The conversion of white adipose to the highly thermogenic beige adipose tissue has been proposed as a potential strategy to counter the unfavorable consequences of obesity. Three regulators of this conversion have recently emerged but information regarding their control is limited, and contradictory. We present two studies examining the control of these regulators. Study 1: In 10 young men, the plasma concentrations of irisin and <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) were determined prior to and during activation of the sympathetic nervous system via hypoxic gas breathing (FIO2 = 0.11). The measurements were performed twice, once with and once without prior/concurrent sympathetic inhibition via transdermal clonidine administration. FGF21 was unaffected by basal sympathetic inhibition (338±113 vs. 295±80 pg/mL; P = 0.43; mean±SE), but was increased during hypoxia mediated sympathetic activation (368±135); this response was abrogated (P = 0.035) with clonidine (269±93). Irisin was unaffected by sympathetic inhibition and/or hypoxia (P>0.21). Study 2: The plasma concentration of irisin and FGF21, and the skeletal muscle protein content of fibronectin type III domain containing 5 (FNDC5) was determined in <em>19</em> young adults prior to and following three weeks of sprint interval training (SIT). SIT decreased FGF21 (338±78 vs. 251±36; P = 0.046) but did not affect FNDC5 (P = 0.79). Irisin was decreased in males (127±18 vs. 90±23 ng/mL; P = 0.045) and increased in females (139±14 vs. 170±18). Collectively, these data suggest a potential regulatory role of acute sympathetic activation pertaining to the browning of white adipose; further, there appears to be a sexual dimorphic response of irisin to SIT.

We isolated the rabbit gene for the 92-kDa matrix metalloproteinase, gelatinase B, and sequenced 1802 contiguous bases covering the first three exons and 522 bases of DNA upstream of the start site for transcription. The DNA between bases -5<em>19</em> and +<em>19</em> is sufficient to drive expression of a reporter gene in early passage cultures of corneal <em>fibroblasts</em> or primary cultures of corneal epithelial cells. Basal activity of the gelatinase B promoter in <em>fibroblasts</em> is lower than a collagenase promotor of 1800 base pairs, but activity of both promotors is similarly stimulated by treatment of transfected cells with phorbol 12-myristate 13-acetate, and stimulation is enhanced by co-treatment with transforming <em>growth</em> <em>factor</em>-beta. In contrast, basal activity of the gelatinase B promotor in epithelial cells is higher than the collagenase promotor. Deletion analysis demonstrated that sequences upstream of base -330 confer cell type-specific activity to the gelatinase B promotor. Site-directed mutagenesis revealed that an AP1-like element within this region is specifically utilized by <em>fibroblasts</em>. This region also contains elements that confer the capacity for activation by AP2, a transcription <em>factor</em> found to be expressed by corneal epithelial cells but not by corneal <em>fibroblasts</em>. In contrast, AP2 does not activate the collagenase promotor. These results provide a molecular basis for the unique cell type-specific expression pattern of gelatinase B as compared to other matrix metalloproteinases.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) have been implicated in the development of numerous malignancies including prostate cancer. In a pilot study it has been shown that FGF8 mRNA is up-regulated in prostate cancer. The aim of the present study was to determine whether aFGF and bFGF were co-expressed with FGF8 in human prostate cancer. Twenty-nine cases of prostate cancer of different histological grades were examined. Immunohistochemical analysis was employed to study aFGF and bFGF expression. In the light of the results, aFGF immunoreactivity was studied in a further 43 cases. aFGF and bFGF immunoreactivity was identified in the cytoplasm of the malignant prostatic epithelium. aFGF was overexpressed in 62/72 (86.1 per cent) cases and bFGF in <em>19</em>/29 (65.5 per cent). High levels of aFGF immunoreactivity were noted in areas of high-grade prostatic intraepithelial neoplasia (PIN). In this series, aFGF immunoreactivity was most commonly observed and correlated closely with Gleason score and tumour stage ( p=0.007 and 0.007, respectively). Co-localization of aFGF, bFGF, and FGF8 was detected in 9/29 (31.0 per cent) cases. There was a significant correlation between aFGF and FGF8 expression. In conclusion, aFGF, bFGF, and FGF8 are co-localized in human prostate cancer; they may have a synergistic effect in prostate cancer <em>growth</em> and progression.

BACKGROUND

Epidemiological data suggest a more favorable outcome of breast carcinoma in women taking cardiac glycosides. This study investigated whether digoxin could inhibit tumor growth in mice.

METHODS

Tumor growth experiments were done in mice grafted with the neuroblastoma cell lines SH-SY5Y, Neuro-2a, colonic cancer cells (LS174T) or Lewis lung cancer cells (LLC). Angiogenesis inhibition was investigated in vitro on fibroblast growth factor-2 (FGF-2)-stimulated bovine endothelial cell (BCE) growth and in vivo in the chick chorioallantoic membrane (CAM) assay.

