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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:737.)
© 2006 American Heart Association, Inc.

Vascular Biology

Transforming Growth Factor Beta 1 Induces Neointima Formation Through Plasminogen Activator Inhibitor-1–Dependent Pathways

Goro Otsuka; Ramtin Agah; Andrew D. Frutkin; Thomas N. Wight; David A. Dichek

From the Department of Medicine (G.O., A.D.F., D.A.D.) University of Washington, Seattle, Wash; the Hope Heart Program (T.N.W.), Benaroya Research Institute at Virginia Mason, Seattle, Wash; and the Gladstone Institute of Cardiovascular Disease (R.A., D.A.D.), University of California, San Francisco, Calif.

Correspondence to David A. Dichek, MD, Department of Medicine, University of Washington, Box 357710, 1959 NE Pacific St, Seattle, WA 98195-7710. E-mail ddichek{at}u.washington.edu

	Abstract

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Objective— The mechanisms through which transforming growth factor (TGF)-ß1 promotes intimal growth, and the pathways through which TGF-ß1 expression is regulated in the artery wall, are incompletely understood. We used a mouse model to investigate mechanisms of TGF-ß1–induced intimal growth.

Methods and Results— Adenovirus-mediated overexpression of TGF-ß1 in uninjured carotid arteries of wild-type mice induced formation of a cellular and matrix-rich intima. Intimal growth appeared primarily due to cell migration and matrix accumulation, with only a negligible contribution from cell proliferation. Overexpression of TGF-ß1 also stimulated expression of plasminogen activator inhibitor type 1 (plasminogen activator inhibitor [PAI]-1) in the artery wall. To test the hypothesis that PAI-1 is a critical downstream mediator of TGF-ß1–induced intimal growth, we transduced carotid arteries of PAI-1–deficient (Serpine1^–/–) mice with the TGF-ß1–expressing vector. Overexpression of TGF-ß1 in Serpine1^–/– arteries did not increase intimal growth, matrix accumulation, cell migration, or proliferation. Moreover, TGF-ß1–transduced arteries of Serpine1^–/– mice secreted 6- to 10-fold more TGF-ß1 than did arteries of wild-type mice that were infused with the same concentration of the TGF-ß1–expressing vector.

Conclusions— PAI-1 is both a critical mediator of TGF-ß1–induced intimal growth and a key negative regulator of TGF-ß1 expression in the artery wall.

We investigated mechanisms of TGF-ß1–induced intimal growth. TGF-ß1 induced neointimal formation through cell migration and matrix accumulation. Overexpression of TGF-ß1 in arteries of PAI-1–deficient mice revealed that PAI-1 is both a critical mediator of TGF-ß1–induced intimal growth and a key negative regulator of TGF-ß1 expression in the artery wall.

Key Words: carotid arteries • gene transfer • intima • plasminogen activator inhibitor-1 • TGF-beta

	Introduction

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Transforming growth factor (TGF)-beta 1 (TGF-ß1) and its cellular receptors are expressed at elevated levels in atherosclerotic and restenotic human arteries and in balloon-injured animal arteries.^1–4 The role of TGF-ß1 in promoting or preventing intimal growth is controversial and may depend on whether TGF-ß1 is acting locally in the artery wall or systemically, as an immunosuppressant.⁵ The effect of elevated TGF-ß signaling on the cells of the artery wall is also controversial. Many studies suggest that TGF-ß1 acts on smooth muscle cells (SMCs) of the artery wall to accelerate intimal growth;^1,4,6–9 however, other studies suggest that TGF-ß1 retards SMC proliferation, migration, and intimal growth.^10–13 In both cases, the precise cellular and molecular pathways through which TGF-ß1 alters intimal growth are not yet defined.

See page 679

To elucidate the role of local TGF-ß1 expression in intimal growth and define the mechanisms through which it acts, we and others expressed a TGF-ß1 cDNA in animal arteries or infused TGF-ß1 protein systemically in animal models of arterial injury.^1,4,6,7 In all cases, TGF-ß1 promoted intimal growth. Other groups reported that TGF-ß1 antagonists decreased intimal growth in animal models of arterial injury.^8,9 Thus, a large amount of in vivo data indicate that TGF-ß1 acts within the artery wall to accelerate intimal growth. Data from these studies suggest that cellular proliferation and matrix accumulation contribute to TGF-ß1–induced intimal growth; however, the relative contributions of cell proliferation and matrix accumulation, and whether cell migration plays any role in TGF-ß1–stimulated intimal growth, remain unclear. Moreover, the molecular pathways that mediate the effects of TGF-ß1 on intimal growth remain unidentified.

