Mason et al, Matrix Metalloproteinase-9 Overexpression…

Materials and Methods

Materials

Male Fischer 344 rats (250-350 g) were obtained from Simonsen Laboratories (Gilroy,

CA). The full length cDNA for rat MMP-9 was obtained from Dr. Paul Basset

(INSERM/ CNRS/ Universite Louis Pasteur, De Strasbourg, France). The recombinant human PDGF-BB was kindly supplied by Zymogenetics, Inc., Seattle, WA, BB-94 by

British Biotech Pharmaceuticals Limited (Oxford, UK), and purified MMP-2 by William

Stetler-Stevenson (NIH). The Vitrogen collagen was purchased from Collagen

Corporation (Fremont, CA) and other chemical reagents were purchased from Sigma

Chemical Co. (St.Louis, MO). Bromodeoxyuridine (BrdU) and antibodies to BrdU and alpha smooth muscle actin were purchased from Boehringer-Mannheim Co. A rabbit antibody against MMP-9 used for western blotting was kindly provided by Dr. L. Jack

Windsor (University of Alabama at Birmingham, Birmingham, AL).

Gene Transfection

Fischer 344 SMCs were prepared by enzymatic digestion of aortas from male Fischer rats as described 1 and propagated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum, 200 g streptomycin/ml, and 200 U penicillin/ml

(GIBCO Laboratories, Grand Island, NY) in 5% C02 at 370C. Fischer rat SMCs at passage thirty were co-transfected with pSV2NEO as a selectable marker, plus a plasmid 2 consisting of tTA protein under the control of a promoter containing the Tet operator sequence, essentially as described by Shockett et al. 2. The tTA protein is a hybrid transcriptional activator that binds to the Tet operator sequence only when tetracycline is not present. The transfected cells express low levels of inactive tTA protein in the presence of tetracycline and high levels of active tTA protein in the absence of tetracycline. After selecting for survival in the presence of G418 (536 g/ml), we evaluated clones by transient transfection assay with a tetracycline dependent - galactosidase construct. We used the clone showing the lowest basal activity in the presence of tetracycline combined with the highest induced activity in the absence of tetracycline, as host for subsequent stable co-transfection with a hygromycin resistance plasmid and Tet-regulated expression vector with both MMP-9 and a nuclear-targeted myc-tagged -galactosidase reporter gene under control of tTA-dependent promoters.

After selection in hygromycin (100 g/ml), we evaluated subclones for tetracycline regulation of MMP-9 using gelatin zymography and western blotting. All studies reported here were performed with the clone [SMC(tTA-MMP-9)], which showed the best regulation by tetracycline.

Cell Culture

Cells were maintained in DMEM with 10% calf serum supplemented with 1 g/ml tetracycline (Tet+). For experiments comparing cells not expressing MMP-9 (Tet+) to those with MMP-9 overexpression (Tet-), cells were washed twice with phosphate buffered saline (PBS) and maintained for 48 hours in medium with serum but without tetracycline to induce MMP-9 . 3

SMC Proliferation

SMC (tTA-MMP-9) cells were plated into 12 well plates at a subconfluent density of 50 cells/mm2 and grown in 10% CS in the presence (Tet+) or absence (Tet-) of tetracycline.

Cells were trypsinized and counted using a hemocytometer daily for 5 days. The assay was repeated three times with four replicates in each experiment.

Zymography/Western Blot Analysis/Reverse Zymography

Individual carotid arteries were pulverized under liquid nitrogen and extracted for 15 minutes on ice using a buffer of 50mmol/L Tris, 0.2% Triton X-100, 10mmol/L CaCl2,

2.0 mol/L guanidine hydrochloride, pH 7.5 3. The extracts were centrifuged 15 minutes at 4oC and the supernatant was dialyzed twice against 10,000 volumes of 0.2% Triton X-

100, 50mmol/L Tris, pH 7.5. Protein was measured using bovine serum albumin as a standard. Equal amounts of protein (10-20g) per lane or of conditioned media from

Tet+ and Tet- cells grown under serum free conditions for 48 hours were loaded and the proteolytic activity assessed by zymography as previously described 4. To exclude the possibility that tetracycline might directly affect metalloproteinase activity, we incubated gels after electrophoresis with 0, 1,10,50, and 100 g/ml of tetracycline in Tris/Calcium buffer before staining. Western blotting was performed as previously described using anti-MMP-9 at 5g/ml 5.

