Keratin (KRT) <em>19</em> is a poor prognostic marker for hepatocellular carcinoma (HCC); however, regulatory mechanisms underlying its expression remain unclear. We have previously reported the presence of fibrous tumor stroma in KRT<em>19</em>-positive HCC, suggesting that cross-talk between cancer-associated <em>fibroblasts</em> (CAF) and tumor epithelial cells could regulate KRT<em>19</em> expression. This was investigated in this study using an in vitro model of paracrine interaction between HCC cell lines (HepG2, SNU423) and hepatic stellate cells (HSC), a major source of hepatic myo<em>fibroblasts</em>. HSCs upregulated transcription and translation of KRT<em>19</em> in HCC cells via paracrine interactions. Mechanistically, hepatocyte <em>growth</em> <em>factor</em> (HGF) from HSCs activated c-MET and the MEK-ERK1/2 pathway, which upregulated KRT<em>19</em> expression in HCC cells. Furthermore, AP1 (JUN/FOSL1) and SP1, downstream transcriptional activators of ERK1/2, activated KRT<em>19</em> expression in HCC cells. In clinical specimens of human HCC (n = 339), HGF and KRT<em>19</em> protein expression correlated with CAF levels. In addition, HGF or MET protein expression was associated with FOSL1 and KRT<em>19</em> expression and was found to be a poor prognostic <em>factor</em>. Analysis of data from The Cancer Genome Atlas also revealed KRT<em>19</em> expression was closely associated with CAF and MET-mediated signaling activities. These results provide insights into the molecular background of KRT<em>19</em>-positive HCC that display an aggressive phenotype.Significance: These findings reveal KRT<em>19</em> expression in hepatocellular carcinoma is regulated by cross-talk between cancer-associated <em>fibroblasts</em> and HCC cells, illuminating new therapeutic targets for this aggressive disease. Cancer Res; 78(7); 16<em>19</em>-31. ©2018 AACR.

OBJECTIVE

Chronic diarrhea caused by primary bile acid diarrhea (PBAD) is a common condition. We have previously shown PBAD is associated with low fasting serum levels of the ileal hormone, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>). FGF<em>19</em> is a negative regulator of hepatic bile acid synthesis and is stimulated by farnesoid X receptor agonists, which produce symptomatic improvement in PBAD. We aimed to assess possible causes for low serum FGF<em>19</em> in patients with PBAD.

METHODS

Patients with PBAD, defined by reduced (75)Se-labelled homocholic acid taurine (SeHCAT) retention, and idiopathic diarrhea controls had measurements of fasting lipids and fasting/post-prandial FGF<em>19</em> serum profiles. Specific functional variants in candidate genes were investigated in exploratory studies. In further groups, basal and bile acid-stimulated transcript expression was determined in ileal biopsies and explant cultures by quantitative PCR.

RESULTS

FGF<em>19</em> profiles in PBAD patients included low fasting and meal-stimulated responses, which were both strongly correlated with SeHCAT. A subgroup of 30% of PBAD patients had fasting hypertriglyceridemia and higher FGF<em>19</em>. No clear significant differences were found for any genetic variant but there were borderline associations with FGFR4 and KLB. SeHCAT retention significantly correlated with the basal ileal transcript expression of FGF<em>19</em> (rs=0.59, P=0.03) and apical sodium-dependent bile acid transporter (ASBT) (rs=0.49, P=0.04), and also with the degree of stimulation by chenodeoxycholic acid at 6 h for transcripts of FGF<em>19</em> (median 184-fold, rs=0.50, P=0.02) and ileal bile acid binding protein (IBABP) (median 2.2-fold, rs=0.47, P=0.04). Median stimulation of FGF<em>19</em> was lower in patients with SeHCAT retention <10% (P=0.01).

CONCLUSIONS

These studies demonstrate a complex, multi<em>factor</em>ial etiology of PBAD, including impairments in ileal FGF<em>19</em> expression and responsiveness.

Viruses are the most abundant biological entities and carry a wide variety of genetic material, including the ability to encode host-like proteins. Here we show that viruses carry sequences with significant homology to several human peptide hormones including insulin, insulin-like <em>growth</em> <em>factors</em> (IGF)-1 and -2, FGF-<em>19</em> and -21, endothelin-1, inhibin, adiponectin, and resistin. Among the strongest homologies were those for four viral insulin/IGF-1-like peptides (VILPs), each encoded by a different member of the family Iridoviridae VILPs show up to 50% homology to human insulin/IGF-1, contain all critical cysteine residues, and are predicted to form similar 3D structures. Chemically synthesized VILPs can bind to human and murine IGF-1/insulin receptors and stimulate receptor autophosphorylation and downstream signaling. VILPs can also increase glucose uptake in adipocytes and stimulate the proliferation of <em>fibroblasts</em>, and injection of VILPs into mice significantly lowers blood glucose. Transfection of mouse hepatocytes with DNA encoding a VILP also stimulates insulin/IGF-1 signaling and DNA synthesis. Human microbiome studies reveal the presence of these Iridoviridae in blood and fecal samples. Thus, VILPs are members of the insulin/IGF superfamily with the ability to be active on human and rodent cells, raising the possibility for a potential role of VILPs in human disease. Furthermore, since only 2% of viruses have been sequenced, this study raises the potential for discovery of other viral hormones which, along with known virally encoded <em>growth</em> <em>factors</em>, may modify human health and disease.

