OPEN
Neovascularization and functional recovery after
intracerebral hemorrhage is conditioned by the Tp53
Arg72Pro single-nucleotide polymorphism
Cristina Rodríguez
1,2
, Tomás Sobrino
3
, Jesús Agulla
1,2
, Verónica Bobo-Jiménez
1,2
, María E Ramos-Araque
1,2
, Juan J Duarte
1
,
José C Gómez-Sánchez
1
, Juan P Bolaños
1,2
, José Castillo
3
and Ángeles Almeida*
,1,2
Intracerebral hemorrhage (ICH) is a devastating subtype of stroke that lacks effective therapy and reliable prognosis.
Neovascularization following ICH is an essential compensatory response that mediates brain repair and modulates the clinical
outcome of stroke patients. However, the mechanism that dictates this process is unknown. Bone marrow-derived endothelial
progenitor cells (E PCs) promote endothelial repair and contribute to ischemia-induced neovascularization. The human Tp53 gene
harbors a common single-nucleotide polymorphism (SNP) at codon 72, which yields an arginine-to-proline amino-acidic
substitution (Arg72Pro) that modulates the apoptotic activity of the p53 protein. Previously, we found that this SNP controls
neuronal susceptibility to ischemia-induced apoptosis in vitro. Here, we evaluated the impact of the Tp53 Arg72Pro SNP on
vascular repair and functional recovery after ICH. We first analyzed EPC mobilization and functional outcome based on the
modified Rankin scale scores in a hospital-based cohort of 78 patients with non-traumatic ICH. Patients harboring the Pro allele of
the Tp53 Arg72Pro SNP showed higher levels of circulating EPC-containing CD34
+
cells, EPC-mobilizing cytokines – vascular
endothelial growth factor and stromal cell-derived factor-1α – and good functional outcome following ICH, when compared with
the homozygous Arg allele patients, which is compatible with increased neovascularization. To assess directly whether Tp53
Arg72Pro SNP regulated neovascularization after ICH, we used the humanized Tp53 Arg72Pro knock-in mice, which were subjected
to the collage nase-induced ICH. The brain endothelial cells of the Pro allele-carrying mice were highly resistant to ICH-mediated
apoptosis, which facilitated cytokine-mediated EPC mobilization, cerebrovascular repair and funct ional recovery. However, these
processes were not ob served in the Arg allele-carrying mice. These results reveal that the Tp53 Arg72Pro SNP determines
neovascularization, brain repair and neurological recovery after ICH. This study is the first in which the Pro allele of Tp53 is linked
to vascular repair and ability to functionally recover from stroke.
Cell Death and Differentiation (2017) 24, 144–154; doi:10.1038/cdd.2016.109; published online 21 October 2016
Non-traumatic intracerebral hemorrhage (ICH) is one of the
most devastating and disabling forms of stroke that account for
10–15% of all cases of stroke hospital admissions.
1,2,3
The
average mortality rate was 50%, most of which occur during
the first days after stroke. Among survivors, only an estimated
20% will regain functional independence at 6 months, whereas
more than one-third of affected patients will not survive the first
year.
1,3
Unfortunately, prediction of functional outcome that
would assist in both preventive and therapeutic interventions
remains elusive.
1,3
Functional outcome after ICH depends on the balance
between brain injury and the activation of yet unknown com-
pensatory response repairing the consequent damage.
4,5
Recent evidences indicate that strategies to enhance the
response following brain injury provide promising opportu-
nities to improve clinical outcomes and brain repair.
5
Then, to
develop effective therapies that promote brain repair, we must
have an understanding of the cellular and molecular events
involved in the recovery from an ischemic insult.
The adult brain vascular system is stable under normal
conditions and is activated in response to pathological
conditions including injury.
6
Neovascularization following
stroke is an essential compensatory response mediating brain
repair, as it stimulates blood flow and metabolism in the
damaged area
7,8
that activate other brain remodeling post-
stroke events leading to improved functional outcome of stroke
patients.
