The heart is not traditionally considered either a target or a site of <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>) production. However, recent findings indicate that FGF<em>21</em> can act as a cardiomyokine; that is, it is produced by cardiac cells at significant levels and acts in an autocrine manner on the heart itself. The heart is sensitive to the effects of FGF<em>21</em>, both systemic and locally generated, owing to the expression in cardiomyocytes of β-Klotho, the key co-receptor known to confer specific responsiveness to FGF<em>21</em> action. FGF<em>21</em> has been demonstrated to protect against cardiac hypertrophy, cardiac inflammation, and oxidative stress. FGF<em>21</em> expression in the heart is induced in response to cardiac insults, such as experimental cardiac hypertrophy and myocardial infarction in rodents, as well as in failing human hearts. Intracellular mechanisms involving PPARα and Sirt1 mediate transcriptional regulation of the FGF<em>21</em> gene in response to exogenous stimuli. In humans, circulating FGF<em>21</em> levels are elevated in coronary heart disease and atherosclerosis, and are associated with a higher risk of cardiovascular events in patients with type 2 diabetes. These findings provide new insights into the role of FGF<em>21</em> in the heart and may offer potential therapeutic strategies for cardiac disease.

Here we evaluated the performance of a large set of serum biomarkers for the prediction of rapid progression of chronic kidney disease (CKD) in patients with type 2 diabetes. We used a case-control design nested within a prospective cohort of patients with baseline eGFR 30-60 ml/min per 1.73 m(2). Within a 3.5-year period of Go-DARTS study patients, 154 had over a 40% eGFR decline and 153 controls maintained over 95% of baseline eGFR. A total of 207 serum biomarkers were measured and logistic regression was used with forward selection to choose a subset that were maximized on top of clinical variables including age, gender, hemoglobin A1c, eGFR, and albuminuria. Nested cross-validation determined the best number of biomarkers to retain and evaluate for predictive performance. Ultimately, 30 biomarkers showed significant associations with rapid progression and adjusted for clinical characteristics. A panel of 14 biomarkers increased the area under the ROC curve from 0.706 (clinical data alone) to 0.868. Biomarkers selected included <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em>, the symmetric to asymmetric dimethylarginine ratio, β2-microglobulin, C16-acylcarnitine, and kidney injury molecule-1. Use of more extensive clinical data including prebaseline eGFR slope improved prediction but to a lesser extent than biomarkers (area under the ROC curve of 0.793). Thus we identified several novel associations of biomarkers with CKD progression and the utility of a small panel of biomarkers to improve prediction.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a stress-inducible hormone that has important roles in regulating energy balance and glucose and lipid homeostasis through a heterodimeric receptor complex comprising FGF receptor 1 (FGFR1) and β-klotho. Administration of FGF<em>21</em> to rodents or non-human primates causes considerable pharmacological benefits on a cluster of obesity-related metabolic complications, including a reduction in fat mass and alleviation of hyperglycaemia, insulin resistance, dyslipidaemia, cardiovascular disorders and non-alcoholic steatohepatitis (NASH). However, native FGF<em>21</em> is unsuitable for clinical use owing to poor pharmacokinetic and biophysical properties. A large number of long-acting FGF<em>21</em> analogues and agonistic monoclonal antibodies for the FGFR1-β-klotho receptor complexes have been developed. Several FGF<em>21</em> analogues and mimetics have progressed to early phases of clinical trials in patients with obesity, type 2 diabetes mellitus and NASH. In these trials, the primary end points of glycaemic control have not been met, whereas substantial improvements were observed in dyslipidaemia, hepatic fat fractions and serum markers of liver fibrosis in patients with NASH. The complexity and divergence in pharmacology and pathophysiology of FGF<em>21</em>, interspecies variations in FGF<em>21</em> biology, the possible existence of obesity-related FGF<em>21</em> resistance and endogenous FGF<em>21</em> inactivation enzymes represent major obstacles to clinical implementation of FGF<em>21</em>-based pharmacotherapies for metabolic diseases.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a hormone-like member of FGF family which controls metabolic multiorgan crosstalk enhancing energy expenditure through glucose and lipid metabolism. In addition, FGF<em>21</em> acts as a stress hormone induced by endoplasmic reticulum stress and dysfunctions of mitochondria and autophagy in several tissues. FGF<em>21</em> also controls stress responses and metabolism by modulating the functions of somatotropic axis and hypothalamic-pituitary-adrenal (HPA) pathway. FGF<em>21</em> is a potent longevity <em>factor</em> coordinating interactions between energy metabolism and stress responses. Recent studies have revealed that FGF<em>21</em> treatment can alleviate many age-related metabolic disorders, e.g. atherosclerosis, obesity, type 2 diabetes, and some cardiovascular diseases. In addition, transgenic mice overexpressing FGF<em>21</em> have an extended lifespan. However, chronic metabolic and stress-related disorders involving inflammatory responses can provoke FGF<em>21</em> resistance and thus disturb healthy aging process. First, we will describe the role of FGF<em>21</em> in interorgan energy metabolism and explain how its functions as a stress hormone can improve healthspan. Next, we will examine both the induction of FGF<em>21</em> expression via the integrated stress response and the molecular mechanism through which FGF<em>21</em> enhances healthy aging. Finally, we postulate that FGF<em>21</em> resistance, similarly to insulin resistance, jeopardizes human healthspan and accelerates the aging process.

