The Role of NOX2-Derived Reactive Oxygen Species in the Induction of Endothelin-Converting Enzyme-1 by Angiotensin II.

Endothelin-1 is a key regulator of vascular tone and blood pressure in health and disease. We have recently found that ET-1 production in human microvascular endothelial cells (HMECs) can be promoted by angiotensin II (Ang II) through a novel mechanism involving octamer-binding transcription factor-1 (Oct-1), NADPH oxidase-2 (NOX2), and superoxide anions. As the formation of bioactive ET-1 also depends on endothelin-converting enzyme-1 (ECE-1), we investigated the transcriptional regulation of the ECE1 gene. We found that exposure of HMECs to Ang II resulted in a concentration- and time-dependent increase in ECE1 mRNA expression. Pharmacological inhibition of ECE-1 reduced Ang II-stimulated ET-1 release to baseline values. The effect of Ang II on ECE1 mRNA expression was associated with Oct-1 binding to the ECE1 promoter, resulting in its increased activity. Consequently, the Ang II-stimulated increase in ECE1 mRNA expression could be prevented by siRNA-mediated Oct-1 inhibition. It could also be abolished by silencing the NOX2 gene and neutralizing superoxide anions with superoxide dismutase. In mice fed a high-fat diet, cardiac expression of Ece1 mRNA increased in wild-type mice but not in Nox2-deficient animals. It can be concluded that Ang II engages Oct-1, NOX2, and superoxide anions to stimulate ECE1 expression in the endothelium.

Modulation of the hippo-YAP pathway by cyclic stretch in rat type 2 alveolar epithelial cells-a proof-of-concept study.

Background: Mechanical ventilation (MV) is a life supporting therapy but may also cause lung damage. This phenomenon is known as ventilator-induced lung injury (VILI). A potential pathomechanisms of ventilator-induced lung injury may be the stretch-induced production and release of cytokines and pro-inflammatory molecules from the alveolar epithelium. Yes-associated protein (YAP) might be regulated by mechanical forces and involved in the inflammation cascade. However, its role in stretch-induced damage of alveolar cells remains poorly understood. In this study, we explored the role of YAP in the response of alveolar epithelial type II cells (AEC II) to elevated cyclic stretch in vitro. We hypothesize that Yes-associated protein activates its downstream targets and regulates the interleukin-6 (IL-6) expression in response to 30% cyclic stretch in AEC II. Methods: The rat lung L2 cell line was exposed to 30% cyclic equibiaxial stretch for 1 or 4 h. Non-stretched conditions served as controls. The cytoskeleton remodeling and cell junction integrity were evaluated by F-actin and Pan-cadherin immunofluorescence, respectively. The gene expression and protein levels of IL-6, Yes-associated protein, Cysteine-rich angiogenic inducer 61 (Cyr61/CCN1), and connective tissue growth factor (CTGF/CCN2) were studied by real-time polymerase chain reaction (RT-qPCR) and Western blot, respectively. Verteporfin (VP) was used to inhibit Yes-associated protein activation. The effects of 30% cyclic stretch were assessed by two-way ANOVA. Statistical significance as accepted at p < 0.05. Results: Cyclic stretch of 30% induced YAP nuclear accumulation, activated the transcription of Yes-associated protein downstream targets Cyr61/CCN1 and CTGF/CCN2 and elevated IL-6 expression in AEC II after 1 hour, compared to static control. VP (2 µM) inhibited Yes-associated protein activation in response to 30% cyclic stretch and reduced IL-6 protein levels. Conclusion: In rat lung L2 AEC II, 30% cyclic stretch activated YAP, and its downstream targets Cyr61/CCN1 and CTGF/CCN2 and proinflammatory IL-6 expression. Target activation was blocked by a Yes-associated protein inhibitor. This novel YAP-dependent pathway could be involved in stretch-induced damage of alveolar cells.

PLAC8-Mediated Activation of NOX4 Signalling Restores Angiogenic Function of Endothelial Colony-Forming Cells in Experimental Hypoxia.

