(–)-Epicatechin Alters Reactive Oxygen and Nitrogen Species Production Independent of Mitochondrial Respiration in Human Vascular Endothelial Cells

Introduction. Vascular endothelial dysfunction is characterised by lowered nitric oxide (NO) bioavailability, which may be explained by increased production of reactive oxygen species (ROS), mitochondrial dysfunction, and altered cell signalling. (-)-Epicatechin (EPI) has proven effective in the context of vascular endothelial dysfunction, but the underlying mechanisms associated with EPI’s effects remain unclear. Objective(s). Our aim was to investigate whether EPI impacts reactive oxygen and nitrogen species (RONS) production and mitochondrial function of human vascular endothelial cells (HUVECs). We hypothesised that EPI would attenuate ROS production, increase NO bioavailability, and enhance indices of mitochondrial function. Methods. HUVECs were treated with EPI (0-20 μM) for up to 48 h. Mitochondrial and cellular ROS were measured in the absence and presence of antimycin A (AA), an inhibitor of the mitochondrial electron transport protein complex III, favouring ROS production. Genes associated with mitochondrial remodelling and the antioxidant response were quantified by RT-qPCR. Mitochondrial bioenergetics were assessed by respirometry and signalling responses determined by western blotting. Results. Mitochondrial superoxide production without AA was increased 32% and decreased 53% after 5 and 10 μM EPI treatment vs. CTRL ( P < 0.001 ). With AA, only 10 μM EPI increased mitochondrial superoxide production vs. CTRL (25%, P < 0.001 ). NO bioavailability was increased by 45% with 10 μM EPI vs. CTRL ( P = 0.010 ). However, EPI did not impact mitochondrial respiration. NRF2 mRNA expression was increased 1.5- and 1.6-fold with 5 and 10 μM EPI over 48 h vs. CTRL ( P = 0.015 and P = 0.001 , respectively). Finally, EPI transiently enhanced ERK1/2 phosphorylation (2.9 and 3.2-fold over 15 min and 1 h vs. 0 h, respectively; P = 0.035 and P = 0.011 ). Conclusion(s). EPI dose-dependently alters RONS production of HUVECs but does not impact mitochondrial respiration. The induction of NRF2 mRNA expression with EPI might relate to enhanced ERK1/2 signalling, rather than RONS production. In humans, EPI may improve vascular endothelial dysfunction via alteration of RONS and activation of cell signalling.

Soluble Guanylate Cyclase-Mediated Relaxation in Aortas from Rats with Renovascular Hypertension

Soluble guanylate cyclase (sGC) plays an important role in nitric oxide (NO)-mediated regulation of vascular tone; however, NO bioavailability is often reduced in diseased blood vessels. Accumulating evidence suggests that a shift of sGC from the NO-sensitive form to the NO-insensitive form could be an underlying cause contributing to this reduction. Herein, we investigated the impact of renovascular hypertension on NO-sensitive and NO-insensitive sGC-mediated relaxation in rat aortas. Renovascular hypertension was induced by partially clipping the left renal artery (2-kidneys, 1-clip; 2K1C) for 10 weeks. Systolic, diastolic, and mean arterial pressures were significantly increased in the 2K1C group when compared with the sham group. In addition, plasma thiobarbituric acid reactive substances and aortic superoxide generation were significantly enhanced in the 2K1C group when compared with those in the sham group. The vasorelaxant response of isolated aortas to the sGC stimulator BAY 41-2272 (NO-sensitive sGC agonist) was comparable between the sham and 2K1C groups. Likewise, the sGC activator BAY 60-2770 (NO-insensitive sGC agonist)-induced relaxation did not differ between the sham and 2K1C groups. In addition, the cGMP mimetic 8-Br-cGMP (protein kinase G agonist) induced similar relaxation in both groups. Furthermore, there were no differences in BAY 41-2272-stimulated and BAY 60-2770-stimulated cGMP generation between the groups. These findings suggest that the balance between NO-sensitive and NO-insensitive forms of sGC is maintained during renovascular hypertension. Therefore, sGC might not be responsible for the reduced NO bioavailability observed during renovascular hypertension.

