Original Article Protein Z-deficiency is associated with enhanced neointima formation and inflammatory response after vascular injury in mice

Abstract

Background:
Protein Z (PZ) is a vitamin K-dependent coagulation factor without catalytic activity. Evidence points towards PZ as an independent risk factor for the occurrence of human atherosclerotic vascular diseases. The aim of this study was to investigate the role of PZ in vascular arterial disease.

Material and methods:
PZ-deficient (PZ(-/-)) mice and their wild-type littermates (PZ(+/+)) were subjected to unilateral carotid artery injury by using ferric chloride and dissected 21 days thereafter for histological analysis. Human aortic smooth muscle cells (SMC) were used for in vitro wound healing assay to assess the influence of PZ on SMC migration and for cell proliferation studies.

Results:
Morphometric analysis of neointima formation revealed a significantly increased area and thickness of the neointima and subsequently increased luminal stenosis in carotid arteries of PZ(-/-) mice compared to PZ(+/+) mice (p < 0.05, n = 9). Immunohistochemical analysis of neointima lesion composition revealed significantly higher numbers of PCNA-positive and α-SMA-positive cells in the neointima of PZ(-/-) mice. Furthermore, PZ showed an anti-migratory potency in in vitro wound healing assay with SMCs, while no effect of PZ on SMC proliferation was detectable. Conclusion: PZ contributes to a reduced neointima formation after vascular injury, underlining the modulatory role of the coagulation cascade in vascular homeostasis.

Full text

Int J Clin Exp Pathol 2014;7(9):6064-6071
www.ijcep.com /ISSN:1936-2625/IJCEP0001572
Original Article
Protein Z-deciency is associated with enhanced
neointima formation and inammatory
response after vascular
injury in mice
Antje Butschkau1, Nana-Maria Wagner2, Laura Bierhansl2, Berit Genz1, Brigitte Vollmar1
1Institute for Experimental Surgery, Rostock University Medical Center, Rostock, Germany; 2Clinic for Anesthesiology
and Critical Care Medicine, Rostock University Medical Center, Rostock, Germany
Rceived July 28, 2014; Acepted August 23, 2014; Epub August 15, 2014; Published September 1, 2014
Abstract: Background: Protein Z (PZ) is a vitamin K-dependent coagulation factor without catalytic activity. Evidence
points towards PZ as an independent risk factor for the occurrence of human atherosclerotic vascular diseases. The
aim of this study was to investigate the role of PZ in vascular arterial disease. Material and methods: PZ-decient
(PZ-/-) mice and their wild-type littermates (PZ+/+) were subjected to unilateral carotid artery injury by using ferric chlo-
ride and dissected 21 days thereafter for histological analysis. Human aortic smooth muscle cells (SMC) were used
for in vitro wound healing assay to assess the inuence of PZ on SMC migration and for cell proliferation studies.
Results: Morphometric analysis of neointima formation revealed a signicantly increased area and thickness of the
neointima and subsequently increased luminal stenosis in carotid arteries of PZ-/- mice compared to PZ+/+ mice (p <
0.05, n = 9). Immunohistochemical analysis of neointima lesion composition revealed signicantly higher numbers
of PCNA-positive and α-SMA-positive cells in the neointima of PZ-/- mice. Furthermore, PZ showed an anti-migratory
potency in in vitro wound healing assay with SMCs, while no effect of PZ on SMC proliferation was detectable.
Conlusion: PZ contributes to a reduced neointima formation after vascular injury, underlining the modulatory role of
the coagulation cascade in vascular homeostasis.
Keywords: Atherosclerosis, critical leg ischemia, thrombosis, coagulation, smooth muscle cells
Introduction
Despite major advances in vascular medicine
over the past decades, cardiovascular diseas-
es remain the main cause of morbidity and
mortality in the Western world [1, 2].
Atherosclerosis constitutes the underlying
pathology of coronary artery disease and poses
the main contributor to death caused by cardi-
ac diseases [3]. The important role of platelets
in atherotrombotic disease has become evi-
dent from many studies investigating platelet
inhibition. However, the impact of coagulation
components and the crosstalk of coagulation
and inammation in atherosclerotic vascular
disease are less well established [4]. Data from
animal models investigating the effects of
hypercoagulability on atherosclerotic disease
revealed aggravated atherogenesis in different
hypercoagulable genotypes such as factor V
Leiden mutation and protein C (PC) deciency
[5].
Human Protein Z (PZ) is a 62 kDa vitamin
K-dependent coagulation glycoprotein identi-
ed in human plasma in 1984 [6]. PZ is charac-
terized by a structural homology with the other
vitamin K-dependent proteins, factors VII, IX, X
and PC [7], but in contrast to these zymogens,
PZ has no catalytic activity [8]. PZ serves as a
cofactor for the protein Z-dependent protease
inhibitor (ZPI), a serpin of 72 kDa which inhibits
factor Xa [9, 10].
In 2007, So et al. observed an association
between low PZ-levels and both the occurrence
and the severity of peripheral arterial disease
(PAD) in a case-control study, hypothesizing
that PZ is linked to the atherosclerotic process
apart from the acute thrombotic event with a

