Analysis of possible factors relating to prognosis in autologous peripheral blood mononuclear cell transplantation for critical limb ischemia

Cytotherapy, 2014; 0: 1e7

Analysis of possible factors relating to prognosis in autologous peripheral blood mononuclear cell transplantation for critical limb ischemia

LINGJIE SUN1, LIHUA WU1, ZHUOQING QIAO2, JINGYI YU1, LIANLIAN LI1, SHANGZHU LI1, QINGGUO LIU1, YIMIN HU1, NING XU1 & PINGPING HUANG1 1

General Medical Center of the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China, and 2Department of Hematopoietic Stem Cell Transplantation, Hospital Affiliated with the Academy of Military Medical Sciences, Beijing, China

Abstract Background aims. Autologous transplantation of granulocyte colony-stimulating factoremobilized peripheral blood mononuclear cells (M-PBMNCs) has been shown to be effective in treating critical limb ischemia (CLI); however, the studies of the possible prognosis predictors after autologous M-PBMNC transplantation are inadequate. The objective of the study was to assess the possible factors affecting the results of M-PBMNC transplantation for CLI. Methods. We reviewed the clinical profiles of 87 patients with CLI who were treated with M-PBMNC implantation in the Blood Diseases Hospital, Chinese Academy of Medical Sciences, between December 2002 and December 2011, and we followed these patients. The patients were divided into a good prognosis group and a poor prognosis group on the basis of whether amputation was performed. The significant differences of clinical variables between two groups were analyzed by means of the Mann-Whitney test and c2 test, and logistic regression analysis was used to study the variables representing the possible prognostic factors for amputation. Results. Of the 87 patients, three patients died and one patient was lost during the follow-up period. We analyzed 83 patients. The diseases included CLI complicated by diabetes mellitus gangrene (35 cases, 42.2%), arteriosclerosis obliterans (31 cases, 37.3%) and thromboangiitis (17 cases, 20.5%). The mean age was 62 years (range, 30e87). The median follow-up time for the surviving patients was 5 years. The 5-year amputation-free rate was 72.2%, and no adverse effects related to MPBMNC transplantation were observed. Conclusions. The significant prognostic factors associated with poor angiogenesis were fibrinogen >4 g/L and fasting blood glucose >6 mmol/L. Key Words: critical limb ischemia, peripheral arterial disease, peripheral blood mononuclear cell transplantation, prognostic factor

Introduction Critical limb ischemia (CLI), which is the most severe form of peripheral arterial disease, has a dismal result for limb salvage and survival. CLI can arise from various types of vasculitis, including diabetes mellitus (DM) gangrene, arteriosclerosis obliterans (ASO) and thromboangiitis (TAO). The goals of treatment for patients with CLI are to relieve exertional symptoms, improve walking capacity, relieve ischemic pain at rest, heal ischemic ulceration, prevent limb loss and improve quality of life (1). A large number of patients still cannot avoid amputation despite recent progress has happened in drug, vascular surgery and interventional radiology for peripheral arterial disease. Since Folkman (2) reported the conception of “therapeutic angiogenesis”

in 1998, more and more animal and clinical studies about cell transplantation for lower-limb ischemia, including bone marrow mononuclear cells (BMMNC)-mobilized and granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (M-PBMNCs) have arisen (3e13). In 2002, Tateishi et al. (4) first proved that autologous implantation of BMMNCs could be safe and effective for achievement of therapeutic angiogenesis. A phase II trial of autologous transplantation of BMMNCs for CLI showed an amputation-free survival rate of 75.2% at 12 months after the treatment (12). Dubsky et al. (13) proved that both autologous BMMNC and M-PBMNC transplantation improved ischemia in patients with diabetic foot, and there were no significant differences

Correspondence: Pingping Huang, MD, General Medical Center of the Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union of Medical College, 288 Nanjing Road, Tianjin 300020, China. E-mail: [email protected] (Received 22 December 2013; accepted 20 March 2014) ISSN 1465-3249 Crown Copyright Ó 2014 Published by Elsevier Inc. on behalf of International Society for Cellular Therapy. All rights reserved. http://dx.doi.org/10.1016/j.jcyt.2014.03.007

