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Department of Medicine, University of California San Diego, La Jolla, California 92093-0623
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Berg, John T., Zhenxing Fu, Ellen C. Breen, Hung-Cuong Tran,
Odile Mathieu-Costello, and John B. West. High lung inflation increases mRNA levels of ECM components and growth factors in lung
parenchyma. J. Appl. Physiol. 83(1):
120-128, 1997.
Remodeling of pulmonary capillaries occurs after
chronic increases in capillary pressure (e.g., mitral stenosis). Also,
remodeling of pulmonary arteries begins within 4 h of increased wall
stress and is endothelium dependent. We have previously shown that high
lung inflation increases wall stress in pulmonary capillaries. This
study was designed to determine whether high lung inflation induces
remodeling of the extracellular matrix (ECM) in lung parenchyma.
Open-chest rabbits were ventilated for 4 h with
9-cmH2O positive end-expiratory pressure (PEEP) on one lung and
1-cmH2O PEEP on the other
(High-PEEP group), or with 2-cmH2O
PEEP on both lungs (Low-PEEP group). An additional untreated control
group was also included. We found increased levels of mRNA in both
lungs of High-PEEP rabbits (compared with both the Low-PEEP and
untreated groups) for 
1(III)
and 2(IV) procollagen,
fibronectin, basic fibroblast growth factor, and transforming growth
factor-
1. In contrast,
2(I) procollagen and vascular
endothelial growth factor mRNA levels were not changed. We conclude
that high lung inflation for 4 h increases mRNA levels of ECM
components and growth factors in lung parenchyma.
extracellular matrix; messenger ribonucleic acid; stress failure; wall tension; vascular remodeling; strength of capillaries
INTRODUCTION THE MAGNITUDE OF MECHANICAL STRESS in a vessel wall is
directly proportional to transmural blood pressure but inversely
proportional to wall thickness. Because of the thinness of pulmonary
capillary walls, it can be calculated that wall stresses in pulmonary
capillaries during conditions of severe exercise are very high and
similar to those in the normal human aorta (31). This suggests that the
blood-gas barrier faces a serious bioengineering dilemma. It has long
been appreciated that the capillary wall must be extremely thin to
allow diffusive gas exchange. However, the capillary wall must also be
immensely strong to withstand periods of very high wall stress.
There is evidence that the capillary wall is just strong enough to
withstand the largest stresses encountered under normal physiological
conditions. However, if capillary pressure becomes abnormally high,
circumferential tension increases in the vessel wall, and stress
failure of pulmonary capillaries occurs with high-permeability edema
(29, 31). Exercise-induced pulmonary hemorrhage, high-altitude
pulmonary edema, and neurogenic pulmonary edema are examples of
conditions in which stress failure is caused by increased capillary
transmural pressure. In addition, high lung inflation can cause stress
failure of pulmonary capillaries by increasing longitudinal tension in
tissue elements in the alveolar wall. Direct evidence that this occurs
comes from morphometric studies where the appearance of breaks in the
capillary wall after high lung inflation was quantified (9).
Additionally, damage to pulmonary capillaries is prevented by encasing
rabbits in whole body plaster casts to prevent expansion of the chest
wall during high peak inspiratory pressure ventilation (10). The
increased capillary permeability observed in some patients after
mechanical ventilation at high lung volumes may also be attributed to
stress failure of pulmonary capillaries (22).
The fact that the blood-gas barrier is just thick enough, and strong
enough, to withstand the maximal wall stresses that develop under
physiological conditions suggests that its structure is being
continuously remodeled in response to capillary pressure. There is some
evidence to support this hypothesis. For example, in mitral stenosis
where capillary pressure is gradually raised over many months or years,
alveolar capillaries show thickening of the endothelial cell basement
membrane, with an increase in the number of cytoplasmic processes of
pericytes (15). Similar changes are observed in pulmonary capillaries
during pulmonary venoocclusive disease where capillary pressure is
raised gradually over long periods of time (14). The importance of the
basement membrane in providing strength to the capillary wall is
supported by several observations including the fact that the thickness of the basement membrane in systemic capillaries increases as the
hydrostatic pressure within these capillaries increases down the body
(32), and glomerular capillaries, which are exposed to higher
hydrostatic pressures than are pulmonary capillaries, have much thicker
basement membranes (30).
