Cell Transplantation, Vol. 21, pp. 1909–1918, 2012 Printed in the USA. All rights reserved. Copyright 2012 Cognizant Comm. Corp.
0963-6897/12 $90.00 + .00 DOI: http://dx.doi.org/10.3727/096368912X636948 E-ISSN 1555-3892 www.cognizantcommunication.com
No Difference in Intra-Arterial and Intramuscular Delivery of Autologous Bone Marrow Cells in Patients With Advanced Critical Limb Ischemia Andrej Klepanec,*† Martin Mistrik,‡ Cestmir Altaner,§ Martina Valachovicova,* Ingrid Olejarova,† Roman Slysko,† Tibor Balazs,† Terezia Urlandova,† Daniela Hladikova,† Branislav Liska,† Jan Tomka,† Ivan Vulev,*† and Juraj Madaric*† *Slovak Medical University, Bratislava, Slovakia †National Cardiovascular Institute, Bratislava, Slovakia ‡Clinic of Haematology and Transfusiology, Faculty Hospital, Bratislava, Slovakia §Institute of Experimental Oncology, Slovak Academy of Science, Bratislava, Slovakia
Stem cell therapy has been proposed to be an alternative therapy in patients with critical limb ischemia (CLI), not eligible for endovascular or surgical revascularization. We compared the therapeutic effects of intramuscular (IM) and intra-arterial (IA) delivery of bone marrow cells (BMCs) and investigated the factors associated with therapeutic benefits. Forty-one patients (mean age, 66 ± 10 years; 35 males) with advanced CLI (Rutherford category, 5 and 6) not eligible for revascularization were randomized to treatment with 40 ml BMCs using local IM (n = 21) or selective IA infusion (n = 20). Primary endpoints were limb salvage and wound healing. Secondary endpoints were changes in transcutaneous oxygen pressure (tcpO2), quality-of-life questionnaire (EQ5D), ankle–brachial index (ABI), and pain scale (0–10). Patients with limb salvage and wound healing were considered to be responders to BMC therapy. At 6-month follow-up, overall limb salvage was 73% (27/37) and 10 subjects underwent major amputation. Four patients died unrelated to stem cell therapy. There was significant improvement in tcpO2 (15 ± 10 to 29 ± 13 mmHg, p < 0.001), pain scale (4.4 ± 2.6 to 0.9 ± 1.4, p < 0.001), and EQ5D (51 ± 15 to 70 ± 13, p < 0.001) and a significant decrease in the Rutherford category of CLI (5.0 ± 0.2 to 4.3 ± 1.6, p < 0.01). There were no differences among functional parameters in patients undergoing IM versus IA delivery. Responders (n = 27) were characterized by higher CD34+ cell counts in the bone marrow concentrate (CD34+ 29 ± 15×106 vs. 17 ± 12×106, p < 0.05) despite a similar number of total nucleated cells (4.3 ± 1.4×109 vs. 4.1 ± 1.2×109, p = 0.66) and by a lower level of C-reactive protein (18 ± 28 vs. 100 ± 96 mg/L, p < 0.05) as well as serum leukocytes (8.3 ± 2.1×109/L vs. 12.3 ± 4.5×109/L, p < 0.05) as compared with nonresponders (10 patients). Both IM and IA delivery of autologous stem cells are effective therapeutic strategies in patients with CLI. A higher concentration of CD34+ cells and a lower degree of inflammation are associated with better clinical therapeutic responses. Key words: Autologous stem cells; Critical limb ischemia; Angiogenesis; Intramuscular delivery; Intra-arterial delivery
INTRODUCTION Critical limb ischemia (CLI) is the end stage of peripheral arterial disease (PAD) characterized by ischemic rest pain, ulcers, or gangrene, with a significant risk of loss of the affected limb. The therapeutic options for patients with failed endovascular or surgical revascularization or in those in which these procedures cannot be carried out (20–30% of CLI patients) are very limited. About 40% of these high-risk patients will require amputation within 6 months of the initial diagnosis, whereas 20% will die (23). The quality of life (QoL) of these patients is poor and comparable to those with terminal cancer (1).
