BACKGROUND

Angiogenesis is regarded as a hallmark in cancer development, and anti-angiogenic treatment is presently used in non-small cell lung cancer (NSCLC) patients. MicroRNAs (miRs) are small non-coding, endogenous, single stranded RNAs that regulate gene expression. In this study we aimed to identify significantly altered miRs related to angiogenesis in NSCLC.

METHODS

From a large cohort of 335 NSCLC patients, paraffin-embedded samples from 10 patients with a short disease specific survival (DSS), 10 with a long DSS and 10 normal controls were analyzed. The miRs were quantified by microarray hybridization and selected miRs were validated by real-time qPCR. The impacts of different pathways, including angiogenesis, were evaluated by Gene Set Enrichment Analysis (GSEA) derived from Protein ANalysis THrough Evolutionary Relationship (PANTHER). One of the most interesting candidate markers, miR-155, was validated by in situ hybridization (ISH) in the total cohort (n = 335) and correlation analyses with several well-known angiogenic markers were done.

RESULTS

128 miRs were significantly up- or down-regulated; normal versus long DSS (n = 68) and/or normal versus short DSS (n = 63) and/or long versus short DSS (n = 37). The pathway analysis indicates angiogenesis-related miRs to be involved in NSCLC. There were strong significant correlations between the array hybridization and qPCR validation data. The significantly altered angiogenesis-related miRs of high interest were miR-<em>21</em>, miR-106a, miR-126, miR-155, miR-182, miR-<em>21</em>0 and miR-424. miR-155 correlated significantly with <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) in the total cohort (r = 0.17, P = 0.002), though most prominent in the subgroup with nodal metastasis (r = 0.34, P<0.001).

CONCLUSIONS

Several angiogenesis-related miRs are significantly altered in NSCLC. Further studies to understand their biological functions and explore their clinical relevance are warranted.

The EFFECT-II study aimed to investigate the effects of dapagliflozin and omega-3 (n-3) carboxylic acids (OM-3CA), individually or combined, on liver fat content in individuals with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD).

This randomised placebo-controlled double-blind parallel-group study was performed at five clinical research centres at university hospitals in Sweden. 84 participants with type 2 diabetes and NAFLD were randomly assigned 1:1:1:1 to four treatments by a centralised randomisation system, and all participants as well as investigators and staff involved in the study conduct and analyses were blinded to treatments. Each group received oral doses of one of the following: 10 mg dapagliflozin (n = <em>21</em>), 4 g OM-3CA (n = 20), a combination of both (n = 22) or placebo (n = <em>21</em>). The primary endpoint was liver fat content assessed by MRI (proton density fat fraction [PDFF]) and, in addition, total liver volume and markers of glucose and lipid metabolism as well as of hepatocyte injury and oxidative stress were assessed at baseline and after 12 weeks of treatment (completion of the trial).

Participants had a mean age of 65.5 years (SD 5.9), BMI 31.2 kg/m2 (3.5) and liver PDFF 18% (9.3). All active treatments significantly reduced liver PDFF from baseline, relative changes: OM-3CA, -15%; dapagliflozin, -13%; OM-3CA + dapagliflozin, -<em>21</em>%. Only the combination treatment reduced liver PDFF (p = 0.046) and total liver fat volume (relative change, -24%, p = 0.037) in comparison with placebo. There was an interaction between the PNPLA3 I148M polymorphism and change in liver PDFF in the active treatment groups (p = 0.03). Dapagliflozin monotherapy, but not the combination with OM-3CA, reduced the levels of hepatocyte injury biomarkers, including alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase (γ-GT), cytokeratin (CK) 18-M30 and CK 18-M65 and plasma <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>). Changes in γ-GT correlated with changes in liver PDFF (ρ = 0.53, p = 0.02). Dapagliflozin alone and in combination with OM-3CA improved glucose control and reduced body weight and abdominal fat volumes. Fatty acid oxidative stress biomarkers were not affected by treatments. There were no new or unexpected adverse events compared with previous studies with these treatments.

Combined treatment with dapagliflozin and OM-3CA significantly reduced liver fat content. Dapagliflozin monotherapy reduced all measured hepatocyte injury biomarkers and FGF<em>21</em>, suggesting a disease-modifying effect in NAFLD.

ClinicalTrials.gov NCT02279407 FUNDING: The study was funded by AstraZeneca.

