A new FGF15/19-mediated gut-to-heart axis controls cardiac hypertrophy.

FGF15 and its human orthologue, FGF19, are members of the endocrine FGF family and are secreted by ileal enterocytes in response to bile acids. FGF15/19 mainly targets the liver, but recent studies indicate that it also regulates skeletal muscle mass and adipose tissue plasticity. The aim of this study was to determine the role(s) of the enterokine FGF15/19 during the development of cardiac hypertrophy. Studies in a cohort of humans suffering from heart failure showed increased circulating levels of FGF19 compared with control individuals. We found that mice lacking FGF15 did not develop cardiac hypertrophy in response to three different pathophysiological stimuli (high-fat diet, isoproterenol, or cold exposure). The heart weight/tibia length ratio and the cardiomyocyte area (as measures of cardiac hypertrophy development) under hypertrophy-inducing conditions were lower in Fgf15-null mice than in wild-type mice, whereas the levels of the cardiac damage marker atrial natriuretic factor (Nppa) were up-regulated. Echocardiographic measurements showed similar results. Moreover, the genes involved in fatty acid metabolism were down-regulated in Fgf15-null mice. Conversely, experimental increases in FGF15 induced cardiac hypertrophy in vivo, without changes in Nppa and up-regulation of metabolic genes. Finally, in vitro studies using cardiomyocytes showed that FGF19 had a direct effect on these cells promoting hypertrophy. We have identified herein an inter-organ signaling pathway that runs from the gut to the heart, acts through the enterokine FGF15/19, and is involved in cardiac hypertrophy development and regulation of fatty acid metabolism in the myocardium. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Identification and experimental validation of druggable epigenetic targets in hepatoblastoma.

BACKGROUND & AIMS: Hepatoblastoma (HB) is the most frequent childhood liver cancer. Patients with aggressive tumors have limited therapeutic options; therefore, a better understanding of HB pathogenesis is needed to improve treatment. HBs have a very low mutational burden; however, epigenetic alterations are increasingly recognized. We aimed to identify epigenetic regulators consistently dysregulated in HB and to evaluate the therapeutic efficacy of their targeting in clinically relevant models. METHODS: We performed a comprehensive transcriptomic analysis of 180 epigenetic genes. Data from fetal, pediatric, adult, peritumoral (n = 72) and tumoral (n = 91) tissues were integrated. Selected epigenetic drugs were tested in HB cells. The most relevant epigenetic target identified was validated in primary HB cells, HB organoids, a patient-derived xenograft model, and a genetic mouse model. Transcriptomic, proteomic and metabolomic mechanistic analyses were performed. RESULTS: Altered expression of genes regulating DNA methylation and histone modifications was consistently observed in association with molecular and clinical features of poor prognosis. The histone methyltransferase G9a was markedly upregulated in tumors with epigenetic and transcriptomic traits of increased malignancy. Pharmacological targeting of G9a significantly inhibited growth of HB cells, organoids and patient-derived xenografts. Development of HB induced by oncogenic forms of ß-catenin and YAP1 was ablated in mice with hepatocyte-specific deletion of G9a. We observed that HBs undergo significant transcriptional rewiring in genes involved in amino acid metabolism and ribosomal biogenesis. G9a inhibition counteracted these pro-tumorigenic adaptations. Mechanistically, G9a targeting potently repressed the expression of c-MYC and ATF4, master regulators of HB metabolic reprogramming. CONCLUSIONS: HBs display a profound dysregulation of the epigenetic machinery. Pharmacological targeting of key epigenetic effectors exposes metabolic vulnerabilities that can be leveraged to improve the treatment of these patients. IMPACT AND IMPLICATIONS: In spite of recent advances in the management of hepatoblastoma (HB), treatment resistance and drug toxicity are still major concerns. This systematic study reveals the remarkable dysregulation in the expression of epigenetic genes in HB tissues. Through pharmacological and genetic experimental approaches, we demonstrate that the histone-lysine-methyltransferase G9a is an excellent drug target in HB, which can also be harnessed to enhance the efficacy of chemotherapy. Furthermore, our study highlights the profound pro-tumorigenic metabolic rewiring of HB cells orchestrated by G9a in coordination with the c-MYC oncogene. From a broader perspective, our findings suggest that anti-G9a therapies may also be effective in other c-MYC-dependent tumors.

Splicing Factor SLU7 Prevents Oxidative Stress-Mediated Hepatocyte Nuclear Factor 4α Degradation, Preserving Hepatic Differentiation and Protecting From Liver Damage.

