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.

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.

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.

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.

Inhibition of the p38 MAP kinase in vivo improves number and functional activity of vasculogenic cells and reduces atherosclerotic disease progression.

Initial trials suggest that bone marrow-derived vasculogenic cells augment neovascularization in patients after myocardial infarction. Moreover, in some experimental settings, they also provide an anti-atherosclerotic effect by maintaining the integrity of the endothelium. Risk factors for coronary artery disease were shown to reduce the number and functional activity of vasculogenic cells and increased cellular p38 MAP kinase activity. Inhibition of p38 MAP kinase increases the number and functional activity of proangiogenic cells in vitro and clinical trials are under way to examine the effect of p38 inhibition in patients with CAD. Here, we examined the effect of systemic p38 MAP kinase inhibition on vasculogenic cells and atherosclerotic disease progression in vivo. Treatment of ApoE(-/-) mice with the p38 inhibitor SB203580 significantly increased the number of pro-angiogenic cells such as Sca-1(+)Flk-1(+) as well as CD11b(low)Flk-1(+) cells and reduced the number of the inflammatory Gr1(+)CD45(+) cells. Moreover, invasion capacity of bone marrow-derived mononuclear cells under basal conditions as well as towards a gradient of SDF-1 was significantly augmented in ApoE-/- mice after p38 inhibition. Finally, treatment of ApoE(-/-) mice with SB203580 for 4 months reduced atheromatous lesion size by 51 +/- 3% (p < 0.05) without affecting the density of vasa vasorum in the plaques. In conclusion, this study demonstrates that systemic p38 MAP kinase inhibition with SB203580 improves the number and function of vasculogenic cells in an animal model of hypercholesterolemia, and reduces atherosclerotic disease progression in ApoE(-/-) mice.

CXCR4 expression determines functional activity of bone marrow-derived mononuclear cells for therapeutic neovascularization in acute ischemia.

OBJECTIVE: Bone marrow-derived mononuclear cells (BMCs) improve the functional recovery after ischemia. However, BMCs comprise a heterogeneous mixture of cells, and it is not known which cell types are responsible for the induction of neovascularization after cell therapy. Because cell recruitment is critically dependent on the expression of the SDF-1-receptor CXCR4, we examined whether the expression of CXCR4 may identify a therapeutically active population of BMCs. METHODS AND RESULTS: Human CXCR4(+) and CXCR4(-) BMCs were sorted by magnetic beads. CXCR4(+) BMCs showed a significantly higher invasion capacity under basal conditions and after SDF-1 stimulation. Hematopoietic or mesenchymal colony-forming capacity did not differ between CXCR4(+) and CXCR4(-) BMCs. Injection of CXCR4(+) BMCs in mice after induction of hindlimb ischemia significantly improved the recovery of perfusion compared to injection of CXCR4(-) BMCs. Likewise, capillary density was significantly increased in CXCR4(+) BMC-treated mice. Because part of the beneficial effects of cell therapy were attributed to the release of paracrine effectors, we analyzed BMC supernatants for secreted factors. Importantly, supernatants of CXCR4(+) BMCs were enriched in the proangiogenic cytokines HGF and PDGF-BB. CONCLUSIONS: CXCR4(+) BMCs exhibit an increased therapeutic potential for blood flow recovery after acute ischemia. Mechanistically, their higher migratory capacity and their increased release of paracrine factors may contribute to enhanced tissue repair.

Pilot trial on determinants of progenitor cell recruitment to the infarcted human myocardium.

