Prevention of radiotherapy-induced arterial inflammation by interleukin-1 blockade

Figures

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  Figure 1. Network of inflammasome-related transcripts in human arteries with or without chronic radiation injury. Biopsies of paired human arteries with and without chronic radiation injury were analysed using Affymetrix Gene Array (n = 20) paired biopsies. (A) Inflammasome-related genes were mapped onto an interaction repository (BioGRID), a subnetwork extracted using the prize-collecting Steiner Forest graph optimization approach (PCSF) to include ‘bridges’ that connect these genes. Genes in the subnetwork are colour-coded to indicate the expression change between irradiated and non-irradiated. Genes associated with macrophages are shown on yellow background, endothelial cells (pink), and with T-cells/smooth muscle cells (grey). (B) Transcript levels of selected inflammasome associated genes. Centrelines correspond to median values. Hinges indicate the 25th and 75th percentiles. Whiskers indicate 1.5 times the interquartile range and observations beyond the whiskers are outliers. Difference between groups were analysed using the Wilcoxon signed rank test. CASP, caspase; CCL, chemokine (C-C motif) ligand; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; NLRP, NLR family Pyrin…

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  Figure 2. Inflammasome-associated transcripts were elevated in human arteries with chronic radiation injury. Biopsies of paired human arteries with and without chronic radiation injury were analysed using qPCR. mRNA levels of CCL2/MCP-1 (A), CCL5 (B), and VCAM1 (C) in human arterial biopsies without (Ctl) and with (XRT) chronic radiation injury (n = 12 patients) are plotted. Data are presented as −ΔΔCt normalized to the geometrical mean of the two endogenous controls. Differences between groups were analysed using the Wilcoxon signed ranked test. CCL, chemokine (C-C motif) ligand; MCP-1, monocyte chemoattractant protein-1; VCAM1, vascular cell adhesion molecule-1.

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  Figure 3. Increased macrophage accumulation in human arteries with chronic radiation injury. Biopsies of paired human arteries with and without chronic radiation injury were sectioned and stained with anti-CD68 antibodies (brown) and haematoxylin QS. (A) Microscopic images of sections from biopsies with or without chronic radiation injury in one representative patient. Insets show higher magnification of the intima and adventitia layers, respectively. (B) Number of CD68+ cells in sections of arterial biopsies (n = 12). Differences between groups were analysed using the Wilcoxon signed ranked test. Ctl, non-irradiated; XRT, irradiated.

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  Figure 4. Elevated, pro-caspase and caspase-1 in human arteries with chronic radiation injury. Protein from paired human arteries with and without chronic radiation injury was analysed for procaspase-1 and caspase-1 using western blot (n = 3) (A and C). Densitometry was measured using the image studio software and a Li-Cor scanner, normalized to total protein content, grouped by absence (Ctl) or presence (XRT) of chronic radiation injury, and plotted (B). kD, kilodalton.

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  Figure 5. A mouse model of anti-inflammatory treatment in upper chest and neck irradiation. (A) Overview of treatment groups, study timeline and treatment protocol. (B) Schematic illustration of the irradiation field in terms of anatomical overview (left), irradiation dose, distribution field, and radiation scatter (right). The X- and Z-axes show distance from the collimator in inches, and the Y-axis shows arbitrary units of radiation exposure. Colours illustrate the dose, ranging from yellow = high (14 Gy) to blue = low (non-detected). Apoe−/−, apolipoprotein E deficient, i.p; inj, intraperitoneal injection; NaCl, sodium chloride; +/−, received/did not receive treatment.

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  Figure 6. Treatment with anakinra reduced arterial inflammation in irradiated Apoe−/− mice. Female Apoe−/− mice were subjected to either a controlled radiation injury or sham treatment with or without a subsequent 2 weeks of daily i.p. injections of anakinra. Tissues were harvested and evaluated at 10 weeks after radiation treatment (A–D). RNA was isolated from thoracic aorta, and levels of Ccl2/Mcp-1 (A) and Ccl5 (B) analysed by qPCR. Aortic roots were cryosectioned and stained for VCAM-1 (C) and I-Ab (MHC class II) (D). The stained area was measured in a section at 250/260 μm (VCAM-1/I-Ab (MHC class II)) from the aortic valve plane and normalized to lesion area. Number of animals per group: Ctl = 9/7/6, XRT = 8/5/5, Ctl/Ak = 10/7/5, XRT/Ak = 9/7/8 for mRNA/VCAM/I-Ab (MHC class II). Differences between groups were analysed using one-way ANOVA followed by Tukey post hoc analysis. Ccl, chemokine (C-C motif) ligand; Ctl, non-irradiated; Mcp-1, monocyte chemoattractant protein-1; MHC class II, major histocompatibility complex class II; VCAM-1, vascular cell adhesion molecule-1; XRT, irradiated.

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  Take home figure. A translational model for therapeutic target discovery in radiation injury. Clinical event: Increased risk for cardiovascular disease at the site of previous radiotherapy. Human biobank: Gene expression array of paired (irradiated and non-irradiated) human arteries retrieved at the same time during microvascular autologous free tissue transfers for reconstructive cancer surgery. Radiotherapy induces a sustained inflammatory response in human arteries. Animal model: Establishment of an animal model of local irradiation with similar inflammatory patterns as observed in the human biobank. Treatment: Treatment with anakinra (IL-1R antagonist) reduced radiation-induced vascular inflammation in our Apoe−/− mouse model of local irradiation. Ctl, non-irradiated; Ccl2/Mcp-1, chemokine (C-C motif) ligand2/monocyte chemoattractant protein-1; DAMP, damage associated molecular pattern; IL-1R, interleukin-1 receptor; ROS, reactive oxygen species; VCAM1, vascular cell adhesion molecule-1; XRT, irradiated. Illustration by Tinna Christersdottir based on published data.