Role Of Caveolin-1in Changes Of Rat Pulmonary Microvascular Endothelial Permeability Induced By TNF-α

BackgroundAcute respiratory distress syndrome (ARDS) is responsible for significant morbidityand mortality in intensive care units, despite improvement in ventilation strategies. Thepathogenesis of ARDS is still poorly understood, and therapeutic options remain limited.As we all know that inflammatory mediators play an important role in the mechanism ofARDS, but it is difficult to achieve the desired effect by blocking or closure ofinflammatory mediators in the treatment of ARDS. Recentlly, more and moreresearchers began to study on the caveolin-1in order to find a novel treatment target ofARDS.Caveolin-1(cav-1), a22-kDa transmembrane scaffolding protein, is the principalstructural component of caveolae. Cav-1regulates critical cell functions includingproliferation, apoptosis, cell differentiation, and transcytosis via diverse signalingpathways. Many studies have provided compelling evidence that caveolin-1binds tosignalling molecules and is involved in regulating endothelial permeability. Previousstudies on the role of caveolin-1in endothelial permeability have reported both positiveand negative effects. Thus, caveolin-1might have a more general role in regulating celljunctions, but its molecular regulation of endothelial cell adhesion and barrier functionneeds to be defined.Inflammatory mediators such as TNF-α, thrombin, and LPS, increase vascularpermeability primarily by formation of intercellular gaps between endothelial cells. Themechanisms of gap formation can be widely contributed to three overlapping categories including; i) injury and/or apoptosis of cells in the endothelial monolayer, ii)internalization or disassembly of intercellular junctions, and iii) remodeling of the actincytoskeleton, leading to a change in cell shape.Rac1, the main GTPase, is known toregulate the cytoskeleton and intercellular junctions via cell spreading, buttress corticalactin rim formation and reorganization of the junction-associated cortical actincytoskeleton.The cortical actin band spans the entire circumference of endothelial cells and iscomposed of F-actin bundles which are thought to be associated with adherens and tightjunctions. Cortactin is a ubiquitously expressed tyrosine kinase target and has beenimplicated in cortical actin assembly and reorganization. Evidence was provided thatRac1strengthening of the cortical actin cytoskeleton via promoting cortactin toaccumulate at cell border could be effective to enhance endothelial barrier properties.Indeed, functional relevance of cortactin for endothelial permeability was revealed byattenuated responses to barrier-protective stimuli following cortactin knockdown.Therefore, it is conceivable that strengthening of the cortical actin cytoskeleton could beeffective to enhance endothelial barrier properties by promoting stabilization ofjunctional proteins.In all, considered the important role of Rac1in controlling cell permeability and theclose relationship between Rac1and caveolin-1, we hypothesized that it was reasonableto explore whether caveolin-1is involved in regulating endothelial permeability inducedby TNF-α through the Rac1signaling pathway.ObjectiveTo construct caveolin-1gene RNA interference lentiviral vector, observing the influenceof down-regulation of caveolin-1on pulmonary microvascular hyperpermeabilityinduced by TNF-ɑ and to explore its mechanism. These may be helpful for treatment ofARDS. Methods1. To construct lentiviral expression vector pLL3.7-caveolin-1shRNA： Caveolin-1gene RNA interference target sequence was choiced and were constructed intolentiviral expression vector pLL3.7-caveolin-1shRNA. Recombinant lentivirusvector were packaged using a four-plasmid transient transfection procedure. VSV-G,RSV-REV, pMDL and pLL3.7lentiviral vector were contransfected into293Tinduced by lipofectamine2000, which produced lentivirus; the supernatant wasconcentrated and diluted into certain ratio.2. The primary RPMVECs were isolated from the lung of rat and successfully culturedin vitro. The biological characteristics of RPMVECs were observed by the invertedmicroscope, staining with FITC-BSI.3. RPMVECs were transfected by Lentiviral vector pLL3.7-caveolin-1shRNA andwere identified: The green fluorescence protein was confirmed by invertedfluorescence microscope. Dilution method of counting were carried to evaluate theefficiency of transfection. The expression of caveolin-1were evaluated by Westernblotting analysis.4. RPMVECs were divided into three categories: normal cells, control