The Insulin-like <em>Growth</em> <em>Factor</em> I (IGF-1) signalling pathway is essential for cell <em>growth</em> and facilitates tumourogenic processes. We recently reported that IGF-1 induces a transcriptional programme for mitochondrial biogenesis, while also inducing expression of the mitophagy receptor BCL2/adenovirus E1B <em>19</em> kDa protein-interacting protein 3 (BNIP3), suggesting that IGF-1 has a key mitochondria-protective role in cancer cells. Here, we investigated this further and delineated the signaling pathway for BNIP3 induction. We established that IGF-1 induced BNIP3 expression through a known AKT serine/threonine kinase 1 (AKT)-mediated inhibitory phosphorylation on Glycogen Synthase Kinase-3β (GSK-3β), leading to activation of Nuclear <em>Factor</em> Erythroid 2-related <em>Factor</em> 2 (NFE2L2/Nrf2) and acting through the downstream transcriptional regulators Nuclear Respiratory <em>Factor</em>-1 (NRF1) and Hypoxia-inducible <em>Factor</em> 1 subunit α (HIF-1α). Suppression of IGF-1 signaling, Nrf2 or BNIP3 caused the accumulation of elongated mitochondria and altered the mitochondrial dynamics. IGF-1R null Mouse Embryonic <em>Fibroblasts</em> (MEFs) were impaired in the BNIP3 expression and in the capacity to mount a cell survival response in response to serum deprivation or mitochondrial stress. IGF-1 signalling enhanced the cellular capacity to induce autophagosomal turnover in response to activation of either general autophagy or mitophagy. Overall, we conclude that IGF-1 mediated a mitochondria-protective signal that was coordinated through the cytoprotective transcription <em>factor</em> Nrf2. This pathway coupled mitochondrial biogenesis with BNIP3 induction, and increased the cellular capacity for autophagosome turnover, whilst enhancing survival under conditions of metabolic or mitochondrial stress.

BACKGROUND

Upregulated <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) expression in human hepatocellular carcinoma (HCC) specimens is associated with tumor progression and poor prognosis. Nonalcoholic steatohepatitis (NASH) patients are at high risk for malignant transformation into HCC.

METHODS

A steatohepatitis-HCC model was established in male C57L/J mice treated with N-nitrosodiethylamine (DEN) and high-fat diet (HFD). A mouse HCC cell line (Hepa1-6) and a mouse hepatocyte line (FL83B) were used to elucidate the mechanism by free fatty acids (FFA) treatment. FGF15, the mouse orthologue of FGF<em>19</em>, and it receptor <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor4 (FGFR4) as well as co-receptor β-klotho were studied. FGF<em>19</em> signaling was also studied in human samples of HCC with steatohepatitis.

RESULTS

HCC incidence and tumor volume were significantly increased in the DEN+HFD group compared to that in the DEN+control diet (CD) group. Increased levels of FGF15/FGFR4/β-klotho, aberrant epithelial-mesenchymal transition (EMT) and Wnt/β-catenin signaling were detected in DEN+HFD mice. Blockage of the FGF15 signal can attenuate cell migration ability and aberrant EMT and Wnt/β-catenin signaling.

CONCLUSIONS

Up-regulated FGF15/FGFR4 signaling promoted the development of HCC by activation of EMT and Wnt/β-catenin signaling in the lipid metabolic disorder microenvironment. Further investigation of FGF<em>19</em>/FGFR4 signaling is important for potential early diagnosis and therapeutic targeting in HCC patients.

OBJECTIVE

The articular cartilage is known to be highly mechanosensitive, and a number of mechanosensing mechanisms have been proposed as mediators of the cellular responses to altered mechanical load. These pathways are likely to be important in tissue homeostasis as well as in the pathogenesis of osteoarthritis. One important injury-activated pathway involves the release of pericellular fibroblast growth factor 2 (FGF-2) from the articular cartilage. Using a novel model of murine cartilage injury and surgically destabilized joints in mice, we examined the extent to which FGF-2 contributes to the cellular gene response to injury.

METHODS

Femoral epiphyses from 5-week-old wild-type mice were avulsed and cultured in serum-free medium. Explant lysates were Western blotted for phospho-JNK, phospho-p38, and phospho-ERK or were fixed for immunohistochemical analysis of the nuclear translocation of p65 (indicative of NF-κB activation). RNA was extracted from injured explants, rested explants that had been stimulated with recombinant FGF-2 or FGF-18, or whole joints from either wild-type mice or FGF-2(-/-) mice. Reverse transcription-polymerase chain reaction was performed to examine a number of inflammatory response genes that had previously been identified in a microarray analysis.

