Endothelial Dysfunction and Claudin 5 Regulation
during Acrolein-Induced Lung Injury
An Soo Jang1,2, Vincent J. Concel1, Kiflai Bein1, Kelly A. Brant1, Shannen Liu1, Hannah Pope-Varsalona1,
Richard A. Dopico, Jr.1, Y. P. Peter Di1, Daren L. Knoell3, Aaron Barchowsky1, and George D. Leikauf1
1Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania;
2Department of Internal Medicine, Soon Chun Hyang University, Bucheon, Korea; and3Dorothy M. Davis Heart and Lung Research Institute and
Department of Pharmacy, The Ohio State University, Columbus, Ohio
An integral membrane protein, Claudin 5 (CLDN5), is a critical
component of endothelial tight junctions that control pericellular
permeability. Breaching of endothelial barriers is a key event in the
development of pulmonary edema during acute lung injury (ALI). A
major irritant in smoke, acrolein can induce ALI possibly by altering
assess susceptibility, 12 mouse strains were exposed to acrolein
(10 ppm, 24 h), and survival monitored. Histology, lavage protein,
and CLDN5 transcripts were measured in the lung of the most
sensitive and resistant strains. CLDN5 transcripts and phosphoryla-
tion status of forkhead box O1 (FOXO1) and catenin (cadherin-
associated protein) beta 1 (CTNNB1) proteins were determined in
control and acrolein-treated human endothelial cells. Mean survival
time (MST) varied more than 2-fold among strains with the suscep-
tible (BALB/cByJ) and resistant (129X1/SvJ) strains (MST, 17.3 6 1.9
lung. Lung CLDN5 transcript and protein increased more in the
resistant strain than in the susceptible strain. In human endothelial
cells, 30 nM acrolein increased CLDN5 transcripts and increased
p-FOXO1 protein levels. The phosphatidylinositol 3-kinase inhibitor
LY294002 diminished the acrolein-induced increased CLDN5 tran-
script. Acrolein (300 nM) decreased CLDN5 transcripts, which were
accompanied by increased FOXO1 and CTNNB1. The phosphoryla-
tion status of these transcription factors was consistent with the
be a novel clinical approach for ALI therapy.
Keywords: ARDS; perivascular edema; vascular permeability; smoke
inhalation; carboxyl stress
Adhesive structures between adjacent cells, including tight and
adherens junctions, enable the establishment of cell polarity,
differentiation, and survival and are critical to the maintenance
of tissue integrity (1). Tight junctions consist of a macromolec-
ular complex of numerous adhesive molecules, including occlu-
adhesion molecules, and claudins (2). Tight junctions are often
located apically with respect to adherens junctions (composed
mainly of cadherins), as in epithelial cells, but can be inter-
mingled throughout cell–cell contact areas, as in endothelial
cells (3). Tight junction strands serve as a physical barrier to
regulate solutes and water movement through the paracellular
space between epithelial or endothelial cells. Compromised
barrier function of adhesive structures is a common event in
several diseases, including ischemic brain disease (4, 5), Crohn’s
disease (6), and acute lung injury (ALI) (7–9).
In spite of considerable medical achievements, treatment for
ALI is limited to supportive care, and recent estimates indicate
that mortality remains high (z 30–40% or 74,500 deaths per
year in the United States) (10). ALI can be induced indirectly
(e.g., by sepsis or trauma) or directly (e.g., by smoke inhalation)
(8). One major histological feature of ALI is pulmonary edema,
which results from increased epithelial and endothelial perme-
ability and decreased clearance of edema fluid by the alveolar
epithelium. These events, when combined with decreased
surfactant-associated protein B synthesis, disrupt surfactant
surface tension and ultimately produce respiratory failure
(11). These barrier functions require adequate control of para-
cellular tight junctions; however, our current understanding of
the structural components and regulation of tight junctions in
the lung is insufficient.
