Vascular Reactive Oxygen Species Production Research Articles

Angiotensin‐(1‐7)/Mas receptor agonist: a disease modifying therapeutic for VCID protects cognitive function and inhibits serum neurofilament light protein

AbstractBackgroundVascular contributions to cognitive impairment and dementia (VCID) and Alzheimer’s disease related dementias (ADRD) are associated with increases in systemic and brain inflammatory cytokines, increased reactive oxygen species (ROS) and decreased brain blood flow. We have completed preclinical development of novel synthetic glycopeptide derivatives of Angiotensin‐(1‐7) (Ang‐1‐7) MasR agonists that have outstanding brain penetration, improved half‐life, reverse cognitive impairment and decreases plasma neurofilament light protein (NfL) in our preclinical model of VCID. Ang‐(1‐7)/MasR mechanisms of action include inhibition of 1) microglia activation, 2) brain cytokines activation and, 3) vascular endothelial ROS production. The purpose of this pilot study was to 1) determine if plasma NfL is increased in coronary artery bypass graft (CABG) surgery patients at‐risk for VCID and 2) determine if treatment with an Ang‐(1‐7)/MasR agonist protects cognitive function post‐surgy and modifies NfL.MethodSubjects were randomly assigned to receive either 200 micrograms/kg/day s.c. (n = 3) or saline‐placebo (n = 2). Injections were begun 2 hours prior to surgery and then daily for 21 days. Cognitive function was measured prior to surgery and at 21‐ and 90‐days post‐surgery. Serum NfL was measured via Quanterix Simoa assay. Statistical analysis was performed using GraphPad and significance tested using Welch’s ANOVA, Dunnett’s post. Memory function was measured using a composite score comprised of memory, executive functioning, language, and processing speed.ResultPlasma levels of NfL prior to surgery were significantly increased by 176% in CABG patients (n = 5) as compared to age‐matched healthy controls (n = 8, p = .003). Treatment with Ang‐(1‐7) for 21‐days via subcutaneous injections protected memory function as measured by both the Face‐Name and Cured Name Retrieval associative memory tests at both 21‐ and 90‐days post ‐surgery and significantly attenuated CABG induced increases in NfL as compared to placebo‐saline treatment (p = .004).ConclusionThese preliminary data suggest that NfL may serve as a biomarker of VCID in CABG surgery patients and for MasR target engagement for VCID‐modifying treatments with Ang‐(1‐7) analogs. We have begun studies to develop NfL as a biomarker for Ang‐(1‐7)/MasR target activation and for patient stratification for treatment of VCID (Supported by NIH U01HL131014, U01AG066623).

Short-term salicylate treatment improves microvascular endothelium-dependent dilation in young adults with major depressive disorder.

Reactive oxygen species (ROS)-mediated reductions in nitric oxide (NO)-dependent dilation are evident in adults with major depressive disorder (MDD); however, the upstream mechanisms remain unclear. Here, we hypothesized that nuclear factor-κB (NF-κB) activation-induced ROS production contributes to microvascular endothelial dysfunction in MDD. Thirteen treatment-naive adults with MDD (6 women; 19-23 yr) and 10 healthy nondepressed adults (HAs; 5 women; 20-25 yr) were tested before and after (open-label design) systemic NF-κB knockdown (nonacetylated salicylate; 3,000-4,500 mg/day × 4 days). Red cell flux (laser Doppler flowmetry) was measured during graded intradermal microdialysis perfusion of the endothelium-dependent agonist acetylcholine (ACh), alone and in combination with NO synthase inhibition [NG-nitro-l-arginine methyl ester (l-NAME)] or ROS scavenging (apocynin). Serum salicylate concentrations following treatment were not different between groups (22.8 ± 7.4 HAs vs. 20.8 ± 4.3 mg/dL MDD; P = 0.46). When compared with HAs, the NO-dependent component of ACh-induced dilation was blunted in adults with MDD before (P = 0.023), but not after (P = 0.27), salsalate treatment. In adults with MDD, the magnitude of improvement in endothelium-dependent dilation following salsalate treatment was inversely related to the degree of functional impairment at baseline (R2 = 0.43; P = 0.025). Localized ROS scavenging improved NO-dependent dilation before (P < 0.01), but not after (P > 0.05), salsalate treatment. Salsalate did not alter systemic concentrations of pro- or anti-inflammatory cytokines (all P > 0.05). These data suggest that NF-κB activation, via increased vascular ROS production, contributes to blunted NO-dependent dilation in young adults with MDD but otherwise free of clinical disease. These data provide the first direct evidence for a mechanistic role of vascular inflammation-associated endothelial dysfunction in human depression.NEW & NOTEWORTHY Our data indicate that short-term treatment with therapeutic doses of the nuclear factor-κB (NF-κB) inhibitor salsalate improved nitric oxide (NO)-mediated endothelium-dependent dilation in adults with major depressive disorder (MDD). In adults with MDD, acute localized scavenging of reactive oxygen species (ROS) with apocynin improved NO-dependent dilation before, but not after, salsalate administration. These data suggest that activation of NF-κB, in part via stimulation of vascular ROS production, contributes to blunted NO-mediated endothelium-dependent dilation in young adults with MDD.

