We have shown previously that epidermal <em>growth</em> <em>factor</em> (EGF) plus <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2) expands the neural precursor cells in the ependyma of the normal adult rat spinal cord in vivo. To investigate the therapeutic effect of these <em>factors</em> on spinal cord injury (SCI), we administered EGF, FGF2, EGF plus FGF2, or artificial cerebrospinal fluid (aCSF) intrathecally (<em>15</em> ng/h of EGF or FGF2) for 3 or 14 days after mild (2.4-g) or moderate (20-g) clip compression injury at T1 in adult rats. Histological and functional assessments were used to evaluate the therapeutic effects. The EGF plus FGF2 group, which received these agents for 14 days, showed better functional recovery than the aCSF group 42 days after moderate SCI (p < 0.05). At 14 days, the EGF plus FGF2 group showed a much greater expansion of ependymal cells and astrocytes compared to the other groups, and there was evidence for extensive migration of ependymal cells into the surrounding injured cord. These mitogens did not significantly enhance nestin expression in the ependymal layer or alter the expansion of oligodendrocyte precursor cells or microglia/macrophages, and dividing cells did not show the neuron-specific marker NeuN except immediately adjacent to the ependyma. The exact mechanism for improved functional recovery after EGF plus FGF2 is not known.

YKL-40 is a <em>growth</em> <em>factor</em> for chondrocytes and <em>fibroblasts</em>. The aim was to evaluate YKL-40 expression in the musculoskeletal system during early human development. We studied sections from <em>15</em> human embryos [weeks 5.5-8; 7- to 31-mm crown-rump length (CRL)] and 68 fetuses (weeks 9-14; 33- to 105-mm CRL) for YKL-40 protein expression by immunohistochemistry. YKL-40 mRNA expression was evaluated in two human embryos (days 41 and 51). Initially YKL-40 is expressed in all germ layers: ecto-, meso-, and endoderm. YKL-40 mRNA and protein expression are found in tissues of the ecto-, meso-, and endoderm, and YKL-40 protein expression is present during development of cartilage, bone, joints, and muscles. At the cellular level, YKL-40 protein expression is high in tissues characterized by rapid proliferation, marked differentiation, and undergoing morphogenetic changes. Examples of rapid cell proliferation include the chondrogenic inner layer of perichondrium and the osteogenic inner layer of periosteum. Differences in YKL-40 expression during differentiation are found in the chondrogenic and osteogenic cell lineages. The initial shaping of cartilage and bone models and joints is concomitant with a strong outline of YKL-40-positive cells. This indicates that YKL-40 is associated with cell proliferation, differentiation, and tissue morphogenesis during development of the human musculoskeletal system.

Bile acids play a critical role in the regulation of glucose, lipid, and energy metabolism through activation of the nuclear bile acid receptor farnesoid X receptor (FXR) and membrane G protein-coupled bile acid receptor-1 (Gpbar-1, aka TGR5). Agonist activation of FXR and TGR5 improves insulin and glucose sensitivity and stimulates energy metabolism to prevent diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD). Bile acids have both pro- and anti-inflammatory actions through FXR and TGR5 in the intestine and liver. In the intestine, bile acids activate FXR and TGR5 to stimulate stimulate <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> and glucagon-like peptide-1 secretion. FXR and TGR5 agonists may have therapeutic potential for treating liver-related metabolic diseases, such as diabetes and NAFLD.

BACKGROUND

Laboratory and clinical data suggest that the anti-angiogenic agent, thalidomide, if combined with cytotoxic agents, may be effective against recurrent glioblastoma multiforme (GBM).

OBJECTIVE

To determine 6-month progression-free survival (6PFS) and toxicity of temozolomide plus thalidomide in adults with recurrent GBM.

METHODS

Eligible patients had recurrent GBM after surgery, radiotherapy, and/or adjuvant chemotherapy. Temozolomide was given at <em>15</em>0-200 mg/m(2)/day on days 1-5 of each 28-day cycle. Thalidomide was given orally at 400 mg at bedtime (days 1-28) and increased to 1,200 mg as tolerated. Patients were evaluated with magnetic resonance imaging scans every 56 days. The study was designed to detect an increase of the historical 6PFS for GBM from 10 to 30%.

RESULTS

Forty-four patients were enrolled, 43 were evaluable for efficacy and safety. The study population included <em>15</em> women, 29 men; median age was 53 years (range 32-84); median Karnofsky performance status was 80% (range 60-100%). Thirty-six (82%) patients were chemotherapy-naïve. There were 57 reports of toxicity of grade 3 or greater. Non-fatal grade 3-4 granulocytopenia occurred in <em>15</em> patients (34%). The objective response rate was 7%. The estimated probability of being progression-free at 6 months with this therapy is 24% [95% confidence interval (C.I.) 12-38%]. The median time to progression is <em>15</em> weeks (95% C.I. 10-20 weeks). There was no observed correlation between serum levels of vascular endothelial <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and IL-8 and the 6PFS outcome.

