Baker et al. [Cancer Res. 46: 1263-1274, 1986] developed an adhesive tumour cell culture system (ATCCS) using a culture surface coated with a cell attachment matrix (CAM). The ability of CAM-coated plates to support the <em>growth</em> of cells from human tumour biopsies has been evaluated. Successful <em>growth</em> was obtained in 9/<em>22</em> samples (41%), but <em>fibroblasts</em>, rather than tumour cells, grew in the majority. Comparison of CAM with other surfaces showed that CAM was no better for establishing tumour cell <em>growth</em> than the presence of feeder cells or an alternative attachment <em>factor</em> vitronectin.

A previous publication (Leith et al., 1992) showed that administration of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2, 0.25 mg kg-1, q.i.d. x 7) to mice bearing xenografted DLD-2 human colon cancers would increase treated tumour <em>growth</em> rates as compared to control neoplasms. Additionally, at the end of the 7 day treatment period, clonogenic excision assays showed that the percentage of hypoxic cells in tumours from mice receiving FGF-2 administration was significantly decreased as compared to control neoplasms (from about 42 to about 19%). The present study was undertaken to better define the kinetics of changes in hypoxic percentages as a function of tumour volume and FGF-2 treatment. In sham-injected control tumours, the hypoxic percentage increased from about 14% at day 15 postimplantation, (i.e. when sham- or FGF-2 injections were begun) to about 42% by day <em>22</em>, and to about 75% at 29 days postimplantation (respective average volumes <em>22</em>0, 910, and 2810 mm3). In contrast, the hypoxic percentages in mice treated with FGF-2 remained at the levels seen in control mice on day 15, not only throughout the 7 day FGF-2 treatment schedule, but for at least 1 week after the cessation of <em>growth</em> <em>factor</em> administration. The hypoxic percentage was 16% on day 29 postimplantation, even though average tumour volumes were about 4325 mm3. These data show that the effect of FGF-2 administration on tumour <em>growth</em> rate and hypoxic percentages in xenografted DLD-2 neoplasms is rapid, and continues for some period of time even after administration is ended. Studies of tumour perfusion with injected 86RbCl at equivalent tumour volumes of about 1800 mm3 indicated that the percentage of cardiac output to FGF-2 treated tumours was 33% greater than in sham-injected control neoplasms.

Endothelial progenitor cells (EPC) can differentiate into both endothelial cells and contractile smooth muscle cells (SMC). Previously we reported that TR-BME2 cells, a model for EPC, developed contractile SMC-like characteristics in culture medium deprived of endothelial cell <em>growth</em> <em>factors</em> (ECGF). The aim of the present study was to clarify the effect of one of these <em>factors</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) on differentiation of EPC. First it was confirmed that bFGF receptor (FGFR-1) mRNA is expressed in TR-BME2 cultured in both ECGF-rich and ECGF-deprived medium. When TR-BME2 cells were cultured in ECGF-deprived medium, they differentiated into contractile SMC. Expression of an undifferentiated state marker, CD133, and proliferation of TR-BME2 were both reduced by ECGF deprivation, but these changes were diminished in the presence of bFGF. mRNA expression of smooth muscle α-actin (SMA) and smooth muscle protein <em>22</em> (SM<em>22</em>), which are contractile SMC markers, was induced by deprivation of ECGF and the induction was suppressed by bFGF. In vascular endothelial cell <em>growth</em> <em>factor</em> (VEGF)-induced tube formation assay, TR-BME2 cells formed tube structures in the presence of bFGF, but not in its absence. Our results indicate that bFGF is essential for the maintenance of EPC phenotype, serving to suppress differentiation to contractile SMC.

OBJECTIVE

To investigate the influence of growth factors on tissue-engineered pediatric human elastic cartilage relative to potential clinical application.

METHODS

Controlled study.

METHODS

Eleven children ranging in age from 5 to 15 years provided auricular elastic cartilage specimens measuring approximately 1 x 1 x 0.2 cm and weighing approximately 100 mg.

METHODS

Three million chondrocytes were plated into 4 groups of Ham F-12 culture medium: group 1, Ham F-12 culture medium only; no growth factors (control group); group 2, Ham F-12 culture medium and basic fibroblast growth factor; group 3, Ham F-12 culture medium and transforming growth actor beta; and group 4, Ham F-12 culture medium and a combination of both growth factors. At 3 weeks, the cells were harvested and mixed with a copolymer gel of polyethylene glycol and polypropylene oxide (Pluronic F-127). The cell solution was injected subcutaneously into athymic mice. The constructs were harvested at up to 22 weeks of in vivo culture and histologically analyzed.

RESULTS

The average number of cells generated in vitro was as follows: group 1, 12 million; group 2, 40 million; group 3, 7 million; and group 4, 35 million. Group 2 in vivo gross specimens were the largest and heaviest. Histologically, the control group and the basic fibroblast growth factor group (groups 1 and 2) exhibited characteristics compatible with normal auricular cartilage; groups 3 and 4 demonstrated cellular disorganization and moderate to severe fibrous tissue infiltration.