Methionine restriction (MR) decreases body weight and adiposity and improves glucose homeostasis in rodents. Similar to caloric restriction, MR extends lifespan, but is accompanied by increased food intake and energy expenditure. Most studies have examined MR in young animals; therefore, the aim of this study was to investigate the ability of MR to reverse age-induced obesity and insulin resistance in adult animals. Male C57BL/6J mice aged 2 and 12 months old were fed MR (0.172% methionine) or control diet (0.86% methionine) for 8 weeks or 48 h. Food intake and whole-body physiology were assessed and serum/tissues analyzed biochemically. Methionine restriction in 12-month-old mice completely reversed age-induced alterations in body weight, adiposity, physical activity, and glucose tolerance to the levels measured in healthy 2-month-old control-fed mice. This was despite a significant increase in food intake in 12-month-old MR-fed mice. Methionine restriction decreased hepatic lipogenic gene expression and caused a remodeling of lipid metabolism in white adipose tissue, alongside increased insulin-induced phosphorylation of the insulin receptor (IR) and Akt in peripheral tissues. Mice restricted of methionine exhibited increased circulating and hepatic gene expression levels of FGF21, phosphorylation of eIF2a, and expression of ATF4, with a concomitant decrease in IRE1α phosphorylation. Short-term 48-h MR treatment increased hepatic FGF21 expression/secretion and insulin signaling and improved whole-body glucose homeostasis without affecting body weight. Our findings suggest that MR feeding can reverse the negative effects of aging on body mass, adiposity, and insulin resistance through an FGF21 mechanism. These findings implicate MR dietary intervention as a viable therapy for age-induced metabolic syndrome in adult humans.

HLA-A2-restricted, CD3+, CD8+, alpha/beta+ cytotoxic T cell (CTL) clones were isolated from peripheral blood (PBL) or tumor infiltrating lymphocytes (TIL) of two HLA-A2+ melanoma patients (9742 and 5810), to evaluate the possible recognition of autologous melanoma and of allogeneic HLA-A2-matched normal melanocytes. These CTL clones lysed not only fresh and cultured autologous melanoma cells, but also allogeneic HLA-A2+, but not HLA-A2-, normal melanocytes. The lysis of autologous neoplastic cells and of melanocytes could be inhibited by an anti-HLA-A2 monoclonal antibody (mAb). Lysis of the normal melanocytes was not dependent on the presence of human or fetal calf serum in the culture medium. HLA-A2-restricted CTL clones recognized not only proliferating melanocytes cultured in complete melanocyte medium, but also melanocytes made quiescent by culture for up to 6 d in a basal medium devoid of exogenous <em>factors</em> such as phorbol ester (O-tetradecanoyl phorbol 13-acetate [TPA]), epidermal <em>growth</em> <em>factor</em>, insulin, and pituitary extracts. Analysis of specificity of four CTL clones (A75, A83, A94, and 119) from patient 9742, performed on a panel of 39 targets, indicated that the three HLA-A2-restricted CTL (A75, A83, and A94) lysed all but one of nine allogeneic melanomas expressing the HLA-A2 molecule with no reactivity on nine HLA-A2- allogeneic melanomas. Only a few instances of borderline reactivity were seen by the same effectors on <em>21</em> targets of nonmelanocyte lineage, including 12 carcinomas of different histology, four Epstein-Barr virus-transformed B cells (lymphoblastoid cell lines [LCL]), including the autologous LCL, four lines of normal <em>fibroblasts</em>, and normal kidney cells. Lack of reactivity on allogeneic targets of nonmelanocyte lineage occurred in spite of expression of HLA-A2 on 14 of these targets as determined by conventional tissue typing and cytofluorimetric analysis with four different anti-HLA-A2 mAb. These data indicate that tissue-related antigens can be expressed on normal and neoplastic cells of the melanocyte lineage and can be recognized in association with HLA-A2 by CTL clones from melanoma patients.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a hormone produced by fat and the liver that plays an important role in lipid metabolism. FGF<em>21</em> expression is induced by peroxisome proliferator-actived receptor alpha in response to physiological conditions requiring increased fatty acid oxidation. Retinoic acid receptor-related receptor alpha (RORalpha) is another nuclear receptor that plays a critical role in lipid metabolism as well as in regulation of the circadian rhythm. In this study we demonstrate that RORalpha directly regulates the expression and secretion of FGF<em>21</em>. A canonical ROR response element was identified in the proximal promoter of the FGF<em>21</em> gene and shown to exhibit functional activity. Overexpression of RORalpha in HepG2 cells resulted in increased expression and secretion of FGF<em>21</em>. Suppression of RORalpha expression caused a decrease in FGF<em>21</em> expression and secretion, suggesting that RORalpha contributes to the basal expression of FGF<em>21</em>. These data suggest that one mechanism by which RORalpha regulates lipid metabolism may be by modulation of FGF<em>21</em> secretion. Furthermore, this study identifies a clear link between RORalpha, a key regulator of the mammalian clock, and FGF<em>21</em>, an important hormone regulating glucose and lipid homeostasis.

High serum concentrations of vascular endothelial <em>growth</em> <em>factor</em> (S-VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (S-bFGF) are associated with unfavorable clinical characteristics in cancer. The combined effect of S-VEGF and S-bFGF on the survival of 200 patients with non-Hodgkin lymphoma (NHL) was studied. High S-VEGF and S-bFGF at diagnosis were associated with poor survival with the medians, the highest tertiles, or the highest quartiles as the cutoff values. The highest prognostic power was obtained when S-VEGF and S-bFGF were examined as a combination. Patients who had both S-VEGF and S-bFGF within the highest quartiles had only a <em>21</em>% 5-year survival rate in contrast to a 64% 5-year survival rate among patients with both <em>factors</em> within the 3 lowest quartiles (P <.0001). Simultaneous elevation of S-VEGF and S-bFGF was associated with poor survival in different grades of lymphomas and in the largest histologic subgroup, the large-cell diffuse and immunoblastic lymphomas. S-VEGF (relative risk [RR], 1.83; P =.019) and S-bFGF (RR, 2.02; P =.0049) had independent influences on survival in multivariate models when tested together with the components of the International Prognostic Index (IPI). Patients with both S-VEGF and S-bFGF within the highest quartiles had nearly 3 times higher risk for death (RR, 2.90; 95% confidence interval [CI], 1.56-5.40; P =.0008) than the rest of the patients. This RR was higher than the relative risks associated with any of the components of the IPI in the same model. The authors conclude that the combination of S-VEGF and S-bFGF is a powerful prognostic variable in NHL. (Blood. 2000;96:3712-3718)

