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1 Cardiac Services, 2 Department of Critical Care Medicine, Flinders Medical Centre, and 3 Department of Human Physiology, Flinders University, 5042 Adelaide, South Australia; and 4 Cardiology Department, Royal Perth Hospital and West Australian Institute of Medical Research, 6000 Perth, Western Australia, Australia
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Chronic heart failure (CHF) is associated with adaptive structural changes at the alveolocapillary barrier that may be associated with altered protein permeability. Bidirectional protein movement across the barrier was studied in anesthetized rats with infarct-induced CHF by following 125I-labeled albumin (125I-albumin) flux into the alveoli and the leakage of surfactant protein (SP)-B from the alveoli into the circulation. Three groups were studied: controls [0% left ventricular (LV) infarction], moderate infarct (25-45% LV infarction), and large infarct (>46% LV infarction). Wet and dry lung weights increased in the large infarct group (both P < 0.001), consistent with increased lung water and solid lung tissue. 125I-albumin flux increased across the endothelial (P < 0.001) and epithelial (P < 0.01) components of the alveolocapillary barrier in the large infarct group. Plasma SP-B increased 23% with moderate infarcts (P < 0.05) and 97% with large infarcts (P < 0.001), independent of alveolar levels. Lavage fluid immune cells (P < 0.01) and myeloperoxidase activity (P < 0.05) increased in the large infarct group, consistent with inflammation. Bidirectional protein movement across the alveolocapillary barrier is increased in CHF, and alveolar inflammation may contribute to this pathophysiological defect.
alveolocapillary barrier permeability; cardiogenic pulmonary edema; surfactant protein B; albumin permeability
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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RAISED PULMONARY MICROVASCULAR PRESSURE (Pmv) challenges the strength of the fragile alveolocapillary barrier (64). Indeed, acute increases in Pmv [as occurs in acute cardiogenic pulmonary edema (17)] can lead to rupture of the alveolocapillary barrier (10, 64), thereby blurring the distinction between the hydrostatic and permeability components of pulmonary edema in heart disease (13, 49).
In chronic heart failure (CHF), chronic Pmv elevation induces adaptive structural changes that serve to thicken the alveolocapillary barrier (24, 33, 37, 57). This provides protection from further high vascular pressure damage (57) and may contribute to the relative resistance to the development of pulmonary edema in CHF patients (6, 28, 57, 58, 63).
Although thickening of the barrier contributes to reduced diffusing capacity for carbon monoxide in CHF (43, 48), permeability to protein may be affected differently. Consistent with alveolar injury (1, 62), type I alveolar epithelial cells are replaced with progenitor cuboidal cells when Pmv is chronically elevated (33, 37). This, coupled with the proliferation of interstitial fibrous tissue (33, 37), changes the normal architecture of the alveolocapillary barrier and may compromise the integrity of its fragile cell layers and their connecting tight junctions. Indeed, fragmentation of the basement membrane and detachment of damaged epithelial cells have been documented in CHF (37).
Although Pmv primarily determines the development of pulmonary edema in CHF (19), abnormal protein permeability of the alveolocapillary barrier may complicate the disease through deleterious effects of excess air space protein (5, 18), which may include macrophage activation and alveolar surfactant dysfunction (30).
Movement of transferrin has been used to study alveolocapillary barrier permeability in CHF, with conflicting results (9, 22, 32). Subsequently, Townsley and coworkers (57) found no change in the pulmonary microvascular osmotic reflection coefficient of protein in dogs after 7 wk of pacing to induce CHF. In contrast, Huang and co-workers (28) demonstrated a reduction in vascular protein permeability in an aortic banding model of CHF.
We hypothesized that the permeability of the alveolocapillary barrier to protein is increased in CHF and investigated this by studying protein movement both into and out of the alveolus. We followed the flux of intravascular 125I-labeled albumin (125I-albumin) across the alveolocapillary barrier into the alveoli and measured the leakage of the pulmonary-specific surfactant protein (SP)-B from the alveoli into the circulation in a rat model of myocardial infarct-induced CHF.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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This study was approved by the Flinders University Animal Welfare Committee.
