Abstract
Purpose :
Peptide amphiphiles (PA) are a class of molecules that combine the structural features of amphiphilic surfactants with the functions of bioactive peptides that assemble into a variety of nanostructures above a critical aggregation concentration (CAC). Here we have investigated the effects of a PA based upon a 13-residue sequence, YEALRVANEVTLN, derived from the C-terminus of the proteoglycan Lumican, attached to a C16 hydrophobic tail (lum-PA). In particular, we focused on the enhanced bioactivity of the lum-PA in supra-molecular form and its mode of action on human corneal fibroblasts (HCF).
Methods :
The lum-PA was dissolved in water below (0.025 wt%) or above (0.1 wt%) its CAC thus existing as a solution of either monomeric molecules or aggregated nanotapes. Further dilution to 0.00125 wt% and 0.0025 wt% allowed the effects of supra-molecular assembly to be tested. Circular dichroism (CD) was performed on the lum-PA solutions.
HCF were extracted from donor corneal-rings and maintained in DMEM/F12, 5% FBS, 1 mM ascorbic acid, then with serum-free media (SFM) supplemented with 1% ITS 3 days prior to experiments. Subsequently 1x104 cells / well were cultured in 0.00125 wt% or 0.0025 wt% solutions of lum-PA in SFM for 7 days or 3 weeks. Cell proliferation and collagen production was quantified by alamarBlue® and Sirius red assays. These experiments were repeated in the presence of an ALK inhibitor SB 431542 at 0.5μM.
Results :
CD confirmed the presence of supra-molecular form following dilution. HCF cultured in 0.0025 wt% and 0.00125 wt% lum-PA showed normal morphology and comparable viability after 7 days in monomeric or diluted-aggregated lum-PA. HCF produced significantly greater (p<0.05) total amounts of collagen compared to the control. Over 21 days, both total mass of collagen and collagen per cell significantly increased (p<0.05) in response to the diluted-aggregated lum-PA solutions. SB 431542 inhibited collagen production in the presence of diluted-aggregated lum-PA.
Conclusions :
A peptide amphiphile has been designed using a biologically active peptide sequence from the C-terminal domain of lumican (lum-PA). The lum-PA’s supra-molecular organisation is not only maintained upon subsequent dilution, but its nano-structure produces a significant stimulatory effect on HCF’s collagen production which appears to be mediated by ALK receptor activity.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.