Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Remote ischemic conditioning wikipedia , lookup
Cardiac contractility modulation wikipedia , lookup
Arrhythmogenic right ventricular dysplasia wikipedia , lookup
Myocardial infarction wikipedia , lookup
Cardiac surgery wikipedia , lookup
Coronary artery disease wikipedia , lookup
Coronary Heart Disease Growth Properties of Cardiac Stem Cells Are a Novel Biomarker of Patients’ Outcome After Coronary Bypass Surgery Domenico D’Amario, MD, PhD; Antonio M. Leone, MD; Antonio Iaconelli, MD; Nicola Luciani, MD; Mario Gaudino, MD; Ramaswamy Kannappan, PhD; Melissa Manchi, MD; Anna Severino, PhD; Sang Hun Shin, PhD; Francesca Graziani, MD; Gina Biasillo, MD; Andrea Macchione, MD; Costantino Smaldone, MD; Giovanni Luigi De Maria, MD; Carlo Cellini, MD; Andrea Siracusano, MS; Lara Ottaviani, MS; Massimo Massetti, MD; Polina Goichberg, PhD; Annarosa Leri, MD; Piero Anversa, MD; Filippo Crea, MD Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Background—The efficacy of bypass surgery in patients with ischemic cardiomyopathy is not easily predictable; preoperative clinical conditions may be similar, but the outcome may differ significantly. We hypothesized that the growth reserve of cardiac stem cells (CSCs) and circulating cytokines promoting CSC activation are critical determinants of ventricular remodeling in this patient population. Methods and Results—To document the growth kinetics of CSCs, population-doubling time, telomere length, telomerase activity, and insulin-like growth factor-1 receptor expression were measured in CSCs isolated from 38 patients undergoing bypass surgery. Additionally, the blood levels of insulin-like growth factor-1, hepatocyte growth factor, and vascular endothelial growth factor were evaluated. The variables of CSC growth were expressed as a function of the changes in wall thickness, chamber diameter and volume, ventricular mass-to-chamber volume ratio, and ejection fraction, before and 12 months after surgery. A high correlation was found between indices of CSC function and cardiac anatomy. Negative ventricular remodeling was not observed if CSCs retained a significant growth reserve. The high concentration of insulinlike growth factor-1 systemically pointed to the insulin-like growth factor-1–insulin-like growth factor-1 receptor system as a major player in the adaptive response of the myocardium. hepatocyte growth factor, a mediator of CSC migration, was also high in these patients preoperatively, as was vascular endothelial growth factor, possibly reflecting the vascular growth needed before bypass surgery. Conversely, a decline in CSC growth was coupled with wall thinning, chamber dilation, and depressed ejection fraction. Conclusions—The telomere-telomerase axis, population-doubling time, and insulin-like growth factor-1 receptor expression in CSCs, together with a high circulating level of insulin-like growth factor-1, represent a novel biomarker able to predict the evolution of ischemic cardiomyopathy following revascularization. (Circulation. 2014;129:157-172.) Key Words: coronary artery disease ◼ receptor, IGF type 1 ◼ stem cells ◼ telomerase ◼ telomere ◼ ventricular remodeling T he therapeutic efficacy of bypass surgery in patients who have severe coronary atherosclerosis and ischemic cardiomyopathy are not easily predictable.1 Although the preoperative clinical conditions of the patients may be similar, the mid- and long-term outcome may differ significantly.2 Following surgery, attenuation in chamber dilation and wall thickening are important positive variables of ventricular remodeling, whereas the opposite effects are the hallmarks of chronic heart failure.3 These changes in cardiac anatomy have profound consequences on patients’ mortality,4,5 emphasizing the need to identify biomarkers that can predict the evolution of the myopathic heart. Editorial see p 136 Clinical Perspective on p 172 In the current report, we raised the possibility that intrinsic factors within the heart may play a major role in preserving the Received October 2, 2013; accepted October 25, 2013. From the Department of Cardiovascular Sciences, Catholic University of the Sacred Heart, Rome, Italy (D.D'A., A.M.L., A.I., N.L., M.G., M. Manchi, A. Severino, F.G., G.B., A.M., C.S., G.L.D.M., C.C., A. Siracusano, L.O., M. Massetti, F.C.); and Departments of Anesthesia and Medicine, and Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA (D.D'A., R.K., S.H.S., P.G., A.L., P.A.). Guest Editor for this article was Joshua M. Hare, MD. The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA. 113.006591/-/DC1. Correspondence to Piero Anversa, MD, Departments of Anesthesia and Medicine, and Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02215 (E-mail [email protected]); or Filippo Crea, MD, Department of Cardiovascular Sciences, Catholic University of the Sacred Heart, Rome, Italy, 00168 (E-mail [email protected]). © 2013 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.113.006591 157 158 Circulation January 14, 2014 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 impact of successful bypass surgery and the improvement in ventricular performance. The recognition that the adult human heart possesses a stem cell compartment capable of differentiating into cardiomyocytes and coronary vessels6,7 suggests that cardiac stem cells (CSCs) and their growth properties may be critically implicated in the protection of the structural and functional integrity of the myocardium. Moreover, circulating cytokines able to promote CSC activation and commitment may be equally relevant in favoring the increase in wall thickness and the decrease in chamber volume, months after the surgical intervention. Based on this premise, the growth kinetics of CSCs and the telomere-telomerase axis were characterized in 38 patients with stable angina who were undergoing bypass surgery. Additionally, the blood level of insulin-like growth factor-1 (IGF-1), hepatocyte growth factor (HGF), stem cell factor (SCF), vascular endothelial growth factor (VEGF), granulocyte-colony stimulating factor (G-CSF), and basic fibroblast growth factor (bFGF) were measured before surgery and 1 year later, when the beneficial effects of revascularization were well established.8 IGF-1, HGF, and SCF have the potential to activate surface receptors in CSCs, inducing CSC division, survival, and migration.9,10 VEGF is the ligand of vascular endothelial growth factor receptor-27; it has the ability to induce angiogenesis and vasculogenesis.11 G-CSF mobilizes hematopoietic stem cells (HSCs) and endothelial progenitor cells, which may expand the coronary microcirculation and transdifferentiate generating cardiomyocytes.12–14 Conversely, bFGF may favor fibroblast proliferation and collagen accumulation, altering the mechanical behavior of the myocardium. Methods An expanded Materials and Methods section is provided in the online-only Data Supplement. Patients and Study Design We enrolled 55 patients undergoing elective on-pump coronary artery bypass surgery (CABG) for multivessel coronary atherosclerosis. The inclusion criteria were as follows: (1) chronic stable angina in the past 6 months, together with evidence of ischemia at stress test; and (2) complete surgical revascularization within 7 days. The exclusion criteria were as follows: (1) evidence of previous myocardial infarct or pathological Q waves on the 12-lead ECG; (2) hospitalization for acute coronary syndrome during the past 6 months; (3) severe valvular heart disease; (4) CABG or percutaneous transluminal angioplasty in the preceding 12 months; (5) life expectancy <1 year; (6) presence of malignancies; (7) psychosocial conditions precluding long-term adherence; (8) pregnancy; (9) enrollment in other clinical research trial; (10) cardiogenic shock; (11) severe comorbidities; and (12) patients unable or unwilling to give informed written consent. Because 17 patients were lost at follow-up, the study was restricted to 38 subjects (Figure I in the online-only Data Supplement). The right atrial appendage was harvested during on-pump CABG for isolation and expansion of CSCs. At the time of enrollment and follow-up, 12±1 months after CABG, patients had complete clinical and physical examinations with collection of cardiovascular risk factors, 2-dimensional echocardiography, and evaluation of New York Heart Association functional class. The serum level of IGF-1, HGF, VEGF, SCF, G-CSF, and bFGF was also measured (Figure I in the online-only Data Supplement). The study was approved by the local ethics committee and conformed to the Declaration of Helsinki on human research. Echocardiography Transthoracic echocardiogram was performed according to the recommendations of the European Society of Echocardiography from a single experienced operator (G.B.) with a second harmonic 2.25-MHz probe to optimize endocardial border visualization. All echocardiograms were digitized and reanalyzed off-line by 3 experienced operators (F.G., A.M., C.S.), blind to the clinical and laboratory data. Temporal changes in left ventricular (LV) volume from baseline to 12±1 months after surgery were computed. An increase in LV enddiastolic volume of ≥20% at follow-up was interpreted as an index of negative LV remodeling (LVR).15 Human CSCs CSCs were isolated from 38 myocardial samples, and their in vitro properties were determined.6,7,16 Aliquots of CSCs were fixed and following incubation with antibodies against surface markers, nuclear transcription factors, and cytoplasmic proteins were analyzed by fluorescence-activated cell sorter (FACSAria or AccuriC6). The presence of epitopes of hematopoietic (CD34, CD45) and mesenchymal (CD90, CD105) stem cells was determined, in combination with the expression of IGF-1 receptors (IGF-1Rs). Myocyte, endothelial cell, and smooth muscle cell markers were also included in this assay (Tables I and II in the online-only Data Supplement). Growth Properties of CSCs CSCs were plated at a low density, and the number of cells per unit area of the culture dish was measured daily for 5 to 7 days. Population-doubling time (PDT) was computed by linear regression of log2 values of cell number.17 The length of telomeres was determined by flow–fluorescent in situ hybridization, and the catalytic activity of telomerase was assessed by quantitative polymerase chain reaction.16–18 Growth Factors A customized multiplex microarray technology based 2-site sandwich enzyme-linked immunosorbent assay was used to measure quantitatively the serum level of IGF-1, HGF, VEGF, SCF, G-CSF, and bFGF. Statistical Analysis Continuous variables were expressed as mean±standard deviation, and dichotomous variables are shown as percentages. Unpaired t test or Mann-Whitney U-test was used for comparison between 2 groups. Categorical variables were compared by using the χ2 test or Fisher exact test, as appropriate. Linear regression analysis was performed to correlate the characteristics of CSCs in vitro with the indices of LVR: in each case the D’Amario et al Cardiac Stem Cells and the Evolution of Ischemic Cardiomyopathy 159 Table 1. Characteristics of the Patient Population Variables Table 1. Continued Patients (38) Clinical characteristics Age, years±SD Female, n (%) Patients (38) Number of anastomosis 69±8 Sex Male, n (%) Variables 33 (87) 5 (13) 4±1 LIMA, n (%) 37 (97.3) BIMA, n (%) 7 (18.4) Cross-clamp, min±SD 61±18 ECC time, min±SD 72±18 Hypertension, n (%) 37 (97) Cardioplegia, mL, DS 19.6±6.2 Current smoker, n (%) 23 (61) Total mEq K+ infused 39.3±12.5 Dyslipidemia, n (%) 30 (79) Ischemic time, min±SD 51.7±15.0 Diabetes mellitus, n (%) 24 (63) Post-CABG atrial fibrillation, n (%) Family history of CAD, n (%) 28 (74) CK-MB (peak; 48 h post-CABG) Body mass index, kg/m2 28±4 LVEF, average±SD 54±11 Patients with LVEF<45%, n (%) 8 (21) NYHA I, n (%) 2 (5) Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 NYHA II, n (%) 21 (55.5) NYHA III, n (%) 15 (39.5) NYHA IV, n (%) 0 (0) Therapy on admission β-Blockers, n (%) 33 (87) ACE inhibitors, n (%) 27 (71) Statins, n (%) 30 (79) Aspirin, n (%) 25 (66) ARBs, n (%) Diuretics, n (%) 1 (2.6) 26 (68) Heparin, n (%) 4 (10.5) Clopidogrel, n (%) 0 (0) Laboratory tests Urea nitrogen, mg/dL 18±7 Uric acid, mg/dL 5.4±2 HbA1c, % ±SD 7±1 Total cholesterol, mg/dL±SD 196±23 LDL, mg/dL±SD 134±31 HDL, mg/dL±SD Triglycerides, mg/dL±SD Creatinine clearance, mL/min±SD 62±9 hsCRP, mg/L 1.9±0.7 Angiographic analysis and preoperative risk stratification Coronary disease extension, n (%) 1 Vessel+LM 2 (5.2) 2 Vessels 10 (26.3) 3 Vessels 26 (68.5) CABG+valve replacement, n (%) Log euroSCORE (average) Log euroSCORE 0–4, n (%) 0 (0) 8.3 19 (50) Log euroSCORE 5–9, n (%) 7 (18.4) Log euroSCORE >10, n (%) 12 (31.6) euroSCORE II (average) 8.2 Intra- and perioperative variables Number of grafts 3±1 (Continued ) 17.3±11.6 ACE indicates angiotensin-converting enzyme; ARB, angiotensin receptor blocker; BIMA, left and right internal mammary artery; CABG, coronary artery bypass surgery; CAD, coronary artery disease; CK-MB, creatine kinase MB; ECC, extra corporeal circulation; HbA1c, hemoglobin A1c; HDL, high-density lipoprotein; hsCRP, high-sensitivity C-reactive protein; LDL, low-density lipoprotein; LIMA, left internal mammary artery; LM, left main stem disease; LV, left ventricle; LVEF, left ventricular ejection fraction; NYHA, New York Heart Association; and SD, standard deviation. best-fit regression line is shown together with the 95% confidence interval, and the P and R2 values. P values of <0.05 were considered significant. Multivariate logistic regression analysis was performed to identify independent predictors of LVR. Variables showing a P value of <0.05 at univariate analysis were included in the models. β-Values and 95% confidence intervals have been reported. Receiver operating characteristic curve was performed to determine the cellular biomarker or growth factor level that best predicted negative LV remodeling and to assess the best cutoff value. The Youden index was introduced to evaluate the sensitivity and specificity of each variable. Statistical comparisons were performed by using SPSS 20.0 (IBM SPSS Statistics, IBM Corporation, Armonk, NY); however, receiver operating characteristic analysis was done with the use of MedCalc (MedCalc, Mariakerke, Belgium).19,20 40±11 156±18 6 (15.8) Results Patients A cohort of 55 consecutive patients affected by chronic coronary artery disease (CAD) with indication for bypass surgery was studied. In all 55 patients, the right atrial appendage was collected at the time of surgery for CSC isolation and characterization. Ten of the 55 patients did not return to the clinic and 5 refused follow-up tests. Two additional patients were excluded because they developed malignant tumors. Thus, 38 patients were included in the final study (Figure I in the online-only Data Supplement). Patients’ characteristics are listed in Table 1. There were 33 men and 5 women. Risk factors included hypertension, hyperlipidemia, family history of cardiovascular disease, type 2 diabetes mellitus, renal dysfunction, and hyperuricemia. Indices of high-risk perioperative outcomes were evaluated: 15 patients were in New York Heart Association class III and 26 had a 3-vessel disease (stenosis ≥70%). The preoperative predictor logistic euroSCORE II was determined; 12 patients 160 Circulation January 14, 2014 were in the highest tertile with euroSCORE II ≥ 10. LV ejection fraction (LVEF) averaged 54%; however, 8 patients had LVEF < 45%. CSC Characterization and Growth Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 A major challenge and potential limitation of this work was related to the successful acquisition of c-kit–positive CSCs in each of the 38 patients, a prerequisite for the comparison to be made with the evolution of the cardiac disease following bypass surgery. In each case, the right atrial appendage was digested, and, following expansion of the small-cell pool, c-kit–positive cells were collected with immunomagnetic beads and cultured; c-kit–positive CSCs were obtained in all cases. At P5 to P6, c-kit–positive CSCs were characterized by fluorescence-activated cell sorter analysis. These cells were negative for the markers of HSCs, CD34, and CD45, and for a cocktail of antibodies against bone marrow–derived cells (Figure IIA in the online-only Data Supplement). The absence of CD45 excluded the presence of mast cells. Additionally, these cells did not express epitopes of mesenchymal stromal cells including CD90 and CD105. Similarly, the myocyte transcription factors GATA4, Nkx2.5, and Mef2C and the myocyte contractile protein α-sarcomeric actin were detected rarely (Figure IIB in the online-only Data Supplement). The endothelial cell transcription factor Ets1 and the smooth muscle cell transcription factor GATA6 were only occasionally seen, as the endothelial cell cytoplasmic protein von Willebrand factor and the smooth muscle cell cytoplasmic protein α-smooth muscle actin (Figure IIB in the online-only Data Supplement). Importantly, the fraction of c-kit–positive cells varied from 80% to 95% (Figure 1A) and did not correlate with any major determinants of LVR (Figure III in the online-only Data Supplement). As discussed later, the first 27 patients (1–27 color-coded green) experienced positive LVR, and the remaining 11 (28–38 color-coded red) experienced negative LVR. This characterization of CSCs allowed us to establish whether age, sex, and the presence of type 2 diabetes mellitus affected the CSC phenotype. Although age, sex, and diabetes mellitus alter the pool size of CSCs in the myocardium,21–23 no significant differences were found in the proportion of undifferentiated and committed CSCs with any of these 3 variables (Figure 1B). These observations suggest that a selection process occurs in vitro during CSC growth with loss or death of the nonfunctionally competent stem cells. This conclusion is supported by the evaluation of the PDT of CSCs, which varied modestly from 23 to 30 hours, averaging 26 hours (Figure 2A and 2B). Moreover, bromodeoxyuridine labeling of CSCs after a single pulse of the halogenated nucleotide was relatively similar in the 12 cases tested (Figure 2C), despite age, sex, and the presence or absence of diabetes mellitus. These 12 cases included patients 1 to 6 with positive LVR and patients 28 to 33 with negative LVR. We have previously shown that functionally competent human CSCs can be successfully obtained from small biopsy samples in extremely sick patients undergoing LV assist device implantation or heart transplant.16 Age, sex, and diabetes mellitus may increase the functional heterogeneity of the CSC pool within the entire myocardium, but do not prevent the acquisition of resident stem cells with significant growth reserve and expandability in vitro. Thus, consistent with previous results,6,17,18 with our protocol, a significant number of undifferentiated c-kit–positive CSCs can be obtained in all patients. CSCs and the Telomere–Telomerase Axis Stem cell growth is regulated by the length of telomeres and telomerase activity, which restores in part the telomeric DNA lost following each cell division24; telomere length provides information on the replicative history of non-postmitotic cells. In intact human CSCs telomere length is ≈9 to 10 kbp.6,17 With each cell division, human CSCs lose 130 bp of telomeric DNA,6 and replicative senescence and irreversible growth arrest occur when telomere length reaches 1.5 to 2 kbp.24 In this study, telomere length was measured by Flow– fluorescent in situ hybridization and was found to vary from a minimum of 6.7 kbp to a maximum of 8.5 kbp, averaging 7.4 kbp (Figure 2D and 2E). Telomerase activity, evaluated by quantitative polymerase chain reaction, was seen to be high in all cases (Figure 2F). Age, sex, and type 2 diabetes mellitus appeared not to influence these 2 fundamental parameters of CSC proliferation (Figure 2G). Telomerase function is regulated in part by the IGF1–IGF-1R system. In mouse cardiomyocytes, IGF-1 stimulates the phosphoinositide-3-kinase–Akt pathway that, in turn, phosphorylates telomerase, because there is a consensus site for Akt phosphorylation in the catalytic subunit of this ribonucleoprotein.25 To test whether a similar process is operative in human CSCs, 3 assays were performed. High levels of phospho-Akt were found by Western blotting in CSC preparations (Figure 3A). The enzymatic activity of Akt was documented by a kinase assay, and the results were consistent with the expression of the phosphorylated protein at Ser473 (Figure 3B). Additionally, telomerase was immunoprecipitated and exposed to an antibody against Ser824 located within the putative Akt consensus site (Figure 3C). Phosphotelomerase was high mimicking the level of telomerase activity in these cases. Importantly, human CSCs expressing IGF-1R have a powerful regenerative capacity following experimental myocardial injury,17 suggesting that the expression of IGF-1R in CSCs conditions favorably the evolution of chronic CAD. In the 38 CSC preparations, IGF-1R expression varied but was present in all cases (Figure 4A through 4C). Consistent with the observations above, there was a significant correlation in the 38 samples of CSCs between IGF-1R expression and telomere length and telomerase activity (Figure 4D and 4E). Thus, the telomere-telomerase axis, together with IGF-1R expression and Akt phosphorylation of the ribonucleoprotein, supports the view of a remarkable growth reserve of human CSCs. CSCs and Ventricular Remodeling Echocardiographic measurements of LV dimension and wall thickness were obtained before bypass surgery and 12±1 months later when optimal revascularization was D’Amario et al Cardiac Stem Cells and the Evolution of Ischemic Cardiomyopathy 161 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Figure 1. Phenotype of CSCs. A, Percentage of c-kit–positive CSCs in each of the 38 preparations. Patients 1 to 27 (color-coded green) experienced positive LVR; patients 28 to 38 (color-coded red) experienced negative LVR. B, Effects of age, sex, and diabetes mellitus on c-kit–positive CSCs and the expression of lineage markers. Data are shown by box plots: the box represents the interquartile range, the horizontal line inside the box marks the median, and whiskers show 5 to 95 percentiles range. CSC indicates cardiac stem cell; EC, endothelial cell; LVR, left ventricular remodeling; and SMC, smooth muscle cell. demonstrated by stress test in all 38 patients. Wall thickness, cavitary diameters, chamber volume, and the ventricular mass-to-chamber volume ratio in systole and diastole were expressed as a function of 4 variables of CSC growth: PDT, telomere length, telomerase activity, and IGF-1R expression. The anatomic parameters were computed as the difference 162 Circulation January 14, 2014 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Figure 2. Continued on next page D’Amario et al Cardiac Stem Cells and the Evolution of Ischemic Cardiomyopathy 163 Figure 2. Growth properties of CSCs. A, Phase-contrast images illustrating CSC division and growth in culture. B and C, Populationdoubling time in the 38 CSC preparations (B), and bromodeoxyuridine (BrdU) labeling in 12 CSC preparations (C). D, Flow-FISH of CSCs and lymphoma cells of known telomere length (internal controls); CSCs were combined with lymphoma cells and incubated without (blank, not shown) or with PNA probe. The histogram represents the intensity of PNA probe binding in gated CSCs (green area), R cells (long telomeres: 48 kbp, blue area), and S cells (short telomeres: 7 kbp, grey area). Lymphoma cells were used to compute average telomere length in kbp. E, Telomere length in each of the 38 CSC preparations. F, Telomerase activity measured by qPCR in each of the 38 CSC samples. G, Effects of age, sex, and diabetes mellitus on telomere length and telomerase activity. Data are shown by box plots: the box represents the interquartile range, the horizontal line inside the box marks the median, and whiskers show 5 to 95 percentiles. CSC indicates cardiac stem cell; FISH, fluorescent in situ hybridization; PNA, peptide nucleic acid; and qPCR, quantitative polymerase chain reaction. Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 (Δ) before and 12±1 months following revascularization, whereas the characteristics of CSCs were defined in samples collected at the time of surgery. This protocol cannot exclude that the improvement in coronary perfusion with surgery may have affected the growth kinetics of resident CSCs. However, a second myocardial biopsy for research purposes only was not justified. Wall thickening, reduction in chamber diameters and volume, and increases in ventricular mass-to-chamber volume ratios were interpreted as significant determinants of successful LVR in view of the critical role that these anatomic indices play in diastolic and systolic wall stress.3 An inverse correlation was found between PDT and the changes in wall thickness. The shorter the PDT of CSCs, the greater was the increase in the anterior and posterior wall thickness at followup. In contrast, the longer the PDT, the larger was the increase in cavitary diameters. Moreover, longer telomeres, higher Figure 3. Akt and telomerase in CSCs. A, Phospho-Akt and total Akt were identified by Western blotting. Optical density (OD) data are shown. B, GSK-3α/β was used as a substrate for the detection of Akt kinase activity. Western blotting of phosphorylated GSK-3α/β at Ser21/9 is shown. Loading, IgG heavy chain band. C, Phosphotelomerase at Ser824 was detected following immunoprecipitation of CSC protein lysates with an antibody against telomerase. CSCs were obtained from patients 4, 6, 8, and 9, or 4, 6, and 9. CSC indicates cardiac stem cell; IgG, immunoglobulin G; and TERT, telomerase reverse transcriptase. telomerase activity, and higher expression of IGF-1R in CSCs showed similar beneficial effects on wall thickening and LV chamber diameters (Figure 5A through 5F). Importantly, the indicators of preserved CSC growth were coupled with a decrease in chamber volume, and with increases in ventricular mass, ventricular mass-to-chamber volume ratio, and LVEF (Figure 5G through 5K; Figure IV in the online-only Data Supplement). However, not all CSC parameters showed a Figure 4. IGF-1R in CSCs. A, Bivariate distribution of c-kit and IGF-1R in CSCs. B, Confocal micrographs illustrating IGF-1R labeling (left, red) in c-kit–positive CSCs (center, green). Right, merge. C, Percentage of IGF-1R–positive CSCs measured by FACS in each of the 38 preparations. Patients 1 through 27 (color-coded green) experienced positive LVR; patients 28 through 38 (color-coded red) experienced negative LVR. D and E, Correlations between the percentage of IGF-1R–positive CSCs and telomere length (D), or telomerase activity (E). These relationships are shown by linear regression; solid lines represent the best-fit regression line associated with 95% confidence interval (dashed lines). P values represent the significance of the relationship expressed by the R2 values. Longer telomeres and higher telomerase activity correlate with positive LVR. CSC indicates cardiac stem cell; FACS, fluorescence-activated cell sorter; IGF-1R, insulin-like growth factor-1 receptor; and LVR, left ventricular remodeling. 164 Circulation January 14, 2014 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Figure 5. Continued on next page D’Amario et al Cardiac Stem Cells and the Evolution of Ischemic Cardiomyopathy 165 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Figure 5. Continued on next page 166 Circulation January 14, 2014 Figure 5. Growth reserve of CSCs and anatomic indices of LVR. A through K, Correlations between PDT, telomere length, telomerase activity, and fraction of IGF-1R–positive (IGF-1Rpos) CSCs and the various parameters of cardiac size, shape, and function. These relationships are shown by linear regression; solid lines represent the best-fit regression line associated with 95% confidence interval (dashed lines). P values represent the significance of the relationship expressed by the R2 values. Ch. indicates left ventricular chamber; CSC, cardiac stem cell; EDV, end-diastolic volume; ESV, end-systolic volume; IGF-1R, insulin-like growth factor-1 receptor; LVR, left ventricular remodeling; and PDT, population-doubling time. Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 strong correlation. The Δ anterior and posterior wall thickness in systole and telomerase activity, the Δ posterior wall thickness in diastole and telomerase activity, the Δ end-systolic diameter and IGF-1R–positive CSCs, and the Δ ventricular mass and telomere length and telomerase activity had P values of 0.04 to 0.05. Subsequently, the 38 patients were divided into 1 groups: (1) group 1 showed preservation, reduction, or increase in LV end-diastolic volume <20%, ie, positive LVR (n=27); and (2) group 2 showed an increase in LV end-diastolic volume ≥20%, ie, negative LVR (n=11). These 2 patient cohorts did not differ in terms of baseline clinical, anatomic, and functional characteristics (Table 2). When the growth properties of CSCs in these 2 patient subsets were compared, PDT was 10% shorter in positively remodeled hearts. Moreover, telomere length was 8% longer, and telomerase activity and IGF-1R expression were, respectively, 25% and 26% higher in this group (Figure 6A). Wall thickening, chamber diameter and volume, LV mass, and LV mass-to-chamber volume ratio varied significantly after surgery. From baseline to 12±1 months following revascularization, Δ end-diastolic and Δ end-systolic LV volume decreased, respectively, 11% and 18%, whereas ΔLVEF increased 9% in patients with positive LVR. Conversely, in patients with negative LVR, Δ end-diastolic volume increased 28%, Δ end-systolic volume increased 20%, and LVEF decreased 14% (Figure 6B and 6C). By multivariate analysis, the 4 variables of CSC growth (ie, PDT, telomere length, telomerase activity, and IGF-1R expression) retained their statistical significant association with LVR (Table III in the online-only Data Supplement). Additionally, the receiver operating characteristic curve indicated that PDT, telomere length, and telomerase activity were significantly related to LVR with an area under the curve of 0.92 (95% confidence interval [CI], 0.78–0.98; P<0.001), 0.85 (95% CI, 0.70–0.94; P<0.001) and 0.75 (95% CI, 0.59–0.88; P<0.003), respectively. Similarly, IGF-1R presented an area under the curve of 0.86 (95% CI, 0.71–0.95; P<0.001) (Table IV in the online-only Data Supplement). Thus, the replication reserve of CSCs, based on PDT, telomere length, telomerase activity, and IGF-1R expression may be viewed as a novel biomarker of clinical outcome in patients requiring bypass surgery for CAD and ischemic cardiomyopathy. The use of 2-dimensional echocardiography as an imaging modality to evaluate LVR has limitations. However, the guidelines of the joint American Society of Echocardiography and European Association of Echocardiography still recommend the Simpson biplane method of discs as the routine preferred 2-dimensional protocol for calculating ventricular dimensions and LVEF. Moreover, based on the Bland-Altman test, the inter- and intraobserver agreement for this analysis was 94% and 96%, respectively. Circulating Growth Factors The blood concentration of IGF-1, HGF, SCF, VEGF, G-CSF, and bFGF was determined at baseline and 12±1 months following revascularization. These growth factors were selected because of their ability to activate CSC division and migration (IGF-1, HGF, SCF), formation of coronary vessels (VEGF), egression of HSCs from the bone marrow into the systemic circulation (SCF, G-CSF), and fibroblast proliferation (bFGF). The ΔIGF-1, ΔHGF, ΔSCF, ΔVEGF, ΔG-CSF, and ΔbFGF failed to correlate with the anatomic parameters of LVR. However, the levels of IGF-1 and HGF were, respectively, 2.5-fold and 27% higher before bypass surgery in patients who experienced positive LVR; but, at 12±1 months, only IGF-1 remained elevated. In these patients, the concentration of VEGF was 17% higher at baseline but decreased significantly at 12±1 months. The circulating amounts of SCF, G-CSF, and bFGF were similar before and after surgery (Figure 6D). Thus, IGF-1, HGF, and VEGF may have a protective effect on the myocardium before revascularization, although IGF-1 may be implicated in the manifestations of positive LVR after surgery. Discussion The results of the current study indicate that the function of resident CSCs provides critical information concerning the recovery of the myocardium following successful revascularization of patients who have chronic CAD. Negative LVR over a period of 12 months after coronary bypass was not observed if the CSC compartment before surgery retained a significant growth reserve. Decline in the replicative potential of CSCs was paralleled by alterations in ventricular wall thickening, together with chamber dilation and reduction in LV mass-to-chamber volume ratio. The correlation between each of the 4 indices of CSC growth and the size and shape of the heart strongly suggest that the behavior of CSCs is implicated in the positive or negative outcome of the surgically treated ischemic cardiomyopathic heart. PDT, telomere length, telomerase activity, and the expression of IGF-1R have dramatic effects on CSC division and survival and in the generation of a differentiated specialized progeny.9,10,17 These variables, in combination with the high concentration of IGF-1 in the circulation before and after cardiac surgery, point to the IGF-1–IGF-1R system as a major player in positive LVR. Although CSCs were isolated from the right atrial appendage and not from the diseased LV myocardium, the growth characteristics of human CSCs have been shown to be comparable in these 2 anatomic regions, and in the right side of the septum and right ventricle, as well.6,7,16,18,26 Caution has to be exercised in the interpretation of these findings. As indicated in the Results section, not all parameters showed a strong correlation, raising the possibility that the D’Amario et al Cardiac Stem Cells and the Evolution of Ischemic Cardiomyopathy 167 Table 2. Characteristics of Patients with Negative and Positive LVR Variables Table 2. Continued Negative LVR Patients 11 (29%) Positive LVR Patients 27 (71%) P Value 65±9 70±8 0.10 Clinical characteristics Age, years±SD Sex Male, n (%) Female, n (%) Hypertension, n (%) 0.29 11 (100) 22 (81.5) 0 (0) 5 (18.5) 11 (100) 26 (96.3) 1.00 Current smoker, n (%) 7 (63.6) 16 (59.2) 1.00 Dyslipidemia, n (%) 8 (72.7) 22 (81.5) 0.67 Diabetes mellitus, n (%) 7 (63.6) 17 (63) 1.00 Family history of CAD, n (%) 10 (91) 18 (66.6) 0.23 Body mass index, kg/m2 29.1±5 27.8±4 0.44 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 NYHA functional class 0.66 NYHA I, n (%) 1(9.0) 1 (3.7) NYHA II, n (%) 5 (45.5) 16(59.3) NYHA III, n (%) 5 (45.5) 10(37) NYHA IV, n (%) 0 (0) 0(0) Therapy on admission β-Blockers, n (%) 8 (72.7) 25 (92.6) 0.13 ACE inhibitors, n (%) 8 (72.7) 19 (70.4) 1.00 Statins, n (%) 9 (81.8) 21 (77.8) 1.00 Aspirin, n (%) 6 (54.5) 19 (70.4) 0.45 ARBs, n (%) 0 1 (3.7) 1.00 Diuretics, n (%) 6 (54.5) Heparin, n (%) 1 (9.1) 3 (11.1) 1.00 0 0 NA 17.63±9 18.3±6 0.82 5.4±2 5.4±2 0.98 7±1 6.9±1 0.77 Clopidogrel, n (%) 20 (74) 0.28 Laboratory tests Urea nitrogen, mg/dL Uric acid, mg/dL HbA1c, %±SD Total cholesterol, mg/dL±SD 173±41 151±49 0.22 LDL, mg/dL±SD 121±32 115±46 0.18 HDL, mg/dL±SD 40±18 44±18 0.24 111±30 95±18 0.28 68±13 70±10 0.22 57.7±11.2 52.8±11 0.23 113.1±37 Triglycerides, mg/dL ±SD Creatinine clearance, mL/min±SD Echocardiography on admission LV ejection fraction, % LV end-diastolic volume, mL 106.5±45.5 LV end-systolic volume, mL 53.9±33.2 Wall Motion Score Index 1.78±1 LV diastolic diameter, mm 48.7±4.7 0.67 59.5±30.6 0.63 1.4±0.3 0.15 47.4±4.4 0.43 LV anterior wall thickness, mm 10±1.2 9.5±0.9 0.21 LV posterior wall thickness, mm 9.9±1 9.4±0.9 0.25 LV mass, g 177±43.8 157.7±40 0.22 LV mass/chamber volume, M/ChVol 1.8±0.6 1.5±0.3 0.10 (Continued ) Variables Negative LVR Patients 11 (29%) Positive LVR Patients 27 (71%) P Value Angiographic analysis and preoperative risk stratification Coronary disease extension, n (%) 1 Vessel+LM 1 (9.1) 1 (3.7) 2 Vessels 2 (18.2) 8 (29.6) 3 Vessels 8 (72.7) 18 (66.7) CABG+valve replacement, n (%) 0 0 Log euroSCORE, average±SD 9.7±11.2 7.8±8.5 Log euroSCORE 0–4, n (%) 5 (45.4) 14 (51.9) Log euroSCORE 5–9, n (%) 3 (27.3) 4 (14.8) Log euroSCORE >10, n (%) 3 (27.3) 9 (33.3) euroSCORE II, average±SD 7.7±7.1 8.5±9.2 Intra- and perioperative variables Number of grafts, n (%) 3±1 3±1 0.95 Number of anastomosis, n (%) 4±1 4±1 0.98 LIMA, n (%) 11 (100) 26 (96.3) 1.00 BIMA, n (%) 2 (18) 5 (18.5) 1.00 Cross-clamp, min±SD 57±16 63±19 0.39 ECC time, min±SD 69±18 73±18 0.55 Cardioplegia, mL, DS 20.5±9.8 19.3±4.4 0.72 Total mEq K+ infused 40.9±19.5 38.7±8.8 0.71 Ischemic time, min±SD 50.3±14.8 52.4±16.4 0.70 Post-CABG atrial fibrillation 14.8±0.4 18.2±0.4 0.81 CK-MB (peak; 48 h post-CABG) 19.4±24.6 16.4±14.5 0.60 Therapy at discharge β-Blockers, n (%) 9 (81.8) 25 (92.6) 0.43 ACE inhibitors, n (%) 8 (72.7) 25 (92.6) 0.20 Statins, n (%) 9 (81.8) 22 (81.5) 0.88 Aspirin, n (%) 10 (90.9) 26 (96.3) 0.88 Diuretics, n (%) 9 (81.8) 21 (77.8) Heparin, n (%) 0 0 Amiodarone, n (%) 2 (18.2) 4 (14.8) 0.59 Digoxin, n (%) 1 (9.1) 2 (7.4) 0.65 Ca2+ antagonist, n (%) 1 (9.1) 2 (7.4) 0.65 0.9 1 ACE indicates angiotensin-converting enzyme; ARB, angiotensin receptor blocker; BIMA, left and right internal mammary artery; CABG, coronary artery bypass surgery; CAD, coronary artery disease; CK-MB, creatine kinase MB; ECC, extra corporeal circulation; HbA1c, hemoglobin A1c; HDL, high-density lipoprotein; LDL, low-density lipoprotein; LIMA, left internal mammary artery; LM, left main stem disease; LV, left ventricle; NYHA, New York Heart Association; and SD, standard deviation. approach used here may have to be extended to a significantly larger number of patients to strengthen the data or to restrict the variables of CSC growth that predict negative and positive LVR following coronary bypass surgery. Moreover, the most appropriate characterization of CSC behavior for clinical outcome would have required sampling of the myocardium after the recovery from the surgical procedure, a protocol that could not be implemented without exposing patients to unnecessary, additional risks. 168 Circulation January 14, 2014 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Figure 6. Continued on next page D’Amario et al Cardiac Stem Cells and the Evolution of Ischemic Cardiomyopathy 169 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Figure 6. CSCs, growth factors, and LVR. A through C, Data are shown by box plots: the box represents the interquartile range, the horizontal line inside the box marks the median, and whiskers show 5 to 95 percentiles. *Indicates P<0.05 vs negative (neg) LV remodeling (A, LVR) or baseline (B and C). Shorter population-doubling time, longer telomeres, and indices of wall thickening, decreased chamber size, and increased myocardial mass were coupled with positive LVR. D, Serum levels of IGF-1, HGF, VEGF, SCF, G-CSF, and bFGF at baseline and follow-up. The increase in IGF-1 was associated with positive LVR. The quantity of each growth factor in patients with positive (pos) and negative (neg) LVR is shown as mean±SD. *P<0.05 vs baseline values in patients who underwent neg LVR at follow-up; **P<0.05 vs follow-up values in patients who underwent neg LVR. bFGF indicates basic fibroblast growth factor; BrdU, bromodeoxyuridine; CSC, cardiac stem cell; G-CSF, granulocyte-colony stimulating factor; HGF, hepatocyte growth factor; IGF1, insulin-like growth factor-1; IGF-1R, insulin-like growth factor-1 receptor; LVR, left ventricular remodeling; SCF, stem cell factor; SD, standard deviation; and VEGF, vascular endothelial growth factor. Increased circulating levels of IGF-1 are characterized by a decreased incidence of heart failure and mortality in elderly individuals.27,28 Additionally, the upregulation of IGF-1 locally in the myocardium favors the spontaneous recovery from advanced heart failure in patients with dilated cardiomyopathy and LV assist device implantation.29,30 In contrast, low IGF-1 concentrations in the general population predict the development of ischemic heart disease and chronic heart failure.31–34 Data in this study support the notion that the systemic increase of IGF-1 in patients with positive LVR may exert its beneficial effect by activating IGF-1R on CSCs and thereby promoting stem cell division and myocyte and vessel formation. This possibility is consistent with a series of experimental findings in which the interaction of IGF-1 with IGF-1R in CSCs contributes, together with HGF, to reverse myocardial aging,10 and rescues myocardial infarcts commonly incompatible with life in small mammals.35 In this regard, cardiac restricted overexpression of IGF-1 prolongs the lifespan in transgenic mice.36 HGF, a powerful mediator of CSC migration,9,10,35 was significantly higher at baseline in subjects who had positive LVR, although comparable concentrations were found in both groups of patients 12 months following revascularization. VEGF behaved as HGF. A small category of human c-kit–positive CSCs express vascular endothelial growth factor receptor-27 and may be activated by VEGF, resulting in vascular growth mostly needed before successful bypass surgery. Chronic coronary artery disease is characterized by a fixed reduction of coronary flow reserve attributable to an epicardial obstruction resulting in inadequate myocardial perfusion following stress. Even in the absence of previous myocardial infarction or acute coronary syndrome, myocyte loss may occur and dysfunctional myocardial areas may be present. However, no correlations were found between preoperative burden of ischemia and indices of LV recovery after CABG (Figure V in online-only Data Supplement). These results suggest that amelioration in coronary flow translates into positive LVR in the presence of functionally competent resident CSCs. CSCs are multipotent and generate parenchymal cells and coronary vessels.6,7,17 CSCs possess the ability to form resistance arterioles and capillary structures, both critical determinants of blood flow regulation and tissue oxygenation. These unique properties provide the necessary substrate for the recovery in structure and function observed here in patients experiencing positive LVR following CABG. Bypass surgery repairs the defects in the conductive coronary arteries, and CSCs may operate at the more distal level of the coronary microcirculation. The recognition that variables intrinsic to the myocardium, ie, CSC growth reserve, and extrinsic to the heart, ie, systemic levels of IGF-1, HGF, and VEGF, are coupled with increases in LVEF, wall thickness in systole and diastole, and significant decreases in systolic and diastolic diameters and chamber volume following revascularization has important clinical implications. The improvement in systolic performance and the likelihood of reduction in wall stress, dictated by these changes in cardiac anatomy,3 have been coupled with decreased morbidity and mortality in patients with ischemic cardiomyopathy.4,5 Conversely, negative LVR is characterized by an opposite response with a decrease in LVEF and 170 Circulation January 14, 2014 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 an expansion in chamber volume, accurate predictors of increased mortality in this patient population.4,5 Recently, stem cells have been introduced as a new experimental treatment for subacute myocardial infarction37 or chronic ischemic cardiomyopathy with severely impaired systolic function.18,38 In both cases, encouraging results have been obtained. Autologous CSCs18,38 induce myocardial regeneration with absolute reduction in infarct scar size and promote dramatic increases in LVEF. Moreover, a significant reduction in New York Heart Association functional class and a remarkable amelioration of quality of life were demonstrated.18 But no clear indication of reverse LVR has been acquired. Our results in a subset of patients with better preserved cardiac function indicate that the diseased myocardium retains the ability to decrease heart size and increase LV mass-to-chamber volume ratio. Whether the implementation of more sophisticated protocols involving new CSC classes will achieve this objective remains a major challenge in stem cell–based therapy of the failing heart. The recognition, nearly 12 years ago, that c-kit–positive HSCs have the inherent ability to repair the infarcted myocardium in experimental models14 has profoundly affected cardiovascular research and clinical cardiology. The unanticipated plasticity of adult HSCs to form cells beyond their own tissue boundary has become the driving force of a series of clinical studies in which bone marrow cells have been introduced as an experimental therapy in the management of the acutely infarcted or chronically failing heart.13,14 A recent meta-analysis strongly supports the view that various classes of bone marrow cells interfere with cardiac dysfunction, infarct size, ventricular remodeling, and mortality in patients