Autologous Bone Marrow Mononuclear Cells Transplant in Patients With Critical Leg Ischemia: Preliminary Clinical Results

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1 ARTIcle Autologous Bone Marrow Mononuclear Cells Transplant in Patients With Critical Leg Ischemia: Preliminary Clinical Results Min Li, 1 Hua Zhou, 2 Xing Jin, 2 Mo Wang, 2 Shiyi Zhang, 2 Lei Xu 2 Abstract Objectives: Stem cell transplant can induce vasculogenesis and improve the blood supply to an ischemic region, offering hope for chronic lower extremity ischemic diseases. Bone marrow mononuclear cells are one of the sources for stem cell transplants. We sought to observe the safety and efficacy of autologous bone marrow mononuclear cells transplant for treating critical limb ischemia. Materials and Methods: Eligible patients were randomized 1:1 to receive placebo (0.9% NaCl) or piece/ml bone marrow mononuclear cell transplant. For 6 months, patients skin ulcers, ankle-brachial index, and rest pain were examined and recorded before and after treatment. Results: Six months after the bone marrow mononuclear cells transplant, clinical symptoms like rest pain and skin ulcers gradually abated (P <.05). Ankle-brachial index also increased after the transplant (P <.01). Conclusions: Autologous bone marrow mono - nuclear cells transplant for treatment of patients with chronic limb ischemia is safe, effective, and feasible. Key words: Bone marrow mononuclear cells, Transplant, Autologous, Leg, Ischemia, Stem cell From the Departments of 1 Cardiology and 2 Vascular Surgery, Provincial Hospital affiliated to Shandong University, Shandong Province, China Acknowledgements: This study was supported by Shandong province young scientist research rewarding foundation [2008bs03011]. We thank the participants and the supporting medical staff for making this study possible. Min Li and Hua Zhou contributed equally to this study. Corresponding author: Mo Wang, Department of Vascular Surgery, Shandong Provincial Hospital affiliated to Shandong University, 324 Jingwu Road, Jinan , Shandong Province, China Phone: Fax: doctorwangmo@126.com Experimental and Clinical Transplantation (2013) 5: Introduction Blood vessel bypass operation and intravascular interventional therapy for chronic lower limb ischemia can be effective. However, this goal often is not achieved for some patients with chronic critical limb ischemia. They are not suitable for bypass surgery or percutaneous transluminal angioplasty because they have an unfavorable anatomy, inadequate bypass conduit, or poor/high operative risk. According to previous studies, stem cell transplant is a novel, therapeutic strategy to rescue tissue from critical ischemia. 1 Stem cell transplant could induce vasculogenesis and improve blood supply to the ischemic region, thereby providing hope in the treatment of chronic lower extremity ischemic diseases. 2 Bone marrow mononuclear cells (BM-MNCs) are one of the sources for stem cell transplants. Bone marrow mononuclear cells contain many cell components such as endothelial progenitor cells and mesenchymal stem cells as well as cytokines. Both in vitro induction and animal experiments show that mesenchymal stem cells can differentiate into vascular endothelial cells and smooth muscle cells, playing an active part in vascular remodeling. 3 In addition, various vascular growth factors and cytokines in mesenchymal stem cells have been shown to promote neovascularization and collateral vascularization. 4 We sought to determine the effectiveness and the adverse events of bone marrow mononuclear cell transplant as a treatment for chronic critical limb ischemia. Materials and Methods The study protocol was approved according to the instructions of the Institutional Review Board of Copyright Başkent University 2013 Printed in Turkey. All Rights Reserved. DOI: /ect

