Am J Physiol Lung Cell Mol Physiol 306: L246 –L259, 2014.
First published November 27, 2013; doi:10.1152/ajplung.00109.2013.
Deregulation of the lysyl hydroxylase matrix cross-linking system in
experimental and clinical bronchopulmonary dysplasia
Thilo J. Witsch,1 Paweł Turowski,1 Elpidoforos Sakkas,1,2 Gero Niess,1,2 Simone Becker,1,2
Susanne Herold,1 Konstantin Mayer,1 István Vadász,1 Jesse D. Roberts, Jr.,3 Werner Seeger,1,2
and Rory E. Morty1,2
1
Department of Internal Medicine (Pulmonology), University of Giessen and Marburg Lung Center (UGMLC), Member of the
German Center for Lung Research (DZL), Giessen, Germany; 2Department of Lung Development and Remodelling, Max
Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; 3Cardiovascular Research Center, Massachusetts
General Hospital, Charlestown, Massachusetts
Submitted 26 April 2013; accepted in final form 20 November 2013
lung development; extracellular matrix; bronchopulmonary dysplasia;
lysyl hydroxylase
(BPD) is a significant complication of premature birth that, since the first description of BPD
by Northway and colleagues in 1967 (28), remains a key cause
of morbidity and mortality in neonatal intensive care units
worldwide. The diagnosis of clinical BPD currently relies
exclusively on the degree of oxygen dependence at a defined
postmenstrual age (21). A key pathophysiological feature of
infants afflicted with BPD is a dysmorphic pattern of lung
development, including an arrest of alveolarization, where
BRONCHOPULMONARY DYSPLASIA
Address for reprint requests and other correspondence: R. E. Morty, Dept. of
Lung Development and Remodelling, Max Planck Institute for Heart and Lung
Research, Parkstrasse 1, D-61231 Bad Nauheim, Germany (e-mail: rory.
[email protected]).
L246
secondary septation is limited, and thus the formation of the
alveolar gas exchange units is impeded (19). The resultant
general reduction in the gas exchange surface area of the lung
has both immediate and long-term consequences for affected
infants that extend beyond childhood (29). Interestingly, improvements in the medical management of premature infants
have led to improved survival of extremely premature infants
and, with that, a concomitant increase in the prevalence of
BPD. However, the pathogenesis of BPD, in particular, the
molecular basis of blunted septation and the consequent impaired alveolarization, is very poorly understood (20, 24).
An emerging area of interest in BPD pathogenesis is the
possible role played by aberrant remodeling of the extracellular
matrix (ECM). In particular, several studies have pointed to
improper deposition and maturation of elastin and collagen
fibers in the developing septa (1, 5, 6, 8, 23, 30, 43). This has
been attributed to two possible phenomena. First, the abundance of total collagen and elastin may be altered in the injured
lung. Abnormal collagen and elastin deposition has been reported in premature lambs (1, 6, 30), in mechanically ventilated newborn mice (5, 8), and mouse pups in which late lung
development has been arrested by exposure to high oxygen
levels (23, 49). Second, a growing body of evidence indicates
that the cellular machinery that is responsible for the posttranslational processing of ECM components (7), in particular, the
proteolytic processing and covalent cross-linking of ECM
structures, is dysregulated in the lungs of patients with BPD, as
well as in animal models of BPD. Notable among these ECM
maturation systems is the lysyl oxidase family of amine oxidases, which catalyze the covalent cross-linking of lysine and
hydroxylysine residues in collagen and elastin and thereby
promote stability of the ECM. Recent reports have indicated
that lysyl oxidase expression and activity are increased both in
lungs of patients affected with BPD (23), as well as in two
rodent models of BPD that rely on mechanical ventilation
and hyperoxia to induce lung damage (5, 23). It has been
proposed that an aberrantly active ECM cross-linking system,
as would be expected with elevated activity of lysyl oxidases,
would generate a lung matrix structure that was “over crosslinked” or too stable, which may resist the normal remodeling
of the developing lung that must take place to allow new
alveolar units to form (23).
Although the lysyl oxidases have been studied in BPD, lysyl
oxidases do not act alone to covalently cross-link ECM structures in the lung and other organs. Lysyl oxidases require
lysine or hydroxylysine residues in substrate ECM molecules,
1040-0605/14 Copyright © 2014 the American Physiological Society
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Witsch TJ, Turowski P, Sakkas E, Niess G, Becker S, Herold S,
Mayer K, Vadász I, Roberts JD Jr, Seeger W, Morty RE. Deregulation of the lysyl hydroxylase matrix cross-linking system in experimental and clinical bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol
Physiol 306: L246–L259, 2014. First published November 27, 2013;
doi:10.1152/ajplung.00109.2013.—Bronchopulmonary dysplasia (BPD) is a
common and serious complication of premature birth, characterized
by a pronounced arrest of alveolar development. The underlying
pathophysiological mechanisms are poorly understood although perturbations to the maturation and remodeling of the extracellular matrix
(ECM) are emerging as candidate disease pathomechanisms. In this
study, the expression and regulation of three members of the lysyl
hydroxylase family of ECM remodeling enzymes (Plod1, Plod2, and
Plod3) in clinical BPD, as well as in an experimental animal model of
BPD, were addressed. All three enzymes were localized to the septal
walls in developing mouse lungs, with Plod1 also expressed in the
vessel walls of the developing lung and Plod3 expressed uniquely at
the base of developing septa. The expression of plod1, plod2, and
plod3 was upregulated in the lungs of mouse pups exposed to 85% O2,
an experimental animal model of BPD. Transforming growth factor
(TGF)-␤ increased plod2 mRNA levels and activated the plod2
promoter in vitro in lung epithelial cells and in lung fibroblasts. Using
in vivo neutralization of TGF-␤ signaling in the experimental animal
model of BPD, TGF-␤ was identified as the regulator of aberrant
plod2 expression. PLOD2 mRNA expression was also elevated in
human neonates who died with BPD or at risk for BPD, compared
with neonates matched for gestational age at birth or chronological
age at death. These data point to potential roles for lysyl hydroxylases
in normal lung development, as well as in perturbed late lung
development associated with BPD.
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LYSYL HYDROXYLASES IN BRONCHOPULMONARY DYSPLASIA
MATERIALS AND METHODS
Mouse model of BPD. All animal procedures were approved by the
Regierungspräsidium Gießen (which houses the functional equivalent
of an Institutional Animal Care and Use Committee in Germany)
under approval 22/2000, for animal studies conducted in Germany.
