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(Circulation Research. 1999;84:498-504.)
© 1999 American Heart Association, Inc.

Original Contribution

TIMP-4 Is Regulated by Vascular Injury in Rats

Clare M. Dollery, Jean R. McEwan, Mingsheng Wang, Qingxiang Amy Sang, Yiliang E. Liu, Y. Eric Shi

From the Hatter Institute (C.M.D., J.R.M.), University College London Hospitals, London; the Departments of Pediatrics (M.W., Y.E.L.) and Pathology (Y.E.S.), Long Island Jewish Medical Centre, the Long Island Campus for the Albert Einstein College of Medicine; and the Department of Chemistry (Q.A.S.), Florida State University.

Correspondence to Clare M. Dollery, MRCP, PhD, Cardiology Department, 4th Floor, Jules Thorn Building, Middlesex Hospital, Mortimer St, London W1N8AA, UK. E-mail c.dollery{at}ucl.ac.uk

	Abstract

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Abstract—The role of basement membrane–degrading matrix metalloproteinases (MMPs) in enabling vascular smooth muscle cell migration after vascular injury has been established in several animal models. In contrast, the role of their native inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs), has remained unproven despite frequent coregulation of MMPs and TIMPs in other disease states. We have investigated the time course of expression and localization of TIMP-4 in rat carotid arteries 6 hours, 24 hours, 3 days, 7 days, and 14 days after balloon injury by in situ hybridization, immunohistochemistry, and Western blot analysis. TIMP-4 protein was present in the adventitia of injured carotid arteries from 24 hours after injury. At 7 and 14 days after injury, widespread immunostaining for TIMP-4 was observed throughout the neointima, media, and adventitia of injured arteries. Western blot analysis confirmed the quantitative increase in TIMP-4 protein at 7 and 14 days. In situ hybridization detected increased expression of TIMP-4 as early as 24 hours after injury and a marked induction in neointimal cells 7 days after injury. We then studied the effect of TIMP-4 protein on the migration of smooth muscle cells through a matrix-coated membrane in vitro and demonstrated a 53% reduction in invasion of rat vascular smooth muscle cells. These data and the temporal relationship between the upregulation of TIMP-4, its accumulation, and the onset of collagen deposition suggest an important role for TIMP-4 in the proteolytic balance of the vasculature controlling both smooth muscle migration and collagen accumulation in the injured arterial wall.

Key Words: tissue inhibitor • artery • muscle, smooth, vascular • vascular injury

	Introduction

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The importance of extracellular matrix stability in maintaining the integrity of the vessel wall in single-gene defect disorders such as Marfan syndrome has been recognized for many years. Recent studies have focused on the role of the extracellular matrix in postprocedural vasculopathies such as angioplasty restenosis.^{1 2 3 4 5 6 7} The restenotic process after vascular injury is a result of normal but unwanted healing that causes a loss of lumen and a return of symptoms in 25% of patients.

After arterial injury, vascular smooth muscle cells (VSMCs) proliferate and migrate, to form a neointima that accumulates extracellular matrix in the late stages of the restenotic process.⁸ VSMCs secrete proteases that digest extracellular matrix through plasminogen-dependent and -independent pathways, which facilitate migration.⁹ The expression of both plasminogen activators and inhibitors has been studied in the rat model of arterial balloon injury and has been shown to correlate with migration of VSMCs.¹⁰ Concomitant secretion of both activators and inhibitors was demonstrated, and the overall proteolytic activity resulted from the balance of the two.

