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Abstract: FR-OR121

Non-HLA Antibodies Targeting Angiotensin II Type 1 Receptor and Endothelin 1 Type A Receptors Induce Endothelial Injury via β2-Arrestin Link to mTOR Pathway

Session Information

Category: Transplantation

  • 1901 Transplantation: Basic

Authors

  • Dragun, Duska, University Hospital Charite, Campus Virchow, Berlin, Germany
  • Catar, Rusan, Charité, Universitätsmedizin Berlin, Berlin, Germany
  • Wischnewski, Oskar, Charite, Berlin, Germany
  • Rutz, Claudia, Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), Berlin, Germany
  • Schülein, Ralf, Leibniz-Forschungsinstitut für Molekulare Pharmakologie, Berlin, Germany
  • Kusch, Angelika, Charité, Universitätsmedizin Berlin, Berlin, Germany
Background

Functional non-HLA antibodies targeting G protein-coupled receptors (GPCR) Angiotensin II Type 1 receptor (AT1R) and Endothelin-1 Type A receptor (ETAR) are implicated in the pathogenesis of transplant vasculopathy. While ERK signaling may represent general cellular response to agonist stimulation, the molecular link between receptor stimulation and development of vascular obliteration has not been fully established yet. We hypothesized the involvement of β-arrestins and PI3K/mTOR signaling and assessed functional consequences of AT1R- and ETAR-activation by non-HLA antibodies.

Methods

Human microvascular endothelial cells (HMEC) were stimulated with AT1R-Ab and ETAR-Ab IgG isolated from kidney transplant patients with chronic vasculopathy. Phospho-specific antibodies against ERK and mTOR downstream targets were used to assess activation of mTORC1 and mTORC2. β-arrestin involvement was investigated using RNA silencing and laser scanning microscopy studies in ARRB2.GFP and ETA.myc.Cherry-transfected HEK293 cells. Scratch assay was employed to study effect of non-HLA-antibodies on endothelial repair. Involvement of AT1R/ETAR activation was addressed by use of specific inhibitors.

Results

Signaling activity of both, mTORC1 and mTORC2, was increased after treatment with patient IgG compared to cells treated with IgG from healthy controls. This effect could be inhibited by specific AT1R-/ETAR- blockers. Activation of mTORC1 and mTORC2 were PI3K-dependent and independent from ERK. mTOR inhibitor rapamycin completely abolished activation of mTORC1 and in addition mTORC2 after long term treatment induced by receptor antibodies. Imaging studies revealed that β2- and not β1-arrestin was recruited to ETAR in response to ET1 and transplant patient IgG. Furthermore, AT1R and ETAR downstream signaling to ERK1/2 and mTORC2 was significantly reduced in β2-arrestin silenced HMECs.
Non-HLA antibodies impaired endothelial repair by AT1R and ETAR induced mTORC2 signaling.

Conclusion

We provide evidence that functional AT1R-/ETAR-Abs induce ERK1/2 and mTOR signaling involving β2-arrestins in human microvascular endothelium. Our data may provide a translational rationale for mTOR inhibitors in combination with receptor blocker in patients with non-HLA receptor recognizing antibodies.