Bone marrow and plasma FGF-23 in heart failure patients: novel insights into the heart-bone axis.

AIMS: Fibroblast growth factor 23 (FGF-23) is known to be elevated in patients with congestive heart failure (CHF). As FGF-23 is expressed in the bone but can also be expressed in the myocardium, the origin of serum FGF-23 in CHF remains unclear. It is also unclear if FGF-23 expressed in the bone is associated with outcome in CHF. The aim of the present study was to investigate FGF-23 levels measured in bone marrow plasma (FGF-23-BM) and in peripheral blood (FGF-23-P) in CHF patients to gain further insights into the heart-bone axis of FGF-23 expression. We also investigated possible associations between FGF-23-BM as well as FGF-23-P and outcome in CHF patients. METHODS AND RESULTS: We determined FGF-23-P and FGF-23-BM levels in 203 CHF patients (85% male, mean age 61.3 years) with a left ventricular ejection fraction (LVEF) ≤45% and compared them with those of 48 healthy controls (48% male, mean age 39.2 years). We investigated the association between FGF-23-BM and FGF-23-P with all-cause mortality in CHF patients, 32 events, median follow-up 1673 days, interquartile range [923, 1828]. FGF-23-P (median 60.3 vs. 22.0 RU/mL, P < 0.001) and FGF-23-BM (median 130.7 vs. 93.1 RU/mL, P < 0.001) levels were higher in CHF patients compared with healthy controls. FGF-23-BM levels were significantly higher than FGF-23-P levels in both CHF patients and in healthy controls (P < 0.001). FGF-23-P and FGF-23-BM correlated significantly with LVEF (r = -0.37 and r = -0.33, respectively), N terminal pro brain natriuretic peptide levels (r = 0.57 and r = 0.6, respectively), New York Heart Association status (r = 0.28 and r = 0.25, respectively), and estimated glomerular filtration rate (r = -0.43 and r = -0.41, respectively) (P for all <0.001) and were independently associated with all-cause mortality in CHF patients after adjustment for LVEF, estimated glomerular filtration rate, New York Heart Association status, and N terminal pro brain natriuretic peptide, hazard ratio 2.71 [confidence interval: 1.18-6.20], P = 0.018, and hazard ratio 2.80 [confidence interval: 1.19-6.57], P = 0.018, respectively. CONCLUSIONS: In CHF patients, FGF-23 is elevated in bone marrow plasma and is independently associated with heart failure severity and all-cause mortality. The failing heart seems to interact via FGF-23 within a heart-bone axis.

Minute Myocardial Injury as Measured by High-Sensitive Troponin T Serum Levels Predicts the Response to Intracoronary Infusion of Bone Marrow-Derived Mononuclear Cells in Patients With Stable Chronic Post-Infarction Heart Failure: Insights From the TOPCARE-CHD Registry.

RATIONALE: Cell-based therapies are a promising option in patients with chronic postinfarction heart failure (ischemic cardiomyopathy [ICM]). However, the responses after intracoronary infusion of autologous bone marrow-derived mononuclear cells (BMCs) are heterogeneous, which may be related to impaired cell retention in patients with ICM. Ischemic injury is associated with upregulation of prototypical chemoattractant cytokines mediating retention and homing of circulating cells. The development of ultrasensitive tests to measure high-sensitive troponin T (hs-TnT) serum levels revealed the presence of ongoing minute myocardial injury even in patients with stable ICM. OBJECTIVE: To test the hypothesis that serum levels of hs-TnT correlate with cell retention and determine the response to intracoronary BMC application in patients with ICM. METHODS AND RESULTS: About 157 patients with stable ICM and no substantial impairment of kidney function received intracoronary BMC administration. Immediately prior to cell application, hs-TnT levels to measure myocardial injury and NT-proBNP levels as marker of left ventricular wall stress were determined. Patients with elevated hs-TnT were older and had more severe heart failure. Importantly, only patients with elevated baseline hs-TnT≥15.19 pg/mL (upper tertile) demonstrated a significant (P=0.04) reduction in NT-proBNP serum levels (-250 [-1465; 33] pg/mL; relative reduction -24%) 4 months after BMC administration, whereas NT-proBNP levels remained unchanged in patients in the 2 lower hs-TnT tertiles. The absolute decrease in NT-proBNP at 4 months was inversely correlated with baseline hs-TnT (r=-0.27, P=0.001). Finally, retention of intracoronarily infused, 111Indium-labeled cells within the heart was closely associated with hs-TnT levels in patients with chronic ischemic heart failure (P=0.0008, n=10, triple measurements). CONCLUSIONS: The extent of ongoing myocardial injury as measured by serum levels of hs-TnT predicts the reduction of NT-proBNP serum levels at 4 months after intracoronary BMC administration in patients with ICM, suggesting that the beneficial effects of BMC application on LV remodeling and wall stress are confined to patients with ongoing minute myocardial injury. CLINICAL TRIAL REGISTRATION: URL: www.clinicaltrials.gov. Unique identifier: NCT00962364.

