While the physiological benefits of the <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) hepatokine are documented in response to fasting, little information is available on Fgf<em>21</em> regulation in a glucose-overload context. We report that peroxisome-proliferator-activated receptor α (PPARα), a nuclear receptor of the fasting response, is required with the carbohydrate-sensitive transcription <em>factor</em> carbohydrate-responsive element-binding protein (ChREBP) to balance FGF<em>21</em> glucose response. Microarray analysis indicated that only a few hepatic genes respond to fasting and glucose similarly to Fgf<em>21</em>. Glucose-challenged Chrebp-/- mice exhibit a marked reduction in FGF<em>21</em> production, a decrease that was rescued by re-expression of an active ChREBP isoform in the liver of Chrebp-/- mice. Unexpectedly, carbohydrate challenge of hepatic Pparα knockout mice also demonstrated a PPARα-dependent glucose response for Fgf<em>21</em> that was associated with an increased sucrose preference. This blunted response was due to decreased Fgf<em>21</em> promoter accessibility and diminished ChREBP binding onto Fgf<em>21</em> carbohydrate-responsive element (ChoRE) in hepatocytes lacking PPARα. Our study reports that PPARα is required for the ChREBP-induced glucose response of FGF<em>21</em>.

Mitochondrial dysfunctions accompany several neurodegenerative disorders and contribute to disease pathogenesis among others in Parkinson's disease (PD). Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a major regulator of mitochondrial functions and biogenesis, and was suggested as a therapeutic target in PD. PGC-1α is regulated by both transcriptional and posttranslational events involving also the action of <em>growth</em> <em>factors</em>. <em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>) is a regulator of glucose and fatty acid metabolism in the body but little is known about its action in the brain. We show here that FGF<em>21</em> increased the levels and activity of PGC-1α and elevated mitochondrial antioxidants in human dopaminergic cells in culture. The activation of PGC-1α by FGF<em>21</em> occurred via the NAD(+)-dependent deacetylase Sirtuin-1 (SIRT1) subsequent to an increase in the enzyme, nicotinamide phosphoribosyltransferase (Nampt). FGF<em>21</em> also enhanced mitochondrial respiratory capacity in human dopaminergic neurons as shown in real-time analyses of living cells. FGF<em>21</em> is present in the brain including midbrain and is expressed by glial cells in culture. These results show that FGF<em>21</em> activates PGC-1α and increases mitochondrial efficacy in human dopaminergic neurons suggesting that FGF<em>21</em> could potentially play a role in dopaminergic neuron viability and in PD.

BACKGROUND

To evaluate the concentrations of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in neovascular or edematous retinal diseases.

METHODS

In the clinical comparative interventional study, VEGF and bFGF concentrations in aqueous humor samples of 35 patients with exudative age-related macular degeneration (AMD), 21 patients with diabetic macular edema and 24 patients of a control group were measured using a solid-phase chemiluminescence immunoassay.

RESULTS

Concentrations of VEGF and bFGF, respectively, were significantly higher in the diabetic group (184.7 +/- 107.0 and 5.0 +/- 10.2 pg/l) than in the AMD group (107.7 +/- 73.0 pg/l, p = 0.002; 2.2 +/- 7.4 pg/l, p = 0.002) and the control group (71.5 +/- 94.7 pg/l, p = 0.001; 0.00 pg/l, p = 0.001). The two latter groups did not vary significantly (p = 0.10).

CONCLUSIONS

VEGF and bFGF are present in considerably higher concentrations in eyes with diabetic macular edema than in eyes with exudative AMD or normal eyes. The differences were more marked for VEGF than for bFGF.

