Deep learning and digital pathology powers prediction of HCC development in steatotic liver disease.

BACKGROUND AND AIMS: Identifying patients with steatotic liver disease who are at a high risk of developing HCC remains challenging. We present a deep learning (DL) model to predict HCC development using hematoxylin and eosin-stained whole-slide images of biopsy-proven steatotic liver disease. APPROACH AND RESULTS: We included 639 patients who did not develop HCC for ≥7 years after biopsy (non-HCC class) and 46 patients who developed HCC <7 years after biopsy (HCC class). Paired cases of the HCC and non-HCC classes matched by biopsy date and institution were used for training, and the remaining nonpaired cases were used for validation. The DL model was trained using deep convolutional neural networks with 28,000 image tiles cropped from whole-slide images of the paired cases, with an accuracy of 81.0% and an AUC of 0.80 for predicting HCC development. Validation using the nonpaired cases also demonstrated a good accuracy of 82.3% and an AUC of 0.84. These results were comparable to the predictive ability of logistic regression model using fibrosis stage. Notably, the DL model also detected the cases of HCC development in patients with mild fibrosis. The saliency maps generated by the DL model highlighted various pathological features associated with HCC development, including nuclear atypia, hepatocytes with a high nuclear-cytoplasmic ratio, immune cell infiltration, fibrosis, and a lack of large fat droplets. CONCLUSIONS: The ability of the DL model to capture subtle pathological features beyond fibrosis suggests its potential for identifying early signs of hepatocarcinogenesis in patients with steatotic liver disease.

Noninvasive graft monitoring using donor-derived cell-free DNA in Japanese liver transplantation.

AIM: To evaluate the use of donor-derived cell-free DNA (dd-cfDNA) in diagnosing graft injuries in Japanese liver transplantation (LTx), including family-related living donors. METHODS: A total of 321 samples from 10 newly operated LTx recipients were collected to monitor the early dynamics of dd-cfDNA levels after LTx. Fifty-five samples from 55 recipients were collected during protocol biopsies (PB), whereas 36 samples from 27 recipients were collected during event biopsies, consisting of 11 biopsy-proven acute rejection (AR), 20 acute dysfunctions without rejection (ADWR), and 5 chronic rejections. The levels of dd-cfDNA were quantified using a next-generation sequencer based on single nucleotide polymorphisms. RESULTS: The dd-cfDNA levels were elevated significantly after LTx, followed by a rapid decline to the baseline in patients without graft injury within 30 days post-LTx. The dd-cfDNA levels were significantly higher in the 11 samples obtained during AR than those obtained during PB (p < 0.0001), which decreased promptly after treatment. The receiver operator characteristic curve analysis of diagnostic ability yielded areas under the curve of 0.975 and 0.897 for AR (rejection activity index [RAI] ≥3) versus PB and versus non-AR (ADWR + PB). The dd-cfDNA levels during AR were elevated earlier and correlated more strongly with the RAI (r = 0.740) than aspartate aminotransferase/alanine aminotransferase. The dd-cfDNA levels were neither associated with graft fibrosis based on histology nor the status of donor-specific antibodies in PB samples. CONCLUSIONS: Donor-derived cell-free DNA serves as a sensitive biomarker for detecting graft injuries in LTx. Further large-scale cohort studies are warranted to optimize its use in differentiating various post-LTx etiologies.

Severe Liver Disorder Following Liver Transplantation in STING-Associated Vasculopathy with Onset in Infancy.

