X-linked hypophosphatemic rickets/osteomalacia (XLH), autosomal dominant hypophosphatemic rickets/osteomalacia (ADHR) and autosomal recessive hypophosphatemic rickets/osteomalacia (ARHR1 or ARHR2) are hereditary <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23)-related hypophosphatemic rickets showing similar clinical features. We here show a patient with hypophosphatemic rickets and widespread ossification of posterior longitudinal ligament (OPLL). The proband is a 62-year-old female. Her parents are first cousins and showed no signs of rickets or osteomalacia. She showed hypophosphatemic rickets with elevated FGF23 level and had been clinically considered to be suffering from XLH. However, direct sequencing of all coding exons and exon-intron junctions of phosphate regulating gene with homologies to endopeptidases on the X chromosome (PHEX), FGF23 and dentin matrix protein 1 (DMP1) genes, responsible genes for XLH, ADHR and ARHR1, respectively, showed no mutation. A novel homozygous splice donor site mutation was found at the exon-intron junction of exon <em>21</em> of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene responsible for ARHR2 (IVS<em>21</em>+1_3(GTA>CACC)). Subsequent analysis of mRNA revealed that this mutation caused skipping of exon <em>21</em> which created a premature stop codon in exon 22. These results indicate that genetic analysis is mandatory for the correct diagnosis of hereditary FGF23-related hypophosphatemic rickets. Because Enpp1 knockout mouse is a model of OPLL, this case also suggests that OPLL is associated with ARHR2.

Calcification of vascular elastin occurs in patients with arteriosclerosis, renal failure, diabetes, and vascular graft implants. We hypothesized that pathological elastin calcification is related to degenerative and osteogenic mechanisms. To test this hypothesis, the temporal expression of genes and proteins associated with elastin degradation and osteogenesis was examined in the rat subdermal calcification model by quantitative real-time reverse transcription-polymerase chain reaction and specific protein assays. Purified elastin implanted subdermally in juvenile rats exhibited progressive calcification in a time-dependent manner along with <em>fibroblast</em> and macrophage infiltration. Reverse transcription-polymerase chain reaction analysis showed that relative gene expression levels of matrix metalloproteinases (MMP-2 and MMP-9) and transforming <em>growth</em> <em>factor</em>-beta1 were increased in parallel with calcification. Gelatin zymography showed strong MMP activities at early time points, which were associated with high levels of soluble elastin peptides. Gene expression of core binding <em>factor</em> alpha-1, an osteoblast-specific transcription <em>factor</em>, increased in parallel with elastin calcification and attained approximately 9.5-fold higher expression at <em>21</em> days compared to 3 days after implantation. Similarly, mRNA levels of the bone markers osteopontin and alkaline phosphatase also increased progressively, but osteocalcin levels remained unchanged. We conclude that degenerative and osteogenic processes may be involved in elastin calcification.

Childhood hypothyroidism causes <em>growth</em> arrest with delayed ossification and <em>growth</em>-plate dysgenesis, whereas thyrotoxicosis accelerates ossification and <em>growth</em>. Thyroid hormone (T(3)) regulates chondrocyte proliferation and is essential for hypertrophic differentiation. <em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) are also important regulators of chondrocyte proliferation and differentiation, and activating mutations of FGF receptor-3 (FGFR3) cause achondroplasia. We investigated the hypothesis that T(3) regulates chondrogenesis via FGFR3 in ATDC5 cells, which undergo a defined program of chondrogenesis. ATDC5 cells expressed two FGFR1, four FGFR2, and one FGFR3 mRNA splice variants throughout chondrogenesis, and expression of each isoform was stimulated by T(3) during the first 6-12 d of culture, when T(3) inhibited proliferation by 50%. FGFR3 expression was also increased in cells treated with T(3) for <em>21</em> d, when T(3) induced an earlier onset of hypertrophic differentiation and collagen X expression. FGFR3 expression was reduced in <em>growth</em> plates from T(3) receptor alpha-null mice, which exhibit skeletal hypothyroidism, but was increased in T(3) receptor beta(PV/PV) mice, which display skeletal thyrotoxicosis. These findings indicate that FGFR3 is a T(3)-target gene in chondrocytes. In further experiments, T(3) enhanced FGF2 and FGF18 activation of the MAPK-signaling pathway but inhibited their activation of signal transducer and activator of transcription-1. FGF9 did not activate MAPK or signal transducer and activator of transcription-1 pathways in the absence or presence of T(3). Thus, T(3) exerted differing effects on FGFR activation during chondrogenesis depending on which FGF ligand stimulated the FGFR and which downstream signaling pathway was activated. These studies identify novel interactions between T(3) and FGFs that regulate chondrocyte proliferation and differentiation during chondrogenesis.

