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Background Cisplatin-based chemotherapy increases the risk of cardiovascular and renal disease. Objectives We aimed to define the time course, pathophysiology, and approaches to prevent cardiovascular disease associated with cisplatin-based chemotherapy. Methods Two cohorts of patients with a history of testicular cancer (n = 53) were recruited. Cohort 1 consisted of 27 men undergoing treatment with: 1) surveillance; 2) 1 to 2 cycles of bleomycin, etoposide, and cisplatin (BEP) chemotherapy (low-intensity cisplatin); or 3) 3 to 4 cycles of BEP (high-intensity cisplatin). Endothelial function (percentage flow-mediated dilatation) and cardiovascular biomarkers were assessed at 6 visits over 9 months. Cohort 2 consisted of 26 men previously treated 1 to 7 years ago with surveillance or 3 to 4 cycles BEP. Vasomotor and fibrinolytic responses to bradykinin, acetylcholine, and sodium nitroprusside were evaluated using forearm venous occlusion plethysmography. Results In cohort 1, the percentage flow-mediated dilatation decreased 24 h after the first cisplatin dose in patients managed with 3 to 4 cycles BEP (10.9 ± 0.9 vs. 16.7 ± 1.6; p < 0.01) but was unchanged from baseline thereafter. Six weeks after starting 3 to 4 cycles BEP, there were increased serum cholesterol levels (7.2 ± 0.5 mmol/l vs. 5.5 ± 0.2 mmol/l; p = 0.01), hemoglobin A1c (41.8 ± 2.0 mmol/l vs. 35.5 ± 1.2 mmol/l; p < 0.001), von Willebrand factor antigen (62.4 ± 5.4 mmol/l vs. 45.2 ± 2.8 mmol/l; p = 0.048) and cystatin C (0.91 ± 0.07 mmol/l vs. 0.65 ± 0.09 mmol/l; p < 0.01). In cohort 2, intra-arterial bradykinin, acetylcholine, and sodium nitroprusside caused dose-dependent vasodilation (p < 0.0001). Vasomotor responses, endogenous fibrinolytic factor release, and cardiovascular biomarkers were not different in patients managed with 3 to 4 cycles of BEP versus surveillance. Conclusions Cisplatin-based chemotherapy induces acute and transient endothelial dysfunction, dyslipidemia, hyperglycemia, and nephrotoxicity in the early phases of treatment. Cardiovascular and renal protective strategies should target the early perichemotherapy period. (Clinical Characterisation of the Vascular Effects of Cis-platinum Based Chemotherapy in Patients With Testicular Cancer [VECTOR], NCT03557177; Intermediate and Long Term Vascular Effects of Cisplatin in Patients With Testicular Cancer [INTELLECT], NCT03557164), Central Illustration

Objective: Transient receptor potential (TRP) melastatin 7 (TRPM7) cation channel, a dual-function ion channel/protein kinase, regulates vascular smooth muscle cell (VSMC) Mg2+ homeostasis and mitogenic signaling. Mechanisms regulating vascular growth effects of TRPM7 are unclear, but epidermal growth factor (EGF) may be important because it is a magnesiotropic hormone involved in cellular Mg2+ regulation and VSMC proliferation. Here we sought to determine whether TRPM7 is a downstream target of EGF in VSMCs and if EGF receptor (EGFR) through TRPM7 influences VSMC function. Approach and results: Studies were performed in primary culture VSMCs from rats and humans and vascular tissue from mice deficient in TRPM7 (TRPM7+/Δkinase and TRPM7R/R). EGF increased expression and phosphorylation of TRPM7 and stimulated Mg2+ influx in VSMCs, responses that were attenuated by gefitinib (EGFR inhibitor) and NS8593 (TRPM7 inhibitor). Co-immunoprecipitation (IP) studies, proximity ligation assay (PLA) and live-cell imaging demonstrated interaction of EGFR and TRPM7, which was enhanced by EGF. PP2 (c-Src inhibitor) decreased EGF-induced TRPM7 activation and prevented EGFR–TRPM7 association. EGF-stimulated migration and proliferation of VSMCs were inhibited by gefitinib, PP2, NS8593 and PD98059 (ERK1/2 inhibitor). Phosphorylation of EGFR and ERK1/2 was reduced in VSMCs from TRPM7+/Δkinase mice, which exhibited reduced aortic wall thickness and decreased expression of PCNA and Notch 3, findings recapitulated in TRPM7R/R mice. Conclusions: We show that EGFR directly interacts with TRPM7 through c-Src-dependent processes. Functionally these phenomena regulate [Mg2+]i homeostasis, ERK1/2 signaling and VSMC function. Our findings define a novel signaling cascade linking EGF/EGFR and TRPM7, important in vascular homeostasis.

Increased generation of reactive oxygen species (ROS) and altered Ca 2+ handling cause vascular damage in hypertension. Mechanisms linking these systems are unclear, but TRPM2 (transient receptor potential melastatin 2) could be important because TRPM2 is a ROS sensor and a regulator of Ca 2+ and Na + transport. We hypothesized that TRPM2 is a point of cross-talk between redox and Ca 2+ signaling in vascular smooth muscle cells (VSMC) and that in hypertension ROS mediated-TRPM2 activation increases [Ca 2+ ] i through processes involving NCX (Na + /Ca 2+ exchanger). VSMCs from hypertensive and normotensive individuals and isolated arteries from wild type and hypertensive mice (LinA3) were studied. Generation of superoxide anion and hydrogen peroxide (H 2 O 2 ) was increased in hypertensive VSMCs, effects associated with activation of redox-sensitive PARP1 (poly [ADP-ribose] polymerase 1), a TRPM2 regulator. Ang II (angiotensin II) increased Ca 2+ and Na + influx with exaggerated responses in hypertension. These effects were attenuated by catalase−polyethylene glycol -catalase and TRPM2 inhibitors (2-APB, 8-Br-cADPR olaparib). TRPM2 siRNA decreased Ca 2+ in hypertensive VSMCs. NCX inhibitors (Benzamil, KB-R7943, YM244769) normalized Ca 2+ hyper-responsiveness and MLC20 phosphorylation in hypertensive VSMCs. In arteries from LinA3 mice, exaggerated agonist (U46619, Ang II, phenylephrine)-induced vasoconstriction was decreased by TRPM2 and NCX inhibitors. In conclusion, activation of ROS-dependent PARP1-regulated TRPM2 contributes to vascular Ca 2+ and Na + influx in part through NCX. We identify a novel pathway linking ROS to Ca 2+ signaling through TRPM2/NCX in human VSMCs and suggest that oxidative stress-induced upregulation of this pathway may be a new player in hypertension-associated vascular dysfunction.