An arrest of alveolarization was induced in mouse pups by exposure
to normobaric hyperoxia (85% O2), exactly as described previously
(3). Mouse pups were randomized to two groups, one group exposed
to normobaric normoxia (21% O2) and the other group exposed to
normobaric hyperoxia (85% O2), within 12 h of birth [postnatal day
(P) 0.5]. This model has previously been described and carefully
characterized by the investigators (3, 23) and other groups (10, 14,
27), where a pronounced arrest of lung development is seen in
response to hyperoxia exposure.
Cells. The A549 cell line was obtained from the American Type
Culture Collection. Primary mouse lung fibroblasts and alveolar type
II cells were isolated from the lungs of adult C57Bl/6J mice, and
primary lung fibroblasts were isolated from human lungs, exactly as
described previously (2, 25). Primary human lung microvascular
endothelial cells and primary human pulmonary artery smooth muscle
cells were purchased from Promocell and maintained as recommended by the manufacturer.
Gene and protein expression analysis. By convention, mouse genes
and proteins are indicated in lower case (for example, plod1 and
Plod1, respectively), and human genes and proteins are indicated in
upper case (for example PLOD1 and PLOD1, respectively). Real-time
RT-PCR was undertaken exactly as described previously (2, 25),
using mouse lung, human lung, and whole cell mRNA pools as a
template, with the primers listed in Table 1. For TGF-␤ stimulation,
cells were exposed to TGF-␤1 (2 ng/ml final concentration; R&D
Systems) for 18 h. This represents a dose well within the standard
range (0.2–10.0 ng/ml) for in vitro TGF-␤1 stimulation studies (3, 23).
Immunoblotting was undertaken exactly as described previously,
using the following primary antibodies: goat anti-Plod1 (SC-50062,
1:200; Santa Cruz Biotechnology), goat anti-Plod2 (SC-50067, 1:200;
Santa Cruz Biotechnology), rabbit anti-Plod3 (11027–1-AP, 1:200;
Protein Tech), and rabbit ␣-tubulin (SC-5286, 1:2,500; Santa Cruz
Biotechnology). Immune complexes were detected with the following
secondary antibodies: donkey anti-goat IgG horseradish peroxidase
conjugate (SC-2020, 1:1,000; Santa Cruz Biotechnology) and goat
anti-rabbit IgG horseradish peroxidase conjugate (31460, 1:3,000;
Pierce), employing enhanced chemiluminescence.
Immunohistochemistry. Lungs from mouse pups were pressure
fixed at 20 cm H2O pressure, embedded in paraffin, and 3-␮m sections
were prepared from mouse and human lungs and were processed for
the detection of lysyl hydroxylases exactly as described previously (2,
Table 1. Primers employed in this study
Gene*
Human genes
PLOD1
PLOD2
PLOD3
HPRT
PLOD2 promoter
Mouse genes
plod1
plod2
plod3
Hprt
18S rRNA
plod2 promoter
Application
Forward Primer†
Reverse Primer†
Expression
Expression
Expression
Expression
Cloning
analysis
analysis
analysis
analysis
5=-AAGCCGGAGGACAACCTTTTA ⫺3=
5=-CATGGACACAGGATAATGGCTG-3=
5=-GACCCGGTCAACCCAGAGA-3=
5=-AAGGACCCCACGAAGTGTTG-3=
5=-GCTAGCCCATCCTGAGGTAGGGTAAGT-3= (NheI)
5=-CCGAAGAGAATGACCAGATCC-3=
5=-AGGGGTTGGTTGCTCAATAAAAA-3=
5=-CTCCACCAACTGTTCGAGCC-3=
5=-GGCTTTGTATTTTGCTTTTCCA-3=
5=-CTCGAGCCACGTCTGGACTGTTTGCTC-3= (XhoI)
Expression
Expression
Expression
Expression
Expression
Cloning
analysis
analysis
analysis
analysis
analysis
5=-GTTTTTCCTGCTGCTGCTGCG-3=
5=-TGGAGCACTACGCCAGTCAGGA-3=
5=-ACACAGACCACCTACACCCAGACC-3=
5=-GATGATCTCTCAACTTTA-3=
5=-AGGGGAGAGCGGGTAAGAGA-3=
5=-GAGCTCCTTCAACCTCACCTACTCAGT-3= (SacI)
5=-CCGTGCTCCGCCAGGAACTG-3=
5=-AGCCCGTCCGCTGCAAAGAC-3=
5=-TCTGCTCGGTCAGCAGTGGGA-3=
5=-AGTCTGGCCTGTATCCAA-3=
5=-GGACAGGACTAGGCGGAACA-3=
5=-CTCGAGGGAAGGCCTGTGGCGAGGACT-3= (XhoI)
*By convention, mouse genes are indicated in lower case and human genes in upper case. †Engineered restriction sites are indicated in bold type, with the
corresponding restriction enzymes indicated in parentheses.
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which serve as cross-linkable residues. Hydroxylysine residues
are generated by another family of enzymes: the lysyl hydroxylase, or procollagen-lysine, 2-oxoglutarate 5-dioxygenase
(PLOD) (EC 1.14.11.4) family (26). The lysyl hydroxylase
family comprises three members, all of which are products of
separate genes, designated PLOD1, PLOD2, and PLOD3,
which are mixed-function oxygenases that catalyze the hydroxylation of peptidyllysine (usually in protocollagen, as well
as other proteins with collagen-like domains) to peptidylhydroxylysine (26). The hydroxylysine residues generated serve
as substrates for lysyl oxidase, which convert hydroxylysine to
hydroxyallysine, which is a precursor for a covalent cross-link.
Additionally, the hydroxylysine residues serve as acceptor sites
for sugars, permitting glycosylation of ECM proteins (26).
Indeed, PLOD3, in addition to lysine hydroxylation activity,
also possesses glucosyltransferase and galactosyltransferase
activities. Thus lysyl hydroxylases promote ECM structural
stability and maturation by promoting inter- and intramolecular
cross-links and the addition of carbohydrate moieties to ECM
molecules (26).
