Gamma-secretase Inhibitor Prevents Proliferation and Migration of Ductus Arteriosus Smooth Muscle Cells through the Notch3-HES1/2/5 Pathway

Abstract

Patent ductus arteriosus (PDA) can cause morbidity and mortality in neonates. Vascular remodeling, characterized by proliferation and migration of smooth muscle cells (SMCs), is an essential process for postnatal DA closure. Notch signaling is an important mediator of vascular remodelling but its role in DA is unkonwn. We investigated the effects and underlying mechanisms of γ-secretase inhibitor DAPT, a Notch signaling inhibitor on angiotensin II (Ang II)-induced proliferation and migration of DASMCs. Proliferation and migration of DASMCs cultured from neonatal Wistar rats were induced by Ang II, with or without DAPT pre-treatment. In addition, potential underlying mechanisms including cell cycle progression, Ca(2+) influx, reactive oxygen species (ROS) production, signal transduction of MAPK and Akt, and Notch receptor with its target gene pathway were examined. We found that DAPT inhibited Ang II-induced DASMCs proliferation and migration dose dependently. DAPT also arrested the cell cycle progression in the G0/G1-phase, and attenuated calcium overload and ROS production caused by Ang II. Moreover, DAPT inhibited nuclear translocation of Notch3 receptor intracellular domain, with decreased expression of its down-stream genes including HES1, HES2 and HES5. Finally, Ang II-activated ERK1/2, JNK and Akt were also counteracted by DAPT. In conclusion, DAPT inhibits Ang II-induced DASMCs proliferation and migration. These effects are potentially mediated by decreased calcium influx, reduced ROS production, and down-regulation of ERK1/2, JNK and Akt, through the Notch3-HES1/2/5 pathway. Therefore, Notch signaling has a role in DA remodeling and may provide a target pathway for therapeutic intervention of PDA.

Keywords: Ductus arteriosus; Notch signaling; remodeling.

Figure 1

Effects of DAPT on Ang II-induced proliferation and DNA synthesis of DASMCs. (A). The cell viability of DASMCs was increased after incubation with Ang II for 48 h. DAPT attenuated Ang II-induced proliferation in a concentration-dependent manner. (B). BrdU incorporation of DASMCs was increased after incubation with Ang II for 48 h. DAPT attenuated Ang II-induced BrdU incorporation in a concentration-dependent manner. Values represent mean ± SEM. N=6. Control: DASMCs were placed in medium with 1% FBS. ^##P < 0.01 and ^### P < 0.001 vs. control group; ^**P < 0.01 and ^***P < 0.001 vs. cells exposed to Ang II alone.

Figure 2

Effects of DAPT on Ang II-induced migration of DASMCs. Ang II induced migration of DASMCs into the upper chamber from the lower chamber at 48 h. DAPT inhibited Ang II-induced migration dose-dependently. The bar graph shows migration activities assayed for the number of cells observed in six high-power fields. Values represent mean ± SEM. N=6. ^##P < 0.01 vs. control group; ^*P < 0.05 and ^**P < 0.01 vs. cells exposed to Ang II alone.

Figure 3

Effects of DAPT on cell cycle progression affected by Ang II. Treatment of Ang II for 48 h significantly decreased the percentage of cells in the G₀/G₁-phase from 78.3 ± 1.2% to 71.4 ± 0.6% (P < 0.05). DAPT counteracted this inhibitory effect and increased the percentage of cells in the G₀/G₁-phase dose-dependently (P < 0.05). Values represent mean ± SEM. N=6.

Figure 4

Effects of DAPT on Ang II-induced elevation of intracellular calcium concentration ([Ca²⁺]i) in DASMCs. (A) In calcium-containing buffer, Ang II increased [Ca²⁺]i, and pretreatment with DAPT attenuated these effects in a dose-dependent fashion. (B) These effects were quantified by the bar graph. Values represent mean ± SEM. N=6. ^*P < 0.05 and ^**P < 0.01 vs. cells exposed to Ang II alone.

Figure 5

Effects of DAPT on Ang II-induced ROS production. Ang II induced significant ROS production in DASMCs as shown by oxidation of intracellular DCFH-DA to fluorescent DCFH determined by flow cytometry. DAPT significantly attenuated the production of ROS induced by Ang II in a concentration-dependent manner. Values represent mean ± SEM. N=6. ^##P < 0.01 vs. control group; ^*P < 0.05 and ^**P < 0.01 vs. cells exposed to Ang II alone.

Figure 6

DAPT inhibited Ang II -induced nuclear translocation of Notch3 ICD but not Notch1 ICD. (A and B) Representative photomicrographs by a confocal microscope showing that DASMCs were unstimulated (control), or stimulated with Ang II 10 nM (Ang II) alone or with DAPT 10 μM (Ang II + DAPT) for 48 h. Cells were stained for α-smooth muscle actin (α-SMA, green) and Notch1/3 ICD (red). DAPI (blue) was used as a nuclear marker. 'Merge' indicates the merged images of α-SMA, DAPI and Notch1/3 ICD. Scale bars: 20μm. (C and D) The bar graphs represent nuclear/cytoplasmic (N/C) ratios of Notch1/3 ICD fluorescent intensities derived from individual nuclei and cytoplasm for each fluorescent channel. Values represent mean ± SEM. N=6. ^##P < 0.01 vs. control; ^**P < 0.01 vs. cells exposed to Ang II alone.

Figure 7

Effects of DAPT on Ang II-induced gene expressions of HES1, HES2 and HES5. Ang II was tested alone or in combination with DAPT for 48 h. RT-PCR analysis demonstrated that Ang II induced relative gene expressions of HES1 (A), HES2 (B) and HES5 (C). DAPT down-regulated expressions of all these genes dose-dependently. Values represent mean ± SEM. N=6. ^#P < 0.05 and ^##P < 0.01 vs. control; ^*P < 0.05 and ^**P < 0.01 vs. cells exposed to Ang II alone.

Figure 8

Effects of DAPT on Ang II-induced ERK1/2, JNK, and Akt demonstrated by Western blotting. Ang II was tested alone or in combination with DAPT for 48 h. (A and B) DAPT down-regulated Ang II-induced ERK1/2 and JNK expression dose-dependently. (C) DAPT down-regulated Ang II-induced phosphorylation of Akt dose-dependently. Densitometric results represent mean ± SEM. N = 6. ^##P < 0.01 vs. control; ^*P < 0.05 and ^**P < 0.01 vs. cells exposed to Ang II alone.