Genetic variants conferring risk for autism spectrum disorder (ASD) have been identified, but the role of post-transcriptional mechanisms in ASD is not well understood. We performed genome-wide microRNA (miRNA) expression profiling in post-mortem brains from individuals with ASD and controls and identified miRNAs and co-regulated modules that were perturbed in ASD. Putative targets of these ASD-affected miRNAs were enriched for genes that have been implicated in ASD risk. We confirmed regulatory relationships between several miRNAs and their putative target mRNAs in primary human neural progenitors. These include hsa-miR-<em>21</em>-3p, a miRNA of unknown CNS function that is upregulated in ASD and that targets neuronal genes downregulated in ASD, and hsa_can_1002-m, a previously unknown, primate-specific miRNA that is downregulated in ASD and that regulates the epidermal <em>growth</em> <em>factor</em> receptor and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor signaling pathways involved in neural development and immune function. Our findings support a role for miRNA dysregulation in ASD pathophysiology and provide a rich data set and framework for future analyses of miRNAs in neuropsychiatric diseases.

<em>Growth</em> <em>factor</em> over-production by responsive cells might contribute to their autonomous proliferation as well as their acquisition of a transformed phenotype in culture. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has been shown to induce transient changes in cell behavior that resemble those encountered in transformed cells. In addition, several types of human tumor cells have been shown to produce bFGF. To determine directly the role that bFGF might play in the induction of the transformed phenotype, we have introduced a human bFGF cDNA expression vector into baby hamster kidney-derived (BHK-<em>21</em>) <em>fibroblasts</em>. One of the BHK transfectants, termed clone 19, expresses the bFGF mRNA and produces biologically active bFGF that accumulates to a high concentration inside the cells. These properties correlate with the ability of the cells to grow in serum-free medium without the addition of exogenous bFGF. Clone 19 cells also proliferated in soft agar, indicating that constitutive expression of the bFGF gene results in a loss of anchorage-dependent <em>growth</em>.

Skeletal muscle is the largest organ determining whole-body insulin sensitivity and metabolic homoeostasis. Adaptive changes of skeletal muscle in response to physical activity include adjustments in the production and secretion of muscle-derived bioactive <em>factors</em>, known as myokines, such as myostatin, IL-4, IL-6, IL-7 and IL-15, myonectin, follistatin-like 1 or leukaemia inhibitory <em>factor</em>. These myokines not only act locally in the muscle in an autocrine/paracrine manner, but also are released to the bloodstream as endocrine <em>factors</em> to regulate physiological processes in other tissues. Irisin, derived from the cleavage of FNDC5 protein, constitutes a myokine that induces myogenesis and fat browning (switch of white adipocytes to brown fat-like cells) together with a concomitant increase in energy expenditure. Besides being a target for irisin actions, the adipose tissue also constitutes a production site of FNDC5. Interestingly, irisin secretion from subcutaneous and visceral fat depots is decreased by long-term exercise training and fasting, suggesting a discordant regulation of FNDC5/irisin in skeletal muscle and adipose tissue. Accordingly, our group has recently reported that the adipokine leptin differentially regulates FNDC5/irisin expression in skeletal muscle and fat, confirming the crosstalk between both tissues. Moreover, irisin secretion and function are regulated by other myokines, such as follistatin or myostatin, as well as by other adipokines, including <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> and leptin. Taken together, myokines have emerged as novel molecular mediators of fat browning and their activity can be modulated by adipokines, confirming the crosstalk between skeletal muscle and adipose tissue to regulate thermogenesis and energy expenditure.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>21</em> is highly expressed in the liver and regulates glucose and lipid metabolism in rodents. The effects of obesity and fatty liver on circulating FGF-<em>21</em> levels have been described mainly in adults. Herein, we measured plasma FGF-<em>21</em> levels in lean and obese adolescents with low and high hepatic fat content (HFF% <5.5% and HFF% ≥ 5.5%, respectively) and explored their relationship with hepatic fat content, measures of hepatic apoptosis, and insulin sensitivity.

METHODS

A total of <em>21</em>7 lean and obese adolescents with both low and high HFF% (lean = 31; obese low HFF% = 107; and obese high HFF% = 79) underwent an oral glucose tolerance test, a fast gradient magnetic resonance imaging to measure the %HFF and abdominal fat distribution. Cytokeratin 18 levels were measured as a biomarker of liver apoptosis. A subset of adolescents underwent a 2-step hyperinsulinemic-euglycemic clamp, and a liver biopsy (N = 14), to assess insulin sensitivity and steatohepatitis, respectively.

RESULTS

Compared to controls, FGF-<em>21</em> levels were higher in obese youth, especially in those with high HFF (P < .001). FGF-<em>21</em> significantly correlated with adiposity indexes (P < .001), visceral fat (r² = 0.240, P < .001), hepatic fat content (r² = 0.278, P < .001), cytokeratin 18 (r² = 0.<em>21</em>7, P < .001), and alanine aminotransferase (r² = .164, P < .001). In subjects with steatoheaptitis, FGF-<em>21</em> levels significantly correlated with the nonalcoholic fatty liver disease activity score (r² = 0.27, P = .04). Stepwise regression analysis indicated that these relationships are independent of body mass index, visceral fat, and insulin sensitivity. An inverse correlation was documented with insulin, hepatic resistance indexes, and adipose resistance indexes, which disappeared after adjusting for hepatic fat content.

