Mitochondrial dynamics is a conserved process by which mitochondria undergo repeated cycles of fusion and fission, leading to exchange of mitochondrial genetic content, ions, metabolites, and proteins. Here, we examine the role of the mitochondrial fusion protein optic atrophy 1 (OPA1) in differentiated skeletal muscle by reducing OPA1 gene expression in an inducible manner. OPA1 deficiency in young mice results in non-lethal progressive mitochondrial dysfunction and loss of muscle mass. Mutant mice are resistant to age- and diet-induced weight gain and insulin resistance, by mechanisms that involve activation of ER stress and secretion of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) from skeletal muscle, resulting in increased metabolic rates and improved whole-body insulin sensitivity. OPA1-elicited mitochondrial dysfunction activates an integrated stress response that locally induces muscle atrophy, but via secretion of FGF<em>21</em> acts distally to modulate whole-body metabolism.

Pulmonary fibrosis is a chronic condition characterized by progressive scarring of lung parenchyma. The aim of this study was to examine the effects of an ultramicronized preparation of palmitoylethanolamide (PEA-um(®)), an endogenous fatty acid amide, in mice subjected to idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis was induced in male mice by a single intratracheal administration of saline with bleomycin sulphate (1mg/kg body weight) in a volume of 100μL. PEA-um(®) was injected intraperitoneally at 1, 3 or 10mg/kg 1h after bleomycin instillation and daily thereafter. Animals were sacrificed after 7 and <em>21</em>days by pentobarbitone overdose. One cohort of mice was sacrificed after seven days of bleomycin administration, followed by bronchoalveloar lavage and determination of myeloperoxidase activity, lung edema and histopathology features. In the <em>21</em>-day cohort, mortality was assessed daily, and surviving mice were sacrificed followed by the above analyses together with immunohistochemical localization of CD8, tumor necrosis <em>factor</em>-α, CD4, interleukin-1β, transforming <em>growth</em> <em>factor</em>-β, inducible nitric oxide synthase and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Compared to bleomycin-treated mice, animals that received also PEA-um(®) (3 or 10mg/kg) had significantly decreased weight loss, mortality, inflammation, lung damage at the histological level, and lung fibrosis at 7 and <em>21</em>days. PEA-um(®) (1mg/kg) did not significantly inhibit the inflammation response and lung fibrosis. This study demonstrates that PEA-um(®) (3 and 10mg/kg) reduces the extent of lung inflammation in a mouse model of idiopathic pulmonary fibrosis.

Liver biopsy still remains the gold standard for diagnosing nonalcoholic steatohepatitis (NASH), but with limitations. There is an urgent need to develop noninvasive tests that accurately distinguish NASH from simple steatosis. The purpose of this meta-analysis was to evaluate the diagnostic value of serum biomarkers including cytokeratin 18 (CK-18), <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>), and combined biomarker panel (CBP) in the diagnosis of NAFLD, especially NASH. A total of 25 studies met the inclusion criteria. Pooled sensitivity and specificity values for chosen serum markers for diagnosing NASH are as follows: CK-18 (M30), 0.75 and 0.77; CK-18 (M65), 0.71 and 0.77; FGF-<em>21</em>, 0.62 and 0.78; and CBP, 0.92 and 0.85. CBP demonstrated better accuracy with higher sensitivity and specificity than those tested individually. Furthermore, the AUROC of CBP was 0.94 (95% CI, 0.92-0.96), compared to CK-18 or FGF-<em>21</em> assay, which showed the most significant ability to distinguish NASH from simple steatosis. The results suggest that increased circulating CK-18 and FGF-<em>21</em> are associated with NASH and may be used for initial assessment, but not enough. Importantly, CBP is potentially used as accurate diagnostic tools for NASH. Further prospective designed studies are warranted to confirm our findings.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a hormone that has insulin-sensitizing properties. Some trials of FGF<em>21</em> analogs show weight loss and lipid-lowering effects. Recent studies have shown that a common allele in the FGF<em>21</em> gene alters the balance of macronutrients consumed, but there was little evidence of an effect on metabolic traits. We studied a common FGF<em>21</em> allele (A:rs838133) in 451,099 people from the UK Biobank study, aiming to use the human allele to inform potential adverse and beneficial effects of targeting FGF<em>21</em>. We replicated the association between the A allele and higher percentage carbohydrate intake. We then showed that this allele is more strongly associated with higher blood pressure and waist-hip ratio, despite an association with lower total body-fat percentage, than it is with BMI or type 2 diabetes. These human phenotypes of variation in the FGF<em>21</em> gene will inform research into FGF<em>21</em>'s mechanisms and therapeutic potential.

