Materials and Methods s16

Materials and Methods

Cells culture

Rat VSMCs cultures were established from the thoracic aorta of 8-week-old male

Sprague-Dawley rats by a modified explant method. Cultures were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical, Tokyo, Japan) supplemented with

10% fetal calf serum (FCS). Cells were identified as VSMCs by immunocytochemistry using a monoclonal antibody that recognizes smooth muscle -actin (DakoCytomation, Kyoto,

Japan). The cells were incubated at 37C in a 5% CO2 humidified atmosphere and maintained at a sub-confluent stage by passaging with 0.025% trypsin / 1 mmol/L EDTA (Gibco-BRL,

Invitrogen, Carlsbad, CA).

Construction of a recombinant TP/PD-ECGF phagemid and gene transfer

We cloned the full length of the human TP/PD-ECGF cDNA, restricted from pBluescript

II SK (+) /TP (a gift from Roche Diagnostics, Tokyo, Japan) as a 1571-bp EcoRI-EcoRI fragment, into the corresponding sites in the PBK-RSV “backbone” of a phagemid expression vector (Stratagene, La Jolla, CA); this construct was named pTP. Either pTP or control PBK-

RSV vector was transfected into VSMCs using DOTAP transfection reagent (Biontex,

Munich, Germany). Expression of TP/PD-ECGF by cells was detected by immunoblot analysis and by a TP/PD-ECGF activity assay. Thirteen clones that overexpressed TP/PD-

ECGF were selected using geneticin (G-418 sulfate, Wako, Osaka, Japan). All of these VSMC clones overexpressing TP/PD-ECGF showed a slower growth rate than empty vector- transfected control cells (pC). Clone 2 (C2) cells and the pC cells were used for the following experiments. 2

Protein isolation, Western blotting, and TP/PD-ECGF activity analysis

Cells were washed twice with ice-cold PBS, and then were lysed with lysis buffer (50 mmol/L Tris-HCl, pH 6.8, 1% Triton X-100, 2 mmol/L phenylmethylsulfonyl fluoride, and

0.02% mercaptoethanol). Protein concentrations were determined using a standard colorimetric protein assay (Bio-Rad Laboratories, Philadelphia, PA). Twenty micrograms of total protein were separated electrophoretically in a 10% sodium dodecyl sulfate (SDS)- polyacrylamide gel, and then transferred electrophoretically to nitrocellulose membranes.

After blocking the membranes with 5% skim milk in Tris-buffered saline containing 0.1%

Tween-20 for 2 hr at room temperature, blots were incubated overnight at 4C with TP/PD-

ECGF antibody (at a 1:400 dilution, 654-1 mouse monoclonal anti-human antibody; a gift from Roche Diagnostics). The membranes were washed and then incubated for 30 min with horseradish peroxidase-conjugated goat anti-mouse antibody (Envision/HRP kit;

DakoCytomation). Blots were developed using enhanced chemiluminescence ECL

(Amersham, Piscataway, NJ); and relative protein content was quantified by scanning densitometry (ATTO, Tokyo, Japan). TP/PD-ECGF activity was detected as described previously.1

MTT assay

Cells were seeded at a density of 1.5 × 103 per well in 96-well plates; nine wells were used for each set of conditions. After treatment with various agents, the cell proliferation rate was assessed by a slightly modified 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT; Sigma Chemical, St. Louis, MO) assay as originally described by Mosmann.2

In brief, at the end of the experiments, cells were washed with phosphate-buffered saline 3

(PBS) three times and MTT (5 mg/mL in PBS) was added to the phenol red- and serum-free medium to result in a final concentration of 0.5 mg/mL; then incubation was continued at

37C for 4 hr. Experimental reactions were terminated, and the chromophore was dissolved by overnight exposure of the reaction mixtures to 100 l acid-isopropanol (0.04 N HCl in isopropanol) in darkness. Absorbance in the wells was measured at 570 nm with illumination at 630 nm using a microtiter plate spectrophotometer (SPECTRA MAX 250, Molecular

Devices, Sunnyvale, CA). Background absorbance was subtracted from all readings.