AIMS NOX-derived reactive oxygen species (ROS) are mediators of signaling pathways implicated in vascular smooth muscle cell (VSMC) dysfunction in hypertension. Among the numerous redox-sensitive kinases important in VSMC regulation is c-Src. However, mechanisms linking NOX/ROS to c-Src are unclear, especially in the context of oxidative stress in hypertension. Here we investigated the role of NOX-induced oxidative stress in VSMCs in human hypertension focusing on NOX5, and explored c-Src, as a putative intermediate connecting NOX5-ROS to downstream effector targets underlying VSMC dysfunction. METHODS AND RESULTS VSMC from arteries from normotensive (NT) and hypertensive (HT) subjects were studied. NOX1,2,4,5 expression, ROS generation, oxidation/phosphorylation of signaling molecules, actin polymerization and migration were assessed in the absence and presence of NOX5 (melittin) and Src (PP2) inhibitors. NOX5 and p22phox-dependent NOXs (NOX1-4) were downregulated using NOX5 siRNA and p22phox-siRNA approaches. As proof of concept in intact vessels, vascular function was assessed by myography in transgenic mice expressing human NOX5 in a VSMC-specific manner. In HT VSMCs NOX5 was upregulated, with associated oxidative stress, hyperoxidation (c-Src, peroxiredoxin, DJ-1) and hyperphosphorylation (PKC, ERK1/2, MLC20) of signaling molecules. NOX5 siRNA reduced ROS generation in NT and HT subjects. NOX5 siRNA, but not p22phox-siRNA, blunted c-Src phosphorylation in HT VSMCs. NOX5 siRNA reduced phosphorylation of MLC20 and FAK in NT and HT. In p22phox- silenced HT VSMCs, Ang II-induced phosphorylation of MLC20 was increased, effects blocked by melittin and PP2. NOX5 and c-Src inhibition attenuated actin polymerization and migration in HT VSMCs. In NOX5 transgenic mice, vascular hypercontractilty was decreased by melittin and PP2. CONCLUSIONS We define NOX5/ROS/c-Src as a novel feedforward signaling network in human VSMCs. Amplification of this system in hypertension contributes to VSMC dysfunction. Dampening the NOX5/ROS/c-Src pathway may ameliorate hypertension-associated vascular injury. TRANSLATIONAL PERSPECTIVE Oxidative stress is a major factor contributing to vascular damage in hypertension. We corroborate experimental evidence that NOX-derived ROS generation is increased in human vascular smooth muscle cells (VSMC) and demonstrate that in human hypertension NOX5 upregulation is a major trigger of VSMC dysfunction. We uncover new regulatory molecular mechanisms of NOX5 and define NOX5/ROS/c-Src as a novel signaling pathway in human VSMCs. This system is augmented in hypertension contributing to abnormal VSMC redox signaling, cytoskeletal disorganization and vascular dysfunction. Modulating the NOX5/ROS/c-Src pathway may have therapeutic potential by targeting redox signaling pathways involved in vascular dysfunction associated with hypertension.

The current COVID-19 pandemic, caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) virus, represents the largest medical challenge in decades. It has exposed unexpected cardiovascular vulnerabilities at all stages of the disease (pre-infection, acute phase, and subsequent chronic phase). The major cardiometabolic drivers identified as having epidemiologic and mechanistic associations with COVID-19 are abnormal adiposity, dysglycemia, dyslipidemia, and hypertension. Hypertension is of particular interest, because components of the renin–angiotensin system (RAS), which are critically involved in the pathophysiology of hypertension, are also implicated in COVID-19. Specifically, angiotensin-converting enzyme-2 (ACE2), a multifunctional protein of the RAS, which is part of the protective axis of the RAS, is also the receptor through which SARS-CoV-2 enters host cells, causing viral infection. Cardiovascular and cardiometabolic comorbidities not only predispose people to COVID-19, but also are complications of SARS-CoV-2 infection. In addition, increasing evidence indicates that acute kidney injury is common in COVID-19, occurs early and in temporal association with respiratory failure, and is associated with poor prognosis, especially in the presence of cardiovascular risk factors. Here, we discuss cardiovascular and kidney disease in the context of COVID-19 and provide recent advances on putative pathophysiological mechanisms linking cardiovascular disease and COVID-19, focusing on the RAS and ACE2, as well as the immune system and inflammation. We provide up-to-date information on the relationships among hypertension, diabetes, and COVID-19 and emphasize the major cardiovascular diseases associated with COVID-19. We also briefly discuss emerging cardiovascular complications associated with long COVID-19, notably postural tachycardia syndrome (POTS). Résumé: La pandémie actuelle de COVID-19 causée par le coronavirus du syndrome respiratoire aigu sévère 2 (SRAS-CoV-2) est le plus grand enjeu médical des dernières décennies. Elle a mis en évidence des vulnérabilités cardiovasculaires imprévues à tous les stades de la COVID-19 (avant l'infection, pendant la phase aiguë et pendant la phase chronique subséquente). Les principaux facteurs cardiométaboliques dont les associations épidémiologiques et mécanistiques avec la COVID-19 ont été avérées comprennent l'adiposité anormale, la dysglycémie, la dyslipidémie et l'hypertension. L'hypertension suscite un intérêt particulier, car certaines composantes du système rénine-angiotensine (SRA), dont le rôle est crucial dans la physiopathologie de l'hypertension, sont également en cause dans la COVID-19. Plus précisément, l'enzyme de conversion de l'angiotensine 2 (ECA2), une protéine multifonctionnelle du SRA faisant partie de l'axe protecteur du SRA, est également le récepteur permettant au virus SRAS-CoV-2 d'entrer dans les cellules hôtes et de provoquer une infection virale. Les affections cardiovasculaires et cardiométaboliques concomitantes ne font pas que prédisposer les personnes qui en sont atteintes à la COVID-19, elles constituent également des complications de l'infection à SRAS-CoV-2. En outre, de plus en plus de données probantes indiquent que l'atteinte rénale aiguë est fréquente en cas de COVID-19, qu'elle survient tôt et fait l'objet d'une association temporelle avec l'insuffisance respiratoire, et qu'elle est associée à un pronostic sombre, notamment en présence de facteurs de risque cardiovasculaires. Nous discutons ici des maladies cardiovasculaires et rénales dans le contexte de la COVID-19, et présentons les progrès récents sur les mécanismes physiopathologiques en cause dans le lien entre les maladies cardiovasculaires et la COVID-19 en nous attardant sur le SRA et l'ECA2, ainsi que sur le système immunitaire et l'inflammation. Nous présentons de l'information à jour sur les liens entre l'hypertension, le diabète et la COVID-19, et soulignons les principales maladies cardiovasculaires associées à la COVID-19. Nous analysons également brièvement les complications cardiovasculaires émergentes associées à la COVID-19 de longue durée, notamment le syndrome de tachycardie orthostatique posturale (STOP).

Introduction: Interferon (IFN) alpha (IFNα) and lambda3 (IFNL3) constitute the first line of immunity against SARS-CoV-2 infection by increasing interferon-stimulated genes (ISGs). IFNs influence the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. Here we hypothesized that in human microvascular endothelial cells (EC) IFNL3 and IFNα influence ACE2 and immune/inflammatory responses mediated by S1P. Methods: EC were stimulated with S1P of SARS-CoV-2 (1 μg/10^6 cells), IFNα (100 ng/mL) or IFNL3 (100 IU/mL). Because ACE2, metalloproteinase domain 17 (ADAM17) and type II transmembrane serine protease (TMPRSS2) are important for SARS-CoV-2 infection, cells were treated with inhibitors of ADAM17 (marimastat, 3.8nM and TAPI-1, 100nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50μM). Expression of ISGs (ISG15, IFIT1, and MX1) was investigated by real-time PCR (5h) and protein expression by immunoblotting (24h). Results: EC stimulated with S1P increased expression of ISGs: ISG15 (2 fold), IFIT1 (6 fold), MX1 (6 fold) (n=12, p Conclusions: In human microvascular endothelial cells, S1P, IFNα and IFNL3 induced an immune response characterized by increased expression of interferon-stimulated genes and IL-6 production, processes that involve ADAM17. Inflammation induced by S1P was amplified by IFNα. Our novel findings demonstrate that S1P induces an endothelial immune/inflammatory response that may be important in endotheliitis associated with COVID-19.

