Extracellular matrix (ECM) stiffening with downstream activation of mechanosensitive pathways is strongly implicated in fibrosis. We previously reported that altered collagen nanoarchitecture is a key determinant of pathogenetic ECM structure-function in human fibrosis (Jones et al., 2018). Here, through human tissue, bioinformatic and ex vivo studies we provide evidence that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of the oxygen status (pseudohypoxia). Whilst TGFβ increased the rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting pyridinoline cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knockdown of Factor Inhibiting HIF (FIH), which modulates HIF activity, or oxidative stress caused pseudohypoxic HIF activation in the normal fibroblasts. By contrast, endogenous FIH activity was reduced in fibroblasts from patients with lung fibrosis in association with significantly increased normoxic HIF pathway activation. In human lung fibrosis tissue, HIF-mediated signalling was increased at sites of active fibrogenesis whilst subpopulations of human lung fibrosis mesenchymal cells had increases in both HIF and oxidative stress scores. Our data demonstrate that oxidative stress can drive pseudohypoxic HIF pathway activation which is a critical regulator of pathogenetic collagen structure-function in fibrosis.

Background Routine follow-up of patients hospitalised with COVID-19 is recommended, however due to the ongoing high number of infections this is not without significant health resource and economic burden. In a previous study we investigated the prevalence of, and risk factors for, persistent chest radiograph (CXR) abnormalities post-hospitalisation with COVID-19 and identified a 5-point composite score that strongly predicted risk of persistent CXR abnormality at 12-weeks. Here we sought to validate and refine our findings in an independent cohort of patients. Methodology A single-centre prospective study of consecutive patients attending a virtual post-hospitalisation COVID-19 clinic and CXR as part of their standard clinical care between 2nd March – 22nd June 2021. Inpatient and follow-up CXRs were scored by the assessing clinician for extent of pulmonary infiltrates (0–4 in each lung) with complete resolution defined as a follow-up score of zero. Results 182 consecutive patients were identified of which 31% had persistent CXR abnormality at 12-weeks. Patients with persistent CXR abnormality were significantly older (p < 0.001), had a longer hospital length of stay (p = 0.005), and had a higher incidence of both level 2 or 3 facility admission (level 2/3 care) (p = 0.003) and ever-smoking history (p = 0.038). Testing our composite score in the present cohort we found it predicted persistent CXR abnormality with reasonable accuracy (area under the receiver operator curve [AUROC 0.64]). Refining this score replacing obesity with Age ≥ 50 years, we identify the SHADE-750 score (1-point each for; Smoking history, Higher-level care (level 2/3 admission), Age ≥ 50 years, Duration of admission ≥ 15 days and Enzyme-lactate dehydrogenase (LDH ≥ 750U/L), that accurately predicted risk of persistent CXR abnormality, both in the present cohort (AUROC 0.73) and when retrospectively applied to our 1st cohort (AUROC 0.79). Applied to both cohorts combined (n = 213) it again performed strongly (AUROC 0.75) with all patients with a score of zero (n = 18) having complete CXR resolution at 12-weeks. Conclusions In two independent cohorts of patients hospitalised with COVID-19, we identify a 5-point score which accurately predicts patients at risk of persistent CXR abnormality at 12-weeks. This tool could be used by clinicians to identify patients in which radiological follow-up may not be required.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease that usually affects elderly people. It has a poor prognosis and there are limited therapies. Since epigenetic alterations are associated with IPF, histone deacetylase (HDAC) inhibitors offer a novel therapeutic strategy to address the unmet medical need. This study investigated the potential of romidepsin, an FDA-approved HDAC inhibitor, as an anti-fibrotic treatment and evaluated biomarkers of target engagement that may have utility in future clinical trials. The anti-fibrotic effects of romidepsin were evaluated both in vitro and in vivo together with any harmful effect on alveolar type II cells (ATII). Bronchoalveolar lavage fluid (BALF) from IPF or control donors was analyzed for the presence of lysyl oxidase (LOX). In parallel with an increase in histone acetylation, romidepsin potently inhibited fibroblast proliferation, myofibroblast differentiation and LOX expression. ATII cell numbers and their lamellar bodies were unaffected. In vivo, romidepsin inhibited bleomycin-induced pulmonary fibrosis in association with suppression of LOX expression. LOX was significantly elevated in BALF of IPF patients compared to controls. These data show the anti-fibrotic effects of romidepsin, supporting its potential use as novel treatment for IPF with LOX as a companion biomarker for evaluation of early on-target effects.