By Chochola, M Pytlik, R; Kobylka, P; Skalicka, L; Kideryova, L; Beran, S; Varejka, P; Jirat, S; Koivanek, J; Aschermann, M; Linhart, A
Aim. The injection of bone marrow mononuclear cells (BMMC) into the gastrocnemius muscle has given promising results in patients with critical limb ischemia (CLI). In this article, we have assessed whether a less invasive procedure, i.e. intravascular BMMC infusion, could be effective in this population of patients. Methods. A total of 28 limbs in 24 patients with CLI were treated. An amount of 276- 700 mL of marrow blood was harvested from posterior iliac crests and BMMC were obtained by standard procedure used for bone marrow transplantation. After performance of digital subtraction angiography, BMMC were injected laterally through a 4 Fr sheet. Primary outcome was efficacy of the procedure measured as heating of defects, frequency of high amputations and change of ischemia grade; among secondary outcomes were safety of the procedure, angiographic changes and changes in quality of life.
Results. One year after treatment, all patients were alive and only 2 patients have undergone high amputation. Eleven of 14 defects have healed (78%) and Fontaine grade of ischemia has changed from median grade 3.5 to median grade 2 (P
Conclusion. Intra-arterial infusion of BMMC can lead to significant and long-lasting subjective and objective improvements in patients with CLI. The results merit validation by randomized controlled studies in patients with less critical limb ischemia.
[Int Angiol 2008;27:281-90]
Key words: Extremities – Ischemia – Neovascularization, physiologic – Bone marrow – Quality of life.
The term chronic critical limb ischemia (CLI) has been used in patients with chronic ischemic pain necessitating a regular administration of analgesics for longer than 2 weeks, with cutaneous defects and distal necrosis due to proven obliterative artery disease and with a high probability of high limb amputation within 6- 12 months if the limb perfusion would not improve. Objective criteria include ankle blood pressures
The accurate incidence of CLI is not known. Worldwide, it is estimated to be about 0.5 million to 1 million new cases every year. The prognosis of patients with CLI is associated with high mortality and is comparable to patients with advanced malignant disease.3 While 5-year mortality of patients with ischemic leg disease with claudications is approximately 30%, in patients with CLI it is up to 50%.4 The principal causes of death in these patients are other cardiovascular diseases (myocardial infarction or stroke).5
This grave prognosis in patients with CLI has prompted the need for new therapeutic modalities, which would lead to limb salvage, a reduction of morbidity and mortality and improvement in their quality of life. The most successful therapeutic strategies are improvement of limb perfusion with surgical or percutaneous revascularization. However, patients with angiographic findings which does not permit either of these treatment modalities are the subjects of conservative treatment attempted at halting or slowing the progression of the disease, the improvement of local and general circulation, pain relief and on the healing of eventual defects. In approximately 20-30% of patients no treatment is effective and amputation is performed.
In preclinical experiments, administration of angiogenic precursor cells into the ischemic limb leads to improved perfusion and higher rate of limb salvage.6,7 Therefore, this method could become an alternative to the conservative treatment of patients with CLI. Several case reports and phase I-II clinical trials seemed to bring promising results with mononuclear bone marrow cells being mostly used as source of angiogenic precursors.8-10 However, the administration of these cells has been usually intramuscular, which could lead to the short-term aggravation of pain and potential risk of infection in these highly susceptible locations. Therefore, we have performed a feasibility and efficacy pilot study of intra- arterial infusion of bone marrow in patients with CLI who were either not eligible for surgical or percutaneous revascularization, refused it, or the revascularization attempt had failed.
Materials and methods
Patients eligibility
Patients 18-85 years old were eligible for the study if they had grade III or IV leg ischemia according to Fontaine classification, with or without defects and without possibility of surgical or transcutaneous revascularization. Patients had to be able to undergo a surgical procedure under general or epidural anesthesia and all the patients had signed informed consent according to Helsinki declaration. The study was approved by the local ethical committee.
Exclusion criteria were: critical stenosis of coronary artery or unstable angina pectoris; previous malignant disease treated with chemotherapy or pelvic radiotherapy; and other uncontrolled medical disorders. For purposes of the endothelial progenitor assay, the patients should have not undergone treatment with statins for the previous 1 month.