CONCLUSIONS

This drug combination was reasonably safe, but with little indication of improvement compared to temozolomide alone.

Previous studies have established the link between aberrant microRNA (miRNA) expression and hypoxia in various neoplasms. However, how these hypoxia-related miRNAs modulate tumor progression is still unclear. Therefore, the patterns of miRNA in colorectal carcinoma cell lines in response to hypoxia or not were first screened and the hypoxia-induced repression of the miR-<em>15</em>-16 cluster was confirmed. Then, this repression was found to be associated with high tumor stage and poor prognosis in colorectal carcinoma and is shown to promote tumor angiogenesis and metastasis by the loss of restriction of its target gene, <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2). Moreover, the general and alterative promoters of the miR-<em>15</em>-16 host (deleted in lymphocytic leukemia 2, DLEU2) were mapped, and three c-Myc/Max binding sites in response to the hypoxia-induced repression of miR-<em>15</em>-16 were further identified. Finally, an enhanced stability of c-Myc/Max heterodimer promoted by increased hypoxia-inducible <em>factor</em>-2α (HIF-2α) was validated, and we also verified that the enhancement contributed to the hypoxia-induced repression of miR-<em>15</em>-16. In brief, the c-Myc-mediated transcriptional repression of miR-<em>15</em>-16 in hypoxia is induced by increased HIF-2α and promoted tumor angiogenesis and hematogenous metastasis by the further loss of post-transcriptional inhibition of FGF2. Our study provides a better understanding of the coping mechanisms in response to tumor hypoxia and may be helpful in developing an effective prognostic marker or treatment target against solid tumors.

The bile acid-activated receptors, nuclear farnesoid X receptor (FXR) and the membrane Takeda G-protein receptor 5 (TGR5), are known to improve glucose and insulin sensitivity in obese and diabetic mice. However, the metabolic roles of these two receptors and the underlying mechanisms are incompletely understood. Here, we studied the effects of the dual FXR and TGR5 agonist INT-767 on hepatic bile acid synthesis and intestinal secretion of glucagon-like peptide-1 (GLP-1) in wild-type, Fxr-/-, and Tgr5-/- mice. INT-767 efficaciously stimulated intracellular Ca2+ levels, cAMP activity, and GLP-1 secretion and improved glucose and lipid metabolism more than did the FXR-selective obeticholic acid and TGR5-selective INT-777 agonists. Interestingly, INT-767 reduced expression of the genes in the classic bile acid synthesis pathway but induced those in the alternative pathway, which is consistent with decreased taurocholic acid and increased tauromuricholic acids in bile. Furthermore, FXR activation induced expression of FXR target genes, including <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em>, and unexpectedly Tgr5 and prohormone convertase 1/3 gene expression in the ileum. We identified an FXR-responsive element on the Tgr5 gene promoter. Fxr-/- and Tgr5-/- mice exhibited reduced GLP-1 secretion, which was stimulated by INT-767 in the Tgr5-/- mice but not in the Fxr-/- mice. Our findings uncovered a novel mechanism in which INT-767 activation of FXR induces Tgr5 gene expression and increases Ca2+ levels and cAMP activity to stimulate GLP-1 secretion and improve hepatic glucose and lipid metabolism in high-fat diet-induced obese mice. Activation of both FXR and TGR5 may therefore represent an effective therapy for managing hepatic steatosis, obesity, and diabetes.

OBJECTIVE

Studies have shown improved survival in cancer patients treated with low molecular weight heparins (LMWHs). Tumors depend on an expanding vasculature, and heparins may affect vessel growth and function. We investigated the effect of heparins differing in Mr on selected endothelial cell properties.

RESULTS

Human umbilical vein endothelial cells were cultured with fibroblast growth factor-2 and heparins differing in Mr. Cell proliferation was assessed by [3H]thymidine incorporation, and vascular organization was assessed by in vitro assays. Maximum inhibition of 94+/-2% was observed with 6-kDa LMWH, greater than the inhibition seen with unfractionated heparin (58+/-8%) or 3-kDa LMWH (60+/-9%, P=0.02 for both). No inhibition of proliferation was observed with heparin tetrasaccharide, octasaccharide, or pentasaccharide (fondaparinux). Three- and 6-kDa fractions decreased endothelial tube formation in Matrigel to 58+/-15% and 67+/-9% (P<0.05), respectively, of that with fibroblast growth factor-2, whereas no inhibition was observed with unfractionated heparin, tetrasaccharide, pentasaccharide, or octasaccharide. LMWH (6 kDa) also inhibited vessel formation in a placental explant.