Prevalence of type 2 diabetes (T2D) and obesity is increasing worldwide. Currently available therapies are not suited for all patients in the heterogeneous obese/T2D population, hence the need for novel treatments. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is considered a promising therapeutic agent for T2D/obesity. Native FGF<em>21</em> has, however, poor pharmacokinetic properties, making gene therapy an attractive strategy to achieve sustained circulating levels of this protein. Here, adeno-associated viral vectors (AAV) were used to genetically engineer liver, adipose tissue, or skeletal muscle to secrete FGF<em>21</em>. Treatment of animals under long-term high-fat diet feeding or of ob/ob mice resulted in marked reductions in body weight, adipose tissue hypertrophy and inflammation, hepatic steatosis, inflammation and fibrosis, and insulin resistance for>> 1 year. This therapeutic effect was achieved in the absence of side effects despite continuously elevated serum FGF<em>21</em>. Furthermore, FGF<em>21</em> overproduction in healthy animals fed a standard diet prevented the increase in weight and insulin resistance associated with aging. Our study underscores the potential of FGF<em>21</em> gene therapy to treat obesity, insulin resistance, and T2D.

Wound contraction is one function of granulation tissue which is critical to repair. This study compares the ability of <em>fibroblast</em>-like cells derived from granulation tissue of various ages to contract a tissue equivalent, or a collagen gel, and examines the influence of <em>growth</em> <em>factors</em> implicated in wound repair on collagen gel contraction by these different cell populations. Cells from older granulation tissue (<em>21</em> and 28 days) have an enhanced ability to contract a tissue equivalent when compared to cells from younger granulation tissue (7 and 14 days) or normal rat skin <em>fibroblasts</em>. Transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1) enhanced contractility most in those cells which had a greater basal contractile ability. While basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) alone had moderately stimulatory effects at low doses (0.1-1.0 ng/ml), higher doses (greater than or equal to 10 ng/ml) inhibited basal contraction. Pretreatment with bFGF followed by exposure to TGF-beta 1, with or without the continued presence of bFGF, delayed gel contraction by cells from skin and early granulation tissue, but bFGF enhanced TGF-beta 1 activity in highly contractile cells. Transforming <em>growth</em> <em>factor</em>-alpha moderately enhanced contraction by cells from older granulation tissue. While both TGF-beta 1 and bFGF enhanced wound repair, their differential effects on the <em>fibroblast</em>-like cell derived from granulation tissue of different ages suggest that phenotypic differences exist between these cell populations. In addition, our results predict significant interactions between polypeptide cytokines at the site of repair.

OBJECTIVE

Fibroblast growth factor receptor 3 (FGFR3) mutations were reported recently at a high frequency in low-grade urothelial cell carcinoma (UCC). We investigated the feasibility of combining microsatellite analysis (MA) and the FGFR3 status for the detection of UCC in voided urine.

