High-density Lipoprotein–based Therapy Reduces the
Hemorrhagic Complications Associated With Tissue
Plasminogen Activator Treatment in Experimental Stroke
Bertrand Lapergue, MD; Bao Quoc Dang, MSc; Jean-Philippe Desilles, MSc;
Guadalupe Ortiz-Munoz, PhD; Sandrine Delbosc, PhD; Stéphane Loyau, BscTech;
Liliane Louedec, BscTech; Pierre-Olivier Couraud, MD, PhD; Mikael Mazighi, MD, PhD;
Jean-Baptiste Michel, MD, PhD; Olivier Meilhac, PhD; Pierre Amarenco, MD
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
Background and Purpose—We have previously reported that intravenous injection of high-density lipoproteins (HDLs)
was neuroprotective in an embolic stroke model. We hypothesized that HDL vasculoprotective actions on the blood–
brain barrier (BBB) may decrease hemorrhagic transformation-associated with tissue plasminogen activator (tPA)
administration in acute stroke.
Methods—We used tPA alone or in combination with HDLs in vivo in 2 models of focal middle cerebral artery occlusion
(MCAO) (embolic and 4-hour monofilament MCAO) and in vitro in a model of BBB. Sprague–Dawley rats were
submitted to MCAO, n=12 per group. The rats were then randomly injected with tPA (10 mg/kg) or saline with or without
human plasma purified-HDL (10 mg/kg). The therapeutic effects of HDL and BBB integrity were assessed blindly 24
hours later. The integrity of the BBB was also tested using an in vitro model of human cerebral endothelial cells under
oxygen–glucose deprivation.
Results—tPA-treated groups had significantly higher mortality and rate of hemorrhagic transformation at 24 hours in both
MCAO models. Cotreatment with HDL significantly reduced stroke-induced mortality versus tPA alone (by 42% in
filament MCAO, P=0.009; by 73% in embolic MCAO, P=0.05) and tPA-induced intracerebral parenchymal hematoma
(by 92% in filament MCAO, by 100% in embolic MCAO; P<0.0001). This was consistent with an improved BBB
integrity. In vitro, HDLs decreased oxygen–glucose deprivation–induced BBB permeability (P<0.05) and vascular
endothelial cadherin disorganization.
Conclusions—HDL injection decreased tPA-induced hemorrhagic transformation in rat models of MCAO. Both in vivo and in
vitro results support the vasculoprotective action of HDLs on BBB under ischemic conditions. (Stroke. 2013;44:699-707.)
Key Words: blood–brain barrier ■ hemorrhagic transformation ■ high-density lipoproteins ■ ischemic stroke
■ tissue plasminogen activator
R
unit (BBB, neurons, and astrocytes) at the acute phase of
stroke.5,7
High-density lipoproteins (HDLs) have been proposed as a
new neuro- and vasculoprotective treatment in IS.8,9 Beyond
their well-documented action of reverse cholesterol transport,
HDLs have pleiotropic effects (ie, anti-inflammatory, antioxidant, and more generally endothelial protective effects).10,11
We recently reported that intravenous administration of HDLs
isolated from human plasma, up to 5 hours after the onset
of ischemia, was effective in reducing the infarct volume by
maintaining BBB integrity in a rat model of embolic stroke.8
ecombinant tissue plasminogen activator (tPA) is the only
effective fibrinolytic treatment at the acute stage of ischemic stroke (IS). However, its use remains restricted to carefully selected patients within 4.5 hours after stroke onset and
it is associated with an increased risk of brain edema and secondary hemorrhagic transformation (HT).1–4 Therefore, there
is a need for development of treatments that would improve
the safety and efficacy of tPA. The disruption of the blood–
brain barrier (BBB) has been shown to be involved in edema
and HT after tPA treatment in IS.5,6 Ischemic injury and tPA
display cumulative deleterious effects on the neurovascular
Received July 21, 2012; accepted November 28, 2012.
From INSERM, UMR698, Paris, France (B.L., B.Q.D., J.-P.D., G.O.M., S.D., S.L., L.L., M.M., J.-B.M., O.M., P.A.); Univ Paris Diderot, Sorbonne
Paris Cité, Paris, France (B.L., B.Q.D., J.-P.D., G.O.M., S.D., S.L., L.L., M.M., J.-B.M., O.M., P.A.); CHU X-Bichat, Paris, France (B.L., B.Q.D., J.-P.D.,
G.O.M., S.D., S.L., L.L., M.M., J.-B.M, O.M., P.A.); Departments of Neurology and Stroke Centre, Paris-Diderot University Hospital, Paris, France (B.L.,
J.-P.D, M.M., O.M., P.A.); and CNRS (UMR 8104), Institut Cochin, Université Paris-Descartes, Paris, France (P.-O.C).
Christopher Kleinschnitz, MD, was guest editor for this article.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.
112.667832/-/DC1.
Correspondence to Pierre Amarenco, MD, INSERM U-698, Department of Neurology and Stroke Centre, Bichat University Hospital, 46 rue Henri
Huchard, 75018 Paris, France. E-mail [email protected]
© 2013 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org
DOI: 10.1161/STROKEAHA.112.667832
699
700 Stroke March 2013
Here, we sought to assess the potential of HDL therapy in
combination with tPA in 2 different models of focal cerebral
ischemia in rats. We speculated that beneficial effects of
HDLs on BBB integrity could allow reduction of edema and
HT induced by tPA injection in IS.
Materials and Methods
Animal Procedures and Experimental Design
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
Animal care and experimental protocols were approved by the
Animal Ethics Committee of the INSERM-University Paris 7, authorization 2010/13/698-0002. Male Sprague–Dawley rats (Janvier,
France), weighing 300 to 350 g, were anesthetized by isoflurane
mixed with air (4% for induction; 1% during surgery), under spontaneous respiration. Two different models of focal cerebral ischemia
were used: embolic middle cerebral artery occlusion (eMCAO) and
transient filament MCAO (fMCAO). Focal cerebral ischemia was
induced by intraluminal occlusion of the middle cerebral artery for
4 hours. eMCAO was induced by injection of a preformed clot at
the origin of MCA as already described.12 Continuous laser Doppler
flowmetry (VMS, Moor Instrument) was used to monitor regional cerebral perfusion to ensure the adequacy of MCA occlusion (perfusion
decreased to <20% of preischemic baselines). Animals were assigned
to 1 of 4 groups (n=12 per group, except for the tPA-treated group,
n=24): control (saline treatment only), tPA alone (10 mg/kg), HDLs
alone (10 mg/kg intravenous, jugular vein) (Figure IV in online-only
Data Supplement), and tPA plus HDLs. Continuous tPA or saline infusion (for 30 minutes; Harvard Apparatus Infusion Pump) started
4 hours after stroke onset, immediately before recanalization (after
withdrawal of the filament in fMCAO), or 4 hours after clot placement in the MCA. One single intravenous injection was performed
according to previously reported pharmacokinetics of HDLs in rats.13
Saline (2.0 mL/kg body weight) with or without recombinant tPA (10
mg/kg body weight; Actilyse, Boehringer-Ingelheim) was administered (10% bolus; 90% continuous infusion) to rats via the right femoral vein. The relatively high dose of tPA was necessary to achieve
a fibrinolytic effect in rats similar to that of thrombolytic therapy
in humans.14 After saline or tPA administration, rats were returned
to their cages. Body temperature was maintained at 37±0.5°C with
a heating pad for the duration of surgery. Glycemia, arterial blood
pressure, and blood gases were also monitored during surgery. The
mortality rate was determined at 24 hours. The neurological deficit
was determined on the remaining rats using a modified Neurological
Severity Score which is a composite of motor, sensory, and balance
tests.8 Computer-based randomization was used to allocate drug regimens to each group. Experiments were blinded and the operator was
unaware of group allocation during surgery and outcome assessment.
