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doi:10.1111/j.1745-7270.2006.00123.x |
Transforming Growth Factor-b1 Induces Transdifferentiation of
Fibroblasts into Myofibroblasts in Hypoxic Pulmonary Vascular Remodeling
Yong-Liang JIANG, Ai-Guo DAI*,
Qi-Fang LI, and Rui-Cheng HU
Department
of Respiratory Medicine, Hunan Institute of Gerontology, Hunan Province
Geriatric Hospital, Changsha 410001, China
Received: July 19,
2005
Accepted: September
5, 2005
This work was supported
by the grants from the Foundation of Hunan Province Health Committee (No.
Y02-081 and No. B2004-137) and the Foundation of Hunan Province Educational
Committee (No. 03C397)
*Corresponding author: Tel, 86-731-4762793; Fax,
86-731-4735215; E-mail, [email protected]
Abstract The muscularization of non-muscular pulmonary arterioles is
an important pathological feature of hypoxic pulmonary vascular remodeling.
However, the origin of the cells involved in this process is still not well
understood. The present study was undertaken to test the hypothesis that
transforming growth factor-b1 (TGF-b1) can induce transdifferentiation of
fibroblasts into myofibroblasts, which might play a key role in the
muscularization of non-muscular pulmonary arterioles. It was found that mean
pulmonary arterial pressure increased significantly after 7 d of hypoxia.
Pulmonary artery remodeling index and right ventricular hypertrophy became evident
after 14 d of hypoxia. The distribution of nonmuscular, partially muscular, and
muscular vessels was significantly different after 7 d of hypoxia.
Immunocytochemistry results demonstrated that the expression of a-smooth muscle actin was increased in intra-acinar
pulmonary arteries with increasing hypoxic time. TGF-b1
mRNA expression in pulmonary arterial walls was increased significantly after
14 d of hypoxia, but showed no obvious changes after 3 or 7 d of hypoxia. In
pulmonary tunica adventitia and tunica media, TGF-b1
protein staining was poorly positive in control rats, but was markedly enhanced
after 3 d of hypoxia, reaching its peak after 7 d of hypoxia. The myofibroblast
phenotype was confirmed by electron microscopy, which revealed microfilaments
and a well-developed rough endoplasmic reticulum. Taken together, our results
suggested that TGF-b1 induces transdifferentiation
of fibroblasts into myofibroblasts, which is important in hypoxic pulmonary
vascular remodeling.
Key words transforming growth factor-b1;
fibroblast; myofibroblast; hypertension; lung
Chronic obstructive pulmonary disease in humans is associated with
chronic hypoxia. The principal medical consequences of chronic hypoxia include
polycythemia and pulmonary hypertension, and finally, cor pulmonale. Laboratory
animals subjected to decreased ambient oxygen concentrations manifest similar
physiological responses, resulting in the occurrence of hypoxic pulmonary
hypertension. The pathophysiology of hypoxic pulmonary hypertension is complex
and involves vasoconstriction as well as neomuscularisation and thickening of
the media and adventitia of pulmonary arterioles. The fibroblast is the most
abundant cell type in normal connective tissues and plays a central role in
synthesis, degradation, and remodeling of the
extracellular matrix in health and disease. The majority of fibroblasts
demonstrate the ability of converting into a-smooth muscle actin (a-SMA)-containing
myofibroblasts in response to specific stimuli. Myofibroblasts are “hyperactivated”
fibroblasts with properties of fibroblasts and muscle cells [1–3] that
facilitate tissue remodeling and wound healing and also play a pathological
role in fibrotic disease. Compared with their precursor cell type
(referred to as stellate transformed [4] or protomyofibroblasts [1]), activated
myofibroblasts have dramatically higher levels of extracellular matrix (ECM)
and cytokine secretion, increased contraction [3], and a trademark stellate
morphology with prominent stress fibers. Histochemically, myofibroblasts are
characterized by the expression of a-SMA. They can also express other contractile proteins, such as the striated-muscle isoforms of myosin heavy
chain. Myofibroblast activation is strictly regulated by cytokines that control
differentiation, proliferation, contraction, ECM secretion, and migration to
the site of wound healing or tissue remodeling [3]. After
completion of remodeling activities, myofibroblasts are eliminated by
apoptosis; however, when the myofibroblast life cycle is not regulated
properly, myofibroblasts persist with continued force generation and ECM
production, resulting in pathological fibrosis, scarring, and fibrocontractile
disease [5].
