Hepatic Hypoxia Research Articles

Effect of hypoxia on aquaporins and hepatobiliary transport systems in human hepatic cells.

Hepatic ischemia and hypoxia are accompanied by reduced bile flow, biliary sludge and cholestasis. Hepatobiliary transport systems, nuclear receptors and aquaporins were studied after hypoxia and reoxygenation in human hepatic cells. Expression of Aquaporin 8 (AQP8), Aquaporin 9 (AQP9), Pregnane X receptor (PXR), Farnesoid X receptor (FXR), Organic anion transporting polypeptide 1 (OATP1), and the Multidrug resistance-associated protein 4 (MRP4) were investigated in induced pluripotent stem cells (iPSCs) derived hepatic cells and the immortalized hepatic line HepG2. HepG2 was subjected to combined oxygen and glucose deprivation for 4 h followed by reoxygenation. Expression of AQP8 and AQP9 increased during differentiation in iPSC-derived hepatic cells. Hypoxia did not alter mRNA levels of AQP8, but reoxygenation caused a marked increase in AQP8 mRNA expression. While expression of OATP1 had a transient increase during reoxygenation, MRP4 showed a delayed downregulation. Knock-down of FXR did not alter the expression of AQP8, AQP9, MRP4, or OATP1. Post-hypoxic protein levels of AQP8 were reduced after 68 h of reoxygenation compared to normoxic controls. Post-transcriptional mechanisms rather than reduced transcription cause reduction in AQP8 protein concentration after hypoxia-reoxygenation in hepatic cells. Expression patterns differed between hepatobiliary transport systems during hypoxia and reoxygenation. Expression of AQP8 and AQP9 increased during differentiation in induced pluripotent stem cells. Expression of hepatobiliary transporters varies during hypoxia and reoxygenation. Post-hypoxic protein levels of AQP8 were reduced after 68 h of reoxygenation. Post-transcriptional mechanisms rather than reduced transcription cause reduction in AQP8 protein concentration after hypoxia-reoxygenation in hepatic cells. Hypoxia and reoxygenation may affect aquaporins in hepatic cells and potentially affect bile composition.

Hif-1α expression targets the TMA/Fmo3/TMAO axis to participate in gallbladder cholesterol stone formation in individuals living in plateau regions

The incidence of gallbladder cholesterol stones (GCS) increases rapidly among people living in high-altitude hypoxic environments compared to those in normoxic areas. Upregulation of hepatic hypoxia inducible factor 1α (Hif-1α) plays a key role in the formation of GCS. High plasma trimethylamine-N-oxide (TMAO) levels are positively correlated with the occurrence of GCS. We hypothesized that HIF-1α may upregulate TMAO levels by promoting the transcription of flavin-containing monooxygenase 3 (Fmo3), which eventually leads to GCS formation. Our study shows that in women, high plasma total cholesterol and apolipoprotein B were positively correlated with cholecystolithiasis and hypoxia. Hif-1α binds to the Fmo3 promoter and promotes Fmo3 expression. Hypoxia and lithogenic diet induce the expression of Hif-1α, Fmo3, TMAO and cholesterol tube transporters in the livers of mice, disturb the proportion of bile and plasma components, and induce the formation of GCS. In cell experiments, silencing Hif-1α downregulates the expression of Fmo3, TMAO and cholesterol tube transporters. In a mouse model of hypoxic cholecystolithiasis, silencing Hif-1α downregulates the expression of related genes, restores the proportion of bile and plasma lipid components, and reduces the formation of GCS. Our study shows that Hif-1α binds to the promoter region of Fmo3 and promotes Fmo3 transcription. Thus, it mediates the transcriptional activation of the TMA/Fmo3/TMAO pathway, upregulates the expression of ATP-binding cassettes (Abc) g5 and g8, and participates in the regulation of the occurrence of GCS in the plateau region.

The Therapeutic Effects of Baicalein on the Hepatopulmonary Syndrome in the Rat Model of Chronic Common Bile Duct Ligation.

Hepatopulmonary syndrome (HPS) is characterized by arterial oxygenation defects due to pulmonary vascular dilation in liver disease. To date, liver transplantation remains the only effective treatment for HPS. This study aimed to explore the preventative role of baicalein in HPS development. Sixty male rats were randomly assigned to three groups: sham, common bile duct ligation (CBDL), and baicalein, receiving intraperitoneal injections of baicalein (40 mg·kg-1·d-1, diluted in saline) for 21 days. Survival rate, liver and kidney function, and bile acid metabolism levels were evaluated. Liver and lung angiogenesis and hepatic glycogen staining were assessed, and the expression of relevant proteins was evaluated by immunohistochemistry. Baicalein improved survival rates and hypoxemia in rats post-CBDL, reducing angiogenic protein levels and enhancing glucose homeostasis. Compared to the untreated group, baicalein suppressed the expression of vascular endothelial growth factor, placental growth factors, matrix metalloprotease 9 and C-X-C motif chemokine 2, and it increased the expression of glycemic regulatory proteins, including dipeptidyl peptidase-4, sirtuin 1, peroxisome proliferator-activated receptor gamma co-activator 1α, and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3. Baicalein significantly improves hepatic function and hypoxia in HPS rats by attenuating pathological angiogenesis in the liver and lungs, showing promise as a treatment for HPS.