CONCLUSIONS

Heparin inhibition of endothelial cell proliferation and organization requires a chain length of >8 saccharide units, with maximal inhibition at Mr of 6 kDa. This Mr dependence differs from that required for anticoagulant activity.

Serum concentrations of angiogenic <em>factors</em> have been reported to correlate with tumour burden and prognosis in metastatic melanoma. The present study was performed to assess the value of angiogenic <em>factors</em> in serum in indicating response or failure to chemotherapy and immunochemotherapy in stage IV melanoma. Thirty-five patients suffering from stage IV melanoma according to the American Joint Committee on Cancer (AJCC) criteria were included in this prospective study. Before and following chemotherapy or immunochemotherapy, serum levels of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), platelet-derived <em>growth</em> <em>factor</em> (PDGF-AB), vascular cell adhesion molecule-1 (VCAM-1) and interleukin-8 (IL-8) were measured. Staging examinations following chemotherapy revealed <em>15</em> patients with response to therapy (complete response, partial response, stable disease), 14 patients with progressive disease and six patients with mixed response. Patients who responded to therapy showed a significant decrease in the serum level of IL-8 at the time of staging examinations, whereas patients with progressive disease did not. Following chemotherapy, serum concentrations of PDGF-AB had significantly decreased in both patients with response and patients with progressive disease. Comparing the VEGF and bFGF levels of responders and non-responders after a single administration of cytostatics showed significantly lower concentrations in patients with response to therapy. In all patients, a high intra- and inter-individual variability of serum values was observed during application of therapy. It can be concluded that low IL-8 serum levels after chemotherapy indicate response to chemotherapy in stage IV melanoma patients. The persistence of elevated serum levels of VEGF and bFGF following the initial cytostatic administration may help to identify patients resistant to chemotherapy. The distinct variability of serum levels indicates that processes other than tumour angiogenesis also influence the serum concentration of the examined angiogenic <em>factors</em>.

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional receptor that undergoes constitutive endocytosis and recycling. To identify LRP-1 in lipid rafts, we biotin-labeled cells using a membrane-impermeable reagent and prepared Triton X-100 fractions. Raft-associated proteins were identified in streptavidin affinity-precipitates of the Triton X-100-insoluble fraction. PDGF beta-receptor was identified exclusively in lipid rafts, whereas transferrin receptor was excluded. LRP-1 distributed partially into rafts in murine embryonic <em>fibroblasts</em> (MEFs) and HT 1080 cells, but not in smooth muscle cells and CHO cells. LRP-1 partitioning into rafts was not altered by ligands, including alpha2-macroglobulin, platelet-derived <em>growth</em> <em>factor</em>-BB, and receptor-associated protein (RAP). To examine LRP-1 trafficking between membrane microdomains, we developed a novel method based on biotinylation and detergent fractionation. Association of LRP-1 with rafts was transient; by <em>15</em> min, nearly all of the LRP-1 that was initially raft-associated exited this compartment. LRP-1 in the Triton X-100-soluble fraction, which excludes lipid rafts, demonstrated complex kinetics, with phases reflecting import from rafts, endocytosis, and recycling. Potassium depletion blocked LRP-1 endocytosis but did not inhibit trafficking of LRP-1 from rafts into detergent-soluble microdomains. Our data support a model in which LRP-1 transiently associates with rafts but does not form a stable pool. Fluid movement of LRP-1 between microdomains may facilitate its function in promoting the endocytosis of other plasma membrane proteins, such as the urokinase receptor, which localizes in lipid rafts.

The design of an aqueous formulation for acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) requires an understanding of the type of compounds that can either directly or indirectly stabilize the protein. To this end, spectrophotometric turbidity measurements were initially employed to screen the ability of polyanionic ligands, less specific compounds, and variations in solution conditions (temperature and pH) to stabilize aFGF against heat-induced aggregation. It was found that in addition to the well-known protection of aFGF by heparin, a surprisingly wide variety of polyanions (including small sulfated and phosphorylated compounds) also stabilizes aFGF. These polyanionic ligands are capable of raising the temperature at which the protein unfolds by <em>15</em>-30 degrees C. Many commonly used excipients were also observed to stabilize aFGF in both the presence and the absence of heparin. High concentrations of some of these less specific agents are also able to increase the temperature of aFGF thermal unfolding by as much as 6-12 degrees C as shown by circular dichroism and differential scanning calorimetry. Other compounds were found which protect the chemically labile cysteine residues of aFGF from oxidation. Aqueous formulations of aFGF were thus designed to contain both a polyanionic ligand that enhances structural integrity by binding to the protein and chelating agents (e.g., EDTA) to prevent metal ion-catalyzed oxidation of cysteine residues. While room-temperature storage (30 degrees C) leads to rapid inactivation of aFGF in physiological buffer alone, several of these aFGF formulations are stable in vitro for at least 3 months at 30 degrees C. Three aFGF topical formulations were examined in an impaired diabetic mouse model and were found to be equally capable of accelerating wound healing.

