Search

Aims/hypothesis Diabetic cardiomyopathy (DCM) is a serious and under-recognised complication of diabetes. The first sign is diastolic dysfunction, which progresses to heart failure. The pathophysiology of DCM is incompletely understood but microcirculatory changes are important. Endothelial glycocalyx (eGlx) plays multiple vital roles in the microcirculation, including in the regulation of vascular permeability, and is compromised in diabetes but has not previously been studied in the coronary microcirculation in diabetes. We hypothesised that eGlx damage in the coronary microcirculation contributes to increased microvascular permeability and hence to cardiac dysfunction. Methods We investigated eGlx damage and cardiomyopathy in mouse models of type 1 (streptozotocin-induced) and type 2 (db/db) diabetes. Cardiac dysfunction was determined by echocardiography. We obtained eGlx depth and coverage by transmission electron microscopy (TEM) on mouse hearts perfusion-fixed with glutaraldehyde and Alcian Blue. Perivascular oedema was assessed from TEM images by measuring the perivascular space area. Lectin-based fluorescence was developed to study eGlx in paraformaldehyde-fixed mouse and human tissues. The eGlx of human conditionally immortalised coronary microvascular endothelial cells (CMVECs) in culture was removed with eGlx-degrading enzymes before measurement of protein passage across the cell monolayer. The mechanism of eGlx damage in the diabetic heart was investigated by quantitative reverse transcription-PCR array and matrix metalloproteinase (MMP) activity assay. To directly demonstrate that eGlx damage disturbs cardiac function, isolated rat hearts were treated with enzymes in a Langendorff preparation. Angiopoietin 1 (Ang1) is known to restore eGlx and so was used to investigate whether eGlx restoration reverses diastolic dysfunction in mice with type 1 diabetes. Results In a mouse model of type 1 diabetes, diastolic dysfunction (confirmed by echocardiography) was associated with loss of eGlx from CMVECs and the development of perivascular oedema, suggesting increased microvascular permeability. We confirmed in vitro that eGlx removal increases CMVEC monolayer permeability. We identified increased MMP activity as a potential mechanism of eGlx damage and we observed loss of syndecan 4 consistent with MMP activity. In a mouse model of type 2 diabetes we found a similar loss of eGlx preceding the development of diastolic dysfunction. We used isolated rat hearts to demonstrate that eGlx damage (induced by enzymes) is sufficient to disturb cardiac function. Ang1 restored eGlx and this was associated with reduced perivascular oedema and amelioration of the diastolic dysfunction seen in mice with type 1 diabetes. Conclusions/interpretation The association of CMVEC glycocalyx damage with diastolic dysfunction in two diabetes models suggests that it may play a pathophysiological role and the enzyme studies confirm that eGlx damage is sufficient to impair cardiac function. Ang1 rapidly restores the CMVEC glycocalyx and improves diastolic function. Our work identifies CMVEC glycocalyx damage as a potential contributor to the development of DCM and therefore as a therapeutic target. Graphical abstract

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.

The vertebrate stress response comprises a suite of behavioural and physiological traits that must be functionally integrated to ensure organisms cope adaptively with acute stressors. Natural selection should favour functional integration, leading to a prediction of genetic integration of these traits. Despite the implications of such genetic integration for our understanding of human and animal health, as well as evolutionary responses to natural and anthropogenic stressors, formal quantitative genetic tests of this prediction are lacking. Here, we demonstrate that acute stress response components in Trinidadian guppies are both heritable and integrated on the major axis of genetic covariation. This integration could either facilitate or constrain evolutionary responses to selection, depending upon the alignment of selection with this axis. Such integration also suggests artificial selection on the genetically correlated behavioural responses to stress could offer a viable non-invasive route to the improvement of health and welfare in captive animal populations.

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.

