Human umbilical cord mesenchymal stem cells alleviate fatty liver ischemia-reperfusion injury by activating autophagy through upregulation of IFNγ.

Liver ischemia-reperfusion injury (IRI) is an important factor affecting the prognosis of liver transplantation, and extended criteria donors (e.g., steatosis donor livers) are considered to be more sensitive to ischemia-reperfusion injury in liver transplantation. Currently, the application of human umbilical cord mesenchymal stem cells (hMSCs) has great promise in the treatment of various injuries in the liver. This study aimed to investigate the therapeutic role and mechanism of hMSCs in fatty liver IRI. After more than 8 weeks of high-fat chow feeding, we constructed a fatty liver mouse model and established ischemic injury of about 70% of the liver. Six hours after IRI, liver injury was significantly alleviated in hMSCs-treated mice, and the expression levels of liver enzyme, inflammatory factor TNF-α, and apoptotic proteins were significantly lower than those of the control group, which were also significant in pathological sections. Transcriptomics analysis showed that IFNγ was significantly upregulated in the hMSCs group. Mechanistically, IFNγ, which activates the MAPK pathway, is a potent agonist that promotes the occurrence of autophagy in hepatocytes to exert a protective function, which was confirmed by in vitro experiments. In summary, hMSCs treatment could slow down IRI in fatty liver by activating autophagy through upregulation of IFNγ, and this effect was partly direct.

Influence of intraoperative blood salvage and autotransfusion on tumor recurrence after deceased donor liver transplantation: a large nationwide cohort study.

BACKGROUND AND AIMS: The practice of intraoperative blood salvage and autotransfusion (IBSA) during deceased donor liver transplantation (DDLT) for hepatocellular carcinoma (HCC) can potentially reduce the need for allogeneic blood transfusion. However, implementing IBSA remains debatable due to concerns about its possible detrimental effects on oncologic recurrence. METHODS: This study retrospectively enrolled nationwide recipients of DDLT for HCC between 2015 and 2020. The focus was on comparing the cumulative recurrence rate and the recurrence-free survival rate. Propensity score matching was conducted repeatedly for further subgroup comparison. Recipients were categorized based on the Milan criteria, macrovascular invasion, and pre-transplant α-Fetoprotein (AFP) level to identify subgroups at risk of HCC recurrence. RESULTS: A total of 6196 and 329 patients were enrolled in the non-IBSA and IBSA groups in this study. Multivariable competing risk regression analysis identified IBSA as independent risk factors for HCC recurrence (P<0.05). Post-matching, the cumulative recurrence rate and recurrence-free survival rate revealed no significant difference in the IBSA group and non-IBSA group (22.4% vs. 16.5%, P=0.12; 60.3% vs. 60.9%, P=0.74). Recipients beyond Milan criteria had higher, albeit not significant, risk of HCC recurrence if receiving IBSA (33.4% vs. 22.5%, P=0.14). For recipients with macrovascular invasion, the risk of HCC recurrence has no significant difference between the two groups (32.2% vs. 21.3%, P=0.231). For recipients with an AFP level<20 ng/mL, the risk of HCC recurrence was comparable in the IBSA group and the non-IBSA group (12.8% vs. 18.7%, P=0.99). Recipients with an AFP level ≥20 ng/mL, the risk of HCC recurrence was significantly higher in the IBSA group. For those with an AFP level≥400 ng/mL, the impact of IBSA on the cumulative recurrence rate was even more pronounced (49.8% vs. 21.9%, P=0.011). CONCLUSIONS: IBSA does not appear to be associated with worse outcomes for recipients with HCC exceeding the Milan criteria or with macrovascular invasion. IBSA could be confidently applied for recipients with a pre-transplant AFP level<20 ng/mL. For recipients with AFP levels≥20 ng/mL, undertaking IBSA would increase the risk of HCC recurrence.

Evaluation of liver regeneration after hemi-hepatectomy by combining computed tomography and post-operative liver function.

