Journal: Journal of Neuroinflammation

Article Title: Ganglion cell-derived LysoPS induces retinal neovascularisation by activating the microglial GPR34-PI3K-AKT-NINJ1 axis

doi: 10.1186/s12974-024-03265-7

Figure Lengend Snippet: Inhibition of LysoPS-activated microglial inflammation ( A , B ) NINJ1 was silenced in HMC3 cells using a lentiviral shNINJ1 and selected by RT-qPCR (A, n = 5), and clone 1 was confirmed by immunoblotting ( B ). ( C ) in addition to NINJ1 silencing, HMC3 cells were treated with a GPR34 antagonist (GPR34-ANT) and a PI3K inhibitor (LY294002). The levels of NINJ1, PI3K, AKT, and their phosphorylated forms were quantified by immunoblotting. ( D – F ) the fold changes in the relative protein levels were calculated ( n = 4). ( G – J ) cell supernatants collected from the treated HMC3 cells were cocultured with HRVEC cells to determine cell phenotypic changes, including cell proliferation (upper panel, 20 μm), cell migration (middle panel, 20 μm), and tube formation (lower panel, 100 μm). The results are presented as the mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, no significance; one-way ANOVA in ( D – F ), two-way ANOVA in ( H – J )

Article Snippet: Simultaneously, they were intravitreally injected with 1 µl of 10 µM LysoPS (0130549, Aladdin, USA), 5 µM GPR34 antagonist (HY-138501, MedChemExpress, USA), or 10 µM PI3K inhibitor LY294002 (HY-10108, MedChemExpress).

Techniques: Inhibition, Quantitative RT-PCR, Western Blot, Migration

Journal: Journal of Neuroinflammation

Article Title: Ganglion cell-derived LysoPS induces retinal neovascularisation by activating the microglial GPR34-PI3K-AKT-NINJ1 axis

doi: 10.1186/s12974-024-03265-7

Figure Lengend Snippet: Prevention of LysoPS-induced inflammation in OIR ( A , B ) AAV-carried shNINJ1 was intravitreally injected into OIR mice, and the efficiency of NINJ1 silencing in retinal tissues was confirmed by immunoblotting ( n = 4). ( C ) following AAV injection, further intravitreal injections of LysoPS, GPR34-ANT, or LY294002 were into OIR mice. Retinal tissue slides were imaged, with yellow areas indicating neovascularisation and white areas representing avascular regions. ( D , E ) the neovascular and avascular areas were quantified ( n = 6). ( F – I ) relative cytokines levels were quantified using relative ELISA kits ( n = 6). The results are presented as the mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001,**** p < 0.0001, ns, no significance; one-way ANOVA in ( B ), two-way ANOVA in ( D – I ); scale bar, 200 μm in ( C )

Article Snippet: Simultaneously, they were intravitreally injected with 1 µl of 10 µM LysoPS (0130549, Aladdin, USA), 5 µM GPR34 antagonist (HY-138501, MedChemExpress, USA), or 10 µM PI3K inhibitor LY294002 (HY-10108, MedChemExpress).

Techniques: Injection, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Neuroinflammation

Article Title: Ganglion cell-derived LysoPS induces retinal neovascularisation by activating the microglial GPR34-PI3K-AKT-NINJ1 axis

doi: 10.1186/s12974-024-03265-7

Figure Lengend Snippet: Suppression of retinal neovascularisation by inhibiting the GPR34-PI3K-AKT-NINJ1 axis ( A , B ) Retinal endothelial cells were isolated from the fibrovascular membranes of PDR patients and cultured in an endothelial-specific medium. The purity of the cultured endothelial cells was analysed using flow cytometry with an endothelial marker CD31 antibody ( n = 4). ( C , D ) the fibrovascular membranes were treated with supernatants collected from LysoPS-treated HMC3 cells. To inhibit the effect of LysoPS, the HMC3 cells were pretreated with GPR34-ANT, LY294002, or shNINJ1. The relative sprouting rate of the endothelial cells within the fibrovascular membranes was evaluated on the specified days, as described in Fig. E, F ( n = 4). ( E ) schematic representation of the proposed mechanistic interaction among ganglion cells, microglia, and endothelial cells in promoting retinal neovascularisation. The results are presented as the mean ± SD, **** p < 0.0001, Student’s t test in ( B ), two-way ANOVA in ( D ); scale bar, 200 μm in ( C )

Article Snippet: Simultaneously, they were intravitreally injected with 1 µl of 10 µM LysoPS (0130549, Aladdin, USA), 5 µM GPR34 antagonist (HY-138501, MedChemExpress, USA), or 10 µM PI3K inhibitor LY294002 (HY-10108, MedChemExpress).

Techniques: Isolation, Cell Culture, Flow Cytometry, Marker