Several studies have addressed the expression of the MMPs after vascular injury^{1 4 5 6 7} and in atherosclerosis,^{11 12 13} but few studies have investigated their endogenous inhibitors, the tissue inhibitors of matrix metalloproteinases (TIMPs).^{7 14} Alteration of the proteolytic balance in the vasculature by inhibition of MMP activity either pharmacologically or by gene transfer has been shown to suppress migration and neointima formation.^{15 16} The upregulation of MMP expression after vascular injury shows similar trends to the changes in the plasminogen activators, which suggests that MMP inhibitors may participate in the control of proteolysis after vascular injury. We have investigated the temporal relationship between vascular injury and the most recently identified endogenous inhibitor of the MMPs, TIMP-4, and studied its effects on smooth muscle cell migration in vitro.¹⁷ TIMP-4 is a 23-kDa protein that inhibits MMP-1, MMP-3, MMP-7, and MMP-9¹⁸ and shows a particular interaction with MMP-2; TIMP-4 binds specifically to its C-terminal domain.¹⁹ We chose to study TIMP-4 because of its specific tissue expression: transcripts are abundant in the human heart but occur at low levels in most other organs, which suggests a possible cardiovascular specificity for this metalloproteinase inhibitor.

We have studied the time course and localization of TIMP-4 protein and mRNA in rat carotid arteries 6 hours, 24 hours, 3 days, 7 days, and 14 days after balloon injury through immunohistochemistry, Western blot analysis, and in situ hybridization. Results obtained show a substantial increase in TIMP-4 immunoreactivity after arterial injury with initial low levels of induction that peak at 7 to 14 days. TIMP-4 protein reduces smooth muscle cell migration in vitro by 53%. This suggests that TIMP-4 may play a key role in the control of cell migration and extracellular matrix accumulation after vascular injury.

	Materials and Methods

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Arterial Injury Model
20 male Wistar rats that weighed 300 to 350 g were anesthetized with an intraperitoneal injection of fentanyl/fluanisone (Hypnorm Jansson) 0.025 mg per 100 g and 0.8 mg/100 g, respectively, and midazolam (Hypnovel, Roche) 0.42 mg/100 g. The left common carotid and the external and internal carotid arteries were exposed by blunt dissection via a midline neck incision. A 2F Fogarty balloon embolectomy catheter (Baxter Healthcare Corp) was introduced into the external carotid artery and passed down the common carotid artery into the aorta. The embolectomy balloon was then inflated with 0.03 mL of sterile saline (0.9% wt/vol) and pulled up the common carotid artery to denude the endothelium and stretch the media of the arterial wall. This procedure was repeated twice, and the catheter was then removed. The external carotid artery was then ligated, and the wound was closed with surgical clips. The animals were anesthetized with fentanyl and midazolam at the time points indicated and exsanguinated via an aortic cannula before pressure perfusion fixation at 120 mm Hg with 2% formaldehyde and 0.2% glutaraldehyde. All arteries were placed in fixative for 24 hours before being processed for paraffin embedding in an automated system. In a subset of animals, fresh tissues were frozen in liquid nitrogen and protein was extracted as previously described; Western blot analysis was performed as described below.⁷ Protein content of the samples was determined according to the method of Bradford, and the concentration was normalized to 0.9 mg/mL.⁷

Western Blot Analysis
Protein extracts of rat carotid arteries were boiled in a SDS-mercaptoethanol sample buffer and electrophoresed in 12% polyacrylamide gel; 10 µg of total protein was loaded for each sample. Gels were blotted onto polyvinylidene membrane in 25 mmol/L Tris/192 mmol/L glycine buffer, pH 8.3, that contained 20% vol/vol methanol. Blots were blocked in 5% BSA (Sigma Chemical Co) for 1 hour. The specific anti–TIMP-4 antibody²⁰ was diluted 1:2000 in TTBS (30 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 0.1% Tween-20). After incubation with the primary antibody overnight at 4°C, the blots were washed 4x for 10 minutes in TTBS, then incubated for 1 hour in goat anti-rabbit IgG-HRP (Sigma) diluted 1:1200 in TTBS. The blots were then washed 4x for 10 minutes in TTBS, and the bands were visualized by chemiluminescence.

