Vascular calcification causes hardening of the arteries and leads to cardiovascular death and currently there are no treatments. Our work has shown that a key driver of vascular calcification is a change in the function of vascular smooth muscle cells (VSMCs) of the vessel wall from a contractile to an osteogenic-like cell. This project will focus on understanding the role of epigenetic factors in mediating this phenotype change. We have shown that the SWI/SNF (SWItch/Sucrose Non-Fermentable), subfamily of ATP-dependent chromatin complexes are deregulated in the vessel wall during calcification and ageing and their manipulation in vitro can block osteogenic change in VSMCs. The aims of this project will be to understand what the key components of the complex are that regulate calcification and to understand the targets of the complex in response to stimuli that induce calcification. We will verify these pathways in human tissue samples and animal models of calcification where the complex has been disrupted.
Techniques and Skills:
Primary human VSMC culture and calcification assays. ‘Cut and Run’ ChIP-seq experiments, ATAC and RNA-seq and downstream bioinformatics analysis to identify transcriptional targets and pathways. ChIP-PCR, RT-PCR, IP (immunoprecipitation) and Western blotting to verify targets. Proteomics Mass-Spec to identify complex components under control and calcifying conditions. Immunohistochemistry (IHC) in human tissue sections from healthy and calcified patients. Analysis of animal models of calcification where the SWI/SNF complex has been disrupted genetically or with drugs
Year 1-2 – Identification of timecourse of expression of candidate SWI/SNF components during calcification and characterisation of complex changes using IP and Mass Spec.
Year 2 – ChIP-seq and RNA-seq experiments and bioinformatics to identify transcriptional targets. Target verification and effects on calcification using siRNA/inhibitors assays.
Year 3 – Selected targets are verified in vivo using IHC and human and animal models.