Ischaemic cardiovascular disease is associated with tissue hypoxia as a significant determinant of angiogenic dysfunction and adverse remodelling. While cord blood-derived endothelial colony-forming cells (CB-ECFCs) hold clear therapeutic potential due to their enhanced angiogenic and proliferative capacity, their impaired functionality within the disease microenvironment represents a major barrier to clinical translation. The aim of this study was to define the specific contribution of NOX4 NADPH oxidase, which we previously reported as a key CB-ECFC regulator, to hypoxia-induced dysfunction and its potential as a therapeutic target. CB-ECFCs exposed to experimental hypoxia demonstrated downregulation of NOX4-mediated reactive oxygen species (ROS) signalling linked with a reduced tube formation, which was partially restored by NOX4 plasmid overexpression. siRNA knockdown of placenta-specific 8 (PLAC8), identified by microarray analysis as an upstream regulator of NOX4 in hypoxic versus normoxic CB-ECFCs, enhanced tube formation, NOX4 expression and hydrogen peroxide generation, and induced several key transcription factors associated with downstream Nrf2 signalling. Taken together, these findings indicated that activation of the PLAC8-NOX4 signalling axis improved CB-ECFC angiogenic functions in experimental hypoxia, highlighting this pathway as a potential target for protecting therapeutic cells against the ischaemic cardiovascular disease microenvironment.

Associations of Tissue and Soluble LOX-1 with Human Abdominal Aortic Aneurysm.

Background Indication for prophylactic surgical abdominal aortic aneurysm (AAA) repair depends on the maximal aortic diameter. The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the major receptor for uptake of oxidized low-density lipoprotein cholesterol and is implicated in atherosclerosis. A soluble form of LOX-1 (sLOX-1) has been discussed as a novel biomarker in coronary artery disease and stroke. Herein, we assessed the regulation of aortic LOX-1 as well as the diagnostic and risk stratification potential of sLOX-1 in patients with AAA. Methods and Results Serum sLOX-1 was assessed in a case-control study in AAA (n=104) and peripheral artery disease (n=104). sLOX-1 was not statistically different between AAA and peripheral artery disease but was higher in AAA (ß=1.28, P=0.04) after adjusting for age, atherosclerosis, type 2 diabetes, prescription of statins, ß-blockers, ACE inhibitors, and therapeutic anticoagulation. sLOX-1 was not associated with the aortic diameter, AAA volume, or the thickness of the intraluminal thrombus. Aortic LOX-1 mRNA expression tended to be higher in AAA when compared with disease, and expression was positively associated with cleaved caspase-3, smooth muscle actin, collagen, and macrophage content. Conclusions In AAA, sLOX-1 was differently affected by age, cardiometabolic diseases, and corresponding medical therapies. Comparison with nonatherosclerotic disease would be beneficial to further elucidate the diagnostic potential of sLOX-1, although it was not useful for risk stratification. Aneurysmal LOX-1 mRNA expression was increased and positively associated with smooth muscle cells and collagen content, suggesting that LOX-1 is eventually not deleterious in human AAA and could counteract AAA rupture.

Regulation of endothelial function by cigarette smoke and next-generation tobacco and nicotine products.

Cigarette smoking is the most important avoidable cardiovascular risk factor. It causes endothelial dysfunction and atherosclerosis and increases the risk of its severe clinical complications like coronary artery disease, myocardial infarction, stroke, and peripheral artery disease. Several next-generation tobacco and nicotine products have been developed to decrease some of the deleterious effects of regular tobacco smoking. This review article summarizes recent findings about the impact of cigarette smoking and next-generation tobacco and nicotine products on endothelial dysfunction. Both cigarette smoking and next-generation tobacco products lead to impaired endothelial function. Molecular mechanisms of endothelial dysfunction like oxidative stress, reduced nitric oxide availability, inflammation, increased monocyte adhesion, and cytotoxic effects of cigarette smoke and next-generation tobacco and nicotine products are highlighted. The potential impact of short- and long-term exposure to next-generation tobacco and nicotine products on the development of endothelial dysfunction and its clinical implications for cardiovascular diseases are discussed.

