Autologous stem cell therapy for peripheral arterial disease

Atherosclerosis 209 (2010) 10–17

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Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis

Review

Autologous stem cell therapy for peripheral arterial disease Meta-analysis and systematic review of the literature Gian Paolo Fadini ∗ , Carlo Agostini, Angelo Avogaro Department of Clinical and Experimental Medicine, University of Padova, Medical School, Padova, Italy

a r t i c l e

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Article history: Received 17 June 2009 Received in revised form 5 August 2009 Accepted 17 August 2009 Available online 21 August 2009 Keywords: Endothelium Angiogenesis Cardiovascular diseases Bone marrow Meta-analysis

a b s t r a c t Background: Peripheral arterial disease (PAD) is a common cause of disability and mortality. Up to one third of patients are not susceptible to traditional revascularization and may benefit from stem cell therapies. Objective: In this meta-analysis, we sought to determine whether autologous cell therapy is effective in the treatment of PAD. Methods: We searched the English literature in Medline, Excerpta Medica and the Cochrane database for trials of autologous cell therapy in patients with PAD published before 31 January 2009. We included controlled and non-controlled, randomized and non-randomized trials using autologous bone marrow or granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood cells to treat PAD. We also collected data from trials of G-CSF monotherapy, as a control treatment. Results: In a meta-analysis of 37 trials, autologous cell therapy was effective in improving surrogate indexes of ischemia, subjective symptoms and hard endpoints (ulcer healing and amputation). On the contrary, G-CSF monotherapy was not associated with significant improvement in the same endpoints. Patients with thromboangiitis obliterans showed some larger benefits than patients with atherosclerotic PAD. The intramuscular route of administration and the use of bone marrow cells seemed somehow more effective than intrarterial administration and the use of mobilized peripheral blood cells. The procedures were well tolerated and generally safe. Conclusion: This meta-analysis indicates that intramuscular autologous bone marrow cell therapy is a feasible, relatively safe and potentially effective therapeutic strategy for PAD patients, who are not candidate for traditional revascularization. Larger, placebo-controlled, randomized multicenter trials need to be planned and conducted to confirm these findings. © 2009 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2.

3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Literature search . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Clinical endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Quality of data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Type of disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Type of cell therapy and doses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Patients characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1. ABI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2. TcO2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author at: Dipartimento di Medicina Clinica e Sperimentale, Divisione di Malattie del Metabolismo, Policlinico Universitario, via Giustiniani, 2, 35100, Padova, Italy. Tel.: +39 049 8212185; fax: +39 049 8212184. E-mail addresses: [email protected], [email protected] (G.P. Fadini). 0021-9150/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2009.08.033

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4. 5.

3.5.3. Pain-free walking distance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.4. Pain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.5. Ulcer healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.6. Amputation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Determinants of efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.1. Cell therapies versus G-CSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.2. ASO versus TAO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.3. Mobilized peripheral blood cells versus bone marrow cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.4. Intramuscular versus intrarterial administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Safety issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Limitations of meta-analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8.1. Publication bias . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8.2. Heterogeneity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8.3. Robustness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix A. Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Peripheral arterial disease (PAD) is a common pathologic condition all over the world. It is associated with a significant burden in terms of morbidity and mortality, due to claudication, rest pain, ulcerations and amputations. The most common causes of PAD are atherosclerosis obliterans (ASO) and thromboangiitis obliterans (TAO). Connective tissue disorders (CTD) are a rarer cause of PAD. ASO is by far the most common cause in the western world, while TAO is highly prevalent in Asia. While ASO involves almost invariably only lower limbs, TAO and CTD often involve both lower and upper limbs. As ASO patients are older and carry frequent co-morbid conditions, especially ischemic heart disease, their prognosis is often poorer [1]. On the other hand, TAO affects young individuals, usually with smokes, but is often very aggressive [2]. The gold-standard treatment of severe PAD is surgical or endovascular revascularization. However, up to 30% of patients are not candidate for such interventions, due to excessive operative risk or unfavorable vascular involvement. The presence of diffuse, multiple and distal arterial stenosis renders successful revascularization sometimes impossible. These “no-option” patients are left to medical therapy, which may slow disease progression at best [3]. During the last decade, a novel therapeutic strategy has been proposed thanks to the advancements of basic research. In 1997, Asahara et al. discovered that a subset of bone marrowderived circulating cells is able to differentiate into endothelium and promote new blood vessel growth [4]. Subsequently, these so-called endothelial progenitor cells (EPCs) proved able to ameliorate tissue perfusion in many experimental models of myocardial and peripheral ischemia, thanks to the stimulation of vasculogenesis [5]. This prompted clinical researchers to explore feasibility of cell therapies in patients with peripheral and coronary artery disease. However, it became soon clear that patients with coronary or peripheral atherosclerosis suffer from a reduction and dysfunction of bone marrow and circulating EPCs, especially in the presence of diabetes mellitus [6,7]. These alterations have been healed as a novel pathogenic mechanism of vascular disease, but they may limit the success of autologous cell therapy [8,9], because dysfunctional autologous cell would be less prone to stimulate blood flow recovery. Despite this limitation, there are numerous published studies suggesting benefits of treating PAD and CAD patients with autologous stem/progenitor cells. A recent meta-analysis showed significant improvement in cardiac remodeling after cell therapy for acute myocardial infarction [10]. Herein,