The claudin (CLDN) family consists of 24 tetraspan trans-
membrane proteins, each of which has a tissue-restricted
expression pattern (12–14). In the normal lung, bronchiolar
epithelial cells mainly express CLDN1, -3, -4, -7, and -18, and
alveolar type II epithelial cells mainly express CLDN3, -4, -7,
and -18 (7, 9, 15). In epithelial cells, transgenic expression of
CLDN1 with CLDN3 increased transepithelial resistance and
decreased paracellular permeability, whereas CLDN4 confers
One of the major histological features of acute lung injury
is pulmonary edema, which results from increased epithe-
lial and endothelial permeability and decreased clearance
of edema fluid by the alveolar epithelium. In contrast to
other claudins (CLDN) in the lung, CLDN5, which is
expressed weakly in the epithelium, is expressed strongly
in endothelium of normal lung. However, its role in acute
lung injury has had modest attention in the past. Acrolein-
induced acute lung injury was marked by perivascular
edema in mice. This is accompanied by a compensatory
increase in CLDN5 transcripts, which was more evident in
a resistant than in a sensitive mouse strain. Acrolein (30 nM)
stimulated phosphorylation of FOXO1 protein and in-
creased CLDN5 transcripts, whereas 300 nM acrolein stim-
ulated FOXO1 and CTNNB1 protein levels and decreased
CLDN5 transcripts. These events are consistent with the
rapid increase in vascular permeability and could provide
a critical target for future pharmacological intervention
during acute lung injury.
(Received in original form October 23, 2009 and in final form May 4, 2010)
This study was supported by NIH grants ES013781 (A.B.); ES015675, HL077763,
and HL085655 (G.L.); HL086981 (D.L.), and HL091938 (P.D.).
Correspondence and requests for reprints should be addressed to George D.
Leikauf, Ph.D., Department of Environmental and Occupational Health, Graduate
School of Public Health, University of Pittsburgh, 100 Technology Drive, Suite
350 Pittsburgh, PA 15219-3130. E-mail: firstname.lastname@example.org
This article has an online supplement, which is accessible from this issue’s table of
contents at www.atsjournals.org
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2009-0391OC on June 4, 2010
Internet address: www.atsjournals.org
Vol 44. pp 483–490, 2011
selective ion transport function without effecting paracellular
solute permeability (16). Recently, Wray and colleagues
reported that CLDN4 inhibition decreased transepithelial re-
sistance without altering paracellular permeability in primary
rat and human epithelial cells (9). In addition, in vivo CLDN4
epithelial expression was an early event in ALI, leading the
authors to conclude that CLDN4 represents a possible mecha-
nism to limit pulmonary edema.
In contrast to other claudins in the lung, CLDN5, while
expressed weakly in the epithelium, is expressed strongly in
endothelium of normal lung and is intense in endothelium in
usual interstitial pneumonia (15). Newborn gene-targeted
Cldn5(2/2)mice die within 10 hours of birth possibly due to
altered permeability of the blood–brain barrier (17). When
CLDN5 was transfected into airway epithelial cells, paracellular
permeability increased even in the presence of excessive
CLDN1 and CLDN3 (18). Moreover, inducing CLDN5 expres-
sion in leaky rat lung endothelial cells can enhance paracellular
barrier function against large (but not small) molecules (19).
One of the key pathognomic features of ALI is perivascular
edema, in part due to endothelial dysfunction (20). In this study,
we examine the endothelial dysfunction and CLDN5 expression
during lung injury induced by acrolein, a key irritant in smoke.
