Original Article
Anti-endothelial cell antibodies in connective tissue diseases associated with pulmonary arterial hypertension Xiao-Dan Liu, Sheng-Yu Guo, Li-Li Yang, Xiao-Li Zhang, Wen-Yi Fu, Xiao-Fei Wang Department of Rheumatology, Shengjing Hospital of China Medical University, Shenyang 110004, China Correspondence to: Professor Xiao-Fei Wang. Department of Rheumatology, Shengjing Hospital of China Medical University, 36 Sanhao Street, Heping District, Shenyang 110004, China. Email: [email protected].
Objective: To investigate the prevalence of anti-endothelial cell antibodies (AECA) in connective tissue diseases (CTD) associated with pulmonary arterial hypertension (PAH) and to corroborate the pathologic function of AECA in PAH-associated CTDs. Methods: AECA were detected by cellular enzyme-linked immunosorbent assay (ELISA) in sera of 19 PAH-associated CTD patients, 22 CTD patients without PAH involvement, and 20 age- and sex-matched healthy individuals as controls. Using IgG purified from the sera of AECA-positive, AECA-negative, and healthy subjects, the effects of AECA on the expression of ICAM-1 and the chemokine regulated upon activation normal T-cell expressed and secreted (RANTES) in cultured endothelial cells were also evaluated. Results: A total of 12 of the 19 (63.2%) CTD patients with PAH, 9 of the 22 (40.9%) CTD patients without PAH, and 1 of the 20 (5%) healthy controls were positive for AECA, which were calculated as ELISA ratio (ER) values. ER values in PAH-associated CTD patients were significantly higher than those with CTD without PAH (3.68±2.05 versus 1.67±1.07, P25 mmHg. A total of 41 CTD patients admitted to the Rheumatology Department of China Medical University between 2008 and 2011 were enrolled in the study, including the study group (PAH-associated CTD group, n=19) and disease control group (no-PAH CTD group, n=22). In the 19 PAH-associated CTD patients, the underlying diseases included systemic sclerosis (SSc, n=8), systemic lupus erythematosis (SLE, n=3), mixed connective tissue disease (MCTD, n=4), primary Sjögren syndrome (pSS, n=2), and undifferentiated connective tissue disease (uCTD, n=2), with no significant distribution difference compared to the diseases of the no-PAH CTD group. Sera samples were collected at the time of diagnosis when none of the patients were under systemic steroid or immunosuppressive therapy. Twenty age- and sex-matched healthy volunteers served as the control group. The study protocol was approved by our institutional review board, and each patient gave informed consent.
© Pioneer Bioscience Publishing Company. All rights reserved.
Cellular enzyme-linked immunosorbent assay (ELISA) for AECA HPAECs (ScienCell Corp.) at passage 4 were seeded onto 96-microculture plates and allowed to reach confluence. AECA were detected using a cyto-ELISA method as described previously (10). In brief, after being fixed with 0.1% glutaraldehyde, sera samples diluted 1:400 were added to triplicate wells for 1 h at 37 ℃. After washing, horseradish peroxidase-conjugated goat anti-human IgG-F(ab') 2 diluted 1 : 1,000 was added for 1 h at 37 ℃. Then 3,3', 5,5'-tetramethylbenzidine was added as a substrate and the color reaction was terminated by the addition of 1 mol/L H2SO4. Optical density was measured at 450 nm and the results are expressed as the ELISA ratio (ER) = (S − A)/(B − A), where S is the absorbance of the sample, and A and B are the absorbances of negative and positive reference serum, respectively. Positivity was defined as ER values > mean +3 standard deviations (SD) of the healthy control values. Intra- and inter-assay coefficients of variation were 5% and 12%, respectively. Sera from six AECA-positive patients, six AECA-negative patients, and five AECA-negative healthy subjects were pooled and purified using Protein G Sepharose (GE Hitrap) chromatography. ICAM-1 and RANTES production by HPAECs To identify the effect of AECA on cultured HPAECs, we incubated arterial endothelial cells forming a monolayer with IgG purified from AECA-positive, AECA-negative, or healthy control sera. ICAM-1 and RANTES levels were measured using a commercial ELISA kit (USCN Life Science Inc., Wuhan, China), following the manufacturer’s instructions. Statistical analysis All data are presented as means ± SD or as percentages. The t-test and χ2 test were used for statistical comparisons. Statistical analysis was performed with the SPSS software version 17.0, and P
Anti-endothelial cell antibodies in connective tissue diseases associated with pulmonary arterial hypertension Xiao-Dan Liu, Sheng-Yu Guo, Li-Li Yang, Xiao-Li Zhang, Wen-Yi Fu, Xiao-Fei Wang Department of Rheumatology, Shengjing Hospital of China Medical University, Shenyang 110004, China Correspondence to: Professor Xiao-Fei Wang. Department of Rheumatology, Shengjing Hospital of China Medical University, 36 Sanhao Street, Heping District, Shenyang 110004, China. Email: [email protected].
