Metalloproteinase Blockade by Local Overexpression of TIMP-1 Increases Elastin Accumulation in Rat Carotid Artery Intima

From the Departments of Surgery (R.F., H.L., E.A., M.C., A.W.C.) and Pathology (D.H.), University of Washington School of Medicine, Seattle; and the Department of Biochemistry (B.S.), University of Texas Health Center at Tyler, Tex.

Correspondence to Dr Alexander W. Clowes, University of Washington, Department of Surgery, Box 356410, Seattle, WA 98195.

	Abstract

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Abstract—We have recently demonstrated that the blockade of matrix metalloproteinases by local overexpression of the intrinsic inhibitor tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) reduces intimal hyperplasia. We now report a major change in the elastin content of the intima of rat carotid arteries seeded with TIMP-1–overexpressing smooth muscle cells. To understand the mechanism responsible for elastin accumulation, synthesis and degradation of elastin in TIMP-1 and control cell–seeded rats were measured. There were no differences in elastin mRNA or elastin synthesis, as documented by ¹⁴[C]proline incorporation between TIMP-1 and control cell–seeded arteries. In contrast, there was an increase in cross-linked elastin in the TIMP-1 group. In addition, in TIMP-1 and control rats, an elastase activity of approximately 28 kD was detected by elastin zymography and was decreased in TIMP-1 cell–seeded vessels. The 28 kD elastolytic activity was inhibited by exogenously added TIMP-1 and EDTA but not by PMSF, suggesting that it was a metalloelastase. Therefore, we have demonstrated that a shift of the proteolytic balance toward protease inhibition by TIMP-1 overexpression does not change elastin synthesis but rather changes posttranslational processing, resulting in increased elastin accumulation.

Key Words: TIMP-1 • elastin • intima • extracellular matrix • zymography

	Introduction

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We have recently demonstrated that the blockade of matrix metalloproteinases by local overexpression of the intrinsic inhibitor tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) reduces intimal hyperplasia.¹ We now report a major change in the elastin constitution in the intima of rat carotid arteries seeded with TIMP-1–overexpressing smooth muscle cells (SMCs), as demonstrated by histochemical and biochemical analysis. To understand the mechanism responsible for elastin accumulation, we looked at changes in synthesis and degradation of elastin between TIMP-1–and control-seeded rats. There were no changes in elastin synthesis, as documented by ¹⁴ [C]proline incorporation. Similarly, there were no changes in elastin mRNA level between TIMP-1 and control cell–seeded arteries. In contrast, there was an increase in cross-linked elastin in the TIMP-1 group, as measured by desmosine content. In addition, characterization of elastolytic activities in TIMP-1 and control rats by use of elastin zymography demonstrated a metalloelastase activity of approximately 28 kD that was decreased in TIMP-1–seeded compared with control cell–seeded vessels. The 28 kD elastolytic activity was inhibited by exogenously added TIMP-1 and EDTA but not by PMSF, suggesting that it is a metalloelastase. Therefore, we have demonstrated that a shift of the proteolytic balance toward protease inhibition by TIMP-1 overexpression does not change elastin synthesis but rather changes posttranslational processing, resulting in increased elastin accumulation.

Elastin is one of the major connective components of the blood vessel wall and is responsible for the elastic properties of the vessel. The mature elastin fibers have an extremely slow turnover rate, so that while elastin is rapidly synthesized in young growing vessels, newly synthesized elastin is virtually nonexistent in older, mature vessels.² It is only during injury or other pathological conditions that elastin synthesis is reactivated. Elastin fibers are susceptible to enzymatic degradation by elastases, a group of nonspecific proteolytic enzymes derived from polymorphonuclear leukocytes,³ macrophages,⁴ and pancreatic cells.⁵ Recently, several members of the matrix metalloproteinase (MMP) family, and in particular MMP-9, MMP-2, and MMP-7, have been shown to posses elastase activity.⁶ Excessive elastin degradation by MMP-9 may contribute to a number of vascular diseases, including abdominal aortic aneurysms.^{7 8} Furthermore, the expression of MMPs as measured by gelatin, casein, and elastin zymography is increased in rat carotid arteries subjected to balloon injury.⁹ In fact, upregulation and activation of MMPs correlates with the vascular SMC proliferation and migration events leading to hyperplastic intima after balloon injury.¹

We recently reported that by disruption of endogenous MMP proteolytic activity through overexpression of the physiological inhibitor TIMP-1, we are able to inhibit intimal hyperplasia in balloon-injured rat carotid artery by 40%.¹ To elucidate further the mechanism of blockade of intimal hyperplasia, we have investigated the contribution of extracellular matrix and have shown a significant accumulation of elastin in rat arteries seeded with SMCs overexpressing TIMP-1.

