Proteomics And Prognosis Of Patients With Pulmonary Hypertension

Part I:Lung Tissue Proteomics Research in Idiopathic Pulmonary Arterial Hypertension Patients and Validation Different Expression of TransgelinObjective:Pulmonary arterial hypertension (PAH) is a disorder of vascular remodeling causing increased resistance to pulmonary blood flow. Idiopathic pulmonary arterial hypertension (IPAH) was defined as PAH unexplained by any secondary cause. At present the pathogenesis of IPAH is not yet clear. Recent developments of proteomics provide powerful tools for studying the key proteins involving in many disease. The expression of proteins in lungs from pulmonary arterial hypertension patients was investigated using iTRAQ coupled liquid chromatography and tandem mass spectrometry(LG MS/MS) to further understand the pathobiology of this disease.Methods:six patients undergoing lung transplant with IPAH were enrolled, and six patients with other diseases who accepted operation on lung or organs near lung were enrolled as control group. The PAH diagnosis was confirmed using right heart catheterization (RHC). The proteins of two groups was analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography-mass spectrometry. Functional enrichment analysis was performed to understand the function classification and molecular activity of all detected differentially expressed proteins. Protein interaction network and signal pathways analysis were performed to known the network and signal pathways differentially expressed proteins involved. The potential proteins were selected based on bioinformatics analysis and related researches.Results:1. In total,112 differentially expressed proteins were identified by TripleTOFTM 5600 Mass spectrometer. Compared to control group, there were 48 protein increased to≥1.5 times in IPAH patients, and 64 proteins decreased to≥1.5 times in IPAH patients.2.The functional enrichment analysis indicated that these detected proteins maybe involved in many biological processes, such as response to wounding, extracellular structure organization, inflammatory response, cytoskeleton organization, et al. The network analysis showed there were main eight network module, as regulation of immune system process, response to endogenous stimulus, system development, response to growth factor, extracellular regulation of signal transduction, cotranslational protein targeting to membrane, mitotic nuclear envelope disassembly, et al. Signal passway analysis demonstrated that differentially expressed proteins were involved in cytoskeleton remodeling, blood coagulation, heme metabolism, development_S1P2 and S1P3 receptors in cell proliferation and differentiation, et al.3. In this study, we found that Transgelin was upregulated in IPAH patients(IPAH/control=5.105, P=0.000). Immunohistochemical demonstrated that Transgelin was expressed mainly in vascular smooth muscle, and western blot showed Transgelin was upregulated in lung tissues of IPAH patients (P=0.017).Conclusions:112 proteins differently expressed in lung tissues from control group and IPAH patients were detected by iTRAQ-LC-MS. Immunohistochemical and western blot verified that Transgelin was pregulated in lung tissues from patients with IPAH.Part Ⅱ:Proteomic Analysis of Lung Tissues from Patients with Pulmonary Arterial Hypertension associated with Congenital Heart DiseaseObjective:Pulmonary arterial hypertension (PAH) is a common complication of congenital heart disease, and is now predominantly among patients with uncorrected left-to-right shunts. There is a growing population characterized by persistent or recurrent PAH after surgical or interventional correction of left-to-right shunts and worse prognosis than other forms of PAH associated with congenital heart disease(PAH-CHD). For patients with PAH-CHD considered borderline for surgery, there are no evidence-based recommendations of how best to proceed. Recent developments of proteomics provide powerful tools for studying key proteins involving in many disease. The expression of proteins in lungs from pulmonary artery hypertension in patients with congenital heart disease(CHD-PAH) was investigated using iTRAQ coupled liquid chromatography and tandem mass spectrometry(LG MS/MS) to further understand the pathobiology of this disease.Methods:CHD-PAH patients undergoing surgical repair of congenital heart disease were enrolled, and six patients with other diseases who accepted operation on lung or organs near lung were enrolled as control group. After surgical, CHD-PAH patients were followed up for one year, and they received reexamination of right heart catheterization (RHC) one year after surgical, patients with mean pulmonary artery pressure<25 mmHg were defined as reversible PAH, and patients with mean pulmonary artery pressure>25 mmHg were defined as irreversible PAH. The proteins of three groups (control group, reversible PAH group, irreversible PAH group) were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography-mass spectrometry. Functional enrichment analysis was performed to understand the function classification and molecular activity of all detected differentially expressed proteins. Protein interaction network and signal passway analysis were performed to known the network and signal passway differentially expressed proteins involved. The