Dissecting the role of neuropeptide CGRP in human lymphatic endothelial cells

Project Description

Calcitonin receptor-like receptor (CLR) is a key G-protein-coupled receptor (GPCR) that mediates the signalling of three peptides - adrenomedullin (AM), calcitonin gene-related peptide (CGRP; neuropeptide) and intermedin (IMD/AM-2). Animal models revealed critical roles for these molecules in the pathogenesis of cardiovascular disease, migraine, lymphoedema and cancer, implying that CLR is a promising target for clinical diagnosis, imaging and therapy. However, CLR properties and function in human tissues, where this GPCR is predominantly expressed by the endothelial cells (EC), remain poorly understood and characterised.

Recently we have discovered that neuropeptide CGRP plays a significant role in human lymphatic EC (LEC) biology. However, in contrast to AM, it does not affect CLR internalisation and induces very different transcriptional response in these cells. These findings suggest that further studies are essential to characterise AM- and CGRP-induced effects, as well as CLR properties and its role in mediating the effects of these peptides, in human LEC. Obtained data would help avoid possible undesirable side-effects of recently developed therapies targeting CGRP signalling, such as anti-migraine drugs utilising CGRP-blocking antibodies, on lymphatic and possibly circulatory systems in human.

In this proposal, we hypothesize that CGRP effects in human LEC are mediated via CLR despite observed differences in AM- and CGRP-induced responses and that this neuropeptide competes with endogenously produced by these cells AM and IMD/AM-2. The overarching aim of this research project is to delineate underlying molecular mechanisms by using primary human LEC and established in our laboratory in Hull methodologies and, whenever possible, utilising antagonists and RNAi technology to address three key objectives:

1. To further characterise and contrast CLR-mediated downstream signalling pathways in response to CGRP and AM.
2. To compare CLR internalisation, trafficking and desensitisation dynamics in response to these two agonists.
3. To confirm the functional relevance of molecular findings using three-dimensional (spheroid and tube formation) assays and hence establishing a solid platform for translational research.