Hepatic bile acid synthesis is subject to complex modes of transcriptional control, in which the bile acid-activated nuclear receptor farnesoid X receptor (FXR) in liver and intestine-derived, FXR-controlled <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (Fgf<em>15</em>) are involved. The Fgf<em>15</em> pathway is assumed to contribute significantly to control of hepatic bile acid synthesis. However, scientific evidence supporting this assumption is primarily based on gene expression data. Using intestine-selective FXR knockout mice (iFXR-KO), we show that contribution of intestinal FXR-Fgf<em>15</em> signalling in regulation of hepatic cholesterol 7α-hydroxylase (Cyp7A1) expression depends on time of the day with increased hepatic Cyp7A1 expression in iFXR-KO mice compared with controls exclusively during the dark phase. To assess the physiological relevance hereof, we determined effects of intestine-selective deletion of FXR on physiological parameters such as bile formation and kinetics of the enterohepatic circulation of bile acids. It appeared that intestinal FXR deficiency leads to a modest but significant increase in cholic acid pool size, without changes in fractional turnover rate. As a consequence, bile flow and biliary bile acid secretion rates were increased in iFXR-KO mice compared with controls. Feeding a bile acid-containing diet or treatment with a bile acid sequestrant similarly affected bile formation in iFXR-KO and control mice and induced similar changes in Cyp7A1 and Cyp8B1 expression patterns. In conclusion, this study is the first to demonstrate the physiological relevance of the contribution of the intestinal FXR-Fgf<em>15</em> signalling pathway in control of hepatic bile acid synthesis. Fgf<em>15</em> contributes to the regulation of hepatic bile acid synthesis in mice mainly during the dark phase. Expansion of the circulating bile acid pool as well as bile acid sequestration diminishes the contribution of intestinal FXR-Fgf<em>15</em> signalling in control of hepatic bile acid synthesis and bile formation.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) influences the differentiation and survival of retinal photoreceptors in vivo and in vitro, but it is not known whether it acts directly on photoreceptor FGF receptors or indirectly through activation of surrounding cells. To clarify the effects of FGF-2 on photoreceptor survival, we developed a purified photoreceptor culture system. The outer nuclear layers of postnatal day 5-<em>15</em> rat retinas were isolated by vibratome sectioning, and the photoreceptor fractions obtained were enzymatically dissociated. Photoreceptors were maintained in monolayer culture for 1 week in a chemically defined medium. Immunocytochemical labeling showed that >99.5% of cells were photoreceptors, and glial contamination represented approximately 0. 2%. Photoreceptors from postnatal day 5-9 retinas survived for at least 24 hr in vitro, whereas cells from postnatal day 10-<em>15</em> retinas died rapidly. Subsequent studies performed with postnatal day 5 photoreceptors showed that their survival was increased in a dose-dependent manner after the addition of FGF-2. In control cultures, 36% of originally seeded photoreceptors were alive after 5 d in vitro, and in the presence of 20 ng/ml FGF-2 this number was doubled to 62%. This increase was not caused by proliferation of photoreceptor precursors. Denaturing or blocking FGF-2 prevented enhancement of survival. Conversely, only 25.5% of photoreceptors survived in the presence of epidermal <em>growth</em> <em>factor</em> (EGF). FGF- and EGF-receptor mRNA and proteins were detected in purified photoreceptors in vitro, and addition of FGF-2 or EGF led to tyrosine phosphorylation of photoreceptor proteins. These data support a direct mechanism of action for FGF-2 stimulation of photoreceptor survival.

