Alterations of Retinal Vasculature in Cystathionine–β-Synthase Heterozygous Mice

Abstract

Mild to moderate hyperhomocysteinemia is prevalent in humans and is implicated in neurovascular diseases, including recently in certain retinal diseases. Herein, we used hyperhomocysteinemic mice deficient in the Cbs gene encoding cystathionine-β-synthase (Cbs(+/-)) to evaluate retinal vascular integrity. The Cbs(+/+) (wild type) and Cbs(+/-) (heterozygous) mice (aged 16 to 52 weeks) were subjected to fluorescein angiography and optical coherence tomography to assess vasculature in vivo. Retinas harvested for cryosectioning or flat mount preparations were subjected to immunofluorescence microscopy to detect blood vessels (isolectin-B4), angiogenesis [anti-vascular endothelial growth factor (VEGF) and anti-CD105], gliosis [anti-glial fibrillary acidic protein (GFAP)], pericytes (anti-neural/glial antigen 2), blood-retinal barrier [anti-zonula occludens protein 1 (ZO-1) and anti-occludin], and hypoxia [anti-pimonidazole hydrochloride (Hypoxyprobe-1)]. Levels of VEGF, GFAP, ZO-1, and occludin were determined by immunoblotting. Results of these analyses showed a mild vascular phenotype in young mice, which progressed with age. Fluorescein angiography revealed progressive neovascularization and vascular leakage in Cbs(+/-) mice; optical coherence tomography confirmed new vessels in the vitreous by 1 year. Immunofluorescence microscopy demonstrated vascular patterns consistent with ischemia, including a capillary-free zone centrally and new vessels with capillary tufts midperipherally in older mice. This was associated with increased VEGF, CD105, and GFAP and decreased ZO-1/occludin levels in the Cbs(+/-) retinas. Retinal vein occlusion was observed in some Cbs(+/-) mouse retinas. We conclude that mild to moderate elevation of homocysteine in Cbs(+/-) mice is accompanied by progressive alterations in retinal vasculature characterized by ischemia, neovascularization, incompetent blood-retinal barrier, and vascular occlusion.

Palm tocotrienol-rich fraction inhibits methionine-induced cystathionine β-synthase in rat liver

“…Many models such as choline deficiency [21], genetic alterations [40], methionine load [30] and folate deficiency [32] have been employed to study hyperhomocysteinaemia in experimental animals. In the present study, dietary methionine load for 10 weeks had increased plasma homocysteine level.…”

“…Therefore, high consumption of dietary protein may increase plasma homocysteine level, which is clinically known as hyperhomocysteinaemia. Hyperhomocysteinaemia is one of the risk factors for cardiovascular diseases [40]. Clinically, folate and vitamin B therapies are used as an intervention for this condition [13,24].…”

Mthfr as a modifier of the retinal phenotype of Crb1 mice

“…Mutations of this gene are associated with increased levels of homocysteine and we have had a longstanding interest in the effects of hyperhomocysteinemia on retinal structure and function. We have examined the consequences on retina when homocysteine was injected into the vitreal cavity, as an acute injury (Moore et al, 2001) and we have assessed the consequences when metabolic defects have led to moderately excessive endogenous homocysteine levels (Ganapathy et al, 2009; Yu et al, 2011; Tawfik et al, 2014 and Markand et al, 2015). Much of our recent work focused on the effects of deficiency of enzymes involved in the homocysteine-methionine pathway.…”

A lipidomic screen of hyperglycemia-treated HRECs links 12/15-Lipoxygenase to microvascular dysfunction during diabetic retinopathy via NADPH oxidase

“…Mice were administered 10 to 20 l 10% fl uorescein sodium and rapid acquisition of fl uorescent images ensued for ‫ف‬ 5 min, as previously described ( 33 ).…”