Erratum: Differential expression of hepatic cancer stemness and hypoxia markers in residual cancer after locoregional therapies for hepatocellular carcinoma.

Erratum: Differential expression of hepatic cancer stemness and hypoxia markers in residual cancer after locoregional therapies for hepatocellular carcinoma.

Sinomenine improves resistance to foodborne enteritis and Anti-bacteria mucosal immunity in grass carp

Based on our previous study in zebrafish, sinomenine was found to relieve foodborne enteritis through immunomodulation. To examine the protective effects of sinomenine on mucosal immunity in farmed fish species, a dietary inclusion study was conducted in grass carp juveniles by adding sinomenine to soybean meal feed. After a 5-week feeding trial to model foodborne enteritis, the gut and liver tissues were examined for histopathology, enteritis-related gene expression, transcriptome, and 16 s rRNA gene sequencing. The results demonstrated that including sinomenine in the diet at the concentration of 2 g/kg could prevent intestinal pathological changes, such as a decrease in the number of goblet cells and increased glycogen, induced by 40% soybean meal substitution. At the molecular level, the downregulated proinflammatory genes, together with upregulated anti-inflammatory genes (such as il10, il13, and tgfβ) were observed. At the omics level, for the DEGs, the downregulated intestinal pathway “phagosome” and term “platelet activation” indicated control of inflammation, while the upregulation of the “PPAR signaling pathway” in both the gut and liver, suggests enhanced immune homeostasis. For the mucosal surface in the sinomenine-supplemented group, we observed not only control of pathogenic Camparilobacterota and Aeromonas but also an increase in Actinobacteria, which may enhance mucosal immunity. Due to the influence of the gut-liver axis, hepatic metabolism was altered, particularly with an upregulation of “fatty acid degradation” and “tryptophan metabolism”. Subsequently, the fish in the sinomenine group exhibited a significantly higher survival rate after a 7-day immersion challenge with Aeromonas hydrophila, with less inflammatory CD4 signals in the gills. Hepatic hypoxia and low respiratory efficiency in the gill may be a side effect. In summary, sinomenine demonstrated a protective effect on the mucosal immunity of the gut and gill, thereby preventing enteritis and subsequent bacteria challenges. Therefore, sinomenine could be employed in aquaculture as phytotherapy, at the very least through dietary supplementation in fish.

AN INFLUENCE OF ACUTE CORONAVIRUS DISEASE ON HEPATIC TISSUE (LITERATURE REVIEW)

Nowadays we have a great number of patients who have suffered from acute coronavirus disease. More than 687 million people at least once had the positive COVID-test. Moreover, more than 27 thousand people are suffering from this disease in Ukraine now. SARS-CoV-2 virus can damage various organs and systems, primarily the respiratory system that will manifest in a dry cough, shortness of breath, general weakness, up to the development of acute respiratory distress syndrome, shock and acute heart failure. The damage of lover by SARS-CoV-2 virus can be both direct and indirect. The presence of liver damage often leads to a more severe course of acute coronavirus disease. In order to treat new cases more effectively and deal with complications of old ones it is necessary to study better the mechanisms of the viral effect on various organs, in particular the liver. The best way to find out this mechanism is to examine main laboratory and instrumental indicators, learn about the changes in histological sections.
 Aim. The aim of the research was to investigate the results of up-to-date researches of the main changes in laboratory and instrumental indicators because of acute coronavirus disease, as well as possible mechanisms of liver damage.
 Materials and methods. Analysis of open publications of Scopus, Web of Science, PubMed for the last 10 years.
 The results. In patients with acute coronavirus disease an increase in the levels of transaminases (in most cases due to alanine aminotransferase), lactate dehydrogenase, bilirubin, prolongation of prothrombin time, an increase in the level of D-dimer, and a decrease in the number of platelets and albumins were often observed. Histological examination of biopsies revealed diffuse lung damage, significant desquamation of pneumocytes and hyaline membranes, extensive interstitial fibrosis with areas of fibrinous exudation and inflammation in the alveolar space. Hepatocyte apoptosis, moderate microvascular and minor macrovascular steatosis were observed in the hepatic tissue. Thus, we can suppose that the possible mechanisms of liver damage are direct virus damage, immunomodulated inflammation, hypercoagulative processes, hepatic hypoxia, drug-induced damage. Virus SARS-CoV-2 can get into the cell using angiotensin-converting enzyme 2 receptors. Direct virus influence can be possible because of high quantity of ACE2 receptors in bile-ducts and hepatocytes. Great amount of them is also present in vascular endothelium. As a result of apoptosis of COVID- infected cells a great amount of cytokines is released. This can lead to immunomodulated damage of different organs including liver. Moreover, inflammation leads to hypercoagulable state that in future can cause hypoxia of hepatic tissue because of thrombosis of liver vessels. Cytokine storm that developed in patients with severe COVID-19 pneumonia can lead to the destruction of tissues in the center of inflammation. The large number of cytokines that are released can increase vascular permeability that leads to dyspnea and respiratory failure because of blood and fluid migration. Researches showed us that some of the drugs that are uses as a treatment for COVID-19 may cause drug-induced damage of liver. Remdesivir is known to increase hepatic enzymes, reducing the level of albumin and increasing the level of bilirubin. LPV/r caused side effects more often than other drugs.
 Conclusions. As a result of the research the main symptoms, clinical and instrumental changes typical for acute coronavirus infection, possible mechanisms of liver damage because of this disease were revealed. However, we don’t have enough information to prescribe a pathogenetically justified treatment so the further investigation is needed.

