CLINICAL PERSPECTIVE

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(Circulation. 2006;114:2679-2684.)
© 2006 American Heart Association, Inc.

Vascular Medicine

Unblinded Pilot Study of Autologous Transplantation of Bone Marrow Mononuclear Cells in Patients With Thromboangiitis Obliterans

Koji Miyamoto, MD; Kazuhiro Nishigami, MD; Noritoshi Nagaya, MD; Koichi Akutsu, MD; Masaaki Chiku, MD; Masataka Kamei, MD; Toshihiro Soma, MD; Shigeki Miyata, MD; Masahiro Higashi, MD; Ryoichi Tanaka, MD; Takeshi Nakatani, MD; Hiroshi Nonogi, MD; Satoshi Takeshita, MD

From the Departments of Medicine (K.M., K.N., K.A., M.C., H.N., S.T.), Regenerative Medicine and Tissue Engineering (N.N.), Anesthesiology (M.K.), Transfusion Medicine (S.M.), Radiology (M.H., R.T.), and Organ Transplantation (T.N.), National Cardiovascular Center, Osaka; and the Department of Clinical Laboratory (T.S.), Osaka Minami Medical Center, Osaka, Japan.

Correspondence to Dr Satoshi Takeshita, MD, FACC, Department of Medicine (Cardiology), National Cardiovascular Center, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan. E-mail stake{at}muse.ocn.ne.jp

Received June 9, 2006; revision received September 19, 2006; accepted September 21, 2006.

	Abstract

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Background— The short-term clinical benefits of bone marrow mononuclear cell transplantation have been shown in patients with critical limb ischemia. The purpose of this study was to assess the long-term safety and efficacy of bone marrow mononuclear cell transplantation in patients with thromboangiitis obliterans.

Methods and Results— Eleven limbs (3 with rest pain and 8 with an ischemic ulcer) of 8 patients were treated by bone marrow mononuclear cell transplantation. The patients were followed up for clinical events for a mean of 684±549 days (range 103 to 1466 days). At 4 weeks, improvement in pain was observed in all 11 limbs, with complete relief in 4 (36%). Pain scale (visual analog scale) score decreased from 5.1±0.7 to 1.5±1.3. An improvement in skin ulcers was observed in all 8 limbs with an ischemic ulcer, with complete healing in 7 (88%). During the follow-up, however, clinical events occurred in 4 of the 8 patients. The first patient suffered sudden death at 20 months after transplantation at 30 years of age. The second patient with an incomplete healing of a skin ulcer showed worsening of the lesion at 4 months. The third patient showed worsening of rest pain at 8 months. The last patient developed an arteriovenous shunt in the foot at 7 months, which spontaneously regressed by 1 year.

Conclusions— In the present unblinded and uncontrolled pilot study, long-term adverse events, including death and unfavorable angiogenesis, were observed in half of the patients receiving bone marrow mononuclear cell transplantation. Given the current incomplete knowledge of the safety and efficacy of this strategy, careful long-term monitoring is required for future patients receiving this treatment.

Key Words: angiogenesis • collateral circulation • endothelium • peripheral vascular diseases

	Introduction

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The clinical consequences of severe peripheral arterial disease or critical limb ischemia include rest pain and the loss of tissue integrity in the distal limb.^1–3 Therapeutic options for such patients are limited. These conditions are often refractory to conservative measures and are typically unresponsive to drug therapy. When vascular obstruction involves a long segment or is widespread, percutaneous revascularization may not be feasible. Surgical therapy, consisting of arterial bypass or amputation, is complicated by variable morbidity and mortality, and its effectiveness depends on the short- and long-term patencies of the conduit employed. Therapeutic angiogenesis thus constitutes a potential alternative treatment strategy for such patients.^4,5

Previous investigators have suggested that endothelial progenitor cells, originating from bone marrow, circulate in adult peripheral blood and participate in postnatal neovascularization.^6–8 Subsequent experiments have shown that bone marrow or bone marrow–derived cells have the potential to stimulate angiogenesis and thereby modulate the hemodynamic deficit in ischemic limbs in vivo.^9,10 The Therapeutic Angiogenesis by Cell Transplantation (TACT) study first demonstrated that the magnitude of angiogenesis stimulated by these cells is sufficient to constitute a therapeutic benefit in patients with critical limb ischemia.¹¹ In that study, the investigators injected bone marrow mononuclear cells (BM-MNCs) into the ischemic limb of patients and documented a significant improvement in the hemodynamic deficit as well as the relief of ischemic symptoms. Although the TACT study established the concept of using BM-MNCs for therapeutic angiogenesis, limited information is available about the long-term safety and efficacy of this strategy.

