Histone deacetylase inhibition enhances self renewal and cardioprotection by human cord blood-derived CD34 cells.

Abstract

Use of peripheral blood- or bone marrow-derived progenitors for ischemic heart repair is a feasible option to induce neo-vascularization in ischemic tissues. These cells, named Endothelial Progenitors Cells (EPCs), have been extensively characterized phenotypically and functionally. The clinical efficacy of cardiac repair by EPCs cells remains, however, limited, due to cell autonomous defects as a consequence of risk factors. The devise of "enhancement" strategies has been therefore sought to improve repair ability of these cells and increase the clinical benefit.
Pharmacologic inhibition of histone deacetylases (HDACs) is known to enhance hematopoietic stem cells engraftment by improvement of self renewal and inhibition of differentiation in the presence of mitogenic stimuli in vitro. In the present study cord blood-derived CD34(+) were pre-conditioned with the HDAC inhibitor Valproic Acid. This treatment affected stem cell growth and gene expression, and improved ischemic myocardium protection in an immunodeficient mouse model of myocardial infarction.
Our results show that HDAC blockade leads to phenotype changes in CD34(+) cells with enhanced self renewal and cardioprotection.

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Histone Deacetylase Inhibition Enhances Self Renewal
and Cardioprotection by Human Cord Blood-Derived
CD34+Cells
Ilaria Burba1, Gualtiero I. Colombo2, Lidia Irene Staszewsky3, Marco De Simone1, Paolo Devanna1,
Simona Nanni4, Daniele Avitabile1, Fabiola Molla3, Simona Cosentino5, Ilaria Russo3, Noeleen De
Angelis3, Annarita Soldo3, Antonella Biondi3, Elisa Gambini1, Carlo Gaetano6, Antonella Farsetti7, Giulio
Pompilio1, Roberto Latini3, Maurizio C. Capogrossi6, Maurizio Pesce1*
1Laboratorio di Biologia Vascolare e Medicina Rigenerativa, Centro Cardiologico Monzino, IRCCS, Milan, Italy, 2Laboratorio di Genomica Funzionale ed Immunologia,
Centro Cardiologico Monzino, IRCCS, Milan, Italy, 3Dipartimento di Scienze Cardiovascolari, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy, 4Istituto di
Patologia Medica, Universita ` Cattolica del Sacro Cuore, Rome, Italy, 5Laboratorio di Aterotrombosi, Centro Cardiologico Monzino, IRCCS, Milan, Italy, 6Laboratorio di
Patologia Vascolare, Istituto Dermopatico dell’ Immacolata, IDI-IRCCS, Rome, Italy, 7Dipartimento di Oncologia Sperimentale, Istituto Regina Elena, Rome, Italy
Abstract
Background: Use of peripheral blood- or bone marrow-derived progenitors for ischemic heart repair is a feasible option to
induce neo-vascularization in ischemic tissues. These cells, named Endothelial Progenitors Cells (EPCs), have been
extensively characterized phenotypically and functionally. The clinical efficacy of cardiac repair by EPCs cells remains,
however, limited, due to cell autonomous defects as a consequence of risk factors. The devise of ‘‘enhancement’’ strategies
has been therefore sought to improve repair ability of these cells and increase the clinical benefit.
Principal Findings: Pharmacologic inhibition of histone deacetylases (HDACs) is known to enhance hematopoietic stem
cells engraftment by improvement of self renewal and inhibition of differentiation in the presence of mitogenic stimuli in
vitro. In the present study cord blood-derived CD34+were pre-conditioned with the HDAC inhibitor Valproic Acid. This
treatment affected stem cell growth and gene expression, and improved ischemic myocardium protection in an
immunodeficient mouse model of myocardial infarction.
Conclusions: Our results show that HDAC blockade leads to phenotype changes in CD34+cells with enhanced self renewal
and cardioprotection.
Citation: Burba I, Colombo GI, Staszewsky LI, De Simone M, Devanna P, et al. (2011) Histone Deacetylase Inhibition Enhances Self Renewal and Cardioprotection
by Human Cord Blood-Derived CD34+Cells. PLoS ONE 6(7): e22158. doi:10.1371/journal.pone.0022158
Editor: Dan Kaufman, University of Minnesota, United States of America
Received December 10, 2010; Accepted June 18, 2011; Published July 18, 2011
Copyright: ? 2011 Burba et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The present work was funded by Thercord EU-P6 funded grant (contract no LSHB-CT-2005-018817), by Ricerca Finalizzata (ex art 56) from the Italian
Ministry of Health, and by Fondazione Monzino. This work was also in part supported by Associazione Italiana Ricerca sul Cancro (AIRC) to Antonella Farsetti. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: maurizio.pesce@ccfm.it
Introduction
Progenitor cells-based ischemic tissues repair is one of the major
endpoints in cardiovascular regenerative medicine. To provide
substantial clinical benefits, progenitor cells should be able to
engraft in sufficient numbers and differentiate into appropriate
cardiovascular cell types, primarily endothelial, vascular smooth
muscle cells or cardiac myocytes, or to promote ischemic tissue
salvage by paracrine interaction with host tissues cells. Since 1997,
the cells primarily deputed to fulfill this role are endothelial
progenitor cells (EPCs) [1]. Repair efficiency of patient-derived
EPCs may be limited by cell autonomous defects caused by
cardiovascular risk factors that greatly reduce EPCs tolerance to
stress conditions [2], their ability to produce differentiated
progenies or to survive into recipient tissues [3]. Various strategies
to restore innate EPC biological activity have been therefore
sought [4], based on pre-treatment with drugs that restore pro-
survival pathways, on culture in the presence of chemotactic
cytokines promoting EPCs migratory activity, or on use of drugs
that limit glucotoxicity or oxidative stress [5,6].
The relevance of the so called ‘‘histone code’’ [7] for epigenetic
control of stem cells differentiation vs. self renewal has been
highlighted by studies showing involvement of specific histone tails
modifications (acetylation, methylation) in establishment of gene
expression signatures specific for pluripotent cells [8], lineage-
committed cells [9] or adult-derived stem cells [10]. Given the
emerging role of epigenetic phenomena as fundamental triggers of
(stem) cell differentiation and plasticity, in the present study we
studied whether modification of the epigenetic landscape by
pharmacologic inhibition of histone deacetylases (HDAC) enzymes
affects CD34+cells growth, stemness, phenotype and gene
expression; we also assessed whether HDACi-treated CD34+cells
have a modified or enhanced regeneration capacity in a mouse
model of myocardial infarction.
