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J. Exp. Med. Vol. 208 No. 1 53-66
Atherosclerosis is a disease of the vasculature
that is characterized by chronic inflammation
of the arterial wall (Hansson and Libby, 2006).
The development of atherosclerosis is initiated
by the activation of endothelial cells (ECs)
leading to expression of adhesion molecules for
inflammatory cells (Berk, 2008). In addition,
these activated ECs facilitate the passage of
lipid components in the plasma, such as low-
density lipoproteins (LDLs; Hansson, 2005).
A critical element in the progression of athero-
sclerosis is the development of an oxidizing
environment caused by the activation of mac-
rophages that become loaded with oxidized
LDL and other lipids. These macrophages
produce reactive oxygen species (ROS) and
secrete cytokines and growth factors that con-
tribute to the progression of atherosclerotic
plaques and promote vulnerable lesions (Weber
et al., 2008).
Cyclophilin A (CyPA) is a ubiquitously
distributed protein belonging to the immuno-
philin family recognized as the intracellular re-
ceptor for the potent immunosuppressive drug
cyclosporine A (CsA; Handschumacher et al.,
1984). CyPA possesses peptidyl-prolyl isom-
erase activity and plays an important role in
protein folding and trafficking (e.g., nuclear
translocation of ERK1/2 [Pan et al., 2008] and
AIF [apoptosis-inducing factor; Zhu et al.,
2007]). Interestingly, it has been shown that
CyPA is a part of a cytosolic trafficking com-
plex consisting of caveolin, heat-shock pro-
tein 56, cyclophilin 40, CyPA, and cholesterol
(Uittenbogaard et al., 1998). Although CyPA
was initially believed to function primarily as
an intracellular protein, recent studies have
revealed that it can be secreted by cells in
Bradford C. Berk:
Abbreviations used: BAEC,
bovine aortic EC; CHX, cyclo-
heximide; CsA, cyclosporine A;
CyPA, cyclophilin A; DCF,
dihydroethidium; EC, endothe-
lial cell; eNOS, endothelial NO
synthase; HUVEC, human
umbilical vein EC; LDL, low-
density lipoprotein; mRNA,
messenger RNA; NO, nitric
oxide; PI, propidium iodide;
ROS, reactive oxygen species;
siRNA, small interfering RNA;
TUNEL, terminal deoxynucle-
otidyl transferase dUTP nick
end labeling; VSMC, vascular
smooth muscle cell.
P. Nigro and K. Satoh contributed equally to this paper.
Cyclophilin A is an inflammatory mediator
that promotes atherosclerosis
in apolipoprotein E–deficient mice
Patrizia Nigro,1 Kimio Satoh,1,3 Michael R. O’Dell,1 Nwe Nwe Soe,1
Zhaoqiang Cui,1 Amy Mohan,1 Jun-ichi Abe,1 Jeffrey D. Alexis,1
Janet D. Sparks,2 and Bradford C. Berk1
1Aab Cardiovascular Research Institute, Department of Medicine and 2Department of Pathology and Laboratory Medicine,
University of Rochester School of Medicine and Dentistry, Rochester, NY 14642
3Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Aoba-ku, Sendai 980-8574, Japan
Cyclophilin A (CyPA; encoded by Ppia) is a ubiquitously expressed protein secreted in
response to inflammatory stimuli. CyPA stimulates vascular smooth muscle cell migration
and proliferation, endothelial cell adhesion molecule expression, and inflammatory cell
chemotaxis. Given these activities, we hypothesized that CyPA would promote athero
sclerosis. Apolipoprotein E–deficient (Apoe/) mice fed a highcholesterol diet for 16 wk
developed more severe atherosclerosis compared with Apoe/Ppia/ mice. Moreover,
CyPA deficiency was associated with decreased lowdensity lipoprotein uptake, VCAM1
(vascular cell adhesion molecule 1) expression, apoptosis, and increased eNOS (endothelial
nitric oxide synthase) expression. To understand the vascular role of CyPA in atherosclerosis
development, bone marrow (BM) cell transplantation was performed. Atherosclerosis was
greater in Apoe/ mice compared with Apoe/Ppia/ mice after reconstitution with
CyPA+/+ BM cells, indicating that vascularderived CyPA plays a crucial role in the progres
sion of atherosclerosis. These data define a role for CyPA in atherosclerosis and suggest
CyPA as a target for cardiovascular therapies.
© 2011 Nigro et al. This article is distributed under the terms of an Attribu-
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The Journal of Experimental Medicine
Cyclophilin A contributes to atherosclerosis | Nigro et al.
Atherosclerosis development is dependent on CyPA
To study the functional role of CyPA in atherogenesis, we
used the Apoe/ mouse, a well-known model of atheroscle-
rosis (Nakashima et al., 1994). We generated Apoe/Ppia/
(double knockout) mice, and fed them a high-cholesterol diet
for 16 wk. To visualize lipid-rich atherosclerotic plaques,
aortas were stained with Oil red O. As shown in Fig. 1 A,
Apoe/Ppia/ mice compared with Apoe/ mice exhibited
significantly less atherosclerosis: aortic coverage of 7.5 ± 2% in
Apoe/Ppia/ versus 19.3 ± 8.2% in Apoe/ (Fig. 1 B).
In another cohort of mice, we quantified plaque area in
hematoxylin- and eosin (H&E)-stained cross sections of the
aortic arch and thoracic aorta. Lesion area was significantly
decreased in both the aortic arch (Fig. 1, C and D) and tho-
racic aorta (Fig. 1, C and E) of Apoe/Ppia/ mice com-
pared with Apoe/ mice. As expected, the elastic lamina was
frequently degraded with large regions exhibiting disruption
in Apoe/ mice compared with Apoe/Ppia/ mice (Fig. S1).
These results demonstrate a remarkable reduction in athero-
sclerosis in CyPA-deficient mice and strongly support our
hypothesis that CyPA contributes to atherosclerosis.
The absence of CyPA decreases the lesion area
and the migration of macrophages in the aortic sinus
The aortic sinus is particularly prone to intimal lesion develop-
ment, and the cusps of the valves provide a useful positional cue
in comparative experiments of sectioned tissues
(Tangirala et al., 1995). As shown in Fig. 2
(A and B), Apoe/Ppia/ mice compared with
Apoe/ mice exhibited significantly less athero-
sclerosis measured by plaque area (H&E staining).
There was also significantly less lipid deposition
and cholesterol clefts in Apoe/Ppia/ mice
(Fig. 2 A, Masson and Trichrome and Oil red
O). Both H&E and Masson and Trichrome
showed a significant reduction in intima forma-
tion in Apoe/Ppia/ mice (Fig. 2 A).
