OBJECTIVE

To date there are only very few data available on the ligamentogenic differentiation capacity of mesenchymal stromal/progenitor cells (MSC) and anterior cruciate ligament (ACL) fibroblasts.

METHODS

We describe the in vitro potential of MSC and ACL cells to undergo ligamentogenic differentiation upon transduction with adenoviral vectors encoding the human cDNA for bone morphogenetic protein (BMP) 12 and BMP13, also known as growth and differentiation factors (GDF) 6 and 7, respectively.

RESULTS

Transgene expression for at least 14 days was confirmed by Western blot analyzes. After 21 days of cell culture within collagen type I hydrogels, histochemical (hematoxylin/eosin (H&E), Azan and van Gieson), immunohistochemical and polymerase chain reaction (PCR) analyzes of the genetically modified constructs of both cell types revealed elongated, viable fibroblast-like cells embedded in a ligament-like matrix rich in collagens, vimentin, fibronectin, decorin, elastin, scleraxis, tenascin, and tenomodulin.

CONCLUSIONS

It appears that both MSC and ACL fibroblasts are capable of ligamentogenic differentiation with these factors. This information may aid in the development of biologic approaches to repair and restore ACL after injury.

Recently <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has been identified as a potent regulator in glucose and lipid homeostasis. Here, we firstly investigated the metabolic role of FGF<em>21</em> in human liver-derived HepG2 cells, and suggested that overexpression of FGF<em>21</em> suppressed triglyceride accumulation by inhibiting the transcription of the gene necessary for de novo lipogenesis. The potential mechanism of FGF<em>21</em> regulating lipogenesis was also explored, which revealed that FGF<em>21</em> repressed the transcription of sterol regulatory element binding protein 1c (SREBP1c), an essential transcription <em>factor</em> promoting expression of lipogenesis-related genes. Overexpression of FGF<em>21</em> ameliorated the up-regulation of SREBP1c and fatty acid synthase (FAS) in HepG2 cells elicited by FFAs treatment. Moreover, FGF<em>21</em> could inhibit the transcriptional levels of the key genes involved in processing and nuclear translocation of SREBP1c, and decrease the protein amount of mature SREBP1c. Unexpectedly, overexpression of SREBP1c in HepG2 cells could also inhibit the endogenous FGF<em>21</em> transcription. Further experiments demonstrated that SREBP1c could significantly attenuate the promoter activity of FGF<em>21</em>. In conclusion, our data identifies a clear link between FGF<em>21</em> and SREBP1c during lipogenesis in hepatocyte in culture.

Plaque vascularity has been implicated in its <em>growth</em> and stability. However, there is a paucity of information regarding the origin of plaque vasculature and the role of vasa vasorum in plaque <em>growth</em>. To inhibit <em>growth</em> of vasa vasorum in atherogenic mice and assess its effect on plaque <em>growth</em>, we used a truncated plasminogen activator inhibitor (PAI)-1 protein, rPAI-1(23), that has significant antiangiogenic activity. Female LDLR(-/-)ApoB-48-deficient mice fed Paigen's diet without cholate for 20 weeks received rPAI-1(23) treatment (n=<em>21</em>) for the last 6 weeks. Plaque size and vasa vasorum density were compared to 2 controls: mice fed Paigen's diet and treated with saline for the last 6 weeks (n=16) and mice fed Paigen's diet until the onset of treatment (n=14). The rPAI-1(23) treatment significantly reduced plaque area and plaque cholesterol in the descending aorta and plaque area in the innominate artery. Measurements of reconstructed confocal microscopy images of vasa vasorum demonstrate that rPAI-1(23) treatment decreased vasa vasorum area and length, which was supported by microCT images. Confocal images provide evidence for vascularized plaque in the saline-treated group but not in rPAI-1(23)-treated mice. The increased vessel density in saline-treated mice is attributable, in part, to upregulated <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 expression, which is inhibited by rPAI-1(23). In conclusion, rPAI-1(23) inhibits <em>growth</em> of vasa vasorum, as well as vessels within the adjacent plaque and vessel wall, through inhibition of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, leading to reduced plaque <em>growth</em> in atherogenic female LDLR(-/-)ApoB-48-deficient mice.

