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. 2019 May 16;26(1):37.
doi: 10.1186/s12929-019-0529-6.

Fibronectin inhibitor pUR4 attenuates tumor necrosis factor α-induced endothelial hyperpermeability by modulating β1 integrin activation

Ting-Hein Lee  1   2   3 , Sung-Tsang Hsieh  4   5   6 , Hou-Yu Chiang  7   8   9
Affiliations

Fibronectin inhibitor pUR4 attenuates tumor necrosis factor α-induced endothelial hyperpermeability by modulating β1 integrin activation

Ting-Hein Lee et al. J Biomed Sci. .

Abstract

Background: The blood-spinal cord barrier (BSCB) is composed of a monolayer of endothelium linked with tight junctions and extracellular matrix (ECM)-rich basement membranes and is surrounded by astrocyte foot processes. Endothelial permeability is regulated by interaction between endothelial cells and ECM proteins. Fibronectin (FN) is a principal ECM component of microvessels. Excessive FN deposition disrupts cell-cell adhesion in fibroblasts through β1 integrin ligation. To determine whether excessive FN deposition contributes to the disruption of endothelial integrity, we used an in vitro model of the endothelial monolayer to investigate whether the FN inhibitor pUR4 prevents FN deposition into the subendothelial matrix and attenuates endothelial leakage.

Methods: To correlate the effects of excessive FN accumulation in microvessels on BSCB disruption, spinal nerve ligation-which induces BSCB leakage-was applied, and FN expression in the spinal cord was evaluated through immunohistochemistry and immunoblotting. To elucidate the effects by which pUR4 modulates endothelial permeability, brain-derived endothelial (bEND.3) cells treated with tumor necrosis factor (TNF)-α were used to mimic a leaky BSCB. A bEND.3 monolayer was preincubated with pUR4 before TNF-α treatment. The transendothelial electrical resistance (TEER) measurement and transendothelial permeability assay were applied to assess the endothelial integrity of the bEND.3 monolayer. Immunofluorescence analysis and immunoblotting were performed to evaluate the inhibitory effects of pUR4 on TNF-α-induced FN deposition. To determine the mechanisms underlying pUR4-mediated endothelial permeability, cell morphology, stress fiber formation, myosin light chain (MLC) phosphorylation, and β1 integrin-mediated signaling were evaluated through immunofluorescence analysis and immunoblotting.

Results: Excessive FN was accumulated in the microvessels of the spinal cord after spinal nerve ligation; moreover, pUR4 inhibited TNF-α-induced FN deposition in the bEND.3 monolayer and maintained intact TEER and endothelial permeability. Furthermore, pUR4 reduced cell morphology alteration, actin stress fiber formation, and MLC phosphorylation, thereby attenuating paracellular gap formation. Moreover, pUR4 reduced β1 integrin activation and downstream signaling.

Conclusions: pUR4 reduces TNF-α-induced β1 integrin activation by depleting ECM FN, leading to a decrease in endothelial hyperpermeability and maintenance of monolayer integrity. These findings suggest therapeutic benefits of pUR4 in pathological vascular leakage treatment.

Keywords: Endothelial permeability; Fibronectin; Stress fiber; β1 integrin.