TGF-ß1 upregulates expression of several molecules that could contribute to intimal growth: plasminogen activator inhibitor (PAI)-1; matrix proteins, including collagen and fibronectin; and growth factors, including insulin growth factor-1 and connective tissue growth factor.^14–16 Among these molecules, we hypothesized that PAI-1 could be a critical mediator through which TGF-ß1 accelerates intimal growth. Overexpression of PAI-1 in the artery wall can increase intimal growth, whereas PAI-1 deficiency can limit intimal growth.^17,18 Moreover, PAI-1 expression can both stimulate cell migration and enhance matrix accumulation, processes that contribute to intimal formation.^19,20 PAI-1 also inhibits plasminogen activation, through which it could limit plasmin-mediated activation of TGF-ß1, thereby acting as a negative regulator of active TGF-ß1 expression.²¹ However, whether PAI-1 affects TGF-ß1 expression and activation in vivo is controversial.^22,23

We previously reported a mouse model of arterial gene transfer that we suggested might be useful for elucidating mechanisms of action of transferred genes.²⁴ Here we report use of this model to elucidate mechanisms through which TGF-ß1 accelerates intimal growth and to test whether PAI-1 is both a critical mediator of TGF-ß1–induced intimal growth and a negative regulator of TGF-ß1 expression in the artery wall.

	Materials and Methods

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For complete Materials and Methods, please see http://atvb.ahajournals.org.

Adenoviral Vectors
We used 2 adenoviral vectors: AdrTGFß, which expresses an active form of rat TGF-ß1, and a control vector AdCMVNull²⁵ (hereafter termed AdNull), which lacks a transgene.

Animal Procedures
Uninjured left common carotid arteries of male C57BL/6J and C57BL/6J Serpine1^–/– mice were infused with either AdrTGFr or AdNull.²⁴ Cell proliferation was detected by either pulse injection or continuous infusion of bromodeoxyuridine (BrdU).

Gene Expression
TGF-ß1 secretion was measured by enzyme-linked immunosorbent assay of media conditioned by explanted arteries.⁷ PAI-1 expression in carotid arteries was measured by quantitative reverse-transcription polymerase chain reaction.

Morphometric Analysis
Arteries were sectioned at 300 µm steps, with serial sections at each step stained with hematoxylin and eosin, Movat pentachrome, and picrosirius red. Intimal and medial areas, lumen circumference, and arterial circumference at the levels of the internal and external elastic laminae (IEL and EEL) were measured by 2 independent observers. Lumen area was calculated.

Immunohistochemistry and Histochemistry
BrdU incorporation was detected using biotinylated anti-BrdU antibodies.¹⁷ SMCs and inflammatory cells were identified with anti-smooth muscle -actin and anti-CD45 monoclonal antibodies, respectively. Collagen and proteoglycan accumulation were analyzed by picrosirius red and Movat pentachrome staining. Hyaluronon was detected with biotinylated hyaluronan binding protein.²⁶ Immunostaining for fibrin(ogen) was performed essentially as described.¹⁷

Electron Microscopy
Transmission and scanning electron microscopy were performed as described.^27,28

Statistical Analysis
Results are reported as mean±SEM or as median (25% to 75%) range for data not normally distributed. The significance of intergroup differences was determined with the t test or the Mann-Whitney rank-sum test, respectively.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
AdrTGFr Increases TGF-ß1 Secretion in Carotid Arteries of Wild-Type Mice
All AdrTGFr arteries secreted active TGF-ß1 on day 3 [180 (160–570) pg/24 hours] and day 7 [100 (70–120) pg/24 hours]. In contrast, active TGF-ß1 was not detected in explant cultures of any of 15 AdNull-infused arteries harvested at these time points (Figure 1A). AdrTGFr arteries also secreted substantial total (active + latent) TGF-ß1: 1900 (910–2200) pg/24 hours on day 3, 550 (430–840) pg/24 hours on day 7, and 210 (180–250) pg/24 hours on day 28. Total TGF-ß1 secretion from AdNull arteries was significantly less (P<0.01) at all time points (Figure 1B).

