Three forms of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), initiated at an AUG (18 kDa) and two CUG (<em>21</em> and 22.5 kDa) start codons, were produced following transfection of COS cells with human hepatoma bFGF cDNA. The subcellular localization of the different forms was investigated directly or by using chimeric genes constructed by fusion of the bFGF and chloramphenicol acetyltransferase open reading frames. The AUG-initiated proteins were cytoplasmic, while the CUG-initiated forms were nuclear. The signal sequence responsible for the nuclear localization of bFGF is contained within 37 amino acid residues between the second CUG and the AUG start codons. Alternative initiation of translation regulates the subcellular localization of bFGF and thus could modulate its role in cell <em>growth</em> and differentiation control.

The effects of insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) and insulin on bone matrix synthesis and bone cell replication were studied in cultured <em>21</em>-day-old fetal rat calvariae. Histomorphometry techniques were developed to measure the incorporation of [2,3-3H]proline and [methyl-3H]thymidine into bone matrix and bone cell nuclei, respectively, using autoradiographs of sagittal sections of calvariae cultured with IGF-I, insulin, or vehicle for up to 96 h. To confirm an effect on bone formation, IGF-I was also studied for its effects on [3H]proline incorporation into collagenase-digestible protein (CDP) and noncollagen protein and on [3H]thymidine incorporation into acid-precipitable material (DNA). IGF-I at 10(-9)-10(-7) M significantly increased the rate of bone matrix apposition and CDP after 24 h by 45-50% and increased cell labeling by 8-fold in the osteoprogenitor cell zone, by 4-fold in the osteoblast cell zone, and by 2-fold in the periosteal <em>fibroblast</em> zone. Insulin at 10(-9)-10(-6) M also increased matrix apposition rate and CDP by 40-50%, but increased cell labeling by 2-fold only at a concentration of 10(-7) M or higher and then only in the osteoprogenitor cell zone. When hydroxyurea was added to IGF-I-treated bones, the effects of IGF-I on DNA synthesis were abolished, but the increase in bone matrix apposition induced by IGF-I was only partly diminished. In conclusion, IGF-I stimulates matrix synthesis in calvariae, an effect that is partly, although not completely, dependent on its stimulatory effect on DNA synthesis.

<AbstractText><em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) 2 gene alterations are involved in the pathogenesis of cholangiocarcinoma. Pemigatinib is a selective, potent, oral inhibitor of FGFR1, 2, and 3. This study evaluated the safety and antitumour activity of pemigatinib in patients with previously treated, locally advanced or metastatic cholangiocarcinoma with and without FGFR2 fusions or rearrangements.</AbstractText><AbstractText>In this multicentre, open-label, single-arm, multicohort, phase 2 study (FIGHT-202), patients aged 18 years or older with disease progression following at least one previous treatment and an Eastern Cooperative Oncology Group (ECOG) performance status of 0-2 recruited from 146 academic or community-based sites in the USA, Europe, the Middle East, and Asia were assigned to one of three cohorts: patients with FGFR2 fusions or rearrangements, patients with other FGF/FGFR alterations, or patients with no FGF/FGFR alterations. All enrolled patients received a starting dose of 13·5 mg oral pemigatinib once daily (<em>21</em>-day cycle; 2 weeks on, 1 week off) until disease progression, unacceptable toxicity, withdrawal of consent, or physician decision. The primary endpoint was the proportion of patients who achieved an objective response among those with FGFR2 fusions or rearrangements, assessed centrally in all patients who received at least one dose of pemigatinib. This study is registered with ClinicalTrials.gov, NCT02924376, and enrolment is completed.</AbstractText><AbstractText>Between Jan 17, 2017, and March 22, 2019, 146 patients were enrolled: 107 with FGFR2 fusions or rearrangements, 20 with other FGF/FGFR alterations, 18 with no FGF/FGFR alterations, and one with an undetermined FGF/FGFR alteration. The median follow-up was 17·8 months (IQR 11·6-<em>21</em>·3). 38 (35·5% [95% CI 26·5-45·4]) patients with FGFR2 fusions or rearrangements achieved an objective response (three complete responses and 35 partial responses). Overall, hyperphosphataemia was the most common all-grade adverse event irrespective of cause (88 [60%] of 146 patients). 93 (64%) patients had a grade 3 or worse adverse event (irrespective of cause); the most frequent were hypophosphataemia (18 [12%]), arthralgia (nine [6%]), stomatitis (eight [5%]), hyponatraemia (eight [5%]), abdominal pain (seven [5%]), and fatigue (seven [5%]). 65 (45%) patients had serious adverse events; the most frequent were abdominal pain (seven [5%]), pyrexia (seven [5%]), cholangitis (five [3%]), and pleural effusion (five [3%]). Overall, 71 (49%) patients died during the study, most frequently because of disease progression (61 [42%]); no deaths were deemed to be treatment related.</AbstractText><AbstractText>These data support the therapeutic potential of pemigatinib in previously treated patients with cholangiocarcinoma who have FGFR2 fusions or rearrangements.</AbstractText><AbstractText>Incyte Corporation.</AbstractText>