CONCLUSIONS

Basic fibroblast growth factor demonstrates the greatest positive influence on the in vitro and in vivo growth of engineered pediatric human auricular cartilage. The results suggest that basic fibroblast growth factor has the potential for clinical application in which a goal will be to generate a large volume of tissue-engineered cartilage from a small donor specimen in a short period of time and of a quality similar to native human elastic cartilage.

To analyze the molecular mechanisms of coronary vessel formation, we performed in vitro experiments on explant cultures of proepicardial organs (PEOs) excised from embryos taken from 9.5-day pregnant mice. When plated on coverglasses coated with rat tail collagen I, fibronectin, or laminin, PEO cells spread and formed an epithelial sheet. When PEOs were cultured on collagen gel in the presence of fetal calf serum (FCS), small projections were seen around the explants 3 days after plating. Around day 6, cord-like structures began to grow from the explants, gradually elongating, increasing in number, and forming a branching network. Histological sections demonstrated that the cells migrated into the gel and formed tube-like structures similar to the vascular channels of the embryonic heart. The cells lining the lumen of the tube-like structures were positive for platelet endothelial cell adhesion molecule (PECAM). Reverse transcriptase-polymerase chain reaction analyses demonstrated that the expression of PECAM, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and smooth muscle <em>22</em>-alpha (SM<em>22</em>alpha) was upregulated in association with the tube formation, whereas the expression of Flk-1, Flt-1, and hepatocyte <em>growth</em> <em>factor</em> (HGF) was gradually downregulated. Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) was continuously expressed during the culture. These changes were not observed when PEOs were explanted without FCS. Furthermore, addition of any one or combinational addition of the <em>growth</em> <em>factors</em>, including bFGF, VEGF, or HGF, did not induce tube formation. These results suggest that PEOs contain precursor cells of coronary vasculature and that vasculogenesis may be simultaneously regulated by multiple <em>factors</em>.

Human neonatal <em>fibroblasts</em> in monolayer culture secrete a number of insulin-like <em>growth</em> <em>factor</em> binding proteins (IGFBPs), including IGFBP-3, which may alter paracrine or autocrine IGF activity. Studies in vitro have demonstrated that exogenous IGFBP-3 can both inhibit and potentiate IGF action in these cells; however, it is not known to what extent there is regulatory interaction between the IGFBPs. In this study we report that exogenous and endogenous IGFBP-3 inhibit production of an IGF inducible IGFBP. When analyzed by SDS-PAGE and [125I]IGF-II ligand blotting, human neonatal <em>fibroblasts</em> secrete IGFBP-3, an IGFBP of 29-31 kDa, and a <em>22</em>-24 kDa IGFBP after treatment with 50 ng/ml IGF-I. When IGF-I treatment was carried out in the presence of increasing concentrations (50-1000 ng/ml) of pure human serum-derived IGFBP-3, there was a dose-dependent decrease in the 29-31 kDa protein. In the presence of excess (250 ng/ml) IGF-I, IGFBP-3 had approximately 20-fold reduced potency in inhibiting 29-31 kDa IGFBP. When endogenous production of IGFBP-3 was increased by treatment with transforming <em>growth</em> <em>factor</em>-beta 1 (TGF beta 1), there was complete inhibition of 29-31 kDa IGFBP, while at high IGF-I concentrations TGF beta 1 had 2 to 3-fold reduced potency. These results demonstrate that <em>fibroblast</em> IGFBP production can be altered by exogenous and endogenous IGFBP-3, and suggest the existence of regulatory interactions between <em>fibroblast</em> IGFBPs.

Currently, inhibiting or reducing neuronal cell death is the main strategy to improve recovery of spinal cord injury (SCI). Therapies using nerve <em>growth</em> <em>factors</em> to treat SCI mainly focused on reducing the area damaged by postinjury degeneration to promote functional recovery. In this report, we investigated the mechanism of ER (endoplasmic reticulum) stress-induced apoptosis and the protective action of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>22</em> (FGF<em>22</em>) <i>in vivo</i>. Our results demonstrated that ER stress-induced apoptosis plays a significant role in injury of SCI model rats. FGF<em>22</em> administration promoted recovery and increased neuron survival in the spinal cord lesions of model mice. The protective effect of FGF<em>22</em> is related to decreased expression of CHOP (C/EBP-homologous protein), GRP78 (glucose-regulated protein 78), caspase-12, X-box binding protein 1 (XBP1), eukaryotic initiation <em>factor</em> 2α (Eif-2α) and Bad which are ER stress-induced apoptosis response proteins. Moreover, FGF<em>22</em> administration also increased the number of neurons and the expression of <em>growth</em>-associated protein 43 (GAP43) which was related to axon regeneration. We also demonstrated that the protective effect of FGF<em>22</em> effectively reduces neuronal apoptosis and promotes axonal regeneration. Our study first illustrated that the function of FGF<em>22</em> is related to the inhibition of ER stress-induced cell death in SCI recovery <i>via</i> activation of downstream signals. This study also suggested a new tendency of FGF<em>22</em> therapy development in central neural system injuries, which involved chronic ER stress-induced apoptosis.