A crucial step in atherogenesis is the infil-
tration of monocytes into the subendothelial
space of large arteries where they differentiate
response to inflammatory stimuli, especially ROS (Jin et al.,
2000; Suzuki et al., 2006; Satoh et al., 2009). Extracellular
CyPA is a potent leukocyte chemoattractant for human
monocytes, neutrophils, eosinophils, and T cells (Sherry et al.,
1992; Xu et al., 1992; Allain et al., 2002; Yurchenko et al.,
2002; Arora et al., 2005; Damsker et al., 2007; Pan et al., 2008),
and it stimulates inflammatory responses when injected
in vivo (Sherry et al., 1992). Most importantly, plasma CyPA
is significantly increased in patients with inflammatory dis-
eases such as rheumatoid arthritis (Kim et al., 2005) and sepsis
(Tegeder et al., 1997). We have shown that ROS promote
secretion of CyPA from vascular smooth muscle cells
(VSMCs; Jin et al., 2000; Liao et al., 2000) and that extracel-
lular CyPA stimulates EC adhesion molecule expression in
vitro (Jin et al., 2004; Suzuki et al., 2006). Furthermore, we
found that CyPA mediates vascular remodeling by promot-
ing inflammation and VSMC proliferation (Satoh et al.,
2008), and it is indispensable for the development of an-
giotensin II–induced aortic aneurysms (Satoh et al., 2009;
Given these functions of CyPA, we hypothesized that
CyPA would contribute to the development of atherosclero-
sis. In this study, we report that CyPA is atherogenic by
enhancing LDL uptake, adhesion molecule expression, and
inflammatory cell migration. Our data suggest that CyPA
and its signaling pathways are novel targets for atheroscle-
Figure 1. CyPA deficiency limits atherosclerosis
formation. (A) Representative photographs showing
Oil red O staining of aortas from Apoe/ and
Apoe/Ppia/ mice fed a high-cholesterol diet for
16 wk. (B) Lesion area was significantly decreased in
Apoe/Ppia/ mice (n = 15) compared with Apoe/
mice (n = 21). (C) Longitudinal cross sections from the
aortic arch and thoracic aorta stained with H&E. Bars:
(left) 25 µm; (right) 200 µm. (D and E) Quantification of
plaque area showed that Apoe/Ppia/ mice (n = 7)
exhibited decreased atherosclerosis compared with
Apoe/ mice (n = 9) in both the aortic arch (D) and
thoracic aorta (E). (B, D, and E) Results are mean ± SD;
*, P < 0.01 compared with Apoe/ mice. Results in A–E
show pooled data from two experiments.
JEM VOL. 208, January 17, 2011
radioactivity as measured by a gamma counter. As shown in
Fig. S3, LDL uptake was significantly reduced in aortas
from Apoe/Ppia/compared with Apoe/ mice. To gain
insight into how CyPA regulates lipid uptake, we examined
expression of several scavenger receptors in aortas after
12 wk of high-cholesterol diet. The expression of lectin-
like oxidized LDL receptor (LOX-1) and CD36 were sig-
nificantly decreased in Apoe/Ppia/ mice compared with
Apoe/ mice, whereas SR-BI was decreased by an equiva-
lent 40% (Fig. 3, C and D). The expression of SR-A did not
differ significantly between Apoe/ and Apoe/Ppia/
mice (Fig. 3, C and D). Collectively, the data demonstrate
that CyPA influences LDL uptake by regulating the expres-
sion of scavenger receptors on the vessel wall.
CyPA deficiency induces the development of a more
proatherogenic lipoprotein profile in Apoe/ mice
Next, we performed phenotypic characterization of Apoe/
and Apoe/Ppia/ mice fed a high-cholesterol diet for
16 wk. Body weights were similar in all the groups of mice
before and after a high-cholesterol diet (Fig. S4 A). There
were no significant differences in plasma cholesterol and tri-
glyceride levels (Fig. S4, B and C). We next examined lipo-
protein profiles by gel filtration chromatography (Fig. S4 D).
into macrophages and become functionally active (Galkina and
Ley, 2009). As shown in Fig. 2 (A and D), the Apoe/Ppia/
mice fed high-cholesterol diet for 16 wk showed significantly
fewer Mac3-positive macrophages compared with the Apoe/
mice. All these data suggest that CyPA is a key protein in-
volved in the atherosclerosis progression and migration of in-
flammatory cells in the lesion area.
CyPA regulates LDL infiltration and the expression
of scavenger receptors
Because it is well established that the transport of LDL cho-
lesterol into the artery wall is the initiating event that trig-
gers atherosclerosis (Glass and Witztum, 2001), we examined
the role of CyPA in this process. To measure LDL uptake
into vessels of Apoe/ and Apoe/Ppia/ mice, we per-
formed ex vivo incubation with fluorescent DiI-labeled
LDLs and en face imaging of the aorta from the EC surface
to a depth of 50 µm into the intima. CyPA deficiency
caused a 43% decrease in DiI-LDL uptake in the lesser cur-
vature of the aortic arch (atherosclerosis-prone area; Fig. 3,
A and B; and Fig. S2). To strengthen this important finding,
we confirmed these data with an independent method.
Apoe/ and Apoe/Ppia/ aortas were incubated with
[125I]LDL for a subsequent determination of incorporated
Figure 2. CyPA deficiency reduces lesion size and inflammatory cell accumulation in the aortic sinus. (A) Representative histological analysis of
the aortic sinus stained with H&E, Masson and Trichrome, Oil red O, Mac3 (a macrophage marker), and -smooth muscle cell actin (-SMA). Insets are
higher magnification images of the areas in the dashed boxes. Bars, 100 µm. (B–D) Quantification of the plaque area (B), lipid accumulation (C), and
macrophage accumulation (D) shows a significant reduction in Apoe/Ppia/ mice (n = 7) compared with Apoe/ (n = 9) mice. Results are mean ± SD;
*, P < 0.01 compared with Apoe/ mice. Results in A–D show pooled data from two experiments.
Cyclophilin A contributes to atherosclerosis | Nigro et al.
To determine the role of BM-derived CyPA in atherosclero-
sis, BM transplantation experiments were performed.
BM cells from Apoe/ and Apoe/Ppia/ mice were
transplanted into 6-wk-old lethally irradiated Apoe/ mice.