Immunohistochemistry and Histomorphometry
Deparaffinized and acid-treated sections (5 µm thick) were rehydrated by passing them through a series of ethanol concentrations (100%, 100%, 95%, 85%, 70%, 50%, and 30%) followed by 2 passes through distilled water. The sections were treated with 0.3% H₂O₂ for 15 minutes, washed with H₂O, and digested with 0.1% trypsin for 20 minutes. After blocking with 10% BSA for 30 minutes, the slides were incubated with the affinity–purified specific anti–TIMP-4 antibody (2 µg/mL) for 1 hour¹⁸ followed by 3 stringent washes. Sections were incubated with biotin-conjugated secondary rabbit anti-mouse antibodies (DAKO). As we previously described,²¹ the colorimetric detection was performed by a standard, indirect streptavidin-biotin immunoreaction method with the use of the DAKO universal LSAB kit according to the manufacturer's instructions. Serial 5-µm histological sections were cut and stained with hematoxylin and eosin, and additional sections were stained with a smooth muscle–actin antibody conjugated with horseradish peroxidase according to the manufacturers instructions (DAKO, EPOS anti-human SM Actin-HRP). Colorimetric detection was achieved with diaminobenzidine (Sigma).

In Situ Hybridization
In situ hybridization was performed as previously described.²¹ Briefly, deparaffinized and acid-treated sections (5 µm thick) were treated with proteinase K, prehybridized, and hybridized overnight with digoxigenin labeled antisense transcripts from a TIMP-4 cDNA insert (obtained from Y.E.S.).¹⁷ The TIMP-4 antisense probe is a 550-bp fragment from nucleic acid 130 to 683. The full-length TIMP-4 cDNA was cut by KPNI and SmaI; the 550-bp insert was subcloned into Bluescript II plasmid, and the resulting plasmid was named Bluescript TIMP-4–B. The 550-bp antisense probe was generated by SmaI digestion of Bluescript TIMP-4–B plasmid followed by application of T7 polymerase. After hybridization, RNase treatment and 3 stringent washes were performed. Sections were incubated with mouse antidigoxigenin antibodies (Boehringer) followed by incubation with biotin-conjugated secondary rabbit anti-mouse antibodies (DAKO). The colorimetric detection was performed by a standard, indirect streptavidin-biotin immunoreaction method with the DAKO universal LSAB kit according to manufacturer's instructions.

Cell Culture and In Vitro Assessment of Migration and Proliferation
Primary rat VSMCs isolated from male Wistar rats with the method of Rennick et al²² were used at passages 4 to 6. Cells were incubated at 37°C in a humidified 5% CO₂/95% O₂ incubator in RPMI media supplemented with 10% FCS, 100 U/mL penicillin, 100/mL streptomycin, and 1% transferrin. Immunohistochemistry with an anti–-smooth muscle–actin antibody (Sigma) confirmed positive staining in 98% of cells. Inhibition of smooth muscle cell migration by purified recombinant human TIMP-4 protein (rTIMP-4) was evaluated with the Matrigel invasion assay with reconstituted basement membrane as previously described.^{18 20} Briefly, 10 µm pore polycarbonate membranes were coated with 4 mg/mL growth factor–reduced Matrigel. The cells were seeded at a density of 30 000 cells/mL per well in DMEM that contained 5% FCS. The medium in the bottom chamber contained 10% FCS. After incubation in a humidified incubator with 5% CO₂ at 37°C for 40 hours, the medium and the cells were removed from the bottom chambers, centrifuged, and counted with a Nikon microscope. To assay cell growth, exponentially growing cultures of SMC were detached with trypsin, and the trypsin was neutralized with DMEM–10% serum. Cells were counted, diluted, and seeded in triplicate at 3000 cells per well (24-well plate) in 1-mL DMEM–5% serum. Cells were treated with 10 nmol/L rTIMP-4 for 2 days, and cell growth was measured by counting cell numbers per well.