After skin wounding, the repair process is initiated by the release of <em>growth</em> <em>factors</em>, cytokines, and bioactive lipids from injured vessels and coagulated platelets. These signal molecules induce synthesis and deposition of a provisional extracellular matrix, as well as <em>fibroblast</em> invasion into and contraction of the wounded area. We previously showed that sphingosine-1-phosphate (S1P) triggers a signal transduction cascade mediating nuclear translocation of the LIM-only protein Fhl2 in response to activation of the RhoA GTPase (Muller, J.M., U. Isele, E. Metzger, A. Rempel, M. Moser, A. Pscherer, T. Breyer, C. Holubarsch, R. Buettner, and R. Schule. 2000. EMBO J. 19:359-369; Muller, J.M., E. Metzger, H. Greschik, A.K. Bosserhoff, L. Mercep, R. Buettner, and R. Schule. 2002. EMBO J. <em>21</em>:736-748.). We demonstrate impaired cutaneous wound healing in Fhl2-deficient mice rescued by transgenic expression of Fhl2. Furthermore, collagen contraction and cell migration are severely impaired in Fhl2-deficient cells. Consequently, we show that the expression of alpha-smooth muscle actin, which is regulated by Fhl2, is reduced and delayed in wounds of Fhl2-deficient mice and that the expression of p130Cas, which is essential for cell migration, is reduced in Fhl2-deficient cells. In summary, our data demonstrate a function of Fhl2 as a lipid-triggered signaling molecule in mesenchymal cells regulating their migration and contraction during cutaneous wound healing.

The three mammalian transforming <em>growth</em> <em>factor</em> (TGF)-beta isoforms (TGF-beta1, TGF-beta2, and TGF-beta3) differ in their putative roles in radiation-induced fibrosis in intestine and other organs. Furthermore, tissue specificity of TGF-beta action may result from temporal or spatial changes in production and/or activation. The present study examined shifts in the cell types expressing TGF-beta mRNA relative to TGF-beta immunoreactivity and histopathological injury during radiation enteropathy development. A 4-cm loop of rat small intestine was locally exposed to O, 12, or <em>21</em>-Gy single doses of x-irradiation. Sham-irradiated and irradiated intestine were procured 2 and 26 weeks after irradiation. Cells expressing the TGF-beta1, TGF-beta2, or TGF-beta3 transcripts were identified by in situ hybridization with digoxigenin-labeled riboprobes. Intestinal wall TGF-beta immunoreactivity was measured using computerized image analysis, and structural radiation injury was assessed by quantitative histopathology. Normal intestinal epithelium expressed transcripts for all three TGF-beta isoforms. Two weeks after irradiation, regenerating crypts, inflammatory cells, smooth muscle cells, and mesothelium exhibited increased TGF-beta1 expression and, to a lesser degree, TGF-beta2 and TGF-beta3 expression. Twenty-six weeks after irradiation, TGF-beta2 and TGF-beta3 expression had returned to normal. In contrast, TGF-beta1 expression remained elevated in smooth muscle, mesothelium, endothelium, and <em>fibroblasts</em> in regions of chronic fibrosis. Extracellular matrix-associated TGF-beta1 immunoreactivity was significantly increased at both observation times, whereas, TGF-beta2 and TGF-beta3 immunoreactivity exhibited minimal postradiation changes. Intestinal radiation injury is associated with overexpression of all three TGF-beta isoforms in regenerating epithelium. Radiation enteropathy was also associated with sustained shifts in the cellular sources of TGF-beta1 from epithelial cells to cells involved in the pathogenesis of chronic fibrosis. TGF-beta2 and TGF-beta3 did not exhibit consistent long-term changes. TGF-beta1 appears to be the predominant isoform in radiation enteropathy and may be more important in the mechanisms of chronicity than TGF-beta2 and TGF-beta3.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>21</em> improves insulin sensitivity, reduces body weight, and reverses hepatic steatosis in preclinical species. We generated long-acting FGF<em>21</em> mimetics by site-specific conjugation of the protein to a scaffold antibody. Linking FGF<em>21</em> through the C terminus decreased bioactivity, whereas bioactivity was maintained by linkage to selected internal positions. In mice, these CovX-Bodies retain efficacy while increasing half-life up to 70-fold compared with wild-type FGF<em>21</em>. A preferred midlinked CovX-Body, CVX-343, demonstrated enhanced in vivo stability in preclinical species, and a single injection improved glucose tolerance for 6 days in ob/ob mice. In diet-induced obese mice, weekly doses of CVX-343 reduced body weight, blood glucose, and lipids levels. In db/db mice, CVX-343 increased glucose tolerance, pancreatic β-cell mass, and proliferation. CVX-343, created by linkage of the CovX scaffold antibody to the engineered residue A129C of FGF<em>21</em> protein, demonstrated superior preclinical pharmacodynamics by extending serum half-life of FGF<em>21</em> while preserving full therapeutic functionality.

OBJECTIVE

Human basic fibroblast growth factor (bFGF) is an endothelial mitogen that stimulates angiogenesis and proliferation of other cells such as fibroblasts and smooth muscle cells. After this peptide was stabilized to acid and pepsin by site-specific mutagenesis, it was tested whether bFGF might accelerate the healing of experimental duodenal ulcers.

METHODS

This mutein peptide (bFGF-CS23) was administered orally in comparison with cimetidine to rats with chronic duodenal ulcers previously induced by cysteamine.

