migration, as evinced by a study showing that thrombocytopenia inhibits intimal thickening in balloon-injured rat carotid arteries but does not affect SMC replication.¹² Indeed, PDGF, a mitogen and chemoattractant for connective tissue cells, has been implicated as an important positive regulator of SMC migration.¹³ Exogenous PDGF-BB increased 20-fold intimal thickening and SMC migration from the media into the intima, with minimal effects on SMC replication in the rat model of balloon angioplasty.¹³ Moreover, a polyclonal antibody to PDGF inhibited the development of an intimal lesion in the carotid artery of athymic nude rats induced by balloon injury deendothelialization¹⁴ ; SMC replication was not affected by the polyclonal antibody to PDGF.¹⁴

The stimulatory effects of PDGF on SMC migration appear to be linked to SMC fibrinolysis through the generation of plasmin, a serine protease with broad specificity.¹⁵ The conversion of plasminogen into plasmin by plasminogen activators in the vessel wall, such as TPA and UPA, results in degradation of ECM and basement membrane proteins.¹⁶ Plasmin is also a potent activator of procollagenase, which facilitates ECM degradation.^{16 17} TPA and UPA may augment vascular SMC migration by enhancing cell surface plasmin activity.^{18 19} Moreover, rat aortic SMCs have been shown to degrade SMC ECM via plasminogen activation.²⁰ Plasmin inhibition has been shown to abrogate SMC migration.²¹ Both a polyclonal antibody to PDGF and thrombocytopenia not only effect a reduction in SMC migration from the media into the intima in the balloon-injured rat carotid artery but also reduce arterial wall TPA activity.²² Furthermore, tranexamic acid, a plasmin inhibitor, inhibits SMC migration in this model, further demonstrating that vessel wall plasmin is necessary for SMC migration.²² Therefore, PDGF may stimulate the expression and synthesis of SMC TPA.²² In addition, serum has been identified as a potent inducer of SMC TPA, probably reflecting the high quantity of PDGF present in serum.²³ PAI-1 is a serpin inhibitor that endogenously binds and inactivates plasminogen activators such as TPA and UPA.¹⁹ PAI-1 is located in the ECM in an active form and is secreted by both endothelial cells and SMCs.¹⁹ PAI-1 mRNA expression is increased in atherosclerotic human arteries.²⁴ PDGF has been shown to increase PAI-1 activity, protein synthesis, and mRNA levels in both bovine vascular SMCs²⁵ and rat cultured vascular SMCs.²⁶

We hypothesized that T cells present in the atherosclerotic plaque may modulate vascular SMC migration from the media into the intima owing to their secretion of lymphokines. In particular, we hypothesized that IL-4 may increase and -IFN may decrease vascular SMC fibrinolysis and migration in parallel, given their effects on monocyte and endothelial cell fibrinolysis. In order to test this hypothesis, we examined the effects of human recombinant IL-4 and -IFN on serum and PDGF induction of human aortic SMC TPA, UPA, and PAI-1 antigens as determined by ELISAs. Moreover, we studied the effects of IL-4 and -IFN on in vitro human aortic SMC migration through a micro–Boyden chamber. Our studies confirm and extend to the in vitro milieu prior observations that PDGF stimulates SMC TPA production in vivo. Furthermore, in the present study, we report for the first time that T-cell lymphokines may modulate vascular SMC migration and fibrinolysis as evidenced by IL-4 augmentation and -IFN inhibition of serum and PDGF induction of in vitro vascular smooth muscle cell fibrinolysis and migration.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
The sources of the agents were as follows: human recombinant IL-4 was obtained from Genzyme (specific activity, 1x10⁷ U/mg by proliferation assay using human peripheral blood T lymphocytes); human recombinant -IFN, PDGF-BB, -AB, and -AA, and anti–PDGF-AB (recognizes PDGF-AB, -AA, and -BB; polyclonal), from Upstate Biotechnology Inc; anti–human IL-4 neutralizing antibody, from R&D Systems; FBS, M199, and DMEM, from GIBCO; tranexamic acid, cycloheximide, actinomycin D, lipopolysaccharide, sodium hydroxide, and hydrogen chloride, from Sigma Chemical Co; and anti-CD23 monoclonal antibody, from Becton Dickinson.

Cell Culture
Human aortic SMCs were obtained from Clonetics and subcultured by using smooth muscle growth medium (Clonetics) containing human epidermal growth factor (10 ng/mL), human fibroblast growth factor (2 ng/mL), dexamethasone (0.39 µg/mL), 5% FBS, gentamicin (50 µg/mL), and amphotericin B (50 ng/mL) at 37°C in a humidified 95% air/5% CO₂ atmosphere. The growth medium was changed every other day until confluence was reached. The cells used for experiments were from passages 5 to 10. Verification of SMC phenotype was performed via positive fluorescent staining for -actin (compared with a known positive control for SMCs) and negative staining for factor VIII antigen. Cell viability was 95%, as determined by trypan blue exclusion at the end of experiments.

Cytokine Treatment
Cells were grown to confluence in 6-well or 12-well tissue culture plates (diameter, 35 and 22.6 mm, respectively) (Costar). Confluent SMCs were washed three times with basal M199 and incubated with serum-free medium (M199 supplemented with 0.2% bovine serum albumin) for 48 hours to obtain quiescent nondividing cells.²⁷ Thereafter, cells were incubated with equal volumes of human recombinant IL-4 at concentrations of 0 to 1000 U/mL in M199 containing either 0% or 2% FBS. In some experiments, cells were not growth-arrested.

At various time points, conditioned media were removed, centrifuged at 1000g to remove debris, and stored at -80°C. Simultaneously, SMCs were rinsed with PBS and lysed with 0.2% Triton X-100. The cellular lysates were then centrifuged at 1000g and stored at -80°C, as previously described.²⁸ Cell numbers were counted in parallel at the end of the experiments with either a hemocytometer or a Coulter counter (model Z1, Coulter Electronics).

