Regional biomechanical and histological characterisation of the passive porcine urinary bladder: Implications for augmentation and tissue engineering strategies

The aim of this study was to identify and quantify potential regional and directional variations in the quasistatic uniaxial mechanical properties of the passive urinary bladder wall. Overall, the lower body and trigone regions demonstrated the highest degree of directional anisotropy, whereas the ventral region demonstrated the least directional anisotropy. Significant regional anisotropy was found only along the apex-to-base direction. The dorsal and ventral regions demonstrated a significantly increased distensibility along the apex-to-base direction compared to the other bladder regions, whereas the trigone and lower body regions demonstrated the least distensibility. The trigone, lower body and lateral regions also demonstrated the highest tensile Strength both at regional and directional levels. The study detected significant regional and directional anisotropy in the mechanical properties of the bladder and correlated this anisotropy to the distended and non-distended tissue histioarchitecture and whole organ mechanics. By elucidating the inhomogeneous nature of the bladder, the results from this study will aid the regional differentiation of bladder treatments in terms of partial bladder replacement with suitable natural or synthetic biomaterials, as well as the development of more realistic constitutive models of bladder wall biomechanics and improved computational simulations to predict deformations in the natural and augmented bladder. (c) 2008 Elsevier Ltd. All rights reserved

Dynamic changes in lung microRNA profiles during the development of pulmonary hypertension due to chronic hypoxia and monocrotaline

<b>Objective</b>: MicroRNAs (miRNAs) are small noncoding RNAs that have the capacity to control protein production through binding "seed" sequences within a target mRNA. Each miRNA is capable of potentially controlling hundreds of genes. The regulation of miRNAs in the lung during the development of pulmonary arterial hypertension (PAH) is unknown.<p></p> <b>Methods and Results</b>: We screened lung miRNA profiles in a longitudinal and crossover design during the development of PAH caused by chronic hypoxia or monocrotaline in rats. We identified reduced expression of Dicer, involved in miRNA processing, during the onset of PAH after hypoxia. MiR-22, miR-30, and let-7f were downregulated, whereas miR-322 and miR-451 were upregulated significantly during the development of PAH in both models. Differences were observed between monocrotaline and chronic hypoxia. For example, miR-21 and let-7a were significantly reduced only in monocrotaline-treated rats. MiRNAs that were significantly regulated were validated by quantitative polymerase chain reaction. By using in vitro studies, we demonstrated that hypoxia and growth factors implicated in PAH induced similar changes in miRNA expression. Furthermore, we confirmed miR-21 downregulation in human lung tissue and serum from patients with idiopathic PAH.<p></p> <b>Conclusion</b>: Defined miRNAs are regulated during the development of PAH in rats. Therefore, miRNAs may contribute to the pathogenesis of PAH and represent a novel opportunity for therapeutic intervention.<p></p&gt

The urothelial transcriptomic response to interferon gamma : implications for bladder cancer prognosis and immunotherapy

Interferon gamma (IFNγ) is central to the inflammatory immune response, such as that entrained by BCG immunotherapy for bladder cancer. However, immune-mediated tumour cell killing is subject to modulation by immunoinhibitory “checkpoint” receptors such as PD-L1. We investigated the effects of IFNγ on barrier-forming in vitro-differentiated normal human urothelium using mRNA-sequencing, and showed canonical upregulation of MHC class I/II and de novo expression of the T cell tropic CXCL9-11 chemokines. Normal urothelium constitutively expressed immunoinhibitory B7 family member VSIR (VISTA), while CD274 (PD-L1) expression was induced/upregulated by IFNγ. We generated a urothelial IFNγ response gene signature. When applied to the unsupervised clustering of non-muscle-invasive bladder cancers, the IFNγ-signature predicted longer recurrence-free survival. In muscle-invasive cancers, the IFNγ-signature split the basal/squamous consensus subtype, with significantly worse overall survival when weak or absent. This study offers novel insights into strategies to enhance immunotherapy via the IFNγ and VISTA/PD-L1 nexus

LPCAT4 knockdown alters barrier integrity and cellular bioenergetics in human urothelium

Urothelium is a transitional, stratified epithelium that lines the lower urinary tract, providing a tight barrier to urine whilst retaining the capacity to stretch and rapidly resolve damage. The role of glycerophospholipids in urothelial barrier function is largely unknown, despite their importance in membrane structural integrity, protein complex assembly, and the master regulatory role of PPARγ in urothelial differentiation. We performed lipidomic and transcriptomic characterisation of urothelial differentiation, revealing a metabolic switch signature from fatty acid synthesis to lipid remodelling, including 5-fold upregulation of LPCAT4. LPCAT4 knockdown urothelial cultures exhibited an impaired proliferation rate but developed elevated trans-epithelial electrical resistances upon differentiation, associated with a reduced and delayed capacity to restitute barrier function after wounding. Specific reduction in 18:1 PC fatty acyl chains upon knockdown was consistent with LPCAT4 specificity, but was unlikely to elicit broad barrier function changes. However, transcriptomic analysis of LPCAT4 knockdown supported an LPC-induced reduction in DAG availability, predicted to limit PKC activity, and TSPO abundance, predicted to limit endogenous ATP. These phenotypes were confirmed by PKC and TSPO inhibition. Together, these data suggest an integral role for lipid mediators in urothelial barrier function and highlight the strength of combined lipidomic and transcriptomic analyses for characterising tissue homeostasis

Comprehensive Rare Variant Analysis via Whole-Genome Sequencing to Determine the Molecular Pathology of Inherited Retinal Disease