Hypertension, vascular dysfunction and downregulation of the renin angiotensin system as sequelae of COVID-19 The long-term CV consequences of COVID are unknown however the potential for ongoing cardiac and vascular inflammation with RAAS alteration may increase the risk of developing hypertension and CV disease. Non-hypertensive patients hospitalised in April-May 2020 with either confirmed COVID19 (cases) or non-COVID (controls) diagnosis were recruited ≥12 weeks post-discharge. All underwent detailed BP and vascular/immune and RAAS phenotyping. The primary outcome was ABPM 24-hr SBP. Paired t-tests and multivariable regression models used to assess differences. Thirty cases and eighteen controls completed the study. Cases were older (51±7 vs 45±9 years) with lower discharge SBP (121±10 vs 128±15 mmHg; p0.01). ABPM at study visit was higher in the cases compared to controls (24-hour SBP (OR[95%CI]: 8.6[0.9-16.3]; p0.03), day-time SBP (8.6[1.5-17.3]; p0.02), day-time DBP (4.6[0.1-9.1]; p

SARS-CoV-2, the virus responsible for COVID19, binds to ACE2, via its spike protein S1 subunit, leading to viral infection and respiratory disease. COVID-19 is associated with cardiovascular disease and systemic inflammation. Since ACE2 is expressed in vascular cells we questioned whether SARS-CoV-2 induces vascular inflammation and whether this is related to viral infection. Human microvascular endothelial cells (EC) were exposed to recombinant S1p (rS1p) 0.66 μg/mL for 10 min, 5h and 24h. Gene expression was assessed by RT-PCR and levels of IL6 and MCP1, as well as ACE2 activity, were assessed by ELISA. Expression of ICAM1 and PAI1 was assessed by immunoblotting. ACE2 activity was blocked by MLN4760 (ACE2 inhibitor) and siRNA. Viral infection was assessed by exposing Vero E6 (kidney epithelial cells; pos ctl) and EC to 10 5 pfu of SARS-CoV-2 where virus titre was measured by plaque assay. Co-IP coupled mass spectrometry protein identification and label free proteomics were used to investigate ACE2-mediated signalling. rS1p increased IL6 mRNA (14.2±2.1 vs. C:0.61±0.03 2^-ddCT) and levels (1221.2±18.3 vs. C:22.77±3.2 pg/mL); MCP1 mRNA (5.55±0.62 vs. C:0.65±0.04 2^-ddCT) and levels (1110±13.33 vs. C:876.9±33.4 pg/mL); ICAM1 (17.7±3.1 vs. C:3.9±0.4 AU) and PAI1 (5.6±0.7 vs. C: 2.9±0.2), pvs. C: 1011±268 RFU, p

Supplemental Digital Content is available in the text., Combined inhibition of NEP (neutral endopeptidase) and ACE (angiotensin-converting enzyme), without unwanted effects, remains an attractive therapeutic strategy in cardiovascular medicine. Omapatrilat, a dual NEP inhibitor–ACE inhibitor, was a promising antihypertensive drug but failed in trials due to angioedema, an effect possibly caused by inhibition of both the N- and C-domains of ACE. Here, we aimed to determine whether lisinopril-tryptophan (lisW-S), a C-domain specific ACE inhibitor that preserves the N-domain catalytic activity, together with sacubitril (NEP inhibitor), differentially influences cardiovascular function and vascular permeability in hypertension compared with omapatrilat and lisinopril+sacubitril which inhibits both the ACE C- and N-domains. Ang II (angiotensin II)–dependent hypertensive mice (transgenic mice expressing active human renin in the liver [also known as LinA3]) received vehicle, sacubitril, lisW-S, lisinopril, lisinopril+sacubitril, or lisW-S+sacubitril for 4 weeks. Systolic blood pressure was increased in LinA3 mice, along with cardiac hypertrophy/dysfunction, impaired endothelium-dependent vasorelaxation, hypercontractile responses, vascular remodeling, and renal inflammation. LisW-S+sacubitril, lisinopril+sacubitril, and omapatrilat reduced systolic blood pressure and normalized cardiovascular remodeling and vascular hypercontractile responses in LinA3 mice. Although lisinopril+sacubitril and omapatrilat improved Ach-induced vasorelaxation, lisW-S+sacubitril had no effect. Endothelial permeability (Evans Blue assessment) was increased in omapatrilat but not in LisW-S+sacubitril–treated mice. In conclusion, lisW-S combined with sacubitril reduced systolic blood pressure and improved cardiac dysfunction in LinA3 mice, similar to omapatrilat but without effects on endothelium-dependent vasorelaxation. Moreover, increased vascular leakage (plasma extravasation) induced by omapatrilat was not evident in mice treated with lisW-S+sacubitril. Targeting ACE C-domain and NEP as a combination therapy may be as effective as omapatrilat in lowering systolic blood pressure, but without inducing vascular permeability and endothelial injury.

No abstract available.

New findings What is the topic of this review? This review provides a comprehensive overview of Nox5 from basic biology to human disease and highlights unique features of this Nox isoform What advances does it highlight? Major advances in Nox5 biology relate to crystallization of the molecule and new insights into the pathophysiological role of Nox5. Recent discoveries have unravelled the crystal structure of Nox5, the first Nox isoform to be crystalized. This provides new opportunities to develop drugs or small molecules targeted to Nox5 in an isoform-specific manner, possibly for therapeutic use. Moreover genome wide association studies (GWAS) identified Nox5 as a new blood pressure-associated gene and studies in mice expressing human Nox5 in a cell-specific manner have provided new information about the (patho) physiological role of Nox5 in the cardiovascular system and kidneys. Nox5 seems to be important in the regulation of vascular contraction and kidney function. In cardiovascular disease and diabetic nephropathy, Nox5 activity is increased and this is associated with increased production of reactive oxygen species and oxidative stress implicated in tissue damage. Abstract Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox), comprise seven family members (Nox1-Nox5 and dual oxidase 1 and 2) and are major producers of reactive oxygen species in mammalian cells. Reactive oxygen species are crucially involved in cell signalling and function. All Noxs share structural homology comprising six transmembrane domains with two haem-binding regions and an NADPH-binding region on the intracellular C-terminus, whereas their regulatory systems, mechanisms of activation and tissue distribution differ. This explains the diverse function of Noxs. Of the Noxs, NOX5 is unique in that rodents lack the gene, it is regulated by Ca2+ , it does not require NADPH oxidase subunits for its activation, and it is not glycosylated. NOX5 localizes in the perinuclear and endoplasmic reticulum regions of cells and traffics to the cell membrane upon activation. It is tightly regulated through numerous post-translational modifications and is activated by vasoactive agents, growth factors and pro-inflammatory cytokines. The exact pathophysiological significance of NOX5 remains unclear, but it seems to be important in the physiological regulation of sperm motility, vascular contraction and lymphocyte differentiation, and NOX5 hyperactivation has been implicated in cardiovascular disease, kidney injury and cancer. The field of NOX5 biology is still in its infancy, but with new insights into its biochemistry and cellular regulation, discovery of the NOX5 crystal structure and genome-wide association studies implicating NOX5 in disease, the time is now ripe to advance NOX5 research. This review provides a comprehensive overview of our current understanding of NOX5, from basic biology to human disease, and highlights the unique characteristics of this enigmatic Nox isoform.

