Biology subjects

Knight, M. M.

Publications and source records attributed to Knight, M. M..

4 recordsLinked to original sources

bioRxiv2025

Human coronary artery tri-culture organ-chip recapitulates anti-inflammatory effect of pulsatile wall strain

Inflammation is a precursor to vascular diseases, including atherosclerosis, and is modulated by the local biomechanical environment. There is an urgent need for improved in vitro models, to advance understanding, and to test new therapeutic approaches.. This study describes the development and characterization of a human coronary artery organ-chip model of vascular inflammation, with physiological biomechanical stimulation. Human coronary artery endothelial cells and smooth muscle cells were cultured on appropriate extracellular matrices in the two adjoining channels of the Chip-S1(R) from Emulate Inc. Both endothelial and smooth muscle cells demonstrated characteristic phenotypic identity, shown by expression of CD31 and -SMA respectively. Application of physiological pulsatile tensile strain induced alignment of both cell types, perpendicular to strain direction, as seen in vivo. Addition of TNF- to the vascular channel drove an inflammatory response in both cell types, shown by upregulation of ICAM-1 and P65, and attachment and invasion of circulating THP-1 monocytes. Strain field analysis revealed pressure-dependent spatial variation with 12% strain in the center of the chip, and 5% towards the ends. Pulsatile tensile strain reduced the inflammatory response to TNF- with a greater localized inflammatory response in areas of lower strain, further replicating in vivo behavior. In conclusion, we present a fully characterized, tri-culture model of the human coronary artery which recapitulates the physiological effects of pulsatile vessel dilation on morphology and localized inflammatory susceptibility. Our model was developed upon a commercially-available, organ-chip platform, allowing for rapid adoption for therapeutic testing, and fundamental discovery science.

bioengineering↗

bioRxiv2022

CD98 is critical for a conserved inflammatory response to diverse injury stimuli relevant to IPF exacerbations and COVID pneumonitis

Progressive fibrosing interstitial lung diseases (PFILDs) cause substantial morbidity and mortality. Antifibrotic agents slow progression, but most of the clinical need remains unmet. The archetypal PFILD is idiopathic pulmonary fibrosis (IPF). Chronic progression is driven by transforming growth factor (TGF-){beta}1 signalling. It is punctuated by inflammatory flares known as acute exacerbations (AE-IPF), which are associated with accelerated decline and high mortality. We hypothesized that acute injury responses underlying exacerbations and the mechanisms of chronic fibrosis overlap at the molecular level, via a cell surface assembly nucleated by galectin-3 that we term the gal-3-fibrosome. We focused upon a putative pro-inflammatory galectin-3 ligand, the CD98:integrin complex. Our data indicate CD98 and {beta}1-integrin co-localise with galectin-3 within epithelial cells in IPF lung tissue, and within 40 nm in human lung tissue treated with TGF-{beta}1 compared to controls. CD98 is required for interleukin (IL-)6 and IL-8 responses to biochemical and biophysical conditions mimicking stimuli of AE-IPF in vivo, ex vivo and in cells, and for an interstitial neutrophilic response in a mouse model. We demonstrate this pathway progresses via intracellular influx of Ca2+ mediated by TRPV4, and NF-{kappa}B activation, operating in positive feedback. Lastly we show the CD98- and galectin-3-dependence of IL-6 and IL-8 responses to the SARS-CoV-2 spike protein receptor binding domain and the conservation of this response pattern between lung epithelial cells and monocyte-derived macrophages. Taken together our findings identify CD98 as a key mediator of both pro-fibrotic and acute inflammatory responses in the lung with relevance to AE- and chronic progression of IPF, and the priming of fibrotic lungs for acute inflammatory responses. They similarly implicate CD98 and galectin-3 as mediators of COVID pneumonitis and worse outcomes in ILD patients with COVID.

cell biology↗

bioRxiv2021

Fibrillar-Level Strain Gradients Across the Calcified Bone-Cartilage Interface

The bone-cartilage unit (BCU) is a universal feature in diarthrodial joints, which is mechanically-graded and subjected to shear and compressive strains. Changes in the BCU have been linked to osteoarthritis progression. Here we report existence of a physiological internal strain gradient (pre-strain) across the BCU at the ultrastructural scale of the extracellular matrix constituents, specifically the collagen fibril. We use X-ray scattering that probes changes in the axial periodicity of fibril-level D-stagger of tropocollagen molecules in the matrix fibrils, as a measure of microscopic pre-strain. We find that mineralized collagen nanofibrils in the calcified plate are in tensile pre-strain relative to the underlying trabecular bone. This behaviour contrasts with the previously accepted notion that fibrillar pre-strain (or D-stagger) in collagenous tissues always reduces with mineralization, via reduced hydration and associated swelling pressure. Within the calcified part of the BCU, a finer-scale gradient in pre-strain (0.6% increase over [~]50m) is observed. The increased fibrillar pre-strain is linked to prior research reporting large tissue-level residual strains under compression. The findings may have biomechanical adaptative significance: higher in-built molecular level resilience/damage resistance to physiological compression, and disruption of the molecular-level pre-strains during remodelling of the BCI may be potential factors in osteoarthritis-based degeneration.

bioengineering↗

bioRxiv2021

Modelling TGFβR and Hh pathway regulation of prognostic matrisome molecules in ovarian cancer

In a multi-level deconstruction of omental metastases, we previously identified a prognostic matrisome gene expression signature in high-grade serous ovarian cancer (HGSOC) and twelve other malignancies. Here, our aim was to understand how six of these extracellular matrix, ECM, molecules, COL11A1, COMP, FN1, VCAN, CTSB and COL1A1, are up-regulated in cancer. Using biopsies, we identified significant associations between TGF{beta}R activity, Hedgehog signalling and these ECM molecules and then studied the associations in mono-, co- and tri-culture. Activated omental fibroblasts produced more matrix than malignant cells, directed by TGF{beta}R and Hedgehog signalling crosstalk. We reconstructed omental metastases in tri-culture of HGSOC cells, omental fibroblasts and adipocytes. This combination was sufficient to generate all six ECM proteins and the matrisome expression signature. TGF{beta}R and Hedgehog inhibitor combinations attenuated fibroblast activation, gel remodelling and ECM remodelling in these models. The tri-culture model reproduces key features of omental metastases and allows study of diseased-associated ECM.

cancer biology↗