Granzyme B Contributes to Choroidal Neovascularization and Age-Related Macular Degeneration Through Proteolysis of Thrombospondin-1.

Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss in the elderly. The pathology of neovascular age-related macular degeneration (nAMD), also known as wet AMD, is associated with an abnormal blood vessel growth in the eye and involves an imbalance of proangiogenic and antiangiogenic factors. Thrombospondin (TSP)-1 and TSP-2 are endogenous matricellular proteins that inhibit angiogenesis. TSP-1 is significantly diminished in eyes with AMD, although the mechanisms involved in its reduction are unknown. Granzyme B (GzmB) is a serine protease with an increased extracellular activity in the outer retina and choroid of human eyes with nAMD-related choroidal neovascularization (CNV). This study investigated whether TSP-1 and TSP-2 are GzmB substrates using in silico and cell-free cleavage assays and explored the relationship between GzmB and TSP-1 in human eyes with nAMD-related CNV and the effect of GzmB on TSP-1 in retinal pigment epithelial culture and an explant choroid sprouting assay (CSA). In this study, TSP-1 and TSP-2 were identified as GzmB substrates. Cell-free cleavage assays substantiated the GzmB proteolysis of TSP-1 and TSP-2 by showing dose-dependent and time-dependent cleavage products. TSP-1 and TSP-2 proteolysis were hindered by the inhibition of GzmB. In the retinal pigment epithelium and choroid of human eyes with CNV, we observed a significant inverse correlation between TSP-1 and GzmB, as indicated by lower TSP-1 and higher GzmB immunoreactivity. In CSA, the vascular sprouting area increased significantly with GzmB treatment and reduced significantly with TSP-1 treatment. Western blot showed significantly reduced expression of TSP-1 in GzmB-treated retinal pigment epithelial cell culture and CSA supernatant compared with that in controls. Together, our findings suggest that the proteolysis of antiangiogenic factors such as TSP-1 by extracellular GzmB might represent a mechanism through which GzmB may contribute to nAMD-related CNV. Future studies are needed to investigate whether pharmacologic inhibition of extracellular GzmB can mitigate nAMD-related CNV by preserving intact TSP-1.

Microbial Community Dynamics During Lake Ice Freezing.

Many freshwater environments experience dramatic seasonal changes with some systems remaining ice-covered for most of the winter. Freshwater systems are also highly sensitive to environmental change. However, little is known about changes in microbial abundance and community composition during lake ice formation and times of persistent ice cover. The goal of this study is to characterize temporal dynamics of microbial communities during ice formation and persistent ice cover. Samples were collected in triplicate, five days per week from surface water in the Keweenaw Waterway between November and April. Environmental conditions along with microbial abundance and microbial community composition was determined. Distinct community composition was found between ice-free and ice-covered time periods with significantly different community composition between months. The microbial community underwent dramatic shifts in microbial abundance and diversity during the transitions into and out of ice cover. The richness of the microbial community increased during times of ice cover. Relatives of microbes involved in nitrogen cycling bloomed during times of ice cover as sequences related to known nitrifying taxa were significantly enriched during ice cover. These results help to elucidate how microbial abundance and diversity change over drastic seasonal transitions and how ice cover may affect microbial abundance and diversity.