The α-glucosidase inhibitor acarbose, which slows carbohydrate digestion and blunts postprandial rises in plasma glucose, has long been used to treat patients with type 2 diabetes or glucose intolerance. Like metformin, acarbose tends to aid weight control, postpone onset of diabetes and decrease risk for cardiovascular events. Acarbose treatment can favourably affect blood pressure, serum lipids, platelet aggregation, progression of carotid intima-media thickness and postprandial endothelial dysfunction. In mice, lifetime acarbose feeding can increase median and maximal lifespan-an effect associated with increased plasma levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) and decreased levels of insulin-like <em>growth</em> <em>factor</em>-I (IGF-I). There is <em>growing</em> reason to suspect that an upregulation of fasting and postprandial production of glucagon-like peptide-1 (GLP-1)-stemming from increased delivery of carbohydrate to L cells in the distal intestinal tract-is largely responsible for the versatile health protection conferred by acarbose. Indeed, GLP-1 exerts protective effects on vascular endothelium, the liver, the heart, pancreatic β cells, and the brain which can rationalise many of the benefits reported with acarbose. And GLP-1 may act on the liver to modulate its production of FGF<em>21</em> and IGF-I, thereby promoting longevity. The benefits of acarbose are likely mimicked by diets featuring slowly-digested 'lente' carbohydrate, and by certain nutraceuticals which can slow carbohydrate absorption. Prebiotics that promote colonic generation of short-chain fatty acids represent an alternative strategy for boosting intestinal GLP-1 production. The health benefits of all these measures presumably would be potentiated by concurrent use of dipeptidyl peptidase 4 inhibitors, which slow the proteolysis of GLP-1 in the blood.

Extremely mild hypothermia to 36.0 °C is not thought to appreciably differ clinically from 37.0 °C. However, it is possible that 36.0 °C stimulates highly sensitive hypothermic signaling mechanism(s) and alters biochemistry. To the best of our knowledge, no such ultra-sensitive pathway/mechanisms have been described. Here we show that cold stress protein RNA binding motif 3 (RBM3) increases in neuron and astrocyte cultures maintained at 33 °C or 36 °C for 24 or 48 h, compared to 37 °C controls. Neurons cultured at 36 °C also had increased global protein synthesis (GPS). Finally, we found that melatonin or <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) augmented RBM3 upregulation in young neurons cooled to 36 °C. Our results show that a 1 °C reduction in temperature can induce pleiotropic biochemical changes by upregulating GPS in neurons which may be mediated by RBM3 and that this process can be pharmacologically mimicked and enhanced with melatonin or FGF<em>21</em>.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is involved in regulating energy metabolism, and it has shown significant promise as a treatment for type II diabetes; however, the native protein has a very short circulating half-life necessitating frequent injections to maintain a physiological effect. Polyethylene glycol (PEG) conjugation to proteins has been used as a method for extending the circulating half-life of many pharmaceutical proteins; however, PEG does carry the risk of vacuole formation, particularly in the renal tubular epithelium. Since renal vacuole formation may be particularly problematic for diabetic patients, we engineered site-directed PEGylated variants of FGF<em>21</em> with sustained potency and minimized vacuole formation. This was accomplished both by probing the site of PEGylation on FGF<em>21</em> as well as by examining various PEG configurations. While the site of PEGylation has a significant impact on the bioactivity of FGF<em>21</em>, it has only a marginal impact on vacuole formation; however, the configuration and number of PEGs conjugated to the protein has a much more profound effect on vacuologenesis.

Glucocorticoids have been shown to accelerate fetal lung type II cell maturation, and this effect appears, in part, to be mediated via <em>fibroblasts</em>. To identify glucocorticoid induced genes in fetal lung <em>fibroblasts</em>, we screened a cDNA library from cortisol-treated fetal lung <em>fibroblasts</em> with a subtracted cDNA probe which was enriched for sequences specific for cortisol-treated fetal lung <em>fibroblasts</em>. Fifty-seven clones were isolated from the cDNA library. One cDNA represented approximately 30% of the 57 clones. Analysis of DNA sequence homology suggested that this cDNA encodes the rat transforming <em>growth</em> <em>factor</em>-beta 3 (TGF beta 3). We found that TGF beta 3 mRNA was expressed in fetal lung <em>fibroblasts</em> but not epithelial cells. Expression of message in fetal lung <em>fibroblasts</em> was developmentally regulated. TGF beta 3 mRNA levels were low during the pseudoglandular stage (day 18), peaked during the early canalicular stage of lung development (day 19), then fell again at days 20 and <em>21</em> (term = 22 days). Exposure of fetal lung <em>fibroblasts</em> to cortisol increased TGF beta 3 mRNA expression in a time- and dose-dependent manner. Maternal administration of dexamethasone also enhanced mRNA expression of TGF beta 3 in fetal lung <em>fibroblasts</em>. These data suggest that glucocorticoids may mediate their stimulatory effect on lung maturation by inducing TGF beta 3 expression in fetal lung <em>fibroblasts</em>.

