Department of Cardiology (T.A., T.S., Y.Y., R.F., Y.A., K.N., S.H., S.S., M.I.), Institute of Medicine, University of Tsukuba, Ibaraki, Japan.
Department of Respiratory Medicine (T.A., N.H.), Institute of Medicine, University of Tsukuba, Ibaraki, Japan.
Arterioscler Thromb Vasc Biol. 2023 Sep;43(9):1668-1683. doi: 10.1161/ATVBAHA.123.319266. Epub 2023 Aug 3.
The mechanisms underlying pulmonary hypertension (PH) remain largely unknown; further, why advanced vascular remodeling preferentially occurs in arterioles is yet to be answered. VEGF (vascular endothelial growth factor) regulates angiogenesis through Flk1 (fetal liver kinase 1) and Flt1 (fms-like tyrosine kinase 1) on endothelial cells (ECs), which may be related to PH pathogenesis. However, spatiotemporal expression patterns of Flk1 and Flt1 in the pulmonary vascular system and the role of endothelial Flk1 in PH development remain poorly understood.
We analyzed multiple reporter mice, including Flk1-GFP (green fluorescent protein) bacterial artificial chromosome transgenic (Tg), Flt1-DsRed bacterial artificial chromosome Tg, and Flk1-GFP/Flt1-DsRed double Tg mice, to determine the spatiotemporal expression of Flk1 and Flt1 in hypoxia-induced PH. We also used Cdh5/Flk1/Tomato (Flk1-KO [knockout]) mice to induce EC-specific Flk1 deletion and lineage tracing in chronic hypoxia.
Flk1 was specifically expressed in the ECs of small pulmonary vessels, including arterioles. Conversely, Flt1 was more broadly expressed in the ECs of large- to small-sized vessels in adult mouse lungs. Intriguingly, Flk1 ECs were transiently increased in hypoxia with proliferation, whereas Flt1 expression was unchanged. Flk1-KO mice did not exhibit pulmonary vascular remodeling nor PH in normoxia; however, the arteriolar ECs changed to a cuboidal shape with protrusion. In hypoxia, Flk1 deletion exacerbated EC dysfunction and reduced their number via apoptosis. Additionally, Flk1 deletion promoted medial thickening and neointimal formation in arterioles and worsened PH. Mechanistically, lineage tracing revealed that neointimal cells were derived from Flk1-KO ECs. Moreover, RNA sequencing in pulmonary ECs demonstrated that Flk1 deletion and hypoxia synergistically activated multiple pathways, including cell cycle, senescence/apoptosis, and cytokine/growth factor, concomitant with suppression of cell adhesion and angiogenesis, to promote vascular remodeling.
Flk1 and Flt1 were differentially expressed in pulmonary ECs. Flk1 deficiency and hypoxia jointly dysregulated arteriolar ECs to promote vascular remodeling. Thus, dysfunction of Flk1 ECs may contribute to the pathogenesis of advanced vascular remodeling in pulmonary arterioles.
肺动脉高压（PH）的发病机制尚不清楚；此外，为什么高级血管重构优先发生在小动脉仍未得到解答。血管内皮生长因子（VEGF）通过内皮细胞（ECs）上的 Flk1（胎肝激酶 1）和 Flt1（fms 样酪氨酸激酶 1）调节血管生成，这可能与 PH 的发病机制有关。然而，Flk1 和 Flt1 在肺血管系统中的时空表达模式以及内皮 Flk1 在 PH 发展中的作用仍知之甚少。
我们分析了多种报告小鼠，包括 Flk1-GFP（绿色荧光蛋白）细菌人工染色体转基因（Tg）、Flt1-DsRed 细菌人工染色体 Tg 和 Flk1-GFP/Flt1-DsRed 双 Tg 小鼠，以确定 Flk1 和 Flt1 在缺氧诱导的 PH 中的时空表达。我们还使用 Cdh5/Flk1/Tomato（Flk1-KO [敲除]）小鼠在慢性缺氧时诱导 EC 特异性 Flk1 缺失和谱系追踪。
Flk1 特异性表达于小肺动脉包括小动脉的 ECs 中。相反，Flt1 在成年小鼠肺中大至小血管的 ECs 中广泛表达。有趣的是，Flk1 ECs 在缺氧时短暂增殖增加，而 Flt1 表达不变。Flk1-KO 小鼠在常氧时不会发生肺血管重构或 PH；然而，小动脉 EC 变为有突起的立方体形。在缺氧时，Flk1 缺失通过凋亡加剧 EC 功能障碍并减少其数量。此外，Flk1 缺失促进小动脉中层增厚和新生内膜形成并加重 PH。在机制上，谱系追踪显示新生内膜细胞来源于 Flk1-KO ECs。此外，肺 ECs 的 RNA 测序表明，Flk1 缺失和缺氧协同激活了多个通路，包括细胞周期、衰老/凋亡和细胞因子/生长因子，同时抑制细胞黏附和血管生成，促进血管重构。
Flk1 和 Flt1 在肺 ECs 中差异表达。Flk1 缺失和缺氧共同失调小动脉 ECs 以促进血管重构。因此，Flk1 ECs 的功能障碍可能导致肺小动脉高级血管重构的发病机制。