Editorial p 2581

Clinical Perspective p 2684

The purpose of the present study was to determine the long-term safety and clinical impact of BM-MNC transplantation for "no-option" patients with thromboangiitis obliterans.

	Methods

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Patients
Eight patients with thromboangiitis obliterans were treated with an autologous transplantation of BM-MNCs between March 2002 and September 2004. The diagnosis of thromboangiitis obliterans was based on the criteria proposed by Olin¹²: (1) onset before age 45; (2) current (recent) history of tobacco use; (3) the presence of distal-extremity ischemia (infrapopliteal or infrabrachial) indicated by claudication, rest pain, ischemic ulcers, or gangrene; (4) exclusion of autoimmune or connective tissue diseases, hypercoagulable states, and diabetes mellitus; (5) exclusion of a proximal source of emboli by echocardiography and arteriography; and (6) consistent arteriographic findings in the clinically involved and noninvolved limbs.

Patients qualified for cell transplantation if they had chronic limb ischemia, with rest pain or a nonhealing ischemic ulcer, present for a minimum of 4 weeks without evidence of improvement in response to conventional drug therapy; showed angiographic evidence of vasculopenia in the affected limb; and were not candidates for percutaneous or surgical revascularization. The exclusion criteria included severe concurrent illness, the presence of proliferative diabetic retinopathy, and a history or clinical evidence of a malignant disorder.

All the patients involved in the present study received continuous medical therapy for >2 months before BM-MNC transplantation to confirm that conventional measures would be insufficient to achieve improvement in rest pain or skin ulcer/gangrene. During this period, no surgical therapies such as bypass grafting, extensive debridement, skin grafting, or limb amputation were performed. In addition, the patients were admitted to the hospital for a minimum of 1 month before BM-MNC transplantation to exclude the likelihood of spontaneous improvement in ischemic symptoms resulting from an enrollment bias. It should be also pointed out that the patients remained in the hospital and received the same therapy for at least 1 month after BM-MNC transplantation to avoid changes in their treatment.

BM-MNC Transplantation
While the patients were under general anesthesia, marrow cells were aspirated from the ileum. BM-MNCs were sorted on an AS-104 blood-cell separator (Fresenius HemoCare, Redmond, Wash) and concentrated to a final volume of 50 mL. After bone marrow cells were sorted on the AS-104 blood-cell separator, a small fraction of the cells was used for BM-MNC counting; the concentration of BM-MNCs in the final product was determined by using a microscope counting chamber after May-Giemsa staining. By using another fraction of cells, the number of CD34⁺ cells in the BM-MNCs was also determined by fluorescence-activated cell sorting (FACS SCAN flow cytometer; Becton Dickinson, San Jose, Calif). The cells were incubated with the FITC-conjugated mouse monoclonal antibody against human CD34 (clone 581; Becton Dickinson) according to manufacturer’s instructions.

For each patient, 100 aliquots of BM-MNCs (0.5 mL per aliquot) were administered via a syringe with a 27-gauge needle. Injection was performed into 9 lower limbs in 7 of the patients and the bilateral hands in 1. Injection sites were arbitrarily selected according to angiographic findings (ie, the degree of vasculopenia) and included calf muscles such as the soleus and gastrocunemius muscles as well as the sole muscles of the foot. For the patient with hand ischemia, injection was performed in palm muscles.

Assessment of Short-Term Outcome
Ischemic pain was assessed with a visual analog pain scale (VAS) with 10 levels. Ischemic ulcers were documented by color photography. Resting ankle-brachial pressure index (ABI) was calculated as the quotient of absolute ankle pressure and brachial pressure (the patient who received BM-MNC transplantation in his hands was excluded from ABI analysis). Angiographic assessment was performed with magnetic resonance angiography, computed tomographic angiography, or digital subtraction angiography. Adverse events were defined as death, limb amputation, pathological angiogenesis, recurrence/worsening of ischemic symptoms (ie, rest pain, skin ulcer, gangrene), myocardial infarction, stroke, and malignant disease.

