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Safety and Efficacy of Therapeutic Angiogenesis as a Novel Treatment in Patients with Critical Limb Ischemia
Safety and Efficacy of Therapeutic Angiogenesis as a Novel Treatment in Patients with Critical Limb Ischemia R. Lara-Hernandez,1 P. Lozano-Vilardell,1 P. Blanes,1 N. Torreguitart-Mirada,1 A. Galmes,2 and J. Besalduch,2 Baleares, Spain
Background: In some patients with critical limb ischemia (CLI) the possibility of revascularizing treatment does not exist. In this case therapeutic angiogenesis (TA) using autologous endothelial progenitor cell (EPC) transplantation could be an alternative. The objective of our study was to evaluate the safety and efficacy of TA using EPC. Methods: Twenty-eight patients with CLI who were not candidates for surgical or endovascular revascularization were included in a prospective study. To mobilize EPCs from the bone marrow, granulocyte colony-stimulating growth factor was injected subcutaneously at doses of 5 mg/kg/ day for 5 days. Apheresis was performed, obtaining 50 mL of blood with a high rate of EPCs (CD34+ and CD133+ cells were counted). EPCs were implanted in the ischemic limb by intramuscular injections. Primary end points were the safety and feasibility of the procedure and limb salvage rate for amputation at 12 months. Other variables studied were improvement in rest pain, healing of ulcers, ankle-brachial pressure index (ABI), and digital plethysmography. All procedures were done pretreatment and every 3 months for a year on average. Postransplantation arteriography was done in selected cases. Results: No adverse effects were observed. Mean follow-up was 14 months. Before treatment, mean basal ABI was 0.35 ± 0.2 and at 18 months postimplantation, 0.72 ± 0.51 ( p ¼ 0.009). There was a mean decrease of five points in pain scale: basal 8.7 ± 1, after TA 3.8 ± 2.9 ( p ¼ 0.01). Seven patients required major amputation. Kaplan-Meier analysis revealed a limb salvage rate of 74.4% after 1 year. Conclusion: Implantation of EPCs in CLI is a safe alternative, improves tissue perfusion, and obtains high amputation-free rates. Nevertheless, this is a small cohort and results should be tested with long randomized trials.
INTRODUCTION Critical limb ischemia (CLI) is the end stage in peripheral arterial occlusive disease (PAOD) of the lower limbs, with a calculated incidence of 30 patients per 100,000 inhabitants/year. It has a great 1 Vascular Surgery Department, Hospital Universitario Son Dureta, Palma de Mallorca, Baleares, Spain. 2 Hematology Department, Hospital Universitario Son Dureta, Palma de Mallorca, Baleares, Spain.
Correspondence to: R. Lara-Hernandez, Vascular Surgery Department, Hospital Universitario Son Dureta, C/ Andrea Doria 55. CP 07015 Palma de Mallorca, Baleares, Spain, E-mail: [email protected] Ann Vasc Surg 2010; 24: 287-294 DOI: 10.1016/j.avsg.2009.10.012 Ó Annals of Vascular Surgery Inc.
impact on the patient and his or her environment because of the severity of the disease, the associated risk factors, and the lack of really effective treatments. In 20-30% of CLI cases, the possibility of revascularizing treatment does not exist and pharmacological treatment constitutes the main therapy. However, despite the painkillers and control of trophic lesions, major limb amputation is usually the last therapy, with limb loss rates that may vary 70-95% per year.1-4 In recent years, different investigations have established that it is possible to use gene therapy (GT), growth factors (GFs), or cellular therapy (CT) to increase the development of collateral vessels into ischemic tissues (generally known as angiogenic therapy [AT]). However, the method of applying treatment, doses, frequency, and administration 287
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route that determine maximal effectiveness, associated with a good range of security, remain to be determined.5-9 Hematopoietic stem cells (HSCs) were the first tissue stem cells to be discovered and well studied. HSC transplantation has been successfully applied to cure a variety of diseases of hematological and immunological systems. The bone marrow contains heterogeneous populations of cells, including endothelial progenitor cells (EPCs), which have been shown to differentiate into endothelial cells and to release several angiogenic factors and thereby enhance neovascularization in animal models of hind limb ischemia. Promising results from various preclinical studies provide the basis for clinical trials using bone marrowederived cells or other cells, like cells from the peripheral blood or other tissues. However, the mechanisms of how these cells exert their positive effects have been poorly understood until now. Bone marrowe derived EPCs were first described over 10 years ago. These cells have also been isolated from peripheral blood and cord blood. Although the underlying mechanisms remain undisclosed, all these findings suggest that bone marrowederived cells, alone or with the help of accessory cells with a paracrine effect, are capable of inducing the formation of new vessels. Based on these previous results, the objective of our study was to evaluate the efficacy and safety of AT in the treatment of nonrevascularizing CLI, with intramuscular implantation of mobilized peripheral blood EPCs.
