Internal Mammary Artery Segments Research Articles

Effect of Vericiguat Pre-treatment on Phenylephrine Constrictor Responses in Isolated Human Internal Mammary Artery Segments With/without Intact Endothelium

Effect of Vericiguat Pre-treatment on Phenylephrine Constrictor Responses in Isolated Human Internal Mammary Artery Segments With/without Intact Endothelium

Effect of Type-2 Diabetes Mellitus on the Expression and Function of Smooth Muscle ATP-Sensitive Potassium Channels in Human Internal Mammary Artery Grafts.

Here we have shown for the first time altered expression of the vascular smooth muscle (VSM) KATP channel subunits in segments of the human internal mammary artery (HIMA) in patients with type-2 diabetes mellitus (T2DM). Functional properties of vascular KATP channels in the presence of T2DM, and the interaction between its subunits and endogenous ligands known to relax this vessel, were tested using the potassium (K) channels opener, pinacidil. HIMA is the most commonly used vascular graft in cardiac surgery. Previously it was shown that pinacidil relaxes HIMA segments through interaction with KATP (SUR2B/Kir6.1) vascular channels, but it is unknown whether pinacidil sensitivity is changed in the presence of T2DM, considering diabetes-induced vascular complications commonly seen in patients undergoing coronary artery bypass graft surgery (CABG). KATP subunits were detected in HIMA segments using Western blot and immunohistochemistry analyses. An organ bath system was used to interrogate endothelium-independent vasorelaxation caused by pinacidil. In pharmacological experiments, pinacidil was able to relax HIMA from patients with T2DM, with sensitivity comparable to our previous results. All three KATP subunits (SUR2B, Kir6.1 and Kir6.2) were observed in HIMA from patients with and without T2DM. There were no differences in the expression of the SUR2B subunit. The expression of the Kir6.1 subunit was lower in HIMA from T2DM patients. In the same group, the expression of the Kir6.2 subunit was higher. Therefore, KATP channels might not be the only method of pinacidil-induced dilatation of T2DM HIMA. T2DM may decrease the level of Kir6.1, a dominant subunit in VSM of HIMA, altering the interaction between pinacidil and those channels.

Systemic and local vascular inflammation and arterial reactive oxygen species generation in patients with advanced cardiovascular diseases.

Systemic inflammation may cause endothelial activation, mediate local inflammation, and accelerate progression of atherosclerosis. We examined whether the levels of circulating inflammatory cytokines reflect local vascular inflammation and oxidative stress in two types of human arteries. Human internal mammary artery (IMA) was obtained in 69 patients undergoing coronary artery bypass graft (CABG) surgery and left anterior descending (LAD) artery was obtained in 17 patients undergoing heart transplantation (HTx). Plasma levels of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were measured using ELISA, high-sensitivity C-reactive protein (hs-CRP) was measured using Luminex, and mRNA expression of proinflammatory cytokines in the vascular tissues was assessed. Furthermore, formation of superoxide anion was measured in segments of IMA using 5 uM lucigenin-dependent chemiluminescence. Vascular reactivity was measured using tissue organ bath system. TNF-α, IL-6 and IL-1β mRNAs were expressed in all studied IMA and LAD segments. Plasma levels of inflammatory cytokines did not correlate with vascular cytokine mRNA expression neither in IMA nor in LAD. Plasma TNF-α and IL-6 correlated with hs-CRP level in CABG group. Hs-CRP also correlated with TNF-α in HTx group. Neither vascular TNF-α, IL-6 and IL-1β mRNA expression, nor systemic levels of either TNF-α, IL-6 and IL-1β were correlated with superoxide generation in IMAs. Interestingly, circulating IL-1β negatively correlated with maximal relaxation of the internal mammary artery (r = -0.37, p = 0.004). At the same time the mRNA expression of studied inflammatory cytokines were positively associated with each other in both IMA and LAD. The positive correlations were observed between circulating levels of IL-6 and TNF-α in CABG cohort and IL-6 and IL-1β in HTx cohort. This study shows that peripheral inflammatory cytokine measurements may not reflect local vascular inflammation or oxidative stress in patients with advanced cardiovascular disease (CVD). Circulating pro-inflammatory cytokines generally correlated positively with each other, similarly their mRNA correlated in the arterial wall, however, these levels were not correlated between the studied compartments.