Exclusion Criteria
Animals were excluded for analysis if the following occurred: subarachnoid hemorrhage (n=1 in eMCAO and 2 in fMCAO) or inadequacy of MCA occlusion (n=3 in eMCAO and 1 in fMCAO). One
death owing to anesthesia or surgery occurred within 4 hours of
stroke induction (1 in fMCAO).
Measurement of Intracranial Hemorrhage and
Infarct Volume
Rats were euthanized 24 hours after induction of focal ischemia with a
lethal dose of isoflurane and perfused through the heart with 10 U/mL
heparin in 0.9% saline. The brain was rapidly removed. Seven coronal sections of the brain (2 mm in thickness) were immediately made
and numeric images were captured for quantification of hemorrhage.
Intracerebral hemorrhage was classified by an examiner, unaware of
the rat’s regimen, into three groups: no hemorrhage (0); hemorrhagic
infarction (H), defined as individual or several petechiae in the core or
at the borders of the ischemic area; and parenchymal hematoma (P),
when a large area of blood was observed within the core of the infarct.4,15 Moreover, areas of HT from these 7 unstained brain sections
were measured (hemorrhage area, mm2) using Photoshop software
by semiautomatic selection of extravasated erythrocytes as previously described.16,17 The slices were then immediately stained with 2%
2,3,5-triphenyltetrazolium chloride (Sigma-Aldrich) for 20 minutes at
room temperature.8 Volume calculation with edema correction was performed blindly using the following formula: 100×(contralateral hemisphere volume−noninfarct ipsilateral hemisphere volume)/contralateral
hemisphere volume. After 2,3,5-triphenyltetrazolium chloride staining,
the slice at +0.70 mm posterior to bregma18 was fixed in paraformaldehyde 3.7%, cryoprotected, embedded in optimal cutting temperature
medium and immediately frozen for immunohistological analysis.
IgG Extravasation
BBB permeability was evaluated 24 hours after stroke by using fluorescence detection of IgG extravasation in the fMCAO model as previously described.19 Using a cryostat, 10-µm sections were prepared
from the coronal slice (at +0.70 mm posterior to bregma) previously
frozen in optimal cutting temperature medium. Sections were incubated overnight at 4°C with donkey antirat IgG antibodies (5 µg/mL,
Molecular Probes) labeled with Alexa Fluor 488. IgG extravasation in
the ischemic area was quantified semiautomatically using morphometry software (Histolab 6.1.5, Microvision Instruments).
Immunohistochemistry
Frozen sections were fixed with 3.7% paraformaldehyde and blocked
with 10% goat serum. Sections were incubated overnight at 4°C with
a rabbit polyclonal to collagen IV (2 µg/mL; Abcam) for detection
of basal lamina of intracerebral vessels. Fluorescein isothiocyanateconjugated Bandeiraea Simplicifolia Lectin I (isolectin B4) (5 µg/
mL; Vector Laboratories) was used to visualize endothelial cells
(ECs), and a polyclonal rabbit antimyeloperoxidase (16.5 µg/mL;
Dako) to detect polymorphonuclear neutrophils (PMNs). The number of immunostained cells was determined semiautomatically using
morphometry software (Histolab 6.1.5, Microvision Instruments).8
Nonimmune rabbit IgG was included in each set of experiments as
primary antibody to test the specificity of the signal. We used AlexaFluor 555 as secondary antibodies. Immunostaining was analyzed
with a fluorescent microscope interfaced with a digital capture system. All immunohistological evaluations were performed by an observer who was blinded to the treatment. For semiquantification of
the collagen type IV expression, 3 fields of each territory (right MCA
and contralateral area) were acquired using a ×10 objective. A threshold of fluorescence intensity, which encompasses positive vessels on
the contralateral image, was applied to each corresponding ipsilateral
image. Data are presented as a percentage of the positive immunoreactivity area of the contralateral area (pixel). Immunohistochemistry
analysis was performed in the fMCAO model.
Isolation of Lipoproteins and In Vivo Tracking
of HDLs
Lipoproteins were isolated from a pool of heparinized plasma of
healthy volunteers by ultracentrifugation.20 In brief, plasma density
was adjusted to d=1.22 with KBr and overlaid with KBr saline solution (d=1.063). Ultracentrifugation was performed at 100 000g for
20 h at 10°C. The density of the bottom fraction containing HDLs
was adjusted to 1.25 with KBr and overlaid with KBr saline solution
(d=1.22). The second ultracentrifugation was performed at 100 000g
overnight at 10°C. After this step, HDL fractions, representing the
top layer of the tube, were recovered as a single band and were then
extensively rinsed with saline and concentrated using a centrifugal
concentrating device (cutoff 10 kDa; Vivascience, Stonehouse, UK).
All fractions were desalted either by dialysis against saline or by centrifugation and 3 washes with saline.
For tracking in vivo, HDLs were labeled with carbocyanines. HDLs
were incubated overnight at 37°C under gentle shaking with 8.5 µg/
mL DiIC18 carbocyanines (Molecular Probes Inc, USA) and then
separated by ultracentrifugation. Labeled HDLs (10 mg apoA1/kg)
were administrated intravenously in the fMCAO model (4 hours of
MCAO) immediately after stroke onset (n=3). Rats were euthanized
Lapergue et al HDLs Reduce tPA-Induced Hemorrhagic Complications in Stroke 701
at 1, 3, and 24 hours after stroke onset. After decapitation, brain
slices were embedded in optimal cutting temperature medium and
immediately frozen. Coronal sections (8 µm) (at +0.70 mm posterior
to bregma)18 were prepared with the use of a cryostat. Isolectin B4
(Vector Laboratories) was used to identify ECs as already described.21
Cell nuclei were stained with 4′,6′-diamidino-2-phenylindole (DAPI)
(0.5 µg/mL for 10 minutes) and the sections were observed under an
epifluorescence microscope.
