Keratinocyte <em>growth</em> <em>factor</em> (KGF) is a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family. Portions of the gene encoding KGF were amplified during primate evolution and are present in multiple nonprocessed copies in the human genome. Nucleotide analysis of a representative sampling of these KGF-like sequences indicated that they were at least 95% identical to corresponding regions of the KGF gene. To localize these sequences to specific chromosomal sites in human and higher primates, we used fluorescence in situ hybridization. In human, using a cosmid probe encoding KGF exon 1, we assigned the location of the KGF gene to chromosome 15q15-<em>21</em>.1. In addition, copies of KGF-like sequences hybridizing only with a cosmid probe encoding exons 2 and 3 were localized to dispersed sites on chromosome 2q<em>21</em>, 9p11, 9q12-13, 18p11, 18q11, <em>21</em>q11, and <em>21</em>q<em>21</em>.1. The distribution of KGF-like sequences suggests a role for alphoid DNA in their amplification and dispersion. In chimpanzee, KGF-like sequences were observed at five chromosomal sites, which were each homologous to sites in human, while in gorilla, a subset of four of these homologous sites was identified; in orangutan two sites were identified, while gibbon exhibited only a single site. The chromosomal localization of KGF sequences in human and great ape genomes indicates that amplification and dispersion occurred in multiple discrete steps, with initial KGF gene duplication and dispersion taking place in gibbon and involving loci corresponding to human chromosomes 15 and <em>21</em>. These findings support the concept of a closer evolutionary relationship of human and chimpanzee and a possible selective pressure for such dispersion during the evolution of higher primates.

Novel beta-sheet-forming peptide 33 mers, beta pep peptides, have been designed by using a combination approach employing basic folding principles and incorporating short sequences or proposed key residues from the beta-sheet domains of interleukin-8 (IL-8), platelet <em>factor</em>-4 (PF4) and bactericidal/permeability increasing protein (B/PI). Since PF4 and B/PI are anti-angiogenic and IL-8 is angiogenic, the library of 30 beta pep peptides was assayed for the ability to affect the <em>growth</em> of endothelial cells. Results indicate that five beta pep peptides (beta pep-2, 7, 8, <em>21</em> and 25) demonstrate greater than 50% anti-proliferative activity at 30 micrograms/ml, and one of those (beta pep-25) is similarly active at 10 micrograms/ml. Insight into the mechanism of action was probed in an apoptosis assay. Anti-proliferative activity was found to be correlated with the induction of apoptosis. For example, at 100 micrograms/ml beta pep-25 induces 85% of endothelial cells to undergo apoptosis within 2 days. These effects from beta pep peptides appear to be selective for endothelial cell (EC) because normal cells (<em>fibroblasts</em> and leukocytes) and various tumor cells are not significantly affected at peptide concentrations used in this study. Comparison of active and inactive beta pep sequences allows structure-function relationships to be deduced. Five hydrophobic residues and two lysines appear to be crucial to activity. This research contributes to the development of novel anti-angiogenic peptides.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) is a heparin-binding protein capable of inducing angiogenesis in multiple animal models of chronic ischemia. The pharmacokinetics and pharmacodynamics of a single dose of recombinant FGF-2 (rFGF-2) administered by intracoronary or intravenous infusion were evaluated in a Phase I trial in 66 patients with severe coronary artery disease. rFGF-2 displayed biphasic elimination with a mean studywide distribution t1/2 of <em>21</em> minutes and a mean apparent terminal elimination t1/2 of 7.6 hours. Systemic exposure to rFGF-2 was comparable following intracoronary or intravenous administration. Peak plasma concentration and area under the concentration-time curve increased proportionally with dose, indicating linear pharmacokinetics over the dose range examined (0.33 to 48.0 micrograms/kg). Greater systemic exposure was observed when heparin was administered closer to rFGF-2 infusion, consistent with slower clearance of heparin/rFGF-2 complexes. Infusion of rFGF-2 was associated with changes in acute hemodynamics. While a clear PK/PD dose-response relationship was not established, a trend toward hypotension and tachycardia with higher rFGF-2 doses was observed.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) receptor of human umbilical vein-derived endothelial (HUE) cells has been identified by affinity labeling. It has an apparent molecular weight of 130,000. It binds both basic and acidic FGF, but not with epidermal <em>growth</em> <em>factor</em>, insulin, or transferrin. The lectin concanavalin-A does not inhibit the binding of 125I-bFGF to HUE cell-surface receptors, whereas it inhibits bFGF binding to BHK-<em>21</em> cell-surface FGF receptor. This suggests that both types of receptors may differ in their degree of glycosylation. In contrast to other cell types, heparin only slightly inhibits the binding of basic FGF to its receptor. Protamine sulfate, which is anti-angiogenic in vivo, and suramin, a drug used in the therapy of trypanosomiasis and onchocerciasis, also inhibit the binding of basic FGF to the receptor.