We examined whether messenger RNA for basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) could be visualized specifically by a fluorescent probe in living cells. A <em>15</em>-nucleotide-long antisense or sense sequence for human bFGF was sandwiched between two complementary 5-nucleotide-long arm sequences. A fluorophore, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), was joined to the 5'-terminal phosphate, while 4-(4'-dimethylaminophenylazo)benzoic acid, quencher for EDANS, was joined to the 3'-terminal hydroxyl group. The probe emitted blue fluorescence only upon hybridization with the complementary 18-nucleotide-long sequence under ultraviolet light. The antisense or sense probe carried with liposome was delivered to human cells, trabecular cells of the eye, in a glass-bottom culture dish placed on the stage of an inverted microscope. Cells with the antisense probe did, but not with the sense probe, show blue fluorescence under ultraviolet light. The present study opens a way to measure the changing levels of a specific messenger RNA in living cells.

Endothelin is a potent vasoconstrictive peptide recently isolated by Yanagisawa, M., Kurihara, H., Kimura, S., Tamobe, Y., Kobayashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988) Nature 332, 411-4<em>15</em>). In order to understand the mechanism of action of endothelin in various cell types we have examined the effects of endothelin on second messenger levels in Rat-1 <em>fibroblasts</em> and A10 smooth muscle cells. Endothelin stimulated a <em>15</em>-fold increase in the accumulation of inositol trisphosphate in Rat-1 cells, with half-maximal stimulation observed at 0.5 nM endothelin. In the A10 vascular smooth muscle cell line, endothelin stimulated phosphatidylinositol turnover more than 3-fold, comparable to the stimulation produced by serum. Concurrent with the increase in inositol phosphate release, endothelin increased diacylglycerol levels by 50% in A10 cells and by more than 3-fold in Rat-1 cells. The increase in diacylglycerol levels in response to endothelin was equal to or greater than the response to serum. Stimulation of phosphatidylinositol turnover by endothelin did not require the presence of extracellular calcium and was not blocked by treatment with EGTA or cobalt. Furthermore, endothelin did not stimulate Ca2+ uptake by either cell type and actually reduced Ca2+ uptake below control levels with increased duration of preincubation. Endothelin stimulated an increase in intracellular Ca2+ levels, from 100 to over 750 nM in Rat-1 cells and from <em>15</em>0 to over 350-nM in A10 cells as measured by fura-2 microspectrofluorimetry. The rise in intracellular Ca2+ concentration was not inhibited by the presence of EGTA or cobalt. These data indicate that endothelin did not act to open Ca2+ channels in either Rat-1 <em>fibroblasts</em> or A10 smooth muscle cells. Cytoplasmic levels of VL30 RNA, a gene independently induced by protein kinase C and by epidermal <em>growth</em> <em>factor</em>, were increased following endothelin treatment, even in protein kinase C-depleted cells. We conclude that endothelin is a very potent stimulus for phosphatidylinositol turnover, diacylglycerol release, and gene transcription. These data may have wide-ranging implications for a number of disease states.

The hallmarks of rheumatoid arthritis (RA) are leukocytic infiltration of the synovium and expansiveness of <em>fibroblast</em>-like synoviocytes (FLS). The abnormal proliferation of FLS and their resistance to apoptosis is mediated, at least in part, by present in RA joints proinflammatory cytokines and <em>growth</em> <em>factors</em>. Because IL-<em>15</em> exerts properties of antiapoptotic and <em>growth</em> <em>factors</em>, and is produced by RA FLS, we hypothesized that IL-<em>15</em> participates in RA FLS activation. To test this hypothesis, we first examined whether RA FLS express chains required for high affinity functional IL-<em>15</em>R. Indeed, RA FLS express IL-<em>15</em>Ralpha at mRNA and protein levels. Moreover, we confirmed the presence of IL-2Rbeta and common gamma-chains. Interestingly, TNF-alpha or IL-1beta triggered significant elevation of IL-<em>15</em>Ralpha chain at mRNA and protein levels. Next, we investigated the effects of exogenous or endogenous IL-<em>15</em> on Bcl-2 and Bcl-x(L) expression, FLS proliferation, and apoptosis. Exogenous IL-<em>15</em> enhanced RA FLS proliferation and increased the level of mRNA-encoding Bcl-x(L). To test the role of endogenous IL-<em>15</em> in the activation of RA FLS, an IL-<em>15</em> mutant/Fcgamma2a protein exerting properties of specific antagonist to the IL-<em>15</em>Ralpha chain was used. We found that blocking IL-<em>15</em> biological activities using this protein substantially reduced endogenous expression of Bcl-2 and Bcl-x(L), and RA FLS proliferation that was reflected by increased apoptosis. Thus, we have demonstrated that a distinctive phenotype of RA FLS, i.e., persistent activation, proliferation, and resistance to apoptosis, is related to the autocrine activation of IL-<em>15</em>Rs by FLS-derived IL-<em>15</em>.