These lysyl hydroxylases have been implicated in connective tissue disorders, exemplified by Ehlers-Danlos Syndrome
type VI, where mutations in the PLOD1 gene cause a heritable
disorder characterized by kyphoscoliosis, joint laxity, skin
fragility, and muscle hypertonia (9, 50). PLOD2 is implicated
in pathological processes, where increased PLOD2 expression
in fibroblasts is associated with systemic sclerosis (dermal
fibroblasts) (39) and fibrosis (fibroblasts isolated from keloid,
hypertrophic scars, and the palmar fascia of patients with
Dupuytren’s disease) (46), and mutations in the PLOD2 gene
are associated with Bruck’s Syndrome, a recessively inherited
ECM disorder presenting with skeletal changes comparable to
osteogenesis imperfecta with contractures of the large joints
(18). More recently, mutations in PLOD3 have been linked to
a severe connective tissue disorder reminiscent of Stickler
Syndrome (36). However, to date, no lysyl hydroxylase has
been implicated in normal or pathological processes in the
lung. Given the emerging importance of perturbed ECM remodeling in arrested lung development, the authors hypothesized that lysyl hydroxylase expression was deregulated during
aberrant late lung development associated with BPD.
L248
LYSYL HYDROXYLASES IN BRONCHOPULMONARY DYSPLASIA
In vivo TGF-␤ neutralization experiments. The inhibition of TGF-␤
signaling in the lungs of mice exposed to normobaric hyperoxia, using
the pan-TGF-␤-neutralizing IgG1 antibody 1D11 (R&D Systems),
along with the isotype-matched nonimmune IgG1 antibody MOPC21
(Sigma) that has been described previously by the investigators (23)
and other groups (27). This treatment partially normalizes lung structure in oxygen-injured lungs (27).
Human patient material. All use of human material was approved
by the Ethik-Kommission (the German equivalent of an institutional
review board) of the University of Giessen School of Medicine under
approval number 189/09. The harvesting of lung material from preterm and term neonates has been previously described in detail (23).
The clinical characteristics of patients afflicted with BPD or at risk for
the development of BPD are provided in Table 2. Additionally, two
control patients groups are also described in Table 3, where one
control group was age matched to the BPD group for chronological
age at death (CAD), whereas the other control group was age matched
to the BPD group for gestational age at birth (GAB).
RESULTS
Lysyl hydroxylase expression was deregulated in the lungs
of neonatal mice exposed to 85% O2. The mRNA expression
levels of plod1 (Fig. 1A), plod2 (Fig. 1B), and plod3 (Fig. 1C)
in the lungs of mouse pups were regulated over the first 2 wk
of normal lung development and were altered by exposure to
85% O2. Over the course of normal late lung development, the
alveolarization process in mouse pups proceeds over the first 2
wk of life and is largely completed by P14, which is followed
by a phase of microvascular maturation. During these first 2 wk
of life, the bulk of the alveolar gas exchange units is formed.
Our data reveal that, over this period, there is a pronounced
downregulation of lysyl hydroxylase gene expression, where
both plod1 (Fig. 1A) and plod3 (Fig. 1C) mRNA levels were
reduced by 0.5 ⌬Ct units by P9.5 (vs. P2.5), and 5 days later the
expression of all three genes, plod1 (Fig. 1A), plod2 (Fig. 1B),
Table 2. Clinical characteristics of patients with BPD or at risk for the development of BPD
Birth
Weight, g
Sex
720
M
29
1,055
M
3*
835
4*
930
5*
6*
Patient Number
1*
2
Median
Mean ⫾ SE
P value vs.† CAD (Table 3)‡
P value vs. GAB (Table 3)†
Duration FiO2
⬎0.50, days
Mechanical
Ventilation, days
Cause of Death/autopsy Diagnosis and
Medication
62
13
62 (c, hf)
32
18
18
18 (c)
M
26
65
27
65 (c, hf)
M
26
99
98
99 (c, hf)
1,250
F
28
34
34
34 (c, hf)
1,220
M
31
35
35
35 (c, hf)
BPD, IRDS, Staphylococcus aureus sepsis.
Drugs: surfactant, inotropes, tobramycin,
flucloxacillin, cortisone
BPD, ventricular septal defect, Edwards
syndrome. Drugs: inotropes
BPD, cerebral bleeding, ductus arteriosus.
Drugs: surfactant, inotropes,
dexamethasone, theophylline
BPD, IRDS, pneumothorax, subependymal
hemorrhage. Drugs: surfactant,
inotropes, dexamethasone, tobramycin,
penicillin, amphotericin
BPD, IRDS, Staphylococcus epidermidis
sepsis. Drugs: surfactant, furosemide,
amoxicillin, erythromycin
BPD, IRDS, right ventricular hypertrophy,
anemia, rickets. Drugs: furosemide,
amoxicillin, vancomycin
18
27 ⫾ 8
33
38 ⫾ 9
930
976 ⫾ 77
0.0016
0.593 (NS)
Gestational
Age, wk
28
28 ⫾ 0.9
0.0039
0.1776 (NS)
CAD, days
33
38 ⫾ 8
02789 (NS)
⬍0.0001
Inotropes included dopamine, dobutamine, and adrenaline. *Patient had clinically-defined BPD. †By unpaired Student t-test. In the case of chronological age
at death (CAD), the postmenstrual ages at death, rounded to the nearest full week, were compared. BPD, bronchopulmonary dysplasia; c, conventional ventilation;
hf, high-frequency ventilation; GAB, gestational age at birth; IRDS, infant respiratory distress syndrome; NS, not significant.
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25). Lysyl hydroxylases were detected using the following primary
antibodies: goat anti-Plod1 (SC-50062, 1:50; Santa Cruz Biotechnology), goat anti-Plod2 (SC-50067, 1:25; Santa Cruz Biotechnology),
rabbit anti-Plod3 (11027–1-AP, 1:25; Protein Tech). Staining specificity was assessed by preadsorbing with a 100-fold molar excess of a
competing peptide C-19 (SC-50062P; Santa Cruz Biotechnology) for
Plod1, competing peptide N-15 (SC-50067P; Santa Cruz Biotechnology), and a glutathione-S-transferase (GST)-Plod3 fusion protein
(Ag1480; Protein Tech) for Plod3. Immune complexes were detected
with biotinylated Histostain secondary antibodies: biotinylated rabbit
anti-mouse (95– 6543B, “ready to use”; Invitrogen) and biotinylated
rabbit anti-goat (A10518, 1:1,000; Invitrogen), followed by a Streptavidin-horseradish peroxidase complex colorimetric detection system.
