Supervisor:  Dr Caroline Pellet-Many, Dr Charlotte Lawson

Department:  Comparative Biomedical Sciences

 

Background

Adult mammals are unable to fully regenerate their heart following myocardial infarction. Yet, the activated epicardium (visceral layer of the pericardium) starts to proliferate and undergoes a phenotypic change into multi-potent epicardial derived cells (EPDCs) via the process of epithelial to mesenchymal transition (EMT). The EMT also leads to the release of trophic factors promoting the survival of cardiomyocytes and supporting the formation of new vessels around the ischemic area of the heart.

Neuropilins (NRP1 and NRP2) are cell surface glycoprotein co-receptors capable of binding a variety of cytokines and their corresponding receptors and thus, are able to mediate several signaling pathways. In cancer cell lines, NRPs knock down is known to impair the EMT process leading to a reduction in cell invasion, and metastasis. We have also recently showed in the zebrafish, an animal model used to study cardiac regeneration, that Nrp1a is required for zebrafish heart regeneration in response to cryoinjury. It promotes both revascularisation, activation and migration of the epicardium by inducing Wt1b (Wilms tumor suppressor, a marker of epicardial activation). Nevertheless, the detailed mechanisms and the signalling pathways implicated with this processes have not been completely characterised. Moreover, little is known about the related isoform, neuropilin 2 (NRP2) which was also significantly upregulated by the epicardium of the zebrafish after cryoinjury (Lowe et al., 2018).

Hypothesis

Using a Rat epicardial cell line, we hypothetise that the depletion of NRP1 and/or NRP2 will similarly impair the EMT process by reducing epicardial cell proliferation and migration as well as stopping the epithelial to mesenchymal phenotypic changes.

Aims

  1. Optimisation of the siRNA transfection techniques and delivery of adenoviral shRNA construct to knock down NRP1 and NRP2 in rat epicardial cells (verified by Western blot and immunofluorescence).  
  2. Phenotypic assessment of the NRPs-depleted cells via Western blot (to assess EMT and epithelial markers), qPCR and immunofluorescence (confocal microscopy).  
  3. Study of the NRP-dependent signalling pathways involved in EMT in epicardial cells.
  4. Validation of new targets identified by RNA sequencing (using qPCR, Western blotting and immunofluorescence).
  5. Validation of the neuropilin-dependent signaling targets using nrp1 and nrp2 zebrafish knock out models used by other lab members.

References:

  1. Lowe V, Wisniewski L, Sayers J, Frankel P, Mercader N, Zachary C, Pellet-Many C (2019) Neuropilin 1 mediates epicardial activation and revascularization in the regenerating zebrafish heart. doi: 10.1101/468504 %J biorxiv (under revision for publication in Development).
  2. Wada A., Smith T, Osler M, Reese D & Bader D (2003) Epicardial/Mesothelial cell line retains vasculogenic potential of embryonic epicardium. Circulation Research, 92, 525-31.
  3. Pellet-Many C, Frankel P, Jia H, Zachary I. (2008) Neuropilins: structure, function and role in disease. Biochemical Journal. 411(2):211-26.

Requirements:

 Applicants should have a first or second class university honours degree in biological sciences, a veterinary or medical degree.

This is a full-time (12 month) project commencing in October 2019, based at RVC's Camden campus.

Partially funded - the lab will be covering some of the project costs, and the MRes student will be expected to meet the outstanding sum required, course fees and their living expenses.

 We welcome informal enquiries - these should be directed to cpelletmany@rvc.ac.uk

Top of page