Jason Covar

Alterations of Retinal Vasculature in Cystathionine-Beta-Synthase Mutant Mice, a Model of Hyperhomocysteinemia

PURPOSE Mice with moderate/severe hyperhomocysteinemia due to deficiency or absence of the cbs gene encoding cystathionine-beta-synthase (CBS) have marked retinal disruption, ganglion cell loss, optic nerve mitochondrial dysfunction, and ERG defects; those with mild hyperhomocysteinemia have delayed retinal morphological/functional phenotype. Excess homocysteine is a risk factor for cardiovascular diseases; however, it is not known whether excess homocysteine alters retinal vasculature. METHODS Cbs(+/+), cbs(+/-), and cbs(-/-) mice (age ∼3 weeks) were subjected to angiography; retinas were harvested for cryosections, flat-mount preparations, or trypsin digestion and subjected to immunofluorescence microscopy to visualize vessels using isolectin-B4, to detect angiogenesis using anti-VEGF and anti-endoglin (anti-CD105) and activated glial cells (anti-glial fibrillary acidic protein [anti-GFAP]) and to investigate the blood-retinal barrier using the tight junction markers zonula occludens-1 (ZO-1) and occludin. Expression of vegf was determined by quantitative RT-PCR (qRT-PCR) and immunoblotting. Human retinal endothelial cells (HRECs) were treated with excess homocysteine to analyze permeability. RESULTS Angiography revealed vascular leakage in cbs(-/-) mice; immunohistochemical analysis demonstrated vascular patterns consistent with ischemia; isolectin-B4 labeling revealed a capillary-free zone centrally and new vessels with capillary tufts midperipherally. This was associated with increased vegf mRNA and protein, CD105, and GFAP in cbs(-/-) retinas concomitant with a marked decrease in ZO-1 and occludin. Homocysteine-treated HRECs showed increased permeability. CONCLUSIONS Severe elevation of homocysteine in cbs(-/-) mutant mice is accompanied by alterations in retinal vasculature (ischemia, neovascularization, and incompetent blood-retinal barrier). The marked disruption of retinal structure and decreased visual function reported in cbs(-/-) mice may reflect vasculopathy as well as neuropathy.

Virus spread and immune response following anterior chamber inoculation of HSV-1 Lacking the Beclin-binding domain (BBD)

The autophagy response induced by HSV-1 infection is antagonized by the Beclin-binding domain (BBD). The purpose of this study was to determine if lack of the BBD affects viral spread and immune response in the eyes and brain. Our results showed that lack of the BBD increases autophagy response and activation of NLRP3 inflammasome, which in turn induces a more rapid innate immune response mediated by macrophage/microglia and NK cells in the injected eye, limiting virus replication and retinal damage. We conclude that autophagy plays a role in controlling HSV-1 infection by more rapid induction of the innate immune response.

Lack of TNF-α Promotes Caspase-3–Independent Apoptosis during Murine Cytomegalovirus Retinitis

PURPOSE Both caspase-dependent and caspase-independent apoptosis contribute to retinal damage during murine cytomegalovirus (MCMV) retinitis, and TNF-α is among the inducers of apoptosis. The aim of this study was to determine the contribution of TNF-α by studying virus replication and apoptosis in immunosuppressed (IS) TNF-α(-/-) mice. METHODS IS TNF-α(-/-) mice or wild-type mice were inoculated with MCMV by the supraciliary route. Injected eyes were examined by plaque assay, electron microscopy, Western blot analysis (caspase-3, caspase-8, caspase-12, Bid, NF-κB, cFlip, XIAP), staining for MCMV early antigen, and TUNEL assay. RESULTS Although the titer of MCMV was similar in both groups, significantly more apoptotic cells were observed in the retinas of IS TNF-α(-/-) mice than in those of wild-type mice. The level of active caspase-3 was similar in both groups; however, more activated proteins for genes involved in the mitochondrial pathway (cleaved caspase-8, tBid) and endoplasmic reticulum (ER) stress (cleaved caspase-12) and, though less active, NF-κB subunits and antiapoptotic proteins (XIAP and cFlip) were detected in the TNF-α(-/-) eyes compared with wild-type mice. CONCLUSIONS Although TNF-α is an inducer of apoptosis, the results of this study suggest that TNF-α is also antiapoptotic by the following mechanism: TNF-α activation of NF-κB promotes the production of the antiapoptosis genes, c-flip or XIAP, which, in turn, inhibit the activation of caspase-8 and the mitochondrial pathway or the activation of caspase-12 and ER stress.