Microglia polarization to the classical M1 activation state is characterized by elevated pro-inflammatory cytokines; however, a full profile has not been generated in the early stages of a sterile inflammatory response recruiting only resident microglia. We characterized the initial M1 state in a hippocampal injury model dependent upon tumor necrosis <em>factor</em> (TNF) receptor signaling for dentate granule cell death. Twenty-one-day-old CD1 male mice were injected with trimethyltin (TMT 2.3 mg/kg, i.p.) and the hippocampus was examined at an early stage (24-h post-dosing) of neuronal death. Glia activation was assessed using a custom quantitative nuclease protection assay. We report elevated mRNA levels for glia response such as ionizing calcium-binding adapter molecule-1 and glial fibrillary acidic protein (Gfap); Fas, hypoxia inducible <em>factor</em> alpha, complement component 1qb, TNF-related genes (Tnf, Tnfaip3, Tnfrsfla); interleukin-1 alpha, Cd44, chemokine (C-C motif) ligand (Ccl)2, Cc14, integrin alpha M, lipocalin (Lcn2), and secreted phosphoprotein 1 (Spp1). These changes occurred in the absence of changes in matrix metalloproteinase 9 and 12, neural cell adhesion molecule, metabotropic glutamate receptor (Grm)3, and Ly6/neurotoxin 1 (Lynx1), as well as, a decrease in neurotrophin 3, glutamate receptor subunit epsilon (Grin)-2b, and neurotrophic tyrosine kinase receptor, type 3. The M2 anti-inflammatory marker, transforming <em>growth</em> <em>factor</em> beta-1 (Tgfb1) was elevated. mRNAs associated with early stage of injury-induced neurogenesis including <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> and Mki67 were elevated. In the "non-injured" temporal cortex receiving projections from the hippocampus, Lynx1, Grm3, and Grin2b were decreased and Gfap increased. Formalin fixed-paraffin-embedded tissue did not generate a comparable profile.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is involved in the regulation of energy balance and adipose metabolism. Our previous genome-wide association study identified genetic variants in the FGF<em>21</em> region associated with macronutrient intake preference. We investigated whether the FGF<em>21</em> genotype modified effects of weight-loss diets varying in macronutrient intake on changes in adiposity in a 2-year randomized diet intervention trial.

We genotyped FGF<em>21</em> rs838147 in 715 overweight or obese individuals who were assigned to one of four diets varying in macronutrient contents. A DEXA scan was performed to evaluate body composition.

We observed a significant interaction between the FGF<em>21</em> genotype and carbohydrate/fat intake on 2-year changes in waist circumference (WC), percentage of total fat mass, and percentage of trunk fat (P = 0.049, P = 0.001, and P = 0.003 for interaction, respectively). In response to the low-carbohydrate/high-fat diet, carrying the carbohydrate intake-decreasing C allele of rs838147 was marginally associated with less reduction in WC (P = 0.08) and significantly associated with less reduction of total fat mass (P = 0.01) and trunk fat (P = 0.02). Opposite genetic associations with these outcomes were observed among the high-carbohydrate/low-fat diet group; carrying the C allele was associated with a greater reduction of WC, total body fat mass, and trunk fat.

Our data suggest that FGF<em>21</em> genotypes may interact with dietary carbohydrate/fat intake on changes in central adiposity and body fat composition. A low-calorie, high-carbohydrate/low-fat diet was beneficial for overweight or obese individuals carrying the carbohydrate intake-decreasing allele of the FGF<em>21</em> variant to improve body composition and abdominal obesity.