CONCLUSIONS

Plasma FGF-<em>21</em> levels are increased in obese adolescents, particularly in those with fatty liver. FGF-<em>21</em> concentrations significantly and independently correlate with hepatic fat content and markers of hepatic apoptosis in obese youths.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is an important feature of atherosclerosis, restenosis, and hypertension. Although multiple mediators of VSMC <em>growth</em> have been identified, few effective pharmacological tools have been developed to limit such <em>growth</em>. Recent evidence indicating an important role for oxidative stress in cell <em>growth</em> led us to investigate the potential role of aldose reductase (AR) in the proliferation of VSMCs. Because AR catalyzes the reduction of mitogenic aldehydes derived from lipid peroxidation, we hypothesized that it might be a potential regulator of redox changes that accompany VSMC <em>growth</em>. Herein we report several lines of evidence suggesting that AR facilitates/mediates VSMC <em>growth</em>. Stimulation of human aortic SMCs in culture with mitogenic concentrations of serum, thrombin, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and the lipid peroxidation product 4-hydroxy-trans-2-nonenal (HNE) led to a 2- to 4-fold increase in the steady-state levels of AR mRNA, a 4- to 7-fold increase in AR protein, and a 2- to 3-fold increase in its catalytic activity. Inhibition of the enzyme by sorbinil or tolrestat diminished mitogen-induced DNA synthesis and cell proliferation. In parallel experiments, the extent of reduction of the glutathione conjugate of HNE to glutathionyl-1,4-dihydroxynonene in HNE-exposed VSMCs was decreased by serum starvation or sorbinil. Immunohistochemical staining of cross sections from balloon-injured rat carotid arteries showed increased expression of AR protein associated with the neointima. The media of injured or uninjured arteries demonstrated no significant staining. Compared with untreated animals, rats fed sorbinil (40 mg. kg(-1). d(-1)) displayed a 51% and a 58% reduction in the ratio of neointima to the media at 10 and <em>21</em> days, respectively, after balloon injury. Taken together, these findings suggest that AR is upregulated during <em>growth</em> and that this upregulation facilitates <em>growth</em> by enhancing the metabolism of secondary products of reactive oxygen species.

Nonalcoholic fatty liver disease (NAFLD) is associated with increased risk of hepatic, cardiovascular, and metabolic diseases. High-protein diets, rich in methionine and branched chain amino acids (BCAAs), apparently reduce liver fat, but can induce insulin resistance. We investigated the effects of diets high in animal protein (AP) vs plant protein (PP), which differ in levels of methionine and BCAAs, in patients with type 2 diabetes and NAFLD. We examined levels of liver fat, lipogenic indices, markers of inflammation, serum levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), and activation of signaling pathways in adipose tissue.

We performed a prospective study of individuals with type 2 diabetes and NAFLD at a tertiary medical center in Germany from June 2013 through March 2015. We analyzed data from 37 subjects placed on a diet high in AP (rich in meat and dairy foods; n = 18) or PP (mainly legume protein; n = 19) without calorie restriction for 6 weeks. The diets were isocaloric with the same macronutrient composition (30% protein, 40% carbohydrates, and 30% fat). Participants were examined at the start of the study and after the 6-week diet period for body mass index, body composition, hip circumference, resting energy expenditure, and respiratory quotient. Body fat and intrahepatic fat were detected by magnetic resonance imaging and spectroscopy, respectively. Levels of glucose, insulin, liver enzymes, and inflammation markers, as well as individual free fatty acids and free amino acids, were measured in collected blood samples. Hyperinsulinemic euglycemic clamps were performed to determine whole-body insulin sensitivity. Subcutaneous adipose tissue samples were collected and analyzed for gene expression patterns and phosphorylation of signaling proteins.

Postprandial levels of BCAAs and methionine were significantly higher in subjects on the AP vs the PP diet. The AP and PP diets each reduced liver fat by 36%-48% within 6 weeks (for AP diet P = .0002; for PP diet P = .001). These reductions were unrelated to change in body weight, but correlated with down-regulation of lipolysis and lipogenic indices. Serum level of FGF<em>21</em> decreased by 50% in each group (for AP diet P < .0002; for PP diet P < .0002); decrease in FGF<em>21</em> correlated with loss of hepatic fat. In gene expression analyses of adipose tissue, expression of the FGF<em>21</em> receptor co<em>factor</em> β-klotho was associated with reduced expression of genes encoding lipolytic and lipogenic proteins. In patients on each diet, levels of hepatic enzymes and markers of inflammation decreased, insulin sensitivity increased, and serum level of keratin 18 decreased.

In a prospective study of patients with type 2 diabetes, we found diets high in protein (either animal or plant) significantly reduced liver fat independently of body weight, and reduced markers of insulin resistance and hepatic necroinflammation. The diets appear to mediate these changes via lipolytic and lipogenic pathways in adipose tissue. Negative effects of BCAA or methionine were not detectable. FGF<em>21</em> level appears to be a marker of metabolic improvement. ClinicalTrials.gov ID NCT02402985.

