Research project objectives/ Research hypothesis

The aim of this project is to investigate the molecular mechanisms associated with the Nrf2 (nuclear factor (erythroid-derived 2) -like 2) transcription factor and microRNA 34a (miR-34a)- dependent regulation of senescence and apoptosis in endothelial cells. Hypotheses of the project are based on our initial results demonstrating that 1) inhibition of Nrf2 expression induces premature senescence of primary human aortic endothelial cells (HAECs) in vitro, 2) Nrf2 deficient HAECs do not show oxidative imbalance or increased DNA damage, which are described as classical inducers of premature senescence, 3) miR-34a acts as a switch between apoptosis and senescence in HAECs with silenced Nrf2. Based on our preliminary data and the current knowledge we have formulated the following hypotheses: 1) premature senescence of Nrf2 deficient endothelial cells is observed in vivo and evokes deleterious effects on blood vessel function, 2) premature senescence of Nrf2 deficient endothelial cells is caused not by DNA damage and oxidative imbalance, but by impaired autophagy, nitrosative stress and/or metabolic dysfunction, 3) miR-34a acts as a switch between senescent and apoptotic endothelial cell fate by direct interactions with proteins regulating these processes, 4) sudden cell death of Nrf2-/- miR-34a-/- endothelial cells is related to mitoptosis, and 5) Nrf2/miR34a axis modulates senescent/apoptotic phenotype of endothelial cells in vivo.

 

Research project methodology

The proposal is based on in vitro, ex vivo and in vivo research, and is divided into six research tasks. Experiments will be carried out in the laboratories of the Department of Medical Biotechnology at the Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University. The vast majority of the methods proposed in the project are methods routinely used in our Department. Laboratories, which we use, or which we have a direct access to, are equipped with research equipment necessary to complete the project. Research model for in vitro experiments are the primary human aortic endothelial cells (HAEC). Ex vivo and in vivo studies will be conducted on mice with total deletion of the Nrf2 and on mice with Nrf2 removed from endothelium. We plan to use mainly methods which have been applied in our earlier studies, including flow cytometry, colorimetric, luminometric and fluorimetric methods, gene expression analysis (qRT-PCR, Western blot, IHC, RNA-seq in sorted cell populations), as well as methods with requiring advanced equipment, e.g. electron microscope, mass spectrometer, laser Doppler, EPR, HPLC, Seahorse or high resolution ultrasonograph.

 

Expected impact of the research project on the development of science, civilization and society

Predicted outcome of proposed study is an indication of the cause of premature senescence of Nrf2 deficient endothelial cells and elucidating the molecular mechanisms of miR-34a dependent switching between senescent and apoptotic cell fate. We are also planning to check: i) whether the Nrf2/miR-34a mechanisms are universal or cell-type specific, ii) whether miR-34a acts as a switch if senescence is evoked independently of Nrf2, and iii) whether double knockout of Nrf2 and miR-34a leads to endothelial cell death also in vivo. We believe that the new knowledge arising from this study will help to better understand the physiology and pathology of vascular aging, what can facilitate development of new therapeutic strategies for age-related cardiovascular diseases. According to our best knowledge, up to date nobody has tried to investigate the role of Nrf2/miR-34a axis in senescence/apoptosis switching.