OBJECTIVE

The hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) exerts diverse, beneficial effects on energy balance and insulin sensitivity when administered systemically to rodents with diet-induced obesity (DIO). The current studies investigate whether central FGF<em>21</em> treatment recapitulates these effects.

METHODS

After preliminary dose-finding studies, either saline vehicle or recombinant human FGF<em>21</em> (0.4 microg/day) was infused continuously for 2 weeks into the lateral cerebral ventricle of male Wistar rats rendered obese by high-fat feeding. Study end points included measures of energy balance (body weight, body composition, food intake, energy expenditure, and circulating and hepatic lipids) and glucose metabolism (insulin tolerance test, euglycemic-hyperinsulinemic clamp, and hepatic expression of genes involved in glucose metabolism).

RESULTS

Compared with vehicle, continuous intracerebroventricular infusion of FGF<em>21</em> increased both food intake and energy expenditure in rats with DIO, such that neither body weight nor body composition was altered. Despite unchanged body fat content, rats treated with intracerebroventricular FGF<em>21</em> displayed a robust increase of insulin sensitivity due to increased insulin-induced suppression of both hepatic glucose production and gluconeogenic gene expression, with no change of glucose utilization.

CONCLUSIONS

FGF<em>21</em> action in the brain increases hepatic insulin sensitivity and metabolic rate in rats with DIO. These findings identify the central nervous system as a potentially important target for the beneficial effects of FGF<em>21</em> in the treatment of diabetes and obesity.

The presence of mRNAs for vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and a VEGF-related protein, placenta <em>growth</em> <em>factor</em> (PIGF) was examined in 29 cases of renal cell carcinoma tissues and adjacent normal kidney tissues and in 4 human renal cell carcinoma cell lines. Northern blot analysis showed that 26 of 27 hypervascular renal cell carcinoma tissues (96%) exhibited a markedly elevated level (3-13 fold) of VEGF mRNA compared to the adjacent normal kidney tissues. Even tumors of small size, whenever they were hypervascular, overexpressed VEGF mRNA. We also demonstrated that mRNA for PIGF was expressed in <em>21</em> of 23 hypervascular renal cell carcinoma tissues (91%) but was not detected in the adjacent normal kidney tissues. Two hypovascular carcinoma tissues neither overexpressed VEGF mRNA nor had PIGF mRNA. VEGF mRNA was detected in four human renal cell carcinoma cell lines, while PIGF mRNA was not. There was no difference in the level of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> mRNA between tumor tissues and normal kidney tissue, although our previous study demonstrated elevated basic <em>fibroblast</em> <em>growth</em> <em>factor</em> protein in the serum of renal cell carcinoma patients (K. Fujimoto et al., Biochem. Biophys. Res. Commun., 180: 386-392, 1991). Taken together, these results suggest that VEGF, PIGF, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> are cooperatively working to increase the angiogenesis in renal cell carcinoma in vivo.

Prematurely born infants who require oxygen therapy often develop bronchopulmonary dysplasia (BPD), a debilitating disorder characterized by pronounced alveolar hypoplasia. Hyperoxic injury is believed to disrupt critical signaling pathways that direct lung development, causing BPD. We investigated the effects of normobaric hyperoxia on transforming <em>growth</em> <em>factor</em> (TGF)-beta and bone morphogenetic protein (BMP) signaling in neonatal C57BL/6J mice exposed to <em>21</em>% or 85% O(2) between postnatal days P1 and P28. <em>Growth</em> and respiratory compliance were significantly impaired in pups exposed to 85% O(2), and these pups also exhibited a pronounced arrest of alveolarization, accompanied by dysregulated expression and localization of both receptor (ALK-1, ALK-3, ALK-6, and the TGF-beta type II receptor) and Smad (Smads 1, 3, and 4) proteins. TGF-beta signaling was potentiated, whereas BMP signaling was impaired both in the lungs of pups exposed to 85% O(2) as well as in MLE-12 mouse lung epithelial cells and NIH/3T3 and primary lung <em>fibroblasts</em> cultured in 85% O(2). After exposure to 85% O(2), primary alveolar type II cells were more susceptible to TGF-beta-induced apoptosis, whereas primary pulmonary artery smooth muscle cells were unaffected. Exposure of primary lung <em>fibroblasts</em> to 85% O(2) significantly enhanced the TGF-beta-stimulated production of the alpha(1) subunit of type I collagen (Ialpha(1)), tissue inhibitor of metalloproteinase-1, tropoelastin, and tenascin-C. These data demonstrated that hyperoxia significantly affects TGF-beta/BMP signaling in the lung, including processes central to septation and, hence, alveolarization. The amenability of these pathways to genetic and pharmacological manipulation may provide alternative avenues for the management of BPD.

Transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) plays a central role in promoting extracellular matrix protein deposition by promoting the transformation of <em>fibroblasts</em> to myo<em>fibroblasts</em>. To gain new insights into the transcriptional programs involved, we profiled human fetal lung <em>fibroblast</em> global gene expression in response to TGF-beta1 up to 24 hours using oligonucleotide microarrays. In this report, we present data for 146 genes that were up-regulated at least twofold at two time points. These genes group into several major functional categories, including genes involved in cytoskeletal reorganization (n = 30), matrix formation (n = 25), metabolism and protein biosynthesis (n = 27), cell signaling (n = <em>21</em>), proliferation and survival (n = 13), gene transcription (n = 9), and of uncertain function (n = <em>21</em>). For 80 of these genes, this is the first report that they are TGF-beta1-responsive. The early induction of two members of the inhibitor of differentiation (ID) family of transcriptional regulators, ID1 and ID3, was followed by the up-regulation of a number of genes that are usually expressed by highly differentiated smooth muscle cells, including smooth muscle myosin heavy chain, basic calponin, and smoothelin. These findings were confirmed at the protein level for primary adult lung <em>fibroblasts</em>. ID1 further behaved like a typical immediate-early gene and, unlike ID3, was expressed and induced at the protein level. Immunohistochemical analysis showed that ID1 was highly expressed by (myo)<em>fibroblasts</em> within fibrotic foci in experimentally induced pulmonary fibrosis. ID1 acts as a dominant-negative antagonist of basic helix-loop-helix transcription <em>factors</em> that drive cell lineage commitment and differentiation. These findings have important implications for our understanding of <em>fibroblast</em> transcriptional programming in response to TGF-beta1 during development, oncogenesis, tissue repair, and fibrosis.

An increased circulating level of <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) is an independent risk <em>factor</em> for mortality, cardiovascular disease, and progression of chronic kidney disease (CKD), but its role in transplant allograft and patient survival is unknown. We tested the hypothesis that increased FGF23 is an independent risk <em>factor</em> for all-cause mortality and allograft loss in a prospective cohort of 984 stable kidney transplant recipients. At enrollment, estimated GFR (eGFR) was 51 ± <em>21</em> ml/min per 1.73 m(2) and median C-terminal FGF23 was 28 RU/ml (interquartile range, 20 to 43 RU/ml). Higher FGF23 levels independently associated with increased risk of the composite outcome of all-cause mortality and allograft loss (full model hazard ratio: 1.46 per SD increase in logFGF23, 95% confidence interval: 1.28 to 1.68, P<0.001). The results were similar for each component of the composite outcome and in all sensitivity analyses, including prespecified analyses of patients with baseline eGFR of 30 to 90 ml/min per 1.73 m(2). In contrast, other measures of phosphorus metabolism, including serum phosphate and parathyroid hormone (PTH) levels, did not consistently associate with outcomes. We conclude that a high (or elevated) FGF23 is an independent risk <em>factor</em> for death and allograft loss in kidney transplant recipients.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>) is a novel endocrine and paracrine regulator of metabolic homeostasis. The aim of our study was to measure its serum concentrations in patients with obesity, obesity and type 2 diabetes mellitus (T2DM) and healthy subjects (C), and to assess the changes of its circulating levels and mRNA expression after dietary and pharmacological interventions.