Interferon-stimulated gene 15 pathway is a novel mediator of endothelial dysfunction and aneurysms development in angiotensin II infused mice through increased oxidative stress.

Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that induces a reversible post-translational modification (ISGylation) and can also be secreted as a free form. ISG15 plays an essential role as host-defence response to microbial infection; however, its contribution to vascular damage associated with hypertension is unknown. Bioinformatics identified ISG15 as a mediator of hypertension-associated vascular damage. ISG15 expression positively correlated with systolic and diastolic blood pressure and carotid intima-media thickness in human peripheral blood mononuclear cells. Consistently, Isg15 expression was enhanced in aorta from hypertension models and in angiotensin II (AngII)-treated vascular cells and macrophages. Proteomics revealed differential expression of proteins implicated in cardiovascular function, extracellular matrix and remodelling, and vascular redox state in aorta from AngII-infused ISG15-/- mice. Moreover, ISG15-/- mice were protected against AngII-induced hypertension, vascular stiffness, elastin remodelling, endothelial dysfunction, and expression of inflammatory and oxidative stress markers. Conversely, mice with excessive ISGylation (USP18C61A) show enhanced AngII-induced hypertension, vascular fibrosis, inflammation and reactive oxygen species (ROS) generation along with elastin breaks, aortic dilation, and rupture. Accordingly, human and murine abdominal aortic aneurysms showed augmented ISG15 expression. Mechanistically, ISG15 induces vascular ROS production, while antioxidant treatment prevented ISG15-induced endothelial dysfunction and vascular remodelling. ISG15 is a novel mediator of vascular damage in hypertension through oxidative stress and inflammation.

Inhibition of Rac1 GTPase Decreases Vascular Oxidative Stress, Improves Endothelial Function, and Attenuates Atherosclerosis Development in Mice.

Aims: Oxidative stress and inflammation contribute to atherogenesis. Rac1 GTPase regulates pro-oxidant NADPH oxidase activity, reactive oxygen species (ROS) formation, actin cytoskeleton organization and monocyte adhesion. We investigated the vascular effects of pharmacological inhibition of Rac1 GTPase in mice.Methods and Results: We treated wild-type and apolipoprotein E-deficient (ApoE−/−) mice with Clostridium sordellii lethal toxin (LT), a Rac1 inhibitor, and assessed vascular oxidative stress, expression and activity of involved proteins, endothelial function, macrophage infiltration, and atherosclerosis development. LT-treated wild-type mice displayed decreased vascular NADPH oxidase activity and ROS production. Therapeutic LT doses had no impact on behavior, food intake, body weight, heart rate, blood pressure, vascular and myocardial function, differential blood count, and vascular permeability. ApoE−/− mice were fed a cholesterol-rich diet and were treated with LT or vehicle. LT treatment led to decreased aortic Rac1 GTPase activity, NADPH oxidase activity and ROS production, but had no impact on expression and membrane translocation of NADPH oxidase subunits and RhoA GTPase activity. LT-treated mice showed improved aortic endothelium-dependent vasodilation, attenuated atherosclerotic lesion formation and reduced macrophage infiltration of atherosclerotic plaques. Concomitant treatment of cholesterol-fed ApoE−/− mice with LT, the specific synthetic Rac1 inhibitor NSC 23766 or simvastatin comparably reduced aortic Rac1 activity, NADPH oxidase activity, oxidative stress, endothelial dysfunction, atherosclerosis development, and macrophage infiltration.Conclusions: These findings identify an important role of the small GTPase Rac1 in atherogenesis and provide a potential target for anti-atherosclerotic therapy.

Inhibition of NOX1 Mitigates Blood Pressure Increases in Elastin Insufficiency.

Elastin (ELN) insufficiency leads to the cardiovascular hallmarks of the contiguous gene deletion disorder, Williams-Beuren syndrome, including hypertension and vascular stiffness. Previous studies showed that Williams-Beuren syndrome deletions, which extended to include the NCF1 gene, were associated with lower blood pressure (BP) and reduced vascular stiffness. NCF1 encodes for p47phox, the regulatory component of the NOX1 NADPH oxidase complex that generates reactive oxygen species (ROS) in the vascular wall. Dihydroethidium and 8-hydroxyguanosine staining of mouse aortas confirmed that Eln heterozygotes (Eln+/- ) had greater ROS levels than the wild-types (Eln+/+ ), a finding that was negated in vessels cultured without hemodynamic stressors. To analyze the Nox effect on ELN insufficiency, we used both genetic and chemical manipulations. Both Ncf1 haploinsufficiency (Ncf1+/- ) and Nox1 insufficiency (Nox1-/y ) decreased oxidative stress and systolic BP in Eln+/- without modifying vascular structure. Chronic treatment with apocynin, a p47phox inhibitor, lowered systolic BP in Eln+/- , but had no impact on Eln+/+ controls. In vivo dosing with phenylephrine (PE) produced an augmented BP response in Eln+/- relative to Eln+/+ , and genetic modifications or drug-based interventions that lower Nox1 expression reduced the hypercontractile response to PE in Eln+/- mice to Eln+/+ levels. These results indicate that the mechanical and structural differences caused by ELN insufficiency leading to oscillatory flow can perpetuate oxidative stress conditions, which are linked to hypertension, and that by lowering the Nox1-mediated capacity for vascular ROS production, BP differences can be normalized.