Induction of Heart Failure
Left ventricular (LV) myocardial infarction was induced in male Sprague-Dawley rats (250-300 g) by the method of Pfeffer and co-workers (42), with minor modifications. Briefly, rats were anesthetized in an anesthetic chamber with inhaled isoflurane (3-4%, Forthrane, Abbott Australasia; Kurnell, Australia), intubated, and then ventilated (model 683 Harvard rodent ventilator; Holliston, MA). Anesthesia was maintained with inhaled enflurane (1-2%, Ethrane, Abbott Australasia) administered through a vaporizer connected to the ventilator. Rats were placed supine on a thermostatically controlled plate to maintain body temperature. The chest wall was shaved, and a left parasternotomy was performed through three costal cartilages. The pericardium was opened, and the left coronary artery was ligated between the pulmonary artery outflow tract and the left atrium with a 6.0 prolene suture. The thorax was closed, and the rats were allowed to recover from anesthesia and extubated. Intraperitoneal buprenorphine (0.02 mg/kg, Temgesic, Reckitt and Colman; West Ryde, Australia) was administered twice daily for up to 4 days postoperatively.Cardiorespiratory Variables
After 7 wk, the rats were anesthetized with intraperitoneal thiopentone (60 mg/kg, Pentothal, Abbott Australasia). The caudal artery and one lateral caudal vein at the base of the tail were cannulated (8) and fixed in place with tissue glue (Loctite 406; Carringbah, Australia). Anesthesia was maintained by arterial infusion of pentobarbitone (21 ml · kg
1 · h1,
Nembutal, Rhone Merieux; Pinkenba, Australia) in heparinized saline (2 U/ml, 2 ml/h). The neck was shaved, and a 15-mm vertical incision was
made on the right side, 5 mm from the midline. The right carotid artery
was mobilized by blunt dissection and intubated with a polyethylene
catheter (outer diameter 1 mm, inner diameter 0.5 mm, Adelab
Scientific; Norwood, Australia). LV end-diastolic pressure (LVEDP) was
monitored by advancing the catheter across the aortic valve into the LV.
Cardiorespiratory variables were monitored for 15 min using a MacLab system 4 analog-to-digital instrument and Chart version 3.4.2 software (ADInstruments; Sydney, Australia). Systemic arterial blood pressure, heart rate, and LV pressures were monitored with disposable pressure transducers (Sorensen Trans Pac, Abbott Critical Care Systems; Chicago, IL). Arterial blood gases were analyzed with an ABL 5 blood gas analyzer (Radiometer; Copenhagen, Denmark).
Triple Radiolabel Study
Our triple radiolabel technique for the study of alveolocapillary barrier permeability has been previously reported (7).Preparation of radiolabeled red blood cells. Approximately 1.2 ml of blood were drawn from male Sprague-Dawley donor rats into a syringe containing heparin (5,000 U/ml, 75 µl) and acid citrate-dextrose (12.25 g glucose, 11 g sodium citrate, and 4 g citric acid/500 ml, 140 µl) and centrifuged at 6,000 rpm for 10 min. The plasma was discarded, and the cells were resuspended in 40 µl of PBS and 40 µl of acid citrate-dextrose. Sodium chromate (51Cr; 15 µCi/100 g of recipient rat) was added, and the cells were incubated at room temperature for 1 h. The 51Cr-labeled red blood cells (51Cr-RBCs) were pelleted as described above, washed three times in 0.7 ml of PBS, and resuspended in PBS to 1 ml.