How, if at all, does one’s intention to realize an end bear on the justification for taking the means to that end? Theories that allow that intending an end directly provides a reason to take the means are subject to a well-known "bootstrapping" objection. On the other hand, "anti-psychologistic" accounts—which seek to derive instrumental reasons directly from the reasons that support adopting the end itself—have unacceptable implications where an agent faces multiple rationally permissible options. An alternative, predictive, role for intention in means-end reasoning is considered and rejected. A new proposal is then developed, according to which instrumental reasons are not merely reasons to perform an act necessary for a given end, but to perform the act for the sake of that end.

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.

The formation and growth of new particles has recently been shown to have a significant influence on Chinese haze pollution, and sulfuric acid has long been recognized as a major contributor to new particle formation. In this study, four comparison groups of experiments related to SO 2 photooxidation, as well as aerosol nucleation and growth, have been conducted in the CAPS-ZJU (Complex Air Pollution Study-Zhejiang University) smog chamber. These were conducted either under SO 2 /NOx or SO 2 /toluene gas-phase environments in the absence of seed particles. During aerosol nucleation and growth process, several physical properties such as mass, size and effective density were measured simultaneously by Scanning Mobility Particle Sizer (SMPS) and Differential Mobility Analyzer-Aerosol Particle Mass Analyzer-Condensation Particle Counter (DMA-APM-CPC). The effective density of new particles decreased from 1.8 to 1.35 g/cm 3 as the particle size increased from 20 to 65 nm. The single particle mass showed good power-law relationship with mobility diameter, with an average mass-mobility exponent of 2.885. A new algorithm and a reference density of 1.38 g/cm 3 based on size-resolved single particle mass (SPM) were proposed to calculate the mass concentration of new particles. Two methods based on Log Normal and Max Concentration were applied to derive particle growth rate (GR), and data merging from both methods was implemented to decrease calculation uncertainty. Meanwhile, both continuous nucleation and inhibition of further growth in sub-20 nm size range were observed in different experiments depending on composition, and possible reasons were analyzed. The presence of NO was found to suppress nucleation and subsequent aerosol growth; while the presence of NO 2 or toluene promoted it. It was concluded that decreasing NOx (NO or NO 2 ) or increasing toluene may promote SO 2 photooxidation, nucleation and subsequent aerosol growth, all of which is significant for deeper understanding of complex air pollution in China.

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

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

© 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.

Highlights • Human coronary artery endothelial cells show a biological response to cigarette smoke. • This response was not seen following exposure to e-cigarette aerosol. • Using e-cigarettes instead of cigarettes may reduce immediate cardiovascular harms., Background It is generally acknowledged that e-cigarettes are unlikely to be as harmful as conventional cigarettes, but there is little data that quantifies their relative harms. We investigated the biological response to e-cigarette aerosol exposure (versus conventional cigarette smoke exposure) at the cellular level, by exposing human coronary artery endothelial cells (HCAEC) to aqueous filtered extracts of e-cigarette aerosol or cigarette smoke and looking at gene expression changes consistent with a stress response. This included genes controlled by the oxidant-stress sensing transcription factor NFR2 (NFE2L2), and cytochrome P450 family members. Methods Cigarette smoke extract (CSE) was created using mainstream smoke from a single cigarette drawn through 10 ml of endothelial cell growth media MV2. Electronic cigarette aerosol extract (eCAE) was created using the same apparatus, using a constant power output of 10.8 w (4.2 V) and 18 mg/ml nicotine solution. eCAE was generated using 5 cycles of 5 s heat with at least 10 s in between each puff to allow the coil to cool, air being drawn through the device at 70 ml/minute. Results HCAEC responded to the noxious components in CSE, resulting in activation of NRF2 and upregulation of cytochrome p450. However, eCAE did not induce NRF2 nuclear localisation, upregulation of NRF2-activated genes, or the upregulation of cytochrome p450. Conclusions The use of e-cigarettes as a substitute for conventional cigarettes is likely to reduce immediate tobacco-related harm, at least with respect to cardiovascular harms.