Angiogenesis is essential in tissue <em>growth</em> and regeneration. There are several <em>factors</em> that are able to stimulate vascular endothelial cell <em>growth</em>, including platelet-derived <em>growth</em> <em>factor</em> (PDGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF). Disc herniation tissue (DHT) contains vascular in<em>growth</em>, which promotes granulation tissue formation. In this study we observed 50 disc herniations for PDGF and VEGF immunoreactivity. PDGF immunopositivity was detected in 38 samples (78%). In 28 samples (56%) there were PDGF immunopositive capillaries, PDGF immunopositive disc cells were detected in 19 samples (38%) and PDGF immunopositive <em>fibroblasts</em> in 6 DHT samples (12%). VEGF immunopositive capillaries were identified in 44 DHT samples (88%). For neither <em>growth</em> <em>factor</em> was immunopositivity dependent on preoperative radicular pain duration. In extrusions (n = 25) VEGF immunopositive capillaries were detected in 23 samples (92%) and PDGF immunopositivity in <em>21</em> samples (84%). PDGF immunopositivity was more commonly associated with capillaries than with nuclei of disc cells. In sequesters (n = 20) VEGF immunopositive capillaries were identified in all samples and PDGF immunopositivity in 16 (80%). As in extrusions, PDGF immunoreaction was more prevalent in capillaries than in disc cells. Patient age did not relate to VEGF expression. In all age groups it was higher than 80%. Thus capillaries in disc herniation tissue are evidently newly formed and our results demonstrate that PDGF and VEGF participate in the neovascularization process. The presence of PDGF in <em>fibroblasts</em> and in disc cells suggests that this <em>growth</em> <em>factor</em> regulates the function of these cells, possibly the proliferation of the cells and the production of extracellular matrix components.

The role of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in the proliferation and differentiation of rat bone marrow cells in culture was studied. bFGF stimulated [3H]thymidine incorporation into these cells by 4-fold at a concentration of 0.3 ng/ml and half-maximal effect was observed at a concentration of 15 pg/ml. In addition to its mitogenic effect, bFGF stimulated alkaline phosphatase activity by 3.6-fold. Continuous treatment with bFGF (for <em>21</em> days) resulted in a 6.3-fold increase in the culture dish surface area covered by bone-like mineralized tissue. Maximal bone-like tissue formation was observed in the presence of 3 ng/ml bFGF with half-maximal effect at a concentration of 0.3 ng/ml. These results indicate the possible role of bFGF in the proliferation of osteogenic rat bone marrow cells and their differentiation into cells of osteoblast-like phenotype.

OBJECTIVE

The events preceding myointimal thickening in vein grafts after vascular reconstructions are not well characterized. Indeed, the injury response associated with vein graft arterialization may be different than that observed in the balloon angioplasty model. Therefore, we used a rat model to study the early cellular response after arterialization of vein grafts.

METHODS

Epigastric veins were placed as femoral artery interposition grafts in 37 male Lewis rats (weight range, 350-400 g). Vein grafts and contralateral epigastric veins were harvested at different time points (6 hours, 1 day, 2 days, 3 days, 7 days, 14 days, <em>21</em> days, 30 days, and 70 days). Tissue specimens were processed for histology and immunohistochemistry with antibodies for the proliferating cell nuclear antigen (PCNA) and for different cell types. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay was used as a means of determining the presence of apoptosis. Electron microscopy was used as means of assessing the integrity of the endothelial cell surface (SEM) and confirming the presence of apoptosis (TEM). Specimens were also snap frozen in liquid nitrogen for RNA isolation and molecular analysis.

RESULTS

At 1 day, endothelial denudation with platelet deposition on the surface was shown by means of SEM. Both apoptosis and necrosis of smooth muscle cells (SMCs) were present in the media, along with monocyte infiltration. Cellular proliferation and apoptosis were most intense within the first week of implantation. PCNA staining was first seen in the adventitial fibroblasts and microvessels, then in the medial SMCs at 3 days. With reverse transcriptase polymerase chain reaction, upregulation of vascular endothelial growth factor (VEGF) messenger RNA (mRNA) was noted at 1 day. Myointimal thickening progressively developed, with no apparent diminution of the luminal area as long as 70 days after implantation. By means of the analysis of the transforming growth factor beta1, mRNA showed expression during intimal thickening and accumulation of extracellular matrix. Reendothelialization was complete at 30 days.

