Clonal haematopoiesis in chronic ischaemic heart failure: prognostic role of clone size for DNMT3A- and TET2-driver gene mutations.

AIMS: Somatic mutations of the epigenetic regulators DNMT3A and TET2 causing clonal expansion of haematopoietic cells (clonal haematopoiesis; CH) were shown to be associated with poor prognosis in chronic ischaemic heart failure (CHF). The aim of our analysis was to define a threshold of variant allele frequency (VAF) for the prognostic significance of CH in CHF. METHODS AND RESULTS: We analysed bone marrow and peripheral blood-derived cells from 419 patients with CHF by error-corrected amplicon sequencing. Cut-off VAFs were optimized by maximizing sensitivity plus specificity from a time-dependent receiver operating characteristic (ROC) curve analysis from censored data. 56.2% of patients were carriers of a DNMT3A- (N = 173) or a TET2- (N = 113) mutation with a VAF >0.5%, with 59 patients harbouring mutations in both genes. Survival ROC analyses revealed an optimized cut-off value of 0.73% for TET2- and 1.15% for DNMT3A-CH-driver mutations. Five-year-mortality was 18% in patients without any detected DNMT3A- or TET2 mutation (VAF < 0.5%), 29% with only one DNMT3A- or TET2-CH-driver mutations above the respective cut-off level and 42% in patients harbouring both DNMT3A- and TET2-CH-driver mutations above the respective cut-off levels. In carriers of a DNMT3A mutation with VAF ≥ 1.15%, 5-year mortality was 31%, compared with 18% mortality in those with VAF < 1.15% (P = 0.048). Likewise, in patients with TET2 mutations, 5-year mortality was 32% with VAF ≥ 0.73%, compared with 19% mortality with VAF < 0.73% (P = 0.029). CONCLUSION: The present study defines novel threshold levels for clone size caused by acquired somatic mutations in the CH-driver genes DNMT3A and TET2 that are associated with worse outcome in patients with CHF.

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.

Association of Mutations Contributing to Clonal Hematopoiesis With Prognosis in Chronic Ischemic Heart Failure.

Importance: Somatic mutations causing clonal expansion of hematopoietic cells (clonal hematopoiesis of indeterminate potential [CHIP]) are increased with age and associated with atherosclerosis and inflammation. Age and inflammation are the major risk factors for heart failure, yet the association of CHIP with heart failure in humans is unknown. Objective: To assess the potential prognostic significance of CHIP in patients with chronic heart failure (CHF) owing to ischemic origin. Design, Setting, and Participants: We analyzed bone marrow-derived mononuclear cells from 200 patients with CHF by deep targeted amplicon sequencing to detect the presence of CHIP and associated such with long-term prognosis in patients with CHF at University Hospital Frankfurt, Frankfurt, Germany. Data were analyzed between October 2017 and April 2018. Results: Median age of the patients was 65 years. Forty-seven mutations with a variant allele fraction (VAF) of at least 0.02 were found in 38 of 200 patients with CHF (18.5%). The somatic mutations most commonly occurred in the genes DNMT3A (14 patients), TET2 (9 patients), KDM6A (4 patients), and BCOR (3 patients). Patients with CHIP were older and more frequently had a history of hypertension. During a median follow-up of 4.4 years, a total of 53 patients died, and 23 patients required hospitalization for heart failure. There was a significantly worse long-term clinical outcome for patients with either DNMT3A or TET2 mutations compared with non-CHIP carriers. By multivariable Cox proportional regression analysis, the presence of somatic mutations within TET2 or DNMT3A (HR, 2.1; 95% CI, 1.1-4.0; P = .02, for death combined with heart failure hospitalization) and age (HR, 1.04; 95% CI, 1.01-1.07 per year; P = .005) but not a history of hypertension remained independently associated with adverse outcome. Importantly, there was a significant dose-response association between VAF and clinical outcome. Conclusions and Relevance: Our data suggest that somatic mutations in hematopoietic cells, specifically in the most commonly mutated CHIP driver genes TET2 and DNMT3A, may be significantly associated with the progression and poor prognosis of CHF. Future studies will have to validate our findings in larger cohorts and address whether targeting specific inflammatory pathways may be valuable for precision medicine in patients with CHF carrying specific mutations encoding for CHIP.

