Research ArticleCardiology

Transcatheter aortic valve replacements alter circulating serum factors to mediate myofibroblast deactivation

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Science Translational Medicine  11 Sep 2019:
Vol. 11, Issue 509, eaav3233
DOI: 10.1126/scitranslmed.aav3233
  • Fig. 1 Pre-TAVR serum activates VICs to a myofibroblast state, whereas post-TAVR serum mediates fibroblast quiescence.

    (A) SOMAscan DNA aptamer array heatmap of individual log2-RFU values representing relative abundances for 283 significantly altered human serum proteins before and after a TAVR procedure (n = 12 pre-TAVR samples and n = 12 post-TAVR samples, paired t test, FDR-adjusted P < 0.05). (B) PCA plot of pre-TAVR and post-TAVR serum samples. (C) Photograph of a PEG hydrogel on a 12-mm-diameter glass coverslip. Scale bar, 12 mm. (D) Schematic of in vitro experiments using pre-TAVR and post-TAVR sera to treat porcine VICs on soft hydrogels (created with BioRender). (E) Representative images of VICs treated with pre- and post-TAVR serum. Green, α-SMA; magenta, cytoplasm; blue, nuclei. Scale bars, 100 μm. (F) Percentage of activated VICs treated with sera from TAVR patients (n = 9 measurements per group, one-way ANOVA with Tukey posttests, means ± SD shown, *P < 0.05, **P < 0.01, ***P < 0.001). (G) Fold change reduction of VIC activation in post-TAVR serum (XPost values) normalized to the mean activation in pre-TAVR serum from the same patient (μPre). Groups with different letters indicate statistical significance (n = 9 measurements per group, one-way ANOVA with Tukey posttests, means ± SD shown, P < 0.05). (H) Pre-TAVR and (I) post-TAVR ontological categorization of serum proteins to identify factors contributing to myofibroblast activation and quiescence, respectively.

  • Fig. 2 Transcriptomics analysis reveals p38 MAPK signaling activity in pre-TAVR–mediated myofibroblast activation.

    (A) MD plot reveals differentially expressed genes in porcine VICs treated with pre-TAVR and post-TAVR sera (n = 8 patients, quantile-adjusted conditional maximum likelihood method in edgeR, FDR-adjusted P < 0.05). (B) Log2-transformed counts per million (CPM) values for key myofibroblast-associated genes (n = 8, all pre-TAVR versus post-TAVR comparisons, FDR-adjusted P < 0.05) and organized into cytoskeleton, ECM remodeling, and fibrocalcification categories. (C) PCA plot of key myofibroblast-associated genes. (D and E) Enriched KEGG signaling pathways for (D) up-regulated and (E) down-regulated genes in pre-TAVR serum relative to post-TAVR serum. (F) Ingenuity Pathway Analysis. Pre-TAVR serum protein candidates are represented by pink nodes and arrows among up-regulated genes.

  • Fig. 3 Pre-TAVR serum factors (BMP-6, CXCL9, and IFN-γ) mediate myofibroblast activation.

    (A) Schematic of valvular activation experiments with BMP-6, CXCL9, and IFN-γ (created with BioRender). Representative images of porcine VICs treated initially with (B) BMP-6, (E) CXCL9, or (H) IFN-γ for 2 days (left column) and then treated with either control media or post-TAVR serum (right column). Stains: green, α-SMA; magenta, cytoplasm; blue, nuclei. Scale bars, 100 μm. Percentage of activated VICs initially treated with (C) BMP-6, (F) CXCL9, or (I) IFN-γ and deactivated with post-TAVR serum from four patients. Patient-specific fold changes in post-TAVR deactivation in VICs initially activated with (D) BMP-6, (G) CXCL9, or (J) IFN-γ. Groups with different letters indicate statistical significance (n = 9 measurements per group, means ± SD shown, one-way ANOVA with Tukey posttests, P < 0.05).

  • Fig. 4 Validation of p38 MAPK signaling in mediating myofibroblast activation on soft hydrogels in the presence of various factors.

    (A) Representative images of porcine VICs treated with pre-TAVR serum in the absence or presence of 20 μM SB203580. Stains: green, α-SMA; magenta, cytoplasm; blue, nuclei. Scale bars, 100 μm. (B) VIC activation in pre-TAVR serum (n = 4 patients) in the absence or presence of SB203580 (n = 9 measurements per group, means ± SD shown, unpaired t test for each patient, *P < 0.05, **P < 0.01). (C) Representative images of VICs treated with BMP-6, CXCL9, and IFN-γ in the absence or presence of 20 μM SB203580. Stains: green, α-SMA; magenta, cytoplasm; blue, nuclei. Scale bars, 100 μm. (D) VIC activation in BMP-6, CXCL9, and IFN-γ in the absence or presence of SB203580 (n = 9 measurements per group, means ± SD shown, two-way ANOVA with Tukey posttests, *P < 0.05, ****P < 0.001).