BACKGROUND: Clinical trials indicate a beneficial effect of intracoronary infusion of progenitor cells on myocardial function in patients with ischemic heart disease. The extent and potential determinants of proangiogenic progenitor cell homing into the damaged myocardium after intracoronary infusion and the underlying mechanisms are still unknown. METHOD AND RESULTS: Circulating proangiogenic progenitor cells isolated from peripheral blood and cultivated for 3 days were labeled with radioactive indium oxine ((111)In-oxine). Radiolabeled proangiogenic progenitor cells (7.6+/-3.0 MBq, mean+/-SD) were administered to patients with previous myocardial infarction and a revascularized infarct vessel at various stages after infarction (5 days to 17 years). Viability of the infarcted myocardium was determined by (18)F-fluorodeoxyglucose-positron emission tomography and microcirculatory function by intracoronary Doppler measurements. One hour after application of progenitor cells, a mean of 6.9+/-4.7% (range, 1% to 19%; n=17) of total radioactivity was detected in the heart, which declined to 2+/-1% after 3 to 4 days. Average activity within the first 24 hours was highest among patients with acute myocardial infarction (14 days to 1 year; 4.5+/-3.2%; n=4) or a chronic stage (infarct age >1 year; 2.5+/-1.6%; n=5). Low viability of the infarcted myocardium and reduced coronary flow reserve were significant (P<0.05) predictors of proangiogenic progenitor cell homing. CONCLUSIONS: In patients after myocardial infarction undergoing intracoronary infusion of (111)In-oxine-labeled proangiogenic progenitor cells, a substantial amount of radioactivity is detected for several days in the heart, indicating homing of progenitor cells to the myocardium. The amount of proangiogenic progenitor cells retained in the heart decreased progressively with time after the acute myocardial infarction. Proangiogenic progenitor cells preferentially home to extensive acute myocardial infarcts characterized by low viability and reduced coronary flow reserve.

The nucleotide analogue 3-deazaadenosine prevents neointima-formation after balloon injury.

We have recently shown that 3-deazaadenosine (c3Ado) inhibits atherogenesis in mice. We studied whether its anti-inflammatory capacity would also affect neointima-formation after balloon injury. Sprague Dawley rats underwent balloon angioplasty. C3Ado was administered orally, starting 5 days prior to the balloon injury and continued for 2 weeks. Fourteen days after balloon injury the intima/media ratio in the c3Ado-treated group was reduced by 67% (p<0.001) and luminal stenosis by 50% (p<0.001). Neointimal cellular density was decreased by 25% (p<0.001) and the induction of c-Jun and ki67 was markedly lower. The reduction of the intima/media ratio was still observed 3 months after balloon injury. Furthermore, a c3Ado-dependent inhibition of PDGF-mediated ERK-activation and proliferation could be demonstrated. Short-term administration of C3Ado inhibits neointima-formation in rats for at least 3 months after injury. The present findings implicate that c3Ado may be useful as an inhibitor of restenosis-formation after balloon angioplasty in humans.

Transcoronary transplantation of functionally competent BMCs is associated with a decrease in natriuretic peptide serum levels and improved survival of patients with chronic postinfarction heart failure: results of the TOPCARE-CHD Registry.

Although intracoronary administration of bone marrow-derived mononuclear progenitor cells (BMCs) may be associated with improved cardiac function in patients with chronic postinfarction heart failure, the impact on prognosis and clinical outcome of these patients is unknown. To identify potential predictors for a favorable clinical outcome, we assessed natriuretic peptide serum levels as objective markers of heart failure and the occurrence of cardiac death in relation to functional capacity of the infused cells in a consecutive series of 121 patients with chronic ischemic heart disease treated with intracoronary infusion of BMCs. Our analyses show that both N-terminal pro-brain natriuretic peptide (NT-proBNP) and N-terminal pro-atrial natriuretic peptide (NT-proANP) serum levels were significantly reduced in patients with established postinfarction heart failure 3 months after transcoronary progenitor cell administration. NT-proBNP serum levels greater than or equal to median (735 pg/mL) at baseline and a high number of infused progenitor cells with colony-forming capacity were the only independent predictors of a favorable response 3 months after intracoronary administration of BMCs. During extended clinical follow-up (577+/-442 days), a total of 14 deaths occurred in the overall patient population. Kaplan-Meier curves for both all cause and cardiac mortality showed that patients receiving a higher number of colony-forming cells were significantly less likely to die than those patients receiving low numbers of colony-forming cells (P=0.01). Most importantly, infusion of a high number of cells with colony-forming capacity was associated with a complete abrogation of increased mortality in patients with elevated NT-proBNP serum levels (> or =735 pg/mL; median) at baseline (P<0.001). Taken together, our results show that patients with objective evidence of postinfarction heart failure demonstrate a significant reduction of both NT-proBNP and NT-proANP serum levels within 3 months following intracoronary infusion of BMCs. Importantly, infusion of progenitor cells with a high functional capacity is associated with a significantly lower mortality during further follow-up.

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.

Glycogen synthase kinase 3beta inhibits myocardin-dependent transcription and hypertrophy induction through site-specific phosphorylation.