MATERIALS AND METHODS We conducted a prospective pilot study on patients with CLI with no possibility of revascularizing treatment. Patients with rest pain and/or trophic lesions with no indication for primary major amputation were included. Limited life expectancy, severe neurological or psychomotor deficit, and neoformative process (hematology included) were exclusion criteria. The study was approved by the Hospital Ethics Committee. All patients gave informed written consent to participation. The primary variable of effectiveness was limb salvage rate after 12 months’ follow-up. Other variables studied were improvement of pain control by means of a visual analogue pain scale (VAPS), ankle perfusion pressure (segmental pressure), anklebrachial index (ABI), and digital photoplethysmography (PPG). Posttransplantation angiography was done in selected cases. Absence of cellular infusion technique-derived complications, adverse reactions to the cellular
Annals of Vascular Surgery
mobilization and sanguine apheresis, as well as pathological manifestations at the local or systemic level that could be attributable to the study were considered safety factors. To mobilize EPCs from the bone marrow, all patients were subjected to ambulatory treatment for 5 days by means of subcutaneous injections of granulocyte colony-stimulating factor (G-CSF, dose of 5 mg/kg/day), with the purpose of increasing the bone marrow EPC production and maturation. At the fifth day of treatment, blood apheresis was carried out for 3 hr (Cell Separator Machine CS3000 PLUS; Bayer, Leverkusen, Germany), obtaining a final volume of 50 mL per apheresis. The final product of each apheresis (50 mL) was analyzed for mononuclear cells, CD34 cells, CD3 cells, natural killer (NK) granulocytes, and platelets using an automated cell counter (Advia 120, Bayer). The viability of mononuclear cells was studied by trypan blue-dye exclusion. We performed flow-cytometric analysis, using anti-CD33 fluorescein isothiocyanate and anti-CD34 phycoerythrin on a flow cytometer (FACScan; Becton Dickinson, Mountain View, CA). The total volume of the inoculum (50 mL) was divided into aliquots of 2 mL and implanted by intramuscular injections 24 hr after apheresis, under epidural anesthesia and surgical aseptic conditions. Injection sites were selected according to angiographic findings, including gastrocnemius, soleus, and anterior tibial muscles, as well as the sole muscles of the foot. Ankle segmental pressures measured with Doppler, ABI, VAPS, and PPG at the sixth week postprocedure and every 3 months in the first year were carried out during the follow-up. Angiography was done in selected cases. Statistical analysis was conducted using SPSS for Windows, version 13.0 (SPSS, Inc., Chicago, IL). Data were expressed as mean ± standard deviation. Statistically significant differences between measures were considered at p < 0.05. Kaplan-Meier analysis was developed to assess limb salvage rate after 1year follow-up.
RESULTS Between January 2002 and July 2008, more than 350 patients with CLI were treated in our institution. We included 28 patients (21 men and seven women) with the established approaches (Table I). In two patients both legs were treated (30 legs). Mean age was 67 years. Mean follow-up was 14.7 ± 5.6 months (range 1-48). All patients had rest pain, and 85% (n ¼ 24) were associated with trophic ulcers at the digital level or in the heel of
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Table I. Inclusion/exclusion criteria and patient characteristics Inclusion criteria
Exclusion criteria Patients/legs Mean age (range, years) Sex (male/female) Clinical presentation CLI Grade III Grade IV Comorbidity Diabetes mellitas Chronic ischemic cardiopathy Chronic renal failure
CLI stage III or IV Buerger diagnosis ABI <0.6 Not candidates for revascularization Limited vital expectancy Active neoplastic disorder 28/30 67 (22-79) 21/7 30 6 24 15 6 4
the foot. Two had a clinical diagnosis of thromboangiitis obliterans (Buerger disease, TO), and 15 had diabetes. Four of the diabetic patients had segmental pressures at the maleolar level >250 mm Hg (Mo¨nkenberg calcinosis). These pressures were not included when statistical analysis was done for perfusion improvement. Major amputation was done in seven patients, in a median of 30 days (range 7161). In three cases, the amputation was done because of uncontrolled pain despite medical treatment. Kaplan-Meier analysis showed a 74.4% limb salvage rate after 18-month follow-up (Fig. 1). The median time to achieve clinical improvement was 51 days (range 34-68). Five patients died during the study due to cardiovascular disease, all of them with major amputation. Mean segmental pressures increased in the first year posttreatment. This finding was correlated with an improvement in ABI (mean measures): before treatment 0.35 ± 0.2 (range 0.05-0.6) and 18-month follow-up 0.72 ± 0.51 (range 0.1-0.8) ( p ¼ 0.01). No differences between patients with distal pressures >250 mm Hg were observed after treatment. There was an improvement from the layout PPG in 80% of patients after 1-year followup, with the exception of those who required forefoot or major amputation (Table II). Rest pain was determined by means of VAPS. All patients were interrogated previous to treatment and during follow-up. All patients with rest pain reported improvement, stressing that this improvement was accented starting from the second month after the cellular implantation. After 1-year
follow-up, 18 patients required occasional analgesic treatment (no opioids), and in no case did the pain impede night rest. These data were correlated with a five-point decrease in the mean score obtained by VAPS: before treatment 8.7 ± 1 points and 1year follow-up 3.8 ± 2.9 points ( p ¼ 0.01). Only three patients did not report improvement in rest pain and suffered major amputation. In the group of patients with ulcers, 80% showed improvement in their lesions. Some type of minor amputation was necessary in three patients (one transmetatarsal, two digital) because of infected trophic lesions. Only in the first six patients was angiography performed at 1 year posttreatment. There seemed to be an increase in the collateral network, but a strong subjectivity factor was thought to influence the evaluation of the images (Fig. 2). We saw a unique case of high blood levels of liver enzymes after the cellular mobilization. It was normalized after 2 weeks. The patient did not have previous liver disease; viral serum tests were negative previous to the inclusion in the study and after the implantation. No other adverse effects were observed during the mobilization, the process of cellular apheresis, or the cellular infusion technique. We analyzed the cellular content of the inoculum in relationship with clinical outcome (improvement of rest pain, healing of ulcer, resting ABI, digital PPG, and limb salvage). We did not find a significant statistical influence between the number of CD34+ cells, CD3+ cells, NK cells, mononuclear cells, and granulocytes infused and clinical outcome.
