Endothelial Reactive Oxygen Species Production Research Articles

Methylglyoxal induces endothelial cell apoptosis and coronary microvascular dysfunction through regulating AR-cPLA2 signaling

ObjectiveSince diabetic patients with coronary microvascular dysfunction (CMD) exhibit high cardiac mortality and women have higher prevalence of non-obstructive coronary artery disease than men, we tried to expand the limited understanding about the etiology and the sex difference of diabetic CMD. Approach and resultsAccumulated methylglyoxal (MGO) due to diabetes promotes vascular damage and it was used for mimicking diabetic status. Flow cytometry analysis and isometric tension measurement were performed to evaluate coronary artery endothelial injury. MGO induced apoptosis of coronary endothelial cells, accompanied by downregulation of androgen receptor (AR). Lentivirus-mediated stable expression of AR in coronary endothelial cells increased anti-apoptotic Bcl-2 expression and attenuated MGO-induced cell apoptosis. cPLA2 activation was the downstream of AR downregulation by MGO treatment. Moreover, MGO also activated cPLA2 rapidly to impair endothelium-dependent vasodilation of coronary arteries from mice. Reactive oxygen species (ROS) overproduction was demonstrated to account for MGO-mediated cPLA2 activation and endothelial dysfunction. Importantly, AR blockade increased endothelial ROS production whereas AR activation protected coronary artery endothelial vasodilatory function from the MGO-induced injury. Although galectin-3 upregulation was confirmed by siRNA knockdown in endothelial cells not to participate in MGO-induced endothelial apoptosis, pharmacological inhibitor of galectin-3 further enhanced MGO-triggered ROS generation and coronary artery endothelial impairment. ConclusionsOur data proposed the AR downregulation-ROS overproduction-cPLA2 activation pathway as one of the mechanisms underlying diabetic CMD and postulated a possible reason for the sex difference of CMD-related angina. Meanwhile, MGO-induced galectin-3 activation played a compensatory role against coronary endothelial dysfunction.

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).

Glutamine Counteracts Glucose‐Mediated Human Endothelial Cell Dysfunction

Vascular disease is the major cause of morbidity and mortality in diabetes mellitus. While the etiology of vascular disease is multifactorial, endothelial cell (EC) dysfunction is a hallmark of diabetes‐associated vascular disease. Substantial evidence indicates that high glucose (HG) concentrations trigger EC dysfunction through the production of reactive oxygen species (ROS). Recently, studies from our laboratory and others identified the conditionally essential amino acid glutamine as a critical regulator of oxidative stress and EC phenotype. In the present study, we investigated the effect of HG concentrations on ECs and determined if glutamine influences the biological actions of HG in these cells. Treatment of human umbilical vein ECs with glucose (5.6‐25mM) resulted in a concentration‐ and time‐dependent increase in the production of reactive oxygen species (ROS), whereas mannitol (an osmotic control) minimally affected ROS formation. HG concentrations (25mM) also evoked a time‐dependent decrease in EC viability that was paralleled by an increase in apoptosis, as reflected by a rise in annexin V binding and caspase‐3 activity. Notably, the administration of glutamine (500μM) inhibits HG‐mediated endothelial ROS production and improves EC viability in response to HG levels. Glutamine also induces the expression of heme oxygenase‐1 (HO‐1) in ECs. However, pharmacological inhibition of HO‐1 activity with tin protoporphyrin‐IX (10μM) reversed the antioxidant and cytoprotective effect of glutamine in HG‐treated ECs. In conclusion, the present study found that glutamine counteracts HG concentration‐mediated ROS generation and EC death. Moreover, it demonstrates that the induction of HO‐1 by glutamine contributes to the beneficial actions of the amino acid and identifies glutamine as a critical regulator of EC survival in diabetes.

