Ioannis K. Toumpoulis, MD, Julia Thom Oxford, PhD, Douglas B

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Differential Expression of Collagen Type V and XI α-1 in Human Ascending Thoracic Aortic Aneurysms Ioannis K. Toumpoulis, MD, Julia Thom Oxford, PhD, Douglas B. Cowan, PhD, Constantine E. Anagnostopoulos, MD, Chris K. Rokkas, MD, Themistocles P. Chamogeorgakis, MD, Dimitrios C. Angouras, MD, Richard J. Shemin, MD, Mohamad Navab, PhD, Maria Ericsson, BSc, Micheline Federman, PhD, Sidney Levitsky, MD, James D. McCully, PhD The Annals of Thoracic Surgery Volume 88, Issue 2, Pages 506-513 (August 2009) DOI: 10.1016/j.athoracsur.2009.04.030 Copyright © 2009 The Society of Thoracic Surgeons Terms and Conditions

Fig 1 Histological analysis using Masson's trichrome (A and B) and Movat's pentachrome (C and D). Representative serial sections of the tunica media of the aortic wall from control (A and C) and ascending thoracic aortic aneurysms (ATAA) (B and D) are shown. Collagen (C), smooth muscle cells (SMC), elastin (E), nuclei (N), and ground substance (GS) are shown. Scale bars represent 50 μm. Representative serial sections of the tunica media of the aortic wall from control (E) and ATAA (F) are shown. In E and F elastin autofluorescence is shown in green and collagen type I is shown in red. Scale bars represent 10 μm. Transmission electron microscopy collagen fibrils are shown in longitudinal and cross sections. Representative serial sections of the tunica media of the aortic wall from control (G) and ATAA (H) are shown. Collagen fibrils in ATAA are fragmented and disorientated as compared with control aorta. Scale bars represent 100 nm. The Annals of Thoracic Surgery 2009 88, 506-513DOI: (10.1016/j.athoracsur.2009.04.030) Copyright © 2009 The Society of Thoracic Surgeons Terms and Conditions

Fig 2 Immunohistological analysis of collagen α1(XI). (A) Control tissue and (B) ascending thoracic aortic aneurysms (ATAA) tissue, at ×200 original magnification. (C) Cystic medial degenerative (CMD) lesion at ×50 original magnification. (D) Control tissue, (E) ATAA tissue, and (F) CMD lesion from ATAA at ×630 original magnification. Tissue sections stained with antibody directed to collagen α1(XI) demonstrated staining within the tunica media in both control tissues and ATAA. Presence of collagen α1(XI) is indicated by brown staining (indicated by arrow CXI). Tissues were counterstained briefly with hematoxylin to stain nuclei (blue, indicated by arrow N). An increase in staining for collagen α1(XI) is shown within regions of CMD lesions. Scale bars in A to F are 100 μm. The Annals of Thoracic Surgery 2009 88, 506-513DOI: (10.1016/j.athoracsur.2009.04.030) Copyright © 2009 The Society of Thoracic Surgeons Terms and Conditions

Fig 3 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Panel A shows primer sets used for qRT-PCR and expected product size (open squares = forward primer; closed squares = reverse primer). Panel B shows a representative 1.5% agarose gel of amplified products from each primer set. Panels C to G show real-time quantitative amplification curves for collagen α1(I), α1(III), α2(V), α1(XI), and β-actin. For C–G, x-axis = cycle; y-axis = fluorescence. In C, measurements are 0–60 fluorescence units at increments of 10 along x-axis and 0–1.2 fluorescence units at increments of 0.1 along y-axis; in D–G, measurements are 0–60 fluorescence units at increments of 10 along x-axis and 0–1.5 fluorescence units at increments of 0.25 along y-axis. Ascending thoracic aortic aneurysm size is indicated. Panel H shows the fold changes versus controls. Significant differences versus control at p less than 0.001 are shown in bold type. The Annals of Thoracic Surgery 2009 88, 506-513DOI: (10.1016/j.athoracsur.2009.04.030) Copyright © 2009 The Society of Thoracic Surgeons Terms and Conditions

Fig 4 Western blot analysis. A representative 10% sodium-dodecyl-sulfate polyacrylamide gel stained with Coomassie brilliant blue for each aneurysm size is shown in panel A. Representative Western immunoblots are shown in panel B. Densitometric analysis of Western blots is shown in panel C. Ascending thoracic aortic aneurysm size is indicated. Significant differences at p less than 0.001 versus control are shown as *. The Annals of Thoracic Surgery 2009 88, 506-513DOI: (10.1016/j.athoracsur.2009.04.030) Copyright © 2009 The Society of Thoracic Surgeons Terms and Conditions

Fig 5 Spearman rank correlations of the messenger ribonucleic acid (A) and protein (B) expression levels of collagens type I, III, V, and α1(XI) with aneurysm size are shown. Control mean diameter was 2.3 cm. (ATAA = ascending thoracic aortic aneurysm.) The Annals of Thoracic Surgery 2009 88, 506-513DOI: (10.1016/j.athoracsur.2009.04.030) Copyright © 2009 The Society of Thoracic Surgeons Terms and Conditions