OBJECTIVE

In rodents, brown (BAT) and white (WAT) adipose tissues are targets and expression sites for <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>). In contrast, human WAT expresses negligible levels of FGF<em>21</em>. We examined FGF<em>21</em> expression in human BAT samples, including the induced BAT found in adult patients with pheochromocytoma, and interscapular and visceral BAT from newborns.

METHODS

The expression of FGF<em>21</em> and uncoupling protein-1 (UCP1, a brown adipocyte marker), was determined by quantitative real-time-PCR and immunoblotting. The transcript levels of marker genes for developmentally-programmed BAT (zinc-finger-protein of the cerebellum-1, ZIC1) and inducible-BAT (cluster of differentiation-137, CD137) were also determined.

RESULTS

FGF<em>21</em> and UCP1 are significantly expressed in visceral adipose tissue from pheochromocytoma patients, but not in visceral fat from healthy individuals. In neonates, FGF<em>21</em> and UCP1 are both expressed in visceral and interscapular fat, and their expression levels show a significant positive correlation. Marker gene expression profiles suggest that inducible BAT is present in visceral fat from pheochromocytoma patients and neonates, whereas developmentally-programmed BAT is present in neonatal interscapular fat.

CONCLUSIONS

Human BAT, but not WAT, expresses FGF<em>21</em>. The expression of FGF<em>21</em> is especially high in inducible, also called beige/brite, neonatal BAT, but it is also found in the interscapular, developmentally-programmed, BAT of neonates.

BACKGROUND

Keloids represent a dysregulated response to cutaneous wounding that result in an excessive deposition of extracellular matrix (ECM), especially types I and III collagen. In keloid scars, the ratio of 'type I to type III collagen' varies compared to normal skin. Transforming growth factor beta (TGF-beta) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of keloid fibroblasts. However, the underlying mechanisms are poorly understood. RNA interference (RNAi) is an evolutionally conserved mechanism for repressing targeted gene expression. In mammalian cells, RNAi is mediated by small interfering RNA (siRNA). In this paper, we examined the function of Sma and Drosophila mothers against decapentaplegic homolog 3 (Smad3), recently characterized as intracellular effector of TGF-beta signalling, in keloid fibroblasts using siRNA.

METHODS

Dermal fibroblasts obtained from one female patient aged 21 years were maintained in Dulbecco's modified Eagle's medium. Cells (<6 passages) were treated with or without Smad3 siRNA and the expression levels of related genes were examined by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blot. Statistical analysis was performed using one-way ANOVA (Dunnett correction) and the Excel 7.0 software, with significance set at p<0.05.

RESULTS

The knockdown of Smad3 expression in mRNA and protein levels was confirmed using RT-PCR and Western Blot. Compared to blank, the mRNA levels of types I and III procollagen were also significantly and uniquely decreased following the reduction of Smad3 by siRNA (p<0.05). The results indicate that Smad3 plays an important role in the TGF-beta induced fibrosis in keloid. Downregulation of Smad3 expression in keloid fibroblasts can significantly decrease procollagen gene expression. SiRNA targeting Smad3 was an efficient reagent to reduce ECM deposition and attenuate process of fibrosis. It could be a new promising therapeutic approach to improve skin wound healing and inhibit progression of fibrotic conditions by interrupting the TGF-beta signal pathway.

Peroxisome proliferator-activated receptor type gamma (PPARgamma) is a subgroup of the PPAR transcription <em>factor</em> family. Recent studies indicate that loss of PPARgamma is associated with the development of pulmonary hypertension (PH). We hypothesized that the endothelial dysfunction associated with PPARgamma inhibition may play an important role in the disease process by altering cellular gene expression and signaling cascades. We utilized microarray analysis to determine if PPARgamma inhibition induced changes in gene expression in pulmonary arterial endothelial cells (PAEC). We identified 100 genes and expressed sequence tags (ESTs) that were upregulated by >1.5-fold and <em>21</em> genes and ESTs that were downregulated by >1.3-fold (P < 0.05) by PPARgamma inhibition. The upregulated genes can be broadly classified into four functional groups: cell cycle, angiogenesis, ubiquitin system, and zinc finger proteins. The genes with the highest fold change in expression: hyaluronan-mediated motility receptor (HMMR), VEGF receptor 2 (Flk-1), endothelial PAS domain protein 1 (EPAS1), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2), and caveolin-1 in PAEC were validated by real time RT-PCR. We further validated the upregulation of HMMR, Flk-1, FGF2, and caveolin-1 by Western blot analysis. In keeping with the microarray results, PPARgamma inhibition led to re-entry of cell cycle at G(1)/S phase and cyclin C upregulation. PPARgamma inhibition also exacerbated VEGF-induced endothelial barrier disruption. Finally we confirmed the downregulation of PPARgamma and the upregulation of HMMR, Flk-1, FGF2, and Cav-1 proteins in the peripheral lung tissues of an ovine model of PH. In conclusion, we have identified an array of endothelial genes modulated by attenuated PPARgamma signaling that may play important roles in the development of PH.