Several preclinical and clinical studies showed that delivery of autologous bone marrow cells (BMCs) can improve blood circulation and tissue perfusion and thus prevent amputation via the induction of collateral and capillary growth in a process called “therapeutic angiogenesis” (2,5,12,18,22,31,35). However, the optimal route of administration of cells remains unclear. Intra-arterial (IA) as well as intramuscular (IM) delivery methods have shown promising results in promoting neoangiogenesis (2,5,12,35), although direct comparison between both routes of BMC deli very is lacking. Accordingly, we conducted a randomized
Received June 13, 2011; final acceptance October 22, 2011. Online prepub date: April 2, 2012. Address correspondence to Juraj Madaric, M.D., Ph.D., Department of Cardiology and Angiology, National Cardiovascular Institute, Pod Krasnou horkou 1, 833 48 Bratislava, Slovakia. Tel: 00421259320276; E-mail: jurmad@hotmail.com
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clinical study to compare IM versus IA delivery of autol ogous BMCs in “no-options” patients with advanced CLI. To understand the underlying mechanism of the therapeutic effects of stem cells, we sought to address factors associated with therapeutic benefit in response to cell therapy. MATERIALS AND METHODS Patients Between October 2009 and August 2010, 41 patients (mean age, 66 ± 10 years; 35 males) with advanced CLI (Rutherford category, 5 or 6) after failed or impossible revascularization were randomized to application of 40 ml of bone marrow concentrate via the local IM route (n = 21) or via selective IA infusion (n = 20). Inclusion Criteria. (1) Patients over 18 years of age with ischemic skin lesions (ulcers or gangrene) with a CLI Rutherford category of 5 or 6 according to the TransAtlantic InterSociety Consensus (TASC) classification (minor or major tissue loss) (23). (2) CLI defined by ankle–brachial index ≤ 0.4, or ankle systolic pressure < 50 mmHg, or toe systolic pressure < 30 mmHg, or transcutaneous oxygen pressure (tcpO2) < 30 mmHg. (3) No option for endovascular or surgical revascularization assessed by a vascular surgeon and interventionalist. (4) Failed revascularization defined as no change of clinical status with the best standard care 4 weeks after endovascular or surgical revascularization. Exclusion Criteria. (1) Life expectancy < 6 months. (2) Evidence of malignancy during last 5 years. (3) Proliferative retinopathy. (4) Critical coronary artery disease or unstable angina pectoris. (5) End-stage kidney disease and patient on dialysis. (6) Bone marrow disease (e.g., myelodysplastic syndrome, severe anemia, leukopenia, and thrombocytopenia). Method of BMC Isolation. Isolation of stem cells was undertaken under analgosedation with propofol. A total of 240 ml of bone marrow from both posterior iliac crests was harvested using a standard disposable needle for bone marrow aspiration. Bone marrow aspirate was processed using a SmartPreP2 Bone Marrow Aspirate Concentrate System (Harvest, Plymouth, MA, USA), which uses gradient density centrifugation to provide 40 ml of bone marrow-rich product for all blood elements within 15 min. The biological potential of this system has been evaluated in a mouse model of hindlimb ischemia with similar or greater functional activity compared with the Ficoll isolation procedure as the current “gold standard” (10) and has also been tested in several trials in patients with CLI (2,25). Administration of Stem Cells. Immediately after the harvesting and centrifugation of stem cells, BMCs were administered by IM (group A) or IA (group B) methods.