DISCUSSION The results of our study suggest that intramuscular implantation of EPCs obtained from peripheral blood (PB), improves tissue perfusion in CLI of the lower limbs, as demonstrated by the increase in segmental pressures, ABI, and the healing of the trophic lesions. It seems also to relieve rest pain. Moreover, the patients who responded to treatment were able to walk a reasonable distance without claudication. Likewise with high limb salvage rates after 1-year follow-up, this could be considered a novel treatment that should be considered in cases with no surgical or endovascular revascularizing options. Different studies have demonstrated the capacity to form new vessels by administration of different growth factors. However, this capacity is limited by an effectiveness-dependent dose and the possibility for developing neoformative processes because
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Fig. 1. Failure-free survival. A Overal limb salvage (OLS). B OLS in function of clinical stage CLI (Leriche-La Fontaine classification).
many of these factors are involved in tumoral angiogenesis.10-15 Different cell lines existing inside the bone marrow maintain the ability to develop into the different cellular components of the arterial wall and, therefore, to be able to give functionality to the newly formed vessels.16-19 EPCs constitute a small group inside the mononucleated cells
(0.05%), which present angioblastic capacity for endothelial differentiation, and they circulate in the PB. Since the discovery of EPCs in adult bone marrow, it has been known that the increase of their formation, maturation, and liberation from the bone marrow to PB is an inducible process (Fig. 3).20 Some studies have indicated that EPC
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Table II. Clinical and hemodynamic behavior of patients after stem cell infusion Posttransplant
Clinical No symptoms Rest pain Ulcer Amputation Exitus Hemodynamic ABI Plane PPG1 Normal PPG Legs
Pretransplant
1 month
d 6 24 d d
9 1 14 6 d
0.35 ± 0.28 30 d 30
0.44 ± 0.24 25 5 30
circulating levels constitute a functionality marker of the vascular system in such a way that even diminished EPC circulating levels are related to a decreased endothelial repair capacity and, therefore, an increased progression of arterial disease.21,22 Moreover, others have identified some alterations in EPC circulating levels and its capacity for diferentiation in atherosclerotic and diabetic patients.23 In different animal models of CLI, administration of peripheral mononucleated cells is able to improve limb perfusion, diminishing the amputation rate.24-27 Tateishi-Yuyama et al.28 carried out the first study in patients with CLI, where the utility of mononucleated cells extracted from PB was compared with that of cells directly extracted from bone marrow. Satisfactory results were shown with both groups compared to the group given placebo. Others have verified this fact,29-31 and moreover, it is possible to obtain EPCs directly from PB without the necessity of carrying out direct punctures for bone marrow.32-34 Good results have been obtained using bone marrow or PB cells, and both methods present advantages and inconveniences; when using PB cells, the main limitation is the time invested in the mobilization and cell populations gathering, as well as the time necessary to obtain results (6-8 weeks). Anyway, and in accordance with the results published by other groups that support our work method, we do not feel justified in subjecting the patient to unnecessarily invasive processes that are not exempt from complications. Mainly two forms of administration have been described: intramuscular injection28,29,31-35 and intra-arterial direct puncture,36,37 the latter being the one used more frequently by all groups and by us. Using this delivery system, we can ensure that EPCs will home into the ischemic tissue. This study, like others, opens a debate to explain the mechanism of neovascularization observed when the cells
p
6 months
p
14 0 7 1 2 0.08
0.50 ± 0.2 18 6 24
18 months
p
18 0 0 d 3 0.02
0.72 ± 0.51 4 17 21
0.009
are implanted. The conclusion is that it is probable that the effect of neovascularization resides both in accessory cells, which secrete cytokines and growth factors, and in the infused hematopoietic stem cells that differentiate in EPCs. Regarding the methods to evaluate the effectiveness of the treatment, one of them could be the use of quality-of-life questionnaires.38 In our work, we did not believe it to be necessary. The primary objective was to determine the effect of the treatment in the trophic limb and, secondly, to determine if that improvement was related with the decrease in analgesic need and with improvement of walking distance. It is certain that it can be a work tool that guides the doctor to control the evolution of the treatment, but we do not think of it as a definitive test of effectiveness. On the other hand, VAPS has been widely used as a good test to evaluate the results of procedures done in patients with cronic pain.39 It could be considered a limitation of our study that we used angiography to determine the increase of the collateral circulation since this technique is known to be limited when evaluating small-caliber vessels (<1 mm diameter)40 and there is observer subjectivity when determining the degree of increase of the collateral circulation. Other techniques have been used (e.g., magnetic resonance), but we did not have this option available in our institution. Serious limitations should be noted: first, the lack of a control group to compare with; second, the small cohort obtained. In this sense, we did a pilot study with the first 10 patients, comparing the results with a historical cohort treated in our institution. The results showed that, in the historical cohort, >90% of the patients treated medically suffered major amputation in less than 1 year. This
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Fig. 2. Angiography. A1, A2 Before transplantation. B1, B2 Posttransplantation with development of new collateral vessels.
could be discussed in terms of statistical significance, but the fact is that there was a difference in terms of limb salvage rate after 1-year follow-up. On the other hand, the reduced number of patients included should indicate that the inclusion criteria were extremely strict, with revascularization attempted as the first option in all patients treated in our institution. The discovery of EPCs in the adult and their functionality inside the vascular system have have led to the study of their possible applications in arterial pathology. The good results obtained in recent
published works31,35 and the use of autologous patient cells make this a new treatment to consider in the future. In accordance with our results, we think that the use of EPCs mobilized from bone marrow and their later implantation into ischemic limbs by intramuscular injection improves tissue perfusion and constitutes a valid and novel treatment that should be considered in selected patients with CLI and no revascularizing options. The main limitations of our study have been discussed, and a randomized study with a higher number of cases that may confirm our results would be necessary.
