- Type of research degree
- 4 year PhD
- Application deadline
- Ongoing deadline
- Country eligibility
- International (outside UK)
- Dr Jian Shi and Dr Piruthivi Sukumar
- Additional supervisors
- Professor David J Beech
- School of Medicine
- Research groups/institutes
- Leeds Institute of Cardiovascular and Metabolic Medicine
Mechanotransduction in biological systems is the conversion of mechanical stimuli into biological signals and is involved in the modulation of diverse cellular functions such as migration, proliferation, differentiation and apoptosis. Evidently, mechanotransduction is essential from embryo- and organo- genesis to growth and homeostasis. A newly discovered mechanosensitive transmembrane protein, Piezo1 is reported to be primarily responsible for mechanotransduction in diverse cells. We and others have identified that Piezo1 is essential for vascular development and health, blood pressure regulation and exercise related vascular contraction/relaxation. Recently, many gain and loss of function mutations in Piezo1 gene have been identified in a range of pathophysiological conditions including lymphatic dysplasia, anaemia and malarial resistance. While Piezo1 expression and importance is clearly shown by many investigators, Piezo1 mutations that can lead to cardiovascular consequences are not yet identified. The primary aim of this PhD study will be to identify single nucleotide polymorphisms (SNPs) in Piezo1 gene that are associated with cardiovascular diseases. About 500,000 imputed genomic data stored in the UK Biobank will be analysed using appropriate bioinformatics tools, to identify the prevalent SNPs. Furthermore, using in silico methods, SNPs will be characterised and suitable candidates will be selected for laboratory studies. Then the patho-physiological consequences of such mutations on cardiovascular health and disease will be explored using over expression system, CRISPR mediated introduction of mutations in in vitro cell culture and in vivo animal models.
<h6>Background</h6> <p>With obesity reaching epidemic proportions worldwide, the resultant increase in diabetes, hypertension, myocardial infarction and stroke are the major causes of morbidity and mortality. Hence the search for better therapeutic targets for these complex, multifactorial diseases is never ending. All living organisms are subjected to mechanical forces from their environment and rely on mechanotransduction for their survival. The inner lining cells of all blood and lymphatic vessels, endothelial cells are constantly exposed to physical force i.e. shear stress produced by the beating heart and flowing blood/lymph. Hence they have pronounced mechanosensitivity which aid in development and maintenance of healthy vasculature. Although it is long known that multiple patho-physiological signalling pathways are regulated by physical forces in endothelial cells, our current knowledge is limited in understanding the sensory pathways of mechanical stimuli and their roles in cardiovascular diseases. </p> <p>A recently discovered mechanosensitive transmembrane protein, Piezo1, expressed widely in various cells, is reported to be the primary mechanosensor protein in these cells too. Functional Piezo proteins were discovered in 2010, in a targeted short interfering RNA (siRNA) knock down analysis of candidate transmembrane proteins against mechanically activated ionic currents in the murine N2A neuroblastoma cell line. Its discovery has created huge opportunities in exploiting mechanotransduction induced signalling as diseases targets. Human Piezo1 encoded by the PIEZO1/FAM38A is a highly polymorphic gene with many coding variants reported in the 1000 Genomes Project database. Piezo1 mutations have been linked to multiple human disorders. Six of them are known to be gain-of-function mutations that slow the inactivation rate and are associated with dehydrated hereditary xerocytosis. Mechanistically, the mutant channels allow excess calcium influx into RBC leading to osmotically-driven dehydration. In contrast, several loss-of-function Piezo1 mutations occur in patients with generalised lymphatic dysplasia. Piezo1 channels are widely expressed throughout the cardiovascular system and have important roles in angiogenesis in developing embryos, blood pressure control and exercise mediated changes in blood flow distribution and hence can potentially have long-term implications for cardiovascular health and disease. Therefore, mutations in the Piezo1 gene affecting the protein function might play an important role in causation/progression of cardiovascular diseases (CVD). No such mutations are described so far in CVD.