In certain experiments, the SMCs were incubated with 100 U/mL of recombinant IL-4 boiled for 45 minutes. In other experiments, the SMCs were incubated with IL-4 (100 U/mL) plus -IFN at a concentration of 10 to 1000 U/mL or 1 µg/mL of polyclonal anti–human IL-4 antibody. In yet other experiments, the SMCs were pretreated with either 10 µg/mL of cycloheximide or 2 µg/mL actinomycin D for 30 minutes before the addition of IL-4.

The SMC-conditioned media and cellular lysates were subsequently assayed for TPA antigen, TPA activity, PAI-1 antigen, and UPA antigen as described below.

Measurement of TPA Antigen and Activity
The TPA antigen concentrations in the SMC-conditioned media and cellular lysates were measured by an ELISA using monoclonal antibodies (Asserachrom, TPA, Diagnostica Stago) and an ELISA card reader. This test system measured the total amount of TPA present (free and complexed TPA, single-chain and double-chain TPA).²⁹ TPA activity was determined by using the Spectrolyse TPA kit (American Diagnostica). The assay is based on the functional parabolic rate assay described by Ranby et al,³⁰ which allows highly specific determination of the fibronolytic activity exerted by human TPA.

Measurement of PAI-1 Antigen
The PAI-1 antigen concentrations in the SMC-conditioned media and cellular lysates were measured by an ELISA (Asserachrom, PAI-1, Diagnostica Stago) with an ELISA card reader.³¹ This procedure measured the total quantity of PAI-1 present (free or complexed with TPA, bound or not to vitronectin, and in active or in inactive form).

Measurement of UPA Antigen
UPA antigen levels in conditioned media and cellular lysates were measured by using an ELISA method (IMUBIND UPA ELISA, American Diagnostica). Single-chain UPA (Sc-UPA, Pro-UPA) and high molecular weight UPA forms of the urokinase-type activator are all recognized by the assay, as is receptor-bound UPA and UPA complexed with PAI-1 and PAI-2.³² The lower detection limit is 10 pg UPA per milliliter of sample.

Measurement of [³H]Thymidine Incorporation
In experiments in which [³H]thymidine incorporation was assessed, 1 µCi [methyl-³H]thymidine (New England Nuclear) was added to each well 48 hours after the addition of reagents. The cells were incubated an additional 24 hours; after which, media were removed, and labeled cells were washed with basal M199, fixed for 30 to 60 minutes with 10% ice-cold trichloracetic acid, and then solubilized with 1N NaOH. Cellular lysates were neutralized with an equal volume of 1N HCL and counted by using an Ultima Gold (Packard) liquid scintillation cocktail in a Packard liquid scintillation counter (model TRI-CARB 4000 series).²⁷

SMC Migration
SMC migration activity was assayed in a modified micro–Boyden chamber^{14 33} by using a polycarbonate filter with an 8.0-µm (diameter) pore size (Costar) to divide the upper and lower well chambers. Cultured human SMCs were trypsinized and suspended at a concentration of 5x10⁵ cells per milliliter in M199 supplemented with 2% FBS. A volume of 1 mL of cell suspension was placed in the upper chamber, and a volume of 2 mL of the same medium containing vehicle, PDGF (50 ng/mL), varying concentrations of IL-4, or IL-4 (100 U/mL) in combination with varying doses of -IFN or tranexamic acid was loaded into the lower chamber of the apparatus. After 48 hours of cytokine incubation (37°C, 5% CO₂ in air), the cells on the upper and lower sides of the filter were trypsinized and counted with a Coulter counter (model Z1, Coulter Electronics). Migration activity was determined by measuring the ratio of the cell number of triplicate counts in the upper and lower chambers of the apparatus and normalizing to the control group.

IL-4 Bioassay
Il-4–responsive Ramos 2G6 B cells (American Type Culture Collection) were used to assess the activity of IL-4 as determined by CD23 upregulation.³⁴ Ramos 2G6 B cells were stimulated with either media alone, IL-4 (100 U/mL) alone, tranexamic acid (100 µm) alone, or IL-4 (100 U/mL) plus tranexamic acid (100 µm) for 24 hours. Fluorescence-activated cell sorting analysis (FACS Star Plus, Becton Dickinson) was then performed on the cells with a monoclonal anti-CD23 antibody or a negative control antibody.³⁴

Statistics
All experiments were conducted in either duplicate or triplicate of different passages of SMCs if not otherwise indicated. Data are presented as mean±SEM of the independent experiments. Statistical significance was determined by one-way ANOVA and Fisher's protected least significant difference test (StatView 4.01, Brain Power, Inc). A value of P<.05 was considered statistically significant between the means.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of IL-4 on SMC TPA, UPA, and PAI-1 Antigen Levels
IL-4 had no direct effect on SMC TPA antigen production (data not shown). Therefore, we examined the effects of increasing concentrations of human recombinant IL-4 in the presence of 2% FBS on SMC TPA antigen levels in conditioned media (Fig 1a). SMC TPA antigen levels in conditioned media after coincubation with 2% FBS and IL-4 at concentrations of 1 U/mL (P<.03), 10 U/mL (P<.003), 100 U/mL (P<.006), and 1000 U/mL (P<.03) were significantly increased over the values observed with control SMCs incubated in media supplemented with 2% FBS alone. The concentrations of IL-4 that stimulated maximal SMC TPA antigen production were between 10 and 100 U/mL (twofold increase). The effects of IL-4 on SMC TPA antigen levels in cellular lysates showed the same dose-dependent response as in conditioned media. SMC TPA antigen levels in cellular lysates after coincubation with IL-4 at concentrations of 1 U/mL (P<.032), 10 U/mL (P<.023), 100 U/mL (P<.012), and 1000 U/mL (P<.043) were significantly greater than the values observed in control SMCs incubated in media supplemented with 2% FBS alone (Fig 1b). IL-4 had no significant effects on SMC TPA antigen levels in the absence of FBS (data not shown). It should be noted that we have duplicated all of our results with IL-4 in experiments with M199 (which contains adenine nucleotides and adenosine) as well as in control experiments with DMEM, which lacks adenosine or adenine nucleotides. Therefore, the effects of IL-4 on SMC fibrinolysis do not appear to be secondary to adenosine or adenine nucleotide effects. We have demonstrated that lipopolysaccharide does not alter SMC TPA levels at concentrations of 0.1 to 100 µg/mL and that IL-4 coincubated with polymyxin B, which inactivates lipopolysaccharide, does not abolish the effects of IL-4 on SMC TPA antigen levels. Thus, the effects of IL-4 on SMC TPA production were not from lipopolysaccharide contamination.