MATERIALS AND METHODS
Twelve inbred mouse strains were exposed to filtered air (control) or
acrolein (10 ppm, 24 h) generated and monitored as described pre-
viously (21–23), and survival time was recorded. Detailed analyses,
which included measurement of lung CLDN5 mRNA and protein, lung
histology, and bronchoalveolar lavage (BAL), contrasted the response
of the most sensitive (BALB/cByJ) and resistant (129X1/SvJ) strains
after exposure to filtered air (0 h, control) or acrolein (10 ppm, 6 or
12 h). Additional tests were performed with a cell line derived from the
fusion of human umbilical vascular endothelial cells with the lung
carcinoma cell line A549 (EA.hy926) or primary human lung micro-
vascular endothelial cells. Confluent cells were washed three times in
Dulbecco’s modified PBS (DPBS), incubated for 30 minutes, and
exposed to acrolein (< 4 h) in DPBS. Assays include measurements
of Claudin 5 transcripts, catenin (cadherin-associated protein), beta 1
(CTNNB1), phosphorylated CTNNB1 (Ser 552) (p-CTNNB1), fork-
head box O1 (FOXO1), phosphorylated FOXO1 (Ser256) (p-FOXO1),
thymoma viral proto-oncogene 1 (AKT), and phosphorylated AKT
(p-AKT). EA.hy926 cells were also treated with vehicle (DMSO), 30
nM acrolein, or 30 nM acrolein and 10 mM LY294002 [2-(4-Morpho-
linyl)-8-phenyl-4H-1-benzopyran-4-one] (LY), a phosphatidylinositol
3-kinase (PI3K) inhibitor. Additional details of the methods are
contained in the online supplement.
Mouse Strains Vary in Sensitivity to Acrolein-Induced ALI
The mean survival time (MST) varied among the 12 mouse strains
exposed to acrolein (10 ppm, 24 h) (Figure 1A). The most susceptible
(BALB/cByJ) and resistant (129X1/SvJ) mouse strains differed by
more than 2-fold (MST 17.3 6 1.9 h vs. 41.4 6 5.1 h, respectively).
Survival curves for the sensitive (BALB/cByJ) and resistant (129X1/
SvJ) mouse strains were significantly different (P , 0.001) (Figure 1B).
Thus, the BALB/cByJ and 129X1/SvJ were selected for subsequent
analyses. Histological assessment of lung tissue from the sensitive
BALB/cByJ strain demonstrated perivascular enlargement present
within 12 hours of acrolein exposure (Figure 2C), as compared with
strain-matched control (Figure 2A). In acrolein-treated BALB/cByJ
mouse lung, leukocytes were present in the perivascular space (Figure
2C) and focal areas in the alveolar interstitium (Figure 2E), with
thickening of alveolar wall (Figure 2E). The perivascular enlargement
included an increased distance between the tunica media and the
tunica adventitia in lung of BALB/cByJ mice (Figure 2G). Neither
perivascular enlargement nor leukocytes was evident in the resistant
129X1/SvJ strain after 17 hours of acrolein exposure (Figures 2D and
2F) compared with strain-matched control mice exposed to filtered air
(Figure 2B). Similarly, BAL protein increased more in sensitive
(BALB/cByJ) mice than in resistant (129X1/SvJ) mice after 12 hours
of acrolein exposure (P , 0.001) (see Figure E1 in the online
Lung CLDN5 Transcript Levels Increased during
Because the endothelial tight junction protein CLDN5 had previously
been associated with endothelial permeability (18, 19), CLDN5 tran-
script and protein levels were measured after 6 and 12 hours of acrolein
exposure (Figure 3). At 12 hours, lung CLDN5 transcript (Figure 3A)
and protein (Figure 3C) increased more in resistant (129X1/SvJ) mice
compared with strain-matched control mice (P , 0.001), whereas the
lung CLDN5 protein increased less (Figure 3C), and transcript levels in
the sensitive (BALB/cByJ) mice were not significantly different from
acrolein exposure. MST varied among mouse strains, with the most
susceptible (BALB/cByJ) and resistant (129X1/SvJ) mouse strains vary-
ing more than 2-fold (MST, 17.3 6 1.9 h vs. 41.4 6 5.1 h,
respectively). Mice (n 5 9 mice per strain) were exposed to acrolein
(10 ppm, 24 h) under specific pathogen-free conditions. (B) Survival
curves for the sensitive (BALB/cByJ) and resistant (129X1/SvJ) mouse
strains. Between-strain survival time was significantly different using the
Kaplan-Meyer method (P , 0.001).
