Y chromosome–linked UTY modulates sex differences in valvular fibroblast methylation in response to nanoscale extracellular matrix cues

Figures

• 

  Fig. 1.. Single-cell sequencing and sex-separated analysis of human patients with AVS reveal clustering of VIC populations. (A) Uniform manifold approximation and projection (UMAP) clustering and cell population distribution in healthy and diseased patient samples for males and females. (B) Distribution of VIC and non-VIC population percentages in healthy and diseased patient samples for males and females. (C to E) Ingenuity pathway analysis for male (C) VIC1, (D) VIC2, and (E) VIC3. IL-17A, interleukin-17A; TNFR2, tumor necrosis factor receptor 2; IL-6, interleukin-6; ID1, inhibitor of differentiation 1; IL-8, interleukin-8; IL-17F, interleukin-17F; NOD1/2, nucleotide-binding oligomerization domain 1/2.

• 

  Fig. 2.. PS-NP hydrogels recapitulate nanoscale features in AV tissue ECM. (A) Scanning electron microscopy (SEM) images of male healthy and diseased and of female healthy and diseased human AV leaflets. Scale bars, 5 μm. (B) Schematic of process to incorporate PS-NPs into PEG-Nb hydrogel precursor solutions to create PS-NP hydrogels adhered to glass coverslips via ultraviolet (UV) polymerization. Figure was drawn with BioRender. (C) Rheological characterization of UV-mediated polymerization of blank/soft, stiff, and PS-NP hydrogels made with 200- and 2000-nm NPs (n = 3 representative measurements) at low and high concentrations (0.02 and 0.7 mg/ml, respectively). (D) Young’s modulus values for blank/soft, stiff, and PS-NP hydrogels (n = 3 gels). Significance determined via one-way analysis of variance (ANOVA; ****P < 0.0001 denoting statistical significance between hydrogel formulations). Means ± SD are shown. (E) SEM of blank and PS-NP hydrogels (200 nm and 2000 nm). Yellow arrows denote PS-NPs. Scale bars, 5 μm.

• 

  Fig. 3.. Male and female VICs exhibit sex-specific myofibroblast and osteoblast-like phenotypes. (A) Schematic of experiments assessing myofibroblast activation with αSMA and osteoblast-like differentiation with runt related (RUNX2). Figure was drawn with BioRender. (B) Representative confocal microscopy images of αSMA stress fibers (green) and nuclei (blue). Scale bars, 50 μm. (C) Normalized αSMA expression for male and female VICs cultured on blank hydrogels with no PS-NPs, 200-nm PS-NPs, or 2000-nm PS-NPs at low and high concentrations (0.02 and 0.7 mg/ml, respectively). (D) Representative confocal microscopy images of RUNX2 expression (red) and nuclei (blue). Scale bars, 50 μm. (E) RUNX2 nuclear to cytoplasmic intensity ratios of male and female VICs cultured on blank hydrogels with no PS-NPs, 200-nm PS-NPs, or 2000-nm PS-NPs at low and high concentrations (0.02 and 0.7 mg/ml, respectively). Significance for [(C) and (E)] was determined via Cohen’s d test (*d < 0.2, **d < 0.5, ***d < 0.8, and ****d < 1.4 denoting significance between biological sex in the provided table; blue asterisk color indicates significant increase in male groups; #d < 0.2, ##d < 0.5, ###d < 0.8, and ####d < 1.4…

• 

  Fig. 4.. Male and female VICs exhibit sex-specific changes in global methylation and acetylation states. (A) Schematic describing experimental workflow for assessing sex-specific methylation and acetylation states in VICs cultured on PS-NP gels. Figure was drawn with BioRender. (B) Representative confocal microscopy images of methylated lysine (yellow) and nuclei (blue). Scale bars, 50 μm. (C) Normalized methylation in male and female VICs cultured on blank hydrogels with no PS-NPs, 200-nm PS-NPs, or 2000-nm PS-NPs at low and high concentrations (0.02 and 0.7 mg/ml, respectively). (D) Representative confocal microscopy images of acetylated lysine (yellow) and nuclei (blue). Scale bars, 50 μm. (E) Normalized acetylation in male and female VICs cultured on control hydrogels with no PS-NPs, 200-nm PS-NPs, or 2000-nm PS-NPs at low and high concentrations (0.02 and 0.7 mg/ml, respectively). Significance for [(C) and (E)] was determined via Cohen’s d test (*d < 0.2, **d < 0.5, ***d < 0.8, and ****d < 1.4 denoting significance between biological sex in the provided table; blue asterisk color indicates significant increase in male groups; #d < 0.2, ##d < 0.5, ###d < 0.8, and ####d < 1.4 denoting…

• 

  Fig. 5.. Y-linked UTY modulates male-specific methylation states in VICs in vivo and in vitro. (A) Fold change (FC) of UTY+ VICs in diseased human male VIC populations VIC1-3 relative to healthy male VICs. Each percentage is shown relative to the total number of VICs in each specified subpopulation. (B and C) UTY relative mRNA expression in VIC populations 1 to 3 in (B) males and (C) females. (D) Real-time quantitative polymerase chain reaction (RT-qPCR) for relative expression of UTY in male and female VICs cultured on blank, 200-nm, and 2000-nm PS-NP hydrogels. (E) Normalized methylation for male VICs with three UTY-targeting siRNAs for exon regions 17 and 5. (F) RT-qPCR to validate partial silencing of UTY in male VICs after introduction of siRNA vector. (G) Representative confocal microscopy images of methylated lysine (red) and nuclei (blue). Scale bars, 50 μm. (H) Normalized methylation in male and female VICs cultured on blank hydrogels with no PS-NPs and 2000-nm PS-NPs. Significance determined via Cohen’s d test (*d < 0.2 and **d < 0.5 denoting significance between biological sex in the provided table; blue asterisk color indicates significant increase in male groups; #d < 0.2,…

• 

  Fig. 6.. Transcriptomics analyses in porcine male VICs after UTY-KD unveils osteoblast-associated pathways. (A and B) Bulk mRNA sequencing shows differentially expressed genes (DEGs) in male porcine VICs (blue, down-regulated; red, up-regulated; gray, not significant) in (A) blank and (B) PS-NP hydrogels using DESeq2. (C and D) Ingenuity pathway analysis of porcine VICs after UTY-KD on (C) blank and (D) PS-NP hydrogels [log2 fold change (log2FC) > 1, log2FC < −1, and P value < 0.05]. (E) Venn diagrams of up-regulated and down-regulated DEGs in porcine VICs cultured on blank and PS-NP hydrogels after UTY-KD. (F) Venn diagrams of up-regulated genes in common between porcine and human transcriptomics datasets.

• 

  Fig. 7.. Proposed mechanism for sex-specific transcriptomic regulation in the presence and absence of nanoscale stiffness cues. At baseline conditions, male VICs maintain higher levels of RUNX2 nuclear localization, while females maintain higher levels of αSMA stress fibers. (A) In the presence of PS-NPs, we observed increased demethylation in male porcine VICs, whereas (B) we only observed decreases in αSMA stress fiber presence in female porcine VICs.