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.

Mediation of endothelial injury following neutrophil adherence to extracellular matrix

1993 ◽  
Vol 264 (4) ◽  
pp. L401-L405 ◽  
Author(s):  
R. A. Kaslovsky ◽  
L. Lai ◽  
K. Parker ◽  
A. B. Malik
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Endothelial Permeability ◽  
Endothelial Injury ◽  
Matrix Proteins ◽  
Extracellular Matrix Components ◽  
The Matrix ◽  
Neutrophil Adherence ◽  
Permeability Increase

Since polymorphonuclear leukocytes (PMN) rapidly migrate across the endothelial barrier and attach to extracellular matrix components, we tested the hypothesis that adhesion of PMN to matrix proteins can mediate endothelial injury following PMN activation. Studies were made using gelatin- and fibronectin-coated polycarbonate microporous filters (10 microns thick) on which confluent monolayers of bovine pulmonary microvessel endothelial cells were grown. PMN were layered either directly onto endothelial cells (at a ratio of 10:1) (“upright system”) or onto gelatin- and fibronectin-coated filters with the endothelial monolayer grown on the underside of the filter without contact between PMN and endothelial cells (“inverted system”). PMN were activated with phorbol 12-myristate 13-acetate (PMA; 5 x 10(-9) M) in both systems. PMN activation increased endothelial permeability to 125I-labeled albumin in upright as well as inverted systems. Pretreatment of PMN with anti-CD18 monoclonal antibodies IB4 or R15.7, which inhibited PMN adherence to matrix constituents as well as to endothelial cells, prevented the permeability increase in both configurations. This effect of anti-CD18 monoclonal antibodies (mAbs) was not ascribed to a reduction in PMN activation, since PMA-induced superoxide generation was unaffected. We conclude that activation of PMN adherent to extracellular matrix proteins increases endothelial permeability to albumin and that this response is dependent on PMN adhesion to the matrix. The results support the concept that PMN-mediated increase in endothelial permeability is the result of “targeted” release of PMN products independent of whether the PMN are adherent to the extracellular matrix or the endothelium.

TNF-α disruption of lung endothelial integrity: reduced integrin mediated adhesion to fibronectin

2002 ◽  
Vol 282 (2) ◽  
pp. L316-L329 ◽  
Author(s):  
Robert F. Rotundo ◽  
Theresa M. Curtis ◽  
Melissa D. Shah ◽  
Baochong Gao ◽  
Anthony Mastrangelo ◽  
...  
Keyword(s):  
Fluid Phase ◽  
Significant Loss ◽  
Compensatory Response ◽  
Rgd Peptides ◽  
Tnf Α ◽  
Coated Surfaces ◽  
Factor Α ◽  
Endothelial Integrity ◽  
Protein Permeability ◽  
Cpae Cells

Tumor necrosis factor-α (TNF-α) causes an increase in transendothelial protein permeability of confluent monolayers of calf pulmonary artery endothelial (CPAE) cells, and the addition of plasma fibronectin (pFn) to the culture medium can attenuate this increase in permeability. We determined if reduced integrin function had a role in decreased endothelial cell adhesion to immobilized Fn after exposure of the endothelial monolayers to TNF-α. TNF-α also causes a reorganization of the subendothelial Fn rich matrix and a significant loss in RGD-dependent adhesion of TNF-α treated CPAE cells to pFn coated surfaces. However, flow cytometry revealed no decrease in α5β1or total β1integrin expression on the surface of the CPAE cells after TNF-α. Reduced CPAE adhesion to immobilized Fn was, in part, due to a loss of β1-integrin function since the β1-integrin blocking antibody mAb 13 significantly ( P < 0.05) prevented the adhesion of normal control CPAE cells but did not further reduce the adhesion of TNF-α-treated cells. In addition, antibodies which activate β1integrins restored ( P < 0.05) adhesion of TNF-α-treated cells to immobilized pFn but did not alter the adhesion of control cells. Despite reduced ability to adhere to immobilized Fn, TNF-α-treated CPAE monolayers demonstrated increased binding and incorporation of fluid-phase pFn into the subendothelial extracellular matrix (ECM) as measured by the analysis of the deoxycholate (DOC) detergent insoluble pool of125I-Fn in the cell layer. In contrast to the RGD-mediated adhesion of CPAE cells to matrix Fn, the increased binding of soluble pFn after TNF-α was not inhibited by RGD peptides or mAb 13. Thus reduced integrin-dependent adhesion of the CPAE cells to matrix Fn as well as disruption of the Fn matrix may contribute to the increased protein permeability of previously confluent endothelial monolayer after TNF-α. In addition, increased ability for the monolayer to incorporate fluid-phase Fn into the ECM after TNF-α via a non-β1- integrin dependent mechanism may be a compensatory response to stabilize the Fn matrix and the endothelial barrier.