Background: Accurate evaluation of postoperative liver regeneration is essential to prevent postoperative liver failure. Aims: To analyze the predictors that affect liver regeneration after hemi-hepatectomy. Method: Patients who underwent hemi-hepatectomy in Hangzhou First People's Hospital and Hangzhou Shulan Hospital from January 2016 to December 2021 were enrolled in this study. The regeneration index (RI) was calculated by the following equation: RI = [(postoperative total liver volume {TLVpost} - future liver remnant volume {FLRV}/FLRV] × 100 %. Hepatic dysfunction was defined according to the "TBilpeak>7" standard, which was interpreted as (peak) total bilirubin (TBil) >7.0 mg/dL. Good liver regeneration was defined solely when the RI surpassed the median with hepatic dysfunction. Logistic regression analyses were performed to estimate prognostic factors affecting liver regeneration. Result: A total of 153 patients were enrolled, with 33 in the benign group and 120 patients in the malignant group. In the entire study population, FLRV% [OR 4.087 (1.405-11.889), P = 0.010], international normalized ratio (INR) [OR 2.763 (95%CI, 1.008-7.577), P = 0.048] and TBil [OR 2.592 (95%CI, 1.177-5.710), P = 0.018] were independent prognostic factors associated with liver regeneration. In the benign group, only the computed tomography (CT) parameter FLRV% [OR, 11.700 (95%CI, 1.265-108.200), P = 0.030] predicted regeneration. In the malignant group, parenchymal hepatic resection rate (PHRR%) [OR 0.141 (95%CI, 0.040-0.499), P = 0.002] and TBil [OR 3.384 (95%CI, 1.377-8.319), P = 0.008] were independent prognostic factors. Conclusion: FLRV%, PHRR%, TBil and INR were predictive factors associated with liver regeneration.

Integrated multi-omic analysis identifies fatty acid binding protein 4 as a biomarker and therapeutic target of ischemia-reperfusion injury in steatotic liver transplantation.

BACKGROUND AND AIMS: Due to a lack of donor grafts, steatotic livers are used more often for liver transplantation (LT). However, steatotic donor livers are more sensitive to ischemia-reperfusion (IR) injury and have a worse prognosis after LT. Efforts to optimize steatotic liver grafts by identifying injury targets and interventions have become a hot issue. METHODS: Mouse LT models were established, and 4D label-free proteome sequencing was performed for four groups: normal control (NC) SHAM, high-fat (HF) SHAM, NC LT, and HF LT to screen molecular targets for aggravating liver injury in steatotic LT. Expression detection of molecular targets was performed based on liver specimens from 110 donors to verify its impact on the overall survival of recipients. Pharmacological intervention using small-molecule inhibitors on an injury-related target was used to evaluate the therapeutic effect. Transcriptomics and metabolomics were performed to explore the regulatory network and further integrated bioinformatics analysis and multiplex immunofluorescence were adopted to assess the regulation of pathways and organelles. RESULTS: HF LT group represented worse liver function compared with NC LT group, including more apoptotic hepatocytes (P < 0.01) and higher serum transaminase (P < 0.05). Proteomic results revealed that the mitochondrial membrane, endocytosis, and oxidative phosphorylation pathways were upregulated in HF LT group. Fatty acid binding protein 4 (FABP4) was identified as a hypoxia-inducible protein (fold change > 2 and P < 0.05) that sensitized mice to IR injury in steatotic LT. The overall survival of recipients using liver grafts with high expression of FABP4 was significantly worse than low expression of FABP4 (68.5 vs. 87.3%, P < 0.05). Adoption of FABP4 inhibitor could protect the steatotic liver from IR injury during transplantation, including reducing hepatocyte apoptosis, reducing serum transaminase (P < 0.05), and alleviating oxidative stress damage (P < 0.01). According to integrated transcriptomics and metabolomics analysis, cAMP signaling pathway was enriched following FABP4 inhibitor use. The activation of cAMP signaling pathway was validated. Microscopy and immunofluorescence staining results suggested that FABP4 inhibitors could regulate mitochondrial membrane homeostasis in steatotic LT. CONCLUSIONS: FABP4 was identified as a hypoxia-inducible protein that sensitized steatotic liver grafts to IR injury. The FABP4 inhibitor, BMS-309403, could activate of cAMP signaling pathway thereby modulating mitochondrial membrane homeostasis, reducing oxidative stress injury in steatotic donors.

