Low Levels of Low-Density Lipoprotein-C Associated With

Proprotein Convertase Subtilisin Kexin 9 Inhibition Do Not

Increase the Risk of Hemorrhagic Transformation
Alexy Tran-Dinh, MD; Anglique Levoye, PhD; Gilles Lambert, PhD; Liliane Louedec, BSc;
Clment Journ, MSc; Olivier Meilhac, PhD; Pierre Amarenco, MD
Background and PurposeLow levels of low-density lipoprotein-cholesterol (LDL-C) are suspected to be associated with
a risk of hemorrhagic transformation after ischemic stroke. We assessed the risk of hemorrhagic transformation after
cerebral ischemia/reperfusion in mice with low levels of LDL-C resulting from proprotein convertase subtilisin kexin 9
(PCSK9) deficiency.
MethodsPCSK9/ and PCSK9+/+ mice were fed with a high-fat/high-cholesterol (21%/0.15%) diet for 1 month. Plasma
lipids were measured using colorimetric assays. PCSK9/ and PCSK9+/+ mice (n=15 per group) were subjected to a 4-hour
intraluminal occlusion of the middle cerebral artery followed by 20 hours of reperfusion. Spontaneous hemorrhagic
transformation was assessed by quantification of hemoglobin in ischemic tissue. In vitro, a cell model of bloodbrain
barrier was used to test endothelial barrier integrity in response to decreasing concentrations of LDL-C from 1 to 0.25g/L
in ischemia/reperfusion conditions.
ResultsPCSK9/ mice had lower LDL-C, high-density lipoprotein-cholesterol, and total cholesterol levels than PCSK9+/+
mice before and after 1 month on the high-fat/high-cholesterol diet. Hemoglobin concentration in ischemic cerebral
tissue was not different between PCSK9/ and PCSK9+/+ mice (31.5 [18.960.1] and 32.8 [14.769.9] ng/mg protein,
respectively; P=0.81). Infarct volume was also similar in both groups (P=0.66). Incubation of human cerebral endothelial
cells with decreasing concentrations of LDL-C under ischemia/reperfusion conditions did not alter bloodbrain barrier
permeability.
ConclusionsLow levels of LDL-C did not increase the risk of hemorrhagic transformation after cerebral ischemia/
reperfusion in mice. Our observations suggest that PCSK9 inhibition, leading to LDL-C lowering, should not increase
hemorrhagic complications after acute ischemic stroke.(Stroke. 2014;45:3086-3088.)
Key Words: hemorrhage low-density lipoprotein cholesterol mice PCSK9 protein stroke

roprotein convertase subtilisin kexin type 9 (PCSK9)

is a natural inhibitor of the low-density lipoprotein
(LDL) receptor pathway. PCSK9 binds to the LDL receptor at the cell surface and targets it for lysosomal degradation after endocytosis.1 As a result, heterozygous carriers
of PCSK9 loss-of-function mutations have naturally low
LDL-C levels and a reduced incidence of cardiovascular
events.2 Pharmacological inhibition of PCSK9, especially
when used in combination with statins, reduces LDL-C to
low levels (0.25g/L).3 A relationship between low LDL-C
levels and the risk of hemorrhagic transformation (HT) after
ischemic stroke has been suggested.4,5 HT occurs in 10% to
40% of patients with ischemic stroke5 and is predictive of
the worst clinical outcomes.6 Our aim was to test whether
LDL-C lowering with chronic PCSK9 inhibition increases
the risk of HT in acute ischemic stroke. We assessed this

risk in a mouse model with low LDL-C levels because of

PCSK9 deficiency.

Methods
Our in vivo protocol was approved by the Animal Ethics Committee
of Inserm/Diderot University-Paris (20122015/698-0098). PCSK9knockout (PCSK9/) and control mice (PCSK9+/+), purchased from
Jackson Laboratory, were fed with a high-fat/high-cholesterol
(21%/0.15%) diet for 1 month. Plasma lipids were measured using
colorimetric assays on an Olympus AU400 chemistry analyzer.
Mice (male; 10 weeks old; 25 g) were anesthetized with isoflurane,
and their body temperature was maintained at 37C with a heating
pad. Cerebral ischemia/reperfusion was induced by a 4-hour intraluminal middle cerebral artery occlusion (MCAO) by introducing a 7-0
silicon-coated monofilament (Doccol Corporation) through the right
common carotid to decrease cerebral blood flow at the bifurcation
of the right MCA and the right internal carotid. After removing the
monofilament, reperfusion was allowed for the next 20 hours. Mice

