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Vascular endothelial growth factor-C derived from CD11b+ cells induces therapeutic improvements in a murine model of hind limb ischemia
From the Society for Vascular Surgery
Vascular endothelial growth factor-C derived from CD11bþ cells induces therapeutic improvements in a murine model of hind limb ischemia Go Kuwahara, MD,a,b Hitomi Nishinakamura, PhD,b Daibo Kojima, MD, PhD,b Tadashi Tashiro, MD, PhD,a and Shohta Kodama, MD, PhD,b Fukuoka, Japan Objective: The use of bone marrow cells (BMCs) in therapeutic angiogenesis has been studied extensively. However, the critical paracrine effects of this treatment are still unclear. Therefore, we studied autotransfusable cells that produce vascular endothelial growth factor (VEGF), especially VEGF-C. Methods: Male C57BL/6 mice with hind limb ischemia were administered intramuscular injections of phosphate-buffered saline as controls, or unsorted BMCs, sorted CD11bD, or CD11bL cells from BMCs, and recombinant VEGF-C. To evaluate the treatments, perfusion was measured by laser Doppler scanning performed on days 0, 1, 3, 7, 14, 21, and 28. A functional assay was performed in parallel, with mice traversing an enclosed walkway. Capillary density was determined by directly counting vessels stained positive with von Willebrand factor at individual time points. Lymphangiogenesis was assessed by LYVE-1 positive cells. Results: Postischemic recovery of hind limb perfusion significantly improved in BMC, CD11bD, and VEGF-C treatment groups compared with the control groups, as assessed by laser Doppler scanning. On early operative days 1 and 3, the blood flow recovery ratio was higher in the CD11bD-treated group compared with BMC and VEGF-C treatment groups. In the functional assay, the VEGF-C group dramatically recovered compared with the control group. The capillary/myofiber ratio in the thigh muscle and number of LYVE-1 positive cells was higher in the CD11bD and VEGF-C groups than in controls. Furthermore, expression of VEGF-A, VEGF-C, and VEGF receptor messenger ribonucleic acid and protein was observed in CD11bD cells. Conclusions: The VEGF-C derived from CD11bD cells play a critical role in angiogenesis and lymphangiogenesis in a murine model of hind limb ischemia. Consequently, treatment with self-CD11bD cells accelerated recovery from ischemia and may be a promising therapeutic strategy for peripheral arterial disease patients. (J Vasc Surg 2013;57:1090-9.) Clinical Relevance: The incidence of peripheral arterial disease is rapidly increasing commensurate with a rapid rise in the elderly population. Conventional therapy has a 5-year prognosis and survival rates <30%; thus, an alternative approach, termed therapeutic angiogenesis, has been developed, consisting of local injection of bone marrow cells (BMCs). However, the precise therapeutic mechanism, including paracrine effects, is unclear. We focused on CD11bD cells, which promote regulation of vascular endothelial growth factor-A and especially vascular endothelial growth factor-C. CD11bD treatment could reduce the total number of cell injections and improve the early operative points of blood flow compared with conventional therapy with BMCs. Consequently, CD11bD treatment accelerates recovery from ischemia and is helpful in reducing the need for repeated aspiration of BMCs.
The incidence of peripheral arterial disease (PAD) is rapidly increasing commensurate with a rapid rise in the elderly population.1 Critical limb ischemia is the end stage of lower extremity PAD, in which severe obstruction of From the Department of Cardiovascular Surgerya and the Department of Regenerative Medicine & Transplantation,b Faculty of Medicine, Fukuoka University. Supported by a Ministry of Education, Culture, Sports, Science and Technology of Japan Grant in-Aid for Scientific Research and Fukuoka University intramural funds. Author conflict of interest: none. Presented at the 2012 Vascular Annual Meeting of the Society for Vascular Surgery, National Harbor, Md, June 7-9, 2012. Reprint requests: Shohta Kodama, MD, PhD, Department of Regenerative Medicine & Transplantation, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City, Fukuoka Pref, 814-0180, Japan (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214/$36.00 Copyright Ó 2013 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2012.08.121
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blood flow results in ischemic rest pain, ulcers, and significant risk of limb loss. In patients with severe limbthreatening ischemia, whether surgical or endovascular revascularization treatment is applied to improve the blood flow must be determined. Approximately 20% to 30% of patients with critical limb ischemia are not considered candidates for vascular or endovascular procedures, which often leaves amputation as the final option.2 Leg amputation due to atherosclerotic PAD results in an acute mortality rate of around 30% and a 5-year prognosis with survival rates <30%.3 Therefore, in addition to conventional therapy, which includes percutaneous intervention and surgical bypass, novel therapies are needed to treat this disorder. Alternative approaches, termed therapeutic angiogenesis, have been developed, which include local intramuscular injection of autologous adult bone marrow cells (BMCs) and growth factors such as vascular endothelial growth factor (VEGF)-A.4,5 Accumulating evidence has indicated that BMCs contribute to neovascularization and tissue repair through paracrine/autocrine mechanisms rather
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than through differentiation to neovessels themselves.6-8 However, the optimal cell type is unknown. The role of VEGF-A in angiogenesis has been studied extensively in vivo and in vitro.9 Despite supporting evidence from animal studies, there has been a discrepancy in translating the angiogenic capacity of growth factors into benefits for individuals with PAD.10 The reason for these disappointing results is the side effects of VEGF-A, which increases permeability, causing significant edema in PAD patients.5,11 Otherwise, VEGF-C is considered an important lymphangiogenic factor that enhances venous return and lymphatic drainage from the ischemic limbs, which may have the potential to improve ischemic limbs in PAD patients. In this report, we focused on bone marrow-derived CD11bþ cells, which produce VEGF-C and VEGF-D,12 and the critical lymphangiogenic factor VEGF-C, which could improve venous congestion and lymphedema. We hypothesized that VEGF-C derived from CD11bþ cells may be important in angio- and lymphangiogenesis of ischemic tissue. Using a murine model of hind limb ischemia, we assessed whether CD11bþ cells promoted angio- and lymphangiogenesis. Furthermore, we demonstrated that lymphangiogenesis induced by VEGF-C through the VEGFR-3 signaling pathway has a synergistic therapeutic effect in a murine model of hind limb ischemia. METHODS Animal model and procedures. Twelve-week-old male C57BL/6N mice were purchased from Charles River Laboratories (Saga, Japan). The care of mice and experimental procedures complied with the “Principles of Laboratory Animal Care” (Guide for the Care and Use of Laboratory Animals, National Institutes of Health publication 86-23, 1985). The experimental protocol was approved by the Animal Care and Use Committee of Fukuoka University. Mouse hind limb ischemia was induced as previously described.13,14 Briefly, the left femoral artery and vein were gently excised from the proximal portion of the femoral artery to the distal portion of the saphenous artery. The remaining arterial branches, including the perforator arteries, also were excised. The contralateral hind limb served as an internal control. Measurement of hind limb blood flow. Blood flow was monitored using a laser Doppler perfusion imager (Moor Instruments Ltd, Devon, United Kingdom). Blood flow was expressed as a ratio of the ischemic and nonischemic hind limbs. In additional experiments, mice were randomized to receive systemic administration of neutralizing antibody to VEGFR-2 (800 mg of DC101; BioXCell, West Lebanon, NH) twice a week for 2 weeks (n ¼ 5 per group).15 Enrichment of CD11bD cells from BMCs and flow cytometric analysis. After 8-week-old male C57BL/6N mice were sacrificed, bone marrow from the femur and tibia was collected. CD11bþ cells that include macrophages and dendritic cells in the BMCs were stained with fluorescein isothiocyanate-conjugated rat anti-mouse CD11b (BD Biosciences, San Jose, Calif) and antifluorescein isothiocyanate MicroBeads (Miltenyi Biotec,
