NOX5-induced uncoupling of endothelial NO synthase is a causal mechanism and theragnostic target of an age-related hypertension endotype.

Hypertension is the most important cause of death and disability in the elderly. In 9 out of 10 cases, the molecular cause, however, is unknown. One mechanistic hypothesis involves impaired endothelium-dependent vasodilation through reactive oxygen species (ROS) formation. Indeed, ROS forming NADPH oxidase (Nox) genes associate with hypertension, yet target validation has been negative. We re-investigate this association by molecular network analysis and identify NOX5, not present in rodents, as a sole neighbor to human vasodilatory endothelial nitric oxide (NO) signaling. In hypertensive patients, endothelial microparticles indeed contained higher levels of NOX5-but not NOX1, NOX2, or NOX4-with a bimodal distribution correlating with disease severity. Mechanistically, mice expressing human Nox5 in endothelial cells developed-upon aging-severe systolic hypertension and impaired endothelium-dependent vasodilation due to uncoupled NO synthase (NOS). We conclude that NOX5-induced uncoupling of endothelial NOS is a causal mechanism and theragnostic target of an age-related hypertension endotype. Nox5 knock-in (KI) mice represent the first mechanism-based animal model of hypertension.

[Therapeutic drug monitoring in neuropsychopharmacology : Summary of the consensus guidelines 2017 of the TDM task force of the AGNP]. / Therapeutisches Drug-Monitoring in der Neuropsychopharmakologie : Zusammenfassung der Konsensusleitlinien 2017 der TDM-Arbeitsgruppe der AGNP.

Therapeutic drug monitoring (TDM) is the quantification and interpretation of drug concentrations in blood serum or plasma to optimize pharmacological therapy. TDM is an instrument with which the high interindividual variability of pharmacokinetics of patients can be identified and therefore enables a personalized pharmacotherapy. In September 2017 the TDM task force of the Working Group for Neuropsychopharmacology and Pharmacopsychiatry (AGNP) published an update of the consensus guidelines on TDM published in 2011. This article summarizes the essential statements for the clinical practice in psychiatry and neurology.

Rutin protects against H2O2-triggered impaired relaxation of placental arterioles and induces Nrf2-mediated adaptation in Human Umbilical Vein Endothelial Cells exposed to oxidative stress.

BACKGROUND: Rutin intake is associated with a reduced risk of cardiovascular disease (CVD). The exact mechanism by which rutin can protect against CVD development is still enigmatic. Since, rutin is a compound with a relatively short half-life, the direct antioxidant effect of rutin cannot explain the long-lasting effect on human health. We hypothesized that rutin next to its direct antioxidant effect that improves endothelial function, may also induce an adaptive response in endogenous antioxidant systems. METHODS AND RESULTS: In Human Umbilical Vein Endothelial Cells (HUVECs), the direct antioxidant effect was confirmed. During scavenging of Reactive Oxygen Species (ROS), rutin is oxidized into a quinone derivative. HUVECs pretreated with rutin quinone became better protected against a second challenge with oxidative stress 3h later. LC-MS/MS analysis indicated that rutin quinone targets cysteine 151 of Keap1. Moreover, we found that the quinone is an inhibitor of the selenoprotein thioredoxin reductase 1. These properties correlated with an activation of Nrf2 and upregulation of Glutamate Cysteine Ligase, the rate-limiting enzyme of glutathione synthesis, while NF-κB and HIF activation became blunted by rutin treatment. Furthermore, rutin was found to prevent hydrogen peroxide from impairing relaxation of human chorionic plate placental vessels, which may help to protect endothelial function. CONCLUSION AND SIGNIFICANCE: Rutin functions as an antioxidant and is oxidized into a quinone that upregulates the Nrf2-mediated endogenous antioxidant response. This mechanism suggests that rutin selectively exerts its protective effects in regions with increased oxidative stress, and explains how rutin reduces the risk of developing CVD. GENERAL SIGNIFICANCE: The newly found mechanism behind the long-term protection of rutin against cardiovascular disease, the selective upregulation of endogenous antioxidant systems, contributes to the further understanding why rutin can reduce the risk on developing cardiovascular disease.

Phosphodiesterase 1 regulation is a key mechanism in vascular aging.

