Chronic NOS inhibition prevents adverse lung remodeling and pulmonary arterial hypertension in caveolin-1 knockout mice.

Abstract

Recently generated caveolin-1 deficient mice (cav-1 ko) suffer from severe lung fibrosis with marked pulmonary hypertension and arterial hypoxemia and may therefore serve as an useful animal model of this devastating human disorder. Accumulating evidence strongly supports the negative regulatory influence of caveolin-1 on endothelial nitric oxide synthase resulting in a constitutive hyperactivation of the nitric oxide (NO) pathway in cav-1 ko. We therefore hypothesized that a disturbed NO signaling is implicated in the evolution of the adverse lung phenotype of cav-1 ko. For this purpose, cav-1 ko of 2 months age were compared with knockout counterparts experiencing 2-month postnatal NO synthase inhibition by NG-nitro-l-arginine methyl ester (L-NAME) treatment. Chronic l-NAME administration prevented adverse lung remodeling in cav-1 ko. Furthermore, l-NAME donation led to a normalized oxygen saturation (91.5+/-1.8% vs. 98.5+/-2.3%, P<0.01, n=10-12), a marked decrease in right ventricular hypertrophy (LV/RV ratio: 4.0+/-0.3 vs. 2.7+/-0.3, P<0.01, n=10-12) and reductions of the elevated pulmonary artery pressure (40.2+/-3.1 mmHg vs. 26.3+/-4.6 mmHg, P<0.01, n=6). Collectively, these improvements resulted in an enhanced exercise capacity of l-NAME-treated cav-1 ko. Finally, we found evidence for enhanced oxidative stress in untreated cav-1 ko which was substantially reduced by chronic l-NAME administration to cav-1 ko. In view of these data, we speculate that a perturbation of NO signaling, together with enhanced O2(-) production originating from NO synthases, may play a pivotal role in the pathogenesis of the adverse pulmonary phenotype seen in cav-1 ko.

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Author’sAccepted Manuscript
Chronic NOS inhibition prevents adverse lung
remodelling and pulmonary arterial hypertension in
caveolin-1 knockout mice
CarstenWunderlich,AlexanderSchmeisser,Christian
Heerwagen,BerndEbner,KristinSchober,Ruediger
C. Braun-Dullaeus, Carsten Schwencke, Michael
Kasper, Henning Morawietz, Ruth H. Strasser
PII:
DOI:
Reference:
S1094-5539(07)00102-2
doi:10.1016/j.pupt.2007.11.005
YPUPT807
To appear in:
Pulmonary Pharmacology & Therapeutics
Received date:
Revised date:
Accepted date:
26 July 2007
8 November 2007
21 November 2007
Cite this article as: Carsten Wunderlich, Alexander Schmeisser, Christian Heerwagen,
Bernd Ebner, Kristin Schober, Ruediger C. Braun-Dullaeus, Carsten Schwencke, Michael
Kasper, Henning Morawietz and Ruth H. Strasser, Chronic NOS inhibition prevents ad-
verse lung remodelling and pulmonary arterial hypertension in caveolin-1 knockout mice,
Pulmonary Pharmacology & Therapeutics (2007), doi:10.1016/j.pupt.2007.11.005
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peer-00499152, version 1 - 9 Jul 2010
Author manuscript, published in "Pulmonary Pharmacology & Therapeutics 21, 3 (2008) 507"
DOI : 10.1016/j.pupt.2007.11.005

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Accepted manuscript
Word count: Abstract, 239; total manuscript, 4733
Chronic NOS inhibition prevents adverse lung remodelling and
pulmonary arterial hypertension in caveolin-1 knockout mice
Carsten Wunderlich1*, Alexander Schmeisser1*, Christian Heerwagen1, Bernd Ebner1,
Kristin Schober1, Ruediger C. Braun-Dullaeus1, Carsten Schwencke1, Michael Kasper2,
Henning Morawietz3, and Ruth H. Strasser1
1University of Technology Dresden, Department of Cardiology, Medical Clinic, Fetscherstr.
76, Dresden 01307, Germany.