PZ deciency in vascular injury
6065 Int J Clin Exp Pathol 2014;7(9):6064-6071
strict relationship to arterial risk factors [11]. In
2009 they could conrm these results by
another case-control study, demonstrating
again a signicant association of low PZ-levels
with the occurrence and severity of PAD [12].
Therefore, the aim of this study was to assess
the role for PZ in arterial vascular diseases, by
using an in vivo model of vascular injury in mice
decient for PZ and their wild-type littermates
as well as established in vitro assays.
Material and methods
Mice
The experiments were conducted in accord-
ance with the guidelines for the Care and Use of
Laboratory Animals and the Institutional Animal
Care and Use Committee (Rostock University
Medical Center, Rostock, Germany; reference
number: 7221.3-1-055/13). PZ-decient mice
(PZ-/-) in a C57Bl/6x129 genetic background, as
described by Yin et al. [13], were compared to
their respective wild-type littermates (PZ+/+).
Male mice were used at an age of 3-6 months
and a body weight of 25-30 g.
Genotyping of PZ mice
All animals were genotyped for presence or
absence of PZ by PCR, as described by Yin et al.
[13] using genomic DNA isolated from the tail
tip.
Vascular injury protocol
Mice were anaesthetized by intraperitoneal
injection of ketamine (75 mg/kg bw) and xyla-
zine (5 mg/kg bw) and subjected to carotid
artery injury using 10% ferric chloride as previ-
ously described [14, 15]. Briey, the left carotid
artery was carefully separated from the accom-
panying nerve and vein and any adventitial tis-
sue, which might prevent diffusion of the ferric
chloride solution, was removed by forceps. The
carotid was injured by placing a 0.5-1.0 mm
strip of lter paper soaked in 10% ferric chlo-
ride solution onto the adventitia for 3 min. The
wound was carefully sutured with prolene 6-0
(Ethicon Johnson & Johnson Medical GmbH,
Norderstedt, Germany) and the mice returned
to their cages.
Histology
Three weeks after injury, mice were anesthe-
tized as described above and carefully per-
fused with physiological saline and xed with
phosphate buffered formalin (4%) through the
left ventricle. Several 5 µm thick cross sections
of the carotid artery were done in 200 µm
intervals.
Morphometric analysis of neointima formation
Neointima formation was quantied per speci-
men in hematoxylin-eosin (HE) stained sec-
tions, in particular by assessing neointima
area, thickness and luminal stenosis using
computerized image analysis software (Image-
Pro Plus; Media Cybernetics, Silver Spring, Md.,
USA), as previously described [15]. Thickness
of neointima was measured from the highest
point of the area to the internal elastic lamina.
Luminal stenosis was calculated by substrac-
tion of the neointima area from the area of the
original lumen and is given in %. The results
were averaged for each animal (n = 9 per
group).
Immunhistochemical analysis of neointima
lesion composition
Parafn sections of carotid arteries at 3 weeks
after arterial injury were analyzed for the pres-
ence of α-actin-positive smooth muscle cells
(α-SMA; abcam ab5694) by analysis of the
α-SMA-positive area in the neointima lesion.
Proliferating cells were detected using anti-pro-
liferating cell nuclear antigen (PCNA; abcam
ab29 [PC10]) antibody. PCNA-positive cells
were manually counted and expressed as the
percentage of total cell nuclei within neointima
lesion.
Cell culture
Human aortic smooth muscle cells (SMC) were
purchased from Lonza (Basel, Switzerland).
After thawing, the cells were seeded into 10 cm
cell culture dishes and cultured according to
the supplier’s recommendations in SmGMTM-
2BulletKitTM (Lonza, Basel, Switzerland) supple-
mented with 10% fetal calf serum (FCS), 0.1%
hEGF, 0.1% insulin, 0.2% hFGF-B and 1% peni-
cillin/streptomycin. The cells were placed in a
humidied incubator at 37°C and 5% CO2 and
used from passage 5 to 10.
In vitro wound healing assay
SMC migration was analyzed employing the in
vitro wound scratch assay [16]. The cells were