After 4 wk of reconstitution, mice were fed with a high-
cholesterol diet for 12 wk. Surprisingly, there were no differ-
ences in atherosclerosis measured by lesion area in the entire
aorta (Fig. 4, A and B; Oil red O) or aortic root (Fig. 4,
C and D; H&E). These data demonstrate that atherosclerosis
in Apoe/Ppia/ mice was not altered by specific CyPA de-
ficiency in BM-derived cells. Note that the atherosclerosis
lesion area was reduced by 50% in Apoe/ mice after irra-
diation (compare Fig. 4 B with Fig. 1 B).
We next assessed the development of high-cholesterol–
induced atherosclerosis in Apoe/Ppia/ chimeric mice
that were transplanted with Apoe/ BM or Apoe/Ppia/
BM. There was no significant difference in atherosclerosis
lesions between the chimeric mice with Apoe/ BM versus
Apoe/Ppia/ BM (unpublished data).
Although there were no differences observed in cholesterol
and triglyceride levels, Apoe/Ppia/ mice demonstrated
increases in the VLDL (very low-density lipoprotein)- and
IDL (intermediate-density lipoprotein)/LDL-sized lipopro-
teins compared with Apoe/ mice. No differences were ob-
served in the HDL (high-density lipoprotein) fraction or in
the levels of plasma apoB-100 and apoB-48 (Fig. S4 E).
Collectively, these results indicate that loss of CyPA, in the
context of the Apoe/ genetic background, leads to the ap-
pearance of a more proatherogenic lipoprotein profile.
BMderived CyPA does not play a significant role
in atherosclerosis formation
CyPA has important roles in the immune system and is a well-
described regulator of T lymphocyte functions (Colgan et al.,
2004). Extracellular CyPA is a potent chemoattractant for in-
flammatory cells (Xu et al., 1992; Allain et al., 2002; Yurchenko
et al., 2002). CyPA has also been reported to stimulate migra-
tion of BM-derived cells in vitro (Khromykh et al., 2007).
Figure 3. CyPA regulates LDL entry into the aortic wall and the expression of the scavenger receptors. (A) En face fluorescence images of
aortic arches from 6-wk-old Apoe/ and Apoe/Ppia/ mice (n = 3 each group) after incubation with DiI–labeled LDL (red) and SYTOX green nucleic
acid stain (green). (B) Quantification of DiI-LDL fluorescence intensity from the en face images. The data are quantified as fluorescence-positive area.
(C) Western blot analyses of aortic extracts from Apoe/ and Apoe/Ppia/ mice fed a high-cholesterol diet for 12 wk. Results for three representative
mice are shown for each genotype. (D) Densitometric analyses of the blots in C show significant decreases in LOX-1, CD36, and SR-BI scavenger receptors
in Apoe/Ppia/ mice versus Apoe/ mice. (B and D) Results are mean ± SD; *, P < 0.01 compared with Apoe/ mice. Results in A–D show pooled
data from two experiments.
JEM VOL. 208, January 17, 2011
much greater in the Apoe/ recipient mice (Fig. 4 E, left)
compared with Apoe/Ppia/ recipient mice (Fig. 4 E,
right) after high-cholesterol diet. High-magnification im-
ages (Fig. 4 G, top) showed the presence of enlarged foam
cells in the Apoe/ mice with GFP+ BM (arrows), which
is consistent with the concept that BM-derived cells dif-
ferentiate into foam cells upon exposure to hyperlipidemia.
Direct demonstration that the BM-derived GFP+ cells
were localized beneath the ECs (and not just adherent to
the luminal surface) was obtained by examination of cross-
sectional images of z-series stacks (Fig. 4 G, bottom). These
results suggested that CyPA present in BM-derived cells
is less important for recruitment to the sites of atheroscle-
rosis lesions than CyPA present in vessels and other non-
To investigate the chemoattractive function of CyPA in
vivo, we studied the trafficking of BM-derived cells into
atherosclerotic lesions. To analyze BM cells that migrated
into lesions, we used donor BM cells harvested from mice
constitutively expressing the GFP protein (GFP+ BM which
are Ppia+/+) and performed BM transplantation in lethally
irradiated Apoe/ and Apoe/Ppia/ mice. There was
no significant difference in the reconstitution ratio (per-
centage of GFP+ cells in the peripheral blood) in GFP+ BM-
transplanted Apoe/Ppia/ mice compared with GFP+
BM-transplanted Apoe/ mice. However, there was still a
significant difference in the atherosclerosis lesion area in the
Apoe/ GFP+ BM mice compared with Apoe/Ppia/
GFP+ BM mice (9.6 ± 1.3% vs. 2.3 ± 0.3%), and migra-
tion of GFP+ BM cells into the lesser curvature region was
Figure 4. BMderived CyPA is not crucial in atherosclerosis. (A) Representative Oil red O staining of aortas from Apoe/ mice transplanted
with Apoe/ BM (n = 7) or Apoe/Ppia/ BM (n = 8) and fed a high-cholesterol diet for 12 wk. (B) Quantification of the lesion area shows that
BM transplant of Apoe/Ppia/ cells does not decrease lesion development in Apoe/ mice. (C and D) Representative histological analysis of aortic
sinus stained with H&E (C) and quantification of the aortic root lesion area (D). (B and D) Results are mean ± SD; P > 0.01. (E) Ppia+/+ BM cells (GFP+)
were transplanted into irradiated Apoe/ (n = 4) or Apoe/Ppia/ (n = 4) mice as described in Materials and methods. Representative PECAM-1
en face staining (Alexa Fluor 546; red) and migration of the GFP+ cells in aortic arch from Apoe/ and Apoe/Ppia/ mice with Ppia+/+ BM under
high-cholesterol diet for 12 wk. (F) Number of migrating GFP+ cells was dramatically higher in the aortic wall of Apoe/ (n = 4) compared with
Apoe/Ppia/ mice (n = 4). Results are mean ± SD; *, P < 0.01 compared with Apoe/ mice. (G) En face confocal microscopy of aortic arch from
Apoe/ mice transplanted with GFP+ cells. Top panel shows foam cells, which were identified by large size and diffuse GFP pattern (arrows). Bottom
panel shows the reconstruction in z axis of the images shown in the top panel. GFP+ cells are clearly present in the subendothelial space defined by the
PECAM-1 positive fluorescence above them. Results in A–G show pooled data from two experiments. Bars: (C) 100 µm; (E and G) 10 µm.