Effect of the Renin-Angiotensin-Aldosterone System Reactivity on Endothelial Function and Modulative Role of Valsartan in Male Subjects with Essential Hypertension

Background: The aim of the study was to evaluate the relationship between renin-angiotensin-aldosterone (RAA) system activity and reactivity, and the endothelial function profile in normotensive subjects (N), and in essential hypertensives (H), followed by analysis of the modulatory role of an angiotensin receptor blocker (ARB): valsartan, administered in the management of hypertension. Methods: A total of 101 male subjects were enrolled to the study: 31H and 70N. The nitric-oxide (NO) bioavailability (l-Arginine, asymmetric dimethylarginine (ADMA)), symmetric dimethylarginine (SDMA), endothelial vasodilative function (flow mediated dilation (FMD)), oxidative-stress markers (malonyldialdehyde (MDA), thiol index (GSH/GSSG), nitrotyrozine (N-Tyr)), and pro-inflammatory/angiogenic parameters (sICAM-1, sVCAM-1, PAI-1, sE-selectin, PAI-1, thromboxane -B2) were assessed at baseline, then after intravenous -l-arginine administration, which was repeated after the 4-day acetylsalicylic acid (ASA) administration (75 mg/24 h). In hypertensives, this whole protocol was repeated following 2 weeks of valsartan therapy. Results: No effect of valsartan and ASA on the flow-mediated vasodilation (FMD) and the NO bioavailability in hypertensives was observed. Administration of valsartan increased plasma renin activity (PRA), but without a decrease in the aldosterone levels. ASA treatment minimized the pre-existing differences between the groups, and increased the PRA in the N-subgroup with the highest ARR values. The blood concentrations of proinflammatory sICAM-1, sE-selectin, sVCAM-1, and PAI-1 were higher, whereas the anti-inflammatory 6-keto-PGF1 alpha level was lower in hypertensive subjects. The levels of angiogenic VEGF did not differ between groups. Conclusions: Our study does not confirm the modulative effect of valsartan on endothelial function. Normotensive men showed an increase in FMD after l-arginine administration, possibly indicating baseline impairment of the NO synthesis.

Membrane estrogen receptor alpha (ERα) participates in flow-mediated dilation in a ligand-independent manner

Estrogen receptor alpha (ERα) activation by estrogens prevents atheroma through its nuclear action whereas plasma membrane-located ERα accelerates endothelial healing. The genetic deficiency of ERα was associated with a reduction in flow-mediated dilation (FMD) in one man. Here, we evaluated ex vivo the role of ERα on FMD of resistance arteries. FMD, but not agonist (acetylcholine, insulin)-mediated dilation, was reduced in male and female mice lacking ERα (Esr1-/- mice) compared to wild-type mice and was not dependent on the presence of estrogens. In C451A-ERα mice lacking membrane ERα, not in mice lacking AF2-dependent nuclear ERα actions, FMD was reduced, and restored by antioxidant treatments. Compared to wild-type mice, isolated perfused kidneys of C451A-ERα mice revealed a decreased flow-mediated nitrate production and an increased H2O2 production. Thus, endothelial membrane ERα promotes NO bioavailability through inhibition of oxidative stress and thereby participates in FMD in a ligand-independent manner.

