Aortic valve stenosis serum IGFBP2 mediates female-specific Evogliptin resistance in valve myofibroblasts

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  Figure 1.. Proteomic profiling reveals distinct serum signatures in patients with AVS (A) Schematic overview of the approach to obtain and supplement serum samples for proteomic analysis. Figure created in BioRender: Aguado, B. (2026) https://BioRender.com/g4omlx4. (B) Hierarchical clustering dendrogram links all serum samples based on proteomic data (N = 43 healthy, N = 42 AVS). (C) Principal component analysis of the serum proteome from healthy and patients with AVS (N = 43 healthy, N = 42 AVS). (D) Volcano plot of 1,512 protein abundances enriched in healthy serum samples (green) or AVS serum samples (purple) (N = 43 healthy, N = 42 AVS). Statistical significance was defined as false-discovery rate (FDR) adjusted p value <0.05 (unpaired t test with Welch’s correction) and fold change greater than 1.2. (E) Venn diagram compares serum biomarkers identified in this study against the serum biomarkers identified in the Atherosclerosis Risk In Communities study.56 Statistical significance was determined by Fisher’s exact test. (F and G) Ingenuity pathway analysis shows the top pathways associated with proteins that are more abundant in (F) AVS serum samples and (G) healthy serum samples.

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  Figure 2.. Clinically relevant doses of antifibrotic drugs prevent male and female VIC myofibroblast activation (A) Schematic overview of the protocol to optimize antifibrotic drug dosing using hydrogel biomaterials. Figure created in BioRender. Aguado, B. (2026) https://BioRender.com/lue0gxl. (B) Representative image of formed hydrogels ready for cell seeding. (C–E) Percent αSMA reduction in male and female VICs cultured on hydrogels with increasing Cmax concentrations of (C) Ataciguat, (D) Colchicine, or (E) Evogliptin (N = 3–4 gels). X indicates complete cell death at that concentration. Statistical significance is indicated as # = p < 0.05 for male VICs and $ = p < 0.05 for female VICs (one-sample t and Wilcoxon tests against a theoretical mean of zero). Data are shown as mean ± standard error of the mean. (F and G) Percent cytotoxicity in (F) male and (G) female VICs in response to increasing Cmax concentrations of Ataciguat, Colchicine, or Evogliptin (N = 3–4 gels). A 5 nM dose of Staurosporine was used as a positive control for cell death. Statistical significance is indicated as **** = p < 0.0001 (one-way ANOVA with Tukey posttests). (H) Representative immunofluorescent images of…

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  Figure 3.. Sex-specific serum biomarkers regulate antifibrotic drug efficacy (A) Schematic overview of the approach to link candidate serum biomarkers with in vitro drug efficacy. Figure created in BioRender. Aguado, B. (2026) https://BioRender.com/9cyhk4j. (B) αSMA gradient mean intensity for male and female VICs cultured on hydrogels with individual sex-matched AVS patient serum samples (N = 16 male serum samples, N = 15 female serum samples). Statistical significance is indicated as *** = p < 0.001 (unpaired two-tailed t test with Welch’s correction). (C) Percent αSMA reduction in male and female VICs cultured on hydrogels with optimized doses of Ataciguat, Colchicine, or Evogliptin (N = 16 male serum samples, N = 15 female serum samples). Statistical significance is indicated as ** = p < 0.01 (unpaired two-tailed t test with Welch’s correction). (D) Heatmaps show the correlation between each candidate serum factor and antifibrotic drug efficacy for male or female VICs cultured with sex-matched AVS serum (N = 16 male, N = 15 female). Statistical significance is indicated as * = p < 0.05 (paired two-tailed t test). (E) Correlation between serum IGFBP2 abundance (relative fluorescence…

