Korean J Physiol Pharmacol.  2015 May;19(3):229-234. 10.4196/kjpp.2015.19.3.229.

Nafamostat Mesilate Inhibits TNF-alpha-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production

Affiliations
  • 1Department of Thoracic and Cardiovascular Surgery, School of Medicine, Chungnam National University, Daejeon 301-721, Korea.
  • 2Department of Neurology, School of Medicine, Chungnam National University, Daejeon 301-721, Korea.
  • 3Department of Endocrinology, School of Medicine, Chungnam National University, Daejeon 301-721, Korea.
  • 4Department of Rehabilitation Medicine, School of Medicine, Chungnam National University, Daejeon 301-721, Korea.
  • 5Department of Internal Medicine, School of Medicine, Chungnam National University, Daejeon 301-721, Korea.
  • 6Department of Pathology, School of Medicine, Chungnam National University, Daejeon 301-721, Korea.
  • 7Department of physiology, School of Medicine, Chungnam National University, Daejeon 301-747, Korea. cskim@cnu.ac.kr

Abstract

Nafamostat mesilate (NM) is a serine protease inhibitor with anticoagulant and anti-inflammatory effects. NM has been used in Asia for anticoagulation during extracorporeal circulation in patients undergoing continuous renal replacement therapy and extra corporeal membrane oxygenation. Oxidative stress is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial function. We investigated whether NM could inhibit endothelial dysfunction induced by tumor necrosis factor-alpha (TNF-alpha). Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha for 24 h. The effects of NM on monocyte adhesion, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) protein expression, p38 mitogen-activated protein kinase (MAPK) activation, and intracellular superoxide production were then examined. NM (0.01~100 microg/mL) did not affect HUVEC viability; however, it inhibited the increases in reactive oxygen species (ROS) production and p66shc expression elicited by TNF-alpha (3 ng/mL), and it dose dependently prevented the TNF-alpha-induced upregulation of endothelial VCAM-1 and ICAM-1. In addition, it mitigated TNF-alpha-induced p38 MAPK phosphorylation and the adhesion of U937 monocytes. These data suggest that NM mitigates TNF-alpha-induced monocyte adhesion and the expression of endothelial cell adhesion molecules, and that the anti-adhesive effect of NM is mediated through the inhibition of p66shc, ROS production, and p38 MAPK activation.

Keyword

ICAM-1; Nafamostat mesilate; p66shc; Reactive oxygen species; VCAM-1

MeSH Terms

Asia
Endothelial Cells*
Extracorporeal Circulation
Human Umbilical Vein Endothelial Cells
Humans
Intercellular Adhesion Molecule-1
Membranes
Mesylates*
Monocytes
Oxidative Stress
Oxygen
p38 Mitogen-Activated Protein Kinases
Phosphorylation
Protein Kinases
Reactive Oxygen Species*
Renal Replacement Therapy
Risk Factors
Serine Proteases
Superoxides
Tumor Necrosis Factor-alpha
Up-Regulation
Vascular Cell Adhesion Molecule-1
Vascular Diseases
Intercellular Adhesion Molecule-1
Mesylates
Oxygen
Protein Kinases
Reactive Oxygen Species
Serine Proteases
Superoxides
Tumor Necrosis Factor-alpha
Vascular Cell Adhesion Molecule-1
p38 Mitogen-Activated Protein Kinases

Figure

  • Fig. 1 Effect of nafamostat mesilate (NM) on endothelial cell viability. (A) Structure of NM. (B) Cell viability was assessed via propidium iodide staining. Cells were treated with various concentrations (0.01~100 µg/mL) of NM for 24 h. Bars represent means±standard error (n=4).

  • Fig. 2 NM inhibits intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression induced by tumor necrosis factor-α (TNF-α). Cells were pretreated with various concentrations (0.3~3 µg/mL) of NM for 30 min and then with 3 ng/mL TNF-α for 8 h in the presence of NM. (A, B) NM inhibits TNF-α-induced ICAM-1 and VCAM-1 protein expression in human umbilical vein endothelial cells (HUVECs). The cells were harvested for Western blot analysis of ICAM-1 and VCAM-1 expression (upper panels of A and B). β-actin was included as a loading control. The levels of ICAM-1 and VCAM-1 were quantified by densitometric analyses (lower panels of A and B). (C, D) NM inhibits TNF-α-induced ICAM-1 and VCAM-1 mRNA expression in HUVECs. All Western blots are representative of three independent experiments. The data are presented as the means±SEM of three independent experiments. *p<0.05 compared with control cells.

  • Fig. 3 Effect of NM on the TNF-α-induced production of reactive oxygen species (ROS) in HUVECs. Cells were treated with 1 ng/mL TNF-α in the presence or absence of 3 µg/mL NM for 8 h. (A) ROS production was measured by dihydroethidine (DHE) staining. Cells were imaged on a fluorescence microscope (upper panel). Quantitative data for TNF-α-induced DHE fluorescence in HUVECs (lower panel) (n=4). *p<0.05 compared to control cells. (B) Hydrogen peroxide levels were measured by Amplex-Red. (n=3). *p<0.05 compared with control cells. (C) The cells were harvested for an analysis of p66shc and p66shc phosphorylation by Western blotting. β-actin was included as a loading control (upper panel). The level of p66shc phosphorylation was examined by densitometric analysis (lower panel) (n=3). *p<0.05 compared with control cells.

  • Fig. 4 NM reduced the levels of p38 mitogen-activated protein kinase (MAPK) induced by TNF-α. Cells were pretreated with 3 µg/mL NM for 30 min and then with 3 ng/mL TNF-α for various times. After pretreatment, TNF-α (15 ng/mL) was added for various times and the effect of NM on p38 phosphorylation was analyzed by Western blot analysis. The level of p66shc phosphorylation was obtained by densitometric analysis (n=3). *p<0.05 compared with control cells.

  • Fig. 5 NM inhibits U937 monocyte adhesion to HUVECs treated with TNF-α. HUVECs treated with TNF-α in the presence or absence of 3 µg/mL NM for 8 h were incubated with U937 monocytes for 30 min. Representative photomicrographs of U937 cells adherent to HUVECs (upper panel) and quantification of the adherent cells (lower panel) are shown (n=3). *p<0.05 compared with untreated cells.


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Nafamostat mesilate promotes endothelium-dependent vasorelaxation via the Akt-eNOS dependent pathway
Sujeong Choi, Hyon-Jo Kwon, Hee-Jung Song, Si Wan Choi, Harsha Nagar, Shuyu Piao, Saet-byel Jung, Byeong Hwa Jeon, Dong Woon Kim, Cuk-Seong Kim
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