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Gladbach, Germany) and enriched by using a magnetic cell sorter (MACS; Miltenyi Biotec). Then the cells were analyzed by flow cytometry (BD FACSCanto II; BD Biosciences) using Cell Quest software. Data were analyzed using FlowJo software (BD Biosciences).16 In vivo tissue implant angiogenesis assay. The mice were randomly divided into five groups (n ¼ 5 per group). Treatment groups received phosphate-buffered saline (PBS; controls), 1 107 unselected BMCs, 1 106 CD11bþ cells, 1 106 CD11b cells, or 5 mg of recombinant human VEGF-C (Cys156Ser; R&D Systems, Minneapolis, Minn) in a total volume of 100 mL in PBS, injected percutaneously into the thigh muscle at three points using a 27-gauge needle. Clinical score of hind limb ischemia in mice. Mice were examined at 7 and 14 days after induction of hind limb ischemia. Functional grading was performed according to the Tarlov scale.17 To precisely evaluate the mobility of mice after limb ischemia, we used a previously described scoring system, where 0 ¼ normal, 1 ¼ pale foot or gait abnormalities, 2 ¼ gangrenous tissue in less than half of the foot without lower limb necrosis, 3 ¼ gangrenous tissue in less than half of the foot with lower limb necrosis, 4 ¼ gangrenous tissue in more than half of the foot, and 5 ¼ loss of half of the lower limb.18 The clinical outcome of all mice was observed and recorded at the same points as blood flow measurement. Gait analysis. Mice were assessed for functional recovery 7 days after hind limb ischemia by determining the extent to which the injured limb was used for walking. Each mouse traversed an enclosed walkway (CatWalk system; Noldus, Wageningen, The Netherlands) three times. In this system, a light-emitting diode from a fluorescent lamp illuminates the inside of a glass floor plate of the walkway. Light is reflected downward at the site of the paw in contact with the upper surface of the glass plate, the intensity of which is proportional to the relative force being exerted by the paw. Light reflection was recorded with a video camera and analyzed to determine the duration of paw contact with the floor and the mean pixel intensity at contact.19 Immunofluorescent staining. Mice were sacrificed at 7 and 14 days after surgery. The thigh adductor muscles of the legs were harvested, fixed in 10% formaldehyde solution, and embedded in paraffin. Muscle fiber number was examined in sections stained with hematoxylineeosin, and the mean counts from five separate fields in four distinct areas in each specimen were calculated. Capillary endothelial cells were identified by immunohistochemical staining with von Willebrand factor antibody (DAKO, Glostrup, Denmark).20 Capillary density was expressed as the ratio of von Willebrand factor positive cells to myofibers per high power field (magnification 400). Furthermore, lymphangiogenesis was assessed by LYVE-1 antibody (Relia Tech GmbH, Wolfenbüttel, Germany).21 Immunohistochemistry. The thigh adductor muscles of native and treated mice were fixed in 10% formaldehyde solution, processed, and embedded in paraffin. The sections
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were stained immunohistochemically with anti-VEGFR-1, anti-VEGFR-2, and anti-VEGFR-3 antibody (Abcam, Tokyo, Japan), then detected by diaminobenzidine staining. Reverse transcription-polymerase chain reaction analysis. Total RNA was isolated from thigh muscle with PureLink RNA Mini kit (Life Technologies, Carlsbad, Calif). Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using high-capacity cDNA reverse transcription kits (Applied Biosystems, Carlsbad, Calif). The following oligonucleotides were used: glyceraldehyde-3-phosphate dehydrogenase (G3PDH): 50 -ACA TCAAGAAGGTAGTGAAGCTGGC-30 , 50 -TTCAAGAGATTAGGGCTCCCTAGG-30 ; VEGFR-1: 50 -TACCTC AAGAGCAAACGTGAC-30 , 50 -CGATGAATGCACTTT CTGG-30 ; VEGFR-2: 50 -CTAAGGGCATGGAGTTCT TG-30 , 50 -CATACAGGAAACAGGTGAGGT-30 ; and VE GFR-3: 50 -CTATGGCTGAGCCCAATGAC-30 , 50 -ACCT TATCAAAGATGCTCTCGG-30 .22 The annealing temperature and the PCR cycles were G3PDH: 60 C, 28 cycles; VEGFR-1: 60.8 C, 35 cycles; VEGFR-2: 58.1 C, 30 cycles; and VEGFR-3: 60.8 C, 35 cycles. Immunoblotting. Thigh muscle and embryo were homogenized at 4 C in lysis buffer (Cell Signaling Technology, Danvers, Mass) with protease inhibitor cocktail (Roche, Mannheim, Germany) and then centrifuged at 13,000 rpm for 15 minutes. The supernatant was collected. Protein concentrations were measured using a bicinchoninate acid protein assay kit (ThermoFisher Scientific, Waltham, Mass). Equal amounts of protein from each experimental group were loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by immunoblotting. The membranes were proved with anti-VEGFR-1 (R&D Systems), anti-VEGFR-2 (Cell Signaling Technology ), anti-VEGFR-3 (eBioscience, San Diego, Calif) and anti-b-actin (Cell Signaling Technology). Membrane signals were detected using the ECL reagent (GE Healthcare, Buckinghamshire, United Kingdom). Enzyme-linked immunosorbent assay. Thigh muscle was homogenized at 4 C in PBS with protease inhibitor cocktail (Roche) and then centrifuged at 13,000 rpm for 15 minutes. The supernatant was collected. Protein levels for VEGF-A and VEGF-C were determined by enzymelinked immunosorbent assay (R&D Systems and Life Science Inc, St. Petersburg, Fla, respectively) as previously described.23 Statistical analysis. Statistical analysis was performed using PASW Statistics 18.0 (IBM Japan, Tokyo, Japan). A two-tailed Student t-test, paired Student t-test, or oneway analysis of variance and Mann-Whitney U test were used for statistical analysis. P < .05 was considered statistically significant. RESULTS Effect of implanted BMCs in ischemic limb muscle. Before the experiments, all mice had similar baseline blood flow in the leg and functional status. In the PBS control group, the blood flow decreased rapidly after limb ischemia and returned slowly, achieving 60% of
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preprocedural flow after 4 weeks (Fig 1, A). Blood flow recovery was significantly improved in mice treated with 1 107 BMCs at 7 and 14 days after the operative procedure. At 21 and 28 days, the BMC-treated group demonstrated a trend toward increased blood flow recovery compared with the control group, but this was not significantly different (Fig 1, A). Recovery of blood flow in the ischemic limbs treated with CD11bD cells. To determine whether BMCs contained enriched CD11bþ cells, we performed flow cytometric analysis of CD11bþ cells from total BMCs. Unseparated BMCs contained 29.59% CD11bþ cells. After MACS sorting, the purity of CD11bþ cells was 95.56% (Fig 1, B). We next measured the recovery blood flow in ischemic limbs treated with purified CD11bþ cells. Mice were treated with 1 106 CD11bþ cells, CD11b cells, or PBS for controls. To determine the cell number of CD11bþ cells, three different numbers of CD11bþ cells (1 104, 1 105, and 1 106) were investigated as a preliminary study. The 1 106 CD11bþ cells had a statistically significant difference as the lowest number in a preliminary study. The control animals were measured at each time point. On the earliest operative days 1 and 3, the blood flow recovery ratio was higher in the CD11bþ-treated group compared with the control and CD11b groups. Moreover, blood flow recovery was significantly improved in the CD11bþ-treated group compared with the control and CD11b groups at 7 and 14 days after the operative procedure. At 21 and 28 days, there was no significant difference between blood flow recovery of the CD11bþ-treated group compared with the control and CD11b groups (Fig 1, C). Thus, mice treated with 1 106 CD11bþ cells showed an improved blood flow, the same as the 1 107 BMC-treated mice at multiple time points (Fig 1, A and C). Protein expression of VEGF-A, VEGF-C, VEGFR1, VEGFR-2, and VEGFR-3. To identify the production of VEGF-A and VEGF-C from CD11bþ cells, we measured levels of VEGF-A and VEGF-C in cultured supernatant from CD11bþ and CD11b cells. Enzyme-linked immunosorbent assay analyses showed significantly increased levels of VEGF-A and VEGF-C in the cultured supernatant of CD11bþ cells compared with CD11b cells. Although VEGF-A was secreted by both CD11bþ and CD11b cells, VEGF-C was not secreted from cultured CD11b cells (Fig 2, A). The VEGFR-1 and VEGFR-3 messenger ribonucleic acid (mRNA) were expressed in CD11bþ cells, but VEGFR-2 mRNA was not detected in either CD11bþ or CD11b cells (Fig 2, B). Furthermore, we confirmed that CD11bþ and CD11b cells expressed VEGFR-1 and VEGFR-3 protein (Fig 2, C). These results suggest the possibility that CD11bþ cells are activated by autocrine VEGF-A and VEGF-C via VEGFR-1 and VEGFR-3, respectively. Recovery of blood flow in ischemic limbs treated with recombinant VEGF-C. We compared treatment of mice with 5 mg of recombinant VEGF-C with PBStreated controls in restoring blood flow to ischemic hind
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Fig 1. Recovery of blood flow in ischemic limbs treated with bone marrow cells (BMCs) and CD11bþ cells derived from BMCs. A and C, Representative laser Doppler perfusion images taken at indicated intervals for the phosphatebuffered saline (PBS) control group, unselected BMCs group, CD11b group, and CD11bþ group. Blood perfusion in the ischemic hind limbs was markedly increased in the unselected BMCs and CD11bþ 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% 6 9.0% vs 14.8 6 6.7%; P < .01; day 14: 72.2% 6 7.4% vs 31.1 6 12.0%; P < .05; n ¼ 5-7). The increase in the ischemic/nonischemic ratio was significantly higher in the CD11bþ group compared with the CD11b group at postoperative days 1, 3, 7, and 14 ( day 1: 17.1% 6 4.0% vs 12.3 61.7%; P < .05; day 3: 26.4% 6 3.1% vs 12.3% 6 7.2%; P < .05; day 7: 48.4% 6 16.0% vs 20.7% 6 8.6%; P < .01; day 14: 85.7% 6 23.3% vs 45.7% 6 4.8%; P < .01; n ¼ 5-7). Data are given as mean 6 SEM, n ¼ 5-7 per group. *P < .05; **P < .01; ***P < .001. B, Flow cytometric analysis of CD11bþ macrophages from BMCs. The purity of subpopulations of the enriched CD11bþ macrophages was approximately 95% according to the FACS analysis.
