Fine tuning between regeneration and anti-apoptosis as a key advantage of using normothermic machine perfusion in intestinal transplantation

Maintaining intestinal barrier integrity is critical to ensuring intestinal transplant (IT) patients’ health. Our research has focused on optimizing allograft viability during the storage period by mimicking a normal physiological environment through normothermic machine perfusion (NMP). In initial studies comparing NMP to traditional cold storage methods, NMP storage reduced apoptotic cells (CC3 ), increased proliferative cells (Ki67 ), and significantly improved recipient animal survival. We therefore hypothesized that NMP improves graft preservation via mechanisms of enhanced epithelial proliferation and reduced apoptosis. Porcine intestine underwent NMP storage for 6h followed by transplantation into a recipient. Biopsies were obtained at donor procurement (T0), following 6h NMP (T6), after 1h of in vivo post-transplant reperfusion (T1RP), and at 48h post-transplant (T48). Mechanisms of epithelial cell apoptosis and regenerative potential were evaluated utilizing transcriptomic and proteomic analyses of crypt derived cells and were confirmed using whole tissue immunofluorescence (IF). Gene biomarker expression of proliferation Ki67 was significantly upregulated in jejunal and ileal crypts at T6 and T1RP with increased KI67 cell counts confirmed by IF. In the ileum, VEGFA upregulation likely enhanced oxygen supply for tissue repair. The intestinal stem cell (ISC) marker ATOH1, critical for secretory lineage differentiation, was downregulated in both the jejunum and ileum, suggesting a shift toward epithelial proliferation over differentiation during early post-transplant phases. Active and reserve ISC markers LGR5 and HOPX, respectively , remained stable in the jejunum and were downregulated in the ileum. Together, this data suggests a role of transit amplifying cell activation in epithelial barrier maintenance. We also found evidence of reduced apoptotic signals at T6 and T1RP, including a significant decrease in ileum CASP3 gene expression and a reduced number of CC3 cells confirmed by IF in jejunum and ileum. Proteomic analyses revealed increased levels of anti-apoptotic proteins such as HYOU1, Regucalcin, and Galectin that regulate stress responses and cell death pathways, promoting cell survival and tissue repair and further corroborating the minimal tissue injury and favorable graft environment of NMP-mediated transplant. This molecular profile of NMP in IT revealed maintenance of epithelial barrier integrity via intracellular mechanisms targeting proliferation and apoptotic pathways. An enhanced renewal characterized by increased proliferation and reduced apoptosis underscores NMP's potential to optimize graft preservation and improve long-term transplantation outcomes. US DOD PR181265, NIH K01OD010199 SERCA, NIH 5T32OD011130-15, R01Al182590-01This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Duke Scholars

Author Andrew Serghios Barbas Surgery, Abdominal Transplant Surgery