The Role Of Cathepsin L In Ox-LDL-induced Increased Of Vascular Endothelial Cell Monolayer Permeability And Its Mechanism

【Objective】To explore the effect of cathepain L (CATL) in Ox-LDL induced HumanUmbilical Vein Endothelial Cells (HUVECs) monolayer permeability change and itsmechanism.【Methods】(1) After treated withg different concentrations of Ox-LDL for24h, transwellmethod was used to determine the change of endothelial cell monolayer permeability.RT-PCR and Western blot were applied to determine CATL, VE-cadherin, Beclin1,LC3, caspase3, caspase9and Bcl-2mRNA and protein levels, respectively. Theactivity of CATL was detected by CATL activity assay kit. The autofluorescentsubstance monodansylcadaverine (MDC) was used to stain autophagic vacuoles(AVs). The apoptosis of HUVECs was analyed by Hochest staining and FlowCytometry (FCM).(2) HUVECs were pretreated with different concentrations of CATL inhibitor for24h, followed by50mg/L Ox-LDL treatment for another24h. The endothelial cellmonolayer permeability and the endothelial cell autophagy and apoptosis weredetected.(3) RT-PCR and Western blot were used to deteted the mRNA and proteinexpression of Ten-Eleven-Translocation2(TET2) induced by Ox-LDL in present ofCATL inhibitor or absence of CATL inhibitor. After transfected with TET2siRNA，Western blot was applied to determine protein expression of Beclin-1, LC3, Caspase-3and VE-cadherin in HUVECs.【Results】(1) After treated with different concentrations (0,25,50and75mg/L) ofOx-LDL for24h, mRNA expression of CATL was no obviously changed (P>0.05),however, the protein expression and activity were significanltly increased (P<0.01).Ox-LDL decreased VE-cadherin protein expression (P<0.05) and increasedendothelia cell monolayer permeability (P<0.05) with a dose-denpendent manner.Beclin-1mRNA and protein expression were up-regulated (P<0.05). The LC3mRNAexpression was no obviously changed, whereas the LC3Ⅰ/LC3Ⅱ valueincreased(P<0.01). MDC staining showed that Ox-LDL up-regulated HUVECsautophagy. The Caspase-3mRNA and protein expression increased(P<0.05), whilethere was no effect on Caspase-9mRNA expression(P>0.05). The Bcl-2mRNAexpression was increased with a dose-denpendent manner(P<0.01). Furthermore,Hochest staining and FCM showed that Ox-LDL promote HUVECs apoptosis.(2) After pretreated with different concentrations of CATL inhibitor, CATLinhibitor up-regulated VE-Cadherin protein expression and inhibited endothelial cellmonolayer permeability increased induced by Ox-LDL (P<0.01). The CATL inhibitorhad no effect on the Beclin-1and LC3mRNA expression, while down-regulatedBeclin-1protein expression and LC3Ⅰ/LC3Ⅱvalue (P<0.01). At the same time, theCATL inhibitor significantly up-regulated Caspase-3mRNA and protein expression(P<0.05), while had no effect on Caspase-9mRNA expression (P>0.05). Theup-regulated of Bcl-2mRNA induced by Ox-LDL was attenunated when pretreatedwith CATL inhibitor (P<0.01). Hochest staining and FCM also showed that theapoptosis of HUVECs induced by Ox-LDL was further promoted by CATL inhibitor.(3) Ox-LDL down-regulated TET2mRNA and protein expression, while CATLinhibitor attenuated the down-regualton of TET2mRNA and protein expression(P<0.05). TET2siRNA up-regulated Beclin-1protein level and LC3Ⅱ/LC3Ⅰvaule(P<0.01), while down-regulated Caspase-3protein expression (P<0.01). TheVE-Cadherin protein expression of HUVECs was no obviously changed after treated with CATL inhibitor.【Conclusions】CATL involved in Ox-LDL-induced autophagy and apoptosis of HUVECs,which contributed to the increase of endothelial cell monolayer permeability responseto Ox-LDL treatment. Moreover, TET2may involve in this process.