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we present an extensive literature review of clinical trials of cell therapy in PAD patients, and results of the first comprehensive meta-analysis. 2. Methods We followed recommendations of the QUOROM statement for meta-analysis of randomized controlled trials and of the MOOSE checklist (Table 1S of data supplement) for meta-analysis of observational studies [11,12]. 2.1. Literature search The authors conducted a search of clinical trials in patients with peripheral arterial disease with the use of autologous stem cells from bone marrow or granulocyte colony stimulating factor (G-CSF) mobilized peripheral blood. G-CSF monotherapy without stem cell transplantation was considered as a control treatment strategy. The search is updated as of 31 January 2009. Search sources were Medline, Embase Excerpta Medica and the Cochrane library. Search terms were “critical limb ischemia” OR “peripheral atherosclerosis” OR “atherosclerosis obliterans” OR “peripheral arterial disease” OR “peripheral vascular disease” AND “bone marrow” OR “stem cells” OR “mononuclear cells” OR “granulocyte colony stimulating factor” OR “G-CSF”. We also checked carefully reference lists of obtained articles. All articles describing the results of human trials with bone marrow- or peripheral bloodderived stem/progenitor cells were considered, independently of sample size, appropriate controls and randomization. Single case reports were included in the literature review, but not in metaanalyses. Studies in which G-CSF was administered as a biological therapy for peripheral arterial disease in the absence of stem cell therapy were also collected. Papers were retrieved through the University of Padova online system and by direct contact with the authors. Non-English manuscripts and unpublished studies were not considered. 2.2. Clinical endpoints We chose the following endpoints to be assessed in the meta-analysis: change in ankle-brachial index (ABI), change in transcutaneous oxygen tension (TcO2 ), change in pain scale (always transformed into a 0–10 scale), change in pain-free walking distance (walking time could not be converted into walking distance),

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incidence of wound healing, and incidence of amputation. Clearly, for endpoint variables which can be assessed before and after cell therapy (e.g. ABI), efficacy meta-analysis was conducted also with non-controlled trials. For categorical endpoints, such as wound healing, meta-analysis was conducted only with controlled trials. 2.3. Statistical analysis Meta-analysis was conducted according to the recent guidelines proposed by Cleophas and Zwinderman [13]. As a minority of trials included appropriate controls, we performed separate metaanalyses of efficacy for all studies (controlled and non-controlled) and for controlled studies only. Efficacy meta-analysis of all trials was based on paired data before and after treatment. Efficacy metaanalysis of controlled trials included also categorical outcomes, such as wound healing and amputation. The summary statistics of continuous data were calculated as size-weighted mean differences with 95% confidence intervals. As rough data were rarely available, standard error of the summary statistics of paired data may be overestimated. The summary statistics of categorical variables was calculated as ratio of the odds (OR) in the natural logarithmic scale. The random-effects estimate was used to calculate weights. Heterogeneity was explored using the chi-square test. Publication bias was assessed using the Funnel plot regression. Statistical significance was accepted at p < 0.05. 3. Results 3.1. Quality of data The search of the literature yielded a total of 108 studies, 42 of which were clinical trials (38 with cell therapies, 3 with G-CSF therapy alone and 1 in which cell therapy and G-CSF were compared). Most were pilot studies, assessing safety and feasibility of cell therapy. One study was excluded as it appeared that its results have been included and expanded in a subsequent publication [14]. There were four single case reports, which were considered for the literature review, but not in the meta-analysis [15–18]. There were six controlled trials (four randomized and two non-randomized), plus four trials in which the non-treated limbs served as internal controls (Fig. 1 and Table 2S of data supplement). Regarding geographic distribution, 26 trials were done in Asian countries (especially Japan and China), 15 in European countries, and 1 in Cuba. In addition to these studies, we found a trial using (allogeneic) umbilical cord blood-derived stem cells to treat peripheral arterial disease [19], which was not considered further, being outside the target of this meta-analysis. 3.2. Type of disease Atherosclerosis obliterans (30 trials) and thromboangiitis obliterans (12 trials) were the two most representative causes of peripheral arterial disease. There were four studies in patients with connective tissue disorders (CTD) and one single case report in compartment syndrome [17], which was not considered further. Seven trials included patients with differing diagnoses: in these cases, outcomes in patients with ASO, TAO and CTD were analyzed separated whenever possible. 3.3. Type of cell therapy and doses Clinical trials used essentially two sources of stem cells: (i) bone marrow aspiration and (ii) apheresis of peripheral blood