View larger version (17K): [in this window] [in a new window]   Figure 1. Effects of human recombinant IL-4 on SMC TPA (t-PA) antigen levels in conditioned media (a) and cellular lysates (b). Human aortic SMCs were incubated with varying concentrations of IL-4 in media containing 2% FBS for 72 hours at 37°C in a humidified 5% CO2 atmosphere. SMC TPA antigen levels in the conditioned media and cellular lysates were determined by an ELISA. Values are mean±SEM (n=6 independent experiments). *P<.05, **P<.01 relative to control SMCs cultured in media supplemented with 2% FBS alone.

Fig 2a represents the time course of the effects of IL-4 on SMC TPA antigen secretion in conditioned media. Basal SMC TPA levels in conditioned media without 2% FBS did not increase over a 72-hour incubation with media alone. Adding 2% FBS into the media significantly induced SMC TPA antigen production at 24 hours (P<.017), 48 hours (P<.003), and 72 hours (P<.002) over the values observed with media alone. The media containing 2% FBS plus IL-4 at a concentration of 100 U/mL significantly augmented SMC TPA antigen levels at 24 hours (P<.01), 48 hours (P<.0001), and 72 hours (P<.0001) over the values observed with media alone or with media supplemented with 2% FBS. The time course of the effect of IL-4 on SMC TPA antigen levels in cellular lysates is shown in Fig 2b. SMC TPA levels in cellular lysates after incubation with media containing 2% FBS and IL-4 at a concentration of 100 U/mL were significantly elevated at 48 hours (P<.001) and 72 hours (P<.0001) over the values observed with media alone or with media containing 2% FBS.

View larger version (19K): [in this window] [in a new window]   Figure 2. Time course of the effects of human recombinant IL-4 on SMC TPA (t-PA) antigen levels in conditioned media (a) and cellular lysates (b). Human aortic SMCs were incubated with IL-4 (100 U/mL) in media containing 2% FBS for 12 to 72 hours at 37°C in a humidified 5% CO2 atmosphere. SMC TPA antigen levels in the conditioned media and cellular lysates were determined by an ELISA. Values are mean±SEM (n=6 independent experiments). *P<.05, **P<.01 relative to control SMCs cultured in media devoid of FBS; P<.05 compared with control SMCs cultured in media supplemented with 2% FBS.

We next examined whether IL-4 affects SMC proliferation. SMC proliferation was determined by [³H]thymidine incorporation and direct determination of cell number using a Coulter counter after the SMCs were incubated in media containing varying doses of IL-4 for 48 to 72 hours. IL-4 at various concentrations ranging from 1 to 1000 U/mL did not increase either [³H]thymidine incorporation or the number of SMCs cultured in either basal media or media containing 2% FBS (data not shown).

We also examined the effects of human recombinant IL-4 on non–growth-arrested SMC TPA antigen production in conditioned media and cellular lysates. TPA antigen levels in conditioned media and cellular lysates of non–growth-arrested SMCs showed the same dose-dependent response to IL-4 as those of 48-hour growth-arrested SMCs (data not shown). The control levels of UPA antigen were <10 pg/10⁵ cells, and IL-4 did not alter SMC UPA production in either conditioned media or cellular lysates of growth-arrested cells in either the presence or absence of 2% FBS (data not shown).

The basal production of human aortic SMC PAI-1 is high (130 ng/10⁵ cells in conditioned media and 25 ng/10⁵ cells in cellular lysates). Both human recombinant IL-4 and -IFN did not alter SMC PAI-1 antigen production in conditioned media and cellular lysates in growth-arrested cells in the presence and absence of 2% FBS (data not shown).

Modulation of the Effect of IL-4 on SMC TPA Antigen Production
Human aortic SMCs were incubated in media containing either 2% FBS alone (control) or 2% FBS with the addition of one of the following: 100 U/mL of IL-4, 100 U/mL of boiled IL-4, 2 µg/mL of actinomycin D alone, 10 µg/mL of cycloheximide alone, 100 U/mL of IL-4 plus 1 µg/mL of a polyclonal goat anti–human IL-4 antibody, 100 U/mL of IL-4 plus 2 µg/mL of actinomycin D, or 100 U/mL of IL-4 plus 10 µg/mL of cycloheximide. All incubations were carried out for 24 hours at 37°C in a humidified 5% CO₂ atmosphere. In the presence of 2% FBS, IL-4 significantly increased the levels of TPA antigen measured in conditioned media (P<.007); coincubation with anti–human IL-4 antibody, actinomycin D, or cycloheximide or boiling of the IL-4 before use abolished this effect (Table). Actinomycin D alone and cycloheximide alone both abrogated TPA antigen induction.

