NO-Donating Aspirin and Aspirin Partially Inhibit Age-Related Atherosclerosis but Not Radiation-Induced Atherosclerosis in ApoE Null Mice

BACKGROUND: We previously showed that irradiation to the carotid arteries of ApoE(-/-) mice accelerated the development of macrophage-rich, inflammatory atherosclerotic lesions, prone to intra-plaque hemorrhage. In this study we investigated the potential of anti-inflammatory and anti-coagulant intervention strategies to inhibit age-related and radiation-induced atherosclerosis. METHODOLOGY/PRINCIPAL FINDINGS: ApoE(-/-) mice were given 0 or 14 Gy to the neck and the carotid arteries and aortic arches were harvested at 4 or 30 weeks after irradiation. Nitric oxide releasing aspirin (NCX 4016, 60 mg/kg/day) or aspirin (ASA, 30 or 300 mg/kg/day) were given continuously in the chow. High dose ASA effectively blocked platelet aggregation, while the low dose ASA or NCX 4016 had no significant effect on platelet aggregation. High dose ASA, but not NCX 4016, inhibited endothelial cell expression of VCAM-1 and thrombomodulin in the carotid arteries at 4 weeks after irradiation; eNOS and ICAM-1 levels were unchanged. After 30 weeks of follow-up, NCX 4016 significantly reduced the total number of lesions and the number of initial macrophage-rich lesions in the carotid arteries of unirradiated mice, but these effects were not seen in the brachiocephalic artery of the aortic arch (BCA). In contrast, high dose ASA lead to a decrease in the number of initial lesions in the BCA, but not in the carotid artery. Both high dose ASA and NCX 4016 reduced the collagen content of advanced lesions and increased the total plaque burden in the BCA of unirradiated mice. At 30 weeks after irradiation, neither NCX 4016 nor ASA significantly influenced the number or distribution of lesions, but high dose ASA lead to formation of collagen-rich "stable" advanced lesions in carotid arteries. The total plaque area of the irradiated BCA was increased after ASA, but the plaque burden was very low compared with the carotid artery. CONCLUSIONS/SIGNIFICANCE: The development and characteristics of radiation-induced atherosclerosis varied between different arteries but could not be circumvented by anti-inflammatory and anti-coagulant therapies. This implicates other underlying mechanistic pathways compared to age-related atherosclerosis

Endoglin Haplo-Insufficiency Modifies the Inflammatory Response in Irradiated Mouse Hearts without Affecting Structural and Mircovascular Changes

<div><p>Background</p><p>It is now widely recognized that radiotherapy of thoracic and chest wall tumors increases the long-term risk of cardiovascular damage although the underlying mechanisms are not fully elucidated. There is increasing evidence that microvascular damage is involved. Endoglin, an accessory receptor for TGF-β1, is highly expressed in damaged endothelial cells and may play a crucial role in cell proliferation and revascularization of damaged heart tissue. We have therefore specifically examined the role of endoglin in microvascular damage and repair in the irradiated heart.</p><p>Materials & Methods</p><p>A single dose of 16 Gy was delivered to the heart of adult Eng^+/+ or Eng^+/− mice and damage was evaluated at 4, 20 and 40 weeks, relative to age-matched controls. Gated single photon emission computed tomography (gSPECT) was used to measure cardiac geometry and function, and related to histo-morphology, microvascular damage (detected using immuno- and enzyme-histochemistry) and gene expression (detected by microarray and real time PCR).</p><p>Results</p><p>Genes categorized according to known inflammatory and immunological related disease were less prominently regulated in irradiated Eng^+/− mice compared to Eng^+/+ littermates. Fibrosis related genes, TGF-β1, ALK 5 and PDGF, were only upregulated in Eng^+/+ mice during the early phase of radiation-induced cardiac damage (4 weeks). In addition, only the Eng^+/+ mice showed significant upregulation of collagen deposition in the early fibrotic phase (20 weeks) after irradiation. Despite these differences in gene expression, there was no reduction in inflammatory invasion (CD45+cells) of irradiated Eng^+/− hearts. Microvascular damage (microvascular density, alkaline phosphatase and von-Willebrand-Factor expression) was also similar in both strains.</p><p>Conclusion</p><p>Eng^+/− mice displayed impaired early inflammatory and fibrotic responses to high dose irradiation compared to Eng^+/+ littermates. This did not result in significant differences in microvascular damage or cardiac function between the strains.</p></div

Representation of the top network and functional pathways using IPA approach.

<p>Top networks for 4, 20 and 40 weeks after 16 Gy irradiation of Eng^+/+ and Eng^+/− mice. Numbers in brackets represent the network score, which is explained in material and methods. The first two functional pathways for 4, 20 and 40 weeks after 16 Gy irradiation of Eng^+/+ and Eng^+/− mice are also shown. Nerv.system devlp. & funct. : Nervous system development and function; Lipid metabl, Molecl. Transp.: Lipid metabolism, Molecular transport; Hematl. system devlp.& funct.: Hematological system development and function; Cell-to-cell signlg.& inter.: Cell-to-cell signaling and interaction; Cardio.system devlp.& funct.: Cardiovascular system development and function.</p

Graphical representation of the top network of differentially regulated genes (40 weeks).

<p>Each network symbolizes the biological functions and/or diseases that were most significantly regulated 40 weeks after cardiac irradiation of Eng ^+/+ mice (n = 4–5) (A) and Eng ^+/− mice (n = 4–7) (B). The genes marked in red represent the upregulated genes and in green the downregulated genes. The solid arrows represent direct interactions and the dotted arrows indirect interactions. Genes circled in dark blue represents central molecules and the light blue lines direct interaction with other genes.</p

Graphical representation of the top network of differentially regulated genes (20 weeks).

<p>Each network symbolizes the biological functions and/or diseases that were most significantly regulated 20 weeks after cardiac irradiation of Eng ^+/+ mice (n = 4–5) (A) and Eng ^+/− mice (n = 5) (B). The genes marked in red represent the upregulated genes and in green the downregulated genes. The solid arrows represent direct interactions and the dotted arrows indirect interactions. Genes circled in dark blue represents central molecules and the light blue lines direct interaction with other genes.</p

Inflammatory and fibrotic changes at 4, 20 and 40 weeks after irradiation or sham treatment.

<p>(A) Quantification of CD45+ cells per section in the myocardium and (B) epicardium. (C) Percentage interstitial collagen content of irradiated heart sections, relative to age-matched unirradiated controls. Values represent mean ± SEM with 3–6 mice in the 4 weeks group, 4–5 mice in the 20 weeks group and 5–7 in the 40 weeks group, *p<0.05 compared to age-matched unirradiated controls.</p

Expression of genes involved in TGFβ pathway measured by RT PCR.

<p>Each bar represents the average expression per group ± SEM. Values of sham-treated animals were set to 1. The graph show the fold-change in gene expression in irradiated mice relative to respective controls at 4 (n = 4–5), 20 (n = 4–5), and 40 (n = 4–7) weeks after 16 Gy irradiation.</p

EDV, ESV, EF and SV measured by gated SPECT at 20 weeks or 40 weeks after irradiation or sham treatment.

<p>Values represent mean ± SEM (7–14 mice in each irradiated group), *p<0.05 compared to age-matched, unirradiated controls.</p