2Department of Anatomy, Fiedlersr. 42, Dresden 01307, Germany
3Department of Microcirculation, Fiedlerstr. 42, Dresden 01307, Germany
* both authors equally contributed to this work
Address for correspondence:
Carsten Wunderlich M.D.
Department of Medicine and Cardiology
University of Technology Dresden
Fetscherstr. 76
01307 Dresden, Germany
Tel.: +49 351 4501700
Fax.: +49 351 4501702
Email: carstenwunderlich@gmx.de
Manuscript information: 22 text pages, 5 figures and 1 table.
Abbreviations: eNOS-endothelial nitric oxide synthase, wt-wild type, cav-1 ko-caveolin-1
knockout animals, L-NAME - NG-nitro-L-arginine methyl ester
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adverse pulmonary phenotype seen in cav-1 ko.
Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice
2
Abstract
Recently generated caveolin-1 deficient mice (cav-1 ko) suffer from severe lung fibrosis with
marked pulmonary hypertension and arterial hypoxemia and may therefore serve as an useful
animal model of this devastating human disorder. Accumulating evidence strongly supports
the negative regulatory influence of caveolin-1 on endothelial nitric oxide synthase resulting
in a constitutive hyperactivation of the nitric oxide (NO) pathway in cav-1 ko. We therefore
hypothesized that a disturbed NO signalling is implicated in the evolution of the adverse lung
phenotype of cav-1 ko. For this purpose cav-1 ko of 2 month age were compared with
knockout counterparts experiencing 2 month postnatal NO synthase inhibition by NG-nitro-L-
arginine methyl ester (L-NAME) treatment. Chronic L-NAME administration prevented
adverse lung remodelling in cav-1 ko. Furthermore, L-NAME donation led to a normalized
oxygen saturation (91.5±1.8 vs. 98.5±2.3 %, P<0.01, n=10-12), a marked decrease in right
ventricular hypertrophy (LV/RV ratio: 4.0±0.3 vs. 2.7±0.3, P<0.01, n=10-12) and reductions
of the elevated pulmonary artery pressure (40.2±3.1 vs. 26.3±4.6 mmHg, P<0.01, n=6).
Collectively, these improvements resulted in an enhanced exercise capacity of L-NAME
treated cav-1 ko. Finally, we found evidence for enhanced oxidative stress in untreated cav-1
ko which was substantially reduced by chronic L-NAME administration to cav-1 ko. In view
of these data we speculate that a perturbation of NO signalling, together with enhanced O2-
production originating from NO synthases, may play a pivotal role in the pathogenesis of the
Key words: caveolin, eNOS, nitric oxide, superoxide, lung fibrosis
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Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice
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Introduction
Caveolae are small invaginations of the plasma membrane abundantly present in most cells of
the cardiopulmonary system. In these cells caveolae function in protein trafficking,
cholesterol homeostasis and signal transduction. Three family members of caveolins are
identified to date. Caveolin-1 (cav-1) and caveolin-2 (cav-2) are coexpressed in adipocytes,
fibroblasts and endothelial cells while caveolin-3 (cav-3) expression is confined to muscle
cells.1 Cav-1 and cav-3 are essential for the biogenesis of caveolae in cells that normally
express these isoforms2-4, whereas cav-2 is not absolutely required.5
Recently, the caveolin proteins have been implicated in structural lung remodelling and
development of pulmonary arterial hypertension (PAH).5-8 Indeed, one prominent feature of
caveolin-1 knockout mice (cav-1 ko) is an adverse lung phenotype with thickened alveolar
septa, smaller alveolar spaces and an interstitial hypercellularity.2;3 Cav-1 ko are further
characterized by a substantial pulmonary arterial hypertension (PAH) which is accompanied
by a hypertrophied right ventricle (RV).9 Moreover, cav-1 ko were shown to suffer from a
dramatic reduction in life span.10 Of note, cav-1 ko also display a near complete ablation of
cav-2 expression. These findings provide the basis for an intense debate on the functional
implications of cav-1 on lung remodelling.8 However, the most direct evidence for a
independent role of cav-1 on pulmonary function versus alveolar thickening is supported by
data documenting that recently generated cav-2 deficient mice (with retained cav-1
expression) do not have PAH, but do exhibit pulmonary hyperplasia.5 Moreover, several
papers reported a decreased expression of pulmonary caveolins in various animal models of
PAH.8;11 These findings also appear to be relevant to human PAH in that decreases in cav-1
gene and protein expression have been recently demonstrated in plexiform lesions of patients
with severe PAH.12 Altogether these studies validate the importance of caveolins in the
development of lung pathologies but the specific role of cav-1 in this process awaits further
clarification.
