| ObjectiveImmune thrombocytopenia(ITP)is an autoimmune hemorrhagic disease without obvious exogenous etiology.which is a common clinical disease.ITP can cause skin,mucous membrane and even visceral hemorrhage,which can be life-threatening in severe cases.The aim of this study was to investigate the damaging of human umbilical vein endothelial cells(HUVEC)induced by antiplatelet GPⅢa(integrin β3)antibodies in ITP patients.This study provide new ideas and theoretical basis for clinical treatment of ITP patients with vascular injury.Methods1.The serum of 36 patients with chronic ITP were collected.The antiplatelet antibodies positive serum was detected by flow cytometry.2.The antiplatelet integrin β3 antibodies positive serum was screened by monoclonal antibody specific immobilization of platelet antigen(MAIPA)assay for follow-up experiments.3.HUVECs were treated with normal serum(NS)and ITP patient serum containing anti-integrin β3 antibodies(PS)respectively,the cell proliferation was detected by colony formation assay.4.After HUVECs were treated with PS for 0,24,48 and 72 hours,the cell damage was detected by lactate dehydrogenase(LDH)assay.5.After HUVECs were treated with NS and PS for 48 hours,apoptosis and mitochondrial membrane potential of HUVECs was detected by flow cytometry,the expression of apoptosis-related gene Bax was detected by Reverse transcription-Quantitative real-time PCR(RT-q PCR),the expressions of Bax,p AKT,t AKT,p ERK1/2 and t ERK1/2 were detected by Western blot.6.HUVECs were treated with NS,PS and PS plus AKT activator SC79 respectively,the damage of HUVECs was detected by LDH assay,apoptosis of HUVECs was detected by flow cytometry(annexin V-FITC staining),the expression of apoptosis-related gene Bax was detected by RT-q PCR.Results1.Twenty three serum samples containing Ig G antiplatelet antibodies were screened from 36 ITP patients’ serum by flow cytometry,Ig M and Ig A antiplatelet antibodies were not detected in these serum.Five serum samples with positive anti integrin β 3 antibody were screened out from these 23 serum samples by MAIPA assay,and antiplatelet GPⅡb,GPⅨ and GPⅠb antibodies were not detected in these five serum samples.2.Cell clone formation assay and LDH assay showed that,comparing with HUVEC cultured with NS,HUVEC treated with PS exhibited significant reduction of proliferation(P <0.05),LDH activity was significantly increased in a time-dependent manner(P <0.05).3.Flow cytometry(annexin V-FITC staining and mitochondrial membrane potential detection)showed that,comparing with HUVEC cultured with NS,HUVEC treated with PS exhibited significant increase of apoptosis(P <0.05),mitochondrial membrane potential was significantly decreased(P <0.05).These results suggest that the apoptosis of HUVEC induced by anti-integrinβ3 antibody may be related to the change of mitochondrial membrane potential.4.RT-q PCR and Western blot showed that,comparing with HUVEC cultured with NS,HUVEC treated with PS exhibited significant increase of both of gene and protein expression of Bax(P <0.05),protein expression of p AKT was inhibited(P <0.05),but protein expression of t AKT,p ERK1/2 and t ERK1/2 were not significant change.These results suggest that anti-integrinβ3 antibody inhibit AKT signaling pathway,increase the expression of Bax and induce endothelial cell apoptosis.5.Comparing to HUVEC cultured with PS alone,HUVEC treated with PS plus SC79 exhibited significant reduction of apoptosis(P <0.05),LDH activity was significantly reduced(P <0.05),and gene expression of Bax was significantly decreased(P <0.05).These results suggest that anti-integrin β3antibody can induce endothelial cell apoptosis through inhibit Akt phosphorylation,and sc79 can reverse endothelial cell apoptosis induced by anti-integrin β3 antibody.ConclusionsAnti-integrin β3 antibody in ITP patients’ serum cause HUVEC impairment and apoptosis through AKT signaling pathway,the apoptotic effects of anti-integrin β3 antibodies on HUVEC was effectively reversed by SC79. |
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