© 1996 American Heart Association, Inc.
Articles |
Contrasting Effects of Plasminogen Activators, Urokinase Receptor, and LDL Receptor–Related Protein on Smooth Muscle Cell Migration and Invasion
the University of Pennsylvania School of Medicine, Philadelphia.
Correspondence to Elliot S. Barnathan, MD, University of Pennsylvania School of Medicine, 524 Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6060.
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Abstract |
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Smooth muscle cell (SMC) migration is an early response to vascular injury and contributes to the development of intimal thickening. Upregulation of several components of the plasminogen activator (PA) system has been documented after vascular injury. Utilizing a Transwell filter assay system and human umbilical vein SMCs, we sought to define the role of four different PA system components on SMC migration and matrix invasion: (1) PAs, (2) plasmin, (3) PA receptors, and (4) PA clearance receptors (ie, low density lipoprotein receptor–related protein [LRP]). Addition of active two-chain urokinase-type PA (UPA) stimulated random migration (192±30% of control, 0.36 nmol/L, P<.001). The stimulation was inhibited by pretreatment with diisopropylfluorophosphate, PA inhibitor type 1 (PAI-1), or aprotinin, a plasmin inhibitor. Augmented migration was also observed with either low-molecular-weight UPA or the amino terminal fragment of UPA (ATF), with the effects being additive. Stimulation by ATF alone, however, was not inhibited by aprotinin. The stimulatory effect was not specific for UPA, in that tissue-type PA (TPA) also increased migration (169±9% of control, 10 nmol/L, P<.001); the augmentation was inhibited by pretreatment with DFP, PAI-1, or aprotinin and was additive to the UPA effect. Antibodies to the UPA receptor but not 5'-nucleotidase (another glycosylphosphatidylinositol-anchored cell surface protein) inhibited baseline and UPA-stimulated migration. Similarly, both UPA and TPA stimulated invasion of a collagen gel; this augmentation was inhibited by aprotinin, whereas antibodies to the UPA receptor reduced baseline invasion. Finally, we tested whether inhibition of LRP function, which mediates internalization of PA/inhibitor complexes, affected either process. Both antibodies to LRP and recombinant receptor associated protein, a known inhibitor of ligand binding to the LRP, significantly inhibited migration but did not affect collagen gel invasion. These data demonstrate the ability of several components of the PA system to modulate SMC migration and invasion in vitro via plasmin-dependent and -independent mechanisms.
Key Words: plasminogen activators • urokinase receptor • LDL receptor–related protein • migration • smooth muscle cell
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Introduction |
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SMC migration from the media to the intima is one of the key initiating events in the development of intimal thickening after vascular injury. It has been suggested that proteases such as UPA and TPA, by their binding to specific cell surface receptors and accretion of plasmin at discrete sites on the cell membrane, play an important role in matrix degradation and tissue invasion.1 As endogenous modulators of PA activity, three important classes of molecule have been described: (1) cell surface receptors that augment PA activity, (2) cell- or matrix-associated inhibitors of PA activity such as PAI-1, and (3) internalization receptors that target the enzymes for lysosomal degradation. A cell surface receptor for UPA (ie, UPAR) has been cloned,2 and Hajjar et al3 have identified annexin II as both a TPA- and a plasminogen-binding protein on endothelial cells. A distinct internalization receptor has been identified for TPA on hepatocytes4 and SMCs5 as the LRP. A 39-kDa RAP is coexpressed with LRP, and binding of RAP to LRP blocks internalization of a variety of ligands, including TPA, TPA/PAI-1, scUPA, and UPA/PAI-1.5 6 7
SMCs express both UPAR and LRP and polarize UPAR in the direction of cell migration in a wound assay.8 Similar polarization to the leading edge of migrating monocytes has also been demonstrated.9 10 When UPAR function was impeded by antibodies that specifically blocked UPAR, monocyte migration was reduced even in the absence of UPA, suggesting that the association between UPAR and cell migration may be unrelated to a mechanism of extracellular matrix proteolysis but may involve signal transduction.9 Other investigators have suggested that UPAR also plays a role in cellular adhesion to the extracellular matrix.11 LRP, on the other hand, is thought to be involved in the clearance of proteases, protease/inhibitor complexes, and other molecules such as growth factors and cytokines,12 13 14 but the actual importance of their clearance by LRP has not been delineated. Because cell migration and invasion may depend on PA, UPAR, and/or LRP functions in different ways, we studied the role of each in well-defined in vitro assays. We demonstrate that both UPA and TPA can independently augment SMC migration and invasion in a plasmin-dependent manner. We also demonstrate a component of migration stimulation for UPA only that is protease independent but requires binding to UPAR. Finally, we demonstrate that inhibition of LRP function with either antibodies or RAP can inhibit migration but not invasion, whereas antibodies to UPAR inhibit both processes.
