The Primary Study Of The Effect Of Glycosylphosphatidylinositol-specific Phospholipase D On Anti-atherosclerosis

Objective: To investigate effect of the overexpression of glycosyl-phosphatidylinositol-specific phospholipase D (GPI-PLD) gene on the artherosclerosis and to provide theoretical foundation for GPI-PLD gene on anti-atherosclerosis, we detected the activities of the GPI-PLD of coronary heart disease patients and the health adults, created overexpression GPI-PLD Human Umbilical Vein Endothelial Cells model. We studied the chang of expression of T-CAD and measured the Cell proliferation, apoptosis, cell cycle, reactive oxygen species.Method:1. The GPI-PLD activities of 32 coronary artery disease patients and 31 health adult were measured, and the mononuclear cells were separated from those peripheral blood, their expressing of GPI-PLD gene was detected by reverse transcription polymerase chain reaction (RT-PCR).2. The GPI-PLD gene eukaryon expression vector pcDNA3.1 (+) /GPI-PLD produced by our laboratory was transfected into HUVEC cell by lipid-media. After screening G418, the single clone was got. Expression of GPI-PLD mRNA in HUVEC was detected by RT-PCR. The transfected HUVEC was induced by Ox-LDL for 24h, and the HUVEC and HUVEC transfected with pcDNA3.1(+) as control. Before and after the induction, the proliferation of cells was detected by MTT, morphological assessment of apoptosis was examined by flow cytometry, GPI-PLD activity levels of cells was determined through Triton X-114 phase partitioning by using GPI anchored placental the alkaline phosphatase (PLAP), produced by our laboratory, as substrate, ET-1 and the GPI anchored protein T-CAD detected by RT-PCR, The activities of Caspase3, MDA and active oxygen were detected by kit.Results:1. RT-PCR results and GPI-PLD activities showed that the expressing and activity of GPI-PLD of 31 health adults were significantly higher than 32 coronary artery disease patients (P<0. 05).2. RT-PCR results and GPI-PLD activities showed that the HUVEC cell line with stably over-expression of GPI-PLD gene was constructed.①Compared HUVEC cell or pcDNA3.1(+)/HUVEC cell, expression of GPI-PLD mRNA in pcDNA3.1(+)/GPI-PLD/HUVEC cell was significantly up-regulated.②GPI-PLD activities in pcDNA3.1(+)/ GPI-PLD/HUVEC cell were obviously increased, which compared with HUVEC or pcDNA3.1(+)/ HUVEC cell, GPI-PLD activities in three groups were 13.5±0.89, 0.40±0.070 and 0.35±0.089, separately.3. The effects of overexpression of GPI-PLD gene on HUVEC after inducing by Ox-LDL were studied.①Compared with induced HUVEC cell or pcDNA3.1(+)/HUVEC cell, the results about MTT and flow cytometer showed that proliferative capacity of pcDNA3.1(+)/ GPI-PLD/HUVEC cell was obviously increased.②Compared with HUVEC cell or pcDNA3.1(+)/HUVEC cell, the anti-lipid peroxidation of pcDNA3.1(+)/GPI-PLD/HUVEC was obviously increased. However, the levels of celluar reactive oxygen species(ROS) and MDA were significantly decreased (P < 0.01) .③Before the induction, there was no difference in expressing of ET-1, NO and T-CAD among three groups of HUVEC; but after induced by Ox-LDL, the level of NO in the stably transfected GPI-PLD HUVEC cell was significantly increased, and the expressions of ET-1 and T-CAD in the stably transfected GPI-PLD HUVEC cell were lower than HUVEC cell and pcDNA3.1(+)/HUVEC cell.④Compared with induced HUVEC cell or pcDNA3.1(+)/HUVEC cell, the apoptosis rate of the stably transfected GPI-PLD HUVEC cell was significantly decreased and the activities of caspase 3 in these cells were significantly decreased (P < 0.05) .Conclusion:1. The expression and activity of GPI-PLD in 31 health adults were significantly higher than in 32 patients with coronary artery disease, therefore, GPI-PLD expressing maybe execute as a blood vessel protective factors.2. The stable HUVEC cell line with over-expression of GPI-PLD gene was constructed.3. This study preliminarily confirmed that GPI-PLD gene has effects of anti-artherosclerosis and antioxidations. GPI-PLD gene up-regulated GPI-PLD activities, then it hydrolyzed the GPI anchored protein from HUVEC cells, GPI-PLD ulteriorly decreased sensitivity of these cells to Ox-LDL, promoted proliferative capacity of cell , depressed apoptosis and prevented disfuntion. This study provide theoretical and experimental foundation that GPI-PLD was beneficial to proliferation of HUVEC and to damage repair, to prevent cell apoptosis. So that, it is possible a new approach to cure artherosclerosis with GPI-PLD.