Autologous Peripheral Blood Mononuclear Cell Implantation for Patients With Peripheral Arterial Disease Improves Limb Ischemia

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1 Circ J 2005; 69: Autologous Peripheral Blood Mononuclear Cell Implantation for Patients With Peripheral Arterial Disease Improves Limb Ischemia Akio Ishida, MD; Yusuke Ohya, MD; Hitoshi Sakuda, MD*; Katsuhiko Ohshiro, MD; Yasushi Higashiuesato, MD**; Moriyasu Nakaema, MD*; Shinobu Matsubara, MD*; Shizuko Yakabi ; Ayano Kakihana, MD; Mika Ueda, MD; Chiharu Miyagi, MD; Nobuhisa Yamane, MD ; Kageharu Koja, MD*; Kimihiro Komori, MD ; Shuichi Takishita, MD Background Implantation of bone marrow mononuclear cells, including endothelial progenitor cells, into ischemic limbs has been shown to improve collateral vessel formation. In the present study the safety and feasibility of autologous peripheral blood mononuclear cells (PBMNCs) implantation after granulocyte-colony stimulating factor (G-CSF)-induced mobilization was investigated in patients with severe peripheral arterial disease. Methods and Results Six cases were enrolled: 5 of thromboangitis obliterans and 1 of arteriosclerosis obliterans. Following administration of G-CSF (10 g kg 1 day 1 ), PBMNCs were harvested and injected intramuscularly (5 legs and 1 arm) for 2 days for the patients with ischemia of the legs. No serious adverse events related to G-CSF administration, harvest or implantation were observed during this study period. Improvement in the ankle brachial pressure index ( ABI: >0.1) was seen in 4 patients at 4 weeks and ischemic ulcers improved in 3 of 3 patients. The mean maximum walking distance significantly increased from 203 m to 559 m (p=0.031) at 4 weeks and was sustained for 24 weeks. Significant improvement was seen in physiological functioning subscale of Short Form-36. Conclusion Implantation of PBMNCs collected after G-CSF administration could be an alternative to therapeutic angioplasty in patients with severe peripheral arterial disease. (Circ J 2005; 69: ) Key Words: Granulocyte-colony stimulating factor; Peripheral arterial disease; Peripheral blood mononuclear cells; Therapeutic angiogenesis (Received January 26, 2005; revised manuscript received June 29, 2005; accepted July 6, 2005) Division of Cardiovascular Medicine, Nephrology and Neurology, *Second Department of Surgery, **Department of Clinical Laboratories, Blood Transfusion Department, University of the Ryukyus School of Medicine, Okinawa and Division of Vascular Surgery, Department of Surgery, Nagoya University, Graduate School of Medicine, Nagoya, Japan Mailing address: Yusuke Ohya, MD, Division of Cardiovascular Medicine, Nephrology and Neurology, University of the Ryukyus School of Medicine, 207 Uehara, Nishihara-cho, Okinawa , Japan. ohya@med.u-ryukyu.ac.jp Bone marrow is a rich reservoir of tissue-specific stem and progenitor cells. Experimental and clinical studies have shown that endothelial progenitor cells are mobilized from bone marrow, migrate to ischemic tissue, and contribute to the neovacularization process in response to tissue ischemia. 1 3 In patients suffering from peripheral arterial disease (PAD), such as arteriosclerosis obliterans (ASO) and thromboangitis obliterans (TAO, Buerger s disease), the implantation of autologous whole bonne marrow mononuclear cells into the gastrocnemius muscle resulted in significant improvements of limb blood flow. 4,5 In hematological and cancer diseases, peripheral blood has largely replaced bone marrow as the source of stem cells in autologous transplantation, because of faster hematologic recovery. The safety of granulocyte-colony stimulating factor (G-CSF) administration and apheresis has already been established, although frequent side-effects of G-CSF administration are myalgias, arthralgias, headache, and fever; however, long-term serious complications have not been reported. 6 Pretreatment with G-CSF can mobilize not only hematopoietic stem cells, but also endothelial progenitor cells from bone marrow to peripheral blood. 2,7,8 Therefore, we conducted an open-labeled clinical trial of implantation of autologous peripheral blood mononuclear cells (PBMNCs) after mobilization with G-CSF in patients with severe PAD. Methods Patients Six cases of PAD were enrolled, all male patients: 5 cases of TAO, and 1 of ASO. All the patients had a history of intermittent claudication, rest pain, non-healing ischemic ulcers, or all three, and were not candidates for surgical revascularisation. None of the patients had responded to conventional medical therapy for at least 8 weeks. Exclusion criteria were: diabetes mellitus with proliferative retinopathy; malignant disease; recent onset (within 3 month) of myocardial infarction or brain infarction; uncontrolled myocardial ischemia; persistent severe heart failure (ejection fraction <30%); hematological disease; current serious infectious disease; aged older than 80 years; and diseases with life expectancy of less than 1 year. We

