1 Department of Physiology and Cell Biology and 2 Department Pathology and Laboratory Medicine, Albany Medical College of Union University, Albany, New York 12208
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasma fibronectin (pFN) can incorporate into the lung extracellular matrix (ECM) as well as enhance hepatic cell phagocytic removal of bloodborne microparticulate debris that can contribute to lung vascular injury. Treatment of human pFN (hFN) with N-ethylmaleimide (NEM) blocks its ECM incorporation but not its ability to augment phagocytosis. Using hFN purified from fresh human plasma cryoprecipitate, we compared the effect of NEM-treated hFN versus normal hFN on lung transvascular protein clearance (TVPC) in postoperative bacteremic sheep to determine whether the ability of hFN to attenuate the increase in lung endothelial permeability required its ECM incorporation. Sheep with lung lymph fistulas were infused with a sublethal dose of Pseudomonas aeruginosa (5 × 108) 48 h after surgery. In the first study, sheep received either FN-rich human cryoprecipitate, FN-deficient cryoprecipitate, FN purified from cryoprecipitate (hFN), FN-deficient cryoprecipitate reconstituted with purified hFN, or the sterile saline diluent. In the second study, sheep received either 200 mg of purified hFN (group I), 200 mg of NEM-treated hFN (group II), or the saline diluent (group III). In the first study, the increase in TVPC after bacterial challenge was attenuated by FN-rich cryoprecipitate, hFN, or reconstituted FN-deficient cryoprecipitate (P < 0.05) but not by saline and FN-deficient cryoprecipitate. In the second study, TVPC increased by 2 h (P < 0.05) and peaked over 4-8 h (P < 0.05) at 380-420% above baseline in postoperative bacteremic sheep given the diluent (group III). In contrast, intravenous infusion of hFN, but not of NEM-treated hFN, significantly (P < 0.05) attenuated this increase of lung protein clearance. Thus the ability for the intravenously infused purified pFN to attenuate the increase in lung endothelial protein permeability in sheep during postsurgical bacteremia appears to require its ECM incorporation into the interstitial ECM of the lung.
lung vascular permeability; fibronectin incorporation; extracellular matrix
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
DISRUPTION OF THE LUNG endothelial barrier contributes to the etiology of lung edema and respiratory failure in septic surgical patients (32, 36). Fibronectin (FN) is both an opsonic and an adhesive glycoprotein that influences hepatic phagocytic removal of bloodborne particulates as well as the adhesion of lung endothelial cells to their FN-rich subendothelial matrix (2, 15, 32). Patients manifest an early acute depletion of plasma FN (pFN) after major surgery, trauma, or burn, which soon normalizes in 1-3 days, only to be followed by a more sustained decrease in pFN (36-38) in association with severe sepsis and pulmonary edema. In sheep, low plasma concentrations of FN can amplify the increase in lung transvascular protein clearance (TVPC) during postoperative bacteremia or intraperitoneal sepsis (26, 37), whereas an elevation in pFN can reduce lung TVPC (10, 32, 37) as well as the fluid requirements needed for hemodynamic support in such septic sheep (18).
Two major concepts have emerged to explain the mechanism by which elevated pFN levels can attenuate the increase in lung protein permeability in sheep during postsurgical bacteremia. One concept is related to the role of FN as an opsonic protein (2, 33, 36) enhancing the hepatic Kupffer cell phagocytic removal of bloodborne actin-containing cytoskeletal debris and collagenous tissue debris as well as fibrin-containing microaggregates resulting from major surgery, severe blunt trauma, thermal injury, or sepsis-induced intravascular coagulation (12, 21, 31, 32, 36, 38). Rapid clearance of such bloodborne microparticulates minimizes their lung microvascular deposition and the associated lung sequestration of activated blood neutrophils and mononuclear macrophages capable of further disrupting the lung matrix and endothelial barrier (27, 36).
However, a second and equally valid concept is that pFN may also
influence lung endothelial integrity by its incorporation into the
subendothelial and interstitial extracellular matrix (ECM) of the lung
(5, 13, 28, 29) where it facilitates endothelial cell adhesion to the
ECM due to the ligation of cell surface
5
1-FN
integrins, with Arg-Gly-Asp (RGD) sites in the matrix localized FN (11,
15, 44, 45). FN is an integral component of the lung interstitial
matrix as well as of both the subepithelial and subendothelial ECMs,
and FN is known to influence cell-cell and cell-substratum interactions
(4, 6, 7, 15, 19, 43).
Soluble pFN infused into the blood will rapidly incorporate into the ECM of many organs including the heart, liver, spleen, and lungs (6, 13, 19, 30). Such incorporation can be prevented (5, 29, 30) by prior treatment of the pFN with N-ethylmaleimide (NEM), which blocks the ability of pFN to bind to cell-associated matrix assembly sites (5, 23, 24, 30) such as those found on fibroblasts and lung endothelial cells (20, 23, 25, 30). However, pFN treated with low concentrations of NEM (<10-20 mM) will still retain its dimeric structure, affinity for gelatin or fibrin, and full ability to augment particle phagocytosis by hepatic Kupffer cells and peritoneal macrophages (5). We used FN purified from fresh normal human plasma cryoprecipitate (3, 5, 18) to determine whether ECM incorporation of infused human pFN (hFN) was essential for it to attenuate the increase in lung endothelial protein permeability in postsurgical bacteremic sheep (10, 32). To accomplish this goal, we first documented that hFN was the active molecule in fresh human plasma cryoprecipitate that mediates its influence on lung TVPC and then compared the effects of an intravenous infusion of normal hFN versus NEM-treated hFN on lung protein clearance in postsurgical bacteremic sheep.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Plasma cryoprecipitate. Plasma
cryoprecipitate has an FN concentration 8-10 times greater than
normal plasma as determined by immunoassay (1, 3, 33, 36). Because
cryoprecipitate (American Red Cross) was the source of purified hFN for
the present study, we first compared the effect of an intravenous
infusion of either fresh human plasma cryoprecipitate, hFN purified
from cryoprecipitate, FN-deficient cryoprecipitate, or FN-deficient cryoprecipitate reconstituted with purified hFN on lung protein clearance in the postsurgical septic sheep model. Only fresh
(<30-day-old) and not outdated (>1-yr-old) cryoprecipitate was used
because prolonged storage of cryoprecipitate (in excess of 2 mo)
results in FN fragments with limited ability to be incorporated into
the ECM and reduced opsonic activity (3). The fresh cryoprecipitate was
stored at 20°C until used.
Isolation of purified hFN. hFN has a
very high affinity for gelatin and was purified from plasma
cryoprecipitate by gelatin-Sepharose affinity chromatography (3, 34,
37) with two repeat passages as previously documented (18). FN was
eluted off the columns with 4 M urea in the column buffer (1.0 M sodium
chloride, 5 mM EDTA, 3 mM benzamidine, and 0.01 M sodium phosphate, pH
7.4) and then dialyzed overnight against 0.2 M sodium phosphate buffer (pH 7.4). Purified hFN was stored at 80°C and quick-thawed
at 37°C on the day of use. Electroimmunoassay as well as gel
electrophoretic analysis coupled with SDS-PAGE analysis confirmed the
full removal of hFN by duplicate chromatographic adsorption, with no
significant residual hFN in the cryoprecipitate (18). The purified hFN, FN-deficient cryoprecipitate, FN-rich cryoprecipitate, and
reconstituted FN-deficient cryoprecipitate were all sterile filtered
before intravenous infusion. Sheep were infused only once with each of the test solutions.