RESULTS

Oral bFGF-CS23 therapy maintained for 21 days at 100 ng/100 g twice daily resulted in (1) significant acceleration of healing of duodenal ulcers, i.e., reduction of mean ulcer area by 83% in the bFGF-CS23-treated rats compared with only 61% for cimetidine therapy and 40% for untreated controls; (2) complete healing with no residual ulcer in 62% of the bFGF-CS23-treated rats compared with only 7% of untreated rats; and (3) a ninefold increase in angiogenesis in the ulcer bed compared with untreated controls. A single dose of the bFGF-CS23 mutein had no effect on gastric output of hydrochloric acid or pepsin, but daily treatment for 2 or 3 weeks resulted in enhanced acid and pepsin outputs.

CONCLUSIONS

Chronic duodenal ulcers can be healed rapidly by stimulating angiogenesis and other wound-healing processes in the ulcer bed without reduction of gastric acid.

BACKGROUND

Many fibroblast growth factor family proteins (FGFs) bind to the heparan sulfate/heparin (HP) subtypes of sulfated glycosaminoglycans (GAGs), and a few have recently been reported to also interact with chondroitin sulfate (CS), another sulfated GAG subtype.

METHODS

To gain additional insight into this interaction, we prepared all currently known FGFs (i.e., FGF1-FGF23) and assessed their affinity for HP, CS-B, CS-D and CS-E. In addition, midkine, hepatocyte growth factor and pleiotrophin were studied as other known HP-binding proteins.

RESULTS

We found that members of the FGF19 subfamily (i.e., FGF15, 19, 21 and 23) had little or no affinity for HP; all of the other secretable growth factors tested had strong affinities for HP, as was indicated by the finding that their elution from HP-Sepharose columns required 1.0-1.5 M NaCl. We also found that FGF3, 6, 8 and 22 had strong affinities for CS-E, while FGF5 had a moderate affinity for CS-D. The interactions between FGFs and GAGs thus appear to be more diverse than previously understood.

CONCLUSIONS

This is noteworthy, as the differential interactions of these growth factors with GAGs may be key determinants of their specific biological activities.

OBJECTIVE

The incidence of the metabolic syndrome and type 2 diabetes mellitus (T2DM) is rising worldwide. Liver-derived <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>21</em> affects glucose and lipid metabolism. The aim of this study was to analyze the predictive value of FGF-<em>21</em> on the incidence of T2DM and the metabolic syndrome.

METHODS

The Metabolic Syndrome Berlin Potsdam (MeSyBePo) recall study includes 440 individuals. Glucose metabolism was analyzed using an oral glucose tolerance test, including insulin measurements. FGF-<em>21</em> was measured using enzyme-linked immunosorbent assay. Primary study outcome was diabetes and the metabolic syndrome incidence and change of glucose subtraits.

RESULTS

During a mean follow-up of 5.30 ± 0.1 years, 54 individuals developed the metabolic syndrome, 35 developed T2DM, and 69 with normal glucose tolerance at baseline progressed to impaired glucose metabolism, defined as impaired fasting glucose, impaired glucose tolerance, or T2DM. FGF-<em>21</em> predicted incident metabolic syndrome (lnFGF-<em>21</em> odds ratio [OR] 2.6 [95% CI 1.5 - 4.5]; P = 0.001), T2DM (2.4 [1.2-4.7]; P = 0.01), and progression to impaired glucose metabolism (2.2 [1.3 - 3.6]; P = 0.002) after adjustment for age, sex, BMI, and follow-up time. Additional adjustment for waist-to-hip ratio, systolic blood pressure, HDL cholesterol, triglycerides, and fasting glucose did not substantially modify the predictive value of FGF-<em>21</em>.

CONCLUSIONS

FGF-<em>21</em> is an independent predictor of the metabolic syndrome and T2DM in apparently healthy Caucasians. These results may indicate FGF-<em>21</em> resistance precedes the onset of the metabolic syndrome and T2DM.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a modulator of energy homeostasis and is increased in human nonalcoholic liver disease (NAFLD) and after feeding of methionine- and choline-deficient diet (MCD), a conventional inducer of murine nonalcoholic steatohepatitis (NASH). However, the significance of FGF<em>21</em> induction in the occurrence of MCD-induced NASH remains undetermined. C57BL/6J Fgf<em>21</em>-null and wild-type mice were treated with MCD for 1 week. Hepatic Fgf<em>21</em> mRNA was increased early after commencing MCD treatment independent of peroxisome proliferator-activated receptor (PPAR) α and farnesoid X receptor. While no significant differences in white adipose lipolysis were seen in both genotypes, hepatic triglyceride (TG) contents were increased in Fgf<em>21</em>-null mice, likely due to the up-regulation of genes encoding CD36 and phosphatidic acid phosphatase 2a/2c, involved in fatty acid (FA) uptake and diacylglycerol synthesis, respectively, and suppression of increased mRNAs encoding carnitine palmitoyl-CoA transferase 1α, PPARγ coactivator 1α, and adipose TG lipase, which are associated with lipid clearance in the liver. The MCD-treated Fgf<em>21</em>-null mice showed increased hepatic endoplasmic reticulum (ER) stress. Exposure of primary hepatocytes to palmitic acid elevated the mRNA levels encoding DNA damage-inducible transcript 3, an indicator of ER stress, and FGF<em>21</em> in a PPARα-independent manner, suggesting that lipid-induced ER stress can enhance hepatic FGF<em>21</em> expression. Collectively, FGF<em>21</em> is elevated in the early stage of MCD-induced NASH likely to minimize hepatic lipid accumulation and ensuing ER stress. These results provide a possible mechanism on how FGF<em>21</em> is increased in NAFLD/NASH.

