The role of NADPH oxidases in airway smooth muscle from asthmatics and COPD patients
Creators: name: Biddle, Michael
Publisher: name: University of Leicester
Year: 2022
Subjects: Uncategorized

description: <strong>Background</strong> Oxidative stress is present in chronic obstructive pulmonary disease (COPD) and asthma, with one major source of reactive oxygen species (ROS) being the NADPH oxidase (Nox) family. Within these diseases airway smooth muscle (ASM) is dysfunctional exhibiting increased: mass, matrix deposition, contraction, inflammatory mediator expression and corticosteroid resistance. Of interest, ROS is implicated in all these characteristics. With the exclusion of Nox4, Nox expression in ASM has not been fully investigated and thus we hypothesised that ASM dysfunction, may be driven by one or more members of the Nox family. <strong>Aims</strong> The overall aim of this project was to investigate the expression and function of Nox enzymes in ASM cells from healthy controls, asthmatics, and COPD patients. This was achieved by generating overexpression plasmids enabling the validation of antibodies, inhibitors and TaqMan assays prior to their use within primary ASM cells. Nox expression and accessory proteins was then assessed in primary human ASM cells from healthy control, asthmatics, and COPD patients at the mRNA and protein level. This expression data was used to decide on further experiments to be performed, regarding the role of Nox enzymes in ASM dysfunction. As Nox4 was the most predominantly expressed isoform, a focus was placed on investigating the effects of Nox4 in ASM cells. However, as there was no difference in the expression of Nox4 across ASM from healthy control, asthmatics and COPD patients’ expression of Nox4 by ASM cells was assessed following treatment with the asthma and COPD relevant cytokine TGFβ1. Following this, the effects of both TGFβ1 and Nox4 overexpression were further assessed, with a focus on mechanisms relevant to ASM mass, with the ultimate aim of assessing the role of Nox4 in the TGFβ1 mediated effects, if the effects of TGFβ1 and Nox4 overexpression mimicked each other. <strong>Methods</strong> Overexpression plasmids were generated to confirm the efficiency of TaqMan assays (by qPCR) and to validate commercial Nox antibodies (by flow cytometry) and inhibitors (by measuring H2O2 with Amplex UltraRed) using overexpressed HEK293T cells. The expression of the Nox family and associated accessory proteins was assessed in cultured primary ASM cells, using qPCR and flow cytometry, with or without 24-hour treatment with TGFβ1. ASM production of H2O2 was assessed using the AmplexUltraRed assay following 24-hour treatment with TGFβ1. Cell count, hypertrophy, and granularity were then assessed following TGFβ1 treatment and/or Nox4 overexpression through lentiviral transduction, and subsequent flow cytometry. <strong>Results</strong> qPCR confirmed the expression of all Nox enzymes (excluding Nox3), with further confirmation of accessory gene expression for Nox1 and Nox4. No difference in expression was observed at the gene or protein level between ASM cells from healthy controls, asthmatics, or COPD patients. Using flow cytometry, Nox4 was confirmed to be localised at the cell surface and within the cell. TGFβ1 treatment increased the expression of Nox4 at the cell surface and within the cell in a dose-dependent manner. Treatment with TGFβ1 also significantly increased the H2O2 production of ASM cells. TGFβ1 treatment and Nox4 overexpression resulted in a reduction in ASM cell number, an increase in cell hypertrophy and an increase in cellular granularity within primary ASM cells. <strong>Conclusion</strong> Nox4 overexpression and TGFβ1 treatment resulted in reduced ASM proliferation and increased ASM cell size and granularity, all features of cellular senescence. Further work is required to confirm the contribution of Nox4 to TGFβ1 effects, their relation to senescence and potential implications for ASM dysfunction.