We have examined the presence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) in normal and in Alzheimer brains, studied the distribution of the mitogen by immunohistochemical techniques, measured the quantities of <em>growth</em> <em>factor</em> in selected areas of the brain (Brodmann areas 10/11 and 20/<em>21</em>), characterized the molecular forms by Western blotting and determined its sites of synthesis by in situ hybridization. Although the same molecular forms of basic FGF are found in control and Alzheimer brains, basic FGF is increased in the brains of Alzheimer's patients. Furthermore, basic FGF is not distributed in an identical fashion to normal and Alzheimer brains, but is found in association with the lesions that characterize this disease. In normal controls (n = 5), basic FGF was found to be widely distributed throughout the three brain regions examined (prefrontal cortex, hippocampus, and hypothalamus). Immunoreactivity was observed within astrocytes in both the grey and white matter, as well as within neuronal perikarya. Brain tissues that were obtained from Alzheimer patients (N = 4) showed a substantial increase in the overall specific staining of astrocytes and neurons, particularly in areas of reactive gliosis. Focal concentration of immunoreactive basic FGF was evident within the neuritic plaques, and could be clearly seen in association with the neurofibrillary tangles present within neuronal perikarya. The possibility that basic FGF expression in the CNS is linked to the pathogenesis of the disease is discussed.

The regulation of galectin-3 expression in skeletal tissues is not completely understood. Our studies indicate that HIF-1 alpha regulates galectin-3 expression by interacting with hypoxia regulatory elements in the promoter region. Finally, we show that galectin-3 serves a prosurvival role in the intervertebral disc.

BACKGROUND

Earlier reports indicated that galectin-3 (gal-3) is highly expressed in the epiphyseal growth plate cartilage and the intervertebral disc. Because these skeletal tissues have a limited vascular supply and the cells reside in a low O2 environment, we determined if the oxemic status modulates gal-3 expression.

METHODS

Cells were cultured in normoxia (21% O2) or hypoxia (2% O2), and gal-3 expression and promoter activity were evaluated. Interaction of hypoxia inducible factor (HIF)-1 alpha with the gal-3 promoter was confirmed by gel shift and site-directed mutagenesis.

RESULTS

There was minimal oxygen-dependent change in HIF-1 alpha levels and no change in gal-3 expression and promoter activity in nucleus pulposus cells. In contrast, hypoxia induced gal-3 mRNA, protein, and promoter activity in HeLa cells and mouse embryonic fibroblasts (MEFs) from HIF-1 alpha wildtype but not HIF-1-null mice. To evaluate the importance of HIF-1 in regulation of gal-3 expression, we overexpressed HIF-1 alpha or constitutively active-HIF-1 alpha in null MEF. An increase in gal-3 promoter activity was observed in both normoxia and hypoxia. Similarly, suppression of HIF-1 alpha in nucleus pulposus cells, and wildtype MEF, using siRNA and pharmacological inhibitors resulted in suppression of gal-3 promoter activity and mRNA levels. Analysis of the gal-3 promoter indicated that it contained two hypoxia response elements (HREs). Gel-shift and chromatin immunoprecipitation analysis confirmed that there was binding of HIF-1 alpha to the gal-3 HRE. Furthermore, site-directed mutagenesis of HRE completely blocked hypoxic induction of gal-3 promoter activity. In nucleus pulposus cells, suppression of gal-3 expression promoted FasL-mediated apoptosis.

CONCLUSIONS

Together, these studies showed that gal-3 is a HIF-1-regulated lectin that plays an important role in nucleus pulposus cell survival.

Recently, mast cell tryptase has been identified as another potent proangiogenic <em>factor</em> in tumors, along with <em>fibroblast</em> and vascular endothelial <em>growth</em> <em>factors</em>. Its role has been studied in a number of cancers, including carcinoma of the uterine cervix, with discordant results. Our aim was to study the expression of tryptase and bFGF in mast cells (MCs) during development of neoangiogenesis in premalignant and malignant lesions of the cervix. Biopsy specimens from <em>21</em> patients without cancer and from 63 patients with dysplasias and squamous cell carcinomas were used. They were stained with Alcian blue-safranin O (ABSO) and immunostained with specific antibodies against <em>factor</em> VIII, CD105, tryptase, and bFGF. Tryptase-positive mast cells increased with tumor progression and were close to newly formed blood vessels. Vascularization showed a linear increase from dysplasia to invasive cancer. We suggest that MC tryptase may upregulate neoangiogenesis in carcinogenesis of the uterine cervix.

