Birgit Assmus

Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging.

Background—Experimental and initial clinical studies suggest that transplantation of circulating blood– (CPC) or bone marrow–derived (BMC) progenitor cells may beneficially affect postinfarction remodeling processes after acute myocardial infarction (AMI). To relate functional characteristics of the infused cells to quantitative measures of outcome at 4-month follow-up, we performed serial contrast-enhanced MRI and assessed the migratory capacity of the transplanted progenitor cells immediately before intracoronary infusion. Methods and Results—In 28 patients with reperfused AMI receiving either BMCs or CPCs into the infarct artery 4.7±1.7 days after AMI, serial contrast-enhanced MRI performed initially and after 4 months revealed a significant increase in global ejection fraction (from 44±10% to 49±10%; P =0.003), a decrease in end-systolic volume (from 69±26 to 60±28 mL; P =0.003), and unchanged end-diastolic volumes (122±34 versus 117±37 mL; P =NS). Infarct size, measured as late enhancement (LE) volume, decreased significantly, from 46±32 to 37±28 mL (P <0.05). There was a significant correlation between the reduction in LE volume and global ejection fraction improvement. The migratory capacity of transplanted cells as assessed ex vivo toward a gradient of vascular endothelial growth factor for CPCs and stromal cell derived factor-1 for BMCs was closely correlated with the reduction of LE volume. By multivariate analysis, migratory capacity remained the most important independent predictor of infarct remodeling. Conclusions—Analysis of serial contrast-enhanced MRI suggests that intracoronary infusion of adult progenitor cells in patients with AMI beneficially affects postinfarction remodeling processes. The migratory capacity of the infused cells is a major determinant of infarct remodeling, disclosing a causal effect of progenitor cell therapy on regeneration enhancement.

Fluid Shear Stress Stimulates Phosphorylation of Akt in Human Endothelial Cells: Involvement in Suppression of Apoptosis

Fluid shear stress alters the morphology and function of the endothelium by activating several kinases. Furthermore, shear stress potently inhibits apoptosis of endothelial cells. Since activation of Akt kinase has been shown to prevent cell death, we investigated the effects of shear stress on Akt phosphorylation. To test the hypothesis that shear stress interacts with the Akt kinase pathway, human umbilical venous endothelial cells were exposed to laminar shear stress (15 dyne/cm2). Western blotting with specific antibodies against the phosphorylated Akt demonstrated a time-dependent stimulation of Akt phosphorylation by shear stress with a maximal increase up to 6-fold after 1 hour of shear stress exposure. The stimulation of Akt phosphorylation by shear stress thereby seemed to be mediated by the phosphoinositide 3-OH kinase (PI3K), as evidenced by the significant inhibition of shear stress-induced Akt phosphorylation by the PI3K inhibitors wortmannin (20 nmol/L) and Ly294002 (10 micromol/L). In addition, pharmacological inhibition of P13K reduced the antiapoptotic effect of shear stress against growth factor depletion-induced apoptosis. Most important, overexpression of a dominant-negative Akt mutant significantly inhibited the apoptosis-suppressive effect of shear stress against serum depletion-induced apoptosis, thus indicating the direct involvement of shear stress-induced Akt phosphorylation for inhibition of endothelial cell apoptosis. These results define a novel shear stress-stimulated signal transduction pathway, namely, activation of the serine/threonine kinase Akt, which may contribute to the profound changes in endothelial morphology and function by shear stress.

