Linking autophagy to endothelial cell immunogenicity in transplant-associated ischemia-reperfusion injury

Abstract

Background: Cold storage (CS) and ischemia-reperfusion injury (IRI) are inevitable consequences of heart transplantation (HTx), primarily affecting the microvascular endothelial cells (ECs) of donor hearts. The nutrient deprivation and oxidative stresses sustained by ECs during CS and IRI activate and trigger alloimmune recognition. ECs adapt to these conditions by utilizing autophagy to maintain cellular homeostasis. Whether autophagy plays a role in EC activation and immunogenicity during CS and IRI in HTx remains unknown.

Methods: Autophagy in murine microvascular cardiac endothelial cells (MCEC) was modulated by genetic (knockout of the autophagy-related gene 5 (Atg5) by CRISPR/Cas9 technology) or pharmacological (rapamycin to induce autophagy and chloroquine to inhibit autophagy) approaches or by nanocarriers encapsulating rapamycin. MCECs were subjected to a previously established cold storage-warm reperfusion (CS-WR) model to mimic CS and IRI. We evaluated a) changes in autophagy by immunoblotting and confocal imaging, b) MCEC activation via pro-inflammatory cytokine and chemokine secretion by ELISA, and c) MCEC immunogenicity by determining T-cell activation in EC-T-cell co-cultures with MCEC-sensitized CD8⁺ T cells. The human relevance of EC autophagy was demonstrated by reanalyzing a recently published single-nucleus RNA sequencing dataset obtained from baseline and cold-preserved human donor hearts.

Results: Autophagy was reduced in MCECs when subjected to CS alone in organ preservation solution and increased early on during WR. The absence of the autophagic machinery in Atg5^-/- MCECs significantly increased MCEC activation and immunogenicity, while pharmacological induction of autophagy with rapamycin significantly mitigated this effect. Additionally, treating MCECs with an organ preservation solution supplemented with rapamycin-loaded nanocarriers during hypothermic CS demonstrated a protective effect in mitigating MCEC immunogenicity. Reanalysis of the single-nucleus RNA sequencing dataset in EC subclusters revealed that cold storage induced an activated EC state characterized by reduced expression of autophagy-related genes, aligning with our in vitro findings.

Conclusions: A novel role of autophagy in MCEC immunogenicity during CS and IRI is implicated. A viable preconditioning approach has also been demonstrated to bolster MCEC autophagy with nanocarriers during CS and mitigate its immunogenicity.

Keywords: Antigen presentation; Autophagy; Endothelial cells; Heart transplantation; Ischemia-reperfusion injury.