Ang II Promotes ET-1 Production by Regulating NOX2 Activity Through Transcription Factor Oct-1.

BACKGROUND: Increasing evidence suggests that superoxide ions produced by NOX (nicotinamide adenine dinucleotide phosphate oxidases) mediate vascular effects of Ang II (angiotensin II) evoked by atherogenic diets. Here, we analyzed the mechanism by which NOX2 contributes to Ang II-induced ET-1 (endothelin 1) production in human microvascular endothelial cells. METHODS: The effects of high-fat diet were compared between WT (wild type) and Nox2 (mouse NOX2 gene)-deficient mice. ET-1 production and NOX2 expression by human microvascular endothelial cells in vitro were analyzed by ELISA, reverse transcription quantitative polymerase chain reaction, electrophoretic mobility shift assay, promoter deletions, RNA interference, and pharmacological inhibition. Production of superoxide anions was visualized by fluorescent cell labeling. RESULTS: Feeding mice high-fat diet for 10 weeks increased cardiac expression and plasma levels of Ang II and ET-1 in WT but not in Nox2-deficient animals. Exposure of human microvascular endothelial cells to Ang II resulted in increased ET-1 production, which could be blocked by silencing NOX2 (human NOX2 gene). Ang II promoted NOX2 expression through induction of the Oct-1 (human/mouse octamer binding transcription factor 1 protein) and activation of the NOX2 promoter region containing Oct-1-binding sites. Stimulation of NOX2 expression by Ang II was associated with increased production of superoxide anions. Inhibition of Oct-1 by small interfering RNA reduced Ang II-induced NOX2 expression and superoxide anion production, and neutralization of superoxide by SOD (superoxide dismutase) abolished Ang II-stimulated ET1 (human ET-1 gene) promoter activity, ET1 mRNA expression, and ET-1 release. CONCLUSIONS: Ang II may promote ET-1 production in the endothelium in response to atherogenic diets through a mechanism that involves the transcription factor Oct-1 and the increased formation of superoxide anions by NOX2.

Comparative study of the effects of cigarette smoke versus next-generation tobacco and nicotine product extracts on inflammatory biomarkers of human monocytes.

Monocytes exhibiting a pro-inflammatory phenotype play a key role in adhesion and development of atherosclerotic plaques. As an alternative to smoking, next-generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their pro-inflammatory effects on monocytes. We investigated cell viability, anti-oxidant and pro-inflammatory gene and protein expression in THP-1 monocytes after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine (nic). Treatment with 3R4F reduced cell viability in a dose-dependent manner, whereas exposure to alternative smoking products showed no difference to control. At the highest non-lethal dose of 3R4F (20%), the following notable mRNA expression changes were observed for 3R4F, HTP, and e-cig respectively, relative to control; HMOX1 (6-fold, < 2-fold, < 2-fold), NQO1 (3.5-fold, < 2-fold, < 2-fold), CCL2 (4-fold, 3.5-fold, 2.5-fold), IL1B (4-fold, 3-fold, < 2-fold), IL8 (5-fold, 2-fold, 2-fold), TNF (2-fold, 2-fold, < 2-fold) and ICAM1 was below the 2-fold threshold for all products. With respect to protein expression, IL1B (3-fold, < 2-fold, < 2-fold) and IL8 (3.5-fold, 2-fold, 2-fold) were elevated over the 2-fold threshold, whereas CCL2, TNF, and ICAM1 were below 2-fold expression for all products. At higher doses, greater inductions were observed with all extracts; however, NGP responses were typically lower than 3R4F. In conclusion, anti-oxidative and pro-inflammatory processes were activated by all products. NGPs overall showed lower responses relative to controls than THP-1 cells exposed to 3R4F AqE.