View this table: [in this window] [in a new window]   Table 1. Modulation of the Effect of Human Recombinant IL-4 on SMC TPA Antigen Production in Conditioned Media

Effects of IL-4 on SMC TPA Activity
The effects of human recombinant IL-4 on SMC TPA activity in conditioned media are depicted in Fig 3a. Human aortic SMCs were incubated with varying concentrations of IL-4 in media containing 2% FBS for 72 hours at 37°C in a humidified 5% CO₂ atmosphere. TPA activity in conditioned media after coincubation of SMCs with IL-4 at concentrations of 10 U/mL (P<.002) and 100 U/mL (P<.029) was significantly increased over the activity observed with control SMCs incubated in media supplemented with 2% FBS alone. The stimulatory effects of IL-4 on SMC TPA activity in cellular lysates occurred in the same dose-dependent fashion as in conditioned media. SMC TPA activity in cellular lysates after coincubation with IL-4 at concentrations of 1 U/mL (P<.018), 10 U/mL (P<.009), 100 U/mL (P<.003), and 1000 U/mL (P<.022) was significantly greater than the values observed in control SMCs incubated in media supplemented with 2% FBS alone (Fig 3b). The discrepancy obtained between SMC TPA antigen production and TPA activity induced by IL-4 doses of 1 and 1000 U/mL in conditioned media may reflect the presence of large quantities of PAI-1 (>50-fold higher than TPA by weight) present in the conditioned media or possibly the presence of other plasmin inhibitors.

View larger version (16K): [in this window] [in a new window]   Figure 3. Effects of human recombinant IL-4 on SMC TPA (t-PA) activity in conditioned media (a) and cellular lysates (b). Human aortic SMCs were incubated with varying concentrations of IL-4 in media containing 2% FBS for 72 hours at 37°C in a humidified 5% CO2 atmosphere. SMC TPA activity in the conditioned media and cellular lysates was determined by the functional parabolic rate assay described by Ranby et al.30 Values are mean±SEM (n=6 independent experiments). *P<.05 relative to control SMCs cultured in media supplemented with 2% FBS alone.

Effects of -IFN on IL-4–Induced SMC TPA Antigen Production
-IFN had no direct effects on SMC TPA antigen production in either conditioned media or cellular lysates (data not shown). Fig 4 shows the effects of human recombinant -IFN on SMC TPA antigen production induced by IL-4 in conditioned media. Human aortic SMCs were incubated with varying concentrations of -IFN in media containing 2% FBS with or without IL-4 (100 U/mL) for 72 hours at 37°C in a humidified 5% CO₂ atmosphere. The levels of TPA antigen in the conditioned media of SMCs incubated in media supplemented with 2% FBS and IL-4 (100 U/mL) were significantly greater than those of SMCs incubated in media alone (P<.0001) and in media containing 2% FBS alone (P<.006). -IFN at a concentration of 1000 U/mL alone did not change SMC TPA antigen production. -IFN at concentrations of 100 U/mL (P<.038), 500 U/mL (P<.005), and 1000 U/mL (P<.014) significantly attenuated IL-4–stimulated production of SMC TPA antigen. -IFN did not diminish SMC TPA antigen production induced by IL-4 in cellular lysates (data not shown).

View larger version (35K): [in this window] [in a new window]   Figure 4. Effects of human recombinant -IFN on SMC TPA (t-PA) antigen production in conditioned media induced by IL-4. Human aortic SMCs were incubated with varying concentrations of -IFN in media containing 2% FBS with or without IL-4 (100 U/mL) or control media containing 0.2% BSA for 72 hours at 37°C in a humidified 5% CO2 atmosphere. SMC TPA antigen levels in the conditioned media were determined by an ELISA. Values are mean±SEM (n=5 independent experiments). *P<.05, **P<.01 relative to SMCs cultured in media containing 2% FBS and 100 U/mL IL-4.

Effects of IL-4 on PDGF-Induced SMC TPA Antigen Levels
We next sought to determine which factor(s) present in 2% FBS was potentiating SMC TPA antigen production by IL-4. The most logical target of the effects of IL-4 appeared to be PDGF, the potent SMC mitogen and chemoattractant, which is the major macromolecule in serum stimulating SMC DNA synthesis and cell growth.^{35 36} However, a polyclonal neutralizing antibody to PDGF failed to diminish the augmentation by IL-4 of 2% FBS–induced SMC TPA antigen production (data not shown). This antibody failed to manifest a diminution in SMC proliferation, thereby suggesting that although it may bind to PDGF, it does not inhibit PDGF functionally. We then examined whether IL-4 could directly potentiate PDGF-induced SMC TPA antigen production by measuring the effects of human recombinant IL-4 on TPA antigen levels of SMCs treated with increasing concentrations of human recombinant PDGF-BB in conditioned media (Fig 5a). SMC TPA antigen levels in conditioned media after incubation with PDGF-BB (100 ng/mL) alone (P<.0001) or coincubated with IL-4 (500 U/mL) and either 10 ng/mL of PDGF-BB (P<.002) or 100 ng/mL of PDGF-BB (P<.0001) were significantly increased over the values observed with control SMCs incubated in media supplemented with 0.2% BSA alone. TPA antigen levels in conditioned media in the SMC group incubated with PDGF-BB (100 ng/mL) plus IL-4 500 (U/mL) were significantly greater than those in the SMC group incubated with PDGF-BB (100 ng/mL) alone (P<.004). The effects of human recombinant IL-4 on TPA antigen levels of SMCs treated with various concentrations of human recombinant PDGF-BB in cellular lysates are shown in Fig 5b. SMC TPA antigen levels in cellular lysates after incubation with PDGF-BB (100 ng/mL) alone (P<.0001) or coincubated with IL-4 (500 U/mL) and either 10 ng/mL of PDGF-BB (P<.005) or 100 ng/mL of PDGF-BB (P<.0001) were significantly increased over control SMCs incubated in media supplemented with 0.2% BSA alone. TPA antigen levels in cellular lysates in the SMC group incubated with PDGF-BB (10 ng/mL) plus IL-4 (500 U/mL) were significantly greater than the values observed in the group incubated with PDGF-BB (100 ng/mL) alone (P<.004). We also tested AA and AB dimeric forms of PDGF alone and in combination with IL-4 (500 U/mL) on human aortic SMC TPA antigen production. Both PDGF-AA and PDGF-AB had effects that were very similar to those of PDGF-BB on SMC TPA antigen production (data not shown). PDGF at 100 ng/mL increased SMC TPA antigen in conditioned media fourfold compared with a onefold increase of SMC PAI-1 antigen (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 5. Effects of human recombinant IL-4 on SMC TPA (t-PA) antigen levels induced by PDGF in conditioned media (CM) (a) and cellular lysates (CLs) (b). Human aortic SMCs were incubated with varying concentrations of PDGF alone or in combination with 500 U/mL of IL-4 in media containing 0.2% BSA for 48 hours at 37°C in a humidified 5% CO2 atmosphere. SMC TPA antigen levels in CM and CLs were determined by an ELISA. Values are mean±SEM (n=6). **P<.01 relative to control SMCs cultured in media alone; P<.05, P<.01 relative to SMCs cultured with PDGF alone.