9,10
Neovascularization requires new vessel forma-
tion from mature endothelial cells and immature CD34
+
progenitor cells that includes endothelial progenitor cells
(EPCs). These progenitor cells are mobilized from the bone
marrow after vascular injury and home to the site of
neovascularization, contributing to regeneration by either
1
Institute of Biomedical Research of Salamanca, University Hospital of Salamanca-University of Salamanca, Salamanca, Spain;
2
Institute of Functional Biology and
Genomics, University of Salamanca-CSIC, Salamanca, Spain and
3
Health Research Institute of Santiago de Compostela, University Clinical Hospital-University of Santiago
de Compostela, A Coruña, Spain
*Corresponding author: A Almeida, Institute of Biomedical Research of Salamanca, University Hospital of Salamanca-University of Salamanca, Calle Zacarías González 2,
Salamanca 37007, Spain. Tel: +34 923 294908; Fax: +34 923 224876; E-mail: aaparra@usal.es
Received 02.4.16; revised 29.8.16; accepted 07.9.16; Edited by M Deshmukh; published online 21.10.16
Abbreviations: Ang-1, angiopoietin-1; EPC, endothelial progenitor cell; ICH, intracerebral hemorrhage; IB4, isolectin-B4; mRS, modified Rankin Scale; SNP, single-
nucleotide polymorphism; SDF-1α, stromal cell-derived factor-1α; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; VEGF, vascular endothelial
growth factor; VEGFR2, VEGF receptor-2
Cell Death and Differentiation (2017) 24, 144–154
Official journal of the Cell Death Differentiation Association
www.nature.com/cdd
direct incorporation into newly forming vascular structures or
indirectly via the secretion of proangiogenic growth factors,
thereby enhancing the overall vascular recovery of ischemic
brain.
11–14
Furthermore, emerging evidences confer to circu-
lating EPC levels a prognostic value in the prediction of
functional outcome in ischemic stroke
15–17
and ICH
18
patients.
The human Tp53 gene harbors a common single-nucleotide
polymorphism (SNP) at codon 72, which yields an arginine-to-
proline amino-acidic substitution (Tp53 Arg72Pro SNP) that
modulates the apoptotic activity of the tumor suppressor
protein p53.
19–22
Previously, we found that the Tp53 Arg72Pro
SNP controls neuronal susceptibility to ischemia-induced
apoptosis in vitro.
23
In this study, we show that the Tp53
Arg72Pro SNP modulates endothelial cell survival after
experimental ICH in vivo, which promotes EPC mobilization
and neovascularization. Furthermore, functional recovery of
patients after ICH is conditioned by the Tp53 Arg72Pro
genotype. Our results thus reveal a novel function of the Tp53
Arg72Pro SNP in cerebrovascular repair and neurological
outcome after stroke.
Results
The Tp53 Pro allele is associated with high levels of
circulating CD34
+
cells and good functional prognosis
after ICH. To assess whether the Tp53 Arg72Pro SNP is
associated with long-term (12 months) prognosis after ICH,
patients (see Table 1 for baseline characteristics) were
matched by functional outcome at discharge based on the
modified Rankin scale (mRS) scores
24
(Table 2). We found
that homozygous Arg allele patients (referred as Arg patients)
with good outcome (mRS ⩽ 2) at discharge suffered progres-
sive disability (mRS42) after ICH. In contrast, patients
harboring the Pro allele (referred as Pro patients) maintained
a favorable outcome, even at long term after ICH (Figure 1a).
Moreover, functional recovery was time-dependently
improved in the Pro patients who had poor functional
outcome (mRS42) at discharge, whereas it worsened in
the Arg patients (Figure 1b). Interestingly, although the lesion
volume on admission (Figure 1c) and perihematoma edema
volume at 48–72 h following ICH (Figure 1d) were similar in
both genotypes, indicating similar initial hemorrhagic
damage, a fourfold lower residual cavity volume was
observed in Pro patients at 6 months after ICH when
compared with Arg patients (Figure 1e). Taken together,
these results indicate that Tp53-codon 72 Pro allele-carrying
subjects are protected against long-term poor functional
outcome after ICH, whereas the homozygous Arg allele is
associated to poor prognosis.