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Figure 1. Growth inhibition andstem cell markers (CD34, CD133)expressionin controlandHDACi-preconditioned stem cells. (A–B) Time
course experiment at 3 and 5 days showing that TSA and VPA dose-dependently inhibited cytokine-induced cellular growth and enhanced the expression
of CD34 marker. R1, R2 and R3 represent the three regions corresponding to CD34neg, CD34dimand CD34brightcells, respectively, as detected by flow
cytometry. (C–D) Quantification of CD34neg, CD34dim, CD34brightcells and CD133neg, CD133dimand CD133brightcells at 5, 14 and 21 days of culture in the
presence or the absence of 2.5 mM VPA by flow cytometry. * indicate P,0.05 by 2 ways ANOVA with Bonferroni post-hoc analysis (n$4).
doi:10.1371/journal.pone.0022158.g001
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HDACi Preconditioning of Human CD34+Cells
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Results
Effects of HDAC inhibition on CD34+cells growth and
proliferation
In the present study, a serum free culture method was used to
expand CD34+cells [11] in the presence of increasing amounts of
Trichostatin A (TSA) and Valproic Acid (VPA), two wide range
HDAC inhibitors. As shown in Figures S1 and S2, morphology of
HDACi-treated cells was dose-dependently affected by treatment
with both drugs. To identify whether specific subpopulations were
affected by treatment with increasing VPA and TSA concentra-
tions, a flow cytometry analysis was performed by grouping cells
into three discrete regions: R1, corresponding to CD34negcells,
R2, corresponding to CD34dimand R3, corresponding to
CD34brightcells; in parallel, cellular growth was assessed by
counting at two consecutive time points. Figure 1A and B show
results of a 5 day time course analysis; increasing doses of TSA and
VPA decreased cellular growth with a dose-dependent shift toward
a homogeneous CD34brightcell population. To assess the time
extension of HDAC inhibition on CD34 and CD133 expression, a
flow cytometry analysis was performed in cells cultured in the
presence of 2.5 mM VPA at 5, 14 and 21 days (Figure 1C, D;
Figure S3).
TSA was not used in subsequent experiments for the reason that
this molecule is not routinely clinically used, such as VPA, for
treatment of diseases [12,13]. On the other hand, dose-response
treatments indicated that 2.5 mM VPA had a marked effect on
CD34 expression enhancement without producing maximal
growth retardation, but it did not have a significant effect on cell
death (trypan blue exclusion test in cells cultured for 7 days: CTR
vs. VPA cells: 13.5%62.7% vs. 15.7%61.77%; P=0.675 paired t-
test) or apoptosis (percent of cells in sub-G1 phase as detected by
flow cytometry in PI-stained cells cultured for 7 days: 4.963.2 vs.
1.260.4; CTR vs. VPA; mean 6 SE, n=3, P=0.38, paired t-test).
VPA 2.5 mM dose was then used in all the following experiments.
To clarify the relationship between stem cells generation time and
retardation in cell cycle progression, Carboxyfluorescein Succini-
midyl ester (CFSE) dye was used to assess CTR and VPA growth
progression at 5 and 7 days and to calculate proliferation index
and cellular generations number in the presence and absence of
VPA [14]. Figure 2A and B show that, at 7 days, proliferation
index was significantly reduced in VPA vs. CTR cells and that
CD34bright/CFSEbrightcells after VPA treatment were increased.
This corresponded to an average 1–2 generations delay in the
presence of VPA (Figure S4A) and to a specific growth retardation
of CD34brightcells (Figure S4B). Cell cycle analysis and double
staining with antibodies recognizing CD34 and Ki67 confirmed
that 7 days VPA treatment caused a significant elongation of G1
phase and a slight, but significant, increase in G0 cells (CD34+/
Ki672; Figure 2 C and D). Finally, as shown in Figure 2E,
expression of CDK inhibitors (p14ARF, p16INK4, p21Cip1/Waf-1)
was increased at the same time point, although at a different
extent, in VPA vs. CTR cells.
Histone hyperacetylation is directly linked to enhanced
CD34+expression and increased self renewal
To determine whether shifting toward a homogeneous
CD34brightphenotype is consequent to histone hyperacetylation
resulting from HDAC inhibition, Valpromide (VPM), a non-
teratogenic VPA derivative with lower HDAC inhibition activity
[15], was used in parallel to VPA in a 5 day time course
experiment. VPA substitution with VPM inhibited at interme-
diate levels cellular growth, while it produced a CD34 expression
profile in R1, R2 and R3 regions similar to CTR cells
(Figure 3A). Western blotting using pan-acetylated histone H4
(H4Ac) and lysine 9 acetylated histone H3 (H3K9Ac) showed
that 7 days VPA treatment specifically enhanced histone tails
acetylation (Figure 3B). An immunoprecipitation (ChIP) exper-
iment was then performed with chromatin extracted by cells
grown for 7 days in the presence or the absence of VPA, using
anti-H4Ac and anti- H3K9Ac antibodies, followed by real time
PCR to detect enrichment of specific CD34 promoter regions. As
shown in Figure 3C, H4Ac and H3K9Ac association to various
regions upstream and downstream of the CD34 promoter
transcriptional start site (TSS) was increased by VPA treatment,
suggesting epigenetic regulation of CD34 expression.
Phenotype analysis, clonogenic activity and gene
profiling of HDACi treated CD34+cells
Figure S5 shows the experimental design adopted for
phenotypic and molecular characterization of HDACi-treated
CD34+cells. Seven days VPA-treated cells were first analyzed to
measure the ability to extrude Rhodamine123 (Rho123) drug, a
typical primitive stem cell feature relative to the MDR-1 gene
product expression [16], and verify expression of Aldehyde
Dehydrogenase (ALDH) [17] (Figures 4A, B and S6). These tests
showed that VPA maintained a higher percentage of CD34bright/
Rho123loand CD34bright/ALDH+cells compared with controls.
