Extended 78% Hepatectomy in a Mouse Surgical Model.

Partial 2/3 hepatectomy in mice is used in research to study the liver's regenerative capacity and explore outcomes of liver resection in a number of disease models. In the classical partial 2/3 hepatectomy in mice, two of the five liver lobes, namely the left and median lobes representing approximately 66% of the liver mass, are resected en bloc with an expected postoperative survival of 100%. More aggressive partial hepatectomies are technically more challenging and hence, have seldom been used in mice. Our group has developed a mouse model of an extended hepatectomy technique in which three of the five liver lobes, including the left, median, and right upper lobes, are resected separately to remove approximately 78% of the total liver mass. This extended resection, in otherwise healthy mice, leaves a remnant liver that cannot always sustain adequate and timely regeneration. Failure to regenerate ultimately results in 50% postoperative lethality within 1 week due to fulminant hepatic failure. This procedure of extended 78% hepatectomy in mice represents a unique surgical model for the study of small-for-size syndrome and the evaluation of therapeutic strategies to improve liver regeneration and outcomes in the setting of liver transplantation or extended liver resection for cancer.

Partial liver resection alters the bile salt-FGF19 axis in patients with perihilar cholangiocarcinoma: Implications for liver regeneration.

BACKGROUND: Extended liver resection is the only treatment option for perihilar cholangiocarcinoma (pCCA). Bile salts and the gut hormone FGF19, both promoters of liver regeneration (LR), have not been investigated in patients undergoing resection for pCCA. We aimed to evaluate the bile salt-FGF19 axis perioperatively in pCCA and study its effects on LR. METHODS: Plasma bile salts, FGF19, and C4 (bile salt synthesis marker) were assessed in patients with pCCA and controls (colorectal liver metastases), before and after resection on postoperative days (PODs) 1, 3, and 7. Hepatic bile salts were determined in intraoperative liver biopsies. RESULTS: Partial liver resection in pCCA elicited a sharp decline in bile salt and FGF19 plasma levels on POD 1 and remained low thereafter, unlike in controls, where bile salts rose gradually. Preoperatively, suppressed C4 in pCCA normalized postoperatively to levels similar to those in the controls. The remnant liver volume and postoperative bilirubin levels were negatively associated with postoperative C4 levels. Furthermore, patients who developed postoperative liver failure had nearly undetectable C4 levels on POD 7. Hepatic bile salts strongly predicted hyperbilirubinemia on POD 7 in both groups. Finally, postoperative bile salt levels on day 7 were an independent predictor of LR. CONCLUSIONS: Partial liver resection alters the bile salt-FGF19 axis, but its derailment is unrelated to LR in pCCA. Postoperative monitoring of circulating bile salts and their production may be useful for monitoring LR.

Molecular Pathways Governing the Termination of Liver Regeneration.

The liver has the unique capacity to regenerate, and up to 70% of the liver can be removed without detrimental consequences to the organism. Liver regeneration is a complex process involving multiple signaling networks and organs. Liver regeneration proceeds through three phases: the initiation phase, the growth phase, and the termination phase. Termination of liver regeneration occurs when the liver reaches a liver-to-body weight that is required for homeostasis, the so-called "hepatostat." The initiation and growth phases have been the subject of many studies. The molecular pathways that govern the termination phase, however, remain to be fully elucidated. This review summarizes the pathways and molecules that signal the cessation of liver regrowth after partial hepatectomy and answers the question, "What factors drive the hepatostat?" SIGNIFICANCE STATEMENT: Unraveling the pathways underlying the cessation of liver regeneration enables the identification of druggable targets that will allow us to gain pharmacological control over liver regeneration. For these purposes, it would be useful to understand why the regenerative capacity of the liver is hampered under certain pathological circumstances so as to artificially modulate the regenerative processes (e.g., by blocking the cessation pathways) to improve clinical outcomes and safeguard the patient's life.

Hepatic recruitment of myeloid-derived suppressor cells upon liver injury promotes both liver regeneration and fibrosis.

