Epigenetic regulation of F2RL3 associates with myocardial infarction and platelet function

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

Cigarette smoke extract profoundly suppresses TNFα-mediated proinflammatory gene expression through upregulation of ATF3 in human coronary artery endothelial cells

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

Cigarette smoke but not electronic cigarette aerosol activates a stress response in human coronary artery endothelial cells in culture

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

The SNX-PX-BAR Family in Macropinocytosis: The Regulation of Macropinosome Formation by SNX-PX-BAR Proteins

Background: Macropinocytosis is an actin-driven endocytic process, whereby membrane ruffles fold back onto the plasma membrane to form large (> 0.2 mu m in diameter) endocytic organelles called macropinosomes. Relative to other endocytic pathways, little is known about the molecular mechanisms involved in macropinocytosis. Recently, members of the Sorting Nexin (SNX) family have been localized to the cell surface and early macropinosomes, and implicated in macropinosome formation. SNX-PX-BAR proteins form a subset of the SNX family and their lipid-binding (PX) and membrane-curvature sensing (BAR) domain architecture further implicates their functional involvement in macropinosome formation

The development and validation of a scoring tool to predict the operative duration of elective laparoscopic cholecystectomy

Background: The ability to accurately predict operative duration has the potential to optimise theatre efficiency and utilisation, thus reducing costs and increasing staff and patient satisfaction. With laparoscopic cholecystectomy being one of the most commonly performed procedures worldwide, a tool to predict operative duration could be extremely beneficial to healthcare organisations. Methods: Data collected from the CholeS study on patients undergoing cholecystectomy in UK and Irish hospitals between 04/2014 and 05/2014 were used to study operative duration. A multivariable binary logistic regression model was produced in order to identify significant independent predictors of long (> 90 min) operations. The resulting model was converted to a risk score, which was subsequently validated on second cohort of patients using ROC curves. Results: After exclusions, data were available for 7227 patients in the derivation (CholeS) cohort. The median operative duration was 60 min (interquartile range 45–85), with 17.7% of operations lasting longer than 90 min. Ten factors were found to be significant independent predictors of operative durations > 90 min, including ASA, age, previous surgical admissions, BMI, gallbladder wall thickness and CBD diameter. A risk score was then produced from these factors, and applied to a cohort of 2405 patients from a tertiary centre for external validation. This returned an area under the ROC curve of 0.708 (SE = 0.013, p  90 min increasing more than eightfold from 5.1 to 41.8% in the extremes of the score. Conclusion: The scoring tool produced in this study was found to be significantly predictive of long operative durations on validation in an external cohort. As such, the tool may have the potential to enable organisations to better organise theatre lists and deliver greater efficiencies in care

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome