Endothelial Function in Response to Topographical Micropatterning

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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.