Development of albumin monitoring system with hepatic hypoxia-on-a-chip

Hypoxia plays an essential role in the pathogenesis of various liver diseases, and albumin is one of the important biomarkers secreted by the liver. In this study, we developed an albumin monitoring system composed of hepatic hypoxia-on-a-chip and an albumin sensor to study liver function change due to hypoxia. In hepatic hypoxia-on-a-chip, we vertically stack an oxygen-scavenging channel on a liver on a chip with a thin gas-permeable membrane in the middle. This unique design of the hepatic hypoxia-on-a-chip can help to induce hypoxia quickly, attaining <5% within 10 min. An electrochemical albumin sensor was fabricated based on the covalent immobilization of antibodies on the Au electrode to monitor albumin secreting function on the hepatic hypoxia-on-a-chip. Standard albumin samples spiked in PBS, and culture media were measured by the electrochemical impedance spectroscopy using the fabricated immunosensor. The LOD was calculated to be 10 ag/mL in both cases. Using the electrochemical albumin sensor, we measured albumin secretion in normoxia and hypoxia in the chips. The albumin concentration decreased to 27% after 24 h in hypoxia compared to normoxia. This response was consistent with physiological studies. With technical refinements, the present albumin monitoring system can be a powerful tool in studying hepatic hypoxia with real-time liver function monitoring.

Ultrasensitive US Microvessel Imaging of Hepatic Microcirculation in the Cirrhotic Rat Liver.

Background Liver microcirculation dysfunction plays a vital role in the occurrence and development of liver diseases, and thus, there is a clinical need for in vivo, noninvasive, and quantitative evaluation of liver microcirculation. Purpose To evaluate the feasibility of ultrasensitive US microvessel imaging (UMI) in the visualization and quantification of hepatic microvessels in healthy and cirrhotic rats. Materials and Methods In vivo studies were performed to image hepatic microvasculature by means of laparotomy in Sprague-Dawley rats (five cirrhotic and five control rats). In vivo conventional power Doppler US and ex vivo micro-CT were performed for comparison. UMI-based quantifications of perfusion, tortuosity, and integrity of microvessels were compared between the control and cirrhotic groups by using the Wilcoxon test. Spearman correlations between quantification parameters and pathologic fibrosis, perfusion function, and hepatic hypoxia were evaluated. Results UMI helped detect minute vessels below the liver capsule, as compared with conventional power Doppler US and micro-CT. With use of UMI, lower perfusion indicated by vessel density (median, 22% [IQR, 20%-28%] vs 41% [IQR, 37%-46%]; P = .008) and fractional moving blood volume (FMBV) (median, 6.4% [IQR, 4.8%-8.6%] vs 13% [IQR, 12%-14%]; P = .008) and higher tortuosity indicated by the sum of angles metric (SOAM) (median, 3.0 [IQR, 2.9-3.0] vs 2.7 [IQR, 2.6-2.9]; P = .008) were demonstrated in the cirrhotic rat group compared with the control group. Vessel density (r = 0.85, P = .003), FMBV (r = 0.86, P = .002), and median SOAM (r = -0.83, P = .003) showed strong correlations with pathologically derived vessel density labeled with dextran. Vessel density (r = -0.81, P = .005) and median SOAM (r = 0.87, P = .001) also showed strong correlations with hepatic tissue hypoxia. Conclusion Contrast-free ultrasensitive US microvessel imaging provided noninvasive in vivo imaging and quantification of hepatic microvessels in cirrhotic rat liver. © RSNA, 2022 Supplemental material is available for this article. See also the editorial by Fetzer in this issue.