METHODS

In a prospective setting, 59 UCC tissues and matched urine samples were obtained, and subjected to MA (23 markers) and FGFR3 mutation analysis (exons 7, 10, and 15). In each case, a clinical record with tumor and urine features was provided. Fifteen patients with a negative cystoscopy during follow-up served as controls.

RESULTS

A mutation in the FGFR3 gene was found in 26 (44%) UCCs of which 22 concerned solitary pTaG1/2 lesions. These mutations were absent in the 15 G3 tumors. For the 6 cases with leukocyturia, 46 microsatellite alterations were found in the tumor. Only 1 of these was also detected in the urine. This was 125 of 357 for the 53 cases without leukocyte contamination. The sensitivity of MA on voided urine was lower for FGFR3-positive UCC (15 of 21; 71%) as compared with FGFR3 wild-type UCC (29 of 32; 91%). By including the FGFR3 mutation, the sensitivity of molecular cytology increased to 89% and was superior to the sensitivity of morphological cytology (25%) for every clinical subdivision. The specificity was 14 of 15 (93%) for the two (molecular and morphological) cytological approaches.

CONCLUSIONS

Molecular urine cytology by MA and FGFR3 mutation analysis enables a highly sensitive and specific detection of UCC. The similarity of molecular profiles in tumor and urine corroborate their clonal relation.

Addition of several arginine-specific serine proteases to culture medium conditioned by <em>fibroblasts</em> results in the proteases being taken into sodium dodecyl sulfate-stable complexes with a secreted <em>factor</em> termed protease nexin (PN) (Baker, J. B., Low, D. A., Simmer, R. L., and Cunningham, D. D. (1980) Cell <em>21</em>, 37-45). PN not only inhibits these degradative enzymes but also mediates their binding, internalization, and degradation by the cells (Low, D. A., Baker, J. B., Koonce, W. C., and Cunningham, D. D. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 2340-2344). Here we describe a simple procedure for purifying milligram quantities of PN from serum-free medium conditioned by human foreskin cells. Accumulation of PN in the medium is increased by using high density microcarrier cultures supplemented with epidermal <em>growth</em> <em>factor</em> and bovine serum albumin. Application of ultrafiltration-concentrated medium to a heparin-Sepharose column followed by extensive washing of the column with buffer containing 0.2 M NaCl and elution with buffer containing 1.0 M NaCl results in the recovery of 60-90% of the input PN in a form that is 90-97% pure. This preparation can be further purified by hydrophobic chromatography on octyl-agarose. Purified PN has a molecular mass of approximately 51 kilodaltons. On nonequilibrium pH gradient electrophoresis it migrates as five bands with isoelectric points between 7.5 and 7.8. Purified PN exhibits all the properties attributed to PN in culture medium. These include: 1) formation of sodium dodecyl sulfate-stable complexes with thrombin, urokinase, and plasmin; 2) inhibition of protease activity; 3) heparin-enhanced inhibition of thrombin; and 4) cellular binding of protease-PN complexes in a heparin-sensitive reaction. When thrombin-PN complexes are dissociated with 1 M hydroxylamine a smaller form of PN (approximately 46 kilodaltons) is detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the complexed PN is proteolytically modified.

Our previous studies showed that degradable dextran hydrogels are rapidly formed in situ upon mixing aqueous solutions of dextran vinyl sulfone (dex-VS) conjugates and tetrafunctional mercapto poly(ethylene glycol) (PEG-4-SH) by Michael addition. The hydrogel degradation time and storage modulus could be controlled by the degree of vinyl sulfone substitution (DS) and dextran molecular weight. The degradation time could further be adjusted by the spacer between the thioether and the ester bond of the dex-VS conjugates (ethyl vs. propyl, denoted as dex-Et-VS and dex-Pr-VS, respectively). In this paper, the release of three model proteins, i.e. immunoglobulin G (d(h)=10.7 nm, IgG), bovine serum albumin (BSA, d(h)=7.2 nm) and lysozyme (d(h)=4.1 nm), as well as basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) from these in situ forming dextran hydrogels is studied. Proteins could be easily loaded into the hydrogels by mixing protein containing solutions of dex-VS and PEG-4-SH. The release of IgG from dex-Et-VS hydrogels followed biphasic release kinetics, with a slow, close to first order release for the first 9 days followed by an accelerated release and over 80% of IgG was released in 12 to 25 days. Interestingly, the release of IgG from dex-Pr-VS hydrogels followed close to zero order kinetics, wherein approximately 95% was released in <em>21</em> days. The release of BSA from dex-Pr-VS hydrogels followed biphasic kinetics, with almost first order release followed by close to zero order release. Approximately 75% of the entrapped BSA could be released from dex-Pr-VS hydrogels in 16 days. Dex-Pr-VS hydrogels released 40% of lysozyme in 14 days, with full preservation of the enzymatic activity of the released lysozyme, as determined by bacteria lysis experiments. The release of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) from dex-Pr-VS hydrogels showed first order kinetics, with quantitative release in 28 days. These results show that the in situ forming degradable dextran hydrogels can be used for the controlled release of proteins.

