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Endothelial regulation of calmodulin expression and eNOS–calmodulin interaction in vascular smooth muscle

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Abstract

Calmodulin (CaM) is a Ca2+ sensor protein that is required for numerous vascular smooth muscle cell (VSMC) functions. Since CaM is not expressed enough for its many target proteins, factors that modulate its expression and interactions with targets in VSMCs can have extensive effects on vascular functions. VSMCs receive many regulatory inputs from endothelial cells (ECs). However, it is unknown if ECs regulate vascular functions via controlling expression of CaM and its interactions in VSMCs. In this work, we tested the hypothesis that ECs also affect VSMC signaling via regulation of CaM expression and interactions with its target proteins in VSMCs. Using ECs and VSMCs isolated from the same vessels and grown in a co-culture system, we observed that the presence of proliferating ECs significantly upregulates total CaM expression in VSMCs. An imaging module was devised to concurrently measure free Ca2+ and CaM levels in VSMCs in co-culture with ECs. Using indo-1/AM and a CaM biosensor built from a modified CaM-binding sequence of endothelial nitric oxide synthase (eNOS), this system revealed that in response to a generic Ca2+ signal, free Ca2+-bound CaM level is enhanced ~ threefold in VSMCs in co-culture with proliferating ECs. Interestingly, VSMCs express eNOS and eNOS–CaM association in response to the same Ca2+ stimulus is also enhanced ~ threefold in VSMCs co-cultured with ECs. Mechanistically, the endothelium-dependent upregulation of CaM in VSMCs is not affected by inhibition of NO production or endothelin receptors but is prevented by inhibition of vascular endothelial growth factor receptors. Consistently, VEGF-A level is upregulated in VSMCs co-cultured with proliferating ECs. These data indicate a new role of the endothelium in regulating vascular functions via upregulating CaM and its interactions in VSMCs.

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Data availability

The data in this study are available from the corresponding author on reasonable request.

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Funding

This study was supported in part by NIH Grant HL112184 and by Iowa Osteopathic and Educational Research Grant #12-14-01 to QK-T.

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Author notes

  1. Michael G. Stencel

    Present address: Department of Urology, University of Pittsburgh, Pittsburgh, PA, USA

  2. Mark VerMeer

    Present address: DGG Biomolecular Analysis Division, Agilent Technologies, Ames, IA, USA

Authors and Affiliations

  1. Department of Physiology & Pharmacology, Des Moines University Osteopathic Medical Center, 3200 Grand Avenue, Des Moines, IA, 50312, USA

    Michael G. Stencel, Mark VerMeer, Jennifer Giles & Quang-Kim Tran

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  1. Michael G. Stencel
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  2. Mark VerMeer
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Contributions

QT-T conceived the study and designed experiments. MGS, MVM, and JG carried out the experiments and data analysis. QK-T wrote the paper. All authors reviewed the manuscript.

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Correspondence to Quang-Kim Tran.

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Stencel, M.G., VerMeer, M., Giles, J. et al. Endothelial regulation of calmodulin expression and eNOS–calmodulin interaction in vascular smooth muscle. Mol Cell Biochem 477, 1489–1498 (2022). https://doi.org/10.1007/s11010-022-04391-7

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  • DOI: https://doi.org/10.1007/s11010-022-04391-7

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