limbs. To determine the dosage of VEGF-C, four different doses of VEGF-C, adjusted to 3, 5, 12.5, and 25 mg, were injected as a preliminary study. The 5 mg of VEGF-C had a statistically significant difference as the lowest dose in a preliminary study. The control animals were measured at each time point. Blood perfusion in the ischemic hind limbs was markedly increased in the 5 mg VEGF-C-treated group compared with controls at multiple time points. The increase in the ischemia/nonischemic ratio was significantly upregulated in the 5 mg VEGF-C-treated group compared with the control group at 7 and 14 days after operative procedure. At 21 and 28 days, the 5 mg VEGF-C-treated group showed a better recovery ratio than the control
group, but there was no significant difference in the recovery of blood flow between the groups (Fig 3, A). Functional recovery following hind limb ischemia by intramuscular injection of VEGF-C. To evaluate more precisely the functional deficits and ischemic grade of ischemic limbs, we tested mice according to the Tarlov score for function and Ischemic score for ischemia. Using the Tarlov score, the VEGF-C-treated group showed a significantly increased functional status at 7 and 14 days compared with the control group. Using the Ischemic scoring system, the VEGF-C group showed a significantly increased level of ischemic tissue grade at day 7 compared with the control group. At 14 days, there were no
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Fig 2. Expression of vascular endothelial growth factor (VEGF)A, VEGF-C, VEGFR-1, VEGFR-2, and VEGFR-3 in CD11b and CD11bþ cells. A, Concentration of VEGF-A and VEGF-C secreted by CD11b or CD11bþ cells. After magnetic cell sorting (MACS), 1 105 CD11b or CD11bþ cells were incubated for 7 days and the supernatant analyzed by enzyme-linked immunosorbent assay (n ¼ 3; mean [standard deviation]; ***P < . 001). B, Representative messenger ribonucleic acid (mRNA) of VEGFR-1, VEGFR-2, and VEGFR-3 from CD11b and CD11bþ cells. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an internal control. Reverse transcriptionpolymerase chain reaction is representative of three separate experiments. C, Representative immunoblotting images for VEGFR-1, VEGFR-2, and VEGFR-3 from CD11b and CD11bþ cells. After MACS sorting, 5 106 CD11b and CD11bþ cells were lysed and examined by immunoblotting analysis. The staining of b-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.
significant differences in tissue grade of ischemic limbs between the VEGF-C and control groups. In addition, there were no significant differences in functional status and in ischemic tissue grade at 7 and 14 days between the VEGF-C and CD11bþ-treated groups (Fig 3, B). Furthermore, we performed gait analysis in the VEGFC-treated and control groups. The control (PBS) group could not use the injured limb (left hind limb) for walking, whereas mice from the VEGF-C-treated group could use both hind limbs for walking. The blue arrowhead in the contact duration map in Fig 3, C, depicts the pixel intensity generated by the mouse paws upon contact with the glass floor. Pixel intensity generated by the three uninjured limbs in the control group was higher compared with the VEGFC-treated group. These findings indicate that functional recovery of ischemic limbs in the VEGF-C-treated group was dramatically improved compared with the control group. In quantitative analysis, paw contact time was
significantly prolonged in ischemic limbs of the VEGFC-treated group compared with the control group (0.17 6 0.13 s vs 0.02 6 0.05 s; P < .05). In addition, paw intensity was significantly higher in the ischemic limbs of the VEGF-C-treated group compared with the control group (71.9 6 22.7 arbitrary units vs 31.6 6 45.0 arbitrary units; P < .01; Fig 3, C). Protein levels of VEGF-A and VEGF-C in thigh muscle. There was no statistical difference in VEGF-A protein expression in the thigh muscle at 7 and 14 days after limb ischemia in the CD11bþ-treated, VEGFC-treated, and control groups (Fig 4, A). However, 7 days after the intramuscular injection, the CD11bþ cell-treated group secreted significantly more VEGF-C protein in the thigh muscle after limb ischemia compared with the other groups (P < .05; Fig 4, B), indicating a crucial role of VEGF-C in angiogenesis. Expression of angiogenesis and lymphangiogenesis marker. After limb ischemia, both the CD11bþ and VEGF-C groups had significantly increased numbers of von Willebrand factor positive cells and LYVE-1 positive cells compared with the control group (Fig 5, A). Capillary density, representing angiogenesis, was significantly higher in the CD11bþ-treated and VEGF-C-treated groups compared with the control group. Furthermore, in lymphangiogenesis, capillary density was significantly upregulated in the CD11bþ-treated and VEGF-C-treated groups compared with the control group (Fig 5, B). These findings suggest increased blood flow and functional recovery after hind limb ischemia when treated with CD11bþ cells or recombinant VEGF-C. In addition, RT-PCR and histologic analysis showed that VEGFR-1, VEGFR-2, and VEGFR-3 mRNA and protein were expressed in ischemic muscle at days 7 and 14 after hind limb ischemia in the control, CD11bþ, and VEGF-C-treated mice (Fig 5, C). Effect of VEGFR-2-neutralizing antibody treatment. Recovery of blood flow in ischemic hind limbs of the CD11bþ and VEGF-C-treated groups was suppressed by systemic administration of anti-VEGFR-2 (DC101) on postoperative day 7. Although recovery of blood flow in ischemic limbs was reduced dramatically, the ischemic limbs of CD11bþ and VEGF-C-treated groups showed augmented recovery of hind limb perfusion, and blood flow returned to the same level of hind limb perfusion before administration of the anti-VEGFR-2 antibody (Fig 5, D and E). These results indicate that VEGF-A via VEGFR-2 signaling may have an important role in angiogenesis, and that VEGF-C may function as a primary or adjunctive agent for therapeutic angiogenesis. DISCUSSION In this study, we analyzed treatment with CD11bþ cells derived from BMCs. Furthermore, our data clearly demonstrate that intramuscular injection of CD11bþ cells that secreted VEGF-C improved recovery of blood flow in a murine model of hind limb ischemia. In addition, CD11bþ treatment could reduce the total number of cell injections and improve the early operative points of blood
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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 phosphatebuffered saline (PBS) control group and the VEGF-C group. Blood perfusion in the ischemic hind limbs was markedly
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flow compared with conventional therapy with BMCs. The novelty and advantage of using CD11bþ cells is the secretion of a distinct combination of the angiogenic factor VEGF-A and the lymphangiogenic factor VEGF-C, and their substantial in vivo capacity to induce neovascularization. In our report, VEGF-A protein was secreted by cultured CD11bþ cells and a few CD11b cells. In contrast, VEGF-C was secreted by cultured CD11bþ cells but not CD11b cells. Although hind limb ischemia has been improved by the administration of VEGF-A in animal models, there has been little improvement in translating the angiogenic capacity of VEGF-A to benefit PAD patients.24,25 Because VEGF-A increases vessel permeability, many patients receiving intramuscular injection of VEGF-A developed complications such as edema.26 Vascular leakage resulting in tissue edema is a major problem in therapeutic angiogenesis. However, coadministration of VEGF and angiopoietin-1 has modified angiogenesis and reduced vascular permeability in ischemic hind limb models.27,28 These combination gene therapies are currently being examined in animal models in preparation for translation to clinical human therapy. Revascularization has good outcome in patients with severe PAD, but it is well known that the addition of chronic venous insufficiency to PAD makes treatment more difficult. Many patients suffer with compromised occlusive arterial disease and venous congestion. Therefore, in patients with critical limb ischemia for whom the current treatment options are not effective, enhancing venous return and lymphatic drainage from the ischemic limb may have the potential to improve ischemic limbs. The VEGF-C is a specific ligand for the endothelial receptor tyrosine kinases VEGFR-2 and VEGFR-3, and it mediates its lymphangiogenic effects through VEGFR-3.29 Furthermore, recent studies showed that blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation,30 and that upregulation of Notch-dependent VEGFR-3 allows angiogenesis without VEGF-A through VEGFR-2 signaling.31 We focused on VEGF-C and evaluated the ectopic application of recombinant VEGF-C to induce potent angiogenic effects in vivo. Interestingly, intramuscular injection of exogenous VEGF-C can enhance the recovery of blood flow in ischemic limbs and the function of walking, indicating that enhancing
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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 ¼ 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 CD11bþ cell-treated mice had significantly increased levels of VEGF-C compared with the other groups (n ¼ 3; mean [standard deviation]; *P < .05).
lymphatic drainage and venous return accelerates the recovery of hind limb ischemia. A critical role of VEGFC in angiogenesis was suggested by Witzenbichler et al,32 who reported that VEGF-C promotes angiogenesis in a rabbit model of hind limb ischemia. In accordance with this finding, VEGF-C induces neovascularization in the mouse cornea micropocket assay.33 Concurrently, Bauer et al34 demonstrated that VEGF-C overexpression,
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% 6 12.0% vs 14.8 6 6.7%; P < .01; day 14: 65.5% 6 11.6% vs 31.1 6 12.0%; P < .05; n ¼ 5-7). Data are expressed as mean 6 SEM, n ¼ 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 6 0.5 vs 1.0 6 0.81; P < .05; day 14: 5.75 6 0.5 vs 3.42 6 2.37; P < .05; n ¼ 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 6 0.5 vs 2.85 6 1.06; P < .01; n ¼ 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.
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Fig 5. Effect of CD11bþ cell and vascular endothelial growth factor (VEGF)-C treatment on capillary density 14 days after the induction of ischemia (n ¼ 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.