Aprotinin does not Impair Vascular Function in Patients Undergoing Coronary Artery Bypass Graft Surgery.

Bleeding is a major complication in coronary artery bypass graft surgery. Antifibrinolytic agents like serine protease inhibitor aprotinin can decrease postoperative bleeding and complications of cardiac surgery. However, the effects of aprotinin on vascular function are not completely elucidated. We compared the ex vivo vascular function of left internal mammary arteries from patients undergoing coronary artery bypass graft surgery with and without intraoperative application of aprotinin using a Mulvany Myograph. Human internal mammary arteries were treated with aprotinin ex vivo and tested for changes in vascular function. We analyzed the impact of aprotinin on vascular function in rat aortic rings. Finally, impact of aprotinin on expression and activity of endothelial nitric oxide synthase was tested in human endothelial cells. Intraoperative application of aprotinin did not impair ex vivo vascular function of internal mammary arteries of patients undergoing coronary artery bypass graft surgery. Endothelium-dependent and -independent relaxations were not different in patients with or without aprotinin after nitric oxide synthase blockade. A maximum vasorelaxation of 94.5%±11.4vs. 96.1%±5.5% indicated a similar vascular smooth muscle function in both patient groups (n=13 each). Long-term application of aprotinin under physiological condition preserved vascular function of the rat aorta. In vitro application of increasing concentrations of aprotinin on human endothelial cells resulted in a similar expression and activity of endothelial nitric oxide synthase. In conclusion, intraoperative and ex vivo application of aprotinin does not impair the endothelial function in human internal mammary arteries and experimental models.

NOX4 mRNA correlates with plaque stability in patients with carotid artery stenosis.

Carotid artery stenosis (CAS) develops from atherosclerotic lesions and plaques. Plaque rupture or stenosis may result in occlusion of the carotid artery. Accordingly, the asymptomatic disease becomes symptomatic, characterized by ischemic stroke or transient ischemic attacks, indicating an urgent need for better understanding of the underlying molecular mechanisms and eventually prevent symptomatic CAS. NOX4, a member of the NADPH oxidase family, has anti-atherosclerotic and anti-inflammatory properties in animal models of early atherosclerosis. We hypothesized that NOX4 mRNA expression is linked to protective mechanisms in CAS patients with advanced atherosclerotic lesions as well. Indeed, NOX4 mRNA expression is lower in patients with symptomatic CAS. A low NOX4 mRNA expression is associated with an increased risk of the development of clinical symptoms. In fact, NOX4 appears to be linked to plaque stability, apoptosis and plaque hemorrhage. This is supported by cleaved caspase-3 and glycophorin C and correlates inversely with plaque NOX4 mRNA expression. Even healing of a ruptured plaque appears to be connected to NOX4, as NOX4 mRNA expression correlates to fibrous cap collagen and is reciprocally related to MMP9 activity. In conclusion, low intra-plaque NOX4 mRNA expression is associated with an increased risk for symptomatic outcome and with reduced plaque stabilizing mechanisms suggesting protective effects of NOX4 in human advanced atherosclerosis.

Pharmacotherapies and Aortic Heme Oxygenase-1 Expression in Patients with Abdominal Aortic Aneurysm.

BACKGROUND: Treatment of cardiovascular risk factors slows the progression of small abdominal aortic aneurysms (AAA). Heme oxygenase-1 (HO-1) is a stress- and hemin-induced enzyme providing cytoprotection against oxidative stress when overexpressed. However, nothing is known about the effects of cardiometabolic standard therapies on HO-1 expression in aortic walls in patients with end-stage AAA. METHODS: The effects of statins, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), calcium channel blockers (CCBs), beta-blockers, diuretics, acetylsalicylic acid (ASA), and therapeutic anticoagulation on HO-1 mRNA and protein expressions were analyzed in AAA patients using multivariate logistic regression analysis and comparison of monotherapy. RESULTS: Analysis of monotherapy revealed that HO-1 mRNA and protein expressions were higher in patients on diuretics and lower in patients on statin therapy. Tests on combinations of antihypertensive medications demonstrated that ACE inhibitors and diuretics, ARBs and diuretics, and beta-blockers and diuretics were associated with increase in HO-1 mRNA expression. ASA and therapeutic anticoagulation were not linked to HO-1 expression. CONCLUSION: Diuretics showed the strongest association with HO-1 expression, persisting even in combination with other antihypertensive medications. Hence, changes in aortic HO-1 expression in response to different medical therapies and their effects on vessel wall degeneration should be analyzed in future studies.