Effect of HDLs on tPA Activity.
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
The potential effect of HDLs on tPA activity was measured in vitro using recombinant tPA or ex vivo on plasma after injection of
tPA±HDLs. The amidolytic activity of tPA (400 µg/mL) was assessed
by using SPECTROZYME chromogenic substrate (methylsulfonyl-d-cyclohexyltyrosyl-glycyl-arginine paranitroaniline acetate;
American Diagnostica) P-444 substrate in the presence or absence
of HDLs (0.4 g/mL). The kinetics of amidolysis by tPA with 1 mM
SPECTROZYME chromogenic substrate at 37°C were monitored
by measuring absorbance at 405 nm for 2 hours with a multiscan
spectrophotometer (BMG, Labtech). Initial rates of tPA activity were
calculated from plots of 405 nm versus time (mDO/min). For ex
vivo experiments, tPA (10 mg/kg) was administered via the femoral
vein (10% bolus and 90% during 20 minutes) in rats with or without
HDLs (10 mg/kg). Blood was sampled from the jugular vein in citrate-containing tubes, 3 minutes after administration of tPA, HDLs,
or tPA+HDLs. Plasma samples were diluted 20-fold with phosphate
buffered saline containing 0.1% human serum albumin (AbCys) and
0.01% Tween 20 in 96-well microtiter plates. tPA amidolytic activity
was determined as described above. Measurements were performed
in triplicate (n=3 per group of rats) and expressed as mean±SD.
(0.385 mg/mL) to that in the collecting wells and plotted against time.
Permeability surface (PS) was calculated from 1/PS=1/PSe−1/PSf,
where PSe is the slope of clearance in the presence of cells and PSf
is the slope for a blank insert (cell free).23 Pe=PS/S, where S is the
surface area of the insert (0.7 cm²).
For cell culture, functionality assay, and mRNA expression analysis, please see online-only Data Supplement.
Immunocytofluorescence
HCMEC/D3 cells were seeded at 5×105 cells/cm² onto collagen-coated labteks in complete endothelial basal medium-2. Cells were grown
for 7 days postconfluence before use. After treatment, cells were fixed
in 3.7% paraformaldehyde for 30 minutes and stored in phosphate
buffered saline at 4°C. Rabbit polyclonal antihuman vascular endothelial (VE) cadherin (1:200; Bender Med Systems) was used as
primary antibody, followed by a secondary antibody conjugated to
Alexa 555 (Invitrogen). Negative controls using nonimmune rabbit
IgGs at the same concentration as anti-VE cadherin were included in
each set of experiments to check for nonspecific staining.
Statistical Analysis
Data are presented as medians (quartiles) for continuous variables,
or mean (±SD) for immunostaining experiments (Figure 3B; Figure
IB in online-only Data Supplement) and percentages for qualitative variables. We analyzed data by a Kruskal–Wallis test followed
by pairwise Mann–Whitney U tests. Comparison of mortality and
intracerebral hemorrhage between groups was performed using the
Fisher exact test. A 2-tailed value of P<0.05 was considered significant. Data were analyzed using JMP 7.0.1. The number of animals
analyzed (n) is provided in the figure legends.
In Vitro BBB Model
Cell Culture
The human brain EC line hCMEC/D3 was kindly provided by Dr
P.O. Couraud.22 Cells were cultured in complete endothelial basal medium-2 (EBM +2.5% of fetal calf serum and supplements containing
hydrocortisone and growth factors).
Sample Size Calculation
The study was designed with 80% power to compare the proportion of
parenchymal hematoma in tPA+HDL-treated group versus tPA alone.
For proportions of 0.7 versus 0.2 (P=0.05), which are similar values
from preliminary experiments, a sample size of 11 would be required.
Cell Treatments
Before each experiment, cells were washed 3 times with phosphate
buffered saline and then incubated with 400 µg/mL of HDL solution.
Oxygen–glucose deprivation (OGD) conditions were obtained by using Dulbecco’s Modified Eagle’s Medium without glucose (Gibco)
versus 1 g/L glucose Dulbecco’s Modified Eagle’s Medium for nonOGD conditions. Oxygen deprivation was obtained by using a hypoxia chamber (Billups-Rothenberg) where atmospheric air was replaced
by a gas mixture (0% O2, 5% CO2, 95% N2, air product). Dulbecco’s
Modified Eagle’s Medium used for OGD conditions was equilibrated
with the same mixture.
In Vitro Permeability Measurements
For permeability experiments, hCMEC/D3 cells were seeded at
5×105 cells/cm² on collagen inserts (pore controllable fiber filters, 0.4
µm pore size, Millicell 24-well plates, Millipore) in complete endothelial basal medium-2. Cells were grown for 10 days postconfluence
before use. Permeability was assessed using fluorescein isothiocyanate-labeled dextran (70 kDa molecular weight). Fluorescein isothiocyanate-dextran (0.385 mg/mL) was added to the inserts, which
were transferred every 15 minutes for 45 minutes to collecting wells
containing 600 µL of fresh medium. Two hundred microliters from
the collecting wells were transferred into a 96-well plate, and the
dextran fluorescence was determined on the microplate reader at 485
nm (excitation) and 538 nm (emission). Fluorescence was converted
to dextran concentration using a standard curve. The volume cleared
was calculated from the ratio of dextran concentration in each sample
to the applied concentration (0.385 mg/mL). The endothelial permeability coefficient (Pe) (cm/min) was calculated by determining the
volumes of clearance of dextran with or without cells. The cleared
volume was calculated from the ratio of initial dextran concentration
Results
In Vivo Experiments
Baseline Characteristics
Physiological characteristics (body temperature, glycemia,
blood pressure, and blood gases) did not differ across groups
throughout the experiments (Table in online-only Data
Supplement).