Metabolism Regulates Cellular Functions of Bone Marrow-Derived Cells used for Cardiac Therapy.

Administration of bone marrow-derived mononuclear cells (BMC) may increase cardiac function after myocardial ischemia. However, the functional capacity of BMC derived from chronic heart failure (CHF) patients is significantly impaired. As modulation of the energy metabolism allows cells to match the divergent demands of the environment, we examined the regulation of energy metabolism in BMC from patients and healthy controls (HC). The glycolytic capacity of CHF-derived BMC is reduced compared to HC, whereas BMC of metabolically activated bone marrow after acute myocardial infarction reveal increased metabolism. The correlation of metabolic pathways with the functional activity of cells indicates an influence of metabolism on cell function. Reducing glycolysis without profoundly affecting ATP-production reversibly reduces invasion as well as colony forming capacity and abolishes proliferation of CD34(+) CD38(-) lin(-) hematopoietic stem and progenitor cells (HSPC). Ex vivo inhibition of glycolysis further reduced the pro-angiogenic activity of transplanted cells in a hind limb ischemia model in vivo. In contrast, inhibition of respiration, without affecting total ATP production, leads to a compensatory increase in glycolytic capacity correlating with increased colony forming capacity. Isolated CD34(+) , CXCR4(+) , and CD14(+) cells showed higher glycolytic activity compared to their negative counterparts. Metabolic activity was profoundly modulated by the composition of media used to store or culture BMC. This study provides first evidence that metabolic alterations influence the functional activity of human HSPC and BMC independent of ATP production. Changing the balance between respiration and glycolysis might be useful to improve patient-derived cells for clinical cardiac cell therapy. Stem Cells 2016;34:2236-2248.

The challenges of autologous cell therapy: systemic anti-thrombotic therapies interfering with serum coagulation may disable autologous serum-containing cell products for therapeutical use.

Cell therapy of acute myocardial infarction (AMI) with bone marrow-derived mononuclear cells (BMC) resulted in a modest improvement of cardiac function, but clinical trial results were heterogeneous. After isolation, BMC are maintained in medium supplemented with complements such as autologous serum to maintain optimal cell viability until administration. In the REPAIR-AMI trial, serum was prepared using tubes containing coagulation accelerators, but the regulatory agency recommended using additive-free tubes for the pivotal BAMI trial. Here, we show that serum obtained from patients with anti-thrombotic therapy in tubes without coagulation accelerators induces clotting, thereby rendering the cell product unsuitable for intra-coronary application. Specifically, systemic treatment of patients with low doses of heparin prevented efficient coagulation ex vivo, and the resulting partially clotted plasma induced cell aggregation within 1-18 h in the cell product. Utmost care has to be taken to test autologous components of cell products before clinical use. The development of media including the appropriate recombinant growth factors for maintaining cell functionality ex vivo may be warranted.

Complications associated with the prehospital use of laryngeal tubes--a systematic analysis of risk factors and strategies for prevention.

OBJECTIVE: With the increasing spread of laryngeal tubes (LT) in emergency medicine, complications and side-effects are observed. We sought to identify complications associated with the use of LTs in emergency medicine, and to develop strategies to prevent these incidents. METHODS: In a prospective clinical study, all patients who had their airways managed in the field with a LT and who were admitted through the emergency department of the Frankfurt University Hospital during a 6 year period were evaluated using anonymised data collection sheets. A team of experts was available 24/7 and was requested whenever a patient was admitted with a LT in place. This team evaluated the condition of the patients with respect to prehospital airway management and was responsible for further advanced airway management. All complications were analysed, and strategies for prevention developed. RESULTS: One hundred eighty nine patients were included and analysed. The initial cuff pressure of the LTs was 10 0 cm H2O on the median. Complications consisted of significant tongue swelling (n=73; 38.6%), resulting in life-threatening cannot ventilate, cannot intubate scenarios in two patients (1.0%) and the need for surgical tracheostomy in another patient, massive distension of the stomach (n=20, 10.6%) with ventilation difficulties when LTs without gastric drainage were used; malposition of the LT in the piriform sinus (n=1, 0.5%) and significant bleeding from soft tissue injuries (n=4, 2.1%). CONCLUSIONS: The prehospital use of LTs may result in severe and even life-threatening complications. Likely, such complications could have been prevented by using gastric drainage and cuff pressure adjustment. Both, prehospital health care providers and emergency department staff should develop a greater awareness of such complications to best avoid them in the future.

