Atypical <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF) <em>21</em> and 19 play a central role in energy metabolism through the mediation of Klotho coreceptor. Contradictory findings are available about the association of FGF<em>21</em> and FGF19 with nonalcoholic fatty liver disease (NAFLD) in humans. We investigated the association of serum FGF<em>21</em>, FGF19 and liver Klotho coreceptor with non-alcoholic steatohepatitis (NASH) and fibrosis in children with NAFLD. Serum FGF<em>21</em> and FGF19 were measured in 84 children with biopsy-proven NAFLD and 23 controls (CTRL). The hepatic expression of Klotho coreceptor was measured in 7 CTRL, 9 patients with NASH (NASH+) and 11 patients without NASH (NASH-). FGF<em>21</em> and FGF19 showed a tendency to decrease from CTRL (median FGF<em>21</em> = 196 pg/mL; median FGF19 = 201 pg/mL) to NASH- (FGF<em>21</em> = 89 pg/mL; FGF19 = 81 pg/mL) to NASH+ patients (FGF<em>21</em> = 54 pg/mL; FGF19 = 41 pg/mL) (p<0.001 for all comparisons) and were inversely associated with the probability of NASH and fibrosis in children with NAFLD. The hepatic expression of Klotho coreceptor was inversely associated with NASH (R(2) = 0.87, p<0.0001) and directly associated with serum FGF<em>21</em> (R(2) = 0.57, p<0.0001) and FGF19 (R(2) = 0.67, p<0.0001). In conclusion, serum FGF19 and FGF<em>21</em> and hepatic Klotho expression are inversely associated with hepatic damage in children with NAFLD and these findings may have important implications for understanding the mechanisms of NAFLD progression.

Skeletal muscle has a pleiotropic role in organismal energy metabolism, for example, by storing protein as an energy source, or by excreting endocrine hormones. Muscle proteolysis is tightly controlled by the hypothalamus-pituitary-adrenal signalling axis via a glucocorticoid-driven transcriptional programme. Here we unravel the physiological significance of this catabolic process using skeletal muscle-specific glucocorticoid receptor (GR) knockout (GRmKO) mice. These mice have increased muscle mass but smaller adipose tissues. Metabolically, GRmKO mice show a drastic shift of energy utilization and storage in muscle, liver and adipose tissues. We demonstrate that the resulting depletion of plasma alanine serves as a cue to increase plasma levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) and activates liver-fat communication, leading to the activation of lipolytic genes in adipose tissues. We propose that this skeletal muscle-liver-fat signalling axis may serve as a target for the development of therapies against various metabolic diseases, including obesity.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has beneficial effects on glucose homeostasis and insulin sensitivity. In the current study, we investigated serum concentrations of FGF<em>21</em> in patients with gestational diabetes mellitus (GDM) as compared with healthy pregnant controls matched for gestational age and fasting insulin. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> was determined by enzyme-linked immunosorbent assay in control (n = 80) and GDM (n = 40) patients and correlated to clinical and biochemical measures of renal function, glucose and lipid metabolism, as well as inflammation in both groups. Median maternal serum FGF<em>21</em> concentrations were not significantly different in subjects with GDM (97.5 ng/L) as compared with healthy pregnant controls (102.9 ng/L). <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> significantly and positively correlated with markers of insulin resistance (increased homeostasis model assessment of insulin resistance, decreased adiponectin) and dyslipidemia (increased triglycerides, decreased high-density lipoprotein cholesterol) in univariate and multivariate analyses. Furthermore, FGF<em>21</em> serum levels were highest in patients in the third tertile of homeostasis model assessment of insulin resistance. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> is independently associated with markers of insulin resistance and an adverse lipid profile but is not dysregulated in GDM if patients are matched with controls for fasting insulin.

OBJECTIVE

The aim of this study was to evaluate the potential use of serum transforming <em>growth</em> <em>factor</em>-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 (TIMP-1), fetuin-A, and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) in the detection of liver fibrosis in patients with chronic hepatitis B (CHB). The value of the noninvasive fibrosis models - that is, the aspartate aminotransferase to platelet ratio index (APRI), the fibrosis index based on the four <em>factors</em> (FIB-4) score, and Forn's index - was also examined.

METHODS

CHB patients who underwent liver biopsy for the evaluation of fibrosis were included in the study. A total of 73 patients were divided into two groups according to their METAVIR scores (F0-1, no/minimal fibrosis; F2-4, significant fibrosis). Serum levels of TGF-β1, TIMP-1, fetuin-A, and FGF<em>21</em> were measured besides APRI, FIB-4, and Forn's scores. The area under the receiver operating characteristic curve was measured for each parameter, followed by calculation of sensitivity, specificity, and positive and negative predictive values.