Impact of Dietary Sodium Reduction on the Development of Obesity and Type 2 Diabetes in db/db Mice.

The impact of dietary sodium reduction on mouse models of type 2 diabetes is not well understood. Therefore, we analyzed the effect of a low-salt diet on obesity and parameters of type 2 diabetes in db/db mice. Five-week-old male db/db and lean db/m mice were fed a normal salt (0.19% Na+, NS) or a low-salt diet (<0.03% Na+, LS) for 5 weeks. Body and organ weight and parameters of glucose and insulin tolerance were analyzed. Plasma levels of steroids were determined by liquid chromatography tandem mass spectrometry. Body weight, glucose, and insulin tolerance were not affected by LS. The amount of gonadal adipose tissue showed a trend to be increased by LS whereas liver, pancreas, kidney, heart, and adrenal weight remained unaffected. LS reduced urinary sodium-to-creatinine ratio but did not affect plasma Na+ levels in both genotypes. Plasma and urinary potassium-to-creatinine ratio did not differ in all groups of mice. Aldosterone as a major determinant of changes in dietary sodium remained unaffected by LS in db/db mice as well as further investigated steroid hormones. The present study showed reduced sodium-to-creatinine ratio, but no additional effects of dietary sodium reduction on major metabolic parameters and steroid levels in obese and hyper-glycemic db/db mice.

Induction of Heme Oxygenase-1 Is Linked to the Severity of Disease in Human Abdominal Aortic Aneurysm.

Background Rupture of abdominal aortic aneurysm (rAAA) is associated with high case fatality rates, and risk of rupture increases with the AAA diameter. Heme oxygenase-1 (gene HMOX1, protein HO-1) is a stress-induced protein and induction has protective effects in the vessel wall. HMOX1-/- mice are more susceptible to angiotensin II-induced AAA formation, but the regulation in human nonruptured and ruptured AAA is only poorly understood. Our hypothesis proposed that HO-1 is reduced in AAA and lowering is inversely associated with the AAA diameter. Methods and Results AAA walls from patients undergoing elective open repair (eAAA) or surgery because of rupture (rAAA) were analyzed for aortic HMOX1/HO-1 expression by quantitative real-time polymerase chain reaction and Western blot. Aortas from patients with aortic occlusive disease served as controls. HMOX1/HO-1 expression was 1.1- to 7.6-fold upregulated in eAAA and rAAA. HO-1 expression was 3-fold higher in eAAA specimen with a diameter >84.4 mm, whereas HO-1 was not different in rAAA. Other variables that are known for associations with AAA and HO-1 induction were tested. In eAAA, HO-1 expression was negatively correlated with aortic collagen content and oxidative stress parameters H2O2 release, oxidized proteins, and thiobarbituric acid reactive substances. Serum HO-1 concentrations were analyzed in patients with eAAA, and maximum values were found in an aortic diameter of 55 to 70 mm with no further increase >70 mm, compared with <55 mm. Conclusions Aortic HO-1 expression was increased in eAAA and rAAA. HO-1 increased with the severity of disease but was additionally connected to less oxidative stress and vasoprotective mechanisms.

Comparative study of the effects of cigarette smoke versus next generation tobacco and nicotine product extracts on endothelial function.

