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1 Department of Anesthesiology and Cardiovascular-Pulmonary Research Laboratory, University of Colorado Health Sciences Center, Denver, Colorado 80262; and Departments of 2 Pathology and 3 Pharmacology, College of Medicine, University of South Alabama, Mobile, Alabama 36688
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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An intact
endothelial cell barrier maintains normal gas exchange in the lung, and
inflammatory conditions result in barrier disruption that produces
life-threatening hypoxemia. Activation of store-operated
Ca2+ (SOC) entry increases the
capillary filtration coefficient
(Kf,c) in the
isolated rat lung; however, activation of SOC entry does not promote
permeability in cultured rat pulmonary microvascular endothelial cells.
Therefore, current studies tested whether activation of SOC entry
increases macro- and/or microvascular permeability in the
intact rat lung circulation. Activation of SOC entry by the
administration of thapsigargin induced perivascular edema in pre- and
postcapillary vessels, with apparent sparing of the microcirculation as
evaluated by light microscopy. Scanning and transmission electron
microscopy revealed that the leak was due to gaps in vessels 
100 µm, consistent with the idea that activation of SOC entry influences
macrovascular but not microvascular endothelial cell shape. In
contrast, ischemia and reperfusion induced microvascular endothelial cell disruption independent of
Ca2+ entry, which similarly
increased Kf,c.
These data suggest that 1)
activation of SOC entry is sufficient to promote macrovascular barrier
disruption and 2) unique mechanisms
regulate pulmonary micro- and macrovascular endothelial barrier functions.
lung; thapsigargin; pulmonary edema; signal transduction; reperfusion injury
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INTRODUCTION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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ENDOTHELIAL CELL (EC) barrier disruption is a cardinal feature of inflammation and has been implicated in the pathogenesis of ischemia-reperfusion (I/R), acute respiratory distress syndrome, and asthma. Endothelial barrier disruption produces an accumulation of extravascular lung water that results in severe abnormalities in gas exchange and hypoxemia. Early paradigms of pulmonary edema predicted that the major site of fluid leakage in both hydrostatic and permeability (inflammatory) edema occurred at the level of alveolar vessels (28). Further investigation has indicated that significant leak can also occur at the level of extra-alveolar vessels (2, 19). Mechanisms that regulate segmental barrier function are not known, although in vitro conduit ECs demonstrate a clear role for increased intracellular Ca2+ concentration ([Ca2+]i) as a catalyst for increased permeability. However, microvascular (e.g., <100 µm) ECs apparently do not respond to increased [Ca2+]i with EC shape changes that promote permeability (18, 29). This suggests that unique mechanisms account for pulmonary arterial EC versus pulmonary microvessel EC barrier disruption.
In conduit EC cultures and isolated microvessels, activation of store-operated Ca2+ (SOC) entry and elevation of intracellular Ca2+ appears sufficient to increase endothelial permeability (6, 8, 13-16, 20, 22, 26). SOC entry or capacitative Ca2+ entry is a phenomenon in which a declining internal store Ca2+ concentration ([Ca2+]) activates Ca2+ entry across the plasmalemma (24). Decreased stored [Ca2+] occurs in response to activation of the inositol 1,4,5-trisphosphate-receptor channel or after inhibition of sarco(endo)plasmic reticulum ATPase (SERCA) activity. In the lung, the plant alkaloid thapsigargin (TG) inhibits SERCA activity and promotes SOC entry, which increases hydraulic conductivity [capillary filtration coefficient (Kf,c)] (4). Oxidants inhibit SERCA function and similarly increase Kf,c (9-12, 31). Therefore, a link may exist between SERCA inhibition, activation of SOC entry, and oxidant-mediated (inflammatory) acute lung injury. Current studies were therefore undertaken to determine whether inhibition of SERCA function and activation of SOC entry is sufficient to increase Kf,c due to EC gap formation in macro- and/or microvascular lung segments.
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METHODS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Isolated perfused lung preparation.
