Citation

  • Authors: Chu H. et al.
  • Year: 2022
  • Journal: Biochem Biophys Res Commun 605 148-153
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell type: HEK-293T
    Description: Human embryonic kidney Fibroblast
    Known as: HEK293T, 293T

Method

Transient transfection in 293T cells was performed using jetOPTIMUS (Polyplus-transfection®, New York, NY)

Abstract

Pigment epithelium-derived factor (PEDF) is a secreted protein that is essential in tissue homeostasis and is involved in multiple functions in the eye, such as antiangiogenesis and neuroprotection. However, short retention in the retinal microenvironment can limit its therapeutic effects. In this study, we modified the amino acid sequence of PEDF to increase its affinity for heparin and hyaluronic acid (HA), which are negatively charged extracellular matrix (ECM) molecules. HA is the major component of the vitreous humor. We selectively converted neutral or anionic residues into cationic residues to obtain engineered PEDF (ePEDF). Using in vitro binding assays, we demonstrate that ePEDF had higher affinity for heparin and HA than wild-type PEDF (wtPEDF). ePEDF exhibited antiangiogenic and retinal survival bioactivities. It inhibited endothelial cell proliferation and tube formation in vitro. In an ex vivo model mimicking retinal degeneration, ePEDF protected photoreceptors from cell death. The findings suggest that protein engineering is an approach to develop active PEDF with higher ECM affinity to potentially improve its retention in the retina microenvironment and in turn make it a more efficient therapeutic drug for retinal diseases.

Pubmed