Abstract 12000: A Novel Human Arterial Transcriptomic Signature Associated With Major Adverse Cardiovascular Events and Oxidative Stress

Introduction: The biology of human vascular wall is implicated in cardiovascular pathology. We applied discovery transcriptomics to internal mammary arteries (IMAs) obtained from cardiac surgery patients, to identify pathways that can be treated therapeutically. Methods: Weighted correlation network analysis was conducted in sequencing data from IMAs RNA in 205 patients (Arm 1). Association with future incidence of major adverse cardiovascular events [MACE: cardiovascular death (CVD), non-fatal myocardial infarction or stroke, or new-onset heart failure] was assessed with Cox regression models (adjusted for age, sex, hypertension, dyslipidaemia, diabetes mellitus, and plasma TNFa). Arm 2 included 497 patients in whom segments of IMA were used for ex-vivo quantification of NADPH-stimulated superoxide by lucigenin chemiluminescence. Results: We identified 10 coexpressed gene ‘modules’ in the human arterial wall, through unsupervised transcriptomic analysis. The “red” module was found to cluster with arterial NADPH oxidase activity, while its eigengene values had the most significant correlation ( a ). Patients with high eigengene values for the “red” module showed a 3-fold higher risk of MACE ( b ), and enrichment analysis revealed top pathways ( c ). The Study Arm 2 population was followed up for a median of 6.35 years [IQR: 3.97-8.38], with 50 CVD and 101 MACE. High NADPH oxidases activity was independently predictive of cardiovascular mortality ( d ) and MACE (adj. hazard ratio 1.58 [1.07-2.36], p=0.02). Conclusion: We demonstrate that arterial oxidative stress leads to high risk of MACE in patients with atherosclerosis. We also identify a novel, redox-related arterial transcriptomic signature that predicts long-term cardiovascular risk. Pathways related with oxidative stress, or glucose metabolism or Hypoxia-Inducible Factor-1 in the human arterial wall, may be rational therapeutic targets to reduce cardiovascular risk in secondary prevention.

A human arterial transcriptomic signature predicts major adverse cardiac events and identifies novel, redox-related therapeutic targets within the vascular wall

Abstract Background The transcriptomic profile of the human vascular wall is implicated in a range of pathologies. RNA sequencing technologies allow for interrogation of gene expression patterns that are associated with clinical outcomes and can guide future research and drug development. Purpose To apply discovery network transcriptomics to internal mammary arteries (IMAs) obtained from patients undergoing cardiac surgery, in order to identify redox-related molecular pathways within the vascular wall that can be treated therapeutically. Methods Arm 1 included 377 patients in whom segments of IMA were used for ex-vivo quantification of NADPH-stimulated superoxide production by lucigenin-enhanced chemiluminescence. Arm 2 included 205 patients in whom bulk RNA sequencing was performed in RNA isolated from IMA, and the WGCNA package used for the analyses. The association with future incidence of major adverse cardiac events (MACE: cardiovascular death, non-fatal myocardial infarction, and stroke) was assessed using Cox regression models (adjusted for age, sex, hypertension, dyslipidaemia, diabetes mellitus, body mass index, smoking, and plasma TNFa). Results Over a median follow-up of 4.84 years [IQR: 2.03–7.14], 38 (11.2%) MACE occurred in Arm 1. High arterial NADPH-stimulated superoxide was independently associated with MACE risk (Adj. HR [95% CI]: 2.62 [1.13–6.07] high group, p=0.02). Unsupervised transcriptomic analysis in Arm 2 allowed identification of 10 coexpressed gene “modules”. Eigengenes summarising modular coexpression signatures were then correlated with NADPH-stimulated superoxide revealing the red module (a) as the most significant (rho=0.19, p=0.01). In survival analysis the red module showed significant correlation with MACE (Adj. HR [95% CI]: 1.40 [1.00–1.95] per SD, p=0.04). For an optimal cut-off, patients with high eigengene values for the red module showed a 4-fold higher risk of MACE (b), and significantly higher arterial oxidative stress (c). Enrichment analysis (performed with Enrichr) of genes in the red module revealed “Electron Transport Chain”, “Oxidative phosphorylation”, “Striated Muscle Contraction Pathway”, and “Glycolysis and Gluconeogenesis” amongst the top enriched pathways (d). Conclusion We present for the first time a novel human arterial transcriptomic signature reflecting changes in redox state, which identifies long-term cardiovascular risk. Targeting pathways in the vasculature related with the mitochondrial electron transport chain, the contractile mechanism, or glucose metabolism may lead to the development of novel therapeutics in cardiovascular disease. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): EPSRC-EP/N509711/1-2119518British Heart Foundation Figure 1

Homocysteine alters vasoreactivity of human internal mammary artery by affecting the KCa channel family.