MicroRNAs in stem cell function and regenerative therapy of the heart.

MicroRNAs are small noncoding RNAs that posttranscriptionally control gene expression by targeting mRNAs. Distinct microRNAs regulate stem and progenitor cell functions, thereby modulating cell survival and homing or controlling differentiation and maturation. Experimental studies additionally show that microRNAs regulate endogenous repair and might potentially be useful to enhance the regeneration of the heart. This review summarizes the current studies that address the use of microRNAs to either improve cellular therapies or that might be targeted for enhancing endogenous tissue repair and regeneration after myocardial infarction.

Effect of shock wave-facilitated intracoronary cell therapy on LVEF in patients with chronic heart failure: the CELLWAVE randomized clinical trial.

IMPORTANCE: The modest effects of clinical studies using intracoronary administration of autologous bone marrow-derived mononuclear cells (BMCs) in patients with chronic postinfarction heart failure may be attributed to impaired homing of BMCs to the target area. Extracorporeal shock wave treatment has been experimentally shown to increase homing factors in the target tissue, resulting in enhanced retention of applied BMCs. OBJECTIVE: To test the hypothesis that targeted cardiac shock wave pretreatment with subsequent application of BMCs improves recovery of left ventricular ejection fraction (LVEF) in patients with chronic heart failure. DESIGN, SETTING, AND PARTICIPANTS: The CELLWAVE double-blind, randomized, placebo-controlled trial conducted among patients with chronic heart failure treated at Goethe University Frankfurt, Germany, between 2006 and 2011. INTERVENTIONS: Single-blind low-dose (n = 42), high-dose (n = 40), or placebo (n = 21) shock wave pretreatment targeted to the left ventricular anterior wall. Twenty-four hours later, patients receiving shock wave pretreatment were randomized to receive double-blind intracoronary infusion of BMCs or placebo, and patients receiving placebo shock wave received intracoronary infusion of BMCs. MAIN OUTCOMES AND MEASURES: Primary end point was change in LVEF from baseline to 4 months in the pooled groups shock wave + placebo infusion vs shock wave + BMCs; secondary end points included regional left ventricular function assessed by magnetic resonance imaging and clinical events. RESULTS: The primary end point was significantly improved in the shock wave + BMCs group (absolute change in LVEF, 3.2% [95% CI, 2.0% to 4.4%]), compared with the shock wave + placebo infusion group (1.0% [95% CI, -0.3% to 2.2%]) (P = .02). Regional wall thickening improved significantly in the shock wave + BMCs group (3.6% [95% CI, 2.0% to 5.2%]) but not in the shock wave + placebo infusion group (0.5% [95% CI, -1.2% to 2.1%]) (P = .01). Overall occurrence of major adverse cardiac events was significantly less frequent in the shock wave + BMCs group (n = 32 events) compared with the placebo shock wave + BMCs (n = 18) and shock wave + placebo infusion (n = 61) groups (hazard ratio, 0.58 [95% CI, 0.40-0.85]; P = .02). CONCLUSIONS AND RELEVANCE: Among patients with postinfarction chronic heart failure, shock wave-facilitated intracoronary administration of BMCs vs shock wave treatment alone resulted in a significant, albeit modest, improvement in LVEF at 4 months. Determining whether the increase in contractile function will translate into improved clinical outcomes requires confirmation in larger clinical end point trials. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00326989.

Impact of intracoronary reinfusion of bone marrow-derived mononuclear progenitor cells on cardiopulmonary exercise capacity in patients with chronic postinfarction heart failure.

