Mechanisms of autophagy activation in endothelial cell and their targeting during normothermic machine liver perfusion

Abstract

Ischaemia-reperfusion injury (IRI) is the leading cause of injury seen in the liver following transplantation. IRI also causes injury following liver surgery and haemodynamic shock. The first cells within the liver to be injured by IRI are the liver sinusoidal endothelial cells (LSEC). Recent evidence suggests that LSEC co-ordinate and regulates the livers response to a variety of injuries. It is becoming increasingly apparent that the cyto-protective cellular process of autophagy is a key regulator of IRI. In particular LSEC autophagy may be an essential gatekeeper to the development of IRI. The recent availability of liver perfusion devices has allowed for the therapeutic targeting of autophagy to reduce IRI. In particular normothermic machine liver perfusion (NMP-L) allow the delivery of pharmacological agents to donor livers whilst maintaining physiological temperature and hepatic flow rates. In this review we summarise the current understanding of endothelial autophagy and how this may be manipulated during NMP-L to reduce liver IRI.

Keywords: Autophagy; Ischaemia-reperfusion injury; Liver transplant; Normothermic machine liver perfusion.

Figure 1

Autophagy signalling pathways. Upstream autophagy activation is regulated by the integration of signalling from a number of pathways including AMPK, PI-3K and the Mitogen-Activated Protein Kinases. Phagophore initiation is directly regulated by the serine/threonine protein kinases ULK1 that forms a complex with Beclin 1. Upon initiation, cytoplasmic constituents are enclosed in a double membraned isolation structure known as an autophagosome that is elongated mainly through the action of two ubiquitin-like conjugation systems. Autophagosomes fuse with lysosomes to form autophagolysosomes, where breakdown of the vesicle contents/cargo takes place along with the autophagosome inner membrane. Autophagy can be activated by many stimuli including starvation, toxins, oxidative stress and infections. (Taken from Shan NN et al Targeting autophagy as a potential therapeutic approach for immune thrombocytopenia therapy. Crit Rev Oncol Hematol 2016; 100: 11-15. DOI: 10.1016/j.critrevonc.2016.01.011).

Figure 2

Autophagy activation in endothelial cells. A number of mechanisms potentially regulate autophagy activation in endothelial cells. A decrease in cellular ATP or reduction in growth factors availability leads to the activation of AMP-activated protein kinase (AMPK). Once activated AMPK can inhibit mTOR leading to the activation of ULK1 and hence autophagy activation. In addition decreases in intracellular calcium can activate CaMKK-β leading to mTOR inhibition and autophagy activation. Moreover, Sirt1 can activate autophagy via deacetylation of ATG5, ATG7, ATG8 and increased transcription of FoxO1 and FoxO3 that then regulate the expression ATGs via deacetylation of Akt. Reactive oxygen species (ROS) and shear stress are important regulators of Sirt1 activity.

Figure 3

Autophagy and liver viability following normothermic machine liver perfusion. Immunohistochemical analysis was performed for the autophagy protein LC3B in liver tissue prior to normothermic machine liver perfusion (NMP-L) and after 6 h of NMP-L. Livers deemed viable after NMP-L demonstrated LC3B immunostaining in the hepatic sinusoids prior to commencing NMP-L and at the end of perfusion. Donor livers not fulfilling viability criteria demonstrated no LC3B Immunostaining.