Cardiac fibrosis is closely related to multiple cardiovascular system diseases, and noncoding RNAs (ncRNAs), including long noncoding RNA (lncRNA) and microRNA (miRNA), have been reported to play a vital role in fibrogenesis. The present study aims to investigate the potential regulatory mechanism of lncRNA H19 and miR-455 on fibrosis-associated protein synthesis in cardiac <em>fibroblasts</em> (CFs). miRNA microarray assay revealed 34 significantly dysregulated miRNAs, including 13 upregulated miRNAs and <em>21</em> downregulated miRNAs. Among these aberrantly expressed miRNAs, we paid attention to miR-455, which was significantly downregulated in diabetic mouse myocardium and Ang II-induced CFs. Loss- and gain-of-function experiments showed that miR-455 expression levels were negatively correlated with collagen I and III expression in Ang II-induced CFs. Bioinformatic prediction programs (TargetScan, miRanda, starBase) predicted that miR-455 targeted connective tissue <em>growth</em> <em>factor</em> (CTGF) and H19 with complementary binding sites at the 3'-untranslated region, which was validated by luciferase reporter assay. Functional validation assay demonstrated that H19 knockdown could enhance the antifibrotic role of miR-455 and attenuate the CTGF expression and further decrease fibrosis-associated protein synthesis (collagen I, III, and α-SMA). The present study reveals a novel function of the H19/miR-455 axis targeting CTGF in cardiac fibrosis, suggesting its potential therapeutic role in cardiac diseases.

BACKGROUND

Flavopiridol is a flavonoid with antiproliferative effects mediated, in part, by inhibition of cyclin-dependent kinases. Clinical manifestations in a previous Phase I trial in patients with refractory malignancies treated with a 72-h flavopiridol infusion included a proinflammatory syndrome consisting of fever, fatigue, and "local" tumor pain with concomitant alterations in plasma acute-phase reactant proteins.

OBJECTIVE

The aim of this study was to determine whether the proinflammatory syndrome observed in this trial was associated with modulation of plasma cytokines.

METHODS

Patients receiving flavopiridol (n = 76) had serial plasma samples drawn preinfusion and during the infusion for evaluation of interleukin (IL)-6, IL-10, IL-12, granulocyte macrophage colony-stimulating factor, basic-fibroblast growth factor, transforming growth factor-beta, and tumor necrosis factor-alpha levels by standard ELISA assays. The Wilcoxon signed rank test was used to test the significance of the difference between the baseline (time 0) plasma cytokine levels compared with the values of each subsequent data collection time points (8, 24, 48, and 72 h).

RESULTS

There was a significant and sustained increase in plasma IL-6 levels at all time points when compared with baseline values. Paired values were used in the statistical analysis. Median plasma (interquartile range) values of IL-6 were elevated from 15.5 (9-52) pg/ml at baseline to 23 (4-48) pg/ml (P < 0.01) at 8 h; from 15 (2-48) pg/ml at baseline to 46 (21-105) pg/ml (P < 0.001) at 24 h; from 16 (9-52) pg/ml at baseline to 61 (32-170) pg/ml (P < 0.001) at 48 h; and from 15.5 (6-48) pg/ml to 68 (40-200) pg/ml (P < 0.001) at 72 h. Significance was maintained even when adjusted for multiple comparisons. The relative increase in IL-6 concentration was dose-dependent. Moreover, IL-6 elevation had a direct correlation with flavopiridol peak plasma concentration, flavopiridol area under the curve, and plasma C-Reactive protein levels. A significant decrease in plasma granulocyte macrophage colony-stimulating factor occurred at the 8-h sampling point: 50 pg/ml (interquartile range 10-205 pg/ml, P < 0.01) when compared with baseline plasma levels and 71 pg/ml (interquartile range 5-152 pg/ml, P < 0.01). No changes in the other pro or anti-inflammatory cytokines were observed. Immunohistochemistry studies in bone marrow aspirates from a prospective group of patients in this trial demonstrated approximately 4-fold induction of IL-6 (compared with baseline), mostly in non-T cells.

CONCLUSIONS

Biochemical analysis of plasma in patients undergoing infusional flavopiridol found a significant dose-dependent induction of IL-6. IL-6 elevation could be a marker for the process leading to the appearance of the proinflammatory syndrome observed in patients treated with infusional flavopiridol. The mechanism(s) underlying IL-6 induction and its significance are still unknown but may influence strategies to modulate flavopiridol's clinical effects.

BACKGROUND

Impaired wound healing in diabetes is related to decreased production of growth factors. Hence, gene therapy is considered as promising treatment modality. So far, efforts concentrated on single gene therapy with particular emphasis on vascular endothelial growth factor-A (VEGF-A). However, as multiple proteins are involved in this process it is rational to test new approaches. Therefore, the aim of this study was to investigate whether single AAV vector-mediated simultaneous transfer of VEGF-A and fibroblast growth factor 4 (FGF4) coding sequences will improve the wound healing over the effect of VEGF-A in diabetic (db/db) mice.