The functions of a large number (>435) of extracellular regulatory proteins are controlled by their interactions with heparan sulfate (HS). In the case of <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs), HS binding determines their transport between cells and is required for the assembly of high affinity signaling complexes with their cognate FGF receptor. However, the specificity of the interaction of FGFs with HS is still debated. Here, we use a panel of FGFs (FGF-1, FGF-2, FGF-7, FGF-9, FGF-18, and FGF-<em>21</em>) spanning five FGF subfamilies to probe their specificities for HS at different levels as follows: binding parameters, identification of heparin-binding sites (HBSs) in the FGFs, changes in their secondary structure caused by heparin binding and structures in the sugar required for binding. For interaction with heparin, the FGFs exhibit K(D) values varying between 38 nM (FGF-18) and 620 nM (FGF-9) and association rate constants spanning over 20-fold (FGF-1, 2,900,000 M(-1) s(-1) and FGF-9, 130,000 M(-1) s(-1)). The canonical HBS in FGF-1, FGF-2, FGF-7, FGF-9, and FGF-18 differs in its size, and these FGFs have a different complement of secondary HBS, ranging from none (FGF-9) to two (FGF-1). Differential scanning fluorimetry identified clear preferences in these FGFs for distinct structural features in the polysaccharide. These data suggest that the differences in heparin-binding sites in both the protein and the sugar are greatest between subfamilies and may be more restricted within a FGF subfamily in accord with the known conservation of function within FGF subfamilies.

The biology of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has evolved through its first decade at a revolutionary pace with dramatic refinements in this relatively short span of time. This field is poised now with a deeper understanding of its specific physiological role, pathological ramifications for its inappropriate function, and a much-enriched context of the complex hormonal network in which it serves to regulate metabolism. As a derivative of these discoveries, the application of FGF<em>21</em> as a medicinal agent has emerged with structurally optimized protein-based analogs being preclinically explored in multiple species, and, more recently, through clinical studies. These novel findings set a foundation for ongoing inquiries that structure future research into this intriguing protein.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em> is an endocrine <em>factor</em> with multiple beneficial effects on glucose and lipid metabolism in animals. This study aimed to investigate the association of serum FGF<em>21</em> levels with type 1 diabetes, latent autoimmune diabetes in adults (LADA) and type 2 diabetes.

METHODS

Serum FGF<em>21</em> levels were determined by ELISA in patients with type 1 diabetes (n = 76), LADA (n = 68), type 2 diabetes (n = 77), and their age- and sex-matched controls. The association of serum FGF<em>21</em> with markers of autoimmunity was studied.

RESULTS

In type 1 diabetic patients, serum FGF<em>21</em> levels were significantly lower than controls [108.3 (61.5-180.1) vs. 196.0 (103.7-330.9) pg/ml, P < 0.001]. In LADA patients, serum FGF<em>21</em> levels were significantly lower than controls after adjustment for body mass index [<em>21</em>0.9 (1<em>21</em>.4-441.6) vs. 268.3 (159.5-443.6) pg/ml, P = 0.003]. By contrast, serum FGF<em>21</em> levels in type 2 diabetic patients were significantly higher than controls [381.2 (244.7-531.3) vs. 301.4 (173.9-444.2) pg/ml, P = 0.006]. FGF<em>21</em> levels increased progressively from type 1 diabetes, LADA, to type 2 diabetes (P < 0.001 for global trend). Furthermore, FGF<em>21</em> levels correlated inversely with titers of glutamic acid decarboxylase and insulinoma-associated protein 2 autoantibodies in type 1 diabetic and LADA patients.

