Yoda1 Only Partially Models Laminar Flow Effects on Endothelial Cells

Introduction: A single layer of endothelial cells (ECs) lines the innermost part of blood vessels. Blood flow in vessels exposes the EC to shear stress, which is essential for vascular health. Endothelial cells have multiple mechanosensors that will convert this mechanical stimulus into biochemical signals that modify cell function. Piezo1 is a mechanosensitive ion channel that opens in response to shear stress. Yoda1, a Piezo1 agonist, can also be used to open the ion channel. However, it remains unclear which aspects of EC response to shear stress are effectively reproduced with Yoda1 treatment. In this study, we therefore examined how EC morphology and function change in response to either Yoda1 treatment or steady laminar flow. We hypothesized that ECs exposed to Yoda1 would increase expression of the transcription factor KLF-2, which would increase EC nitric oxide production; however, ECs exposed to Yoda1 would not elongate or decrease glycolytic flux, as observed in ECs exposed to laminar flow.

Materials and

Methods: Human umbilical vein endothelial cells (HUVECs) were cultured in Endothelial Growth Media-2. Cells were treated with 2 μM of Yoda1 for 0, 0.5, 1, 3, 6, or 24 hours. Cells treated with DMSO solvent alone in static culture were the controls. qPCR was used to measure gene expression changes. RNA was isolated, then converted into cDNA. Gene expression was quantified using the QuantStudio 7 Flex qPCR System. We used a primer for Krüppel-like Factor 2 (KLF2) with ribosomal protein lateral stalk subunit P0 (RPLP0) as the housekeeper gene. Western blot was used to measure protein levels. Cells were lysed in RIPA buffer, after which proteins were separated on NuPage 4-12% Bis-Tris protein gels, transferred to PVDF membranes, and incubated with primary antibodies for endothelial nitric oxide synthase (eNOS) and p-eNOS, with GAPDH for normalization. Membranes were imaged and band intensity was analyzed with AlphaView software. EC morphology was quantified by fixing cells with 4% paraformaldehyde and labeling their nuclei with Hoechst. Cells were imaged at 20x magnification, and nuclear aspect ratio was quantified using ImageJ. To measure glycolysis, we collected 200 µL of treatment media at 0 and 24 hours and analyzed glucose and lactate concentrations on a YSI 2950 Bioanalyzer. Statistics were done in GraphPad Prism.

Results, Conclusions, and Discussions: ECs exposed to Yoda1 had a statistically significant increase in KLF2 expression after 0.5 and 1 hour of treatment, as compared to the control (p = 0.0018 and 0.0023, respectively). Similarly, ECs exposed to Yoda1 for 30 minutes had a statistically significant increase in eNOS phosphorylation, as compared to the control (p < 0.0001). Shear stress is known to increase KLF2 expression and eNOS phosphorylation. This suggests that Yoda1 treatment does model these aspects of shear stress exposure. However, ECs exposed to Yoda1 did not show a change in nuclear elongation, which has been show to increase with shear stress. Our YSI data showed an increase in glucose uptake and a statistically significant increase in lactate production with Yoda1 treatment, suggesting that Yoda1 treatment actually increases glycolysis instead of decreasing glycolysis, as occurs in EC exposed to shear stress. Thus, our data suggest that Yoda1 treatment models some, but not all EC changes in response to shear stress.
In the future, we will explore additional EC functions, including inflammation, and reactive oxygen species. We will also elucidate which pathways are differently activated by shear stress and Yoda1. This study will help us understand which EC functions are modulated through which mechanosensors, which is essential to understanding EC mechanobiology and developing mechanotherapies for cardiovascular disease.

Acknowledgements (Optional): This work is supported by NIH R01HL165193 and Clark Doctoral Fellowship.

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