Effect of the Combined Treatment With tPA
and HDLs on Mortality, Infarct Volume, and
Neurological Deficit
To test whether HDL infusion may be effective in preventing
deleterious effects of tPA injected at the reperfusion stage,
tPA±HDL solution was administered 4 hours after stroke
onset and compared with control groups (saline or HDLs
alone), in two models of stroke in rats. tPA increased mortality
and infarct volume in both filament and embolic models as
detailed in Figure 1. The mortality rate observed in the tPAtreated group was higher in the fMCAO than in the eMCAO
group (86 versus 56%; P=0.09). Additional treatment with
HDLs relative to tPA treatment alone significantly decreased
infarct volume: −59.8% in eMCAO, P=0.005 and −46.4%
in fMCAO (P<0.0001), and mortality: −72.7% in eMCAO,
P=0.05 and −41.9% in fMCAO (P=0.009) (Figure 1). HDL
treatment alone significantly reduced cerebral infarct volume
702 Stroke March 2013
Filament MCAO
B
*
100
*
Stroke-induced
mortality at 24hours
S
Aer ischemia (%dead)
SStroke-induced mortality at 24hours
Aer ischemia (%dead)
A
NS
75
50
25
0
HDL
Saline
tPA
**
*
*
75
NS
50
25
0
Saline
**
80
70
60
50
40
30
20
10
0
HDL
tPA
HDL
tPA
HDL+tPA
**
*
Infarcon Volume
(% of contralateral hemisphere)
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
Infarcon Volume
(% of contralateral hemisphere)
100
HDL+tPA
90
Saline
Embolic MCAO
HDL+tPA
90
80
70
60
50
40
30
20
10
0
Saline
l
compared with saline in both stroke models (P<0.05) but
only a trend for decreased mortality was observed (P=0.29).
Combined treatment also improved the neurological outcome
relative to tPA treatment alone in both models (fMCAO,
P=0.01; eMCAO, P=0.01; data not shown).
Effects of HDL Treatment on HT and Cerebral
Edema Induced by tPA
Because the major complications associated with tPA treatment are HT and edema, we tested the effect of HDLs in
our models of stroke on these particular endpoints. Only
the groups treated by tPA alone exhibited a high percentage of parenchymal hematoma (P) (fMCAO, 62%; eMCAO,
46%), which was strongly associated with mortality (P=0.02;
Figure 2). No significant difference was observed for the rate
of petechial hemorrhage between groups. Interestingly, the
combined treatment of tPA and HDLs markedly decreased the
incidence of parenchymal hematoma by >90% in both models
compared with both tPA alone and saline treatment (P<0.001;
Figure 2A). The quantitative analysis of hemorrhage area was
also markedly reduced in the tPA+HDL-treated group relative
to tPA alone in both MCAO models, 4.33 (interquartile range
[IQR], 0–17.56) versus 44.22 (IQR, 29.30–65.49) for fMCAO
and 0 (IQR, 0–3.79) versus 41.12 (IQR, 19.23–99.58) for
eMCAO, P<0.001 (Figure 2B).
Effects of Combined Treatment on BBB Integrity
To assess the effect of the combined treatment on BBB integrity, cerebral edema and IgG extravasation were evaluated as a
surrogate marker of BBB disruption during acute stroke.
Large areas of edema surrounding ischemic cerebral vessels
were observed only in the tPA-treated group compared with
the tPA+HDL group (Figure 3A). Increased IgG extravasation
was shown in the ipsilateral ischemic versus the contralateral
hemisphere across the different groups. Combined treatment
HDL
tPA
PA
Figure 1. Effects of high-density lipoproteins (HDL) and tissue plasminogen activator (tPA) on mortality and infarct volume
in focal stroke models (A, transient filament middle cerebral artery occlusion,
fMCAO; B, embolic MCAO, eMCAO). A,
tPA increased mortality rate compared
with the 3 other groups (*P<0.05). HDLs
significantly decreased stroke-induced
mortality in the tPA+HDL-treated group
compared with tPA alone in both models.
B, HDL injection significantly decreased
infarct volume at 24 hours after stroke
versus saline and in combination with tPA
versus tPA alone (n=12 per group, except
for tPA-treated group, n=24). *P<0.05,
**P<0.001. NS indicates not statistically
significant.
HDL+tPA
HDL PA
with tPA and HDLs significantly reduced IgG extravasation
relative to tPA alone, respectively, 39±3% versus 88±1% of
the ischemic area, P=0.04 (Figure 3B). We then performed
immunostaining for collagen IV, a major component of the
basal lamina of cerebral microvessels. Twenty-four hours
after stroke, the number of collagen IV immunoreactive vessels in the infarcted area was decreased compared with the
contralateral hemisphere in the saline group and even more in
the tPA-treated group. Combined tPA and HDL therapy was
associated with a significant increase in collagen IV immunoreactive vessels relative to tPA alone, suggesting an improved
BBB integrity, respectively, 91±3% versus 45±8%, P=0.02
(Figure I in online-only Data Supplement).
HDLs taken up by ECs reduced the expression
of adhesion molecules and PMN recruitment
To produce a significant effect on tPA-associated complications, we hypothesized that injected HDLs should rapidly
reach the ischemic area and be taken up by cerebral ECs. HDL
particles were internalized as early as 1 hour after injection
(Figure 4) and remained detectable within ECs for 24 hours.
HDLs decreased adhesion molecule expression by cerebral
ECs in vitro and limited PMN recruitment in the ischemic area
in vivo.
Given the anti-inflammatory effect of HDLs on ECs,
we tested their capacity to reduce ICAM-1 and VCAM-1
expression in human cerebral EC culture, in response to
tumor necrosis factor-α. These 2 major adhesion molecules
play a critical role in PMN diapedesis and infiltration into
ischemic tissues.24 We show that HDLs decreased tumor
necrosis factor-α–induced expression of ICAM-1 and
VCAM-1 by ECs in a dose-dependent manner (Figure 2
in online-only Data Supplement). We then assessed PMN
recruitment in the ischemic area in vivo, in our experimental
groups. PMN recruitment was significantly decreased in
Lapergue et al HDLs Reduce tPA-Induced Hemorrhagic Complications in Stroke 703
A
Macroscopic scoring of intracerebral hemorrhage
incidence of hemorrhage (%)
No Hemorrhage
Incidence of hemorrhage (%)
Saline
Saline
HDL
HDL
**
TYPE H
hemorrhagic infarcon
**
tPA
tPA
**
0
TYPE P
parenchymal hematoma
**
tPA+HDL
tPA+HDL
25
100
75
50
0
100
Quanficaon of intracerebral hemorrhage
**
**
150
Hem
morrhage area (mm
m 2)
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
Hem
morrhage area (mm
m2)
75
50
Embolic MCAO
Filament MCAO
B
25
100
50
0
Saline
HDL
tPA
HDL+tPA
Filament MCAO
**
150
**
100
50
0
physio
HDL
tPA
tPA+HDL
Embolic MCAO
Figure 2. Effect of high-density lipoproteins (HDL) and tissue plasminogen activator (tPA) on intracerebral hemorrhage in focal stroke
models. A, left Representative brain sections indicating classification of intracerebral hemorrhages. Right Incidence of intracerebral hemorrhage in filament and embolic middle cerebral artery occlusion (MCAO) according to macroscopic classification across the groups (%).