BACKGROUND AND AIMS: Hepatocellular dedifferentiation is emerging as an important determinant in liver disease progression. Preservation of mature hepatocyte identity relies on a set of key genes, predominantly the transcription factor hepatocyte nuclear factor 4α (HNF4α) but also splicing factors like SLU7. How these factors interact and become dysregulated and the impact of their impairment in driving liver disease are not fully understood. APPROACH AND RESULTS: Expression of SLU7 and that of the adult and oncofetal isoforms of HNF4α, driven by its promoter 1 (P1) and P2, respectively, was studied in diseased human and mouse livers. Hepatic function and damage response were analyzed in wild-type and Slu7-haploinsufficient/heterozygous (Slu7+/- ) mice undergoing chronic (CCl4 ) and acute (acetaminophen) injury. SLU7 expression was restored in CCl4 -injured mice using SLU7-expressing adeno-associated viruses (AAV-SLU7). The hepatocellular SLU7 interactome was characterized by mass spectrometry. Reduced SLU7 expression in human and mouse diseased livers correlated with a switch in HNF4α P1 to P2 usage. This response was reproduced in Slu7+/- mice, which displayed increased sensitivity to chronic and acute liver injury, enhanced oxidative stress, and marked impairment of hepatic functions. AAV-SLU7 infection prevented liver injury and hepatocellular dedifferentiation. Mechanistically we demonstrate a unique role for SLU7 in the preservation of HNF4α1 protein stability through its capacity to protect the liver against oxidative stress. SLU7 is herein identified as a key component of the stress granule proteome, an essential part of the cell's antioxidant machinery. CONCLUSIONS: Our results place SLU7 at the highest level of hepatocellular identity control, identifying SLU7 as a link between stress-protective mechanisms and liver differentiation. These findings emphasize the importance of the preservation of hepatic functions in the protection from liver injury.

Dual Targeting of G9a and DNA Methyltransferase-1 for the Treatment of Experimental Cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. APPROACH AND RESULTS: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. CONCLUSIONS: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.

Epigenetic mechanisms and metabolic reprogramming in fibrogenesis: dual targeting of G9a and DNMT1 for the inhibition of liver fibrosis.

OBJECTIVE: Hepatic stellate cells (HSC) transdifferentiation into myofibroblasts is central to fibrogenesis. Epigenetic mechanisms, including histone and DNA methylation, play a key role in this process. Concerted action between histone and DNA-mehyltransferases like G9a and DNMT1 is a common theme in gene expression regulation. We aimed to study the efficacy of CM272, a first-in-class dual and reversible G9a/DNMT1 inhibitor, in halting fibrogenesis. DESIGN: G9a and DNMT1 were analysed in cirrhotic human livers, mouse models of liver fibrosis and cultured mouse HSC. G9a and DNMT1 expression was knocked down or inhibited with CM272 in human HSC (hHSC), and transcriptomic responses to transforming growth factor-ß1 (TGFß1) were examined. Glycolytic metabolism and mitochondrial function were analysed with Seahorse-XF technology. Gene expression regulation was analysed by chromatin immunoprecipitation and methylation-specific PCR. Antifibrogenic activity and safety of CM272 were studied in mouse chronic CCl4 administration and bile duct ligation (BDL), and in human precision-cut liver slices (PCLSs) in a new bioreactor technology. RESULTS: G9a and DNMT1 were detected in stromal cells in areas of active fibrosis in human and mouse livers. G9a and DNMT1 expression was induced during mouse HSC activation, and TGFß1 triggered their chromatin recruitment in hHSC. G9a/DNMT1 knockdown and CM272 inhibited TGFß1 fibrogenic responses in hHSC. TGFß1-mediated profibrogenic metabolic reprogramming was abrogated by CM272, which restored gluconeogenic gene expression and mitochondrial function through on-target epigenetic effects. CM272 inhibited fibrogenesis in mice and PCLSs without toxicity. CONCLUSIONS: Dual G9a/DNMT1 inhibition by compounds like CM272 may be a novel therapeutic strategy for treating liver fibrosis.

Splicing events in the control of genome integrity: role of SLU7 and truncated SRSF3 proteins.

