Role of caveolin-1 in pulmonary microvascular endothelial cells injury induced by lipopolysaccharide in rat

Abstract

To investigate the role of caveolin-1 (Cav-1) in the modulation of rat pulmonary microvascular endothelial cells (RPMVEC) injury induced by lipopolysaccharide (LPS). Cultured RPMVEC were randomly divided into time-dependent injury group induced by LPS and intervention group in which cells were pretreated by protein kinase A inhibitor (PKI). In the time-dependent injury group, monolayers of cells were constructed to determine permeability changes after 10 μg/mL LPS challenge for 0, 1, 3, 6, 12 and 24 hours with the method of Evans blue-labeled albumin flux across the monolayer (Pd). Western blotting was used to determine the Cav-1 expression after LPS stimulation and the phosphorylation-Cav-1 (p-Cav-1) expression after LPS challenge for 0, 10, 30, 60, 90, 120 minutes. In the intervention group, after pre-treatment with 10 μmol/L PKI for 30 minutes, RPMVECs were challenged with 10 μg/mL LPS, and the expression of p-Cav-1 was determined 30 minutes after LPS challenge, the permeability and the Cav-1 protein expression were assessed by Pd and Western blotting, respectively. Non-stimulation group and single PKI simulation group served as controls. Western blotting revealed that the expression of Cav-1 protein was elevated at 1 hour (2.97 ± 0.07), peaking at 3 hours (3.77 ± 0.37), then it lowered gradually, but it was still higher at 24 hours (1.45 ± 0.18) when compared with 0 hour group (1.12±0.08) with significant differences (F=178.047, P=0.000). After RPMVEC monolayers were challenged by LPS for different periods (0, 1, 3, 6, 12 and 24 hours), there were significant increases in a time-dependent manner in Cav-1 expression in the permeability as measured by Pd [(99.67 ± 4.32)%, (118.17 ± 2.32)%, (159.00 ± 2.61)%, (141.17 ± 2.64)%, (120.17 ± 2.79)% and (108.83 ± 2.04)%, F=345.869, P=0.000] which was similar to the changes in Cav-1 expression. LPS also increased Cav-1 phosphorylation in a time-dependent manner occurring at 10 minutes (2.41 ± 0.11), peaking at 30 minutes (2.83 ± 0.10), and then it decreased gradually, finally returned to basal levels (1.03±0.04) at 120 minutes (1.04 ± 0.04) after LPS treatment with significant difference (F=519.417, P=0.000). When PKI was pre-treated with RPMVEC the expression of Cav-1 was significantly increased (5.07 ± 0.22 vs. 3.81 ± 0.23, P<0.01), and p-Cav-1 (3.93 ± 0.23 vs. 2.77±0.10, P<0.01), and RPMVEC monolayers permeability was enhanced [(184.17 ± 5.49)% vs. (151.50 ± 3.08)%, P<0.01] after being challenged. Up-regulated expression of Cav-1 and phosphorylation-Cav-1 that may be modulated by protein kinase A signal pathway plays an important role in RPMVEC permeability injury as induced by LPS.