Aflibercept and Faricimab Equipotently Restore Endothelial Barrier Function

Figures

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  Figure 1.. Experimental design, HUVEC monolayer permeability, and non-inferiority testing by setting. (A) Schematic representation of the assay setup to investigate the effect of VEGF-A165 on HUVEC monolayer integrity comparing co-addition of VEGF-A165 (0.26 nM) and 1.7 nM AFL or 2.0 nM FAR (preventive) or the addition of either agent after 24 hours of exposure to exogenous VEGF-A165 (therapeutic/rescue). The asterisk sign denotes the time at which the cells or culture supernatants were harvested for analysis. (B) Cellular impedance curves reflecting HUVEC monolayer permeability. A higher cell index indicates intact intercellular tight junctions and monolayer integrity, whereas a lower cell index indicates higher monolayer permeability. The means ± SD of three technical replicates per donor are shown. Green arrows denote the timeline for the preventive setting; blue arrows denote the timeline for the therapeutic setting; black arrows denote VEGF-A165 addition. The dotted vertical line denotes the addition of 0.26 nM VEGF-A165 at 24 hours. (C) Non-inferiority testing for the preventive setting. (D) Non-inferiority testing for the therapeutic/rescue setting. Red and green dashed lines…

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  Figure 2.. Differential gene expression profiles in response to VEGF-A165 in the presence and absence of AFL or FAR. (A) Venn diagram summarizing the number of differentially expressed genes in 0.26 nM VEGF-A165–exposed HUVEC with and without 1.7 nM AFL or 2.0 nM FAR. Both the preventive and therapeutic/rescue settings are considered for the three donors combined. (B) Volcano plot of differentially expressed genes driven by VEGF-A165 exposure, highlighting some of the genes that are most differentially expressed. (C) Volcano plot depicting reversal of VEGF-A165–driven differentially expressed genes by AFL. (D) Volcano plot depicting reversal of VEGF-A165–driven differentially expressed genes by FAR. Significant differential expression of genes was defined by the following criteria: (i) FDR <0.1, (ii) log2 fold change >log2(1.5), and (iii) mean expression (baseMean) >50. AFL, aflibercept; FDR, false discovery rate; FAR, faricimab; VEGF-A, vascular endothelial growth factor-A.

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  Figure 3.. ANGPT2 gene expression and ANG2 protein concentrations in HUVEC culture supernatants from three independent donors. (A) ANGPT2 gene expression and (B) ANG2 protein concentrations in the preventive as well as therapeutic/rescue settings. VEGF-A165 concentration was 0.26 nM, AFL concentration was 1.7 nM, and FAR concentration was 2.0 nM. Error bars indicate mean ± SD of technical triplicates of cells (RNA-Seq) or culture supernatant samples (protein measurement) from three separate HUVEC-containing wells per donor. Significance was assessed in a combined analysis of the three donors. *P < 0.05; **P < 0.01; ***P < 0.001. ns: P > 0.05. AFL, aflibercept; FAR, faricimab; HUVEC, human umbilical vein endothelial cell; ns, not significant; RLU, relative light units; TPM, transcripts per million; VEGF-A, vascular endothelial growth factor-A.

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  Figure 3.. Continued.

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  Figure 4.. TEK gene expression and soluble Tie2 protein concentrations in HUVEC culture supernatants from three independent donors. (A) TEK gene expression and (B) soluble Tie2 protein concentrations in the preventive setting as well as therapeutic/rescue settings. VEGF-A165 concentration was 0.26 nM, AFL concentration was 1.7 nM, and FAR concentration was 2.0 nM. Error bars: mean ± SD of technical triplicates of cells (RNA-Seq) or culture supernatant samples (protein measurement) from three separate HUVEC-containing wells per donor. Significance was assessed in a combined analysis of the three donors. *P < 0.05; **P < 0.01; ***P < 0.001; ns: P > 0.05. AFL, aflibercept; FAR, faricimab; HUVEC, human umbilical vein endothelial cell; ns, not significant; RLU, relative light units; TEK, Tie2 receptor-encoding gene; TPM, transcripts per million; VEGF, vascular endothelial growth factor.

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  Figure 4.. Continued.