2 436 Min Li et al /Experimental and Clinical Transplantation (2013) 5: Exp Clin Transplant Shandong Province Hospital. Patients gave written informed consent for their participation. All protocols conformed with the ethical guidelines of the 1975 Helsinki Declaration. Patients Patients with critical limb ischemia enrolled in the study were in-patients receiving therapy in the vascular surgery ward of Shandong Provincial Hospital. Critical limb ischemia was defined according to the Transatlantic Inter-Society Consensus document. 5 Evidence of critical limb ischemia had to be present, including rest pain and/or nonhealing ischemic ulcers for a minimum of 4 weeks without improvement, in response to conventional therapies. The ratio of systolic blood pressure at the ankle-brachial index was < 0.6, or toe systolic blood pressure < 30 mm Hg in the affected limb. Patients were not optimal candidates for surgical or percutaneous revascularization according to angiographic evidence of superficial femoral artery or infrapopliteal disease in the affected limb. Exclusion criteria were a history of malignancy, evidence of possible malignancies after evaluation with carcinoembryonic antigen levels, chest radiographs, computerized tomographic scans, and mammography in women or prostate examination in men. Study design This was a single blinded study aimed at analyzing the effect of BM-MNCs transplant on the clinical evolution of patients with chronic critical limb ischemia unresponsive to standard revascularization treatment. Eligible patients were randomized 1:1 to receive placebo (0.9% NaCl) or piece/ml BM-MNC transplant. Patients were followed for 6 months with subsequent measures (month 6 for safety assessment only). Transplant of mesenchymal stem cells Bone marrow (50 ml in IU of heparin) was extracted from the posterior superior iliac spine. Bone marrow mononuclear cells were isolated from the bone marrow by density gradient centrifugation with lymphocyte separating fluid. Cell counting was performed. Isolated BM-MNCs were diluted into 50 to 120 ml suspensions to be transplanted. At the same time, the patient was taken to the operating suite and prepared for transplant. We chose epidural anesthesia or general anesthesia, depending on the condition of the patient. Bone marrow mononuclear cells were injected into the muscles of the ischemia areas with a 16 gauge needle. Suspensions (0.5 ml) were injected into every point (6 rows, cm deep, 1 cm distance). Safety assessment The primary objective was safety assessment performed according to American Cancer Society guidelines before the start of the study and during follow-up. Safety assessments included adverse events, physical examination, cancer screening, echocardiographs, blood chemistry, hematology, and urinalysis. efficacy assessments Response was defined as hemodynamic improvement, improvement of skin ulcers and rest pain, and limb survival. 6 A hemodynamic improvement was defined as an absolute increase of > 15% in the ankle-brachial index, which is considered significant improvement. 7,8 Rest ankle-brachial index was calculated as the quotient of the ankle pressures and the simultaneously measured brachial pressure. A technician who was unaware of the patient treatment performed this examination. Ischemic skin defects were documented by color photography. Shrinkage of the ulcerated area to less than half of the baseline area was defined as improvement. Assessment of ischemic rest pain was performed using a visual analogue scale for pain scores and by the amounts of analgesic medications used. Improvement of pain was defined as a > 50% decrease in pain scores as assessed with the visual analogue scale at different time points (baseline to months 1, 3, and 6). Limb survival was defined as the absence of a major amputation. A major amputation is an amputation proximal to level of the ankle. 6 Statistical analyses Baseline characteristics and response rate comparisons between the groups were analyzed using the chi-square test, corrected for continuity. For continuous variables, the independent t test was used. Differences between the groups were measured using the Mann-Whitney U test. The same tests were used for analysis of laboratory parameters. Survival analysis was calculated according to Kaplan Meier. P values <.05 were considered statistically significant.