The research protocol was approved by the University of Colorado
(Denver) Health Sciences Center Animal Care and Use Committee. Adult
male Sprague-Dawley rats (
250-300 g; SASCO, Omaha, NE) were
anesthetized with pentobarbital sodium (60 mg/kg ip), and a tracheotomy
catheter was inserted. The lungs were ventilated at inspiratory and
expiratory pressures of 6.0 and 2.0 cmH2O, respectively, with room air
until the heart was cannulated, after which they were ventilated with
21% O2-5% CO2-74%
N2. After a median sternotomy,
heparin (60 units) was administered via the left ventricle and allowed
to circulate for 3 min. Pulmonary arterial and double-lumen left
ventricular catheters were inserted and secured with sutures. The lungs
were perfused (Gilson Minipuls 3, Gilson, Middleton, WI) at a constant
flow of 0.04 ml · g body
wt
1 · min1
with a bicarbonate-buffered physiological salt solution
(Krebs-Henseleit buffer, Sigma) that contained (in mM)
11.1 D-glucose, 1.2 MgSO4, 1.2 KH2PO4,
4.7 KCl, 118.1 NaCl, and 1.5 CaCl2
(680 mM stock; Fujisawa) or 10 µM
CaCl2 (20 mM stock; Sigma) and was
osmotically stabilized with 4% bovine serum albumin (Sigma). The lungs
and heart were removed en bloc, suspended from a force transducer (Grass FT03, Grass Instruments, Quincy, MA), and placed in a chamber with 100% humidity at 37°C. The first 20 ml of perfusate were used
to flush the lungs. An additional 60 ml of perfusate were used for
recirculation. Pulmonary arterial and venous
(Pv) pressures were continuously
monitored (TSD 104, Biopac, Goleta, CA) and captured to a Macintosh
computer with an analog-to-digital converter (MP100A, Biopac). Zone 3 conditions were maintained in all experiments (mean: arterial > venous > alveolar pressures).
Assessment of permeability. Kf,c is a measurement of hydraulic conductivity (permeability) and was estimated with a gravimetric method modified from Drake et al. (7). In the isolated perfused lung, perfusion and colloid oncotic pressures are constant. Edema formation (net filtration) was estimated by lung weight gain, and pulmonary capillary pressure (Pc) was estimated by double occlusion. When lung weight is stable (weight equilibrium), hydrostatic forces are equally opposed by oncotic forces. The weight equilibrium was disturbed by increasing Pv from 3.5 to 9.0 mmHg for 15 min. Pc was estimated before and after Pv was increased. A rapid rise in weight due to vascular distension (recruitment and distension) was followed by gradual weight gain (net filtration). Over the period of measurement, it was assumed that changes in interstitial pressure and oncotic forces were minimal. The rate of gradual weight gain from 5 to 15 min was analyzed with linear regression. Kf,c was estimated by determining the ratio of the rate of gradual weight gain to the change in Pc. Kf,c measurements were normalized to left lung dry weight and are expressed as milliliters per minute per millimeter of mercury per 10 g of lung tissue. Left lung dry weight was measured after the lung was dried in a desiccator for 48 h. In all experiments, isolated lungs were allowed to equilibrate and attain isogravimetric conditions for 20 min, after which baseline Kf,c was determined.
TG dose response and determination of EC50. To determine the optimal dose of TG in subsequent experimental protocols, dose-response studies were conducted in the isolated lung preparation. Ten minutes after the baseline Kf,c determination, TG in a range of doses (0-100 nM) was added to the perfusate reservoir. A second Kf,c value was determined 20 min after treatment.
Lung fixation. Lungs from selected experiments were fixed for light and scanning (SEM) and transmission (TEM) electron microscopy 20 min posttreatment with TG (30 or 50 nM) or vehicle (DMSO). An additional group of lungs (perfusate Ca2+ 1.5 mM) subjected to 45 min of ischemia followed by 30 min of reperfusion was also
fixed. Baseline
Kf,c was
determined, but neither posttreatment nor reperfusion
Kf,c was
determined. At the appropriate time point, ventilation was
discontinued, and the lungs were inflated with 2.5 ml of room air. The
lungs were then flushed (gravity feed) via the pulmonary artery with 25 ml of a glutaraldehyde mixture [3% glutaraldehyde (Fisher) in
0.2 M cacodylic acid (Sigma) buffer containing 16 mM HCl, pH
7.2-7.4]. Specimens were stored in the glutaraldehyde
mixture at 4°C.
Light microscopy. To evaluate
segmental differences in edema accumulation, lungs were examined with
light microscopy. The lungs were cut in the sagital plane through the
middle of the apex. The lateral parts were routinely processed in
paraffin, and 5-µm slices were stained with hematoxylin and eosin and
examined with light microscopy at ×140 magnification.