BACKGROUND

High expression of vascular endothelial growth factor (VEGF) induces subepithelial fibrosis associated with angiogenesis in patients with asthma. Thrombin is recognized as a new candidate mediating airway remodeling. Therefore, this study was designed to determine the role of up-regulated thrombin activity induced by VEGF on airway remodeling in patients with asthma.

METHODS

Levels of biochemical parameters in induced sputum were examined in 21 asthmatic patients and 11 normal control subjects.

RESULTS

Thrombin activity in induced sputum was significantly higher in asthmatic patients than in normal control subjects: median, 3.67 U/mL; range, 1.15 to 10.2 U/mL; vs median, 1.26 U/mL; range, 0.93 to 2.42 U/mL (p < 0.0001). In contrast, protein C activity in induced sputum was lower in asthmatic patients than in normal control subjects: median, 20%; range, 5 to 30%; vs 41%; range, 30 to 59% (p < 0.0001). VEGF level in induced sputum was positively correlated with thrombin activity in asthmatic patients (r = 0.55, p = 0.02), while inversely correlated with protein C activity (r = - 0.57, p = 0.01). Levels of basic fibroblast growth factor (bFGF), a major profibrotic factor, were also significantly higher in asthmatic patients than in normal control subjects. Moreover, thrombin activity was significantly correlated with bFGF level in asthmatic patients (r = 0.67, p = 0.003).

CONCLUSIONS

Increase in VEGF level leads to up-regulation of thrombin activity in asthmatic airways, and this elevated thrombin activity induces elevation of bFGF level. It will become to be a new strategy of asthma therapy to attenuate thrombin activity for the regulation of airway remodeling.

Perilipin 5 (PLIN5) is a lipid droplet protein and is highly expressed in oxidative tissue. Expression of the PLIN5 gene is regulated by peroxisome proliferator-activated receptor-α, fasting, and exercise. However, the effect of increased muscle PLIN5 expression on whole-body energy homeostasis remains unclear. To examine this, we developed a mouse line with skeletal muscle PLIN5 overexpression (MCK-Plin5). We show that MCK-Plin5 mice have increased energy metabolism and accumulate more intramyocellular triacylglycerol but have normal glucose and insulin tolerance. MCK-Plin5 mice fed high-fat chow manifest lower expression of inflammatory markers in their liver and increased expression of "browning" factors in adipose tissue. This muscle-driven phenotype is, at least in part, mediated by myokines; the MCK-Plin5 mice have 80-fold higher FGF21 gene expression in muscle and increased serum FGF21 concentration. The increase in FGF21 occurs mainly in muscles with a predominance of fast-twitch fibers, suggesting that fiber type-specific lipid storage may be part of the mechanism conferring metabolic protection in MCK-Plin5 mice. In conclusion, upregulating the PLIN5 level in skeletal muscle drives expression of the FGF21 gene in fast-twitch fibers and is metabolically protective. These findings provide insight into the physiology of PLIN5 and the potential contribution of its upregulation during exercise.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 9 (FGF9) is a member of secreted polypeptide families and involved in many important biological processes, including implantation and morphogenesis during embryogenesis and adult life. Recently, Fgf9-knockout mice exhibited male-to-female sex reversal, demonstrating a novel function for FGF9 in testicular development. We hypothesized that FGF9 is involved in sex development at an early embryonic stage in humans. In this study, we systematically screened sequences of the FGF9 gene in <em>21</em> XY females and 72 XX females and XY males to examine whether sequence variants of the FGF9 gene play a pathophysiological role on human gonadal dysgenesis. No mutation was identified, but a single nucleotide variant and two microsatellites were found. The allelic distribution of polymorphic microsatellite in the 3'-UTR of FGF9 between patients and controls was slightly different with Bonferroni correction (P=0.06). We further applied reporter gene system and quantitative RT-PCR to study the function of this microsatellite motif and results demonstrated that the (TG)(n) motif modulated gene expression at both pre- and post-transcriptional levels. The (TG)(14) allele, which showed a potential association with male-to-female sex reversal (odds ratio=6.08, 95% confidence interval=1.39-26.63), displayed the strongest promoter activity and longest mRNA stability. These data demonstrated that 3'-UTR microsatellite of the FGF9 is a functional polymorphism that plays dual roles in regulating FGF9 expression. Although our preliminary result suggested a possible association between FGF9 and human gonadal dysgenesis, the major limitation of small dataset in this study points out the requirement for further investigation.

