Proteomic and metabolomic profiling in the stroke-prone spontaneously hypertensive rat and chromosome 2 congenic strains

Essential hypertension (EH) is considered one of the major contributors to the present pandemic of cardiovascular disease (CVD). EH has a largely obscure aetiology, which lies upon both environmental risk factors and underlying genetic traits. The stroke-prone spontaneously hypertensive rat (SHRSP) is an excellent model of human EH and exhibits salt sensitivity. Two quantitative trait loci (QTL) for blood pressure (BP) regulation have been identified on rat chromosome 2 (chr.2). On this basis, previous work in our laboratory focused on construction of chr.2 congenic strains, on both the SHRSP and Wistar-Kyoto (WKY) genetic backgrounds. In combination with microarray gene expression profiling in kidney from salt-loaded rats, two positional candidate genes for salt-sensitive hypertension were identified. Sphingosine-1-phosphate receptor 1 (S1pr1) and vascular adhesion molecule (Vcam1) lie on the chr.2 congenic interval implicated in salt-sensitivity. Additionally, studies on vascular smooth muscle cells (VSMC) demonstrated enhanced S1PR1-mediated sphingosine signalling in SHRSP compared to WKY. Finally, glutathione S-transferase mu 1 (Gstm1) was identified as another chr.2 candidate gene for BP regulation, lying outside the region implicated in salt-sensitivity. This project attempts to comprehensively investigate the potential role of altered S1PR1 signalling in BP regulation and salt-sensitivity, through comparative proteomic and metabolomic profiling in WKY, SHRSP and chromosome 2 congenic and transgenic stains (WKY.SPGla2a, SP.WKYGla2a, SP.WKYGla2k and Gstm1-transgenic). Characterisation of S1PR1 expression in renal and vascular tissue from 21 week-old salt-loaded rats, demonstrated below detection protein levels across parental and congenic strains. To further investigate the effect of the congenic interval and Gstm1 on salt-sensitivity and BP regulation and identify putative biomarkers, high-throughput metabolomic screening of urine and plasma was conducted in parental, SP.WKYGla2k congenic and Gstm1-transgenic strains, on a normal-salt and high-salt diet. In both urine and plasma, salt-loading affected processes implicated in CVD, including inflammatory response, free radical scavenging and lipid metabolism. In urine, oleic acid, implicated in regulation of renin levels, was increased in the SHRSP and transgenic salt-sensitive strains compared to the WKY and 2k congenic salt-resistant strains, upon salt-loading. In plasma, known biomarkers of CVD were altered in SHRSP compared to the other three strains, at normal-salt, including L-proline and linoleic acid. Upon salt-loading, glutathione disulfide and sphingosine-1-phosphate (S1P) were identified in high levels in the salt-sensitive strains. However, at normal-salt S1P was decreased in SHRSP compared to WKY and 2k congenic strains. Therefore, characterisation of the impact of S1P/S1PR1 signalling in the vasculature across the different strains was further investigated. Initially, structure, mechanical properties and vascular reactivity of mesenteric resistance arteries (MRA) were studied in 16 week-old parental and reciprocal 2a congenic strains (WKY.SPGla2a and SP.WKYGla2a). There was no significant remodelling observed across the strains. However, SHRSP vessels were stiffer and this phenotype was under the control of the congenic segment. SHRSP exhibited hypercontractility, which was mediated by RhoA/Rock signalling pathway and was corrected by the transfer of the congenic interval in SP.WKYGla2a. SHRSP also displayed endothelial dysfunction, which was related to reduced nitric oxide (NO) bioavailability and was not improved by the congenic interval. The predominant regulatory mechanisms of contraction and relaxation in MRAs from WKY and WKY.SPGla2a were demonstrated to be different compared to SHRSP. Subsequently, representation of these physiological differences in MRAs, at the molecular level, was investigated along with the effect of S1P-signalling in HTN. Comprehensive, high-throughput proteome profiling of S1P-stimulated primary mesenteric VSMCs from parental and 2a-reciprocal congenic strains, was achieved through triple stable isotope labelling (SILAC), LC-MS/MS analysis and MaxQuant quantification. Detection of few abundant phosphorylated proteins was attributed to lack of enrichment for phosphoproteome. Therefore, focus was placed on proteins whose differential expression between SHRSP and WKY was genetically regulated. These proteins mapped to pathways implicated in BP-regulation, including oxidative stress, vascular tone regulation and vascular remodelling. Glutathione S-transferase mu 1 (GSTM1) was upregulated in SHRSP, as opposed to down-regulated NAD(P)H oxidase quinone 1 (NQO1) and heme oxygenase 1 (HMOX1), suggesting different antioxidant mechanisms in health and disease. Natriuretic peptide receptor C (NPR3) which is implicated in vascular relaxation was increased in SHRSP, along with activators of RhoA contractile mechanism, such as caveolin1 (CAV1). Furthermore, RhoA/Rock signalling pathway was highly altered in SHRSP. Finally, differentially expressed proteins were related to sphingosine signalling, including superoxide dismutase 2 (SOD2) and collagen type III, alpha 1 (COL3A1). To further investigate the metabolic effect of sphingosine signalling across the strains, and assess the contribution of the congenic interval, metabolomic profiling of primary mesenteric VSMCs from parental and SP.WKYGla2a congenic strains, was performed at basal conditions and upon S1P-stimulation. A labelling-free, untargeted approach was employed, using HILIC-MS analysis and data processing through IDEOM. The effect of the congenic interval on the metabolic profile of SP.WKYGla2a was more profound under basal conditions. S1P-stimulation induced greater responses in SHRSP than WKY, indicating altered signalling. Furthermore the responses were different in each strain, suggesting a combined effect of the genetic background and the congenic interval on S1P signalling regulation. Inosine, which is implicated in purine metabolism, was significantly decreased in SHRSP compared to SP.WKYGla2a, at basal conditions, but was increased upon-S1P stimulation, implying that this S1P effect depends on the congenic interval. Moreover, tyramine, which has vasodilatory properties, was increased in stimulated SHRSP compared to basal conditions, indicating potential relation of sphingosine signalling with BP-regulation. This study has combined high-throughput proteomic and metabolomic screenings with congenic and transgenic strains to capture a clearer picture of the pathophysiological processes that underlie HTN in SHRSP. Individual metabolites and proteins or pathways and processes identified to be altered in HTN, through this work, can be used for generation of new testable hypothesis towards the development of new therapeutic approaches against HTN.