Sofia Tsiropoulou

Oxidative Stress and Human Hypertension: Vascular Mechanisms, Biomarkers, and Novel Therapies

Hypertension is a major cardiovascular risk factor. Of the many processes involved in the pathophysiology of hypertension, vascular damage due to oxidative stress (excess bioavailability of reactive oxygen species [ROS]) is particularly important. Physiologically, ROS regulate vascular function through redox-sensitive signalling pathways. In hypertension, oxidative stress promotes endothelial dysfunction, vascular remodelling, and inflammation, leading to vascular damage. Vascular ROS are derived primarily by nicotinamide adenine dinucleotide phosphate oxidases, which are prime targets for therapeutic development. Although experimental evidence indicates a causative role for oxidative stress in hypertension, human data are less convincing. This might relate, in part, to suboptimal methods to accurately assess the redox state. Herein we review current knowledge on oxidative stress in vascular pathobiology and implications in human hypertension. We also discuss biomarkers to assess the redox state in the clinic, highlight novel strategies to inhibit ROS production, and summarize how lifestyle modifications promote vascular health by reducing oxidative stress.

Redox signaling, Nox5 and vascular remodeling in hypertension

Purpose of reviewExtensive 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 findingsOxidative 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. SummaryIn 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.

Adipocyte-specific mineralocorticoid receptor overexpression in mice is associated with metabolic syndrome and vascular dysfunction - role of redox-sensitive PKG-1 and Rho kinase.

Mineralocorticoid receptor (MR) expression is increased in adipose tissue from obese individuals and animals. We previously demonstrated that adipocyte-MR overexpression (Adipo-MROE) in mice is associated with metabolic changes. Whether adipocyte MR directly influences vascular function in these mice is unknown. We tested this hypothesis in resistant mesenteric arteries from Adipo-MROE mice using myography and in cultured adipocytes. Molecular mechanisms were probed in vessels/vascular smooth muscle cells and adipose tissue/adipocytes and focused on redox-sensitive pathways, Rho kinase activity, and protein kinase G type-1 (PKG-1) signaling. Adipo-MROE versus control-MR mice exhibited reduced vascular contractility, associated with increased generation of adipocyte-derived hydrogen peroxide, activation of vascular redox-sensitive PKG-1, and downregulation of Rho kinase activity. Associated with these vascular changes was increased elastin content in Adipo-MROE. Inhibition of PKG-1 with Rp-8-Br-PET-cGMPS normalized vascular contractility in Adipo-MROE. In the presence of adipocyte-conditioned culture medium, anticontractile effects of the adipose tissue were lost in Adipo-MROE mice but not in control-MR mice. In conclusion, adipocyte-MR upregulation leads to impaired contractility with preserved endothelial function and normal blood pressure. Increased elasticity may contribute to hypocontractility. We also identify functional cross talk between adipocyte MR and arteries and describe novel mechanisms involving redox-sensitive PKG-1 and Rho kinase. Our results suggest that adipose tissue from Adipo-MROE secrete vasoactive factors that preferentially influence vascular smooth muscle cells rather than endothelial cells. Our findings may be important in obesity/adiposity where adipocyte-MR expression/signaling is amplified and vascular risk increased.

Vascular dysfunction in obese diabetic db/db mice involves the interplay between aldosterone/mineralocorticoid receptor and Rho kinase signaling

Activation of aldosterone/mineralocorticoid receptors (MR) has been implicated in vascular dysfunction of diabetes. Underlying mechanisms are elusive. Therefore, we investigated the role of Rho kinase (ROCK) in aldosterone/MR signaling and vascular dysfunction in a model of diabetes. Diabetic obese mice (db/db) and control counterparts (db/+) were treated with MR antagonist (MRA, potassium canrenoate, 30 mg/kg/day, 4 weeks) or ROCK inhibitor, fasudil (30 mg/kg/day, 3 weeks). Plasma aldosterone was increased in db/db versus db/+. This was associated with enhanced vascular MR signaling. Norepinephrine (NE)-induced contraction was increased in arteries from db/db mice. These responses were attenuated in mice treated with canrenoate or fasudil. Db/db mice displayed hypertrophic remodeling and increased arterial stiffness, improved by MR blockade. Vascular calcium sensitivity was similar between depolarized arteries from db/+ and db/db. Vascular hypercontractility in db/db mice was associated with increased myosin light chain phosphorylation and reduced expression of PKG-1α. Vascular RhoA/ROCK signaling and expression of pro-inflammatory and pro-fibrotic markers were exaggerated in db/db mice, effects that were attenuated by MRA. Fasudil, but not MRA, improved vascular insulin sensitivity in db/db mice, evidenced by normalization of Irs1 phosphorylation. Our data identify novel pathways involving MR-RhoA/ROCK-PKG-1 that underlie vascular dysfunction and injury in diabetic mice.

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.

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], IRE1&agr; [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 IRE1&agr; 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 hypertension.

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.

