Aims Potential loss-of-function variants of ATP13A3, the gene encoding a P5B-type transport ATPase of undefined function, were recently identified in pulmonary arterial hypertension (PAH) patients. ATP13A3 is implicated in polyamine transport but its function has not been fully elucidated. Here, we sought to determine the biological function of ATP13A3 in vascular endothelial cells and how PAH-associated variants may contribute to disease pathogenesis. Methods and Results We studied the impact of ATP13A3 deficiency and overexpression in endothelial cell (EC) models (human pulmonary ECs, blood outgrowth ECs (BOECs) and HMEC-1 cells), including a PAH patient-derived BOEC line harbouring an ATP13A3 variant (LK726X). We also generated mice harbouring an Atp13a3 variant analogous to a human disease-associated variant to establish whether these mice develop PAH. ATP13A3 localised to the recycling endosomes of human ECs. Knockdown of ATP13A3 in ECs generally reduced the basal polyamine content and altered the expression of enzymes involved in polyamine metabolism. Conversely, overexpression of wild-type ATP13A3 increased polyamine uptake. Functionally, loss of ATP13A3 was associated with reduced EC proliferation, increased apoptosis in serum starvation and increased monolayer permeability to thrombin. Assessment of five PAH-associated missense ATP13A3 variants (L675V, M850I, V855M, R858H, L956P) confirmed loss-of-function phenotypes represented by impaired polyamine transport and dysregulated EC function. Furthermore, mice carrying a heterozygous germ-line Atp13a3 frameshift variant representing a human variant spontaneously developed a PAH phenotype, with increased pulmonary pressures, right ventricular remodelling and muscularisation of pulmonary vessels. Conclusion We identify ATP13A3 as a polyamine transporter controlling polyamine homeostasis in ECs, deficiency of which leads to EC dysfunction and predisposes to PAH. This suggests a need for targeted therapies to alleviate the imbalances in polyamine homeostasis and EC dysfunction in PAH.

中文翻译：

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ATP13A3 变异通过破坏多胺转运促进肺动脉高压

目的 最近在肺动脉高压 (PAH) 患者中发现了 ATP13A3 的潜在功能丧失变异，ATP13A3 是编码功能未定义的 P5B 型转运 ATP 酶的基因。 ATP13A3 参与多胺转运，但其功能尚未完全阐明。在这里，我们试图确定 ATP13A3 在血管内皮细胞中的生物学功能以及 PAH 相关变异如何促进疾病发病机制。方法和结果我们研究了 ATP13A3 缺乏和过度表达在内皮细胞 (EC) 模型（人肺 EC、血液生长 EC (BOEC) 和 HMEC-1 细胞）中的影响，包括来自 PAH 患者的含有 ATP13A3 变异的 BOEC 系（ LK726X）。我们还培育了携带 Atp13a3 变体（类似于人类疾病相关变体）的小鼠，以确定这些小鼠是否会患上 PAH。 ATP13A3 定位于人类 EC 的回收内体。 EC 中 ATP13A3 的敲低通常会降低基础多胺含量并改变参与多胺代谢的酶的表达。相反，野生型 ATP13A3 的过度表达会增加多胺的摄取。从功能上来说，ATP13A3 的缺失与 EC 增殖减少、血清饥饿时细胞凋亡增加以及凝血酶单层通透性增加有关。对五种 PAH 相关错义 ATP13A3 变体（L675V、M850I、V855M、R858H、L956P）的评估证实了以多胺转运受损和 EC 功能失调为代表的功能丧失表型。此外，携带代表人类变异的杂合种系 Atp13a3 移码变异的小鼠自发地形成 PAH 表型，伴有肺压升高、右心室重塑和肺血管肌肉化。结论 我们确定 ATP13A3 是控制内皮细胞内多胺稳态的多胺转运蛋白，其缺乏会导致内皮细胞功能障碍并易患 PAH。这表明需要靶向治疗来缓解 PAH 中多胺稳态失衡和 EC 功能障碍。