Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Rembrandt Institute for Cardiovascular Sciences Background Venous bypass grafts may have limited patency due to excessive intimal hyperplasia and accelerated atherosclerosis. YAP-TAZ signaling regulates cellular responses to biomechanical cues, such as disturbed flow or vessel distention as seen in vein grafts. YAP activation leads to inflammation and angiogenesis, which both contribute to vein graft atherosclerosis. Inhibition of YAP-TAZ attenuates naive atherogenesis, but its potential to inhibit intraplaque angiogenesis and reduce vein graft atherosclerosis has not been explored. Methods Saphenous vein progenitor cells (SVPs) were subjected to mechanical strain in vitro to assess YAP activation. IHC to assess YAP-expression was performed on vein grafts from pigs as well as ex vivo cultured human saphenous veins. Furthermore, YAP-IHC was performed on murine vein grafts from hypercholesterolemic ApoE3*Leiden mice harvested at early, mid- and late-stage disease timepoints to assess optimal treatment window. In a separate experiment, mice (n=11-13/group) underwent bypass surgery and were treated with the FDA-approved YAP-TAZ inhibitor Verteporfin (50 mg/kg) or vehicle 3 times/week from day 10 until sacrifice (day 28). Ultrasound was used to longitudinally quantify vascular remodeling. Vein grafts were harvested and processed for morphometric and compositional analysis. Results Mechanical strain induced activation of YAP and its downstream targets (Ctgf, Cyr61) in SVPs, which was inhibited by Verteporfin (confirmed by WB, IP and qPCR). Moreover, YAP was abundantly expressed in pig vein graft lesions, whilst Verteporfin blocked YAP expression in ex vivo cultured human saphenous veins after exposure to disturbed flow. In addition, YAP was increasingly expressed during vein graft lesion development in mice, reaching its peak around day 14, which coincides with the initiation of intraplaque angiogenesis. Furthermore, YAP was predominantly expressed in luminal and adventitial endothelial cells. Analysis of ultrasound demonstrated that Verteporfin prevented vein graft thickening in mice over time. End-point histology revealed that the intima/media ratio was significantly decreased by Verteporfin treatment (37%, p=0.001). Intraplaque angiogenesis was also reduced (34%, p=0.002), whilst neovessel maturation was improved upon verteporfin treatment. The vessel wall ACTA2 content was reduced (42%, p=0.023), whilst collagen was not altered between both groups, indicating that Verteporfin diminished the fibroproliferative response without affecting vessel wall stability. Conclusion Verteporfin inhibited YAP-TAZ activation induced by mechanical strain in vitro (SVPs) as well as ex vivo (cultured human saphenous veins). In vivo, Verteporfin treatment resulted in a significant decrease of vein graft atherosclerosis and intraplaque angiogenesis. Therefore, therapeutic targeting of YAP-TAZ using Verteporfin might be a new candidate to improve vein graft patency.

中文翻译：

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生物力学激活的 YAP-TAZ 信号传导的治疗性抑制可预防静脉移植动脉粥样硬化


资金致谢 资金来源类型： 公共补助金 – 仅国家预算。主要资金来源：伦勃朗心血管科学研究所 背景 由于内膜过度增生和加速动脉粥样硬化，静脉旁路移植术的通畅性可能有限。 YAP-TAZ 信号传导调节细胞对生物力学信号的反应，例如静脉移植物中出现的血流紊乱或血管扩张。 YAP 激活会导致炎症和血管生成，这两者都会导致静脉移植物动脉粥样硬化。 YAP-TAZ 的抑制作用可减弱幼稚动脉粥样硬化形成，但其抑制斑块内血管生成和减少静脉移植物动脉粥样硬化的潜力尚未被探索。方法 对隐静脉祖细胞 (SVP) 进行体外机械应变以评估 YAP 激活。对猪的静脉移植物以及离体培养的人隐静脉进行 IHC 评估 YAP 表达。此外，对在早期、中期和晚期疾病时间点收获的高胆固醇血症 ApoE3*Leiden 小鼠的鼠静脉移植物进行 YAP-IHC，以评估最佳治疗窗口。在一项单独的实验中，小鼠（n=11-13/组）接受了旁路手术，并使用 FDA 批准的 YAP-TAZ 抑制剂维替泊芬（50 mg/kg）或载体进行治疗，从第 10 天开始每周 3 次，直至处死（第28）。超声用于纵向量化血管重塑。收获静脉移植物并进行处理以进行形态测定和成分分析。结果机械应变诱导 SVP 中 YAP 及其下游靶标（Ctgf、Cyr61）的激活，而维替泊芬可抑制这种激活（经 WB、IP 和 qPCR 证实）。 此外，YAP 在猪静脉移植病变中大量表达，而维替泊芬在体外培养的人隐静脉暴露于扰动的血流后阻断 YAP 表达。此外，YAP 在小鼠静脉移植病变发展过程中表达不断增加，在第 14 天左右达到峰值，这与斑块内血管生成的开始一致。此外，YAP 主要在管腔内皮细胞和外膜内皮细胞中表达。超声分析表明，随着时间的推移，维替泊芬可以防止小鼠静脉移植物增厚。终点组织学显示，维替泊芬治疗后内膜/中膜比率显着降低（37%，p=0.001）。斑块内血管生成也减少（34%，p=0.002），同时维替泊芬治疗后新血管成熟得到改善。血管壁 ACTA2 含量降低（42%，p=0.023），而两组之间的胶原蛋白没有改变，表明维替泊芬减少了纤维增殖反应，而不影响血管壁稳定性。结论 维替泊芬抑制体外（SVP）和离体（培养的人隐静脉）机械应变诱导的 YAP-TAZ 活化。在体内，维替泊芬治疗导致静脉移植动脉粥样硬化和斑块内血管生成显着减少。因此，使用维替泊芬进行 YAP-TAZ 治疗靶向可能是改善静脉移植物通畅的新候选者。