Materials and Methods

Morphometry: Mice were anesthetized by ketaset (80 mg/kg) and xylazine (5 mg/kg) and were perfused with saline. For aortic tree analysis, the entire aorta from the heart to 5-10 mm below the bifurcation of the iliac arteries and including the subclavian, right and left carotid arteries, were removed, dissected and fixed in 3% paraformaldehyde (PFA). The aorta was evaluated for lesion development by en face Oil Red O staining and morphometry of digital images of the stained aortas was performed using the software Adobe Photoshop 7.0. For aortic root analysis, the hearts were removed and fixed in 3%PFA. Paraffin embedded sections of 10 m thickness were obtained from the region of the proximal aorta to the level of the aortic leaflet and were stained using hematoxylin and eosin. Digital microscopic images were analyzed using image analysis software for Apple Macintosh Computers (NIH Image 1.63). Lesion size for each mouse was calculated as the average lesion size in 10-15 sections over a distance of 200-300 m in the aortic root.

Bone marrow transplantation studies: Bone marrow cells were collected from the femora and tibias of donor mice and was reconstituted in Iscove’s modified Dulbecos media (Gibco Invitrogen Corp, Carlsbad, CA), as previously described1. Recipient mice were lethally irradiated with a total dose of 900 rads. After 4 hours, the recipient mice received bone marrow from either Trk B(+/+)|Apo E(-/-) or Trk B(+/-)|Apo E(-/-) mice by tail vein injection. Each mouse received 2 x 106 bone marrow cells in 200 ml media. After a 4 week recovery, surviving mice were placed on the high fat Western diet described in the manuscript. After 12 weeks, the hearts were obtained for analysis of lesion development in the aortic root as described above.

In a separate cohort of mice, four weeks following bone marrow transplantation, mice were injected intraperitoneally with 3% Brewer Thioglycollate Medium containing 0.3 mM thioglycollate (DIFCO, Detroit, MI) to induce peritoneal accumulation of macrophages, as previously described2. After 4 days, peritoneal macrophages were isolated, genomic DNA was extracted, and PCR analysis for the Trk B positive allele and the Trk B negative allele was performed, using primers designed by Jackson Laboratories.

Immunohistochemistry: Murine Hearts were removed and cryopreserved in 30% sucrose:OCT (Sakura Finetechnical, Torrence, CA) (1:1). Serial frozen sections were obtained by cryostat sectioning at the level of the aortic leaflet. Human and murine frozen sections were air-dried onto microscopic slides and endogenous peroxidase activity was quenched with 0.1% H2O2 in methanol for 30 minutes. Adjacent sections were incubated with either anti-Trk B antibody directed against the extracellular domain (rabbit polyclonal, H-181, Santa Cruz Biotechnology, Santa Cruz, CA), anti-BDNF (chick polyclonal, R&D, Minneapolis, MN), anti-smooth muscle cell a-actin (mouse monoclonal, clone 1A4, Dako Corporation, Carpinteria, CA or R&D), anti-human macrophage (monoclonal, clone Ham-56, Dako Corp) or anti-mouse monocytes/macrophages (Rat monoclonal, MOMA-2, Chemicon International, Temecula, CA). For studies of murine atherosclerotic lesions, the anti-smooth muscle cell -actin antibody was biotinylated using the Animal Research kit (BT-anti-smooth muscle cell -actin; DAKO Corporation) prior to use. Sections were incubated with purified IgG or IgY at comparable concentrations. Following incubation with the appropriate biotinylated secondary antibody (Vector Laboratories, Burlingame, CA), immunoreactive proteins were detected using avidin-biotin based horseradish peroxidase system, using Vector VIP as a chromogenic substrate (Vector laboratories), followed by counterstaining with hematoxylin. Quantification of smooth muscle cell, macrophage and collagen content was performed using the software Adobe Photoshop 7.