Tobacco smoking and hemodynamic forces are key stimuli for the development of endothelial dysfunction. As an alternative to smoking, next generation tobacco and nicotine products (NGP) are now widely used. However, little is known about their potential pro-inflammatory and atherogenic effects on the endothelium. In this study, we analyzed key parameters of endothelial function after exposure to aqueous smoke extracts (AqE) of a heated tobacco product (HTP), an electronic cigarette (e-cig), a conventional cigarette (3R4F) and pure nicotine. All experiments were performed under atheroprotective high laminar or atherogenic low flow with primary human endothelial cells. Treatment with 3R4F, but not alternative smoking products, reduced endothelial cell viability and wound healing capability via the PI3K/AKT/eNOS(NOS3) pathway. Laminar flow delayed detrimental effects on cell viability by 3R4F treatment. 3R4F stimulation led to activation of NRF2 antioxidant defense system at nicotine concentrations ≥0.56 µg/ml and increased expression of its target genes HMOX1 and NQO1. Treatment with HTP revealed an induction of HMOX1 and NQO1 at dosages with ≥1.68 µg/ml nicotine, whereas e-cig and nicotine exposure had no impact. Analyses of pro-inflammatory genes revealed an increased ICAM1 expression under 3R4F treatment. 3R4F reduced VCAM1 expression in a dose-dependent manner; HTP treatment had similar but milder effects; e-cig and nicotine treatment had no impact. SELE expression was induced by 3R4F under static conditions. High laminar flow prevented this upregulation. Stimulation with laminar flow led to downregulation of CCL2 (MCP-1). From this downregulated level, only 3R4F increased CCL2 expression at higher concentrations. Finally, under static conditions, all components increased adhesion of monocytes to endothelial cells. Interestingly, only stimulation with 3R4F revealed increased monocyte adhesion under atherosclerosis-prone low flow. In conclusion, all product categories activated anti-oxidative or pro-inflammatory patterns. NGP responses were typically lower than in 3R4F exposed cells. Also, 3R4F stimulation led to an impaired endothelial wound healing and induced a pro-inflammatory phenotype compared to NGP treatment.

Autophagy unleashes noncanonical microRNA functions.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression which act by guiding AGO (argonaute) proteins to target RNA transcripts in the RNA-induced silencing complex (RISC). This macromolecular complex includes multiple additional components (e.g., TNRC6A) that allow for interaction with enzymes mediating inhibition of translation or RNA decay. However, miRNAs also reside in low-molecular weight complexes without being engaged in target repression, and their function in this context is largely unknown. Our recent findings show that endothelial cells exposed to protective high-shear stress or MTORC inhibition activate the macroautophagy/autophagy machinery to sustain viability by promoting differential trafficking of MIR126 strands and by enabling unconventional features of MIR126-5p. Whereas MIR126-3p is degraded upon autophagy activation, MIR126-5p interacts with the RNA-binding protein MEX3A to form a ternary complex with AGO2. This complex forms on the autophagosomal surface and facilitates its nuclear localization. Once in the nucleus, MIR126-5p dissociates from AGO2 and establishes aptamer-like interactions with the effector CASP3 (caspase 3). The binding to MIR126-5p prevents dimerization and proper active site formation of CASP3, thus inhibiting proteolytic activity and limiting apoptosis. Disrupting this pathway in vivo by genetic deletion of Mex3a or by specific deficiency of endothelial autophagy aggravates endothelial apoptosis and exacerbates the progression of atherosclerosis. The direct inhibition of CASP3 by MIR126-5p reveals a non-canonical mechanism by which miRNAs can modulate protein function and mediate the autophagy-apoptosis crosstalk.

Noncanonical inhibition of caspase-3 by a nuclear microRNA confers endothelial protection by autophagy in atherosclerosis.

MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.

NOX4 is a major regulator of cord blood-derived endothelial colony-forming cells which promotes post-ischaemic revascularization.

