Download Pulmonary Hypertension in Transgenic Mice Expressing a Dominant

Pulmonary Hypertension in Transgenic Mice Expressing a
Dominant-Negative BMPRII Gene in Smooth Muscle
James West, Karen Fagan, Wolfgang Steudel, Brian Fouty, Kirk Lane, Julie Harral,
Marloes Hoedt-Miller, Yuji Tada, John Ozimek, Rubin Tuder, David M. Rodman
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Abstract—Bone morphogenetic peptides (BMPs), a family of cytokines critical to normal development, were recently
implicated in the pathogenesis of familial pulmonary arterial hypertension. The type-II receptor (BMPRII) is required
for recognition of all BMPs, and targeted deletion of BMPRII in mice results in fetal lethality before gastrulation. To
overcome this limitation and study the role of BMP signaling in postnatal vascular disease, we constructed a smooth
muscle–specific transgenic mouse expressing a dominant-negative BMPRII under control of the tetracycline gene
switch (SM22-tet-BMPRIIdelx4⫹ mice). When the mutation was activated after birth, mice developed increased
pulmonary artery pressure, RV/LV⫹S ratio, and pulmonary arterial muscularization with no increase in systemic arterial
pressure. Studies with SM22-tet-BMPRIIdelx4⫹ mice support the hypothesis that loss of BMPRII signaling in smooth
muscle is sufficient to produce the pulmonary hypertensive phenotype. (Circ Res. 2004;94:1109-1114.)
Key Words: artery 䡲 bone morphogenetic peptide 䡲 hypertension 䡲 smooth muscle 䡲 vascular
A
ssociation between abnormal bone morphogenetic peptide (BMP) signaling and pulmonary vascular disease
was suggested by genetic studies of familial pulmonary
arterial hypertension (PAH), a disorder characterized by the
pathological development of increased pressure and structural
remodeling in the pulmonary circulation later in life.1,2
Fifteen to 25% of cases of PAH occur in families, with
autosomal-dominant inheritance, and in the year 2000, two
groups independently identified the presence of mutations in
the BMRII gene in familial PAH.3,4 Subsequently, approximately 25% of sporadic cases of PAH were also found to be
associated with mutations in BMPRII.5
Dissecting the function of BMPII using transgenic mice
has identified a critical role for BMP signaling in development.6 Although there is redundancy at the level of ligands,
type I receptors, intra- and extracellular inhibitors and downstream signaling via SMADs, functional BMPRII is an
absolute requirement for BMP signaling. Consequently, BMPRII⫺/⫺ mice die early in development, before gastrulation,
whereas BMPRII⫹/⫺ mice develop normally and have no
apparent phenotype.7 To overcome the developmental lethality identified by traditional transgenic approaches, we constructed a conditional, tissue-specific BMPRII transgenic
mouse, using a smooth muscle cell–specific promotor and a
dominant-negative BMPRII identified in a family with PAH.8
Using these mice, we asked two questions: (1) is expression
of a strong loss-of-function mutation in BMPRII sufficient to
produce pulmonary hypertension, and (2) is loss of BMPRII
function in smooth muscle alone sufficient to produce pulmonary hypertension?
Materials and Methods
Construction of SM22-tet-BMPRIIdelx4ⴙ
Transgenic Mice
The mice were generated at the University of Cincinnati Transgenic
Mouse Science from plasmids we provided. The murine SM22
promotor (488 bp starting 340 bp before the start site and extending
148 bp into the 5⬘ UTR) was used to drive expression of the reverse
tetracycline transactivator (rtTA), and a septad of the tetracycline
response element and minimal CMV promotor were used to drive
expression of the mutant BMPRII mutation on the FVB/N background (Figure 1).9,10 The BMPRII mutation, identified by Machado
et al in family UK21, was insertion of a “T” at base 504 in the kinase
(4th) domain of the protein, resulting in a premature stop 18 amino
acids into the kinase domain.8 We refer to this as BMPRIIdelx4⫹ to
indicate that it is a deletion from the 4th domain onward. After birth,
mothers were fed doxycycline in food (1 mg/g) while nursing, and
after weaning, SM22-tet-BMPRIIdelx4⫹ mice were fed doxycycline
until age 8 weeks when phenotyping was performed. Control mice
were double transgenic littermates not fed doxycycline. All procedures were approved by the University of Colorado institutional
animal use committee.