BackgroundHyperhomocysteinemia is an independent risk factor for atherosclerotic heart disease. We previously demonstrated that disruption of calcium-activated potassium (KCa) channel activity is involved in homocysteine-induced dilatory dysfunction of porcine coronary arteries. Recently we reported that the KCa channel family, including large-, intermediate-, and small-conductance KCa (BKCa, IKCa, and SKCa) subtypes, are abundantly expressed in human internal mammary artery (IMA). In this study, we further investigated whether homocysteine affects the expression and functionality of the KCa channel family in this commonly used graft for coronary artery bypass surgery (CABG).MethodsResidual IMA segments obtained from patients undergoing CABG were studied in a myograph for the role of KCa subtypes in both vasorelaxation and vasoconstriction. The expression and distribution of KCa subtypes were detected by Western blot and immunohistochemistry.ResultsBoth the BKCa channel activator NS1619 and the IKCa/SKCa channel activator NS309 evoked significant IMA relaxation. Homocysteine exposure suppressed NS1619-induced relaxation whereas showed no influence on NS309-induced response. Blockade of BKCa but not IKCa and SKCa subtypes significantly suppressed acetylcholine-induced relaxation and enhanced U46619-induced contraction. Homocysteine compromised the vasodilating activity of the BKCa subtype in IMA, associated with a lowered protein level of the BKCa β1-subunit. Homocysteine potentiated the role of IKCa and SKCa subtypes in mediating endothelium-dependent relaxation without affecting the expression of these channels.ConclusionsHomocysteine reduces the expression of BKCa β1-subunit and compromises the vasodilating activity of BKCa channels in IMA. Unlike BKCa, IKCa and SKCa subtypes are unessential for IMA vasoregulation, whereas the loss of BKCa functionality in hyperhomocysteinemia enhances the role of IKCa and SKCa subtypes in mediating endothelial dilator function. Targeting BKCa channels may form a strategy to improve the postoperative graft performance in CABG patients with hyperhomocysteinemia who receive IMA grafting.

The effects of botulinum toxin A and papaverine on human saphenous vein and internal mammary artery grafts: an in vitro study.

Autologous saphenous vein (SV) and internal mammary artery (IMA) are used as bypass conduits during coronary artery bypass graft surgery. Vasospasm of the arterial and venous grafts may constitute a significant clinical problem. Pretreatment with a vasodilator drug of the graft ex vivo or intraluminal injection before implantation may be used for spasm prophylaxis. This in vitro study was designed to assess the vasoactive effects and time-dependent changes of botulinum toxin A (BTX-A) and papaverine pretreatment on vasospasm of human SV and IMA grafts. Also, histomorphology of the vessels was assessed. SV and IMA segments were suspended in organ baths, and isometric contraction responses to 2 different concentrations of 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1) were recorded after incubation with 2 different concentrations of BTX-A and papaverine at 2 time points (0 h and 2 h). The results revealed the following: 1) incubation with BTX-A and papaverine relaxes both SV and IMA rings contracted with 5-HT and ET-1; 2) the duration of the relaxant effect of BTX-A lasts longer than papaverine; and 3) no apparent histomorphological changes were observed in the grafts under light microscopy. This study demonstrates that in human SV and IMA grafts, pretreatment with both BTX-A and papaverine are safe and have a potent inhibitory effect depending on the vessel and vasoconstrictor agent. The long-lasting vasodilatory effect of BTX-A on vascular smooth muscle may provide promising results in the prevention of venous and arterial graft spasm.

Coronary-Coronary bypass graft as a safe and an alternative technique for revascularization of coronary arteries

Coronary–coronary bypass graft was first performed by Rowland and Grooters. This technique can be performed between two segments of the same coronary artery using saphenous vein grafts or free arterial grafts in on/off-pump coronary artery bypass grafting, also can be an alternative safe technique in some cases as calcified ascending aorta, porcelain aorta and insufficient graft length. The coronary– coronary bypass graft can provide nearly the same flow rate as conventional coronary artery bypass graft, another advantage this technique is that we can use to decrease sternal and respiratory morbidity. we performed this technique to a 55-year-old woman to whom coronary angiography showed critical three-vessel disease, using a free segment of right internal mammary artery, combined to conventional coronary artery bypass graft. The postoperative course was uneventful with the absence of ischemic lesions and the grafts were patent at 6 months after procedure. Aim: This aim of this article is to describe this technique and suggest the ways that we can use it.

