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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1251-1257

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:1251-1257.)
© 1997 American Heart Association, Inc.

Articles

Bimodal Effects of Angiotensin II on Migration of Human and Rat Smooth Muscle Cells

Direct Stimulation and Indirect Inhibition Via Transforming Growth Factor-ß1

Guizben Liu; Emma Espinosa; Barry S. Oemar; ; Thomas F. Lüscher

From the Division of Cardiology, Cardiovascular Research, University Hospital/Inselspital, Bern, Switzerland.

Correspondence to Thomas F. Lüscher, MD, Cardiology, University Hospital, CH-8091 Zürich, Switzerland.


*    Abstract
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 *Abstract
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Abstract Angiotensin II may be an important mediator of neointima formation in vascular disease. This study was designed to examine the mechanisms involved in angiotensin II-stimulated migration of human and rat aortic vascular smooth muscle cells (VSMCs). VSMCs were seeded in one corner of Nunc four-well culture chambers; angiotensin II within filter paper was glued onto the wall of the opposite side. After 48 hours of incubation in serum-free medium containing growth-arresting factor, migrated cells were counted using a light microscope. Angiotensin II (2x10-11 to 2x10-8 mol/L) increased migration of VSMCs in a concentration-dependent manner. Interestingly, at higher concentrations of angiotensin II (up to 2x10-6 mol/L), migration was reduced to levels comparable with control levels. Losartan, an AT1 receptor antagonist, prevented migration, while PD123319, an AT2 receptor antagonist, had no significant inhibitory effect. Transforming growth factor-ß1 (TGF-ß1; 0.01 to 10.0 pg/mL) inhibited migration induced by angiotensin II (2x10-8 mol/L) in a concentration-dependent manner. A neutralizing TGF-ß antibody unmasked migratory effects of high concentrations of angiotensin II. Furthermore, angiotensin II (10-6 mol/L) upregulated TGF-ß1 mRNA levels fivefold in rat and fourfold in human VSMCs; this effect was prevented by losartan but not by PD123319. Thus, the effects of angiotensin II on migration of VSMCs are bimodal, ie, both migratory and antimigratory pathways are activated. Autocrine release of TGF-ß1 induced by angiotensin II exerts an antimigratory effect in rat and human VSMCs. The AT1 receptor is involved in regulation of both pathways.


Key Words: angiotensin II • losartan • transforming growth factor-ß1 • migration • smooth muscle cells


*    Introduction
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 *Introduction
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Arterial intimal thickening occurs in atherosclerosis, restenosis, and hypertension and has generally been attributed to proliferation of VSMCs.1 2 3 4 Because VSMCs in a normal artery are found mainly in the media, migration of VSMCs into the intima has recently been recognized as a prerequisite for arterial intimal thickening.5 6 Moreover, because approximately half the cells that migrate into lesions never divide,2 5 migration alone without proliferation accounts for part of the accumulation of VSMCs in the intima in vascular disease. Therefore, understanding of the cellular mechanisms involved in this response and of potential pharmacological tools able to inhibit VSMC migration may lead to new forms of therapy for atherosclerosis, restenosis, and hypertension.

Among a number of cardiovascular mediators, angiotensin II may be particularly important.7 8 Angiotensin II is a potent growth factor for VSMCs in vitro as well as in vivo.1 2 9 10 11 12 Angiotensin II accelerates myointimal proliferation in the rat, at least after balloon-induced vascular injury.1 2 AT1-receptor blockade with losartan reduces DNA synthesis of medial VSMCs and inhibits injury-induced intimal hyperplasia and proliferation.13 14 15 These data suggest that angiotensin II is involved in the intimal response to vascular injury. However, at the cellular level, little is known about the modulatory effects of angiotensin II on VSMC migration and whether the response is mediated by a single receptor or multiple receptors, or whether other migratory factors and/or inhibitors are involved. Furthermore, it is uncertain whether similar responses occur in human VSMCs.

