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Apixaban Downregulates Endothelial Inflammatory and Prothrombotic Phenotype in an In Vitro Model of Endothelial Dysfunction in Uremia

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Abstract

Purpose

Chronic kidney disease (CKD) associates with inflammatory and prothrombotic phenotypes, resulting in higher cardiovascular risk. Factor Xa displays functions beyond coagulation, exhibiting proinflammatory effects. The aim of the present study was to investigate whether a direct FXa inhibitor protects from the endothelial dysfunction (ED) caused by uremia.

Methods

Macro (HUVEC) and microvascular (HMEC) endothelial cells (ECs) were exposed to serum from uremic patients or healthy donors, in absence and presence of apixaban (60 ng/ml). We evaluated changes in surface VCAM-1 and ICAM-1, intracellular eNOS, reactive oxygen species (ROS), and von Willebrand Factor (VWF) production by immunofluorescence, reactivity of the extracellular matrix (ECM) towards platelets, and intracellular signaling.

Results

ECs exposed to uremic serum triggered dysregulation of all the parameters. Presence of apixaban resulted in decreased expression of VCAM-1 (178 ± 14 to 89 ± 2% on HMEC and 324 ± 71 to 142 ± 25% on HUVEC) and ICAM-1 (388 ± 60 to 111 ± 10% on HMEC and 148 ± 9% to 90 ± 7% on HUVEC); increased eNOS (72 ± 8% to 95 ± 10% on HMEC); normalization of ROS levels (173 ± 21 to 114 ± 13% on HMEC and 165 ± 14 to 127 ± 7% on HUVEC); lower production of VWF (168 ± 14 to 92 ± 4% on HMEC and 151 ± 22 to 99 ± 11% on HUVEC); and decreased platelet adhesion onto ECM (134 ± 22 to 93 ± 23% on HMEC and 161 ± 14 to 117 ± 7% on HUVEC). Apixaban inhibited p38MAPK and p42/44 activation in HUVEC (139 ± 15 to 48 ± 15% and 411 ± 66 to 177 ± 57%, respectively) (p < 0.05 vs control for all parameters).

Conclusion

Anti-FXa strategies, such as apixaban, prevented ED caused by the uremic milieu, exhibiting anti-inflammatory and antioxidant properties and modulating the reactivity of the ECM.

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Data Availability

The data that support the findings of this study are available from the corresponding author, M.D-R, upon reasonable request.

Abbreviations

FXa:

Activated factor X

DOACs:

Direct oral anticoagulants

PARs:

Protease-activated receptors

ECs:

Endothelial cells

ED:

Endothelial dysfunction

CKD:

Chronic kidney disease

ESRD:

End-stage renal disease

ECM:

Extracellular matrix

HUVECs:

Human umbilical vein endothelial cells

HMEC-1:

Human dermal microvascular endothelial cells

C:

Control condition

U:

Uremic condition

A:

Apixaban treatment

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Acknowledgments

We would like to thank the Primary Hemostasis laboratory group from Hospital Clínic de Barcelona for their technical support. We would also like to thank the collaboration of the nurses from Hospital de la Maternitat from Barcelona for collecting and providing us with human umbilical cords from which we obtained primary endothelial cell cultures.

Funding

This study was partially supported by Bristol Myers-Squibb (ERISTA 15), German José Carreras Leukaemia Foundation (grant 11R/2016), Integrated Project in Health Institutes (PIE15/00027), Technology Development Projects in Health 2016 (DTS/00133) and FIS PI19/00888 from the Instituto de Salud Carlos III, Spanish Government; 2017-SGR671 from Generalitat de Catalunya, and CERCA Programme of the Generalitat de Catalunya. CIBEREHD is funded by Instituto de Salud Carlos III.

Author information

Author notes

  1. Sergi Torramade-Moix and Marta Palomo contributed equally to this work.

Authors and Affiliations

  1. Pathology Department, CDB, Hospital Clínic, IDIBAPS, University of Barcelona, Barcelona, Spain

    Sergi Torramade-Moix, Marta Palomo, Didac Jerez, Ana Belen Moreno-Castaño, Gines Escolar & Maribel Diaz-Ricart