Objective: The mechanisms involved in NOX5 activation in atherosclerotic processes are not completely understood. The present study tested the hypothesis that lysophosphatidylcholine (LPC), a proatherogenic component of oxLDL, induces endothelial calcium influx, which drives NOX5-dependent reactive oxygen species (ROS) production, oxidative stress, and endothelial cell dysfunction. Approach: Human aortic endothelial cells (HAEC) were stimulated with LPC (10−5 M, for different time points). Pharmacological inhibition of NOX5 (Melittin, 10−7 M) and NOX5 gene silencing (siRNA) was used to determine the role of NOX5-dependent ROS production in endothelial oxidative stress induced by LPC. ROS production was determined by lucigenin assay and electron paramagnetic spectroscopy (EPR), calcium transients by Fluo4 fluorimetry, and NOX5 activity and protein expression by pharmacological assays and immunoblotting, respectively. Results: LPC increased ROS generation in endothelial cells at short (15 min) and long (4 h) stimulation times. LPC-induced ROS was abolished by a selective NOX5 inhibitor and by NOX5 siRNA. NOX1/4 dual inhibition and selective NOX1 inhibition only decreased ROS generation at 4 h. LPC increased HAEC intracellular calcium, important for NOX5 activation, and this was blocked by nifedipine and thapsigargin. Bapta-AM, selective Ca2+ chelator, prevented LPC-induced ROS production. NOX5 knockdown decreased LPC-induced ICAM-1 mRNA expression and monocyte adhesion to endothelial cells. Conclusion: These results suggest that NOX5, by mechanisms linked to increased intracellular calcium, is key to early LPC-induced endothelial oxidative stress and pro-inflammatory processes. Since these are essential events in the formation and progression of atherosclerotic lesions, the present study highlights an important role for NOX5 in atherosclerosis.

A link between oxidative stress and hypertension has been firmly established in multiple animal models of hypertension but remains elusive in humans. While initial studies focused on inactivation of nitric oxide by superoxide, our understanding of relevant reactive oxygen species (superoxide, hydrogen peroxide, and peroxynitrite) and how they modify complex signaling pathways to promote hypertension has expanded significantly. In this review, we summarize recent advances in delineating the primary and secondary sources of reactive oxygen species (nicotinamide adenine dinucleotide phosphate oxidases, uncoupled endothelial nitric oxide synthase, endoplasmic reticulum, and mitochondria), the posttranslational oxidative modifications they induce on protein targets important for redox signaling, their interplay with endogenous antioxidant systems, and the role of inflammasome activation and endoplasmic reticular stress in the development of hypertension. We highlight how oxidative stress in different organ systems contributes to hypertension, describe new animal models that have clarified the importance of specific proteins, and discuss clinical studies that shed light on how these processes and pathways are altered in human hypertension. Finally, we focus on the promise of redox proteomics and systems biology to help us fully understand the relationship between ROS and hypertension and their potential for designing and evaluating novel antihypertensive therapies.

Magnesium regulates numerous cellular functions and enzymes and abnormal magnesium homeostasis contributes to vascular dysfunction and the development of hypertension. The transient receptor potential melastatin 7 (TrpM7) has emerged as a key player in cardiovascular magnesium homeostasis. This bifunctional channel/kinase is ubiquitously expressed and regulates embryonic development. Its integral membrane ion channel domain regulates transmembrane movement of divalent cations, primarily Ca 2+ , Mg 2+ and Zn 2+ , and its kinase domain controls gene expression via histone phosphorylation. TrpM7 not only localizes on the cell surface to serve as a critical regulator of transmembrane Mg 2+ flux, but also forms an intracellular Zn 2+ release channel in vesicles of unknown origin. Palmitoylation is a dynamic reversible posttranslational modification, which regulates ion channel activity, stability, and subcellular localization. We found that TrpM7 is palmitoylated in multiple cell types. Here we sought to identify palmitoylated cysteines and the functional consequences of TrpM7 palmitoylation in HEK cells. Mutation of Cysteines 1143, 1144 and 1146 on TrpM7 (TrpM7-AAA) to alanines reduced its palmitoylation by 68.4±8% (n=13; P P P P P

TRPM7 has cation channel and kinase properties, is permeable to Mg 2+ , Ca 2+ , and Zn 2+ and is protective in the cardiovascular system. Hyperaldosteronism, which induces hypertension and cardiovascular fibrosis, is associated with Mg 2+ wasting. Here we questioned whether TRPM7 plays a role in aldosterone- induced hypertension and fibrosis and whether it influences cation regulation. Wild-type (WT) and TRPM7-deficient (M7+/Δ) mice were treated with aldosterone (600μg/Kg/day) and/or 1% NaCl (drinking water) (aldo, salt or aldo-salt) for 4 weeks. Blood pressure (BP) was evaluated by tail-cuff. Vessel structure was assessed by pressure myography. Molecular mechanisms were investigated in cardiac fibroblasts (CF) from WT and M7+/Δ mice. Protein expression was assessed by western-blot and histology. M7+/Δ mice exhibited reduced TRPM7 expression (30%) and phosphorylation (62%), levels that were recapitulated in WT aldo-salt mice. M7+/Δ exhibited increased BP by aldo, salt and aldo-salt (135-140mmHg) vs M7+/Δ-veh (117mmHg) (p2+ -dependent phosphatase, was reduced (3-fold) only in M7+/Δ mice. M7+/Δ CF showed reduced proliferation (30%) and PPM1A (4-fold) and increased expression of TGFβ, IL-11 and IL-6 (2-3-fold), activation of Stat1 (2-fold), Smad3 (9-fold) and ERK1/2 (8-fold) compared with WT. Mg 2+ supplementation normalized cell proliferation and reduced protein phosphorylation in M7+/Δ CF (p2+ -dependent mechanisms.

Nox5 is the major ROS-generating Nox isoform in human vascular smooth muscle cells (VSMC). The role of Nox5 in oxidative stress and redox signaling underlying vascular dysfunction in hypertension is unclear. We examined molecular processes that regulate VSMC Nox5-induced ROS generation, focusing on c-Src. VSMC isolated from small arteries from normotensive (NT) and hypertensive (HT) subjects were studied. Nox5 expression and phosphorylation (immunoblotting, immunoprecipitation); ROS generation (chemiluminescence); activation of contractile signaling pathways (immunoblotting), Ca 2+ influx (Cal-520AM fluorescence), reversible protein oxidation (cysteine sulfenic acid probe BCN-E-BCN), actin polymerization (phalloidin staining) and migration (wound healing assay) were assessed in absence/presence of Nox5 (melittin) and Src (PP2) inhibitors. To study Nox5-specific effects, we used p22phox-silenced VSMCs (siRNA). Vascular function in VSMC-specific Nox5 transgenic mice was studied by wire myography. In HT, ROS levels (139±27%), Nox5 expression (103±23%) and phosphorylation were increased (77±17.93%) (p20 (416±71%) and Ang II-induced Ca 2+ influx (574±44 vs NT:451±26) were also increased in HT (p20 (89±22% vs Ctl) phosphorylation, effects blocked by melittin and PP2 (p2+ influx, actin polymerization and migration in HT. Hypercontractility observed in Nox5 mice was abolished by melittin and PP2. Our findings demonstrate that Nox5 is upregulated in human hypertension. This is associated with activation of c-Src, increased redox signaling and VSMC cytoskeletal reorganization, migration and vascular contraction. We define a novel Nox5-ROS-c-Src signaling pathway that may play a role in vascular remodeling/dysfunction in hypertension.

