Research On The Role Of Neutrophils In Identifying The Properties Of Coronary Artery Plaque And In Thrombosis

Predictive values of neutrophil to lymphocyte ratio for the presence of non-calcified or mixed coronary atherosclerotic plaquesBackground:Atherosclerosis is the main cause of coronary heart disease,which is one of the leading causes of death in the world.Acute coronary events are dangerous and life-threatening.Studies have shown that about 75%of acute coronary events are caused by rupture of unstable plaques.Atherosclerosis is a ehronic inflammatory disease.Inflammatory cells and factors plays an important role in the plaque formation,development,rupture and thrombosis.Neutrophils are the main cells in peripheral blood,which can be recruited into plaque tissue and participate in plaque development,rupture and thrombosis formation by secreting inflammatory factors.Clinical studies have demonstrated that there is an association between neutrophil to lymphocyte ratio(NLR)and the severity,non-calcified plaque burden and coronary artery calcium score of coronary atherosclerosis.Several studies have confirmed that patients with non-calcified or mixed plaques of coronary artery had a higher risk of poor outcomes compared with those without plaques or who had calcified plaques.However,there is no published data on the relationship between NLR and presence of non-calcified or mixed coronary atherosclerotic plaques in Chinese population.Aim:To investigate the association of NLR level with the presence of non-calcified or mixed coronary atherosclerotic plaques.Methods:We retrospectively collected the clinical and laboratory data of 598 consecutive subjects undergoing CCTA with chest pain.Coronary plaques were classified as calcified,non-calcified and mixed.If the patients had one more type of plaques,the most stenotic plaque was recorded.In this study,the subjects were divided to three groups according to the type of plaques.There were 168 subjects who had no coronary plaques(NP),219 patients with calcified plaques(CP),212 patients with non-calcified plaques(NCP)and mixed plaques(MP).Patients with NCPs or MPs had a high risk of poor outcomes;therefore,the two groups were combined(NCP/MP)for analysis.Results:NLR were significant higher in patients with NCP/MP,compared with the subjects with NP or CR To investigate the relationship between the NLR and NCP/MP,we divided our participants into 3 groups according to NLR tertiles.The prevalence of NCP/MP increased significantly from 28.6%in tertile 1 to 42.7%in textile 3 group and patients with NP were more likely to be in tertile 1 and textile 2 groups.Spearman correlation analysis showed that NLR was positively correlated with high-sensitivity C-reactive protein(hs-CRP)(r=0.177,p<0.001).Multiple logistic regression analysis showed that NLR was an independent risk factor for the presence of NCP/MP(odds ratio=1.195;95%Cl:1.020-1.400;p=0.028),and subjects with high level of NLR had a higher risk of NCP/MP compared with subjects with low level of NLR.Conclusions:NLR is independently associated with the presence of NCP/MP.The findings highlight the potential value of these easily and cheap measured factors in predicting the presence of non-calcified and mixed plaques,and monitoring the development of atherosclerosis.Lysophosphatidic acid is associated with thrombus stability by inducing rapid neutrophil extracellular traps formation via a peptidylarginine.deiminase 4-dependent pathwayBaekground:Thrombosis is a potentially fatal disease eaused by the formation of blood clots in arteries or veins.The prevention and degradation of thrombosis remains the focus of medical research.Neutrophil extracellular traps(NETs),the new weapon of neutrophil,can bind red blood cells,platelets,plasma proteins et al.Previous studies have demonstrated that NETs play an important role in the formation and development of thrombosis.In contrast to fibrin thrombus,NETs-present thrombus cannot be completely dissolved by tissue plasminogen activator(tPA)and addition of DNase I can further accelerate the lysis of thrombus,which may point out the cause of poor therapeutic of routine thrombolytic therapy and the new target in clinic.Lysophosphatidic acid(LPA),a bioactive phospholipid,can platelet sharp change and aggregation,which may play an important