CONCLUSIONS

These observations indicate that the cellular composition in our vein graft model is similar to human stenotic explants. Endothelial denudation is observed in rat vein grafts with complete regeneration by 30 days. VEGF mRNA is upregulated at 1 day, followed by proliferation of microvessel endothelial cells in the adventitia. Cellular proliferation and apoptosis are minimal after <em>21</em> days, with progressive intimal thickening likely to be the result of matrix accumulation.

In the symposium entitled "Transcriptional controls of energy sensing," the authors presented recent advances on 1) AMP kinase, an intracellular energy sensor; 2) PGC-1α (peroxisome proliferator-activated receptor γ co-activator 1α), a transcriptional co-activator that has powerful effects on mitochondria; 3) methylation and demethylation in response to metabolic fluctuations; and 4) FGF<em>21</em> (<em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>) as an emerging hormone-like intercellular metabolic coordinator. This introduction places these advances within a broad overview of energy sensing and energy balance, with a focus on human evolution and disease. Four key elements of human biology are analyzed: 1) elevated body temperature; 2) complex prolonged reproductive pathways; 3) emergence of 4 large, well-defined fat depots, each with its own functional role; and 4) an immune system that is often up-regulated by nutrition-related signals, independent of the actual presence of a pathogen. We propose that an overactive immune system, including the "metabolic syndrome," was adopted evolutionarily in the distant past to help hold out against unconquerable infections such as tuberculosis, malaria, and trypanosomiasis. This immune activation is advantageous in the absence of other disease management methods, especially under conditions in which life expectancy is short. The inflammation has become a major agent of pathology in wealthy populations in whom the pathogens are a minor threat and life expectancy is long. The "Conclusions" section sketches cautiously how understanding the molecules involved in energy sensing and energy balance may lead to specific therapies for obesity and diabetes and for their complications.

Incubation of BHK-<em>21</em> hamster <em>fibroblasts</em> in a serum- and amino acid-deficient medium caused a 3-fold increase in the degradation of endogenous protein, a doubling of the degradation of endocytosed epidermal <em>growth</em> <em>factor</em>, and an eightfold increase in the degradation of endocytosed alpha 2-macroglobulin. 3-Methyladenine (3MA) inhibited the deprivation-induced lysosomal degradation of both endogenous and endocytosed protein, but had no effect on basal (non-induced) degradation. 3MA also inhibited deprivation-induced protein degradation in human IMR-90 <em>fibroblasts</em>. Some inhibition of protein synthesis and of endocytic uptake of alpha 2-macroglobulin was observed in 3MA-treated BHK-<em>21</em> cells, whereas cellular ATP levels were unaffected. These results are different from those obtained with isolated hepatocytes, and suggest that in some cells both endogenous and endocytic protein degradation may be accelerated as part of a general deprivation response.

Pancreatic adenocarcinoma is known for early aggressive local invasion, high metastatic potential, and a low 5-year survival rate. Matrix metalloproteinases (MMPs) play important roles in tumor <em>growth</em> and invasion. Earlier studies on pancreatic cancer have found increased expression of certain MMPs to correlate with poorer prognosis, short survival time or presence of metastases. We studied the expression of MMP-<em>21</em>, -26, and tissue inhibitor of matrix metalloproteinases (TIMP)-4 in 50 tissue samples, including 25 adenocarcinomas, seven other malignant pancreatic tumors, and 18 control samples of non-neoplastic pancreatic tissue with immunohistochemistry. The regulation of MMP-<em>21</em>, -26, and TIMP-4 mRNAs by cytokines was studied with RT-PCR in pancreatic cancer cell lines PANC-1, BxPC-3, and AsPC-1. MMP-<em>21</em>, -26, and TIMP-4 were detected in cancer cells in 64, 40, and 60% of tumors, respectively. MMP-<em>21</em> expressed in well-differentiated cancer cells and occasional <em>fibroblasts</em>, like TIMP-4, tended to diminish in intensity from grade I to grade III tumors. Patients with metastatic lymph nodes had increased expression of MMP-26 in actual tumor samples. All cultured cancer cell lines expressed MMP-<em>21</em> basally at low levels, and presence of the protein was confirmed immunohistochemically in cultured cells. MMP-<em>21</em> expression was induced by epidermal <em>growth</em> <em>factor</em> (EGF) in PANC-1 cells. MMP-26 was neither expressed basally nor induced by tumor necrosis <em>factor</em> alpha, transforming <em>growth</em> <em>factor</em> beta-1 (TGFbeta1), EGF, or interferon gamma. Basal TIMP-4 expression was lowest in the poorly differentiated cancer cell line PANC-1 compared to better-differentiated BxPC-3 and AsPC-1 cells. TIMP-4 expression was induced by TGFbeta1 in PANC-1 cells and by EGF in BxPC-3 cells. Our findings suggest that MMP-<em>21</em> is not a marker of invasiveness, but rather of differentiation, in pancreatic cancer and it may be upregulated by EGF. The putative role of MMP-26 as a marker of metastases warrants further studies. Unlike other TIMPs, TIMP-4 was not upregulated in relation to aggressiveness of pancreatic cancer.