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between cav-1 and eNOS in normal physiology we questioned whether a perturbation of NO
Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice
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Within the vascular endothelium endothelial nitric oxide synthase (eNOS) targets to caveolae
of the plasma membrane where it is tonically inhibited by interaction with cav-1 resulting in a
constitutive hyperactivation of the nitric oxide (NO) pathway in cav-1 ko.3;9;13 There is
mounting evidence that eNOS can be converted to an important ‘reactive oxygen species’
generator within the vessel wall.14;15 Within the so-called “uncoupled” eNOS enzyme
electrons flow to molecular oxygen and generate superoxide (O2-) instead of NO. This
scenario results in a reduced NO production by eNOS while this enzyme additionally
produces O2-. The superoxide anion avidly reacts with vasodilating NO to form the oxidant
species peroxynitrite.16 Accordingly, the NO bioavailability is further reduced with concurrent
increased oxidant stress.
PAH is a common finding in human lung fibrosis.17 Despite recent advances in symptomatic
treatment this disease is characterized by a progressive malfunction associated with a poor
prognosis of affected individuals. Unfortunately, no current treatment option cures this
devastating condition. Various molecules have been implicated as putative candidates of its
pathogenesis including endothelial dysfunction due to impaired NO signalling.17 In this
context recently generated cav-1 ko may serve as an useful model of this disorder and help to
further unveil the molecular mechanism of this disease and to uncover potential targets for
future therapies.
Given the adverse pulmonary phenotype of cav-1 ko and the close regulatory interaction
signalling with consecutive enhanced oxidative stress may be involved in the genesis of the
pathological lung phenotype in cav-1 ko. Therefore we treated respective animals with the
NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) for 2 month and studied
the exercise capacity, pulmonary function, lung morphology and the O2- production in related
tissues thereafter.
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home-made saline filled catheter connected to a pressure transducer (ADInstruments,
Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice
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Materials and Methods
Animals
Age and gender matched mice were bred and housed in a barrier facility at the Institute for
Animal Studies of the University of Technology, Dresden, Germany. After birth one group of
animals (cav-1 ko and age/gender matched wild-type littermates) received standard care with
a normal rodent chow and drinking water ad libitum. A second group was additionally treated
with oral L-NAME (50 mg/kg, dissolved in drinking water) beginning after birth. Mice of two
months age were included in the study. Animal experiments conformed to the Guide for the
Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH
Publication No. 85-23, revised 1996). The generation of the transgenic mice has been
described elsewhere.3
Hemodynamic measurements
Mice were injected with 250 units heparin intraperitoneally (i.p.) and anesthetized by
injection of 12 mg/kg xylazine i.p. and 80 mg/kg ketamine i.p. and placed ona warming table
to keep the body temperature at 37 °C throughout the experiment. Subsequent to anesthesia,
the mice were orally intubated, connected to a rodent ventilator (TSE Animal Respirator, Bad
Homburg, Germany) and ventilated in a pressure-controlled manner at a rate of 110 breaths
per minute with the fraction of inspired oxygen being 21%. After shaving and disinfection a
Powerlab, MLT844, Spechbach, Germany) was inserted into the jugular vein and advanced to
the pulmonary artery for hemodynamic studies. The central venous pressure and the
pulmonary artery pressure was evaluated at end expiration. Simultaneously, the left
ventricular end diastolic pressure (LVEDP) was measured with a home-made saline filled
catheter connected to a pressure transducer (ADInstruments, Powerlab, MLT844, Spechbach,
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was determined according to the following formula [(wet wt- dry wt/ wet wt) x 100]. For
Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice
6
Germany) after retrograde passage of the aortic valve. We applied the following formula for
calculation of the transpulmonary gradient (TPG=PAPmean-LVEDP). All data were
measured continuously by using a PC with dedicated software (ADInstruments, Chart,
Spechbach, Germany).
