Investigating the mechanisms through which lung MSCs regulate endothelial functionality in COPD using distal lung organoid models

<bold>Background:</bold> Reduced repair and regeneration in COPD drives disease progression. The regenerative capacity of resident lung MSCs (LMSCs) in health and in COPD is poorly understood. Prior work has shown that LMSCs from COPD patients secrete lower levels of hepatocyte growth factor (HGF) compared to non-COPD donors. Hence, we tested the role of LMSC-derived HGF using distal lung organoid models and explored mechanisms of HGF regulation in LMSCs by altering DNA methylation levels. <bold>Methods:</bold> LMSCs were isolated from lung tissues of non-COPD and COPD patients (n=5/group) as previously described (PMID:36681830). HGF levels in cell supernatants were quantified by ELISA. Supernatants were added to HPMECs to assess tube formation capacity. Specific siRNA or HGF receptor antagonist were used to inhibit HGF signalling. 5-azacytidine, an inhibitor of DNA methyltransferase I, was used to target DNA hypermethylation. Distal lung organoids were produced by self-aggregation of small airway epithelial cells (SAECs), LMSCs, and HPMECs in Matrigel for 21 days before analysis by confocal microscopy. <bold>Results:</bold> 5-azacytidine significantly improved HGF secretion in COPD LMSCs and restored endothelial tube formation ability to that of non-COPD controls. siHGF transfection or treatment with HGF inhibitor of non-COPD LMSC removed their ability to support tube formation. COPD and siHGF transfected control LMSCs formed smaller organoids and did not support endothelial cells, unlike non-COPD controls. Pre-treatment of COPD LMSCs with 5-azacytidine partially mitigated these effects. <bold>Conclusion:</bold> LMSC secretion of HGF supports endothelial cells in human lung tissue and is impaired in COPD.