Cyclophilin A contributes to atherosclerosis | Nigro et al.
eNOS function is critical for vascular homeostasis via
generation of NO, and its loss is proatherogenic (Knowles
et al., 2000; Chen et al., 2001; Kuhlencordt et al., 2001;
Kawashima and Yokoyama, 2004). Furthermore, the pro-
gression of atherosclerosis is associated with decreases in both
eNOS expression (Oemar et al., 1998; Handa et al., 2008)
and NO production (Li and Förstermann, 2009). Therefore,
we compared eNOS expression in aortas from Apoe/ and
Apoe/Ppia/ mice by en face staining. As shown in Fig. 6 A,
eNOS protein expression was significantly higher in the
Apoe/Ppia/ mice compared with Apoe/ mice. In addi-
tion, the location of eNOS differed, being predominantly
perinuclear in the Apoe/ mice and diffuse (especially mem-
brane associated) in the Apoe/Ppia/ mice. To define the
mechanisms responsible for decreased eNOS expression, we
studied the effect of altering CyPA levels in cultured human
umbilical vein ECs (HUVECs). To increase eNOS expression
and stimulate cellular responses that are atheroprotective,
cells were placed in a cone and plate viscometer, and steady
laminar s-flow at physiological shear stress (12 dyn/cm2; termed
s-flow here) was applied. As shown in Fig. 6 (B and C), s-flow–
mediated induction of eNOS was significantly increased by
CyPA regulates endothelial nitric oxide (NO) synthase
The aforementioned results suggest that the athero protec-
tion observed in the Apoe/Ppia/ was caused by decreased
inflammation mediated by the absence of CyPA. The vascu-
lar endothelium by virtue of its strategic location between the
plasma and the underlying tissue is endowed with a large
array of functions that are vital for the initiation of atheroscle-
rosis. Therefore, we performed an extensive examination of
the endothelium of aortic arch and thoracic aorta by using
PECAM-1 en face staining to visualize ECs. Apoe/Ppia/
mice showed a decreased EC disorganization in both the
aortic segments when compared with Apoe/ mice (Fig. S5,
A and B).
To evaluate further the mechanisms by which CyPA pro-
motes inflammation, we measured VCAM-1 (vascular cell
adhesion molecule 1) expression, which is highly expressed
in activated ECs and promotes atherosclerosis (Cybulsky and
Gimbrone, 1991; Nakashima et al., 1998; Cybulsky et al., 2001).
En face staining (Fig. 5, A and B) of aortic tissues showed
that VCAM-1 expression was significantly reduced in mice
Figure 5. VCAM1 expression is significantly downregulated in Apoe/Ppia/ mice compared with Apoe/ mice. (A) Representative
en face staining for VCAM-1 expression (Alexa Fluor 546; red) in the aortic arch. EC morphology was changed in the atherosclerosis regions where ECs
are stretched and may have lost PECAM-1 staining (Alexa Fluor 488; green) at some cell junctions. Bars, 10 µm. (B) Densitometric analysis of the en face
staining also shows a significant reduction in VCAM-1 expression in Apoe/Ppia/ mice compared with Apoe/ mice (n = 4 each group). Results are
mean ± SD; *, P < 0.01 compared with Apoe/ mice. Results in A and B show pooled data from two experiments.
JEM VOL. 208, January 17, 2011
levels in HUVECs (Fig. 7 F). These
findings illustrate a novel mechanism
by which CyPA promotes atheroscle-
rosis, through suppression of KLF2
expression and consequent repression
of eNOS transcription.
CyPA affects eNOS expression
by a ROSdependent mechanism
ROS are key mediators of signaling pathways that underlie
vascular inflammation in atherogenesis. We have previously
shown that CyPA is a key determinant for ROS generation,
contributing to the onset of vascular inflammation during
aortic aneurism formation (Satoh et al., 2009). Therefore, we
evaluated whether ROS are downstream mediators of CyPA
in the pathophysiologic context of inflammation we ob-
served. We first overexpressed CyPA in ECs, and then we
evaluated the basal level of ROS by dichlorofluorescein
(DCF; Fig. 8 A) and dihydroethidium (DHE; Fig. 8 B)
staining. ROS production was significantly higher in ECs
overexpressing CyPA compared with cells transfected with
the vector control (4.2- and 1.6-fold increase in DCF and
DHE fluorescence, respectively). These data suggest that CyPA
plays a critical role in ROS generation in ECs similar to our
findings in VSMCs (Satoh et al., 2009).
CyPA small interfering RNA (siRNA). Also, CyPA knock-
down in HUVECs increased eNOS promoter activity and
eNOS messenger RNA (mRNA) levels (Fig. 6 D). Parmar
et al. (2006) have shown that the s-flow induction of the KLF2
(Kruppel-like factor 2) transcription factor stimulates eNOS
mRNA expression through the ERK5–MEF2–KLF2 path-
way. Therefore, we examined whether CyPA could regulate
KLF2 expression. As expected, s-flow increased KLF2 pro-
moter activity and KLF2 mRNA levels (Fig. 6 E). These effects
were significantly increased by CyPA knockdown. To fur-
ther substantiate the role of CyPA in the regulation of eNOS
expression, we transfected HUVECs with WT CyPA (CyPA-
WT). Overexpression of CyPA was accompanied by down-
regulation of eNOS at protein (Fig. 7, A and B) and mRNA
levels (Fig. 7 C). Consistent with the expression data, over-
expression of CyPA decreased eNOS promoter activity
in HUVECs (Fig. 7 D). Finally, overexpression of CyPA
decreased KLF2 promoter activity (Fig. 7 E) and mRNA
Figure 6. CyPA deficiency and sflow are
associated with increased eNOS expres
sion. (A) Aortas from 12-wk Apoe/ and
Apoe/Ppia/ mice were harvested for
qualitative analysis of eNOS expression (Alexa
Fluor 546; red) by en face dual staining im-
munofluorescence. PECAM-1 staining (Alexa
Fluor 488; green) was used to identify the EC
layer. Note that these images were obtained
from the thoracic aorta, a region of s-flow.
eNOS protein expression was higher in
Apoe/Ppia/ mice compared with Apoe/
mice aortas (relative fluorescence of 147 ±
18.48 in Apoe/Ppia/ vs. 100 ± 13.61 in
Apoe/; n = 3 each group). Bars, 10 µm.
(B and C) HUVECs were transfected with
CyPA siRNA or scrambled siRNA for 48 h as
described in Materials and methods and then
exposed to s-flow for 24 h. Western blot analysis
shows significantly greater eNOS expression
after depletion of CyPA. (D and E) HUVECs
were treated for 48 h with CyPA siRNA or
scrambled siRNA and then transfected with
eNOS (D) or KLF2 (E) promoter. Cells were
then exposed to s-flow or maintained in
static condition. To evaluate the promoter
function, luciferase activity was measured
after 24 h. To measure eNOS and KLF2 mRNA
levels, a quantitative PCR analysis was per-
formed. (C–E) Results are mean ± SD of three
independent experiments performed in tripli-
cate. *, P < 0.01 versus scrambled siRNA.
