The Role of P38a MAPK in Atherosclerosis Figure 4. P38a ablation in macrophages does not affect atherosclerosis development. (A) Southern blotting on protein extracts from p38aMY-KO/ApoE2/2 and ApoE2/2 BMDMs to analyse p38a deletion efficiency in macrophages. P38aMY-KO/ApoE2/2 mice, n = 3; ApoE2/2, n = 3. (B) LPS (1 mg/ml, Sigma) stimulation and immunoblotting of protein extracts from p38aMY-KO/ApoE2/2 and ApoE2/2 BMDMs. (C) Quantification of lesion size on whole aorta from male p38aMY-KO/ApoE2/2 and ApoE2/2 mice. En face Sudan IV staining of lesions. P38aMY-KO/ApoE2/2 males, n = 9; ApoE2/2 males, n = 9. Scale bar 2 mm. (D) Quantification of lesion area of atherosclerotic plaques at the aortic sinus of p38aMY-KO/ApoE2/2 and ApoE2/2 mice. Plaques are marked by arrows on aortal cross sections at the height of the aortic sinus. P38aMY-KO/ApoE2/2 males, n = 9; ApoE2/2 males, n = 15; p38aMY-KO/ ApoE2/2 females, n = 8; ApoE2/2, n = 9. Scale bar, 0.2 mm. doi:10.1371/journal.pone.0021055.g004 Serum cholesterol level measurements were performed after overnight fasting using the PAD-CHOL reagent (Roche) and reading absorbance at 500 nm, according to manufacturer’s instructions. Atherosclerotic lesion analysis Consecutive 7 mm sections of the heart, at the level of the atrioventricular valves, were collected and stained with toluidine blue for morphometric analysis and atherosclerosis quantification, as previously described [7]. Toluidine stained sections were also used for the quantification of the necrotic core area, which was defined as a clear area that was toluidine free [8]. En face analysis of atherosclerotic lesions was performed by Sudan IV staining (red) of whole aortas, as described previously [7]. Plaque areas, in both the atrioventricular valves and the whole aortas, but also the necrotic core area, were quantified using Adobe Photoshop. Histology and immunostainings For MOMA2 (anti-mouse macrophages/monocytes MCA519GT, Serotec) immunostainings, frozen sections of the aortic root were fixed in ice-cold acetone, blocked in 4% FCS with Avidin D solution (Avidin/Biotin Blocking Kit; Vector Laborato- ries) for 30 min and incubated with primary antibody (MOMA2, rat, 1:1000) for 60 min in 4% FCS with Biotin (Avidin/Biotin Blocking Kit; Vector Laboratories). Sections were then incubated with biotin-conjugated rabbit anti-rat/biotinylated antibody (E0468, 1:100, DakoCytomation) in 4%FCS/2% NMS for 60 min, incubated in Avidin/Biotin solution in PBS (Vectastain ABC Kit, Vector Laboratories) and color was developed with AEC substrate (Vector Laboratories). The sections were then counter- stained with haematoxylin and mounted with Entellon (MERCK). Immunostaining for collagen on frozen sections of the aortic root were performed by using the Masson Trichrome staining kit, which stains keratin and muscle fibers red, collagen green or blue, cytoplasm light red or pink, and cell nuclei dark brown to black (Sigma Aldrich), according to the manufacturer’s instructions. Macrophage isolation Bone marrow-derived macrophages (BMDM) were obtained according to standard procedures [7]. In brief, bone marrow cells isolated from the femurs of mice, were subjected to red blood cell (RBC) lysis and plated on 10 cm bacterial Petri dishes (Greiner) in RPMI Glutamax (Invitrogen) supplemented with 10% FCS, penicillin/streptomycin and 20% L929 conditioned medium. The cells were cultured for 8 days and starved in RPMI without FCS for 3 hrs, before experiments were performed. Thioglycolate-elicited peritoneal macrophages (PM) were isolat- ed as described elsewhere [31]. After RBC lysis, cells were washed