O2197: Annexin-A2+ hepatocytes modulate intestinal failure associated liver disease after small bowel resection in murine model

Background: Short gut syndrome (SGS) is a recognized complication occurring after extensive small bowel resection (SBR). SGS is associated with the development of intestinal failure associated liver disease (IFALD), a leading indication for multivisceral transplantation in pediatric SGS. Advanced stage disease is associated with increased liver-related and all-cause mortality. Annexin A2 is a pleiotropic protein, meaning that its function is context-dependent, and is involved in diverse cellular processes such as cell motility, endocytosis, fibrinolysis, ion channel formation, and cell matrix interactions. It also helps to organize exocytosis of intracellular proteins to the extracellular domain. Previously, we identified a cluster of annexin A2+ (ANXA2) hepatocytes with transcriptional alterations associated with fibrosis using single nuclear RNA sequencing in a TPN-independent preclinical model of SBR. The purpose of this study is to directly investigate the role of ANXA2 in the pathogenesis of IFALD.

Methods: 8 week old C57BL/6J (wild type control, WT) female mice and B6J.129X1-Anxa2tm1Kah/Mmmh (ANXA2 knockout, KO) female mice underwent sham (n=6 WT) or 75% proximal SBR (n=7 WT, n=5 ANXA2 KO). Mice were sacrificed at 10 weeks post operatively with collection of serum for serology and livers for histology and RNA isolation for quantitative PCR. Immunofluorescence co-staining was performed with antibodies against ANXA2 (D11G2 at 1:50 dilution, Cell Signaling Technology, Danvers, MA) and albumin (Alb) (A90134 at 1:50 dilution, ThermoFischer, Waltham, MA).

Results: When compared with sham-operated controls, SBR livers showed accumulation of ANXA2+ Alb+ hepatocytes distributed around the central veins as detected by immunofluorescence, while sparing the periportal region (Figure 1A). WT SBR mice demonstrated significant transaminitis (ALT, AST elevations) and increased expression of fibrosis markers collagen (Col1a1) and smooth muscle actin (SMA), while ANXA2 KO SBR mice showed a significant reduction in both transaminase levels and fibrosis markers when compared to WT controls (Figure 1B, 1C). WT SBR livers demonstrated increased expression of superoxide reductase gene NAD(P)H quinone dehydrogenase (NQO1) and ubiquitin binding protein/oxidative stress sensor sequestosome 1 (p62) by q-RT-PCR when compared to sham, suggesting that SBR leads to oxidative stress injury and an ER stress response. By contrast, ANXA2 KO SBR livers revealed an increase in NQO1 expression and decreased p62 expression compared to B6 SBR mice, suggesting that ER stress is attenuated with loss of ANXA2, but not oxidative stress (Figure 1D).

Conclusion: Annexin A2 is increasingly expressed in hepatocytes in the pericentral region in SBR induced liver injury in WT control mice. Germline knockout of ANXA2 reduces transaminitis and fibrosis markers while increasing expression of NQO1 and decreasing expression of p62. These findings suggest that ANXA2 may regulate responses to oxidative and ER stress in SBR-induced liver injury.