Western diet triggers Toll-like receptor 4 signaling-induced endothelial dysfunction in female Wistar rats.

Overconsumption of a diet rich in fat and carbohydrates, called the Western diet, is a major contributor to the global epidemic of cardiovascular disease. Despite previously documented cardiovascular protection exhibited in female rats, this safeguard may be lost under certain metabolic stressors. We hypothesized that female Wistar rats challenged by a Western diet composed of 21% fat and 50% carbohydrate (34.1% sucrose) for 17 wk would develop endothelial dysfunction via endothelial Toll-like receptor 4 (TLR4) signaling. Western diet-fed female rats exhibited dysregulation of metabolism, revealing increased body weight and abdominal fat, decreased expression of adiponectin in white adipose tissue, glucose intolerance, and impaired insulin sensitivity. Western diet exposure increased hepatic triglycerides and cholesterol alongside hepatic steatosis, categorizing nonalcoholic fatty liver disease. Moreover, a Western diet negatively affected vascular function, revealing hypertension, impaired endothelium-dependent vasorelaxation, aortic remodeling, and increased reactive oxygen species (ROS) production. Aortic protein expression of TLR4 and its downstream proteins were markedly increased in the Western diet-fed group in association with elevated serum levels of free fatty acids. In vitro experiments were conducted to test whether free fatty acids contribute to vascular ROS overproduction via the TLR4 signaling pathway. Cultured endothelial cells were stimulated with palmitate in the presence of TAK-242, a TLR4 signaling inhibitor. Palmitate-induced overgeneration of ROS in endothelial cells was abolished in the presence of TAK-242. Our data show that a Western diet induced endothelial dysfunction in female rats and suggest that endothelial TLR4 signaling may play a key role in abolishing female cardiovascular protection. NEW & NOTEWORTHY A Western diet induced elevated levels of free fatty acids, produced nonalcoholic fatty liver disease, and provoked endothelial dysfunction in female rats in association with Toll-like receptor 4 signaling-mediated vascular reactive oxygen species production. Limited consumption of a Western diet in premenopausal women may decrease their risk of cardiovascular complications.

Methylene blue alleviates endothelial dysfunction and reduces oxidative stress in aortas from diabetic rats.

Endothelial dysfunction and the related increase in reactive oxygen species (ROS) production are important events in the pathophysiology of diabetes mellitus (DM). Methylene blue (MB) has been systematically investigated for its protective effects against refractory hypotension and mitochondrial dysfunction. We have previously demonstrated that MB improved mitochondrial respiration and partially decreased oxidative stress in diabetic rat hearts. The present study was aimed to investigate whether MB modulates vascular function and ROS production in thoracic aortic rings isolated from rats with streptozotocin-induced DM (after 4 weeks of hyperglycemia). The effects of MB (0.1 μM, 30 min ex vivo incubation) on vascular reactivity in organ chamber (phenylephrine-induced contraction, acetylcholine-induced relaxation) and H2O2 production (assessed by ferrous iron xylenol orange oxidation assay) were investigated in vascular preparations with intact endothelium and after denudation. DM elicited a significant alteration of vascular function: increased contractility to phenylephrine, attenuation of acetylcholine-dependent relaxation, and augmented H2O2 generation. Ex vivo incubation with MB partially reversed all these changes (by approximately 70%) in vascular segments with intact endothelial layer (but not in denuded vessels). In conclusion, MB might be useful in alleviating endothelial dysfunction and mitigating endothelial oxidative stress, observations that clearly require further investigation in the setting of cardiometabolic disease.

SET8 is involved in the regulation of hyperglycemic memory in human umbilical endothelial cells.

Hyperglycemic memory occurs in diabetic cardiovascular complications, but the underlying mechanism remains to be elucidated. Although the depletion of SET8 leads to increased mitochondrial oxidative stress via increasing cellular reactive oxygen species (ROS) production, the role of SET8 in hyperglycemic memory-induced mitochondrial dysfunction is not well understood. Here, we investigated the role of SET8 in this setting. Our results showed that high glucose-induced vascular inflammation, ROS production and apoptosis remained at high levels even when glucose returned to normal level. Elevated glucose reduced SET8 expression, which also remained at low level after returning to normoglycemia. SET8 overexpression protected cells from elevated glucose and hyperglycemic memory-induced endothelial injury by blocking ROS accumulation, attenuating vascular inflammation, and restoring nitric oxide production. Thus, our results suggest that SET8 may be a key mediator in hyperglycemic memory.