Using modeling of heparin-<em>fibroblast</em> <em>growth</em> <em>factor</em> interactions, we replaced four basic residues of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) with neutral glutamine residues by site-specific mutagenesis to give the mutants K128Q, K138Q, K128Q-K138Q, R129Q, K134Q, and R129Q-K134Q. The FGF mutants were characterized for their receptor and heparin binding affinities, mitogenic and cell proliferation activities, and their ability to induce plasminogen activator (PA) production and in vitro angiogenesis by cultured endothelial cells. Heparin binding properties and biological activities of the three mutants involving R129 and K134 remained essentially unchanged; however, significant changes for three mutants involving K128 and K138 were found. The KD values for heparin binding for K128Q and K138Q mutants were increased about 10-fold, and that for the K128Q-K138Q double mutant was increased by about 100-fold. The mutant K128Q-K138Q required a 10-fold higher concentration of heparin to promote binding to heparan sulfate proteoglycan (HSPG)-deficient CHO cells transfected with <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-1 (FGFR1) or to induce DNA synthesis in HSPG-deficient myeloid cells transfected with FGFR1. Binding affinities of the mutants to cell surface receptors on BHK-<em>21</em> cells, however, were similar to that of wild-type FGF-2. In endothelial cell proliferation assays the activities of K128Q and K128Q-K138Q were about 10-fold lower than that of the wild-type protein, whereas the K138Q mutant exhibited wild-type activity. In addition, the K128Q-K138Q mutant displayed a markedly lowered capacity to induce PA activity in cultured endothelial cells and to form capillary-like structures in an in vitro angiogenesis model.(ABSTRACT TRUNCATED AT 250 WORDS)

The E5 protein of bovine papillomavirus is a 44-amino acid, Golgi-resident, type II transmembrane protein that efficiently transforms immortalized mouse <em>fibroblasts</em>. The transmembrane (TM) domain of E5 is not only critical for biological activity, it also regulates interactions with cellular targets including the platelet derived <em>growth</em> <em>factor</em> receptor (PDGF-R) and the 16-kDa subunit of the vacuolar proton ATPase (V-ATPase). In order to define the specific TM amino acids essential for E5 biological and biochemical activity, we performed scanning alanine mutagenesis on 25 of the 30 potential TM residues and genetically mapped discrete alpha-helical domains which separately regulated the ability of E5 to bind PDGF-R, activate PDGF-R, and to form oligomers. Alanine substitutions at positions 17, <em>21</em>, and 24 (which lie on the same helical face) greatly inhibited E5 association with the PDGF-R, suggesting that this region comprises the receptor binding site. PDGF-R activation also mapped to a specific but broader domain in E5; mutant proteins with alanines on one helical face (positions 8, 9, 11, 16, 19, 22, and 23) continued to induce PDGF-R tyrosine phosphorylation, whereas mutant proteins with alanines on the opposite helical face (positions 7, 10, 13, 17, 18, <em>21</em>, 24, and 25) did not, indicating that the latter helical face was critical for mediating receptor transphosphorylation. Interestingly, these "activation-defective" mutants segregated into two classes: 1) those that were unable to form dimers but that could still form higher order oligomers and transform cells, and 2) those that were defective for PDGF-R binding and were transformation-incompetent. These findings suggest that the ability of E5 to dimerize and to bind PDGF-R is important for receptor activation. However, since several transformation-competent E5 mutants were defective for binding and/or activating PDGF-R, it is apparent that E5 must have additional activities to mediate cell transformation. Finally, alanine substitutions also defined two separate helical faces critical for E5/E5 interactions (homodimer formation). Thus, our data identify distinct E5 helical faces that regulate homologous and heterologous intramembrane interactions and define two new classes of biologically active TM mutants.