with ischemic heart disease.39 Importantly, transcatheter, intramyocardial injections of autologous bone marrow mononuclear cells or mesenchymal stromal cells improve the regional contractility of a chronic myocardial scar and reverse ventricular remodeling in animals and humans.40–44 A relevant issue to be emphasized relates to the protocol we have developed and improved over the years concerning the isolation and expansion of human CSCs. From an initial rate of success of ≈60%,6 we are currently able to obtain large quantities of CSCs with significant growth reserve in essentially 100% of the cases as documented previously18 and in the present report. Here, the fraction of undifferentiated CSCs varied from a minimum of 80% to a maximum of 95%. Twenty-nine of the 38 preparations had values of c-kit–positive CSCs ranging from 86% to 95%. This level of consistency was essential for the evaluation of the properties of CSCs to be correlated with the parameters of cardiac anatomy and function of patients with ischemic cardiomyopathy. Unfortunately, this degree of accuracy is difficult to achieve when mixed cell populations are used and the proportion of 1 cell type versus the other may change significantly in different samples. Collectively, our results indicate that the telomere-telomerase axis, PDT, and IGF-1R expression in CSCs represent novel biomarkers able to predict the evolution of the ischemic cardiomyopathic heart following revascularization. This conviction becomes particularly relevant because CSCs can be easily isolated, expanded, and carefully characterized from endomyocardial biopsies,16 avoiding the surgical approach used here. Additionally, the circulating levels of IGF-1, HGF, and VEGF are routinely measured offering, together with the properties of CSCs, a rather unique perspective of bypass surgery in patients with chronic CAD. Although the analysis of CSCs in each patient needed cell expansion in vitro for the multiple assays, the cultured CSCs retained properties that correlated with the severity and unfavorable or favorable evolution of the cardiac disease. Three criteria have been postulated for the definition of a biomarker: its accuracy, its ability to give information that cannot be obtained from clinical assessment, and its relevance on medical decision.45 The CSC phenotypes defined in the current report fulfill these 3 criteria; they are highly reproducible, provide an understanding at the fundamental cellular level of the pathological heart, and suggest new therapeutic strategies. Sources of Funding This work was supported by a grant from the Italian Ministry for University and Research (PRIN 2010/2011/2010S7CET4) and by National Institutes of Health grants. Disclosures Dr Anversa is a member of Analogous, LLC. The other authors report no conflicts. References 1. Rizzello V, Poldermans D, Boersma E, Biagini E, Schinkel AF, Krenning B, Elhendy A, Vourvouri EC, Sozzi FB, Maat A, Crea F, Roelandt JR, Bax JJ. Opposite patterns of left ventricular remodeling after coronary revascularization in patients with ischemic cardiomyopathy: role of myocardial viability. Circulation. 2004;110:2383–2388. 2. Schinkel AF, Poldermans D, Rizzello V, Vanoverschelde JL, Elhendy A, Boersma E, Roelandt JR, Bax JJ. Why do patients with ischemic cardiomyopathy and a substantial amount of viable myocardium not always recover in function after revascularization? J Thorac Cardiovasc Surg. 2004;127:385–390. 3. Pfeffer MA, Braunwald E. Ventricular remodeling after myocardial infarction. Experimental observations and clinical implications. Circulation. 1990;81:1161–1172. 4. Pfeffer MA, Braunwald E, Moyé LA, Basta L, Brown EJ Jr, Cuddy TE, Davis BR, Geltman EM, Goldman S, Flaker GC. Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction. Results of the survival and ventricular enlargement trial. The SAVE Investigators. N Engl J Med. 1992;327:669–677. 5. Verma A, Meris A, Skali H, Ghali JK, Arnold JM, Bourgoun M, Velazquez EJ, McMurray JJ, Kober L, Pfeffer MA, Califf RM, Solomon SD. Prognostic implications of left ventricular mass and geometry following myocardial infarction: the VALIANT (VALsartan In Acute myocardial iNfarcTion) Echocardiographic Study. JACC Cardiovasc Imaging. 2008;1:582–591. 6. Bearzi C, Rota M, Hosoda T, Tillmanns J, Nascimbene A, De Angelis A, Yasuzawa-Amano S, Trofimova I, Siggins RW, Lecapitaine N, Cascapera S, Beltrami AP, D’Alessandro DA, Zias E, Quaini F, Urbanek K, Michler RE, Bolli R, Kajstura J, Leri A, Anversa P. Human cardiac stem cells. Proc Natl Acad Sci U S A. 2007;104:14068–14073. 7. Bearzi C, Leri A, Lo Monaco F, Rota M, Gonzalez A, Hosoda T, Pepe M, Qanud K, Ojaimi C, Bardelli S, D’Amario D, D’Alessandro DA, Michler RE, Dimmeler S, Zeiher AM, Urbanek K, Hintze TH, Kajstura J, Anversa P. Identification of a coronary vascular progenitor cell in the human heart. Proc Natl Acad Sci U S A. 2009;106:15885–15890. 8. Vanoverschelde JL, Depré C, Gerber BL, Borgers M, Wijns W, Robert A, Dion R, Melin JA. Time course of functional recovery after coronary artery bypass graft surgery in patients with chronic left ventricular ischemic dysfunction. Am J Cardiol. 2000;85:1432–1439. D’Amario et al Cardiac Stem Cells and the Evolution of Ischemic Cardiomyopathy 171 Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 9. Linke A, Müller P, Nurzynska D, Casarsa C, Torella D, Nascimbene A, Castaldo C, Cascapera S, Böhm M, Quaini F, Urbanek K, Leri A, Hintze TH, Kajstura J, Anversa P. Stem cells in the dog heart are self-renewing, clonogenic, and multipotent and regenerate infarcted myocardium, improving cardiac function. Proc Natl Acad Sci U S A. 2005;102:8966–8971. 10.Gonzalez A, Rota M, Nurzynska D, Misao Y, Tillmanns J, Ojaimi C, Padin-Iruegas ME, Müller P, Esposito G, Bearzi C, Vitale S, Dawn B, Sanganalmath SK, Baker M, Hintze TH, Bolli R, Urbanek K, Hosoda T, Anversa P, Kajstura J, Leri A. Activation of cardiac progenitor cells reverses the failing heart senescent phenotype and prolongs lifespan. Circ Res. 2008;102:597–606. 11. Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature. 2011;473:298–307. 12. Leone AM, Valgimigli M, Giannico MB, Zaccone V, Perfetti M, D’Amario D, Rebuzzi AG, Crea F. From bone marrow to the arterial wall: the ongoing tale of endothelial progenitor cells. Eur Heart J. 2009;30:890–899. 13. Anversa P, Kajstura J, Rota M, Leri A. Regenerating new heart with stem cells. J Clin Invest. 2013;123:62–70. 14. Leri A, Anversa P. Stem cells: bone-marrow-derived cells and heart failure–the debate goes on. Nat Rev Cardiol. 2013;10:372–373. 15.Bolognese L, Cerisano G, Buonamici P, Santini A, Santoro GM, Antoniucci D, Fazzini PF. Influence of infarct-zone viability on left ventricular remodeling after acute myocardial infarction. Circulation. 1997;96:3353–3359. 16. D’Amario D, Fiorini C, Campbell PM, Goichberg P, Sanada F, Zheng H, Hosoda T, Rota M, Connell JM, Gallegos RP, Welt FG, Givertz MM, Mitchell RN, Leri A, Kajstura J, Pfeffer MA, Anversa P. Functionally competent cardiac stem cells can be isolated from endomyocardial biopsies of patients with advanced cardiomyopathies. Circ Res. 2011;108:857–861. 17.D’Amario D, Cabral-Da-Silva MC, Zheng H, Fiorini C, Goichberg P, Steadman E, Ferreira-Martins J, Sanada F, Piccoli M, Cappetta D, D’Alessandro DA, Michler RE, Hosoda T, Anastasia L, Rota M, Leri A, Anversa P, Kajstura J. Insulin-like growth factor-1 receptor identifies a pool of human cardiac stem cells with superior therapeutic potential for myocardial regeneration. Circ Res. 2011;108:1467–1481. 18.Bolli R, Chugh AR, D’Amario D, Loughran JH, Stoddard MF, Ikram S, Beache GM, Wagner SG, Leri A, Hosoda T, Sanada F, Elmore JB, Goichberg P, Cappetta D, Solankhi NK, Fahsah I, Rokosh DG, Slaughter MS, Kajstura J, Anversa P. Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial. Lancet. 2011;378:1847–1857. 19. Berenson ML, Levine DM, Rindskopf D. Applied Statistics. Englewood Cliffs, NJ: Prentice Hall; 1988. 20. Fawcett T. An introduction to ROC analysis. Pattern Recognition Lett. 2006;27:861–874 21. Rota M, LeCapitaine N, Hosoda T, Boni A, De Angelis A, Padin-Iruegas ME, Esposito G, Vitale S, Urbanek K, Casarsa C, Giorgio M, Lüscher TF, Pelicci PG, Anversa P, Leri A, Kajstura J. Diabetes promotes cardiac stem cell aging and heart failure, which are prevented by deletion of the p66shc gene. Circ Res. 2006;99:42–52. 22.Kajstura J, Gurusamy N, Ogórek B, Goichberg P, Clavo-Rondon C, Hosoda T, D’Amario D, Bardelli S, Beltrami AP, Cesselli D, Bussani R, del Monte F, Quaini F, Rota M, Beltrami CA, Buchholz BA, Leri A, Anversa P. Myocyte turnover in the aging human heart. Circ Res. 2010;107:1374–1386. 23. Cesselli D, Beltrami AP, D’Aurizio F, Marcon P, Bergamin N, Toffoletto B, Pandolfi M, Puppato E, Marino L, Signore S, Livi U, Verardo R, Piazza S, Marchionni L, Fiorini C, Schneider C, Hosoda T, Rota M, Kajstura J, Anversa P, Beltrami CA, Leri A. Effects of age and heart failure on human cardiac stem cell function. Am J Pathol. 2011;179:349–366. 24.Aubert G, Lansdorp PM. Telomeres and aging. Physiol Rev. 2008;88:557–579. 25. Torella D, Rota M, Nurzynska D, Musso E, Monsen A, Shiraishi I, Zias E, Walsh K, Rosenzweig A, Sussman MA, Urbanek K, Nadal-Ginard B, Kajstura J, Anversa P, Leri A. Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression. Circ Res. 2004;94:514–524. 26. Itzhaki-Alfia A, Leor J, Raanani E, Sternik L, Spiegelstein D, Netser S, Holbova R, Pevsner-Fischer M, Lavee J, Barbash IM. Patient characteristics and cell source determine the number of isolated human cardiac progenitor cells. Circulation. 2009;120:2559–2566. 27.Perkel D, Naghi J, Agarwal M, Morrissey RP, Phan A, Willix RD Jr, Schwarz ER. The potential effects of IGF-1 and GH on patients with chronic heart failure. J Cardiovasc Pharmacol Ther. 2012;17:72–78. 28. Arcopinto M, Bobbio E, Bossone E, Perrone-Filardi P, Napoli R, Sacca L, Cittadini A. The GH/IGF-1 axis in chronic heart failure. Endocr Metab Immune Disord Drug Targets. 2013;13:76–91. 29. Fazio S, Sabatini D, Capaldo B, Vigorito C, Giordano A, Guida R, Pardo F, Biondi B, Saccà L. A preliminary study of growth hormone in the treatment of dilated cardiomyopathy. N Engl J Med. 1996;334:809–814. 30. Barton PJ, Felkin LE, Birks EJ, Cullen ME, Banner NR, Grindle S, Hall JL, Miller LW, Yacoub MH. Myocardial insulin-like growth factor-I gene expression during recovery from heart failure after combined left ventricular assist device and clenbuterol therapy. Circulation. 2005;112(9 suppl):I46–I50. 31. Rosén T, Bengtsson BA. Premature mortality due to cardiovascular disease in hypopituitarism. Lancet. 1990;336:285–288. 32. Juul A, Scheike T, Davidsen M, Gyllenborg J, Jørgensen T. Low serum insulin-like growth factor I is associated with increased risk of ischemic heart disease: a population-based case-control study. Circulation. 2002;106:939–944. 33. Saccà L. Heart failure as a multiple hormonal deficiency syndrome. Circ Heart Fail. 2009;2:151–156. 34.Cittadini A, Saldamarco L, Marra AM, Arcopinto M, Carlomagno G, Imbriaco M, Del Forno D, Vigorito C, Merola B, Oliviero U, Fazio S, Saccà L. Growth hormone deficiency in patients with chronic heart failure and beneficial effects of its correction. J Clin Endocrinol Metab. 2009;94:3329–3336. 