Epigenetic regulation of vascular NADPH oxidase expression and reactive oxygen species production by histone deacetylase-dependent mechanisms in experimental diabetes

Reactive oxygen species (ROS) generated by up-regulated NADPH oxidase (Nox) contribute to structural-functional alterations of the vascular wall in diabetes. Epigenetic mechanisms, such as histone acetylation, emerged as important regulators of gene expression in cardiovascular disorders. Since their role in diabetes is still elusive we hypothesized that histone deacetylase (HDAC)-dependent mechanisms could mediate vascular Nox overexpression in diabetic conditions. Non-diabetic and streptozotocin-induced diabetic C57BL/6J mice were randomized to receive vehicle or suberoylanilide hydroxamic acid (SAHA), a pan-HDAC inhibitor. In vitro studies were performed on a human aortic smooth muscle cell (SMC) line. Aortic SMCs typically express Nox1, Nox4, and Nox5 subtypes. HDAC1 and HDAC2 proteins along with Nox1, Nox2, and Nox4 levels were found significantly elevated in the aortas of diabetic mice compared to non-diabetic animals. Treatment of diabetic mice with SAHA mitigated the aortic expression of Nox1, Nox2, and Nox4 subtypes and NADPH-stimulated ROS production. High concentrations of glucose increased HDAC1 and HDAC2 protein levels in cultured SMCs. SAHA significantly reduced the high glucose-induced Nox1/4/5 expression, ROS production, and the formation malondialdehyde-protein adducts in SMCs. Overexpression of HDAC2 up-regulated the Nox1/4/5 gene promoter activities in SMCs. Physical interactions of HDAC1/2 and p300 proteins with Nox1/4/5 promoters were detected at the sites of active transcription. High glucose induced histone H3K27 acetylation enrichment at the promoters of Nox1/4/5 genes in SMCs. The novel data of this study indicate that HDACs mediate vascular Nox up-regulation in diabetes. HDAC inhibition reduces vascular ROS production in experimental diabetes, possibly by a mechanism involving negative regulation of Nox expression.

Absence of Nicotinamide Nucleotide Transhydrogenase in C57BL/6J Mice Exacerbates Experimental Atherosclerosis

Background: Mitochondrial reactive oxygen species (ROS) contribute to inflammation and vascular remodeling during atherosclerotic plaque formation. C57BL/6N (6N) and C57BL/6J (6J) mice display distinct mitochondrial redox balance due to the absence of nicotinamide nucleotide transhydrogenase (NNT) in 6J mice. We hypothesize that differential NNT expression between these animals alters plaque development. Methods: 6N and 6J mice were treated with AAV8-PCSK9 (adeno-associated virus serotype 8/proprotein convertase subtilisin/kexin type 9) virus leading to hypercholesterolemia, increased low-density lipoprotein, and atherosclerosis in mice fed a high-fat diet (HFD). Mice were co-treated with the mitochondria-targeted superoxide dismutase mimetic MitoTEMPO to assess the contribution of mitochondrial ROS to atherosclerosis. Results: Baseline and HFD-induced vascular superoxide is increased in 6J compared to 6N mice. MitoTEMPO diminished superoxide in both groups demonstrating differential production of mitochondrial ROS among these strains. PCSK9 treatment and HFD led to similar increases in plasma lipids in both 6N and 6J mice. However, 6J animals displayed significantly higher levels of plaque formation. MitoTEMPO reduced plasma lipids but did not affect plaque formation in 6N mice. In contrast, MitoTEMPO surprisingly increased plaque formation in 6J mice. Conclusion: These data indicate that loss of NNT increases vascular ROS production and exacerbates atherosclerotic plaque development.

Aluminum exposure at human dietary levels promotes vascular dysfunction and increases blood pressure in rats: A concerted action of NAD(P)H oxidase and COX-2.

