Endothelial Cell Elongation Impacts Thrombogenicity Independent of Fluid Shear Stress.
Authors: Vartanian Keri B , Blakley Brad J , McCarty Owen JT , Hanson Stephen , Hinds Monica T 
Published: April 2020

Abstract
Atherosclerotic vascular disease and dysfunction of endothelial cells (ECs), which form the continuous lining of blood vessels, preferentially develop in regions where blood vessels are bifurcated and curved. In these regions, ECs are exposed to low, oscillatory fluid shear stress (FSS), are cobblestone in morphology, and have an athero-prone phenotype. In contrast, in regions where FSS is high and unidirectional, ECs are elongated parallel to the direction of flow and have an athero-protective phenotype. Although previous research has correlated FSS with EC morphology and phenotype, the effects of dramatic changes in cell morphology alone, i.e., in the absence of FSS differences, on EC functions remain largely unknown. To determine the role of EC shape on cell function, we investigated the regulation of EC hemostatic functions, an important measure of EC dysfunction and atherosclerosis, by elongated and cobblestone ECs (with shape independent of FSS). To separate EC shape from FSS-induced effects, surface engineering was used to create elongated ECs on micropatterned collagen I lanes (25 μm wide with 100 μm spacing). By 24 hrs, ECs elongated on these micropatterned lanes had a comparable shape index and cytoskeletal alignment as ECs elongated by exposure to 24 hrs of 12.5 dyn/cm2 FSS. qtPCR was used to determine the gene expression of the following markers of coagulant/hemostatic functions: tissue factor (TF), tissue factor pathway inhibitor (TFPI), endothelial nitric oxide synthase (eNOS), thrombomodulin (TM), and von Willebrand Factor (vWF). PCR results indicated that EC elongation alone upregulated expression of TF (1.41 ± 0.28) and decreased expression of eNOS (0.78 ± 0.07). vWF was downregulated (0.59 ± 0.11). Micropattern elongated ECs expressed TFPI and TM at levels comparable to cobblestoneappearing ECs (1.04 ± 0.08 and 1.12 ± 0.04, respectively). To determine whether these changes in gene expression had functional consequences, the generation of thrombin (factor X activation, FXa) and platelet adhesion were studied. Micropattern elongated ECs were able to convert more FX to FXa per cell compared to cobblestone ECs (0.88 ± 0.20 and 0.065 ± 0.01 pg/cell, respectively), indicating an increase in TF activity. This data is consistent with the increased TF gene expression seen in micropattern elongated ECs. Platelet adhesion studies also suggested a thrombogenic phenotype for micropattern elongated ECs, with more platelets adhering and spreading per cell on elongated (8.82 ± 1.47) versus cobblestone (4.64 ± 1.49) ECs. Overall, these findings suggest that EC shape is an independent variable that can regulate cell hemostatic functions, such as thrombotic potential. Surprisingly, elongated ECs exhibited a more thrombogenic phenotype, findings that contrast results obtained with FSS-elongated ECs, both in vitro and in vivo. Thus, both cell shape and FSS may play important and in some instances opposing roles in regulating EC hemostatic functions in the maintenance of vascular integrity.