Reduced nitric oxide (NO)/cGMP signalling is observed in age-related vascular disease. We hypothesize that this disturbed signalling involves effects of genomic instability, a primary causal factor in aging, on vascular smooth muscle cells (VSMCs) and that the underlying mechanism plays a role in human age-related vascular disease. To test our hypothesis, we combined experiments in mice with genomic instability resulting from the defective nucleotide excision repair gene ERCC1 (Ercc1(d/-) mice), human VSMC cultures and population genome-wide association studies (GWAS). Aortic rings of Ercc1(d/-) mice showed 43% reduced responses to the soluble guanylate cyclase (sGC) stimulator sodium nitroprusside (SNP). Inhibition of phosphodiesterase (PDE) 1 and 5 normalized SNP-relaxing effects in Ercc1(d/-) to wild-type (WT) levels. PDE1C levels were increased in lung and aorta. cGMP hydrolysis by PDE in lungs was higher in Ercc1(d/-) mice. No differences in activity or levels of cGMP-dependent protein kinase 1 or sGC were observed in Ercc1(d/-) mice compared with WT. Senescent human VSMC showed elevated PDE1A and PDE1C and PDE5 mRNA levels (11.6-, 9- and 2.3-fold respectively), which associated with markers of cellular senescence. Conversely, PDE1 inhibition lowered expression of these markers. Human genetic studies revealed significant associations of PDE1A single nucleotide polymorphisms with diastolic blood pressure (DBP; ß=0.28, P=2.47×10(-5)) and carotid intima-media thickness (cIMT; ß=-0.0061, P=2.89×10(-5)). In summary, these results show that genomic instability and cellular senescence in VSMCs increase PDE1 expression. This might play a role in aging-related loss of vasodilator function, VSMC senescence, increased blood pressure and vascular hypertrophy.

Impaired autonomic regulation of resistance arteries in mice with low vascular endothelial growth factor or upon vascular endothelial growth factor trap delivery.

BACKGROUND: Control of peripheral resistance arteries by autonomic nerves is essential for the regulation of blood flow. The signals responsible for the maintenance of vascular neuroeffector mechanisms in the adult, however, remain largely unknown. METHODS AND RESULTS: Here, we report that VEGF( partial differential/ partial differential) mice with low vascular endothelial growth factor (VEGF) levels suffer defects in the regulation of resistance arteries. These defects are due to dysfunction and structural remodeling of the neuroeffector junction, the equivalent of a synapse between autonomic nerve endings and vascular smooth muscle cells, and to an impaired contractile smooth muscle cell phenotype. Notably, short-term delivery of a VEGF inhibitor to healthy mice also resulted in functional and structural defects of neuroeffector junctions. CONCLUSIONS: These findings uncover a novel role for VEGF in the maintenance of arterial neuroeffector function and may help us better understand how VEGF inhibitors cause vascular regulation defects in cancer patients.

Hypoxia-induced mitochondrial abnormalities in cells of the placenta.

INTRODUCTION: Impaired utero-placental perfusion is a well-known feature of early preeclampsia and is associated with placental hypoxia and oxidative stress. Although aberrations at the level of the mitochondrion have been implicated in PE pathophysiology, whether or not hypoxia-induced mitochondrial abnormalities contribute to placental oxidative stress is unknown. METHODS: We explored whether abnormalities in mitochondrial metabolism contribute to hypoxia-induced placental oxidative stress by using both healthy term placentae as well as a trophoblast cell line (BeWo cells) exposed to hypoxia. Furthermore, we explored the therapeutic potential of the antioxidants MitoQ and quercetin in preventing hypoxia-induced placental oxidative stress. RESULTS: Both in placental explants as well as BeWo cells, hypoxia resulted in reductions in mitochondrial content, decreased abundance of key molecules involved in the electron transport chain and increased expression and activity of glycolytic enzymes. Furthermore, expression levels of key regulators of mitochondrial biogenesis were decreased while the abundance of constituents of the mitophagy, autophagy and mitochondrial fission machinery was increased in response to hypoxia. In addition, placental hypoxia was associated with increased oxidative stress, inflammation, and apoptosis. Moreover, experiments with MitoQ revealed that hypoxia-induced reactive oxygen species originated from the mitochondria in the trophoblasts. DISCUSSION: This study is the first to demonstrate that placental hypoxia is associated with mitochondrial-generated reactive oxygen species and significant alterations in the molecular pathways controlling mitochondrial content and function. Furthermore, our data indicate that targeting mitochondrial oxidative stress may have therapeutic benefit in the management of pathologies related to placental hypoxia.

Twenty-four-hour exposure to altered blood flow modifies endothelial Ca2+-activated K+ channels in rat mesenteric arteries.

