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Suppression of graft regeneration, not ischemia/reperfusion injury, is the primary cause of small-for-size syndrome after partial liver transplantation in mice.

ABSTRACT: BACKGROUND: Ischemia/reperfusion injury (IRI) is commonly considered to play a crucial role in the pathogenesis of small-for-size syndrome (SFSS) after liver transplantation. Rapid regeneration is also considered essential for the survival of SFS grafts. METHODS: Mouse models of full-size orthotopic liver transplantation, 50% partial liver transplantation and 30% partial liver transplantation were established. Survival rate and serum alanine aminotransferase were observed. IRI was assessed by hepatic pathologic alterations, apoptosis and necrosis. Regeneration response was detected by mitotic index, BrdU incorporation and PCNA, Cyclin D1 and Cyclin E expression. The expression of mTOR, AKT, ERK, JNK2 and p70S6K, also involved in regeneration signaling pathways, were analyzed as well. RESULTS: 30% partial liver graft resulted in a significantly low 7-day survival rate (P = 0.002) with no marked difference in tissue injury compared with the 50% partial graft group. Serum alanine aminotransferase levels were not significantly different between partial transplantation and full-size transplantation. Western blot analysis of caspase-3 and TUNEL staining also indicated no significant difference in apoptosis response between 30% partial transplantation and half-size or full-size transplantation (P = 0.436, P = 0.113, respectively). However, liver regeneration response indicators, mitotic index (P<0.0001) and BrdU (P = 0.0022), were markedly lower in 30% LTx compared with 50% LTx. Suppressed expression of PCNA, cyclin D1, cyclin E, mTOR, JNK2, AKT, ERK and p70S6K was also detected by western blot. CONCLUSIONS: Liver regeneration is markedly suppressed in SFSS, and is more likely the primary cause of SFSS, rather than ischemia/reperfusion injury. Therapy for recovering graft regeneration could be a potentially important strategy to reduce the incidence of SFSS.

SUBMITTER: Pan N

PROVIDER: S-EPMC3977924 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMuscle is severely affected by ischemia/reperfusion injury (IRI). Quiescent satellite cells differentiating into myogenic progenitor cells (MPC) possess a remarkable regenerative potential. We herein established a model of local application of MPC in murine hindlimb ischemia/reperfusion to study cell engraftment and differentiation required for muscle regeneration.MethodsA clamping model of murine (C57b/6 J) hindlimb ischemia was established to induce IRI in skeletal muscle. After 2 h (h) warm ischemic time (WIT) and reperfusion, reporter protein expressing MPC (TdTomato or Luci-GFP, 1 × 106 cells) obtained from isolated satellite cells were injected intramuscularly. Surface marker expression and differentiation potential of MPC were analyzed in vitro by flow cytometry and differentiation assay. In vivo bioluminescence imaging and histopathologic evaluation of biopsies were performed to quantify cell fate, engraftment and regeneration.Results2h WIT induced severe IRI on muscle, and muscle fiber regeneration as per histopathology within 14 days after injury. Bioluminescence in vivo imaging demonstrated reporter protein signals of MPC in 2h WIT animals and controls over the study period (75 days). Bioluminescence signals were detected at the injection site and increased over time. TdTomato expressing MPC and myofibers were visible in host tissue on postoperative days 2 and 14, respectively, suggesting that injected MPC differentiated into muscle fibers. Higher reporter protein signals were found after 2h WIT compared to controls without ischemia, indicative for enhanced growth and/or engraftment of MPC injected into IRI-affected muscle antagonizing muscle damage caused by IRI.ConclusionWIT-induced IRI in muscle requests increased numbers of injected MPC to engraft and persist, suggesting a possible rational for cell therapy to antagonize IRI. Further investigations are needed to evaluate the regenerative capacity and therapeutic advantage of MPC in the setting of ischemic limb injury.