Antibodies were then used to detect expression of stem cell
(CD34, CD133, KDR) hematopoietic (CD3, CD4, CD8, CD14,
CD38, CD45, CD48), endothelial (CD31, CD105, CD144,
CD146, LDL uptake), mesenchymal (CD73, CD90, CD130,
CD200) and integrin (a2b1, a4b1, a5b1) markers in multi-
parametric flow cytometry in CTR and VPA cells at 7 days.
Results (Figure 4C) indicated that VPA significantly enhanced
CD34, CD38, CD48, CD133 and KDR expression, but also
caused de novo expression of CD90, CD130 and CD146. Data
obtained by prolonging the culture time up to 14 days (Figure
S7) confirmed increased CD34, CD133 and KDR expression,
induction of CD146 and increased CD31, and maintenance of a
higher LDL uptake ability. Interestingly, expression of CD14
was significantly inhibited at both time points.
Recently, long term proliferating EPCs from cord blood cells
characterized by CD34, CD38, CD45 and CD133 markers have
been obtained using clonal culture conditions [18]. These cells
resemble those named Endothelial Colony Forming Cells (ECFCs)
in previous definitions of human EPCs identity [19]. To assess
Figure 2. Analysis of CD34+cells proliferation in the presence and the absence of VPA by flow cytometry. (A) CFSE staining profiles of
control and VPA-treated cells at 5 and 7 days of culture. Note that the fluorescence intensity reduction as a consequence in cell proliferation was less
pronounced in VPA vs. CTR cells at both time points. Proliferation index at 7 days was significantly reduced. (B) Seven days VPA treated cells had a
higher frequency of slow dividing immature (CD34bright) stem cells (blue area in contour plots), as detected by co-staining with CFSE and CD34
antibody; plots on the left indicate the fluorescence profile of cells stained with CFSE and CD34 isotype antibody (iso). (C) Cell cycle analysis in 7 days
CTR and VPA-treated cells revealed a higher frequency of cells in the G0–G1 and a lower percentage in either S and G2-M phases. (D) The percentage
of cells specifically arrested in G0 was also increased, as detected by co-staining with anti Ki67 and CD34 antibodies at the same time point. * indicate
P,0.05 by paired t-test (n$3). (E) Quantification of the relative expression level (22DDCtmethod) of small cyclin/CDK inhibitors (p14ARF, p16INK4,
p21Cip1/waf1and p27) by qRT-PCR analysis. * indicates P,0.05 by unpaired t-test (n$3).
doi:10.1371/journal.pone.0022158.g002
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Figure 3. Effect of VPA is directly related to HDAC inhibition. (A) The VPA structural analogue Valpromide (VPM) reduced CD34+cells proliferation
at lower extent compared to continuous treatment with VPA in a 5 day time course experiment. By contrast, the CD34 expression profile was identical to
that of control cells, as detected by flow cytometry. (B) Western blotting showing that a 7 day VPA treatment induced hyper acethylation on H4 and H3 (at
lysine residue 9) histones. * indicate P,0.05 by two ways ANOVA with Bonferroni post hoc analysis (n$3). (C) Representative ChIP experiment showing an
increased enrichment of various sites in the CD34 gene promoter (upstream and downstream of the TSS), as a result of chromatin hyper-acetylation (pan-
H4Ac, H3K9Ac) due to VPA treatment. Data are expressed as relative enrichment calculated by real time PCR amplification.
doi:10.1371/journal.pone.0022158.g003
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whether VPA-treated CD34+cells have a higher ability to form
ECFC clusters, these cells were plated under ECFC inducing
conditions [20]. Results showed that derivation of ECFC clusters
was higher from VPA cells compared with CTR and freshly
isolated CD34+cells. Despite this, morphologic appearance,
expression of ECFC markers and the ability to form capillary-
like structures onto Matrigel was not different in CTR vs. VPA
cells-derived ECFCs (Figure 4D).
To investigate possible gene expression changes in 7-days
cultured VPA vs. CTR cells, a survey of stem cells-specific
transcripts expression was performed using qRT-PCR low density
arrays (Figure 5A). Unsupervised clustering analysis recognized
coherently upregulated or downregulated stem cell-associated
genes, allowing discriminate between VPA vs. CTR cells
signatures. Statistical analysis revealed 34 transcripts significantly
modified by VPA. Of these, 6 were down-modulated while 18
were up-regulated. Finally, 10 genes that were not detectable or
were at detection limit by qRT-PCR, were expressed de novo by
HDAC inhibition (Table 1). A microRNA (miRNA) profiling was
finally performed on high-throughput sorted CTR and VPA
CD34brightcellular populations (Figure S8). Remarkably, unsu-
pervised clustering clearly distinguished miRNA profiles of
CD34brightcells in VPA vs. CTR conditions (Figure 5B). Results
from statistical analysis identified 44 miRNAs whose expression
was significantly modified. Of these, 6 were downregulated, 31
were upregulated, while 7 were expressed de novo (Table 2).
Heart repair potency of control and HDACi-treated
CD34+cells
Seven days cultured CTR and VPA cells were injected in the
ischemic left ventricle of SCIDbeigemice 15 min after coronary
artery ligation (CAL). Six weeks after CAL and cell injection, mice
that survived and reached the end of the follow up period were
analyzed for heart function and morphometry. Mortality data
during the follow up period (Figure 6A) showed a dramatic
difference between animals receiving VPA cells compared to those
injected with CTR cells or saline. Echocardiography revealed that
in animals injected with VPA cells Left Ventricular Ejection
Fraction (LVEF) was significantly increased (33% improvement)
compared with saline-injected mice; in addition VPA cells reduced
Left Ventricular End Diastolic/Systolic volumes (LVEDV,
LVESV) compared with CTR cells and saline (Figure 6B, Tables
S1, Figure S9). To further assess efficacy of CTR and VPA cells,
diastole-arrested hearts from sham operated, saline-, CTR- and
VPA cells-injected mice were analyzed by histology to obtain LV
morphometric parameters and by fluorescent lectin staining to
measure capillary density (Figure 6C, D; Table S2). Surprisingly,
VPA-treated cells neither reduced infarct size nor increased
capillary density, compared with CTR cells.
