Screening Techniques For HPS Patients And The Study Of Serum-induced Directed Migration Of HPMVEC Population

Part1 A study on IPVD screening technology for HPS patients based on machine learningBackgroundHepatopulmonary syndrome(HPS)is a serious pulmonary complication caused by intrapulmonary vascular dilation(IPVD)and hypoxemia based on chronic liver disease.which significantly increased perioperative lung infections,respiratory failure,and even death.It is important for HPS patients to screen and treat in early stage;contrast enhanced echocardiography(CEE)is an important diagnostic methods,but which is invasive and expensive,and difficult to implement in large quantities patients.In particular,there are many liver disease patients in China,but the diagnosis of HPS is only considered when breathing difficulties come to particularly significant.To date,there is still a lack of rapid and non-invasive HPS screening and diagnosis technology.Based on the previous research,we cooperated with the Chongqing Institute of Green and Intelligent Technology to carry out the research on the IPVD screening strategy of HPS patients based on machine learning.It is helpful for the rapid and effective screening of HPS patients,and reducing the incidence of various pulmonary complications during the perioperative period,with important clinical significance.Methods1.The study protocol was approved by the institutional ethics committee of the First Affiliated Hospital of Third Military Medical University(No.2017(35),and registered in clinicaltrials.gov(NCT03435406).patients with liver cirrhosis underwent elective surgery in the First Affiliated Hospital of Army Medical University from July 27,2017 to April 1,2018 were selected to participate in the study2.All enrolled patients underwent CEE,ABG,and physical examination;and biochemical indicators were obtained from electronic medical records(EMR)3.Three screening strategies were used for analysis,namely clinical symptoms strategy(N strategy),clinical symptoms+blood gas analysis strategy(CA strategy),clinical symptoms+blood gas analysis+biochemical index outcome strategy(CAS strategy);4.combined with the Chongqing Institute of Green and Intelligent Technology,four ML algorithms:logistic regression(LR),adaptive boosting(adaptive boosting,AdaBoost),support vector machines(SVM),and gradient boosting A decision tree(gradient boosting decision tree,GBDT)were used for model construction and prediction.The sensitivity,specificity,positive predictive value,F1-SCORE,and area under curve(AUC)were used to evaluate the screening diagnosis effectResults1.A total of 306 eligible patients were included,after eliminated for a variety of reasons,193 were finally included for further research.Of all the 193 patients,117(60.6%)were associated with IPVD,and 76 were not associated with(39.4%)IPVD.There was no significant difference in age,gender,BMI,prevalence of hypertension and diabetes,MELD score,drinking and smoking index among the two groups of patients2.Compared with the patients without IPVD,IPVD patients have higher Child-Pugh score,IBIL,and alveolar-arterial oxygen partial pressure difference;ALB,Pa02,Sp02 are lower,and prothrombin time,PT)longer3.Clinical indicators significantly associated with IPVD include clubbing fingers,palms of the liver,spider moles,dyspnea,ascites,and hepatic encephalopathy4.The four algorithms modeled the three strategies respectively.The results suggest that GBDT is overfitting and LR is underfitting.The AUC of the SVM(0.82,95%Cl:0.72-0.92)is significantly smaller than AdaBoost(0.88).,95%CI:0.80-0.96)5.Comparison of the three strategies in AdaBoost,the results suggest that there is no statistical difference in AUC between the three strategies of N,CA,and CAS.The specificity of N and CA is the same(81.8%vs.81.8%),but the false negative rate is higher(30.6%vs..18.2%)6.The IPVD screening strategy:AdaBoost can be used for initial screening of the N strategy;if the predicted value is less than 0.538,then the CA strategy is re-evaluatedConclusionAmong the N,CA and CAS strategy,AdaBoost algorithm is significantly better than SVM,LR,GBDT;AdaBoost-based cirrhosis IPVD screening technology is significantly better than the current single index prediction method,which can be used for rapid perioperative screening of HPS patients and has clinical significance.Recommended The two-step strategy based on Adaboost with clinical features and ABG analysis may prove highly promising for preoperative screening cirrhotic patients’ presence of IPVDPart2 Cyclooxygenase-2 regulates HPS patient serum induced-directional collective HPMVEC migration via PKC/Rac signaling pathwayBackgroundThe mechanism of IPVD in HPS is still unknown,result to difficult prevention and treatment.In recent years,it has been found that pathological pulmonary angiogenesis(PPA)promotes the occurrence and development of IPVD and leads to the deterioration of hypoxemia.In this process,there is a significant reverse collective migration of the human pulmonary microvascular