Neural stem/progenitor cells (NSCs) can spontaneously differentiate into neurons and glial cells in the absence of mitogen <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) or epidermal <em>growth</em> <em>factor</em> (EGF) in medium and the spontaneous differentiation of NSCs is mediated partially by endogenous ciliary neurotrophic <em>factor</em> (CNTF). This study examined the relationship of FGF-2 and CNTF in the spontaneous differentiation of adult hippocampal progenitor cells (AHPs). AHPs were cultured in the medium containing different concentration of FGF-2 (1-100 ng/mL). Western blotting and immunofluorescence staining were applied to detect the expression of the astrocytic marker GFAP, the neuronal marker Tuj1, the oligodendrocytic marker CNPase and, Nestin, the marker of AHPs. The expression of endogenous CNTF in AHPs at early (passage 4) and late stage (passage <em>22</em>) was also measured by Western blotting. The results showed that FGF-2 increased the expression of Nestin, dramatically inhibited the expression of GFAP and Tuj1 and slightly suppressed the expression of CNPase. FGF-2 down-regulated the expression of endogenous CNTF in AHPs at both early (passage 4) and late stage (passage <em>22</em>). These results suggested that FGF-2 could inhibit the spontaneous differentiation of cultured AHPs by negatively regulating the expression of endogenous CNTF in AHPs.

Introduction: Muscle sympathetic nerve activity (MSNA) may play a role in insulin resistance in obesity. However, the direction and nature of the relationship between MSNA and insulin resistance in obesity remain unclear. We hypothesized that resting MSNA would correlate inversely with both muscle and liver insulin sensitivity and that it would be higher in insulin-resistant vs. insulin-sensitive subjects. Materials and methods: Forty-five non-diabetic obese subjects were studied. As no significant relationships were found in women, the data presented in on <em>22</em> men aged 48 ± 12 years. Two-step (15 and 80 mU/m2/min) hyperinsulinaemic-euglycaemic clamps were performed using deuterated glucose to determine liver and muscle insulin sensitivity. Clinical and metabolic parameters were assessed. MSNA was measured via a microelectrode inserted percutaneously into the common peroneal nerve. Results: MSNA burst frequency correlated inversely with liver insulin sensitivity (r = -0.53, P = 0.02) and positively with the hepatokines C-reactive protein (CRP) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-19 (r = 0.57, P = 0.006, and r = -0.47, P = 0.03, respectively). MSNA burst frequency was lower in Liversen compared to Liverres (27 ± 5 vs. 38 ± 2 bursts per minute; P = 0.03). Muscle insulin sensitivity was unrelated to MSNA. Discussion: Sympathetic neural activation is related to liver insulin sensitivity and circulating hepatokines CRP and FGF-19 in non-diabetic obese men. These results suggest a potential hepato-endocrine-autonomic axis. Future studies are needed to clarify the influence of MSNA on liver insulin sensitivity in men.

<AbstractText>There are no validated markers that predict response or resistance in patients with metastatic clear-cell renal cell carcinoma (mccRCC) treated with vascular endothelial <em>growth</em> <em>factor</em> receptor tyrosine kinase inhibitors such as sunitinib and pazopanib. Recently, single nucleotide polymorphism (SNP) rs2981582 in <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> Receptor 2 (FGFR2) was found to be associated with clinical outcome in patients with mccRCC treated with pazopanib and sunitinib. We aimed to validate these findings in patients treated with sunitinib.</AbstractText><AbstractText>Germline DNA was collected in patients with mccRCC starting first-line systemic therapy with sunitinib. SNP rs2981582 in FGFR2 C>T was genotyped. Association of the genotype with response rate, tumor shrinkage, median progression-free survival (mPFS), and median overall survival (mOS) was studied.</AbstractText><AbstractText>We collected clinical data from 154 patients with available germline DNA. Baseline prognostic markers were well-balanced between both subgroups. Patients with the TT genotype had a poorer outcome compared with patients with the CT/CC genotype. The median shrinkage of selected tumor target lesions during treatment with sunitinib was -16% versus -31% (P = .002), mPFS was 8 versus 15 months (P = .0007), and mOS was <em>22</em> versus 33 months (P = .04), respectively. On multivariate analysis, rs2981582 remained an independent predictor of PFS (hazard ratio, 2.858; 95% confidence interval, 1.659-4.923; P < .0001) and OS (hazard ratio, 1.795; 95% confidence interval, 1.003-3.212; P = .049).</AbstractText><AbstractText>Polymorphism rs2981582 in FGFR2 is correlated to PFS and OS in patients with mccRCC treated with sunitinib. Prospective validation of the impact of this SNP is warranted.</AbstractText>