BACKGROUND

The energy-burning capacity of brown adipose tissue (BAT) makes it an attractive target for use in anti-obesity therapies. Moreover, due to its ability to oxidize glucose and lipids, BAT activation has been considered a potential therapy to combat type 2 diabetes and atherogenesis.

CONCLUSIONS

BAT is mainly regulated by the sympathetic nervous system (SNS); yet, recent findings have shown a group of novel activators that act independently of the stimulation of the SNS such as cardiac natriuretic peptides, irisin, interleukin-6, β-aminoisobutyric acid and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> that could influence BAT metabolism. Several strategies are being examined to activate and recruit BAT with no side effects. In this review, we postulate that exercise might activate and recruit human BAT through the activation of SNS, heart and skeletal muscle.

UNASSIGNED

Epidemiological and well-designed exercise-based randomized controlled studies are needed to clarify if exercise is able to activate BAT in humans.

BACKGROUND

Little information is yet available on the signals involved in progenitor cell migration that precede reparative dentin synthesis. Our aim was to investigate the effect of the controlled release of fibroblast growth factor (FGF)-2 and transforming growth factor β1 (TGF-β1) on permanent teeth pulp cell proliferation and progenitor cell migration.

METHODS

FGF-2 and TGF-β1 were encapsulated into a biodegradable polymer matrix of lactide and glycolide. Human pulp cells were prepared from third molars, and progenitor cells were sorted by STRO-1. The synthesized microsphere toxicity was checked with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. The growth factor release kinetics were checked by an enzyme-linked immunosorbent assay while maintaining their biological activity and were evaluated by investigating their effects on pulp cell proliferation. Their chemotactic potential was investigated on STRO-1-sorted cells in a migration chamber on Matrigel (Cambrex Bio Science, Walkersville, MD).

RESULTS

The cell viability was unaffected by the presence of microspheres. The released amount of FGF-2 and TGF-β1 from the microspheres was maintained after 21 days. Increasing the FGF-2-loaded microsphere concentration or the release period significantly increased dental pulp cell proliferation. TGF-β1 acted as a potent chemotactic factor of STRO-1-sorted cells.

CONCLUSIONS

Encapsulating TGF-β1 and FGF-2 in a biodegradable polymer of lactide and glycolide microsphere allowed a sustained release of growth factors and provided a protection to their biological activities. Our results clearly show the usefulness of growth factor controlled release in investigating the early events of pulp/dentin regeneration. It provides additional data on the signals required for vital pulp therapy and future tissue engineering.

BACKGROUND

Lung hypoplasia in congenital diaphragmatic hernia (CDH) seems to involve impaired alveolar septation. We hypothesized that disturbed deposition of elastin and expression of fibroblast growth factor 18 (FGF18), an elastogenesis stimulus, occurs in CDH.

OBJECTIVE

To document FGF18 and elastin in human CDH and ovine surgical and rat nitrofen models and to use models to evaluate the benefit of treatments.

METHODS

Human CDH and control lungs were collected post mortem. Diaphragmatic hernia was created in sheep at 85 days; fetal lungs were collected at 139 days (term = 145 days). Pregnant rats received nitrofen at 12 days; fetal lungs were collected at 21 days (term = 22 days). Some of the sheep fetuses with hernia underwent tracheal occlusion (TO); some of the nitrofen-treated pregnant rats received vitamin A. Both treatments are known to promote lung growth.

