Materials and Methods
Periadventitial collar injury of carotid artery
Wild type and iNOS knockout mice (B6/129 background, male, 5 weeks of age, 18 to 20 g, Jackson Laboratory, Bar Harbor, Me) were fed regular chow throughout the duration of the experiment. At the age of 25 weeks, mice were anesthetized with Enflurane inhalation and right carotid artery was dissected and a non-occlusive, soft and flexible plastic cuff (3 mm long, 0.51 mm internal diameter; Cole-Parmer Instrument Co., Vernon Hills, Il) was placed around the carotid artery. Mice were sacrificed at 3, 7 or 21 days after injury and carotid artery was perfused with 0.9% saline for 10 minutes and frozen at –70 OC after embedding in O.C.T. compound (Tissue-Tekâ, Allegiance). Contralateral non-injured carotid artery was used as control. Serial 4 mm-thick arterial sections were used for staining. This experimental protocol was approved by the Institutional Animal Care and Use Committee. Intravascular thrombosis after H & E staining was observed in only one wild type mouse.
Morphometric measurement
Sections from middle half of injured segment were collected. This is because the anatomy of the sections from cut ends was occasionally distorted when vessel was removed from the cuff. Serial 4 mm-thick sections with averaged 4 mm intervals were obtained. Four sections were collected on each slide and 20 – 25 slides were collected for each arterial segment. Slides were sequentially divided into 3 groups and first three slides from each group were stained with hematoxylin and eosin. Computer-assisted morphometric analysis was done with an image analysis software (Optima 5.1, Bioscan) on these sections. Measurement of sections from these slides from each animal were averaged and analyzed.
Isolation and culture of vascular smooth muscle cells
Vascular smooth muscle cells isolated from the aorta of male Sprague-Dawley rats were grown at 37oC in an humidified incubator with 5% CO2 in DMEM/F-12 medium supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine and 1% penicillin/streptomycin. After confluency, culture medium was changed to serum free DMEM/F-12 containing 0.1% albumin, 2 mmol/L L-glutamine and 1% penicillin/streptomycin 48 hours prior to injury.
In vitro Injury protocol
For in vitro cell injury experiment, a sterile rubber tube was gently pressed onto the surface of cultured VSMC for 10 seconds.14 For Western blot analysis and immunohisto-chemical staining for iNOS protein, injured cells were harvested at 24 or 48 hours after injury. For electrophoretic mobility shift assay (EMSA) and determination of cytosolic levels of NF-kB, cells were injured at 30 minutes, 2 hours and 6 hours prior to harvest.
Cytosolic and nucleoprotein extraction
Cytosolic and nuclear protein were extracted as described previously1. Protein concentrations were determined by Coomassie Plus Protein Assay (Pierce, Rockford, Il.).
Western blot analysis
For detection of iNOS protein after cell injury, cytosolic protein was subjected to electrophoresis on 7.5% SDS-PAGE gels. For the detection of p50, p65 and IkB protein, cytosolic protein underwent electrophoresis udsing 12% SDS-PAGE gels. The membrane was probed with rabbit polyclonal iNOS, p50, p65 or IkB antibody (Santa Cruz Biotechnology, Santa Cruz, CA) followed by HRP-conjugated anti-rabbit antibody. Detection was performed according to the ECL protocol (Amersham, England). Peptide blocking experiments were performed to confirm specificity.
Electrophoretic mobility shift assay
The oligodeoxynucleotide used in EMSA incorporated the published sequence between –113 and –92 bp in the rat iNOS promoter containing the NF-kB binding site (underlined, 5’-3’ CCTACTGGGGACTCTCCCTTTG).15 The oligodeoxynucleotide was annealed with its complementary sequence and labeled with [32P] ATP and T4 kinase. Six mg of nuclear extracts were used for EMSA as described previously.1 Specificity was determined by adding 100x excess of unlabelled oligodeoxynucleotide. The DNA-protein complexes were separated on a 6% polyacrylamide gel in low strength buffer and the dried gel exposed to film. For supershift assay the antibody against NF-kB p65 subunit or p50 NLS subunit (15mg/ml, Santa Cruz Biotechnology) was added to the extracts for 10 min before addition of radiolabelled probe.
Immunohistochemical staining
VSMC or frozen sections of arterial segments were fixed in 2% paraformaldehyde in PBS for one hour or in ice cold 100% acetone for 5 minutes, respectively. For PCNA immunoreactivity, arterial sections were fixed in 1% paraformaldehyde in PBS for 2 minutes, and in methanol at –20oC for 10 minutes. Immunohistochemical studies were performed using standard technique with the following antibodies: Anti-iNOS, anti-eNOS antibody, anti-PCNA (all rabbit polyclonal and from Santa Cruz Biotechnology unless indicated), rat monoclonal anti-MOMA-2 (Serotec, Raleigh, NC) and rat monoclonal anti-mouse VCAM-1 antibody (Pharmingen, San Diego, CA). Negative controls included non-immune isotype antibody or omission of the primary antibody. Mucosal cells in sections of intestinal villi from wild type mice were used as positive control for PCNA staining.
For immunohistochemical staining, arterial sections were collected similarly to the collection of sections for neointimal morphometric analysis. Semi-quantitative measurement of VCAM-1 or MOMA-2 antibody stained area from 4 sections per animal was done with computer-assisted analysis as described previously.16 Data were presented as averaged percentage of VCAM-1 or MOMA-2 positive area standardized against medial area. Proliferating cells in the medial layer were identified by PCNA positive nuclei counted in each section and expressed as stained cells per section. Two observers were blinded to the animal group labels and the inter-observer variability was less than 5%.