Stress- and Nutrition-Sensing Transcription Factors : Looking for Biomarkers in Atherosclerosis - A Medium Throughput Screening Prospective Study

Coordinating Institution: Université du Luxembourg
From: 01/04/2009
To: 31/03/2011
Budget: 251,000.00€
Contact(s): Bueb Jean-Luc

Summary

Among various other diseases where chronic inflammation, hence cellular stress are involved, Alzheimer’s disease, atherosclerosis and cancer have been recognized as the leading causes of mortality within the developed world. Diseases occur not only from an internal dysfunction of the complex constitutive interactions between genes, their expression products and their functions, but also from numerous external disturbing interferences. Cell and organism functions depend on the dynamic interaction of a collection of transcription factors through the networks that link them. The transcription factor NF-κB is a central sensor of cellular stress, e.g. exposure to cytokines. In parallel, nuclear receptors bind with a variety of environmental factors, including nutrients originating from food intake, e.g. the nuclear receptor ‘vitamin D receptor’ (VDR) with the micronutrient vitamin D. These transcription factors may serve as mediators in the communication between cellular stress, nutrition and the human genome. Our starting hypothesis is that a misbalance of the functional interactions between nutrient-sensing nuclear receptors and stress-sensing transcription factors, induced by a lifetime exposure to micronutrients and cytokines, is a central molecular process towards atherosclerosis. During this feasibility study based on a Medium Throughput Screening concept, genomic, transcriptomic, simple proteomic and functional data from wet lab in vitro cellular inflammation processes will be collected in order to evidence new genetic biomarkers in atherosclerosis. The project is subdivided into the three following objectives, of which we are presently addressing the first one. 1. Identify and validate primary target genes of nuclear receptor ‘Vitamin D receptor’ and NF-κB in human endothelial cells (HUVEC) and macrophages (THP-1) cell lines. Additionally to the easy growing THP-1 cells, HUVEC cultures have already been established.

Among the different cell lines HUVEC-C, -CS and EA.hy926, cultured under various combinations of conditions (supports, media and coatings), EA.hy926 was the most suited to the study. Macroarrays of TNFa- and/or VitD-stimulated cells are now being analyzed. 2. Characterize the functional consequences of these target gene expressions on the cellular behaviour of HUVEC and THP-1 cells, using live cell-based medium throughput screening with siRNA against target genes for cellular signaling responses (apoptosis, necrosis, cAMP elevation, Ca2+ homeostasis, release of cytokines). 3. Unify data derived from our own experiments and others reported in literature or databases with clinical data, using systems biology approaches, to generate in silico models of nuclear receptor and NF-κB pathways in metabolism, cellular regulation and inflammation, and to validate new potential highlighted atherosclerosis biomarkers.

Figure 1: EA.hy926 Cells in Culture

Figure 2: Polyvalent Liquid Handling robot