Fig 1: HG exposure modulated the transcript expression of complement mediators. (A) Scatterplot displaying a slight positive correlation between C3aR1 and C5aR1 after 15 mM HG exposure for all time points investigated. Bar graphs show the transcript expression of complement receptors C3aR1 (B) and C5aR1 (C), complement activators C3 (D), C5 (E) and C9 (F), and finally complement modulators CFH (G), CD55 (H) and CD59 (I) in CECs exposed to 15 mM HG (white bars) or 30 mM HG (grey bars) over 1, 3 and 5 days. Expression ratios were calculated relative to untreated control cells and data are shown as mean ± SEM. Pearson correlation coefficient (r) and significance are shown inside panel (A). Statistical comparisons were performed with 2-way ANOVA Dunnett’s post hoc analysis for time-point and group comparisons (B–I), and significance were * p < 0.05; ** p < 0.01; *** p < 0.001. The red dashed lines were included as an additional stricter cutoff identifying biologically relevant changes. Legend: C3aR1, Complement C3a Receptor 1; C5aR1, Complement C5a Receptor 1; C3, Complement component 3; C5, Complement component 5; C9, Complement component 9; CFH, Complement Factor H; CD55, Cluster of Differentiation 55; CD59, Cluster of Differentiation 59.
Fig 2: Acute and chronic HG exposure increased CFH and C5b-9 protein levels. CFH (A) and C5b-9 (B) protein levels were measured respectively in conditioned media and cell pellet from 15 mM HG or 30 mM HG upon acute (day 1) and chronic (day 5) exposure, in comparison to control cells (CTR; ELISA). Data are expressed as mean ± SEM (pg/mL) from four independent experiments performed in triplicate. Two-way ANOVA followed by a Tukey–Kramer post hoc test analysis (glucose vs. timing) provided the statistical differences shown in the panels by asterisk (* p < 0.05; ** p < 0.01). (C,D) Scatterplot displaying the relationship between CFH and C5b-9 protein levels after acute (C) and chronic (D) HG exposure. Pearson correlation coefficient (r) and significance are shown in the panel. Legend: CTR, control cells; CFH, complement factor H; C5b-9, Membrane Attack Complex (MAC).
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