Molecular and Cellular Responses of Human Endothelial Cells to Low-Dose Radiation
Authors:
Rebecca Weinberg* ([email protected]), Sara Forrester, Justin Podowski, Abraham Stroka, Thomas S. Brettin, Dan Schabacker, Rick Stevens (PI)
Institutions:
Argonne National Laboratory
Abstract
Radiation exposure has a wide spectrum of impacts on human health, notably in carcinogenesis, but also in neurological and cardiovascular disorders. While acute toxicity from high doses of radiation is well characterized, understanding the range of outcomes following exposure to low-dose radiation is more challenging. This research team is establishing new experimental workflows that will enable high throughput experiments across molecular and cellular scales to facilitate more comprehensive modeling. In the pilot study, a monolayer of human umbilical vessel endothelial cells (HUVECs) was exposed to a point source of 137Cs at a low-dose rate (6 milligrays/hour). Cells were exposed for one week in culture (a total dose of 1,008 milligrays), and then harvested for RNA or replated for Cell Painting staining. Cell Painting is a streamlined multiparameter approach to fluorescence microscopy that provides rich feature data of cell structure and function.
A major advantage of Cell Painting is a robust, publicly available dataset spanning thousands of small molecular and genomic perturbations produced by a collaborative team (the JUMP Consortium). The scale of characterized phenotypes has facilitated development of predictive models that incorporate chemical structural information, biological mechanism of action, and gene expression, which this team will expand into the realm of radiation exposure. With Cell Painting, features can be extracted based on staining of nuclear, endoplasmic reticulum, plasma membrane and Golgi, actin, nucleoli, and mitochondria. Principle component analysis of control and irradiated cells provided a proof-of-principle demonstration that Cell Painting enables detection of features impacted by irradiation. The team’s transcriptome analysis revealed that in endothelial cells, radiation robustly induced cell response pathways integral to cytokine and chemokine pathways, such as the Tumor Necrosis Factor pathway. Underscoring the relevancy of HUVECs to cardiovascular disease, pathways associated with “lipid” and “atherosclerosis” were also activated. Two Kyoto Encyclopedia of Genes and Genomes terms shed light on the molecular mechanisms of these processes, namely the HIF-1 and NF-kappa B signaling pathways.
To compare these results to previously obtained studies of low-dose radiation exposure, the team compared its data with gene expression datasets from the RadBioBase, a publicly available comprehensive transcriptome repository of irradiated mammalian samples. Researchers selected datasets that used human cells and doses below 0.5 grays to identify 235 genes impacted by radiation across four published datasets. Of these, 35 genes were also seen in this study’s data, notably the inflammatory cytokines IL6 and IL1B, as well as the genes PTGS2 (COX2) and CXCL12, which are involved in inflammatory processes underlying cardiovascular disease.
To overcome the limitations (variable dose field, high activity) of the point radiation source in the pilot study, a major goal of the next phase of this project is to prototype and deploy new source geometries (96-well plate format) for high-throughput experimental exposures. New source geometries will require minimal activity, provide uniform dose fields, and allow for multiple dose rate exposures in parallel. Researchers will then assess the impact of low-dose radiation harnessing molecular (multiomic) and cellular (Cell Painting) assays that can be used to develop advanced multiscale models of the impacts of low-dose radiation.