Rationale The pathogenesis of PAH remains unclear. The four microRNAs representing the miR-143 and miR-145 stem loops are genomically clustered. Objective To elucidate the transcriptional regulation of the miR-143/145 cluster, and the role of miR-143 in PAH. Methods and Results We identified the promoter region that regulates miR-143/145 miRNA expression in pulmonary artery smooth muscle cells (PASMCs). We mapped PAH-related signalling pathways, including estrogens receptor (ER), liver X factor/retinoic X receptor (LXR/RXR), TGF-β (Smads), and hypoxia (HRE) that regulated levels of all pri-miR stem loop transcription and resulting miRNA expression. We observed that miR-143-3p is selectively upregulated compared to miR-143-5p during PASMC migration. Modulation of miR-143 in PASMCs significantly altered cell migration and apoptosis. In addition, we found high abundance of miR-143-3p in PASMCs-derived exosomes. Using assays with pulmonary arterial endothelial cells (PAECs) we demonstrated a paracrine pro-migratory and pro-angiogenic effect of miR-143-3p enriched exosomes from PASMC. Quantitative PCR and in situ hybridisation showed elevated expression of miR-143 in calf models of PAH as well as in samples from PAH patients. Moreover, in contrast to our previous findings that had not supported a therapeutic role in vivo, we now demonstrate a protective role for miR-143 in experimental PH in vivo in miR-143−/− and antimiR143-3p-treated mice exposed to chronic hypoxia in both preventative and reversal settings. Conclusions miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while inhibition of miR-143-3p blocked experimental PH. Taken together these findings confirm an important role for the miR-143/145 cluster in PAH pathobiology.

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Adenoviruses are common pathogens, mostly targeting ocular, gastrointestinal and respiratory cells, but in some cases infection disseminates, presenting in severe clinical outcomes. Upon dissemination and contact with blood, coagulation factor X (FX) interacts directly with the adenovirus type 5 (Ad5) hexon. FX can act as a bridge to bind heparan sulphate proteoglycans, leading to substantial Ad5 hepatocyte uptake. FX “coating” also protects the virus from host IgM and complement-mediated neutralisation. However, the contribution of FX in determining Ad liver transduction whilst simultaneously shielding the virus from immune attack remains unclear. In this study, we demonstrate that the FX protection mechanism is not conserved amongst Ad types, and identify the hexon hypervariable regions (HVR) of Ad5 as the capsid proteins targeted by this host defense pathway. Using genetic and pharmacological approaches, we manipulate Ad5 HVR interactions to interrogate the interplay between viral cell transduction and immune neutralisation. We show that FX and inhibitory serum components can co-compete and virus neutralisation is influenced by both the location and extent of modifications to the Ad5 HVRs. We engineered Ad5-derived HVRs into the rare, native non FX-binding Ad26 to create Ad26.HVR5C. This enabled the virus to interact with FX at high affinity, as quantified by surface plasmon resonance, FX-mediated cell binding and transduction assays. Concomitantly, Ad26.HVR5C was also sensitised to immune attack in the absence of FX, a direct consequence of the engineered HVRs from Ad5. In both immune competent and deficient animals, Ad26.HVR5C hepatic gene transfer was mediated by FX following intravenous delivery. This study gives mechanistic insight into the pivotal role of the Ad5 HVRs in conferring sensitivity to virus neutralisation by IgM and classical complement-mediated attack. Furthermore, through this gain-of-function approach we demonstrate the dual functionality of FX in protecting Ad26.HVR5C against innate immune factors whilst determining liver targeting.

This work was to assess possible impacts of novel insoluble fiber 8% bacterial cellulose (BC), soluble fiber 8% konjac glucomannan (KGM), and their mixture (4% BC/4% KGM) on fatty acid metabolism and intestinal microbiota of C57BL/6J mice fed with a high-fat diet (HFD). HFD-fed mice receiving the dietary fibers (DFs) for 16 weeks exhibited an improvement in lipid-associated cytokines and a decrease in inflammation factors, which was associated with the improved hepatic and serum fatty acid composition. The DFs, notably the mixed BC/KGM, elevated the HFD-caused decrease in the contents of acetic acid (from 23.9 ± 0.85 to 32.2 ± 0.84 mM/g; p < 0.05), propionic acid (from 6.53 ± 0.28 to 12.8 ± 0.58 mM/g; p < 0.05), and butyric acid (from 7.73 ± 0.43 to 13.5 ± 0.47 mM/g; p < 0.05). Furthermore, the mixed BC/KGM significantly decreased the abundance of Firmicutes (from 90.4 to 67.6%) and Mucispirillum (from 4.77 to 1.58%) and dramatically increased the abundance of Bacteroidetes (from 7.83 to 25.0%) and Akkermansia (from 0.69 to 2.80%) in the gut of HFD-fed mice at the genus level. Moreover, correlation analysis revealed that the multiplicity of gut microbiota was useful in sustaining colonic integrity through producing short-chain fatty acids to some extent. This finding suggests that a mixture of insoluble BC and soluble KGM has positive effects on modulation of the intestinal microecosystem in mice.