Comparing Effects of Short- and Long-Term Exposure of Cigarette Smoke Extract on Human Vocal Fold Fibroblasts

Abstract

To explore the effects of short- and long-term cigarette smoke extract (CSE) stimulation on the expression of extracellular matrix (ECM) components and inflammatory cytokines in an in vitro model for studying Reinke's edema using human vocal fold fibroblasts (hVFF). Experimental pilot study using intervention with CSE in vitro. Immortalized hVFF were pretreated with 5% CSE or control medium over a period of 2 or 8weeks, followed by a final 3-day incubation time. We evaluated cell proliferation and examined gene and protein expression of control- and CSE-treated cells using quantitative polymerase chain reaction, Western Blot and enzyme linked immunosorbent assay. Cell numbers of CSE-treated hVFF strongly decreased after 8weeks and limited the overall duration of the experiment. We observed significant upregulations in gene expression and protein levels of inflammatory markers (cyclooxygenase COX1, COX2) and ECM components (decorin, matrix metalloproteinase 1, transglutaminase 2, gremlin 2) induced by CSE after 2 and 8weeks. Interleukin 1 receptor 1, prostaglandin I2 synthase, collagen- and hyaluronan-related gene expression showed minor upregulations. The majority of the observed genes were similarly regulated at both time points. However, the CSE-induced mRNA level of COX1 was ablated after 8weeks. Long-term treatment did not yield results significantly different from the short-term protocol. Therefore, we propose that prolonged CSE exposure is not superior to short-term settings, which save both time and materials.