METHODS

Leptin receptor-deficient db/db mice were randomized to receive intradermal injections of PBS or AAVs carrying β-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A), FGF-4 (AAV-FGF4-IRES-GFP) or both therapeutic genes (AAV-FGF4-IRES-VEGF-A). Wound healing kinetics was analyzed until day 21 when all animals were sacrificed for biochemical and histological examination.

RESULTS

Complete wound closure in animals treated with AAV-VEGF-A was achieved earlier (day 19) than in control mice or animals injected with AAV harboring FGF4 (both on day 21). However, the fastest healing was observed in mice injected with bicistronic AAV-FGF4-IRES-VEGF-A vector (day 17). This was paralleled by significantly increased granulation tissue formation, vascularity and dermal matrix deposition. Mechanistically, as shown in vitro, FGF4 stimulated matrix metalloproteinase-9 (MMP-9) and VEGF receptor-1 expression in mouse dermal fibroblasts and when delivered in combination with VEGF-A, enhanced their migration.

CONCLUSIONS

Combined gene transfer of VEGF-A and FGF4 can improve reparative processes in the wounded skin of diabetic mice better than single agent treatment.

Epoxyeicosatrienoic acids (EETs), the metabolites of arachidonic acid derived from the cytochrome P450 (CYP450) epoxygenases, are mainly metabolized by soluble epoxide hydrolase (sEH) to their corresponding diols. EETs but not their diols, have anti-inflammatory properties and inhibition of sEH might provide protective effects against inflammatory fibrosis. We test the effects of a selected sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), on bleomycin-induced pulmonary fibrosis (PF) in mice. A mouse model of PF was established by intratracheal injection of bleomycin and TPPU was administered for <em>21</em> days after bleomycin injection. We found TPPU treatment improved the body weight loss and survival rate of bleomycin-stimulated mice. Histological examination showed that TPPU treatment alleviated bleomycin-induced inflammation and maintained the alveolar structure of the pulmonary tissues. TPPU also decreased the bleomycin-induced deposition of collagen and the expression of procollagen I mRNA in lung tissues of mice. TPPU decreased the transforming <em>growth</em> <em>factor</em>-β1 (TGF-β1), interleukin-1β (IL-1β) and IL-6 levels in the serum of bleomycin-stimulated mice. Furthermore, TPPU inhibited the proliferation and collagen synthesis of mouse <em>fibroblasts</em> and partially reversed TGF-β1-induced α-smooth muscle actin expression. Our results indicate that the inhibition of sEH attenuates bleomycin-induced inflammation and collagen deposition and therefore prevents bleomycin-induced PF in a mouse model.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a promising drug candidate to combat metabolic diseases. However, high-level expression and purification of recombinant FGF<em>21</em> (rFGF<em>21</em>) in Escherichia coli (E. coli) is difficult because rFGF<em>21</em> forms inclusion bodies in the bacteria making it difficult to purify and obtain high concentrations of bioactive rFGF<em>21</em>. To overcome this problem, we fused the FGF<em>21</em> with SUMO (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and expressed the fused gene in E. coli BL<em>21</em>(DE3).

RESULTS

By inducing with IPTG, SUMO-FGF<em>21</em> was expressed at a high level. Its concentration reached 30% of total protein, and exceeded 95% of all soluble proteins. The fused protein was purified by DEAE sepharose FF and Ni-NTA affinity chromatography. Once cleaved by the SUMO protease, the purity of rFGF<em>21</em> by high performance liquid chromatography (HPLC) was shown to be higher than 96% with low endotoxin level (<1.0 EU/ml). The results of in vivo animal experiments showed that rFGF<em>21</em> produced by using this method, could decrease the concentration of plasma glucose in diabetic rats by streptozotocin (STZ) injection.

CONCLUSIONS

This study demonstrated that SUMO, when fused with FGF<em>21</em>, was able to promote its soluble expression of the latter in E. coli, making it more convenient to purify rFGF<em>21</em> than previously. This may be a better method to produce rFGF<em>21</em> for pharmaceutical research and development.