Non-alcoholic fatty liver disease (NAFLD) describes a spectrum of liver conditions from simple steatosis, steatohepatitis to end-stage liver disease. The prevalence of NAFLD has been on the rise in many parts of the world, including Asia, and NAFLD is now the liver disease associated with the highest mortality, consequent to the increased risk of cardiovascular diseases and hepatocellular carcinoma. Whereas NAFLD is an independent risk <em>factor</em> for type 2 diabetes, increased hepatic and peripheral insulin resistance contribute to the pathogenesis of both NAFLD and diabetes, which are associated with enhanced cardiovascular risk. Studies in humans and animal models have suggested obesity as the common link of these two diseases, likely mediated by adipose tissue inflammation and dysregulated adipokine production in obesity. In the present review, we discuss recent advances in our understanding of the role of several novel adipokines (adiponectin, adipocyte fatty acid binding protein and <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em>) in the pathophysiology of NAFLD and diabetes, as well as their use as potential biomarkers and therapeutic targets for dysglycemia in NAFLD patients.

OBJECTIVE

Angiotensin II (Ang II) induces fibroblast proliferation and collagen synthesis in the myocardium, but its precise mechanisms of action in human hearts are still unknown. Therefore, we investigated whether Ang II directly affects the collagen mRNA content in the human myocardium and in isolated human cardiac fibroblasts or whether the growth factors TGFbeta-1 and osteopontin are involved in this process.

UNASSIGNED

In a first set of experiments, the direct effect of Ang II on collagen I, TGFbeta-1 and osteopontin mRNA content in fresh samples of human atrial myocardium was determined by the use of a short stimulation period. After 4 h, Ang II-stimulated atrial samples gave a significantly higher expression of both TGFbeta-1 (183+/-21% of control, p<0.05) and osteopontin mRNA (275+/-58%, p<0.02) than the controls. In contrast, the expression of collagen I mRNA was unchanged (95+/-8%). Stimulation with TGFbeta-1 led to an increase in collagen I and III mRNA (127+/-10%, p<0.05; 140+/-15%, p<0.02).

UNASSIGNED

In a second protocol, to assess the effects of longer stimulation periods, we determined the effects of Ang II and its potential mediator TGFbeta-1 on collagen I, III and fibronectin mRNA expression and on proliferation of cultured human cardiac fibroblasts. Ang II caused a dose-dependent stimulation of proliferation but did not affect collagen I, II or fibronectin mRNA content after 24 h. In contrast, TGFbeta-1 stimulation significantly increased collagen I and III mRNA expression (124+/-5% and 128+/-5%, p<0.002).

CONCLUSIONS

In the human heart, Ang II does not directly increase collagen or fibronectin mRNA, but it does increase TGFbeta-1 and osteopontin mRNA expression. Since TGFbeta-1 induces collagen I and III mRNA in atrial samples and in isolated cardiac fibroblasts, it may represent a necessary mediator of the Ang II effects in the human heart.

The regulation of linear <em>growth</em> by nutritional and inflammatory influences is examined in terms of <em>growth</em>-plate endochondral ossification, in order to better understand stunted <em>growth</em> in children. Linear <em>growth</em> is controlled by complex genetic, physiological, and nutrient-sensitive endocrine/paracrine/autocrine mediated molecular signalling mechanisms, possibly including sleep adequacy through its influence on <em>growth</em> hormone secretion. Inflammation, which accompanies most infections and environmental enteric dysfunction, inhibits endochondral ossification through the action of mediators including proinflammatory cytokines, the activin A-follistatin system, glucocorticoids and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>). In animal models linear <em>growth</em> is particularly sensitive to dietary protein as well as Zn intake, which act through insulin, insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1) and its binding proteins, triiodothyronine, amino acids and Zn2+ to stimulate <em>growth</em>-plate protein and proteoglycan synthesis and cell cycle progression, actions which are blocked by corticosteroids and inflammatory cytokines. Observational human studies indicate stunting to be associated with nutritionally poor, mainly plant-based diets. Intervention studies provide some support for deficiencies of energy, protein, Zn and iodine and for multiple micronutrient deficiencies, at least during pregnancy. Of the animal-source foods, only milk has been specifically and repeatedly shown to exert an important influence on linear <em>growth</em> in both undernourished and well-nourished children. However, inflammation, caused by infections, environmental enteric dysfunction, which may be widespread in the absence of clean water, adequate sanitation and hygiene (WASH), and endogenous inflammation associated with excess adiposity, in each case contributes to stunting, and may explain why nutritional interventions are often unsuccessful. Current interventions to reduce stunting are targeting WASH as well as nutrition.

This study investigated the potential use of synovium-derived stem cells (SDSCs) as a cell source for cartilage tissue engineering. Harvested SDSCs from juvenile bovine synovium were expanded in culture in the presence (primed) or absence (unprimed) of <em>growth</em> <em>factors</em> (1 ng/mL transforming <em>growth</em> <em>factor</em>-β(1), 10 ng/mL platelet-derived <em>growth</em> <em>factor</em>-ββ, and 5 ng/mL basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-2) and subsequently seeded into clinically relevant agarose hydrogel scaffolds. Constructs seeded with <em>growth</em> <em>factor</em>-primed SDSCs that received an additional transient application of transforming <em>growth</em> <em>factor</em>-β(3) for the first <em>21</em> days (release) exhibited significantly better mechanical and biochemical properties compared to constructs that received sustained <em>growth</em> <em>factor</em> stimulation over the entire culture period (continuous). In particular, the release group exhibited a Young's modulus (267±96 kPa) approaching native immature bovine cartilage levels, with corresponding glycosaminoglycan content (5.19±1.45%ww) similar to native values, within 7 weeks of culture. These findings suggest that SDSCs may serve as a cell source for cartilage tissue engineering applications.

