Inducible changes in cell size and attachment area due to expression of a mutant SWI/SNF chromatin remodeling enzyme

The SWI/SNF enzymes belong to a family of ATP-dependent chromatin remodeling enzymes that have been functionally implicated in gene regulation, development, differentiation and oncogenesis. BRG1, the catalytic core subunit of some of the SWI/SNF enzymes, can interact with known tumor suppressor proteins and can act as a tumor suppressor itself. We report that cells that inducibly express ATPase-deficient versions of BRG1 increase in cell volume, area of attachment and nuclear size upon expression of the mutant BRG1 protein. Examination of focal adhesions reveals qualitative changes in paxillin distribution but no difference in the actin cytoskeletal structure. Increases in cell size and shape correlate with over-expression of two integrins and the urokinase-type plasminogen activator receptor (uPAR), which is also involved in cell adhesion and is often over-expressed in metastatic cancer cells. These findings demonstrate that gene expression pathways affected by chromatin remodeling enzymes can regulate the physical dimensions of mammalian cell morphology

Effects of tranexamic acid on death, disability, vascular occlusive events and other morbidities in patients with acute traumatic brain injury (CRASH-3): a randomised, placebo-controlled trial

Background Tranexamic acid reduces surgical bleeding and decreases mortality in patients with traumatic extracranial bleeding. Intracranial bleeding is common after traumatic brain injury (TBI) and can cause brain herniation and death. We aimed to assess the effects of tranexamic acid in patients with TBI. Methods This randomised, placebo-controlled trial was done in 175 hospitals in 29 countries. Adults with TBI who were within 3 h of injury, had a Glasgow Coma Scale (GCS) score of 12 or lower or any intracranial bleeding on CT scan, and no major extracranial bleeding were eligible. The time window for eligibility was originally 8 h but in 2016 the protocol was changed to limit recruitment to patients within 3 h of injury. This change was made blind to the trial data, in response to external evidence suggesting that delayed treatment is unlikely to be effective. We randomly assigned (1:1) patients to receive tranexamic acid (loading dose 1 g over 10 min then infusion of 1 g over 8 h) or matching placebo. Patients were assigned by selecting a numbered treatment pack from a box containing eight packs that were identical apart from the pack number. Patients, caregivers, and those assessing outcomes were masked to allocation. The primary outcome was head injury-related death in hospital within 28 days of injury in patients treated within 3 h of injury. We prespecified a sensitivity analysis that excluded patients with a GCS score of 3 and those with bilateral unreactive pupils at baseline. All analyses were done by intention to treat. This trial was registered with ISRCTN (ISRCTN15088122), ClinicalTrials.gov (NCT01402882), EudraCT (2011-003669-14), and the Pan African Clinical Trial Registry (PACTR20121000441277). Results Between July 20, 2012, and Jan 31, 2019, we randomly allocated 12 737 patients with TBI to receive tranexamic acid (6406 [50·3%] or placebo [6331 [49·7%], of whom 9202 (72·2%) patients were treated within 3 h of injury. Among patients treated within 3 h of injury, the risk of head injury-related death was 18·5% in the tranexamic acid group versus 19·8% in the placebo group (855 vs 892 events; risk ratio [RR] 0·94 [95% CI 0·86-1·02]). In the prespecified sensitivity analysis that excluded patients with a GCS score of 3 or bilateral unreactive pupils at baseline, the risk of head injury-related death was 12·5% in the tranexamic acid group versus 14·0% in the placebo group (485 vs 525 events; RR 0·89 [95% CI 0·80-1·00]). The risk of head injury-related death reduced with tranexamic acid in patients with mild-to-moderate head injury (RR 0·78 [95% CI 0·64-0·95]) but not in patients with severe head injury (0·99 [95% CI 0·91-1·07]; p value for heterogeneity 0·030). Early treatment was more effective than was later treatment in patients with mild and moderate head injury (p=0·005) but time to treatment had no obvious effect in patients with severe head injury (p=0·73). The risk of vascular occlusive events was similar in the tranexamic acid and placebo groups (RR 0·98 (0·74-1·28). The risk of seizures was also similar between groups (1·09 [95% CI 0·90-1·33]). Interpretation Our results show that tranexamic acid is safe in patients with TBI and that treatment within 3 h of injury reduces head injury-related death. Patients should be treated as soon as possible after injury. Funding National Institute for Health Research Health Technology Assessment, JP Moulton Charitable Trust, Department of Health and Social Care, Department for International Development, Global Challenges Research Fund, Medical Research Council, and Wellcome Trust (Joint Global Health Trials scheme)

Resistin increases monolayer permeability of human coronary artery endothelial cells.