Among the 22 fibroblast growth factors (FGFs), FGF21 has now emerged as a key metabolic regulator. However, the mechanism whereby FGF21 mediates its metabolic actions per se remains largely unknown. Here, we show that FGF21 represses mammalian target of rapamycin complex 1 (mTORC1) and improves insulin sensitivity and glycogen storage in a hepatocyte-autonomous manner. Administration of FGF21 in mice inhibits mTORC1 in the liver, whereas FGF21-deficient mice display pronounced insulin-stimulated mTORC1 activation and exacerbated hepatic insulin resistance (IR). FGF21 inhibits insulin- or nutrient-stimulated activation of mTORC1 to enhance phosphorylation of Akt in HepG2 cells at both normal and IR condition. TSC1 deficiency abrogates FGF21-mediated inhibition of mTORC1 and augmentation of insulin signaling and glycogen synthesis. Strikingly, hepatic βKlotho knockdown or hepatic hyperactivation of mTORC1/ribosomal protein S6 kinase 1 abrogates hepatic insulin-sensitizing and glycemic-control effects of FGF21 in diet-induced insulin-resistant mice. Moreover, FGF21 improves methionine- and choline-deficient diet-induced steatohepatitis.

FGF21 acts as an inhibitor of mTORC1 to control hepatic insulin action and maintain glucose homeostasis, and mTORC1 inhibition by FGF21 has the therapeutic potential for treating IR and type 2 diabetes. (Hepatology 2016;64:425-438).

Ingestion of very low-carbohydrate ketogenic diets (KD) is associated with weight loss, lowering of glucose and insulin levels and improved systemic insulin sensitivity. However, the beneficial effects of long-term feeding have been the subject of debate. We therefore studied the effects of lifelong consumption of this diet in mice. Complete metabolic analyses were performed after 8 and 80weeks on the diet. In addition we performed a serum metabolomic analysis and examined hepatic gene expression. Lifelong consumption of KD had no effect on morbidity or mortality (KD vs. Chow, 676 vs. 630days) despite hepatic steatosis and inflammation in KD mice. The KD fed mice lost weight initially as previously reported (Kennnedy et al., 2007) and remained lighter and had less fat mass; KD consuming mice had higher levels of energy expenditure, improved glucose homeostasis and higher circulating levels of β-hydroxybutyrate and triglycerides than chow-fed controls. Hepatic expression of the critical metabolic regulators including <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> were also higher in KD-fed mice while expression levels of lipogenic enzymes such as stearoyl-CoA desaturase-1 was reduced. Metabolomic analysis revealed compensatory changes in amino acid metabolism, primarily involving down-regulation of catabolic processes, demonstrating that mice eating KD can shift amino acid metabolism to conserve amino acid levels. Long-term KD feeding caused profound and persistent metabolic changes, the majority of which are seen as health promoting, and had no adverse effects on survival in mice.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF-<em>21</em>) is a new member of the FGF super-family and an important endogenous regulator of glucose and lipid metabolism. It has been proposed as a therapeutic target for diabetes and obesity. Its function in the central nervous system (CNS) remains unknown. Previous studies from our laboratory demonstrated that aging primary neurons are more vulnerable to glutamate-induced excitotoxicity, and that co-treatment with the mood stabilizers lithium and valproic acid (VPA) induces synergistic neuroprotective effects. This study sought to identify molecule(s) involved in these synergistic effects. We found that FGF-<em>21</em> mRNA was selectively and markedly elevated by co-treatment with lithium and VPA in primary rat brain neurons. FGF-<em>21</em> protein levels were also robustly increased in neuronal lysates and culture medium following lithium-VPA co-treatment. Combining glycogen synthase kinase-3 (GSK-3) inhibitors with VPA or histone deacetylase (HDAC) inhibitors with lithium synergistically increased FGF-<em>21</em> mRNA levels, supporting that synergistic effects of lithium and VPA are mediated via GSK-3 and HDAC inhibition, respectively. Exogenous FGF-<em>21</em> protein completely protected aging neurons from glutamate challenge. This neuroprotection was associated with enhanced Akt-1 activation and GSK-3 inhibition. Lithium-VPA co-treatment markedly prolonged lithium-induced Akt-1 activation and augmented GSK-3 inhibition. Akt-1 knockdown markedly decreased FGF-<em>21</em> mRNA levels and reduced the neuroprotection induced by FGF-<em>21</em> or lithium-VPA co-treatment. In addition, FGF-<em>21</em> knockdown reduced lithium-VPA co-treatment-induced Akt-1 activation and neuroprotection against excitotoxicity. Together, our novel results suggest that FGF-<em>21</em> is a key mediator of the effects of these mood stabilizers and a potential new therapeutic target for CNS disorders.