BACKGROUND

Angiogenesis is a target in the treatment of ovarian cancer. Nintedanib, an oral triple angiokinase inhibitor of VEGF receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, has shown activity in phase 2 trials in this setting. We investigated the combination of nintedanib with standard carboplatin and paclitaxel chemotherapy in patients with newly diagnosed advanced ovarian cancer.

METHODS

In this double-blind phase 3 trial, chemotherapy-naive patients (aged 18 years or older) with International Federation of Gynecology and Obstetrics (FIGO) IIB-IV ovarian cancer and upfront debulking surgery were stratified by postoperative resection status, FIGO stage, and planned carboplatin dose. Patients were randomly assigned (2:1) via an interactive voice or web-based response system to receive six cycles of carboplatin (AUC 5 mg/mL per min or 6 mg/mL per min) and paclitaxel (175 mg/m(2)) in addition to either 200 mg of nintedanib (nintedanib group) or placebo (placebo group) twice daily on days 2-21 of every 3-week cycle for up to 120 weeks. Patients, investigators, and independent radiological reviewers were masked to treatment allocation. The primary endpoint was investigator-assessed progression-free survival analysed in the intention-to-treat population. This trial is registered with ClinicalTrials.gov, number NCT01015118.

RESULTS

Between Dec 9, 2009, and July 27, 2011, 1503 patients were screened and 1366 randomly assigned by nine study groups in 22 countries: 911 to the nintedanib group and 455 to the placebo group. 486 (53%) of 911 patients in the nintedanib group experienced disease progression or death compared with 266 (58%) of 455 in the placebo group. Median progression-free survival was significantly longer in the nintedanib group than in the placebo group (17·2 months [95% CI 16·6-19·9] vs 16·6 months [13·9-19·1]; hazard ratio 0·84 [95% CI 0·72-0·98]; p=0·024). The most common adverse events were gastrointestinal (diarrhoea: nintedanib group 191 [21%] of 902 grade 3 and three [<1%] grade 4 vs placebo group nine [2%] of 450 grade 3 only) and haematological (neutropenia: nintedanib group 180 [20%] grade 3 and 200 (22%) grade 4 vs placebo group 90 [20%] grade 3 and 72 [16%] grade 4; thrombocytopenia: 105 [12%] and 55 [6%] vs 21 [5%] and eight [2%]; anaemia: 108 [12%] and 13 [1%] vs 26 [6%] and five [1%]). Serious adverse events were reported in 376 (42%) of 902 patients in the nintedanib group and 155 (34%) of 450 in the placebo group. 29 (3%) of 902 patients in the nintedanib group experienced serious adverse events associated with death compared with 16 (4%) of 450 in the placebo group, including 12 (1%) in the nintedanib group and six (1%) in the placebo group with a malignant neoplasm progression classified as an adverse event by the investigator. Drug-related adverse events leading to death occurred in three patients in the nintedanib group (one without diagnosis of cause; one due to non-drug-related sepsis associated with drug-related diarrhoea and renal failure; and one due to peritonitis) and in one patient in the placebo group (cause unknown).

CONCLUSIONS

Nintedanib in combination with carboplatin and paclitaxel is an active first-line treatment that significantly increases progression-free survival for women with advanced ovarian cancer, but is associated with more gastrointestinal adverse events. Future studies should focus on improving patient selection and optimisation of tolerability.