Differential expression of hepatic cancer stemness and hypoxia markers in residual cancer after locoregional therapies for hepatocellular carcinoma.

Transarterial chemoembolization (TACE) and transarterial radioembolization (TARE) treatment to hepatocellular carcinoma (HCC) are effective tools to control tumor growth, prolong survival, palliate symptoms, and improve quality of life for patients with intermediate‐stage HCC. Nevertheless, there is high variability of local HCC responses to locoregional therapies; therefore, better and personalized prediction of tumor response to TACE is necessary for management of patients with HCC, especially when these modalities of treatment are used to bridge patients for liver transplant. Here, we investigated differential expression of hepatic cancer stem cell and hypoxia in residual HCC after TACE treatment in comparison with TARE. A publicly available gene data set was screened for differentially expressed genes (DEGs) in TACE_Response compared with TACE_Non‐response HCC. Analysis of the GSE104580 data set displayed a total of 406 DEGs, including 196 down‐regulated and 210 up‐regulated DEGs. Of the 196 down‐regulated DEGs, three hepatic cancer stem cell (CSC) markers and 11 hypoxia‐related genes were identified. Immunohistochemical staining of hepatic CSC and hypoxia markers on explant liver tissues exhibited more intense positive staining of hepatic CSC markers (CD24, EpCAM) and hypoxia marker carbonic anhydrase 9 (CA9) in residual tumor nodule from patients with HCC treated with TACE compared with nontreated patients. Furthermore, Pearson's correlation analysis revealed the significant correlation between hepatic CSC markers and hypoxia marker, CA9. Conclusion: Hepatic CSC and hypoxia markers predict nonresponse to TACE and are differentially expressed in residual tumor after TACE compared with TARE. In the long term, TACE‐induced hypoxia may select an aggressive HCC phenotype.

Berberine reduces hepatic ceramide levels to improve insulin resistance in HFD-fed mice by inhibiting HIF-2α

Several studies have documented the effects of hypoxia and ceramides on lipid and glucose metabolism, resulting in insulin resistance. However, the roles of ceramide in hepatic hypoxia and hepatic insulin resistance remain to be clarified. This study aimed to explore the relationship between hypoxia, ceramide synthesis, and hepatic insulin resistance in high-fat diet (HFD)-fed mice. Given the interaction of hypoxia-inducible factors 2α(HIF-2α) and berberine determined using molecular docking, this study also assessed the pharmacological effects of berberine on the HIF-2α-ceramide-insulin resistance pathway. In the preliminary phase of the study, gradually aggravated hepatic hypoxia and varying levels of ceramides were observed with the development of type 2 diabetes mellitus (T2DM) due to increasing HIF-2α accumulation. Lipidomic analyses of animal and cell models revealed that berberine reduced hypoxia-induced ceramide production and attenuated ceramide-induced insulin resistance. This research provides timely and necessary evidence for the role of ceramide in hypoxia and insulin resistance in the liver. It also contributes to a better understanding of the pharmacological effects of berberine on ameliorating hypoxia and insulin resistance in T2DM therapy.

Endothelial dysfunction in pathological processes of chronic liver disease during aging.

Aging is associated with gradual changes in liver structure and physiological/pathological functions in hepatic cells including hepatocytes, cholangiocytes, Kupffer cells, hepatic stellate cells (HSCs), and liver sinusoidal endothelial cells (LSECs). LSECs are specialized hepatic endothelial cells that regulate liver homeostasis. These cells actively impact the hepatic microenvironment as they have fenestrations and a thin morphology to allow substance exchange between circulating blood and the liver tissue. As aging occurs, LSECs have a reduction in both the number and size of fenestrations, which is referred to as pseudocapillarization. This along with the aging of the liver leads to increased oxidative stress, decreased availability of nitric oxide, decreased hepatic blood flow, and increased inflammatory cytokines in LSECs. Vascular aging can also lead to hepatic hypoxia, HSC activation, and liver fibrosis. In this review, we described the basic structure of LSECs, and the effect of LSECs on hepatic inflammation and fibrosis during aging process. We briefly summarized the changes of hepatic microcirculation during liver inflammation, the effect of aging on the clearance function of LSECs, the interactions between LSECs and immunity, hepatocytes or other hepatic nonparenchymal cells, and the therapeutic intervention of liver diseases by targeting LSECs and vascular system. Since LSECs play an important role in the development of liver fibrosis and the changes of LSEC phenotype occur in the early stage of liver fibrosis, the study of LSECs in the fibrotic liver is valuable for the detection of early liver fibrosis and the early intervention of fibrotic response.

