1. Vascular endothelial growth factor-C derived from CD11b+ cells induces therapeutic improvements in a murine model of hind limb ischemia 
Go Kuwahara, MD, Hitomi Nishinakamura, PhD, Daibo Kojima, MD, PhD, Tadashi Tashiro, MD, PhD, Shohta Kodama, MD, PhD 
Journal of Vascular Surgery 
Volume 57, Issue 4, Pages (April 2013)
DOI: /j.jvs
Copyright © 2013 Society for Vascular Surgery Terms and Conditions

2. Fig 1
Recovery of blood flow in ischemic limbs treated with bone marrow cells (BMCs) and CD11b+ cells derived from BMCs. A and C, Representative laser Doppler perfusion images taken at indicated intervals for the phosphate-buffered saline (PBS) control group, unselected BMCs group, CD11b− group, and CD11b+ group. Blood perfusion in the ischemic hind limbs was markedly increased in the unselected BMCs and CD11b+ groups compared with the control and CD11b− groups, respectively. Quantitative analysis of hind limb blood perfusion demonstrated an increase in the ischemic/nonischemic ratio, which was significantly higher in the BMCs group compared with the control group at postoperative days 7 and 14 (day 7: 38.9% ± 9.0% vs 14.8 ± 6.7%; P < .01; day 14: 72.2% ± 7.4% vs 31.1 ± 12.0%; P < .05; n = 5-7). The increase in the ischemic/nonischemic ratio was significantly higher in the CD11b+ group compared with the CD11b− group at postoperative days 1, 3, 7, and 14 ( day 1: 17.1% ± 4.0% vs 12.3 ±1.7%; P < .05; day 3: 26.4% ± 3.1% vs 12.3% ± 7.2%; P < .05; day 7: 48.4% ± 16.0% vs 20.7% ± 8.6%; P < .01; day 14: 85.7% ± 23.3% vs 45.7% ± 4.8%; P < .01; n = 5-7). Data are given as mean ± SEM, n = 5-7 per group. *P < .05; **P < .01; ***P < B, Flow cytometric analysis of CD11b+ macrophages from BMCs. The purity of subpopulations of the enriched CD11b+ macrophages was approximately 95% according to the FACS analysis.
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2013 Society for Vascular Surgery Terms and Conditions

3. Fig 2
Expression of vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGFR-1, VEGFR-2, and VEGFR-3 in CD11b− and CD11b+ cells. A, Concentration of VEGF-A and VEGF-C secreted by CD11b− or CD11b+ cells. After magnetic cell sorting (MACS), 1 × 105 CD11b− or CD11b+ cells were incubated for 7 days and the supernatant analyzed by enzyme-linked immunosorbent assay (n = 3; mean [standard deviation]; ***P < . 001). B, Representative messenger ribonucleic acid (mRNA) of VEGFR-1, VEGFR-2, and VEGFR-3 from CD11b− and CD11b+ cells. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an internal control. Reverse transcription-polymerase chain reaction is representative of three separate experiments. C, Representative immunoblotting images for VEGFR-1, VEGFR-2, and VEGFR-3 from CD11b− and CD11b+ cells. After MACS sorting, 5 × 106 CD11b− and CD11b+ cells were lysed and examined by immunoblotting analysis. The staining of β-actin was used as an internal control. Blots are representative of three separate experiments. E11 and E13 used for positive control means embryonic day 11 and 13.
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2013 Society for Vascular Surgery Terms and Conditions