RESULTS

Coincidental with the onset of secondary septation, FGF18 protein increased threefold in control human lungs, which failed to occur in CDH. FGF18 labeling was found in interstitial cells of septa. Elastin staining demonstrated poor septation and markedly decreased elastin density in CDH lungs. Consistently, lung FGF18 transcripts were diminished 60 and 83% by CDH in sheep and rats, respectively, and elastin density and expression were diminished. TO and vitamin A restored FGF18 and elastin expression in sheep and rats, respectively. TO restored elastin density.

CONCLUSIONS

Impaired septation in CDH is associated with decreased FGF18 expression and elastic fiber deposition. Simultaneous correction of FGF18 and elastin defects by TO and vitamin A suggests that defective elastogenesis may result, at least partly, from FGF18 deficiency.

BACKGROUND

Pulmonary pleomorphic carcinoma (PPC) is a rare type of lung cancer characterized by the poor response to conventional chemotherapy and subsequent disappointing outcomes. Therefore, it is paramount to delineate the molecular characteristics of this disease entity.

METHODS

In this study, we retrospectively examined the surgical specimens of 61 patients who underwent lung surgery. Mutational or gene amplification statuses of epidermal growth factor receptor (EGFR), k-ras, c-kit, c-met, and fibroblast growth factor receptor (FGFR) were examined using genomic DNA sequencing, real-time PCR and/or fluorescence in situ hybridization (FISH).

RESULTS

The median age was 61 years, and 50 patients were men and 11 were women. In the histologic review of epithelial component, adenocarcinoma were in 44 cases (72%), squamous cell carcinoma in 15 (25%) and large cell carcinoma in 2 patients (3%). Overall, 30 cases (49%) had any molecular alterations. Nine patients (15%) possessed EGFR deletion in exon 19 (n = 8) or L858R mutations in exon 21 (n = 1), while 3 other cases having atypical EGFR mutations. Six patients (9.8%) had k-ras mutations in exon 12, and 3 had c-kit mutations. High gene copy number of c-met was found in 11 patients (18.0%) and that of FGFR was in 6 patients (9.8%). No significant relationships were identified among the occurrence and type of mutations and patient survival or any other clinicopathological variables.

CONCLUSIONS

Given the diverse repertoire of mutational profiles observed in PPC samples, clinical trials based on accurate cancer-genotyping should be considered as a legitimate treatment scheme for this rare disease entity in the future.

Extracellular Tat protein, the transactivating <em>factor</em> of the human immunodeficiency virus type 1 (HIV-1), modulates gene expression, <em>growth</em>, and angiogenic activity in endothelial cells by interacting with the vascular endothelial <em>growth</em> <em>factor</em> (VEGF) receptor-2 (Flk-1/KDR). Recombinant Tat protein, produced as glutathione-S-transferase chimera (GST-Tat), activates mitogen-activated protein kinase (MAPK) ERK(1/2) in human, murine, and bovine endothelial cells whereas GST is ineffective. In bovine aortic endothelial cells, GST-Tat and the 165 amino acid VEGF isoform (VEGF165) induce transient ERK(1/2) phosphorylation with similar potency and kinetics. The synthetic peptide Tat(41-60), but not peptides Tat(1-<em>21</em>) and Tat(71-86), causes ERK(1/2) phosphorylation, thus implicating Tat/KDR interaction in the activation of this signalling pathway. Accordingly, GST-Tat induces ERK(1/2) phosphorylation in KDR-transfected porcine aortic endothelial cells but not in parental cells. MAPK kinase inhibitors PD098059 and U0126 prevent ERK(1/2) phosphorylation by Tat. However, they do not affect the angiogenic activity exerted by Tat in the murine Matrigel plug and chick embryo chorioallantoic membrane assays. Blocking of MAPK kinase activity impairs instead the angiogenic response to VEGF165 and to <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2). Our data demonstrate that ERK(1/2) activation following the interaction of HIV-1 Tat protein with endothelial cell Flk-1/KDR receptor does not represent an absolute requirement for a full angiogenic response to this <em>growth</em> <em>factor</em> that appears to utilize mechanism(s) at least in part distinct from those triggered by other prototypic angiogenic <em>growth</em> <em>factors</em>.