PFKFB3 Mediated Changes in ROS/NO Balance Contribute to Endothelial Dysfunction in Obesity

Obesity is a strong risk factor for cardiovascular disease, in large part due to an altered metabolic state. One of the earliest consequences of cardiovascular dysfunction in obesity is the loss of endothelial function and impaired nitric oxide (NO) signaling. In blood vessels, NO is synthesized by endothelial nitric oxide synthase (eNOS). The biological actions of NO can be compromised by rapid inactivation by reactive oxygen species (ROS) such as superoxide that is mainly generated by NADPH oxidases (NOXs). However, the signals coordinating the increased superoxide production leading to loss of NO with altered metabolism remain unknown.We have found that the expression of 6‐phosphofructo‐2‐kinase/fructose‐2, 6‐biphosphatase 3 (PFKFB3), which is a key glycolytic regulatory enzyme, is significantly increased in endothelial cells of obese animals. Overexpression of PFKFB3 within the endothelium of blood vessels is accompanied by the loss of vascular relaxation and in decreased NO production as a result of increased eNOS phosphorylation on the inhibitory T495 residue. PFKFB3 overexpression also blunted Akt‐S473 phosphorylation, reducing stimulus‐dependent phosphorylation and activation of eNOS at S1177. While PFKFB3 exerted negative effects on NO signaling, it increased the activity and mRNA levels of NOX1, a major contributor to endothelial dysfunction. Further, we have also found that pharmacological inhibition of NOX1 attenuates the decreased vasodilator responses in PFKFB3 overexpressing aortic rings.These results demonstrate a novel functional relationship between endothelial metabolism, ROS and NO production that may contribute to endothelial dysfunction in obesity.

Galectin‐3 Regulates Oxidant Stress in Obesity

ObjectiveInvestigate the mechanism by which overexpression of Galectin‐3 (GAL3) in the endothelium of obese mice contributes to excess reactive oxygen species (ROS) production and subsequent vascular dysfunction.Methodsdb/db mice, a well‐characterized model of human obesity, were crossed with mice lacking GAL3, a receptor for advanced glycation end products, to generate lean and obese mice with and without GAL3 deletion. Metabolic and cardiovascular endpoints were assessed by mRNA and protein expression in freshly isolated microvascular endothelial cells, microvascular reactivity assessed by pressure myography, and ROS production assessed by dihydroethidium (DHE) staining. Other previously characterized db/db colonies were utilized to further elucidate the mechanistic contributions of GAL3, including mice harboring deletions of NADPH Oxidase I (NOX1), Myostatin (MSTN), and Protein Tyrosine Phosphatase 1B (PTP1B).ResultsDeletion of GAL3 in obese mice had no observable effect on anatomical, metabolic, or plasma phenotype, except for normalizing levels of plasma ROS as evidenced by decrease in thiobarbituric acid reactive substances (TBARS). Obese mice exhibited profound endothelial dysfunction, which was rescued by deletion of GAL3. Our data demonstrate that NOX1, the predominant source of endothelial superoxide production, is significantly downregulated by GAL3 deletion, thereby restoring endothelial function and ameliorating endothelial ROS production in obesity. Furthermore, we demonstrate that GAL3‐mediated NOX1 overexpression is amenable to improvements in metabolic status, such as lowering blood glucose with metformin, improving glucose handling by augmenting muscle mass through deletion of MSTN, or improving insulin signaling through deletion of PTP1B.ConclusionTaken together, these data demonstrate that the overproduction of superoxide by endothelial NOX1 is regulated by the metabolic sensor GAL3 in obesity, leading to endothelial dysfunction and subsequent cardiovascular disease. This pathway presents an attractive target for therapeutic intervention to break the link between aberrant metabolism in obesity and its corresponding vascular pathologies.