Cloning of the mouse plod2 and human PLOD2 promoters. The
mouse plod2 promoter was cloned by PCR amplification of a 2,767
base-pair fragment using the forward and reverse primers: 5=GAGCTCCTTCAACCTCACCTACTCAGT-3= (forward; SacI) and
5=-CTCGAGGGAAGGCCTGTGGCGAGGACT-3= (reverse; XhoI),
containing built-in restriction sites (in bold type) and mouse lung
genomic DNA as a template. The mouse plod2 promoter sequence has
been deposited in the GenBank database under accession number
FJ416599. Similarly, the human PLOD2 promoter was cloned by PCR
amplification of a 3,196 base-pair fragment using forward and reverse
primers: 5=-GCTAGCCCATCCTGAGGTAGGGTAAGT-3= (forward; NheI) and 5=-CTCGAGCCACGTCTGGACTGTTTGCTC-3=
(reverse; XhoI), respectively, and human lung genomic DNA as a
template. The human PLOD2 promoter sequence has been deposited
in the GenBank database under accession number KC788822. Sequences were initially T/A cloned into pGEM T-Easy (Promega) and
then subcloned into pGL3-basic (Promega) using the restriction sites
built into the primers to create pGL3-plod2 (for mouse plod2 promoter) and pGL3-PLOD2 (for human PLOD2 promoter).
Dual luciferase reporter assay. The dual luciferase reporter assay
was performed exactly as described previously (2, 25), using the
firefly luciferase-expressing pGL3-plod2 and pGL3-PLOD2 constructs described above, along with the Renilla luciferase-expressing
pRLTK (Promega) normalization construct.
LYSYL HYDROXYLASES IN BRONCHOPULMONARY DYSPLASIA
L249
Table 3. Clinical characteristics of control patients
Patient Number
Birth Weight,
Gestational Age, CAD, Duration FiO2
Mechanical
g
Sex
wk
days ⬎0.50, days Ventilation, days
7
8
9
10
11
Median
Means ⫾ SE
1,625
2,350
1,740
1,800
1,190
1,740
1,741 ⫾ 186
Cause of Death/autopsy Diagnosis and Medication/material Available
Control group age matched to the BPD group (Table 2) for CAD
M
M
F
M
M
33
35
34
32
31
33
33 ⫾ 0.7
3
⬍1
⬍1
5
⬍1
0
0
0
0
0
0
⬍1 h
0
5
0
Congenital heart malformation. Drugs: atropine, prostaglandin A1 (r)
Perinatal asphyxia. Drugs: atropine, adrenaline (h⫹r)
Intrauterine death (ventriculomegaly) (r)
Meningoencephalitis (h⫹r)
Placental abruption (h⫹r)
Control group matched to the BPD group (Table 2) for birth weight and GAB
954
914
826
852
995
758
914
929 ⫾ 55
M
M
M
M
M
M
26
27
29
27
27
26
27
27 ⫾ 0.4
⬍1
⬍1
⬍1
⬍1
⬍1
⬍1
0
0
0
0
0
0
0
0
0
0
0
0
Intracranial hemorrhage (h⫹r)
DiGeorge syndrome (22q11.2 deletion); intrauterine infection (h⫹r)
Hydrocephalus, Arnold Chiari malformation (h⫹r)
Hypoxic-ischemic encephalopathy (r)
Placental abruption (h⫹r)
Intrauterine infection (h⫹r)
h, histological sections; r, RNA.
and plod3 (Fig. 1C) was dramatically downregulated by 3– 4
⌬Ct units, comparing P14.5 with P9.5 (comparing normoxiatreated groups only, for normally developing lungs). In sum,
lysyl hydroxylase gene expression was progressively downregulated in normally developing lungs of mouse pups over the
first 14 days of life. This trend is strongly impacted (and to a
degree, reversed) by exposure to 85% O2, where levels of all
three genes were comparable between the 21% O2 and 85% O2
groups at P2.5, but by P9.5 both plod1 (Fig. 1A) and plod3
(Fig. 1C) mRNA levels were increased in the lungs of mouse
pups exposed to 85% O2. By P14.5, the levels of plod1 (Fig.
1A), plod2 (Fig. 1B), and plod3 (Fig. 1C) mRNA were all
elevated by 2– 4 ⌬Ct units in the 85% O2 group, compared with
the 21% O2 group. The ability of 85% O2 to maintain elevated
lysyl oxidase expression was also evident in immunoblots,
where an increase in Plod1 and Plod2 protein levels was
evident in the lungs of 85% O2-exposed mouse pups, compared
with 21% O2-exposed littermates (Fig. 1D). In sum, although
lysyl hydroxylase expression is gradually downregulated over
the course of normal alveolarization, exposure to 85% O2 leads
to abnormally elevated levels of lysyl hydroxylases, over the
course of aberrant alveolarization in mouse pups.
Fig. 1. Expression of lysyl hydroxylases during normal and aberrant late lung development. Expression levels of mRNA encoding plod1 (A), plod2 (B), and plod3
(C) were assessed by real-time RT-PCR in mRNA pools from lung homogenates from mouse pups at postnatal days (P) 2.5, 9.5, and 14.5, after exposure to 21%
O2 () or 85% O2 (o) from P0.5. The 18S rRNA was used as a reference. Data reflect means ⫾ SE (n ⫽ 5). The P values, indicated above pairs of data sets,
were assessed by 1-way ANOVA with Tukey’s post hoc test. D: protein expression of Plod1, Plod2, and Plod3 was assessed by immunoblot of protein extracts
from lung homogenates from mouse pups over the course of late lung development, during exposure to 21% O2 or 85% O2 from P0.5. A single representative
series is illustrated that is representative of at least 2 other series.
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12
13
14
15
16
17
Median
Means ⫾ SE
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LYSYL HYDROXYLASES IN BRONCHOPULMONARY DYSPLASIA
Lysyl hydroxylases have specific expression patterns in the
developing mouse lung. To assess where deregulated lysyl
hydroxylase expression may impact late lung development, the
localization of the three lysyl hydroxylases was determined by
immunohistochemistry. Plod1 could be localized to the parenchyma, where Plod1 staining was observed in developing septa
of mouse pups at P7 exposed to 21% O2 (Fig. 2A) and 85% O2
(Fig. 2B). Plod1 staining was also observed in the blood vessel
walls, appearing to localize to the muscular layer of the vessel
wall in the lungs of mouse pups exposed to 21% O2 (Fig. 2C)
and 85% O2 (Fig. 2D). Staining for Plod1 in the lung vessel
walls appeared to be more intense in the 85% O2-treated mouse
pups (Fig. 2D) compared with the 21% O2-treated mouse pups
(Fig. 2C) although the authors do not consider the relative
intensity of immunohistochemical staining to be quantitative.