A novel arterial redox-specific machine learning-derived radiomic signature of perivascular adipose tissue predicts cardiac mortality from routine CCTA

Abstract Background Vascular oxidative stress is involved in inflammation and atherogenesis. Vascular inflammation induces spatial changes in perivascular adipose tissue (PVAT) composition, which can be detected by radiomic analysis of coronary computed tomography angiography (CCTA) images. Purpose To explore the association of arterial oxidative stress with long-term risk of major adverse cardiovascular events (MACE). To develop a radiomic signature to identify high oxidative stress non-invasively using CCTA. Finally, to assess the ability of this signature to predict future cardiac risk. Methods Arm 1 included 272 patients undergoing cardiac surgery. Segments of internal mammary artery (IMA) were used for ex-vivo quantification of NADPH-stimulated and Vas2870 (pan-NOX inhibitor) inhibitable superoxide production by lucigenin-enhanced chemiluminescence. Eighty-two of these patients with CCTA scans available constituted Arm 2. Peri-IMA PVAT was segmented and used to extract 1,616 radiomic features, which, after filtering (40 final features), were utilised to train extreme gradient boosting, a machine learning algorithm, to predict high arterial oxidative stress. Arm 3 included a nested cohort of 308 participants (41 suffering cardiac death and 267 matched controls) from the CRISP-CT (Cardiovascular RISk Prediction using Computed Tomography) study to externally validate the redox-specific signature developed in Arm 2 for cardiac risk prediction. Results Over a median follow-up of 40 months, 18 (6.6%) MACE (cardiovascular death, non-fatal myocardial infarction, and stroke) occurred in Arm 1. High arterial NADPH-stimulated superoxide was independently associated with MACE risk (Adj. HR[95% CI]: 1.61 [1.04–2.53] per SD, p=0.03, adjusted for age, sex, diabetes, hypertension, hyperlipidemia, smoking, obesity, and plasma TNFa). Unsupervised hierarchical clustering of radiomic features from peri-IMA PVAT segments in Arm 2 identified two distinct clusters (A) that differed in NADPH stimulated (p=0.01) and Vas2870 inhibitable (p=0.04) IMA superoxide (B), supporting the hypothesis that PVAT mapping can capture changes corresponding to differential levels of underlying vascular redox state. This prompted the development of a radiomic signature specific to PVAT alterations associated with high vascular oxidative stress, which was validated in Arm 3 (AUC:0.61, p=0.026, C). The novel signature was able to stratify cardiac risk in the validation set, independently of the Fat Attenuation Index, epicardial adipose tissue volume, high-risk plaque features, and obstructive CAD (Adj. HR [95% CI]:2.56 [1.35–4.87], p=0.004, D). Conclusion Increased arterial oxidative stress predicts cardiac risk in patients with advanced atherosclerosis. We present for the first time a novel, non-invasive CCTA imaging biomarker reflecting changes in vascular redox state by radiomic phenotyping of perivascular space, which stratifies cardiac risk beyond standard and newer risk assessment methods. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation, National Institute of Health Research, Oxford Biomedical Research Centre

Single Endothelial Cell mRNA Sequencing Better Captures the Severity of Coronary Artery Disease Than Targeted Total Arterial Markers of Inflammation and Senescence

Endothelial dysfunction is the initial step towards atherosclerotic plaque development and coronary artery disease (CAD). Senescent endothelial cells (SnEC) have been linked to the atherosclerotic burden. In particular, the senescent‐associated secretory phenotype protein angiopoietin‐like 2 (ANGPTL2) was shown to be elevated in the plasma of CAD patients and related to the senescent cellular load in human internal mammary artery (IMA) segments discarded during coronary artery bypass grafting (CABG) surgery. We tested the hypothesis that the accumulation of vascular SnEC causes endothelial dysfunction and precedes the appearance of atherosclerotic lesions.Discarded atheroma‐free IMA segments from 12 patients (11 men and 1 woman, 69±3 years) were collected during consecutive elective CABG surgeries. Arterial rings of IMA segments were mounted in a wire myograph to record isometric changes in tension: arteries were pre‐contracted with U46619, a synthetic analogue of PGH2, and endothelium‐dependent relaxations to increasing concentrations of acetylcholine (ACh) were recorded. The maximal relaxation (Emax) and the concentration of ACh inducing 50% of relaxation (EC50) were calculated. Afterwards total mRNA was extracted from the IMA segments. Vascular gene expression of ANGPTL2 and p21 (senescence markers), and CD68 and PAI‐1 (inflammatory markers) were assessed by RT‐qPCR. In parallel, single‐cell RNA sequencing was performed in IMA segments from two age‐matched male patients obtained either during elective (stable CAD) or emergency (unstable CAD, with numerous risk factors) procedures, and differential gene expression was specifically analyzed in vascular EC.Endothelium‐dependent relaxations were characterized by Emax (56±7 %, [18–100%], n=12) and pD2 (‐log EC50: 6.9±0.1 [5.8–7.3], n=12). Patients were divided into 2 groups according to their Emax (< or > to 50%) to define low and high endothelial function; vascular gene expression of the 4 senescence and inflammatory markers were similar between the two groups, demonstrating that global arterial wall senescence and inflammation did not distinguish severity of endothelial dysfunction. We then used single‐cell mRNA sequencing and analysed the unbiased differential gene expression specifically in vascular EC. From the total IMA cellular counts, EC represented ~2%. Nine genes were identified that were differentially overexpressed in EC from unstable CAD patient that have been associated with senolytic drug targets and cardiovascular diseases (Table ), potentially attesting to the severity of endothelial dysfunction and CAD.In conclusion, unlike total arterial wall mRNA quantification, unbiased single‐cell mRNA sequencing identified differentially upregulated endothelial pathways that may contribute to the severity of the CAD by inducing precocious endothelial dysfunction and senescence. Top genes differentially expressed in endothelial cells between a stable and unstable CAD patient. Percentages represent the percent of endothelial cells expressing the gene in each patient dataset. P‐values are Bonferroni corrected. Stable CAD patient Unstable CAD patient p‐value Gene Associated CVD disorder or drug target 0.0% 41.7% 0.00061 TGFB2 Atrial fibrillation 0.0% 41.7% 0.00061 SQLE Statins 2.9% 58.3% 0.00090 RPPH1 Quercetin (senolytic drug) 7.1% 66.7% 0.00339 ARL14EP Systolic blood pressure 25.7% 100.0% 0.00914 RPL17 Myocardial ischemia 17.1% 83.3% 0.01556 IGFBP7 Ruptured aneurysm 0.0% 33.3% 0.01699 TMEM8B Heart valve disorders 0.0% 33.3% 0.01699 ZNF675 Congestive heart failure 8.6% 66.7% 0.04848 SELL Vaso‐occlusive crisis