Butyrate protects endothelial function through PPARδ/miR-181b signaling

Reports of the beneficial roles of butyrate in cardiovascular diseases, such as atherosclerosis and ischemic stroke, are becoming increasingly abundant. However, the mechanisms of its bioactivities remain largely unknown. In this study, we explored the effects of butyrate on endothelial dysfunction and its potential underlying mechanism. In our study, ApoE-/- mice were fed with high-fat diet (HFD) for ten weeks to produce atherosclerosis models and concurrently treated with or without sodium butyrate daily. Thoracic aortas were subsequently isolated from C57BL/6 wild-type (WT), PPARδ-/-, endothelial-specific PPARδ wild-type (EC-specific PPARδ WT) and endothelial-specific PPARδ knockout (EC-specific PPARδ KO) mice were stimulated with interleukin (IL)-1β with or without butyrate ex vivo. Our results demonstrated that butyrate treatment rescued the impaired endothelium-dependent relaxations (EDRs) in thoracic aortas of HFD-fed ApoE-/- mice. Butyrate also rescued impaired EDRs in IL-1β-treated thoracic aorta ring ex vivo. Global and endothelial-specific knockout of PPARδ eliminated the protective effects of butyrate against IL-1β-induced impairment to EDRs. Butyrate abolished IL-1β-induced reactive oxygen species (ROS) production in endothelial cells while the inhibitory effect was incapacitated by genetic deletion of PPARδ or pharmacological inhibition of PPARδ. IL-1β increased NADPH oxidase 2 (NOX2) mRNA and protein expressions in endothelial cells, which were prevented by butyrate treatment, and the effects of butyrate were blunted following pharmacological inhibition of PPARδ. Importantly, butyrate treatment upregulated the miR-181b expression in atherosclerotic aortas and IL-1β-treated endothelial cells. Moreover, transfection of endothelial cells with miR-181b inhibitor abolished the suppressive effects of butyrate on NOX2 expressions and ROS generation in endothelial cells. To conclude, butyrate prevents endothelial dysfunction in atherosclerosis by reducing endothelial NOX2 expression and ROS production via the PPARδ/miR-181b pathway.

Nitric Oxide Donors as Potential Drugs for the Treatment of Vascular Diseases Due to Endothelium Dysfunction.

Endothelial dysfunction and consequent vasoconstriction are a common condition in patients with hypertension and other cardiovascular diseases. Endothelial cells produce and release vasodilator substances that play a pivotal role in normal vascular tone. The mechanisms underlying endothelial dysfunction are multifactorial. However, enhanced reactive oxygen species (ROS) production and consequent vasoconstriction instead of endothelium-derived relaxant generation and consequent vasodilatation contribute to this dysfunction considerably. The main targets of the drugs that are currently used to treat vascular diseases concerning enzyme activities and protein functions that are impaired by endothelial nitric oxide synthase (eNOS) uncoupling and ROS production. Nitric oxide (NO) bioavailability can decrease due to deficient NO production by eNOS and/or NO release to vascular smooth muscle cells, which impairs endothelial function. Considering the NO cellular mechanisms, tackling the issue of eNOS uncoupling could avoid endothelial dysfunction: provision of the enzyme cofactor tetrahydrobiopterin (BH4) should elicit NO release from NO donors, to activate soluble guanylyl cyclase. This should increase cyclic guanosine-monophosphate (cGMP) generation and inhibit phosphodiesterases (especially PDE5) that selectively degrade cGMP. Consequently, protein kinase-G should be activated, and K+ channels should be phosphorylated and activated, which is crucial for cell membrane hyperpolarization and vasodilation and/or inhibition of ROS production. The present review summarizes the current concepts about the vascular cellular mechanisms that underlie endothelial dysfunction and which could be the target of drugs for the treatment of patients with cardiovascular disease.

Sex-specific alterations in blood-borne factors in physically inactive individuals are detrimental to endothelial cell functions.

Mechanisms underlying the protective effects of both habitual endurance exercise and the female sex on vascular function are incompletely understood. Blood-borne circulating factors, such as circulating microRNAs (ci-miRs), may partially explain these effects. Blood samples were obtained from young, healthy men and women who either habitually performed endurance exercise (endurance trained) or were relatively inactive (sedentary). Women were tested during the early follicular phase of the menstrual cycle or the placebo pill phase of oral contraceptive to control for estrogen. Cultured human umbilical vein endothelial cells (HUVECs) were exposed to participants' serum in migration, proliferation, and reactive oxygen species (ROS) assays. Real-time quantitative polymerase chain reaction was used to quantify an initial array of 84 cardiovascular disease (CVD)-related ci-miRs, followed by validation of 10 ci-miRs. All participants were devoid of traditional CVD risk factors, and circulating estradiol concentration was not different between groups. Serum of endurance-trained women induced greater HUVEC migration compared with serum of sedentary women. HUVEC ROS production was greater in response to serum of sedentary men compared with serum of endurance-trained men and sedentary women. There were sex effects on the levels of nine ci-miRs, with greater levels in men, while ci-miRs-140-5p and 145-5p were also higher in sedentary compared with endurance-trained men and/or women. In a sex-specific manner, habitual endurance exercise was associated with beneficial effects of serum on HUVECs. Thus, alterations in circulating factors may contribute to the protective effects of habitual endurance exercise on vascular health. Additionally, sex had a greater impact than habitual activity level on the levels of vascular-related ci-miRs.NEW & NOTEWORTHY Serum from sedentary women caused impaired endothelial migration, whereas serum from sedentary men elicited increased endothelial reactive oxygen species production as compared with serum from their endurance-trained counterparts. Select CVD-related circulating microRNAs (ci-miRs) were higher in men than women, while ci-miRs-140-5p and 145-5p were also higher in sedentary versus trained men and/or women. Our data suggest that alterations in circulating factors may contribute to the protective effects of habitual exercise and sex on vascular health.