HMG-CoA Reductase Inhibitors Reduce Senescence and Increase Proliferation of Endothelial Progenitor Cells via Regulation of Cell Cycle Regulatory Genes

Abstract— Endothelial progenitor cells (EPCs) play an important role in postnatal neovascularization of ischemic tissue. Ex vivo expansion of EPCs might be useful for potential clinical cell therapy of myocardial ischemia. However, cultivation of primary cells leads to cellular aging (senescence), thereby severely limiting the proliferative capacity. Therefore, we investigated whether statins might be able to prevent senescence of EPCs. EPCs were isolated from peripheral blood and characterized. After ex vivo cultivation, EPCs became senescent as determined by acidic &bgr;-galactosidase staining. Atorvastatin or mevastatin dose-dependently inhibited the onset of EPC senescence in culture. Moreover, atorvastatin increased proliferation of EPCs as assessed by BrdU incorporation and colony-forming capacity. Whereas geranylgeranylpyrophosphate or farnesylpyrophosphate reduced the senescence inhibitory effect of atorvastatin, NO synthase inhibition, antioxidants, or Rho kinase inhibitors had no effect. To get further insights into the underlying downstream effects of statins, we measured telomerase activity and determined the expression of various cell cycle regulatory genes by using a microarray assay. Whereas telomerase activity did not change, atorvastatin modulated expression of cell cycle genes including upregulation of cyclins and downregulation of the cell cycle inhibitor p27Kip1. Taken together, statins inhibited senescence of EPCs independent of NO, reactive oxygen species, and Rho kinase, but dependent on geranylgeranylpyrophosphate. Atorvastatin-mediated prevention of EPC senescence appears to be mediated by the regulation of various cell cycle proteins. The inhibition of EPC senescence and induction of EPC proliferation by statins in vitro may importantly improve the functional activity of EPCs for potential cell therapy.

Clinical Outcome 2 Years After Intracoronary Administration of Bone Marrow–Derived Progenitor Cells in Acute Myocardial Infarction

Background—The aim of this study was to investigate the clinical outcome 2 years after intracoronary administration of autologous progenitor cells in patients with acute myocardial infarction (AMI). Methods and Results—Using a double-blind, placebo-controlled, multicenter trial design, we randomized 204 patients with successfully reperfused AMI to receive intracoronary infusion of bone marrow–derived progenitor cells (BMC) or placebo medium into the infarct artery 3 to 7 days after successful infarct reperfusion therapy. At 2 years, the cumulative end point of death, myocardial infarction, or necessity for revascularization was significantly reduced in the BMC group compared with placebo (hazard ratio, 0.58; 95% CI, 0.36 to 0.94; P=0.025). Likewise, the combined end point death and recurrence of myocardial infarction and rehospitalization for heart failure, reflecting progression toward heart failure, was significantly reduced in the BMC group (hazard ratio, 0.26; 95% CI, 0.085 to 0.77; P=0.015). Intracoronary administration of BMC remained a significant predictor of a favorable clinical outcome by Cox regression analysis when adjusted for classical predictors of poor outcome after AMI. There was no evidence of increased restenosis or atherosclerotic disease progression after BMC therapy nor any evidence of increased ventricular arrhythmias or neoplasms. In addition, regional left ventricular contractility of infarcted segments, as assessed by MRI in a subgroup of patients at 2-year follow-up, was significantly higher in the BMC group compared with the placebo group (P<0.001). Conclusions—Intracoronary administration of BMC is associated with a significant reduction of the occurrence of major adverse cardiovascular events maintained for 2 years after AMI. Moreover, functional improvements after BMC therapy may persist for at least 2 years. Larger studies focusing on clinical event rates are warranted to confirm the effects of BMC administration on mortality and progression of heart failure in patients with AMIs. Clinical Trial Registration—clinicaltrials.gov. Identifier: NCT00279175.

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 (≥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.

