Ralf W. Sprengers

Autologous bone marrow-derived cell therapy in patients with critical limb ischemia: a meta-analysis of randomized controlled clinical trials.

Background: Critical Limb Ischemia (CLI) is the most advanced stage of peripheral arterial disease and is usually treated with bypass surgery or endovascular revascularization. However, a considerable proportion of CLI patients are not eligible to these treatment strategies and amputation is often the only option left. In the past decade, research has focused on bone marrow (BM)–derived cell-based strategies that aim at neovascularization to improve limb perfusion. Individual studies did not convincingly prove efficacy of BM-derived cell therapy in CLI patients thus far. Objectives: Perform a meta-analysis of all randomized controlled trials (RCTs) available that studied BM-derived cell therapy compared to standard care with or without placebo in CLI patients and provide summary efficacy data on this approach. Methods: A systematic search in the electronic databases of Medline, Embase, and the Cochrane Controlled Trials Register was performed. All studies were critically appraised and data were extracted and meta-analyzed using a random-effects model. Major amputation and amputation-free survival were considered as the primary endpoints. Results: A total of 12 RCTs jointly including 510 CLI patients were identified and analyzed. The meta-analysis showed beneficial effects of BM-derived cell therapy on both subjective and surrogate objective endpoints, that is, pain score, pain-free walking distance, ankle-brachial index, and transcutaneous oxygen measurements (all P < 0.00001). Overall, the RCTs showed reduced amputation rates in the therapeutic arms of the included trials with a relative risk (RR) on major amputation of 0.58 [95% confidence interval (CI), 0.40–0.84; P = 0.004]. However, when only the placebo-controlled RCTs were considered, the beneficial effect on major amputation rates was considerably reduced and nonsignificant (RR = 0.78; 95% CI, 0.40–1.51; P = 0.46). Amputation-free survival did not significantly differ between the BM treated and the control group (RR = 1.16; 95% CI, 0.92–1.48; P = 0.22). Conclusions: This meta-analysis underlines the promising potential of BM-derived cell therapy in CLI patients. Importantly, the results of placebo-controlled and non–placebo-controlled RCTs seem to diverge, which stresses the necessity to use placebo in the control arms of these trials. Future well-designed larger placebo-controlled RCTs are needed and should include long-term follow-up data to assess durability of treatment effects.

Effect of Repetitive Intra-Arterial Infusion of Bone Marrow Mononuclear Cells in Patients With No-Option Limb Ischemia The Randomized, Double-Blind, Placebo-Controlled Rejuvenating Endothelial Progenitor Cells via Transcutaneous Intra-arterial Supplementation (JUVENTAS) Trial