4. Fig 3
Vascular endothelial growth factor (VEGF)-C promotes neovascularization and functional recovery following hind limb ischemia. A, Representative laser Doppler perfusion images taken at the indicated intervals for the phosphate-buffered saline (PBS) control group and the VEGF-C group. Blood perfusion in the ischemic hind limbs was markedly increased in the VEGF-C group compared with the control group. Quantitative analysis of hind limb blood perfusion demonstrated an increase in the ischemic/nonischemic ratio, which was significantly higher in the VEGF-C group compared with control group at postoperative days 7 and 14 (day 7: 39.8% ± 12.0% vs 14.8 ± 6.7%; P < .01; day 14: 65.5% ± 11.6% vs 31.1 ± 12.0%; P < .05; n = 5-7). Data are expressed as mean ± SEM, n = 5-7 per group. B, The Tarlov score was significantly higher in the VEGF-C group compared with the control group at postoperative days 7 and 14 (day 7: 5.25 ± 0.5 vs 1.0 ± 0.81; P < .05; day 14: 5.75 ± 0.5 vs 3.42 ± 2.37; P < .05; n = 5). Ischemic scores show that the tissue grade was significantly higher in the VEGF-C group compared with the control group at day 7 postoperation (4.75 ± 0.5 vs 2.85 ± 1.06; P < .01; n = 5). C, Video analysis of mice traversing an illuminated glass walkway. The period of time during which each of the paws was in contact with the glass is illustrated as a contact duration map. The mean paw contact time and paw intensity per step for each limb, based on five separate experiments each with three replicates, are quantified in the graph below the map. *P < .05; **P < .01; ***P < .001.
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2013 Society for Vascular Surgery Terms and Conditions

5. Fig 4
Protein levels of vascular endothelial growth factor (VEGF)-A and VEGF-C in thigh muscle at postoperative days 7 and 14. A, There was no statistically significant difference in the level of VEGF-A expression between all groups (n = 3; mean [standard deviation]). B, The amount of VEGF-C from the thigh muscle after limb ischemia, 7 days after the intramuscular injection, revealed that CD11b+ cell-treated mice had significantly increased levels of VEGF-C compared with the other groups (n = 3; mean [standard deviation]; *P < .05).
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2013 Society for Vascular Surgery Terms and Conditions

6. Fig 5
Effect of CD11b+ cell and vascular endothelial growth factor (VEGF)-C treatment on capillary density 14 days after the induction of ischemia (n = 5 animals in each group). A, In these representative photomicrographs (original magnification ×400), von Willebrand factor positive and LYVE-1 positive cells are stained green and red, respectively. B, During angiogenesis, capillary density was significantly higher in the CD11b+ and VEGF-C groups than in the control group (1.88 ± 0.34 vs 0.36 ± 0.09; P < .001; 0.88 ± 0.29 vs 0.36 ± 0.09; P < .001). Furthermore, in lymphangiogenesis, capillary density was significantly upregulated in the CD11b+ and VEGF-C groups compared with control group (1.89 ± 0.26 vs 0.22 ± 0.12; P < .001; 1.19 ± 0.37 vs 0.22 ± 0.12; P < .001). *P < .05; **P < .01; ***P < C, Reverse transcription-polymerase chain reaction demonstrates VEGFR-1, VEGFR-2, and VEGFR-3 messenger ribonucleic acid (mRNA) expression in thigh muscle at postoperative days 7 and 14. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as the internal control. Representative images are shown (n = 3). Photomicrographs (original magnification ×400) show representative diaminobenzidine staining for VEGFR-1, VEGFR-2, and VEGFR-3. The thigh muscle of each group was isolated 14 days after induction of ischemia. Photomicrographs are representative of observations made in three animals from each group. D and E, Recovery of blood flow in the ischemic hind limb of the CD11b+ and VEGF-C groups was suppressed by administration of anti-VEGFR-2 antibody (DC101). The recovery of blood flow in ischemic limbs in the CD11b+ group treated with VEGFR-2-neutralizing antibody was significantly suppressed compared with the CD11b+-treated group at postoperative days 3 and 7. In the VEGF-C group treated with VEGFR-2-neutralizing antibody, blood flow recovery was significantly suppressed compared with the VEGF-C group at postoperative day 7. †P < .05; ‡P < .01.
Journal of Vascular Surgery  , DOI: ( /j.jvs )
Copyright © 2013 Society for Vascular Surgery Terms and Conditions