Genome instability is related to disease development and carcinogenesis. DNA lesions are caused by genotoxic compounds but also by the dysregulation of fundamental processes like transcription, DNA replication and mitosis. Recent evidence indicates that impaired expression of RNA-binding proteins results in mitotic aberrations and the formation of transcription-associated RNA-DNA hybrids (R-loops), events strongly associated with DNA injury. We identify the splicing regulator SLU7 as a key mediator of genome stability. SLU7 knockdown results in R-loops formation, DNA damage, cell-cycle arrest and severe mitotic derangements with loss of sister chromatid cohesion (SCC). We define a molecular pathway through which SLU7 keeps in check the generation of truncated forms of the splicing factor SRSF3 (SRp20) (SRSF3-TR). Behaving as dominant negative, or by gain-of-function, SRSF3-TR impair the correct splicing and expression of the splicing regulator SRSF1 (ASF/SF2) and the crucial SCC protein sororin. This unique function of SLU7 was found in cancer cells of different tissue origin and also in the normal mouse liver, demonstrating a conserved and fundamental role of SLU7 in the preservation of genome integrity. Therefore, the dowregulation of SLU7 and the alterations of this pathway that we observe in the cirrhotic liver could be involved in the process of hepatocarcinogenesis.

Liver Expression of a MiniATP7B Gene Results in Long-Term Restoration of Copper Homeostasis in a Wilson Disease Model in Mice.

Gene therapy with an adeno-associated vector (AAV) serotype 8 encoding the human ATPase copper-transporting beta polypeptide (ATP7B) complementary DNA (cDNA; AAV8-ATP7B) is able to provide long-term copper metabolism correction in 6-week-old male Wilson disease (WD) mice. However, the size of the genome (5.2 kilobases [kb]) surpasses the optimal packaging capacity of the vector, which resulted in low-yield production; in addition, further analyses in WD female mice and in animals with a more advanced disease revealed reduced therapeutic efficacy, as compared to younger males. To improve efficacy of the treatment, an optimized shorter AAV vector was generated, in which four out of six metal-binding domains (MBDs) were deleted from the ATP7B coding sequence, giving rise to the miniATP7B protein (Δ57-486-ATP7B). In contrast to AAV8-ATP7B, AAV8-miniATP7B could be produced at high titers and was able to restore copper homeostasis in 6- and 12-week-old male and female WD mice. In addition, a recently developed synthetic AAV vector, AAVAnc80, carrying the miniATP7B gene was similarly effective at preventing liver damage, restoring copper homeostasis, and improving survival 1 year after treatment. Transduction of approximately 20% of hepatocytes was sufficient to normalize copper homeostasis, suggesting that corrected hepatocytes are acting as a sink to eliminate excess of copper. Importantly, administration of AAVAnc80-miniATP7B was safe in healthy mice and did not result in copper deficiency. Conclusion: In summary, gene therapy using an optimized therapeutic cassette in different AAV systems provides long-term correction of copper metabolism regardless of sex or stage of disease in a clinically relevant WD mouse model. These results pave the way for the implementation of gene therapy in WD patients.

Dual Targeting of Histone Methyltransferase G9a and DNA-Methyltransferase 1 for the Treatment of Experimental Hepatocellular Carcinoma.

Epigenetic modifications such as DNA and histone methylation functionally cooperate in fostering tumor growth, including that of hepatocellular carcinoma (HCC). Pharmacological targeting of these mechanisms may open new therapeutic avenues. We aimed to determine the therapeutic efficacy and potential mechanism of action of our dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitor in human HCC cells and their crosstalk with fibrogenic cells. The expression of G9a and DNMT1, along with that of their molecular adaptor ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was measured in human HCCs (n = 268), peritumoral tissues (n = 154), and HCC cell lines (n = 32). We evaluated the effect of individual and combined inhibition of G9a and DNMT1 on HCC cell growth by pharmacological and genetic approaches. The activity of our lead compound, CM-272, was examined in HCC cells under normoxia and hypoxia, human hepatic stellate cells and LX2 cells, and xenograft tumors formed by HCC or combined HCC+LX2 cells. We found a significant and correlative overexpression of G9a, DNMT1, and UHRF1 in HCCs in association with poor prognosis. Independent G9a and DNMT1 pharmacological targeting synergistically inhibited HCC cell growth. CM-272 potently reduced HCC and LX2 cells proliferation and quelled tumor growth, particularly in HCC+LX2 xenografts. Mechanistically, CM-272 inhibited the metabolic adaptation of HCC cells to hypoxia and induced a differentiated phenotype in HCC and fibrogenic cells. The expression of the metabolic tumor suppressor gene fructose-1,6-bisphosphatase (FBP1), epigenetically repressed in HCC, was restored by CM-272. Conclusion: Combined targeting of G9a/DNMT1 with compounds such as CM-272 is a promising strategy for HCC treatment. Our findings also underscore the potential of differentiation therapy in HCC.