Assessment of Long-Term Outcome
The mean length of follow-up was 684±549 days (range 103 to 1466). Patients were followed up by history analysis, physical examination, routine blood testing, ABI, and angiography at prescribed intervals during the first year, after which they were contacted at an outpatient clinic or by telephone to track events.

Data Analysis
All data are presented as mean±SD (range) or frequencies (percentage).

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

	Results

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Diagnosis
The diagnosis of thromboangiitis obliterans was made according to the criteria described above. Among the 8 patients, only patient 6 did not completely fulfill the criteria; ie, this patient had no history of tobacco use (Table 1). Laboratory screening excluded the possibility of other underlying diseases, however, including autoimmune and connective tissue diseases. It should be also pointed out that patient 6 had diabetes mellitus at the time of cell transplantation but not at the onset of thromboangiitis obliterans. With the typical characteristic angiographic findings of thromboangiitis obliterans, such as multiple segmental arterial involvement (skip lesions) and "cork-screw" collateral vessels, we diagnosed patient 6 as having thromboangiitis obliterans, even though the patient did not have a history of tobacco use.

View this table: [in this window] [in a new window]   TABLE 1. Patient Characteristics

Patient Characteristics
The demographic and clinical data of the 8 patients are shown in Table 1. The mean age of the patients enrolled was 46±14 years (range 28 to 63). Seven patients (88%) were male. One patient had undergone prior femoral-tibial artery bypass grafting, and 1 had undergone sympathetic ganglion block. These treatments were performed >1 year before BM-MNC transplantation. Seven patients (88%) had a history of smoking, all of whom stopped smoking at least 1 month before transplantation.

Short-Term Outcome
The total volume of cells aspirated from the ileum was 728±72 mL (range 600 to 800) per patient, and the total volume of injected BM-MNCs was 45±7 mL (range 30 to 50) per patient. Total number of injected BM-MNCs was 3.5±0.8x10⁹ (range 2.0 to 4.7x10⁹), and that of CD34⁺ cells was 6.8±2.6x10⁷ (range 2.4 to 9.7x10⁷).

Angiographic assessment at 4 weeks after transplantation revealed an apparent increase in limb vascularity in 3 of the 8 (38%) patients (4 of the 11 limbs) (Figure 1). Hemodynamic assessment also failed to document evidence of improved collateral development. Specifically, an increase in ABI (>0.1) was observed in 2 of 7 (29%) patients (2 of 8 limbs), whereas a decrease in ABI (>0.1) was observed in 2 of 7 (29%) patients (2 of 8 limbs). As a result, mean ABI measured at 4 weeks (0.71±0.30) did not differ from that at the baseline (0.70±0.27). Because 2 patients had sites of arterial occlusion distal to the ankle, they showed normal ABIs before treatment. Even after the exclusion of these 2 patients, ABI showed no changes between before (0.55±0.15) and after transplantation (0.55±0.24).

View larger version (141K): [in this window] [in a new window]   Figure 1. Digital subtraction angiography at (A) baseline and (B) 1 month after cell transplantation. Arrows indicate newly visible collateral vessels at the calf level.

In contrast to the angiographic and hemodynamic results, improvement in limb status was observed in all 8 patients (100%). Improvement in VAS was observed in all 11 limbs, with a decrease from a mean of 5.1±0.7 to 1.5±1.3. Furthermore, complete pain relief was achieved in 4 of the 11 limbs (36%). Improvement in skin ulcers was also observed in all 8 limbs (100%), with complete healing in 7 (88%). Although surgical amputations of the distal limb were performed in 2 patients at 1 month, these operations were intentionally scheduled to be performed after transplantation with the expectation of sufficiently improving the limb perfusion to distally advance the site of amputation (Table 2; Figure 2A and 2B).

View this table: [in this window] [in a new window]   TABLE 2. Adverse Outcomes After Autologous Transplantation of BM-MNCs in Patients With Thromboangiitis Obliterans

View larger version (53K): [in this window] [in a new window]   Figure 2. Skin ulcer at (A) 1 month, (B) 2 months, and (C) 4 months after cell transplantation. The patient had far-advanced gangrene, and total limb integrity could not be fully preserved. The patient received a prescheduled amputation of the distal limb at 1 month, and the skin ulcer continued to improve thereafter. At 4 months, however, the skin lesion began to enlarge.