The insulin-like <em>growth</em> <em>factor</em> (IGF) axis is a multi-component network of molecules involved in the regulation of cell <em>growth</em>. The axis includes two major ligands, (IGF-I and IGF-II), cell surface receptors, (the type I IGF receptor family as well as the type II IGF receptor), a family of high affinity binding proteins which regulate IGF availability to the receptors, (the IGFBPs), and a group of IGFBP proteases which cleave IGFBBPs and modulate IGF action. Human seminal plasma contains IGF-I and -II, IGFBP-2 and -4, as well as IGFBP-3 fragments and IGFBP-3 protease activity. A prostatic source for these IGF-axis molecules is likely. We have demonstrated the human prostate to contain all the elements of a functional IGF system. Prostate <em>fibroblasts</em> in primary culture (PC-F) express mRNA for IGF-II and produce IGF-II peptide in biologically active concentrations. This IGF-II appears to be of a high molecular weight (<em>15</em> kD) species. Prostate epithelial cells in primary culture (PC-E) express the type I IGF receptor. These cells also produce IGFBP-2 and -4, (on both mRNA and peptide levels), while PC-F secrete IGFBP-2, -3 and -4. PC-E are exquisitely sensitive to the mitogenic effects of IGFs. Finally, prostate specific antigen (PSA), secreted from PC- and found in seminal plasma, can function as a potent IGFBP-3 protease. This IGFBP-3 protease activity can remove the inhibitory effects of IGFBP-3 on IGF-I induced PC-3 <em>growth</em>.(ABSTRACT TRUNCATED AT 250 WORDS)

Skeletal muscle is the largest organ determining whole-body insulin sensitivity and metabolic homoeostasis. Adaptive changes of skeletal muscle in response to physical activity include adjustments in the production and secretion of muscle-derived bioactive <em>factors</em>, known as myokines, such as myostatin, IL-4, IL-6, IL-7 and IL-<em>15</em>, myonectin, follistatin-like 1 or leukaemia inhibitory <em>factor</em>. These myokines not only act locally in the muscle in an autocrine/paracrine manner, but also are released to the bloodstream as endocrine <em>factors</em> to regulate physiological processes in other tissues. Irisin, derived from the cleavage of FNDC5 protein, constitutes a myokine that induces myogenesis and fat browning (switch of white adipocytes to brown fat-like cells) together with a concomitant increase in energy expenditure. Besides being a target for irisin actions, the adipose tissue also constitutes a production site of FNDC5. Interestingly, irisin secretion from subcutaneous and visceral fat depots is decreased by long-term exercise training and fasting, suggesting a discordant regulation of FNDC5/irisin in skeletal muscle and adipose tissue. Accordingly, our group has recently reported that the adipokine leptin differentially regulates FNDC5/irisin expression in skeletal muscle and fat, confirming the crosstalk between both tissues. Moreover, irisin secretion and function are regulated by other myokines, such as follistatin or myostatin, as well as by other adipokines, including <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 and leptin. Taken together, myokines have emerged as novel molecular mediators of fat browning and their activity can be modulated by adipokines, confirming the crosstalk between skeletal muscle and adipose tissue to regulate thermogenesis and energy expenditure.

BACKGROUND

Bile acid diarrhoea is a common, under-diagnosed cause of chronic watery diarrhoea, responding to specific treatment with bile acid sequestrants. We previously showed patients with bile acid diarrhoea have lower median levels compared with healthy controls, of the ileal hormone fibroblast growth factor 19 (FGF19), which regulates bile acid synthesis.

OBJECTIVE

To measure serum FGF19 and SeHCAT retention prospectively in patients with chronic diarrhoea.

METHODS

One hundred and fifty-two consecutive patients were grouped according to (75) Se-homocholic acid taurine (SeHCAT) 7-day retention: normal (>15%) in 72 (47%) diarrhoea controls; ≤15% in 54 (36%) with primary bile acid diarrhoea, and in 26 (17%) with secondary bile acid diarrhoea. Fasting blood was assayed for FGF19, 7α-hydroxy-4-cholesten-3-one (C4) and total bile acids.

RESULTS

FGF19 was significantly lower in the primary bile acid diarrhoea group compared with the diarrhoea control group (median 147 vs. 225 pg/mL, P < 0.001), and also in the secondary group (P < 0.006). FGF19 and SeHCAT values were positively correlated (rs = 0.44, P < 0.001); both were inversely related to C4. Other significant relationships included SeHCAT and body mass index (BMI)(P = 0.02), and FGF19 with age (P < 0.01). The negative and positive predictive values of FGF19 ≤ 145 pg/mL for a SeHCAT <10% were 82% and 61%, respectively, and were generally improved in an index including BMI, age and C4. In a subset of 28 primary patients, limited data suggested that FGF19 could predict response to sequestrant therapy.