	Results

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Histology and Immunohistochemistry
Examination of histological sections 6 and 24 hours after balloon injury showed an intact endothelium in the control right carotid arteries and endothelial denudation in the injured left carotid arteries. Seven days after injury, a small neointima was visible in all injured arteries and developed into the characteristic mature lesion by 14 days.

Control immunocytochemical staining was performed in serial sections with mouse immunoglobulins to evaluate background staining. The antibody used was originally raised against human TIMP-4, and its ability to recognize both human and rat TIMP-4 was confirmed in samples of human and rat myocardium that showed identical immunostaining (data not shown). Selected sections were incubated with an excess of rTIMP-4 protein to block antibody binding, and no immunostaining was seen.

The immunostaining of the right carotid artery of the same animal at the same time point was used as a control in all qualitative assessment of injured arteries (n=3 for all time points). At all time points, background levels of TIMP-4 were seen in the uninjured carotids, but after 24 hours, additional levels of TIMP-4 immunoreactivity were detected in the adventitia of the injured arteries (Figure 1A to1D). This staining was localized in the adventitia of injured arteries at 3 days after balloon injury but remained at background levels in controls. Staining of serial sections for smooth muscle actin did not show positive cells in the adventitia. At 7 days after injury, extensive positive staining for TIMP-4 was seen in the media and developing neointima, with some reduction in adventitial staining compared with control right carotid arteries (Figure 2B). This substantial increase in TIMP-4 immunoreactivity was maintained in the mature lesion at 14 days, with the densest staining occurring in the neointima and a progressive reduction through the media to the adventitia (Figure 2D). Staining of serial sections for smooth muscle actin showed positive cells in the media at 7 days and in both the media and neointima at 14 days (Figure 2C and 2D).

View larger version (103K): [in this window] [in a new window]   Figure 1. TIMP-4 immunostaining after carotid artery injury. A, Control artery shows weak immunoreactivity 24 hours after contralateral injury. B, Injured artery at 6 hours after ballooning shows a similar level of TIMP-4. C, At 24 hours after injury, the adventitia shows a small increase in TIMP-4 (brown stain). D, At 3 days after injury, the adventitia continues to demonstrate increased levels of TIMP-4. Bar=50 µm.

View larger version (104K): [in this window] [in a new window]   Figure 2. TIMP-4 and smooth muscle–actin immunostaining of balloon injured vessels 7 to 14 days after carotid injury. A, Injured artery 7 days after injury showing positive smooth muscle–actin staining in the media and occasional neointimal cells. B, At 7 days after injury, intense TIMP-4 staining is present in the media and neointima. C, Actin staining is present in the neointima and media 14 days after injury; TIMP-4 staining at 14 days (D) is intense in the neointima and is gradually reduced toward the adventitia in the injured vessel. A indicates adventitia; M, media; and I, intima. Bar=50 µm.

TIMP-4 Protein Expression After Vascular Injury
To further confirm the immunohistochemical staining of the increased TIMP-4 expression in the injured vessel, we performed Western blot analyses for TIMP-4 expression at the same time points after vascular injury. As shown in Figure 3, low levels of TIMP-4 expression can be detected in control vessels or vessels at 6 hours, 1 day, and 3 days after vascular injury. TIMP-4 protein expression was substantially increased at day 7 and day 14 after vascular injury. Although the secreted rTIMP-4 protein was recognized as a 23-kDa band in the Western blot, the rat TIMP-4 protein in the tissue extract migrated to the slightly higher molecular weight band. The molecular-weight difference may reflect variations in TIMP-4 in different species. Alternatively, the high-molecular-weight form of rat TIMP-4 may represent the nonsecreted intracellular TIMP-4 precursor.¹⁷

View larger version (18K): [in this window] [in a new window]   Figure 3. Western blot analysis of TIMP-4 expression in arterial extracts with a specific anti–TIMP-4 antibody. Lane 1, 1 ng of purified human rTIMP-4. Lane 2, 10 ng protein extracted from rat myocardium. Lanes 3 to 7, Samples of injured rat carotid arteries: lane 3, 6 hours after injury; lane 4, 1 day after injury; lane 5, 3 days after injury; lane 6, 7 days after injury; and lane 7, 14 days after injury. Lane 8, Control noninjured vessel from a rat 14 days after balloon injury to the contralateral vessel.