Blood analysis
After anesthesia and injection with Heparin (250 units i.p.), the mice were ventilated in a
pressure-controlled manner at a rate of 110 breaths/min only with room air. A midline skin
incision at the lower xiphoid was performed, and the skin was retracted laterally. Afterwards,
a substernal abdominal midline incision was done to approach the abdominal surface of the
diaphragm, which then was incised to visualize the hearts apex. The oxygenated blood was
collected from left ventricle with a heparin-covered syringe. The hemoglobin and hematocrit
content and the oxygen saturation were measured with a Micros 60 blood analyzer (Horiba
Diagnostics, Irvine, California, USA).
Lung and liver weight
For determination of the lung weight, fresh lungs were flushed in saline to remove
contaminating blood and weighed immediately (wet weight). The lungs were then placed in a
drying oven at 80 °C until a constant weight was obtained (dry weight). Tissue water content
measurement of the liver weight the organ was harvested, flushed in saline to remove
contaminating blood and weighed immediately (liver weight). To normalize differences in
lungs/liver weight which can be attributed to the overall size of the mouse we then choose to
express the results in relation to the total body weight.
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tolerance of mice. The mouse was gently placed inthe water and carefully observed. The time
Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice
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Assessment of right ventricular hypertrophy
The heart was isolated and placed under a dissecting microscope. Attached vessels and both
atria were dissected and removed. The right ventricular wall was cut out, blotted, and
weighed; then the left ventricular wall and septum were treated the same way and weighed.
Measurement of plasma NO metabolites
After anesthesia and injection with Heparin (250 units i.p.), mouse blood was collected by
direct cardiac puncture. The plasma total NO was quantitatively determined based on the
enzymatic conversion of nitrate to nitrite by nitrate reductase according with the manufactures
instructions and as described previously.18
Tissue histology
Lungs from untreated and L-NAME treated animals were fixed and stained with hematoxylin
and eosin, essentially as described elsewhere.13 All images were taken using a computerized
analyzing system (Leica DMRB microscope, Wetzlar, Germany; Sony Power HAD camera,
Tokyo, Japan).
Assessment of exercise tolerance
A 4-liter beaker filled with water (34°C) was used as a swimming pool to assess the exercise
at which the mousewas initially unable to maintain complete buoyancy was recorded,and the
mouse was immediately removed from the pool. No micewere injured in these experiments.
Measurement of superoxide production
Superoxide production was measured using the superoxide dismutase-inhibitable (SOD-
inhibitable) cytochrome c reduction assay. Animals were sacrificed by cervical dislocation.
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probability value of less than 0.05.
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Aortas, pulmonary arteries and lungs were rapidly removed, cleaned of adventitial tissue and
placed into chilled modified Krebs-buffer (composition in mmol/l: 117.48 NaCl, 4.74 KCl,
2.50 CaCl2, 1.19 MgSO4, 1.19 KH2PO4, 24.76 NaHCO3 and 10 glucose [pH 7.4]). After
cyotchrom c (50 µM; SIGMA C-4186, Sigma-Aldrich, St. Louis, Missouri, USA) were added,
the samples were incubated at 37°C for 60 minutes with and without SOD
(125 U/ml). Cytochrome c reduction was calculated using absorbanceat 550 nm corrected for
background readings at 540 and 560 nm. Superoxide production was quantified in picomoles
per milligram of the respective tissue (dry weight) from the difference between absorbance
with or withoutSOD.
Materials
All compounds used in this study were either analytical grade or of the highest purity
available. Heparin was obtained from ratiopharm (Ulm, Germany), Ketamin from CuraMED
Pharma (Karlsruhe, Germany) and xylazine from medistar (Holzwickede, Germany). All other
reagents were obtained from Merck (Darmstadt, Germany).
