Study On Anti-endothelial Cell Antibodies In Patients With Pulmonary Arterial Hypertension Associated With Connective Tissue Diseases

BackgroundPulmonary arterial hypertension (PAH) is defined as a group of diseases characterized by a progressive increase of pulmonary vascular resistance leading to right ventricular failure. It may also be associated with connective tissue diseases (CTD) such as systemic sclerosis (SSc), mixed connective tissue diseases, and systemic lupus erythematosus. PAH associated with CTD is now considered as a main problem of rheumatologists because of its unique clinical features, difficulty of early diagnosis and poor prognosis. It is a leading cause of death. Several studies had indicated that anti-endothelial cell antibodies (AECA) might be closely related to CTD associated PAH in recent years. A higher prevalence of AECA had been observed in CTD patients with PAH than those without PAH. A study has observed SSc patients have a high incidence in AECA-75KD by immunoblotting. While most AECAs were detected by ELISA on fixed layers of the human endothelial cell line previously, it is hard to define more precisely specific antigens in the group of heterophile antibodies. Recently, with the development of proteomics, it is helpful to investigate the specific antibodies and propose pathogenesis for the diseases by mass spectrometry techniques. The present study was therefore designed to identify novel auto-antibodies in CTD associated PAH by immunoblotting (focused on the target proteins with molecule weight 70～80KD) , isolate and identify the specific auto-antigen by matrix-assisted laser desorption ionization, time-of-flight mass spectrometry (MALDI-TOF MS), then investigate its role in the pathogenic process of CTD associated PAH.Objective1. To detect the positive rate of anti-endothelial cell antibody(AECA) in patients with pulmonary arterial hypertension (PAH) associated with Connective Tissue diseases (CTD) , and analyze their clinical significance.2. To identify the specific endothelial cell auto-antigen in PAH associated with CTD by MALDI-TOF MS.3. Establish antibody-mediated vascular endothelial injury model and evaluate its ability to induce the apoptosis of endothelial cell.4. Recombinate the recognized antigen by the specific antibody. Then express and purify the recombinant protein. Screen antibodies in sera from patients of experimental group and control groups, then confirm the presence of antibodies specific to the recombinant protein.Method1. To detect AECA by Cyto-ELISA and immunoblotting, then to analyze their clinical significance.1) With EA. hy926 (endothelial cell line) as substrate cell, sera from experimental group and control groups were screened for the presence of AECA by fixed cell-ELISA. The experimental group is composed of 68 patients with PAH associated with CTD. These patients were complicated only with PAH, without interstitial lung disease, glomerulonephritis, nervous system or other systemic involvement. Sera of 61 CTD patients without PAH (including 21 with glomerulonephritis, 20 with interstitial lung disease (ILD), and 20 patients without systemic involvement), 20 with idiopathic pulmonary arterial hypertension (IPAH), 20 with chronic obstructive pulmonary diseases and pulmonary arterial hypertension (COPD-PAH) , and 20 healthy persons were collected as controls. The results were analyzed with their relationships with the disease activity and clinical manifestation. 2) Extract membrane protein of the endothelial cell line EA. hy926 and Western blotting was performed to detect specific AECA in sera of the experimental group and controls mentioned above.2. Isolate and identity the auto-antigen of AECA related to PAH associated with CTD.1) The proteomics technique, with sodium dodecyl sulphonate denatured polyacryamide gel electrophoresis (SDS-PAGE) and MALDI-TOF MS in combination with western blotting were used to isolate and identity the auto-antigen of AECA related to PAH associated with CTD.2) Extract membrane protein of the Human Pulmonary Microvascular Endothelial Cells (HPMEC). Then undertake Two-Dimensional Gel Electrophoresis of two kinds of membrane (EA. hy926 and HPMEC) respectively, compare the two gels and analyze differences of the membrane protein by MS.3) Repeat Western blotting as mentioned above by positive serum IgG. Analyze the specific auto-antigen by MALDI-TOF MS.3. To obtain the monoclonal AECA specific to the antigen. Then incubate HPMEC with the mAb and observe the changes of function and structure in order to