The etiology of hypertension involves complex interactions among genetic, environmental, and pathophysiologic factors that influence many regulatory systems. Hypertension is characteristically associated with vascular dysfunction, cardiovascular remodelling, renal dysfunction, and stimulation of the sympathetic nervous system. Emerging evidence indicates that the immune system is also important and that activated immune cells migrate and accumulate in tissues promoting inflammation, fibrosis, and target-organ damage. Common to these processes is oxidative stress, defined as an imbalance between oxidants and antioxidants in favour of the oxidants that leads to a disruption of oxidation-reduction (redox) signalling and control and molecular damage. Physiologically, reactive oxygen species (ROS) act as signalling molecules and influence cell function through highly regulated redox-sensitive signal transduction. In hypertension, oxidative stress promotes posttranslational modification (oxidation and phosphorylation) of proteins and aberrant signalling with consequent cell and tissue damage. Many enzymatic systems generate ROS, but NADPH oxidases (Nox) are the major sources in cells of the heart, vessels, kidneys, and immune system. Expression and activity of Nox are increased in hypertension and are the major systems responsible for oxidative stress in cardiovascular disease. Here we provide a unifying concept where oxidative stress is a common mediator underlying pathophysiologic processes in hypertension. We focus on some novel concepts whereby ROS influence vascular function, aldosterone/mineralocorticoid actions, and immunoinflammation, all important processes contributing to the development of hypertension.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) leads to premature stroke and vascular dementia. Mechanism-specific therapies for this aggressive cerebral small vessel disease are lacking. CADASIL is caused by NOTCH3 mutations that influence vascular smooth muscle cell (VSMC) function through unknown processes. We investigated molecular mechanisms underlying the vasculopathy in CADASIL focusing on endoplasmic reticulum (ER) stress and RhoA/Rho kinase (ROCK). Peripheral small arteries and VSMCs were isolated from gluteal biopsies of CADASIL patients and mesentery of TgNotch3R169C mice (CADASIL model). CADASIL vessels exhibited impaired vasorelaxation, blunted vasoconstriction, and hypertrophic remodeling. Expression of NOTCH3 and ER stress target genes was amplified and ER stress response, Rho kinase activity, superoxide production, and cytoskeleton-associated protein phosphorylation were increased in CADASIL, processes associated with Nox5 upregulation. Aberrant vascular responses and signaling in CADASIL were ameliorated by inhibitors of Notch3 (γ-secretase inhibitor), Nox5 (mellitin), ER stress (4-phenylbutyric acid), and ROCK (fasudil). Observations in human CADASIL were recapitulated in TgNotch3R169C mice. These findings indicate that vascular dysfunction in CADASIL involves ER stress/ROCK interplay driven by Notch3-induced Nox5 activation and that NOTCH3 mutation–associated vascular pathology, typical in cerebral vessels, also manifests peripherally. We define Notch3-Nox5/ER stress/ROCK signaling as a putative mechanism-specific target and suggest that peripheral artery responses may be an accessible biomarker in CADASIL., ER stress and Rho kinase are potentially druggable targets in CADASIL, the most aggressive form of small vessel disease and dementia.

Background and Purpose: Inflammation plays a key role in atherosclerosis. The protective role of angiotensin 1–7 (Ang-(1–7)) in vascular pathologies suggested the therapeutic use of low MW, non-peptide Ang-(1–7) mimetics, such as AVE0991. The mechanisms underlying the vaso-protective effects of AVE0991, a Mas receptor agonist, remain to be explored. Experimental Approach: We investigated the effects of AVE0991 on the spontaneous atherosclerosis in apolipoprotein E (ApoE)−/− mice, in the context of vascular inflammation and plaque stability. Key Results: AVE0991 has significant anti-atherosclerotic properties in ApoE−/− mice and increases plaque stability, by reducing plaque macrophage content, without effects on collagen. Using the descending aorta of chow-fed ApoE−/− mice, before significant atherosclerotic plaque develops, we gained insight to early events in atherosclerosis. Interestingly, perivascular adipose tissue (PVAT) and adventitial infiltration with macrophages and T-cells precedes atherosclerotic plaque or the impairment of endothelium-dependent NO bioavailability (a measure of endothelial function). AVE0991 inhibited perivascular inflammation, by reducing chemokine expression in PVAT and through direct actions on monocytes/macrophages inhibiting their activation, characterized by production of IL-1β, TNF-α, CCL2 and CXCL10, and differentiation to M1 phenotype. Pretreatment with AVE0991 inhibited migration of THP-1 monocytes towards supernatants of activated adipocytes (SW872). Mas receptors were expressed in PVAT and in THP-1 cells in vitro, and the anti-inflammatory effects of AVE0991 were partly Mas dependent. Conclusions and Implications: The selective Mas receptor agonist AVE0991 exhibited anti-atherosclerotic and anti-inflammatory actions, affecting monocyte/macrophage differentiation and recruitment to the perivascular space during early stages of atherosclerosis in ApoE−/− mice. Linked Articles: This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc

Patients with coronary microvascular dysfunction (CMD), a potential cause of heart ischemia, have systemic vascular dysfunction, characterized by increased vascular contraction to ET-1 and a thromboxane A2 analogue (U46619). Nox5 regulates vascular contraction and is involved in cardiovascular diseases. In our study, we questioned whether Nox5 plays a role in systemic vascular dysfunction in heart ischemia. As Nox5 expression has been described in the cardiovascular system of rabbits, a model of ischaemic cardiomyopathy (IC) was used. Coronary artery ligation was performed in Male New Zealand White rabbits. After 8 weeks, skin and mesenteric arteries were isolated and vascular function assessed by wire myography. Vascular contraction to NA (EMax %KCl: 122±4 vs sham 97±3.7) and U46619 (EMax %KCl: 82±3 vs sham 67±4) were exacerbated in skin arteries from IC (p2+ channels, but an increase in RyR was observed (2^-ddCT: 1.67±0.15 vs sham 0.98±0.08) in VSMCs isolated from IC animals. Peroxiredoxin (Prdx), antioxidant, mRNA was increased in IC (2^-ddCT: 1.95±0.4 vs sham 0.88±0.1, p11 /mL: 4.8±0.6 vs control 1.75±0.2), where Nox5 expression was also increased (AU: 0.11±0.02 vs control MP 0.03±0.006) (p

TRPM7 is a chanzyme that influences cellular Mg 2+ homeostasis and vascular signaling. We demonstrated that aldosterone mediates cellular effects through TRPM7-dependent signaling pathways. Since hyperaldosteronism causes hypertension and Mg 2+ wasting, we questioned whether TRPM7 plays a role in aldosterone-induced hypertension. Wild-type (WT) and TRPM7-deficient (M7+/Δ) mice were treated with aldosterone (600μg/Kg/day) and/or 1% NaCl (drinking water) (aldo, salt or aldo/salt) for 4 weeks. Blood pressure (BP) was evaluated by tail-cuff. Vessel function was investigated in mesenteric arteries by wire and pressure myography. Protein expression was assessed by western-blot and histology. Cardiac fibroblasts (CF) were isolated from WT and M7+/Δ. M7+/Δ exhibited increased BP by aldo (140mmHg), salt (135mmHg) and aldo/salt (137mmHg) vs M7+/Δ-veh (117mmHg) (p

