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First published online January 26, 2006
Stem Cells Vol. 24 No. 5 May 2006, pp. 1194 -1200
doi:10.1634/stemcells.2005-0349; www.StemCells.com
© 2006 AlphaMed Press

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TRANSLATIONAL AND CLINICAL RESEARCH

Angiogenesis Facilitated by Autologous Whole Bone Marrow Stem Cell Transplantation for Buerger’s Disease

Dong-Ik Kima, Mi-Jung Kimd, Jin-Hyun Joha, Sung-Wook Shinb, Young-Soo Dob, Ji-Young Moona, Na-Ri Kima, Joung-Eun Limf, Ae-Kyeong Kimf, Hyun-Seon Eof, Byung-Soo Kime, Seung-Woo Chog, Seung-Hye Yangd, Chan-Jeoung Parkd, Jong-Sup Shimc

a Division of Vascular Surgery and
b Departments of Radiology and
c Orthopedic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea;
d Department of Laboratory Medicine, University of Ulsan College of Medicine, Asan Institute for Life Sciences, Seoul, Korea;
e Department of Bioengineering, Hanyang University, Seoul, Korea;
f Samsung Biomedical Research Institute,
g School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea

Key Words. Angiogenesis • Bone marrow • Stem cells

Correspondence: Dong-Ik Kim, M.D., Ph.D., Division of Vascular Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwondong, Kangnamku, Seoul 135-710, Korea. Telephone: 82-2-3410-3467; Fax: 82-2-3410-0040; e-mail: dikim{at}smc.samsung.co.kr or Mi-Jung Kim, Department of Laboratory Medicine, University of Ulsan College of Medicine, Asan Institute for Life Sciences, 388 Pungnap-2 dong, Songpa-gu, Seoul 138-736, Korea. Telephone: 82-2-3010-4147; Fax: 82-2-3010-4182; E-mail: mijkim{at}amc.seoul.kr

Received July 29, 2005; accepted for publication January 17, 2006.

   ABSTRACT
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
We hypothesized that angiogenesis can be triggered by autologous whole bone marrow stem cell transplantation. Twenty-seven patients (34 lower limbs) with Buerger’s disease, who were not candidates for surgical revascularization or radiologic intervention, were enrolled in this study. Six sites of the tibia bone were fenestrated using a 2.5-mm-diameter screw under fluoroscopic guidance. Clinical status and outcome were determined using the "Recommended Standards for Reports." To mobilize endothelial progenitor cells (EPCs) from bone marrow, recombinant human granulocyte colony-stimulating factor (r-HuG-CSF) was injected subcutaneously as a dose of 75 µg, preoperatively. During the follow-up period (19.1 ± 3.5 months), one limb showed a markedly improved outcome (+3), and 26 limbs showed a moderately improved outcome (+2). Thirteen limbs among 17 limbs with nonhealing ulcers healed. Postoperative angiograms were obtained for 22 limbs. Two limbs showed marked (+3), five limbs moderate (+2), and nine limbs slight (+1) collateral development. However, six limbs showed no collateral development (0). Peripheral blood and bone marrow samples were analyzed for CD34 and CD133 molecules to enumerate potential EPCs, and EPC numbers were found to be increased in peripheral blood and in bone marrow after r-HuG-CSF injection. In conclusion, the transplantation of autologous whole BMCs by fenestration of the tibia bone represents a simple, safe, and effective means of inducing therapeutic angiogenesis in patients with Buerger’s disease.


   INTRODUCTION
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
Therapeutic angiogenesis has been studied for the treatment of patients with peripheral arterial occlusive disease who are not candidates for surgical revascularization or radiologic intervention. Angiogenesis can be achieved by introducing growth factors [1], angiogenic genes [2], or stem cells containing endothelial progenitor cells (EPCs) and mesenchymal cells. Bone marrow contains many kinds of immature cells and EPCs that secrete several growth factors. However, the majority of recently published studies have focused on bone marrow mononuclear cell (BM-MNC) transplantation [3], and the isolation of BM-MNCs from whole bone marrow cells is highly complex and expensive and presents the potential danger of contamination. Also, unknown cells and growth factors required for angiogenesis might be removed during the isolation process. We hypothesized that an angiogenic effect could be achieved by employing autologous whole bone marrow stem cell transplantation via fenestration of the tibia bone. The primary outcomes of our study were safety and the feasibility of treatment, as defined by ischemic symptoms and wound healing improvements.