The expression of autocrine growth factors, such as TGF-ß, has been noted in vessels from hypertensive animals, in human vascular restenotic lesions, and in injury-induced proliferation in rats.16 17 18 TGF-ß1 exerts a bimodal function on VSMCs as a growth inhibitor as well as growth promoter.19 20 Angiotensin II increases TGF-ß1 gene expression and promotes the conversion of latent TGF-ß1 to its biologically active form.21 22 23 Furthermore, in rat VSMCs, angiotensin II can activate both proliferative and antiproliferative pathways in which TGF-ß1 is considered to mediate the inhibitory response. The effects of angiotensin II and TGF-ß1 on migration of both human and rat VSMCs and their interaction during the migration process, however, have not been explored.

In the present study, we investigated (1) the effects of angiotensin II on migration of human and rat VSMCs in a four-well chamber system, (2) the type of AT receptor involved, and (3) the modulatory effects of TGF-ß1 on migration.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
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Materials and Reagents
Male Wistar-Kyoto rats (8 to 13 weeks old) were purchased from IFFA GREDO (L'Arbresle, France). Rat fibronectin, agarose, the Nunc four-well chamber, trizol, and all reagents for cell culture were from Life Technologies (Basel, Switzerland). Filter paper was from Schleicher and Schuell (Switzerland). The Coulter Counter was from Coulter Electronics Ltd, Luton, Beds (United Kingdom). Cytosine-ß-D-arabinofuranoside (cytosine), hematoxylin, human angiotensin II, and purified mouse IgG1 were purchased from Sigma (Buchs, Switzerland). Human recombinant TGF-ß1 and neutralizing monoclonal mouse anti-TGF-ß1 antibody were obtained from Genzyme (Basel, Switzerland). Losartan was kindly supplied by DuPont Merck (Wilmington, Del), and PD123319 was a gift of Parke-Davis (Ann Arbor, Mich).

Cell Culture
Rat VSMCs were isolated from rat thoracic aortas, and human VSMCs were obtained from ascending thoracic aortas of patients undergoing coronary bypass surgery. The VSMCs were isolated by the modified explant method.24 Explants and subsequent cellular outgrowths were maintained in DMEM supplemented with fetal calf serum (10% for rat VSMCs and 20% for human VSMCs), 25 mmol/L HEPES buffer, penicillin (100 µ/mL), streptomycin (100 mg/mL), and glutamine at 37°C in a humidified 5% CO2/95% air atmosphere. At confluence, these cells formed a hill-and-valley pattern typical of cultured SMCs. Cells were passaged with trypsin/EDTA, and medium was replaced twice per week. The purity of VSMCs was characterized by indirect immunofluorescence staining with specific anti–smooth muscle {alpha}-actin monoclonal antibodies. Experiments were performed with rat VSMCs from 5 to 7 rats at cell passages 3 to 6, and with human VSMCs from 4 to 5 patients at passages 3 to 9.

Cytosine Assay
Cytosine, a cytostatic drug, was used to arrest cell growth in migration studies. In separate experiments, cells were seeded in 24-well plates and allowed to attach in 10% fetal calf serum culture medium; they were then trypsinized and counted with a Coulter counter to measure baseline cell number before treatment. Cultures were then treated with human angiotensin II (2x10-8 mol/L) and cytosine at final concentrations of 0, 10, 25, and 50 µmol/L for 8, 24, 48, and 72 hours, respectively. The cells were washed three times with PBS, harvested with trypsin/EDTA, and counted. VSMCs treated with angiotensin II (2x10-8 mol/L) plus 50 µmol/L of cytosine maintained a stable cell number (13.6±1.4x105 and 2.9±2.9x105 for rat and human VSMCs, respectively) similar to that obtained at baseline (13.8±1.5x105 and 3.3±2.6x105, respectively; three independent experiments in triplicate, NS). However, after 72 hours, the number of cells tended to decrease (data not shown). Therefore, in all subsequent experiments, SMCs were treated with cytosine for 48 hours only.