BACKGROUND: The liver regeneration is a highly complicated process depending on the close cooperations between the hepatocytes and non-parenchymal cells involving various inflammatory cells. Here, we explored the role of myeloid-derived suppressor cells (MDSCs) in the processes of liver regeneration and liver fibrosis after liver injury. METHODS: We established four liver injury models of mice including CCl4-induced liver injury model, bile duct ligation (BDL) model, concanavalin A (Con A)-induced hepatitis model, and lipopolysaccharide (LPS)-induced hepatitis model. The intrahepatic levels of MDSCs (CD11b+Gr-1+) after the liver injury were detected by flow cytometry. The effects of MDSCs on liver tissues were analyzed in the transwell co-culture system, in which the MDSCs cytokines including IL-10, VEGF, and TGF-ß were measured by ELISA assay and followed by being blocked with specific antibodies. RESULTS: The intrahepatic infiltrations of MDSCs with surface marker of CD11b+Gr-1+ remarkably increased after the establishment of four liver injury models. The blood served as the primary reservoir for hepatic recruitment of MDSCs during the liver injury, while the bone marrow appeared play a compensated role in increasing the number of MDSCs at the late stage of the inflammation. The recruited MDSCs in injured liver were mainly the M-MDSCs (CD11b+Ly6G-Ly6Chigh) featured by high expression levels of cytokines including IL-10, VEGF, and TGF-ß. Co-culture of the liver tissues with MDSCs significantly promoted the proliferation of both hepatocytes and hepatic stellate cells (HSCs). CONCLUSIONS: The dramatically and quickly infiltrated CD11b+Gr-1+ MDSCs in injured liver not only exerted pro-proliferative effects on hepatocytes, but also accounted for the activation of profibrotic HSCs.

Amelioration of NAFLD by sleeve gastrectomy-triggered hepatocyte regeneration in mice - experimental research.

BACKGROUND: Sleeve gastrectomy (SG) is known to alleviate non-alcoholic fatty liver disease (NAFLD) and restore liver function; however, its underlying mechanism remains unclear. MATERIALS AND METHODS: We investigated the effect of SG on the metabolic phenotype of diet-induced obese (DIO) mice. Postoperative stained liver images were analyzed to determine the hepatocyte proliferation phenotype. Single-cell RNA sequencing was used to characterize the regeneration signals of the liver after SG in DIO mice, and real-time quantitative reverse transcription PCR was performed to verify the above results. We employed Olink proteomics to capture serum element changes and investigated the role of Yes1 protein in liver regeneration and carcinogenesis through the Hippo-YAP pathway. DIO mice were treated with YAP inhibitor verteporfin after SG mice to clarify whether SG-induced liver regeneration is related to the YAP signaling pathway. RESULTS: SG significantly reduced NAFLD-associated dysfunction in hepatocytes and replaced them with fully functional hepatocytes, which have a high regenerative capacity across the entire liver. SG also enhanced the hepatic regenerative capacity, as demonstrated by SG combined with hepatic lobectomy in healthy mice. Yes1 protein was identified as the signaling molecule most closely related to classical regeneration signals. Our study showed that SG-enhanced proliferation and improved metabolism did not depend on YAP signaling. CONCLUSION: SG can enhance hepatic regenerative capacity and improve liver metabolism. This study provides a better understanding of the mechanisms underlying SG-induced metabolic improvements.

Sarcopenia does not affect liver regeneration and postoperative course after a major hepatectomy. A prospective study on 125 patients using CT volumetry and HIDA scintigraphy.