Reduced caveolae density in arteries of SHR contributes to endothelial dysfunction and ROS production

Caveolae are plasma membrane invaginations enriched with high cholesterol and sphingolipid content; they also contain caveolin proteins in their structure. Endothelial nitric oxide synthase (eNOS), an enzyme that synthesizes nitric oxide (NO) by converting L-arginine to L-citrulline, is highly concentrated in plasma membrane caveolae. Hypertension is associated with decreased NO production and impaired endothelium-dependent relaxation. Understanding the molecular mechanisms that follow hypertension is important. For this study, we hypothesized that spontaneously hypertensive rat (SHR) vessels should have a smaller number of caveolae, and that the caveolae structure should be disrupted in these vessels. This should impair the eNOS function and diminish NO bioavailability. Therefore, we aimed to investigate caveolae integrity and density in SHR aortas and mesenteric arteries and the role played by caveolae in endothelium-dependent relaxation. We have been able to show the presence of caveolae-like structures in SHR aortas and mesenteric arteries. Increased phenylephrine-induced contractile response after treatment with dextrin was related to lower NO release. In addition, impaired acetylcholine-induced endothelium-dependent relaxation could be related to decreased caveolae density in SHR vessels. The most important finding of this study was that cholesterol depletion with dextrin induced eNOS phosphorylation at Serine1177 (Ser1177) and boosted reactive oxygen species (ROS) production in normotensive rat and SHR vessels, which suggested eNOS uncoupling. Dextrin plus L-NAME or BH4 decreased ROS production in aorta and mesenteric arteries supernatant’s of both SHR and normotensive groups. Human umbilical vein endothelial cells (HUVECs) treated with dextrin confirmed eNOS uncoupling, as verified by the reduced eNOS dimer/monomer ratio. BH4, L-arginine, or BH4 plus L-arginine inhibited eNOS monomerization. All these results showed that caveolae structure and integrity are essential for endothelium-dependent relaxation. Additionally, a smaller number of caveolae is associated with hypertension. Finally, caveolae disruption promotes eNOS uncoupling in normotensive and hypertensive rat vessels and in HUVECs.

Inter-α inhibitor proteins maintain neutrophils in a resting state by regulating shape and reducing ROS production

AbstractThe plasma levels of inter-α inhibitor proteins (IAIPs) are decreased in patients with sepsis and the reduced levels correlate with increased mortality. In the present study, we examined the effects of IAIPs on human neutrophils to better understand the beneficial effects of IAIPs in the treatment of sepsis. We demonstrated that IAIPs induced a spherical shape that was smaller in size with a smooth cellular surface in a concentration-dependent manner. These changes were inhibited by a specific antibody against IAIPs. In contrast, bikunin, light chain of IAIP, had no effect on neutrophil morphology. The neutrophils treated with IAIPs could easily pass through the artificial microcapillaries and were prevented from entrapment inside the capillaries. Coincubation of human blood neutrophils with a confluent human vascular endothelial monolayer showed that adhesion of neutrophils on endothelial cells was suppressed by treatment with IAIPs. IAIPs inhibited the spontaneous release of reactive oxygen species (ROS) in a concentration-dependent fashion. ROS inhibition was associated with reductions in p47phox phosphorylation on Ser328. These results suggest that IAIP-induced morphological changes that render neutrophils quiescent, facilitate passage through the microvasculature, and reduce adhesion to vascular endothelial cells and production of ROS. Thus, IAIP plays a key role in controlling neutrophil activation.