   PATIENTS AND METHODS
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients
Twenty seven patients (34 lower limbs) with Buerger’s disease (26 men and one woman; mean age, 37.6 ± 6.9 years) and severe claudication, resting pain, or nonhealing ischemic wounds for a minimum of 3 months (without evidence of improvement in response to conventional medical therapy), who were not candidates for surgical revascularization or radiologic intervention, were enrolled in the present study. Patients with malignant disorders, those more than 60 years old, and those with any other significant medical condition, including diabetes mellitus, hypertension, and hyperlipidemia, were excluded. All patients were allowed to continue previous medications.

Clinical statuses and outcomes were assessed using the "Recommended Standards for Reports," published by the Society for Vascular Surgery/North American Chapter and the International Society for Cardiovascular Surgery [4]. Digital subtraction angiography was performed at 5 days before and at more than 3 months after treatment. All angiograms were performed using the same protocol. The force of contrast injection, the amount of contrast injected, and patient and catheter tip positions were strictly fixed. Status of angiogenesis on radiological findings were graded as +0 (no collateral development), +1 (slight collateral development), +2 (moderate collateral development), or +3 (rich collateral development) by one radiologist.

This study was approved by the Institutional Review Board and the Institutional Biosafety Committee of Samsung Medical Center. Written informed consent for participation in the study was obtained from all the patients.

Procedures
Ankle-brachial indexes, toe pressures, and treadmill test results were serially examined preoperatively and postoperatively. The treatment schedule is detailed in Table 1Go. The blood samples were obtained from antecubital veins at 6 a.m. for 5 days. Flow cytometric analysis was done after staining mononuclear cells with CD34-fluorescein isothiocyanate and CD133-phosphatidylethanolamine monoclonal antibodies (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen). Recombinant human granulocyte colony-stimulating factor (r-HuG-CSF; Cheil Jedang, Korea) was injected subcutaneously (s.c.) daily at 6 p.m. for 5 days. r-HuG-CSF doses were adjusted daily according to leukocyte numbers in peripheral blood. r-HuG-CSF was injected s.c. at 75 µg daily until leukocyte numbers reached 20,000 cells/ml but was reduced to 50 µg when leukocyte numbers exceeded this figure.


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  		Table 1. Study protocol

 
Fenestration through the tibia bone was performed under general or spinal anesthesia. Six tibial sites were fenestrated with a 2.5-mm-diameter screw under fluoroscopic guidance (Fig. 1Go). This procedure allowed bone marrow stem cells to be released into ischemic calf muscle through fenestrated posterior tibia holes. Approximately 10 ml of bone marrow cells was aspirated through one of the fenestrated tibial holes for analysis of the angiogenic precursor cells within the bone marrow stem cells.


Figure 1
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  		Figure 1. Fenestration procedure through the tibia. Six tibial sites were fenestrated with a 2.5-mm-diameter screw under fluoroscopic guidance. Abbreviations: F, fibular bone; T, tibia bone.

 

   RESULTS
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
The mean follow-up period was 19.1 ± 3.5 months (range, 12.4 to ~25 months). All of the 34 limbs had symptoms that were more than category 3 (severe claudication) as noted during pretreatment examination. Among 34 limbs, 25 (92.5%) limbs had symptoms such as ischemic rest pain or tissue loss. During the follow-up period, 23 (67.6%) limbs had symptoms of category 2 (moderate claudication). One limb achieved a +3 (markedly improved) outcome, 26 (76.5%) a +2 (moderately improved) outcome, and 7 (20.6%) limbs were unchanged. However, no deterioration was observed in the clinical status of any patient (Table 2Go). Thirteen of 17 limbs with nonhealing ulcer (Fontaine classification, category 5) healed during follow-up (Fig. 2Go). Postoperative angiograms were obtained in 22 limbs (Fig. 3Go); the other subjects refused angiogram due to the invasive nature of the angiogram studies. Two limbs showed an angiogenic status of +3 (rich collateral development), five limbs showed +2 (moderate collateral development), and nine limbs showed +1 (slight collateral development). However, six limbs showed an angiogenic status of 0 (no collateral development).