Fig. 1. Literature search. Results of the literature search and flow diagram of metaanalysis according to the QUOROM statement.

after stimulation with G-CSF. In two cases of peripheral blood and in one case of bone marrow usage, CD34+ cells were selectively enriched and used for cell therapy. In all other cases unselected mononuclear cells were used. Despite this, the number of CD34+ cells used for cell therapy is often reported in detail, suggesting that most studies acknowledged the importance of these cells. The mean number of mononuclear cells implanted or infused was 3.56 ± 2.81 × 109 , while the mean number of CD34+ cells was 5.0 ± 1.48 × 107 , indicating that about 1.4% of transplanted cells were CD34+. The cumulative doses of G-CSF used to stimulate mobilization of stem/progenitor cells from bone marrow into peripheral blood varied considerably, ranging from 200 to 4900 ␮g (median 1400). The most common protocol was daily 5 ␮g/kg of G-CSF for 4–5 consecutive days (to calculate and compare cumulative doses, a fixed ideal 70 kg weight was used). In one G-CSF trial, the authors performed also tibial bone fenestration to allow stem/progenitor cells to exit bone marrow and reach directly the ischemic site [20]. The route of cell administration was intramuscular in 33 trials, intrarterial in 4 trials and combined intrarterial plus intramuscular in 1 trial; 1 trial compared intramuscular versus intramuscular plus intrarterial cell administration [21]. Intramuscular administration is usually done via multiple injections at the level of gastrocnemius muscles, while intrarterial infusion is usually done through a classic femoral access. Concomitant treatment consisted of maximal medical therapy plus wound debridement (if needed) in most studies; in one study, two patients were also subjected to by-pass surgery [22]; in 2 studies skin grafts were used [16,17].

3.4. Patients characteristics The weighted average clinical characteristics of patients subjected to these therapies are reported in Table 1. Patients are also divided according to disease type: TAO patients were considerably younger and have a lower prevalence of diabetes, ischemic heart disease and cerebrovascular disease than ASO patients. CTD patients represented a much smaller group.

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Table 1 Patients’ characteristics. Characteristic

All patients

ASO

Number Age Male sex (%) Diabetes (%) Hemodialysis (%) Ischemic heart disease (%) Cerebrovascular disease (%)

701 60.1 (25.6) 70.9 37.6 17.0 27.4 12.6

545 64.8 (22.5) 68.9 46.7 25.0 42.2 22.4

Disease stage I (%) II (%) III (%) IV (%)

0.9 5.0 30.1 64.0

1.1 6.4 30.6 61.9

TAO 147 45.1 (9.4) 80.6 11.4 0 2.0 0 0 0 29.9 70.1

CTD 9 52.0 (2.8) 33.3 0 0 0 0 0 0 0 100.0

p – <0.001 0.007 <0.001 <0.001 <0.001 <0.001 0.20 0.002 0.87 0.07

Pooled characteristics of patients in the cell therapy trials. Percentages or mean (SD). p-values are referred to the comparison between ASO and TAO patients. Disease stage according to Leriche–Fontain.

