Endothelial microparticles released by activated protein C protect beta cells through EPCR/PAR1 and annexin A1/FPR2 pathways in islets.

Guillaume Kreutter,Philippe Baltzinger,Mohamad Kassem,Laurence Kessler,Lamia Amoura,Florence Toti,Zobairi Fatiha,Geneviève Ubeaud‐Sequier,Blandine Yver,Jean Peluso,Julie Boisrame‐Helms,Ali El Habhab,Malak Abbas

doi:10.1111/jcmm.13191

Guillaume Kreutter, Philippe Baltzinger + Show 11 more

Open Access

https://doi.org/10.1111/jcmm.13191

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Abstract

Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox‐sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin‐secreting Rin‐m5f β‐cells and its role in autocrine and paracrine MP‐mediated cell crosstalk under conditions of oxidative stress. MP from aPC‐treated primary endothelial (EC) or β‐cells were applied to H2O2‐treated Rin‐m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26‐stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR‐1 expression in EC and to a lesser extent in β‐cells. MP from aPC‐treated EC (eMaPC) exhibited high EPCR and annexin A1 content, protected β‐cells, restored insulin secretion and were captured by 80% of β cells in a phosphatidylserine and ANXA1‐dependent mechanism. eMP activated EPCR/PAR‐1 and ANXA1/FPR2‐dependent pathways and up‐regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H2O2‐treated rat islets with increased viability (62% versus 48% H2O2), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets.

Highlights

Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine
Cells were washed three times and incubated with 0.75 mM S2366 and absorbance was measured at 37°C using a spectrophotometer equipped with a kinetics software (Versamax, molecular Device, UK). activated protein C (aPC) activity was calculated by reference to a standard curve (Xigrisâ, 0.2–30 nM)
Drop in insulin secretion, concentrations in supernatant returning to significantly higher values from 0.7 Æ 0.1 ng/ml/100,000 cells in H2O2-treated b-cells to 10 Æ 0.5 ng/ml/100,000 cells (P < 0.001, n = 4, Fig. 1B)

Summary

Introduction

Infused islets and residual exocrine cells from the islet preparation trigger platelet activation, thrombin generation, the initiation of the complement cascade, the production of ROS and cytokines by leukocytes recruited at the vicinity of the islets [7, 9,10,11]. Under such pro-inflammatory conditions, the expression of tissue factor (TF), the cellular initiator of coagulation, is induced in leukocytes, endothelial and insulinsecreting cells that all shed MP bearing the active form of TF thereby promoting coagulation close to the islets [10, 12, 13]. Regardless of the eventual presence of TF, all MP are procoagulant because they expose phosphatidylserine, an anionic phospholipid that constitutes the catalytic surface for blood coagulation complexes and that potentiates TF activity [14]

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