</p> <p>The UK Biobank (http://www.ukbiobank.ac.uk/), a health research resource, recruited over 500,000 people aged 40 to 69 years in 2006–2010 across the UK. In July 2017, the genetic information from 501,708 samples was released which combined with the extensive information on medical history and lifestyle choices recorded gives an enormous opportunity to investigate how genetics and other factors impact the onset and development of diseases. This study aims to access the data from UK Biobank, screen for the Piezo1 gene variants/mutations which have associations with a range of CVD and then characterise the functional properties of these mutations, which may aid in the development of therapeutic agents targeting the mutants.</p> <h6>Hypothesis</h6> <p>Mutations in mechanosensing nonselective cation permeable ion channel Piezo1 can lead to cardiovascular disease development/progression.</p> <h6>Aim</h6> <p>Identifying SNPs associated with cardiovascular diseases and further characterisation of candidate SNPs using in silico methods and, in vitro and in vivo models.</p> <h6>Objectives and Experimental Plans</h6> <p>1. Identifying and characterising SNPs associated with cardiovascular diseases in UK Biobank <br /> The genetic data from UK Biobank will be analysed using genomic data analysis software packages (e.g. PLINK) using whole genome association analysis toolset that is designed to perform a range of basic, large-scale analyses in a computationally efficient manner. The focus will be purely on analysis of genotype/phenotype data. SNP index files will be procured from UK Biobank. Using an appropriate confidence interval, SNPs occurring in the locus of Piezo1 gene will be extracted and further downstream analysis will be performed. Then genetic data will be filtered on the basis of whether the subjects are suffering from any CVD or if they acquire any CVD later. Further, the Piezo1 SNPs in CVD patients with ischemic heart disease and hypertension and age matched controls will be analysed and the most frequent Piezo1 mutations or variants in CVD patients will be identified.<br /> <br /> In the next stage, clinical and life style data such as blood pressure, biochemical markers, diet and exercise habits, will be analysed to find any associations between particular genetic variants and a wide range of CVD. Next, a variety of bioinformatics tools and approaches will be employed to predict functional effects of the identified SNPs. Bioinformatics tools include - PROVEAN (Protein Variation Effect Analyzer), PhD-SNP (Predictor of human Deleterious Single Nucleotide Polymorphisms), MutPred2 (for predicting pathogenicity of amino acid substitution), I-mutant (to estimate changes in stability arising due to single point mutation) and structural modelling methods. They will be utilised to predict and select the SNPs that would be of potential functional consequences for further investigation.</p> <p>2. Elucidating the functional impact of selected SNPs in vitro<br /> Using the above mentioned methods, 3-5 SNPs will be selected for further characterisation. By site directed mutagenesis, we will create those SNPs individually in human Piezo1 construct. HEK 293 cells in which native Piezo1 is knocked out stably will be used for over-expressing mutant Piezo1 and study the functional effects. Primarily, calcium influx using radiometric calcium indicator fluorescent dye and single channel current recordings using path-clamp technique will be used for functional studies. Based on those results, protein expression and trafficking studies will be conducted. Taking further the study into cardiovascular context, the selected mutations will be introduced into endothelial cells too by double transfection method (knocking out native Piezo1 by siRNA/shRNA and transfecting the mutant construct temporarily) or mutating the native Piezo1 using CRISPR technology. After checking the knockout and over-expression or mutations by mRNA/protein expression/sequence analysis, calcium response and current measurement, functional characteristics of endothelial cells such as proliferation, migration, tube formation and secretory activity will be studied.