METHODS

We measured biochemical parameters, serum FGF<em>21</em>, adiponectin, leptin and insulin levels by commercial ELISA and RIA kits, and mRNA expression in the liver, subcutaneous and visceral fat by RT PCR in 26 obese patients, 11 T2DM patients and 32 control subjects. The interventions were acute hyperinsulinaemia during isoglycaemic-hyperinsulinaemic clamp, very low calorie diet (VLCD) and 3 months treatment with PPAR-alpha agonist fenofibrate.

RESULTS

Baseline serum FGF<em>21</em> levels were significantly higher in both obese and T2DM patients relative to healthy controls. FGF<em>21</em> levels in obesity did not significantly differ from T2DM group. Both 3 weeks of VLCD and 3 months of fenofibrate treatment significantly increased FGF<em>21</em> levels. FGF<em>21</em> mRNA expression in visceral fat was twofold higher in obesity relative to C group, while it did not differ in subcutaneous fat. VLCD significantly increased FGF<em>21</em> mRNA expression in subcutaneous fat of obesity. 3-h hyperinsulinaemia during the clamp increased FGF<em>21</em> levels in T2DM but not in C group.

CONCLUSIONS

An increase in FGF<em>21</em> levels after VLCD and fenofibrate treatment may contribute to positive metabolic effect of these interventions and suggests the possibility of direct positive metabolic effects of FGF<em>21</em> in humans.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is an atypical member of the FGF family that functions as an endocrine hormone. Pharmacologic studies show that FGF<em>21</em> has broad metabolic actions in obese rodents and primates that include enhancing insulin sensitivity, decreasing triglyceride concentrations, and causing weight loss. In lean rodents, FGF<em>21</em> expression is strongly induced in liver by prolonged fasting through a mechanism that involves the nuclear receptor peroxisome proliferator-activated receptor alpha. FGF<em>21</em>, in turn, induces the transcriptional coactivator protein peroxisome proliferator-activated receptor gamma coactivator protein 1alpha and stimulates hepatic gluconeogenesis, fatty acid oxidation, and ketogenesis. FGF<em>21</em> also blocks somatic <em>growth</em> and sensitizes mice to a hibernation-like state of torpor. Thus, FGF<em>21</em> plays a key role in eliciting and coordinating the adaptive starvation response. Interestingly, FGF<em>21</em> expression is induced in white adipose tissue by peroxisome proliferator-activated receptor gamma, which suggests that it also regulates metabolism in the fed state. This article highlights recent advances in our understanding of FGF<em>21</em>'s pharmacologic and physiologic actions.

Fibroblast growth factor (FGF21) plays an important role in regulating hepatic oxidation of fatty acids and gluconeogenesis in response to fasting and during consumption of a ketogenic diet. However, the metabolic pathways through which FGF21 regulates hepatic function are not well defined. To identify the effects of FGF21 on the liver in vivo, we administered FGF21 to mice and analyzed acute effects on signaling and gene expression. We found that FGF21 acts directly on the liver to stimulate phosphorylation of fibroblast growth factor receptor substrate 2 and ERK1/2. Acute FGF21 treatment induced hepatic expression of key regulators of gluconeogenesis, lipid metabolism, and ketogenesis including glucose-6-phosphatase, phosphoenol pyruvate carboxykinase, 3-hydroxybutyrate dehydrogenase type 1, and carnitine palmitoyltransferase 1α. In addition, injection of FGF21 was associated with decreased circulating insulin and free fatty acid levels. FGF21 treatment induced mRNA and protein expression of peroxisome proliferator-activated receptor-γ coactivator (PGC-1α), suggesting that PGC-1α may play a role in regulating FGF21 action. However, studies using mice with liver-specific ablation of PGC-1α revealed the same regulation of gluconeogenic gene expression by FGF21 as seen in wild-type mice, indicating that PGC-1α is not necessary for the effect of FGF21 on glucose metabolism. These data demonstrate that FGF21 acts directly on the liver to modulate hepatic metabolism. The direct effects we examined are not dependent on PGC-1α. In addition, FGF21 treatment is associated with decreased serum insulin levels that my affect hepatic function.

