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AimsEndothelial erosion of plaques is responsible for ∼30% of acute coronary syndromes (ACS). Smoking is a risk factor for plaque erosion, which most frequently occurs on the upstream surface of plaques where the endothelium experiences elevated shear stress. We sought to recreate these conditions in vitro to identify potential pathological mechanisms that might be of relevance to plaque erosion.Methods and resultsCulturing human coronary artery endothelial cells (HCAECs) under elevated flow (shear stress of 7.5 Pa) and chronically exposing them to cigarette smoke extract (CSE) and tumour necrosis factor-alpha (TNFα) recapitulated a defect in HCAEC adhesion, which corresponded with augmented Nrf2-regulated gene expression. Pharmacological activation or adenoviral overexpression of Nrf2 triggered endothelial detachment, identifying Nrf2 as a mediator of endothelial detachment. Growth/Differentiation Factor-15 (GDF15) expression was elevated in this model, with protein expression elevated in the plasma of patients experiencing plaque erosion compared with plaque rupture. The expression of two Nrf2-regulated genes, OSGIN1 and OSGIN2, was increased by CSE and TNFα under elevated flow and was also elevated in the aortas of mice exposed to cigarette smoke in vivo. Knockdown of OSGIN1&2 inhibited Nrf2-induced cell detachment. Overexpression of OSGIN1&2 induced endothelial detachment and resulted in cell cycle arrest, induction of senescence, loss of focal adhesions and actin stress fibres, and disturbed proteostasis mediated in part by HSP70, restoration of which reduced HCAEC detachment. In ACS patients who smoked, blood concentrations of HSP70 were elevated in plaque erosion compared with plaque rupture.ConclusionWe identified a novel Nrf2-OSGIN1&2-HSP70 axis that regulates endothelial adhesion, elevated GDF15 and HSP70 as biomarkers for plaque erosion in patients who smoke, and two therapeutic targets that offer the potential for reducing the risk of plaque erosion.

DNA hypomethylation at theWe conducted multiple independent experiments to assess whether DNA hypomethylation atObservationally, DNA methylation atSmoking-induced epigenetic DNA hypomethylation at

Background: DNA hypomethylation at the F2RL3 (F2R like thrombin or trypsin receptor 3) locus has been associated with both smoking and atherosclerotic cardiovascular disease; whether these smoking-related associations form a pathway to disease is unknown. F2RL3 encodes protease-activated receptor 4, a potent thrombin receptor expressed on platelets. Given the role of thrombin in platelet activation and the role of thrombus formation in myocardial infarction, alterations to this biological pathway could be important for ischemic cardiovascular disease. Methods: We conducted multiple independent experiments to assess whether DNA hypomethylation at F2RL3 in response to smoking is associated with risk of myocardial infarction via changes to platelet reactivity. Using cohort data (N=3205), we explored the relationship between smoking, DNA hypomethylation at F2RL3 , and myocardial infarction. We compared platelet reactivity in individuals with low versus high DNA methylation at F2RL3 (N=41). We used an in vitro model to explore the biological response of F2RL3 to cigarette smoke extract. Finally, a series of reporter constructs were used to investigate how differential methylation could impact F2RL3 gene expression. Results: Observationally, DNA methylation at F2RL3 mediated an estimated 34% of the smoking effect on increased risk of myocardial infarction. An association between methylation group (low/high) and platelet reactivity was observed in response to PAR4 (protease-activated receptor 4) stimulation. In cells, cigarette smoke extract exposure was associated with a 4.9% to 9.3% reduction in DNA methylation at F2RL3 and a corresponding 1.7-(95% CI, 1.2–2.4, P =0.04) fold increase in F2RL3 mRNA. Results from reporter assays suggest the exon 2 region of F2RL3 may help control gene expression. Conclusions: Smoking-induced epigenetic DNA hypomethylation at F2RL3 appears to increase PAR4 expression with potential downstream consequences for platelet reactivity. Combined evidence here not only identifies F2RL3 DNA methylation as a possible contributory pathway from smoking to cardiovascular disease risk but from any feature potentially influencing F2RL3 regulation in a similar manner.

