Abstract
Purpose: :
Homocysteine (Hcy) is a non-proteinogenic amino acid whose elevation has been implicated in human visual disorders. Recently, we investigated the retinal phenotype of mice with moderate/severe hyperhomocysteinemia due to deficiency/absence, respectively, of the gene encoding cystathionine-beta-synthase (CBS). We reported marked retinal disruption, ganglion cell loss, overstimulation of the NMDA receptor, optic nerve mitochondrial dysfunction and ERG defects in cbs-/- mice and a delayed morphological/functional phenotype in cbs+/- mice (Ganapathy et al, 2009, 2011; Yu et al, 2011). Excess Hcy is an independent risk factor for human systemic cardiovascular diseases such as hypertension; however it is not known whether excess Hcy alters retinal vasculature. We used cbs mutant mice to address this.
Methods: :
Retinas of cbs+/+ (n =21), cbs +/- (n = 34), cbs -/- (n =16) mice (age 2 wks) were harvested for wholemount preparation or cryosections and subjected to immunofluorescence microscopy to (1) visualize vessels via the endothelial cell marker isolectin-B4; to detect (2) angiogenesis (VEGF and CD 105 (endoglin)), (3) activated glial cells (GFAP), (4) inflammation (TNF-α) and (5) superoxide production (DHE). Retinal wholemounts were examined by LSCM; cryosections were evaluated using a Zeiss Axioplan-2 microscope and the AxioVision program.
Results: :
Retinal vasculature of cbs+/- mice was similar to WT mice, consistent with a normal phenotype at this age (Ganapathy et al, 2009). However, cbs-/- mice demonstrated vascular patterns consistent with ischemia: isolectin B4 labeling revealed a marked capillary-free zone in the central retina and new vessels with capillary tufts in the mid-peripheral area. This altered vascular pattern was associated with increased VEGF and CD105 levels. In retinal cryosections, marked increase in GFAP, consistent with glial cell activation was observed in cbs -/- mice, as were increased levels of TNFα and superoxide.
Conclusions: :
The findings suggest that severe elevation of Hcy, as seen in the cbs-/- mouse, is accompanied by alterations in retinal vasculature. The retinal disruption and decreased visual function reported previously in cbs-/- mice may reflect vasculopathy as well as the neuropathy.
Keywords: neovascularization • retina • vascular endothelial growth factor