Endothelial erosion of atherosclerotic plaques and resulting thrombosis causes approximately 30% of acute coronary syndromes (ACS). As changes in the haemodynamic environment strongly influence endothelial function and contribute to plaque development, we reconstructed the coronary artery geometries of plaques with thrombi overlying intact fibrous caps from 17 ACS patients and performed computational fluid dynamic analysis. The results demonstrated that erosions frequently occur within areas of stenosis exposed to elevated flow. We recapitulated this flow environment in vitro, exposing human coronary artery endothelial cells to elevated flow and modelled smoking (a risk factor for erosion) by exposure to a combination of aqueous cigarette smoke extract and TNFα. This treatment induced endothelial detachment, which increased with pharmacological activation of the antioxidant system controlled by transcription factor Nrf2 (encoded by NFE2L2). The expression of Oxidative Stress Growth INhibitor genes OSGIN1 and OSGIN2 increased under these conditions and also in the aortas of mice exposed to cigarette smoke. Sustained high level expression of OSGIN1+2 resulted in cell cycle arrest, induction of senescence, loss of focal adhesions and actin stress fibres, and dysregulation of autophagy. Overexpression of either Nrf2 or OSGIN1+2 induced cell detachment, which did not depend on apoptosis, and could be partially rescued by inhibition of HSP70 using VER-155008, or AMP kinase activation using metformin. These findings demonstrate that under elevated flow, smoking-induced hyperactivation of Nrf2 can trigger endothelial cell detachment, highlighting a novel mechanism that could contribute to ACS involving endothelial erosion overlying stenotic plaques.

Protease-activated receptor 4 (PAR4) is a potent thrombin receptor. Epigenetic control of theF2RL3locus (which encodes for PAR4) via DNA methylation is associated with both smoking and cardiovascular disease. We examined the association between DNA hypomethylation atF2RL3and risk of cardiovascular disease, focusing on acute myocardial infarction (AMI) (n=853 cases / 2,352 controls). We usedin vitrocell models to dissect the role of DNA methylation in regulating expression ofF2RL3.We investigated the interplay betweenF2RL3DNA methylation and platelet function in human (n=41). Lastly, we used Mendelian randomization to unify observational and functional work by assessing evidence for causal relationships using data from UK Biobank (n=407,141) and CARDIoGRAMplusC4D (n=184,305). Observationally, one standard deviation (SD) decrease in DNA methylation atF2RL3was associated with a 25% increase in the odds of AMI.In vitro, short-term exposure of cells to cigarette smoke reducedF2RL3DNA methylation and increased gene expression. Transcriptional assays flagged a role for a CEBP recognition sequence in modulating the enhancer activity ofF2RL3exon 2. Lower DNA methylation atF2RL3was associated with increased platelet reactivity in human. The estimated casual odds ratio of ischaemic heart disease was 1.03 (95% CI: 1.00, 1.07) per 1 SD decrease inF2RL3DNA. In conclusion, we show that DNA methylation-dependent platelet activation is part of a complex system of features contributing to cardiovascular health. Tailoring therapeutic intervention to new knowledge ofF2RL3/PAR4 function should be explored to ameliorate the detrimental effects of this risk factor on cardiovascular health.One sentence summaryDNA methylation-dependent platelet activation is a likely causal contributor to cardiovascular health.

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.

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.

Raised endothelial shear stress is protective against atherosclerosis but such protection may be lost at sites of inflammation. We found that four splice variants of the peptidase inhibitor 16 (PI16) mRNA are among the most highly shear stress regulated transcripts in human coronary artery endothelial cells (HCAECs), in vitro but that expression is reduced by inflammatory mediators TNFα and IL-1β. Immunohistochemistry demonstrated that PI16 is expressed in human coronary endothelium and in a subset of neointimal cells and medial smooth muscle cells. Adenovirus-mediated PI16 overexpression inhibits HCAEC migration and secreted matrix metalloproteinase (MMP) activity. Moreover, PI16 inhibits MMP2 in part by binding an exposed peptide loop above the active site. Our results imply that, at high endothelial shear stress, PI16 contributes to inhibition of protease activity; protection that can be reversed during inflammation.