  • Fig. 5 Post-TAVR serum deactivates valvular myofibroblasts activated with pre-TAVR serum on soft and stiff hydrogels.

    (A) Schematic of myofibroblast deactivation experiments (created with BioRender). (B) Representative images and (C) percentage of activated porcine VICs on soft hydrogels treated initially with pre-TAVR serum and subsequently with either pre-TAVR or post-TAVR serum. (D) Fold change of VIC deactivation on soft hydrogels in post-TAVR serum (XPost values) normalized to the mean activation in pre-TAVR serum from the same patient (μPre). (E) Scatter plot of fold change in VIC deactivation on soft hydrogels versus aortic valve area measurements. (F) Representative images and (G) percentage of activated VICs on stiff hydrogels treated initially with pre-TAVR serum and subsequently with either pre-TAVR or post-TAVR serum. (H) Fold change of VIC deactivation on stiff hydrogels in post-TAVR serum (XPost values) normalized to the mean activation in pre-TAVR serum from the same patient (μPre). (I) Scatter plot of fold change in VIC deactivation on stiff hydrogels versus aortic valve area measurements. Stained images: green, α-SMA; magenta, cytoplasm; blue, nuclei. Scale bars, 100 μm. For all bar graphs, n = 9 measurements per group, means ± SD shown, and significance tested with one-way ANOVA for all data and indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, or groups with different letters indicate statistical significance (P < 0.05).

  • Fig. 6 Post-TAVR serum deactivates cardiac myofibroblasts activated with pre-TAVR serum on soft and stiff hydrogels.

    (A) Representative images and (B) percentage of activated ARVFs on soft hydrogels treated initially with pre-TAVR serum and subsequently with either pre-TAVR or post-TAVR serum. (C) Fold change of ARVF deactivation on soft hydrogels in post-TAVR serum (XPost values) normalized to the mean activation in pre-TAVR serum from the same patient (μPre). (D) Representative images and (E) percentage of activated ARVFs on stiff hydrogels treated initially with pre-TAVR serum and subsequently with either pre-TAVR or post-TAVR serum. (F) Fold change of ARVF deactivation on stiff hydrogels in post-TAVR serum (XPost values) normalized to the mean activation in pre-TAVR serum from the same patient (μPre). (G and H) Scatter plots of fold change in ARVF deactivation on (G) soft and (H) stiff hydrogels versus patient STS scores. (I and J) Scatter plots of patient fold change in ARVF deactivation on soft hydrogels versus (I) LVIDs and (J) LVIDd measurements. (K and L) Scatter plots of fold change in ARVF deactivation on stiff hydrogels versus patient (K) LVIDs and (L) LVIDd measurements. Stained images: green, α-SMA; yellow, cytoplasm; blue, nuclei. Scale bars, 100 μm. For all bar graphs, n = 9 measurements per group, means ± SD shown, and significance tested with one-way ANOVA for all data and indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, or groups with different letters indicate statistical significance (P < 0.05).

  • Fig. 7 Inflammatory macrophage factors identified in post-TAVR serum mediate myofibroblast deactivation on stiff hydrogels.

    (A) Schematic of myofibroblast deactivation experiments with IL-1β or TNF-α (created with BioRender). (B) Representative images and (C) percentage of activated porcine VICs treated with pre-TAVR serum (n = 4 different patients), followed by treatments with control media, IL-1β (10 ng/ml), or TNF-α (10 ng/ml). (D and E) Patient-specific fold changes in (D) IL-1β– or (E) TNF-α–mediated deactivation in VICs initially activated with pre-TAVR serum (n = 4 patients). (F) Representative images and (G) percentage of activated VICs treated with proinflammatory M1 macrophage conditioned media at varying dilutions. (H) IL-1β and TNF-α concentrations in control and M1 macrophage conditioned media measured with ELISA. Stained images: green, α-SMA; magenta, cytoplasm; blue, nuclei. Scale bars, 100 μm. For all bar graphs, n = 9 measurements per group, means ± SD shown, and significance tested with one-way ANOVA for all data and indicated as *P < 0.05, **P < 0.01, ****P < 0.0001, or groups with different letters indicate statistical significance (P < 0.05).

  • Fig. 8 VIC and ARVF response to pre-TAVR and post-TAVR sera from male and female patients with AVS.