3.5. Efficacy Median follow-up was 6 months (interquartile range 3–12). For efficacy meta-analyses, trials using bone marrow or peripheral blood cells were first pooled together, while trials using G-CSF alone were considered separately. Results for cell therapy trials are reported in Fig. 2. 3.5.1. ABI Considering all trials of cell therapy, ABI improved from 0.46 ± 0.04 before therapy to 0.63 ± 0.04 after therapy (p = 0.011). Considering only controlled trials of cell therapies, ABI improved by 0.115 ± 0.060 (p = 0.054). In trials with G-CSF monotherapy, ABI was not significantly increased, considering both all studies (from 0.41 ± 0.09 to 0.59 ± 0.11; p = 0.30) and controlled studies (difference 0.049 ± 0.22; p = 0.83), although this result may be influence by the low cumulative sample size. 3.5.2. TcO2 TcO2 increased from 22.8 ± 2.8 to 35.8 ± 2.9 (p = 0.0002) considering all trials of cell therapy, and increased by 12.8 ± 7.0 (p = 0.069) considering only controlled trials. In trials with G-CSF monotherapy, data on TcO2 was too scant to be meta-analyzed. 3.5.3. Pain-free walking distance Walking capacity increased significantly considering all trials (from 75.7 ± 19.4 to 402.3 ± 70.9 m; p < 0.0001) and controlled trials (by 311.7 ± 128.1 m; p = 0.020) of cell therapy. This endpoint was reported in detail only in one trial of G-CSF therapy and there was no significant difference versus placebo [23]. 3.5.4. Pain For cell therapy, pain (on a 0–10 scale) was significantly reduced both when all trials (from 6.35 ± 0.43 to 2.11 ± 0.37; p < 0.0001) and controlled trials (by −2.39 ± 1.01; p = 0.019) were considered. In G-CSF trials pain was not significantly reduced (all trials: from 7.61 ± 1.45 to 4.78 ± 2.0; p = 0.33; one controlled trial −1.75 ± 5.94; p = 0.29). 3.5.5. Ulcer healing In controlled trials of cell therapy the incidence of ulcer healing or improvement was significantly better in the active treatment group as compared with the control group (OR 3.54, 95% C.I. 1.09–11.51; p = 0.032). In one controlled trial of G-CSF therapy, in which these data are reported in detail, there was no evidence of improved ulcer healing versus placebo (OR 2.57; 95% C.I. 0.21–31.7; p = 0.45).

3.5.6. Amputation This outcome was explored in detail only in two controlled trials of cell therapy, one randomized [24] and one non-randomized [25], showing a significant benefit in terms of limb salvage as compared to control treatment (OR for amputation 0.09; 95% C.I. 0.02–0.44; p = 0.0005). Incidence of amputation was not assessed in controlled G-CSF trials. 3.6. Determinants of efficacy 3.6.1. Cell therapies versus G-CSF One small controlled trial directly compared cell therapy with G-CSF alone, showing similar benefits of the two treatments in terms of change in ABI and TcO2 [26]. The study was however largely underpowered to detect significant differences between the two treatments (power = 5% for ABI and 6% for TcO2 ). Our metaanalysis indicates that G-CSF monotherapy leads to non-significant improvements in ABI, pain-free walking distance, rest pain and ulcer healing. This is compliant with the results of a double-blinded, randomized, placebo-controlled study showing no superiority of GCSF over placebo [23]. However, it should be noted that there were very few studies testing G-CSF monotherapy; therefore, the conclusions are limited. On the contrary, meta-analysis of cell therapy trials indicates significant benefits in all pre-specified endpoints. Therefore, cell therapy, either with bone marrow cells or G-CSF mobilized peripheral blood mononuclear cells, should be considered superior to G-CSF monotherapy in patients with PAD. 3.6.2. ASO versus TAO There were no controlled trials in TAO patients, therefore we compared all trials in ASO and in TAO patients. The benefit of cell therapy was significantly larger in patients with TAO than in patients with ASO as for change in ABI (p = 0.021), TcO2 (p = 0.03), pain scale (p = 0.003) and pain-free walking distance (p = 0.019). The same conclusion is confirmed by a long-term study of safety, survival and incidence of amputation in 74 ASO and 41 TAO patients treated with intramuscular bone marrow cells [27]. Given the impressive differences between TAO and ASO patients in terms of cardiovascular risk profile (Table 1), this finding cannot be interpreted that TAO is more amenable to cell therapy than ASO. 3.6.3. Mobilized peripheral blood cells versus bone marrow cells One study that addressed directly this issue in a randomized fashion showed better improvement in ABI and pain scale with mobilized peripheral blood [28]. Upon meta-analysis, mobilized peripheral blood cell therapy was consistently associated with slightly and not significantly better improvements in ABI and TcO2 and pain-free walking distance. Pain scale reduction was significantly better with mobilized peripheral blood than with bone