CONCLUSIONS

Serum FGF<em>21</em> level is increased in type 2 diabetes but decreased in type 1 diabetes and LADA. In autoimmune diabetes, the reduction in circulating FGF<em>21</em> is closely associated with markers of pancreatic β-cell autoimmunity.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF<em>21</em> is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF<em>21</em> analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF<em>21</em> fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF<em>21</em> via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF<em>21</em>. Two specific point mutations were introduced into FGF<em>21</em>. The leucine to arginine substitution at position 98 (L98R) suppressed FGF<em>21</em> aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF<em>21</em> identified in mice and cynomolgus monkeys. The derived Fc-FGF<em>21</em>(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF<em>21</em>(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF<em>21</em> or Fc-FGF<em>21</em> wild type. A single administration of Fc-FGF<em>21</em>(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF<em>21</em> or Fc-FGF<em>21</em> lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF<em>21</em> analog with a favorable pharmaceutical property for potential clinical development.

Hormones such as <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) and glucocorticoids (GCs) play crucial roles in coordinating the adaptive starvation response. Here we examine the interplay between these hormones. It was previously shown that FGF<em>21</em> induces corticosterone levels in mice by acting on the brain. We now show that this induces the expression of genes required for GC synthesis in the adrenal gland. FGF<em>21</em> also increases corticosterone secretion from the adrenal in response to ACTH. We further show that the relationship between FGF<em>21</em> and GCs is bidirectional. GCs induce Fgf<em>21</em> expression in the liver by acting on the GC receptor (GR). The GR binds in a ligand-dependent manner to a noncanonical GR response element located approximately 4.4 kb upstream of the Fgf<em>21</em> transcription start site. The GR cooperates with the nuclear fatty acid receptor, peroxisome proliferator-activated receptor-α, to stimulate Fgf<em>21</em> transcription. GR and peroxisome proliferator-activated receptor-α ligands have additive effects on Fgf<em>21</em> expression both in vivo and in primary cultures of mouse hepatocytes. We conclude that FGF<em>21</em> and GCs regulate each other's production in a feed-forward loop and suggest that this provides a mechanism for bypassing negative feedback on the hypothalamic-pituitary-adrenal axis to allow sustained gluconeogenesis during starvation.

The thermogenic activity of brown adipose tissue (BAT) and browning of white adipose tissue are important components of energy expenditure. Here we show that GPR120, a receptor for polyunsaturated fatty acids, promotes brown fat activation. Using RNA-seq to analyse mouse BAT transcriptome, we find that the gene encoding GPR120 is induced by thermogenic activation. We further show that GPR120 activation induces BAT activity and promotes the browning of white fat in mice, whereas GRP120-null mice show impaired cold-induced browning. Omega-3 polyunsaturated fatty acids induce brown and beige adipocyte differentiation and thermogenic activation, and these effects require GPR120. GPR120 activation induces the release of <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>) by brown and beige adipocytes, and increases blood FGF<em>21</em> levels. The effects of GPR120 activation on BAT activation and browning are impaired in FGF<em>21</em>-null mice and cells. Thus, the lipid sensor GPR120 activates brown fat via a mechanism that involves induction of FGF<em>21</em>.

Ampelopsis grossedentata, a medicinal and edible plant, has been widely used in China for hundreds of years, and dihydromyricetin is the main active ingredient responsible for its various biological actions. We investigated the effects of dihydromyricetin on glucose and lipid metabolism, inflammatory mediators and several biomarkers in nonalcoholic fatty liver disease. In a double-blind clinical trial, sixty adult nonalcoholic fatty liver disease patients were randomly assigned to receive either two dihydromyricetin or two placebo capsules (150 mg) twice daily for three months. The serum levels of alanine, aspartate aminotransferase, γ-glutamyl transpeptidase, glucose, low-density lipoprotein-cholesterol and apolipoprotein B, and the homeostasis model assessment of insulin resistance (HOMA-IR) index were significantly decreased in the dihydromyricetin group compared with the placebo group. In the dihydromyricetin group, the serum levels of tumor necrosis <em>factor</em>-alpha, cytokeratin-18 fragment and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> were decreased, whereas the levels of serum adiponectin were increased at the end of the study. We conclude that dihydromyricetin supplementation improves glucose and lipid metabolism as well as various biochemical parameters in patients with nonalcoholic fatty liver disease, and the therapeutic effects of dihydromyricetin are likely attributable to improved insulin resistance and decreases in the serum levels of tumor necrosis <em>factor</em>-alpha, cytokeratin-18, and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>.

