For in vivo experiments we utilized transplantation

For in vivo experiments, we utilized transplantation of SHh-pretreated Gm-CD34+ mao inhibitors rather than the co-administration of Gm-CD34+ cells (EPCs) and SHh protein or SHh gene transfer (Palladino et al., 2012) into ischemic hindlimbs, and could detect both EC marker and VSMC marker expressing cells, possibly derived from transplanted Gm-CD34+ cells in the microvasculature sprouting toward the center of healing during histological analysis. This morphogenetic effect of transplanted Gm-CD34+ cells was likely attributed to their direct contribution to vasculogenesis/arteriogenesis (Supplemental Fig. 4S, left side) as well as an indirect paracrine effect via secreted pro-angiogenic growth factors/cytokines from the Gm-CD34+ cells activated by SHh (Supplemental Fig. 4S, right side). Therefore, even though the number of transplanted Gm-CD34+ cells (105 per hindlimb) was lower than that in previous reports (Madeddu et al., 2004; O et al., 2011; Zhou et al., 2007), the pre-treatment of Gm-CD34+ cells with SHh could exhibit immediate paracrine effect at the time of transplantation and direct contribution to neovascularization differentiating into ECs and SMCs by endogenous SHh protein production from ischemic tissue in late phase, resulting in sufficient outcome compared with non-treated Gm-CD34+ cells. Although SHh expression in normal adult organs/tissues is extremely limited, it is expressed constitutively in perivascular and interstitial tissues, and induced incidentally in ischemic tissues (Kusano et al., 2005; Pola et al., 2003). This indicates that constitutive or inducible SHh expression may play a key role during cell fate decision and functional regulation of circulating CD34+ cells (EPCs) in situ. As recent papers demonstrated, resident vascular progenitors are located in adventitia tissues and can contribute to vascular repair or remodeling by promoting a vasculogenic zone in the adventitia (Ergun et al., 2011). The induced SHh expression in the perivascular area could encourage the recruitment of circulating CD34+ cells, which in turn could increase their vasculogenic capacity and enhance vascular repair by the local enrichment of growth factors, and an increase in the paracrine effect on resident ECs (Supplemental Fig. 5S, right side). The adventitia as a putative source for SHh could thus foreordain CD34+ cells into an endothelial phenotype while promoting the smooth muscle phenotype in the outer vascular tissue leading to neovascularization (Supplemental Fig. 5S, left side).
Conclusions SHh signaling was activated only in human G-CSF mobilized (Gm-) CD34+ cells but not in normal CD34+ cells and the trend appeared to be evident in the patient\'s Gm-CD34+ cells. The SHh signaling activation enhanced certain cell functions that are essential for neovascularization including vasculogenesis/arteriogenesis demonstrating an increased differentiation potential of these cells into ECs/VSMCs, with the activated CD34+ cells contributing to vascular regeneration in ischemic tissues. Since Gm-CD34+ cells with activated SHh signaling are used in ongoing clinical trials of autologous CD34+ cell transplantation therapy for non-option patients with ischemic cardiomyopathy (Losordo et al., 2011), critical limb ischemia including Burger disease and ASO (Kawamoto et al., 2009; Kudo et al., 2003), the modification/pre-treatment of Gm-CD34+ cells with SHh may make sense and therefore enhance the therapeutic outcome. In terms of inducing stable vascular regeneration in ischemic tissues, not only angiogenesis with just ECs but also arteriogenesis with both ECs and pericytes would be critical aspects of a successful Gm-CD34+ cell transplantation. Thus, an efficient direct induction system for human Gm-CD34+ cell differentiation into ECs and pericytes/VSMCs would be required in Gm-CD34+ cell transplantation for ischemic diseases. Although further pre-clinical studies will be warranted, our findings may give rise to a novel and simple strategy for the next generation-CD34+ cell therapy in patients with ischemic diseases.