Endothelial Function in Response to Topographical Micropatterning

项目摘要 心血管疾病影响近一半的美国成年人，是全球死亡的主要原因。 晚期病例通常通过血管移植来绕过闭塞的血管进行治疗。人造血管移植物 由于血栓形成和新生内膜生长阻碍材料的 小直径应用的长期功能。因此，对于改进的系统存在关键的未满足的需求。 生物相容性小直径血管移植物，以支持长期患者结局并减少再狭窄。 干预程序。在合成生物材料上的内皮层的体外建立已经被证明是可行的。 由于内皮细胞（EC）具有防止血栓形成的稳态能力， 形成和限制免疫原性。因此，支持EC生长的血管移植材料表面和 功能是一个重要的临床需求。EC积极响应材料表面线索和局部 血流动力学流体剪切应力（FSS）通过以下途径平衡止血、免疫保护和抗血栓性 各种机械感受器和机械转换器。而单向FSS诱导形态学完整细胞 延伸和排列以及细胞骨架成分的排列，振荡的FSS（位于末端到 血管移植物的侧支化）诱导具有随机细胞骨架排列的鹅卵石状EC形态。 Krüppel样因子2（KLF 2）和Yes相关蛋白（雅普）是两种高度敏感的转录因子 细胞形状和机械应力。KLF 2和雅普已被证明可调节EC功能和表型。 KLF 2在单向FSS下上调，导致抗炎表型。雅普是超- 在振荡FSS下激活并诱导免疫倾向表型，而在振荡FSS下保持失活。 单向FSS然而，在缺乏FSS的情况下，转录因子在EC形态中的调节作用 并且细胞骨架排列驱动的免疫保护是未知的。EC形态和细胞骨架排列 可以使用地形微图案化技术来控制，而不依赖于血液动力学流动。我们和 其他人已经表明在静态培养中地形微图案化驱动抗炎EC表型， 使其成为合成移植物表面的有前途的工具。微图案化EC的研究已经显示出添加剂 图案化到单向FSS的益处和在正交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EC可能对微图案化表面的抗氧化性更强。 振荡流的免疫原性效应;但这从未被直接研究过。拟议的工作旨在（1） 阐明转录因子调节内皮形态驱动功能的机制 独立于血流动力学效应和（2）确定振荡流对微图案EC的影响 转录因子调节和免疫原性。确定EC如何响应地形 微图案化在设计用于小直径应用的改进的合成血管移植物中是关键的。

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.