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Methods |
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Reagents
rTPA was kindly supplied by Genentech, Inc. rscUPA, HMW tcUPA, LMW UPA, and ATF (amino acids 1 to 143) were obtained from Abbott Laboratories (kind gifts from A. Mazar and J. Henkin). DIP-TPA and DIP-tcUPA were prepared by the use of DFP (Sigma Chemical Co) as previously described.5 Complexes of PAI-1 with TPA and tcUPA were prepared by incubation with guanidine-activated PAI-1 (a kind gift of C. Reilly, Merck Sharpe and Dohme) as previously described.5 Polyclonal rabbit anti-LRP antibody and RAP were obtained from the American Red Cross (kind gifts of M. Kounnas and D. Strickland), and a polyclonal rabbit antiserum to 5'-nucleotidase (1:300 dilution) was kindly provided by C. Widnell (University of Pittsburgh). Both murine monoclonal (No. 3936) and rabbit polyclonal (No. 399R) anti-UPAR antibodies (purified immunoglobulin used at 25 µg/mL) were supplied by American Diagnostica (kind gifts of J. Bognacki and R. Hart).
SMC Cultures
SMC cultures were prepared and characterized as described previously.5 In brief the media of human umbilical veins was isolated surgically and the tissues were minced into small pieces. These pieces were plated onto a Petri dish covered with fibronectin. The cells were grown in SMC medium (DMEM/Ham's F-12 mixture with 10% heat-inactivated FCS, penicillin, and streptomycin) and passed when there was adequate proliferation of cells beyond the explants. Cells from passages 1 and 2 were used for experiments. Cells were identified as SMCs by their typical spindle shape, "hill-and-valley" appearance in culture, and immunohistochemical staining with antibodies to SM-specific isoforms of 
-actin (HHF-35, Enzo Diagnostics; IA4, Dako Corp). Cell viability, as assessed by the lack of trypan blue uptake at the end of experiments with and without added agents, was generally >99%. The cells were always cultured in medium containing 10% FCS and were therefore in a "proliferative" medium before and during migration assays. However, as a control to assess the amount of proliferation during the 10-hour migration assays, experiments were performed with or without hydroxyurea (1 to 10 mmol/L), and total cell count was assessed at 10 hours by manual cell counting.
Transwell Filter Migration Assay
Transwell filters (Costar) with 12-µm pores were coated for 12 hours with fibronectin at 37°C. SMCs were seeded onto the filters at a density of 2.5x104 cells per well and allowed to migrate for 10 hours with SMC medium in both chambers. Unless otherwise noted, all Transwell filter migration assays were performed with equal concentrations of FCS and agent(s) in question in the upper and lower chambers, so that random migration or chemokinesis could be measured. In some experiments a "checkerboard" analysis was performed to determine whether the agent in question possessed chemoattractant capabilities. These experiments were designed with the agent in question either present (+) or absent (-) in the top/bottom chamber in the following four combinations: +/+, +/-, -/+, and -/-. In all experiments, after 10 hours the SMCs were washed three times in PBS, fixed in 10% formalin-acetate buffer, washed twice with acetate buffer and twice with deionized water, air dried, and then stained with toluidine blue. The upper surface of the filter was scraped free of cells, and the number of cells that had migrated through the pores was manually counted per high-power field for each condition (12 fields for each filter). Data are reported as the number of SMCs counted per 12 high-power fields, expressed as a percentage of control cells that were not treated with the agent(s) in question. All experiments were performed at least three times. In selected experiments the incubation time after plating was varied to assess temporal effects on migration. Finally, to enable comparison with a known enhancer of chemokinesis, parallel experiments were performed with FGF-2 (10 ng/mL) added to both chambers.
