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Kaylene Carter, Anne Zijing Ye, Gabriella Fernandes-Cunha, Jennifer R Cochran, David Myung; An engineered dimeric fragment of hepatocyte growth factor improves corneal epithelial wound healing in vitro. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3206.
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© ARVO (1962-2015); The Authors (2016-present)
Hepatocyte growth factor (HGF) is a naturally occurring mitogen which plays a critical role in corneal wound healing. Our study examined the wound healing properties of an engineered dimeric fragment of HGF, (termed eNK1 dimer), on primary corneal epithelial cells (CECs) through migration and proliferation assays. We tested the hypothesis that CEC migration and proliferation is improved with eNK1 and that it performs similarly to recombinant HGF (rHGF).
NK1 is a fragment of HGF that weakly activates the c-Met receptor. NK1 was previously engineered for increased stability and recombinant expression yield using directed evolution. Disulfide-linked NK1 homodimers were created through the introduction of an N-terminal cysteine residue. Migration and proliferation assays were conducted on immortalized and primary CECs, respectively. Immortalized CEC migration was evaluated by scratch assay with 100 ng/mL treatments of eNK1 and rHGF. Wound closure was monitored at 6 and 12 hours under inverted microscopy. Primary CEC proliferation and metabolic activity were evaluated after 48 hours. Cells were first starved in growth factor free medium and then treated with 100 ng/mL of eNK1 or rHGF. Cell proliferation and metabolic activity were quantified using Click-iT EdU and MTT assays, respectively.
The engineered NK1 covalent dimers exhibit nearly an order of magnitude improved agonistic activity compared to wild-type NK1, approaching the activity of full-length HGF. CD spectroscopy showed the dimer to have increased thermal stability over wild type NK1. Scratch assay showed that both rHGF and eNK1-treated CECs achieved significantly greater wound closure compared to the negative control (p < 0.01). EdU assay showed that CECs treated with eNK1 and rHGF were significantly more EdU-positive than untreated cells, indicating increased levels of DNA synthesis and therefore proliferation (p < 0.05). MTT assay showed that CECs treated with eNK1 and rHGF had improved metabolic activity compared to untreated cells (p < 0.05).
Our findings support our hypothesis that eNK1 is a stable protein which influences corneal epithelial cell migration, proliferation, and metabolic activity to levels similar to full-length, recombinant HGF. The results suggest that eNK1 improves corneal epithelial wound healing and may have potential as a therapeutic alternative to recombinant HGF.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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