The response of pulmonary capillaries and small parenchymal blood
vessels to increased wall stress has not previously been evaluated at
the molecular level. However, there has been extensive work on the
response of large vessels to increased pressure in the pulmonary
circulation. For example, Meyrick and Reid (20) used a hypoxic rat
model to increase pulmonary arterial pressure for periods up to 52 days. After 2 days of hypoxia, they observed new smooth muscle in small
pulmonary arteries, and by the third day they noted thickening of
capillary endothelial cells. After 10 days of hypoxia, there was a
doubling in thickness of the media and adventitia in the main pulmonary
artery due to increased smooth muscle, collagen, elastin, and edema.
The molecular biology of the response of pulmonary arteries to
increased wall stress has been studied by several groups. Mecham et al.
(19) looked at the response of pulmonary arteries to hypoxia in newborn
calves. They observed a two- to fourfold increase in the number of
medial smooth muscle cells and similar increases in elastin production
and elastin mRNA level. Poiani et al. (24) exposed rats to hypoxia and
also observed an increase in elastin synthesis in the pulmonary artery.
In addition, they found increases in collagen synthesis and in the
level of mRNA for In this study, we postulated that vascular remodeling in the pulmonary
artery in response to increased wall stress [as observed by Tozzi
et al. (28)] represents a generalized property of the pulmonary
vasculature, including pulmonary capillaries. Subjecting pulmonary
capillaries and other vessels in the parenchyma to high lung inflation,
and thus increased wall stress, may, therefore, initiate remodeling of
the extracellular matrix (ECM) including basement membrane. To test
this hypothesis, the right and left lungs of open-chest rabbits were
independently cannulated so that lung volume in each lung could be
selectively controlled by application of positive end-expiratory
pressure (PEEP) in the expectation that the opposite lung would be an
internal control. We restricted our study to parenchymal tissues
because parenchyma has the greatest concentration of small blood
vessels and we were specifically interested in the response of small
pulmonary vessels to high lung inflation. We found increased levels of
mRNA for ECM components and growth factors in lung parenchyma after
ventilation at high lung volume. Unexpectedly, the response occurred on
both sides of the lung.
METHODS
1(I)
procollagen within 3 days of exposure to hypoxia. Tozzi et al. (28)
applied mechanical tension to explant pulmonary artery rings from rats and showed increases in mRNA for
1(I) procollagen within 4 h that were endothelium dependent.
1 · h1,
respectively). A tracheal tube was inserted for temporary ventilation of both lungs (20-ml tidal volume, 45 breaths/min), and the lungs were
sighed to open collapsed airways (peak airway pressure of sighs
throughout the experiment = 25-30
cmH2O). The right carotid artery
was then cannulated for determination of blood gases and systemic blood
pressure. Finally, ventilator tidal volume was reduced to 10 ml,
the left chest was opened at the 4th intercostal space, and the
left main bronchus was cannulated to allow independent ventilation of
the left lung. The separate cannulations also allowed independent
monitoring of expired PO2,
PCO2, airway pressure, and lung
volume in each side of the lung.
32P]dCTP cDNA
probes, which have a specific activity of at least 1 × 109
disintegrations · min1 · µg1
(Prime-It II kit, Stratagene, La Jolla, CA). The membrane was then
prehybridized and hybridized in 50% formamide 5× saline sodium citrate (SSC) (20× SSC is 0.3 M sodium chloride, 0.3 M sodium citrate), 10× Denhardt's solution (100× Denhardt's
solution is 2% Ficoll, 2% polyvinyl pyrrolidone, 2% bovine serum
albumin, Factor V), 50 mM
NaH2PO4
(pH = 6.5), and 100-250 µg/ml salmon sperm at 37 or 42°C.
Blots were washed with 2× SSC, 0.1% sodium dodecyl sulfate (SDS)
at room temperature and with 0.1-0.5× SSC, 0.1% SDS at
50-65°C and were then exposed to X-ray film with the use of a
Cronex Lightening Plus intensifier screen at 
70°C.
Autoradiographs were quantitated by densitometry within the
linear range of signals and normalized to ribosomal 18S RNA
levels.
Source of recombinant plasmids. Rabbit
2(I) and
2(IV) procollagen cDNAs were
provided by Y. Ninomiya (Okayama University Medical School, Okayama,
Japan). Mouse 1(III)
procollagen cDNA was cloned by Liau et al. (18). Rat fibronectin cDNA
was cloned by Schwartzbauer et al. (27). Human basic fibroblast growth factor (bFGF) cDNA was cloned by Kurokawa et al. (16). Human transforming growth factor-1
(TGF-1) cDNA was cloned by
Qian et al. (25), and human vascular endothelial growth factor (VEGF) cDNA was cloned by Leung et al. (17).
