Translational studies in models of cardiovascular disease; investigating genetic determinants of complex polygenic disorders

Cardiovascular disease is estimated to kill an average of 17 million people each year and is regarded as being the single largest risk for mortality both in developed and developing countries. A major factor that contributes to this mortality rate is hypertension (high blood pressure), which accounts for nearly two-thirds of all strokes and a half of all heart disease. Approximately 50% of blood pressure variation in the human population is genetically determined and therefore the identification of the genes responsible for hypertension and its major complications is key to understanding the underlying disease processes. The complexity of this disease and the inability to conduct genetic studies in humans necessitate the use of animal models which closely mimic human disease characteristics. Rodent models such as spontaneously hypertensive rats and genetically modified knock-out mice provide an invaluable resource for studies which will ultimately lead to improved understanding, better treatment and prevention of human hypertension and its cardiovascular complications.

Congenic and transgenic strategies for functional analysis of candidate genes in the SHRSP rat:
Utilising a range of congenic strains derived from the stroke-prone spontaneously hypertensive rat (SHRSP) combined with microarray expression profiling we have identified several important candidate genes for baseline blood pressure regulation (i.e. Gstm1) [1,2] and response to salt (i.e. Edg1, Vcam1) [3] on rat chromosome 2. In addition, we have identified a candidate gene for cardiac hypertrophy and fibrosis on rat chromosome 14 (i.e. Spp1), and through congenic substrain production have also identified a congenic interval on rat chromosome 3 harbouring genes for pulse pressure regulation and salt-induced renal injury.
• To confirm the causal role of Gstm1 in blood pressure regulation we have performed a transgenic rescue experiment resulting in the generation of two novel SHRSP transgenic lines, which express wild type Gstm1.These strains demonstrate significantly reduced haemodynamic profile and reduced oxidative stress parameters downstream of the antioxidant glutathione. Translational studies to assess human Gstm1 orthologues in human kidneys are currently ongoing.
• To further investigate the role of Edg1 and Vcam1 candidate genes we have carried out analysis of Edg1 functional networks utilising cell based assays in SHRSP and WKY vascular smooth muscle cells. These studies have confirmed the importance of Edg1 in the mechanism of hypertension development [4].
High-resolution mapping of multiple quantitative trait loci in Heterogeneous Stock rats:
In collaboration with the EURATools and EURATRANS consortia we have combined sequence-based and genetic mapping analysis in heterogeneous stock (outbred) rats [5] identifying 31 genes involved in 27 different phenotypes, implicating novel genes in models of anxiety, heart disease and multiple sclerosis. We are currently carrying out functional validation of the implicated cardiovascular genes (e.g. Shank2 and heart weight).
Pharmacological intervention studies:
We have identified a novel mitochondria-targeted antioxidant, MitoQ10, which improves cardiovascular function and attenuates left ventricular hypertrophy in the SHRSP [6]. Our studies demonstrate that MitoQ10 has potential as a unique complementary therapeutic intervention to current antihypertensive drugs.
Functional analysis of the human candidate gene, uromodulin (Umod):
In a previous GWAS study we identified a locus on human chromosome 16 in the 5’ region of the uromodulin gene, which is associated with hypertension [7]. Uromodulin (also known as Tam Horsfall Protein or THP) is a specific kidney protein that is synthesised in the thick ascending limb (TAL) of Henle’s loop and is the most abundant protein excreted in the urine of normal mammals. The role of uromodulin in blood pressure regulation and renal salt handling is being investigated in the THP (umod) knock-out mouse. These mice demonstrate significantly lower blood pressure and increased salt excretion during 2% salt loading. The underlying mechanisms are being investigated in primary thick ascending limb cells isolated from THP mice.
Additional collaborative projects:
• Angiotensin 1-9 and angiotensin 1-7: assessment of their mechanisms of action as counter-regulatory renin angiotensin system peptides in cardiovascular disease.
In collaboration with Dr S Nicklin (PI) and Dr C Loughrey (BHF project grant).
• The role of AMP-activated protein kinase in adipocyte glucose transport and insulin signalling.
In collaboration with Ian Salt (PI) (Diabetes UK research grant).

