Abstract：
Vascular smooth muscle cells (VSMCs) are the main stromal cells in the medial layer of the vascular wall. These cells produce the extracellular matrix (ECM) and are involved in many pathological changes in the vascular wall. Semicarbazide-sensitive amine oxidase (SSAO) and lysyl oxidase (LOX) are vascular enzymes associated with the development of atherosclerosis. In the vascular smooth muscle cells, increased SSAO activity elevates reactive oxygen species (ROS) and induces VSMCs death; increased LOX induces chemotaxis through hydrogen peroxide dependent mechanisms; and decreased LOX contributes to endothelial dysfunction. This study investigates the relationship between SSAO and LOX in VSMCs by studying their activity, protein, and mRNA levels during VSMCs passaging and after silencing the LOX gene, while using their respective substrates and inhibitors. At the basal level, LOX activity decreased with passage and its protein expression was maintained between passages. βAPN abolished LOX activity (** p < 0.01 for 8 vs. 3 and * p < 0.05 for 5 vs. 8) and had no effect on LOX protein and mRNA levels. MDL72527 reduced LOX activity at passage 3 and 5 (## p < 0.01) and had no effect on LOX protein, and mRNA expression. At the basal level, SSAO activity also decreased with passage, and its protein expression was maintained between passages. MDL72527 abolished SSAO activity (**** p < 0.0001 for 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 5 (** p < 0.01) and 8 (**** p < 0.0001), and Aoc3 mRNA levels at passage 8 (* p < 0.05). βAPN inhibited SSAO activity (**** p < 0.0001 for 5 vs. 3 and 8 vs. 3 and * p < 0.05 for 5 vs. 8), VAP-1 expression at passage 3 (* p < 0.05), and Aoc3 mRNA levels at passage 3 (* p < 0.05). Knockdown of the LOX gene (**** p < 0.0001 for Si6 vs. Sictrl and *** p < 0.001 for Si8 vs. Sictrl) and LOX protein (** p < 0.01 for Si6 and Si8 vs. Sictrl) in VSMCs at passage 3 resulted in a reduction in Aoc3 mRNA (#### p < 0.0001 for Si6 vs. Sictrl and ### p < 0.001 for Si8 vs. Sictrl) and VAP-1 protein (# p < 0.05 for Si8 vs. Sictrl). These novel findings demonstrate a passage dependent decrease in LOX activity and increase in SSAO activity in rat aortic VSMCs and show an association between both enzymes in early passage rat aortic VSMCs, where LOX was identified as a regulator of SSAO activity, protein, and mRNA expression.
摘要：
血管平滑肌细胞(VSMCs)是血管壁中层的主要基质细胞。这些细胞产生细胞外基质（ECM），并参与血管壁的许多病理变化。氨基脲敏感的胺氧化酶（SSAO）和赖氨酰氧化酶（LOX）是与动脉粥样硬化发展相关的血管酶。在血管平滑肌细胞中，SSAO活性的增加会提高活性氧（ROS）并诱导VSMCs死亡；LOX的增加通过过氧化氢依赖性机制诱导趋化性；LOX的减少有助于内皮功能障碍。本研究通过研究SSAO和LOX在VSMCs中的活性，蛋白质，和mRNA水平在VSMC传代和沉默LOX基因后，同时使用各自的底物和抑制剂。在基础水平，LOX活性随着传代而降低，其蛋白表达在传代之间得以维持。βAPN消除了LOX活性（8与8的**p<0.013和*p<0.05为5vs.8），对LOX卵白和mRNA程度无影响。MDL72527在第3代和第5代降低LOX活性(##p<0.01),对LOX蛋白无影响,和mRNA表达。在基础水平，SSAO活性也随着传代而下降，并且其蛋白表达在传代之间保持。MDL72527取消了SSAO活动(*****p<0.0001，为83和*p<0.05为5vs.8),第5代（**p<0.01）和第8代（***p<0.0001）时的VAP-1表达，和Aoc3mRNA水平在第8代(*p<0.05)。βAPN抑制SSAO活性（****p<0.0001，5vs.3和8vs.3和*p<0.05为5vs.8),第3代VAP-1表达（*p<0.05），和Aoc3mRNA水平在第3代(*p<0.05)。敲除LOX基因（Si6与Si6的****p<0.0001Si8与Si8的Sictrl和***p<0.001Sictrl）和LOX蛋白（Si6和Si8的**p<0.01与第3代VSMC中的Sictrl）导致Aoc3mRNA减少（Si6与###p<0.0001Si8与Si8相比，Sictrl和###p<0.001Sictrl）和VAP-1蛋白（Si8与Sictrl）。这些新发现证明了大鼠主动脉VSMC中LOX活性的传代依赖性降低和SSAO活性的增加，并显示了早期传代大鼠主动脉VSMC中两种酶之间的关联。其中LOX被确定为SSAO活性的调节剂，蛋白质，和mRNA表达。