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Therapeutic angiogenesis for CLI 293
Basics on Angiogenesis Bone Marow M ro Ma r w qui qu iesce escen nt quiescent
MMP-9
EPC
+
eNOS
Vascular System mobilization (Peripheral blood)
Bone Marrow proliferative
Ischemic Tissue Infarction
New vessels recruitment
Trauma ↓ VEGF, GM-CSF EPO
Zammaretti P, Zisch AH. Adult endothelial progenitor cells. Renewing Vasculature. Int J Biochem Cell Biol 2005 ; 37 : 493-503
MMP-9 : metalo-proteinase 9, eNOS : nitric oxide synthetase, EPC : endotelial progenitor cell, GM-CSF: Granulo-monocyte colony stimulating factor, VEGF: vascular endotelial growth factor, EPO: eritropoietin Fig. 3. Basic mechanism in angiogenesis. MMP-9, metalloproteinase 9; eNOS, endothelial nitric oxide synthetase; EPC, endotelial progenitor cell; GM-CSF, granulocyte
colony-stimulating factor; VEGF, vascular endothelial growth factor; EPO, erythropoietin.
REFERENCES
10. Lederman RJ, Mendelsohn F, Anderson RD, et al. Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (the TRAFFIC study): a randomized trial. Lancet 2002;359:2053-2058. 11. Rajagopalan S, Mohler E, Lederman RJ, et al. Regional angiogenesis with vascular endothelial growth factor (VEGF) in peripheral arterial disease: design of the RAVE trial. Am Heart J 2003;145:1114-1118. 12. Baffour R, Berman J, Garb J, Rhee S, Kaufman J, Friedmann P. Enhanced angiogenesis and growth of collaterals by in vivo administration of recombinant bFGF in a rat model of acute lower limb ischaemia: dose response effect of bFGF. J Vasc Surg 1992;2:181-191. 13. Morishita R, Nakamura S, Hayashi S, et al. Therapeutic angiogenesis induced by human recombinant hepatocyte growth factor in a rabbit hind limb ischaemia model as cytokine supplement therapy. Hypertension 1999;33:13791384. 14. Henry T, Rocha-Singh K, Isner J, et al. Intracoronary administration of recombinant human VEGF to patients with coronary artery disease. Am Heart J 2001;142:872-880. 15. Henry T, Annex B, McKendall G, et al. The VIVA trial: VEGF in Ischaemia for Vascular Angiogenesis. Circulation 2003;107: 1359-1365. 16. Cooper L, Hiatt W, Creager M, et al. Proteinuria in a placebo-controlled study of bFGF for intermittent claudication. Vasc Med 2001;6:235-239. 17. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM. Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 2002;109:337-346.
1. Dormandy J, Mahir M, Ascady G, et al. Fate of the patient with chronic leg ischaemia. A review article. J Cardiovasc Surg 1989;30:50-57. 2. Lozano-Vilardell P, Corominas-Roura C, Gomez-Ruiz FT, Manuel-Rimbau E, Julia-Montoya J. Evoluci on natural de la isquemia crıtica de los miembros inferiores. Angiologıa 2003;55(Suppl. 1):S38-S46. 3. Inter-Society Consensus for the Management of Peripheral Arterial Disease (TASC II). Eur J Vasc Endovasc Surg 2007;33:S5-S75. 4. Wolfe JH, Wyatt MG. Critical and subcritical ischaemia. Eur J Vasc Endovasc Surg 1997;13:578-582. 5. Risau W, Sariola H, Zerwes HG, et al. Vasculogenesis and angiogenesis in embryonic stem-cell-derived embryoid bodies. Development 1988;102:471. 6. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med 1971;285:1182. 7. Oren M, Tepper BA, Galiano RD, Kalka C, Gurtner GC. Endothelial progenitor cells: the promise of vascular stem cells for plastic surgery. Plast Reconstr Surg 2003;111: 846-854. 8. Collinson DJ, Donnelly R. Therapeutic angiogenesis in peripheral arterial disease: can biotechnology produce an effective collateral circulation? Eur J Vasc Endovasc Surg 2004;28:9-23. 9. Yoshida WB. Angiogenesis, arteriogenesis and vasculogenesis: treatment of the future for lower limb critical ischemia? Braz Vasc J. 2005;4:209-211.