Role of COVID-19 in Hepatic Damage and Its Cellular Entrance and Mechanism of Action: A Mini-Review

Context: COVID-19 induces damage to the liver in addition to the pulmonary system. This event can alter hepatic biochemical and histological variables. Therefore, the present mini-review study investigated the effect of COVID-19 on hepatic damages and its cellular mechanism of action. Evidence Acquisition: In this mini-review study, the effects of COVID-19 on liver cells were sought in PubMed without restriction. The related components were searched in the Me SH term system and also used in two groups of COVID-19 and hepatocytes in combination with each other. Then, the titles and abstracts of the selected articles were assessed, and unrelated articles were removed. Results and Conclusion: COVID-19 leads to liver tissue damage and hepatocyte apoptosis by inducing increased cytokines, ischemia, and hepatic hypoxia following cardiorespiratory failure, coagulation disorders, and intracellular calcium. The SARS-CoV-2 virus exerts this effect through the ACE2 receptor and increasing its expression in cholangiocytes.

HFD and HFD-provoked hepatic hypoxia act as reciprocal causation for NAFLD via HIF-independent signaling

BackgroundThe occurrence of non-alcoholic fatty liver disease (NAFLD) is found to be higher in patients with obstructive sleep apnea (OSA), which is characterized by intermittent hypoxia. Activation of hypoxia-inducible factors has been shown in the development and progression of NAFLD, implying a cause and effects relationship between NAFLD and hypoxia. The present study was designed to investigate the interaction of lipotoxicity and hypoxia in the pathogenesis of NAFLD using mice model with high-fat diet (HFD) feeding or hypoxic treatment.MethodsNAFLD model was induced in mice by HFD feeding, and in cultured primary hepatocytes by administration of palmitate acid. Mouse hypoxic model was produced by placing the mice in a Animal incubator with oxygen concentration at 75% followed by a 21% oxygen supplement. Hypoxic condition was mimicked by treating the hepatocytes with cobalt chloride (CoCl2) or 1% oxygen supply. Pimonidazole assay was conducted to evaluate hypoxia. Lipid metabolic genes were measured by real-time polymerase-chain reaction. HIF-1α and HIF-2α genes were silenced by siRNA.ResultsHFD feeding and palmitate acid treatment provoked severe hepatic hypoxia along with TG accumulation in mice and in cultured primary hepatocytes respectively. Conversely, hypoxia induced hepatic TG accumulation in mice and in cultured primary hepatocytes. Hypoxic treatment inhibited the expression of lipolytic genes, while increased the expression of lipogenicgenes in mice. Although both lipotoxicity and hypoxia could activate hepatic hypoxia-induced factor 1α and 2α, while neither lipotoxicity- nor hypoxia- induced hepatic steatosis was affected when HIF was knocked down.ConclusionsHFD resulted in hepatic TG accumulation and concomitant hypoxia. Conversely, hypoxia induced hepatic TG accumulation in mice and in cultured heptocytes. Thus lipotoxicity and hypoxia might work as reciprocal causation and orchestrate to promote the development of NAFLD.

Oxygen therapy alleviates hepatic steatosis by inhibiting hypoxia-inducible factor-2α.

Non-alcoholic fatty liver disease (NAFLD) is difficult to manage due to the lack of effective treatments. Increased oxygen consumption caused by overnutrition, along with reduced oxygen delivery to liver cells induces hepatic steatosis. Here, we investigated the efficacy of oxygen therapy (OT) to alleviate hepatic steatosis. The effect of OT on hepatic steatosis was evaluated in high-fat-diet (HFD)-fed mice and palmitic acid (PA)-treated primary hepatocytes. Liver biopsy tissue samples were used to determine the relationship between the expression of hypoxia-inducible factor-2α (HIF-2α) and the progression of NAFLD. The role of HIF-2α in the OT group was determined based on the overexpression of HIF-2α in vitro. OT safely alleviated hepatic hypoxia and improved hepatic steatosis by inhibiting hepatic de novo lipogenesis in HFD-fed mice and PA-treated primary hepatocytes, and this was accompanied by reduced expression of HIF-2α and hepatic de novo lipogenesis. The analysis of liver tissues from individuals with or without NAFLD revealed a positive correlation between hepatic HIF-2α expression and NAFLD progression. Overexpression of HIF-2α in vitro inhibited the beneficial effect of OT against hepatic lipogenesis and steatosis. OT might be a viable treatment option for NAFLD and functions by alleviating hypoxia and inhibiting the liver HIF-2α signaling pathway.

Utility of N-acetylcysteine in ischemic hepatitis in cirrhotics with acute variceal bleed: a randomized controlled trial.

