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(Circulation. 2001;103:597.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Endothelial Cells Inhibit Flow-Induced Smooth Muscle Cell Migration

Role of Plasminogen Activator Inhibitor-1

Eileen M. Redmond, PhD; John P. Cullen, PhD; Paul A. Cahill, PhD; James V. Sitzmann, MD; Steingrimur Stefansson, PhD; Daniel A. Lawrence, PhD; S. Steve Okada, MD

From the Department of Surgery, University of Rochester Medical Center, Rochester, NY (E.M.R., J.P.C., P.A.C., J.V.S., S.S.O.), and Department of Biochemistry, Holland Laboratory, Rockville, Md (S.S., D.A.L.).

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—The endothelium may play a pivotal role in hemodynamic force–induced vascular remodeling. We investigated the role of endothelial cell (EC) plasminogen activator inhibitor-1 (PAI-1) in modulating flow-induced smooth muscle cell (SMC) migration.

Methods and Results—Human SMCs cocultured with or without human ECs were exposed to static (0 mL/min) or flow (26 mL/min; shear stress 23 dyne/cm²) conditions for 24 hours in a perfused capillary culture system. SMC migration was then assessed with a Transwell migration assay. In the absence but not in the presence of ECs, pulsatile flow significantly increased the migration of SMCs (264±26%) compared with SMCs under static conditions, concomitant with a 3- and 4-fold increase in PAI-1 mRNA and protein, respectively, in cocultured ECs. In the presence of PAI-1-/- ECs, flow increased wild-type SMC migration (226±25%), an effect that was reversed by exogenous PAI-1. To determine whether the antimigratory activity of PAI-1 was dependent primarily on inhibition of PAs or its association with vitronectin, experiments were conducted with PAI-1R (a mutant PAI-1 that binds to vitronectin but does not inhibit PA) and PAI-1K (a mutant that inhibits PA but has reduced affinity for vitronectin). PAI-1R inhibited both basal and flow-induced migration, whereas PAI-1K inhibited flow-induced migration in the absence of any effect on baseline migration.

Conclusions—Flow-induced EC PAI-1 inhibits flow-induced SMC migration in vitro. EC PAI-1 expression may be one of the predominant mechanisms responsible for controlling the process of vascular remodeling.

Key Words: endothelium • stress • atherosclerosis • muscle, smooth • plasminogen activators

	Introduction

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Mechanical forces associated with blood flow (hemodynamic forces) play an important role in the control of blood vessel structure and function.^{1 2 3} These hemodynamic forces can be resolved into 2 components: shear stress, the tangential frictional force acting at the endothelial cell surface, and pressure stretch, acting perpendicular to the vascular wall.^{1 2 3}

Neointimal hyperplasia, observed during native-vessel atherosclerosis, is also an undesirable consequence of many iatrogenic forms of vascular injury, such as arterialization of saphenous vein grafts after CABG. Initially, when an autologous vein graft is placed in the arterial circulation, the change in environment from the venous to the arterial circulation is accompanied by a significant increase in shear stress.⁴ The wall stress is initially very high and has a tendency to normalize as the vein graft wall thickens, partly as a result of smooth muscle cell (SMC) accumulation.⁵ Therefore, some of the wall thickening is beneficial, because it leads to normalization of wall stress in the graft.^{4 5} In humans, however, this response to vein grafting often becomes pathological, resulting in focal narrowings or graft closure, which occurs far more frequently with vein grafts than when arterial grafts (eg, mammary artery) are used.⁶ In favorable outcomes, the intimal growth reaches a steady state in which luminal area and functional integrity of the vein graft are maintained. In this setting, vessel wall homeostasis may be maintained by the intact endothelium, which exerts its regulatory influence on the process of neointimal thickening by production of soluble factors. The precise mechanisms by which the endothelium inhibits intimal thickening are currently unknown.