Received April 29, 2014; final revision received July 22, 2014; accepted July 23, 2014.
From the Inserm UMR1148, Paris7 University, Xavier Bichat Hospital, Paris, France (A.T.-D., A.L., L.L., C.J., O.M., P.A.); Paris 7 University, Universit
Paris Diderot, Paris, France (A.T.-D., P.A.); CHU Bichat Stroke Center, Paris, France (P.A.); CHU de La Runion, Saint-Pierre, France (O.M.); Paris 13
University, Villetaneuse, France (A.L.); UMR PhAN Laboratory, Nantes, France (G.L.); and University of Nantes Medical School, Nantes, France (G.L.).
Guest Editor for this article was Costantino Iadecola, MD.
Correspondence to Amarenco Pierre, MD, Bichat Hospital and Medical School, Paris, France. E-mail pierre.amarenco@bch.aphp.fr
2014 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org

DOI: 10.1161/STROKEAHA.114.005958

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3086 at INSERM - DISC on March 18, 2015

Tran-Dinh et al PCSK9 Inhibition in Ischemic Stroke 3087

Figure 1. Hemorrhagic transformation (HT) at 24 hours after cerebral ischemia/reperfusion in PCSK9/ (n=14) and PCSK9+/+ (n=13) mice. A,
Hemoglobin concentration (ng/mg protein) in ischemic cerebral tissue. PCSK9/ (n=12) and PCSK9+/+ (n=13). B, Infarct volume (cm3). PCSK9/
(n=14) and PCSK9+/+ (n=13). C, Representative TTC staining showing the infarct area (white) and HT (red hematomas and petechiae, arrows).
PCSK9 indicates proprotein convertase subtilisin kexin 9.

were euthanized, and intravascular washout was performed by intracardiac perfusion of heparinized saline. The brain was removed and
cut into 1-mm coronal slices using a brain matrix mold for evaluation of infarct volume and HT. HT was macroscopically scored on
coronal brain slices by 3 independent observers blinded to the group
status and confirmed by Masson trichrome staining. Hemoglobin was
measured to assess HT in homogenates of ischemic brain tissue quantitatively (Hemoglobin Mouse ELISA Kit-Abcam). Infarct volume
was determined using Image J software from coronal brain sections
stained with 2,3,5-triphenyltetrazolium chloride.7
In vitro, a cell model of bloodbrain barrier, consisting of human
cerebral microvascular endothelial cells/D3 donated by P.O. Couraud
(Institut Cochin, Paris, France), was used to test the endothelial barrier integrity in response to decreasing concentrations of LDL-C.
Bloodbrain barrier integrity was measured as transendothelial
electric resistance using the xCELLigence system (Roche, Basel,
Switzerland) as follows: cells were seeded at 15103 cells per well
onto E-plates coated with collagen I. When transendothelial electric
resistance reached a maximal plateau, the confluent cell monolayer
was incubated with decreasing concentrations of LDL-C (isolated
from human plasma), from 1 to 0.25 g/L, for 24 hours and subjected to oxygen-glucose deprivation for 4 hours. Glucose and oxygen
were then resupplied to the cells for 20 hours to mimic reperfusion.
Changes in transendothelial electric resistance are attributed to resistance variations because of modifications of paracellular junction
tightness. Kinetics of transendothelial electric resistance were displayed as Cell Index (arbitrary units).
Data were analyzed using a MannWhitney test, and P values were
2-sided, with a significance level of 0.05. Results are expressed as
medians (minmax).

Results
As anticipated,1 PCSK9/ mice had lower LDL-C, high-density lipoprotein-cholesterol, and total cholesterol levels than
PCSK9+/+ mice before (not shown) and after 1 month on the

high-fat/high-cholesterol diet (Figure1). Transient intraluminal MCAO was performed on 15 PCSK9/ and 15 PCSK9+/+
mice. One mouse of each genotype died overnight after cerebral ischemia. Twenty-four hours after MCAO, hemoglobin
concentration in ischemic cerebral tissue was not different
between PCSK9/ and PCSK9+/+ mice (31.5 [18.960.1]
and 32.8 [14.769.9] ng/mg protein, respectively; P=0.81;
Figure2). Macroscopic and histological (Masson trichrome)
qualitative evaluations of HT by blinded multiple-observer
analysis confirmed this observation. Infarct volumes were also
similar in PCSK9/ and PSCK9+/+ mice (0.86 [0.701.07] and
0.90 [0.591.26] cm3, respectively; P=0.66).
In vitro, the transendothelial electric resistance of the
human cerebral microvascular endothelial cells/D3 monolayer
was unaffected by decreasing LDL-C concentrations under
oxygen-glucose deprivation conditions (Figure3).