To understand the mechanism responsible for the long-term
difference in prognosis of both alleles, we focused on
neovascularization, a late-onset process that facilitates the
neurogenesis, synaptogenesis and synaptic plasticity respon-
sible for the long-term repair of the ischemic brain.
9
Neovascularization after cerebral ischemia is promoted by
circulating CD34
+
progenitor cells
11,13,14,25
and, although the
mechanism is unknown, it correlates with functional outcome
in ischemic stroke
15–17
and ICH
18
patients. We first corrobo-
rated this correlation in our ICH cohort (Figure 2a). Thus,
levels of circulating CD34
+
progenitor increased in patients
with good functional outcome, whereas remained unchanged
in those with poor prognosis (Figure 2a). To elucidate whether
the Tp53 Arg72Pro SNP influenced neovascularization, we
next matched patients by genotype and determined the levels
of circulating CD34
+
cells. As shown in Figure 2b, whilst levels
of CD34
+
cells at admission were similar in both genotypes,
they significantly increased as soon as 7 days following ICH in
Pro, but not in Arg patients. Interestingly, the patients showing
the highest levels of CD34
+
cells at 7 days after ICH
corresponded with the Pro genotype and good prognosis
(mR ⩽ 2 at 3 months); in contrast, the patients with the lowest
CD34
+
levels were those of the Arg genotype and poor
prognosis (mRS42 at 3 months) (Figure 2c). Flow cytometry
analysis of the CD34
+
progenitor cells revealed that the
observed differences in CD34
+
cell levels were not due to cell
survival changes (Figure 2d). Next, we analyzed vascular
endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and
stromal cell-derived factor-1α (SDF-1α) for their known roles in
Table 1 Baseline demographic and clinical features of patients
Variables ICH (n = 78)
Age (years) 70.4 ± 11.1
Gender
Females, n (%) 24 (30.8)
Males, n (%) 54 (69.2)
Time from ICH onset (h) 5.1 ± 3.7
Diagnosis
Etiologic
Hypertensive, n (%) 36 (46.2)
Amyloid, n (%) 26 (33.3)
Anticoagulants, n (%) 3 (3.8)
Undetermined, n (%) 13 (16.7)
Topographic
Deep, n (%) 48 (61.5)
Lobar, n (%) 30 (38.5)
Ventricular extension, n (%) 23 (29.5)
NIHSS on admission 10 [5, 16]
Lesion volume on admission (ml) 28.8 ± 27.9
Residual lesion volume at 6 months (ml) 11.7 ± 17.7
Poor prognosis at 3 months, n (%) 42 (53.8)
mRS at 3 months
0, n (%) 10 (12.8)
1, n (%) 17 (21.8)
2, n (%) 9 (11.5)
3, n (%) 12 (15.9)
4, n (%) 9 (11.5)
5, n (%) 3 (3.8)
6, n (%) 18 (23.1)
Tp53 Arg72Pro SNP
Pro (Arg/Pro+Pro/Pro), n (%) 31 (39.8)
Arg (Arg/Arg), n (%) 47 (60.2)
Circulating CD34
+
progenitor cells
At admission, cells per 2.5 × 10
5
events 203.9 ± 90.7
At day 7, cells per 2.5 × 10
5
events 359.9 ± 138.4
Abbreviations: ICH, intracerebral hemorrhage; mRS, modified Rankin Scale;
NIHSS, National Institute of Health Stroke Scale; SNP, single-nucleotide
polymorphism.
Patients were admitted at the University Hospital of Santiago de Compostela
(Galicia, Spain). Data are shown as percentage (n, %), mean (S.D.) or medians
(quartiles).