OBJECTIVE

Evaluate the safety and tolerability of beloranib, a fumagillin-class methionine aminopetidase-2 (MetAP2) inhibitor, in obese women over 4 weeks.

METHODS

Thirty-one obese (mean BMI 38 kg/m2) women were randomized to intravenous 0.1, 0.3, or 0.9 mg/m2 beloranib or placebo twice weekly for 4 weeks (N = 7, 6, 9, and 9).

RESULTS

The most frequent AEs were headache, infusion site injury, nausea, and diarrhea. Nausea and infusion site injury occurred more with beloranib than placebo. The most common reason for discontinuation was loss of venous access. There were no clinically significant abnormal laboratory findings. In subjects completing 4 weeks, median weight loss with 0.9 mg/m2 beloranib was -3.8 kg (95% CI -5.1, -0.9; N = 8) versus -0.6 kg with placebo (-4.5, -0.1; N = 6). Weight change for 0.1 and 0.3 mg/m2 beloranib was similar to placebo. Beloranib (0.9 mg/m2) was associated with a significant 42 and 18% reduction in triglycerides and LDL-cholesterol, as well as improvement in C-reactive protein and reduced sense of hunger. Changes in β-hydroxybutyrate, adiponectin, leptin, and <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> were consistent with the putative mechanism of MetAP2 inhibition. Glucose and blood pressure were unchanged.

CONCLUSIONS

Beloranib treatment was well tolerated and associated with rapid weight loss and improvements in lipids, C-reactive protein, and adiponectin.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2) is expressed in isoforms of different molecular masses from one mRNA species by alternative start of translation. The higher molecular mass isoforms (FGF-2(<em>21</em>) and (23)) contain an arginine-rich N-terminus organized in RG-motifs followed by the 18 kDa FGF-2 (FGF-2(18)) core which is common to all isoforms. Both isoforms localize differentially to the nucleus. Here, we analyzed the nuclear localization of FGF-2(<em>21</em>). Surprisingly, the lack of one RG-motif in FGF-2(<em>21</em>) resulted in the nucleolar distribution characteristic of FGF-2(18). We have previously shown that 23 kDa FGF-2 (FGF-2(23)) interacts specifically with the survival of motoneuron (SMN) protein, an assembly protein for small nuclear ribonucleoprotein particles. For this assembly, Sm-proteins methylated by protein arginine methyltransferase 5 (PRMT5) are required. In our study, we aimed to analyze whether FGF-2(23) is also a substrate for symmetrical methylation by PRMT5. We could confirm that both proteins exist in a common complex. Moreover, PRMT5 methylates FGF-2(23) in vitro, whereas mutated inactive PRMT5 does not. FGF-2(23) is therefore a new substrate of PRMT5. With regard to function, inhibition of methyltransferase activity in HEK293T cells leads to cytoplasmic enrichment of FGF-2, indicating the importance of arginine methylation for shuttling of FGF-2(23) to the nucleus.

OBJECTIVE

To investigate the role of urinary measurements of an angiogenic factor, basic fibroblast growth factor (bFGF), in the assessment of patients with bladder cancer.

METHODS

Urine from 83 patients was assayed using a commercially available ELISA for bFGF. Thirty-eight patients had a bladder tumour and 21 had a history of bladder cancer but no disease at the time of testing. Twenty-four patients acted as controls, 16 of whom were about to undergo transurethral resection of the prostate (TURP) for benign prostatic hypertrophy (BPH) and eight who had no urological disease.

RESULTS

Median urinary bFGF was higher in patients with active bladder cancer than in those with a clear cystoscopy (5.20 and 2.13 ng/g creatinine, respectively; P < 0.005). Median urinary bFGF was also elevated in patients about to undergo TURP (4.52 ng/g creatinine). Using a threshold value of 6.0 ng/g creatinine, the sensitivity of the test for detecting cancer was 42% and specificity was 88%. At a threshold value of 4.0 ng/g the sensitivity was 62% and the specificity 70%.