RESULTS

APRI, FIB-4, and Forn's index scores were significantly higher in patients with significant fibrosis (P<0.05). There was no difference between no/minimal fibrosis and significant fibrosis groups in terms of serum levels of TGFβ-1, TIMP-1, fetuin-A, and FGF<em>21</em> (P>0.05). The areas under the receiver operating characteristic curve for TGF-β1, TIMP-1, fetuin-A, FGF<em>21</em>, APRI, FIB-4, and Forn's index were 0.445, 0.483, 0.436, 0.585, 0.662, 0.687, and 0.680, respectively.

CONCLUSIONS

Our results suggest that serum TGF-β1, TIMP-1, fetuin-A, and FGF<em>21</em> are not useful for the assessment of the extent of liver fibrosis in CHB in this patient group. However, APRI, FIB-4, and Forn's index have a better diagnostic value in patients with significant fibrosis than in those with no/minimal fibrosis.

BACKGROUND

Retinoic acid (RA) is present in the chick limb bud, and excess RA induces limb duplications. Here, we have investigated the role of endogenous RA during chick limb development by preventing the synthesis of RA and testing the effect on various genes expressed during limb initiation and outgrowth.

RESULTS

We demonstrate that the stage 20/<em>21</em> limb bud synthesizes didehydroretinoic acid (ddRA), and that the posterior half of the limb bud synthesizes ddRA at a higher rate than the anterior half. Disulphiram inhibits this synthesis at micromolar concentrations. Administering disulphiram to embryos prior to limb bud out<em>growth</em> (stages 12-18) abolishes out<em>growth</em>, and no limb develops in the majority of cases. Disulphiram treatment also prevents the expression of Sonic hedgehog (Shh), but the expression of the <em>fibroblast</em> <em>growth</em> <em>factor</em>-8 gene (Fgf-8) appears as normal in the ectoderm over the prospective limb bud. The application of a bead soaked in RA can rescue Shh expression. Disulphiram treatment of later limb buds (stages 20-23) similarly down-regulates Shh, and also Fgf-4, expression, whereas the expression of Fgf-8, as at earlier stages, is initially unaffected. Again, RA can rescue the expression of Shh in these limb buds.

CONCLUSIONS

RA, in conjunction with Fgf-8, may be needed for the induction of the chick limb bud and the induction of Shh and Fgf-4 expression. The expression of Shh and Fgf-4 remains dependent upon the continued synthesis of RA within the limb bud. Didehydroretinoic acid is the major active retinoid in the stage 20 chick limb bud.

Drugs that inhibit <em>growth</em> of tumours and their blood supply could have considerable therapeutic potential. 2-Methoxyoestradiol-3,17-O,O-bis-sulphamate (2-MeOE2bisMATE) has been shown to inhibit the proliferation of MCF-7 (ER+) breast cancer cells and angiogenesis in vitro. 2-MeOE2bisMATE and its analogue, 17-Cym-2-MeOE2MATE, were investigated for their ability to inhibit in vivo angiogenesis and tumour <em>growth</em>. The mouse Matrigel plug assay for angiogenesis was used to investigate the effect of compounds on neovascularisation and was quantified using a FITC-dextran injection technique. Nude mice bearing tumours derived from MCF-7 cells were used to assess efficacy on tumour <em>growth</em>. Tumour sections were stained for VEGFR-2 and Ki67 to assess tumour angiogenesis and cell proliferation respectively. Matrigel plugs supplemented with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> resulted in increased neovascularisation over 7 days. Oral administration of 2-MeOE2bisMATE for 7 days at 10 or 50 mg kg(-1) significantly reduced neovascularisation to or below control levels respectively. 17-Cym-2-MeOE2MATE at 20 mg kg(-1) was equally effective. 2-MeOE2bisMATE, dosed daily for <em>21</em> days, caused a 52% reduction in tumour <em>growth</em> at 5 mg kg(-1) and 38% regression at 20 mg kg(-1). 17-Cym-2-MeOE2MATE (20 mg kg(-1)) reduced tumour <em>growth</em> by 92%. Immunohistochemistry revealed a reduction in angiogenesis and proliferation. Matrigel plug and tumour imaging after FITC-dextran injection indicated that 2-MeOE2bisMATE caused a marked disruption of vasculature. These sulphamoylated oestrogen derivatives have been shown to be potent inhibitors of angiogenesis in vivo. This, together with their ability to inhibit tumour <em>growth</em>, indicates the potential of this new class of drugs for further development for cancer therapy.