	Methods

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TIMP-1 Overexpression in Rat
Cloning of baboon TIMP-1 cDNA, construction of recombinant retrovirus, preparation of retrovirally infected Fischer 344 rat SMCs, and ballooning/seeding of Fischer 344 rat common carotid arteries are described in detail elsewhere.^{1 10} Briefly, the left carotid was surgically exposed and a 2F balloon catheter was used to denude and injure the artery wall. Immediately after injury, a catheter was inserted into the lumen of the carotid and 10⁵ SMCs were infused in the lumen and allowed to attach for 15 minutes. Nonadherent SMCs were flushed out, and the arteriotomy was ligated. One day after cell seeding, 18% of the original SMCs remained attached to the artery wall. Histology at day 1 shows roughly a two-cell-layer thickness of seeded SMCs covering 75% of the internal elastic lamina. This declined at 1 week to 45% of the day 1 value, and then remained stable at 2 weeks (data not shown). Animals were cared for according to the Principles of Laboratory Animal Care (formulated by the National Society for Medical Research) and the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86 to 23, revised 1985).

Histochemistry
Seeded rats were killed and the carotid vessels perfusion fixed with 4% (wt/vol) paraformaldehyde in PBS, pH 7.4. Histological sections (4 µm) were cut from paraffin embedded rat carotid arteries prepared and placed on poly-L-lysine–coated microscope slides. The slides were stained with hematoxylin and eosin or modified Movat's stain, which uses Weigert's hematoxylin (alcoholic hematoxylin, ferric chloride, iodine) to stain elastic fibers black.

Northern Analysis
Total RNA was extracted from 2-week control- and TIMP-1–seeded rat carotid arteries by using 4.2 mol/L guanidine isothiocyanate¹¹ (six arteries per group). Twenty micrograms of each RNA sample was size fractionated by electrophoresis, transferred to a nylon membrane, and hybridized with a [³²P]-labeled rat tropoelastin cDNA (a generous gift of Dr C. Boyd, University of Medicine & Dentistry of New Jersey, New Brunswick). Hybridization and washes were carried out at 65°C according to Church and Gilbert.¹² Blots were hybridized in 1% bovine serum albumin, 7% SDS, 0.5 mol/L sodium phosphate (pH 7.0), 1 mmol/L EDTA, 20% deionized formamide, 200 µg/mL denatured salmon sperm, 10⁶ cpm/mL probe at 65°C for 18 hours. The blots were washed to final stringency in 1% SDS, 40 nmol/L sodium phosphate (pH 7.0), 1 mmol/L EDTA at 65°C, analyzed on the PhosphorImager (Markey Molecular Medicine Center), and quantified with ImageQuant Version 3.3 (Molecular Dynamics).

Elastin Synthesis in Organ Culture
Rats were killed at various times postseeding and their seeded carotids removed. Subsequently, these vessels were stripped of their adventitial layers and incubated individually in Dulbecco's modified Eagle's medium with 10% fetal calf serum (Hyclone) containing ¹⁴[C]proline (0.5 µCi/mL medium, DuPont–New England Nuclear), and ascorbic acid (0.25 mg/mL medium, ICN Biomedical) for 6 hours. After this labeling period, vessels were removed from the media and incubated individually with 50 mg/mL cyanogen bromide (prepared in 70% formic acid) under N₂ for 24 hours. Insoluble elastin was hydrolyzed in 6N HCl at 110°C for 24 hours and counted in a scintillation counter.¹³ Results were normalized to vessel length at time of harvest.

Desmosine Measurement
Vessel lengths were measured, and the tissue was freeze-dried and weighed. After hydrolyses in 6N HCl at 105°C for 24 hours, the hydrolysates were evaporated to dryness and redissolved in 1 mL of distilled water. Aliquots from each sample were analyzed for hydroxyproline on a Beckman 6300 amino acid analyzer. Desmosine content was measured by radioimmunoassay as described previously.¹⁴