Impact of preoperative infection on the outcomes of liver transplant recipients: a national propensity score-matched retrospective cohort study in China.

BACKGROUND: Impact of preoperative infection on liver transplantation (LT) needs further investigation. MATERIALS AND METHODS: From 1 January 2015 to 31 December 2022, 24 122 eligible patients receiving LT were enrolled from the China Liver Transplant Registry database. The outcomes of LT were compared after using the propensity score-matched analysis. RESULTS: Compared to the noninfection group, patients in the infection group were more likely to have postoperative effusion, infection, abdominal bleeding, and biliary complications (all P <0.01), and they had shorter 30-day, 90-day survival, and overall survival (all P <0.01). Cox proportional hazards regression analysis revealed that MELD score and cold ischemia time were risk factors for the overall survival in the infection group (both P <0.05). Besides, compared to the nonpulmonary group, patients in the pulmonary group were more likely to have postoperative effusion and infection (both P <0.0001), and less likely to have postoperative abscess and early allograft dysfunction (both P <0.05). Patients in the nonabdominal group also had a higher proportion of postoperative infection than those in the abdominal group ( P <0.05). Furthermore, compared to the number=1 group, patients in the number ≥2 group were more prone to postoperative effusion and infection (both P <0.01), and they also had shorter 30-day and 90-day survival (both P <0.05). CONCLUSION: Preoperative infection can result in a higher incidence of early postoperative complications and shorter survival in liver transplant recipients. The types and number of infection sites will also influence the prognosis of liver transplant recipients.

Arouse potential stemness: Intrinsic and acquired stem cell therapeutic strategies for advanced liver diseases.

Liver diseases are a major health issue, and prolonged liver injury always progresses. Advanced liver disorders impair liver regeneration. Millions of patients die yearly worldwide, even with the available treatments of liver transplantation and artificial liver support system. With its abundant cell resources and significant differentiative potential, stem cell therapy is a viable treatment for various disorders and offers hope to patients waiting for orthotopic liver transplantation. Considering such plight, stem cell therapeutic strategies deliver hope to the patients. Moreover, we conclude intrinsic and acquired perspectives based on stem cell sources. The properties and therapeutic uses of these stem cells' specific types or sources were then reviewed. Owing to the recent investigations of the above cells, a safe and effective therapy will emerge for advanced liver diseases soon.

Identification of a brand intratumor microbiome signature for predicting prognosis of hepatocellular carcinoma.

PURPOSE: Given that prognosis of hepatocellular carcinoma (HCC) differs dramatically, it is imperative to uncover effective and available prognostic biomarker(s). The intratumor microbiome plays a significant role in the response to tumor microenvironment, we aimed to identify an intratumor microbiome signature for predicting the prognosis of HCC patients accurately and investigate its possible mechanisms subsequently. METHODS: The TCGA HCC microbiome data (TCGA-LIHC-microbiome) was downloaded from cBioPortal. To create an intratumor microbiome-related prognostic signature, univariate and multivariate Cox regression analyses were used to quantify the association of microbial abundance and patients' overall survival (OS), as well as their diseases specific survival (DSS). The performance of the scoring model was evaluated by the area under the ROC curve (AUC). Based on the microbiome-related signature, clinical factors, and multi-omics molecular subtypes on the basis of "icluster" algorithm, nomograms were established to predict OS and DSS. Patients were further clustered into three subtypes based on their microbiome-related characteristics by consensus clustering. Moreover, deconvolution algorithm, weighted correlation network analysis (WGCNA) and gene set variation analysis (GSVA) were used to investigate the potential mechanisms. RESULTS: In TCGA LIHC microbiome data, the abundances of 166 genera among the total 1406 genera were considerably associated with HCC patients' OS. From that filtered dataset we identified a 27-microbe prognostic signature and developed a microbiome-related score (MRS) model. Compared with those in the relatively low-risk group, patients in higher-risk group own a much worse OS (P < 0.0001). Besides, the time-dependent ROC curves with MRS showed excellent predictive efficacy both in OS and DSS. Moreover, MRS is an independent prognostic factor for OS and DSS over clinical factors and multi-omics-based molecular subtypes. The integration of MRS into nomograms significantly improved the efficacy of prognosis prediction (1-year AUC:0.849, 3-year AUC: 0.825, 5-year AUC: 0.822). The analysis of microbiome-based subtypes on their immune characteristics and specific gene modules inferred that the intratumor microbiome may affect the HCC patients' prognosis via modulating the cancer stemness and immune response. CONCLUSION: MRS, a 27 intratumor microbiome-related prognostic model, was successfully established to predict HCC patients overall survive independently. And the possible underlying mechanisms were also investigated to provide a potential intervention strategy.