35. Urbanek K, Rota M, Cascapera S, Bearzi C, Nascimbene A, De Angelis A, Hosoda T, Chimenti S, Baker M, Limana F, Nurzynska D, Torella D, Rotatori F, Rastaldo R, Musso E, Quaini F, Leri A, Kajstura J, Anversa P. Cardiac stem cells possess growth factor-receptor systems that after activation regenerate the infarcted myocardium, improving ventricular function and long-term survival. Circ Res. 2005;97:663–673. 36. Li Q, Ren J. Influence of cardiac-specific overexpression of insulin-like growth factor 1 on lifespan and aging-associated changes in cardiac intracellular Ca2+ homeostasis, protein damage and apoptotic protein expression. Aging Cell. 2007;6:799–806. 37. Makkar RR, Smith RR, Cheng K, Malliaras K, Thomson LE, Berman D, Czer LS, Marbán L, Mendizabal A, Johnston PV, Russell SD, Schuleri KH, Lardo AC, Gerstenblith G, Marbán E. Intracoronary cardiosphere-derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial. Lancet. 2012;379:895–904. 38. Chugh AR, Beache GM, Loughran JH, Mewton N, Elmore JB, Kajstura J, Pappas P, Tatooles A, Stoddard MF, Lima JA, Slaughter MS, Anversa P, Bolli R. Administration of cardiac stem cells in patients with ischemic cardiomyopathy: the SCIPIO trial: surgical aspects and interim analysis of myocardial function and viability by magnetic resonance. Circulation. 2012;126(11 suppl 1):S54–S64. 39.Jeevanantham V, Butler M, Saad A, Abdel-Latif A, Zuba-Surma EK, Dawn B. Adult bone marrow cell therapy improves survival and induces long-term improvement in cardiac parameters: a systematic review and meta-analysis. Circulation. 2012;126:551–568. 40. Williams AR, Trachtenberg B, Velazquez DL, McNiece I, Altman P, Rouy D, Mendizabal AM, Pattany PM, Lopera GA, Fishman J, Zambrano JP, Heldman AW, Hare JM. Intramyocardial stem cell injection in patients with ischemic cardiomyopathy: functional recovery and reverse remodeling. Circ Res. 2011;108:792–796. 41. Zhao Y, Li T, Wei X, Bianchi G, Hu J, Sanchez PG, Xu K, Zhang P, Pittenger MF, Wu ZJ, Griffith BP. Mesenchymal stem cell transplantation improves regional cardiac remodeling following ovine infarction. Stem Cells Transl Med. 2012;1:685–695. 42. Hare JM, Fishman JE, Gerstenblith G, DiFede Velazquez DL, Zambrano JP, Suncion VY, Tracy M, Ghersin E, Johnston PV, Brinker JA, Breton E, Davis-Sproul J, Schulman IH, Byrnes J, Mendizabal AM, Lowery MH, Rouy D, Altman P, Wong Po Foo C, Ruiz P, Amador A, Da Silva J, McNiece IK, Heldman AW, George R, Lardo A. Comparison of allogeneic vs autologous bone marrow–derived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial. JAMA. 2012;308:2369–2379. 43. Williams AR, Suncion VY, McCall F, Guerra D, Mather J, Zambrano JP, Heldman AW, Hare JM. Durable scar size reduction due to allogeneic 172 Circulation January 14, 2014 mesenchymal stem cell therapy regulates whole-chamber remodeling. J Am Heart Assoc. 2013;2:e000140. 44. Bartunek J, Behfar A, Dolatabadi D, Vanderheyden M, Ostojic M, Dens J, El Nakadi B, Banovic M, Beleslin B, Vrolix M, Legrand V, Vrints C, Vanoverschelde JL, Crespo-Diaz R, Homsy C, Tendera M, Waldman S, Wijns W, Terzic A. Cardiopoietic stem cell therapy in heart failure: the C-CURE (Cardiopoietic stem Cell therapy in heart failURE) multicenter randomized trial with lineage-specified biologics. J Am Coll Cardiol. 2013;61:2329–2338. 45.Braunwald E. Biomarkers in heart failure. N Engl J Med. 2008; 358:2148–2159. Clinical Perspective Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 The efficacy of bypass surgery in improving ventricular function in patients with severe coronary atherosclerosis and ischemic cardiomyopathy cannot be easily predicted. The preoperative conditions may be similar, but the mid- and long-term evolution of cardiac pathology may differ significantly, emphasizing the need to identify determinants that may help anticipating clinical outcome. Successful revascularization and positive left ventricular remodeling may depend on factors intrinsic to the heart or extrinsic to the myocardium. Our findings suggest that variables intrinsic to the myocardium, ie, cardiac stem cell growth reserve, and extrinsic to the heart, ie, the systemic level of insulin-like growth factor-1, positively correlate with increases in ejection fraction, wall thickness in systole and diastole, and decreases in systolic and diastolic diameters and chamber volume following revascularization. Importantly, the growth kinetics of cardiac stem cells included shorter population-doubling time, longer telomeres, higher telomerase activity, and enhanced insulin-like growth factor-1 receptor expression. Conversely, attenuated cardiac stem cell growth and a decline in insulin-like growth factor-1 level in the circulation were characterized by an opposite response with a decrease in ejection fraction and an expansion in chamber volume, both accurate predictors of increased mortality in this patient population. Three criteria define a biomarker: its accuracy, its ability to give information that cannot be obtained from clinical assessment, and its relevance on medical decision. The cardiac stem cell phenotypes defined in the current report fulfill these 3 criteria; they are highly reproducible, provide an understanding at the fundamental cellular level of the pathological heart, and suggest new therapeutic strategies. Downloaded from http://circ.ahajournals.org/ by guest on June 17, 2017 Growth Properties of Cardiac Stem Cells Are a Novel Biomarker of Patients' Outcome After Coronary Bypass Surgery Domenico D'Amario, Antonio M. Leone, Antonio Iaconelli, Nicola Luciani, Mario Gaudino, Ramaswamy Kannappan, Melissa Manchi, Anna Severino, Sang Hun Shin, Francesca Graziani, Gina Biasillo, Andrea Macchione, Costantino Smaldone, Giovanni Luigi De Maria, Carlo Cellini, Andrea Siracusano, Lara Ottaviani, Massimo Massetti, Polina Goichberg, Annarosa Leri, Piero Anversa and Filippo Crea Circulation. 2014;129:157-172; originally published online November 18, 2013; doi: 10.1161/CIRCULATIONAHA.113.006591 Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2013 American Heart Association, Inc. All rights reserved. Print ISSN: 0009-7322. Online ISSN: 1524-4539 The online version of this article, along with updated information and services, is located on the World Wide Web at: http://circ.ahajournals.org/content/129/2/157 An erratum has been published regarding this article. Please see the attached page for: /content/130/7/e65.full.pdf /content/132/22/e363.full.pdf Data Supplement (unedited) at: http://circ.ahajournals.org/content/suppl/2013/11/15/CIRCULATIONAHA.113.006591.DC1 Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Circulation can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Circulation is online at: http://circ.ahajournals.org//subscriptions/ Correction In the article by D’Amario et al, “Growth Properties of Cardiac Stem Cells Are a Novel Biomarker of Patients’ Outcome After Coronary Bypass Surgery,” which was published in the January 14, 2014 issue of the journal (Circulation. 2014;129:157–172), a correction is needed. During the course of an ongoing investigation, questions were raised about Figure 4B in the article. In their attempt to respond to this concern, the authors were unable to locate the original file for the figure, and they were unable to verify its accuracy. Accordingly, the authors have generated a new figure using data from the same sample. The new figure demonstrates the same findings as the original figure, and it does not change the conclusions of the published article. Figure 4B in the online version of the article has been updated. The authors regret this circumstance. (Circulation. 2014;130:e65.) © 2014 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIR.0000000000000092 e65 Correction In the article by D’Amario et al, “Growth Properties of Cardiac Stem Cells Are a Novel Biomarker of Patients’ Outcome After Coronary Bypass Surgery,” which published online before print November 18, 2013, and appeared in the January 14, 2014, issue of the journal (Circulation. 2014;129:157–172. DOI: 10.1161/CIRCULATIONAHA.113.006591), a correction was needed. Piero Anversa, MD, discloses that he is a member of Analogous, LLC. The author regrets this omission. This correction has been made to the current online version of the article, which is available at http://circ.ahajournals.org/content/129/2/157.full (Circulation. 2015;132:e363. DOI: 10.1161/CIR.0000000000000341.) © 2015 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIR.0000000000000341 e363 Online Data Supplement Methods Patients Fifty-five consecutive patients affected by chronic coronary artery disease were included in the study (Figure I in the online-only Data Supplement). Patients underwent elective on-pump CABG for multi-vessel coronary atherosclerosis at the Policlinico A. Gemelli, Catholic University, in Rome. An initial screening was performed prior to surgery to determine the eligibility of patients to participate in the study. The inclusion criteria were: 1. chronic stable angina in the last 6 months, together with evidence of ischemia at stress test; and 2. complete surgical revascularization within 7 days. The exclusion criteria were: 1. evidence of previous myocardial infarct or pathologic Q waves on the 12-leads ECG; 2. hospitalization for acute coronary syndrome during the last 6 months; 3. severe valvular heart disease; 4. coronary artery by-pass graft (CABG) or percutaneous transluminal angioplasty in the preceding 12 months; 5. life expectancy less than 1-year; 6. presence of malignancies; 7. psychosocial conditions precluding long-term adherence; 8. pregnancy; 9. enrollment in other clinical research trial; 10. cardiogenic shock; 11. severe comorbidities; and 12. patients unable or unwilling to give informed written consent. Enrolled patients, who met the specified criteria, were subjected to harvesting of the right atrial appendage during on-pump CABG. A follow-up visit was scheduled 12±1 months after CABG. At the time of enrollment and follow-up, patients had complete clinical and physical examination with collection of cardiovascular risk factors, 2D echocardiography, and evaluation of NYHA functional class. Moreover, the number of diseased vessels and the localization and degree of coronary artery stenosis were evaluated together with the risk stratification score and intra- and peri-operative details (Table 1). Blood samples were drawn at the time of surgery and 12±1 months later to measure the level of IGF-1, HGF, VEGF, SCF, G-CSF, and bFGF. The study was approved by the local ethics committee and conformed to the Declaration of Helsinki on human research. Clinical Evaluation and Cardiovascular Risk Factors In each patient, age, sex, common cardiovascular risk factors, therapy at admission and discharge were recorded. The angiographic assessment of coronary artery disease was also performed. Hypercholesterolemia, diabetes mellitus, and hypertension were considered present if diagnosed during hospitalization or if patients were assuming drugs for these conditions prior to admission. Diagnosis of hypercholesterolemia was made when total serum cholesterol concentration was >200 mg/dL. Patients were considered diabetic if fasting glycemia was >126 mg/dL ≥2 in occasions and hypertensive when blood pressure values were >140/90 mmHg in >4 occasions. Smokers had to consume >3 cigarettes per day at admission. A family history of ischemic heart disease involved