Interferon regulatory factor-1 together with reactive oxygen species promotes the acceleration of cell cycle progression by up-regulating the cyclin E and CDK2 genes during high glucose-induced proliferation of vascular smooth muscle cells

BACKGROUND: The high glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. In a previous study, we confirmed that Interferon regulatory factor-1 (Irf-1) is a positive regulator of the high glucose-induced proliferation of VSMCs. However, the mechanisms remain to be determined. METHODS: The levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions. Subsequently, cells were treated with high glucose/NAC, normal glucose/H(2)O(2), high glucose/U0126 or normal glucose/H(2)O(2)/U0126 during an incubation period. Then proliferation, cyclin/CDK expression and cell cycle distribution assays were performed to determine whether ROS/Erk1/2 signaling pathway was involved in the Irf-1-induced regulation of VSMC growth under high glucose conditions. RESULTS: We found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions. In high glucose conditions, Irf-1 overexpression led to an up-regulation of cyclin E/CDK2 and an acceleration of cell cycle progression, whereas silencing of Irf-1 suppressed the expression of both proteins and inhibited the cell cycle during the high glucose-induced proliferation of VSMCs. Treatment of VSMCs with antioxidants prevented the Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression in high glucose conditions. In contrast, under normal glucose conditions, H(2)O(2) stimulation and Irf-1 overexpression induced cell proliferation, up-regulated cyclin E/CDK2 expression and promoted cell cycle acceleration. In addition, overexpression of Irf-1 promoted the activation of Erk1/2 and when VSMCs overexpressing Irf-1 were treated with U0126, the specific Erk1/2 inhibitor abolished the proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression under high glucose or normal glucose/H(2)O(2) conditions. CONCLUSIONS: These results demonstrate that the downstream effectors of Irf-1 are cyclin E/CDK2 during the high glucose-induced proliferation of VSMCs, whereas they are cyclin D1/CDK4 in normal glucose conditions. The Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression are associated with ROS/Erk1/2 signaling pathway under high glucose conditions

In vitro and in vivo delivery of a sustained release nanocarrier-based formulation of an MRTF/SRF inhibitor in conjunctival fibrosis

Abstract Background Sustained drug delivery is a large unmet clinical need in glaucoma. Here, we incorporated a Myocardin-Related Transcription Factor/Serum Response Factor inhibitor, CCG-222740, into slow release large unilamellar vesicles derived from the liposomes DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and tested their effects in vitro and in vivo. Results The vesicles were spherical particles of around 130 nm and were strongly cationic. A large amount of inhibitor could be incorporated into the vesicles. We showed that the nanocarrier CCG-222740 formulation gradually released the inhibitor over 14 days using high performance liquid chromatography. Nanocarrier CCG-222740 significantly decreased ACTA2 gene expression and was not cytotoxic in human conjunctival fibroblasts. In vivo, nanocarrier CCG-222740 doubled the bleb survival from 11.0 ± 0.6 days to 22.0 ± 1.3 days (p = 0.001), decreased conjunctival scarring and did not have any local or systemic adverse effects in a rabbit model of glaucoma filtration surgery. Conclusions Our study demonstrates proof-of-concept that a nanocarrier-based formulation efficiently achieves a sustained release of a Myocardin-Related Transcription Factor/Serum Response Factor inhibitor and prevents conjunctival fibrosis in an established rabbit model of glaucoma filtration surgery.https://deepblue.lib.umich.edu/bitstream/2027.42/146540/1/12951_2018_Article_425.pd

ROCK Inhibitor Y-27632 Suppresses Dissociation-Induced Apoptosis of Murine Prostate Stem/Progenitor Cells and Increases Their Cloning Efficiency

Activation of the RhoA/ROCK signaling pathway has been shown to contribute to dissociation-induced apoptosis of embryonic and neural stem cells. We previously demonstrated that approximately 1 out of 40 Lin−Sca-1+CD49fhigh (LSC) prostate basal epithelial cells possess the capacities of stem cells for self-renewal and multi-lineage differentiation. We show here that treating LSC cells with the ROCK kinase inhibitor Y-27632 increases their cloning efficiency by 8 fold in an in vitro prostate colony assay. Y-27632 treatment allows prostate colony cells to replate efficiently, which does not occur otherwise. Y-27632 also increases the cloning efficiency of prostate stem cells in a prostate sphere assay and a dissociated prostate cell regeneration assay. The increased cloning efficiency is due to the suppression of the dissociation-induced, RhoA/ROCK activation-mediated apoptosis of prostate stem cells. Dissociation of prostate epithelial cells from extracellular matrix increases PTEN activity and attenuates AKT activity. Y-27632 treatment alone is sufficient to suppress cell dissociation-induced activation of PTEN activity. However, this does not contribute to the increased cloning efficiency, because Y-27632 treatment increases the sphere-forming unit of wild type and Pten null prostate cells to a similar extent. Finally, knocking down expression of both ROCK kinases slightly increases the replating efficiency of prostate colony cells, corroborating that they play a major role in the Y-27632 mediated increase in cloning efficiency. Our study implies that the numbers of prostate cells with stem/progenitor activity may be underestimated based on currently employed assays, supports that dissociation-induced apoptosis is a common feature of embryonic and somatic stem cells with an epithelial phenotype, and highlights the significance of environmental cues for the maintenance of stem cells