Long-Term Outcome
The mean follow-up period was 684±549 days (range 103 to 1466). At the final follow-up, VAS score remained unchanged from that observed at 1 month after transplantation in 5 of the 8 patients (63%). The mean VAS score at follow-up also remained low (2.3±1.9) compared with that observed at baseline (5.1±0.7).

In contrast to the pain scale results, adverse events were observed in as many as 4 patients (50%) (Table 2). At age 30 years, patient 4 suddenly died of an unknown cause at 20 months after transplantation. This patient had previously been a smoker, but had stopped smoking before cell transplantation. He had no history of diabetes, hypertension, or hyperlipidemia. Furthermore, ²⁰¹thallium myocardial scan performed before BM-MNC transplantation showed no signs of myocardial ischemia. After cell transplantation, his limb pain disappeared within 1 week and his skin ulcer resolved by 1 month. Thereafter, he was completely free of limb symptoms. Twenty months after cell transplantation, however, he was found dead at his home. He had never experienced chest pain up to the time of his death. Because no autopsy was performed, the cause of his death remains unknown.

Patient 6 showed worsening of an ischemic ulcer at 4 months. The patient had far-advanced gangrene, and total limb integrity could not be fully preserved. The patient underwent a prescheduled amputation of the distal limb at 1 month (see Short-Term Outcome) (Figure 2A), and the skin ulcer continued to improve thereafter (Figure 2B). At 4 months, however, the skin lesion began to increase in size (Figure 2C). The patient subsequently received a second round of cell therapy.

In patient 7, despite complete healing of the skin ulcer, rest pain did not completely resolve after transplantation, with a VAS score of 3 at 1 month. At 8 months, the patient experienced worsening of rest pain (VAS score=4). After a combination of exercise training and maximal drug therapy, the pain improved and became well tolerated.

Patient 8 experienced swelling and recurrence of the skin ulcer in his foot at 7 months. Computed tomographic angiography documented an early venous return of contrast material in his right limb (Figure 3B) that was not observed at the baseline (Figure 3A). Ultrasound examination disclosed an arterialized waveform in the dorsal vein at the base of his third toe, suggesting the presence of an arteriovenous shunt. By 1 year, the swelling and skin ulcer had spontaneously regressed. The systolic pulsatile component in the venous waveform was found to be diminished on ultrasound examination, and early venous filling had disappeared on computed tomographic angiography (Figure 3C).

View larger version (49K): [in this window] [in a new window]   Figure 3. Computed tomographic angiography at (A) baseline, (B) 7 months, and (C) 1 year after cell transplantation. Early venous return of contrast material observed at 7 months spontaneously regressed by 1 year.

	Discussion

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In the present unblinded and uncontrolled pilot study, we documented that the transplantation of BM-MNCs was associated with an improvement in ischemic symptoms for up to 4 years. Indeed, VAS scores improved from 5.1±0.7 to 2.3±1.9 at follow-up. Furthermore, skin ulcers remained completely healed in 6 of 7 patients. In this regard, the present findings extend previous observations¹¹ by establishing the potential long-term benefit of BM-MNC transplantation for the treatment of arterial insufficiency.

It should be noted, however, that half of the patients suffered adverse events during follow-up. Such a high rate of adverse events cannot be explained by the natural course of the disease itself. In general, the prognosis of patients with thromboangiitis obliterans is directly related to tobacco use.^12,13 Patients who are able to stop smoking avoid the recurrence of the disease and amputation.¹⁴ In addition, unlike those with atherosclerosis of the extremities, these patients rarely show involvement of visceral vessels and do not appear to be at an increased risk of stroke or myocardial infarction. The mortality rates are thus not higher than those of age- and sex-matched populations.^15,16 It is entirely possible that the high rate of adverse events observed in our patients may have been directly related to BM-MNC transplantation rather than to the progression of the disease itself.