Endothelial erosion of atherosclerotic plaques is the underlying cause of approximately 30% of acute coronary syndromes (ACS). As the vascular endothelium is profoundly affected by the haemodynamic environment to which it is exposed, we employed computational fluid dynamic (CFD) analysis of the luminal geometry from 17 patients with optical coherence tomography (OCT)-defined plaque erosion, to determine the flow environment permissive for plaque erosion. Our results demonstrate that 15 of the 17 cases analysed occurred on stenotic plaques with median 31% diameter stenosis (interquartile range 28–52%), where all but one of the adherent thrombi located proximal to, or within the region of maximum stenosis. Consequently, all flow metrics related to elevated flow were significantly increased (time averaged wall shear stress, maximum wall shear stress, time averaged wall shear stress gradient) with a reduction in relative residence time, compared to a non-diseased reference segment. We also identified two cases that did not exhibit an elevation of flow, but occurred in a region exposed to elevated oscillatory flow. Our study demonstrates that the majority of OCT-defined erosions occur where the endothelium is exposed to elevated flow, a haemodynamic environment known to evoke a distinctive phenotypic response in endothelial cells.

BACKGROUND:Attenuation-compensated (AC) technique was recently introduced to improve the plaque characterization of optical coherence tomography (OCT). Histological validation demonstrated promising results but the efficacy and reproducibility of this technique for assessing in-vivo tissue composition remains unclear.Methods and Results:OCT images portraying native (n=200) and stented (n=200) segments and 31 histological cross-sections were analyzed. AC-OCT appeared superior to conventional (C)-OCT in detecting the external elastic lamina (EEM) borders (76% vs. 65.5%); AC-OCT enabled larger EEM arc detection compared with C-OCT (174.2±58.7° vs. 137.5±57.9°; P

Ammonia (NH3) is a major contributor to secondary aerosol in the atmosphere and can alter the kinetics of their formation. However, systematic studies related to the role of NH3 in aerosol nucleation processes and further particle size growth under complex scenarios are lacking. In this study, we conducted 16 experiments in the CSIRO smog chamber under dry conditions using aromatic hydrocarbons (toluene, o-/m-/p-xylene) and different concentrations of NH3. The presence of NH3 did not change the gas-phase chemistry or nucleation onset time, but slowed the nucleation rate (5%–94%) once it began. From the response of nitrogen oxides (NOx) measurement and mechanism modeling results, we hypothesised that the surface reaction between NH3 and nitric acid played a central role in aerosol nucleation and further growth. After nucleation, the subsequently formed ammonium nitrate and organic condensation vapours may partition together into the initial growth process of new particles, thus increasing the aerosol initial growth rate (8%–90%) and size growth potentials (7%–108%), and leading to high aerosol mass formation. Further investigation implied that the initial growth and further growth rate determine the aerosol mass concentration, rather than the nucleation rate. We conclude that both the initial NOx concentration and volatile organic compound (VOC)/NOx ratio are crucial for the initial and further growth, and aerosol mass of new particles, when NH3 levels are high. Our results provide crucial insights into the complex chemistry of VOC/NOx/NH3 in the atmosphere, and highlight the importance of NH3 reduction for particulate matter control.