NEM alkylation of hFN to prevent its matrix
incorporation. Purified hFN was diluted in 10 mM
3-(cyclohexylamino)-1-propanesulfonic acid (CAPS)-0.15 M NaCl
(CAPS-buffered saline) and adjusted to a pH of 11. Alkylation of FN
involved exposure of hFN to solid NEM (Pierce Chemical, Rockford, IL)
at a concentration of 20 mM or less for 4 h (5, 30). After 4 h, hFN was
dialyzed against CAPS buffer and then against 0.2 M sodium phosphate
buffer (pH 7.4). Normal hFN was handled in a similar manner but was not
exposed to NEM. SDS-PAGE analysis of hFN after its purification from
cryoprecipitate or subsequent treatment with NEM verified that it was
not fragmented and had retained a 440-kDa molecular mass
expected of the intact FN dimer (Fig. 1).
Although not photographed, no lower FN fragments were observed at the
bottom of the gel. These gels were run under nonreduced conditions. The
protein was stained with Coomassie blue.
|
Brien et al. (5) have recently documented that hFN treated with 10-20 mM NEM (as used in the present study) retains full ability to support particle phagocytosis by hepatic Kupffer cells and peritoneal macrophages. These recent immunofluorescent findings from our laboratory (5) were part of a sequence of studies (7, 29) designed to compare the ability of hFN versus NEM-treated hFN to incorporate in a fibrillar deposition pattern, especially within the interstitial ECM of the lungs in sheep, a comparison essential for the present analysis of their effects on lung protein permeability in postsurgical septic sheep.
Assay of FN matrix assembly ability with cultured fibroblasts. An in vitro assay with cultured fibroblasts is commonly used to evaluate the ECM assembly of FN (23, 24, 30). This in vitro technique can be done as either a competitive inhibition assay performed over 30-60 min or a 125I-FN deposition assay usually performed over 24 h. For the deposition assay, parallel quantitation of 125I-FN in both the deoxycholate detergent (DOC)-soluble (pool I) and DOC-insoluble (pool II) fractions of the cell layer is done at the end of incubation (25, 30). The pool I fraction reflects FN bound to the cell layer but not incorporated, whereas the pool II fraction primarily reflects FN incorporated into the ECM (23, 24, 29, 30). NEM-treated rat FN and hFN cannot bind to such cell layers and become incorporated into their ECMs (23, 24), a finding now documented with lung tissue slices (29) and intact animals (5, 29). Before intravenous injection, we analyzed the purified hFN preparations to confirm that the NEM-alkylated hFN had lost its ability to be incorporated into the ECM.
In the competitive inhibition assay, we studied the ability for either unlabeled (cold) hFN or unlabeled NEM-treated hFN to inhibit the binding of radiolabeled 125I-hFN to cell-associated matrix assembly sites (17, 24, 30). A1-F human foreskin fibroblasts were cultured in Ham's F-12 medium supplemented with 10% fetal bovine serum (FBS; HyClone, Logan UT), 100 U/ml of penicillin, and 100 µg/ml of streptomycin (GIBCO BRL, Life Technologies, Grand Island, NY). Between passages 9 and 12, the fibroblasts were seeded at a density of 5 × 104 fibroblasts/well and used 1-4 days postseeding. Briefly, the cell layers were washed with phosphate-buffered saline (PBS) and supplemented with medium containing 0.2% bovine serum albumin (instead of FN-deficient FBS). To each well, we then added a tracer amount of 125I-hFN ranging between 1 and 3 µg to achieve adequate counts per minute depending on specific activity over the course of the study. The tracer dose was small, and the response was similar over the 1- to 3-µg range. Then unlabeled hFN or NEM-treated hFN was added to the medium in excess up to 100 µg/ml. The cell layers were then incubated at 37°C for 30 min, gently washed with cold PBS, and extracted with 1 N NaOH before measurement of cell-associated 125I-hFN activity.
Assay of FN matrix assembly ability with cultured endothelial cells. We also used the matrix incorporation assay with endothelial monolayers (11, 44, 45) to compare aliquots of 125I-hFN and NEM-treated 125I-hFN. Calf pulmonary artery endothelial (CPAE) cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL) supplemented with 20% FBS (HyClone), 10 mM nonessential amino acids (GIBCO BRL), 100 U/ml of penicillin, and 100 µg/ml of streptomycin. The cells were split 1:2 every fourth day. Experiments were performed between passages 19 and 23. The endothelial cells were seeded at a density of 75,000 cells/well and allowed to achieve confluence in 3-4 days as previously described by our laboratory (11, 44, 45).
Matrix assembly receptors are primarily located on the basolateral
surface of adherent endothelial cells in culture (20), and the
confluent nature of the CPAE monolayer tends to restrict the rapid
access of fluid-phase hFN or NEM-treated hFN to these receptors. Tumor
necrosis factor (TNF)-, a cytokine released with gram-negative
bacteremia or endotoxemia, appears to be an important mediator
contributing to the increase in lung protein permeability in sheep with
postsurgical sepsis (32, 36). Previous experiments by our laboratory
(11, 44, 45) demonstrated that TNF-
will increase the protein
permeability of such previously confluent CPAE monolayers based on
direct measurements of transendothelial 125I-albumin clearance. Thus we
performed the present study on lung endothelial monolayers with and
without adding recombinant human TNF-
to the culture medium (400 U/ml) for a period of 18 h before our comparative analysis of the
matrix incorporation of 125I-hFN
versus NEM-treated 125I-hFN.
After exposure to TNF- or diluent for 18 h, the endothelial
monolayers were washed with PBS and supplemented with 1.0 ml of fresh
culture medium containing FN-deficient FBS prepared by passage of FBS
over a gelatin-Sepharose affinity column (11, 44, 45). Tracer amounts
(1-3 µg) of either 125I-hFN
or NEM-treated 125I-hFN were again
added, depending on the specific activity, and the CPAE cell layers
were incubated for 24 h at 37°C under a gas phase of 5%
O2-95%
CO2. After a 24-h incubation, the
cell layers were washed three times with PBS, scraped into 1 M NaOH,
and assayed for 125I-FN activity
in both the DOC-soluble (pool
I) and DOC-insoluble (pool
II) fractions to quantify incorporation into the ECM.
