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Late inflammatory and thrombotic changes in irradiated hearts of C57BL/6 wild-type and atherosclerosis-prone ApoE-deficient mice

Späte inflammatorische und thrombotische Veränderungen in bestrahlten Herzen von C57BL/6-Wildtyp- und atherosklerosegefährdeten ApoE-defizienten Mäusen

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Abstract

Background and purpose

Radiation-induced heart disease represents a late complication of thoracic radiotherapy. We investigated the inflammatory and thrombotic response after local heart irradiation in wild-type and atherosclerosis-prone mice.

Material and methods

Atherosclerosis-prone ApoE−/− and C57BL/6 wild-type mice were sacrificed 20, 40, and 60 weeks after irradiation with 0.2, 2, 8, or 16 Gy. The expression of CD31, vascular cell adhesion molecule-1 (VCAM-1), thrombomodulin (TM), and CD45 were quantified by immunofluorescence staining of heart tissue sections.

Results

Microvascular density decreased at 40 weeks after 16 Gy in C57BL/6 but not in ApoE−/− mice. CD31 expression declined in C57BL/6 mice at 40 weeks (8 Gy), but increased in ApoE−/− mice at 20 (2/8/16 Gy) and 60 weeks (16 Gy). Capillary area decreased in C57BL/6 at 40 weeks (8/16 Gy) but increased in ApoE−/− mice at 20 weeks (16 Gy). Endocardial VCAM-1 expression remained unchanged. TM-positive capillaries decreased at 40 weeks (8/16 Gy) in C57BL/6 and at 60 weeks (2/16 Gy) in ApoE−/− mice. Leukocyte infiltration transiently rose 40 weeks after 8 Gy (only ApoE−/−) and 16 Gy. After receiving a low irradiation dose of 0.2 Gy, no significant changes were observed in any of the mouse models.

Conclusion

This study demonstrated that local heart irradiation affects microvascular structure and induces inflammatory/thrombotic responses in mice in a dose- and time-dependent manner. Thereby, significant prothrombotic changes were found in both strains, although they were progressive in ApoE−/− mice only. Proinflammatory responses, like the increase of adhesion molecules and leukocyte infiltration, were more pronounced and occurred at lower doses in ApoE−/− vs. C57BL/6 mice. These findings indicate that metabolic risk factors, such as decreased ApoE lipoproteins, may lead to an enhanced proinflammatory and prothrombotic late response in locally irradiated hearts.

Zusammenfassung

Hintergrund und Zielsetzung

Strahlungsinduzierte kardiovaskuläre Erkrankungen sind als Spätfolgen der thorakalen Radiotherapie bekannt. In dieser Arbeit untersuchten wir inflammatorische und thrombotische Marker nach lokaler Bestrahlung des Herzens in zu Atherosklerose neigenden und Wildtyp-Mäusen.

Material und Methoden

Atherosklerosegefährdete ApoE-defiziente (ApoE−/−) und C57BL/6-Wildtyp-Mäuse wurden mit 0,2 Gy, 2 Gy, 8 Gy oder 16 Gy bestrahlt und 20, 40 und 60 Wochen später euthanasiert. Die Proteinexpression von CD31, VCAM-1 (vascular endothelial adhesion molecule-1), Thrombomodulin (TM) und CD45 wurde mittels Immunfluoreszenzfärbung in Herzgewebeschnitten quantifiziert.

Ergebnisse

Die Kapillardichte nahm 40 Wochen nach 16-Gy-Bestrahlung in C57BL/6-, nicht aber in ApoE−/−-Mäusen ab. Die Expression von CD31 verminderte sich in C57BL/6-Mäusen nach 40 Wochen (8 Gy), stieg jedoch in ApoE−/−-Mäusen nach 20 (2/8/16 Gy) und 60 Wochen (16 Gy) an. Die Kapillargröße reduzierte sich in C57BL/6 40 Wochen nach Bestrahlung (8/16 Gy), vergrößerte sich aber in ApoE−/−-Mäusen 20 Wochen nach 16-Gy-Bestrahlung. Die endokardiale VCAM-1-Expression blieb nach Bestrahlung unverändert. Die Anzahl TM-positiver Kapillaren verminderte sich nach 40 Wochen (8/16 Gy) in C57BL/6- und nach 60 Wochen (2/16 Gy) in ApoE−/−-Mäusen. Die Infiltration von Leukozyten war 40 Wochen nach 8-Gy- (nur ApoE−/−) und 16-Gy-Bestrahlung vorübergehend erhöht. In beiden Mausstämmen konnten nach einer Bestrahlung mit einer niedrigen Dosis von 0,2 Gy keine signifikanten Veränderungen beobachtet werden.

Schlussfolgerung

In dieser Studie wurde gezeigt, dass die lokale Bestrahlung des Herzens die mikrovaskuläre Struktur beeinflusst und eine dosis- und zeitabhängige inflammatorische/thrombotische Reaktion in Mäusen hervorruft. Dabei treten signifikante prothrombotische Veränderungen in beiden untersuchten Mausstämmen auf, wobei diese nur in ApoE−/−-Mäusen progressiv verlaufen. Proinflammatorische Reaktionen, wie der Anstieg von Adhäsionsmolekülen und die Einwanderung von Leukozyten, treten vorwiegend in ApoE−/−-Mäusen und dort bereits bei niedrigeren Dosen, verglichen mit C57BL/6-Mäusen, auf. Diese Daten weisen daraufhin, dass metabolische Risikofaktoren, wie erniedrigte ApoE-Lipoproteine, zu einer verstärkten proinflammatorischen/prothrombotischen Spätreaktion in lokal bestrahlten Herzen führen könnten.

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Compliance with ethical guidelines

Acknowledgments

We are grateful to Britt Rosin for technical support with the preparation of tissue sections and immunofluorescence staining. We thank Gabriela Aust and fellow workers for technical support with tissue cutting. We are grateful to Tobias Polte and colleagues for technical support with the TissueFax fluorescence microscope unit.

The project was funded by the European commission grant FP7, 211403, CARDIORISK.

Conflict of interest

I. Patties, J. Haagen, W. Dörr, G. Hildegrandt, and A. Glasow state that there are no conflicts of interest. All national guidelines on the care and use of laboratory animals have been followed and the necessary approval was obtained from the relevant authorities.

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Authors and Affiliations

  1. Department of Radiation Therapy, University of Leipzig, Stephanstrasse 9a, 04103, Leipzig, Germany

    I. Patties & A. Glasow

  2. Department of Radiotherapy and Radiation Oncology, Medical Faculty Carl Gustav Carus, University of Technology, Dresden, Germany

    J. Haagen & W. Dörr

  3. Department of Radiation Oncology & Christian Doppler Laboratory for Medical Radiation Research for Radiooncology, CCC, Medical University/AKH, Vienna, Austria

    W. Dörr

  4. Department of Radiotherapy and Radiation Oncology, University of Rostock, Rostock, Germany

    G. Hildebrandt

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  1. I. Patties
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Correspondence to I. Patties.

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Patties, I., Haagen, J., Dörr, W. et al. Late inflammatory and thrombotic changes in irradiated hearts of C57BL/6 wild-type and atherosclerosis-prone ApoE-deficient mice. Strahlenther Onkol 191, 172–179 (2015). https://doi.org/10.1007/s00066-014-0745-7

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