In group A, 40 ml of BMCs were administered under analgosedation with propofol by deep injections with a 23-G needle into the muscles of the affected limb along the crural arteries, with each injection being ~1 ml. In group B, IA injection of 40 ml of BMCs was undertaken from a percutaneous retrograde contralateral femoral approach or antegrade femoral approach under local anesthesia at the site of arterial occlusion of the affected limb using a 4-F catheter at 800 ml/h. The duration of procedures in both groups was ~1 h. Preprocedure Assessment and Follow-up. All patients were examined before, 90 days, and 6 months after BMC delivery. Peripheral blood tests such as blood count and basal serological parameters (including C-reactive protein, CRP) were assessed. The total concentration of mononuclear cells (BMMCs/MNCs) and CD34+ cells in bone marrow concentrates was evaluated. Digital subtraction angiography (DSA) was undertaken 1 day before transplantation and 6 months after transplantation with strictly fixed parameters of the amount of contrast medium, constant speed of injection of contrast medium, catheter size, and position of catheter tip. Two experienced operators evaluated the development of new vessels in a blinded fashion by semiquantitative analyses as reported elsewhere (31). New collateral vessels were assessed as +0 (no development of collateral vessels), +1 (slight), +2 (moderate), or +3 (rich). Measurement of the resting ankle–brachial index (ABI) was done according to validated standards (27). It was calculated as the quotient of the highest ankle pressure and highest brachial systolic blood pressure (normal values, 0.95–1.2). Transcutaneous oxygen pressure (tcpO2) of the affected limb was assessed using a TCM400 Mk2 monitor (Radiometer Medical ApS, Copenhagen, Denmark). TcpO2 was measured at the forefoot in the supine position with an electrode temperature of 44°C. Wound characteristics were documented by digital photography. Wound healing was evaluated by two independent physicians. Pain scale was measured with a visual analog scale (VAS) graded form 0 to 10. Patients were discharged the day after the procedure on dual antiplatelet therapy (aspirin and clopidogrel) and statin therapy. All patients received conventional wound care during follow-up. QoL before, 3 months, and 6 months after BMC application was assessed by the EuroQol questionnaire (6). Using a VAS, patients rated their overall health status from 0 (“worst”) to 100 (“best”) imaginable health. Endpoints. The primary endpoint was limb salvage and improvement in wound healing within 6-month follow-up. Patients with limb salvage and wound healing were considered to be responders to BMC therapy. Secondary
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endpoints were change in tcpO2, Rutherford category, QoL, and pain VAS after BMC transplantation. The study design was approved by the local ethical committee of National Cardiovascular Institute, Bratislava. All included patients were informed about the nature of the study and gave their written informed consent. Statistical Analyses Data evaluation was undertaken using a statistical software package SPSS 13.0 (SPSS Inc., Chicago, IL, USA). Discrete variables are presented as counts and percentages. Continuous variables are presented as mean values ± SD. Gaussian distributions of data were tested by the Kolmogorov–Smirnov test. The paired t test was used to compare values before and after BMC transplantation. The frequencies of categorical variables were compared using Fisher‘s exact test. Mean values for continuous parameters were compared using the Student‘s t test and Mann–Whitney test as appropriate. Multivariate logistic regression analysis (binary logistic regression) was used to study predictors of clinical benefit after BMC application. For all analyses, p < 0.05 was considered significant. RESULTS Baseline Characteristics and Overall Results The baseline characteristics of both groups are given in Table 1. In all 41 patients, the underlying cause of obstructive arterial disease was atherosclerosis. During
