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

Redox signaling, Nox5 and vascular remodeling in hypertension

Purpose of review: Extensive data indicate a role for reactive oxygen species (ROS) and redox signaling in vascular damage in hypertension. However, molecular mechanisms underlying these processes remain unclear, but oxidative post-translational modification of vascular proteins is critical. This review discusses how proteins are oxidatively modified and how redox signaling influences vascular smooth muscle cell growth and vascular remodeling in hypertension. We also highlight Nox5 as a novel vascular ROS-generating oxidase. Recent findings: Oxidative stress in hypertension leads to oxidative imbalance that affects vascular cell function through redox signaling. Many Nox isoforms produce ROS in the vascular wall, and recent findings show that Nox5 may be important in humans. ROS regulate signaling by numerous processes including cysteine oxidative post-translational modification such as S-nitrosylation, S-glutathionylation and sulfydration. In vascular smooth muscle cells, this influences cellular responses to oxidative stimuli promoting changes from a contractile to a proliferative phenotype. Summary: In hypertension, Nox-induced ROS production is increased, leading to perturbed redox signaling through oxidative modifications of vascular proteins. This influences mitogenic signaling and cell cycle regulation, leading to altered cell growth and vascular remodeling in hypertension

Vascular Nox (NADPH oxidase) compartmentalization, protein hyperoxidation, and endoplasmic reticulum stress response in hypertension

Vascular Nox (NADPH oxidase)-derived reactive oxygen species and endoplasmic reticulum (ER) stress have been implicated in hypertension. However, relationships between these processes are unclear. We hypothesized that Nox isoforms localize in a subcellular compartment-specific manner, contributing to oxidative and ER stress, which influence the oxidative proteome and vascular function in hypertension. Nox compartmentalization (cell fractionation), O2− (lucigenin), H2O2 (amplex red), reversible protein oxidation (sulfenylation), irreversible protein oxidation (protein tyrosine phosphatase, peroxiredoxin oxidation), and ER stress (PERK [protein kinase RNA-like endoplasmic reticulum kinase], IRE1α [inositol-requiring enzyme 1], and phosphorylation/oxidation) were studied in spontaneously hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). VSMC proliferation was measured by fluorescence-activated cell sorting, and vascular reactivity assessed in stroke-prone SHR arteries by myography. Noxs were downregulated by short interfering RNA and pharmacologically. In SHR, Noxs were localized in specific subcellular regions: Nox1 in plasma membrane and Nox4 in ER. In SHR, oxidative stress was associated with increased protein sulfenylation and hyperoxidation of protein tyrosine phosphatases and peroxiredoxins. Inhibition of Nox1 (NoxA1ds), Nox1/4 (GKT137831), and ER stress (4-phenylbutyric acid/tauroursodeoxycholic acid) normalized SHR vascular reactive oxygen species generation. GKT137831 reduced IRE1α sulfenylation and XBP1 (X-box binding protein 1) splicing in SHR. Increased VSMC proliferation in SHR was normalized by GKT137831, 4-phenylbutyric acid, and STF083010 (IRE1–XBP1 disruptor). Hypercontractility in the stroke-prone SHR was attenuated by 4-phenylbutyric acid. We demonstrate that protein hyperoxidation in hypertension is associated with oxidative and ER stress through upregulation of plasmalemmal-Nox1 and ER-Nox4. The IRE1–XBP1 pathway of the ER stress response is regulated by Nox4/reactive oxygen species and plays a role in the hyperproliferative VSMC phenotype in SHR. Our study highlights the importance of Nox subcellular compartmentalization and interplay between cytoplasmic reactive oxygen species and ER stress response, which contribute to the VSMC oxidative proteome and vascular dysfunction in hypertensio

The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general

The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general.</p

Assessment of protein carbonylation and protein tyrosine phosphatase (PTP) oxidation in vascular smooth muscle cells (VSMCs) using immunoblotting approaches