Background —Patients with severe limb ischemia may not be eligible for conventional therapeutic interventions. Pioneering clinical trials suggest that bone marrow (BM)-derived cell therapy enhances neovascularization, improves tissue perfusion, and prevents amputation. The objective of this trial was to determine whether repetitive intra-arterial infusion of BM mononuclear cells (BMMNC) in patients with severe, non-revascularizable limb ischemia can prevent major amputation. Methods and Results —The Juventas-trial is a randomized, double-blind, placebo-controlled clinical trial in 160 patients with severe, non-revascularizable limb ischemia. Patients were randomized to repetitive (3x; 3-weeks interval) intra-arterial infusion of BMMNC or placebo. No significant differences were observed for the primary outcome, i.e. major amputation at 6 months, with major amputation rates of 19% in the BMMNC vs. 13% in the placebo group (Relative risk [RR] 1.46; 95% confidence interval [CI] 0.62 to 3.42). The safety outcome (all-cause mortality, occurrence of malignancy or hospitalization due to infection) was not significantly different between the groups (RR 1.46; 95%CI 0.63 to 3.38), neither was all-cause mortality at six months with 5% vs. 6% (RR 0.78; 95%CI 0.22 to 2.80). Secondary outcomes quality of life, rest pain, ankle-brachial index, and transcutaneous oxygen pressure improved during follow-up, but there were no significant differences between the groups. Conclusions —Repetitive intra-arterial infusion of autologous BMMNC into the common femoral artery did not reduce major amputation rate in patients with severe, non-revascularizable limb ischemia compared to placebo. The general improvement in secondary outcomes during follow-up in both the BMMNC as well as the placebo group underlines the essential role for placebo-controlled design of future trials. Clinical Trial Registration Information —ClinicalTrials.gov. Identifier: [NCT00371371][1]. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00371371&atom=%2Fcirculationaha%2Fearly%2F2015%2F01%2F07%2FCIRCULATIONAHA.114.012913.atomBackground— Patients with severe limb ischemia may not be eligible for conventional therapeutic interventions. Pioneering clinical trials suggest that bone marrow–derived cell therapy enhances neovascularization, improves tissue perfusion, and prevents amputation. The objective of this trial was to determine whether repetitive intra-arterial infusion of bone marrow mononuclear cells (BMMNCs) in patients with severe, nonrevascularizable limb ischemia can prevent major amputation. Methods and Results— The Rejuvenating Endothelial Progenitor Cells via Transcutaneous Intra-arterial Supplementation (JUVENTAS) trial is a randomized, double-blind, placebo-controlled clinical trial in 160 patients with severe, nonrevascularizable limb ischemia. Patients were randomly assigned to repetitive (3 times; 3-week interval) intra-arterial infusion of BMMNC or placebo. No significant differences were observed for the primary outcome, ie, major amputation at 6 months, with major amputation rates of 19% in the BMMNC versus 13% in the placebo group (relative risk, 1.46; 95% confidence interval, 0.62–3.42). The safety outcome (all-cause mortality, occurrence of malignancy, or hospitalization due to infection) was not significantly different between the groups (relative risk, 1.46; 95% confidence interval, 0.63–3.38), neither was all-cause mortality at 6 months with 5% versus 6% (relative risk, 0.78; 95% confidence interval, 0.22–2.80). Secondary outcomes quality of life, rest pain, ankle-brachial index, and transcutaneous oxygen pressure improved during follow-up, but there were no significant differences between the groups. Conclusions— Repetitive intra-arterial infusion of autologous BMMNCs into the common femoral artery did not reduce major amputation rates in patients with severe, nonrevascularizable limb ischemia in comparison with placebo. The general improvement in secondary outcomes during follow-up in both the BMMNC and the placebo group, as well, underlines the essential role for placebo-controlled design of future trials. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00371371.

Progenitor cell therapy in patients with critical limb ischemia without surgical options.

Objective:To provide a review on progenitor cell therapy for critical limb ischemia. Summary Background Data:Critical limb ischemia is estimated to develop in 500 to 1000 individuals per million persons per year and has a major impact on the quality of life. Despite recent advances in surgical and radiologic vascular procedures, a large number of patients (∼40%) are not eligible for these revascularization procedures. New strategies for revascularization need to be explored. Recent evidence indicates that bone marrow-derived mononuclear cells are a potential new therapeutic target. Methods:A comprehensive review of all published literature on progenitor cell therapy in patients with critical limb ischemia was performed. Results:Twenty-five clinical studies that reported on the use of mononuclear cells or progenitor cells for the treatment of patients with critical limb ischemia have been published, of which 18 were included in this review. Seven studies were published in Chinese, Japanese, or Polish and, therefore, not included. Conclusions:Pioneering clinical studies report promising results for progenitor cell-based therapies for chronic limb ischemia, but no definite proof is available because the clinical studies thus far have been small and lacked double-blinded controls. Larger, randomized, blinded, placebo-controlled trials to investigate the potential clinical effects of bone marrow progenitor cell administration in patients with critical limb ischemia are needed.

Effect of Repetitive Intra-Arterial Infusion of Bone Marrow Mononuclear Cells in Patients With No-Option Limb Ischemia: The Randomized, Double-Blind, Placebo-Controlled JUVENTAS Trial

