HIMF and Stem Cells visualized with an Olympus-BHS microscope attached to a QImaging Retiga 4000RV digital camera. Images were captured with ImagePro Plus (version 5.1) software. camera, analyzed by NIH ImageJ software, and reported as the number of positively stained pixels versus the total number of image pixels. Quantification of GFP-positive cells To quantify the number of GFP+ cells associated with the pulmonary vasculature, the GFP-stained lung sections were examined with a 406 objective lens. Peripheral pulmonary arteries associated with alveolar sacs and alveolar ducts with an external diameter between 25 and 100 mm were identified, and the associated GFP+ cells were counted. GFP+ cells were quantified in approximately 50 consecutive vessels per animal. The values are expressed as the mean number of GFP+ cells per vessel (mean 6 SEM). Assessment of pulmonary vascular remodeling Pulmonary vascular remodeling of the mice was analyzed as we have previously described [7,15–18]. The murine lung sections were initially evaluated after being stained with hematoxylin and eosin. Lung sections were also dual stained with von Willebrand Factor (endothelium) and a-SMA (vascular smooth muscle) as we have described [7,17,18]. Upon completion of the dual-stain, approximately 100 randomly selected arteries were examined under an Olympus-BHS microscope attached to a QImaging Retiga 4000RV digital camera. Only arteries with an internal diameter of ,80 mm were examined. These vessels were classified as non-muscular (NM), partially-muscular (PM), or fully-muscular (FM), according to a-SMA staining. Vessels that had at least one a-SMA+ cell but lacked a continuous layer were considered PM. FM vessels had a continuous a-SMA band. These vessels were then analyzed as we have described [7,17,18]. Images of dual- stained sections were captured as stated above. Immunofluorescence microscopy Frozen lung sections were air dried for 30 min, permeabilized with 0.2% Triton X-100/PBS, and then blocked with 2% BSA/ PBS. The sections were exposed to primary antibody (c-kit, sca-1, CD31, CD34, a-SMA, or HIMF) followed by the appropriate secondary antibody (cy3-donkey anti-rabbit IgG, cy3-donkey anti- rat IgG, FITC-donkey anti-mouse IgG, or cy5-donkey anti-mouse IgG). Nuclei were stained with 50 ng/mL 49,69-diamidino-2- phenylindole dilactate (DAPI) for 5 min. Staining was imaged with a Zeiss 510 Meta confocal microscope via a 206 lens. When four channels were used, cy5 was labeled with pink. Differential interference contrast imaging was used to show the tissue structure. In vitro Cell Migration Assay HMSCs were purchased from Lonza (Walkersville, MD) and cultured according to the manufacturer’s specifications. Only HMSCs from passages 3–5 were used. Costar 24-well cell migration plates with polycarbonate membranes with 8-mm pore size (Costar Corporation, Cambridge, MA) were used for this assay. The lower chamber was filled with 0.6 mL of medium with or without 100 nM recombinant HIMF. Then, 100 mL of HMSC suspension (105 cells) was added to the upper chamber. In some experiments, the cells were pretreated for 30 min with vehicle (0.1% DMSO) or a pharmacological kinase inhibitor [U0126 (10 mM) or LY294002 (10 mM)]. After 24 h at 37uC, the cells were removed from the top surface of the membrane. Migrated cells on the bottom surface were stained with Coomassie blue. The average number of cells per field was evaluated under an Olympus-BHS microscope. Images were captured with a QImaging Retiga 4000RV digital Western Blot Analysis HMSCs were cultured to approximately 70% confluence and then serum- and growth factor- starved overnight. Then they were treated with vehicle or 100 nM HIMF for various time periods in the presence or absence U0126 (10 mM) or LY294002 (10 mM). The HMSCs were collected in equal volumes of Laemlli’s sample buffer, resolved by 4–20% gradient sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad), and transferred to nitrocellulose membranes (Bio-Rad). The blots were blocked with 5% non-fat milk-TBS-T and incubated with either rabbit anti-phospho-Akt (Ser473/Thr308) or rabbit anti-phospho- ERK1/2 (Thr202/Tyr204) antibody. The blots were then incubated with anti-rabbit IgG conjugated to HRP antibodies, developed with enhanced chemiluminescence (ECL) and exposed to X-ray film (Denville Scientific; Metuchen, NJ). To ensure equal protein loading and transfer, the blots were stripped using the Blot Restore kit according to the manufacturer’s instructions (Millipore; Billerica, MA), reprobed with mouse anti-b-actin antibodies and processed as stated above. Statistical analysis A student’s t-test was used to compare mean responses between individual experimental and control groups. ANOVA was used to compare the mean responses among experimental and control groups in experiments with multiple groups. The Dunnett and Scheffe F test was used to determine between which groups significant differences existed. A P-value ,0.05 was considered significant for all experiments. Results Hypoxia and pulmonary HIMF gene transfer induce HIMF expression in bone marrow transplant recipient mice To determine the expression pattern of HIMF in our bone marrow transplant recipients, we evaluated lung sections from mice exposed to normoxia (7d, 20.8% O2), hypoxia (7d, 10.0% O2), AAV-null (14d, 2.561010 VP), or AAV-HIMF (14d, 2.561010 VP) by immunohistochemistry. Both hypoxia and HIMF pulmonary gene transfer led to HIMF expression in the lungs of bone marrow transplant recipients (Figure 1). In normoxic lungs, HIMF staining was not apparent (Figure 1A), but HIMF was strongly expressed in the airway epithelium, pulmonary inflammatory cells, and pulmo- nary vasculature of hypoxic animals (Figure 1B). In the HIMF gene transfer experiments, HIMF staining was absent in AAV-null treated lungs (Figure 1C) but strong in airway epithelium and the pulmonary vasculature (Figure 1D). The introduction of AAV-null virus into the lungs did not change HIMF expression compared to normoxic control (Figure 1A, C). BMD cells are localized to the pulmonary vasculature Immunostaining for GFP with subsequent quantitative analysis showed that in both hypoxic and AAV-HIMF-treated bone marrow transplant recipients, increased numbers of GFP+ cells became associated with the vasculature (Figure 2) compared to appropriate simultaneous controls. Visual examination of both hypoxic and AAV-HIMF-treated lung sections revealed GFP+ cells associated with the vasculature; in comparison, relatively few GFP+ cells were associated with the pulmonary vasculature of normoxic or AAV-null treated mice (Figure 2A–D). Notably, mice from all groups had some GFP+ cells evenly distributed throughout PLoS ONE | www.plosone.org 3 June 2010 | Volume 5 | Issue 6 | e11251