Aluminum (Al) is a non-essential metal and a significant environmental contaminant and is associated with a number of human diseases including cardiovascular disease. We investigated the effects of Al exposure at doses similar to human dietary levels on the cardiovascular system over a 60day period. Wistar male rats were divided into two major groups and received orally: 1) Low aluminum level - rats were subdivided and treated for 60days as follows: a) Untreated - ultrapure water; b) AlCl3 at a dose of 8.3mg/kg bw for 60days, representing human Al exposure by diet; and 2) High aluminum level - rats were subdivided and treated for 42days as follows: C) Untreated - ultrapure water; d) AlCl3 at 100mg/kg bw for 42days, representing a high level of human exposure to Al. Effects on systolic blood pressure (SBP) and vascular function of aortic and mesenteric resistance arteries (MRA) were studied. Endothelium and smooth muscle integrity were evaluated by concentration-response curves to acetylcholine (ACh) and sodium nitroprusside. Vasoconstrictor responses to phenylephrine (Phe) in the presence and absence of endothelium and in the presence of the NOS inhibitor L-NAME, the potassium channels blocker TEA, the NAD(P)H oxidase inhibitor apocynin, superoxide dismutase (SOD), the non-selective COX inhibitor indomethacin and the selective COX-2 inhibitor NS 398 were analyzed. Vascular reactive oxygen species (ROS), lipid peroxidation and total antioxidant capacity, were measured. The mRNA expressions of eNOS, NAD(P)H oxidase 1 and 2, SOD1, COX-2 and thromboxane A2 receptor (TXA-2 R) were also investigated. Al exposure at human dietary levels impaired the cardiovascular system and these effects were almost the same as Al exposure at much higher levels. Al increased SBP, decreased ACh-induced relaxation, increased response to Phe, decreased endothelial modulation of vasoconstrictor responses, the bioavailability of nitric oxide (NO), the involvement of potassium channels on vascular responses, as well as increased ROS production from NAD(P)H oxidase and contractile prostanoids mainly from COX-2 in both aorta and mesenteric arteries. Al exposure increased vascular ROS production and lipid peroxidation as well as altered the antioxidant status in aorta and MRA. Al decreased vascular eNOS and SOD1 mRNA levels and increased the NAD(P)H oxidase 1, COX-2 and TXA-2 R mRNA levels. Our results point to an excess of ROS mainly from NAD(P)H oxidase after Al exposure and the increased vascular prostanoids from COX-2 acting in concert to decrease NO bioavailability, thus inducing vascular dysfunction and increasing blood pressure. Therefore, 60-day chronic exposure to Al, which reflects common human dietary Al intake, appears to pose a risk for the cardiovascular system.

Enhanced Mitochondrial Transient Receptor Potential Channel, Canonical Type 3-Mediated Calcium Handling in the Vasculature From Hypertensive Rats.

BackgroundMitochondrial Ca2+ homeostasis is fundamental to the regulation of mitochondrial reactive oxygen species (ROS) generation and adenosine triphosphate production. Recently, transient receptor potential channel, canonical type 3 (TRPC3), has been shown to localize to the mitochondria and to play a role in maintaining mitochondrial calcium homeostasis. Inhibition of TRPC3 attenuates vascular calcium influx in spontaneously hypertensive rats (SHRs). However, it remains elusive whether mitochondrial TRPC3 participates in hypertension by increasing mitochondrial calcium handling and ROS production.Methods and ResultsIn this study we demonstrated increased TRPC3 expression in purified mitochondria in the vasculature from SHRs, which facilitates enhanced mitochondrial calcium uptake and ROS generation compared with Wistar‐Kyoto rats. Furthermore, inhibition of TRPC3 by its specific inhibitor, Pyr3, significantly decreased the vascular mitochondrial ROS production and H2O2 synthesis and increased adenosine triphosphate content. Administration of telmisartan can improve these abnormalities. This beneficial effect was associated with improvement of the mitochondrial respiratory function through recovering the activity of pyruvate dehydrogenase in the vasculature of SHRs. In vivo, chronic administration of telmisartan suppressed TRPC3‐mediated excessive mitochondrial ROS generation and vasoconstriction in the vasculature of SHRs. More importantly, TRPC3 knockout mice exhibited significantly ameliorated hypertension through reduction of angiotensin II–induced mitochondrial ROS generation.ConclusionsTogether, we give experimental evidence for a potential mechanism by which enhanced TRPC3 activity at the cytoplasmic and mitochondrial levels contributes to redox signaling and calcium dysregulation in the vasculature from SHRs. Angiotensin II or telmisartan can regulate [Ca2+]mito, ROS production, and mitochondrial energy metabolism through targeting TRPC3.

Abstract 591: Absence of Nicotinamide Nucleotide Transhydrogenase Exacerbates Atherosclerosis in Mice

Oxidant stress contributes to endothelial cell injury and inflammation that are hallmarks of early stage atherosclerosis. Emerging evidence implicates mitochondrial reactive oxygen species (ROS) as important contributors to this oxidant stress and differences in mitochondrial function may augment this process. We have shown that variation in mitochondrial function and ROS production associated with ethnicity contributes to endothelial and vascular dysfunction. To model these distinct mitochondrial redox phenotypes we used C57Bl/6N (6N) and C57Bl/6J (6J) mice that also display unique mitochondrial functional properties due to the differential expression nicotinamide nucleotide transhydrogenase (NNT). Mice were treated with adeno-associated virus encoding a gain-of-function form of proprotein convertase subtilisin/kexin type 9 (PCSK9) that leads to hypercholesterolemia, increased LDL levels, and atherosclerosis in mice. PCSK9 treatment and 8 weeks of high fat diet led to increases in plasma lipids in both 6N and 6J mice. However, 6J animals displayed significantly higher levels of fat deposition in the vasculature and increased plaque size in the carotid sinus. 6N mice co-treated with the mitochondria targeted superoxide dismutase mimetic MitoTEMPO for the final 4 weeks of the experiment displayed reduced plasma lipids, but no impact on fat deposition or plaque size was observed. In contrast, MitoTEMPO increased vascular fat deposition and plaque size in 6J mice consistent with a more severe atherosclerotic phenotype. Increased mitochondrial ROS was confirmed by demonstrating elevated vascular superoxide in 6J versus 6N animals and that this difference is exacerbated on high fat diet. MitoTEMPO diminished vascular superoxide production to near baseline levels in both groups, yet increased plaque size and fat deposition in 6J mice suggests a role for hydrogen peroxide in this process. These data indicate that loss of NNT and changes in mitochondrial function increase vascular ROS production and exacerbate lipid deposition and plaque development in the early stages of atherosclerosis.