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follow-up, four patients died (two due to heart failure, one due to myocardial infarction, and one as a result of pneumonia). At 6-month follow-up, the combined primary endpoint of limb salvage and wound healing was met in 27 of the surviving 37 patients (73%). However, in 10 patients, major amputation was required due to CLI progression. Importantly, most cases of amputation (7/10) happened during the first month after BMC delivery. Table 2 shows the functional results of BMC application in patients with limb salvage at 6 months in all patients and that in the IA group and IM group. There was significant improvement in wound healing, tcpO2, pain scoring, and QoL, as well as in CLI Rutherford category at 3-month follow-up, and the results were sustained or even more pronounced at 6 months (Figs. 1 and 2). DSA did not reveal detectable development of new collateral vessels after 6 months compared with baseline angiograms (grades 0 to +3: 0.21 ± 0.43) (Fig. 3). Intramuscular Versus Intra-Arterial Application Table 3 shows a comparison of functional outcomes after IM and IA delivery of BMCs in patients with limb salvage. Both procedures were well tolerated without periprocedural complications. There were no differences in IM versus IA application in either endpoint. The prevalence of limb salvage at 6 months was 72% in the IM group compared with 74% in the IA group ( p = 0.94). Similarly, wound healing was observed in 13 patients (72%) in the IM group compared with 14 patients (74%)
Table 1. Baseline Characteristics of Patients
Age (years) Sex (males) Diabetes mellitus Arterial hypertension Hyperlipidemia BMI LVEF (%) Smoking Rutherford category Creatinine (µmol/L) CRP (mg/L) Leu (109/L) Fbg (g/L) BMMC count (109 cells) CD34+ count (106 cells) Previous PTA/surgery Previous CABG/PCI Post-MI
All Patients (n = 41)
Group A (IM, n = 21)
Group B (IA, n = 20)
p (IM vs. IA)
66 ± 10 35 (85%) 28 (68%) 33 (80%) 21 (51%) 28 ± 4 56 ± 8 17 (41%) 5.0 ± 0.2 94 ± 49 38 ± 60 9.2 ± 3.3 4.1 ± 0.9 4.2 ± 1.4 26 ± 14 29 (71%) 6 (15%) 13 (32%)
66 ± 10 17 (81%) 15 (71%) 17 (81%) 13 (62%) 29 ± 4 60 ± 9 9 (43%) 5.0 99 ± 66 44 ± 73 9.2 ± 3.3 3.9 ± 1.0 4.4 ± 1.5 28 ± 17 14 (67%) 3 (14%) 6 (29%)
66 ± 11 18 (90%) 13 (65%) 16 (80%) 8 (40%) 26 ± 4 55 ± 7 8 (40%) 5.1 ± 0.3 88 ± 19 31 ± 42 9.2 ± 3.5 4.3 ± 0.7 4.0 ± 1.3 23 ± 11 15 (75%) 3 (15%) 7 (35%)
0.86 0.41 0.67 0.62 0.96 0.06 0.43 0.85 0.16 0.47 0.50 0.99 0.11 0.45 0.30 0.73 1.0 0.74
BMI, body mass index; BMMC, bone marrow mononuclear cells; CABG, coronary artery bypass grafting; CRP, C-reactive protein; Fbg, fibrinogen; IA, intra-arterial; IM, intramuscular; Leu, leukocyte level in peripheral blood; LVEF, left ventriclar ejection fraction; MI, myocardial infarction; PCI, percutaneous coronary intervention.
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Table 2. Outcomes of BMC Delivery After 6 Months in Patients With Limb Salvage Overall Group (n = 27) tcpO2 (mmHg) ABIa Pain scale (0–10) QoL (0–100) Rutherford category (0–6) Wound size (cm2)
Base
6 Months
15 ± 10 0.9 ± 0.4 4.4 ± 2.6 51 ± 15 5.0 ± 0.2
29 ± 13 0.9 ± 0.2 0.9 ± 1.4 70 ± 13 4.3 ± 1.6
8.2 ± 6.8
2.8 ± 6.3
Group A (IM, n = 13)
Group B (IA, n = 14)
Base
6 Months
p
Base
6 Months
p
<0.001 0.88 <0.001 <0.001 <0.01
13 ± 10 0.9 ± 0.4 3.8 ± 2.5 55 ± 17 5.0
30 ± 12 0.9 ± 0.2 0.6 ± 1.0 69 ± 12 3.6 ± 1.4
<0.01 0.34 <0.001 <0.05 <0.01
15 ± 11 0.9 ± 0.4 4.8 ± 2.6 49 ± 14 5.1 ± 0.3
28 ± 14 0.9 ± 0.2 0.8 ± 1.3 72 ± 13 3.7 ± 1.4
<0.01 0.22 <0.001 <0.01 <0.01
<0.001
7.1 ± 4.8
2.3 ± 2.1
<0.001
8.9 ± 8.1
3.2 ± 8.1
<0.001
p
ABI, ankle–brachial index; BMC, bone marrow cells; QoL, quality-of-life; tcpO2, transcutaneous oxygen pressure; IA, intra-arterial; IM, intramuscular; p = 6 months versus baseline a Mediasclerosis was noted in 22/27 patients (81%).