The implantation of a blastocyst into a receptive uterus is associated with a series of events, namely the attachment reaction followed by decidualization of the stroma. Previous studies established that the gene encoding heparin-binding EGF-like <em>growth</em> <em>factor</em> (HB-EGF) is expressed in the luminal epithelium solely at the site of blastocyst apposition preceding the attachment reaction. We report here the expression during implantation of <em>21</em> genes encoding other signaling proteins, including those belonging to the Bone morphogenetic protein (BMP), <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), WNT, and Hedgehog (HH) pathways. We find that the attachment reaction is associated with a localized stromal induction of genes encoding BMP-2, FGF-2, and WNT-4. Despite efforts by many investigators, a simple in vitro model of implantation is not yet available to study either the hierarchy of the events triggered in the uterus by the embryo or the function of individual signaling proteins. We have therefore approached these questions by introducing beads loaded with purified <em>factors</em> into the receptive uterus. We show that beads soaked in HB-EGF or insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1), but not other proteins, induce many of the same discrete local responses elicited by the blastocyst, including increased localized vascular permeability, decidualization, and expression of Bmp2 at the sites of the beads. By contrast, the expression domains of Indian hedgehog (Ihh), patched, and noggin become restricted as decidualization proceeds. Significantly, beads containing BMP-2 do not themselves elicit an implantation response but affect the spacing of implantation sites induced by blastocysts cotransferred with the beads.

Peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor expressed in tissues with high oxidative activity that plays a central role in metabolism. In this work, we investigated the effect of hepatocyte PPARα on non-alcoholic fatty liver disease (NAFLD).

We constructed a novel hepatocyte-specific PPARα knockout (Pparα(hep-/-)) mouse model. Using this novel model, we performed transcriptomic analysis following fenofibrate treatment. Next, we investigated which physiological challenges impact on PPARα. Moreover, we measured the contribution of hepatocytic PPARα activity to whole-body metabolism and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> production during fasting. Finally, we determined the influence of hepatocyte-specific PPARα deficiency in different models of steatosis and during ageing.

Hepatocyte PPARα deletion impaired fatty acid catabolism, resulting in hepatic lipid accumulation during fasting and in two preclinical models of steatosis. Fasting mice showed acute PPARα-dependent hepatocyte activity during early night, with correspondingly increased circulating free fatty acids, which could be further stimulated by adipocyte lipolysis. Fasting led to mild hypoglycaemia and hypothermia in Pparα(hep-/-) mice when compared with Pparα(-/-) mice implying a role of PPARα activity in non-hepatic tissues. In agreement with this observation, Pparα(-/-) mice became overweight during ageing while Pparα(hep-/-) remained lean. However, like Pparα(-/-) mice, Pparα(hep-/-) fed a standard diet developed hepatic steatosis in ageing.

Altogether, these findings underscore the potential of hepatocyte PPARα as a drug target for NAFLD.

Peroxisome proliferator-activated receptor gamma (PPARgamma) activity is regulated through association with ligands that include the thiazolidinedione class of antidiabetic drugs, as well as derivatives of polyunsaturated fatty acids. Induction of PPARgamma target gene expression involves ligand-dependent reconfiguration of the ligand-binding domain (LBD), followed by recruitment of specific transcriptional coactivators. In this study, we have identified an amino acid (F372) within helix 7 of the LBD that is required for the response of PPARgamma to endogenous ligands. Additionally, the data show that this amino acid is also required for expression of a novel subset of adipocyte genes (group 2), including <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), and that the FGF<em>21</em> gene is a direct target of PPARgamma. Expression of the group 2 genes is selectively repressed by the NAD-dependent deacetylase SIRT1 in mature 3T3-L1 adipocytes, since knockdown of SIRT1 through the constitutive expression of a corresponding RNA interference enhances their expression without affecting the expression of classic adipogenic genes, such as adiponectin and FABP4/aP2. It appears that many of the group 2 genes repressed by SIRT1 in mature adipocytes correspond to the same set of genes that are selectively activated by treatment of fat cells with the PPARgamma ligand, troglitazone. These data support a role for helix 7 of the LBD of PPARgamma in regulating adipocyte function and suggest that inhibition of SIRT1 in adipocytes induces the same insulin-sensitizing action as PPARgamma ligands.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> is an endocrine <em>factor</em>, secreted mainly by the liver, that exerts metabolic actions that favour glucose metabolism. Its role in the heart is unknown. Here we show that Fgf<em>21</em>(-/-) mice exhibit an increased relative heart weight and develop enhanced signs of dilatation and cardiac dysfunction in response to isoproterenol infusion, indicating eccentric hypertrophy development. In addition, Fgf<em>21</em>(-/-) mice exhibit enhanced induction of cardiac hypertrophy markers and pro-inflammatory pathways and show greater repression of fatty acid oxidation. Most of these alterations are already present in Fgf<em>21</em>(-/-) neonates, and treatment with <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> reverses them in vivo and in cultured cardiomyocytes. Moreover, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> is expressed in the heart and is released by cardiomyocytes. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> released by cardiomyocytes protects cardiac cells against hypertrophic insults. Therefore, the heart appears to be a target of systemic, and possibly locally generated, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>, which exerts a protective action against cardiac hypertrophy.

