Inflammation as an important player in the pathogenesis of hyperhomocysteinemia-induced retinopathy

Abstract

Purpose : Homocysteine (Hcy) is a sulfur-containing amino acid which requires vitamins B12 & folic acid for its metabolism and their deficiencies lead to elevated Hcy levels (HHcy). Vitamins B12 and folic acid deficiencies are common nutritional deficiencies in diabetic and aging population and were linked to the development of diabetic retinopathy (DR) and age-related macular degeneration (AMD). Our previous work reported that HHcy disrupts inner and outer blood-retinal barriers (BRBs), induces retinal ischemia, neovascularization, disrupts retinal pigmented cells (RPE) structure & function, activates oxidative & ER stresses and induces epigenetic modifications. HHcy was reported in patients with DR and AMD in many clinical studies. The goal of the current study was to explore inflammation as an underlying mechanism of HHcy-induced retinopathy.

Methods : living mice with HHcy due to lack of the enzyme cystathionine-β-synthase (cbs^+/-) and wild-type (C57Bl6) mice were evaluated for leukocytes adhesion using intravital microscopy and microglia activation in retinal flat mounts using Iba1 immunofluorescence (IF). Human Retinal Endothelial cells (HRECs), RPE and human monocyte cell line U937 treated with/without Hcy (50μM) for 24 hours were evaluated for inflammatory cytokines, and NFkB activation using multiplex assay and western blot analysis respectively.

Results : HHcy induces an inflammatory response in mouse retina, cultured HRECs, RPE and microglia cells. Mice with HHcy showed significant elevation of leukocyte adhesion and Iba1 immunoreactivity suggesting microglia activation. In addition, NFκB was activated and cytokine array analysis revealed a significant increase in most of the pro-inflammatory cytokines such as IP-10, TNFα, IL-Β and IL-6 and downregulation of anti-inflammatory cytokine (IL-4) in cultured HRECs, RPE and human monocyte cell line U937 treated with Hcy for 24 hours.

Conclusions : The current study shows strong evidence that HHcy induces inflammatory responses in mouse retina and cultured retinal cells. The retinal inflammatory response to HHcy could play a role in retinal dysfunction in DR and AMD. Thus, therapeutic strategies that aim to eliminate excess homocysteine are promising interventions in DR and AMD.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.