S. Duarte1, Z. Huo2, U. Kim2, A. Coito1, A. Zarrinpar2 1University Of California – Los Angeles,Surgery,Los Angeles, CA, USA 2University Of Florida,Surgery,Gainesville, FL, USA
Introduction:
Hepatic ischemia/reperfusion injury (IRI) is a major risk factor for acute rejection and early graft dysfunction in liver transplantation. The large and complex landscape of regulatory signaling events in IRI has not been systematically characterized. While many of the factors responsible have been identified, no potent pharmacologic treatments have been developed. To expand the understanding of the hepatic transcriptional regulatory changes during the early stages of IRI, we used RNASeq-based analysis in a mouse warm hepatic IRI model.
Methods:
C57BL/6 mice were subjected to 70% partial warm ischemia for 90min, followed by 15min, 2h, or 6h of reperfusion. This was done in each of the three technical replicates. The baseline was developed using naïve mice. After ischemia and reperfusion injury, hepatic tissues were collected from euthanized mice, homogenized, and RNA was extracted. Generation of cDNA followed by next-generation sequencing allowed the construction of a whole-genome transcriptome map. These reads were then analyzed using HISAT2, StringTie, and Ballgownto align, assemble, and compute the abundance of the transcripts.
Results:
Using this method 29,832 unique reference sequences were identified from 20,565 unique genes. Principal component analysis resulted in clusters of the technical replicates, as well as gradual progression of the transcriptional profile from ischemia to 2 hours after IRI. However, after 6 hours the transcriptional profile was markedly different from the very early stages, indicating a shift in the activated signaling pathways. (Figure) Several characteristic patterns emerged in the transcriptional activity profiles. Oxidative Stress Response and JAK-STAT pathways are highly active in ischemic tissues and then their activity decreases upon reperfusion. Adipogenesis, fatty acid beta oxidation, and the unfolded protein response pathways demonstrate increased activity as early as 15 minutes after reperfusion. After 2 hours the adipogenesis pathway is still highly active but there is an upregulation of macrophage signaling. Finally at 6 hours after reperfusion the transcriptional activity profile is characterized by significantly more pronounced activity of the acute phase response, the sirtuin signaling, and IL-6 signaling pathways.
Conclusion:
This study substantiates the complex activity of regulatory signaling pathways that have important roles in the progression of hepatic IRI. It identifies both previously unexamined pathways and corroborates previously implicated signals that are valuable potential targets for drug therapy. Enhancing or inhibiting these pathways has the potential to rescue IRI to a great extent and act as novel potential drug targets.