AIMS: Cord blood-derived endothelial colony-forming cells (CB-ECFCs) are a defined progenitor population with established roles in vascular homeostasis and angiogenesis, which possess low immunogenicity and high potential for allogeneic therapy and are highly sensitive to regulation by reactive oxygen species (ROS). The aim of this study was to define the precise role of the major ROS-producing enzyme, NOX4 NADPH oxidase, in CB-ECFC vasoreparative function. METHODS AND RESULTS: In vitro CB-ECFC migration (scratch-wound assay) and tubulogenesis (tube length, branch number) was enhanced by phorbol 12-myristate 13-acetate (PMA)-induced superoxide in a NOX-dependent manner. CB-ECFCs highly-expressed NOX4, which was further induced by PMA, whilst NOX4 siRNA and plasmid overexpression reduced and potentiated in vitro function, respectively. Increased ROS generation in NOX4-overexpressing CB-ECFCs (DCF fluorescence, flow cytometry) was specifically reduced by superoxide dismutase, highlighting induction of ROS-specific signalling. Laser Doppler imaging of mouse ischaemic hindlimbs at 7 days indicated that NOX4-knockdown CB-ECFCs inhibited blood flow recovery, which was enhanced by NOX4-overexpressing CB-ECFCs. Tissue analysis at 14 days revealed consistent alterations in vascular density (lectin expression) and eNOS protein despite clearance of injected CB-ECFCs, suggesting NOX4-mediated modulation of host tissue. Indeed, proteome array analysis indicated that NOX4-knockdown CB-ECFCs largely suppressed tissue angiogenesis, whilst NOX4-overexpressing CB-ECFCs up-regulated a number of pro-angiogenic factors specifically-linked with eNOS signalling, in parallel with equivalent modulation of NOX-dependent ROS generation, suggesting that CB-ECFC NOX4 signalling may promote host vascular repair. CONCLUSION: Taken together, these findings indicate a key role for NOX4 in CB-ECFCs, thereby highlighting its potential as a target for enhancing their reparative function through therapeutic priming to support creation of a pro-reparative microenvironment and effective post-ischaemic revascularization.

NADPH oxidase 4 mediates the protective effects of physical activity against obesity-induced vascular dysfunction.

AIMS: Physical activity is one of the most potent strategies to prevent endothelial dysfunction. Recent evidence suggests vaso-protective properties of hydrogen peroxide (H2O2) produced by main endothelial NADPH oxidase isoform 4 (Nox4) in the vasculature. Therefore, we hypothesized that Nox4 connects physical activity with vaso-protective effects. METHODS AND RESULTS: Analysis of the endothelial function using Mulvany Myograph showed endothelial dysfunction in wild-type (WT) as well as in C57BL/6J/ Nox4-/- (Nox4-/-) mice after 20 weeks on high-fat diet (HFD). Access to running wheels during the HFD prevented endothelial dysfunction in WT but not in Nox4-/- mice. Mechanistically, exercise led to an increased H2O2 release in the aorta of WT mice with increased phosphorylation of eNOS pathway member AKT serine/threonine kinase 1 (AKT1). Both H2O2 release and phosphorylation of AKT1 were diminished in aortas of Nox4-/- mice. Deletion of Nox4 also resulted in lower intracellular calcium release proven by reduced phenylephrine-mediated contraction, whilst potassium-induced contraction was not affected. H2O2 scavenger catalase reduced phenylephrine-induced contraction in WT mice. Supplementing H2O2 increased phenylephrine-induced contraction in Nox4-/- mice. Exercise-induced peroxisome proliferative-activated receptor gamma, coactivator 1 alpha (Ppargc1a), as key regulator of mitochondria biogenesis in WT but not Nox4-/- mice. Furthermore, exercise-induced citrate synthase activity and mitochondria mass were reduced in the absence of Nox4. Thus, Nox4-/- mice became less active and ran less compared with WT mice. CONCLUSIONS: Nox4 derived H2O2 plays a key role in exercise-induced adaptations of eNOS and Ppargc1a pathway and intracellular calcium release. Hence, loss of Nox4 diminished physical activity performance and vascular protective effects of exercise.

