Tissue Derivation and Biological Sex Uniquely Mediate Endothelial Cell Protein Expression, Redox Status, and Nitric Oxide Synthesis

Figures

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  Figure 1. Expression of sex receptors in male and female endothelial cells. HAECs, HUVECs and HMVECs were cultured in starvation medium at ~80% confluency for 24 h with or without TNF-α (20 ng/mL). Protein expression of AR (A–F), ERα (J–O), and ERβ (P–U) were determined by Western blot. Quantification was performed using Image Lab (Bio-Rad Laboratories, Inc.). Protein was normalized to respective control bands (β-actin). Gene expression of AR (G–I) were determined with qPCR. Genes of interest were normalized to housekeeping gene cyclophilin. Data are expressed as mean ± SD from 3–6 experiments. Data were statistically analyzed with one-way ANOVA followed by Tukey post hoc multiple comparison analysis between groups. Values that do not share the same letter are significantly different from each other (p ≤ 0.05).

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  Figure 2. Expression of pathological RAS components in male and female endothelial cells. HAECs, HUVECs and HMVECs were cultured in starvation medium at ~80% confluency for 24 h with or without TNF-α (20 ng/mL). Protein expression of ACE1 (A–F) were determined by Western blot. Quantification was performed using Image Lab (Bio-Rad Laboratories, Inc.). Protein was normalized to respective control bands (β-actin or GAPDH). Gene expression of AT1R (G–I) were determined with qPCR. Genes of interest were normalized to housekeeping gene cyclophilin. Data are expressed as mean ± SD from 3–6 experiments. Data were statistically analyzed with one-way ANOVA followed by Tukey post hoc multiple comparison analysis between groups. Values that do not share the same letter are significantly different from each other (p ≤ 0.05).

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  Figure 3. Expression of protective RAS components in male and female endothelial cells. HAECs, HUVECs and HMVECs were cultured in starvation medium at ~80% confluency for 24 h with or without TNF-α (20 ng/mL). Protein expression of ACE2 (A–I) were determined by Western blot. Quantification was performed using Image Lab (Bio-Rad Laboratories, Inc.). Protein was normalized to respective control bands (β-actin or GAPDH). Gene expression of AT2R (J–L) and Mas receptor (M–O) were determined with qPCR. Genes of interest were normalized to housekeeping gene cyclophilin. Data are expressed as mean ± SD from 4–6 experiments. Data were statistically analyzed with one-way ANOVA followed by Tukey post hoc multiple comparison analysis between groups. Values that do not share the same letter are significantly different from each other (p ≤ 0.05).

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  Figure 4. Expression of pro-oxidant NOX isoforms in male and female endothelial cells. HAECs, HUVECs and HMVECs were cultured in starvation medium at ~80% confluency for 24 h with or without TNF-α (20 ng/mL). Protein expression of NOX2 (A–F), NOX4 (G–L) and NOX5 (M–R) were determined by Western blot. Quantification was performed using Image Lab (Bio-Rad Laboratories, Inc.). Protein was normalized to respective control bands (β-actin or GAPDH). Data are expressed as mean ± SD from 4–6 experiments. Data were statistically analyzed with one-way ANOVA followed by Tukey post hoc multiple comparison analysis between groups. Values that do not share the same letter are significantly different from each other (p ≤ 0.05).

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  Figure 5. Expression of SOD isoforms in male and female endothelial cells. HAECs, HUVECs and HMVECs were cultured in starvation medium at ~80% confluency for 24 h with or without TNF-α (20 ng/mL). Protein expression of SOD1 (A–F), SOD2 (G–L) and SOD3 (M–R) were determined by Western blot. Quantification was performed using Image Lab (Bio-Rad Laboratories, Inc.). Protein was normalized to respective control bands (β-actin or GAPDH). Data are expressed as mean ± SD from 4–6 experiments. Data were statistically analyzed with one-way ANOVA followed by Tukey post hoc multiple comparison analysis between groups. Values that do not share the same letter are significantly different from each other (p ≤ 0.05).

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  Figure 6. Expression of peroxidases in male and female endothelial cells. HAECs, HUVECs and HMVECs were cultured in starvation medium at ~80% confluency for 24 h with or without TNF-α (20 ng/mL). Protein expression of CAT (A–F) and GPx1 (G–L) were determined by Western blot. Quantification was performed using Image Lab (Bio-Rad Laboratories, Inc.). Protein was normalized to respective control bands (β-actin or GAPDH). Data are expressed as mean ± SD from 4–6 experiments. Data were statistically analyzed with one-way ANOVA followed by Tukey post hoc multiple comparison analysis between groups. Values that do not share the same letter are significantly different from each other (p ≤ 0.05).