Alteration of KCa Channel Function by Homocysteine in Human Internal Mammary Artery

ObjectivesHyperhomocysteinemia is an independent risk factor for cardiovascular disease. We previously in porcine coronary arteries demonstrated that disruption of KCa channel activity is involved in homocysteine‐induced impairment of dilatory function. Whether homocysteine affects KCa channels in human arteries remains uninvestigated. In the present study, we aimed to investigate the effect of homocysteine on the expression and functionality of the members of KCa channel family, including large‐ (BKCa), intermediate‐ (IKCa) and small‐ (SKCa) conductance KCa channels in human internal mammary artery (IMA).MethodsResidual IMA segments collected from patients undergoing coronary artery bypass grafting (CABG) were used for myography study for KCa subtype‐mediated relaxation and contraction. Western blot and immunohistochemistry were employed to detect the expression and distribution of KCa subtypes in IMA.ResultsBKCa subtype in the KCa channel family significantly regulated the vasoreactivity of IMA. Blockade of BKCa channels inhibited acetylcholine‐induced relaxation and enhanced U46619‐induced contraction. In comparison, both IKCa and SKCa subtypes played a minor role in the control of relaxation and contraction of IMA. Exposure to homocysteine compromised the vasodilating activity of the BKCa subtype in IMA, evidenced by the suppressed relaxant response to the BKCa channel opener and the decreased impact of the BKCa channel blocker on acetylcholine‐induced relaxation and U46619‐evoked contraction. The functional loss of BKCa was associated with a downregulation of the protein expression of BKCa β1 subunits in the smooth muscle of IMA. Inhibition of endoplasmic reticulum (ER) stress showed protective effect against homocysteine on the function and expression of BKCa channels. In contrast, the role of IKCa and SKCa subtypes in mediating endothelium‐dependent relaxation was augmented by homocysteine though no alterations in the regulatory capacity on vasoconstriction were observed. Homocystine did not alter the protein expression level of IKCa and SKCa subtypes.ConclusionsHomocysteine compromises the vasodilating activity of BKCa channels in IMA, which may be attributed to the loss of BKCa β1 subunits mediated by ER stress. Unlike BKCa, IKCa and SKCa subtypes are unessential for vasoregulation in IMA, the loss of BKCa functionality in hyperhomocysteinemia enhances the role of IKCa and SKCa in mediating endothelial dilator function. Targeting BKCa may form a strategy to improve the postoperative graft performance in CABG patients with hyperhomocysteinemia who receives IMA grafting.Support or Funding InformationSupported by NSFC 81870227 & 81870288, Tianjin Science and Technology Commission (18PTZWHZ00060) & Binhai New Area Health Bureau (2018BWKZ005), and Non‐profit Central Research Institute Fund of Chinese Academy of Medical Sciences 2018TX31002 & 2019XK310001.