Elastin Zymography
Seeded arteries were homogenized with a polytron homogenizer in an extraction buffer containing 50 mmol/L Tris, 0.2% Triton X-100, 10 mmol/L CaCl₂, and 2 mol/L guanidine hydrochloride, pH 7.5.¹⁵ The extracts were centrifuged at 14 000g for 5 minutes and the supernatants dialyzed overnight against 0.2% Triton X-100, 50 mmol/L Tris, pH 7.5. The protein content of the extract was determined using the BCA method according to the manufacturer's recommendations (Pierce Inc). Samples containing an equal amount of protein (20 µg per lane) were electrophoresed on a 10% SDS-polyacrylamide gel containing 1 mg/mL soluble elastin (ETNA-Elastin; Elastin Products) under nonreducing conditions.¹⁶ After electrophoresis, the gels were washed in 2.5% Triton X-100 for 30 minutes to remove SDS. The gels were then incubated at 37°C in 50 mmol/L Tris buffer, pH 7.8, containing 10 mmol/L CaCl₂ for 69 hours. The gels were stained with 0.002% Coomassie brilliant blue (Sigma Chemical Co).¹⁷

	Results

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Histochemical Examination
Movat's staining of cross-sections of balloon-injured/seeded rat carotid arteries at 2 weeks demonstrated markedly increased elastin staining in the intimas of TIMP-1 cell-seeded animals (Fig 1). We did not detect any difference in elastin staining in the intima at 2 days after seeding; however, there was a small increase in elastin staining at 7 days postseeding (data not shown). The neointima in this model of balloon injury/seeding is a mixture of seeded SMCs overexpressing the gene of interest, TIMP-1, and SMCs originating from the medial compartment following balloon injury.¹⁸

View larger version (150K): [in this window] [in a new window]   Figure 1. Elastin accumulation in 2-week balloon-injured rat carotid arteries seeded with control (A) or TIMP-1 (B) cells. Sections were stained with Movat's stain containing Weigert's hematoxylin, which has been used historically to stain elastin fibers black. Arrows indicate internal elastic lamina (magnification x125).

Measurement of Elastin mRNA and Rate of Elastin Synthesis
To assess the tropoelastin mRNA expression in TIMP-1 and control vessel, we performed Northern analysis on extracts from seeded arteries by using a rat tropoelastin cDNA as the probe. There were no significant differences in tropoelastin mRNAs at 14 days between the two groups (Fig 2A) or at 2 days and 7 days (data not shown), suggesting that constitutive and local overexpression of TIMP-1 does not regulate steady state levels of tropoelastin mRNA.

View larger version (35K): [in this window] [in a new window]   Figure 2. A, Expression of tropoelastin in seeded rat carotid arteries. Northern analysis of total RNA isolated from arteries at 2 weeks after seeding with control (lane 1) or TIMP-1 (lane 2) cells and probed with a cDNA encoding rat tropoelastin (upper panel). The membrane was reprobed with a cDNA encoding 28S ribosomal RNA to normalize for RNA loading (lower panel). PhosphorImager quantification of blot shows no significant difference in tropoelastin mRNA expression. B, Elastin synthesis per 1-cm segment of seeded rat carotid arteries. Newly synthesized elastin was measured at different times postseeding, as described in "Methods." There was no significant difference in the rate of newly synthesized elastin between the experimental (TIMP-1) and control groups (Control) at indicated times. P>.05, n=9, mean±SD.

The effects on elastin synthesis of balloon injury and subsequently seeding the vessels with TIMP-1 or control cells were measured using organ cultures of arteries pulsed with ¹⁴[C]proline (Fig 2B).¹³ Balloon injury/seeding resulted in a significant increase in elastin synthesis over the uninjured/unseeded carotid arteries for at least 28 days postseeding. The rate of elastin synthesis at various times following balloon injury/seeding was not different between the TIMP-1–seeded and the control-seeded vessels.

Measurement of Elastin
Elastin was measured using a radioimmunoassay specific for desmosine, an amino acid cross-link present only in mature elastin. The effect of balloon injury/seeding on the desmosine content of arteries 14 days postoperation is shown in the Table. Vessels that were injured but not seeded with cells showed an elevated but not a statistically significant increase in desmosine content compared with uninjured vessels. Vessels that were injured and seeded with control cells showed the same increase in desmosine that was observed in the injured but not seeded vessels. Vessels that were injured and seeded with TIMP-1 cells showed a significant increase in desmosine content compared with control vessels. Changes in intimal elastin content was obtained by subtracting the uninjured right carotid media elastin from the total for the seeded vessel (Table), since the elastin content of normal right and left carotid arteries were the same (data not shown). Based on this calculation, we conclude that TIMP-1 overexpression was associated with at least a doubling of intimal elastin.

Elastase Activity
The presence of elastase was determined in arterial extracts using SDS gels containing soluble elastin. While we observed bands of elastase activity at 72 kD (MMP-2) in both the control and TIMP-1–seeded vessels, there was no apparent quantitative difference between the two groups at either 2 days or 14 days post-seeding (Fig 3). An elastolytic band at 28 kD with much higher activity in the control-seeded than the TIMP-1–seeded vessels at 2 days after seeding. This low-molecular-weight enzyme was completely inhibited by 10 µg/mL TIMP-1, as well as 10 mmol/L EDTA treatment, but not 2 mmol/L PMSF, a serine protease inhibitor (data not shown).