Preparation and infusion of radiolabeled albumin and diethylenetriamine pentaacetic acid. Human serum albumin labeled with 125I (125I-albumin, ICN Biomedicals Australasia; Sydney, Australia) and 99mTc-labeled diethylenetriamine pentaacetic acid (99mTc-DTPA, gift from the Department of Nuclear Medicine, Flinders Medical Centre; Adelaide, Australia), 1 and 20 µCi, respectively, per 100 g of recipient rat, were added to the 51Cr-RBCs (1.2 µl/g body wt) and infused over 10 s via the caudal vein, 10 min before the rats were euthanized by anesthetic overdose.
Compartmentalization of radiolabels. The lungs were ventilated with air via a tracheal catheter at 60 cycles/min with a tidal volume of 7 ml/kg (Flexivent small animal ventilator, SCIREQ; Montreal, Canada). The thorax was rapidly opened through a parasternotomy, and 4-5 ml of whole blood were sampled from the LV. Plasma was separated by centrifugation at 6,000 rpm for 10 min. The lungs and heart were removed from the thorax, and the right upper lobe was resected. The remaining lung was degassed at 0.5 atm for 60 s and lavaged at 2°C with three separate 32 ml/kg volume aliquots of cold saline, with each volume instilled and withdrawn three times.
Radiolabels were counted in the whole blood, plasma, lavage, and right upper lobe with a Cobra 5003 gamma counter (125I, 15-75 keV; 99mTc, 90-190 keV; and 51Cr, 240-400 keV; Auto-gamma 5000 series, Packard Instruments; Downers Grove, IL). Because 99mTc interferes with the counting of 125I and 51Cr, the later two labels were recounted
3 days later, after the 99mTc
had decayed (half-life 6 h).
Quantification of Heart Failure
At the termination of the experiment, the right ventricle (RV) and LV were dissected, separated, and weighed (the RV was dissected off the septum). The LV was preserved in formaldehyde before being cut into four transverse sections for planimetric determination of total circumferential infarct size as a percentage of total LV circumference (42). As the left coronary artery cannot be visualized directly during surgery, there was no myocardial infarction in some animals. Infarcts were therefore graded as follows: no infarct (0% LV infarction, controls); moderate infarct (25-45% LV infarction); and large infarct (>46% LV infarction).Lavage Fluid Total Protein
Total protein concentration in the lavage fluid was determined by a modification of the Lowry method (11).Markers of Air Space Inflammation
The lavage fluid was centrifuged at 2°C for 5 min at 800 rpm. A cell count was performed on the pellet using a hemacytometer (Improved Neubauer BS 748, Weber Scientific International; Teddington, UK), and a differential cell count was performed by staining with Diff-Quik and Papanicolaou stains (7).Myeloperoxidase activity. Bronchoalveolar lavage fluid myeloperoxidase activity was quantified using the method of Schneider and Issekutz (45). The optical density at 450 nm was measured at 5 min using a Dynatech plate reader (Dynatech Laboratories; Chantilly, VA).
SP-B Assay in Plasma and Lavage
To free SP-B from any associated plasma or surfactant components, aliquots were first treated with EDTA, SDS, and Triton X-100 as previously described (13). SP-B was determined using a human-based ELISA inhibition assay (13). The antibody reacts strongly with rat SP-B (66). All samples were assayed in duplicate at four serial dilutions. Standards, assayed in quadruplicate, were included in each ELISA plate at eight serial dilutions (ranging from 7.8 to 1,000 ng/ml, r > 0.99).Measurement of Intra- and Extravascular Lung Water
The right upper lobe and an aliquot of whole blood were counted for 51Cr-RBCs; both were then frozen, dried (
50°C,
Maxi-dry, FTS Systems; Stone Ridge, NY), and weighed. Intravascular
lung water per right upper lobe (IVLW), extravascular lung water per
right upper lobe (EVLW), and blood-free dry lung weight (DLW) were
calculated using a modification (35) of the method
described by Pearce and coworkers (41) to allow expression
of EVLW/DLW as milliliters per gram of blood-free dry weight.