(A) Mean survival time (MST) of 12 mouse strains after
484AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 442011
Acrolein Alters CLDN5 Transcript Levels in EA.hy926 Hybrid
Cells and Human Lung Microvascular Endothelial Cells
To begin to determine a mechanism by which acrolein altered CLDN5
transcripts, we exposed EA.hy926 cells (a hybrid cell line) or human
lung microvascular endothelial cells to acrolein and measured CLDN5
transcript levels (Figure 4). Within 1 hour of exposure to 30 nM
acrolein, CLDN5 transcripts increased and were significantly (P ,
0.001) different from control (DPBS) in EA.hy926 cells (Figure 4A).
This effect was dose dependent (Figure 4B), with CLDN5 mRNA
increasing at doses from 1 to 30 nM acrolein (4 h) and decreasing at
doses of 100 or 300 nM as compared with cells exposed to DPBS
(control) alone. Similarly, in human lung microvascular endothelial
cells, CLDN5 mRNA increased after 30 nM acrolein and CLDN5
mRNA decreased after 100 or 300 nM acrolein treatment (Figure 4C).
Acrolein Increased p-FOXO1 Protein in EA.hy926 Cells
Acrolein could activate a variety of signaling pathways that regulate
the expression of junctional proteins. CLDN5 expression is normally
enhanced by CTNNB1 or repressed by nonphosphorylated FOXO1
and CTNNB1 binding at sites in the proximal promoter (28). To
determine whether acrolein could alter FOXO1 or CTNNB1 status,
time- and dose-dependent effects on FOXO1 and CTNNB1 and their
corresponding phosphorylated forms were measured in cells treated
with acrolein. After 4 hours of exposure to 30 nM acrolein, p-FOXO1
protein increased, whereas FOXO1 protein levels were not signifi-
cantly different from DPBS (control)-treated EA.hy926 cells (Figure
5A). Thus, the FOXO1 to p-FOX1 protein ratio decreased after 30 nM
acrolein as compared with control (P , 0.001) (Figure 5C). Western
immunoblots for CTNNB1 or p-CTNNB1 protein were unchanged by
treatment with 30 nM acrolein (Figures 5A and 5B).
Acrolein Increased FOXO1 or CTNNB1, whereas p-FOXO1 or
p-CTNNB1 Was Unchanged after Acrolein in EA.hy926 Cells
After 1 to 4 hours of exposure to 300 nM acrolein, FOXO1 protein
increased, whereas p-FOXO1 protein levels were not significantly
different from DPBS (control)-treated EA.hy926 cells (Figure 6). Cell
viability as measured by a MTS-dehydrogenase enzyme assay of
EA.hy926 cells treated with 3,000 nM acrolein or less was not significantly
different from vehicle control (Figure E2). Similarly, CTNNB1 but not p-
CTNNB1 increased with treatment (Figure 6B), and the ratio of
CTNNB1 to p-CTNNB1 increased as compared with control (P ,
0.001) (Figure 6B). The ratio of FOXO1 to p-FOXO1 increased as
compared with control (P , 0.001) (Figure 6C).
The Phosphatidylinositol 3-Kinase Inhibitor Diminishes
Acrolein-Induced Increased CLDN5 Transcript
in EA.hy926 Cells
Induction of CLDN5 and p-FOXO1 was observed in EA.Hy926 cells
treated with 30 nM acrolein (see Figures 4B and 5C). PI3K phosphor-
ylates AKT, which in turn phosphorylates FOXO1 (and other tran-
scription factors), and this signaling pathway can be inhibited by LY.