Up-regulation of NADPH oxidase-mediated redox signaling contributes to the loss of barrier function in KRIT1 deficient endothelium

The intracellular scaffold KRIT1/CCM1 is an established regulator of vascular barrier function. Loss of KRIT1 leads to decreased microvessel barrier function and to the development of the vascular disorder Cerebral Cavernous Malformation (CCM). However, how loss of KRIT1 causes the subsequent deficit in barrier function remains undefined. Previous studies have shown that loss of KRIT1 increases the production of reactive oxygen species (ROS) and exacerbates vascular permeability triggered by several inflammatory stimuli, but not TNF−α. We now show that endothelial ROS production directly contributes to the loss of barrier function in KRIT1 deficient animals and cells, as targeted antioxidant enzymes reversed the increase in permeability in KRIT1 heterozygous mice as shown by intravital microscopy. Rescue of the redox state restored responsiveness to TNF-α in KRIT1 deficient arterioles, but not venules. In vitro, KRIT1 depletion increased endothelial ROS production via NADPH oxidase signaling, up-regulated Nox4 expression, and promoted NF-κB dependent promoter activity. Recombinant yeast avenanthramide I, an antioxidant and inhibitor of NF-κB signaling, rescued barrier function in KRIT1 deficient cells. However, KRIT1 depletion blunted ROS production in response to TNF-α. Together, our data indicate that ROS signaling is critical for the loss of barrier function following genetic deletion of KRIT1.

Reactive Oxygen Species, Mitochondria, and Endothelial Cell Death during In Vitro Simulated Dives.

Excessive reactive oxygen species (ROS) is considered a consequence of hyperoxia and a major contributor to diving-derived vascular endothelial damage and decompression sickness. The aims of this work were: 1) to directly observe endothelial ROS production during simulated air dives as well as its relation with both mitochondrial activity and cell survival; and 2) to determine which ambient factor during air diving (hydrostatic pressure or oxygen and/or nitrogen partial pressure) is responsible for the observed modifications. In vitro diving simulation was performed with bovine arterial endothelial cells under real-time observation. The effects of air diving, hydrostatic, oxygen and nitrogen pressures, and N-acetylcysteine (NAC) treatment on mitochondrial ROS generation, mitochondrial membrane potential and cellular survival during simulation were investigated. Vascular endothelial cells performing air diving simulation suffered excessive mitochondrial ROS, mitochondrial depolarization, and cell death. These effects were prevented by NAC: after NAC treatment, the cells presented no difference in damage from nondiving cells. Oxygen diving showed a higher effect on ROS generation but lower impacts on mitochondrial depolarization and cell death than hydrostatic or nitrogen diving. Nitrogen diving had no effect on the inductions of ROS, mito-depolarization, or cell death. This study is the first direct observation of mitochondrial ROS production, mitochondrial membrane potential and cell survival during diving. Simulated air SCUBA diving induces excessive ROS production, which leads to mitochondrial depolarization and endothelial cell death. Oxygen partial pressure plays a crucial role in the production of ROS. Deleterious effects of hyperoxia-induced ROS are potentiated by hydrostatic pressure. These findings hold new implications for the pathogenesis of diving-derived endothelial dysfunction.

Reactive oxygen species in endothelial function - from disease to adaptation - .

Endothelial function is largely dictated by its ability to rapidly sense environmental cues and adapt to these stimuli through changes in vascular tone, inflammation/immune recruitment, and angiogenesis. When any one of these abilities is compromised, the endothelium becomes dysfunctional, which ultimately leads to disease. Reactive oxygen species (ROS) have been established at the forefront of endothelial dysfunction; however, more careful examination has demonstrated that ROS are fundamental to each of the sensing/signaling roles of the endothelium. The purpose of this review is to document endothelial ROS production in both disease and physiological adaptation. Through understanding new endothelial signaling paradigms, we will gain insight into more targeted therapeutic strategies for vascular diseases.

Inhibition of phosphodiesterase 5 restores endothelial function in renovascular hypertension.

