The Mechanism And Contributions Of Clathrin And Caveolae To LPS-induced Vascular Hyperpermeability

Vascular hyperpermeability, which is induced by endothelial barrier dysfunction, is acommon pathogenic process in cases of severe trauma and sepsis and can result inprotein-rich tissue edema, abnormality of the internal environment, abdominal compartmentsyndrome (ACS) and multiple organ dysfunction syndrome (MODS). Lipopolysaccharide(LPS) is the major pathogenic factor in inflammation and sepsis and is also an importantvascular permeabilizing agent. According to the previous reports, LPS regulates endothelialpermeability via the two main pathways: transcellular and paracellular. The paracellularpathway is created due to the contraction of stress fibers after LPS stimuli, and isconsidered as the predominant pathway involved in the regulation of vascularhyperpermeability. However, other studies have revealed that the polymerization of F-actinand the formation of stress fibers occur only early after LPS treatment and thedepolymerization occurs after prolonged LPS treatment, which suggests that othermechanisms might be involved.According to the previous studies, interendothelial junctions (IEJs) are important formaintaining the endothelial barrier. Adherens junctions (AJs) represent the majority of IEJsthat comprise the endothelial barrier, and vascular endothelial cadherin (VE-cad) is themain component of AJs at the IEJs of vascular endothelial cells. The amount of VE-cad inthe plasma membrane can directly modulate the strength of AJs, consequently affecting theendothelial barrier function and the vascular permeability. The significant downregulationof VE-cad expression in the plasma membrane has been shown to be coincident withLPS-induced hyperpermeability of the endothelial cells. Previous studies have alsosuggested that the expression of cadherin in the plasma membrane and AJs is determined byendocytosis, and clathrin-mediated endocytosis is the pathway that is generally accepted tobe involved in the internalization of VE-cad and regulation of hyperpermeability. Someevidence has suggested that the internalization of epithelial cadherin (E-cad) can also occurvia clathrin-independent, caveolae-mediated pathways in some epithelial tumor cell types, which contributes to the disassembly of adherens junctions and tumor cell invasion.However, it is unknown if clathrin-mediated and/or caveolae-mediated endocytosis ofVE-cad also contributes to LPS-induced vascular hyperpermeability, if they contribute insimilar or different manners, and which mechanisms may be relevant in these processes.To elucidate these problems, the experiments were conducted in three parts:①toobserve the role of clathrin-mediated and caveolae-mediated endocytosis of VE-cad inLPS-induced vascular hyperpermeability;②to investigate the mechanisms underlying thedifferent contributions of the two endocytotic pathways on vascular hyperpermeability;③to investigate the mechanisms regulating these two endocytosis pathways switching.Methods:Part I. To observe the role of clathrin-mediated and caveolae-mediated endocytosis ofVE-cad in LPS-induced vascular hyperpermeability1. Clathrin-mediated endocytosis of VE-cad after LPS treatment, and its role invascular hyperpermeability1) The human vascular endothelial cell line CRL-2922was adopted to observe theVE-cad expression in plasma membrane and the monolayer cell permeability at differenttime after LPS (10μg/mL) treatment (1h,2h,4h and6h).2) The CRL-2922cell line was adopted to observe the co-immunoprecipitation andco-localization of VE-cad with clathrin at different time after LPS treatment (1h,2h,4h and6h).3) The clathrin-mediated endocytosis inhibitor CPZ (100μmol/L) and clathrin heavychain siRNA (50nmol/L) was adopted to observe its effect on the co-immunoprecipitationof VE-cad with clathrin, the VE-cad expression in plasma membrane and the monolayercell permeability after LPS treatment (1h and4h).2. Caveolae-mediated endocytosis of VE-cad after LPS treatment and its role invascular hyperpermeability1) The CRL-2922cell line was adopted to observe the protein expression andphosphorylation (Tyr14) of Cav1, and the co-immunoprecipitation and co-localization ofVE-cad with Cav1at different time after LPS treatment (1h,2h,4h and6h).2) The inhibitor of caveolae filipin (5μg/mL) and Cav-1siRNA (50nmol/L) wasadopted to observe