Lung perfusion, tissue sampling, and transmission
electron microscopy. After a 4-h ventilation, the
animals were euthanized, and the lungs were briefly inflated to 25 cmH2O of positive pressure before
being deflated to 5 cmH2O for
perfusion with saline solution (11.06 g NaCl/l, 350 mosM
and 1,000 U heparin/100 ml) after cannulation of the pulmonary artery
(inflow) and left atrium (outflow), as previously described (9).
Perfusion (capillary hydrostatic pressure = 22.5 cmH2O) was carried out for 3 min
to remove blood from the pulmonary circulation, followed by fixative
(phosphate-buffered 2.5% glutaraldehyde; total osmolarity 500 mosM, pH 7.4) for 10 min. The upper level of the liquid in
each reservoir was adjusted to maintain the preset perfusion pressure
of 22.5 cmH2O throughout all
perfusions. After fixation, the lungs were excised and stored in
glutaraldehyde at 4°C.
Tissue preparation and morphometric procedures were identical to those
used in previous studies (9). Briefly, one slab (~0.5 cm thick) was
taken perpendicularly to the cranial-caudal axis at about one-third the
distance from the bottom of either lower lobe in each animal. A thin
vertical slice was then obtained from each slab and cut into smaller
blocks (~1.5 × 1.5 × 2.0 mm), which were rinsed overnight
in 0.1 M phosphate buffer adjusted to 350 mosM with NaCl (pH 7.4) and
embedded in Araldite. Five tissue blocks were selected randomly from
each vertical slice. Ultrathin sections (50-70 nm) were cut from
each block with a LKB Ultratome III, contrasted with uranyl acetate and
bismuth subnitrate (9), and micrographs for morphometry were taken on
70-mm films with a Zeiss 10 electron microscope. Micrographs of a
carbon-grating replica (Fullam, Schenectady, NY) were recorded for
calibration on each film.
For morphometry, we analyzed 12 micrographs from each block, yielding a
total of 57-60 micrographs from each slab in each animal.
Measurements were performed at a final magnification of ×11,000
with a Videometric 150 image analyzer (American Innovision), after
electronic positive reversal of the 70-mm negative films. As in
previous studies (9), a print of each micrograph was also available and
systematically examined during the measurements for unequivocal
identification of small endothelial and epithelial disruptions as well
as the presence (or absence) of basement membrane at all sites of
rupture. The frequency of disruptions of the blood-gas barrier was
quantified as the number of breaks per unit endothelial and epithelial
boundary length in the sections after the contour of capillary (inner
endothelial) and alveolar (outer epithelial) boundary segments in each
field of view were traced and the number of endothelial and epithelial
disruptions were counted. The presence, and extent, of interstitial
edema was assessed by measuring the thickness (profile width) of each
layer of the blood-gas barrier (endothelium, interstitium, and
epithelium). One to five sites were systematically sampled in each
micrograph (total 150-241 barrier sites/animal), and measurements
were made at right angles to the barrier at random points,
systematically determined by the image analyzer via electronically
generated test lines intersecting the barrier, as described previously
(9).
Statistical analysis. Statistical
comparisons between groups were made by using one-way analysis of
variance and the Student-Newman-Keuls test. Comparisons between the
right and left lungs in the same rabbit were made by using a paired
t-test. Values are expressed as means ± SE, and a value of P < 0.05 was considered significant.
RESULTS
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1(III) procollagen was
threefold higher in the lungs of High-PEEP rabbits than in Low-PEEP
rabbits, fibronectin mRNA was increased twofold, and
2(IV) procollagen mRNA was
increased by 50% (Fig. 2, Northern blot
analysis; and Fig. 3, densitometric analysis). Levels of mRNA for
2(I) and
1(III) procollagens and fibronectin in the lungs of Low-PEEP rabbits were also significantly increased compared with values in unventilated rabbits (Table 2, Unventilated group). However, the
significance of these differences is unclear because of uncontrolled
variables (e.g., absence of both surgical intervention and 4-h infusion
of anesthesia during ventilation in unventilated rabbits) that may
contribute to the observed differences between the Low-PEEP and
unventilated groups. For this reason, we have focused our analysis on
the comparison between the Low and High-PEEP groups of rabbits and
consider these differences to most accurately reflect the response of
parenchymal blood vessels to high lung inflation.
2(I) procollagen
[2(I)], 1(III) procollagen [1(III)], and
2(IV) procollagen
[2(IV)] and fibronectin (FN) in parenchymal lung tissue
from lungs of high-positive-end-expiratory-pressure (PEEP) rabbits that
were ventilated for 4 h with
1-cmH2O PEEP on one side of lung
(open bars) and 9-cmH2O on the
other (solid bars). mRNA levels are expressed in arbitrary
densitometric units normalized for loading. Values are means ± SE
(n = 5 rabbits). No differences were
statistically significant.
