Department of Metabolism, Digestion and Reproduction, School of Medical Sciences, Imperial College, London SW7 2AZ, UK.
Department of Clinical, Pharmaceutical and Biological Science, School of Life and Medical Sciences, University of Hertfordshire, Hatfield AL10 9AB, UK.
Int J Mol Sci. 2023 Mar 3;24(5):4946. doi: 10.3390/ijms24054946.
Semicarbazide-sensitive amine oxidase (SSAO) is both a soluble- and membrane-bound transmembrane protein expressed in the vascular endothelial and in smooth muscle cells. In vascular endothelial cells, SSAO contributes to the development of atherosclerosis by mediating a leukocyte adhesion cascade; however, its contributory role in the development of atherosclerosis in VSMCs has not yet been fully explored. This study investigates SSAO enzymatic activity in VSMCs using methylamine and aminoacetone as model substrates. The study also addresses the mechanism by which SSAO catalytic activity causes vascular damage, and further evaluates the contribution of SSAO in oxidative stress formation in the vascular wall. SSAO demonstrated higher affinity for aminoacetone when compared to methylamine (Km = 12.08 µM vs. 65.35 µM). Aminoacetone- and methylamine-induced VSMCs death at concentrations of 50 & 1000 µM, and their cytotoxic effect, was reversed with 100 µM of the irreversible SSAO inhibitor MDL72527, which completely abolished cell death. Cytotoxic effects were also observed after 24 h of exposure to formaldehyde, methylglyoxal and HO. Enhanced cytotoxicity was detected after the simultaneous addition of formaldehyde and HO, as well as methylglyoxal and HO. The highest ROS production was observed in aminoacetone- and benzylamine-treated cells. MDL72527 abolished ROS in benzylamine-, methylamine- and aminoacetone-treated cells (**** < 0.0001), while βAPN demonstrated inhibitory potential only in benzylamine-treated cells (* < 0.05). Treatment with benzylamine, methylamine and aminoacetone reduced the total GSH levels (**** < 0.0001); the addition of MDL72527 and βAPN failed to reverse this effect. Overall, a cytotoxic consequence of SSAO catalytic activity was observed in cultured VSMCs where SSAO was identified as a key mediator in ROS formation. These findings could potentially associate SSAO activity with the early developing stages of atherosclerosis through oxidative stress formation and vascular damage.
氨甲酰基水解酶（SSAO）既是一种可溶性的又是一种膜结合的跨膜蛋白，在血管内皮细胞和血管平滑肌细胞中表达。在血管内皮细胞中，SSAO 通过介导白细胞黏附级联反应，促进动脉粥样硬化的发展；然而，其在血管平滑肌细胞中动脉粥样硬化发展中的贡献尚未得到充分探讨。本研究使用甲胺和氨基丙酮作为模型底物，研究了 VSMCs 中的 SSAO 酶活性。该研究还探讨了 SSAO 催化活性导致血管损伤的机制，并进一步评估了 SSAO 在血管壁氧化应激形成中的作用。与甲胺相比，SSAO 对氨基丙酮的亲和力更高（Km = 12.08 μM 对 65.35 μM）。在浓度为 50 和 1000 μM 时，氨基丙酮和甲胺诱导 VSMCs 死亡，而 100 μM 的不可逆 SSAO 抑制剂 MDL72527 可逆转其细胞毒性作用，完全消除细胞死亡。在 24 小时暴露于甲醛、甲基乙二醛和 HO 后也观察到细胞毒性作用。同时添加甲醛和 HO 以及甲基乙二醛和 HO 后，检测到更强的细胞毒性作用。在氨基丙酮和苄胺处理的细胞中观察到最高的 ROS 产生。MDL72527 消除了苄胺、甲胺和氨基丙酮处理的细胞中的 ROS（**** < 0.0001），而 βAPN 仅在苄胺处理的细胞中显示出抑制潜力（* < 0.05）。用苄胺、甲胺和氨基丙酮处理后，总 GSH 水平降低（**** < 0.0001）；添加 MDL72527 和 βAPN 未能逆转此作用。总之，在培养的 VSMCs 中观察到 SSAO 催化活性的细胞毒性后果，其中 SSAO 被确定为 ROS 形成的关键介质。这些发现可能将 SSAO 活性与通过氧化应激形成和血管损伤导致的动脉粥样硬化早期发展阶段联系起来。