Effects of -IFN on PDGF-Induced SMC TPA Antigen Levels
We examined the effects of increasing concentrations of human recombinant -IFN on PDGF-induced SMC TPA antigen levels in conditioned media (Fig 6a). SMC TPA antigen levels in conditioned media after incubation with PDGF (50 U/mL) (P<.0001) were significantly increased over the values observed with control SMCs incubated in media supplemented with 0.2% BSA alone. -IFN by itself did not alter SMC TPA antigen levels in conditioned media. PDGF stimulation of SMC TPA antigen production was significantly attenuated by -IFN at concentrations of 100 U/mL (P<.03) and 1000 U/mL (P<.003). The effects of human recombinant -IFN on PDGF-induced SMC TPA antigen levels in cellular lysates are depicted in Fig 6b. SMC TPA antigen levels in cellular lysates after incubation with PDGF (50 U/mL) (P<.0001) were significantly increased over control SMCs incubated in media supplemented with 0.2% BSA alone. In the same dose-dependent fashion as in the conditioned media, PDGF-stimulated SMC TPA antigen production in cellular lysates was significantly attenuated by -IFN at concentrations of 100 U/mL (P<.008) and 1000 U/mL (P<.0001). -IFN by itself did not alter SMC TPA antigen levels in cellular lysates.

View larger version (16K): [in this window] [in a new window]   Figure 6. Effects of human recombinant -IFN on SMC TPA (t-PA) antigen levels induced by PDGF in conditioned media (CM) (a) and cellular lysates (CLs) (b). Human aortic SMCs were incubated with varying concentrations of media alone, PDGF alone, or PDGF in combination with varying concentrations of -IFN in media containing 0.2% BSA for 48 hours at 37°C in a humidified 5% CO2 atmosphere. SMC TPA antigen levels in the CM and CLs were determined by an ELISA. Values are mean±SEM (n=6). **P<.01 relative to control SMCs cultured in media alone; P<.05, P<.01 relative to SMCs cultured with PDGF alone.

Effects of -IFN on PDGF-Induced and IL-4–Induced SMC TPA Antigen Production
Fig 7a shows the modulation of the effects of increasing concentrations of human recombinant -IFN on human aortic SMC TPA antigen levels induced by the combination of PDGF and IL-4 in conditioned media. SMC TPA antigen levels in conditioned media after incubation with either PDGF (100 ng/mL) alone (P<.0001) or in combination with IL-4 (500 U/mL) (P<.0001) were significantly increased over control SMCs incubated in media supplemented with 0.2% BSA alone. -IFN at concentrations of 10 U/mL (P<.0003), 100 U/mL (P<.0001), 1000 U/mL (P<.0001), and 5000 U/mL (P<.0001) significantly diminished SMC TPA antigen production in conditioned media induced by the combination of PDGF and IL-4. -IFN by itself did not alter SMC TPA antigen levels in conditioned media. The effects of human recombinant -IFN on PDGF-induced and IL-4–induced SMC TPA antigen levels in cellular lysates are depicted in Fig 7b. SMC TPA antigen levels in cellular lysates after incubation with either PDGF (100 U/mL) (P<.0001) alone or in combination with IL-4 (500 U/mL) were significantly increased over control SMCs incubated in media supplemented with 0.2% BSA alone. In the same dose-dependent fashion as in the conditioned media, human aortic SMC TPA antigen levels in the cellular lysates stimulated by the combination of PDGF and IL-4 were significantly attenuated by -IFN at concentrations of 100 U/mL (P<.006), 1000 U/mL (P<.002), and 5000 U/mL (P<.001). -IFN by itself did not alter SMC TPA antigen levels in cellular lysates.

View larger version (22K): [in this window] [in a new window]   Figure 7. Effects of human recombinant -IFN on TPA (t-PA) antigen levels of SMCs incubated with both PDGF and IL-4 in conditioned media (CM) (a) and cellular lysates (CL) (b). Human aortic SMCs were incubated with PDGF alone or in combination with varying concentrations of -IFN plus 500 U/mL of IL-4 in media containing 0.2% BSA for 48 hours at 37°C in a humidified 5% CO2 atmosphere. SMC TPA antigen levels in the CM and CLs were determined by an ELISA. Values are mean±SEM (n=6). **P<.01 relative to control SMCs cultured in media containing 0.2% BSA alone; P<.05, P<.01 relative to SMCs cultured with PDGF plus IL-4.