The accumulation of SMCs in the intima of arteries is one of the most prominent features of atherosclerosis and of the intimal hyperplastic lesions that cause restenosis after angioplasty.^{7 8} It is increasingly recognized that migration of SMCs from the media is a key event in progressive intimal thickening, leading to atherosclerosis and restenosis.^{7 8 9} Several groups have reported the involvement of the plasminogen activator (PA) system in SMC migration.^{10 11 12 13 14 15} The inactive proenzyme plasminogen is activated to the proteolytic enzyme plasmin by 2 PAs, tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). The system is regulated by a series of PA inhibitors, of which plasminogen activator inhibitor type 1 (PAI-1) is thought to be the most physiologically important. PAI-1 has been shown to inhibit SMC migration,¹⁶ and inactivation of the PAI-1 gene results in exuberant neointimal thickening.¹⁷

We have previously demonstrated that pulse pressure due to pulsatile flow increases SMC migration by a mechanism mediated, in part, by uPA.¹⁸ Here, we demonstrate that endothelial cells protect against flow-induced SMC migration. In addition, we present evidence that flow-induced EC PAI-1 plays a crucial role in mediating this protection.

	Methods

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Animals
PAI-1-/- mice (C57 Bl/6J) were purchased from Jackson Laboratories (Bar Harbor, Me). Mice were housed in specific pathogen-free rooms and fed a normal mouse laboratory diet. All procedures were approved by the Rochester University Animal Care and Use Committee.

Vascular SMCs
SMC cultures were prepared from (1) human umbilical veins (HUSMCs) and (2) murine venae cavae (MSMCs) by the explant technique.¹⁹ Briefly, the media of the vein was isolated surgically and the tissue minced into small pieces. The pieces were plated onto a fibronectin-coated Petri dish and cultured in DMEM/Ham’s F-12 medium with 10% heat-inactivated FCS, glutamine, and 100 U/mL penicillin/100 µg/mL streptomycin in a humidified atmosphere of 5% CO₂, 95% air. Cells were subcultured with 0.125% trypsin-EDTA when there was adequate proliferation of cells beyond the explants. SMCs displayed the typical spindle-shaped morphology and "hill-and-valley" pattern of growth in culture and were further characterized by immunohistochemical staining for smooth muscle–specific -actin. Cells between passages 2 and 4 were used in the perfused transcapillary cultures.

Endothelial Cells
EC cultures were prepared from (1) human umbilical veins (HUVECs) and (2) murine venae cavae (MECs) of both wild-type and PAI-1–knockout animals by established methods as previously described.¹⁹ Briefly, HUVECs were harvested from human umbilical veins by addition of 0.1% collagenase (Gibco Laboratories) for 30 minutes. The cells were grown to confluence in Medium 199 (Gibco) supplemented with 10% heat-inactivated FCS (Flow Laboratories, Inc), penicillin-streptomycin (Gibco), Fungizone (Gibco), and endothelial cell growth factor. MECs were isolated from the inferior venae cavae of mice (20 PAI-1–knockout mice and 20 wild-type mice were used) by a multistep isolation scheme previously described by Ojeifo et al.²⁰ Cells were assessed for endothelial cell phenotype by morphology, expression of von Willebrand factor antigen, and platelet and endothelial cell adhesion molecule. ECs between passages 2 and 5 were used in the coculture system as described below.

Perfused Transcapillary Cultures
Perfused cocultures of SMCs and ECs were established as described in detail previously.^{18 21 22} The Cellmax Quad artificial capillary cell culture system (Cellco, Inc) was used. This apparatus consisted of an enclosed bundle of 50 permeable, Pronectin-F–coated polypropylene capillaries (capillary length, 13 cm; ID, 330 µm; wall thickness, 150 µm; pore size, 0.3 µm; extracapillary surface area, 100 cm²; luminal surface area, 70 cm²) through which medium from a reservoir is pumped in a pulsatile fashion, at a chosen flow rate, via silicone rubber tubing. Pronectin-F is a synthetic protein polymer that incorporates multiple copies of the RGD cell attachment ligand of human fibronectin. In the present study, unless otherwise specified, the intraluminal flow rate used was 25 mL/min, corresponding to a shear stress of 23 dyne/cm², an intraluminal pulse pressure of 149/48 mm Hg experienced by ECs grown in the intraluminal space, and an extraluminal pulse pressure of 106/50 mm Hg with a frequency of 110 cycles/min experienced by SMCs grown in the extracapillary space.