Pulmonary oxygen toxicity plays an important role in the lung injury process that leads to the development of bronchopulmonary dysplasia. Connective tissue <em>growth</em> <em>factor</em> (CTGF) is a <em>fibroblast</em> mitogen and promoter of collagen deposition. We investigated the effects of postnatal hyperoxia on lung collagen and CTGF expression in rats. Rat pups were exposed to 7 d of >95% O2 and a further 3 wk of 60% O2. CTGF mRNA and protein expression increased after hyperoxia treatment, and the values were significantly higher in hyperoxia-exposed rats on postnatal d 7 and 14. Lung collagen levels increased as rats aged, and the values were comparable between room air-exposed and hyperoxia-exposed rats on postnatal d 7 and 14 and were significantly higher in hyperoxia-exposed rats on postnatal d <em>21</em> and 28. Increases in CTGF mRNA and protein expressions preceded the onset of increased lung collagen. These data demonstrate that CTGF is up-regulated at time points preceding the fibrotic phase of the lung injury adding credence to the hypothesis that CTGF seems to be involved in the pathogenesis of hyperoxia-induced lung fibrosis and an anti-CTGF strategy might attenuate hyperoxia-induced lung fibrosis.

BACKGROUND

Data from studies in patients with nonalcoholic steatohepatitis (NASH) suggest an increased hepatic fatty acid oxidation. We have previously shown higher fasting plasma bile acid concentrations in patients with NASH. In-vivo and in-vitro studies suggest that bile acids by binding to peroxisome proliferator-activated receptor α activate <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) and increase hepatic fatty acid oxidation.

METHODS

Plasma bile acid levels were quantified in healthy controls (n=38) and patients with biopsy-proven NASH (n=36). Plasma concentration of fatty acids, β-hydroxybutyrate, insulin, glucose, leptin, alanine aminotransferase, FGF<em>21</em>, and 8-hydroxydeoxyguanosine, a measure of oxidative stress, were measured in 16 healthy controls and 10 patients with NASH in the fasted state and in response to 3 h of infusion of intralipid. In a subgroup of these patients (n=6 each), plasma ceramide subspecies were quantified.

RESULTS

Fasting plasma bile acids, FGF<em>21</em>, and leptin concentrations were significantly higher in patients with NASH. In response to intralipid infusion there was an increase in plasma β-hydroxybutyrate and free fatty acid levels in both controls and NASH; however, the ratio of β-hydroxybutyrate/free fatty acid was higher in NASH (P=0.02). Plasma FGF<em>21</em> concentration increased in response to intralipid in patients with NASH only (P<0.01). Plasma leptin, insulin, glucose, and alanine transferase concentrations did not change in either group after infusion of intralipid. Increase in total ceramides in response to intralipid was greater in NASH.

CONCLUSIONS

Elevated bile acids and FGF<em>21</em> may be responsible for the higher hepatic fatty acid oxidation in NASH.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a novel member identified and was reported to express predominantly in pancreas, liver and adipose tissue, and relatively less in other organs, such as the testis. However, the role of FGF<em>21</em> in the testis has never been addressed. The present study examined FGF<em>21</em> expression at mRNA level by real-time RT-PCR assay in the testis of fasting and non-fasting mice or mice with type 1 diabetes that was induced with streptozotocin. We also examined the effect of Fgf<em>21</em> gene deletion or supplementation of the exogenous FGF<em>21</em> on the testicular apoptotic cell death spontaneously or induced by type 1 diabetes in FGF<em>21</em> knockout (FGF<em>21</em>-KO) mice. Deletion of Fgf<em>21</em> gene does not affect testicular cell proliferation, but significantly increases the spontaneous incidence of testicular TUNEL positive cells with increases in the Bax/Bcl2 expression ratio and apoptosis-inducing <em>factor</em> (AIF) expression. Diabetes induced significant increases in testicular TUNEL positive cells, Bax/Bcl2 expression ratio, AIF expression, CHOP and cleaved caspase-12 expression, and oxidative damage, but did not change the expression of cleaved caspase-3 and caspase-8. Deletion of Fgf<em>21</em> gene also significantly enhances diabetes-induced TUNEL positive cells along with the increased expression of Bax/Bcl2 ratio, AIF, CHOP, cleaved caspase-12, and oxidative damage, which was significantly prevented by the supplementation of exogenous FGF<em>21</em>. These results suggest that Fgf<em>21</em> gene may involve in maintaining normal spermatogenesis and also protect the germ cells from diabetes-induced apoptotic cell death probably via the prevention of diabetes-induced oxidative damage.