The Epidermal Growth Factor Receptor Ligand Amphiregulin Protects From Cholestatic Liver Injury and Regulates Bile Acids Synthesis.

Intrahepatic accumulation of bile acids (BAs) causes hepatocellular injury. Upon liver damage, a potent protective response is mounted to restore the organ's function. Epidermal growth factor receptor (EGFR) signaling is essential for regeneration after most types of liver damage, including cholestatic injury. However, EGFR can be activated by a family of growth factors induced during liver injury and regeneration. We evaluated the role of the EGFR ligand, amphiregulin (AREG), during cholestatic liver injury and regulation of AREG expression by BAs. First, we demonstrated increased AREG levels in livers from patients with primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). In two murine models of cholestatic liver injury, bile duct ligation (BDL) and alpha-naphthyl-isothiocyanate (ANIT) gavage, hepatic AREG expression was markedly up-regulated. Importantly, Areg-/- mice showed aggravated liver injury after BDL and ANIT administration compared to Areg+/+ mice. Recombinant AREG protected from ANIT and BDL-induced liver injury and reduced BA-triggered apoptosis in liver cells. Oral BA administration induced ileal and hepatic Areg expression, and, interestingly, cholestyramine feeding reduced postprandial Areg up-regulation in both tissues. Most interestingly, Areg-/- mice displayed high hepatic cholesterol 7 α-hydroxylase (CYP7A1) expression, reduced serum cholesterol, and high BA levels. Postprandial repression of Cyp7a1 was impaired in Areg-/- mice, and recombinant AREG down-regulated Cyp7a1 mRNA in hepatocytes. On the other hand, BAs promoted AREG gene expression and protein shedding in hepatocytes. This effect was mediated through the farnesoid X receptor (FXR), as demonstrated in Fxr-/- mice, and involved EGFR transactivation. Finally, we show that hepatic EGFR expression is indirectly induced by BA-FXR through activation of suppressor of cytokine signaling-3 (SOC3). Conclusion: AREG-EGFR signaling protects from cholestatic injury and participates in the physiological regulation of BA synthesis.

Revision hip arthroplasty with a rectangular tapered cementless stem: a retrospective study of the SLR-Plus stem at a mean follow-up of 4.1 years.

PURPOSE: Demand for revision total hip arthroplasty (THA) is growing, and this type of surgery remains challenging for orthopedic surgeons. Our objectives were to assess clinical and radiographic outcomes, survivorship and complications with the SLR-Plus stem in revision THA. METHODS: We retrospectively reviewed 65 patients (66 hips) who had undergone revision THA with the SLR-Plus stem between 2008 and 2015 at two medical institutions with a minimum 2-year follow-up. The clinical outcome was assessed using the Harris hip score and the Merlé D'Aubigné score preoperatively and at final follow-up. A postoperative visual analogue scale for pain and satisfaction was also used. Radiographic subsidence and fixation, Kaplan-Meier survivorship and complications were analyzed. RESULTS: The mean follow-up was 4.1 years (SD 2.1). Aseptic loosening (57.6%) was the main indication for surgery. The mean Harris Hip Score improved from 50.4 (SD 16.5) to 83 (SD 12.7) (p < 0.001) and mean Merlé D'Aubigné score improved from 9.5 (SD 2.7) to 14.3 (SD 2.2) (p < 0.001). A total of 98.4% of stems showed radiographically stable fixation. No aseptic loosening of the stem was seen. Radiolucent lines > 1 mm were observed in 33.3% of stems. Three stems were re-revised: two due to infection and one due to instability. At 7 years, estimated stem survival was 95.5% for revision for any reason and 100% for revision for aseptic loosening. Dislocation occurred in 7.6% of hips. CONCLUSION: We have shown significant clinical improvement, 98.4% of stable fixation and 100% stem survivorship for aseptic loosening in revision THA with the SLR-Plus stem.

X-box Binding Protein 1 Regulates Unfolded Protein, Acute-Phase, and DNA Damage Responses During Regeneration of Mouse Liver.

