Hyperhomocysteinemia Alters Retinal Endothelial Cells Barrier Function and Angiogenic Potential via Activation of Oxidative Stress

Riyaz Mohamed,Mohamed Al-Shabrawey,Isha Sharma,Sylvia B Smith,Ahmed S Ibrahim,Nehal M Elsherbiny,Sadanand Fulzele,Khaled Elmasry,Heba Saleh,Amany Tawfik

doi:10.1038/s41598-017-09731-y

Riyaz Mohamed, Mohamed Al-Shabrawey + Show 8 more

Open Access

https://doi.org/10.1038/s41598-017-09731-y

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Abstract

Hyperhomocysteinemia (HHcy) is associated with several human visual disorders, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). Breakdown of the blood-retinal barrier (BRB) is linked to vision loss in DR and AMD. Our previous work revealed that HHcy altered BRB in retinal endothelial cells in vivo. Here we hypothesize that homocysteine (Hcy) alters retinal endothelial cell barrier function and angiogenic potential via activation of oxidative stress. Human retinal endothelial cells (HRECs) treated with and without different concentrations of Hcy showed a reduction of tight junction protein expression, increased FITC dextran leakage, decreased transcellular electrical resistance and increased angiogenic potential. In addition, HRECs treated with Hcy showed increased production of reactive oxygen species (ROS). The anti-oxidant N-acetyl-cysteine (NAC) reduced ROS formation and decreased FITC-dextran leakage in Hcy treated HRECs. A mouse model of HHcy, in which cystathionine-β-synthase is deficient (cbs−/−), was evaluated for oxidative stress by dichlolorofluorescein (DCF), dihydroethidium (DHE) staining. There was a marked increase in ROS production and augmented GSH reductase and antioxidant regulator NRF2 activity, but decreased antioxidant gene expression in retinas of hyperhomocysteinemic mice. Our results suggest activation of oxidative stress as a possible mechanism of HHcy induced retinal endothelial cell dysfunction.

Highlights

A large body of evidence emphasizes the significant role of oxidative stress in retinal vascular hyperpermeability and barrier dysfunction
To confirm the role of excess Hcy in altering the barrier function of Human retinal endothelial cells (HRECs) monolayers, we explored whether HHcy treatment induces permeability changes to Fluorescein isothiocyanate (FITC) dextran flux through the confluent monolayer
We showed that elevated Hcy resulted in altered retinal vasculature as evidenced by ischemia, neovascularization, vascular leakage and a deficient blood-retinal barrier[18,19,20]

Summary

Introduction

A large body of evidence emphasizes the significant role of oxidative stress in retinal vascular hyperpermeability and barrier dysfunction. The direct effect of Hcy on retinal endothelial cells and the underlying mechanisms involved in HHcy induced vascular dysfunction have not been explored. We investigated the role of HHcy in BRB dysfunction to determine whether elevated Hcy directly affects retinal endothelial cells (RECs) by mechanisms that involve oxidative stress.

Results

Conclusion