Discussion
There are some inherent limitations to the experimental models used in the present study. Notably, mice are primarily an
high-density lipoprotein-cholesterol species, whereas humans
are LDL predominates. We have limited this important bias
by feeding the animals a high-fat/high-cholesterol diet to
increase their LDL-C levels by 100%. Another important limitation is that PCSK9 gene invalidation does not mimic the
transient pharmacological inhibition of circulating PCSK9
in vivo. PCSK9 knockout mice may, for instance, adapt to
the permanent lack of PCSK9 in a way that may affect the
cell response to ischemia. Unfortunately, specific antimouse
PCSK9 monoclonal inhibitors are not commercially available.

Figure 2. Plasma lipids of PCSK9/ and

PCSK9+/+ mice after a high fat/high cholesterol diet. A, Low-density lipoproteincholesterol (LDL-C) levels in plasma (g/L).
B, LDL-C, high-density lipoprotein, and
total cholesterol levels in plasma (g/L).
Results are medians [minmax] and P
values. HDL-C indicates high-density
lipoprotein-cholesterol; LDL-C, low-density
lipoprotein-cholesterol; PCSK9 indicates
proprotein convertase subtilisin kexin 9; TC,
total cholesterol; and TTC, triphenyltetrazolium chloride.

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3088StrokeOctober 2014
14

Control
LDL-C 0.25 mg/ml
LDL-C 0.5 mg/ml
LDL-C 1 mg/ml

12

OGD + LDL

Cell Index

10
8

Reperfusion

6
4
2
0

13

26

39

52

65

78

91

104

117

Time (hours)
Figure 3. Effect of low-density lipoprotein (LDL) concentration on bloodbrain barrier (BBB) permeability under oxygen-glucose deprivation (OGD)/reperfusion conditions. Human cerebral endothelial cells were treated with decreasing concentrations of LDL. Plots show Cell
Index (CI). Results are mean CI valuesSEM from 3 independent experiments performed in duplicate.

In addition, this experimental model does not account for the

cumulative LDL-lowering effects resulting from the combined inhibition of PCSK9 by monoclonal antibodies and of
hydroxymethylglutaryl-CoA reductase (HMGCoA) reductase by statins. Transient MCAO procedure is a clinically
relevant model to study HT after cerebral ischemia/reperfusion that has been previously validated in rats.8 The blood
brain barrier human cerebral microvascular endothelial cells/
D3 monolayer permeability model has also been validated in
our laboratory9 and is appropriate to assess the functionality
of brain endothelium.10 Thus, the low circulating lipid levels
of PCSK9 knockout mice do not aggravate HT after MCAO.
Likewise, low levels of LDL-C do not alter bloodbrain barrier permeability. Concentrations of LDL-C from 1 to 0.25
mg/dL are in the same range as those observed in patients
treated with PCSK9 inhibitors. Despite the inherent limitations mentioned above, our observations suggest that PCSK9
inhibition leading to LDL-C lowering should not increase
hemorrhagic complications after acute ischemic stroke. The
effects of PCSK9 inhibitors alone or in combination with
statins on the risk of HT after ischemic stroke should, however, be carefully monitored in the large outcome clinical trials currently underway.

Acknowledgments
We thank Olivier Thibaudeau (UMR Inserm 1152, Plateau de
Morphologie) for histology.

Sources of Funding
This research was supported by the Agence Nationale de la Recherche:
Project Grant ANR-JCJC1105 to O Meilhac and Project Grant ANRBlanc-Bnfices Thrapeutiques d'une Nouvelle Cible Thrapeutique
hypolipmiante (BCNCT) to G. Lambert.

Disclosures
Dr Lambert has received honoraria and research funding from
Sanofi-Regeneron, Amgen, and Pfizer. Dr Amarenco has received

consulting fees from AstraZeneca, Bristol-Myers Squibb, Daiichi,

Eli Lilly, GlaxoSmithKline, Guerbet, Negma, Novartis, Pfizer,
Sankyo, Sanofi-Aventis, and Servier; lecture fees from AstraZeneca
Bayer, Boehringer-Ingelheim, Bristol-Myers Squibb, Merck, Otsuka
Pharmaceutical, Pfizer, Sanofi-Aventis, and Servier and grant support
from Boehringer-Ingelheim, Bristol-Myers Squibb, Eisai, Merck,
Astra-Zeneca, Pfizer, and Sanofi-Aventis. The other authors report no
conflicts.

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Low Levels of Low-Density Lipoprotein-C Associated With Proprotein Convertase

Subtilisin Kexin 9 Inhibition Do Not Increase the Risk of Hemorrhagic Transformation
Alexy Tran-Dinh, Anglique Levoye, Gilles Lambert, Liliane Louedec, Clment Journ, Olivier
Meilhac and Pierre Amarenco
Stroke. 2014;45:3086-3088; originally published online August 14, 2014;
doi: 10.1161/STROKEAHA.114.005958
Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright 2014 American Heart Association, Inc. All rights reserved.
Print ISSN: 0039-2499. Online ISSN: 1524-4628

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