RESULTS

SH-SY5Y and Neuro-2a grafts were inhibited by 44% (p=0.008) and 19% (p=0.007), respectively, whereas the colonic cancer xenografts and LLC syngrafts were less responsive. The neuroblastoma specificity was confirmed in vitro. Digoxin also inhibited angiogenesis in the CAM assay and the BCE cell survival in vitro was 50% at 53 ng/ml.

CONCLUSIONS

Our data suggest that digoxin may be a specific neuroblastoma growth inhibitor and an unspecific inhibitor of angiogenesis.

The present study demonstrated that the vessel number in bone marrow biopsies from acute myeloid leukaemia (AML) patients (n = 23) was significantly increased at diagnosis compared with normal bone marrow (P = 0.0<em>19</em>) and was restored to normal levels after achieving complete remission (P = 0.03). The in vitro angiogenic potential of culture supernatant of AML cells was assessed using endothelial cell (EC) migration and proliferation assays. Increased EC migration and EC proliferation was induced in 7/20 and <em>19</em>/20 AML supernatents respectively. The degree of in vivo neovascularization did not correlate with the ability of AML cells to stimulate in vitro endothelial cell migration and/or proliferation. This might be in part a result of the heterogeneous pattern of angiogenic <em>factors</em> produced by AML cells. The expression of different angiogenic <em>factors</em> was studied using reverse transcription polymerase chain reaction. Cells from 17/20 AML patients showed wide variation in spontaneous vascular endothelial <em>growth</em> <em>factor</em> (VEGF) expression, 4/<em>19</em> expressed varied spontaneous blastic <em>fibroblast</em> <em>growth</em> <em>factor</em> mRNA levels and all patient samples showed spontaneous interleukin 8 mRNA expression. All AML samples expressed matrix metalloproteinase (MMP)-2 and/or MMP-9. VEGF mRNA expression correlated well with protein level (P = 0.006). A correlation was found between the degree of VEGF expression and neoangiogenesis (correlation coefficient = 0.448, P = 0.05). These results suggest that malignant cell proliferation, angiogenesis and VEGF expression are linked in AML and might contribute to the <em>growth</em> advantage of the malignant counterpart as a result of the paracrine production of <em>growth</em> <em>factors</em> produced by the surrounding endothelial cells.

The pathways involved in the maintenance of human embryonic stem (hES) cells remain largely unknown, although some signaling pathways have been identified in mouse embryonic stem (mES) cells. <em>Fibroblast</em> feeder layers are used to maintain the undifferentiated <em>growth</em> of hES cells and an examination of the conditioned media (CM) of human neonatal <em>fibroblasts</em> (HNFs) could provide insights into the maintenance of hES cells. The neonatal foreskin <em>fibroblast</em> line (HNF02) used in this study was shown to have a normal 2n = 46, XY chromosomal complement and to support the undifferentiated <em>growth</em> of the Embryonic Stem Cell International Pte. Ltd.-hES3 cell line. The CM of HNF02 was examined using two-dimensional liquid chromatography-tandem mass spectrometry (2-D LCMS) and two-dimensional electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (2-DE/MALDI). A total of 102 proteins were identified, <em>19</em> by 2-DE/MALDI, 53 by 2-D LCMS and 30 by both techniques. These proteins were classified into 15 functional groups. Proteins identified in the extracellular matrix and differentiation and <em>growth</em> <em>factor</em> functional categories were considered most likely to be involved in the maintenance of hES cell <em>growth</em>, differentiation and pluripotency as these groups contained proteins involved in a variety of events including cell adhesion, cell proliferation and inhibition of cell proliferation, Wnt signaling and inhibition of bone morphogenetic proteins.

The increase in the amount of airway smooth muscle in the bronchial wall associated with asthma is partly due to hyperplasia. It is therefore important to determine which <em>factors</em> regulate <em>growth</em> and especially proliferation. In this study, we describe the effect of interleukin-4 (IL-4), a mast cell- and T lymphocyte-derived cytokine, on human airway smooth muscle proliferation as determined by [3H]thymidine uptake in the presence of fetal bovine serum (FBS), platelet-derived <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and thrombin. IL-4 (5, 15, 50, and 150 ng/ml) significantly decreased 10% FBS-induced proliferation by 50, 73, 43, and 46%, respectively. The proliferative responses to platelet-derived <em>growth</em> <em>factor</em> (20 and 40 ng/ml), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (30 ng/ml), and thrombin (1 and 10 U/ml) were significantly reduced by <em>19</em>, 21, 37, 36, and 57% respectively in the presence of 50 ng/ml of IL-4. We investigated the effect of IL-4 and other known inhibitors of smooth muscle proliferation, namely PGE2, heparin, and forskolin, on intracellular cAMP concentrations. IL-4 (50 ng/ml) and heparin (100 U/ml) did not alter intracellular cAMP levels when cells were treated with 1 or 10% FBS. PGE2 (1 microM) and forskolin (10 microM) significantly increased cAMP concentration above the control value in nonproliferating cells (1% FBS treated) by 7- and 37-fold, respectively. The effect of IL-4 (50 ng/ml), PGE2 (1 microM), and forskolin (10 microM) on cyclin D1 protein expression in 10% FBS-stimulated human airway smooth muscle cells was also examined. PGE2 and forskolin did not significantly inhibit cyclin D1 expression. However, IL-4 decreased cyclin D1 expression by 21%. These results provide evidence that IL-4 decreases human airway smooth muscle cell proliferation via a mechanism that is cAMP independent and mediated, in part, by a decrease in cyclin D1 protein expression.