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18. Kalka C, Masuda H, Takahashi T, et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc Natl Acad Sci USA 2000;97: 3422-3427. 19. Kinnaird T, Stabile E, Burnett MS, Epstein SE. Bonemarrow-derived cells for enhancing collateral development: mechanisms, animal data, and initial clinical experiences. Circ Res. 2004;95:354-363. 20. Zammaretti P, Zisch AH. Adult endothelial progenitor cells. Renewing vasculature. Int J Biochem Cell Biol 2005;37: 493-503. 21. 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: 1449-1457. 22. Hill JM, Zalos G, Halcox JP, et al. Circulating endothelial progenitor cells, vascular function and cardiovascular risk. N Engl J Med 2003;348:593-600. 23. Vasa M, Fichtlscherer S, Adler K, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001;89:1-7. 24. Al-Khaldi A, Al-Sabti H, Galipeau J, Lachapelle K. Therapeutic angiogenesis using autologous bone marrow stromal cells: improved blood flow in a chronic limb ischemia model. Ann Thorac Surg 2003;75:204-209. 25. Iba O, Matsubara H, Nozawa Y, et al. Angiogenesis by implantation of peripheral blood mononuclear cells and platelets into ischemic limbs. Circulation 2002;106:2019-2025. 26. Tan Y, Shao H, Eton D, et al. Stromal cell-derived factor-1 enhances pro-angiogenic effect of granulocyte-colony stimulating factor. Cardiovasc Res 2007;73:823-832. 27. Li S, Zhou B, Han ZC. Therapeutic neovascularization by transplantation of mobilized peripheral blood mononuclear cells for limb ischemia. A comparison between CD34+ and CD34e mononuclear cells. Thromb Haemost 2006;95: 301-311. 28. Tateishi-Yuyama E, Matsubara H, Murohara T, 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: 427-435. 29. Higashi Y, Kimura M, Hara K, et al. Autologous bonemarrow cell implantation improves endothelium-dependent vasodilation in patients with limb ischemia. Circulation 2004;109:1215-1218.
Annals of Vascular Surgery
30. Esato K, Hamano K, Li TS, et al. Neovascularization induced by autologous bone marrow cell implantation in peripheral arterial disease. Cell Transplant 2002;11:747-752. 31. Durdu S, Akar AR, Arat M, Sancak T, Eren NT, Ozyurda U. Autologous bone-marrow mononuclear cell implantation for patients with rutherford grade IIeIII thromboangiitis obliterans. J Vasc Surg 2006;44:732-739. 32. Huang P, Li S, Han M, Xiao Z, Yang R, Han ZC. Autologous transplantation of granulocyte colony-stimulating factor mobilized peripheral blood mononuclear cells improves critical limb ischemia in diabetes. Diabetes Care 2005;28: 2155-2160. 33. Ishida A, Ohya Y, Sakuda H, et al. Autologous peripheral blood mononuclear cell implantation for patients with peripheral arterial disease improves limb ischemia. Circ J 2005;69:1260-1265. 34. Korbling M, Reuben JM, Gao H, et al. Recombinant human granulocyteecolony-stimulating factoremobilized and apheresis-collected endothelial progenitor cells: a novel blood cell component for therapeutic vasculogenesis. Transfusion 2006; 46:1795-1802. 35. Kawamura A, Horie T, Tsuda I, et al. Clinical study of therapeutic angiogenesis by autologous peripheral blood stem cell transplantation in 92 patients with critically ischemic limbs. J Artif Organs 2006;9:226-233. 36. Yoshida M, Horimoto H, Mieno S, et al. Intra-arterial bone marrow cell transplantation induces angiogenesis in rat hindlimb ischemia. Eur Surg Res 2003;35:86-91. 37. Hirsch A, Nijveldt R, van der Vleuten PA, et al. Intracoronary infusion of autologous mononuclear bone marrow cells or peripheral mononuclear blood cells after primary percutaneous coronary intervention: rationale and design of the HEBE trialda prospective, multicenter randomized trial. Am Heart J 2006;152:434-441. 38. Morgan MB, Crayford T, Murrin B, Fraser SC. Developing the Vascular Quality of Life Questionnaire: a new disease-specific quality of life measure for use in lower limb ischemia. J Vasc Surg 2001;33:679-687. 39. Boonstra AM, Schiphorst Preuper HR, Reneman MF, Posthumus JB, Stewart RE. Reliability and validity of the visual analogue scale for disability in patients with chronic musculoskeletal pain. Int J Rehabil Res 2008;31:165-169. 40. Takeshita S, Isshiki T, Mori H, et al. Use of synchrotron radiation microangiography to assess development of small collateral arteries in a rat model of hindlimb ischemia. Circulation 1997;95:805-808.
Background: In some patients with critical limb ischemia (CLI) the possibility of revascularizing treatment does not exist. In this case therapeutic angiogenesis (TA) using autologous endothelial progenitor cell (EPC) transplantation could be an alternative. The objective of our study was to evaluate the safety and efficacy of TA using EPC. Methods: Twenty-eight patients with CLI who were not candidates for surgical or endovascular revascularization were included in a prospective study. To mobilize EPCs from the bone marrow, granulocyte colony-stimulating growth factor was injected subcutaneously at doses of 5 mg/kg/ day for 5 days. Apheresis was performed, obtaining 50 mL of blood with a high rate of EPCs (CD34+ and CD133+ cells were counted). EPCs were implanted in the ischemic limb by intramuscular injections. Primary end points were the safety and feasibility of the procedure and limb salvage rate for amputation at 12 months. Other variables studied were improvement in rest pain, healing of ulcers, ankle-brachial pressure index (ABI), and digital plethysmography. All procedures were done pretreatment and every 3 months for a year on average. Postransplantation arteriography was done in selected cases. Results: No adverse effects were observed. Mean follow-up was 14 months. Before treatment, mean basal ABI was 0.35 ± 0.2 and at 18 months postimplantation, 0.72 ± 0.51 ( p ¼ 0.009). There was a mean decrease of five points in pain scale: basal 8.7 ± 1, after TA 3.8 ± 2.9 ( p ¼ 0.01). Seven patients required major amputation. Kaplan-Meier analysis revealed a limb salvage rate of 74.4% after 1 year. Conclusion: Implantation of EPCs in CLI is a safe alternative, improves tissue perfusion, and obtains high amputation-free rates. Nevertheless, this is a small cohort and results should be tested with long randomized trials.
