Figure 1.
Characterization of LPS-induced activation of pulmonary EC.
Human pulmonary EC were grown on 2.8(200 ng/ml). A - Time-dependent phosphorylation of MYPT and MLC and expression of ICAM-1, VCAM-1, fibronectin and LOX was determined by western blot analysis. Equal protein loading was confirmed by membrane re-probing with β-actin antibody. B – Analysis of fibronectin and collagen 1A (Col1A) mRNA levels after 12-hr LPS treatment was performed by RT-PCR; C – Analysis of LOX mRNA levels and LOX activity in conditioned medium from control and LPS-stimulated (12 hrs) cells; *p<0.05.
Figure 2.
Effect of LOX inhibition on LPS-induced EC inflammatory activation.
Human pulmonary EC grown on 2.8(300 µM), and then stimulated with LPS (200 ng/ml) for 48 hrs with or without BAPN. A – IL-8 production by EC stimulated with or without BAPN was evaluated in conditioned medium by ELISA assay; *P<0.05. B – Expression of ICAM-1 and VCAM-1 was determined by western blot analysis with specific antibodies. C – ICAM-1 expression was examined by immunofluorescence staining of stimulated EC using ICAM-1 antibody (green). Counterstaining with DAPI (blue) was used to visualize cell nuclei. D – HPAEC were transfected with non-specific (nsRNA) or LOX-specific siRNA (si-LOX). ICAM-1 expression was determined by western blot. Equal protein loading was confirmed by membrane re-probing with β-actin antibody.
Figure 3.
Effect of substrate stiffness and LOX inhibition on LPS-induced EC activation.
A - Pulmonary EC were grown on polyacrylamide gels of different stiffness (1.5 kPa, and 40 kPa) and treated with TNFα (2 ng/ml) or LPS (200 ng/ml) for 6 hrs. Left panel: Expression of ICAM-1 and VCAM-1 was determined by western blot analysis. Equal protein loading was confirmed by membrane re-probing with β-actin antibody. Bar graphs depict the quantitative analysis of western blot densitometry data; *P<0.05 vs. 1.5 kPa; n = 4. Right panel: Analysis of fibronectin mRNA levels after 12-hr treatment with LPS or TNFα was performed by RT-PCR; *p<0.05. B – Pulmonary EC were cultured on 2.8 kPa substrates for 3 days in the presence of LPS (200 ng/ml) with or without BAPN (300 µM). After cell detachment, fresh EC were plated on deposited extracellular matrix and stimulated with TNFα (2 ng/ml, 6 hrs). ICAM-1 expression and MYPT phosphorylation was analyzed by western blot. Equal protein loading was confirmed by membrane re-probing with β-actin antibody. Bar graphs depict the quantitative analysis of western blot densitometry data; *P<0.05 vs. LPS+BAPN treatment; n = 4. C - IL-8 production in response to TNFα (2 ng/ml, 6 hrs) was evaluated by ELISA assay. *P<0.05.
Figure 4.
Effect of LOX inhibition on LPS-induced ICAM-1 expression and IL-8 production in human lung slices.
Human lung slices were cultured during 48(200 ng/ml) with or without BAPN (300 µM). A - ICAM-1 expression was analyzed by western blot. Equal protein loading was confirmed by membrane re-probing with β-actin antibody. Bar graphs depict the quantitative analysis of western blot densitometry data; *P<0.05 vs. LPS; n = 4. B - IL-8 production was evaluated by ELISA assay of conditioned medium. *P<0.05.
Figure 5.
Characterization of LPS-induced expression of GEF-H1.
