Targeting of cathepsin S reduces cystic fibrosis-like lung disease.

Cathepsin S (CatS) is upregulated in the lungs of patients with cystic fibrosis (CF). However, its role in CF lung disease pathogenesis remains unclear.In this study, ß-epithelial Na+ channel-overexpressing transgenic (ßENaC-Tg) mice, a model of CF-like lung disease, were crossed with CatS null (CatS-/-) mice or treated with the CatS inhibitor VBY-999.Levels of active CatS were elevated in the lungs of ßENaC-Tg mice compared with wild-type (WT) littermates. CatS-/-ßENaC-Tg mice exhibited decreased pulmonary inflammation, mucus obstruction and structural lung damage compared with ßENaC-Tg mice. Pharmacological inhibition of CatS resulted in a significant decrease in pulmonary inflammation, lung damage and mucus plugging in the lungs of ßENaC-Tg mice. In addition, instillation of CatS into the lungs of WT mice resulted in inflammation, lung remodelling and upregulation of mucin expression. Inhibition of the CatS target, protease-activated receptor 2 (PAR2), in ßENaC-Tg mice resulted in a reduction in airway inflammation and mucin expression, indicating a role for this receptor in CatS-induced lung pathology.Our data indicate an important role for CatS in the pathogenesis of CF-like lung disease mediated in part by PAR2 and highlight CatS as a therapeutic target.

Single-Cell RNA Sequencing Reveals Cardiac Fibroblast-Specific Transcriptomic Changes in Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is the most common cause of heart failure, with a complex aetiology involving multiple cell types. We aimed to detect cell-specific transcriptomic alterations in DCM through analysis that leveraged recent advancements in single-cell analytical tools. Single-cell RNA sequencing (scRNA-seq) data from human DCM cardiac tissue were subjected to an updated bioinformatic workflow in which unsupervised clustering was paired with reference label transfer to more comprehensively annotate the dataset. Differential gene expression was detected primarily in the cardiac fibroblast population. Bulk RNA sequencing was performed on an independent cohort of human cardiac tissue and compared with scRNA-seq gene alterations to generate a stratified list of higher-confidence, fibroblast-specific expression candidates for further validation. Concordant gene dysregulation was confirmed in TGFß-induced fibroblasts. Functional assessment of gene candidates showed that AEBP1 may play a significant role in fibroblast activation. This unbiased approach enabled improved resolution of cardiac cell-type-specific transcriptomic alterations in DCM.

Integrin beta-like 1 is regulated by DNA methylation and increased in heart failure patients.

AIMS: Dynamic alterations in cardiac DNA methylation have been implicated in the development of heart failure (HF) with evidence of ischaemic heart disease (IHD); however, there is limited research into cell specific, DNA methylation sensitive genes that are affected by dysregulated DNA methylation patterns. In this study, we aimed to identify DNA methylation sensitive genes in the ischaemic heart and elucidate their role in cardiac fibrosis. METHODS: A multi-omics integrative analysis was carried out on RNA sequencing and methylation sequencing on HF with IHD (n = 9) versus non-failing (n = 9) left ventricular tissue, which identified Integrin beta-like 1 (ITGBL1) as a gene of interest. Expression of Itgbl1 was assessed in three animal models of HF; an ischaemia-reperfusion pig model, a myocardial infarction mouse model and an angiotensin-II infused mouse model. Single nuclei RNA sequencing was carried out on heart tissue from angiotensin-II infused mice to establish the expression profile of Itgbl1 across cardiac cell populations. Subsequent in vitro analyses were conducted to elucidate a role for ITGBL1 in human cardiac fibroblasts. DNA pyrosequencing was applied to assess ITGBL1 CpG methylation status in genomic DNA from human cardiac tissue and stimulated cardiac fibroblasts. RESULTS: ITGBL1 was >2-fold up-regulated (FDR adj P = 0.03) and >10-fold hypomethylated (FDR adj P = 0.01) in human HF with IHD left ventricular tissue compared with non-failing controls. Expression of Itgbl1 was up-regulated in three isolated animal models of HF and showed conserved correlation between increased Itgbl1 and diastolic dysfunction. Single nuclei RNA sequencing highlighted that Itgbl1 is primarily expressed in cardiac fibroblasts, while functional studies elucidated a role for ITGBL1 in cardiac fibroblast migration, evident in 50% reduced 24 h fibroblast wound closure occurring subsequent to siRNA-targeted ITGBL1 knockdown. Lastly, evidence provided from DNA pyrosequencing supports the theory that differential expression of ITGBL1 is caused by DNA hypomethylation. CONCLUSIONS: ITGBL1 is a gene that is mainly expressed in fibroblasts, plays an important role in cardiac fibroblast migration, and whose expression is significantly increased in the failing heart. The mechanism by which increased ITGBL1 occurs is through DNA hypomethylation.

