Abstract
Purpose :
Collagen is one of the main proteins of the extracellular matrix throughout the body. In the eye, its intact structure is fundamental for homeostatic maintenance of both neural and non-neural tissues. Collagen mimetic peptides (CMPs) bind and, in so doing, repair collagen cleaved by metalloproteinases (MMPs). We have shown that CMPs protect ARPE-19 cells damaged by MMPs in vitro and neurons subjected to ocular stress using in vivo models of glaucoma, optic nerve injury, and dry eye. Here, our purpose was to evaluate changes over small time scales in thickness, mass, and viscosity of collagen I during adsorption, cleavage by MMP-1, and treatment with a type I CMP using the Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D) platform. This platform measures real-time changes in frequency and dissipation of the quartz sensor related to the deposited mass and associated viscoelastic properties.
Methods :
A QSense Analyzer equipped with standard flow modules was used. Collagen I at 10µg/ml was adsorbed on gold-coated sensors in acetate buffer pH 5.6. Intact collagen was cleaved by MMP-1 at 10ng/µl, and the interaction between applied CMP and cleaved collagen was tested at 1mg/ml CMP in DIW. Temperature was kept constant at 37°C. CMP binding to either intact collagen or bare gold-coated sensors were used as negative controls. Changes in frequency and dissipation were recorded using a QSoft401 software and the Kelvin-Voigt viscoelastic model was used to analyze changes in thickness, mass and viscosity of collagen I, using the QSense data analysis software.
Results :
The shifts in dissipation and frequency upon CMP binding to cleaved collagen was related to an increase in thickness and mass when compared to cleaved collagen. The observed changes in viscosity are suggestive of restoration of collagen I structure due to the interaction with CMP. No binding of CMP was observed to either intact collagen nor to bare gold-coated sensor.
Conclusions :
Our results using QCM-D strongly suggest that CMP specifically binds to cleaved collagen and restores its native structure. The present data provide further support that CMPs represent novel collagen-targeting approaches for diseases that challenge the integrity of collagen in the extraceulllar matrix.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.