To examine PI3K inhibition after acrolein treatment of EA.hy926 cells,
cells were treated with 10 mM LY (30 min) and exposed to 30 nM
acrolein (4 h). LY treatment diminished acrolein-induced CLDN5
mRNA (Figure 7). We also examined whether LY could inhibit p-AKT
formation after acrolein treatment of EA.hy926. Acrolein increased
p-AKT, and this effect was inhibited by LY addition (Figure E3).
Figure 2. Mouse strains vary in pathophysiological response to acro-
lein-induced lung pathology. Histological assessment of lung tissue
from (A) control (filtered air) BALB/cByJ mice, (B) control (filtered air)
129X1/SvJ mice, (C, E) acrolein (10 ppm, 12 h) exposed BALB/cByJ
mice, or (D, F) acrolein (10 ppm, 17 h) exposed 129X1/SvJ mice.
Compare (arrow) area around blood vessel from (C) acrolein-treated
with (A) control. In the sensitive BALB/cByJ strain, perivascular enlarge-
ment was present at 12 hours of exposure. Leukocytes were present in
the (C) perivascular space and (E) alveolus in acrolein-treated BALB/
cByJ mouse lung. (D) Perivascular enlargement was not as evident in
the lungs of the resistant 129X1/SvJ strain as compared with the BALB/
cByJ strain after acrolein exposure. (F) Leukocytes present in alveolus
were less in the 129X1/SvJ strain as compared with the BALB/cByJ
strain. Bar indicates magnification of original image obtained from 5-
mM sections prepared with hematoxylin and eosin stain (50 mm in A–D
and 10 mm in E and F). (G) The perivascular interstitial space increased
more in lung of BALB/cByJ than of 129X1/SvJ mouse strains after
acrolein exposure. After acrolein exposure, the length of the distance
between the tunica media and the tunica adventitia (median with 25
and 75% confidence intervals in parentheses) increased from control 5
2.6 (1.8–3.3) mm to exposed 5 16.6 (11.3–24.7) mm in the lung of
BALB/cByJ mice and from control 5 1.9 (1.4–2.9) mm to exposed 5 4.1
(2.8–5.8) mm in the lung of 129X1/SvJ mice. Values plotted indicate
the median (line in box) with 25 and 75% confidence intervals (borders
of the box) and 95% confidence intervals (error bars). *Statistically (P ,
0.001) different from strain-matched control mice (filtered air) as
determined by Kruskal-Wallis ANOVA on ranks followed by pairwise
comparison with the Tukey method.
Soo Jang, Concel, Bein, et al.: Claudin 5 in Acute Lung Injury 485
A major adverse event after smoke inhalation is delayed
pulmonary edema and respiratory failure due to ALI (24). In
fire victims surviving carbon monoxide poisoning, progressive
pulmonary failure and cardiovascular dysfunction are impor-
tant determinants of morbidity and mortality (25). ALI is
marked by perivascular edema (20). This study focused on
acrolein, which previously has been demonstrated to be the
chemical responsible for pulmonary edema in smoke inhala-
tion (26, 27). Endothelial junctional proteins play critical
roles in tissue integrity and can regulate vascular permeabil-
ity, leukocyte diapedesis, and angiogenesis (28). Endothelial
cells express cell type–specific transmembrane junction pro-
teins, including CLDN5 in tight junctions (15, 19) and
cadherin 5, type 2 (vascular endothelium) (CDH5, a.k.a.
VE-cadherin) at adhesion junctions (28). In concert with
CDH5 (1, 29, 30) and other junctional proteins, CLDN5
controls vascular permeability in vitro and in vivo (12–14).
In the normal lung, CLDN5 is expressed strongly in endothe-
lium and is considered a major contributor to the formation of
tight junctions in these cells (15). Inducing CLDN5 expression
in leaky rat lung endothelial cells can help to restore para-
cellular barrier function (19). The objective of this study was
to determine the cell signal mechanism controlling endothe-
lial CLDN5 expression during ALI.