Migration Assays
Migration assays were performed in Nunc four-well glass culture chambers (2x1x1 cm per well), which allowed directed movement of cells on a defined two-dimensional surface without damage to the cells. The chambers were precoated with rat fibronectin (5 µg/mL) to facilitate cell attachment. This concentration of fibronectin has only negligible migratory effects25 and was present both in control cells and those treated with angiotensin II or other agonists. VSMCs (3x105) were seeded in 100 µL of DMEM supplemented with 10% or 20% fetal calf serum in one corner of the chamber and incubated overnight to allow cell attachment. The cells were then washed with serum-free medium containing 0.2% bovine serum albumin, and a start line was drawn along the edge of the attached cells. At the opposite side of the chamber, an 8-mm2 piece of filter paper preincubated in 0.1% agarose containing angiotensin II (2x10-12 to 2x10-6 mol/L) was glued onto the opposite wall of the chamber using preheated (50°C) 0.5% agarose. Serum-free medium (800 µL) supplemented with 50 µmol/L cytosine was then added to each chamber. Cytosine was used as a growth-arresting treatment to exclude any contributing effects of proliferation on the results. The cells were incubated for another 48 hours, which in pilot experiments proved to be the optimal time for VSMC migration under these experimental conditions; at the end of the migration assay, cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with hematoxylin. The migration of VSMCs was then assessed by blinded counting of the cells observed across the start line using light microscopy by one of the authors (E.E.).

To investigate the effects of losartan and PD123319 on angiotensin II-induced VSMC migration, the cells were pretreated with these compounds for 24 hours. This time frame allowed sufficient time for the antagonists to bind and to occupy their specific receptors. The concentrations of the drugs given represent final molar concentrations in the medium of the culture chamber during preincubation. The migration assays were then performed with angiotensin II at the optimal stimulatory concentration (2x10-8 mol/L per filter paper).

To test the hypothesis that the reduction of VSMC migration occurring at high concentrations of angiotensin II (2x10-6 mol/L) may be due to increased release of TGF-ß1, the effects of TGF-ß1 alone or TGF-ß1 plus angiotensin II on VSMC migration were investigated. The effects of a neutralizing TGF-ß antibody were assessed by adding the antibody (25 µg/mL) to VSMCs together with angiotensin II (2x10-6 mol/L) or conditioned medium obtained from the supernatant of VSMCs treated previously with angiotensin II (2x10-6 mol/L). In separate experiments. the effects of this TGF-ß antibody were compared with those of an equal concentration of IgG on cell migration. To confirm that the potentiated migratory response to coincubation with angiotensin II and TGF-ß antibody was related to selective neutralization of active TGF-ß, the TGF-ß antibody (25 µg/mL) was preincubated with TGF-ß1 (0.5 ng/mL) before administration to the culture medium. The effect of coincubation of angiotensin II with the TGF-ß antibody prebound to TGF-ß1 on migration was compared with the response to coincubation of angiotensin II with TGF-ß antibody alone or to cells treated with IgG.

Northern Blot Analysis
Cells grown in petri dishes (100x20 mm) were harvested by lysis in 1 mL of trizol reagent (Life Technologies). Total cellular RNA was isolated according to the manufacturer's instructions. The RNA was electrophoresed in 1% formaldehyde–agarose gels, stained with ethidium bromide to prove RNA integrity, and transferred to hybond-N (Amersham) filters. Filters were hybridized with a random primed, 32P-labeled human TGF-ß1 cDNA (No. 59955, American Type Culture Collection, Bethesda, MD) and a glyceraldehyde-3-phosphate dehydrogenase cDNA probe as a control for loading. Filters were washed in 0.2x standard saline citrate and 0.1% sodium dodecyl sulfate at 65°C and exposed to Kodak XRP x-ray film for 24 hours at -70°C.