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)

pH and temperature modulate norepinephrine-dependent changes in endothelial permeability

1994 ◽  
Vol 76 (6) ◽  
pp. 2760-2764 ◽  
Author(s):  
M. P. Griffin ◽  
J. R. Moorman
Keyword(s):  
Septic Shock ◽  
High Temperature ◽  
Endothelial Permeability ◽  
Low Ph ◽  
Ph Values ◽  
Endothelial Monolayers ◽  
Pulmonary Arterial

To evaluate the role of pH and temperature in norepinephrine (NE)-mediated decreases in endothelial permeability, we studied bovine pulmonary arterial endothelial monolayers at pH values of 4–9 and at temperatures of 35–39 degrees C. Only extremes of pH-modulated endothelial permeability and temperature had no effect. Although both NE and 3-isobutyl-1-methylxanthine decreased endothelial permeability, their effects were diminished by low pH and high temperature. Fever and acidosis may contribute to the edema seen in septic shock by a novel mechanism: attenuation of the barrier-improving function of NE.

scholarly journals Role for anions in pulmonary endothelial permeability

1997 ◽  
Vol 83 (2) ◽  
pp. 615-622 ◽  
Author(s):  
M. Pamela Griffin
Keyword(s):  
Phosphodiesterase Inhibitor ◽  
Endothelial Permeability ◽  
Anion Transport ◽  
Transport Processes ◽  
Disulfonic Acid ◽  
Channel Blockers ◽  
Adrenergic Stimulation ◽  
Endothelial Monolayers ◽  
Wide Range ◽  
Pulmonary Arterial

Griffin, M. Pamela. Role for anions in pulmonary endothelial permeability. J. Appl. Physiol. 83(2): 615–622, 1997.—β-Adrenergic stimulation reduces albumin permeation across pulmonary artery endothelial monolayers and induces changes in cell morphology that are mediated by Cl− flux. We tested the hypothesis that anion-mediated changes in endothelial cells result in changes in endothelial permeability. We measured permeation of radiolabeled albumin across bovine pulmonary arterial endothelial monolayers when the extracellular anion was Cl−, Br−, I−, F−, acetate (Ac−), gluconate (G−), and propionate (Pr−). Permeability to albumin ( P albumin) was calculated before and after addition of 0.2 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), which reduces permeability. In Cl−, the P albumin was 3.05 ± 0.86 × 10−6 cm/s and fell by 70% with the addition of IBMX. The initial P albumin was lowest for Pr− and Ac−. Initial P albumin was higher in Br−, I−, G−, and F− than in Cl−. A permeability ratio was calculated to examine the IBMX effect. The greatest IBMX effect was seen when Cl− was the extracellular anion, and the order among halide anions was Cl− > Br− > I− > F−. Although the level of extracellular Ca2+ concentration ([Ca2+]o) varied over a wide range in the anion solutions, [Ca2+]odid not systematically affect endothelial permeability in this system. When Cl− was the extracellular anion, varying [Ca2+]ofrom 0.2 to 2.8 mM caused a change in initial P albumin but no change in the IBMX effect. The anion channel blockers 4-acetamido-4′-isothiocyanotostilbene-2,2′-disulfonic acid (0.25 mM) and anthracene-9-carboxylic acid (0.5 mM) significantly altered initial P albumin and the IBMX effect. The anion transport blockers bumetanide (0.2 mM) and furosemide (1 mM) had no such effects. We conclude that extracellular anions influence bovine pulmonary arterial endothelial permeability and that the pharmacological profile fits better with the activity of anion channels than with other anion transport processes.

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.

scholarly journals Bone marrow-derived progenitor cells prevent thrombin-induced increase in lung vascular permeability

2010 ◽  
Vol 298 (1) ◽  
pp. L36-L44 ◽  
Author(s):  
Yidan D. Zhao ◽  
Hiroshi Ohkawara ◽  
Stephen M. Vogel ◽  
Asrar B. Malik ◽  
You-Yang Zhao
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Fixed Ratio ◽  
Endothelial Permeability ◽  
Vascular Endothelial ◽  
Endothelial Monolayers ◽  
Thrombin Activation ◽  
Endothelial Integrity ◽  
Mlc Phosphorylation ◽  
Interfering Rna

Since thrombin activation of endothelial cells (ECs) is well-known to increase endothelial permeability by disassembly of adherens junctions (AJs) and actinomyosin contractility mechanism involving myosin light chain (MLC) phosphorylation, we investigated the effects of bone marrow-derived progenitor cells (BMPCs) on the thrombin-induced endothelial permeability response. We observed that addition of BMPCs to endothelial monolayers at a fixed ratio prevented the thrombin-induced decrease in transendothelial electrical resistance, a measure of AJ integrity, and increased mouse pulmonary microvessel filtration coefficient, a measure of transvascular liquid permeability. The barrier protection was coupled to increased vascular endothelial cadherin expression and increased Cdc42 activity in ECs. Using small interfering RNA (siRNA) to deplete Cdc42 in ECs, we demonstrated a key role of Cdc42 in signaling the BMPC-induced endothelial barrier protection. Endothelial integrity induced by BMPCs was also secondary to inhibition of MLC phosphorylation in ECs. Thus BMPCs interacting with ECs prevent thrombin-induced endothelial hyperpermeability by a mechanism involving AJ barrier annealing, inhibition of MLC phosphorylation, and activation of Cdc42.