We have analyzed by circular dichroism (CD) and proton nuclear magnetic resonance (NMR) the helical propensity of the all-beta protein acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) and two peptides corresponding to beta-strand 8 (beta8 peptide, amino acids 95-107) and the beta-strand 8/turn/beta-strand 9 hairpin (beta8/9 peptide, amino acids 95-114), which has been involved in receptor binding. A secondary structure prediction of aFGF carried out by several procedures labels the 95-104 sequence as predominantly alpha-helical. A titration of aFGF with 2,2,2-trifluoroethanol (TFE) induces a change in the far-UV CD spectrum of the protein giving rise to a prominent alpha-helical shape (<em>22</em>% alpha-helix). The cooperativity of the transition and the moderate TFE concentrations used (midpoint at 24%) suggest that the effect of TFE is specific. Moreover, a titration performed at pH 2 yields a higher amount of alpha-helix (55%) at a smaller TFE concentration. Synthetic peptides containing the beta8 and beta8/9 sequences display a random coil conformation at pH 7 but acquire alpha-helical structure in the presence of TFE, methanol, and SDS micelles. At pH below 3.0 a significant amount (20-30%) of alpha-helical conformation is present in both the beta8 and beta8/9 peptides even in the absence of other solvent additives. The secondary structure of the peptides was determined by proton nuclear magnetic resonance (1H NMR). These results suggest that the 95-114 sequence of aFGF has helical propensity and that the protein may fold nonhierarchically in the early steps of folding, acquiring its final beta-structure by a later interaction with the rest of the polypeptide.

OBJECTIVE

To investigate the effects of transforming growth factor (TGF)-beta(1) on fibroblasts differentiation into myofibroblasts, and the change of Egr-1 expression in the course of differentiation in BALB/c 3T3 fibroblasts.

METHODS

BALB/c 3T3 fibroblasts of the experiment group were treated with 10 ng/ml TGF-beta(1) and those of the control group were treated without TGF-beta(1). The expression of alpha-smooth muscle actin (SMA) was detected by flow cytometer after incubation for 24, 48, and 72 h. The expression of Egr-1 was detected by immunocytochemistry and Western blot method after 10 ng/ml TGF-beta(1) incubation for 15, 30, 60, 90, 120, 180, and 240 min.

RESULTS

The percentage of alpha-SMA positive cells was (6.65 +/- 0.48)% when cells were treated with 10 ng/ml TGF-beta(1) for 24 h, and the control group was (5.53 +/- 0.62)%, which showed no statistical significance. However the percentage was up to (28.38 +/- 3.60)% after treated with TGF-beta(1) for 48 h, and the control group was (9.49 +/- 0.21)%; At 72 h, the percentage was (36.04 +/- 0.73)% and the control group was (15.23 +/- 0.33)%; The difference was statistically significant. Immunocytochemistry showed that Egr-1 protein positive signal was brown in color. The color was light in the control group. The gray-level of the control group was 38 +/- 18 measured by Western blot when cells were treated without TGF-beta(1). After treated with 10 ng/ml TGF-beta(1) for 15 min, the level was up to 55 +/- 26, which had no statistical significance. The level was increased with longer incubation. It was 115 +/- 14 at 60 min and reached its peak level 129 +/- 22 at 90 min, which was significantly higher than the control group. The level was decreased after 90 min and reached neap 39 +/- 7 at 240 min.

CONCLUSIONS

TGF-beta(1) increased the expression of alpha-SMA in BALB/c 3T3 fibroblasts and affected the Egr-1 protein expression during the early course of fibroblasts differentiation into myofibroblasts.

Increased levels and activity of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) have been documented in a variety of diseases, including ischemia. Both acute coronary syndromes and exercise are situations that stimulate FGF release. Since experimental studies have demonstrated that FGFs are involved in myocardial preconditioning, it has been suggested that cardiac and circulating FGFs may play a cardioprotective role in ischemic diseases. However, the profile of basic FGF (bFGF) release during transient myocardial ischemia remains uncertain. We sought to determine whether circulating bFGF might be changed in patients with demonstrated coronary artery disease and evidence of ischemia in exercise scintigraphy (Isch +; n = 21). Serum from <em>22</em> age-matched patients with no coronary artery disease and no isotopic ischemia (Isch-) were used as controls. Three blood samples were obtained to determine bFGF at different times: baseline (bFGF-A); maximal exercise (bFGF-B), and isotopic redistribution (bFGF-C). An enzyme-linked immunoassay specific for bFGF was used (limit of detection, 1.0 pg/ml). Circulating bFGF was increased at maximal exercise in both Isch + and control patients. However, serum levels of bFGF were elevated up to more than two-fold in Isch-patients compared to Isch+ patients (8.67 +/- 2.10 pg/ml in Isch+ vs 17.83 +/- 2.97 pg/ml in Isch- patients; p<0.01). According to previous data, these findings suggest that bFGF serum levels could be considered more likely a marker of endothelial dysfunction occurring in patients with coronary artery disease, rather than a marker of acute ischemia. This situation could be different in the clinical setting of chronic myocardial ischemia.