Publications

1. McBride MW, Brosnan MJ, Mathers J, McLellan LI, Miller WH, Graham D, Hanlon N, Hamilton CA, Polke JM, Lee WK, Dominiczak AF. Reduction of Gstm1 expression in the SHRSP contributes to increased oxidative stress. Hypertension 2005;45:786-92.

 2. Graham D, Koh-Tan HHC, Hamilton CA, Nicoll G, McBride MW, Young B, Dominiczak AF. Renal and vascular glutathione s-transferase mu is not affected by pharmacological intervention to reduce systolic blood pressure. J Hypertension 2009;27:1575-1584.

3. Graham D, McBride MW, Gaasenbeek M, Gilday K, Beattie E, Miller WH, McClure JD, Polke JM, Montezano A, Touyz RM, Dominiczak AF. Candidate genes that determine response to salt in the stroke-prone spontaneously hypertensive rat: congenic analysis. Hypertension 2007;50:1134-41.

4. Yogi A, Callera GE, Aranha AB, Graham D, McBride MW, Dominiczak A.F, Touyz R. Sphingosine-1-phosphate-induced inflammation involves receptor tyrosine kinase transactivation in vascular cells: upregulation in hypertension. Hypertension 2011;57:809-18.

5. Johannesson M, Lopez-Aumatell R, Stridh P, Diez M, Tuncel J, Blázquez G, Martinez-Membrives E, Cañete T, Vicens-Costa E, Graham D, Copley RR, Hernandez-Pliego P, Beyeen AD, Ockinger J, Fernández-Santamaría C, Gulko PS, Brenner M, Tobeña A, Guitart-Masip M, Giménez-Llort L, Dominiczak A, Holmdahl R, Gauguier D, Olsson T, Mott R, Valdar W, Redei EE, Fernández-Teruel A, Flint J.  A resource for the simultaneous high-resolution mapping of multiple quantitative trait loci in rats: The NIH heterogeneous stock. Genome Res. 2009;19:150-8.

6. Graham D, Huynh NN, Hamilton CA, Koh-Tan HHC, Smith RAJ, Cocheme HM, Murphy MP, Dominiczak AF. The mitochondria targeted antioxidant MitoQ10 improves cardiovascular function and attenuates left ventricular hypertrophy. Hypertension 2009;54:322-328.

7. Padmanabhan S, Melander O, Johnson T, Di Blasio AM, Lee WK, Gentilini D, Hastie CE, Menni C, Monti MC, Delles C, Laing S, Corso B, Navis G, Kwakernaak AJ, van der Harst P, Bochud M, Maillard M, Burnier M, Hedner T, Kjeldsen S, Wahlstrand B, Sjögren M, Fava C, Montagnana M, Danese E, Torffvit O, Hedblad B, Snieder H, Connell JM, Brown M, Samani NJ, Farrall M, Cesana G, Mancia G, Signorini S, Grassi G, Eyheramendy S, Wichmann HE, Laan M, Strachan DP, Sever P, Shields DC, Stanton A, Vollenweider P, Teumer A, Völzke H, Rettig R, Newton-Cheh C, Arora P,  Zhang F, Soranzo N, Spector TD, Lucas G, Kathiresan S, Siscovick DS, Luan J, Loos RJ, Wareham NJ, Penninx BW, Nolte IM, McBride M, Miller WH, Nicklin SA, Baker AH, Graham D, McDonald RA, Pell JP, Sattar N, Welsh P; Global BPgen Consortium, Munroe P, Caulfield MJ, Zanchetti A, Dominiczak AF. Genomewide Association Study of Blood Pressure Extremes Identifies Variant near UMOD Associated with Hypertension. PLoS Genetics. 2010;6:e1001177.