3 Min Li et al /Experimental and Clinical Transplantation (2013) 5: Results Patient demographics Between September 2008 and January 2010, one hundred five patients were screened: 58 were found to be eligible and were randomized. They were randomized and received placebo, or BM-MNC transplant. Table 1 summarizes the demographics and baseline characteristics of subjects assigned to the treatment groups. There were no significant differences in baseline characteristics between the 2 groups. Table 1. Baseline Characteristics Placebo BM-MNCs Significance Group (n=29) Group (n=29) Age (y); mean ± SD 63 ± ± 9 NS Females 6 7 NS Smoking Current 5 6 NS Former NS Never 5 6 NS Hypertension NS Diabetes NS Coronary artery disease NS Critical limb ischemia disease status Pain NS Skin ulcer NS Ankle-brachial index 0.4 ± ± 0.3 NS Prior vascular reconstruction NS Percutaneous angioplasty Prior amputation 4 5 NS No significant differences were found by chi-square corrected for continuity. For continuous variables, the independent t test was performed. Safety assessment Bone marrow mononuclear cells transplant by intramuscular injections was well tolerated. Adverse events are summarized in Table 2. Adverse events were similar in the placebo and the BM-MNC groups. There were no significant differences in the incidence of adverse events among the groups. Four patients died during follow-up (2 in the placebo group, 2 in the BM-MNC group). These deaths were not considered related to treatment. In the placebo group, 1 patient died 2 weeks after a major amputation, and the other patient died owing to a cerebrovascular accident. The causes of death in the BM-MNC group were myocardial infarction in 1 patient and respiratory failure in the other patient. efficacy assessments Sixteen patients could not be evaluated for hemodynamic assessment because of extensive Table 2. Safety Analysis During 6-Month Follow-Up Placebo BM-MNCs Significance Group (n=29) Group (n=29) Fever 1 3 NS Allergies 0 0 NS Myocardial 0 1 NS Stoke 1 0 NS Malignancy 0 0 NS Death 2 2 NS Data are the number of patients. No significant differences were found by the Fisher exact test. ulceration that made ankle pressure assessment not feasible. An absolute increase of > 15% in anklebrachial index on at least 2 time points occurred in 12 of 23 evaluable BM-MNC patients. In the control group, 1 patient of the 19 evaluable patients showed a hemodynamic increase of > 15%. Bone marrow mononuclear cells treatment tended to improve pressure parameters (P =.002). Median time to improvement (day 0 to first increase of > 15%) was 1.5 months and this improvement was still present at month 6. Skin ulcers were evaluated in all 36 patients with ulcers (Table 1). Of 19 evaluable BM-MNC patients there were 5 responders, whereas none of the control patients showed an improvement of ulceration (P =.047). Ulcer healing of more than 50% occurred after a median 2 months after injection and was still present at month 6. Fifteen patients were not evaluated for pain scores owing to minor surgical intervention just before the transplant or they could not understand the VAS scale. Two of 17 control patients had > 50% decrease in pain score versus 11 of 26 BM-MNC patients (P =.045). A major amputation was done in 5 of the control patients and 3 BM-MNC treated patients (P =.706; Table 3). Overall, 20 patients responded. As some patients responded in more than 1 category (hemodynamic or clinical symptoms), there was a total of 25 responses. In the BM-MNC treated patients, there were 7 patients with improvement in more than Table 3. Treatment Results Endpoint Placebo BM-MNCs Significance Group n (%) Group n (%) P Value Hemodynamic improvement 1/19 (5%) 12/23 (52%).002 Improvement in skin ulcer 0/17 (0%) 5/19 (26%).047 Decrease in pain 2/17 (12%) 11/26 (42%).045 Major amputations 5/29 (17%) 3/29 (10%).706 Responding patients 3/29 (10%) 17/29 (59%).000 Data are the number of patients (percentage of evaluable patients).