Human cells survival at 6 wks after transplantation was
evaluated using a qPCR method tailored to detect a human
polymorphism with an efficiency of 0.1–1 equivalent human cell
genome (0.6–6 pg) in an unrelated DNA mixture [21] (Figure
S10). By this, it was found that survival of human cells in vivo was
low (about 102cells out of the 1.56105injected cells) and
comparable in VPA and CTR cells-injected mice.
It has been proposed that cytokines released from cells injected
into the ischemic myocardium may interfere, at least in part, with
progression of cell death due to hypoxia or may metabolically
sustain ischemic myocardium [22]. To better characterize effects
of VPA vs. CTR cells, release of inflammatory/pro-angiogenic
cytokines was measured in conditioned medium. This tests showed
a significantly higher expression of 11 out of 15 tested cytokines in
VPA vs. CTR cells (Figure 7A, B). To assess whether modified
cytokine expression in VPA vs. CTR-treated CD34+cells protects
against hypoxia-induced apoptosis, HL-1 cardiomyocyte-like cells
[23] were exposed to medium conditioned from VPA and CTR
cells in an in vitro hypoxia model. As shown in Figure 7C, VPA
cells conditioned medium exerted higher protection of these cells
from cell death consequent to hypoxia.
Increased paracrine activity of injected cells might interfere with
myocardial adverse remodeling following infarction. To verify this,
collagen deposition and myofibroblasts number were determined
by picrosirius red staining [24,25] and a-smooth muscle actin (a-
SMA) immunofluorescence [26,27] in hearts injected with saline,
CTR and VPA-treated cells. Results (Figure 8) showed that
collagen deposition and presence of a-SMA+myofibroblasts were
(although not significantly) reduced in VPA cells-injected com-
pared with CTR cells and saline-injected mice, suggesting lower
adverse remodelling in hearts injected with VPA-treated cells.
Discussion
Direct and pleiotropic roles of HDACi blockade on CD34+
cells markers expression, cell cycle and phenotype
For years recognized as an useful treatment for inducing
differentiation of transformed cells [28], histone deacetylase
Figure 4. Phenotype and stem cell function in control and 7-days VPA treated cells by flow cytometry. (A) quantification of cells
actively extruding Rhodamine123 dye as a result of ABCG2 gene product MDR-1, a typical activity of immature stem cells. Contour plots on the
left show the staining profile in the presence of the MDR-1 pump inhibitor Verapamil, plus the CD34 isotype (ISO), while those on the right
show the shift toward the left of a CD34brightcells fraction (blue area) in VPA-treated cells (V). Bar graph on the right indicates quantification of
CD34bright/Rho123locells in C and VPA conditions; dotted line indicate the percentage of CD34bright/Rho123locells immediately after isolating
CD34+cells from cord blood. (B) Quantification of ALDH expressing cells. Contour plots on the left show the fluorescence profile of cells
treated with the ALDH inhibitor DEAB (used as a negative control) and CD34 isotype antibody (i+D), while those on the right show the results
of specific staining with CD34 antibody and fluorescent detection of ALDH activity. Note that in the presence of VPA a higher percentage
of CD34bright/ALDH+cells was present (blue area), while in control cells ALDH staining was lower in the CD34brightgating and present in
CD34dim/negcells (areas in magenta color). Bar graph on the right indicates quantification of CD34bright/ALDH+cells in C and VPA conditions;
dotted line indicate the percentage of these cells immediately after isolating CD34+cells from cord blood. * indicate P,0.05 by paired t-test
(n=4). (C) phenotype analysis of control and VPA cells at 7 days of culture by multiparametric flow cytometry experiments. Upregulation of
stem cells markers CD34, CD133, CD38 and KDR were found along with enhanced expression of mesenchymal markers CD90, CD146 and
CD130. Consistent with an effect of VPA on myeloid differentiation inhibition, CD14 was inhibited. * indicate P,0.05 by paired t-test (n$3). (D)
Derivation of ECFCs from fresh, CTR and VPA CD34+cells. Formation of clusters was observed three weeks after plating these cells onto FN
coated dishes. Histogram represents number of ECFC clusters observed in three independent experiments; * indicates P,0.05 by one way
ANOVA with Neuman Keuls post-hoc. Pictures on the upper right show the morphology of ECFCs derived from CTR and VPA CD34+cells and
their ability to form capillary-like structures, when plated onto matrigel. The amount of these latter structures formed by either CTR or VPA
cells was not different compared with HUVEC cells that were used as a positive control (not shown). Plots in the lower part of the panel show
expression of typical ECFC markers [19].
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(HDAC) pharmacologic inhibition has been more recently
indicated to promote self renewal for expanding the number of
immature stem cells in bone marrow repopulation assays
[14,29,30,31,32]. In line with these investigations, our experiments
show that CD34+cells were functionally modified by HDACi
pretreatment and induced to maintain a ‘‘slow dividing’’
phenotype [33] compared with CTR cells. Increased immaturity
and retarded growth were not the unique consequences of VPA
treatment. In fact we observed: 1) negative and positive
modulation of various stem cells and differentiated cells markers
such as CD14, VEGFR2/KDR, CD31, CD38, CD48 and uptake
ability of Ac-LDL (Figures 4, S7), 2) de novo expression of
mesenchymal (CD90, CD130) and ECFCs/mature endothelial
cells (CD146) [19,34,35] markers (Figures 4, S7) and, 3) enhanced
ability of VPA CD34+cells to produce ECFCs clones (Figure 4).
Taken together, these results suggest that HDAC blockade causes
a phenotype change of CD34+cells due to activation of different,
but likely interdependent, molecular pathways targeting at the
same time cell proliferation and clonogenicity. It is striking that
upregulation of stem cells markers (CD34, CD38, CD133 and
KDR) occurred in concert with de novo expression of markers
typical of differentiated (CD48, CD146) or mesenchymal (CD90,
CD130) cells. This is in line with the evidence that exposure to
drugs with genome-wide effects such as generalized histone
acetylation has global consequences for cell (re)programming
[36,37], but also for fate determination of primitive cells
[38,39,40].
mRNA and miRNA profiling of HDACi-treated CD34+cells
reveals a streamlined epigenetic supervision of immature
CD34+cells phenotype
The clusterization of mRNAs and miRNAs expressed in VPA-
treated cells (Figure 5) allowed clearly recognize specific gene
expression signatures distinguishing them from CTR cells. For
mRNAs it was possible to derive functional annotation charts
describing occurrence of HDACi-regulated mRNAs into various
BIOCARTA/KEGG categories (Table 3). This identified the
canonical Wnt- (FZD1, WNT1), Notch- (NOTCH1, DLL3,
DLL4) and Hedgehog-activated signaling (DHH, BMP2) as
crucial nodes in the generation of the VPA-treated cells
phenotype. This is important, as convergence of canonical-Wnt
and Notch signaling is recognized to maintain primitive stem cells
self renewal in the bone marrow stem cell niche [41], while Notch
signaling has specific roles in EPCs neo-vascularization activity
[42].