INTRODUCTION: Intracoronary infusion of bone marrow-derived progenitor cells (BMC) in patients with chronic ischaemic heart failure (CHF) is associated with improvement in left ventricular ejection fraction (LVEF), reduction of NT-proBNP levels and improved prognosis. However, effects of this therapy on cardiopulmonary exercise capacity have not been investigated separately so far. PATIENTS AND METHODS: One hundred and fifty-four patients with ischaemic heart failure (mean LVEF 40.3 ± 10.9 %, NT-proBNP 1,103 ± 1,436 pg/ml) underwent cardiopulmonary exercise capacity testing (CPX) before and 3 months after intracoronary infusion of autologous BMC. Thirty patients with a potential bias on the CPX course as concomitant coronary intervention, bypass surgery, new onset of arrhythmias or implantation of cardiac resynchronization devices were excluded from further analysis. RESULTS: The remaining 124 patients showed an increase in exercise time and peak workload by 16.8 and 6 %. Peak oxygen uptake and oxygen uptake efficiency slope also improved by 2.9 and 12.9 %, whereas other parameters like peak oxygen pulse and the slope of minute ventilation versus CO2 elimination remained unchanged. Analysis of patients with poor, moderate and conserved CPX results prior to cell therapy documented that patients in tertiles with lowest initial exercise capacity showed the largest improvements in CPX after therapy. The differences in response to cell therapy were detectable in all investigated CPX parameters and became significant for exercise time, peak oxygen uptake and peak oxygen pulse. These findings indicate that intracoronary BMC therapy improves exercise capacity in CHF patients with more advanced heart failure.

Comparison of the Seattle heart failure model and cardiopulmonary exercise capacity for prediction of death in patients with chronic ischemic heart failure and intracoronary progenitor cell application.

BACKGROUND: Despite many therapeutic advances, the prognosis of patients with chronic heart failure (CHF) remains poor. Therefore, reliable identification of high-risk patients with poor prognosis is of utmost importance. Cardiopulmonary exercise testing (CPET) provides important prognostic information by peak O2 uptake (peak VO2 ), maximal oxygen pulse (O2 Pmax), O2 uptake efficiency slope (OUES), and VE/VCO2 slope (VE/VCO2 ). A different approach for prognostic assessment is the Seattle Heart Failure Model (SHFM), which is based on clinical data and calculates the estimated annual mortality. HYPOTHESIS: Comparison of the prognostic value of the Seattle Heart Failure Score and cardiopulmonary excercis testing in patients with chronic heart failure. METHODS: One hundred fifty-seven patients with ischemic heart failure and recent intracoronary progenitor cell application were analyzed for mortality during a follow-up of 4 years. CPET (peak VO2 , O2 Pmax, OUES, VE/VCO2 ) was performed in all patients at baseline. The SHFM score was calculated for every patient, with data obtained at the time of CPET. RESULTS: During follow-up, 24 patients died (15.2%). Nonsurvivors had significantly worse initial CPET results and a higher SHFM score compared to survivors. Receiver operating characteristics curve analysis of sensitivity and specificity revealed the largest area under the curve value for the SHFM score, followed by VE/VCO2 slope. Kaplan Meier analysis using cutoff points of SHFM and VE/VCO2 slope with highest sensitivity and specificity resulted in significant discrimination of survivors and nonsurvivors. By multivariate analysis, only the SHFM score persisted as independent predictor of mortality in these patients. CONCLUSIONS: These data indicate superior prognostic power of the SHFM score compared to CPET in patients with chronic ischemic heart failure.

Procedural safety and predictors of acute outcome of intracoronary administration of progenitor cells in 775 consecutive procedures performed for acute myocardial infarction or chronic heart failure.

BACKGROUND: Cell-based therapies are a promising option in patients with acute myocardial infarction or chronic heart failure (CHF). However, administration of cells requires intracoronary or intracardiac instrumentation, which is potentially associated with periprocedural risks. Therefore, we analyzed periprocedural complications and 30-day outcome in 775 consecutive procedures of intracoronary administration of progenitor cells using the stop-flow technique. METHODS AND RESULTS: Indications for cell administration were acute myocardial infarction (n=126) and CHF of ischemic (n=562) or nonischemic (n=87) etiology. Vessel injury was observed in a total of 9 procedures (1.2%) and could be promptly managed by additional progenitor cell injection (PCI) in all but 1 case. No procedural deaths were observed. A periprocedural increase in troponin T was observed in 3.2% of the CHF procedures, in which no concomitant PCI was performed and troponin levels were not elevated before the procedure. Independent significant predictors of troponin T increase were higher New York Heart Association (NYHA) class (NYHA I versus NYHA IV; P=0.01; NYHA I versus III; P=0.19; NYHA I versus II; P=0.55), concomitant revascularization (P<0.01), presence of elevated troponin T before the procedure (P<0.01), and peripheral occlusive disease (P=0.04). At 30 days, there were 4 deaths (0.5%), 1 stroke (0.13%), 8 acute myocardial infarctions (1%), and 5 hospitalizations for exacerbation of heart failure (0.64%). CONCLUSIONS: Intracoronary infusion of progenitor cells can be performed with adequate safety in patients with acute myocardial infarction or CHF, because the safety profile was similar to what is usually expected from a coronary angiogram in the present cohort. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00962364, NCT00284713, and NCT00289822.

