Unvaporized e-liquid toxicity elevates CD44-dependent hyaluronan catabolic gene expression and triggers inflammation in human vocal fold fibroblasts

Electronic (e)-cigarette and e-liquid exposure have been linked to vocal fold inflammation and dysphonia, yet no targeted non-surgical therapies currently exist. Hyaluronan, a key extracellular matrix component essential for vocal fold structure, repair, and function, is known to be dysregulated in inflammatory conditions; however, its metabolic gene response to e-liquid exposure in human vocal fold fibroblasts (hVFFs) remains uncharacterized. Hence, it is critical to understand hyaluronan metabolic gene expression under e-liquid toxicity to develop novel drug discovery strategies for vocal fold inflammation. To avoid confounding effects from thermal degradation and aerosol variability in conventional vapor models, hVFFs were exposed to nicotine-containing unvaporized e-liquid (0.125-1 mg/mL) for 24 h, revealing concentration-dependent changes in cell morphology and viability (p < 0.05). The lethal concentration 50 (LC₅₀) was determined to be 0.437 mg/mL and used for short-term (24 h) and extended (72-96 h) exposures. Extended exposure induced intracellular reactive oxygen species (ROS), inflammation, suppressed collagenolysis, and increased the collagen 1 A: collagen 3 A ratio, suggesting fibrotic remodeling. Short-term exposure downregulated hyaluronan synthases (HAS1, HAS2, HAS3) and catabolic genes (HYAL2, CD44), reducing extracellular hyaluronan levels. In contrast, extended exposure repressed HAS1 and HAS2 while upregulating HAS3, CD44, and HYAL2, indicating enhanced hyaluronan degradation and accumulation of proinflammatory low molecular weight hyaluronan. CD44 silencing reduced <i>IL-8</i> mRNA expression, confirming its role in hVFF inflammation. These findings provide the first mechanistic insight into unvaporized e-liquid-induced dysregulation of hyaluronan metabolism in hVFFs, offering a foundation for biomarker identification and therapeutic development targeting e-cigarette-associated vocal fold inflammation.