S147 Understanding the role of endothelial senescence and endothelial to mesenchymal transition in development of atherosclerosis in patients with chronic lung disease

Background

Cellular senescence drives the pathophysiology of age-related disorders. Cardiovascular disease (CVD) is a leading cause of morbidity and mortality in both chronic obstructive pulmonary disease (COPD) and in idiopathic pulmonary fibrosis (IPF). Senescent endothelial cells are dysfunctional, exhibit a proinflammatory 'senescence-associated-secretory-phenotype' (SASP) and promote CVD. TGFβ signaling plays a key role in COPD, IPF and development of endothelial to mesenchymal transition (EndMT) promoting atherosclerosis. Recent literature suggests evidence of EndMT in COPD and IPF. We hypothesized that endothelial senescence promotes endothelial dysfunction and EndMT, contributing to the development of atherosclerosis in COPD and IPF.

Methods

Endothelial colony forming cells (ECFC) are circulating endothelial progenitors and provide non-invasive access to endothelial cells. We have provided evidence of accelerated endothelial senescence in COPD patients due to epigenetic dysfunction that can be targeted pharmacologically (Paschalaki et al, Stem Cells 2013; Thorax 2022). We isolated ECFC from COPD cohort (n=16), IPF (n=5) and age-matched healthy non-smokers (n=11). ECFC were characterised for endothelial markers, proliferation, senescence and selected SASP mediators by immunofluorescence (IF) and western blotting. We used a high-throughput 'organ-on-a-chip' microfluidic platform (OrganoPlate-MIMETAS) that allows formation of microvessels for functional and IF analysis.

Results

TGF-β2 treatment of endothelial cells (ECFC and human umbilical endothelial cells: HUVEC) induced premature senescence (increased senescence-associated-β-galactosidase activity: SA-β-gal activity, p21 and p16), increased expression of proinflammatory (IFN-γ-inducible-protein-10: IP-10), prothrombotic (von Willebrand Factor: vWF) and fibrotic mediators (plasminogen activator inhibitor-1: PAI-1), as well as markers of EndMT (increased expression of the mesenchymal marker SM22α). ECFC from COPD and IPF patients exhibited reduced proliferation (Ki-67), increased expression of senescence markers (SA-β-gal activity, p21, p16), IP-10, PAI-1 and vWF compared to ECFC from healthy non-smokers. In 3D cultures maintained for 14 days, microvessels formed with patients' ECFC showed increased permeability, increased expression of markers of senescence and disruption of endothelial junctions.

Conclusion

ECFC from COPD and IPF patients exhibit a dysfunctional, senescent and prothrombotic phenotype that may contribute to the increased risk of cardiovascular comorbidities. TGF-β signaling pathway promotes endothelial senescence and EndMT and may drive vascular ageing and CVD in patients with chronic lung disease.