Abstract
Angiotensin II (AngII) causes endothelial dysfunction. Eucommia ulmoides extract (EUE) is documented to manipulate AngII, but its impact on cardiac microvascular endothelial cell (CMVEC) function remains unknown. This study determines the effects of EUE on AngII-treated CMVECs. CMVECs were treated with different concentrations of AngII or EUE alone and/or the p53 protein activator, WR-1065, before AngII treatment, followed by examinations of the apoptotic, migratory, proliferative, and angiogenic capacities and nitric oxide (NO), p53, von Willebrand factor (vWF), endothelin (ET)-1, endothelial NO synthase (eNOS), manganese superoxide dismutase (MnSOD), hypoxia-inducible factor (HIF)-1α, and vascular endothelial growth factor (VEGF) levels. AngII induced CMVEC dysfunction in a concentration-dependent manner. EUE enhanced the proliferative, migratory, and angiogenic capacities and NO, MnSOD, and eNOS levels but repressed apoptosis and vWF and ET-1 levels in AngII-induced dysfunctional CMVECs. Moreover, AngII increased p53 mRNA levels, p-p53 levels in the nucleus, and p53 protein levels in the cytoplasm and diminishes HIF-1α and VEGF levels in CMVECs; however, these effects were counteracted by EUE treatment. Moreover, WR-1065 abrogated the mitigating effects of EUE on AngII-induced CMVEC dysfunction by activating p53 and decreasing HIF-1α and VEGF expression. In conclusion, EUE attenuates AngII-induced CMVEC dysfunction by upregulating HIF-1α and VEGF levels via p53 inactivation.
Keywords:
Eucommia ulmoides extract. Angiotensin II; Cardiac microvascular endothelial cell; p53 activation; Dysfunction
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Notes: (A) The morphology of CMVECs was observed with a microscope (Olympus MK, Tokyo, Japan). (B) CD31 expression was measured by flow cytometry. CMVECs were treated with different concentrations of AngII. (C) The morphology of CMVECs was observed. (D) Cell viability was examined by MTT assay. (E) Cell migratory capacity was assessed by scratch assay. (F) Angiogenic capacity of CMVECs was evaluated by tubule formation assay. (G) Cell apoptotic capacity was determined by flow cytometry. (H) NO levels were tested with a kit. (I) vWF, ET-1, MnSOD, and eNOS protein expression was detected by western blot. Data were displayed as mean ± standard deviation, N = 3.*P < 0.05 compared with the control group. AngII, angiotensin II; CMVEC, cardiac microvascular endothelial cell; NO, nitric oxide; vWF, von Willebrand factor; ET-1, endothelin-1; MnSOD, manganese superoxide dismutase; eNOS, endothelial NO synthase.
Notes: CMVECs were treated with different concentrations of EUE and AngII. (A) The morphology of CMVECs was observed. (B) Cell viability was tested by MTT assay. (C) Cell migration was determined by scratch assay. (D) Angiogenic ability of cells was evaluated by tubule formation assay. (E) Apoptosis rate was measured by flow cytometry. (F) NO levels were assessed with a kit. (G) vWF, ET-1, MnSOD, and eNOS protein expression was examined by western blot. Data were displayed as mean ± standard deviation, N = 3. *P < 0.05 compared with the saline group. AngII, angiotensin II; CMVEC, cardiac microvascular endothelial cell; EUE, Eucommia ulmoides extract; NO, nitric oxide; vWF, von Willebrand factor; ET-1, endothelin-1; MnSOD, manganese superoxide dismutase; eNOS, endothelial NO synthase.
Notes: CMVECs were treated with AngII. (A) p53 mRNA levels were determined by RT-qPCR. (B) p53 and p-p53 protein expression was examined by western blot. CMVECs were treated with 1 mg/mL EUE and AngII. (C) p53 mRNA levels were determined by RT-qPCR; (D) p53 and p-p53 protein expression was examined by western blot. Data were displayed as mean ± standard deviation, N = 3. *P < 0.05 compared with the control group; # P < 0.05 compared with the saline group. AngII, angiotensin II; CMVEC, cardiac microvascular endothelial cell; EUE, Eucommia ulmoides extract.
Notes: CMVECs were treated with EUE alone or with p53 protein activator WR-1065 before AngII induction. (A) p53 mRNA levels were examined by RT-qPCR and p53 and p-p53 protein expression was examined by western blot. (B) The morphology of CMVECs was observed. (C) Cell viability was evaluated by MTT assay. (D) Cell migration was tested by scratch assay. (E) Angiogenic ability of CMVECs was assessed by tubule formation assay. (F) Apoptosis of CMVECs was determined by TUNEL staining. (G) NO levels were measured with a kit. (H) vWF, ET-1, MnSOD, and eNOS protein expression was determined by western blot. Data were displayed as mean ± standard deviation, N = 3. # P < 0.05 compared with the EUE group. AngII, angiotensin II; CMVEC, cardiac microvascular endothelial cell; EUE, Eucommia ulmoides extract; NO, nitric oxide; vWF, von Willebrand factor; ET-1, endothelin-1; MnSOD, manganese superoxide dismutase; eNOS, endothelial NO synthase.
Note: (A) Hif-1α and VEGF expression was assessed by RT-qPCR in CMVECs. (B) Hif-1α and VEGF expression was assessed by western blot in CMVECs before and after AngII treatment. (C) Hif-1α and VEGF expression was assessed by RT-qPCR in AngII-induced CMVECs after treatment with normal saline, EUE, or EUE + WR-1065. (D) Hif-1α and VEGF expression was assessed by western blot in AngII-induced CMVECs after treatment with normal saline, EUE, or EUE + WR-1065. Data were displayed as mean ± standard deviation, N = 3. *P < 0.05 compared with the control or saline group; # P < 0.05 compared with the EUE group. AngII, angiotensin II; CMVEC, cardiac microvascular endothelial cell; EUE, Eucommia ulmoides extract; Hif-1α, hypoxia-inducible factor-1α; VEGF, vascular endothelial growth factor.