We tested the hypothesis that atrial fibrosis and atrial fibrillation (AF) evoked by angiotensin II (AII) could be prevented by the induction of heat-shock protein 72 (HSP72) by hyperthermia (HT). In cultured atrial <em>fibroblasts</em> isolated from male Sprague-Dawley rats, HT (42 degrees C) was applied for 30 min. AII (100 nmol/L) was added to the medium 8 h later. HT induced the expression of HSP72, which was associated with the attenuation of AII-induced extracellular signal-regulated kinase (ERK1/ERK2) phosphorylation, alpha-smooth muscle actin (alpha-SMA) expression, transforming <em>growth</em> <em>factor</em>-beta(1) secretion, collagen synthesis, and expression of collagen type I and tissue inhibitor of metalloproteinases-1. A small interfering RNA targeting HSP72 abolished these anti-fibrotic effects of HT. In male Sprague-Dawley rats in vivo, an osmotic mini-pump was subcutaneously implanted for continuous infusion of AII (400 ng/kg/min). Whole-body HT (43 degrees C, 20 min) was applied 24 h before and 7, 14, and <em>21</em> days after the start of the AII infusion. Repeated HT led to the induction of HSP72 expression, which resulted in an attenuation of AII-induced left atrial fibrosis. In an electrophysiological study using isolated perfused heart, continuous AII caused slowing of interatrial conduction without affecting atrial refractoriness. In AII-treated hearts, extrastimuli from the right atrial appendage resulted in a high incidence of repetitive atrial responses, which were suppressed by treatment with HT. Our results suggest that HT treatment is effective in suppressing AII-mediated atrial fibrosis and AF via induction of HSP72 at least in parts, and is thus expected to be a novel strategy for prevention of AF.

OBJECTIVE

Hepatokine <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em> takes part in the regulation of lipid metabolism in the liver and adipose tissue. We investigated whether exendin-4 regulates the expression of FGF<em>21</em> in the liver, and whether the effects of exendin-4 on the regulation of FGF<em>21</em> expression are mediated via silent mating type information regulation 2 homolog (SIRT) 1 or SIRT6.

METHODS

The C57BL/6J mice were fed a low fat diet, high fat diet, or high fat diet with 1 nmol/kg/day exendin-4 intraperitoneal injection for 10 weeks. HepG2 used in vitro study was treated with palmitic aicd (0.4 mM) with or without exendin-4 (100 nM) and FGF<em>21</em> (50 nM) for 24 hours. The change of FGF<em>21</em> and its receptors expression by exendin-4 were measured using quantitative real-time RT-PCR and Western blot. The intracellular lipid content in HepG2 cells was evaluated by Oil Red O staining. Inhibition of FGF<em>21</em>, SIRT1 and SIRT6, by 10 nM siRNA was performed to establish the signaling pathway of exendin-4 action in hepatic lipid metabolism.

RESULTS

Exendin-4 increased the expression of FGF<em>21</em> and its receptors in high fat diet-induced obese mice. In addition, recombinant FGF<em>21</em> treatment reduced lipid content in palmitic acid-treated HepG2 cells. We also observed significantly decreased expression of peroxisomal proliferator-activated receptor (PPAR) α and medium-chain acyl-coenzyme A dehydrogenase (MCAD) in hepatocytes transfected with FGF<em>21</em> siRNA. In cells treated with exendin-4, inhibition of SIRT1, but not SIRT6, by siRNA significantly repressed the expression of FGF<em>21</em> mRNA, whereas decreased SIRT1 expression by inhibition of FGF<em>21</em> was not observed.

CONCLUSIONS

These data suggest that exendin-4 could improve fatty liver by increasing SIRT1-mediated FGF<em>21</em>.

In order to elucidate the effects of the different basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) isoforms on vascular smooth muscle, we examined aorta-derived vascular smooth muscle cells from transgenic mice expressing the human isoforms of bFGF. Four cell lines were examined from mice in which transgene expression was driven by the ubiquitous phosphoglycerate kinase promoter. Overexpression and cellular localization was confirmed by Western blot analysis in vascular smooth muscle cells from mice expressing: all four human bFGF isoforms (24, 22, <em>21</em>, and 18 kDa); all three nuclear targeted isoforms (24, 22, and <em>21</em> kDa); only the 24 kDa isoform; and the only secreted/non-nuclear targeted isoform, 18 kDa. All lines showed approximate four-fold increases in bFGF expression, nuclear localization of all nuclear targeted bFGF isoforms, and cytosolic localization of only the 18 kDa bFGF. Measurement of [3H]thymidine incorporation into quiescent cells stimulated with increasing concentrations of serum, showed increased DNA synthesis in cell lines expressing any bFGF isoform when compared to non-transgenic control cells, and a further increase in DNA synthesis in cells expressing the nuclear targeted isoforms (24, 22, and <em>21</em> kDa) over the 18 kDa bFGF expressing cell line at any concentration of serum. All cells showed equal label incorporation when stimulated with 10 ng/ml of platelet-derived <em>growth</em> <em>factor</em> confirming an equal potential for DNA synthesis. Neutralizing the bFGF antibody markedly decreased serum-stimulated DNA synthesis, but only in the cell lines overexpressing the secreted/non-nuclear targeted 18 kDa isoform. These results suggest amplification of DNA synthesis through synergistic intracrine and autocrine effects of the nuclear targeted and non-nuclear targeted bFGF isoforms in vascular smooth muscle cells.

