[Role of caveolin-1 in pulmonary microvascular endothelial cells injury induced by lipopolysaccharide in rat].
OBJECTIVE
To investigate the role of caveolin-1 (Cav-1) in the modulation of rat pulmonary microvascular endothelial cells (RPMVEC) injury induced by lipopolysaccharide (LPS).


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Department of Respiratory Medicine, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, Anhui, China. Corresponding author: Sun Geng-yun, Email: sungengyun@tom.com.
Qing-hai
QH
You
Dan
D
Zhang
Geng-yun
GY
Sun
Yang
Y
Yue
Xiu-juan
XJ
Xu
D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects*; D051242:Caveolin 1 / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D007249:Inflammation; D008070:Lipopolysaccharides / Q000009:adverse effects*; D008297:Male; D010085:Oxidative Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley
C495537:Cav1 protein, rat; D051242:Caveolin 1; D008070:Lipopolysaccharides