BACKGROUND

Boehringer Ingelheim.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), a 181 amino acid circulating protein, is a member of the FGF superfamily, with relevant metabolic actions. It acts through the interaction with specific FGF receptors and a co<em>factor</em> called β-Klotho, whose expression is predominantly detected in metabolically active organs. FGF<em>21</em> stimulates glucose uptake in adipocytes via the induction of glucose transporter-1. This action is additive and independent of insulin. β-Cell function and survival are preserved, and glucagon secretion is reduced by this protein, thus decreasing hepatic glucose production and improving insulin sensitivity. Lipid profile has been shown to be improved by FGF<em>21</em> in several animal models. FGF<em>21</em> increases energy expenditure in rodents and induces weight loss in diabetic nonhuman primates. It also exerts favorable effects on hepatic steatosis and reduces tissue lipid content in rodents. Adaptive metabolic responses to fasting, including stimulation of ketogenesis and fatty acid oxidation, seem to be partially mediated by FGF<em>21</em>. In humans, serum FGF<em>21</em> concentrations have been found elevated in insulin-resistant states, such as impaired glucose tolerance and type 2 diabetes. FGF<em>21</em> levels are correlated with hepatic insulin resistance index, fasting blood glucose, HbA1c, and blood glucose after an oral glucose tolerance test. A relationship between FGF<em>21</em> levels and long-term diabetic complications, such as nephropathy and carotid atheromatosis, has been reported. FGF<em>21</em> levels decreased in diabetic patients after starting therapy with insulin or oral agents. Increased FGF<em>21</em> serum levels have also been found to be associated with obesity. In children, it is correlated with BMI and leptin levels, whereas in adults, FGF<em>21</em> levels are mainly related to several components of the metabolic syndrome. Serum FGF<em>21</em> levels have been found to be elevated in patients with ischemic heart disease. In patients with renal disease, FGF<em>21</em> levels exhibited a progressive increase as renal function deteriorates. Circulating FGF<em>21</em> levels seem to be related to insulin resistance and inflammation in dialysis patients. In summary, FGF<em>21</em> is a recently identified hormone with antihyperglycemic, antihyperlipidemic, and thermogenic properties. Direct or indirect potentiation of its effects might be a potential therapeutic target in insulin-resistant states.

BACKGROUND

This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.

METHODS

Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to <em>21</em> cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis <em>factor</em> (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory <em>factor</em> (LIF), adiponectin, leptin, monocyte chemotactic <em>factor</em> (MCP)1, RANTES, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), hepatocyte <em>growth</em> <em>factor</em> (HGF), vascular <em>growth</em> <em>factor</em> (VEGF).

RESULTS

In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤ 0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P < 0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤ 0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P < 0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P < 0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.

CONCLUSIONS

Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.

Bleomycin-induced lung injury causes increased <em>fibroblast</em> numbers in the lung and pulmonary fibrosis. Studies of <em>fibroblasts</em> isolated from such injured lungs have revealed evidence of increased intrinsic proliferative capacity, but the mechanism is unknown. Telomerase catalyzes the addition of telomeric DNA repeats onto chromosomal ends, which is associated with increased cellular life span or immortality. To examine whether telomerase might play a role in regulating <em>fibroblast</em> proliferative capacity in pulmonary fibrosis, lung <em>fibroblasts</em> were isolated from rats treated with endotracheal injections of phosphate-buffered saline or bleomycin. At selected time points, the rats were killed and lung <em>fibroblasts</em> isolated. The isolated cells and lung tissue were then used in experiments for measurement of telomerase activity. The results show undetectable telomerase activity in <em>fibroblasts</em> isolated from control uninjured lungs, or in the control lung tissue extracts. Similar results were obtained in cells and lung tissue from Days 1, 3, and 28 bleomycin-injured lungs. However, significant telomerase activity was detected in <em>fibroblasts</em> and tissue extracts isolated from Days 7, 14, and <em>21</em> bleomycin-treated rat lungs, with maximal activity observed in the Day 14 samples. Analysis of the isolated cells for telomerase messenger RNA or reverse transcriptase expression, combined with alpha-smooth-muscle actin expression by immunohistochemistry, revealed that telomerase expression localized primarily to nonmyo<em>fibroblasts</em>. These findings suggest that in addition to elevated <em>growth</em> <em>factor</em> expression, the injured lung <em>fibroblast</em> population may contain cells with increased life span, which could contribute to the observed overall increase in lung <em>fibroblast</em> numbers.

BACKGROUND

Anorexia nervosa (AN), a state of chronic nutritional deprivation, is characterized by GH resistance with elevated GH levels and decreased levels of IGF-I. <em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>21</em>, a hormone produced in the liver and adipocytes, is induced in the liver by fasting and peroxisome proliferator-activated receptor-alpha agonists. In a transgenic mouse model, FGF-<em>21</em> reduces IGF-I levels by inhibiting signal transducer and activator of transcription-5, a mediator of the intracellular effects of GH.

OBJECTIVE

The objective of the study was to investigate the relationship between FGF-<em>21</em>, GH, and IGF-I in AN.

METHODS

This was a cross-sectional study.

METHODS

The study was conducted at a clinical research center.

METHODS

Patients included 23 girls: 11 with AN (16.5 +/- 0.6 yr) and 12 normal-weight controls (15.7 +/- 0.5 yr).

METHODS

There were no interventions.

