E2 treatment counteracts Notch inhibition- induced vascular sprouting in collagen-based aortic ring explants.

<p>Aortic ring explants were embedded in collagen gels and cultured for 5 days in 2.5% csFBS medium containing 30 ng/ml of VEGF-A and treated with 1 nM E2, 5 µM DAPT or 1 nM E2 plus 5 µM DAPT. Treatment with DMSO (V) was used as control. Vascular sprouting was quantified by digital microscopy after 5 days of treatment by measuring the greatest length of sprouts from the body of the aortic ring at three distinct points per ring and on three rings per treatment. One representative picture of three different experiments is shown (A) with the respective sprout lengths (B). Data are expressed as mean ± SEM. **P<0.01, DAPT significantly different from the control, §P<0.05, E2 plus DAPT significantly different from DAPT.</p

Effect of 17β-estradiol treatment on Notch pathway components in HUVECs.

<p>(A) HUVECs were treated with 1 nM E2 or DMSO (V) for 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Cell lysates were electrophoresed and immunoblotted with antibodies for Notch2, Notch4, Jagged1, Dll1. β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071440#pone.0071440.s006" target="_blank">Figure S6B</a>. (B) HUVECs were treated with 1 nM E2 or DMSO (V) for 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Total RNA was extracted and qRT-PCR analysis of Notch2, 4 and Jagged1 genes expression was performed. Relative changes in mRNA expression levels were calculated according to the 2^−ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate. **P<0.01, significantly different from the control. (C) Western blot analysis for Notch4 in HUVECs after Notch4 (si)RNA treatment for 48 hours. Lysates were immunoblotted with Notch4 antibody. β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071440#pone.0071440.s006" target="_blank">Figure S6C</a>. (D) qRT-PCR analyses were performed to detect reduction of Notch4 mRNA levels in HUVECs after Notch4 (si)RNA treatment for 48 hours. Relative changes in mRNA expression levels were calculated according to the 2^−ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate. ***P<0.001, significantly different from the control.</p

E2 treatment counteracts Notch inhibition- induced endothelial branching <i>in vitro</i>.

<p>HUVECs were hormone-stripped and treated overnight with 1 nM E2, 5 µM DAPT and a combination of the two. Treatment with DMSO (V) was used as control. The following day 9×10⁴ cells were seeded on 400 µl of Matrigel and treatment continued for additional 8 hours. Number of closed circles was quantified in eight fields after 8 hours of network formation. One representative picture of three different experiments is shown (A) with the respective counts (B). Data are expressed as mean ± SEM. **P<0.01, DAPT significantly different from the control, §P<0.05 E2 plus DAPT significantly different from DAPT.</p

Effect of 17β-estradiol treatment on Notch1 processing in HUVECs under different experimental conditions.

<p>(A) HUVECs were treated with 1 nM 17β-estradiol (E2) or DMSO (V) for 24 hours under different experimental conditions as described in the Materials and Methods section. Cell lysates were electrophoresed and immunoblotted with Notch1 (C-20) antibody to detect changes in the transmembrane form of Notch1 (Notch1TM). β-actin antibody was used to ensure equal loading. (B) Densitometric analysis of Western blot assay to quantify Notch1TM protein levels. Results are expressed as mean ± SEM of three independent experiments, **P<0.01, ***P<0.001 significantly different from the control. (C) HUVECs at different confluence (70% vs. 90%) were treated with 1 nM E2 or DMSO (V) for 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Cell lysates were electrophoresed and immunoblotted with Notch1 (C-20) antibody to detect changes in the precursor (Notch1 precursor) and the transmembrane (Notch1TM) form. β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071440#pone.0071440.s006" target="_blank">Figure S6A</a>. (D) HUVECs were treated with 1 nM E2 or DMSO (V) for 3, 6 and 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Total RNA was extracted and qRT-PCR analysis of Notch1 gene expression was performed. Relative changes in mRNA expression levels were calculated according to the 2^−ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate.</p

Proposed model for E2-mediated activation of VEGF-A-Dll4-Notch1 signalling and consequences on sprouting angiogenesis in HUVECs.

<p>According to a widely accepted model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071440#pone.0071440-Sainson1" target="_blank">[5]</a> Vascular Endothelial Growth Factor A (VEGF-A), by binding to VEGF Receptor-2 (VEGF-R2), promotes sprouting on endothelial cells and induces expression of Dll4 which, by activating Notch1 in adjacent cells leads to reduction of VEGF-R2 and inhibition of sprouting (A). E2 treatment enhances VEGF-A-mediated increase of the active form of Notch1 (N1IC), effect antagonized by the estrogen receptor antagonist ICI 182.780. In the presence of active Notch1 signalling, a further increase of N1IC induced by E2 does not affect sprouting (B). Notch1 inhibition by treatment with DAPT causes increased sprouting (C). Co-treatment with E2 counteracts DAPT-induced increase in vascular sprouting through still undetermined mechanisms (D).</p

Effect of 17β-estradiol treatment on Notch1 processing in presence of Delta-like ligand 4 in HUVECs.

