Fig 1: A1AT ameliorates RPE/BrM and outer retinal thickening in HTRA1 transgenic mice. (A) Quantification of intracellular, apical, and basal elastase activity in primary RPE cell monolayers derived from HTRA1 when compared to CD1 control mice (n = 3). (B) Elastase activity in retina and (C) RPE/choroid tissue extracts of 6 months old CD1 and HTRA1 mice (n = 3). (D) Tropoelastin (TE) protein levels present in the RPE/Choroid of 6 months old HTRA1 and CD1 mice. (E) OCT quantification of RPE/BrM, (F) outer retinal (IS-RPE) and (G) outer nuclear (ONL) layer thicknesses in CD1 and HTRA1 mice compared to HTRA1 mice treated with A1AT (n = 8–16 eyes per group). Statistics used: t-test for single comparison, one-way ANOVA, Tukey’s MC. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. Data represent the mean ± SEM. Abbreviations: TE: tropoelastin, IS-RPE: inner segments to RPE distance measured.
Fig 2: Assessing the effect of M42 recombinant proteins on phagocytosis of apoptotic SH‐SY5Y cells in human induced pluripotent stem cell (hiPSC)‐derived macrophages. (A) Summary graph showing fold change in total phagocytosis assessed over 24 h relative to control (vehicle‐treated cells) across all tested proteins at three concentrations: 3 µg/mL, 1 µg/mL, and 100 ng/mL. (B–F) Phagocytosis following treatment with HTRA1 (B), DAG1 ectodomain (C), TMEFF2 ectodomain (D), MDK (E), and GPC5 (F). Left panels show comparison between phagocytosis levels in vehicle‐ and protein‐treated hiPSC macrophages quantified by area under the curve (AUC). Right panels show a representative trace from one biological repeat (n = 3 technical replicates). Levels of phagocytosis are measured as total integrated intensity of pHrodo‐labeled apoptotic neurons normalized to the confluency of macrophage layer (TII/confluency). Cytochalasin D (CytoD, 10 µM) treatment was included in every experiment as a negative control. N = 3 to 4 independent biological experiments (one or two independent macrophage factory set‐ups; three technical replicates/biological repeat). Error bars correspond to mean ± SD. Repeated measures one‐way ANOVA followed by Dunnett's multiple comparisons test. *p < 0.05; **p < 0.01.
Fig 3: Assessing the effect of M42 protein treatment on viability of hiPSC derived macrophages. Human iPSC macrophages were treated with HTRA1 (A), DAG1 ectodomain (B), TMEFF2 ectodomain (C), MDK (D), GPC5 (E), SDC4 ectodomain (F), SFRP1 (G), SPON1 (H), and PTN (I) at 3 µg/mL, 1 µg/mL, and 100 ng/mL for 24 h. Viability was assessed and expressed as relative luminescence units (RLU). N = 2 or 3 independent biological experiments (one independent macrophage factory set‐up; three technical replicates/biological repeat). Error bars correspond to mean ± standard deviation (SD). Repeated measures one‐way ANOVA followed by Dunnett's multiple comparisons test.
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