Transglutaminase-mediated fibronectin multimerization in lung endothelial matrix in response to TNF-alpha.

Exposure of lung endothelial monolayers to tumor necrosis factor (TNF)-alpha causes a rearrangement of the fibrillar fibronectin (FN) extracellular matrix and an increase in protein permeability. Using calf pulmonary artery endothelial cell layers, we determined whether these changes were mediated by FN multimerization due to enhanced transglutaminase activity after TNF-alpha (200 U/ml) for 18 h. Western blot analysis indicated that TNF-alpha decreased the amount of monomeric FN detected under reducing conditions. Analysis of (125)I-FN incorporation into the extracellular matrix confirmed a twofold increase in high molecular mass (HMW) FN multimers stable under reducing conditions (P < 0.05). Enhanced formation of such HMW FN multimers was associated with increased cell surface transglutaminase activity (P < 0.05). Calf pulmonary artery endothelial cells pretreated with TNF-alpha also formed nonreducible HMW multimers of FN when layered on surfaces precoated with FN. Inhibitors of transglutaminase blocked the TNF-alpha-induced formation of nonreducible HMW multimers of FN but did not prevent either disruption of the FN matrix or the increase in monolayer permeability. Thus increased cell surface transglutaminase after TNF-alpha exposure initiates the enhanced formation of nonreducible HMW FN multimers but did not cause either the disruption of the FN matrix or the increase in endothelial monolayer permeability.

Increased recycling of (alpha)5(beta)1 integrins by lung endothelial cells in response to tumor necrosis factor.

Tumor necrosis factor (alpha) (TNF-(alpha) can change the interaction of lung endothelial cell monolayers with their extracellular matrix in association with an increase in endothelial monolayer protein permeability. Using immunofluorescence microscopy and flow cytometry, we determined if exposure of calf pulmonary artery endothelial monolayers to TNF-(alpha) may influence cell-matrix interactions by altering the clustering as well as internalization of the (&agr;)5(beta)1 integrins (or fibronectin receptors) on the surface of endothelial cells. Immunofluorescence microscopy revealed that TNF-(alpha) caused an increase in the intracellular staining of (alpha)5(alpha)1 integrins within structures similar to endocytic vesicles as well as an increase in antibody-induced clustering of the integrins at the cell periphery. Flow cytometric analysis of endothelial cells incubated at 37 degrees C after antibody-labeling of their surface (alpha)5(beta)1 integrins at 4 degrees C confirmed an increase in the rate of (alpha)5(beta)1 integrin internalization which was at least 3 times greater after TNF-(&agr;) exposure, based on the half-life for antibody-labeled surface integrins to reach equilibrium with non-labeled integrins within the intracellular pool. Interestingly, the total cell surface expression of (alpha)5(beta)1 integrins was relatively constant after TNF-(alpha) exposure despite the enhanced rate of internalization, suggesting an accelerated recycling of the internalized (alpha)5(beta)1 integrins back to the cell surface. This response was confirmed by the measurement of labeled integrin recycling, which showed a significant (P<0.01) increase in the rate of recycling of the internalized integrins in TNF-treated endothelial cells. Enhanced internalization and subsequent recycling of (alpha)5(beta)1 integrins by endothelial monolayers exposed to TNF-(alpha) may facilitate the redistribution of cell-surface integrins in response to this inflammatory cytokine and may also modify cell-matrix interactions leading to reduced integrity and increased protein permeability of the lung endothelial monolayers.

Hepatic fibronectin matrix turnover in rats: involvement of the asialoglycoprotein receptor.

Fibronectin (Fn) is a major adhesive protein found in the hepatic extracellular matrix (ECM). In adult rats, the in vivo turnover of plasma Fn (pFn) incorporated into the liver ECM is relatively rapid, i.e., <24 h, but the regulation of its turnover has not been defined. We previously reported that cellular Fn (cFn) and enzymatically desialylated plasma Fn (aFn), both of which have a high density of exposed terminal galactose residues, rapidly interact with hepatic asialoglycoprotein receptors (ASGP-R) in association with their plasma clearance after intravenous infusion. With the use of adult male rats (250-350 g) and measurement of the deoxycholate (DOC)-insoluble (125)I-labeled Fn in the liver, we determined whether the ASGP-R system can also influence the hepatic matrix retention of various forms of Fn. There was a rapid deposition of (125)I-pFn, (125)I-aFn, and (125)I-cFn into the liver ECM after their intravenous injection. Although (125)I-pFn was slowly lost from the liver matrix over 24 h, more than 90% of the incorporated (125)I-aFn and (125)I-cFn was cleared within 4 h (P < 0.01). Intravenous infusion of excess nonlabeled asialofetuin to competitively inhibit the hepatic ASGP-R delayed the rapid turnover of both aFn and cFn already incorporated within the ECM of the liver. ECM retention of both (125)I-aFn and (125)I-cFn was also less than (125)I-pFn (P < 0.01) as determined in vitro using liver slices preloaded in vivo with either tracer form of Fn. The hepatic ASGP-R appears to participate in the turnover of aFn and cFn within the liver ECM, whereas a non-ASGP-R-associated endocytic pathway apparently influences the removal of normal pFn incorporated within the hepatic ECM, unless it becomes locally desialylated.

