Acadesine inhibits tissue factor induction and thrombus formation by activating the phosphoinositide 3-kinase/Akt signaling pathway

Abstract

Objective: Acadesine, an adenosine-regulating agent and activator of AMP-activated protein kinase, has been shown to possess antiinflammatory activity. This study investigated whether and how acadesine inhibits tissue factor (TF) expression and thrombus formation.

Methods and results: Human umbilical vein endothelial cells and human peripheral blood monocytes were stimulated with lipopolysaccharide to induce TF expression. Pretreatment with acadesine dramatically suppressed the clotting activity and expression of TF (protein and mRNA). These inhibitory effects of acadesine were unchanged for endothelial cells treated with ZM241385 (a specific adenosine A(2A) receptor antagonist) or AMP-activated protein kinase inhibitor compound C, and in macrophages lacking adenosine A(2A) receptor or alpha1-AMP-activated protein kinase. In endothelial cells and macrophages, acadesine activated the phosphoinositide 3-kinase/Akt signaling pathway, reduced the activity of mitogen-activated protein kinases, and consequently suppressed TF expression by inhibiting the activator protein-1 and NF-kappaB pathways. In mice, acadesine suppressed lipopolysaccharide-mediated increases in blood coagulation, decreased TF expression in atherosclerotic lesions, and reduced deep vein thrombus formation.

Conclusion: Acadesine inhibits TF expression and thrombus formation by activating the phosphoinositide 3-kinase/Akt pathway. This novel finding implicates acadesine as a potentially useful treatment for many disorders associated with thrombotic pathology, such as angina pain, deep vein thrombosis, and sepsis.

Figure 1

Acadesine suppresses LPS-induced or cytokine-induced TF expression in HUVEC and human monocytes. HUVEC (A–C) or monocytes (D–F) were pretreated with acadesine for 1 hour at various concentrations, followed by stimulation with LPS at 1 μg/mL for 4 hours. A and D, TF activity of cell lysates was examined with a 1-stage clotting assay. B and E, Total cellular RNA was extracted, and TF mRNA expression was analyzed by real-time reverse-transcription polymerase chain reaction. C and F, TF expression at the protein level was analyzed by Western blotting. Data are shown as means±SEM of 6 independent experiments. ^*P<0.05. ^**P<0.01.

Figure 2

AMPK and A_2AR are not involved in the inhibitory effect of acadesine on TF expression. HUVEC were treated with LPS (1 μg/mL), LPS and acadesine (1 mmol/L), or LPS and acadesine in the presence of AMPK inhibitor compound C (10 μmol/L) or A_2AR antagonist ZM241385 (5 μmol/L). A and D, TF activity of cell lysate was examined with a 1-stage clotting assay. B and E, Total cellular RNA was extracted and TF mRNA expression analyzed by real-time reverse-transcription polymerase chain reaction. GAPDH was used as normalization control. C and F, TF expression at the protein level was analyzed by Western blotting. Data are shown as means±SEM of 6 independent experiments. ^*P<0.05. ^**P<0.01.

Figure 3

Role of the PI3K/Akt pathway in acadesine-downregulated TF expression in endothelial cells. A-C, HUVEC were treated with either acadesine (1 mmol/L) or LPS (1 μg/mL) and acadesine for different periods and lysed. A, PI3Kα was immunoprecipitated from cell lysates and its activity was assayed using a commercial enzyme-linked immunosorbent assay kit. Akt and phospho-Akt (B, C) were examined by Western blotting. D, Akt, GSK3β, extracellular signal-regulated kinase, and their phosphorylated forms were examined by Western blotting at 30 minutes after treatment with LPS (1 μg/mL), acadesine (1 mmol/L), or LPS and acadesine with or without wortmannin pretreatment (50 nM). E, TF in HUVEC was examined by Western blotting at 4 hours after treatment with LPS (1 μg/mL) or LPS and acadesine (1 mmol/L) with or without wortmannin pretreatment (50 nM) or LY294002 (10 μmol/L).

Figure 4

Role of the AP-1 and NF-κB pathways in acadesine-downregulated TF expression in endothelial cells. Nuclear protein was extracted from HUVEC or human monocytes at 1 hour after treatment with LPS (1 μg/mL) or LPS and acadesine (1 mmol/L) with or without wortmannin pretreatment (50 nM). F–I, Electrophoretic mobility shift assays were performed using P-oligonucleotides corresponding to the AP-1 and NF-κB DNA recognition sequences. Results for AP-1 in HUVEC (A) and monocytes (C), respectively. Results for NF-κB (B, D). The intensity of each AP-1 or NF-κB band was quantified by densitometry and expressed as folds of untreated control cells. Data are shown as means±SEM of 4 independent experiments. ^*P<0.05.

Figure 5

Acadesine inhibits TF production in the mouse models of sepsis, atherosclerosis, and thrombosis. C57 Bl/6J mice (male, 25–30 grams) were injected intraperitoneally with acadesine (500 mg/kg) and LPS (2 mg/kg). At 6 or 24 hours after injections, livers were collected for RNA extraction or processed for Western blots and histochemical analysis. A, TF mRNA expression was analyzed by real-time reverse-transcription polymerase chain reaction. B, Liver lysates were prepared and fibrin expression at the protein level was analyzed by Western blotting. C, Liver sections were immunostained with AP-conjugated antibody against fibrin to detect fibrin on the endothelium of venules (arrows; ^*P<0.05; ^** P<0.01). D and E, Apolipoprotein E^−/− mice fed a western diet for 3 months were intraperitoneally injected with vehicle (0.9% saline) or acadesine (500 mg/kg) daily for 5 days. The atherosclerotic arteries were collected and processed for immunostaining and RNA extraction (^*P<0.05; ^**P<0.01). D, The stainings of TF in atherosclerotic lesions of mice treated with the vehicle and acadesine were quantified and compared. E, The levels of TF mRNA were measured with real-time reverse-transcription polymerase chain reaction and compared between 2 groups. F, C57BL/6J mice (male, 25–30 grams) were injected intraperitoneally with vehicle (saline) or acadesine (500 mg/kg) and then underwent a surgical procedure to initiate thrombus formation in the inferior vena cava. The injections were performed daily for 3 days. The thrombosed inferior vena cava was weighed and the length of thrombus was measured. The size of the thrombus was quantified as mg/cm. Three representative thrombosed inferior vena cava from 10 mice in each group and the quantitative data for thrombosed inferior vena cava are shown. ^*P<0.05. ^**P<0.01.