It is widely believed that abnormal production of polypeptide <em>growth</em> <em>factors</em>, together with other molecular alterations, play an important role in neoplastic development. Transforming <em>growth</em> <em>factor</em> alpha (TGFalpha), hepatocyte <em>growth</em> <em>factor</em> (HGF) and acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) are the three major <em>growth</em> <em>factors</em> that contribute to liver regeneration occurring via both hepatocyte replication and oval cell proliferation. It is not clear, however, whether and to what extent these <em>growth</em> <em>factors</em> are also involved in hepatocarcinogenesis. In the present study, the gene expression of TGFalpha, HGF and aFGF and their corresponding receptors was examined by Northern blotting and in situ hybridization during hepatocarcinogenesis induced by the Solt-Farber protocol. All three <em>growth</em> <em>factor</em>/receptor systems, TGFalpha/epidermal <em>growth</em> <em>factor</em> receptor (EGFR), HGF/c-met and aFGF/FGF receptors (flg and bek) were significantly elevated at early time points when oval cells were proliferating. Their respective expression decreased after 1 month and remained at a low level until the development of liver tumors. In all hepatocellular carcinomas (HCC) examined, the transcripts of TGFalpha and aFGF were highly expressed, while those of HGF were low. With regard to the receptor expression in the tumors, EGFR was present at varying levels, c-met was expressed at higher levels and flg increased significantly, whereas bek remained at low levels. These data suggest that TGFalpha and aFGF are the major <em>growth</em> <em>factors</em> involved in the progression of HCC, and that the signal of aFGF is mainly transduced by the receptor flg in HCC. Furthermore, HCC cells were phenotypically very similar to oval cells with regard to the gene expression of <em>growth</em> <em>factor</em>/receptor systems. These results, along with the finding that all the HCC cells are positive for the oval cell antigen OV6, and that cytokeratin <em>19</em> is heavily expressed in both tumor and oval cells, strongly suggest that at least some of the HCC induced by the Solt-Farber protocol may be derived from oval cells.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGF), and in particular FGF8, have been strongly implicated in prostate carcinogenesis. This study investigated the expression of Sef, a key inhibitory regulator of FGF signalling, in prostate cancer. In a panel of cell lines, hSef was detected in both androgen-dependent and independent cells but was significantly reduced in highly metastatic derivative clones. hSef expression was not influenced by androgenic stimulation. Forced downregulation of hSef by siRNA increased FGF8b induced cell migration (P=0.02) and invasion (P=0.007). Reduced hSef levels also enhanced FGF8b stimulated expression of MMP9 (P=0.005). mRNA in situ hybridization revealed hSef expression in 80% (8/10) of benign biopsies but in only 69% (23/33) of Gleason sum 4-7 and 35% (10/28) of Gleason sum 8-10 cancer biopsies (P=0.004). Quantitative PCR of microdissected glands confirmed this trend (P=0.001). hSef was expressed in 69% (27/39) of non-metastatic tumours but in only 18% (2/11) of metastatic tumours (P=0.004, n=50). hSef expression was next correlated with earlier data on FGF8b expression in a subgroup of cancers. In this cohort, 86% (<em>19</em>/22) of high-grade cancers expressed FGF8 but only 31% (7/22) expressed hSef. Positive FGF8 expression but a loss of hSef was observed in 88% (7/8) of metastatic tumours. In contrast, metastasis was evident in only 10% (1/10) of tumours, which co-expressed both FGF8 and hSef (P<0.001). These results suggest evidence that hSef is downregulated in advanced prostate cancer and might facilitate an enhanced tumorigenic response to FGFs. Further research into the role of hSef in cancer cell signalling and the mechanism of its downregulation may contribute to more effective targeting of <em>growth</em> <em>factors</em> in prostate cancer.

BACKGROUND

Adipose tissue hypoxia and endoplasmic reticulum (ER) stress may link the presence of chronic inflammation and macrophage infiltration in severely obese subjects. We previously reported the up-regulation of TNF-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible 14 (Fn14) axis in adipose tissue of severely obese type 2 diabetic subjects.

OBJECTIVE

The objective of the study was to examine TWEAK and Fn14 adipose tissue expression in obesity, severe obesity, and type 2 diabetes in relation to hypoxia and ER stress.

