The Role of Endothelial Cell Morphology and Alignment in the Coordinated Regulation of AKT-mediated and Calcium-mediated eNOS Activation
Authors:   , Bhattacharyya Aparna , Barbee Kenneth A 
Published: November 2022

Dysfunctional regulation of endothelial nitric oxide synthase (eNOS) is a hallmark of atherosclerosis which often emerges preferentially in arterial branches and curvatures where endothelial cells (ECs) are exposed to low, non-laminar shear stress. In contrast to their prototypical aligned and elongated morphology, ECs residing in branched and curved segments display a polygonal morphology and random orientation and produce lower levels of the atheroprotective molecule, nitric oxide (NO). In vitro investigations of topography-aligned ECs have demonstrated their capacity to reproduce some of the salient features of aligned ECs in vivo. Many of these investigations are limited by their focus on cell behavior individually rather than in a monolayer. Additionally, there is a paucity of data on the mechanisms driving morphology-mediated EC phenotypes in monolayer cultures. In this study, we sought to identify the mechanisms by which endothelial cell (EC) morphology and alignment modulate AKT-mediated and calcium-mediated activation of eNOS. Using grooved polydimethylsiloxane substrates, ECs were aligned and elongated into a monolayer to resemble the atheroprotective morphology of healthy endothelium. VEGF-stimulated eNOS, AKT and FAK phosphorylation were concurrently upregulated to a greater extent in aligned ECs compared with unaligned ECs. By inhibiting myosin II-generated cytoskeletal tension, we established a role for contractility as a prerequisite to VEGF-stimulated eNOS phosphorylation. Basal and stimulated responses to VEGF in the presence of ROCK inhibitor were dissimilar between unaligned and aligned ECs pointing to a fluid role for ROCK in eNOS regulation. Suppression of FAK tyr397 phosphorylation proved to be detrimental to VEGF-stimulated AKT and eNOS activation. This established a scheme in which the actions of VEGF on eNOS act first via induction of cytoskeletal contraction, second via activation of FAK, and finally through activation of AKT. We next investigated the role of EC morphology and alignment in the regulation of Ca²⁺ mobilization and Ca²⁺-mediated eNOS activation. Relative to unaligned cells, aligned cells produced a more robust Ca²⁺ influx and eNOS activation in response to Ca²⁺ stimulating agonists: VEGF and ATP. Aligned cells were more sensitive to the absence of extracellular calcium, displaying a significant reduction in peak and sustained Ca²⁺ in response to ATP and VEGF. This suggested a more prominent role for store-operated calcium entry in aligned cells. We then measured VEGF-stimulated phosphorylation changes in eNOS, AKT and FAK in the absence of extracellular Ca²⁺ or in the presence of a Ca²⁺ chelator to identify coordinated effects of AKT-mediated signaling and Ca²⁺-mediated signaling. Intracellular Ca²⁺ was shown to be a requirement for the activation of all components of the phosphorylation cascade. The absence of extracellular Ca²⁺ had a moderate effect on reduction of peak phosphorylation of eNOS, AKT and FAK. Together these data support a model in which VEGF cooperatively stimulates activation of eNOS in aligned ECs, via heightened contractility and enhanced Ca²⁺ mobilization. The interplay between contractility and Ca²⁺ likely creates a positive feedback system and coordinately sustains eNOS activation in a heightened state relative to that in unaligned cells. Intriguingly, aligned ECs displayed some morphological and phenotypic features similar to those reported in investigations of flow-adapted ECs. These include prominent ventral stress fiber formation, upregulation of VEGFR2 expression, long-term elevation of stimulated eNOS-phosphorylation, and polarization of caveolar signaling domains. This study provides the first investigation of the mechanisms through which ECs aligned in a monolayer by topography respond to chemical stimuli. For the first time, it is shown that aligned ECs modulate VEGF-stimulated eNOS activation through robust Ca²⁺ influx and cytoskeletal contractility-mediated FAK activation. This work also highlights the utility of topographically regulating EC phenotype in order to better inform drug development and vascular stent design for the treatment of cardiovascular diseases.