BackgroundThe clipping of an artery supplying one of the two kidneys (2K1C) activates the renin-angiotensin (Ang) system (RAS), resulting in hypertension and endothelial dysfunction. Recently, we demonstrated the intrarenal beneficial effects of sildenafil on the high levels of Ang II and reactive oxygen species (ROS) and on high blood pressure (BP) in 2K1C mice. Thus, in the present study, we tested the hypothesis that sildenafil improves endothelial function in hypertensive 2K1C mice by improving the NO/ROS balance.Methods2K1C hypertension was induced in C57BL/6 mice. Two weeks later, they were treated with sildenafil (40 mg/kg/day, via oral) or vehicle for 2 weeks and compared with sham mice. At the end of the treatment, the levels of plasma and intrarenal Ang peptides were measured. Endothelial function and ROS production were assessed in mesenteric arterial bed (MAB).ResultsThe 2K1C mice exhibited normal plasma levels of Ang I, II and 1–7, whereas the intrarenal Ang I and II were increased (~35% and ~140%) compared with the Sham mice. Sildenafil normalized the intrarenal Ang I and II and increased the plasma (~45%) and intrarenal (+15%) Ang 1–7. The 2K1C mice exhibited endothelial dysfunction, primarily due to increased ROS and decreased NO productions by endothelial cells, which were ameliorated by treatment with sildenafil.ConclusionThese data suggest that the effects of sildenafil on endothelial dysfunction in 2K1C mice may be due to interaction with RAS and restoring NO/ROS balance in the endothelial cells from MAB. Thus, sildenafil is a promising candidate drug for the treatment of hypertension accompanied by endothelial dysfunction and kidney disease.

Endothelial cell-specific reactive oxygen species production increases susceptibility to aortic dissection.

Increased production of reactive oxygen species (ROS) throughout the vascular wall is a feature of cardiovascular disease states, but therapeutic strategies remain limited by our incomplete understanding of the role and contribution of specific vascular cell ROS to disease pathogenesis. To investigate the specific role of endothelial cell (EC) ROS in the development of structural vascular disease, we generated a mouse model of endothelium-specific Nox2 overexpression and tested the susceptibility to aortic dissection after angiotensin II (Ang II) infusion. A specific increase in endothelial ROS production in Nox2 transgenic mice was sufficient to cause Ang II-mediated aortic dissection, which was never observed in wild-type mice. Nox2 transgenic aortas had increased endothelial ROS production, endothelial vascular cell adhesion molecule-1 expression, matrix metalloproteinase activity, and CD45(+) inflammatory cell infiltration. Conditioned media from Nox2 transgenic ECs induced greater Erk1/2 phosphorylation in vascular smooth muscle cells compared with wild-type controls through secreted cyclophilin A (CypA). Nox2 transgenic ECs (but not vascular smooth muscle cells) and aortas had greater secretion of CypA both at baseline and in response to Ang II stimulation. Knockdown of CypA in ECs abolished the increase in vascular smooth muscle cell Erk1/2 phosphorylation conferred by EC conditioned media, and preincubation with CypA augmented Ang II-induced vascular smooth muscle cell ROS production. These findings demonstrate a pivotal role for EC-derived ROS in the determination of the susceptibility of the aortic wall to Ang II-mediated aortic dissection. ROS-dependent CypA secretion by ECs is an important signaling mechanism through which EC ROS regulate susceptibility of structural components of the aortic wall to aortic dissection.

Endothelial G Protein–Coupled Receptor Kinase 2 Regulates Vascular Homeostasis Through the Control of Free Radical Oxygen Species

The role of endothelial G protein-coupled receptor kinase 2 (GRK2) was investigated in mice with selective deletion of the kinase in the endothelium (Tie2-CRE/GRK2(fl/fl)). Aortas from Tie2-CRE/GRK2(fl/fl) presented functional and structural alterations as compared with control GRK2(fl/fl) mice. In particular, vasoconstriction was blunted to different agonists, and collagen and elastic rearrangement and macrophage infiltration were observed. In primary cultured endothelial cells deficient for GRK2, mitochondrial reactive oxygen species was increased, leading to expression of cytokines. Chronic treatment with a reactive oxygen species scavenger in mice corrected the vascular phenotype by recovering vasoconstriction, structural abnormalities, and reducing macrophage infiltration. These results demonstrate that GRK2 removal compromises vascular phenotype and integrity by increasing endothelial reactive oxygen species production.