Seeding of SMCs and EC Experimental Protocol
Human or mouse SMCs (5x10⁶ cells) in DMEM/Ham’s F-12 supplemented with 10% FBS and antibiotics (SMC medium) were seeded into the extracapillary space via the side ports. The SMCs were allowed to adhere and establish themselves on the outer surface of the capillaries, which were perfused at the lowest flow rate, 0.3 mL/min. For cocultures, human or mouse ECs (2x10⁶ cells) were seeded intraluminally via the end ports essentially as described previously.^{21 22}

A series of perfused transcapillary cultures were examined in parallel. The cultures were designated as either static or flow. After the 48-hour stabilization period, the static group cultures were disconnected from the pulsatile pump and experienced static conditions (0 mL/min), whereas the flow group cultures were exposed to a single-step increase in flow up to 25 mL/min and maintained for 24 hours. Where indicated, recombinant forms of PAI-1 at a final concentration of 500 nmol/L were added to the medium reservoir during this 24-hour period. The specific forms of PAI-1 used were as follows: PAI-1S, a stable analogue of wild-type PAI-1 that has been described previously (clone 14-1b²³ ) and was obtained from Molecular Innovations. This form of PAI-1 has a >70-fold stabilization compared with wild-type PAI-1 (145 versus 2.0 hours) but otherwise is indistinguishable from wild-type PAI-1 with respect to inhibitory activity or vitronectin binding.^{23 24} The second mutant, PAI-1K, was also constructed on the stable clone 14-1b background and has stability and inhibitory activity similar to that of PAI-1S but has the additional mutation of Gln 123 to Lys. Mutants with this latter substitution have previously been shown to retain wild-type inhibitory activity but to not bind vitronectin.²⁵ The third mutant, PAI-1R, is a double mutant with Thr 333 to Arg and Ala 335 to Arg (residues P₁₄ and P₁₂ of the reactive center loop); it was constructed on the wild-type PAI-1 background and has no inhibitory activity but binds to vitronectin with wild-type affinity (manuscript in preparation). This mutant is similar to a previously described PAI-1 mutant that had a single substitution of Thr 333 to Arg and was shown to have no inhibitory activity.²⁶ At the end of the experimental period, either the SMCs were harvested with trypsin and used in the migration assay or total EC RNA and protein were extracted after treatment of cultures with Trizol (Gibco) for Northern blot and Western blot analyses.

Transwell Filter Migration Assay
Fibronectin-coated Transwell filters, 12-µmol/L pore size (Costar), were used for migration assays as described previously.¹⁸ SMCs harvested from the transcapillary cultures were seeded at a density of 1.0x10⁴ cells/filter. Cells were allowed to migrate for 10 hours with conditioned media from the respective transcapillary cultures in both upper and lower chambers. In this way, random migration or chemokinesis was being measured, because there was no concentration gradient between the upper and lower chambers. The number of cells per high-power field (x20 magnification) that had migrated through the filter pores was counted manually with a microscope (Nikon Diaphot). Data are reported as the number of SMCs counted per 12 high-power fields, expressed as percentage of control, where control is SMC monocultures exposed to static conditions unless otherwise stated.

Northern Blot Analysis
Total RNA was isolated from static and flow group ECs with TRIzol Reagent (Gibco BRL) as described previously.¹⁸ Aliquots (10 µg) of the total RNA samples were separated on formaldehyde-agarose gels. The RNAs were transferred, UV–cross-linked to nylon membranes, and hybridized with ³²P-labeled cDNA probes for human PAI-1 and GAPD (ATCC) that were prepared by random priming. Transcripts were quantified by NIH Image 1.60 and normalized with GAPD levels used for equal loading.

Immunoblots
Cell lysates were prepared and analyzed for PAI-1 expression by Western blot analysis essentially as described previously.¹⁸ Rabbit anti-human recombinant PAI-1 (Molecular Innovations) was used at 1 to 5000 dilution.