Plod2 was also detected in the developing septa of mouse pups
at P7 (Fig. 3, A and B), with staining again appearing more
intense in the 85% O2-treated group (Fig. Fig. 3B) compared
with the 21% O2-treated group (Fig. 3A). In contrast to Plod1,
no Plod2 staining was evident in the vessel wall of mouse pups
at P7 exposed to 21% O2 (Fig. 3C) and 85% O2 (Fig. 3D).
Staining for Plod3 revealed a very specific and discrete expres-
21% O2
85% O2
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A
Plod1 (Parenchyma)
B
aw
C
D
Plod1 (Vessel wall)
v
aw
v
F
E
Plod1 (competing
peptide control)
v
v
aw
aw
v
200 µm
Fig. 2. Localization of Plod1 expression in the parenchyma (A and B) and vessel walls (C and D) of the lungs of mouse pups at P7.5, after exposure to 21%
O2 or 85% O2 from P0.5. The airways (aw) and vessels (v) are indicated. E: staining for Plod1 in a neonatal mouse lung at P7.5, after exposure to 85% O2 from
P0.5, after preadsorption of the primary anti-Plod1 antibody with a competing peptide, to demonstrate specificity. F: low magnification staining for Plod1 in a
neonatal mouse lung at P7.5 after exposure to 85% O2 from P0.5.
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LYSYL HYDROXYLASES IN BRONCHOPULMONARY DYSPLASIA
21% O2
85% O2
B
C
D
aw
v
Plod2 (competing peptide control)
v
E
F
v
v
aw
aw
200 µm
Fig. 3. Localization of Plod2 expression in the parenchyma (A and B) and vessel walls (C and D) of the lungs of mouse pups at P7.5, after exposure to 21%
O2 or 85% O2 from P0.5. The airways and vessels are indicated. E: staining for Plod2 in a neonatal mouse lung at P7.5, after exposure to 85% O2 from P0.5,
after preadsorption of the primary anti-Plod2 antibody with a competing peptide, to demonstrate specificity. F: low magnification staining for Plod2 in a neonatal
mouse lung at P7.5 after exposure to 85% O2 from P0.5.
sion pattern, specifically at the base of the developing septa
(Fig. 4, A and B), which localizes Plod3 to a place of intense
activity in the developing lung and, hence, in a position where
Plod3 may influence alveolar development. No appreciable
staining for Plod3 was evident in the muscular layer of the
vessel walls in the lungs of mouse pups at P7 exposed to 21%
O2 (Fig. 4C) and 85% O2 (Fig. 4D) although staining in the
endothelium of the latter group was evident (Fig. 4D). Together, these data reveal that lysyl hydroxylases have discrete
expression patterns in the developing mouse lung and that the
detection of all three lysyl hydroxylases in the developing septa
places all three members of this family of matrix cross-linking
enzymes in a position to influence secondary septation and,
hence, alveolarization.
TGF-␤ regulates mRNA levels of lysyl hydroxylases in
constituent cells types of the developing lung. TGF-␤ is acknowledged as a key regulator of late lung development and
alveolarization. TGF-␤ is also a potent profibrotic growth
factor and has been ascribed a key role in the pathogenesis
of BPD. Furthermore, we identified multiple Smad-binding
elements (which confer TGF-␤ responsiveness) in the plod2
and PLOD2 promoters, by in silico analysis. For these
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Plod2 (Vessel wall)
Plod2 (Parenchyma)
A
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LYSYL HYDROXYLASES IN BRONCHOPULMONARY DYSPLASIA
21% O2
85% O2
B
Plod3 (Parenchyma)
A
v
D
Plod3 (Vessel wall)
v
aw
aw
Plod3 (competing antigen control)
v
E
F
v
aw
aw
200 µm
v
Fig. 4. Localization of Plod3 expression in the parenchyma (A and B) and vessel walls (C and D) of the lungs of mouse pups at P7.5, after exposure to 21%
O2 or 85% O2 from P0.5. The airways and vessels are indicated. E: staining for Plod3 in a neonatal mouse lung at P7.5, after exposure to 85% O2 from P0.5,
after preadsorption of the primary anti-Plod3 antibody with a competing antigen, to demonstrate specificity. F: low magnification staining for Plod3 in a neonatal
mouse lung at P7.5 after exposure to 85% O2 from P0.5.
reasons, the ability of TGF-␤ to impact lysyl hydroxylase
expression in constituent cell types of the developing alveolus was assessed. TGF-␤ exhibited a broad spectrum of
effects on lysyl hydroxylase gene expression, where an 18-h
exposure of A549 cells, a commonly used model of the human
lung epithelium, to a dose of 2 ng/ml TGF-␤, upregulated PLOD2
expression (Fig. 5A). In contrast, in primary mouse alveolar type
II cells, TGF-␤ was without effect on plod2 mRNA levels but did
upregulate plod3 mRNA expression (Fig. 5B). TGF-␤ dramatically increased PLOD2 mRNA levels in primary human lung
fibroblasts (Fig. 5C) and dramatically increased plod2 mRNA
levels in primary mouse lung fibroblasts (Fig. 5D). TGF-␤
also upregulated PLOD3 mRNA levels in primary human
lung microvascular endothelial cells (Fig. 5E) although
TGF-␤ was without any impact on lysyl hydroxylase mRNA
levels in primary human pulmonary artery smooth muscle
cells (Fig. 5F). Thus TGF-␤ appears to be a mediator of
plod2 and plod3 expression in constituent cells types of the
alveolus.
TGF-␤ regulated PLOD2 promoter activity in human lung
fibroblasts and epithelial cells. TGF-␤ increased the activity of
the human PLOD2 promoter in human lung fibroblasts (Fig.
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LYSYL HYDROXYLASES IN BRONCHOPULMONARY DYSPLASIA
L253
6A) and in A549 cells (Fig. 6B). The specificity of the effect of
TGF-␤ was confirmed by the observation that a pan-TGF-␤neutralizing antibody (1D11) was able to abrogate the effects
of TGF-␤, while an isotype-matched control antibody
(MOPC21) was not (Fig. 6, A and B). These data support the
observation that TGF-␤ increased the abundance of PLOD2
mRNA in human lung fibroblasts (Fig. 5C) and in A549 cells
(Fig. 5A) and support the contention that TGF-␤ is a regulator
of PLOD2 expression in constituent cells types of the developing alveolus.