shRNA cellsand caveolin-1shRNA cells. The RPMVECs were grouped according to theprogram of administer. The RPMVECs were seeded on transwell polyestermembranes to construct models of RPMVECs monolayer in vitro and the changesof transendothelial electrical resistance (TER) and FITC-BSA flux across themonolayer were recorded. Moreover, RPMVECs were double immunofluorescencestained for filamentous actin (F-actin) and cortactin then cells were scaned with thelaser scanning confocal microscope.The translocation of cortactin was analysisedby immunoblotting. Pull-down assays and immunoblotting was used to determinethe Rac1-GTPase expression. Results1. Caveolin-1shRNA lentiviral vector pLL3.7-caveolin-1was successfully constructed,which was confirmed by digestion and sequencing. After concentration, the titer ofpackaged lentivirus is2×108TU/ml.2. RPMVECs were isolated and cultured in vitro finely and identified by the invertedmicroscope, staining with FITC-BSI.3. Lentiviral vector was transfected into RPMVECs successfully. The greenfluorescence protein was confirmed by inverted fluorescence microscope.caveolin-1shRNA reduced caveolin-1expression to60±10%at48h and tominimum of10±2%after72h. No changes in expression of caveolin-1wereobserved with the control shRNAs.4. Effect of down-regulation of caveolin-1on RPMVECs with or without TNF-ɑchallenging.1) Effect of down-regulation of caveolin-1on TER of RPMVEC monolayer.Exposure to TNF-α (100ng/ml) for2hour, TER decreased to63±5%of baselinelevels and the flux of FITC-BSA was significantly increased to205±23%ofbaseline. Pre-incubation with O-Me-cAMP could prevent the decrease of TER or theincrease of the flux of FITC-BSA induced by TNF-α. Down-regulation ofcaveolin-1expression led to an increase in the mean baseline TER of RPMVECsmonolayer by116±10%compared with control shRNA cell monolayers. At thesame time, the flux of FITC-BSA in Cav-1-deficient cells decreased. Moreover,down-regulation of caveolin-1could prevent the decrease of TER or the increase ofthe flux of FITC-BSA induced by TNF-α. For example, challenged with TNF-α for2h, the TER of Cav-1-deficient cells monolayer was decreased to78±7%ofbaseline and it was59±4%in control shRNA monolayer. The flux of FITC-BSA inCav-1-deficient cells was increased to122±11%of baseline and it was197±27%in control shRNA monolayer. But inhibition of Rac1activity abolished thebarrier-protective effect induced by down-regulation of caveolin-1in response toTNF-α in RPMVECs.2) Effect of down-regulation of caveolin-1on cytoskeleton rearrangement andcortactin distribution in RPMVECs.RPMVECs exposed to TNF-α for2hours demonstrated a dramatic cytoskeletonrearrangement, which was characterized by the formation of central actin stressfibers and the disappearance of cortactin from the cell periphery. Down-regulationof caveolin-1expression led to lamellipodia formation, cortactin translocation andincreases in peripheral F-actin levels. RPMVECs challenged with TNF-α led toobvious formation of lamellipodia, however, the disappearance of cortactin from thecell periphery was not obvious nor was a decrease in the levels of central actin stressfiber.3) Effect of down-regulation of caveolin-1on Rac1activity in RPMVECs.In our study, we observed that shRNA-mediated caveolin-1loss enhanced Rac1activity in the resting state and that the loss of caveolin-1also attenuated decreasedRac1activity induced by TNF-α stimulation in primary RPMVECs. Caveolin-1deficient cells showed a significant increase in GTP-bound Rac1compared withcells treated with the control shRNA (2.5±0.2-fold increase, p＜0.001) and afterTNF-α stimulation for2hours (2.7±0.4-fold increase, p＜0.01) suggesting thatcaveolin-1acts as a negative modulator of Rac1.Conclusions1. The model of down-regulation caveolin-1in the RPMVECs was successfullyconstructed.2. Inhibition the expression of caveolin-1attenuated TNF-α-induced breakdown of monolayer integrity in RPMVECs.3. TNF-α induced barrier breakdown in cultured RPMVEC was a direct result ofimpaired Rac1signaling. Inhibitting expression of caveolin-1increased Rac1activity in RPMVECs, resulting in cortactin translocation and increases in peripheralF-actin levels and leading to buttress cortical actin rim formation and reorganizationof the junction-associated cortical actin cytoskeleton. These morphology changescould enhance the endothelia barrier function and protect endothelial barrier frominjury due to TNF-α.4. Inhibition of Rac1activity attenuated the barrier-protective effect induced byinhibitting expression of caveolin-1in response to TNF-α in RPMVECs.