OBJECTIVE

To examine the expression and regulation of interleukin-<em>21</em> (IL-<em>21</em>) and IL-<em>21</em> receptor (IL-<em>21</em>R) in patients with systemic sclerosis (SSc; scleroderma).

METHODS

Skin biopsy specimens were obtained from 23 patients with SSc and 15 healthy controls. IL-<em>21</em>/IL-<em>21</em>R messenger RNA (mRNA) was quantified using real-time polymerase chain reaction (PCR). The expression pattern of IL-<em>21</em>/IL-<em>21</em>R was analyzed by in situ hybridization and Western blotting. Stimulation experiments were performed with cultured dermal fibroblasts from patients with SSc and healthy controls as well as with keratinocytes, using IL-1beta, platelet-derived growth factor BB, monocyte chemoattractant protein 1, transforming growth factor beta, and IL-<em>21</em>. The SCID-hu skin mouse model was used for in vivo experiments.

RESULTS

IL-<em>21</em>R mRNA was detected in all biopsy specimens from patients with SSc and controls, with a 4.7-fold increase observed in SSc samples. In situ hybridization and immunohistochemical analysis showed an up-regulation of IL-<em>21</em>R in samples of epidermis from SSc patients, whereas no signal was detected in skin specimens from healthy controls. These results were confirmed in vitro, in that cultured keratinocytes expressed significant levels of IL-<em>21</em>R, whereas no signal was observed in fibroblasts. Interestingly, mRNA for IL-<em>21</em> could not be detected by real-time PCR and in situ hybridization. Various concentrations of key cytokines in the pathogenesis of SSc did not stimulate the expression of IL-<em>21</em>R mRNA in cultured keratinocytes and dermal fibroblasts. In the SCID mouse transplantation model, the overexpression of IL-<em>21</em>R mRNA in SSc keratinocytes remained unchanged after transplantation.

CONCLUSIONS

The up-regulation of IL-<em>21</em>R in keratinocytes indicates that, similar to fibroblasts and endothelial cells, the expression pattern is altered in SSc. Moreover, the expression of IL-<em>21</em>R appears to be independent of key cytokines that are operant in SSc.

Recent studies have indicated that TNF can promote activation of the coagulation mechanism by modulating coagulant properties of endothelial cells. In this report, we demonstrate that infusion of low concentrations of TNF (3 micrograms/animal) into mice bearing meth A fibrosarcomas leads to localized fibrin deposition with formation of occlusive intravascular thrombi in close association with the endothelial cell surface. Studies with 125I-fibrinogen showed tenfold enhanced accumulation of radioactivity in tumor within 2 h after TNF infusion. Western blots of tumor extracts subjected to SDS-PAGE and visualized with a fibrin-specific mAb indicated that fibrin forms in the tumor after the TNF infusion. Electron microscopic studies demonstrated fibrin strands, based on the characteristic <em>21</em>-nm periodicity, which appeared to be adherent to the endothelial cell surface. Further ultrastructural studies indicated that fibrin formation, first evident within 30 min of the TNF infusion, led to occlusive thrombi limited to the tumor vascular bed (i.e., not in the normal mouse vasculature) within 2 h and was associated with an 80% reduction in tumor perfusion based on studies with Evans blue. In view of previous work concerning TNF induction of endothelial cell procoagulant activity, the hypothesis that tumor cell products prime the response of endothelium to this cytokine was tested. Supernatants of cultured meth A fibrosarcomas obtained serum-free conditions, which had no intrinsic procoagulant activity, considerably enhanced tissue <em>factor</em> induction in endothelium in response to submaximal concentrations of TNF. The <em>factor</em>(s) in the tumor-conditioned medium appeared to be distinct from IL-1, <em>fibroblast</em> <em>growth</em> <em>factor</em>, IFN-gamma, TNF, endotoxin, TGF-alpha, and TGF-beta. These studies delineate a novel model of localized clot formation in which thrombosis is initiated by a pathophysiologic mediator, TNF, and provides an opportunity to examine mechanisms in the microenvironment directing clot formation to the tumor vascular bed.