Measurement of Extravascular Albumin and DTPA Flux
To allow permeability conclusions from right upper lobe radiolabel concentrations, changes in intravascular volume were excluded as follows (31). Intravascular 125I-albumin was calculated by multiplying the IVLW by (1 fraction RBCs) by plasma 125I-albumin [in
counts/min (cpm)]. Extravascular 125I-albumin was
then determined by subtracting intravascular 125I-albumin
from total right upper lobe 125I-albumin (in cpm).
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(1) |
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Measurement of Epithelial Albumin and DTPA Permeability
Epithelial permeability to erythrocytes, albumin, and DTPA was expressed as lavage tracer relative to blood or plasma tracer, as follows
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(3) |
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(4) |
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Statistical Analysis
Data are presented as means ± SE unless otherwise indicated. Normally distributed data (Kolmogorov-Smirnov test of normality) were analyzed using one-way ANOVA and post hoc pairwise t-tests against the control group with modified Bonferroni corrections (61). The remaining data were analyzed using the Kruskal-Wallis test and post hoc comparisons with the Mann-Whitney U-test with modified Bonferroni corrections (61). Statistical significance was defined as P < 0.05.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Of the 39 rats studied, LV histology revealed 15 rats with no myocardial infarction (control group). Seven rats had large infarcts (55 ± 4% LV circumference), and seventeen rats had moderate infarcts (35 ± 2% LV circumference). Rat weights were similar at the terminal experiment (controls, 427 ± 1 g; moderate infarct, 448 ± 1 g; large infarct, 435 ± 3 g; P = 0.64).
Physiological Evidence of CHF
Heart rate and mean arterial blood pressure were similar between the three groups; however, LVEDP increased progressively with infarct size (P < 0.001; Table 1). Although there was no difference in RV weight between the controls and the moderate infarct group, RV weight increased 73% in the large infarct group (P < 0.001). In contrast, LV weight was unchanged over the three groups.
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Although the arterial PO2 fell as infarct size increased (P < 0.01; Table 1), there was no difference in the pH or arterial PCO2 between the groups.
Lung Composition
Whereas the wet lung weight was not changed in the moderate infarct group, it was elevated 152% in the large infarct group (P < 0.001; Fig. 1A), and this increase was matched by an increase in dry lung weight (P < 0.001; Fig. 1B), such that the wet-to-dry lung weight ratio was unchanged across groups (Fig. 1C). Derived values of lung water (35, 41) were consistent with these weight changes in the large infarct group, with a 215% increase in EVLW (P < 0.001; Fig. 2B), partially offset by a 20% reduction of IVLW (P < 0.05; Fig. 2A). EVLW/DLW increased 23% in the large infarct group (P < 0.001; Fig. 2C).
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Extravascular and Lavage Radiolabel Compartmentalization
51Cr-RBCs.
Lavage erythrocytes remained constant, and at very low levels, over the
three groups (controls, 3.3 × 105 ± 0.4 × 105; moderate infarct, 3.5 × 105 ± 0.7 × 105; large infarct,
4.0 × 105 ± 0.4 × 105;
P = 0.51).
125I-albumin.
There was a 247% increase in extravascular
125I-albumin flux in the large infarct group
(P < 0.001; Fig.
3A). The lavage
125I-albumin also increased markedly (243%) in this group
(P < 0.01; Fig. 3B).
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99mTc-DTPA. There was an 88% increase in extravascular 99mTc-DTPA flux in the large infarct group (P < 0.001; Fig. 3C), whereas lavage 99mTc-DTPA increased 60% in this group (P < 0.025; Fig. 3D).
Total Protein in Lavage Fluid
Lavage fluid total protein was similar in the control (0.14 ± 0.01 mg/ml) and moderate infarct groups (0.15 ± 0.02 mg/ml). However, there was a 350% increase in the large infarct group (0.644 ± 0.17 mg/ml; P < 0.001).Surfactant Protein-B
Plasma SP-B increased progressively with infarct size (P < 0.001). There was a 23% increase in the moderate infarct group (P < 0.05) and a further 60% increase in the large infarct group (P < 0.001) (Fig. 4A). Lavage SP-B was unchanged in the moderate infarct group but did increase slightly (9%) in the large infarct group (P < 0.05; Fig. 4B).