Our observations also reveal a possible interplay between
positive and negative stimuli controlling the immature and slow
dividing phenotype of VPA-treated cells. In fact, VPA caused
downregulation of CDH1 (E-cadherin) and upregulation of FDZ1,
WNT1, CCND1, DLL1/3 and mir-9 in VPA-treated CD34+cells,
suggesting enhancement of b-catenin-mediated transactivation
and positive effects on CD34+cells proliferation [43]. On the other
hand, HDAC inhibition also determined coherent up- or down-
modulation of several miRNAs directly involved in positive (e.g.
mir-129-3p, mir-193b, mir-370) or negative (e.g. mir-196b, mir-
335, mir-370) control of cell cycle [44,45,46,47,48,49], thus
suggesting the existence of an epigenetically regulated negative
loop protecting CD34+cells from unrepressed cellular growth, and
reinforcing the anti proliferative effect exerted by small cyclin/
CDK inhibitors such as p14ARF, p16INK4and p21Cip1/Waf1gene
products (Figure 2E). Negative effects on cell proliferation may
also depend on an HDACi-related modification of the DNA
methylation status. This is suggested by the finding that VPA
induced mir148, a miRNA targeting DNMT3b methyltransferase,
and by the evidence that several of the miRNAs over- or under-
expressed in VPA-treated cells are transcriptionally regulated by
CpG islands methylation [50,51,52].
Cardiac protection by HDACi-treated CD34+cells is
independent of CD34+cells regeneration enhancement
HDAC inhibitors potently reduce in vitro and in vivo angiogenesis
by repressing the ability of mature endothelial cells to form
vascular structures [53] or by inhibiting EPCs maturation into
endothelium [54]. In addition, HDAC genes targeting (e.g. SIRT-
1 or HDAC4,7) impairs vascular development [55,56,57].
Therefore, preconditioning with HDACi should reduce and not
increase CD34+cells pro-angiogenic function into ischemic tissues.
Injection of CTR and VPA-treated cells in the ischemic heart
(Figure 6) showed a remarkable effect of HDACi cellular
preconditioning on the survival of treated mice. This was
associated to a significant enhancement of cardiac function but,
surprisingly, neither corresponded to a more efficient reduction of
the infarct size, nor to a significant improvement of myocardial
tissue regeneration compared with CTR cells (Figure 6, Tables S1,
S2). Furthermore, as shown by survival of similar, but low, amount
of living human cells in the host myocardium (Figure S10), this
effect was not due to a higher engraftment ability of VPA-treated
cells.
How to reconcile these data? Our results call for a generalized
increase of CD34+cells cardioprotection ability or an improved
‘‘paracrine effect’’ that may sustain cardiac contractility or
interfere with myocardial cells apoptosis as short times after
infarction. In support of this hypothesis is the finding that VPA-
treated cells showed an enhanced expression of several pro-
angiogenic/pro-inflammatorycytokines
CXCL12/SDF-1) or protein (bFGF, IL-8, VEGF, Ang-2, IFN-c,
TNFa) levels, of cardioprotective factors such as Follistatin [58]
and of resident progenitors activating factors such as HGF [59].
Therefore, generalized upregulation of all these gene products in
the secretome of CD34+cells may lead to functional preservation
of the left ventricle by sustaining cardiac metabolism and
contractility, even in the absence of a net reduction of infarct
at mRNA(FGF2,
Figure 5. Unsupervised hierarchical cluster analysis and statistical analysis of mRNA and miRNA profiling in CTR vs. 7 days VPA
treated CD34+cells (A and B, respectively). The analyses were performed initially using the whole datasets of genes (panel A, left heat map) or
miRNAs (panel B, left heat map) that passed the quality assurance and filtering criteria (see Supplementary Methods), to assess whether expression
profiles discriminates treatment groups. A second round of unsupervised hierarchical clustering was done on differentially expressed genes (panel B,
right heat map) or miRNAs (panel C, right heat map), as selected by significance analysis (see Supplementary Methods), to identify biologically
relevant co-expressed gene clusters. The mean centered level of expression of each gene/miRNA in each sample is represented with green, black, and
red colour scales (green indicates below mean; black, equal to mean; and red, above mean). The dendrograms on top of each heat map display the
unsupervised clustering of control and VPA-treated CD34+cells using the whole or the differentially expressed gene/miRNA lists. The dendrograms
on the left side of each heat map show the unsupervised clustering of the genes. Correlation coefficients are reported for both. See Supplementary
Methods for data transformation and adjustments, distance metrics and linkage methods.
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size compared with control CD34+cells (discussed in [22]). The in
vitro data showing significantly higher rescue of HL-1 cardiac
myocytes from hypoxia in the presence of VPA cells conditioned
medium (Figure 7C) supports this conclusion.
Presence of bone marrow-derived or resident mesenchymal
cells-derived myofibroblasts during early stages after infarction is
Table 1. Identity of stem cell genes coherently modulated by
7-days VPA treatment in CD34+cells.