Collagen Gel Invasion Assay
Collagen gels were prepared according to the methods of Boudreau et al.15 In brief 28.8 mL of purified type I bovine dermal collagen (3 mg/mL; Vitrogen 100, Celtrix Pharmaceuticals, Inc), 3.6 mL of 10x medium 199 (GIBCO BRL), and 12.6 mL of 1x medium 199 (GIBCO BRL) were mixed at 4°C to a final concentration of 2 mg/mL (pH 7.4). Agents that were hypothesized to inhibit or stimulate SMC invasion were mixed into the collagen gel. Aliquots of 1.25 mL were added to 12-well plates and allowed to polymerize overnight in a 37°C CO2 incubator. Gels were then rinsed with medium 199 with 10% FCS three times before use. SMC medium (1 mL) containing appropriate concentration of agent to be tested (scUPA, TPA, etc) and 1.5x104 SMCs were then plated onto the surface of each gel. Invasion of the collagen gel matrix was assessed 10 hours after seeding in five randomly selected fields by phase-contrast microscopy (Nikon Diaphot, x20 objective). With the calibrated fine focus used to measure depth of invasion, only those SMCs that had invaded the gel >10 µm were counted as invading SMCs and expressed as a percentage of the total number of cells counted.
Statistics
Comparisons between treated and control cells in migration analyses were performed with unpaired two-tailed Student's t tests, with P<.05 considered significant unless multiple comparisons were made. When more than two groups were present, ANOVA (factorial design) was used (Statview). Unless otherwise noted, all values are expressed as mean±SE.
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Results |
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Effect of UPA on SMC Migration
Using a Transwell assay system we first determined the effect of scUPA on SMC migration. Time-course studies were performed, with migration assessed 2 to 10 hours after plating in the presence of scUPA (0.36 nmol/L). FGF-2 (10 ng/mL) was used as a biologically relevant positive control, and antibody to 5'-nucleotidase (a GPI-anchored cell surface protein found on SMCs) served as a negative control to ensure that simple antibody binding itself did not limit migration. As shown in Fig 1
, significant numbers of cells appeared under the filter by 4 hours. Both scUPA and FGF-2 but not 5'-nucleotidase antibody stimulated migration at each subsequent point up to 10 hours. To control for possible confounding effects due to cell proliferation at the 10-hour point, additional experiments were performed to measure changes in cell number during a 10-hour period in the presence or absence of hydroxyurea. No significant increase in cell number occurred in either condition (data not shown). Therefore, 10 hours was used for all subsequent experiments.
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, control) or with scUPA (
, 0.36 nmol/L), FGF-2 (
, 10 ng/mL), or antibody (ab) to 5'-nucleotidase (
, 1:300 dilution), a glycoprotein I–anchored protein known to be expressed on the SMC surface. Compounds were added to upper and lower chambers in the presence of medium containing 10% FCS. Degree of migration was assessed by counting the number of migrated cells in 12 high-power fields (HPF). Each point represents the mean of triplicate filters±SEM. At 6, 8, and 10 hours both FGF-2 and scUPA were significantly increased (by ANOVA) compared with control.
