Fig 1.
Effects of hypoxia on chemerin protein expression in human coronary artery endothelial cells (HCAECs).
(A) A representative western blot of chemerin in HCAECs at different degrees (2.5%, 5% and 10% O2) of hypoxia for 4 h. (B) Quantitative analysis of chemerin protein levels (n = 4 per group); (C) Representative western blots for chemerin in cells subjected to 2.5% O2 hypoxia for various periods. (D, E) Quantitative analysis of protein and mRNA levels of chemerin. The values from hypoxic HCAECs were normalized to α-tubulin data and then expressed as a ratio of the normalized values to mRNA in control cells (n = 4 per group); *p < 0.01 compared to control.
Fig 2.
The ERK pathway mediates the hypoxia-induced increase in chemerin expression in HCAECs.
(A and B) The hypoxia-induced increase in chemerin expression was blocked by an ERK inhibitor (PD98059), ERK siRNA, or an anti-TNF antibody. Addition of exogenous TNF—alpha during normoxia increased chemerin expression (n = 4 per group); *p < 0.01 compared to control.
Fig 3.
Phosphorylation of ERK was responsible for the hypoxia-induced chemerin expression in HCAECs, which was blocked by PD98059 or ERK siRNA.
(A and B) HCAECs were subjected to normoxia or hypoxia for different periods in the presence or absence of the inhibitors. Cell lysates were collected for western blot analysis with an antibody for total ERK and phospho-ERK. T-ERK = total ERK, P-ERK = phosphorylated ERK (n = 4 per group); *p < 0.01 compared to control.
Fig 4.
Binding of chemerin promoter with the SP1 transcription factor and transcriptional activity of the SP1-binding site of the chemerin promoter are increased in hypoxic HCAECs.
(A) The electrophoretic mobility shift assay (EMSA) showed an increase in binding of chemerin promoter and SP1 in HCAECs during 2.5% O2 hypoxia. (B and C) The luciferase reporter assay revealed that hypoxia at 2.5% O2 increased the transcriptional activity of SP1 on the chemerin promoter as compared to the chemerin mutant. Transcriptional activity was suppressed by PD98059 and an anti-TNF-alpha antibody (Ab; n = 3 per group); *p < 0.01 compared to control, §p < 0.01 compared to time point 1 h.
Fig 5.
Hypoxia increases proliferation of HCAECs.
Incorporation of [3H]thymidine into HCAECs increased during 2.5% O2 hypoxia and after addition of exogenous chemerin or TNF-alpha during normoxia for 2 to 4 h. Hypoxia-induced incorporation of [3H]thymidine into HCAECs was suppressed by chemerin siRNA, PD98059, or by the anti-TNF-alpha antibody (Ab; n = 4 per group); *p < 0.01 compared to control, §p < 0.01 compared to hypoxia. Cpm: counts per minute.
Fig 6.
Effects of hypoxia on tube formation by HCAECs.
(A) A representative image of a tube formed by HCAECs. (B) Quantitative branching points analysis (n = 4 per group); *p < 0.01 compared to control, §p < 0.01 compared to hypoxia.