Background —Patients with severe limb ischemia may not be eligible for conventional therapeutic interventions. Pioneering clinical trials suggest that bone marrow (BM)-derived cell therapy enhances neovascularization, improves tissue perfusion, and prevents amputation. The objective of this trial was to determine whether repetitive intra-arterial infusion of BM mononuclear cells (BMMNC) in patients with severe, non-revascularizable limb ischemia can prevent major amputation. Methods and Results —The Juventas-trial is a randomized, double-blind, placebo-controlled clinical trial in 160 patients with severe, non-revascularizable limb ischemia. Patients were randomized to repetitive (3x; 3-weeks interval) intra-arterial infusion of BMMNC or placebo. No significant differences were observed for the primary outcome, i.e. major amputation at 6 months, with major amputation rates of 19% in the BMMNC vs. 13% in the placebo group (Relative risk [RR] 1.46; 95% confidence interval [CI] 0.62 to 3.42). The safety outcome (all-cause mortality, occurrence of malignancy or hospitalization due to infection) was not significantly different between the groups (RR 1.46; 95%CI 0.63 to 3.38), neither was all-cause mortality at six months with 5% vs. 6% (RR 0.78; 95%CI 0.22 to 2.80). Secondary outcomes quality of life, rest pain, ankle-brachial index, and transcutaneous oxygen pressure improved during follow-up, but there were no significant differences between the groups. Conclusions —Repetitive intra-arterial infusion of autologous BMMNC into the common femoral artery did not reduce major amputation rate in patients with severe, non-revascularizable limb ischemia compared to placebo. The general improvement in secondary outcomes during follow-up in both the BMMNC as well as the placebo group underlines the essential role for placebo-controlled design of future trials. Clinical Trial Registration Information —ClinicalTrials.gov. Identifier: [NCT00371371][1]. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00371371&atom=%2Fcirculationaha%2Fearly%2F2015%2F01%2F07%2FCIRCULATIONAHA.114.012913.atomBackground— Patients with severe limb ischemia may not be eligible for conventional therapeutic interventions. Pioneering clinical trials suggest that bone marrow–derived cell therapy enhances neovascularization, improves tissue perfusion, and prevents amputation. The objective of this trial was to determine whether repetitive intra-arterial infusion of bone marrow mononuclear cells (BMMNCs) in patients with severe, nonrevascularizable limb ischemia can prevent major amputation. Methods and Results— The Rejuvenating Endothelial Progenitor Cells via Transcutaneous Intra-arterial Supplementation (JUVENTAS) trial is a randomized, double-blind, placebo-controlled clinical trial in 160 patients with severe, nonrevascularizable limb ischemia. Patients were randomly assigned to repetitive (3 times; 3-week interval) intra-arterial infusion of BMMNC or placebo. No significant differences were observed for the primary outcome, ie, major amputation at 6 months, with major amputation rates of 19% in the BMMNC versus 13% in the placebo group (relative risk, 1.46; 95% confidence interval, 0.62–3.42). The safety outcome (all-cause mortality, occurrence of malignancy, or hospitalization due to infection) was not significantly different between the groups (relative risk, 1.46; 95% confidence interval, 0.63–3.38), neither was all-cause mortality at 6 months with 5% versus 6% (relative risk, 0.78; 95% confidence interval, 0.22–2.80). Secondary outcomes quality of life, rest pain, ankle-brachial index, and transcutaneous oxygen pressure improved during follow-up, but there were no significant differences between the groups. Conclusions— Repetitive intra-arterial infusion of autologous BMMNCs into the common femoral artery did not reduce major amputation rates in patients with severe, nonrevascularizable limb ischemia in comparison with placebo. The general improvement in secondary outcomes during follow-up in both the BMMNC and the placebo group, as well, underlines the essential role for placebo-controlled design of future trials. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00371371.

Quality of life in patients with no-option critical limb ischemia underlines the need for new effective treatment.

OBJECTIVE To provide a solid baseline reference for quality of life (QoL) in patients with no-option critical limb ischemia (CLI). CLI is associated with surgery, endovascular interventions, hospitalization, and a poor prognosis. An increasing number of clinical trials are, therefore, investigating new treatment strategies (eg, therapeutic neovascularization) in patients with CLI. QoL serves as an important secondary endpoint in many of these trials, but solid reference QoL data for patients with no-option CLI are lacking. METHODS The Medical Outcomes Study Short Form 36 (SF-36) and the EuroQol-5D (EQ-5D) questionnaires were used to obtain baseline QoL scores from 47 patients with no-option CLI participating in a therapeutic neovascularization trial. To allow for easy comparability, a norm-based scoring (NBS) method was used to report the results of the SF-36. Scores of patients with CLI were furthermore compared with scores of patients with milder forms of peripheral arterial disease (PAD) and with patients with cardiovascular risk factors only. Determinants of QoL in patients with PAD were identified using multiple linear regression methods. RESULTS Patients with no-option CLI reported QoL scores below the general population mean on every health dimension of the SF-36. Physical functioning, role physical functioning, and bodily pain were affected most intensively. These poor physical QoL scores were further underlined when compared with other patients with milder forms of PAD or patients with cardiovascular risk factors only. Patients with CLI scored poorly on the pain/discomfort and the usual activities domain of the EQ-5D. Diabetes, female gender, body mass index, and the ankle-brachial index at rest were significant determinants of the QoL in PAD on multivariate analysis. CONCLUSION The QoL data of patients with no-option CLI using NBS methods for the SF-36 provide a baseline reference for ongoing clinical trials on new treatment strategies. Our data stress the need for new revascularization therapies in patients with no-option CLI.

