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The P2-receptor-mediated Ca2+ signalosome of the human pulmonary endothelium - implications for pulmonary arterial hypertension

  • Jan K. HennigsEmail author
  • Nicole Lüneburg
  • Annett Stage
  • Melanie Schmitz
  • Jakob Körbelin
  • Lars Harbaum
  • Christiane Matuszcak
  • Julia Mienert
  • Carsten Bokemeyer
  • Rainer H. Böger
  • Rainer Kiefmann
  • Hans Klose
Original Article

Abstract

Dysfunction of the pulmonary endothelium is associated with most lung diseases. Extracellular nucleotides modulate a plethora of endothelial functions in the lung such as vessel integrity, vasodilatation, inflammatory, and thrombotic responses as well as survival and DNA repair, mostly via Ca2+ signaling pathways. However, a comprehensive analysis of the molecular components of the underlying P2 receptor-mediated Ca2+ signaling pathways in the lung has not been conducted so far. Therefore, our aim was to identify the principal P2 receptor Ca2+ signalosome in the human pulmonary endothelium and investigate potential dysregulation in pulmonary vascular disease. Comparative transcriptomics and quantitative immunohistochemistry were performed on publicly available RNA sequencing and protein datasets to identify the specific expression profile of the P2-receptor Ca2+ signalosome in the healthy human pulmonary endothelium and endothelial cells (EC) dysfunctional due to loss of or defective bone morphogenetic protein receptor (BMPR2). Functional expression of signalosome components was tested by single cell Ca2+ imaging. Comparative transcriptome analysis of 11 endothelial cell subtypes revealed a specific P2 receptor Ca2+ signalosome signature for the pulmonary endothelium. Pulmonary endothelial expression of the most abundantly expressed Ca2+ toolkit genes CALM1, CALM2, VDAC1, and GNAS was confirmed by immunohistochemistry (IHC). P2RX1, P2RX4, P2RY6, and P2YR11 showed strong lung endothelial staining by IHC, P2X5, and P2Y1 were found to a much lesser extent. Very weak or no signals were detected for all other P2 receptors. Stimulation of human pulmonary artery (HPA) EC by purine nucleotides ATP, ADP, and AMP led to robust intracellular Ca2+ signals mediated through both P2X and P2Y receptors. Pyrimidine UTP and UDP-mediated Ca2+ signals were generated almost exclusively by activation of P2Y receptors. HPAEC made dysfunctional by siRNA-mediated BMPR2 depletion showed downregulation of 18 and upregulation of 19 P2 receptor Ca2+ signalosome genes including PLCD4, which was found to be upregulated in iPSC-EC from BMPR2-mutant patients with pulmonary arterial hypertension. In conclusion, the human pulmonary endothelium expresses a distinct functional subset of the P2 receptor Ca2+ signalosome. Composition of the P2 receptor Ca2+ toolkit in the pulmonary endothelium is susceptible to genetic disturbances likely contributing to an unfavorable pulmonary disease phenotype found in pulmonary arterial hypertension.

Keywords

ATP UTP CD39 Endothelial dysfunction Purinoceptor Cardiovascular disease Lung Remodeling Proliferation 

Notes

Funding information

This work was supported, in part, by research scholarships to JKH from “Hubertus Wald Tumorzentrum-University Cancer Center Hamburg” and from the “Clinician Scientist Program” of the University Medical Center Hamburg Eppendorf.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Jan K. Hennigs
    • 1
    • 2
    • 3
    Email author
  • Nicole Lüneburg
    • 4
  • Annett Stage
    • 2
    • 4
  • Melanie Schmitz
    • 1
    • 2
    • 3
  • Jakob Körbelin
    • 1
    • 2
    • 3
  • Lars Harbaum
    • 1
    • 2
    • 3
  • Christiane Matuszcak
    • 1
    • 2
    • 3
  • Julia Mienert
    • 1
    • 2
    • 3
  • Carsten Bokemeyer
    • 3
  • Rainer H. Böger
    • 4
  • Rainer Kiefmann
    • 5
  • Hans Klose
    • 1
    • 2
    • 3
  1. 1.Department of PneumologyUniversity Medical Center Hamburg-EppendorfHamburgGermany
  2. 2.Center for Pulmonary Arterial Hypertension HamburgUniversity Medical Center Hamburg-EppendorfHamburgGermany
  3. 3.II. Department of Internal Medicine, Center of OncologyUniversity Medical Center Hamburg-EppendorfHamburgGermany
  4. 4.Institute of Clinical Pharmacology, Center of Experimental MedicineUniversity Medical Center Hamburg-EppendorfHamburgGermany
  5. 5.Department of Anesthesiology, Center of Anesthesiology and Critical Care MedicineUniversity Medical Center Hamburg-EppendorfHamburgGermany

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