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Lavage Cytology
Although unchanged in the moderate infarct group, the lavage cell count was increased in the large infarct group (P < 0.01; Fig. 5A), with increased numbers of neutrophils (P < 0.01; Fig. 5B). Furthermore, myeloperoxidase activity was increased in the cell-free lavage fluid from the large infarct group (P < 0.05; Fig. 5C).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We demonstrated increased flux of 125I-albumin across both the pulmonary microvascular endothelium and alveolar epithelium in the setting of infarct-induced CHF in the rat. This, coupled with the novel finding of increased leakage of pulmonary-specific SP-B into the circulation from the alveoli, demonstrates increased bidirectional protein movement across the alveolocapillary barrier in CHF. We propose that the adaptive structural changes in the lung parenchyma that accompany CHF are associated with increased protein permeability of the alveolocapillary barrier.
Justification of Methods
CHF model. LV dysfunction was demonstrated by the progressively increased LVEDP as infarct size increased and the markedly increased RV weight in the large infarct group. Consistent with the original description of the coronary artery ligation technique, hemodynamic abnormalities were related to increased RV weight and infarct size (42). These changes, together with the increase in wet lung weight, are consistent with CHF in the large infarct group, whereas the moderate infarct group represents LV dysfunction without CHF but with reduced peak cardiac performance (42).
Measurement of permeability. Care is required in the interpretation and the strength of conclusions regarding permeability drawn without considerations of the pulmonary microvascular surface area (55). Despite an increase in LVEDP favoring pulmonary vascular congestion in the large infarct group, we found a reduction in IVLW, a paradox that may result from fibrotic pulmonary parenchymal structural changes in CHF, which tend to limit vascular distension (2, 43). Because it is unlikely that the pulmonary vascular surface area is increased in our large infarct group, changes in protein flux are likely to predominantly reflect changes in permeability or hydrostatic pressure.
Lung Composition in CHF
In CHF, Pmv is chronically elevated, and adaptive structural changes are thought to evolve to protect the alveolocapillary barrier from further vascular pressure injury (57). Such changes include the thickening of basement membranes (33, 37, 57), interstitial fibrosis of alveolar walls (33, 37), and replacement of type I alveolar epithelial cells with cuboidal type II progenitor cells (33, 37). These structural changes, which alter the composition of the lung parenchyma, must be considered to allow appropriate interpretation of tracer flux within the lungs in CHF.Wet lung weight increased 152% in the large infarct group. Total lung water can be divided into intra- and extravascular compartments (IVLW and EVLW), and, as mentioned above, IVLW may be variably affected by CHF. We found a small reduction in IVLW in the large infarct group, making the increase in EVLW more striking. However, when expressed per weight of blood-free dry lung, EVLW/DLW was increased only 23%, reflecting a marked increase in lung solid tissue in the large infarct group. Indeed, the proportional increase in dry lung weight in the large infarct group (144%) resulted in a normal wet-to-dry weight ratio.
Past studies have also documented increases in dry lung weight in CHF (28, 57, 58), with increased total lung protein content (58) and reduced pulmonary vascular compliance (58). This major change in lung composition is consistent with an adaptive increase in pulmonary connective tissue in response to chronically elevated Pmv. In summary, our large infarct (CHF model) rat lungs were heavier with more solid tissue and EVLW, despite a minor reduction in IVLW.
Compartmentalization of Radiolabels
The permeability of the normal alveolocapillary barrier and the location and nature of leakage sites are not clearly defined. The epithelial component of the alveolocapillary barrier provides the major obstacle to protein movement (5, 18), having a protein reflection coefficient (
) near unity (39) and tight junction effective pore radii of 0.5-0.9 nm (54).