2(I),
1(III), and
2(IV) procollagen and
fibronectin. 18S, ribosomal RNA.
2(I),
1(III), and
2(IV) procollagen and FN in
parenchymal lung tissue from Low-PEEP (open bars) or High-PEEP (solid
bars) animals for 4 h. mRNA levels are expressed in arbitrary
densitometry units normalized for loading
(* P < 0.05 vs. other group).
Values represent means ± SE (n = 10 lungs).
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1
were increased fourfold in the lungs of High-PEEP rabbits compared with
Low-PEEP rabbits, whereas mRNA levels for bFGF increased by 60% (Fig.
5, Northern blot analysis; and Fig.
6, densitometric analysis). In contrast,
the level of VEGF mRNA was not altered by high lung inflation. Levels
of mRNA for bFGF were also higher in the lungs of Low-PEEP rabbits than
in unventilated controls (Table 2).
1
(TGF-1), or vascular
endothelial growth factor (VEGF) in parenchymal lung tissue from lungs
of High-PEEP rabbits that were ventilated for 4 h with
1-cmH2O PEEP on one side of lung
(open bars) and 9-cmH2O on the
other (solid bars). mRNA levels are expressed in arbitrary densitometry
units normalized for loading. Values represent means ± SE
(n = 5 rabbits). No differences were
statistically significant.
1,
or VEGF.
1, or VEGF in
parenchymal lung tissue from Low-PEEP (open bars) or High-PEEP (solid
bars) animals for 4 h. mRNA levels are expressed in arbitrary
densitometry units normalized for loading.
* P < 0.05 vs. other group.
Values represent means ± SE (n = 10 lungs).
DISCUSSION
High lung inflation increases longitudinal tension in tissue elements of the alveolar wall and stretches both the blood-gas barrier and associated cells in the periphery of the lung. There is substantial literature on the response of cells to mechanical stretch (7). However, most studies have focused on the response of tissues such as bone, skeletal muscle, myocardium, systemic blood vessels and pulmonary arteries, and relatively few studies have focused on cells in peripheral lung parenchyma.
We have previously demonstrated that high lung inflation causes stress failure of pulmonary capillaries (9). In that study, we found that the mean number of breaks in pulmonary capillaries increased tenfold (from 0.7 to 7.1 breaks/mm in the endothelium and from 0.9 to 8.5 breaks/mm in the epithelium) when airway pressure was raised from 5 to 20 cmH2O at a constant capillary transmural pressure of 32.5 cmH2O (see Fig. 4 in Ref. 9). In addition, there was considerable edema formation (mean thickness of the capillary interstitium increased from 0.17 to 0.49 µm).
The present study was designed to determine whether high lung inflation induces vascular remodeling of pulmonary capillaries and small parenchymal blood vessels in vivo. Because our protocol required physiological maintenance of rabbits during 4-h ventilation, it was necessary to select PEEP levels sufficiently high to increase wall stress but not excessively high to cause lung injury or edema and compromise gas exchange. As shown in Table 1, capillaries in the two groups of rabbits were normal and exhibited no evidence of stress failure other than the development of mild interstitial thickening on the side of the lung in High-PEEP rabbits that received 9-cmH2O PEEP ventilation. In addition, the basement membrane remained continuous along each of the few capillary breaks observed in all samples (9 breaks out of 358 micrographs in the Low-PEEP group; 1 break out of 357 micrographs in the High-PEEP group). These results, and the fact that all rabbits maintained gas exchange in both lungs during 4-h ventilation, suggest that stress failure of pulmonary capillaries did not occur. The observed interstitial thickening in capillaries of High-PEEP rabbits (Table 1) implies that high lung inflation caused stretching of the blood-gas barrier and fluid accumulation in the interstitial space. In total, the morphometric data suggest that 9-cmH2O PEEP provided a modest increase in wall stress without causing gross lung injury. The lack of either gross injury or edema in the lungs of Low-PEEP rabbits, and in the 1-cmH2O-PEEP lung of High-PEEP rabbits, further illustrates that wall stress remained low in these lungs during ventilation.
Vascular remodeling. Vascular remodeling is a dynamic process that involves both synthesis and degradation of collagen molecules (33). Functionally, types I, III, and IV collagen provide tensile strength to the vessel wall. Types I and III collagen are synthesized by fibroblasts, myofibroblasts, and smooth muscle cells and are distributed diffusely throughout the media and adventitia of small vessels and on the thick side of the capillary wall. In contrast, type IV collagen is synthesized by endothelial and epithelial cells and is found mainly in the basement membrane where it provides major support for the thin side of the blood-gas barrier (30).