P2709Wnt5a contributes to human atherosclerosis via novel USP17 redox signalling

Abstract Background Wnt5a is a non-canonical Wnt ligand with potential vascular effects, but its mechanistic role in atherosclerosis progression and the underlying downstream mechanisms are poorly explored. Purpose To address the hypothesis that Wnt5a induces vascular NADPH-oxidases activity, endothelial dysfunction and detrimental downstream redox signalling which could propagate atherosclerosis in humans. Methods Study 1 included 70 patients with coronary artery disease (CAD) versus age- and sex-matched non-CAD controls. Study 2 included 1,003 CAD patients undergoing cardiac surgery; internal mammary artery (IMA) and saphenous vein (SV) segments were harvested and used for ex vivo experiments. Study 3 included 68 individuals undergoing two cardiac computed tomography scans 3–5 years apart; calcified plaque burden was assessed by coronary calcium score (CCS). Superoxide (O2·−) generation was measured by lucigenin chemiluminescence with NADPH 100μM stimulation as indicator of NADPH-oxidases activity. Activation of Rac1, a key NADPH-oxidases subunit, was evaluated by a commercially available kit. Primary vascular smooth muscle cells (VSMCs) and HeLa cells were used for in vitro experiments. Circulating Wnt5a and Sfrp5 (a Wnt5a antagonist) were measured by ELISA in fasting plasma samples. Results In Study 1, the presence of CAD was independently linked with increased circulating Wnt5a bioavailability (A), which was, in turn, associated with increased IMA NADPH-oxidases activity in Study 2 (B). Recombinant Wnt5a directly stimulated NADPH-oxidases activity (C) via Rac1 activation (not shown) in human IMA, while inducing endothelial dysfunction evidenced by impaired SV endothelium-dependent acetylcholine (Ach) vasorelaxations (D). Transcriptomic analysis in Wnt5a-treated primary VSMCs versus controls identified USP17, a deubiquitinating enzyme implicated in Rac1 activation, as the top differentially regulated hit (not shown). Indeed, Wnt5a stimulated USP17 upregulation in VSMCs which was reversed by PEGylated superoxide dismutase (peg-SOD) 300U/mL (E), suggesting a redox sensitive effect. USP17 knockdown abolished the ability of Wnt5a to induce Rac1 activation in HeLa cells (F). At a clinical level, plasma Wnt5a was a predictor of plaque progression (defined as ΔCCS≥1, G) and new onset calcification (H) in Study 3. Conclusions We demonstrate for the first time that Wnt5a is elevated in CAD and causally associated with increased vascular oxidative stress and endothelial dysfunction in humans. We further reveal USP17 to be a novel, previously undescribed, link between Wnt5a, Rac1 activation and NADPH-oxidase activity induction in humans. We finally propose that circulating Wnt5a may have a clinically relevant role in predicting atherosclerosis progression. Our findings identify Wnt5a as rational therapeutic target in vascular disease. Acknowledgement/Funding British Heart Foundation; Alexandros S Onassis Public Benefit Foundation

Propofol-Induced Vasodilation in Human Internal Mammary Artery: Role of Potassium Channels

The aim of this study was to investigate the vascular effects and mechanisms of propofol in the human internal mammary artery (IMA). In vitro experimental study. The study was conducted inthe research laboratory of a pharmacology department. IMA segments were obtained from 52 patients undergoing coronary artery bypass surgery. The IMA rings were suspended in isolated organ baths, and the changes in the tension were isometrically recorded. The antagonistic effect of propofol (1 μM, 10 μM, and 100 μM) on contractions induced by potassium chloride (45 mM), phenylephrine (1 μM), 5-hydroxytryptamine (30 μM), and calcium chloride (10 μM-10 mM) was investigated. The relaxations induced by propofol also were tested in the presence of the nitric oxide synthase inhibitor, nitro-L-arginine methyl ester (100 mM); the cyclooxygenase inhibitor, indomethacin (10 mM); and the potassium ion channel inhibitors, tetraethylammonium (1 mM), iberiotoxin (20 nM), glibenclamide (10 µM), 4-aminopyridine (1 mM), and barium chloride (30 µM). Propofol caused a significant concentration-dependent vasorelaxation, which was endothelium independent. It inhibited the contractions induced by potassium chloride, phenylephrine, 5-hydroxytryptamine, and calcium chloride (p < 0.001), but it did not affect the basal tension. Propofol-induced relaxation was significantly inhibited by iberiotoxin and tetraethylammonium (p < 0.001); however, it was not affected by 4-aminopyridine, glibenclamide, and barium chloride. This study clearly reveals that propofol relaxes the IMA, and propofol-induced vasodilation may be related to large conductance calcium ion-activated potassium ion channel activation. Propofol use in coronary artery bypass surgery can be valuable via its favorable vasodilator effect to overcome perioperative vasospasm of IMA.