In contrast, the pulmonary endothelium is closer to 0.9 (39) [radii 6.5-7.5 nm (54)]. Under
normal circumstances, molecules the size of albumin (atomic radius
3.5 nm, molecular mass = 67 kDa) are, to some extent, able to
cross the endothelial layer of the alveolocapillary barrier, but the
epithelial layer is much less permeable (54).
We have previously shown that intravenous 125I-albumin takes several hours to reach a steady state in control rats, whereas 99mTc-DTPA equilibrates in all compartments within minutes (7). Thus, whereas tissue 99mTc-DTPA reflects the total extracellular fluid volume, and tissue 51Cr-RBCs reflect the vascular volume, tissue 125I-albumin reflects the amount of albumin flux in 10 min. Because plasma proteins reach the alveoli by both convection and passive diffusion (14), the 125I-albumin flux at 10 min depends on its concentration gradient, the hydrostatic perfusion pressure, and the permeability of the alveolocapillary barrier.
The three radiolabels were measured in both the lavage fluid and the resected lobe to provide differential information on the relative permeability of each component of the alveolocapillary barrier to erythrocytes, protein, and fluid. Given that the lung endothelium is considerably more permeable than the epithelium and that the interstitial fluid compartment is normally much larger than that of the alveolus (3), then the radiolabel changes in the lavage fluid must reflect epithelial flux, whereas those in the resected lobe primarily reflect flux across the endothelium.
Vascular volume. As previously noted, pulmonary vascular volume was reduced slightly in the large infarct group. There was no increase in lavage 51Cr-RBCs, consistent with morphological studies of CHF lungs, which do not show sufficient epithelial breaks for cellular movement, despite raised Pmv (37, 57).
Lung fluid. Extravascular 99mTc-DTPA flux was increased 88% in whole lung tissue, consistent with the increase in wet lung weight. However, the attenuated increase in EVLW relative to the blood-free dry lung (EVLW/DLW) of only 23% illustrated the success of the pulmonary structural changes of CHF in resisting pulmonary fluid accumulation. Previously, Townsley and co-workers (57) demonstrated a 50% reduction in transcapillary fluid flux in CHF lungs compared with control lungs in response to high vascular pressure. Furthermore, the same investigators later documented changes in interstitial function in CHF lungs (58), which also acts to reduce pulmonary fluid accumulation in CHF (56).
Our initial assumption of tissue tracer reflecting predominantly endothelial permeability requires maintenance of a small alveolar fluid space relative to the interstitial fluid space. Concerns regarding the validity of this assumption in CHF, where there is alveolar edema, are allayed because alveolar fluid increased only 60%, whereas EVLW increased 215%, so that the relative differences in interstitial and alveolar fluid volumes were maintained, and in fact increased, in CHF. Lavage 99mTc-DTPA was also increased in the large infarct group, reflecting increased hydrostatic pressure-driven fluid flux across the epithelial component of the alveolocapillary barrier. However, this attenuated increase in fluid flux across the epithelium (60%) vs. the endothelium (88%) may reflect the fact that epithelial permeability is normally much less than endothelial permeability and the success of the pulmonary edema safety factors in CHF. These safety factors include a reduction in interstitial osmotic pressure (15) and pooling of fluid in proximal interstitial spaces (39), but most importantly increased pulmonary lymphatic drainage in CHF (38).Albumin flux. There was a marked (247%) increase in extravascular 125I-albumin flux in the large infarct group. Furthermore, this must underestimate the true albumin flux across the endothelium, as it does not account for the elevated lymphatic clearance of labeled tracer from the interstitium in CHF (29, 38).