Strong evidence that mechanical forces cause remodeling of the
pulmonary vasculature comes from studies in which pulmonary artery
rings were subjected to mechanical stretch. Tozzi et al. (28) applied static mechanical stretch to pulmonary artery
segments for 4 h and observed increased synthesis of collagen
and elastin at the protein level and increased gene expression of mRNA
for 1(I) procollagen and
v-sis [a protooncogene encoding
a peptide homologous to the B chain of platelet-derived growth factor
(PDGF)]. These changes occurred without a proliferative response
as assayed by
[3H]thymidine
incorporation. Because the response was not present in arterial
segments devoid of endothelium, the authors conclude that
endothelium-derived factors are involved in stretch-induced production
of ECM proteins. Because of the increase in
v-sis mRNA, they further proposed that
PDGF (or a PDGF-like peptide) and, possibly, TGF- and bFGF may be
involved. Alternatively, work by others (1) suggests that cell
interactions between endothelial cells and smooth muscle cells or
pericytes may be required for growth factor activation.
Studies by Poiani et al. (24) provide additional in vivo evidence that
remodeling of pulmonary arteries occurs after increased wall stress.
These investigators exposed rats to normobaric hypoxia (10% oxygen)
for up to 14 days and observed progressive increases in mean right
ventricular pressure during the first 10 days of exposure. Increased
amounts of collagen and elastin were also observed in the pulmonary
artery during this time period, and these increases paralleled the
development of pulmonary hypertension. Interestingly, levels of mRNA
for 1(I) procollagen increased in the pulmonary artery during hypoxia but not in the aorta, where blood pressure remained constant. In addition, collagen and elastin levels in the pulmonary artery returned to normal within 7 days after
return of rats to normoxia. In total, these observations imply that
increases in transmural pressure increase wall stress to alter gene
expression of ECM proteins in the pulmonary artery.
In this study, we observed a threefold increase in
1(III) procollagen mRNA after
high lung inflation (Fig. 3). This observation is consistent with the
possibility that parenchymal blood vessels adapt to increased wall
stress by increasing type III collagen synthesis, thereby increasing
the tensile strength of the vessel wall. We also observed a smaller
(50%) increase in level of mRNA for
2(IV) procollagen, which may
suggest that vascular remodeling also occurs in the basement membrane
of parenchymal blood vessels. In addition, fibronectin mRNA levels were
twofold greater in High-PEEP rabbits than in Low-PEEP rabbits. Because
fibronectin provides a scaffolding for cell attachment and plays an
important role in relaying the transmission of force throughout the
cytoskeleton, this observation provides further evidence that high lung
inflation causes vascular remodeling. Thus, in our preparation, type
III procollagen and fibronectin are the main ECM components expressed as a result of 4-h high lung inflation with smaller increases in
2(IV) procollagen.
Role of growth factors. In this study,
we also determined mRNA levels for several growth factors that have the
potential to regulate remodeling in the capillary wall, either by
increasing cell proliferation or by increasing ECM deposition.
TGF-1 mRNA levels were
increased fourfold in response to high lung inflation, and levels of
bFGF mRNA were increased by 61% (Fig. 6).
TGF-1 plays a central role in
regulating the synthesis and degradation of ECM collagens and is
produced by most cells, particularly macrophages, fibroblasts,
endothelial cells, and platelets (26). bFGF also participates in
regulation of these processes (6). In vitro studies have demonstrated
that TGF-1 elevates synthesis
and secretion of type I and III procollagen and fibronectin (12).
TGF-1 is abundantly stored in
the ECM and is released in an active form via both autocrine and
paracrine pathways (1). Direct evidence that
TGF-1 participates in the
regulation of vascular remodeling is provided by an in vivo study by
Nabel et al. (21). These investigators transfected the human
TGF-1 gene into the iliofemoral artery of pigs and demonstrated that
TGF-1 gene expression is associated with hyperplasia of the vessel wall and increased synthesis of type I procollagen. They also found that the response is specific for TGF-1, since transfer of
the human PDGF- gene did not alter procollagen levels in the
arterial wall. Our results support these studies and imply that
TGF-1 participates in the early
response of parenchymal cells to mechanically induced capillary
remodeling.
