Sex-Specific Response to Combinations of Shear Stress and Substrate Stiffness by Endothelial Cells In Vitro

Abstract

By using a full factorial design of experiment, the combinatorial effects of biological sex, shear stress, and substrate stiffness on human umbilical vein endothelial cell (HUVEC) spreading and Yes-associated protein 1 (YAP1) activity are able to be efficiently evaluated. Within the range of shear stress (0.5-1.5 Pa) and substrate stiffness (10-100 kPa), male HUVECs are smaller than female HUVECs. Only with sufficient mechanical stimulation do they spread to a similar size. More importantly, YAP1 nuclear localization in female HUVECs is invariant to mechanical stimulation within the range of tested conditions whereas for male HUVECs it increases nonlinearly with increasing shear stress and substrate stiffness. The sex-specific response of HUVECs to combinations of shear stress and substrate stiffness reinforces the need to include sex as a biological variable and multiple mechanical stimuli in experiments, informs the design of precision biomaterials, and offers insight for understanding cardiovascular disease sexual dimorphisms. Moreover, here it is illustrated that different complex mechanical microenvironments can lead to sex-specific phenotypes and sex invariant phenotypes in cultured endothelial cells.

Keywords: cell-material interaction; mechanobiology; precision medicine; sex differences.

Figure 1.

Visual representation of the experimental design. Corner points in black. Center points in red.

Figure 2.

Representative fluorescent images of cytoskeletal F-actin staining at each factor-level combination. Scale bar 50 μm.

Figure 3.

Representative confocal fluorescent images of YAP1 immunostaining at each factor-level combination. Scale bar 50 μm.

Figure 4.

Violin plots for each factor-level combination of A) cell area, B) nuclear area, and C) YAP1 nuclear-to-cytoplasm ratio. The large dashed lines indicate the median value and the small dashed lines indicate the 25th and 75th quartile values. Plots for male HUVECs are shown in blue (left of pairs) and plots for female HUVECs are shown in red (right of pairs).

Figure 5.

Surface plots of the median A) cell area, D) nuclear area, and G) YAP1 nuclear-to-cytoplasm ratio for male HUVECs at each factor-level combination of shear stress and substrate stiffness. Surface plots of the median B) cell area, E) nuclear area, and H) YAP1 nuclear-to-cytoplasm ratio for female HUVECs at each factor-level combination of shear stress and substrate stiffness. The colormap indicates regions of high (yellow), mid (green), and low (blue) values. Comparison surface plots combining the male and female plots together for median C) cell area, F) nuclear area, and I) YAP1 nuclear-to-cytoplasm ratio. Female plots are colored red and male plots are colored blue.

Figure 6.

A,B,G) Pairwise comparisons of cell area, B,E,H) nuclear area, and C,F,I) YAP1 nuclear-to-cytoplasm ratio between the high and low levels for each factor. Each response was transformed by taking its natural logarithm to satisfy normality conditions for evaluation by three-way ANOVA and post-hoc Šidák multiple comparison tests. Comparisons were made without the center points. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns p ≥ 0.05. Data presented as mean ± standard deviation of the natural logarithmically transformed response.

Figure 7.

Main effect plots. Plots of the median show the individual influence of A) sex, B) shear stress, and C) stiffness on cell area. Plots of the median show the individual influence of D) sex, E) shear stress, and F) stiffness on nuclear area. Plots of the median show the individual influence of G) sex, H) shear stress, and I) stiffness on YAP1 nuclear-to-cytoplasm ratio. Each factor had a statistically significant effect (p < 0.0001) except shear stress on nuclear area as determined from the ANOVA table for the model fit when transformed according to Table S4 in the Supporting Information. Center points (in red) can indicate nonlinearity in the response to the different factors if they are far away from the trend line between corner points and can indicate linearity if they are near the trend line between corner points.

Figure 8.

Interaction effect plots. Plots of the median show the interactive influence of A) sex and shear stress), B) sex and stiffness, and C) shear stress and stiffness on cell area. Plots of the median show the interactive influence of D) sex and shear stress, E) sex and stiffness, and F) shear stress and stiffness on nuclear area. Plots of the median show the interactive influence of G) sex and shear stress, H) sex and stiffness, and I) shear stress and stiffness on YAP1 nuclear-to-cytoplasm ratio. Each factor combination had a statistically significant effect (p < 0.0001) as calculated from the ANOVA table for the model fit when transformed according to Table S4 in the Supporting Information. Center points (in red) can indicate nonlinearity in the response to the different factors if they are far away from the trend line between corner points and can indicate linearity if they are near the trend line between corner points.

Figure 9.

Plot of the median YAP1 nuclear-to-cytoplasm ratio versus the median cell area for each sex. Bubbles enclose the maximal interquartile area of the two measurables. The two male conditions clustering with the female conditions were those stimulated by the center point level of shear stress and substrate stiffness (1.0 Pa and 55 kPa) and the corner point with high levels for both shear stress and substrate stiffness (1.5 Pa and 100 kPa).