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Fig. 2. Results of meta-analysis of cell therapy trials in patients with peripheral arterial disease. Bars indicate 95% confidence intervals. Box sizes are proportional to study size.

marrow cells (p = 0.006). On the contrary, bone marrow cell therapy significantly improved a hard endpoint such ulcer healing (OR 7.23; p = 0.038), while mobilized peripheral blood cells did not (OR 2.24; p = 0.13). There were no significant differences in the clinical characteristics between patients treated with mobilized peripheral blood cells or bone marrow cells (not shown). 3.6.4. Intramuscular versus intrarterial administration The effects of intrarterial or intrarterial plus intramuscular cell administration were compared to the effects of intramuscular cell administration. One study that specifically addressed this issue showed no difference between these two strategies [21], but it was underpowered to detect differences in terms of ulcer healing, ABI

and walking distance improvement (power = 15–20%). Upon metaanalysis, we found that ABI and TcO2 were significantly improved after intramuscular cell therapy, while they were not after intrarterial cell therapy. Both significantly improved pain and pain-free walking distance and there were no difference between the two. Intramuscular cell therapy significantly improved ulcer healing (OR 2.62; p = 0.029), while this could not be assessed in details in trials of intrarterial cell therapy. There were no significant differences in the clinical characteristics between patients treated with intrarterial or intrarterial plus intramuscular cells (not shown). Fig. 3 reports differences in the continuous endpoints in ASO versus TAO patients, intramuscular versus intrarterial administration, and use of bone marrow cells versus mobilized peripheral blood

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Fig. 3. Determinants of efficacy of cell therapy. Different change in the continuous endpoints in ASO versus TAO patients, intramuscular (IM) versus intrarterial (IA) administration, and use of bone marrow cells (BMC) versus mobilized peripheral blood cells (M-PBC). Bars indicate standard deviations. *p < 0.05.

cells. We did not find any other significant determinant of efficacy. The identification of determinants of efficacy should be considered as hypothesis-generating rather than definite, especially when available randomized controlled trials do not support the results of the meta-analysis. 3.7. Safety issues Safety data were described in 32/41 studies. Bone marrow aspiration was well tolerated, the most frequent adverse reaction being local pain, responsive to non-steroidal anti-inflammatory drugs. Another less common adverse event was mild anemia. G-CSF stimulation was generally well tolerated, with prevalently minor side effects, including flu-like symptoms, myalgia, fever, and bone pain. Three patients had to discontinue high-dose G-CSF because of chest pain, muscle pain and anaphylaxis, respectively [23]; there was also one ventricular fibrillation (recovered after cardioversion) [26] and one minor retinal bleeding with G-CSF [29]. Long-term safety has been questioned by a trial of intramuscular bone marrow cell therapy [30], in which, out of eight patients, one died suddenly 30 months after the procedure, two had ulcer worsening, and one had incompetent angiogenesis [31]. This prompted other researchers to report the long-term status of their patients: a total of 21 deaths (20 in the cell therapy group plus 1 in the G-CSF group) were reported between 2 months and 3 years after therapy out of 761 patients treated. No controlled trial

reported mortality rates in the experimental versus control groups. Based on these data, it appears that no worrying safety concerns exist in relation to this type of cell therapy. However, most studies were not properly designed to assess safety in comparison with control treatment, and systematic reporting of adverse events was rare. Therefore, larger randomized controlled trials are needed to definitely answer the open question of safety. 3.8. Limitations of meta-analysis 3.8.1. Publication bias We cannot exclude that a publication bias affects reliability of the present meta-analysis. Particularly, some small negative studies may have not been published as full reports. However, given the high amount of resources needed for such studies, it is unlikely that larger trials have not been reported because of negative results. Exclusion of non-English language papers might be another source of publication bias. Slops of the Funnel plot regression were not significantly different from zero for ABI, TcO2 , pain-scale and walking capacity. 3.8.2. Heterogeneity Chi-square test for summary statistics among studies of cell therapy revealed no significant heterogeneity for ABI (p = 0.99), TcO2 (p = 0.99), pain-free walking distance (p = 0.59), and rest pain (0.90).