RESULTS

Murine cartilage avulsion injury resulted in rapid activation of the 3 MAP kinase pathways as well as NF-κB. Almost all genes identified in murine joints following surgical destabilization were also regulated in cartilage explants upon injury. Many of these genes, including those for activin A (Inhba), tumor necrosis factor-stimulated gene 6 (Tnfaip6), matrix metalloproteinase 19 (Mmp19), tissue inhibitor of metalloproteinases 1 (Timp1), and podoplanin (Pdpn), were significantly FGF-2 dependent following injury to cartilage in vitro and to joint tissues in vivo.

CONCLUSIONS

FGF-2-dependent gene expression occurs in vitro and in vivo in response to cartilage/joint injury in mice.

Epidermal <em>growth</em> <em>factor</em> is a polypeptide that stimulates proliferation and differentiation of a variety of cell types, including the developing intestinal epithelium; it is the agent in human milk that induces mitosis in human <em>fibroblast</em> culture. We systematically evaluated the EGF content of milk from 20 women delivering prematurely and from 11 women delivering at term. In preterm mothers, the concentration of EGF was 70 +/- 5 ng/ml (mean +/- SEM), with no significant change during seven weeks of lactation. EGF concentration in milk of term mothers was 68 +/- <em>19</em> ng/ml (mean +/- SEM). No diurnal variation in the concentration was found. Total EGF content was closely correlated with the volume of milk expressed, suggesting a passive transport from the circulation. These observations confirm that a substantial amount of EGF is present in human milk and that EGF concentrations are not affected by duration of gestation, time of day, or duration of lactation.

The identification of the key regulators of bile acid (BA) synthesis and transport within the enterohepatic circulation has revealed potential targets for pharmacological therapies of cholestatic liver diseases. Novel drug targets include the bile BA receptors, farnesoid X receptor and TGR5, the BA-induced gut hormones, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> and glucagon-like peptide 1, and the BA transport systems, apical sodium-dependent bile acid transporter and Na+ -taurocholate cotransporting polypeptide, within the enterohepatic circulation. Moreover, BA derivatives undergoing cholehepatic shunting may allow improved targeting to the bile ducts. This review focuses on the pathophysiological basis, mechanisms of action, and clinical development of novel pharmacological strategies targeting BA transport and signaling in cholestatic liver diseases. (Hepatology 2017;65:1393-1404).

OBJECTIVE

The initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) results from the interplay of genetic and epigenetic events. While the genetic alterations of PDAC have been well characterized, epigenetic pathways regulating PDAC remain, for the most part, elusive. The goal of this study was to identify novel epigenetic regulators contributing to the biology of PDAC.

METHODS

In vivo pooled shRNA screens targeting 118 epigenetic proteins were performed in two orthotopic PDAC xenograft models. Candidate genes were characterized in <em>19</em> human PDAC cell lines, heterotopic xenograft tumor models, and a genetically engineered mouse (GEM) model of PDAC. Gene expression, IHC, and immunoprecipitation experiments were performed to analyze the pathways by which candidate genes contribute to PDAC.

RESULTS

In vivo shRNA screens identified BRD2 and BRD3, members of the BET family of chromatin adaptors, as key regulators of PDAC tumor growth. Pharmacologic inhibition of BET bromodomains enhanced survival in a PDAC GEM model and inhibited growth of human-derived xenograft tumors. BET proteins contribute to PDAC cell growth through direct interaction with members of the GLI family of transcription factors and modulating their activity. Within cancer cells, BET bromodomain inhibition results in downregulation of SHH, a key mediator of the tumor microenvironment and canonical activator of GLI. Consistent with this, inhibition of BET bromodomains decreases cancer-associated fibroblast content of tumors in both GEM and xenograft tumor models.

CONCLUSIONS

Therapeutic inhibition of BET proteins offers a novel mechanism to target both the neoplastic and stromal components of PDAC. Clin Cancer Res; 22(16); 4259-70. ©2016 AACR.

The role of the intestine in the maintenance of cholesterol homeostasis increasingly is recognized. Fecal excretion of cholesterol is the last step in the atheroprotective reverse cholesterol transport pathway, to which biliary and transintestinal cholesterol excretion (TICE) contribute. The mechanisms controlling the flux of cholesterol through the TICE pathway, however, are poorly understood. We aimed to identify mechanisms that regulate and stimulate TICE.