METHODS

In the obesity study, 19 lean, 28 overweight, and 15 obese nondiabetic subjects were studied. In the severe obesity study, 23 severely obese and 35 control subjects were studied. In the type 2 diabetes study, 11 type 2 diabetic and 36 control subjects were studied. The expression levels of the following genes were analyzed in paired samples of sc and visceral adipose tissue: Fn14, TWEAK, VISFATIN, HYOU1, FIAF, HIF-1a, VEGF, GLUT-1, GRP78, and XBP-1. The effect of hypoxia, inflammation, and ER stress on the expression of TWEAK and Fn14 was examined in human adipocyte and macrophage cell lines.

RESULTS

Up-regulation of TWEAK/Fn14 and hypoxia and ER stress surrogate gene expression was observed in sc and visceral adipose tissue only in our severely obese cohort. Hypoxia modulates TWEAK or Fn14 expression in neither adipocytes nor macrophages. On the contrary, inflammation up-regulated TWEAK in macrophages and Fn14 expression in adipocytes. Moreover, TWEAK had a proinflammatory effect in adipocytes mediated by the nuclear factor-kappaB and ERK but not JNK signaling pathways.

CONCLUSIONS

Our data suggest that TWEAK acts as a pro-inflammatory cytokine in the adipose tissue and that inflammation, but not hypoxia, may be behind its up-regulation in severe obesity.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF; FGF-2) is one of <em>19</em> related members of a <em>growth</em> <em>factor</em> family with mitogenic and hormone-regulatory functions. In Xenopus laevis oocytes, a 1.5-kb FGF-2 antisense (GFG) RNA complementary to the third exon and 3'-untranslated region (UTR) of FGF-2 mRNA has been implicated in FGF-2 mRNA editing and stability. The human homolog has been cloned, and we localized this gene by yeast artificial chromosome (YAC), somatic cell, and radiation hybrid panels to the same chromosomal site as FGF-2 (chromosome 4, JO4513 adjacent to D4S430), confirming this as a human endogenous antisense gene. The full-length GFG antisense RNA encodes a 35-kDa protein, which is highly homologous with the MutT family of antimutator nucleosidetriphosphatases (NTPases). We show that human pituitary tumors express FGF-2 and its endogenous antisense partner GFG. While normal pituitary expresses GFG but not FGF-2, pituitary adenomas express FGF-2 and have reduced levels of GFG; aggressive and recurrent adenomas expressed more FGF than GFG mRNA. To examine the effects of this antisense gene in the pituitary, we transfected the pituitary-derived GH4 mammosomatotroph cell line with constructs encoding the full-length human GFG cDNA. Transiently and stably transfected cells expressed the 35-kDa GFG protein that was localized to the cytoplasm. These cells exhibited enhanced PRL expression as documented by transiently transfected PRL-luciferase reporter assay and by endogenous PRL protein. GFG expression in these cells did not alter endogenous FGF-2 expression but increased the proportion of the higher molecular mass 22-kDa form of GH. Moreover, GFG expression inhibited cell proliferation as shown by [(3)H]thymidine incorporation, proliferating cell nuclear antigen (PCNA) nuclear staining, and cell cycle analysis. We conclude that the GFG-encoded protein has divergent hormone-regulatory and antiproliferative actions in the pituitary that are independent of FGF-2 expression. GFG represents a novel mechanism involved in restraining pituitary tumor cell <em>growth</em> while promoting hormonal activity.