Surgical preparation of the lung lymph fistula. The lung lymph fistula model in sheep (37, 40) has been extensively used to measure in vivo lung protein clearance as an index of pulmonary endothelial protein permeability in postoperative bacteremic endotoxic sheep (7, 10, 26, 32). Healthy adult male sheep (n = 29) with a narrow weight range (25-35 kg) were fasted of solid food for 48 h before surgery. General anesthesia was induced with 2.5% Pentothal Sodium (15 mg/kg; Abbott Laboratories, Chicago, IL). The animals were endotracheally intubated and mechanically ventilated (Harvard Apparatus, Millis, MA) at a rate of 18 breaths/min and a tidal volume of 12 ml/kg. The sheep were maintained on inhalational 78% N2O, 20% O2, and 1-1.5% halothane. Surgical preparation of the lung lymph fistula required cannulation of the efferent duct from the caudal mediastinal lymph node and was performed under sterile conditions as previously described (10, 18, 26, 37, 40). Each sheep received 2 liters of 0.9% sterile saline intravenously during surgery.
The operative procedure consisted of a standard right thoracotomy via the seventh intercostal space. The operative procedure was approved by the Institutional Animal Care and Use Committee. The posterior portion of the node was ligated to minimize contamination of lung lymph by diaphragmatic lymphatics (10, 26, 32, 37). The efferent duct of the node was cannulated with a Silastic catheter (0.047 inch OD, 0.025 inch ID) impregnated with a 2% tridodecylmethylammonium chloride (TDMAC)-heparin complex (Polysciences, Warrington, PA). The catheter was secured with 4-0 silk, tunneled under the parietal pleura, and positioned to exit the thoracic cavity at the level of the ninth intercostal space. This corresponds to the level of the caudal mediastinal lymph node when sheep are standing because the protocol is done with the unanesthetized sheep model. The fistula was secured with a cannula holder to the skin. The ribs were approximated with cable ties, and the incision was closed with multiple layers of 0 silk suture material. All sheep were quickly mobile and given intramuscular butorphanol for analgesia. The sheep recovered in a special postoperative metabolic cage. They were continuously hydrated intravenously with sterile normal saline for 2 days after surgery before being studied. They were allowed free access to food and water by 24 h postsurgery.
Hemodynamics. Experiments were done 48 h after surgery. The right common carotid artery was cannulated with a polyethylene catheter (PE-240) impregnated with a 2% TDMAC-heparin complex for blood sampling (10, 26). An 8.5-Fr Cordis introducer (Cordis Laboratories, Miami, FL) was placed in the right internal jugular vein for infusions (10, 18, 26). On the morning of the experiment, a Swan-Ganz catheter (Edwards, Santa Ana, CA) was inserted into each sheep, and baseline hemodynamic measurements were recorded for 2 h to verify stability of lymph flow. Statham P23AC pressure transducers and a Grass model 7B polygraph were used to measure mean systemic arterial pressure, mean pulmonary arterial pressure, central venous pressure, and pulmonary capillary wedge pressure. Cardiac output was measured at 30-min intervals by thermodilution with a cardiac output computer (Edwards model 7510A). Systemic vascular resistance was a calculated variable.
Protocol for FN-rich and FN-deficient cryoprecipitate infusion study. Before infusion, six units of cryoprecipitate were rapidly thawed at 37°C, pooled, and diluted to a total volume of 240 ml with sterile normal saline. Sheep were then infused intravenously with either FN-rich cryoprecipitate, purified hFN, FN-deficient cryoprecipitate, reconstituted FN-deficient cryoprecipitate, or the saline diluent; all infusions were started 2.5 h before the start of the intravenous bacterial challenge. Based on the hFN concentration in the pooled human cryoprecipitate, we infused an average of 225 mg of hFN (200- to 250-mg range) suspended in 240 ml of sterile saline into each sheep in the FN-rich cryoprecipitate, hFN, or reconstituted FN-deficient cryoprecipitate groups. One-third of the total 240-ml volume, i.e., 80 ml, was infused over the initial half hour and the remaining 160 ml were infused at a rate of 40 ml/h for the next 4 h. Thus the total treatment infusion interval lasted 4.5 h. At time 0, i.e., 2.5 h into the treatment infusion, each sheep was given a single sublethal dose of live, washed Pseudomonas aeruginosa (5 × 108; American Type Culture Collection) suspended in sterile saline (50 ml) and injected intravenously over a 60-min interval (6, 10). Each sheep was used only once in the protocol, and each sheep received only one infusion of the FN-rich or FN-deficient cryoprecipitate test solution.
Protocol for comparative hFN versus NEM-treated hFN infusion study. For the comparison of normal hFN versus NEM-treated hFN in the second study, we injected each sheep with a standard 200-mg dose of purified hFN, the lowest dose that appeared able to consistently attenuate TVPC in this postoperative bacteremic sheep model. Three groups of postsurgical septic sheep were analyzed. Group I received 200 mg of purified hFN diluted in 240 ml of sterile saline, group II received 200 mg of NEM-treated purified hFN diluted in 240 ml of sterile saline, and group III received only 240 ml of sterile saline diluent. Infusion of the 240-ml volume of the test solutions was again started 2.5 h before bacterial challenge at a rate of an 80-ml bolus over the initial 30 min followed by continuous infusion of the remaining 160 ml of the test treatment solutions (hFN, NEM-treated hFN, and diluent) at a rate of 40 ml/h for the next 4 h, identical to the cryoprecipitate protocol. Again, each sheep was used only once in the study and each sheep received only one infusion of the hFN or NEM-treated hFN test solution. All sheep again received only a single sublethal 5 × 108 dose of live P. aeruginosa infused over a 60-min interval starting at time 0, which corresponded to 2.5 h after the beginning of the infusion of the test solutions. There was no repeat intravenous bacterial challenge to avoid the possibility of development of antibodies to the bacteria.
Determination of lung TVPC. Lung TVPC
was used as an index of lung vascular permeability as previously
described (10, 26, 32, 40). It was calculated as the product of lymph
flow (L; in
ml/h) × the lymph-to-plasma total protein concentration ratio, which was measured at 30-min intervals. Lymph was collected in graduated tubes containing 100 µl of an antiproteolytic solution consisting of 7.5% EDTA, 3 mM benzamidine, and 2 mM iodoacetic acid.
Blood samples (2 ml) were drawn from the arterial line and anticoagulated with 100 µl of the antiprotease solution. Lymph and
blood samples were centrifuged at 6,000 g for 10 min, and the supernatants
were assayed in duplicate for total protein by a modified Biuret method
(6, 10) with an automated plate reader (Bio-Tek EL312e).
Determination of FN levels in plasma and lymph. The concentration of sheep FN (sFN) and hFN in plasma and lymph samples was determined by Laurel rocket electroimmunoassay (1, 37, 38) with species-specific antibodies (5, 6, 10). Rabbit antibodies against sFN (which do not react with hFN) as well as sheep antibodies against hFN (which do not react with sFN) were used. As recently reported (5), the NEM treatment of hFN renders the protein less detectable in sheep plasma by electroimmunoassay, especially with an antibody against hFN prepared in sheep. Thus the blood levels of hFN versus NEM-treated hFN were not measured in the second protocol. A recently available goat antibody to hFN can apparently be used with more confidence (5).