2 Therapeutic Angiogenesis by PBMNCs Implantation 1261 Table 1 Patient and Procedure Characteristics Case no. Age Implanted Total harvested MNC Total CD34+ Diagnosis Fontaine stage (years) lesion ( ) ( 10 8 ) 1 53 TAO Arm (R) TAO Leg (R) TAO Leg (R) ASO Leg (R) TAO Leg (L) TAO Leg (R) Case 1 and 5 are the same patient. MNC, mononuclear cell; TAO, thromboangitis obliterans; ASO, arteriosclerosis obliterans. Fig 1. Changes in the white blood cell (WBC) count after granulocyte-colony stimulating factor (G-CSF) administration. Administration was initiated on day 0. Harvest and implantation of peripheral blood mononuclear cells (PBMNCs) were done for 1 day for the arm (patient no. 1), and 2 days for the leg (patients nos. 2 6). G-CSF administration was initiated 4 days before PBMNCs harvest and implantation for cases 1 and 2, and 3 days before for the other patients (arrowhead indicates the day of PBMNCs harvest and implantation for patients nos. 1 and 2). obtained written informed consent from all patients and the Medical Ethics Committee of University of the Ryukyus School of Medicine approved the protocol. Our study started in February 2003, at the University of the Ryukyus. By September 2004, 17 patients had been screened for this therapy, but 11 patients were excluded because of indication and exclusion criteria. Procedure After administration of 10 g kg 1 day 1 G-CSF (filgrastim, Kirin Brewery Co, Tokyo, Japan) by subcutaneous injection for 4 5 days, PBMNCs were harvested using an AS104 cell separator (Fresenius Medical Care, Tokyo, Japan). During the preparation of the PBMNCs, the sedimentation process reduced the volume of PBMNCs to approximately ml. Within a few hours after PBMNCs harvest, patients were injected with ml of mononuclear cells in each of sites in the ischemic limb muscles, under epidural anesthesia. Injections were spaced 2 3 cm apart, using a 25-gauge needle. PBMNCs harvest and injection were done for 2 days for lower limb ischemia. On the second day, approximately 50ml of PBMNCs were injected in between the first injection sites. We assessed ischemic status by measuring the ankle brachial pressure index (ABI), transcutaneous oxygen pressure (TcPO2), and exercise treadmill test (2.4 km/h, 12% incline, pain-free walking distance, maximal walking distance). The healthrelated quality of life of the patients was assessed using the Short Form (SF)-36 questionnaire. 9 Statistical Analysis Data are presented as mean (SD). We used a paired t test for comparison of continuous variables. Statistical significance was assumed at a value of p<0.05. Statistical analysis was done with Statview version J-5.0 (Hulinks, Tokyo, Japan). Results The characteristics and number of harvested cells of the patients are shown in Table 1. Cases 1 and 5 are the same patient; although the improvement of the ulcer and pain in his right thumb was sustained for 1 year after the first PBMNCs implantation, an ischemic ulcer of his left leg worsened despite conventional therapy and he underwent PBMNCs implantation in the affected leg 14 months after the first implantation. No serious adverse reactions related to G-CSF administration occurred during the periprocedural period: 4 of the patients complained of mild lumbago, which was easily controlled with analgesic and completely relieved when the G-CSF administration ceased. No thromboembolic complications, such as coronary or cerebrovascular events, occurred during G-CSF administration or follow-up. The maximum white blood cell (WBC) counts of the 6 patients were 19,500 46,900 (mean 31,800) / l (Fig 1). Because the maximum WBC counts were obtained on the third day after G-CSF administration for patients nos. 1 and 2, G-CSF administration was initiated 3 days before apher-

3 1262 ISHIDA A et al. Fig 2. Number of (a) mononuclear cells and (b) CD34+ cells collected during apheresis. Although the number of harvested peripheral blood mononuclear cells (PBMNCs) was slightly low in all patients on second day of harvest (2.35 [0.99] vs 1.96 [0.76] p=0.061), a comparable number of CD34+ cells (1.12 [0.36] 10 8 vs 1.07 [0.47] 10 8, p=0.697) were collected on the second PBMNCs harvest day. Fig 3. Changes in (a) ankle-brachial pressure index (ABI) and (b) transcutaneous oxygen pressure (TcPO2) after the treatment. Fig 4. Change in walking capacity after the treatment. (a) Pain-free walking distance and (b) maximal walking distance were assessed by treadmill exercise test at 2.4 km/h and 12% incline. *p<0.05 vs baseline. esis for the other patients. Although the number of harvested PBMNCs in all patients was slightly low on the second day of harvest, we could collect a comparable number of CD34+ cells on that day (Fig 2). The remainder of the collected peripheral blood cells was returned to the patient by intravenous continuous infusion. No adverse events occurred during PBMNCs harvest. Patients were injected with approximately 30 or 100 ml of