Atherosclerosis and aneurysms are leading causes of mortality worldwide. MicroRNAs (miRs) are key determinants of gene and protein expression, and atypical miR expression has been associated with many cardiovascular diseases; although their contributory role to atherosclerotic plaque and abdominal aortic aneurysm stability are poorly understood.To investigate whether miR-181b regulates tissue inhibitor of metalloproteinase-3 expression and affects atherosclerosis and aneurysms.Here, we demonstrate that miR-181b was overexpressed in symptomatic human atherosclerotic plaques and abdominal aortic aneurysms and correlated with decreased expression of predicted miR-181b targets, tissue inhibitor of metalloproteinase-3, and elastin. Using the well-characterized mouse atherosclerosis models of ApoeOur findings suggest that the management of miR-181b and its target genes provides therapeutic potential for limiting the progression of atherosclerosis and aneurysms and protecting them from rupture.

AimsThe oxidized low-density lipoprotein receptor LOX-1 is up-regulated on activated endothelial cells, for example, the endothelium of atherosclerosis-prone sites, in both human and animal models. We examined whether endothelial LOX-1 overexpression may contribute to atherogenesis.MethodsAdenoviral vectors expressing LOX-1 or LOXIN (a splice variant of LOX-1 with inhibitory function) were created and used to transduce the normally lesion-free common carotid artery, in high fat-fed female ApoE−/− mice. Mice were placed on high-fat diet for 4 weeks prior to gene transfer with either LOX-1 or a combination of LOX-1 and LOXIN, and assessment of plaque development analyzed 6 weeks following gene transfer.ResultsCompared to controls, LOX-1 transduction induced a significant increase in plaque coverage within the common carotid artery to 91% compared to 50% after RAd66 control virus infection (P≤.05). This was inhibited by co-expression of LOXIN (62%).ConclusionsThese results demonstrate that up-regulation of LOX-1 promotes atherogenesis, highlighting LOX-1 function as a target for intervention. In addition, this study further demonstrated the inhibitory function of LOXIN.

Most acute coronary events occur in the upstream region of stenotic atherosclerotic plaques that experience laminar shear stress (LSS) elevated above normal physiological levels. Many studies have described the atheroprotective effect on endothelial behavior of normal physiological LSS (approximately 15 dynes/cm2) compared to static or oscillatory shear stress (OSS), but it is unknown whether the levels of elevated shear stress imposed by a stenotic plaque would preserve, enhance or reverse this effect. Therefore we used transcriptomics and related functional analyses to compare human endothelial cells exposed to laminar shear stress of 15 (LSS15-normal) or 75 dynes/cm2 (LSS75-elevated). LSS75 upregulated expression of 145 and downregulated expression of 158 genes more than twofold relative to LSS15. Modulation of the metallothioneins (MT1-G, -M, -X) and NADPH oxidase subunits (NOX2, NOX4, NOX5, and p67phox) accompanied suppression of reactive oxygen species production at LSS75. Shear induced changes in dual specificity phosphatases (DUSPs 1, 5, 8, and 16 increasing and DUSPs 6 and 23 decreasing) were observed as well as reduced ERK1/2 but increased p38 MAP kinase phosphorylation. Amongst vasoactive substances, endothelin-1 expression decreased whereas vasoactive intestinal peptide (VIP) and prostacyclin expression increased, despite which intracellular cAMP levels were reduced. Promoter analysis by rVISTA identified a significant over representation of ATF and Nrf2 transcription factor binding sites in genes upregulated by LSS75 compared to LSS15. In summary, LSS75 induced a specific change in behavior, modifying gene expression, reducing ROS levels, altering MAP kinase signaling and reducing cAMP levels, opening multiple avenues for future study. J. Cell. Physiol. 226: 2841–2848, 2011. © 2011 Wiley-Liss, Inc.

