Dental pediatric imaging: an investigation towards low dose radiation induced risks

Belmans Niels

Promoter

Lambrichts Ivo, (UHasselt), ivo.lambrichts@uhasselt.be

SCK•CEN Mentor

Moreels Marjan
marjan.moreels@sckcen.be
+32 14 33 28 16

SCK•CEN Co-mentor

Baatout Sarah
sarah.baatout@sckcen.be
+32 14 33 27 29

Expert group

Radiobiology

PhD started

2015-10-01

Short project description

Growing epidemiological evidence indicates an excess risk of late occurring cardiovascular diseases (CVD) at much lower doses of ionizing radiation than previously thought. Until now, these epidemiological data are suggestive rather than persuasive due to a lack of statistical power and fairly limited knowledge of the underlying biological and molecular mechanisms. As a result, cardiovascular effects of low dose ionizing radiation still constitute a black box resulting in a possible improper radiation protection. In this project we will focus on atherosclerosis as a possible mechanism of radiation-induced CVD.

Atherosclerosis is a progressive disease initiated by endothelial cell (EC) dysfunction, and characterized by inflammation and apoptosis (cell death), both nurtured by oxidative stress. Recent evidence demonstrates that ionizing radiation can increase oxidative stress by the production of reactive oxygen species (ROS) and the occurrence of Ca2+ overload, both leading to apoptosis.

Cellular effects occur not only in directly irradiated, but also in adjacent non-irradiated cells, a process known as the bystander effect. Two main routes are reported to mediate bystander responses: cell-cell communication through gap junctions (GJs) and paracrine signaling. Connexin proteins (Cxs) play a major role in both pathways. They form (i) GJ channels that connect the cytoplasm of neighboring cells thereby providing a direct cell-cell communication pathway as well as (ii) plasma membrane hemichannels (‘half of a GJ’) which contribute to paracrine communication.

Several Cx subtypes have been shown to be present in ECs with a role in the development of atherosclerosis. In addition, Cx channels are critical modulators of inflammatory as well as cell death processes via the intercellular propagation of cytoplasmic Ca2+ elevation (Ca2+ waves). Although it has been demonstrated that Cx expression and channel activity are highly sensitive to ionizing radiation and GJs can modulate bystander responses, its role in radiation-induced atherosclerosis has never been investigated, and is subject of this PhD project. By understanding better the effects of radiation on the onset and development of atherosclerosis, we aim to contribute to a better radiation protection.

Objective

Objectives

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?

 

Methodology

  • 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).