TP/PD-ECGF effect on proliferation of VSMC

Cells were seeded and cultured for 24 hr, followed by serum-withdrawal for 48 hr to synchronize them. The media were then changed to media containing 0%, 2.5%, 5%, or 10%

FSC in the presence or absence of 200 µmol/L of an inhibitor of TP/PD-ECGF (TPI; TAIHO

Pharmaceutical, Tokyo, Japan) for further incubation for 24 hr. The serum-stimulated proliferation of cells was quantified with the MTT assay. In another experiment, wild type

VSMC were cultured for 24 hr followed by synchronization for 48 hr. The media were then changed to media containing 10% FCS, and containing 100, 50, 25, 12.5 or 6.25 mg/ml of cell lysate (C2, pC or wild VSMC lysate), and incubated for further 24 hr, the proliferation was quantified with the MTT assay. To determine the effect of HO activity on VSMC proliferation, cells were seeded at a density of 1.5 x 104 cells per well in 6-well plates and cultured for 24 hr, following synchronization for 48 hr. The media were then changed to media containing 10% FCS in the presence or absence of 10 µmol/L tin-mesoporphyrin

(SnMP, Porphyrin Products, Logan, UT), an inhibitor of HO activity, and incubated for further

24 hr. The cells were counted using the trypan blue exclusion assay. 4

Proliferation of VSMC during hypoxia and analysis of apoptosis

When cells reached 60% to 70% of confluence, media were changed to media containing

10% FCS and 0, 75, 150, 300, 600, or 1200 µmol/L of CoCl2 (Sigma) in the presence or absence of 200 µmol/L TPI, for further incubation for 24 hr. The proliferation of cells was assessed by the MTT assay. To assay for apoptosis, cells were seeded at a density of 7.5 x 105 per 10-cm plate and allowed to grow to 60% to 70% of confluence. Media then were changed to media containing 10% FCS containing 0, 150, 300, 600, or 1200 µmol/L of CoCl2 for further incubation for 24 hr. Cells were harvested with trypsin-EDTA, and DNA was isolated for DNA ladder detection using an apoptotic DNA ladder kit (Roche Diagnostics GmbH,

Mannheim, Germany) as described by the manufacturer.

VSMC migration and wound regeneration assay

Cell migration assays were performed using QCMTM-FN kit according to the protocol from

Chemicon (Chemicon International, Temecula, CA) with minor modification. Cells (2.5 

104) were added to the top of each Boyden chamber (8 m pores) containing fibronectin- coated membranes and with the lower chamber containing 300 l of migration buffer

(DMEM containing 5% bovine serum albumin). Cells were allowed to migrate through the 8

m size pores to the other side of the membrane for 24 hr, and migratory cells were then stained. The stain was extracted from the cells using an extraction buffer and the absorbance at 550 nm was determined. In another study, 5 parallel horizontal lines and 5 parallel vertical lines were drawn on the bottom of 10-cm plates, and the 16 squares in the center of the plate were numbered. Cells were seeded at a density of 7.5 × 105 cells per 10-cm plates in DMEM media supplemented with 10% FCS. When the cells reached confluence, the medium was 5 refreshed with serum free DMEM and maintained for 24 hr to synchronize the cells. The confluent monolayer of synchronized cells was scratched with a pipette tip, thereby causing four parallel lesions, each about 670 m wide, on the monolayer. Following the injury, the plates were washed with serum free media three times to dislodge any cellular debris, and then images of the numbered denuded areas were captured with a Nikon Diaphot inverted microscope using a 4 x objective, coupled to a CCD, and acquired using Q500 Leica software.

