Characterization of the inflammatory and apoptotic cells in the aortas of patients with ascending thoracic aortic aneurysms and dissections Rumin He,

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Presentation transcript:

Characterization of the inflammatory and apoptotic cells in the aortas of patients with ascending thoracic aortic aneurysms and dissections Rumin He, MD, Dong-Chuan Guo, PhD, Anthony L. Estrera, MD, Hazim J. Safi, MD, Tam T. Huynh, MD, Zhengnan Yin, MD, PhD, Shi- Nian Cao, MD, Jing Lin, MD, Thomas Kurian, L. Maximillian Buja, MD, Yong-Jian Geng, MD, PhD, Dianna M. Milewicz, MD, PhD The Journal of Thoracic and Cardiovascular Surgery Volume 131, Issue 3, Pages 671-678.e2 (March 2006) DOI: 10.1016/j.jtcvs.2005.09.018 Copyright © 2006 The American Association for Thoracic Surgery Terms and Conditions

Figure 1 T-lymphocyte infiltrates in TAA and TAD. For orientation, lumen of aorta is at top with adventitia at bottom. A through C, Immunohistochemical staining demonstrates that CD3+ cells (arrow) are rarely present in control aorta. D, F, and G, Media from patients with TAA and TAD demonstrate CD3+ cells (arrows) throughout media of aorta. E, Adventitia from patient with TAA demonstrates CD3+ cells throughout adventitia. H through J, CD4+ and CD8+ cells are present in aortic media from patients with TAA and TAD. K and L, Media from patient with TAA demonstrates that CD1α+ and CD68+ cells are present in aneurysmal aortic media. Monoclonal anti-CD3 antibody used in D was obtained from DAKO Corporation, Carpinteria, Calif; antibodies in panels A through C and E through F were obtained from Zymed Laboratories Inc, South San Francisco, Calif, for comparison. Original magnification 400×. The Journal of Thoracic and Cardiovascular Surgery 2006 131, 671-678.e2DOI: (10.1016/j.jtcvs.2005.09.018) Copyright © 2006 The American Association for Thoracic Surgery Terms and Conditions

Figure 2 CD3+ cells present in media from control, aneurysmal, and dissected aortas. CD3+ cells in 1 mm2 of aortic media were counted from aortic tissue sections from control subjects, patients with TAAs, and patients with TADs. Total areas examined were 2.2 mm2. Box-whisker plots display CD3. Central horizontal bars indicate median, upper and lower ends of boxes indicate 75th and 25th percentiles, and error bars indicate maximum and minimum values of CD3 values. P values were determined by Mann-Whitney Wilcoxon rank-sum tests. The Journal of Thoracic and Cardiovascular Surgery 2006 131, 671-678.e2DOI: (10.1016/j.jtcvs.2005.09.018) Copyright © 2006 The American Association for Thoracic Surgery Terms and Conditions

Figure 3 Increased TUNEL-positive and FasL staining in media of aneurysmal and dissected aortas. Top: A, TUNEL-positive cells (arrows) are rarely present in control aorta. B and C, Media from patients with TAA and TAD demonstrated TUNEL-positive cells (arrows). D through F, Double staining with combination of TUNEL and immunohistochemical assay with α-actin, CD3, and CD68 antibodies demonstrated that SMCs, T lymphocytes, and macrophages were undergoing apoptosis (arrows) in aortic media from patient with TAA. TUNEL staining in panels A through C and E used DeadEnd Colorimetric TUNEL System; that in panels D and F used DeadEnd Fluorescence TUNEL System. Bottom: A and C, Adventitia and media in aneurysmal aorta indicate influx of CD3+ cells in walls of vasa vasorum and aortic media. B and D, Same vasa vasorum and media were stained with anti-FasL antibody, which indicated colocation of CD3+ and FasL-positive cells. Magnification 400×. The Journal of Thoracic and Cardiovascular Surgery 2006 131, 671-678.e2DOI: (10.1016/j.jtcvs.2005.09.018) Copyright © 2006 The American Association for Thoracic Surgery Terms and Conditions

Figure 4 TUNEL-positive cells present in media from control, aneurysmal, and dissected aortas. TUNEL-positive cells in 1 mm2 of the aortic media were counted from aortic tissue sections from control patients, patients with TAAs, and patients with TADs. Total areas examined were 2.2 mm2. Box-whisker plots display TUNEL. Central horizontal bars indicate median, upper and lower ends of boxes indicate 75th and 25th percentiles, and error bars indicate maximum and minimum values of TUNEL values. P values were determined by Mann-Whitney Wilcoxon rank-sum tests. The Journal of Thoracic and Cardiovascular Surgery 2006 131, 671-678.e2DOI: (10.1016/j.jtcvs.2005.09.018) Copyright © 2006 The American Association for Thoracic Surgery Terms and Conditions

Figure E1 Medial degeneration of aorta associated with loss of SMCs and elastic fibers. Representative cross-sections of human control and aneurysmal aortic tissue were stained with hematoxylin and eosin (A-C) or with monoclonal antibody directed against SMC α-actin (D-F). A and D, Media from control aorta demonstrates nuclei and elastic fibers throughout media. B and E, Media from patient with TAA reveals loss of cell nuclei in band in media, but elastic fibers remain. C and F, Media in different field from same patient as in B and E displays areas with loss of both cells and nuclei. Magnification 100×. The Journal of Thoracic and Cardiovascular Surgery 2006 131, 671-678.e2DOI: (10.1016/j.jtcvs.2005.09.018) Copyright © 2006 The American Association for Thoracic Surgery Terms and Conditions

Figure E2 Immunoblot analysis showed levels of Fas, FasL, and activated caspase-3 to be increased in aneurysmal and dissected aortas. Levels of Fas and FasL were assessed relative to actin. Increased levels of Fas antigen were detected in aneurysmal (0.82 ± 0.39) and dissected (0.68 ± 0.62) tissues relative to control tissues (0.07). Increased levels of FasL antigen were detected in aneurysmal (0.71 ± 0.23) and dissected (0.42 ± 0.31) tissues relative to control tissues (0.15). Total protein loaded into each lane was 50 μg. After separation by sodium dodecylsulfate–polyacrylamide gel electrophoresis, membranes were blocked and incubated with primary antibody against Fas, FasL, and activated caspase-3. Membranes were incubated with horseradish peroxidase–linked secondary antibodies, followed by visualization with SuperSignal West Femto kit (Pierce Biotechnology, Inc, Rockford, Ill). Bands correspond to following molecular weighs of proteins: Fas, 40 kd; FasL, 37 kd; activated caspase-3, 20.4 kd. Results are consistent with results obtained with immunohistochemical testing. The Journal of Thoracic and Cardiovascular Surgery 2006 131, 671-678.e2DOI: (10.1016/j.jtcvs.2005.09.018) Copyright © 2006 The American Association for Thoracic Surgery Terms and Conditions