Angiographic demonstration of neoangiogenesis after intra-arterial infusion of autologous bone marrow mononuclear cells in diabetic patients with critical limb Ischemia

Critical limb ischemia in diabetic patients is associated with high rates of morbidity and mortality. Suboptimal responses to the available medical and surgical treatments are common in these patients, who also demonstrate limited vascular homeostasis. Neovasculogenesis induced by stem cell therapy could be a useful approach for these patients. Neovasculogenesis and clinical improvement were compared at baseline and at 3 and 12 months after autologous bone marrow-derived mononuclear cell (BMMNC) transplantation in diabetic patients with peripheral artery disease. We conducted a prospective study to evaluate the safety and efficacy of intra-arterial administration of autologous BMMNCs (100–400 × 106 cells) in 20 diabetic patients with severe below-the-knee arterial ischemia. Although the time course of clinical effects differed among patients, after 12 months of follow-up all patients presented a notable improvement in the Rutherford-Becker classification, the University of Texas diabetic wound scales, and the Ankle-Brachial Index in the target limb. The clinical outcome was consistent with neovasculogenesis, which was assessed at 3 months by digital subtraction angiography and quantified by MetaMorph software. Unfortunately, local cell therapy in the target limb had no beneficial effect on the high mortality rate in these patients. In diabetic patients with critical limb ischemia, intra-arterial perfusion of BMMNCs is a safe procedure that generates a significant increase in the vascular network in ischemic areas and promotes remarkable clinical improvement.

Bone Marrow Alterations and Lower Endothelial Progenitor Cell Numbers in Critical Limb Ischemia Patients

Background Critical limb ischemia (CLI) is characterized by lower extremity artery obstruction and a largely unexplained impaired ischemic neovascularization response. Bone marrow (BM) derived endothelial progenitor cells (EPC) contribute to neovascularization. We hypothesize that reduced levels and function of circulating progenitor cells and alterations in the BM contribute to impaired neovascularization in CLI. Methods Levels of primitive (CD34+ and CD133+) progenitors and CD34+KDR+ EPC were analyzed using flow cytometry in blood and BM from 101 CLI patients in the JUVENTAS-trial (NCT00371371) and healthy controls. Blood levels of markers for endothelial injury (sE-selectin, sICAM-1, sVCAM-1, and thrombomodulin), and progenitor cell mobilizing and inflammatory factors were assessed by conventional and multiplex ELISA. BM levels and activity of the EPC mobilizing protease MMP-9 were assessed by ELISA and zymography. Circulating angiogenic cells (CAC) were cultured and their paracrine function was assessed. Results Endothelial injury markers were higher in CLI (P<0.01). CLI patients had higher levels of VEGF, SDF-1α, SCF, G-CSF (P<0.05) and of IL-6, IL-8 and IP-10 (P<0.05). Circulating EPC and BM CD34+ cells (P<0.05), lymphocytic expression of CXCR4 and CD26 in BM (P<0.05), and BM levels and activity of MMP-9 (P<0.01) were lower in CLI. Multivariate regression analysis showed an inverse association between IL-6 and BM CD34+ cell levels (P = 0.007). CAC from CLI patients had reduced paracrine function (P<0.0001). Conclusion CLI patients have reduced levels of circulating EPC, despite profound endothelial injury and an EPC mobilizing response. Moreover, CLI patients have lower BM CD34+-cell levels, which were inversely associated with the inflammatory marker IL-6, and lower BM MMP-9 levels and activity. The results of this study suggest that inflammation-induced BM exhaustion and a disturbed progenitor cell mobilization response due to reduced levels and activity of MMP-9 in the BM and alterations in the SDF-1α/CXCR4 interaction contribute to the attenuated neovascularization in CLI patients.