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3.8.3. Robustness The present meta-analysis includes both good quality (controlled, randomized and/or blinded) and low quality (uncontrolled, unblinded and/or non-randomized) trials. Therefore, results might be influenced by low quality studies. Moreover, most were pilot studies not properly designed to assess safety and efficacy, and placebo effects cannot be definitely ruled out. Two surrogate objective endpoints (ABI and TcO2 ) were found to be significantly improved when all trials were considered, while they were marginally improved in pooled controlled trials. Hard objective endpoints (ulcer healing and amputation) were significantly improved in controlled and non-controlled trials. Subjective endpoints (such as pain scale and pain-free walking distance), which are more susceptible to placebo effect, showed significant improvement in controlled and non-controlled trials. Indeed, elimination of poor-quality studies did not affect the overall result, arguing against a lack of robustness in this meta-analysis.

somewhat more effective than G-CSF mobilized peripheral blood cells in inducing ulcer healing. This is in compliance with the observation that mobilized cells are transiently dysfunctional due to clivage of the chemokine receptor CXCR4, which is directly involved in stem cell homing [34]. On the basis of study results, the intramuscular route of administration seems preferable, maybe because cells could hardly reach the target tissue when infused intrarterially in severely compromised arterial beds. To date, the mechanisms by which transplanted cells improve the patients’ clinical status are still unclear. Experimental animal studies indicate that bone marrow-derived cells contribute to vascular and muscle regeneration, by physically integrating into the tissue and/or by secreting growth factors [5,35]. In most studies analyzing patients’ tissue samples, the local physical contribution of transplanted cells is rather low, and can hardly explain the observed clinical benefit. Therefore, the paracrine effect appears to be more plausible. 4.1. Future directions

4. Discussion Herein, we provide the most comprehensive review and metaanalysis of cell therapy trials in patients with peripheral arterial disease. About 700 patients without revascularization options were treated in these trials. The result is that cell therapy significantly improved ABI, TcO2 , rest pain, pain-free walking distance, ulcer healing and limb salvage. On the contrary, G-CSF monotherapy was not associated with significant improvement of these endpoints, but the number of trials testing G-CSF was limited and conclusion cannot be definite. It is generally agreed that high quality controlled trials yield more reliable inference in meta-analyses. In this study, we included also non-controlled studies because the number of available high quality studies was very limited. Moreover, comparison of results obtained in the meta-analysis of all studies to results obtained in the meta-analysis of controlled trials only showed good consistency, thus providing evidence for robustness. Evaluation of ischemia in patients with peripheral arterial disease usually relies on surrogate quantitative indexes, such as ABI and TcO2 , while the disease burden essentially derives from pain, disability, ulceration and amputation. Our analysis shows that cell therapy improved objective surrogate indexes (ABI and TcO2 ), more subjective symptoms (pain scale and walking capacity), as well as hard endpoints (ulcer healing and amputation). Remarkably, when the meta-analysis was restricted to controlled trials, there was still evidence of improvement in pain scale and pain-free walking distance after cell therapy, thus minimizing the chance that placebo effect and study enrollment fictitiously altered these results. The procedures appeared to be generally safe and well tolerated, most adverse reactions occurring in patients treated with G-CSF (in monotherapy or for stem cell mobilization). Incident deaths were expected given the severe underlying disease and could not be directly attributed to cell therapy. Although there is no evidence in favor or against an increased long-term mortality of treated patients, larger randomized controlled trials are needed to definitely answer this question. Patients with ASO showed a weaker improvement as compared to TAO patients, but this result may be influenced by a placebo effect because there were no controlled trials in TAO patients. Nonetheless, it is known that advanced age and concomitant cardiovascular risk factors, which are more prevalent in ASO than in TAO, negatively influence function of bone marrow progenitor cells [32,33], possibly limiting results of autologous stem cell therapy. Thus, a better outcome after cell therapy in TAO than in ASO patients could be attributable to their healthier status, rather than to the specific characteristics of the underlying disease. Bone marrow cells seemed to be

