HIF–VEGF Pathways Are Critical for Chronic Otitis Media in Junbo and Jeff Mouse Mutants

Otitis media with effusion (OME) is the commonest cause of hearing loss in children, yet the underlying genetic pathways and mechanisms involved are incompletely understood. Ventilation of the middle ear with tympanostomy tubes is the commonest surgical procedure in children and the best treatment for chronic OME, but the mechanism by which they work remains uncertain. As hypoxia is a common feature of inflamed microenvironments, moderation of hypoxia may be a significant contributory mechanism. We have investigated the occurrence of hypoxia and hypoxia-inducible factor (HIF) mediated responses in Junbo and Jeff mouse mutant models, which develop spontaneous chronic otitis media. We found that Jeff and Junbo mice labeled in vivo with pimonidazole showed cellular hypoxia in inflammatory cells in the bulla lumen, and in Junbo the middle ear mucosa was also hypoxic. The bulla fluid inflammatory cell numbers were greater and the upregulation of inflammatory gene networks were more pronounced in Junbo than Jeff. Hif-1a gene expression was elevated in bulla fluid inflammatory cells, and there was upregulation of its target genes including Vegfa in Junbo and Jeff. We therefore investigated the effects in Junbo of small-molecule inhibitors of VEGFR signaling (PTK787, SU-11248, and BAY 43-9006) and destabilizing HIF by inhibiting its chaperone HSP90 with 17-DMAG. We found that both classes of inhibitor significantly reduced hearing loss and the occurrence of bulla fluid and that VEGFR inhibitors moderated angiogenesis and lymphangiogenesis in the inflamed middle ear mucosa. The effectiveness of HSP90 and VEGFR signaling inhibitors in suppressing OM in the Junbo model implicates HIF–mediated VEGF as playing a pivotal role in OM pathogenesis. Our analysis of the Junbo and Jeff mutants highlights the role of hypoxia and HIF–mediated pathways, and we conclude that targeting molecules in HIF–VEGF signaling pathways has therapeutic potential in the treatment of chronic OM

A mutation in Nischarin causes otitis media via LIMK1 and NF-κB pathways

Otitis media (OM), inflammation of the middle ear (ME), is a common cause of conductive hearing impairment. Despite the importance of the disease, the aetiology of chronic and recurrent forms of middle ear inflammatory disease remains poorly understood. Studies of the human population suggest that there is a significant genetic component predisposing to the development of chronic OM, although the underlying genes are largely unknown. Using N-ethyl-N-nitrosourea mutagenesis we identified a recessive mouse mutant, edison, that spontaneously develops a conductive hearing loss due to chronic OM. The causal mutation was identified as a missense change, L972P, in the Nischarin (NISCH) gene. edison mice develop a serous or granulocytic effusion, increasingly macrophage and neutrophil rich with age, along with a thickened, inflamed mucoperiosteum. We also identified a second hypomorphic allele, V33A, with only modest increases in auditory thresholds and reduced incidence of OM. NISCH interacts with several proteins, including ITGA5 that is thought to have a role in modulating VEGF-induced angiogenesis and vascularization. We identified a significant genetic interaction between Nisch and Itga5; mice heterozygous for Itga5-null and homozygous for edison mutations display a significantly increased penetrance and severity of chronic OM. In order to understand the pathological mechanisms underlying the OM phenotype, we studied interacting partners to NISCH along with downstream signalling molecules in the middle ear epithelia of edison mouse. Our analysis implicates PAK1 and RAC1, and downstream signalling in LIMK1 and NF-κB pathways in the development of chronic OM

Percutaneous revascularization for ischemic left ventricular dysfunction: Cost-effectiveness analysis of the REVIVED-BCIS2 trial