Current Status and Prospect of Delivery Vehicle Based on Mesenchymal Stem Cell-Derived Exosomes in Liver Diseases.

With the improvement of the average life expectancy and increasing incidence of obesity, the burden of liver disease is increasing. Liver disease is a serious threat to human health. Currently, liver transplantation is the only effective treatment for end-stage liver disease. However, liver transplantation still faces unavoidable difficulties. Mesenchymal stem cells (MSCs) can be used as an alternative therapy for liver disease, especially liver cirrhosis, liver failure, and liver transplantation complications. However, MSCs may have potential tumorigenic effects. Exosomes derived from MSCs (MSC-Exos), as the important intercellular communication mode of MSCs, contain various proteins, nucleic acids, and DNA. MSC-Exos can be used as a delivery system to treat liver diseases through immune regulation, apoptosis inhibition, regeneration promotion, drug delivery, and other ways. Good histocompatibility and material exchangeability make MSC-Exos a new treatment for liver diseases. This review summarizes the latest research on MSC-Exos as delivery vehicles in different liver diseases, including liver injury, liver failure, liver fibrosis, hepatocellular carcinoma (HCC), and ischemia and reperfusion injury. In addition, we discuss the advantages, disadvantages, and clinical application prospects of MSC-Exos-based delivery vectors in the treatment of liver diseases.

Prediction of tumor recurrence by distinct immunoprofiles in liver transplant patients based on mass cytometry.

Recurrence of hepatocellular carcinoma (HCC) after liver transplantation (LT) is a marker of poor prognosis. However, the reliable biomarkers of post-LT HCC recurrence remain to be identified. In this study, serial peripheral blood samples from the LT recipients with and without HCC recurrence were collected at five time points. Single-cell mass cytomertry (CyTOF) was utilized for the in-depth analysis of peripheral blood monocellular cells (PBMCs). CyTOF analysis showed that at 3 weeks post-LT, the activated immune cell population was increased, while the fraction of immune cells with suppressive functions (myeloid-derived suppressive cells) was reduced. The post-LT immune composition in patients with LT for HCC was enormously different from that in patients with LT for causes other than HCC. Furthermore, at 3 weeks after LT, compared with patients without recurrence, the patients with HCC recurrences were high in two subsets of T cells: CD57+ HLA-DR+ CD8+ and CD28+γδ. The CD57+ HLA-DR+ CD8+ T cells presented high levels of perforin, granzyme B, and Ki-67 and displayed a highly cytotoxic and proliferative phenotype, while the CD28+γδ T cells had reduced levels of IFN-γ and, hence, were less activated compared to CD28- cells. Based on these findings, we concluded that analyzing the PBMCs of LT recipients by CyTOF can predict the post-LT HCC recurrence. The distinct immune features can stratify patients with the risk of HCC recurrence at 3 weeks after LT, which will help clinician in further management plan and improve the prognosis of patients.

Cellular crosstalk during liver regeneration: unity in diversity.