endothelial cells(HPMVEC),which become an important early intervention target for HPS,and is worthy of further study.Cyclooxygenase(COX)is a key rate-limiting enzyme in the conversion of arachidonic acid to prostaglandins;it consists of two subtypes,including COX 1 and COX-2.In our previous studies,it has been shown that the upregulation of COX-2 in PMVECs promotes the accumulation of activated CD68(+)large phages in the lung microvessels of CBD rats,which may promote the reverse collective migration of HPMVEC and accelerate the process of PPA.In this study,we investigated the effect of COX-2 on the directed collective migration of HPMVEC under the serum of HPS patients,and whether this effect depends on the activation of the PKC/Rac signaling pathway,in order to find better intervention targets for the prevention and treatment of HPSMethods1.Collect serum from 9 HPS patients and 12 healthy people for research;2.Identification when resuscitate the HPMVEC cell line by conventional methods after inoculation and climbing3.Transwell,endothelial cell tube formation test and scratch test were used to detect the effect of HPS patients induced by directional migration of HPMVEC;4.Western blot was used to detect the expression of COX2 in HPMVEC;ELISA was used to detect the expression of PGE2 in HPMVEC medium;scratch test was used to detect the control group,HPS group,control+parecoxib group,HPS+Directional migration of cells in the parecoxib group;5.Western blot was used to detect the changes of P-PKC in normal serum and HPS patient serum after stimulation of HPMVEC.Immunofluorescence was used to detect the change of PKC expression after Si-RNA PKC was transcribed into HPMVEC.Scratch test was used to detect the control group,HPS group,Differential cell migration in control+Si-RNA PKC group and HPS+Si-RNA PKC group;6.Pull-down method was used to detect the expression level of GTP-Rac;Si-RNA PKC was transcribed into HPMVEC,and Western blot was used to detect the expression levels of PKC and P-PKC;Si-RNA PKC was transcribed into HPMVEC and pulled-down method Detection of GTP-Rac expression levelResults1.The survival rate of HPMVEC recovery is about 90%,the shape is long spindle or polygon,the cells are tightly connected,and the appearance is "paving stone”;2.The results of Trans well showed that HPS serum significantly promoted the migration of HPMVEC in the upper chamber to the lower chamber;the results of tube formation experiments suggested that the serum of HPS patients promoted the angiogenesis of HPMVEC;the results of the scratch test suggested that the serum of HPS patients promoted the endothelial cells in Migration in the wound area;calculations with Matlab software showed that HPMVEC migrated for a longer distance,a wider area,and a longer duration under the stimulation of serum from HPS patients;3.The serum of the control group had no significant effect on the expression of COX-2;Serum has no significant effect on the expression of PGE2,while serum of HPS patients up-regulates the expression of PGE2 in the medium in a time-dependent manner;In the absence of parecoxib,compared with the control group,the consistency of the scratch growth distance and the movement direction of the HPS group is more obvious;after adding parecoxib,compared with the control group,the consistency of the scratch growth distance and the movement direction of the HPS group The difference was not statistically significant;4.Compared with normal serum,HPS serum stimulated HPMVEC,PKC phosphorylation increased significantly in a time-dependent manner;COX-2 selective antagonist Parecoxib inhibited PKC activation in serum of HPS patients,and exogenous PGE2 could be reversed Inhibition of PKC activation by Parecoxib;after 12 hours,the length of the scratch growth was the longest in the HPS group,the edges were the most complete,and the direction of movement was consisted;the number of outlier cells was the largest in the HPS+Si-PKC group;the proportion of cells that sustained motion was highest in control+Si-PKC In the grou;5.After 36 hours,the content of GTP-Rac in the HPS group increased with time and was significantly higher than the normal group;there was no significant difference between the HPS+Si-RNA PKC group and the control+Si-RNA PKC group;but the contents of PKC and P-PKC in the HPS+Si-RNA empty group were significantly increased when PKC phosphorylation was not interference;the results showed that there was no significant difference in GTP-Rac between the control+Si-RNA empty vector group and the control+Si-RNA PKC group;both were lower than HPS+Si-RNA empty vector group,HPS+Si-RNA PKC group,of which HPS+Si-RNA empty vector group has the highest contentConclusionCOX-2 activates the PKC/Rac signaling pathway,thereby promoting the HPMVEC significant population-oriented migration,leading to PPA progression,which may be one of the important reasons for the development of IPVD during HPS.Which means COX-2 specific inhibitors have a significant perioperative lung protection in patients with HPS.