Hypoxic Culture Maintains Cell Growth of the Primary Human Valve Interstitial Cells with Stemness

Figures

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  Figure 1. VICs exhibit increased growth under hypoxia. (a) Schematic of experimental strategy for RNA-sequencing. (b) Phase-contrast micrographs of VICs cultured under normoxia and 2% hypoxia at 0, 7, 14, and 28 d. Bar = 100 μm. (c) Quantifications of proliferation in hypo- and normo-VICs. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

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  Figure 2. Gene expression in hypo- and normo-VICs. (a) Scatter plots of RNA-sequencing data (log10 TMM) at 7, 14, and 28 d. (b) Graphical summaries of IPA pathway analysis at 7, 14, and 28 d. Genes significantly upregulated in normo-VICs and hypo-VICs are denoted in blue and orange, respectively.

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  Figure 3. Expression of oxidative stress and cell-cycle regulators in normo- and hypo-VICs. (a) Top 30 candidates from canonical pathways (ratio and significance) from IPA analysis. Red arrowheads indicate canonical pathways pertaining to redox. (b) IPA pathway maps for glutathione redox reactions I and superoxide radical degradation. The rhombus colored in green denotes genes downregulated in hypo-VICs relative to normo-VICs. (c) Heatmap of expression of oxidative stress- and cell cycle-related genes from RNA-sequencing data (TMM). (d) Western blot detection of markers for oxidative stress and cell-cycle regulation. (e) Fluorescence micrographs of ROS. Bar = 100 μm (left panel). ROS quantification (right panel). ** p < 0.01.

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  Figure 4. Expression of mesenchymal and hematopoietic stem cell markers in hypo- and normo-VICs. (a) Quantifications of mRNA levels for THY1, CD34, Oct4, CD29, ENG, VCAM1, CD44, MCAM, and ALCAM (n = 3). Expression levels were normalized to those of GAPDH. Data are presented as means ± SE. (b). Validation of CD29, VCAM1, and CD34 upregulation in hypo-VICs by western blotting. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. n.s.: Not significant.

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  Figure 5. Differentiation of hypo- and normo-VICs into osteoblasts. (a) Photographs of DMEM- and osteogenic medium (OGM)-treated normo- and hypo-VICs stained by Alizarin red. (b) Calcium content quantified using cetylpyridinium chloride; absorbance was measured at 560 nm. Data are represented as means ± SE. (c) Western blot detection of RUNX2 in DMEM- and OGM-treated normo- and hypo-VICs (left panels). Beta-actin expression was used for normalization. RUNX2 protein expression in DMEM- and OGM-treated normo- and hypo-VICs (right panels). (d) Quantitative PCR data for RUNX2 mRNA expression in DMEM- and OGM-treated normo- and hypo-VICs. ** p < 0.01, *** p < 0.001, **** p < 0.0001. n.s.: Not significant.

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  Figure 6. Expression of the oxidative stress marker SOD2 is increased in VICs from calcified AS tissues (cVICs). (a) 2DGE from lysates of cultured cVICs and ncVICs from patient 1. (b) Left panels, enlargements of the region surrounded by broken lines in (a) (patient 1). Middle and right panels show the same regions enlarged from 2DGE images of patients 2 and 3. (c) Immunohistochemical detection of SOD2 in AS and normal valves. Lower panels show enlarged views of areas 1–3 (boxes in upper panels). (d) Quantification of SOD2-stained areas; data are represented as means ± SE (n = 6). ** p < 0.01.