Non-alcoholic fatty liver disease (NAFLD) includes a cluster of liver disorders ranging from simple fatty liver to non-alcoholic steatohepatitis (NASH) and cirrhosis. Due to its liver and vascular complications, NAFLD has become a public health problem with high morbidity and mortality. The pathogenesis of NAFLD is considered a "multi-hit hypothesis" that involves lipotoxicity, oxidative stress, endoplasmic reticulum stress, a chronic inflammatory state and mitochondrial dysfunction. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family with multiple metabolic functions. FGF<em>21</em> directly regulates lipid metabolism and reduces hepatic lipid accumulation in an insulin-independent manner. Several studies have shown that FGF<em>21</em> can ameliorate the "multi-hits" in the pathogenesis of NAFLD. The administration of FGF<em>21</em> reverses hepatic steatosis, counteracts obesity and alleviates insulin resistance in rodents and nonhuman primates. Using several strategies, we show that the reversal of simple fatty liver and NASH is mediated by activation of the FGF<em>21</em> signaling pathway. In this review, we describe the molecular mechanisms involved in the onset and/or progression of NAFLD, and review the current literature to highlight the therapeutic procedures associated with the FGF<em>21</em> signaling pathway for simple fatty liver and NASH, which are the two most important types of NAFLD.

Omentin-1, also known as intelectin-1, is a recently identified novel adipocytokine of 313 amino acids, which is expressed in visceral (omental and epicardial) fat as well as mesothelial cells, vascular cells, airway goblet cells, small intestine, colon, ovary, and plasma. The level of omentin-1 expression in (pre)adipocytes is decreased by glucose/insulin and stimulated by <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> and dexamethasone. Several lines of experimental evidence have shown that omentin-1 plays crucial roles in the maintenance of body metabolism and insulin sensitivity, and has anti-inflammatory, anti-atherosclerotic, and cardiovascular protective effects via AMP-activated protein kinase/Akt/nuclear <em>factor</em>-κB/mitogen-activated protein kinase (ERK, JNK, and p38) signaling. Clinical studies have indicated the usage of circulating omentin-1 as a biomarker of obesity, metabolic disorders including insulin resistance, diabetes, and metabolic syndrome, and atherosclerotic cardiovascular diseases. It is also possible to use circulating omentin-1 as a biomarker of bone metabolism, inflammatory diseases, cancers, sleep apnea syndrome, preeclampsia, and polycystic ovary syndrome. Decreased omentin-1 levels are generally associated with these diseases. However, omentin-1 increases to counteract the acute phase after onset of these diseases. These findings indicate that omentin-1 may be a negative risk <em>factor</em> for these diseases, and also act as an acute-phase reactant by its anti-inflammatory and atheroprotective effects. Therapeutic strategies to restore omentin-1 levels may be valuable for the prevention or treatment of these diseases. Weight loss, olive oil-rich diet, aerobic training, and treatment with atorvastatin and antidiabetic drugs (metformin, pioglitazone, and exenatide) are effective means of increasing circulating omentin-1 levels. This review provides insights into the potential use of omentin-1 as a biomarker and therapeutic target for these diseases. © 2017 American Physiological Society. Compr Physiol 7:765-781, 2017.

Type 2 diabetes is frequently complicated with atherogenic dyslipidemia. This study aimed to evaluate the efficacy and safety of pemafibrate (K-877) in patients with type 2 diabetes comorbid with hypertriglyceridemia.

Patients were randomly assigned to three groups and received placebo (n = 57), 0.2 mg/day pemafibrate (n = 54), or 0.4 mg/day pemafibrate (n = 55) for 24 weeks (treatment period 1). Subsequently, the patients received follow-up treatment for another 28 weeks (treatment period 2), in which the placebo was switched to 0.2 mg/day pemafibrate. This article presents the results of treatment period 1, which were the primary objectives.

The pemafibrate groups showed significantly reduced fasting serum triglyceride levels by ∼45% compared with the placebo group (P < 0.001). Additionally, the pemafibrate groups displayed significant decreases in non-HDL and remnant lipoprotein cholesterol, apolipoprotein (Apo) B100, ApoB48, and ApoCIII levels and significant increases in HDL cholesterol and ApoA-I levels. LDL cholesterol levels were not considerably altered in the pemafibrate groups. Furthermore, the 0.2 mg/day pemafibrate group showed a significantly reduced HOMA-insulin resistance score compared with the placebo group; however, no significant changes compared with placebo were found in fasting plasma glucose, fasting insulin, glycoalbumin, or HbA1c levels. The pemafibrate groups also showed significantly increased <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> levels compared with the placebo group. All groups displayed comparable rates of adverse events and drug reactions.