Cyclophilin A contributes to atherosclerosis | Nigro et al.
effect was significantly decreased to 6.65 ± 0.07% by using
CyPA siRNA (Fig. 9 A). To better discriminate between
apoptosis and necrosis (sub-G1 cell count might also include
necrotic cells), we performed the double staining with an-
nexin V (FITC) and propidium iodide (PI), followed by flow
cytometry analysis. Representative cytograms in Fig. 9 B
show that cells transfected with scrambled siRNA and treated
with TNF plus CHX increased both the percentage of cells
gated in the top right quadrant (annexin+/PI+; late apoptotic/
necrotic cells) and in the bottom right quadrant (annexin+/
PI; early apoptosis). Transfection of CyPA siRNA sig-
nificantly attenuated this effect. Moreover, ECs lacking
CyPA showed less cleaved caspase-3 expression by Western
blot analysis (Fig. 9 C). Because EC apoptosis has been sug-
gested as an initial step in atherogenesis, we studied EC apop-
tosis in the early stages of the lesion development in mice fed
with a high-cholesterol diet for only 1 mo. As shown in Fig. 9
(D and E), there was a marked decrease in apoptotic ECs
(detected by terminal deoxynucleotidyl transferase dUTP
nick end labeling [TUNEL] staining) in aortic lesions from
Apoe/Ppia/ mice compared with littermate control mice.
These data underline the crucial role of CyPA during the
apoptotic process that is an important early event for the de-
velopment of atherosclerosis.
The major finding of this study is that CyPA deficiency in
vivo decreases atherosclerotic lesion burden in a mouse model
of atherosclerosis. We characterized five important patholog-
ical mechanisms by which vascular CyPA promotes athero-
sclerosis. First, CyPA increases LDL uptake in the vessel wall
by regulating the expression of scavenger receptors. Second,
CyPA increases EC activation and inflammation by increas-
ing VCAM-1 expression. Third, CyPA decreases eNOS ex-
pression through KLF2 transcriptional repression. Fourth,
CyPA is a key determinant for TNF-induced EC apoptosis.
Finally, CyPA stimulates recruitment of inflammatory cells
derived from the BM to the aortic wall. All of these mecha-
nisms, while promoting an environment of oxidative stress,
are likely to contribute to the increased atherosclerosis ob-
served in the Apoe/Ppia+/+ mice.
The decrease in Oil red O staining is consistent with the
decrease of multiple lipid uptake receptors in vessels of
Apoe/Ppia/ mice. It is likely that CyPA regulates scaven-
ger receptor expression by multiple mechanisms. Because
CyPA is a chaperone protein, it may be necessary for appro-
priate folding and/or transport of LOX-1 and CD36 to the
cell membrane. CD36 is mainly expressed in caveolae, and
CyPA is essential for caveolae formation via transport of cho-
lesterol and caveolin-1 (Uittenbogaard et al., 1998; Everson
and Smart, 2001). In addition, both LOX-1 and CD36 are
highly regulated by ROS and cytokines, which are markedly
reduced in the Apoe/Ppia/ mice.
The decrease in VCAM-1 expression and increase in
eNOS expression observed in Apoe/Ppia/ mice likely
contributes to less inflammation. The decrease in VCAM-1
To demonstrate that ROS are key determinants in CyPA-
mediated inflammation, we evaluated whether CyPA de-
creases eNOS expression by a ROS-dependent mechanism.
Both the antioxidants N-acetyl-cysteine and Tiron, by rebal-
ancing excessive ROS production induced by CyPA overex-
pression, reversed the CyPA-mediated inhibition of eNOS
(Fig. 8 C) and KLF2 promoter activity (Fig. 8 D). These data
demonstrated that CyPA induces inflammation through
ROS-dependent mechanisms. Based on these results, we be-
lieve that CyPA, acting as a proinflammatory cytokine, syn-
ergistically augments ROS production, contributing to vascular
inflammation and atherogenesis.
Deletion of CyPA decreases EC apoptosis
To ascertain whether intracellular CyPA may participate in
the EC apoptosis, we knocked down CyPA in bovine aortic
ECs (BAECs) by siRNA and measured hypodiploidia (sub-
G1 cell count) after treatment with TNF plus cycloheximide
(CHX) as apoptogens. TNF promoted 11.95 ± 0.07% cell
death in cells transfected with scrambled siRNA, and this
Figure 7. CyPA overexpression decreases eNOS and KLF2 expres
sion. (A) HUVECs were transfected with CyPA-WT for 24 h and then ex-
posed to static and s-flow condition for a further 24 h. (A–F) Western blot
data and quantitative PCR analysis revealed that CyPA overexpression
inhibited s-flow–induced eNOS protein (A and B), eNOS mRNA (C), and
also KLF2 mRNA (F) levels. HUVECs were cotransfected with CyPA-WT and
with either eNOS (D) or KLF2 (E) promoter. The cells were exposed to static
or s-flow condition, and the luciferase activity was measured after 24 h.
Overexpression of CyPA decreases eNOS and KLF2 promoter activity. Data
are the mean values ± SD of three independent experiments performed in
triplicate. *, P < 0.01 versus control pcDNA3.1.
JEM VOL. 208, January 17, 2011
regulated KLF2 function remains to be elucidated, although
a nuclear function for CyPA has been suggested for CXCR4
signaling (Pan et al., 2008).
An essential role for CyPA in apoptosis has become in-
creasingly apparent. We showed that extracellular CyPA
promoted EC apoptosis in association with increased JNK
(c-Jun N-terminal kinase) and p38 activities (Jin et al., 2004).
In this study, we found that knockdown of CyPA in ECs
reduced TNF-induced apoptosis in vitro, and CyPA defi-
ciency was associated with a marked decrease in EC apoptosis
in early stages of atherosclerosis. A likely mechanism for
CyPA-mediated apoptosis is binding and nuclear transloca-
tion of AIF (Zhu et al., 2007). In addition, the increase in the
vascular oxidative stress requires CyPA (Satoh et al., 2009)
and thereby sensitizes ECs to apoptosis.