Data Analysis
All results are expressed as means ? S.D. For multiple comparisons, ANOVA was applied,
followed by the Bonferroni correction. Differences were assumed to be significant with a
Results
Lowered systemic nitric oxide in L-NAME treated cav-1 ko
Previous studies have unequivocally shown that cav-1 can bind and negatively regulate eNOS
activity. We therefore assayed systemic levels the two major oxidation products of nitric oxide
(NO), nitrate and nitrite (=NOx) in untreated and treated mice of both strains. Systemic NOx
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corroborated by marked reductions in lung weight to body weight ratios in L-NAME treated
Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice
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is generally regarded as a surrogate marker of systemic NO levels. In plasma obtained from
untreated cav-1 ko NOx concentration was significantly higher than in WT mice (cav-1 ko:
8.85±1.43 vs. WT: 3.99±0.77 µM, n=10, P<0.01). Several groups have recently shown by
western blot analysis that no overt change in expression levels of the three major NO
synthases (i.e., inducible, endothelial and neuronal NO synthases) was evident in cav-1 ko as
compared to WT animals. Particularly, the inducible NO synthase was undetectable in cav-1
ko.9;13 Given the close regulatory interaction between cav-1 and eNOS these findings suggest
that the high NOx levels are most likely caused by an elevated eNOS activity in cav-1 ko.
After L-NAME application systemic NOxlevels fell significantly in both strains (cav-1 ko:
3.81±1.33 vs. WT: 2.31±0.86 µM, n=10, P<0.01 vs. untreated). Notably, NOxconcentration in
L-NAME treated cav-1 ko approached the level of untreated WT mice.
Normalized lung structure in L-NAME treated cav-1 ko
One prominent feature of cav-1 ko is the adverse lung phenotype with thickened alveolar
septa, smaller alveolar spaces and an interstitial hypercellularity.2;3 All these findings are
highly reminiscent of a human disorder named lung fibrosis.1 Given the hyperactivated NO
pathway in cav-1 ko we studied the influence of chronic NOS inhibition by L-NAME on lung
structure of cav-1 ko. In L-NAME-treated cav-1 ko we observed improvements of the lung
architecture since the alveolar spaces appeared significantly wider and alveolar septa were
thinner as compared with untreated cav-1 ko (Figure 1 C+D). These findings are further
cav-1 ko (Figure 2 A). No differences were evident between untreated and L-NAME treated
WT mice.
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similar to that seen in other pulmonary hypertension models. Additionally, the central venous
Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice
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Improved lung function in L-NAME treated cav-1 ko
In view of the adverse lung fibrosis in cav-1 ko we looked in detail on the oxygen saturation
with and without L-NAME administration. Without chronic NOS inhibition cav-1 ko
displayed a significantly reduced oxygen saturation which was completely normalized after L-
NAME treatment (Table 1). As compared to WT littermates hemoglobin and hematocrit
levels revealed a polyglobic blood count in untreated cav-1 ko which is most probably a
sequel of hypoxic erythropoetin stimulation. As depicted in Table 1 L-NAME application
substantially lowered these pathological values although no complete normalization was
achieved.
Reversal of the pulmonary hypertension, right ventricular hypertrophy and right
ventricular heart failure in L-NAME treated cav-1 ko
Cav-1 ko are additionally characterized by markedly hypertrophied right ventricles (RV). The
RV to body weight ratio was significantly increased in untreated cav-1 ko (1.44±0.2, n=10) as
compared to WT littermates (1.06±0.2, n=12, P<0.05) and was accompanied by a profound
pulmonary hypertension (Figure 3 A). The left ventricular end diastolic pressure (LVEDP)
was elevated in untreated cav-1 ko (15.5±1.9 mmHg) as compared to WT mice (9.0±1.9
mmHg, n=10-12, P<0.05). The same was true for the transpulmonary gradient (TPG – Figure
3 B). Of note, the left ventricular (LV) to RV ratio was dramatically reduced (Figure 2 B) and
pressure was significantly elevated in cav-1 ko as compared to WT counterparts (cav-1 ko:
17.4±3.5 vs. WT: 4.36±2.4 mmHg, n=7, P<0.01). The liver to body weight ratio was
dramatically increased in cav-1 ko as compared to WT mice (cav-1 ko: 57.77±5.52 vs. WT:
44.36±5.65 mg/g, n=8-12, P<0.01).
