Effects Of Tryptase Down-regulation In Mast Cells On Synthesis And Release Of Inflammatory Mediators In Vascular Endothelial Cells

Atherosclerosis is a kind of chronic inflammatory disease.In the pathogenesis of vascular inflammation of atherosclerosis,monocytes migrate into the vascular intima, become macrophages and release many cytokines,which can cause leukocytes recruitment,accumulation and migration into the intima.Activated leukocytes produce and secrete plentiful cytokines and inflammatory mediators,which result in the damage function of vascular endothelial cells.In the inflammatory process,mast cells are populated from peripheral blood into the intima.Activated mast cells can undergo degranulation to release many secretory mediators including tryptase, chymase,histamine and heparin.Tryptase is an inartificial protease with multiplicate biological functions.In cardiovascular system,tryptase,as the activator of PAR-2 on vascular smooth muscle cell,vascular endothelial cell and cardiac myocyte cell, affects on blood vessels relaxation and contraction,cells mitogenesis,Ca²⁺ concentration increasing,von Willebrand factor release and NO release.Tryptase is nearly concerned with the pathogenesis of atherosclerosis by causing neutrophil recruitment to promote inflammation and stimulating endothelial cells to synthesize and release of IL-1βand IL-8 that induce leukocytes infiltration.Vascular endothelial cells dysfunction is an early pathological event in vascular inflammation of atherosclerosis.In the process of the event,mononuclear cells, vascular smooth muscle cells and vascular endothelial cells synthesize and release many inflammatory mediators,which can damage the function of vascular endothelial cells,increase the inflammatory infiltration in vessel wall and accelerate the development of vascular inflammation.Among these inflammatory mediators,IL-6 and TNF-αplay an important role in mediating the vascular inflammation of atherosclerosis.IL-6 mediates acute-phase response of inflammation and primarily induces the hepatic production of C-reactive protein.TNF-αis the most important initiation factor of inflammation in vascular system.TNF-αcan activate NF-κB to regulate gene transcription of many inflammatory mediators and lead to cascading inflammation reactions.It was confirmed that tryptase stimulated peripheral blood monocytes produce and secrete IL-6,TNF-αand IL-1βby activating PAR-2 on cell membrane,so as to affect the occurrence and development of vascular inflammation of atherosclerosis.We found that tryptase up-regulated the synthesis and release of endothelial IL-6 and TNF-αby activating PAR-2 in concentration- dependent manner and time-dependent manner.In the present study,two siRNA for tryptase were designed and transfected into P815 cells.Tryptase mRNA expression in P815 cells was measured using RT-PCR. The result showed the significantly inhibited tryptase expression induced by RNAi. On the base of founding inhibiting tryptase expression model in mast cells,the P815-conditioned medium,in which tryptase was normally expressed,and the siRNA-P815-conditioned medium,in which tryptase expression was inhibited,were prepared to culture bend.3 cells.Released tryptase in these two conditioned medium was detected by ELISA.The result showed that tryptase was significantly lower in the siRNA-P815-conditioned medium than the P815-conditioned medium.After bEnd.3 cells were cultured with the different conditioned medium,mRNA expression level and protein release level of IL-6 and TNF-αin bend.3 cells were measured using RT-PCR and ELISA,respectively.The results showed that the mRNA and the protein levels of IL-6 and TNF-αin bEnd.3 cells were significantly lower cultured in the siRNA-P815-conditioned medium than cultured in the P815-conditioned medium,and a little higher when they were compared to levels in the DMEM medium.The present study demonstrated that tryptase secreted by mast cells might stimulate vascular endothelial cells to synthesize and release IL-6 and TNF-α;inhibited expression of tryptase induced by RNAi could dramatically down-regulate synthesis and release of IL-6 and TNF-αin vascular endothelial cells.Effects of tryptase on cardiovascular diseases especially atherosclerosis are widely regarded now.The present study confirmed the effects of trypase on the expression of two important inflammatory mediators IL-6 and TNF-αin vascular endothelial cells, and implied the role of tryptase in the pathological process of vascular inflammation of atherosclerosis.The result suggested that decreased tryptase expression induced by RNAi may inhibit vascular inflammation of atherosclerosis by decreasing downstream inflammatory factors.Thus,inhibiting tryptase expression in mast cells may be a new therapeutic strategy for atherosclerosis. Hyperlipidemia is a disease that the concentration of lipid in blood preponderates over normal level.When hyperlipidemia occurs,T-CHO,TG and LDL increases in serum,as well as HDL decreases.Hyperlipidemia may lead to the damage of vascular endothelial cells,which can produce and secrete many endogenous protective enzymes,such as eNOS and HO-1,to inhibit vascular inflammation,keep the stabilization of vessel structure and the balance of angiokinesis.eNOS generates NO upon the conversion of L-arginine to L-citrulline.NO is a kind of vasodilator in vivo, which can keep vessel tensility,regulate blood pressure,and inhibit platelet agglomeration and leucocytes accumulation.It is important to decelerate the development of inflammatory vascular diseases.HO-1 decomposes haemachrome to CO,biliverdin and Fe²⁺.These products play the roles on combating oxidative stress, preventing inflammatory damage,inhibiting cells proliferation,blocking apoptosis and regulating angiokinesis.High lipid concentration in blood and endothelial cells dysfunction induced by hyperlipidemia may influence the expressions of eNOS and HO-1.As a result,the pathogenesis of some chronic inflammatory cardiovascular diseases will be affected by their influenced down-stream products,such as NO and CO.In the present study,C57BL/6 mice were fed a high-fat diet as high-fat diet group and fed a normal diet as control.18 weeks later,serum lipid profiles of the mice including T-CHO,TG,HDL and LDL were detected.The results showed that, compared to normal diet group,the levels of serum T-CHO,TG and LDL clearly increased in high-fat diet group,while HDL decreased.It suggest that the high-fat diet could lead to hyperlipidemia in vivo.After detecting serum lipid profiles of the mice, mRNA and protein expression levels of eNOS and HO-1 in heart and lung tissues of two groups were measured using Real-Time PCR and immunohistochamistry, respectively.The results showed that mRNA and protein expression of eNOS in heart and lung tissues reduced in high-fat diet group than them in normal diet group,while HO-1 increased.The present study proved that high-fat diet could lead to hyperlipidemia in mice,which might down-regulate eNOS expression and up-regulate HO-1 expression in heart and lung tissues.Down-regulated eNOS induced by hyperlipidemia reduces the protective effects of NO on vascular endothelial cells,so as to accelerate the pathological process of inflammatory vascular diseases.The up-regulated HO-1 expression may play a compensatory role in the accelerative process.More discussion and researches are needed to explore the mechanism of this compensatory role.When self-compensation is destroied by continual stimulation of hyperlipidemia,the damage of cells and organs will appear and the manufacture of endogenous protective enzymes will decrease.Therefore,it may be valuable for inputting or inducing protective enzymes such as eNOS and HO-1 to intervene the development of inflammatory vascular diseases.