Aims Vascular smooth muscle cell (VSMC) apoptosis can lead to thinning of the fibrous cap and plaque instability. We previously showed that cell–cell contacts mediated by N-cadherin reduce VSMC apoptosis. This study aimed to determine whether matrix-degrading metalloproteinase (MMP)-dependent N-cadherin cleavage causes VSMC apoptosis. Methods and results Induction of human VSMC apoptosis using different approaches, including 200 ng/mL Fas ligand (Fas-L) and culture in suspension, caused N-cadherin cleavage and resulted in the appearance of a C-terminal fragment of N-cadherin (∼35 kDa). Appearance of this fragment during apoptosis was inhibited by 47% with the broad-spectrum MMP inhibitor BB-94. We observed retarded cleavage of N-cadherin after treatment with Fas-L in aortic mouse VSMCs lacking MMP-7. Furthermore, VSMC apoptosis, measured by quantification of cleaved caspase-3, was 43% lower in MMP-7 knockout mouse VSMCs compared with wild-type VSMCs following treatment with Fas-L. Addition of recombinant active MMP-7 increased the amount of N-cadherin fragment by 82% and augmented apoptosis by 53%. The involvement of MMP-7 was corroborated using human cells, where a MMP-7 selective inhibitor reduced the amount of fragment formed by 51%. Importantly, we observed that treatment with Fas-L increased levels of active MMP-7 by 80%. Finally, we observed significantly increased cleavage of N-cadherin, MMP-7 activity, and apoptosis in human atherosclerotic plaques compared with control arteries, and a significant reduction in apoptosis in atherosclerotic plaques from MMP-7 knockout mice. Conclusion This study demonstrates that MMP-7 is involved in the cleavage of N-cadherin and modulates VSMC apoptosis, and may therefore contribute to plaque development and rupture.

Background—Gene therapy offers an unprecedented opportunity to treat diverse pathologies. Adeno-associated virus (AAV) is a promising gene delivery vector for cardiovascular disease. However, AAV transduces the liver after systemic administration, reducing its usefulness for therapies targeted at other sites. Because vascular endothelial cells (ECs) are in contact with the bloodstream and are heterogeneous between organs, they represent an ideal target for site-specific delivery of biological agents.Methods and Results—We isolated human venous EC-targeting peptides by phage display and genetically incorporated them into AAV capsids after amino acid 587. Peptide-modified AAVs transduced venous (but not arterial) ECs in vitro, whereas hepatocyte transduction was significantly lower than with native AAV. Intravenous infusion of engineered AAVs into mice produced reduced vector accumulation in liver measured 1 hour and 28 days after injection and delayed blood clearance rates compared with native AAV. Peptide-modified AAVs produced enhanced uptake of virions in the vena cava with selective transgene expression. Retargeting was dose dependent, and coinfusion of either heparin or free competing peptides indicated that uptake was principally independent of native AAV tropism and mediated via the peptide.Conclusions—AAV tropism can be genetically engineered by use of phage display–derived peptides to generate vectors that are selective for the vasculature.

Atherosclerotic plaque composition can be imaged using the optical attenuation coefficient derived from intravascular optical coherence tomography (OCT) data. The relation between optical properties and tissue type has been established on autopsy tissues. In this study, we validate an ex-vivo model for the effect of temperature and tissue fixation on optical parameters. We studied the optical attenuation of human coronary arteries at three temperatures, before and after formalin fixation. We developed an en-face longitudinal display of attenuation data of the OCT pullbacks. Using the unfixed, body-temperature condition image as a standard, and after extensive registration with other condition images, we quantify the differences in optical attenuation and the backscattered intensity. The results suggest that tissue fixation and temperature do not introduce systematic errors in studies of arterial optical properties. (C) 2014 Optical Society of America

