Download Coronary Heart Disease

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