Effects Of Caveolin-1 In LPS And Visfatin Induced Pulmonary Microvascular Cells Hyperpermeability

Objective Pulmonary microvascular endothelial hyperpermeability and proterin-rich tissue edema is a key hallmark of acute respiratory distress syndrome(ARDS). Transport of the most abundant plasma protein, albumin, occurs by means of transcellular and praracellular pathways. Under physiological condition, because of restrictive endothelial cell-cell contacts, transcellular transport of albumin from the endothelial lumen to the abluminal perivascular interstitium via caveolae is a primary determinant of basal endothelial permeability. Other studies showed that pulmonary vascular hyperpermeability induced by activation of neutrophils adherent to the vessel wall is dependent on signaling via Cav-1 phosphorylation and subsequently increased caveolae-mediated transcytosis. It is well known that LPS induce the hyperpermeability of PMVECs during severe sepsis. Although the precise mechanisms have not been completely elucidated, studies have implicated an increase in paracellular permeability via opening the interendothelial junctions by activating signaling mechanism. However, whether and how Cav-1 and caveolae participates in the regulation of albumin transcytosis and endothelial hyperpermeability induced by LPS remains important and unkown. Visfatin is a new factor from visceral adipose tissue, which conducts complex biological functions, such as lowering blood glucose with an insulin-like effect as well as acting as inflammatory mediators in the inflammatory response in vivo, is potential risk factor involved in the endothelial dysfunction. Some studies observed that visfatin induced aggregation of “lipid rafts” in the cell membrane, increased permeability of the endothelial monolayer cells by promoting oxidative stress, andcaveolae just is the most important type of "lipid rafts". Clinical study show that visfatin is an important inflammatory mediator involved in both the development and severity of ARDS. But, whether visfatin can directly lead to PMVECs injury and its specific mechanism are still unclear. The aim of this study is investigate the effects of LPS on RPMVECs uptake(endocytosis) and transcytosis of albumin firstly, and assess the role of Cav-1 and caveolae in this prossess, then determine the effect and possible mechanism of visfatin on RPMVECs permeability.Methods Primary cultured RPMVECs will be used in this study; stimulate cells with LPS, detect cell albumin endocytosis by A488-labeled albumin; seed RPMVECs in Transwell, determine transcytosis of albulmin absorbance of EBA at 620 nm in the lower compartment, assess the paracellular permeability(endothelial barrier) by transendothelial electrical resisitance(TER); at the same time, the phosphorylation and translocation of Cav-1 assayed by western blot and immunohistochemistry; then give PP2 or methyl-β-cyclodextrin(MβCD) in preprocessing to suppress Src kinase activity or destruction of caveolae structure, use the same method to detect the effect of pretreatment on LPS. In addition, we stimulate RPMVECs with visfatin, LPS or visfatin + LPS, understand the change of PKC activation and phosphorylation of Cav-1, ERM by Western bolt; detect the absorbance of EBA through the cell monolayer, assess the TER; and observate cell F-actin cytoskeleton bound with FITC-phalloidin; finally, measure the TER to reflect the effect of BIM(PKC inhibitor) on the permeability change induced by visfatin.Results 1. In vitro, cells isolated from the pulmonary microvascular endothelium of SD rats were cultured successfully. These cells were further confirmed by typical morphology and CD34, FITC-BSI binding assay.2. 10μg/ml LPS induced phophorylation of Cav-1 in a time-dependent manner. This phosphorylation increased within 5min, peaked at 30 min, then declined till 60min(beyond the base), total Cav-1 did not changed during the same time. 3. 10μg/ml LPS induced translocation of Cav-1 from triton-soluble domain to triton-insoluble domain and from membrane to cytoplasm, these translocation began within 5min, peaked at 15 min after incubated with LPS, gradually decreased but still continued to 60 min. 4. 10μg/ml LPS caused increase in albumin endotytosis and transendothelial transport in RPMVECs at 15 min, but did not disrupt the endothelial barrier, because TER of RPMVECs started decrease at 30 min after incubated with LPS. 5. 15μmol/L PP2 pretreatment significantly decreased LPS-induced Cav-1 phosphorylation; 15μmol/L PP2 and 2.0mmol/L MβCD pretreatment both inhibited translocation of Cav-1 induced by LPS. 6. 15μmol/L PP2 or 2.0mmol/L MβCD inhibited LPS-induced A488-labeled albumin endocytosis significantly, reduced the increasing of EBA absorbance in stimulated 15 min by LPS, above effects were partly blocked by pretreatment with PP2 or MβCD. 7. 0.5μg/ml visfatin couldn’t increase the absorbance of EBA in lower compartment lonely at 15 min, but aggravated this effect of LPS. 8. 0.5μg/ml visfatin stimulated RPMVECs lonely, the TER decline could be detected from 30 minutes until at least 180 min, and derangement of F-actin skeleton could be observed, these effects were similar but mild to LPS, visfatin + LPS co-damage were more significantly.9. 0.5μg/ml visfatin induced translocation of PKC(cytosol-to-membrane), and phosphorylation of Cav-1 and ERM at 30 min. 10. 1 mmol/L BIM pretreatment significantly eased the visfatin-induced TER reduction.Conclusion 1. LPS increased phophorylation and translocation of Cav-1 in a time-dependent manner. 2. The phophorylation of Cav-1 was dependent on Src kinase partly, inhibite Src kinase or destroy caveolae structure could restrain the translocation of Cav-1. 3. The increase of albumin endotytosis and transendothelial transport were early than the decrease of TER, in this process, phophorylation and translocation of Cav-1 were the key steps. 4. visfatin failed to increase albumin transcellular permeability early, but after that caused skeleton of RPMVECs cells reorganize, and increased paracellular permeability of RPMVECs, these damage would be more significant if incubated cells with LPS+visfatin. 5. Hyperpermeability of RPMVECs induced by visfatin was dependent on the activation of PKC, and may be related with phosphorylation of Cav-1 and ERM.