Effects Of Asymmetric Dimethylarginine On The Function Of Human Umbilical Vein Endothelial Cells

Cardiovascular disease is a serious disease which threatens the human's health. Multiple investigations indicate endothelial dysfunction has a close correlation with cardiovascular disease in its happening and developing, especially atherosclerosis, hypertension, heart failure, etc. Recently, investigations show that asymmetric dimethylarginine (ADMA) is closely related with endothelial dysfunction. It has been regarded as not only a marker of endothelial dysfunction, but also a potential novel cardiovascular risk factor. ADMA is an endogenous competitive inhibitor of nitric oxide synthase (NOS), synthesized by endothelial cells. ADMA can inhibit NOS activity and make a decrease of endothelium- derived mediator as nitric oxide (NO), result in endothelium-dependent vasodilatation impairment. It also leads to intima thicker and inhibition of angiogenesis. Endothelial dysfunction is a systemic disorder; it is characterized by a reduction of the bioavailability of vasodilators, in particular, nitric oxide (NO), whereas endothelium-derived contracting factors increased. In addition, endothelial dysfunction induced the adhesion of monocytes and leukocytes to endothelium, characterized by increasing expression of adhesion molecular, mainlyintercellular adhesion molecule (1CAM-1) and vascular adhesion molecule (VCAM). Adhesion molecule plays an important role during the development of coronary heart disease (CHD). NO, endothelin-1 (ET-1) and ICAM-1 are often regarded as markers of endothelial function in clinical. L-arginine (L-arg) is a kind of half-necessity amino acid and substrate of NOS. It is also the main substance of NO synthesis. Supplemental L-arg can effectively antagonize the effects of ADMA and improve the endothelial function. The study was designed to determine the effects of ADMA on the expression of NO, soluble intercellular adhesion molecule-1 (sICAM-1) and ET-1 in cultured human umbilical vein endothelial cells (HUVCEs), and observe whether L-arg of different dosage can antagonize the effects of ADMA.Methods HUVECs were cultured in medium DMEM-HG containing 15% fetal calf serum (FCS), taking 6~10th passage cells for the experiment. The cells were seeded at a density of 105 /ml in 96-well culture plates and 24-well culture plates and grew to confluence. Upon confluence, the medium was rejected and replaced with different concentrations of intervention substance after the culture plates were gently washed by PBS two times and DMEM-HG one time. The experiment was divided into two stages: Stage 1 investigated the effects of ADMA on the expression of NO, sICAM-1 and ET-1 in HUVECs after being treated for 24h. It included six groups: ? Control group(C group), with equivalence DMEM-HG medium lacking intervention substance; ?ADMA group I , with medium containing ADMA 1/imol/L; (3)ADMA group II, with medium containing ADMA 4/imol/L; ? ADMA group III, with medium containing ADMA 8/zmol/L; ?ADMA group IV, with medium containing ADMA 12/zmol/L; ? ADMA group V, with medium containing ADMA 16,amol/L. Stage 2 compared the differences of NO, sICAMl and ET-1 after being treated by ADMA16//mol/L or ADMA 16//mol/L with different dosage of L-arg for 24h. It included six groups: ? ADMA group, with medium containing ADMA 16^mol/L; (2) ADMA+L-arg group I , with medium containing ADMA 16^mol/L plus L-arg 0.2mmol/L ;? ADMA+L-arg group II, with medium containing ADMA 16/zmol/L plus L-arg 0.4mmol/L; ? ADMA+L-arg groupIII, with medium containing ADMA16/zmol/Lplus L-arg 0.8mmol/L; ?ADMA+L-arg group IV, with medium containing ADMA 16/zmol/L plus L-arg 1.6mmol/L; ? ADMA+L-arg group V, with medium containing ADMA 16/imol/L plus L-arg 3.2mmol/L. Namely from ADMA+L-arg group I to ADMA+L-arg group V, the added L-arg/ADM A ratios were 12.5:1, 25:1, 50:1, 100:1, 200:1, respectively. The levels of NO, sICAM-1 and ET-1 in conditioned medium were measured by means of Griess Assay, Enzyme-Linked Immunosorbent and radioimmunosorbent respectively. Statistical evaluation of the data was performed using SPSS10.0. F<0.05 was regarded as statistically significance.Results (J) ADMA increased HUVECs to express sICAM-1, ET-1 and decreased HUVECs to generate NO in a concentration-dependent manner, but these effects were gradually dismissed after giving the increasing dosage of L-arg; (2) Compared with the control group, ADMA in a low level, namely ADMA group I had no significant difference in the expression of NO, sICAM-1 and ET-1 (P>0.05). This may be because the level of ADMA is close to physiological condition. Except this group, the other four ADMA groups all had significant difference compared with the control group (P<0.01, respectively). And among the five ADMA groups, there was significant difference each other (P<0.05, respectively); (3) Along with the L-arg/ADMA ratio increasing, the levels of NO, sICAM-1, ET-1 were gradually got normal. Each ADMA+L-arg group all had significant difference compared with the ADMA group (F<0.01, respectively); (4)There was no significant difference between ADMA+L-arg group IV and ADMA+L-arg group V(P>0.05), this may be because the added L-arg/ADMA ratio is big enough to compete with ADMA; (5) The levels of NO, sICAM-1 and ET-1 in conditioned medium were significantly correlated with the levels of ADMA (r=-0.940, 0.941, 0.943, F<0.01, respectively).Conclusions ? ADMA can induce endothelial dysfunction by means of increasing to express sICAM-1, ET-1 and decreasing to generate NO of endothelial cells. The degree of endothelial dysfunction was significantly correlated with the levels of ADMA; (2) Give exogenous L-arg can antagonize the effects of ADMA, but when the added L-arg/ADMA ratio is big enough, the endothelial function basicallyreturn to normal level and can not be improved any more; (3) The new aim to improve endothelial function and treat the cardiovascular disease may be take effective measures to regulate the plasma level of ADM A.