CyPA may play an important role in several stages of ath-
erosclerosis. During fatty streak formation, it may play a role
in lipid uptake via its effect on scavenger receptors. In all
may be causally related to increased eNOS expression be-
cause it was previously shown that eNOS can down-regulate
VCAM-1 expression (Kawashima et al., 2001). Consequently,
there will be reduced monocyte adhesion to the endothe-
lium, transmigration into the subendothelial space, and
inflammation. It may be relevant to note that VCAM-1 ex-
pression appears to precede lesion formation (Nakashima et al.,
1998; Cybulsky et al., 2001), suggesting an important role
for CyPA in the initiation of atherosclerotic lesions. More-
over, we found that CyPA decreased eNOS expression by
repressing KLF2 at the transcriptional level. This finding is of
significant interest because KLF2 is a central regulator of EC
biology (Atkins and Jain, 2007), and it has recently been
shown that hemizygous deficiency of KLF2 increased high-
cholesterol diet–induced atherosclerosis in Apoe/ mice
(Atkins et al., 2008). KLF2 also protects ECs from oxidative
stress–mediated injury and subsequent apoptosis (Parmar
et al., 2006). The mechanism by which CyPA negatively
Figure 8. CyPA increases ROS levels in ECs. (A and B) HUVECs were transfected with CyPA-WT and vector control. After 48 h of incubation, ROS
levels were measured by determining DCF (A) and DHE (B) fluorescence. CyPA overexpression significantly increased ROS content in cells. Data are the
representation of three independent experiments performed in triplicate. *, P < 0.01 vs. control pcDNA3.1. (C and D) HUVECs were transfected with
CyPA-WT and vector control. After 24 h, cells were treated with 1 mM N-acetyl-cysteine and 10 mM Tiron. 16 h later, eNOS (C) and KLF2 (D) promoter
activity were measured by luciferase assay. Data are the mean values ± SD of three independent experiments performed in triplicate. *, P < 0.01 versus
Cyclophilin A contributes to atherosclerosis | Nigro et al.
We would like to point out that studies in animals and
humans, although contradictory and not conclusive, have re-
ported that CsA, the immunosuppressive drug that inhibits
CyPA, increases hyperlipidemia and risk of atherosclerosis
(Fernández-Miranda et al., 1998; Ojo, 2006; Kockx et al.,
2010). We also reported that decreasing cholesterol content
in caveolae by CsA is a potentially important pathogenic
mechanism for CsA-induced endothelial dysfunction and
atherosclerosis (Lungu et al., 2004). These findings appear to
contradict our data. However, the diversity of the pathways
affected by CsA does not allow for simple conclusions to be
drawn. CsA also affects many cellular pathways not associated
stages, it may play a role in inflammation by promoting
monocyte adhesion and recruitment as well as contributing
to an oxidative environment. The data from Seizer et al.
(2010) showing that CyPA is secreted from foam cells suggest
an important role in later stages of atherosclerosis. Finally, we
have recently shown an essential role for CyPA for matrix
metalloproteinase activation (Satoh et al., 2009), suggesting
that CyPA may also play an important role in plaque rup-
ture. Altogether, our data suggest that the agents that inhibit
CyPA expression or agents that block CyPA receptors might
be candidates that may regulate atherosclerotic plaque and
Figure 9. CyPA deficiency reduces EC apoptosis in vitro and in vivo. (A) BAECs were transfected with CyPA siRNA or scrambled siRNA for 48 h and
then treated with 10 ng/ml TNF + 10 µg/ml CHX for 6 h. Apoptotic cells were determined by their hypochromic, subdiploid staining profiles (sub-G1 popula-
tion). The data represent the mean ± SD of triplicate samples repeated in three separate experiments. *, P < 0.01 versus scrambled siRNA. (B) Dot-plot dia-
grams of FITC-conjugated annexin V– and PI-stained cells after treatment with TNF + CHX for 3 h. Results are representative of two separate experiments
that gave similar results. (C) Western blot analyses of cleaved caspase-3 in BAECs treated with TNF + CHX for 6 h. Data represent the mean ± SD of three
separate experiments. *, P < 0.01 versus scrambled siRNA. (D) Whole-mount en face dual immunofluorescence staining of Apoe/ and Apoe/Ppia/ mice
aortas after 4 wk of a high-cholesterol diet. Note that these images were obtained from the inner curvature of the aortic arch, which is a region of disturbed
flow. PECAM-1 staining shows polygonal ECs that are not aligned with flow. TUNEL staining shows increased fluorescence in Apoe/ mice. Bars, 10 µm.
(E) Quantitative data show that Apoe/Ppia/ mice aortas have a significant decrease in apoptotic cells in the lesion area. The data are expressed as the
percentage of TUNEL-positive cells per field (four fields per three mice each group). *, P < 0.01 compared with Apoe/ mice. Results are the mean ± SD and
show pooled data from two experiments.
JEM VOL. 208, January 17, 2011
digital camera (SPOT Insight 2; Diagnostic Instruments, Inc.). Vessel areas
and densitometric analysis were measured with Image-Pro Plus software.
En face analysis. Immunofluorescence staining of mouse aortic ECs was
performed as described previously (Won et al., 2007). Aortas were perfused
with PBS followed by 2% paraformaldehyde for 10 min. After fixation, the
aortas were cut in small segments and incubated in blocking buffer contain-
ing 2% BSA. Primary antibody incubations were performed overnight at
4°C. The primary antibodies used were VCAM-1 (1:200; Santa Cruz Bio-
technology, Inc.), eNOS (1:100; BD), and PECAM-1 (1:200; BD). After
washing the aortic segments three times, secondary antibodies were added
and incubated for 1 h. Negative controls included addition of nonimmune
goat or rabbit IgG. After washing, aortic specimens were opened, placed on
a glass slide with the luminal side up, and then mounted for confocal micros-
copy (FLUOVIEW; Olympus).
Ex vivo LDL tissue uptake. Apoe/ and Apoe/Ppia/ mice were
anesthetized and euthanized. Aortas were dissected, cut in small fragments,
and incubated in 50 µg/ml DiI-LDL. After 2 h, aortas were washed with
PBS and fixed using 4% paraformaldehyde. Then, aortic segments were
opened and stained with SYTOX green nucleic acid stain (dilution 1:10,000;
Invitrogen). After washing, the aortic specimens were prepared for en face
analysis by confocal microscopy (FLUOVIEW). Analysis of different images
was performed using Image-Pro Plus software. The results are expressed in
terms of fluorescence-positive areas for each animal (n = 3 each group,
4 fields each animal). Human LDL was isolated by ultracentrifugation (1.019
g/ml < d < 1.063 g/ml) and dialyzed against 0.15 M NaCl/1 mM EDTA
overnight at 4°C using a 50,000 MWCO dialysis membrane. The LDL
(250 µg) was directly iodinated using iodobeads (Thermo Fisher Scientific)
as described by the manufacturer, and unincorporated 125I was removed by
two rounds of gel filtration on PD10 columns (GE Healthcare) using PBS,
pH 7.0, as elution buffer. The 125I-labeled LDL was adjusted to a specific
activity of 500 dpm/ng by addition of unlabeled LDL and then filter steril-
ized using 0.2 µm Acrodisk syringe filter (Pall Life Sciences). More than 98%
of the radioactivity in the [125I]LDL preparation was precipitable with tri-
chloroacetic acid. For [125I]LDL uptake experiments, aortas (n = 4 each
group) were incubated with 10 µg/ml [125I]LDL in serum-free medium.