PTH induces the synthesis of insulin-like <em>growth</em> <em>factor</em> I (IGF-I) and regulates the expression of IGF-binding proteins (IGFBP) in osteoblast cultures. IGFBP-related protein-1 (IGFBP-RP-1), the product of the mac25 gene, binds IGF-I, IGF-II, and insulin. We tested the actions of PTH on the expression of mac25/IGFBP-RP-1 in cultures of osteoblast-enriched cells from <em>22</em>-day-old fetal rat calvariae (Ob cells). PTH at 0.1-10 nM for 6-48 h increased mac25/IGFBP-RP-1 messenger RNA (mRNA) levels in Ob cells, an effect not altered by cycloheximide. PGE2 increased mac25/IGFBP-RP-1 mRNA levels, but indomethacin did not modify basal or PTH-stimulated mac25/IGFBP-RP-1 expression. The decay of mac25/IGFBP-RP-1 mRNA in transcriptionally arrested Ob cells was not modified by PTH, and PTH increased the rate of IGFBP-RP-1 transcription. GH, insulin, bone morphogenetic protein-2, <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, platelet-derived <em>growth</em> <em>factor</em> BB, IGF-I, and IGF-II did not modify mac25/IGFBP-RP-1 expression, whereas transforming <em>growth</em> <em>factor</em>-beta1 was modestly stimulatory. In conclusion, PTH stimulates mac25/IGFBP-RP-1 transcription in osteoblasts, an effect that could be relevant to the actions of PTH in bone.

Intestinal epithelial cell wound healing involves cell migration, proliferation, and differentiation. Although numerous studies have analyzed the migration of absorptive epithelial cells during wound healing, it remains unclear how goblet cells restitute and how MUC2 mucin production affects this process. In this study, we examined the role of high MUC2 production in goblet cell migration during wound healing and demonstrated that during high MUC2 output, goblet cells migrated slower because of impaired production of wound healing <em>factors</em> and endoplasmic reticulum (ER) stress. Two goblet cell lines, HT29-H and HT29-L, that produced high and low MUC2 mucin, respectively, were used. HT29-L healed wounds faster than HT29-H cells by producing significantly higher amounts of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 1, FGF2, vascular endothelial <em>growth</em> <em>factor</em>-C, and matrix metallopeptidase 1. Predictably, treatment of HT29-H cells with recombinant FGF2 significantly enhanced migration and wound healing. High MUC2 biosynthesis in HT29-H cells induced ER stress and delayed migration that was abrogated by inhibiting ER stress with tauroursodeoxycholic acid and IL-<em>22</em>. FGF2- and IL-<em>22</em>-induced wound repair was dependent on STAT1 and STAT3 signaling. During wound healing after dextran sulfate sodium-induced colitis, restitution of Math1M1GFP+ goblet cells occurred earlier in the proximal colon, followed by the middle and then distal colon, where ulceration was severe. We conclude that high MUC2 output during colitis impairs goblet cell migration and wound healing by reducing production of <em>growth</em> <em>factors</em> critical in wound repair.

Fibrotic disease is a major cause of morbidity and mortality and is characterized by the transition of resident <em>fibroblast</em> cells into active myo<em>fibroblast</em>s, identified by their expression of alpha smooth muscle actin. Myo<em>fibroblast</em> differentiation is regulated by <em>growth</em> <em>factor</em> signaling and mechanical signals transduced through integrins, which converge at focal adhesion proteins (Src and FAK) and MAPK signaling, but lead to divergent outcomes. While details are known about individual pathways, little is known about their interactions. To this end, an ODE-based model of this cell signaling network was developed in parallel with <i>in vitro</i> experiments to analyze potential mechanisms of crosstalk and regulation of <i>α</i>SMA production. We found that cells lacking Src or FAK produce significantly less or more <i>α</i>SMA than wild type cells, respectively. Transforming <em>growth</em> <em>factor</em> beta 1 and <em>fibroblast</em> <em>growth</em> <em>factor</em> signal through ERK and MAPK p38 with different dynamic profiles to increase or decrease <i>α</i>SMA expression, respectively. Our model effectively recreated <i>α</i>SMA expression levels across a set of <em>22</em> experimental conditions and matched some features of transient phosphorylation of ERK and p38. These results support a potential mechanism for regulation of <em>fibroblast</em> differentiation: <i>α</i>SMA production is promoted by active p38 and Src and opposed by ERK.

Keywords: Alpha-smooth muscle actin (αSMA); Extracellular signaling-related kinase (ERK); Fibroblast growth factor (FGF); Focal adhesion kinase (FAK); Integrin; Src; Transforming growth factor beta (TGF-β1); p38.

Objective: Our lab recently identified the centrally circulating α-klotho protein as a novel hypothalamic regulator of food intake and glucose metabolism in mice. The current study aimed to investigate novel molecular effectors of central α-klotho in the arcuate nucleus of the hypothalamus (ARC), while further deciphering its role regulating energy balance in both humans and mice.

<strong class="sub-title"> Methods: </strong> Cerebrospinal fluid (CSF) was collected from <em>22</em> adults undergoing lower limb orthopedic surgeries and correlations between body weight and α-klotho were determined using an α-klotho ELISA kit. To investigate the effects of α-klotho on energy expenditure (EE), 2-day intracerebroventricular (ICV) treatment was performed in diet-induced obesity (DIO) mice housed in the TSE Phenomaster indirect calorimetry metabolic cages. Immunohistochemical staining for cFOS and patch clamp electrophysiology were used to determine the effects of central α-klotho on proopiomelanocortin (POMC) and tyrosine hydroxylase (TH) neurons. Additional stains were performed to determine novel roles for central α-klotho to regulate non-neuronal cell populations in the ARC. Lastly, ICV pretreatment with <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) or PI3kinase inhibitors was performed to determine the intracellular signaling involved in α-klotho-mediated regulation of ARC nuclei.