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  		Table 2. Clinical and angiographic findings

 

Figure 2
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  		Figure 2. Angiogram. (A, C, E): Preoperative findings. (B, D, F): Postoperative findings in the respective patients. Radiologic angiogenic statuses were graded as +0 (no collateral development), +1 (slight collateral development), +2 (moderate collateral development), or +3 (rich collateral development). (B): Typical finding of +1 (slight collateral development). (D): Typical finding of +2 (moderate). (F): Typical finding of +3 (rich).

 

Figure 3
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  		Figure 3. Gangrene of the right big toe healed 6 months after autologous whole bone marrow stem cell transplantation. Left, pre-treatment; right, 6 months after treatment.

 
To identify the correlations between angiographic findings and an improved outcome or wound healing, Spearman’s correlation analysis was performed. However, no statistically significant relations were found (p = .1945 vs. improved outcome; p = .4389 vs. wound healing).

Peripheral blood mononuclear cell composition was divided into three regions (R1, R2, and R3), by flow cytometric dot plot analysis according to forward and side scatter. Lymphocytes constitute the major cell population in R1, monocytes in R2, and granulocytes in R3. On day 0, prior to the r-HuG-CSF injection, cells numbers were highest in R1 and lowest in R3, but the R2 and R3 populations were gradually increased after r-HuG-CSF injection (day 1 to ~day 4) (Fig. 4Go). CD34-positive and CD133-positive cells were increased in R1 after r-HuG-CSF injection (Fig. 5Go), whereas although CD34-negative/CD133-positive cells in R2 were increased at day 1 post-injection by more than threefold, they then gradually decreased, to maintain a level of 1.5 times that of the day 0 value. CD133-positive cells also were increased twofold versus the day 0 value and then gradually decreased from day 3 (Fig. 6Go). On the other hand, the R3 cell population showed a wide range of phenotypes and no significant differences among days 0, 1, 2, 3, and 4 in terms of the numbers of EPCs.


Figure 4
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  		Figure 4. Flow-cytometric dot-plot analysis of peripheral blood (day 0 to ~day 4 postinjection of recombinant human granulocyte colony-stimulating factor [r-HuG-CSF[) and bone marrow (day 4). Cellular populations in R3 gradually increased after r-HuG-CSF injection. Abbreviations: BM, bone marrow; FSC-H, forward scatter height; R, region; SSC-H, side scatter height.

 

Figure 5
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  		Figure 5. Analysis of the endothelial progenitor cell phenotype-positive population in R1: CD34+ and CD133+ cell percentages increased after injection of recombinant human granulocyte colony-stimulating factor. Abbreviation: BM, bone marrow.

 

Figure 6
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  		Figure 6. Analysis of the endothelial progenitor cell phenotype-positive population in R2. Numbers of CD34-D133+ cells were elevated by threefold 1 day after injection of recombinant human granulocyte colony-stimulating factor and then gradually decreased, but numbers were maintained at 1.5-fold for the day 0 value. CD133+ cell numbers were also increased twofold at day 1 to ~day 2 versus baseline, but then they gradually reduced to baseline at day 3 to ~day 4. Abbreviation: BM, bone marrow.

 
Peripheral blood and bone marrow samples vary in terms of cellularity. Here, we analyzed the total mononuclear cell population regardless of sample volume or cellularity, and we present data on a cell number percentage basis. Tables 3Go and 4Go represent average absolute numbers of total mononuclear cells and EPCs in regions 1 and 2 (cell numbers in parentheses are calculated per 1 x 106 mononuclear cells).