PZ deciency in vascular injury
6066 Int J Clin Exp Pathol 2014;7(9):6064-6071
cultured in 12-well plates and a cross scratch
wound was created in the center of the cellular
monolayer by gentle removal of the attached
cells with a sterile plastic pipette tip. The cells
were then gently washed with PBS to remove
single not-adherent cells and incubated with PZ
(3 µg/ml; South Bend, IN, USA) or TNF-α (10 ng/
ml; R&D Systems, Minneapolis, MN, USA) in
serum- and growth-factor reduced medium
(containing 1% FCS) in duplicate. Non-
stimulated cells served as control (ctrl). Images
of the cells were taken at 12 h after wound
scratching. The images were captured using a
uorescence microscope (Leica, Germany) and
wound closure was quantied employing imag-
ing software (ImageProPlus Software, CA, USA)
Figure 1. Morphometric analysis of neointima formation in PZ mice. (A) Representative images of HE-stained cross
sections from carotids of PZ+/+ and PZ-/- mice. (B) Quantication of neointima area, (C) neointima thickness and (D)
luminal stenosis in PZ+/+ mice and PZ-/- mice after carotid artery injury using 10% ferric chloride. Data are given as
box plots indicating the median with the 25th and 75th percentiles. Mann-Whitney rank-sum test, *P value of less
than 0.05 versus PZ+/+ mice; n = 9.
Figure 2. Cellular composition of neointima formation in PZ mice. A. Representative images of immunohistochemi-
cal detection of PCNA within vascular lesions of PZ+/+ and PZ-/- mice (brown signal). B. Quantication of PCNA-
positive cells in percent of all cells in the neointimal lesion. PCNA, proliferating cell nuclear antigen; Data are given
as box plots indicating the median with the 25th and 75th percentiles. Mann-Whitney rank-sum test, *P value of less
than 0.05 versus PZ+/+ mice; n = 9. C. Representative images of immunohistochemical detection of α-SMA positive
SMCs within the neointimal lesion (pink signal). D. Quantication of α-SMA positive SMCs within the neointima.
α-SMA, α-smooth muscle actin; Data are given as box plots indicating the median with the 25th and 75th percentiles.
Mann-Whitney rank-sum test, *P value of less than 0.05 versus PZ+/+ mice; n = 9.

PZ deciency in vascular injury
6067 Int J Clin Exp Pathol 2014;7(9):6064-6071
by counting migrated cells into the wound in 8
high power elds per wound.
Cell proliferation studies
Cell proliferation was analyzed by measuring
DNA synthesis with a colorimetric bromodeoxy-
uridine (BrdU) enzyme-linked immunosorbent
assay kit (Roche Diagnostics, Basel, Swi-
tzerland), according to the manufacturer’s
instructions. Briey, 1 × 104 cells were seeded
into a 96-well microplate and starved 5 hours
before stimulation with PZ (3 µg/ml; South
Bend, IN, USA) or TNF-α (50 ng/ml; R&D
Systems, Minneapolis, MN, USA) in serum- and
growth-factor reduced medium (containing 1%
FCS). After 24 hours of stimulation the cells
were then labelled with BrdU labeling reagent
for 10 hours. After xation, the cells were incu-
bated with anti-BrdU antibody for 90 min. After
washing, 100 µl of substrate (tetramethylbenzi-
dine) was added to each well and the plates
were incubated at room temperature for 30
min. The absorbance was measured at 450 nm
Figure 3. Inuence of PZ on migration of SMCs
in wound healing assay in vitro. A. Representa-
tive images of scratch wound closures after 12
hours of incubation with PZ (ctrl corresponds
to untreated cells). 50-fold magnication. B.
Quantication of migration of PZ stimulated
SMCs after 12 hours of stimulation; Data
are given as box plots indicating the median
with the 25th and 75th percentiles. ANOVA on
Ranks, #P value of less than 0.05 versus ctrl,
§p value of less than 0.05 versus TNF-α; n = 6
independent experiments.