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between the two groups. Recently, a meta-analysis of all randomized, controlled trials on cell therapy in patients with CLI showed reduced amputation rates in the therapeutic arms, with a relative risk of major amputation of 0.58 (14). Gupta et al. (15) also proved the safety of the use of allogeneic bone marrowederived mesenchymal stromal cells (BMMSCs) in patients with CLI and showed positive trends toward improvement in ankle-brachial index through a randomized, double-blinded, placebocontrolled multicenter phase I/II trial (15). In 2004, we evaluated the clinical efficacy of M-PBMNC in five patients with severe ASO of the lower extremities. The results suggested for the first time that the autologous transplantation of M-PBMNCs is a practical, safe and effective treatment for lower-limb ischemia in China (6). Our team also conducted a randomized trial and demonstrated that the autologous transplantation of M-PBMNCs or BMMNCs significantly improves limb ischemia in patients with lower-limb arteriosclerosis obliterans 12 weeks after cell implantation; the improvement in the anklebrachial index, skin temperature and resting pain was more significant in the patients in the M-PBMNC transplantation group than in the BMMNC group, and there was no significant difference between the two groups in pain-free walking distance, transcutaneous oxygen pressure, ulceration or rate of lower-limb amputation (7). The possible mechanism of therapeutic angiogenesis is that endothelial progenitor cells from bone marrow and peripheral blood could incorporate into the existing vasculature to increase capillary density and that a fraction of these cells support angiogenesis and vasculogenesis through the paracrine effects (8). However, studies about long-term clinical outcome of autologous M-PBMNC transplantation for lower-limb ischemia were limited. In addition, the studies about the prognostic factors show contradictory results for M-PBMNC implantation. Our study aims to assess the possible prognostic factors regarding the long-term outcome for angiogenesis after autologous M-PBMNC transplantation in patients with chronic lower-limb ischemia in China.

Methods Patients and variables This clinical study was approved by the ethical committee board of the Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences & Peking Union of Medical College. Clinical profiles of 87 patients treated with autologous MPBMNC transplantation for lower-limb ischemia in the Institute of Hematology & Hospital of Blood

Diseases, Chinese Academy of Medical Sciences & Peking Union of Medical College from December 2002 through December 2011 were retrospectively studied. One patient with polycythemia vera was excluded. The 87 patients were selected to undergo M-PBMNC implantation because they were unresponsive to medication or surgical or endovascular procedures were deemed inappropriate. All patients received antiplatelet or anticoagulant drugs before transplantation. Angiography of all patients was analyzed, and no substantial heterogeneity was detected. The patients were followed up through telephone interviews, outpatient examinations or home visits. The variables studied included the following: the patient’s sex, patient’s age at first transplantation, Rutherford classification, vascular complications (including high blood pressure, ischemic cardiomyopathy and cerebral vascular disease), history of smoking, leukocyte counts after mobilization, blood concentrations of hemoglobin, fibrinogen, liver enzymes (including alanine aminotransferase and aspartate aminotransferase), creatinine, blood urea nitrogen, fasting blood glucose on first admission and times of transplantation. We evaluated the curative effect according to amputation (3). In this study, “lower-limb amputation” refers to any part of lower-limb amputation. Patients who avoided amputation were classified in the good prognosis group, and other patients were classified into the poor prognosis group. We retrieved all of the clinical records of the patients who received autologous M-PBMNC transplantation for lowerlimb ischemia. The contents of the follow-up included whether death and amputation occurred and the cause of death. M-PBMNC isolation and implantation The method of transplantation was as previously described (5). The patients received 600 mg/d of recombinant human G-CSF (Kirin Pharmaceuticals, Tokyo, Japan) by subcutaneous injection for 5 days to mobilize the stem/progenitor cells. Meanwhile, a perfusion of 10,000 units/d heparin for 5 days by intravenous drip was used to avoid the possible risks of embolism. Approximately 300 mL of a suspension of blood circulating M-PBMNCs was collected from the patients treated with G-CSF. The superfluous cells were stored in liquid nitrogen for further use. Three hours later, each diseased lower limb was intramuscularly injected (40 sites, 3
3 cm distance, 1e1.5 cm deep, 7.5
108 M-PBMNC per site) into the thigh and leg, with a total of 3
109 mobilized PBMNCs. Every 40 days after transplantation, the severely diseased lower limb was given an additional transplantation of the same number of the cells frozen in liquid nitrogen.

Prognostic analysis of cell therapy for CLI Table I. Categorical variables.