Clopidogrel Plus Aspirin vs Aspirin Alone in Patients With Acute Mild to Moderate Stroke

Importance Dual antiplatelet therapy has been demonstrated to be superior to single antiplatelet in reducing recurrent stroke among patients with transient ischemic attack or minor stroke, but robust evidence for its effect in patients with mild to moderate ischemic stroke is lacking. Objective To evaluate whether dual antiplatelet therapy is superior to single antiplatelet among patients with mild to moderate ischemic stroke. Design, Setting, and Participants This was a multicenter, open-label, blinded end point, randomized clinical trial conducted at 66 hospitals in China from December 20, 2016, through August 9, 2022. The date of final follow-up was October 30, 2022. The analysis was reported on March 12, 2023. Of 3065 patients with ischemic stroke, 3000 patients with acute mild to moderate stroke within 48 hours of symptom onset were enrolled, after excluding 65 patients who did not meet eligibility criteria or had no randomization outcome. Interventions Within 48 hours after symptom onset, patients were randomly assigned to receive clopidogrel plus aspirin (n = 1541) or aspirin alone (n = 1459) in a 1:1 ratio. Main Outcomes and Measures The primary end point was early neurologic deterioration at 7 days, defined as an increase of 2 or more points in National Institutes of Health Stroke Scale (NIHSS) score, but not as a result of cerebral hemorrhage, compared with baseline. The superiority of clopidogrel plus aspirin to aspirin alone was assessed based on a modified intention-to-treat population, which included all randomized participants with at least 1 efficacy evaluation regardless of treatment allocation. Bleeding events were safety end points. Results Of the 3000 randomized patients, 1942 (64.6%) were men, the mean (SD) age was 65.9 (10.6) years, median (IQR) NIHSS score at admission was 5 (4-6), and 1830 (61.0%) had a stroke of undetermined cause. A total of 2915 patients were included in the modified intention-to-treat analysis. Early neurologic deterioration occurred in 72 of 1502 (4.8%) in the dual antiplatelet therapy group vs 95 of 1413 (6.7%) in the aspirin alone group (risk difference −1.9%; 95% CI, −3.6 to −0.2; P = .03). Similar bleeding events were found between 2 groups. Conclusions and Relevance Among Chinese patients with acute mild to moderate ischemic stroke, clopidogrel plus aspirin was superior to aspirin alone with regard to reducing early neurologic deterioration at 7 days with similar safety profile. These findings indicate that dual antiplatelet therapy may be a superior choice to aspirin alone in treating patients with acute mild to moderate stroke.Trial RegistrationClinicalTrials.gov Identifier: NCT0286900

Structural and Functional Neural Correlates in Individuals with Excessive Smartphone Use: A Systematic Review and Meta-Analysis

Background: Despite known association of internet addiction with a reduced brain volume and abnormal connectivity, the impact of excessive smartphone use remains unclear. Methods: PubMed, Embase, ClinicalTrial.gov, and Web of Science databases were systematically searched from inception to July 2022 using appropriate keywords for observational studies comparing differences in brain volumes and activations between excessive smartphone users and individuals with regular use by magnetic resonance imaging. Results: Of the 11 eligible studies retrieved from 6993 articles initially screened, seven and six evaluated brain volumes and activations, respectively. The former enrolled 421 participants (165 excessive smartphone users vs. 256 controls), while the latter recruited 276 subjects with 139 excessive smartphone users. The results demonstrated a smaller brain volume in excessive smartphone users compared to the controls (g = −0.55, p < 0.001), especially in subcortical regions (p < 0.001). Besides, the impact was more pronounced in adolescents than in adults (p < 0.001). Regression analysis revealed a significant positive association between impulsivity and volume reduction. Regarding altered activations, the convergences of foci in the declive of the posterior lobe of cerebellum, the lingual gyrus, and the middle frontal gyrus were noted. Conclusions: Our findings demonstrated a potential association of excessive smartphone use with a reduced brain volume and altered activations

Prickly waterlily and rigid hornwort genomes shed light on early angiosperm evolution

Angiosperms represent one of the most spectacular terrestrial radiations on the planet(1), but their early diversification and phylogenetic relationships remain uncertain(2-5). A key reason for this impasse is the paucity of complete genomes representing early-diverging angiosperms. Here, we present high-quality, chromosomal-level genome assemblies of two aquatic species-prickly waterlily (Euryale ferox; Nymphaeales) and the rigid hornwort (Ceratophyllum demersum; Ceratophyllales)-and expand the genomic representation for key sectors of the angiosperm tree of life. We identify multiple independent polyploidization events in each of the five major clades (that is, Nymphaeales, magnoliids, monocots, Ceratophyllales and eudicots). Furthermore, our phylogenomic analyses, which spanned multiple datasets and diverse methods, confirm that Amborella and Nymphaeales are successively sister to all other angiosperms. Furthermore, these genomes help to elucidate relationships among the major subclades within Mesangiospermae, which contain about 350,000 species. In particular, the species-poor lineage Ceratophyllales is supported as sister to eudicots, and monocots and magnoliids are placed as successively sister to Ceratophyllales and eudicots. Finally, our analyses indicate that incomplete lineage sorting may account for the incongruent phylogenetic placement of magnoliids between nuclear and plastid genomes. Genome assemblies of two aquatic species, prickly waterlily and rigid hornwort, clarify the early diversification and phylogeny of plants. Multiple independent polyploidization events are inferred in each of the five major angiosperm clades