We performed studies with C57Bl/6J mice, as well as with mice with intestine-specific knockout of the farnesoid X receptor (FXR), mice that express an FXR transgene specifically in the intestine, and ABCG8-knockout mice. Mice were fed a control diet or a diet supplemented with the FXR agonist PX20606, with or without the cholesterol absorption inhibitor ezetimibe. Some mice with intestine-specific knockout of FXR were given daily injections of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>19</em>. To determine fractional cholesterol absorption, mice were given intravenous injections of cholesterol D5 and oral cholesterol D7. Mice were given 13C-acetate in drinking water for measurement of cholesterol synthesis. Bile cannulations were performed and biliary cholesterol secretion rates were assessed. In a separate set of experiments, bile ducts of male Wistar rats were exteriorized, allowing replacement of endogenous bile by a model bile.

In mice, we found TICE to be regulated by intestinal FXR via induction of its target gene Fgf15 (FGF<em>19</em> in rats and human beings). Stimulation of this pathway caused mice to excrete up to 60% of their total cholesterol content each day. PX20606 and FGF<em>19</em> each increased the ratio of muricholate:cholate in bile, inducing a more hydrophilic bile salt pool. The altered bile salt pool stimulated robust secretion of cholesterol into the intestinal lumen via the sterol-exporting heterodimer adenosine triphosphate binding cassette subfamily G member 5/8 (ABCG5/G8). Of note, the increase in TICE induced by PX20606 was independent of changes in cholesterol absorption.

Hydrophilicity of the bile salt pool, controlled by FXR and FGF15/<em>19</em>, is an important determinant of cholesterol removal via TICE. Strategies that alter bile salt pool composition might be developed for the prevention of cardiovascular disease. Transcript profiling: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=irsrayeohfcntqx&acc=GSE74101.

Bile acid diarrhoea results from imbalances in the homoeostasis of bile acids in the enterohepatic circulation. It can be a consequence of ileal disease/dysfunction, associated with other GI pathology or can be idiopathic.

To summarise the different types of bile acid diarrhoea and discuss the currently available diagnostic methods and treatments.

Bile acid diarrhoea is found in up to 40% of patients diagnosed as having functional diarrhoea/IBS-D, and in up to 80% of patients who have undergone ileal resection. It is likely under-diagnosed and under-treated. In idiopathic disease, errors in regulation feedback of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> contribute to the development of the condition. Clinical therapeutic trials for bile acid diarrhoea have been used to diagnose it, but the 75 SeHCAT test is the primary current method. It is sensitive, specific and widely available, though not in the USA. Other diagnostic methods (such as serum measurement of the bile acid intermediate 7α-hydroxy-4-cholesten-3-one, or C4) have less widespread availability and documentation, and some (such as faecal measurement of bile acids) are significantly more complex and costly. First-line treatment of bile acid diarrhoea is with the bile acid sequestrant cholestyramine, which can be difficult to administer and dose due to gastrointestinal side effects. These side effects are less prominent in newer agents such as colesevelam, which may provide higher efficacy, tolerability and compliance.

Bile acid diarrhoea is common, and likely under-diagnosed. Bile acid diarrhoea should be considered relatively early in the differential diagnosis of chronic diarrhoea.

Bile acids (BA) modulate lipid and glucose metabolism in a feedback loop through production of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>19</em> in the terminal ileum. Changes in BA after bariatric surgery may lead to improvements in the metabolic syndrome, including fatty liver disease. This study investigated the relationship between BA and metabolic and inflammatory profiles after laparoscopic sleeve gastrectomy (LSG).

Patients undergoing LSG had fasting blood samples taken pre-operatively and 6 months post-surgery. Liver injury was measured using cytokeratin (CK) 18 fragments. BA were measured using liquid chromatography tandem-mass spectrometry. FGF-<em>19</em> was measured using enzyme-linked immunosorbent assay.

The study included 18 patients (12 females), with mean age 46.3 years (SEM ± 2.9) and BMI 60.1 kg/m(2) (±2.6). After 6 months, patients lost 39.8 kg (±3.1; p < 0.001). Fourteen patients (78 %) had steatosis. FGF-<em>19</em> increased from median 128.1 (IQR 89.4-210.1) to 177.1 (121.8-288.9, p = 0.045) at 6 months. Although total BA did not change, primary glycine- and taurine-conjugated BA, cholic acid decreased, and secondary BA, glycine-conjugated urodeoxycholic acid increased over the study period. These changes are associated with reduction in insulin resistance, pro-inflammatory cytokines and CK-18 levels.

The profile of individual BA is altered after LSG. These changes occur in the presence of reductions in inflammatory cytokines and markers of liver injury. This study supports evidence from recent animal models that LSG may have an effect on fatty liver through changes in BA metabolism.