Plasma samples analyzed for sFN were diluted to 10% before the assay, whereas lymph samples analyzed for sFN were only diluted to 20% before the assay because the endogenous FN concentration in sheep lung lymph is less than that in sheep plasma (6, 10). In contrast, plasma samples assayed for their content of infused hFN were diluted 50%, whereas lymph samples assayed for hFN content were not diluted due to the limited amount of hFN antigen in each sample (6). Purified sFN or hFN standards were included on each electrophoretic plate. Samples were electrophoresed (LKB multiphor) at 80 V for 20 h at 15-17°C. After staining with Coomassie blue, rocket heights were measured in millimeters, and a double-reciprocal plot was defined with the sFN or hFN standards (1, 6, 10). FN concentrations are expressed as micrograms per milliliter.
Statistical analysis. Data are presented as means ± SE. Differences between groups over time were assessed with a two-way analysis of variance. Differences between time points were analyzed with an unpaired Student's t-test with a Bonferroni correction. With a confidence level of 95%, P < 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Table 1 presents some of the hemodynamic
variables in the first protocol. In saline control sheep, postsurgical
infusion of bacteria caused a slight decline in arterial blood pressure to 80.0 ± 2.4 mmHg and a slight increase in cardiac output to 6.8 ± 0.4 l/min at 8 h. We also observed a transient early rise in
pulmonary arterial pressure after bacterial infusion
(P < 0.05), which typically returned
toward baseline over 4-5 h, but the slight variation in pulmonary
arterial pressure between the five experimental groups was not
different (P > 0.05). The relatively
stable hemodynamic pattern over 4-8 h in this sublethal
postsurgical bacteremic sheep model (6, 7, 10) was especially ideal
because our focus was the effect of hFN on lung TVPC over the same
interval. In this regard, we were especially interested in the
"increased permeability phase" of the response to a sublethal (5 × 108) intravenous
Pseudomonas challenge in the
postsurgical sheep model (10, 32, 36) when elevated lung protein
clearance existed in association with relatively normal pulmonary
arterial pressures. This phase of increased lung endothelial protein
permeability typically exists over 4-10 h (10, 32).
|
In designing the first study, we infused a volume of hFN-rich
cryoprecipitate estimated to provide an amount of hFN somewhat equivalent to or slightly greater than the anticipated depletion of
endogenous pFN after bacterial challenge to achieve a normal or
slightly elevated total FN concentration (endogenous sFN plus infused
hFN). As verified by analysis of variance with repeated measures, there
was a gradual elevation (P < 0.05)
in total pFN antigen (sFN plus hFN) after infusion of either purified
hFN, hFN-rich cryoprecipitate, or FN-deficient cryoprecipitate
reconstituted with purified hFN (Fig. 2).
Actually, the total FN concentration was typically elevated by
~15-20% above preinfusion levels in these three groups by the
end of the infusion, suggesting a need to slightly lower the hFN dose
for the second protocol. We observed no consistent major differences in
total FN levels between the FN-rich cryoprecipitate, hFN, or
reconstituted FN-deficient cryoprecipitate groups.
|
There was a pronounced three- to fourfold increase
(P < 0.01) in TVPC in
those postsurgical bacteremic sheep treated with either FN-deficient
cryoprecipitate or the saline diluent (Fig. 3). In contrast, the increase in lung TVPC
was significantly attenuated over 5-8 h in sheep treated with
either FN-rich cryoprecipitate, hFN, or reconstituted FN-deficient
cryoprecipitate (P < 0.05) as
statistically verified by analysis of variance with repeated measures.
Post hoc statistical analysis with Bonferroni correction also confirmed
that lung TVPC in the FN-rich cryoprecipitate, hFN, and reconstituted
FN-deficient cryoprecipitate groups were similar to each other, but
each was significantly different from both the saline and FN-deficient
cryoprecipitate groups (P < 0.05), whereas lung TVPC values in the saline- and FN-deficient
cryoprecipitate-infused groups were not different from each other
(P > 0.05). Thus FN appeared to be
the active molecule in fresh plasma cryoprecipitate that contributed to
its ability to attenuate the typical increase in lung TVPC in
postoperative bacteremic sheep.
|
For the second study, hFN was first purified from human plasma
cryoprecipitate and then radioiodinated. Aliquots of the iodinated hFN
were then subjected to either sham alkylation or NEM alkylation with
low concentrations of NEM (10-20 mM). Paired aliquots of hFN and
NEM-treated hFN were verified to be nonfragmented (Fig. 1) and analyzed
for matrix incorporation activity with cell layers of lung endothelial
cells or fibroblasts (5, 23-25, 30). Using fibroblasts in the
competitive inhibition assay, we observed that excess unlabeled hFN
added to the culture medium caused a dose-dependent inhibition of the
binding of 125I-hFN to the
fibroblast monolayers (P < 0.05;
Fig. 4), whereas excess soluble NEM-treated
hFN failed to inhibit 125I-hFN
binding (P > 0.05). Using
fibroblasts in the matrix incorporation assay, we also observed (data
not shown) progressive deposition of
125I-purified hFN into the ECM of
the fibroblast cell layers over 24 h
(P < 0.01) but negligible ECM
deposition of NEM-treated
125I-hFN, consistent with the
pattern recently documented with fibroblasts from our laboratory (30).
|
We then compared the matrix incorporation of
125I-hFN versus NEM-treated
125I-hFN using confluent lung
endothelial monolayers, some of which were pretreated with TNF- to
increase their protein permeability to allow more rapid access of the
soluble hFN added to the culture medium to the basolateral region of
the cell layer (11, 20, 44, 45). As shown in Fig.
5, in both normal and TNF-
-treated CPAE
monolayers, we observed little binding (pool
I; data not shown) or incorporation
(pool II) of NEM-treated
125I-hFN into the DOC-insoluble
fraction of the cell layer. In contrast, deposition of
125I-hFN was at least fourfold
greater in normal control CPAE cell layers
(P < 0.001) and further elevated in
those endothelial monolayers pretreated with TNF-
(P < 0.05; Fig. 5). Thus even after
TNF-
treatment to increase CPAE monolayer permeability, NEM
(10-20 mM)-alkylated hFN still displayed little matrix
incorporation. As recently documented (5), alkylation of hFN with NEM
at 10-20 mM does not appear to block its opsonic activity as
tested by several phagocytic bioassays.
|
In the second protocol (Fig. 6), sheep
infused with hFN appeared to have a slight early upward trend in the
endogenous concentration of sFN over the 2 h to
time 0 interval, but, based on
previous studies by our laboratory (5, 27, 32, 34), this ~5%
increase is not sufficient to influence either hepatic phagocytic
function or lung protein clearance. After bacterial challenge, the
typical gradual decline (P < 0.05)
in the plasma level of endogenous sFN in saline-infused control sheep
was attenuated (P < 0.05) over 3-8 h with an intravenous infusion of purified hFN but not by an
infusion of NEM-treated hFN (Fig. 6), suggesting that the extra hFN may
have minimized the plasma loss and ECM deposition of circulating endogenous FN with bacterial sepsis.
|
In the first protocol with FN-rich cryoprecipitate, we were able to use a rocket electroimmunoassay (1, 3, 5, 33, 34, 38) with a sheep antibody to hFN to measure the hFN in the sheep's plasma after its intravenous infusion. However, during the second study, we observed that hFN alkylated with NEM is less detectable in sheep plasma by electroimmunoassay with an antibody made in sheep against hFN. Accordingly, in Fig. 6, we only plotted the endogenous sFN levels. However, in a recent preliminary follow-up study with antibodies against hFN that were made in goats (not in sheep), we observed that the half-life of the plasma clearance of a smaller 100-mg bolus intravenous dose of either hFN or NEM-treated hFN in postsurgical bacteremic sheep is ~5-6 h. Thus both groups likely had very similar levels of opsonically active hFN circulating in their blood when given the bacteria.