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using fibroblasts, stimulates multiple biologic processes known to impact ischemic wound healing, such as collagen constriction in three-dimensional gels, capillary sprouting, and endothelial progenitor cell invasion and migration through the extracellular matrix. These studies further enhance the evidence for VEGF-C as an important angiogenic factor as well as a lymphangiogenic factor. To examine the autocrine effects of CD11bþ cells, we analyzed cultured CD11bþ and CD11b cells by RT-PCR and Western blotting. Our data show that VEGFR-1 and VEGFR-3 mRNA and protein, but not VEGFR-2, were detected in cultured CD11bþ and CD11b cells. These results indicate that the autocrine effects of CD11bþ cells are not the main regulator in promoting angiogenesis through VEGFR-2 for self-renewal. However, not only VEGFR-1 and VEGFR-3 but also VEGFR-2 was detected by RT-PCR analysis and immunohistochemical analysis in ischemic hind limbs. These results indicate that the VEGF-A and VEGF-C produced by CD11bþ cells or recombinant VEGF-C activate endothelial cells or migrated macrophages through VEGFR signaling transduction. In addition, we showed that neutralization of VEGFR-2 inhibits the recovery of blood flow in ischemic hind limbs in CD11bþ cell and VEGF-C group mice until 7 days postoperation. In the CD11bþ cell group treated with VEGFR-2-neutralizing antibody, these results indicate that VEGF-C derived from CD11bþ cell through VEGFR-3 signaling contributes to the recovery of blood flow including endogenous VEGF-C secretion. On the other hand, in the VEGF-C group, recombinant VEGFC through VEGFR-3 signaling has a role in angiogenesis in blood flow recovery in a murine model of hind limb ischemia including endogenous VEGF-C secretion, as it was previously reported that VEGF-C (Cys156) induced a specific signal through VEGFR-3 but not VEGFR-2.35 These results demonstrate that VEGF-C signaling through VEGFR-3 has a synergistic action with VEGF-C signaling through VEGFR-2 in angiogenesis. Accordingly, CD11bþ cells that secrete both VEGF-A and VEGF-C have a greater potential to accelerate restoration from ischemia. Furthermore, VEGF-C signaling through VEGFR-3 plays a critical role in lymphangiogenesis in improving hind limb ischemia, even if the VEGFR-2
inhibitor suppressed the VEGF-A or VEGF-C secreted from CD11bþ cells. Although further investigations of the molecular cascades involved in VEGF-C-mediated effects in ischemia and in vivo confirmation are needed, these data offer insights into novel methods to promote angiogenesis and lymphangiogenesis during ischemia. Collectively, by focusing on CD11bþ cells and VEGF-C as novel targets, these findings might contribute to the development of new therapeutic strategies for limb ischemia. AUTHOR CONTRIBUTIONS Conception and design: GK, HN Analysis and interpretation: GK, HN, DK, SK Data collection: GK, HN, DK Writing the article: GK, HN, SK Critical revision of the article: GK, HN, Final approval of the article: GK, HN, DK, TT, SK Statistical analysis: GK, HN Obtained funding: TT, SK Overall responsibility: SK GK and HN contributed equally to this work. REFERENCES 1. Aronow H. Peripheral arterial disease in the elderly: recognition and management. Am J Cardiovasc Drugs 2008;8:353-64. 2. Lawall H, Bramlage P, Amann B. Treatment of peripheral arterial disease using stem and progenitor cell therapy. J Vasc Surg 2011;53:445-53. 3. Adam DJ, Beard JD, Cleveland T, Bell J, Bradbury AW, Forbes JF, et al. Bypass versus angioplasty in severe ischaemia of the leg (BASIL): multicentre, randomised controlled trial. Lancet 2005;366:1925-34. 4. Losordo DW, Dimmeler S. Therapeutic angiogenesis and vasculogenesis for ischemic disease: part II: cell-based therapies. Circulation 2004;109:2692-7. 5. Masaki I, Yonemitsu Y, Yamashita A, Sata S, Tanii M, Komori K, et al. Angiogenic gene therapy for experimental critical limb ischemia: acceleration of limb loss by overexpression of vascular endothelial growth factor 165 but not of fibroblast growth factor-2. Circ Res 2002;90:966-73. 6. Gupta R, Losordo DW. Cell therapy for critical limb ischemia: moving forward one step at a time. Circ Cardiovasc Interv 2011;4:2-5. 7. Shireman PK. The chemokine system in arteriogenesis and hind limb ischemia. J Vasc Surg 2007;45(Suppl A):A48-56. 8. Silvestre JS, Mallat Z, Tedgui A, Levy BI. Post-ischaemic neovascularization and inflammation. Cardiovasc Res 2008;78:242-9. 9. Tammela T, Enholm B, Alitalo K, Paavonen K. The biology of vascular endothelial growth factors. Cardiovasc Res 2005;65:550-63.
B, During angiogenesis, capillary density was significantly higher in the CD11bþ and VEGF-C groups than in the control group (1.88 6 0.34 vs 0.36 6 0.09; P < .001; 0.88 6 0.29 vs 0.36 6 0.09; P < .001). Furthermore, in lymphangiogenesis, capillary density was significantly upregulated in the CD11bþ and VEGF-C groups compared with control group (1.89 6 0.26 vs 0.22 6 0.12; P < .001; 1.19 6 0.37 vs 0.22 6 0.12; P < .001). *P < .05; **P < .01; ***P < .001. 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-3phosphate dehydrogenase (G3PDH) was used as the internal control. Representative images are shown (n ¼ 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 CD11bþ and VEGF-C groups was suppressed by administration of anti-VEGFR-2 antibody (DC101). The recovery of blood flow in ischemic limbs in the CD11bþ group treated with VEGFR2-neutralizing antibody was significantly suppressed compared with the CD11bþ-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. yP < .05; zP < .01.
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10. Losordo DW, Dimmeler S. Therapeutic angiogenesis and vasculogenesis for ischemic disease. Part I: angiogenic cytokines. Circulation 2004;109:2487-91. 11. Jain RK. Molecular regulation of vessel maturation. Nat Med 2003;9: 685-93. 12. Cursiefen C, Chen L, Borges LP, Jackson D, Cao J, Radziejewski C, et al. VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J Clin Invest 2004;113:1040-50. 13. Murohara T, Asahara T, Silver M, Bauters C, Masuda H, Kalka C, et al. Nitric oxide synthase modulates angiogenesis in response to tissue ischemia. J Clin Invest 1998;101:2567-78. 14. Takeshita K, Satoh M, Ii M, Silver M, Limbourg FP, Mukai Y, et al. Critical role of endothelial Notch1 signaling in postnatal angiogenesis. Circ Res 2007;100:70-8. 15. Baluk P, Tammela T, Ator E, Lyubynska N, Achen MG, Hicklin DJ, et al. Pathogenesis of persistent lymphatic vessel hyperplasia in chronic airway inflammation. J Clin Invest 2005;115:247-57. 16. Kodama S, Kuhtreiber W, Fujimura S, Dale EA, Faustman DL. Islet regeneration during the reversal of autoimmune diabetes in NOD mice. Science 2003;302:1223-7. 17. Westvik TS, Fitzgerald TN, Muto A, Maloney SP, Pimiento JM, Fancher TT, et al. Limb ischemia after iliac ligation in aged mice stimulates angiogenesis without arteriogenesis. J Vasc Surg 2009;49: 464-73. 18. Yu J, deMuinck ED, Zhuang Z, Drinane M, Kauser K, Rubanyi GM, et al. Endothelial nitric oxide synthase is critical for ischemic remodeling, mural cell recruitment, and blood flow reserve. Proc Natl Acad Sci U S A 2005;102:10999-1004. 19. Masocha W, Parvathy SS. Assessment of weight bearing changes and pharmacological antinociception in mice with LPS-induced monoarthritis using the Catwalk gait analysis system. Life Sci 2009;85: 462-9. 20. Iwasaki K, Kojima K, Kodama S, Paz AC, Chambers M, Umezu M, et al. Bioengineered three-layered robust and elastic artery using hemodynamically-equivalent pulsatile bioreactor. Circulation 2008;118: S52-7. 21. Hirakawa S, Brown LF, Kodama S, Paavonen K, Alitalo K, Detmar M. VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites. Blood 2007;109:1010-7. 22. Seo S, Fujita H, Nakano A, Kang M, Duarte A, Kume T. The forkhead transcription factors, Foxc1 and Foxc2, are required for arterial specification and lymphatic sprouting during vascular development. Dev Biol 2006;294:458-70.
