HIMF and Stem Cells Figure 7. HIMF-stimulated HMSC migration is PI-3K-dependent. A: HMSCs (105 cells) were cultured in the upper chamber of a transwell plate; the lower chamber held medium containing BSA (control) or HIMF (100 nM). After 24 h, the cells were fixed and stained by Coomasie blue solution. B: HMSCs were grown as described in A, but cells were pretreated with vehicle, U0126 (10 mM), or LY294002 (10 mM). Migrated cells were quantified and results were reported as mean (6SEM) of area (pixels). *Significant increase vs. vehicle control at P,0.05. {Significant decrease vs. HIMF stimulation alone at P,0.05. C, D: HMSCs were cultured to approximately 70% confluence, serum and growth factor starved overnight, and then exposed to HIMF (100 nM) or vehicle for up to 60 min in the presence or absence of ERK1/2 MAPK inhibitor U0126 (10 mM) or PI-3K inhibitor LY294002 (10 mM). Cells were lysed and proteins were resolved with SDS-PAGE and transferred to nitrocellulose membranes. The membranes were probed with rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (C) or rabbit anti-phospho-Akt (Ser473/Thr308) (D), followed by HRP-conjugated anti-rabbit IgG antibodies, and developed with ECL. To ensure equal loading and transfer, blots were stripped and reprobed with anti-b-actin. doi:10.1371/journal.pone.0011251.g007 Our finding that HIMF was chemotactic for HMSCs in this study raises the possibility that HIMF directly recruits mesenchy- mal stem cells to the remodeling pulmonary vasculature. Previous studies have demonstrated the existence of circulating BMD smooth muscle cells and the potential vascular engraftment of these cells in human disease [21,23]. In our system, we showed that these cells are of mesenchymal lineage and directly engraft into the vascular wall. It is important to note the possibility that HIMF is driving these recruited cells to the mesenchymal lineage. HIMF has been shown to play a key role in the transition of fibroblasts to myofibroblasts in experimentally-induced pulmonary fibrosis [8]. It is likely that the cells that are currently c-kit+ and sca-I+ will transition into vascular smooth muscle cells in the remodeling vasculature. A receptor for HIMF or its related molecules has not yet been described, although a few signaling pathways have been identified. We have previously reported that HIMF activates the PI-3K pathway in pulmonary vascular smooth muscle cells in a dose- and time-dependent manner [3]. We have also demonstrated that HIMF activates the Akt/PI-3K pathway in endothelial cells; this activation plays a critical role in HIMF-induced endothelial migration and tubule formation [6]. Other groups also have shown this pathway to be activated in primary lung fibroblasts as well as endothelial and lung epithelial cell lines [9,10]. Here we showed that HIMF activates the PI-3K pathway in a time-dependent manner in primary HMSCs and that this pathway is involved in HIMF stimulated cell migration. One signaling pathway that activates the Akt/PI-3K pathway in several cellular systems is BTK [26]. Our previous studies have shown that HIMF is a binding partner for BTK as well as an activating agent in murine BMD cells [5]; intracellular movement of BTK to the migrating cell process is essential to HIMF-induced cell migration of these cells. It is possible that HIMF is activating Akt/PI-3K through the BTK pathway in this system. HIMF also activated ERK1/2 MAPK in a time-dependent manner in HMSCs, but this pathway did not appear to be involved in the HIMF-stimulated cell migration process. In summary, the current study demonstrates that pulmonary gene transfer of HIMF induces pulmonary vascular remodeling and the recruitment of BMD cells to the pulmonary vasculature similar to that of chronic hypoxia. Cells that were recruited to the vasculature were c-kit+, sca-1+, and a-SMA + , but CD312 and CD342; these results suggest that these recruited cells are BMD and mesenchymal in origin or have the potential to differentiate into mesenchymal-like cells that participate in pulmonary vascular remodeling. The study also shows that HIMF has direct action on PLoS ONE | www.plosone.org 9 June 2010 | Volume 5 | Issue 6 | e11251