Resistin has been linked to obesity, insulin resistance, atherosclerosis, and the development of cardiovascular disease. Nevertheless, the effects and the molecular mechanisms of resistin on endothelial permeability, a key event in the development of atherosclerosis, inflammation, and vascular disease, are largely unknown. In order to determine the effect of resistin on endothelial permeability, human coronary artery endothelial cells (HCAECs) were treated with clinically relevant concentrations of resistin and the endothelial permeability was measured using the Transwell system with a Texas-Red-labeled dextran tracer. The permeability of HCAEC monolayers treated with resistin (80 ng/mL) was 51% higher than the permeability of control monolayers (P<0.05). The mRNA levels of tight junction proteins zonula occludens-1 (ZO-1) and occludin in resistin-treated cells were 37% and 42% lower, respectively, than the corresponding levels in untreated cells. The protein levels of these molecules in resistin-treated cells were significantly reduced by 35% and 37%, respectively (P<0.05), as shown by flow cytometry and Western blot analysis. Superoxide dismutase (SOD) mimetic MnTBAP effectively blocked the resistin-mediated reduction of ZO-1 and occludin levels in HCAECs. In addition, superoxide anion production was increased from 21% (untreated cells) to 55% (cells treated with 40 ng/mL resistin), and 64% (resistin, 80 mg/mL) (P<0.05). The natural antioxidant Ginkgolide A effectively inhibited resistin-induced increase in permeability and the increase in superoxide anion production in HCAECs. Furthermore, resistin treatment significantly activated p38 MAPK, but not ERK1/2. Pretreatment of HCAECs with a p38 inhibitor effectively blocked resistin-induced permeability. These results provide new evidence that resistin may contribute to the vascular lesion formation via increasing endothelial permeability through the mechanism of oxidative stress and the activation of p38 MAPK

Ginsenoside Rb1 Blocks Ritonavir-Induced Oxidative Stress and eNOS Downregulation through Activation of Estrogen Receptor-Beta and Upregulation of SOD in Human Endothelial Cells

We have previously shown that ritonavir (RTV), a highly active anti-retroviral therapy (HAART) drug, can cause endothelial dysfunction through oxidative stress. Several antioxidants including ginsenoside Rb1, a compound with antioxidant effect, can effectively block this side effect of RTV in endothelial cells. In the current study, we explored a mechanism by which ginsenoside Rb1 could protect these cells via binding of estrogen receptors (ERs). We found that several human endothelial cell lines differentially expressed ER-&beta; and had very low levels of ER-&alpha;. RTV treatment significantly increased the production of reactive oxygen species (ROS) and decreased the expression of endothelial nitric oxidase synthase (eNOS) and superoxide dismutase (SOD) in HUVECs, while Rb1 effectively blocked these effects of RTV. These effects of Rb1 were effectively inhibited by silencing ER-&beta;, indicating that ginsenoside Rb1 requires ER-&beta; for its antioxidant activity in inhibiting the deleterious effect of RTV in human endothelial cells. Furthermore, Rb1 specifically activated ER-&beta; transactivation activity by ER-&beta; luciferase reporter assay. Rb1 competitively bound to ER-&beta;, which was determined by the high sensitive fluorescent polarization assay

Effects of resistin on mRNA and protein levels of junctional molecules in HCAECs.

<p>(<b>A</b>)<b>.</b> HCAECs were treated with resistin (20, 40, and 80 ng/mL) for 24 hours, or pretreated with Ginkgolide A (5 µM) for 30 minutes before resistin treatment (40 ng/mL) for 24 hours. The mRNA levels of junction molecules (VE-cadherin, ZO-1, and occluding) were determined by real time PCR. The relative mRNA levels of each gene were normalized to the expression of a house keeping gene β-actin. The results of the resistin-treated cells were compared with the results of the control cells (n = 3, *<i>P</i><0.05). The results of the cells that were pretreated with Ginkgolide A for 30 minutes and then treated with resistin (40 ng/mL) for 24 hours were compared with results of the resistin-treated cells (n = 3, ^#<i>P</i><0.05). (<b>B</b>)<b>.</b> The protein levels of VE cadherin, ZO-1, and occludin were determined by Western blot analysis after resistin treatment and compared with controls. Equal loading control was monitored by reprobing the blot with anti-β-actin antibody. Western blot band density ratio for each tight junction protein and control β-actin was measured with ImageJ (1.47) software (NIH). To determine the effect of adding an antioxidant, cells were pretreated with MnTBAP (2 µM) for 30 minutes, and then incubated with resistin.</p

Flow cytometry analysis of junctional molecules in HCAECs.

<p>HCAECs were pretreated with Ginkgolide A (GA) for 30 minutes (or left untreated), and then treated with resistin (80 ng/mL) for 24 hours. The protein levels of junction molecules were determined by flow cytometry analysis. (<b>A</b>)<b>.</b> These representative histograms show the percentage of positively stained cells for each specific antibody against each junction protein, including VE-cadherin, ZO-1, and occludin. (<b>B</b>)<b>.</b> Bar diagram showing the average percentage of positively stained cells in three separate experiments. The results of the resistin-treated cells were compared with results of control cells (n = 3, *<i>P</i><0.05).</p

Effect of resistin on the permeability of HCAECs.

<p>Endothelial monolayer permeability was measured using the Costar Transwell permeability system with a fluorescence-labeled dextran tracer. HCAECs were treated with different concentrations of resistin (40 and 80 ng/mL), with or without Ginkgolide A (GA, 5 µM) pre-treatment, for 24 hours. Cells treated with TNF-α (2 ng/mL) served as a positive control. The results of the resistin-treated cells were compared with the results of control cells (n = 3, *<i>P</i><0.05). The results of the cells that were pretreated with Ginkgolide A for 30 minutes before incubating them with resistin for 24 hours, were compared with results of the resistin-treated cells (n = 3, ^#<i>P</i><0.05). The experiment was repeated thrice.</p

Potential molecular mechanisms (hypothesis) of resistin-induced endothelial permeability.

<p>Resistin may interact with toll-like receptor 4 (TLR4), inducing a TLR4-mediated signaling cascade such as the activation of p38, NADPH oxidase and transcriptional factor CREB, which directly and/or indirectly reduces the expression of ZO-1 and occludin at transcriptional and/or post-transcriptional levels.</p