Fig. 6. Regulation of PLOD2 promoter activity by TGF-␤. The activity of the human
PLOD2 promoter was assessed by dual luciferase assay (DLR) in primary human lung
fibroblasts (A), as well as human lung A549
cells (B), as a transfectable model of the
alveolar epithelium, after preincubation (30
min, 37°C; in stimulation medium) of
TGF-␤ with a pan-TGF-␤-neutralizing antibody (1D11) or an isotype-matched control
(MOPC21) antibody. Data reflect means ⫾
SD (n ⫽ 5). The P values (above the horizontal line) represented compare vehicle- vs.
TGF-␤-treated groups, or MOPC21 vs.
1D11 control groups and were assessed by
unpaired Student’s t-test.
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Fig. 5. Regulation of lysyl hydroxylase
mRNA levels by TGF-␤. PLOD1, PLOD2,
and PLOD3 expression was assessed by
real-time RT-PCR using the primers listed in
Table 1, in mRNA pools from human A549
cells (A), as well as in primary human lung
fibroblasts (C), microvascular endothelial
cells (E), and pulmonary artery smooth muscle cells (F) after stimulation with vehicle
alone or TGF-␤ (2 ng/ml; 16 h). The HPRT
gene was used as a reference. Similarly,
plod1, plod2, and plod3 expression was assessed by real-time RT-PCR using the primers listed in Table 1, in mRNA pools from
primary mouse lung alveolar type II cells
(B), and mouse lung fibroblasts (D) after
stimulation with vehicle alone or TGF-␤ (2
ng/ml; 16 h). The hprt gene was used as a
reference. Data reflect means ⫾ SD (n ⫽ 3).
The P values (above the horizontal line)
represented compare vehicle- vs. TGF-␤treated groups and were assessed by unpaired Student’s t-test.
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LYSYL HYDROXYLASES IN BRONCHOPULMONARY DYSPLASIA
Exposure to 85% O2 drove aberrant plod2 expression in the
developing mouse lung via TGF-␤. Neutralization of TGF-␤
signaling in vivo in mouse pups that were exposed to 85% O2
partially normalized elastin structures in the developing septa
(Fig. 7C; arrowheads) because these structures largely resembled elastin structures in the developing septa of mouse lungs
exposed to 21% O2 (Fig. 7A; arrowheads), compared with the
perturbed elastin structures seen in the septa of developing
lungs in mice that were exposed to 85% O2 that received a
control MOPC21 antibody (Fig. 7B; arrows). These data support previous observations (23, 27) that neutralization of
TGF-␤ signaling in the hyperoxia model of BPD partially
restores normal structure to the developing lung.
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PBS
21% O2
A
50 µm
MOPC21
B
C
1D11
85% O2
50 µm
50 µm
Fig. 7. Impact of in vivo neutralization of TGF-␤ signaling on hyperoxiainduced changes to elastin structure in the developing septa. Sections of
developing mouse lungs at P10.5 were stained for elastin using Hart’s stain,
after treatment with phosphate-buffered saline (PBS) and exposure to 21% O2
(A), with a control MOPC21 antibody and exposure to 85% O2 (B), or
treatment with a pan-TGF-␤-neutralizing antibody and exposure to 85% O2
(C). Arrowheads indicate the normal, punctate elastin structures, whereas
arrows indicate the disturbed “brush-like” elastin structures in the septa
associated with aberrant late lung development.
Fig. 8. Impact of in vivo neutralization of TGF-␤ signaling on hyperoxiainduced lysyl hydroxylase expression. Expression levels of mRNA encoding
plod1 (A), plod2 (B), and plod3 (C) were assessed by real-time RT-PCR in
mRNA pools from lung homogenates from mouse pups at P10.5, after
exposure to 21% O2 (open bars) or 85% O2 (closed bars) from P0.5 on, with
concomitant administration of a pan-TGF-␤-neutralizing antibody (1D11) or
an isotype-matched control (MOPC21) antibody. The 18S rRNA was used as
a reference. Data reflect means ⫾ SD (n ⫽ 6, per group). The P values
(presented below the horizontal brackets) were assessed by 1-way ANOVA
with Tukey’s post hoc test.
Neutralization of TGF-␤ signaling in vivo in mouse pups
that were exposed to 85% O2 did not impact plod1 expression
in the lungs of mouse pups (Fig. 8A); however, TGF-␤ neutralization did restore normal plod2 expression in the lungs of
mouse pups (Fig. 8B). As with plod1, no impact of TGF-␤
neutralization on plod3 expression was noted (Fig. 8C). Al-
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LYSYL HYDROXYLASES IN BRONCHOPULMONARY DYSPLASIA
21% O2
L255
85% O2
B
MOPC21
A
50 m
50 m
1D11
D
50 m
50 m
Fig. 9. Impact of in vivo neutralization of TGF-␤ signaling on hyperoxia-induced changes to Plod2 localization. Sections of developing mouse lungs at P10.5
were stained for Plod2 after treatment with a control MOPC21 antibody and exposure to 21% O2 (A) or 85% O2 (B), or after treatment with a
pan-TGF-␤-neutralizing antibody and exposure to 21% O2 (C) or 85% O2 (D).
though the authors do not consider the intensity of immunohistochemical staining to be quantitative, staining intensity for
Plod2 in the developing septa of mouse pup lungs exposed to
85% O2 that received a TGF-␤-neutralizing antibody (Fig. 9D)
appeared less intense when compared with mouse pup lungs
exposed to 85% O2 that received a control (nonimmune)
MOPC21 antibody (Fig. 9B). Together, these data suggest that
TGF-␤ mediated the effects of hyperoxia exposure on Plod2
expression in the aberrantly developing mouse lung.
Elevated PLOD2 expression is associated with BPD. The
mRNA levels of PLOD1 (Fig. 10A), PLOD2 (Fig. 10B), and
PLOD3 (Fig. 10C) were assessed in mRNA pools from the
lungs of patients that had died with BPD or at risk for BPD, as
well as in two control groups, one control group age matched
to the BPD group for gestational age at birth (GAB) and
another control group age matched to the BPD group for
chronological age at death (CAD). No appreciable differences
were observed comparing the expression of PLOD1 (Fig. 10A)
Fig. 10. Expression of lysyl hydroxylases in human neonates with normal lung development or in neonates afflicted with, or at risk for, bronchopulmonary
dysplasia (BPD). PLOD1 (A), PLOD2 (B), and PLOD3 (C) expression was assessed by real-time RT-PCR using the primers listed in Table 1, in mRNA pools
from patients afflicted with or at risk for BPD (Table 2), from control patients matched to the BPD group for gestational age at birth (GAB; Table 3), or from
control patients matched to the BPD group for chronological age at death (CAD; Table 3). The HPRT gene was used as a reference. The bars represent the data
range, the boxes represent the lower and upper quartiles, and the line within the quartile box indicates the median. No outliers were noted by Grubbs’ test. The
P values represented compare the 3 patient groups and were assessed by 1-way analysis of variance with the Newman-Keuls post hoc test (see Tables 2 and 3
for number of patients per group).