Variable

Table II. Baseline characteristics of patients. Good Poor Subgroup prognosis prognosis

>60 <60 Sex Male Female Rutherford classification 1, 2, 3, 4 5, 6 Vascular complications Absence (including high blood pressure, Presence ischemic cardiomyopathy and cerebral vascular disease) History of smoking Absence Presence Blood concentrations of Normal hemoglobin (normal range: Anemia female: 110e150 g/L male: 120e160 g/L) Leukocytes after mobilization >40 (
109/L) <40 Fibrinogen (normal range: >4 2e4 g/L) <4 ALT (normal range: 0e40 U/L) >40 <40 AST (normal range: 0e40 U/L) >40 <40 Creatinine (normal range: >115 40e115 mmol/L) >115 Blood urea nitrogen (normal >7.5 range: 2.9e7.5 mmol/L) <7.5 Glucose (normal range: >6.0 3.9e6.1 mmol/L) <6.0 Times of transplantation >3 <3 Age (years)

3

45 15 43 17 22 38 23 37

12 11 19 4 7 16 11 12

27 33 44 16

7 16 19 4

29 31 15 45 6 54 3 57 2 58 7 53 16 44 36 24

11 12 13 10 2 21 2 21 2 21 3 20 12 11 13 10

ALT, alanine aminotransferase; AST, aspartate aminotransferase.

Data processing The data management and statistical analysis were performed with SPSS version 16.0 for Windows (SPSS, Chicago, IL, USA). The Mann-Whitney test was used to analyze the significant differences for the continuous variables between the two prognosis groups. All variables were then classified into categorical variables (Table I). The normal range of all laboratory tests were referred to the calibration values of laboratory tests in Institute of Hematology & Hospital of Blood Diseases. The c2 test was performed in categorical variables. Finally, logistic regression analysis was used to study the categorical variables representing the possible prognostic factors for the unsatisfied effect. A value of P < 0.05 was considered significant. Variables showing statistic significance in all of three tests were considered as possible prognostic factors.

Results Of the 87 patients, two patients died of ischemic heart disease, one patient died of cerebrovascular

Characteristic Age (years) Median Range Sex Male Female Rutherford classification 1e4 5, 6 Vascular complications Absence Presence History of smoking Absence Presence Blood concentrations of hemoglobin (g/L) Median Range Leukocyte after mobilization (
109/L) Median Range Fibrinogen (g/L) Median Range ALT Median Range AST Median Range Creatinine (mmol/L) Median Range Blood urea nitrogen (mmol/L) Median Range Glucose (mmol/L) Median Range Times of transplantation Median Range

Good prognosis Poor prognosis group group 64 33e87

61 30e84

43 17

19 4

22 38

7 16

23 37

11 12

27 33

7 16

131.5 73e161

127 79e148

39.65 17.0e70.45

39.46 22.14e69.41

3.52 1.38e6.9

4.09 2.17e7.15

19 5e120

15 2e103

20 7e60

18 10e61

70.15 41e122

71.3 48e145

4.85 1.2e11.8

4.76 3.1e20

5.17 3.38e12.5

6.28 4.2e16

3 1e5

3 1e4

ALT, alanine aminotransferase; AST, aspartate aminotransferase.

disease and one patient was lost during the follow-up period. We analyzed 83 patients. The overall mean age was 62 years (range, 30e87), and 75% of the patients were male (62 men, 21 women). The diseases included CLI complicated by DM (35 cases, 42.2%), ASO (31 cases, 37.3%) and TAO (17 cases, 20.5%). All patients received angiography before MPBMNC transplantation. Angiography showed that all patients had multiple arterial obstructions, and no substantial heterogeneity was detected between the three diseases. The Rutherford classifications were 1 (1/83), 2 (4/83), 3 (8/83), 4 (14/83), 5 (10/83) and 6 (46/83); 57.8% (48/83) of patients underwent

