An investigation into the TRPV1-CGRP signalling pathway in vessel development

Bates, Adam

Abstract

TRPV1 is a non-selective cation channel which is activated through various different factors including, but not limited to, raised temperature and acidic pH. It has been linked in the past to endothelial cell proliferation in acidic conditions, but a direct link between TRPV1-CGRP signalling in endothelial cells in development has not been highlighted before. The main goal of this thesis is to explore the effects of manipulating TRPV1 on vessel formation.
The main three overarching hypotheses are that CGRP, which is released by TRPV1 activation will cause transcriptional changes in endothelial cells in culture, the knockdown of TRPV1 will cause transcriptional changes to a developing zebrafish embryo and that TRPV1 knockdown will have detrimental effects to a developing zebrafish embryo and its vascular system.
Chapter 2 analyses the effects of human dermal lymphatic endothelial cells (HDLECs) transcriptomic response to being stimulated by CGRP, a molecule which is released by TRPV1 activation. It was concluded that lymphatic endothelial cells were more prone to being stimulated than blood endothelial cells, and that a neuropeptide CGRP causes transcriptional changes to genes with functions relating to vessel development of HDLECs.
Results showed that CGRP induced changes to the expression of 144 genes, compared to 23 DEGs upon AM stimulation. The HDLECs experiments results were then explored in the in vivo model of a developing zebrafish embryo in the two subsequent chapters.
Chapter 3 explores the hypothesis that TRPV1 knockdown will have detrimental effects to the developing embryo, causing a change to the transcriptome. The embryo was injected with TRPV1 targeting morpholino in combination with the TRPV1 agonist 2-APB. Swim responses of MO injected embryos was half of the control upon response to heat stimuli (0.52cm/sec compared to 1.02cm/sec). RNA-seq results show that the genes which are significantly differentially expressed upon TRPV1 knockdown have endothelial cell related functions and these results are supported through the use of antibody staining to identify the vessels in the developing embryo.
Chapter 4 investigates whether the changes to gene expression identified in the previous chapter are sustained when the embryo is exposed to different stressors, individually and in combination. Bevacuzimab was included to be a comparison of TRPV1 knockdown as it is known to affect the blood vessel development of zebrafish, by targeting the VEGFA pathway. Gene expression was measured using qLAMP and a novel analysis method in combination with developmental and survival rates of the embryo.
The results of the LAMP reactions showed that both bevacizumab injection and TRPV1 knockdown had a significant effect on the gene expression relating to vessel formation. When APB was used in combination with the knockdown, there was an even more significant change to overall gene expression (p<0.0001). Antibody staining also showed that TRPV1 is expressed in olfactory bulbs in the developing embryo which had been injected with bevacizumab.
These results show a novel in vivo link between TRPV1 and vessel development in zebrafish which may have implications in pathophysiological conditions such as cancer, hypertension and chronic pain as well as environmental conditions such as increasing heat.

Citation

Bates, A. An investigation into the TRPV1-CGRP signalling pathway in vessel development. (Thesis). University of Hull. https://hull-repository.worktribe.com/output/4865918

• Thesis Type: Thesis
• Deposit Date: Oct 15, 2024
• Publicly Available Date: Oct 29, 2024
• Keywords: Biology
• Public URL: https://hull-repository.worktribe.com/output/4865918
• Additional Information: Department of Biological Science; University of Hull
• Award Date: Jul 17, 2024