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  		Table 3. Average absolute numbers of total MNCs and CD34+/CD133+/CD34–CD133+/CD34+CD133+ cells in region 1

 

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  		Table 4. Average absolute numbers of total MNCs and CD34+/CD133+/CD34–CD133+/CD34+CD133+ cells in region 2

 

   DISCUSSION
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
The therapeutic effectiveness of angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor, administered as gene therapies has been previously described. Moreover, bone marrow is known to contain many kinds of immature cells, including the EPCs, and to have the potential to induce angiogenesis. In addition, the transplantation of autologous bone marrow stem cells (BMSCs) was reported to induce angiogenesis and to improve a deteriorated exercise capacity in a rat ischemic hind limb model [5]. In the clinical field, the autologous transplantation of BM-MNCs was found to improve ischemic status [6]. Most investigators have selected the iliac bone as a source of BMSCs and have used BM-MNCs for transplantation.

In one study, approximately 500 ml of bone marrow cells was aspirated from iliac bone, and mononuclear cells were sorted and concentrated to a final volume of approximately 30 ml. These BM-MNCs were then transplanted by intramuscular injection into an ischemic lower limb, for example, into the gastrocnemius muscle, in a procedure that took approximately 3–4 hours [6]. However, this procedure has some disadvantages. First, the sorting of BM-MNCs is done in vitro, which introduces the potential danger of contamination. Moreover, this process is expensive and time-consuming. Second, unknown cells and growth factors that positively influence angiogenesis are probably removed during the sorting process.

Several studies have suggested that EPCs are enriched in peripheral blood in the CD133+ cell population [7–10]. Moreover, multipotent adult progenitor cells and CD34–VE-cadherin–AC133+Flk+ cells can differentiate into CD34+VE-cadherinFlk+cells, which is an angioblast phenotype in VEGF culture conditions [11]. However, numerous unidentified cell markers remain to be identified. Although CD34 and CD133 are not unique markers of EPCs, additional specific markers are also used to characterize EPCs. Much in vitro and in vivo data suggest that CD34-positive and CD133-positive cells are EPCs and that CD34 and CD133 could be used as broad EPC markers.

Complications such as cerebral and myocardial infarction after r-HuG-CSF administration have been reported, and the primary mechanisms involved were presumed to be acute arterial occlusion by thrombus. The thrombogenic properties of peripheral blood are the result of an increased viscosity due to the mobilizations of granulocytes, immature cells (including endothelial progenitor cells), and mesenchymal cells. To prevent thrombus formation, nadroparin calcium (low molecular weight heparin) 2,850 IU was injected s.c. daily during the r-HuG-CSF injection period. In the present study, no complications such as cerebral or myocardial infraction occurred.

Compared with the method based on BM-MNC sorting described above, our method has several advantages. First, we selected autologous BMSCs for transplantation. This process involves the transplantation of abundant immature cells and large amounts of growth factors that might be removed during any sorting procedure. Moreover, because the efficacy of angiogenesis is dependent upon the number of transplanted cells, whole bone marrow stem cells might have a more potent angiogenic effect than BM-MNCs. Second, we selected tibial bone as a BMSCs source. Fenestration of the tibia releases automatically autologous BMSCs into ischemic lower limbs and presents no potential risk for infection or contamination; in addition, it shortens procedure time. Third, r-HuG-CSF provides a synergic angiogenic effect. The efficacy of BMSC-induced angiogenesis is dependent upon the number of immature cells, for example, EPCs and mesenchymal cells. Moreover, it has been reported that r-HuG-CSF mobilizes EPCs from the bone marrow and contributes to angiogenesis in ischemic tissues [12]. In the present study, r-HuG-CSF administration clearly modified cell populations in peripheral blood. As shown in Figure 4Go, cell populations in R2 and R3 changed remarkably following r-HuG-CSF administration. However, patients’ responses to r-HuG-CSF differed. In the present study, we characterized phenotypic changes in R3 by using CD34, CD133, CD33, and CD14 in a single patient. CD34+ cell percentages in R3 in this patient increased from 1.97% (day 0) to 5.42% (day 4); CD133+ cells also increased from 0.38% (day 0) to 3.38% (day 4). CD33+ cells in R3 gradually reduced after r-HuG-CSF treatment, whereas CD14+ cells were elevated on days 1 and 2 and then reduced to reach baseline on days 3–5. These observed phenotypic changes were similar to those observed in R2, although this region was more affected by r-HuG-CSF treatment. Generally in normal peripheral blood flow cytometric analysis, R2 and R3 are regarded as monocytic and granulocytic, respectively. However, after r-HuG-CSF treatment, R2 and R3 cannot be described as mature monocytic or granulocytic lineage cells. In fact, our results indicate that post-treatment cells in these regions are of a stem/progenitor cell phenotype. In the present study, because we expected better results for combined therapy in the described clinical setting, we did not separately examine the effect of r-HuG-CSF treatment without fenestration.

