Interstitial cells in calcified aortic valves have reduced differentiation potential and stem cell-like properties

Figure 1. (a) Microscopic visualization (10 x objective) of calcification by Alizarin Red staining of interstitial cells isolated from healthy (n = 7) and calcified (n = 7) aortic valves and cultured for 21 days in standard growth medium (control) or osteogenic medium, as indicated. (b) Quantification of Alizarin Red staining by absorbance at 405 nm. Groups were compared by Wilcoxon matched-pairs signed rank test (control vs osteogenic medium+) or Kolmogorov-Smirnov test (healthy vs calcified). Lines in scatter plots represent the median.

Figure 2. Relative gene expression, as measured by quantitative reverse transcription PCR, of calcification- and myofibroblast-related genes: (a) ACTA2 (alpha–smooth muscle actin 2), (b) CNN1 (calponin), (c) TAGLN (transgelin), (d) BMP2 (bone morphogenetic protein 2), (e) OPG (osteoprotegrin), (f) POSTN (periostin) and (g) TSP-1 (thrombospondin 1) in interstitial cells isolated from healthy (n = 6–7) or calcified (n = 5–7) aortic valves and cultured for 21 days in standard growth medium (control) or osteogenic medium. Groups were compared by Student’s t-test (parametric) or Wilcoxon matched-pairs signed rank test (non-parametric) for paired data (control vs osteogenic medium+) and unpaired Student’s t-test (parametric) or Mann-Whitney test (non-parametric) for unpaired data (healthy vs calcified). Lines in scatter plots represent the median.

Figure 3. Relative gene expression, as measured by quantitative reverse transcription PCR, of calcification- and myofibroblast-related genes: (a) ACTA2 (alpha–smooth muscle actin 2), (b) CNN1 (calponin), (c) TAGLN (transgelin), (d) BMP2 (bone morphogenetic protein 2), (e) OPG (osteoprotegrin), (f) POSTN (periostin) and (g) TSP-1 (thrombospondin 1) in interstitial cells isolated from healthy (n = 5–7) or calcified (n = 5–7) aortic valves and cultured for 4 and 14 days in standard growth medium (control) or myofibroblastic medium. Groups were compared by Student’s t-test (parametric) or Wilcoxon matched-pairs signed rank test (non-parametric) for paired data (control vs myofibroblastic medium+) and unpaired Student’s t-test (parametric) or Mann-Whitney test (non-parametric) for unpaired data (healthy vs calcified). Lines in scatter plots represent the median.

Figure 4. (a) Representative pictures (10 x objective) of immunofluorescence staining for myofibroblastic markers: alpha-smooth muscle actin (αSMA) and calponin in interstitial cells isolated from healthy (n = 6) and calcified (n = 5) aortic valves and cultured for 14 days in standard growth medium (control) or myofibroblastic medium. αSMA (red), calponin (green), cell nuclei (Hoechst 3342/blue). (b–e) Flow cytometry analysis of αSMA and calponin expression in interstitial cells isolated from healthy (n = 6) or calcified (n = 5) aortic valves and cultured for 4 or 14 days with standard medium (control) or myofibroblastic medium. (b,c) Data are presented as normalized mean fluorescence intensity (nMFI). Groups were compared by Student’s t-test for paired data (control vs myofibroblastic medium+) and unpaired Student’s t-test for unpaired data (healthy vs calcified). Lines in scatter plots represent the median. (d,e) Data are presented as histogram overlays of individual samples from healthy (green curves) or calcified aortic valves (red curves). (f) Comparison of collagen gel contraction between interstitial cells isolated from healthy (n = 4) and calcified (n = 4) aortic valves cultured in…

Figure 5. Relative gene expression, as measured by quantitative reverse transcription PCR, of chondrogenic markers: (a) ACAN (aggrecan), (b) COL2A1 (collagen type II alpha 1 chain), in interstitial cells isolated from healthy (n = 6) and calcified (n = 6) aortic valves and cultured for 21 days in standard growth medium (control) or chondrogenic medium. Groups were compared by Wilcoxon matched-pairs signed rank test for paired data (control vs chondrogenic medium+) and Mann-Whitney test for unpaired data (healthy vs calcified). Lines in scatter plots represent the median.

Figure 6. (a) Microscopic visualization (20 x objective) of lipid accumulation by Oil Red O staining of interstitial cells isolated from healthy (n = 6) and calcified (n = 6) aortic valves and cultured for 21 days in standard growth medium (control) or adipogenic medium, as indicated. (b) Quantification of Oil Red O staining by absorbance at 490 nm. Groups were compared by Wilcoxon matched-pairs signed rank test (control vs adipogenic medium+) or Mann-Whitney test (healthy vs calcified). Lines in scatter plots represent the median. (c-f) Relative gene expression, as measured by quantitative reverse transcription PCR, of adipogenic markers: (c) PPARG (peroxisome proliferator-activated receptor gamma), (d) CFD (complement factor D), (e) LPL (lipoprotein lipase) and (f) CEBPA (CCAAT enhancer binding protein alpha) in interstitial cells isolated from healthy (n = 6) and calcified (n = 5–6) aortic valves and cultured for 4 and 21 days in standard growth medium (control) or adipogenic medium. Groups were compared by Student’s t-test (parametric) or Wilcoxon matched-pairs signed rank test (non-parametric) for paired data (control vs adipogenic medium+) and unpaired Student’s t-test (parametric)…

Figure 7. (a,b) Flow cytometry analysis of surface expression of the mesenchymal stem cell-associated marker CD106 in interstitial cells isolated from healthy (n = 5) or calcified (n = 5) aortic valves. (a) The relative percentage of CD106bright cells from healthy or calcified valves are shown as a scatter plot. Groups were compared by Student’s t-test. Lines in scatter plot represent the median. (b) Histogram overlays of individual samples from healthy (green curves) or calcified (red curves) aortic valves. (c,d) Integral aldehyde dehydrogenase (ALDH) activity in interstitial cells isolated from healthy (n = 5) and calcified (n = 5) aortic valves analyzed by flow cytometry following single-cell staining using an enzymatic fluorescent assay. (c) The relative percentage of ALDHbright cells from healthy or calcified valves are shown as a scatter plot. Groups were compared by Student’s t-test. Lines in scatter plot represent the median. (d) Representative dot plots showing populations of interstitial cells isolated from healthy and calcified valves that are determined as ALDHdim (in red) and ALDHbright (in green).

Figure 8. Proliferation of interstitial cells isolated from healthy (n = 5) and calcified (n = 5) aortic valves measured as mean number of cells in triplicates on 0, 2, 4 and 6 days of culturing in growth medium. Data were analyzed by two-way ANOVA with repeated measures. Differences between healthy and calcified donors were determined with Sidak’s multiple comparison post-test and significant p-value is shown on 6th day of measurement. Values are expressed as mean ± SD. Overall p-value from two-way ANOVA is shown in bold.