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C
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LYSYL HYDROXYLASES IN BRONCHOPULMONARY DYSPLASIA
DISCUSSION
The proper deposition and posttranslational modification
and remodeling of the lung ECM is critical to normal lung
development, including the formation of the alveolar air sacs,
the principal gas exchange unit of the lung, which takes place
during late lung development. Perturbations to the proper
deposition, processing, and remodeling of the lung ECM are
credited as being a key underlying cause of disturbed late lung
development. Clinically, disturbed late lung development is
A
exemplified in humans by BPD, also called chronic lung
disease of early infancy. BPD results from the mechanical
ventilation and/or oxygen supplementation of premature infants, which then exhibit a pronounced arrest of late lung
development (19), where alveolar simplification is evident, as
well as blunted lung microvascular development (41) and
dysangiogenesis (13). The pathomechanisms at play in the
lung of affected infants are not well understood. However,
several histopathological observations have pointed to dramatically malformed matrix structures in patients with BPD,
including collagen fibers in the alveolar septa that were
described to be “thickened, tortuous, and disorganized”
(43). These observations have also been made examining
lungs of experimental animals where BPD has been modeled, including in ventilated preterm lambs (1, 6, 30),
ventilated mouse pups (5, 8), and mouse pups exposed to
elevated oxygen concentrations (23, 49).
To date, little information is available about how and why
ECM structures are deformed in lungs in which alveolarization
has been disturbed and whether these perturbed matrix structures play a causal role in arresting the alveolarization process.
One theory put forward is that the matrix processing and
maturations systems, which remodel the ECM structures, may
be disturbed. Along these lines, deregulated expression and
activity of matrix metalloproteinases, which remodel ECM
structures, have been implicated in BPD. Another theory for-
B
s
aw
100 μm
a
VSM
v
v
s
100 μm
100 μm
C
D
aw
v
100 μm
100 μm
Endo
s
100 μm
s
a
a
v
100 μm
Fig. 11. Plod expression in the developing lungs of human neonates. The tissue localization of Plod1 (A and B; patient 8), Plod2 (C; patient 10), and Plod3 (D;
patient 8) was assessed by immunohistochemistry, and specificity controls were performed with competing peptides for Plod1 (B; inset) and Plod2 (C, inset),
and with a competing antigen for Plod3 (D, inset). The airways, alveolar airspaces (a), endothelium (Endo), vascular smooth muscle (VSM), alveolar septa (s),
and vessels are indicated. Staining patterns are representative for patterns observed in at least 2 other series.
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and PLOD3 (Fig. 10C) across all three groups. However, the
mRNA levels of PLOD2 were appreciably elevated in the
lungs of patients with or at risk for BPD, compared with both
the GAB and CAD groups (Fig. 10C). These data demonstrate
that the expression of lysyl hydroxylases, namely the PLOD2
gene, is deregulated in the lungs of infants with or at risk for
BPD. All three Plod proteins were detected in the developing
lungs of human neonates by immunohistochemistry, with
Plod1 evident in the airway epithelium, vascular smooth muscle, and in the septa (Fig. 11, A and B). Plod2 was evident in
the endothelium, airway epithelium, and developing septa (Fig.
11C), whereas Plod3 was evident in the septa (Fig. 11D). It is
evident from these data that Plod2, which has emerged from
this study as a candidate player in the pathogenesis of BPD, is
present at sites of intensive ECM production and remodeling in
the developing human lung.
LYSYL HYDROXYLASES IN BRONCHOPULMONARY DYSPLASIA
Curiously, Plod3 exhibited the most restricted pulmonary
expression of all the lysyl hydroxylases and was localized to
the base of the developing septa, which is a region of intense
tissue remodeling during the alveolarization process. Plod3 is a
bifunctional enzyme that catalyzes the formation of hydroxylysine in collagen polypeptides and can also transfer sugars to
these residues by virtue of glucosyltransferase and galactosyltransferase activities. Unlike Plod1 and Plod2, Plod3 is present
both intracellularly and is also secreted (47) and has a critical
function in the glycosylation of type IV and type VI collagen
that is undertaken by the galactosylhydroxylysyl glucosyltransferase (GGT) activity of Plod3, which regulates type IV and
type VI collagen secretion and assembly (31, 40). Disrupting
the GGT activity of Plod3 disrupts the localization of type IV
collagen and, hence, formation of the basement membrane,
leading to embryonic lethality of plod3⫺/⫺ mice at embryonic
day (E) 9.5 (34). The plod3⫺/⫺ mouse embryos exhibited a
30% reduction in the number of hydroxylysine residues in the
type IV and type V collagen fractions of mouse embryo lungs
(and kidneys) although the levels of hydroxylysine in type I,
type II, and type III collagen fractions of mouse embryo lungs
(and kidneys) remained normal (34). These data point to a role
for Plod3 in the formation of the lung epithelial basement
membrane. Particularly interesting is that the lung exhibits the
highest Plod3 protein abundance and the highest Plod3 GGT
activity of any organ (38). As such, perturbations to Plod3
expression, such as those described to occur during the arrested
lung development in the mouse lungs we describe in this study,
may have a pronounced impact on secondary septation and
alveolar development.