Gene Main functionP value
Fold
Change
V vs. C FDR
S100B Muscle/Neuronal
Differentiation
0,000157
29,620,002788
CDH1 E-Cadherin, cell adhesion 0,039466
28,680,084911
TERTStemness0,000418
25,82 0,004244
CCNA2Cell cycle 0,008564
22,970,028954
MYST1HAT 0,007580
21,70 0,029901
BGLAP MSCs bone differentiation0,023935
21,340,054819
ADARARI RNA editing0,0427391,330,089249
CCND2 Cell cycle0,0083731,470,029725
CD44 MSCs marker0,0045312,060,018922
CCNE1 Cell cycle0,0028912,450,015789
HDAC2Histone deacethylase0,0185232,530,045350
PARD6ACell polarity0,016895 3,010,042842
TUBB3Cell polarity0,0021733,190,014024
FZD1 Wnt pathway0,0025433,200,015048
CCND1 Cell Cycle0,0014933,310,011784
FGFR1Angiogenesis0,0106713,730,031569
FGF2Angiogenesis0,0042974,86 0,019068
COL2A1Extracellular Matrix0,0142159,980,038819
ALDH1A1Stemness0,00009810,010,002320
DLL1Notch pathway0,00314310,710,015942
DHH Mesenchyme differentiation 0,01953312,44 0,046228
CDH2 N-cadherin, neurogenesis0,011550 13,61 0,032801
CXCL12Angiogenesis0,003988 25,480,018877
COL1A1Extra cellular matrix0,00079232,270,007033
GeneMain function
P value
Fold
Induction
V vs. CFDR
BMP2Mesenchyme differentiation0,0086083,680,027781
ABCG2Stemness0,0512525,340,106239
T
(Brachyury)
Mesoderm programming0,0078267,56 0,029243
CD8AHematopoietic marker0,0004149,610,004902
DLL3Notch pathway0,0000719,910,002516
WNT1 Wnt pathway0,0159759,970,042008
GDF2 Mesenchyme differentiation0,00196212,240,013927
GDF3 Stem cells self renewal0,01030325,430,031806
MSX1 Mesenchyme differentiation0,00000179,960,000078
NCAM1Neurogenesis0,000253 142,310,003599
P values are calculated by a multivariate paired t-test, using 100 permutations
and limiting the false discovery rate (FDR) proportion to ,0.1 (see
Supplementary Methods). C: CD34+control cells; V: VPA-treated CD34+cells.
doi:10.1371/journal.pone.0022158.t001
Table 2. Identity of miRNAs modulated by 7-days VPA
treatment in CD34brightcells.
miRNAP valueFold change V vs. C FDR
miR-196b0,000833
29,79 0,0288
miR-133a0,006061
23,65 0,0589
miR-509-3p0,013360
23,490,105
miR-1500,022776
22,740,122
miR-1860,027408
22,640,139
miR-628-5p0,020783
22,570,121
miR-1450,0460012,130,181
miR-99b0,0453192,250,181
miR-135a*0,0316872,340,144
miR-376c0,0389892,570,165
miR-487b0,0311112,660,144
miR-30e*0,0362572,970,161
miR-5650,0180843,240,119
miR-5390,0223683,410,122
miR-4330,0096033,46 0,0791
miR-296-5p0,0068393,46 0,0592
miR-4320,0186953,640,119
miR-532-5p0,0166993,80 0,116
miR-532-3p 0,0047573,90 0,0514
miR-4110,0284074,070,139
miR-485-3p0,0161604,110,116
miR-371-3p0,0389704,260,165
miR-768-3p0,0068414,30 0,0592
miR-886-3p0,0210244,590,121
miR-409-3p0,0405904,800,167
miR-323-3p0,0191854,860,119
miR-193a-5p0,0061315,36 0,0589
miR-148a 0,0033565,420,0447
miR-335 0,0044025,430,0508
miR-379 0,0233205,640,122
miR-127-3p0,0019985,84 0,0339
miR-3650,0019808,05 0,0339
miR-30a*0,00143510,20 0,0339
miR-193b0,00215311,21 0,0339
miR-6420,00202312,34 0,0339
miR-483-5p0,00327416,96 0,0447
miR-1490,00144922,48 0,0339
miRNA
P valueFold induction V vs. CFDR
miR-3750,0290184,19 0,139
miR-370 0,01411110,550,106
miR-654-3p0,004255 12,120,0508
miR-193b*0,000377 21,25 0,0163
miR-90,00001964,59 0,0011
miR-129-3p0,000002343,250,000329
miR-935 0,000006717,680,000554
P values are calculated using a random-variance model for univariate
significance paired t-test, based on all available permutations. The maximum
proportion of FDR was ,0.2 (see Supplementary Methods). C: CD34+control
cells; V: VPA-treated CD34+cells.
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an important component of the innate immunity response to
ischemia [60,61,62]. By contrast, chronic permanence of these
cells after MI [63], or their increased number due to aging [64], is
associated to enhanced fibrosis and ventricular dysfunction. In the
present study, specific experiments to assess the dynamics of
myofibroblasts accumulation at early time points after MI were
not performed. However, the lower amount of collagen and the
reduced myo-fibroblasts number observed at six weeks in the
infarct zone (Figure 8), suggest that injection of VPA-treated cells
partially prevented chronic scarring of the myocardial tissue, thus
justifying the observed reduction of ventricular dysfunction. Again,
this may depend on enhanced cytokine secretion observed in
VPA-treated vs. CTR cells.
In summary, the data shown in the present study identify a
possible novel strategy to enhance the protective ability of human
CD34+cells against the consequences of myocardial infarction.
Future studies will be needed to clarify the molecular mechanisms
underlying the interplay between VPA-treated CD34+cells and
the ischemic micro-environment, thus allowing to draw a clear
scenario for their enhanced repair function.
Materials and Methods
Ethics statement
Collection of cord blood samples was performed upon written
consent and on a voluntary basis. An Institutional Review Board
formal approval for cord blood collection at Melzo Hospital was
obtained to this aim (December 12, 2008; authorization
no 843). All animal studies conformed to national and interna-
tional ‘‘Guide for the Care and Use of Laboratory Animals’’ and
the Helsinki declaration. The protocols were reviewed and
approved by the Animal Care and Use Committee at Mario
Negri Institute, and by the Italian Health Ministry (protocol
0804 base1); they were further compliant with European
directives and guidelines (Legislative Decree September 19,
1994, n. 626 (89/391/CEE, 89/654/CEE, 89/655/CEE,
89/656/CEE, 90/269/CEE, 90/270/CEE, 90/394/CEE,
90/679/CEE).
Cord blood collection, expansion and phenotype analysis
of CD34+cells
Isolation and culture of CD34+cells were performed using a
magnetic beads-based method (MINI-MACS) and a serum-free
expansion medium [11] as detailed in Materials and Methods S1.