View larger version (12K): [in this window] [in a new window]   Figure 1. TGF-ß1 secretion after gene transfer in wild-type mice. Active TGF-ß1 secretion (A) and total TGF-ß1 secretion (B) by arteries transduced with AdrTGFß (white circles) or AdNull (black circles) and explanted 3, 7, or 28 days after gene transfer. Data points represent individual arteries; bars are group medians. Dotted lines indicate the lower limit of detection of the assay (50 pg/vessel per 24 hours).

AdrTGFß Causes Intimal Formation in Wild-Type Arteries
Pilot studies revealed that intimal growth occurred between days 14 and 28 after AdrTGFß infusion (Figure I, available online at http://atvb.ahajournals.org). Therefore, we measured arterial intimas and determined their composition on day 28. The intimas of AdNull-infused arteries were essentially limited to a single layer of endothelium (Figure 2A and 2B). In contrast, AdrTGFß arteries had significant neointimal growth (5200±1300 µm² versus 930±210 µm² for AdNull; P=0.006; Figures 2D to 2E and 3A). Medial areas of AdrTGFr and AdNull arteries were not significantly different (30 000±3100 versus 24 000±2300 µm²; P=0.13; Figure 3B). The intimal-to-medial area ratios were also significantly larger in AdrTGFr arteries (0.16±0.032 versus 0.04±0.007, P=0.002; Figure 3C). Luminal area and IEL and EEL circumferences did not differ between AdrTGFß and AdNull arteries (Table I, available online at http://atvb.ahajournals.org).

View larger version (72K): [in this window] [in a new window]   Figure 2. AdNull-transduced and AdrTGFß-transduced arteries at day 28. A and B, Intimas of AdNull-transduced wild-type arteries consisted only of a single layer of endothelial cells. D and E, AdrTGFß-transduced wild-type arteries developed a cellular and matrix-rich neointima. Matrix accumulation was also noted in the media. C and F, In Serpine1–/– mice, relatively little neointima was present in either AdNull or AdrTGFß-transduced arteries. A, C, D, and F, Hematoxylin and eosin stain. B and E, Movat stain. Arrowheads: internal elastic lamina. Scale bars: 50 µm

View larger version (15K): [in this window] [in a new window]   Figure 3. Intimal growth in wild-type and Serpine1–/– arteries 28 days after gene transfer. A, Intimal area. B, Medial area. C, Intima/media (I/M) area ratio. Data are mean±SEM of 6 or 7 arteries per group.

The majority of cells in 28-day AdrTGFß neointimas stained positive for smooth muscle actin, whereas only rare intimal cells were CD45⁺ (Figure II, available online at http://atvb.ahajournals.org). Collagen, proteoglycan, and hyaluronan accumulation were also significantly higher in the medias and intimas of 28-day AdrTGFß arteries (Figure 2; Figure III, available online at http://atvb.ahajournals.org). Transmission electron microscopy of intimas of 28-day AdrTGFß arteries confirmed accumulation of matrix that contained collagen fibrils and proteoglycans (Figure IVA and IVB, available online at http://atvb.ahajournals.org). To determine whether an early increase in fibrin matrix in AdrTGFß arteries might contribute to intimal growth, we measured fibrin(ogen) accumulation in the intima and media of arteries harvested 7 days after gene transfer. There was no difference between AdrTGFß and AdNull arteries [1.1 (0.2 to 7.7%) versus 2.3 (1.5 to 10.1%) positive area for fibrin(ogen); P=0.5].

To investigate the remote possibility that acute vector-related toxicity with endothelial loss²⁹ might account for intimal growth in AdrTGFß arteries, we harvested 3 wild-type arteries 1 day after infusion of AdrTGFß and examined the luminal surfaces with SEM. The luminal endothelium appeared nearly completely intact with <3% of the surfaces covered by platelets (Figure IVC).

AdrTGFß Increases PAI-1 Expression
Three days after AdrTGFß infusion, carotid artery PAI-1 mRNA was increased by 2.4-fold (4.5±0.74 versus 1.9±0.47 arbitrary units in AdNull arteries; P=0.008; Figure 4).