Immunosenescence-associated microRNAs in age and heart failure.

AIMS: Ageing of the immune system, immunosenescence, is characterized by impaired lymphopoiesis, especially B-lymphocyte maturation, and is a hallmark of chronic heart failure (CHF). MicroRNAs (miRNAs) are non-coding, small RNAs, which post-transcriptionally control gene expression of multiple target genes. The miR-181 family is known to control haematopoietic lineage differentiation. Here, we study the role of the miR-181 family in immunosenescence and CHF. METHODS AND RESULTS: We conducted a clinical study analysing peripheral blood (PB) for miRNA expression and leucocyte distribution of young healthy controls (25 ± 4 years; n = 30), aged healthy controls (64 ± 5 years; n = 13), and age-matched CHF patients (64 ± 11years; n = 18). The expression of miR-181 family members was reduced, whereas miR-34a was increased in PB of aged individuals. In particular, miR-181c was further reduced in age-matched CHF patients. In PB, we observed reduced numbers of lymphocytes, in particular cytotoxic T cells and B cells, with rising age, and the expression of miR-181 correlated with the number of B cells. Notably, in CHF patients, ischaemic heart failure was associated with a further reduction of total B cells as well as their subpopulations, such as memory B cells, compared with age-matched healthy volunteers. CONCLUSIONS: Ageing- and CHF-associated changes in PB leucocyte subsets are paralleled by alterations in the expression of miRNAs involved in lymphopoiesis, which might play an important role in the age-related and CHF-mediated dysregulation of immune functions resulting in immunosenescence. Furthermore, miR-181c may serve as a marker for reduced immune functions in CHF patients.

Simplistic approach to prognosis after cardiopulmonary resuscitation-value of pH and lactate.

PURPOSE: The purpose was to determine the prognostic value of broadly available clinical parameters such as pH and lactate for early stratification of outcome after cardiopulmonary resuscitation. METHODS: We analyzed patients with status postresuscitation who were admitted to the Frankfurt University hospital. Arterial pH and serum lactate levels were dichotomized as "high" or "low." Primary end point was a combination of death or severe hypoxic brain damage within 30 days. RESULTS: In total, 67% of 206 patients (66 ± 12 years) reached the primary end point. Univariate analysis showed that age more than 65 years (hazard ratio [HR] 1.7; 95% confidence interval (CI), 1.2-2.4), high lactate (>6.94 mmol/L [62.5 mg/dL)]; HR 2.8; 95% CI 2.0-3.9), and low pH (<7.21; HR 2.7; 95% CI 1.9-3.8) on admission were associated with the end point. Upon multivariate analysis, age more than 65 years, high lactate, and low pH on admission remained as independent predictors. Specificity and sensitivity to detect patients with an end point were 0.51 and 1.0 for the combination of low pH, high lactate, and age more than 65 years (negative predictive value, 1.0). CONCLUSION: A combination of clinical data and broadly available parameters can help to stratify prognosis of patients after cardiopulmonary resuscitation with sufficiently high predictive value. Interestingly, a combination of the 3 parameters-age more than 65 years, high lactate, and low pH upon admission-had a sensitivity of 1.0 for a poor outcome after return of spontaneous circulation.

Does the integration of personalized ultrasound change patient management in critical care medicine? Observational trials.

Objective. To test the influence of personalized ultrasound (PersUS) on patient management in critical care. Design of the Study. Prospective, observational, and critical care setting. Four substudies compared PersUS and mobile ultrasound, work distribution, and diagnostic and procedural quality. Patients and Interventions. 640 patient ultrasound exams including 548 focused diagnostic exams and 92 interventional procedures. Main Outcome Measures. Number of studies, physician's judgement of feasibility, time of usage per patient, and referrals to echo lab. Results. Randomized availability of PersUS increased its application in ICU work shifts more than twofold from 33 to 68 exams mainly for detection and therapy of effusions. Diagnostic and procedural quality was rated as excellent/very good in PersUS-guided puncture in 95% of cases. Integrating PersUS within an initial physical examination of 48 randomized cases in an emergency department, PersUS extended the examination time by 100 seconds. Interestingly, PersUS integration into 53 randomized regular ward rounds of 1007 patients significantly reduced average contact time per patient by 103 seconds from 8.9 to 7.2 minutes. Moreover, it lowered the patient referral rate to an echo lab from 20% to 2% within the study population. Conclusions. We propose the development of novel ultrasound-based clinical pathways by integration of PersUS.