Pretreatment and post-treatment evaluation procedures
DIGITAL SUBTRACTION ANGIOGRAPHY
Digital subtraction angiography (DSA) was performed by retrograde puncture of the femoral artery in the involved leg. The site of the puncture was infiltrated with 10 mL of 1-2% Mesocain. For DSA, iodine contrast (Iomeron, Bracco, Germany) was applied through 4 Fr sheet with constant speed of 10 mL/min. Documentation was performed in at least 6 segments of the limb, with speed of 1-3 images/s, to document both the arterial and venous phase of angiography. DSA was performed as part of the evaluation procedures before and after implantation of marrow mononuclear cells and after 1 year. Evaluation of the development of collateral vessels was performed in a blinded fashion by two independent angiologists on a semiquantitative scale, where the worsening of angiography was scored as -1, no change as 0, slight improvement as 1 and significant improvement as 2. Evaluation was performed both on positive and negative images.
ANKLE-ARM INDEX MEASUREMENT
Ankle-arm index (AAI) was performed according to Rutherford et al.11 For measurements, an 8 MHz Doppler probe was used to localize the pulse signal at the posterior tibial artery or dorsalis pedis. The cuff was placed on the distal thigh of patients lying in the supine position. The measured pressure was compared with the pressure measured at the brachial artery. AAI measurements were performed before mononuclear cell implantation, after 6 and after 12 months.
TRANSCUTANEOUS OXYGEN PRESSURE MEASUREMENT
Transcutaneous oxygen pressure measurement (tcpO^sub 2^) was performed on the dorsum of the involved foot between 1st and 2nd metatarsal.12 This non-invasive method indirectly evaluates skin perfusion. This measurement was performed with Clark’s probe, working on polarographic principle and measuring the partial pressure of oxygen diffusing through skin. The TCM400 Mk2 (Radiometer Copenhagen, Copenhagen, Denmark) was used for all measurements.
QUALITY OF LIFE OUTCOMES
Quality of life (QoL) was prospectively measured with the SF-36 questionnaire. This is the most extensively utilized tool for QoL assessment, is not specific for any given disease and has been successfully used in many diseases, including cardiovascular diseases and ischemic leg disease. The questionnaire was filled in before cell implantation and at 1 year after the implantation. The questionnaire consists of 36 questions aggregated into 8 domains (1. Physical functioning; 2. Role-physical; 3. Role-emotional; 4. Social functioning; 5. Bodily pain; 6. Mental health; 7. Vitality; 8. General health), which then can be used for calculation of two composite scores (physical composite score [PCS] and mental composite score [MCS]). The questionnaire was evaluated according to appropriate instructions13 and physical and MCSs were calculated with the online NBS calculator (www.sf-36.org).
OTHER EXAMINATIONS
Ischemic defects in all patients were photo-documented before treatment and then at 6-month intervals. Usual preoperation blood tests, electrocardiogram (ECG), blood sugar and lipid measurement were performed before stem cell harvest. In the afternoon and on the day after harvest, ECG, blood count and set of cardiology enzymes to detect possible myocardial damage were performed. Treadmill exercises were not performed, because more than 50% of patients had either ischemic pain at rest or foot defects making the pretreatment test impossible.
