Investigating the effect of telomere length on endothelial cell function

Telomeres are protein bound DNA repeat structures at the ends of eukaryotic chromosomes, Telomere length (TL) has been proposed as a marker of biological ageing and shorter TL associates with several age related diseases, including coronary artery disease (CAD). Recent genetic studies have provided evidence that shorter TL is causal to CAD, but how TL shortening increases disease risk is not yet understood. In addition, a previous study shows that statin treatment specifically benefits individuals who have shorter TL, suggesting that TL could be used to identify patients who would most benefit from this treatment.

Endothelial cells are one of two major cell types in the vessel wall and endothelial dysfunction is an early stage of the disease process. This project will aim to understand how TL influences disease risk via a potential effect on endothelial function using a cellular model of telomere shortening to investigate how TL may influence endothelial cell physiology and how statin treatment may alter the effect of TL in this cell type.

The student will have the opportunity to learn a broad range of cellular and molecular techniques, including cell culture and functional assays (cell adherence and migration), gene expression analysis (qPCR, microarray) and protein expression analysis (western blot and flow-cytometry). The student will also attend postgraduate training courses offered by the University.

Background

Telomere length (TL) has been proposed as a marker of biological ageing and shorter TL associates with several age related diseases, including coronary artery disease (CAD).1-3 Recently, through large-scale genetic analysis and downstream Mendelian randomisation studies we have provided evidence towards a causal relationship between shorter TL and increased risk of CAD4. In additionally, there is evidence that individuals with shorter TL specifically benefit from statin therapy but not via modulation of plasma lipids.2 Therefore, mechanistic studies to establish how TL influences risk could identify therapeutic targets and support potential patient stratification for treatment.

Proposed study

We have been working to establish a cellular model to create isogenic lines which differ only in TL in the absence of traditional confounding factors. Preliminary finding suggest that TL influences expression of ICAM1 and E-selectin in endothelial cells.

This study will firstly utilise the model to explore potential effects of TL on the early stages of endothelial dysfunction and will aim to:

1) Confirm the effect of TL on adhesion molecule expression and test if this translates to monocyte adhesion in-vitro.
2) Investigate if TL alters endothelial nitric oxide production.
3) Explore whether cells with different TL react differently to pro-atherogenic stimuli (e.g. TNFα).
4) Establish whether any observed effects are driven via induction of senescence.

As there is some evidence that statins may influence endothelial function5 the above effects will be examined under the influence of statins.

We will assess differences in gene expression which are driven by TL shortening at either the global level or by targeted pathway analysis.

Telomere shortening influences the expression of genes proximal to the end of the chromosomes, through a mechanism termed the telomere position effect (TPE).6 This model will be used to investigate gene expression changes influenced by TL by microarray. We will then assess what influence statin treatment may have on identified genes/pathways.

Hypothesis

Telomere length modulates CAD risk via reduced endothelial function

Experimental Methods and Research Plan:

Methods will include a range of cellular and molecular techniques, such as:
Culture of primary endothelial cells (HUVEC) carrying a removable TERT transgene, transient transfection, gene expression analysis by qPCR, microarray and/or pathway array, protein expression by flow-cytometry and western blotting, cellular adhesion assays, colorimetric assay for eNOS activity.

Years 1&2 Generation of “long”, “intermediate” and “short” TL isogenic lines (N=3) from three separate TERT transgenic parental lines (N=9). They will be used for functional characterisation of the effect of TL on eNOS activity and adhesion molecule expression and in-vitro cell adhesion. Experiments will be performed with and without stimulation by pro-inflammatory cytokines or oxidative stress.

Year 3 The student will investigate statin influence on the cellular response to pro-inflamatory stimulation in a TL dependent manner. They will also perform microarray analysis to identify which genes/pathways are altered in response to TL and identify any which are altered by statin treatment.

Entry requirements:

Applicants are required to hold/or expect to obtain a UK Bachelor Degree 2:1 or better in a relevant subject. The University of Leicester English language requirements apply where applicable (https://le.ac.uk/study/research-degrees/entry-reqs/eng-lang-reqs)

How to apply:

You should submit your application using our online application system - https://www2.le.ac.uk/research-degrees/phd/applyphd

Apply for a PhD in Cardiovascular Sciences
In the funding section of the application please indicate you wish to be considered for a LPMI/BRC studentship
In the proposal section please provide the name of the supervisor and project you want to be considered for.
You do not need to submit a proposal but please include a personal statement detailing your interest in this project

Project / Funding Enquiries:
Dr Veryan Codd [Email Address Removed]
Prof Nilesh Samani [Email Address Removed]

Application enquiries to [Email Address Removed]

Closing date for applications: 27th January