Transforming growth factor-b1 (TGF-b1) is involved in tissue
repair by modulating the growth of mesenchymal cells, augmenting the synthesis
of several ECM proteins, and facilitating the differentiation of fibroblasts
[6]. Increased expression of TGF-b1 has been demonstrated in
human restenotic lesions. In experimental settings systemic administration of
TGF-b1 resulted in the formation of a neointima rich in ECM proteins [7].
Conversely, anti-TGF-b1 neutralizing antibodies reduced ECM proteins, further pointing to the important role of TGF-b1 in vascular repair [8]. However,
the roles of TGF-b1 and myofibroblasts in the development of hypoxia-induced pulmonary
hypertension and the accompanying vascular remodeling are incompletely
understood. In this study, we investigate the dynamic expression of TGF-b1 and myofibroblast
formation in a rat model of hypoxia.
Materials and
methods
Animals and hypoxia model
The protocol for exposure of rats to hypoxia and normoxia was
identical to that reported previously by our laboratory [9]. In the present
study, we used 40 male Wistar rats purchased from the Animal Experimental
Center of Central South University, Changsha, China. The animals weighed 220+/–10 g and the
average age was 6–8 weeks. They were randomly divided into five groups (eight rats in
each group). Each group of hypoxic rats was exposed for a specified time period
(3, 7, 14, or 21 d) for 8 h per day intermittently to normobaric hypoxia
(10.0%+/–0.5% oxygen) in a ventilated chamber. Age- and weight-matched
control rats were maintained in normobaric 21% oxygen (fresh air). To establish
the hypoxic conditions the chamber was flushed with a mixture of room air and
nitrogen from a liquid nitrogen reservoir. An oxygen analyser (HT-6101; Kanda
Electrical, Chengdu, China) was used to monitor the chamber environment. Carbon
dioxide was removed with soda lime, excess humidity removed by anhydrous
calcium chloride, and boric acid was used to keep ammonia levels within the
chamber to a minimum. The normoxic control rats were not kept in the chamber
but they were housed in the same room and treated in the same fashion as the
hypoxic rats.
Mean pulmonary arterial
pressure (mPAP) measurement
mPAP was measured as described
previously [10]. After rats were anesthetized with pentobarbital sodium (40
mg/kg intraperitoneally), a specially designed single-lumen catheter was
inserted into the main pulmonary artery through the right jugular vein, at
which point the position of the catheter was judged by the waveform of the
pressure signal. The mPAP was measured with PowerLab monitoring
equipment (AD Instruments, Milford, USA).
Right ventricular hypertrophy
index (RVHI)
After the measurement of mPAP, the rats were killed and their
lungs were collected for morphometry analysis, in situ hybridization and
immunohistochemical examination; their hearts were collected for measurement of
RVHI. For right ventricular hypertrophy measurement, hearts were excised
and atria were removed. The right ventricular free wall was dissected, and each
chamber weighed. The ratio of right ventricular weight (RV) to weight of
left ventricle (LV) plus septum (S) [RV/(LV+S)]
was used as an index of right ventricular hypertrophy.
Vessel morphometric analysis and
constituent ratio of the three types of pulmonary vessels in the intra-acinus
pulmonary artery
Four micrometer lung sections were embedded in paraffin, stained
with hematoxylin-eosin, then examined using light microscopy. At least five
representative pulmonary arterioles (outer diameter approximately 100–150 mm), chosen from
three different sections from each animal, were independently examined. The
images of the arterioles were captured and analysed with PIPS-2020 Image
software (Chongqing Tianhai Company, Chongqing, China). To evaluate hypoxic
pulmonary vascular remodeling, the ratio of vascular wall area to external
diameter (WA%), the ratio of vascular lumen area to total area (LA%),
the number of smooth muscle cell nuclei in pulmonary arteriole tunica media (SMC;
per 1000 mm2) and pulmonary artery media thickness were obtained. The total
number of intra-acinus pulmonary artery of unit area (25 mm2) was counted. Moreover, the constituent ratio of
nonmuscular, partially muscular, and muscular arteries was obtained.
