Hypothesis: Peripheral senescent keratocytes act as ECM‑organizing chaperones via a transient, TGF‑β‑biased SASP that fibers collagen lattice, and their premature removal accelerates stromal disorganization in aging cornea

Mechanistic Basis

Recent work shows senescent keratocytes accumulate in the peripheral stroma after injury and secrete COL6A2, COL8A1, COL12A1 through their SASP (4). In young tissue, these cells display a SASP enriched in TGF‑β1, PDGF‑AA and low levels of IL‑6/CXCL8, promoting fibroblast‑to‑keratocyte differentiation and orderly collagen fibrillogenesis (2). With age, oxidative stress shifts the SASP toward a pro‑inflammatory profile (↑CXCL1/8, MMP‑9) while reducing TGF‑β output, converting the chaperone signal into a matrix‑degrading cue (1). Thus, the functional state of senescent keratocytes hinges on the balance between TGF‑β‑driven ECM organization and inflammation‑driven remodeling.

Testable Predictions

1. In young adult mice (2‑3 mo), peripheral p16⁺ keratocytes will exhibit higher TGF‑β1/SMAD2/3 signaling and lower NF‑κB activity than their aged counterparts (18‑20 mo).
2. Genetic ablation of p16⁺ cells in young mice will lead to decreased stromal collagen fibril diameter uniformity and increased light scattering within 2 weeks post‑clearance.
3. Pharmacological supplementation with exogenous TGF‑β1 will rescue the stromal disorganization phenotype after senolytic treatment in young mice.
4. In aged mice, senolytic clearance will reduce stromal haze because the deleterious SASP outweighs any chaperone benefit.

Experimental Approach

• Use p16‑3MR mice to visualize and selectively eliminate senescent keratocytes via ganciclovir.
• Perform second‑harmonic generation (SHG) imaging to quantify collagen fibril organization and optical coherence tomography (OCT) for stromal thickness/haze.
• Isolate stromal cells from young and aged mice, sort p16⁺/p16⁻ fractions, and assay SASP components by multiplex ELISA and phospho‑SMAD2/3 vs phospho‑p65 Western blots.
• Treat cleared young mice with recombinant TGF‑β1 (1 ng/µL, topical) or vehicle and assess rescue of SHG metrics.
• Statistical analysis: two‑way ANOVA (age × treatment) with post‑hoc Tukey; n ≥ 6 per group.

If predictions 1‑3 hold, senescent keratocytes function as age‑dependent chaperones whose loss precipitates ECM disarray, challenging the notion that senolytic clearance is universally beneficial in the cornea.