At the indicated time points, cells were incubated with suitable
combinations of monoclonal antibodies recognizing stem cell-
specific, endothelial, hematopoietic or mesenchymal specific
markers (1–10 mg/ml final concentration). Cell growth was
assessed by incubating cells with CFSE or Propidium Iodide
staining and by Ki-67 specific antibodies. To assess stem cell
phenotype of VPA vs. control treated CD34+cells, Rhodamine123
extrusion by MDR-1 gene product and ALDH enzyme activity
were measured by flow cytometry. Methodology and reagents used
for these tests are described in the Materials and Methods S1.
ECFC Clonogenic expansion
ECFC clonogenic assay was performed as already described
[20]. Briefly, fresh, CTR and VPA-treated CD34+cells were
plated at low density into collagen-coated dishes. Clusters of
rapidly expanding endothelial-like cells were counted and further
expanded for phenotype and functional analyses, as described in
Materials and Methods S1.
Chromatin immunoprecipitation
Chromatin immunoprectipitation was performed as described in
Nanni et al., 2009 [65], using ChIP-IT Express Enzymatic kit,
according manufacture’s instruction (Active Motif). Enrichment of
DNA sequences associated to segments of the CD34 gene promoter
was tested using a quantitative method based on real-time PCR.
Further details are provided in Materials and Methods S1.
Transcript and miRNA profiling
For detection of stem cells-associated transcripts, total RNA was
extracted from control vs. VPA-treated cells after which the RT2
Profiler PCR Array system (human stem cell-specific card,
catalogue PAHS-405E,Version 4.26; SABiosciences) was used.
For microRNAs profiling, total RNA was extracted from high
throughput-sorted CD34brightcells by flow cytometry. Profiling
was performed by TaqMan Human MicroRNA A and B Arrays,
version 2.0 (Applied Biosystems, USA). Both procedures were
performed in a 7900HT (Applied Biosystems, USA) fast real time
cycler. A complete description of cell sorting and RNA extraction
procedures as well as of raw data (Tables S3, S4 and S5)
normalization and statistical handling is provided in Materials and
Methods S1.
Animal studies
A SCIDbeige
permanent left coronary artery ligation was used. Control and
VPA-treated cells (1.56105cells/animal) were injected in the left
ventricle (LV) at the infarct border zone 15minafter CAL. As
controls, sham operated and saline-injected animals were used.
After a 6 weeks follow up period, heart functional analyses were
performed by transthoracic ultra-imaging echocardiography
(VisualSonics, Vevo 770), followed by mice sacrifice for histolog-
ical analysis and immunofluorescence. Further details are provided
in the Materials and Methods S1.
mouse model of myocardial infarction by
Cytokine, chemokine and growth factor detection;
apoptosis in HL-1 cell line
Bio-Plex assay (Bio-Rad Laboratories, Italy), a bead-based
multiplex immunoassay, was used to quantify cytokines, chemo-
kines and growth factors secreted in culture supernatant by
Control and VPA-treated cells. Results are expressed as pg/ml/
Figure 6. Effect of 7 days cultured CTR and VPA-treated CD34+cells on survival, left ventricle function neo-vascularization and
ventricular remodeling in an immunodeficient mouse model of myocardial infarction. (A) Mortality Kaplan-Meier curve of sham operated,
saline-injected, control and VPA CD34+cells-injected mice. The mortality in mice injected with VPA-treated CD34+cells was not significantly different
from that of sham operated mice (see enclosed table). Significance calculated by log-rank (Mantel-Cox) test. (B) Echocardiographic assessment of
ejection fraction, left ventricular end-diastolic and end-systolic volumes showed an improved ventricular function in VPA treated CD34+cells, but not
in control CD34+cells-injected animals. (C) Representative images of transversal sections of diastole-arrested hearts, used to evaluate the infarct scar
size and LV morphometric values. * indicate P,0.05 by one way ANOVA with Newman Keuls post hoc analysis (n$7). (D) Representative images of
Rhodamine-labeled Griffonia simplicifolia Lectin 1 staining of LV histological sections for capillary density determination. The bar graph indicates the
density of these vessels at the infarct border zone.
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Figure 7. Enhanced cytokine release and higher hypoxia suppression by 7 days VPA-treated CD34+cells conditioned medium. (A)
Analysis of pro-inflammatory and pro-angiogenic factors present in the control and VPA-treated CD34+cells secretome. Table shows means and
standard error of each cytokine released in the culture supernatants. Heat maps indicate a treatment-related coherent upregulation of most of the
secreted factors in four independent CD34+cells samples. (B) Statistical analysis of the cytokine concentrations in culture supernatants by paired t-
test. The fold change in the release of these cytokines from VPA-treated vs. CTR CD34+cells is shown. With the exception of Leptin, IL-8, VEGF, and
bFGF (orange color), all the other cytokines were significantly up-regulated in HDACi preconditioned cells, indicating an enhancement of their
paracrine effect. (C) Effect of CTR and VPA CD34+cells conditioned medium on rescue from apoptosis of HL-1 cardiomyocytes cell line exposed to
hypoxia conditions. Data in the graph represent the percent variation in apoptotic death of HL-1 cells exposed to hypoxia in the presence of VPA
treated CD34+cells conditioned medium in comparison with medium conditioned by CTR cells; * indicate P,0.05 by paired t-test (n=5).
doi:10.1371/journal.pone.0022158.g007
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105cells. Conditioned medium from these cells was also used to
assess protection from hypoxia-induced apoptosis in HL-1
cardiomyocyte cell line [23]. Further details are provided in
Materials and Methods S1.
Cardiac engraftment analysis
Analysis of CD34+cells engraftment was performed using a
qPCR method allowing detection of a human-specific polymor-
phism in an excess mouse DNA [21]. Further details are provided
in Materials and Methods S1.
Data analysis
All results are expressed as mean 6 standard error. Statistical
significance was determined with paired or unpaired Student’s t-
test, one-way Anova with Newman-Keuls post-hoc analysis or
Two-ways Anova with Bonferroni post-hoc analysis.