During the 2016 Hangzhou G20 Summit, the chemical composition of submicron particles (PM1) was measured by a High-Resolution Time-of-Flight Aerosol Mass Spectrometer (HR-ToF-AMS) along with a suite of collocated instruments. The campaign was undertaken between August 5 and September 23, 2016. The impacts of emission controls and meteorological conditions on PM1 chemical composition, diurnal cycles, organic aerosol (OA) source apportionment, size distribution and elemental ratios were characterized in detail. Excluding rainy days, the mean PM1 mass concentration during G20 was 30.3 μg/m3, similar to that observed before G20 (28.6 μg/m3), but much lower than that after G20 (42.7 μg/m3). The aerosol chemistry during the three periods was substantially different. Before G20, high PM1 loading mostly occurred at daytime, with OA accounting for 60.1% of PM1, followed by sulfate (15.6%) and ammonium (9.1%). During G20, the OA fraction decreased from 60.1% to 44.6%, whereas secondary inorganic aerosol (SIA) increased from 31.8% to 49.5%. After G20, SIA dominated high PM1 loading, especially at nighttime. Further analysis showed that the nighttime regional transport might play an unfavorable role in the slight increase of secondary PM1 during G20, while the strict emissions controls were implemented. The OA (O/C = 0.58) during G20 was more aged, 48.7% and 13.7% higher than that before and after G20 respectively. Our study highlighted that the emission controls during G20 were of great success in lowering locally produced aerosol and pollutants, despite of co-existence of nighttime regional transport containing aerosol high in low-volatile organics and sulfate. It was implied that not only are emissions controls on both local and regional scale important, but that the transport of pollutants needs to be sufficiently well accounted for, to ensure the successful implementation of air pollution mitigation campaigns in China.

Introduction Endothelial erosion of atherosclerotic plaques and resulting thrombosis causes approximately 30% of acute coronary syndromes (ACS). Plaque erosion is most frequently observed in smokers, which induces endothelial dysfunction, partially through elevated circulating mediators of inflammation, such as tumour necrosis factor-alpha (TNFα), as well as free radical, oxidative and chemically induced damage. We have previously demonstrated that fresh aqueous cigarette smoke extract (CSE) increases the expression of Nrf2-target genes in human coronary artery endothelial cells, which was further increased by TNFα in a shear stress-dependent manner. Methods The haemodynamic environment significantly regulates both plaque development and endothelial function, therefore we determined the haemodynamic environment permissive for plaque erosion. We reconstructed the coronary artery geometries from 17 heart attack patients with thrombi overlying intact fibrous caps (OCT-defined erosion) and performed computational fluid dynamic analysis. We created an in vitro model of erosion by culturing human coronary artery endothelial cells under elevated flow and exposing them to CSE and TNFα. Results We identified that in 14 cases of OCT-defined erosion occurred in areas of stenosis, with the preeminent flow feature being elevated flow. Exposing human coronary artery endothelial cells to elevated flow, CSE and TNFα induced significant endothelial detachment, which was enhanced by pharmacological activation of the antioxidant system controlled by transcription factor Nrf2. The Oxidative Stress Growth INhibitor genes OSGIN1 and OSGIN2 were both maximally upregulated under these conditions and also in the aortas of mice exposed to cigarette smoke. Adenoviral overexpression of OSGIN1+2 in static culture resulted in cell cycle arrest in S-phase (5.5-fold increase, p= 0.003), with a significant increase in the number of multinucleated cells (4.5-fold, p= Conclusions In summary, we have defined the haemodynamic environment in which endothelial erosion occurs and identified that smoking-induced hyperactivation of Nrf2 may promote endothelial cell detachment, contributing to plaque erosion overlying stenotic plaques, through the combined upregulation of OSGIN1 and OSGIN2. This highlights a completely novel mechanism potentially contributing to 30% of ACS and suggests possible therapeutic avenues for further investigation. Conflict of Interest none