BACKGROUND & AIMS: Liver regeneration after partial hepatectomy (PH) increases the protein folding burden at the endoplasmic reticulum of remnant hepatocytes, resulting in induction of the unfolded protein response. We investigated the role of the core unfolded protein response transcription factor X-box binding protein 1 (XBP1) in liver regeneration using genome-wide chromatin immunoprecipitation analysis. METHODS: We performed studies with C57Bl6-J (control) and interleukin 6-knockout mice. Mice underwent PH or sham surgeries. In some mice, hepatic expression of XBP1 was knocked down by injection of adenoviral vectors encoding small hairpin RNAs against Xbp1 messenger RNA. Liver tissues were collected before surgery and at 6 and 48 hours after surgery and analyzed by chromatin immunoprecipitation followed by sequencing. We also performed functional analyses of HepG2 cells. RESULTS: Expression of XBP1 by hepatocytes increased immediately after PH (priming phase of liver regeneration) in control mice, but this effect was delayed in interleukin 6-deficient mice. In mice with knockdown of XBP1, we observed of liver tissue persistent endoplasmic reticulum stress, defects in acute-phase response, and increased hepatocellular damage, compared with control mice. Chromatin immunoprecipitation analyses of liver tissue showed that at 6 hours after PH, liver XBP1 became bound to a large set of genes implicated in proteostasis, the acute-phase response, metabolism, and the DNA damage response (DDR). At this time point, XBP1 bound the promoter of the signal transducer and activator of transcription 3 gene (Stat3). Livers of XBP1-knockdown mice showed reduced expression of STAT3 and had lower levels of STAT3 phosphorylation at Ser727, a modification that promotes cell proliferation and the DDR. Regenerating livers from XBP1-knockdown mice expressed high levels of a marker of DNA double-strand breaks, phosphorylated histone 2A, member X (H2AX), compared with control mice. The inhibition of XBP1 expression caused a reduced up-regulation of DDR messenger RNAs in regenerating hepatocytes. CONCLUSION: In livers of mice, we found that PH induces expression of XBP1, and that this activity requires interleukin 6. XBP1 expression regulates the unfolded protein response, acute-phase response, and DDR in hepatocytes. In regenerating livers, XBP1 deficiency leads to endoplasmic reticulum stress and DNA damage.

Bile acids, FGF15/19 and liver regeneration: From mechanisms to clinical applications.

The liver has an extraordinary regenerative capacity rapidly triggered upon injury or resection. This response is intrinsically adjusted in its initiation and termination, a property termed the "hepatostat". Several molecules have been involved in liver regeneration, and among them bile acids may play a central role. Intrahepatic levels of bile acids rapidly increase after resection. Through the activation of farnesoid X receptor (FXR), bile acids regulate their hepatic metabolism and also promote hepatocellular proliferation. FXR is also expressed in enterocytes, where bile acids stimulate the expression of fibroblast growth factor 15/19 (FGF15/19), which is released to the portal blood. Through the activation of FGFR4 on hepatocytes FGF15/19 regulates bile acids synthesis and finely tunes liver regeneration as part of the "hepatostat". Here we review the experimental evidences supporting the relevance of the FXR-FGF15/19-FGFR4 axis in liver regeneration and discuss potential therapeutic applications of FGF15/19 in the prevention of liver failure. This article is part of a Special Issue entitled: Cholangiocytes in Health and Disease edited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Interaction of glucocorticoids with FXR/FGF19/FGF21-mediated ileum-liver crosstalk.

At high doses, glucocorticoids (GC) have been associated with enhanced serum bile acids and liver injury. We have evaluated the effect of GC, in the absence of hepatotoxicity, on FXR/FGF91(Fgf15)/FGF21-mediated ileum-liver crosstalk. Rats and mice (wild type and Fxr-/-, Fgf15-/- and int-Gr-/- strains; the latter with GC receptor (Gr) knockout selective for intestinal epithelial cells), were treated (i.p.) with dexamethasone, prednisolone or budesonide. In both species, high doses of GC caused hepatotoxicity. At a non-hepatotoxic dose, GC induced ileal Fgf15 down-regulation and liver Fgf21 up-regulation, without affecting Fxr expression. Fgf21 mRNA levels correlated with those of several genes involved in glucose and bile acid metabolism. Surprisingly, liver Cyp7a1 was not up-regulated. The expression of factors involved in transcriptional modulation by Fxr and Gr (p300, Drip205, CBP and Smrt) was not affected. Pxr target genes Cyp3a11 and Mrp2 were not up-regulated in liver or intestine. In contrast, the expression of some Pparα target genes in liver (Fgf21, Cyp4a14 and Vanin-1) and intestine (Vanin-1 and Cyp3a11) was altered. In mice with experimental colitis, liver Fgf21 was up-regulated (4.4-fold). HepG2 cells transfection with FGF21 inhibited CYP7A1 promoter (prCYP7A1-Luc2). This was mimicked by pure human FGF21 protein or culture in medium previously conditioned by cells over-expressing FGF21. This response was not abolished by deletion of a putative response element for phosphorylated FGF21 effectors present in prCYP7A1. In conclusion, GC interfere with FXR/FGF19-mediated intestinal control of CYP7A1 expression by the liver and stimulate hepatic secretion of FGF21, which inhibits CYP7A1 promoter through an autocrine mechanism.