B, The area of hemorrhagic transformation was measured by semiautomatic detection of extravasated erythrocytes (hemorrhage area,
mm2). Parenchymal hematoma incidence and hemorrhage area were significantly increased in tPA-treated groups versus saline groups in
both models (**P<0.001). Combined treatment with HDL (10 mg/kg) markedly decreased parenchymal hematoma incidence and hemorrhage area (**P<0.001, n=12 per group, except for tPA-treated group, n=24). 0, absence of hemorrhage; H, hemorrhagic infarction, individual or border petechiae; P, parenchymal hematoma.
the HDL+tPA group compared with tPA alone (P=0.035)
(Figure 5).
HDLs Prevented tPA-induced BBB Injury In Vitro
We hypothesized that the beneficial in vivo effects of HDLs at
the acute phase of stroke may be, at least in part, owing to protection of the BBB. We thus assessed BBB integrity using an
in vitro model subjected to OGD with or without HDLs. In our
conditions (4 hours of stimulation), OGD induced an increased
BBB permeability: 7.8×10−3 (IQR, 6.2–9.4×10−3) versus
5.6×10−3 (IQR, 5.5–6.4×10−3) in control, P=0.04. Interestingly,
coincubation with HDLs decreased OGD-induced BBB permeability by 38.5%, P=0.002 (Figure 6A). In addition, we
looked at VE cadherin, given that it is a pivotal junction protein
involved in the maintenance of the restricted permeability of the
endothelial barrier.25 Immunofluorescent staining for VE cadherin, performed on hCMEC/D3 cells 7 days postconfluence,
showed that OGD induced a disorganization of intercellular
junctions characterized by intracellular patches of VE cadherin.
Supplementation with HDLs reduced, at least partially, this
phenotype by maintaining the cell–cell junctions (Figure 6B).
In Vitro and Ex Vivo Assessment of HDLs on tPA
Activity
Because HDLs display antiprotease properties, we tested the
hypothesis they may inhibit tPA activity in vitro and ex vivo.
Using a selective substrate of tPA, we showed that HDLs
did not modify tPA proteolytic activity, in vitro, HDL+tPA:
216±17 versus tPA: 244±35, nonsignificant (Figure IIIA in
online-only Data Supplement). These results were further supported ex vivo in plasma of rats that had received intravenous
administration of tPA and HDLs. The proteolytic activity of
tPA from rat plasma was not affected by concomitant injection
of HDLs, HDL+tPA: 216±16 versus tPA: 225±53, nonsignificant (Figure IIIB in online-only Data Supplement) suggesting that a combined treatment is possible without interference
with the fibrinolytic action of tPA.
Discussion
In the present study, using 2 models of focal stroke in rats, we
showed that HDL injection prevented the risk of HT induced
by tPA administration in the acute stage of IS. Both in vivo
and in vitro results support the vasculoprotective action of
HDLs on BBB under ischemic conditions.
1.HDL administration limits HT induced by tPA.
To address whether HDL treatment may decrease HT
induced by tPA, we used 2 stroke models with tPA characterized by a high incidence of parenchymal hematoma and associated mortality,26 as observed in humans with delayed tPA
injection.27 Combination therapy with HDLs decreased the risk
of hematoma by at least 90% in both models. These results are
704 Stroke March 2013
A
tPA
HDL+ tPA
% of ischemic area (meansSD)
B
*
*
*
100
90
80
70
60
50
40
30
20
10
0
IgG extravasation
Saline
HDL
t‐PA
t‐PA+HDL
of major importance given that in human patients, hematoma
subsequent to tPA treatment are associated with clinical deterioration and poor clinical outcome.28 Our results on HT are
consistent in 2 different models (eMCAO and fMCAO) which
is in accordance with Stroke Therapy Academic Industry
Roundtable recommendations.29 In the filament model of
MCAO, tPA was injected at the time of reperfusion, 4 hours
after occlusion, highlighting the (extra) vascular deleterious
effects of this potent serine protease without any effect of
tPA on clot fibrinolysis. This condition may be similar when
patients with acute cerebral artery occlusion are recanalized
by thrombectomy.30 In the embolic stroke model, HT induced
by tPA injection was the consequence of both clot dissolution
and extravascular action during the reperfusion. It is worth
noting that the infarct volume of tPA-treated group did not
differ significantly from the saline-treated group. In fact, late
administration of tPA (4 hours) has been shown to have no
effect on infarct volume of focal IS in rodents.31 The percentage of hematoma and death differed significantly between the
2 models probably because of the difference in MCA occlusion time. As we have shown in a duplex sonography study, in
a similar embolic stroke model, 29% of rats with confirmed
MCAO occlusion had undergone recanalization between
1 and 4 hours after stroke onset,32 whereas MCA occlusion
induced by a filament lasted exactly 4 hours.
2.Effects of combined treatment on BBB integrity in vivo
and in vitro.
Pleiotropic protective effects of HDLs on ECs have been
demonstrated in clinical and preclinical settings.10,11,20,33,34 Our
in vivo and in vitro data support this beneficial effect on cerebral ECs.
A
Saline
B
tPA
15000
MPO-po
osive cells
per ischemiic hemisphere
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
Figure 3. Effect of high-density lipoproteins (HDL) and tissue
plasminogen activator (tPA) on brain edema and IgG extravasation. A, Representative brain sections showing large areas of
edema surrounding ischemic cerebral vessels (white arrows)
in the tPA-treated group relative to the tPA+HDL group. Scale
bar=100 µm. B, Effect of HDL and tPA on IgG extravasation
(% of fluorescent pixels in the ischemic area). tPA significantly
increased IgG extravasation in the ischemic hemisphere versus
saline. Combined treatment with HDLs significantly prevented
this increase (*P<0.05, n=4 per group).
Figure 4. In vivo uptake of high-density lipoproteins (HDLs) in
cerebral endothelial cells 1 hour after injection. HDL particles were
labeled by carbocyanine (red) and colocalize with endothelial cells
(fluorescein isothiocyanate-conjugated isolectin B4, green). Nuclei
were counterstained by 4′,6′-diamidino-2-phenylindole (blue).
Scale bar=100 µm.
tPA+HDL
*
*
10000
*
5000
0
Saline
HDL
tPA
tPA+HDL
Figure 5. Assessment of polymorphonuclear neutrophil (PMN) recruitment in the ischemic area in
vivo. PMN recruitment was significantly decreased
in the high-density lipoprotein (HDL)+tissue plasminogen activator (tPA) group compared to tPA
alone (P<0.05). A, Immunostaining for myeloperoxidase (MPO) on brain sections 24 hours after stroke.
Scale bar=200 µm. B, Quantification of MPO immunostaining signal (n=4 per group; *P=0.035).