Angiogenesis is controlled by a balance between stimulators and inhibitors. We propose that the balance, as well as the general sensitivity of the endothelium to these <em>factors</em>, varies from individual to individual. Indeed, we have found that individual mouse strains have dramatically different responses to <em>growth</em> <em>factor</em>-induced neovascularization. Quantitative trait loci (QTLs), which influence the extent of corneal angiogenesis induced by vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2), were previously identified by our laboratory. To investigate the genetic contribution to choroidal neovascularization (CNV), a leading cause of blindness, we have undertaken a similar mapping approach to identify QTLs that influence laser-induced CNV in the BXD series of recombinant inbred mouse strains. Composite interval mapping identified new angiogenic QTLs on chromosomes 2 and <em>19</em>, in addition to confirming our previous corneal neovascularization QTLs of AngVq1 and AngFq2. The new QTLs are named AngCNVq1 and AngCNVq2. The newly mapped regions contain several candidate genes involved in the angiogenic process, including thrombospondin 1, delta-like 4, BclII modifying <em>factor</em>, phospholipase C, beta 2, adrenergic receptor, beta 1, actin-binding LIM protein 1 and colony stimulating <em>factor</em> 2 receptor, alpha. Differences in these regions may control individual susceptibility to CNV.

BACKGROUND

Arterial thickening contributes to elevated cardiovascular risk in patients on maintenance renal replacement therapy. The common carotid artery intima-media thickness (CCA-IMT) is an early atherosclerotic marker and may be used to assess the stratification of atherosclerotic advancement and resultant arterial calcification.

OBJECTIVE

The aim of the study was to evaluate the associations between atherosclerotic changes in the common carotid arteries expressed as the CCA-IMT and the body mass index (BMI), serum lipid levels, C‑reactive protein (CRP), and selected bone metabolism parameters including phosphorus, calcium, intact parathormone (iPTH), alkaline phosphatase, osteopontin, osteoprotegerin, osteocalcin, fetuin A, and fibroblast growth factor‑23 (FGF‑23) in patients treated with peritoneal dialysis.

METHODS

The study included 67 patients with chronic kidney disease (36 men and 31 women) aged 53 ±13 years (range, 19-75 years) treated with peritoneal dialysis for 30 ±24 months. The CCA‑IMT was assessed by ultrasonography using Acuson 128/10 XP. The BMI was calculated using the Quetelet formula. Serum lipid levels, phosphorus, calcium, iPTH, alkaline phosphatase, and CRP were measured using standard laboratory methods, while fetuin A, osteocalcin, osteoprotegerin, osteopontin, and FGF‑23 using commercial enzyme‑linked immunosorbent assay kits.

RESULTS

Positive correlations were observed between CCA-IMT and age (r = 0.54, P <0.0001), BMI (r = 0.39, P = 0.003), and osteoprotegerin (r = 0.38, P = 0.004). In a multiple regression analysis, age (r = 0.41, P = 0.01), osteocalcin (r = 0.34, P = 0.04), and log‑transformed osteoprotegerin values (r = 0.38, P = 0.02) remained independently associated with the CCA-IMT. The highest CCA‑IMT values (0.85 ±0.21) were observed in patients with osteoprotegerin concentrations in the upper tertile. Osteoprotegerin concentrations strongly and positively correlated with the duration of dialysis treatment (r = 0.55, P <0.0001).

CONCLUSIONS

The CCA‑IMT has been shown to be a reliable noninvasive measure of subclinical atherosclerosis and, therefore, of associated increased vascular risk. Elevated serum osteoprotegerin levels may be useful as a prognostic marker of cardiovascular risk in dialyzed patients.

We have previously shown that the ethanol-mediated elevation of lipocaline-2 (LCN2) is closely associated with the development of alcoholic fatty liver disease (AFLD) in mice. Herein, we aimed to understand the functional significance of LCN2 induction by ethanol and to explore its underlying mechanisms. We evaluated the effects of LCN2 in an in vitro cellular alcoholic steatosis model and in an animal study using wild-type and LCN2 knockout mice fed for 4 weeks with an ethanol-supplemented Lieber-DeCarli diet. In the cellular model of alcoholic steatosis, recombinant LCN2 or overexpression of LCN2 exacerbated ethanol-induced fat accumulation, whereas knocking down LCN2 prevented steatosis in hepatocytes exposed to ethanol. Consistently, removal of LCN2 partially but significantly alleviated alcoholic fatty liver injury in mice. Mechanistically, LCN2 mediates detrimental effects of ethanol in the liver via disrupted multiple signaling pathways, including aberrant nicotinamide phosphoribosyltransferase-sirtuin 1 axis, perturbed endocrine metabolic regulatory <em>fibroblast</em> <em>growth</em> <em>factor</em> 15/<em>19</em> signaling, and impaired chaperone-mediated autophagy. Finally, compared with healthy human livers, liver samples from patients with AFLD had lower gene expression of several LCN2-regualted molecules. Our study demonstrated a pivotal and causal role of LCN2 in the development of AFLD and suggested that targeting the LCN2 could be of great value for the treatment of human AFLD.