In conclusion, the described transplantation of autologous whole BMSCs by fenestration of tibial bone was found to provide a straightforward, safe, and effective means of inducing therapeutic angiogenesis in Buerger’s disease. Moreover, increased number of cells with the EPC phenotype were observed in peripheral blood and in bone marrow after r-HuG-CSF; we believe that this effect might be used synergistically to promote therapeutic angiogenesis.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 
This research was supported by the Samsung Biomedical Research Institute (grants C-A5-105-1 and C-A5-105-2) and Stem Cell Research Center of the 21st Century Frontier Research Program (grants SC3280 and SC3220) funded by the Ministry of Science and Technology, Republic of Korea.

DISCLOSURES
The authors indicate no potential conflicts of interest.


   REFERENCES
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

  1. Takeshita S, Zheng LP, Brogi E et al. Therapeutic angiogenesis: A single intraarterial bolus of vascular endothelial growth factor augments revas-cularization in a rabbit ischemic hindlimb model. J Clin Invest 1994;93: 662–670.[Medline]

  2. Isner JM, Pieczek A, Schainfield R et al. Clinical evidence of angiogensis after arterial gene transfer of phVEGF165 in patients with an ischemic limb. Lancet 1996;348:370–374.[CrossRef][Medline]

  3. Higashi Y, Kimura M, Hara K et al. Autologous bone-marrow mononuclear cell implantation improves endothelium-dependent vasodilation in patients with limb ischemia. Circulation 2004;109:1215–1218.[Abstract/Free Full Text]

  4. Rutherford RB, Baker JD, Ernst C et al. Recommended standards for reports dealing with lower extremity ischemia. Revised version. J Vasc Surg 1997;26:517–538.[CrossRef][Medline]

  5. Ikenaga S, Hamano K, Nishida M et al. Autologous bone marrow implantation induced angiogenesis and improved deteriorated exercise capacity in a rat ischemic hindlimb model. J Surg Res 2001;96:277–283.[CrossRef][Medline]

  6. Tateishi-Yuyama E, Matsubara H, Murohara T et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet 2002;360:427–435.[CrossRef][Medline]

  7. Asahara T, Murohara T, Sullivan A et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–967.[Abstract/Free Full Text]

  8. Peichev M, Naiyer AJ, Pereira D et al. Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors. Blood 2000;95:952–958.[Abstract/Free Full Text]

  9. Powell TM, Paul JD, Hill JM et al. Granulocyte colony-stimulating factor mobilizes functional endothelial progenitor cells in patients with coronary artery disease. Arterioscler Thromb Vasc Biol 2005;25:296–301.[Abstract/Free Full Text]

  10. Quirici N, Soligo D, Caneva L et al. Differentiation and expansion of endothelial cells form human bone marrow CD133(+) cells. Br J Haematol 2001;115:186–194.[CrossRef][Medline]

  11. Reyes M, Dudek A, Jahagirdar B et al. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 2002;109:337–346.[CrossRef][Medline]

  12. Takahashi T, Kalka C, Masuda H et al. Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 1999;5:434–438.[CrossRef][Medline]




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