TRPM6 and 7 are channels important in Mg 2+ and Ca 2+ homeostasis. We demonstrated that TRPM7 is influenced by angiotensin II. TRPM6 is influenced by EGF and cancer patients treated with EGFR inhibitors exhibit hypomagnesemia and hypertension by unclear mechanisms. Whether growth factors influence vascular TRPM7 is unknown. Here we questioned if VEGF/EGF mediate vascular effects through TRPM7. Studies were performed in human VSMC, wild type (WT) and TRPM7-deficient (M7+/Δ) mice. VSMC were stimulated with VEGF or EGF (50ng/ml) in the absence/presence of vatalanib, gefitinib (1μM), 2APB (30μM) and NS8593 (40μM), inhibitors of VEGFR, EGFR and TRPM7 respectively. Ca 2+ and Mg 2+ levels were assessed by Cal-520 and Mg-green. VEGF/EGF signaling was assessed by immunoblotting and vascular function by myography in mesenteric arteries from WT and M7+/Δ mice and treated with EGF or VEGF (50ng/ml). TRPM7 expression in aortas and kidneys from WT treated with vatalanib or gefitinib (100mg/Kg/day, 2 weeks) was assessed by immunoblotting. VEGF and EGF increased TRPM7 expression (50% and 67% respectively) and phosphorylation (2-fold), promoted influx of Ca 2+ (8% and 10%) and Mg 2+ (8%), effects that were reduced by vatalanib, gefitinib, NS8593, and 2-APB. EGF but not VEGF increased phosphorylation of PKC (43%), p38MAPK (47%), and ERK1/2 (120%). These responses were reduced by gefitinib, however only ERK1/2 phosphorylation was inhibited by NS8593, and 2-APB. Mice treated with vatalanib or gefitinib showed reduced expression of TRPM7 in aortas (50% and 74% respectively) and kidneys (36% and 66% respectively). Vessels exposed to EGF were less responsive to acetylcholine (ACh)-induced relaxation, [Emax %: WT (veh 97±3 vs EGF 63±10, p2+ and Ca 2+ are important.

Growing evidence indicates that cells are able to communicate with neighbouring and distant cells in the body by production of extracellular vesicles (EV). EV are classified according to their size and mechanisms of formation. Exosomes and microparticles are the most extensively studied clinically relevant forms of EV, and they often reflect the activation status of the parent cell, by carrying similar surface markers and cargo. Because of these molecular characteristics, EV are considered to be mediators of cell activation by transferring molecules (e.g., proteins, lipids, and nucleic acids) to neighbouring or distant cell populations. Increased levels of circulating EV have been observed in various diseases, including hypertension, atherosclerosis, kidney diseases, and cancer. In this chapter, we will address the formation of different EV and their importance in cell-cell communication, controlling basic cellular functions in homeostatic and pathologic conditions associated with cardiovascular diseases. In addition, we highlight their role as biomarkers and discuss the potential of EV as therapeutic tools.

Objectives: To assess the acute effects of nicotine-containing electronic cigarettes versus tobacco smoking on vascular and respiratory function and circulating microparticles, particularly platelet microparticles (PMPs, biomarker of haemostasis/thrombosis) and endothelial microparticles (EMPs, biomarker of endothelial function).\ud \ud Methods: Heart rate (HR), blood pressure, reactive hyperaemia index (RHI, microvascular reactivity), augmentation index (arterial stiffness) and respiratory function were assessed in 20 smokers immediately before and after electronic cigarettes use and tobacco smoking. The number of microparticles was determined by flow cytometry using counting beads as a reference. Labelling with Annexin-V was used to detect the total microparticle fraction. EMPs were characterized as CD31+CD42− and PMPs as CD31+CD42+.\ud \ud Results: HR increased after electronic cigarettes use and tobacco smoking (P < 0.001), whereas blood pressure remained unchanged (P > 0.05). RHI (P = 0.006), augmentation index (P = 0.010) but not augmentation index standardized to HR 75 bpm (P > 0.05) increased with electronic cigarettes use but not with tobacco smoking. Following tobacco smoking, there was a significant increase in total microparticles (P < 0.001), EMPs (P < 0.001) and PMPs (P < 0.001). In contrast, electronic cigarettes were only associated with an increase in PMPs (P < 0.001), with no significant changes in the total microparticle fraction or EMPs (all P > 0.05). Peak expiratory flow significantly decreased following electronic cigarettes use (P = 0.019).\ud \ud Conclusion: Our results demonstrate that acute exposure to tobacco smoking as well as electronic cigarettes influences vascular and respiratory function. Where tobacco smoking significantly increased microparticle formation, indicative of possible endothelial injury, electronic cigarettes use induced vasoreactivity and decreased peak expiratory flow. These findings suggest that both electronic cigarettes and tobacco smoking negatively impact vascular function.

No abstract available.

We previously demonstrated that TRPM7, a Mg 2+ /cation channel fused to an α-kinase domain, is regulated by vasoactive mediators and plays a protective role in hypertension. Here we questioned whether TRPM7-kinase influences vascular inflammation and fibrosis. We used Wild-type (WT) and heterozygote mutant mice for TRPM7-kinase (M7+/-). Vascular inflammatory responses were assessed ex vivo by intravital microscopy. Immune cells were investigated by flow cytometry. Fibrosis was investigated by sirius-red staining. Bone-marrow derived macrophages (BMDM) and Cardiac fibroblasts (CF) were obtained from WT and M7+/. [Mg 2+ ]i in cardiac tissue, cardiac macrophages and circulating monocytes was significantly reduced (30-50%) in M7+/- vs WT mice. In small arteries studied by intravital microscopy, leukocytes from M7+/- showed reduced velocity (47%), increased adhesion (222%) and transmigration (480%). Expression of vascular pro-inflammatory markers including VCAM-1(33-fold), iNOS (12-fold), and IL-12 (6.8-fold) was increased in M7+/- vs WT. Cardiac galectin-3 (Gal-3) levels (16.6±3.6 vs WT 9.2±1.2 cells/field), collagen area (6.7% vs WT 2.6%), infiltration of CD45+ cells (6±0.6% vs WT 4±0.4%) and protein expression of fibronectin (280%), TGFβ (125%), and p-Smad3 (66%), were increased in M7+/- mice. BMDM macrophages from M7+/- exhibited increased levels of Gal-3 (2.6±0.05 vs WT 2.1±0.09ng/mL), IL-10 (807±92 vs WT 305±37 pg/mL) and IL-6 (84±8 vs WT 13±5 pg/mL). A similar profile was demonstrated in resident peritoneal macrophages. CF treated with supernatant of macrophages from M7+/- increased fibronectin (43%) and PCNA (36%) vs WT. To evaluate whether these processes are Mg 2+ -sensitive, we examined effects of Mg 2+ treatment and demonstrated that Mg 2+ ameliorated pro-fibrotic and pro-inflammatory signalling evident in TRPM7+/- mice. In conclusion, TRPM7-kinase deficiency is associated with cardiac and vascular inflammation and fibrosis, processes associated with cellular Mg 2+ deficiency. Our findings highlight an important cardiovascular protective role of TRPM7 and Mg 2+ .