Ischemic hepatitis (IH) following acute variceal bleed (AVB) carries an ominous prognosis. N-Acetylcysteine (NAC), a potent anti-oxidant, may prevent IH by improving tissue oxygen delivery and improving hepatic hypoxia. Consecutive cirrhotics with AVB were prospectively randomized to receive either standard of care (SOC) plus NAC intravenously for 72h(at 150mg/kg/h for 1h followed by 12.5mg/kg/h for 4h, followed by 6.25mg/kg for 67h) (Group A, n = 107) or SOC alone (Group B, n = 107). Baseline characteristics were comparable. IH developed more frequently in Gr.B 25(23%) than A-15(14%); p = 0.08). Incidence of IH increased with severity of liver disease. Binary logistic regression analysis showed reduced incidence of IH in Gr.A than B [odds ratio (OR) 0.33, 0.11-0.93] patients after controlling for other significant factors. The incidence of acute kidney injury (AKI) was also reduced in Gr.A [OR 0.34, 0.15-0.75]. Development of IH was significantly associated with increased deaths due to liver failure at 6weeks [subdistribution hazard ratio (SHR) 21.6, 7.4-62.8]. On multivariate competing risk analysis, significantly lower deaths due to liver failure (SHR 0.33, 0.11-0.97) were noted in Gr.A than B. One in five patients with acute variceal bleed develops ischemic hepatitis which is associated with worse outcomes. NAC therapy averts deaths due to liver failure by preventing IH and reduces AKI and is, therefore, recommended for cirrhotics with acute variceal bleed. Clinicaltrials.gov no: NCT02015403.

NCOA4 Expression in Hepatic Cells Is Upregulated Under Physiological and Pathophysiological Conditions Associated with Hypoxia

The ubiquitously expressed intracellular protein NCOA4 mediates the degradation of ferritin in vitro (Mancias et al., Nature 2014; Dowdle et al., Nat Cell Biol 2014) and its loss disrupts systemic iron balance in mice (Bellelli et al., Cell Rep 2016). While a mechanism for increased NCOA4 protein turnover under high iron conditions has been reported in transformed cell lines of non-hepatic origin (Mancias et al., Elife, 2015), factors that regulate NCOA4 mRNA levels have not yet been described. Here we investigate stimuli that induce NCOA4 mRNA expression in hepatoma cells as well as in liver, the major iron depot of the body. We found that treating the human hepatoma cell line, Hep3B, for 18 hrs with increasing concentrations of the iron chelator desferrioxamine (DFO; 25-100μM) resulted in significantly higher NCOA4 mRNA (ANOVA P&amp;lt;.0001; 1.8 fold increase at the highest dose). Additionally, the murine hepatoma cell line, Hepa1-6, responded to 18 hrs of DFO treatment (25-100μM) with a dose-dependent rise in Ncoa4 mRNA levels (ANOVA P&amp;lt;.0001; 2.7 fold increase at the highest dose). DFO treatment of Hep3B and Hepa1-6 cells also increased mRNA levels of the transferrin receptor, confirming induction of iron deficiency. Because DFO has also been shown to stabilize hypoxia inducible factor (HIF), we examined the effects of other chemical hypoxia mimetics on hepatoma cell lines. Hep3B cells treated for 18 hrs with CoCl2 (25-75 μM), which is known to enhance the stability of HIF1α, showed a 2-fold increase in NCOA4 mRNA (ANOVA; P&amp;lt;.0001). Similarly, treatment of Hep3B cells with dimethyloxalylglycine (DMOG; 1mM), a competitive inhibitor of HIF prolyl-hydroxylases that promote HIF-α degradation, resulted in a 3-fold increase in NCOA4 mRNA at 12 hrs, which was further enhanced at 18 hrs. These results suggest that hepatoma cells in culture may respond to decreased iron availability and/or hypoxia by upregulating NCOA4 mRNA levels. We also investigated if Ncoa4 mRNA expression is upregulated in mouse livers under conditions associated with depletion of hepatic iron stores and/or hypoxia. C57BL/6N mice raised on a 45 p.p.m. iron diet were subjected to a large-volume phlebotomy (500μl; accompanied by intraperitoneal saline volume replacement) and fed an iron-deficient diet (2-6 p.p.m.) after bleeding. Seven days after phlebotomy, mice showed significantly lower blood hemoglobin levels (Student's T test P&amp;lt;.0001) and hepatic non-heme iron concentrations (P&amp;lt;.0001). However, the phlebotomized mice exhibited 1.8-fold higher hepatic Ncoa4 mRNA levels (P&amp;lt;.01) compared to non-phlebotomized controls. Additionally, we examined if hepatic Ncoa4 expression changed during normal pregnancy, a state in which extra-hepatic iron demands are increased by both the expanding maternal blood volume and the growing fetus. Compared to non-pregnant females, C57BL/6N mice at day 18.5 of pregnancy showed significantly lower liver non-heme iron concentrations (P&amp;lt;.01), while hepatic Ncoa4 mRNA levels were 3.5-fold higher (P&amp;lt;.01) in pregnant females compared to non-pregnant controls. By mining a published murine gene expression dataset (GEO accession GDS5818), we found that hepatic Ncoa4 mRNA levels are relatively high in early life (embryonic day 18 and postnatal day 5) and decline by adulthood (postnatal day 56), suggesting a relatively greater hepatic requirement for NCOA4 function during periods of high growth. Collectively, these in vivo data show that Ncoa4 upregulation correlates with conditions in which mobilizing iron for systemic use takes precedence over building hepatic iron stores. In summary, we show for the first time that Ncoa4 mRNA rises significantly in physiological and pathophysiological states in which increased extra-hepatic iron demands induce a reduction in hepatic iron stores. Additionally, we show that Ncoa4 mRNA levels in cultured cells of hepatic origin rise in response to hypoxia mimetics. Because NCOA4 functions as a cargo receptor that traffics ferritin to the autolysosome, NCOA4 protein is expected to be consumed during the process of ferritinophagy. We suggest that upregulation of hepatic Ncoa4 mRNA in response to hypoxia may represent a physiological adaptation to replenish NCOA4 protein that is needed to support continued mobilization of iron from the liver during periods of increased systemic iron demands. Disclosures No relevant conflicts of interest to declare.