Aldosterone is thought to modulate renal fibrosis, in part, through increasing plasminogen activator inhibitor type 1 (PAI-1), a major inhibitor of ECM degradation. The present study investigated aldosterone effects on PAI-1 and transforming <em>growth</em> <em>factor</em> (TGF)-beta(1) and asked whether PAI-1 effects were TGF-beta mediated and whether aldosterone and TGF-beta(1) acted synergistically to increase PAI-1 and decrease ECM degradation. Rat mesangial cells (MCs) and <em>fibroblast</em> cells [normal rat kidney (NRK)-49F] were used. (3)H-labeled ECM was produced by MCs. The effect of aldosterone and TGF-beta on ECM degradation by newly plated MCs or NRK-49F was measured by the release of (3)H into medium. Aldosterone markedly increased PAI-1 mRNA and protein in both cell types, increases completely blocked by spironolactone and partially blocked by TGF-beta neutralizing antibody. Adding both aldosterone and TGF-beta(1) produced PAI-1 mRNA and protein increases higher than the sum of increases seen with either compound alone. Aldosterone or TGF-beta(1) alone inhibited matrix degradation by 39 and 49% in MCs and <em>21</em> and 23% in NRK-49F, respectively. When both compounds were added, matrix degradation was further decreased by 93% in MCs and 61% in NRK-49F. The results indicate that aldosterone-induced PAI-1 increases are partially mediated by TGF-beta(1) and lead to decreased ECM degradation. While aldosterone alone induced TGF-beta(1) weakly, aldosterone and TGF-beta(1) added together produced dramatic synergistic effects on PAI-1 production and subsequent ECM accumulation. Thus the elevated aldosterone induced by renin-angiotensin-aldosterone system activation may amplify renin-angiotensin-aldosterone system profibrotic actions.

Patients with end-stage renal disease (ESRD) suffer exceptionally high mortality rates in their first year of chronic hemodialysis. Both vitamin D and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-23 levels correlate with survival in these patients. Klotho is a protein in the vitamin D/FGF-23 signaling pathway that has been linked with accelerated aging and early mortality in animal models. We therefore hypothesized that genetic variation in the Klotho gene might be associated with survival in subjects with ESRD. We tested the association between 12 single nucleotide polymorphisms (SNPs) in the Klotho gene and mortality in a cohort of ESRD patients during their first year on hemodialysis (n = 1307 white and Asian). We found a significant association between the CC genotype of one tag SNP, rs577912, and increased risk for 1-yr mortality (RR, 1.76; 95% CI, 1.19-2.59; p = 0.003). This effect was even more marked among patients who were not treated with activated vitamin D supplementation (HR, 2.51; 95% CI, 1.18-5.34; p = 0.005). In lymphoblastoid cell lines derived from HapMap subjects, the CC genotype was associated with a 16-<em>21</em>% lower Klotho expression compared with the AA/AC genotype. Our data suggest that a specific Klotho variant (rs577912) is linked to survival in ESRD patients initiating chronic hemodialysis and that therapy with activated vitamin D may modify this risk.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) has been shown to enhance the survival and neurite extension of various types of neurons including spinal ganglion neurons. In addition, endogenous FGF-2 and FGF receptors are upregulated following peripheral nerve lesion in ganglia and at the lesion site. FGF-2 protein is expressed in different isoforms (18 kDa, <em>21</em> kDa, 23 kDa) and differentially regulated after nerve injury. In the rat we analyzed the regenerative capacity of the high molecular weight (HMW) FGF-2 isoforms (<em>21</em>/23 kDa) to support the regeneration of the axotomized adult sciatic nerve across long gaps. The nerve stumps were inserted into the opposite ends of a silicone chamber resulting in an interstump gap of 15 mm. Silicone tubes were filled with Matrigel or a mixture of Schwann cells (SC) and Matrigel. SC were prepared from newborn rats and transfected to overexpress HMW FGF-2. Four weeks after the operation procedure, channels were analyzed with regard to tissue cables bridging both nerve stumps and myelinated axons distal to the original proximal nerve stump. Peripheral nerves interposed with HMW Schwann cells displayed significantly enhanced nerve regeneration, with the greatest number of tissue cables containing myelinated axons and the highest number of myelinated axons. These results suggest that a cellular substrate together with a source of a trophic <em>factor</em> could be a promising tool to promote nerve regeneration and, therefore, become useful also for a clinical approach to repair long gaps.

