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Acta Biochim Biophys
Sin 2006, 38: 15-21
doi:10.1111/j.1745-7270.2006.00131.x
Effect of L-Arginine on
Pulmonary Artery Smooth Muscle Cell Apoptosis in Rats with Hypoxic Pulmonary Vascular
Structural Remodeling
Ingrid Karmane Sumou1, Jun-Bao DU1,3*, Bing Wei1, Chun-Yu ZHang1, Jian-Guang Qi1, and Chao-Shu Tang2,3
1 Department of
Pediatrics, Peking University First Hospital, Beijing 100034, China;
2 Institute of Cardiovascular
Diseases, Peking University First Hospital, Beijing 100034, China
3 Key Laboratory of
Molecular Cardiovascular Diseases, Ministry of Education, Beijing 100034, China
Received: August 9,
2005
Accepted: November
11, 2005
This work was supported
by the grants from the National Natural Science Foundation of China (30425010
and 30571971) and the Major Basic Research Program of China (G2000056905)
*
Corresponding author: Tel, 86-10-66171122; Fax, 86-10-66134261; E-mail,
Abstract This study investigated the effect of L-arginine (L-Arg) on
the apoptosis of pulmonary artery smooth muscle cells (PASMC) in rats with
hypoxic pulmonary vascular structural remodeling, and its mechanisms. Seventeen
Wistar rats were randomly divided into a control group (n=5), a hypoxia
group (n=7), and a hypoxia+L-Arg group (n=5). The morphologic
changes of lung tissues were observed under optical microscope. Using the
terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin
nick end labeling assay, the apoptosis of PASMC was examined. Fas expression in
PASMC was examined using immunohistochemistry. The results showed that the
percentage of muscularized artery in small pulmonary vessels, and the relative
medial thickness and relative medial area of the small and median pulmonary
muscularized arteries in the hypoxic group were all significantly increased.
Pulmonary vascular structural remodeling developed after hypoxia. Apoptotic
smooth muscle cells of the small and median pulmonary arteries in the hypoxia
group were significantly less than those in the control group. After 14 d of
hypoxia, Fas expression by smooth muscle cells of median and small pulmonary
arteries was significantly inhibited. L-Arg significantly inhibited hypoxic
pulmonary vascular structural remodeling in association with an augmentation of
apoptosis of smooth muscle cells as well as Fas expression in PASMC. These
results showed that L-Arg could play an important role in attenuating hypoxic
pulmonary vascular structural remodeling by upregulating Fas expression in
PASMC, thus promoting the apoptosis of PASMC.
Key words hypoxia; pulmonary artery; arginine; apoptosis
Hypoxic pulmonary vascular
structural remodeling is an important pathologic basis of hypoxic pulmonary
hypertension. The proliferation and hypertrophy of pulmonary vascular smooth
muscle cells, the thickening of pulmonary artery media, the muscularization of
small peripheral vessels and the increase in extracellular matrix are the main
pathologic features of pulmonary vascular structural remodeling [1]. Previous
studies showed that L-arginine (L-Arg)/nitric oxide (NO) could alleviate the
development of hypoxic pulmonary vascular structural remodeling and hypoxic pulmonary
hypertension. However, the molecular mechanism by which L-Arg attenuates
hypoxic pulmonary vascular structural remodeling remains unknown [2,3].
Apoptosis and proliferation are two important factors that determine the
biological behavior of vascular smooth muscle cells, hence the balance between
them influences the vascular structure. In vitro studies showed that NO
could induce apoptosis of smooth muscle cells [4]. Recent studies showed that
supplemental L-arg partially
inhibited pulmonary vascular structural remodeling that occurred secondarily to
increase pulmonary pressure due to cold exposure, and NO-induced apoptosis in
artery smooth muscle cells might contribute to its regulatory effect on cold
exposure-induced pulmonary vascular structural changes. Therefore, we can
assume that the effects of NO on pulmonary smooth muscle cell apoptosis might
be involved in the mechanisms by which L-Arg modulates hypoxic pulmonary
vascular structural remodeling. However, sufficient evidence to demonstrate such
effects has been lacking.
The present study was designed to
examine the influence of the NO precursor, L-Arg, on the apoptosis of pulmonary
artery smooth muscle cells (PASMC) and the expression of Fas protein by
pulmonary arteries of rats with hypoxic pulmonary vascular structural
remodeling.