<p>(A) HUVECs were treated with 1 nM E2, 0.1 µM ICI 182.780, 5 µM DAPT, 1 nM E2 plus 0.1 µM ICI 182.780, 5 µM DAPT plus 1 nM E2 or DMSO (V) for 24 hours under M5 experimental conditions (2% csFBS-EGM-2). Cell lysates were electrophoresed and immunoblotted with cleaved Notch1 (Val1744) antibody. β-actin antibody was used to ensure equal loading. (B) Densitometric analysis of Western blot assay to quantify Notch1IC protein levels. Results are expressed as mean ± SEM of three independent experiments, **P<0.01, ***P<0.001 significantly different from the control. (C) HUVECs were treated with 1 nM E2 for 24 hours under M5 experimental conditions (2% csFBS-EGM-2). Total RNA was extracted and qRT-PCR analysis of Hes1, Hes4, Hey1, Hey2 and Dll4 genes expression was performed. Relative changes in mRNA expression levels were calculated according to the 2^−ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate. * P<0.05; ** P<0.01; *** P<0.001, significantly different from control.</p

Expression of Estrogen Receptors in HUVECs.

<p>(A) Cell lysates obtained from different pools of commercially available HUVECs (1–6) and HUVECs obtained from a single donor (D) were electrophoresed and immunoblotted with ERα and ERβ antibodies. β-actin antibody was used to ensure equal loading. ERα⁺: estrogen receptor α positive cells (MCF-7 cells). (B) Densitometric analysis of Western blot assay to quantify ERα and ERβ protein levels. Results are expressed as mean ± SEM of three independent experiments, ***P<0.001 significantly different from the ERα levels in MCF7 cells.</p

Effect of 17β-estradiol treatment on Notch canonical target genes in HUVECs.

<p>(A) HUVECs were treated with 1 nM E2 or DMSO (V) for 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Total RNA was extracted and qRT-PCR analysis of Hes1, Hey1, Hey2 and HeyL genes expression was performed. Relative changes in mRNA expression levels were calculated according to the 2^−ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate. (B) HUVECs were treated with 5 µM DAPT for 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Cell lysates were electrophoresed and immunoblotted with Notch1 (C-20), Notch2 (clone C651.6DbHN) and Notch4 (H-225) antibodies to detect the transmembrane form of Notch1 (Notch1TM), the active form of Notch2 (Notch2IC) and the active form of Notch4 (Notch4IC). β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071440#pone.0071440.s006" target="_blank">Figure S6D</a>. (C) HUVECs were treated with 5 µM DAPT for 24 hours under M4 experimental conditions (2% FBS overnight followed by 20% csFBS). Total RNA was extracted and qRT-PCR analysis of Hes1, Hey1 and Hey2 genes expression was performed. Relative changes in mRNA expression levels were calculated according to the 2^−ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate. **P<0.01, significantly different from the control.</p

Effect of 17β-estradiol treatment on Notch ligands in HUVECs under different experimental conditions.

<p>(A) HUVECs were treated with 1 nM E2 or DMSO (V) for 24 hours under M4 (2% FBS overnight followed by 20% csFBS) or M5 (2% csFBS-EGM-2) experimental conditions. Cell lysates were electrophoresed and immunoblotted with Jagged1, Dll4 and Dll1 antibodies. β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071440#pone.0071440.s006" target="_blank">Figure S6E</a>. (B) HUVECs were exposed to different VEGF-A concentrations (20 ng/ml and 50 ng/ml) for 24 hours under M5 experimental conditions (2% csFBS-EGM-2). Cell lysates were electrophoresed and immunoblotted with Dll4 antibody. β-actin antibody was used to ensure equal loading. Densitometric analysis of Western blot assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071440#pone.0071440.s006" target="_blank">Figure S6F</a>. (C) HUVECs were treated with VEGF-A (20 ng/ml and 50 ng/ml) for 24 hours under M5 experimental conditions. Total RNA was extracted and qRT-PCR analysis of Dll4 gene expression was performed. Relative changes in mRNA expression levels were calculated according to the 2^−ΔΔCt method using RPL13A as reference gene. Results are expressed as mean ± SEM of three independent experiments, each performed in triplicate. *** P<0.001, significantly different from control.</p