<AbstractText>The sodium glucose cotransporter 2 (SGLT-2) inhibitor dapagliflozin is a novel drug for the treatment of diabetes mellitus. Recent studies suggest that SGLT-2 inhibitors affect phosphate homeostasis, but their effects on phosphate-regulating hormones in patients with diabetic kidney disease are still unclear.</AbstractText><p><div><b>DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS</b></div>We performed a <i>post-hoc</i> analysis of a double-blind, randomized, crossover trial in patients with type 2 diabetes with early-stage diabetic kidney disease on stable renin-angiotensin-aldosterone system blockade, with an albumin-to-creatinine ratio between 100 and 3500 mg/g, eGFR≥45 ml/min per 1.73 m², and glycosylated hemoglobin≥7.2% and <11.4%. Patients were randomized to dapagliflozin 10 mg/d or placebo during consecutive 6-week study periods, separated by a 6-week wash-out. We investigated effects on circulating phosphate, calcium, parathyroid hormone (PTH), <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23), 25-hydroxyvitamin D (25[OH]D), and 1,25-dihydroxyvitamin D (1,25[OH]₂D) levels.</p><p><div><b>RESULTS</b></div>Thirty-one patients (age 62 years; 23% female) were analyzed. Compared with placebo, dapagliflozin increased serum phosphate by 9% (95% confidence interval, 4% to 15%; <i>P</i>=0.002), PTH increased by 16% (3% to 30%; <i>P</i>=0.01), FGF23 increased by <em>19</em>% (0.3% to 42%; <i>P</i>=0.05), and serum 1,25(OH)₂D decreased by -12% (-25% to 4%; <i>P</i>=0.12). Calcium and 25(OH)D were unaffected. We found no correlation between changes in markers of phosphate homeostasis and changes in eGFR or 24-hour albumin excretion during dapagliflozin treatment.</p><AbstractText>Dapagliflozin increases serum phosphate, plasma PTH, and FGF23. This effect was independent of concomitant changes in eGFR or 24-hour albumin excretion.</AbstractText>

Basal-like breast cancer is an aggressive tumor subtype with poor prognosis. The discovery of underlying mechanisms mediating tumor cell survival, and the development of novel agents to target these pathways, is a priority for patients with basal-like breast cancer. From a functional screen to identify key drivers of basal-like breast cancer cell <em>growth</em>, we identified <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 (FGFR4) as a potential mediator of cell survival. We found that FGFR4 mediates cancer cell survival predominantly via activation of PI3K/AKT. Importantly, a subset of basal-like breast cancer cells also secrete <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>), a canonical ligand specific for FGFR4. siRNA-mediated silencing of FGF<em>19</em> or neutralization of extracellular FGF<em>19</em> by anti-FGF<em>19</em> antibody (1A6) decreases AKT phosphorylation, suppresses cancer cell <em>growth</em> and enhances doxorubicin sensitivity only in the FGFR4+/FGF<em>19</em>+ breast cancer cells. Consistently, FGFR4/FGF<em>19</em> co-expression was also observed in 82 out of 287 (28.6%) primary breast tumors, and their expression is strongly associated with AKT phosphorylation, Ki-67 staining, higher tumor stage and basal-like phenotype. In summary, our results demonstrated the presence of an FGFR4/FGF<em>19</em> autocrine signaling that mediates the survival of a subset of basal-like breast cancer cells and suggest that inactivation of this autocrine loop may potentially serve as a novel therapeutic intervention for future treatment of breast cancers.

Immunohistochemical studies were performed using specific polyclonal antibodies to transforming <em>growth</em> <em>factor</em> (TGF)-beta 1 and TGF-beta 2 to determine their presence and cellular localization in human ovarian tissues of various reproductive states. In the small ovarian follicles, the immunostaining for TGF-beta 1 was present in oocytes, follicle cells, and granulosa and theca cell layers. The level of immunostaining associated with granulosa and theca cell layers intensified as the size of the follicles increased. In the luteal tissue, both the small and large luteal cells immunostained for TGF-beta 1 and their intensities were similar to theca and granulosa cell layers, respectively. The patterns of immunostaining were similar in early (days 14-<em>19</em>), mid (days 22-25), and late (days 26-29) luteal phases; however, the intensity was highest at mid and decreased at late luteal phase. Corpus albicans showed a very weak immunostaining for TGF-beta 1, whereas ectopic pregnancy small luteal cells immunostained relatively intensely. The ovarian stromal, luteal tissue <em>fibroblasts</em>, and arterioles endothelial and smooth muscle cells were also immunostained for TGF-beta 1. The immunostaining of the ovarian tissues for TGF-beta 2 indicated that the theca cell layers were the exclusive cells in the follicles with intense immunostaining, which increased in the larger follicles. A low immunostaining was also observed in granulosa cell of the large follicles. In the luteal tissues, only small luteal cells showed intense immunostaining for TGF-beta 2, which was similar in intensity to that in the theca cells; however, the large luteal cells showed a low level of immunostaining at midluteal phase. The small luteal cells in corpus albicans and ectopic pregnancy luteal tissues retained their immunostaining for TGF-beta 2, but with lower intensity. Endothelial and smooth muscle cells of arterioles also immunostained for TGF-beta 2, but not ovarian stromal cells. Atretic follicles showed very low or no detectable immunostaining for TGF-beta 1 or TGF-beta 2. The results of present studies show that human ovarian tissue at all the reproductive states locally produces TGF-beta 1 and TGF-beta 2, and although TGF-beta 1 is present in most major ovarian cell types, TGF-beta 2 is only produced by theca cells in the follicles and small luteal cells in luteal tissues.