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implantation more than two times. The baseline characteristics of the patients and the details of the clinical features between the two groups are shown in Table II. No adverse effect in the period from GCSF administration to 1 month after cell implantation, including gastrointestinal bleeding, anemia, seizure and acute myocardial infarction (3), was observed. The patients were followed up through telephone interviews (63 patients), outpatient examinations (23 patients) or home visits (one patient). The median follow-up time for the surviving patients was 5 years (range, 1e10 years). Seventy-nine patients received ongoing antiplatelet or anticoagulant drugs after transplantation; 62.65% (52/83) of the patients claimed improvement in clinical manifestation，including walking capacity, ischemic pain at rest and ischemic ulceration. No transplantationrelated side reaction was observed. The overall 5year amputation-free rate was 72.2% (60/83). The amputation-free rates for the three diseases were77.1%, 72% and 64.7%, respectively. The Mann-Whitney test showed that there were significant differences in fibrinogen (P ¼ 0.0065) and fasting blood glucose (P ¼ 0.0215) between the good prognosis group and the poor prognosis group. By use of the c2 test, we observed that the patients with age 60 years, fasting blood glucose >6.0 mmol/L and fibrinogen >4 g/L showed higher amputation rates, with the P value of 0.043, 0.027 and 0.008, respectively. Finally, logistic regression analysis was used to study the categorical variables, and variables with a value of P < 0.05 were considered prognostic factors. The results showed that age 60 years (P ¼ 0.007), fasting blood glucose >6.0 mmol/L (0.009) and fibrinogen >4 g/L (P ¼ 0.017) might predict poor prognosis after autologous M-PBMNC transplantation. According to our study design, only fasting blood glucose >6.0 mmol/L and fibrinogen >4 g/L were considered as possible prognostic predictors. The results above clarified that transplantation of M-PBMNCs was effective for lower-limb ischemia. Blood glucose >6.0 mmol/L and fibrinogen >4 g/L were possible factors related to poor angiogenesis in autologous M-PBMNC transplantation for lowerlimb ischemia.

Discussion The clinical application of hematopoietic stem cells in ischemic diseases is rapidly expanding in medical practice. Encouraging results have been shown by several studies (3e14,16e19). However, reports about prognostic factors of autologous stem cell transplantation are controversial. With the use of an

ischemic hind-limb model, Li et al. (20) found that the natural recovery from induced limb ischemia in Zucker fatty rats was significantly worse than that in lean rats. Complications from coronary artery disease (20,21), severity of resting pain and repeated bypass surgery (10) have been shown as factors that negatively affect angiogenesis and limb salvage in animal experiments and clinical settings. Horie et al. (3) demonstrated that the significant negative prognostic factors associated with overall survival were ischemic heart disease and the collection of a small number of CD34-positive cells. The factors associated with time-to-amputation and amputation-free survival were a combination of the Fontaine classification and lower-limb gangrene as well as a history of dialysis. A clinical study from Cuba (22) found that the factors identified as possibly predictive of poor angiogenesis after hematopoietic stem cell autograft for lower-limb ischemia were as follows: a final leukocyte count <20
109/L after mobilization with factor-CSF, age 60 years, a pain scale score of 10, glycemia of >6 mmol/L and triglycerides of >1.8 mmol/L. In contrast, we found that fasting blood glucose >6.0 mmol/L and fibrinogen >4 g/L were possible predictors of poor angiogenesis, which is partly in accordance with prior studies. These results indicate that patients with CLI, especially those with DM, should control blood glucose in the normal range; in addition, patients with fibrinogen >4 g/L might pay attention to stronger antiplatelet or anticoagulant therapy after M-PBMNC transplantation. In this study, we designed and advanced possible predictors for autologous M-PBMNC transplantation for patients with CLI in China. Fibrinogen is a glycoprotein synthesized in liver. It is a marker of thrombus formation and plays a critical role in maintaining the balance between thrombosis and hemostasis (23). It is also known as an acute phase reactant. In many prior studies, high fibrinogen levels were proved to be associated with ischemic diseases (24e26). In addition, a systematic review of meta-analyses on levels of serological biomarkers for atherothrombosis found that fibrinogen was associated with primary stroke (27). In our study, we found that 81.8% of the patients with fibrinogen 4 g/L avoided amputation versus only 53.6% of the patients with fibrinogen >4 g/L (P < 0.05), which confirmed previous reports (28). Endothelial progenitor cells (EPCs) are adult stem cells generated from bone marrow (29), and some factors, such as ischemia, G-CSF and stromalderived factor 1 (SDF1), can induce EPCs to mobilize to peripheral circulation and are able to colonize in endothelium (30). EPCs are believed to participate in endothelial repair and postnatal angiogenesis because of their abilities of differentiating into endothelial cells and secreting protective