In view of these findings we studied the influence of L-NAME treatment on these distinct
parameters. While the RV to body weight ratio in L-NAME treated cav-1 ko significantly
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is contrasted by significant improvements in the exercise capacity of L-NAME treated cav-1
Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice
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decreased (0.96±0.1, n=9, P<0.05 vs. untreated cav-1-ko) it did not change in L-NAME
treated WT animals (0.94±0.04, n=13). As shown in Figure 2 B L-NAME donation in cav-1
ko resulted in an increased LV/RV ratio. Furthermore, after L-NAME treatment LVEDP
decreased in both strains mice (cav-1 ko: 6.8±1.9 P<0.01 vs. untreated cav-1-/- , WT: 7.0±2.8
mmHg, n=8-12). Likewise, as depicted in Figure 3, we observed a reduction of both the
pulmonary artery hypertension and the TPG after chronic NOS inhibition with L-NAME. As
a sign of the reduced right ventricular congestion both the central venous pressure (cav-1 ko:
7.2±4.3 P<0.01 vs. untreated cav-1 ko , WT: 5.7±3.6 mmHg, n=6, P<0.01) and the liver/body
weight ratio dramatically decreased after L-NAME treatment (cav-1 ko: 52.22±2.26 P<0.01
vs. untreated cav-1-/-, WT: 46.37±1.54 mg/g, n=8-12).
L-NAME treatment in cav-1 ko results in improved exercise capacity
Cav-1 ko have been found to exhibit a profound exercise intolerance.3 This observation may
be attributed to the severe lung fibrosis and the pulmonary arterial hypertension. Since we
observed substantial regressions in both findings after L-NAME administration we grossly
assessed the possible physical consequences of L-NAME treatment by subjecting these
animals to a swimming test. In this way, we could directly compare the exercise tolerance of
WT, cav-1 ko, and L-NAME treated mice of both strains. As displayed in figure 4 WT
animals were able to swim forup to 35 min, while cav-1 ko were exhausted after 13 min. This
ko (Figure 4).
Enhanced superoxide generation in cav-1 ko is reduced after L-NAME treatment
A possible explanation for the adverse pulmonary phenotype is enhanced oxidant stress in
cav-1 ko. To test this hypothesis we harvested aortas, pulmonary arteries and lung tissue from
untreated animals and performed a cytochrome c reduction assay for measurement of O2-
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prevented the evolution of the pulmonary abnormalities and alleviated the oxidative stress in
Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice
12
release. We observed striking disparities between WT and cav-1 ko in aorta, pulmonary artery
(data not shown, but almost identical as compared to aortic tissue) and lung tissue with a
markedly increased O2-production in knockout animals (Figure 5 A, B). Given the
aforementioned hyperactivity of the eNOS-NO pathway in cav-1 ko we next treated animals
of both strains with oral L-NAME and measured ROS generation in these animals. As shown
in Figure 5 L-NAME-supplementation resulted in a decreased O2- production in lungs and
aortas taken from cav-1 ko.
Discussion
Several groups have independently generated cav-1 ko, and these animals were repeatedly
shown to suffer from clear and reproducible vascular and pulmonary phenotypes including a
severe lung fibrosis with pulmonary arterial hypertension (PAH).2;3;9 These phenotypes,
driven by the global loss of cav-1, may be caused by the deficiency of the gene in cells that
normally express this protein, including endothelial cells, adipocytes, fibroblasts, epithelial
cells and smooth muscle cells. A plethora of biochemical and genetic data support the role of
cav-1 as a negative regulator of agonist-mediated eNOS activation.3;7;9;13 Accordingly,
constitutive eNOS hyperactivation was observed in cav-1 ko. The present study argues that
major lung pathologies are caused at least in part by dysregulated NO synthases resulting in
enhanced oxidative stress since chronic treatment with the NO synthase inhibitor L-NAME
cav-1 ko. These findings are inasmuch of paramount importance since they underline the
central role of a balanced NO signalling in normal pulmonary physiology.