SEM. Two-millimeter-thick slices with
surfaces of 1 cm2 were taken from
the middle of lungs. Specimens were washed in 0.1 M cacodylate buffer,
dehydrated in alcohol and dried in
CO2 at critical point, mounted on a stand, and coated with 20 nm of
gold-palladium. The samples were viewed in a Philips XL 20 SEM at 10 kV, and each segment of the vasculature was thoroughly investigated for
EC layer changes. Gaps in interendothelial junctions were counted from
video prints of scans taken from one vessel representing an area of 1 × 104
µm2 at ×1,500
magnification. The number of lesions and their size were evaluated and
are expressed as means ± SE.
TEM. Tissue samples 1 mm3 in size from different areas
of the glutaraldehyde perfusion-fixed lungs were washed in 0.1 M
cacodylate buffer and postfixed for 1 h in 1%
OsO4. After being washed in cacodylate buffer and dehydrated in alcohol, the samples were embedded
in Poly/Bed 812 Resin (Polysciences, Warrington, PA). Semithin 1-µm
sections were stained with toluidine blue to identify the
representative specimens containing vascular structures. Thin sections
(60-80 nm) were placed on copper grids, stained with lead citrate
and uranyl acetate, and systematically viewed in a Philips CM 100 TEM.
Changes in EC junctions in vessels > 100 µm in diameter were
counted at ×3,000 magnification and are expressed as a percentage
of all junctional complexes counted (minimum of 30 junctions) in one
type of vessel from at least three grids.
I/R ± TG pretreatment.
A previous study (4) demonstrated that lowering
Ca2+ entry attenuates TG-induced
(Ca2+ entry-mediated) permeability
increases. To test the effects of decreasing
Ca2+ entry on I/R-induced lung
leak, low [Ca2+]
perfusate studies were conducted. A select group of experiments was
pretreated with TG to deplete Ca2+
stores and limit Ca2+ release. All
experiments were conducted with a low
[Ca2+] perfusate
(10 M). After measurement of baseline
Kf,c and 10 min
before the onset of ischemia, TG (100 nM) or vehicle (DMSO) was
added to the perfusate reservoir. Ventilation and flow were interrupted
for 45 min (warm ischemia). Ventilation and flow were reinstituted to their original settings on reperfusion.
Kf,c was determined at 30 min of reperfusion.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Activation of SOC entry increases
Kf,c.
TG increases permeability in the isolated rat lung as assessed by
Kf,c (4). To more
completely characterize the hemodynamic influence of TG in the
pulmonary circulation, studies were conducted to determine the
EC50 (Fig.
1). Data indicated that TG induced a
dose-dependent increase in
Kf,c, with an
EC50 of 30 nM. Doses > 100 nM
generated unmeasurable responses because of severe vasoconstriction and
unstable weight tracings; therefore, a 100 nM dose was utilized to
generate the "maximal" response in
Kf,c in this
preparation (Fig. 1).
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100 µm. TG produced
a dramatic increase in apparent gaps within pre- and postcapillary
vessels, and, occasionally, large defects in the endothelial layer
could be observed (Fig. 3B). As
suggested by light-microscopic sections, intercellular gaps were absent
in the microcirculation (e.g., 30 µm; Fig.
3D). To confirm that the gaps
apparent on SEM represented a complete paracellular pathway for
transudation of fluid and macromolecules, we performed TEM studies
using buffer-perfused lungs (Fig. 4). In
control lungs, endothelial cell-cell contact was associated with tight
junctional complexes (Fig. 4A),
whereas cell-matrix tethering was accompanied by clear association
between ECs and the underlying basement membrane. These characteristics
were apparent in macro- and microvascular sections, although alteration
of cell-cell contact was occasionally seen in ECs of larger vessels
(data not shown). TG caused a prominent increase in the number of EC
gaps (Figs. 5 and
6), characterized by
distension of the intercellular spaces, in most cases limited by
diminished, although still present, tight junctional complexes (Fig.
4B). Gaps were accompanied by fluid
accumulation in the subendothelial tissues, apparent by an increased
distance between the endothelium and the muscle layer, as well as by
enlarged spaces between the adjunct smooth muscle cells (Fig.
4B). The loss of tight cell-cell
contact was apparent in vessels 100 µm, indicating that this was
the probable cause of perivascular cuffing. ECs of capillaries in
control and TG-treated tissue samples were connected by unchanged tight
junctions (Fig. 4C). These findings
were consistent with the idea that activation of SOC entry influences
macrovascular but not microvascular EC gaps.
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Ca2+ entry is
not required for the initiation of I/R-induced lung leak.
