Phenotypic and functional characterization of primary murine endothelial cells after in vivo irradiation

The inner cellular lining of all blood vessels consists of a monolayer of endothelial cells (ECs) that have a low proliferation rate in healthy tissue. Under pathological conditions such as wound healing, inflammation and in growing tumors, the proliferation rate of ECs is elevated. Radiotherapy is commonly used for the local control of solid tumors. During radiotherapy of patients with thoracic tumors, the surrounding healthy tissue including ECs of heart and lung may become damaged which in turn can change the proliferation rate of these resting ECs. This damage may increase the risk to develop cardiac diseases in patients after thoracic irradiation at later time-points. It is hypothesized that the protein profile of resting and growing primary ECs is different. To address this question, viable primary ECs are required. Established methods allow the isolation of ECs only from very young mice at low yields and purities. The analysis of late radiation-induced effects on primary ECs requires the availability of primary ECs from old mice. I succeeded to establish a novel method for the isolation of viable primary ECs at high purity from non-proliferating (heart from young and old mice), proliferating benign (repair blastema) and malignant (tumor) tissues at high yields. These ECs were characterized phenotypically, functionally and with respect to their gene expression profiling under static and physiological flow conditions. The expression density of proliferation markers such as endoglin and VE-cadherin is higher on isolated ECs of proliferating tissues from repair blastema and tumor compared to non-proliferating normal tissues from heart and lung. The expression density of the progenitor marker mucosialin is elevated on tumor-derived ECs, but not on those of repair blastemas. The inflammatory markers PECAM-1, ICAM-1 and ICAM-2 were found to be elevated on ECs of repair blastema and tumor compared to ECs from heart and lung. Further, I could show that tumor ECs are larger, have a significantly higher migration capacity and distribute in a more chaotic pattern in cell culture compared to ECs derived from normal tissues. Tube formation assays showed that tumor ECs have a smaller number of branching points and loops compared to that of normal ECs. In contrast to normal tissue ECs, tumor-derived ECs show no tendency to align under flow conditions. The results suggest that increased expression of surface molecules on ECs in proliferating tissues contributes to a loss of EC function that might be responsible for a chaotic tumor vasculature. In contrast to the short-term effects in tumors and repair blastemas induced by proliferation, irradiation can cause long-term effects in heart and lung ECs. Local thorax irradiation of mice resulted in a temporary and differential up-regulation of proliferation markers such as HCAM, integrin β3, endoglin, VE-cadherin and VEGFR-2 on ECs after 8 Gy at later time-points. The progenitor marker mucosialin is increased on lung ECs 15 to 20 weeks after irradiation. Inflammatory markers such as PECAM-1, ICAM-1, ICAM-2 and VCAM-1 started to increase 10 weeks after thorax irradiation with 8 Gy. Interestingly, ICAM-1 and VCAM-1 remained up-regulated even 20 weeks after thorax irradiation on heart and lung ECs. The persistent increase of both markers ICAM-1 and VCAM-1 after irradiation may suggest a predisposition for the development of atherosclerotic plaques in heart and lung ECs at later time points. Shear stress on ECs induced by blood flow is disturbed in heart capillaries with atherosclerotic plaques as well as in tumor vasculature. mRNA expression analysis of genes from heart ECs under normal flow conditions and pathological static conditions in vitro show significant changes related to extracellular organisation, cell membrane function, signaling, hemostasis, metabolism and smooth muscle contraction. Moreover, it was observed that mRNA expression of the inflammation markers Pecam1, Icam1 and Icam2 was higher on heart ECs under static conditions. The protein expression of these inflammatory markers was also elevated on ECs from pathologic conditions. These results provide the basis for subsequent investigations on the role of irradiation on gene expression profiles of ECs derived from normal and tumor tissues.