Osteopontin activity modulates sex‐specific calcification in engineered valve tissue mimics

Figures

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  FIGURE 1. Human valve tissue from patients with aortic valve disease shows potential sex differences in size and distribution of calcification. (a) A representative image of a histological section from a female diseased valve stained with Von Kossa (VK) shows small, positive punctae of VK staining. (b) A representative image of a male histological section of diseased valve shows large regions of positive VK staining. (c) Normalized integrated density of total positive VK staining between female and male tissue sections. (d) Radial autocorrelation profiles of positive VK staining in human diseased valve tissue, and (e) correlation length analysis per patient sample. Female data are presented in red, males in blue. For patient‐specific details, refer to Supporting Information Material S1 (n = 3 females, 2 males: seven images per patient sample minimum; *p < 0.05 as determined via one‐way ANOVA; scale bar: 100 μm).

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  FIGURE 2. Female human diseased valve tissue sections have increased COL1A and OPN staining relative to male tissue sections. A representative image of RUNX2 (red) and nuclei (blue) staining in female (a) and male (b) diseased valve tissue. (c) Quantification of RUNX2 mean fluorescence intensity indicates no significant difference in intensity between female and male samples. (d) Representative image of a female diseased valve tissue section stained for COL1A1 (green) and nuclei (blue), relative to male tissue (e). (f) Quantification of COL1A1 mean fluorescence intensity indicated a significant increase in staining in female valve tissue relative to male. (g) Representative image of female diseased valve section stained for OPN (red) and nuclei (blue) relative to male tissue (h). (i) Quantification of the OPN mean fluorescence intensity shows increased OPN staining in the female valve tissue sections compared to male (n = 3 females, 2 males; 10 images per patient sample minimum. *p < 0.05 as determined via unpaired t test; scale bar: 100 μm).

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  FIGURE 3. VICs encapsulated within 3D PEG‐based hydrogels preserve increased OPN observed in female human tissue. (a) Experimental schematic shows sex‐separated VICs encapsulated within degradable hydrogels synthesized from PEG + collagen type I (Col) IPN and exposed to osteogenic medium (OM; 10 mM βGP, 50 mg/ml ASC, and 1 mM DEX) for 15 days. (b) RUNX2 mRNA expression of female and male VICs, relative to L30 control. (c) OPN mRNA expression increased in female VICs relative to males, relative to L30 control (n = 3 biological and technical replicates for qPCR analysis). (d) Full‐length OPN protein (66 kDa) expression from encapsulated VICs in female and male samples (n = 3 biological replicates run in same blot). (e) Representative western blot lanes for OPN (66 kDa) and normalization protein (HIS3, 17 kDa) for female and male samples. (f) Representative image of a histological section from female (left) sample stained with Von Kossa (VK), relative to stained male section (right), which displayed no discrete positive staining punctae in either sex. (g) Quantification of normalized total integrated density of positive VK staining between female and male sectioned samples shows no…

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  FIGURE 4. PEG + Col hydrogels in calcifying medium show reduced calcification in female samples. (a) Experimental schematic shows female or male porcine VICs encapsulated within degradable hydrogels synthesized from PEG + Col and exposed to calcifying medium (CM, OM [osteogenic medium] + 1 mg/ml CaCl2) for 12 days. (b) Representative images of histological sections stained with Von Kossa (VK) for female and male VICs within the PEG + Col hydrogel in CM relative to OM. Females (top) displayed small punctae of positive VK staining with CM, where males (bottom) showed large areas of positive punctae in samples treated with CM. (c) Integrated density of VK staining for female and male encapsulated VICs in OM or CM after 12 days in culture. (d) Correlation length analysis of VK staining in individual female and male samples treated for 12 days in OM, or CM. (e) Sex‐averaged correlation lengths (*p < 0.05, ***p < 0.001 as determined via one‐way ANOVA, scale bar = 100 μm)

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  FIGURE 5. OPN distribution shifts from the ECM to nuclear regions in encapsulated female VICs cultured in calcifying medium. (a) Representative histological sections with immunofluorescent (IF) staining for OPN (red) and nuclei (blue) for female (top) and male (bottom) conditions, in OM (osteogenic medium) or CM (calcifying medium). (b) Quantification of OPN mean fluorescence intensity indicates no significant differences in OPN intensity in calcified samples of either sex. (c) OPN fluorescence intensity was measured as a function of nuclear distance. Results indicate a significant increase in the cell:ECM ratio for OPN signal in female samples treated with CM (*p < 0.05 as determined with one‐way ANOVA, scale bar: 100 μm).

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  FIGURE 6. ET‐1 treatment reduces calcification while increasing OPN gene expression. (a) Experimental schematic shows female or male porcine VICs encapsulated within degradable hydrogels synthesized from PEG + Col and exposed to calcifying medium (CM, OM [osteogenic medium] + 1 mg/ml CaCl2) supplemented with exogenous ET‐1. (b) Representative images of histological sections stained with Von Kossa (VK) for control (CM only) versus ET‐1 (CM + 100 nM ET‐1) treated female or male samples after 12 days in culture. (c) Quantification of VK staining for female or male histological sections of samples cultured for 12 days in CM + indicated concentration of ET‐1 (n = 2 biological replicates minimum, >14 FOV per replicate). (d) OPN gene expression of female or male samples in CM + ET‐1 for 3 days, relative to L30 control (n = 3 biological and technical replicates). (e) Quantification of OPN cell:ECM ratio for female or male histological sections stained for OPN after 12 days in culture in CM + indicated concentration of ET‐1 (n = 3 biological replicates, >5 images per replicate) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; scale bar = 100 μm).