PZ deciency in vascular injury
6068 Int J Clin Exp Pathol 2014;7(9):6064-6071
with a multilabel plate reader (VICTOR X, Perkin
Elmer, Waltham, MA, USA).
Statistical analysis
All data are given as median and interquartile
range (IQR; the 25% and 75% percentiles).
Differences between groups were calculated
using Mann-Whitney rank-sum test, followed by
Bonferroni correction. Overall statistical signi-
cance was dended as a P-value of < 0.05. The
statistical power was calculated for each sig-
nicance at α = 0.05 with a level of 80%.
Statistics, power calculation and graphics were
performed using the software packages
SigmaStat software version 3.5 and SigmaPlot
software version 12.5 (Jandel Corporation, San
Rafael, CA, USA).
Results
Murine PZ-deciency is accompanied by in-
creased neointima formation
Focal arterial inammation that results in
remodeling of the vascular wall is an initial step
leading to arterial disease. Using the neointima
lesion model in PZ+/+ and PZ-/- mice we morpho-
metrically analyzed the developed lesions. Area
of the neointima induced by ferric chloride was
signicantl y increased in PZ-/- mice compared to
their wild-type littermates (Figure 1A, 1B), with
the vascular lumen found almost occluded in
PZ-/- mice. Also neointima thickness was signi-
cantly higher in PZ-/- mice with a median of 145
µm compared to 93 µm in PZ+/+ mice (Figure
1A, 1C). As a consequence of increased neo-
intima area and thickness, PZ-/- mice showed a
signicantly higher degree of luminal stenosis
(57% in PZ-/- mice vs. 38% in PZ+/+ mice; P <
0.05; Figure 1A, 1D). Five mice out of 14 PZ-/-
mice died within a few days after induction of
the neointima lesion, while no loss could be
registered in the PZ+/+ mice group, which con-
ceivably underlines the increased susceptibility
proliferation. Within the neointimal lesion PZ-/-
mice exhibited signicantly more PCNA-positive
cells compared to their wild-type littermates
PZ+/+ (21% PZ-/- mice vs. 9% in PZ+/+ mice; P <
0.05; Figure 2A, 2B), indicating an accelerated
proliferative phenotype of PZ-/- mice. Migration
and proliferation of SMCs in response to endo-
thelial activation are thought to be major
pathomechanisms underlying intimal hyperpla-
sia [17]. Immunohistochemical analysis reve-
aled an increased area of α-actin-positive
SMCs in neointimal lesions of PZ-/- mice (medi-
an 37%) vs. PZ+/+ mice (median 21%; Figure 2C,
2D), indicating an increased proliferation of
SMC in PZ-/- mice.
PZ diminishes SMC migration in vitro
To analyze the inuence of PZ on SMC migra-
tion in vitro, a conuent monolayer of cells was
scratched and incubated with PZ or TNF-α and
were compared to unstimulated cells serving
as control. Following 12 hours of incubation
quantication of migrated cells revealed a
trend towards increased migration of cells upon
TNF-α stimulation. SMCs stimulated with PZ
showed signicantly diminished migration com-
pared to TNF-α and ctrl (Figure 3A, 3B).
PZ has no inuence on SMC proliferation in
vitro
Analyzing the proliferation of SMC by measur-
ing BrdU incorporation, there was no signicant
inuence of PZ on SMC proliferation compared
to unstimulated control SMCs (Table 1). TNF-α
stimulated SMCs showed a slight increase of
proliferation.
Discussion
In the present model, placement of FeCl3
soaked lter paper on a very small section of
the carotid artery induced vascular injury with
denudation of the inner vascular wall, resulting
in vascular healing processes with an inam-
Table 1. Inuence of PZ on SMC proliferation in vitro
ctrl PZ TNF-α
Median (IQR) Median (IQR) Median (IQR)
OD x-fold vs. ctrl 1.0 (1.0-1.0) 1.1 (1.1-1.2) 1.2 (1.2-1.3)
Data are given as median and IQR. ANOVA on Ranks; n = 3 independ-
ent experiments. Abbreviations: Ctrl, control; OD, optical density; PZ,
protein Z; TNF-α, tumor necrosis factor alpha; IQR, interquartile range.
of PZ-/- mice to arterial vascular disease-
related complications (data not shown).
Cellular composition of neointima for-
mation reects an increased inamma-
tory response in PZ-/- mice
In order to analyse arterial lesion com-
position carotids were stained for PCNA,
which is an established marker for cell