Elevated levels of the mediator of catabolic bone remodeling RANKL in the bone marrow environment link chronic heart failure with osteoporosis.

BACKGROUND: Chronic heart failure (CHF) is associated with a 4-fold increased risk for osteoporotic fractures. Therefore, we sought to identify the pathophysiological link between chronic heart failure and catabolic bone remodeling. METHODS AND RESULTS: In a total cohort of 153 subjects (123 patients with CHF, 30 patients with coronary artery disease and preserved cardiac function) as well as mice with heart failure, bone marrow (BM) plasma levels of the catabolic receptor activator of NF-κB ligand (RANKL), and its antagonist, osteoprotegerin were measured. The osteoclast inducing activity of BM plasma was tested in cell culture. BM plasma levels of RANKL and of the ratio RANKL/osteoprotegerin were significantly elevated in patients with CHF. On multivariate regression analysis, parameters of severity and duration of heart failure were independent determinants of elevated BM plasma RANKL levels. BM plasma levels of RANKL were directly correlated with the systemic marker of bone turnover C-telopeptide of type 1 collagen (r=0.6; P<0.001). Alterations in BM plasma levels of RANKL/osteoprotegerin were confirmed in a mouse model of postinfarction heart failure. Stimulation of human mesenchymal cells with BM plasma obtained from CHF patients increased the formation of osteoclasts, and this effect was blocked by the RANKL inhibition. CONCLUSIONS: CHF is associated with a profound and selective elevation of the bone resorption stimulating RANKL within the BM microenvironment. These data for the first time disclose a direct pathophysiological pathway linking CHF with catabolic bone remodeling associated with an increased osteoporotic fracture risk. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifiers: NCT 00289822, NCT 00284713, NCT 00326989, NCT 00962364.

G-CSF stimulation and coronary reinfusion of mobilized circulating mononuclear proangiogenic cells in patients with chronic ischemic heart disease:five-year results of the TOPCARE-G-CSF trial.

Prognosis of patients with heart failure remains poor despite improved conventional and interventional treatment regimens. The improvement of neovascularization and repair processes by administration of bone marrow-derived cells modestly improved the recovery after acute myocardial infarction. However, circulating patient-derived cells are reduced in number and function particularly in chronic heart failure. Therefore, we tested the hypothesis whether the mobilization of circulating mononuclear proangiogenic cells (CPCs) by G-CSF may overcome some of these limitations. In the present pilot study, 32 patients with at least 3-month-old myocardial infarction were randomized to G-CSF alone (G-CSF group) or intracoronary infusion of G-CSF-mobilized and cultured CPCs into the infarct-related artery (G-CSF/CPC group). Primary endpoint of the study was safety. Efficacy parameters included serial assessment of LV function, NT-proBNP levels, and cardiopulmonary exercise testing. G-CSF effectively mobilized circulating CD34(+)CD45(+) cells after 5 days in all patients (408 ± 64%) without serious adverse events. At 3 months, NYHA class and global LV function did not show significant improvements in both treatment groups (G-CSF: ΔLVEF 1.6 ± 2.4%; p = 0.10; G-CSF/CPC: ΔLVEF 1.4 ± 4.1%; p = 0.16). In contrast, target area contractility improved significantly in the G-CSF/CPC group. During 5-year follow-up, one patient died after rehospitalization for worsening heart failure. Eleven patients underwent further revascularization procedures. NT-proBNP levels, cardiopulmonary exercise capacity, and NYHA class remained stable in both treatment groups. The results from our pilot trial indicate that administration of G-CSF alone or G-CSF-mobilized and cultured CPCs can be performed safely in patients with chronic ischemic heart disease. However, only minor effects on LV function, NT-proBNP levels, and NYHA classification were observed during follow-up, suggesting that the enhancement of CPCs by G-CSF alone does not substantially improve intracoronary cell therapy effects in patients with chronic ischemic heart failure.