Lung matrix incorporation of plasma fibronectin reduces vascular permeability in postsurgical bacteremia.

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 x 10(8)) 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.

Circulating cellular fibronectin may be a natural ligand for the hepatic asialoglycoprotein receptor: possible pathway for fibronectin deposition and turnover in the rat liver.

It has been postulated that the in vivo removal of many plasma glycoproteins after desialylation is mediated by their interaction with a specific endocytic receptor on hepatocytes called the asialoglycoprotein receptor (ASGP-R), which is known to have a high affinity for specific carbohydrate residues, such as galactose. However, this mechanism has never been proven in vivo, nor has a naturally occurring ligand for the ASGP-R been identified. We investigated the influence of the terminal galactose residues on plasma fibronectin (pFn) on its liver deposition and turnover in adult rats, using neuraminidase to remove sialic acid residues to expose galactose residues. We also tested the hypothesis that the normal presence of a large amount of terminal galactose residues in cellular Fn (cFn) may allow cFn to serve as a natural ligand readily able to interact with the ASGP-R. In contrast to the slow clearance of normal pFn from the blood, cFn and desialylated pFn (aFn) displayed a rapid plasma clearance (P < .001) with greater than 50% of both the 125I-cFn or 125I-aFn depositing in the liver within 15 minutes. The enhanced plasma removal and liver deposition of both 125I-cFn and 125I-aFn was competitively inhibited (P < .01) by prior intravenous infusion of excess asialofetuin, which can selectively bind to the ASGP-R. The enzymatic addition of terminal sialic acid residues onto cFn to "mask" or "cap" the normally exposed galactose residues delayed the rapid plasma removal of cFn. Accelerated degradation of 125I-aFn and 125I-cFn as compared with 125I-pFn was demonstrated in vitro by both primary cultures of normal rat hepatocytes or incubated (37 degrees C) tissue slices of livers harvested from normal rats after in vivo preloading with tracer 125I-Fn forms. Thus, the ASGP-R appears to directly participate in the rapid in vivo removal of cFn from the blood, while native pFn may be removed by an alternative pathway unless it can become desialylated in vivo. These findings suggest that cFn may be a naturally occurring ligand that does not require desialylation before removal by the ASGP-R on hepatocytes.

TNF-alpha-induced matrix Fn disruption and decreased endothelial integrity are independent of Fn proteolysis.

Exposure of confluent pulmonary arterial endothelial monolayers to tumor necrosis factor (TNF)-alpha causes both a reorganization and/or disruption of fibronectin (Fn) in the extracellular matrix and an increase in transendothelial protein permeability. However, the factors initiating this response to TNF-alpha have not been defined. Because TNF-alpha can induce proteinase expression in endothelial cells, we determined whether proteinases cause both the alteration of the Fn matrix and the permeability increase as is often speculated. Incubation of calf pulmonary arterial endothelial monolayers with TNF-alpha (200 U/ml) for 18 h caused a disruption of the Fn matrix and an increase in transendothelial protein permeability. A reduced colocalization of cell-surface alpha5beta1-Fn integrins with the Fn fibers in focal contacts was also observed. TNF-alpha treatment of endothelial monolayers with matrices prelabeled with 125I-human Fn (hFn) did not cause the release of Fn fragments or alter the content of Fn antigen in the medium as analyzed by SDS-PAGE coupled with autoradiography. Both the content and fragmentation pattern of Fn within the cell layer and the insoluble Fn matrix also appeared unchanged after TNF-alpha exposure as confirmed by Western immunoblot. Fn-substrate zymography revealed that TNF-alpha increased the expression of two proteinases within the conditioned medium in which activity could be blocked by aprotinin but not by EDTA, 1,10-phenanthroline, leupeptin, or pepstatin. However, inhibition of the Fn proteolytic activity of these two serine proteinases did not prevent either the TNF-alpha-induced disruption of the Fn matrix or the increase in permeability. Thus the reorganization and/or disruption of the Fn matrix and the temporally associated increase in endothelial permeability caused by TNF-alpha appear not to be due to proteolytic degradation of Fn within the extracellular matrix. In contrast, decreased alpha5beta1-Fn integrin interaction with Fn fibers in the matrix may be important in the response to TNF-alpha exposure.