These images served as time zero (T0) images. The cells were refreshed with DMEM containing 10% FCS and 1 g/mL actinomycin-D and cultured for an additional 24 hr. At 24 hr post injury, the experiments were stopped and the same areas were re-photographed and the images served as time 24 hr (T24) images. The experiments were repeated three times. Ten numbered fields were selected randomly for statistical analysis. The lesion area of each field was measured using ImageJ software (ImageJ 1.32j, Wayne Rasband, NIH), and the percentage of regeneration was calculated as (T0-T24)/T0. 3

Protein isolation and Western analysis

Cells were allowed to grow to 60% to 70% of confluence, and treated with various agents as outlined in the Results. Cells were washed twice with ice-cold PBS, lysed in electrophoresis buffer containing 125 mmol/L Tris at pH 6.8, 12.5% glycerol, 2% SDS, and a trace amount of bromophenol blue, and boiled for 5 min. Protein concentrations were determined, and 20 g of total protein was separated electrophoretically and then transferred to nitrocellulose membranes. Blots were incubated overnight at 4C with rabbit anti-HO-1 antibody (1:200 dilution; Cat. No., 374089, Calbiochem-Novabiochem, San Diego, CA) or antibodies against heat shock protein (HSP) 25 (1:400 dilution; rabbit anti-HSP25 polyclonal antibody, Stressgen Biotechnologies, Victoria, BC, Canada), p27KIP1 (1:200 dilution; BD 6

Biosciences, San Jose, CA), or cyclin D1 (1:200 dilution; BD Biosciences), and developed as described above.

HO activity assay

Cells were seeded at a density of 105 cells per well in 6-well plates, and allowed to grow for 24 hr. After the cells were synchronized for 24 hr, the media were changed to 600 l of the media as described in the Results, and incubated for another 48 hr. An aliquot of the media

(500 l) was used for HO activity analysis. The extraction of bilirubin was determined as a

1 1 difference in absorbance between 450 and 600 nm (ε450: 27.3 mM− , cm− ) as described by

Turcanu et al.4

Cell cycle analysis

Cells were seeded at a density of 105 cells/ 25-cm2 flask and allowed to grow for 48 hr before media were exchanged for serum-free media. Cells then were incubated for an additional 24 hr. These synchronized cells then were treated with media containing 10% FCS for 24 hr, and then dispersed with trypsin / EDTA, washed in PBS, and centrifuged at 1000 rpm for 5 min to collected cells. The PBS was removed, and cells were re-suspended in 0.2%

Triton X-100 in PBS to expose nuclei, and filtrated through a nylon mesh filter. A final concentration of 0.5 mg/mL RNase was added, and nuclei were stained with 25 g/ml propidium iodide. DNA contents were analyzed by flow cytometery (EPICS XL, Beckman

Coulter, Miami, FL).

In vivo experiments 7

Fourteen male Sprague-Dawley rats (290 to 310 g; Sankyo Labo Service, Toyama, Japan) were anesthetized with pentobarbital (50 mg/kg, IP), and the right carotid artery was isolated through a middle cervical incision, suspended on ties, and stripped of adventitia. The distal right common carotid artery and region of the bifurcation were exposed. A 2F Fogarty balloon catheter (Edwards Lifesciences, Irvine, CA) was introduced through the external carotid artery, and advanced into the aorta. The balloon was inflated with 0.2 ml of air to distend the common carotid artery and pulled back to the external carotid artery. This procedure was repeated 3 times. This procedure completely removes the endothelium, and there is some injury to the medial smooth muscle layers throughout the common carotid artery. After removal of the catheter, the external carotid artery was ligated. For gene transfection, a chitosan hydrogel containing pCIhTP or pCILacZ plasmid and heparin was placed around the common carotid arteries and the wound closed. The left carotid artery was not damaged and served as a control. Rats were killed by an intraperitoneal overdose of pentobarbital 14 days later for measurement of neointima formation. The use of animals was in compliance with the

Guidelines of the Institutional Animal Care and Use Committee of the Faculty of Medical

Sciences, University of Fukui, and conforms to the Guide for the Care and Use of Laboratory

Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). 8

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