4 438 Min Li et al /Experimental and Clinical Transplantation (2013) 5: Exp Clin Transplant 1 category as opposed to none in the control group. Three responders received placebos and 17 received BM-MNCs. The advantage of BM-MNCs compared with placebo was significant (P =.000; Table 3). Discussion The goals of treatment of chronic lower extremity ischemia are to relieve ischemic pain and prevent limb loss. Treatment options include medication, vascular bypass transplant, and intravascular interventional therapy. Patients with distal outflow, defected cases, or patients who cannot tolerate bypass surgery or interventional therapy cannot be operated on. With research regarding neovascularization induced by stem cell transplants, we see significant hope for these patients. According to previous studies, BM-MNCs contain endothelial progenitor cells, mesenchymal stem cells, and various growth factors and cytokines, which can contribute to the growth of blood vessels and improve ischemia status. 9,10 Tateishi-Yuyama and associates 11 reported that gastrocnemius injection of autologous BM-MNCs is an effective treatment option for lower extremity ischemic conditions. They claimed that autologous BM-MNC transplant could improve the blood supply to the ischemic region. Our study demonstrated the clinical symptoms like rest pain and skin ulcers were gradually relieved within 6 months of BM-MNC transplant. Anklebrachial index also increased after transplant. These data suggest that BM-BNC transplant could increase the blood supply to the affected extremity and alleviate the ischemic symptoms. The data also show that 2 or 3 days after BM-MNC transplant, a patient s clinical symptoms were greatly relieved, but these symptoms tended to become aggravated 1 week postoperatively. The above phenomenon can be explained as follows: Newly transplanted BM-MNCs cannot produce positive results in the 2 to 3 days after the operation; however, the early relief of the major symptoms can be attributed to epidural anesthesia performed during the operation and the inflammatory reaction caused by the BM-MNC suspension injected into the ischemic region. Epidural anesthesia could alleviate the arterial spasm in the lower extremity and increase the blood supply. However, when the above factors are not present, a patients symptoms could recur. Only after 1 to 3 months, would the endothelial progenitor cells, mesenchymal stem cells, and other cell components differentiate into vascular endothelial cells and smooth muscle cells, and build a capillary network and collateral circulation system in ischemic regions, resulting in the patient experiencing long-term relief from the symptoms and increased blood supply. Although much research has been done regarding the safety and efficacy of BM-MNC transplant in treatment of lower extremity ischemic disease, 12 this research is preclinical trials, with small sample size and with no placebo control. A case study has been reported with 8 patients with thromboangiitis obliterans, in which 1 sudden death occurred after intramuscular implantation of BM-MNCs. 13 The authors concluded that careful long-term monitoring was needed for future patients receiving this treatment, but the absence of a postmortem examination failed to give an accurate cause of death. Hirsch and associates 14 commented that it is impossible to evaluate whether this death is attributable to a direct effect of BM-MNC transplant. In our study, no death related to BM-MNC transplant was observed in 6 months monitoring. Our findings would be useful to predict the efficacy of BM-MNC transplant. In conclusion, autologous bone marrow mononuclear cells transplant for treatment of patients with chronic limb ischemia is safe, effective, and feasible. References 1. Isner JM, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest. 1999;103(9): Al-Khaldi A, Al-Sabti H, Galipeau J, Lachapelle K. Therapeutic angiogenesis using autologous bone marrow stromal cells: improved blood flow in a chronic limb ischemia model. Ann Thorac Surg. 2003;75(1): Tang J, Xie Q, Pan G, Wang J, Wang M. Mesenchymal stem cells participate in angiogenesis and improve heart function in rat model of myocardial ischemia with reperfusion. Eur J Cardiothorac Surg. 2006;30(2): Takahashi M, Li TS, Suzuki R, et al. Cytokines produced by bone marrow cells can contribute to functional improvement of the infarcted heart by protecting cardiomyocytes from ischemic injury. Am J Physiol Heart Circ Physiol. 2006;291(2):H886-H Dormandy JA, Rutherford RB. Management of peripheral arterial disease (PAD). TASC Working Group. TransAtlantic Inter-Society Consensus (TASC). J Vasc Surg. 2000;31(1 Pt 2):S1-S Kusumanto YH, van Weel V, Mulder NH, et al. Treatment with intramuscular vascular endothelial growth factor gene compared with placebo for patients with diabetes mellitus and critical limb ischemia: a double-blind randomized trial. Hum Gene Ther. 2006;17(6): Rutherford RB, Becker GJ. Standards for evaluating and reporting the results of surgical and percutaneous therapy for peripheral arterial disease. Radiology. 1991;181(1):

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