Effects of IL-4 and -IFN on SMC Migration
SMC migration studies were performed with a modified micro–Boyden chamber method using a polycarbonate filter in a transwell apparatus containing upper and lower well chambers. SMC migration activity after a 48-hour cytokine incubation (37°C, 5% CO₂ in air) of SMCs in the upper chambers was determined by measuring the ratio of the cell number of triplicate counts in the upper and lower chambers of the apparatus and normalizing to the media alone control group. Neither IL-4 nor -IFN had direct effects on SMC migration (data not shown). The effect of IL-4 in the presence of 2% FBS on SMC migration is shown in Fig 8. PDGF (50 ng/mL) was used as a positive control. PDGF induced SMC migration from the upper chamber to the lower chamber (229±24%), which was significantly greater than the induction of SMC migration by the group incubated with media alone (control) (P<.0001) and the groups incubated with varying doses of IL-4 (P<.01). IL-4 at concentrations of 10 U/mL (147±12%, P<.05) and 100 U/mL (173±25%, P<.001) significantly increased SMC migration over the values observed with the group incubated with media alone (control) (n=6). -IFN at concentrations of 100 U/mL (P<.001, n=9) and 1000 U/mL (P<.01, n=9) attenuated SMC migration induced by IL-4 in a dose-dependent manner (Fig 9). Fig 10 demonstrates the effects of -IFN on human aortic SMC migration induced by PDGF. PDGF (50 ng/mL) significantly induced SMC migration from the upper chamber to the lower chamber (P<.0002). -IFN at concentrations of 100 U/mL (P<.03) and 1000 U/mL (P<.01) significantly inhibited SMC migration induced by PDGF.

View larger version (25K): [in this window] [in a new window]   Figure 8. Effects of IL-4 on SMC migration. SMC migration studies were performed by using a modified micro–Boyden chamber with a polycarbonate filter in a transwell apparatus containing upper and lower chambers. Human aortic SMCs (5x105/mL) were added into the upper chamber of the transwell, while varying concentrations of IL-4 or PDGF (50 ng/mL) in media containing 2% FBS were added to the lower chamber. SMC migration was determined by using a Coulter counter after a 48-hour cytokine incubation at 37°C in a humidified 5% CO2 atmosphere. Migration activity was determined by measuring the ratio of the cell number of triplicate counts in the upper and lower chambers of the apparatus and normalizing to the control. Values are mean±SEM (n=6 independent experiments). *P<.05, **P<.01 relative to control SMCs cultured in media supplemented with 2% FBS alone; P<.05, P<.01 relative to SMCs cultures with PDGF.

View larger version (25K): [in this window] [in a new window]   Figure 9. Effects of -IFN on SMC migration induced by IL-4. SMC migration studies were performed by using a modified micro–Boyden chamber method. Human aortic SMCs (5x105/mL) were added into the upper chamber of the transwell, while IL-4 alone, IL-4 in combination with varying concentrations of -IFN, or PDGF in media containing 2% FBS was added to the lower chamber. SMC migration was determined using a Coulter counter after a 48-hour cytokine incubation at 37°C in a humidified 5% CO2 atmosphere. Migration activity was determined by measuring the ratio of the cell number of triplicate counts in the upper and lower chambers of the apparatus and normalizing to the control. Values are mean±SEM (n=9 independent experiments). *P<.05, **P<.01 relative to control SMCs cultured in media supplemented with 2% FBS alone; P<.05, P<.01 relative to SMCs cultured with IL-4 (100 U/mL) alone.

View larger version (17K): [in this window] [in a new window]   Figure 10. Effects of -IFN on SMC migration induced by PDGF. SMC migration studies were performed by using a modified micro–Boyden chamber method. Human aortic SMCs (5x105/mL) were added into the upper chamber of the transwell, while PDGF alone or PDGF in combination with varying concentrations of -IFN in media containing 0.2% BSA was added to the lower chamber. SMC migration was determined by using a Coulter counter after a 48-hour cytokine incubation at 37°C in a humidified 5% CO2 atmosphere. Migration activity was determined by measuring the ratio of the cell number of triplicate counts in the upper and lower chambers of the apparatus and normalizing to the control. Values are mean±SEM (n=4 independent experiments). *P<.05, **P<.01 relative to control SMCs cultured in media supplemented with 2% FBS alone; P<.01 relative to SMCs cultured with IL-4 (100 U/mL) alone.

Effects of Tranexamic Acid on IL-4–Induced Migration
In order to further establish the role of plasmin in IL-4–induced SMC migration, we examined the effects of tranexamic acid, a plasmin inhibitor, on SMC migration induced by IL-4. IL-4 at concentrations of 100 U/mL in the presence of 2% FBS significantly increased SMC migration compared with media alone. Tranexamic acid at concentrations of 1, 10, and 100 µmol/L (P<.01, n=4) attenuated SMC migration induced by IL-4 in a dose-dependent manner (Fig 11). In order to ensure that tranexamic acid did not block IL-4 function directly (rather, indirectly, through plasmin activity), we measured the effects of tranexamic acid on IL-4 upregulation of CD23 on Ramos 2G6 human B cells, an established bioassay for the measurement of human IL-4 function.³⁴ Tranexamic acid had no effect on IL-4–dependent upregulation of CD23 (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 11. Effects of tranexamic acid (TA) on SMC migration induced by IL-4. SMC migration studies were performed by using a modified micro–Boyden chamber method. Human aortic SMCs (5x105/mL) were added into the upper chamber of the transwell, while IL-4 alone or IL-4 in combination with varying concentrations of tranexamic acid in media containing 2% FBS was added to the lower chamber. SMC migration activity was determined by measuring the ratio of the cell number of triplicate counts in the upper and lower chambers of the transwell apparatus and normalizing to the control. Values are mean±SEM (n=4 independent experiments). *P<.05, **P<.01 relative to control SMCs cultured in media supplemented with 2% FBS alone; P<.01 relative to SMCs cultured with IL-4 (100 U/mL) alone.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we confirm and extend to the in vitro milieu prior observations that PDGF increases SMC TPA production in vivo.²² Moreover, our study is the first demonstration that T cells may influence vascular SMC migration owing to T-cell lymphokine modulation of SMC TPA production. In particular, we reveal for the first time that the T-cell lymphokines IL-4 and -IFN manifest opposing effects on in vitro human aortic SMC fibrinolysis and migration. Indeed, IL-4 potentiates SMC TPA antigen production induced by 2% FBS as well as PDGF without affecting PAI-1 antigen production. Moreover, IL-4 increases in vitro SMC migration. In contrast, -IFN inhibits PDGF induction of SMC TPA antigen and IL-4 potentiation of the latter process. Furthermore, -IFN attenuates the promigratory effects of both IL-4 and PDGF. The SMC promigratory effects of IL-4 appear to be plasmin dependent, as evinced by their abrogation by tranexamic acid, a plasmin inhibitor.