Non-alcoholic fatty liver disease, which is considered a hepatic manifestation of metabolic syndrome, independently increases the risks of developing cardiovascular disease (CVD) and type 2 diabetes mellitus. Recent emerging evidence suggests that a group of predominantly liver-derived proteins called hepatokines directly affect the progression of atherosclerosis by modulating endothelial dysfunction and infiltration of inflammatory cells into vessel walls. Here, we summarize the role of the representative hepatokines <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>, fetuin-A, and selenoprotein P in the progression of CVD.

The endocrine <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) requires both <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) and β-Klotho for signaling. In this study, we sought to understand the inter-molecular physical interactions in the FGF<em>21</em>/FGFR/β-Klotho complex by deleting key regions in FGFR1c or FGF<em>21</em>. Deletion of the D1 and the D1-D2 linker (the D1/linker region) from FGFR1c led to β-Klotho-independent receptor activation by FGF<em>21</em>, suggesting that there may be a direct interaction between FGF<em>21</em> and the D1/linker region-deficient FGFR1c. Consistent with this, the extracellular portion of FGFR1c lacking the D1/linker region blocked FGF<em>21</em> action in a reporter assay, presumably by binding to and sequestering FGF<em>21</em> from acting on cell surface receptor complex. In addition, the D1/linker region-deficient FGFR1c had enhanced interaction with β-Klotho. Further, we demonstrated that deletion of the D1/linker region enhanced the formation of the FGF<em>21</em>/β-Klotho/FGFR1c ternary complex in both Biacore and asymmetrical flow field flow fractionation studies. Finally, we found that the N-terminus of FGF<em>21</em> is involved in the interaction with FGFR1c and FGF<em>21</em>/β-Klotho/FGFR1c ternary complex formation. Taken together, our data suggest that the D1/linker region regulates both the FGF<em>21</em>/FGFR1c and FGFR1c/β-Klotho interaction, and a direct interaction of FGF<em>21</em> with FGFR1c may be an important step in receptor-mediated FGF<em>21</em> signaling.

The alarming prevalence of obesity has led to a better understanding of the molecular mechanisms controlling energy homeostasis. Regulation of energy intake and expenditure is more complex than previously thought, being influenced by signals from many peripheral tissues. In this sense, a wide variety of peripheral signals derived from different organs contributes to the regulation of body weight and energy expenditure. Besides the well-known role of insulin and adipokines, such as leptin and adiponectin, in the regulation of energy homeostasis, signals from other tissues not previously thought to play a role in body weight regulation have emerged in recent years. The role of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), insulin-like <em>growth</em> <em>factor</em> 1 (IGF-I), and sex hormone-binding globulin (SHBG) produced by the liver in the regulation of body weight and insulin sensitivity has been recently described. Moreover, molecules expressed by skeletal muscle such as myostatin have also been involved in adipose tissue regulation. Better known is the involvement of ghrelin, cholecystokinin, glucagon-like peptide 1 (GLP-1) and PYY(3-36), produced by the gut, in energy homeostasis. Even the kidney, through the production of renin, appears to regulate body weight, with mice lacking this hormone exhibiting resistance to diet-induced obesity. In addition, the skeleton has recently emerged as an endocrine organ, with effects on body weight control and glucose homeostasis through the actions of bone-derived <em>factors</em> such as osteocalcin and osteopontin. The comprehension of these signals will help in a better understanding of the aetiopathology of obesity, contributing to the potential development of new therapeutic targets aimed at tackling excess body fat accumulation.

OBJECTIVE

Systemic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>21</em> levels and hepatic FGF<em>21</em> production are increased in non-alcoholic fatty liver disease patients, suggesting FGF<em>21</em> resistance. We examined the effects of exenatide on FGF<em>21</em> in patients with type 2 diabetes and in a diet-induced mouse model of obesity (DIO).

METHODS

Type 2 diabetes mellitus patients (n = 24) on diet and/or metformin were randomised (using a table of random numbers) to receive additional treatment consisting of pioglitazone 45 mg/day or combined therapy with pioglitazone (45 mg/day) and exenatide (10 μg twice daily) for 12 months in an open label parallel study at the Baylor Clinic.