Atherosclerotic plaque instability is precipitated by vascular smooth muscle cell apoptosis in the fibrous cap, weakening it and leading to plaque rupture. We previously showed that reducing smooth muscle cell apoptosis with soluble N-cadherin (SNC) increased features of plaque stability. We have now identified the active site of SNC and examined whether a truncated form containing this site retains the antiapoptotic effect. SNC was mutated to prevent interaction with N-cadherin or fibroblast growth factor receptor (FGFR). Interaction with FGFR in the extracellular (EC) 4 domain of SNC was essential for the antiapoptotic effect. Therefore, we made a truncated form consisting of the EC4 domain. EC4 significantly reduced smooth muscle cell, macrophage, and endothelial cell apoptosis in vitro by ~70%, similar to SNC. Elevation of plasma levels of EC4 in male apolipoprotein E–deficient mice with existing atherosclerosis significantly reduced apoptosis in brachiocephalic artery plaques by ~50%. EC4 reduced plaque size and the incidence of buried fibrous layers and the macrophage:smooth muscle cell ratio (surrogate markers of plaque instability). Interaction of EC4 with FGFR induced potent antiapoptotic signaling in vitro and in vivo. EC4 modulates atherosclerosis in mice demonstrating its therapeutic potential for retarding plaque size and instability.

Increasing the level and duration of transgene expression and restricting expression to vascular cells are important goals for clinically useful gene therapy vectors. We evaluated several promoters, enhancers and introns in endothelial, smooth muscle and liver cells in tissue culture and in vivo, comparing local delivery to the carotid artery with intravenous delivery to the liver. A 1800-bp fragment of the oxidized LDL receptor (LOX-1) promoter showed highest in vivo activity in the carotid artery, achieving 39% the activity of the reference cytomegalovirus promoter, with 188-fold greater specificity for carotid artery over liver. An enhancer from the Tie2 gene in combination with the intracellular adhesion molecule-2 promoter improved endothelial specificity of plasmid vectors, increased the expression from adenoviral vectors in cultured endothelial cells and doubled the specificity for carotid artery over liver in vivo. Adding a short intron to expression cassettes increased expression in both endothelial and smooth muscle cells in vitro; however, the eNOS enhancer failed to consistently increase the expression or endothelial specificity of the vector. In conclusion, elements from the LOX-1 promoter and Tie2 enhancer together with an intron can be used to improve vectors for vascular gene transfer.

Sequestration of adenovirus serotype 5 (Ad5) in liver restricts its use for gene delivery to other target sites in vivo. To date, no studies have systematically assessed the impact of genetic capsid modifications on in vivo tropism in rats, an important preclinical model for many disease types. We evaluated a panel of Ad5 vectors with capsid mutations or pseudotyped with the short fiber from serotype 41 (Ad41s) for infectivity in Wistar Kyoto rats in vitro and systemically in vivo. In vitro studies demonstrated that both coxsackie and adenovirus receptor (CAR) and heparan sulfate proteoglycan (HSPG) binding were predominant predictors of Ad5 tropism. In vivo, neither CAR nor integrin mutations alone affected liver transduction. The HSPG-binding mutation alone moderately reduced rat liver transgene levels by 2-fold (P0.05). This was further substantially decreased by additional mutation of CAR binding (95-fold). Combining CAR and integrin mutations reduced transgene levels by99% (509-fold, P0.01), an effect not observed in parallel experiments in mice and highly variable when studied further in an additional two strains of rat. Ad41s mediated very low liver transduction (58-fold lower than AdCTL). Moreover, CAR-binding mutants (KO1-containing) or pseudotyping 41s eliminated hemagglutination of rat and human red blood cells in vitro. This highlights some important potential species and strain differences dictating Ad5 tropism in vivo and identifies vectors that are substantially detargeted from rat liver in vivo.