The present study is distinct in that this is the first demonstration that T-cell lymphokines modulate vascular SMC migration through their effects on vascular SMC TPA production. The opposing effects of the lymphokines IL-4 and -IFN on vascular SMC fibrinolysis and migration are noteworthy in view of the previously established modulatory roles exerted by various cytokines on endothelial cell fibrinolysis and migration. Indeed, basic fibroblast growth factor upregulates endothelial cell plasminogen activator activity, whereas transforming growth factor-ß, which is activated by plasmin, is a potent inhibitor of endothelial cell plasminogen activator activity and a very potent inducer of PAI-1.^{37 38}

Prior studies have indicated that PDGF increases SMC TPA activity²² as well as PAI-1 activity.^{25 26} The present study demonstrates that PDGF augments net fibrinolytic activity, as evidenced by a PDGF-induced fourfold increase in SMC TPA antigen compared with only a onefold increase in SMC PAI-1 antigen. Thus, PDGF may serve to modulate SMC fibrinolytic activity, and its effects on SMC fibrinolysis may be regulated by its interactions with other cytokines, such as the lymphokines IL-4 and -IFN. Our results confirm the importance of SMC TPA for SMC migration and corroborate the prior in vivo studies demonstrating that PDGF stimulates SMC TPA activity and SMC migration in parallel in the rat carotid balloon injury model.²² The regulation of plasminogen activator activity may modulate in vivo SMC migration.^{19 39} TPA may have a more profound role than UPA on in vivo plasmin generation and fibrinolysis, as evinced by the finding that inactivation of the TPA gene impairs clot lysis in mice, whereas inactivation of the UPA gene results in occasional fibrin deposition.⁴⁰ SMCs express UPA during mitogenesis and TPA during migration in the injured rat carotid artery.³⁹ Both TPA mRNA and protein increase during SMC migration in the rat carotid model.^{19 39} In addition, heparin, an SMC migration inhibitor, selectively inhibits the transcription of TPA in baboon arterial SMCs during mitogenesis²³ and in balloon-injured rat carotid SMCs during migration.³⁹ The conversion of plasminogen to plasmin by plasminogen activators such as TPA and UPA can result in activation of the matrix metalloproteinases.⁴¹ In particular, the expression of the matrix metalloproteinase gelatinase facilitates SMC migration in the rat carotid artery balloon injury model.⁴²

In the present study, IL-4 had no direct effect on SMC fibrinolysis and migration; however, in the presence of 2% FBS, IL-4 effected a twofold maximal increase in TPA antigen and activity. In contrast, balloon injury in the in vivo rat carotid model induced more than a threefold increase in TPA activity.²² This may reflect the fact that cultured SMCs may have a higher PAI-1 content than SMCs in the in vivo state. It should be noted that our time-course studies of the effects of IL-4 on SMC TPA antigen production induced by 2% FBS reveal a maximal effect by 48 to 72 hours. This is consistent with the in vivo studies of Jackson et al,²² who revealed that TPA activity and SMC migration in the rat carotid artery cannot be detected until 3 to 4 days after injury, when SMC migration commences. Therefore, TPA may be necessary but not sufficient for SMC migration, as evidenced by the revelation that lisinopril, an angiotensin-converting enzyme inhibitor, inhibits SMC migration after rat carotid balloon injury without altering plasminogen-activator activity.¹⁵ Thus, there appear to be both plasmin-dependent and plasmin-independent mechanisms of SMC migration. Our studies demonstrating that tranexamic acid, a plasmin inhibitor, abrogates the stimulatory effects of IL-4 on in vitro SMC migration corroborate tranexamic acid inhibition of in vivo SMC migration.²² In the present study, tranexamic acid completely blocked the effects of IL-4 on SMC migration, although the level of SMC migration attained was similar to that of 2% FBS alone. Our results are consistent with those of Jackson et al,²² who demonstrated that tranexamic acid inhibited in vivo SMC migration by 75%, once again suggesting that there are plasmin-dependent and plasmin-independent mechanisms of SMC migration. In addition, we have unequivocally demonstrated that tranexamic acid does not block IL-4 function directly (rather, indirectly, through plasmin activity) by control experiments indicating that tranexamic acid has no effect on IL-4 upregulation of CD23 on Ramos 2G6 human B cells, a bioassay for the measurement of human IL-4 function.³⁴

-IFN, which usually opposes the actions of IL-4, possesses several antiatherogenic properties. -IFN inhibits the induction by IL-4 of monocyte 15-lipoxygenase.^{43 44} -IFN inhibits both the size and extent of atherogenic lesions in rabbits with hypercholesterolemia.⁴⁵ -IFN inhibits SMC proliferation in tissue culture and also inhibits in vivo formation of arterial proliferative lesions after rat carotid balloon injury.⁴⁶ Our in vitro findings that -IFN has no direct effect by itself on SMC fibrinolysis and migration but that -IFN attenuates the effects of IL-4 on SMC fibrinolysis and migration are consistent with other studies demonstrating an antifibrinolytic effect of -IFN. Indeed, -IFN has been shown to antagonize tumor necrosis factor–mediated induction of endothelial cell UPA proteolysis of ECM.⁹ Although we did not find a direct effect of -IFN on SMC PAI-1 production, -IFN has been demonstrated to be an inducer of PAI-1 in human orbital fibroblasts.⁴⁷ Furthermore, administration of recombinant -IFN to human subjects decreases total fibrinolytic activity and significantly increases plasminogen activator inhibitor levels.⁴⁸

In the present study, -IFN manifested potent pleiotropic properties with respect to the inhibition of SMC TPA antigen production. Indeed, -IFN inhibition of SMC TPA antigen production was not specific for IL-4, as evinced by -IFN attenuation of PDGF induction of SMC TPA antigen in the absence of IL-4. -IFN decreased IL-4 induction of SMC TPA antigen in conditioned media but not cellular lysates. This may reflect the ability of -IFN to block SMC TPA secretion without altering SMC intracellular TPA content.