RESULTS

Twenty-one patients completed the entire study and were included in the analysis. Pioglitazone treatment (n = 10) reduced hepatic fat as assessed by magnetic resonance spectroscopy, despite a significant increase in body weight (Δ = 3.7 kg); plasma FGF<em>21</em> levels did not change (1.9 ± 0.6 to 2.2 ± 0.6 ng/ml [mean ± SEM]). However, combined pioglitazone and exenatide therapy (n = 11) was associated with a significant reduction of FGF<em>21</em> levels (2.3 ± 0.5 to 1.1 ± 0.3 ng/ml) and a greater decrease in hepatic fat. Besides weight gain observed in the pioglitazone-treated patients, lower extremity oedema was observed as a side effect in two of the ten patients. Three patients who received pioglitazone and exenatide combination therapy complained of significant nausea that was self-limiting and did not require them to leave the study. In DIO mice, exendin-4 for 4 weeks significantly reduced hepatic triacylglycerol content, decreased hepatic FGF<em>21</em> protein and mRNA, and enhanced phosphorylation of hepatic AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase, although no significant difference in weight and body fat was observed. Hepatic FGF<em>21</em> correlated inversely with hepatic AMPK phosphorylation

CONCLUSIONS

In type 2 diabetes mellitus, combined pioglitazone and exenatide therapy is associated with a reduction in plasma FGF<em>21</em> levels, as well as a greater decrease in hepatic fat than that achieved with pioglitazone therapy. In DIO mice, exendin-4 treatment reduces hepatic triacylglycerol and FGF<em>21</em> protein, and enhances hepatic AMPK phosphorylation, suggesting an improvement of hepatic FGF<em>21</em> resistance.

BACKGROUND

ClinicalTrials.gov NCT 01432405.

Wound healing is a dynamic process, and a variety of <em>growth</em> <em>factors</em> have a significant impact on the process. Although the WNT family has a multitude of effects on the state of various physiological pathways, the expression and role of WNT in wounded tissue have remained an enigma. The aim of this study was to assess the expression and localization of WNTs in a murine model of wound healing. RNA isolated from full-thickness cutaneous wounds from day 1 to day <em>21</em> postwounding were subjected to reverse transcription-polymerase chain reaction, and expression of WNT3, 4, 5a, and 10b were observed. Immunohistochemistry localized WNT10b to regenerating epithelial cells on day 1 and 3, and WNT4 on day 3 and 5. WNT4 also reacted with <em>fibroblast</em>-like cells beneath the epithelium. The cytoplasmic staining of beta-catenin, a WNT signaling molecule, in the epithelial cells indicates an activation of the WNT signaling pathway. Among target genes downstream of the pathway, matrix metalloproteinases (MMPs) degrade and remodel the extracellular matrix during wound healing. Gelatin zymography showed that MMP9 was expressed from day 1 to day 5. MMP-2 was continuously expressed, but maximally up-regulated at day 5. Activation of MMP-2 coincided with expression of membrane-type 1 MMP, suggesting an involvement of WNTs in this proteolytic cascade. Therefore, WNTs may contribute to the process of wound healing in a spatiotemporal manner.

The aryl hydrocarbon receptor (AHR), also known as the dioxin receptor, was originally characterized as a xenobiotic receptor that senses xenotoxicants. We investigated the endobiotic and hepatic role of AHR in fatty liver and energy metabolism and identified the endocrine <em>factor</em> that mediates the metabolic function of AHR. Wild-type and liver-specific constitutively activated human AHR transgenic mice were used to investigate the role of AHR in fatty liver and energy homeostasis. Adenovirus expressing short hairpin RNA targeting <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) were used to determine the involvement of FGF<em>21</em> in the metabolic effect of AHR. We showed that, despite their severe fatty liver, the transgenic mice were protected from diet-induced obesity and type 2 diabetes. We identified the endocrine hormone FGF<em>21</em> as a mediator for the metabolic benefit of AHR and established FGF<em>21</em> as a direct transcriptional target of AHR. Interestingly, the transactivation of FGF<em>21</em> by AHR contributed to both hepatic steatosis and systemic insulin hypersensitivity, both of which were largely abolished upon FGF<em>21</em> knockdown.

CONCLUSIONS

The AHR-FGF<em>21</em> endocrine signaling pathway establishes AHR as a pivotal environmental modifier that integrates signals from chemical exposure in the regulation of lipid and energy metabolism.

OBJECTIVE

The aim of the present study was to evaluate the pharmacokinetics/pharmacodynamics (PK/PD), safety and tolerability of single intravenous (IV) doses of PF-05231023, a long acting <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) analogue being developed for the treatment of type 2 diabetes mellitus (T2DM).