Activation of retinoic acid receptor (RAR) with all-trans-retinoic acid (RA) ameliorates glucose intolerance and insulin resistance in obese mice. The recently discovered <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a hepatocyte-derived hormone that restores glucose and lipid homeostasis in obesity-induced diabetes. However, whether hepatic RAR is linked to FGF<em>21</em> in the control of lipid metabolism and energy homeostasis remains elusive. Here we identify FGF<em>21</em> as a direct target gene of RARβ. The gene transcription of Fgf<em>21</em> is increased by the RAR agonist RA and by overexpression of RARα and RARβ, but it is unaffected by RARγ in HepG2 cells. Promoter deletion analysis characterizes a putative RA-responsive element (RARE) primarily located in the 5'-flanking region of the Fgf<em>21</em> gene. Disruption of the RARE sequence abolishes RA responsiveness. In vivo adenoviral overexpression of RARβ in the liver enhances production and secretion of FGF<em>21</em>, which in turn promotes hepatic fatty acid oxidation and ketogenesis and ultimately leads to increased energy expenditure in mice. The metabolic effects of RA or RARβ are mimicked by FGF<em>21</em> overexpression and largely abolished by FGF<em>21</em> knockdown. Moreover, hepatic RARβ is bound to the putative RAREs of the Fgf<em>21</em> promoter in a fasting-inducible manner in vivo, which contributes to FGF<em>21</em> induction and the metabolic adaptation to prolonged fasting. In addition to other nuclear receptors, such as peroxisome proliferator-activated receptor α and retinoic acid receptor-related receptor α, RAR may act as a novel component to induce hepatic FGF<em>21</em> in the regulation of lipid metabolism. The hepatic RAR-FGF<em>21</em> pathway may represent a potential drug target for treating metabolic disorders.

Oncostatin M (OSM) is a multifunctional cytokine, a member of the interleukin-6/leukemia inhibitory <em>factor</em> (IL-6/LIF) family, that can regulate a number of connective-tissue cell types in vitro including cartilage and synovial tissue-derived <em>fibroblasts</em>, however its role in joint inflammation in vivo is not clear. We have analyzed murine OSM (muOSM) activity in vitro and in vivo in mouse joint tissue, to determine the potential role of this cytokine in local joint inflammation and pathology. The effects of muOSM and other IL-6/LIF cytokines on mouse synovial <em>fibroblast</em> cultures were assessed in vitro and showed induction of monocyte chemotactic protein-1, interleukin-6, and tissue inhibitor metalloproteinase-1, as well as enhancement of colony <em>growth</em> in soft agarose culture. Other IL-6/LIF cytokines including IL-6, LIF, or cardiotrophin-1, did not have such effects when tested at relatively high concentrations (20 ng/ml). To assess effects of muOSM in articular joints in vivo, we used recombinant adenovirus expressing muOSM cDNA (AdmuOSM) and injected purified recombinant virus (10(6) to 10(8) pfu) intra-articularly into the knees of various mouse strains. Histological analysis revealed dramatic alterations in the synovium but not in synovium of knees treated with the control virus Ad-dl70 or knees treated with Adm-IL-6 encoding biologically active murine IL-6. AdmuOSM effects were characterized by increases in the synovial cell proliferation, infiltration of mononuclear cells, and increases in extracellular matrix deposition that were evident at day 4, but much more marked at days 7, 14, and <em>21</em> after administration. The synovium took on characteristics similar to pannus and appeared to contact and invade cartilage. Collectively, these results provide good evidence that OSM regulates synovial <em>fibroblast</em> function differently than other IL-6-type cytokines, and can induce a proliferative invasive phenotype of synovium in vivo in mice on overexpression. We suggest that OSM may contribute to pathology in arthritis.

