[논문]Nafamostat Mesilate Inhibits TNF-<tex>${\\alpha}$</tex>-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production

Kang, Min-Woong; Song, Hee-Jung; Kang, Shin Kwang; Kim, Yonghwan; Jung, Saet-Byel; Jee, Sungju; Moon, Jae Young; Suh, Kwang-Sun; Lee, Sang Do; Jeon, Byeong Hwa; Kim, Cuk-Seong

doi:10.4196/kjpp.2015.19.3.229

Nafamostat Mesilate Inhibits TNF-${\\alpha}$-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production 원문보기

The Korean journal of physiology & pharmacology : official journal of the Korean Physiological Society and the Korean Society of Pharmacology, v.19 no.3, 2015년, pp.229 - 234

Kang, Min-Woong (Department of Thoracic and Cardiovascular Surgery, School of Medicine, Chungnam National University) , Song, Hee-Jung (Department of Neurology, School of Medicine, Chungnam National University) , Kang, Shin Kwang (Department of Thoracic and Cardiovascular Surgery, School of Medicine, Chungnam National University) , Kim, Yonghwan (Department of Thoracic and Cardiovascular Surgery, School of Medicine, Chungnam National University) , Jung, Saet-Byel (Department of Endocrinology, School of Medicine, Chungnam National University) , Jee, Sungju (Department of Rehabilitation Medicine, School of Medicine, Chungnam National University) , Moon, Jae Young (Department of Internal Medicine, School of Medicine, Chungnam National University) , Suh, Kwang-Sun (Department of Pathology, School of Medicine, Chungnam National University) , Lee, Sang Do (Department of physiology, School of Medicine, Chungnam National University) , Jeon, Byeong Hwa (Department of physiology, School of Medicine, Chungnam National University) , Kim, Cuk-Seong (Department of physiology, School of Medicine, Chungnam National University)

Abstract ▼ AI-Helper

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 mitogenactivated protein kinase (MAPK) activation, and intracellular superoxide production were then examined. NM ($0.01{\sim}100{\mu}g/mL$) 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.

주제어

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가설 설정

(A) Structure of NM. (B) Cell viability was assessed via propidium iodide staining. Cells were treated with various concentrations (0.

제안 방법

, Seongnam, South Korea). Real-time PCR was performed using the Prism 7000 Sequence Detection System (Applied Biosys tems, Foster City, CA, USA) and the Super Script III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen, Carlsbad, CA, USA). The primers used for human VCAM-1 were as follows: (sense) 5'-CAT GGA ATT CGA ACC CAA ACA-3' and (antisense) 5'-GGC TGA CCA AGA CGG TTG TATC-3'.

대상 데이터

Total RNA from cells was isolated using the acid guanidinium thiocyanate-phenol-chloroform method. Complementary DNA was prepared from total RNA using the MAXIME RT Premix Kit (iNtRON Biotechnology Inc., Seongnam, South Korea). Real-time PCR was performed using the Prism 7000 Sequence Detection System (Applied Biosys tems, Foster City, CA, USA) and the Super Script III Platinum SYBR Green One-Step qRT-PCR Kit (Invitrogen, Carlsbad, CA, USA).
Sub-confluent, proliferating HUVECs at passages 2∼8 were used. U937 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM supplemented with 10% fetal bovine serum and antibiotics. The effects of NM on HUVEC viability were examined using an ADAM-MC automatic cell counter (Digital Bio, Seoul, South Korea), which assesses propidium iodide (PI) staining [14].

데이터처리

Data in which two conditions were compared were evaluated using one-way analysis of variance followed by Tukey’s post-hoc test; p-values ＜0.05 were considered significant.
All data are expressed as means±standard deviations. Statistical analysis was performed using Sigma Stat (Systat Software, La Jolla, CA, USA). Data in which two conditions were compared were evaluated using one-way analysis of variance followed by Tukey’s post-hoc test; p-values ＜0.

이론/모형

The results were interpreted by the relative quantity method (ΔΔCt).
Total RNA from cells was isolated using the acid guanidinium thiocyanate-phenol-chloroform method. Complementary DNA was prepared from total RNA using the MAXIME RT Premix Kit (iNtRON Biotechnology Inc.

성능/효과

In conclusion, our findings show that the inhibitory effects of NM on TNF-α-induced monocyte adhesion to endothelial cells are likely to have relevance for inhibiting not only VCAM-1 and ICAM-1 expression but also ROS production through decreased p66shc expression, which improves vascular endothelial cell function and prevents endothelial dysfunction related to vascular inflammation.

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