METHODS

T2DM subjects (glycosylated haemoglobin: 7.0-10.5%; on stable metformin therapy and/or diet and exercise) were randomized to receive a single dose of placebo or PF-05231023 (0.5-200 mg). Safety evaluations were performed up to 14 days after dosing. PK and PD endpoints were measured and a PK/PD model was developed for triglyceride - an early marker of drug activity.

RESULTS

No antidrug antibody or serious adverse events (AEs) were observed. The most frequent AEs were gastrointestinal but were generally mild. Plasma PF-05231023 levels peaked immediately post-IV dosing, with mean terminal half-lives of 6.5-7.7 h and 66.5- 96.6 h for intact C- and N-termini, respectively. Intact C-terminus exposures increased proportionally with increasing dose, whereas N-terminus exposures appeared to trend higher than dose-proportionally. Although no apparent effect on plasma glucose was seen, dose-dependent decreases in triglyceride were observed, with a maximum reduction of 48.5 ± 10.0% (mean ± standard deviation) for the 200 mg dose compared with a reduction of 19.1 ± 26.4% for placebo, demonstrating proof of pharmacology. Moreover, a reduction in total cholesterol and low-density lipoprotein cholesterol and an increase in high-density lipoprotein cholesterol were observed in the high-dose groups.

CONCLUSIONS

Single IV doses of PF-05231023 up to 200 mg were generally safe and well tolerated by subjects with T2DM. The observed early sign of pharmacology supports further clinical testing of PF-05231023 upon repeated administration.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) promotes insulin sensitivity but causes bone loss. It elevates bone resorption by an undefined non-osteoclast-autonomous mechanism. We have detected a pro-osteoclastogenic activity in the hepatic secretome that is increased by FGF<em>21</em> and largely attributed to insulin-like <em>growth</em> <em>factor</em> binding protein 1 (IGFBP1). Ex vivo osteoclast differentiation and in vivo bone resorption are both enhanced by recombinant IGFBP1 but suppressed by an IGFBP1-blocking antibody. Anti-IGFBP1 treatment attenuates ovariectomy-induced osteoporosis and abolishes FGF<em>21</em>-induced bone loss while maintaining its insulin-sensitizing metabolic benefit. Mechanistically, IGFBP1 functions via its RGD domain to bind to its receptor integrin β1 on osteoclast precursors, thereby potentiating RANKL-stimulated Erk-phosphorylation and NFATc1 activation. Consequently, osteoclastic integrin β1 deletion confers resistance to the resorption-enhancing effects of both IGFBP1 and FGF<em>21</em>. Therefore, the hepatokine IGFBP1 is a critical liver-bone hormonal relay that promotes osteoclastogenesis and bone resorption as well as an essential mediator of FGF<em>21</em>-induced bone loss.

OBJECTIVE

Intestinal fibrosis is a potentially serious complication of inflammatory bowel disease and its pathophysiology is still unclear. This review will discuss recent developments relating to sources of fibroblasts in intestinal inflammation, mediators that modulate fibroblast activation and function, as well as new clinical, laboratory, endoscopic and radiological studies aimed at improving diagnosis and management of intestinal fibrosis in inflammatory bowel disease.

RESULTS

The fibroblast remains the central cell responsible for intestinal fibrosis in inflammatory bowel disease and transforming growth factor-beta1 is still the most potent pro-fibrogenic cytokine. Novel mediators, however, are being identified that modulate fibroblast function, such as interleukin-13, interleukin-21, galectin-3, osteopontin, Wnt and toll-like receptor ligands, and anti-tumor necrosis factor-alpha agents. New fibroblast sources are being identified, such as fibrocytes, and new mechanisms of fibroblast generation, like epithelial- and endothelial-to-mesenchymal transition. Animal models of intestinal fibrosis are still few, but new ways to induce gut fibrosis are being explored. Serological markers indicating a clinically complicated course that includes intestinal fibrosis are promising and are being tested in adult and pediatric populations, particularly in Crohn's disease. Video capsule endoscopy, the Given Patency capsule, double balloon enteroscopy, and computed tomographic enteroscopy are some of the new modalities being developed to assess the risk and improve the diagnosis of intestinal fibrosis. Novel therapeutic approaches include endoscopic balloon dilatation with conventional and double balloon enteroscopy, and local injection of glucocorticoids and tumor necrosis factor-alpha blockers, showing partial but encouraging success.

CONCLUSIONS

More studies are needed to improve knowledge of the pathophysiology of intestinal fibrosis if better preventive, diagnostic and therapeutic measures are to be expected in the near future.