IL-6, which is also known as IFN-beta 2, hybridoma <em>growth</em> <em>factor</em>, hepatocyte-stimulating <em>factor</em>, and B cell differentiation <em>factor</em>, mediates acute phase responses including fever, has lymphocyte-stimulating capacities, and antiviral activity. IL-6 is produced by monocytes, <em>fibroblasts</em>, certain lymphocytes, and various tumor cells. The present study demonstrates that this multifunctional cytokine is released also by normal human epidermal cells (EC) and human epidermoid carcinoma cell lines (A431, KB). Accordingly, supernatants derived from freshly isolated EC, long term keratinocyte cultures, A431, or KB cells stimulated the proliferation of a hybridoma <em>growth</em> <em>factor</em>/IL-6-dependent plasmacytoma cell line (B9). IL-6 constitutively was produced in the presence of serum proteins. The addition of IL-1 alpha, IL-1 beta, or the tumor promoter PMA significantly enhanced the synthesis and release of EC-derived IL-6 (EC-IL 6). Like monocyte or <em>fibroblast</em>-derived IL-6, EC-IL-6 exhibited Mr microheterogeneity within <em>21</em> and 28 kDa. Similarly in Western blotting experiments an antiserum directed against human rIFN-beta 2/IL-6 detected the different Mr forms of EC-IL-6. Moreover, this antiserum was able to block the B9 cell <em>growth</em>-promoting capacity of EC-IL-6 strongly suggesting that this EC-derived mediator is closely related, if not identical with IL-6. This was further confirmed by Northern blot analysis detecting IL-6 specific mRNA both in long term cultured keratinocytes and A431 cells by hybridization with a cDNA fragment encoding for B cell differentiating <em>factor</em> 2/IL-6. Therefore, in addition to the production of other cytokines as previously reported, EC and in particular keratinocytes also synthesize and release IL-6. This further supports the important regulatory role of the epidermis during the pathogenesis of inflammatory, autoimmune, and neoplastic diseases.

OBJECTIVE

To investigate renal elimination of the adipokine <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) by determining circulating FGF<em>21</em> levels in patients on chronic hemodialysis (CD) as compared with control subjects with a glomerular filtration rate (GFR) >50 ml/min.

METHODS

FGF<em>21</em> was determined by enzyme-linked immunosorbent assay in control (n = 60) and CD (n = 60) patients and correlated to clinical and biochemical measures of renal function, glucose and lipid metabolism, and inflammation in both groups.

RESULTS

Median serum FGF<em>21</em> levels were >15-fold higher in CD patients (3,710.6 ng/l) than in subjects with a GFR >50 ml/min (201.9 ng/l) (P < 0.001). Furthermore, serum creatinine positively and GFR negatively predicted FGF<em>21</em> concentrations in multiple regression analyses in control subjects (P < 0.05).

CONCLUSIONS

FGF<em>21</em> serum levels increase in CD patients and are related to markers of renal function in control subjects.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>) signaling requires the presence of beta-Klotho, a co-receptor with a very short cytoplasmic domain. Here we show that FGF<em>21</em> binds directly to beta-Klotho through its C-terminus. Serial C-terminal truncations of FGF<em>21</em> weakened or even abrogated its interaction with beta-Klotho in a Biacore assay, and led to gradual loss of potency in a luciferase reporter assay but with little effect on maximal response. In contrast, serial N-terminal truncations of FGF<em>21</em> had no impact on beta-Klotho binding. Interestingly, several of them exhibited characteristics of partial agonists with minimal effects on potency. These data demonstrate that the C-terminus of FGF<em>21</em> is critical for binding to beta-Klotho and the N-terminus is critical for <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) activation.

After acute exposure of cells to arsenic, reactive oxygen species mediate changes in cell behavior, including activation of proliferative signaling. For chronic exposure to arsenic, however, the function of reactive oxygen species in cell transformation remains poorly understood. Although microRNA-<em>21</em> (miR-<em>21</em>) has been implicated in various aspects of carcinogenesis, its functions and molecular mechanisms in carcinogen-induced tumorigenesis are unclear. The purpose of this study was to determine if miR-<em>21</em> is involved in arsenite-induced malignant transformation and to characterize the associated signaling pathways. During arsenite-induced transformation of human embryo lung <em>fibroblast</em> (HELF) cells, miR-<em>21</em> was upregulated, and the extracellular signal-regulated kinase (ERK)/nuclear <em>factor</em>-κB (NF-κB) signal pathway was activated. Moreover, superoxide radical dismutase (a scavenger of superoxide) and catalase (a scavenger of hydroperoxides) blocked the arsenite-induced effects in HELF cells and mouse embryonic <em>fibroblasts</em>. Blockage of ERK by the inhibitor U0126 or inhibition of NF-κB p65 by siRNA or Bay 11-7082 prevented the increases in miR-<em>21</em> and the decreases in Spry1, Pten, and Pdcd4, the target proteins of miR-<em>21</em>, induced by arsenite. As determined by a ChIP-qPCR assay, NF-κB p65 regulated miR-<em>21</em> expression by binding directly to the promoter of miR-<em>21</em>. Further, anti-miR-<em>21</em> downregulated miR-<em>21</em> expression and prevented the arsenite-induced activation of ERK via the increase in Spry1, indicating that miR-<em>21</em> has a feedback effect in regulating ERK activation. Overexpression of miR-<em>21</em> with an miR-<em>21</em> mimic and feedback activation of ERK and NF-κB via the decrease in Spry1 promoted the malignancy of HELF cells exposed to arsenite, but knockdown of miR-<em>21</em> with anti-miR-<em>21</em> and feedback blockage of ERK and NF-κB activation through an increase in Spry1 decreased anchorage-independent <em>growth</em> of arsenite-transformed cells. Thus, the transformation of HELF cells induced by chronic exposure to arsenite is mediated by increased miR-<em>21</em> expression, which, in turn, is mediated by reactive oxygen species activation of the ERK/NF-κB pathway.