investigate the possible mechanism of HPMEC's injury.4. Amplify the gene CDS of specific antigen by polymerase chain reaction(PCR). Combine the product with expression vector, analyze the recombinant protein by Mass spectrometry. Screen the sera from patients of experimental group and control group, to identify novel auto-Abs of the patients by western blotting, and discuss the clinical significance.Results1. The prevalence of AECA by EA. hy926 cyto-ELISA was 77.25% in CTD patients, significantly higher than those patients without CTD(0.06%) (P<0.01%), while there is no significantly difference in groups with CTD patients. The positive rates of CTD with PAH, CTD with glomerulonephritis, CTD with ILD, CTD without systemic involvement are as follows respectively: 76.4%(52/68), 81.0% (17/21) , 85.0% (17/20), 70.0% (14/20).2. With membrane protein abstracted from EA. hy926 to screen AECA in sera of different groups. We found that the specific molecular size of AECA was 78kDa. The 78kDa AECA-positive rate of CTD patients with PAH was 79. 4% (54/68), not notably different from that of CTD with glomerulonephritis (71.4%, 15/21), but significantly higher than those of CTD with ILD (15.0%, 3/20) and CTD without systemic involvement (P< 0.01 and P< 0.05 respectively), also higher than those of IPAH (8. 3%. 1/12). The 78KD-AECA was negative in COPD-PAH and healthy controls.3. A higher positive rates of AECA-78KD were related to Raynaud's phenomenon, long course with more than 5-year duration and severity of PAH. While AECA-78KD positive rate has no correlation to patients' anti-U1RNP antibody, anti-SSA antibody and inflammation index including erythrocyte sedimentation rate, CRP, complement and leukocytes.4. The 78KD protein of endothelial cell extract was identified to be moesin by the combination of immunoblotting, Two-Dimensional Gel Electrophoresis and MS. 5. Moesin can be detacted by IIF on the membrane of HPMEC. Under the effect of TNF-α, HPMEC can express moesin and the monoclonal antibody of moesin will injury HPMEC by changing the structure of cytoskeleton protein and inducing early apoptosis.6. The CDS of human moesin gene was subcloned to expression vector. Recombinant human moesin was expressed in E. coli and purified. The recombinant protein was demonstrated to be human moesin by Mass spectrometry. The recombinant protein can be bind to monoantibody of moesin through Western blotting.7. Taking recombinant moesin(r-moesin) as substrate, we screen the sera from patients of experimental group and control group by immunoblotting analysis. The positive rates are as follows: 44. 1% (30/68) in CTD with PAH, 66.7% (14/21) in CTD with glomerulonephritis, 8.3% (1/12) in IPAH, 20.0%(4/20) CTD without systemic involvement and 15.0% (3/20) in CTD with ILD. The results have poor correlation to those of 78-KD AECA. As a diagnosis method of PAH in CTD patients, the sensitivity, specificity, positive predictive value and negative predictive value of anti r-moesin antibody are as follows respectively: 44.1%, 65.6%, 58.8%, and 51. 3%. The diagnosis of anti-recombinant moesin antibody has been 69.8% accordance to ultrasound cardiogram (UCG).Conclusion1. AECA could be found more frequently in patients with CTD than those without CTD by cyto-ELISA. CTD patients with different target organ involved has different 78kDa AECA-positive rate.2. Immunoblotting analysis indicated that CTD patients with different target organ involved has different 78kDa AECA-positive rate. AECA-78KD could be found more frequently in CTD patients with PAH and those with glomerulonephritis than CTD patients with ILD and patients without systemic involvement. AECA-78KD has correlation with Raynaud's phenomenon, course period of CTD and severity of PAH.3. Furthermore, AECA against the EC antigen (endothelial cell protein with molecular size of 78KD) with same molecular size could be found in a variety of CTD with PAH and CTD with glomerulonephritis. It was found to be moesin by the combination of immunoblotting and proteomics technology.4. The protein moesin can be detacted on HPMEC membrane. Under the effect of TNF-α, HPMEC can express moesin and the monoclonal antibody of moesin will injury HPMEC by changing the structure of cytoskeleton protein and inducing early apoptosis.5. Moesin is one of the auto-antigen recognized by AECA. The positive rate of anti r-moesin antibody is much higher in CTD with PAH and CTD with glomerulonephritis than CTD with ILD and CTD without systemic involvement. But the prediction value to PAH in CTD patients is limited.