Inherited retinal disease is a common cause of visual impairment and represents a highly heterogeneous group of conditions. Here, we present findings from a cohort of 722 individuals with inherited retinal disease, who have had whole-genome sequencing (n = 605), whole-exome sequencing (n = 72), or both (n = 45) performed, as part of the NIHR-BioResource Rare Diseases research study. We identified pathogenic variants (single-nucleotide variants, indels, or structural variants) for 404/722 (56%) individuals. Whole-genome sequencing gives unprecedented power to detect three categories of pathogenic variants in particular: structural variants, variants in GC-rich regions, which have significantly improved coverage compared to whole-exome sequencing, and variants in non-coding regulatory regions. In addition to previously reported pathogenic regulatory variants, we have identified a previously unreported pathogenic intronic variant in $\textit{CHM}$ in two males with choroideremia. We have also identified 19 genes not previously known to be associated with inherited retinal disease, which harbor biallelic predicted protein-truncating variants in unsolved cases. Whole-genome sequencing is an increasingly important comprehensive method with which to investigate the genetic causes of inherited retinal disease.This work was supported by The National Institute for Health Research England (NIHR) for the NIHR BioResource – Rare Diseases project (grant number RG65966). The Moorfields Eye Hospital cohort of patients and clinical and imaging data were ascertained and collected with the support of grants from the National Institute for Health Research Biomedical Research Centre at Moorfields Eye Hospital, National Health Service Foundation Trust, and UCL Institute of Ophthalmology, Moorfields Eye Hospital Special Trustees, Moorfields Eye Charity, the Foundation Fighting Blindness (USA), and Retinitis Pigmentosa Fighting Blindness. M.M. is a recipient of an FFB Career Development Award. E.M. is supported by UCLH/UCL NIHR Biomedical Research Centre. F.L.R. and D.G. are supported by Cambridge NIHR Biomedical Research Centre

Phenotypic Characterization of EIF2AK4 Mutation Carriers in a Large Cohort of Patients Diagnosed Clinically With Pulmonary Arterial Hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 (BMPR2) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene (EIF2AK4) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH. METHODS: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured. RESULTS: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% [interquartile range, 30%-35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival. CONCLUSIONS: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation

Phenotypic Characterization of <i>EIF2AK4</i> Mutation Carriers in a Large Cohort of Patients Diagnosed Clinically With Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 ( BMPR2 ) are the commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene ( EIF2AK4 ) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH. Methods: Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource–Rare Diseases study. Heterozygous variants in BMPR2 and biallelic EIF2AK4 variants with a minor allele frequency of &lt;1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and sorting intolerant from tolerant predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured. Results: Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in BMPR2 were identified in 130 patients (14.8%). Biallelic mutations in EIF2AK4 were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic EIF2AK4 mutations. These patients had a reduced transfer coefficient for carbon monoxide (K co ; 33% [interquartile range, 30%–35%] predicted) and younger age at diagnosis (29 years; interquartile range, 23–38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without EIF2AK4 mutations. However, radiological assessment alone could not accurately identify biallelic EIF2AK4 mutation carriers. Patients with PAH with biallelic EIF2AK4 mutations had a shorter survival. Conclusions: Biallelic EIF2AK4 mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low K co and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation. </jats:sec

Comprehensive Cancer-Predisposition Gene Testing in an Adult Multiple Primary Tumor Series Shows a Broad Range of Deleterious Variants and Atypical Tumor Phenotypes.

Multiple primary tumors (MPTs) affect a substantial proportion of cancer survivors and can result from various causes, including inherited predisposition. Currently, germline genetic testing of MPT-affected individuals for variants in cancer-predisposition genes (CPGs) is mostly targeted by tumor type. We ascertained pre-assessed MPT individuals (with at least two primary tumors by age 60 years or at least three by 70 years) from genetics centers and performed whole-genome sequencing (WGS) on 460 individuals from 440 families. Despite previous negative genetic assessment and molecular investigations, pathogenic variants in moderate- and high-risk CPGs were detected in 67/440 (15.2%) probands. WGS detected variants that would not be (or were not) detected by targeted resequencing strategies, including low-frequency structural variants (6/440 [1.4%] probands). In most individuals with a germline variant assessed as pathogenic or likely pathogenic (P/LP), at least one of their tumor types was characteristic of variants in the relevant CPG. However, in 29 probands (42.2% of those with a P/LP variant), the tumor phenotype appeared discordant. The frequency of individuals with truncating or splice-site CPG variants and at least one discordant tumor type was significantly higher than in a control population (χ2 = 43.642; p ≤ 0.0001). 2/67 (3%) probands with P/LP variants had evidence of multiple inherited neoplasia allele syndrome (MINAS) with deleterious variants in two CPGs. Together with variant detection rates from a previous series of similarly ascertained MPT-affected individuals, the present results suggest that first-line comprehensive CPG analysis in an MPT cohort referred to clinical genetics services would detect a deleterious variant in about a third of individuals.JW is supported by a Cancer Research UK Cambridge Cancer Centre Clinical Research Training Fellowship. Funding for the NIHR BioResource – Rare diseases project was provided by the National Institute for Health Research (NIHR, grant number RG65966). ERM acknowledges support from the European Research Council (Advanced Researcher Award), NIHR (Senior Investigator Award and Cambridge NIHR Biomedical Research Centre), Cancer Research UK Cambridge Cancer Centre and Medical Research Council Infrastructure Award. The University of Cambridge has received salary support in respect of EM from the NHS in the East of England through the Clinical Academic Reserve. The views expressed are those of the authors and not necessarily those of the NHS or Department of Health. DGE is an NIHR Senior Investigator and is supported by the all Manchester NIHR Biomedical Research Centre