CONCLUSIONS

The relationship between urinary basic FGF and the presence of bladder cancer was significant. The test is not sufficiently sensitive or specific to use as a screening test for bladder cancer but may be very useful in monitoring the effectiveness of systemic therapies in bladder cancer. Elevated levels of bFGF in the urine of patients about to undergo TURP suggests a role for bFGF in the pathogenesis of BPH.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF-<em>21</em>) has been proposed as a novel metabolic regulator, and animal experiments suggested that FGF-<em>21</em> may affect energy balance. In humans, FGF-<em>21</em> was correlated with obesity. Until now, no data exist regarding the relationship of FGF-<em>21</em> and weight reduction in humans. We therefore investigated whether FGF-<em>21</em> is modified by a moderate intended weight loss in a human trial. Thirty obese individuals (24 female, 6 male) participated in a weight reduction program for 6 months. In addition to several anthropometric and metabolic parameters, FGF-<em>21</em> was measured before and after weight loss. Baseline serum FGF-<em>21</em> was independently associated with markers of lipid metabolism and waist circumference. The multimodal intervention induced a moderate weight loss (97.4 ± 3.1 vs 92.2 ± 3.1 kg, P < .001), which was accompanied by a significant improvement of lipid and glucose metabolism. However, FGF-<em>21</em> levels were not modified by moderate weight reduction; and FGF-<em>21</em> levels at baseline were not a predictive marker for subsequent weight loss. The results presented here confirmed that FGF-<em>21</em> levels are associated with markers of lipid metabolism and an estimate of abdominal adiposity. The finding that moderate weight loss did not induce changes of FGF-<em>21</em> levels in humans suggests that FGF-<em>21</em> is not directly regulated by fat mass under those conditions.

The liver plays a critical role in glucose metabolism and communicates with peripheral tissues to maintain energy homeostasis. Obesity and insulin resistance are highly associated with nonalcoholic fatty liver disease (NAFLD). However, the precise molecular details of NAFLD remain incomplete. The p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) regulate liver metabolism. However, the physiological contribution of MAPK phosphatase 1 (MKP-1) as a nuclear antagonist of both p38 MAPK and JNK in the liver is unknown. Here we show that hepatic MKP-1 becomes overexpressed following high-fat feeding. Liver-specific deletion of MKP-1 enhances gluconeogenesis and causes hepatic insulin resistance in chow-fed mice while selectively conferring protection from hepatosteatosis upon high-fat feeding. Further, hepatic MKP-1 regulates both interleukin-6 (IL-6) and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>). Mice lacking hepatic MKP-1 exhibit reduced circulating IL-6 and FGF<em>21</em> levels that were associated with impaired skeletal muscle mitochondrial oxidation and susceptibility to diet-induced obesity. Hence, hepatic MKP-1 serves as a selective regulator of MAPK-dependent signals that contributes to the maintenance of glucose homeostasis and peripheral tissue energy balance. These results also demonstrate that hepatic MKP-1 overexpression in obesity is causally linked to the promotion of hepatosteatosis.

<AbstractText>This study investigated the mechanism through which <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) protects against angiotensin II (Ang II)-induced cardiac hypertrophy and dysfunction.</AbstractText><AbstractText>Male silent information regulator 1 (SIRT1) flox/flox and cardiomyocyte-specific inducible SIRT1 knockout mice (SIRT1-iKO) were generated and treated with Ang II (1.1 mg/kg/day for 4 weeks) at the age of 8-12-week-old. FGF<em>21</em> treatment [2.5 mg/kg/day for 4 weeks by intraperitoneal (i.p.) injection] was initiated at the same time as the Ang II infusion. For in vitro studies, neonatal rat cardiomyocytes (NRCMs), H9c2 rat cardiomyocytes and isolated adult mouse cardiomyocytes were treated with Ang II (1 μM) and FGF<em>21</em> (20 nM) for 24 h with or without SIRT1 silencing.</AbstractText><AbstractText>FGF<em>21</em> treatment significantly attenuated Ang II-induced cardiac hypertrophy and dysfunction. SIRT1 knockout abolished the ability of FGF<em>21</em> to prevent Ang II-induced cardiac hypertrophy, fibrosis, and apoptosis, without affecting the beneficial effects of FGF<em>21</em> in Ang II-induced hypertension, and did not influence the hypertension itself. FGF<em>21</em> markedly increased the deacetylase activity of SIRT1 and promoted the interaction of SIRT1 with liver kinase B1 (LKB1) and forkhead box protein O1 (FoxO1), resulting in decreased acetylation of these SIRT1 target proteins. Consequently, FGF<em>21</em> promoted the activation of the LKB1 target adenosine monophosphate-activated protein kinase (AMPK) and altered the transcriptional activity of FoxO1 on its downstream target genes catalase (Cat), MnSOD (Sod2), and Bim, resulting in reduced reactive oxygen species (ROS) accumulation and cardiomyocyte apoptosis.</AbstractText><AbstractText>FGF<em>21</em> improves cardiac function and alleviates Ang II-induced cardiac hypertrophy in a SIRT1-dependent manner.</AbstractText>