INTRODUCTION Critical limb ischemia (CLI) is the end stage in peripheral arterial occlusive disease (PAOD) of the lower limbs, with a calculated incidence of 30 patients per 100,000 inhabitants/year. It has a great 1 Vascular Surgery Department, Hospital Universitario Son Dureta, Palma de Mallorca, Baleares, Spain. 2 Hematology Department, Hospital Universitario Son Dureta, Palma de Mallorca, Baleares, Spain.
Correspondence to: R. Lara-Hernandez, Vascular Surgery Department, Hospital Universitario Son Dureta, C/ Andrea Doria 55. CP 07015 Palma de Mallorca, Baleares, Spain, E-mail: [email protected] Ann Vasc Surg 2010; 24: 287-294 DOI: 10.1016/j.avsg.2009.10.012 Ó Annals of Vascular Surgery Inc.
impact on the patient and his or her environment because of the severity of the disease, the associated risk factors, and the lack of really effective treatments. In 20-30% of CLI cases, the possibility of revascularizing treatment does not exist and pharmacological treatment constitutes the main therapy. However, despite the painkillers and control of trophic lesions, major limb amputation is usually the last therapy, with limb loss rates that may vary 70-95% per year.1-4 In recent years, different investigations have established that it is possible to use gene therapy (GT), growth factors (GFs), or cellular therapy (CT) to increase the development of collateral vessels into ischemic tissues (generally known as angiogenic therapy [AT]). However, the method of applying treatment, doses, frequency, and administration 287
288 Lara-Hernandez et al.
route that determine maximal effectiveness, associated with a good range of security, remain to be determined.5-9 Hematopoietic stem cells (HSCs) were the first tissue stem cells to be discovered and well studied. HSC transplantation has been successfully applied to cure a variety of diseases of hematological and immunological systems. The bone marrow contains heterogeneous populations of cells, including endothelial progenitor cells (EPCs), which have been shown to differentiate into endothelial cells and to release several angiogenic factors and thereby enhance neovascularization in animal models of hind limb ischemia. Promising results from various preclinical studies provide the basis for clinical trials using bone marrowederived cells or other cells, like cells from the peripheral blood or other tissues. However, the mechanisms of how these cells exert their positive effects have been poorly understood until now. Bone marrowe derived EPCs were first described over 10 years ago. These cells have also been isolated from peripheral blood and cord blood. Although the underlying mechanisms remain undisclosed, all these findings suggest that bone marrowederived cells, alone or with the help of accessory cells with a paracrine effect, are capable of inducing the formation of new vessels. Based on these previous results, the objective of our study was to evaluate the efficacy and safety of AT in the treatment of nonrevascularizing CLI, with intramuscular implantation of mobilized peripheral blood EPCs.
MATERIALS AND METHODS We conducted a prospective pilot study on patients with CLI with no possibility of revascularizing treatment. Patients with rest pain and/or trophic lesions with no indication for primary major amputation were included. Limited life expectancy, severe neurological or psychomotor deficit, and neoformative process (hematology included) were exclusion criteria. The study was approved by the Hospital Ethics Committee. All patients gave informed written consent to participation. The primary variable of effectiveness was limb salvage rate after 12 months’ follow-up. Other variables studied were improvement of pain control by means of a visual analogue pain scale (VAPS), ankle perfusion pressure (segmental pressure), anklebrachial index (ABI), and digital photoplethysmography (PPG). Posttransplantation angiography was done in selected cases. Absence of cellular infusion technique-derived complications, adverse reactions to the cellular
Annals of Vascular Surgery
mobilization and sanguine apheresis, as well as pathological manifestations at the local or systemic level that could be attributable to the study were considered safety factors. To mobilize EPCs from the bone marrow, all patients were subjected to ambulatory treatment for 5 days by means of subcutaneous injections of granulocyte colony-stimulating factor (G-CSF, dose of 5 mg/kg/day), with the purpose of increasing the bone marrow EPC production and maturation. At the fifth day of treatment, blood apheresis was carried out for 3 hr (Cell Separator Machine CS3000 PLUS; Bayer, Leverkusen, Germany), obtaining a final volume of 50 mL per apheresis. The final product of each apheresis (50 mL) was analyzed for mononuclear cells, CD34 cells, CD3 cells, natural killer (NK) granulocytes, and platelets using an automated cell counter (Advia 120, Bayer). The viability of mononuclear cells was studied by trypan blue-dye exclusion. We performed flow-cytometric analysis, using anti-CD33 fluorescein isothiocyanate and anti-CD34 phycoerythrin on a flow cytometer (FACScan; Becton Dickinson, Mountain View, CA). The total volume of the inoculum (50 mL) was divided into aliquots of 2 mL and implanted by intramuscular injections 24 hr after apheresis, under epidural anesthesia and surgical aseptic conditions. Injection sites were selected according to angiographic findings, including gastrocnemius, soleus, and anterior tibial muscles, as well as the sole muscles of the foot. Ankle segmental pressures measured with Doppler, ABI, VAPS, and PPG at the sixth week postprocedure and every 3 months in the first year were carried out during the follow-up. Angiography was done in selected cases. Statistical analysis was conducted using SPSS for Windows, version 13.0 (SPSS, Inc., Chicago, IL). Data were expressed as mean ± standard deviation. Statistically significant differences between measures were considered at p < 0.05. Kaplan-Meier analysis was developed to assess limb salvage rate after 1year follow-up.