Pemafibrate significantly ameliorated lipid abnormalities and was well tolerated in patients with type 2 diabetes comorbid with hypertriglyceridemia.

In recent years, an important secretory role of brown adipose tissue (BAT) has emerged, which is consistent, to some extent, with the earlier recognition of the important secretory role of white fat. The so-called brown adipokines or "batokines" may play an autocrine role, which may either be positive or negative in the thermogenic function of brown adipocytes. Additionally, there is a <em>growing</em> recognition of the signalling molecules released by brown adipocytes that target sympathetic nerve endings (such as neuregulin-4 and S100b protein), vascular cells (e.g., bone morphogenetic protein-8b), and immune cells (e.g., C-X-C motif chemokine ligand-14) to promote the tissue remodelling associated with the adaptive BAT recruitment in response to thermogenic stimuli. Moreover, existing indications of an endocrine role of BAT are being confirmed through the release of brown adipokines acting on other distant tissues and organs; a recent example is the recognition that BAT-secreted <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> and myostatin target the heart and skeletal muscle, respectively. The application of proteomics technologies is aiding the identification of new members of the brown adipocyte secretome, such as the extracellular matrix or complement system components. In summary, BAT can no longer be considered a mere producer of heat in response to environment or dietary challenges; it is also an active secretory tissue releasing brown adipokines with a relevant local and systemic action. The identification of the major brown adipokines and their roles is highly important for the discovery of novel candidates useful in formulating intervention strategies for metabolic diseases.

OBJECTIVE

Micro ribonucleic acid (miR) expression is altered in urologic malignancies, including bladder cancer (BC). Individual miRs have been shown to modulate multiple signaling pathways that contribute to BC. We reviewed the primary literature on the role of miRs in BC; we provide a general introduction to the processing, regulation, and function of miRs as tumor suppressors and oncogenes and critically evaluate the literature on the implications of altered miR expression in BC.

METHODS

We searched the English language literature for original and review articles in PubMed from 1993 to March 2013, using the terms "microRNA" and "bladder cancer," "transitional cell carcinoma," or "urothelial carcinoma." This search yielded 133 unique articles with more than 85% of them published within the last 3 years.

RESULTS

To date, the majority of miR studies in BC use profiling to describe dynamic changes in miR expression across stage and grade. Generalized down-regulation of miRs, including those that target the <em>fibroblast</em> <em>growth</em> <em>factor</em> 3 pathway, such as miR-145, miR-101, miR-100, and miR-99a, has been observed in low-grade, non-muscle invasive BC. In contrast, generalized increased expression of miRs is observed in high-grade, muscle-invasive BC compared with adjacent normal bladder urothelium, including miRs predicted to target p53, such as miR-<em>21</em> and miR-373. Furthermore, p53 suppresses transcriptional <em>factors</em> that promote mesenchymal differentiation, ZEB-1 and ZEB-2, through regulation of the miR200 family.

CONCLUSIONS

Aberrations in miR expression identified between non-muscle invasive BC and muscle-invasive BC provide insight into the molecular alterations known to distinguish the two parallel pathways of bladder carcinogenesis. The heterogeneity of tumor specimens and research methods limits the reproducibility of changes in miR expression profiles between studies and underscores the importance of in vivo validation in a field that utilizes in silico miR target-prediction models.