View larger version (12K): [in this window] [in a new window]   Figure 4. PAI-1 expression in transduced arteries. Arteries were harvested 3 days after infusion of either AdNull or AdrTGFß. PAI-1 mRNA was quantified by reverse-transcription polymerase chain reaction, normalized to GAPDH expression, and expressed in arbitrary units (AU). Data points represent individual arteries; bars are group means.

Intimal Growth in Serpine1^–/– Mice
To test whether PAI-1 is a critical downstream mediator of TGF-ß1–induced intimal growth, we repeated the carotid gene transfer experiments in Serpine1^–/– mice. Infusion of AdrTGFß in arteries of Serpine1^–/– mice did not increase intimal growth (2500±360 versus 1800±290 µm² in AdNull-infused arteries of Serpine1^–/– mice; P=0.2; Figures, 2C, 2F, and 3A). AdrTGFß also had no effect on medial area, intimal-to-medial area ratio, IEL and EEL circumference, or luminal area (Figure 3B and 3C; Table I). Notably, however, intimas of AdNull-infused arteries of Serpine1^–/– mice were twice as large as intimas of AdNull-infused arteries of wild-type mice (1800 versus 930 µm²; P=0.026; Figure 3A).

TGF-ß1 Secretion in Transduced Arteries of Serpine1^–/– Mice
Because AdrTGFß did not cause intimal growth in Serpine1^–/– arteries, we were concerned that we had not achieved adequate levels of TGF-ß1 expression in these arteries. However, AdrTGFß-infused Serpine1^–/– arteries harvested 3 days after vector infusion secreted particularly high amounts of active TGF-ß1: 1900 (840–7100) versus <50 pg/24 hours for AdNull-infused Serpine1^–/– arteries (P=0.004; Figure 5A). Moreover, AdrTGFß-infused Serpine1^–/– arteries secreted 8-fold more active TGF-ß1 than AdrTGFß-infused wild-type arteries (P=0.004; Figure 5A). Secretion of total TGF-ß1 followed the same pattern: 14 000 (10 000–100 000) pg/24 hours in AdrTGFß-infused Serpine1^–/– arteries versus 230 (110–310) pg/24 hours in AdNull-infused Serpine1^–/– arteries and 2300 (1900–3800) pg/24 hours in AdrTGFß-infused wild-type arteries (P=0.004 for both; Figure 5B). Measurement of total TGF-ß1 secretion by explanted AdNull arteries also revealed that PAI-1 deficiency increased endogenous (ie, not vector-driven) TGF-ß1 expression: endogenous TGF-ß1 expression was significantly greater in AdNull-infused Serpine1^–/– arteries than in AdNull-infused wild-type arteries (P=0.04; Figure 5B). Thus, TGF-ß1 expression in Serpine1^–/– arteries was substantial and PAI-1 is a negative regulator of both vector-driven and endogenous TGF-ß1 expression in the artery wall.

View larger version (16K): [in this window] [in a new window]   Figure 5. TGF-ß1 secretion after gene transfer in Serpine1–/– mice, compared with wild-type mice. A, Active TGF-ß1 secretion. B, Total TGF-ß1 secretion. Arteries were explanted 3 days after vector infusion. AdrTGFß-transduced wild-type arteries are different arteries from those in Figure 1. AdNull-transduced wild-type arteries, included here for reference, are the same arteries as in Figure 1. Data points represent individual arteries; bars are group medians. Dotted lines indicate the lower limit of detection of the assay (50 pg/vessel per 24 hours).

Mechanisms of Intimal Growth
To further elucidate the mechanisms of TGF-ß1–induced intimal formation, we measured intimal cell proliferation, cell accumulation, and cell density in wild-type and Serpine1^–/– arteries. We measured cell proliferation during the period of intimal growth (ie, 14 to 28 days after gene transfer; Figure IA). In a pilot experiment in wild-type mice, BrdU pulse labeling on day 21 revealed similar, low rates of intimal proliferation in both AdrTGFß and AdNull arteries (&1%; Figure ID). To increase the assay sensitivity, in the remaining experiments we infused BrdU continuously from days 14 to 28. In wild-type mice, intimal BrdU incorporation was 5-fold higher in AdrTGFß than AdNull arteries [3.1 (2.8% to 4.5%) versus 0.6 (0% to 1.2%); P=0.014; Figure 6A]. In Serpine1^–/– mice, however, AdrTGFß did not increase intimal BrdU incorporation [3.1 (1.8% to 8.2%) versus 2.2 (0% to 2.5%); P=0.4]. Intimal BrdU incorporation was not increased in AdNull-infused arteries of wild-type versus Serpine1^–/– mice (P=0.4; Figure 6A).

