Fig. 5: Sirt3 depletion alters the metabolic and angiogenic landscape of the neovascular niche.
a–d Human retinal astrocytes (35000 cells/well) under hypoxia were treated with scrambled siRNA (scrRNA) or siRNA against Sirt3 (siSIRT3) and characterized using a Seahorse metabolic analyzer. a Glycolysis stress tests measured changes in extracellular acidification rates (ECAR, mean ± SEM) to calculate b glycolysis and glycolytic capacity between control (scrRNA, n = 5 wells) and Sirt3-depleted astrocytes (siSIRT3, n = 5 wells). c Mitochondrial stress tests measured changes in oxygen consumption rates (OCR, mean ± SEM) between control (scrRNA, n = 13 wells) and Sirt3-depleted astrocytes (siSIRT3, n = 9 wells). d Energy map of ATP-linked respiration and the glycolytic capacity of scrRNA and siSIRT3 astrocytes (mean ± SEM) from a–c. e Circos Plot from NicheNet analysis showing the differential communication between WT and Sirt3-/- neovascular ligands and tip and tuft EC receptors in OIR retinas based on scRNAseq data (P14-P17 combined). Selected ligands were differentially expressed between WT and Sirt3-/- neovascular cells (log2FC > 0.25, % expression > 40%). Selected receptors were differentially expressed between WT and Sirt3-/- tip (left) or tuft (right) ECs (log2FC > 0.25, % expression > 10%). f Dot plot representing the differential expression of selected ligands from WT and Sirt3-/- retinal neovascular cells based on NicheNet analysis. g Bar graph depicting log2 FC of Vegfa expression between Sirt3-/- versus WT cell types of the neovascular unit based on scRNAseq data (P14/P17 combined). h Lectin-stained retinal flat-mount of P17 Sirt3lox/lox mice treated with intravitreal injection of PBS (n = 7 retinas), pAAV.GFAP.GFP (n = 6 retinas) or pAAV.GFAP.Cre (n = 13 retinas) and exposed to the OIR model. Littermate Sirt3lox/lox pups were injected intravitreously at P7, one eye with pAAV.GFAP.cre and the other eye with either PBS or pAAV.GFAP.GFP. Bar graph of VO (blue) and NV (yellow) areas relative to the total retinal area (mean ± SEM). Scale: 500 μm. i Graphical representation of the neovascular niche in proliferative retinopathy. Statistical significance was established using two-tailed Student’s t-test (b) and one-way ANOVA (h) with Tukey post hoc analysis. ns: not significant.