BACKGROUND

Thermal ablation by radiofrequency or laser is used increasingly for the treatment of colorectal liver metastases. Recurrence after thermal ablation is common and occurs both locally and at distant sites. One possible cause of this recurrence may be a result of growth stimulation of micrometastases in the remaining liver. This study examined the impact of thermal ablation on growth patterns of hepatic micrometastases.

METHODS

Colorectal liver metastases were induced in male CBA-strain mice via an intrasplenic injection of a murine-derived cancer cell line. Subtotal thermal ablation of the left posterior lobe of the liver (30% of total liver volume) was performed by neodymium yttrium-aluminum-garnet laser 7 days after induction of metastases. The distribution, number, cross-sectional diameter, volume, and proliferation rate of established neoplasms were compared with controls at 21 days after tumor induction. The effect of thermal ablation of 7% of the total liver volume by laser on the expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor 2 (FGF-2), transforming growth factor beta, and cellular proliferation (Ki-67 antigen) adjacent to the ablated site was assessed by immunohistochemistry in separate groups of animals at specific time points after therapy.

RESULTS

Thermal ablation did not alter the overall volume, number, size, and proliferation rate of neoplasms 21 days after laser ablation. There were no extrahepatic metastases after therapy. The number of neoplasms in the regenerated posterior lobe was equivalent to control despite subtotal ablation (29 +/- 2 vs 27 +/- 2; P = NS). A greater amount of metastases occupied the regenerated thermal-ablated lobe compared with controls (55% +/- 4% vs 29% +/- 3%; P < .04). Thermal ablation stimulated liver proliferation adjacent to the treatment site at 12 hours compared with untreated controls. Stimulation peaked at 72 hours (20% +/- 1% vs 1% +/- 1%; P < .001) and persisted to 21 days after therapy. FGF-2 and VEGF expression increased in liver tissue adjacent to the ablation site compared with baseline, peaking at 12 hours (112% +/- 2% vs 102% +/- 1%; P < .001) and 72 hours (114% +/- 2% vs 101% +/- 1%; P < .001), respectively.

CONCLUSIONS

Thermal ablation promotes the progression of micrometastases to form macroscopically detectable neoplasms in treated regenerating liver. This effect may relate to an increased expression of VEGF and FGF-2 adjacent to the treatment site.

Studies on the direct effects of hormones and <em>growth</em> <em>factors</em> on bone alkaline phosphatase have been limited to parathyroid hormone (PTH) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and have not been compared to other parameters of bone formation. Insulin, PTH, 1,25(OH)2D3, epidermal and <em>fibroblast</em> <em>growth</em> <em>factors</em> (EGF, FGF) were examined for their effects on alkaline phosphatase activity and type I, [alpha 1 (I)]2 alpha 2, collagen synthesis in cultures of <em>21</em>-day fetal rat calvariae. After 24 hr and 96 hr of treatment, insulin increased whereas PTH, 1,25(OH)2D3, EGF and FGF inhibited calvarial alkaline phosphatase activity and the incorporation of 3H-proline into collagenase-digestible protein and type I collagen. The agents tested did not affect the release of alkaline phosphatase into the culture medium. Although type I collagen was the only collagen detected, a small amount of another collagen might have been also synthesized. The hormonal effects on alkaline phosphatase activity and type I collagen synthesis were of greater magnitude after 96 hr than after 24 hr of continuous exposure to the agents tested and the two parameters correlated well (r = 0.88 after 96 hr and r = 0.97 after 24 hr of treatment. These studies indicate that insulin increases bone alkaline phosphatase activity and type I collagen synthesis in calvariae whereas PTH, 1,25(OH)2D3, EGF and FGF have an inhibitory effect. The results suggest that these agents affect osteoblastic function.

Culturing cells in 3-dimensional (3D) systems is important in tissue engineering and in fundamental studies of cellular mechanisms that are sensitive or specific to the 3D environment. To guide the engineering of artificial vocal fold lamina propria tissue, we developed 3D cultures containing human vocal fold <em>fibroblasts</em> (hVFFs) dispersed in a synthetic peptide hydrogel matrix. <em>Growth</em> <em>factors</em> were added to the culture to examine their influence on extracellular matrix (ECM) synthesis, cell proliferation, and matrix contraction. The hVFF-hydrogel constructs were treated with transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta1), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), or hepatocyte <em>growth</em> <em>factor</em> (HGF), and the culture was maintained for <em>21</em> days. TGF-beta1 induced matrix contraction and enhanced collagen and sulfated glycosaminoglycan production, bFGF effectively increased cell proliferation, and HGF stimulated synthesis of hyaluronic acid and elastin with less collagen accumulation than other conditions. Of the <em>growth</em> <em>factors</em> tested, HGF appears to be most useful for stimulating essential tissue components for restoring vocal fold pliability. The results also suggest that multiple <em>growth</em> <em>factors</em> might be employed sequentially or in combination to program the makeup of cell-hydrogel constructs for vocal fold tissue repair.