Statistical Analysis
All results are expressed as the mean±SEM. The number of rats or patients from whom blood vessels were obtained is represented by n. Statistical analysis of the data was performed using a paired Student's t test and ANOVA when appropriate. A value of P<.05 was considered significant.


*    Results
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 up arrowMethods
 *Results
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Effects of Angiotensin II on VSMC Migration
Angiotensin II caused a significant increase in migration of both rat and human aortic VSMCs (Fig 1Down, P<.001 and P<.005 versus control, respectively). These cells migrated as far as the border of the filter paper located at the other end of the chamber ({approx}1.5 cm). The migration induced by angiotensin II occurred in a concentration-dependent manner. The maximal effect was observed at 2x10-8 mol/L of angiotensin II and increased threefold in rat (Fig 1ADown) and fivefold in human VSMCs (Fig 1BDown) compared with untreated controls. PDGF-BB (5 to 10 ng/mL) used as positive control had a similar effect on migration (data not shown) as angiotensin II (up to 2x10-8 mol/L). However, at a higher concentration of angiotensin II (2x10-6 mol/L), cell migration was reduced to levels comparable with the control level (Fig 1Down; NS versus control); under these conditions, migration was significantly decreased compared with a lower concentration of angiotensin II (2x10-8 mol/L; P<.05).



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  		Figure 1. Bar graphs showing the effects of angiotensin II on migration of cultured rat (A, n=5) and human (B, n=5) aortic SMCs. Cells were seeded and grown on one side of four-well chambers; angiotensin II within a filter paper was glued to the opposite side of the chamber. Angiotensin II significantly stimulated migration of rat and human SMCs after 48 hours of incubation up to a concentration of 2x10-8 mol/L (*P<.05, **P<.005, ***P<.001 versus control).

Effects of Angiotensin Receptor Antagonists
Losartan, an AT1-receptor antagonist, inhibited angiotensin II-induced migration of VSMCs in a concentration-dependent manner (2.5x10-14 to 2.5x10-9 mol/L). The number of migrated rat VSMCs was already reduced at 2.5x10-11 mol/L of losartan (P<.05) and was maximally inhibited at 2.5x10-10 mol/L (P<.001) (Fig 2ADown). Angiotensin II-induced migration in human VSMCs was prevented by losartan (P<.05) at a concentration of 2.5x10-9 mol/L (Fig 2BDown). PD123319, an AT2-receptor antagonist, at concentrations up to 10-6 mol/L had no significant inhibitory effect (Fig 3ADown and 3BDown). Migration of both rat and human VSMCs at higher concentrations of angiotensin II was also not affected (data not shown).



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  		Figure 2. Bar graphs showing the effects of increasing concentrations of the AT1-receptor antagonist losartan on angiotensin II-induced migration of rat (A, n=7) and human (B, n=4) aortic SMCs (*P<.005, **P<.001 versus angiotensin II alone).



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  		Figure 3. Bar graphs showing the lack of effect of increasing concentrations of the AT2-receptor antagonist PD123319 on angiotensin II-induced migration of aortic SMCs (A, rat, n=5; B, human, n=4; NS versus angiotensin II alone).

Effects of TGF-ß1 on VSMC Migration
TGF-ß1 (0.0001 to 10 ng/mL) enhanced migration of rat SMCs at 0.01 ng/mL but had no significant effects at higher concentrations (Fig 4ADown; P<.05). In human VSMCs, migration was stimulated only at 1 ng/mL (Fig 4BDown; P<.05).



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  		Figure 4. Bar graphs showing the effects of TGF-ß1 alone on rat (A, n=5) and human (B, n=5) aortic SMC migration (*P<.05 versus control).

Pretreatment of the cells with human recombined TGF-ß1 (0.01 to 10 pg/mL) for 2 hours inhibited angiotensin II- induced (2x10-8 mol/L) cell migration in a concentration-dependent manner (Fig 5ADown and B). The effect of angiotensin II on migration was totally prevented by TGF-ß1 at a concentration of 1 pg/mL in rat VSMCs (Fig 5ADown) and at 10 pg/mL in human VSMCs (Fig 5BDown).



