Combination of stromal-derived factor-1α and vascular endothelial growth factor gene- modified endothelial progenitor cells is more effective for ischemic neovascularization  Jian-Xing Yu, MD, PhD, Xue-Fei Huang, MD, PhD, Wei-Ming Lv, MD, PhD, Cai-Sheng Ye, MD, PhD, Xin-Zhi Peng, MD, Hui Zhang, MD, Long-Bin Xiao, MD, Shen-Ming Wang, MD, PhD  Journal of Vascular Surgery  Volume 50, Issue 3, Pages 608-616 (September 2009) DOI: 10.1016/j.jvs.2009.05.049 Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 1 Flow chart of animal protocol. EBM-2, endothelial cell basal medium-2; EPC, endothelial progenitor cells; PBS, phosphate-buffered saline; SDF, stromal-derived factor. Journal of Vascular Surgery 2009 50, 608-616DOI: (10.1016/j.jvs.2009.05.049) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 2 Histologic identification of incorporation of endothelial progenitor cells (EPCs) into ischemic hind limb neovasculature. A, Representative photomicrographs show double fluorescence in ischemic limb muscles at day 7. Transplanted human EPCs labeled with DiI were identified in tissue sections by red fluorescence, and host mouse vasculature was identified by green fluorescence. Transplanted EPC-derived vasculature and mouse vasculature were analyzed quantitatively in the same microscopic field. B, Quantitative analysis of incorporated EPCs showed the density of DiI-labeled EPCs in tissue sections of hind limb muscles was greater in the Td/V-EPCs plus stromal-derived factor-1α (SDF-1α) group than any of the other five groups (*P < .001). C, Quantitative analysis of host endothelial cells. The density of Bandeiraea simplicifolia lectin-1 (BS-1) lectin–positive vasculature in tissue sections of hind limb muscles was greater in the Td/V-EPCs plus SDF-1α group than in the other five groups (*P < .001). Journal of Vascular Surgery 2009 50, 608-616DOI: (10.1016/j.jvs.2009.05.049) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 3 Physiologic and histologic evidence for enhanced neovascularization in ischemic hind limb. A, Laser Doppler perfusion imaging (LDPI) disclosed profound differences in the limb perfusion 28 days after induction of limb ischemia. In these digital color-coded images, red hue indicates regions with maximum perfusion, yellow indicates medium perfusion values, and blue designates the lowest perfusion. B, In quantitative analysis of perfusion recovery measured by LDPI, the ratio of ischemic/normal blood flow in the group that received Td/V-endothelial progenitor cells (EPCs) plus stromal-derived factor-1α (SDF-1α) was greater than in the other five groups (10 in each group; *P < .01). C, Quantitative analysis of capillary density in light microscopic sections at day 28 showed capillary density of the Td/V-EPCs plus SDF-1α group was significantly higher than other groups (10 in each group; *P < .01). Journal of Vascular Surgery 2009 50, 608-616DOI: (10.1016/j.jvs.2009.05.049) Copyright © 2009 Society for Vascular Surgery Terms and Conditions

Fig 4 Stromal-derived factor-1α (SDF-1α) and vascular endothelial growth factor (VEGF) synergize to promote angiogenic function of endothelial progenitor cells (EPCs) in vitro. A and B, SDF-1α induced a notable migration of EPC that was significantly more efficient in the presence of VEGF expression in Td/V-EPCs groups compared with Td/V-EPCs or SDF-1α alone. SDF-1α-mediated cell migration in Td/V-EPCs group was completely inhibited by AMD3100, the CXCR4-specific antagonist (**P < .01, ***P < .001). EPCs, Td/V-EPCs or Td/p-EPCs were seeded for transwell migration assay in the presence of SDF-1α respectively. After 24 hours of incubation, migration rates were quantified by counting the migrated cells in five random fields (original magnification × 100.) Data summarize three independent experiments. One representative of three independent experiments was shown. The range bars show the standard error of the mean. C, Reverse transcription-polymerase chain reaction analysis of messenger RNA transcripts for CXCR4 in EPCs, Td/V-EPCs, or Td/p-EPCs respectively. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. D, SDF-1α and VEGF synergistically protect EPC apoptosis in the Td/V-EPCs plus SDF-1α group. EPCs, Td/V-EPCs, or Td/p-EPCs were cultured with medium containing 3% and 20% fetal bovine serum (FBS), respectively, for 24 hoursy. SDF-1α (100 ng/mL) was added into the culture. Cellular apoptosis was analyzed by annexin V and propidium iodide staining and expressed as the percentage of apoptotic cells. Range bars designate the standard error of the mean. Journal of Vascular Surgery 2009 50, 608-616DOI: (10.1016/j.jvs.2009.05.049) Copyright © 2009 Society for Vascular Surgery Terms and Conditions