We hypothesized that human ocular surface squamous neoplasia (OSSN) may result from the continuous <em>growth</em> stimulation of corneal epithelial progenitor cells. In the present study, we analyzed the effects of excess <em>fibroblast</em> <em>growth</em> <em>factor</em>-7 (FGF-7) on both the proliferation and differentiation of corneal epithelium in a novel Krt12-rtTA/tet-O-FGF-7 double transgenic mouse model in which cornea-specific FGF-7 overexpression is achieved by doxycycline (Dox) treatment. When such adult mice were exposed to Dox, they exhibited epithelial hyperplasia with increases in phospho-extracellular signal-regulated kinase 1/2-, nuclear beta-catenin-, and 5-bromo-2'-deoxyuridine-labeled cells and altered keratin (K) 14 (K14) expression pattern, a normal K12 expression pattern, and the normal absence of K10. Hyperplasia of the adult cornea was fully reversible 2 weeks after the removal of Dox from chow. In contrast, double transgenic embryos that were exposed to Dox from embryonic day 0.5 to postnatal day <em>21</em> developed papillomatous tumors in the cornea, resembling human OSSN, and ectopic gland-like structures in the limbus, accompanied by the down-regulation of K12 and the up-regulation of K14, Pax6, and p63. These epithelial anomalies observed in young experimental mice were not fully resolved after the termination of Dox induction. Taken together, Krt12-rtTA/tet-O-FGF-7 mice may be a suitable animal model for the study of the molecular and cellular mechanisms of human OSSN.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGF) regulate bone <em>growth</em>, but their expression in human cartilage is unclear. Here, we determined the expression of entire FGF family in human fetal <em>growth</em> plate cartilage. Using reverse transcriptase PCR, the transcripts for FGF1, 2, 5, 8-14, 16-19, and <em>21</em> were found. However, only FGF1, 2, 17, and 19 were detectable at the protein level. By immunohistochemistry, FGF17 and 19 were uniformly expressed within the <em>growth</em> plate. In contrast, FGF1 was found only in proliferating and hypertrophic chondrocytes whereas FGF2 localized predominantly to the resting and proliferating cartilage. In addition, only the 18 kD isoform of FGF2 was found in resting chondrocytes while proliferating chondrocytes also synthesized 22 kD and 24 kD FGF2, similar to in vitro cultivated chondrocytes. In cell <em>growth</em> experiments, FGF1, 2, and 17 but not FGF19 inhibited the proliferation of FGFR3-expressing rat chondrosarcoma chondrocytes (RCS) with relative potency FGF2>>) FGF1 = FGF17. We conclude that FGF1, 2, 17, and 19 are the predominant FGF ligands present in developing human cartilage that are, with the exception of FGF19, experimentally capable of inhibiting chondrocyte proliferation.

Large tympanic membrane perforations usually require a surgical tympanoplasty for closure. Reducing surgical costs and risks has encouraged investigators to examine nonsurgical office procedures for healing these perforations. <em>Growth</em> accelerators are the most promising agents. We study here the closure of large acute perforations using weekly applications of 1 mg of 1% hyaluronic acid (HA), 0.4 microg basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), or 1.0 microg epidermal <em>growth</em> <em>factor</em> (EGF) directly to the tympanic membranes of the experimental ears. Control ears were treated with 0. 1 mL Vasocidin. Complete closure was obtained in 100% of the ears treated with HA and EGF and 85.7% of those treated with bFGF by day <em>21</em>, compared with 63.6% of the controls by day 32. Moderate-to-severe ipsilateral and contralateral external canal hypertrophy was noted in 14.2% and 37.5% of the ears treated with bFGF and HA, respectively, but was not seen in ears treated with EGF or in the control group.

Three-dimensional (3D) collagen hydrogels have been extensively used for cell culture experiments and are more closely representative of in vivo conditions than monolayer (2D) culture. Here we cultured rat bone marrow-derived mesenchymal stem cells (MSCs) in collagen hydrogels containing varying concentrations of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) to examine the effect of bFGF on MSC proliferation and osteogenic differentiation in 3D culture. The optimal bFGF concentration that promoted the greatest degree of cell proliferation and expression of the early osteogenic induction marker alkaline phosphatase was also determined. Subsequent quantitative real-time polymerase chain reaction analysis of gene expression demonstrated that bFGF promoted significant upregulation of the bone-related genes: collagen type I, osteopontin (OPN), bone sialoprotein (BSP), and osteocalcin (OCN) for periods of up to <em>21</em> days. Immunofluorescence staining and fluorescence-activated cell sorting analysis further supported the enhanced osteogenic differentiation of cells as a greater proportion of cells were found to express OPN. Matrix mineralization within the collagen hydrogels was enhanced in the presence of bFGF, as assessed by calcium detection using von Kossa staining. These results clearly demonstrate a positive effect of bFGF on proliferation and osteogenic induction of MSCs in 3D collagen hydrogels when applied at the appropriate concentration. Moreover, collagen hydrogel constructs containing MSCs and appropriate <em>growth</em> <em>factor</em> stimulus might be a potentially useful biological tool for 3D bone tissue engineering.