We tested the hypothesis that changes in arterial blood flow modify the function of endothelial Ca2+-activated K+ channels [calcium-activated K+ channel (K(Ca)), small-conductance calcium-activated K+ channel (SK3), and intermediate calcium-activated K+ channel (IK1)] before arterial structural remodeling. In rats, mesenteric arteries were exposed to increased [+90%, high flow (HF)] or reduced blood flow [-90%, low flow (LF)] and analyzed 24 h later. There were no detectable changes in arterial structure or in expression level of endothelial nitric-oxide synthase, SK3, or IK1. Arterial relaxing responses to acetylcholine and 3-oxime-6,7-dichlore-1H-indole-2,3-dione (NS309; activator of SK3 and IK1) were measured in the absence and presence of endothelium, NO, and prostanoid blockers, and 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo [b,n] [1,5,12,16]tetraazacyclotricosine-5,13-diium dibromide (UCL 1684; inhibitor of SK3) or 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; inhibitor of IK1). In LF arteries, endothelium-dependent relaxation was markedly reduced, due to a reduction in the endothelium-derived hyperpolarizing factor (EDHF) response. In HF arteries, the balance between the NO/prostanoid versus EDHF response was unaltered. However, the contribution of IK1 to the EDHF response was enhanced, as indicated by a larger effect of TRAM-34 and a larger residual NS309-induced relaxation in the presence of UCL 1684. Reduction of blood flow selectively blunts EDHF relaxation in resistance arteries through inhibition of the function of K(Ca) channels. An increase in blood flow leads to a more prominent role of IK1 channels in this relaxation.

Placental hypoxia-induced alterations in vascular function, morphology, and endothelial barrier integrity.

Preeclampsia (PE) is a pregnancy-related disorder characterized by hypertension and proteinuria that affects 3-10% of all pregnancies. Although its pathophysiology remains obscure, placental hypoxia-induced oxidative stress and alterations in vascular function, morphology, and endothelial barrier integrity are considered to play a key role in the development of preeclampsia. In this study, placental villous explants of noncomplicated placentae and BeWo cells were subjected to hypoxia. The effect of placental hypoxic-conditioned medium (HCM) on intraluminal-induced contraction and endothelial barrier integrity in chorionic arteries was investigated using pressure myography. The impact of BeWo cell HCM on endothelial cell viability, reactive oxygen species formation and inflammation was also determined. Alterations in arterial morphology and contractile responsiveness to the thromboxane A2 analog (U46619) after exposure to placental HCM were examined immunohistochemically and by wire myography, respectively. Intraluminal administration of placental HCM induced vasoconstriction and increased the endothelial permeability for KCl, which was concentration-dependently prevented by quercetin. Placental and BeWo cell HCMs decreased endothelial cell viability, increased the production of reactive oxygen species and enhanced the secretion of IL-6 and IL-8. The cross-sectional area of the arterial media was increased upon exposure to placental HCM, which was associated with increased vascular proliferation and contractile responsiveness to U46619, and all of these effects were prevented by the antioxidants quercetin and RRR-α-tocopherol. This study is the first to comprehensively demonstrate the link between factors secreted by placental cells in response to hypoxia and vascular abnormalities and paves the way for new diagnostic approaches and therapies to better protect the maternal vasculature during and after a preeclampsia-complicated pregnancy.

The direct and sustained consequences of severe placental hypoxia on vascular contractility.

INTRODUCTION: Preeclampsia is a major health problem in human pregnancy, severely complicating 5-8% of all pregnancies. The emerging molecular mechanism is that conditions like hypoxic stress trigger the release of placental messengers into the maternal circulation, which causes preeclampsia. Our objective was to develop an in vitro model, which can be used to further elucidate the molecular mechanisms of preeclampsia and which might be used to find a remedy. METHODS: Human non-complicated term placentas were collected. Placental explants were subjected to severe hypoxia and the conditioned media were added to chorionic arteries that were mounted into a myograph. Contractile responses of the conditioned media were determined, as well as effects on thromboxane-A2 (U46619) induced contractility. To identify the vasoactive compounds present in the conditioned media, specific receptor antagonists were evaluated. RESULTS: Factors released by placental explants generated under severe hypoxia induced an increased vasoconstriction and vascular contractility to thromboxane-A2. It was found that agonists for the angiotensin-I and endothelin-1 receptor released by placental tissue under severe hypoxia provoke vasoconstriction. The dietary antioxidant quercetin could partially prevent the acute and sustained vascular effects in a concentration-dependent manner. DISCUSSION: Both the acute vasoconstriction, as well as the increased contractility to U46619 are in line with the clinical vascular complications observed in preeclampsia. Data obtained with quercetin supports that our model opens avenues for e.g. nutritional interventions aimed at treating or preventing preeclampsia.