In the second protocol, we again measured various hemodynamic
parameters in the sheep after infusion of either purified HFN, NEM-treated HFN, or diluent (Table 2).
Again, we observed that systemic arterial pressure and cardiac output
were minimally altered in this sublethal unanesthetized bacteremic
model, but pulmonary arterial pressure transiently increased over the
1- to 3-h interval (P < 0.05) and
then began to typically normalize over 3-4 h. This pattern was not
significantly different in the hFN, NEM-treated hFN, and diluent groups
(P > 0.05; Table 2). Without
bacterial challenge, the hemodynamic and lung fluid balance parameters
in these sheep are very stable over the 8-h experimental interval (data
not shown), thus confirming previous reports that the large increase in
TVPC is secondary to the bacterial challenge (6, 7, 10, 26,
32).
|
We then calculated pulmonary TVPC
(L × lymph-to-plasma total protein concentration ratio) in all sheep based
on an analysis of
L (in ml/h) as
well as the plasma and lymph total protein concentrations in serial
samples collected before and over the 8-h postbacterial challenge
interval. In postsurgical sheep injected with diluent, the sustained
400+% elevation in lung TVPC after the bacterial challenge was
significantly reduced by infusion of purified hFN
(P < 0.05; Fig.
7). In contrast, infusion of an identical
dose of NEM-treated hFN, which had opsonic activity but limited ability
to be incorporated normally into the interstitial matrix of the
sheep's lung (5) or the ECM of cultured endothelial cells or
fibroblasts as seen in the present study (Figs. 4 and 5), was unable to
attenuate the increase in lung protein clearance (Fig. 7). The changes
in TVPC in the NEM-treated hFN group were identical to the TVPC pattern
measured in the diluent-treated control sheep.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
FN is an adhesive molecule that can influence cell-cell interaction and
cell adhesion to a substratum (11, 15, 16, 25, 32). FN can be locally
synthesized in tissues by many cell types including fibroblasts,
hepatocytes, epithelial cells, and endothelial cells, but the plasma
pool of FN appears to be the reservoir for much of the FN rapidly
deposited at sites of tissue injury or incorporated within the normal
ECM (9, 12-14, 19, 21, 28, 32). Incorporation of soluble pFN into
the ECM (13) is a cell-dependent process that appears to require the
interaction of the amino-terminal 27-kDa domain of FN with
cell-associated matrix assembly sites, an interaction essential for the
subsequent polymerization of soluble FN into fine FN fibers in the ECM
(17, 23, 24). Once in the ECM, the RGD site in FN is believed to
interact with cell surface
5
1-FN
integrins to influence cell adhesion and cell behavior (11, 25, 32,
44). In the lung, FN is found in the interstitial matrix as well as
under both the endothelial and alveolar epithelial cell layers (4,
6, 41, 43). These cells can also locally synthesize and incorporate FN
into their ECM via a cell-dependent polymerization process (20, 23, 44,
45) potentially influenced by disulfide exchange in the amino-terminal
portion of FN (23, 24).
Mononuclear macrophages, especially hepatic Kupffer cells, play an important role in the systemic host defense response to injury and shock (19, 32). Their phagocytic function is vital to the removal of abnormal bloodborne nonbacterial collagenous and membranous microparticulate tissue debris generated as a result of intravascular coagulation, burn injury, or soft tissue trauma (21, 31, 34, 36). Mononuclear macrophages infiltrating an area of tissue injury can also remove cellular debris in a process apparently amplified by soluble FN (14, 31), which has an affinity for actin, fibrin, denatured collagen (gelatin), C1q-containing immune complexes, and intracellular membranous debris (reviewed in Refs. 32, 36).
Septic patients who develop pulmonary edema and respiratory distress
after surgery, trauma, or burn often also manifest prolonged suboptimal
concentrations of soluble FN in their blood (14, 33, 37, 38). Low
levels of pFN are believed to delay hepatic Kupffer cell clearance of
bloodborne microparticulates (27, 32-34), thus contributing to
lung microembolization and pulmonary vascular injury (27, 36).
Sustained bacteremia and/or endotoxemia can activate hepatic Kupffer
cells (8, 22) as well as monocytes/macrophages sequestered in the lung,
a response that can contribute to lung vascular injury by the release
of inflammatory cytokines such as TNF-. TNF-
can increase the
protein permeability of lung endothelial monolayers as well as
disrupt their FN-rich subendothelial matrix (11, 44,
45).
The ongoing deposition of pFN into the ECM of various tissues (6, 12,
13, 19, 28-30) is an important pathway for its normal turnover
(13). Such plasma loss is greatly accelerated in sheep (5-7, 10)
and humans (33, 37, 38) during gram-negative bacteremia or after major
surgery, trauma, or burn (12, 14, 21, 36, 42). The concept that a
reduced plasma pool of FN might limit the ECM content of FN in the lung
and thus contribute to lung vascular integrity is supported by the
finding that experimental depletion of pFN in rats reduces the
heparin-urea extractable pool of FN within the lung matrix (35).
Conversely, an elevation in the FN concentration in the perfusate of
isolated perfused rabbit lungs will influence its capillary filtration
coefficient (39), whereas raising pFN in postsurgical bacteremic sheep
can attenuate the increase in lung endothelial permeability (6, 10, 32,
36). Indeed, addition of purified hFN to the culture medium of lung endothelial monolayers can prevent as well as reverse the TNF--induced increase in protein permeability (11, 44, 45), but
such a protective response is not seen after the addition of
160/180-kDa (44, 45) fragments of hFN that cannot be incorporated into
the subendothelial ECM.
The
5
1-integrins
appear to be especially important to the integrity of lung endothelial
monolayers because the TNF-
-induced increase in protein permeability
and disruption of the FN-rich subendothelial matrix can be mimicked by
the addition of either antibodies to
5
1-FN
integrins or soluble RGD peptides [but not to Arg-Gly-Glu (RGE)
peptides] to the culture medium (11, 44, 45). Specificity is
apparent because the TNF-
-induced increase in lung endothelial
monolayer protein permeability cannot be prevented by adding either
human plasma vitronectin or human plasma fibrinogen, both of which have
RGD binding sites but cannot get covalently assembled into the ECM (44,
45), nor is it seen by adding alkylated hFN (NEM-treated hFN) (44, 45).
These observations suggested that it may be the ability of pFN to
incorporate into the ECM of the lung in vivo and not its ability to
support hepatic phagocytic function that allows it to attenuate the
increase in lung protein permeability in sheep during postsurgical bacteremia.