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  Figure 4.. IGFBP2 mediates Evogliptin resistance in female VICs cultured with female AVS serum (A) Serum IGFBP2 concentrations quantified using an ELISA kit (N = 31 AVS, N = 5 healthy). Statistical significance is indicated as **** = p < 0.0001 (unpaired two-tailed t test with Welch’s correction). (B and C) Percent αSMA reduction in (B) female and (C) male VICs cultured on hydrogels with FBS and treated with Evogliptin and/or recombinant human IGFBP2 protein (N = 6 gels). Statistical significance is indicated as * = p < 0.05 (one-sample t and Wilcoxon test against a theoretical mean of zero). (D) Serum IGFBP2 levels quantified using an ELISA kit with and without IGFBP2 neutralizing antibody (IGFBP2 Ab), IgG control antibody, and recombinant human IGFBP2 protein (N = 3 male, N = 3 female). Statistical significance is indicated as *** = p < 0.001 (one-way ANOVA with Tukey posttests). (E) Percent αSMA reduction in male and female VICs cultured on hydrogels with sex-matched AVS patient serum samples, Evogliptin, IgG, IGFBP2 Ab, or recombinant human IGFBP2 protein (N = 3 male, N = 3 female). Statistical significance between groups is indicated as *** = p < 0.001, and significant interactions…

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  Figure 5.. RNA sequencing shows that IGFBP2 acts through mechanosensing pathways to promote Evogliptin resistance in female VICs (A) Principal component analysis clustering samples based on RNA sequencing results (N = 3). (B and C) Volcano plot of differential gene expression from RNA sequencing between (B) Vehicle and Evogliptin groups or (C) Vehicle and Evogliptin plus IGFBP2 neutralizing antibody groups (N = 3). Statistical significance was defined as FDR-adjusted p value <0.05 (unpaired two-tailed t test with Welch’s correction) and fold change greater than 2. (D) Venn diagram compares differentially expressed genes in the Evogliptin and Evogliptin plus IGFBP2 neutralizing antibody groups relative to vehicle control (N = 3). (E) Volcano plot of differential gene expression from RNA sequencing between Evogliptin and Evogliptin plus IGFBP2 neutralizing antibody groups (N = 3). Statistical significance was defined as FDR-adjusted p value <0.05 (unpaired t test with Welch’s correction) and fold change greater than 2. (F) Heatmap of the top 20 up- and down-regulated genes in the Evogliptin plus IGFBP2 neutralizing antibody group relative to vehicle control (N = 3). (G and H) Top 15…

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  Figure 6.. IGFBP2 drives female-specific Evogliptin resistance through Rho/ROCK and focal adhesion kinase signaling (A) Schematic overview of the approach to validate the mechanism of IGFBP2-mediated Evogliptin resistance. Figure Created in BioRender. Aguado, B. (2026) https://BioRender.com/2c3itp7. (B) Percent αSMA reduction in female VICs cultured on hydrogels with female AVS serum and treated with various doses of H-1152 with and without Evogliptin (N = 3 gels). For H-1152: + = 0.1 μM; ++ = 0.5 μM; +++ = 5 μM. Statistical significance is indicated as * = p < 0.05 (unpaired two-tailed t test with Welch’s correction). (C) Percent αSMA reduction in female VICs cultured on hydrogels with AVS serum and treated with various doses of PF-573228 with and without Evogliptin (N = 3 gels). For PF-573228: + = 0.1 μM; ++ = 1 μM; +++ = 10 μM. Statistical significance is indicated as * = p < 0.05 (unpaired two-tailed t test with Welch’s correction). (D and E) Percent αSMA reduction in (D) female or (E) male VICs cultured on hydrogels with sex-matched AVS serum and treated with various combinations of Evogliptin, IGFBP2 neutralizing antibody, H-1152, and PF-573228 (N = 3 serum samples per sex).…

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  Figure 7.. Proposed mechanism for IGFBP2 driving sex-specific Evogliptin resistance Summary mechanistic figure illustrates how elevated IGFBP2 in female serum modulates Evogliptin efficacy in female VICs. Figure created in BioRender. Aguado, B. (2026) https://BioRender.com/8zc6m9e.