Additional investigations are required to determine the cellular source
of the changes in mRNA observed in the present study. Likely candidates
are the fibroblast, myofibroblast, or pericyte. These cells are
abundant in the small vessels and capillaries of the outer lung
parenchyma. The myofibroblast, in particular, produces abundant amounts
of fibronectin and is highly activated by
TGF-1 (8). In addition, several
pathological conditions involving increased mechanical stress or
tension correlate with the presence of myofibroblasts in the lung. For
example, Kapanci et al. (13) observed activated myofibroblasts in
patients with venoocclusive disease, postcapillary pulmonary
hypertension secondary to heart failure, or mitral stenosis.
Mechanism of information transfer to the non-PEEP lung. A very interesting finding in this study is that the observed changes in mRNA were identical in both the 9-cmH2O-PEEP and 1-cmH2O-PEEP lungs of High-PEEP rabbits (Figs. 1 and 4). This observation suggests that a generalized organ-specific response occurred after the localized (unilateral) application of mechanical force. Although it is speculative, one possibility is that information is transferred via the circulation from the 9-cmH2O-PEEP lung to the 1-cmH2O-PEEP lung in High-PEEP rabbits. Precedents for signaling throughout the lung via the circulation come from transplantation studies by Hislop et al. (11). In that study, immature left lung was transplanted into an adult rat. Normally, compensatory growth in the adult rat lung after pneumonectomy occurs through an increase in size, rather than number, of alveoli (4). The alveoli in the remaining lung increase in size until the total lung volume is sufficient to replace the resected tissue. However, Hislop et al. (11) found that the presence of transplanted immature lung caused the contralateral adult lung to revert to an immature pattern of compensatory growth (i.e., growth occurred through an increase in number of alveoli rather than through an increase in size). Because all connections to the left lung were severed during transplantation, Hislop et al. concluded that the immature lung must release blood-borne factors that influence growth in the mature lung.
When one follows this line of reasoning, it is possible that activation
of inflammatory cells (e.g., alveolar or interstitial macrophages),
either in direct response to stretch or on contact with exposed
basement membranes after high lung inflation (9), stimulates the
release of quick-acting circulatory mediators. In this regard, it has
recently been demonstrated that ventilation of isolated perfused mouse
lungs with end-expiratory lung volumes similar to those experienced by
the High-PEEP rabbits in this study (2.5-fold increase) causes the
release of prostacyclin, tumor necrosis factor-, and interleukin-6
into the perfusate after 30-min ventilation (3). Alternatively,
endothelial and epithelial cells as well as fibroblasts and
myofibroblasts also respond to stretch and thus may play a role.
An additional possible factor in this study is that the frequency of sighs differed between groups of lungs. Both lungs of Low-PEEP rabbits and the 9-cmH2O-PEEP lung of High-PEEP rabbits received, on average, 1 sigh/51 min, whereas the low-PEEP lung in High-PEEP rabbits required more frequent sighing (1 sigh every 15 min) to prevent compression of airways and maintain gas exchange. It is, therefore, possible that differences in the frequency of sighs, and not a blood-borne mediator, caused the observed increases in expression of mRNA in the 1-cmH2O-PEEP lung of High-PEEP rabbits. Although this possibility cannot be ruled out, it seems unlikely that parenchymal blood vessels would selectively increase gene expression in response to sighs at 15-, but not at 51-min, intervals. Also, during normal ventilation, humans average 1 sigh/6 min when awake (2) and 1 sigh/36 min while sleeping, with considerable variation being observed between people (23). Data are not available on the frequency of sighs in rabbits. Thus, although data from humans suggest that the frequency of sighs used in this study may be within normal physiological limits, further study is required to resolve this question.
It is also possible that application of high PEEP narrows capillaries and increases vascular resistance to redistribute blood to the other lung. In this case, the increased blood flow to the lung receiving 1-cmH2O PEEP ventilation would increase vascular pressure and wall stress that might cause vascular remodeling. Although this process may occur to some extent, the normal pulmonary circulation has such reserves that even directing the whole cardiac output through one lung causes only a small rise in vascular pressures. The increase in wall stress will, therefore, be small. In addition, gas exchange in the 9-cmH2O-PEEP lung of High-PEEP rabbits was similar to gas exchange in the lung receiving 1-cmH2O PEEP ventilation. This observation implies that blood flow was not abolished in the high PEEP lung during 4-h ventilation.
In summary, we found that high lung inflation for 4 h stimulates
increased expression of 1(III)
and 2(IV) procollagen mRNAs in
lung parenchyma. On the assumption that these increases are translated
into increased protein for these collagens, the result would be to
strengthen the capillary wall. We also observed increases in
fibronectin mRNA. A fibronectin-rich ECM allows increased cell attachment and transduction of forces from the extracellular
environment through cytoskeletal elements.
