Alterations of retinal vasculature in cystathionine-beta-synthase heterozygous mice, a model of mild-moderate hyperhomocysteinemia (HHcy).

Abstract

Purpose: HHcy is implicated in neurovascular diseases including certain retinal diseases such as diabetic retinopathy and age-related macular degeneration. Recently, we analyzed the retinal vasculature of mice completely lacking cystathionine β−synthase (CBS), which have 40-fold increase in homocysteine (Hcy), and observed severely altered retinal vasculature mimicking ischemic retinopathy (Tawfik et al, IOVS, 2013). The purpose of the current study was to assess the retinal vascular phenotype in mice with mild-moderate HHcy, which is much more prevalent in the human population.

Methods: HHcy mice deficient in the cbs gene (cbs^+/-) were used to evaluate retinal vascular integrity. Cbs^+/+ (n=56) and cbs ^+/- (n-47) mice (age: 16 - 52 wks) were subjected to fluorescein angiography (FA) and optical coherence tomography (OCT) to assess retinal vasculature in vivo. Retinas harvested for cryosectioning or flatmount preparations were subjected to immunofluorescence microscopy (IMF) to detect blood vessels (isolectin-B4), angiogenesis (anti-VEGF, anti-CD105), gliosis (anti-GFAP), blood-retinal barrier integrity (anti-ZO-1, anti-occludin) and hypoxia (anti-HP-1). Levels of VEGF, GFAP, ZO-1 and occludin were determined by immunoblotting.

Results: FA revealed neovascularization and vascular leakage in cbs^+/- mice that was progressive (mild at 16 wks, marked by 52 wks); OCT confirmed new vessels in the vitreous chamber by 1 yr. IMF demonstrated vascular patterns consistent with ischemia including a capillary-free zone centrally and new vessels with capillary tufts mid-peripherally in 36 and 52 wk mice. This was associated with increased VEGF, CD105 and GFAP and decreased ZO-1/occludin levels in the cbs^+/- retinas. Western blotting showed a 2-fold increase in VEGF and GFAP. Retinal vein occlusion was observed in some cbs^+/- mouse retinas.

Conclusions: Our earlier studies clearly showed a link between severe HHcy and retinal vascular disease. The current studies provide strong evidence that even mild-moderate elevation of Hcy as seen in cbs^+/- mice is accompanied by progressive alterations in retinal vasculature including ischemia, neovascularization, incompetent blood-retinal barrier and vascular occlusion. Studies are underway to determine mechanisms by which HHcy triggers retinal vascular alterations.

Keywords: 688 retina • 700 retinal neovascularization • 592 metabolism