1. Interstitial lung disease is a common complication of systemic sclerosis. The mechanism by which excess collagen is deposited in the lung is poorly understood, but is thought to involve release of mediators which activate lung <em>fibroblasts</em>. In this study we investigated and partially characterized the <em>fibroblast</em> proliferative activity of bronchoalveolar lavage fluid from 29 patients with systemic sclerosis, <em>19</em> with and 10 without evidence of lung disease assessed by thin-section computed tomography. 2. Bronchoalveolar lavage fluid from both groups of patients stimulated <em>fibroblast</em> proliferation compared with control subjects: systemic sclerosis with normal computed tomography, 27.7 (range 10.5-57.9)% above control; systemic sclerosis with abnormal computed tomography, 26.7 (range 5.0-47.8)% above control, P < 0.02 in both cases. 3. The activity was reduced by about one-third by neutralizing antibodies to insulin-like <em>growth</em> <em>factor</em>-1 but not platelet-derived <em>growth</em> <em>factor</em>. Levels of insulin-like <em>growth</em> <em>factor</em>-1 of bronchoalveolar fluid were increased in patients with systemic sclerosis [2.10 (range 1.10-3.48) ng/ml of bronchoalveolar lavage fluid] compared with controls [1.45 (range 1.10-2.05) ng/ml; P < 0.01]. When patients were subdivided into those with abnormal computed tomography [2.10 (range 1.20-3.48) ng/ml] and those with normal computed tomography [1.85 (range 1.10-2.90) ng/ml] only the values for the group with evidence of lung disease were increased compared with control subjects (P < 0.02). Platelet-derived <em>growth</em> <em>factor</em> could not be detected in bronchoalveolar lavage fluid from any group. Fractionation of bronchoalveolar lavage fluid demonstrated activity in several fractions consistent with the molecular masses of insulin-like <em>growth</em> <em>factor</em>-1 associated with binding proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanisms by which mechanical forces promote fetal lung development are not fully understood. Here, we investigated differentiation of fetal type II epithelial cells via the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) in response to mechanical strain. First, we showed that incubation of embryonic day (E) <em>19</em> fetal type II cells with recombinant heparin-binding EGF-like <em>growth</em> <em>factor</em> (HB-EGF) or transforming <em>growth</em> <em>factor</em> (TGF)-alpha, but not with amphiregulin (AR), betacellulin (BTC) or epiregulin (EPR), increased fetal type II cell differentiation, as measured by surfactant protein B/C mRNA and protein levels. Next, we demonstrated that 5% cyclic stretch of E<em>19</em> monolayers transfected with plasmid encoding alkaline phosphatase (AP)-tagged ligands shed mature HB-EGF and TGF-alpha into the supernatant and promoted type II cell differentiation. Release of these ligands was also observed in E<em>19</em> cells subjected to higher degrees of cyclic strain, but not in cells exposed to continuous stretch. Interestingly, the addition of <em>fibroblasts</em> to type II cell cultures did not enhance release of HB-EGF. Whereas HB-EGF shedding was also detected in E18 cells exposed to 5% cyclic stretch, release of this ligand after 2.5% sustained stretch was restricted to cells isolated on E18 of gestation. In addition, mechanical stretch released EGF, AR and BTC. We conclude that mechanical stretch promotes fetal type II cell differentiation via ectodomain shedding of HB-EGF and TGF-alpha. The magnitude of shedding varied depending on gestational age, ligand, and strain protocol. These studies provide novel mechanistic information potentially relevant to fetal lung development and to mechanical ventilation-induced lung injury.

Sorafenib, a multi-kinase inhibitor, is used as a standard therapy for advanced hepatocellular carcinoma (HCC). However, complete remission has not been achieved and the molecular basis of HCC resistance to sorafenib remains largely unknown. Previous studies have shown that <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) expression correlates with tumor progression and poor prognosis of HCC. Here, we demonstrate the novel role of FGF<em>19</em> in HCC resistance to sorafenib therapy.

FGF<em>19</em> Knockdown cells were achieved by lentiviral-mediated interference, and FGFR4 knockout cells were achieved by CRISPR-Cas9. Protein levels of FGF<em>19</em>, FGFR4 and c-PARP in various HCC cell lines were measured by Western blotting analysis. Cell viability was determined by MTS assay, apoptosis was determined by DAPI nuclear staining and Western blot of c-PRAP, and ROS generation was determined by DCFH-DA staining and electrochemical biosensor.

We showed that FGF<em>19</em>, when overexpressed, inhibited the effect of sorafenib on ROS generation and apoptosis in HCC. In contrast, loss of FGF<em>19</em> or its receptor FGFR4 led to a remarkable increase in sorafenib-induced ROS generation and apoptosis. In addition, knockdown of FGF<em>19</em> in sorafenib-resistant HCC cells significantly enhanced the sensitivity to sorafenib. Importantly, targeting FGF<em>19</em>/FGFR4 axis by ponatinib, a third-generation inhibitor of chronic myeloid leukemia, overcomes HCC resistance of sorafenib by enhancing ROS-associated apoptosis in sorafenib-treated HCC.

Our results provide the first evidence that inhibition of FGF<em>19</em>/FGFR4 signaling significantly overcomes sorafenib resistance in HCC. Co-treatment of ponatinib and sorafinib may represent an effective therapeutic approach for eradicating HCC.

BACKGROUND

Venous hypertension is regarded as an important factor in the pathogenesis of dural arteriovenous fistula (DAVF). We investigate histologic reaction of dural sinus under the condition of venous hypertension using a rat venous hypertension model to present hemodynamic and immunohistochemical effect in the development of DAVF.

METHODS

Twenty-four Sprague-Dawley male rats were divided into venous hypertension and control groups. Venous hypertension was induced with a left common carotid artery-external jugular vein anastomosis and an occlusion of a right posterior facial vein. Measurements of systemic mean arterial pressure, draining vein pressure (DVP), and cerebral perfusion pressure (CPP) were conducted on the next day, at 7 days, and at 28 days after surgery, and the rats were killed for histologic examinations.

RESULTS

Postoperative DVP increased significantly in venous hypertension group compared to control group (35 +/- 5 vs 13 +/- 2 mm Hg, P < .05). Increased DVP remained above 30 mm Hg throughout the observation period. Postoperative CPP decreased significantly in venous hypertension group compared to control group (49 +/- 8 vs 86 +/- 9 mm Hg, P < .05). In venous hypertension group, there was a significant difference between days 1 and 28 (49 +/- 8 vs 64 +/- 8 mm Hg, P < .05). Histologic examination revealed thickening of connective tissues, proliferation of fibroblasts, and strong expression of vascular endothelial growth factor (VEGF) in endothelium under venous hypertension condition. Immunostained VEGF cells decreased significantly from day 7 to day 28 (100 +/- 16 vs 72 +/- 19 cells, P < .05). A positive correlation was observed between DVP and VEGF expression (Pearson correlation coefficient; r = 0.671, P = .0017). There was a negative correlation between CPP and immunostained VEGF cells (r = -0.702, P = .0089).

