Figure 1.
A, Confluent CMEC monolayer presents cobblestone morphology (scale bar:20 mm). B¸ CMECs uptook Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) (red, Dil-Ac-LDL; blue, DAPI). C, CMECs express CD31.
Figure 2.
Hypoxia increases ROS production and apoptosis of CMECs.
A: Representative immunofluorescence imagines of ROS formation (red fluorescent), apoptosis (TUNEL, green fluorescent) and DAPI (blue fluorescence) in CMECs under hypoxia condition for the indicated time points. (Scale bars: 20 µm) B: The average fluorescence intensity of DHE in each group. C: Quantification of the apoptotic CMECs was presented as the percentage of apoptotic cells. (n = 5,*p<0.05 vs. control).
Figure 3.
Hypoxia increases the expressions of HIF-1α, decreased the activity of Akt and regulates the expressions of FoxOs in CMECs.
A: Representative Western blots of HIF-1α, pAkt, Akt, FoxO1, FoxO3a, pFoxO3a in CMECs subjected to hypoxic injury. B: Semiquantitative analysis of HIF-1α. C: Semiquantitative analysis of FoxO1 and FoxO3a at the indicated time point. D: The ratios of p-Akt/total Akt at the indicated time point. E: The ratios of p-Akt/total Akt at the indicated time point. (n = 5,*p<0.05 vs. control, # p<0.05, NS: not significant).
Figure 4.
Hypoxia promoted FoxO3a nuclear location.
A: Representative immunofluorescence staining for subcellular localization of FoxO3a (green fluorescent) in CMECs under hypoxia condition for the indicated time points. (Scale bars: 20 µm) B: Quantification of FoxO3 nuclear or cytoplasmic localization in CMECs (n = 5).
Figure 5.
HIF-1α modulated the hypoxia-induced FoxO3a expression in CMECs.
A: CMECs were incubated with siRNA directed against HIF-1α (HIF-1α siRNA) or scrambled oligonucleotide control RNA (Crt siRNA) and then subjected to hypoxia for 6 h. Western blot assay was performed to evaluate the expressions of HIF-1α, p-Akt, Akt, FoxO1, p-FoxO3a, FoxO3a, BimEl and Bclxl. The semiquantitative analysis of expressions of HIF-1α (B), FoxO3a (C), FoxO1 (F), BimEl (G) and Bclxl (H). D: the ratio of p-Akt/total Akt. E: the ratio of p-FoxO3a/total FoxO3a. The protein expression in CMECs treated with control siRNA (Crt siRNA) under normoxia condition was set as 100%.(n = 5,*p<0.05, NS: not significant).
Figure 6.
HIF-1α regulates the hypoxia-induced ROS accumulation and apoptosis in CMECs.
A: Representative immunofluorescence imagines of ROS formation (red fluorescent) and DAPI (blue fluorescence) in CMECs incubated with HIF-1α siRNA or control siRNA (Crt siRNA) under normoxia or hypoxia condition for 6h. (Scale bars: 20 µm); B: Confocal imaging of cell apoptosis determined by TUNEL assay. Apoptotic nuclei were identified as TUNEL positive (green fluorescent) and total nuclei by DAPI counters taining (blue fluorescent). Scale bar represents 50 µm. C: The average fluorescence intensity of DHE in each group. D: Quantification of the apoptotic CMECs was presented as the percentage of apoptotic cells. (n = 5, *p<0.05 vs. Crt siRNA plus normoxia, # p<0.05 vs. Crt siRNA plus hypoxia).
Figure 7.
FOXO3a affects the expressions of Bcl-2 family proteins in CMECs.
CMECs were incubated with siRNA directed against FoxO3a (FoxO3a siRNA) or scrambled oligonucleotide control RNA (Crt siRNA) and then subjected to hypoxia for 6 h. A: Representative Western blot of HIF-1α, FoxO1, FoxO3a and Bcl-2 proteins, including Bim EL, Bcl xl, Bcl-2 and Bax. The semiquantitative analysis of HIF-1α (B), FoxO1 (C), FoxO3a (D), Bim EL (E), Bcl xl (F), Bcl-2 (G) and Bax (H). The protein expression in CMECs treated with control siRNA (Crt siRNA) under normoxia condition was set as 100%.(n = 5, *p<0.05).
Figure 8.
FoxO3 silencing suppresses hypoxia-induced ROS production and apoptosis in CMECs.
A: Representative immunofluorescence imagines of ROS formation (red fluorescent) and DAPI (blue fluorescence) in CMECs incubated with FoxO3a siRNA or control siRNA (Crt siRNA) under normoxia or hypoxia condition for 6 h. (Scale bars: 20 µm); B: Representative confocal imaging of TUNEL (green fluorescent) and DAPI (blue fluorescent) staining assay. (Scale bars: 20 µm) C: The average fluorescence intensity of DHE in each group. D: Quantification of the apoptotic CMECs was presented as the percentage of apoptotic cells. (n = 5, *p<0.05 vs. Crt siRNA plus normoxia, # p<0.05 vs. Crt siRNA plus hypoxia).
Figure 9.
Proposed scheme for the mechanism that regulates hypoxia-induced injury in CMECs.
Hypoxia stress increases HIF-1α expression, which subsequently promotes the transcription of FoxO3a and directly stimulates ROS formation. Meanwhile, hypoxia also inhibits the activation of Akt which results in decreased phosphorylation and degradation of FoxO3a. Thus, hypoxia increases FoxO3a translocation to nucleus and the transcription activities, which results in the elevated ROS accumulation and Bcl-2 protein family disturbance. In parallel, increased ROS formation also promotes the activity of FoxO3a which increases the pro-apoptotic proteins Bim and Bax and decreased the anti-apoptotic proteins Bcl-xL and Bcl-2. The disturbance of anti-apoptosis proteins and pro-apoptosis proteins induced by oxidative stress subsequently triggers the apoptosis of CMECs. Accordingly, FoxO3a plays a central role in hypoxia-induced ROS formation and apoptosis in CMECs.