Long-Acting FGF21 Inhibits Retinal Vascular Leakage in In Vivo and In Vitro Models.

Yohei Tomita,William Britton,Ye Sun,Ann Hellström,Zhongjie Fu,Zhongxiao Wang,Steve S Cho,Bertan Cakir,Lois E.H Smith,Saswata Talukdar

doi:10.3390/ijms21041188

Abstract

The aim of the current study was to investigate the impact of long-acting fibroblast growth factor 21 (FGF21) on retinal vascular leakage utilizing machine learning and to clarify the mechanism underlying the protection. To assess the effect on retinal vascular leakage, C57BL/6J mice were pre-treated with long-acting FGF21 analog or vehicle (Phosphate Buffered Saline; PBS) intraperitoneally (i.p.) before induction of retinal vascular leakage with intravitreal injection of mouse (m) vascular endothelial growth factor 164 (VEGF164) or PBS control. Five hours after mVEGF164 injection, we retro-orbitally injected Fluorescein isothiocyanate (FITC) -dextran and quantified fluorescence intensity as a readout of vascular leakage, using the Image Analysis Module with a machine learning algorithm. In FGF21- or vehicle-treated primary human retinal microvascular endothelial cells (HRMECs), cell permeability was induced with human (h) VEGF165 and evaluated using FITC-dextran and trans-endothelial electrical resistance (TEER). Western blots for tight junction markers were performed. Retinal vascular leakage in vivo was reduced in the FGF21 versus vehicle- treated mice. In HRMECs in vitro, FGF21 versus vehicle prevented hVEGF-induced increase in cell permeability, identified with FITC-dextran. FGF21 significantly preserved TEER compared to hVEGF. Taken together, FGF21 regulates permeability through tight junctions; in particular, FGF21 increases Claudin-1 protein levels in hVEGF-induced HRMECs. Long-acting FGF21 may help reduce retinal vascular leakage in retinal disorders and machine learning assessment can help to standardize vascular leakage quantification.

Highlights

Diabetic retinopathy (DR) is a major cause of vision impairment with increasing prevalence worldwide [1]
The machine learning program was trained over multiple trials such that every object in an image was individually categorized with a different color
We found that receptors for fibroblast growth factor 21 (FGF21) were expressed in human (h) primary retinal microvascular endothelial cells (HRMECs) in vitro: Fibroblast growth factor receptor 1 (FGFR1) at a high level, FGFR3 and co-receptor β-KLOTHO at moderate levels, and FGFR2 and FGFR4 at low levels (Supplementary Figure S1A–E)

Summary

Introduction

Diabetic retinopathy (DR) is a major cause of vision impairment with increasing prevalence worldwide [1]. Diabetic macular edema (DME), which occurs secondary to retinal vascular leakage, is the leading cause of vision loss in DR. Anti-VEGF and steroid therapies are widely used to treat DME. These therapeutic approaches have improved the visual prognosis of DME, many cases are non-responders. These treatments carry risks, such as cataract formation, glaucoma, and endophthalmitis [4,5,6]. It is important to develop alternative therapeutic approaches to treat DME, optimally with an intervention that suppresses both neovascularization and vascular leakage

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