Background Intravascular imaging modalities are used clinically to investigate ambiguous angiographic coronary lesions, guide and optimise stent deployment, and assess stent-related complications. Both intravascular ultrasound (IVUS) and optical coherence tomography (OCT) facilitate characterisation of plaque components, although a lack of adequate spatial resolution and depth of penetration, respectively, limit their clinical application. Adaptation of the existing technologies and novel techniques are in development but require further validation. We have developed an ex-vivo whole heart cadaveric model that facilitates multi-modality imaging and accurate comparison with histology. Methods We have developed a model for fluoroscopic and invasive assessment of coronary arteries within “whole heart” cadaveric specimens. Hearts are provided by the West of England heart valve bio-bank, following harvesting of valves for homograft production. The coronary ostium is dissected and mobilised to allow cannulation with a modified 6F coronary guiding catheter (Abstract 023 figure 1A), secured with sutures. The cadaveric specimen is held within a purpose-built perspex container, with adaptors on both sides of the container9s lid allowing connection of the guide catheter internally, and a Y-connector and pressure/injector manifold externally, see Abstract 023 figure 1 panel B. Cadaveric specimens undergo angiography (see Abstract 023 figure 1C,D), placement of a 0.014″ guide wire and imaging catheter manipulation with the artery held at physiological temperature and pressure. In collaboration with the Department of Bioengineering, Erasmus MC, Rotterdam, we are using this model for assessment and validation of optical attenuation analysis as a tool to accurately delineate areas of macrophage infiltration, a major marker of plaque vulnerability. Optical attenuation governs the signal drop-off associated with tissue penetration. It is derived by fitting the following functional relation to the OCT data, I (z) = I 0 exp(- μ t z), where μ t (z) is the local optical attenuation, the parameter of interest. The local signal intensity, I 0 , is also a free parameter in the fit, but is currently not analysed. The data are fitted in windows of 200 μm length, after appropriate processing to reduce speckle noise. Results Comparison of OCT, IVUS, and VH-IVUS against histology confirm the challenges in characterising plaque (Abstract 023 figure 2A–D: arrows indicate calcium). Analysis of optical attenuation appears to correlate with areas of macrophage infiltration (arrows in Abstract 023 figure 2E–H). Conclusions Our ex-vivo whole heart cadaveric model facilitates accurate comparison of imaging modalities against histology. Developments in the imaging technologies are necessary to facilitate plaque characterisation as a clinical application. Optical attenuation may offer additional information regarding the macrophage content and “vulnerability” of plaque, validation work using our cadaveric model is ongoing.

Purpose: Foam cell macrophage (FCM) formation is an early event in atherosclerosis that could contribute to fibrous cap development, although it has also been strongly implicated in cap rupture and thrombus formation. To understand this apparent paradox we compared the transcriptome of FCM and non-foamy macrophages (NFM) produced in vivo using Illumina bead chips. Methods: Sponges were implanted into fat-fed ApoE null or chow-fed C57Bl6 mice to produce FCM or NFM, respectively. Cells were purified based on their buoyant density and/or differential adherence. Gene expression was examined using Ingenuity Pathway Analysis, GO annotation and clustering (DAVID Bioinformatics Resources). Data was confirmed using q-PCR and IHC. Results: The functions enriched/upregulated in FCM included connective tissue development, function and disorders as well as immune response, cell signalling. The primary canonical pathway upregulated in the FCM was liver X receptor/retinoid X receptor (LXR/RXR) activation, consistent with a recent study using peritoneal FCM from LDLr knockout mice (Spann et al Cell 2012;151:138). We confirmed that FCM had more mRNA for LXR, RXR and target genes using qRT-PCR, and used IHC to identify LXRα containing FCM in the sponges. GO identified hepatic fibrosis pathways as highly upregulated in the FCM and Ingenuity suggested a role for transforming growth factor β1 (TGFβ1). For example, there was an increase in mRNA for many extracellular matrix proteins (including collagens 1, 4, 6, 8, biglycan and decorin), connective tissue growth factor (CTGF), BMP-1 and Fos/FosB/Jun/JunB. FCM also had more mRNA for matrix proteases (including cathepsins C and E and matrix metalloproteinases 2 and 23), without a corresponding difference in their inhibitors. Using IHC we established that many cells within the sponges and brachiocephalic arteries from fat-fed ApoE mice contained CTGF. As TGFβ1-induced CTGF expression is known to be regulated via SMADs, we examined these tissues for the presence of phosphoSMAD2 (pSMAD2) by IHC and found that it was present in the cytoplasm and nucleus of sponge FCM and plaque cells. Conclusions: Our data confirm the paradox introduced by Spann and colleagues showing that FCMs are anti-inflammatory via LXR activation. We have extended this by showing that FCM can contribute to fibrosis and matrix deposition, possibly due to exposure to TGFβ1. Clearly additional factors, including mediators of innate and acquired immunity, must be responsible for converting FCM from an anti-inflammatory/pro-fibrotic phenotype into cells capable of mediating plaque rupture.

