HIMF and Stem Cells Figure 2. Chronic hypoxia and pulmonary HIMF gene transfer increase the number of BMD cells associated with the pulmonary vasculature. A–D: Paraffin-embedded lung sections from mice exposed to normoxia (7d, 20.8% O2) (A), hypoxia (7d, 10.0% O2) (B), AAV-null (14d, 2.561010 VP) (C), or AAV-HIMF (14d, 2.561010 VP) (D) were probed with polyclonal antibodies raised against GFP. Arrows indicate GFP+ cells within the vasculature. Scale bar: 50 mm. E, F: Quantification of GFP+ cells within the pulmonary vasculature. GFP+ cells within the pulmonary vasculature are shown as mean 6 SEM of GFP+ cells/vessel. * P,0.05, ** P,0.01 vs. control. doi:10.1371/journal.pone.0011251.g002 hematopoietic lineage and have multipotent potential; CD31 and CD34 are prominent markers for cells of endothelial lineage. Finally, a-SMA indicates cells of mesenchymal origin. As shown in Figure 5, GFP+ BMD cells recruited to the vasculature were sca-1+ (Figure 5B, D) and c-kit+ (Figure 5F, H). Some sca-1+ and c-kit+ cells contained no GFP signal; these cells are likely unlabeled BMD cells, as not all of the BMD cells from the GFP transgenic donor express GFP [13]. Surprisingly, none of the GFP+ cells associated with the vasculature were also CD34+ (Figure 5J, L). To determine at which layer of the vasculature the GFP+ cells were located, we performed immunohistochemical staining for both a- SMA (vascular smooth muscle) and CD31 (endothelium). As shown in Figure 6, the GFP+ cells appeared to be recruited to the smooth muscle layer of the vasculature. GFP and a-SMA signals colocalized in many vascular cells that anatomically appeared to associate with the smooth muscle layer (Figure 6C, D; arrows). These signals also co-localized with HIMF (Figure 6B). There was no apparent co-localization between GFP and the endothelial cell marker, CD31 (Figure 6A, B). HIMF-stimulated HMSC chemotaxis is Akt/PI-3K-dependent To examine if HIMF has a direct effect on mesenchymal stem cells, we performed a cell migration assay using HMSCs. For these experiments, HMSCs were cultured in transwell plates in the presence of vehicle or recombinant HIMF (100 nM) for 24 h. HIMF increased HMSC migration approximately 2-fold (Figure 7). We have previously shown that HIMF can induce activation of both the Akt/PI-3K pathway and the ERK1/2 MAPK pathway in vitro [3]. To determine if HIMF activated these pathways in HMSCs, we treated cultured HMSCs that had been serum and growth factor starved overnight with vehicle or HIMF (100 nM) for 15 or 60 min. The addition of HIMF activated both the PI-3K and ERK1/2 MAPK pathways in a time-dependent manner (Figure 7C, D). Because HIMF induced cell migration and activated these signaling pathways in HMSCs, we wanted to determine if one or both of these pathways were involved in HIMF-induced cell migration. Preincubation of HMSCs with the PI-3K inhibitor LY294002 (10 mM) returned HIMF-induced cell migration to baseline levels (Figure 7B); inhibition of ERK1/2 MAPK with the pharmacological inhibitor U0126 (10 mM) had a slight, but not statistically significant effect (Figure 7B). In Figure 7C and 7D, we demonstrate that both LY294002 and U0126 successfully entered the HMSCs and blocked the appropriate signaling pathway. Discussion In the current study, we examined the possibility that HIMF/ FIZZ1/RELMa acts as a chemokine to induce BMD cell recruitment to the remodeling pulmonary vasculature. To address this question, we transplanted bone marrow from GFP+ transgenic mice [13] into lethally irradiated wild-type recipients, subjected the recipients to models of PH, and tracked BMD cell migration [14] in the pulmonary vasculature. The results show that both chronic hypoxia and AAV-HIMF introduction induce PH-like vascular remodeling and the engraftment of BMD cells to the pulmonary vasculature. Further analysis of the recruited BMD cells in AAV-HIMF-treated mice revealed that many of these cells expressed c-kit, sca-1, and a-SMA and lacked expression of CD31 PLoS ONE | www.plosone.org 5 June 2010 | Volume 5 | Issue 6 | e11251