(—)-Epicatechin alters reactive oxygen and nitrogen species production independent of mitochondrial respiration in human vascular endothelial cells

Introduction: Vascular endothelial dysfunction is characterised by lowered nitric oxide (NO) bioavailability, which may be explained by increased production of reactive oxygen species (ROS), mitochondrial dysfunction and altered cell signalling. (—)-Epicatechin (EPI) has proven effective in the context of vascular endothelial dysfunction, but the underlying mechanisms associated with EPIs effects remain unclear. Objective(s): Our aim was to investigate whether EPI impacts reactive oxygen and nitrogen species (RONS) production and mitochondrial function of human vascular endothelial cells (HUVECs). We hypothesised that EPI would attenuate ROS production, increase NO bioavailability, and enhance indices of mitochondrial function. Methods: HUVECs were treated with EPI (0-20 μM) for up to 48 h. Mitochondrial and cellular ROS were measured in the absence and presence of antimycin A (AA), an inhibitor of the mitochondrial electron transport protein complex III, favouring ROS production. Genes associated with mitochondrial remodelling and the antioxidant response were quantified by RT-qPCR. Mitochondrial bioenergetics were assessed by respirometry and signalling responses determined by western blotting. Results: Mitochondrial superoxide production without AA was increased 32% and decreased 53% after 5 and 10 μM EPI treatment vs. CTRL (P<0.001). With AA, only 10 μM EPI increased mitochondrial superoxide production vs. CTRL (25%, P<0.001). NO bioavailability was increased by 45% with 10 μM EPI vs. CTRL (P=0.010). However, EPI did not impact mitochondrial respiration. NRF2 mRNA expression was increased 1.5- and 1.6-fold with 5 and 10 μM EPI over 48 h vs. CTRL (P=0.015 and P=0.001, respectively). Finally, EPI transiently enhanced ERK1/2 phosphorylation (2.9 and 3.2-fold over 15 min and 1 h vs. 0 h, respectively; P=0.035 and P=0.011). Conclusion(s): EPI dose dependently alters RONS production of HUVECs but does not impact mitochondrial respiration. The induction of NRF2 mRNA expression with EPI might relate to enhanced ERK1/2 signalling, rather than RONS production. In humans, EPI may improve vascular endothelial dysfunction via alteration of RONS and activation of cell signalling.

Nitric Oxide Pathways in Neurovascular Coupling Under Normal and Stress Conditions in the Brain: Strategies to Rescue Aberrant Coupling and Improve Cerebral Blood Flow

The brain has impressive energy requirements and paradoxically, very limited energy reserves, implying its huge dependency on continuous blood supply. Aditionally, cerebral blood flow must be dynamically regulated to the areas of increased neuronal activity and thus, of increased metabolic demands. The coupling between neuronal activity and cerebral blood flow (CBF) is supported by a mechanism called neurovascular coupling (NVC). Among the several vasoactive molecules released by glutamatergic activation, nitric oxide (•NO) is recognized to be a key player in the process and essential for the development of the neurovascular response. Classically, •NO is produced in neurons upon the activation of the glutamatergic N-methyl-D-aspartate (NMDA) receptor by the neuronal isoform of nitric oxide synthase and promotes vasodilation by activating soluble guanylate cyclase in the smooth muscle cells of the adjacent arterioles. This pathway is part of a more complex network in which other molecular and cellular intervenients, as well as other sources of •NO, are involved. The elucidation of these interacting mechanisms is fundamental in understanding how the brain manages its energy requirements and how the failure of this process translates into neuronal dysfunction. Here, we aimed to provide an integrated and updated perspective of the role of •NO in the NVC, incorporating the most recent evidence that reinforces its central role in the process from both viewpoints, as a physiological mediator and a pathological stressor. First, we described the glutamate-NMDA receptor-nNOS axis as a central pathway in NVC, then we reviewed the link between the derailment of the NVC and neuronal dysfunction associated with neurodegeneration (with a focus on Alzheimer’s disease). We further discussed the role of oxidative stress in the NVC dysfunction, specifically by decreasing the •NO bioavailability and diverting its bioactivity toward cytotoxicity. Finally, we highlighted some strategies targeting the rescue or maintenance of •NO bioavailability that could be explored to mitigate the NVC dysfunction associated with neurodegenerative conditions. In line with this, the potential modulatory effects of dietary nitrate and polyphenols on •NO-dependent NVC, in association with physical exercise, may be used as effective non-pharmacological strategies to promote the •NO bioavailability and to manage NVC dysfunction in neuropathological conditions.

