HIMF and Stem Cells Figure 4. Both chronic hypoxia and pulmonary HIMF gene transfer induce pulmonary vascular remodeling. A–D: Paraffin-embedded lung sections were double-stained with antibodies to von Willebrand factor (black) and a-smooth muscle actin (red). Arrows indicate small pulmonary vessels. Scale bar: 50 mm. E, F: Percent muscularization of small pulmonary arteries in mouse lungs. NM, non-muscularized; PM, partially muscularized; FM, fully muscularized. *Significantly decreased vs. control at P,0.05. {Significantly increased vs. control at P,0.05. doi:10.1371/journal.pone.0011251.g004 receive bone marrow transplants display engraftment of BMD cells within their vasculature [23]. In our study, we demonstrate that GFP+ BMD cells are recruited to the pulmonary vasculature following chronic hypoxia or overexpression of HIMF in the lung. In fact, there are nearly twice as many GFP+ BMD cells associated with the vessels from mice exposed to chronic hypoxia or AAV- HIMF. In pulmonary vessels from normoxia/AAV-null control mice, there is almost never more than one GFP+ cell associated with an individual vessel. These GFP+ cells are rarely incorporated into the media layer in the vessels of these mice. In the experimental groups (chronic hypoxia or AAV-HIMF), it is common for some pulmonary vessels to have multiple associated GFP+ cells with these cells frequently localizing to the medial layer of the vessel. In several cases, these BMD cells aggregate around previously endothelial-only capillary vessels; neomuscularization of these vessels are a key component of the pathogenesis of PH. Recent evidence has suggested a potential role for the recruitment of BMD progenitor cells to the remodeled pulmonary vasculature associated with PH [11,12,24]. An initial study by Davie et al. [11] revealed an increased number of c-kit-expressing cells in the circulation and in the pulmonary vasculature of hypoxic calves. C-kit, also known as CD117, is the receptor for a cytokine called stem cell factor; it is expressed on the surface of BMD cells with multipotent potential [25]. A subsequent study by Hayashida et al. [24] demonstrated that mice exposed to chronic hypoxia for 4 or 8 weeks displayed increased infiltration of BMD cells in the lung and lung vasculature compared with normoxic control. Many of these recruited BMD cells were a-SMA+. Hypoxia does not seem to be the only stimulus for this process; in the monocrotaline inflammatory model of PH, many BMD cells were recruited to the pulmonary vasculature, some of which were a-SMA+ [12]. A detailed examination of AAV-HIMF-treated lungs showed that many of the recruited cells stained positive for the cellular markers c-kit and sca-1. This finding would indicate that these BMD cells have multipotent potential, including the ability to differentiate into mesenchymal-like cells; these differen- tiated cells could then participate in the observed pulmonary vascular remodeling. The fact that these cells were negative for the endothelial progenitor markers CD31 and CD34 strengthens the possibility that these cells will likely differentiate into mesenchy- mal-like cells. Several of the GFP+ cells associated with the pulmonary blood vessels also expressed a-SMA, indicating a mesenchymal lineage and suggesting the possible transition to myofibroblasts and vascular smooth muscle cells. The fact that many of the smallest neomuscularized vessels expressed only GFP+ cells in their new medial layer, strongly suggests a functional role for these cells. This work supports the ability of HIMF to recruit BMD cells to the vascular wall in the remodeling associated with the development of PH. We have recently demonstrated that HIMF is chemotactic for murine BMD cells in culture [5] and that the mechanism involves HIMF binding to BTK, resulting in BTK autophosphorylation and intracellular movement of BTK to the migrating cell process. Here, we showed that HIMF expression in the lung can recruit BMD cells to the remodeling pulmonary vasculature and that HIMF induces chemotaxis of HMSCs in culture. These results are consistent with our earlier findings and those of previously published reports [11,24] in which chronic hypoxia induced vascular remodeling. Intranasal AAV-HIMF treatment elicited similar results. PLoS ONE | www.plosone.org 7 June 2010 | Volume 5 | Issue 6 | e11251