4 Therapeutic Angiogenesis by PBMNCs Implantation 1263 Fig 5. Improvement in ischemic ulcers after the treatment. Non-healing ischemic ulcer on the right thumb (case 1), left big toe (case 5), and right big toe improved 4 weeks (cases- 1, 6) and 8 weeks (case 5) after peripheral blood mononuclear cell implantation. PBMNCs in the arm or leg, respectively. Local edema and muscle pain, except flare reaction and local fever, at the injection sites were observed in all patients from the day following implantation, but abated within 1 week. At 4 weeks follow-up, there was an increase in ABI ( ABI >0.1) in 4 of 5 patients who received PBMNCs implantation in the leg, however, it nearly returned to the baseline level at 24 weeks. The TcPO2 level increased in 2 patients and was unchanged in 2 patients (Fig 3). The pain-free walking distance improved from 72.1± 33.5 m to 189.3±108.9 m (p=0.053) at 4 weeks. After PBMNCs implantation, leg pain did not affect continuous walking and as a result, the maximum walking distance significantly increased from 203±95.4 m to 559±318 m (p=0.031) at 4 weeks (Fig4) and continued to improve up to 24 weeks (858±305.9 m, p=0.019). Ischemic ulcers of the thumb (patient no. 1) and big toe (patient nos. 5, 6) showed improvement either at 4 weeks (patient nos. 1, 6) or 8 weeks (patient no. 5) after implantation (Fig 5). Pain relief occurred within 1 week after implantation and daily use of analgesics was reduced (patient nos. 1, 5, 6). The bodily pain subscale rating on the SF-36 improved from 47±29.5 to 64±29.1 at 4 weeks in all except 1 patient (no. 4) and the physical functioning subscale significantly improved from 51±17.7 to 70±10.5 (p=0.032) at 4 weeks and to 81±4.8 (p=0.032) at 24 weeks (data not shown). Discussion We here report the results of 6 cases of PAD for which we used autologous PBMNCs implantation, in order to demonstrate the safety and feasibility of this procedure. There was improvement in the ABI, ulcer healing, TcPO2, maximum walking distance, and health-related quality of life, without any serious adverse reactions, similar to previous reports of the use of bone marrow cells. 4,5 The numbers of mononuclear cells and CD34+ cells are lower in peripheral blood than in bone marrow, 5 but G-CSF administration mobilizes CD34+ cells, including endothelial progenitor cells, from the bone marrow to the peripheral blood. 7,8 The mobilizing effect of G-CSF was sustained for several days, and we could collect comparable numbers of CD34+ cells on the second day of PBMNCs harvest (Fig 2). In this study, PBMNCs were harvested and implanted into ischemic leg muscles over 2 successive days to enhance neovascularization. The average total number of harvested mononuclear cells and CD34+ cells for each patient were , and cells, respectively, which is equivalent to or more than those harvested from bone marrow in previous studies. 5,10 Although the minimum number of mononuclear cells or CD34+ cells for improvement of the tissue ischemia has not been established, repeatability is one of the advantages of PBMNCs implantation over bone marrow mononuclear cells implantation. In a recent report the number of implanted CD34+ cells positively correlated with the efficacy of bone-marrowderived cells implantation in PAD patients, 11 which supports our method. It is unclear whether the composition of mononuclear cells from peripheral blood harvested after G-CSF administration is similar to that from bone marrow. Tateishi- Yuyama et al have showed the advantages of bone marrow cells over peripheral blood cells without G-CSF administration for improvement of ischemic status in patients with PAD, 5 but a recent study performed in patients with myocardial infarction demonstrated intracoronary infusion of PBMNCs mobilized with G-CSF, essentially following the identical procedure used in the present study, resulted in a similar improvement of left ventricular systolic function as with bone marrow cell implantation. 10 Unfortunately, there is not yet an established method for evaluating neovascularization after cell implantation. The most important objectives of any intervention in patients with PAD are symptomatic relief, limb salvage, and functional improvement. In the present study subjective symptoms such as ischemic pain were greatly reduced within 1 week, and analgesic use was reduced. In 3 patients there