We determined that the sensitivity of 12 inbred mouse
strains to acrolein-induced ALI varied. The polar strains
(sensitive: BALB/cByJ; resistant: 129X1/SvJ) differed more
than 2-fold in survival time. After acrolein exposure, histolog-
ical changes included perivascular edema and increased BAL
protein, features consistent with ALI. These signs occurred
earlier and were demonstrably greater in the sensitive strain
compared with the resistant strain. This finding strongly sup-
ports the likelihood that an underlying genetic difference is
linked to these phenotypes, much like we and others have found
for other models of ALI (11, 31–41). Future studies to expand
the number of strains examined for single nucleotide poly-
morphisms mapping or to use crosses from these strains for
quantitative trait loci analysis are clearly warranted.
Having obtained histological evidence that acrolein altered
vascular permeability, we next measured CLDN5 transcripts in
mouse lung and confluent endothelial cells. In vitro assays with
human lung microvascular endothelial cells or EA.hy926 cells
indicated that the CLDN5 transcript regulation was concentration
dependent. At 30 nM acrolein, CLDN5 transcript levels increased.
A similar increase was noted in mouse lung, which was greater in
the resistant strain than in the sensitive strain. This initial CLDN5
induction can be considered a compensatory mechanism to mend
junctional complexes and restore barrier function. At 300 nM
acrolein in vitro, CLDN5 transcripts decreased, which would be
detrimental to the maintenance of tight junction function.
To better understand the mechanism of CLDN5 regulation,
we examined FOXO1 and CTNNB1 at 30 or 300 nM acrolein
concentrations. FOXO1 integrates various cell signals critical to
endothelial cell function at the transcriptional level (42). CLDN5 is
expressed in the absence of nuclear accumulation of FOXO1
transcription factor. This is controlled by PI3K-mediated phos-
phorylation of AKT, which in turn mediates downstream phos-
phorylation of FOXO1 (p-FOXO1). p-FOXO1 can be retained in
the cytoplasm or degraded after ubiquitination mediated, in
part, by ring finger and WD repeat domain 2 (43). Consistent
with this mechanism, treatment with a PI3K inhibitor (LY)
diminished acrolein-induced AKT phosphorylation and increased
CLDN5 transcript levels. This signaling can be enhanced by
growth factors, including VEGFA, which has previously been
more in the resistant mouse strain (129X1/SvJ) than in the sensitive
mouse strain (BALB/cByJ) after acrolein exposure. (A) CLDN5 transcript
increased more at 12 hours in 129X1/SvJ than in BALB/cByJ mouse
lung. Mice were exposed to control (filtered air, 0 h) or 10 ppm
acrolein for 6 or 12 hours, and lung mRNA was analyzed by qRT-PCR.
Values are mean 6 SE (n 5 8 mice per group). At 12 hours, lung
CLDN5 transcript levels increased in resistant (129X1/SvJ) mice com-
pared with strain-matched control mice, whereas the lung CLDN5
transcript levels in the sensitive (BALB/cByJ) mice were not significantly
different from control mice. *Significantly different (P , 0.0001) from
strain-matched control mice as determined by ANOVA with an all
pairwise multiple comparison procedure (Holm-Sidak method). (B)
CLDN5 protein increased more at 12 hours in 129X1/SvJ than in
BALB/cByJ mouse lung as determined by Western blot. Each lane
represents protein from a single mouse. (C) CLDN5 protein increased
more at 12 hours in 129X1/SvJ than in BALB/cByJ mouse lung. Each test
was repeated four times and quantified using ImageQuant 5.2 software
(Typhoon 9410; GE Healthcare, Piscataway, NJ). Values are mean 6 SE
(n 5 4) normalized to b-actin. *Significantly different (P , 0.05) from
strain-matched control mice (0 h) as determined by ANOVA with an all
pairwise multiple comparison procedure (Holm-Sidak method). **Sig-
nificantly different (P , 0.001) between 12 hour–exposed 129X1/SvJ
and 12 hour–exposed BALB/cByJ mice as determined by ANOVA with
an all pairwise multiple comparison procedure (Holm-Sidak method).