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23. Huggenberger R, Siddiqui SS, Brander D, Ullmann S, Zimmermann K, Antsiferova M, et al. An important role of lymphatic vessel activation in limiting acute inflammation. Blood 2011;117:4667-78. 24. Simons M, Ware JA. Therapeutic angiogenesis in cardiovascular disease. Nat Rev Drug Discov 2003;2:863-71. 25. Simons M. Angiogenesis: where do we stand now? Circulation 2005;111:1556-66. 26. Weis S, Shintani S, Weber A, Kirchmair R, Wood M, Cravens A, et al. Src blockade stabilizes a Flk/cadherin complex, reducing edema and tissue injury following myocardial infarction. J Clin Invest 2004;113: 885-94. 27. Chae JK, Kim I, Lim ST, Chung MJ, Kim WH, Kim HG, et al. Coadministration of angiopoietin-1 and vascular endothelial growth factor enhances collateral vascularization. Arterioscler Thromb Vasc Biol 2000;20:2573-8. 28. Yamauchi A, Ito Y, Morikawa M, Kobune M, Huang J, Sasaki K, et al. Pre-administration of angiopoietin-1 followed by VEGF induces functional and mature vascular formation in a rabbit ischemic model. J Gene Med 2003;5:994-1004. 29. Tammela T, Alitalo K. Lymphangiogenesis: molecular mechanisms and future promise. Cell 2010;140:460-76. 30. Tammela T, Zarkada G, Wallgard E, Murtomaki A, Suchting S, Wirzenius M, et al. Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature 2008;454:656-60. 31. Benedito R, Rocha SF, Woeste M, Zamykal M, Radtke F, Casanovas O, et al. Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling. Nature 2012;484:110-4. 32. Witzenbichler B, Asahara T, Murohara T, Silver M, Spyridopoulos I, Magner M, et al. Vascular endothelial growth factor-C (VEGF-C/ VEGF-2) promotes angiogenesis in the setting of tissue ischemia. Am J Pathol 1998;153:381-94. 33. Cao Y, Linden P, Farnebo J, Cao R, Eriksson A, Kumar V, et al. Vascular endothelial growth factor C induces angiogenesis in vivo. Proc Natl Acad Sci U S A 1998;95:14389-94. 34. Bauer SM, Bauer RJ, Liu ZJ, Chen H, Goldstein L, Velazquez OC. Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels. J Vasc Surg 2005;41:699-707. 35. Joukov V, Kumar V, Sorsa T, Arighi E, Weich H, Saksela O, et al. A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities. J Biol Chem 1998;273:6599-602. Submitted Jun 14, 2012; accepted Aug 29, 2012.
Vascular endothelial growth factor-C derived from CD11bþ cells induces therapeutic improvements in a murine model of hind limb ischemia Go Kuwahara, MD,a,b Hitomi Nishinakamura, PhD,b Daibo Kojima, MD, PhD,b Tadashi Tashiro, MD, PhD,a and Shohta Kodama, MD, PhD,b Fukuoka, Japan Objective: The use of bone marrow cells (BMCs) in therapeutic angiogenesis has been studied extensively. However, the critical paracrine effects of this treatment are still unclear. Therefore, we studied autotransfusable cells that produce vascular endothelial growth factor (VEGF), especially VEGF-C. Methods: Male C57BL/6 mice with hind limb ischemia were administered intramuscular injections of phosphate-buffered saline as controls, or unsorted BMCs, sorted CD11bD, or CD11bL cells from BMCs, and recombinant VEGF-C. To evaluate the treatments, perfusion was measured by laser Doppler scanning performed on days 0, 1, 3, 7, 14, 21, and 28. A functional assay was performed in parallel, with mice traversing an enclosed walkway. Capillary density was determined by directly counting vessels stained positive with von Willebrand factor at individual time points. Lymphangiogenesis was assessed by LYVE-1 positive cells. Results: Postischemic recovery of hind limb perfusion significantly improved in BMC, CD11bD, and VEGF-C treatment groups compared with the control groups, as assessed by laser Doppler scanning. On early operative days 1 and 3, the blood flow recovery ratio was higher in the CD11bD-treated group compared with BMC and VEGF-C treatment groups. In the functional assay, the VEGF-C group dramatically recovered compared with the control group. The capillary/myofiber ratio in the thigh muscle and number of LYVE-1 positive cells was higher in the CD11bD and VEGF-C groups than in controls. Furthermore, expression of VEGF-A, VEGF-C, and VEGF receptor messenger ribonucleic acid and protein was observed in CD11bD cells. Conclusions: The VEGF-C derived from CD11bD cells play a critical role in angiogenesis and lymphangiogenesis in a murine model of hind limb ischemia. Consequently, treatment with self-CD11bD cells accelerated recovery from ischemia and may be a promising therapeutic strategy for peripheral arterial disease patients. (J Vasc Surg 2013;57:1090-9.) Clinical Relevance: The incidence of peripheral arterial disease is rapidly increasing commensurate with a rapid rise in the elderly population. Conventional therapy has a 5-year prognosis and survival rates <30%; thus, an alternative approach, termed therapeutic angiogenesis, has been developed, consisting of local injection of bone marrow cells (BMCs). However, the precise therapeutic mechanism, including paracrine effects, is unclear. We focused on CD11bD cells, which promote regulation of vascular endothelial growth factor-A and especially vascular endothelial growth factor-C. CD11bD treatment could reduce the total number of cell injections and improve the early operative points of blood flow compared with conventional therapy with BMCs. Consequently, CD11bD treatment accelerates recovery from ischemia and is helpful in reducing the need for repeated aspiration of BMCs.
The incidence of peripheral arterial disease (PAD) is rapidly increasing commensurate with a rapid rise in the elderly population.1 Critical limb ischemia is the end stage of lower extremity PAD, in which severe obstruction of From the Department of Cardiovascular Surgerya and the Department of Regenerative Medicine & Transplantation,b Faculty of Medicine, Fukuoka University. Supported by a Ministry of Education, Culture, Sports, Science and Technology of Japan Grant in-Aid for Scientific Research and Fukuoka University intramural funds. Author conflict of interest: none. Presented at the 2012 Vascular Annual Meeting of the Society for Vascular Surgery, National Harbor, Md, June 7-9, 2012. Reprint requests: Shohta Kodama, MD, PhD, Department of Regenerative Medicine & Transplantation, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka City, Fukuoka Pref, 814-0180, Japan (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214/$36.00 Copyright Ó 2013 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2012.08.121
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blood flow results in ischemic rest pain, ulcers, and significant risk of limb loss. In patients with severe limbthreatening ischemia, whether surgical or endovascular revascularization treatment is applied to improve the blood flow must be determined. Approximately 20% to 30% of patients with critical limb ischemia are not considered candidates for vascular or endovascular procedures, which often leaves amputation as the final option.2 Leg amputation due to atherosclerotic PAD results in an acute mortality rate of around 30% and a 5-year prognosis with survival rates <30%.3 Therefore, in addition to conventional therapy, which includes percutaneous intervention and surgical bypass, novel therapies are needed to treat this disorder. Alternative approaches, termed therapeutic angiogenesis, have been developed, which include local intramuscular injection of autologous adult bone marrow cells (BMCs) and growth factors such as vascular endothelial growth factor (VEGF)-A.4,5 Accumulating evidence has indicated that BMCs contribute to neovascularization and tissue repair through paracrine/autocrine mechanisms rather
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than through differentiation to neovessels themselves.6-8 However, the optimal cell type is unknown. The role of VEGF-A in angiogenesis has been studied extensively in vivo and in vitro.9 Despite supporting evidence from animal studies, there has been a discrepancy in translating the angiogenic capacity of growth factors into benefits for individuals with PAD.10 The reason for these disappointing results is the side effects of VEGF-A, which increases permeability, causing significant edema in PAD patients.5,11 Otherwise, VEGF-C is considered an important lymphangiogenic factor that enhances venous return and lymphatic drainage from the ischemic limbs, which may have the potential to improve ischemic limbs in PAD patients. In this report, we focused on bone marrow-derived CD11bþ cells, which produce VEGF-C and VEGF-D,12 and the critical lymphangiogenic factor VEGF-C, which could improve venous congestion and lymphedema. We hypothesized that VEGF-C derived from CD11bþ cells may be important in angio- and lymphangiogenesis of ischemic tissue. Using a murine model of hind limb ischemia, we assessed whether CD11bþ cells promoted angio- and lymphangiogenesis. Furthermore, we demonstrated that lymphangiogenesis induced by VEGF-C through the VEGFR-3 signaling pathway has a synergistic therapeutic effect in a murine model of hind limb ischemia. METHODS Animal model and procedures. Twelve-week-old male C57BL/6N mice were purchased from Charles River Laboratories (Saga, Japan). The care of mice and experimental procedures complied with the “Principles of Laboratory Animal Care” (Guide for the Care and Use of Laboratory Animals, National Institutes of Health publication 86-23, 1985). The experimental protocol was approved by the Animal Care and Use Committee of Fukuoka University. Mouse hind limb ischemia was induced as previously described.13,14 Briefly, the left femoral artery and vein were gently excised from the proximal portion of the femoral artery to the distal portion of the saphenous artery. The remaining arterial branches, including the perforator arteries, also were excised. The contralateral hind limb served as an internal control. Measurement of hind limb blood flow. Blood flow was monitored using a laser Doppler perfusion imager (Moor Instruments Ltd, Devon, United Kingdom). Blood flow was expressed as a ratio of the ischemic and nonischemic hind limbs. In additional experiments, mice were randomized to receive systemic administration of neutralizing antibody to VEGFR-2 (800 mg of DC101; BioXCell, West Lebanon, NH) twice a week for 2 weeks (n ¼ 5 per group).15 Enrichment of CD11bD cells from BMCs and flow cytometric analysis. After 8-week-old male C57BL/6N mice were sacrificed, bone marrow from the femur and tibia was collected. CD11bþ cells that include macrophages and dendritic cells in the BMCs were stained with fluorescein isothiocyanate-conjugated rat anti-mouse CD11b (BD Biosciences, San Jose, Calif) and antifluorescein isothiocyanate MicroBeads (Miltenyi Biotec,