Previous investigators have found that >92% of the resistance to albumin flux across the alveolocapillary barrier lies in the epithelium (20). We have shown markedly increased (243%) albumin movement into the alveoli and, consistent with this, a more than threefold increase in lavage total protein in the large infarct group. Although increased albumin flux across the alveolocapillary barrier in CHF may be the result of altered permeability, it may also simply represent solvent drag after hydrostatically driven convective flow across the barrier. The latter is less likely given that lavage 99mTc-DTPA increased proportionally less than lavage 125I-albumin and that circulating SP-B was increased.SP-B in CHF
We demonstrated for the first time an increase in circulating SP-B in an infarct model of CHF.SPs as biological markers of alveolocapillary barrier damage.
SP-B is uniquely suited to act as a biomarker of alveolocapillary
barrier damage (14, 25). It is synthesized exclusively by
type II alveolar epithelial cells (14, 25) and
compartmentalized in the alveoli by only apical secretion (14,
25). In its two immunoreactive forms, SP-B (26 and 46 kDa)
(13) is slightly smaller than albumin (67 kDa), and the
healthy lung maintains an epithelial lining fluid-to-plasma gradient of
~1,500:1 (14). However, when the alveolocapillary
barrier is damaged, SP-B is no longer effectively partitioned, and
increased amounts leak into the bloodstream (14, 25). The
short systemic half-life of SP-B (3-13 min) and higher arterial
than venous concentrations suggest that its circulating levels acutely
reflect changes in lung permeability (12). Indeed, plasma
levels closely reflect markers of lung function (13)
rather than hepatic or renal clearance markers (13, 26).
Although precisely how SPs breach the alveolocapillary barrier remains
uncertain, plasma levels are an early and sensitive indicator of lung
injury from a variety of etiologies (13, 34, 36, 27, 21, 52,
4), including raised Pmv secondary to acute LV
failure (10).
Determinates of increased SP-B in CHF. The increase in circulating SP-B in CHF may be a reflection of the increased concentration gradient across the alveolocapillary barrier, lymphatic drainage into the systemic circulation, or increased alveolocapillary barrier protein permeability.
The complex respiratory (6) and neuroendocrine consequences of CHF may favor increased production of SPs by alveolar type II epithelial cells. Indeed, in vitro, there is an ~30% increase in rat alveolar type II epithelial cell SP-D secretion in response to exposure to neurohormonal mediators of CHF (40). We found a 9% increase in lavage SP-B in the large infarct group, which cannot account for the doubling of circulating SP-B. Furthermore, in the moderate infarct group, where there was no change in lavage SP-B, a modest increase in circulating SP-B was noted. Indeed, the ratio of circulating-to-lavage SP-B progressively increased across the three groups (Fig. 2C), supporting a progressive increase in alveolocapillary barrier permeability. Although lung lymph flow is increased in CHF (38), the lymphatics do not play a major role in alveolar surfactant clearance (53). Furthermore, because SP-B is only released into the alveoli by type II epithelial cells (60, 65), interstitial SP-B (required to allow lymphatic drainage) represents increased permeability of the epithelial component of the alveolocapillary barrier, which is the dominant barrier to protein movement (20).Mechanism of Altered Alveolocapillary Barrier Protein Permeability in CHF
Although previous in vitro studies of protein permeability in CHF have yielded conflicting results (57, 28), our study is unique in being performed in the intact animal with all potential confounding physiological systems active. Furthermore, protein movement was documented at chronic resting levels of high Pmv rather than after an acute elevation of Pmv, thereby more accurately reflecting the human CHF state. The mechanism of increased alveolocapillary barrier protein permeability in infarct-induced CHF remains unclear, and the in vivo design of this study does not allow for the determination of precise transport mechanisms, only speculation through previous observations. Extensive rupture of individual components of the alveolocapillary barrier has not been observed morphologically in CHF (37, 57). However, the structural changes seen in the lungs in CHF may themselves alter the integrity of the alveolocapillary barrier, particularly at the intercellular tight junctions, where the pores that allow protein movement are situated. In support of this, studies of diseases of primary pulmonary parenchymal fibrosis have revealed increased permeability to both fluid (23, 44, 50) and protein (36, 51) despite the thickened alveolocapillary barrier.An alternative explanation involves pulmonary parenchymal inflammation. Pulmonary remodeling in response to pressure injury requires recruitment and activation of inflammatory cells at the alveolocapillary barrier, which may potentiate alveolocapillary barrier damage. We found that lavage fluid inflammatory cell numbers were increased in the CHF model with an increase in neutrophil numbers. Furthermore, the neutrophil granule enzyme myeloperoxidase was increased in the lavage fluid of the large infarct group.