Double immunofluorescence: Cryopreserved sections were air dried and incubated with the anti-Trk B antibody (rabbit IgG as the negative control) or the anti-BDNF antibody (chicken IgY as the negative control) as described above. Following incubation with the appropriate biotinylated secondary antibodies, immunoreactive Trk B or BDNF were detected using rhodamine-conjugated avidin. After reblocking with the avidin/biotin blocking kit (Vector Laboratories), murine sections were incubated with either BT-anti-smooth muscle cell -actin or a FITC-conjugated anti-mouse macrophages/monocytes antibody (rat monoclonal, MOMA-2; Serotec Ltd. Oxford, UK). Sections were incubated with either BT-mouse IgG (Santa Cruz Biotechnology) or a FITC-conjugated rat IgG (Serotech Ltd) as a negative control, as indicated. Immunoreactive smooth muscle cell a-actin was detected using FITC-conjugated avidin. After incubation with rhodamine/avidin, human sections were incubated with either anti-smooth muscle cell a-actin, anti-human macrophage (HAM56), or mouse IgG. Immunoreactive smooth muscle cell a-actin or Ham-56 antigen were detected using FITC-conjugated anti-mouse IgG (Vector Laboratories). Immunoreactive proteins were visualized by fluorescent microscopy and digital images were obtained using an Olympus microscope. Quantification of colocalization of Trk B with either smooth muscle cell a-actin or the MOMA antigen in murine atherosclerotic lesions was performed using the software Adobe Photoshop 7.

Migration Assay: Temperature-sensitive mouse smooth muscle cells 3 were transfected with a plasmid expressing the cDNA for Trk B using the Amaxa nuclear Transfection system. Control cells were transfected with plasmid alone. The cells were cultured overnight at 33oC, then moved to 39.5oC and quiesced for 18 hours by culturing in 0.5% FCS. Migration assays were performed using a transwell filter 48-well chamber microchemotaxis apparatus (Neuroprobe Inc.) as previously described3. Briefly, a cell suspension was obtained by incubating the cells in PBS/EDTA for 5 min, after centrifugation, the cells were resuspended in DMEM containing 0.5% FCS at a concentration of 300,000 cells/ml. BDNF (10 and 25 ng/ml; murine; Promega) or PDGF-BB (10 and 30 ng/ml; recombinant human; R&D) were resuspended in 0.5% FCS and placed in the lower chamber. Cells (50 ml) were added to the upper chamber, over a polycarbonate polyvinylpyrrolidone-free 8-mm pore membrane (Poretics Corp) coated with nondenatured rat tail collagen (Biomedical Technologies), and incubated for 4 hours at 37oC in 95% air/5% CO2. Cells attaching to the upper surface were removed by scraping, and cells migrating through the pores were fixed with 3% PFA. Trk B immunoreactivity was detected by incubation with a Pan-Trk Antibody (Rabbit IgG, C-14, Santa Cruz Biotechnology), followed by incubation with a biotinylated anti-Rabbit IgG and Rhodamine-conjugated avidin. The cells were counterstained with DAPI. Migrating cells were visualized by fluorescence microscopy and manually counted. The results are expressed as the number of total cells/well, with 3 replicates per experimental group.

Immunoprecipitation/Western blot analysis: Aortas from Apo E (-/-) mice wild type and haplodeficient for Trk B expression were homogenized with radioimmune precipitation buffer containing phenylmethylsulfonyl fluoride (1 mM), aprotinin (1 mg/ml), leupeptin (10 mg/ml), and sodium vanadate (1 mmol/liter). Protein lysates were immunoprecipitated with the Pan-Trk antibody using protein A-sepharose. Immunoreactive proteins were separated by SDS-PAGE and Western blot analysis was performed using an anti-Trk B antibody (monoclonal Clone 47; BD Biosciences). Immunoreactive proteins were detected using enhanced Chemiluminescence (Amersham) with an anti-mouse IgG conjugated to horseradish peroxidase (Chemicon Corp).


REFERENCES

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2. Khan KM, Howe LR, Falcone DJ. Extracellular matrix-induced cyclooxygenase-2 regulates macrophage proteinase expression. J. Biol. Chem. 2004;279:22039-22046.

3. Kraemer R, March KL, Hempstead BL. NGF activates similar intracellular signaling pathways in vascular smooth muscle cells as PDGF-BB but elicits different biological respones. Arteriosclerosis, Thrombosis and Vascular Biology. 1999;19:1041-1050.