After a 3-h incubation at 37°C, aortas were washed three times with 2 ml
PBS and assayed for cell-associated label and protein content (Podrez et al.,
1999). Measurement of radioactivity was performed in a gamma counter
(Wallac 1470 Automatic Gamma Counter; EG&G Wallac).
Western blot analysis. Aortic tissue samples were frozen with liquid nitro-
gen, crushed, and lysed in cell lysis buffer (Cell Signaling Technology) with
protease inhibitor cocktail (Sigma-Aldrich). HUVECs and BAECs were
washed twice with PBS and harvested on ice in radioimmunoprecipitation
assay lysis buffer (50 mmol/l Hepes, 10 mmol/l EDTA, 150 mmol/l NaCl,
1% NP-40, 0.5% Na deoxycholate, and 0.1% SDS, pH 7.4) supplemented
with the protease inhibitor cocktail. Total cell lysates were loaded on SDS-
PAGE and electrotransferred into nitrocellulose membrane followed by
blocking 1 h at room temperature in 5% nonfat dry milk in PBS/0.1%
Tween 20. After being washed three times with PBS/0.1% Tween 20, the
blots were incubated overnight at 4°C with the appropriate primary anti-
body. The primary antibodies used were CyPA (1:5,000 dilution; Enzo Life
Sciences, Inc.), LOX-1 (1:1,000 dilution; Abcam), SR-BI (1:1,000 dilution;
Abcam), CD36 (1:1,000 dilution; Santa Cruz Biotechnology, Inc.), VCAM-1
(1:2,000 dilution; Santa Cruz Biotechnology, Inc.), actin (1:5,000 dilution;
Santa Cruz Biotechnology, Inc.), eNOS (1:1,000 dilution; BD), -tubulin
(1:5,000 dilution; Sigma-Aldrich), Flag (1:2,000 dilution; Sigma-Aldrich),
and caspase-3 (1:1,000 dilution; Cell Signaling Technology). ApoB isoforms
present in serum were determined by immunoblotting with rabbit anti–rat
apoB antibodies prepared in our laboratory (Chirieac et al., 2000). The mem-
branes were incubated with peroxidase-conjugated secondary antibodies for
1 h. Signals were visualized using the enhanced chemiluminescence Western
blotting detection system (GE Healthcare). Images were acquired with a
with immunosuppression, several of which can be linked to
its cardiovascular side effects. Moreover, CsA is not a selec-
tive drug for CyPA. In fact, CyPA, -B, and -C all bind with
high affinity to CsA in vitro (Bram et al., 1993). Therefore,
the proatherogenic effect of CsA could be related to the in-
hibition of other cyclophilin isoforms. In addition, CsA could
promote atherosclerosis by a CyPA-independent mechanism.
For example, CsA directly binds to LDL and affects LDL
metabolism at several levels (López-Miranda et al., 1993;
Vaziri et al., 2000). We believe that the discovery of more
uniquely selective and specific inhibitors of CyPA will be a
good therapeutic approach for atherosclerosis.
MATERIALS AND METHODS
Animal procedures. All animal experiments were conducted in accor-
dance with experimental protocols that were approved by the University
Committee on Animal Resources at the University of Rochester. Ppia/
mice were purchased from The Jackson Laboratory and were backcrossed to
C57BL/6J mice for 10 generations. The Apoe/ mice on a C57BL/6J back-
ground were obtained from The Jackson Laboratory. Double knockout
Apoe/Ppia/ mice were generated by crossing Ppia/ mice with Apoe/
mice. The F1 generation was backcrossed with Apoe/ mice to fix the
Apoe/ genotype, and littermates were crossed. All mice were genotyped
by PCR on tail clip samples, and all experiments were performed with gen-
erations F4–F6 using littermate Apoe/Ppia+/+ as controls. Animals were
housed under a 12-h light and 12-h dark regimen. Accelerated atherosclero-
sis was induced by feeding the mice for 16 wk with a high-cholesterol diet
containing 1.25% cholesterol (Research Diets D12108C).
Atherosclerotic lesion analysis. Mice were anesthetized with an intra-
peritoneal injection of 80 mg kg1 ketamine and 5 mg kg1 xylazine. Hearts
were perfused through the left ventricle with PBS followed by 10% buffered
formaldehyde. After fixation overnight in 10% formaldehyde, the aortas
were thoroughly cleaned under a dissecting microscope. All adventitial fat
and connective tissue was carefully removed. The vessels were then cut open
longitudinally through the inner curvature and the ventral portions of the
thoracic and abdominal sections. Aortas were rinsed with 5 ml of 60% iso-
propanol for 5 min. Staining was performed with 5 ml of filtered Oil red
O solution (Poly Scientific) for 15 min. Vessels were then rinsed with 60%
isopropanol for 15 min, followed by a final rinse with distilled water. After
staining, the greater curvature of the aorta was cut to divide the arch in half.
The vessel was precisely pinned to black wax in PBS to reveal the entire
luminal surface area. Images were obtained using SPOT version 4.1.1, a
camera (SPOT Insight 4; Diagnostic Instruments, Inc.) connected to a
microscope (MZ12.5; Leica). Plaques were analyzed in Photoshop 8.0
(Adobe), and lesion area was quantified using Image-Pro Analyzer 6.2
Histological analysis. Mice were anesthetized and euthanized. For mor-
phological analysis, aortas were perfused with normal saline and fixed with
10% phosphate-buffered formalin at physiological pressure for 5 min (Satoh
et al., 2008). The whole aortas and hearts were harvested, fixed for 24 h, and
embedded in paraffin, and 5-µm cross sections were prepared. Paraffin
sections were stained with H&E or Masson and Trichrome or used for
immunostaining. Analyses were performed by using Image-Pro Plus soft-
ware (Media Cybernetics).