The ability of TGF-␤ to regulate lysyl hydroxylase activity
in constituent cells of the lung alveolus was interesting, given
that TGF-␤ signaling is dynamically regulated during lung
alveolarization (3, 48). Furthermore, excessive TGF-␤ signaling has been associated with BPD (15) because elevated levels
of TGF-␤ are detected in bronchoalveolar lavage fluid of
patients with BPD (4, 22) and that TGF-␤ has been causally
implicated in the pathogenesis of experimental BPD, where
inhibition of TGF-␤ signaling by elafin (16, 17), curcumin
(35), and neutralizing antibodies (23, 27) attenuated the deleterious impact of hyperoxia on lung structure and function, and
other avenues for the attenuation of TGF-␤ signaling in fetal
sheep lungs, such as antenatal glucocorticoids, are currently
being explored (11, 12). Additionally, TGF-␤ signaling is
upregulated in the lungs of mouse pups in which alveolarization has been arrested by exposure to hyperoxia (2). Consistent
with the findings that Plod2 is upregulated in a TGF-␤dependent manner in synovial fibrosis (32), and in dermal
fibroblasts (44), TGF-␤ dramatically upregulated PLOD2 expression and activated the PLOD2 promoter in primary human
lung fibroblasts and A549 cells. Similarly, plod2 expression
was upregulated by TGF-␤ in primary mouse lung fibroblasts.
These data suggested that TGF-␤ may drive plod2 expression
in the BPD rodent model, with the caveat that these studies
were performed in cell lines or primary cells derived from
adult, not neonatal mice. To address this, TGF-␤ signaling was
neutralized in the lungs of hyperoxia-exposed mouse pups.
TGF-␤ neutralization, which partially normalized alveolarization in the BPD model, normalized plod2 gene expression,
identifying TGF-␤ as the mediator of aberrant plod2, but not
plod1 or plod3 expression, in hyperoxia-exposed mouse pup
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warded is that the ECM stabilization systems, which cross-link
collagen and elastin polypeptides to permit the formation of
stable collagen and elastin fibers, respectively, may be impacted in BPD. These systems include the concerted action of
lysyl hydroxylases, which catalyzes the hydroxylation of peptidyllysine to peptidylhydroxylysine, and generate a substrate
for lysyl oxidases, which convert hydroxylysine to hydroxyallysine, which forms part of the lysyl oxidase-generated covalent cross-links in ECM structures. Indeed, lysyl oxidases have
been studied in the context of clinical and experimental BPD,
where the expression of two members of the lysyl oxidase
family, namely, Lox and LoxL1, is elevated in both patients
that died with BPD, as well as in the lungs of mice in a BPD
rodent model. These data make a strong case for a role for
perturbed matrix cross-linking in arrested lung development.
In the present study, the authors addressed the expression
and regulation of a second family of ECM cross-linking enzymes, that of the lysyl hydroxylases, or procollagen-lysine,
2-oxoglutarate 5-dioxygenases, which comprises three members in humans: PLOD1, PLOD2, and PLOD3. All three
enzymes generate hydroxylysine residues from lysine residues
in collagen peptides (as well as in other peptides with a
collagen-like domain) and provide a key substrate for subsequent intra- and intermolecular cross-linking mediated by the
lysyl oxidases. Additionally, PLOD3 catalyzes the glycosylation of collagen and other molecules.
No roles for any lysyl hydroxylase in lung physiology have
been described to date although several lines of investigation
point at possible pulmonary functions for lysyl hydroxylases.
All three lysyl hydroxylases are abundantly expressed in the
lung (33, 37). The data presented here reveal that the expression of all three lysyl hydroxylases was deregulated in a rodent
model of BPD and that this deregulation took place over the
critical period of late lung development when alveolarization
takes place. Additionally, the localization studies reveal that all
three lysyl hydroxylases are ideally positioned to influence late
lung development because, in the developing mouse lung,
Plod1, Plod2, and Plod3 were all detected in the developing
septa.
Plod1 is dispensable in mice because plod1⫺/⫺ mice are
viable. However, Plod1 appears to play a role in the lung
because the amounts of hydroxylysine residues (per collagen
triple helix) in the lungs of plod1⫺/⫺ mice were reduced by
14% compared with wild-type (plod1⫹/⫹) mice, and the
amounts of hydroxylysylpyridinoline cross-links in the lungs
of plod1⫺/⫺ mice were decreased by 66% (42). In the present
study, Plod1 was detected in the developing septa, as well as in
the lung vessel walls, placing Plod1 in a position that could
influence secondary septation and vascular development.
As with Plod1, no role for Plod2 has been described in the
lung. However, Plod2 is widely implicated in fibrotic processes
and, in particular, with TGF-␤-driven synovial fibrosis associated with osteoarthritis (32), where TGF-␤ has also been
demonstrated to drive Plod2 expression in dermal fibroblasts
(44), and increased PLOD2 expression is seen in fibrotic skin
lesions from patients with systemic sclerosis (45). Plod2 was
detected in the present study in developing septal walls although not in the blood vessel walls. Additionally, Plod2
exhibited the highest expression levels of all three lysyl hydroxylases in human and mouse lung epithelial cells and was
induced by TGF-␤.
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LYSYL HYDROXYLASES IN BRONCHOPULMONARY DYSPLASIA
4.
5.
6.
7.
8.
9.
10.
11.
ACKNOWLEDGMENTS
The authors acknowledge the constructive discussions and provision of
reagents by Richard D. Bland, Thomas J. Mariani, Robert Mecham, Richard A.
Pierce, Dick Tibboel, and David Warburton.
12.
GRANTS
This study was supported by the German Research Foundation through
Grant Mo 1789/1 (to R. Morty) and Excellence Cluster 147 “Cardio-Pulmonary System” (to S. Herold, K. Mayer, I. Vadász, W. Seeger, and R. Morty) as
well as the Federal Ministry of Higher Education, Research and the Arts of the
State of Hessen LOEWE Program (to S. Herold, K. Mayer, I. Vadász, W.
Seeger, and R. Morty), the German Center for Lung Research (Deutche
Lungenforschungszentrum; all German authors), and U.S. National Heart,
Lung, and Blood Institute Grant HL-094608 (to J. Roberts).
13.
14.
15.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
16.
Author contributions: T.J.W., P.T., E.S., W.S., and R.E.M. conception and
design of research; T.J.W., P.T., E.S., G.N., S.B., K.M., and J.D.R. performed
experiments; T.J.W., P.T., G.N., S.H., K.M., I.V., J.D.R., W.S., and R.E.M.
analyzed data; T.J.W., S.H., I.V., W.S., and R.E.M. interpreted results of
experiments; T.J.W., P.T., E.S., G.N., S.B., S.H., K.M., I.V., J.D.R., W.S., and
R.E.M. approved final version of manuscript; E.S., G.N., and R.E.M. prepared
figures; S.H., I.V., and R.E.M. drafted manuscript; K.M., I.V., and R.E.M.
edited and revised manuscript.
17.
18.
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The development of specific inhibitors or floxed alleles for
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