Accumulating studies suggest that overnutrition-associated obesity may lead to development of type 2 diabetes mellitus and metabolic syndromes (MetS). MetS and its components are important risk <em>factors</em> of mild cognitive impairment, age-related cognitive decline, vascular dementia, and Alzheimer's disease. It has been recently proposed that development of a disease-course modification strategy toward early and effective risk <em>factor</em> management would be clinically significant in reducing the risk of metabolic disorder-initiated cognitive decline. In the present study, we propose that <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a novel candidate for the disease-course modification approach. Using a high-fat diet (HFD) consumption-induced obese mouse model, we tested our hypothesis that recombinant human FGF<em>21</em> (rFGF<em>21</em>) administration is effective for improving obesity-induced cognitive dysfunction and anxiety-like behavior, by its multiple metabolic modulation and anti-pro-inflammation actions. Our experimental findings support our hypothesis that rFGF<em>21</em> is protective to HFD-induced cognitive impairment, at least in part by metabolic regulation in glucose tolerance impairment, insulin resistance, and hyperlipidemia; potent systemic pro-inflammation inhibition; and improvement of hippocampal dysfunction, particularly by inhibiting pro-neuroinflammation and neurogenesis deficit. This study suggests that FGF<em>21</em> might be a novel molecular target of the disease-course-modifying strategy for early intervention of MstS-associated cognitive decline.

Danhong Injection (DHI), a Chinese medicine for treatment of patients with coronary heart disease, inhibits primary abdominal aortic aneurysms in apoE deficient (apoE(-/-)) mice. Formation of microaneurysms plays an important role in the development of diabetic retinopathy and nephropathy. It remains unknown if DHI can reduce these diabetic complications. In this study, diabetic db/db mice in two groups were injected with saline and DHI, respectively, for 14 weeks. Blood and tissue samples were collected to determine serum glucose, lipids and tissue structure. DHI reduced diabetes-induced body weight gain, serum cholesterol and glucose levels. In retinas, DHI blocked the shrink of whole retina and retinal sub-layers by inhibiting expression of caspase 3, matrix metalloproteinase 2 (MMP-2) and MMP-9, accumulation of carbohydrate macromolecules and formation of acellular capillaries. DHI improved renal functions by inhibiting mesangial matrix expansion, expression of vascular endothelial <em>growth</em> <em>factor</em> A, fibronectin and advanced glycation end products in kidneys. Mechanistically, DHI induced expression of glucokinase, AMPKα/phosphorylated AMPKα, insulin receptor substrate 1, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> and peroxisome proliferator-activated γ. Expression of genes responsible for energy expenditure was also activated by DHI. Therefore, DHI inhibits diabetic retinopathy and nephropathy by ameliorating glucose metabolism and demonstrates a potential application in clinics.

Perinatal asphyxia often results in neonatal cerebral hypoxia-ischemia (HI), which is associated with high mortality and severe long-term neurological deficits in newborns. Currently, there are no effective drugs to mitigate the functional impairments post-HI. Previous studies have shown that <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has a potential neuroprotective effect against brain injury. However, the effect of FGF<em>21</em> on neonatal HI brain injury is unclear. In the present study, both in vivo and in vitro models were used to assess whether recombinant human FGF<em>21</em> (rhFGF<em>21</em>) could exert a neuroprotective effect after HI and explore the associated mechanism. The results showed that the rhFGF<em>21</em> treatment remarkably reduced the infarct volume, ameliorated the body weight and improved the tissue structure after HI in neonatal rats. In addition, the rhFGF<em>21</em> treatment lengthened the running endurance times in the rotarod test and decreased the mean escape latencies and increased the number of platform crossings in the Morris water maze test at <em>21</em> d post-HI insult. In contrast, the FGFR1 inhibitor PD173074 and PI3K inhibitor LY294002 partially reversed these therapeutic effects. In isolated primary cortical neurons, the rhFGF<em>21</em> treatment protected primary neurons from oxygen-glucose deprivation (OGD) insult by inhibiting neuronal apoptosis and promoting neuronal survival. Both our in vivo and in vitro results reveal that rhFGF<em>21</em> could inhibit neuronal apoptosis by activating the PI3K/Akt signaling pathway via FGF<em>21</em>/FGFR1/β-klotho complex formation. Therefore, rhFGF<em>21</em> may be a promising therapeutic agent for promoting functional recovery after HI-induced neonatal brain injury.