its effect on the co-immunoprecipitation of VE-cad with Cav1, theVE-cad expression in plasma membrane and the monolayer cell permeability after LPS treatment (1h and4h).3) The CRL-2922cell line was adopted to observe the protein expression of Src afterLPS treatment, the inhibitors of Src, SU6656(2μmol/L), and TLR4, CLI-095(5μg/mL)was adopted to observe its effect on the phosphorylation (Tyr14) of Cav1, theco-immunoprecipitation of VE-cad with Cav1, the co-immunoprecipitation of VE-cad withP-Cav1, the VE-cad expression in plasma membrane and the monolayer cell permeabilityafter LPS treatment (1h and4h).Part II. To investigate the mechanisms underlying the different contributions of thetwo endocytotic pathways on vascular hyperpermeability1. The CRL-2922cell line was adopted to observe the co-immunoprecipitation ofVE-cad with Rab11, a marker of the recycling endosomes, and the co-immunoprecipitationof VE-cad with LAMP2, a marker of the late endosomes/lysosomes, after LPS treatment(1h,2h,4h and6h).2. The clathrin-mediated endocytosis inhibitor CPZ and the inhibitor of caveolae filipinwas adopted to observe their effects on the co-immunoprecipitation of VE-cad with Rab11andthe co-immunoprecipitation of VE-cad with LAMP2after LPS treatment (1h and4h).Part III. To investigate the mechanisms regulating these two endocytosis pathwaysswitching1. The CRL-2922cell line was adopted to observe the dynamic change of cytoskeletonafter LPS treatment (1h,2h,4h and6h).2. The cytoskeleton depolymerizing agent, Cyt D (2μmol/L) and the cytoskeletonstabilizer Jasp (1μmol/L) was adopted to observe their effects on theco-immunoprecipitation of VE-cad with clathrin, the co-immunoprecipitation of VE-cadwith Rab11, the co-immunoprecipitation of VE-cad with Cav1,the co-immunoprecipitationof VE-cad with LAMP2, the VE-cad expression in plasma membrane and the monolayercell permeability after LPS treatment (1h and4h).Results:1. The role of clathrin-mediated and caveolae-mediated endocytosis of VE-cad inLPS-induced vascular hyperpermeability1) Clathrin-mediated endocytosis of VE-cad after LPS treatment, and its role invascular hyperpermeability Normal cells displayed strong VE-cad expression in the plasma membrane, and thedecreased plasma membrane VE-cad expression was found after LPS (10μg/mL) treatment(P<0.05), the total protein expression of VE-cad was also decreased after LPS treatment(P<0.05). The permeability of the monolayer cells increased gradually in a time-dependentmanner after LPS treatment (P<0.05). The monolayer cell permeability and the location ofVE-cad in the plasma membrane were negatively correlated.The amount of clathrin that co-immunoprecipitated with VE-cad was low in thenormal control, increased1h after LPS treatment, and decreased as time went on.Accordingly, the co-localization of VE-cad and clathrin was altered in a similar pattern. Theclathrin-mediated endocytosis of VE-cad did not correlate well with the location of VE-cadin the plasma membrane and the monolayer cell permeability after LPS treatment.The clathrin-mediated endocytosis inhibitor CPZ (100μmol/L) and clathrin heavychain siRNA (50nmol/L) could decrease the co-immunoprecipitation of VE-cad andclathrin (P<0.05), increase the localization of VE-cad in the plasma membrane (P<0.05),and improve the monolayer cell permeability (P<0.05)1h after LPS treatment. However,these changes were absent4h after LPS treatment. These results suggest thatclathrin-mediated endocytosis of VE-cad occurs1h after LPS treatment, leading to thelimited decrease of plasma membrane-localized VE-cad and monolayer cellhyperpermeability. In contrast, the activity of this endocytosis pathway was reduced4hafter LPS treatment and was not responsible for the internalization of VE-cad and thesevere monolayer cell hyperpermeability at that time.2) Caveolae-mediated endocytosis of VE-cad after LPS treatment and its role invascular hyperpermeabilityThe protein expression of Cav1, a main structural protein of caveolae, did notsignificantly change after LPS treatment, whereas the phosphorylation (Tyr14) of Cav1wassignificantly increased after LPS treatment (P<0.05). The co-immunoprecipitation andco-localization of VE-cad with Cav1was negligible in the normal control and wasincreased in a time dependent manner after LPS treatment (P<0.05), which was negativelycorrelated with the localization of VE-cad in the