Lung matrix deposition of normal and alkylated plasma fibronectin: response to postsurgical sepsis.

Plasma fibronectin (Fn) can both enhance phagocytic clearance of microparticulate debris by macrophages as well as incorporate it into the lung extracellular matrix (ECM). The goal of this study was to document that N-ethylmaleimide (NEM)-treated human plasma Fn (HFn) would lose its ability to incorporate into the lung ECM in vivo even though it would retain its ability to stimulate test particle phagocytosis and bind to fibrin. Using dual-label immunofluorescence, we compared the lung deposition of purified normal HFn and NEM-alkylated HFn (NEM-HFn) after their intravenous injection into postoperative nonbacteremic and bacteremic sheep in relationship to the localization of endogenous sheep Fn. Two days after a sterile surgical thoracotomy, sheep were infused with either 5 x 10(8) Pseudomonas aeruginosa (postsurgical bacteremic model) or the diluent (nonbacteremic model). They also received a bolus 100-mg injection (5 min) of either HFn or NEM-HFn. Analysis of serial lung biopsies harvested at 2-h intervals demonstrated little deposition of NEM-HFn compared with HFn in the lung interstitial matrix of postoperative nonbacteremic sheep. In contrast, enhanced deposition of both HFn and NEM-HFn was observed in the lungs of postoperative bacteremic sheep. However, in the lungs of bacteremic sheep, HFn displayed a diffuse fibrillar deposition pattern in the lung characteristic of ECM incorporation, whereas the enhanced NEM-HFn deposition, especially in the interstitial ECM region of the lung, was primarily focal and punctate, with very little fibrillar incorporation. Immunofluorescent analysis with antibodies specific to fibrinogen, Fn, and lung macrophage surface antigens coupled with immunoperoxidase staining for HFn antigen revealed that the punctate fluorescence pattern was due to both the binding of HFn to fibrin and its colocalization with inflammatory cells. Thus treatment of plasma Fn with low concentrations of NEM will limit its normal in vivo fibrillar incorporation into the interstitial ECM region of the lung.

Reduced in vivo plasma fibronectin content of lung matrix during postoperative sepsis.

Sepsis after surgery, trauma, or burn contributes to altered lung endothelial permeability and respiratory failure. Fibronectin (Fn), an opsonic and adhesive glycoprotein, exists in both a soluble form in plasma and an insoluble form in the extracellular matrix (ECM). Recent studies [E. M. Wheatley, P. J. McKeown-Longo, P. A. Vincent, and T. M. Saba, Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9): L148-L157, 1993] suggest that the ECM content of Fn may influence lung vascular permeability. We evaluated the incorporation of plasma-derived Fn (pFn) into the ECM of the lung during postoperative sepsis. Postoperative nonseptic and postoperative septic rats were compared, using a model of laparotomy followed by cecal ligation and puncture. To label the pFn pool, rats received intravenously 3 micrograms of purified rat 125I-labeled Fn/100 g body weight 6 h after surgery (laparotomy). 125I-Fn in the deoxycholate detergent-insoluble fraction of tissues was used to quantify matrix-incorporated Fn at 4 h after infusion with 125I-Fn. Septic rats exhibited a peripheral leukopenia as well as reduction in plasma volume, Fn halflife, and total pFn pool. Incorporation of pFn in the liver and spleen of postsurgical septic rats was not different (P > 0.05) from sham-operated (postsurgical nonseptic) rats, but incorporation was significantly decreased (P < 0.05) in vivo in the lung. However, under controlled in vitro conditions, lung tissue harvested from septic or sham-operated rats demonstrated a similar tissue incorporation of soluble 125I-pFn as well as similar rates of retention/turnover of ECM 125I-Fn, based on pulse-chase experiments. These data suggest that the in vivo inflammatory environment in the lung during postoperative sepsis, which cannot be reproduced in vitro, may alter the Fn content of the ECM of the lung. Such reduced levels of pFn in the lung ECM may be a factor influencing lung vascular integrity during postoperative sepsis.