Antiangiogenic therapy is a potent cancer treatment, however, the possibility of recurrence and resistance to this approach remains. Here we show that hypoxia and low-nutrition double-deprivation stress induces reversible tumor aggressiveness. In a stress-cycle-dependent manner, murine melanoma cells showed morphological changes, up-regulated phospho-Akt, and abnormal regulation of multiple genes including <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em>, a metabolic regulator, resulting in increased cell proliferation in vitro, and increased tumorigenesis and invasive potential in vivo. In this system, altered cellular metabolism participates in the adaptation of tumor to the double-deprivation stress. Our results suggest the targeting of a minor population of cancer cells resistant to both hypoxia and low nutrition to be an effective new antitumor strategy in combination with antiangiogenic therapy.

The present study aimed to investigate the actions of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) on energy balance in a natural model of relative fatness, the Siberian hamster. Hamsters were studied under long days (LD) to promote weight gain, or short days to induce weight loss, and treated with rhFGF<em>21</em> (3 mg/kg/day) via s.c. minipumps for 14 days. On days 7-9, detailed assessments of ingestive behaviour, metabolic gas exchange and locomotor activity were made. FGF<em>21</em> caused substantial (P < 0.0001) weight loss in the fat LD state but not in the lean SD state: at the end of the study, FGF<em>21</em>-treated hamsters in LD lost 18% of body weight compared to vehicle controls, which is comparable to the natural body weight loss observed in SD. Epididymal fat pads, a correlate of total carcass fat content, were reduced by 19% in FGF<em>21</em> treated hamsters in LD, whereas no difference was found in SD. Body weight loss in LD was associated with a reduction in food intake (P < 0.001) and a decreased respiratory exchange ratio (P < 0.001), indicating increased fat oxidation. Treatment with FGF<em>21</em> maintained the normal nocturnal increase in oxygen consumption and carbon dioxide production into the early light phase in hamsters in LD, indicating increased energy expenditure, although locomotor activity was unaffected. These data suggest a greater efficacy of FGF<em>21</em> in hamsters in LD compared to those in SD, which is consistent with both the peripheral and possibly central actions of FGF<em>21</em> with respect to promoting a lean phenotype. The observed differences in FGF<em>21</em> sensitivity may relate to day length-induced changes in adipose tissue mass.

Transforming <em>growth</em> <em>factor</em> beta1 (TGFβ1) is a pleiotropic <em>growth</em> <em>factor</em> with a very broad spectrum of effects on wound healing. Chronic non-healing wounds such as diabetic foot ulcers express reduced levels of TGFβ1. On the other hand, our previous studies have shown that the microRNA miR-<em>21</em> is differentially regulated in diabetic wounds and that it promotes migration of <em>fibroblast</em> cells. Although interplay between TGFβ1 and miR-<em>21</em> are studied in relation to cancer, their interaction in the context of chronic wounds has not yet been investigated. In this study, we examined if TGFβ1 could stimulate miR-<em>21</em> in <em>fibroblasts</em> that are subjected to high glucose environment. MiR-<em>21</em> was, in fact, induced by TGFβ1 in high glucose conditions. The induction by TGFβ1 was dependent on NFκB activation and subsequent ROS generation. TGFβ1 was instrumental in degrading the NFκB inhibitor IκBα and facilitating the nuclear translocation of NFκB p65 subunit. EMSA studies showed enhanced DNA binding activity of NFκB in the presence of TGFβ1. ChIP assay revealed binding of p65 to miR-<em>21</em> promoter. NFκB activation was also required for the nuclear translocation of Smad 4 protein and subsequent direct interaction of Smad proteins with primary miR-<em>21</em> as revealed by RNA-IP studies. Our results show that manipulation of TGFβ1-NFκB-miR-<em>21</em> pathway could serve as an innovative approach towards therapeutics to heal diabetic ulcers.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) can regulate glucose and lipid metabolism. The placenta actively synthesizes and secretes many hormones, but it is unknown whether this includes FGF<em>21</em>. This study aimed to analyze the placental expression of FGF<em>21</em> in women with or without gestational diabetes mellitus (GDM).