Prolyl-isomerase-1 (pin1) mediates hyperglycemia-induced oxidative stress, endothelial function and vascular inflammation: insights in patients with type 2 diabetes

Purpose: Prolyl-isomerase-1 (Pin1) regulates function of protein substrates through isomerization of peptide bonds that link phosphoserine or phosphothreonine to proline. Pin1 triggers reactive oxygen species (ROS) production and inflammation in human cancer. Whether Pin1 is involved in cardiovascular disease remains largely unknown. This study investigates the role of Pin1 in diabetes-related vascular dysfunction. Methods: Human aortic endothelial cells (HAECs) were exposed to normal (NG, 5mmol/L) or high glucose concentrations (HG, 25 mmol/L) in the presence or in the absence of Pin1 pharmacological inhibitor Juglone or siRNA-mediated knockdown. Diabetes was induced in C57/B6 male mice (aged 4-6 months) by streptozocin and animals were treated i.v. with Pin1 siRNA or Juglone i.p for 30 days. Endothelial function was assessed by dose-response curve with acetylcoline. Protein expression was assessed by immunoblotting. Mitochondrial ROS were measured by ESR spectroscopy. Immunoprecipitation was performed to show the interaction of Pin1 with phosphorylated p66Shc and NFkB subunit p65. In parallel, Pin1 gene expression was assessed in peripheral blood monocytes (PBM) of 30 patients with type 2 diabetes (T2DM) and 18 healthy age-matched controls and correlated with flow-mediated vasodilation (FMD) of the brachial artery, urinary 8-isoprostaglandinF2α (8-isoPGF2α) as a marker of oxidative stress, and plasma adhesion molecules VCAM-1, ICAM-1 and MCP-1. Results: Pin1 expression markedly increased in HAECs exposed to HG (289±22% vs NG, p<0.01) and aortas of diabetic mice (216±32% vs controls, p<0.05). Immunoprecipitation showed that Pin1 recognizes phosphoserine motifs of the pro-oxidant mitochondrial adaptor p66Shc as well as NFkB subunit p65. Interestingly, Juglone or Pin1siRNA prevented p66Shc-induced ROS production and suppressed upregulation of adhesion molecules VCAM-1, ICAM-1 and MCP-1 via inhibition of p65 nuclear translocation. In vivo knockdown of Pin1 or Juglone in diabetic mice protected against hyperglycemia-induced endothelial dysfunction, ROS production and vascular inflammation. Of note, Pin1 mRNA was significantly upregulated in PBM of T2DM patients as compared with healthy controls (370±97 vs. 25±28, p<0.01) and correlated with glycated haemoglobin (r=0.44, p<0.05), FMD (r=-0.36,p<0.01), urinary 8-isoPGF2α (r=0.39, p<0.05), as well as VCAM-1 (r=0.56, p<0.05) and ICAM-1 (r=0.53, p<0.05). Conclusions: This study shows for the first time that Pin1 may critically contribute to oxidative stress, endothelial dysfunction and vascular inflammation in patients with type 2 diabetes.