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  		Figure 5. Bar graphs showing the effects of TGF-ß1 on angiotensin II-induced cell migration. Pretreatment of rat (A, n=5) or human (B, n=5) SMCs with increasing concentrations of TGF-ß1 induced a concentration-dependent inhibition of angiotensin II-stimulated cell migration (n=5; *P<.05, **P<.005, ***P<.001 versus angiotensin II alone).

IgG alone did not affect VSMC migration. When coincubated with a high concentration of angiotensin II (2x10-6 mol/L), VSMC migration also remained unaltered compared with angiotensin II (2x10-6 mol/L) alone (NS). However, in the presence of a monoclonal TGF-ß antibody (25 µg/mL), migratory effects of a high concentration of angiotensin II (2x10-6 mol/L) were unmasked. Under these conditions, a high concentration of angiotensin II increased VSMC migration 3-fold (rat VSMCs; Fig 6ADown; P<.05) or 6-fold (human VSMCs; Fig 6BDown; P<.05) compared with angiotensin II (2x10-6 mol/L) alone. Similarly, conditioned medium obtained from VSMCs treated with angiotensin II (2x10-6 mol/L) induced migration only in the presence of the TGF-ß antibody. Under these conditions, conditioned medium from VSMCs treated with angiotensin II (2x10-6 mol/L) increased migration 2.5-fold (rat SMCs) and more than 5-fold (human SMCs) (Fig 6Down). Preincubation of TGF-ß antibody with TGF-ß1 abolished the effects of the antibody on the migration of cells treated with angiotensin II (2x10-6 mol/L). Incubation with TGF-ß antibody or control IgG alone did not affect VSMC migration (data not shown).



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  		Figure 6. Bar graphs showing the effects of neutralizing TGF-ß antibody (TGF-AB) on rat (A, n=3) and human (B, n=4) SMC migration. A high concentration (2x10-6 mol/L) of angiotensin II (Ang II) alone did not stimulate migration as compared with the control (far left). In the presence of a TGF-AB (fifth from left), migration of VSMCs stimulated with Ang II (2x10-6 mol/L) was increased as compared with Ang II (2x10-6 mol/L) alone (fourth from left), control, or IgG plus Ang II (far right). Similarly, in SMCs exposed to conditioned medium (CM; second from left) plus TGF-AB (third from left), migration was enhanced (*P<.05, **P<.01, ANOVA).

Effects of Angiotensin II on TGF-ß1 Gene Expression
Cultured quiescent human VSMCs expressed low levels of TGF-ß1 mRNA. Administration of angiotensin II (10-6 mol/L) for 24 hours resulted in a fivefold increase in mRNA levels in rat VSMCs (Fig 7Down, upper panel) and a threefold increase in human VSMCs. This effect was prevented by losartan (Fig 7Down, lower panel) but not by PD123319 (data not shown).



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  		Figure 7. Angiotensin II (top panel: lane 2, 10-6 mol/L; lane 3, 10-8 mol/L) increased TGF-ß1 mRNA levels in rat SMCs measured 24 hours after stimulation as compared with vehicle alone (lane 1: control). TGF-ß1 mRNA levels corrected for reduced glyceraldehyde-phosphate dehydrogenase increased fivefold after stimulation with angiotensin II. Quiescent human VSMCs expressed low levels of TGF-ß1 mRNA (lower panel: lane 1, control). Angiotensin II (lane 2, 10-6 mol/L) induced TGF-ß1 mRNA expression threefold in human VSMCs. This effect was abolished by losartan (lane 3, 10-8 mol/L).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
This study demonstrates that angiotensin II is a migratory factor that stimulates directed movement not only of rat aortic SMCs but also human aortic SMCs. Losartan, an AT1-receptor antagonist, prevented angiotensin II-induced migration, while the AT2-receptor antagonist PD123319 had no significant inhibitory effect. Interestingly, in this experimental setup the migratory effects of angiotensin II were particularly pronounced at lower concentrations and reached a maximum at around 10-8 mol/L, while at higher concentrations this migratory effect decreased to reach levels comparable with control levels at the highest concentration of angiotensin II (2x10-6 mol/L). TGF-ß1 inhibited angiotensin II-induced migration in a concentration-dependent manner in both human and rat aortic SMCs. On the other hand a neutralizing antibody for TGF-ß markedly increased the migratory effects of angiotensin II, particularly at high concentrations. Moreover, angiotensin II stimulated the expression of mRNA for TGF-ß1, and this was blocked by losartan.