Activation of Peroxisome Proliferator Activator Receptor β/δ Improves Endothelial Dysfunction and Protects Kidney in Murine Lupus.

Women with systemic lupus erythematosus exhibit a high prevalence of hypertension, endothelial dysfunction, and renal injury. We tested whether GW0742, a peroxisome proliferator activator receptor β/δ (PPARβ/δ) agonist, ameliorates disease activity and cardiovascular complications in a female mouse model of lupus. Thirty-week-old NZBWF1 (lupus) and NZW/LacJ (control) mice were treated with GW0742 or with the PPARβ/δ antagonist GSK0660 plus GW0742 for 5 weeks. Blood pressure, plasma double-stranded DNA autoantibodies and cytokines, nephritis, hepatic opsonins, spleen lymphocyte populations, endothelial function, and vascular oxidative stress were compared in treated and untreated mice. GW0742 treatment reduced lupus disease activity, blood pressure, cardiac and renal hypertrophy, splenomegaly, albuminuria, and renal injury in lupus mice, but not in control. GW0742 increased hepatic opsonins mRNA levels in lupus mice and reduced the elevated T, B, Treg, and Th1 cells in spleens from lupus mice. GW0742 lowered the higher plasma concentration of proinflammatory cytokines observed in lupus mice. Aortae from lupus mice showed reduced endothelium-dependent vasodilator responses to acetylcholine and increased nicotinamide adenine dinucleotide phosphate oxidase-driven vascular reactive oxygen species production, which were normalized by GW0742 treatment. All these effects of GW0742 were inhibited by PPARβ/δ blockade with GSK0660. Pharmacological activation of PPARβ/δ reduced hypertension, endothelial dysfunction, and organ damage in severe lupus mice, which was associated with reduced plasma antidouble-stranded DNA autoantibodies and anti-inflammatory and antioxidant effects in target tissues. Our findings identify PPARβ/δ as a promising target for an alternative approach in the treatment of systemic lupus erythematosus and its associated vascular damage.

CD14+CD16++ "nonclassical" monocytes are associated with endothelial dysfunction in patients with coronary artery disease.

Endothelial dysfunction and inflammation are key mechanisms of vascular disease. We hypothesised that heterogeneity of monocyte subpopulations may be related to the development of vascular dysfunction in coronary artery disease (CAD). Therefore, we examined the relationships between monocyte subsets (CD14++CD16- "classical - Mon1", CD14++CD16+ "intermediate - Mon2" and CD14+CD16++ "nonclassical - Mon3"), endothelial function and risk factor profiles in 130 patients with CAD undergoing coronary artery bypass grafting. This allowed for direct nitric oxide (NO) bioavailability assessment using isometric tension studies ex vivo (acetylcholine; ACh- and sodium-nitropruside; SNP-dependent) in segments of internal mammary arteries. The expression of CD14 and CD16 antigens and activation markers were determined in peripheral blood mononuclear cells using flow cytometry. Patients with high CD14+CD16++ "nonclassical" and low CD14++CD16- "classical" monocytes presented impaired endothelial function. High frequency of CD14+CD16++ "nonclassical" monocytes was associated with increased vascular superoxide production. Furthermore, endothelial dysfunction was associated with higher expression of activation marker CD11c selectively on CD14+CD16++ monocytes. Nonclassical and classical monocyte frequencies remained independent predictors of endothelial dysfunction when major risk factors for atherosclerosis were taken into account (β=0.18 p=0.04 and β=-0.19 p=0.03, respectively). In summary, our data indicate that CD14+CD16++ "nonclassical" monocytes are associated with more advanced vascular dysfunction measured as NO- bioavailability and vascular reactive oxygen species production.

Ellagic Acid Reduces High Glucose-Induced Vascular Oxidative Stress Through ERK1/2/NOX4 Signaling Pathway

Background/Aims: Elevated production of reactive oxygen species (ROS) is linked to endothelial dysfunction and is one of the key contributors to the pathogenesis of diabetic vascular complications. Emerging evidence has indicated that ellagic acid (EA), a polyphenol found in fruits and nuts, possesses numerous biological activities including radical scavenging. However, whether EA exerts a vasculo-protective effect via antioxidant mechanisms in blood vessels exposed to diabetic conditions remains unknown. Accordingly, the goal of this current study was to determine whether EA decreases vascular ROS production and thus ameliorates endothelial dysfunction in the diabetic milieu. Methods: Intact rat aortas and human aortic endothelial cells (HAEC) were stimulated with 30mM high glucose (HG) with and without EA co-treatment. Endothelium-dependent vasodilation was measured using a wire myograph. Gene and protein expression of non-phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 4 (NOX4) were detected using RT-PCR and western blotting, respectively. Oxidative stress was determined by measuring ROS levels using dihydroethidium (DHE) staining. Results: Intact aortas exposed to HG condition displayed exacerbated ROS production and impairment of endothelium-dependent vasodilation, characterizing endothelial dysfunction. These effects were markedly reduced with EA treatment. HG enhanced ROS production in HAEC, paralleled by increased ERK1/2 activation and NOX4 expression. EA treatment blunted the increase of ROS generation, ERK1/2 activation and decreased NOX4. Conclusions: EA significantly decreases endothelial ROS levels and ameliorates the impairment of vascular relaxation induced by HG. Our results suggest that EA exerts a vasculo-protective effect under diabetic conditions via an antioxidant effect that involves inhibition of ERK1/2 and downregulation of NOX4.

