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

1993 ◽  
Vol 265 (2) ◽  
pp. L148-L157 ◽  
Author(s):  
E. M. Wheatley ◽  
P. J. McKeown-Longo ◽  
P. A. Vincent ◽  
T. M. Saba
Keyword(s):  
Extracellular Matrix ◽  
Human Plasma ◽  
Tnf Alpha ◽  
Plasma Fibronectin ◽  
Endothelial Monolayer ◽  
Necrosis Factor Alpha ◽  
The Matrix ◽  
Monolayer Permeability ◽  
Adhesive Proteins ◽  
Protein 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.

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

1993 ◽  
Vol 264 (1) ◽  
pp. R90-R96 ◽  
Author(s):  
E. M. Wheatley ◽  
P. A. Vincent ◽  
P. J. McKeown-Longo ◽  
T. M. Saba
Keyword(s):  
Extracellular Matrix ◽  
Tumor Necrosis Factor ◽  
Vascular Permeability ◽  
Human Plasma ◽  
Tumor Necrosis ◽  
Plasma Fibronectin ◽  
Endothelial Monolayers ◽  
Lung Vascular Permeability ◽  
Protein Permeability ◽  
Necrosis Factor

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)

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

1995 ◽  
Vol 269 (2) ◽  
pp. L248-L260 ◽  
Author(s):  
T. M. Curtis ◽  
P. J. McKeown-Longo ◽  
P. A. Vincent ◽  
S. M. Homan ◽  
E. M. Wheatley ◽  
...  
Keyword(s):  
Tissue Injury ◽  
Endothelial Permeability ◽  
Rgd Peptide ◽  
Tnf Alpha ◽  
Rabbit Serum ◽  
Endothelial Monolayer ◽  
Necrosis Factor Alpha ◽  
Rgd Peptides ◽  
Subendothelial Matrix ◽  
Protein Permeability

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.

A 96-kDa gelatinase induced by TNF-alpha contributes to increased microvascular endothelial permeability

1993 ◽  
Vol 265 (5) ◽  
pp. L438-L447 ◽  
Author(s):  
C. A. Partridge ◽  
J. J. Jeffrey ◽  
A. B. Malik
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Permeability ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Tnf Alpha ◽  
Vascular Endothelial ◽  
Type Iv Collagenase ◽  
Necrosis Factor Alpha ◽  
Extracellular Matrix Components ◽  
Monolayer Permeability ◽  
Microvascular Endothelial

Tumor necrosis factor-alpha (TNF-alpha) may increase vascular endothelial permeability through alteration of the extracellular matrix (ECM). Incubation of bovine pulmonary microvascular endothelial (BPMVE) cells grown to confluence on microporous filters with 10(4) U/ml TNF-alpha for 24 h increased monolayer permeability to 125I-labeled albumin two- to threefold. TNF-alpha treatment also induced expression of a 96-kDa gelatinolytic metalloproteinase that was present in the medium and bound to the ECM. The induced 96-kDa metalloproteinase was purified from conditioned medium and found to cleave fibronectin, laminin, types IV and V collagens, and gelatins from types I and III collagens, suggesting identity as a type IV collagenase-gelatinase. Incubation of BPMVE cells with the 96-kDa gelatinase increased monolayer permeability, an effect prevented by inclusion of either tissue inhibitor of metalloproteinase (TIMP) or 1,10-phenanthroline. When BPMVE cells were incubated with the 96-kDa gelatinase or 10(4) U/ml TNF-alpha and then stripped from the filters, the remaining ECM displayed increased permeability to 125I-albumin compared with matrix from untreated BPMVE. The ECM extracts from both TNF-alpha- and enzyme-treated cells were found to contain less fibronectin, whereas their total protein contents were similar to those of untreated controls. These results suggest that the 96-kDa metalloproteinase induced by TNF-alpha contributes to increased vascular endothelial permeability through the degradation of specific extracellular matrix components.

scholarly journals Human plasma fibronectin as a substrate for human urokinase

1989 ◽  
Vol 262 (2) ◽  
pp. 529-534 ◽  
Author(s):  
L I Gold ◽  
R Schwimmer ◽  
J P Quigley
Keyword(s):  
Extracellular Matrix ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Early Event ◽  
Plasma Fibronectin ◽  
Proteolytic Digestion ◽  
Enzyme Substrate ◽  
Tumour Cell Invasion ◽  
Substrate Ratio ◽  
Dose Dependent