Lung Claudin 5 (CLDN5) transcript and protein increased
486AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 44 2011
demonstrated to increase markedly and may initiate edema
during ALI (29, 44–46).
In addition, CTNNB1 can be released from adhesion com-
plexes during stress (47), relocates to the nucleus, and activates
numerous critical survival events, including the transcription of
CLDN5 (1). At 30 nM acrolein, CLDN5 transcripts and the
ratio of p-FOXO1 to FOXO1 increased in EA.hy926 cells,
which is consistent with such an outcome. With mounting stress,
FOXO1 and CTNNB1 proteins accumulate in the cell, trans-
locate to the nucleus, and partner to regulate target genes that
promote stress resistance, cell cycle arrest, or apoptosis (48–51).
At 300 nM acrolein in vitro, CLDN5 transcripts decreased and
FOXO1 and CTNNB1 increased in EA.hy926 cells, which is
consistent with more severe outcome. The ultimate loss of
CLDN5 in tight junctions by this mechanism can lead to
disassembly of adhesive structures, endothelial barrier dysfunc-
tion, and ultimately increased vascular permeability.
The acrolein levels used in this study, 10 ppm in vivo and 1 to
300 nM in vitro, are relevant to human exposure. As an
a,b-unsaturated 2-alkenal, acrolein is highly reactive in biolog-
ical systems and can be extremely irritating (e.g., 0.06 ppm can
cause eye irritation within 5 min) (52–54). Acrolein can rapidly
bind with macromolecules and disrupt critical cellular functions
(55–60). Acrolein is generated by combustion and is the major
irritant in grassland and forest fires, high temperature cooking
with oils (especially in woks), and diesel exhaust (26, 27, 61, 62).
Over 30 million nonsmokers in the United States are exposed
to acrolein concentrations in indoor air ranging from 0.8 to
1.5 ppm, and levels between 0.1 to 10 ppm have been detected
in bars and restaurants (63–67). Acrolein levels are elevated in
second-hand smoke compared with mainstream smoke because
side-stream smoke is generated at lower combustion tempera-
tures (61, 64, 68–70). The nanomolar acrolein concentrations
used in this study also are relevant to endogenously generated
levels in injured tissues (55, 56), which can result from amine
oxidase–mediated catabolism of spermine or spermidine (71–
75), myeloperoxidase catabolism of threonine (76–78), or, albeit
less likely, oxidative degradation of membrane fatty acids (61,
79–81). Acrolein-protein adducts accumulate in ischemic tissue
(81, 82) and in atherosclerotic lesions (78, 83).
In summary, acrolein can induce ALI with perivascular
edema in mice. This is accompanied by a compensatory increase
in CLDN5 transcript and protein, which was more evident in
a resistant than a sensitive mouse strain. In vitro, 30 nM acrolein
stimulated phosphorylation of FOXO1 protein and increased
CLDN5 transcripts, whereas 300 nM acrolein stimulated
FOXO1 and CTNNB1 protein levels and decreased CLDN5
transcripts. These events are consistent with the rapid increase
in vascular permeability and could provide a critical target for
future pharmacological intervention during ALI.
Figure 4. Acrolein alters CLDN5 transcript in vitro. (A) Time course of
acrolein-induced CLDN5 transcript increases in EA.hy926 cells. Cells
were exposed to 30 nM acrolein for the indicated times, and mRNA
was analyzed by qRT-PCR. Tests were repeated on three occasions.
Values are mean 6 SE (n 5 12 dishes). *Significantly different (P ,
0.001) from Dulbecco’s PBS (control) as determined by ANOVA with
an all pairwise multiple comparison procedure (Holm-Sidak method).
