Nano-Scaffold for Therapeutic Angiogenesis Figure 5. Collateral vessel formations in hind limb ischemic model. (A) 3D-CT angiography of mice (BALB/cAJcl) was performed immediately after the operation (a) and 7 days after implantation of vehicle alone (b), NS alone (c), BMNCs alone (d), LA+BMNCs (e) or NS+BMNCs (f). Representative 3D-CT angiograms are presented. PLLA microspheres containing magnetite (PLA-Particles-M) were used as core NS and LA to be detected by X-ray 3D-CT. Detected NS and LA by 3D-CT were visualised as green particles. (B) Quantitative volume analysis of collateral vessels in the ischemic area used the arterial phase 3D-CT angiogram data (n = 3 in each group). Data are shown as means (SD). *P,0.05 for the NS+BMNCs group compared to the vehicle, NS alone, BMNCs alone, and LA+BMNCs groups. Abbreviations: NS, nano-scaffolds; LA, unmodified PLLA microspheres; BMNCs, bone marrow mononuclear cells. doi:10.1371/journal.pone.0035199.g005 In the present study, NS markedly enhanced therapeutic angiogenesis and arteriogenesis, which showed good correlations with increased VEGF and FGF-2 levels in ischemic muscle. VEGF is a potent stimulator of endothelial cell mitogenesis and cell migration [3,7]. FGF-2 is also crucial for arteriogenesis with efficient blood flow through the stabilisation of newly formed endothelial tubules by recruiting pericytes and smooth muscle cells [3,7]. Our observations further suggest that the clusters containing NS, implanted cells, and recruited host cells may form a microenvironment containing secreted cytokines and growth factors, such as VEGF and FGF-2 (Fig. 7), which could also contribute to reduce apoptotic cell death [7,11]. Transfection of donor cells with antiapoptotic transgenes, e.g., VEGF, FGF-2, Akt and Bcl-2, has been reported to enhance cell survival, resulting in augmentation of the proangiogenic cytokine production [6,7]. Meanwhile, we for the first time demonstrated that NS-mediated prolonged localization of unmodified donor cells as a cluster in ischemic tissue could change the local environment, through accelerated expression of proangiogenic factors, improve cell survival and behaviour, and augment neovascularisation, without gene manipulation or artificial extracellular matrix. The increase in tissue VEGF and FGF-2 levels shown in Fig. 6 does not appear to be as large as compared to the marked alleviation of apoptosis of transplanted BMNCs by NS (Fig. 8). Because tissue cytokine levels were measured using the tissue extracts from whole thigh muscle, the levels of these cytokines shown in Fig. 6 are the average of the entire ischemic muscle. Meanwhile, these cytokines were distributed in high concentra- tions in the area where the implanted cells and NS formed clusters (Fig. 7). Therefore, a significantly high cytokine level measured as the average for the entire muscle indicated that the effect of NS in amplifying the cytokine level in the implanted area was much larger than that shown in Fig. 6. Exposure to such high levels of cytokines in this area appears to markedly alleviate apoptosis (Fig. 8), and such a cytokine concentration gradient appears to be useful to induce efficient neovascularization in the implanted area [3]. Similar to VEGF and FGF-2, IL-1b, MCP-1, and SDF-1 are known as proangiogenic factors induced by ischemia [3,4,40–42]. In fact, increase in the expression of all proangiogenic factors was induced by ischemia alone 3 days after ischemic induction in the present study (Fig. 6, vehicle group). Although we observed PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35199