Abstract
Purpose :
TIMPs mediate cellular matrix remodelling, which is necessary for cell migration, proliferation, and differentiation. However, their roles in corneal injury and inflammation are not well defined. Here, the expression of different TIMPs was characterized in the corneal epithelial cells in homeostatic and inflammatory conditions.
Methods :
Immortalized human corneal limbal epithelial cells were cultured in a monolayer for 75% confluency. Two inflammatory conditions were utilized in the experiments. For experiment 1, HCLE cells were stimulated with 100 ng/mL of recombinant human IL-1β (rhIL-1β), and in experiment 2, a scratch wound was created on the cell monolayer. Both conditions were incubated overnight in 5% CO2 at 37oC. Lysates and supernatants of the culture were collected, and mRNA expression and protein levels of the TIMP family were assessed using RT-qPCR and protein array, respectively. Statistical analysis was performed assuming a significant difference at p<0.05 using 2-way ANOVA analysis.
Results :
TIMP-1, -2 and -3 but not -4 were constitutively expressed by human corneal epithelial cells. TIMP-2 transcript level was higher following rhIL-1β stimulation, compared to untreated control (p=0.05). Similarly, an upward trend of 12.5% in the expression of TIMP-3 was observed following rhIL-1β stimulation over the untreated control. This upward trend in the transcript level was similar to the scratch-wound experiment. The constitutive expression of TIMP-1 and -2 was confirmed at the protein level. Interestingly, in both conditions (rhIL-1β stimulated and scratch-wound), expression of TIMP-2 was substantially higher than TIMP-1; yet TIMP-4 expression remained extremely low. Within the group, the scratch wound elevated the TIMP-2 level more significantly than the untreated cells (p=0.01) and showed more than a 30% increase in TIMP-1, compared to the untreated cells.
Conclusions :
Human corneal epithelial cells persistently secrete elevated levels of TIMP-2, which is upregulated under inflammatory conditions, suggesting a role of TIMP-2 in the extracellular matrix remodelling of the corneal epithelium during inflammation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.