Materials and Methods
Animals and methods of hypoxia
Male Wistar rats weighing between
210 g and 300 g were randomly divided into a control group (n=7), a
hypoxia group (n=5), and a hypoxia with L-Arg group (n=5). For
hypoxic challenge, the rats of the hypoxia group and the hypoxia+L-Arg group
were put into a normobaric hypoxic chamber (Chinese Academy of Medical
Sciences, Beijing, China) with an oxygen concentration of 10.0%+/–0.5%. The animals underwent continuous
hypoxic challenge of 6 h per day for 14 d. For rats in the hypoxia+L-Arg group,
L-Arg was given intraperitoneally at a dose of 500 mg/kg each day before
hypoxic challenge. An equal volume of normal saline was injected
intraperitoneally in rats of the control and hypoxia groups. The rats in the
control group breathed room air. In these three groups, the routine breeding,
feeding and drinking conditions were the same.
Morphologic observation of the
pulmonary vessels
After 14 d of treatment, the rats
were anesthetized with an intraperitoneal injection of 10% (W/V)
urethane and fixed on the operating table. The thoracic cavity was exposed. One
side of a lung lobe was removed, fixed in 10% (W/V) formalin and
dehydrated in an ascending gradient of alcohol. After the lung tissues were
made transparent with dimethylbenzene, they were embedded in paraffin and
routinely processed into sections of 5 mm for elastic straining, and weighed and
counterstained with Van Gieson stain.
Determination of the
percentage of three types of small pulmonary vessels
Observed
under 40?
optical microscope CHC-212 (Olympus, Tokyo, Japan), the number of small
pulmonary vessels including muscularized arteries, partially muscularized
arteries and non-muscularized vessels with external diameters between 15 mm and 50 mm were counted and the percentage of each
type of vessel was determined.
Observed
under 40?
optical microscope CHC-212 (Olympus, Tokyo, Japan), the number of small
pulmonary vessels including muscularized arteries, partially muscularized
arteries and non-muscularized vessels with external diameters between 15 mm and 50 mm were counted and the percentage of each
type of vessel was determined.
Determination of relative
medial thickness (RMT) and relative medial area (RMA) of small and median
pulmonary arteries
An Image Processing & Analyzing
System Q550 LW (Leica, Wetzlar, Germany) was used for the analysis of small and
median muscularized pulmonary arteries with clearly defined internal elastic
laminae and regular shapes. The following parameters were measured: the
external diameter of external elastic lamina along the longest and shortest
axes (D1 and D2), the length of the internal elastic lamina
(LIEL) and the area enclosed by the
internal and external elastic lamina (AIEL and AEEL). The RMT and RMA of pulmonary arteries with
different cut angles a
and the conditions of either contraction or relaxation were computed from those
measurements using Barth’s method [5]. For each rat, 5–10 pulmonary median muscularized arteries
measuring 50–150
mm in external diameter, and
pulmonary small vessels measuring 15–50 mm in
external diameter were assessed. RMT and RMA of the median and small pulmonary
muscularized arteries were determined and the average values were computed.
Analysis of apoptosis
Using the terminal
deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate
(dUTP)-biotin nick end-labeling assay,
the apoptotic cells were detected. The paraffin-embedded sections of the lung
tissues were used for analysis. The sections were routinely dewaxed and
hydrated, then blocked in H2O2 and digested for 10–20 min in 100 mg/ml proteinase K at room temperature, and
dehydrated with 90% ethanol. Then 4 ml of 5?TdT
buffer (Promega, Madison, USA), 1 ml of 2 mM dUTP (Sigma, St. Louis, USA), 15 ml of water and 0.4 ml of TdT was added. The total reaction system
remained 20 ml.
The slides were put into a humidified chamber for 2 h at 37 ?C and the
reaction fluid was thoroughly washed out. The sample was blocked in horse serum
(1:100) and kept at room temperature for 20 min, then horseradish peroxidase
labeled with avidin and biotin was added. We incubated the sample in a
humidified chamber at 37 ?C for 1 h, rinsed it three times, for 5 min
each, and developed the color for 2–3 min using 3,3‘-diaminobenzidine-tetrachloride (DAB)-H2O2, then stained it with hematoxylin. The cells
with brown granules in the nucleus were defined as apoptotic cells. For each
rat, 15–20 pulmonary
arteries were detected, and the numbers of apoptotic smooth muscle cells and
total smooth muscle cells were counted. The ratio of numbers of apoptotic cells
to smooth muscle cells in median and small pulmonary arteries was calculated.
Immunohistochemical analysis
After they were dewaxed by dimethylbenzene,
the sections of lung tissue were put into water and the antigens were
heat-processed (92 ?C–98 ?C) for 10 min. Then the slides were
washed twice (5 min each cycle) in phosphate-buffered saline (PBS), blocked in
sheep (goat) serum working solution for 10 min, then incubated at 37 ?C for 1 h
with anti-Fas antibodies (1:100) (Santa Cruz Biotechnology, Santa Cruz, USA).