the documentation of acute coronary syndrome before 60 years of age in at least one firstdegree relative. The New York Heart Association (NYHA) functional class was determined in all patients before and after CABG. Patients in NYHA functional class I had asymptomatic cardiac disease. Patients in NYHA functional class II showed mild symptoms of cardiac disease, which involved modest shortness of breath and/or angina, and minimal limitations in regular activity. Patients in NYHA class III had marked limitations in activity, e.g. walking short distances (20–100 m) and were comfortable only at rest. Patients in NYHA class IV experienced symptoms at rest and had severe limitations in activity. Predicted operative mortality risk was calculated using Euroscore II and logistic Euroscore. Euroscore values for each patient were measured using the interactive online calculator (www.euroscore.org/calc). Echocardiography At enrollment and follow-up, patients were evaluated echocardiographically. Transthoracic echocardiograms were performed according to the guidelines of the European Society of Echocardiography by the same experienced operator (G.B.) using 2 commercially available equipment (TA700, Artida, Toshiba Medical Systems, Japan) with a second harmonic 2.25-MHz probe to optimize endocardial border visualization. All echocardiograms were digitized and re-analyzed off-line by three experienced operators (FG, AM, CS), who were blinded to clinical and laboratory data. Left ventricular (LV) anterior and posterior wall thickness, and LV end-systolic and enddiastolic diameters were measured from the parasternal long axis view and fractional shortening was calculated. LV end-systolic and end-diastolic volumes were computed, and ejection fraction (EF) was calculated from the apical 4- and 2-chamber views using a modified Simpson’s biplane method. LV mass was calculated using the Devereux equation. All measurements were obtained on three cardiac cycles and average values derived.1-3 Inter- and intra-observer agreement for this analysis was 94% and 96%, respectively. Regional wall motion was scored as follows: 1= normal; 2= hypokinesia; 3= akinesia; and 4= dyskinesia.4 The wall motion score index was calculated as the sum of the score of all segments divided by the total number of segments. Changes in all these parameters were calculated as Δ from baseline to 12±1 months following CABG. The presence of negative LV remodeling (LVR) was claimed when, with respect to baseline, there was an increase ≥20% in LV end-diastolic volume 12±1 months after surgery.5 Isolation of Cardiac Stem Cells (CSCs) The right atrial appendage was harvested and CSCs were isolated as previously described.6-8 Following removal of the fibrotic and fat tissue, the specimens weighted 25 to 50 mg. Each sample was kept in a sterile container filled with growth medium; the tissue was minced in small pieces, ~0.2-1 mg each. Subsequently, tissue fragments were transferred into a 100 mm culture dish, washed twice with saline, minced, placed in 50 ml tubes and allowed to sediment. The supernatant was discarded and samples were resuspended in Ham’s F12K medium containing 1-2 mg/ml collagenase; enzymatic digestion was performed at 37°C. The supernatant containing the small cell pool was 3 transferred into a 15 ml tube filled with growth medium, centrifuged and suspended. Undigested pieces were allowed to sediment and the protocol was repeated 2-3 times. The unfractionated cell population was plated in the presence of growth medium, which was changed twice weekly. Following 2-3 passages, cells were trypsinized, washed with PBS, and centrifuged. The cell pellet was suspended in 300 µl PBS-EDTA containing microbeads conjugated with mouse monoclonal c-kit antibody (CD117 Microbead kit Human, Miltenyi). Following incubation for 20 minutes at 4°C on a 360° swinging rotor, the cell suspension was transferred to columns in the Mini-MACS system. Columns were washed 3 times, the washing solution collected, and the depleted c-kit-negative cells discarded. The column was then detached from the Magnetic System and the plunger applied to the column in order to separate the c-kit-positive cells. These cells were flushed into a 15 ml tube; c-kit-positive cells were centrifuged, re-suspended in growth medium, and plated in culture dishes. Following magnetic separation, c-kitpositive cells were analyzed by flow-cytometry to measure the presence of epitopes specific for myocytes, endothelial cells (ECs), and smooth muscle cells (SMCs). FACS Analysis Aliquots of CSCs at P5-P6 were fixed in 4% paraformaldehyde for 15 minutes at room temperature and incubated with antibodies against transcription factors, cytoplasmic proteins, and surface antigens (Table I in the online-only Data Supplement). The presence of epitopes of bone marrow hematopoietic and mesenchymal stem cells was examined together with the expression of myocyte-specific (GATA4, Nkx2.5, MEF2C, αsarcomeric actin), SMC-specific (GATA6, α-smooth muscle actin) and EC-specific (Ets1, von Willebrand factor) markers. In all cases, CSCs were incubated with the primary antibody for 45 minutes at 37°C. Flow cytometry was performed with FACSAria (Becton Dickinson) or Accuri C6 (Accuri Cytometers) instruments. Cellular debris and aggregates were gated out based on forward and side scatter. Gating on the signal of the nuclear stain DAPI was employed to exclude dead cells and additional artifacts. Isotype- 4 matched negative controls were utilized to define the threshold for each specific signal and establish the appropriate gate for positive cells.6-10 Data were analyzed with the instrument software. Population Doubling Time (PDT) Following characterization by FACS, CSCs were plated at low density, 100 cells per cm2, and the number of cells per unit area of the culture dish was measured daily for 5-7 days. PDT was computed by linear regression of log2 values of cell number. Only values in the exponential growth phase were used.6,8,9 Telomere Length Telomere length in CSCs was measured by flow-FISH.7,11,12 Calibration of the flow cytometer, cell fixation, staining protocol, and normalization were performed utilizing mouse lymphoma cells with known telomere length. Approximately 5 x 105 CSCs and mouse lymphoma cells with long telomeres (L5178Y-R) were washed in hybridization buffer and re-suspended in hybridization solution containing formamide and 0.3 µg/ml FITC-conjugated with a telomere specific PNA probe. Control samples were incubated in hybridization solution in the absence of the PNA probe. Lymphoma cells were distinguished from CSCs by immunolabeling with CD45 antibody. DNA content was established by propidium iodide staining. Based on DNA content, cells were gated at G0/G1 and the fluorescence intensity of telomeres was calculated. Measurements were performed by FACSAria. Telomerase Activity The catalytic activity of telomerase in CSCs was assessed by quantitative PCR.7-10,13 CSCs were homogenized in CHAPS buffer and centrifuged at 4°C. Two different protein concentrations, 0.5 and 1 μg, were employed for the assay. CSC extracts were incubated in a solution containing reverse transcriptase reaction mix and Taq polymerase (TRAPEZE RT Telomerase Detection Kit, Chemicon) at 30°C for 30 minutes. HeLa cells were used as positive control and serial dilutions of control template TSR8 were employed for quantification. CHAPS buffer in the absence of protein lysates was used as 5 negative control. PCR cycling conditions were as follows: 1 cycle of 95°C for 2.0 minutes; 40 cycles of 94°C for 15 seconds, and 59°C for 60 seconds. Data were collected at the 59°C stage of each cycle. Western Blotting and Immunoprecipitation Protein lysates of cultured CSCs were obtained using CHAPS buffer (Millipore) and protease inhibitors (Complete tablets, Roche). Equivalents of 5-10 μg of proteins were separated on 4-20% SDS-PAGE, transferred onto PVDF membranes (Bio-Rad) and subjected to Western blotting with rabbit polyclonal anti-Akt (Cell Signaling) and rabbit polyclonal anti-phospho-AktSer473 (Cell Signaling) diluted 1:1500 in TBST containing 5% BSA overnight at 4°C.9,14 Proteins were detected by chemiluminescence (ECL Plus, Thermopierce). Optical density was measured and the quantification was performed as ratio of phospho-Akt to total Akt. Loading conditions were determined by the expression of GAPDH (Cell Signaling). Equivalents of 50 µg of protein extracts were incubated with rabbit polyclonal anti-telomerase antibody (Abcam) overnight at 4°C and subsequently were exposed to magnetic protein G (Millipore) for 1 hour at room temperature.15-17 Immunoprecipitated proteins were eluted by adding 2X loading buffer and heating at 95°C for 5 minutes. Proteins were separated on 4-20% SDS-PAGE and transferred onto a PVDF membrane. Blots were incubated overnight at 4°C with rabbit polyclonal anti phospho-telomerase at Ser824 (Abcam). Optical density was measured and the quantification was performed as ratio of phospho-telomerase to total telomerase. Akt Kinase Assay This assay was performed according to the manufacturer’s protocol (Cell Signaling). Protein lysates were immunoprecipitated using phospho-Akt antibody conjugated with Sepharose beads overnight at 4°C. After washing, GSK-3α/β substrate was added and the enzymatic reaction was carried on for 30 minutes at 30°C. The reaction was stopped by adding loading buffer and heating at 95°C for 5 minutes. Following centrifugation, the supernatant was loaded on 4-20% SDS-PAGE and transferred onto a PVDF membrane. 6 Blots were incubated overnight at 4°C with rabbit polyclonal anti phospho-GSK-3α/β. The detection of a band of the correct molecular weight was indicative of Akt kinase activity in the sample.18 Growth Factors To obtain an accurate, efficient, and quantitative determination of the concentration of multiple human growth factors in the patient’s serum, a multiplex microarray technology based on two-site sandwich ELISA was employed. Capture antibodies specific for target growth factors are pre-spotted into each well of a 96 well microplate so that the growth factors present in the samples are bound by the immobilized antibodies. This approach allowed us to detect simultaneously 6 growth factors in the same sample of serum. Standards and samples were added to the wells. Following washing of the unbound proteins, biotinylated detection antibodies were used. Streptavidin-HRP and, subsequently, chemiluminiscent substrate reagents were added to the wells. A signal proportional to the amount of each growth factor bound to its specific antibody was produced. Plates were read using a digital camera imaging system, and pixel intensity was measured using an analytical software package. A standard curve was created for each analyte by plotting the median pixel intensity for each standard on the y-axis and the concentration of the standard on the x-axis. Data were linearized by plotting the log of the concentrations versus the log of the pixel intensity and the best fit line was determined by regression analysis and corrected for the dilution factor.19 Statistical Analysis Continuous variables were expressed as mean±SD, while dichotomous variables are shown as percentages. Unpaired T-test or Mann Whitney U-test was used for comparison between two groups. Categorical variables were compared using the chi-square test or Fisher’s exact test, as appropriate. Linear regression analysis was performed to correlate the characteristics of CSCs in vitro with the indices of LVR: in each case the best-fit regression line is showed together with the 95% confidence interval, and the P and R2 values. P values less than 0.05 were considered significant. 