BACKGROUND: Percutaneous coronary intervention (PCI) is frequently undertaken in patients with ischemic left ventricular systolic dysfunction. The REVIVED (Revascularization for Ischemic Ventricular Dysfunction)-BCIS2 (British Cardiovascular Society-2) trial concluded that PCI did not reduce the incidence of all-cause death or heart failure hospitalization; however, patients assigned to PCI reported better initial health-related quality of life than those assigned to optimal medical therapy (OMT) alone. The aim of this study was to assess the cost-effectiveness of PCI+OMT compared with OMT alone. METHODS: REVIVED-BCIS2 was a prospective, multicenter UK trial, which randomized patients with severe ischemic left ventricular systolic dysfunction to either PCI+OMT or OMT alone. Health care resource use (including planned and unplanned revascularizations, medication, device implantation, and heart failure hospitalizations) and health outcomes data (EuroQol 5-dimension 5-level questionnaire) on each patient were collected at baseline and up to 8 years post-randomization. Resource use was costed using publicly available national unit costs. Within the trial, mean total costs and quality-adjusted life-years (QALYs) were estimated from the perspective of the UK health system. Cost-effectiveness was evaluated using estimated mean costs and QALYs in both groups. Regression analysis was used to adjust for clinically relevant predictors. RESULTS: Between 2013 and 2020, 700 patients were recruited (mean age: PCI+OMT=70 years, OMT=68 years; male (%): PCI+OMT=87, OMT=88); median follow-up was 3.4 years. Over all follow-ups, patients undergoing PCI yielded similar health benefits at higher costs compared with OMT alone (PCI+OMT: 4.14 QALYs, £22 352; OMT alone: 4.16 QALYs, £15 569; difference: −0.015, £6782). For both groups, most health resource consumption occurred in the first 2 years post-randomization. Probabilistic results showed that the probability of PCI being cost-effective was 0. CONCLUSIONS: A minimal difference in total QALYs was identified between arms, and PCI+OMT was not cost-effective compared with OMT, given its additional cost. A strategy of routine PCI to treat ischemic left ventricular systolic dysfunction does not seem to be a justifiable use of health care resources in the United Kingdom

Arrhythmia and death following percutaneous revascularization in ischemic left ventricular dysfunction: Prespecified analyses from the REVIVED-BCIS2 trial

BACKGROUND: Ventricular arrhythmia is an important cause of mortality in patients with ischemic left ventricular dysfunction. Revascularization with coronary artery bypass graft or percutaneous coronary intervention is often recommended for these patients before implantation of a cardiac defibrillator because it is assumed that this may reduce the incidence of fatal and potentially fatal ventricular arrhythmias, although this premise has not been evaluated in a randomized trial to date. METHODS: Patients with severe left ventricular dysfunction, extensive coronary disease, and viable myocardium were randomly assigned to receive either percutaneous coronary intervention (PCI) plus optimal medical and device therapy (OMT) or OMT alone. The composite primary outcome was all-cause death or aborted sudden death (defined as an appropriate implantable cardioverter defibrillator therapy or a resuscitated cardiac arrest) at a minimum of 24 months, analyzed as time to first event on an intention-to-treat basis. Secondary outcomes included cardiovascular death or aborted sudden death, appropriate implantable cardioverter defibrillator (ICD) therapy or sustained ventricular arrhythmia, and number of appropriate ICD therapies. RESULTS: Between August 28, 2013, and March 19, 2020, 700 patients were enrolled across 40 centers in the United Kingdom. A total of 347 patients were assigned to the PCI+OMT group and 353 to the OMT alone group. The mean age of participants was 69 years; 88% were male; 56% had hypertension; 41% had diabetes; and 53% had a clinical history of myocardial infarction. The median left ventricular ejection fraction was 28%; 53.1% had an implantable defibrillator inserted before randomization or during follow-up. All-cause death or aborted sudden death occurred in 144 patients (41.6%) in the PCI group and 142 patients (40.2%) in the OMT group (hazard ratio, 1.03 [95% CI, 0.82–1.30]; P =0.80). There was no between-group difference in the occurrence of any of the secondary outcomes. CONCLUSIONS: PCI was not associated with a reduction in all-cause mortality or aborted sudden death. In patients with ischemic cardiomyopathy, PCI is not beneficial solely for the purpose of reducing potentially fatal ventricular arrhythmias. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01920048

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Supporting data for: Dietary inference from dental topographic analysis of feeding tools in diverse animals

Data for Stockey et al. Dietary inference from dental topographic analysis of feeding tools in diverse animals.Data include 2.5D mandible models and raw topographic metric values.- .txt files containing the edited 2.5D functional surfaces of the left mandibles of orthopteran species (Genus species, specimen ID number)- .xlsx files containing raw topographic metric values for both orthopteran mandibles and published mammal molars,</div

Mild to moderate hearing impairments in <i>Nisch</i>^{<i>edsn/edsn</i>}, <i>Nisch</i>^{<i>V33A/V33A</i>} and <i>Nisch</i>^{<i>edsn/V33A</i>} mice due to otitis media.

