The inhibitory effects of CSS are independent of the presence of sex and thyroid hormones.

<p>ECs were incubated in Standard Medium (SM, solid bars), CSS-medium (CSS-M, open bars), CSS-M + E2 (1 nM, spotted bars), CSS-M + DHT (10 nM, diagonal bars), or CSS-M + T3 (10 nM, squared bars). MTT absorbance (<b>A, C, E</b>) or cell number (<b>B, D, F</b>) were measured after 48h of incubation. Cell number was expressed as percent of control, <i>i</i>.<i>e</i>. cells cultured in SM, set at 100%. In (<b>A</b>), (<b>C</b>), and (<b>E</b>), *p<0.05, **p<0.01 (CSS-M <i>vs</i> SM), ns (CSS-M <i>vs</i> CSS-M + E2, DHT or T3), n = 3, <i>t</i> test. In (<b>B</b>), (<b>D</b>), and (<b>F</b>), *p<0.05, **p<0.01, ***p<0.001 (CSS-M <i>vs</i> SM), ns (CSS-M <i>vs</i> CSS-M + E2, DHT or T3), n = 3, <i>t</i> test. (<b>G</b>) CSS-M was replaced with CSS-M (open bars) or SM (diagonal bars), and MTT was measured after further 48h of incubation. Solid bars: SM replaced with SM. ***p<0.001 <i>vs</i> SM, °°°p<0.001 <i>vs</i> CSS-M, n = 3, <i>t</i> test. F- and M-ECs are orange and blue, respectively.</p

Palmitic acid and acetate rescue the CSS-induced inhibition of cell number.

<p>Cell number was measured in F-ECs (<b>A</b>, orange bars) and M-ECs (<b>B</b>, blue bars) after 48h of incubation in SM (solid bars), CSS-M + vehicle (ethanol/BSA, cross-hitched bars), CSS-M + palmitic acid (250 μM, vertical bars), CSS-M (open bars), or CSS-M + acetate (20 mM, horizontal bars). In (<b>A</b>) and (<b>B</b>), **p<0.01 (CSS-M <i>vs</i> SM), *p<0.05 (CSS-M <i>vs</i> CSS-M + palmitic or CSS-M + acetate), n = 3–4 for F- and M-ECs, respectively, <i>t</i> test.</p

CSS reduces cell number in human F- and M-ECs.

<p>MTT absorbance (<b>A</b>), cell number (<b>B</b>), and ATP luminescence (<b>C</b>) were measured after 48h of incubation in Standard Medium (SM, solid bars) or CSS-medium (CSS-M, open bars). Mean values were compared by Student’s <i>t</i> test. In (<b>A</b>), *p<0.05, n = 14–11 for F- and M-ECs, respectively; in (<b>B</b>), ***p<0.001, n = 3; in (<b>C</b>), *p<0.05, **p<0.01, n = 11–10 for F- and M-ECs, respectively. In (<b>D</b>), ATP luminescence was normalized to the corresponding protein levels. F- and M-ECs are orange and blue, respectively.</p

Effect of CSS on eNOS expression in F- and M-ECs.

<p>(<b>A</b>) Total eNOS protein was evaluated by immunoblotting in F- and M-EC lysates prepared after 48 h of incubation in SM (solid bars) or in CSS-M (open bars). β-actin was used as a loading control. A representative blot and the densitometric analysis of eNOS protein expression normalized to β-actin are shown. **p<0.01 <i>vs</i> SM-cultured M-ECs, <i>t</i> test, n = 13–12 for F- and M-ECs, respectively. (<b>B</b>) F-EC lysates were prepared after 48 h of incubation in SM (solid bar), CSS-M (open bar), or CSS-M + E2 (10 nM, spotted bar). β-actin was used as a loading control. A representative blot and the densitometric analysis of eNOS protein expression normalized to β-actin are shown. n = 4.</p

CSS drastically impairs <i>in vitro</i> angiogenesis.

<p>(<b>A</b>) Representative images of spheroids from F-ECs (upper panels) or M-ECs (lower panels) embedded in collagen gels in the presence of SM (left panels) or CSS-M (right panels). Photographs were taken 24 h later. Quantification of the cumulative length (<b>B</b>), the average length (<b>C</b>), and the number of sprouts (<b>D</b>) emerging from F- or M-EC spheroids incubated in the presence of SM (solid bars) or CSS-M (open bars). *p<0.05, **p<0.01, ***p<0.001 <i>vs</i> SM; ^§p<0.05<i>vs</i> F-ECs cultured in CSS-M, n = 7, <i>t</i> test. F- and M-ECs are orange and blue, respectively.</p

Lack of endocrine response in F- and M-ECs cultured in FA-reconstituted CSS-medium.

<p>E2 (1 nM) or DHT (10 nM) were added to CSS-M in the absence (open bars) or in the presence (solid bars) of palmitic acid (250 μM), and cell number was measured after 48 of incubation in F-ECs (<b>A</b>, orange bars) and M-ECs (<b>B</b>, blue bars). In (<b>A</b>), ***p<0.001; in (<b>B</b>), **p<0.01, n = 3, Two-way ANOVA. The presence of E2 or DHT did not significantly affect the results. (<b>C</b>) E2 (10 nM) was added to CSS-M in the absence (open bars) or in the presence (solid bars) of palmitic acid (250 μM), and the cumulative length of sprouts emerging from F- and M-ECs was measured after 24 h of incubation. ***p<0.001, n = 3, Two-way ANOVA. The presence of E2 did not significantly affect the results. F- and M-ECs are orange and blue, respectively.</p

Palmitic acid and acetate rescue the CSS-induced inhibition of <i>in vitro</i> angiogenesis.

<p>(<b>A</b>) Representative images of spheroids from F-ECs incubated for 24 h in SM, CSS-M, CSS-M + palmitic acid, or CSS-M + acetate. The cumulative (<b>B</b>, <b>D</b>) and the average (<b>C, E</b>) length of sprouts emerging from F-ECs (<b>B</b>, <b>C</b>) and M-ECs (<b>D</b>, <b>E</b>) were measured after 24h of incubation. Treatments and bars are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189528#pone.0189528.g005" target="_blank">Fig 5</a>: SM, solid bars; CSS + vehicle (ethanol/BSA, cross-hitched bars), CSS + palmitic acid (250 μM, vertical bars); CSS-M, open bars; CSS-M + acetate (20 mM, horizontal bars). Data are expressed as percent of control, <i>i</i>.<i>e</i>. the cumulative and the average length of sprouts from cells cultured in SM, set at 100%. In (<b>B</b>) and (<b>D</b>), ***p<0.001 (CSS-M + vehicle <i>vs</i> SM), *p<0.05, **p<0.01 (CSS-M + vehicle <i>vs</i> CSS-M + palmitic acid), n = 3; °°°p<0.001 (CSS-M <i>vs</i> SM), °p<0.05 (CSS-M <i>vs</i> CSS-M + acetate), n = 4, <i>t</i> test. In (<b>C</b>) and (<b>E</b>), **p<0.01 (CSS-M + vehicle <i>vs</i> SM), *p<0.05, ***p<0.001 (CSS-M + vehicle <i>vs</i> CSS-M + palmitic acid), n = 3; °p<0.05, °°°p<0.001 (CSS-M <i>vs</i> SM), °°p<0.01, °°°p<0.001 (CSS-M <i>vs</i> CSS-M + acetate), n = 4, <i>t</i> test. F- and M-ECs are orange and blue, respectively.</p