Sex-specific eNOS activity and function in human endothelial cells

Figure 1. eNOS expression and activation are higher in F- than in M-ECs. (A) eNOS RNA was measured by RT-qPCR and normalized to the housekeeping gene 18 S. ***p < 0.001, n = 3. (B) Total eNOS protein was evaluated by immunoblotting on male and female lysates. β-actin was used as a loading control. A representative blot (showing 4 independent male and female lysates, 10 µg/lane) and the densitometric analysis of eNOS protein expression normalized to β-actin are shown. **p < 0.01; n = 14. (C) Upper panel, eNOS protein evaluated on lysates from twin male and female ECs at P1 passage (in vitro, left panel) or collected immediately after the flushing from cords (ex vivo, right panel). Representative blots out of two are shown. Lower panel, eNOS RNA measured by RT-qPCR on total RNA extracted from twin M- and F-ECs at P1 in vitro passage. Data are normalized to the housekeeping gene ACTB. *p < 0.05; n = 3. (D) eNOS dimers and monomers were separated by LT-PAGE as described in the Methods section. A representative blot and the densitometric analysis of the dimers-to-monomers ratio are shown. *p < 0.05; n = 6. (E) A representative blot (showing 2 independent male and female lysates, 20 µg/lane) and…

Figure 2. EC proliferation is eNOS-independent in both sexes. (A) M- and F-ECs were cultured for the indicated times, and WST-8 absorbance was measured after 4 h of incubation. n = 3. (B) Cell cycle distribution was analyzed by FACS 48 h after plating. The percent of cells in each phase of the cycle is shown (solid, G0/G1; open, S; vertical, G2/M). n = 3–4 for M- and F-ECs, respectively. (C) M- and F-ECs were cultured in the absence (solid bars) or in the presence of L-NAME (100 µM, horizontal bars), and WST-8 was added for the last 4 h of incubation. Data are expressed as the percent of absorbance measured in the corresponding vehicle-treated cells. n = 3. M- and F-ECs are green and red, respectively.

Figure 3. F-ECs show higher eNOS-dependent migratory properties than M-ECs: correlation with lamellipodia and eNOS localization. (A) Chemotaxis toward 10% FBS was performed in the absence (solid bars) or in the presence of L-NAME (100 µM, horizontal bars). Results are expressed as the number of migrating cells, and are the mean ± s.e.m of 25 counts from a representative experiment, repeated 3 times with similar results. ***p < 0.001 vs vehicle, &p < 0.05 vs M-ECs. (B) Results are expressed as the percent of change in the number of migrating F-ECs over M-ECs in the presence of vehicle (**p < 0.01, n = 4) or L-NAME (**p < 0.01, n = 3). (C and D) Representative images of FITC-labeled phalloidin-stained male (left panel) and female (right panel) showing lamellipodia (white arrows), and quantification of the area occupied by lamellipodia expressed as the percent of the total cell area. *p < 0.05, n = 3. Scale bar, 20 µm. (E) Forward scatter height (FSC-H) and side scatter height (SSC-H) of M- and F-ECs. n = 8 for M-ECs, and n = 9–10 for F-ECs. (F, G and H) Analysis of spontaneous cell-motility tracks by time-lapse imaging in M- and F-ECs, showing quantification of the cumulative distance,…

Figure 4. 2-D wound healing assay reveals eNOS-dependent migration in female ECs. (A) Representative photographs of male and female ECs taken at the time of wounding (t 0) and after a 16- h incubation in 20% FBS (vehicle) or in 20% FBS supplemented with L-NAME (100 µM) or mytomicin (MitoC, 500 ng/ml). Images of male and female ECs transfected with eNOS siRNA 48 h before wounding and then incubated for 16 h in 20% FBS are also shown. Scale bar, 100 µm. (B) The percentage of wound closure at 16 h is shown. Vehicle, solid bars; L-NAME, horizontal bars; siRNA, squared bars; MitoC, vertical bars; L-NAME + MitoC, diagonal bars. **p < 0.01; ***p < 0.001 vs the corresponding vehicle, n = 3–4 for M- and F-ECs, respectively.

Figure 5. eNOS is essential for in vitro angiogenesis in female ECs. (A) Representative images of spheroids from male or female ECs embedded in collagen gels in the absence (vehicle) or in the presence of L-NAME (1 mM) or MitoC (500 ng/ml). Also eNOS siRNA-transfected spheroids are shown. Photographs were taken 18 h later. Scale bar, 100 µm. (B) Quantification of in vitro angiogenesis. Results are expressed as the percent of the total sprout length measured in vehicle–treated cells, set at 100%. Vehicle, solid bars; L-NAME, horizontal bars; siRNA, squared bars; MitoC, vertical bars; L-NAME + MitoC, diagonal bars. ***p < 0.001 vs the corresponding vehicle, £p < 0.05 vs MitoC-treated F-ECs, n = 4–7 for M- and F-ECs, respectively. (C) Representative fluorescence photograph of a mosaic spheroid containing a 1:1 mixture of GFP-M-ECs and Cherry-F-ECs embedded in collagen gels. Photographs were taken 18 h later. Arrows indicate F-ECs located as tip cells. Scale bar, 100 µm. (D) Quantification of the percentage of tip cell contribution in sprouts containing both GFP-M-ECs and Cherry-F-ECs emerging from vehicle- or L-NAME-treated spheroids. *p < 0.05, n = 17.