Human pulmonary EC were on 2.8(200 ng/ml). A - Time-dependent expression of GEF-H1 was determined by western blot analysis. Equal protein loading was confirmed by membrane re-probing with β-actin antibody. B – Time-dependent analysis of LPS-induced GEF-H1 mRNA expression in human lung macrovascular EC (left panel) and microvascular EC (right panel) was performed by RT-PCR; *p<0.05; C – Human pulmonary macrovascular EC (left panel) or microvascular EC (right panel) grown on 2.8 kPa substrate were treated for 24 hrs with vehicle or BAPN (300 µM), and then stimulated with LPS (200 ng/ml) with or without BAPN. After 48 hrs, expression of GEF-H1 was determined by western blot analysis. Bar graphs depict the quantitative analysis of western blot densitometry data; *P<0.05 vs. LPS; n = 3. D – Effect of si-RNA induced LOX knockdown (72 hrs) on LPS-induced GEF-H1 expression. Bar graphs depict the quantitative analysis of western blot densitometry data; *P<0.05 vs. non-specific RNA; n = 3. E – Human lung slices were cultured during 48 hrs in the presence of LPS (200 ng/ml) with or without BAPN (300 µM). GEF-H1 expression was analyzed by Western blot. Equal protein loading was confirmed by membrane re-probing with β-actin antibody. Bar graphs depict the quantitative analysis of western blot densitometry data; *P<0.05 vs. LPS; n = 4.
Figure 6.
Effect of substrate stiffness and LOX inhibition on LPS-induced GEF-H1 expression.
A - Pulmonary EC were grown on polyacrylamide gels of different stiffness (1.5 kPa, and 40 kPa) and treated with LPS (200 ng/ml) for 6 hrs. GEF-H1 expression determined by western blot analysis. Bar graphs depict the quantitative analysis of western blot densitometry data; *P<0.05 vs. 1.5 kPa; n = 4. B – Pulmonary EC were cultured on 2.8 kPa substrates for 3 days in the presence of LPS (200 ng/ml) with or without BAPN (300 µM). After cell detachment, fresh EC were plated on deposited extracellular matrix and stimulated with LPS (200 ng/ml, 6 hrs). GEF-H1 expression was analyzed by western blot. Bar graphs depict the quantitative analysis of western blot densitometry data; *P<0.05 vs. LPS+BAPN treatment; n = 4. C – Pulmonary EC grown on 2.8 kPa substrates and treated with nonspecific or GEF-H1-specific siRNA for 72 hrs were stimulated with TNFα (2 ng/ml) or LPS (200 ng/ml) for 6 hrs, and ICAM-1 expression was analyzed by western blot. GEF-H1 depletion was confirmed by membrane reprobing with GEF-H1 antibody. Equal protein loading was confirmed by membrane re-probing with β-actin antibody. Bar graphs depict the quantitative analysis of western blot densitometry data; *P<0.05 vs. non-specific RNA; n = 3.
Figure 7.
Effect of LOX inhibitor on LPS-induced lung inflammation and barrier dysfunction.
C57BL/6J mice were challenged with vehicle or LPS (0.63 mg/kg, i/t) with or without BAPN treatment (100 mg/kg). Control animals were treated with sterile saline solution. A – Total cell count, PMN cell count and protein concentration were determined in bronchoalveolar lavage fluid collected 48 hrs after treatments. B - Evans blue dye (30 ml/kg, i/v) was injected 2 hr before termination of the experiment. Lung vascular permeability was assessed by Evans blue accumulation in the lung tissue. The quantitative analysis of Evans blue labeled albumin extravasation was performed by spectrophotometric analysis of Evans blue extracted from the lung tissue samples; *p<0.05. C – Histological analysis of lung tissue by hematoxilin & eosin staining (×40 magnification); D - Expression of ICAM-1 and GEF-H1 in lung tissue samples evaluated by western blot analysis. Equal protein loading was confirmed by membrane re-probing with β-actin antibody.
Figure 8.
Enhancement of LPS-induced inflammation and acute lung injury via stiffness-dependent stimulation of GEF-H1.
LPS or other inflammatory interventions activate inflammatory cascades in endothelial cells manifested by cytokine production and expression of leukocyte adhesion molecules. In addition, LPS stimulates expression of ECM proteins and LOX leading to local tissue stiffening and stiffness-induced expression of GEF-H1. GEF-H1 - Rho signaling pathway then provides a positive feedback mechanism leading to escalation of vascular inflammation and acute lung injury.