Insulin-Mediated Stimulation of Protein Kinase Akt: A Potent Survival Signaling Cascade for Endothelial Cells

Insulin exerts potent antiapoptotic effects in neuronal cells and has been suggested to promote angiogenesis. Therefore, we investigated whether insulin inhibits tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). Because insulin has been shown to stimulate the protein kinase Akt, we investigated whether activation of Akt contributes to the apoptosis-suppressive effect of insulin and characterized the downstream signaling pathway. Incubation with insulin dose-dependently prevented apoptosis induced by TNF-alpha (50 ng/mL). The extent of apoptosis suppression by insulin was similar to the effect of vascular endothelial growth factor. Pharmacological inhibition of Akt activation or overexpression of a dominant-negative Akt mutant prevented the antiapoptotic effect of insulin. Furthermore, we investigated the effect of TNF-alpha on Akt phosphorylation by Western blot analysis with the use of a phosphospecific Akt antibody. Incubation of HUVECs with TNF-alpha induced a marked dephosphorylation of Akt. Insulin counteracted this TNF-alpha-induced dephosphorylation of Akt. Furthermore, we investigated the downstream signaling events. Akt has been shown to mediate its apoptosis-suppressive effects via phosphorylation of Bad or caspase-9. However, incubation with insulin did not lead to enhanced phosphorylation of Bad at Ser 136 or Ser 112. In contrast, insulin inhibited caspase-9 activity and prevented caspase-9-induced apoptosis. Mutation of the Akt site within caspase-9 significantly reduced the apoptosis-suppressive effect of insulin. The present study demonstrates an important role for insulin-mediated Akt activation in the prevention of endothelial cell apoptosis, which may importantly contribute to cell homeostasis and the integrity of the endothelium. In endothelial cells, Akt seems to mediate its antiapoptotic effect, at least in part, via phosphorylation of caspase-9 rather than Bad.

Transcoronary Concentration Gradients of Circulating MicroRNAs

Background— Circulating levels of microRNA (miR) have been proposed as biomarkers for cardiovascular disease. To identify the heart as a potential source for miRs released into the circulation, we measured concentration gradients across the coronary circulation for muscle-enriched (miR-133a, miR-499, miR-208a), vascular (miR-126, miR-92a), leukocyte-related (miR-155), and platelet-enriched (miR-223) miRs. Methods and Results— Circulating miRs were measured by TaqMan polymerase chain reaction in EDTA-plasma simultaneously obtained from the aorta and the coronary venous sinus in patients without coronary artery disease (n=7), with stable coronary artery disease (n=31), and with troponin-positive acute coronary syndromes (n=19). Circulating levels of the muscle-enriched miR-499 (>20-fold; P<0.01), miR-133a (11-fold; P<0.01), and miR-208a (5-fold; P<0.01) were significantly elevated in the aorta of troponin-positive acute coronary syndrome patients compared with patients with coronary artery disease. Importantly, there was a significant increase in circulating levels of miR-499 and miR-133a across the coronary circulation in troponin-positive acute coronary syndrome patients, suggestive of a release into the coronary circulation during myocardial injury. Indeed, miR-499 concentration gradients were significantly correlated with the extent of myocardial damage as measured by high-sensitivity troponin T (r=0.70, P<0.01). In contrast, circulating levels of miR-126 (P=0.16) decreased during transcoronary passage in patients with evidence of myocardial injury, suggesting consumption during transcoronary passage. Conclusions— Muscle-enriched miR-499 and miR-133a are released from the heart into the coronary circulation on myocardial injury, whereas the vascular miR-126 is consumed during transcoronary passage. The differential regulation of circulating miRs during the transcoronary passage might provide important insights to exploit their role as cardiac biomarkers. Clinical Trial Registration— URL: http://www.germanctr.de. Unique identifier: DRKS00000207; in German Clinical Trials Registry.

