Abstract
Purpose :
Characterized by tear film insufficiency and/or instability and ocular surface irritation and inflammation, dry eye disease can lead to corneal damage and loss of visual acuity. Based on a recent report that a single fragment from the carboxy terminus of human Lacritin, called Lacripep, promotes tear secretion and restores ocular surface integrity through binding Syndecan-1, we explored effects of multivalent Lacripep fusion proteins to expand the future therapeutic applications of Lacritin.
Methods :
Three consecutive copies of the gene encoding Lacripep (LP, the last 21 amino acids of the Lacritin C-term) were expressed as a fusion with the Elastin-like polypeptide (ELP) named A96 (a.a sequence=(Val-Pro-Gly-Ala-Gly)96) and termed 3LP-A96. 3LP-A96 was compared side-by-side with 1LP-A96 (single copy of LP fused to A96), synthesized Lacripep or recombinant Lacritin for biological activity. Peptide constructs were exposed to cells in culture media and evaluated for their ability to evoke calcium signaling in human corneal epithelial cell cultures (HCE-T) using Fluo-4 (calcium indicator) and as secretagogues in cultured primary rabbit lacrimal gland acinar cells (LGACs), measuring beta-hexosaminidase levels released in the culture media.
Results :
When added to HCE-T cells, 3LP-A96 induced a reproducible homogenous increase in calcium flux signals with duration >7 min, compared to the heterogenous 15~25 sec transient calcium flux signals evoked by Lacripep. 3LP-A96 significantly increased beta-hexosaminidase secretion from primary rabbit LGACs at a magnitude comparable to recombinant Lacritin, and at a higher level than that elicited by 1LP-A96 (2-fold, p<0.001) or non-treated controls (3-fold, p<0.001, n=6). Live cell imaging using confocal fluorescence microscopy confirmed that FITC-labeled 3LP-A96 showed stronger binding to primary rabbit LGACs compared to the low-level binding of FITC-1LP-A96.
Conclusions :
We generated a multivalent nanoparticle using Lacripep, a promising agent being tested in clinical trials as an ophthalmic emulsion. While equipotent as a secretagogue in primary rabbit LGACs, the prolonged calcium signals evoked by 3LP-A96 on HCE-T cells suggest that it may be more potent at inducing downstream signaling, perhaps by residing longer on the ocular surface compared to Lacripep. This feature may enhance its therapeutic properties.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.