Ketogenesis and ketolysis are central metabolic processes activated during the response to fasting. Ketogenesis is regulated in multiple stages, and a nuclear receptor peroxisome proliferator activated receptor α (PPARα) is one of the key transcription <em>factors</em> taking part in this regulation. PPARα is an important element in the metabolic network, where it participates in signaling driven by the main nutrient sensors, such as AMP-activated protein kinase (AMPK), PPARγ coactivator 1α (PGC-1α), and mammalian (mechanistic) target of rapamycin (mTOR) and induces hormonal mediators, such as <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>). This work describes the regulation of ketogenesis and ketolysis in normal and malignant cells and briefly summarizes the positive effects of ketone bodies in various neuropathologic conditions.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is an endocrine hormone that is primarily expressed in the liver and exerts beneficial effects on obesity and related metabolic diseases. In addition to its remarkable pharmacologic actions, the physiological roles of FGF<em>21</em> include the maintenance of energy homeostasis in the body in conditions of metabolic or environmental stress. The expression of FGF<em>21</em> is induced in multiple organs in response to diverse physiological or pathological stressors, such as starvation, nutrient excess, autophagy deficiency, mitochondrial stress, exercise, and cold exposure. Thus, the FGF<em>21</em> induction caused by stress plays an important role in adaptive response to these stimuli. Here, we highlight our current understanding of the functional importance of the induction of FGF<em>21</em> by diverse stressors as a feedback mechanism that prevents excessive stress.

A key tenet of tissue engineering is the principle that the scaffold can perform the dual roles of biomechanical and biochemical support through presentation of the appropriate mediators to surrounding tissue. While <em>growth</em> <em>factors</em> have been incorporated into scaffolds to achieve sustained release, there are a limited number of studies investigating release of biologically active molecules from reactive two-component polymers, which have potential application as injectable delivery systems. In this study, we report the sustained release of platelet-derived <em>growth</em> <em>factor</em> (PDGF) from a reactive two-component polyurethane. The release of PDGF was bi-phasic, characterized by an initial burst followed by a period of sustained release for up to <em>21</em> days. Despite the potential for amine and hydroxyl groups in the protein to react with the isocyanate groups in the reactive polyurethane, the in vitro bioactivity of the released PDGF was largely preserved when added as a lyophilized powder. PUR/PDGF scaffolds implanted in rat skin excisional wounds accelerated wound healing relative to the blank PUR control, resulting in almost complete healing with reepithelization at day 14. The presence of PDGF attracted both <em>fibroblasts</em> and mononuclear cells, significantly accelerating degradation of the polymer and enhancing formation of new granulation tissue as early as day 3. The ability of reactive two-component PUR scaffolds to promote new tissue formation in vivo through local delivery of PDGF may present compelling opportunities for the development of novel injectable therapeutics.

Pulmonary arterial hypertension (PAH) is characterized by widespread loss of pulmonary microvasculature. Therefore we hypothesized that angiogenic gene therapy would reverse established PAH, in part restoring the lung microcirculation. Three weeks after monocrotaline (MCT) treatment, Fisher 344 rats were randomized to receive a total of either 1.5 x 10(6) syngeneic <em>fibroblasts</em> (FB) transfected with vascular endothelial <em>growth</em> <em>factor</em> A (VEGF), endothelial NO synthase (eNOS), or null-plasmid transfected FBs. Right ventricular systolic pressure (RVSP) was similarly increased in all MCT-treated groups at the time of gene transfer. Animals receiving the null-vector progressed to severe PAH by Day 35 (P < 0.001). In contrast, eNOS gene transfer significantly reduced RVSP at Day 35 compared with Day <em>21</em>, whereas VEGF prevented further increases in RVSP over the subsequent 2 wk but did not reverse established PAH. RV hypertrophy was significantly reduced in both the eNOS-treated and VEGF-treated groups compared with the null-transfected controls. Fluorescent microangiography revealed widespread occlusion of the pre-capillary arterioles <em>21</em> d after MCT treatment, and animals receiving eNOS gene transfer exhibited the greatest improvement in the arteriolar architecture and capillary perfusion at Day 35. Cell-based eNOS gene transfer was more effective than VEGF in reversing established PAH, associated with evidence of regeneration of pulmonary microcirculation.

Tissue repair is driven by migratory macrophages and <em>fibroblasts</em> that infiltrate injury sites and secrete <em>growth</em> <em>factors</em>. We now report the enhancement of skeletal muscle repair by targeting transgene delivery to these repair cells using matrix-immobilized gene vectors. Plasmid and adenovirus vectors immobilized in collagen-gelatin admixtures were delivered to excisional muscle wounds, and when encoding either <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2) or FGF6 transgenes, produced early angiogenic responses that subsequently remodeled into arteriogenesis. FGF2 gene delivery enhanced the number of CD31(+) endothelial cells present at treatment sites>> 6-fold by day 14, and muscular arteriole density up to 11-fold by day <em>21</em> (P<0.0001). Muscle repair was also enhanced, as FGF gene-treated wounds filled with regenerating myotubes expressing the marker CD56 (an average 20-fold increase in CD56 expression versus controls, P<0.0001). These responses required transfection of a threshold level of repair cells, achievable only in injured muscles, and were transgene-driven, as neither platelet-derived <em>growth</em> <em>factor</em>-B (PDGFB) gene nor FGF2 protein delivery produced equivalent responses. In conclusion, using biomatrices to direct gene delivery to repair cells allows for relatively complex regenerative processes such as arteriogenesis and myogenesis, and therefore represents a promising approach to treating injured and ischemic muscle.