BMPRII Activity Assay In Vitro
Semiconfluent human pulmonary artery smooth muscle cells
(SMCs) (Clonetics) were cotransfected (lipofectamine PLUS) with a
CMV-driven BMPRIIdelx4⫹ plasmid (or empty vector) and a SMAD-
Original received November 13, 2003; resubmission received January 22, 2004; revised resubmission received March 5, 2004; accepted March 9, 2004.
From the University of Colorado Health Sciences Center, Center for Genetic Lung Disease, Division of Pulmonary Sciences and Critical Care Medicine
and Department of Anesthesia, Denver, Colo; Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt University, Nashville, Tenn; and
the Department of Pathology, Johns Hopkins University, Baltimore, Md.
This manuscript was sent to Donald D. Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Correspondence to David M. Rodman, Center for Genetic Lung Disease, UCHSC Box B133, 4200 E Ninth Ave, Denver, CO 80262. E-mail
[email protected]
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000126047.82846.20
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Circulation Research
April 30, 2004
Results
Validation of Transgene Function and
Tissue Distribution
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Figure 1. Construction of the SM22-tet-BMPRIIdelx4⫹ transgenic
mouse. Transgenic mouse #1 used a 488-bp fragment of the
murine smooth muscle–specific SM22-␣ promotor to drive
expression of the reverse tetracycline transactivator gene (rtTA).
Transgenic mouse #2 used a septad of the tetracycline
response element (TetO7) and minimal CMV promotor to drive
expression of the BMPRIIdelx4⫹ mutant, encoding a receptor with
a premature stop codon early in the kinase domain. Double
transgenic mice have copies of each transgene in all cells. In
SMCs expressing the required transactivating transcription factors, the rtTA gene is transcribed and binds to TetO7 and, in the
presence of doxycycline, the BMPRIIdelx4⫹ mutant gene is
transcribed.
responsive luciferase construct.11 Every well also received a
␤-galactosidase expression plasmid, for normalization to transfection
efficiency. Whereas transfection efficiency was only 15% to 20%,
because the two plasmids were mixed and transfected together, cells
expressing the reporter also expressed the mutant BMP, and thus the
luciferase measurements were not significantly affected by transfection efficiency. Forty-eight hours after transfection, either recombinant human BMP2 (Sigma) or vehicle was added to wells, and after
3 hours, cells were assayed for luciferase activity.
SM22-Driven Transgene Localization In Vivo
SM22-driven rtTA transgenic mice were crossbred with TetO7CMVLuciferase mice (Jackson Labs) or TetO7-lacZ mice (gift of L.
Chodosh, University of Pennsylvania School of Medicine, Philadelphia, Pa). Eight-week-old mice were fed doxycycline in food (1
mg/g) for 4 days, euthanized, and approximately 20 mg of tissue
from each organ homogenized for use with the promega luciferase
assay system, or formalin-fixed tissue used for lacZ
immunohistochemistry.
Phenotyping
Hemodynamic profiling was performed on mice anesthetized and
kept under anesthesia with repetitive intravenous injections of
ketamine (80 mg/kg BW) and xylazine (12 mg/kg BW) while
spontaneously breathing 100% oxygen through a small face mask.
For measurement of RV pressure, the right jugular vein was
surgically exposed and a microtip pressure transducer catheter (0.8 F,
Milar Instruments) was inserted via the jugular vein into the right
ventricle and continuously monitored. For measurement of LV
pressure, the right carotid artery was exposed, and the Milar catheter
advanced into the LV.12 The degree of right ventricular hypertrophy
was assessed using RV/LV⫹S ratio. To do so, immediately after
euthanasia the heart was removed and the right ventricle separated
from the septum, after which tissue was immediately weighed before
significant desiccation occurred. Morphometry, using standard techniques, was performed on multiple formalin-fixed lung sections as
previously described.13
To confirm that the BMPRIIdelx4⫹ mutation encoded a
dominant-negative receptor, we coexpressed BMPRIIdelx4⫹
along with a second expression plasmid encoding a SMAD
response element driving the luciferase reporter gene to
assess BMP receptor activity. As Figure 2 shows, cells
expressing the delx4⫹ mutation had markedly reduced
SMAD activity, indicative of a dominant-negative phenotype.