RESULTS Between January 2002 and July 2008, more than 350 patients with CLI were treated in our institution. We included 28 patients (21 men and seven women) with the established approaches (Table I). In two patients both legs were treated (30 legs). Mean age was 67 years. Mean follow-up was 14.7 ± 5.6 months (range 1-48). All patients had rest pain, and 85% (n ¼ 24) were associated with trophic ulcers at the digital level or in the heel of
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Table I. Inclusion/exclusion criteria and patient characteristics Inclusion criteria
Exclusion criteria Patients/legs Mean age (range, years) Sex (male/female) Clinical presentation CLI Grade III Grade IV Comorbidity Diabetes mellitas Chronic ischemic cardiopathy Chronic renal failure
CLI stage III or IV Buerger diagnosis ABI <0.6 Not candidates for revascularization Limited vital expectancy Active neoplastic disorder 28/30 67 (22-79) 21/7 30 6 24 15 6 4
the foot. Two had a clinical diagnosis of thromboangiitis obliterans (Buerger disease, TO), and 15 had diabetes. Four of the diabetic patients had segmental pressures at the maleolar level >250 mm Hg (Mo¨nkenberg calcinosis). These pressures were not included when statistical analysis was done for perfusion improvement. Major amputation was done in seven patients, in a median of 30 days (range 7161). In three cases, the amputation was done because of uncontrolled pain despite medical treatment. Kaplan-Meier analysis showed a 74.4% limb salvage rate after 18-month follow-up (Fig. 1). The median time to achieve clinical improvement was 51 days (range 34-68). Five patients died during the study due to cardiovascular disease, all of them with major amputation. Mean segmental pressures increased in the first year posttreatment. This finding was correlated with an improvement in ABI (mean measures): before treatment 0.35 ± 0.2 (range 0.05-0.6) and 18-month follow-up 0.72 ± 0.51 (range 0.1-0.8) ( p ¼ 0.01). No differences between patients with distal pressures >250 mm Hg were observed after treatment. There was an improvement from the layout PPG in 80% of patients after 1-year followup, with the exception of those who required forefoot or major amputation (Table II). Rest pain was determined by means of VAPS. All patients were interrogated previous to treatment and during follow-up. All patients with rest pain reported improvement, stressing that this improvement was accented starting from the second month after the cellular implantation. After 1-year
follow-up, 18 patients required occasional analgesic treatment (no opioids), and in no case did the pain impede night rest. These data were correlated with a five-point decrease in the mean score obtained by VAPS: before treatment 8.7 ± 1 points and 1year follow-up 3.8 ± 2.9 points ( p ¼ 0.01). Only three patients did not report improvement in rest pain and suffered major amputation. In the group of patients with ulcers, 80% showed improvement in their lesions. Some type of minor amputation was necessary in three patients (one transmetatarsal, two digital) because of infected trophic lesions. Only in the first six patients was angiography performed at 1 year posttreatment. There seemed to be an increase in the collateral network, but a strong subjectivity factor was thought to influence the evaluation of the images (Fig. 2). We saw a unique case of high blood levels of liver enzymes after the cellular mobilization. It was normalized after 2 weeks. The patient did not have previous liver disease; viral serum tests were negative previous to the inclusion in the study and after the implantation. No other adverse effects were observed during the mobilization, the process of cellular apheresis, or the cellular infusion technique. We analyzed the cellular content of the inoculum in relationship with clinical outcome (improvement of rest pain, healing of ulcer, resting ABI, digital PPG, and limb salvage). We did not find a significant statistical influence between the number of CD34+ cells, CD3+ cells, NK cells, mononuclear cells, and granulocytes infused and clinical outcome.
DISCUSSION The results of our study suggest that intramuscular implantation of EPCs obtained from peripheral blood (PB), improves tissue perfusion in CLI of the lower limbs, as demonstrated by the increase in segmental pressures, ABI, and the healing of the trophic lesions. It seems also to relieve rest pain. Moreover, the patients who responded to treatment were able to walk a reasonable distance without claudication. Likewise with high limb salvage rates after 1-year follow-up, this could be considered a novel treatment that should be considered in cases with no surgical or endovascular revascularizing options. Different studies have demonstrated the capacity to form new vessels by administration of different growth factors. However, this capacity is limited by an effectiveness-dependent dose and the possibility for developing neoformative processes because
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Fig. 1. Failure-free survival. A Overal limb salvage (OLS). B OLS in function of clinical stage CLI (Leriche-La Fontaine classification).