OBJECTIVE

Reports of increased circulating <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) levels in obesity indicate that FGF<em>21</em> may be implicated in body weight homeostasis. We sought to investigate the existence of FGF<em>21</em> in human cerebrospinal fluid (CSF) and, if present, the relationship between CSF FGF<em>21</em> with body adiposity and metabolic parameters.

METHODS

CSF and corresponding plasma FGF<em>21</em> were measured by an enzyme-linked immunosorbent assay (18 men and 20 women, aged 19-80 years, and BMI 16.2-38.1 kg/m(2)) and correlated to body adiposity and metabolic parameters.

RESULTS

CSF and plasma FGF<em>21</em> increased in particular with rising BMI and fat mass. In CSF, FGF<em>21</em> was detectable at concentrations ~40% that of plasma levels. CSF and plasma FGF<em>21</em> levels were significantly positively correlated with BMI and fat mass, body weight, plasma insulin, and homeostasis model assessment of insulin resistance. Plasma FGF<em>21</em> levels were significantly negatively correlated with plasma adiponectin. When subjected to multiple regression analysis, only fat mass was predictive of plasma FGF<em>21</em> (β = 0.758; P = 0.004) and CSF FGF<em>21</em> (β = 0.767; P = 0.007). The CSF-to-plasma FGF<em>21</em> ratio was significantly negatively correlated with BMI, fat mass, and plasma FGF<em>21</em>. Subjects in the highest plasma FGF<em>21</em> quintile had a lower CSF-to-plasma FGF<em>21</em> ratio (12.7% [9.7-14.9%]) compared with those in the lowest plasma FGF<em>21</em> quintile (94.7% [37.3-99.8%]) (P < 0.01).

CONCLUSIONS

Our observations have important implications with respect to the potential central actions of FGF<em>21</em>. Future research should seek to clarify whether FGF<em>21</em> would be beneficial in the management of obesity and its metabolic complications.

BACKGROUND

The excess and persistent accumulation of <em>fibroblasts</em> due to aberrant tissue repair results in fibrotic diseases such as idiopathic pulmonary fibrosis. Recent reports have revealed significant changes in microRNAs during idiopathic pulmonary fibrosis and evidence in support of a role for microRNAs in myofibroblast differentiation and the epithelial-mesenchymal transition in the context of fibrosis. It has been reported that microRNA-<em>21</em> is up-regulated in myo<em>fibroblasts</em> during fibrosis and promotes transforming <em>growth</em> <em>factor</em>-beta signaling by inhibiting Smad7. However, expression changes in microRNA-<em>21</em> and the role of microRNA-<em>21</em> in epithelial-mesenchymal transition during lung fibrosis have not yet been defined.

METHODS

Lungs from saline- or bleomycin-treated C57BL/6 J mice and lung specimens from patients with idiopathic pulmonary fibrosis were analyzed. Enzymatic digestions were performed to isolate single lung cells. Lung epithelial cells were isolated by flow cytometric cell sorting. The expression of microRNA-<em>21</em> was analyzed using both quantitative PCR and in situ hybridization. To induce epithelial-mesenchymal transition in culture, isolated mouse lung alveolar type II cells were cultured on fibronectin-coated chamber slides in the presence of transforming <em>growth</em> <em>factor</em>-β, thus generating conditions that enhance epithelial-mesenchymal transition. To investigate the role of microRNA-<em>21</em> in epithelial-mesenchymal transition, we transfected cells with a microRNA-<em>21</em> inhibitor. Total RNA was isolated from the freshly isolated and cultured cells. MicroRNA-<em>21</em>, as well as mRNAs of genes that are markers of alveolar epithelial or mesenchymal cell differentiation, were quantified using quantitative PCR.