Angiotensin II is known to induce proliferation and migration of rat and bovine aortic SMCs.2 9 26 27 The increase in migration may be related to the substantial increase in cell-associated u-PA activity in migrating SMCs during angiotensin II administration.26 The present study demonstrates that angiotensin II is also a migratory factor for human aortic SMCs. The potency of angiotensin II is comparable to PDGF, suggesting that it might be an important mediator in this process. Angiotensin II can exert its biological effects via a growing number of angiotensin receptor subtypes. Experiments with the selective AT1-receptor antagonist losartan and the AT2-receptor antagonist PD123319 demonstrated that in both rat and human aortic SMCs, migration is mediated via the AT1-receptor subtype.

Migration of cells can be studied under different experimental conditions. Boyden chambers are used most commonly,27 while in this study we used Nunc four-well chambers, in which the cells were seeded in one corner of the chamber, and the migratory stimulus, such as angiotensin II or PDGF-BB, on the other side of the chamber. This model allowed us to study directed movement of growth-arrested cells on a defined two-dimensional surface along a concentration gradient of a given migratory stimulus without cell wounding. Compared with other models, such as mechanical debridement, this model has the advantage that no cell debris and intracellular mediators are present at the site of migration as it occurs after wounding.

Interestingly, under these experimental conditions we observed a bimodal action of angiotensin II whereby lower concentrations markedly stimulated migration of the cells toward angiotensin II, while at higher concentrations the migratory responses were reduced as compared with responses at lower concentrations of angiotensin II. Both effects were mediated by AT1 receptors, since AT2-receptor blockade by PD123319 had no significant effect on angiotensin II-induced migration, nor did it prevent the reduction of this response at higher concentrations of angiotensin II.

Although in our experiments only VSMCs were used (as evidenced by the typical hill-and-valley pattern and {alpha}-smooth muscle actin staining), we cannot conclude that our observations are necessarily true for all VSMC subtypes. Indeed, it is possible that during cell culture, VSMC subtypes with a particular propensity to migrate and/or proliferate are selected. However, such cells may also be particularly important in the development of atherosclerotic plaque.28

TGF-ß1 is a molecule with diverse effects on VSMCs. Indeed, TGF-ß1 alone in vitro either inhibits or stimulates VSMC proliferation depending on the cell passage, cell density, culture cofactors, and the concentration of TGF-ß1 used.29 30 Indeed, in bovine and rat aortic SMCs in culture, TGF-ß1 alone increases migration31 32 33 but inhibits PDGF-induced migration in a concentration-dependent manner.33 Hence, to explore the phenomenon of the bimodal effects of angiotensin II on migration of rat and human aortic SMCs, we examined the potential role of this growth factor. Increasing concentrations of TGF-ß1 alone transiently enhanced migration of human or rat SMCs only at one concentration used. In cells that were pretreated with exogenous TGF-ß1, the migratory effects of angiotensin II, however, were potently inhibited in a concentration-dependent manner. Moreover, a monoclonal antibody directed against TGF-ß also unmasked migratory effects of high concentrations of angiotensin II. Similar results were obtained when cells were incubated with conditioned medium obtained from SMCs treated with a high concentration of angiotensin II, suggesting that the release of a transferable factor, most likely TGF-ß, is involved. In line with this interpretation, angiotensin II upregulated TGF-ß1 gene expression in SMCs, and this was prevented by losartan. The effects of angiotensin II on TGF-ß1 gene expression were already noted at 10-8 mol/L with no further increase at 10-6 mol/L, possibly because gene expression does not strictly reflect the amount of protein formed. This may explain why TGF-ß1 gene expression induced by the two concentrations of angiotensin II was similar, yet the antimigratory effects were most pronounced at the high concentration of angiotensin II. Hence, together these data indicate that angiotensin II at higher concentrations induces the expression and release of TGF-ß1 from SMCs via activation of the AT1 receptor.