Role of cGMP and EDHF Pathways in Hydrogen Sulfide‐induced Vasorelaxation in the Human Artery

BackgroundEndogenous H2S generated by mercaptopyruvate sulfurtransferase (MPST) coupled with cysteine aminotransferase (CAT) in endothelium as a physical vasodilator and blood pressure regulator may play an important protective role in the pathogenesis of coronary heart disease. The present study aimed to investigate the role of cGMP and endothelium‐derived hyperpolarizing factor (EDHF) in H2S‐induced vasorelaxation in the human internal mammary artery (IMA).MethodsIn the human IMA segments from 54 patients undergoing coronary artery bypass grafting, the acetylcholine (ACh)‐induced relaxation and the effect of CAT inhibitor (aminooxyacetic acid [AOAA] and L‐aspartate) and KCa channel blocker or KATP channel blocker were studied in a myograph. Enzyme‐linked Immunosorbent Assay for PDE5A and western blotting analysis for eNOS and p‐eNOSser1177 were performed. Methylene blue assay was used to quantify the contents of H2S.ResultsThe maximal relaxation induced by ACh was significantly attenuated by AOAA or L‐aspartate, Iberiotixin (BKCa channel blocker) (P&lt;0.01), or IKCa channel blocker plus SKCa channel blocker (TRAM‐34+Apamin) (P&lt;0.01), or KATP channel blocker (Glibenclamide) (P&lt;0.01). The H2S donor NaHS increased eNOS phosphorylation at its activating site S1177 and down‐regulated PDE5A activity. Incubation with ACh for 4 hours significantly increased the H2S concentration.ConclusionsWe for the first time demonstrated in the human IMA that endogenous H2S could be produced under the stimulation of ACh and that CAT is one of the key enzymes in production of H2S in the endothelial cell of the IMA. H2S may activate eNOS to produce NO that activates PKG and may also inhibit PDE5A activity; both enhance the cGMP pathway‐mediated relaxation. Further, H2S as a potential EDHF directly stimulates KCa channels to induce vasodilatation. These findings may provide new therapeutical targets for the treatment of vasospasm of coronary artery bypass grafting vessels and provide a pharmacological basis for the development of new vasodilator drugs.Support or Funding InformationSupported by grants from the National Natural Science Foundation of China (81170148 &amp; 81641017), Zhejiang Provincial Natural Science Foundation (LY15H020008), &amp; Tianjin Binhai Key Platform for Creative Research Program (2012‐BK110004), Binhai New Area Health Bureau (2012BWKZ008, 2016BWKY007, 2016BWKZ003).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects Of Endothelin-1 On Intracellular Tetrahydrobiopterin Levels In Vascular Tissue

Objective: Tetrahydrobiopterin (BH4) is the essential cofactor of endothelial nitric oxide synthase (eNOS) and intracellular levels of BH4 is regulated by oxidative stress. The aim of this paper was to describe the influence of exogenous endothelin-1 on intracellular BH4 and its oxidation products dihydrobiopterin (BH2) and biopterin (B) in a wide range of vascular tissue.Design: Segments of internal mammary artery (IMA) and human saphenous vein (SV) from 41 patients undergoing elective surgery were incubated in ET-1 (0.1 μM). Aorta and lung from transgenic mice overexpressing ET-1 in the endothelium (ET-TG) were analysed with regards to intracellular biopterin levels. Human umbilical vein endothelial cells (HUVEC) were incubated in ET-1 (0.1 μM) and intracellular biopterin levels were analysed. From 6 healthy women undergoing caesarean section, subcutaneous fat was harvested and the resistance arteries in these biopsies were tested for ET-mediated endothelial dysfunction.Results: In HUVEC, exogenous ET-1 (0.1 μM) did not significantly change intracellular BH4, 1.54 ± 1.7 vs 1.68 ± 1.8 pmol/mg protein; p = .8. In IMA and SV, exogenous ET-1(0.1 μM) did not change intracellular BH4 n = 10, p = .4. In aorta from wild type vs ET-TG mice there was no significant difference in intracellular BH4 between the groups: 1.3 ± 0.49 vs 1.23 ± 0.3 pmol/mg protein; p = .6. In resistance arteries (n = 6) BH4 together with DTE (an antioxidant) was not able to prevent ET-mediated endothelial dysfunction.Conclusion: ET-1 did not significantly alter intracellular tetrahydrobiopterin levels in IMA, SV, HUVEC or aorta from ET-TG mice. These findings are important for future research in ET-1 mediated superoxide production and endothelial dysfunction.

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

Background and aimsObesity 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. MethodsThe 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. ResultsMaximum 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. ConclusionOur 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.

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.

Effects of levosimendan on isolated human internal mammary artery and saphenous vein: concurrent use with conventional vasodilators.