METHODS

We measured fasting FGF-<em>21</em>, glucose, insulin, IGF-I, and total area under the curve for GH (GH-AUC) and leptin during 12-h overnight frequent sampling.

RESULTS

FGF-<em>21</em> levels were significantly higher in AN compared with controls, and there was a positive correlation between FGF-<em>21</em> and GH-AUC (P = 0.03) after controlling for percent body fat and insulin resistance. In subjects with elevated FGF-<em>21</em> levels, there was a strong inverse association between FGF-<em>21</em> and IGF-I (R = -0.88, P = 0.004). FGF-<em>21</em> strongly correlated with total area under the curve for leptin (R = 0.67, P = 0.02).

CONCLUSIONS

FGF-<em>21</em> levels are higher in AN independent of the effects of percent body fat and insulin resistance. The positive association between FGF-<em>21</em> and GH-AUC and the inverse association between elevated FGF-<em>21</em> levels and IGF-I suggests that above the normal range, FGF-<em>21</em> may mediate a state of GH resistance in AN.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor that regulates the expression of genes associated with lipid metabolism. Recent studies have suggested that the expression of PPARalpha-dependent <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) plays important roles in adaptation to fasting, such as lipolysis and ketogenesis. We found that a nighttime injection of bezafibrate, a ligand of PPARalpha, effectively induced FGF<em>21</em> expression, whereas a daytime injection did not affect it. Furthermore, bezafibrate-induced circadian FGF<em>21</em> expression was abolished in PPARalpha-deficient mice. These observations suggest that bezafibrate-induced circadian FGF<em>21</em> expression is due to circadian variations in the responsiveness of the PPARalpha system in the liver.

The current treatments used for osteoarthritis from cartilage damage have their disadvantages of donor site morbidity, complicated surgical interventions and risks of infection and graft rejection. Recent advances in tissue engineering have offered much promise in cartilage repair but the best cell source and in vitro system have not as yet been optimised. Human bone marrow mesenchymal stem cells (hBMSCs) have thus far been the cell of choice. However, we derived a unique stem cell from the human umbilical cord Wharton's jelly (hWJSC) that has properties superior to hBMSCs in terms of ready availability, prolonged stemness characteristics in vitro, high proliferation rates, wide multipotency, non-tumorigenicity and tolerance in allogeneic transplantation. We observed enhanced cell attachment, cell proliferation and chondrogenesis of hWJSCs over hBMSCs when grown on PCL/Collagen nanoscaffolds in the presence of a two-stage sequential complex/chondrogenic medium for <em>21</em> days. Improvement of these three parameters were confirmed via inverted optics, field emission scanning electron microscopy (FESEM), MTT assay, pellet diameters, Alcian blue histology and staining, glycosaminglycans (GAG) and hyaluronic acid production and expression of key chondrogenic genes (SOX9, Collagen type II, COMP, FMOD) using immunohistochemistry and real-time polymerase chain reaction (qRT-PCR). In separate experiments we demonstrated that the 16 ng/ml of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) present in the complex medium may have contributed to driving chondrogenesis. We conclude that hWJSCs are an attractive stem cell source for inducing chondrogenesis in vitro when grown on nanoscaffolds and exposed sequentially first to complex medium and then followed by chondrogenic medium.

Because therapeutical options for advanced urological cancers are limited, the understanding of key elements responsible for invasion and metastasis is very important. It has been hypothesized that progression to malignant <em>growth</em> is associated with a dysregulation of <em>growth</em> <em>factors</em> and/or their receptors. In the last few years, signaling pathways of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family have been subject to intense investigation. <em>Fibroblast</em> <em>growth</em> <em>factors</em> constitute one of the largest families of <em>growth</em> and differentiation <em>factors</em> for cells of mesodermal and neuroectodermal origin. The family comprises two prototypic members, acidic FGF (aFGF) and the basic FGF (bFGF), as well as <em>21</em> additionally related polypeptide <em>growth</em> <em>factors</em> that have been identified to date. FGFs are involved in many biological processes during embryonic development, wound healing, hematopoesis, and angiogenesis. In prostate, bladder, and renal cancers, FGFs regulate the induction of metalloproteinases (MMP) that degrade extracellular matrix proteins, thus facilitating tumor metastasis. Probably due to their potent angiogenic properties, aFGF and bFGF have received the most attention. However, there is increasing evidence that other FGFs also play crucial roles in tumors of the prostate, bladder, kidney, and testis. This review will discuss the different elements involved in FGF signaling and summarize the present knowledge of their biological and clinical relevance in urological cancers.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a hepatic metabolic regulator with pleotropic actions. Its plasma concentrations are increased in obesity and diabetes; states associated with an increased incidence of cardiovascular disease. We therefore investigated the direct effect of FGF<em>21</em> on cardio-protection in obese and lean hearts in response to ischemia.