Patatin-like phospholipase domain containing 3 variants differentially impact metabolic traits in individuals at high risk for cardiovascular events.

Single nucleotide polymorphism (SNP) rs738409 C>G in the patatin-like phospholipase domain containing 3 (PNPLA3) gene results in an amino acid exchange from isoleucin to methionine at position I148M of PNPLA3. The expression of this loss-of-function mutation leads to impaired hepatocellular triglyceride hydrolysis and is associated with the development of liver steatosis, fibrosis, and hepatocellular carcinoma. In contrast to these well-established associations, the relationship of the PNPLA3 rs738409 variant with other metabolic traits is incompletely understood. We therefore assessed the association of the PNPLA3 rs738409 genotype with relevant metabolic traits in a prospective study of patients at high risk for cardiovascular events, i.e., patients undergoing coronary angiography. In a total of 270 patients, known associations of the PNPLA3 rs738409 GG genotype with nonalcoholic steatohepatitis and liver fibrosis were confirmed. In addition, we found an association of the PNPLA3 rs738409 G allele with the presence of diabetes (22% versus 28% versus 58% for CC versus CG versus GG genotype, respectively; P = 0.02). In contrast to its association with nonalcoholic fatty liver disease, liver fibrosis, and diabetes, the minor G allele of PNPLA3 rs738409 was inversely associated with total serum cholesterol and low-density lipoprotein serum levels (P = 0.003 and P = 0.02, respectively). Finally, there was a trend toward an inverse association between the presence of the PNPLA3 rs738409 G allele and significant coronary heart disease. Comparable trends were observed for the transmembrane 6 superfamily member 2 (TM6SF2) 167 K variant, but the sample size was too small to evaluate this rarer variant. Conclusion: The PNPLA3 rs738409 G allele is associated with liver disease but also with a relatively benign cardiovascular risk profile. (Hepatology Communications 2018;2:798-806).

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.

A Decade of Transapical Aortic Valve Implantation.

BACKGROUND: Transcatheter-based aortic valve procedures have undergone tremendous evolution during the past decade and have led to great changes in the treatment of valvular heart disease. The Hospital of the Johann Wolfgang Goethe University, Frankfurt am Main, Germany is one of the three pioneering centers that started performing transapical transcatheter aortic valve implantation (TA-TAVI) back in 2005, and this study reviews the 10-year institutional experience with this approach. METHODS: From January 2005 through January 2015, 312 consecutive high-risk patients underwent TA-TAVI. Echocardiographic follow-up at discharge, at 6 and 12 months, and yearly thereafter was 100% complete. Structural behavior of the balloon-expandable valves in 11 patients with a mean follow-up time beyond 8 years was additionally evaluated at latest follow-up using computed tomography measurements. RESULTS: The age of the patients in this study was 79.8 ± 5.8 years, and the mean logistic EuroSCORE II and The Society of Thoracic Surgeons score were 23.9% ± 17.2% and 9.8% ± 8.6%, respectively. Perioperative, 30-day, and in-hospital mortality rates were 1.3%, 8.2%, and 9.5%, respectively, with a decrease in 30-day mortality to 4.2% in 2014. The incidence of neurologic complications was 3.2%. Mean length of hospital stay was 8.7 ± 4.3 days. Echocardiographic results demonstrated a significant and persistent increase of effective aortic valve orifice area (preoperative: 0.69 ± 0.1 cm(2) vs. late-follow-up: 1.52 ± 0.2 cm(2); p = 0.04) and a decrease in mean transvalvular gradient (preoperative: 49.5 ± 8.2 mm Hg vs. late-follow-up: 13.8 ± 4.3 mm Hg; p = 0.03) after a mean follow-up time of 4.1 ± 2.3 years. Overall survival rates were 73% ± 2% and 56% ± 6% at 3 and 5 years, respectively. Computed tomography measurements have not shown any signs of stress fracture of balloon-expandable stents up to 8 years of follow-up. CONCLUSIONS: A decade after clinical introduction of TA-TAVI, procedural and technical advances have made it an established alternative to classic aortic valve replacement in high-risk patients with aortic valve stenosis. Despite limited worldwide data on hemodynamic and structural valve behavior beyond 8 years, 11 patients from our early experience who were followed up for 8 years in the current report did not have any signs of structural valve dysfunction.