To validate use of the SM22 promotor, SM22-tet-luciferase
mice were generated and, as Figure 2 shows, when the double
transgenic progeny were fed doxycycline, they demonstrated
increased luciferase activity in both pulmonary (lung) and
systemic (aorta) beds. Cellular distribution was evaluated by
generating SM22-tet-lacZ transgenic mice. Figure 2 shows an
example of lacZ immunostaining in these mice, which revealed immunoreactive protein in the media of the vasculature. However, the level of intensity of immunostaining was
generally weak, particularly in small vessels, indicating that
the 488-bp SM22-␣ promotor sequence produced a relatively
low level of expression. Normal sized litters resulted from the
SM22-rtTA X TetO7-BMPRIIdelx4⫹ cross with the expected
proportion of double transgenic progeny. The resulting double transgenic mice, which we have termed SM22-tetBMPRIIdelx4⫹, were assayed at age 8 weeks for transgene
expression using quantitative RT/PCR. As Figure 2 shows,
dominant-negative BMPRII RNA was detected in the lungs
of SM22-tet-BMPRIIdelx4⫹ mice fed doxycycline from birth,
with little expression detected in littermates not fed
doxycycline.
Phenotyping SM22-tet-BMPRIIdelx4ⴙ Mice
The cardiovascular phenotype of SM22-tet-BMPRIIdelx4⫹
mice was evaluated using in vivo hemodynamic measurements. The Table shows average data for hemodynamic
parameters. Heart rate and LV systolic pressure did not differ
in doxycycline-fed and -unfed mice. RV and LV diastolic
pressure was near zero in all mice (data not shown). As
Figure 3 shows, right ventricular (RV) systolic pressure was
nearly 2-fold increased in doxycycline-fed transgenic mice,
compared with non– doxycycline-fed littermates. Measurement of RV relative weight confirmed the presence of
sustained pulmonary hypertension, with a 30% increase in
RV/LV⫹septum weight in doxycycline-fed transgenics.
There was a modest increase in hematocrit in doxycycline-fed
transgenic mice, although not of a magnitude capable of
increasing blood viscosity or pressure.14
To evaluate the effect of hypoxia, arterial blood gas studies
in mice spontaneously breathing room air were performed.
Arterial PO2 (PaO2) did not differ between groups (dox⫺,
74⫾5 versus dox⫹, 78⫾3 mm Hg; P⫽NS, n⫽4), and PCO2
and pH were normal (data not shown). To evaluate the
possibility that mice expressing BMPRIIdelx4⫹ were more
susceptible to the mild effects of hypoxia in Denver (PaO2 75
to 80 mm Hg versus 110 to 120 mm Hg at sea level), two
pregnant double transgenic mice were placed into hyperbaric
chambers that maintained normal sea level ambient pressure
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Figure 2. Validation of transgenic constructs. A, BMPRIIdelx4⫹ functions as a
dominant-negative: coexpression of
BMPRIIdelx4⫹ along with a reporter construct expressing luciferase under transcriptional control of an SMAD response
element. BMPRIIdelx4⫹ prevented transcriptional activation stimulated by
BMP2, confirming dominant-negative
BMPRII expression. B, Distribution of
transgene in SM22-tet-luciferase transgenic mice: SM22-rtTA mice were crossbred with tetO7-luciferase mice, and the
progeny fed doxycycline. Using luciferase assay, lung expressed the transgene to a comparable level as did aorta,
suggesting that the SM22 promotor was
activating expression in both pulmonary
and systemic vessels. C, Localization of
transgene in the lungs of SM22-tet-lacZ
transgenic mice: SM22-rtTA mice were
crossbred with tetO7-lacZ mice and
progeny fed doxycycline. The localization of reporter gene expression was
assayed by ␤-gal immunostaining. In the
photomicrograph (40⫻ objective), the
arrow points to ␤-gal expression in
smooth muscle of a large (L) pulmonary
artery. Little immunostaining was seen in
the capillary endothelial cells and epithe-lial cells of the alveolar wall (A). D, Control of BMPRIIdelx4⫹ expression by the tetracycline gene
delx4⫹
delx4⫹
switch in SM22-tet-BMPRII
mice. SM22-rtTA mice were crossbred with tetO7-BMPRII
mice and lungs from double transgenic
progeny homogenized for RNA isolation and quantitative RT/PCR determination of BMPRIIdelx4⫹ expression. Open circles are lung tissue
from mice not fed doxycycline, and filled circles are mice fed doxycycline. In transgenic mice fed doxycycline, there was a strong correlation between SM22-driven rtTA expression and the amount of BMPRIIdelx4⫹ transgene detected (P⬍0.001). These experiments
establish that SM22-tet-BMPRIIdelx4⫹ transgenic mice are viable and express a dominant-negative BMPRII receptor in pulmonary and
systemic vascular smooth muscle only when fed doxycycline.