CONCLUSIONS

Reduced fibroblast growth factor 19 is a feature of bile acid diarrhoea. Further studies will fully define its role in predicting the response of these patients to therapy.

OBJECTIVE

The ectopically expressed and deregulated <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) results from a t(4;14) chromosomal translocation that occurs in approximately <em>15</em>% of multiple myeloma (MM) patients and confers a particularly poor prognosis. This study assesses the antimyeloma activity of CHIR-258, a small-molecule inhibitor of multiple receptor tyrosine kinases that is currently in phase I trials, in a newly developed FGFR3-driven preclinical MM animal model.

METHODS

We developed an orthotopic MM model in mice using a luciferase-expressing human KMS-11-luc line that expresses mutant FGFR3 (Y373C). The antimyeloma activity of CHIR-258 was evaluated at doses that inhibited FGFR3 signaling in vivo in this FGFR3-driven animal model.

RESULTS

Noninvasive bioluminescence imaging detected MM lesions in nearly all mice injected with KMS-11-luc cells, which were mainly localized in the spine, skull, and pelvis, resulting in frequent development of paralysis. Daily oral administration of CHIR-258 at doses that inhibited FGFR3 signaling in KMS-11-luc tumors in vivo resulted in a significant inhibition of KMS-11-luc tumor growth, which translated into a significant improvement in animal survival.

CONCLUSIONS

Our data provide a relevant preclinical basis for clinical trials of CHIR-258 in FGFR3-positive MM patients.

Phosphaturic mesenchymal tumours (PMTs) are uncommon soft tissue and bone tumours that typically cause hypophosphataemia and tumour-induced osteomalacia (TIO) through secretion of phosphatonins including <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23). PMT has recently been accepted by the World Health Organization as a formal tumour entity. The genetic basis and oncogenic pathways underlying its tumourigenesis remain obscure. In this study, we identified a novel FN1-FGFR1 fusion gene in three out of four PMTs by next-generation RNA sequencing. The fusion transcripts and proteins were subsequently confirmed with RT-PCR and western blotting. Fluorescence in situ hybridization analysis showed six cases with FN1-FGFR1 fusion out of an additional 11 PMTs. Overall, nine out of <em>15</em> PMTs (60%) harboured this fusion. The FN1 gene possibly provides its constitutively active promoter and the encoded protein's oligomerization domains to overexpress and facilitate the activation of the FGFR1 kinase domain. Interestingly, unlike the prototypical leukaemia-inducing FGFR1 fusion genes, which are ligand-independent, the FN1-FGFR1 chimeric protein was predicted to preserve its ligand-binding domains, suggesting an advantage of the presence of its ligands (such as FGF23 secreted at high levels by the tumour) in the activation of the chimeric receptor tyrosine kinase, thus effecting an autocrine or a paracrine mechanism of tumourigenesis.

Many <em>growth</em> <em>factors</em> and cytokines bind to more than one receptor, but in many cases the different roles of the separate receptors in signal transduction are unclear. Intracellular sorting of ligand-receptor complexes may modulate the signalling, and we have here studied the intracellular trafficking of ligand bound to receptors for <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs). For this purpose, we transfected HeLa cells with any one of the four tyrosine kinase FGF receptors (FGFR1-4). In cells expressing any one of these receptors, externally added FGF1 was localized to sorting/early endosomes after <em>15</em> minutes at 37 degrees C. After longer incubation times, FGF1 internalized in cells expressing FGFR1 was localized mainly to late endosomes/lysosomes, similarly to EGF. By contrast, FGF1 internalized in cells expressing FGFR4 followed largely the same intracellular pathway as the recycling ligand, transferrin. In cells expressing FGFR2 or FGFR3, sorting of FGF1 to lysosomes was somewhat less efficient than that observed for FGFR1. Furthermore, FGF1 was more slowly degraded in cells expressing FGFR4 than in cells expressing FGFR1-3 and in addition, internalized FGFR4 as such was more slowly degraded than the other receptors. The data indicate that after endocytosis, FGFR4 and its bound ligand are sorted mainly to the recycling compartment, whereas FGFR1-3 with ligand are sorted mainly to degradation in the lysosomes. Alignment of the amino acid sequence of the intracellular part of the four FGFRs revealed several lysines conserved in FGFR1-3 but absent in FGFR4. Lysines are potential ubiquitylation sites and could thus target a receptor to lysosomes for degradation. Indeed, we found that FGFR4 is less ubiquitylated than FGFR1, which could be the reason for the different sorting of the receptors.