Although estrogens can stimulate the <em>growth</em> of uterine epithelial cells in vivo, there is no clear effect of estrogens on the in vitro <em>growth</em> of epithelial cells from reproductive tract tissues; thus, we have established a defined culture system for mouse uterine epithelial cells. Pieces of uteri from immature CD-1 mice (<em>21</em>-23 days of age) were treated with trypsin, and the epithelial fragments were separated, enriched by Percoll gradient centrifugation, and seeded on collagen gels prepared from rat tail tendon. Initially, the cells were cultured in a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's Medium supplemented with epidermal <em>growth</em> <em>factor</em> (EGF; 10 ng/ml), insulin (10 micrograms/ml), transferrin (10 micrograms/ml), hydrocortisone (0.1 micrograms/ml), and vitamin A (10 ng/ml). The cells formed a monolayer on the collagen gel within 1-2 days, but with time, cells began to detach from the gel. Further studies revealed that the attachment and <em>growth</em> of these cells on collagen were markedly influenced by the calcium concentration. It was found that lowering the calcium concentration from 1.05 to 0.05-0.1 mM dramatically suppressed cell detachment; the number of cells doubled after 7 days of culture. Proliferation of uterine epithelial cells was enhanced by EGF, but not by <em>fibroblast</em> <em>growth</em> <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>, nerve <em>growth</em> <em>factor</em>, multiplication-stimulating activity, or somatomedin-C. The uterine epithelial cells exhibited a single class of high affinity binding sites for [125I]iodo-EGF (Kd, approximately 1.8 nM), with approximately 5 X 10(4) receptors/cell; binding was inhibited by EGF but not by the other polypeptides. This cell culture system will aid in our investigations on hormonal effects on the <em>growth</em> and differentiation of estrogen target cells.

Thyroid hormone is critical for the proper development of the central nervous system. However, the specific role of thyroid hormone on brain angiogenesis remains poorly understood. Treatment of rats from birth to postnatal day <em>21</em> (P<em>21</em>) with propylthiouracil (PTU), a reversible blocker of triiodothyronine (T3) synthesis, resulted in decreased brain angiogenesis, as indicated by reduced complexity and density of microvessels. However, when PTU was withdrawn at P22, these parameters were fully recovered by P90. These changes were paralleled by an altered expression of vascular endothelial <em>growth</em> <em>factor</em> A (Vegfa) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (Fgf2). Physiologic concentrations of T3 and thyroxine (T4) stimulated proliferation and tubulogenesis of rat brain-derived endothelial (RBE4) cells in vitro. Protein and mRNA levels of VEGF-A and FGF-2 increased after T3 stimulation of RBE4 cells. The thyroid hormone receptor blocker NH-3 abolished T3-induced Fgf2 and Vegfa upregulation, indicating a receptor-mediated effect. Thyroid hormone inhibited the apoptosis in RBE4 cells and altered mRNA levels of apoptosis-related genes, namely Bcl2 and Bad. The present results show that thyroid hormone has a substantial impact on vasculature development in the brain. Pathologically altered vascularization could, therefore, be a contributing <em>factor</em> to the neurologic deficits induced by thyroid hormone deficiency.

The absence of long term bone marrow cultures for studying the <em>growth</em> and differentiation of human B cell precursors (BCP) has placed restrictions on the ability to analyze the early stages of human B cell ontogeny. We now describe a bone marrow-derived adherent cell microenvironment that maintains human BCP for several weeks in vitro. The adherent cells are maintained in a serum-free tissue culture medium, and consist of a predominant population of CD10+ <em>fibroblast</em>-like cells and a minor population of CD10+/nonspecific esterase+ macrophages. Adherent cell cultures seeded with fresh or cryopreserved fetal bone marrow, or purified CD10+/surface IgM- cells, provide a supportive microenvironment for lymphoid cells with a predominant phenotype of CD10+/CD19+/HLA-DR+/surface IgM-. Supplementation of the adherent cell cultures with human IL-7 induces active <em>growth</em> of BCP during the first 14 to <em>21</em> days of culture. However, the expansion of these cells does not continue past <em>21</em> days, and the cultures undergo a steady decline in BCP. Analysis of adherent cell conditioned medium revealed the presence of an unidentified soluble <em>factor</em> (or <em>factors</em>) that acts in concert with IL-7 to promote the <em>growth</em> of CD10+/surface IgM- cells. This culture system will be useful in elucidating the patterns of gene expression and <em>growth</em> <em>factor</em> requirements that characterize normal human B cell ontogeny, and perturbations of normal B cell ontogeny that lead to immunodeficiency and leukemia.

