Cigarette smoke and air pollution induce dysfunctional pulmonary microvascular endothelial repair

Abstract

<b>Background:</b> Dysfunctional microvascular endothelial cells (ECs) drive microangiopathies in respiratory diseases where tobacco smoking and air pollution are two major risk factors. We aimed to assess the effect of cigarette smoke (CS) and diesel exhaust particles (DEP) on pulmonary microvascular endothelial repair <i>in vitro</i>. <b>Methods:</b> Immortalized human pulmonary microvascular endothelial cells (HPMECs) were serum-starved for 24 h followed by treatments with either control, CS extract (CSE) (1%, 2.5%, 5%) or DEP (50, 100, 200 µg/ml) for 24h/48h. Post-analyses comprised scratch wound healing and organoid assays. Lung organoids were generated by co-culturing murine epithelial progenitors (CD31-/CD45-/Epcam+), fibroblasts, and HPMECs in Matrigel. Furthermore, RNA sequencing (RNA-seq) was performed on ECs (CD31+ cells) isolated from mice exposed to air or CS. <b>Results:</b> HPMECs rapidly closed scratch wounds in controls, yet were inhibited, both by 2.5% CSE and 100 µg/ml DEP. In the lung organoid assay, the presence of HPMECs increased the formation of alveolar epithelial organoids. This stimulatory effect was inhibited by pre-treatment of HPMECs with 2.5% CSE or DEP (100 µg/ml). RNA-seq analysis on ECs obtained from smoke-exposed mice showed several differentially expressed gene pathways including VEGFA-VEGFR2, IL-18, vitamin D receptor, circadian rhythm, and NRF2 signaling pathways, which were all upregulated in response to CS exposure. <b>Conclusions:</b> CSE or DEP exposure impairs the wound repair function of HPMECs. CS induces a wide range of transcriptomic changes on pulmonary microvascular ECs, which provide novel guidance for mechanisms and future therapeutic strategies.