An early event in malignant transformation is the increased expression of proteases, such as plasminogen activator, which can degrade surrounding extracellular matrices, thereby conferring an advantage for tumour cell invasion and metastasis. The present studies provide evidence that plasma fibronectin (Fn), which is a component of the extracellular matrix, is a direct substrate for the plasminogen activator urokinase (UK). Human plasma Fn was incubated with human UK under plasminogen-free conditions. Fn cleavage was both time- and dose-dependent and was evident within 30 min. The proteolytic digestion was limited and complete within 12 h at an enzyme/substrate ratio of 1:20. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native dimeric 440 kDa structure of Fn with the concomitant appearance of three proteolytic fragments of 210, 200 and 25 kDa. Since two large fragments of similar size to the 220 kDa monomeric chains of Fn were obtained following proteolysis, it is proposed that UK cleaves Fn at two sites, one towards the N-terminal and one close to the C-terminal, but N-terminal to its interchain disulphide bonds. These studies suggest that the local proteolytic digestion and release of Fn from the extracellular matrix by tumour cells possessing high levels of UK may involve the direct proteolytic breakdown of Fn by UK.

Hypoxia and reoxygenation stimulate biphasic changes in endothelial monolayer permeability

1995 ◽  
Vol 269 (1) ◽  
pp. L52-L58 ◽  
Author(s):  
C. A. Partridge
Keyword(s):  
Extracellular Matrix ◽  
Type I Collagen ◽  
Stress Fibers ◽  
Type I ◽  
Endothelial Monolayer ◽  
Hypoxic Conditions ◽  
Endothelial Monolayers ◽  
Monolayer Permeability ◽  
Hypoxic Incubation ◽  
Intercellular Gaps

Incubation of bovine pulmonary microvascular endothelial (BPMVE) cells in low O2 content (95% N2-5% CO2) for 4 h increased monolayer permeability to dextran almost twofold and also increased the incidence of intercellular gaps and intracellular actin stress fibers. Hypoxic incubation decreased the extracellular matrix contents of fibronectin and vitronectin, proteins that serve as anchorage points for the endothelial cells. This state was reversed after 24 h of hypoxic incubation, and the BPMVE monolayer permeability to dextran was less than that of normoxic controls. The monolayer had fewer intercellular gaps and stress fibers, and the extracellular matrix contained increased amounts of fibronectin, vitronectin, and type I collagen. These alterations stimulated by 24 h of hypoxic incubation were resolved within 4 h of reoxygenation in room air supplemented with 5% CO2. These studies indicate that incubation of endothelial monolayers in hypoxic conditions first increases and then decreases monolayer permeability, through increased and decreased formation of intercellular gaps.

Influence of extracellular matrix in tumor necrosis factor-induced increase in endothelial permeability

1992 ◽  
Vol 263 (6) ◽  
pp. L627-L633 ◽  
Author(s):  
C. A. Partridge ◽  
C. J. Horvath ◽  
P. J. Del Vecchio ◽  
P. G. Phillips ◽  
A. B. Malik
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cell ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Endothelial Permeability ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
Necrosis Factor ◽  
Intercellular Gaps

We examined the possibility that alterations of the extracellular matrix (ECM) contribute to the tumor necrosis factor-alpha (TNF-alpha)-induced increase in endothelial monolayer permeability. Endothelial permeability to 125I-labeled albumin was determined using bovine pulmonary microvessel endothelial cell (BPMVE) monolayers grown to confluence on microporous (0.8 microns diam) gelatin- and fibronectin-coated polycarbonate filters. Treatment of BPMVE with TNF-alpha (10(2) to 10(4) U/ml for 4–24 h) produced concentration- and time-dependent increases in endothelial permeability that paralleled the changes in morphology from cobblestone to elongated cells and the formation of prominent intercellular gaps and actin stress fibers. We examined the role of ECM in these changes using filters coated with ECM made by the BPMVE. Fresh BPMVE seeded onto filters coated with ECM produced by TNF-alpha-treated BPMVE had two- to threefold higher 125I-albumin permeability values than BPMVE monolayers seeded onto filters coated with ECM from control cells (P < 0.05). BPMVE seeded onto ECM from TNF-alpha-treated BPMVE also developed intercellular gaps and centralized actin filaments characteristic of the TNF-alpha-treated BPMVE. This effect was not attributable to TNF-alpha adsorbed to ECM. Polyacrylamide gel electrophoresis of ECM extracted from BPMVE treated with TNF-alpha showed decreased fibronectin. These findings suggest that the TNF-alpha-induced increase in endothelial permeability involves the loss of fibronectin and remodeling of the ECM. The increase in endothelial permeability may be secondary to decreased endothelial cell-ECM contacts resulting in elongation of cells and formation of intercellular gaps.