OBJECTIVE

We examined the relationship between brachial-ankle pulse wave velocity (baPWV) reflecting arterial stiffness and the levels of novel hepatokines <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) and fetuin-A. In addition, we evaluated the effect of a 3-month combined aerobic and resistance exercise programme on FGF<em>21</em> and fetuin-A levels as well as arterial stiffness in obese women.

METHODS

Forty nondiabetic, obese women (body mass index = 27·6 ± 2·4 kg/m(2) ) were included in the study and were compared before and after a 3-month exercise programme, which was composed of 45 min of aerobic exercise at an intensity of 60-75% of the age-predicted maximum heart rate (300 kcal/session) and 20 min of resistance training (100 kcal/session) five times a week. All exercise sessions were supervised by a professional exercise physiologist.

RESULTS

At baseline, baPWV levels were correlated with age, body mass index (BMI), systolic blood pressure (SBP), high density lipoprotein cholesterol, fasting glucose and serum FGF<em>21</em> levels. In a multiple stepwise regression analysis using baPWV as a dependent variable, baPWV levels were associated with age, BMI, SBP, FGF<em>21</em> and fetuin-A levels (R(2) = 0·744). After the exercise programme, BMI, waist circumference, SBP, diastolic blood pressure and triglyceride levels were significantly decreased. Moreover, baPWV values were significantly improved (P < 0·001) along with modest decrease in FGF<em>21</em> levels (P = 0·043). However, fetuin-A levels were not changed significantly (P = 0·202).

CONCLUSIONS

A 3-month combined exercise programme decreases the FGF<em>21</em> levels as well as arterial stiffness in obese Korean women.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a potent antidiabetic and triglyceride-lowering hormone whose hepatic expression is highly responsive to food intake. FGF<em>21</em> induction in the adaptive response to fasting has been well studied, but the molecular mechanism responsible for feeding-induced repression remains unknown. In this study, we demonstrate a novel link between FGF<em>21</em> and a key circadian output protein, E4BP4. Expression of Fgf<em>21</em> displays a circadian rhythm, which peaks during the fasting phase and is anti-phase to E4bp4, which is elevated during feeding periods. E4BP4 strongly suppresses Fgf<em>21</em> transcription by binding to a D-box element in the distal promoter region. Depletion of E4BP4 in synchronized Hepa1c1c-7 liver cells augments the amplitude of Fgf<em>21</em> expression, and overexpression of E4BP4 represses FGF<em>21</em> secretion from primary mouse hepatocytes. Mimicking feeding effects, insulin significantly increases E4BP4 expression and binding to the Fgf<em>21</em> promoter through AKT activation. Thus, E4BP4 is a novel insulin-responsive repressor of FGF<em>21</em> expression during circadian cycles and feeding.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a novel metabolic regulator that represents a promising target for the treatment of several metabolic diseases. Administration of recombinant wild type FGF<em>21</em> to diabetic animals leads to a dramatic improvement in glycaemia and ameliorates other systemic measures of metabolic health. Here we report the pharmacologic outcomes observed in non-human primates upon administration of a recently described FGF<em>21</em> analogue, LY2405319 (LY). Diabetic rhesus monkeys were treated subcutaneously with LY once daily for a period of seven weeks. The doses of LY used were 3, 9 and 50 mg/kg each delivered in an escalating fashion with washout measurements taken at 2, 4, 6 and 8 weeks following the final LY dose. LY therapy led to a dramatic and rapid lowering of several important metabolic parameters including glucose, body weight, insulin, cholesterol and triglyceride levels at all doses tested. In addition, we observed favorable changes in circulating profiles of adipokines, with increased adiponectin and reduced leptin indicative of direct FGF<em>21</em> action on adipose tissue. Importantly, and for the first time we show that FGF<em>21</em> based therapy has metabolic efficacy in an animal with late stage diabetes. While the glycemic efficacy of LY in this animal was partially attenuated its lipid lowering effect was fully preserved suggesting that FGF<em>21</em> may be a viable treatment option even in patients with advanced disease progression. These findings support continued exploration of the FGF<em>21</em> pathway for the treatment of metabolic disease.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a liver-secreted endocrine <em>factor</em> with multiple beneficial effects on obesity-related disorders. It enhances glucose uptake by inducing the expression of glucose transporter-1 (GLUT1) in adipocytes. Here we investigated the signaling pathways that mediate FGF<em>21</em>-induced GLUT1 expression and glucose uptake in vitro and in animals. Quantitative real-time PCR and a luciferase reporter assay showed that FGF<em>21</em> induced GLUT1 expression through transcriptional activation. The truncation of the GLUT1 promoter from -3145 to -3105 bp, which contains two highly conserved serum response element (SRE) and E-Twenty Six (ETS) binding motif, dramatically decreased FGF<em>21</em>-induced promoter activity of the GLUT1 gene. A chromatin immunoprecipitation assay demonstrated that the transcription <em>factors</em> serum response <em>factor</em> (SRF) and Ets-like protein-1 (Elk-1) were recruited to the GLUT1 promoter upon FGF<em>21</em> stimulation. The siRNA-mediated knockdown of either SRF or Elk-1 resulted in a marked attenuation in FGF<em>21</em>-induced GLUT1 expression and glucose uptake in adipocytes. In C57 lean mice, a single intravenous injection of FGF<em>21</em> induced phosphorylation of Elk-1 at Ser(383) and SRF at Ser(103) and also led to the recruitment of Elk-1 and SRF to the GLUT1 promoter in epididymal fats. By contrast, such effects of in vivo FGF<em>21</em> administration were blunted in high fat diet-induced obese mice. In conclusion, FGF<em>21</em> induces GLUT1 expression and glucose uptake through sequential activation of ERK1/2 and SRF/Elk-1, which in turn triggers the transcriptional activation of GLUT1 in adipocytes. The impairment in this signaling pathway may contribute to FGF<em>21</em> resistance in obese mice.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>21</em>, and possibly FGF19, protect against type 2 diabetes mellitus (T2DM) and obesity in rodents. We investigated the circulating levels of FGF<em>21</em> and FGF19 in obese patients with varying degrees of abnormal glucose homeostasis, and we determined gene expression for FGF receptors (FGFR1-4) and the co-receptor β-Klotho, in liver and adipose tissues.