Aldehyde-terminated self-assembled monolayers (SAMs) on gold surfaces were modified with proteins and employed to capture intact living cells through specific ligand-cell surface receptor interactions. In our model system, the basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) binding receptor was targeted on baby hamster kidney (BHK-<em>21</em>) cells. Negative control and target proteins were immobilized on a gold surface by coupling protein primary amines to surface aldehyde groups. Cell-binding was monitored by phase contrast microscopy or surface plasmon resonance (SPR) imaging. The specificity of the receptor-ligand interaction was confirmed by the lack of cell binding to the negative control proteins, cytochrome c and insulin, and by the disruption of cell binding by treatment with heparitinase to destroy heparan sulfate which plays an essential role in the binding of bFGF to FGF receptors. This approach can simultaneously probe a large number of receptor-ligand interactions in cell populations and has potential for targeting and isolating cells from mixtures according to the receptors expressed on their surface.

Epidermal <em>growth</em> <em>factor</em> (EGF) responsive precursors isolated from the developing mouse striatum could be continually expanded in culture as free-floating spheres of cells for over 50 days. Under identical conditions, EGF-responsive precursors from the developing rat striatum could only be expanded for between <em>21</em> and 28 days, after which crisis ensued and there was a reduction in cell number at each passage. The outer regions of 28-day-old rat spheres contained a heterogeneous population of both dividing and dying cells while the cores were full of dying cells, many of which showed features consistent with apoptosis. <em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) alone did not lead to an expansion in rat striatal precursor cell number under the conditions used here. EGF combined with FGF-2 acted synergistically on cell <em>growth</em>, but did not prevent the final senescence and death of the rat precursors.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) was recently discovered as stress-induced myokine during mitochondrial disease and proposed as key metabolic mediator of the integrated stress response (ISR) presumably causing systemic metabolic improvements. Curiously, the precise cell-non-autonomous and cell-autonomous relevance of endogenous FGF<em>21</em> action remained poorly understood.

METHODS

We made use of the established UCP1 transgenic (TG) mouse, a model of metabolic perturbations made by a specific decrease in muscle mitochondrial efficiency through increased respiratory uncoupling and robust metabolic adaptation and muscle ISR-driven FGF<em>21</em> induction. In a cross of TG with Fgf<em>21</em>-knockout (FGF<em>21</em>(-/-)) mice, we determined the functional role of FGF<em>21</em> as a muscle stress-induced myokine under low and high fat feeding conditions.

RESULTS

Here we uncovered that FGF<em>21</em> signaling is dispensable for metabolic improvements evoked by compromised mitochondrial function in skeletal muscle. Strikingly, genetic ablation of FGF<em>21</em> fully counteracted the cell-non-autonomous metabolic remodeling and browning of subcutaneous white adipose tissue (WAT), together with the reduction of circulating triglycerides and cholesterol. Brown adipose tissue activity was similar in all groups. Remarkably, we found that FGF<em>21</em> played a negligible role in muscle mitochondrial stress-related improved obesity resistance, glycemic control and hepatic lipid homeostasis. Furthermore, the protective cell-autonomous muscle mitohormesis and metabolic stress adaptation, including an increased muscle proteostasis via mitochondrial unfolded protein response (UPR(mt)) and amino acid biosynthetic pathways did not require the presence of FGF<em>21</em>.

CONCLUSIONS

Here we demonstrate that although FGF<em>21</em> drives WAT remodeling, the adaptive pseudo-starvation response under elevated muscle mitochondrial stress conditions operates independently of both WAT browning and FGF<em>21</em> action. Thus, our findings challenge FGF<em>21</em> as key metabolic mediator of the mitochondrial stress adaptation and powerful therapeutic target during muscle mitochondrial disease.