In Situ Hybridization
Low levels of TIMP-4 expression were seen in normal vessels, indicated by brown staining (Figure 4A). Upregulation of TIMP-4 was initially seen 3 days after injury in the medial smooth muscle cells (Figure 4C). Extensive TIMP-4 expression was seen in the neointimal cells from the vessels harvested 7 days after injury with some expression seen in the media (Figure 4D). Expression of TIMP-4 with the use of colorimetric detection showed a diffuse pattern because of the large proportion of cytoplasm relative to the number of nuclei in 5-µm sections. Seven days after injury, the section of artery was also hybridized with the sense probe, and no detectable background staining was observed at the same conditions used for the antisense probe (Figure 4B).

View larger version (96K): [in this window] [in a new window]   Figure 4. In situ hybridization analysis of TIMP-4 expression in rat coronary vessels. Cells labeled with brown indicate TIMP-4 gene expression. All sections were counterstained lightly with hematoxylin to view negatively stained cells. A, Background staining of TIMP-4 in an uninjured vessel. B, Section of artery 7 days after injury hybridized with the sense probe; no detectable background staining was observed at the same conditions used for the anti–sense probe. C, Positive staining of TIMP-4 in the smooth muscle cells from the vessel 3 days after injury. D, Strong positive TIMP-4 expression in the neointimal cells from the vessel 7 days after injury. Sections of the vessel 7 days after injury were also hybridized with the sense probe, and no detectable background staining was observed under the same conditions for the antisense probe. All sections presented in the figure were derived from the same experiment.

Inhibition of Migration of Rat Smooth Muscle Cells
The increased TIMP-4 expression in the injured vessel may play a major role in inhibiting ECM degradation and the subsequent cell migration. The effect of purified rTIMP-4 on the migration of smooth muscle cells was investigated. Rat smooth muscle cells were moderately invasive. At the end of a 40-hour incubation, 1% of the cells had migrated across the Matrigel barrier. A 53% reduction in migration was noted when rTIMP-4 was added at a concentration of 10 nmol/L (281±28 and 133±19 cells per high-power field in control and TIMP-4–treated cells, respectively, P=0.03) (Figure 5). No significant difference in growth rate was observed between the control and rTIMP-4–treated cells assessed by counting cell number (data not shown). The different migration results between the control cells and rTIMP-4–treated cells are not therefore caused by an effect of TIMP-4 on smooth muscle cell proliferation.

View larger version (14K): [in this window] [in a new window]   Figure 5. Inhibition of cell migration by rTIMP-4. The bottom wells of the invasion chamber were filled with DMEM that contained 10% serum. Rat smooth muscle cells were seeded at a density of 30 000 cells/mL per well with or without 10 nmol/L of rTIMP-4 at the DMEM that contained 5% FCS. Cell migrations were analyzed 40 hours later. Mean±SEM of 3 cultures are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
MMPs are thought to be critical to the migration of smooth muscle cells into the arterial neointima after vascular balloon injury.¹ We have shown that one of the cardiospecific molecules capable of inhibiting the MMPs, TIMP-4, is upregulated by vascular injury. The substantial rise in TIMP-4 showed a distinct temporal and spatial pattern, with initial small increases in the adventitia followed by strongly positive immunostaining in the media and neointima. Western blot analysis established the substantial quantitative increase in TIMP-4 protein between 7 and 14 days after injury. Background levels of TIMP-4 were present in control right carotid arteries at all time points when analyzed by either immunohistochemistry or Western blotting. In situ hybridization studies confirmed that intimal smooth muscle cells express TIMP-4 seven days after injury with lower levels of expression also detectable in the media. The expression of TIMP-4 in the neointima appears to precede the detection of smooth muscle actin, which suggests that these cells still exhibit a secretory phenotype typical of this time point after injury.²³ At 14 days after injury, both intima and media cells express TIMP-4 and smooth muscle actin. The time course of these changes in both TIMP-4 protein and mRNA parallel the cessation of smooth muscle cell migration and the accumulation of extracellular matrix in the neointima, which suggests that TIMP-4 may play a key role in arresting smooth muscle cell migration and in controlling the laying down of matrix. Investigation of the effect of TIMP-4 protein on vascular smooth muscle cell migration in vitro yielded further evidence in support of this hypothesis: a 53% reduction in the ability of the cells to cross a membrane barrier in the presence of TIMP-4 was indicated.