Figure 1. Nonhealing ulcers before and 6 months after intramuscular (A) and intra-arterial (B) delivery of BMCs. BMCs, bone marrow cells; tcpO2, transcutaneous oxygen pressure.
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Figure 2. Improvement in trancutaneous oxygen pressure and pain scale 3 and 6 months after BMC delivery: IM versus IA group. BMC, bone marrow cells; IA, intra-arterial; IM, intramuscular; tcpO2, transcutaneous oxygen pressure. *p = NS IM versus IA; †p < 0.005 baseline versus 6 months.
in the IA group (p = 0.94). There were no procedurerelated complications in either group. Responders Versus Nonresponders to Cell Therapy Table 4 shows the characteristics of responders versus nonresponders to BMC therapy. Responders (n = 27) were characterized by a higher CD34+ cell count in the BMC product (29 ± 15 × 106 vs. 17 ± 12 × 106, p < 0.05) despite having a similar number of total nucleated cells (4.3 ± 1.4 × 109 vs. 4.1 ± 1.2 × 109, p = 0.66). Responders had lower CRP levels (18 ± 28 vs. 100 ± 96 mg/L, p < 0.05) and peripheral blood leukocyte count (8.3 ± 2.1 × 109/L vs. 12.3 ± 4.5 × 109/L, p < 0.05) as compared with nonresponders (10 patients). Upon univariate analysis, a CD34+ cell count >20 × 106 was associated with a positive therapeutic response to stem cell therapy [p = 0.015, odds ratio (OR) 4.7, 95% confidence interval (CI) 1.15–19.24], and peripheral blood leukocyte count >10 × 109/L was associated with a negative therapeutic response (p = 0.006, OR 2.1, 95% CI 1.06–4.1), similar to the effect of CRP level >10 mg/L ( p = 0.038, OR 1.54, 95% CI 1.01–2.32). Upon multivariate analysis (binary logistic regression), the number of administrated CD34+ cells >20 × 106 emerged as an independent predictor of clinical benefit after BMC application ( p = 0.03). A peripheral leukocyte count >10 × 109/L was shown to be an independent predictor of negative therapeutic response to cell therapy ( p = 0.048). According to the receiver operating characteristic (ROC) analysis, a cutoff limit for CD34+ cells of 20 × 106 in delivered bone marrow concentrate was predictive for a positive clinical response with 80% specificity and 65% sensitivity (Fig. 4).
DISCUSSION The present study investigates the effects of two methods of delivery of autologous BMCs on the progression of advanced CLI. The main findings can be summarized as follows: (1) IM and IA delivery routes are effective and comparable in inducing a therapeutic effect in CLI and (2) higher CD34+ cell counts and a lower degree of inflammation are associated with a better clinical response to BMC administration. Delivery of Autologous BMCs in Patients With CLI Several authors have reported the clinical benefit of administration of autologous BMCs in patients with CLI (2,5,12,18,22,31,35). BMC delivery has been associated with limb salvage, increase in tcpO2, blood flow perfusion, or the ABI. The results of the present study corroborate the positive effects of BMC therapy on wound healing, tcpO2, pain scoring, Rutherford category, as well as QoL. Conversely, the ABI did not differ after 6 months in the present study, similar to the findings in the PROVASA trial (35). Of note, most of our patients suffered from diabetes mellitus, with the high prevalence of mediasclerosis [22/27 subjects (81%) in the responders group], where noncompressible ankle arteries precluded meaningful determination of the ABI. Importantly, the prevalence of limb salvage 73% in the present study was similar to that of other studies reporting the effects of cell therapy at 6 months (15,37). Intramuscular Versus Intra-Arterial Routes of BMC Delivery There is ongoing discussion about the optimal method of cell delivery in CLI. IM as well as IA methods of
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Figure 3. Digital subtraction angiography before and 6 months after BMC delivery: IM (A) versus IA (B) group.
administration of stem cells have shown promising results in achieving therapeutic benefit (2,5,12,25,35). One potential advantage of IM delivery is creation of “local depots” of implanted cells with increased local paracrine activity in the ischemic area. However, the prevalence of cell retention and survival times after IM injection into ischemic limb muscle is not known. For IA delivery, the homing of administrated BMCs in the zone of ischemia is crucial (4). With selective IA delivery, stem cells can reach the border zone of maximum ischemia by blood flow, although the degree of cell uptake from the circulation and from engraftment is unknown.