The pro-inflammation <em>factor</em> high-mobility group box protein 1 (HMGB1) has been implicated in the pathogenesis of asthma. In this study, we used a murine model of chronic asthma to evaluate the effects of HMGB1 on airway remodeling. Female BALB/c mice were randomly divided into four groups: control, ovalbumin (OVA) asthmatic, OVA+isotype antibody and OVA+anti-HMGB1 antibody. Anti-HMGB1 antibody therapy was started on day <em>21</em> and was administered three times per week for 6 weeks before intranasal challenge with OVA. In this mouse model, HMGB1 expression is significantly elevated. The anti-HMGB1 antibody group exhibited decreased levels of immunoglobulin E (IgE) and inflammatory mediators and reduced inflammatory cell accumulation, airway hyperresponsiveness (AHR), mucus synthesis, smooth muscle thickness and lung collagen content compared with the OVA groups. Treatment with HMGB1 increased proliferation, migration, collagen secretion and α-smooth muscle actin (SMA) expression in MRC-5 cells. Treatment with the HMGB1/IL-1β complex significantly increased the expression and secretion of transforming <em>growth</em> <em>factor</em> (TGF-β1), matrix metalloproteinase (MMP)-9 and vascular endothelial <em>growth</em> <em>factor</em> (VEGF). Altogether, these results suggest that blocking HMGB1 activity may reverse airway remodeling by suppressing airway inflammation and modulating lung <em>fibroblast</em> phenotype and activation.

OBJECTIVE

To investigate neurological effects of transplanting bone marrow-derived mesenchymal stem cells (BMSCs) transfected with the basic fibroblast growth factor (bFGF) gene in spinal cord-injured rats.

METHODS

Ninety-six male adult Sprague-Dawley rats were randomized into four groups: (1) pcDNA3.1-bFGF group; (2) pcDNA3.1 group; (3) BMSCs group; and (4) vehicle control (DMEM) group. After the rat model of acute spinal cord injury (SCI) was established, 1×10(6) BMSCs or cells transfected with pcDNA3.1-bFGF or pcDNA3.1 were injected into rats of groups 1-3. At days 1, 7, 14, and 21 after injection, the Basso-Beattie-Bresnahan (BBB) locomotor rating scale was used to evaluate recovery of motor function. Expression changes of bFGF, myelin basic protein (MBP), and NF200 were examined by immunohistochemistry.

RESULTS

The BBB score of DMEM group was significantly lower than those of groups 1-3 (P<0.05), but the score of pcDNA3.1-bFGF group was significantly higher than that of BMSCs group or pcDNA3.1 group at day 14 or 21 after injection (P<0.01). The number of bFGF-positive neurons in rats of pcDNA3.1-bFGF group was significantly higher than those of groups 1-3 at any time point (P<0.05). The optical density values of NF200-positive neurons and MBP-positive MBP axons in rats of pcDNA3.1-bFGF group were significantly higher than those of groups 1-3 at day 7 or 14 after injection (P<0.05).

CONCLUSIONS

bFGF gene-modified BMSCs not only effectively promoted axonal outgrowth but also enhanced recovery of neurological function after SCI in rats, and may be a good candidate to evaluate gene therapy of SCI in man.