plasma membrane and positivelycorrelated with monolayer cell permeability after LPS treatment.Filipin (5μg/mL), an inhibitor of caveolae, and Cav-1siRNA (50nmol/L) significantly reduced the co-immunoprecipitation of VE-cad with Cav1(P<0.05), increased the plasmamembrane expression of VE-cad (P<0.05), and improved the monolayer cell permeability4h after LPS treatment (P<0.05). These results suggest that caveolae-mediated endocytosisof VE-cad takes place4h after LPS treatment and contributes to the internalization ofVE-cad, thus aggravating monolayer cell hyperpermeability.The protein expression of Src was gradually increased after LPS treatment (P<0.05),and its increased expression was decreased by the inhibitor of TLR4, CLI-095(5μg/mL)(P<0.05). The inhibitors of Src, SU6656(2μmol/L), and TLR4, CLI-095, significantlydecreased the phosphorylation (Tyr14) of Cav1(P<0.05), reduced theco-immunoprecipitation of VE-cad with Cav1and the co-immunoprecipitation of VE-cadwith P-Cav1(P<0.05), increased the plasma membrane expression of VE-cad (P<0.05), andimproved the monolayer cell permeability4h after LPS treatment (P<0.05).2. The mechanisms underlying the different contributions of the two endocytoticpathways on vascular hyperpermeabilityThe co-immunoprecipitation of VE-cad with Rab11, a marker of the recyclingendosomes, was increased1h after LPS treatment, and decreased with prolongation of LPStreatment (P<0.05). In contrast, the co-immunoprecipitation of VE-cad with LAMP2, amarker of the late endosomes/lysosomes, was gradually increased after LPS treatment(P<0.05).The increased co-immunoprecipitation of VE-cad with Rab111h after LPS treatmentcould be decreased by the clathrin-mediated endocytosis inhibitor, CPZ (P<0.05). However,it was not significantly changed by the inhibitor of caveolae, filipin. The increasedco-immunoprecipitation of VE-cad with LAMP24h after LPS treatment could bedecreased by the caveolae inhibitor filipin (P<0.05) but not by CPZ, the inhibitor ofclathrin-mediated endocytosis.3. The mechanisms regulating these two endocytosis pathways switchingThe cytoskeleton showed a dynamic change after LPS treatment. Specifically,1h afterLPS treatment, F-actin polymerized and stress fibers formed. However,4h after LPStreatment, they had depolymerized.The cytoskeleton depolymerizing agent, Cyt D (2μmol/L), could decrease theco-immunoprecipitation of VE-cad with clathrin (P<0.05), as well as the co-immunoprecipitation of VE-cad with Rab11(P<0.05). In contrast, it increased theco-immunoprecipitation of VE-cad with Cav1(P<0.05) and the co-immunoprecipitation ofVE-cad with LAMP2(P<0.05). As a result,1h after LPS treatment, VE-cad was lost fromthe plasma membrane (P<0.05) and the monolayer cell hyperpermeability (P<0.05) becamemore severe.The cytoskeleton stabilizer Jasp (1μmol/L), which was applied1h after LPStreatment, did not significantly change the co-immunoprecipitation of VE-cad with clathrin(P<0.05) and the co-immunoprecipitation of VE-cad with Rab114h after LPS treatment(P<0.05). In contrast, it decreased the co-immunoprecipitation of VE-cad with Cav1(P<0.05) and the co-immunoprecipitation of VE-cad with LAMP24h after LPS treatment(P<0.05). As a result, the location of VE-cad in the plasma membrane (P<0.05) andmonolayer cell permeability (P<0.05, Figure6H) was improved4h after LPS treatment.Conclusions:1. Both the clathrin-mediated and caveolae-mediated endocytosis pathways participatein the plasma membrane loss of VE-cad and vascular hyperpermeability after LPStreatment. Clathrin-mediated endocytosis is dominant early after LPS treatment,caveolae-mediated endocytosis of VE-cad is dominant hours after LPS treatment andactivated through LPS-TLR4-Src signal pathway.2. The clathrin-mediated endocytosis results in the localization of VE-cad in recyclingendosomes after endocytosis, which limits the loss of VE-cad present at the plasmamembrane and monolayer cell hyperpermeability. In contrast, caveolae-mediatedendocytosis results in the localization of VE-cad in lysosomes after endocytosis, whichaggravates the loss of VE-cad present at the plasma membrane and monolayer cellhyperpermeability.3. Actin cytoskeleton shifts from polymerization to depolymerization after LPS treatment, andthe structural changes in the actin cytoskeleton regulates the switch betweenclathrin-mediated and caveolae-mediated endocytosis of VE-cad.