Treatment
BONE MARROW MONONUCLEAR CELL HARVEST AND PROCESSING
Bone marrow cell harvest was performed under standard aseptic conditions under general or epidural anesthesia. Approximately 350- 400 mL of bone marrow blood in 3-4 mL portions was harvested from one or more skin punctures from one or both posterior iliac crests with standard single-use needle for bone marrow harvest (Somatex, Teltow, Germany). The marrow blood clotting was prevented with the normal saline-heparin solution. The marrow blood was collected in the Bone Marrow Collection Kit with Pre-Filter and Inline Filters (Baxter, Deerfield, IL, USA) which have indwelling filters for removal of large marrow particles. After the filtration of the blood from the collection bag to the transport and to the processing bag, the blood was transferred onto the Cell Therapy Unit of the Institute of Hematology and Blood Transfusion for further processing. The appropriate amount of Gelofusin (B. Braun, Melsungen, Germany) was added directly to the processing bag and repeated red cell sedimentation was performed according to the standard operational procedure for approximately 2 h. The supernatant plasma, containing mainly nuclear cells and only a minimum amount of erythrocytes, was segregated by plasma-extractor and centrifuged again. Clear plasma was then transferred back to the sedimentation bag and the process was repeated. Then, the resulting mononuclear cell fraction was resuspended in approximately 30 mL of autologous plasma and prepared for intra-arterial infusion on the same day. Neither autologous nor allogeneic red cell transfusions were given routinely and the volume balance was kept with crystalloid solutions. EVALUATION OF STEM CELL CONTENTT IN THE GRAFT AND OF CIRCULATING ENDOTHELIAL PRECURSOR CELLS
For stem cell content of the graft evaluation, CD34 measurements were performed according to accepted standards.14,15 Colony-forming units, granulocyte-macrophage (CFU-GM) and burstforming units, erythroid (BFU-E) cultivations were performed on MethoCult(TM) (StemCell Technologies, Vancouver, Canada) according to the recommendation of the manufacturer. Briefly, 2×10^sup 4^ bone marrow mononuclear cells (BMMC) after hydroxyethylstarch sedimentation was seeded in 1 mL of complete MethoCult(TM) media in 35 mm Petri dishes. Each experiment was run in triplicate. Before placing in the incubator, the 35 mm Petri dishes were placed in larger 100 mm dishes to decrease the possibility of contamination and to allow for keeping the humidity. CFU-GM and BFU-E were then counted under inverted microscope according to their typical morphology.
BONE MARROW MONONUCLEAR CELL IMPLANTATION
Approximately 4 h after stem cell harvest, DSA was performed. After DSA, mononuclear cells were infused intra-arterially via lateral access into a 4 Fr sheet in a rate 900 mL/hod. Control angiography was performed after cell implantation. The patient was then monitored in the Angiology Intensive Care Unit, where the sheet was removed and puncture site compressed. The patient was allowed to walk after 8 h. After control examinations, the patient was discharged the day after the procedure.
Follow-up
Patients were regularly followed every 6 months after the procedure or as needed. Trophical defects were treated according to institutional guidelines.
Definition of outcomes
The primary endpoint was efficacy of the procedure evaluated 1 year after treatment. Measured outcomes were a change of ischemia grade according to Fontaine classification, healing of trophic defects and frequency of treatment failures, defined as high amputation, major cardiovascular accident (stroke or myocardial infarction) and death from any cause. Minor surgery for removal of necrotic defects already present before treatment was not considered as treatment failure.
Secondary endpoints were safety of the procedure, improvements in collateral vessel development assessed on angiographie measurements, improvements in blood perfusion of the treated limb, assessed as AAI and tcpO^sub 2^ measurement, and improvement of quality of life outcomes.
Statistical analysis
For comparison of continuous variables, a Mann-Whitney U-test was used for comparison of two non-paired samples and a paired t-test for two paired samples. For comparison of multiple samples containing continuous variables, a Kruskal-Wallis version of ANOVA test was performed with post-test calculation for trend. Correlations were calculated with Spearman correlation coefficient. P
Results
Patient characteristics
From 1 September 2004 to 31 December 2006, we performed 28 autologous intra-arterial infusions of BMMC in 24 patients. Median age was 68 years (range: 26-85 years), 13 patients were males and 11 females. In 4 patients, two procedures were performed and BMMC were infused to both legs in 5-13 month intervals between the two procedures. According to the Fontaine classification, grade III ischemia was present in 14 legs and grade IV ischemia in 14 legs. Fourteen patients (50%) had ischemic defects. In 21 patients (88%), the cause of arterial obliteration was atherosclerosis, while thromboangiitis obliterans was diagnosed in 3 patients (12%). Other characteristics are shown in Table I.
Bone marrow harvest
Median 400 mL (range: 276-700 mL) of mixture of bone marrow blood and heparin in normal saline was obtained in 28 procedures. Median number of 64.3 x 10^sup 8^ BMMC were harvested (range: 13.2-131.1 x 10^sup 8^). After processing, median numher of 34.9×10^sup 6^ (range: 3.96-135.4×10^sup 6^) CD34+ cells, 873 CFU-GM (range: 174-3 489) and 551 (128-2 512) BFU-E was infused. Other details of bone marrow harvest are shown in Table II. Of the pretreatment characteristics, we have found significant negative correlation between age and number of CD34+ cells (r=-0.41; P=0.04) and between age and number of BFU-E (r=-0.44; P=0.03). Also, a lower number of infused CD34+ cells was found in patients with concomitant ischemic heart disease (median: 27×10^sup 6^ vs 53×10^sup 6^ without ischemic heart disease; P=0.019) and a lower percentage (but not lower total number) of CD34+ cells was noted in patients with hyperlipidemia (0.64% vs 0.77% in patients without hyperlipidemia; P=0.03).