PURPOSE: STING-associated vasculopathy with onset in infancy (SAVI) is a type-I interferonopathy, characterized by systemic inflammation, peripheral vascular inflammation, and pulmonary manifestations. There are three reports of SAVI patients developing liver disease, but no report of a SAVI patient requiring liver transplantation. Therefore, the relevance of liver inflammation is unclear in SAVI. We report a SAVI patient who developed severe liver disorder following liver transplantation. METHODS: SAVI was diagnosed in a 4-year-old girl based on genetic analysis by whole-exome sequencing. We demonstrated clinical features, laboratory findings, and pathological examination of her original and transplanted livers. RESULTS: At 2 months of age, she developed bronchitis showing resistance to bronchodilators and antibiotics. At 10 months of age, she developed liver dysfunction with atypical cholangitis, which required liver transplantation at 1 year of age. At 2 years of age, multiple biliary cysts developed in the transplanted liver. At 3.9 years of age, SAVI was diagnosed by whole-exome sequencing. Inflammatory cells from the liver invaded the stomach wall directly, leading to fatal gastrointestinal bleeding unexpectedly at 4.6 years of age. In pathological findings, there were no typical findings of liver abscess, vasculitis, or graft rejection, but biliary cysts and infiltration of inflammatory cells, including plasmacytes around the bile duct area, in the transplanted liver were noted, which were findings similar to those of her original liver. CONCLUSION: Although further studies to clarify the mechanisms of the various liver disorders described in SAVI patients are needed, inflammatory liver manifestations may be amplified in the context of SAVI.

Neonatal systemic juvenile Xanthogranuloma with Hydrops diagnosed by Purpura skin biopsy: a case report and literature review.

BACKGROUND: Systemic juvenile xanthogranuloma is a very rare disease typically presents as skin lesions with yellow papules or nodules and is sometimes fatal. We report a case of congenital neonatal systemic juvenile xanthogranuloma with atypical skin appearance that made the diagnosis difficult. CASE PRESENTATION: A preterm Japanese female neonate with prenatally diagnosed fetal hydrops in-utero was born with purpuric lesions involving the trunk and face. Since birth, she had hypoxemic respiratory failure, splenomegaly, anemia, thrombocytopenia, coagulopathy, and was transfusion dependent for red blood cells, fresh frozen plasma, and platelets. Multiple cystic lesions in her liver, part of them with vascular, were detected by ultrasound. A liver biopsy was inconclusive. A skin lesion on her face similar to purpura gradually changed to a firm and solid enlarged non-yellow nodule. Technically, the typical finding on skin biopsy would have been histiocytic infiltration (without Touton Giant cells) and immunohistochemistry results which then would be consistent with a diagnosis of systemic juvenile xanthogranuloma, and chemotherapy improved her general condition. CONCLUSIONS: This case report shows that skin biopsies are necessary to detect neonatal systemic juvenile xanthogranuloma when there are organ symptoms and skin eruption, even if the skin lesion does not have a typical appearance of yellow papules or nodules.

Outcome for Pediatric Recipients of Macrosteatotic Liver Grafts From Living Donors.

In deceased donor liver transplantation, a donor liver with moderate (>30%) macrosteatosis used to be considered inappropriate for grafting. We examined the outcomes of children who underwent living donor liver transplantation (LDLT) at the National Center for Child Health and Development whose donor livers had moderate-to-severe macrosteatosis. Twelve children were enrolled who had received a moderate-to-severe macrosteatotic liver graft and underwent liver biopsy soon after LDLT. The primary diseases were biliary atresia in 7 patients, acute liver failure in 3 patients, glycogen storage disease type 1 in 1 patient, and primary sclerosing cholangitis in 1 patient. Median age was 11 months. There were 4 recipients who received grafts from their fathers, and 8 received grafts from their mothers. Median donor age was 35.5 years. We compared the degree of donor liver steatosis with the results of graft liver biopsies that were collected 4-105 days after LDLT. The levels of donor liver macrovesicular steatosis were moderate (34%-66%) in 9 patients and severe (>66%) in 3 patients. The nonalcoholic fatty liver disease activity score was 3 in 7 patients and 4 in 5 patients. Shortly after LDLT, 11 of 12 patients showed improvement in steatosis compared with the donor livers. One biopsy specimen taken 22 days after LDLT showed 60% macrosteatosis, which was the same as that in the donor liver. However, this patient was alive and well 6 years after LDLT. One patient died after LDLT because of infection and respiratory failure. The levels of steatosis of the donor liver grafts improved soon after LDLT in children, and the outcomes of children receiving a moderate-to-severe macrosteatotic liver from their parents were excellent.

Longterm Outcomes of Living Donor Liver Transplantation for Glycogen Storage Disease Type 1b.