View larger version (72K): [in this window] [in a new window]   Figure 3. Elastase activity in seeded rat carotid arteries. Elastin zymography shows MMP-2 (upper arrow) and 28-kD metalloelastase (lower arrow). Control, 2 days (lane 1); TIMP-1, 2 days (lane 2); Control, 2 weeks (lane 3); TIMP-1, 2 weeks (lane 4); cell-seeded arteries.

	Discussion

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We have previously described pharmacological experiments in which SMCs overexpressing TIMP-1 were used to control intimal hyperplasia after balloon catheterization and aneurysmal rupture after xenograft implantation.^{18 18A} In both instances, histological evidence suggested an increase in elastin in the TIMP-1 vessels compared with control vessels. These observations prompted us to test the hypothesis that MMPs induced by physical manipulation of vascular wall affect intimal elastin turnover and that this effect could in turn be modulated by elevated levels of TIMP-1.

The histochemical evidence in the present study showed a marked increase in elastin fibers in the TIMP-1–seeded vessels that was evident by 14 days postseeding. These observations were confirmed by the quantitative analysis of desmosine, which also showed a significant accumulation of elastin in the TIMP-1–seeded vessels after 14 days. In vitro studies showed that control-seeded vessels increased in the rate of elastin synthesis to the same extent as the TIMP-1–seeded cells compared with unseeded vessels. Tropoelastin message levels were also comparable between control-seeded and TIMP-1–seeded vessels. These findings indicate that injury to the intima of arteries accelerates elastogenesis. Under normal physiological repair conditions, catabolism of elastin by proteolytic enzymes maintains the elastin content of the vessel wall at near normal levels. However, when the intimal cells overexpress TIMP-1, the enzymatic repair process is attenuated and the elastin content of the vessel increases.

The molecular weight, kinetics of appearance, and EDTA sensitivity, as well as evidence showing that approximately 1% of the cells in balloon-denuded rat carotid artery are comprised of leukocytes including macrophages,¹⁹ suggest that the elastase involved in the vessel repair process is a macrophage metalloelastase. A polyclonal antibody against MMP-12 (generous gift of Dr S. Shapiro, Washington University, St Louis, Mo) gave a positive Western blot at 53 kD, corresponding to the reported size of the MMP-12 proenzyme.²⁰ However, this band did not change over time in experimental and control arteries, and the lower-molecular-weight form of MMP-12 (28 kD) was not identified on the blots (data not shown). Since both the secreted and processed forms of MMP-12 contain the catalytic domain, both bands should have been detected on the elastin zymogram. In a similar model of injury alone, we also have observed a low-molecular-weight MMP and have ruled out MMP-1, -3, -7, and -12 by various methods.⁹ Nonetheless, pure TIMP-1 was able to block the elastin-degrading property of the 28-kD molecule.

TIMP-1 overexpression might inhibit intimal thickening by blocking MMP activity needed for the migration of SMCs residing in the blood vessel wall. There may also be indirect effects on SMC migration and growth through the increased accumulation of elastin. Studies by Ooyama et al²¹ have shown that elastin can influence the biological responses of SMCs. Soluble elastin peptides induce a chemotactic response in SMCs, whereas the same preparation of elastin peptides in a filter-bound format inhibits SMC migration. This inhibitory effect was shown to be specific for SMCs, as the filter-bound elastin failed to block migration of different cell types, including polymorphonuclear leukocytes.²² These investigators also found that coating the cell-culture dishes with elastin blocked the phenotypic change of SMCs from the quiescent contractile to the proliferative synthetic type. Therefore, it is possible that intimal elastin could function to inhibit medial and intimal SMC migration and growth, thereby blocking intimal hyperplasia.

Taken together, these results support the hypothesis that TIMP-1 overexpression in balloon-injured arterial intima modulates proteolytic activity, resulting in an accumulation of matrix elastin, which contributes to the attenuation of intimal hyperplasia.

	Acknowledgments

 
This research was supported by NIH grants HL18645 and HL07312–15. We are grateful to Dr David Eyer, Department of Orthopedics, University of Washington, for helpful discussion. We also thank Dr C. Boyd for tropoelastin cDNA probe and Dr S. Shapiro for the antibody to MMP-12.

Received September 15, 1997; accepted December 15, 1997.

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