Pre-Eclampsia Biomarkers for Women With Type 1 Diabetes Mellitus: A Comprehensive Review of Recent Literature.

Background: Pre-eclampsia is a serious consideration for women with type 1 diabetes mellitus (T1DM) planning pregnancy. Risk stratification strategies, such as biomarkers measured in the first trimester of pregnancy, could help identify high-risk women. The literature on T1DM-specific pre-eclampsia biomarkers is expanding. We aimed to provide a narrative review of recently published evidence to identify the most promising biomarker candidates that could be targeted for clinical implementation in existing PE models. Methods: A search using MeSH terms was carried out of Medline, EMBASE, Maternity and Infant Care, Web of Science, and Scopus for relevant papers published since 2015 inclusive and in English. The time limit was applied from the publication of the preceding systematic review in this field. Included studies had pre-eclampsia as a primary outcome, measured one or more serum, plasma or urine biomarkers at any time during pregnancy, and had a distinct group of women with T1DM who developed pre-eclampsia. Studies with pre-eclampsia as a composite outcome were not considered. No restrictions on study types were applied. A narrative synthesis approach was adopted for analysis. Results: A total of 510 records were screened yielding 18 eligible studies relating to 32 different biomarkers. Higher first-trimester levels of HbA1c and urinary albumin were associated with an increased risk of pre-eclampsia development in women with T1DM. Urinary neutrophil gelatinase-associated lipocalin and adipokines were novel biomarkers showing moderate predictive ability before 15 gestational weeks. Two T1DM-specific pre-eclampsia prediction models were proposed, measuring adipokines or urinary neutrophil gelatinase-associated lipocalin together with easily attainable maternal clinical characteristics. Contradicting previous literature, pre-eclampsia risk in women with T1DM was correlated with vitamin D levels and atherogenic lipid profile in the context of haptoglobin phenotype 2-2. Pregnancy-associated plasma protein-A and soluble endoglin did not predict pre-eclampsia in women with T1DM, and soluble Fms-like tyrosine kinase 1 only predicted pre-eclampsia from the third trimester. Conclusion: Maternally derived biomarkers reflecting glycemic control, insulin resistance and renal dysfunction performed better as PE predictors among women with T1DM than those derived from the placenta. These biomarkers could be trialed in current PE prediction algorithms to tailor them for women with T1DM.

Effects of Epinephrine, Detomidine, and Butorphanol on Assessments of Insulin Sensitivity in Mares.