Glycogen storage disease (GSD) type 1b (Online Mendelian Inheritance in Man [OMIM] 232220) is an autosomal recessive inborn error of carbohydrate metabolism caused by defects in glucose-6-phosphate translocase. GSD1b patients have severe hypoglycemia with several clinical manifestations of hepatomegaly, obesity, a doll-like face, and neutropenia. Liver transplantation (LT) has been indicated for severe glucose intolerance, poor metabolic control (PMC), and poor growth (PG). We retrospectively reviewed 11 children with GSD1b who underwent living donor liver transplantation (LDLT) at the National Center for Child Health and Development in Tokyo, Japan. Between November 2005 and December 2018, 495 children underwent LDLT with an overall 10-year patient and graft survival of 90.6% and 88.9%, respectively. Of these, LT was indicated for 11 patients with GSD1b. All patients are doing well with the stabilization of glucose intolerance and decreased hospitalization for infectious complications. Demand for granulocyte colony-stimulating factor significantly decreased. However, although LT stabilized the blood glucose level, the platelet function was not improved. The posttransplant developmental quotient (DQ) remained similar to the pretransplant DQ without deterioration. LDLT is a feasible procedure for GSD1b patients with regard to the longterm prognosis. LT should be considered for patients with severe glucose intolerance to protect the cognitive function against hypoglycemic encephalopathy and to ameliorate PMC and PG.

Etiology of liver dysfunction after liver transplantation in children with metabolic disorders.

AIM: It has been reported that the long-term outcome for children with metabolic disorders after liver transplantation (LT) is excellent. However, there are several reports citing LT patients with metabolic disorders developing liver dysfunction early after LT. We examined the pathogenesis of liver dysfunction observed after LT in recipients with metabolic disorders at the National Center for Child Health and Development. METHODS: Of 106 children (aged <18 years) with metabolic disorders who underwent LT at our center, 36 patients who underwent liver biopsy within 60 days after LT were enrolled. The underlying diseases were urea cycle disorders (14 patients), methylmalonic acidemia (11 cases), Wilson's disease (3 patients), mitochondrial hepatopathy (3 patients), and others (5 patients). The median age was 1 year 2 months at LT. The reasons for biopsy were liver dysfunction (31 patients) and ascites (5 patients). RESULTS: The main findings of graft liver biopsy were diffuse steatosis (21 patients), rejection (8 patients), infection (3 patients), and others (4 patients). Of 21 patients who received graft biopsy showing steatosis, all the donor livers originally showed no steatosis or only mild steatosis. The liver function improved immediately after biopsy in 18 of 21 patients that showed diffuse steatosis. CONCLUSIONS: The major cause of liver dysfunction after LT in recipients with metabolic disorders was steatosis and the risk of rejection was low. It is important to take a liver biopsy and examine the cause of liver dysfunction to avoid administration of excess immunosuppressant, and select the right therapy for recipients with metabolic disorders.

Late T cell-mediated rejection may contribute to poor outcomes in adolescents and young adults with liver transplantation.

Although poor long-term graft survival in LT in AYA is recognized, detailed epidemiological data are still lacking. L-TCMR may have poor outcomes. This study aimed to provide a detailed, epidemiological assessment of the association between AYA age and rejection. L-TCMR was defined in this study as TCMR with central vein or perivenular inflammation occurring later than 3 months after LT. A total of 342 patients who survived for at least 3 months after LT between 2005 and 2015 were enrolled. The AYA group (10-24 years) was compared with the C group (less than 10 years), and the incidence and outcomes of L-TCMR were analyzed. In total, 342 patients had LT; 38 of these were AYA with the mean follow-up period of 6.7 years. A total of 304 patients in C group had a mean follow-up period of 6.3 years (P = .28). The incidence of L-TCMR in AYA group was significantly higher than in C group (15.8% vs 4.6%, P = .006). The time to L-TCMR after LT was significantly shorter in AYA group (P = .01). Neither patient survival nor the incidence of non-adherence differed significantly between the groups (P = .18 and P = .89). The number of additional immunosuppressants after L-TCMR was significantly higher in the AYA group (P = .04). A high incidence of L-TCMR was observed in AYA group irrespective of non-adherence. AYA patients with L-TCMR should be followed carefully due to the poor results of post-treatment biopsy and the need for intensive immunosuppressive therapy.