We addressed the question of whether hypertrophy induced by compensatory overload differs according to innervation status, and how <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF) and hepatocyte <em>growth</em> <em>factor</em> (HGF) mRNAs are expressed in the rat plantaris muscle during overload (OL) and/or denervation. Male Wistar rats were divided into four groups (Normal-Cont, Normal-OL, Denervated-Cont, and Denervated-OL). according to the plantaris denervation and/or overload. Three weeks later, plantaris weight in Denervated-Cont and Denervated-OL was significantly lower than in the Normal-Cont. The muscle weights in the Normal-OL were higher than in the Normal-Cont. The muscle weights in the Denervated-OL were higher than in the Denervated-Cont. Three days after the treatment, FGF-2, FGF-6, FGF-7 and HGF mRNAs in the Normal-OL were significantly higher than those in the Normal-Cont. FGF-2, FGF-6, FGF-7 and HGF mRNAs in the Denervated-OL were also significantly higher after 3 days than those in the Denervated-Cont. After 7 days, FGF-2, FGF-5, FGF-6, FGF-7 and HGF mRNAs were significantly higher in the Normal-OL than those in the Normal-Cont. At <em>21</em> days, FGF-1, FGF-6 and HGF mRNA levels were significantly increased. In the Denervated-OL, FGF-2, FGF-7 and HGF mRNAs at 7 days, and FGF-2 mRNA at <em>21</em> days were significantly higher than those in the Denervated-Cont. FGF-2 and FGF-6 mRNA levels decreased significantly following denervation; however, FGF-1, FGF-5, FGF-7 and HGF mRNA levels increased and maintained this increase for the <em>21</em>-days treatment period. Muscle hypertrophy was thus induced by compensatory overload irrespective of innervation status, possibly in association with certain FGFs and HGF. The differential mRNA expression patterns of FGFs and HGF observed following compensatory overload and/or denervation suggest distinct roles for individual FGFs and HGF in muscle hypertrophy and/or atrophy.

It was demonstrated that estrogen deficiency and consuming high fat (HF) diet enhanced orexigenic activity of ghrelin. Therefore, we hypothesized that antagonizing of ghrelin action would attenuate food intake and body weight in mice obese both from ovariectomy (OVX) and feeding a HF diet. Ghrelin receptor antagonist [D-Lys(3)]GHRP-6 after seven days of subcutaneous treatment markedly decreased food intake in OVX mice fed both HF and standard diets; furthermore, it reduced body weight and blood glucose, insulin and leptin, and increased β-hydroxybutyrate level and uncoupling-protein-1 mRNA in brown adipose tissue. Pair-feeding revealed that effect of [D-Lys(3)]GHRP-6 was primary anorexigenic. Estrogen supplementation reduced anorexigenic effects of [D-Lys(3)]GHRP-6. OVX [D-Lys(3)]GHRP-6 treatment in mice on HF diet resulted in markedly increased circulating level and liver expression of a major metabolic regulator, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>. Our data suggest that ghrelin antagonists could be especially beneficial in individuals with common obesity combined with estrogen deficiency.

Endothelial cells chronically reside in low-O2 environments in vivo (2%-13% O2), which are believed to be critical for cell homeostasis. To elucidate the roles of this physiological chronic normoxia in human endothelial cells, we examined transcriptomes of human umbilical vein endothelial cells (HUVECs), proliferation and migration of HUVECs in response to <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) and vascular endothelial <em>growth</em> <em>factor</em> A (VEGFA), and underlying signaling mechanisms under physiological chronic normoxia. Immediately after isolation, HUVECs were cultured steadily under standard cell culture normoxia (SCN; <em>21</em>% O2) or physiological chronic normoxia (PCN; 3% O2) up to 25 days. We found that PCN up-regulated 41 genes and down-regulated <em>21</em> genes, 90% of which differed from those previously reported from HUVECs cultured under SCN and exposed to acute low O2. Gene ontology analysis indicated that PCN-regulated genes were highly related to cell proliferation and migration, consistent with the results from benchtop assays that showed that PCN significantly enhanced FGF2- and VEGFA-stimulated cell proliferation and migration. Interestingly, preexposing the PCN cells to <em>21</em>% O2 up to 5 days did not completely diminish PCN-enhanced cell proliferation and migration. These PCN-enhanced cell proliferations and migrations were mediated via augmented activation of MEK1/MEK2/ERK1/ERK2 and/or PI3K/AKT1. Importantly, these PCN-enhanced cellular responses were associated with an increase in activation of VEGFR2 but not FGFR1, without altering their expression. Thus, PCN programs endothelial cells to undergo dramatic changes in transcriptomes and sensitizes cellular proliferative and migratory responses to FGF2 and VEGFA. These PCN cells may offer a unique endothelial model, more closely mimicking the in vivo states.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a powerful endocrine hormone modulating glucose and lipid metabolism and represents a promising drug for type 2 diabetes. The present study was to determine the effect of FGF<em>21</em> on high glucose-induced damage and dysfunction in endothelial cells.