View larger version (13K): [in this window] [in a new window]   Figure 6. Mechanisms of TGFß-induced intimal growth. A, Percent of intimal cells (including endothelial cells) incorporating BrdU. BrdU was infused systemically from 14 to 28 days after gene transfer; arteries were harvested on day 28. B, Cell accumulation in 28-day intimas (total intimal nuclei per 5-µm-thick section, including endothelial cells). C, Cell density in 28-day intimas (total intimal nuclei per total intimal area). Data points represent individual arteries; bars are group medians.

To examine the contributions of cell and matrix accumulation to TGF-ß1–induced intimal growth, we counted intimal cells and calculated intimal cell density in 28-day AdrTGFß and AdNull arteries of wild-type and Serpine1^–/– mice. AdrTGFß increased intimal cell number in wild-type mice [65 (42–98) versus 32 (27–34) intimal cells/section in AdNull arteries, P=0.005; Figure 6B]. AdrTGFß did not increase intimal cell number in Serpine1^–/– mice [71 (47–77) versus 55 (41–72) intimal cells/section in AdNull arteries; P=0.5]. However, intimal cell number was significantly higher in AdNull-infused Serpine1^–/– arteries than in AdNull-infused wild-type arteries (P=0.005; Figure 6B). AdrTGFß infusion also decreased intimal cell density, but only in wild-type mice. Intimal cell density was decreased by nearly 60% in AdrTGFß versus AdNull-infused wild-type arteries [0.016 (0.015 to 0.018) versus 0.037 (0.034 to 0.10) cells/µm² P=0.005; Figure 6C]. Intimal cell densities did not differ between AdrTGFß and AdNull-infused arteries of Serpine1^–/– mice [0.032 (0.028 to 0.045) nuclei/µm² versus 0.038 (0.036 to 0.042), respectively; P=0.3]. There was also no difference in intimal cell density between AdNull-infused arteries of wild-type and Serpine1^–/– mice (P=1.0; Figure 6C).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We used a mouse model to investigate mechanisms of TGF-ß1–induced intimal growth and to determine whether PAI-1 is a critical mediator of TGF-ß1–induced intimal growth. Our major findings were: (1) TGF-ß1 stimulates growth of a SMC-rich and matrix-rich intima between 2 and 4 weeks after peak TGF-ß1 expression; (2) TGF-ß1 increases intimal cell proliferation; however, this increase is insufficient—by far—to account for TGF-ß1–induced intimal growth; (3) TGF-ß1 appears to induce neointima formation by stimulating cell migration from the media; (4) TGF-ß1 upregulates PAI-1 expression in the artery wall; (5) expression of PAI-1 is required for TGF-ß1–induced intimal growth; (6) PAI-1 is an important negative regulator of TGF-ß1 expression in the artery wall; (7) PAI-1 expression is required for TGF-ß1–induced intimal cell proliferation, intimal cell accumulation, and matrix accumulation.

The biological effects of TGF-ß1 are context-specific and cell type-specific.³⁰ Therefore, to interpret our results and relate them to other studies of TGF-ß1 and intimal growth, it is important to emphasize the biological context of our experiments and identify the cell types that are exposed to TGF-ß1 in our model. Our experiments involve local overexpression of TGF-ß1 from endothelium of a normal uninjured artery of a normolipidemic animal.²⁴ Cells exposed to TGF-ß1 are almost exclusively endothelial cells and SMCs. This model contrasts with other models used to study the role of TGF-ß1 in intimal growth, including balloon injury, hyperlipidemia, or systemic delivery of TGF-ß1 or its antagonists.^1,4,5,8,9 Thus, our experiments most directly address mechanisms through which TGF-ß1 acts on endothelial cells or SMCs to stimulate intimal growth in an uninjured artery. Our experiments may also clarify the effects of local TGF-ß1 expression on SMCs during intimal growth in a balloon-injured artery, whereas they are less likely to reveal the role of increased TGF-ß1 expression in lipid-induced atherosclerosis, in which TGF-ß1 actions on the immune system may be critical.⁵ When extrapolated to humans—with the caveat that experiments in one vascular bed may not predict the response to TGF-ß1 in another bed³¹—our experiments are more likely to clarify the role of TGF-ß1 in diffuse intimal thickening and postangioplasty restenosis (ie, SMC-rich lesions) than in the development of the macrophage-rich lesions of hyperlipidemia-induced atherosclerosis.

