| BackgroundChronic thromboembolic pulmonary hypertension (CTEPH) constitutes Group 4 of the Dana Point classification of pulmonary hypertension. It develops in 0.4% to 5.1% of all acute pulmonary thromboembolism(PE) patients. The characteristics of CTEPH is the presence of unresolved thromboemboliundergoing fibrotic organisation. The pathophysiological mechanism of CTEPH remain unclear. Previous studies have shown that,1) ironoverload plays important role in thrombotic diseases; 2) typical plexogenic lesions indistinguishable from idiopathic pulmonary arterial hypertension are believed to occur in normal-flowdistal pulmonary vascular bed of CTEPH patients; 3) differential proteomics is a robust technique to clarify pathogenesis and find probable specific target proteins or signal transduction pathways; 4) management of acute PE patients could be improved by adding sensitive predictors. Herein, we focused on iron metabolism, gene muatution, proteomics changes in CTEPH patients in this study to investigate the pathophysiological mechanism. And we also conducted cardiopulmonary exercise testing in CTEPH patients and patients recovered from acute PE patients to find a sensitive and reliable predictor for CTEPH.Part 1 The relationship between iron status and CTEPHObjectTo explore whether or not iron status participate in the development of CTEPH.MethodsA case-control study was conducted.45 CTEPH patients were enrolled as cases and 36 age, sex-frequency matched chronic PE patients without pulmonary hypertension were selected as controls. Levels of free iron, soluble transferrin receptor (sTfR), ferritin, sTfR/ferritin ratio, hepcidin-25, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, and malondialdehyde (MDA) were compared between the two groups. logistic regression analysis was carried out to estimate odds ratios (ORs).ResultsThere was no difference of the levels of free iron, hepcidin-25, sTfR, ferritin, sTfR/ ferritin ratio, TNF-a and MDA between CTEPH patients and the controls. Levels of sTfR and ferritin in both groups were within the normal limits. Levels of IL-6 in CTEPH patients were significantly higher than that in the controls. A negative correlation was observed between hepcidin-25 and sTfR (Spearman’s r=-0.438, p< 0.001), and a positive correlation was observed between hepcidin-25 and ferritin (Spearman’s r=0.503, p<0.001). In the univariate logistic regression model, there was no association observed between CTEPH and free iron, hepcidin-25, sTfR, ferritin, sTfR/ferritin ratio, TNF-α, IL-6 and MDA.Part 2:The frequency of gene mutation in Chinese patients with CTEPHObjectTo investigate whether PAH-causing gene mutations exist in some CTEPHpatients, and act as a background to facilitate the development of CTEPH.MethodsForty-nine CTEPH patients and 17 patients recovered from acute PE and without PH were tested of gene mutation for BMPR2, ACVRL1, ENG, SMAD9, CAV1, KCNK3, and CBLN2 by direct sequencing and multiple ligation probe amplification (MLPA) analysis.ResultsA total of 40 mutations were identified, including 31 point mutations and 9 large size rearrangements. Among them,5 mutations were synonymous (3 in CTEPH patients,2 in PE without PH patients).Thirteen mutations were predicted to be probably damaging,2 mutation was predicted to be possibly damaging, and 2 mutations were predicted to be benign. Three patients had multiple point mutations.The nonsynonymous mutation rate in CTEPH patients is significantly higher than that in PE without PH patients (25 out of 49 (51%) CTEPH patients vs. 3 out of the 17 patients (18%); p=0.022).Twelve SNPs which already recorded in the public dbSNP database were detected among all the 66 patients. The allele of SNP rs3739817 and SNP rs55805125 were significantly correlated with CTEPH.Part 3:Proteomic analysis of proliferative tunica intima obtained from pulmonary thromboendarterectomyin patients with CTEPHObjectTo find "specific proteins" or "specific signal transduction pathways"for CTEPH.MethodsThe proliferative tunica intima tissue obtained from pulmonary thromboendar-terectomy in 7 patients with CTEPH was analyzed by high-performance liquid chromatography- mass spectrometry. The proteins were compared with mixture of cultured human pulmonary artery endothelial cells, human pulmonary artery smooth musle cells and human pulmonary fibroblast. GO and KEGG analysis was performed to understand the function classification and molecular activity of all the tissure "specific proteins" and "specific siginal transduction pathways".Results679 tissue specific proteins were detected. The GO analysis indicated that these proteins maybe involved in many biological process, such as response to wounding, immune response, acute inflammatory response, complement activation, blood coagulation and so on. KEGG analysis showed that these specific proteins might be related with many transduction pathways, including complement and coagulation cascades, systemic lupus erythematosus, ECM-receptor interaction, cell adhesion molecules, Fc epsilon R1 signaling pathway, viral myocarditis, prion disease pathway, Fc gamma R-mediated phagocytosis.Part 4:The role of cardiopulmonary exercise testing inprediction of CTEPHObjectTo find a sensitive and reliable ventilatory efficiency parameter during cardiopulmonary exercise testing (CPET) to monitor the process and predict CTEPH.MethodsFifteen patients rehabilitated from acute PE (total resolution of thrombi),44 patients with chronic PE (with residual thrombi),66 patients with CTEPH, and 36 sedentary healthy controls performed incremental CPET.ResultsThe lowest VE/VCO2 was higher in CTEPH patients than that in chronic PE and rehabilitated patients (43.4 L/min vs 29.9 L/min vs 27.1 L/min, p<0.005). The VE/VCO2 slope (48.4 L/min/L/min vs 29.9 L/min/L/min vs 28.0 L/min/L/min, p<0.005) and oxygen uptake efficiency plateau (OUEP) (37.1 L/min vs 27.0 L/min vs 25.2 L/min, p<0.005) had the similar changes. In logistic regression analysis, the lowest VE/VCO2 ≥34.35 L/min was the best predictor of CTEPH (OR 159.0, 95% CI 36.0-702.3, p<0.001). The lowest VE/VCO2 was higher in chronic PE patients compared with the controls (29.9 L/min vs 26.5 L/min, p<0.05), but there was no difference between the rehabilitated patients and the controls. In multiple linear regression analysis, the percentage of vascular obstruction by ventilation-perfusion lung scanning (PVO) was the most significant independent predictor for indices of ventilatory efficiency in chronic PE and rehabilitated patients.Conclusions1. CTEPH has no association with Iron overload. The iron status evaluated by sTfR and ferritin is within the normal limits in this CTEPH population.2. The high frequency of PAH-causing genes mutations might be associated with CTEPH in Chinese population.3. The mechanism of CTEPH might be involved with immune response, acute inflammatory response, complement activation, blood coagulation and ECM-receptor interaction, cell adhesion and migrations.4. CTEPH is associated with weakened ventilatory efficiency. The lowest VE/VCO2 ratio has the best capability to predict CTEPH. Ventilatory inefficiency improves along with recovery of acute PE. |
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