Embedding and Backscattered Scanning Electron Microscopy (EM-BSEM) Is Preferential over Immunophenotyping in Relation to Bioprosthetic Heart Valves

Figures

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  Figure 1. Histological examination of calcified AVs and SVD-affected BHVs. (A) AVs excised from the patients with CAS because of critical leaflet thickening and severe calcification. Aside from calcification, the fibrotic ECM retained its integrity. Macrophage spread was limited to areas with a degraded ECM around the calcium deposits (demarcated by a red contour; macrophages are indicated by black arrows), whilst the AV surface (demarcated by a green contour) was covered by VECs (indicated by black arrows) and the fibrotic ECM (demarcated by a blue contour) was populated by VICs (indicated by black arrows). The fibrosa layer (aortic, or outflow side) is on the top, whereas the ventricularis layer (ventricular, or inflow side) is at the bottom of the section; (B) In comparison with AVs, BHVs suffered from ECM disintegration and the fibres were loosened. Immune cells were located at the base of the leaflets and near the mineral deposits (demarcated by a red contour; immune cells are indicated by black arrows). Areas devoid of host cells (indicated by a green contour) alternated with cellular infiltrations associated with perforations and tears (indicated by a blue contour; immune cells are…

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  Figure 2. Side-by-side comparison of ultrastructural (EM-BSEM) and immunohistochemical analysis of calcified AVs and SVD-affected BHVs. (A) ECs had distinct appearance in AVs and BHVs: AVs (left images) were covered with a monolayer of VECs (indicated by red arrows) that had an elongated shape and a large nucleus oriented towards the flow, and were positive for the canonical EC marker CD31, whilst BHVs (right images) were lined with endothelial-like cells (indicated by blue arrows) that had a bigger size, lower nucleus-to-cytoplasm ratio, and contained noticeable inclusions, being also slightly positive for CD31. Note the concordance of ultrastructural and immunohistochemical analysis in the AVs and the detailed rather than vague phenotype of endothelial-like cells in the BHVs when examined by EM-BSEM, but not immunohistochemical staining. EM-BSEM: top images, immunohistochemical staining: bottom images; (B) Mesenchymal cells were abundant in the AVs (VICs, left images, indicated by red arrows) and were positive for α-SMA, but were rarely encountered in the BHVs, where they showed lower α-SMA expression (myofibroblasts, right images, indicated by blue arrows). EM-BSEM: top images,…

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  Figure 3. High-magnification ultrastructural analysis of calcified AVs and degenerated BHVs. (A) VECs at the surface of the AVs (left images) had an elongated shape and formed a continuous monolayer by contacting through the tight junctions, whilst endothelial-like cells lining the BHVs (right images) were associated with adhering monocytes and their layer was intermittent; (B) VICs within the AVs (left images) generally had a high nucleus-to-cytoplasm ratio and were devoid of any inclusions, whereas the sparse fibroblasts or myofibroblasts frequently had lipid droplets (indicated by red arrows) and digested ECM fragments; (C) Digesting activity of the macrophages was indicated by internalised and partitioned ECM fragments (indicated by red arrows) and by the lysed or loosened ECM surrounding such macrophages. Such phenomenon was infrequent in the AVs (left images) but common for the BHVs (right images). The right images within each group (demarcated by red contour, ×5000 magnification, scale bar: 10 μm) are close-ups of the corresponding left images (overview, ×1000 magnification, scale bar: 50 μm).

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  Figure 4. Immunofluorescence staining of AVs and BHVs with antibodies to EC (CD31 and VEGFR2), contractile (α-SMA and VIM), and immune-cell (CD45 and CD68) markers. (A) AVs, staining for CD31 (green) and VEGFR2 (red), and ECs (CD31+VEGFR2+ cells) are indicated by yellow arrows; (B) AVs, staining for α-SMA (green) and VIM (red), and VICs (α-SMA+VIM+ cells) are indicated by yellow arrows; (C) AVs, staining for CD68 (green) and CD45 (red), and CD45+CD68- cells are indicated by white arrows, and CD45+CD68+ cells are indicated by yellow arrows; (D) BHVs, staining for CD31 (green) and VEGFR2 (red), and CD31+VEGFR2- cells are indicated by white arrows; (E) BHVs, staining for α-SMA (green) and VIM (red), and α-SMA+VIM- cells indicated by white arrows; (F) BHVs, staining for CD68 (green) and CD45 (red), and CD45+CD68− and CD68+CD45− cells are indicated by white arrows, and CD45+CD68+ cells are indicated by yellow arrows. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue). Magnification: ×400, scale bar: 50 μm.