We next investigated the dose dependence of this stimulatory effect. Augmentation of migration by scUPA was maximal at 0.36 nmol/L (160±12% versus control, n=12, P<.001; Fig 2). Parallel experiments with equimolar concentrations of ATF showed a similar increase, which was also maximal at 0.36 nmol/L (143±14% of control, n=9, P
.001). A checkerboard analysis with 0.36 nmol/L scUPA did not show any evidence of chemoattractant capabilities by scUPA (data not shown). These data initially suggested that stimulation of migration might occur by simple binding of UPA to its receptor, UPAR, without the need for plasmin generation. To more specifically define the contribution of plasmin generation to the enhancement of migration, we compared the effect of several forms of UPA, with and without aprotinin, an inhibitor of plasmin activity. Active tcUPA stimulated migration the most (192±30% of control, P<.001), but LMW UPA also had an enhancing effect (147±26%, P=.003). The enhancing effects of LMW UPA and ATF were fully additive and equal to those of tcUPA (Fig 3). The importance of plasmin generation to augmented cell migration detected in this system was demonstrated by the ability of aprotinin to inhibit the stimulatory effects of scUPA and LMW UPA (Fig 3). In contrast, enhancement due to ATF was not inhibited by aprotinin, nor did aprotinin alone affect baseline levels of migration. Finally, we sought to determine whether inhibition of PA activity itself could limit the effect. Preincubation of tcUPA with either DFP or PAI-1 completely abrogated the stimulatory effect (Fig 3). These data suggested that augmentation of SMC migration in this system by all forms of UPA, except ATF, was dependent on PA activity that generated plasmin.
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) or ATF (
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Effect of TPA on SMC Migration
Since augmented SMC migration appeared to be dependent on plasmin generation but at least in some cases was independent of ligand binding to UPAR, we next tested whether TPA, another PA that does not interact with UPAR, might also stimulate migration. TPA enhanced SMC migration in a dose-dependent fashion, with maximal stimulation at 10 nmol/L (169±9%, n=9, P<.00; Fig 4A). A checkerboard analysis with 10 nmol/L TPA did not show any evidence of chemoattractant capabilities (data not shown). The stimulatory effect of TPA was inhibited by DFP, PAI-1, and aprotinin (Fig 4B). There was no significant difference in the degree of stimulation between TPA or scUPA alone at maximally effective concentrations. However, simultaneous addition of TPA and scUPA at maximally effective concentrations had an additive effect, resulting in a 2.4±0.4-fold increase in migration. By ANOVA, this increase was significantly higher than that seen with scUPA, ATF, or TPA alone.
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Effect of Anti-UPAR Antibodies on SMC Migration
To further investigate the ability of UPAR to modulate SMC migration, we performed blocking experiments with and without added UPA in the presence or absence of antibodies to UPAR. We tested two antibodies, No. 3936 and No. 399R (American Diagnostica), which are known to inhibit binding of UPA to UPAR.16 As shown in Fig 5, monoclonal antibody No. 3936 and polyclonal antibody No. 399R inhibited SMC migration compared with control antibodies (39±18% of control, n=6; 19±9% of control, n=8, respectively; P<.001). Addition of anti-UPAR antibody (No. 3936) completely inhibited the stimulation by scUPA or ATF and furthermore decreased the amount of migration to a level observed with that of anti-UPAR antibody alone. These data suggest that UPAR plays an important function in signal transduction, augmentation of plasmin generation, or both. Since antibodies to UPAR substantially inhibit migration in the absence of added UPA, despite the fact that very little endogenous UPA is generated by these SMCs (S.S.O. and E.S.B., unpublished data, 1992), these data suggest that UPAR may have a role in migration independent of that of UPA, as others have suggested.9 11
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Effect of LRP Inhibition on SMC Migration