Objective: To investigate the prevalence of anti-endothelial cell antibodies (AECA) in connective tissue diseases (CTD) associated with pulmonary arterial hypertension (PAH) and to corroborate the pathologic function of AECA in PAH-associated CTDs. Methods: AECA were detected by cellular enzyme-linked immunosorbent assay (ELISA) in sera of 19 PAH-associated CTD patients, 22 CTD patients without PAH involvement, and 20 age- and sex-matched healthy individuals as controls. Using IgG purified from the sera of AECA-positive, AECA-negative, and healthy subjects, the effects of AECA on the expression of ICAM-1 and the chemokine regulated upon activation normal T-cell expressed and secreted (RANTES) in cultured endothelial cells were also evaluated. Results: A total of 12 of the 19 (63.2%) CTD patients with PAH, 9 of the 22 (40.9%) CTD patients without PAH, and 1 of the 20 (5%) healthy controls were positive for AECA, which were calculated as ELISA ratio (ER) values. ER values in PAH-associated CTD patients were significantly higher than those with CTD without PAH (3.68±2.05 versus 1.67±1.07, P25 mmHg. A total of 41 CTD patients admitted to the Rheumatology Department of China Medical University between 2008 and 2011 were enrolled in the study, including the study group (PAH-associated CTD group, n=19) and disease control group (no-PAH CTD group, n=22). In the 19 PAH-associated CTD patients, the underlying diseases included systemic sclerosis (SSc, n=8), systemic lupus erythematosis (SLE, n=3), mixed connective tissue disease (MCTD, n=4), primary Sjögren syndrome (pSS, n=2), and undifferentiated connective tissue disease (uCTD, n=2), with no significant distribution difference compared to the diseases of the no-PAH CTD group. Sera samples were collected at the time of diagnosis when none of the patients were under systemic steroid or immunosuppressive therapy. Twenty age- and sex-matched healthy volunteers served as the control group. The study protocol was approved by our institutional review board, and each patient gave informed consent.
© Pioneer Bioscience Publishing Company. All rights reserved.
Cellular enzyme-linked immunosorbent assay (ELISA) for AECA HPAECs (ScienCell Corp.) at passage 4 were seeded onto 96-microculture plates and allowed to reach confluence. AECA were detected using a cyto-ELISA method as described previously (10). In brief, after being fixed with 0.1% glutaraldehyde, sera samples diluted 1:400 were added to triplicate wells for 1 h at 37 ℃. After washing, horseradish peroxidase-conjugated goat anti-human IgG-F(ab') 2 diluted 1 : 1,000 was added for 1 h at 37 ℃. Then 3,3', 5,5'-tetramethylbenzidine was added as a substrate and the color reaction was terminated by the addition of 1 mol/L H2SO4. Optical density was measured at 450 nm and the results are expressed as the ELISA ratio (ER) = (S − A)/(B − A), where S is the absorbance of the sample, and A and B are the absorbances of negative and positive reference serum, respectively. Positivity was defined as ER values > mean +3 standard deviations (SD) of the healthy control values. Intra- and inter-assay coefficients of variation were 5% and 12%, respectively. Sera from six AECA-positive patients, six AECA-negative patients, and five AECA-negative healthy subjects were pooled and purified using Protein G Sepharose (GE Hitrap) chromatography. ICAM-1 and RANTES production by HPAECs To identify the effect of AECA on cultured HPAECs, we incubated arterial endothelial cells forming a monolayer with IgG purified from AECA-positive, AECA-negative, or healthy control sera. ICAM-1 and RANTES levels were measured using a commercial ELISA kit (USCN Life Science Inc., Wuhan, China), following the manufacturer’s instructions. Statistical analysis All data are presented as means ± SD or as percentages. The t-test and χ2 test were used for statistical comparisons. Statistical analysis was performed with the SPSS software version 17.0, and P
![[Pulmonary arterial hypertension associated with connective tissue diseases].](/assets/img/hold.png)