Vascular injury initiates a combination of events. Initially, VSMCs from the media migrate into the neointima, and rapid growth of these cells produces a characteristic lesion of fibrocellular intimal hyperplasia.^{24 25} As the resultant lesion matures, there is a greater preponderance of extracellular matrix.⁸ Recent clinical studies with intravascular ultrasound have shown transmural injury and repair and have revealed a change in total vessel dimensions after angioplasty,²⁶ although laboratory studies of porcine coronary support the importance of vascular remodeling after balloon injury.²⁷

There is considerable evidence that smooth muscle cells, macrophages, and fibroblasts can degrade extracellular matrix by upregulation of MMP activity, but few parallel studies of the endogenous inhibitors have been performed.^{28 29} In the rat carotid model used in this study, 92-kDa gelatinase B is induced from 1 to 7 days after injury, whereas 72-kDa gelatinase A is constitutively expressed with some induction 4 to 5 days after angioplasty.^{1 7} Studies of the plasminogen-activator system after balloon injury in both the rat and rabbit models show acute upregulation of urokinase plasminogen activator activity,^{30 31} which could activate the MMP cascade. It is clear that the smooth muscle cell can alter its surrounding matrix if stimulated by the appropriate cytokines, but it is unlikely that the MMPs are the sole regulators of this process. A cascade of events that incorporate both plasminogen activators and inhibitors and MMPs and TIMPs probably exists.

TIMPs are secreted multifunctional proteins that have anti-MMP activity as well as erythroid-potentiating and cell growth–promoting activities.^{32 33 34 35 36} To date, 4 mammalian TIMPs have been cloned, characterized, and sequenced. These are classified by their structural homology and their ability to inhibit MMPs, but evidence exists for specific physiological roles of individual members of the group. For example, a specific complex forms between MMP-2 and TIMP-2 and MMP-2³⁷ and TIMP-4.^{18 19} Inducibility of expression is also varied within the group with TIMP-3 subject to cell-cycle regulation.³⁸ Only 2 studies to date have examined the expression of TIMPs after vascular injury.^{7 14} Hasenstab et al¹⁴ studied TIMPs 1 to 3 and identified an increase in TIMP-2 mRNA at 24 hours, which peaked at 7 days (at which time the change was statistically significant). No modulation of TIMP-1 mRNA was detected, and no TIMP-3 expression was observed. TIMP-2 protein localized to the neointima 7 days after injury, and reverse zymography showed MMP inhibitory activity at days 3 to 5, with low levels of expression in control arteries and reduction by 14 days. In contrast, our own group identified TIMP-1 message peaking at 24 hours after injury by semiquantitative reverse transcriptase polymerase chain reaction.⁷ Both these studies raise the important concept that the balance between MMPs and TIMPs determines proteolysis in the vessel wall, but neither study has identified changes in TIMP-1, -2, or -3 expression on the same scale as MMP induction after vascular injury. However, our results suggest that TIMP-4 may be the predominant inhibitor of the MMPs from 7 to 14 days after vascular injury.