Post-translational modification of proteins, such as phosphorylation and oxidation, plays a major role in cellular signaling by influencing protein structure and function. In vascular cells, in addition to influencing phosphorylation, angiotensin II (Ang II) induces oxidation of proteins, important in redox signaling in the cardiovascular and renal systems. The present chapter describes immunoblotting approaches to assess irreversible protein carbonylation and protein tyrosine phosphatase (PTPs) oxidation status in the proteome of vascular smooth muscle cells (VSMC).Protein carbonylation is generally measured using the OxyBlot™ approach, whereby derivatization of protein carbonyl groups (C = O) on oxidized amino acids by dinitrophenylhydrazine (DNPH) results in the formation of a stable dinitrophenyl (DNP) hydrazone product. The samples are analyzed by SDS-PAGE and a primary antibody raised against the DNP moiety is used to determine levels of irreversible protein carbonylation in the sample by immunoblotting.Oxidation of PTPs can be evaluated using a monoclonal antibody against the "hyperoxidized" (SO3H) catalytic site of these enzymes. The described methodology offers the ability to discriminate between irreversible (SO3H) and reversible (SOH) PTP oxidation states. Initially, the free unmodified PTP-thiols (S(-)) are alkylated and the sample is split into two. One part is used to assess the PTP-SO3H form. In the other part reversibly modified PTP-thiols are first reduced and then hyperoxidized by pervanadate (PV). Both untreated and PV-treated samples are analyzed by SDS-PAGE and "hyperoxidized" PTPs are detected by immunoblotting. The proportion of reversibly oxidized PTP-SOH fraction is determined by the difference between the signals in untreated and the PV-treated samples.The above immunoassays provide general approaches to detect and quantify global levels of irreversible protein oxidation and of irreversibly/reversibly oxidized PTPs in any (patho)physiological context. Characterization of the global redox status is essential to better understand the redox-sensitive mechanisms underlying chronic diseases associated with oxidative stress. This is particularly important in systems influenced by the renin angiotensin system, because Ang II is a potent inducer of oxidative stress and redox signaling

Urine metabolomics in hypertension research

Functional genomics requires an understanding of the complete network of changes within an organism by extensive measurements of moieties from mRNA, proteins, and metabolites. Metabolomics utilizes analytic chemistry tools to profile the complete spectrum of metabolites found in a tissue, cells, or biofluids using a wide range of tools from infrared spectroscopy, fluorescence spectroscopy, NMR spectroscopy, and mass spectrometry. In this protocol, we outline a procedure for performing metabolomic analysis of urine samples using liquid chromatography–mass spectrometry (LC-MS). We outline the advantages of using this approach and summarize some of the early promising studies in cardiovascular diseases using this approach

Biomarkers of oxidative stress in human hypertension

Hypertension is a major cardiovascular risk factor. Of the many processes involved in the pathophysiology of hypertension, cardiac, vascular and renal damage due to oxidative stress (excess bioavailability of reactive oxygen species (ROS)) is important. Physiologically, ROS regulate cell function through redox-sensitive pathways. In hypertension, oxidative stress promotes endothelial dysfunction, vascular remodeling and inflammation, leading to vascular damage. While experimental evidence indicates a causative role for oxidative stress in hypertension, human data are less convincing. This may relate to sub-optimal approaches to accurately measure ROS in humans. Various methods have been developed to assess the extent and nature of oxidative stress, including markers of protein oxidation, lipid oxidation, and anti-oxidant status. These approaches are, in general, indirect and measure indices of redox state. While large clinical studies to establish whether biomarkers of oxidative stress accurately predict disease risk are still needed, oxidative biomarkers have provided important mechanistic insights regarding redox-sensitive processes of hypertension. Here we briefly describe the importance of ROS in redox signaling and hypertension and discuss biomarkers of oxidative stress in human hypertension

Nonlinear functionals of wavelet expansions - adaptive reconstruction and fast evaluation

This paper is concerned with the efficient evaluation of nonlinear expressions of wavelet expansions obtained through an adaptive process. In particular, evaluation covers here the computation of inner products of such expressions with wavelets which arise, for instance, in the context of Galerkin or Petrov Galerkin schemes for the solution of differential equations. The central objective is to develop schemes that facilitate such evaluations at a computational expense exceeding the complexity of the given expansion, i.e., the number of nonzero wavelet coefficients, as little as possible. The following issues are addressed. First, motivated by previous treatments of the subject, we discuss the type of regularity assumptions that are appropriate in this context and explain the relevance of Besov norms. The principal strategy is to relate the computation of inner products of wavelets with compositions to approximations of compositions in terms of possibly few dual wavelets. The analysis of these approximations finally leads to a concrete evaluation scheme which is shown to be in a certain sense asymptotically optimal. We conclude with a simple numerical example. (orig.)Available from TIB Hannover: RN 8680(160) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Biomarkers of vascular inflammation and cardiovascular disease

Cardiovascular disease is the major cause of morbidity and mortality globally. As such better approaches for early detection and mechanism-targeted therapies are key priorities in cardiovascular research. Growing evidence indicates that vascular inflammation and oxidative stress may play an important role in the genesis and progression of cardiovascular disease. Accordingly identification of markers reflecting these processes may be useful early predictors of vascular damage and could provide insights into mechanisms, risk and targeted treatment. The present chapter provides a brief overview of vascular damage in cardiovascular disease and discusses recently identified novel biomarkers of vascular inflammation and oxidative stress. The potential clinical relevance is also highlighted