FGF21 stimulates FGFR1c activity in cells that co-express Klothoβ (KLB); however, relatively little is known about the interaction of these receptors at the plasma membrane. We measured the dynamics and distribution of fluorescent protein-tagged KLB and FGFR1c in living cells using fluorescence recovery after photobleaching and number and brightness analysis. We confirmed that fluorescent protein-tagged KLB translocates to the plasma membrane and is active when co-expressed with FGFR1c. FGF21-induced signaling was enhanced in cells treated with lactose, a competitive inhibitor of the galectin lattice, suggesting that lattice-binding modulates KLB and/or FGFR1c activity. Fluorescence recovery after photobleaching analysis consistently revealed that lactose treatment increased KLB mobility at the plasma membrane, but did not affect the mobility of FGFR1c. The association of endogenous KLB with the galectin lattice was also confirmed by co-immunoprecipitation with galectin-3. KLB mobility increased when co-expressed with FGFR1c, suggesting that the two receptors form a heterocomplex independent of the galectin lattice. Number and brightness analysis revealed that KLB and FGFR1c behave as monomers and dimers at the plasma membrane, respectively. Co-expression resulted in monomeric expression of KLB and FGFR1c consistent with formation of a 1:1 heterocomplex. Subsequent addition of FGF21 induced FGFR1 dimerization without changing KLB aggregate size, suggesting formation of a 1:2 KLB-FGFR1c signaling complex. Overall, these data suggest that KLB and FGFR1 form a 1:1 heterocomplex independent of the galectin lattice that transitions to a 1:2 complex upon the addition of FGF21.

OBJECTIVE

Vascular endothelial growth factor (VEGF) causes widespread retinal vascular dilation, produces breakdown of the blood-retinal barrier, and is implicated in ocular neovascularization (NV). Basic fibroblast growth factor (bFGF) also has been implicated in the production of ocular NV. This study was performed to investigate the ability of simultaneous sustained intravitreal release of both VEGF and bFGF to induce robust retinal NV in the rabbit.

METHODS

Intravitreal implantation of sustained-release Hydron polymeric pellets containing both 20 microg of VEGF and 20 microg of bFGF was performed on adult male Dutch belted rabbits. In other animals either 20 microg or 50 microg bFGF-containing pellets was implanted intravitreally; also, either 20 microg VEGF or 50 microg VEGF-containing pellets was implanted. Control rabbits received either blank polymeric pellets or a pellet containing 30 microg bovine serum albumin. Eyes were examined by indirect ophthalmoscopy after surgery at 24 hrs, 48 hrs, 4 days, 7 days, 14 days, 21 days, and 28 days. Findings were documented by color fundus photography and fluorescein angiography (FA). Eyes were enucleated and prepared for histologic analysis at 28 days following intravitreal implantation of the VEGF/bFGF-containing pellets.

RESULTS

In all eyes implanted with VEGF/bFGF pellets, dilation and tortuosity of existing blood vessels were observed within 48 hrs after pellet implantation. The progression of retinal vascular changes was rapid and occurred over the entire optic disk and medullary rays between 4 and 7 days. Hemorrhage occurred as early as 14 days after VEGF/bFGF pellet implantation. In eyes with massive hemorrhage, total traction retinal detachment developed after the second week. The presence of abnormal tissues at the vitreo-retinal interface within 28 days was demonstrated by light microscopy while FA showed profuse leakage of dye from anomalous vessels within the first week. Neither bFGF-exposed eyes nor control eyes showed any vascular changes. Eyes that received only VEGF-containing pellets exhibited tortuosity of existing vessels, but neither hemorrhaging nor retinal detachment occurred.

CONCLUSIONS

These results demonstrate that retinal vascular changes leading to hemorrhaging is produced rapidly in the rabbit by simultaneous intravitreal release of both VEGF and bFGF. Understanding how these growth factors induce retinal NV may suggest novel therapeutic treatment strategies.