7 Multivariate logistic regression analysis was performed in order to identify independent predictor of LVR. Variables showing a p value less than 0.05 at univariate analysis were included in the models. Beta value and 95% confidence interval have been reported. Receiver Operating Characteristic (ROC) curve was performed to determine the cellular biomarker or growth factor level that best predicted negative LVR, and to assess the best cut-off value. The Youden index was introduced to evaluate the sensitivity and specificity of each variable. Statistical comparisons were performed using SPSS 20.0 (SPSS, Inc Chicago, Illinois); however, ROC analysis was done using MedCalc (MedCalc, Mariakerke, Belgium).20,21 The magnitude of sampled cells is listed in Table II in the online-only Data Supplement. References 1. Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, Pellikka PA, Picard MH, Roman MJ, Seward J, Shanewise J, Solomon S, Spencer KT, St John Sutton M, Stewart W. Recommendations for chamber quantification. Eur J Echocardiogr. 2006; 7:79-108. 2. Schiller NB, Shah PM, Crawford M, DeMaria A, Devereux R, Feigenbaum H. Recommendations for quantitation of the left ventricle by two-dimensional echocardiography. American Society of Echocardiography Committee on Standards, Subcommittee on Quantitation of Two-Dimensional Echocardiograms. J Am Soc Echocardiogr. 1989; 2:358-367. 3. Lang RM, Bierig M, Devereux RB, Flachskampf FA, Foster E, Pellikka PA, Picard MH, Roman MJ, Seward J, Shanewise JS, Solomon SD, Spencer KT, Sutton MS, Stewart WJ; Chamber Quantification Writing Group; American Society of Echocardiography's Guidelines and Standards Committee; European Association of Echocardiography. Recommendations for chamber quantification: a report from the American Society of Echocardiography's Guidelines and 8 Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology. J Am Soc Echocardiogr. 2005; 18:14401463. 4. Cerqueira MD, Weissman NJ, Dilsizian V, Jacobs AK, Kaul S, Laskey WK, Pennell DJ, Rumberger JA, Ryan T, Verani MS; American Heart Association Writing Group on Myocardial Segmentation and Registration for Cardiac Imaging. Standardized myocardial segmentation and nomenclature for tomographic imaging of the heart. A statement for healthcare professionals from the Cardiac Imaging Committee of the Council on Clinical Cardiology of the American Heart Association. Circulation. 2002; 105:539-542 5. Bolognese L, Cerisano G, Buonamici P, Santini A, Santoro GM, Antoniucci D, Fazzini PF. Influence of infarct-zone viability on left ventricular remodeling after acute myocardial infarction. Circulation. 1997; 96:3353-3359. 6. Bearzi C, Rota M, Hosoda T, Tillmanns J, Nascimbene A, De Angelis A, Yasuzawa-Amano S, Trofimova I, Siggins RW, Lecapitaine N, Cascapera S, Beltrami AP, D'Alessandro DA, Zias E, Quaini F, Urbanek K, Michler RE, Bolli R, Kajstura J, Leri A, Anversa P. Human cardiac stem cells. Proc Natl Acad Sci USA. 2007; 104:14068-14073. 7. Bolli R, Chugh AR, D'Amario D, Loughran JH, Stoddard MF, Ikram S, Beache GM, Wagner SG, Leri A, Hosoda T, Sanada F, Elmore JB, Goichberg P, Cappetta D, Solankhi NK, Fahsah I, Rokosh DG, Slaughter MS, Kajstura J, Anversa P. Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): initial results of a randomised phase 1 trial. Lancet. 2011; 378:1847-1857. 8. Kajstura J, Bai Y, Cappetta D, Kim J, Arranto C, Sanada F, D'Amario D, Matsuda A, Bardelli S, Ferreira-Martins J, Hosoda T, Leri A, Rota M, Loscalzo J, Anversa P. Tracking chromatid segregation to identify human cardiac stem cells that regenerate extensively the infarcted myocardium. Circ Res. 2012; 111:894-906. 9 9. D'Amario D, Cabral-Da-Silva MC, Zheng H, Fiorini C, Goichberg P, Steadman E, Ferreira-Martins J, Sanada F, Piccoli M, Cappetta D, D'Alessandro DA, Michler RE, Hosoda T, Anastasia L, Rota M, Leri A, Anversa P, Kajstura J. Insulin-like growth factor-1 receptor identifies a pool of human cardiac stem cells with superior therapeutic potential for myocardial regeneration. Circ Res. 2011; 108:1467-1481. 10. D'Amario D, Fiorini C, Campbell PM, Goichberg P, Sanada F, Zheng H, Hosoda T, Rota M, Connell JM, Gallegos RP, Welt FG, Givertz MM, Mitchell RN, Leri A, Kajstura J, Pfeffer MA, Anversa P. Functionally competent cardiac stem cells can be isolated from endomyocardial biopsies of patients with advanced cardiomyopathies. Circ Res. 2011; 108:857-861. 11. Canela A, Vera E, Klatt P, Blasco MA. High-throughput telomere length quantification by FISH and its application to human population studies. Proc Natl Acad Sci USA. 2007; 104:5300-5305. 12. Baerlocher GM, Vulto I, de Jong G, Lansdorp PM. Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat Protoc. 2006; 1:23652376. 13. Herbert BS, Hochreiter AE, Wright WE, Shay JW. Non radioactive detection of telomerase activity using the telomeric repeat amplification protocol. Nature Protocols. 2006; 1:1583-1599. 14. Torella D, Rota M, Nurzynska D, Musso E, Monsen A, Shiraishi I, Zias E, Walsh K, Rosenzweig A, Sussman MA, Urbanek K, Nadal-Ginard B, Kajstura J, Anversa P, Leri A. Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression. Circ Res. 2004; 94:514-524. 15. Leri A, Barlucchi L, Limana F, Deptala A, Darzynkiewicz Z, Hintze TH, Kajstura J, Nadal-Ginard B, Anversa P. Telomerase expression and activity are coupled with myocyte proliferation and preservation of telomeric length in the failing heart. Proc Natl Acad Sci USA. 2001; 98:8626-8631. 10 16. Boni A, Urbanek K, Nascimbene A, Hosoda T, Zheng H, Delucchi F, Amano K, Gonzalez A, Vitale S, Ojaimi C, Rizzi R, Bolli R, Yutzey KE, Rota M, Kajstura J, Anversa P, Leri A. Notch1 regulates the fate of cardiac progenitor cells. Proc Natl Acad Sci USA. 2008;105: 15529-15534. 17. Goichberg P, Kannappan R, Cimini M, Bai Y, Sanada F, Sorrentino A, Kajstura J, Rota M, Anversa P, Leri A. Age-associated defects in EphA2 signaling impair the migration of human cardiac stem cells. Circulation. Epub ahead of print, 2013. 18. Thimmaiah KN, Easton J, Huang S, Veverka KA, Germain GS, Harwood FC, Houghton PJ. Insulin-like growth factor I-mediated protection from rapamycininduced apoptosis is independent of Ras-Erk1-Erk2 and phosphatidylinositol 3'kinase-Akt signaling pathways. Cancer Res. 2003; 63:364-74. 19. Fitzgerald JP, Nayak B, Shanmugasundaram K, Friedrichs W, Sudarshan S, Eid AA, DeNapoli T, Parekh DJ, Gorin Y, Block K. Nox4 mediates renal cell carcinoma cell invasion through hypoxia-induced interleukin 6- and 8- production PLoS One. 2012; 7:e30712. 20. Berenson ML, Levine DM, Rindskopf D. Applied Statistics. 1988. Englewood Cliffs, NJ. 21. Fawcett T. An introduction to ROC analysis. Pattern Recognition Letters. 2006; 27:861-874 11 Table I. List of Antibodies _______________________________________________________________________ Epitope Manufacturer Host Animal Labeling _________________________________________________________________________ Surface Markers c-kit DAKO rabbit direct, indirect, F, T CD34 BD Pharmigen mouse direct, PE CD45 BD Pharmigen mouse direct, APC Lineage Markers BD Pharmigen mouse indirect, Cy5 CD90 BD Pharmigen mouse direct, PE CD105 BD Pharmigen mouse direct, PE Transcription Factors GATA4 Nkx2.5 MEF2C Ets1 GATA6 Santa Cruz Santa Cruz Santa Cruz BD Pharmingen Santa Cruz rabbit goat goat rabbit rabbit direct, indirect, F, T direct, indirect, F, T indirect, F, T indirect F, T indirect, F, T Cytoplasmic Proteins α-sarcomeric actin von Willebrand factor α-smooth muscle actin Sigma DAKO Sigma mouse rabbit mouse indirect, F, T indirect, F, T, Cy5 indirect, F, T, Cy5 Other Epitopes BrdU Roche mouse indirect, F,Cy5 ________________________________________________________________________ F: fluorescein isothiocyanate; T: tetramethyl rhodamine isothiocyanate; Cy5: cyanine 5. 12 Table II. Magnitude of Sampling ______________________________________________________________________ Parameter n value Aggregate Sample size sample size (mean±SD) ______________________________________________________________________ FACS Analysis c-kit CD34 CD45 CD133 Lineage markers CD90 CD105 38 38 38 38 38 38 38 675,336 (1) 658,510 (1) 656,686 (1) 644,030 (1) 661,068 (1) 654,563 (1) 655,693 (1) 13,766 ± 5,795 15,851 ± 3,424 15,668 ± 1,761 14,403 ± 1,641 16,108 ± 2,393 15,456 ± 2,510 15,563 ± 1,665 38 38 38 38 38 49,875 (2) 49,032 (2) 47,598 (2) 223,088 (1) N/A 1,012 ± 39 1,452 ± 349 1,380 ± 229 6,154 ± 1307 N/A Growth and Death PDT BrdU TdT Telomere Length Telomerase Activity Lineage Commitment GATA4 38 336,972 (1) 7,121 ± 2,775 8,594 ± 2,104 Nkx2.5 38 424,753 (1) (1) MEF2C 38 439,668 9,958 ± 4,122 α-SA 38 394,177 (1) 7,397 ± 3,539 (1) GATA6 38 387,377 8,447 ± 4,248 α-SMA 38 387,014 (1) 8,376 ± 3,841 (1) 5,804 ± 2,734 CD31 38 436,439 vWf 38 432,858 (1) 9,107 ± 4,458 ______________________________________________________________________ (1) Number of cells analyzed by FACS; (2) Number of cells examined by confocal microscopy. 13 Table III. Predictors of LV Remodeling PDT IGF-1R positive CSC (%) Telomere Length (kbp) Telomerase Act. (n° copies 1x 105) IGF-1 at baseline (ng/ml) IGF-1 at follow-Up HGF at baseline (pg/ml) VEGF at baseline (pg/ml) 26.0 35.5 7.0 1.6 35.4 70.6 884.6 253.9 14 24.8-28.5 31.2-39.6 6.8-7.2 1.4-1.8 19.2-51.6 47.4-93.7 811.6-1257.5 231.4-397.6 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 =0.0921 =0.0873 Table IV. ROC Analysis. 15 Legend to Figures Figure I. Patient population and study design. Figure II. Phenotype of CSCs. A, Bivariate distribution of c-kit, epitopes of hematopoietic stem cells (CD34, CD45), mast cells (CD45), mesenchymal stromal cells (CD90, CD105), and cocktail of bone marrow cell lineages. B, Bivariate distribution of ckit and epitopes of cardiomyocytes (GATA4, Nkx2.5, MEF2C, α-SA), ECs (Ets1, vWf) and SMCs (GATA6, α-SMA). Figure III. CSCs and anatomical indices of LVR. Correlations between c-kit expression in CSCs (showed in percentage) and indices of cardiac shape (Δ end-systolic volume and Δ end-diastolic volume) and function (Δ ejection fraction). These relationships are shown by linear regression. Figure IV. Growth reserve of CSCs and anatomical indices of LVR. Analysis of PDT, telomere length, telomerase activity and fraction of IGF-1R-postive (IGF-1Rpos) CSCs in patients divided in tertiles according to the variation (∆) of parameters of cardiac shape (end-systolic volume and end-diastolic volume) and function (ejection fraction). Data are shown as mean±SD. ESV, end-systolic volume; EDV, end-diastolic volume; EF, left ventricular ejection fraction. *Indicates P<0.05 vs. I tertile values; **Indicates P<0.05 vs. II tertile values. Figure V. Ischemic burden and anatomical indices of LVR. Correlations between preoperative burden of ischemia, expressed as double product values at 1 mm ST-segment depression during stress test and indices of cardiac shape (end-systolic volume and enddiastolic volume) and function (ejection fraction). These relationships are shown by linear regression. 16 17 18 19 20 21