BACKGROUND & OBJECTIVES: Sarcopenia is a morbi-mortality risk factor in digestive surgery, though its impact after major hepatectomy (MH) remains unknown. This prospective pilot study investigated whether volume and function of a regenerating liver is influenced by body composition. METHODS: From 2011 to 2016, 125 consecutive patients had computed tomography and 99mTc-labelled-mebrofenin SPECT-scintigraphy before and after MH at day 7 and 1 month for measurements of liver volumes and functions. L3 vertebra muscle mass identified sarcopenia. Primary endpoint was the impact of sarcopenia on regeneration capacities (i.e. volume/function changes and post-hepatectomy liver failure (PHLF) rate). Secondary endpoint was 3-month morbi-mortality. RESULTS: Sarcopenic patients (SP; N = 69) were significantly older than non-sarcopenic (NSP), with lower BMI and more malignancies, but with comparable liver function/volume at baseline. Postoperatively, SP showed higher rates of ISGLS_PHLF (24.6 % vs 10.9 %; p = 0.05) but with comparable rates of severe morbidity (23.2 % vs 16.4 %; p = 0.35), overall (8.7 % vs 3.6 %; p = 0.3) and PHLF-related mortality (8,7 % vs 1.8 %; p = 0.075). After matching on the extent of resection or using propensity score, regeneration and PHLF rates were similar. CONCLUSION: This prospective study using first sequential SPECT-scintigraphy showed that sarcopenia by itself does not affect liver regeneration capacities and short-term postoperative course after MH.

Role of HNF4α-cMyc Interaction in CDE Diet-Induced Liver Injury and Regeneration.

Hepatocyte nuclear factor 4 alpha (HNF4α) is a nuclear factor essential for liver function that regulates the expression of cMyc and plays an important role during liver regeneration. This study investigated the role of the HNF4α-cMyc interaction in regulating liver injury and regeneration using the choline-deficient and ethionine-supplemented (CDE) diet model. Wild-type (WT), hepatocyte-specific HNF4α-knockout (KO), cMyc-KO, and HNF4α-cMyc double KO (DKO) mice were fed a CDE diet for 1 week to induce subacute liver injury. To study regeneration, normal chow diet was fed for 1 week after CDE diet. WT mice exhibited significant liver injury and decreased HNF4α mRNA and protein expression after CDE diet. HNF4α deletion resulted in significantly higher injury with increased inflammation, fibrosis, proliferation, and hepatic progenitor cell activation compared with WT mice after CDE diet but indicated similar recovery. Deletion of cMyc lowered liver injury with activation of inflammatory genes compared with WT and HNF4α-KO mice after CDE diet. DKO mice had a phenotype comparable to that of the HNF4α-KO mice after CDE diet and a complete recovery. DKO mice exhibited a significant increase in hepatic progenitor cell markers both after injury and recovery phase. Taken together, these data show that HNF4α protects against inflammatory and fibrotic changes after CDE diet-induced injury, which is driven by cMyc.

Liver regeneration after portal and hepatic vein embolization improves overall survival compared with portal vein embolization alone: mid-term survival analysis of the multicentre DRAGON 0 cohort.

BACKGROUND: The purpose of this study was to compare 3-year overall survival after simultaneous portal (PVE) and hepatic vein (HVE) embolization versus PVE alone in patients undergoing liver resection for primary and secondary cancers of the liver. METHODS: In this multicentre retrospective study, all DRAGON 0 centres provided 3-year follow-up data for all patients who had PVE/HVE or PVE, and were included in DRAGON 0 between 2016 and 2019. Kaplan-Meier analysis was undertaken to assess 3-year overall and recurrence/progression-free survival. Factors affecting survival were evaluated using univariable and multivariable Cox regression analyses. RESULTS: In total, 199 patients were included from 7 centres, of whom 39 underwent PVE/HVE and 160 PVE alone. Groups differed in median age (P = 0.008). As reported previously, PVE/HVE resulted in a significantly higher resection rate than PVE alone (92 versus 68%; P = 0.007). Three-year overall survival was significantly higher in the PVE/HVE group (median survival not reached after 36 months versus 20 months after PVE; P = 0.004). Univariable and multivariable analyses identified PVE/HVE as an independent predictor of survival (univariable HR 0.46, 95% c.i. 0.27 to 0.76; P = 0.003). CONCLUSION: Overall survival after PVE/HVE is substantially longer than that after PVE alone in patients with primary and secondary liver tumours.

Hepatic Snai1 and Snai2 promote liver regeneration and suppress liver fibrosis in mice.