Tp53 ArgPro SNP dictates vascularization after ICH
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Cell Death and Differentiation
the activation and mobilization of progenitor cells from the
bone marrow in response to peripheral tissue hypoxia after
stroke,
12,26
and migration of EPC to injured tissues
8,27
to
promote neovascularization. Serum levels of VEGF
(Figure 1e), Ang-1 (Figure 1f) and SDF-1α (Figure 1g) were
significantly higher in Pro patients when compared with Arg
patients, at 72 h after ICH. Altogether, these data indicate that
the Tp53 Arg72Pro SNP determines the release of progenitor
cell-activating factors and EPC mobilization from the bone
marrow as a compensatory response to promote brain repair
and functional recovery after ICH in humans.
The p53 Pro variant increases endothelial cell survival
and triggers efficient EPC mobilization via VEGF and
SDF-1α leading to neovascularization after experimental
ICH. To investigate directly the impact of the Tp53 Arg72Pro
SNP on neovascularization after ICH, we next subjected
humanized Tp53 knock-in (KI) mice models of both Arg72Pro
codon variants
28
to a previously validated experimental
ICH.
29
As shown in Figures 3a and b, the lesion volume
observed at 6 h after experimental ICH was similar in both
genotypes, indicating similar initial hemorrhagic insult; how-
ever, the time-dependent decrease in lesion volume was
significantly faster in Pro than in Arg allele-carr ying mice.
Next, we evaluated functional recovery of mice after ICH by
testing motor coordination and limb strength using the
accelerated rotarod test.
30,31
Whereas at 24 h after ICH
motor impairment was similar in both genotypes, mice
expressing the Pro variant showed better motor performance
than those with the Arg variant at both 48 h and 7 days after
the hemorrhagic insult, which reflects a different level of
functional recovery. Furthermore, mice carrying the Pro allele
reached basal (before ICH) motor performance at 7 days
Table 2 Modified Rankin Scale
Grade Criteria
0 No symptoms at all
1 No significant disability: despite symptoms, able to carry
out all usual duties and activities
2 Slight disability: unable to perform all previous activities
but able to look after own affairs without assistance
3 Moderate disability: requiring some help but able to walk
without assistance
4 Moderately severe disability: unable to walk without
assistance and unable to attend to own bodily needs
without assistance
5 Severe disability: bedridden, incontinent and requiring
constant nursing care and attention
6 Death
Modified from Banks and Marotta
24
Figure 1 The Tp53 Arg72Pro SNP modulates long-term functional outcome after ICH. Patients were admitted at the University Clinical Hospital of Santiago de Compostela
(Galicia, Spain). The study included 78 (Arg/Arg: 47, referred as Arg; Arg/Pro and Pro/Pro: 31, referred as Pro) patients with non-traumatic ICH. (a and b) Modified Rankin Scale
(mRS) was used to evaluate the functional outcome of ICH patients. Patients were matched by (a) good (mRS ⩽ 2) and (b) poor (mRS42) functional outcome at discharge and
mRS scores were evaluated at 3 and 12 months after ICH with indicated Tp53 Arg72Pro genotypes. Boxplots show median values (horizontal line inside the box) and quartiles
(box boundaries) (Mann–Whitney test). (c) Lesion volume on admission, (d) perihematoma edema volume at 48–72 h after ICH, and (e) residual lesion volume at 6 months after
ICH were measured in patients with indicated Tp53 Arg72Pro genotypes. Data are mean ± S.D. (Student’s t-test). **Po0.0001 versus Pro patients
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Cell Death and Differentiation
after ICH; however, this effect was not obser ved in the Arg
allele-carrying mice (Figure 3c), indicating poorer functional
recover y than the Pro mice. All these findings confirm the
results observed in humans (Figure 1), supporting that the
humanized Tp53 Arg72Pro SNP KI mice variants functionally
recapitulate the human phenotypes.