RESULTS

FGF<em>21</em>, FGF<em>21</em>-receptor 1 (FGFR1) and beta-Klotho (βKlotho) were expressed in rodent, human hearts and primary rat cardiomyocytes. Cardiac FGF<em>21</em> was expressed and secreted (real time RT-PCR/western blot and ELISA) in an autocrine-paracrine manner, in response to obesity and hypoxia, involving FGFR1-βKlotho components. Cardiac-FGF<em>21</em> expression and secretion were increased in response to global ischemia. In contrast βKlotho was reduced in obese hearts. In isolated adult rat cardiomyocytes, FGF<em>21</em> activated PI3K/Akt (phosphatidylinositol 3-kinase/Akt), ERK1/2(extracellular signal-regulated kinase) and AMPK (AMP-activated protein kinase) pathways. In Langendorff perfused rat [adult male wild-type wistar] hearts, FGF<em>21</em> administration induced significant cardio-protection and restoration of function following global ischemia. Inhibition of PI3K/Akt, AMPK, ERK1/2 and ROR-α (retinoic-acid receptor alpha) pathway led to significant decrease of FGF<em>21</em> induced cardio-protection and restoration of cardiac function in response to global ischemia. More importantly, this cardio-protective response induced by FGF<em>21</em> was reduced in obesity, although the cardiac expression profiles and circulating FGF<em>21</em> levels were increased.

CONCLUSIONS

In an ex vivo Langendorff system, we show that FGF<em>21</em> induced cardiac protection and restoration of cardiac function involving autocrine-paracrine pathways, with reduced effect in obesity. Collectively, our findings provide novel insights into FGF<em>21</em>-induced cardiac effects in obesity and ischemia.

Scleral <em>fibroblasts</em> are involved in scleral remodeling during axial elongation in myopia. Mechanical load is a potent stimulator of gene expression. This study seeks to identify changes in gene expression of scleral <em>fibroblasts</em> in response to mechanical load and speculate on possible mechanisms of scleral remodeling in the development of myopia. Human scleral <em>fibroblasts</em> (HSFs) were mechanically stretched for 30 min and 24 hr. A gene microarray analysis was used to measure changes in gene expression. A total of 237 genes revealed differential and significant changes in expression (P<0.01) after 30 min of stretching. Of these, 28 unexpressed genes began to be expressed (turned on), while 31 expressed genes were no longer expressed (turned off). After 24 hr, 308 genes showed reproducible changes in expression (P<0.01), while 29 genes were turned on and 17 genes were turned off. After 30 min, 25 genes showed at least a threefold change in expression. These included genes for cell receptors, protein kinases, cell <em>growth</em>/differentiation <em>factors</em>, extracellular matrix (ECM) proteins, lipid metabolism, protein metabolism, transcription <em>factors</em>, binding proteins and water channels. After 24 hr, <em>21</em> genes showed at least a threefold change in expression. These included genes for cell receptors, protein kinases, cell <em>growth</em>/differentiation <em>factors</em>, lipid metabolism, ECM proteins, transcription <em>factors</em>, and carbohydrate metabolism. RT-PCR and Southern blotting confirmed the changes in expression of selected genes. In this study we identified a large number of early and late mechanical response genes in HSFs. These changes in gene expression will provide potential candidate genes that might be involved in scleral remodeling during axial elongation in myopia.

OBJECTIVE

In the current study, we used a model of limb ischemia to determine whether nicotine could enhance arteriogenesis, to compare the magnitude of this effect to the angiogenic factor basic fibroblast growth factor (bFGF), and to investigate the mechanisms of the effect.

BACKGROUND

We have shown previously that nicotine stimulates angiogenesis via stimulation of endothelial nicotinic cholinergic receptors. Stimulation of endothelial nicotinic cholinergic receptors causes endothelial cell proliferation, migration, and formation of capillary networks in vitro and angiogenesis in vivo in conditions of ischemia and inflammation.

METHODS

New Zealand White rabbits (n = 85) underwent unilateral femoral artery occlusion and were randomized to nicotine (0.05 to 5.0 microg/kg/day), bFGF (10 microg/kg/day), or vehicle delivered intra-arterially via osmotic minipumps. At day 21, morphologic changes were assessed by immunohistochemistry and angiography. Blood flow in the ischemic limb was determined by intra-arterial Doppler flow measurements and microsphere distribution.

RESULTS

Nicotine enhanced capillary density in the ischemic hind-limb to a similar extent as bFGF. Nicotine also increased angiographic score, calf blood pressure ratio, intra-arterial Doppler flow, and microsphere distribution. In vitro, nicotine stimulated monocyte adhesion and transmigration. Nicotine increased by two- to three-fold the expression of monocyte adhesion molecules CD11b and CD11a; the expression of the endothelial adhesion molecule intercellular adhesion molecule-1; and the endothelial release of monocyte chemoattractant protein-1.

