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Influence of Angiotensin-(1-7) on the Expression of Tissue Factor Induced by LPS and Its Mechanism in Vascular Endothelial Cells

Author: HuJing
Tutor: WenZhiBin
School: Central South University
Course: Physiology
Keywords: Ang-(1-7) tissue factor vascular endothelial cell LPS cell signal transduction
CLC: R363
Type: Master's thesis
Year: 2008
Downloads: 125
Quote: 1
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Abstract


Physiological coagulation and pathological thrombus are both started with the tissue factor pathway.Tissue factor（TF）is a single strand transmembrane glycoprote"m with 47 kD Mol Wt which is composed with 263 amino acids.It is not only the receptor of FⅦ/FⅦa but also functions as a cofactor of FⅦa.Tissue factor pathway is concerned with the coagulation initiated by TF,in which TF binds with FⅦa,then the compound FⅦa/TF activates FⅩand FⅨwith the existence of Ca2+, eventually leading to thrombin generation and coagulation.Lipopoly Saccharide（LPS）is a complex glucolipid which composed the Gram-negative bacteria（G-）exinous.It has wide biological effects, such as thermaogenic reaction,alteration of leukocyte count, disseminated intravascular coagulation（DIC）and shock.LPS is the most effective stimulant which induces the generation of mediators of inflammation.It can directly or indirectly stimulate vascular endothelial cell（VEC）to generate inflammatory factors,such as TNF-α、IL-1α、IL-1βand IL-6,as well as to generate TF,adherence factors,growth factors and PAF.Renin-angiotensin system（RAS）is one of the main regulative systems in human body,and plays an important role in maintaining homeostasis,especially in cardiovascular system.Recently,many reports indicated that a new member of the system called angiotensin-（1-7）, which is the product of angiotensinⅠand/or angiotensinⅡ,consists of 7 amino acids,not only relaxes blood vessel,but also inhibits cell proliferation.It was reported that LPS induces the expression of TF,but until now it remains unclear whether angiotensin-（1-7）can also inhibit the expression of TF induced by LPS in VEC.AIM The purposes of this study were to observe the effects of angiotensin-（1-7）on the expression of TF induced by LPS in human umbilical vein endothelium derived cell line（HUVEC）s and to explore the inhibitory mechanisms of angiotensin-（1-7）on the expression of TF induced by LPS.METHODS HUVECs were cultured in DMEM.Cell activity was measured by MTT.TF activity was determined with one-stage clotting assay measuring total cellular pro-coagulant activity（PCA）.TF antigen was measured by ELISA Kit.TF mRNA was examined by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR）.Immunofluorescence analysis was performed to evaluate the activation of NF-κB.RESULTS①A gradual increase in PCA and TF mRNA were observed in HUVECs stimulated with increasing concentration of LPS （0.01～100 mg/L）（r=0.963,P＜0.05）,and 10mg/L was the peak.②Ang-（1-7）(10-8～10-5mol/L)alone could not affect the expression of TF in HUVECs（P＞0.05）.When pretreated with Ang-（1-7）(10-8～10-5 mol/L),it can inhibit the effects of LPS on PCA,TF antigen and mRNA in dose-dependent manner（r=-0.947,P＜0.05）,and 10-6mol/L was the strongest concentration.10-6mol/L Ang-（1-7）decreased TF activity and TF antigen in HUVECs in a time dependent manner,reaching a maximum level after 8 h.③L-NAME alone,which is the inhibitor of NOS,had no marked effects on TF activity and TF mRNA in HUVECs, but L-NAME significantly inhibited the effects of Ang-（1-7）on TF expression induced by LPS（P＜0.05）.④Immtmofluorescence analysis demonstrated that NF-κB translocation from cytoplasm to the nucleus after treatment of HUVECs with LPS and the effect of NF-κB translocation induced by LPS was partly inhibited by Ang-（1-7）.CONCLUSION The present data suggest that:①Ang-（1-7）can inhibit the expression of TF induced by LPS in HUVECs at mRNA level.②NO pathway participates in the inhibitory effects of Ang-（1-7）on the expression of TF induced by LPS.③NF-κB translocation that plays an important role in LPS-induced TF expression can be inhibited by LPS.

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