many of these factors are involved in tumoral angiogenesis.10-15 Different cell lines existing inside the bone marrow maintain the ability to develop into the different cellular components of the arterial wall and, therefore, to be able to give functionality to the newly formed vessels.16-19 EPCs constitute a small group inside the mononucleated cells
(0.05%), which present angioblastic capacity for endothelial differentiation, and they circulate in the PB. Since the discovery of EPCs in adult bone marrow, it has been known that the increase of their formation, maturation, and liberation from the bone marrow to PB is an inducible process (Fig. 3).20 Some studies have indicated that EPC
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Table II. Clinical and hemodynamic behavior of patients after stem cell infusion Posttransplant
Clinical No symptoms Rest pain Ulcer Amputation Exitus Hemodynamic ABI Plane PPG1 Normal PPG Legs
Pretransplant
1 month
d 6 24 d d
9 1 14 6 d
0.35 ± 0.28 30 d 30
0.44 ± 0.24 25 5 30
circulating levels constitute a functionality marker of the vascular system in such a way that even diminished EPC circulating levels are related to a decreased endothelial repair capacity and, therefore, an increased progression of arterial disease.21,22 Moreover, others have identified some alterations in EPC circulating levels and its capacity for diferentiation in atherosclerotic and diabetic patients.23 In different animal models of CLI, administration of peripheral mononucleated cells is able to improve limb perfusion, diminishing the amputation rate.24-27 Tateishi-Yuyama et al.28 carried out the first study in patients with CLI, where the utility of mononucleated cells extracted from PB was compared with that of cells directly extracted from bone marrow. Satisfactory results were shown with both groups compared to the group given placebo. Others have verified this fact,29-31 and moreover, it is possible to obtain EPCs directly from PB without the necessity of carrying out direct punctures for bone marrow.32-34 Good results have been obtained using bone marrow or PB cells, and both methods present advantages and inconveniences; when using PB cells, the main limitation is the time invested in the mobilization and cell populations gathering, as well as the time necessary to obtain results (6-8 weeks). Anyway, and in accordance with the results published by other groups that support our work method, we do not feel justified in subjecting the patient to unnecessarily invasive processes that are not exempt from complications. Mainly two forms of administration have been described: intramuscular injection28,29,31-35 and intra-arterial direct puncture,36,37 the latter being the one used more frequently by all groups and by us. Using this delivery system, we can ensure that EPCs will home into the ischemic tissue. This study, like others, opens a debate to explain the mechanism of neovascularization observed when the cells
p
6 months
p
14 0 7 1 2 0.08
0.50 ± 0.2 18 6 24
18 months
p
18 0 0 d 3 0.02
0.72 ± 0.51 4 17 21
0.009
are implanted. The conclusion is that it is probable that the effect of neovascularization resides both in accessory cells, which secrete cytokines and growth factors, and in the infused hematopoietic stem cells that differentiate in EPCs. Regarding the methods to evaluate the effectiveness of the treatment, one of them could be the use of quality-of-life questionnaires.38 In our work, we did not believe it to be necessary. The primary objective was to determine the effect of the treatment in the trophic limb and, secondly, to determine if that improvement was related with the decrease in analgesic need and with improvement of walking distance. It is certain that it can be a work tool that guides the doctor to control the evolution of the treatment, but we do not think of it as a definitive test of effectiveness. On the other hand, VAPS has been widely used as a good test to evaluate the results of procedures done in patients with cronic pain.39 It could be considered a limitation of our study that we used angiography to determine the increase of the collateral circulation since this technique is known to be limited when evaluating small-caliber vessels (<1 mm diameter)40 and there is observer subjectivity when determining the degree of increase of the collateral circulation. Other techniques have been used (e.g., magnetic resonance), but we did not have this option available in our institution. Serious limitations should be noted: first, the lack of a control group to compare with; second, the small cohort obtained. In this sense, we did a pilot study with the first 10 patients, comparing the results with a historical cohort treated in our institution. The results showed that, in the historical cohort, >90% of the patients treated medically suffered major amputation in less than 1 year. This
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Fig. 2. Angiography. A1, A2 Before transplantation. B1, B2 Posttransplantation with development of new collateral vessels.
could be discussed in terms of statistical significance, but the fact is that there was a difference in terms of limb salvage rate after 1-year follow-up. On the other hand, the reduced number of patients included should indicate that the inclusion criteria were extremely strict, with revascularization attempted as the first option in all patients treated in our institution. The discovery of EPCs in the adult and their functionality inside the vascular system have have led to the study of their possible applications in arterial pathology. The good results obtained in recent
published works31,35 and the use of autologous patient cells make this a new treatment to consider in the future. In accordance with our results, we think that the use of EPCs mobilized from bone marrow and their later implantation into ischemic limbs by intramuscular injection improves tissue perfusion and constitutes a valid and novel treatment that should be considered in selected patients with CLI and no revascularizing options. The main limitations of our study have been discussed, and a randomized study with a higher number of cases that may confirm our results would be necessary.
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Basics on Angiogenesis Bone Marow M ro Ma r w qui qu iesce escen nt quiescent
MMP-9
EPC
+
eNOS
Vascular System mobilization (Peripheral blood)
Bone Marrow proliferative
Ischemic Tissue Infarction
New vessels recruitment
Trauma ↓ VEGF, GM-CSF EPO
Zammaretti P, Zisch AH. Adult endothelial progenitor cells. Renewing Vasculature. Int J Biochem Cell Biol 2005 ; 37 : 493-503
MMP-9 : metalo-proteinase 9, eNOS : nitric oxide synthetase, EPC : endotelial progenitor cell, GM-CSF: Granulo-monocyte colony stimulating factor, VEGF: vascular endotelial growth factor, EPO: eritropoietin Fig. 3. Basic mechanism in angiogenesis. MMP-9, metalloproteinase 9; eNOS, endothelial nitric oxide synthetase; EPC, endotelial progenitor cell; GM-CSF, granulocyte
colony-stimulating factor; VEGF, vascular endothelial growth factor; EPO, erythropoietin.
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