Extracellular matrix incorporation of normal and NEM-alkylated fibronectin: liver and spleen deposition.

The incorporation of plasma fibronectin (pFn) into the extracellular matrix (ECM) is believed to influence tissue integrity, wound repair, and vascular permeability. In vitro, matrix assembly of Fn requires the binding of soluble Fn to cell-associated matrix assembly sites. Alkylation of human pFn (HFn) with N-ethylmaleimide (NEM) prevents the initial binding of Fn to matrix assembly sites as well as its in vitro incorporation into the ECM as reflected by detergent-insoluble 125I-labeled Fn (pool II Fn). We determined the kinetics of Fn matrix incorporation in tissue and whether NEM treatment of rat pFn (NEM-RFn) would limit its in vivo incorporation into ECM by analysis of pool I [deoxycholate (DOC) soluble] and pool II (DOC insoluble) 125I-Fn in tissues after its intravenous injection into rats. After intravenous injection, tissue incorporation of normal rat 125I-pFn was especially intense in liver and spleen, in agreement with the large amount of endogenous Fn detected in the matrices of these organs. Tissue deposition of plasma-derived 125I-RFn in liver and spleen peaked by 4 h, with significant (P < 0.01) loss over 24 h, indicating turnover of matrix Fn. Tissue localization of normal 125I-RFn in liver, lung, spleen, heart, and intestine was greater (P < 0.05) than 125I-NEM-RFn at 4 h. Normal HFn, but not NEM-HFn, was incorporated into tissues and colocalized with endogenous Fn in the matrix. To identify the cells mediating the intense incorporation of pFn into liver ECM, we compared matrix assembly of 125I-HFn by cultured fibroblasts, hepatocytes, and hepatic Kupffer cells. With fibroblasts, 125I-HFn in pool I reached steady state by 3 h, whereas 125I-HFn in pool II exceeded that in pool I by 6 h and continued to increase over 24 h. With hepatocytes, pool I 125I-HFn reached steady state by 1 h, and a progressive increase (P < 0.05) of 125I-HFn in pool II was observed over 24 h. Kupffer cells were not able to incorporate significant amounts of 125I-HFn into matrix. NEM-HFn displayed limited incorporation into ECM by both fibroblast and hepatocyte cultures. These novel observations suggest that the interaction of soluble pFn with matrix assembly sites is necessary to its in vivo incorporation into the ECM.

Fibronectin attenuates increased endothelial monolayer permeability after RGD peptide, anti-alpha 5 beta 1, or TNF-alpha exposure.

Endothelial permeability can be altered by tumor necrosis factor-alpha (TNF-alpha), a cytokine released in association with inflammation-induced tissue injury. In the subendothelial matrix, fibronectin (Fn) influences endothelial cell adhesion by the interaction of integrins with RGD and non-RGD attachment sites in Fn. We compared the effect of TNF-alpha, RGD-containing peptides (GRGDSP), or antibody to alpha 5 beta 1-integrins on the protein permeability of bovine lung endothelial monolayers as assessed by transendothelial 125I-labeled albumin clearance. We also examined the influence of purified human plasma fibronectin (hFn) on this permeability response. TNF-alpha, RGD peptides, and antibodies to alpha 5 beta 1-integrins elicited a dose- and time-dependent increase in protein permeability as well as a reorganization and/or disruption of the endogenous Fn matrix. A control RGE peptide (GRGESP) as well as immunoglobulin G purified from nonimmune rabbit serum did not increase endothelial protein permeability or disrupt the endogenous fibrillar Fn pattern in the matrix. Likewise, a LDV peptide derived from the alternatively spliced type III connecting segment (IIICS) within bovine Fn (bFn) was unable to increase permeability of the bovine endothelial monolayer. Co-incubation of purified soluble hFn (300 or 600 micrograms/ml) with either TNF-alpha, the RGD peptide, or the antibody to alpha 5 beta 1-integrins prevented the increase in endothelial permeability. This protective effect was also observed when the purified hFn (600 micrograms/ml) was added after the TNF-alpha-induced increase in endothelial permeability had taken place. Immunofluorescent analysis confirmed the incorporation of the hFn into the subendothelial matrix and its co-localization with the endogenous bFn. The similar alteration of the subendothelial matrix after exposure to RGD peptides, anti-alpha 5 beta 1-antibodies, or TNF-alpha, coupled with the ability for hFn to attenuate the permeability increase typically elicited by all three agents, suggests that disruption of cell-matrix interactions may be the mechanism by which TNF-alpha alters endothelial permeability.