METHODS

We analyzed 35 lean healthy (71% men) and 61 obese patients (49% men, median body mass index (BMI): 40.5 kg m(-2), interquartile range: 34.7-46.2). Among obese patients, 36 were normoglycemic, 15 showed impaired glucose tolerance and 10 had T2DM. Biopsies from liver and visceral and subcutaneous fat from a subset of obese patients and controls were analyzed. FGF19 and FGF<em>21</em> levels were measured using enzyme-linked immunosorbent assay, and tissue mRNA and protein levels by reverse transcription-polymerase chain reaction and immunoblotting.

RESULTS

FGF<em>21</em> serum levels were significantly increased in obese patients compared with controls (P<0.001), whereas FGF19 levels were decreased (P < 0.001). FGF<em>21</em> levels were positively correlated with homeostasis model assessment of insulin resistance (P = 0.0002, r = 0.37) and insulin (P = 0.001, r = 0.32), whereas FGF19 levels were negatively correlated (P = 0.007, r = -0.27; P=0.003, r = -0.28; respectively). After adjusting for BMI, the correlations of FGF<em>21</em> and FGF19 levels with indicators of abnormal glucose homeostasis were not significant. In obese patients, the hepatic expression of FGF<em>21</em> was increased. (P = 0.04). β-Klotho transcript levels in visceral fat (P = 0.002) and β-Klotho protein levels in subcutaneous (P = 0.03) and visceral fat (P = 0.04) were significantly reduced in obese patients, whereas hepatic levels for β-Klotho (P = 0.03), FGFR1 (P = 0.04) and FGFR3 (P = 0.001) transcripts were significantly increased.

CONCLUSIONS

Obesity is characterized by reciprocal alterations in FGF19 (decrease) and FGF<em>21</em> (increase) levels. Although worsened in diabetic obese patients, obesity itself appears as the predominant determinant of the abnormalities in FGF<em>21</em> and FGF19 levels. Opposite changes in β-Klotho expression in fat and liver indicate potential tissue-specific alterations in the responsiveness to endocrine FGFs in obesity.

Angiogenesis, the development of new blood vessels, is an important process in tissue development and wound healing but becomes pathologic when associated with solid tumor <em>growth</em>, proliferative retinopathies, and rheumatoid arthritis. To date, there has not been a physiologically relevant in vitro model for human angiogenesis that can be used to screen for enhancers and inhibitors of human angiogenesis and allow further investigation of this process. Initially, culture conditions were established for the induction of human angiogenesis in vitro using fragments of human placental blood vessel. Once the assay was validated, it was examined for its ability to detect known inhibitors and enhancers of angiogenesis. The role of endogenous acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) in the angiogenic response was also assessed by performing RT-PCR on both the parent vessel and microvessel out<em>growth</em>s. In addition, neutralizing antibodies against the three <em>growth</em> <em>factors</em> were used to quantify the relative importance of each <em>growth</em> <em>factor</em> in the angiogenic response. A fragment of human placental blood vessel was embedded in a fibrin gel in microculture plates and was found to give rise to a complex network of microvessels during a period of 7 to <em>21</em> days in culture. The response did not require the addition of exogenous <em>growth</em> <em>factors</em>, and thus provides a convenient system for testing substances for their ability to stimulate or inhibit a human in vitro angiogenic response. The ability of the well known angiogenesis antagonist, hydrocortisone, in the presence and absence of heparin, and suramin to significantly inhibit the angiogenic response indicated that the model could be used as an efficient in vitro assay for screening inhibitors of human angiogenesis. The presence of mRNA for aFGF, bFGF, and three isoforms of VEGF, as well as their receptors, FGFR1, FGFR2, Flt-1, and KDR, in vessel out<em>growth</em>s and the parent vessel, as identified by RT-PCR, strongly implicated aFGF, bFGF, and VEGF as having an important role in this neovascularization response. This was further confirmed by the ability of neutralizing antibodies to aFGF, bFGF, and VEGF to inhibit the angiogenic response to varying extent. Furthermore, the response could be enhanced by the addition of these <em>growth</em> <em>factors</em> in serum-starved cultures. Finally, a stimulatory effect was observed when matrigel was incorporated into the fibrin gel, which indicates that components of the extracellular matrix also play an important role in governing the strength of the angiogenic response. A physiologic angiogenic response relevant to wound healing can be generated by culturing fragments of human placental blood vessels in fibrin gels. The <em>growth</em> <em>factors</em> aFGF, bFGF, and VEGF were shown to play an important role in stimulating this spontaneous angiogenic response. This assay, which can be performed in microcultures, was also shown to be an excellent method for screening for potential inhibitors and enhancers of human angiogenesis.