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  Figure 7. Expression of endogenous antioxidant regulators in male and female endothelial cells. HAECs, HUVECs and HMVECs were cultured in starvation medium at ~80% confluency for 24 h with or without TNF-α (20 ng/mL). Protein expression of SIRT1 (A–F), NRF2 (G–L) and HO-1 (M–R) and NQO1 (S–X) were determined by Western blot. Quantification was performed using Image Lab (Bio-Rad Laboratories, Inc.). Protein was normalized to respective control bands (β-actin or GAPDH). Data are expressed as mean ± SD from 4–6 experiments. Data were statistically analyzed with one-way ANOVA followed by Tukey post hoc multiple comparison analysis between groups. Values that do not share the same letter are significantly different from each other (p ≤ 0.05).

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  Figure 8. Redox activity in male and female endothelial cells. HAECs, HUVECs and HMVECs were cultured at ~80% confluency for 24 h in starvation medium with or without TNF-α (20 ng/mL). H2DCFDA or DHE dissolved in dimethyl sulfoxide were added to wells (10 µM final concentration) followed by 30 min incubation (37 °C and 5% CO2). Phenol red-free starvation medium supplemented with NucBlue™ (1 drop/mL) was added. Fluorescence was read at the following Ex/Em: 495/527 (A–C; H2DCFDA), 518/606 (D–F; DHE; O2−) 480/576 (G–I; DHE; non-specific radicals) and 360/460 (Hoechst 33342 with NucBlue™). Fluorometric values for H2DCFDA and DHE were normalized to Hoechst 33342. Cells were also fixed on poly-L-lysine cover slips and stained with DAB diluent following incubation with nitrotyrosine primary antibody (J–O). Quantification was performed with Image J. The scale bar is 50 µm and objective magnification is 20×. Data are expressed as mean ± SD from 3–6 experiments. Data were statistically analyzed with one-way ANOVA followed by Tukey post hoc multiple comparison analysis between groups. Values that do not share the same letter are significantly different from each other (p ≤ 0.05).

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  Figure 9. Regulation of eNOS in male and female endothelial cells. HAECs, HUVECs and HMVECs were cultured in starvation medium at ~80% confluency for 24 h with or without TNF-α (20 ng/mL). Protein expression of t-eNOS (A–F), p-akt (G–L), p-eNOSSer1177 (M–R) and p-eNOSThr495 (S–X) were determined by Western blot. Quantification was performed using Image Lab (Bio-Rad Laboratories, Inc.). Band expression of t-eNOS and t-akt were normalized to respective control bands (β-actin or GAPDH). For phosphorylated proteins that were not probed on the same membrane as their respective total proteins, they were first normalized to control proteins (β-actin or GAPDH) followed by normalization with their respective normalized total proteins from a separate membrane. Data are expressed as mean ± SD from 4–6 experiments. Data were statistically analyzed with one-way ANOVA followed by Tukey post hoc multiple comparison analysis between groups. Values that do not share the same letter are significantly different from each other (p ≤ 0.05).

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  Figure 10. Nitric oxide bioavailability in male and female endothelial cells. HAECs, HUVECs and HMVECs were cultured in starvation medium at ~80% confluency for 24 h with or without TNF-α (20 ng/mL). DAF-2DA was detected by fluorescence microscopy (A,C,E) at 485/528 (Ex/Em). Fluorescent intensity of cells was quantified with Image J (B,D,F). Data are expressed as mean ± SD from 4–6 experiments. Data were statistically analyzed with one-way ANOVA followed by Tukey post hoc multiple comparison analysis between groups. Values that do not share the same letter are significantly different from each other (p ≤ 0.05).

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  Figure 11. Expression of inflammatory markers in male and female endothelial cells. HAECs, HUVECs and HMVECs were cultured in starvation medium at ~80% confluency for 24 h with or without TNF-α (20 ng/mL). Protein expression of phospho-p65 (A–F) and iNOS (J–O) were determined by Western blot. Quantification was performed using Image Lab (Bio-Rad Laboratories, Inc.). Protein was normalized to respective control bands (β-actin). Phosphorylated proteins were normalized to respective total proteins. Gene expression of MCP-1 (G–I) were determined with qPCR. Genes of interest were normalized to housekeeping gene cyclophilin. Data are expressed as mean ± SD from 4–6 experiments. Data were statistically analyzed with one-way ANOVA followed by Tukey post hoc multiple comparison analysis between groups. Values that do not share the same letter are significantly different from each other (p ≤ 0.05).

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  Figure 12. Proposed schematic presentation of sex-specific differences in protein expression between endothelial cells that may mediate sex- and cell-specific discrepancies in NO bioavailability.