ESPN 50th Annual Meeting

ESPN 2017


 
CALCINEURIN INHIBITOR-INDUCED ENDOTHELIAL CELL INJURY AND DYSFUNCTION - A ROLE FOR COMPLEMENT
CHIA WEI TEOH 1 MAGDALENA RIEDL 1 VALENTINA BRUNO 2 Jun Li 1 LISA ROBINSON 1 CHRISTOPH LICHT 1

1- THE HOSPITAL FOR SICK CHILDREN, UNIVERSITY OF TORONTO, TORONTO, ONTARIO, CANADA
2- UNIVERSITY OF NAPLES FEDERICO II, NAPLES, ITALY
 
Introduction:

Calcineurin inhibitors (CNI) are associated with nephrotoxicity, endothelial cell (EC) dysfunction and thrombotic microangiopathy (TMA). Evolving evidence suggests a central role for complement dysregulation in the pathogenesis of CNI-induced TMA. However, the exact mechanism of CNI-induced complement-mediated injury remains unknown. We hypothesize that CNIs impair complement regulation on EC cell surfaces and induce complement-mediated EC injury.

Material and methods:

Complement activation/regulation were assessed by flow cytometry for C3c and surface complement regulators CD46, CD55 and CD59. Complement factor H (CFH) surface binding was assessed by flow cytometry, and its activity assessed by surface CFH cofactor assay. EC cytotoxicity was assessed via LDH assay. EC repair was assessed by scratch wound assay. Blood outgrowth endothelial cells (BOECs) were incubated with cyclosporine (CsA) and subsequently exposed to 50% normal human serum (NHS) or heat inactivated serum (HIS). A previously established method of complement fixation on EC using (blocking) antibody specific to CD59 was utilized.

Results:

The sequence of CsA incubation and 50% NHS resulted in a dose- and time-dependent enhancement of EC complement (C3c) deposition and cell death, exacerbated by serum starvation and anti-CD59 antibody alone. An optimal balance of EC survival and CNI effect was obtained with CsA 10 mcg/ml for 24 hours. CsA led to upregulation of CD46, CD55 and CD59, and decereased CFH surface cofactor activity. Scratch wound healing over 6 hours was significantly impaired by CsA.

Conclusions:

CsA causes dose- and time-dependent increase in complement activation on EC with increased cell death and impaired repair. This effect was enhanced by serum starvation and by anti-CD59 antibody. We found that CsA led to decreased surface CFH cofactor activity and upregulation of surface-bound complement regulators CD46, CD55 and CD59. Our findings suggest a role for CsA-induced, complement-mediated EC injury.