ApoC-III is a novel inducer of calcification in human aortic valves

Figures

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  Figure 1. Specific apolipoproteins predominate in calcific aortic valve disease (CAVD) tissues.A, hierarchical cluster analysis (multigroup comparison; q ≤ 0.3) of the CAVD disease stage proteome (n = 9 donors; N = 1238 proteins with three or more unique peptides). Inset, is a zoomed view of apolipoproteins enrichment in CAVD tissue. B, sum-normalized apolipoprotein abundances across CAVD stages from the global proteomics data (upper panel; n = 9 donors) and from the parallel-reaction monitoring data (lower panel; n = 5 donors; averaged sum-normalized signal of one to three peptides per donor). C, fold change of gene expression of apolipoproteins between fibrotic and calcific or nonfibrotic/noncalcific and CAVD tissues from RNA-Seq. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA with Bonferroni multiple comparison test or Kruskal–Wallis test with Dunn's multiple comparison test for nonparametric data. C, calcific tissue; F, fibrotic tissue; N, nonfibrotic/noncalcific tissue.

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  Figure 2. Aortic valve apolipoprotein localization and association with calcification. Negative control 1 and 2: staining without primary antibody, but horseradish peroxidase–conjugated antimouse and anti-rabbit secondary antibodies; representative immunohistochemistry images for apolipoproteins on human aortic valve tissue in the fibrosa layer (top panel, fibrosa facing up), around calcification (middle panel), and on nondiseased controls. The scale bar represents 100 μm.

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  Figure 3. Tissue apolipoprotein distribution.A, coimmunofluorescence for apoJ and vimentin (left panel) and apoJ and αSMA (right panel). B, coimmunofluorescence for apoD and CD68; yellow arrows indicate double-positive cells. The scale bar represents 100 μm. Observations were confirmed in three independent donors. C, immunofluorescence for apoC-III, vimentin (valvular interstitial cells), and calcification (Osteosense 680; near-infrared calcium tracer [Perkin Elmer, USA]) on fibrotic and calcific human aortic valve sections. D, oil red O staining of calcific human aortic valve tissue and apoC-III immunohistochemistry on serial sections identifying areas of lipid-poor apoC-III. αSMA, α-smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole.

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  Figure 4. ApoC-III is a contributor to valvular interstitial cell calcification.A, representative alizarin red staining of valvular interstitial cells incubated with normal media (NM), procalcifying media (PM), PM with 100 μg/ml human apoC-III (PM + apoC-III), and PM with 100 μg/ml human apoA-I (PM + apoA-I) at day 7 after first incubation; and alizarin red absorbance quantification (n = 4 independent donors, all passage 2). B, representative alizarin red staining of valvular interstitial cells incubated with NM, PM, PM + apoC-III, and PM + apoA-I at day 14 after first incubation; and alizarin red absorbance quantification (n = 4 independent donors, all passage 2).

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  Figure 5. ApoC-III drives mitochondrial dysfunction and oxidative stress.A, principal component analysis of 2773 proteins with ≥2 unique peptides. B, heat map of 1365 proteins with q ≤ 0.1 between normal medium (NM), procalcifying medium (PM), and PM + apoC-III. Red box highlights enriched proteins in PM + apoC-III versus NM and PM. C–D, apoC-III and superoxide dismutase 2 (SOD-2) are significantly enriched in PM + apoC-III quantified by MS, respectively. E, differentially enriched proteins in NM versus PM + apoC-III. F, differentially enriched proteins in PM versus PM + apoC-III. n = 4 donors, day 7 and day 14 data.

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  Figure 6. ApoC-III may act through mitochondrial stress–mediated inflammation.A, proteins with q ≤ 0.1 between normal medium (NM), procalcifying medium (PM), and PM + apoC-III increased in PM + apoC-III versus NM with proteins of major pathways highlighted (n = 4 donors, day 7 and day 14 data). B, proteins with q ≤ 0.1 between NM, PM, and PM + apoC-III increased in PM + apoC-III versus PM with proteins of major pathways highlighted (n = 4 donors, day 7 and day 14 data). C, ApoC-III incubation increased interleukin-6 (IL-6) mRNA expression (n = 5 independent donors, apoC-III or apoA-I incubation for 48 h). D, bone morphogenetic protein 2 (BMP-2) mRNA was highly expressed in valvular interstitial cells incubated with 100 μg/ml apoC-III (n = 5 independent donors, apoC-III or apoA-I incubation for 48 h). Data are expressed as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA with Bonferroni multiple comparison test or Kruskal–Wallis test with Dunn's correction. E, potential role of ApoC-III in valvular calcification. ApoC-III induces mitochondrial dysfunction–driven inflammation that promotes calcification-associated processes.