Background NADPH Oxidase 5 (Nox5) is a calcium‐sensitive superoxide‐generating Nox. It is present in lower forms and higher mammals, but not in rodents. Nox5 is expressed in vascular cells, but the functional significance remains elusive. Given that contraction is controlled by calcium and reactive oxygen species, both associated with Nox5, we questioned the role of Nox5 in pro‐contractile signaling and vascular function. Methods and Results Transgenic mice expressing human Nox5 in a vascular smooth muscle cell–specific manner (Nox5 mice) and Rhodnius prolixus , an arthropod model that expresses Nox5 endogenoulsy, were studied. Reactive oxygen species generation was increased systemically and in the vasculature and heart in Nox5 mice. In Nox5‐expressing mice, agonist‐induced vasoconstriction was exaggerated and endothelium‐dependent vasorelaxation was impaired. Vascular structural and mechanical properties were not influenced by Nox5. Vascular contractile responses in Nox5 mice were normalized by N ‐acetylcysteine and inhibitors of calcium channels, calmodulin, and endoplasmic reticulum ryanodine receptors, but not by GKT 137831 (Nox1/4 inhibitor). At the cellular level, vascular changes in Nox5 mice were associated with increased vascular smooth muscle cell [Ca 2+ ] i , increased reactive oxygen species and nitrotyrosine levels, and hyperphosphorylation of pro‐contractile signaling molecules MLC 20 (myosin light chain 20) and MYPT 1 (myosin phosphatase target subunit 1). Blood pressure was similar in wild‐type and Nox5 mice. Nox5 did not amplify angiotensin II effects. In R. prolixus , gastrointestinal smooth muscle contraction was blunted by Nox5 silencing, but not by VAS 2870 (Nox1/2/4 inhibitor). Conclusions Nox5 is a pro‐contractile Nox isoform important in redox‐sensitive contraction. This involves calcium‐calmodulin and endoplasmic reticulum–regulated mechanisms. Our findings define a novel function for vascular Nox5, linking calcium and reactive oxygen species to the pro‐contractile molecular machinery in vascular smooth muscle cells.

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), O 2 − (lucigenin), H 2 O 2 (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 hypertension.

Hyperaldosteronism and hypertension were unexpected side effects observed in trials of torcetrapib, a cholesteryl ester-transfer protein (CETP) inhibitor that increases high-density lipoprotein. Given that CETP inhibitors are lipid soluble, accumulate in adipose tissue, and have binding sites for proteins involved in adipogenesis, and that adipocytes are a source of aldosterone, we questioned whether CETP inhibitors (torcetrapib, dalcetrapib, and anacetrapib) influence aldosterone production by adipocytes. Studies were performed using human adipocytes (SW872), which express CETP, and mouse adipocytes (3T3-L1), which lack the CETP gene. Torcetrapib, dalcetrapib, and anacetrapib increased expression of CYP11B2, CYP11B1, and steroidogenic acute regulatory protein, enzymes involved in mineralocorticoid and glucocorticoid generation. These effects were associated with increased reactive oxygen species formation. Torcetrapib, dalcetrapib, and anacetrapib upregulated signal transducer and activator of transcription 3 (STAT3) and peroxisome proliferation-activated receptor-γ, important in adipogenesis, but only torcetrapib stimulated production of chemerin, a proinflammatory adipokine. To determine mechanisms whereby CETP inhibitors mediate effects, cells were pretreated with inhibitors of Nox1/Nox4 [GKT137831; 2-(2-chlorophenyl)-4-[3-(dimethylamino)phenyl]-5-methyl-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione], Nox1 (ML171 [2-acetylphenothiazine]), mitochondria (rotenone), and STAT3 (S3I-201 [2-hydroxy-4-(((4-methylphenyl)sulfonyloxy)acetyl)amino)-benzoic acid]). In torcetrapib-stimulated cells, Nox inhibitors, rotenone, and S3I-201 downregulated CYP11B2 and steroidogenic acute regulatory protein and reduced aldosterone. Dalcetrapib and anacetrapib effects on aldosterone were variably blocked by GKT137831, ML171, rotenone, and S3I-201. In adipocytes, torcetrapib, dalcetrapib, and anacetrapib inhibit enzymatic pathways responsible for aldosterone production through Nox1/Nox4- and mitochondrial-generated reactive oxygen species and STAT3. CETP inhibitors also influence adipokine production. These processes may be CETP independent. Our findings identify novel adipocyte-related mechanisms whereby CETP inhibitors increase aldosterone production. Such phenomena may contribute to hyperaldosteronism observed in CETP inhibitor clinical trials.

See related article, pp 461–468 Tissue fibrosis, defined as an excessive accumulation of extracellular matrix (ECM) components leading to the destruction of organ architecture and impaired function, affects virtually every tissue and organ in the body, including the arteries. Vascular fibrosis of small and large arteries contributes to arterial remodeling, important in the development and complications of hypertension.1 Fibrogenesis is an active process that involves accumulation of structural proteins (collagen and fibronectin) and adhesion proteins (laminin and fibronectin), expression of adhesion molecules and integrins, and remodeling of the ECM.2 Healthy arteries are surrounded by perivascular adventitial tissue comprising collagens I and III in the intima, media, and adventitia, with collagen types I, III, IV, and V in the endothelial and vascular smooth muscle cell basement membranes.3 These fibrillar proteins maintain vascular integrity and normal vascular tone and function. In hypertension, accumulation of collagen and fibronectin and ECM reorganization lead to increased stiffness of the vessel wall.4 Initially, these processes are adaptive and reversible and may compensate for higher blood pressures, but with time and progressive increases in blood pressure, this becomes maladaptive and decompensated, leading to arterial stiffness that contributes to hypertension-associated target organ damage. These events have been demonstrated in many experimental models of hypertension and in hypertensive patients and have been attributed to activation of ERK1/2, p38mitogen-activated protein kinase, transforming growth factor-β, SMAD pathways, oxidative stress, and dysregulation of matrix metalloproteinases.2 Decreased activation of matrix metalloproteinases and increased activity of tissue inhibitors of metalloproteinase leads to reduced collagen turnover and consequent accumulation, with thickening and remodeling of the vascular wall. Vascular fibrosis is a dynamic and active phenomenon, where a proinflammatory, oxidative milieu, triggered by prohypertensive stimuli, lays the foundation for fibrosis and activation of ECM-producing cells. Until recently the process seemed fairly …

Although VEGF (vascular endothelial growth factor) inhibitors (VEGFIs), are effective anticancer therapies, they cause hypertension through unknown mechanisms. We questioned whether changes in vascular redox state may be important, because VEGF signaling involves nitric oxide (NO) and reactive oxygen species. Molecular mechanisms, including NOS, NADPH oxidase (Nox)–derived reactive oxygen species, antioxidant systems, and vasoconstrictor signaling pathways, were probed in human endothelial cells and vascular smooth muscle exposed to vatalanib, a VEGFI. Vascular functional effects of VEGFI were assessed ex vivo in mouse arteries. Cardiovascular and renal in vivo effects were studied in vatalanib- or gefitinib (EGFI [epidermal growth factor inhibitor])-treated mice. In endothelial cells, vatalanib decreased eNOS (Ser 1177 ) phosphorylation and reduced NO and H 2 O 2 production, responses associated with increased Nox-derived O 2 − and ONOO − formation. Inhibition of Nox1/4 (GKT137831) or Nox1 (NoxA1ds), prevented vatalanib-induced effects. Nrf-2 (nuclear factor erythroid 2–related factor 2) nuclear translocation and expression of Nrf-2–regulated antioxidant enzymes were variably downregulated by vatalanib. In human vascular smooth muscles, VEGFI increased Nox activity and stimulated Ca 2+ influx and MLC 20 phosphorylation. Acetylcholine-induced vasodilatation was impaired and U46619-induced vasoconstriction was enhanced by vatalanib, effects normalized by N-acetyl-cysteine and worsened by L-NAME. In vatalanib-, but not gefitinib-treated mice vasorelaxation was reduced and media:lumen ratio of mesenteric arteries was increased with associated increased cardiovascular and renal oxidative stress, decreased Nrf-2 activity and downregulation of antioxidant genes. We demonstrate that inhibition of VEGF signaling induces vascular dysfunction through redox-sensitive processes. Our findings identify Noxs and antioxidant enzymes as novel targets underling VEGFI-induced vascular dysfunction. These molecular processes may contribute to vascular toxicity and hypertension in VEGFI-treated patients.