Exosomes released from skeletal muscle cells play important roles in myogenesis and muscle development via the transfer of specific signal molecules. In this study, we investigated whether exosomes secreted during myotube differentiation from human skeletal myoblasts (HSkM) could induce a cellular response from human adipose-derived stem cells (HASCs) and enhance muscle regeneration in a muscle laceration mouse model. The exosomes contained various signal molecules including myogenic <em>growth</em> <em>factors</em> related to muscle development, such as insulin-like <em>growth</em> <em>factors</em> (IGFs), hepatocyte <em>growth</em> <em>factor</em> (HGF), <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2), and platelet-derived <em>growth</em> <em>factor</em>-AA (PDGF-AA). Interestingly, exosome-treated HASCs fused with neighboring cells at early time points and exhibited a myotube-like phenotype with increased expression of myogenic proteins (myosin heavy chain and desmin). On day <em>21</em>, mRNAs of terminal myogenic genes were also up-regulated in exosome-treated HASCs. Moreover, in vivo studies demonstrated that exosomes from differentiating HSkM reduced the fibrotic area and increased the number of regenerated myofibers in the injury site, resulting in significant improvement of skeletal muscle regeneration. Our findings suggest that exosomes act as a biochemical cue directing stem cell differentiation and provide a cell-free therapeutic approach for muscle regeneration.

Phyllodes tumors of breast, even histologically diagnosed as benign, can recur locally and have metastatic potential. Histologic markers only have limited value in predicting the clinical behavior of phyllodes tumors. It remains unknown what drives the malignant progression of phyllodes tumors. We found that the expression of myo<em>fibroblast</em> markers, α-smooth muscle actin (α-SMA), <em>fibroblast</em> activation protein (FAP), and stromal cell-derived <em>factor</em>-1 (SDF-1), is progressively increased in the malignant progression of phyllodes tumors. Microarray showed that miR-<em>21</em> was one of the most significantly upregulated microRNAs in malignant phyllodes tumors compared with benign phyllodes tumors. In addition, increased miR-<em>21</em> expression was primarily localized to α-SMA-positive myo<em>fibroblast</em>s. More importantly, α-SMA and miR-<em>21</em> are independent predictors of recurrence and metastasis, with their predictive value of recurrence better than histologic grading. Furthermore, miR-<em>21</em> mimics promoted, whereas miR-<em>21</em> antisense oligos inhibited, the expression of α-SMA, FAP, and SDF-1, as well as the proliferation and invasion of primary stromal cells of phyllodes tumors. The ability of miR-<em>21</em> to induce myo<em>fibroblast</em> differentiation was mediated by its regulation on Smad7 and PTEN, which regulate the migration and proliferation, respectively. In breast phyllodes tumor xenografts, miR-<em>21</em> accelerated tumor <em>growth</em>, induced myo<em>fibroblast</em> differentiation, and promoted metastasis. This study suggests an important role of myo<em>fibroblast</em> differentiation in the malignant progression of phyllodes tumors that is driven by increased miR-<em>21</em>.

BACKGROUND

Therapeutic neovascularization using high growth factor concentrations may lead to transient vessel formation and abnormal microvascular structure. The goal of this study was to quantify temporal and concentration effects of fibroblast growth factor-1 (FGF-1) on the persistence and morphology of microvascular networks.

METHODS

Endothelial cells were incubated in suspension culture forming aggregates that were embedded in fibrin glue (FG) and stimulated with varying concentrations of FGF-1 with of heparin. Capillary networks formed were quantified for 21 days.

RESULTS

High FGF-1 concentrations resulted in rapid and intense sprout formation, with excessive branching. At later times, these vessels regressed, with cellular debris in former vessel locations. At later times, the 1-ng/mL group surpassed the high concentration groups with continuous sprout growth and complete FG vascularization by 23 days.

CONCLUSIONS

Sustained low levels of FGF-1 maintained a persistent microvascular network response, whereas higher levels resulted in abnormal phenotype followed by vessel regression.

Antley-Bixler syndrome (ABS, MIM 207410) is a skeletal abnormality syndrome primarily affecting head and limbs. Little is known of the origin of the condition but inactivating mutations in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR2) has been found in some patients. Genital ambiguity is seen occasionally in this condition, suggesting possible disordered steroidogenesis in early pregnancy. We report the steroid excretion of eight patients diagnosed with the syndrome and one with a related condition, a mild phenotype of the disorder since skeletal and genital abnormalities were not evident. The steroid excretion pattern was consistent and very distinctive in all nine patients. Metabolites of the two primary precursors of steroid hormones, pregnenolone and progesterone, were elevated as were the classical diagnostic metabolites for 17- and <em>21</em>-hydroxylase deficiencies. Cortisol production was typically within the normal range but generally had blunted response to ACTH. Androgen metabolite excretion tends to be low in patients over 2 months of age, but may be elevated in the newborn period. The metabolome suggested attenuated steroid hydroxylation (including 17,20-lyase activity) although underlying cause is yet to be established. Mutations in CYP17 and CYP<em>21</em> have not been found and currently the prime suspect is an abnormality in an essential redox partner (P450 oxidoreductase). This paper proposes use of the distinctive steroid metabolome as the primary biochemical parameter for diagnosis of ABS, at least the form not associated with FGFR2 mutations.