Living donor liver transplantation for congenital hepatic fibrosis in children.

Congenital hepatic fibrosis (CHF) often accompanies autosomal recessive polycystic kidney disease (ARPKD), which stems from a PKHD1 gene mutation. The aim of this study was to clarify the prognosis of children with CHF who received living donor liver transplantation (LDLT) from donors who might be heterozygous carriers of a hepatorenal fibrocystic disease. Fourteen children with CHF who underwent LDLT at our center were enrolled. Eight and two patients had ARPKD and nephronophthisis, respectively. Eight of the donors were the recipients' fathers, and six donors were their mothers. We examined the histological and radiological findings of the donor livers and complications in the recipients following the liver transplantation. Seven of the donor livers presented morphological abnormalities of the bile ducts. Abdominal computed tomography revealed liver cysts in eight donors. One recipient underwent re-LT for graft failure due to rejection. Three patients presented with rejection, and one presented with sepsis. The overall survival rate was 100% and the original graft survival rate was 93%. In conclusion, the prognosis of recipients who received a LDLT from their parents for CHF was excellent. However, the morphology of half the donor livers was abnormal. Careful follow-up is needed to ensure long-term graft survival.

A Novel Construction of Lentiviral Vectors for Eliminating Tumorigenic Cells from Pluripotent Stem Cells.

The risk of tumor formation poses a challenge for human pluripotent stem cell (hPSC)-based transplantation therapy. Specific and total elimination of tumorigenic hPSCs by suicide genes (SGs) has not been achieved because no methodology currently exists for testing multiple candidate transgene constructs. Here, we present a novel method for efficient generation of tumorigenic cell-targeting lentiviral vectors (TC-LVs) with diverse promoters upstream of a fluorescent protein and SGs. Our two-plasmid system achieved rapid and simultaneous construction of different TC-LVs with different promoters. Ganciclovir (GCV) exerted remarkable cytotoxicity in herpes simplex virus thymidine kinase-transduced hPSCs, and high specificity for undifferentiated cells was achieved using the survivin promoter (TC-LV.Surv). Moreover, GCV treatment completely abolished teratoma formation by TC-LV.Surv-infected hPSCs transplanted into mice, without harmful effects. Thus, TC-LV can efficiently identify the best promoter and SG for specific and complete elimination of tumorigenic hPSCs, facilitating the development of safe regenerative medicine. Stem Cells 2018;36:230-239.

ABO-incompatible liver transplantation for children under 2 years of age: A case report and a single-center review.

Desensitization with RTX has been broadly introduced in adult LT across the ABO blood type barrier. For pediatric LT, the prophylactic use of RTX has not been standardized, especially for children under 2 years of age. A 20-month-old girl with BA underwent living donor LT from her ABO-I mother. On POD 6, she developed combined T cell-mediated and AMRs. Steroid bolus injection was immediately introduced, followed by antibody-depleting therapy with PE and IVIG. Based on a peripheral blood lymphocyte analysis by fluorescence-activated cell sorting, ATG and RTX were introduced for refractory rejection. Although she recovered from the combined rejections, IHBCs were inevitable as a consequence. We recommend extending the desensitization protocol to cover children under 2 years of age in order to prevent life-threatening complications.

Acute generalized pustular bacterid concomitant with erythema nodosum, polyarthritis, and Achilles tendinitis.

We report a case of acute generalized pustular bacterid (AGPB) concomitant with erythema nodosum (EN), polyarthritis, and Achilles tendinitis. The patient was admitted with a complaint of fever, widespread plural pustules, erythema, and polyarthralgia. Histopathological examination of the skin lesions demonstrated features of AGBP and EN. Although arthralgia and AGPB can be recognized together, EN and Achilles tendinitis are rare manifestations seen in patients with AGPB. In this case report, we suggest arthralgia, EN, and Achilles tendinitis could coexist with AGPB.

