title: Lipopolysaccharide-induced caveolin-1 phosphorylation-dependent increase in transcellular permeability precedes the increase in paracellular permeability.
abstract: BACKGROUND
  Lipopolysaccharide (LPS) was shown to induce an increase in caveolin-1 (Cav-1) expression in endothelial cells; however, the mechanisms regarding this response and the consequences on caveolae-mediated transcellular transport have not been completely investigated. This study aims to investigate the role of LPS-induced Cav-1 phosphorylation in pulmonary microvascular permeability in pulmonary microvascular endothelial cells (PMVECs).
  
  
  METHODS
  Rat PMVECs were isolated, cultured, and identified. Endocytosis experiments were employed to stain the nuclei by DAPI, and images were obtained with a fluorescence microscope. Permeability of endothelial cultures was measured to analyze the barrier function of endothelial monolayer. Western blot assay was used to examine the expression of Cav-1, pCav-1, triton-insoluble Cav-1, and triton-soluble Cav-1 protein.
  
  
  RESULTS
  The LPS treatment induced phosphorylation of Cav-1, but did not alter the total Cav-1 level till 60 min in both rat and human PMVECs. LPS treatment also increased the triton-insoluble Cav-1 level, which peaked 15 min after LPS treatment in both rat and human PMVECs. LPS treatment increases the intercellular cell adhesion molecule-1 expression. Src inhibitors, including PP2, PP1, Saracatinib, and Quercetin, partially inhibited LPS-induced phosphorylation of Cav-1. In addition, both PP2 and caveolae disruptor MβCD inhibited LPS-induced increase of triton-insoluble Cav-1. LPS induces permeability by activating interleukin-8 and vascular endothelial growth factor and targeting other adhesion markers, such as ZO-1 and occludin. LPS treatment also significantly increased the endocytosis of albumin, which could be blocked by PP2 or MβCD. Furthermore, LPS treatment for 15 min significantly elevated Evans Blue-labeled BSA transport in advance of a decrease in transendothelial electrical resistance of PMVEC monolayer at this time point. After LPS treatment for 30 min, transendothelial electrical resistance decreased significantly. Moreover, PP2 and MβCD blocked LPS-induced increase in Evans Blue-labeled BSA level.
  
  
  CONCLUSIONS
  Our study demonstrates that LPS-induced Cav-1 phosphorylation may lead to the increase of transcellular permeability prior to the increase of paracellular permeability in a Src-dependent manner. Thus, LPS-induced Cav-1 phosphorylation may be a therapeutic target for the treatment of inflammatory lung disease associated with elevated microvascular permeability.
authors.affiliation: Department of Respiration, The First Affiliated Hospital of Anhui Medical University, Hefei, People's Republic of China ; Department of Emergency, The First Affiliated Hospital of Anhui Medical University, Hefei, People's Republic of China.
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.affiliation: Department of Respiration, The First Affiliated Hospital of Anhui Medical University, Hefei, People's Republic of China ; Department of Emergency, The First Affiliated Hospital of Anhui Medical University, Hefei, People's Republic of China.
authors.forename: Dan
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Department of Respiration, The First Affiliated Hospital of Anhui Medical University, Hefei, People's Republic of China.
authors.forename: Gengyun
authors.initials: G
authors.lastname: Sun
authors.affiliation: Department of Respiration, The First Affiliated Hospital of Anhui Medical University, Hefei, People's Republic of China ; Department of Emergency, The First Affiliated Hospital of Anhui Medical University, Hefei, People's Republic of China.
authors.forename: Hong
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Respiration, The First Affiliated Hospital of Anhui Medical University, Hefei, People's Republic of China.
authors.forename: Qinghai
authors.initials: Q
authors.lastname: You
authors.affiliation: Department of Respiration, The First Affiliated Hospital of Anhui Medical University, Hefei, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Shao
authors.affiliation: Department of Respiration, The First Affiliated Hospital of Anhui Medical University, Hefei, People's Republic of China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yue
mesh_terms: D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects*; D051242:Caveolin 1 / Q000378:metabolism*; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008168:Lung / Q000098:blood supply*; D055806:Microvessels / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
chemical_list: C495540:CAV1 protein, human; C508606:CXCL8 protein, human; C495537:Cav1 protein, rat; D051242:Caveolin 1; C573420:ICAM1 protein, human; D016209:Interleukin-8; D008070:Lipopolysaccharides; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C467498:vascular endothelial growth factor A, rat; D018799:Intercellular Adhesion Molecule-1; D019061:src-Family Kinases
keywords: caveolin-1; paracellular permeability; phosphorylation; pulmonary microvascular permeability; transcellular permeability