CONCLUSIONS

These results suggest that venous hypertension is associated with increased expression of VEGF, and a decreased CPP may have a potential effect in VEGF expression under venous hypertension condition. These factors are speculated to play an important role in progression of DAVF.

Perlecan is a modular heparan sulphate and/or chondroitin sulphate substituted proteoglycan of basement membrane, vascular tissues and cartilage. Perlecan acts as a low affinity co-receptor for <em>fibroblast</em> <em>growth</em> <em>factors</em> 1, 2, 7, 9, binds connective tissue <em>growth</em> <em>factor</em> and co-ordinates chondrogenesis, endochondral ossification and vascular remodelling during skeletal development; however, relatively little is known of its distribution in these tissues during ageing and development. The aim of the present study was to immunolocalise perlecan in the articular and epiphyseal <em>growth</em> plate cartilages of stifle joints in 2-day to 8-year-old pedigree merino sheep. Perlecan was prominent pericellularly in the stifle joint cartilages at all age points and also present in the inter-territorial matrix of the newborn to <em>19</em>-month-old cartilage specimens. Aggrecan was part pericellular, but predominantly an extracellular proteoglycan. Perlecan was a prominent component of the long bone <em>growth</em> plates and displayed a pericellular as well as a strong ECM distribution pattern; this may indicate a so far unrecognised role for perlecan in the mineralisation of hypertrophic cartilage. A significant age dependant decline in cell number and perlecan levels was evident in the hyaline and <em>growth</em> plate cartilages. The prominent pericellular distribution of perlecan observed indicates potential roles in cell-matrix communication in cartilage, consistent with <em>growth</em> <em>factor</em> signalling, cellular proliferation and tissue development.

Human <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) is an enterohepatic hormone that is involved in the regulation of hepatic metabolism of bile acids, lipids, and glucose. Farnesoid X receptor (Fxr)-null mice exhibit steatosis-like symptoms, showing higher hepatic lipid levels than with the wild-type mice. We investigated the influence of FGF<em>19</em> treatment on hepatic lipogenesis in Fxr-null mice. Recombinant FGF<em>19</em> treatment (400 µg/kg/d) for 3 d prevented the accumulation of lipid droplets and decreased serum alanine aminotransferase activity and hepatic lipid levels, including those of triglycerides and free fatty acids. The treatment significantly decreased the hepatic mRNA levels of acetyl-CoA carboxylase 1 (Acc1), Cd36, and sterol regulatory element-binding protein-1c (Srebp-1c) as well as those of acetyl-CoA carboxylase 2 (Acc2), stearoyl CoA desaturase 1 (Scd1), and Cyp7a1. FGF<em>19</em> treatment (4 µg/kg/d) for 3 d also decreased the hepatic free fatty acid levels and mRNA levels of Acc1, Cd36, and Srebp-1c. These results indicate that FGF<em>19</em>-mediated signaling ameliorates disrupted hepatic lipogenesis in Fxr-null mice.

The t(4;14) translocation detected by fluorescence in situ hybridization (FISH) is an independent prognostic <em>factor</em> for an adverse outcome of multiple myeloma (MM). Because t(4;14) uniquely results in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) expression, decalcified, paraffin-embedded bone marrow biopsies were immunostained for FGFR3, and its expression was correlated with the t(4;14) status. FISH detected t(4;14) in 16 (<em>19</em>%) of 85 MM patient specimens, and immunocytochemistry detected aberrant FGFR3 expression in 13 (15%). Twelve (75%) t(4;14)-positive cases expressed FGFR3, and 12 (92%) FGFR3-positive cases harbored a t(4;14). FGFR3 expression and t(4;14) were strongly correlated (P < .001). FGFR3 expression by immunohistochemistry was associated with the immunoglobulin A (IgA) isotype (P < .001), a shorter progression-free survival (median, 11.5 versus 25.8 months; P < .001), and a shorter overall survival (median, <em>19</em>.2 versus 46.3 months; P < .001).

OBJECTIVE

The purpose is to determine dose-limiting toxicity, pharmacokinetics,pharmacodynamics, and immunobiology after i.p. injections of recombinant human IL-12 (rhIL-12).

METHODS

rhIL-12 was administered to 29 previously treated patients with peritoneal carcinomatosis from Müllerian carcinomas, gastrointestinal tract carcinomas and peritoneal mesothelioma in a Phase I trial. rhIL-12 doses were increased from 3 to 600 ng/kg. Three or more patients at each level received weekly i.p. injections of rhIL-12.

