Novel engineered, membrane-localized variants of vascular endothelial growth factor (VEGF) protect retinal ganglion cells: a proof-of-concept study

Junhui Shen,Jeffrey Bair,Fang Wang,Petr Baranov,Yin Shan Eric Ng,Darlene Dartt,Ru Xiao,Luk H Vandenberghe

doi:10.1038/s41419-018-1049-0

Abstract

Endogenous vascular endothelial growth factor (VEGF-A) can protect retinal ganglion cells (RGC) from stress-induced cell death in ocular hypertensive glaucoma. To exploit the neuroprotective function of VEGF-A for therapeutic application in ocular disorders such as glaucoma while minimizing unwanted vascular side effects, we engineered two novel VEGF variants, eVEGF-38 and eVEGF-53. These variants of the diffusible VEGF-A isoform VEGF121 are expressed as dimeric concatamers and remain tethered to the cell membrane, thus restricting the effects of the engineered VEGF to the cells expressing the protein. For comparison, we tested a Myc-tagged version of VEGF189, an isoform that binds tightly to the extracellular matrix and heparan sulfate proteoglycans at the cell surface, supporting only autocrine and localized juxtacrine signaling. In human retinal endothelial cells (hREC), expression of eVEGF-38, eVEGF-53, or VEGF189 increased VEGFR2 phosphorylation without increasing expression of pro-inflammatory markers, relative to VEGF165 protein and vector controls. AAV2-mediated transduction of eVEGF-38, eVEGF-53, or VEGF189 into primary mouse RGC promoted synaptogenesis and increased the average total length of neurites and axons per RGC by ~ 12-fold, an increase that was mediated by VEGFR2 and PI3K/AKT signaling. Expression of eVEGF-38 in primary RGC enhanced expression of genes associated with neuritogenesis, axon outgrowth, axon guidance, and cell survival. Transduction of primary RGC with any of the membrane-associated VEGF constructs increased survival both under normal culture conditions and in the presence of the cytotoxic chemicals H2O2 (via VEGFR2/PI3K/AKT signaling) and N-methyl-d-aspartate (via reduced Ca2+ influx). Moreover, RGC number was increased in mouse embryonic stem cell-derived retinal organoid cultures transduced with the eVEGF-53 construct. The novel, engineered VEGF variants eVEGF-38 and eVEGF-53 show promise as potential therapeutics for retinal RGC neuroprotection when delivered using a gene therapy approach.

Highlights

Vascular endothelial growth factor (VEGF-A) is best known for its effects on vascular permeability and angiognenesis
The eVEGF variants, eVEGF-38 and eVEGF-53, are expressed by target cells as membrane-anchored dimers To restrict the effects of the eVEGF variants to the targeted retinal ganglion cells (RGC), a glycophosphatidylinositol (GPI) anchor was used to tether the protein to the plasma membrane, an established protein engineering approach for this objective[12]
The cDNAs for eVEGF-38, eVEGF-53, and VEGF189 were subcloned into an adeno-associated virus (AAV)-based expression vector for transfection and transduction, and the corresponding vector containing complementary DNA for green fluorescent protein (GFP) was used as a negative control[16]

Summary

Introduction

Vascular endothelial growth factor (VEGF-A) is best known for its effects on vascular permeability and angiognenesis. Shen et al Cell Death and Disease (2018)9:1018 for the VEGF164 and VEGF188 isoforms[2], in addition to VEGF receptor 2 (VEGFR2)[3,4]. VEGF-A maintains homeostasis in retinal ganglion cells (RGC), which express VEGFR23,5, and contributes to retinal disease states[6]. VEGF-A produced by RGC mediates protection from stress-induced cell death, including that from ocular hypertensive glaucoma and ischemia–reperfusion injury[5,7]. Both exogenous and endogenous VEGF-A have been shown to have potent neuroprotective effects on RGC in mouse models of neovascular age-related macular degeneration and diabetic retinopathy[8]. VEGF-A antagonism reduces axonal transport by RGC8, suggesting that VEGF-A can modulate function as well as survival of these retinal neurons[9]

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Results

Conclusion