The idea that the gene(s) that cause diabetes mellitus can be expressed in extrapancreatic cells has been examined by tissue culture techniques. Skin biopsies were obtained from 25 normal subjects (N), 26 overt diabetics (D), 16 of juvenile onset (JOD) and 9 of maturity onset (MOD), and <em>21</em> subjects genetically predisposed to diabetes (P) on the basis of maturity-onset diabetes in both parents. Each biopsy was subdivided, multiple skin fragments were explanted in vitro, and several parameters of cellular out<em>growth</em> were monitored in primary and secondary cultures until cell division ceased because of senescence. In general, the rank order of <em>growth</em> vigor was N greater than P greater than D although differences were often marginal and statistically significant between N and JOD and(or) MOD. Out<em>growth</em> of epithelial cells was more vigorous in N explants in early stages, but later, JOD and MOD cells grew better than those of N. Out<em>growth</em> of <em>fibroblast</em> cells from N explants was more vigorous both at early and later stages and required less time to achieve maximum percent out<em>growth</em>. In secondary cultures, N cells grew faster than the other three groups so that fewer days elapsed between subcultures but significant differences were only seen between N and one or two of the other groups over some of the first seven subcultures. The onset of cellular senescence occurred earlier in P and JOD cultures both in mean population doublings and calendar time. N cultures had a higher percent surviving clones after picking than MOD, and a shorter recloning time than clones of JOD. The replicative life-spans of cultures (mean population doublings +/- SE) were N = 52.54 +/- 2.24, P = 47.84 +/- 2.43, JOD = 47.12 +/- 2.99, and MOD = 46.40 +/- 4.04, but differences did not reach significance for N vs the other three groups. The data demonstrate that cellular <em>growth</em> is impaired in both JOD and MOD types of cultures and to a generally lesser extent in P cultures. This is consistent with intrinsic genetic defects but the possibility that persistent deleterious effects of in vivo pathophysiology contribute alone or in combination cannot be ruled out. Therefore, the diabetic defect(s) can be expressed in extrapancreatic cells of mesenchymal origin. This system should prove useful in exploring the interplay between genetic and environmental <em>factors</em> in diabetes, the mechanisms(s) of hyperglycemia and other metabolic derangements, and the propensity that affected individuals have to develop degenerative diseases.

Formation of new capillary blood vessels, termed angiogenesis, is essential for the <em>growth</em> and development of tissues and underlies a variety of diseases including tumor <em>growth</em>. Members of the prolactin hormonal family bind to endothelial cell receptors and have direct effects on cell proliferation, migration and tube formation. Because many angiogenic and antiangiogenic <em>factors</em> are produced by endothelial cells, we investigated whether endothelial cells expressed the prolactin gene. Here we show that bovine brain capillary endothelial cells (BBCEC) in culture express the full-length prolactin messenger RNA, in addition to a novel prolactin transcript, lacking the third exon of the gene. In addition cultures of BBCEC synthesize and secrete prolactin-like immunoreactive proteins with apparent molecular masses of 23, <em>21</em> and 14 kDa. The prolactin-like nature of these proteins in supported by the observation that Nb2-cells, a prolactin-responsive cell line, were stimulated to proliferate when co-cultured with endothelial cells and this stimulation was neutralized with prolactin-directed antibodies. Finally, consistent with a possible autocrine effect of endothelial-derived prolactins, polyclonal and monoclonal prolactin antibodies specifically inhibited basal and basis <em>fibroblast</em> <em>growth</em>-<em>factor</em>-stimulated <em>growth</em> of endothelial cells. Taken together, the present findings support the hypothesis of the prolactin gene being expressed in endothelial cells as proteins that could act in an autocrine fashion to regulate cell proliferation.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a metabolic <em>factor</em> involved in glucose and lipid metabolism. However, little is known about the physiological role of FGF<em>21</em> during a dietary challenge in humans.

METHODS

Twenty healthy low birth weight (LBW) with known risk of type 2 diabetes and 26 control (normal birth weight (NBW)) young men were subjected to 5 days of high-fat (HF) overfeeding (+50%). Basal and clamp insulin-stimulated serum FGF<em>21</em> levels were examined before and after the diet, and FGF<em>21</em> mRNA expression was measured in muscle and fat biopsies respectively.

RESULTS

Five days of HF overfeeding diet significantly (P<0.001) increased fasting serum FGF<em>21</em> levels in both the groups (P<0.001). Furthermore, insulin infusion additionally increased serum FGF<em>21</em> levels to a similar extent in both the groups. Basal mRNA expression of FGF<em>21</em> in muscle was near the detection limit and not present in fat in both the groups before and after the dietary challenge. However, insulin significantly (P<0.001) increased FGF<em>21</em> mRNA in both muscle and fat in both the groups during both diets.

