Abstract
Induction of posterior vitreous detachment (PVD) by pharmacologic vitreolysis has been largely attempted through the use of enzymatic reagents. Ocriplasmin has been the only FDA-approved clinical reagent so far. Several adverse effects of ocriplasmin have emerged, however, and the search for alternative PVD-inducing reagents continues. Since i) collagen forms an important structural component of the vitreous, and ii) strong vitreo-retinal adhesions exist between the cortical vitreous and the internal limiting membrane (ILM) of the retina, an effective PVD-inducing reagent would require both, vitreous liquefaction, and concurrent dehiscence of vitreoretinal adhesion, without being toxic to retinal cells. We designed a combination of two reagents to achieve these two objectives; a triple helix-destabilizing collagen binding domain (CBD), and a fusion of RGD (integrin-binding) tripeptide with CBD (RCBD) to facilitate separation of posterior cortical vitreous from retinal surface. Based on in vitro, ex-vivo, and in vivo experiments, we show that a combination of CBD and RCBD displays potential for safe pharmacologic vitreolysis. Our findings assume significance in light of the fact that synthetic RGD-containing peptides have already been used for inhibition of tumor cell invasion. Proteins such as variants of collagen binding domains could have extended therapeutic uses in the future.
Highlights
Induction of posterior vitreous detachment (PVD) by pharmacologic vitreolysis has been largely attempted through the use of enzymatic reagents
Following cloning of genes for collagen binding domain (CBD) and RGD-fused CBD (RCBD) (Supplementary Fig. S1), the proteins were expressed and purified (Fig. 1B; Supplementary Fig. S2); zymography revealed that CBD and RCBD have no gelatinolytic activity, unlike the collagenase, Vibrio mimicus collagenase (VMC), from which these are derived (Fig. 1C)
The stability of CBD and RCBD as well as combination of CBD and RCBD was tested over 48 hours of incubation in the presence of cadaver-derived vitreous; it was observed in each case, that more than 80% of the proteins remained intact and were not degraded over this time period (Supplementary Fig. S3A,B)
Summary
Induction of posterior vitreous detachment (PVD) by pharmacologic vitreolysis has been largely attempted through the use of enzymatic reagents. Non-enzymatic reagents developed to achieve vitrectomy offer the added benefit of safety without any collateral damage to the retinal structures In this direction, the RGD (Arg-Gly-Asp) containing peptides have been used to induce PVD9,10. Since collagen is the major contributor to ocular viscoelasticity and vitreoretinal adhesions, and interlinking collagen fibrils along with Hyaluronic Acid (HA) form the gel-like consistency of vitreous[3], we chose to use the collagen-binding domain (CBD) motif derived from Vibrio mimicus collagenase (VMC) (Fig. 1A) for this purpose. The rationale of using CBD domain was that the CBD would bind to the collagen triple helix and cause its destabilization (without collagen degradation) potentially by loosening up the triple helical structure of collagen, as has been demonstrated in a non-ophthalmological context[17] This is the first attempt at using CBD (rather than the whole collagenase molecule) in inducing PVD. Our data shows that together, the combination of CBD and RCBD can cause PVD through structural perturbation of the collagen fibrils, as well as through decreased adhesion of collagen with the cells of the ILM
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
