The Effects Of Different Trans Fatty Acids Isomers On PRMT/ADMA/DDAH And NOS/NO In HUVEC

Part I The influence of trans fatty acids isomers on survival and apoptosis of human umbilical vein endothelial cells in vitroObjective:Many epidemiological studies indicated that coronary heart disease (CHD) is associated with high intake of trans fatty acids (TFA), but this correlation is dependent on the sources of TFA. Industrially produced TFA (IP-TFA) has been found to correspond to CHD, but ruminant TFA (R-TFA) does not. At present, the mechanisms for the different cardiovascular effects of various TFAs were still unclear. Because the damage of vascular endothelium is the initiating process of CHD, and TFA isomers of IP-TFA are different from those of R-TFA, we give a hypothesis that the TFA isomers contained in IP-TFA may have stronger damage effects on HUVEC than those from R-TFA. The aim of the first part of this study was to investigate whether there were different impacts of five kinds of TFA isomers on the survival and apoptosis of human umbilical vein endothelial cell (HUVEC) in vitro.Method:Five TFA isomers, including trans palmitoleic acid (T-C16:1), trans oleic acid (△6T-C18:1;△9T-C18:1;△11T-C18:1), trans linoleic acid (T-C18:2) were investigated in this study. Six groups were divided according to the TFA isomers used as:control group HUVECs were cultured in general media; T-C16:1group;△6T-C18:1group;△9T-C18:1group;△11T-C18:1group and T-C18:2groups. The latter five groups were treated with corresponding TFA isomer respectively. For evaluating the impacts of TFA isomers on the survival rate of HUVEC, four concentrations of TFA isomers (50umol/1,100umol/1,200umol/1and500umol/1) were used and the HUVEC counts were detected at before (0) and6,12,24hours after TFA intervention.Based on the results of above experiment, TFA of200umol/1was selected as the most proper intervention concentration. The survival and apoptosis rates of HUVEC were detected at0,4,8,12hours.Results:1. Compared with the control group, the survival rates of HUVEC in five TFA treated groups in50umol/1were similar. When TFA of500umol/1concentration was used, the HUVECs in all five treatment groups were almost death at15hours. Significant time and dose-dependent relation was seen in the100umol/l and200umol/l TFA treated groups, and as the TFA of200umol/1is similar to the level in human, TFA of200umol/1was selected as the intervention concentration.2. In200umol/l concentration, five TFA isomers significantly reduced HUVEC survival rate of HUVECs. The survival rate of HUVECs was significantly lower in T-C18.2group than in T-C16:1group at12hours. At24hours, similar changes were observed between△6T-C18:1or A9T-C18:1and T-C16:1groups, but the survival rates between△11T-C18:1group and T-C16:1group were similar.3. In200umol/1concentration, the HUVEC apoptosis rate significantly increased in five TFA isomers groups, but obvious group variations were found. On the decline sequence of apoptosis rate, the order was T-C18:2,△6T-C18:1,△9T-C18:1,△11T-C18:1and T-C16:1. Compared with the T-C16:1group, the apoptosis rates of T-C18:2group,△6T-C18:1and△9T-C18:1group were obviously higher, but no significant difference was seen between△11T-C18:1group and T-C16:1group.Conclusions:1. In100umol/1and200umol/1concentration,T-C16:1,△6T-C18:1,△9T-C18:1,△11T-C18:1and T-C18:2could reduce the survival rate of HUVECs in vitro through increasing apoptosis rate, with time and dose-dependent way.2. In200umol/1concentration, compared with the T-C16:1group, four18-carbon TFA isomers induced significantly higher apoptosis rates of HUVEC, with the order from strong to weak: T-C18:2,△6T-C18:1,△9T-C18:1and△11T-C18:1. It was clear showed that the apoptogenic effects on HUVEC of the four18-carbon TFA isomers were significantly different. 3. Based on above-mentioned data, in it reasonable to suggested that the difference of biological effects of TFA isomers was related to the number and position of the trans double bond.4. Although the trans double bond of△9T-C18:1and T-C16:1molecules located in the same position, their different effects on HUVEC apoptosis rate suggested that the carbon chain length might be a influencing factor of the apoptogenic effect.Part II The influence of trans fatty acids isomers on NOS-NO system of human umbilical vein endothelial cells in vitroObjective:Nitric Oxide (NO) is not only a strong vasodilator molecule, but also an important anti-atherosclerotic molecule. In body, proper NO concentration is needed to maintain the normal function of the cardiovascular system. Nitric oxide synthase (NOS) is key link for NO secretion. So NOS-NO system play an important role in keeping cardiovascular system health. As the apoptogenic effects on HUVEC of the different TFA isomers varied, we give a hypothesis the difference of their apoptogenic effects is associated with the influence of TFA isomers on the NOS-NO system. The aim of the second part of this study was to investigate whether there were different influences of five TFA isomers on NOS-NO in HUVEC in vitro.Method:The experimental groups were the same as in the first part of this study, and the200umol/1concentration of TFA isomers was used also. The NO concentration and total NOS activity in HUVEC culture medium were detected before (0) and6,12,24hours after intervention. The expression levels of endothelial nitric oxide synthase (eNOS) mRNA and protein were detected at24hours by real-time PCR and western blotting respectively. The relation between NO concentration and the apoptosis rate of HUVEC were analyzed by linear correlation. Results:1. Compared with the control group and the basic state, the five TFA isomers significantly reduced NO concentration of HUVEC culture medium. The NO concentration was significantly lower in T-C18:2group