Overall strategy

Local irradiation of the hearts and of the two peripheral arteries of different strains of mice and long-term follow-up of the irradiated animals is at the core of this programme (WP 02). The methods of local irradiation of the peripheral arteries are well established. Using modern imaging and focussing techniques, the established method of local heart irradiation in rats has been adapted to the specific anatomical situation in mice A range of four to six different radiation doses between 0.01 Gy (as lowest dose) up to a maximal dose of 14 Gy (carotid arteries) or 20 Gy (a.saphena) will be given.
Functional imaging of the microvasculature of the heart and the vascular patency of the irradiated arteries (WP 03, WP 04) will be performed in a subgroup of irradiated animals by microSPECT/CT, CT, OCT and MRI. After low and moderate radiation doses (<2 Gy), investigations will be performed 6, 12 and 18 months, at shorter intervals after high radiation doses.

 Histopathological investigations will be performed by TUM, TUD, NKI, MSCCI, UL and CARIM (WP 03, WP 04). Main criterion in the hearts will be the microvascular density and evidence for focal hypoxia in relation to the changes observed by functional imaging. Additionally, in particular at early times after irradiation, changes in inflammatory and thrombotic gene expression will be studied by qRT-PCR and changes in endothelial cell function, protein localisation and abundance by immunohistochemistry (WP 05). Main criterion in the arteries will be the nature of potential atheromatous plaques (inflammatory and thrombotic features) (WP03).

 Ex vivo investigations of the irradiated arteries (WP 06) will investigate intercellular signalling and, in particular the endothelium-leukocyte interactions and pro-thrombotic alterations of the endothelium. In addition, in frozen sections, the expression of adhesion molecules and thrombomodulin will be studied in cooperation with the NOTE integrated project.

 In vitro investigations. Endothelial cells will be isolated at different times after local irradiation. (WP 02) After establishing the primary cultures, cells will be shipped to those partners who will study the different late post-radiation effects in microvascular endothelial cells which are considered to play a role in cardiovascular radiation risks after low dose irradiation, in particular stress responses, changes in intercellular communication, changes in three-dimensional remodelling and migration, in particular in co-culture with cardiomyocytes, changes in endothelial permeability and cytoskeleton structure, pro-inflammatory and prothrombotic changes and changes in endothelial cell/cardiomyocyte interactions (WP 05, WP 06). As a rule, each partner will receive samples from the same primary cell preparation for his/her study, each time.
 In vitro investigations of the function and of molecular alterations of the myocardial cells will be performed with special emphasis on the Rho/ROCK pathway in radiation-induced cardiac remodelling. Specifically, the long-term persistence of phenotypical alterations of endothelial cells will be related to the function of cardiomyocytes isolated from irradiated hearts up to 18 months after irradiation (WP 06).

Proteomics investigations will identify changes in the protein constitution of irradiated cells and hearts. We will subsequently study critical these target proteins from fresh tissue sections of irradiated hearts and in the myocardial cells and endothelial cells isolated from irradiated hearts (WP 07).