When recombinant human bone morphogenetic protein-2 (rhBMP-2) is implanted in soft tissues, bony tissue is induced during the course of endochondral ossification. The relationship between endochondral ossification and vascularization is important in bone formation, and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) is considered to play an important role in this process. In this study, the immunohistological localization of VEGF was investigated in rhBMP-2-induced ectopic endochondral ossification in the calf muscle of rats. In addition, the characteristics of anti-VEGF antibody-reactive cells were histologically investigated using electron microscopy to examine the cause of endochondral ossification induced by recombinant human bone morphogenetic protein-2. The role of VEGF in rhBMP-2-induced osteoinduction and vascular induction was studied by observing the relationship between the localizations of anti-VEGF antibody-reactive cells and vascularization. During the process of rhBMP-2-induced ectopic endochondral ossification, <em>fibroblast</em>-like cells, which were located at the margin of the implant and reactive to BMP-2 at 5 days, were positive for VEGF immunostaining. Hypertrophic chondrocytes appeared 9 days and osteoblasts appeared 14 to <em>21</em> days after implantation, and all these cells were reactive with anti-VEGF antibody. Bony trabeculae subsequently appeared in the muscle, and new blood vessels were formed alongside the trabeculae. When VEGF was added to rhBMP, more new blood vessels and bone were formed in the induced bone. These findings suggested that rhBMP-2 induced the differentiation of undifferentiated mesenchymal cells to chondrocytes and osteoblasts, and these differentiated cells expressed VEGF, creating an advantageous environment for vascularization in bony tissue.

OBJECTIVE

Although rodent models provide insight into the mechanisms underlying type 2 diabetes mellitus (T2DM), they are limited in their translatability to humans. The nonhuman primate (NHP) shares important metabolic similarities with the human, making it an ideal model for the investigation of type 2 diabetes and use in preclinical trials. This review highlights the key contributions in the field over the last year using the NHP model.

RESULTS

The NHP has not only provided novel insight into the normal and pathological processes that occur within the islet, but has also allowed for the preclinical testing of novel pharmaceutical targets for obesity and T2DM. Particularly, administration of <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> in the NHP resulted in weight loss and improvements in metabolic health, supporting rodent studies and recent clinical trials. In addition, the NHP was used to demonstrate that a novel melanocortin-4 receptor agonist did not cause adverse cardiovascular effects. Finally, this model has been used to provide evidence that glucagon-like peptide-1-based therapies do not induce pancreatitis in the healthy NHP.

CONCLUSIONS

The insight gained from studies using the NHP model has allowed for a better understanding of the processes driving T2DM and has promoted the development of well tolerated and effective treatments.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is an important regulator of glucose/lipid metabolism. Although there are studies examining the relationship between serum FGF<em>21</em> levels and glucose homeostasis, the role of FGF<em>21</em> remains unclear. The objective of this study was to examine whether serum FGF<em>21</em> levels are associated with metabolic parameters in subjects with varying degrees of obesity and glucose tolerance and with complications in subjects with type2 diabetes mellitus (T2DM).