<em>Growth</em> <em>factors</em> and hormones may play an autocrine/paracrine role in mechanical stress-induced cardiac hypertrophy. Using an in vitro model of mechanical stress, i.e. stretch of cardiomyocytes and cardiac <em>fibroblasts</em>, we tested the involvement of <em>growth</em> <em>factors</em> and hormones in this process. We found that conditioned medium (CM) derived from 4 h cyclicly (1 Hz) stretched cardiomyocytes increased the rate of protein synthesis in static cardiomyocytes by 8 +/- 3%. Moreover, CM derived from 2 h stretched <em>fibroblasts</em> increased the rate of protein synthesis in static <em>fibroblasts</em> as well as in static cardiomyocytes by 8 +/- 2 and 6 +/- 2%, respectively. Analysis of CM using size-exclusion HPLC showed that cardiomyocytes and <em>fibroblasts</em> released at least three <em>factors</em> with MW < or = 10 kD, their quantities being time-dependently increased by stretch. Subsequent analyses using immunoassays revealed that cardiomyocytes released atrial natriuretic peptide (ANP) and transforming <em>growth</em> <em>factor</em>-beta1 (TGFbeta1) being increased by 45 +/- 17 and <em>21</em> +/- 4% upon 4 h of stretch, respectively. <em>Fibroblasts</em> released TGFbeta1 and very low quantity of endothelin-1 (ET-1). The release of TGFbeta1 was significantly increased by 18 +/- 4% after 24 h of stretch in <em>fibroblasts</em>. Both cell types released no detectable amount of angiotensin II (Ang II). In conclusion, upon cyclic stretch cardiomyocytes and <em>fibroblasts</em> secrete <em>growth</em> <em>factors</em> and hormones which induce <em>growth</em> responses in cardiomyocytes and <em>fibroblasts</em> in an autocrine/paracrine way. TGFbeta secreted by cardiomyocytes and <em>fibroblasts</em>, and ANP secreted by cardiomyocytes are likely candidates. We found no evidence for the involvement of Ang II and ET-1 in autocrine/paracrine mechanisms between cardiac cell types.

Alterations of the extracellular matrix (ECM) with its major component collagen are increasingly discussed as possible risk <em>factors</em> implicated in the development of abdominal-wall herniation. Because of the widespread use of alloplastic meshes for the surgical repair of hernias, an animal study was performed to analyze the influence of various mesh materials on the quantity and quality of collagen deposition. In 60 male Sprague-Dawley rats an abdominal replacement was performed using three different kinds of mesh materials: polyester (PE), a pure polypropylene (PP), and a composite mesh made of polypropylene and polyglactin (PG). A simple fascia suture repair served as control. The count of <em>fibroblasts</em>, the collagen/protein ratio, the type I/III collagen ratio, and the expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (b-FGF) at the interface were analyzed after 7, <em>21</em>, and 90 days. The ratio of collagen to overall protein (microg/mg) showed significant differences comparing different mesh materials (sham controls 38.44 +/- 16.33 microg/mg, PE 68.5 +/- 23.8 microg/mg, PP 101.6 +/- 32.3 microg/mg, PG 49.6 +/- 11.6 microg/mg at day 90). The ratio of collagen type I/III increased over time in all groups. However, 90 days after mesh implantation the ratio was always significantly lowered compared to the controls. No significant difference was found comparing different mesh materials. The alteration of the scar composition is closely connected to an increased b-FGF expression. b-FGF and count of <em>fibroblasts</em> highly correlated (r =.95) and showed significant elevated levels compared to simple suture repair. The results of our study strongly support the notion that wound healing is affected by mesh implantation. The quality of the ECM deposition as determined by collagen type I/III ratio is impaired in general, whereas the quantity of ECM deposition is markedly influenced by the kind of mesh material.

Individual neural progenitors, derived from the external germinal layer of neonatal murine cerebellum, were previously immortalized by the retrovirus-mediated transduction of avian myc (v-myc). C17-2 is one of those clonal multipotent progenitor cell lines (Snyder et al., 1992, Cell 68: 33-51; Ryder et al., 1990, J. Neurobiol. <em>21</em>:356-375). When transplanted into newborn mouse cerebellum (CB), the cells participate in normal CB development; they engraft in a cytoarchitecturally appropriate, nontumorigenic manner and differentiate into multiple CB cell types (neuronal and glial) similar to endogenous progenitors (Snyder et al., 1992, as above). They also appear to engraft and participate in the development of multiple other structures along the neural axis and at multiple other stages (Snyder et al., 1993, Soc. Neurosci. Abstr. 19). Thus conclusions regarding these immortalized progenitors may be applicable to endogenous neural progenitors in vivo. To help identify and analyze <em>factors</em> that promote differentiation of endogenous progenitors, we first investigated the ability to maintain C17-2 cells in a defined, serum-free medium (N2). The cells survive in vitro in N2 but undergo mitosis at a very low rate. Addition of epidermal <em>growth</em> <em>factor</em> (EGF), however, either from mouse submaxillary gland or the human recombinant protein, appreciably stimulates thymidine incorporation and cell division approximately threefold. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is an even more potent mitogen, promoting thymidine incorporation, cell division, and a net increase in cell number equal to that in serum. Both EGF and bFGF are active at very low nanomolar concentrations, suggesting that they interact with their respective receptors rather than a homologous receptor system. The findings demonstrate that C17-2 cells can be maintained and propagated in a fully defined medium, providing the basis for analysis of other <em>growth</em> and differentiation <em>factors</em>. That EGF and particularly bFGF are mitogenic for these cells is in accord with recent observations on primary neural tissue (Reynolds and Weiss, 1992, Science 255:1707-1710; Kilpatrick and Bartlett, 1993, Neuron 10:255-265; Ray et al., 1993, Proc. Natl. Acad. Sci. USA 90:3602-3606) suggesting that bFGF and EGF responsiveness may be fundamental properties of neural progenitors.