Statistical Analysis
Results are expressed as mean±SEM. n is the number of individual perfused transcapillary cultures from which cells were harvested. Experimental points were performed in triplicate with a minimum of 3 independent experiments. Experimental and control cells in migration assays were compared by use of unpaired 2-tailed Student’s t tests. When >2 groups were present, an ANOVA (factorial design) was used (Statview). A probability value of P<0.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of ECs on Flow-Induced SMC Migration
Monocultured HUSMCs (ie, HUSMCs cultured in the absence of HUVECs) significantly increased (264±26%, n=11) their migration after exposure of cells to pulsatile flow (25 mL/min) compared with monocultured HUSMCs under static (0 mL/min) conditions (Figure 1). In contrast, when HUSMCs were cocultured in the presence of HUVECs, there was no effect of pulsatile flow on migration (Figure 1).

View larger version (19K): [in this window] [in a new window]   Figure 1. Effect of pulsatile flow on migration of monocultured or cocultured HUSMCs. Random migration or chemokinesis of HUSMCs cultured in absence or presence of HUVECs was studied by use of a Transwell migration assay as described in Methods after cells were exposed to static or pulsatile flow (25 mL/min, corresponding to a shear stress of 23 dyne/cm2; intraluminal pulse pressure 149/48 mm Hg; extraluminal pulse pressure 106/50 mm Hg) conditions for 24 hours. HUSMC migration is expressed as number of cells that migrated through filter/10 high-power fields (hpf). Data are mean±SEM, n=11 (monocultures); n=4 (cocultures). *P<0.05 vs static monocultures.

Effect of Pulsatile Flow on EC PAI-1 mRNA and Protein Levels
Compared with the no-flow static control group, HUVECs cultured in the absence or presence of HUSMCs and exposed to pulsatile flow demonstrated a significant increase in PAI-1 mRNA expression: 1.79±0.19- and 3.4±0.78-fold increases (n=4) for monocultured and cocultured ECs, respectively (Figure 2). Interestingly, PAI-1 mRNA expression, both at basal levels and after exposure to pulsatile flow, was significantly greater in HUVECs cocultured with HUSMCs than in those cultured in the absence of HUSMCs (Figure 2). Similarly, Western blot analysis revealed 3- and 4-fold increases in EC PAI-1 protein levels under flow conditions for monocultures and cocultures, respectively (Figure 3).

View larger version (28K): [in this window] [in a new window]   Figure 2. Effect of pulsatile flow on EC PAI-1 mRNA. a, Northern blot analysis. HUVECs in absence (monocultures) or presence (cocultures) of HUSMCs were exposed to pulsatile flow (25 mL/min) or static (no-flow) conditions for 24 hours. Total HUVEC RNA was isolated and Northern blots were performed as described in Methods. Representative blots are shown. b, Normalized graph of PAI-1 mRNA level. PAI-1 mRNA levels were quantified with optical densitometry and image analysis software (Image 1.60, NIH). Arbitrary values are expressed after normalization for minor differences in loading with GAPD mRNA level and are expressed relative to static monoculture. Data are mean±SEM, n=3. *P<0.05 vs respective static control; #P<0.05 vs corresponding monoculture.

View larger version (29K): [in this window] [in a new window]   Figure 3. Effect of pulsatile flow on EC PAI-1 protein levels. HUVECs in absence (monocultures) or presence (cocultures) of HUSMCs were exposed to pulsatile flow or static conditions for 24 hours as described in Methods. Top, Representative Western blot with cumulative densitometric data of 3 separate experiments. *P<0.05 vs respective static control; #P<0.05 vs corresponding monoculture.