Isoproterenol decreases protein permeability in edematous isolated rabbit lungs: estimation of PS and sigma.

The objective of the present study was to determine whether the ability of the beta-adrenergic agonist isoproterenol to attenuate pulmonary edema occurs via a permeability and/or hemodynamic mechanism. In isolated perfused rabbit lungs, the restrictive property of the vascular barrier to the movement of fluid and protein was assessed by measurements of the capillary filtration coefficient (Kf) and the transvascular clearance of 125I-labeled albumin, respectively. Regression analysis of albumin clearance vs. transvascular fluid flux was performed to estimate the permeability-surface area product (PS) and the reflection coefficient (sigma) by use of the linear or nonlinear flux equation. Arterial, capillary, and venous pressures and resistances, weight gain, and the wet-to-dry weight ratio were also assessed. Isoproterenol (8 ng.ml-1.min-1) attenuated the arachidonic acid (4 mg)-induced increases in fluid flux, wet-to-dry weight ratio, albumin clearance, and PS and the decrease in sigma. Isoproterenol had no effect on the increase in Kf, and there was no correlation between capillary pressure and fluid flux in any of the four groups. Regression analysis revealed that the non-linear flux equation provided estimates of PS and sigma that more accurately described the statistical differences in albumin clearance among the groups studied than the linear flux equation. These findings demonstrate that isoproterenol attenuated the increased transvascular flux of albumin in edematous lungs by modifying the protein permeability of the vascular barrier.

Delayed elevation of ED1-cellular fibronectin in plasma following postsurgical bacteremia.

Fibronectin (Fn) exists in both a soluble form in plasma and lymph as well as an insoluble form in the extracellular matrix. Matrix-localized cellular fibronectin (cFn) contains extra domains (ED1 and/or ED2) not found in plasma Fn (pFn). Very little (< 1-2%) ED1-containing cFn exists in normal blood, and its rapid release into plasma and/or lymph is believed to reflect acute vascular injury. We used a polyclonal antibody to sheep pFn and a monoclonal antibody to ED1 domain of cFn to measure both pFn and ED1-cFn in relationship to lung lymph flow (QL), lung lymph-to-plasma (L/P) total protein concentration ratio, and lung protein clearance (LPC). Unanesthetized sheep (n = 7) were injected intravenously with Pseudomonas aeruginosa (5 x 10(8)) at both 2 and 7 days following surgical preparation of a lung lymph fistula. After both bacterial challenges, we observed an early increase in QL and a small decline in the L/P ratio (0-2 h), reflecting increased fluid filtration in the presence of an intact vascular barrier. This was followed by a further increase (P < 0.05) in QL; an elevation in the L/P ratio; and a marked (P < 0.05) increase in LPC over 3-6 h, indicative of an increase in lung endothelial protein permeability. Before the first bacterial infusion, ED1-cFn in plasma was 9.97 micrograms/ml or approximately 2% of the total Fn antigen in plasma; whereas ED1-cFn in lung lymph was 6-8% of total lymph Fn.(ABSTRACT TRUNCATED AT 250 WORDS)

Release of ED1 fibronectin from matrix of perfused lungs after vascular injury is independent of protein synthesis.

Fibronectin (Fn) is an adhesive protein found in the plasma and extracellular tissue matrix. Locally synthesized tissue or cellular Fn (cFn) has extra domains (ED1 and ED2) not present in liver synthesized plasma Fn (pFn). In the lung, Fn is found in the endothelial and epithelial basement membranes, as well as in the interstitial matrix. Utilizing murine monoclonal antibodies to ED1 of cFn, we studied the release of total Fn as well as ED1-Fn into the plasma-free perfusate of the isolated perfused rabbit lung in relation to changes in lung weight due to fluid accumulation after oxidant (H2O2) challenge. Both parameters were also studied after addition of cycloheximide (20 micrograms/ml perfusate) to the perfusion medium to inhibit lung protein synthesis. After continuous H2O2 challenge (11 nmol.ml buffer-1.min-1), there was a 2.25 +/- 0.62 g increase in lung weight over 60 min. Measurement of 125I-labeled albumin clearance at 20 min after the start of H2O2 infusion confirmed an increase in lung endothelial protein permeability after H2O2 treatment. Fn antigen was released into the perfusate as early as 15 min after oxidant challenge. By 60 min, total perfusate Fn increased in H2O2-treated lungs (n = 6) to 2.10 +/- 0.48 micrograms/ml compared with only 0.35 +/- 0.09 micrograms/ml in normal control lungs (n = 5). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of nonreduced samples revealed that the Fn released consisted of primarily intact (440 kDa) Fn as well as Fn fragments. A rapid release of ED1-Fn paralleled the increased release of total Fn.(ABSTRACT TRUNCATED AT 250 WORDS)

Incorporation of fibronectin into matrix decreases TNF-induced increase in endothelial monolayer permeability.

