Noregen Promotes Norrin Target Gene Expression and Recovery of Barrier Resistance in Retinal Endothelial Cells Post-VEGF Challenge

Document Type

Conference Proceeding

Publication Date

6-2025

Publication Title

Investigative Ophthalmology and Visual Science

Abstract

Purpose : This study aimed to characterize the effects of Noregen on Norrin target gene expression and barrier formation in primary human retinal microvascular endothelial cells (HRMECs).

Methods : Primary HRMECs were cultured and treated with escalating doses of Noregen (0, 2, 10, 20, 100, or 200 ng/mL). Total RNA was extracted, and first-strand cDNA was made with the LunaScript RT SuperMix Kit (New England Biolabs). Quantitative PCR was performed using the Luna Universal Probe qPCR Master Mix (New England Biolabs) to measure the expression of the Norrin target gene AXIN2. ECIS (Electric Cell-Substrate Impedance Sensing) experiments were conducted using a Z-Theta ECIS instrument to evaluate the impact of Noregen on barrier resistance. HRMECs were grown to a monolayer in 96-well ECIS plates using two different concentrations of hydrocortisone. Cells were then challenged with a high concentration of VEGF165a (50 ng/mL) and treated with varying doses of Noregen to assess recovery of barrier integrity.

Results : Noregen resulted in a dose-dependent increase of basal AXIN2 gene expression in HRMECs, 24 hours after treatment. ECIS analysis revealed that at higher hydrocortisone (HC) concentrations, VEGF165a treatment caused resistance to drop 31 percent within 24 hours. Co-treatment with Noregen (40 ng/mL) restored barrier function above baseline by 48 hours and demonstrated continued improvement by 72 hours. At lower HC concentrations, VEGF165a caused resistance disruption, which did not recover with VEGF alone. However, co-treatment with Noregen significantly improved resistance above baseline after 72 hours.

Conclusions : Noregen induced a dose-dependent increase of AXIN2 gene expression (3.5 fold) in primary HRMECs, consistent with activation of the Norrin-Wnt signaling pathway. Additionally, Noregen promoted recovery of endothelial barrier resistance following disruption by VEGFA165a treatment. The effects of Noregen on primary Human Microvascular Endothelial Cells support its therapeutic potential for repair of the retinal vasculature.

Volume

66

Issue

8

First Page

3053

Comments

Association for Research in Vision and Ophthalmology ARVO Annual Meeting, May 4-8, 2025, Salt Lake City, UT

Last Page

3053

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