Effect of EC PAI-1 Gene Deletion and Exogenous PAI-1 Addition on Pulsatile Flow–Induced SMC Migration
To further investigate the role of EC PAI-1 in inhibiting flow-induced SMC migration, we determined the effect of pulsatile flow on the migration of wild-type MSMCs cultured in the absence or presence of wild-type or PAI-1–knockout MECs. Like HUSMCs, wild-type MSMC monocultures responded to pulsatile flow by increasing their migration (260±15%, n=4), an effect that was completely blocked by the presence of wild-type MECs (Figure 4). However, in the presence of PAI-1–knockout MECs, flow significantly increased MSMC migration (305±21%, n=4) (Figure 4), an effect that was inhibited by addition of exogenous PAI-1S (500 nmol/L) (Figure 5). To determine whether the antimigratory activity of PAI-1 was dependent primarily on inhibition of PA or its association with vitronectin, experiments were carried out with PAI-1R, a mutant PAI-1 that binds to vitronectin but does not inhibit PA, or PAI-1K, a mutant that inhibits PA but has reduced affinity for vitronectin. The exogenous addition of either PAI-1S or PAI-1R (500 nmol/L) inhibited both baseline (flow-independent) and flow-dependent MSMC migration (Figure 5). The PAI-1K mutant significantly inhibited the flow-induced migratory response in the absence of any effect on baseline migration (Figure 5).

View larger version (19K): [in this window] [in a new window]   Figure 4. Effect of EC PAI-1 gene deletion on flow-induced SMC migration. Wild-type MSMCs (WT MSMC) cultured in absence or presence of either wild-type MECs (WT MEC) or PAI-1–knockout ECs (PAI-1 KO MEC) were exposed to either static or pulsatile flow conditions as described in Methods. Data are expressed as mean±SEM, n=4. *P<0.05 vs WT MSMC static.

View larger version (20K): [in this window] [in a new window]   Figure 5. Effect of exogenous PAI-1 addition on flow-induced SMC migration. Wild-type MSMCs (WT MSMC) cocultured with murine PAI-1–knockout ECs (PAI-1 KO MEC) were exposed to either static or pulsatile flow conditions in absence or presence of PAI-1S, PAI-1R, or PAI-1K (500 nmol/L). Data are expressed as mean±SEM, n=3. *P<0.05 vs respective static or flow in absence of PAI-1; #P<0.05 vs flow in presence of PAI-1S.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major finding of the present in vitro study is that ECs, by a mechanism involving PAI-1, protect SMCs from the stimulatory effects of pulsatile flow on migration.

Under normal circumstances, arterial SMCs in the presence of ECs are subjected to mechanical forces without significant vascular remodeling. After endothelial dysfunction, injury, or denudation, however, dramatic changes in SMC migration and proliferation can occur.⁷ In support of such studies, we have previously demonstrated that monocultured SMCs respond to changes in pulse pressure due to increases in pulsatile flow by increasing their migration response.¹⁸ The flow-induced migration response occurred via uPA- and matrix metalloproteinase–dependent mechanisms.¹⁸ However, as demonstrated in the present study, when SMCs were cocultured with ECs, there was no effect of pulsatile flow on SMC migration. Furthermore, endothelial cell inhibition of the flow-induced migration response was dependent on PAI-1 gene expression, because pulsatile flow increased EC PAI-1 mRNA, and ECs deficient in the PAI-1 gene (ie, PAI-1-/- ECs) did not affect the migration response. The changes in PAI-1 mRNA correlated with increased PAI-1 protein expression. The experiments using PAI-1-/- ECs also demonstrate that it is not just a barrier effect of a monolayer of cells that is protecting the SMCs from hemodynamic forces. In parallel cultures, exogenous addition of PAI-1S inhibited flow-induced SMC migration.

Although several previous studies have shown that mechanical forces modulate PA expression in ECs, with increased shear stress and/or cyclic strain increasing tPA expression,²⁷ the effect on PAI-1 is less clear. Diamond et al²⁸ found that EC PAI-1 secretion was unaffected by shear stress, whereas Iba et al²⁹ reported no change in PAI-1 levels after repetitive stretch. Others have demonstrated a shear stress–dependent decrease in PAI-1 mRNA.³⁰ In our study, ECs exposed to pulsatile flow demonstrated a significant increase in PAI-1 mRNA and protein expression compared with the static group. Differences in experimental conditions (eg, time of exposure to flow, level of shear stress, configuration of cells, monocultures versus cocultures) may explain these different results. Of note in our study, EC PAI-1 expression appeared to be influenced by the presence of SMCs, because under both static and flow conditions, PAI-1 mRNA expression and protein levels were greater in cocultured than in monocultured ECs. In addition, the induction of EC PAI-1 was more robust in ECs cocultured in the presence of SMCs. Precisely how the SMCs are affecting EC gene expression requires further investigation.