Liver injury stimulates hepatocyte replication and hepatic stellate cell (HSC) activation, thereby driving liver regeneration. Aberrant HSC activation induces liver fibrosis. However, mechanisms underlying liver regeneration and fibrosis remain poorly understood. Here, we identify hepatic Snai1 and Snai2 as important transcriptional regulators for liver regeneration and fibrosis. Partial hepatectomy or CCl4 treatment increases occupancies of Snai1 and Snai2 on cyclin A2 and D1 promoters in the liver. Snai1 and Snai2 in turn increase promoter H3K27 acetylation and cyclin A2/D1 expressions. Hepatocyte-specific deletion of both Snai1 and Snai2, but not one alone, suppresses liver cyclin A2/D1 expression and regenerative hepatocyte proliferation after hepatectomy or CCl4 treatments but augments CCl4-stimulated HSC activation and liver fibrosis. Conversely, Snai2 overexpression in the liver enhances hepatocyte replication and suppresses liver fibrosis after CCl4 treatment. These results suggest that hepatic Snai1 and Snai2 directly promote, via histone modifications, reparative hepatocyte replication and indirectly inhibit liver fibrosis.

Angiotensin II type-2 receptor signaling facilitates liver injury repair and regeneration via inactivation of Hippo pathway.

The angiotensin II type 2 receptor (AT2R) is a well-established component of the renin-angiotensin system and is known to counteract classical activation of this system and protect against organ damage. Pharmacological activation of the AT2R has significant therapeutic benefits, including vasodilation, natriuresis, anti-inflammatory activity, and improved insulin sensitivity. However, the precise biological functions of the AT2R in maintaining homeostasis in liver tissue remain largely unexplored. In this study, we found that the AT2R facilitates liver repair and regeneration following acute injury by deactivating Hippo signaling and that interleukin-6 transcriptionally upregulates expression of the AT2R in hepatocytes through STAT3 acting as a transcription activator binding to promoter regions of the AT2R. Subsequently, elevated AT2R levels activate downstream signaling via heterotrimeric G protein Gα12/13-coupled signals to induce Yap activity, thereby contributing to repair and regeneration processes in the liver. Conversely, a deficiency in the AT2R attenuates regeneration of the liver while increasing susceptibility to acetaminophen-induced liver injury. Administration of an AT2R agonist significantly enhances the repair and regeneration capacity of injured liver tissue. Our findings suggest that the AT2R acts as an upstream regulator in the Hippo pathway and is a potential target in the treatment of liver damage.

Approaching Thrombospondin-1 as a Potential Target for Mesenchymal Stromal Cells to Support Liver Regeneration after Partial Hepatectomy in Mouse and Humans.

Extended liver resection carries the risk of post-surgery liver failure involving thrombospondin-1-mediated aggravation of hepatic epithelial plasticity and function. Mesenchymal stromal cells (MSCs), by interfering with thrombospondin-1 (THBS1), counteract hepatic dysfunction, though the mechanisms involved remain unknown. Herein, two-thirds partial hepatectomy in mice increased hepatic THBS1, downstream transforming growth factor-ß3, and perturbation of liver tissue homeostasis. All these events were ameliorated by hepatic transfusion of human bone marrow-derived MSCs. Treatment attenuated platelet and macrophage recruitment to the liver, both major sources of THBS1. By mitigating THBS1, MSCs muted surgery-induced tissue deterioration and dysfunction, and thus supported post-hepatectomy regeneration. After liver surgery, patients displayed increased tissue THBS1, which is associated with functional impairment and may indicate a higher risk of post-surgery complications. Since liver dysfunction involving THBS1 improves with MSC treatment in various animal models, it seems feasible to also modulate THBS1 in humans to impede post-surgery acute liver failure.

Effect of Sarcopenia on the Increase in Liver Volume and Function After Portal Vein Embolization.

