Nuclear factor erythroid 2-related factor 2 (NRF2) is a negative regulator of tissue plasminogen activator synthesis in cultured human vascular endothelial EA.hy926 cells

Abstract

Blood coagulation and the fibrinolytic system contribute to vascular lesions. Fibrinolysis in normal circulating blood strongly depends on the balance between tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) secreted from vascular endothelial cells; however, the mechanisms by which endothelial fibrinolysis is regulated remain to be fully understood. In the present study, human vascular endothelial EA.hy926 cells were transfected with small interfering RNA for nuclear factor erythroid 2-related factor 2 (NRF2) and the expression of t-PA and PAI-1 and fibrinolytic activity in the conditioned medium were examined. EA.hy926 cells were also treated with sulforaphane, an NRF2 activator, and fibrinolytic activity was examined to confirm the NRF2 signaling pathway's effect. Enhanced fibrinolytic activity in the conditioned medium was observed in association with increased expression and secretion levels of t-PA in NRF2 knockdown EA.hy926 cells. However, sulforaphane inhibited fibrinolytic activity and t-PA synthesis in EA.hy926 cells without any cell damage. The expression level of PAI-1 did not change in either NRF2 knockdown or sulforaphane treated cells. These results suggest that transcription factor NRF2 may play a role in down-regulating endothelial t-PA synthesis and fibrinolytic activity.

Protease-Activated Receptor 2 (PAR-2) Antagonist AZ3451 Mitigates Oxidized Low-Density Lipoprotein (Ox-LDL)-Induced Damage and Endothelial Inflammation

“…Human endothelial-like immortalized EA.hy926 cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 U/mL). The cells were cultured at 37 °C in a humidified 5% CO 2 atmosphere, and the medium was exchanged for a fresh medium every 2 days . For the experiment in Figure , the cells were treated with 100, 200, and 300 μg/mL of ox-LDL (#20605ES05, Yeasen Biotechnology, China) for 24 h. In subsequent experiments, the cells were treated with 300 μg/mL of ox-LDL or AZ3451 (#SML2050, Sigma-Aldrich, USA) for 24 h.…”

Nucleolin positively regulates spontaneous cell proliferation but is not involved in inhibition of proliferation by lead in cultured bovine aortic endothelial cells

“…Vascular endothelial cells were transfected with siR-NAs using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA), in accordance with the manufacturer's protocol, as we performed previously (Takahashi et al, 2020). Briefly, double-stranded siRNA (final siRNA concentration: 5 pmol) was added to RNAiMAX transfection reagent, followed by incubation for 10 min at room temperature to allow siRNA/RNAiMAX complex formation.…”

“…Western blot analysis was performed as described previously with minor modifications (Takahashi et al, 2020). Briefly, after incubation, the cell layers were washed with cold CMF-PBS and lysed with RIPA buffer.…”

Nucleolin Knockdown Enhances Cadmium Cytotoxicity in Cultured Vascular Endothelial Cells

“…tured cells and subsequent real-time RT-PCR were performed as described previously. 23) Briefly, after incubation, the culture medium was removed and the cell layer was washed twice with cold calcium-and magnesium-free phosphate-buffered saline (CMF-PBS, Nissui Pharmaceutical Co., Ltd.) and cold ISOGEN II reagent (Nippon Gene, Tokyo, Japan) was added to each culture well. Cells were collected by scraping and homogenized by pipetting.…”

“…Western Blotting Western blot analysis was performed as described previously with small modifications. 23) Confluent cultures of vascular endothelial cells were treated with cadmium chloride (1, 2, or 5 µM) in serum-free DMEM for 24 h. After incubation, the cell layers were washed twice with cold CMF-PBS and lysed with RIPA buffer (1 mM Tris-HCl [pH 7.4], 1% NP-40, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 150 mM sodium chloride, 1 mM EDTA, 1 μg/mL leupeptin, 1 μg/mL pepstatin, and 1 mM PMSF). After collection, the solution was centrifuged at 15,000 × g at 4°C for 5 min.…”