Wang Ping, Zheng Lin, Yang Yusi, Yue Xinyang, Liu Jie, Fan Keyi, Zhou Haonan, Dong Honglin
Department of Vascular Surgery, The Second Hospital of Shanxi Medical University, Taiyuan, People's Republic of China.
Department of Cardiology, Third Hospital of Shanxi Medical University, Shanxi Bethune Hospital, Shanxi Academy of Medical Sciences Tongji Shanxi Hospital, Taiyuan, People's Republic of China.
Vasc Health Risk Manag. 2025 Mar 20;21:139-152. doi: 10.2147/VHRM.S487327. eCollection 2025.
Aquaporin 1 (AQP1), a transmembrane water channel protein, has been implicated in the regulation of necroptosis. However, its specific role in atherosclerotic plaque stability through the modulation of necroptosis remains unclear. Therefore, in this study, we aim to investigate whether AQP1 influences necroptosis in atherosclerosis by binding to receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and decreasing the expression of receptor-interacting serine/threonine-protein kinase 3 (RIPK3) and mixed lineage kinase domain-like pseudokinase (MLKL).
The gene expression of AQP1 and necroptosis-associated genes significantly differ between atherosclerosis and normal groups. Genes linked to necroptosis were screened to influence the AS identified by weighted gene coexpression network analysis (WGCNA). Then we collected femoral atherosclerosis and normal aortic samples, further conducted single-cell sequencing and spatial transcriptomic methods to confirm the potential function and pathway of AQP1 in endothelial cells. Meanwhile, we overexpressed AQP1 in ox-LDL-treated endothelial cells in vitro.
Firstly, via single-sample Gene Set Enrichment Analysis (ssGSEA) scores, we found that necroptosis plays the most important role among all ways of programmed cell death in two kinds of atherosclerosis. AQP1, RIPK1, RIPK3 and MLKL express differently in normal and atherosclerosis tissue by differentially expressed gene (DEG) analysis and Western Blot (WB). WGCNA analysis indicates that AQP1, MLKL and RIPK3 were significantly related to the AS. The area under the curve of the above hub genes was greater than 0.8 (AQP1 0.946, RIPK1 0.908, RIPK3 0.988, MLKL 0.863). We found AQP1 highly enriched in endothelial cells (ECs) by single-cell analysis. We sequenced the samples by spatial transcriptome and found that AQP1 was also mainly enriched in ECs both in expression and spatial location. With AQP1 overexpression in ECs, it significantly inhibited the expression of MLKL and RIPK3 and stimulated EC proliferation.
Our study identified that AQP1 suppresses atherosclerotic necroptosis by inhibiting the expression of RIPK3 and MLKL in ECs which might indicates that AQP1 plays a role in atherosclerosis. This new mechanism contributes to improving the diagnostic, prognostic, and therapeutic outcomes of atherosclerosis.
水通道蛋白1（AQP1）是一种跨膜水通道蛋白，与坏死性凋亡的调控有关。然而，其通过调节坏死性凋亡在动脉粥样硬化斑块稳定性中的具体作用仍不清楚。因此，在本研究中，我们旨在探讨AQP1是否通过与受体相互作用的丝氨酸/苏氨酸蛋白激酶1（RIPK1）结合并降低受体相互作用的丝氨酸/苏氨酸蛋白激酶3（RIPK3）和混合谱系激酶结构域样假激酶（MLKL）的表达来影响动脉粥样硬化中的坏死性凋亡。
动脉粥样硬化组和正常组之间AQP1和坏死性凋亡相关基因的基因表达存在显著差异。筛选与坏死性凋亡相关的基因，以影响通过加权基因共表达网络分析（WGCNA）鉴定的动脉粥样硬化。然后我们收集股动脉粥样硬化和正常主动脉样本，进一步采用单细胞测序和空间转录组学方法来确认AQP1在内皮细胞中的潜在功能和途径。同时，我们在体外氧化型低密度脂蛋白（ox-LDL）处理的内皮细胞中过表达AQP1。
首先，通过单样本基因集富集分析（ssGSEA）评分，我们发现在两种动脉粥样硬化中，坏死性凋亡在所有程序性细胞死亡方式中起最重要作用。通过差异表达基因（DEG）分析和蛋白质免疫印迹（WB），AQP1、RIPK1、RIPK3和MLKL在正常组织和动脉粥样硬化组织中的表达不同。WGCNA分析表明，AQP1、MLKL和RIPK3与动脉粥样硬化显著相关。上述核心基因的曲线下面积大于0.8（AQP1 0.946、RIPK1 0.908、RIPK3 0.988、MLKL 0.863）。通过单细胞分析，我们发现AQP1在内皮细胞（ECs）中高度富集。我们通过空间转录组对样本进行测序，发现AQP1在表达和空间位置上也主要富集在ECs中。随着ECs中AQP1的过表达，它显著抑制了MLKL和RIPK3的表达并刺激了ECs增殖。
我们的研究发现，AQP1通过抑制ECs中RIPK3和MLKL的表达来抑制动脉粥样硬化性坏死性凋亡，这可能表明AQP1在动脉粥样硬化中发挥作用。这种新机制有助于改善动脉粥样硬化的诊断、预后和治疗效果。