METHODS

FGF<em>21</em> and peroxisome proliferator-activated receptor (PPAR)-α mRNA and protein expression were measured in the placentae of 20 women with and 18 without GDM. mRNA expression of PPARα, FGF receptors 1-4, the coreceptor β-klotho, and glucose transporter (GLUT)-1, -3, and -4 was investigated. Maternal and fetal circulating FGF<em>21</em> levels were assessed in 10 mother-baby dyads per condition.

RESULTS

FGF<em>21</em> was expressed in the placenta and its mRNA expression increased in women with GDM [10.75 (interquartile range 3.28-125.6 AU)] vs control [0.83 (0.22-4.78), P < .001], as is its protein expression [GDM 2.89 (1.44-5.10)] vs control [0.42 (0.05-1.98), P < .05]. PPARα mRNA but not protein expression was increased in GDM [2.94 (0.70-7.26)] vs control [0.99 (0.43-2.17), P < .05] and was positively correlated to FGF<em>21</em> mRNA expression (ρ = 0.43, P < .01). Placental mRNA expression of FGF receptors and GLUT1 was unchanged, and β-klotho, GLUT3, and GLUT4 showed increased expression in GDM. Maternal circulating FGF<em>21</em> levels were similar [GDM 323 (75-9<em>21</em>) vs control 269 (49-731) pg/mL, P = .81]. FGF<em>21</em> was undetected in fetal cord blood.

CONCLUSIONS

FGF<em>21</em> is expressed in the placenta and its expression is increased in GDM. The absence of FGF<em>21</em> in fetal cord blood suggests that neither placental FGF<em>21</em> nor maternal circulating FGF<em>21</em> is secreted into the fetal circulation. Placental FGF<em>21</em> may be a regulator of placental metabolism.

The purpose of the present work was to assess whether upregulation of trophic <em>factors</em> and protection from damage induced in the retina by optic nerve section are associated with changes in the flash electroretinogram (ERG). We have examined the ERG in adult pigmented rat at different survival times over a period of 3 months following section of the optic nerve. The a-wave was analyzed using the Lamb-Pugh model and the parameters of best fit were estimated in control animals and at successive survival times. The amplitudes of the a- and b-waves were reduced over the first 7 days after nerve section. The a-wave recovered its relative amplitude by <em>21</em> days, but the b-wave remained depressed 5 weeks following nerve section. Analysis of the a-wave indicated a 20-30% reduction in the dark current of sectioned eyes at 7 days survival. A significant reduction of the amplification constant was observed in both nerve-sectioned and nerve-intact eyes, relative to normal and sham-operated controls. This reduction persisted to the longest survival time examined. The reduction of the a-wave at 7 days after nerve section coincides with a period of upregulation of ciliary nerve trophic <em>factor</em>. The amplification <em>factor</em> is influenced over a longer time course, which corresponds with a period of up-regulation of basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. These changes in <em>growth</em> <em>factor</em> expression and ERG parameters are in turn associated with protection of photoreceptors against light damage. Present results suggest that the sensitivity of the retina to light may be regulated by mechanisms which protect photoreceptors against stress.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a key regulator in glucose and lipid metabolism and its plasma levels have been shown to be increased not only in humans in different situations such as type 2 diabetes, obesity, and nonalcoholic fatty liver disease but also in animal models of sepsis and pancreatitis. FGF<em>21</em> is considered as a pharmacological candidate in conditions associated with insulin resistance. The aim of this study was to compare FGF<em>21</em> plasma levels in patients with sepsis, in patients with systemic inflammatory response syndrome (SIRS), and in healthy controls. We measured FGF<em>21</em> plasma concentrations in 22 patients with established sepsis, in 11 with SIRS, and in 12 healthy volunteers. Here, we show that FGF<em>21</em> levels were significantly higher in plasma obtained from patients with sepsis and SIRS in comparison with healthy controls. Also, FGF<em>21</em> levels were significantly higher in patients with sepsis than in those with noninfectious SIRS. FGF<em>21</em> plasma levels measured at study entry correlated positively with the APACHE II score, but not with procalcitonin levels, nor with C-reactive protein, classical markers of sepsis. Plasma concentrations of FGF<em>21</em> peaked near the onset of shock and rapidly decreased with clinical improvement. Taken together, these results indicate that circulating levels of FGF<em>21</em> are increased in patients presenting with sepsis and SIRS, and suggest a role for FGF<em>21</em> in inflammation. Further studies are needed to explore the potential role of FGF<em>21</em> in sepsis as a potential therapeutic target.