The <em>21</em>-kDa protein is an extracellular matrix (ECM) component whose synthesis is stimulated transiently during oncogenic transformation of chicken embryo <em>fibroblasts</em> (CEF) or after treatment of normal cells with the tumor promoter phorbol 12-myristate 13-acetate. Biochemical characterization indicates that the protein is related, but not identical, to two members of the family of tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2. The cDNA of the <em>21</em>-kDa protein was recently cloned, and based upon its deduced amino acid sequence and other supporting data we propose that it is another member of this family, a TIMP-3. We now report electrophoretic purification of sufficient quantities of this protein to determine its function. The protein promotes the detachment of transforming cells from the ECM. Although its presence in the matrix may be necessary for cell release it is not the only <em>factor</em> involved because it does not influence the adhesive properties of nontransformed cells. It also appears to accelerate the morphological changes associated with cell transformation and stimulates the proliferation of <em>growth</em>-retarded, nontransformed cells maintained under low serum conditions. Based on these data we hypothesize that the <em>21</em>-kDa protein promotes the development of the transformed phenotype in cultured cells.

BACKGROUND

Low vitamin D concentrations are prevalent in patients with chronic kidney disease (CKD). We investigated the relationship between plasma 25-hydroxyvitamin D (25[OH]D) or 1,25-dihydroxyvitamin D (1,25[OH](2)D) concentrations with death, cardiovascular events, and dialysis therapy initiation in patients with advanced CKD.

METHODS

The HOST (Homocysteinemia in Kidney and End Stage Renal Disease) Study was a randomized double-blind trial evaluating the effects of high doses of folic acid on death and long-term dialysis therapy initiation in patients with advanced CKD (stages 4 and 5 not yet on dialysis therapy). 25(OH)D and 1,25(OH)(2)D were measured in stored plasma samples obtained 3 months after trial initiation and evaluated at clinically defined cutoffs (<10, 10-30, and >30 ng/mL) and tertiles (<15, 15-22, and >22 pg/mL), respectively. Cox proportional hazard models were used to examine the association between vitamin D concentrations and clinical outcomes.

METHODS

1,099 patients with advanced CKD from 36 Veteran Affairs Medical Centers.

METHODS

25(OH)D and 1,25(OH)(2)D concentrations.

RESULTS

Death, cardiovascular events, and time to initiation of long-term dialysis therapy.

RESULTS

After a median follow-up of 2.9 years, 41% (n = 453) died, whereas 56% (n = 615) initiated dialysis therapy. Mean 25(OH)D and 1,25(OH)(2)D concentrations were <em>21</em> ± 10 ng/mL and 20 ± 11 pg/mL, respectively. After adjustment for potential confounders, the lowest tertile of 1,25(OH)(2)D was associated with death (HR, 1.33; 95% CI, 1.01-1.74) and initiation of long-term dialysis therapy (HR, 1.78; 95% CI, 1.40-2.26) compared with the highest tertile. The association with death and initiation of dialysis therapy was moderately attenuated after adjustment for plasma <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF-23) concentrations (HRs of lower tertiles of 1.20 [95% CI, 0.91-1.58] and 1.56 [95% CI, 1.23-1.99], respectively, compared with highest tertile). There was no association between 25(OH)D concentrations and outcomes.

CONCLUSIONS

Participants were mostly men.

CONCLUSIONS

Low plasma 1,25(OH)(2)D concentrations are associated with death and initiation of long-term dialysis therapy in patients with advanced CKD. FGF-23 level may attentuate this relationship.

BACKGROUND

BIBF 1120 is an oral potent inhibitor of vascular endothelial growth factor receptor, fibroblast growth factor receptor and platelet-derived growth factor receptor, the three key receptor families involved in angiogenesis. This phase I, open-label dose-escalation study investigated BIBF 1120 combined with paclitaxel (Taxol) and carboplatin in first-line patients with advanced (IIIB/IV) non-small-cell lung cancer.

METHODS

Patients received BIBF 1120 (starting dose 50 mg b.i.d.) on days 2-21 and paclitaxel (200 mg/m2) and carboplatin [area under curve (AUC)=6 mg/ml/min] on day 1 of each 21-day cycle. Primary end points were safety and BIBF 1120 maximum tolerated dose (MTD) in this combination. Pharmacokinetics (PK) profiles were evaluated.

RESULTS

Twenty-six patients were treated (BIBF 1120 50-250 mg b.i.d.). BIBF 1120 MTD was 200 mg b.i.d. in combination with paclitaxel and carboplatin. Six dose-limiting toxicity events occurred during treatment cycle 1 (liver enzyme elevations, thrombocytopenia, abdominal pain, and rash). Best responses included 7 confirmed partial responses (26.9%); 10 patients had stable disease. BIBF 1120 200 mg b.i.d. had no clinically relevant influence on the PK of paclitaxel 200 mg/m2 and carboplatin AUC 6 mg/ml/min and vice versa.

CONCLUSIONS

BIBF 1120 MTD was 200 mg b.i.d when given with paclitaxel and carboplatin; this combination demonstrated an acceptable safety profile. No relevant changes in PK parameters of the backbone chemotherapeutic agents or BIBF 1120 were observed.