and PAO2 during pregnancy and weaning of 10 pups that were
raised in the chambers until 8 weeks of age, when they were
catheterized. The Table shows that in these doxycycline-fed
transgenic mice, RV systolic pressure was intermediate to
Denver altitude doxycycline-fed and control mice. RV/LV⫹S
was also intermediate, being 0.315⫾0.015 in sea level
doxycycline-fed mice versus 0.360⫾0.013 in Denver altitude
doxycycline-fed mice and 0.242⫾0.023 in control mice
(P⬍0.01 all groups).
To evaluate structural abnormalities, we performed immunohistochemistry, identifying muscular pulmonary arteries by
␣-SMC actin staining and assessing the degree of muscularization by quantitative morphometry. General aspects of lung
development were unaffected by expression of the transgene,
with normal-appearing alveoli and airways, indicating that
unlike TNF-␣ transgenic mice and Fawn-hooded rats, which
also develop pulmonary hypertension, loss of lung tissue and
Hemodynamics
⫺Dox
(n⫽8)
⫹Dox
(n⫽15)
Heart rate, bpm
266⫾43
262⫾25
LV systolic, mm Hg
110⫾5
116⫾5
RV systolic (Denver), mm Hg
32.7⫾1.3
55.1⫾2.2*
ND
44.0⫾2.0*† (n⫽10)
38.3⫾2.4
43.9⫾1.2‡
RV systolic (sea level), mm Hg
Hematocrit, %
Values are mean⫾SEM. *P⬍0.01 from Denver⫺Dox; †P⬍0.01 from
Denver⫹Dox, ‡P⬍0.05 from ⫺Dox. ND indicates not determined.
abnormal development are not the reason for the pulmonary
hypertensive phenotype in these mice.15,16
Using quantitative morphometry, we found increased medial thickness of muscular pulmonary arteries in doxycyclinefed SM22-tet-BMPRIIdelx4⫹ mice. In addition, there was
increased muscularization of small pulmonary arteries at the
level of alveolar ducts. These abnormalities recapitulate one
of the primary lesions seen in humans with PAH and suggest
that an effect of loss of SMC BMPRII signaling is the
development of increased muscularization of pulmonary arteries, leading either directly or via associated vasoconstriction to increased pulmonary vascular resistance. Figure 4
demonstrates the morphological findings in SM22-tetBMPRIIdelx4⫹ mice.
Discussion
Reports that mutations in BMPRII were associated with
selective pulmonary arterial hypertension in humans suggested a link between BMP signaling and the occurrence of
the disease but did not establish whether mutations were
sufficient to produce the disease phenotype or simply permissive. Further, as the mutations were somatic and BMPRII is
ubiquitously expressed, they did not establish the relevant cell
types in which abnormal BMP signaling leads to the disease
phenotype.
The first goal of our study was to test the hypothesis that
abnormal BMP signaling resulting from expression of a
loss-of-function mutation was sufficient to produce the disease phenotype and, consistent with this hypothesis, SM22-
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Figure 3. Hemodynamic phenotyping of SM22-tet-BMPRIIdelx4⫹
transgenic mice. Left ventricular (LV) pressure (mm Hg) (A) and
right ventricular (RV) pressure (mm Hg) (B) measured in vivo
using a Milar (transducer-tipped) catheter. Representative tracings from a transgenic fed doxycycline for 8 weeks (thin line)
and a littermate not fed doxycycline (thick line) have been
superimposed and synchronized, demonstrating no difference in
LV pressure and significantly elevated RV systolic pressure in
the transgenic mice fed doxycycline. C, Scatter plot showing RV
pressure obtained from 17 doxycycline-fed transgenics and 8
control littermates (some symbols overlap and are not visible).