RESULTS

The lung epithelial cells isolated from the bleomycin-induced lung fibrosis model system had decreased expression of epithelial marker genes, whereas the expression of mesenchymal marker genes was increased. MicroRNA-<em>21</em> was significantly upregulated in isolated lung epithelial cells during bleomycin-induced lung fibrosis and human idiopathic pulmonary fibrosis. MicroRNA-<em>21</em> was also upregulated in the cultured alveolar epithelial cells under the conditions that enhance epithelial-mesenchymal transition. Exogenous administration of a microRNA-<em>21</em> inhibitor prevented the increased expression of vimentin and alpha-smooth muscle actin in cultured primary mouse alveolar type II cells under culture conditions that induce epithelial-mesenchymal transition.

CONCLUSIONS

Our experiments demonstrate that microRNA-<em>21</em> is increased in lung epithelial cells during lung fibrosis and that it promotes epithelial-mesenchymal transition.

Myocardial ischemia (MI) activates innate cardioprotective mechanisms, enhancing cardiomyocyte tolerance to ischemia. Here, we report a MI-activated liver-dependent mechanism for myocardial protection. In response to MI in the mouse, hepatocytes exhibited 6- to 19-fold upregulation of genes encoding secretory proteins, including α-1-acid glycoprotein (AGP)2, bone morphogenetic protein-binding endothelial regulator (BMPER), chemokine (C-X-C motif) ligand 13, <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>21</em>, neuregulin (NRG)4, proteoglycan 4, and trefoil <em>factor</em> (TFF)3. Five of these proteins, including AGP2, BMPER, FGF<em>21</em>, NRG4, and TFF3, were identified as cardioprotective proteins since administration of each protein significantly reduced the fraction of myocardial infarcts (37 ± 9%, 34 ± 7%, 32 ± 8%, 39 ± 6%, and 31 ± 7%, respectively, vs. 48 ± 7% for PBS at 24 h post-MI). The serum level of the five proteins elevated significantly in association with protein upregulation in hepatocytes post-MI. Suppression of a cardioprotective protein by small interfering (si)RNA-mediated gene silencing resulted in a significant increase in the fraction of myocardial infarcts, and suppression of all five cardioprotective proteins with siRNAs further intensified myocardial infarction. While administration of a single cardioprotective protein mitigated myocardial infarction, administration of all five proteins furthered the beneficial effect, reducing myocardial infarct fractions from PBS control values from 46 ± 6% (5 days), 41 ± 5% (10 days), and 34 ± 4% (30 days) to 35 ± 5%, 28 ± 5%, and 24 ± 4%, respectively. These observations suggest that the liver contributes to cardioprotection in MI by upregulating and releasing protective secretory proteins. These proteins may be used for the development of cardioprotective agents.

A newly identified member of the tumor necrosis <em>factor</em> receptor (TNFR) superfamily shows activities associated with osteoclastogenesis inhibition and <em>fibroblast</em> proliferation. This new member, called TR1, was identified from a search of an expressed sequence tag database, and encodes 401 amino acids with a <em>21</em>-residue signal sequence. Unlike other members of TNFR, TR1 does not contain a transmembrane domain and is secreted as a 62 kDa glycoprotein. TR1 gene maps to chromosome 8q23-24.1 and its mRNA is abundantly expressed on primary osteoblasts, osteogenic sarcoma cell lines, and primary <em>fibroblasts</em>. The receptors for TR1 were detected on a monocytic cell line (THP-1) and in human <em>fibroblasts</em>. Scatchard analyses indicated two classes of high and medium-high affinity receptors with a kD of approximately 45 and 320 pM, respectively. Recombinant TR1 induced proliferation of human foreskin <em>fibroblasts</em> and potentiated TNF-induced proliferation in these cells. In a coculture system of osteoblasts and bone marrow cells, recombinant TR1 completely inhibited the differentiation of osteoclast-like multinucleated cell formation in the presence of several bone-resorbing <em>factors</em>. TR1 also strongly inhibited bone-resorbing function on dentine slices by mature osteoclasts and decreased 45Ca release in fetal long-bone organ cultures. Anti-TR1 monoclonal antibody promoted the formation of osteoclasts in mouse marrow culture assays. These results indicate that TR1 has broad biological activities in <em>fibroblast</em> <em>growth</em> and in osteoclast differentiation and its functions.