Treatment outcomes
PRIMARY OUTCOMES
In 1 year post-treatment, all patients were alive. Two of them had undergone high amputation of the treated limb at 3 months after treatment, both of them having Fontaine stage IV disease at the entry of the study. There were no cerebrovascular accidents. No patient had undergone high amputation beyond 1 year. One patient had died 2 years after the study entry from unrelated cause (after femoral neck fracture). The 1-year event free survival is 92%. Three patients had undergone minor surgical procedures to remove the ischemie tissue which was already present before treatment. Otherwise, 11 of 14 defects had healed in 1 year (78%, Figure 1).
According to Fontaine classification, the grade of ischemia had improved from a median value 3.5 to median value 2 (P
SECONDARY OUTCOMES
Safety.-The procedure was extremely well tolerated. There was nonsignificant decrease in hemoglobin levels 24 h after bone marrow harvest with no aggravation in ischemic symptoms. No red cell transfusions had to be administered. Patients were discharged at a median of 1 day (range: 1-3 days) after procedure. Only one episode of grade II bleeding developed from the site of the harvest, which was controlled by compression.
Angiography results.-One year after stem cell infusion, the mean improvement of angiographic findings in the treated extremity was 0.732 points in the site of puncture, 0.652 points in leg, 0.692 points in knee, 1.13 points in the calf and 1.3 points in the foot, measured on a semiquantitative scale as described above. There was a significant trend towards higher scoring in distal parts of the extremity (P=0.0001 on repeated measures ANOVA, test for trends). These results are shown in Figure 2. Representative pictures of pretreatment and posttreatment angiography are shown in Figure 3. However, there were no significant correlations between improvement in any of the measured sites and number of BMMC1 CD34+ cells, CFU- GM or BFU-E, nor there were any correlations between angiographic results and other objective measures of leg ischemia (i.e., AAI and tcpO2). We were also unable to find any pretreatment characteristics correlating with the changes in the angiographie findings. The Fontaine score did not correlate with the score of any specific site. However, if the mean value was calculated for all angiographie changes in a given patient, there was a significant correlation both between this mean value and the pretreatment Fontaine score (r=- 0.41; P=0.048) and between the mean value and post-treatment Fontaine score (r=-0.43; P=0.038).
Ankle-arm index and transcutaneous oxygen pressure measurement.- Both AAI and tcpO^sub 2^ increased substantially 1 year after treatment (P
Quality of life.-After treatment, there was significant improvement in all 8 domains of the SF36 questionnaire when compared with the pretreatment values (P0.95; P
The apparent success of preclinical studies of stem-cell induced angiogenesis has prompted the rapid implementation of this research into clinical practice. As most of the preclinical research has been performed on ischemic heart disease models and some of these trials implied that not only the coronary vessels, but also myocardium, could be repaired with stem cells,16,17 several well-designed randomized trials of cellular treatment of myocardial infarction or chronic ischemic heart disease have been conducted. Today, when the results of these trials have become available, it is clear that despite of statistically significant improvement in various clinical or laboratory outcomes in most of the studies, the clinical benefit is only short-term or of uncertain significance.18-23
Given the fact that skeletal muscle is much more resistant to hypoxia than cardiac muscle, it is interesting that only a few randomized trials of cellular treatment of ischemic leg disease are presently available. It is even more surprising given the fact that results of all of the published pilot studies or case series have been very optimistic.80-10, 24-34 However, most of these trials enrolled only a small number of patients and a publication bias cannot be excluded.