Group means are shown by the thick horizontal bar, with SEM
shown by the thin lines, demonstrating an increase from 32⫾1.3
to 56⫾2.2 mm Hg (P⬍0.01). D, Scatter plot showing the degree
of right ventricular hypertrophy in transgenic mice expressed as
RV mass divided by left ventricular⫹septum (LV⫹S) mass,
showing significant RV hypertrophy in doxycycline-fed transgenic mice (0.24⫾.04 vs 0.37⫾.06%; P⬍0.01).
tet-BMPRIIdelx4⫹ mice developed pulmonary hypertension by
8 weeks of age. Like other type II receptors in the TGF␤
superfamily, signaling function of BMPRII requires binding
to ligand, dimerization with, and phosphorylation of a type I
receptor and, finally, phosphorylation and activation of a
signaling SMAD.17 The mutation we chose for generation of
the transgenic has a premature stop codon with truncation
early in the kinase domain. Functionally the mutation resulted
in dominant-negative inhibition of receptor kinase activity (as
assessed by the inability of BMP to activate SMAD signaling). Recently, it was also reported that the N-terminal
portion of BMPRII binds to, and inactivates the cytoskeletal
regulator, LIM-kinase.18 On dimerization with a type I
receptor, BMPRII releases LIM-kinase, allowing it to phosphorylate its substrates. Although we did not test this property
directly, overexpression of delx4⫹ would also function as a
dominant-negative in this regard, as endogenously expressed
wild-type BMPRII that did not bind BMPRI (due to competition from BMPRIIdelx4⫹) would continue to inactivate LIMkinase. Therefore, although our studies support the hypothesis that loss-of-function of BMPRII produces pulmonary
arterial hypertension, additional studies will be required to
determine if defective receptor kinase activity, inactivation of
LIM-kinase, or a combination of the two are responsible for
the phenotype.
While our studies support the hypothesis that a mutation
resulting in sufficient loss of BMPRII function is capable of
producing the disease phenotype, they do not rule out the
possibility that disease occurrence in individuals with less
severe BMPRII mutations, such as haploinsufficiency, requires the presence of an environmental or genetic costimulus. Consistent with the hypothesis that mutations in BMPRII
can synergize with environmental factors, SM22-tetBMPRIIdelx4⫹ mice raised in Denver developed more severe
pulmonary hypertension than those raised at sea level PAO2,
although significant pulmonary hypertension was seen even
in the “low altitude” group. This suggests that, like humans
with idiopathic PAH, even mild hypoxia exacerbates the
phenotype of SM22-tet-BMPRIIdelx4⫹ mice.19
The second goal of our study was to test the hypothesis that
inducing abnormal BMPRII function selectively in smooth
muscle was sufficient to produce the disease phenotype, and
our findings support that hypothesis as well. BMPRII is
expressed in a number of lung cell types, including smooth
muscle, endothelium, epithelium, and macrophages.20 Prior
studies showed that BMPs could control smooth muscle cell
proliferation in vitro.21,22 Therefore, we chose to target the
mutation to smooth muscle. Morphometric analysis showed
that there was increased medial smooth muscle thickness and
a tendency for distal muscularization of small pulmonary
arteries. These findings recapitulate one of the pathological
hallmarks of PAH. However, the magnitude of the change
was small, relative to the large increase in pulmonary artery
pressure and, unlike advanced PAH, no intimal lesions were
seen. A potential explanation for this paradox is the possibility that a component of the pulmonary hypertension in
8-week-old SM22-tet-BMPRIIdelx4⫹ mice was due to enhanced vasoconstriction. This is consistent with a two-phase
hypothesis for the pathogenesis of PAH in which early
West et al
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Figure 4. Morphometric phenotyping of
SM22-tet-BMPRIIdelx4⫹ transgenic mice. A,
Hematoxylin and eosin staining of lung from
an SM22-tet-BMPRIIdelx4⫹ transgenic mouse
fed doxycycline from birth to age 8 weeks
(20⫻ objective). B indicates the lumen of a
bronchus; AD, alveolar ducts. Remainder of
the field shows individual alveoli. Lung morphology is grossly normal. B, Serial section
stained with anti-SMC ␣-actin to identify
small pulmonary arteries. Arrows point to the
small, precapillary pulmonary arteries identified by ␣-actin staining that were used for
morphometric analysis. C, Quantitative analysis of lung development. Number of alveoli
per high power field and the number of
␣-actin–positive vessels per alveoli were
compared between doxycycline-fed and
-unfed transgenic mice. No difference was
seen, confirming relatively normal lung development. D, Quantitative analysis of pulmonary artery muscularity. An increase in
medial thickness of muscular pulmonary
arteries from 46% to 54% of total vessel
diameter was found (P⬍0.05). In addition,
the number of fully muscularized (FM) precapillary pulmonary arteries and the proportion of precapillary pulmonary arteries that
were fully muscularized (versus partially muscularized, PM) tended to be increased in
doxycycline-fed transgenic mice (P⫽0.06).