Lapergue et al HDLs Reduce tPA-Induced Hemorrhagic Complications in Stroke 705
A
B
0.015
*
**
0.010
OGD
0.005
H
D
L
D
G
O
G
D
O
H
D
L
0.000
D
M
EM
Perm eability coeffic
cient (x10-3 cm/min)
No OGD
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
Indeed, in vivo, intravenously injected HDLs were taken up
by cerebral ECs within 1 hour after stroke onset (Figure 4),
even when the MCA was occluded by the monofilament and
could thus exert their cytoprotective effects (antiapoptotic,
antiprotease, and anti-inflammatory effects).10,11 We speculate
that this may be owing to corticocortical collateral vasculature. HDL particles could be detected up to 24 hours after
injection, suggesting a potential prolonged effect.
As already reported in various experimental models, HDLs
exert potent anti-inflammatory effects and limit PMN recruitment in injured tissues,35 including in our IS model.8 Herein,
we demonstrate that HDLs limited PMN transmigration in our
model of hemorrhagic complications induced by recombinant
tPA. In addition, we showed in vitro that HDLs reduced the
expression of ICAM-1 and VCAM-1 induced by tumor necrosis factor-α. Tumor necrosis factor-α stimulation is classically
used to assess the anti-inflammatory potential of HDLs. This
cytokine has also been shown to play a critical role in the
acute stage of IS.36
Our in vitro study also supports the beneficial effect of
HDLs on the BBB. In vitro, using a well-described model of
BBB, OGD for 4 hours induced an increase in BBB permeability and VE cadherin disorganization, both prevented by
HDLs. We have intentionally chosen a 4-hour OGD period to
conform to the timing of our in vivo model and, more importantly, to the therapeutic window in humans. Our data frame
well with the previously reported direct action of HDLs or
reconstituted HDLs (rHDLs) on ECs.11 The precise mechanisms of HDL protection on ECs are not completely understood but apolipoprotein A1, lipids surrounding the protein
core, sphingosine-1-phosphate, or other proteins associated
with HDLs, including antielastase, may provide explanations
for their beneficial actions on the BBB. By limiting BBB permeability in OGD conditions, HDLs may prevent tPA extravasation and limit its cytotoxic effects within brain tissue.37
BBB disruption in response to aggression by tPA and ischemic conditions plays a pivotal role in the initiation of blood
component leakage into the cerebral compartment. In humans,
OGD + HDL
Figure 6. A, In vitro effects high-density lipoproteins (HDLs) on permeability measured in a
blood-brain barrier model (hCMEC/D3 cells) under
normoxic or oxygen-glucose deprivation (OGD)
conditions. Cells were incubated with HDLs (0.4g/L)
for 4 hours (n=6 for each condition). Results are
presented as box plots in which the median is
shown (*P<0.05). B, Immunofluorescent staining
of vascular endothelial (VE) cadherin (A, red) after
treatment with HDLs (0.4g/L) for 4 hours under
OGD conditions. OGD induced a disorganization of
intercellular junctions characterized by intracellular
patches of VE cadherin, which was prevented by
supplementation with HDLs. Results are representative of three independent experiments. DMEM
indicates Dulbecco’s Modified Eagle’s Medium.
the disruption of the BBB assessed by gadolinium extravasation after brain MRI was strongly correlated with the risk
of symptomatic HT after tPA administration.5,6 HDL-based
therapy may have beneficial effects on cerebral ECs and thus
reduce complications associated with BBB disruption as we
have shown with a decrease in IgG extravasation and collagen
IV degradation when HDLs were coinjected with tPA versus
tPA alone.
3.In vitro and ex vivo assessment of HDL effects on tPA
activity.
Finally, we have shown that HDL did not interact with the
proteolytic action of tPA, a serine protease. We assessed a
putative interaction between HDLs and tPA given that some
proteins carried by HDLs are protease inhibitors.38 Both tPA
and HDL concentrations used in our study are in the range of
those used in humans. Our results suggest that the beneficial
actions of HDLs in combination with tPA treatment decrease
HT without interference with the fibrinolytic effect of tPA.
4.Acute HDL-based therapy in stroke.
Our study highlights the potential of HDL-based therapy
as an original neuro- and vasculoprotective agent in the acute
treatment of stroke. HDL-raising drugs, such as niacin, have
been suggested to be neuroprotective in experimental stroke.39
HDL-based therapy is supported by numerous epidemiological studies that showed that low HDL-cholesterol levels were
associated with cerebrovascular events.40 Recently, among
489 patients with acute IS treated with tPA within 3 hours of
stroke onset, high HDL-C levels were significantly associated with a good outcome at 3 months.41 This suggests that
HDL supplementation may be beneficial at the acute stage of
stroke. Raising HDL levels encompasses 2 different strategies: HDL infusion and HDL-raising drugs. A single rapid
rHDL infusion immediately restored endothelial function in
hypercholesterolemic patients or in subjects with low HDLC.33,42 In addition, infusion of rHDLs decreased the lipid
burden and the concentration of inflammatory markers in
706 Stroke March 2013
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
atherosclerotic plaques.43 Chronic administration of HDLraising drugs, such as cholesterylester transfer protein inhibitors or niacin, is currently being evaluated in secondary
vascular prevention but the first results seem to be disappointing.44,45 Nevertheless, to obtain an effect of HDLs in the acute
phase of cerebral ischemia, HDL infusion seems to be more
appropriate.
Prophylactic injection of rHDL (120 mg/kg) has been
shown to be neuroprotective in 2 different focal stroke models.9
This dose contrasts with the lower dose (10 mg/kg of plasma
HDLs) used in our study. This discrepancy may be explained
by the composition of HDLs that is different between HDLs
isolated from plasma and rHDLs, composed only of phosphatidyl-choline and ApoA1. The HDL-associated proteome
and lipidome encompass >50 proteins and 200 lipids. Several
potentially protective proteins (acute phase–response proteins,
protease inhibitors, etc) or lipids (sphingosine-1-phosphate)
could be transported by HDL isolated from plasma of healthy
volunteers that render these particles immediately efficient
under acute conditions.38,46 rHDLs may need to take up some
proteins from the plasma after injection to be as effective as
native HDLs.
HDLs consist of numerous subpopulations of particles,
which are functionally heterogeneous. Raising HDL levels by
pharmacological agents in patients with a metabolic syndrome
or other risk factors for stroke may lead to production of dysfunctional HDLs. Indeed, studies on HDL function among
type 2 diabetes or coronary heart disease patients have shown
that isolated HDLs were dysfunctional or even proinflammatory.47,48 Global HDL-raising strategies are still debated owing
to functionality issues.49 We thus hypothesize that rapid infusion of functional HDLs may be beneficial in acute cerebral
ischemia.
Limits
We did not use long-term follow-up for our in vivo study
and we used HDLs isolated from human plasma. Thus, this
is a proof of concept study. Although we have validated our
results in 2 different models of IS, they should be replicated
by independent research groups. Further studies are needed,
especially with rHDLs or HDL mimetics in acute stroke in
humans.