OBJECTIVE

The purpose is to identify gene expression patterns induced by docetaxelin head and neck squamous carcinoma (HNSCC) cells using high throughput techniques.

METHODS

HNSCC cells were treated with docetaxel or solvent. After mRNA extraction, cDNA fluorescent (Cy3 or Cy5)-labeled probes were synthesized. Then, Cy3 and Cy5-labeled samples were hybridized onto a microarray slide. The fluorescent images were scanned and analyzed for quantification. PowerBlot immunoblotting technique was used to measure protein expression level. Using this dual approach, we focused on genes in established pathways (cell cycle, apoptosis, angiogenesis, and signal transduction) of tumorigenesis and confirmed these results with conventional techniques.

RESULTS

Using cDNA microarray, we found that docetaxel altered the expression of >100 genes in HNSCC cells. A total of 153 of 1191 genes was found to have altered expression in either HN12 (n = 102), HN30 (n = 72), or both (n = <em>21</em>) by docetaxel. For the PowerBlot analysis, a subset of genes (n = 46) in the cDNA microarray analysis and an additional 98 genes in the cell cycle, apoptosis, angiogenesis, and signal transduction pathways were chosen. We found that PowerBlot data agreed with cDNA microarray in 65% of genes examined. The expression of a cell cycle inhibitor (p19) and promoters (cyclin A, cyclin B1, and cyclin E2F) were increased and decreased, respectively. Apoptosis induced by docetaxel was independent of p53 and, in part, related to increased Fas expression. Both vascular endothelial <em>growth</em> <em>factor</em> secretion and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> expression were inhibited by docetaxel, whereas thrombospondin-1 expression was increased by docetaxel. Epidermal <em>growth</em> <em>factor</em> receptor, activated epidermal <em>growth</em> <em>factor</em> receptor, and activated c-Jun NH(2)-terminal kinase expression was lowered by docetaxel. Activated extracellular signal-regulated kinase was elevated by docetaxel, but not total extracellular signal-regulated kinase levels.

CONCLUSIONS

The identification of altered gene expression induced by docetaxel demonstrates additional biological activity in HNSCC cells, and the altered expression of these genes may serve as potential biomarkers to both predict clinical activity and provide information regarding potential efficacy of adding novel agents.

The aim of this study was to examine the mechanism of interferon alpha (IFN-alpha) on inhibition of metastasis and recurrence of hepatocellular carcinoma (HCC). Nude mice bearing human HCC xenografts with high metastatic potential (LCI-D20) underwent curative resection of tumors on postimplant day 11. IFN-alpha was begun the next day at different dosages given subcutaneously for 35 consecutive days; normal saline solution was injected into the control mice. The mice were killed 48 hours after the final treatment, and the parameters were evaluated. The HCC intrahepatic recurrence rate, the size of the recurrent lesions, the rate of lung metastasis, the serum vascular endothelial <em>growth</em> <em>factor</em> level, and the microvessel density (immunohistochemistry) were as follows: 100%, <em>21</em>36+/-794 mm(3)(mean+/-standard deviation), 100%, 265.7+/-154.7 pg/ml, and 144+/-37/HP, respectively, in the control mice, whereas these same values were 62.5%, 89+/-45 mm(3), 12.5%, 53.3+/-9.9 pg/ml, and 86+/-25/HP, respectively, in the IFN-alpha 1.5 x 10(7)U/kg treatment group (P<0.05) and 26.7%, 46+/-<em>21</em> mm(3), 0%, 65.2+/-17.9 pg/ml, and 39+/-14/HP in the IFN-alpha 3 x 10(7)U/kg treatment group, respectively (P<0.05). However, a significant difference was not found in the serum levels of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> among the control and IFN-alpha treatment groups. IFN-alpha inhibits metastasis and recurrence of human HCC after curative resection in nude mice mediated by antiangiogenesis through downregulating expression of vascular endothelial <em>growth</em> <em>factor</em> but not basic <em>fibroblast</em> <em>growth</em> <em>factor</em>.