Decrease of murine cytomegalovirus-induced retinitis by intravenous delivery of immediate early protein-3-specific siRNA.

PURPOSE Retinitis induced by both human and murine cytomegaloviruses following immunosuppression is characterized by progressive loss of retinal architecture, due to necrosis of virus-infected cells as well as widespread apoptosis of uninfected bystander cells. Because small inhibitory RNA molecules (siRNA) can reduce murine cytomegalovirus (MCMV) gene expression and thereby inhibit virus replication in vitro, we tested siRNAs directed against MCMV immediate early protein-3 (IE-3) to determine if MCMV-induced retinitis could be alleviated in vivo. METHODS Immunosuppressed Balb/c mice (2.0 mg methylprednisolone acetate every 3 days beginning on day -2) were infected with 5 × 10(3) pfu of the K181 strain of MCMV via the supraciliary route. At day 2 post infection, mice were treated with various doses of IE-3-specific siRNA ranging from 0.1 nmol to 10 nmol, in a volume of 20 μL PBS via tail vein injection. Injected eyes were collected at various times post inoculation and subjected to plaque assay for virus titer, MCMV antigen staining, H&E staining, TUNEL assay, and Western blot for MCMV IE-3 protein. RESULTS Small but significant amounts of fluorescently labeled IE-3-specific siRNA localized to the RPE layer 48 hours after intravenous injection. IE-3-specific siRNA significantly reduced virus titers at all concentrations tested (ranging from 0.1 nmol to 10 nmol), but the most potent effect of siRNA was observed at a dose of 1 nmol. We also observed that IE-3-specific siRNA produced a substantial decrease in MCMV titers and a substantial reduction in bystander cell apoptosis over the time course of virus infection. CONCLUSIONS Systemic administration of IE-3-specific siRNA could alleviate MCMV retinitis by inhibiting virus replication and subsequent death of uninfected retinal cells.

Role of Bax in Death of Uninfected Retinal Cells During Murine Cytomegalovirus Retinitis

PURPOSE Extensive death of uninfected bystander neuronal cells is an important component of the pathogenesis of cytomegalovirus retinitis. Our previous results have shown that caspase 3-dependent and -independent pathways are involved in death of uninfected bystander cells during murine cytomegalovirus (MCMV) retinitis and also that Bcl-2, an important inhibitor of apoptosis via the Bax-mediated mitochondrial pathway, is downregulated during this process. The purpose of this study was to determine whether Bax-mediated mitochondrial damage has a significant role in the death of uninfected retinal cells. METHODS BALB/c mice, Bax(-/-) mice, or Bax(+/+) mice were immunosuppressed with methylprednisolone and infected with 5 × 10(3) plaque-forming units (PFU) of the K181 strain of MCMV via the supraciliary route. Injected eyes were analyzed by plaque assay, electron microscopy, hematoxylin and eosin (H&E) staining, TUNEL assay, Western blot (for caspase 3, caspase 12, Bax, receptor interacting protein-1 [RIP1] and receptor interacting protein-3 [RIP3]), as well as immunohistochemical staining for MCMV early antigen and cleaved caspase 3. RESULTS Significantly more Bax was detected in mitochondrial fractions of MCMV-infected eyes than in mitochondrial fractions of mock-infected control eyes. Furthermore, the level of cleaved caspase 3 was significantly lower in MCMV-infected Bax(-/-) eyes than in MCMV-infected Bax(+/+) eyes. However, more caspase 3-independent cell death of uninfected bystander retinal cells and more cleaved RIP1 were observed in Bax(-/-) than in Bax(+/+) eyes. CONCLUSIONS During MCMV retinitis, Bax is activated and has an important role in death of uninfected bystander retinal cells by caspase 3-dependent apoptosis. Although the exact mechanism remains to be deciphered, active Bax might also prevent death of some types of uninfected retinal cells by a caspase 3-independent pathway.