A proportion of chronic wounds fail to heal in response to standard therapy. For venous leg ulcers, a correlation exists between longer duration before treatment initiation and poor healing response to compression therapy. Differences identified between the healing wound microenvironment and that of the non healing chronic wound suggests that many potential mechanisms exist to impair healing. One contributory mechanism may be inhibition of <em>fibroblast</em> proliferation and induction of a stress-induced premature senescence phenotype by the continuing inflammation found in chronic wounds. Senescent <em>fibroblasts</em> exhibit an extracellular matrix degradative phenotype that contributes to wound chronicity. Accumulation of greater than <em>15</em>% senescent <em>fibroblasts</em> has been described as a threshold beyond which wounds become hard to heal. The ratio of senescent : non senescent cells is therefore critical to determining response to treatment, and adjunctive therapies that modulate this ratio in favour of non senescent cells are likely to enhance therapeutic healing rates. A number of tissue-engineered dermal replacements contain non senescent <em>fibroblasts</em> and can donate cells to the wound environment additional to releasing <em>growth</em> <em>factors</em> and reversing the antiproliferative activity of chronic wound exudate. Recognition of the role of <em>fibroblast</em> senescence in wound chronicity may allow for identification of those wounds that will respond positively to these products.

Collagen type I serves as an abundant structural and signalling component of skin. It is also an established target gene of the transcription <em>factor</em>, c-Myb. When c-myb-/- embryos were examined it was observed that their skin was markedly thinner than normal. Importantly, immunohistochemical investigation showed complete absence of collagen type I. Although these homozygous knock-out embryos fail to develop beyond day <em>15</em>, <em>fibroblasts</em> established from these embryos (mouse embryonic <em>fibroblasts</em> [MEFs]) show defective proliferative responses. Furthermore, in vitro scratch wound assays demonstrated that these c-myb-/- MEFs also exhibit slower closure than their wild-type counterparts. Embryonic lethality has meant that examination of the role of c-Myb in adult mouse skin has not been reported to date. However, in view of the abundance of collagen type I in normal skin, its role in skin integrity and the in vitro data showing proliferative and migration defects in c-myb-/- MEFs, we investigated the consequences of heterozygous c-myb loss in adult mice on the complex process of skin repair in response to injury. Our studies clearly demonstrate that heterozygous c-myb deficiency has a functional effect on wound repair, collagen type I levels and, in response to wounding, transforming <em>growth</em> <em>factor</em>-beta1 (an important collagen stimulating <em>factor</em>) induction expression is aberrantly high. Manipulation of c-Myb may therefore provide new therapeutic opportunities for improving wound repair while uncontrolled expression may underpin some fibrotic disorders.

OBJECTIVE

Angiotensin II (Ang II) induces fibroblast proliferation and collagen synthesis in the myocardium, but its precise mechanisms of action in human hearts are still unknown. Therefore, we investigated whether Ang II directly affects the collagen mRNA content in the human myocardium and in isolated human cardiac fibroblasts or whether the growth factors TGFbeta-1 and osteopontin are involved in this process.

UNASSIGNED

In a first set of experiments, the direct effect of Ang II on collagen I, TGFbeta-1 and osteopontin mRNA content in fresh samples of human atrial myocardium was determined by the use of a short stimulation period. After 4 h, Ang II-stimulated atrial samples gave a significantly higher expression of both TGFbeta-1 (183+/-21% of control, p<0.05) and osteopontin mRNA (275+/-58%, p<0.02) than the controls. In contrast, the expression of collagen I mRNA was unchanged (95+/-8%). Stimulation with TGFbeta-1 led to an increase in collagen I and III mRNA (127+/-10%, p<0.05; 140+/-15%, p<0.02).

UNASSIGNED

In a second protocol, to assess the effects of longer stimulation periods, we determined the effects of Ang II and its potential mediator TGFbeta-1 on collagen I, III and fibronectin mRNA expression and on proliferation of cultured human cardiac fibroblasts. Ang II caused a dose-dependent stimulation of proliferation but did not affect collagen I, II or fibronectin mRNA content after 24 h. In contrast, TGFbeta-1 stimulation significantly increased collagen I and III mRNA expression (124+/-5% and 128+/-5%, p<0.002).

CONCLUSIONS

In the human heart, Ang II does not directly increase collagen or fibronectin mRNA, but it does increase TGFbeta-1 and osteopontin mRNA expression. Since TGFbeta-1 induces collagen I and III mRNA in atrial samples and in isolated cardiac fibroblasts, it may represent a necessary mediator of the Ang II effects in the human heart.