Only two randomized studies have been published so far. Tateishi- Yuyama et al. enrolled 47 patients with CLI and 22 of them with bilateral leg ischemia were randomly injected with BMMC in one leg and peripheral blood-mononuclear cells in the other. After both 4 and 24 weeks, there was a significant improvement of the AAI, transcutaneous oxygen pressure and leg pain in legs injected with the bone marrow cells.8 More recently, Huang et al. randomized 28 diabetic patients with CLI to a “transplant” group, which have received treatment with G-CSF mobilized peripheral progenitor cells and to a control group who had received a conservative treatment.31 Three months after treatment, laser Doppler blood perfusion and AAI were significantly improved in the “transplant” group and more limb ulcers were healed (14 of 18 vs. 7 of 18; P=0.016). Otherwise, we have identified only three other studies published in English which accrued more than 20 patients, two of them exclusively treating patients with Buerger’s disease.32-34
We have treated 28 ischemic legs in 24 patients with intra- arterial infusion of BMMC and we have evaluated the results of treatment after 1 year. In other studies, intramuscular injections into gastrocnemius muscle were usually used, possibly because of concern that in limbs with occluded vessels, the angiogenic cells might not reach the periphery and the results were typically evaluated early (3-6 months) after treatment. We are aware of one case series which have used intraarterial infusion of G-CSF mobilized peripheral blood mononuclear cells with results similar to our own.35 However, we have preferred BMMC to G-CSF mobilized peripheral blood progenitor cells mainly because G-CSF treatment may induce hypercoagulable state by increasing levels of FVIII:C and thrombin,36 and its use was accompanied with aggravation of ischemic syndromes in patients with known coronary disease,37 a high risk of in-stent restenosis,38 and even with major arterial thromboses in patients without previously diagnosed atherosclerosis.39 In addition, BMMC differ in their content from the mobilized peripheral blood progenitor cells and even the CD34+ cells mobilized to peripheral circulation have a different quality. As we have found before that the granulocyte-monocytic colonies (CFU-GM), and especially erythroid burst colonies (BFU-E) produce angiogenic factors,40 we considered bone marrow blood as a more appropriate source of cells for cellular-induced vasculogenesis. The fact that we did not find any correlations between the graft content of CD34+ cells or colony forming cells and outcome in this study, is not surprising in view of the predominating opinion that injected mononuclear cells are rather vascular factors producing cells than endothelial precursors,41 and the cytokine and chemokine network needed to induce angiogenesis is extremely complex.
To our knowledge, this is the first study of cellinduced angiogenesis utilizing the complex quality of life assessment tool to study the treatment impact on this outcome. The SF-36 questionnaire had been used in a variety of diseases including ischemie leg disease and it has unequivocally shown that quality of life in these patients deteriorates with the grade of ischemia.42 It has also been shown that a successful revascularization procedure can improve quality of life in many of these patients.43,44 Therefore, it is extremely important that our treatment had been able to improve quality of life of patients, where further revascularization cannot be performed.
Conclusions
In conclusion, our study contributed to the growing evidence of the effectivity of cell-mediated angiogenic treatment in patients with peripheral arterial disease. It has also shown that the benefit of the treatment lasts beyond 3 or 6 months, which had not been adequately assessed in previous studies. As the tolerability of the procedure by these critically ill patients was excellent and no significant adverse events have occurred, we suggest it might be justified to perform controlled studies also in patients with less critical ischemia, i.e. in the stage of claudications.
Fundings.-This work has been sponsored exclusively with the grant from Ministry of Public Health, Czech Republic (IGA MZCR NR-8047/ 3).
Acknowledgements.-Special thanks to Thomas O’Hearn, II., for the language revision of the manuscript.
Received on March 6, 2007; accepted for publication on June 15, 2007.
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M. CHOCHOLA1, R. PYTLIK 2, P. KOBYLKA 3, L. SKALICKA 1, L. KIDERYOVA 2,
S. BERAN 1, P. VAREJKA 1, S. JIRAT 1, J. KOIVANEK4, M. ASCHERMANN 1, A. LINHART 1
1 2nd Department of Medicine, Cardiology and Angiology, General University Hospital, Prague, Czech Republic
2 Laboratory of Experimental Cellular Therapy, 1st Department of Medicine-Hematooncology,
General University Hospital, Prague, Czech Republic
3 Institute of Hematology and Blood Transfusion, Prague, Czech Republic
4 Department of Radiology, General University Hospital, Prague, Czech Republic
Address reprint requests to: M. Chochola, 2nd Department of Medicine, Cardiology and Angiology, General University Hospital, U nemocnice 2, 128 08 Praha 2, Czech Republic.
E-mail [email protected]
Copyright Edizioni Minerva Medica Aug 2008
(c) 2008 International Angiology. Provided by ProQuest LLC. All rights Reserved.
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