After being washed twice in PBS (5 min each cycle), the slides were incubated
with biotinylated secondary antibodies and then with avidin-biotin complex at
37 ?C for 30 min. Subsequently the sample was washed in PBS, incubated in
DAB for 20–30 min and
stained in hematoxylin. Sections were dehydrated through an ascending gradient
of ethanol, made transparent with dimethylbenzene and observed with a
microscope. The brown granules in pulmonary smooth muscle cells were defined as
the positive signals.
The Fas expression in PASMC was
examined using a semiquantitative method. We defined the level of expression as
negative (–) if there were
no positive Fas signals in PASMC, as (+) if 1%–50% cells showed Fas expression, and as (++)
if 51%–100% cells
showed Fas expression. At least 10 median and small pulmonary arteries were
examined for each sample. For the convenience of understanding and statistical
procedures, the content of Fas protein expressed in pulmonary arteries was
calculated as follows: the percentage of pulmonary arteries with a certain
extent of reaction intensity was multiplied by the weighed values of their
reaction intensity. The weighed value of reaction intensity was 0, 0.5 and 1.0,
if the reaction intensity was –, + and ++, respectively.
Statistical analysis
Data were presented as the mean+/–SD. The percentage of these three
types of pulmonary small vessels, RMT and RMA of the pulmonary muscular
arteries, and the integral values of Fas expression in the PASMC were compared
using ANOVA. The Q test was used for intergroup comparison. The ratio of
apoptotic cells to total PASMCs was analyzed using the Kruskal-Wallis test.
Results
changes in the percentage of
three types of pulmonary small vessels
Under an optical microscope, the
muscularized artery has continuous external and internal elastic lamina, the partially
muscularized artery has a continuous external elastic lamina and a
discontinuous internal elastic lamina, and the non-muscularized vessel has only
one single elastic lamina. The percentage of the three types of pulmonary small
vessels was significantly different between any two groups (P<0.01; Table 1). In the
hypoxia group, the percentage of muscularized arteries in pulmonary small
vessels was significantly higher than that in the control group (P<0.01). In the hypoxia+L-Arg group,
the percentage of muscularized arteries in pulmonary small vessels was
significantly lower than that in the hypoxia group (P<0.01). The percentage of
non-muscularized vessels in pulmonary small vessels of rats in the hypoxia
group was significantly lower than that of the control group (P<0.01). The percentage of
non-muscularized vessels in small pulmonary vessels in the hypoxia+L-Arg group
was significantly higher compared with that of the hypoxia group (P<0.01).
Changes in the RMT and RMA of
the median and small muscularized pulmonary arteries
Fig. 1 shows significant differences in RMT and RMA
of median and small muscularized pulmonary arteries in rats in each of the
three groups. The quantitative results are shown in Table 1. The RMT and
RMA of median muscularized pulmonary arteries in the hypoxia group were both
significantly increased compared with those of the control group (P<0.01), and also higher than those
of small muscular pulmonary arteries (P<0.01). In the hypoxia+L-Arg group,
the RMT and RMA of median muscularized pulmonary arteries were both
significantly lower than those in the hypoxia group (P<0.05). But there was no
significant variation in these parameters between the hypoxia+L-Arg group and
the control group (P>0.05). The RMT and RMA of small muscularized pulmonary arteries in the
hypoxia+L-Arg group were significantly lower than those in the hypoxia group (P<0.01).
Apoptosis of PASMC
The apoptosis of PASMC is shown in
Fig. 2. There was a difference in the ratio of apoptotic smooth muscle cells
to smooth muscle cells in the small and median pulmonary arteries between any
two groups (P<0.05)
(Table 2). The ratio of apoptotic smooth muscle cells to smooth muscle
cells of the median and small pulmonary arteries in the hypoxia group was lower
than that in the control group (P<0.05). In the hypoxia+L-Arg group,
the ratio of apoptotic smooth muscle cells to total smooth muscle cells of the
median and small pulmonary arteries was higher than that of the hypoxia group (P<0.05). However, there was no
significant difference in the ratio between the hypoxia+L-Arg group and the
control group (P>0.05).
Fas expression in smooth
muscle cells of pulmonary arteries
As shown in Table 3, the level
of Fas expression in the hypoxia group was significantly lower than in the
control group (P<0.05).
The level of Fas expression in the hypoxia+L-Arg group was significantly higher
than in the hypoxia group (P<0.05). Fig. 3 shows the results of Fas expression in smooth
muscle cells of median pulmonary arteries of the different experimental groups.