For reverse zymography, gelatin (1mg/ml) and MMP-2 (0.30 g/ml) were incorporated into the polyacrylamide gel 6. The gel was washed and incubated as for 4 regular zymography. TIMP-1 and TIMP-2 activity was visualized as undigested dark bands stained by Coomassie blue against a background of lysis with baboon TIMP-1 serving as a standard 7.

SMC Migration - In vitro

Smooth muscle cell migration was assayed in two ways. The first assay was conducted in a 48-well microchemotaxis chamber (Neuro Probe Inc., Cabin John, MD) using polycarbonate filters with 10m diameter pores (Poretics Products, Livermore CA) and

PDGF-BB (10ng/ml) as a chemoattractant as previously described8. The filters were precoated with basement membrane gel matrix by submerging the filter overnight in a

solution of DMEM with diluted Matrigel at 130g/ml in 0.5X PBS (5mmol/L Na2HPO4,

1.5mmol/L KH2PO4, 60mmol/L NaCl). For conditioned media experiments, non- transduced Fischer rat SMCs were suspended in serum free media conditioned in the presence (Tet+) or absence (Tet-) of tetracycline for 48 hours by SMC(tTA-MMP-9) cells. The metalloproteinase inhibitor BB-94 () was used in the top chamber in some experiments 9. The chamber was incubated at 370 C for 5 hours. The cells which had migrated to the bottom portion of the filter were counted at 400X and expressed as percent of control to quantify migration. Experiments were repeated three times.

For longer term migration studies through a thick matrix, a 10% F-127 pluronic gel (Sigma) containing PDGF-BB (20ng/ml; 2ml/380mm2 well) was laid beneath a collagen (1ml Vitrogen) matrix . Approximately 90% of the pluronic gel contents are released in 51 hours 10. The pluronic gel was allowed to harden at 370 C for two hours 5 before adding the chilled Vitrogen (1ml) which was allowed to polymerize for 2 hours at

370 C. SMC (tTA-MMP-9) cells were trypsinized (0.05% trypsin), counted and suspended in 10% calf serum (Tet+ or Tet-) with 5mmol/L hydroxyurea (Sigma) to inhibit proliferation. BB-94 () was used in some experiments with DMSO controls.

Cells were plated on the surface of the Vitrogen at a density of 1.0x103cells/ mm2. After

48 hours, the media was removed and each well fixed with 100% methanol and stained with Diff-Quick stain. The number of cells which had migrated into the collagen was determined by phase microscopy in four random fields (100X) per well. The experiment was performed in duplicate eight times.

SMC Migration - In vivo

Male Fischer 344 rats (250-350) grams were anesthetized with an intramuscular solution

(1.0ml/kg) of ketamine hydrochloride (50mg/ml;Ketaset, Bristol Laboratories, Syracuse,

NY), xylazine (5mg/ml; Xylaject, Phoenix Pharmaceuticals, MI) and acepromazine

(1mg/ml, Fermenta Animal Health Co., Kansas City, MO). A segment of the left carotid artery extending from the most proximal portion of the extrathoracic common carotid to the bifurcation was isolated circumferentially and the adventitia gently stripped. Heparin

(100U/kg IV) was administered and an arteriotomy was made in the external carotid artery and the isolated segment flushed with saline. Parafilm was placed under the carotid artery to protect the surrounding tissue and a stainless steel micro-spatula chilled in liquid nitrogen was applied to the carotid artery for two seconds. Freezing was confirmed visually and 30 seconds allowed for thawing. This procedure was repeated three times to destroy all the vascular wall cells. The vessel was flushed through the 6 arteriotomy in the external carotid, which was then ligated and flow restored through the common carotid. A 0.5 ml suspension (2.5x106 cells/ml) of transduced cells or media alone was flooded over the vessel. The cells or vehicle alone were left undisturbed for 15 minutes at which point the wound was closed. One week following freeze/thaw treatment there are no endgenous cells left. To avoid lateral migration of SMC from uninjured regions, histological sections were only taken from the center of the injured area. Animal care and procedures were conducted at the University of Washington Medical Center in accordance with state and federal laws and under protocols approved by the University of

Washington Animal Care and Use Committee. Animal care complied with the

Principles of Laboratory Animal Care as formulated by the National Society for Medical

Research and the Guide for the Care and Use of Laboratory Animals issued by the

National Institute of Health (US Department of Health and Human Services, NIH

Publication No. 80-23, revised 1985).

