Noregen Promotes Growth to Barrier Formation of Retinal Endothelial Cells and Activates FZD4 Variants

Authors

Kenneth P. Mitton
Wendy A. Dailey
Cecille Pinnock
Victoria Jobczyk
Kimberly A. Drenser, Corewell Health

Document Type

Conference Proceeding

Publication Date

6-2024

Publication Title

Investigative Ophthalmology and Visual Science

Abstract

Purpose : NoregenTM is a recombinantly produced protein therapeutic based on the human Norrin protein. To establish cell-based models for investigation and functional analysis of Noregen, primary human retinal microvascular endothelial cells were adopted for functional barrier analysis with ECIS, and co-transfection of HEK293 cells was employed for dose-response analysis with normal and disease-causing variants of FZD4.

Methods : Noregen was supply-run material provided by Caeregen Therapeutics. Primary Human Retinal Microvascular Endothelial Cells (HRMECs) were cultured for real-time barrier modeling using Electric Cell-substrate Impedance Sensing (ECIS). HRMECs were seeded at low concentrations in ECIS 96-well plates and treated with Noregen. Impedance data was fit to a monolayer model with ECIS software (Applied Biophysics). HEK293 cells were co-transfected with expression plasmids for LRP5, FZD4, or FZD4M105V, and a beta-Catenin reporter-plasmid expressing Firefly Luciferase. At 24-hrs post-transfection, cells were treated with multiple doses of Noregen, and dose-response activation data were fit to a 4-parameter log-logistic dose-response model using the DRC package of R.

Results : ECIS-based analysis showed that Norgen caused a dose-dependent acceleration of growth to barrier formation with primary HRMECs. ECIS modeling also demonstrated barrier formation following proliferation to monolayer. Dose-response analysis of beta-Catenin activation demonstrated a sigmoidal dose-response curve from activation by Noregen. The EC50 values were greater for the FEVR-related variant FZD4(M105V) , 115 ng/mL Noregen, compared to FZD4, 16 ng/mL Noregen. Increased activation of FZD4(M105V) was possible to a maximum level of about 32% of the maximum activation with FZD4.

Volume

65

Issue

7

First Page

3139

Comments

Association for Research in Vision and Ophthalmology Annual Meeting, ARVO 2024, May 5-9, 2024, Seattle, WA

Recommended Citation

Mitton KP, Dailey WA, Pinnock C, Jobczyk V, Drenser KA. Noregen promotes growth to barrier formation of retinal endothelial cells and activates FZD4 variants. Invest Ophthalmol Vis Sci. 2024 Jun;65(7):3139.