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Am J Pathol. 2014 Sep;184(9):2573-85. doi: 10.1016/j.ajpath.2014.05.018. Epub 2014 Jul 10.

Alterations of retinal vasculature in cystathionine-β-synthase heterozygous mice: a model of mild to moderate hyperhomocysteinemia.

Author information

1
Department of Cellular Biology and Anatomy, Medical College of Georgia, Georgia Regents University, Augusta, Georgia; James and Jean Culver Vision Discovery Institute, Georgia Regents University, Augusta, Georgia.
2
James and Jean Culver Vision Discovery Institute, Georgia Regents University, Augusta, Georgia; Department of Oral Biology and Anatomy, Medical College of Georgia, Georgia Regents University, Augusta, Georgia; Department of Ophthalmology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia.
3
Department of Biological Sciences, Idaho State University, Pocatello, Idaho.
4
James and Jean Culver Vision Discovery Institute, Georgia Regents University, Augusta, Georgia; Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia.
5
Department of Cellular Biology and Anatomy, Medical College of Georgia, Georgia Regents University, Augusta, Georgia; James and Jean Culver Vision Discovery Institute, Georgia Regents University, Augusta, Georgia; Department of Ophthalmology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia. Electronic address: sbsmith@gru.edu.

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.

PMID:
25016930
PMCID:
PMC4188277
DOI:
10.1016/j.ajpath.2014.05.018
[Indexed for MEDLINE]
Free PMC Article

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