Plasma fibronectin, a dimeric adhesive protein in blood, incorporates into the subendothelial and interstitial matrix in the lung especially during vascular injury. Fibronectin in the matrix is believed to influence cell-cell interaction and endothelial cell adhesion to the collagen-rich extracellular matrix. We previously observed that addition of purified soluble human plasma fibronectin (hFn) to cultured pulmonary endothelial monolayers attenuates the increase in protein permeability of such monolayers exposed to tumor necrosis factor-alpha (TNF-alpha). In the current study, we determined the specificity of this permeability response to fibronectin by comparing hFn to two other purified adhesive proteins in human plasma, i.e., vitronectin (Vn) and fibrinogen (Fg). We also determined whether matrix incorporation was essential for this hFn-mediated protective response by comparing normal intact hFn to either hFn alkylated with N-ethylmaleimide (NEM) or to purified 160/180-kDa hFn fragments, since these alternate forms of fibronectin are believed to exhibit limited ability to incorporate into matrix. Calf pulmonary artery endothelial (CPAE) monolayers (3-4 days postseeding) were exposed to human recombinant TNF-alpha for 18 h at a medium concentration of 200 U/ml followed by assessment of protein permeability using transendothelial 125I-labeled albumin clearance. Dimeric hFn (600 micrograms/ml) significantly (P < 0.05) reduced the TNF-induced increase in endothelial monolayer permeability. Vn or Fg, added at equal molar concentrations to the hFn, were unable to attenuate endothelial permeability. Immunofluorescent analysis utilizing antibodies specific to either hFn, human Vn, or human Fg revealed incorporation of the exogenous hFn into the extracellular matrix, but no matrix incorporation of Vn or Fg. Both NEM-treated dimeric hFn as well as purified 160/180-kDa fragments of hFn, which cannot incorporate into the matrix, were also unable to prevent the TNF-induced increase in protein permeability. Thus the ability for soluble hFn to reduce the TNF-induced increase in lung endothelial monolayer permeability was specific and dependent on its incorporation into the extracellular matrix.

Immunofluorescent analysis of plasma fibronectin incorporation into the lung during acute inflammatory vascular injury.

Incorporation of plasma fibronectin into tissues is believed to influence endothelial cell-cell interaction, as well as endothelial cell adhesion to matrix. We used immunofluorescent microscopy coupled with tissue extraction of noncovalently incorporated fibronectin to delineate the time course for matrix incorporation of soluble plasma-derived fibronectin into the lung of sheep during postoperative bacteremia. Adult sheep were surgically prepared with both lung and peripheral lymph fistulas. Sheep were anesthetized 2 days following surgery and injected intravenously with a sublethal dose of live Pseudomonas aeruginosa, which consisted of 5 x 10(8) live organisms suspended in 0.9% saline. Bacterial infusion elicited a 300% increase in lung transvascular protein clearance but no increase in peripheral transvascular protein clearance. Purified dimeric human plasma fibronectin (hFn), used as an "immunologic marker," was then infused intravenously (100 mg/sheep) into two additional groups of sheep (nonbacteremic control group and bacteremic experimental group) and allowed to mix with the plasma pool of endogenous soluble sheep fibronectin (sFn). Incorporation of the plasma-derived hFn into the lung matrix and its distribution in relation to endogenous sheep fibronectin in the matrix was assessed by dual-label immunofluorescence using antibodies specific to either sFn or hFn. Human fibronectin from the vascular compartment codistributed with endogenous sheep fibronectin in the lung matrix. Moreover, its deposition into the lung was markedly increased in postoperative bacteremic sheep compared with nonbacteremic control sheep. Increased hFn deposition in the lung with bacteremia was clearly apparent within 2 h. The hFn deposited in the lung was nonextractable using a heparin-urea tissue extraction buffer, suggesting its rapid covalent cross-linking and incorporation into the lung matrix. Microscopic analysis of serial lung biopsies revealed focal areas of inflammation with an intense mononuclear infiltrate into the lungs by 2 h in the bacteremic sheep. Interstitial edema and vascular endothelial injury were observed by 4 h, with alveolar edema apparent over 6 to 8 h. Thus, postoperative bacteremia results in a rapid incorporation of plasma fibronectin into the lung matrix. This may be a physiologic mechanisms to stabilize the integrity of the lung vascular barrier.

