Abstract
Purpose :
Roughly 425 million people are diagnosed with diabetes, which is the leading cause of adult-onset blindness. Despite the prevalence of diabetic retinopathy, 70% of adult diabetic patients develop visually debilitating corneal complications, including impaired wound healing. Unfortunately, treatment for diabetes-induced corneal damage remains limited. The current project seeks to begin investigating a peptide-based combination therapy, thymosin β4 and vasoactive intestinal peptide (Tβ4/VIP), to restore high glucose-induced damage to the corneal epithelium.
Methods :
The electric cell-substrate impedance sensing (ECIS®) system was used for real-time monitoring of barrier function and wound healing of human telomerase-immortalized corneal epithelial cells (HUCLs). HUCLs were seeded (60,000 cells/well) in DMEM/F12 media supplemented with 10% FBS, 1% pen/strep in a 96-well array plate. Upon confluency, cells were serum starved for 12-16 h before treatment with normal glucose (5mM; NG) or high glucose (25mM; HG) + Tβ4 (0.1%) and VIP (5nM). Total resistance of the HUCL monolayer across a range of frequencies (250-64,000 Hz) was measured over time to assess barrier integrity. For the wound healing assay, a precise electrical wound (250 µm diameter) was induced. Cell migration was monitored by recording the time required to reform a monolayer. To confirm these findings, Western blot was used to assess protein levels of select markers (ZO-1, occludin and claudin-1) associated with the corneal epithelial tight junction complex.
Results :
Barrier integrity was significantly impaired under HG conditions and restored with Tβ4/VIP treatment; whereas, Tβ4 and VIP monotherapies were not effective. This was further supported by reduced levels of tight junction proteins after HG exposure compared to NG, which were enhanced with Tβ4/VIP treatment. Likewise, wound healing was significantly impaired under HG conditions as evidenced by reduced migration velocity. Remarkably, Tβ4/VIP significantly improved cell migration velocity despite HG conditions and restored barrier function similar to that observed under NG conditions.
Conclusions :
These results support the premise that Tβ4 works synergistically with VIP to enhance the therapeutic activity with potential for significant impact regarding the treatment of diabetes-induced complications of the cornea.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.