CONCLUSIONS

Short-term HF overfeeding markedly increased serum FGF<em>21</em> levels in healthy young men with and without LBW but failed to increase muscle or fat FGF<em>21</em> mRNA levels. This suggests that the liver may be responsible for the rise of serum FGF<em>21</em> levels during overfeeding. In contrast, the increase in serum FGF<em>21</em> levels during insulin infusion may arise from increased transcription in muscle and fat. We speculate that increased serum FGF<em>21</em> levels during HF overfeeding may be a compensatory response to increase fatty acid oxidation and energy expenditure.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has a significant role in the regulation of energy metabolism, e.g., in the control of systemic glucose and lipid metabolism. For instance, FGF<em>21</em> enhances insulin sensitivity, increases glucose uptake, and thus can decrease serum hyperglycemia, while it also increases lipid oxidation and inhibits lipogenesis. AMP-activated protein kinase (AMPK) is a tissue energy sensor involved in maintaining the energy balance and tissue integrity. It is known that AMPK signaling generates an energy metabolic profile which displays a remarkable overlap with that of FGF<em>21</em>. There is convincing evidence that endocrine FGF<em>21</em> signaling activates the AMPK pathway, either directly through FGFR1/β-klotho signaling or indirectly by stimulating the secretion of adiponectin and corticosteroids, which consequently can activate AMPK signaling in their target tissues. By activating AMPK, FGF<em>21</em> can promote a healthy aging process and thus extend mammalian lifespan. We will examine the signaling mechanisms through which FGF<em>21</em> can activate the AMPK pathway and then discuss the significance of the close connection between FGF<em>21</em> and AMPK signaling in the control of metabolic disorders and the aging process.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em> belongs to a hormone-like subgroup within the FGF superfamily. The members of this subfamily, FGF19, FGF<em>21</em> and FGF23, are characterized by their reduced binding affinity for heparin that enables them to be transported in the circulation and function in an endocrine manner. It is likely that FGF<em>21</em> also acts in an autocrine and paracrine fashion, as multiple organs can produce this protein and its plasma concentration seems to be below the level necessary to induce a pharmacological effect. FGF<em>21</em> signals via FGF receptors, but for efficient receptor engagement it requires a co<em>factor</em>, membrane-spanning βKlotho (KLB). The regulation of glucose uptake in adipocytes was the initial biological activity ascribed to FGF<em>21</em>, but this hormone is now recognized to stimulate many other pathways in vitro and display multiple pharmacological effects in metabolically compromised animals and humans. Understanding of the precise physiology of FGF<em>21</em> and its potential medicinal role has evolved exponentially over the last decade, yet numerous aspects remain to be defined and others are a source of debate. Here we provide a historical overview of the advances in FGF<em>21</em> biology focusing on the uncertainties in the mechanism of action as well as the differing viewpoints relating to this intriguing protein.

OBJECTIVE

Serum concentrations of the hepatokine <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em> are elevated in obesity, type-2 diabetes, and the metabolic syndrome. We asked whether FGF<em>21</em> levels differ between subjects with metabolically healthy vs. unhealthy obesity (MHO vs. MUHO), opening the possibility that FGF<em>21</em> is a cross-talker between liver and adipose tissue in MUHO. Furthermore, we studied the effects of chronic FGF<em>21</em> treatment on adipocyte differentiation, lipid storage, and adipokine secretion.

METHODS

In 20 morbidly obese donors of abdominal subcutaneous fat biopsies discordant for their whole-body insulin sensitivity (hereby classified as MHO or MUHO subjects), serum FGF<em>21</em> was quantified. The impact of chronic FGF<em>21</em> treatment on differentiation, lipid accumulation, and adipokine release was assessed in isolated preadipocytes differentiated in vitro.

RESULTS

Serum FGF<em>21</em> concentrations were more than two-fold higher in MUHO as compared to MHO subjects (457 ± 378 vs. <em>21</em>1 ± 123 pg/mL; p < 0.05). FGF<em>21</em> treatment of human preadipocytes for the entire differentiation period was modestly lipogenic (+15%; p < 0.05), reduced the expression of key adipogenic transcription <em>factor</em>s (PPARG and CEBPA, -15% and -40%, respectively; p < 0.01 both), reduced adiponectin expression (-20%; p < 0.05), markedly reduced adiponectin release (-60%; p < 0.01), and substantially increased leptin (+60%; p < 0.01) and interleukin-6 (+50%; p < 0.001) release.

CONCLUSIONS

The hepatokine FGF<em>21</em> exerts weak lipogenic and anti-adipogenic actions and marked adiponectin-suppressive and leptin and interleukin-6 release-promoting effects in human differentiating preadipocytes. Together with the higher serum concentrations in MUHO subjects, our findings reveal FGF<em>21</em> as a circulating <em>factor</em> promoting the development of metabolically unhealthy adipocytes.

OBJECTIVE

To evaluate the abilities of recombinant adenovirus carrying the basic fibroblast growth factor (bFGF) gene to (1) produce bFGF protein in vitro and (2) rescue retinal photoreceptors in Royal College of Surgeons (RCS) rats in vivo.

