Abstract
Purpose :
Osteopontin (OPN) is a proinflammatory glycoprotein shown to be elevated in both human and mouse retinas in the early stages of diabetic retinopathy. Our study was designed to examine the role of OPN in the development of ocular surface complications in Type-2 Diabetic (T2D) mice.
Methods :
C57BL/6 wild-type (WT) and OPN-knockout (OPN-/-) mice, aged 3-4 weeks, were fed a high-fat-diet (HFD) for 3 weeks, followed by streptozotocin (STZ) injections for 5 consecutive days to induce T2D. Diabetes (blood glucose > 200 mg/dL) was confirmed 1-2 weeks post-STZ treatment. Post-diagnosis, mice were maintained on HFD with regular STZ injections and blood glucose monitoring. ELISA assays were performed targeting epithelial junctional proteins and fluorescein and wheat germ agglutinin (WGA) staining were used to highlight corneal surface irregularities. These assays were conducted at 4, 6, 8, and 16-weeks post-diabetes onset, with results being compared among diabetic WT and OPN-/- mice, and non-diabetic control mice. One-Way ANOVA was used for statistical analysis with P < 0.05 considered significant.
Results :
WT and OPN-/- mice developed T2D within 2 and 3 weeks, respectively, of HFD+STZ treatment. Corneas of WT diabetic mice had significantly higher OPN levels at 4 weeks compared to non-diabetic controls (427.3 ± 5.8 vs. 187.6 ± 1.2 pg/mL; P < 0.0001) and remained significantly elevated at 16 weeks (634.0 ± 20.1 vs. 211 ± 12.3 pg/mL; P < 0.0001). Enhanced fluorescein and WGA staining of corneal irregularities appeared in WT mice at 8 weeks and in OPN-/- mice at 10 weeks post-T2D induction. ELISA analysis over 4-8 weeks of hyperglycemia showed no significant change in ZO-1, ZO-2, Claudin-1, Claudin-4, Claudin-10, and E-Cadherin expression in corneal tissue, irrespective of OPN status. However, at 16 weeks T2D, WT mice exhibited ~1.5, ~1.7, ~1.4, and 1.2-fold significant (P < 0.001) reductions in ZO-1, Claudin-1, Claudin-4, and E-Cadherin respectively, compared to non-diabetic controls and this trend was absent in OPN-/- mice.
Conclusions :
Elevated OPN levels in WT corneas were noted at 4 weeks post T2D, without initial subclinical changes. While significant corneal epithelial cell irregularities appeared at 8 weeks, a pattern delayed in OPN-/- mice. Our findings underscore OPN’s involvement in diabetic eye surface complications, pointing to the potential of OPN-targeted therapies in both managing and preventing such issues.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.