potential proteins were selected based on bioinformatics analysis and related researches.Results:1.In total,160 differentially expressed proteins were identified by TripleTOFTM 5600 Mass spectrometer. Compared to reversible PAH group, there were 85 protein increased to≥1.5 times in irreversible PAH group, and 75 proteins decreased to≥1.5 times in irreversible PAH group.2.The functional enrichment analysis indicated that these detected proteins maybe involved in many biological processes, such as cell adhesion, biological adhesion, response to wounding, positive regulation of endocytosis, actin filament-based process, cell-substrate adhesion, cytoskeleton organization, actin cytoskeleton organization, acute inflammatory response, translational elongation, blood coagulation, hemostasis, cholesterol transport, et al. The network analysis showed there were 15 network module, as regulation of immune system process, immune response-regulating cell surface receptor signaling pathway, cellular component organization, system development, muscle contraction, positive regulation of protein metabolic process, viral transcription, positive regulation of biological process, complement activation, positive regulation of immune system process, response to growth factor, et al. Signal passway analysis demonstrated that differentially expressed proteins were involved in Immune response_classical complement pathway, Cell adhesion_entegrin-mediated cell adhesion and migration, Cytoskeleton remodeling, Cell adhesion_endothelial cell contacts by non-junctional mechanisms, Blood coagulation, Transport_HDL-mediated reverse cholesterol transport, Stimulation of TGF-beta signaling in lung cancer, Cytoskeleton remodeling_Integrin outside-in signaling et al.3.Based on bioinformatics analysis and related researches, we selected Caveolin-1, Filamin A and Apolipoprotein A-I as potential proteins related to the irreversible PAH of CHD patients.Conclusions:160 proteins differently expressed between lung tissues from control group and IPAH patients were detected by iTRAQ-LC-MS,and Caveolin-1, Filamin A and Apolipoprotein A-I may be potential proteins related to the irreversible PAH of CHD patients.Part III:Survival Advantages of Excess Body Mass Index in Patients with Idiopathic Pulmonary Arterial HypertensionObjective:An obesity paradox, a "paradoxical" decrease in morbidity and mortality with increasing body mass index (BMI), has been shown in patients with heart failure. However, the impact of BMI in patients with IPAH has not been studied. This study aims to find out whether BMI is a prognostic factor in idiopathic pulmonary arterial hypertension (IPAH).Methods and results:We analysed 173 patients with IPAH. The patients were subclassified into categories of BMI defined as:underweight (< 18.5 kg/m2), normal weight (18.5 to 24.9 kg/m2), overweight and obese (25 to 34.9 kg/m2). The three BMI groups had similar profiles in terms of haemodynamic parameters assessed by right heart catheterization and level of NT-pro BNP. The overweight and obese group had older age, and better WHO functional class, larger left ventricular end-diastolic dimensions (LVEDDs) than the other two groups. The Kaplan-Meier survival curves for the three BMI categories demonstrated that the overweight and obese group had a significantly higher survival rate than the normal weight and underweight groups (log-rank test, P= 0.027, P= 0.000, respectively). In a stepwise forward regression, lower BMI, higher WHO functional class, lower cardiac index, smaller LVEDDs and absence of targeted medication remained independent predictors of mortality.Conclusions:Excess body mass is a protective factor for death in patients with IPAH.Part IV:High levels of serum lactate dehydrogenase correlate with the severity and mortality of idiopathic pulmonary arterial hypertensionObjective:Liver dysfunction refects the status of heart failure, and previous studies have demonstrated that serum lactate dehydrogenase (S-LDH) levels are increased in patients exhibiting heart failure and liver dysfunction. Right heart failure is a main characteristic of idiopathic pulmonary arterial hypertension (IPAH). The aim of the present study was to assess the prognostic signifcance of S-LDH levels in patients with PAH.Methods:S-LDH levels were determined in 173 patients with IPAH, and patients were subclassifed into two groups according to a defined upper reference limit of S-LDH (250 IU/L). Right heart catheterization was performed in all patients.Results:A total of 53 patients were found to have elevated S-LDH≥250 IU/L. In a mean follow-up period of 31.2± 17.9 months,57 patients succumbed. In the group with lower S-LDH levels (S-LDH<250 IU/L),16.7% of the patients succumbed, compared with 69.8% of patients in the group with higher S-LDH levels (S-LDH≥250 IU/L). The Kaplan-Meier survival curves demonstrated that patients with higher S-LDH levels had a signifcantly lower survival rate than did those with lower S-LDH levels (log-rank test, P<0.001). Cox proportional hazard analyses identifed reduced body mass index, reduced cardiac index, elevated World Health Organization functional class, elevated S-LDH and an absence of PAH-targeted therapy as signifcant predictors of adverse outcomes.Conclusion:elevated S-LDH is a risk factor for mortality in patients with IPAH.