Endothelial function and gene expression in perivascular adipose tissue from internal mammary arteries of obese patients with coronary artery disease.

BACKGROUND AND AIMS: Obesity is a risk factor for endothelial dysfunction and atherosclerosis. However, perivascular adipose tissue can release adipokines and other unknown adipose-derived relaxing factors. Therefore, we investigated the impact of obesity on vascular function and expression of genes in perivascular adipose tissue from internal mammary arteries of patients with coronary artery disease undergoing coronary artery bypass grafting. METHODS: The vessel function was compared between groups of patients with a body-mass index (BMI) between 25 and 30 kg/m2. The groups did not differ in age, gender (males), and ejection fraction. Vascular segments of internal mammary arteries were examined in a Mulvany myograph. Following preconstriction with noradrenaline, dose-response curves were assessed for relaxation with acetylcholine and sodium nitroprusside. RESULTS: Maximum contraction in response to potassium and noradrenaline was increased in obese patients with a BMI >30 kg/m2. EC50 of endothelium-dependent relaxation was impaired in patients with a BMI above 25, but below 30 kg/m2. Sodium nitroprusside-mediated maximal relaxation was not different between study groups. Integrin alpha X chain (ITGAX/CD11c) and macrophage mannose receptor (MRC1/CD206) expression was reduced in perivascular adipose tissue of patients with a BMI above 30 kg/m2, while adiponectin (ADPQ) expression was increased in the same tissue. CONCLUSION: Our data suggest a partially reduced endothelial function in internal mammary arteries of adipose patients with a BMI between 25 and 30 kg/m2 undergoing coronary artery bypass grafting surgery. Increased adiponectin expression in perivascular tissue might contribute to maintenance of endothelial function in obese patients with a BMI above 30 kg/m2.

Lectin-like oxidized low-density lipoprotein receptor-1 promotes endothelial dysfunction in LDL receptor knockout background.

OBJECTIVE: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is the major receptor for oxidized LDL in endothelial cells. LOX-1 is highly expressed in atherosclerotic plaques. The impact of LOX-1 on development of endothelial dysfunction in large vessels in absence or presence of atherosclerosis-prone conditions has not been studied to date. METHODS: Mice with endothelial cell-specific LOX-1 overexpression (bLOX-1tg) were analyzed. Wild-type (WT) mice served as controls. In addition, bLOX-1tg mice were crossed with LDL receptor knockout (Ldlr-/-) mice. All mice were fed a western-type diet (WD) or control diet (CD) for 20 weeks. Afterwards, endothelial function was analyzed ex vivo in thoracic aortas using a Mulvany myograph. RESULTS: WD induced hypertriglyceridemia (bLOX-1tg: 1.6-fold; WT: 1.4-fold) and hypercholesterolemia (P < 0.0001) in bLOX-1tg and WT mice without HDL-elevation in bLOX-1tg mice. Gonadal fat pad weight was 1.7 and 1.2-fold increased on CD and WD in bLOX-1tg mice compared to WT. LOX-1 overexpression impaired endothelial function by 15-16% (P < 0.05) on CD and WD. Crossing bLOX-1tg mice into Ldlr-/- background strongly elevated total (∼6-fold) and LDL-cholesterol (∼9-fold) compared to WT and bLOX-1tg mice on WD. Endothelial function in response to WD was impaired in bLOX-1tg/Ldlr-/- mice (Effmax: 56.7 ± 23.0%) compared to WT (Effmax: 88.2 ± 15.8%, P < 0.001), bLOX-1tg (Effmax: 76.7 ± 12.9%, P < 0.05) and Ldlr-/- mice (Effmax: 70.1 ± 13.1%, P < 0.05). No differences between WT, bLOX-1tg and Ldlr-/- mice were detectable when comparing all genotypes. CONCLUSION: Endothelial LOX-1 overexpression in an atherosclerosis-prone background impairs endothelial function, proving its importance in the development of atherosclerosis.