Evidence that I/R induces microvascular leak, whereas TG does not,
suggests that control of pulmonary macro- and microvascular EC shape is
via distinct mechanisms. We therefore determined whether limiting
Ca2+ entry in the setting of I/R
would alter the permeability response as determined by changes in
Kf,c. Studies
were conducted with low-Ca2+
perfusate (10 µM), sufficient to prevent the TG-induced increase in Kf,c (4).
Lowering perfusate Ca2+ had no
protective effect on I/R-induced increases in
Kf,c (188 ± 33% increase in
Kf,c for I/R vs.
39 ± 26% for time control; P < 0.05; Fig. 9), consistent with
the idea that, unlike TG, I/R-induced lung dysfunction occurs via a
Ca2+ entry-independent mechanism.
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Depletion of Ca2+ stores attenuates I/R-induced permeability changes. Because activation of SOC entry does not appear to have an effect on the microvascular barrier, we hypothesized that I/R-induced permeability may represent a Ca2+ entry-independent event. To test whether TG-sensitive Ca2+ stores are required for I/R-induced Kf,c changes, lungs were perfused with a low-Ca2+ buffer and pretreated with TG to produce Ca2+ store depletion before the onset of I/R. Pretreatment with 100 nM TG significantly attenuated the I/R-induced increase in Kf,c (39 ± 13% increase in Kf,c for time control vs. 38 ± 12% increase in Kf,c for thapsigargin-I/R; P = not significant; Fig. 9).
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Disruption of the pulmonary endothelial barrier as occurs in acute respiratory distress syndrome and I/R injury results in noncardiogenic pulmonary edema that alters gas exchange and produces life-threatening hypoxemia. Specific mechanisms responsible for EC shape change and diminished endothelial barrier function are poorly understood. The novel findings from our study are that 1) SOC entry induces EC gap formation in the macrovascular circulation, with no apparent effect in the microvascular circulation; 2) I/R-induced microvascular lung leak, as assessed by estimation of Kf,c, occurs independent of SOC entry; 3) Ca2+ release from internal storage sites may be an early critical event for the initiation of I/R-induced injury; and 4) EC shape change limited to the extra-alveolar circulation can produce permeability increases, as estimated by Kf,c, comparable to that seen in microvascular barrier dysfunction (e.g., I/R).
SERCA inhibition activated SOC entry. Inhibition of SERCA function with TG induces permeability changes in the isolated rat lung; to further characterize this observation, we performed dose-response studies and determined the EC50. TG, a highly specific and irreversible inhibitor of SERCA function, has the highest inhibitory potency of all SERCA inhibitors (32). SERCA activity assays and Ca2+ uptake studies demonstrate an apparent EC50 of 10-20 nM in COS cells (21). Our studies demonstrated a similar EC50, consistent with selective SERCA inhibition; therefore, nonspecific effects of this compound at concentrations presently utilized are unlikely.
Activation of SOC entry induces extra-alveolar edema. Several studies (6, 8, 13-16, 20, 26) suggested that Ca2+ entry is critical for conduit endothelial gap formation and altered barrier function; many of these observations have been made in either cell culture or isolated systemic microvessel preparations. Recently, Kelly et al. (18) demonstrated that activation of SOC entry is sufficient to produce gap formation in cultured pulmonary arterial ECs; however, activation of SOC entry was not sufficient to increase permeability in cultured pulmonary microvascular ECs (e.g., <100 µm). Thus, in contrast to conduit ECs, microvascular ECs express a unique phenotype that favors enhanced barrier function associated with attenuated agonist-induced Ca2+ influx (18, 29). We determined whether similar differences in EC gaps occurred in macrovascular (>100 µm) compared with microvascular segments in the intact pulmonary circulation. Consistent with results in cultured cells, histological examination revealed that the macrovascular circulation demonstrated a unique response to activation of SOC entry, exhibiting significant cell shape changes, whereas microvascular segments were unaffected.