TDM in psychiatry and neurology: A comprehensive summary of the consensus guidelines for therapeutic drug monitoring in neuropsychopharmacology, update 2017; a tool for clinicians.

OBJECTIVES: Therapeutic drug monitoring (TDM) combines the quantification of drug concentrations in blood, pharmacological interpretation and treatment guidance. TDM introduces a precision medicine tool in times of increasing awareness of the need for personalized treatment. In neurology and psychiatry, TDM can guide pharmacotherapy for patient subgroups such as children, adolescents, pregnant women, elderly patients, patients with intellectual disabilities, patients with substance use disorders, individuals with pharmacokinetic peculiarities and forensic patients. Clear indications for TDM include lack of clinical response in the therapeutic dose range, assessment of drug adherence, tolerability issues and drug-drug interactions. METHODS: Based upon existing literature, recommended therapeutic reference ranges, laboratory alert levels, and levels of recommendation to use TDM for dosage optimization without specific indications, conversion factors, factors for calculation of dose-related drug concentrations and metabolite-to-parent ratios were calculated. RESULTS: This summary of the updated consensus guidelines by the TDM task force of the Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie offers the practical and theoretical knowledge for the integration of TDM as part of pharmacotherapy with neuropsychiatric agents into clinical routine. CONCLUSIONS: The present guidelines for TDM application for neuropsychiatric agents aim to assist clinicians in enhancing safety and efficacy of treatment.

Prenatal stress changes rat arterial adrenergic reactivity in a regionally selective manner.

A suboptimal fetal environment has been linked to increased risk of cardiovascular disease in adulthood. We investigated whether intrauterine stress (IUS) alters the development of adrenergic reactivity in different types of rat arteries. Intrauterine stress was induced by ligation of the uterine arteries at day 13 of pregnancy in Wistar rats. First-order mesenteric, renal, femoral and saphenous arteries of the 21-day-old male offspring were studied in a myograph. IUS in the rat changes arterial adrenergic reactivity in a regionally selective manner. Adrenoceptor-mediated responses are altered in the renal artery. Maximal contractile responses to phenylephrine were increased, while sensitivity to the alpha(1)-adrenoceptor agonist was decreased. Intrauterine stress significantly reduced contractile responses to norepinephrine and enhanced relaxing responses to isoproterenol in the renal artery. Adrenergic responses were not modified in mesenteric, femoral and saphenous arteries. In the kidneys the densities of [(3)H]prazosin binding sites, periarterial adrenergic nerves and of the glomeruli were not altered after intrauterine stress at day 13 of gestation. The observed regionally selective alterations in arterial reactivity might link a suboptimal fetal environment to the development of cardiovascular disease in the adult.

Impaired flow-induced arterial remodeling in DOCA-salt hypertensive rats.

Arteries from young healthy animals respond to chronic changes in blood flow and blood pressure by structural remodeling. We tested whether the ability to respond to decreased (-90%) or increased (+100%) blood flow is impaired during the development of deoxycorticosterone acetate (DOCA)-salt hypertension in rats, a model for an upregulated endothelin-1 system. Mesenteric small arteries (MrA) were exposed to low blood flow (LF) or high blood flow (HF) for 4 or 7 weeks. The bioavailability of vasoactive peptides was modified by chronic treatment of the rats with the dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor SOL1. After 3 or 6 weeks of hypertension, the MrA showed hypertrophic arterial remodeling (3 weeks: media cross-sectional area (mCSA): 10±1 × 10(3) to 17±2 × 10(3) µm(2); 6 weeks: 13±2 × 10(3) to 24±3 × 10(3) µm(2)). After 3, but not 6, weeks of hypertension, the arterial diameter was increased (Ø: 385±13 to 463±14 µm). SOL1 reduced hypertrophy after 3 weeks of hypertension (mCSA: 6 × 10(3)±1 × 10(3) µm(2)). The diameter of the HF arteries of normotensive rats increased (Ø: 463±22 µm) but no expansion occurred in the HF arteries of hypertensive rats (Ø: 471±16 µm). MrA from SOL1-treated hypertensive rats did show a significant diameter increase (Ø: 419±13 to 475±16 µm). Arteries exposed to LF showed inward remodeling in normotensive and hypertensive rats (mean Ø between 235 and 290 µm), and infiltration of monocyte/macrophages. SOL1 treatment did not affect the arterial diameter of LF arteries but reduced the infiltration of monocyte/macrophages. We show for the first time that flow-induced remodeling is impaired during the development of DOCA-salt hypertension and that this can be prevented by chronic NEP/ECE inhibition.