disease is characterized by enhanced vasoconstriction and
minimal remodeling and later disease by progressive remodeling and little vasoconstriction.23 Further studies will be
needed to define the mechanisms underlying the increase in
tone and determine if remodeling becomes more pronounced
as the mice age.
Interestingly, even though the SM22 promotor we used
resulted in expression in systemic as well as pulmonary
smooth muscle, phenotypic abnormalities were only seen in
the lung. Although we were not able to quantify the relative
amount of mutant receptor protein expressed in systemic
versus pulmonary resistance arteries, luciferase expression in
the aortas of SM22-tet-luc mice suggested that, consistent
with the literature on SM22 expression, the promotor was
active in systemic arteries.9 Thus, it appears that under our
experimental conditions, BMP signaling played a more significant role in maintaining normal tone and structure of the
pulmonary than systemic circulation. It is possible that in
other circumstances, such as systemic atherosclerosis, BMP
signaling may modulate systemic vascular disease as well.24
Although we do not have an explanation for the pulmonary
vascular specificity of the phenotype, it is consistent with the
lack of systemic vascular disease in patients with pulmonary
arterial hypertension and suggests that BMP signaling plays a
unique role in controlling tone and structure in the pulmonary
circulation.
Although our studies support the hypothesis that loss of
BMPRII function in smooth muscle is sufficient to produce
pulmonary arterial hypertension, we cannot exclude a modulatory role for BMP signaling in other cell types. The
magnitude of structural changes in pulmonary arteries was
small in SM22-tet-BMPRIIdelx4⫹ mice, and it is possible that
loss of BMPRII signaling in endothelium could augment
vascular pathology, and even result in intimal (“plexiform”)
lesions, which were absent in SM22-tet-BMPRIIdelx4⫹ mice.
Additional transgenic mice that utilize an endothelial-specific
promoter are being constructed to address this possibility.
In conclusion, our studies support the hypothesis that
BMPRII mutations are sufficient to produce pulmonary
arterial hypertension and that the mutation need only be
expressed in smooth muscle to produce the phenotype.
Furthermore, the lack of a major systemic vascular phenotype
in these mice suggests that BMPRII signaling is uniquely
important in the pulmonary circulation. SM22-tetBMPRIIdelx4⫹ mice should provide a useful model to study the
molecular and physiological abnormalities in pulmonary
arterial hypertension and facilitate the discovery and preclinical testing of potential new therapies.
Acknowledgments
Funding sources included HL48038, HL57282, and HL71596
(D.M.R.) from the NIH National Heart, Lung, and Blood Institute
and Mentored Scientist Training (J.W.) and Established Investigator
Awards (K.F.) from the American Heart Association. We thank Ivan
McMurtry and Jack Reeves for critiquing this manuscript and Jeffrey
Whitsett for advice in constructing the conditional transgenic mice.
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Pulmonary Hypertension in Transgenic Mice Expressing a Dominant-Negative BMPRII
Gene in Smooth Muscle
James West, Karen Fagan, Wolfgang Steudel, Brian Fouty, Kirk Lane, Julie Harral, Marloes
Hoedt-Miller, Yuji Tada, John Ozimek, Rubin Tuder and David M. Rodman
Circ Res. 2004;94:1109-1114; originally published online March 18, 2004;
doi: 10.1161/01.RES.0000126047.82846.20
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