Rationale and design of the JUVENTAS trial for repeated intra-arterial infusion of autologous bone marrow-derived mononuclear cells in patients with critical limb ischemia

Critical limb ischemia (CLI) continues to form a substantial burden on Western healthcare. Many patients still face amputation as a last treatment option. Autologous bone marrow (BM)-derived cell administration has emerged as a potential new treatment, but proof for sustainable clinical effects of BM-derived cell therapy in CLI is still lacking. The JUVENTAS (reJUVenating ENdothelial progenitor cells via Transcutaneous intra-Arterial Supplementation) trial is the first randomized, placebo-controlled, double-blinded clinical trial on repeated intra-arterial BM mononuclear cell (MNC) infusion in 110 to 160 CLI patients, designed to provide definite proof for the efficacy of stem cell therapy. Primary outcome is the incidence of major amputation at 6 months. Inclusion of patients is well underway. If BM-MNC cells therapy is beneficial, it could become a novel treatment to prevent amputation in patients with CLI.

Prediction rule for cardiovascular events and mortality in peripheral arterial disease patients: Data from the prospective Second Manifestations of ARTerial disease (SMART) cohort study

BACKGROUND Patients with peripheral arterial disease (PAD) are at high risk of secondary cardiovascular death and events such as myocardial infarction or stroke. To minimize this elevated risk, cardiovascular risk factors should be treated in all PAD patients. Secondary risk management may benefit from a prediction tool to identify PAD patients at the highest risk who could be referred for an additional extensive workup. Stratifying PAD patients according to their risk of secondary events could aid in achieving optimal therapy compliance. To this end we developed a prediction model for secondary cardiovascular events in PAD patients. METHODS The model was developed using data from 800 PAD patients who participated in the Second Manifestations of ARTerial disease (SMART) cohort study. From the baseline characteristics, 13 candidate predictors were selected for the model development. Missing values were imputed by means of single regression imputation. Continuous predictors were truncated and transformed where necessary, followed by model reduction by means of backward stepwise selection. To correct for over-fitting, a bootstrapping technique was applied. Finally, a score chart was created that divides patients in four risk categories that have been linked to the risk of a cardiovascular event during 1- and 5-year follow-up. RESULTS During a mean follow-up of 4.7 years, 120 events occurred (27% nonfatal myocardial infarction, 21% nonfatal stroke, and 52% mortality from vascular causes), corresponding to a 1- and 5-year cumulative incidence of 3.1% and 13.2%, respectively. Important predictors for the secondary risk of a cardiovascular event are age, history of symptomatic cardiovascular disease, systolic blood pressure, high-density lipoprotein cholesterol, smoking behavior, ankle-brachial pressure index, and creatinine level. The risk of a cardiovascular event in a patient as predicted by the model was 0% to 10% and 1% to 28% for the four risk categories at 1- and 5-year follow-up, respectively. The discriminating capacity of the prediction model, indicated by the c statistic, was 0.76 (95% confidence interval, 0.71-0.80). CONCLUSION A prediction model can be used to predict secondary cardiovascular risk in PAD patients. We propose such a prediction model to allow for the identification of PAD patients at the highest risk of a cardiovascular event or cardiovascular death, which may be a viable tool in vascular secondary health care practice.

Stem Cell Therapy in PAD

Critical limb ischemia (CLI) continues to form a substantial burden on Western health care. Despite recent advances in surgical and radiological vascular techniques, a large number of patients is not eligible for these revascularisation procedures and faces amputation as their ultimate treatment option. Growth factor therapy and stem cell therapy - both therapies focussing on augmenting postnatal neovascularisation - have raised much interest in the past decade. Based on initial pre-clinical and clinical results, both therapies appear to be promising strategies to augment neovascularisation and to reduce symptoms and possibly prevent amputation in CLI patients. However, the underlying mechanisms of postnatal neovascularisation are still incompletely understood. Both fundamental research as well as large randomised trials are needed for further optimisation of these treatment options, and will hopefully lead to needed advances in the treatment of no-option CLI patients in the near future.