Placental Ischemia Says “NO” to Proper NOS-Mediated Control of Vascular Tone and Blood Pressure in Preeclampsia

In this review, we first provide a brief overview of the nitric oxide synthase (NOS) isoforms and biochemistry. This is followed by describing what is known about NOS-mediated blood pressure control during normal pregnancy. Circulating nitric oxide (NO) bioavailability has been assessed by measuring its metabolites, nitrite (NO2) and/or nitrate (NO3), and shown to rise throughout normal pregnancy in humans and rats and decline postpartum. In contrast, placental malperfusion/ischemia leads to systemic reductions in NO bioavailability leading to maternal endothelial and vascular dysfunction with subsequent development of hypertension in PE. We end this article by describing emergent risk factors for placental malperfusion and ischemic disease and discussing strategies to target the NOS system therapeutically to increase NO bioavailability in preeclamptic patients. Throughout this discussion, we highlight the critical importance that experimental animal studies have played in our current understanding of NOS biology in normal pregnancy and their use in finding novel ways to preserve this signaling pathway to prevent the development, treat symptoms, or reduce the severity of PE.

Omega-3 fatty acids differentially alter the expression of detoxification enzymes and nitric oxide bioavailability in endothelial cells during IL-6 exposure

Background Atherosclerotic plaques can elaborate reactive oxygen species (ROS) that reduce nitric oxide (NO) bioavailability. Cellular detoxification enzymes including various peroxiredoxin (PRDX) and superoxide dismutase (SOD) isoforms can inactivate ROS. The omega-3 fatty acid (n3-FA) eicosapentaenoic acid (EPA) reduced cardiovascular (CV) events in high-risk patients (REDUCE-IT), a benefit not observed with mixed n3-FAs containing docosahexaenoic acid (DHA). Purpose The purpose of this study was to compare the effects of EPA and DHA on NO bioavailability and expression of detoxification enzymes in the vascular endothelium in vitro. Methods Human umbilical vein endothelial cells (HUVECs) were pretreated with EPA or DHA at equimolar levels (10 μM) for 2 h, then challenged with IL-6 at 12 ng/ml for 24 h. Proteomic analysis was performed using LC/MS to measure relative protein expression. Only significant (p&lt;0.05) changes between treatment groups &gt;1-fold were analyzed. Cells were stimulated with calcium ionophore to measure NO and peroxynitrite (ONOO-) release using a porphyrinic nanosensor. Results EPA, but not DHA, augmented PRDX-2 and SOD1 expression in HUVECs relative to IL-6 alone (1.2-fold and 1.6-fold, respectively, p=0.03). EPA also significantly lowered other isoforms unlike DHA. Either EPA or DHA increased thioredoxin expression by 1.5-fold (p=0.001) and 1.3-fold (p=0.02), respectively and decreased SOD2 expression by 1.5-fold (p=8.75E-11) and 1.6-fold (p=6.03E-9), respectively. IL-6 alone only increased expression of 6 detoxification enzymes by at least 1.2-fold, relative to vehicle. Unlike DHA, EPA also increased the NO to ONOO- release ratio by 36% (p&lt;0.05) relative to IL-6 alone, without changes in NO synthase (eNOS) expression. Conclusions n3-FAs differentially influenced NO bioavailability and expression of ROS detoxification proteins, including peroxiredoxin and SOD isoforms. The net benefits of EPA on eNOS function and ROS detoxification may contribute to reduced atherothrombotic risk compared to DHA. FUNDunding Acknowledgement Type of funding sources: Private company. Main funding source(s): Amarin Pharma Inc., Elucida Research