PURPOSE: Sarcopenia is associated with a decreased kinetic growth rate (KGR) of the future liver remnant (FLR) after portal vein embolization (PVE). However, little is known on the increase in FLR function (FLRF) after PVE. This study evaluated the effect of sarcopenia on the functional growth rate (FGR) after PVE measured with hepatobiliary scintigraphy (HBS). METHODS: All patients who underwent PVE at the Amsterdam UMC between January 2005 and August 2017 were analyzed. Functional imaging by HBS was used to determine FGR. Liver volumetry was performed using multiphase contrast computed tomography (CT). Muscle area measurement to determine sarcopenia was taken at the third lumbar level (L3). RESULTS: Out of the 95 included patients, 9 were excluded due to unavailable data. 70/86 (81%) patients were sarcopenic. In the multivariate logistic regression analysis, sarcopenia (p = 0.009) and FLR volume (FRLV) before PVE (p = 0.021) were the only factors correlated with KGR, while no correlation was found with FGR. 90-day mortality was similar across the sarcopenic and non-sarcopenic group (4/53 [8%] versus 1/11 [9%]; p = 1.000). The resection rates were also comparable (53/70 [75%] versus 11/16 [69%]; p = 0.542). CONCLUSION: FGR after PVE as measured by HBS appears to be preserved in sarcopenic patients. This is in contrast to KGR after PVE as measured by liver volumetry which is decreased in sarcopenic patients. LEVEL OF EVIDENCE: Level 3b, cohort and case control studies.

Heterogeneity of hepatocyte dynamics restores liver architecture after chemical, physical or viral damage.

Midlobular hepatocytes are proposed to be the most plastic hepatic cell, providing a reservoir for hepatocyte proliferation during homeostasis and regeneration. However, other mechanisms beyond hyperplasia have been little explored and the contribution of other hepatocyte subpopulations to regeneration has been controversial. Thus, re-examining hepatocyte dynamics during regeneration is critical for cell therapy and treatment of liver diseases. Using a mouse model of hepatocyte- and non-hepatocyte- multicolor lineage tracing, we demonstrate that midlobular hepatocytes also undergo hypertrophy in response to chemical, physical, and viral insults. Our study shows that this subpopulation also combats liver impairment after infection with coronavirus. Furthermore, we demonstrate that pericentral hepatocytes also expand in number and size during the repair process and Galectin-9-CD44 pathway may be critical for driving these processes. Notably, we also identified that transdifferentiation and cell fusion during regeneration after severe injury contribute to recover hepatic function.

Kupffer cells abrogate homing and repopulation of allogeneic hepatic progenitors in injured liver site.

BACKGROUND: Allogeneic hepatocyte transplantation is an emerging approach to treat acute liver defects. However, durable engraftment of the transplanted cells remains a daunting task, as they are actively cleared by the recipient's immune system. Therefore, a detailed understanding of the innate or adaptive immune cells-derived responses against allogeneic transplanted hepatic cells is the key to rationalize cell-based therapies. METHODS: Here, we induced an acute inflammatory regenerative niche (3-96 h) on the surface of the liver by the application of cryo-injury (CI) to systematically evaluate the innate immune response against transplanted allogeneic hepatic progenitors in a sustained micro-inflammatory environment. RESULTS: The resulting data highlighted that the injured site was significantly repopulated by alternating numbers of innate immune cells, including neutrophils, monocytes and Kupffer cells (KCs), from 3 to 96 h. The transplanted allo-HPs, engrafted 6 h post-injury, were collectively eliminated by the innate immune response within 24 h of transplantation. Selective depletion of the KCs demonstrated a delayed recruitment of monocytes from day 2 to day 6. In addition, the intrasplenic engraftment of the hepatic progenitors 54 h post-transplantation was dismantled by KCs, while a time-dependent better survival and translocation of the transplanted cells into the injured site could be observed in samples devoid of KCs. CONCLUSION: Overall, this study provides evidence that KCs ablation enables a better survival and integration of allo-HPs in a sustained liver inflammatory environment, having implications for rationalizing the cell-based therapeutic interventions against liver defects.

LiverZap: a chemoptogenetic tool for global and locally restricted hepatocyte ablation to study cellular behaviours in liver regeneration.