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.

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.

Stem cell compartmentalization in diabetes and high cardiovascular risk reveals the role of DPP-4 in diabetic stem cell mobilopathy.

Bone marrow (BM) derived stem and progenitor cells contribute to cardiovascular homeostasis and are affected by cardiovascular risk factors. We devised a clinical data-driven approach to test candidate stem cell mobilizing mechanisms in pre-clinical models. We found that PB and BM CD34+ cell counts were directly correlated, and that most circulating CD34+ cells were viable, non-proliferating and derived from the BM. Thus, we analyzed PB and BM CD34+ cell levels as a two-compartment model in 72 patients with or without cardiovascular disease. Self-organizing maps showed that disturbed compartmentalization of CD34+ cells was associated with aging and cardiovascular risk factors especially diabetes. High activity of DPP-4, a regulator of the mobilizing chemokine SDF-1α, was associated with altered stem cell compartmentalization. For validation of these findings, we assessed the role of DPP-4 in the BM mobilization response of diabetic rats. Diabetes differentially affected DPP-4 activity in PB and BM and impaired stem/progenitor cell mobilization after ischemia or G-CSF administration. DPP-4 activity in the BM was required for the mobilizing effect of G-CSF, while in PB it blunted ischemia-induced mobilization. Indeed, DPP-4 deficiency restored ischemia (but not G-CSF)-induced stem cell mobilization and improved vascular recovery in diabetic animals. In conclusion, the analysis of stem cell compartmentalization in humans led us to discover mechanisms of BM unresponsiveness in diabetes determined by tissue-specific DPP-4 dysregulation.

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.

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.

Regulation of cardiac microRNAs by bone marrow mononuclear cell therapy in myocardial infarction.

BACKGROUND: Cell therapy with bone marrow-derived mononuclear cells (BMCs) can improve recovery of cardiac function after ischemia; however, the molecular mechanisms are not yet fully understood. MicroRNAs (miRNAs) are key regulators of gene expression and modulate the pathophysiology of cardiovascular diseases. METHODS AND RESULTS: We demonstrated that intramyocardial delivery of BMCs in infarcted mice regulates the expression of cardiac miRNAs and significantly downregulates the proapoptotic miR-34a. In vitro studies confirmed that the supernatant of BMC inhibited the expression of H(2)O(2)-induced miR-34a and cardiomyocytes apoptosis. These effects were blocked by neutralizing antibodies directed against insulin-like growth factor-1 (IGF-1). Indeed, IGF-1 significantly inhibited H(2)O(2)-induced miR-34a expression, and miR-34a overexpression abolished the antiapoptotic effect of IGF-1. Likewise, inhibition of IGF-1 signaling in vivo abolished the BMC-mediated inhibition of miR-34 expression and the protective effect on cardiac function and increased apoptosis and cardiac fibrosis. IGF-1 specifically blocked the expression of the precursor and the mature miR-34a, but did not interfere with the transcription of the primary miR-34a demonstrating that IGF-1 blocks the processing of miR-34a. CONCLUSIONS: Together, our data demonstrate that the paracrine regulation of cardiac miRNAs by transplanted BMCs contributes to the protective effects of cell therapy. BMCs release IGF-1, which inhibits the processing of miR-34a, thereby blocking cardiomyocyte apoptosis.

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.