Mapping hepatic blood oxygenation by quantitative BOLD (qBOLD) MRI.

Abnormalities in hepatic oxygen delivery and oxygen consumption may serve as a significant indicator of hepatic cellular dysfunction and may predict treatment response. However, conventional and oxygen-enhanced hepatic BOLD MRI can only provide semiquantitative assessment of hepatic oxygenation. A hepatic quantitative BOLD (qBOLD) model was proposed for noninvasive mapping of hepatic venous blood oxygen saturation (Yv ) and deoxygenated blood volume (DBV) in human subjects. The validity and the estimation bias of the proposed model were evaluated by Monte Carlo simulations. Eight healthy subjects were scanned after written consent with institutional review board approval. Monte Carlo simulations demonstrated that the proposed single-compartment hepatic qBOLD model leads to significant deviation of the predicted T2* decay profile from the simulated signal due to high hepatic blood volume fraction. Small relative estimation bias for hepatic Yv and significant overestimation for hepatic DBV were observed, which can be corrected by applying the calibration curves established from simulations. After correction, the mean hepatic Yv in human subjects was 56.8 ± 6.8%, and the mean hepatic DBV was 0.190 ± 0.035, consistent with measurements from other invasive approaches. Except in regions with significant vascular contamination, the maps for hepatic Yv and DBV were relatively homogenous. With estimation bias correction, the hepatic qBOLD approach enables noninvasive mapping of hepatic blood volume and oxygenation in human subjects. The established protocol may be used to quantitatively assess hepatic tissue hypoxia in multiple liver diseases.

The potential role of vascular alterations and subsequent impaired liver blood flow and hepatic hypoxia in the pathophysiology of non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) covers a spectrum of disease ranging from steatosis to steatohepatitis (NASH) and fibrosis, but the underlying pathophysiological mechanisms remain largely unknown. As there is currently no approved pharmacological therapy and the prevalence of NAFLD keeps increasing, understanding of its pathophysiology is crucial. We hypothesise that vascular alterations in early NAFLD play a role in the progression of the disease by inducing an increased intrahepatic vascular resistance and consequently relative hypoxia in the liver. Evidence of the detrimental effects of hypoxia in NAFLD has already been observed in liver surgery, where the outcomes of steatotic livers after ischaemia-reperfusion are worse than in healthy livers, and in obstructive sleep apnoea, which is an independent risk factor of NAFLD. Moreover, early histological damage in NAFLD is situated in the pericentral zone, which is also the first zone to be affected by a decreased oxygen tension because of the unique hepatic vacsular anatomy that causes the pericentral oxygen tension to be the lowest. Angiogenesis is also a characteristic of NAFLD, driven by hypoxia-induced mechanisms, as demonstrated in both animal models and in humans with NAFLD. Relative hypoxia is most probably induced by impaired blood flow to the liver, caused by increased intrahepatic vascular resistance. An increased intrahepatic vascular resistance early in the development of disease has been convincingly demonstrated in several animal models of NAFLD, whereas an increased portal pressure, a consequence of increased intrahepatic vascular resistance, has been proven in patients with NAFLD. Animal studies demonstrated a decreased intrahepatic effect of vasodilators and an increased reactivity to vasoconstrictors that results in an increased intrahepatic vascular resistance, thus the presence of a functional component. Pharmacological products that target vasoregulation can hence improve the intrahepatic vascular resistance and this might prevent or reverse progression of NAFLD, representing an important therapeutic option to study. Some of the drugs currently under evaluation in clinical trials for NASH have interesting properties related to the hepatic vasculature. Some other interesting drugs have been tested in animal models but further study in patients with NAFLD is warranted.In summary, in this paper we summarise the evidence that leads to the hypothesis that an increased intrahepatic vascular resistance and subsequent parenchymal hypoxia in early NAFLD is an important pathophysiological driving mechanism for the progression of the disease.