Patient 4 suddenly died at 20 months after transplantation at the age of 30 years. The deaths of patients receiving BM-MNC transplantation were previously reported in the TACT study, in which 2 of 25 patients died of acute myocardial infarction within 24 weeks after transplantation.¹¹ The patients’ backgrounds in the TACT study, in terms of age and comorbidity, may have been totally different from those of our study, in which only patients with thromboangiitis obliterans were recruited. As mentioned above, in patients with thromboangiitis obliterans, coronary involvement is rare, and they usually do very well as long as they discontinue smoking.^12–16 Patient 4 had no risk factors for atherosclerosis and stopped smoking before BM-MNC transplantation. Furthermore, ²⁰¹thallium scintigraphy performed before transplantation documented no sign of myocardial ischemia. Considering the patient’s background and the natural course of the disease,^12–16 the possibility that his death was related to BM-MNC transplantation cannot be excluded. In this regard, several studies have suggested the possible role of BM-MNCs in atherogenesis. A recent report by Silvestre et al,¹⁷ for example, demonstrated that the transplantation of BM-MNCs into ischemic limbs of apolipoprotein E–knockout mice led to a significant increase in atherosclerotic plaque size at a distant site. More recently, George et al¹⁸ have also shown that an intravenous injection of bone marrow cells into apolipoprotein E–knockout mice results in an increase in atherosclerotic lesion size, whereas an injection of endothelial progenitor cells influences plaque stability. These reports indicate that attempts to enhance neovascularization by using BM-MNCs could also enhance unwanted plaque growth and instability, thus suggesting the possibility that our young patient died of an acute coronary event due to accelerated atherogenesis after BM-MNC transplantation.

We also encountered the development of an arteriovenous shunt, which could be a potential consequence of BM-MNC transplantation. Indeed, concerns have been raised about the potential adverse effects of cell transplantation, ie, unregulated differentiation and proliferation. Wakitani et al¹⁹ reported that teratoma formation could occur after embryonic stem cell transplantation. Yoon et al²⁰ documented intramyocardial calcification after the transplantation of bone marrow cells in rats. Our observations may provide another cautionary example of unregulated differentiation and proliferation. Although the arteriovenous shunt in our case was self-limited, it may represent unwanted angiogenesis; thus, careful monitoring is warranted for future patients who receive BM-MNC transplantation.

Worsening or recurrence of ischemic symptoms was observed in 3 patients. The short-term outcome at 1 month was poor in these patients except in the patient with an arteriovenous shunt. The improvement in rest pain was not substantial in 1 patient. Healing of the skin ulcer was incomplete in another. It is anticipated that a poor response 1 month after BM-MNC transplantation could result in a poor long-term outcome. It is important to note that, in the latter patient with incomplete healing of the skin ulcer, the angiographic improvement of the collateral network at 1 month remained unchanged at 5 months when worsening of the skin ulcer was observed. It is suggested that the temporal sequence of improvement in ischemic limb status does not necessarily parallel the temporal evolution of collateral development.

	Conclusions

Top Abstract Introduction Methods Results Discussion Conclusions References

 
In this unblinded and uncontrolled pilot study, long-term adverse events after BM-MNC transplantation, including death and unfavorable angiogenesis, were observed in half of the patients with thromboangiitis obliterans. Given the current incomplete knowledge of the safety and efficacy of this strategy, careful long-term monitoring is required for future patients receiving BM-MNC transplantation.

	Acknowledgments

 
Sources of Funding

This work was supported by Health and Labor Sciences Research Grants (H16-009, H16-017, H17-009), by Ministry of Health, Labor and Welfare Research Grants for Cardiovascular Disease (16C-6, 18C-4), and by grants from the New Energy and Industrial Technology Development Organization and the Japan Cardiovascular Research Foundation.

Disclosures

None.

	References

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CLINICAL PERSPECTIVE

The favorable short-term outcome of bone marrow mononuclear cell transplantation (BM-MNC) transplantation has been established in patients with critical limb ischemia. However, the long-term outcome of this treatment strategy has not been determined yet. In our case series, we documented that long-term adverse events, including death and unfavorable angiogenesis, were observed in 4 of 8 patients receiving BM-MNC transplantation. The first patient suffered sudden death at 20 months after transplantation at 30 years of age. The second patient with incomplete healing of a skin ulcer showed worsening of the lesion at 4 months. The third patient had worsening of rest pain at 8 months. The last patient developed an arteriovenous shunt in the foot at 7 months, which spontaneously regressed by 1 year. Given the current incomplete knowledge on the safety and efficacy of this strategy, it is suggested that careful long-term monitoring is required in patients receiving BM-MNC transplantation. To our knowledge, this is the first report on the long-term outcome of transplantation of BM-MNCs for critical limb ischemia, and the first that documents the development of unfavorable angiogenesis and sudden death after therapeutic angiogenesis.

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