Hepatic removal of 125I-DLT gelatin after burn injury: a model of soluble collagenous debris that interacts with plasma fibronectin.

The decline of plasma fibronectin after surgery, trauma, and burn, as well as during severe sepsis after injury, appears to limit hepatic Kupffer cell phagocytic activity. Intravenous infusion of gelatin-coated particles to simulate blood-borne particulate collagenous tissue debris in the circulation after injury also depletes plasma fibronectin. We used soluble gelatin conjugated with 125I-labeled dilactitol tyramine (DLT-gelatin) as a model of soluble collagenous tissue debris. We studied its blood clearance as well as organ localization in normal and postburn rats. Fibronectin-deficient plasma harvested early after burn exhibited limited ability to support in vitro phagocytic uptake of the gelatinized microparticles by Kupffer cells in liver tissue from normal rats. However, Kupffer cells in liver tissue from normal and postburn rats phagocytized the test particles at a normal rate when incubated in normal plasma. The DLT-gelatin ligand bound to fibronectin in a dose-dependent manner as verified by its capture with anti-fibronectin coated plastic wells when coincubated with purified fibronectin. By gel filtration chromatography, the binding of fibronectin with the DLT-gelatin ligand was readily detected, resulting in the formation of a high-molecular-weight complex. In normal animals the plasma clearance and liver localization of 125I-DLT-gelatin was competitively inhibited by infusion of excess nonradioactive gelatin. The blood clearance and liver localization of the soluble gelatin ligand were also impaired after burn injury during periods of fibronectin deficiency similarly to the pattern observed with gelatin-coated microparticles. By autoradiography, the cellular site for the uptake of the 125I-DLT-gelatin was primarily but not exclusively hepatic Kupffer cells; 125I-DLT-asialofetuin and 125I-DLT-ovalbumin were removed by hepatocytes and sinusoidal endothelial cells, respectively. Thus, gelatin conjugated with 125I-DLT can be used to simulate blood-borne soluble collagenous tissue debris after burn. It rapidly binds to plasma fibronectin before its hepatic Kupffer cell removal, and its blood clearance is markedly delayed after burn injury during periods of plasma fibronectin deficiency.

ED1-containing cellular fibronectin release into lung lymph during lung vascular injury with postoperative bacteremia.

Fibronectin (Fn) exists in both a soluble and insoluble form. Soluble Fn in plasma and lymph is an opsonic molecule that enhances phagocytic host defense. Insoluble Fn in the subendothelial and extracellular matrix is an adhesive molecule that mediates cell adhesion to substratum. The extracellular matrix of tissues such as the lung contains a mixture of both plasma-derived fibronectin (pFn) as well as locally synthesized cellular fibronectin (cFn). cFn is antigenically related to pFn, but cFn has extra domains (ED1 and ED2) that do not exist in liver synthesized pFn. The purpose of this study was to determine whether ED1-Fn was released into lung lymph before an increase in lung vascular permeability following postoperative bacteremia. Male sheep (n = 8) with surgically prepared lung lymph fistulae were infused intravenously with a sublethal dose (5 x 10(8)) of Pseudomonas aeruginosa 2 days following surgery. Lymph flow (QL), lymph-to-plasma (L/P) total protein ratio, lung protein clearance (QL x L/P), and hemodynamics were measured over 48 h following bacterial challenge. The lymph and plasma ED1-Fn concentrations were determined by enzyme-linked immunosorbent assay (ELISA) using a murine monoclonal antibody specific to the ED1 region of human cFn. There was a rapid rise of ED1-Fn flux in lung lymph which was evident 60 min after the start of bacterial infusion, resulting in a maximum three- to fourfold increase (P < 0.05) in this parameter. In contrast, the ED1-Fn concentration in plasma before bacterial infusion was less than lung lymph and it did not increase over the initial 6 h following bacterial infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of fibronectin on permeability of normal and TNF-treated lung endothelial cell monolayers.