    (A) Porcine VIC activation on soft hydrogels in the presence of female and male pre-TAVR and post-TAVR sera. (B) Fold change of VIC activation on soft hydrogels in post-TAVR serum (XPost values) normalized to the mean activation in pre-TAVR serum from female and male patients (μPre). (C) Total number of differentially abundant proteins in pre-TAVR serum relative to post-TAVR serum for male and female patients (paired t test, P < 0.05). (D) Total number of differentially expressed genes in VICs treated with pre-TAVR serum relative to post-TAVR serum from male and female patients (quantile-adjusted conditional maximum likelihood method in edgeR, FDR-adjusted P < 0.05). (E) VIC deactivation and (F) deactivation fold change on soft hydrogels in the presence of female and male post-TAVR serum. (G) ARVF deactivation and (H) deactivation fold change on soft hydrogels in the presence of female and male post-TAVR serum. (I) VIC deactivation and (J) deactivation fold change on stiff hydrogels in the presence of female and male post-TAVR serum. (K) ARVF deactivation and (L) deactivation fold change on stiff hydrogels in the presence of female and male post-TAVR serum. For all bar graphs, n = 36 measurements were pooled together per group, means ± SD shown, with significance tested using unpaired t tests or one-way ANOVA and indicated as *P < 0.05 and **P < 0.01.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/509/eaav3233/DC1

    Materials and Methods

    Fig. S1. VIC activation on TCPS.

    Fig. S2. Materials characterization of PEG hydrogels.

    Fig. S3. VIC activation on soft hydrogels in healthy human serum.

    Fig. S4. Individual factors identified in pre-TAVR serum mediate myofibroblast activation.

    Fig. S5. Inhibition of p38 MAPK in VICs cultured on TCPS.

    Fig. S6. Myofibroblast deactivation on TCPS.

    Fig. S7. VIC activation on soft and stiff hydrogels.

    Fig. S8. ARVF activation in control FBS and healthy human serum on soft and stiff hydrogels.

    Fig. S9. Correlation between ARVF and VIC deactivation fold changes on soft hydrogels.

    Fig. S10. Validation of post-TAVR serum factors mediating VIC deactivation on stiff hydrogels.

    Fig. S11. Serum from male and female patients with AVS is altered after a TAVR procedure.

    Data file S1 contains the following supplementary tables:

    Table S1. Patient information.

    Table S2. SOMAscan proteomic results.

    Table S3. GO analysis for pre-TAVR serum factors.

    Table S4. GO analysis for post-TAVR serum factors.

    Table S5. Transcriptomics results for VICs treated with pre-TAVR versus post-TAVR serum.

    Table S6. KEGG enrichment for up-regulated genes in pre-TAVR samples.

    Table S7. KEGG enrichment for down-regulated genes in pre-TAVR samples.

    Table S8. Sex-specific analysis of proteomic data.

    Table S9. Sex-specific analysis of transcriptomic data.

    Data file S2. Individual subject-level raw values for all experiments (Excel file).

    Reference (73)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. VIC activation on TCPS.
    • Fig. S2. Materials characterization of PEG hydrogels.
    • Fig. S3. VIC activation on soft hydrogels in healthy human serum.
    • Fig. S4. Individual factors identified in pre-TAVR serum mediate myofibroblast activation.
    • Fig. S5. Inhibition of p38 MAPK in VICs cultured on TCPS.
    • Fig. S6. Myofibroblast deactivation on TCPS.
    • Fig. S7. VIC activation on soft and stiff hydrogels.
    • Fig. S8. ARVF activation in control FBS and healthy human serum on soft and stiff hydrogels.
    • Fig. S9. Correlation between ARVF and VIC deactivation fold changes on soft hydrogels.
    • Fig. S10. Validation of post-TAVR serum factors mediating VIC deactivation on stiff hydrogels.
    • Fig. S11. Serum from male and female patients with AVS is altered after a TAVR procedure.
    • Legends for tables S1 to S9
    • Legend for data file S2
    • Reference (73)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 contains the following supplementary tables:
    • Table S1 (Microsoft Excel format). Patient information.
    • Table S2 (Microsoft Excel format). SOMAscan proteomic results.
    • Table S3 (Microsoft Excel format). GO analysis for pre-TAVR serum factors.
    • Table S4 (Microsoft Excel format). GO analysis for post-TAVR serum factors.
    • Table S5 (Microsoft Excel format). Transcriptomics results for VICs treated with pre-TAVR versus post-TAVR serum.
    • Table S6 (Microsoft Excel format). KEGG enrichment for up-regulated genes in pre-TAVR samples.
    • Table S7 (Microsoft Excel format). KEGG enrichment for down-regulated genes in pre-TAVR samples.
    • Table S8 (Microsoft Excel format). Sex-specific analysis of proteomic data.
    • Table S9 (Microsoft Excel format). Sex-specific analysis of transcriptomic data.
    • Data file S2 (Microsoft Excel format). Individual subject-level raw values for all experiments.

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