Project description:Recruitment of liver sinusoidal endothelial cell progenitor cells (sprocs) from the bone marrow by vascular endothelial growth factor-stromal cell-derived factor-1 (VEGF-sdf-1) signaling promotes recovery from injury and drives liver regeneration. Matrix metalloproteinases (MMPs) can proteolytically cleave VEGF, which might inhibit progenitor cell recruitment, but systemic matrix metalloproteinase inhibition might prevent efflux of progenitors from the bone marrow. The hypothesis for this study was that liver-selective MMP-9 inhibition would protect the hepatic VEGF-sdf-1 signaling pathway, enhance bone marrow sproc recruitment, and thereby ameliorate liver injury and accelerate liver regeneration, whereas systemic MMP inhibition would impair bone marrow sproc mobilization and therefore have less benefit or be detrimental. We found that liver-selective MMP-9 inhibition accelerated liver regeneration after partial hepatectomy by 40%, whereas systemic MMP inhibition impaired liver regeneration. Liver-selective MMP-9 inhibition largely abolished warm ischemia-reperfusion injury. In the extended hepatectomy model, liver-selective MMP-9 inhibition restored liver sinusoidal endothelial cell integrity, enhanced liver regeneration, and reduced ascites. Liver-selective MMP-9 inhibition markedly increased recruitment and engraftment of bone marrow sprocs, whereas systemic MMP inhibition impaired mobilization of bone marrow sprocs and their hepatic engraftment. Hepatic MMP-9 proteolytically cleaved VEGF after partial hepatectomy. Liver-selective MMP-9 inhibition prevented VEGF cleavage and doubled protein expression of VEGF and its downstream signaling partner sdf-1. In contrast, systemic MMP inhibition enhanced recruitment and engraftment of infused allogeneic progenitors. Conclusion: Liver-selective MMP inhibition prevents proteolytic cleavage of hepatic VEGF, which enhances recruitment and engraftment of bone marrow sprocs after liver injury. This ameliorates injury and accelerates liver regeneration. Liver-selective MMP-9 inhibition may be a therapeutic tool for liver injury that damages the vasculature, whereas systemic MMP inhibition can enhance the benefit of stem cell therapy with endothelial progenitor cells.

Project description:Primary graft dysfunction (PGD) is a major cause of morbidity and mortality after lung transplantation. Ischemia-reperfusion injury (IRI) is a key event that contributes to PGD, though complex interactions affect donor lungs status, such as preceding brain death (BD), hemorrhagic shock (HS), and pre-engraftment lung management, the latter recognized as important risk factors for PGD. We hypothesized that a multi-hit isogenic mouse model of lung transplantation is more closely linked to PGD than IRI alone. Left lung transplants were performed between inbred C57BL/6 mice. A one-hit model of IRI was established by inducing cold ischemia (CI) of the donor lungs at 0°C for 1, 72, or 96 hours before engraftment. Multi-hit models were established by inducing 24 hours of HS and/or 3 hours of BD before 24 hours of CI. The recipients were killed at 24 hours after transplant and lung graft samples were analyzed. In the one-hit model of IRI, up to 72-hour CI time resulted in minimal cellular infiltration near small arteries after 24-hour reperfusion. Extension of CI time to 96 hours led to increased cellular infiltration and necroptotic pathway activation, without evidence of apoptosis, after 24-hour reperfusion. In a multi-hit model of PGD, "HS + BD + IRI" demonstrated increased lung injury, cellular infiltration, and activation of necroptotic and apoptotic pathways compared with IRI alone. Treatment with an inhibitor of receptor-interacting protein kinase 1 kinase, necrostatin-1, resulted in a significant decrease of downstream necroptotic pathway activation in both single- and multi-hit models of IRI. Thus, activation of necroptosis is a central event in IRI after prolonged CI, though it may not be sufficient to cause PGD alone. Pathological evaluation of donor lungs after CI-induced IRI, in conjunction with pre-engraftment donor lung factors in our multi-hit model, demonstrated early evidence of lung injury consistent with PGD. Our findings support the premise that pre-existing donor lung status is more important than CI time alone for inflammatory pathway activation in PGD, which may have important clinical implications for donor lung retrieval.

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Suppression of graft regeneration, not ischemia/reperfusion injury, is the primary cause of small-for-size syndrome after partial liver transplantation in mice.

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