METHODS

The study consisted of <em>21</em>3 subjects who were lean and had normal glucose tolerance (lean NGT), were overweight with NGT, had impaired glucose tolerance (IGT) or had T2DM. Serum FGF<em>21</em> levels and their associations with the parameters of adiposity, glucose tolerance and the presence of diabetic complications were examined.

RESULTS

The serum FGF<em>21</em> levels in T2DM were higher than in lean NGT. Serum FGF<em>21</em> levels showed a positive correlation with the urine albumin-to-creatinine ratio (ACR) in all subjects except for the T2DM subjects, who showed a correlation after adjustment of age, gender and body mass index. Moreover, the subjects with carotid artery plaque showed higher serum FGF<em>21</em> levels than those without complications.

CONCLUSIONS

Serum FGF<em>21</em> levels were associated with the urine ACR and diabetic complications including carotid artery plaque.

To comprehensively screen for genetic events underlying colorectal cancer, we performed suppression subtraction hybridization analysis on an advanced colon cancer. Because Dickkopf-4, a member of the Dickkopf family acting as a Wnt-signaling modulator, was identified as one of the upregulated genes in this specimen, we investigated expression profiles of all the Dickkopf family members in 55 colorectal tumors (<em>21</em> cancers and 34 adenomas). We also investigated mechanisms regulating the expression of Dickkopf-4 in these cancers in vitro and in vivo. Compared with normal adjacent mucosae, Dickkopf-4 (median 27.4, P < 0.01) and -2 (median 51.4, P < 0.01) were strongly expressed in colorectal cancers. The level of Dickkopf-4 was positively correlated with <em>fibroblast</em> <em>growth</em> <em>factor</em>-20 (r(s) = 0.61, P = 0.00017), a representative beta-catenin transcriptional target gene, and with the degree of nuclear accumulation of beta-catenin in colorectal tumors. Dickkopf-4 was induced by activated beta-catenin in vitro. Reciprocally, recombinant Dickkopf-4 significantly inhibited T-cell <em>factor</em>/lymphocyte enhancer <em>factor</em> reporter activity stimulated by recombinant Wnt3a in human embryonic kidney 293 cells. We conclude that Dickkopf-4 and -2 are significantly upregulated in most colorectal tumors, and that Dickkopf-4 upregulation reflects activation of the Wnt/canonical pathway.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are a family of at least <em>21</em> heparin-binding proteins involved in many biological processes, both during development and in the adult, including cell proliferation, differentiation, and angiogenesis. FGFs mediate their effects through high-affinity tyrosine kinase receptors (FGFRs), which are encoded by four genes. The aims of the present study were to localize FGFR-1 through FGFR-3 in the normal adult rat kidney and to determine which functional FGFR variants and FGFs were expressed.

METHODS

Avidin-biotin-enhanced horseradish peroxidase immunohistochemistry was used on paraffin sections of rat kidney to localize FGFR-1 through FGFR-3, whereas reverse transcriptase-polymerase chain reaction was used to examine expression of the receptor variants and also of FGF-1 through FGF-10 in cortex, outer medulla, and inner medulla.

RESULTS

By immunohistochemistry, each receptor was localized to distinct and overlapping nephron segments, such that one or more FGFRs were localized to all nephron and collecting duct epithelia. FGFR-1 and FGFR-3 were localized to glomeruli, FGFR-3 to proximal tubules and FGFR-1 to thin limbs. FGFR-1 through FGFR-3 were localized to distal straight tubules, with FGFR-1 and FGFR-3 localized to distal convoluted tubules. FGFR-1 and FGFR-3 were localized to medullary collecting ducts. In addition, FGFR-1 was localized to the smooth muscle of renal arteries. All seven FGFR variants were expressed in the cortex and outer medulla, with fewer FGFRs in the inner medulla. FGF-1, FGF-2, FGF-7, FGF-8, and FGF-9 were expressed in the kidney, with FGF-10 expression found only in the cortex.