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Gladbach, Germany) and enriched by using a magnetic cell sorter (MACS; Miltenyi Biotec). Then the cells were analyzed by flow cytometry (BD FACSCanto II; BD Biosciences) using Cell Quest software. Data were analyzed using FlowJo software (BD Biosciences).16 In vivo tissue implant angiogenesis assay. The mice were randomly divided into five groups (n ¼ 5 per group). Treatment groups received phosphate-buffered saline (PBS; controls), 1 107 unselected BMCs, 1 106 CD11bþ cells, 1 106 CD11b cells, or 5 mg of recombinant human VEGF-C (Cys156Ser; R&D Systems, Minneapolis, Minn) in a total volume of 100 mL in PBS, injected percutaneously into the thigh muscle at three points using a 27-gauge needle. Clinical score of hind limb ischemia in mice. Mice were examined at 7 and 14 days after induction of hind limb ischemia. Functional grading was performed according to the Tarlov scale.17 To precisely evaluate the mobility of mice after limb ischemia, we used a previously described scoring system, where 0 ¼ normal, 1 ¼ pale foot or gait abnormalities, 2 ¼ gangrenous tissue in less than half of the foot without lower limb necrosis, 3 ¼ gangrenous tissue in less than half of the foot with lower limb necrosis, 4 ¼ gangrenous tissue in more than half of the foot, and 5 ¼ loss of half of the lower limb.18 The clinical outcome of all mice was observed and recorded at the same points as blood flow measurement. Gait analysis. Mice were assessed for functional recovery 7 days after hind limb ischemia by determining the extent to which the injured limb was used for walking. Each mouse traversed an enclosed walkway (CatWalk system; Noldus, Wageningen, The Netherlands) three times. In this system, a light-emitting diode from a fluorescent lamp illuminates the inside of a glass floor plate of the walkway. Light is reflected downward at the site of the paw in contact with the upper surface of the glass plate, the intensity of which is proportional to the relative force being exerted by the paw. Light reflection was recorded with a video camera and analyzed to determine the duration of paw contact with the floor and the mean pixel intensity at contact.19 Immunofluorescent staining. Mice were sacrificed at 7 and 14 days after surgery. The thigh adductor muscles of the legs were harvested, fixed in 10% formaldehyde solution, and embedded in paraffin. Muscle fiber number was examined in sections stained with hematoxylineeosin, and the mean counts from five separate fields in four distinct areas in each specimen were calculated. Capillary endothelial cells were identified by immunohistochemical staining with von Willebrand factor antibody (DAKO, Glostrup, Denmark).20 Capillary density was expressed as the ratio of von Willebrand factor positive cells to myofibers per high power field (magnification 400). Furthermore, lymphangiogenesis was assessed by LYVE-1 antibody (Relia Tech GmbH, Wolfenbüttel, Germany).21 Immunohistochemistry. The thigh adductor muscles of native and treated mice were fixed in 10% formaldehyde solution, processed, and embedded in paraffin. The sections
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were stained immunohistochemically with anti-VEGFR-1, anti-VEGFR-2, and anti-VEGFR-3 antibody (Abcam, Tokyo, Japan), then detected by diaminobenzidine staining. Reverse transcription-polymerase chain reaction analysis. Total RNA was isolated from thigh muscle with PureLink RNA Mini kit (Life Technologies, Carlsbad, Calif). Reverse transcription-polymerase chain reaction (RT-PCR) was carried out using high-capacity cDNA reverse transcription kits (Applied Biosystems, Carlsbad, Calif). The following oligonucleotides were used: glyceraldehyde-3-phosphate dehydrogenase (G3PDH): 50 -ACA TCAAGAAGGTAGTGAAGCTGGC-30 , 50 -TTCAAGAGATTAGGGCTCCCTAGG-30 ; VEGFR-1: 50 -TACCTC AAGAGCAAACGTGAC-30 , 50 -CGATGAATGCACTTT CTGG-30 ; VEGFR-2: 50 -CTAAGGGCATGGAGTTCT TG-30 , 50 -CATACAGGAAACAGGTGAGGT-30 ; and VE GFR-3: 50 -CTATGGCTGAGCCCAATGAC-30 , 50 -ACCT TATCAAAGATGCTCTCGG-30 .22 The annealing temperature and the PCR cycles were G3PDH: 60 C, 28 cycles; VEGFR-1: 60.8 C, 35 cycles; VEGFR-2: 58.1 C, 30 cycles; and VEGFR-3: 60.8 C, 35 cycles. Immunoblotting. Thigh muscle and embryo were homogenized at 4 C in lysis buffer (Cell Signaling Technology, Danvers, Mass) with protease inhibitor cocktail (Roche, Mannheim, Germany) and then centrifuged at 13,000 rpm for 15 minutes. The supernatant was collected. Protein concentrations were measured using a bicinchoninate acid protein assay kit (ThermoFisher Scientific, Waltham, Mass). Equal amounts of protein from each experimental group were loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by immunoblotting. The membranes were proved with anti-VEGFR-1 (R&D Systems), anti-VEGFR-2 (Cell Signaling Technology ), anti-VEGFR-3 (eBioscience, San Diego, Calif) and anti-b-actin (Cell Signaling Technology). Membrane signals were detected using the ECL reagent (GE Healthcare, Buckinghamshire, United Kingdom). Enzyme-linked immunosorbent assay. Thigh muscle was homogenized at 4 C in PBS with protease inhibitor cocktail (Roche) and then centrifuged at 13,000 rpm for 15 minutes. The supernatant was collected. Protein levels for VEGF-A and VEGF-C were determined by enzymelinked immunosorbent assay (R&D Systems and Life Science Inc, St. Petersburg, Fla, respectively) as previously described.23 Statistical analysis. Statistical analysis was performed using PASW Statistics 18.0 (IBM Japan, Tokyo, Japan). A two-tailed Student t-test, paired Student t-test, or oneway analysis of variance and Mann-Whitney U test were used for statistical analysis. P < .05 was considered statistically significant. RESULTS Effect of implanted BMCs in ischemic limb muscle. Before the experiments, all mice had similar baseline blood flow in the leg and functional status. In the PBS control group, the blood flow decreased rapidly after limb ischemia and returned slowly, achieving 60% of
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preprocedural flow after 4 weeks (Fig 1, A). Blood flow recovery was significantly improved in mice treated with 1 107 BMCs at 7 and 14 days after the operative procedure. At 21 and 28 days, the BMC-treated group demonstrated a trend toward increased blood flow recovery compared with the control group, but this was not significantly different (Fig 1, A). Recovery of blood flow in the ischemic limbs treated with CD11bD cells. To determine whether BMCs contained enriched CD11bþ cells, we performed flow cytometric analysis of CD11bþ cells from total BMCs. Unseparated BMCs contained 29.59% CD11bþ cells. After MACS sorting, the purity of CD11bþ cells was 95.56% (Fig 1, B). We next measured the recovery blood flow in ischemic limbs treated with purified CD11bþ cells. Mice were treated with 1 106 CD11bþ cells, CD11b cells, or PBS for controls. To determine the cell number of CD11bþ cells, three different numbers of CD11bþ cells (1 104, 1 105, and 1 106) were investigated as a preliminary study. The 1 106 CD11bþ cells had a statistically significant difference as the lowest number in a preliminary study. The control animals were measured at each time point. On the earliest operative days 1 and 3, the blood flow recovery ratio was higher in the CD11bþ-treated group compared with the control and CD11b groups. Moreover, blood flow recovery was significantly improved in the CD11bþ-treated group compared with the control and CD11b groups at 7 and 14 days after the operative procedure. At 21 and 28 days, there was no significant difference between blood flow recovery of the CD11bþ-treated group compared with the control and CD11b groups (Fig 1, C). Thus, mice treated with 1 106 CD11bþ cells showed an improved blood flow, the same as the 1 107 BMC-treated mice at multiple time points (Fig 1, A and C). Protein expression of VEGF-A, VEGF-C, VEGFR1, VEGFR-2, and VEGFR-3. To identify the production of VEGF-A and VEGF-C from CD11bþ cells, we measured levels of VEGF-A and VEGF-C in cultured supernatant from CD11bþ and CD11b cells. Enzyme-linked immunosorbent assay analyses showed significantly increased levels of VEGF-A and VEGF-C in the cultured supernatant of CD11bþ cells compared with CD11b cells. Although VEGF-A was secreted by both CD11bþ and CD11b cells, VEGF-C was not secreted from cultured CD11b cells (Fig 2, A). The VEGFR-1 and VEGFR-3 messenger ribonucleic acid (mRNA) were expressed in CD11bþ cells, but VEGFR-2 mRNA was not detected in either CD11bþ or CD11b cells (Fig 2, B). Furthermore, we confirmed that CD11bþ and CD11b cells expressed VEGFR-1 and VEGFR-3 protein (Fig 2, C). These results suggest the possibility that CD11bþ cells are activated by autocrine VEGF-A and VEGF-C via VEGFR-1 and VEGFR-3, respectively. Recovery of blood flow in ischemic limbs treated with recombinant VEGF-C. We compared treatment of mice with 5 mg of recombinant VEGF-C with PBStreated controls in restoring blood flow to ischemic hind
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Fig 1. Recovery of blood flow in ischemic limbs treated with bone marrow cells (BMCs) and CD11bþ cells derived from BMCs. A and C, Representative laser Doppler perfusion images taken at indicated intervals for the phosphatebuffered saline (PBS) control group, unselected BMCs group, CD11b group, and CD11bþ group. Blood perfusion in the ischemic hind limbs was markedly increased in the unselected BMCs and CD11bþ 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% 6 9.0% vs 14.8 6 6.7%; P < .01; day 14: 72.2% 6 7.4% vs 31.1 6 12.0%; P < .05; n ¼ 5-7). The increase in the ischemic/nonischemic ratio was significantly higher in the CD11bþ group compared with the CD11b group at postoperative days 1, 3, 7, and 14 ( day 1: 17.1% 6 4.0% vs 12.3 61.7%; P < .05; day 3: 26.4% 6 3.1% vs 12.3% 6 7.2%; P < .05; day 7: 48.4% 6 16.0% vs 20.7% 6 8.6%; P < .01; day 14: 85.7% 6 23.3% vs 45.7% 6 4.8%; P < .01; n ¼ 5-7). Data are given as mean 6 SEM, n ¼ 5-7 per group. *P < .05; **P < .01; ***P < .001. B, Flow cytometric analysis of CD11bþ macrophages from BMCs. The purity of subpopulations of the enriched CD11bþ macrophages was approximately 95% according to the FACS analysis.