Cryoprecipitate is the protein fraction precipitated from fresh frozen
plasma when it is chilled between 1 and 6°C at defined intervals.
Compared with normal plasma, several plasma proteins are greatly
concentrated in the cryoprecipitate including fibrinogen, factor
VIII:C, von Willebrand factor, and factor XIII as well as
cold-insoluble globulin (3), which was discovered to be identical to
opsonic 2-surface-binding
glycoprotein (1, 2, 32) or pFN (1, 2, 36). Our present data indicate
that an infusion of fresh (<1-mo-old) FN-rich cryoprecipitate can
attenuate the increase in lung protein clearance in postoperative
bacteremic sheep and that its FN content is essential for this
response. However, outdated (>12-mo-old) plasma cryoprecipitate or
hFN purified from outdated cryoprecipitate will likely not attenuate
the permeability increase because FN is highly susceptible to
proteolytic fragmentation and FN fragments can form in cryoprecipitate
even after 2 mo of storage at
80°C (3).
Our comparison of the effect of hFN versus NEM-treated hFN on lung protein clearance in postoperative bacteremic sheep allowed us to probe the mechanism of the in vivo protective response because hFN treated with low concentrations of NEM (10-20 mM) retains its opsonic activity (5) but has limited ability to be incorporated in vivo into the lung interstitial ECM (5) as recently confirmed by dual-label immunofluorescent analysis of lung tissue harvested from rats and sheep (5-7, 29, 30). In these studies, endogenous sFN was found in a fibrillar deposition pattern throughout the interstitial matrix of the sheep's lung (5-7). The intravenously infused hFN became colocalized with the fibrillar endogenous sFN matrix in the lung interstitium, but very little fibrillar deposition of NEM-hFN was detected in this interstitial matrix (5, 6). Bacteremia after surgery in sheep enhanced the fibrillar deposition of hFN in the lung interstitial ECM, but most of the NEM-treated hFN deposited in the lung appeared mainly in a focal or punctate pattern, unexpectedly colocalized with inflammatory macrophages and fibrin microaggregates in the lung (5, 6).
In the present study, we observed that the typical increase in lung protein clearance in sheep with postoperative bacteremia was significantly (P < 0.05), although not totally, attenuated within 2-3 h after the intravenous infusion of 200 mg of purified hFN was finished. Infusion of a larger 500-mg dose of purified hFN to elevate the total pFN level (sFN plus hFN) by ~40% to 900-950 µg/ml can cause an even greater and more rapid attenuation in lung protein clearance (10). These findings suggest that the plasma concentration of FN may actually influence the in vivo content of FN in the tissue ECM (12, 13) as previously speculated (32). Indeed, the data of Rebres and colleagues (29, 30) derived from rat organ tissue slices incubated with varying concentrations of rat 125I-pFN directly support this concept. It is not likely that the small, early 5% elevation in endogenous sFN in the blood of those sheep infused with pure hFN influenced TVPC because it appears that infusion of a lower 100-mg dose of hFN will not by itself alter lung protein clearance (5-7). Nor is it likely that such a small 5% increase in circulating hFN provided them with an advantage relative to hepatic Kupffer cell function because the normal blood level of FN is more than adequate to support efficient hepatic clearance of circulating test particles, and pFN apparently needs to decline by ~20% to have an adverse effect on phagocytic function (27, 32, 34).
Using an intravenous infusion of metabolically labeled [75Se]selenomethionine in rats, Deno et al. (13) documented that the relatively rapid deposition of pFN in various tissues including the lung was an important part of the normal loss of soluble FN from plasma, a loss accelerated with local tissue injury (12, 14, 21). The plasma loss of intravenously injected hFN in rats or sheep (6, 13) is best described by a two-compartment model in which the initial rapid disappearance reflects its equilibration between the plasma and rapidly exchangeable extravascular compartment, whereas the secondary removal phase reflects the combined loss or degradation from both compartments (13). Subsequently, Charash et al. (6) reported that although the early 30-min distribution phase for intravenously injected hFN in postsurgical sheep was not directly altered with the Pseudomonas infusion, its secondary phase of enhanced removal over 0.5-8 h was likely due to both increased opsonic consumption associated with microaggregate clearance as well as its deposition at sites of vascular and tissue injury (12, 14, 21), especially in the lungs.
In the present study, the enhanced loss of endogenous sFN from the blood after bacterial challenge was reduced by infusion of normal hFN but not of NEM-treated hFN, a response consistent with our hypothesis. For example, if altered integrity of the vascular barrier contributes, in part, to the more rapid loss of FN from the plasma in septic sheep (12, 14, 21, 32), then the hFN infusion may have reduced endogenous pFN loss by improving endothelial barrier integrity, a concept directly supported by our measurements of TVPC. Alternatively, the incorporation of endogenous sheep pFN into the tissue matrices of various organs, including the interstitial matrix of septic and injured lungs (5-7, 19, 29), may have been "reduced or spared," in part, by the parallel ECM deposition of the infused hFN. In rats and sheep, soluble FN is removed from the plasma with a half-life of ~18-21 h (6, 10, 13), with a parallel production of soluble FN by ongoing hepatic synthesis (13). In humans, ~35-36% of the plasma pool of FN is removed every 24 h and matched by ongoing hepatic synthesis of pFN to yield a constant pFN level (42). Based on these findings, the slight upward trend of sFN in the blood with a continuous infusion of purified hFN, even before bacterial challenge, may simply reflect a reduced loss of endogenous pFN coupled with continued hepatic production of pFN (12, 13, 42).
NEM treatment of pFN at pH 11 alkylates lysine and histidine residues in the 27-kDa amino-terminal region of FN in addition to blocking sulfhydryl groups on FN. This amino-terminal domain of FN apparently mediates its binding to cell-associated matrix assembly sites (17, 25), and it is the modification of this region of the FN molecule after treatment with NEM that is believed to be responsible for its inability to bind to cell layers and become incorporated into the ECM (24, 30). These in vitro findings with cultured cells have now been verified with both lung and liver tissue slices as well as in vivo isotopic distribution studies with labeled FN and NEM-treated FN (12, 13, 24, 29, 30). Such observations coupled with our direct analysis of the DOC-insoluble pool of 125I-hFN versus NEM-treated 125I-hFN in tissues as well as their immunofluorescent deposition patterns (5, 29, 30) emphasize the limited ability of NEM-treated pFN to be assembled in a fibrillar form within the interstitial matrix in rats and sheep (5, 29, 30).
In summary, our comparative findings with hFN versus NEM-treated hFN
suggest that the ability for large-dose intravenous infusion of
purified hFN to attenuate the increase in lung transvascular protein
clearance in postsurgical gram-negative bacteremic sheep was
functionally dependent on its normal incorporation into the lung
interstitial ECM. This conclusion is consistent with recent in vitro
studies by our laboratory (11, 44, 45) with bovine lung endothelial
cell monolayers that also compared the effect of soluble hFN versus
soluble NEM-treated hFN on the TNF--induced increase in
transendothelial 125I-albumin
flux. Disturbances in this cell-mediated incorporation process may
contribute to the etiology of prolonged increases in lung endothelial
protein permeability in gram-negative bacteremic patients after surgery
or trauma (32, 36).