Impact of graft thickness reduction of left lateral segment on outcomes following pediatric living donor liver transplantation.

Reducing graft thickness is essential to prevent large-for-size graft problems in pediatric living donor liver transplantation (LDLT). However, long-term outcomes of LDLT using reduced-thickness left lateral segment (LLS) grafts are unclear. In 89 patients who underwent LDLT using reduced LLS grafts between 2005 and 2017, short-term and long-term outcomes were compared between a nonanatomically reduced LLS (NAR-LLS) graft group and a reduced-thickness LLS graft group. Estimated blood loss was lower and abdominal skin closure was less needed in the recipient operation in the reduced-thickness LLS graft group. Postoperatively, portal vein (PV) flow was significantly decreased in the NAR-LLS graft group, and there was shorter intensive care unit (ICU) stay and fewer postoperative complications, especially bacteremia, in the reduced-thickness LLS graft group. Graft survival at 1 and 3 years after LDLT using reduced-thickness LLS grafts was 95.2% and 92.4%, respectively, which was significantly better than for NAR-LLS grafts. Multivariate analysis revealed that fulminant liver failure, hepatofugal PV flow before LDLT, and NAR-LLS graft were associated with poor graft survival. In conclusion, LDLT using reduced-thickness LLS grafts is a safe and feasible option with better short- and long-term outcomes in comparison with NAR-LLS grafts.

Surgical treatment strategy for advanced hepatoblastoma: Resection versus transplantation.

BACKGROUND: Excellent outcomes of the extreme procedure of liver resection (LR) for advanced hepatoblastoma (HB) have been achieved in recent reports. However, liver transplantation (LT) remains the only surgical treatment for patients with unresectable HB. The aim of this study was to evaluate our retrospective data for cases of advanced HB necessitating surgical intervention and analyze the prognostic factors of recurrence by comparing patients with tumors resected by LR and LT. PATIENTS AND METHODS: We retrospectively reviewed 24 children with PRETEXT II/III/IV tumors that required consideration for LT between August 2011 and September 2016. RESULT: The staging at the time of the diagnosis was PRETEXT II/III/IV in 1/13/10 patients, respectively, while the preoperative staging after neoadjuvant chemotherapy was POSTTEXT II/III/IV in 5/17/2 patients. Among those 24 patients, complete resection of the primary tumor was achieved with LT in 12 patients and LR in 12 patients. A high serum level of alpha-fetoprotein (AFP) at the time of surgery, no significant decrease in the rate of change of AFP, and low tumor shrinkage rate were related to the risk of tumor recurrence, and patients with tumors resected by LR with those risks had a higher recurrence rate than those without them. The overall survival was higher in patients with tumors resected by LT (100%) than in patients with tumors resected by LR. CONCLUSION: Patients with advanced HB with a poor response to chemotherapy should definitively be prioritized for primary LT, given the possibility of vascular invasion and microscopic residual tumor.

A severely cholestatic liver graft can be successfully used in deceased donor liver transplantation.

The shortage of deceased organs is still a serious issue in Japan. A proactive approach to using liver grafts from extended criteria donors (ECDs) may be one way of expanding the donor pool; however, if it is recklessly attempted, a recipient receiving such a marginal graft can be at risk of mortality due to primary non-function or delayed graft function. We herein report the successful outcome of a recipient receiving a severely cholestatic graft that was considered transplantable because it lacked features characteristic of a long duration of "cholestasis" according to the precise interpretation of a donor biopsy. Plasma exchange was intentionally introduced to prevent toxic insult by hyperbilirubinemia immediately after transplant. Despite transient acute kidney injury immediately after transplant, the patient's renal impairment was well managed with a renal-sparing immunosuppressive regimen consisting of basiliximab and mycophenolate mofetil. Although the use of liver grafts from ECDs still needs to be discussed, especially regarding graft selection and allocation policies, efforts not to discard valuable grafts should be undertaken in our country.

Necrotizing enterocolitis in the setting of milk allergy after pediatric living donor liver transplantation.