  2. Hospital Clinic, Josep Carreras Leukaemia Research Institute, University of Barcelona, Barcelona, Spain

    Marta Palomo

  3. Barcelona Endothelium Team (BET), Josep Carreras Leukemia Research Institute, Barcelona, Spain

    Marta Palomo & Maribel Diaz-Ricart

  4. Nephrology Department, Hospital Clinic, IDIBAPS, University of Barcelona, Barcelona, Spain

    Manel Vera & Aleix Cases

  5. Cardiovascular Institute, Icahn School of Medicine at Mount Sinai, New York, USA

    M Urooj Zafar

  6. Laboratori Experimental de Nefrologia i Trasplantament (LENIT), Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), Barcelona, Spain

    Jordi Rovira & Fritz Diekmann

  7. Red de Investigacion Renal (REDINREN), Madrid, Spain

    Jordi Rovira & Fritz Diekmann

  8. Department of Nephrology and Kidney Transplantation, Institut Clinic de Nefrologia i Urologia (ICNU), Hospital Clinic de Barcelona, Barcelona, Spain

    Fritz Diekmann

  9. Barcelona Hepatic Hemodynamic Laboratory, Liver Unit, Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain

    Joan Carles Garcia-Pagan

  10. Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Universidad de Barcelona, Barcelona, Spain

    Joan Carles Garcia-Pagan

  11. Health Care Provider of the European Reference Network on Rare Liver Disorders (ERN-Liver), University of Barcelona, Barcelona, Spain

    Joan Carles Garcia-Pagan

Authors
  1. Sergi Torramade-Moix
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  2. Marta Palomo
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  3. Manel Vera
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  4. Didac Jerez
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  5. Ana Belen Moreno-Castaño
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  6. M Urooj Zafar
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  9. Joan Carles Garcia-Pagan
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  10. Gines Escolar
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  11. Aleix Cases
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  12. Maribel Diaz-Ricart
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Contributions

S Torramade-Moix and M Palomo designed the research, performed the experiments, analyzed the results, and wrote the paper. M Vera, D Jerez-Dolz, and AB Moreno performed the experiments and analyzed the results. MU Zafar, J Rovira, and F Diekmann revised the manuscript. JC Garcia-Pagan, G Escolar, A Cases, and M Diaz-Ricart designed the research and revised the manuscript.

Corresponding author

Correspondence to Maribel Diaz-Ricart.

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Conflict of Interest

S.T-M., M.P., M.V., D.J-D., A.B.M-C, M.U.Z., and J.R. have nothing to disclose. F.D. has received lecture fees from Alexion, Chiesi, Novartis, Mallinckrodt, Pfizer, Transplant Biomedical, and Neovii; travel support from Chiesi and Novartis; and grants from TEVA, Chiesi, Pfizer, Novartis, Astellas, and Transplant Biomedical. J.C.G-P. has received speaker fees from GORE, grants from Novartis and Theravance, and has been part of advisory board for Shionogi and Cook. G.E. has received honoraria/consultant fees from Bayer, Bristol Myers-Squibb, Boehringer Ingelheim, CSL Behring, Novo Nordisk, and Pfizer. A.C. has been part of the advisory board for Daiichi Sankyo, has received grants from Bristol Myers-Squibb, and has had lecture fees from Bayer, Bristol Myers-Squibb, and Boehringer. M.D-R. has received grants from Bristol Myers-Squibb and Jazz Pharmaceuticals, and lecture fees from JAZZ pharmaceuticals and Siemens Healthineers.

Ethics Approval

All patients gave their written informed consent to participate in the study, and the study was approved by the Hospital Clinic Ethical Committee of Clinical Investigation.

Consent to Participate

Written informed consent was obtained from all individual participants included in the study.

Consent for Publication

Not applicable.

Code Availability

Density of labeling was calculated by computerized morphometric analysis using image processing and analysis in Java (ImageJ, National Institutes of Health, Bethesda, MD, USA).

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Summary Table

What is known on this topic:

- Targetable factor Xa is involved in thrombogenicity, inflammation, and cellular remodeling.

- Endothelial cells in culture exposed to uremic sera extracted from chronic kidney disease patients exhibit a proinflammatory and prothrombotic phenotype

What this paper adds:

- Apixaban exhibits anti-inflammatory and antioxidant properties on the endothelium under uremic conditions.

- Apixaban modulates uremia-induced endothelial damage.

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Torramade-Moix, S., Palomo, M., Vera, M. et al. Apixaban Downregulates Endothelial Inflammatory and Prothrombotic Phenotype in an In Vitro Model of Endothelial Dysfunction in Uremia. Cardiovasc Drugs Ther 35, 521–532 (2021). https://doi.org/10.1007/s10557-020-07010-z

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