Heparin disrupts the CXCR4/SDF-1 axis and impairs the functional capacity of bone marrow-derived mononuclear cells used for cardiovascular repair.

RATIONALE: Cell therapy is a promising option for the treatment of acute or chronic myocardial ischemia. The intracoronary infusion of cells imposes the potential risk of cell clotting, which may be prevented by the addition of anticoagulants. However, a comprehensive analysis of the effects of anticoagulants on the function of the cells is missing. OBJECTIVE: Here, we investigated the effects of heparin and the thrombin inhibitor bivalirudin on bone marrow-derived mononuclear cell (BMC) functional activity and homing capacity. METHODS AND RESULTS: Heparin, but not bivalirudin profoundly and dose-dependently inhibited basal and stromal cell-derived factor 1 (SDF-1)-induced BMC migration. Incubation of BMCs with 20 U/mL heparin for 30 minutes abrogated SDF-1-induced BMC invasion (16±8% of control; P<0.01), whereas no effects on apoptosis or colony formation were observed (80±33% and 100±44% of control, respectively). Pretreatment of BMCs with heparin significantly reduced the homing of the injected cells in a mouse ear-wound model (69±10% of control; P<0.05). In contrast, bivalirudin did not inhibit in vivo homing of BMCs. Mechanistically, heparin binds to both, the chemoattractant SDF-1 and its receptor, chemokine receptor 4 (CXCR4), blocking CXCR4 internalization as well as SDF-1/CXCR4 signaling after SDF-1 stimulation. CONCLUSIONS: Heparin blocks SDF-1/CXCR4 signaling by binding to the ligand as well as the receptor, thereby interfering with migration and homing of BMCs. In contrast, the thrombin inhibitor bivalirudin did not interfere with BMC homing or SDF-1/CXCR4 signaling. These findings suggest that bivalirudin but not heparin might be recommended as an anticoagulant for intracoronary infusion of BMCs for cell therapy after cardiac ischemia.

Micro-RNA-34a contributes to the impaired function of bone marrow-derived mononuclear cells from patients with cardiovascular disease.

OBJECTIVES: This study evaluated the regulation and function of micro-RNAs (miRs) in bone marrow-mononuclear cells (BMCs). BACKGROUND: Although cell therapy with BMCs may represent a therapeutic option to treat patients with heart disease, the impaired functionality of patient-derived cells remains a major challenge. Small noncoding miRs post-transcriptionally control gene expression patterns and play crucial roles in modulating cell survival and function. METHODS: Micro-RNAs were detected by miR profiling in BMCs isolated from healthy volunteers (n = 6) or from patients with myocardial infarction (n = 6), and the results were confirmed by polymerase chain reaction (PCR) in a larger cohort (n = 37). The function of selected miRs was determined by gain-of-function studies in vitro and by locked nuclear acid (LNA) modified inhibitors in vitro and in vivo. RESULTS: We identified several miRs that are up-regulated in BMCs from patients with myocardial infarction compared with BMCs from healthy controls, including the pro-apoptotic and antiproliferative miR-34a and the hypoxia-controlled miR-210. Inhibition of miR-34 by LNA-34a significantly reduced miR-34a expression and blocked hydrogen peroxide-induced cell death of BMC in vitro, whereas overexpression of miR-34a reduced the survival of BMCs in vitro. Pre-treatment of BMCs with LNA-34a ex vivo significantly increased the therapeutic benefit of transplanted BMCs in mice after acute myocardial infarction (AMI). CONCLUSIONS: These results demonstrate that cardiovascular disease modulates the miR expression of BMCs in humans. Reducing the expression of the pro-apoptotic miR-34a improves the survival of BMCs in vitro and enhances the therapeutic benefit of cell therapy in mice after AMI.

Early remodeling processes as predictors of diastolic function 5 years after reperfused acute myocardial infarction and intracoronary progenitor cell application.