Cardiomyocyte hypertrophy is transcriptionally controlled and inhibited by glycogen synthase kinase 3beta (GSK3beta). Myocardin is a muscle-specific transcription factor with yet unknown relation to hypertrophy. Therefore, we investigated whether myocardin is sufficient to induce cardiomyocyte hypertrophy and whether myocardin is regulated by GSK3beta through site-specific phosphorylation. Adenoviral myocardin overexpression induced cardiomyocyte hypertrophy in neonatal rat cardiomyocytes, with increased cell size, total protein amount, and transcription of atrial natriuretic factor (ANF). In vitro and in vivo (HEK 293 cells) kinase assays with synthetic peptides and full-length myocardin demonstrated that myocardin was a "primed" GSK3beta substrate, with serines 455 to 467 and 624 to 636 being the major GSK3beta phosphorylation sites. Myocardin-induced ANF transcription and increase in total protein amount were enhanced by GSK3beta blockade (10 mmol/L LiCl), indicating that GSK3beta inhibits myocardin. A GSK3beta phosphorylation-resistant myocardin mutant (8xA) activated ANF transcription twice as potently as wildtype myocardin under basal conditions with GSK3beta being active. Conversely, a GSK3beta phospho-mimetic myocardin mutant (8xD) was transcriptionally repressed after GSK3beta blockade, indicating that GSK3beta phosphorylation at the sites identified inhibits myocardin transcriptional activity. GAL4-myocardin fusion constructs demonstrated that GSK3beta phosphorylation reduced the intrinsic myocardin transcriptional activity. A cell-permeable (Antennapedia protein transduction tag) peptide containing the mapped myocardin GSK3beta motifs 624 to 636 induced hypertrophy of cultured cardiomyocytes, suggesting that the peptide acted as substrate-based GSK3beta inhibitor in cardiomyocytes. Therefore, we conclude that the GSK3beta-myocardin interaction constitutes a novel molecular control of cardiomyocyte hypertrophy. Phosphorylation by GSK3beta comprises a novel post-transcriptional regulatory mechanism of myocardin.

p38 mitogen-activated protein kinase downregulates endothelial progenitor cells.

BACKGROUND: Transplantation of endothelial progenitor cells (EPCs) improves neovascularization after ischemia, but patients with coronary artery disease (CAD) or diabetes mellitus show a reduced number of EPCs and impaired functional activity. Therefore, we investigated the effects of risk factors, such as glucose and TNF-alpha, on the number of EPCs in vitro to elucidate the underlying mechanisms. METHODS AND RESULTS: EPCs of patients or healthy subjects were isolated from peripheral blood. Incubation with glucose or TNF-alpha dose-dependently reduced the number of EPCs (79.9+/-1.3% and 74.3+/-8.1% of control; P<0.05, respectively). This reduction was not caused by apoptosis. TNF-alpha and glucose induced a dose- and time-dependent activation of the p38 MAP kinase, the downstream kinase mitogen- and stress-activated kinase 1, and the transcription factor cAMP-responsive element-binding protein (CREB), in EPCs. Moreover, EPCs from CAD patients had significantly higher basal p38-phosphorylation levels (1.83+/-0.2-fold increase; P<0.05) compared with healthy subjects. The inhibition of the p38-kinase by SB203580 or infection with a dominant negative p38 kinase adenovirus significantly increased basal number of EPCs (136.7+/-6.3% and 142.9+/-18% versus control, respectively). Likewise, ex vivo cultivation of EPCs from patients with CAD with SB203580 significantly increased the number of EPCs and partially reversed the impaired capacity for neovascularization of EPCs in vivo (relative blood flow: 0.40+/-0.03 versus 0.64+/-0.08, P<0.05). The increased numbers of EPCs by SB203580 were associated with an augmentation of EPC proliferation and a reduction of cells expressing the monocytic marker proteins CD14 and CD64, suggesting that p38 regulates proliferation and differentiation events. CONCLUSIONS: These results demonstrate that p38 MAP kinase plays a pivotal role in the signal transduction pathways regulating the number of EPCs ex vivo. SB203580 can prevent the negative effects of TNF-alpha and glucose on the number of EPCs and may be useful to improve the number of EPCs for potential cell therapy.

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.

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.

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.

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.