extensively using ice-cold PBS and seeded on 6-well bacterial plates (Greiner) in RPMI Glutamax medium supplemented with 10% FCS. After 2 hrs, non-adherent cells were washed out with PBS and cells were starved for 48 hrs before experiments were performed. Mouse lung endothelial cell isolation (MLECs) Lungs were dissected from PBS perfused mice, sterilized in 75% ethanol and placed in fresh DMEM (Gibco). Lungs were then cut into small pieces and incubated in 0.2% collagenase in DPBS+ CaCl2 (Gibco) for 1 hr at 37uC. Remaining tissue was broken down through a 19G needle and strained through a 70 mM cell strainer. The cell suspension was then centrifuged 1500 rpm/5 min and the pellet was resuspended in endothelial cell medium (DMEM low glucose: Ham’s F-12 1:1, 20% FCS, 50 mg/ml endothelial mitogen, 25 mg/ml heparin, 100 U/ml penicillin/streptomycin and 2 mM glutamine) and plated on a previously coated (0.1% gelatin, 10 mg/ ml human fibronectin, 30 mg/ml bovine collagen type I) T75 flask. Endothelial cells were enriched by negative (LEAF rat anti-mouse CD16/32, 1:2000 dilution, Biolegend) and positive (CD102 rat anti- mouse, 1.5:2000 dilution, BD Biosciences) selection using Dyna- beads (sheep anti-rat IgG, Invitrogen) during passages 1–2. Purity of the cell population was assessed by CD146 FITC (Miltenyi Biotec) staining and flow cytometric analysis. At passage 8, MLECs were treated with His-TAT-NLS-Cre (HTNC) to induce recombination of p38a loxP flanked alleles [21], overnight (16 hrs), in medium containing DMEM (low glucose): RPMI: PBS (1:1:2 ratio) and 100 U/ml penicillin/streptomycin. The HTNC medium was then replaced with endothelial cell medium. The cells were allowed to reach confluence and passaged 1–2 times more before experiments were performed. MACS sorting of MLECS MLECS were isolated from female p38aEC-KO/ApoE2/2 and ApoE2/2 littermates after a 10 week HCD. Collagenase dissociated lungs were passed through a 19G needle to form a cell suspension, which was subsequently passed through a 100 mm, a 70 mm and a 40 mm cell strainer. After washing twice with PBS, debris removal and cell enrichment of the remaining cell suspension was achieved by mixing with a 30% Histodenz solution (Sigma-Aldrich) and centrifuging 15006g/2 mins to form a gradient. Low-density cells at the interface were harvested and washed twice in degassed MACS buffer (PBS pH 7.2, 0.5% BSA and 2 mM EDTA). Cells were resuspended in MACS buffer (90 ml/107 cells) and labeled with Anti-LSEC magnetic MicroBe- ads (beads coupled to the mouse endothelial cell marker CD146, Miltenyi Biotec) by adding 10 ml beads/107 cells and incubating 15 mins/4uC. After washing, the cells were resuspended in 500 ml buffer/108 cells. Magnetic separation of the cells was performed on a MACS column (MS, Miltenyi Biotec), where CD146 positive endothelial cells were retained on the column and eluted only upon removal from the magnetic field. CD146 sorting of lung endothelial cells was confirmed through flow cytometry (FITC), after staining with a CD146-FITC conjugated antibody (Miltenyi Biotec), according to manufacturer’s instructions. Cells obtained from MACS sorting were used to confirm efficient ablation of p38a in endothelial cells, by immunoblotting. Oxidized low-density lipoprotein stimulation (OxLDL) CuSO4 oxidation of human LDL (AppliChem) was performed according to standard protocols [6]. DiI-labeling was achieved by further incubation of the oxidized products with 300 mg DiI reagent/mg LDL (Molecular Probes) for 18 hrs at 37uC, before dialysis. PLoS ONE | www.plosone.org 9 June 2011 | Volume 6 | Issue 6 | e21055