Clearance of protease-inhibitor complexes from the cell surface has been postulated to be a critical cell function that may enable migration by returning unoccupied urokinase receptors to the cell surface. Since UPA/PAI-1, TPA/PAI-1, and scUPA5 7 13 have been demonstrated to be internalized via the LRP, we hypothesized that LRP function may be necessary for SMC migration. Furthermore, we postulated that if LRP function is associated with UPAR or UPA, blocking LRP function alone might be sufficient to reduce the stimulatory effect on SMC migration, even in the presence of ligand for UPAR. We first incubated SMCs with RAP, a protein that is known to inhibit binding of ligands to the LRP and that prevents internalization of TPA/PAI-1 in SMCs.5 We observed dose-dependent inhibition of migration (Fig 6A). Since RAP may bind to membrane receptors other than the LRP, we confirmed that inhibition of LRP specifically could inhibit migration by using anti-LRP antibodies that reduced migration by more than half (43±4% of control; Fig 6B). Both anti-LRP antibody and RAP were capable of diminishing the stimulatory effects of scUPA or ATF on SMC migration compared with the effects of either ATF or scUPA alone, although RAP (200 nmol/L) was a somewhat less efficient inhibitor than anti-LRP antibody. Treatment with anti-LRP antibody and RAP combined (33±9% of control) did not significantly change the amount of SMC migration of either inhibitor alone. The fact that both anti-LRP antibody and RAP could block the stimulation of SMC migration due to ATF or scUPA, without interference from their interaction with UPAR, suggests that the importance of LRP function in SMC migration may be to enable the return of functional unoccupied UPARs to the cell surface.
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Effect of PA System Modulation on SMC Invasion of Collagen Gels
Although migration of SMCs through pores in Transwell filters might be considered analogous to SMC migration through breaks in the internal elastic lamina after injury, SMCs are also required to invade three-dimensional extracellular matrix barriers as well. As a model of SMC invasion, we used a collagen gel invasion assay to delineate the potential influence of PA system components on SMC invasion. Both TPA and scUPA augmented invasion by 
2.5-fold compared with untreated cells (Fig 7). tcUPA and LMW UPA also stimulated invasion, but in contrast to the findings for migration, ATF had no effect. The importance of plasmin generation was evident, in that inactivated TPA or tcUPA had no effect on invasion, and aprotinin completely inhibited the stimulatory effect of TPA and several forms of UPA (Fig 7). Aprotinin treatment alone was comparable with control, suggesting that in this model system, baseline invasion is not significantly dependent on plasmin generation. Finally, we determined the effect of inhibition of UPAR or LRP function on SMC invasion. Similar to the results in migration studies, a monoclonal antibody to UPAR (No. 3936) inhibited invasion (41±9% of control). However, neither RAP nor anti-LRP antibody inhibited SMC invasion (Fig 7).
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Discussion |
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Plasminogen, PAs, and their respective receptors have been demonstrated to play modulatory roles in migration and invasion in a variety of cells.1 The viability of mice deficient in TPA, UPA, PAI-1, plasminogen, and UPAR suggests that none of these molecules is essential for development.17 18 19 20 Nevertheless, when challenged, these proteins may play an important modulatory role in the response to particular stresses or stimuli. In this study we explored the role of the PA system in modulating SMC migration and invasion in two well-defined in vitro assays. In both assays aprotinin alone had no effect, suggesting that baseline plasmin generation is not necessary for either process to occur. However, in both assays plasmin generation by exogenous PAs caused stimulation that was inhibited either by inactivating the PAs prior to their addition or by inhibiting the generated plasmin. These data demonstrate a direct stimulatory effect of plasmin on human SMC migration, as has been suggested by in vivo studies in rats.21
In addition to the stimulatory effects of plasmin, these studies demonstrate an important modulatory role for UPAR in both SMC migration and invasion. Antibodies to UPAR were able to inhibit both processes even when aprotinin could not, suggesting that UPAR may modulate migration/invasion in a plasmin-independent fashion. Since both antibodies inhibit UPA binding to UPAR, it is not clear whether this capability is critical to its inhibition of cell movement. SMCs in culture produce very low levels of PAs and most (>95%) UPARs are unoccupied (S.S.O. and E.S.B., unpublished data, 1992). Therefore, it is not clear whether inhibition of cell movement is related to inhibition of low levels of receptor occupancy or to inhibition of UPAR interactions with extracellular matrix components, such as vitronectin, as has been suggested by others.11 It does seem clear the UPAR can mediate a portion of the augmentation by UPA, in that anti-UPAR antibodies blocked augmented migration by scUPA or ATF. The addition of scUPA to SMCs in the presence of serum and fibronectin has been shown to accelerate early adhesion to plastic cell culture plates (S.S.O. and E.S.B., unpublished data, 1996) and could contribute to increased random migration, as measured in our assays by making a