We previously detected elevated transforming <em>growth</em> <em>factor</em> beta-1 (TGF-beta1) serum levels in patients with invasive bladder carcinomas. In this study, we therefore investigated whether elevated serum levels correlate with enhanced TGF-beta expression in human bladder tumours. mRNA levels of TGF-beta1, -beta2 and -beta3 were reduced in bladder tumour tissue to 86%, 68% and 56%, respectively, of the levels in normal urothelium. On the other hand, TGF-beta1 protein levels were found to be higher in superficial tumours (Ta-T1) (mean level of 0.153 ng mg(-1)) and in invasive T2/T3 tumours (mean level of 0.104 ng mg(-1)) compared with normal urothelium (mean level of 0.065 ng mg(-1)). Invasive T4 tumours, however, contained only low amounts of TGF-beta1 (mean level of 0.02 ng mg(-1)). Neither in mean nor in individual patients were serum and tissue TGF-beta levels correlated with each other. Cell culture experiments on primary bladder cells revealed a 57% decrease in TGF-beta1 mRNA levels in tumour compared with normal epithelial cells. Tumour epithelial cells contained about two times higher levels of TGF-beta2 and TGF-beta3 mRNA than normal epithelial cells. <em>Fibroblasts</em> expressed about the same amount of TGF-beta1 or TGF-beta2 as epithelial cells. Yet, <em>fibroblasts</em> released only <em>19</em>% and 13% of the amount secreted by tumour epithelial cells into the supernatant. TGF-beta3, on the other hand, was expressed by <em>fibroblasts</em> with higher levels than by epithelial cells. TGF-beta1 was the predominent isoform in bladder tissue and cells at protein as well as on mRNA levels indicating that TGFs-beta2 and -beta3 are of minor importance in bladder cancer. In summary, there is a lack of correlation between TGF-beta serum levels and TGF-beta expression in tumour tissue in bladder cancer.

OBJECTIVE

Vascular endothelial growth factor and basic fibroblast growth factor are potent stimulators of angiogenesis. Children with cyanotic congenital heart disease often experience the development of widespread formation of collateral blood vessels, which may represent a form of abnormal angiogenesis. We undertook the present study to determine whether children with cyanotic congenital heart disease have elevated serum levels of vascular endothelial growth factor and basic fibroblast growth factor.

METHODS

Serum was obtained from 22 children with cyanotic congenital heart disease and 19 children with acyanotic heart disease during cardiac catheterization. Samples were taken from the superior vena cava, inferior vena cava, and a systemic artery. Vascular endothelial growth factor and basic fibroblast growth factor levels were measured in the serum from each of these sites by enzyme-linked immunosorbent assay.

RESULTS

Vascular endothelial growth factor was significantly elevated in the superior vena cava (P =.04) and systemic artery (P =.02) but not in the inferior vena cava (P =.2) of children with cyanotic congenital heart disease compared to children with acyanotic heart disease. The mean vascular endothelial growth factor level, determined by averaging the means of all 3 sites, was also significantly elevated (P =.03). Basic fibroblast growth factor was only significantly elevated in the systemic artery (P =.02).

CONCLUSIONS

Children with cyanotic congenital heart disease have elevated systemic levels of vascular endothelial growth factor. These findings suggest that the widespread formation of collateral vessels in these children may be mediated by vascular endothelial growth factor.

OBJECTIVE

We investigated to which extent disturbances in mineral metabolism predict 90-day clinical outcome in end-stage heart failure patients.

METHODS

Among numerous biochemical parameters, we measured serum levels of sodium and magnesium, the calciotropic hormones parathyroid hormone and 1,25-dihydroxyvitamin D as well as fibroblast growth factor-23 (a phosphaturic hormone) in 305 cardiac transplant candidates. Primary endpoint was a composite of the need of mechanical circulatory support (MCS), transplantation, or death.

RESULTS

Of the study cohort, 33.4% reached the primary endpoint. In detail, 19% were transplanted (the vast majority was listed "high urgent"), 8.8% died and 5.6% received MCS implants. As determined by logistic regression analysis, all aforementioned biochemical parameters were independently related to the primary endpoint. Results did not change substantially when transplanted patients were censored. A risk score (0-5 points) was developed. Of the patients who scored 5 points 89.5% reached the primary endpoint whereas of the patients with a zero score only 3.8% reached the primary endpoint.

