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Selective Src-Family B Kinases Inhibition Promotes Pulmonary Artery Endothelial Cell Dysfunction

View ORCID ProfileAdam M. Andruska, View ORCID ProfileMd Khadem Ali, Xuefei Tian, View ORCID ProfileEdda Spiekerkoetter
doi: https://doi.org/10.1101/2021.09.27.462034
Adam M. Andruska
1Division of Pulmonary, Allergy, and Critical Care, Stanford Medical School, Stanford, California
2Vera Moulton Wall Center for Pulmonary Vascular Disease, Stanford University, Stanford, California
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Md Khadem Ali
1Division of Pulmonary, Allergy, and Critical Care, Stanford Medical School, Stanford, California
2Vera Moulton Wall Center for Pulmonary Vascular Disease, Stanford University, Stanford, California
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Xuefei Tian
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Edda Spiekerkoetter
1Division of Pulmonary, Allergy, and Critical Care, Stanford Medical School, Stanford, California
2Vera Moulton Wall Center for Pulmonary Vascular Disease, Stanford University, Stanford, California
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  • For correspondence: eddas@stanford.edu
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Abstract

Protein tyrosine kinases (PTKs) are essential for eukaryotic signaling. By targeting select PTKs, the group of drugs known as Tyrosine kinase inhibitors (TKIs) have proven to be effective for treating multiple diseases ranging from cancer to pulmonary fibrosis. However, some TKIs also paradoxically lead to the development of adverse conditions such as pulmonary arterial hypertension (PAH) by promoting endothelial cell dysfunction (ECD). We hypothesize that (1) subsets of PTKs may disproportionately modulate signaling pathways critical for endothelial homeostasis, such as BMPR2 signaling, and (2) inhibiting those pro-endothelial PTKs can promote the development of ECD. Herein we use an agnostic high-throughput siRNA screen to investigate how PTKs affect the canonical BMPR2 signaling pathway. Our major finding is that within the Src-family of non-receptor PTKs, the Src-B family promotes canonical BMPR2 signaling while the Src-A family suppresses it. We focus on two representative members of each family, Lck (for Src-B) and Fyn (for Src-A) that are the strongest activators or inhibitors of BMPR2 signaling in the screen. We confirm that Lck is expressed in the endothelium of pulmonary arteries and show that Lck knockout (termed si-Lck) in pulmonary artery endothelial cells (PAECs) suppresses canonical BMPR2 signaling while Fyn knockout (termed si-Fyn) promotes canonical BMPR2 signaling. Furthermore, Lck and Fyn are responsible for opposing functional behaviors in PAECs: si-Lck promotes apoptosis and interferes with tube formation while si-Fyn suppresses apoptosis and promotes tube formation. After analyzing the whole-transcriptome signature of si-Lck and si-Fyn PAECs we find that in addition to BMPR2 signaling suppression, si-Lck (and not si-Fyn) increases a broad number of ECD markers and increases canonical NF-κβ signaling. In summary, for the first time we show that Src-A and B Family of PTKs exert differential control over key endothelial cell signaling pathways resulting in direct phenotypic consequences. This knowledge may help to guide the design of more precise TKIs which avoid adverse drug reactions brought about through endothelial cell dysfunction.

Competing Interest Statement

The authors have declared no competing interest.

  • Abbreviations

    ALK1
    Activin receptor-Like Kinase 1
    BMPR2
    Bone Morphogenetic Protein Receptor Type 2
    BRE
    BMP Response Element
    DEG
    Differentially Expressed Gene
    EC
    Endothelial Cell
    ECD
    Endothelial Cell Dysfunction
    FYN
    FYN Proto-Oncogene
    HTS
    High Throughput siRNA Screen
    ID1,2, and 3
    Inhibitor of Differentiation 1, 2, and 3
    LCK
    Lymphocyte specific protein tyrosine kinase
    PAEC
    Pulmonary Artery Endothelial Cell
    PAH
    Pulmonary Arterial Hypertension
    PBMC
    Peripheral Blood Mononuclear Cell
    PDGFR
    Platelet derived growth factor receptor
    pSMAD1
    Phosphorylated SMAD-1
    PBS
    Phosphate Buffered Saline
    PTK
    Protein Tyrosine Kinase
    ROS
    Reactive Oxygen Species
    scRNA-seq
    Single Cell RNA Seq
    siRNA
    Small Interfering RNA
    si-Fyn
    PAECs subject to Fyn knockout with si-RNA
    si-Lck
    PAECs subject to Lck knockout with si-RNA
    SRC
    Cellular Sarcoma Related*
    TCR
    T-Cell Receptor
    TKI
    Tyrosine Kinase Inhibitor
    VEGFR
    Vascular Endothelial Growth Factor Receptor
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    Selective Src-Family B Kinases Inhibition Promotes Pulmonary Artery Endothelial Cell Dysfunction
    Adam M. Andruska, Md Khadem Ali, Xuefei Tian, Edda Spiekerkoetter
    bioRxiv 2021.09.27.462034; doi: https://doi.org/10.1101/2021.09.27.462034
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    Selective Src-Family B Kinases Inhibition Promotes Pulmonary Artery Endothelial Cell Dysfunction
    Adam M. Andruska, Md Khadem Ali, Xuefei Tian, Edda Spiekerkoetter
    bioRxiv 2021.09.27.462034; doi: https://doi.org/10.1101/2021.09.27.462034

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