IL-4 increased SMC TPA antigen production induced by either 2% FBS or PDGF; -IFN inhibited the potentiation by IL-4 of SMC TPA antigen production in the presence of either 2% FBS or PDGF. However, it should be noted that there are important differences in the results obtained with -INF and 2% FBS as opposed to those with -INF and PDGF. Although -IFN had no effect on TPA levels of cellular lysates induced by IL-4 and 2% FBS, -IFN reduced TPA levels of cellular lysates in PDGF-stimulated and IL-4 plus PDGF–stimulated experiments (Fig 7b). Furthermore, -IFN in the presence of PDGF reduced TPA levels below PDGF alone levels (Fig 6a and 6b), whereas -IFN in the presence of 2% FBS decreased TPA levels to the initial levels (2% FBS alone) (Fig 4). These data suggest that cytokine(s) other than PDGF in 2% FBS may be inhibiting the effects of -IFN or that other growth factors in 2% FBS may stimulate TPA induction without being susceptible to -IFN inhibition. Further studies are in progress to determine whether other cytokine(s) in 2% FBS influence IL-4 and -IFN modulation of SMC TPA production and migration.

PDGF-induced SMC migration can be inhibited by protein tyrosine kinase inhibitors, such as methyl 2,5-dihydroxycinnamate and genistein.⁴⁹ Moreover, these two tyrosine kinase inhibitors also inhibit directional cell locomotion owing to the reorganization of microtubules and stress fibers.⁴⁹ Our results demonstrating that IL-4 augments and -IFN inhibits PDGF-mediated SMC migration are intriguing in view of the signal transduction effected by IL-4 in other cellular lines. IL-4 stimulated mitogenesis in hematopoietic cells entails tyrosine phosphorylation of IRS-1, the principal substrate of the insulin receptor.⁵⁰ Indeed, IL-4, insulin, and insulin growth factor-1 all stimulate tyrosine phosphorylation of the IRS-1–related protein, 4PS.⁵⁰ Moreover, insulin stimulation has been demonstrated to effect association of the vß₃ integrin (a vitronectin receptor) with IRS-1, which may be a mechanism for integrin-mediated cell adhesion and motility using ECM receptors.⁵¹

In the present study, the effects of IL-4 on SMC TPA antigen levels in conditioned media and cellular lysates were similar in both growth-arrested and non–growth-arrested SMCs. Thus, the profibrinolytic effects of IL-4 on SMC TPA antigen production do not appear to be dependent on SMC proliferation. Therefore, IL-4 may play a useful role in further examining the cellular mechanisms operative in SMC migration as distinct from those in SMC proliferation. We have demonstrated that the stimulatory effects of IL-4 on SMC TPA antigen production can be inhibited by actinomycin D as well as cycloheximide coincubation. Whereas cycloheximide abolished protein synthesis, the combination of IL-4 and actinomycin D failed to manifest any effect on SMC TPA antigen production during the 12-hour period of protein synthesis. These data suggest a possible transcriptional effect of IL-4 on SMC TPA mRNA production. Further studies are in progress to ascertain the signal transduction and possible transcriptional effects of IL-4 and -IFN on SMC fibrinolysis and migration. Interestingly, not only cycloheximide alone but also actinomycin D alone in the absence of IL-4 abrogated SMC TPA antigen induction by 2% FBS, suggesting that other factors in FBS may also transcriptionally induce SMC TPA antigen. We are presently investigating the possibility that cytokine(s) in 2% FBS other than PDGF such as basic fibroblast growth factor induce SMC TPA antigen with IL-4 augmentation and -IFN inhibition of these effects in a manner similar to that seen with PDGF.

In summary, IL-4 potentiates human aortic SMC TPA antigen production induced by 2% FBS as well as PDGF. Moreover, IL-4 augments in vitro SMC migration. -IFN inhibits PDGF induction of SMC TPA antigen and the potentiation by IL-4 of the latter process. -IFN inhibits the SMC promigratory effects of both IL-4 and PDGF. Tranexamic acid, a plasmin inhibitor, abrogates the SMC migration effected by IL-4. The present study is the first demonstration that T-cell lymphokines may play a role in the regulation of vascular SMC fibrinolysis and migration. Indeed, our findings provide a strong argument in favor of further studies of T cell–smooth muscle cell interactions, which may have ramifications for a variety of pathological entities, including atherosclerosis, angioplasty restenosis, and vein graft intimal hyperplasia.

	Selected Abbreviations and Acronyms

 

-IFN = gamma interferon BSA = bovine serum albumin ECM = extracellular matrix ELISA = enzyme-linked immunosorbent assay FBS = fetal bovine serum IL-4 = interleukin-4 IRS-1 = insulin receptor substrate-1 M199 = medium 199 PAI-1 and -2 = plasminogen activator inhibitor-1 and -2, respectively PDGF = platelet-derived growth factor SMC = smooth muscle cell TPA = tissue-type plasminogen activator UPA = urokinase-type plasminogen activator

	Acknowledgments

 
This study was supported by National Heart, Lung, and Blood Institute Physician Scientist Award K11 HL-O2578 (Dr Rabbani) and grant HL-21006 (Dr Cannon) from the National Institutes of Health, Bethesda, Md, and by the Milstein Family Foundation. The authors thank Dr M. Yellin for his assistance in performing the control experiments evaluating the effects of tranexamic acid on IL-4 upregulation of CD23 on Ramos 2G6 human B cells.

Received January 18, 1995; accepted August 15, 1995.

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