Discussion
The results of this study showed
that pulmonary arteries were remodeled after two weeks of normobaric hypoxia.
The degree of muscularization of the pulmonary small vessels was significantly
enhanced. The RMT and RMA of the median and small pulmonary arteries were
significantly increased. We found that the smooth muscle cell apoptosis in
pulmonary arteries and the expression of Fas, the protein of an
apoptosis-related gene, were significantly suppressed during the process of
pulmonary vascular remodeling. Cellular apoptosis and proliferation are two
important processes determining the biological behavior of vascular smooth
muscle cells, therefore the imbalance between them would influence vascular
structure. Our findings suggested that suppression of PASMC apoptosis might be
involved in the mechanisms of hypoxic pulmonary vascular remodeling.
The present study revealed that
the administration of L-Arg, a NO precursor, alleviated the degree of
muscularization of small pulmonary vessels in hypoxic rats. The RMT and RMA of
the pulmonary median and small muscularized arteries were decreased. These
facts implied that L-Arg alleviated the development of hypoxic pulmonary
vascular remodeling, which was consistent with the previous findings [2].
However, the cellular or molecular
mechanism by which L-Arg alleviated hypoxic pulmonary vascular remodeling is
still not clear. It was found that NO had direct suppressive effects on
vascular smooth muscle cell proliferation [6], which may be one of the
mechanisms by which L-Arg modulated hypoxic pulmonary vascular remodeling. In
addition, NO could modulate hypoxic pulmonary vascular remodeling by inhibiting
the synthesis and secretion of smooth muscle cell proliferation promoting factors, such as
endothelins, platelet-derived growth factor and fibroblast growth factor,
therefore indirectly suppressing cell proliferation [7–9]. Recent studies showed that, in
a pattern contrary to cell mitogenesis (mitosis), apoptosis along with
proliferation determined the number of vascular smooth muscle cells, and the
balance between apoptosis and proliferation maintained the constancy of the
total number of cells. Vascular smooth muscle remodeling is probably the result
of cooperative modulation of proliferation and apoptosis. It was noted that an
increase in cell apoptosis resulted in a decrease in the number of vascular
smooth muscle cells, thereby altering the structure of the vascular wall [10].
An in vitro study on the effect of NO on apoptosis of cultured PASMC in
rats by Smith and co-workers [4] proved that NO could promote PASMC apoptosis.
Recent studies on the effects of NO precursor L-Arg on pulmonary vascular
structural remodeling in broilers with pulmonary hypertension induced by cold
exposure drew the conclusion that supplemental L-Arg partially inhibited
pulmonary vascular structural remodeling that occurred secondary to cold
manipulation [11]. Therefore, it could be assumed that the smooth muscle cell
apoptosis induced by L-Arg might be involved in the mechanisms responsible for
the development of hypoxic pulmonary vascular structural remodeling. It was
also found in another recent study that L-Arg could reduce the synthesis of
extracellular matrix-collagen and increase its degradation, thus having an
important modulating effect on pulmonary vascular matrix remodeling induced by
high pulmonary blood flow [12]. However, no experimental data exists on the
mechanism by which L-Arg influences the apoptosis of PASMC in rats with hypoxic
pulmonary vascular structural remodeling. Our results indicated that the smooth
muscle cell apoptosis of pulmonary small and median arteries was suppressed
apparently along with pulmonary vascular structural remodeling after chronic
hypoxia. Exogenous administration of L-Arg, NO promoted the apoptosis of PASMC
and alleviated the hypoxic pulmonary vascular structural remodeling. Hence, the
above results suggested that the inducement of smooth muscle cell apoptosis by
L-Arg might be involved in the regulation of hypoxic pulmonary vascular
structural remodeling and hypoxic pulmonary hypertension, which agreed with the
previous findings [13].
The mechanism by which L-Arg
modulates pulmonary artery smooth muscle cell apoptosis is still unknown.
Apoptosis-related genes might be involved in this process. Fas is a kind of
uni-transmembrane glycosylated receptor protein on the surface of cells and
belongs to the family of tumor necrosis factor and nerve growth factor
receptors. It could induce cell apoptosis when conjugated with Fas ligand [14].
The results of our study showed that smooth muscle cell apoptosis was
suppressed along with the downregulation of Fas expression in PASMC after
hypoxia. PASMC apoptosis was promoted along with the stimulation of Fas
expression when exogenous L-Arg was administered. Therefore, it is suggested
that Fas expression induced by PASMC might be one of the mechanisms by which
L-Arg modulates smooth muscle cell apoptosis and therefore attenuates hypoxic
pulmonary vascular structural remodeling in rats.
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