<p><b>(A-C)</b> Click-evoked ABR thresholds across a time course show a mild to moderate progressive hearing impairment in (A) <i>Nisch</i>^{<i>edsn/edsn</i>}, (B) <i>Nisch</i>^{<i>V33A/V33A</i>} and (C) <i>Nisch</i>^{<i>V33A/edsn</i>} mice. Expected ABR threshold range for normal hearing was between 15–30 dB SPL (dashed red lines). * <i>P</i> < 0.05; *** <i>P</i> < 0.001. (A) <i>Nisch</i>^<i>+/+</i>, n = 10–33; <i>Nisch</i>^{<i>edsn/+</i>}, n = 12–33; <i>Nisch</i>^{<i>edsn/edsn</i>}, n = 14–25. (B) <i>Nisch</i>^<i>+/+</i>, n = 10; <i>Nisch</i>^{<i>V33A/+</i>}, n = 13; <i>Nisch</i>^{<i>V33A/V33A</i>}, n = 10. (C) <i>Nisch</i>^<i>+/+</i>, n = 5; <i>Nisch</i>^{<i>edsn/+</i>}, n = 6; <i>Nisch</i>^{<i>V33A/+</i>}, n = 4; <i>Nisch</i>^{<i>V33A/edsn</i>}, n = 6. Error bars indicate standard error of mean. A Kruskall-Wallis test was performed followed by Dunn’s multiple comparison tests for post-hoc analysis. <b>(D)</b> Visual inspection of the tympanic membrane is a simple method for diagnosing OM. Wild-type (<i>Nisch</i>^<i>+/+</i>) mice have no visible fluid behind the tympanic membrane and the malleus is easily recognizable, while affected <i>Nisch</i>^{<i>edsn/edsn</i>} mice have fluid behind the tympanic membrane and the malleus is obscured. <b>(E)</b> The incidence of bilateral and unilateral OM increases in prevalence with age in <i>Nisch</i>^{<i>edsn/edsn</i>} mice. <b>(F, G)</b> At 12 wk, an increased proportion of (F) <i>Nisch</i>^{<i>V33A/V33A</i>} and (G) <i>Nisch</i>^{<i>V33A/edsn</i>} mice display bilateral and unilateral OM.</p

The <i>Nisch</i> gene is mutated in <i>edison</i> mice.

<p><b>(A)</b> SNP mapping of 10 <i>edsn</i> mutants identified an approximately 9 Mb interval on chromosome 14 delineated by marker rs30778552 and rs46823676. The grayed box indicates the chromosomal interval bearing the <i>edsn</i> mutation. <b>(B)</b> Sequence analysis of the <i>Nisch</i> locus in <i>Nisch</i>^<i>+/+</i> and <i>Nisch</i>^{<i>edsn/edsn</i>} DNA. A c.3079T>C transition is detected in <i>Nisch</i>^{<i>edsn/edsn</i>} mutants that is not present in <i>Nisch</i>^<i>+/+</i> DNA. <b>(C)</b> Conservation of the mutated leucine residue across species. <i>Mus musculus</i>, ENSMUSG00000021910; <i>Homo sapiens</i>, ENSG00000010322; <i>Pan troglodytes</i>, ENSPTRG00000015001; <i>Sus scrofa</i>, ENSSSCG00000011442; <i>Gallus gallus</i>, ENSGALG00000043825; <i>Anolis carolinensis</i>, ENSACAG00000006939. <b>(D)</b> Schematic of the full length NISCH peptide (1593 amino acids). The molecule consists of a phox-homology (PX) domain, six leucine-rich repeats, a coiled-coil (CC) domain and an alanine/proline-rich region. Both the PX and CC domains of Nischarin are essential for endosomal targeting and interaction with phosphatidylinositol 3-phosphate (PI3P) in PI3P-enriched endosomes. Amino acids 709–807 of Nischarin interact with the integrin α5 (ITGA5) cytoplasmic tail. Both LIMK1 and LKB1 interact with positions 661–869 of Nischarin. Residues 246–1047 of Nischarin interact with PAK1. Rab14 interacts with amino acids 1190–1593. Finally, Rac1 interacts with two regions of Nischarin, amino acids 246–1047 and 1190–1593. The positions of the <i>Nisch</i>^<i>edsn</i> and <i>Nisch</i>^<i>V33A</i> mutations are also indicated.</p