scholarly journals Regulation of Endothelial Permeability by Glutathione S-Transferase Pi Against Actin Polymerization

2018 ◽  
Vol 45 (1) ◽  
pp. 406-418 ◽  
Author(s):  
Yang Yang ◽  
Fangyuan Yin ◽  
Qiyun Hang ◽  
Xiaoliang Dong ◽  
Jiao Chen ◽  
...  
Keyword(s):  
Endothelial Permeability ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Transferase Activity ◽  
Glutathione S Transferase ◽  
Actin Remodeling ◽  
Stress Fiber Formation ◽  
Tnf Α ◽  
Permeability Increase ◽  
Cytoskeleton Rearrangement

Background/Aims: Inflammation-induced injury of the endothelial barrier occurs in several pathological conditions, including atherosclerosis, ischemia, and sepsis. Endothelial cytoskeleton rearrangement is an important pathological mechanism by which inflammatory stimulation triggers an increase of vascular endothelial permeability. However, the mechanism maintaining endothelial cell barrier function against inflammatory stress is not fully understood. Glutathione S-transferase pi (GSTpi) exists in various types of cells and protects them against different stresses. In our previous study, GSTpi was found to act as a negative regulator of inflammatory responses. Methods: We used a Transwell permeability assay to test the influence of GSTpi and its transferase activity on the increase of endothelial permeability induced by tumor necrosis factor alpha (TNF-α). TNF-α-induced actin remodeling and the influence of GSTpi were observed by using laser confocal microscopy. Western blotting was used to test the influence of GSTpi on TNF-α-activated p38 mitogen-activated protein kinase (MAPK)/MK2/heat shock protein 27 (HSP27). Results: GSTpi reduced TNF-α-induced stress fiber formation and endothelial permeability increase by restraining actin cytoskeleton rearrangement, and this reduction was unrelated to its transferase activity. We found that GSTpi inhibited p38MAPK phosphorylation by directly binding p38 and influenced downstream substrate HSP27-induced actin remodeling. Conclusion: GSTpi inhibited TNF-α-induced actin remodeling, stress fiber formation and endothelial permeability increase by inhibiting the p38MAPK/HSP27 signaling pathway.

Cadherin-5 redistribution at sites of TNF-α and IFN-γ-induced permeability in mesenteric venules

1999 ◽  
Vol 276 (2) ◽  
pp. H736-H748 ◽  
Author(s):  
Raymond K. Wong ◽  
Ann L. Baldwin ◽  
Ronald L. Heimark
Keyword(s):  
Endothelial Permeability ◽  
Intercellular Junctions ◽  
Interferon Γ ◽  
Permeability Barrier ◽  
Rat Mesentery ◽  
Tnf Α ◽  
Ifn Γ ◽  
Factor Α ◽  
Endothelial Integrity ◽  
Dose Dependent

The response of the endothelial permeability barrier in microvascular networks of the rat mesentery to perfused immune inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was examined. TNF-α (12.5 U/ml) treatment did not change albumin permeability, but in combination with IFN-γ (20 U/ml), there was a marked increase in the number of sites of extravascular albumin in postcapillary venules. Endothelial integrity was characterized by cadherin-5 immunoreactivity, which was localized to the continuous intercellular junctions of endothelium in arterioles, capillaries, and venules. Perfusion with the combined cytokines showed that the increased albumin permeability was dose dependent and correlated with the focal disorganization of cadherin-5 at intercellular junctions of venular endothelium. No correlation was found between the increase in albumin permeability and the localization of intravascular leukocytes or extravascular mast cells. These results show that the combination of TNF-α and IFN-γ induces an endothelial phenotype with focal loss of cadherin-5 intercellular adhesion, which, in part, facilitates passage of blood macromolecules and cells to the interstitium.

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

2000 ◽  
Vol 279 (1) ◽  
pp. L161-L174 ◽  
Author(s):  
Ruihua Chen ◽  
Baochong Gao ◽  
Cancan Huang ◽  
Byron Olsen ◽  
Robert F. Rotundo ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Surface ◽  
Pulmonary Artery ◽  
Transglutaminase Activity ◽  
Endothelial Monolayers ◽  
Reducing Conditions ◽  
Tnf Α ◽  
Cell Layers ◽  
Monolayer Permeability ◽  
Necrosis Factor

Exposure of lung endothelial monolayers to tumor necrosis factor (TNF)-α 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-α (200 U/ml) for 18 h. Western blot analysis indicated that TNF-α decreased the amount of monomeric FN detected under reducing conditions. Analysis of 125I-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-α also formed nonreducible HMW multimers of FN when layered on surfaces precoated with FN. Inhibitors of transglutaminase blocked the TNF-α-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-α 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.

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