role in thrombosis.Multiple cell ty^es have a response to the stimulation of LPA,such as endothelial cells,smooth muscle cells,T-lymphocytes,monocytes,macrophages and platelets.However,there are few published data on the relationship between the LPA and NETs in thrombosis.Aimsi To investigate whether LPA signaling regulate neutrophils to release NETs and clarify the effect of LPA on thrombosis development.Methods:The laboratory data and plasma of patients with/without acute pulmonary embolism(APE)were collected.The plasma level of markers of NETs and LPA levels in patients with/without APE were detected using modified ELISA,high-performance-liquid-chromatography electrospray ionization tandem mass spectrometry.In addition,intrapulmonary thrombus specimens were collected from patients with chronic thromboembolic pulmonary hypertension(CTEPH)undergoing surgery.Immunofluorescence was used to detect the expression of NETs and autotaxin(ATX)in human intrapulmonary thrombus.Real time polymerase chain reaction(PCR)was used to detected the expression level of LPA1-6 in neutrophils.The survival and migration of neutrophils were detected by using the method of MTT and Transwell assays.Meanwhile,Sj^toxGreen fluorescent dye was used to the release of NETs from neutrophils under the stimulation of LPA.The signaling mechanism of LPA regulating the release of NETs was investigated by using western blot assay.All data analyses were performed using GraphPad Prism software(Version 7.0c).A two-sided P<0.05 was considered significant.Results:Patients with APE showed elevated levels of neutrophils,the markers of NETs(dsDNA,citrullinated histone H3 and nucleosomes)and LPA,compared to control subjects.Histological analysis showed that NETs presented in intrapulmonary thrombus from CTEPH patients,which were marked by the expression of citH3 and MPO,In addition,ATX was also detected in the same location and NETs were surrounded with ATX.Fluorescence observation showed that LPA induced the release of web like dsDNA from neutrophils which was labelled by SytoxGreen.After incubation with DNase I,spread dsDNA was digested.Furthermore,the NETs formation was confirmed by immunofluorescence with citH3 and MPO antibodies.Then,we tested LPA and PMA in the same system and found that the NETs induced by LPA and PMA followed distinct kinetics.The time course analysis showed that LPA gave a more rapid response after 30 min compared with PMA.In vitro,we found that LPA2 receptor was highly expressed in neutrophils,followed by LPA6 receptor.However,blocking LPA 1-5 receptors did not inhibit the secretion of NETs induced by LPA,which indicated that LPA6 might mediate the secretion of NETs induced by LPA.Furthermore,LPA-induced NETs were significantly inhibited by the inhibitors of mTOR,PKA,PKC and peptidylarginine deiminase 4(PAD4).Meanwhile,western bolt analysis showed that LPA significantly induced histone H3 citrullination and PAD4 inhibitor GSK484 almost completely abolished the LPA-induced citrullination of histone H3.In addition,inhibition of mTOR,PKA and PKC signaling also significantly inhibited the citrullination of histone H3,suggesting that LPA could activate PAD4 through multiple signaling pathways.Next,we explore the function of LPA-induced NETs and found that LPA-induced NETs provided a scaffold for the plasma proteins binding,such as von willebrand factor(vWF),fibrinogen and fibronectin,which are able to stabilize the thrombus.In addition,LPA-induced NETs could generate a tissue plasminogen activator(tPA)-resistant thrombus.In tPA-resistant clots,most areas were filled with NETs and NETs interacted closely with fibronectin.Addition of DNase I could further accelerated the lysis of NETs-presented thrombus.Furthermore,we found that DNase I in addition to tPA substantially accelerated the lysis of human intrapulmonary thrombi ex vivo.Meanwhile,we found that LPA-induced NETs could further induce platelets to secrete LPA,which indicated that there might be a cascade amplification of LPA-NETs signaling in thrombosis.Conclusions:We are the first to implicate LPA in regulating the stability of thrombus by inducing rapid NETs release in vitro and ex vivo.These findings provide a new insight into the prevention and therapy of venous thromboembolic disease in which targeting LPA-NETs signaling pathway.