Conclusions
Our results suggest that HDL therapy may limit the deleterious effects of tPA in acute IS, such as HT. This could have
a strong clinical impact. Reconstituted human HDLs should
now be tested in IS patients.
Funding Sources
This research was supported by the Fondation de France, the Fondation
Coeur et Artère, and the Agence Nationale de la Recherche (ANR
JCJC 1105). B. Lapergue was supported by grants from INSERM,
the Lilly Institute, the Fondation pour la Recherche Médicale, and the
Société Française de Neurovasculaire.
None.
Disclosures
Acknowledgments
We are indebted to Ashfaq Shuaib who trained BL for the embolic
model of cerebral ischemia in the rat. We thank Julien Labreuche for
his statistical advice and Mary Osborne-Pellegrin for help in editing
the manuscript.
References
1. Khatri P, Wechsler LR, Broderick JP. Intracranial hemorrhage associated
with revascularization therapies. Stroke. 2007;38:431–440.
2. Fitzpatrick AE, Noone I, O’Shea DD. Thrombolysis 3 to 4.5 hours after
acute ischemic stroke. N Engl J Med. 2008;359:2840; author reply 2841.
3. Hacke W, Kaste M, Bluhmki E, Brozman M, Dávalos A, Guidetti D, et
al.; ECASS Investigators. Thrombolysis with alteplase 3 to 4.5 hours
after acute ischemic stroke. N Engl J Med. 2008;359:1317–1329.
4. del Zoppo GJ, Poeck K, Pessin MS, Wolpert SM, Furlan AJ, Ferbert A,
et al. Recombinant tissue plasminogen activator in acute thrombotic and
embolic stroke. Ann Neurol. 1992;32:78–86.
5. Hjort N, Wu O, Ashkanian M, Sølling C, Mouridsen K, Christensen S, et
al. MRI detection of early blood–brain barrier disruption: parenchymal
enhancement predicts focal hemorrhagic transformation after thrombolysis. Stroke. 2008;39:1025–1028.
6. Neumann-Haefelin C, Brinker G, Uhlenküken U, Pillekamp F, Hossmann
KA, Hoehn M. Prediction of hemorrhagic transformation after thrombolytic therapy of clot embolism: an MRI investigation in rat brain. Stroke.
2002;33:1392–1398.
7. Benchenane K, López-Atalaya JP, Fernández-Monreal M, Touzani O,
Vivien D. Equivocal roles of tissue-type plasminogen activator in strokeinduced injury. Trends Neurosci. 2004;27:155–160.
8. Lapergue B, Moreno JA, Dang BQ, Coutard M, Delbosc S, Raphaeli
G, et al. Protective effect of high-density lipoprotein-based therapy in a
model of embolic stroke. Stroke. 2010;41:1536–1542.
9. Paternò R, Ruocco A, Postiglione A, Hubsch A, Andresen I, Lang MG.
Reconstituted high-density lipoprotein exhibits neuroprotection in two
rat models of stroke. Cerebrovasc Dis. 2004;17:204–211.
10. Argraves KM, Gazzolo PJ, Groh EM, Wilkerson BA, Matsuura BS,
Twal WO, et al. High density lipoprotein-associated sphingosine
1-phosphate promotes endothelial barrier function. J Biol Chem.
2008;283:25074–25081.
11. Mineo C, Deguchi H, Griffin JH, Shaul PW. Endothelial and antithrombotic actions of HDL. Circ Res. 2006;98:1352–1364.
12. Wang CX, Yang T, Shuaib A. An improved version of embolic model of
brain ischemic injury in the rat. J Neurosci Methods. 2001;109:147–151.
13. Ponsin G, Pulcini T, Sparrow JT, Gotto AM Jr, Pownall HJ. High density
lipoprotein interconversions in rat and man as assessed with a novel nontransferable apolipopeptide. J Biol Chem. 1993;268:3114–3119.
14. Korninger C, Collen D. Studies on the specific fibrinolytic effect of
human extrinsic (tissue-type) plasminogen activator in human blood and
in various animal species in vitro. Thromb Haemost. 1981;46:561–565.
15. Henninger N, Bratane BT, Bastan B, Bouley J, Fisher M. Normobaric
hyperoxia and delayed tPA treatment in a rat embolic stroke model.
J Cereb Blood Flow Metab. 2009;29:119–129.
16. Copin JC, Merlani P, Sugawara T, Chan PH, Gasche Y. Delayed matrix
metalloproteinase inhibition reduces intracerebral hemorrhage after
embolic stroke in rats. Exp Neurol. 2008;213:196–201.
17. Tejima E, Katayama Y, Suzuki Y, Kano T, Lo EH. Hemorrhagic transformation after fibrinolysis with tissue plasminogen activator: evaluation
of role of hypertension with rat thromboembolic stroke model. Stroke.
2001;32:1336–1340.
18. Paxinos GW, C. The rat brain in stereotaxic coordinates. 6th ed. New
York, NY: Academic Press; 1998.
19. Kelly MA, Shuaib A, Todd KG. Matrix metalloproteinase activation and
blood–brain barrier breakdown following thrombolysis. Exp Neurol.
2006;200:38–49.
20. Ortiz-Muñoz G, Houard X, Martín-Ventura JL, Ishida BY, Loyau S,
Rossignol P, et al. HDL antielastase activity prevents smooth muscle cell anoikis, a potential new antiatherogenic property. FASEB J.
2009;23:3129–3139.
21. Hess DC, Hill WD, Martin-Studdard A, Carroll J, Brailer J, Carothers J.
Bone marrow as a source of endothelial cells and NeuN-expressing cells
After stroke. Stroke. 2002;33:1362–1368.
22. Weksler BB, Subileau EA, Perrière N, Charneau P, Holloway K, Leveque
M, et al. Blood–brain barrier-specific properties of a human adult brain
endothelial cell line. FASEB J. 2005;19:1872–1874.
Lapergue et al HDLs Reduce tPA-Induced Hemorrhagic Complications in Stroke 707
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
23. Dehouck MP, Jolliet-Riant P, Brée F, Fruchart JC, Cecchelli R, Tillement
JP. Drug transfer across the blood-brain barrier: correlation between in
vitro and in vivo models. J Neurochem. 1992;58:1790–1797.
24. Frijns CJ, Kappelle LJ. Inflammatory cell adhesion molecules in ischemic cerebrovascular disease. Stroke. 2002;33:2115–2122.
25. Harris ES, Nelson WJ. VE-cadherin: at the front, center, and sides
of endothelial cell organization and function. Curr Opin Cell Biol.
2010;22:651–658.
26. Liu W, Hendren J, Qin XJ, Liu KJ. Normobaric hyperoxia reduces the
neurovascular complications associated with delayed tissue plasminogen activator treatment in a rat model of focal cerebral ischemia. Stroke.