Medium-term outcomes of the S-ROM modular femoral stem in revision hip replacement.

INTRODUCTION: The objective of this study was to analyse results achieved with the S-ROM modular stem in revision surgery. MATERIALS AND METHODS: A retrospective observational study was conducted from 2007 to 2015 including 51 patients who had a follow-up of ≥ 2 years and complete medical history. The mean age was 66.5 years old (34-87). The main reason for revision was aseptic loosening (38 cases, 74.5%), followed by infection (10, 19.6%), instability (2, 3.9%) and an adverse reaction associated with a metal-on-metal hip implant (1, 2%). Using the Paprosky classification, there were 22 cases of type I (43.1%), 27 of type II (52.9%) and 2 of type IIIA (4%). At the end of the follow-up, radiological parameters were assessed using Engh's criteria. Pre- and postoperative clinical status was assessed using the Harris Hip Score, a visual analogue scale and the Merle D'Aubigné score. RESULTS: The mean follow-up period was 5.7 years (2-10). The mean Harris Hip Score improved from 45.5 points (22-65) to 85.8 (55-100) (p < 0.001), and the final mean Merle D'Aubigné scores were 5.2, 4.6 and 5.6 for pain, ability to walk and mobility, respectively. Osseointegration was confirmed in all except one patient with fibrous non-union. No aseptic loosening has been recorded. Postoperative complications were deep infection in four cases (7.8%) and dislocation in three (5.9%). CONCLUSION: This study indicates good medium-term outcomes using a modular hip replacement system with porous-coated proximal sleeves in revision surgery in patients with Paprosky type I and II defects.

Fibroblast growth factor 15/19 (FGF15/19) protects from diet-induced hepatic steatosis: development of an FGF19-based chimeric molecule to promote fatty liver regeneration.

OBJECTIVE: Fibroblast growth factor 15/19 (FGF15/19), an enterokine that regulates synthesis of hepatic bile acids (BA), has been proposed to influence fat metabolism. Without FGF15/19, mouse liver regeneration after partial hepatectomy (PH) is severely impaired. We studied the role of FGF15/19 in response to a high fat diet (HFD) and its regulation by saturated fatty acids. We developed a fusion molecule encompassing FGF19 and apolipoprotein A-I, termed Fibapo, and evaluated its pharmacological properties in fatty liver regeneration. DESIGN: Fgf15-/- mice were fed a HFD. Liver fat and the expression of fat metabolism and endoplasmic reticulum (ER) stress-related genes were measured. Influence of palmitic acid (PA) on FGF15/19 expression was determined in mice and in human liver cell lines. In vivo half-life and biological activity of Fibapo and FGF19 were compared. Hepatoprotective and proregenerative activities of Fibapo were evaluated in obese db/db mice undergoing PH. RESULTS: Hepatosteatosis and ER stress were exacerbated in HFD-fed Fgf15-/- mice. Hepatic expression of Pparγ2 was elevated in Fgf15-/- mice, being reversed by FGF19 treatment. PA induced FGF15/19 expression in mouse ileum and human liver cells, and FGF19 protected from PA-mediated ER stress and cytotoxicity. Fibapo reduced liver BA and lipid accumulation, inhibited ER stress and showed enhanced half-life. Fibapo provided increased db/db mice survival and improved regeneration upon PH. CONCLUSIONS: FGF15/19 is essential for hepatic metabolic adaptation to dietary fat being a physiological regulator of Pparγ2 expression. Perioperative administration of Fibapo improves fatty liver regeneration.

Fibroblast Growth Factor 15/19 in Hepatocarcinogenesis.