Immunohistochemistry. Formaldehyde-fixed paraffin sections were in-
cubated with primary antibody overnight at 4°C. The primary antibodies
used were Mac3 (clone M3/84; 1:200 dilution; BD) and -smooth muscle
actin (clone 1A4; 1:1,000 dilution; Sigma-Aldrich). As a negative control,
species- and isotype-matched IgG were used in place of the primary anti-
body. Slides were viewed with a microscope (BX41; Olympus) and with
Cyclophilin A contributes to atherosclerosis | Nigro et al.
with CyPA-WT and vector control for 48 h, HUVECs were washed with
PBS and loaded with 10 µM 2,7-DCF diacetate (H2DCF-DA; Invitrogen)
or 5 µM DHE (Invitrogen) for 30 min at 37°C. ROS levels were measured
using flow cytometry (FACSCanto II). Data from 20,000 events per sample
were collected, and forward light scatter characteristics were evaluated to
exclude cell debris from the analysis.
Apoptosis analysis. Apoptotic cells were determined by analyzing their
subdiploid staining profiles (sub-G1 population). BAECs were cultured in
the presence of TNF + CHX and then washed in PBS, resuspended (5 × 105
cells/ml) in PI hypotonic solution (0.1% Na citrate, 0.1% Triton X-100, and
50 µg/ml PI), and left at 4°C for 30 min in the dark. Data from 20,000
events per sample were collected by flow cytometry (FACSCanto II). For-
ward light scatter characteristics were used so that cell debris could be ex-
cluded from the analysis. For the estimation of apoptotic cells, cells were
collected and double labeled with FITC-conjugated annexin V and PI ac-
cording to the manufacturer’s instructions (Trevigen). Green (FITC-conjugated
annexin V) and red (PI) fluorescence of individual cells were measured by
flow cytometry (FACSCanto II). Electronic compensation was required to
exclude overlapping of the two emission spectra. For the detection of apop-
tosis on the whole aortic mount, FITC-labeled dUTP nick end labeling
(TUNEL) method was applied to the aortic segments by using an In Situ
Apoptosis Detection kit-POD according to the manufacturer’s instruction
(Roche). Images were acquired by confocal microscopy (FLUOVIEW).
Statistical analyses. Quantitative results are expressed as mean ± SD.
Comparisons of parameters among two groups were made by the unpaired
Student’s t test. Comparisons of parameters among the three groups were
made by one-way analysis of variance, and comparisons of different parame-
ters between the two genotypes were made by two-way analysis of variance,
followed by a post hoc analysis using the Bonferroni test. Statistical signifi-
cance was evaluated with StatView (StatView 5.0; SAS Institute, Inc.).
A value of P < 0.05 was considered to be statistically significant.
Online supplemental material. Fig. S1 shows that CyPA deficiency pre-
vents atherosclerosis formation. Fig. S2 shows that CyPA deficiency de-
creases LDL uptake in the lesser curvature of the mouse aorta. Fig. S3 shows
that CyPA regulates LDL uptake. Fig. S4 shows metabolic parameters in the
absence of CyPA. Fig. S5 shows that CyPA promotes EC damage and disor-
ganization. Online supplemental material is available at http://www.jem
We are grateful to the Aab Cardiovascular Research Institute members for useful
suggestions and Mary A. Georger, Joanne Cianci, and Chelsea Wong for technical
assistance. We are also grateful to Eiichiro Yamamoto for assisting with data analysis.
This work was supported by National Institutes of Health grant HL49192 (to B.C.
Berk), Internal Grant of the University of Salerno (to P. Nigro), Japan Heart Foundation/
Bayer Yakuhin Research Grant Abroad (to K. Satoh), Japan Heart Foundation/Novartis
Grant for Research Award on Molecular and Cellular Cardiology (to K. Satoh),
AstraZeneca Research Grant (to K. Satoh), and grants-in-aid from the Japanese
Ministry of Education, Culture, Sports, Science and Technology (to K. Satoh).
The authors declare no competing financial interests.
Submitted: 11 June 2010
Accepted: 23 November 2010
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Lipids analysis and lipoprotein profiles measurement. Mice were
anesthetized, and blood samples were collected from the left ventricle.
Plasma was prepared and stored at 80°C. Plasma cholesterol and triglycer-
ides were enzymatically measured using the Cholesterol E kit (Wako Chem-
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ously (Satoh et al., 2006). In brief, recipient mice were lethally irradiated and re-
ceived an intravenous injection of 5 × 106 donor BM cells suspended in 100 µl
of calcium- and magnesium-free PBS with 2% FBS. After transplantation, the
mice were placed on a regular chow diet for 6 wk followed by high-cholesterol
diet for 12 wk. Transgenic mice ubiquitously expressing GFP were obtained
from The Jackson Laboratory. The chimeric rate assessed by reconstitution with
GFP+ BM cells was >99% by FACS analysis (FACSCanto II; BD).
BM-derived cell recruitment assays. Quantitative numbers or percent-
ages of the migrating GFP+ cells was analyzed by en face staining as reported
in En face analysis. PECAM-1 antibody was used to identify the EC by using
a FLUOVIEW confocal microscope.
Cell culture and transfection. HUVECs were obtained from collagenase-
digested umbilical veins and collected in M200 medium supplemented with low
serum growth supplement (Invitrogen), 5% fetal calf serum (Invitrogen), 50 U/
ml penicillin, and 50 µg/ml streptomycin. BAECs were cultured in M199 me-
dium (Invitrogen) supplemented with 10% fetal clone III (Hyclone), MEM–
amino acids, 50 U/ml penicillin, and 50 µg/ml streptomycin. HUVECs as well
as BAECs were cultured on 2% gelatin–precoated dishes. For transient expres-
sion experiments, 80% confluent cells were transfected using Opti-MEM and
Lipofectamine 2000 (Invitrogen). 3 h after transfection, Opti-MEM was re-
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siRNA-driven depletion of CyPA, HUVECs and BAECs were transiently
transfected with 100 nM scrambled siRNA or CyPA siRNA using Lipo-
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Steady laminar flow (s-flow) protocol. Confluent cells cultured in
100-mm dishes were exposed to flow in a cone and plate viscometer placed
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California, San Francisco, San Francisco, CA) or KLF2 promoter (provided by
J. Lingrel, University of Cincinnati, Cincinnati, OH) and -galactosidase using
Lipofectamine 2000. After 8 h, the cells were exposed to flow. 24 h later, cells
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conducted by using TaqMan reverse transcription reagents (Applied Biosys-
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mRNAs were obtained using the comparative Ct method and were normal-
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ROS measurement. The evaluation of ROS production was performed as
described previously (Griendling and FitzGerald, 2003). After transfection
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