Prognostic analysis of cell therapy for CLI cytokines and growth factors (31). However, studies have shown that high concentrations of blood glucose can impair EPCs in many aspects, such as the circulating EPC numbers, migration ability and endocrine function (32e34). EPCs have been found to be reduced in pre-diabetic states (impaired fasting glucose and impaired glucose tolerance), with further significant reductions in number at the clinical onset of diabetes and after w20 years of disease (33). In our study, we found that the patients with normal blood glucose concentrations had relatively more successful angiogenesis than those with high blood glucose concentrations, with statistical significance. The results were in accord with a prior study (22). The exact mechanism of EPC dysfunction is not yet clarified. The relationship between DM and SDF-1a has been established. A study showed the downregulation of SDF-1a in diabetic cutaneous wounds might cause the deficient of EPCs in recruitment and neovascularization, resulting in delayed healing. (35). Coculture bone marrowederived stem cells with SDF-1a significantly promoted wound healing, neovascularization and EPC recruitment (36). MicroRNA (miR), an emerging class of highly conserved, noncoding small RNA, is also studied in patients with DM. Meng et al. (37) found that expression of miR-126, miR-27b, miR-130a, miR-21 and miR-27a was less than in controls. AntiemiR126 inhibited EPC proliferation and migration and enhanced apoptosis. Restored miR-126 expression in EPCs from DM promoted EPC proliferation and migration and inhibited EPC apoptosis ability (37). In our study, we proved that intramuscular administration of M-PBMNCs was feasible and safe and had long-term beneficial clinical effect. The overall 5-year amputation-free rate was 72.2%, which was comparable to the result from intra-arterial infusion from published data (11). The amputationfree rates for the DM, ASO, and TAO were77.1%, 72% and 64.7%, respectively, which was partly in accordance with prior studies (3). Horie et al. (3) showed the 1-year amputation-free rates for ASO, TAO and DM as 70%, 79% and 75%, respectively. Studies showed that the effectiveness of stem cell transplantation in TAO is probably more pronounced and probably more durable as well (14). However, in our study, we found that patients with TAO had the highest ratio of amputation (6/17). Two reasons might explain the result. For one thing, the number of patients with TAO was small. In addition, patients with TAO were younger than patients with DM and patients with ASO, with the mean age of 42 years, and several of the patients with TAO refused amputation before cell therapy. Analyzing the angiography of all patients before transplantation, we found that four in 11 patients

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with TAO refused to have surgical revascularization, even though they were advised to do so. Among the four patients, three underwent amputation 1 week after the first transplantation; however, their clinical manifestation, including walking capacity, ischemic pain at rest and ischemic ulceration, had improved since 1 month after transplantation. According to the study design, these patients were still considered to have poor prognosis. The result implied that patients with impending amputation might not derive any benefit from M-PBMNC administration. These could also explain that patients with age 60 years might have poor prognosis after autologous MPBMNC transplantation in our study, which was contradictory to prior study (3,22). A majority of our patients received transplantation more than twice, and we did not observe a difference in the prognoses between the patients who received transplantation three or more or fewer than three times. This finding might suggest that patients do not obtain additional benefit from transplantation more than three times. There are limitations to our study. The sample size of the study is small. Larger studies should be conducted to identify the predictors of poor angiogenesis after autologous M-PBMNC transplantation, and prospective studies are needed. The proportion of men to women in our study is unbalanced, which should be ameliorated in a future study. We compared the post-mobilization leukocyte counts between the two groups; however, we did not study the proportion of the CD34þ cells, which was related to the number of EPCs. We only assessed fibrinogen as one of the main parameters and predictors, but levels of C-reactive protein, D-dimers, protein C, protein S and anti-thrombin III were not detected. In conclusion, transplantation of M-PBMNCs was effective for lower-limb ischemia. Blood glucose >6.0 mmol/L and fibrinogen >4 g/L were the possible factors related to an unsatisfactory prognosis in autologous M-PBMNC transplantation for lowerlimb ischemia. In the future, larger-sample and prospective studies should be performed to identify the possible predictors of poor angiogenesis after autologous M-PBMNC transplantation for lowerlimb ischemia. Acknowledgments This work was supported by grants of the Key Science and Technology Project of Tianjin City (13ZCZDSY02200), Teaching Research Project from Peking Union of Medical College (2011zlgc0110) and 863 project (2011AA020115) from the Ministry of Science & Technology of China. The study sponsors were not involved in the study design, collection, analysis and interpretation of data; they also did not

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participate in the writing of the manuscript and in the decision to submit the manuscript for publication. We sincerely thank Drs Donglei Zhang, Rongfeng Fu and Xian Zhang for writing assistance. We express our gratitude to all people who participated in the study. Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article.

