Endothelial Function in Response to Topographical Micropatterning

项目摘要 心血管疾病影响着近一半的美国成年人，是全球主要的死亡原因。 晚期病例通常通过血管移植绕过闭塞血管进行治疗。人造血管移植物 材料由于血栓形成和新生内膜生长而遭受通畅并发症，阻碍了材料的 适用于小直径应用的长期功能。因此，有一种迫切的未得到满足的改进需求。 生物相容的小直径血管移植物，以支持患者的长期结果，并减少复发 干预程序。在人工合成的生物材料上体外建立内皮层 建议作为一种解决方案，因为内皮细胞(ECs)具有防止血栓的动态平衡能力 形成并限制免疫原性。因此，支持EC生长和生长的血管移植材料表面 功能是一个重要的临床需求。ECS积极响应材料表面提示和局部提示 血流动力学流体切应力(FSS)通过以下途径平衡止血、免疫保护和血栓抵抗 各种机械感受器和换能器。而单向FSS诱导形态全细胞 细胞骨架组件的伸长和对齐以及对齐、振荡FSS(位于端到端 血管移植物的侧吻合)诱导细胞骨架随机排列的鹅卵石状EC形态。 KRüppel样因子2(KLF2)和YAP是高度敏感的转录因子 与细胞形状和机械应力有关。KLF2和YAP已被证明调节EC的功能和表型。 KLF2在单向FSS作用下表达上调，导致抗炎表型。YAP是超级- 在振荡FSS下激活，并诱导免疫倾向表型，而在以下条件下保持失活 单向FSS。然而，在缺乏FSS的情况下，转录因子调控在EC形态中的作用 而细胞骨架排列驱动的免疫保护是未知的。EC形态与细胞骨架排列 可以使用地形微模式技术进行控制，而不依赖于血流动力学。我们和 其他研究表明，在静态培养中，地形微图案驱动了抗炎EC表型， 使其成为合成嫁接表面的一种很有前途的工具。对微图案化ECs的研究表明添加了 图案化对单向FSS的好处以及在正交条件下保持细胞伸长的能力 单向流动。这表明微图案化表面上的内皮细胞可能更耐受 振荡流的免疫原性作用；然而，这从未被直接研究过。拟开展的工作旨在(1) 阐明转录因子调控内皮细胞形态驱动功能的机制 与血流动力学效应无关以及(2)确定振荡流对微图案化EC的影响 转录因子调节和免疫原性。确定ECS对地形的响应方式 微图案化是设计用于小直径应用的改进合成血管移植物的关键。

Project Summary Cardiovascular disease affects nearly half of all U.S. adults and is the leading cause of death worldwide. Advanced cases are often treated through vascular grafting to bypass occluded vessels. Synthetic vasculargraft materials suffer from patency complications due to thrombosis and neointimal growth impeding the materials’ long-term function for small-diameter applications. Thus, there is a critical unmet need for improved biocompatible small-diameter vascular grafts in order to support long-term patient outcomes and reduce re- intervention procedures. The in vitro establishment of an endothelial layer on synthetic biomaterials has been suggested to be a solution due to the endothelial cells’ (ECs) homeostatic capabilities to prev ent thrombus formation and limit immunogenicity. Therefore, vascular graft material surfaces which support EC growth and function are a significant clinical need. ECs actively respond to both material surface cues and local hemodynamic fluid shear stress (FSS) to balance hemostasis, immuno-protection, and thrombo-resistance via a variety of mechano-receptors and -transducers. While unidirectional FSS induces morphological whole cell elongation and alignment as well as alignment of cytoskeletal components, oscillatory FSS (located at end-to- side anastomoses of vascular grafts) induces a cobblestoneEC morphology with randomcytoskeletal alignment. Krüppel-like factor 2 (KLF2) and Yes-associated protein (YAP) are transcription factors that are highly sensitive to cell shape and mechanical stresses. KLF2 and YAP have been shown to regulate EC function and phenotype. KLF2 is upregulated under unidirectional FSS, resulting in an anti-inflammatory phenotype. YAP is hyper- activated under oscillatory FSS and induces an immuno-prone phenotype, while remaining inactivated under unidirectional FSS. However, in the absence of FSS, the role of transcription factor regulation in EC morphology and cytoskeletal alignment driven immuno-protection is unknown. EC morphology and cytoskeletal alignment can be controlled using techniques of topographical micropatterning, independent of hemodynamic flow. We and others have shown that topographical micropatterning drivesan anti-inflammatory EC phenotypein static culture, making it a promising tool for synthetic graft surfaces. Studies of micropatterned ECs have shown additive benefits of patterning to unidirectional FSS and the ability to maintain cellular elongation under orthogonal unidirectional flow. This suggests that ECs on micropatterned surfaces may be more resistant to the immunogenic effects of oscillatory flow; yet this has never been directly studied. The proposed work aims to (1) elucidate the mechanism by which transcription factors regulate endothelial morphology driven functions independent of hemodynamic effects and (2) determine the effect of oscillatory flow on micropatterned EC transcription factor regulation and immunogenicity. Determining how ECs respond to topographical micropatterning is critical in designing improved synthetic vascular grafts for small-diameter applications.