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge the technical assistance of Edward Lewis and the secretarial assistance of Deborah Moran and Maureen Davis.
![]() |
FOOTNOTES |
---|
This study was supported primarily by National Institute of General Medical Sciences (NIGMS) Grant GM-21447.
M. Resnikoff was a National Institutes of Health Postdoctoral Research Fellow in Physiology and Cell Biology supported by NIGMS Grant T32-GM-07033 and a Fellow in Vascular Surgery. T. Brien was a National Institutes of Health Postdoctoral Research Fellow in Physiology and Cell Biology supported by NIGMS Grant T32-GM-07033 and is currently a Resident in Pathology and Laboratory Medicine. R. F. Rotundo was a National Institutes of Health Postdoctoral Research Fellow in Physiology and Cell Biology supported by NIGMS Grant T32-GM-07033 and is currently a Research Associate.
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 and other correspondence: T. M. Saba, Dept. of Physiology and Cell Biology (MC-134), Albany Medical College, 47 New Scotland Ave., Albany, NY 12208 (E-mail: Thomas_Saba{at}ccgateway.amc.edu).
Received 1 March 1999; accepted in final form 4 June 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Blumenstock, F. A.,
T. M. Saba,
and
P. Weber.
Purification of 2-opsonic glycoprotein from human serum and its measurement by immunoassay.
J. Reticuloendothel. Soc.
23:
119-134,
1978[Medline].
2.
Blumenstock, F. A.,
T. M. Saba,
P. Weber,
and
R. Laffin.
Biochemical and immunological characterization of human opsonic 2-SB glycoprotein: its identity with cold-insoluble globulin.
J. Biol. Chem.
253:
4287-4291,
1978[Abstract].
3.
Blumenstock, F. A.,
C. R. Valeri,
T. M. Saba,
E. Cho,
A. Melaragno,
A. Gray,
and
M. Lewis.
Progressive loss of fibronectin-mediated opsonic activity in plasma cryoprecipitate with storage. Role of fibronectin fragmentation.
Vox Sang.
54:
129-137,
1988[Medline].
4.
Bray, B. A.
Cold-insoluble globulin (fibronectin) in connective tissues of adult human lung and in trophoblast basement membrane.
J. Clin. Invest.
62:
745-752,
1978[Medline].
5.
Brien, T. P.,
P. P. Reddy,
P. A. Vincent,
E. P. Lewis,
J. S. Ross,
and
T. M. Saba.
Lung matrix deposition of normal and alkylated plasma fibronectin: response to postsurgical sepsis.
Am. J. Physiol.
274 (Lung Cell. Mol. Physiol. 18):
L432-L443,
1998
6.
Charash, W. E.,
P. A. Vincent,
P. J. McKeown-Longo,
T. M. Saba,
E. Lewis,
and
M. A. Lewis.
Kinetics of plasma fibronectin: increased lung tissue incorporation after postoperative bacteremia.
Am. J. Physiol.
260 (Regulatory Integrative Comp. Physiol. 29):
R553-R562,
1991
7.
Charash, W. E.,
P. A. Vincent,
T. M. Saba,
F. L. Minnear,
P. J. McKeown-Longo,
J. A. Migliozzi,
M. A. Lewis,
E. Lewis,
and
C. Giunta.
Immunofluorescent analysis of plasma fibronectin incorporation into the lung during acute inflammatory vascular injury.
Am. Rev. Respir. Dis.
148:
467-476,
1993[Medline].
8.
Chaudry, I. H.,
R. Zellweger,
and
A. Ayala.
The role of bacterial translocation of Kupffer cell immune function following hemorrhage.
Prog. Clin. Biol. Res.
392:
209-218,
1995[Medline].
9.
Clark, R. A. F.,
H. J. Winn,
H. F. Dvorak,
and
R. B. Colvin.
Fibronectin beneath re-epithelializing epidermis in vivo. Sources and significance.
J. Invest. Dermatol.
80:
26s-30s,
1983[Medline].
10.
Cohler, L. F.,
T. M. Saba,
E. P. Lewis,
P. A. Vincent,
and
W. E. Charash.
Plasma fibronectin therapy and lung protein clearance with bacteremia after surgery.
J. Appl. Physiol.
63:
623-633,
1987
11.
Curtis, T. M.,
P. J. McKeown-Longo,
P. A. Vincent,
S. M. Homan,
E. M. Wheatley,
and
T. M. Saba.
Fibronectin attenuates increased endothelial monolayer permeability after RGD peptide, anti-5
1, or TNF-
exposure.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L248-L260,
1995
12.
Deno, D. C.,
M. H. McCafferty,
T. M. Saba,
and
F. A. Blumenstock.
Mechanism of acute depletion of plasma fibronectin following thermal injury in rats: appearance of a gelatin-like ligand in plasma.
J. Clin. Invest.
73:
20-34,
1984[Medline].
13.
Deno, D. C.,
T. M. Saba,
and
E. P. Lewis.
Kinetics of endogenously labeled plasma fibronectin: incorporation into tissues.
Am. J. Physiol.
245 (Regulatory Integrative Comp. Physiol. 14):
R564-R575,
1983[Medline].
14.
Gauperaa, T.,
and
R. Seljelid.
Plasma fibronectin is sequestered into tissue damaged by inflammation and trauma.
Acta Chir. Scand.
152:
85-90,
1986[Medline].
15.
Gold, L. I.,
and
E. Pearlstein.
Fibronectin-collagen binding and requirement during cellular adhesion.
Biochem. J.
186:
551-559,
1980[Medline].
16.
Hayman, E. G.,
and
E. Ruoslahti.
Distribution of fetal bovine serum fibronectin and endogenous rat cell fibronectin in extracellular matrix.
J. Cell Biol.
83:
255-259,
1979[Abstract].
17.
Hocking, D. C.,
J. Sottile,
and
P. J. McKeown-Longo.
Fibronectin's III-1 module contains a conformation-dependent binding site for the amino-terminal region of fibronectin.
J. Biol. Chem.
269:
19183-19191,
1994
18.
Holman, J. M.,
T. M. Saba,
and
E. Lewis.
Effect of fibronectin-rich human cryoprecipitate on fluid volume requirements in sheep during post-operative sepsis.
J. Trauma
28:
571-581,
1988[Medline].
19.
Jin, H.-M.,
P. A. Vincent,
W. E. Charash,
T. M. Saba,
P. J. McKeown-Longo,
F. A. Blumenstock,
and
E. Lewis.
Incorporation of circulating fibronectin into various tissues during sepsis: colocalization with endogenous tissue fibronectin.
Exp. Mol. Pathol.
55:
203-216,
1991[Medline].
20.
Kowalczyk, A. P.,
R. H. Tulloh,
and
P. J. McKeown-Longo.
Polarized fibronectin secretion and localized matrix assembly sites correlate with subendothelial matrix formation.