The significant increases in TGF-ß1 expression 3 days after infusion of either AdrTGFß or AdNull into Serpine1^–/– versus wild-type arteries (Figure 5) establish that PAI-1 is a negative regulator of TGF-ß1 in the artery wall. The mechanism through which PAI-1 limits TGF-ß1 expression in wild-type arteries is unclear but may involve inhibition of plasminogen activator/plasmin conversion of latent to active TGF-ß1, which in turn limits auto-induction of TGF-ß1 transcription by active TGF-ß1^21,32 (Figure V, available online at http://atvb.ahajournals.org). PAI-1 might also regulate TGF-ß1 expression through interactions with integrins.¹⁹

In agreement with studies in other animal models,^1,4,6,7 TGF-ß1 overexpression in mouse carotids caused intimal growth. We attempted to identify (among the candidate mechanisms of enhanced matrix accumulation, cell proliferation, and cell migration)^1,4,6,7 the mechanism(s) that are responsible for TGF-ß1–induced intimal growth. Our data indicate a trivial role for cell proliferation, with major roles for matrix accumulation and cell migration.

AdrTGFß induced substantial intimal matrix accumulation in wild-type arteries. TGF-ß1 induction of collagen and proteoglycan synthesis in vivo is well documented experimentally.^6,7 This report is the first, to our knowledge, that provides a causal link between TGF-ß1 expression and hyaluronan accumulation in the artery wall. Accumulation of medial proteoglycans and hyaluronan may contribute to "loosening" of medial matrix structure, which could facilitate SMC migration from the media to the intima.³³ Consistent with this model, we observed proteoglycan accumulation in the media of AdrTGFß arteries 2 weeks after gene transfer, before the onset of intimal growth (data not shown).

Previous studies of TGF-ß1–induced intimal growth do not yield a consensus regarding a role for cell proliferation.^1,7–9 Here, we focused on measurement of cell proliferation during the period of maximal intimal growth (2 to 4 weeks after gene transfer) and instead of pulse labeling^1,7–9 we used the more sensitive technique of continuous BrdU infusion. We found that AdrTGFß increased the intimal cell BrdU index from <1% to 3% during the entire 2-week period of intimal growth. This increase was statistically significant; however, it is vastly inadequate to account for the 100% increase in intimal cells in AdrTGFß arteries (Figure 6B).

We also considered a role for altered apoptosis in TGF-ß1–induced neointimal growth. However, no neointimal cells accumulate in AdNull arteries of wild-type mice; there is only endothelium. Therefore, it is not possible to calculate an apoptotic rate for AdNull neointimas and test whether this rate is higher in AdrTGFß neointimas. If no neointimal cells arrive in AdNull arteries, decreased apoptosis cannot be a mechanism through which they accumulate in AdrTGFß arteries.

Because neither increased proliferation nor decreased apoptosis can explain TGF-ß1–induced neointimal growth in wild-type mice, cell migration must be the mechanism for cell accumulate in AdrTGFß neointimas. Because the neointima forms largely between 3 to 4 weeks after gene transfer (Figure IA) and at 4 weeks neointimal cells stain overwhelmingly positive for smooth muscle -actin (Figure II), the vascular media is the most likely source of neointimal cells. Extravascular or adventitial sources for some neointimal cells remain possible; however, our data suggest that medial SMCs are the principal cellular contributors to the TGF-ß1–induced neointima. Direct measurement of cell migration into the intima remains a challenge. Here, as in other studies,³⁴ we identified migration as a mechanism of intimal growth by excluding other mechanisms, such as cell proliferation.