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Figures

Fig. 1
Fig. 1
Spinal nerve ligation induces excessive FN deposition around microvessels in the spinal cord. The L5 spinal nerve distal to the dorsal root ganglion of rats was tightly ligated. a Representative photomicrograph depicting immunofluorescence analysis for FN on the operated and contralateral sides of the L5 segment of the spinal cord 7 days after surgery. b Regional magnification of the boxed area in (a); FN+ microvessel-like profiles are indicated by arrows. c Percentages of the FN+ area in the total area on the operated and contralateral sides of the L5 segment of the spinal cord were analyzed (n = 3). Data are presented as means ± standard errors of means. **P < 0.01, Student’s t test. d and e Representative images at low (d) and high (e) magnification showing immunocytochemistry of FN in the L5 dorsal part of the spinal cord. Arrows indicate FN+-microvessel-like profiles on the operated side of the spinal cord. f Immunoblotting for FN expression in the pooled L5 dorsal spinal cord on the operated and contralateral sides in five male Sprague Dawley rats. Equal protein loading was confirmed with α-tubulin. Quantification of immunoblotting of FN normalized to α-tubulin in tissues is shown. g–i Confocal microscopic images of FN+-microvessel-like profiles (red; g) and collagen IV+ capillaries (green; h) in the L5 dorsal part of the spinal cord; merged images (i) showing the colocalization of FN and collagen IV (yellow) in the capillaries are indicated with arrowheads
Fig. 2
Fig. 2
TNF-α-induced FN deposition in bEND.3 cells is inhibited by pUR4. After being incubated with 1000 nM scrambled peptide, 1000 nM pUR4, or PBS for 16 h, bEND.3 cells underwent treatment with TNF-α (20 ng/mL) or PBS for 24 h. a–f Immunofluorescence analysis for FN was performed on nonpermeabilized cells to evaluate FN fibrils accumulated outside bEND.3 cells. g–h To assess the extracellular matrix FN deposited by bEND.3 cells after the various treatments, cellular components were extracted using the lysis buffer, as described in the Methods section, and the assembled ECM FN by bEND.3 cells (g) and FN in the corresponding conditioned medium (h) were immunoblotted. Quantification of protein band intensity was determined (n = 3). Data are presented as means ± standard deviations. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, two-way ANOVA followed by Tukey’s multiple comparison test and Sidak’s multiple comparison test
Fig. 3
Fig. 3
TNF-α-induced monolayer hyperpermeability was prevented by pUR4. After being grown to confluence on Transwell inserts, bEND.3 cells were preincubated with 1000 nM scrambled peptide, 1000 nM pUR4, or PBS for 16 h and then underwent treatment with TNF-α (20 ng/mL) or PBS control for 24 h. a TEER assay was performed 24 h after TNF-α or PBS treatment to determine bEND.3 monolayer integrity (n = 3 for each experimental group). Data are presented as means ± standard deviations. **P < 0.01 and ****P < 0.0001, two-way ANOVA followed by Tukey’s multiple comparison test and Sidak’s multiple comparison test. b and c 40-kDa FITC–dextran (final concentration = 1 mg/mL; b) and sodium fluorescein (final concentration = 10 ng/mL; c) were loaded into the upper chamber for 1 h after 24-h treatment with TNF-α or PBS control, and the FI of the medium in the lower chamber was measured. Permeability was determined through FI60min/FI0min and normalized to the PBS control (n = 3). Data are presented as means ± standard deviations. **P < 0.01, ***P < 0.001, and ****P < 0.0001, two-way ANOVA followed by Tukey’s multiple comparison test and Sidak’s multiple comparison test
Fig. 4
Fig. 4
Tight junction protein expression is not altered by pUR4. After being pretreated with 1000 nM scrambled peptide or 1000 nM pUR4 for 16 h, bEND.3 cells were stimulated with 20 ng/mL TNF-α for the indicated durations. The protein expression of ZO-1 (a), claudin-5 (c), and occludin (e) in bEND.3 cells 24 h after TNF-α treatment was evaluated through immunoblotting; the quantitative analysis results for of ZO-1, claudin-5, and occludin are normalized to GAPDH or α-tubulin (n = 3). ZO-1 (b), claudin-5 (d), and occludin (f) transcript expression in bEND.3 cells treated with TNF-α for 16 and 36 h was evaluated using quantitative real-time PCR (n = 3). Data are presented as means ± standard deviations. **P < 0.01 and ****P < 0.0001, one-way ANOVA followed by Tukey’s multiple comparison test