METHODS

The protein expression of β-klotho was examined in human umbilical vein endothelial cell (HUVECs) using immunofluorescence and Western blotting. HUVECs were cultured in medium with normal glucose (NG), high glucose (HG) and HG + FGF<em>21</em> (30 nM). Cell viability, migration, reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase, nitric oxide (NO) production, intracellular cyclic guanosine monophosphate (cGMP) and endothelial nitric oxide synthase (eNOS) phosphorylation at Ser-1177/Ser-633 sites were measured.

RESULTS

β-klotho, the anchor protein of FGF<em>21</em>, is expressed in HUVECs. Administration of FGF<em>21</em> prevented HG-induced impairment of cell viability, migration, oxidant stress, NO production and intracellular cGMP levels in HUVECs. FGF<em>21</em> also rescued HG-induced decrease of eNOS phosphorylation at Ser-1177 and Ser-633. HG and FGF<em>21</em> had no effects on eNOS phosphorylation at Ser-617 and Thr-495. Inhibition of AMP-activated protein kinase (AMPK), but not Akt or Ca(2+)/calmodulin-dependent protein kinase II, abolished the protective effect of FGF<em>21</em> on eNOS phosphorylation at Ser-1177. The protective effect of FGF<em>21</em> on eNOS phosphorylation at Ser-633 was also abolished by inhibition of AMPK but not by Akt or cAMP-dependent protein kinase A.

CONCLUSIONS

Our results provide the first evidence that FGF<em>21</em> protects against high glucose induced cell damage and eNOS dysfunction in an AMPK-dependent manner in HUVECs, and suggest that FGF<em>21</em> may be a promoting therapeutic agent for vascular complications in diabetes.

Fibrous capsule formation around implants can inhibit solute exchange between implantable devices and the circulation. Parylene-n coated polycarbonate disks surrounded with <em>growth</em> <em>factor</em> reduced Matrigel (MG) or several gelatin-based matrices were implanted intramuscularly into rats for <em>21</em> or 50 days. MG supplemented with vascular endothelial <em>growth</em> <em>factor</em> (VEGF) or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) increased capsule microvascular density at <em>21</em> days (p < 0.05) when compared to bare parylene-coated polycarbonate disks (control). The increased microvascular density around VEGF- and bFGF-treated implants regressed by 50 days and was no longer significantly different from controls. The microvascular density induced by the gelatin-based matrices was not significantly different from controls at <em>21</em> days, but was increased at 50 days (p < 0.05), suggesting a slower, long-term effect. Disks treated with MG and gelatin-based matrices had thinner capsules at <em>21</em> days (p < 0.05). By 50 days, the capsule thicknesses around these implants were no longer statistically thinner than controls. The capsule thickness around implants treated with VEGF, bFGF, and essential gelatin-based matrix was thinner than controls at 50 days (p < 0.05). These results indicate that it is possible to increase functional microvascular density within fibrous capsules using angiogenic <em>growth</em> <em>factors</em> and gelatin-based matrices. However, this effect may be short-lived, requiring chronic administration of <em>growth</em> <em>factors</em>.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs) play an important role in development and tumorigenesis. Mutations in FGFR2 cause more than five craniosynostosis syndromes. The FGFR2 genomic structure is the largest of the FGFR family. We have refined and extended the genomic organization of the FGFR2 gene by sequencing more than 119 kb of PACs, cosmids, and PCR products and assembling a region of approximately 175 kb. Although the gene structure has been reported to include only 20 exons, we have verified the presence of at least 22 exons, some of which are alternatively spliced. The sizes of six exons differed from those reported previously. Comparison of our sequence and those in the NCBI database detected more than 300 potential single nucleotide polymorphisms (SNPs). However, sequencing regions containing 52 of these potential SNPs verified only 14 in PCR products generated from 16 CEPH alleles. In contrast, direct sequencing of the CEPH DNAs revealed <em>21</em> other polymorphisms. Only one SNP was found in the 2,926 bp of coding sequence. Twenty-seven SNPs, two insertion polymorphisms and five microsatellite polymorphisms are contained in approximately 16.6 kb of non-coding sequence. These data yield an average of one polymorphism for approximately 488 bp of non-coding sequence examined. This collection of SNP, insertion, and repeat polymorphisms will aid future association studies between the FGFR2 gene and human disease and will enhance mutation detection.