CONCLUSIONS

Mapping of these receptors is critical to the determination of the effects of FGF ligands in discrete regions of the kidney. The distributions of the FGFRs in the normal adult kidney and the restricted expression of FGF ligands suggest that specific FGFs have distinct and important roles in the maintenance of normal kidney structure and function.

Young (4 month) and aged (15-18 months) mice were given intranasal saline or γ--herpesvirus-68 infection. After <em>21</em> days, aged, but not young mice, showed significant increases in collagen content and fibrosis. There were no differences in viral clearance or inflammatory cells (including fibrocytes) between infected aged and young mice. Enzyme-linked immunosorbent assays showed increased transforming <em>growth</em> <em>factor</em>-β in whole lung homogenates of infected aged mice compared with young mice. When <em>fibroblasts</em> from aged and young mice were infected in vitro, aged, but not young, <em>fibroblasts</em> upregulate alpha-smooth muscle actin and collagen I protein. Infection with virus in vivo also demonstrates increased alpha-smooth muscle actin and collagen I protein and collagen I, collagen III, and fibronectin messenger RNA in aged <em>fibroblasts</em>. Furthermore, evaluation revealed that aged <em>fibroblasts</em> at baseline have increased transforming <em>growth</em> <em>factor</em>-β receptor 1 and 2 levels compared with young <em>fibroblasts</em> and are resistant to apoptosis. Increased responsiveness to transforming <em>growth</em> <em>factor</em>-β was verified by increased collagen III and fibronectin messenger RNA after treatment in vitro with transforming <em>growth</em> <em>factor</em>-β.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is an endocrine-member of the FGF family. It is synthesized mainly in the liver, but it is also expressed in adipose tissue, skeletal muscle, and many other organs. It has a key role in glucose and lipid metabolism, as well as in energy balance. FGF<em>21</em> concentration in plasma is increased in patients with obesity, insulin resistance, and metabolic syndrome. Recent findings suggest that such increment protects tissue from an increased oxidative stress environment. Different types of physical stress, such as strenuous exercising, lactation, diabetic nephropathy, cardiovascular disease, and critical illnesses, also increase FGF<em>21</em> circulating concentration. FGF<em>21</em> is now considered a stress-responsive hormone in humans. The discovery of an essential response element in the FGF<em>21</em> gene, for the activating transcription <em>factor</em> 4 (ATF4), involved in the regulation of oxidative stress, and its relation with genes such as NRF2, TBP-2, UCP3, SOD2, ERK, and p38, places FGF<em>21</em> as a key regulator of the oxidative stress cell response. Its role in chronic diseases and its involvement in the treatment and follow-up of these diseases has been recently the target of new studies. The diminished oxidative stress through FGF<em>21</em> pathways observed with anti-diabetic therapy is another clue of the new insights of this hormone.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>)-mediated weight loss and improvements in glucose metabolism correlate with increased uncoupling protein 1 (Ucp1) levels in adipose tissues, suggesting that UCP1-dependent thermogenesis may drive FGF<em>21</em> action. It was reported that FGF<em>21</em> is equally effective at reducing body weight and improving glucose homeostasis without UCP1. We find while FGF<em>21</em> can lower body weight in both wild-type and Ucp1 knockout mice, rapid clearance of glucose by FGF<em>21</em> is defective in the absence of UCP1. Furthermore, in obese wild-type mice there is a fall in brown adipose tissue (BAT) temperature during glucose excursion, and FGF<em>21</em> improves glucose clearance while preventing the fall in BAT temperature. In Ucp1 knockout mice, the fall in BAT temperature during glucose excursion and FGF<em>21</em>-mediated changes in BAT temperature are lost. We conclude FGF<em>21</em>-mediated improvements in clearance of a glucose challenge require UCP1 and evoke UCP1-dependent thermogenesis as a method to increase glucose disposal.

OBJECTIVE

The purpose of this study was to characterize cell cultures and xenografts derived from patients with ovarian cancer.

METHODS

Ninety specimens from 67 patients were plated in RPMI 1640 or inoculated in nude mice. Growth characteristics of cell cultures and xenografts were determined. Expression of receptors for estrogen, progesterone, androgen, epithelial growth factor, fibroblast growth factor, HER-2/erbB-2/c-neu proto-oncogene, and the P53 expression were characterized by immunocytochemistry in 28 cell cultures.