larger number of cells available for earlier migration. The binding of scUPA to UPAR may also augment cell-associated plasminogen activation, as has been demonstrated for endothelial cells.22 However, the augmentation of migration by ATF seems unrelated to plasmin generation and may have been due to direct signal transduction mediated by UPAR. ATF has been shown to enhance adhesion in monocytoid cells,23 stimulate migration in a melanocyte cell line that expresses high levels of UPAR,9 and increase diacylglycerol production while stimulating migration in human epidermal cells.24 ATF has also been shown to stimulate endothelial cell migration in a wound assay25 and tube formation in an in vitro angiogenesis model.26 In our study of SMC migration, treatment with DIP-tcUPA, tcUPA/PAI-1, or scUPA/aprotinin, unlike that with ATF, had no stimulatory effect, despite the fact that in each case the ligand bound with high affinity to UPAR. This suggests that simple binding to UPAR alone may be inadequate to initiate signal transduction. It is also possible that removal of the C-terminal portion of UPA, which occurs when ATF is generated, exposes a cryptic binding site or causes a conformational change that enables or augments signal transduction. Several groups have directly addressed the role of UPAR in cell migration. Inhibition of UPAR expression by antisense oligonucleotide or of its function by antibodies resulted in diminished monocyte migration independent of UPA.27 Our results also agree with a recent study, which demonstrated that an anti-UPAR antibody inhibited SMC migration in response to wounding in culture.28
Clowes et al29 originally demonstrated that levels of TPA (and UPA) were higher in the rat carotid artery after injury at a time concurrent with SMC migration. The present study demonstrates for the first time that TPA directly augments human SMC migration and that the effect is additive to that observed with scUPA. Similar stimulation of migration by TPA was previously reported in wounded cultures of endothelial cells, wherein the effect was also abolished by PAI-1.30 TPA has been shown to be mitogenic in serum-starved human aortic SMCs in culture and thus may also activate a signal transduction pathway.31 Whether any of these signals are mediated via binding to cell surface receptors, such as annexin II, is not clear. In our studies all augmentation by TPA could be attributed to its enzymatic activity and subsequent plasmin generation.
These studies are also the first to demonstrate a role for LRP in cell migration. This concept was supported by experiments that demonstrated dose-dependent inhibition of migration by RAP and by antibody inhibition studies. Early studies of LRP gene inactivation in mice suggested that a possible defect in trophoblast implantation might be related to the lack of clearance of UPA/PAI-1 complexes from the cell surface.32 Although some embryos were later found to implant,33 LRP inactivation is still embryonically lethal, but the exact cause is still under investigation. Whether the effect of RAP and/or antibodies is related to diminished unoccupied UPAR or other reasons remains to be investigated. Paradoxically, we found no effect of either RAP, anti-LRP antibodies, or aprotinin on baseline invasion. These findings may be due to the fact that in a pure collagen invasion assay, there may be greater dependence on metalloproteinases to facilitate cell invasion at baseline, which may require neither LRP function nor plasmin activity. This notion is supported by the finding that type IV collagenase activity is required for SMC invasion of basement membrane barriers in vitro.34
Our current working model of UPA/receptor interactions at the cell surface is depicted in Fig 8. We propose four possible mechanisms whereby UPAR may modulate SMC migration and invasion. First, UPA may bind to UPAR, activate cell-associated plasminogen, and generate plasmin, thereby locally facilitating proteolysis and enabling migration/invasion. Second, binding of UPA or its derivatives (ATF) may cause direct intracellular signaling, resulting in augmented SMC migration. Third, direct interactions between UPAR and matrix components such as vitronectin may increase cellular adhesion and thus facilitate migration. Fourth, LRP may aid migration by clearing UPA and UPA/PAI-1 complexes and thus enable free UPARs to return to the cell surface to participate in the first three mechanisms. Anti-UPAR antibodies compete with UPA for the UPAR binding site, thus interfering with plasmin generation but also possibly blocking the signaling pathway by binding to the receptor or interfering with receptor-matrix interactions. Inhibition of LRP by antibodies or RAP may diminish clearance of cell surface PA/inhibitor complexes, potentially disabling one or more of the first three mechanisms and leading to reduced cell migration.