To understand how ongoing next-generation trials of cell therapy for peripheral arterial disease are evolving, we searched the NIH ClinicalTrials.gov, a large and widely available database of clinical trials. We found 23 registered trials, 21 with various forms of stem cells or bone marrow cells, 1 with G-CSF monotherapy and 1 comparing bone marrow cells versus G-CSF. According to the declared sample size, it appears that about 1100 patients are going to be treated in the next couple of years. These will be distributed in 8 phase I, 11 phase II and 3 phase III studies from all over the world (1 non-specified). Ten trials are going to be conducted in the United States, seven in Europe, five in Asia and one in Latin America. One of the most important innovation is that private industries will be directly involved in 8/23 trials (35%), usually by providing the cell product, expanding the possibility that more centers will have access to this therapeutic option. Results of these ongoing trials will likely provide stronger efficacy data of cell therapy. 5. Conclusions Results of this meta-analysis should be cautiously interpreted because most studies were not adequately designed to assess safety and efficacy, and a definite evidence of efficacy awaits confirmation by the ongoing randomized controlled trials. Nonetheless, the current literature supports that intramuscular bone marrow cell administration is a relatively safe, feasible and possibly effective therapy for patients with peripheral arterial disease, who are not susceptible to conventional revascularization. The private industry is robustly coming into second-generation trials, which could make this novel therapeutic option more widely available. Larger, placebo-controlled, randomized multicenter trials need to be planned and conducted to confirm safety and efficacy of this therapy. If these expectations were met, cell therapy would enlarge our therapeutic armamentarium against advanced PAD. Acknowledgement Partly supported by the Heart Failure and Cardiac Repair consortium (LSHM-CT-2005-018630, www.heartrepair.eu) and by the Italian non-profit organization Stem for Life. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.atherosclerosis.2009.08.033.

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References [1] Hirsch AT, Criqui MH, Treat-Jacobson D, et al. Peripheral arterial disease detection, awareness, and treatment in primary care. JAMA 2001;286:1317–24. [2] Olin JW. Thromboangiitis obliterans (Buerger’s disease). N Engl J Med 2000;343:864–9. [3] Hankey GJ, Norman PE, Eikelboom JW. Medical treatment of peripheral arterial disease. JAMA 2006;295:547–53. [4] Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science 1997;275:964–7. [5] Fadini GP, Agostini C, Sartore S, Avogaro A. Endothelial progenitor cells in the natural history of atherosclerosis. Atherosclerosis 2007;194:46–54. [6] Fadini GP, Miorin M, Facco M, et al. Circulating endothelial progenitor cells are reduced in peripheral vascular complications of type 2 diabetes mellitus. J Am Coll Cardiol 2005;45:1449–57. [7] Fadini GP, Sartore S, Albiero M, et al. Number and function of endothelial progenitor cells as a marker of severity for diabetic vasculopathy. Arterioscler Thromb Vasc Biol 2006;26:2140–6. [8] Fadini GP, Sartore S, Agostini C, Avogaro A. Significance of endothelial progenitor cells in subjects with diabetes. Diabetes Care 2007;30:1305–13. [9] Werner N, Nickenig G. Influence of cardiovascular risk factors on endothelial progenitor cells: limitations for therapy? Arterioscler Thromb Vasc Biol 2006;26:257–66. [10] Lipinski MJ, Biondi-Zoccai GG, Abbate A, et al. Impact of intracoronary cell therapy on left ventricular function in the setting of acute myocardial infarction: a collaborative systematic review and meta-analysis of controlled clinical trials. J Am Coll Cardiol 2007;50:1761–7. [11] Moher D, Cook DJ, Eastwood S, et al. Improving the quality of reports of metaanalyses of randomised controlled trials: the QUOROM statement. Quality of Reporting of Meta-analyses. Lancet 1999;354:1896–900. [12] Stroup DF, Berlin JA, Morton SC, et al. Meta-analysis of observational studies in epidemiology: a proposal for reporting. Meta-analysis Of Observational Studies in Epidemiology (MOOSE) group. JAMA 2000;283:2008–12. [13] Cleophas TJ, Zwinderman AH. Meta-analysis. Circulation 2007;115:2870–5. [14] Kawamura A, Horie T, Tsuda I, et al. Prevention of limb amputation in patients with limbs ulcers by autologous peripheral blood mononuclear cell implantation. Ther Apher Dial 2005;9:59–63. [15] Kirana S, Stratmann B, Lammers D, et al. Wound therapy with autologous bone marrow stem cells in diabetic patients with ischaemia-induced tissue ulcers affecting the lower limbs. Int J Clin Pract 2007;61:690–2. [16] Sugihara S, Yamamoto Y, Matsubara K, et al. Autoperipheral blood mononuclear cell transplantation improved giant ulcers due to chronic arteriosclerosis obliterans. Heart Vessels 2006;21:258–62. [17] Umemura T, Nishioka K, Igarashi A, et al. Autologous bone marrow mononuclear cell implantation induces angiogenesis and bone regeneration in a patient with compartment syndrome. Circ J 2006;70:1362–4. [18] Ozawa T, Kato K, Sanada H, et al. Marked decrease of plasma VEGF after implantation of autologous bone marrow mononuclear cells in a patient with critical limb ischemia—a case report. Angiology 2006;57:235–9. [19] Kim SW, Han H, Chae GT, et al. Successful stem cell therapy using umbilical cord blood-derived multipotent stem cells for Buerger’s disease and ischemic limb disease animal model. Stem Cells 2006;24:1620–6.