Blood
75:
2335-2342,
1990[Abstract].
21.
La Celle, P.,
F. A. Blumenstock,
C. McKinley,
T. M. Saba,
P. A. Vincent,
and
V. Gray.
Blood-borne collagenous debris complexes with plasma fibronectin after thermal injury.
Blood
75:
470-478,
1990[Abstract].
22.
Mainous, M. R.,
W. Ertel,
I. H. Chaudry,
and
E. A. Deitch.
The gut: a cytokine-generating organ in systemic inflammation.
Shock
4:
193-199,
1995[Medline].
23.
McKeown-Longo, P. J.,
and
D. F. Mosher.
Binding of plasma fibronectin to cell layers of human skin fibroblasts.
J. Cell Biol.
97:
466-472,
1983[Abstract].
24.
McKeown-Longo, P. J.,
and
D. F. Mosher.
Mechanism of formation of disulfide-bonded multimers of plasma fibronectin in cell layers of cultured human fibroblasts.
J. Biol. Chem.
259:
12210-12215,
1984
25.
McKeown-Longo, P. J.,
and
D. F. Mosher.
The assembly of the fibronectin matrix in cultured human fibroblast cells.
In: Fibronectin, edited by D. F. Mosher. San Diego, CA: Academic, 1989, p. 163-179.
26.
Niehaus, G. D.,
P. T. Schumacker,
and
T. M. Saba.
Influence of opsonic fibronectin deficiency on lung fluid balance during bacterial sepsis.
J. Appl. Physiol.
49:
693-699,
1980
27.
Niehaus, G. D.,
P. T. Schumacker,
and
T. M. Saba.
Reticuloendothelial clearance of blood-borne particulates: relevance to experimental lung microembolization and vascular injury.
Ann. Surg.
191:
479-487,
1980[Medline].
28.
Oh, E.,
M. Pierschbacher,
and
E. Ruoslahti.
Deposition of plasma fibronectin in tissues.
Proc. Natl. Acad. Sci. USA
78:
3218-3221,
1981[Abstract].
29.
Rebres, R. A.,
E. Cho,
R. F. Rotundo,
and
T. M. Saba.
Reduced incorporation of plasma fibronectin into the lung matrix during postoperative sepsis.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L409-L418,
1996
30.
Rebres, R. A.,
P. J. McKeown-Longo,
P. A. Vincent,
E. Cho,
and
T. M. Saba.
Extracellular matrix incorporation of normal and NEM-alkylated fibronectin: liver and spleen deposition.
Am. J. Physiol.
269 (Gastrointest. Liver Physiol. 32):
G902-G912,
1995
31.
Rovin, B.,
J. Molnar,
D. Chevalier,
and
P. Ng.
Interaction of plasma fibronectin (pFN) with membranous constituents of peritoneal exudate cells and pulmonary macrophages.
J. Leukoc. Biol.
36:
601-620,
1984[Abstract].
32.
Saba, T. M.
Kinetics of plasma fibronectin: relationship to phagocytic function and lung vascular injury.
In: Fibronectin, edited by D. F. Mosher. San Diego, CA: Academic, 1989, p. 395-439.
33.
Saba, T. M.,
F. A. Blumenstock,
D. M. Shah,
R. H. Landaburu,
M. E. Hrinda,
D. C. Deno,
J. M. Holman, Jr.,
E. Cho,
C. Phelan,
and
P. M. Cardarelli.
Reversal of opsonic deficiency in surgical, trauma, and burn patients by infusion of purified human plasma fibronectin.
Am. J. Med.
80:
229-240,
1986[Medline].
34.
Saba, T. M.,
and
E. Cho.
Reticuloendothelial systemic response to operative trauma as influenced by cryoprecipitate or cold-insoluble globulin therapy.
J. Reticuloendothel. Soc.
26:
171-186,
1979[Medline].
35.
Saba, T. M.,
E. Cho,
and
F. A. Blumenstock.
Effect of acute plasma fibronectin depletion on tissue fibronectin levels: analysis by a new fluorescent immunoassay.
Exp. Mol. Pathol.
41:
81-95,
1984[Medline].
36.
Saba, T. M.,
J. B. Fortune,
and
J. R. Wallace.
Microaggregation hypothesis of multiple system organ failure.
In: Multiple System Organ Failure, edited by D. E. Fry. St. Louis, MO: Mosby Year Book, 1992, p. 25-41.
37.
Saba, T. M.,
G. D. Niehaus,
W. A. Scovill,
F. A. Blumenstock,
J. C. Newell,
J. Holman,
and
S. R. Powers.
Lung vascular permeability after reversal of fibronectin deficiency in septic sheep: correlation with patient studies.
Ann. Surg.
198:
654-662,
1983[Medline].
38.
Scovill, W. A.,
S. J. Annest,
T. M. Saba,
F. A. Blumenstock,
J. C. Newell,
H. H. Stratton,
and
S. R. Powers.
Cardiovascular hemodynamics after opsonic -2-surface binding glycoprotein therapy in injured patients.
Surgery
86:
284-293,
1979[Medline].
39.
Seeger, W.,
D. Walmrath,
N. Heimburger,
and
H. Neuhof.
Fibronectin decreases pulmonary vascular permeability under baseline conditions and after administration of arachidonic acid in rabbit lungs.
Thromb. Res.
44:
135-146,
1986[Medline].
40.
Staub, N. C.,
R. Bland,
K. Brigham,
R. Demling,
A. J. Erdman,
and
W. C. Woolverton.
Preparation of chronic lung lymph fistulas in sheep.
J. Surg. Res.
19:
315-320,
1975[Medline].
41.
Stenman, S.,
and
A. Vaheri.
Distribution of a major connective tissue protein, fibronectin, in normal human tissues.
J. Exp. Med.
147:
1054-1064,
1978[Abstract].
42.
Thompson, C.,
F. A. Blumenstock,
T. M. Saba,
J. E. Kaplan,
P. J. Feustel,
J. B. Fortune,
L. Hough,
and
V. Gray.
Plasma fibronectin synthesis in normal and injured humans as determined by stable isotope incorporation.
J. Clin. Invest.
84:
1226-1235,
1989[Medline].
43.
Torikata, C.,
B. Villiger,
C. Kuhn,
and
J. A. McDonald.
Ultrastructural distribution of fibronectin in normal and fibrotic human lung.
Lab. Invest.
52:
399-408,
1985[Medline].
44.
Wheatley, E. M.,
P. J. McKeown-Longo,
P. A. Vincent,
and
T. M. Saba.
Incorporation of fibronectin into matrix decreases the TNF-induced increase in endothelial monolayer permeability.
Am. J. Physiol.
265 (Lung Cell. Mol. Physiol. 9):
L148-L157,
1993
45.
Wheatley, E. M.,
P. A. Vincent,
P. J. McKeown-Longo,
and
T. M. Saba.
Effect of fibronectin on permeability of normal and TNF-treated lung endothelial cell monolayers.
Am. J. Physiol.
264 (Regulatory Integrative Comp. Physiol. 33):
R90-R96,
1993