CONCLUSIONS

In the short term, nicotine promotes angiogenesis and arteriogenesis in the setting of ischemia. The effect of nicotine may be mediated in part by activation of endothelial-monocyte interactions involved in arteriogenesis.

We recently established that two midgestation-derived stromal clones--UG26-1B6, urogenital ridge-derived, and EL08-1D2, embryonic liver-derived--support the maintenance of murine adult bone marrow and human cord blood hematopoietic repopulating stem cells (HSCs). In this study, we investigate whether direct HSC-stroma contact is required for this stem cell maintenance. Adult bone marrow ckit+ Ly-6C- side population (K6-SP) cells and stromal cells were cocultured under contact or noncontact conditions. These experiments showed that HSCs were maintained for at least 4 weeks in culture and that direct contact between HSCs and stromal cells was not required. To find out which <em>factors</em> might be involved in HSC maintenance, we compared the gene expression profile of EL08-1D2 and UG26-1B6 with four HSC-nonsupportive clones. We found that EL08-1D2 and UG26-1B6 both expressed <em>21</em> genes at a higher level, including the putative secreted <em>factors</em> <em>fibroblast</em> <em>growth</em> <em>factor</em>-7, insulin-like <em>growth</em> <em>factor</em>-binding proteins 3 and 4, pleiotrophin, pentaxin-related, and thrombospondin 2, whereas 11 genes, including GPX-3 and HSP27, were expressed at a lower level. In summary, we show for the first time long-term maintenance of adult bone marrow HSCs in stroma noncontact cultures and identify some secreted molecules that may be involved in this support.

Tumor angiogenesis is stimulated by a pro-angiogenic shift in both inducers and inhibitors of endothelial <em>growth</em>. To study this shift, we measured serum and plasma levels of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), endostatin, and thrombospondin 1 (TSP1) in <em>21</em> advanced non-small cell lung cancer (NSCLC) patients and 46 healthy control subjects. In addition, we assessed the relevance of these levels to disease outcome. Cytokine levels were prospectively measured in plasma and serum by enzyme-linked immunosorbent assay at three times: before chemotherapy and at 1 and 12 weeks following initiation of chemotherapy. In NSCLC patients, serum VEGF levels (sVEGF) were elevated (p<0.001), whereas serum and plasma TSP1 levels were lower (p=0.012 and p=0.004, respectively) than in healthy control subjects. Pretreatment plasma endostatin and serum bFGF levels were higher in NSCLC patients than in healthy controls (p=0.05 and 0.01, respectively). Change in sVEGF at week 12 after initiation of chemotherapy correlated with response to therapy (p=0.002). Patients with pretreatment sVEGF levels <500 pg/mL had a median survival of 11 months, but those with sVEGF >500 pg/mL had only a 6 months' median survival (p < 0.03). In NSCLC patients, VEGF levels are increased, whereas TSP1 levels are decreased, which may trigger and sustain tumor angiogenesis. High levels of serum VEGF at the time of presentation with NSCLC may predict worse survival.

Fibrinogen (FBG) assembles into matrix fibrils of <em>fibroblasts</em>, lung and mammary epithelial cells, but not endothelial cells. Furthermore, cryptic beta15-<em>21</em> residues are exposed in FBG fibrils with no evidence of thrombin or plasmin proteolysis. Herein, the effects of FBG on migration and proliferation of wounded dermal <em>fibroblasts</em> were investigated. FBG preassembled into matrix prior to scrape-wounding induced 3H-thymidine incorporation 8-fold and shortened the time to wound closure 1.6-fold +/- 0.1-fold. FBG added immediately after wounding did not enhance either response. <em>Fibroblast</em> <em>growth</em> <em>factor</em>-2/platelet-derived <em>growth</em> <em>factor</em> (FGF-2/PDGF) stimulated cell proliferation 2.2-fold for FGF-2 and 3.2-fold for PDGF and wound closure 1.5-fold +/- 0.1-fold in the absence of matrix-FBG. Surprisingly, exogenous <em>growth</em> <em>factors</em> had negligible effect on wound closure and cell proliferation already enhanced by matrix-FBG. Matrix-FBG-enhanced wound closure required active assembly of an FBG-fibronectin matrix, engagement of alphavbeta3, and FBG Aalpha-RGDS572-575 integrin recognition sites; Aalpha-RGDF95-98 sites were not sufficient for matrix-FBG assembly, enhanced wound closure, or cell proliferation. Although Bbeta1-42 was not necessary for matrix assembly, it was required for matrix-FBG-enhanced cell migration. These data indicate that FBG serves as an important matrix constituent in the absence of fibrin formation to enhance wound repair and implicate Bbeta1-42 as a physiologic inducer of signal transduction to promote an intermediate state of cell adhesion and a migratory cell phenotype.