Results: Obese/overweight human subjects had significantly lower CSF α-klotho concentrations compared to lean counterparts (1044±251 vs. 1616±218 pmol/L respectively). Additionally, 2 days ICV α-klotho treatment increased EE in DIO mice. α-Klotho had no effects on TH neuron activity, but elicited varied responses in POMC neurons, with ∼44% experiencing excitatory and ∼56% inhibitory effects. Inhibitor experiments identified an α-klotho→FGFR→PI3kinase signaling mechanism in the regulation of ARC POMC and NPY/AgRP neurons. Acute ICV α-klotho treatment also increased phosphorylated ERK in ARC astrocytes via FGFR signaling.

Conclusion: Our human CSF data provides the first evidence that impaired central α-klotho function may be involved in the pathophysiology of obesity. Furthermore, results in mouse models identify ARC POMC neurons and astrocytes as novel molecular effectors of central α-klotho. Overall, the current study highlights prominent roles of α-klotho→FGFR→PI3kinase signaling in the homeostatic regulation of ARC neurons and whole-body energy balance.

Keywords: PI3kinase; POMC neuron; fibroblast growth factor; metabolism; obesity; α-klotho.

BACKGROUND

Nocturnal eating have recently increased. Serum phosphorus levels and regulators of phosphorus have circadian variations, so it is suggested that the timing of eating may be important in controlling serum phosphorus levels. However, there have been no reports on the effects of nocturnal eating on phosphorus metabolism. The objective was to evaluate the effects of nocturnal eating on phosphorus metabolism.

METHODS

Fourteen healthy men participated in two experimental protocols with differing dinner times. The design of this study was a crossover study. The subjects were served test meals three times (breakfast; 07:30 h, lunch; 12:30 h, dinner; 17:30 or <em>22</em>:30 h) a day. Blood and urine samples were collected to assess diurnal variation until the following morning.

RESULTS

The following morning, fasting serum phosphorus levels in the late dinner group were markedly higher than those in the early dinner group (p < 0.001), although serum calcium levels were maintained at approximately constant levels throughout the day in both groups. Fluctuations in urinary calcium excretion were synchronized with the timing of dinner eating, however, fluctuations in urinary phosphorus excretion were not synchronized. Urinary phosphorus excretions at night were inhibited in the late dinner group. In the late dinner group, intact parathyroid hormone levels didn't decrease, and they were significantly higher in this group compared with the early dinner group at 20:00 h (p = 0.004). The following morning, fasting serum fibroblast growth factor 23 levels in the late dinner group had not changed, but those in the early dinner group were significantly increased (p = 0.003). Serum free fatty acid levels before dinner were significantly higher in the late dinner group compared with the early dinner group.

CONCLUSIONS

Our results indicate that nocturnal eating inhibits phosphorus excretion. It is suggested that nocturnal eating should be abstained from to manage serum phosphorus levels to within an adequate range.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has significant properties in wound healing, tissue regeneration and ulcer repair of the upper digestive tract. The purpose of the present study was to identify and analyse <em>factors</em> affecting the concentration of bFGF in saliva from healthy human individuals. A commercially available enzyme-linked immunosolvent assay kit (ELISA) was used for the analyses of bFGF in saliva. In total, 56 samples were collected from 28 healthy subjects, 15 male and 13 female. Determination of bFGF was performed by spectrophotometer (wavelength 490 nm). bFGF was detected in all samples. Mean bFGF concentration was 0.87 pg/ml (SD 0.49) and the concentration ranged from 0.3 to 1.9 pg/ml. In subjects aged <em>22</em> to 49 years, no age-dependent variation in bFGF was present, females did not differ from males, and no difference was evident between European and Asian subjects. Smokers had significantly higher saliva concentrations of bFGF than non-smokers. Since bFGF, together with other <em>growth</em> <em>factors</em>, is involved in wound healing and tissue repair, we suggest that bFGF in saliva is involved in the reparative processes of mucous membranes.

OBJECTIVE

We sought to elucidate the levels of fibroblast growth factor 2 (FGF-2) in synovial fluid taken from internally deranged human temporomandibular joints (TMJs) and to discuss the role of FGF-2 in the pathogenesis of internal derangement.

METHODS

Through the use of a pumping procedure, diluted synovial fluid was collected from the upper joint compartment of 22 TMJs with evidence of internal derangement (21 patients) and 8 TMJs with no such evidence (5 control subjects). Two of the control subjects were patients who had habitual dislocation, and three were healthy volunteers. The level of FGF-2 in the synovial fluid was assessed by means of an enzyme-linked immunosorbent assay.

RESULTS

FGF-2 levels were at detectable levels in 15 of the 22 TMJs (68%) with internal derangement. The mean concentration of FGF-2 was 24 pg/mL. In the control group, FGF-2 levels were detectable in only 1 of 8 joints (13%), for a concentration of 3 pg/mL. The mean concentration of FGF-2 in the synovial fluid was significantly higher in the internal derangement group than in the control group (P =.02).

