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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.

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.

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.

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.