Introduction Endothelial glycocalyx (eGlx) contributes to the microvascular permeability barrier and its dysfunction correlates with albuminuria in diabetic nephropathy. Albuminuria is a potent risk factor for cardiovascular disease. We therefore hypothesised that coronary microvascular eGlx damage also occurs in diabetic cardiomyopathy (DCM). Methods Diabetes was induced in FVB mice with streptozotocin (STZ). DCM was assessed with echocardiography by E/A ratio. A group of diabetic FVB mice received Angiopoietin 1 (Ang1) after DCM development. Results FVB mice developed DCM at 7 weeks post STZ injection. Labelling with MAL-I, a specific lectin that binds to eGlx, was reduced in diabetic heart capillaries (DCM vs. ctrl: 1.64±0.27 vs. 2.70±0.27). Electron microscopy of diabetic heart capillaries showed decreased eGlx depth (DCM vs. ctrl: 14.54±0.79 vs. 27.88±5.82nm), increased perivascular space (DCM vs. ctrl: 2.08±0.22 vs. 0.54±0.11fold) and thickened endothelial cells (DCM vs. ctrl: 0.30±0.04 vs. 0.22±0.01μm). Partial depletion of eGlx in rat hearts with the combination of heparanase and chondroitinase led to decreased cardiac output (enzymes vs. ctrl: 63.47±10.14% vs. 91.68±9.82%). Ang1 improved diastolic function of FVB mice with DCM (DCM vs. DCM+Ang1: 1.05±0.08 vs. 1.35±0.09 fold relative to pre-treatment). In Ang1-treated diabetic mice, eGlx thickness in heart capillaries (DCM vs. DCM+Ang1: 13.87±0.87 vs. 24.55±2.02nm) and eGlx coverage (DCM vs. DCM+Ang1: 48.08±3.07% vs. 82.44±5.43%) were improved, and the increased perivascular space due to oedema in DCM normalised (DCM vs. DCM+Ang1: 2.08±0.13 vs. 0.23±0.03fold). Conclusion We have shown DCM development is associated with eGlx damage and that injury to eGlx impairs cardiac function. Recovered heart function with Ang1 treatment parallels reversal of eGlx loss. As such, correction of eGlx damage may have therapeutic potential for DCM and other diabetic vascular complications. Conflict of interest None

© 2016, The Author(s). Lipid-core atherosclerotic plaques are associated with disease progression, procedural complications, and cardiac events. Coronary plaque lipid can be quantified in optical coherence tomography (OCT) pullbacks by measurement of lipid arcs and lipid lengths; parameters frequently used in clinical research, but labor intensive and subjective to analyse. In this study, we investigated the ability of quantitative attenuation, derived from intravascular OCT, to detect plaque lipid. Lipid cores are associated with a high attenuation coefficient. We compared the index of plaque attenuation (IPA), a local quantitative measure of attenuation, to the manually measured lipid score (arc and length) on OCT images, and to the plaque characterization ex-vivo. We confirmed a correlation between the IPA and lipid scores (r 2 > 0.7). Comparison to histology shows that high attenuation is associated with fibroatheroma, but also with macrophage presence. IPA is a robust, reproducible, and user-independent measure that facilitates quantification of coronary lipid, a potential tool in clinical research and in guiding percutaneous coronary intervention.

SummaryMyocardial infarction is a prevalent, life-threatening consequence of athero-thrombosis. Post-mortem histology and intravascular imaging in live patients have shown that approximately one third of myocardial infarctions are caused by a thrombus overlying an intact, non-ruptured atherosclerotic plaque. Histology identifies erosion of luminal endothelial cells from smooth muscle and proteoglycan-rich, thick fibrous cap atheromas as the underlying pathology. Unlike plaque ruptures, endothelial erosions tend to occur on thick-capped atherosclerotic plaques and may or may not be associated with inflammation. Smoking and female gender are strong risk factors for erosion. Multiple mechanisms may contribute to endothelial erosion, including endothelial dysfunction, TLR signalling, leukocyte activation and modification of sub-endothelial matrix by endothelial or smooth muscle cells, which may trigger loss of adhesion to the extracellular matrix or endothelial apoptosis. Diagnosis of endothelial erosion by intravascular imaging, especially high resolution optical coherence tomography, may influence treatment strategies, offering prognostic value and utility as an endpoint in trials of agents designed to preserve an intact coronary endothelium.