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Our data suggest that SMC migration and invasion in vitro can be significantly modulated by alterations in expression or function of several components of the PA system. Altered in vivo expression of one or more of these components may also significantly effect the vascular response to injury. Jackson and Reidy21 demonstrated that UPA and TPA activity increased in injured rat carotid arteries and that tranexamic acid (an inhibitor of plasmin) reduced SMC migration from the media to the intima. Watanabe et al35 demonstrated upregulation of LRP mRNA in aortas of rabbits that were fed a high-cholesterol diet, and Lupu et al36 detected both mRNA and protein for LRP in normal and atherosclerotic human arteries. Recently, Noda-Heiny et al28 demonstrated increased vessel wall expression of UPAR in cholesterol-fed rabbits and human atherosclerotic arteries. All of these data suggest that alterations in the expression of PA system components that favor higher PA or PA receptor expression may augment or modulate the vascular response to injury. This hypothesis has been challenged by studies of apolipoprotein(a)-transgenic mice, which are susceptible to atherosclerosis and in which lower levels of active plasmin were reported in their artery walls,37 presumably due to displacement of plasminogen from its receptors.38 39 These investigators proposed that vessel wall plasmin is "antiatherogenic," in that it activates latent transforming growth factor-ß, which in turn suppresses SMC proliferation39 and migration.40 However, a recent series of experiments in knockout mice by Carmeliet and coworkers41 42 43 has called this hypothesis into question, at least in terms of the response to direct vascular injury. Mice deficient in UPA or plasminogen had less intimal thickening in the carotid artery after either electrical or mechanical injury, whereas mice deficient in PAI-1 had intimal thickening well above control levels. Unexpectedly, intimal thickening in UPAR-deficient mice appeared to be similar to that in wild-type mice, thus perhaps emphasizing the importance of UPA activity rather than its interaction with UPAR. Nevertheless, these animals presumably still retained cell surface receptors for plasminogen and could still localize plasmin at specific sites. Furthermore, the lack of effect without UPAR does not negate a potential modulatory role if the receptors are present or upregulated. Recent evidence demonstrates a restricted spatial and temporal upregulation of TPA, UPA, UPAR, and PAI-1 after balloon injury in rats.43 The precise role that plasmin, PAs, their receptors, and inhibitors play in modulating human atherosclerosis or the vascular response to iatrogenic injury remains to be determined.
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Acknowledgments |
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This work was supported in part by grants from the National Institutes of Health (NIH), Nation's Capital Affiliate American Heart Association Grant-In-Aid, and American Diagnostica (HL02870 and American Heart Association GIA DC95-GS-3 to S.S.O.; HL47839 and American Diagnostica to E.S.B.). Dr Okada is a recipient of an NIH-Clinician Investigator Development Award. Dr Okada is now an Assistant Professor of Medicine in the Division of Cardiology at Georgetown University, Washington, DC. Dr Grobmyer was a recipient of a Sarnoff Society Medical Student Fellowship.
Received October 23, 1995;
revision received June 27, 1996;
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