that BMD progenitor cells are recruited during this remodeling process [11,12]. Davie et al. [11] demonstrated that BMD c-kit+ cells were localized within the pulmonary artery walls of chronically hypoxic calves, and Spees et al. [12] reported that a- smooth muscle actin (a-SMA)+ BMD cells became engrafted into the pulmonary vasculature in an inflammatory model of PH. These studies suggest the interesting possibility that pulmonary vascular remodeling may involve cells of multiple origins, possibly including multipotent ‘‘stem cells.’’ In the current study, we demonstrate in mice that both chronic hypoxia and pulmonary gene transfer of HIMF induce BMD cell recruitment to the remodeling pulmonary vasculature; many of these cells localize to the newly formed media of previously non- muscularized capillary-like vessels. Both mouse models led to significant pulmonary vascular remodeling consistent with our prior demonstration of structural and hemodynamic PH. We describe several of these cells to be stem cell antigen (sca)-1+ and c-kit+ as well as CD312 and CD342. The BMD cells located within the vessel walls are likely of mesenchymal origin as they + are a-SMA . We also show that HIMF induces migration of human mesenchymal stem cells (HMSCs) in a PI-3K-dependent manner in vitro. All of these data suggest that HIMF/FIZZ1/ RELMa recruits BMD cells to the remodeling pulmonary vasculature. Materials and Methods Experimental animals/bone marrow transplantation Female C57/BL6 mice (6–8 weeks old; Charles River Laboratories, Wilmington, MA) were used as bone marrow transplant recipients for all experiments. Four- to 6-week-old male transgenic enhanced green fluorescent protein (EGFP) mice on a C57BL/6 background (Jackson Laboratories, Bar Harbor, ME; stock number: 003291) were used as bone marrow donors [13]. Animal housing and experimental protocols were approved by the Animal Care and Use Committee of the Johns Hopkins University (Protocol #: MO08M424). The mice were given free access to food and water and were maintained in a room with a 12:12 hour light-dark cycle between 20–24uC. Bone marrow transplants were performed as previously stated [14]. Briefly, whole bone marrow was collected from the transgenic EGFP mice, and 26106 bone marrow cells were transplanted into lethally irradiated (1,050 cGy) recipient mice through intravenous injection. The transplant recipients were then allowed to recover for 4–6 weeks. Antibodies and Inhibitors Rabbit anti-mouse HIMF polyclonal antibodies were prepared as we have described [3]. Polyclonal rabbit anti-GFP antibodies were purchased from Invitrogen (Carlsbad, CA). Goat anti-mouse RELMa (HIMF), rat anti-mouse c-kit, and rat anti-mouse sca-1 antibodies were purchased from R&D Systems (Minneapolis, MN). The rat anti-mouse CD31 and the mouse anti-b-actin monoclonal antibodies were purchased from BD Biosciences (San Jose, CA). The rabbit anti-CD34 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse anti-a- SMA antibody was purchased from DakoCytomation (Carpin- teria, CA). Fluorescein isothiocyanate (FITC), rhodamine, cy3- and cy5-labeled secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA). The rabbit anti- phospho-Akt (Ser473/Thr308) and rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) polyclonal antibodies as well as the pharmaco- logical inhibitors U0126 and LY294002 were purchased from Cell Signaling Technologies, Inc. (Beverly, MA). HIMF and Stem Cells Intranasal instillation of viral vectors To selectively induce HIMF expression in the lungs, we used a recombinant adeno-associated virus (AAV) vector that expresses murine HIMF (AAV-HIMF) as stated [7]. This viral vector contains the ubiquitous CB promoter and was prepared by the University of Florida Vector Core Laboratory. To control for the possibility of viral effects, we used a similar empty AAV vector (AAV-null). Intranasal instillation of the AAV vectors was performed as follows. First, female bone marrow transplant recipient mice were lightly anesthetized with isoflurane. Then, a gel loading tip primed with 50 mL of solution that contained either 2.561010 viral particles (VP) AAV-HIMF [with 5 mL lipofecta- mine 2000 (Invitrogen)] or 2.561010 VP AAV-null (with 5 mL lipofectamine 2000) was placed directly on the nasal passage and the solution expelled. Mice were sacrificed 14 days after intranasal instillation of the vector by isoflurane overdose, and tissue was processed as stated [7]. Briefly, the heart and lungs were removed en bloc. The right lung was tied off and the left lung was inflated with 1% low-melting point agarose with constant pressure and then placed on ice. The right lung was removed and split into individual lobes. A portion of the right lung was frozen in liquid N2 and stored at 280uC for use in Western blot analysis. The agarose-filled lung tissue was fixed in 4% paraformaldehyde and then processed for either paraffin or frozen sections as stated [3,7]. The efficiency of instillation was measured by Western blotting or immunohistochemistry for HIMF. Chronic hypoxia model of PH Female mice receiving bone marrow transplantation were exposed to either normal room air (20.8% O2) or 10% O2 for 7 days as we have described [3,7,15–18]. The fractional concentra- tion of O2 was monitored and controlled with a Pro:Ox model 350 unit (Biospherix, Redfield, NY) by infusion of N2 (Roberts Oxygen, Rockville, MD) balanced against an inward leak of air through holes in the chamber. The chambers were continuously scavenged for CO2 and ammonia. At the end of the 7-day period, mice were sacrificed and processed as stated above. Immunohistochemistry Paraffin blocks of lungs from mice exposed to room air (20.8%O2), hypoxia (10.0% O2), AAV-null, or AAV-HIMF were cut into 6-mm sections and placed onto clean glass slides. The slides were then deparaffinized and rehydrated as described previously [3,7]. For antigen retrieval, the slides were submerged in antigen unmasking solution (Vector Laboratories, Burlingame, CA) and heated at 95uC for 20 min. Endogenous peroxidase activity was blocked by treatment with 3% H2O2 in PBS for 10 min at room temperature (RT). We then blocked endogenous avidin and biotin for 15 min each at RT using the Avidin/Biotin Blocking Kit (Vector Laboratories). Nonspecific protein binding was blocked by treatment with either normal goat or horse serum for 30 min at RT. After the blocking steps, the sections were treated with polyclonal rabbit anti-GFP antibodies, polyclonal goat anti-HIMF antibodies, or antibody diluent alone for 2 h at RT. The slides were then washed with PBS and treated with either goat anti-rabbit or donkey anti-goat biotinylated secondary antibodies (Vector Laboratories) in PBS for 30 min at RT. Then, the lung sections were exposed to an ABC horseradish peroxidase (HRP) reagent (Vector Laboratories) for 30 min at RT. The GFP/ HIMF signal was developed with the Peroxidase Substrate Kit DAB (Vector Laboratories). The sections were counterstained with hematoxylin as we have described [7]. Finally, the sections were dehydrated, cleared, and mounted with Cytoseal 60 (Richard- Allan Scientific, Kalamazoo, MI). The stained sections were PLoS ONE | www.plosone.org 2 June 2010 | Volume 5 | Issue 6 | e11251