The liver restores its mass and architecture after injury. Yet, investigating morphogenetic cell behaviours and signals that repair tissue architecture at high spatiotemporal resolution remains challenging. We developed LiverZap, a tuneable chemoptogenetic liver injury model in zebrafish. LiverZap employs the formation of a binary FAP-TAP photosensitiser followed by brief near-infrared illumination inducing hepatocyte-specific death and recapitulating mammalian liver injury types. The tool enables local hepatocyte ablation and extended live imaging capturing regenerative cell behaviours, which is crucial for studying cellular interactions at the interface of healthy and damaged tissue. Applying LiverZap, we show that targeted hepatocyte ablation in a small region of interest is sufficient to trigger local liver progenitor-like cell (LPC)-mediated regeneration, challenging the current understanding of liver regeneration. Surprisingly, the LPC response is also elicited in adjacent uninjured tissue, at up to 100 µm distance to the injury. Moreover, dynamic biliary network rearrangement suggests active cell movements from uninjured tissue in response to substantial hepatocyte loss as an integral step of LPC-mediated liver regeneration. This precisely targetable liver cell ablation tool will enable the discovery of key molecular and morphogenetic regeneration paradigms.

Region-specific cellular and molecular basis of liver regeneration after acute pericentral injury.

Liver injuries often occur in a zonated manner. However, detailed regenerative responses to such zonal injuries at cellular and molecular levels remain largely elusive. By using a fate-mapping strain, Cyp2e1-DreER, to elucidate liver regeneration after acute pericentral injury, we found that pericentral regeneration is primarily compensated by the expansion of remaining pericentral hepatocytes, and secondarily by expansion of periportal hepatocytes. Employing single-cell RNA sequencing, spatial transcriptomics, immunostaining, and in vivo functional assays, we demonstrated that the upregulated expression of the mTOR/4E-BP1 axis and lactate dehydrogenase A in hepatocytes contributes to pericentral regeneration, while activation of transforming growth factor ß (TGF-ß1) signaling in the damaged area mediates fibrotic responses and inhibits hepatocyte proliferation. Inhibiting the pericentral accumulation of monocytes and monocyte-derived macrophages through an Arg-Gly-Asp (RGD) peptide-based strategy attenuates these cell-derived TGF-ß1 signalings, thus improving pericentral regeneration. Our study provides integrated and high-resolution spatiotemporal insights into the cellular and molecular basis of pericentral regeneration.

Ten-eleven translocation-2-mediated macrophage activation promotes liver regeneration.

BACKGROUND: The remarkable regenerative capacity of the liver enables recovery after radical Hepatocellular carcinoma (HCC) resection. After resection, macrophages secrete interleukin 6 and hepatocyte growth factors to promote liver regeneration. Ten-eleven translocation-2 (Tet2) DNA dioxygenase regulates pro-inflammatory factor secretion in macrophages. In this study, we explored the role of Tet2 in macrophages and its function independent of its enzymatic activity in liver regeneration. METHODS: The model of liver regeneration after 70% partial hepatectomy (PHx) is a classic universal model for studying reparative processes in the liver. Mice were euthanized at 0, 24, and 48 h after PHx. Enzyme-linked immunosorbent assays, quantitative reverse transcription-polymerase chain reaction, western blotting, immunofluorescence analysis, and flow cytometry were performed to explore immune cell infiltration and liver regenerative capability. Molecular dynamics simulations were performed to study the interaction between Tet2 and signal transducer and activator of transcription 1 (Stat1). RESULTS: Tet2 in macrophages negatively regulated liver regeneration in the partial hepatectomy mice model. Tet2 interacted with Stat1, inhibiting the expression of proinflammatory factors and suppressing liver regeneration. The Tet2 inhibitor attenuated the interaction between Stat1 and Tet2, enhanced Stat1 phosphorylation, and promoted hepatocyte proliferation. The proliferative function of the Tet2 inhibitor relied on macrophages and did not affect hepatocytes directly. CONCLUSION: Our findings underscore that Tet2 in macrophages negatively regulates liver regeneration by interacting with Stat1. Targeting Tet2 in macrophages promotes liver regeneration and function after a hepatectomy, presenting a novel target to promote liver regeneration and function.