(B-79) Red Blood Cell Mechanical Stimulation via Piezo1 as a Novel Target To Enhance Vasculoprotective Nitric Oxide Bioavailability

Introduction::
Diminished nitric oxide bioavailability is the initial hallmark of cardiovascular disease, the leading cause of worldwide death.Nitric oxide is released by both endothelial and red blood cells (RBCs) to promote healthy vascular function [1]. Piezo1 is a novel mechanosensitive Ca²⁺ ion channel found in endothelial and RBCs that triggers nitric oxide production. Endothelial and RBCs under shear stress open piezo1, increasing endothelial nitric oxide synthase (eNOS) phosphorylation and subsequently generating nitric oxide [2-3]. Both endothelial and RBCs contain functional eNOS [1], but little is known about the molecular pathways through which piezo1 stimulation triggers eNOS phosphorylation. Here, we mechanistically elucidate how piezo1 stimulation increases endothelial and RBC eNOS phosphorylation. Understanding how piezo1 facilitates eNOS phosphorylation will inspire novel therapeutics that enhance vascular nitric oxide bioavailability. We hypothesized that piezo1 stimulation increases eNOS phosphorylation through different molecular mechanisms in endothelial and RBCs.




Materials and Methods:: Freshly isolated RBCs were resuspended, and human umbilical vein endothelial cells (HUVECs) were cultured in endothelial basal media-2 (EBM-2) supplemented with 1% penicillin-streptomycin and 1% glutamine. HUVEC and RBCs were then treated with 2µM and 10µM piezo1-agonist yoda1 for 30 minutes. Untreated cells served as controls. We lysed the cells in RIPA buffer containing protease and phosphatase inhibitors and EDTA, separated proteins in 4-12% Bis-Tris gels and then transferred proteins to polyvinylidene difluoride (PVDF) membranes. PVDF membranes were probed using primary antibodies for eNOS, and upstream kinases that phosphorylate eNOS, including protein kinase A (PKA), Akt, protein kinase C (PKC), AMP-activated protein kinase (AMPK), calmodulin-dependent protein kinase II (CamKII), and Erk. GAPDH was used as a housekeeper. We then performed an additional set of Western blots to identify the specific kinase that augments piezo1-induced eNOS phosphorylation. Specifically, HUVEC were treated with 1µM Akt inhibitor MK2206 and 1µM Erk inhibitor SCH772984 before yoda1 treatment, as well as 1µM Akt agonist SC79 without yoda1 treatment. Finally, RBCs were treated with 10µM PKC inhibitor Gö6983 before yoda1 treatment and 1µM PKC agonist phorbol 12-myristate 13-acetate (PMA) without yoda1 treatment.


Results, Conclusions, and Discussions:: Both HUVEC and RBCs treated with piezo1-agonist yoda1 increased p-eNOS (Fig 1A-B; p< 0.05). Yoda1 did not increase p-PKA, p-CamKII, and p-AMPK in either HUVEC or RBCs. We found that yoda1 increased p-Akt and p-Erk1/2 in HUVEC and p-PKC in RBCs (Fig 1A-B; p< 0.05). Interestingly, we did not observe an increase in p-PKC in HUVEC or p-Akt in RBCs. These data collectively, suggest that piezo1 stimulation increases HUVEC and RBCs p-eNOS through different mechanisms. Furthermore, treating HUVEC with Akt agonist SC79 increased p-eNOS, but inhibiting yoda1-induced p-Akt using MK2206 did not diminish p-eNOS (Fig 1C). Erk1/2 inhibition using SCH772984 diminished p-Erk1/2 but did not decrease p-eNOS (Fig 1C). This data suggests that piezo1 stimulation does not increase endothelial p-eNOS via Akt or Erk. RBCs treated with PKC agonist PMA increased p-eNOS, and inhibiting RBC yoda1-induced p-PKC using Gö6983 diminished p-eNOS (Fig 1D). This data suggests that piezo1 stimulation increases RBC p-eNOS via PKC.


We show that piezo1 increases RBC, but not endothelial, p-eNOS via PKC. RBCs are a promising therapeutic target because they may produce nitric oxide through different mechanisms than endothelial cells. Future work will manipulate the RBC piezo1-PKC-eNOS pathway to control nitric oxide production, and elucidate how piezo1 stimulation increases p-eNOS in endothelial cells.




Acknowledgements (Optional): : Funding was provided to G.S.S by the AHA Postdoctoral Fellowship (916512), C.M.W. through the National Science Foundation Graduate Research Fellowship Program (DGE 1840340), and A.M.C by NIH (R01HL140239-0).

References (Optional): : [1] Leo, F., Circulation, 114(2021):870-889, [2] Davies, J. E., Biochem Pharmacol, 168 (2019): 473-480. [3] Kuck., L., Am. J. Physiol. Heart Circ. Physiol, 323.1 (2022): H24-H37