In situ hybridization of TGF-b1
In situ hybridization was performed
using a detection kit (Wuhan Boster Biological Technology, Wuhan, China). The
oligonucleotide probes (Wuhan Boster Biological Technology) were designed according
to the TGF-b1 sequences of rat. The sequences of probes against TGF-b1 mRNA were: 5‘-ACCTGCAAGACCATCGACATGGAGCTGGTG-3‘;
5‘-TGTACAACAGCACCCGCGACCGGGTGGCAG-3‘; 5‘-CTACCAGAAATATAGCAACAATTCCTGGCG-3‘.
Hybridization was performed on serial sections of formalin-fixed
(containing 0.1% diethylpyrocarbonate) paraffin-embedded lung tissues according
to manufacturer’s instructions. Briefly, sections were digested with pepsin for
20 min at 37 ºC. After 2 h of prehybridization, sections were incubated with digoxin-labeled
single-stranded oligonucleotide probes for 16 h at 38 ºC (negative controls
were incubated with blank probe solution). After unbound probes were washed
off, sections were incubated with rabbit antibodies against digoxin and with
biotinylated goat-antirabbit secondary antibodies. Afterwards, sections were
incubated with streptavidin-horseradish peroxidase (HRP) and visualized by a
color reaction with diaminobenzidine (Wuhan Boster Biological Technology).
Brown and yellow colours indicated positive results. Finally, the sections were
counterstained with hematoxylin and mounted. Expression levels of mRNA were
quantified by a pathology image analysis system (PIPS-2020).
Immunohistochemistry analysis
of TGF-b1 and a-SMA
A streptavidin-biotin complex kit (Wuhan Boster Biological
Technology) was used for immunohistochemisty, which was performed similar to
that described previously with minor modifications. Briefly, serial sections of
formalin-fixed paraffin-embedded lung tissues were digested with 3% H2O2 for 20 min at room temperature, then preincubated with 10%
non-immunized serum. Sections were incubated with rabbit anti-TGF-b1 or anti-a-SMA polyclonal
antibody (at a working dilution of 1:100) overnight at 4 ºC (negative controls
were incubated with phosphate-buffered saline only). After unbound antibodies
were washed off, the sections were incubated with biotinylated goat-antirabbit
secondary antibodies and thereafter incubated with streptavidin-HRP.
Subsequently, sections were visualized by a color reaction with
diaminobenzidine as the substrate. Brown and yellow colours indicated positive
results (mainly cytoplasm). Finally, the sections were counterstained with
hematoxylin (resulting in blue nuclei) and mounted. Expression levels of
protein were quantified by a pathology image analysis system (PIPS-2020).
Electron microscopy
For electron microscopy, tissue cultures were fixed in cold 2.5%
glutaraldehyde in 0.1 M of sodium cacodylate buffer and postfixed in a solution
of 1% osmium tetroxide, dehydrated, and embedded in a standard fashion. The
specimens were then embedded, sectioned, and stained by routine means for a
JEO-1200 electron microscope (JEOL, Tokyo, Japan).
Statistical analysis
Data were expressed as mean+/–SD. The group t-test
was used to compare data between two groups. ANOVA
was used to determine statistically significant differences among
multiple groups, with the Newman-Keuls test comparing statistical significance
between two groups. Categorical data were expressed as frequency distribution
and the c2 test was used. P<0.05
was considered as statistically significant.
Results
Chronic hypoxia increased mPAP
mPAP was measured as an indicator of
pulmonary artery pressure in conscious rats. mPAP in normoxic rats was
14.02+/–0.41 mmHg. As expected, the hypoxic animals developed pulmonary
hypertension after 7 d of exposure to hypoxia (P<0.05), reaching its
peak level after 14 d of hypoxia, then remained on the high level (Table 1).
Chronic hypoxia leads to
hypoxic pulmonary vascular remodeling and right ventricular hypertrophy
As shown in Table 1, pulmonary arterioles in normoxic animals
were thin, whereas after 7 d of hypoxic exposure, they developed increased medial
thickness characteristic of pulmonary hypertension. Quantification of these
structural changes in several lung sections of all of the animals exposed to
each of the different hypoxia time periods (3, 7, 14 or 21 d) revealed
significantly increased medial thickness of pulmonary arterioles in hypoxic
animals in comparison with normoxic controls. Right ventricular hypertrophy
resulting from right ventricle pressure overload is a hallmark of pulmonary
hypertension. After 14 d of hypoxia, RVHI was significantly increased in
comparison with the control (P<0.05). RVHI had increased
further after 21 d of hypoxia. This result indicated right ventricular
hypertrophy had developed after 14 d of exposure to hypoxia.