RESULTS

Forty-nine percent of samples were cultured successfully in vitro. Ascitic and pleural effusion specimens were more likely to produce a cell culture or a xenograft than solid tissue specimens (P < 0.005). All of the cell cultures had an epithelial morphology, and 89% were aneuploid with a mean DNA index of 1.6 (range, 0.9-3.0). Of 54 and 61 specimens inoculated into nude mice i.p. and s.c., 15 (28%) and 18 (30%) produced a xenograft, respectively, with two-thirds of these xenografts being reproducibly tumorigenic. The median time to first passage was 21 weeks for cell cultures and 8-12 weeks for xenografts. Expression of epithelial growth factor receptor, HER-2/erbB-2/c-neu proto-oncogene, fibroblast growth factor receptor, estrogen, progesterone, and androgen was seen in 24, 21, 31, 17, 43, and 18%, respectively. P53 was overexpressed in 62% of cell cultures analyzed.

CONCLUSIONS

Ovarian cancer cells collected from effusions are easier to grow in vitro than in vivo. The only characteristic that may be associated with tumorigenicity was abnormal P53 expression. This panel of ovarian cancer materials provides useful models for biological or therapeutical studies.

OBJECTIVE

The feasibility of using a biodegradable hydrogel incorporating basic fibroblast growth factor (bFGF) to induce bone regeneration at the site of a skull defect in monkeys was investigated.

METHODS

Basic fibroblast growth factor was incorporated into a bioabsorbable hydrogel, which was prepared through glutaraldehyde crosslinking of gelatin. Following treatment of monkey skull defects measuring 6 mm in diameter (six defects/experimental group) with gelatin hydrogel incorporating bFGF, skull bone regeneration was evaluated using soft x-ray studies, dual x-ray absorptometry, and histological examinations. The water content of the hydrogels varied according to the glutaraldehyde concentration in the hydrogel preparation. Gelatin hydrogels incorporating 100 microg of bFGF significantly promoted bone regeneration and the skull defect was completely closed 21 weeks after implantation. This is in marked contrast with the effect of the same dose of bFGF in solution form. Bone mineral density (BMD) measured at the sites of skull defect was enhanced by the bFGF-incorporating hydrogels. The BMD enhancement was more prominent at lower water contents of hydrogel. Empty gelatin hydrogels neither induced nor interfered with skull bone regeneration.

CONCLUSIONS

The findings of this study indicate that bFGF coupled with bioabsorbable hydrogel is a very promising tool to assist in the regrowth of bone at the site of a skull defect, which clinically has been recognized as almost impossible.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a key metabolic regulator that is induced by fasting and starvation, and its expression is thought to be regulated by the circadian clock in the liver. To evaluate the functional role of FGF<em>21</em> in the circadian regulation of physiology and behavior, we examined the temporal expression profiles of Fgf<em>21</em> and circadian clock genes in addition to behavioral activity rhythms under adlibitum feeding (ALF) and time-imposed restricted feeding (RF) in mice. Four hours of daily restricted feeding during the daytime induced over an 80-fold increase in feeding-dependent rhythmic Fgf<em>21</em> mRNA expression in epididymal white adipose tissue (eWAT), although the expression levels were continuously increased 10-fold in the liver of wild-type (WT) mice. Refeeding subsequent to transient fasting revealed that refeeding but not fasting remarkably induces Fgf<em>21</em> expression in eWAT, although fasting-induced hepatic Fgf<em>21</em> expression is completely reversed by refeeding. The free-running period of locomotor activity rhythm under ALF and the food anticipatory activity (FAA) under RF remained intact in Fgf<em>21</em> knockout (KO) mice, suggesting that FGF<em>21</em> is dispensable for both the central clock in the suprachiasmatic nucleus (SCN) and the food-entrainable oscillator that governs the FAA. Temporal expression profiles of circadian genes such as mPer2 and BMAL1 were essentially identical in both tissues between WT and Fgf<em>21</em> KO mice under RF. The physiological role of the refeeding-induced adipose Fgf<em>21</em> expression remains to be elucidated.

FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders.