HIMF and Stem Cells Figure 1. HIMF expression in murine lung. Paraffin-embedded lung sections from normoxic (7d, 20.8% O2) (A), hypoxic (7d, 10.0% O2) (B), AAV- null-treated (14d, 2.561010 VP) (C), and AAV-HIMF-treated (14d, 2.561010 VP) (D) mice were rehydrated and stained with goat anti-mouse HIMF polyclonal antibodies. Pa: pulmonary artery. Aw: airway. Scale bar: 50 mm. doi:10.1371/journal.pone.0011251.g001 the lung parenchyma, most likely as a result of irradiation injury [19] or possibly representing normal distribution of inflammatory BMD cells in lung. Quantification of the GFP+ cells associated with individual pulmonary vessels confirmed the visual findings. Our analysis revealed approximately twice as many GFP+ cells/ vessel in hypoxic mice (1.1360.09 GFP+ cells/vessel) compared with normoxic mice (0.6360.05 GFP+ cells/vessel; P = 0.0005; Figure 2E). Similarly, the number of GFP+ cells/vessel in AAV- HIMF-treated mice (1.0960.14 GFP+ cells/vessel) was greater than that of AAV-null-treated mice (0.5860.09 GFP+ cells/vessel; P = 0.0266; Figure 2F). The result of AAV-HIMF treatment looks strikingly similar with hypoxia; HIMF gene transfer was sufficient to induce the recruitment of BMD cells to the pulmonary vasculature (Figure 2). To gain a different visual perspective of these lung sections, we performed immunofluorescence microsco- py using lung sections from each group. Figure 3A–I shows that after either hypoxia or AAV-HIMF treatment, GFP+ BMD cells formed an organized layer that surrounded the blood vessel. A cross sectional view is shown in Figure 3J–U. A higher magnification image of a small vessel from an AAV-HIMF treated mouse revealed that the entire media layer of the neomuscularized small vessel was made up of GFP+ BMD cells (Figure 3M, Q, U). Most of the recruitment of GFP+ cells was observed in small vessels approximately 20–80 mm in diameter, including small capillary sized vessels normally without any muscle cells. This is strong evidence of a functional role of these cells in the remodeling pulmonary vasculature and the development of PH. Hypoxia and AAV-HIMF treatment similarly increased pulmonary vascular remodeling In the bone marrow transplant recipients, muscularization of the small pulmonary arteries was increased by both hypoxia and AAV-HIMF treatment compared to the corresponding controls (Figure 4A–D). After exposure to 10.0% O2 for 7 days, the percentage of vessels that were FM increased (30.3163.46% vs. 7.9664.30%; P = 0.0018), while the percentage of vessels that were NM decreased (30.5264.78% vs. 57.46611.19%; P = 0.0392) compared with control animals exposed to room air (Figure 4E). Examination of the lung sections 14 days after intranasal instillation of AAV-HIMF revealed increased percent- ages of FM vessels (15.05611.19%) when compared to simulta- neously treated AAV-null controls (5.2962.64%; P = 0.0187; Figure 4F). BMD cells are recruited to the smooth muscle layer of pulmonary vessels To determine the phenotype of recruited cells in AAV-HIMF- treated lungs, we used the markers sca-1, c-kit, CD34, CD31, and a-SMA. The markers sca-1 and c-kit indicate that the cells are of PLoS ONE | www.plosone.org 4 June 2010 | Volume 5 | Issue 6 | e11251