Biology subjects

Ridley, R. A.

Publications and source records attributed to Ridley, R. A..

3 recordsLinked to original sources

bioRxiv2021

Pseudohypoxic HIF pathway activation dysregulates collagen structure-function in human lung fibrosis

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 show that hypoxia-inducible factor (HIF) pathway activation is a critical pathway for this process regardless of oxygen status (pseudohypoxia). Whilst TGF{beta} increased rate of fibrillar collagen synthesis, HIF pathway activation was required to dysregulate post-translational modification of fibrillar collagen, promoting bone-type cross-linking, altering collagen nanostructure, and increasing tissue stiffness. In vitro, knock down of Factor Inhibiting HIF (FIH) or oxidative stress caused pseudohypoxic HIF activation in normal fibroblasts. In 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 IPF lung 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.

cell biology↗

bioRxiv2020

Label-free and Multimodal Second Harmonic Generation Light Sheet Microscopy

Light sheet microscopy (LSM) has emerged as one of most profound three dimensional (3D) imaging tools in the life sciences over the last decade. However, LSM is currently performed with fluorescence detection on one- or multi-photon excitation. Label-free LSM imaging approaches have been rather limited. Second Harmonic Generation (SHG) imaging is a label-free technique that has enabled detailed investigation of collagenous structures, including its distribution and remodelling in cancers and respiratory tissue, and how these link to disease. SHG is generally regarded as having only forward- and back-scattering components, apparently precluding the orthogonal detection geometry used in Light Sheet Microscopy. In this work we demonstrate SHG imaging on a light sheet microscope (SHG-LSM) using a rotated Airy beam configuration that demonstrates a powerful new approach to direct, without any further processing or deconvolution, 3D imaging of harmonophores such as collagen in biological samples. We provide unambiguous identification of SHG signals on the LSM through its wavelength and polarisation sensitivity. In a multimodal LSM setup we demonstrate that SHG and two-photon signals can be acquired on multiple types of different biological samples. We further show that SHG-LSM is sensitive to changes in collagen synthesis within lung fibroblast 3D cell cultures. This work expands on the existing optical methods available for use with light sheet microscopy, adding a further label-free imaging technique which can be combined with other detection modalities to realise a powerful multi-modal microscope for 3D bioimaging.

biophysics↗

bioRxiv2020

A novel isoform of ACE2 is expressed in human nasal and bronchial respiratory epithelia and is upregulated in response to RNA respiratory virus infection

Angiotensin-converting enzyme 2 (ACE2) is the main entry point in the airways for SARS-CoV-2. ACE2 binding to SARS-CoV-2 protein Spike triggers viral fusion with the cell membrane, resulting in viral RNA genome delivery into the host. Despite ACE2s critical role in SARS-CoV-2 infection, an understanding of ACE2 expression, including in response to viral infection, remains unclear. Until now ACE2 was thought to encode five transcripts and one 805 amino acid protein. Here we identify a novel short isoform of ACE2. Short ACE2 is expressed in the airway epithelium, the main site of SARS-CoV-2 infection; it is substantially upregulated in response to interferon stimulation and RV infection, but not in response to SARS-CoV-2 infection, and it shows differential regulation in asthma patients. This short isoform lacks SARS-CoV-2 spike glycoprotein high-affinity binding sites and altogether, our data are consistent with a model where short ACE2 may influence host susceptibility to SARS-CoV-2 infection.

molecular biology↗