NEC is an idiopathic intestinal mucosal injury that may progress to transmural bowel necrosis without mesenteric ischemia. NEC usually affects 7- to 10-day-old neonates following enteral feeding. A 10-month-old girl with no history of laparotomy underwent LDLT for acute liver failure. After starting enteral feeding on postoperative day 5, she developed abdominal distention. Diffuse PVG and PI were detected by radiologic modalities. Exploratory laparotomy revealed patchy necrosis of the intestine without perforation. The microscopic findings of a resected specimen revealed transmural coagulative necrosis with multiple small thromboses compatible with neonatal NEC features, and eosinophil infiltration was also observed. Subsequently, after the resumption of enteral feeding with cow's milk, she developed severe diarrhea, the symptoms of which were eliminated after the administration of cow's milk was stopped. These clinical and pathological findings support the speculation that NEC might have been induced by a CMA. Food allergies, which can be induced by immunosuppressive agents, should be considered as a potential cause of NEC in the setting of pediatric liver transplantation.

Free cholesterol accumulation in liver sinusoidal endothelial cells exacerbates acetaminophen hepatotoxicity via TLR9 signaling.

BACKGROUND & AIMS: Although obesity is a risk factor for acute liver failure, the pathogenic mechanisms are not yet fully understood. High cholesterol (HC) intake, which often underlies obesity, is suggested to play a role in the mechanism. We aimed to elucidate the effect of a HC diet on acetaminophen-induced acute liver injury, the most frequent cause of acute liver failure in the USA. METHODS: C57BL/6 Toll-like receptor 9 (TLR9) knockout (Tlr9-/-) mice and their Tlr9+/+ littermates were fed an HC diet for fourweeks and then treated with acetaminophen. Liver sinusoidal endothelial cells (LSECs) were isolated from the mice for in vivo and in vitro analyses. RESULTS: The HC diet exacerbated acetaminophen-induced acute liver injury in a TLR9/inflammasome pathway-dependent manner. LSECs played a major role in the cholesterol loading-induced exacerbation. The accumulation of free cholesterol in the endolysosomes in LSECs enhanced TLR9-mediated signaling, thereby exacerbating the pathology of acetaminophen-induced liver injury through the activation of the TLR9/inflammasome pathway. The accumulation of free cholesterol in LSEC endolysosomes induced a dysfunction of the Rab7 membrane trafficking recycling mechanism, thus disrupting the transport of TLR9 from late endosomes to the lysosomes. Consequently, the level of active TLR9 in the late endosomes increased, thereby enhancing TLR9 signaling in LSECs. CONCLUSIONS: HC intake exaggerated acetaminophen-induced acute liver injury via free cholesterol accumulation in LSECs, demonstrating a novel role of free cholesterol as a metabolic factor in TLR9 signal regulation and pathologies of acetaminophen-induced liver injury. Therapeutic approaches may target this pathway. Lay summary: High cholesterol intake exacerbated acetaminophen-induced acute liver injury via the accumulation of free cholesterol in the endolysosomes of liver sinusoidal endothelial cells. This accumulation enhanced Toll-like receptor 9 signaling via impairment of its membrane trafficking mechanism. Thus, free cholesterol accumulation, as an underlying metabolic factor, exacerbated the pathology of acetaminophen-induced liver injury through activation of the TLR9/inflammasome pathway.

The optimal immunosuppressive protocol for the portal vein infusion of PGE1 and methylprednisolone in pediatric liver transplantation for fulminant hepatic failure of unknown etiology.

The outcome of LTx in pediatric patients with FHF of unknown etiology remains inferior to that of LTx in pediatric patients with cholestatic diseases. A higher incidence of steroid-resistant severe rejection has been increasingly recognized among the responsible factors. We assessed the efficacy of the administration of steroids and PGE1 via PVI in the management of LTx for FHF in pediatric patients. In our early cohort (1995-2007), seven patients who underwent LTx for FHF of unknown etiology were treated with conventional immunosuppressive therapy (calcineurin inhibitor and a steroid). Seven of eight grafts (one patient underwent re-LTx) sustained CV and/or CPV associated with ACR, and four patients died of a graft failure or infectious complications that were associated with the treatment for rejection. Of note, the pathological incidence of CV/CPV was significantly higher in recipients with FHF of unknown etiology than in recipients with biliary cholestatic disease during the same study period (87.5% vs. 13.7%, p < 0.00001). From 2008, three patients underwent LTx for cryptogenic FHF with PVI and conventional IS. PVI was well tolerated, and no relevant severe complications were observed. More strikingly, the patients who received PVI overcame biopsy-proven immunological events and are all currently doing well with excellent graft function after more than five yr. We conclude that PVI is technically safe and effective for preventing severe rejection in pediatric patients who undergo LTx for FHF of unknown etiology and that it does not increase the risk of fatal infectious complications.