Proprotein convertase subtilisin/kexin type 9 links inflammation to vascular endothelial cell dysfunction

Vascular endothelial cell (EC) dysfunction is a pathological mediator of the development, progression, and clinical manifestations of atherosclerotic disease. Inflammation is associated with EC dysfunction, but the responsible mechanisms are not well characterized. There is substantial evidence that serum proprotein convertase subtilisin/kexin type 9 (PCSK9) is increased in pro-inflammatory states and that elevated PCSK9 levels are associated with adverse cardiovascular outcomes after controlling for traditional risk factors, including low-density lipoprotein (LDL) cholesterol. Here we investigate PCSK9 as a novel link between inflammation and vascular EC dysfunction, as assessed by nitric oxide (NO) bioavailability. Tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine, increased PCSK9 mRNA expression (1.98 [0.7, 3.4]-fold increase, p=0.02 vs. control) and PCSK9 protein levels (1.52±0.1-fold increase, p&lt;0.01 vs. control) in isolated human aortic ECs. This was accompanied by reduced phosphorylated endothelial nitric oxide synthase (eNOS) protein which was 56% ± 5.6% of that in the controls (p&lt;0.01) and NO bioavailability, which was reduced by 29% ± 22.1% compared to that in the controls (p&lt;0.01). Finally, genetic PCSK9 reduction utilizing a PCSK9 specific siRNA in human aortic ECs resulted in the rescue of eNOS phosphorylation and NO bioavailability. Our results demonstrate that PCSK9 is increased in human aortic ECs exposed to a pro-inflammatory stimulus and that this increase is associated with EC dysfunction. Silencing of TNFα-mediated augmentation of PCSK9 protein expression utilizing a small interfering RNA against PCSK9 rescued the inflammation-induced EC dysfunction. These results indicate that PCSK9 is a causal link between inflammation and EC dysfunction, a potent driver of atherosclerotic cardiovascular disease. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Career Development Award from the American Heart Association, OAIC pilot Award

Intravital Assessment of Pre-capillary Pulmonary Arterioles of Type-1 Diabetic Mice Shows Oxidative Damage and Increased Tone in Response to NOS Inhibition

Microvascular dilation, important for peripheral tissue glucose distribution, also modulates alveolar perfusion and is inhibited by loss of bioavailable nitric oxide (NO) in diabetes mellitus (DM). We hypothesized that DM-induced oxidative stress decreases bioavailable NO and pulmonary pre-capillary arteriolar diameter, causing endothelial injury. We examined sub-pleural pulmonary arterioles after acute NO synthase (NOS) inhibition with L-NAME in streptozotocin (STZ) and saline (CTRL)-treated C57BL/6J mice. Microvascular changes were assessed by intravital microscopy in the right lung of anesthetized mice with open-chest and ventilated lungs. Arteriolar tone in pulmonary arterioles (27.2 to 48.7 µm diameter), increased in CTRL mice (18.0 ± 11% constriction p=0.034, n=5) but decreased in STZ (13.6 ± 7.5% dilation p= 0.009, n=5), after L-NAME. Lung tissue DHE fluorescence (superoxide), inducible NOS expression, and protein nitrosylation (3-nitrotyrosine) increased in STZ mice and correlated with increased glucose levels (103.8 ± 8.8 mg/dL). Fluorescently-labeled fibrinogen administration and fibrinogen immunostaining showed fibrinogen adhesion, indicating endothelial injury in STZ mice. In CTRL mice, vasoconstriction to L-NAME was likely due to the loss of bioavailable NO. Vasodilation in STZ mice may be due to decreased formation of a vasoconstrictor or emergence of a vasodilator. These findings provide novel evidence that DM targets the pulmonary microcirculation and that decreased NO bioavailability and increased precapillary arteriolar tone could potentially lead to ventilation-perfusion abnormalities, exacerbating systemic DM complications.