CONCLUSIONS

FGF-2 levels are elevated in the human synovial fluid of TMJs with internal derangement.

OBJECTIVE

The purpose of this study was to test whether basic fibroblast growth factor (bFGF) participates in arterialized vein graft remodeling.

METHODS

Rabbits underwent in vivo gene transfer and carotid interposition vein grafting. Segments of external jugular vein were infected with an adenovirus that expressed antisense bFGF RNA (Ad.ASbFGF) at 1 x 10(10) PFU/mL to inhibit new synthesis of bFGF by cells in the vein graft wall. Control rabbits were treated with either adenovirus that encoded beta-galactosidase (Ad.lacZ) at 1 x 10(10) PFU/mL or vehicle (phosphate-buffered saline solution [PBS]). At 3 days, 3 grafts per treatment group were harvested for the determination of gene expression of ASbFGF RNA by reverse transcriptase-polymerase chain reaction. Rabbits were killed, and perfusion was fixed 2 months after the grafting. Total wall thickness and lumen circumference of vein grafts and normal arteries were measured in cross sections. Calculated mean tangential stress (+/-SD) for the ASbFGF-treated group and controls was compared for significance. Grafts were immunohistochemically stained to assess bFGF protein production.

RESULTS

Only the grafts infected with the Ad.ASbFGF gene expressed ASbFGF RNA. Grafts that were treated with Ad.ASbFGF displayed lower tangential stress (10.9 +/- 2.3 dynes/cm(2)) than PBS alone (22 +/- 2.8 dynes/cm(2)) or Ad. lacZ-treated controls (20.6 +/- 5.4 dynes/cm(2); P <.001). Tangential stress in the Ad.ASbFGF group was comparable to a normal carotid artery (13.9 +/- 2.1 dynes/cm(2)). The difference in mean total wall thickness was significant among the 3 treatment groups: Ad.ASbFGF, 164 +/- 3.4 microm); Ad.lacZ, 100 +/- 3.3 microm; and PBS, 96 +/- 3.6 microm; P <.01). Luminal circumference was not different among the groups. The Ad.ASbFGF-treated vein graft wall was composed of thick layers of concentric smooth muscle cells and elastin fibers in contrast to the sponge-like appearance observed in control arterialized vein grafts. Reduction in bFGF protein was noted only in the Ad.ASbFGF-treated group.

CONCLUSIONS

Inhibition of bFGF synthesis in vivo with the use of adenoviral gene transfer of antisense RNA to bFGF promotes a vein graft with decreased tangential stress while maintaining the luminal area. The vein graft wall is remodeled and qualitatively resembles an artery so that wall tangential stress in Ad.ASbFGF and normal artery are not significantly different. The lack of significant difference in lumen circumference among groups suggests that wall thickening in the Ad. ASbFGF grafts is not at the expense of luminal narrowing. Our results suggest that ASbFGF RNA expression may represent an effective strategy in limiting the failure of arterialized venous conduits.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)13, a nonsecreted, X-linked, FGF homologous <em>factor</em>, is differentially expressed in adipocytes in response to diet, yet <i>Fgf13</i>'s role in metabolism has not been explored. Heterozygous <i>Fgf13</i> knockouts fed normal chow and housed at <em>22</em>°C showed hyperactivity accompanying reduced core temperature and obesity when housed at 30°C. Those heterozygous knockouts showed defects in thermogenesis even at 30°C and an inability to protect core temperature. Surprisingly, we detected trivial FGF13 in adipose of wild-type mice fed normal chow and no obesity in adipose-specific heterozygous knockouts housed at 30°C, and we detected an intact brown fat response through exogenous β3 agonist stimulation, suggesting a defect in sympathetic drive to brown adipose tissue. In contrast, hypothalamic-specific ablation of <i>Fgf13</i> recapitulated weight gain at 30°C. Norepinephrine turnover in brown fat was reduced at both housing temperatures. Thus, our data suggest that impaired CNS regulation of sympathetic activation of brown fat underlies obesity and thermogenesis in <i>Fgf13</i> heterozygous knockouts fed normal chow.-Sinden, D. S., Holman, C. D., Bare, C. J., Sun, X., Gade, A. R., Cohen, D. E., Pitt, G. S. Knockout of the X-linked <i>Fgf13</i> in the hypothalamic paraventricular nucleus impairs sympathetic output to brown fat and causes obesity.

OBJECTIVE

To investigate the characteristics of the expression of transforming growth factor-beta(1) (TGF-beta(1)) and basic fibroblast growth factor (bFGF) in the apical articular process cartilages of adolescent idiopathic scoliosis (AIS) patients.

