CD44-targeting small-molecule inhibitors restore hyaluronan homeostasis and reduce profibrotic matrix remodeling in vocal fold fibroblasts exposed to cigarette smoke extract.

Vocal fold scarring (VFS) is a debilitating fibrotic disorder lacking molecular biomarkers for early detection or for monitoring toxicant-induced injury. Cigarette smoke toxicity (CST), a major environmental driver of vocal fold fibrosis, disrupts extracellular matrix (ECM) homeostasis; however, the mechanisms linking CST to fibrotic remodeling remain poorly understood. Here, we identify CD44, a hyaluronan receptor, as a key regulator of CST-induced VFS and healthy human vocal fold fibroblast (hVFF) homeostasis. CD44 depletion reduced elastin, collagen, and lysyl oxidase mRNA expression and increased conditioned medium viscosity, demonstrating its essential role in maintaining ECM composition and viscoelastic properties. Cigarette smoke extract (CSE; 2.5-20%) induced concentration-dependent morphological alterations and cytotoxicity in hVFFs. At the median lethal concentration (LC₅₀, 5% CSE), exposure increased collagen synthesis, NHE1 expression, oxidative stress, and p53-mediated apoptosis. CSE promoted degradation of high-molecular-weight hyaluronan (500-1100kDa) into low-molecular-weight fragments (30.6-550kDa) within the ECM via CD44 and hyaluronidase but not through CEMIP, leading to glycocalyx disruption, impaired wound repair and profibrotic remodeling. These findings identify the CD44-dependent hyaluronan-catabolic pathway as a central mediator of CST-induced ECM remodeling. CD44 exhibited a context-dependent dual role in hVFF responses to CSE, acting protectively during short-term exposure (5%, 24h) but promoting detrimental cellular responses with increasing CSE concentration (≥5%) and prolonged exposure (≥72h). A synergistic pair of small-molecule inhibitors targeting CD44 signaling and structural integrity reduced oxidative stress, normalized intracellular pH, suppressed p53 expression, restored CD44 expression, attenuated inflammation, and improved wound healing. Collectively, these findings identify CD44 as a promising molecular indicator of early VFS-associated changes and a potential therapeutic target for CST-induced profibrotic remodeling.