In the clinical setting, the question of the optimal route of BMC delivery has been addressed only in small studies (8,32). Our results from direct head-to-head comparison of different administration routes indicate that IM as well as IA methods of BMC delivery are effective in limb salvage and wound healing, with no significant differences in various functional surrogate endpoints between the techniques. Our clinical observations corroborate experimental findings of a similar level of angiogenic activity after IM and IA injections in the rat ischemic hind-limb model (36). Likewise, they are in agreement with a recent study showing no differences in the extent of perfusion improvement after IM
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Table 3. Six-month Comparison of Functional Outcomes of BMC Delivery in Patients With Limb Salvage
∆ tcpO2 (mmHg) ∆ tcpO2 > 15% ∆ ABI ∆ Pain scale (0–10) ∆ QoL (0–100) ∆ Rutherford category (1–6) ∆ Wound size (cm2)
Group A (IM, n = 13)
Group B (IA, n = 14)
p (IM vs. IA)
13.4 ± 12.4 7 (70%) 0.05 ± 0.23 –3.7 ± 2.1 18 ± 13 –1.28 ± 1.70
13.5 ± 13.4 9 (69%) –0.12 ± 0.30 –3.6 ± 2.8 22 ± 17 –1.36 ± 1.43
0.66 1.0 0.25 0.63 0.56 0.46
–4.8 ± 3.2
–5.8 ± 5.4
0.59
ABI, ankle–brachial index; BMC, bone marrow cells; QoL, qualityof-life; tcpO2, transcutaneous oxygen pressure; IA, intra-arterial; IM, intramuscular; ∆, baseline value at 6 months.
administration or combined IM and IA administration in patients with CLI (32). There are various techniques of IM or IA administration of stem cells. For IA methods, slow delivery over 3 min upon injection into the superficial femoral artery (19) or IA infusion at 900 ml/h (5) has been described. We undertook selective IA delivery of BMCs at a controlled rate of 800 ml/h at the site of arterial occlusion. For IM administration, in most clinical trials, injection has usually been done using a symmetric grid with a fixed number of injections (2,25). In the present study, BMC administration into the muscles of the affected limb was carried out along the crural arteries. Functional improvement after IM and IA administration was observed in the absence of macrovascular changes of visible collaterals or arteriogenesis of the affected limb upon DSA. This finding supports the concept that the therapeutic effects of BMCs in CLI are primarily exerted at the microcirculation and that DSA is not a suitable method for the evaluation of therapeutic angiogenesis (33). Predictors of Therapeutic Responses to Delivery of Autologous BMCs The potential predictors of the therapeutic response to cell-based therapy in CLI have not been clearly elucidated. In the present study, two factors were predictive of the response to cell therapy. The number of administrated CD34+ cells, but not the total number of nucleated cells (bone marrow mononuclear cells, BMMCs), was strongly related to clinical benefit. This result partially contradicts recent findings in the PROVASA trial in which CD34+ cells and BMMC numbers were independent predictors of improved ulcer healing (35). Conversely, they are consistent with various studies demonstrating the superior effects of enriched CD34+ cells as compared with BMMCs. In fundamental studies, the surface expression of CD34, CD133 and vascular endothelial growth factor receptor-2
[VEGFR-2/kinase insert domain receptor (KDR)] identified a population of endothelial progenitor cells (EPCs) with enhanced potency for neovascularization of ischemic tissue (7,14,16,24). Likewise, CD34+ cells could restore the microcirculation and improve tissue perfusion in preclinical models (13,14) as well as in clinical series (21,28) that appeared to be superior to BMMCs alone (13). Furthermore, enriched CD133+ progenitor cells demonstrated positive functional effects in patients with chronic as well as recently infarcted myocardium (3,29). Evidence that CD34+ cells may be pivotal for therapeutic benefits is supported by the notion that mononuclear cells depleted of CD34+ cells do not improve myocardial function in a murine infarct model (16). Nevertheless, it is likely that nonhematopoietic stem cells (mesenchymal or stromal cells), with their high paracrine ability, could also contribute to the beneficial effect of cell therapy. Mesenchymal cells, although found in low numbers in mononuclear cell fractions, are a potent source of trophic cytokines and have been shown to exert pro-angiogenic effects regardless of bone marrow or adipose origin (9,17). In addition, the composition of the bone marrow concentrate used in the present study included a small portion of an erythrocyte layer together with a high number of platelets. Platelets, as a rich source of paracrine activity, have been shown to augment the formation of collateral vessels in ischemic tissue in the presence of mononuclear cells (11). Second, elevated inflammatory markers (e.g., leukocyte count, CRP levels) were predictive of a negative therapeutic response to cell delivery. CRP is considered to be one of the strongest predictors of vascular death (26) and appears to be an important mediator of atherogenesis (30). It can exert a harmful effect on EPC function, resulting in impaired repair of vessels and impaired neovascularization of ischemic tissues (34). Accordingly, a reduced CRP level has been shown to be a major predictor of successful outcome in percutaneous transluminal angioplasty in diabetic patients with infected foot ulcers (20). Our findings
Table 4. Characteristics of Responders Versus Nonresponders to BMC Delivery
BMMC (109) CD34+ (106) CRP (mg/L) Leu (109/L) Tr (109/L) tcpO2 baseline (mmHg) Wound size (cm2)
Responders (n = 27)
Nonresponders (n = 10)
p
4.3 ± 1.4 29 ± 15 18 ± 28 8.3 ± 2.1 290 ± 107 15 ± 10 8.2 ± 6.8
4.1 ± 1.2 17 ± 12 100 ± 96 12.3 ± 4.5 352 ± 163 9 ± 8 14.1 ± 10.3
0.66 0.03 0.03 0.02 0.33 0.07 0.12
BMC, bone marrow cells; BMMC, bone marrow mononuclear cells; CRP, C-reactive protein; Leu, leukocytes in peripheral blood; tcpO2, transcutaneous oxygen pressure; Tr, thrombocytes in peripheral blood.
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Figure 4. Receiver operating characteristic of CD34+ cells, BMMC count, CRP, and leukocyte levels for the prediction of the BMC therapeutic response. Area under the receiver operating characteristic (ROC) curve: CD34+ = 0.75 (CI 0.57–0.94, p = 0.02); mononuclear cell (MNC) = 0.55 (CI 0.35–0.75, p = 0.65); c-reactive protein (CRP) = 0.26 (CI 0.04–0.47, p = 0.03); leukocyte (leu) = 0.21 (CI 0.01–0.4, p = 0.007)
corroborate the clinical results of the PROVASA trial whereby patients with Rutherford classification of 6 CLI (gangrene or major loss of tissue) at baseline (typical by the highest inflammatory burden) did not respond to cell therapy (35). Advanced local inflammation of ischemic tissue acts as a hostile environment for delivered stem cells. Hence, the question of appropriate timing of stem cell therapy with regard to the deleterious inflammatory setting is one of the key factors of clinical success. LIMITATIONS A relatively small number of patients in individual groups could be considered to be one of the limitations; the findings of the present study need to be confirmed in larger, prospectively designed cohorts. Second, absence of a control group cannot exclude the possibility of spontaneous improvement in some patients. However, the significant improvements in tcpO2 as an objective parameter in patients with no options for surgical or endovascular revascularization are unlikely to be the result of placebo or spontaneous improvement. CONCLUSIONS Both intramuscular and intra-arterial delivery of autologous BMCs is effective and a comparable therapeutic strategy for patients with CLI who are not suitable for endovascular
or surgical revascularization. A higher concentration of CD34+ cells and a lower degree of inflammation are associated with a better therapeutic response to BMC therapy. ACKNOWLEDGMENTS: This study was sponsored with a grant from European Regional Development Funding (ITMS code: 26240220023). Authors declare no conflicts of interest.
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