In vivo terminal deoxynucleotidyl transferase dUTP nick-
end labeling (TUNEL) staining revealed significantly less
apoptotic cell death in the perihematoma area in the mice
harboring the Pro allele than in those with the Arg allele
(Figure 3d). Among the affected cells, we found not only
neurons (TUNEL
+
NeuN
+
cells) (Figure 3e) but also endothe-
lial cells (TUNEL
+
CD31
+
cells) (Figure 3f), which both
underwent less apoptotic death in the Pro mice than in the
Arg one. In vitro ischemia (oxygen and glucose deprivation)
confirmed higher survival of cultured brain endothelial cells
obtained from the Pro mice (Figure 3g). We therefore
hypothesized whether endothelial cells’ survival against ICH
dictated the production and release of angiogenic and EPC-
mobilizing factors, including VEGF, Ang-1 and SDF-1α that led
to the EPC mobilization, cerebrovascular repair and functional
prognosis observed in patients (Figures 1a and 2e–g). To test
this, we measured circulating levels of CD34
+
progenitor cells
in both the Tp53 Pro and Arg SNP variant KI mice after
experimental ICH. In good agreement with the data observed
in ICH patients (Figure 2b), the levels of CD34
+
progenitor
cells were significantly higher in the Pro mice when compared
with the Arg one, 24 h after the insult (Figure 4a). Given that
CD34 is not exclusively present in EPC, but also on mature
endothelial cells – although at a lower level – specific EPC
identification should be demonstrated by showing the pre-
sence of the endothelial marker VEGF receptor-2 (VEGFR2)
and the absence of the pan-leukocyte marker CD45.
12,16,32,33
We therefore analyzed CD34
+
/VEGFR2
+
/CD45
−
cells by
FACS as a bona fide marker of EPC. As shown in Figure 4b,
experimental ICH in the Tp53 Arg72Pro mice triggered a t
ime-dependent increase in the circulating levels of EPC
in the Pro allele-carrying mice, reaching the maximum level
at 24 h after the insult; however, this effect was significantly
attenuated in mice with the Arg allele (Figure 4b).
Such difference was not because of a putative differential
rate of EPC proliferation at the light of their same BrdU
Figure 2 The Tp53 Arg72Pro SNP dictates levels of circulating CD34
+
progenitor cells after ICH. Patients were admitted at the University Clinical Hospital of Santiago de
Compostela (Galicia, Spain). The study included 78 (Arg/Arg: 47, referred as Arg; Arg/Pro and Pro/Pro: 31, referred as Pro) patients with non-traumatic ICH. (a) Levels of
circulating CD34
+
progenitor cells in patients with good (mRS ⩽ 2; 36 patients) and poor (mRS42; 42 patients) functional outcome at 3 months after ICH (one-way analysis of
variance (ANOVA) followed by Bonferroni’s test). (b) Patients were matched by genotype (Arg and Pro) and we studied circulating levels of CD34
+
progenitor cells at 7 days after
ICH in relation to mRS scores at 3 months after ICH. (c) Levels of circulating CD34
+
progenitor cells in patients with indicated Tp53 Arg72Pro genotypes (one-way ANOVA
followed by Bonferroni’s test). (d) Apoptosis (annexin V-APC-stained cells that were 7-AAD-negative) were determined in CD34
+
progenitor cells by flow cytometry at day 7
following ICH. (g–i) Serum levels of (g) VEGF (pg/ml), (h) Ang-1 (ng/ml) and (i ) SDF-1α (ng/ml) at 72 h after ICH with indicated Tp53 Arg72Pro genotypes (Student’s t-test). Data
are mean ± S.D. *Po0.001; **Po0.0001 versus (a) good functional outcome or (b–g) Pro patients
Tp53 ArgPro SNP dictates vascularization after ICH
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Cell Death and Differentiation
incorporation rate (Figure 4c) and cell cycle phase distribution
(Figure 4d) in both genotypes. Furthermore, levels of
EPC-mobilizing cytokines, VEGF (Figure 4e) and SDF-1α
(Figure 4f), sharply increased in the Pro mice after the
ICH insult, whereas this effect was significantly weakened in
the Arg mice. Altogether, these results strongly suggest
that the regulation of EPC mobilization by these
cytokines dictates the different functional outcomes
of the Tp53 Pro and Arg alleles in mice and patients
after ICH.
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