5 1264 ISHIDA A et al. was improvement of the ulcers and limbs were salvaged after PBMNCs implantation. The SF-36 is responsive to thereapeutic interventions and improvements in PAD symptoms, particularly the physical functioning subscale, 13,14 and in the present study that showed significant improvement after PBMNCs implantation and was maintained during 24 weeks of follow-up. Although the increased ABI nearly returned to baseline level, the improvement in maximum walking distance was maintained during the 24 weeks follow-up. We do not know the exact reason for this discrepancy. In patient no. 5, the ABI level did not decrease at the baseline and there was no difference after PBMNCs implantation, although his big toe ulcer was obviously improved at 8 weeks after treatment, which suggests that the ABI level does not accurately reflect improvement in peripheral circulation. Isner et al reported that in diabetic neuropathy ischemic change resulting from a decrease of vasa nervorum can be restored by administration of vascular endothelial growth factor (VEGF), 15,16 and Iba et al showed that implantation of PBMNCs, which have a high concentration of VEGF and various angiogenic factors, into ischemic limbs enhanced collateral vessel formation. 17 It could be hypothesized that sustained improvement of microvascular ischemia around the peripheral nerves in ischemic tissue after PBMNCs implantation may restore peripheral nerve function and produce the improvement in the subjective signs and symptoms. G-CSF administration may promote leukocytosis, hypercoagulability or plaque vulnerability, and induce acute vascular events; however, none occurred during the follow-up period in this study. Risk factors for coronary artery disease were entirely controlled in all the present patients. We assessed the carotid and coronary artery by ultrasonography, and 201 Tl-dipyridamole scintigraphy or coronary angiography in all patients before G-CSF administration. There is a hypothetical concern that implanted cells may differentiate into mesenchymal cells, such as osteoblasts, fibroblasts, smooth muscle cells and myogenic cells, instead of endothelial cells in the injected tissue, suggesting that selective implantation of endothelial lineage cells might be suitable for therapeutic angiogenesis. In fact, Inaba et al demonstrated that implantation of selected CD34+ cells is also effective as angiogenic therapy for PAD patients. 10 However, recent studies showed the potential of bone marrow cells to promote secretion of angiogenic cytokines, such as VEGF and basic fibroblast growth factor (bfgf), and suggest that it is these secreted cytokines rather than cell incorporation that promote functional recovery. 5,17,21,22 Not only CD34+ cells but also the CD34 fraction in bone marrow mononuclear cells secrete angiogenic cytokines, and enhance angiogenesis. It has been reported that PBMNCs also secrete several angiogenic cytokines such as VEGF, bfgf, platelet-derived growth factor-ab, and transforming growth factor- 17 Therefore, we implanted the entire mononuclear cell population obtained after G-CSF administration. The present results demonstrate that implantation of autologous PBMNCs mobilized with G-CSF is a safe and feasible strategy for therapeutic angiogenesis in patient with PAD. The major limitation of this study is the lack of a control group, such as PBMNCs implantation alone or a G-CSF alone group. It is difficult to obtain equal number of PBMNCs and CD34+ cells without G-CSF administration and G-CSF per se may increase the peripheral blood flow by mobilization of endothelial progenitor cells from bone marrow to peripheral artery. However, Kang et al reported that G-CSF-mobilized peripheral blood cells improved cardiac function in patients with acute myocardial infarction, though G-CSF alone had no effect on cardiac function. 14 Interestingly, the improvement in the finger ulcer in patient no. 1 was sustained at least for 14 month after PBMNCs implantation, although an ulcer of the big toe developed 12 months later (=patient no. 5), suggesting that the effect of G-CSF per se is not as effective as that of PBMNCs implantation. Further follow-up examinations of the patients are needed to define whether the beneficial effects will be sustained long-term. The advantage of PBMNCs over bone marrow mononuclear cells is its repeatability, which thereby enhances the formation of collateral vessels, and it is a procedure that does not require general anesthesia. Recently, potent angiogenic effects of PBMNCs without mobilization with G- CSF were reported, 23 so randomized trials are required to compare the clinical outcome of PBMNCs with or without G-CSF administration versus the use of bone marrow mononuclear cells. Acknowledgments This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No ). References 1. Asahara T, Murohara T, Sullivan A, Silver M, van der Zee R, Li T, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997; 275: Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med 2003; 9: Sata M. Molecular strategies to treat vascular diseases: Circulating vascular progenitor cell as a potential target for prophylactic treatment of atherosclerosis. Circ J 2003; 67: Esato K, Hamano K, Li TS, Furutani A, Seyama A, Takenaka H, et al. 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