OBJECTIVE

To investigate the spontaneous occurrence of circulating mesenchymal stem cells (MSC) and angiogenic factors in patients with ST elevation acute myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (PCI).

METHODS

In 20 patients with STEMI, blood samples were obtained on days 1, 3, 7, 14, 21, and 28 after the acute PCI. Fifteen patients with a normal coronary angiography formed a control group. MSC (CD45-/CD34-), plasma stromal derived factor 1 (SDF-1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2) were measured by multiparametric flow cytometry and enzyme linked immunosorbent assay (ELISA).

RESULTS

Circulating CD45-/CD34- cells were significantly decreased on day 7 compared with day 3. Cell counts normalised one month after the acute onset of STEMI. The changes were mainly seen in patients with a large infarction. Plasma SDF-1 increased significantly from day 3 to day 28, and VEGF-A and FGF-2 increased significantly from day 7 to day 28.

CONCLUSIONS

Spontaneous sequential fluctuations in MSC and the increase in vascular growth factor concentrations after STEMI suggest that the optimal time for additional stem cell therapy is three weeks after a myocardial infarction to obtain the maximum effects by stimulating endogenous growth factors on the delivered stem cells.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) has been implicated in the progression of human tumours via both autocrine and paracrine (angiogenic) activities. We investigated the expression of FGF-2 and FGF receptors (FGFR-1 to -4) in NSCLC cell lines (N = 16), NSCLC surgical specimens (N = <em>21</em>) and 2 control cell lines. Our data show that almost all NSCLC cells produce elevated levels of FGF-2 and FGFR in vitro and in vivo. FGF-2 expression did correlate with a short doubling time as well as with potent anchorage-independent <em>growth</em> of NSCLC cell lines. In contrast with control cells, NSCLC cells did not secrete considerable amounts of FGF-2 into the extracellular space. Expression levels of FGFR-1 and -2 in NSCLC cell lines correlated with FGF-2 production. FGFR were located at the plasma membranes in some low FGF-2-producing NSCLC and control cell lines. These cells were sensitive to the proliferative effect of recombinant FGF-2 (rFGF-2). In NSCLC cell lines with an enhanced FGF-2 production, representing the majority studied, FGFR localisation was predominantly intracellular. These cells were insensitive to both the proliferative effect of rFGF-2 and <em>growth</em> inhibition by FGF-2-neutralising antibodies. In contrast, several agents antagonised FGF-2 intracellularly impaired <em>growth</em> of almost all NSCLC cell lines. Our data suggest a role of FGF-2 and FGFR in the <em>growth</em> stimulation of NSCLC cells possibly via an intracrine mechanism.

OBJECTIVE

The diagnosis of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) is limited by the need for liver biopsy. We aimed at testing the accuracy of cytokeratin-18 fragment (CK-18), adipocyte fatty acid binding protein (AFABP) and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) for the diagnosis of NAFLD and NASH.

METHODS

146 patients with biopsy-proven NAFLD and 74 age- and gender-matched healthy controls were included. Serum CK-18, AFABP and FGF<em>21</em> levels were determined by enzyme-linked immunosorbent assay.

RESULTS

Serum CK-18, AFABP, and FGF<em>21</em> increased in a stepwise fashion in control subjects (median 103 U/L, 15.4 ng/ml, and 104 pg/ml), patients with non-NASH NAFLD (263 U/L, 18.9 ng/ml, and 249 pg/ml) and NASH (418 U/L, 19.4 ng/ml, and 354 pg/ml) (p<0.001, 0.060, and 0.016, respectively). The area under receiver-operating characteristics curve to diagnose NAFLD and NASH was 0.91 and 0.70 for CK-18, 0.66 and 0.59 for AFABP, and 0.84 and 0.62 for FGF<em>21</em>. At cut-offs of 203 and 670 U/L, CK-18 had 71% negative predictive value (NPV) and 77% positive predictive value (PPV) to exclude and diagnose NASH. A 2-step approach measuring CK-18 followed by FGF<em>21</em> further improved the NPV to 74% and PPV to 82%. In a validation cohort of 51 patients with paired liver biopsies, the NPV and PPV of the 2-step approach were 67% and 78%, respectively.

CONCLUSIONS

CK-18 is the most accurate biomarker for NAFLD and NASH. A two-step approach using CK-18 and FGF<em>21</em> further improves the accuracy in diagnosing NASH.