TGF-1 and bFGF mRNA levels were also significantly elevated after high lung inflation, but the level of
VEGF mRNA did not change. In conclusion, our results show that mRNA
levels of ECM components and growth factors increase in lung parenchyma
after high lung inflation. Although further studies, using in situ
hybridization techniques, are required to precisely identify the
anatomical location of the observed changes, these observations are
consistent with the possibility that increased wall stress initiates
vascular remodeling in peripheral lung parenchyma.
ACKNOWLEDGEMENTS
We thank Jeff Struthers and Nick Busan for technical assistance.
FOOTNOTES
Address for reprint requests: J. B. West, UCSD, Dept. of Medicine 0623A, 9500 Gilman Dr., La Jolla, CA 92093-0623.
Received 23 September 1996; accepted in final form 7 March 1997.
REFERENCES
| 1. |
Antonelli-Orlidge, A.,
K. B. Saunders,
S. R. Smith,
and
P. A. D'Amore.
An activated form of transforming growth factor beta is produced by cocultures of endothelial cells and pericytes.
Proc. Natl. Acad. Sci. USA
86:
4544-4548,
1989 |
| 2. |
Bendixen, H. H.,
G. M. Smith,
and
J. Mead.
Pattern of ventilation in young adults.
J. Appl. Physiol.
19:
195-198,
1964 |
| 3. | Bethmann, A. V., F. Brasch, K. M. Müller, and S. Uhlig. Barotrauma induced cytokin- and eicosanoid-release from the isolated perfused and ventilated mouse lung (Abstract). Am. J. Respir. Crit. Care Med. 153: A530, 1996. |
| 4. |
Buhain, W. J.,
and
J. S. Brody.
Compensatory growth of the lung following pneumonectomy.
J. Appl. Physiol.
35:
898-902,
1973 |
| 5. | Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline]. |
| 6. |
Davidson, J. M.,
M. Klagsbrun,
K. E. Hill,
A. Buckley,
R. Sullivan,
P. S. Brewer,
and
S. C. Woodward.
Accelerated wound repair, cell proliferation, and collagen accumulation are produced by a cartilage-derived growth factor.
J. Cell Biol.
100:
1219-1227,
1985 |
| 7. |
Davies, P. F.,
and
S. C. Tripathi.
Mechanical stress mechanisms and the cell. An endothelial paradigm.
Circ. Res.
72:
239-245,
1993 |
| 8. |
Desmouliere, A.,
A. Geinoz,
F. Gabbiani,
and
G. Gabbiani.
Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts.
J. Cell Biol.
122:
103-111,
1993 |
| 9. |
Fu, Z.,
M. L. Costello,
K. Tsukimoto,
R. Prediletto,
A. R. Elliott,
O. Mathieu-Costello,
and
J. B. West.
High lung volume increases stress failure in pulmonary capillaries.
J. Appl. Physiol.
73:
123-133,
1992 |
| 10. |
Hernandez, L. A.,
K. J. Peevy,
A. A. Moise,
and
J. C. Parker.
Chest wall restriction limits high airway pressure-induced lung injury in young rabbits.
J. Appl. Physiol.
66:
2364-2368,
1989 |
| 11. |
Hislop, A. A.,
M. Rinaldi,
R. Lee,
C. G. McGregor,
and
S. G. Haworth.
Growth of immature lung transplanted into an adult recipient.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L60-L65,
1993 |
| 12. |
Ignotz, R. A.,
and
J. Massague.
Transforming growth factor-beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix.
J. Biol. Chem.
261:
4337-4345,
1986 |
| 13. | Kapanci, Y., S. Burgan, G. G. Pietra, and G. Gabbiani. Modulation of actin isoform expression in alveolar myofibroblasts (contractile interstitial cells) during pulmonary hypertension. Am. J. Pathol. 136: 881-889, 1990[Abstract]. |
| 14. | Kay, J. M., D. J. De Sa, and J. F. K. Mancer. Ultrastructure of lung in pulmonary veno-occlusive disease. Hum. Pathol. 14: 451-456, 1983[Medline]. |
| 15. | Kay, J. M., and F. R. Edwards. Ultrastructure of the alveolar-capillary wall in mitral stenosis. J. Pathol. 111: 239-245, 1973[Medline]. |
| 16. | Kurokawa, T., R. Sasada, M. Iwane, and K. Igarashi. Cloning and expression of cDNA encoding human basic fibroblast growth factor. FEBS Lett. 213: 189-194, 1987[Medline]. |
| 17. |
Leung, D. W.,
G. Cachianes,
W. J. Kuang,
D. V. Goeddel,
and
N. Ferrara.
Vascular endothelial growth factor is a secreted angiogenic mitogen.