In the moderate infarct group, there was an increase in circulating SP-B and a trend toward increased lung tissue 125I-albumin (P = 0.14). This is intriguing, as this group did not appear to have evidence of either pulmonary remodeling or inflammation to explain an increase in permeability. Circulating SP levels have shown themselves to be very sensitive markers of alveolocapillary barrier damage, examples of which include elevated levels in smokers (34), the prediction of acute respiratory distress syndrome development in acute respiratory failure patients (4), and differentiation of the alveolitis stage of idiopathic pulmonary fibrosis (51). Elevated SP-B in LV dysfunction without CHF could reflect structural changes or alveolocapillary barrier injury below the threshold of detection with our relatively crude measures. Although the absolute elevation in LVEDP was modest (3.7mmHg) in the moderate infarct group, these readings were made in the anesthetized animal, minimizing hemodynamic derangements in the setting of LV dysfunction. This finding may suggest that as well as alveolocapillary barrier remodeling in CHF causing increased permeability, mechanical hydrostatic changes such as pore stretching (47, 64) in milder degrees of hemodynamic overload may also lead to detectable changes in protein permeability, even before they are of sufficient magnitude to induce adaptive structural changes.
Clinical Relevance
We found a marked increase in lavage fluid total protein in a rat model of CHF, consistent with increased alveolocapillary barrier protein permeability. Although the alveoli are unusual among epithelial surfaces (e.g., gastrointestinal) in not normally being a particularly hostile environment for proteins (25), alveolar nonpulmonary specific protein accumulation may have several unfavorable consequences, particularly in CHF. These include surfactant dysfunction (5) with consequent increased work of breathing, induction of inflammation through altered macrophage activity (59), and potentially providing a favorable milieu for microorganisms (46), perhaps contributing to the high incidence of pneumonia in CHF patients (16, 19). From a therapeutic viewpoint, circulating SP-B may identify a window, before the gross pulmonary consequences of CHF, where disease modifying or preventive treatment strategies may be most effective.In conclusion, we demonstrated increased bidirectional movement of protein across the alveolocapillary barrier in an infarct-induced CHF model in the rat. Despite the marked structural changes in CHF lungs, which provide increased strength, the protein segregating function of the alveolocapillary barrier appears to decrease. Although the exact mechanism of this physiological defect is uncertain, there is evidence of pulmonary parenchymal inflammation in CHF, which may play a role.
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ACKNOWLEDGEMENTS |
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This study was supported by a scholarship from the Lions Medical Research Foundation and by a National Heart Foundation of Australia "J" Trust Fund grant.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C. G. De Pasquale, Cardiac Services, Flinders Medical Centre, Flinders Dr., Bedford Park, 5042 Adelaide, South Australia, Australia (E-mail: carmine.depasquale{at}fmc.sa.gov.au).
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. Section 1734 solely to indicate this fact.
First published February 6, 2003;10.1152/ajpheart.00875.2002
Received 4 October 2002; accepted in final form 27 January 2003.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Adamson, IY,
and
Bowden DH.
The type II cell as progenitor of alveolar epithelial regeneration: a cytodynamic study in mice after exposure to oxygen.
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P < 0.001). C:
EVLW/DLW increased with infarct size (P < 0.001) and
was higher in the large infarct group compared with controls
(
P < 0.05) and the large infarct group
(§P < 0.025) compared with controls.
