Hypoxia-induced OPN5 expression is associated with matrix-preserving responses in human scleral fibroblasts

Myopia-associated scleral remodeling is characterized by altered extracellular matrix (ECM) turnover and progressive biomechanical weakening, processes that have been increasingly linked to scleral hypoxia. Opsin 5 (OPN5), a violet light-sensitive opsin, has been implicated in ocular growth regulation, but its role in scleral fibroblasts remains largely unknown. This study investigated the expression of OPN5 in human scleral fibroblasts (HSFs) under hypoxic conditions and examined its association with remodeling-related cellular responses.

Primary HSFs were cultured under hypoxia (2% O₂) and exposed to violet light (360-400 nm, 400 μW/cm²). Expression of OPN5 and remodeling-associated markers, including COL1A1, MMP2, TIMP2, and α-SMA, was assessed by quantitative real-time PCR and Western blotting. OPN5 gain- and loss-of-function models were generated to evaluate associations between OPN5 expression and ECM-related responses. Transcriptomic profiling was performed in OPN5-overexpressing cells to explore potentially associated biological pathways.

Exposure to hypoxic conditions resulted in progressive upregulation of OPN5 at both the transcript and protein levels and was accompanied by reduced COL1A1 and TIMP2 expression together with increased MMP2 and α-SMA expression, consistent with a remodeling-associated phenotype. Violet light further enhanced OPN5 expression under hypoxic conditions and was associated with partial restoration of COL1A1 and TIMP2 expression, together with reduced MMP2 and α-SMA expression levels. OPN5 overexpression was associated with increased COL1A1 and TIMP2 expression and decreased MMP2 and α-SMA expression, whereas CRISPR/Cas9-mediated reduction of endogenous OPN5 was associated with decreased COL1A1 and TIMP2 together with increased α-SMA expression. Transcriptomic analysis suggested potential associations between OPN5 expression and pathways related to extracellular matrix regulation, metabolism, Wnt signaling, and cytokine responses.

OPN5 expression was increased in human scleral fibroblasts under hypoxic conditions and was associated with molecular signatures consistent with extracellular matrix preservation and reduced fibroblast activation. Violet light further enhanced OPN5 expression and was associated with partial attenuation of hypoxia-associated remodeling changes. These findings suggest that OPN5 is a hypoxia-responsive factor in scleral fibroblasts and support a context-dependent association between OPN5 signaling and extracellular matrix homeostasis under conditions relevant to myopia-associated scleral remodeling.