We studied TIMP-4, the most recently cloned and characterized TIMP, because of its high level of expression in the human heart, which may be associated with a physiological role in the cardiovascular system. With the use of purified rTIMP-4, enzymatic kinetic studies show values concentration at 50% inhibition IC₅₀ of 19, 3, 45, 8, and 83 nmol/L for MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9, respectively.¹⁹ In addition, overexpression of TIMP-4 in human breast cancer cells inhibits invasion in vitro¹⁹ and tumor growth and metastasis in vivo.²⁰ Interestingly, the highest level of TIMP-4 expression is in the myocardium,¹⁷ in which cancer metastasis rarely occurs. The current results demonstrate the ability of TIMP-4 to inhibit smooth muscle cell invasion. The increase in TIMP-4 protein on days 7 to 14 after injury may curtail the migration of smooth muscle cells into neointima, which typically is greatest from days 5 to 8 in this model of vascular injury.³⁹

The temporal change in TIMP-4 raises the possibility that it may contribute to the overall increase in collagen content in the 14-day lesion. Matrix production has been shown to contribute to intimal thickening predominantly in the second week after injury,⁸ and fibronectin and collagens I, III, and VIII transcripts are increased from 7 days after ballooning in the rat model.⁴⁰ Other models of vascular injury have shown a later increase in collagen content; for example, at 4 to 12 weeks after arterial injury in the rabbit.¹⁵ This occurs despite a decrease in collagen synthesis over this period and may reflect a reduced breakdown of collagen that could be mediated by the action of TIMPs. Our results show increased TIMP-4 throughout the arterial wall at 7 to 14 days; therefore, the proteolytic balance at this point would be in favor of the laying down of collagen to form a mature lesion.^{1 7}

The rat model of carotid balloon injury has been widely debated because of its inability to predict response to therapy in human coronary restenosis.⁴¹ Recent studies in the porcine coronary artery have implicated adventitial fibroblasts in extracellular matrix synthesis after balloon injury.⁴² Activated adventitial fibroblasts express procollagen I and some translocate to the intima of the artery. The authors noted that despite increased intracellular procollagen type I, mature type I collagen did not appear in the adventitia for 1 week after injury. They suggest that this may be due to the excess of MMPs and plasminogen activators up to this point. Our studies show an initial small adventitial increase in TIMP-4 followed by strongly positive immunostaining throughout the artery. This may reflect the important role of the adventitia in vascular injury and the accumulation of extracellular matrix. Preliminary findings suggest that translocation of adventitial fibroblasts also occurs in the rat model of vascular injury. The pattern of TIMP-4 protein accumulation appears to move from the adventitia to the media and then to predominate in the neointima. Expression of TIMP-4 mRNA occurs principally in the media and neointima. Proteins may be produced in the neointima, diffuse through the arterial wall, and be trapped in the adventitia. However, TIMP-4 is produced by perivascular fibroblasts in the human coronary artery, and this correlation with the findings of Shi et al requires further study.

The response of the arterial wall to balloon injury is incompletely understood, and the clinical problem of angioplasty restenosis is unresolved. This study implicates TIMP-4, a novel, heart-specific MMP inhibitor, in the response to vascular injury and shows a temporal correlation with the cessation of smooth muscle cell migration and the onset of collagen deposition. The potential application of TIMP-4 in the prevention of angioplasty restenosis warrants further investigation.

	Acknowledgments

 
Dr Dollery was supported by the Medical Research Council of Great Britain and the Jean Shanks Fund. Work in the laboratory of Dr McEwan was supported by The British Heart Foundation. Work in the laboratory of Dr Shi was supported by grant CA68064-01 from the National Institutes of Health and by Helen and Irving Schneider. This research was supported in part by Grant-in-Aid AHA 9601457 from the American Heart Association, Florida Affiliate, to Dr Q.X. Sang.

Received July 16, 1998; accepted December 16, 1998.

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