Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Health Research Fund (Fondo de Investigación Sanitaria [FIS]) from the Instituto de Salud Carlos III Introduction Endothelial dysfunction (ED) is pivotal in Chronic Thromboembolic Pulmonary Hypertension (CTEPH) due to structural and functional alterations in endothelial. Previous studies have shown the critical function of cytoskeletal elements, particularly actin microfilaments and microtubules, along with associated cell adhesion complexes, in the endothelial integrity. Nevertheless, precise alterations in the cytoskeletal elements of endothelial cells (ECs) in CTEPH patients remain unclear. Purpose This study aims to investigate the cytoskeleton changes in CTEPH-ECs compared to healthy ECs, characterizing morphological and structural differences. Objectives involve assessing microtubules and microfilaments expression, and their influence on ECs migratory capacity. Finally, our study aims to gain novel insights into the spatial organization of cytoskeleton components in CTEPH patients. Methods CTEPH-ECs were isolated from freshly resected pulmonary endarterectomy specimens of CTEPH patients, while human pulmonary artery ECs (HPAE) were used as healthy controls. Fluorescence immunocytochemistry assessed phenotypic and compositional characteristics of the cytoskeleton. Morphometric analysis described morphology and filament organization of cytoskeletal proteins using a custom-built pipeline. X-ray microscopy examined the phenotype and cytoskeletal components distribution. The migration assay evaluated how these structural differences affected ECs migratory capacity and the cytoskeletal elements expression. Results CTEPH-ECs exhibited larger surface area (1352 vs 662µm2, p<0.01) and a more stellate morphology (0.365 vs 0.289, p<0.01) than HPAE-ECs. Immunostaining indicated altered cytoskeletal composition in CTEPH-ECs with significantly higher tubulin expression, expressed in microtubules, (3327 vs 2238AU, p<0.01) and F-actin microfilaments (2068 vs 1300AU, p<0.01) compared to HPAE. Similarly, CTEPH-ECs showed reduced G-actin expression (3002 vs 7461AU, p<0.01). Morphometric analysis demonstrated that microfilaments were thinner (0.623 vs 0.831AU, p<0.01), more straight (0.135 vs. 0.172, p<0.01) and more aligned (0.132 vs. 0.156, p<0.01) in CTEPH-ECs. Microtubule thickness decreased compared to HPAE-ECs (0.341 vs 1.371AU, p<0.01). CTEPH-ECs showed impaired wound healing (60 vs 89% closure, p<0.05). CTEPH group showed an increase in microtubules and microfilaments expression during wound healing, but with irregular patterns, unlike the constant levels in HPAE. TXM-technology revealed spatial organization differences, especially expansive microtubules distribution in CTEPH-ECs (4.2*10^7 vs. 8.7*10^6 nm³, p<0.05). Conclusion These preliminary findings highlight significant structural and compositional differences in CTEPH-ECs, along with altered wound healing capacity, suggesting dynamic cytoskeletal and cellular changes in CTEPH. Understanding these alterations is crucial for advancing CTEPH knowledge and identifying potential therapeutic targets.

中文翻译：

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慢性血栓栓塞性肺动脉高压的内皮细胞表现出细胞骨架改变


资金致谢 资金来源类型：公共拨款 - 欧盟资金。主要资金来源：健康研究基金 (Fondo de Investigación Sanitaria [FIS])，来自 Instituto de Salud Carlos III 简介 由于内皮细胞结构和功能的改变，内皮功能障碍 (ED) 在慢性血栓栓塞性肺动脉高压 (CTEPH) 中至关重要。先前的研究表明，细胞骨架元件，特别是肌动蛋白微丝和微管，以及相关的细胞粘附复合物在内皮完整性中发挥着关键作用。然而，CTEPH 患者内皮细胞 (EC) 细胞骨架成分的精确变化仍不清楚。目的 本研究旨在研究 CTEPH-EC 与健康 EC 相比的细胞骨架变化，表征形态和结构差异。目标涉及评估微管和微丝表达及其对 EC 迁移能力的影响。最后，我们的研究旨在获得对 CTEPH 患者细胞骨架成分空间组织的新见解。方法从CTEPH患者新鲜切除的肺动脉内膜切除标本中分离CTEPH-ECs，并以人肺动脉ECs（HPAE）作为健康对照。荧光免疫细胞化学评估了细胞骨架的表型和组成特征。形态计量分析使用定制的管道描述了细胞骨架蛋白的形态和丝组织。 X 射线显微镜检查表型和细胞骨架成分分布。迁移测定评估了这些结构差异如何影响 EC 迁移能力和细胞骨架元件表达。 结果 CTEPH-EC 比 HPAE-EC 表现出更大的表面积（1352 vs 662μm2，p<0.01）和更多的星形形态（0.365 vs 0.289，p<0.01）。免疫染色表明，与 HPAE 相比，CTEPH-EC 中的细胞骨架组成发生了改变，微管蛋白表达显着升高（3327 vs 2238AU，p<0.01）和 F-肌动蛋白微丝（2068 vs 1300AU，p<0.01）。类似地，CTEPH-EC 显示 G-肌动蛋白表达降低（3002 vs 7461AU，p<0.01）。形态分析表明，CTEPH-EC 中的微丝更细（0.623 vs 0.831AU，p<0.01）、更直（0.135 vs. 0.172，p<0.01）且更加对齐（0.132 vs. 0.156，p<0.01）。与 HPAE-EC 相比，微管厚度降低（0.341 vs 1.371AU，p<0.01）。 CTEPH-EC 显示伤口愈合受损（60% 与 89% 闭合，p<0.05）。 CTEPH 组在伤口愈合过程中显示微管和微丝表达增加，但模式不规则，与 HPAE 中的恒定水平不同。 TXM 技术揭示了空间组织差异，尤其是 CTEPH-EC 中广泛的微管分布（4.2*10^7 与 8.7*10^6 nm3，p<0.05）。结论 这些初步研究结果强调了 CTEPH-EC 的显着结构和成分差异，以及伤口愈合能力的改变，表明 CTEPH 中细胞骨架和细胞的动态变化。了解这些改变对于推进 CTEPH 知识和确定潜在的治疗靶点至关重要。