Changes of constituent ratios
of three types of pulmonary arterioles after hypoxia
We observed the distribution of arterioles by wall structure in
hypoxia-induced pulmonary hypertension. The distribution of nonmuscular,
partially muscular, and muscular arterioles was significantly different (P<0.05)
from 7 d in alveolar wall arterioles; muscular and partially muscular arteries
increased at the expense of nonmuscular arterioles (Fig. 1).
Dynamic analysis of a-SMA in alveolar wall arterioles
In both normal and hypoxic lungs, bronchial smooth muscle cells and
the smooth muscle cells of pre-acinar vessels associated with bronchioli and terminal bronchioli expressed a-SMA. In the
normal lung, in the thick-walled oblique muscular
artery, and in arterioles associated with respiratory bronchioli at the
entrance to the acinus, cells expressing a-SMA were evident [Table
2 and Fig. 2(A)]. Alveolar duct and alveolar wall arterioles with
cells expressing a-SMA were rare. Initially in hypoxia (3 d), the distribution of a-SMA cells was
similar to that throughout the normal lung [Table 2 and Fig. 2(B)].
By 7 d, increased numbers of a-SMA cells were evident in pre-acinar arterioles of the hypoxic
lung, but most intra-acinar arterioles were still negative. At 14 d, alveolar
arterioles with a-SMA cells increased and septal cells at the entrance
to alveolar ducts were also positive [Table 2 and Fig. 2(C)]. By 21 d, the number and intensity of a-SMA cells had increased
and small thick-walled alveolar wall and duct arterioles with
these cells were evident, including arterioles with
muscular walls as well as partially muscular arterioles.
All of the distal thick-walled arterioles had a-SMA cells.
Hypoxia induces TGF-b1 mRNA and protein expression in
pulmonary arterial walls
Table 3 shows control pulmonary
arterioles displayed low-level expression of TGF-b1 transcripts in medial SMCs.
Adventitial fibroblasts also showed a paucity of TGF-b1 transcripts,
TGF-b1 mRNA expression in pulmonary arterial walls was increased
significantly after 14 and 21 d of hypoxia, but showed no obvious changes after
3 or 7 d of hypoxia [Fig. 3(A–C)]. TGF-b1 mRNA was
located predominantly in tunica adventitia and tunica media. In pulmonary
arterioles tunica adventitia and tunica media, TGF-b1 protein staining was
poorly positive in control rats, but was markedly enhanced after 3 and 7 d of
hypoxia, then weakened after 14 and 21 d of hypoxia [Fig. 4(A–C)].
Ultrastructural characteristics
of adventitial myofibroblast and analysis of cell phenotype in alveolar wall
arterioles
To examine the ultrastructural changes associated with TGF-b1 induction, the
smallest vessels were examined by electron microscopy. In the normal lung, they
were constituted in alveolus and endothelial cells [Fig. 5(A)], but at
the late stage of hypoxia (21 d), myofibroblast phenotype was organized between
elastic laminae. Aligning fibroblasts were associated with the wall.
Myofibroblasts formed the walls of the smallest vessels [Fig. 5(B)]. All
of these cells were well-developed microfilaments.
Discussion
Hypoxic pulmonary vascular remodeling is an important pathological
feature of hypoxic pulmonary hypertension, leading to increased pulmonary vascular
resistance and reduced compliance. It involves thickening of all three layers
of the blood vessel wall (due to hypertrophy and/or hyperplasia of the
predominant cell type within each layer), as well as extracellular matrix
deposition. Neomuscularisation of non-muscular arteries and neo-intimal lesions
also occur [11]. But the cell origin is unclear for the muscularization of
non-muscular pulmonary arterioles in hypoxic pulmonary vascular remodeling. Our
results demonstrate that the hypoxic animals developed pulmonary hypertension
after 7 d of exposure to hypoxia; after 14 and 21 d, hypoxic animals showed
significantly increased medial thickness of pulmonary arterioles and the
muscularization of non-muscular pulmonary arterioles compared with normoxic
controls.
