Sustained Inflammation Due to Nuclear Factor-Kappa B Activation in Irradiated Human Arteries
Martin Halle, MD, PhD*, ,*,
Anders Gabrielsen, MD, PhD, ,
Gabrielle Paulsson-Berne, PhD,
Caroline Gahm, MD, PhD ,
Hanna E. Agardh, BA,
Filip Farnebo, MD, PhD* and
Per Tornvall, MD, PhD,
* Department of Molecular Medicine and Surgery, Section of Reconstructive Plastic Surgery, Karolinska Institute, Stockholm, Sweden
Department of Medicine, Center for Molecular Medicine, Karolinska Institute, Stockholm, Sweden
Department of Medicine, Section of Cardiology, Karolinska Institute, Stockholm, Sweden
Department of ORL, Head and Neck Surgery, Karolinska Institute, Stockholm, Sweden
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Figure 1 Box Plots of Data for Candidate Genes Derived From Analysis of Irradiated Arteries Compared With Nonirradiated Internal Controls
Box plots of data for candidate genes (shaded) derived from Taqman low-density array analysis of irradiated arteries compared with nonirradiated internal controls for all patients (n = 13). Whiskers represent maximum and minimum values that are no more than 1.5 times of the interquartile range. Outliers are indicated if present. Differential gene expression displayed as log-values of relative quantification (RQ). Data were normalized against the housekeeping gene 18S. Two additional housekeeping genes were included as controls (striped).
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Figure 3 Immunohistochemical Stainings of Nonirradiated Human Radial Forearm Arteries (Controls) and Irradiated Human Cervical Arteries
Irradiated arteries showed positive nuclear factor-kappa B (B), CD68 (macrophages) (D), and CD3 (T-cells) (H) staining compared with controls (A, C, G). Irradiated (F) and control (E) arteries both presented a morphologically intact endothelium shown by staining for von Willebrand factor. Images from a patient, radiated 4 years before harvest of biopsies, are representative for stainings of control and irradiated arteries in 3 patients. Bar = 50 µm.
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Figure 4 Immunohistochemical Stainings of Nonirradiated Human Radial Forearm Arteries (Controls) and Irradiated Human Cervical Arteries
Increased staining for matrix metalloproteinase (MMP)-1 in radiated arteries (B) compared with controls (A) was confined to macrophages visualized by CD68-staining in consecutive sections of radiated (D) and control (C) arteries, representative of paired biopsies from 3 patients. Bar = 25 µm.
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Figure 5 Staining of p65 Subunit Superimposed on Photographs of DAPI Nuclear Staining
Staining of p65 subunit (red fluorescence) superimposed on photographs of 4',6-diamidino-2-phenylindole (DAPI) nuclear staining (blue fluorescence) indicating nuclear factor- B (NF-B)-activation by nuclear translocation seen with co-localization of p65 and DAPI in the cell nucleus (purple) in irradiated arteries (B, D) compared with nonirradiated arteries (A, C). Co-staining for CD68 (green) showed that the p65 subunit was localized to the nuclei in macrophages but only present in radiated arteries. Single channel stainings of the merged photographs (C and D) are visualized for nonirradiated (E, G, I) and irradiated (F, H, J) arteries from the same patient, irradiated 4 years before harvest of biopsies, representative for stainings of nonirradiated and irradiated arteries in 3 patients (arrows = p65 nuclear stains; arrowhead = p65 expression in the cytoplasm; arrowhead asterisk = p65 nuclear staining positive cells co-expressed with CD68 in the cytoplasm). Bar = 10 µm.
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