Free cholesterol accumulation in hepatic stellate cells: mechanism of liver fibrosis aggravation in nonalcoholic steatohepatitis in mice.

UNLABELLED: Although nonalcoholic steatohepatitis (NASH) is associated with hypercholesterolemia, the underlying mechanisms of this association have not been clarified. We aimed to elucidate the precise role of cholesterol in the pathophysiology of NASH. C57BL/6 mice were fed a control, high-cholesterol (HC), methionine-choline-deficient (MCD), or MCD+HC diet for 12 weeks or a control, HC, high-fat (HF), or HF+HC diet for 24 weeks. Increased cholesterol intake accelerated liver fibrosis in both the mouse models without affecting the degree of hepatocellular injury or Kupffer cell activation. The major causes of the accelerated liver fibrosis involved free cholesterol (FC) accumulation in hepatic stellate cells (HSCs), which increased Toll-like receptor 4 protein (TLR4) levels through suppression of the endosomal-lysosomal degradation pathway of TLR4, and thereby sensitized the cells to transforming growth factor (TGF)ß-induced activation by down-regulating the expression of bone morphogenetic protein and activin membrane-bound inhibitor. Mammalian-cell cholesterol levels are regulated by way of a feedback mechanism mediated by sterol regulatory element-binding protein 2 (SREBP2), maintaining cellular cholesterol homeostasis. Nevertheless, HSCs were sensitive to FC accumulation because the high intracellular expression ratio of SREBP cleavage-activating protein (Scap) to insulin-induced gene (Insig) disrupted the SREBP2-mediated feedback regulation of cholesterol homeostasis in these cells. HSC activation subsequently enhanced the disruption of the feedback system by Insig-1 down-regulation. In addition, the suppression of peroxisome proliferator-activated receptor γ signaling accompanying HSC activation enhanced both SREBP2 and microRNA-33a signaling. Consequently, FC accumulation in HSCs increased and further sensitized these cells to TGFß-induced activation in a vicious cycle, leading to exaggerated liver fibrosis in NASH. CONCLUSION: These characteristic mechanisms of FC accumulation in HSCs are potential targets to treat liver fibrosis in liver diseases including NASH.

High-salt in addition to high-fat diet may enhance inflammation and fibrosis in liver steatosis induced by oxidative stress and dyslipidemia in mice.

BACKGROUND: It is widely known that salt is an accelerating factor for the progression of metabolic syndrome and causes cardiovascular diseases, most likely due to its pro-oxidant properties. We hypothesized that excessive salt intake also facilitates the development of nonalcoholic steatohepatitis (NASH), which is frequently associated with metabolic syndrome. METHODS: We examined the exacerbating effect of high-salt diet on high-fat diet-induced liver injury in a susceptible model to oxidative stress, apoE knockout and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) transgenic mice. RESULTS: High-salt diet led to NASH in high-fat diet-fed LOX-1 transgenic/apoE knockout mice without affecting high-fat diet-induced dyslipidemia or hepatic triglyceride accumulation. Additionally, a high-salt and high-fat diet stimulated oxidative stress production and inflammatory reaction to a greater extent than did a high-fat diet in the liver of LOX-1 transgenic/apoE knockout mice. CONCLUSIONS: We demonstrated that high-salt diet exacerbated NASH in high-fat diet-fed LOX-1 transgenic /apoE knockout mice and that this effect was associated with the stimulation of oxidative and inflammatory processes; this is the first study to suggest the important role of excessive salt intake in the development of NASH.