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

1998 ◽  
Vol 275 (1) ◽  
pp. L126-L138 ◽  
Author(s):  
Theresa M. Curtis ◽  
Robert F. Rotundo ◽  
Peter A. Vincent ◽  
Paula J. McKeown-Longo ◽  
Thomas M. Saba
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Permeability ◽  
Fragmentation Pattern ◽  
Endothelial Monolayers ◽  
Tnf Α ◽  
The Matrix ◽  
Pulmonary Arterial ◽  
Permeability Increase ◽  
Endothelial Integrity ◽  
Protein Permeability

Exposure of confluent pulmonary arterial endothelial monolayers to tumor necrosis factor (TNF)-α 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-α have not been defined. Because TNF-α 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-α (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 α5β1-Fn integrins with the Fn fibers in focal contacts was also observed. TNF-α treatment of endothelial monolayers with matrices prelabeled with125I-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-α exposure as confirmed by Western immunoblot. Fn-substrate zymography revealed that TNF-α 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-α-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-α appear not to be due to proteolytic degradation of Fn within the extracellular matrix. In contrast, decreased α5β1-Fn integrin interaction with Fn fibers in the matrix may be important in the response to TNF-α exposure.

scholarly journals Ultrastructural immunogold localization of tumor necrosis factor-alpha to the matrix compartment of eosinophil secondary granules in patients with idiopathic hypereosinophilic syndrome.

1993 ◽  
Vol 41 (11) ◽  
pp. 1611-1615 ◽  
Author(s):  
W J Beil ◽  
P F Weller ◽  
D M Tzizik ◽  
S J Galli ◽  
A M Dvorak
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis Factor Alpha ◽  
Tumor Necrosis ◽  
Hypereosinophilic Syndrome ◽  
Tnf Alpha ◽  
Necrosis Factor Alpha ◽  
The Matrix ◽  
Matrix Compartment ◽  
Factor Alpha ◽  
Necrosis Factor

Peripheral blood eosinophils from two normal donors and two patients with the hypereosinophilic syndrome (HES) were analyzed with a post-embedding immunogold method to detect the substructural location of tumor necrosis factor-alpha (TNF-alpha). In eosinophils of HES patients, TNF-alpha was localized to the matrix compartment of 64% of the specific secondary granules. Other structures in the HES eosinophils were unlabeled. No TNF-alpha was detected in eosinophils of normal donors. These studies document the first ultrastructural subcellular localization of any cytokine within the major population of secretory granules in human eosinophils and support other lines of evidence indicating that the expression of TNF-alpha may be greater in the eosinophils of HES patients than in those of normal donors.

scholarly journals Synthesis of fibronectin by cultured human endothelial cells.

1978 ◽  
Vol 147 (6) ◽  
pp. 1779-1791 ◽  
Author(s):  
E A Jaffe ◽  
D F Mosher
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Human Plasma ◽  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Basement Membranes ◽  
Plasma Fibronectin ◽  
Human Endothelial Cells ◽  
Cultured Endothelial Cells

Plasma fibronectin is probably the major nonimmune particulate opsonin in blood and is cross-linked to fibrin during the final stage of blood coagulation. Fibronectin also occurs in an insoluble form in basement membranes especially those underlying endothelial cells and in loose connective tissue. Fibronectin was demonstrated in cultured human endothelial cells and in the surrounding extracellular matrix by immunofluorescence microscopy by using antibody to human plasma fibronectin. Cultured human endothelial cells released fibronectin into the culture medium which was immunologically identical to the fibronectin in human plasma. Cultured human endothelial cells were labeled with [3H] leucine. The radioactive fibronectin present in the endothelial postculture medium and in urea extracts of cellular monolayers was isolated with either anti-fibronectin coupled to Protein A-Sepharose or double antibody immunoprecipitation and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When reduced, the [3H] fibronectin synthesized by cultured endothelial cells had the same mol wt (approximately 200,000) as plasma fibronectin. Unreduced, the [3H] fibronectin synthesized by endothelial cells migrated as a dimer, as did plasma fibronectin. Fibronectin accounted for approximately 15% of the protein synthesized and released by endothelial cells into the culture medium. Thus, cultured endothelial cells synthesize fibronectin, secrete it into the culture medium, and incorporate it into extracellular matrix. The results suggest that the endothelial cell is potentially a major site of synthesis of circulating plasma fibronectin. In addition, fibronectin derived from endothelial cells may be an important structural component of the subendothelium.

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