Graft spasm is a common problem in coronary artery bypass grafting (CABG). In this study, we aimed to investigate the interaction of levosimendan, a novel inodilator, with vasodilator agents that are clinically used for the treatment of graft spasm and with endogenous vasoconstrictors that are thought to play a role in graft vasospasm, in human internal mammary artery (IMA) and saphenous vein (SV). Isolated human IMA and SV segments derived from patients undergoing CABG were suspended in an organ bath. Responses to cumulative concentrations of noradrenaline (NA), serotonin (5-HT), papaverine, nitroglycerin (NG), and diltiazem were recorded before and after 10(-5) m levosimendan incubation (30 min). In addition, cumulative levosimendan responses were taken in vessels precontracted with NA or 5-HT. 10(-5) m levosimendan reduced NA Emax and sensitivity in IMA and SV, and 5-HT Emax responses in IMA. Moreover, levosimendan caused concentration-dependent relaxation in both grafts. Papaverine Emax or sensitivity was not altered by levosimendan neither in IMA nor in SV. Levosimendan diminished NG sensitivity in IMA and Emax responses in SV and decreased diltiazem Emax responses both in IMA and SV. Our results suggest that levosimendan may be used alone for prevention or treatment of graft spasm in IMA or in combination with papaverine in IMA and SV grafts. However, as concurrent administration with diltiazem or NG causes a reduction in relaxation in vitro, we suggest caution should be exercised when using levosimendan in combination with these agents.

Endothelial Denudation of Isolated Human Internal Mammary Artery Segments

Background: Endothelial denudation is an important approach to evaluate the role of the endothelium in vascular reactivity studies. Although approaches to remove the endothelium are well established in animal models, these methods have proved difficult to effectively translate to remnants of human Internal Mammary Artery (IMA) obtained during coronary bypass. This study sought to identify the optimal technique for endothelial denudation of IMA while preserving vascular contractile responses. Methods: IMA segments were subject to endothelial denudation using one of the following techniques: (1) surface abrasion, rubbing with a stainless steel wire, (2) vasoconstriction abrasion or (3) shear abrasion via infusion of an effervescent solution. Following intervention, IMA segments were evaluated by: (1) histochemistry to quantify structural damage and endothelial cell abundance and (2) functional endothelium-dependent vasodilator response using vascular myography in an organ bath preparation. Results: Vasoconstriction abrasion removed endothelial cells and caused disruption of the internal elastic lamina, these vessels failed to respond to the vasoconstrictor Phenylephrine (PE) or the endothelium-dependent vasodilator A23187. Surface abrasion alone was incomplete in removing endothelial cells, vessel vasodilated partially when challenged with A23187 in the presence of PE. Shear abrasion removed endothelial cells most effectively, as these pre-constricted vessels did not relax to A23187 but demonstrated increased sensitivity to PE. Conclusions: In this controlled comparative study assessing both structural and functional endpoints of endothelial denudation techniques, we have demonstrated that shear abrasion by infusion of an effervescent solution is the optimal technique to remove the endothelium and preserve vascular function in human IMA.

Adiponectin as a link between type 2 diabetes and vascular NADPH oxidase activity in the human arterial wall: the regulatory role of perivascular adipose tissue.

Oxidative stress plays a critical role in the vascular complications of type 2 diabetes. We examined the effect of type 2 diabetes on NADPH oxidase in human vessels and explored the mechanisms of this interaction. Segments of internal mammary arteries (IMAs) with their perivascular adipose tissue (PVAT) and thoracic adipose tissue were obtained from 386 patients undergoing coronary bypass surgery (127 with type 2 diabetes). Type 2 diabetes was strongly correlated with hypoadiponectinemia and increased vascular NADPH oxidase-derived superoxide anions (O2˙(-)). The genetic variability of the ADIPOQ gene and circulating adiponectin (but not interleukin-6) were independent predictors of NADPH oxidase-derived O2˙(-). However, adiponectin expression in PVAT was positively correlated with vascular NADPH oxidase-derived O2˙(-). Recombinant adiponectin directly inhibited NADPH oxidase in human arteries ex vivo by preventing the activation/membrane translocation of Rac1 and downregulating p22(phox) through a phosphoinositide 3-kinase/Akt-mediated mechanism. In ex vivo coincubation models of IMA/PVAT, the activation of arterial NADPH oxidase triggered a peroxisome proliferator-activated receptor-γ-mediated upregulation of the adiponectin gene in the neighboring PVAT via the release of vascular oxidation products. We demonstrate for the first time in humans that reduced adiponectin levels in individuals with type 2 diabetes stimulates vascular NADPH oxidase, while PVAT "senses" the increased NADPH oxidase activity in the underlying vessel and responds by upregulating adiponectin gene expression. This PVAT-vessel interaction is identified as a novel therapeutic target for the prevention of vascular complications of type 2 diabetes.