Top of pageAbstract Successful gene transfer to vascular tissue requires gene expression cassettes that are capable of achieving both high levels and sustained transgene expression. Regulation of gene expression is achieved at multiple levels: transcription, mRNA processing, nuclear export, translation and post-translational processing. To achieve maximal expression of transgenes, expression cassettes need to be optimised to ensure all of these processes are operating efficiently. The overall design of this project has two steps: (I) in vitro assessment of individual non-viral transcription elements with the aim of maximising expression; (II) construction of adenoviral vectors containing optimised constructs to assess tropism, magnitude and longevity of expression in vitro and in vivo. To this end a number of expression cassettes have been constructed to individually assess selected promoters (ICAM-2, ICAM-1, Lox-1250/1800, Flt-1274/1031, Tie-2 and CMV), enhancers (Tie-2 and eNOS), introns, exonic splicing enhancers and matrix attachment regions to initiate and augment gene expression in primary endothelial cells. Novel findings of this study have identified exonic splicing enhancers (ESEs) as critical elements for achieving high-level transgene expression in primary human umbilical vein endothelial cells (HUVECs). ESEs are multifunctional regulators of mRNA metabolism with diverse roles that couple the process of splicing, mRNA export and translation. The Serine-Arginine rich (SR) proteins that bind to these splicing enhancers are important in regulating splice site usage and nuclear export, regulating alternative splicing in a cell-type specific manner and in response to both inter and intracellular signalling. In addition, at least one member of this family (SF2/ASF) stays associated with the mature mRNA and increases translation efficiency. To assess the ability of these splicing enhancers to increase expression of transgenes, naturally occurring and consensus binding sites for a number of SR proteins (SF2/ASF, SC35, SRp40 and SRp55) were introduced after the splice acceptor site of a short intron. Expression cassettes containing the modified introns showed only modest variations in transgene expression compared to the original intron in HeLa cells. In HUVECs however, all but one of the modified introns demonstrated an increase in expression, by between 20 and 40 fold. In addition, short novel matrix attachment regions (MARs) isolated from the Tie-2 intergenic region, have shown the ability to augment transcription by up to 3-fold in HeLa. 12 different adenoviral vectors are currently in production to assess these constructs in a range of other cell-types as well as in vivo. This study underlines the need to include elements within gene expression cassettes capable of positively influencing all steps that control expression.

The application of a microcomputer-based simulation model for analyzing the operation of a parking system is described as well as the model's results and sensitivity in coping with various uncertainties posed by the project's decision makers. Additional sensitivities were investigated based on an extrapolation of the natural growth of traffic flows and variations in vehicle arrival rates. The simulation model was designed to handle time headway, speed, intersections and stop signs, and peak traffic flows. The operational performance measures used for evaluating the system were the number of cars waiting at certain key intersections and at the entrances and exits of the parking structure, and the average amount of time that these cars spent waiting.