Alterations of retinal vasculature in cystathionine-β-synthase heterozygous mice

A model of mild to moderate hyperhomocysteinemia

Amany Mohamed Tawfik, Shanu Markand, Mohamed Al-Shabrawey, Jamie N. Mayo, Jason Reynolds, Shawn E. Bearden, Vadivel Ganapathy, Sylvia B Smith

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)2573-2585
Number of pages13
JournalAmerican Journal of Pathology
Volume184
Issue number9
DOIs
StatePublished - Jan 1 2014

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Cystathionine
Hyperhomocysteinemia
Zonula Occludens-1 Protein
Occludin
Blood Vessels
Glial Fibrillary Acidic Protein
Blood-Retinal Barrier
Vascular Endothelial Growth Factor A
Retina
Retinal Vessels
Fluorescein Angiography
Optical Coherence Tomography
Fluorescence Microscopy
Cryoultramicrotomy
Ischemia
Retinal Vein Occlusion
Retinal Diseases
Pericytes
Gliosis
Homocysteine

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Medicine(all)

Cite this

Alterations of retinal vasculature in cystathionine-β-synthase heterozygous mice : A model of mild to moderate hyperhomocysteinemia. / Tawfik, Amany Mohamed; Markand, Shanu; Al-Shabrawey, Mohamed; Mayo, Jamie N.; Reynolds, Jason; Bearden, Shawn E.; Ganapathy, Vadivel; Smith, Sylvia B.

In: American Journal of Pathology, Vol. 184, No. 9, 01.01.2014, p. 2573-2585.

Research output: Contribution to journalArticle

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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.",
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