BACKGROUND: Advanced hepatocellular carcinoma (HCC) is a neoplastic disease with a very bad prognosis and increasing worldwide incidence. HCCs are resistant to conventional chemotherapy and the multikinase inhibitor sorafenib is the only agent that has shown some clinical efficacy. It is therefore important to identify key molecular mechanisms driving hepatocarcinogenesis for the development of more efficacious therapies. However, HCCs are heterogeneous tumors and different molecular subclasses have been characterized. This heterogeneity may underlie the poor performance of most of the targeted therapies so far tested in HCC patients. The fibroblast growth factor 15/19 (FGF15/19), FGF receptor 4 (FGFR4) and beta-Klotho (KLB) correceptor signaling system, a key regulator of bile acids (BA) synthesis and intermediary metabolism, is emerging as an important player in hepatocarcinogenesis. Key Messages: Aberrant signaling through the FGF15/19-FGFR4 pathway participates in the neoplastic behavior of HCC cells, promotes HCC development in mice and its overexpression has been characterized in a subset of HCC tumors from patients with poorer prognosis. Pharmacological interference with FGF15/19-FGFR4 signaling inhibits experimental hepatocarcinogenesis, and specific FGFR4 inhibitors are currently being tested in selected HCC patients with tumoral FGF19-FGFR4/KLB expression. CONCLUSIONS: Interference with FGF19-FGFR4 signaling represents a novel strategy in HCC therapy. Selection of candidate patients based on tumoral FGF19-FGFR4/KLB levels as biomarkers may result in increased efficacy of FGFR4-targeted drugs. Nevertheless, attention should be paid to the potential on target toxic effects of FGFR4 inhibitors due to the key role of this signaling system in BA metabolism.

Matrix metalloproteinase 10 contributes to hepatocarcinogenesis in a novel crosstalk with the stromal derived factor 1/C-X-C chemokine receptor 4 axis.

UNLABELLED: Matrix metalloproteinases (MMPs) participate in tissue repair after acute injury, but also participate in cancer by promoting a protumorigenic microenvironment. Previously, we reported on a key role for MMP10 in mouse liver regeneration. Herein, we investigated MMP10 expression and function in human hepatocellular carcinoma (HCC) and diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis. MMP10 was induced in human and murine HCC tissues and cells. MMP10-deficient mice showed less HCC incidence, smaller histological lesions, reduced tumor vascularization, and less lung metastases. Importantly, expression of the protumorigenic, C-X-C chemokine receptor-4 (CXCR4), was reduced in DEN-induced MMP10-deficient mice livers. Human HCC cells stably expressing MMP10 had increased CXCR4 expression and migratory capacity. Pharmacological inhibition of CXCR4 significantly reduced MMP10-stimulated HCC cell migration. Furthermore, MMP10 expression in HCC cells was induced by hypoxia and the CXCR4 ligand, stromal-derived factor-1 (SDF1), through the extracellular signal-regulated kinase 1/2 pathway, involving an activator protein 1 site in MMP10 gene promoter. CONCLUSION: MMP10 contributes to HCC development, participating in tumor angiogenesis, growth, and dissemination. We identified a new reciprocal crosstalk between MMP10 and the CXCR4/SDF1 axis contributing to HCC progression and metastasis. To our knowledge, this is the first report addressing the role of a MMP in hepatocarcinogenesis in the corresponding genetic mouse model.

Ileal FGF15 contributes to fibrosis-associated hepatocellular carcinoma development.

Fibroblast growth factor 15 (FGF15), FGF19 in humans, is a gut-derived hormone and a key regulator of bile acids and carbohydrate metabolism. FGF15 also participates in liver regeneration after partial hepatectomy inducing hepatocellular proliferation. FGF19 is overexpressed in a significant proportion of human hepatocellular carcinomas (HCC), and activation of its receptor FGFR4 promotes HCC cell growth. Here we addressed for the first time the role of endogenous Fgf15 in hepatocarcinogenesis. Fgf15(+/+) and Fgf15(-/-) mice were subjected to a clinically relevant model of liver inflammation and fibrosis-associated carcinogenesis. Fgf15(-/-) mice showed less and smaller tumors, and histological neoplastic lesions were also smaller than in Fgf15(+/+) animals. Importantly, ileal Fgf15 mRNA expression was enhanced in mice undergoing carcinogenesis, but at variance with human HCC it was not detected in liver or HCC tissues, while circulating FGF15 protein was clearly upregulated. Hepatocellular proliferation was also reduced in Fgf15(-/-) mice, which also expressed lower levels of the HCC marker alpha-fetoprotein (AFP). Interestingly, lack of FGF15 resulted in attenuated fibrogenesis. However, in vitro experiments showed that liver fibrogenic stellate cells were not direct targets for FGF15/FGF19. Conversely we demonstrate that FGF15/FGF19 induces the expression of the pro-fibrogenic and pro-tumorigenic connective tissue growth factor (CTGF) in hepatocytes. These findings suggest the existence of an FGF15-triggered CTGF-mediated paracrine action on stellate cells, and an amplification mechanism for the hepatocarcinogenic effects of FGF15 via CTGF production. In summary, our observations indicate that ileal FGF15 may contribute to HCC development in a context of chronic liver injury and fibrosis.