</p> <p>3. Elucidating the functional impact of selected SNPs in vivo<br /> Based on the results from the above objectives, one point mutation will be selected for in vivo studies. Using CRISPR technology, a transgenic line will be generated in which the mutation is introduced. Basic phenotyping and cardiovascular system specific phenotyping of the transgenic line will be performed to elucidate the functional impact of the mutation on whole body cardiovascular physiology and diseases development. In/ex vivo experiments will include blood pressure measurement, exercise testing, analysing propensity for atherosclerosis and vascular contraction studies. Depending on the findings, further studies on downstream signalling will be performed.</p> <p>Short Methodology for non-routine experiments:<br /> Fluorescence based measurement of calcium. For determining the Piezo1 mediated calcium entry in transfected HEK 293/endothelial cells, the raise in intracellular calcium ([Ca2+]i) level upon addition of Piezo1 modulators (e.g. Yoda1-activator; Dooku1-inhibitor) will be measured using calcium sensitive fluorescent indicator dye Fura-2 AM and state of the art automated plate reader called Flex Station (www.moleculardevices.com/systems/microplate-readers/multi-mode-readers/flexstation-3-multi-mode-microplate-reader). Cells will be loaded with 2 ?M Fura-2 AM for 1 hour at 37°C and then washed in standard bath solution for 30 minutes at 37°C. Cells will be imaged in Flex Station with appropriate filters. Fura-2 is excitable with 340/380 nm light and emits at 510 nm and the ratio between its emission intensity at the two excitation wavelengths is directly proportional to [Ca2+]i. Flex station will be programmed to apply Piezo1 modulators as per the experimental plan at appropriate concentrations.</p> <h6>Patch-clamp recordings of Piezo1 channels in HEK 293 cells and endothelial cells</h6> <p>To determine the characteristics of Piezo1 channels overexpressed in HEK293 cells or endogenously expressed in endothelial cells, whole-cell and single-channel patch-clamp recordings will be used. Piezo1 plasmids will be transfected into the HEK 293 cells with lipofectamine transfection reagent. Endothelial cells will be freshly isolated from mouse second-order mesenteric arteries. The arteries will be enzymatically digested in dissociation solution containing collagenase (1 mg/ml) and then be pipetted gently to release the endothelial cells. For the patch-clamp recordings on these cells, heat-polished patch pipettes with tip resistances between 3 and 5 MΩ will be used and high resistance seals (≥5 GΩ) will be achieved through slight suction. The Piezo1-mediated non-selective cation currents on these cells will be recorded with Axopatch-200B amplifier equipped with Digidata 1550B and pCLAMP 10.6 software using whole-cell and single-channel configurations. The currents will be evoked by either the channel agonist Yoda1 or the mechanical force such as negative pressure. Then the current amplitude/channel kinetics and channel open probability will be analysed. </p> <h6>CRISPR for in vitro mutation studies.</h6> <p>For introducing point mutation in endothelial cells, CRISPR technology will be employed. Either commercially obtained appropriate gRNA with Cas9 protein or plasmids with relevant inserts will be used to transfect the cells. The clonal selection and expansion will be done to get a stable cell line with required mutation.</p> <h6>References</h6> <p>1. Li, J., Hou, B., Tumova, S., Muraki, K., Bruns, A., Ludlow, M.J., Sedo, A., Hyman, A.J., McKeown, L., Young, R.S., et al. (2014). Piezo1 integration of vascular architecture with physiological force. Nature 515, 279–282.<br /> 2. Rode, B., Shi, J., Endesh, N., Drinkhill, M.J., Webster, P.J., Lotteau, S.J., Bailey, M.A., Yuldasheva, N.Y., Ludlow, M.J., Cubbon, R.M., et al. (2017). Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance. Nat. Commun. 8, 350.<br /> 3. Beech, D.J., and Xiao, B. (2018). Piezo channel mechanisms in health and disease: Editorial. J. Physiol. 596, 965–967.<br /> 4. Hyman, A.J., Tumova, S., and Beech, D.J. (2017). Piezo1 Channels in Vascular Development and the Sensing of Shear Stress. Curr. Top. Membr. 79, 37–57. </p>
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<h3 class="heading heading--sm">Linked research areas</h3>