RESULTS

Dose-limiting toxicity (elevated transaminase) occurred in 2 of 4 patients at the 600 ng/kg dose. More frequent toxicities included fever, fatigue, abdominal pain, nausea, and catheter-related infections. Ten patients received 300 ng/kg with acceptable frequency and severity of side effects. Two patients (one with ovarian cancer and one with mesothelioma) had no remaining disease at laparoscopy. Eight patients had stable disease and <em>19</em> progressive disease. At 300 ng/kg i.p., IL-12 was cleared from peritoneal fluid in a biphasic manner with a terminal-phase half-life of 18.7 h; peritoneal fluid levels of IL-12 5 min after i.p. injection were 100-200 pg/ml, and serum levels reached approximately 10 pg/ml between 24 and 36 h. IL-1-alpha, IL-2, IL-10, tumor necrosis <em>factor</em> alpha, and IFN-gamma were determined in serum and peritoneal fluid. IFN-gamma, IL-10, and tumor necrosis <em>factor</em> alpha were detected most frequently. Immunobiological effects included peritoneal tumor cell apoptosis, decreased tumor cell expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and vascular endothelial <em>growth</em> <em>factor</em>, elevated IFN-gamma and IFN-inducible protein 10 transcripts in peritoneal exudate cells, and increased proportions of peritoneal CD3(+) relative to CD14(+) cells.

CONCLUSIONS

rhIL-12 at 300 ng/kg by weekly i.p. injection is biologically active and adequately tolerated for Phase II studies.

OBJECTIVE

We sought to determine whether serum concentrations of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) - an ileum-derived enterokine which plays a role in the control of glucose and lipid homeostasis - are altered in patients with biopsy-proven nonalcoholic fatty liver disease (NAFLD).

METHODS

Serum levels of FGF<em>19</em> were measured using enzyme-linked immunosorbent assay in 91 patients with biopsy-proven NAFLD and 74 controls.

RESULTS

FGF<em>19</em> levels were significantly lower in patients with biopsy-proven NAFLD (median: 130pg/mL) than in controls (median: 210pg/mL, P<0.001). Serum FGF<em>19</em> levels were significantly but modestly associated with hepatocyte ballooning scores in univariate analysis (r=-0.25, P<0.05) but not after adjustment for potential confounders (β=-0.18; t=1.78, P=0.08).

CONCLUSIONS

This pilot study suggests that serum FGF<em>19</em> levels are decreased in patients with NAFLD but are not independently associated with liver histology findings.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) represses cholesterol 7α-hydroxylase (Cyp7α1) and inhibits bile acid synthesis in vitro and in vivo. Previous studies have shown that anti-FGF<em>19</em> antibody treatment reduces <em>growth</em> of colon tumor xenografts and prevents hepatocellular carcinomas in FGF<em>19</em> transgenic mice and thus may be a useful cancer target. In a repeat dose safety study in cynomolgus monkeys, anti-FGF<em>19</em> treatment (3-100 mg/kg) demonstrated dose-related liver toxicity accompanied by severe diarrhea and low food consumption. The mechanism of anti-FGF<em>19</em> toxicity was investigated using in vitro and in vivo approaches. Our results show that anti-FGF<em>19</em> antibody had no direct cytotoxic effect on monkey hepatocytes. Anti-FGF<em>19</em> increased Cyp7α1, as expected, but also increased bile acid efflux transporter gene (bile salt export pump, multidrug resistant protein 2 [MRP2], and MRP3) expression and reduced sodium taurocholate cotransporting polypeptide and organic anion transporter 2 expression in liver tissues from treated monkeys and in primary hepatocytes. In addition, anti-FGF<em>19</em> treatment increased solute transporter gene (ileal bile acid-binding protein, organic solute transporter α [OST-α], and OST-β) expression in ileal tissues from treated monkeys but not in Caco-2 cells. However, deoxycholic acid (a secondary bile acid) increased expression of FGF<em>19</em> and these solute transporter genes in Caco-2 cells. Gas chromatography-mass spectrometry analysis of monkey feces showed an increase in total bile acids and cholic acid derivatives. These findings suggest that high doses of anti-FGF<em>19</em> increase Cyp7α1 expression and bile acid synthesis and alter the expression of bile transporters in the liver resulting in enhanced bile acid efflux and reduced uptake. Increased bile acids alter expression of solute transporters in the ileum causing diarrhea and the enhanced enterohepatic recirculation of bile acids leading to liver toxicity.

BACKGROUND

CYR61 is an extracellular matrix-associated protein that promotes adhesion, migration, and proliferation of endothelial cells and fibroblasts. Prostate enlargement, which frequently causes the urethral compression, is often histologically observed as stromal and epithelial hyperplasia in an enlarged gland. To determine whether or not CYR61 has relevance to the progression of benign prostatic hyperplasia (BPH), we investigated the induction of CYR61, and also examined its function in both prostatic stromal and epithelial cells.