A GAPDH-mediated trans-nitrosylation pathway is required for feedback inhibition of bile salt synthesis in rat liver.

BACKGROUND & AIMS: Bile salts inhibit their own production by inducing the nuclear receptor small heterodimer partner (SHP) (encoded by NR0B2), which contributes to repression of the gene encoding cholesterol 7α-hydroxylase (CYP7A1), a key enzyme for the control of bile salt synthesis. On the other hand, bile salts stimulate hepatic synthesis of nitric oxide. We investigated the role of nitric oxide signaling in the control of CYP7A1 expression and the involvement in this process of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which participates in intracellular propagation of nitric oxide signals. METHODS: We studied the effects of inhibitors of nitric oxide synthesis (L-NG-nitroarginine methyl ester [L-NAME]) or protein nitrosylation (via dithiothreitol) on bile salt homeostasis in male Wistar rats placed on a cholate-rich diet for 5 days and in cultured primary hepatocytes. S-nitrosylation of GAPDH was assessed using a biotin-switch assay. Interacions of SHP with other proteins and with the Cyp7a1 promoter sequence were studied using immunoprecipitation and chromatin immunoprecipitation (ChIP) assays. We reduced the GAPDH levels in H35 cells with small interfering RNAs. GAPDH nitrosylation was assessed in normal and cholestatic rat and human livers. RESULTS: Rats placed on cholate-rich diets and given L-NAME had increased intrahepatic and biliary levels of bile salts, and deficiency in repression of CYP7A1 (at the messenger RNA and protein levels) in liver tissue, despite preserved induction of SHP. In cultured hepatocytes, L-NAME or dithiothreitol blocked cholate-induced down-regulation of CYP7A1 without impairing SHP up-regulation. In hepatocytes, cholate promoted S-nitrosylation of GAPDH and its translocation to the nucleus, accompanied by S-nitrosylation of histone deacetylase 2 (HDAC2) and Sirtuin 1 (SIRT1), deacetylases that participate, respectively, in the formation of Cyp7a1 and Shp repressor complexes. Knockdown of GAPDH prevented repression of CYP7A1 by cholate, and blocking nuclear transport of nitrosylated GAPDH reduced cholate-induced nitrosylation of HDAC2 and SIRT1; this effect was accompanied by abrogation of Cyp7a1 repression. Cholate induced binding of SHP to HDAC2 and its recruitment to the Cyp7a1 promoter; these processes were inhibited by blocking nitric oxide synthesis. Levels of nitrosylated GAPDH and nitrosylated HDAC2 were increased in cholestatic human and rat livers reflecting increased concentrations of bile salts in these conditions. CONCLUSIONS: In rat liver, excess levels of bile salts activate a GAPDH-mediated transnitrosylation cascade that provides feedback inhibition of bile salt synthesis.

Matrix metalloproteinase-10 expression is induced during hepatic injury and plays a fundamental role in liver tissue repair.

BACKGROUND & AIMS: Upon tissue injury, the liver mounts a potent reparative and regenerative response. A role for proteases, including serine and matrix metalloproteinases (MMPs), in this process is increasingly recognized. We have evaluated the expression and function of MMP10 (stromelysin-2) in liver wound healing and regeneration. METHODS: The hepatic expression of MMP10 was examined in two murine models: liver regeneration after two-thirds partial hepatectomy (PH) and bile duct ligation (BDL). MMP10 was detected in liver tissues by qPCR, western blotting and immunohistochemistry. The effect of growth factors and toll-like receptor 4 (TLR4) agonists on MMP10 expression was studied in cultured parenchymal and biliary epithelial cells and macrophages respectively. The role of MMP10 was evaluated by comparing the response of Mmp10+/+ and Mmp10-/- mice to PH and BDL. The intrahepatic turnover of the extracellular matrix proteins fibrin (ogen) and fibronectin was examined. RESULTS: MMP10 mRNA was readily induced after PH and BDL. MMP10 protein was detected in hepatocytes, cholangiocytes and macrophages. In cultured liver epithelial cells, MMP10 expression was additively induced by transforming growth factor-ß and epidermal growth factor receptor ligands. TLR4 ligands also stimulated MMP10 expression in macrophages. Lack of MMP10 resulted in increased liver injury upon PH and BDL. Resolution of necrotic areas was impaired, and Mmp10-/- mice showed increased fibrogenesis and defective turnover of fibrin (ogen) and fibronectin. CONCLUSIONS: MMP10 expression is induced during mouse liver injury and participates in the hepatic wound healing response. The profibrinolytic activity of MMP10 may be essential in this novel hepatoprotective role.