limbs. To determine the dosage of VEGF-C, four different doses of VEGF-C, adjusted to 3, 5, 12.5, and 25 mg, were injected as a preliminary study. The 5 mg of VEGF-C had a statistically significant difference as the lowest dose in a preliminary study. The control animals were measured at each time point. Blood perfusion in the ischemic hind limbs was markedly increased in the 5 mg VEGF-C-treated group compared with controls at multiple time points. The increase in the ischemia/nonischemic ratio was significantly upregulated in the 5 mg VEGF-C-treated group compared with the control group at 7 and 14 days after operative procedure. At 21 and 28 days, the 5 mg VEGF-C-treated group showed a better recovery ratio than the control
group, but there was no significant difference in the recovery of blood flow between the groups (Fig 3, A). Functional recovery following hind limb ischemia by intramuscular injection of VEGF-C. To evaluate more precisely the functional deficits and ischemic grade of ischemic limbs, we tested mice according to the Tarlov score for function and Ischemic score for ischemia. Using the Tarlov score, the VEGF-C-treated group showed a significantly increased functional status at 7 and 14 days compared with the control group. Using the Ischemic scoring system, the VEGF-C group showed a significantly increased level of ischemic tissue grade at day 7 compared with the control group. At 14 days, there were no
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Fig 2. Expression of vascular endothelial growth factor (VEGF)A, VEGF-C, VEGFR-1, VEGFR-2, and VEGFR-3 in CD11b and CD11bþ cells. A, Concentration of VEGF-A and VEGF-C secreted by CD11b or CD11bþ cells. After magnetic cell sorting (MACS), 1 105 CD11b or CD11bþ cells were incubated for 7 days and the supernatant analyzed by enzyme-linked immunosorbent assay (n ¼ 3; mean [standard deviation]; ***P < . 001). B, Representative messenger ribonucleic acid (mRNA) of VEGFR-1, VEGFR-2, and VEGFR-3 from CD11b and CD11bþ cells. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an internal control. Reverse transcriptionpolymerase chain reaction is representative of three separate experiments. C, Representative immunoblotting images for VEGFR-1, VEGFR-2, and VEGFR-3 from CD11b and CD11bþ cells. After MACS sorting, 5 106 CD11b and CD11bþ cells were lysed and examined by immunoblotting analysis. The staining of b-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.
significant differences in tissue grade of ischemic limbs between the VEGF-C and control groups. In addition, there were no significant differences in functional status and in ischemic tissue grade at 7 and 14 days between the VEGF-C and CD11bþ-treated groups (Fig 3, B). Furthermore, we performed gait analysis in the VEGFC-treated and control groups. The control (PBS) group could not use the injured limb (left hind limb) for walking, whereas mice from the VEGF-C-treated group could use both hind limbs for walking. The blue arrowhead in the contact duration map in Fig 3, C, depicts the pixel intensity generated by the mouse paws upon contact with the glass floor. Pixel intensity generated by the three uninjured limbs in the control group was higher compared with the VEGFC-treated group. These findings indicate that functional recovery of ischemic limbs in the VEGF-C-treated group was dramatically improved compared with the control group. In quantitative analysis, paw contact time was
significantly prolonged in ischemic limbs of the VEGFC-treated group compared with the control group (0.17 6 0.13 s vs 0.02 6 0.05 s; P < .05). In addition, paw intensity was significantly higher in the ischemic limbs of the VEGF-C-treated group compared with the control group (71.9 6 22.7 arbitrary units vs 31.6 6 45.0 arbitrary units; P < .01; Fig 3, C). Protein levels of VEGF-A and VEGF-C in thigh muscle. There was no statistical difference in VEGF-A protein expression in the thigh muscle at 7 and 14 days after limb ischemia in the CD11bþ-treated, VEGFC-treated, and control groups (Fig 4, A). However, 7 days after the intramuscular injection, the CD11bþ cell-treated group secreted significantly more VEGF-C protein in the thigh muscle after limb ischemia compared with the other groups (P < .05; Fig 4, B), indicating a crucial role of VEGF-C in angiogenesis. Expression of angiogenesis and lymphangiogenesis marker. After limb ischemia, both the CD11bþ and VEGF-C groups had significantly increased numbers of von Willebrand factor positive cells and LYVE-1 positive cells compared with the control group (Fig 5, A). Capillary density, representing angiogenesis, was significantly higher in the CD11bþ-treated and VEGF-C-treated groups compared with the control group. Furthermore, in lymphangiogenesis, capillary density was significantly upregulated in the CD11bþ-treated and VEGF-C-treated groups compared with the control group (Fig 5, B). These findings suggest increased blood flow and functional recovery after hind limb ischemia when treated with CD11bþ cells or recombinant VEGF-C. In addition, RT-PCR and histologic analysis showed that VEGFR-1, VEGFR-2, and VEGFR-3 mRNA and protein were expressed in ischemic muscle at days 7 and 14 after hind limb ischemia in the control, CD11bþ, and VEGF-C-treated mice (Fig 5, C). Effect of VEGFR-2-neutralizing antibody treatment. Recovery of blood flow in ischemic hind limbs of the CD11bþ and VEGF-C-treated groups was suppressed by systemic administration of anti-VEGFR-2 (DC101) on postoperative day 7. Although recovery of blood flow in ischemic limbs was reduced dramatically, the ischemic limbs of CD11bþ and VEGF-C-treated groups showed augmented recovery of hind limb perfusion, and blood flow returned to the same level of hind limb perfusion before administration of the anti-VEGFR-2 antibody (Fig 5, D and E). These results indicate that VEGF-A via VEGFR-2 signaling may have an important role in angiogenesis, and that VEGF-C may function as a primary or adjunctive agent for therapeutic angiogenesis. DISCUSSION In this study, we analyzed treatment with CD11bþ cells derived from BMCs. Furthermore, our data clearly demonstrate that intramuscular injection of CD11bþ cells that secreted VEGF-C improved recovery of blood flow in a murine model of hind limb ischemia. In addition, CD11bþ treatment could reduce the total number of cell injections and improve the early operative points of blood
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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 phosphatebuffered saline (PBS) control group and the VEGF-C group. Blood perfusion in the ischemic hind limbs was markedly
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flow compared with conventional therapy with BMCs. The novelty and advantage of using CD11bþ cells is the secretion of a distinct combination of the angiogenic factor VEGF-A and the lymphangiogenic factor VEGF-C, and their substantial in vivo capacity to induce neovascularization. In our report, VEGF-A protein was secreted by cultured CD11bþ cells and a few CD11b cells. In contrast, VEGF-C was secreted by cultured CD11bþ cells but not CD11b cells. Although hind limb ischemia has been improved by the administration of VEGF-A in animal models, there has been little improvement in translating the angiogenic capacity of VEGF-A to benefit PAD patients.24,25 Because VEGF-A increases vessel permeability, many patients receiving intramuscular injection of VEGF-A developed complications such as edema.26 Vascular leakage resulting in tissue edema is a major problem in therapeutic angiogenesis. However, coadministration of VEGF and angiopoietin-1 has modified angiogenesis and reduced vascular permeability in ischemic hind limb models.27,28 These combination gene therapies are currently being examined in animal models in preparation for translation to clinical human therapy. Revascularization has good outcome in patients with severe PAD, but it is well known that the addition of chronic venous insufficiency to PAD makes treatment more difficult. Many patients suffer with compromised occlusive arterial disease and venous congestion. Therefore, in patients with critical limb ischemia for whom the current treatment options are not effective, enhancing venous return and lymphatic drainage from the ischemic limb may have the potential to improve ischemic limbs. The VEGF-C is a specific ligand for the endothelial receptor tyrosine kinases VEGFR-2 and VEGFR-3, and it mediates its lymphangiogenic effects through VEGFR-3.29 Furthermore, recent studies showed that blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation,30 and that upregulation of Notch-dependent VEGFR-3 allows angiogenesis without VEGF-A through VEGFR-2 signaling.31 We focused on VEGF-C and evaluated the ectopic application of recombinant VEGF-C to induce potent angiogenic effects in vivo. Interestingly, intramuscular injection of exogenous VEGF-C can enhance the recovery of blood flow in ischemic limbs and the function of walking, indicating that enhancing
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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 ¼ 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 CD11bþ cell-treated mice had significantly increased levels of VEGF-C compared with the other groups (n ¼ 3; mean [standard deviation]; *P < .05).
lymphatic drainage and venous return accelerates the recovery of hind limb ischemia. A critical role of VEGFC in angiogenesis was suggested by Witzenbichler et al,32 who reported that VEGF-C promotes angiogenesis in a rabbit model of hind limb ischemia. In accordance with this finding, VEGF-C induces neovascularization in the mouse cornea micropocket assay.33 Concurrently, Bauer et al34 demonstrated that VEGF-C overexpression,
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% 6 12.0% vs 14.8 6 6.7%; P < .01; day 14: 65.5% 6 11.6% vs 31.1 6 12.0%; P < .05; n ¼ 5-7). Data are expressed as mean 6 SEM, n ¼ 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 6 0.5 vs 1.0 6 0.81; P < .05; day 14: 5.75 6 0.5 vs 3.42 6 2.37; P < .05; n ¼ 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 6 0.5 vs 2.85 6 1.06; P < .01; n ¼ 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.