Potential explanations for the differences in regional EC shape changes in response to SERCA inhibition and activation of SOC entry include segmental differences in 1) SERCA expression, 2) SERCA-Ca2+ store coupling to Ca2+ entry, and 3) sensitivity to Ca2+ in mechanisms responsible for cell shape changes. At least five SERCA isotypes have been identified, of which SERCA2b and -3 have been associated with nonexcitable cells. However, the isotypes relevant to lung endothelial cells have yet to be determined. TG inhibits all SERCA isotypes with equal potency (21), and, therefore, regional differences in SERCA isotype expression may not explain observed regional differences in EC shape changes. Interestingly, there is evidence that inhibition of SERCA function induces an attenuated Ca2+ entry response in microvascular endothelial cells, suggesting that regional differences in Ca2+ store-Ca2+ entry coupling may exist (29), but insensitivity to elevations in [Ca2+]i in control of pulmonary microvascular EC shape argues against this idea as the cause of segmental control of permeability (18). Finally, little is known about phenotypic differences in macro- vs. microvascular cell cytoskeletal structures relevant to agonist-mediated EC shape changes. Morphological characterizations of pulmonary conduit and microvascular ECs, however, demonstrate that microvascular cells possess fewer actinlike filaments, consistent with the possibility that Ca2+-dependent signals, such as Ca2+/calmodulin stimulation of centripetal tension development or tethering of critical cell-cell and cell-matrix adhesion proteins to actin microfilaments, is uniquely regulated in microvascular cells to establish enhanced barrier properties.
Ca2+ release may be an early event in I/R-induced microvascular permeability. Oxidant species have been shown to inhibit SERCA function. Therefore, oxidants may induce permeability changes via activation of SOC entry that, based on the currently described TG-induced lesions, could be limited to macrovascular lung segments. In support of this idea, Pietra and Johns (23) demonstrated the H2O2-induced pulmonary edema was selectively limited to the pulmonary arterial endothelium (macrovascular circulation). However, I/R-induced oxidant generation produces pulmonary edema and alveolar flooding clearly due to microvascular endothelial disruption (1). Our studies verified that I/R-induced lung injury involves a significant microvascular component, with EC shape changes, interstitial edema, and alveolar flooding (3).
Because a predominant feature of I/R-induced lung injury involves microvessels (alveolar-capillary vessels), but cultured pulmonary microvascular ECs do not respond to increased [Ca2+]i with intercellular gap formation that promotes permeability, we next determined whether I/R-induced lung leak requires Ca2+ entry. Whereas reduced extracellular [Ca2+] prevents SOC entry-induced increases in Kf,c in perfused lungs (4) and shape changes in cultured conduit cells (18, 30), similar reductions in extracellular Ca2+ (10 µM) did not attenuate I/R-induced
Kf,c increases.
Thus these data support the idea that I/R-induced lung leak largely
represents a Ca2+
entry-independent alteration in barrier function.
Initiation of intracellular Ca2+
signals (e.g., after receptor-agonist binding) involves two distinct
phases: release of Ca2+ from
intracellular storage sites (initial phase) followed by activation of
Ca2+ entry mechanisms, which serve
to sustain elevations in intracellular Ca2+. Because I/R-induced lung
leak may involve a Ca2+
entry-independent mechanism, we questioned whether
Ca2+ release might be linked to
the initiation of permeability changes associated with I/R-induced lung
leak. To test this idea, stored Ca2+ was depleted by pretreatment
with TG. Low-Ca2+ perfusate (10
µM) was utilized to limit Ca2+
entry and the TG-induced permeability response. Depletion of Ca2+ stores eliminated the
increase in Kf,c
associated with I/R. These data suggest that release of
Ca2+ from internal storage sites
may be an early critical event that initiates I/R-induced permeability
changes and would be consistent with previous observations by Shasby et
al. (27), who demonstrated in ECs that extracellular
H2O2
was accompanied by generation of inositol 1,4,5-trisphosphate.
Furthermore, these observations are consistent with those of
Siflinger-Birnboim et al. (28), who demonstrated that
H2O2-induced
permeability in bovine pulmonary microvessel EC monolayers required
Ca2+ release independent of
Ca2+ entry. Future studies will be
required to determine Ca2+
release-sensitive mechanisms that regulate microvascular EC barrier function.
Structure versus function: models of
permeability. Taken together, our present findings
provide unique information regarding how morphological changes in EC
shape translate into functional increases in biophysical measurements
of fluid flux. An important finding from our present study was that
although the threefold increase in
Kf,c produced by
an EC50 dose of TG was equivalent to the permeability alteration produced by I/R, most other mechanistic and morphological features of these models differed. Previous physiological characterization of the pulmonary vascular barrier indicated that the endothelial lining behaves as a heteroporous membrane, possessing a 1)
large-abundance, small-pore population (water and albumin conductance),
2) small-abundance, large-pore population (macromolecules such as immunoglobulin), and
3) aquaporins (water-only channels)
(25). Water, albumin, and other small solutes readily pass through
small pores at relatively low lymph flows (i.e., low vascular pressures
and filtration rates), whereas evidence of significant large-pore
conductance in the noninflamed lung is only obtained at high vascular
pressures with high filtration rates. There is general agreement that
the anatomic features responsible for the so-called small-pore system
are due to properties of interendothelial junctional complexes, with
the degree and "tightness" of these junctions dictating size
selectivity of the pore. However, no structural correlate to the
putative physiological large-pore system has been identified. Our
experiments revealing the increase in both the number and size of gaps
within extra-alveolar vessels suggest that the increased measured
permeability and interstitial edema formation in response to TG may
have resulted from an increase in large-pore conductance.