Recent insights into the physiological roles of caveolae and caveolins have been dissected in
genetically modified mice. It was revealed that cav-1 is essential for the formation of caveolae
in fibroblasts, endothelial cells, adipocytes.2;3 Additionally, it was shown that the loss of cav-
1, which is the primary isoform in the vascular system, results in multiple alterations with a
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hematocrit values. Elevated hemoglobin and hematocrit levels are a common finding in
Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice
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dysregulation in NO synthesis3;9, vascular permeability6, mechanotransduction19 and
pulmonary function.9 These studies unequivocally validate the in vivo importance of cav-1
beyond cells based studies which are hampered by operational definitions of biochemical
fractions containing caveolins and the lack of specificity inherent in reagents that extract
cholesterol from cells.6
PAH is a common finding in hypoxic lung disorders like lung fibrosis. Pulmonary
hypertension arises when vasoconstriction and adverse remodelling of pulmonary arterioles
results in increased pulmonary vascular resistance. The subsequent rise in pressure load
promotes right ventricular hypertrophy, which often progresses to premature death from right
heart failure.17 Cav-1 has been suggested to function as a key regulator of adverse lung
remodelling and the development of PAH.5-8 Indeed, cav-1 ko were shown to suffer from a
severe lung fibrosis with thickened alveolar septa, smaller alveolar spaces and an interstitial
hypercellularity.2;3 Our findings demonstrate that loss of cav-1 in lung parenchyma also
results in PAH and right ventricular hypertrophy which validates former findings.9 All these
features are highly reminiscent of the human lung fibrosis which is frequently associated with
PAH. Moreover, we found signs of right ventricular heart failure as evidenced by dramatically
increased liver to body weight ratio in cav-1 ko. To the best of our knowledge this study is the
first to show that cav-1 ko additionally display a significantly reduced arterial oxygen
saturation accompanied by a polyglobic blood count with increased hemoglobin and
chronic lung disorders and are regarded as a sequel of enhanced hypoxic erythropoetin
stimulation. Given these characteristics evident in untreated cav-1 ko these animals may be
viewed as an useful model of PAH in lung fibrosis.
In normal lung tissue, endothelial cells and type I pneumocytes, abundantly express cav-1.
Therefore it seems easily conceivable that histological and functional abnormalities develop
in lungs of cav-1 ko. Nevertheless the specific role of cav-1 and caveolae in this process is
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ko. The biological sequelae of the so-called eNOS “uncoupling” on NO bioavailability may
Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice
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still a matter of discussion. Cav-1 is also regarded as a candidate tumor suppressor. In line
with this notion, Razani et al. found dramatically increased markers of cellular proliferation in
lungs of cav-1 ko.2 Therefore they suggested that disruption of cav-1 leads to profound
aberrations in cells cycle checkpoints resulting in the abovementioned pathologies.
In view of the close interaction of cav-1 and eNOS in normal physiology we questioned
whether a perturbation in NO signalling is involved in adverse lung remodelling in cav-1 ko.
There is mounting evidence that validates the negative regulatory influence of cav-1 on eNOS
function.3;7;9;13 For instance, enhanced NO-mediated vascular relaxation in isolated aortic
rings3 together with increased systemic NOx levels9 and enhanced eNOS activity in vitro was
reported.13 Since it was repeatedly demonstrated by western blots that no change in
expression level of the major NO synthases (i.e., eNOS, inducible NOS, neuronal NOS) in
vessels and lungs of cav-1 ko occurred7;9;13 these findings suggest that the high NO levels we
and others have measured are most likely caused by an elevated eNOS activity. Since eNOS
derived NO, through potent vasodilator and antiproliferative effects on vascular smooth
muscle cells is critical in maintaining normal pulmonary vascular tone and structure it seems
difficult to relate eNOS activity to the adverse pulmonary phenotype of cav-1 ko. But
accumulating evidence indicates that in some circumstances eNOS can be converted into a
important source of radicals within the vessel wall.14;15 In line with this concept we measured
a markedly increased O2-generation in vessels and lung tissue harvested from untreated cav-1
be profound, because NO production is reduced and O2- generation is increased. Since
superoxide is known to act as a potent scavenger of bioactive NO this scenario favours
significant reductions in the bioavailability of vasodilating NO. Of note, increased O2-
generation has been observed in other experimental models of PAH20 and markers of
oxidative stress are increased in patients with PAH.17 Superoxide mediates pulmonary
vasoconstriction and stimulates pulmonary smooth muscle proliferation.21 In view of our data
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