Fibronectin is found in a soluble form in plasma and lymph and in an insoluble form in the extracellular matrix. Plasma fibronectin can incorporate into the tissue pool of fibronectin where its adhesive properties may influence cell-cell interaction, cell adhesion to a collagenous matrix, and vascular integrity. Elevation of plasma fibronectin can attenuate the increase in lung vascular permeability in sheep during postoperative gram-negative bacteremia, and plasma fibronectin deficiency can magnify the increase in lung vascular permeability with postoperative sepsis. Using pulmonary endothelial monolayers, we determined if exogenous human plasma fibronectin (pFn) would influence the protein permeability of pulmonary endothelial monolayers as determined by transendothelial clearance (microliters/min) of 125I-albumin after they were exposed to human recombinant tumor necrosis factor-alpha. Treatment of endothelial monolayers with tumor necrosis factor (TNF) (200 U/ml) for 18 h resulted in a significant (P < 0.05) increase in protein permeability. Addition of intact purified human plasma fibronectin to normal confluent endothelial monolayers to yield a medium concentration of 300, 600, and 900 micrograms/ml for 18 h had no effect on baseline protein permeability. In contrast, whereas addition of lower amounts of human plasma fibronectin (300 micrograms/ml) did not attenuate the TNF-induced increase in monolayer permeability, the higher concentrations of 600 or 900 micrograms pFn/ml significantly decreased (P < 0.05) protein permeability. The ability of soluble plasma fibronectin to attenuate the TNF-induced increase in endothelial protein permeability required an incubation time of at least 2-3 h, perhaps due to a lag time required for its incorporation into the extracellular matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous measurement of fluid and protein permeability in isolated rabbit lungs during edema.

Fluid conductance and protein permeability have been studied in isolated perfused lung models of pulmonary edema. However, previous studies have not investigated changes of both fluid conductance and protein permeability in the same isolated lung preparation after injury. Arachidonic acid (AA) metabolites are involved in the inflammatory processes that lead to the development of pulmonary edema. The hemodynamic effects of AA have been well established; however, controversy exists concerning the ability of AA to alter the permeability of the pulmonary microvasculature to fluid and protein. The purpose of this study was to simultaneously determine whether transvascular fluid conductance and protein permeability are increased in isolated perfused rabbit lungs with pulmonary edema induced by AA. Indomethacin (80 microM) was added to the perfusate to inhibit the hemodynamic effects of AA and produce a pressure-independent model of pulmonary edema. Fluid conductance was assessed by determination of the capillary filtration coefficient (Kf), and protein permeability was evaluated by measurement of 125I-albumin clearance. The injection of AA (3 mg/200 ml of perfusate) into the pulmonary arterial catheter resulted in an increase in lung weight over the remaining 30-min experimental period. Kf (microliter.s-1 x cmH2O-1 x g dry lung-1) was increased (P < 0.05) in AA-treated lungs at 10 and 30 min post-AA injection when compared with control lungs and baseline values (determined 10 min before AA injection). Albumin clearance was also greater (P < 0.05) in lungs that received AA. 125I-albumin clearance was measured at different rates of fluid flux produced by elevation of venous pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Rebound elevation of fibronectin after tissue injury and ischemia: role of fibronectin synthesis.

Plasma fibronectin (pFn) stimulates macrophage phagocytosis of tissue debris; pFn deposition in tissues may influence vascular integrity. Although the acute depletion of pFn after surgery and/or injury has been described, less attention has been given to the rebound hyperfibronectinemia presumably "triggered" by the early pFn depletion. Using a model that compartmentalized the site of tissue injury and thus attenuated the initial pFn depletion, we studied this rebound elevation of pFn in anesthetized rats (250-350 g) after the surgical trauma of groin dissection alone (sham group) or surgery coupled with 4 h of hindlimb ischemia (experimental group). Nonoperated control rats were also anesthetized. Shams had baseline (preoperative) 6-, 8-, and 22-h postoperative pFn levels of 573 +/- 61, 598 +/- 62, 695 +/- 57, and 929 +/- 87 micrograms/ml, respectively. In the surgery-ischemia group, pFn also elevated to 1,117 +/- 40 micrograms/ml at 22 h postsurgery. Nonoperated control rats (only anesthetized) had no elevation of pFn. Intravenous infusion of gelatin-coated lipid particles (50 mg/100 g) depleted pFn by 89.3% but was unable to prevent the rebound elevation of pFn. The blood clearance of 125I-labeled pFn was very similar in control, sham, and experimental rats. In contrast, pFn synthesis over the 22-h period was dramatically altered and equal to 2.12 +/- 0.16, 3.40 +/- 0.56, and 4.49 +/- 0.17 mg pFn synthesized/100 g body wt, in control, sham, and experimental rats respectively. Thus a rapid increase in pFn synthesis contributes to the rebound hyperfibronectinemia after sublethal surgical injury.(ABSTRACT TRUNCATED AT 250 WORDS)