545 INVESTIGATING THE RISK OF CARDIAC FIBROSIS IN RESPONSE TO HEAT-NOT-BURN CIGARETTES THROUGH HUMAN CARDIAC STROMAL CELLS

Abstract

Abstract Background The use of alternative smoking devices, such as heat-not-burn cigarettes (HNBC), is increasing on a global scale, and their impact on health is still uncertain. Objective To investigate the effects of circulating molecules in HNBC chronic smokers on the fibrotic specification and paracrine function of cardiac stromal cells (CSCs). Methods Resident CSCs were isolated from the atrial tissue of non-smokers patients with cardiovascular diseases, and exposed to the serum pools derived from 60 young healthy subjects, stratified in exclusive HNBC smokers, traditional combustion cigarette (TCC) smokers, or non-smokers (NS) as reference. Results CSCs treated with TCC serum versus NS displayed impaired 3D growth (NS 568±24 vs. TCC 442±27 spheroids/well, p≤0,01) and reduced migration after 6h (NS 0,62±0,04 vs. TCC 0,80±0,03 normalized scratch area versus t0, p≤0,01) and 10h (NS 0,49±0,03 vs. TCC 0,36±0,03 normalized scratch area versus t0, p≤0,05), as well as significantly increased expression (IL-6, IL-8) and/or release of pro-inflammatory (e.g CRP, PAI-1, CD40L, CXCL4) and pro-fibrotic cytokines (PDGFA, MMP1). CSCs cultured with HNBC serum showed significantly increased mRNA levels of pro-fibrotic genes (PDGFA, THY1, COL1A1, p<0,01), and significantly reduced expression of the gap junction protein CX43 (p<0,05). Nonetheless, both TCC and HNBC sera reduced the release of angiogenic and protective factors (e.g. VEGF, Adiponectin, CHI3L1) from CSCs, as assessed by protein arrays and ELISA. In fact, their paracrine support to tube-formation by endothelial cells (NS 63,29±4,72 vs. TCC 41,43±6,64 vs. HNBC 37,29±5,67 mesh number per well, p≤0,05) and to preserved cell viability of neonatal rat cardiomyocytes (NS 0,60±0,04 vs. TCC 0,40±0,01 vs. HNBC 0,47±0,01 OD490 normalized OD versus t0, p≤0,001) were significantly impaired. Treatment with the sera of both types of smokers also increased the expression of NOX isoforms and the release of H2O2 (NS 1,76±0,09 vs. TCC 3,84±0,25 vs. HNBC 2,90±0,35 µM, p≤0,05) and significantly reduced the release of NO (NS 6,98±0,43 vs. TCC 4,65±0,07 vs. HNBC 5,06±0,13 µM, p≤0,01) by CSCs. Conclusion The circulating molecules in the serum of chronic HNBC smokers induce fibrotic specification in CSCs. They also reduce the beneficial paracrine effects of stromal cells on endothelial cells and cardiomyocytes, albeit to a reduced extent for some features. These results point to a potential risk for atrial fibrosis development triggered by chronic HNBC use.