Primary culture of vascular smooth muscle cells is an important in vitro model for the dissection of molecular mechanisms related to a specific physiological or pathological response at the cellular level. Cultured cells also provide an excellent model to study cell biology. This chapter describes a user-friendly and practical protocol for isolation of vascular smooth muscle cells from small and large vessels by enzymatic dissociation, which can be applied to vessels from different species, including rodents and humans.

The endothelium, which is at the interface between circulating blood and the vascular wall, comprises a simple squamous layer of cells that lines the inner surface of all blood vessels. Endothelial cells are highly metabolically active and play an important role in many physiological functions, including control of vasomotor tone, blood cell trafficking, vascular permeability, and maintenance of vascular integrity (Mensah, Vascul Pharmacol 46(5):310-314, 2007; Yetik-Anacak and Catravas, Vascul Pharmacol 45(5):268-276, 2006). Endothelial cells are characteristically 'quiescent' in that they do not actively proliferate, with the average lifespan of an endothelial cell being >1 year. The endothelium is very sensitive to mechanical stimuli (stretch, shear stress, pressure), humoral agents (angiotensin II (Ang II), endothelin-1 (ET-1), aldosterone, bradykinin, thromoxane) and chemical factors (glucose, reactive oxygen species (ROS)) and responds by releasing endothelial-derived mediators, such as nitric oxide (NO), prostacyclin (PGI2), platelet-activating factor (PAF), C-type atrial natriuretic peptide (ANP), and ET-1 to regulate vascular tone, prevent thrombosis and inflammation, and maintain structural integrity. Primary culture of endothelial cells is an important tool in dissecting the role of the endothelium in many physiological or pathological responses. This chapter describes the explant method for culture of endothelial cells from large vessels. Cells derived by the protocol described here can be used for cell biology and molecular biology studies in hypertension and other cardiovascular diseases where endothelial function may be impaired.

Macrophages play a major role in inflammation, wound healing, and tissue repair. Infiltrated monocytes differentiate into different macrophage subtypes with protective or pathogenic activities in vascular lesions. In the heart and vascular tissues, pathological activation promotes cardiovascular inflammation and remodeling and there is increasing evidence that macrophages play important mechanisms in this environment. Primary murine macrophages can be obtained from: bone marrow by different treatments (granulocyte-macrophage colony-stimulating factor-GM-CSF, macrophage colony-stimulating factor-M-CSF or supernatant of murine fibroblast L929), peritoneal cavity (resident or thioglycolate elicit macrophages), from the lung (alveolar macrophages) or from adipose tissue. In this chapter we describe some protocols to obtain primary murine macrophages and how to identify a pure macrophage population or activation phenotypes using different markers.

Magnesium is an essential cation critically involved in cell viability and physiological regulation of all systems and organs. In the cardiovascular system it modulates mechanical, electrical, and structural functions of cardiac and vascular cells, and small changes in extracellular magnesium levels and/or intracellular free magnesium concentration may have profound effects on cardiac excitability, vascular tone, contractility, and reactivity. Magnesium homeostasis is tightly regulated through intestinal absorption and renal excretion and involves specific magnesium transporters, including transient receptor potential melastatin cation channels-6 and -7 and magnesium transporter subtype 1. Perturbations in magnesium metabolism may lead to cellular magnesium deficiency, which has been associated with various pathologies, including cardiovascular disease. This chapter discusses the cellular, physiological, and pharmacological roles of magnesium in the regulation of vascular function and implications of altered magnesium homeostasis in cardiovascular diseases, focusing on hypertension.

Objective: Sports practice alters the homeostasis of athletes. To achieve homeostatic equilibrium, the integrated action of the neuroendocrine and immune systems is necessary. Here we studied the relation between cytokines, hormones and mood states in marathon runners. Methods: A total of 20 male recreational marathon runners (mean age = 35.7 ± 9 years) and 20 male sedentary individuals (mean age = 35.5 ± 7 years) were recruited. We compared the serum levels of growth hormone (GH), cortisol and interleukins 8 and 10 and the amounts of these two cytokines spontaneously produced by peripheral blood mononuclear cells. Blood samples of the sedentary group were collected at rest. Blood from the marathon runners was collected at rest (baseline: 24 h before the race), immediately after a marathon and 72 h after a marathon. Mood state analysis in both groups was performed using the 24-item Brunel Mood Scale (BRUMS). Results: Our results showed that, at rest, levels of interleukins 8 and 10 in the supernatant of culture cells, the serum concentration of GH, and tension and vigour (evaluated using the BRUMS), were significantly higher in athletes compared to sedentary people. Immediately after the race all serum parameters analysed were statistically higher than baseline values. At 72 h after the marathon, serum levels of hormones and interleukins returned to values at rest, but the concentrations of interleukins in the supernatant of culture cells showed a significant reduction compared to values at rest. Conclusion: The higher serum levels of GH in athletes at rest and the higher production of cytokines in culture without previous stimulus suggest that marathon runners present mechanisms that may be associated with preparing the body to perform prolonged strenuous exercise, such as a marathon.

Atherosclerosis is an inflammatory disease associated with the activation of innate immune TLRs and nucleotide-binding oligomerization domain-containing protein (NOD)like receptor pathways. However ...

We have previously shown that the Platelet-Activating Factor Receptor (PAFR) engagement in murine macrophages and dendritic cells (DCs) promotes a tolerogenic phenotype reversed by PAFR-antagonists treatment in vitro. Here, we investigated whether a PAFR antagonist would modulate the immune response in vivo. Mice were subcutaneously injected with OVA or OVA with PAFR-antagonist WEB2170 on days 0 and 7. On day 14, OVA–specific IgG2a and IgG1 were measured in the serum. The presence of WEB2170 during immunization significantly increased IgG2a without affecting IgG1 levels. When WEB2170 was added to OVA in complete Freund’s adjuvant, enhanced IgG2a but not IgG1 production was also observed, and CD4+ FoxP3+ T cell frequency in the spleen was reduced compared to mice immunized without the antagonist. Similar results were observed in PAFR-deficient mice, along with increased Tbet mRNA expression in the spleen. Additionally, bone marrow-derived DCs loaded with OVA were transferred into naïve mice and their splenocytes were co-cultured with fresh OVA-loaded DCs. CD4+ T cell proliferation was higher in the group transferred with DCs treated with the PAFR-antagonist. We propose that the activation of PAFR by ligands present in the site of immunization is able to fine-tune the adaptive immune response.