ABSTRACT
AIM:To investigate the effects of brain-derived neurotrophic factor(BDNF)on extracellular proteolytic enzymes including matrix metalloproteinases(MMPs)and serine proteases,in particular,the urokinase-type plasminogen activator(uPA)-plasmin system in a human umbilical vein endothelial cell(HUVEC)model.METHODS:The HUVEC was activated with different doses of BDNF(25-200 ?g/L)for different time period(6-48 h).Reverse transcriptase-polymerase chain reaction(RT-PCR)was used to assay MMP-2,MMP-9,TIMP-1,and TIMP-2 mRNA in HUVEC.The cultured conditioned medium was analyzed for MMP and uPA activity by gelatin zymography and fibrin zymography,respectively.uPA,PAI-1,TIMP-1,and TIMP-2 were quantified by Western blotting analysis.RESULTS:The stimulation of serum-starved HUVECs with BDNF caused marked increase in MMP-2 and MMP-9 mRNA expression and induced the pro-MMP-2 and pro-MMP-9 activation without significant differences in proliferation.However,BDNF had no effect on TIMP-1 and TIMP-2 production.BDNF increased uPA and PAI-1 production in a dose dependent manner up to 100 ?g/L,while effects of 200 ?g/L were approximately equal to those of 100 ?g/L.BDNF stimulated uPA and PAI-1 production beyond that in control cultures from 12 h until 48 h after BDNF addition.Protease activity for uPA was also increased by BDNF in a dose dependent manner.CONCLUSION:BDNF activates MMP and uPA/PAI-1 proteolytic network in HUVEC.