The liver is unique in its ability to regenerate from a wide range of injuries and diseases. Liver regeneration centers around hepatocyte proliferation and requires the coordinated actions of nonparenchymal cells, including biliary epithelial cells, liver sinusoidal endothelial cells, hepatic stellate cells and kupffer cells. Interactions among various hepatocyte and nonparenchymal cells populations constitute a sophisticated regulatory network that restores liver mass and function. In addition, there are two different ways of liver regeneration, self-replication of liver epithelial cells and transdifferentiation between liver epithelial cells. The interactions among cell populations and regenerative microenvironment in the two modes are distinct. Herein, we first review recent advances in the interactions between hepatocytes and surrounding cells and among nonparenchymal cells in the context of liver epithelial cell self-replication. Next, we discuss the crosstalk of several cell types in the context of liver epithelial transdifferentiation, which is also crucial for liver regeneration. Video abstract.

CLIC1 Drives Angiogenesis in Hepatocellular Carcinoma by Modulating VEGFA.

Background: Chloride intracellular channel 1 (CLIC1) is upregulated in hepatocellular carcinoma (HCC). The present study aimed to investigate the role of CLIC1 in HCC angiogenesis. Materials and Methods: Immunohistochemistry (IHC) was used to test the expression of CLIC1 and CD34 in 67 pairs of HCC and paracarcinoma tissues. The prognosis data of the patients were used to analyze the clinical relevance of CLIC1. We built a coculture system of HCC cells and endothelial cells to explore the migration of endothelial cells. Conditioned media (CMs) from HCC cells was then collected to assess endothelial cell migration. Experiments were then conducted to confirm the relationship between CLIC1 and angiogenesis in a subcutaneous tumor model. Results: CLIC1 expression was higher in HCC tumor tissues than in paracarcinoma tissues. Patients with increased CLIC1 expression showed a higher microvascular density (MVD; P = .013). Kaplan-Meier curves indicated that patients with lower expression of CLIC1 had better overall survival (P < .001) and recurrence-free survival (P = .046). Vascular endothelial growth factor A (VEGFA) in CMs from CLIC1-knockdown cells was lower than in the control group, while VEGFA in CMs from CLIC1 overexpression cells was higher than in the control group. CMs from CLIC1 overexpression cell lines promote the in vitro migration of EA.hy926 cells. Meanwhile, adding Bevacizumab to CMs from CLIC1 overexpression cells significantly inhibited this migration. The growth of xenograft tumors derived from CLIC1-knockdown Huh7 cells was restrained compared with the control group (P < .001). IHC staining showed MVD was higher in tumors with CLIC1 overexpression. Conclusion: CLIC1 is a promising biomarker for predicting the prognosis of HCC patients, and expression of CLIC1 correlates with angiogenesis in HCC through regulating VEGFA.

Quantitative proteomic profiling of hepatocellular carcinoma at different serum alpha-fetoprotein level.

PURPOSE: Hepatocellular carcinoma (HCC) is characterized by a poor long-term prognosis and high mortality rate. Serum alpha-fetoprotein (AFP) levels show great prognostic value in patients undergoing hepatectomy. This study aims to explore proteomic profiling in HCC samples based on AFP subgroups and identify potential key targets involved in HCC progression. METHODS: Twelve paired tumor and adjacent noncancerous tissue samples were collected from patients with HCC who underwent primary curative resection from January 2012 to December 2013. Clinical information was curated from four tissue microarrays to conduct survival analysis based on serum AFP levels. TMT-based quantitative proteomic analyses and bioinformatics analyses were performed to comprehensively profile molecular features. Immunohistochemistry was carried out to validate protein expression of identified targets. Kaplan-Meier survival analysis was performed to assess the overall survival and recurrence-free survival based on protein expressions. RESULTS: AFP (400 ng/mL) was a turning point in prognosis, metabolic- and invasion-associated pathways. The mass spectrometry analysis yielded a total of 5573 identified proteins. Annotations of 151 differentially expressed proteins in tumors and 95 proteins in paracancerous tissues (1.2-fold) showed similarities in biological processes, cellular components, molecular functions. Furthermore, differentially expressed hub proteins with five innovatively nominated druggable targets (C1QBP, HSPE1, GLUD2 for tumors and CHDH, ITGAL for paracancerous tissues), of which four (C1QBP, HSPE1, CHDH, ITGAL) targets were associated with poor overall survival (all Log-rank P < 0.05). CONCLUSIONS: Our quantitative proteomics analyses identified four key prognostic biomarkers in HCC and provide opportunities for translational medicine and new treatment.