Aluminum exposure for one hour decreases vascular reactivity in conductance and resistance arteries in rats

AimsAluminum (Al) is an important environmental contaminant; however, there are not enough evidences of Al-induced cardiovascular dysfunction. We investigated the effects of acute exposure to aluminum chloride (AlCl3) on blood pressure, vascular reactivity and oxidative stress. Methods and resultsMale Wistar rats were divided into two groups: Untreated: vehicle (ultrapure water, ip) and AlCl3: single dose of AlCl3 (100mg/kg,ip). Concentration-response curves to phenylephrine in the absence and presence of endothelium, the nitric oxide synthase inhibitor l-NAME, the potassium channel blocker tetraethylammonium, and the NADPH oxidase inhibitor apocynin were performed in segments from aortic and mesenteric resistance arteries. NO released was assessed in aorta and reactive oxygen species (ROS), malondialdehyde, non-protein thiol levels, antioxidant capacity and enzymatic antioxidant activities were investigated in plasma, aorta and/or mesenteric arteries. After one hour of AlCl3 exposure serum Al levels attained 147.7±25.0μg/L. Al treatment: 1) did not affect blood pressure, heart rate and vasodilator responses induced by acetylcholine or sodium nitroprusside; 2) decreased phenylephrine-induced vasoconstrictor responses; 3) increased endothelial modulation of contractile responses, NO release and vascular ROS production from NADPH oxidase; 4) increased plasmatic, aortic and mesenteric malondialdehyde and ROS production, and 5) decreased antioxidant capacity and affected the antioxidant biomarkers non-protein thiol levels, glutathione peroxidase, glutathione-S-transferase, superoxide dismutase and catalase enzymatic activities. ConclusionAlCl3-acute exposure reduces vascular reactivity. This effect is associated with increased NO production, probably acting on K+ channels, which seems to occur as a compensatory mechanism against Al-induced oxidative stress. Our results suggest that Al exerts toxic effects to the vascular system.

PP.LB02.10] 60-DAY ALCL3 EXPOSURE AT HUMAN DIETARY LEVELS INCREASES VASCULAR REACTIVITY IN CONDUCTANCE AND RESISTANCE ARTERIES IN RATS

Objective: To investigate the effects of a 60-day aluminum (Al) exposure at doses similar to human dietary levels on cardiovascular system. Design and method: 20 three-month-old male Wistar rats ( ± 300 g) were divided into two groups and received for 60 days in drinking water: a) Control - ultrapure water; b) AlCl3 - aluminum chloride at a dose of 8.3 mg/kg bw. Systolic blood pressure (SBP) was measured by plethysmography. Vascular function was studied in aortic and mesenteric resistance arteries (MRA) in isolated organ bath (J Pharmacol Exp Ther 321: 381–388, 2007 and Circ Res 41:19–26, 1977). Concentration-response curves to acetylcholine (ACh) and sodium nitroprusside were performed. Vasoconstrictor response to phenylephrine (PHE) in presence and absence of endothelium and in presence of NOS inhibitor (L-NAME), potassium channels blocker (TEA), NAD (P)H oxidase inhibitor (apocynin), superoxide dismutase (SOD), non-selective COX inhibitor (indomethacin), selective COX-2 inhibitor (NS 398), and AT1 selective receptor blocker (losartan), were analyzed. Systemic and vascular reactive oxygen species (ROS), lipid peroxidation and total antioxidant capacity, were measured. Results were expressed as mean and SEM, compared by t–test and ANOVA followed by Bonferroni test (*P < 0.05). Ethics Committee Approval 028/2014 - Unipampa. Results: AlCl3-exposure at low doses increased SBP (Ct: 120.3 ± 1.17 vs Al:126.9 ± 1*mmHg, n = 8), decreased ACh induced concentration-dependent relaxation, increased vasoconstrictor response to PHE (MRA - Rmax % to KCl Ct: 111.7 ± 5.4 % vs AlCl3: 126.1 ± 11.4 %*, Aorta - Ct: 89.7 ± 17.4 % vs AlCl3: 111 ± 22.4 %* n = 10), decreased the endothelium vasoconstrictor – modulation, nitric oxide (NO) bioavailability, potassium channels involvement, as well as increased ROS production from NAD (P)H oxidase and contractile prostanoids mainly from COX-2. Al exposure at low levels increased plasmatic and vascular ROS production and lipid peroxidation as well as altered the antioxidant status in plasma, aorta and MRA. Conclusions: Our results demonstrate that 60-day chronic exposure to low doses of AlCl3, which reflect common human dietary Al intake induces endothelial dysfunction probably as a result of the decreased NO bioavailability induced by increases on oxidative stress.