The concentration of active TGF-ß1 induced by angiotensin II has been studied previously by other investigators.22 23 In the defined serum-free medium, angiotensin II at the concentration of 10-6 mol/L induced a 10-fold increase in TGF-ß1 activity from a basal level of 6±4 to 62±8 pmol/L in normal rat VSMCs.22 In SHR VSMCs, angiotensin II released up to 5x10-12 mol/L of TGF-ß1 compared with a baseline level of 10-13 mol/L.33 Furthermore, angiotensin II also stimulates production of plasminogen activator by cultured SMCs,26 which in turn is able to proteolytically activate latent TGF-ß1. Hence, angiotensin II is able to stimulate both the production of latent TGF-ß1 in rat SMCs and its conversion to an active form in serum-free medium. Interestingly, TGF-ß1 has different effects on SMC proliferation in SHRs than in normotensive Wistar-Kyoto rats. SHR VSMCs replicate more rapidly than Wistar-Kyoto rat cells,34 35 36 37 and TGF-ß1 potentiates rather than inhibits growth factor-stimulated proliferation of VSMCs in the SHR.38 Hence, the autocrine inhibitory pathway described herein may be lost in SHRs.

The results of these studies may help us to understand the complex mechanisms involved in angiotensin II-mediated migration of SMCs. The induction of TGF-ß1 by angiotensin II may serve as a negative feedback mechanism, preventing excessive migration at sites where angiotensin II is produced at high levels. It has to be considered, however, that the induction of TGF-ß1 may serve to initiate a second step in the response to vascular injury in which migration and proliferation are followed by matrix formation. Indeed, TGF-ß1 has been shown to take part in the latter process.17 18 39 40 41 Interestingly, TGF-ß1 differentially modulates extracellular matrix production and cellular proliferation in the arterial wall in vivo,42 suggesting that TGF-ß1 could help to promote healing but limit extensive cellular intimal hyperplasia induced by other growth factors such as PDGF-BB or angiotensin II. Hence, the results of this study, ie, that autocrine TGF-ß1 inhibits angiotensin II-induced cell migration, may prove useful in the design of gene therapy to limit restenosis. The observation that the angiotensin AT1-receptor antagonist losartan is able to prevent migration induced by angiotensin II may have important clinical implications, because it suggests that this new class of drugs may be effective in interfering not only with proliferation but also with migration, both of which contribute importantly to structural vascular changes occurring in atherosclerosis, restenosis, and hypertension.


*    Selected Abbreviations and Acronyms
 
IgG = immunoglobulin G
NS = not significant
PDGF = platelet-derived growth factor
SHR = spontaneously hypertensive rat
SMC = smooth muscle cell
TGF = transforming growth factor
VSMC = vascular smooth muscle cell


*    Acknowledgments
 
This work was supported by grants from the Swiss National Research Foundation (No. 32-32541. 91/2) and Merck, Sharp, and Dohme-Chibret, Glattbrugg, Switzerland (G.L.). We thank V. Gafner for excellent technical assistance.


*    Footnotes
 
This work was presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form (Circulation. 1995;92[suppl I]:I-422).

Received November 14, 1995; accepted May 29, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

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