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  Figure 5. Immunofluorescence staining of AVs with antibodies to: (A) CD31 (green) and α-SMA (red), α-SMA+CD31− cells are indicated by white arrows, and CD31+α-SMA+ are indicated by yellow arrows; (B) CD31 (green) and CD45 (red), and CD31+CD45− and CD45+CD31− cells are indicated by white arrows; (C) CD68 (green) and CD31 (red), CD31+CD68− cells are indicated by white arrows, and CD31+CD68+ cells are indicated by yellow arrows; (D) α-SMA (green) and CD45 (red), and α-SMA+CD45− and CD45+α-SMA− are indicated by white arrows; (E) CD68 (green) and α-SMA (red), CD68+α-SMA− cells are indicated by white arrows, and CD68+α-SMA+ cells are indicated by yellow arrows. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Magnification: ×400, scale bar: 50 μm.

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  Figure 6. Immunofluorescence staining of BHVs with antibodies to: (A) CD31 (green) and α-SMA (red), α-SMA+CD31− cells are indicated by white arrows, and CD31+α-SMA+ are indicated by yellow arrows; (B) CD31 (green) and CD45 (red), CD31+CD45− and CD45+CD31− cells are indicated by white arrows, and CD31+CD45+ cells are indicated by yellow arrows; (C) CD68 (green) and CD31 (red), CD68+CD31− cells are indicated by white arrows, and CD68+CD31+ cells are indicated by yellow arrows; (D) α-SMA (green) and CD45 (red), α-SMA+CD45− and CD45+α-SMA− cells are indicated by white arrows, and α-SMA+CD45+ cells are indicated by yellow arrows; (E) CD68 (green) and α-SMA (red), α-SMA+CD68− cells are indicated by white arrows, and CD68+α-SMA+ cells are indicated by yellow arrows. Nuclei are counterstained with DAPI (blue). Magnification: ×400, scale bar: 50 μm.

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  Figure 7. Quantitative image analysis (count of positive cells) of immunofluorescence stainings performed in the AVs and BHVs. (A) CD31 and α-SMA staining, the proportions of CD31+α-SMA−, CD31−α-SMA+, and CD31+α-SMA+ cells on the images are represented by green dots, red squares, and black inverted triangles, respectively; (B) CD31 and CD45 staining, the proportions of CD31+CD45−, CD31−CD45+, and CD31+CD45+ cells on the images are represented by green dots, red squares, and black inverted triangles, respectively; (C) CD68 and CD31 staining, the proportions of CD68+CD31−, CD68−CD31+, and CD68+CD31+ cells on the images are represented by green dots, red squares, and black inverted triangles, respectively; (D) α-SMA and CD45 staining, the proportions of α-SMA+CD45−, α-SMA−CD45+, and α-SMA+CD45+ cells on the images are represented by green dots, red squares, and black inverted triangles, respectively; (E) CD68 and α-SMA staining, the proportions of CD68+α-SMA−, CD68−α-SMA+, and CD68+α-SMA+ cells on the images are represented by green dots, red squares, and black inverted triangles, respectively; Colours of the dots on each plot (green, red, and black) correspond to the antigen indicated in the…

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  Figure 8. Quantitative image analysis (calculation of the proportions of CD31+/CD45+, α-SMA+/CD45+, CD68+/CD31+, and CD68+/α-SMA+ to all CD45+ and CD68+ cells) of immunofluorescence stainings performed in the AVs and BHVs. (A) CD31/CD45 and α-SMA/CD45 stainings showing the proportions of CD31+CD45+ cells (left image) and α-SMA+CD45+ cells (right image) in AVs (red inverted triangles) and BHVs (blue inverted triangles) to all CD45+ cells; (B) CD31/CD68 and α-SMA/CD68 stainings showing the proportions of CD31+CD68+ cells (left image) and α-SMA+CD68+ cells (right image) in AVs (red inverted triangles) and BHVs (blue inverted triangles) to all CD68+ cells. Each inverted triangle on the plots represents one image (n = 9–10 measurements per group). Whiskers of the 25th–75th percentiles and centre lines indicate the median. The p values are provided above the plots, and statistically significant p values are marked by asterisks; Mann–Whitney U-test.