than in T-C16:1group at12hours. At24hours, similar changes were also observed in△6T-C18:1,△9T-C18:1and T-C18:2groups, but the NO concentration between△11T-C18:1group and T-C16:1group was similar.2. Compared with the control group and the basic state, the five TFA isomers could reduce total NOS activity of HUVEC. But in time point, the change of total NOS activity was earlier than the time of NO. We could find total NOS activity was significantly lower in△6T-C18:1、△9T-C18:1and T-C18:2groups than in T-C16:1group at6hours, while this change was similar between△11T-C18:1and T-C16:1group.3. There was a negative correlation between the NO concentration and the apoptosis rate of HUVEC (r=-0.949,P<0.05).4. Compared with the control group, five TFA isomers significantly down-regulated the expression of eNOS mRNA. If the expression level of eNOS mRNA in the control group was taken as the reference of100%, the expression level of eNOS mRNA was32%in T-C16:1group;23%in△11T-C18:1group;13%in△6T-C18:1group;8%in△9T-C18:1group;3%only in T-C18:2group. Meanwhile, the expression level of eNOS mRNA was significantly lower in△6T-C18:1、△9T-C18:1and T-C18:2groups than in T-C16:1group. However, the expression level of eNOS mRNA was similar between△11T-C18:1and T-C16:1group.5. Five TFA isomers used in this study significantly down-regulated the expression of eNOS protein. Compared with T-C16:1group, the expression level of eNOS protein were significantly lower in△6T-C18:1,△9T-C18:1and T-C18:2groups. But expression levels of eNOS protein of both△11T-C18:1and T-C16:1groups were similar.Conclusions: 1. In vitro culture conditions, T-C16:1,△6T-C18:1,△9T-C18:1,△11T-C18:1and T-C18:2significantly reduced NO concentration and total NOS activity in HUVEC culture medium, as well as down-regulate the expression of eNOS mRNA and protein.2. Different TFA isomers had different inhibitory effect on NOS-NO system in HUVEC. The inhibitory effect of T-C18:2was the strongest, then the△6T-C18:1and△9T-C18:1, while△11T-C18:1and T-C16:1was the weakest.3. Because there was a negative relation between NO concentration and HUVEC apoptosis rate, it suggested that the various apoptogenic effects of different TFA isomers was based on the different inhibitory effects on NOS-NO system.Part III The influence of trans fatty acids isomers on PRMT/ADMA/DDAH of human umbilical vein endothelial cells in vitroObjective:Asymmetric dimethylarginine (ADMA) plays a very important role in regulating NOS-NO system and is called "the endogenous NOS inhibitor". Protein-arginine methyl-transferase I (PRMT I) is a rate-limiting enzyme of ADMA generation. More than90%of ADMA is metabolized by Dimethylarginine-dimethylamino-hydrolase (DDAH). In the second part of our research, we found that five TFA isomers had different effects on NOS-NO system in HUVEC. So we give a hypothesis that the different effects of five TFA isomers on NOS-NO system is associated with the influences of them on the PRMT-ADMA-DDAH system. The aim of the third part of this study was to investigate whether there were different influences of five TFA isomers on PRMT-ADMA-DDAH in HUVEC.Method:The experimental groups were the same as in the second part of this study, and the200umol/1concentration of TFA isomers was used also. The ADMA concentration and DDAH activity in HUVEC culture medium were detected before (0) and6,12,24hours after intervention. The mRNA and proteins’expression levels of PRMT I, DDAH I and DDAH II were detected at24hours by real-time PCR and western blotting respectively. And the relations between DDAH activity and ADMA concentration, as well as ADMA concentration and NOS activity were analyzed by linear correlation.Results:1. Compared with the control group and the basic state, ADMA concentration of HUVEC culture medium was significantly increased in five TFA groups. At12and24hours, ADMA concentration was significantly higher in△6T-C18:1,△9T-C18:1and T-C18：2groups than in T-C16:1group. But there were difference in the levels of ADMA concentration among five TFA groups, with the order from high to low:T-C18:2,△6T-C18:1,△9T-C18:1,△11T-C18:1and T-C16:1.2. Compared with the control group and the basic state, the DDAH activity of HUVEC culture medium was reduced in five TFA groups. And a negative relation was found between DDAH activity and ADMA concentration (r=-0.975, P<0.05), as well as a negative relation was also found between ADMA concentration and total NOS activity (r=-0.918, P<0.05).3. The mRNA and protein expression levels of PRMT I were similar in five TFA groups.4. Compared with the control group, the mRNA and protein expression levels of DDAH I and DDAH II were down-regulated in five TFA groups. The mRNA and protein expression levels of DDAH I and DDAH II in△6T-C18:1,△9T-C18:1and T-C18:2groups were significantly lower than in T-C16:1group, but the mRNA and protein expression levels of DDAH I and DDAH II were similar between△11T-C18:1and T-C16:1groups.Conclusions:1. T-C16:1,△6T-C18:1,△9T-C18:1,△11T-C18:1and T-C18:2could significantly increase ADMA concentration and decrease DDAH activity of HUVEC.2. The mRNA and protein expression levels of PRMT I were similar in five TFA groups, it suggested that the change of ADMA concentration of HUVEC was not derived from the ADMA synthase.3. The mRNA and protein expression levels of DDAH I and DDAH ⅡI were down-regulated in T-C16:1,△6T-C18:1,△9T-C18:1,△11T-C18:1and T-C18:2groups. So it suggested that the change of ADMA concentration of HUVEC was derived from the decline of ADM A degradation.4. Five TFA isomers had different effects on PRMT-ADMA-DDAH system by increasing ADMA concentration and decreasing DDAH activity. But obvious group variations were found, T-C18:2was the strongest, then the△6T-C18:1and△9T-C18:1followed, while△11T-C18:1and T-C16:1were the weakest.