METHODS

The specimens of articular processes of 22 AIS patients and 18 congenital scoliosis (CS) patients were collected during operation. The pathology of the processes was observed with H.E staining. Immunohistochemistry and in situ hybridization were adopted to detect the expression of TGF-beta(1) and bFGF. The differences in the pathological changes, and expression of TGF-beta(1) and bFGF between the apical processes and the end processes, the convex processes and the concave processes, and the AIS processes and CS processes. The images of immunohistochemistry and in situ hybridization were input into the image analysis system and were analyzed semi-quantitatively. The SAS (8.01) software was adopted and (128.1 +/- 50.6 vs 165.4 +/- 59.2, 126.5 +/- 47.0 vs 168.3 +/- 46.8; 76.6 +/- 29.9 vs 96.4 +/- 28.4, 73.7 +/- 31.6 vs 101.8 +/- 39.4; 77.1 +/- 52.2 vs 114.4 +/- 59.4, and 69.5 +/- 40.1 vs 109.8 +/- 51.0 (P < 0.05) was defined as the significant levels.

RESULTS

The expression of TGF-beta(1) and the expression of bFGF were not significantly different between the AIS patients and the CS patients. The TGF-beta(1) expression in the concave side of the apical vertebrae of the AIS group was 165.4 +/- 59.2, significantly higher than that in the convex sides (128.1 +/- 50.6, P = 0.03), and the TGF-beta(1) expression in the concave side of the apical vertebrae of the CS group was 168.3 +/- 46.8, significantly higher than that of the convex side (126.5 +/- 47.0, P = 0.02). However, there was no statistically significant differences in the TGF-beta(1) expression between the concave and convex sides of the upper and lower end vertebrae and between the upper and lower end vertebrae. The bFGF expression in the concave side of the apical vertebrae of the AIS group, and the bFGF expression in the concave side of the apical vertebrae of the CS group was 101.8 +/- 39.4, significantly higher than that of the convex side (73.7 +/- 31, P = 0.02). However, there were no statistically significant differences in the bFGF expression between the concave and convex sides of the upper and lower end vertebrae and between the upper and lower end vertebrae.

CONCLUSIONS

The cartilages of the apical processes show some signs of regression and hypoplasia in AIS patients, especially at the concave side in comparison with the convex side. Increase of TGF-beta(1) and bFGF in the concave sides of apical processes in AIS may be the results of reconstruction of extracellular matrix and the compensation reactions which are caused by abnormal biomechanical forces, especially compressive stresses.

OBJECTIVE

To investigate the relationship of transforming growth factor beta1 (TGFbeta1) and basic fibroblast growth factor (bFGF) to detrusor underactivity following bladder outlet obstruction (BOO).

METHODS

Female Wistar rats underwent ligation of the urethra to establish BOO models and were divided into BOO model 2-week group (11 rats) and BOO model 6-week group (10 rats). 8 rats underwent sham operation as control group. The detrusor urine was taken out and stimulated by carbachol to measure the detrusor contraction force (DCF). RT-PCR method was employed to measure the mRNA expression of TGFbeta1 and bFGF in the detrusor urine. Urine TGFbeta1 and bFGF were determined by ELISA.

RESULTS

The maximum DCF levels of the BOO 2-week group under the 1 x 10(-4) mmol/L and 1 x 10(-3) mmol/L carbachol concentrations were 0.96 g +/- 0.11 g and 1.98 g +/- 0.21 g respectively, both significantly higher than those of the sham operation group (0.85 g +/- 0.18 g and 1.82 g +/- 0.19 g respectively, both P < 0.05). The maximum DCF levels of the BOO 6-week group under the 1 x 10(-5), 1 x 10(-4), 1 x 10(-3) and 1 x 10 (-2) mmol/L carbachol concentrations were 0.19 g +/- 0.02 g, 0.65 g +/- 0.06 g, 1.12 g +/- 0.08 g, and 1.40 g +/- 0.19 g respectively, all significantly lower than those of the BOO 2-week group (0.24 g +/- 0.03 g, 0.96 g +/- 0.11 g, 1.98 g +/- 0.21 g, and 2.16 g +/- 0.21 g respectively, all P < 0.05) and those of the sham operation group (0.23 g +/- 0.04 g, 0.85 g +/- 0.18 g, 1.82 g +/- 0.19 g, and 2.12 g +/- 0.26 g respectively, all P < 0.05). The mRNA expression of TGFbeta1 of the BOO 6-week group, BOO 2-week group, and sham operation group was 0.72 +/- 0.21, 0.34 +/- 0.10, and 0.32 +/- 0.01 respectively, there was a significant difference between the BOO 6-week group and the BOO 2-week group (P < 0.01). The mRNA expression level of bFGF of the BOO 6-week group was 0.38 +/- 0.13, significantly higher than those of the BOO 2-week group and sham operation group (0.21 +/- 0.07 and 0.10 +/- 0.05 respectively, both P <0.05). DCF was negatively correlated with the mNRA expression of TGFbeta1 and the mNRA expression bFGF in detrusor (both P < 0.05). The urine TGFbeta1 of the BOO 6-week group was (606 +/- 216) microg/mol Cr, significantly higher than that of the BOO 2-week group [(131 +/- 49) microg/mol Cr] and that of the sham operation group [(107 +/- 22) microg/mol Cr, both P <0.05].

CONCLUSIONS

With the progression of BOO, there is a sustained rise of bFGF mRNA expression in detrusor; however, the TGFbeta1 mRNA expression only increases during the decompensation stage. Urine TGFbeta1 level is very high 6 weeks after BOO, which may help predict the contraction function of bladder after BOO.