181 Nox compartmentalization and protein oxidation in vascular smooth muscle cells – implications in vascular dysfunction in hypertension

NADPH oxidases (Noxs) are a major source of reactive oxygen species (ROS) in vascular cells. ROS are important signalling molecules with diverse actions. Mechanisms underlying differential ROS effects may relate, in part, to subcellular localization and Nox isoform specificity. We investigated the compartmentalization of Noxs and ROS in vascular smooth muscle cells (VSMC) and questioned whether these phenomena are altered in hypertension. VSMCs isolated from mesenteric arteries of Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) were studied. Subcellular compartmentalization of Noxs was evaluated by immunoblotting after organelle fractionation. ROS levels were measured by chemiluminescence (O2-) and amplex red (H2O2) in the absence or presence of of ML171 (Nox1 inhibitor), GKT136901 (Nox1/4 inhibitor), mito-tempol (mitochondrial-targeted antioxidant) and 4-PBA (ER stress inhibitor). Protein oxidation was assessed using the fluorescent probe DCP-Rho1 for protein sulfenylation and the oxyblot assay for protein carbonylation. Oxidation of protein tyrosine phosphatases (PTP) was evaluated by immunoblotting and Peroxiredoxin (Prx) oxidation was assessed by one-dimensional isoelectric focusing. Vascular reactivity was assessed by myography ± DTT (reducing agent) and peroxiredoxin inhibitor (Conoidin A). Expression of Nox1, Nox2 and Nox4 was greater in total cell homogenates from SHRSP versus WKY. Nox isoforms were detected in plasma membrane, ER and nucleus in both strains, but not in the mitochondria. Basal ROS generation was increased in SHRSP cells. In WKY only Nox1 inhibition decreased Ang II-induced ROS generation. Inhibition of Nox1 and Nox4 decreased basal and Ang II-induced ROS in SHRSP. Additionally, mito-tempol and 4-PBA reduced basal ROS generation in SHRSP. Analysis of protein oxidation revealed increased protein carbonylation and PTP oxidation in SHRSP. Furthermore, oxidation of the antioxidant enzymes Prxs was increased in SHRSP. Prx2, localised in the cytosol, and mitochondrial Prx3 were more oxidised in SHRSP cells than WKY cells. Noradrenaline-induced vascular contraction was reduced by DTT and Conoidin A. Our data demonstrate that Noxs are expressed in an organelle-specific manner, with Nox1,2,4 present in plasma membrane, ER and nucleus, but not in mitochondria. In SHRSP VSMCs Nox expression, ROS generation and protein oxidation are increased. Inhibition of oxidation attenuated vascular reactivity. These findings suggest an important role for Nox1/4 in oxidative stress and post-translational modification of proteins, processes that may play an important role in vascular dysfunction in hypertension.

PS 07-14 VASCULAR PROTEIN OXIDATION AND REDOX PROTEOMICS IN HYPERTENSION

Objective: Oxidative stress is implicated in hypertension (HTN) through redox-sensitive processes causing vascular damage. It remains unclear exactly how ROS cause vascular injury. We hypothesise that in HTN, increased ROS levels promote a shift of oxidative post-translational protein modifications from reversible into irreversible forms, leading to aberrant redox signalling and vascular injury. Design and Method: VSMCs from normotensive and hypertensive rats (WKY and SHRSP) were stimulated with AngII (10–7 M). Protein carbonylation was assessed by oxyblot. Protein tyrosine phosphatase (PTP)-oxidation was assessed by immunoblotting. Protein sulfenylation was detected using the DCP-Rho1 cell permeable, fluorescent probe. Differential gel electrophoresis (DiGE) and CyDye thiol labelling were employed for screening of reversibly oxidised proteome. Results: Irreversible protein carbonylation and PTP-hyperoxidation were increased in SHRSP compared to WKY (fold change (FC) = 1.29 and FC = 1.31, p < 0.05, respectively). AngII-stimulation induced PTP-hyperoxidation but not protein carbonylation in VSMCs from WKY rats (FC = 1.32 at 15 min, p < 0.05), with no alterations observed in SHRSP. On the contrary, reversible oxidation was reduced in SHRSP versus WKY, as demonstrated through thiol-proteome oxidation (13.6% (253 spots) decreased versus 6.7% (124 spots) increased oxidation), protein sulfenylation (FC = −1.78, p < 0.05) and PTP-oxidation (FC = −1.24, p < 0.05). AngII-stimulation tended to further decrease sulfenylation and PTP-oxidation levels in WKY. Proteomic data, filtered for FC > 2, detected 1777 spots with 377 (21%) being differentially oxidised between WKY and SHRSP. Candidate proteins exhibiting consistent changes across three replicates included &bgr;-actin (FC = 2.42), annexin A1 (−2.29), galectin-1 (1.83) and GAPDH (2.67). Conclusions: Our findings demonstrate that redox status in HTN is characterised by increased protein hyperoxidation and decreased levels of reversible oxidation. AngII is able to shift the balance between regulatory oxidation and hyperoxidation towards a hypertensive profile. Our findings identify differentially oxidised proteins in VSMCs in SHRSP vs WKY. These phenomena may be important in aberrant vascular signaling/function, contributing to oxidative vascular injury in HTN and associated target organ damage.

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.