METHODS

Recombinant CYR61 protein was used for the examination of the activity of CYR61 as to cell adhesion and proliferation. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was utilized to screen for inducers of the CYR61 gene in prostatic cells. Finally, the effects of an anti-sense oligonucleotide, which could reduce the production of CYR61, on the morphology and growth of prostatic cells were also examined.

RESULTS

Recombinant CYR61 protein promotes prostatic cell adhesion and proliferation. The mRNA for CYR61, a growth factor-inducible immediate early gene, was markedly induced by fetal bovine serum (FBS) within 1 hr, and strongly induced by transforming growth factor-beta1 (TGF-beta) for at least 19 hr following stimulation. The suppression of CYR61 production with an anti-sense oligonucleotide causes obvious morphological changes of prostatic cells. Furthermore, we have shown that CYR61 is necessary, at least in part, for FBS-induced prostatic cell proliferation, because dramatic inhibition of cellular growth was caused by the suppression of CYR61 production with the addition of the anti-sense oligonucleotide before FBS stimulation.

CONCLUSIONS

In this study, we demonstrate that serum growth factors induce the CYR61 gene in both stromal and epithelial cells, and that CYR61 plays functional roles in cell adhesion, morphology, and proliferation, supporting its involvement in benign prostatic enlargement. These results strongly suggest that CYR61 is a key molecule, and therefore could be a potential therapeutic target in prostatic hyperplastic growth.

OBJECTIVE

Since <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF-<em>19</em>) is a potent metabolic regulator that influences glucose and lipid homeostasis, our aim was to develop an ELISA assay for measuring FGF-<em>19</em> in human serum and to investigate its concentrations in healthy volunteers and patients suffering from metabolic syndrome.

METHODS

A sandwich ELISA method was developed for quantitative determination of human FGF-<em>19</em> in serum samples. Blood pressure, waist circumference, FGF-21 serum levels, serum cholesterol, triacylglycerols, HDL-cholesterol, LDL-cholesterol, insulin, glucose, adiponectin, uric acid, creatinine, hs-CRP and calculated BMI and Quicki insulin sensitivity index were measured in 153 healthy volunteers and 66 persons with metabolic syndrome.

RESULTS

Neither sex nor age influenced FGF-<em>19</em> serum concentration in the healthy volunteers. Probands with metabolic syndrome had 65 % lower FGF-<em>19</em> serum values than the healthy ones (medians 158.6 versus 242.4 ng/L; p<0.01). FGF-<em>19</em> correlated with glucose (r = -0.35, p<0.01), HDL (r = 0.24, p = 0.045), triacylglycerols (r = -0.<em>19</em>, p = 0.05) and with a number of other risk <em>factor</em>s for metabolic syndrome (r = -0.28, p = 0.01). When adjusted to the concentrations of triacylglycerols, BMI and glucose, and finally to all data pertinent to FGF-<em>19</em> (according to correlation analysis), our data indicate that FGF-<em>19</em> is an independent marker of metabolic syndrome.

CONCLUSIONS

The present study demonstrates the analytical properties of the ELISA FGF-<em>19</em> assay and its usefulness when studying the metabolic syndrome. Serum concentrations of FGF-<em>19</em> could be new key predictors of metabolic syndrome and thereby even a new negative risk <em>factor</em> of atherosclerosis.

BACKGROUND

Adrenocortical cancer (ACC) is a rare cancer with poor prognosis and scant treatment options. In ACC, no personalized approach has emerged but no extensive molecular screening has been performed to date.

OBJECTIVE

The objective of the study was to evaluate the presence of a large number of potentially targetable molecular events in a large cohort of advanced ACC.

METHODS

We used hot spot gene sequencing (Ion Torrent, 40 patients) and comparative genomic hybridization (CGH; 28 patients; a subset of the entire cohort) in adult stage III-IV ACC samples to screen for mutations and copy number abnormalities of potential interest for therapeutic use in 46 and 130 genes, respectively.

RESULTS

At least one copy number alteration or mutation was found in <em>19</em> patients (47.5%). The most frequent mutations were detected on TP53, ATM, and CTNNB1 [6 of 40 (15%), 5 of 40 (12.5%), and 4 of 40 (10%), respectively]. The most frequent copy number alterations identified were: amplification of the CDK4 oncogene (5 of 28; 17.9%) and deletion of the CDKN2A (4 of 28; 14.3%) and CDKN2B (3 of 28; 10.7%) tumor suppressor genes. Amplifications of FGFR1, FGF9, or FRS2 were discovered in three subjects (10.7%). Associated alterations were: deletions of CDKN2A, CDKN2B with ATM mutations, and TP53 mutations with CTNNB1 mutations.

CONCLUSIONS

No simple targetable molecular event emerged. Drugs targeting the cell cycle could be the most relevant new therapeutic approach for patients with advanced ACC. Inhibitors of the fibroblast growth factor receptor pathway could also be a therapeutic option in a subset of patients, whereas other targeted therapies should be considered on a case-by-case basis.