Angiotensin II (ANG II), generated by activation of local renin-angiotensin systems, is believed to play an important role in tissue repair and remodeling, in part via transforming <em>growth</em> <em>factor</em>-beta (TGF-beta). Angiotensin-converting enzyme (ACE) inhibitors have been shown to abrogate experimental lung injury via a number of potential mechanisms; however, the potentially fibroproliferative role for ANG II in the lung has not been characterized. We hypothesized that, after lung injury, ANG II would stimulate <em>fibroblast</em> procollagen synthesis and promote lung collagen deposition in rats. In vitro, ANG II was a potent inducer of procollagen production in human lung <em>fibroblasts</em> via activation of the type 1 receptor and, at least in part, via the autocrine action of TGF-beta. After bleomycin-induced lung injury, an increase in lung ANG II concentration was observed by day 3 that preceded increases in lung collagen and was maintained until death at day <em>21</em>. Administration of an ACE inhibitor (ramipril) reduced ACE activity, ANG II concentration, TGF-beta expression, and collagen deposition. Losartan (an ANG II type 1 receptor antagonist) also attenuated the increase in TGF-beta expression and lung collagen deposition. These observations suggest that ANG II, possibly generated locally within the lung, may play an important role in the fibrotic response to acute lung injury, at least in part via the action of TGF-beta. ACE inhibitors and receptor antagonists, already widely used clinically, should be assessed as potential new therapies for fibrotic lung disease.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>) is a pleiotropic protein involved in glucose, lipid metabolism and energy homeostasis, with main tissues of expression being the liver and adipose tissue. Brown adipose tissue (BAT) is responsible for cold-induced thermogenesis in rodents. The role of FGF<em>21</em> in BAT biology has not been investigated. In the present study, wild-type C57BL/6J mice as well as a brown adipocyte cell line were used to explore the potential role of cold exposure and β3-adrenergic stimulation in the expression of FGF<em>21</em> in BAT. Our results demonstrate that short-term exposure to cold, as well as β3-adrenergic stimulation, causes a significant induction of FGF<em>21</em> mRNA levels in BAT, without a concomitant increase in FGF<em>21</em> plasma levels. This finding opens new routes for the potential use of pharmaceuticals that could induce FGF<em>21</em> and, hence, activate BAT thermogenesis.

Site-specific delivery of angiogenic <em>growth</em> <em>factors</em> from tissue-engineered devices should provide an efficient means of stimulating localized vessel recruitment to the cell transplants and would ensure cell survival and function. In the present article, we describe the construction of a novel porous alginate scaffold that incorporates tiny poly (lactic-co-glycolic acid) microspheres capable of controlling the release of angiogenic <em>factors</em>, such as basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The microspheres are an integral part of the solid alginate matrix, and their incorporation does not affect the scaffold porosity or pore size. In vitro, bFGF was released from the porous composite scaffolds in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of cardiac <em>fibroblasts</em>. The controlled delivery of bFGF from the three-dimensional scaffolds accelerated the matrix vascularization after implantation on the mesenteric membrane in rat peritoneum. The number of penetrating capillaries into the bFGF-releasing scaffolds was nearly fourfold higher than into the control scaffolds (those incorporating microspheric BSA and heparin but not bFGF). At day 10 posttransplantation, capillary density in the composite scaffolds was 45 +/- 3/mm(2) and it increased to 70 +/- 7/mm(2) by day <em>21</em>. The released bFGF induced the formation of large and matured blood vessels, as judged by the massive layer of mural cells surrounding the endothelial cells. The control over bFGF delivery and localizing its effects to areas of need, may aid in the wider application of bFGF in therapeutic angiogenesis as well as in tissue engineering.