Rats were treated with tetracycline (1mg/ml in 2.5% sucrose; 100mg/kg/day) in their drinking water or with vehicle alone. The metalloproteinase inhibitor BB-94, was given as a daily intraperitoneal injection (30mg/kg/day) 8 following adventitial cell seeding. Control animals received vehicle alone. Animals were sacrificed at seven days with an overdose of sodium pentobarbital (Anthony Products Co., Arcadia, CA) followed by perfusion fixation with 10% formalin. Paraffin-embedded cross-sections from the central portion of the injured segment were stained with Hematoxylin/Eosin or prepared for immunohistochemistry. The total number of cells which had migrated into the intima and media of the vessel were counted under high power. Immunohistochemistry using an 7 antibody to smooth muscle alpha-actin (Boehringer Mannheim Corp., Indianapolis, IN) was used to confirm that the migrated intimal and medial cells were SMCs.

SMC Seeding of Rat Carotid Artery Lumen

SMC seeding was performed as previously described1. Briefly, after administration of heparin (100U/kg IV), the left common carotid artery was injured with three passes of a 2

French Fogarty balloon catheter (Baxter Healthcare Corp., Irvine, CA). Approximately

1x105 transduced cells were infused into the isolated segment through a silastic catheter and the animal placed prone for 2 minutes and supine for 15 minutes. Previous experiments of seeding efficiency using SMCs labeled with 3H-thymidine have demonstrated that 19% of the infused cells (1.9x105) remain in the carotid segment at 1 day after seeding (unpublished data). These cells are distributed in 2-3 layers on the lumen surface. The catheter was removed, blood flushed through the external carotid to remove any free floating cells and the external carotid ligated. Blood flow was then restored through the common carotid artery. Rats were treated with tetracycline (1mg/ml in 2.5% sucrose; 100mg/kg/day) in their drinking water (Tet+) or with vehicle alone

(Tet-) beginning 2 days prior to surgery and were sacrificed at 7, 14 or 28 days. One group was treated with tetracycline for 14 days and then tetracycline was removed for the subsequent 14 days (Tet+/Tet-).

Morphometry

Animals were euthanized with an overdose of sodium pentobarbital and the carotid segments carefully dissected free of surrounding tissue. These were immediately frozen 8 for protein analysis and zymography or rinsed with Ringer’s Lactate and perfusion fixed in situ using 10% formalin at 110 mmHg. Cross sections of the paraffin embedded vessels were stained with Hematoxylin/Eosin and morphometric analysis performed using a camera lucida linked to a computer-driven digitizing pad and software (OPELCO,

Washington, DC) or used for immunohistochemistry. The intimal, medial, lumenal areas and wall thickness were calculated for each cross section as well as lumenal index

(lumenal diameter/cross sectional wall thickness).

Fields from four separate regions of intima per cross section were examined under high power (X1000). Two of these fields were adjacent to the basement membrane and two adjacent to the lumen. The average nuclear density for the neointima for each animal was determined. The total number of intimal cells was calculated by multiplying the intimal area by the nuclear density. Movat’s staining was performed to evaluate the extracellular matrix composition.

For electron microscopy, carotid arteries were perfusion fixed in 4% paraformaldehyde at 110 mmHg and embedded in epoxy resin. Transmission electron photomicrographs of non-overlapping fields at a final magnification of 7,000X were taken in three random sections of each intima from the luminal to abluminal margins as described previously 11. The point-hit method was used to determine the amount of carotid intima occupied by either SMCs or surrounding extracellular matrix as previously described 12. A grid with 120 points of intersection was placed over the photographs.

The number of points which lay over SMCs or extracellular matrix was counted and the area of SMCs or extracellular matrix was determined by the equation: % Matrix =[points matrix/(points Matrix+ Points SMC)] x 100. 9

Measurement of SMC Proliferation in Vivo

A 50 mg tablet containing BrdU (Boehringer Mannheim Corp.) was placed subcutaneously in the lumenally seeded animals 24 hours prior to their sacrifice at 7 days.

The BrdU labeling index (fraction of labeled nuclei x 100) was determined by use of a monoclonal antibody against BrdU (Boehringer Mannheim Corp.) on histologic cross sections as previously described 13.

Statistical Analysis

Results were expressed as mean + SEM. Statistical significance was evaluated by the

Wilcoxon Signed Rank Test for in vitro comparisons of migration between Tet+ and

Tet- groups. The Mann-Whitney nonparametric test was used to evaluate all in vivo experiments. Differences of P<0.05 were considered significant.

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