LBPS 01-03 ENDOTHELIN-1 OVEREXPRESSION EXAGGERATES DIABETES-INDUCED ENDOTHELIAL DYSFUNCTION BY ALTERING OXIDATIVE STRESS BALANCE

Objective: Increased endothelin (ET)-1 expression has been shown to cause endothelial dysfunction and oxidative stress. Plasma ET-1 is increased in patients with diabetes mellitus. Since endothelial dysfunction often precedes vascular complications in diabetes, we sought to determine whether ET-1 contributes to diabetes-induced endothelial dysfunction. We hypothesized that overexpression of ET-1 in the endothelium will exaggerate diabetes-induced endothelial dysfunction. Design and Method: Diabetes was induced by streptozotocin treatment (STZ, 55 mg/kg/day, ip) for 5 days in 6 week-old male wild-type (WT) mice and in mice overexpressing human ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Endothelial function and vascular remodeling using pressurized myography, reactive oxygen species (ROS) production by dihydroethidium staining and mRNA expression by reverse transcription-quantitative PCR were assessed in mesenteric arteries (MA). Results: MA endothelium-dependent vasodilatory responses to acetylcholine were reduced 24% by diabetes in WT, and further decreased by 12% in eET-1. Diabetes decreased MA media/lumen in WT and eET-1 whereas ET-1 overexpression increased MA media/lumen to a similar extent in diabetic and non-diabetic WT mice. Vascular ROS production in MA was increased 2-fold by diabetes in WT and further augmented 1.7-fold in eET-1. Diabetes reduced endothelial nitric oxide synthase (eNOS, Nos3) mRNA expression in eET-1 by 31% but not in WT. Induction of diabetes caused a 52% increase in superoxide dismutase 1 (Sod1) and a 32% increase in Sod2 (32%) mRNA expression in WT but not in eET-1. Conclusions: Increased expression of ET-1 exaggerates diabetes-induced endothelial dysfunction. This may be caused by a decrease in eNOS expression, an increase in vascular oxidative stress and a decrease in antioxidant capacity.

Abstract P226: Endothelin-1 Overexpression Exaggerates Diabetes-induced Endothelial Dysfunction by Altering Oxidative Stress

Objective: Increased endothelin (ET)-1 expression has been shown to cause endothelial dysfunction and oxidative stress. Plasma ET-1 is increased in patients with diabetes mellitus. Since endothelial dysfunction often precedes vascular complications in diabetes, we sought to determine whether ET-1 contributes to diabetes-induced endothelial dysfunction. We hypothesized that overexpression of ET-1 in the endothelium will exaggerate diabetes-induced endothelial dysfunction. Methods: Diabetes was induced by streptozotocin IP injections (STZ, 55 mg/kg/day) for 5 days in 6-week-old male wild-type (WT) mice and in mice overexpressing human ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Endothelial function and vascular remodeling using pressurized myography, reactive oxygen species (ROS) production by dihydroethidium staining and mRNA expression by reverse transcription-quantitative PCR were assessed in mesenteric arteries (MA). Results: MA endothelium-dependent vasodilatory responses to acetylcholine were reduced 24% by diabetes in WT (E max : 61±6 vs 84±3%, P &lt;0.05), and further decreased by 12% in eET-1 (E max : 49±5, P &lt;0.05). Diabetes decreased MA media/lumen in WT (2.4±0.1% vs 3.3±0.2%, P &lt;0.05) and eET-1 (2.9±0.2% vs 4.0±0.2%, P &lt;0.05), whereas ET-1 overexpression increased MA media/lumen to a similar extent in diabetic and non-diabetic WT mice ( P &lt;0.05). Vascular ROS production in MA was increased 2-fold by diabetes in WT (5.0±0.5 vs 2.5±0.3 relative fluorescence units [RFU]/μm 2 , P &lt;0.05) and further augmented 1.7-fold in eET-1 (8.5±1.2 RFU/μm 2 , P &lt;0.05). Diabetes reduced endothelial nitric oxide synthase (eNOS, Nos3) mRNA expression in eET-1 by 50% (0.7±0.1 vs 1.4±0.2, P &lt;0.05) but not in WT. Induction of diabetes caused a 50% increase in superoxide dismutase 1 ( Sod1 , 1.5±0.2 vs 1.0±0.0, P &lt;0.05) and a 30% increase in Sod2 (1.3±0.1 vs 1.0±0.0, P &lt;0.05) mRNA expression in WT but not in eET-1. Conclusions: Increased expression of ET-1 exaggerates diabetes-induced endothelial dysfunction. This may be caused by an increase in vascular oxidative stress, a decrease in eNOS expression and a decrease in antioxidant capacity.