Meta-Analysis of Cell-based CaRdiac stUdiEs (ACCRUE) in Patients With Acute Myocardial Infarction Based on Individual Patient Data

RATIONALE The meta-Analysis of Cell-based CaRdiac study is the first prospectively declared collaborative multinational database, including individual data of patients with ischemic heart disease treated with cell therapy. OBJECTIVE We analyzed the safety and efficacy of intracoronary cell therapy after acute myocardial infarction (AMI), including individual patient data from 12 randomized trials (ASTAMI, Aalst, BOOST, BONAMI, CADUCEUS, FINCELL, REGENT, REPAIR-AMI, SCAMI, SWISS-AMI, TIME, LATE-TIME; n=1252). METHODS AND RESULTS The primary end point was freedom from combined major adverse cardiac and cerebrovascular events (including all-cause death, AMI recurrance, stroke, and target vessel revascularization). The secondary end point was freedom from hard clinical end points (death, AMI recurrence, or stroke), assessed with random-effects meta-analyses and Cox regressions for interactions. Secondary efficacy end points included changes in end-diastolic volume, end-systolic volume, and ejection fraction, analyzed with random-effects meta-analyses and ANCOVA. We reported weighted mean differences between cell therapy and control groups. No effect of cell therapy on major adverse cardiac and cerebrovascular events (14.0% versus 16.3%; hazard ratio, 0.86; 95% confidence interval, 0.63-1.18) or death (1.4% versus 2.1%) or death/AMI recurrence/stroke (2.9% versus 4.7%) was identified in comparison with controls. No changes in ejection fraction (mean difference: 0.96%; 95% confidence interval, -0.2 to 2.1), end-diastolic volume, or systolic volume were observed compared with controls. These results were not influenced by anterior AMI location, reduced baseline ejection fraction, or the use of MRI for assessing left ventricular parameters. CONCLUSIONS This meta-analysis of individual patient data from randomized trials in patients with recent AMI revealed that intracoronary cell therapy provided no benefit, in terms of clinical events or changes in left ventricular function. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01098591.

Effects of Granulocyte Colony Stimulating Factor on Functional Activities of Endothelial Progenitor Cells in Patients With Chronic Ischemic Heart Disease

Objective—Bone marrow–derived circulating endothelial progenitor cells (EPCs) may contribute to regeneration of infarcted myocardium and enhance neovascularization. Granulocyte colony-stimulating factor (G-CSF) is well-established to mobilize hematopoietic stem cells (HSCs) and might, thereby, also increase the pool of endogenously circulating EPC. Therefore, we investigated the effects of G-CSF administration on mobilization and functional activities of blood-derived EPC in patients with chronic ischemic heart disease (CIHD). Methods and Results—Sixteen patients with CIHD received 10 &mgr;g/kg per day subcutaneous G-CSF injection for 5 days. Leukocyte counts, the number of HSCs and EPCs, and the migratory response to VEGF and SDF-1 were analyzed before and after G-CSF-therapy. At day 5 of G-CSF treatment, the number of circulating leukocytes, CD34+CD45+ and CD34+CD133+ cells was significantly increased. Likewise, G-CSF treatment augmented the numbers of colony forming units with endothelial cell morphology (EC-CFU). However, the functional activity of the EPC as assessed by the migratory response to VEGF and SDF-1 was significantly reduced after G-CSF treatment (P<0.01). Because G-CSF was previously shown to cleave the CXCR4 receptor, we determined the surface expression of the 6H8 epitope of the CXCR4 receptor by fluorescence-activated cell sorter (FACS) analysis. Consistent with the reduced migratory capacity, the surface expression of the functionally active CXCR4 receptor was significantly reduced. To test the functional activity of the cultivated EPCs in vivo, cells were intravenously infused in nude mice after hind limb ischemia. EPCs, which were cultivated before G-CSF administration, increased blood flow recovery and prevented limb necrosis. However, infusion of EPCs, which were isolated 5 days after G-CSF treatment from the same patient, showed a reduced capacity to augment blood flow recovery and to prevent necrosis by 27%. Conclusion—G-CSF treatment effectively mobilizes HSCs and EPCs. However, the migratory response to SDF-1 and in vivo capacity of G-CSF-mobilized EPCs was significantly reduced.

