Pretective Effects Of Doxycycline On Endothelial Cells And Asymmetric Dimethylarginine

Background Asymmetric dimethylarginine (ADMA) is an endogenous inhibitorof nitric oxide synthase(NOS) which facilitates the onset of artherosclerosis(AS). It isalso a new cardiovascular risk factor. Matrix metalloproteinases(MMPs) is a familyof zinc proteases which htdrolyzes components of the extracellular matrix and hasimportant influences on the progress of AS. Doxycycline is one kind of tetracyclineswhich can inhibit MMPs. In present study we investigated the protective effects ofDoxycycline on ox-LDL-induced endothelial cells injury and explored the potentialmechanisms.Method (1) to investigate the dose-effect (0,25,50,75,100,200μg/ml, 24 h) andthe time-effect (50μg/ml, 12,24,48 h) of ox-LDL on endothelial cells injury viameasuring cell viability (MTT). (2) to investigate the effects of doxcycline on ox-LDL-induced MMP-2 expression and ADMA level: HUEVCs were incubated withox-LDL (50μg/ml) and different concentration of Doxycycline (0,5,10,20,40μg/ml)for 24 h. Cell viability (MTT), MMP-2 expression (western blot) and ADMA level(High Performance Liquid Chromatography, HPLC) were determined. (3) toinvestigate the effects of ADMA on MMP-2 expression and ROS level in endothelialcells: HUEVCs were incubated with different concentration of ADMA (0,1,3,10,30μmol/L) for 24 h. Cell viability (MTT), MMP-2 expression (western blot) andreactive oxygen species (ROS) level were determined. The counteraction ofintracellular antioxidant PDTC (30μmol/L) and NOS substrate L-Arginine(0.5mmol/L) was also investigated.Results (1) ox-LDL induced endothelial cell injury in a time- and dose- dependent manner. (2) Compared with blanked control, ox-LDL (50μg/ml) inducedmarked elevation of MMP-2 expression and ADMA level (P＜0.01). Doxycycline(5～20μg/ml) significantly attenuated ox-LDL-induced decrease of endothelial cellsviability and increased MMP-2 expression and ADMA level (when the concentrationof Doxycycline was 5μg/ml P＜0.05, when the concentration of Doxycycline was 10,20μg/ml P＜0.01). (3) ADMA (3～30μmol/L) decreased endothelial cell viability(P＜0.01) in a dose-dependent manner and increased ROS level and MMP-2expression (when the concentration of ADMA was 3μmol/L P＜0.05, when theconcentration of ADMA wass 10,30μmol/L P＜0.01). L-Arginine (0.5 mmol/L) orPDTC (10μmol/L) completely abolished ADMA (30μmol/L)-induced increase ofROS level and MMP-2 expression (P＜0.01).Conclusions (1) ox-LDL-induced endothelial injury is related to MMP-2expression, (2) Doxycyline protects endothelial cells by inhibiting MMP-2 expressionwhich may be related to decreasing ADMA level, (3) The effect of ADMA on MMP-2 expression may be related to oxidative stress.