Myofibroblasts represent highly specialized mesenchymal cells that
play a central role in tissue repair. However, very little is known about the
important role myofibroblast differentiation might play in hypoxic pulmonary
vascular remodeling. We designed our experiments to specifically characterize
the myofibroblast phenotype, its induction by TGF-b1, and the potential
contribution to pathological changes in hypoxic pulmonary vascular remodeling. Here,
we demonstrate that myofibroblast formation contributes to muscularization of
non-muscular pulmonary arterioles and TGF-1 is capable of inducing
myofibroblast differentiation. Their formation is marked by the development of
bundles of microfilaments (stress fibers) and
abundant connections with the surrounding ECM. One of the most prominent
features of TGF-b1-inducing fibroblasts is the appearance of a-SMA-positive
myofibroblasts. Our results demonstrate that in hypoxic pulmonary vascular
remodeling, the predominant contribution to myofibroblast
generation comes from resident fibroblasts, and others have identified TGF-b1 as the key
molecular switch in myofibroblast generation [12,13]. Moreover, hypoxia might
also induce differentiation of pulmonary artery adventitial fibroblasts into myofibroblasts [14]. These characteristics are consistent with the primary role of newly-formed
myofibroblasts to close an open wound by means of ECM protein synthesis (e.g.,
collagens) and contraction. Subsequent studies have confirmed the
presence of myofibroblasts in a wide range of other pathological conditions
that are associated with fibrogenesis and organ remodeling [15,16].
TGF-b1 is a member of the TGF-b cytokine superfamily that coordinates
differentiation of mesenchymal stem cells during such distinct processes as
organogenesis, bone and neuronal tissue formation, and myofibroblast activation
[17]. In the present study, hypoxia induced dynamic changes in TGF-b1 expression, with the initial changes involving the adventitia and
media, as reflected by in situ hybridization and
immunohistochemistry findings. The increase in TGF-b1 mRNA in adventitial and
medial cells was apparent as early as 3 d after hypoxia. The question can be
raised as to the mechanism(s) of TGF-b1 induction after vascular hypoxia, inasmuch
as normal adventitial fibroblasts are devoid of this cytokine. The ability of
TGF-b1 to induce its own expression suggests that its release from
degranulated platelets and activated macrophages might initiate TGF-b1 upregulation
in adventitial fibroblasts [18]. Furthermore, platelet-derived growth factor
released from platelets early after vascular insult could contribute to the
induction of TGF-b1 [19]. Interestingly, the TGF-b1 mRNA was not
significantly increased until 14 d of hypoxia, yet TGF-b1 protein was markedly
increased after only 3 d of hypoxia. Moreover, after 14 and 21 d of hypoxia,
when the TGF-b1 mRNA levels were at their highest, protein levels started to drop.
This suggests a major alteration in post-transcriptional regulation.
In vivo and in vitro studies
indicate that TGF-b1 is one of the main inducers of the differentiation of fibroblasts
into myofibroblasts [12,13]. Prior studies have shown that TGF-b1 induces
adventitial myofibroblast differentiation using a protein kinase Ca-dependent process [20].
The origin of myofibroblasts in various tissues has been a subject of controversy [21]. The augmented synthetic function of
myofibroblasts derived from various tissues, with the accompanying deposition
of collagens and fibronectin, is consistent with the profibrotic effects of
TGF-b1 [22]. Furthermore, myofibroblasts are capable of mediating
contraction of their surrounding environment.
Hypoxia might initiate a series of cellular events leading to hypoxic
pulmonary vascular remodeling. Intervention at this stage of the disease might
prevent the development of structural changes and the progression to hypoxic
pulmonary vascular remodeling. Through elucidation of the pathologic importance
of TGF-b1 in fibroblast-differentiated myofibroblasts, we expect the
availability of specific TGF-b1 antagonists. The present study provides new information about
myofibroblast formation and its contribution to the muscularization of
non-muscular pulmonary arterioles. Further elucidation of the origin of
myofibroblasts might reveal clues for approaches to curing hypoxic pulmonary
hypertension.
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