In preparation for studies on the <em>growth</em> <em>factor</em> requirements of normal and transformed human <em>fibroblasts</em>, we have developed a serum-free medium that supports vigorous long-term serial subculture of diploid human <em>fibroblasts</em> and allows them to form large-sized colonies with high efficiency (40 to 60%) when plated at cloning density (2 to 5 cells/cm2). This medium, which is a modification of Ham's MCDB 110 base medium with its serum replacement supplements, is relatively easy to prepare and the cost of the serum replacements is approximately the same as that of fetal bovine serum supplied at 10%. The ingredients of "Supplement B" of MCDB 110 medium were added in an ethanol solution, rather than in the form of liposomes, and were combined with bovine serum albumin (0.5%), a lipid carrier. Gelatin and fetuin were included as attachment <em>factors</em> instead of polylysine. Bioassays indicated that none of the ingredients in the medium were contaminated with either epidermal <em>growth</em> <em>factor</em> or platelet-derived <em>growth</em> <em>factor</em>. In this modified serum-free medium, which we have designated McM+SR1, diploid human <em>fibroblasts</em> grew for <em>21</em> days at the same rate as in the base medium, McM, supplemented with 10% FBS (i.e., <em>21</em> population doublings). During the next 20 days, they underwent 15 population doublings which was 75% of the rate of cells <em>growing</em> in the medium containing serum.

Hepatic metabolic gene networks were studied in dairy cattle fed control (CON, 1.34 Mcal/kg) or higher energy (overfed (OVE), 1.62 Mcal/kg) diets during the last 45 days of pregnancy. A total of 57 target genes encompassing PPARα-targets/co-regulators, hepatokines, <em>growth</em> hormone (GH)/insulin-like <em>growth</em> <em>factor</em> 1 (IGF-1) axis, lipogenesis, and lipoprotein metabolism were evaluated on -14, 7, 14, and 30 days around parturition. OVE versus CON cows were in more negative energy balance (NEB) postpartum and had greater serum non-esterified fatty acids (NEFA), β-hydroxybutyrate (BHBA), and liver triacylglycerol (TAG) concentrations. Milk synthesis rate did not differ. Liver from OVE cows responded to postpartal NEB by up-regulating expression of PPARα-targets in the fatty acid oxidation and ketogenesis pathways, along with gluconeogenic genes. Hepatokines (<em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), angiopoietin-like 4 (ANGPTL4)) and apolipoprotein A-V (APOA5) were up-regulated postpartum to a greater extent in OVE than CON. OVE led to greater blood insulin prepartum, lower NEFA:insulin, and greater lipogenic gene expression suggesting insulin sensitivity was not impaired. A lack of change in APOB, MTTP, and PNPLA3 coupled with upregulation of PLIN2 postpartum in cows fed OVE contributed to TAG accumulation. Postpartal responses in NEFA and FGF<em>21</em> with OVE support a role of this hepatokine in diminishing adipose insulin sensitivity.

The effect of PTH on chondrocyte proliferation as a function of cartilage age was examined. PTH[1-34] induced a 12- to 15-fold increase in the efficiency of colony formation in soft agar by chondrocytes from embryonic 13- to 19-d-old chickens and fetal 25-d-old rabbits with a 10-fold increase in their DNA content. It also caused a 2.5-fold increase in [3H]thymidine incorporation into DNA in fetal 25-d-old rabbit chondrocytes. No mitogenic responses to PTH were observed, however, in postnatal 7- to <em>21</em>-d-old chick chondrocytes or postnatal <em>21</em>-d-old rabbit chondrocytes. This age dependency was observed only with PTH: <em>fibroblast</em> <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>, and insulin stimulated chondrocyte proliferation irrespective of cartilage age. The absence of a mitogenic effect in postnatal chondrocytes was not due to a decrease in number or a reduction in affinity of receptors for PTH. PTH also increased [35S]sulfate incorporation into proteoglycans and the cyclic AMP level in fetal and postnatal chondrocytes, but at 100-fold higher concentrations (10(-8)-10(-7) M) than those (10(-10)-10(-9) M) required for the stimulation of cell division. These results suggest that PTH is a potent mitogen for embryonic chondrocytes, and that its mitogenic effect disappears selectively after birth.