Abstract
Purpose :
Tissue inhibitors of metallopeptidases (TIMPs) regulate extracellular matrixes (ECM), impacting homeostasis, cell migration, & proliferation. Altered TIMPs expression under pathological conditions worsens disease progression. Yet, TIMP functions in corneal wound healing remain unclear. Here, we investigated the expression of TIMPs in human corneal epithelial cells (HCLE) and examined their biological functions in the context of corneal epithelial injury.
Methods :
We profiled TIMP1, 2, 3, & 4 expression and secretion in HCLE under IL-1β stimulation (inflammatory milieu) and scratch-wound conditions. The highest secreted TIMP was selected for further analysis. HCLE at >90% confluence was scratch-wounded and treated with 2.0 μg/mL recombinant (r) TIMP. At three times daily, corneal epithelial injury in mice (C57BL/6, 6-8 wks, male, n = 5) was treated with rTIMP at 0.1 mg/mL. Cells and cornea were harvested for quantitative & qualitative analyses. RT-PCR was used to quantify the injury-induced MMPs and inflammatory cytokines. Microscopically, we assessed the rate of wound closure, cell proliferation, corneal re-epithelization, transparency, & healing. A 2-way ANOVA analysis with a significant mean difference at p<0.05 was performed, and data were presented as mean±SEM.
Results :
HCLE showed upregulated mRNA expression and secretion of TIMP1 and 2 under inflammatory and scratch-wound conditions. However, TIMP2 expression was significantly 3.4-fold higher at the mRNA level (p<0.0001) and 2.6-fold higher at the protein level (p<0.0001) compared to TIMP1. In the scratch wound assay, adding 2.0 μg/mL rTIMP2 significantly (3-fold) promoted wound closure compared to untreated (p<0.0001) and TIMP2-neutralizing antibody-treated cells (p<0.001). Furthermore, rTIMP2 significantly suppressed inflammatory cytokines (IL-1β, IL-6, IL-8, & TNFα; p<0.05) and MMPs (MMP3, 9, & 13; p<0.05). Finally, topical treatment with 0.1 mg/mL rTIMP2, in comparison to protein control-treated injuries, significantly enhanced corneal re-epithelialization within 48 hrs (30% vs 60%, p<0.01) and suppressed the expression of IL-1β, IL-8, MMP2, MMP3, MMP9, & MMP13 (p<0.05).
Conclusions :
Corneal epithelial cells predominantly secrete TIMP2. Topical treatment with rTIMP2 promotes corneal epithelial wound healing and mitigates inflammation, suggesting a potential therapeutic application in corneal inflammatory diseases.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.