Science
246:
1306-1309,
1989 |
| 18. |
Liau, G.,
Y. Yamada,
and
B. de Crombrugghe.
Coordinate regulation of the levels of type III and type I collagen mRNA in most but not all mouse fibroblasts.
J. Biol. Chem.
260:
531-536,
1985 |
| 19. |
Mecham, R. P.,
L. A. Whitehouse,
D. S. Wrenn,
W. C. Parks,
G. L. Griffin,
R. M. Senior,
E. C. Crouch,
K. R. Stenmark,
and
N. F. Voelkel.
Smooth muscle-mediated connective tissue remodeling in pulmonary hypertension.
Science
237:
423-426,
1987 |
| 20. | Meyrick, B., and L. Reid. The effect of continued hypoxia on rat pulmonary arterial circulation. An ultrastructural study. Lab. Invest. 38: 188-200, 1978[Medline]. |
| 21. |
Nabel, E. G.,
L. Shum,
V. J. Pompili,
Z. Y. Yang,
H. San,
H. B. Shu,
S. Liptay,
L. Gold,
D. Gordon,
and
R. Derynck.
Direct transfer of transforming growth factor beta 1 gene into arteries stimulates fibrocellular hyperplasia.
Proc. Natl. Acad. Sci. USA
90:
10759-10763,
1993 |
| 22. |
Parker, J. C.,
M. I. Townsley,
B. Rippe,
A. E. Taylor,
and
J. Thigpen.
Increased microvascular permeability in dog lungs due to high peak airway pressures.
J. Appl. Physiol.
57:
1809-1816,
1984 |
| 23. | Perez-Padilla, R., P. West, and M. H. Kryger. Sighs during sleep in adult humans. Sleep 6: 234-243, 1983[Medline]. |
| 24. |
Poiani, G. J.,
C. A. Tozzi,
S. E. Yohn,
R. A. Pierce,
S. A. Belsky,
R. A. Berg,
S. Y. Yu,
S. B. Deak,
and
D. J. Riley.
Collagen and elastin metabolism in hypertensive pulmonary arteries of rats.
Circ. Res.
66:
968-978,
1990 |
| 25. |
Qian, S. W.,
P. Kondaiah,
A. B. Roberts,
and
M. B. Sporn.
cDNA cloning by PCR of rat transforming growth factor beta-1.
Nucleic Acids Res.
18:
3059,
1990 |
| 26. | Roberts, A. B., U. I. Heine, K. C. Flanders, and M. B. Sporn. Transforming growth factor-beta. Major role in regulation of extracellular matrix. Ann. NY Acad. Sci. 580: 225-232, 1990[Medline]. |
| 27. | Schwarzbauer, J. E., J. W. Tamkun, I. R. Lemischka, and R. O. Hynes. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell 35: 421-431, 1983[Medline]. |
| 28. | Tozzi, C. A., G. J. Poiani, A. M. Harangozo, C. D. Boyd, and D. J. Riley. Pressure-induced connective tissue synthesis in pulmonary artery segments is dependent on intact endothelium. J. Clin. Invest. 84: 1005-1012, 1989. |
| 29. |
Tsukimoto, K.,
N. Yoshimura,
M. Ichioka,
N. Tojo,
I. Miyazato,
F. Marumo,
O. Mathieu-Costello,
and
J. B. West.
Protein, cell, and LTB4 concentrations of lung edema fluid produced by high capillary pressures in rabbit.
J. Appl. Physiol.
76:
321-327,
1994 |
| 30. | West, J. B., and O. Mathieu-Costello. Stress failure of pulmonary capillaries: role in lung and heart disease. Lancet 340: 762-767, 1992[Medline]. |
| 31. |
West, J. B.,
K. Tsukimoto,
O. Mathieu-Costello,
and
R. Prediletto.
Stress failure in pulmonary capillaries.
J. Appl. Physiol.
70:
1731-1742,
1991 |
| 32. | Williamson, J. R., N. J. Vogler, and C. Kilo. Regional variations in the width of the basement membrane of muscle capillaries in man and giraffe. Am. J. Pathol. 63: 359-370, 1971[Medline]. |
| 33. | Winlove, C. P., J. E. Bishop, R. C. Chambers, and G. J. Laurent. The structure and function of extracellular matrix in the pulmonary vasculature. In: Pulmonary Vascular Remodelling, edited by J. E. Bishop, J. T. Reeves, and G. J. Laurent. London: Portland, 1995, p. 21-46. |
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