References 1. Hiatt WR. Medical treatment of peripheral arterial disease and claudication. N Engl J Med. 2001;344:1608e21. 2. Folkman J. Therapeutic angiogenesis in ischemic limbs. Circulation. 1998;97:1108e10. 3. Horie T, Onodera R, Akamastu M, Ichikawa Y, Hoshino J, Kaneko E, et al. Long-term clinical outcomes for patients with lower limb ischemia implanted with G-CSF-mobilized autologous peripheral blood mononuclear cells. Atherosclerosis. 2010;208:461e6. 4. Tateishi-Yuyama E, Matsubara H, Murohara T, Ikeda U, Shintani S, Masaki H, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet. 2002;360:427e35. 5. Huang P, Li S, Han M, Xiao Z, Yang R, Han ZC. Autologous transplantation of granulocyte colony-stimulating factormobilized peripheral blood mononuclear cells improves critical limb ischemia in diabetes. Diabetes Care. 2005;28: 2155e60. 6. Huang PP, Li SZ, Han MZ, Xiao ZJ, Yang RC, Qiu LG, et al. Autologous transplantation of peripheral blood stem cells as an effective therapeutic approach for severe arteriosclerosis obliterans of lower extremities. Thromb Haemost. 2004;91: 606e9. 7. Huang PP, Yang XF, Li SZ, Wen JC, Zhang Y, Han ZC. 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;98:1335e42. 8. Volz KS, Miljan E, Khoo A, Cooke JP. Development of pluripotent stem cells for vascular therapy. Vasc Pharmacol. 2012;56:288e96. 9. Silvestre JS. Pro-angiogenic cell-based therapy for the treatment of ischemic cardiovascular diseases. Thromb Res. 2012; 130(Suppl 1):S90e4. 10. Matoba S, Tatsumi T, Murohara T, Imaizumi T, Katsuda Y, Ito M, 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: 1010e8. 11. Walter DH, Krankenberg H, Balzer JO, Kalka C, Baumgartner I, Schluter M, et al. Intraarterial administration of bone marrow mononuclear cells in patients with critical limb ischemia: a randomized-start, placebo-controlled pilot trial (PROVASA). Circ Cardiovasc Intervent. 2011;4:26e37. 12. Schiavetta A, Maione C, Botti C, Marino G, Lillo S, Garrone A, et al. A phase II trial of autologous transplantation of bone marrow stem cells for critical limb ischemia: results of the Naples and Pietra Ligure Evaluation of Stem Cells study. Stem Cells Transl Med. 2012;1:572e8.

