This research proposal focuses at characterizing and targeting intercellular signaling mechanisms in vitro and in vivo that modulate EC damage which strongly links to the onset and development of atherosclerosis.
The following objectives will be addressed:
Do Cxs and their channels contribute to radiation-induced cell death/inflammatory responses in ECs in vitro and in vivo?
Is the ROS/Ca2+ signaling axis a central player in the radiation-induced cell death/inflammatory responses in ECs in vitro?
Are Cx-mediated Ca2+ waves involved in the cell-cell propagation of ROS production?
In vitro experimental set-up:
We will use commercially available endothelial cell lines and optimize a protocol to isolate primary ECs from mice. Cell cultures will be exposed to X-rays after which different endpoints will be investigated: cell death, proliferation, Cx expression, Cx channel activity, NF-κB activation, EC activation and cytokine production. In addition, we will study the role of Cx channels and the ROS/Ca2+ signaling axis via (i) the application of channel blockers and interference with Cx expression, (ii) time lapse imaging of intracellular Ca2+ and ROS levels and (iii) interfering with ROS/Ca2+ signaling. Techniques that will be applied are RT-PCR, western blot, immunocytochemistry, various biological assays (to investigate cell death, proliferation and inflammation), ELISA and fluorescence microscopy.
In vivo experimental setup:
The in vivo model will consist of the targeted thoracic irradiation of mice using a Small Animal Radiation Research Platform (SARRP). This device allows highly localized treatment planning (1 mm precision beams), dose calculation and verification. It mimics the isocentric external-beam equipment that is used to deliver image-guided radiotherapy in humans. Heart sections will be used for further histological analysis of Cx expression, cell death and inflammation in ECs. These experiments are anticipated to yield a difference in Cx expression in vivo pre- and post-irradiation, and, as such, provide specific Cx targets for further investigation using specific peptide inhibitors and knock-out mice. In addition, atherosclerotic plaques in the main vessels will be visualized using oil red o staining. Finally, blood analysis of circulating endothelial microparticles (EMPs), markers of endothelial damage, will be analysed using fluorescence-activated cell sorting (FACS).