Inducible nitric oxide synthase is present in human abdominal aortic aneurysm and promotes oxidative vascular injury Jian Zhang, MD, Jan Schmidt, MD,

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Inducible nitric oxide synthase is present in human abdominal aortic aneurysm and promotes oxidative vascular injury Jian Zhang, MD, Jan Schmidt, MD, PhD, Eduard Ryschich, MD, Martina Mueller-Schilling, MD, Hardy Schumacher, MD, Jens Rainer Allenberg, MD, PhD Journal of Vascular Surgery Volume 38, Issue 2, Pages 360-367 (August 2003) DOI: 10.1016/S0741-5214(03)00148-4

Fig 1 Expression and cellular localizations of iNOS mRNA in AAA by in situ hybridization and immunohistochemistry. In situ hybridization with digoxingenin labeled iNOS probe showing strong expression of iNOS mRNA in inflammatory cells (A) and inflammatory infiltrating zone (B) in the media and adventitia of AAA (fast red as a chromogen, yielding a red reaction product in positive cells). C through E, serial sections were stained by in situ hybridization for iNOS mRNA, and combined with immunohistochemistry of anti-CD3, anti-CD20, or anti-CD68 on the same sections. DAB was used as chromogen for immunohistochemistry, yielding a brown reaction product at the cell membrane or/and cytoplasma. The expressions of iNOS mRNA were localized in T lymphocytes (C), B lymphocytes (D) and macrophages (E) (black arrows). F [top], we also observed an iNOS mRNA expression in medial mesenchymal cells. With parallel sections immunostained by anti-SMC actin (DAB as substrate), we identified that these medial mesenchymal cells were SMCs (F [bottom]). Journal of Vascular Surgery 2003 38, 360-367DOI: (10.1016/S0741-5214(03)00148-4)

Fig 1 Expression and cellular localizations of iNOS mRNA in AAA by in situ hybridization and immunohistochemistry. In situ hybridization with digoxingenin labeled iNOS probe showing strong expression of iNOS mRNA in inflammatory cells (A) and inflammatory infiltrating zone (B) in the media and adventitia of AAA (fast red as a chromogen, yielding a red reaction product in positive cells). C through E, serial sections were stained by in situ hybridization for iNOS mRNA, and combined with immunohistochemistry of anti-CD3, anti-CD20, or anti-CD68 on the same sections. DAB was used as chromogen for immunohistochemistry, yielding a brown reaction product at the cell membrane or/and cytoplasma. The expressions of iNOS mRNA were localized in T lymphocytes (C), B lymphocytes (D) and macrophages (E) (black arrows). F [top], we also observed an iNOS mRNA expression in medial mesenchymal cells. With parallel sections immunostained by anti-SMC actin (DAB as substrate), we identified that these medial mesenchymal cells were SMCs (F [bottom]). Journal of Vascular Surgery 2003 38, 360-367DOI: (10.1016/S0741-5214(03)00148-4)

Fig 2 Identification of iNOS protein expression in AAA. A representative AAA section immunostained with a monoclonal anti-iNOS antibody is shown (A through C). DAB was used as sustrate, yielding a brown color in positive cells. iNOS expression was observed in medial or adventitial inflammatory infiltration areas colocalized with lymphocytes (A) and macrophages (B) (black arrows). SMCs (C) also showed strong expression of iNOS protein (black arrows). D, Normal aortic tissue sections did not show any positive immunohistostaining for iNOS. Magnification × 400. Journal of Vascular Surgery 2003 38, 360-367DOI: (10.1016/S0741-5214(03)00148-4)

Fig 3 In situ hybridization for iNOS mRNA and immunohistochemistry for iNOS protein showed that several neovascular channels located in the inflamed adventitia of AAA were intensively labeled with iNOS probe (A, red color) and with iNOS antibody (B, brown color) (black arrows). Magnification × 400. Journal of Vascular Surgery 2003 38, 360-367DOI: (10.1016/S0741-5214(03)00148-4)

Fig 4 Representative AAA sections immunostained with anti-nitrotyrosine antibody. The brown reaction product indicates a specific antibody. The brown reaction product indicates a specific antibody. Staining was seen in cells morphologically consistent with macrophages (A) and SMCs (B), with a distribution that parallels to that of iNOS (black arrows). C, Normal aortic tissue sections did not show any immunoreactivity. Magnification ×400. Journal of Vascular Surgery 2003 38, 360-367DOI: (10.1016/S0741-5214(03)00148-4)