13. Dubsky M, Jirkovska A, Bem R, Fejfarova V, Pagacova L, Sixta B, et al. Both autologous bone marrow mononuclear cell and peripheral blood progenitor cell therapies similarly improve ischaemia in patients with diabetic foot in comparison with control treatment. Diabetes Metab Res Rev. 2013; 29:369e76. 14. Teraa M, Sprengers RW, van der Graaf Y, Peters CE, Moll FL, Verhaar MC. Autologous bone marrow-derived cell therapy in patients with critical limb ischemia: a meta-analysis of randomized controlled clinical trials. Ann Surg. 2013;258: 922e9. 15. Gupta PK, Chullikana A, Parakh R, Desai S, Das A, Gottipamula S, et al. A double blind randomized placebo controlled phase I/II study assessing the safety and efficacy of allogeneic bone marrow derived mesenchymal stem cell in critical limb ischemia. J Transl Med. 2013;11:143. 16. Amato B, Compagna R, Della Corte GA, Martino G, Bianco T, Coretti G, et al. Peripheral blood mono-nuclear cells implantation in patients with peripheral arterial disease: a pilot study for clinical and biochemical outcome of neoangiogenesis. BMC Surg. 2012;12(Suppl 1):S1. 17. Bartsch T, Brehm M, Zeus T, Kogler G, Wernet P, Strauer BE. Transplantation of autologous mononuclear bone marrow stem cells in patients with peripheral arterial disease (the TAM-PAD study). Clin Res Cardiol. 2007;96:891e9. 18. Durdu S, Akar AR, Arat M, Sancak T, Eren NT, Ozyurda U. Autologous bone-marrow mononuclear cell implantation for patients with Rutherford grade II-III thromboangiitis obliterans. J Vasc Surg. 2006;44:732e9. 19. Grochot-Przeczek A, Dulak J, Jozkowicz A. Therapeutic angiogenesis for revascularization in peripheral artery disease. Gene. 2013;525:220e8. 20. Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003;348:593e600. 21. Li TS, Furutani A, Takahashi M, Ohshima M, Qin SL, Kobayashi T, et al. Impaired potency of bone marrow mononuclear cells for inducing therapeutic angiogenesis in obese diabetic rats. Am J Physiol Heart Circ Physiol. 2006; 290:H1362e9. 22. Gomez RA, Fernandez JD, Cabrera M, Marrero I, Ramirez N, Alvarez I. Possible predictors of poor angiogenesis after hematopoietic stem cell autograft for lower limb ischemia. MEDICC Rev. 2012;14:31e6. 23. Saldanha C. Fibrinogen interaction with the red blood cell membrane. Clin Hemorheol Microcirc. 2013;53:39e44. 24. Meade TW, Mellows S, Brozovic M, Miller GJ, Chakrabarti RR, North WR, et al. Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart Study. Lancet. 1986;2:533e7. 25. Kannel WB, Wolf PA, Castelli WP, D’Agostino RB. Fibrinogen and risk of cardiovascular disease: the Framingham Study. JAMA. 1987;258:1183e6. 26. Danesh J, Lewington S, Thompson SG, Lowe GD, Collins R, Kostis JB, et al. Plasma fibrinogen level and the risk of major cardiovascular diseases and nonvascular mortality: an individual participant meta-analysis. JAMA. 2005;294:1799e809. 27. van Holten TC, Waanders LF, de Groot PG, Vissers J, Hoefer IE, Pasterkamp G, et al. Circulating biomarkers for predicting cardiovascular disease risk: a systematic review and comprehensive overview of meta-analyses. PloS One. 2013;8: e62080. 28. Kulier A, Levin J, Moser R, Rumpold-Seitlinger G, Tudor IC, Snyder-Ramos SA, et al. Impact of preoperative anemia on outcome in patients undergoing coronary artery bypass graft surgery. Circulation. 2007;116:471e9.

Prognostic analysis of cell therapy for CLI 29. Asahara T, Masuda H, Takahashi T, Kalka C, Pastore C, Silver M, et al. Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res. 1999;85:221e8. 30. Tilling L, Chowienczyk P, Clapp B. Progenitors in motion: mechanisms of mobilization of endothelial progenitor cells. Br J Clin Pharmacol. 2009;68:484e92. 31. Zhao YH, Yuan B, Chen J, Feng DH, Zhao B, Qin C, et al. Endothelial progenitor cells: therapeutic perspective for ischemic stroke. CNS Neurosci Therapeut. 2013;19:67e75. 32. Avogaro A, Fadini GP, Gallo A, Pagnin E, de Kreutzenberg S. Endothelial dysfunction in type 2 diabetes mellitus: nutrition, metabolism, and cardiovascular diseases. Nutr Metab Cardiovasc Dis. 2006;16(Suppl 1):S39e45. 33. Fadini GP, Boscaro E, de Kreutzenberg S, Agostini C, Seeger F, Dimmeler S, et al. Time course and mechanisms of circulating progenitor cell reduction in the natural history of type 2 diabetes. Diabetes Care. 2010;33:1097e102.

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34. Yue WS, Lau KK, Siu CW, Wang M, Yan GH, Yiu KH, et al. Impact of glycemic control on circulating endothelial progenitor cells and arterial stiffness in patients with type 2 diabetes mellitus. Cardiovasc Diabetol. 2011;10:113. 35. Gallagher KA, Liu ZJ, Xiao M, Chen H, Goldstein LJ, Buerk DG, et al. Diabetic impairments in NO-mediated endothelial progenitor cell mobilization and homing are reversed by hyperoxia and SDF-1 alpha. J Clin Invest. 2007; 117:1249e59. 36. Castilla DM, Liu ZJ, Tian R, Li Y, Livingstone AS, Velazquez OC. A novel autologous cell-based therapy to promote diabetic wound healing. Ann Surg. 2012;256: 560e72. 37. Huang F, Zhu X, Hu XQ, Fang ZF, Tang L, Lu XL, et al. Mesenchymal stem cells modified with miR-126 release angiogenic factors and activate Notch ligand Delta-like-4, enhancing ischemic angiogenesis and cell survival. Int J Mol Med. 2013;31:484e92.