The loss of glycocalyx integrity impairs complement factor H binding and contributes to cyclosporine-induced endothelial cell injury

Chia Wei Teoh; Magdalena Riedl Khursigara; Carolina G Ortiz-Sandoval; Jee Woo Park; Jun Li; Arlette Bohorquez-Hernandez; Valentina Bruno; Emily E Bowen; Spencer A Freeman; Lisa A Robinson; Christoph Licht

doi:10.3389/fmed.2023.891513

The loss of glycocalyx integrity impairs complement factor H binding and contributes to cyclosporine-induced endothelial cell injury

Front Med (Lausanne). 2023 Feb 13:10:891513. doi: 10.3389/fmed.2023.891513. eCollection 2023.

Authors

Chia Wei Teoh^{1

2

3

4}, Magdalena Riedl Khursigara^{1

2

3}, Carolina G Ortiz-Sandoval³, Jee Woo Park³, Jun Li³, Arlette Bohorquez-Hernandez³, Valentina Bruno^{1

3

5}, Emily E Bowen⁶, Spencer A Freeman^{3

7}, Lisa A Robinson^{1

2

3

4}, Christoph Licht^{1

2

3

4

8}

Affiliations

¹ Division of Nephrology, The Hospital for Sick Children, Toronto, ON, Canada.
² Department of Paediatrics, University of Toronto, Toronto, ON, Canada.
³ Cell Biology Program, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada.
⁴ Institute of Medical Science, University of Toronto, Toronto, ON, Canada.
⁵ Division of Paediatric Nephrology, Santobono-Pausilipon Children's Hospital, Naples, Italy.
⁶ Bristol Renal, School of Clinical Sciences, University of Bristol, Bristol, United Kingdom.
⁷ Department of Biochemistry, University of Toronto, Toronto, ON, Canada.
⁸ Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada.

Abstract

Background: Calcineurin inhibitors (CNIs) are associated with nephrotoxicity, endothelial cell dysfunction, and thrombotic microangiopathy (TMA). Evolving evidence suggests an important role for complement dysregulation in the pathogenesis of CNI-induced TMA. However, the exact mechanism(s) of CNI-induced TMA remain(s) unknown.

Methods: Using blood outgrowth endothelial cells (BOECs) from healthy donors, we evaluated the effects of cyclosporine on endothelial cell integrity. Specifically, we determined complement activation (C3c and C9) and regulation (CD46, CD55, CD59, and complement factor H [CFH] deposition) as these occurred on the endothelial cell surface membrane and glycocalyx.

Results: We found that exposing the endothelium to cyclosporine resulted in a dose- and time-dependent enhancement of complement deposition and cytotoxicity. We, therefore, employed flow cytometry, Western blotting/CFH cofactor assays, and immunofluorescence imaging to determine the expression of complement regulators and the functional activity and localization of CFH. Notably, while cyclosporine led to the upregulation of complement regulators CD46, CD55, and CD59 on the endothelial cell surface, it also diminished the endothelial cell glycocalyx through the shedding of heparan sulfate side chains. The weakened endothelial cell glycocalyx resulted in decreased CFH surface binding and surface cofactor activity.

Conclusion: Our findings confirm a role for complement in cyclosporine-induced endothelial injury and suggest that decreased glycocalyx density, induced by cyclosporine, is a mechanism that leads to complement alternative pathway dysregulation via decreased CFH surface binding and cofactor activity. This mechanism may apply to other secondary TMAs-in which a role for complement has so far not been recognized-and provide a potential therapeutic target and an important marker for patients on calcineurin inhibitors.

Keywords: alternative pathway; calcineurin inhibitors; complement; cyclosporine; endothelium; heparan sulfate; proteoglycans; thrombotic microangiopathy.

Grants and funding

CWT was funded in part by Restracomp and the Transplant & Regenerative Medicine Centre Fellowship at the Hospital for Sick Children, Toronto, Ontario, Canada.