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Gideon Obasanmi, Matthew R Zeglinski, Erika Yuan Tian, Eleanor To, Jing Z Cui, David Granville, Joanne A Matsubara; Granzyme B Proteolyzes Thrombospondin-1 in RPE Cells and Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2022;63(7):473 – A0010.
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© ARVO (1962-2015); The Authors (2016-present)
An imbalance of pro and anti-angiogenic factors contributes to the pathology of neovascular AMD (nAMD). Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis induced by pro-angiogenic factors such as vascular endothelial growth factor (VEGF). Granzyme B (GrB) is a serine protease whose extracellular activity is increased in the outer retina and choroid of human eyes with choroidal neovascularization (CNV) from nAMD. This study determines the effects of GrB on TSP-1 in silico, and in cell-free cleavage assays, human eyes, RPE culture, and an explant choroid sprouting assay (CSA).
Mapping of human TSP-1 protein sequences for potential GrB cleavage sites was performed with GraBCas while the prediction of cleavage product size was done with the Compute pI/Mw tool from ExPASy. To confirm predictions, cell-free cleavage assays were done by digesting rhTSP-1 with rhGrB in 50 mM Tris, pH 7.5 for 2 hours at 37oC. Electrophoresis was performed on the digestion products, followed by Coomassie Brilliant Blue staining and imaging. CNV donor eye tissue sections were probed by immunohistochemistry for GrB and TSP-1. RPE cells were stimulated with GrB. CSA using RPE-choroid-sclera explants from 3-month-old C57BL/6J mice was established and cultured with rhTSP-1 (1 µg) or rhGrB (100nM) treatments. Images of CSA explants were captured and sprouting morphometry was quantified Western blot (WB) was used to assess TSP-1 expression in RPE cell culture and CSA supernatant.
In silico analysis predicted six potential GrB cleavage sites in the N- and C-termini and within type III repeats of TSP-1. Cell-free cleavage assays shows that GrB cleaves TSP-1 with double fragments ranging ~100kDa and triple fragments between 63-75kDa. An inverse relationship of GrB and TSP-1 was confirmed in RPE and choroid of CNV eyes, with higher GrB and lower TSP-1 immunolabelling. WB showed significantly reduced expression of TSP-1 in GrB-treated RPE cell culture (p<0.05) and CSA supernatant (p<0.05) compared with controls. Vascular sprouting area was significantly reduced in TSP-1 treated CSA (p<0.05) compared with controls.
GrB cleavage of antiangiogenic factors such as TSP-1 may contribute to CNV development in nAMD via pro-angiogenesis. Future studies are required to investigate whether the pharmacological inhibition of extracellular GrB could mitigate CNV in nAMD by conserving intact TSP-1.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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