br Methods br Results br Discussion

Methods
Results
Discussion Originally, human bone marrow-derived CD34+ adenosine receptor agonist were defined as hematopoietic stem/progenitor-rich cells, while endothelial progenitor cells (EPCs), which play an important role in post-natal angiogenesis/vasculogenesis in ischemic tissues, have also been shown to be associated with CD34+ cells (Asahara et al., 1997). CD34+ cells in the human peripheral blood have recently been used as a tool for therapeutic angiogenesis of ischemic diseases, with several lines of evidence accumulated in clinical/animal studies underlining the therapeutic efficacy of CD34+ cell transplantation. However, some previous reports described that the preferential mechanism of CD34+ cell action is likely an indirect contribution to angiogenesis, namely, a paracrine effect by production of a variety of cytokines and angiogenic growth factors rather than a direct contribution of these cells to angiogenesis/vasculogenesis in ischemic tissues (Wang et al., 2010). In this study, we assessed whether human CD34+ cells could commit to the vascular lineage upon activation of SHh signaling, and have demonstrated that 1) Hedgehog signaling was activated by SHh stimulation in G-CSF mobilized- (Gm-) CD34+ cells, 2) SHh promoted the functional activity of Gm-CD34+ cells including their paracrine effects, 3) SHh promoted Gm-CD34+ cell commitment to vascular lineage, exhibiting a vasculogenic activity both in vitro and in vivo, and 4) TGFβ signaling pathway might be involved in the SHh-induced vascular differentiation of Gm-CD34+ cells. Pola et al. discovered that SHh triggered neovascularization through SHh/PTCH signaling as an indirect angiogenic agent specifically present in mesenchymal cells i.e. fibroblasts for the first time (Pola et al., 2001), and that the inhibition of SHh signaling was sufficient to decrease ischemia-induced local angiogenesis and upregulate VEGF in skeletal fibroblasts (Pola et al., 2003). Also, Renault et al. reported that SHh does not activate Gli-dependent transcription in ECs because of the absence of enhanced PTCH mRNA expression in SHh treated ECs (Renault et al., 2010). A notable finding of this study is that human CD34+ cells do indeed express a receptor for SHh, PTCH, and that the response to SHh protein was also confirmed by upregulation of mRNA expression of not only the downstream molecule Gli1 but also SHh and SMO, suggesting that SHh induced and activated the SHh signaling pathway in G-CSF stimulated CD34+ cells but not non-stimulated CD34+ cells via an autocrine mechanism. The angiogenic growth factor gene and vascular gene expressions are relatively low in non-mobilized CD34+ cells compared with G-CSF mobilized CD34+ cells, and the response to SHh treatment was small except for VEGF-A and eNOS. A possible reason for the small response to SHh in CD34+ cells is due to the short time (24h) treatment with SHh in vitro, while the transplanted SHh-treated CD34+ cells exhibited pro-angiogenic effect, perhaps, with long term exposure to endogenous SHh production in ischemic tissue, or only a small subpopulation of CD34+ cells might be endothelial lineage. Based on the evidence that SHh is one of the notochord-derived morphogens which plays a crucial role during embryonic vascular development (Carmeliet and Tessier-Lavigne, 2005) and has also been shown to be involved in postnatal neovascularization, we hypothesized that the SHh signaling pathway might promote Gm-CD34+ cell function and increase differentiation into the vascular lineages. To explore the direct effects of SHh on Gm-CD34+ cells, we performed a series of experiments in vitro and in vivo. In contrast to recent studies in which there are no direct effects of SHh on cellular responses, such as proliferation, migration, and serum deprived survival, in cultured ECs (Kanda et al., 2003; Pola et al., 2001), we could demonstrate a direct effect of SHh on GM-CD34+ cell functions including an increase in proliferation, adhesion, migration, tube formation, and an upregulation of SHh signaling-related genes. We further confirmed the SHh-induced differentiation potential of GM-CD34+ cells into ECs and VSMCs with marker expressions of eNOS/CD31 and calponin/SMα-actin, respectively. In hematopoietic stem cells (HSCs) including CD34+ cells, it has been reported that Notch and Wnt signaling played a role in the self-renewal and Smad signaling with TGFβ negatively regulated the growth while Smad signaling with bone morphogen (BMPs) regulated the development (Blank et al., 2008). We therefore focused on these signaling to explore underlying mechanism for SHh-induced vascular differentiation of Gm-CD34+ cells. Apart from critical signaling pathway in HSCs, TGFβ-Smad signaling pathway was highly activated and SHh (1.0μg/mL) pre-treatment further upregulated the signaling synchronized with a variety of vascular transcription factor gene expressions. Organ including vascular network development is regulated by differential SHh concentration (Scherz et al., 2007). Although it would be difficult to translate the in vitro concentration of SHh to in vivo settings, we assumed that 1.0μg/mL of SHh would be critical in the contribution of endogenous CD34+ cells to vascular development or post-neonatal angiogenesis. Also, SHh-induced TGFβ1 in CD34+ cells might directly promote angiogenesis (Ferrari et al., 2009) and arteriogenesis (van Royen et al., 2002) influencing resident ECs and VSMCs in ischemic tissue. On the other hand, BMP-Smad signaling pathway was downregulated (Fig. 5) and Notch/Wnt signaling pathway was not affected (data not shown) by SHh (1.0μg/mL) pre-treatment in CD34+ cells.