Endothelial erosion of plaques is the underlying mechanism of approximately 30% of heart attacks. It describes a pathology where endothelial detachment from an intact fibrous cap (normally over a highly-stenotic plaque) precipitates thrombosis, triggering an acute coronary syndrome. We have developed an in vitro model to explore potential mechanisms involved in endothelial erosion, by mimicking the combined effects of endothelial dysfunction and elevated flow/shear (ESS) experienced over stenotic atherosclerotic plaques. Human coronary artery endothelial cells (HCAEC) exposed to 5 ng/ml TNF-α and aqueous cigarette smoke extract (CSE) suffered ~30% cell loss when adapted to elevated shear stress (ESS), with no cell loss observed under oscillatory shear stress. Treatment with apoptosis inhibitor (Z-VAD-FMK) or matrix metalloproteinase inhibitor (GM6001) did not prevent cell loss. A robust activation of Nrf2-regulated genes was observed by CSE, which was amplified under ESS by TNF-α. Inclusion of Nrf2 activators sulforaphane (2.5 µM) or isoliquiritigenin (10 µM) triggered ~80% cell loss at elevated shear stress with TNF-α and CSE, implying that hyperactivation of the Nrf2 system, may promote, rather than protect from cell detachment. Expression of both OSGIN1 and OSGIN2 were maximally increased under conditions where cells were detaching, and both were upregulated by Nrf2 activation. To investigate their role in detachment, they were overexpressed using adenoviral vectors. OSGIN1 +2 overexpression in static culture resulted in cell cycle arrest in S-phase (control –CTL v OSGIN1 +2: 5.5-fold, p=0.003), with a significant increase in the number of multinucleated cells (CTL v OSGIN1 +2: 4.5-fold, p=

Free radicals, reactive oxygen/nitrogen species (ROS/RNS), hydrogen sulphide, and hydrogen peroxide play an important role in both intracellular and intercellular signaling; however, their production and quenching need to be closely regulated to prevent cellular damage. An imbalance, due to exogenous sources of free radicals and chronic upregulation of endogenous production, contributes to many pathological conditions including cardiovascular disease and also more general processes involved in aging. Nuclear factor erythroid 2-like 2 (NFE2L2; commonly known as Nrf2) is a transcription factor that plays a major role in the dynamic regulation of a network of antioxidant and cytoprotective genes, through binding to and activating expression of promoters containing the antioxidant response element (ARE). Nrf2 activity is regulated by many mechanisms, suggesting that tight control is necessary for normal cell function and both hypoactivation and hyperactivation of Nrf2 are indicated in playing a role in different aspects of cardiovascular disease. Targeted activation of Nrf2 or downstream genes may prove to be a useful avenue in developing therapeutics to reduce the impact of cardiovascular disease. We will review the current status of Nrf2 and related signaling in cardiovascular disease and its relevance to current and potential treatment strategies.

Proliferation and migration of vascular smooth muscle cells (VSMCs) or endothelial cell (ECs) promote or inhibit, respectively, restenosis after angioplasty, vein graft intimal thickening and atherogenesis. Here we investigated the effects of cAMP-induced cytoskeletal remodelling on the serum response factor (SRF) co-factors Megakaryoblastic Leukemia-1 and -2 (MKL1 and MKL2) and their role in controlling VSMC and EC proliferation and migration. Elevation of cAMP using forskolin, dibutyryl-cAMP (db-cAMP), BAY60-6583 or Cicaprost induced rapid cytoskeleton remodelling and inhibited proliferation and migration in VSMCs but not EC. Furthermore, elevated cAMP inhibited mitogen-induced nuclear-translocation of MKL1 and MKL2 in VSMCs but not ECs. Forskolin also significantly inhibited serum response factor (SRF)-dependent reporter gene (SRE-LUC) activity and mRNA expression of pro-proliferative and pro-migratory MKL1/2 target genes in VSMCs but not in ECs. In ECs, MKL1 was constitutively nuclear and MKL2 cytoplasmic, irrespective of mitogens or cAMP. Pharmacological or siRNA inhibition of MKL1 significantly inhibited the proliferation and migration of VSMC and EC. Our new data identifies and important contribution of MKL1/2 to explaining the strikingly different response of VSMCs and ECs to cAMP elevation. Elucidation of these pathways promises to identify targets for specific inhibition of VSMC migration and proliferation.