Adipose-resident invariant natural killer T (iNKT) cells are key players in metabolic regulation. iNKT cells are innate lipid sensors, and their activation, using their prototypic ligand α-galactosylceramide (αGalCer), induces weight loss and restores glycemic control in obesity. Here, iNKT activation induced <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) production and thermogenic browning of white fat. Complete metabolic analysis revealed that iNKT cell activation induced increased body temperature, V02, VC02, and fatty acid oxidation, without affecting food intake or activity. FGF<em>21</em> induction played a major role in iNKT cell-induced weight loss, as FGF<em>21</em> null mice lost significantly less weight after αGalCer treatment. The glucagon-like peptide 1 (GLP-1) receptor agonist, liraglutide, also activated iNKT cells in humans and mice. In iNKT-deficient mice, liraglutide promoted satiety but failed to induce FGF<em>21</em>, resulting in less weight loss. These findings reveal an iNKT cell-FGF<em>21</em> axis that defines a new immune-mediated pathway that could be targeted for glycemic control and weight regulation.

Adult stem cells gradually lose their stemness when plated in monolayer culture after isolation from their in vivo niche. In this study, we hypothesized that the in vitro microenvironment can be optimized by modulating oxygen tension and mitotic signal in a tissue-specific extracellular matrix (ECM) deposited by synovium-derived stem cells (SDSCs) to rejuvenate expanded SDSC proliferation and chondrogenic potential. Passage 3 SDSCs were plated on either SDSC-derived ECM or plastic flask and incubated in either hypoxia (5% O(2)) or normoxia (<em>21</em>% O(2)) with or without the supplementation of 10 ng/mL of basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) for 7 days, followed by pellet culture in a serum-free chondrogenic medium for 14 days. Our data showed that, compared with the mitotic effect of FGF-2 on SDSCs, ECM expansion greatly enhanced SDSC proliferation while retaining SDSC stem cell characteristics. More importantly, ECM pretreatment yielded SDSC pellets with a comparable chondrogenic index to FGF-2 pretreatment, both of which were much higher than SDSC expansion on plastic flask alone. FGF-2 pretreatment led to the highest glycosaminoglycans and DNA content; intriguingly, it also contributed to the highest expression level of hypertrophic marker genes. Surprisingly, the hypertrophic marker genes could be downregulated if the pretreatment was combined with hypoxia or ECM. The combination of hypoxia, FGF-2, and SDSC-derived ECM contributed to the highest cell number in SDSC expansion. Our study indicates that the three-dimensional microenvironment for ex vivo expansion can be optimized to provide high-quality stem cells for stem cell-based cartilage defect repair.

Inappropriate fibroblast growth factor (FGF) signaling is involved in most tissue-specific pathologies including cancer. Previously we showed that inappropriate expression and chronic activity of FGF receptor (FGFR) 1 in hepatocytes accelerated diethylnitrosamine (DEN)-initiated hepatocarcinogenesis. Here we showed that although widely expressed FGF1 and FGF2 are frequently upregulated in hepatocellular carcinoma (HCC), germline deletion of both FGF1 and FGF2 had no effect on DEN-initiated hepatocarcinogenesis. Thus overexpression of FGF1 or FGF2 may be a consequence rather than contributor to hepatoma progression. FGF21 is the first of 22 homologues whose expression has been reported to be preferentially in the liver. We showed that similar to FGF1 and FGF2, FGF21 mRNA was upregulated in neoplastic and regenerating liver after partial hepatectomy (PH) and CCl4 administration. In situ hybridization analysis confirmed that in contrast to FGF1 and FGF2, expression of FGF21 mRNA was limited to hepatocytes. Forced overexpression of FGF21 in hepatocytes by gene targeting had no apparent impact on normal liver development and compensatory response to injury. Surprisingly, overexpression of FGF21 delayed the appearance of DEN-induced liver tumors. At 8 and 10 mo, only 10% and 30% of transgenic mice, respectively, developed adenomas compared to 50% (all adenomas) and 80% (60% adenoma/20% HCC) in the wild-type (WT) mice. However, the incidence and burden of HCC at 10 mo and later was equal in the FGF21 transgenic and WT mice. We propose that FGF21 may delay development of adenomas through activation of resident hepatocyte FGFR4 at early times, but counteracts the delay by acceleration of progression to HCC through interaction with ectopic FGFR1 once it appears in hepatoma cells. This indicates a dual function of FGF21 that may reflect changes in FGFR isotype during progression of differentiated hepatoma cells.

BACKGROUND

Iron deficiency anemia and serum phosphate levels>> 4.0mg/dL are relatively common in chronic kidney disease stages 3 to 5 and are associated with higher risks of progressive loss of kidney function, cardiovascular events, and mortality.

METHODS

Double-blind, placebo-controlled, randomized trial.

METHODS

149 patients with estimated glomerular filtration rates < 60 mL/min/1.73 m(2), iron deficiency anemia (hemoglobin, 9.0-12.0 g/dL; transferrin saturation [TSAT]≤ 30%, serum ferritin ≤ 300 ng/mL), and serum phosphate levels ≥ 4.0 to 6.0mg/dL. Use of intravenous iron or erythropoiesis-stimulating agents was prohibited.

METHODS

Randomization to treatment for 12 weeks with ferric citrate coordination complex (ferric citrate) or placebo.