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Fig 5. Effect of CD11bþ cell and vascular endothelial growth factor (VEGF)-C treatment on capillary density 14 days after the induction of ischemia (n ¼ 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.
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using fibroblasts, stimulates multiple biologic processes known to impact ischemic wound healing, such as collagen constriction in three-dimensional gels, capillary sprouting, and endothelial progenitor cell invasion and migration through the extracellular matrix. These studies further enhance the evidence for VEGF-C as an important angiogenic factor as well as a lymphangiogenic factor. To examine the autocrine effects of CD11bþ cells, we analyzed cultured CD11bþ and CD11b cells by RT-PCR and Western blotting. Our data show that VEGFR-1 and VEGFR-3 mRNA and protein, but not VEGFR-2, were detected in cultured CD11bþ and CD11b cells. These results indicate that the autocrine effects of CD11bþ cells are not the main regulator in promoting angiogenesis through VEGFR-2 for self-renewal. However, not only VEGFR-1 and VEGFR-3 but also VEGFR-2 was detected by RT-PCR analysis and immunohistochemical analysis in ischemic hind limbs. These results indicate that the VEGF-A and VEGF-C produced by CD11bþ cells or recombinant VEGF-C activate endothelial cells or migrated macrophages through VEGFR signaling transduction. In addition, we showed that neutralization of VEGFR-2 inhibits the recovery of blood flow in ischemic hind limbs in CD11bþ cell and VEGF-C group mice until 7 days postoperation. In the CD11bþ cell group treated with VEGFR-2-neutralizing antibody, these results indicate that VEGF-C derived from CD11bþ cell through VEGFR-3 signaling contributes to the recovery of blood flow including endogenous VEGF-C secretion. On the other hand, in the VEGF-C group, recombinant VEGFC through VEGFR-3 signaling has a role in angiogenesis in blood flow recovery in a murine model of hind limb ischemia including endogenous VEGF-C secretion, as it was previously reported that VEGF-C (Cys156) induced a specific signal through VEGFR-3 but not VEGFR-2.35 These results demonstrate that VEGF-C signaling through VEGFR-3 has a synergistic action with VEGF-C signaling through VEGFR-2 in angiogenesis. Accordingly, CD11bþ cells that secrete both VEGF-A and VEGF-C have a greater potential to accelerate restoration from ischemia. Furthermore, VEGF-C signaling through VEGFR-3 plays a critical role in lymphangiogenesis in improving hind limb ischemia, even if the VEGFR-2
inhibitor suppressed the VEGF-A or VEGF-C secreted from CD11bþ cells. Although further investigations of the molecular cascades involved in VEGF-C-mediated effects in ischemia and in vivo confirmation are needed, these data offer insights into novel methods to promote angiogenesis and lymphangiogenesis during ischemia. Collectively, by focusing on CD11bþ cells and VEGF-C as novel targets, these findings might contribute to the development of new therapeutic strategies for limb ischemia. AUTHOR CONTRIBUTIONS Conception and design: GK, HN Analysis and interpretation: GK, HN, DK, SK Data collection: GK, HN, DK Writing the article: GK, HN, SK Critical revision of the article: GK, HN, Final approval of the article: GK, HN, DK, TT, SK Statistical analysis: GK, HN Obtained funding: TT, SK Overall responsibility: SK GK and HN contributed equally to this work. REFERENCES 1. Aronow H. Peripheral arterial disease in the elderly: recognition and management. Am J Cardiovasc Drugs 2008;8:353-64. 2. Lawall H, Bramlage P, Amann B. Treatment of peripheral arterial disease using stem and progenitor cell therapy. J Vasc Surg 2011;53:445-53. 3. Adam DJ, Beard JD, Cleveland T, Bell J, Bradbury AW, Forbes JF, et al. Bypass versus angioplasty in severe ischaemia of the leg (BASIL): multicentre, randomised controlled trial. Lancet 2005;366:1925-34. 4. Losordo DW, Dimmeler S. Therapeutic angiogenesis and vasculogenesis for ischemic disease: part II: cell-based therapies. Circulation 2004;109:2692-7. 5. Masaki I, Yonemitsu Y, Yamashita A, Sata S, Tanii M, Komori K, et al. Angiogenic gene therapy for experimental critical limb ischemia: acceleration of limb loss by overexpression of vascular endothelial growth factor 165 but not of fibroblast growth factor-2. Circ Res 2002;90:966-73. 6. Gupta R, Losordo DW. Cell therapy for critical limb ischemia: moving forward one step at a time. Circ Cardiovasc Interv 2011;4:2-5. 7. Shireman PK. The chemokine system in arteriogenesis and hind limb ischemia. J Vasc Surg 2007;45(Suppl A):A48-56. 8. Silvestre JS, Mallat Z, Tedgui A, Levy BI. Post-ischaemic neovascularization and inflammation. Cardiovasc Res 2008;78:242-9. 9. Tammela T, Enholm B, Alitalo K, Paavonen K. The biology of vascular endothelial growth factors. Cardiovasc Res 2005;65:550-63.
B, During angiogenesis, capillary density was significantly higher in the CD11bþ and VEGF-C groups than in the control group (1.88 6 0.34 vs 0.36 6 0.09; P < .001; 0.88 6 0.29 vs 0.36 6 0.09; P < .001). Furthermore, in lymphangiogenesis, capillary density was significantly upregulated in the CD11bþ and VEGF-C groups compared with control group (1.89 6 0.26 vs 0.22 6 0.12; P < .001; 1.19 6 0.37 vs 0.22 6 0.12; P < .001). *P < .05; **P < .01; ***P < .001. 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-3phosphate dehydrogenase (G3PDH) was used as the internal control. Representative images are shown (n ¼ 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 CD11bþ and VEGF-C groups was suppressed by administration of anti-VEGFR-2 antibody (DC101). The recovery of blood flow in ischemic limbs in the CD11bþ group treated with VEGFR2-neutralizing antibody was significantly suppressed compared with the CD11bþ-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. yP < .05; zP < .01.
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23. Huggenberger R, Siddiqui SS, Brander D, Ullmann S, Zimmermann K, Antsiferova M, et al. An important role of lymphatic vessel activation in limiting acute inflammation. Blood 2011;117:4667-78. 24. Simons M, Ware JA. Therapeutic angiogenesis in cardiovascular disease. Nat Rev Drug Discov 2003;2:863-71. 25. Simons M. Angiogenesis: where do we stand now? Circulation 2005;111:1556-66. 26. Weis S, Shintani S, Weber A, Kirchmair R, Wood M, Cravens A, et al. Src blockade stabilizes a Flk/cadherin complex, reducing edema and tissue injury following myocardial infarction. J Clin Invest 2004;113: 885-94. 27. Chae JK, Kim I, Lim ST, Chung MJ, Kim WH, Kim HG, et al. Coadministration of angiopoietin-1 and vascular endothelial growth factor enhances collateral vascularization. Arterioscler Thromb Vasc Biol 2000;20:2573-8. 28. Yamauchi A, Ito Y, Morikawa M, Kobune M, Huang J, Sasaki K, et al. Pre-administration of angiopoietin-1 followed by VEGF induces functional and mature vascular formation in a rabbit ischemic model. J Gene Med 2003;5:994-1004. 29. Tammela T, Alitalo K. Lymphangiogenesis: molecular mechanisms and future promise. Cell 2010;140:460-76. 30. Tammela T, Zarkada G, Wallgard E, Murtomaki A, Suchting S, Wirzenius M, et al. Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation. Nature 2008;454:656-60. 31. Benedito R, Rocha SF, Woeste M, Zamykal M, Radtke F, Casanovas O, et al. Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling. Nature 2012;484:110-4. 32. Witzenbichler B, Asahara T, Murohara T, Silver M, Spyridopoulos I, Magner M, et al. Vascular endothelial growth factor-C (VEGF-C/ VEGF-2) promotes angiogenesis in the setting of tissue ischemia. Am J Pathol 1998;153:381-94. 33. Cao Y, Linden P, Farnebo J, Cao R, Eriksson A, Kumar V, et al. Vascular endothelial growth factor C induces angiogenesis in vivo. Proc Natl Acad Sci U S A 1998;95:14389-94. 34. Bauer SM, Bauer RJ, Liu ZJ, Chen H, Goldstein L, Velazquez OC. Vascular endothelial growth factor-C promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels. J Vasc Surg 2005;41:699-707. 35. Joukov V, Kumar V, Sorsa T, Arighi E, Weich H, Saksela O, et al. A recombinant mutant vascular endothelial growth factor-C that has lost vascular endothelial growth factor receptor-2 binding, activation, and vascular permeability activities. J Biol Chem 1998;273:6599-602. Submitted Jun 14, 2012; accepted Aug 29, 2012.