2009;40:2526–2531.
27. The NINDS t-PA Stroke Study Group. Intracerebral hemorrhage after
intravenous t-pa therapy for ischemic stroke. Stroke. 1997;28:2109–2118.
28. Derex L, Nighoghossian N. Intracerebral haemorrhage after thrombolysis for acute ischaemic stroke: an update. J Neurol Neurosurg Psychiatr.
2008;79:1093–1099.
29.Fisher M, Feuerstein G, Howells DW, Hurn PD, Kent TA, Savitz
SI, et al.; STAIR Group. Update of the stroke therapy academic
industry roundtable preclinical recommendations. Stroke. 2009;40:
2244–2250.
30. Smith WS, Sung G, Saver J, Budzik R, Duckwiler G, Liebeskind DS,
et al.; Multi MERCI Investigators. Mechanical thrombectomy for
acute ischemic stroke: final results of the Multi MERCI trial. Stroke.
2008;39:1205–1212.
31. Zhu H, Fan X, Yu Z, Liu J, Murata Y, Lu J, et al. Annexin A2 combined
with low-dose tPA improves thrombolytic therapy in a rat model of focal
embolic stroke. J Cereb Blood Flow Metab. 2010;30:1137–1146.
32. Lapergue B, Deroide N, Pocard M, Michel JB, Meilhac O, Bonnin
P. Transcranial duplex sonography for monitoring circle of Willis
artery occlusion in a rat embolic stroke model. J Neurosci Methods.
2011;197:289–296.
33. Bisoendial RJ, Hovingh GK, Levels JH, Lerch PG, Andresen I, Hayden
MR, et al. Restoration of endothelial function by increasing high-density lipoprotein in subjects with isolated low high-density lipoprotein.
Circulation. 2003;107:2944–2948.
34. Cockerill GW, Rye KA, Gamble JR, Vadas MA, Barter PJ. High-density
lipoproteins inhibit cytokine-induced expression of endothelial cell adhesion molecules. Arterioscler Thromb Vasc Biol. 1995;15:1987–1994.
35. Cockerill GW, McDonald MC, Mota-Filipe H, Cuzzocrea S, Miller
NE, Thiemermann C. High density lipoproteins reduce organ injury
and organ dysfunction in a rat model of hemorrhagic shock. FASEB J.
2001;15:1941–1952.
36. Sairanen T, Carpén O, Karjalainen-Lindsberg ML, Paetau A, Turpeinen
U, Kaste M, et al. Evolution of cerebral tumor necrosis factor-alpha production during human ischemic stroke. Stroke. 2001;32:1750–1758.
37. Chen ZL, Strickland S. Neuronal death in the hippocampus is promoted
by plasmin-catalyzed degradation of laminin. Cell. 1997;91:917–925.
38. Vaisar T, Pennathur S, Green PS, Gharib SA, Hoofnagle AN, Cheung
MC, et al. Shotgun proteomics implicates protease inhibition and complement activation in the antiinflammatory properties of HDL. J Clin
Invest. 2007;117:746–756.
39.Shehadah A, Chen J, Cui Y, Zhang L, Roberts C, Lu M, et al.
Combination treatment with low-dose Niaspan and tissue plasminogen
activator provides neuroprotection after embolic stroke in rats. J Neurol
Sci. 2011;309:96–101.
40. Amarenco P, Goldstein LB, Messig M, O’Neill BJ, Callahan A 3rd,
Sillesen H, et al.; SPARCL Investigators. Relative and cumulative effects
of lipid and blood pressure control in the Stroke Prevention by Aggressive
Reduction in Cholesterol Levels trial. Stroke. 2009;40:2486–2492.
41. Makihara N, Okada Y, Koga M, Shiokawa Y, Nakagawara J, Furui E, et
al. Effect of serum lipid levels on stroke outcome after rt-PA therapy:
SAMURAI rt-PA registry. Cerebrovasc Dis. 2012;33:240–247.
42. Spieker LE, Sudano I, Hürlimann D, Lerch PG, Lang MG, Binggeli C,
et al. High-density lipoprotein restores endothelial function in hypercholesterolemic men. Circulation. 2002;105:1399–1402.
43. Shaw JA, Bobik A, Murphy A, Kanellakis P, Blombery P, Mukhamedova
N, et al. Infusion of reconstituted high-density lipoprotein leads to acute
changes in human atherosclerotic plaque. Circ Res. 2008;103:1084–1091.
44. Landmesser U, von Eckardstein A, Kastelein J, Deanfield J, Lüscher
TF. Increasing high-density lipoprotein cholesterol by cholesteryl ester
transfer protein-inhibition: a rocky road and lessons learned? The early
demise of the dal-HEART programme. Eur Heart J. 2012;33:1712–1715.
45.Boden WE, Probstfield JL, Anderson T, Chaitman BR, DesvignesNickens P, Koprowicz K, et al. Niacin in patients with low hdl cholesterol levels receiving intensive statin therapy. N Engl J Med.
2011;365:2255–2267
46. Kontush A, Chapman MJ. Lipidomics as a tool for the study of lipoprotein metabolism. Curr Atheroscler Rep. 2010;12:194–201.
47.Nobécourt E, Jacqueminet S, Hansel B, Chantepie S, Grimaldi A,
Chapman MJ, et al. Defective antioxidative activity of small dense
HDL3 particles in type 2 diabetes: relationship to elevated oxidative
stress and hyperglycaemia. Diabetologia. 2005;48:529–538.
48. Smith JD. Dysfunctional HDL as a diagnostic and therapeutic target.
Arterioscler Thromb Vasc Biol. 2010;30:151–155.
49. Tall AR, Yvan-Charvet L, Wang N. The failure of torcetrapib: was
it the molecule or the mechanism? Arterioscler Thromb Vasc Biol.
2007;27:257–260.
High-density Lipoprotein−based Therapy Reduces the Hemorrhagic Complications
Associated With Tissue Plasminogen Activator Treatment in Experimental Stroke
Bertrand Lapergue, Bao Quoc Dang, Jean-Philippe Desilles, Guadalupe Ortiz-Munoz, Sandrine
Delbosc, Stéphane Loyau, Liliane Louedec, Pierre-Olivier Couraud, Mikael Mazighi,
Jean-Baptiste Michel, Olivier Meilhac and Pierre Amarenco
Downloaded from http://stroke.ahajournals.org/ by guest on June 15, 2017
Stroke. 2013;44:699-707; originally published online February 19, 2013;
doi: 10.1161/STROKEAHA.112.667832
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2013 American Heart Association, Inc. All rights reserved.
Print ISSN: 0039-2499. Online ISSN: 1524-4628
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://stroke.ahajournals.org/content/44/3/699
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Stroke 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 Stroke is online at:
http://stroke.ahajournals.org//subscriptions/