Liver hypertrophy: Underlying mechanisms and promoting procedures before major hepatectomy

Various procedures can promote hypertrophy of the future liver remnant (FLR) before major hepatectomy to prevent postoperative liver failure. The pathophysiological situation following portal vein embolization (PVE), hepatic artery ligation/embolization or hepatectomy remains unclear. On one hand, the main mechanisms of hepatic regeneration appear to be driven by hepatic hypoxia (involving the hepatic arterial buffer response), an increased portal blood flow inducing shear stress and the involvement of several mediators (inflammatory cytokines, vasoregulators, growth factors, eicosanoids and several hormones). On the other hand, several factors are associated with impaired liver regeneration, such as biliary obstruction, malnutrition, diabetes mellitus, male gender, age, ethanol and viral infection. All these mechanisms may explain the varying degrees of hypertrophy observed following a surgical or radiological procedure promoting hypertrophy the FLR. Radiological procedures include left and right portal vein embolization (extended or not to segment 4), sequential PVE and hepatic vein embolization (HVE), and more recently combined PVE and HVE. Surgical procedures include associated liver partition and portal vein ligation for staged hepatectomy, and more recently the combined portal embolization and arterial ligation procedure. This review aimed to clarify the pathophysiology of liver regeneration; it also describes radiological or surgical procedures employed to improve liver regeneration in terms of volumetric changes, the feasibility of the second step and the benefits and drawbacks of each procedure.

Hepatocyte specific deletion of HIF‐1α affects gut microbiota composition in HFD fed mice

Hepatic hypoxia inducible factor‐1α (HIF‐1α) is a mediator of chronic liver diseases including alcoholic and nonalcoholic fatty liver disease in mice. Our previous study has shown that HIF‐1α signaling pathway is engaged in exercise‐induced protection against cholestasis. Bile acids are synthesized from cholesterol in the liver and further metabolized by the gut microbiota. High‐fat diet (HFD) feeding causes intestinal hyperpermeability and microbial dysbiosis. Therefore, we hypothesized that hepatic HIF‐1α is engaged in the bile acid metabolism and intestinal innate immunity in response to HFD feeding. Albumin Cre mediated, hepatocyte specific deletion of Hif1a (Hif1aLKO) mice were generated. Hepatic metabolism related genes expression patterns were different between the control (Hif1afl/fl) and Hif1aLKO mice. Next, we fed Hif1aLKO and Hif1afl/fl mice (male, 6‐week, n=6 per group) with normal or high‐fat diet (60% fat) for 8‐week. The accumulation of lipid in liver was detected by microCT and Oil Red O staining. Bile acid profile was analyzed by high performance liquid chromatography. The body weight, liver weight, fasting plasma glucose (FPG), serum biomarkers related to liver function and lipid metabolism were also evaluated. Expression levels of bile acid metabolism and intestinal antimicrobial responses related genes were determined using PCR array. Feces were collected on the 0th, 3th and 8th week of HFD feeding. The fecal microbial DNA was extracted and sequenced on an Illumina MiSeq platform. After HFD challenge, Hif1aLKO mice developed greater levels of fat in liver and enhanced FPG (8.5±0.8 vs. 6.1±0.5 mmol/L, P&lt;0.05). Hepatocyte HIF‐1α deletion increased expression of genes involved in bile acid synthesis and affected bile acid profile in liver. HFD‐fed Hif1aLKO mice also exhibited severer intestinal inflammation compare with Hif1afl/fl, and alterations of intestinal gene profile related to antimicrobial responses. Community richness of Hif1aLKO gut microbiome was lower than that of Hif1afl/fl before HFD feeding (ACE index: 328.5±36.6 vs. 403.1±22.3, P&lt;0.05; and Chao1 index: 336.0±34.9 vs. 414.5±26.8, P&lt;0.05). In Figure 1, principal coordinates analysis (PCoA) based on unweighted Unifrac distances highlighted a clear clustering of the microbial populations of Hif1aLKO away from that of Hif1afl/fl. The relative abundances of the given genus within each sample were presented in Heatmap (Figure 2). Notably, there were significantly lower proportions of Akkermansia, Alloprevotella, Allobaculum and Prevotella in Hif1aLKO mice as compared to Hif1afl/fl. Taken together, these results demonstrated that hepatic HIF‐1α is involved in HFD feeding induced changes in gut microbiota composition.Support or Funding InformationPeijie Chen: National Natural Science Foundation of China (31471135). Beibei Luo: National Natural Science Foundation of China (31701040), China Postdoctoral Science Foundation (2015M572679), Shanghai Sailing Program (17YF1418000) and Chenguang Program (16CG57).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.