High sodium intake increases blood pressure and alters renal function in intrauterine growth-retarded rats.

A suboptimal fetal environment increases the risk to develop cardiovascular disease in the adult. We reported previously that intrauterine stress in response to reduced uteroplacental blood flow in the pregnant rat limits fetal growth and compromises renal development, leading to an altered renal function in the adult offspring. Here we tested the hypothesis that high dietary sodium intake in rats with impaired renal development attributable to intrauterine stress, results in increased blood pressure, altered renal function, and organ damage. In rats, intrauterine stress was induced by bilateral ligation of the uterine arteries at day 17 of pregnancy. At the age of 12 weeks, the offspring was given high-sodium drinking water (2% sodium chloride). At the age of 16 weeks, rats were instrumented for monitoring of blood pressure and renal function. After intrauterine stress, litter size and birth weight were reduced, whereas hematocrit at birth was increased. Renal blood flow, glomerular filtration rate, and the glomerular filtration fraction were increased significantly after intrauterine stress. High sodium intake did not change renal function and blood pressure in control animals. However, during high sodium intake in intrauterine stress offspring, renal blood flow, glomerular filtration rate, and the filtration fraction were decreased, and blood pressure was increased. In addition, these animals developed severe albuminuria, an important sign of renal dysfunction. Thus, a suboptimal fetal microenvironment, which impairs renal development, results in sodium-dependent hypertension and albuminuria.

Reduced uteroplacental blood flow alters renal arterial reactivity and glomerular properties in the rat offspring.

Fetal malnutrition and hypoxia may modify organ system maturation and result in cardiovascular diseases in the adult. We tested whether intrauterine stress (IUS) leads to persistent alterations of renal biology. In rats, intrauterine stress was induced by ligation of the uterine arteries at day 17 of pregnancy. Renal arteries of the 21-day-old male offspring were isolated to study pharmacological reactivity. Kidneys were dissected to analyze renal structure and beta-adrenoceptor expression. At 21 days of age, half of the animals underwent unilateral left nephrectomy. At the age of 12 weeks, rats were instrumented for blood pressure monitoring, blood sampling, and renal function measurements. After IUS, litter size and birth weight were reduced, whereas the hematocrit was increased. Renal arterial responses to beta-adrenergic stimulation and sensitivity to adenylyl cyclase activation were increased, along with the renal expression of beta2-adrenoceptors. At 21 days and at 6 months of age, the number and density of the glomeruli were reduced, whereas their size was increased. The filtration fraction and urinary albumin concentration were increased 12 weeks after intrauterine stress. In control rats, removal of the left kidney at 21 days of age did not affect kidney function and blood pressure. However, after IUS, the remaining right kidney failed to compensate for the loss of the left kidney, and blood pressure was increased. In conclusion, prenatal stress transiently modifies renal arterial reactivity and results in long-lasting adverse effects on renal structure and function and on renal compensatory mechanisms.

Role of the Rhoa/Rho kinase system in flow-related remodeling of rat mesenteric small arteries in vivo.

In small arteries, a chronic blood flow reduction leads to inward hypotrophic remodeling, while a chronic blood flow elevation induces outward hypertrophic remodeling. The RhoA/Rho kinase system was shown to be modulated by shear stress, and to be involved in other kinds of vascular remodeling. The aim of this study was to investigate the role of RhoA/Rho kinase in flow-related small artery remodeling. Rat mesenteric small arteries were subjected to flow-modifying surgery. After 1, 2, 4, 16, and 32 days, the animals were sacrificed and small arteries were harvested. Messenger RNA was isolated and amplified. Using cDNA microarray analysis, the differential expression of >14,000 genes was analyzed, part of which was confirmed by RT-PCR. In vivo treatment with fasudil (3 mg/kg/day s.c.) was used to test the effect of Rho kinase inhibition. The main findings are that: (1) blood flow alteration modified the expression of approximately 5% of the genes by >2-fold, (2) flow reduction downregulated many RhoA-related cytoskeletal markers of smooth muscle cell phenotype, (3) many RhoA-related genes were rapidly (<1 day) regulated and (4) fasudil treatment potentiated the inward hypotrophic remodeling in response to chronically reduced flow. These results indicate the importance of the RhoA/Rho kinase system in flow-related small artery remodeling.