CONCLUSIONS

Our data demonstrate that in addition to the well-known predictive value of disturbed sodium metabolism, derangements in calcium, phosphate, and magnesium metabolism also predict midterm clinical outcome in end-stage heart failure patients.

BACKGROUND

Protein products of klothoβ (KLB) and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 (FGFR4) impact <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em>-mediated feedback inhibition of hepatic bile acid (BA) synthesis. Variants of KLB and FGFR4 influence colonic transit (CT) in diarrhea-predominant irritable bowel syndrome (IBS-D).

OBJECTIVE

The purpose of this study was to test the hypothesis that colesevelam's slowing effects on CT in IBS-D patients is influenced by genetic variants in KLB and FGFR4.

METHODS

We examined pharmacogenetic effects of KLB and FGFR4 coding variants (SNPs) on scintigraphic CT response to the BA sequestrant, colesevelam 1.875 g b.i.d. versus placebo (PLA) for 14 days in 24 female IBS-D patients.

RESULTS

FGFR4 rs351855 and KLB rs497501 were associated with differential colesevelam effects on ascending colon (AC) half-emptying time (t(1/2), P = 0.046 and P = 0.085 respectively) and on overall CT at 24 h (geometric center, GC24: P = 0.073 and P = 0.042, respectively), with slower transit for rs351855 GA/AA (but not for GG) and rs497501 CA/AA (but not CC) genotypes.

CONCLUSIONS

FGFR4 rs351855 and KLB rs4975017 SNPs may identify a subset of IBS-D patients with beneficial response to colesevelam.

OBJECTIVE: To explore the safety, efficacy, and biomarkers of bevacizumab with gemcitabine and oxaliplatin in women with recurrent platinum-sensitive ovarian carcinoma. METHODS: The patients received bevacizumab (10 mg/kg), gemcitabine (1000 mg/m(2)), and oxaliplatin (65 mg/m(2)) on days 1 and 15 in 28-day cycles. The patients with safely accessible tumor underwent intratumoral fluid pressure (IFP) measurements and positron-emission tomographies immediately and 2 weeks after treatment. Blood biomarkers were evaluated at 5 time points. RESULTS: The trial was closed after enrolling <em>19</em> of the 53 projected patient accrual. Thirteen (68.5%) of <em>19</em> patients showed a response (1 complete response, 12 partial responses), and 6 patients showed stable disease (31.6%). Median progressive-free survival was 36.9 weeks (258.3 days), and the median overall survival was 112.3 weeks (633 days, not reached). Toxicity was acceptable, and there were no arterial thromboses, serious bleeding, gastrointestinal perforations, or complications from the invasive procedures. Bevacizumab with chemotherapy induced a substantial drop in tumor IFP after treatment. The regimen induced sustained elevation in circulating plasma vascular endothelial <em>growth</em> <em>factor</em> (VEGF), placental <em>growth</em> <em>factor</em> (PlGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), soluable vascular endothelial <em>growth</em> <em>factor</em> receptor 2 (sVEGFR2), and circulating progenitor cells. Plasma PlGF, VEGFR2(+) monocytes, and urinary matrix metalloproteinase 2 activity showed differential associations with treatment outcome when evaluated at baseline and after 14 days of treatment. CONCLUSIONS: Despite early termination of the study, the results indicate that the regimen was well tolerated and demonstrated activity in platinum-sensitive ovarian cancer. Biomarker evaluations showed that bevacizumab with chemotherapy significantly changed the levels of several circulating cellular and molecular biomarkers. The increases in plasma PlGF and VEGFR2(+) monocytes showed correlations with outcome. These exploratory data should be further evaluated in future studies of bevacizumab in ovarian cancer.

The 22 members of the mouse/human <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family of proteins contain a conserved cysteine residue at position 83 (numbering scheme of the 140-residue form of FGF-1). Sequence and structure information suggests that this position is a free cysteine in 16 members and participates as a half-cystine in at least 3 (and perhaps as many as 6) other members. While a structural role as a half-cystine provides a stability basis for possible selective pressure, it is less clear why this residue is conserved as a free cysteine (although free buried thiols can limit protein functional half-life). To probe the structural role of the free cysteine at position 83 in FGF-1, we constructed Ala, Ser, Thr, Val, and Ile mutations and determined their effects on structure and stability. These results show that position 83 in FGF-1 is thermodynamically optimized to accept a free cysteine. A second cysteine mutation was introduced into wild-type FGF-1 at adjacent position Ala66, which is known to participate as a half-cystine with position 83 in FGF-8, FGF-<em>19</em>, and FGF-23. Results show that, unlike position 83, a free cysteine at position 66 destabilizes FGF-1; however, upon oxidation, a near-optimal disulfide bond is formed between Cys66 and Cys83, resulting in approximately 14 kJ/mol of increased thermostability. Thus, while the conserved free cysteine at position 83 in the majority of the FGF proteins may have a principal role in limiting functional half-life, evidence suggests that it is a vestigial half-cystine.