Ischemia-Reperfusion Injury in a Simulated Lung Transplant Setting Differentially Regulates Transcriptomic Profiles between Human Lung Endothelial and Epithelial Cells

Abstract

Current understanding of mechanisms of ischemia-reperfusion-induced lung injury during lung preservation and transplantation is mainly based on clinical observations and animal studies. Herein, we used cell and systems biology approaches to explore these mechanisms at transcriptomics levels, especially by focusing on the differences between human lung endothelial and epithelial cells, which are crucial for maintaining essential lung structure and function. Human pulmonary microvascular endothelial cells and human lung epithelial cells were cultured to confluent, subjected to different cold ischemic times (CIT) to mimic static cold storage with preservation solution, and then subjected to warm reperfusion with a serum containing culture medium to simulate lung transplantation. Cell morphology, viability, and transcriptomic profiles were studied. Ischemia-reperfusion injury induced a CIT time-dependent cell death, which was associated with dramatic changes in gene expression. Under normal control conditions, endothelial cells showed gene clusters enriched in the vascular process and inflammation, while epithelial cells showed gene clusters enriched in protein biosynthesis and metabolism. CIT 6 h alone or after reperfusion had little effect on these phenotypic characteristics. After CIT 18 h, protein-biosynthesis-related gene clusters disappeared in epithelial cells; after reperfusion, metabolism-related gene clusters in epithelial cells and multiple gene clusters in the endothelial cells also disappeared. Human pulmonary endothelial and epithelial cells have distinct phenotypic transcriptomic signatures. Severe cellular injury reduces these gene expression signatures in a cell-type-dependent manner. Therapeutics that preserve these transcriptomic signatures may represent new treatment to prevent acute lung injury during lung transplantation.

Cellular and molecular mechanisms of cell damage and cell death in ischemia–reperfusion injury in organ transplantation

“…Unsurprisingly, treatment with the PKC inhibitor, δV1-1, during prolonged cold ischemic time inhibited mitochondrial translocation of PKC and p53, and thus prevented apoptosis, while siRNA reduced cytokine production and further inhibited apoptosis [ 21 ]. These reports were later confirmed in human lung epithelial and endothelial cells in a simulated lung transplant setting [ 139 ] as well as in rat models of liver and lung transplantation in which diannexin (a recombinant human annexin V homodimer) was used [ 140 , 141 ]. Following these promising experimental results, treatment with diannexin proved protective in a phase II clinical trial (NCT00615966) in kidney transplant recipients by reducing the incidence of DGF and days on dialysis compared to placebo-treated control group [ 142 ].…”

Peptide Sodium Channels Modulator Mu-Agatoxin-Aa1a Prevents Ischemia-Reperfusion Injury of Cells

“…Reperfusion injury is a multifactorial process leading to extensive tissue destruction [ 12 ]. Although the main tissues and cells affected by IRI are tissues of the cardiovascular and nervous systems, cells of epithelial origin, such as renal tubular epithelium and lung epithelial cells, are also highly susceptible to IRI [ 3 , 13 ]. In our study, we used the CHO-K1 cell culture, which refers to epithelial cells that endogenously express voltage-gated sodium channels which can serve as a basis for studying the mechanisms of prevention of IRI in tissues and organs [ 14 ].…”

Acellular ex vivo lung perfusate silences pro-inflammatory signaling in human lung endothelial and epithelial cells

“…The IR and EVLP cell culture models have been described in detail [ 16 , 24 ]. Briefly, 30,000 cells/well were cultured in 6-well plates until sub-confluent in serum-containing DMEM + 10% FBS (D10) at 37 °C with 5% CO 2 , and then cold preservation was applied by replacing D10 with 4 °C Perfadex ® solution (Vitrolife; Englewood, CO) with 0.3 ml/L Tham and 0.6 ml/L CaCl 2 , and cells were stored in a sealed chamber filled with 50% O 2 for 18 h to mimic cold ischemic time (CIT).…”

“…Over the past two decades, cell culture models have been developed to simulate the static cold ischemic storage and warm reperfusion, and they have been used to investigate molecular mechanisms of lung IR injury [ 14 – 16 ] and to test therapeutic interventions [ 17 – 21 ]. Some novel potential therapeutics examined with cell culture models have further been validated with animal studies [ 18 , 21 – 23 ].…”

“…Some novel potential therapeutics examined with cell culture models have further been validated with animal studies [ 18 , 21 – 23 ]. Saren et al showed distinct gene expression profiles between human lung endothelial and epithelial cells, which were altered during simulated IR in a cell-type-specific manner, especially after prolonged cold ischemia of 18 h [ 16 ]. Recently, these cell culture models were modified to simulate EVLP, and it has been found that adding L-alanyl-L-glutamine to the commonly used EVLP perfusate, Steen solution, can improve basic cellular function and protect porcine lungs during EVLP to yield stable lung function for a prolonged time [ 24 ].…”