Proteomics-based identification of the role of osteosarcoma amplified-9 in hepatocellular carcinoma recurrence.

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies; its recurrence is associated with high mortality and poor recurrence-free survival and is affected by multisystem and multilevel pathological changes. To identify the key proteins associated with tumor recurrence and the underlying mechanisms, proteomic profiling of tumor specimens from early recurrence and nonrecurrence patients was performed in this study. Proteomics was applied to identify differentially expressed proteins during the early recurrence of HCC after surgery. Osteosarcoma amplified-9 (OS-9) was discovered, and the correlation between OS-9 expression and the clinicopathological characteristics of patients was analyzed. Invasion and migration were examined in SMMC-7721 cells with and without OS-9 overexpression. Proteomics was performed once again using SMMC-7721 cells with OS-9 overexpression to further analyze the proteins with altered expression. OS-9 was overexpressed in the early recurrence group, and OS-9 overexpression was associated with high serum alpha-fetoprotein levels and poor recurrence-free survival in 196 patients with HCC. The invasion and migration abilities of SMMC-7721 cells were enhanced in the OS-9 overexpression group. Bioinformatic functional enrichment methods, including Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis, revealed that the hypoxia-inducible factor 1 (HIF-1) and tumor necrosis factor (TNF) signaling pathways were activated in the OS-9 overexpression group. The migration and invasion capacities of OS-9 overexpressed HCC cell line were weakened while treated with HIF-1α or TNF-α inhibitors. Conclusion: Our results suggest that the overexpression of OS-9 is related to HCC recurrence, thereby contributing to the migration and invasion capacities of HCC cell line by regulating the HIF-1 and TNF pathways.

Development of a Novel Prognostic Nomogram for High Model for End-Stage Liver Disease Score Recipients Following Deceased Donor Liver Transplantation.

Background: A high model of end-stage liver disease (MELD) score (>30) adversely affects outcomes even if patients receive prompt liver transplantation (LT). Therefore, balanced allocation of donor grafts is indispensable to avoid random combinations of donor and recipient risk factors, which often lead to graft or recipient loss. Predictive models aimed at avoiding donor risk factors in high-MELD score recipients are urgently required to obtain satisfactory outcomes. Method: Data of patients with MELD score >30 who underwent LT at three transplantation institutes between 2015 and 2018 were retrospectively reviewed. Early allograft dysfunction (EAD), length of intensive care unit (ICU) stay, and graft loss were recorded. Corresponding independent risk factors were analyzed using stepwise multivariable regression analysis. A prediction model of graft loss was developed, and discrimination and calibration were measured. Results: After applying the exclusion criteria, 778 patients were enrolled. The incidence of EAD was 34.8% (271/778). Donor graft macrovesicular steatosis, graft-to-recipient weight ratio (GRWR), warm ischemia time (WIT), cold ischemia time (CIT), and ABO blood incompatibility, together with donor serum albumins, were independent predictors of EAD. The incidence of ICU stay over 10 days was 64.7% (503/778). Donor age, recipient's MELD score, Child score, and CIT were independent predictors of ICU stay. The 3-year graft survival rates (GSRs) in the training and validation cohorts were 64.2 and 59.3%, respectively. The independent predictors of graft loss were recipient's Child score, ABO blood type incompatibility, donor serum total bilirubin over 17.1 µmol/L, and cold CIT. A nomogram based on these variables was internally and externally validated and showed good performance (area under the receiver operating characteristic curve = 70.8 and 66.0%, respectively). For a recipient with a high MELD score, the avoidance of ABO blood type incompatibility and CIT ≥6 h would achieve a 3-year GSR of up to 78.4%, whereas the presence of the aforementioned risk factors would decrease the GSR to 35.4%. Conclusion: The long-term prognosis of recipients with MELD scores >30 could be greatly improved by avoiding ABO blood type incompatibility and CIT ≥6 h.