METHODS

Cultured human retinal pigment epithelial cells were infected with one of the following two replication-deficient adenoviral vectors that drive inserted genes by beta-actin promoter with cytomegalovirus enhancer: AxCAJSbFGF, which expresses the human bFGF gene, and AxCAlacZ, carrying the cDNA of bacterial beta-galactosidase as a viral control. These viruses and recombinant bFGF protein were also injected into the subretinal space of RCS rats at the age of 21 days. The production of bFGF was evaluated by an immunohistochemical method in vitro and in vivo. The secretion of bFGF produced in vitro was quantified by an enzyme-linked immunosorbent assay. The thickness of the outer nuclear layer (ONL) as a marker of photoreceptor cell rescue was estimated at 2, 28, and 56 days after the injections.

RESULTS

AxCAJSbFGF produced human bFGF protein effectively both in vitro and in vivo. The semiquantitative analysis of ONL thickness revealed a significant protective effect of AxCAJSbFGF and the recombinant bFGF protein injection up to 56 days after injection.

CONCLUSIONS

These results demonstrate that a recombinant adenoviral vector can achieve the transfer of bFGF gene in vitro and have a protective effect for photoreceptor cells in vivo. Gene therapy with a bFGF-expressing recombinant adenoviral vector may provide a new strategy with which to target retinal degenerative diseases.

BACKGROUND

Neuronal plasticity is associated with depression, probably as a result of modified expression of proteins important for cellular resiliency. It is therefore important to establish if and how antidepressant drugs may be able to regulate these mechanisms in order to achieve relevant clinical effects.

OBJECTIVE

We investigated the effects of chronic treatment with agomelatine (an MT(1)/MT(2) receptor agonist and 5-HT(2C) receptor antagonist) on the brain-derived neurotrophic factor (BDNF), fibroblast growth factor (FGF-2), and activity-regulated cytoskeleton-associated protein (Arc).

METHODS

Animals were treated for 21 days with agomelatine, venlafaxine, or a vehicle and sacrificed 1 h (6 p.m.) or 16 h after the last injection (9 a.m.) to evaluate the messenger RNA (mRNA) and protein expression of these neuroplastic markers in the hippocampus and prefrontal cortex.

RESULTS

Agomelatine, but not venlafaxine, produced major transcriptional changes in the hippocampus, where significant up-regulations of BDNF and FGF-2 were observed. Both drugs up-regulated the Arc transcription levels. No effects were observed in the prefrontal cortex. Instead, the levels of BDNF protein were elevated by agomelatine in both regions: the effects of the drug on mRNA levels in the hippocampus and cortex are different, while the effects on the protein seem to have the same cumulative result, suggesting different modulatory mechanisms in the two regions.

CONCLUSIONS

Our data provide new information regarding the molecular mechanisms that contribute to the chronic effects of the new antidepressant agomelatine on brain function. The ability of agomelatine to modulate the expression of these neuroplastic molecules, which follows a circadian rhythm, may contribute to its antidepressant action.

The serum levels of several cytokines were determined in 94 patients with Adamantiades-Behçet's disease (ABD), aged 36.1+/-11.0 years, during the active stage (n = 75) and the inactive stage (n = 19) of the disease. A group of 75 healthy individuals matched for age and sex served as controls. Cytokine levels were determined using commercially available ELISA kits. Of the 75 patients with active disease and 19 with inactive disease, 38 (51%) and 4 (<em>21</em>%), respectively, and 23 healthy controls (31%) were found to have detectable levels of interleukin 8 (IL-8) in their serum (P < 0.05). Also, increased IL-8 serum levels were found in patients with active disease (median 12 pg/ml, P = 0.010) compared to patients with inactive disease (< or = 10 pg/ml) and to healthy controls (< or = 10 pg/ml). In particular, patients with oral aphthous ulcers (n = 51, 34 pg/ml) and neurological features (n = 4, 71 pg/ml) exhibited increased IL-8 levels. In contrast, there was no correlation between disease activity and the serum levels of IL-1alpha, IL-1beta, tumor necrosis <em>factor</em> alpha (TNF-alpha), soluble intercellular adhesion molecule-1 or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). In a second set of experiments, the involvement of dermal microvascular endothelial cells in IL-8 secretion was investigated. Immortalized human dermal microvascular endothelial cells (HMEC-1 cells) were maintained for 4 h in vitro with serum from 18 ABD patients or with IL-1beta, a known stimulator of IL-8 synthesis, TNF-alpha or their combination at five- to tenfold higher concentrations than those found in the serum of ABD patients. Increased IL-8 secretion was found after incubation with ABD patients' serum (median 20 pg/ml), but IL-1beta, TNF-alpha and IL-1beta + TNF-alpha failed to induce IL-8 secretion by HMEC-1 cells (< or = 1-1.2 pg/ml) in biologically relevant concentrations. Our study showed increased IL-8 serum levels in ABD patients with active oral and neurological manifestations. Human microvascular endothelial cells may, at least partially, be responsible for the enhanced IL-8 secretion in the active stage of the disease.