A Pilot Trial to Assess Potential Effects of Selective Intracoronary Bone Marrow–Derived Progenitor Cell Infusion in Patients With Nonischemic Dilated Cardiomyopathy Final 1-Year Results of the Transplantation of Progenitor Cells and Functional Regeneration Enhancement Pilot Trial in Patients With Nonischemic Dilated Cardiomyopathy

Background— Intracoronary administration of bone marrow–derived progenitor cells (BMC) was shown to improve coronary microvascular function in ischemic heart disease. Because coronary microvascular dysfunction is implicated in the pathogenesis and prognosis of nonischemic dilated cardiomyopathy (DCM), we investigated the effects of intracoronary BMC administration in patients with DCM. Methods and Results— Intracoronary infusion of BMC was performed in 33 patients with DCM by using an over-the-wire balloon catheter. Left ventricular contractility at baseline and after 3 months was assessed by analysis of left ventricular angiograms. Coronary hemodynamics were determined by intracoronary Doppler wire measurements. After 3 months, regional wall motion of the target area (contractility from −1.08±0.39 to −0.97±0.47 SD/chord, P =0.029) and global left ventricular ejection fraction (from 30.2±10.9 to 33.4±11.5%, P <0.001) were improved. Increase of regional contractile function was directly related to the functionality of the infused cells as measured by their colony-forming capacity. Minimal vascular resistance index was significantly reduced in the BMC-treated vessel after 3 months (from 1.53±0.63 to 1.32±0.61 mm Hg · s/cm; P =0.002, n=24), whereas no changes were observed in the reference vessel (from 1.60±0.45 to 1.49±0.45 mm Hg · s/cm; P =0.133, n=13). Twelve months after BMC infusion, N-terminal prohormone brain natriuretic peptide (NT-proBNP) serum levels were decreased, suggesting a beneficial effect on left ventricular remodeling processes (from 1610±993 to 1473±1147 pg/mL; P =0.038 for logNT-proBNP, n=26). Conclusions— Intracoronary administration of BMC seems to be associated with improvements in cardiac contractile and microvascular function in patients with DCM. Thus, randomized blinded studies are warranted to evaluate potential clinical benefits of intracoronary BMC administration in patients with DCM. Received August 26, 2008; accepted June 24, 2009.Background—Intracoronary administration of bone marrow–derived progenitor cells (BMC) was shown to improve coronary microvascular function in ischemic heart disease. Because coronary microvascular dysfunction is implicated in the pathogenesis and prognosis of nonischemic dilated cardiomyopathy (DCM), we investigated the effects of intracoronary BMC administration in patients with DCM. Methods and Results—Intracoronary infusion of BMC was performed in 33 patients with DCM by using an over-the-wire balloon catheter. Left ventricular contractility at baseline and after 3 months was assessed by analysis of left ventricular angiograms. Coronary hemodynamics were determined by intracoronary Doppler wire measurements. After 3 months, regional wall motion of the target area (contractility from −1.08±0.39 to −0.97±0.47 SD/chord, P=0.029) and global left ventricular ejection fraction (from 30.2±10.9 to 33.4±11.5%, P<0.001) were improved. Increase of regional contractile function was directly related to the functionality of the infused cells as measured by their colony-forming capacity. Minimal vascular resistance index was significantly reduced in the BMC-treated vessel after 3 months (from 1.53±0.63 to 1.32±0.61 mm Hg · s/cm; P=0.002, n=24), whereas no changes were observed in the reference vessel (from 1.60±0.45 to 1.49±0.45 mm Hg · s/cm; P=0.133, n=13). Twelve months after BMC infusion, N-terminal prohormone brain natriuretic peptide (NT-proBNP) serum levels were decreased, suggesting a beneficial effect on left ventricular remodeling processes (from 1610±993 to 1473±1147 pg/mL; P=0.038 for logNT-proBNP, n=26). Conclusions—Intracoronary administration of BMC seems to be associated with improvements in cardiac contractile and microvascular function in patients with DCM. Thus, randomized blinded studies are warranted to evaluate potential clinical benefits of intracoronary BMC administration in patients with DCM.