17

[20] Kim DI, Kim MJ, Joh JH, et al. Angiogenesis facilitated by autologous whole bone marrow stem cell transplantation for Buerger’s disease. Stem Cells 2006;24:1194–200. [21] Van Tongeren RB, Hamming JF, Fibbe WE, et al. Intramuscular or combined intramuscular/intra-arterial administration of bone marrow mononuclear cells: a clinical trial in patients with advanced limb ischemia. J Cardiovasc Surg (Torino) 2008;49:51–8. [22] Nakayama M, Asari Y. Angiogenesis achieved by granulocyte colonystimulating factor in combination with bypass surgery in 2 cases of critical limb ischemia. Circ J 2008;72:1385–7. [23] van Royen N, Schirmer SH, Atasever B, et al. START Trial: a pilot study on STimulation of ARTeriogenesis using subcutaneous application of granulocytemacrophage colony-stimulating factor as a new treatment for peripheral vascular disease. Circulation 2005;112:1040–6. [24] Huang P, Li S, Han M, et al. Autologous transplantation of granulocyte colonystimulating factor-mobilized peripheral blood mononuclear cells improves critical limb ischemia in diabetes. Diabetes Care 2005;28:2155–60. [25] Napoli C, Farzati B, Sica V, et al. Beneficial effects of autologous bone marrow cell infusion and antioxidants/L-arginine in patients with chronic critical limb ischemia. Eur J Cardiovasc Prev Rehabil 2008;15:709–18. [26] Arai M, Misao Y, Nagai H, et al. Granulocyte colony-stimulating factor: a noninvasive regeneration therapy for treating atherosclerotic peripheral artery disease. Circ J 2006;70:1093–8. [27] Matoba S, Tatsumi T, Murohara T, et al. Long-term clinical outcome after intramuscular implantation of bone marrow mononuclear cells (Therapeutic Angiogenesis by Cell Transplantation [TACT] trial) in patients with chronic limb ischemia. Am Heart J 2008;156:1010–8. [28] Huang PP, Yang XF, Li SZ, et al. Randomised comparison of G-CSF-mobilized peripheral blood mononuclear cells versus bone marrow-mononuclear cells for the treatment of patients with lower limb arteriosclerosis obliterans. Thromb Haemost 2007;98:1335–42. [29] Hoshino J, Ubara Y, Hara S, et al. Quality of life improvement and longterm effects of peripheral blood mononuclear cell transplantation for severe arteriosclerosis obliterans in diabetic patients on dialysis. Circ J 2007;71: 1193–8. [30] Hirsch AT. Critical limb ischemia and stem cell research: anchoring hope with informed adverse event reporting. Circulation 2006;114:2581–3. [31] Miyamoto K, Nishigami K, Nagaya N, et al. Unblinded pilot study of autologous transplantation of bone marrow mononuclear cells in patients with thromboangiitis obliterans. Circulation 2006;114:2679–84. [32] Heiss C, Keymel S, Niesler U, et al. Impaired progenitor cell activity in agerelated endothelial dysfunction. J Am Coll Cardiol 2005;45:1441–8. [33] Fadini GP, de Kreutzenberg SV, Coracina A, et al. Circulating CD34+ cells, metabolic syndrome, and cardiovascular risk. Eur Heart J 2006;27: 2247–55. [34] Honold J, Lehmann R, Heeschen C, et al. Effects of granulocyte colony simulating factor on functional activities of endothelial progenitor cells in patients with chronic ischemic heart disease. Arterioscler Thromb Vasc Biol 2006;26:2238–43. [35] Heil M, Ziegelhoeffer T, Mees B, Schaper W. A different outlook on the role of bone marrow stem cells in vascular growth. bone marrow delivers software not hardware. Circ Res 2004;94:573–4.