ET1 acts as a potential plasma biomarker and therapeutic target in deep venous thrombosis rat model

Abstract

Deep venous thrombosis (DVT) is the third leading cause of death in cardiovascular disease, following heart attacks and strokes. Early diagnosis and intervention are crucial for effective DVT therapy. We aim to investigate whether endothelin-1 (ET-1) could serve as an early diagnostic marker or a potential therapeutic target in a DVT rat model. CCK8 assay, invasion assay, and flow cytometry were used to detect the proliferation, migration and apoptosis of HUVECs, respectively. Elisa assay was used to detect ET-1 and coagulation factor VII in cell supernatant and rat?s plasma. Western blot was used to detect antioxidant signaling protein. Inferior vena cava stenosis was used to construct the DVT rat model. Lentivirus mediated overexpression of ET-1 in HUVECs impaired the cell proliferation and migration, increased cell apoptosis, inhibited the antioxidant signaling pathway proteins expression (e.g., NQO1, GCLC, Nrf-2), and upregulated coagulation factor VII. Furthermore, overexpression of ET-1 further impaired antioxidant signaling pathway protein in response to H₂O₂ treatment. However, lentivirus mediated ET-1 knockdown and BQ123 (an ET-1 inhibitor), showed the opposite results with ET-1 overexpression. We then established a DVT rat model by inferior vena cava stenosis. The stenosis induced early expression of ET-1 and coagulation factor VII in plasma at day 1 and restore their level at day 10. BQ123 could downregulate the coagulation factor VII to ameliorate the stenosis effects. Our findings suggest that ET-1 might serve as an early diagnostic marker for DVT rat model and a potential therapeutic target for treating DVT.

Keywords: BQ123; Deep venous thrombosis; Diagnostic marker; Endothelin-1; The inferior vena cava stenosis.

Fig. 1

The effect of ET-1 on HUVECs proliferation. (A, B) qRT-PCR validation of ET-1 expression in HUVEC by lentivirus mediated ET-1 overexpression (A) and lentivirus mediated shRNA-knockdown (B). (C, D) Cell morphologies at day 1, 3 and 5, and CCK8 assay at 0 h, 24 h, 72 h and 120 h, showing ET-1 overexpression significantly inhibited HUVECs proliferation, while ET-1 knockdown and BQ123 (an ET-1 inhibitor) could rescue cell proliferation compared to HUVECs control. Scale bars, 50 μm

Fig. 2

The effect of ET-1 on HUVECs migration and apoptosis. (A, B) Cell invasion assay and statistical analysis showing ET-1 overexpression significantly decreased cell migration, and ET-1 knockdown and BQ123 increased cell migration. Scale bars, 50 μm. (C, D) Flow cytometry and statistical analysis showing ET-1 overexpression significantly increased the apoptosis of HUVEC, while ET-1 knockdown and BQ123 administration did not affect HUVEC’s apoptosis

Fig. 3

The effect of ET-1 on antioxidant signaling pathway proteins. (A) qRT-PCR analysis of mRNA level of the antioxidant signaling protein, Nqo1, Gclc and Nrf2. (B) Western blot and statistical analysis showing ET-1 overexpression significantly decreased NQO1, GCLC and Nrf2 protein level, while ET-1 knockdown and BQ123 could upregulate NQO1, GCLC and Nrf2 protein level. (C) To further investigate the effect of ET-1 on antioxidant signaling pathway protein, after constructing the stable HUVEC with overexpressed ET-1 or knockdown ET-1, and simultaneously treating with 1% H2O2, followed by detecting NQO1, GCLC and Nrf2 protein level

Fig. 4

The effect of ET-1 on the secretion of ET-1 and coagulation factors VII. (A, B) Elisa assay showing ET-1 overexpression significantly increased the secreted level of ET-1 (A), and coagulation factor VII (B) in cell culture supernatant, while ET-1 knockdown and BQ123 significantly inhibited those two factors secretion

Fig. 5

The ET-1 might acts as an early diagnostic marker in plasma of DVT rat model. (A) Schematic illustration of construction of DVT rat model through inferior vena cava stenosis. (B) The cut off thrombosis (∼2 cm) showing the success of constructing DVT rat model. (C) H&E staining showing the thrombosis formation blocked the blood vessel. (D, E) Elisa assay showing ET-1 and coagulation factor VII was significantly increased in plasma at early day 1, while BQ123 administration (3 mg/kg) could significantly decrease both of two factors to ameliorate DVT syndrome