METHODS

Coprimary end points were change in TSAT and serum phosphate level from baseline to end of study. Secondary outcomes included change from baseline to end of treatment in values for ferritin, hemoglobin, intact fibroblast growth factor 23 (FGF-23), urinary phosphate excretion, and estimated glomerular filtration rate.

RESULTS

Ferric citrate treatment increased mean TSAT from 22% ± 7% (SD) to 32% ± 14% and reduced serum phosphate levels from 4.5 ± 0.6 to 3.9 ± 0.6 mg/dL, while placebo exerted no effect on TSAT (21% ± 8% to 20% ± 8%) and less effect on serum phosphate level (4.7 ± 0.6 to 4.4 ± 0.8 mg/dL; between-group P<0.001 for each). Ferric citrate increased hemoglobin levels (from 10.5 ± 0.8 to 11.0 ± 1.0 g/dL; P<0.001 vs placebo), reduced urinary phosphate excretion 39% (P<0.001 vs placebo), and reduced serum intact FGF-23 levels from a median of 159 (IQR, 102-289) to 105 (IQR, 65-187) pg/mL (P=0.02 vs placebo). The incidence and severity of adverse effects were similar between treatment arms.

CONCLUSIONS

The study is limited by relatively small sample size and short duration and by having biochemical rather than clinical outcomes.

CONCLUSIONS

Short-term use of ferric citrate repletes iron stores, increases hemoglobin levels, and reduces levels of serum phosphate, urinary phosphate excretion, and FGF-23 in patients with chronic kidney disease stages 3 to 5.

Reduced caloric intake in mammals causes reduced skeletal <em>growth</em> and GH insensitivity. However, the underlying molecular mechanisms are not fully elucidated. The aim of this study was to determine whether the increased activity of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) during chronic undernutrition in mice causes GH insensitivity and <em>growth</em> failure. After 4 wk of food restriction, fgf<em>21</em> knockout (KO) mice exhibited greater body and tibial <em>growth</em> than their wild-type (WT) littermates. Daily injections of recombinant human FGF<em>21</em> in a subgroup of food-restricted fgf<em>21</em> KO mice prevented these differences in body and tibial <em>growth</em>. At the end of the 4-wk food restriction, GH binding and GH receptor expression were reduced in the liver and in the <em>growth</em> plate of food-restricted WT mice (compared to WT mice fed ad libitum), whereas they were similar between food-restricted and ad libitum KO mice. In addition, a single injection of GH induced greater liver signal transducer and activator of transcription 5 phosphorylation and IGF-I mRNA in food-restricted KO mice than in WT mice. Lastly, in the tibial <em>growth</em> plate of food-restricted WT mice, FGF<em>21</em> mRNA and protein expression was greater than that of WT mice fed ad libitum. In contrast, the IGF-I mRNA and protein expression was smaller. Our findings support a causative role for FGF<em>21</em> in the inhibition of skeletal <em>growth</em> during prolonged undernutrition. Such role may be mediated by the antagonistic effect of FGF<em>21</em> on GH action in the liver and, possibly, in the <em>growth</em> plate.

OBJECTIVE

This study investigated <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>)-mediated cardiac protection against apoptosis caused by diabetic lipotoxicity and explored the protective mechanisms involved.

METHODS

Cardiac Fgf<em>21</em> mRNA expression was examined in a diabetic mouse model using real-time PCR. After pre-incubation of palmitate-treated cardiac H9c2 cells and primary cardiomyocytes with FGF<em>21</em> for 15 h, apoptosis and Fgf<em>21</em>-induced cell-survival signalling were investigated using small interfering (si)RNA and/or pharmacological inhibitors. We also examined the cardiac apoptotic signalling and structural and functional indices in wild-type and Fgf<em>21</em>-knockout (Fgf<em>21</em>-KO) diabetic mice.

RESULTS

In a mouse model of type 1 diabetes, cardiac Fgf<em>21</em> expression was upregulated about 40-fold at 2 months and 3-1.5-fold at 4 and 6 months after diabetes. FGF<em>21</em> significantly reduced palmitate-induced cardiac apoptosis. Mechanistically, palmitate downregulated, but FGF<em>21</em> upregulated, phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2, mitogen-activated protein kinase 14 (p38 MAPK) and AMP-activated protein kinase (AMPK). Inhibition of each kinase with its inhibitor and/or siRNA revealed that FGF<em>21</em> prevents palmitate-induced cardiac apoptosis via upregulating the ERK1/2-dependent p38 MAPK-AMPK signalling pathway. In vivo administration of FGF<em>21</em>, but not FGF<em>21</em> plus ERK1/2 inhibitor, to diabetic or fatty-acid-infused mice significantly prevented cardiac apoptosis and reduced inactivation of ERK1/2, p38 MAPK and AMPK and prevented cardiac remodelling and dysfunction. The Fgf<em>21</em>-KO mice were more susceptible to diabetes-induced cardiac apoptosis, and this could be prevented by administration of FGF<em>21</em>. Deletion of Fgf<em>21</em> did not further exacerbate cardiac dysfunction.

CONCLUSIONS

These results demonstrate that FGF<em>21</em> prevents lipid- or diabetes-induced cardiac apoptosis by activating the ERK1/2-p38 MAPK-AMPK pathway. FGF<em>21</em> may be a therapeutic target for the treatment of diabetes-related cardiac damage.