Figure 8. Effect of VPA and CTR cells on myocardial healing. (A) Representative images of a-SMA staining (red fluorescence) of the infarct
zone to reveal the presence of myo-fibroblasts. (B) Representative low and high power views of picrosirius red staining of the myocardium to reveal
the collagen deposition. Pictures on the right show polarized light imaging of the same microscopic fields in the center of the panel, to show that
birefringence of collagen bundles was not affected by saline or CTR and VPA CD34+cells treatments. (C–D) Quantification of Collagen deposition and
myofibroblasts. Collagen data are shown as percentage of the areas containing collagen normalized to total area sections, while myofibroblasts were
determined by counting the number of a-SMA+cells in the infarct zone (n$6). Statistical analysis of these data by one-way ANOVA with Newman-
Keuls post-hoc test did not reveal differences between treatment groups, although lower amount of Collagen and smaller myofibroblasts number
were found in VPA cells-injected mice.
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Supporting Information
Morphology of CD34+cells cultured in the
absence (Control) and the presence of 2.5 mM VPA at 7
days of culture. Note the presence of blast-colonies indicative of
rapid proliferation/differentiation events in control condition
(green arrows) and that of elongated and mild adherent cells
(red arrows) in VPA-treated cells.
(TIF)
Figure S1
Figure S2
side scatter (SSC) increase of CD34+cells at 7 days of
culture. Note progressive shift toward high SSC values of
HDACi-treated cells (open black histogram) overlaid onto control
cells plots. Statistical evaluation by Kolomogorov-Smirnov test
showed significant divergence (D$0.20) of the histogram plots for
TSA at 25 ng/ml and VPA at 2.5 and 5 mM concentrations.
(TIF)
Effect of increasing doses of VPA and TSA on
Figure S3
and VPA-treated cells at 5 days in culture. The three
regions corresponding to CD133neg, CD133dimand CD133bright
cells are shown.
(TIF)
CD133 antigen expression profile in control
Figure S4
software of CFSE profiles in three independent experi-
ments of CD34+cells culture in the presence and the
absence of VPA. The different generations are indicated by
different colours. Histogram plots on the right indicate cells
distribution in the various cellular generations at days 7 of culture.
(B) Example of CFSE profile in the CD34brightgating of seven days
cultured CTR and VPA cells. It is evident that VPA-treated cells
were shifted toward the right side of the plot, indicating brighter
CFSE fluorescence and consistent growth retardation. Inset shows
the result of Kolmogorov-Smirnov test, indicating a statistically
significant (D.0.20) divergence of the two curves.
(TIF)
(A) Mathematical deconvolution by ModFit
Figure S5
actions and time points of the phenotype and in vivo
function analyses of control and VPA-treated CD34+ +
cells.
(TIF)
Experimental flowchart describing the main
Figure S6
isolation from cord blood. Contour plots are designed as in
Figure 4.
(TIF)
Marker analysis in CTR and VPA CD34+cells
at 14 days of culture. As observed at day 7, a number of stem
cell (CD34, CD133 and KDR), endothelial (CD31, CD146, LDL
uptake) and mesenchymal (CD90) markers were upregulated.
(TIF)
CD34 expression profile in CD34+ +
cultured for 7 days in the presence or the absence of
VPA. Contour plots on the top show CD34 expression in cultured
cells before high throughput sorting by flow cytometry; plots on
the bottom indicate the purity control after sorting.
(TIF)
Stem cell activity in CD34+ +
cells after
Figure S7
Figure S8
cells
Figure S9
axis (pSAX, right) views by echocardiography. Left side of
the figure: end diastolic (left) and end-systolic frames (right) of
sham operated or, Saline, CTR CD34+cells and VPA-treated
cells-injected mice. Ao: aorta, LA: left atrium, LV: left ventricle,
MV: mitral valve, IVS: inter-ventricular septum, PW: posterior
wall. Arrows indicate the extension of the infarcted wall. Note the
increased wall thinning, chamber dilatation, and systolic expan-
sion of saline injected compared to CD34+cells (CTR or VPA)
injected mice. Right side of the figure. M-mode echocardiogram of
the left ventricle of the same mice shown in pLAX view. LV: left
ventricle, ASW: anteroseptal wall, IPW: inferior-posterior wall.
Arrows indicate the infarcted wall.
(TIF)
Parasternal long axis (pLAX, left) and short-
Figure S10
the mouse heart. qPCR was performed by a Taqman
amplification protocols to detect the human SNP C/T (rs6625561
Reference: NCBI SNP). Amplification plots in the upper right show
the threshold cycle of increasing (106 higher at each dilution)
amounts of human DNA into a fixed amount of mouse DNA, while
the other amplification plots are derived from amplification of DNA
extracted by saline injected or cells injected mice. Graph on the
bottom shows the approximate linearity between threshold cycle and
106increasing amount human of human DNA into the fixed mouse
DNA excess. Red and green lines indicate the CT values for
Determination of the human cells survival in
Table 3. Gene-enrichment analysis and functional annotation clustering of cell stem related genes in 7-days VPA-treated CD34+
cells.
CATEGORYPATHWAY SPECS (TERM)COUNT%P-valueGENES INVOLVED
BIOCARTAh_ps1Pathway:Presenilin action in
Notch and Wnt signaling
38,823529 0,009788WNT1,FZD1,DLL1
BIOCARTA h_cellcyclePathway:Cyclins and Cell
Cycle Regulation
3 8,8235290,0300447CCNE1,CCND1,CCND2
BIOCARTA h_wntPathway:WNT Signaling
Pathway
38,8235290,0300447 WNT1,CCND1,FZD1
KEGG_PATHWAY hsa04110:Cell cycle5 14,70588 0,0029498CCNE1,CCND1,HDAC2,CCND2,CCNA2
KEGG_PATHWAY hsa04330:Notch signaling pathway3 8,8235290,0219152 HDAC2,DLL3,DLL1
KEGG_PATHWAYhsa04340:Hedgehog signaling
pathway
3 8,8235290,0303929DHH,WNT1,BMP2
KEGG_PATHWAY hsa04310:Wnt signaling pathway411,764710,0365996WNT1,CCND1,CCND2,FZD1
KEGG_PATHWAY hsa04115:p53 signaling pathway38,823529 0,04338CCNE1,CCND1,CCND2
doi:10.1371/journal.pone.0022158.t003
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