(B) Dose response of acrolein-induced CLDN5 transcript in hybrid
EA.hy926 cells. Cells were exposed to acrolein for 4 hours, and mRNA
expression levels were analyzed by qRT-PCR. Tests were repeated on
three occasions. Values are mean 6 SE (n 5 12 dishes). *Significantly
different (P , 0.001) from control as determined by ANOVA with an all
pairwise multiple comparison procedure (Holm-Sidak method). (C)
Dose response of acrolein-induced CLDN5 transcript in human lung
microvascular endothelial cells. Cells were exposed to acrolein for
4 hours, and mRNA was analyzed by qRT-PCR. Tests were repeated on
three occasions. Values are mean 6 SE (n 5 12 dishes). *Significantly
different (P , 0.001) from control as determined by ANOVA with an all
pairwise multiple comparison procedure (Holm-Sidak method).
Soo Jang, Concel, Bein, et al.: Claudin 5 in Acute Lung Injury487
Figure 5. Phospho-forkhead box O1 (p-FOXO1) increased after 30 nM
of acrolein treatment. (A) Western immunoblot with catenin (cadherin-
associated protein), beta 1 (CTNNB1), p-CTNNB1, FOXO1,p-FOXO1,
or b-actin antibody in EA.hy926 cells treated with Dulbecco’s PBS
(control) or 30 nM acrolein. p-FOXO1 increased after 4 hours of 30 nM
acrolein treatment. (B) Mean transcription factor CTNNB1 protein
levels after 30 nM acrolein treatment. Open bar: CTNNB1. Closed
bar: p-CTNNB1. Hatched bar: CTNNB1/p-CTNNB1 ratio. (C) Mean
transcription factor FOXO1 protein levels after 30 nM acrolein
treatment. Open bar: FOXO1. Closed bar: p-FOXO1; Hatched bar:
FOXO1/p-FOXO ratio. Each test was repeated four times and
quantified using ImageQuant 5.2 software (Typhoon 9410; GE
Healthcare). Values are mean 6 SE (n 5 4) normalized to b-actin.
*Significantly different (P , 0.001) from control as determined by
ANOVA with an all pairwise multiple comparison procedure (Holm-
Figure 6. Catenin (cadherin-associated protein), beta 1 (CTNNB1), and
Forkhead box O1 (FOXO1) increased, whereas phospho-FOXO1 (p-
FOXO1) and phospho-CTNNB1 (p-CTNNB1) were unchanged after
treatment with 300 nM acrolein. (A) Western immunoblot with CTNNB1,
p-CTNNB1, FOXO1,p-FOXO1, or b-actin antibody in EA.hy926 cells
treated with Dulbecco’s PBS (control) or 30 nM acrolein. CTNNB1 and
FOXO1 increased at 1, 2, or 4 hours of 300-nM acrolein treatment. (B)
Mean transcription factor CTNNB1 proteins after 300 nM acrolein
treatment. Open bar: CTNNB1. Closed bar: p-CTNNB1. Hatched bar:
CTNNB1/p-CTNNB1 ratio. (C) Mean transcription factor FOXO1 proteins
after 300 nM acrolein treatment. Open bar: FOXO1. Closed bar: p-FOXO1.
Hatched bar: FOXO1/p-FOXO ratio. Each test was repeated four times and
quantified using ImageQuant 5.2 software (Typhoon 9410; GE Health-
care). Values are mean 6 SE (n 5 4) normalized to b-actin. *Significantly
different (P , 0.01) from control as determined by ANOVA with an all
pairwise multiple comparison procedure (Holm-Sidak method).
488 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 44 2011
Author Disclosure: D.K. has received a sponsored grant from the NIH (more than
$100,000). G.L. has served on the advisory board for the NIH (less than $1,000).
None of the other authors has a financial relationship with a commercial entity
that has an interest in the subject of this manuscript.
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