PZ deciency in vascular injury
6069 Int J Clin Exp Pathol 2014;7(9):6064-6071
matory component. Employing this model in
PZ-decient mice for the rst time, our results
contribute to the so far incompletely under-
stood function of PZ in vascular homeostasis
and disease.
The potential contribution of coagulation pro-
teins to the process of atherosclerosis and
thrombosis is so far incompletely understood
[5]. Regarding animal studies analyzing the
impact of mechanisms of the coagulation cas-
cade on atherosclerosis, Loeffen et al. conclud-
ed that although there were variations in ani-
mal age, diet and atherosclerosis model in
these studies, overall hypercoagulability on an
atherogenic background increased the devel-
opment and progression of atherosclerosis [5].
One of these studied coagulation factors is PC,
a functional homologue of PZ [7]. PC+/- mice
subjected to copper/silicona arterial cuff dis-
played enhanced focal arterial inammation
and thrombosis, leading to larger neointima for-
mation and subsequent localized occlusion, as
compared to their WT counterparts [18]. Zorio
et al. found a signicant association between
low circulating APC levels and the extent and
severity of coronary atherosclerosis, which
might be related to the anticoagulatory and
anti-inammatory properties of APC [19]. It has
also been reported that the presence of athero-
sclerotic plaques decreased the expression of
thrombomodulin and endothelial PC receptor
by endothelial cells [20]. Further, the activity of
the PC anticoagulant pathway may be impaired
in atherosclerotic arteries because of the com-
bination of decreased HDL-cholesterol and
increased LDL-cholesterol [21]. Thus, it is most
likely that the benecial effect of PC in vascular
disease is due to a combination of anti-coagu-
lant and anti-inammatory properties. The
strength of PC appears to be attributed to its
capacity to restore the regulation of coagula-
tion and inammation at the endothelial site
[22].
With respect to the structural homology of PZ
and PC, comparable mechanisms may underlie
the action prole of PZ, though not studied yet
in full detail. In the current study PZ-deciency
is accompanied by increased neointima forma-
tion and addition of PZ to wound scratched
SMCs limited cell migration. In an earlier study
of our group it was shown, that murine
PZ-deciency in endotoxin-induced generalized
Shwartzman reaction, displaying disseminated
intravascular coagulation, is accompanied by
an increased inammatory response as reect-
ed by excessive cy tokine release and inltra-
tion of inammatory cells into tissue [23].
Cesari et al. reported a strong positive correla-
tion between PZ and interleukin-6 at baseline
and three months after an acute coronary
artery event and concluded that PZ may be an
acute phase marker with a longer latency time
[24]. This strengthens a possible role of PZ in
inammation-related atherosclerotic diseases.
Further on, PZ was found histologically in
human macrovascular endothelial cells of
arteries from healthy and atherosclerotic
patients, where the proliferating subendothelial
space in atherosclerotic vascular lesions
showed signicant immunopositivity for PZ
[25]. It is not clear so far, whether PZ repre-
sents a causal role in these atherosclerotic
lesions or is rather a contributor to the local
wound healing response. As PZ was only immu-
nologically detected, the positivity of PZ could
be either due to PZ biosynthesis by endothelial
cells, or due to PZ antigen deposition. Due to
the fact that in the current study PZ revealed
anti-migratory potency on SMCs in vitro and
that PZ-/- mice showed increased neointima for-
mation, it can be hypothesized that PZ has
probably a compensatory function at the vascu-
lar wall in this model of vascular injury. Other
groups also found PZ immunolocalized in the
endothelium of arterial and venous vessel sec-
tions, which implies the binding of PZ to a pos-
tulated, but so far not identied endothelial
receptor [26] and points towards a paracrine
function of PZ independent of its function in the
coagulation cascade.
Thus, based on the observations of this study,
it is obvious that PZ activity is of importance in
the development of neointimal lesions and con-
tributes to vascular healing by coagulation-
independent pathways. The further underlying
mechanisms of the contribution of PZ in arterial
vascular disease remain to be elucidated.
Acknowledgements
This work was supported by a grant of the
Deutsche Forschungsgemeinschaft (VO 450/
11-1).
Disclosure of conict of interest
None.

PZ deciency in vascular injury
6070 Int J Clin Exp Pathol 2014;7(9):6064-6071
Address correspondence to: Antje Butschkau,
Institute for Experimental Surgery, Rostock
University Medical Center, Schillingallee 69a,
Rostock D-18057, Germany. Tel: +49381-4942504;
Fax: +49381-4942502; E-mail: antje.butschkau@
uni-rostock.de
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