Macromolecular permeability estimates (e.g., osmotic reflection
coefficient and/or solvent drag coefficient) are
necessary to determine whether these gaps in the extra-alveolar segment
are in fact analogous to the large-pore system. TG caused an increase
in both the number and size of porelike structures in extra-alveolar
arterioles and venules. Although protein permeability estimates
(osmotic reflection coefficient and/or solvent drag coefficient) and physiological pore size calculations were not performed, it is likely that the porelike gaps induced by TG in the
extra-alveolar segments were responsible for the measured global
increases in lung vascular permeability and that these gaps in the
extra-alveolar segment may be analogous to a large-pore system.
Even with the measured increase in solvent permeability
(Kf,c) and the
appearance of gaps 100-µm vessels, alveoli remained edema-free
after TG challenge. This observation suggests that a specific
compartmentalization of edema fluid occurred when the extra-alveolar
vessels became leaky. Perivascular cuffing and swollen vessel
musculature with apparent "tributaries" of fluid coursing between
smooth muscle cells were observed. According to previous models, this
pattern of fluid compartmentalization could have resulted from bulk
fluid transport from the interstitial spaces directly surrounding the
alveolar microvessels and filling of the perivascular compartment,
provided TG did not affect alveolar epithelial integrity (34). If TG
had no effect on alveolar epithelial integrity, then this pattern of
compartmentalization could have resulted from bulk fluid movement from
all microvascular segments, including alveolar capillaries, and filling
of the perivascular compartment from the perimicrovascular compartment
(34). However, it is unlikely that hydration of the smooth muscle
surrounding the larger extra-alveolar vessels would have been so
extensive, especially in the presence of an increased intravascular
pressure. If the perivascular space had been filled with fluid
transported from the alveolar perimicrovascular compartment after TG
challenge, it is therefore likely that the perivascular compartment
filled directly from increased conductance of large pores in the
endothelial membrane lining the extra-alveolar vessels.
The possibility of a significant extra-alveolar vessel contribution to
pulmonary edema development has been previously proposed (17, 19, 33).
In response to acid aspiration injury in rabbit lungs, arterial and
venous extra-alveolar vessel permeabilities were shown to increase
~2.3- and 4.6-fold, respectively, as assessed by segmental
measurements of the
Kf,c.
Additionally, glass bead (50 µm) embolization injury to in situ
canine lungs results in a measured increase in the large-pore
population in the endothelial membrane putatively occurring upstream
from the embolization site. Our study provides morphological and
biophysical data, which agree with these previous observations. In
addition, our data uniquely suggest that endothelial SOC-channel
activation, produced by mediators released in response to particulate
embolization or acid aspiration, may be an important component of the
mechanism leading to pulmonary endothelial barrier disruption with
these types of pulmonary insults.
In conclusion, the present studies demonstrate that SOC entry can
mediate gap formation and alter barrier function of the macrovascular
pulmonary circulation. An increasing body of evidence suggests that at
least a component (e.g., oxidant-mediated inhibition of SERCA function)
of clinical forms of acute lung injury may involve an SOC
entry-dependent mechanism and warrants further investigation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ivan F. McMurtry for invaluable advice and help in preparation of the manuscript.
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FOOTNOTES |
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This research was supported by a grant from the Foundation for Anesthesia Education and Research; a Society of Cardiovascular Anesthesiology Young Investigator Award (both to P. M. Chetham); National Heart, Lung, and Blood Institute Grants HL-56050 and HL-60024 (both to T. Stevens); a Parker B. Francis Pulmonary Fellowship (to T. Stevens); and an American Heart Association Southern Research Consortium Fellowship (to T. M. Moore).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: P. M. Chetham, Dept. of Anesthesiology, B113, Univ. of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262.
Received 22 July 1998; accepted in final form 25 September 1998.
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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/
baseline
Kf,c)/maximal
response
Kf,c] × 100.