Fig. 5
Fig. 5
TNF-α-induced alteration of endothelial morphology is prevented by pUR4. After being preincubated with 1000 nM scrambled peptide or 1000 nM pUR4 for 16 h, bEND.3 cells were treated with TNF-α for 24 h. PBS-treated control and TNF-α-treated bEND.3 cells were immunostained with anti-ZO-1 antibody to depict the cell boundaries. Three independent experiments were performed, and each experiment was repeated with similar results. Representative photomicrographs at low (a)–(c) and high (d)–(f) magnification show the endothelial cell shapes. Paracellular gaps are marked by arrows. Cell width:length ratio (width/length ratio) (g) and quantitative analysis of the cell area delineated by ZO-1 (h) derived from a single representative experiment are shown (n = 7–9). Data are expressed as means ± standard deviations. **P < 0.01 and ***P < 0.001, one-way ANOVA followed by Tukey’s multiple comparison test
Fig. 6
Fig. 6
TNF-α-induced stress fiber formation and MLC phosphorylation are attenuated by pUR4. After being pretreated with 1000 nM scrambled peptide or 1000 nM pUR4 for 16 h, bEND.3 cells were stimulated with 20 ng/mL TNF-α for 24 and 6 h. a–i Cytoskeletal remodeling in response to TNF-α treatment for 24 h was analyzed through immunofluorescence analysis for ZO-1 (red) and phalloidin staining for F-actin (green) in PBS control and TNF-α-treated bEND.3 cells. Arrows indicate paracellular gaps. j Quantitative analysis of the F-actin+ area in the total area in PBS control and TNF-α-treated bEND.3 cells with scrambled peptide or pUR4 preincubation (n = 3). Data are expressed as means ± standard deviations. *P < 0.05 and **P < 0.01 by one-way ANOVA followed by Tukey’s multiple comparison test. k and l After exposure to TNF-α for 6 h, MLC phosphorylation was examined by immunoblotting with phospho-MLC (T18/S19)-specific antibody (pMLC), and total MLC (MLC) antibody on the same membrane after stripping (k). Quantification of immunoblotting of pMLC normalized to MLC in the bEND.3 cells (l; n = 3). Data are represented as means ± standard deviations. **P < 0.01, one-way ANOVA followed by Tukey’s multiple comparison test
Fig. 7
Fig. 7
TNF-α-induced β1 integrin activation and clustering to focal adhesions (FAs) are diminished by pUR4. After being pretreated with 1000 nM scrambled peptide or 1000 nM pUR4 for 16 h, bEND.3 cells were stimulated with TNF-α (20 ng/mL) for 24 h. PBS-treated control and TNF-α-treated bEND.3 cells were costained with antibodies to the activated state of β1 integrin (red) and FAK (green). Three independent experiments were performed, and each experiment was repeated with similar results. a–c Representative immunofluorescence images showing activated β1 integrin in control and bEND.3 cells treated with the scrambled peptide and pUR4.d–f The merged images show colocalization (yellow) of activated state of β1 integrin (red) and FAK (green). g, h, and i Reginal enlargement of the FAs in the boxed area in (d), (e), and (f), respectively. (j) Quantification of FI of activated β1 integrin in PBS control and bEND.3 cells treated with the scrambled peptide and pUR4 was derived from a representative experiment (n = 3). Data are represented as means ± standard deviations. *P < 0.05, one-way ANOVA followed by Tukey’s multiple comparison test. (k) Line scan graphs showing the immunofluorescence intensity along the freely positioned arrows in bEND.3 cells treated with scrambled peptide (e) and pUR4 (f)
Fig. 8
Fig. 8
TNF-α-induced FAK phosphorylation is decreased by pUR4. After being pretreated with 1000 nM scrambled peptide or 1000 nM pUR4 for 16 h, bEND.3 cells were stimulated with TNF-α (20 ng/mL) for 6 h. FAK phosphorylation at Tyr-397 (a) and Tyr-576 (c) were determined through immunoblotting. Quantitative analysis of phosphorylated FAK at Tyr-397 (b) and Tyr-576 (d) normalized to total FAK (n = 4). Data are represented as means ± standard deviations. *P < 0.05 and **P < 0.01, one-way ANOVA followed by Tukey’s multiple comparison test
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