AIMS: Ischemia-induced left ventricular (LV) diastolic dysfunction (DD) is increasingly recognized as a therapeutic challenge. While DD during acute myocardial infarction (AMI) determines patients' prognosis, it is unknown how LV remodeling after AMI affects the development of DD. Therefore, we aimed to identify AMI characteristics, which determine diastolic function after 5 years. METHODS AND RESULTS: 41 patients with reperfused AMI and intracoronary infusion of progenitor cells were included into the present analysis of the TOPCARE-AMI trial. At 5-year follow-up, we determined LV diastolic function including LV-filling index (E/E') by echocardiography. Diastolic function was normal in 21 patients (DD class 0), impaired in 14 patients (DD class 1) and pseudonormal in 6 patients (DD class 2). E/E' increased from DD class 0 to 2 (6.6 ± 1.3 vs. 9.0 ± 2.4 vs. 12.1 ± 6.2; p < 0.01). E/E' correlated with the maximal creatine kinase activity during AMI (CKMB(max) r = 0.73, p < 0.01), the change in end-diastolic or end-systolic LV volumes between AMI and 4 months (∆LVEDV r = 0.67, p < 0.01; ∆LVESV r = 0.58, p < 0.01), ejection fraction at 5 years (r = -0.47, p < 0.01) and NT-proBNP serum levels at 5 years (r = 0.37, p < 0.05). Multivariate analysis revealed CKMB(max) (ß = 0.56, p < 0.01) and ∆LVEDV (ß = 0.38, p < 0.01) as independent predictors for E/E' 5 years after AMI. CONCLUSION: Adverse early remodeling processes (reflected by LV dilatation between infarction and 4 months) determine long-term diastolic function in patients after reperfused AMI and progenitor cell therapy.

Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI): final 5-year results suggest long-term safety and efficacy.

BACKGROUND: Limited data is available for investigating the long-term safety and effects of intracoronary progenitor cell therapy in patients with acute myocardial infarction (AMI). OBJECTIVE: To assess the clinical course, NT-proBNP and MRI data as objective markers of cardiac function of the TOPCARE-AMI patients at 5-year follow-up. DESIGN: The TOPCARE-AMI trial was the first randomized study investigating the effects of intracoronary infusion of circulating (CPC) or bone marrow-derived progenitor cells (BMC) in 59 patients with successfully reperfused AMI. RESULTS: Five-year follow-up data were completed in 55 patients, 3 patients were lost to follow-up. None of the patients showed any signs of intramyocardial calcification or tumors at 5 years. One patient died during the initial hospitalization, no patient was rehospitalized for heart failure and 16 patients underwent target vessel revascularization (TVR). Only two TVRs occurred later than 1 year after cell administration making it very unlikely that the infused cells accelerate atherosclerotic disease progression. Serum levels of NT-proBNP remained significantly reduced at the 5-year follow-up indicating the absence of heart failure. MRI subgroup analysis in 31 patients documented a persistent improvement of LV ejection fraction (from 46 ± 10% at baseline to 57 ± 10% at 5 years, p < 0.001)). Simultaneously, there was a reduction (p < 0.001) in functional infarct size measured as late enhancement volume normalized to LV mass. However, whereas LV end-systolic volume remained stable, LV end-diastolic volume increased significantly. CONCLUSIONS: The 5-year follow-up of the TOPCARE-AMI trial provides reassurance with respect to the long-term safety of intracoronary cell therapy and suggests favorable effects on LV function.

Kruppel-like factor 2 improves neovascularization capacity of aged proangiogenic cells.

AIMS: Coronary artery disease (CAD) patients have less circulating proangiogenic cells (PACs), formerly known as endothelial progenitor cells, which exhibit impaired neovascularization properties. Inverse correlations were also found between PAC function and risk factors like age. Krüppel-like factor 2 (KLF2) is expressed by mature endothelial cells (ECs), is induced by both shear stress and statins, and provokes endothelial functional differentiation. The aim of this study is to identify whether KLF2 can reverse negative effects of ageing on PAC function. METHODS AND RESULTS: We describe that progenitor cells in the bone marrow and PACs also express KLF2 at a comparable level to mature ECs and that senescence decreases KLF2 levels. To study the effects of ageing on KLF2 levels, we compared progenitor cells of 4 weeks and 16- to 18-month-old C57BL/6 mice. In addition to the three-fold reduction of circulating Sca1(+)/c-Kit(+)/Lin(-) progenitor cells and the 15% reduction of Sca1(+)/Flk1(+) endothelial-committed progenitor cells, the spleen-derived PACs and bone marrow-derived progenitor cells isolated from aged mice showed a lower level of KLF2 when compared with young mice. Lentiviral overexpression of KLF2 increased human PAC numbers and endothelial nitric oxide synthase expression by 60% during in vitro culture. Endothelial lineage-specific KLF2 overexpression in aged bone marrow-derived mononuclear cells strongly augments neovascularization in vivo in a murine hind-limb ischaemia model. CONCLUSION: These results imply that KLF2 is an attractive novel target to rejuvenate PACs before autologous administration to CAD patients.