177 THE EFFECTS OF SHORT CHAIN FATTY ACIDS ON HIGH LEVELS OF GLUCOSE-INDUCED ENDOTHELIAL OXIDATIVE STRESS

Hyperglycaemia is a hallmark of type 2 diabetes and plays a prominent role in oxidative stress-induced endothelial cell dysfunction and the development of cardiovascular diseases. Recently, a diet enriched with short-chain fatty acids (SCFAs) i.e. acetate, propionate and butyrate, has been found to improve insulin sensitivity and reduce blood pressure in clinical studies; however, the underlying mechanism remains elusive. In this study we investigated the effect of SCFAs on high glucose-induced endothelial dysfunction <i>in vitro</i>. Human pulmonary microvascular endothelial cells (HPMECs) were pre-incubated with acetate (5mM), propionate (4mM) or butyrate (4mM) for 1 hour before adding high glucose (30mM) and cultured for 24 h in the presence of SCFAs. Compared to cells cultured in the normal glucose medium (5mM), high glucose induced a significant increase (48.5±12.6%; p&lt;0.05) in endothelial reactive oxygen species (ROS) production as measured by both NADPH-dependant lucigenin-chemiluminescence and DHE fluorescence. Increased ROS production was accompanied with a significant increase in cell death (30.66±8.3%; p&lt;0.05) as measured by both MTT and trypan blue exclusion assays, and capillary structure breakdown of HPMEC cultured on the metrigels. We then examined the effects of SCFAs on endothelial ROS production, and found that SCFAs slightly increased the levels of ROS production in HPMEC cultured in 5mM glucose medium. However, in the presence of high glucose, SCFAs significantly inhibited high glucose-induced ROS production, reduced cell death and preserved capillary structure on the metrigels. In conclusion, SCFAs inhibit high glucose-mediated endothelial oxidative stress and improve endothelial cell function. SCFAs may represent an effective therapeutic strategy as a food supplement for cardiovascular complications linked to hyperglycaemia and diabetes.

Alteration of endothelial CaMKII in AngII‐induced hypertensive mice

Endothelial dysfunction found in AngII‐induced hypertension is characterized by an alteration of the delicate balance between reactive oxygen species (ROS) and nitric oxide (NO). Loss of endothelial Ca2+ homeostasis has also been shown in hypertension. Recent evidences suggest that CaMKII is activated by Ca2+ pulsars, a localized spontaneous Ca2+ signal and is involved in endothelial function. Since CaMKII is also sensitive to intracellular ROS, endothelial CaMKII and Ca2+ pulsars in an AngII‐induced hypertension mouse model was studied. Real‐time confocal imaging showed that acute exposure to AngII stimulated both Ca2+ pulsars and production of ROS. As expected, an increase in endothelial ROS production was found with dihydroethidium (DHE) in the AngII hypertensive compared to normotensive mice. Moreover, AngII‐induced hypertension appears to recruit activated CaMKII (T286) at the myoendothelial projection (MEP), sites of Ca2+ pulsars. Consistently, clustering of all endothelial CaMKII isoforms (α, β and δ) within the MEP has showed a similar distribution. Our data suggest that CaMKII activity is modulated by an increased ROS production and Ca2+ pulsars stimulation in AngII‐induced hypertensive mice. Supported by CIHR, FRQS, CFI and HSFC.

The effect of inorganic arsenic on endothelium-dependent relaxation: Role of NADPH oxidase and hydrogen peroxide

Chronic arsenic ingestion predisposes to vascular disease, but underlying mechanisms are poorly understood. In the present study we have analyzed the effects of short-term arsenite exposure on vascular function and endothelium-dependent relaxation.Endothelium-dependent relaxations, nitric oxide (NO) and endothelium derived hyperpolarizing factor (EDHF)-type, were studied in rabbit iliac artery and aortic rings using the G protein-coupled receptor agonist acetylcholine (ACh) and by cyclopiazonic acid (CPA), which promotes store-operated Ca2+ entry by inhibiting the endothelial SERCA pump. Production of reactive oxygen species (ROS) in the endothelium of rabbit aortic valve leaflets and endothelium-denuded RIA and aortic rings was assessed by imaging of dihydroethidium.In the iliac artery, exposure to 100μM arsenite for 30min potentiated EDHF-type relaxations evoked by both CPA and ACh. Potentiation was prevented by catalase, the catalase/superoxide dismutase mimetic manganese porphyrin and the NADPH oxidase inhibitor apocynin. By contrast in aortic rings, that exhibited negligible EDHF-type responses, endothelium-dependent NO-mediated relaxations evoked by CPA and ACh were unaffected by arsenite. Arsenite induced apocynin-sensitive increases in ROS production in the aortic valve endothelium, but not in the media and adventitia of the iliac artery and aorta.Our results suggest that arsenite can potentiate EDHF-type relaxations via a mechanism that is dependent on hydrogen peroxide, thus demonstrating that dismutation of the superoxide anion generated by NADPH oxidase can potentially offset loss of NO bioavailability under conditions of reduced eNOS activity. By contrast, selective increases in endothelial ROS production following exposure to arsenite failed to modify relaxations mediated by endogenous NO.