Maeda Toshinaga, Takeuchi Keisuke, Xiaoling Pang, P Zankov Dimitar, Takashima Naoyuki, Fujiyoshi Akira, Kadowaki Takashi, Miura Katsuyuki, Ueshima Hirotsugu, Ogita Hisakazu
Division of Molecular Medical Biochemistry, Department of Biochemistry and Molecular Biology, Shiga University of Medical Science.
J Atheroscler Thromb. 2014;21(8):839-53. doi: 10.5551/jat.21386. Epub 2014 Apr 8.
Mutations in lipoprotein-associated phospholipase A2 (Lp-PLA2) are related to atherosclerosis. However, the molecular effects of Lp-PLA2 on atherosclerosis have not been fully investigated. Therefore, this study attempted to elucidate this issue.
Monocytes were isolated from randomly selected healthy male volunteers according to each Lp-PLA2 genotype (wild-type Lp-PLA2 [Lp-PLA2 (V/V)], the heterozygous V279F mutation [LpPLA2 (V/F)] and the homozygous V279F mutation [Lp-PLA2 (F/F)]) and differentiated into macrophages. The level of apoptosis in the macrophages following incubation without serum was measured using the annexin V/propidium iodide double staining method, and the underlying mechanisms were further examined using a culture cell line.
The average plasma Lp-PLA2 concentration [Lp-PLA2 (V/V): 129.4 ng/mL, Lp-PLA2 (V/F): 70.7 ng/mL, Lp-PLA2 (F/F): 0.4 ng/mL] and activity [Lp-PLA2 (V/V): 164.3 nmol/min/mL, LpPLA2 (V/F): 100.9 nmol/min/mL, Lp-PLA2 (F/F): 11.6 nmol/min/mL] were significantly different between each genotype, although the basic clinical characteristics were similar. The percentage of apoptotic cells was significantly higher among the Lp-PLA2 (F/F) macrophages compared with that observed in the Lp-PLA2 (V/V) macrophages. This induction of apoptosis was independent of the actions of acetylated low-density lipoproteins. In addition, the transfection of the expression plasmid of V279F mutant Lp-PLA2 into Cos-7 cells or monocyte/macrophage-like U937 cells promoted apoptosis. The knockdown of Lp-PLA2 also increased the number of apoptotic cells. Among the cells expressing mutant Lp-PLA2, the caspase-7 activity was increased, while the activated Akt level was decreased.
The V279F mutation of Lp-PLA2 positively regulates the induction of apoptosis in macrophages and Cos-7 cells. An increase in the caspase-7 activity and a reduction in the activated Akt level are likely to be involved in this phenomenon.
脂蛋白相关磷脂酶A2（Lp-PLA2）突变与动脉粥样硬化有关。然而，Lp-PLA2对动脉粥样硬化的分子作用尚未得到充分研究。因此，本研究试图阐明这一问题。
根据每种Lp-PLA2基因型（野生型Lp-PLA2 [Lp-PLA2 (V/V)]、杂合V279F突变型[LpPLA2 (V/F)]和纯合V279F突变型[Lp-PLA2 (F/F)]）从随机选取的健康男性志愿者中分离单核细胞，并将其分化为巨噬细胞。采用膜联蛋白V/碘化丙啶双染法检测无血清培养后巨噬细胞的凋亡水平，并使用培养细胞系进一步研究其潜在机制。
尽管基本临床特征相似，但各基因型之间的平均血浆Lp-PLA2浓度[Lp-PLA2 (V/V)：129.4 ng/mL，Lp-PLA2 (V/F)：70.7 ng/mL，Lp-PLA2 (F/F)：0.4 ng/mL]和活性[Lp-PLA2 (V/V)：164.3 nmol/min/mL，LpPLA2 (V/F)：100.9 nmol/min/mL，Lp-PLA2 (F/F)：11.6 nmol/min/mL]存在显著差异。与Lp-PLA2 (V/V)巨噬细胞相比，Lp-PLA2 (F/F)巨噬细胞中的凋亡细胞百分比显著更高。这种凋亡诱导与乙酰化低密度脂蛋白的作用无关。此外，将V279F突变型Lp-PLA2的表达质粒转染到Cos-7细胞或单核细胞/巨噬细胞样U937细胞中可促进凋亡。敲低Lp-PLA2也会增加凋亡细胞数量。在表达突变型Lp-PLA2的细胞中，半胱天冬酶-7活性增加，而活化的Akt水平降低。
Lp-PLA2的V279F突变正向调节巨噬细胞和Cos-7细胞中凋亡的诱导。半胱天冬酶-7活性增加和活化的Akt水平降低可能与这一现象有关。