Localized expression of PA activity has been postulated to be important in a wide variety of normal and pathological conditions. This localized expression in turn may stimulate or inhibit the process of neointimal thickening. The process of SMC migration requires loosening of intercellular junctions and degradation of the pericellular matrix, possibly by 1 or more proteases, such as plasmin. Plasmin is formed by proteolysis of Glu- or Lys-plasminogen by tPA or uPA. Plasmin degrades noncollagenous matrix components, directly activates procollagenase, and regulates the activation of mitogens such as interleukin-1.^{31 32} The system is regulated by PAI-1.¹⁷ The imbalance between the endogenous expression of profibrinolytic (uPA, tPA) and antifibrinolytic (PAI-1) molecules in the vessel wall determines the long-term arterial wall mass changes.¹⁴ Increased PAI-1 expression has been demonstrated in some atherosclerotic arteries.^{33 34 35} Others have reported increased PA activity and decreased PAI activity in fibrous plaques.³⁶ Furthermore, coronary arteries with a wide range of vascular pathology express uPA, uPA receptor, tPA, and PAI-1, suggesting an abundance of antifibrinolytic potential with enhanced local expression of profibrinolytic proteins, mainly within atherosclerotic plaques.³⁷ Carmeliet et al³⁸ recently reported that mice deficient in uPA alone or both uPA and tPA showed markedly reduced intimal thickening, but PAI-1–null mice demonstrated exaggerated vascular response to injury.¹⁷ However, it has recently been suggested that plasmin generation may not be the only mechanism by which the neointimal thickening process is controlled. For example, it has been demonstrated that PAI-1 has a direct inhibitory effect on SMC migration by blocking the binding of vitronectin to the vitronectin receptor.¹⁶ In addition, PAI-1 can inhibit cell migration by interfering with the binding of uPA receptor to vitronectin, independent of its function as a PAI.³⁹ These data suggest that alterations in the expression of PA system components may modulate intimal thickening via plasmin-dependent and plasmin-independent mechanisms. We have previously shown that the flow-induced SMC migratory response is uPA-dependent in vitro.¹⁸ In agreement with our previous study, we demonstrated that PAI-1K, a mutant that inhibits PA activity, significantly inhibited flow-dependent MSMC migration without affecting baseline (flow-independent) migration. These experiments, using recombinant forms of PAI-1 with either PA-inhibiting activity or vitronectin-binding ability, suggest that the ability of PAI-1 to inhibit plasmin formation is important for inhibition of flow-dependent MSMC migration. Moreover, the association of PAI-1 with vitronectin is also necessary for inhibition of both basal and flow-induced migration.

In conclusion, these studies demonstrate for the first time that flow-induced endothelial cell PAI-1 inhibits flow-induced SMC migration in vitro and thus support a role for the endothelium in "protecting" the underlying SMCs against hemodynamic forces. It is therefore tempting to speculate that modulation of EC PAI-1 gene expression by pulsatile flow in vivo may represent an important mechanism whereby hemodynamic forces regulate SMC migration and thus vascular remodeling.

	Acknowledgments

 
This work was supported in part by grants from the National Institutes of Health and the American Heart Association (AA-12610 and DK-09223 to Dr Redmond; HL-59696 to Dr Cahill; DK-47067 to Dr Sitzmann; and HL-02870, HL-64971, and AHA 9740143N to Dr Okada). Dr Okada is a recipient of a National AHA Established Investigator Award.

	Footnotes

 
Reprint requests to Eileen M. Redmond, PhD, University of Rochester Medical Center, Department of Surgery, Box SURG, 601 Elmwood Ave, Rochester, NY 14642-8410.

Received June 9, 2000; revision received July 26, 2000; accepted July 28, 2000.

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