During the 2016 Hangzhou G20 Summit, the chemical composition of submicron particles (PM

Objective— Our recent studies have highlighted membrane type-1 matrix metalloproteinase (MMP)-14 as a selective marker for an invasive subset of macrophages potentially related to atherosclerotic plaque progression. Moreover, colony stimulating factors (CSF) may exert divergent effects on macrophage MMP expression, possibly through microRNAs. We, therefore, aim to identify and test the pathophysiological role of microRNAs, which modulate macrophage MMP-14 expression in atherosclerotic plaque progression. Approach and Results— Compared with macrophage CSF–differentiated macrophages, granulocyte/macrophage CSF–matured macrophages exhibited reduced MMP-14 mRNA levels but increased protein expression and activity, which resulted in heightened macrophage invasion. MicroRNA-24, identified to target MMP-14, was accordingly increased in macrophage CSF compared with granulocyte/macrophage CSF macrophages. Silencing microRNA-24 in macrophage CSF macrophages significantly increased MMP-14 expression and enhanced their invasive capacity, mimicking granulocyte/macrophage CSF macrophages, and suggesting that granulocyte/macrophage CSF modulates MMP-14 protein expression and subsequent macrophage invasion in a microRNA-24–dependent manner. In human coronary atherosclerotic plaques, increased MMP-14 protein expression in foam cell macrophages was associated with lesions exhibiting histological characteristics associated with an unstable phenotype. Furthermore, microRNA-24 expression in these atherosclerotic plaques was inversely related to MMP-14 protein expression. Moreover, stable plaques contained higher microRNA-24 levels than unstable plaques, and microRNA-24 colocalized with foam cell macrophages that exhibited low MMP-14 protein expression. Finally, in atherosclerotic mice (apolipoprotein E-deficient), microRNA-24 inhibition increased plaque size and macrophage MMP-14 expression. Conclusions— Taken together, our data demonstrates that downregulation of microRNA-24 promotes an invasive macrophage subset and plays a novel regulatory role in MMP-14 proteolytic activity and, therefore, plaque stability, highlighting its therapeutic potential.

Histological images from the longitudinal section of four diseased coronary arteries were used, for the first time, to study the pulsatile blood flow distribution within the lumen of the arteries by means of computational fluid dynamics (CFD). Results indicate a strong dependence of the hemodynamics on the morphology of atherosclerotic lesion. Distinctive flow patterns appear in different stenosed regions corresponding to the specific geometry of any artery. Results show that the stenosis affects the wall shear stress (WSS) locally along the diseased arterial wall as well as other adjacent walls. The maximum magnitude of WSS is observed in the throat of stenosis. Moreover, high oscillatory shear index (OSI) is observed along the stenosed wall and the high curvature regions. The present study is capable of providing information on the shear environment in the longitudinal section of the diseased coronary arteries, based on the models created from histological images. The computational method may be used as an effective way to predict plaque forming regions in healthy arterial walls.

Endothelial dysfunction caused by the combined action of disturbed flow, inflammatory mediators and oxidants derived from cigarette smoke is known to promote coronary atherosclerosis and increase the likelihood of myocardial infarctions and strokes. Conversely, laminar flow protects against endothelial dysfunction, at least in the initial phases of atherogenesis. We studied the effects of TNFα and cigarette smoke extract on human coronary artery endothelial cells under oscillatory, normal laminar and elevated laminar shear stress for a period of 72 hours. We found, firstly, that laminar flow fails to overcome the inflammatory effects of TNFα under these conditions but that cigarette smoke induces an anti-oxidant response that appears to reduce endothelial inflammation. Elevated laminar flow, TNFα and cigarette smoke extract synergise to induce expression of the transcriptional regulator activating transcription factor 3 (ATF3), which we show by adenovirus driven overexpression, decreases inflammatory gene expression independently of activation of nuclear factor-κB. Our results illustrate the importance of studying endothelial dysfunction in vitro over prolonged periods. They also identify ATF3 as an important protective factor against endothelial dysfunction. Modulation of ATF3 expression may represent a novel approach to modulate proinflammatory gene expression and open new therapeutic avenues to treat proinflammatory diseases.