Sympathoadrenal stimulation may perturb results of endocrine tests performed on fractious horses. Sedation may be beneficial; however, perturbation of results may preclude useful information. Four experiments were designed to 1) determine the effects of epinephrine on insulin response to glucose (IR2G), 2) assess the effects of detomidine (DET), alone or combined with butorphanol (DET/BUT), on IR2G and glucose response to insulin (GR2I), and 3) assess the effects of BUT alone on IR2G. In Experiment 1, mares were administered saline or epinephrine (5 µg/kg BW) immediately before infusion of glucose (100 mg/kg BW). Glucose stimulated (P < .05) insulin release in controls at 5 minutes that persisted through 30 minutes; insulin was suppressed (P < .05) by epinephrine from 5 to 15 minutes, rising gradually through 30 minutes. Experiments 2 (IR2G) and 3 (GR2I) were conducted as triplicated 3 × 3 Latin squares with the following treatments: saline (SAL), DET, and DET/BUT (all administered at .01 mg/kg BW). Glucose stimulated (P < .05) insulin release that persisted through 30 minutes in SAL mares; DET and DET/BUT severely suppressed (P < .0001) the IR2G. Sedation did not affect resting glucose and had inconsistent effects on the GR2I when mares were treated with 50 mIU/kg BW recombinant human insulin. Butorphanol had no effect on IR2G. In conclusion, adrenergic agonists severely suppress the IR2G and cannot be used for sedation for this test. The use of DET did not alter the GR2I, and therefore may be useful for conducting this test in fractious horses.

Sea snake cathelicidin (Hc-cath) exerts a protective effect in mouse models of lung inflammation and infection.

We investigated the anti-inflammatory and antibacterial activities of Hc-cath, a cathelicidin peptide derived from the venom of the sea snake, Hydrophis cyanocyntus, using in vivo models of inflammation and infection. Hc-cath function was evaluated in in vitro, in vivo in the wax moth, Galleria mellonella, and in mouse models of intraperitoneal and respiratory Pseudomonas aeruginosa infection. Hc-Cath downregulated LPS-induced pro-inflammatory responses in macrophages and significantly improved the survival of P. aeruginosa infected G. mellonella over a 5-day period. We also demonstrated, for the first time, that Hc-cath can modulate inflammation in a mouse model of LPS-induced lung inflammation by significantly reducing the release of the pro-inflammatory cytokine and neutrophil chemoattractant, KC, resulting in reduced cellular infiltration into the lungs. Moreover, Hc-cath treatment significantly reduced the bacterial load and inflammation in mouse models of P. aeruginosa intraperitoneal and respiratory infection. The effect of Hc-cath in our studies highlights the potential to develop this peptide as a candidate for therapeutic development.

Inflammation and host-pathogen interaction: Cause and consequence in cystic fibrosis lung disease.

Cystic Fibrosis (CF) lung disease is associated with dysregulation of host defence systems, which ultimately disrupts the balance between inflammation and resolution and leaves the host susceptible to repeated infection. However, the mechanisms underlying these defects are complex and continue to garner significant interest among the CF research community. This review explores emerging data on novel aspects of innate host defence with promising biomarker and therapeutic potential for CF lung disease. Improved understanding of inflammation and host defence against pathogens in patients and animal models during the progression of CF lung disease is pivotal for the discovery of new therapeutics that can limit and/or prevent damage from birth.

Cystic fibrosis epithelial cells are primed for apoptosis as a result of increased Fas (CD95).

BACKGROUND: Previous work suggests that apoptosis is dysfunctional in cystic fibrosis (CF) airways with conflicting results. We evaluated the relationship between dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) and apoptosis in CF airway epithelial cells. METHODS: Apoptosis and associated caspase activity were analysed in non-CF and CF tracheal and bronchial epithelial cell lines. RESULTS: Basal levels of apoptosis and activity of caspase-3 and caspase-8 were significantly increased in CF epithelial cells compared to controls, suggesting involvement of extrinsic apoptosis signalling, which is mediated by the activation of death receptors, such as Fas (CD95). Increased levels of Fas were observed in CF epithelial cells and bronchial brushings from CF patients compared to non-CF controls. Neutralisation of Fas significantly inhibited caspase-3 activity in CF epithelial cells compared to untreated cells. In addition, activation of Fas significantly increased caspase-3 activity and apoptosis in CF epithelial cells compared to control cells. CONCLUSIONS: Overall, these results suggest that CF airway epithelial cells are more sensitive to apoptosis via increased levels of Fas and subsequent activation of the Fas death receptor pathway, which may be associated with dysfunctional CFTR.