Our experiments in Serpine1^–/– mice identify PAI-1 as an essential mediator of TGF-ß1-induced intimal growth. AdrTGFß-infused Serpine1^–/– arteries are resistant to the effects of increased TGF-ß1, because proliferation, intimal cell number, or intimal cell density are not altered by TGF-ß1 overexpression in Serpine1^–/– arteries. Well-established activities of PAI-1 that could mediate TGF-ß1–induced intimal growth are precisely those suggested by our studies: enhancement of cell migration and matrix accumulation. PAI-1 can enhance cell migration through a "de-adhesive" activity that detaches cells from matrix.¹⁹ PAI-1 can promote matrix accumulation by inhibiting the urokinase plasminogen activator (uPA)/plasmin/matrix metalloproteinase (MMP) system, a proteolytic cascade that contributes to matrix degradation.³⁵ PAI-1 could also contribute to intimal formation by enhancing fibrin matrix accumulation;³⁶ however, we found no evidence for this.

PAI-1 is clearly a downstream mediator of TGF-ß1–induced intimal growth; however, comparison of intimal growth in AdNull-infused arteries of Serpine1^–/– and wild-type mice (Figures 3 and 6) suggests that PAI-1 can also inhibit intimal growth. Specifically, intimas of AdNull-infused Serpine1^–/– arteries were larger (Figure 3) and had increased cell number (but unaltered cell proliferation and density) compared with AdNull-infused wild-type arteries (Figure 6). These findings indicate that cell migration is responsible for increased intimal growth in AdNull-infused arteries of Serpine1^–/– mice versus wild-type mice. This seemingly paradoxical finding, that absence of PAI-1 increased cell migration and intimal growth in AdNull arteries, is in agreement with a study reporting increased SMC migration and intimal growth in injured arteries of Serpine1^–/– versus wild-type mice.³⁷

How can the observation that absence of PAI-1 increases intimal growth (2-fold; Figure 3A) and cell migration (Figure 6) in AdNull arteries be reconciled with the major finding of this study, that in wild-type arteries TGF-ß1 acts via PAI-1 to increase intimal growth (5-fold; Figure 3A) and cell migration (Figure 6)? We speculate that PAI-1 increases cell migration when TGF-ß1 and PAI-1 expression are high (ie, in wild-type AdrTGFß arteries), whereas PAI-1 inhibits cell migration when TGF-ß1 and PAI-1 expression are low (ie, in AdNull arteries). Disparate effects of PAI-1 on cell migration and intimal growth have been reported elsewhere^18,36–38 and have been challenging to reconcile.³⁹ Based on the present study, we hypothesize that the effect of PAI-1 on cell migration and intimal growth depends on whether TGF-ß1 expression is low or high (Figure VI, available online at http://atvb.ahajournals.org). When TGF-ß1 expression is elevated either experimentally or because of thrombus deposition (platelets are a major source of TGF-ß1 in vivo) PAI-1 would promote migration and intimal growth.^17,18,36 When TGF-ß1 and PAI-1 expression are low (for example in thrombus-free arteries or in the AdNull arteries of the present study), PAI-1 would inhibit migration and intimal growth.^37,38 Dependence of PAI-1 activity on TGF-ß1 expression could be caused by TGF-ß1–induced alterations in matrix composition⁴⁰ (eg, alterations in the availability of PAI-1-binding matrix proteins such as vitronectin) or concentration-dependent actions of PAI-1 on cell–matrix interactions⁴¹ (eg, PAI-1 may promote cell detachment only when present above a threshold level).

Because the role of PAI-1 in regulating intimal growth appears context-specific and dependent on the level of TGF-ß1 expression, one could ask which context is most relevant to human disease: low or high TGF-ß1? Unlike normal mouse arteries, in which TGF-ß1 expression is low (Figure 5, and data not shown), diseased human arteries express high levels of TGF-ß1.^2,3 Therefore, our data suggest that PAI-1 promotes cell migration and intimal growth in human arteries and that strategies that block vascular PAI-1 activity could prevent TGF-ß1–induced intimal growth in diseased human arteries while potentially leaving intact the salutary suppressive effects of TGF-ß1 on arterial inflammation.⁵

	Acknowledgments

 
This study was supported by National Institutes of Health grants to D.A.D. (HL69063) and T.N.W. (HL18645). R.A. and A.D.F. were supported by National Institutes of Health National Research Service Awards (T32HL07731 and T32HL07828). We thank Angela Buchholtz, Erlan Toulegenov, Stephanie Lara, and Margo Weiss for administrative and technical assistance. We are grateful to Dr Renu Virmani for the Movat staining.

Received July 6, 2005; accepted December 7, 2005.

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