Controlled release of human vascular endothelial <em>growth</em> <em>factor</em> (VEGF) or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) from hydrogels composed of chemically modified hyaluronan (HA) and gelatin (Gtn) was evaluated both in vitro and in vivo. We hypothesized that inclusion of small quantities of heparin (Hp) in these gels would regulate <em>growth</em> <em>factor</em> (GF) release over an extended period, while still maintaining the in vivo bioactivity of released GFs. To test this hypothesis, HA, Gtn, and Hp (15 kDa) were modified with thiol groups, then co-crosslinked with poly (ethylene glycol) diacrylate (PEGDA). Either VEGF or bFGF was incorporated into the gels before crosslinking with PEGDA. Release of these GFs in vitro could be sustained over 42 days by less than 1% Hp content, and was found to decrease monotonically with increasing Hp concentration. As little as 0.03% Hp in the gels reduced the released VEGF fraction from 30% to <em>21</em>%, while 3% Hp reduced it to 19%. Since the minimum Hp concentration capable of effective controlled GF release in vitro was found to be 0.3% (w/w), this concentration was selected for subsequent in vivo experiments. To evaluate the bioactivity of released GFs in vivo, gel samples were implanted into the ear pinnas of Balb/c mice and the resulting neovascularization response measured. In the presence of Hp, vascularization was sustained over 28 days. GF release was more rapid in vitro from gels containing Gtn than from gels lacking Gtn, though unexpectedly, the in vivo neovascularization response to Gtn-containing gels was decreased. Nevertheless significant numbers of neovessels were generated. The ability to stimulate localized microvessel <em>growth</em> at controlled rates for extended times through the release of GFs from covalently linked, Hp-supplemented hydrogels will ultimately provide a powerful therapeutic tool.

Angiogenesis plays a key role in tumor <em>growth</em> and metastasis. The transforming <em>growth</em> <em>factor</em> alpha (TGF-alpha)-epidermal <em>growth</em> <em>factor</em> receptor (EGFR) autocrine pathway controls in part the production of angiogenic <em>factors</em> such as vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in cancer cells. In this study, we have evaluated the antiangiogenic and antitumor activity of monoclonal antibody (MAb) C225, an anti-EGFR chimeric human-mouse MAb, alone and in combination with a human VEGF antisense (AS) <em>21</em>-mer phosphorothioate oligonucleotide (VEGF-AS) in human GEO colon cancer cells. MAb C225 treatment determined a dose-dependent inhibition of VEGF, bFGF, and TGF-alpha production by GEO cells in vitro. Treatment with VEGF-AS caused a selective inhibition in VEGF expression by GEO cells in vitro. Treatment of immunodeficient mice bearing established, palpable GEO xenografts for 3 weeks with VEGF-AS or with MAb C225 determined a cytostatic reversible inhibition of tumor <em>growth</em>. In contrast, a prolonged inhibition of tumor <em>growth</em> was observed in all mice treated with the two agents, in combination with a significant improvement in mice survival compared with controls (P < .001), to MAb C225 (P < .001), or to VEGF-AS (P < .001) treated mice. All mice died within 4, 6, and 8 weeks after tumor cell injection in the control, VEGF-AS and MAb C225 groups, respectively. In contrast, 50% of mice treated with the combination of VEGF-AS and MAb C225 were alive at 13 weeks. Ten % of mice treated with VEGF-AS plus MAb C225 were alive at 20 weeks and had no histological evidence of GEO tumors. Immunohistochemical analysis of GEO tumor xenografts demonstrated a significant reduction of VEGF expression after treatment with VEGF-AS with a parallel reduction in microvessel count. MAb C225 treatment determined a reduction in the expression of VEGF, bFGF, and TGF-alpha with a reduction in microvessel count. Finally, a significant potentiation in inhibition of VEGF expression and little or no microvessels were observed in GEO tumors after the combined treatment with the two agents.

Mitochondrial respiratory chain (RC) deficiency is among the most common causes of inherited metabolic disease, but its physiological consequences are poorly characterized. We studied the skeletal muscle gene expression profiles of mice with late-onset mitochondrial myopathy. These animals express a dominant patient mutation in the mitochondrial replicative helicase Twinkle, leading to accumulation of multiple mtDNA deletions and progressive subtle RC deficiency in the skeletal muscle. The global gene expression pattern of the mouse skeletal muscle showed induction of pathways involved in amino acid starvation response and activation of Akt signaling. Furthermore, the muscle showed induction of a fasting-related hormone, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (Fgf<em>21</em>). This secreted regulator of lipid metabolism was also elevated in the mouse serum, and the animals showed widespread changes in their lipid metabolism: small adipocyte size, low fat content in the liver and resistance to high-fat diet. We propose that RC deficiency induces a mitochondrial stress response, with local and global changes mimicking starvation, in a normal nutritional state. These results may have important implications for understanding the metabolic consequences of mitochondrial myopathies.

The emergence of the myo<em>fibroblast</em> phenotype (characterized by alpha-smooth muscle actin expression) in wound healing and in tissues undergoing fibrosis is thought to be responsible for the increased contractility of the affected tissues. In bleomycin-induced pulmonary fibrosis, the myo<em>fibroblast</em> is also responsible for the observed increase in collagen gene expression. To evaluate further these phenotypic changes in lung <em>fibroblasts</em>, contractile and other phenotypic properties of <em>fibroblasts</em> isolated from lungs of rats with bleomycin-induced fibrosis were compared with those of normal rats using in vitro models. Pulmonary fibrosis was induced in rats by endotracheal injection on day 0, and 7 and 14 days later the animals were sacrificed and lung <em>fibroblasts</em> isolated. Using immunofluorescence, < 10% of <em>fibroblasts</em> from control animals express alpha-smooth muscle actin when cultured as a monolayer. In contrast, 19% and <em>21</em>% of cells from day 7 and day 14 bleomycin-treated animals, respectively, expressed this actin and with greater intensity than in control lung cells. This increase in actin expression was associated with enhanced contractility when evaluated using a three-dimensional cell culture model consisting of <em>fibroblast</em>-populated collagen gels. This enhanced contractility was abolished by treatment with antibody to transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), whereas exogenous TGF-beta 1 and serum-stimulated contraction of control lung <em>fibroblasts</em>. TGF-beta 1 gene expression was greater in cells from bleomycin-treated animals than those from control lungs. These results show that cells with the myo<em>fibroblast</em> phenotype are more abundant in fibrotic lung, and that these cells possess greater contractile capacity in vitro at least partly by virtue of their enhanced endogenous TGF-beta 1 gene expression.

Pancreatic cancer is characterized by excessive desmoplastic reaction and by a hypoxic microenvironment within the solid tumor mass. Chronic pancreatitis is also characterized by fibrosis and hypoxia. <em>Fibroblasts</em> in the area of fibrosis in these pathological settings are now recognized as activated pancreatic stellate cells (PSCs). Recent studies have suggested that a hypoxic environment concomitantly exists not only in pancreatic cancer cells but also in surrounding PSCs. This study aimed to clarify whether hypoxia affected the cell functions in PSCs. Human PSCs were isolated and cultured under normoxia (<em>21</em>% O(2)) or hypoxia (1% O(2)). We examined the effects of hypoxia and conditioned media of hypoxia-treated PSCs on cell functions in PSCs and in human umbilical vein endothelial cells. Hypoxia induced migration, type I collagen expression, and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) production in PSCs. Conditioned media of hypoxia-treated PSCs induced migration of PSCs, which was inhibited by anti-VEGF antibody but not by antibody against hepatocyte <em>growth</em> <em>factor</em>. Conditioned media of hypoxia-treated PSCs induced endothelial cell proliferation, migration, and angiogenesis in vitro and in vivo. PSCs expressed several angiogenesis-regulating molecules including VEGF receptors, angiopoietin-1, and Tie-2. In conclusion, hypoxia induced profibrogenic and proangiogenic responses in PSCs. In addition to their established profibrogenic roles, PSCs might play proangiogenic roles during the development of pancreatic fibrosis, where they are subjected to hypoxia.

Clinical use of recombinant <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) for the treatment of type 2 diabetes and other disorders linked to obesity has been proposed; however, its clinical development has been challenging owing to its poor pharmacokinetics. Here, we describe an alternative antidiabetic strategy using agonistic anti-FGFR1 (FGF receptor 1) antibodies (R1MAbs) that mimic the metabolic effects of FGF<em>21</em>. A single injection of R1MAb into obese diabetic mice induced acute and sustained amelioration of hyperglycemia, along with marked improvement in hyperinsulinemia, hyperlipidemia, and hepatosteatosis. R1MAb activated the mitogen-activated protein kinase pathway in adipose tissues, but not in liver, and neither FGF<em>21</em> nor R1MAb improved glucose clearance in lipoatrophic mice, which suggests that adipose tissues played a central role in the observed metabolic effects. In brown adipose tissues, both FGF<em>21</em> and R1MAb induced phosphorylation of CREB (cyclic adenosine 5'-monophosphate response element-binding protein), and mRNA expression of PGC-1α (peroxisome proliferator-activated receptor-γ coactivator 1α) and the downstream genes associated with oxidative metabolism. Collectively, we propose FGFR1 in adipose tissues as a major functional receptor for FGF<em>21</em>, as an upstream regulator of PGC-1α, and as a compelling target for antibody-based therapy for type 2 diabetes and other obesity-associated disorders.