A method for establishing primary cultures of smooth muscle cells (SMCs) from the porcine coronary artery without either microdissection and/or enzymatic dispersion was developed using selective migration of cells from coronary explants in vitro. This culture method relies on the heterogeneity of cell types and differences in their migration and adherence ability to separate SMC from contaminating <em>fibroblasts</em> or endothelial cells. The cell type was determined by immunohistochemical staining with monoclonal antibodies to SM alpha-actin, SM myosin, h-caldesmon and von Willebrand <em>factor</em>. The first wave of migration (1-7 days) consisted of a mixture of <em>fibroblasts</em> and SMCs. Only SMCs were present in the second wave of migration (7-14 days). Endothelial cells, which exhibited a lower capacity for migration and adherence, were restricted to the third wave of migration (14-<em>21</em> days). Cells obtained from the second wave of migration exhibited the characteristic single-layered, aligned, hill-and-valley pattern of SMCs when confluent. Quiescence was attained 4-5 days after removal of serum, as established by [3H]-thymidine incorporation. Stimulation of the quiescent SMCs with 20% FBS resulted in a synchronous re-entry into the cell-cycle with S phase reached 15-18 h later. The SMCs prepared using this protocol thus exhibit the structural markers and capacity to undergo phenotypic modulation that are characteristic of SMCs in vivo. This approach to establishing primary cultures of SMCs offers the advantage of selecting for the subpopulation of cells capable of migration in response to injury or <em>growth</em> <em>factor</em> stimulation.

Protamine sulfate, an inhibitor of angiogenesis in vivo, markedly inhibits the ability of angiogenic <em>factors</em> such as acidic or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF, bFGF) to stimulate the proliferation in vitro of either BHK-<em>21</em> cells or vascular endothelial cells. The inhibition is reversible, and cells remain viable even after prolonged exposure to protamine sulfate. Protamine sulfate inhibits the mitogenic effects of both <em>growth</em> <em>factors</em> by preventing them from binding to their common cell surface receptors. It also inhibits the mitogenic activity of the extracellular matrix produced by bovine corneal endothelial cells. This substrate has been shown in previous studies to replace the requirement for FGF of many cell types. In contrast, protamine sulfate potentiates the mitogenic activity of epidermal <em>growth</em> <em>factor</em> (EGF). This indicates that protamine sulfate also acts at cellular sites which are not associated with FGF receptors.

Tumor necrosis <em>factor</em>-alpha (TNF-alpha) has been shown to enhance the synthesis of interleukin-6 (IL-6) and collagenase in human omental microvascular endothelial (HOME) cell (Mawatari, M., Kohno, K., Mizoguchi, H., Matsuda, T., Asoh, K., Van Damme, J. V., Welgus, H. G., and Kuwano, M. (1989) J. Immunol. 143, 1619-1627). In the present study, we have examined whether the TNF-alpha-induced synthesis of IL-6 or collagenase in HOME cells is mediated by an inducible <em>growth</em> <em>factor</em>. Among several <em>growth</em> <em>factors</em> examined, we found that the expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) mRNA was the one most prominently enhanced when HOME cells were treated with TNF-alpha. The increase of bFGF mRNA by TNF-alpha in HOME cells was observed in both a dose- and time-dependent manner when assayed by Northern blot analysis. The induction of bFGF mRNA was observed by 3 h after incubation with TNF-alpha, and the maximal increase of 5-fold was obtained after 12 h of incubation with 100 units/ml TNF-alpha. Western blot analysis confirmed the enhanced synthesis of bFGF by TNF-alpha. Metabolic labeling and immunoprecipitation assays of bFGF showed that exposure to TNF-alpha enhanced secretion of bFGF into culture medium and also that TNF-alpha stimulated the production of bFGF molecules with molecular masses of 18, <em>21</em>, and 22.5 kDa in HOME cells. TNF-alpha induced the expression of collagenase mRNA and IL-6 mRNA in HOME cells as well, and the coadministration of neutralizing anti-bFGF antibody almost completely blocked the effects of TNF-alpha. The treatment of HOME cells with exogenous bFGF significantly stimulated the expression of collagenase mRNA and IL-6 mRNA in HOME cells. Therefore, the biological effects of TNF-alpha on HOME cells may be mediated, at least in part, by TNF-alpha-induced bFGF.

Experiments were carried out to determine the specificity of <em>growth</em> <em>factor</em> action on maturation of the oocyte-cumulus cell complex in vitro. Cumulus cell-enclosed oocytes (CEO) from primed mice were maintained in meiotic arrest in vitro with hypoxanthine (HX) and treated with one of ten different <em>growth</em>-promoting <em>factors</em>. The percentage of germinal vesicle breakdown (GVB) in the HX controls ranged from 44 to 64.7% after <em>21</em>-22 h. Oocytes responded to treatment with <em>growth</em>-promoting <em>factors</em> in one of three ways: (1) no response; (2) low response; or (3) high response. The nonresponding groups included transforming <em>growth</em> <em>factor</em>-beta, platelet-derived <em>growth</em> <em>factor</em>, bombesin, sodium orthovanadate, nerve <em>growth</em> <em>factor</em>, and insulin-like <em>growth</em> <em>factors</em> I and II, each of which had no statistically significant effect on GVB. Insulin and <em>fibroblast</em> <em>growth</em> <em>factor</em> were members of the low response group and stimulated increases in GVB of <em>21</em>.2 24.9%. Epidermal <em>growth</em> <em>factor</em> (EGF) was the only <em>factor</em> that produced a high frequency of maturation in the CEO; 100% of the arrested CEO were stimulated to undergo GVB in response to EGF treatment (a 51% increase over controls). No interaction was observed when EGF was tested with follicle-stimulating hormone (FSH) on hormone-induced GVB. When tested for an action on cumulus cell expansion, EGF was the only <em>growth</em>-promoting <em>factor</em> that triggered this response and did so more effectively than FSH. Heparin suppressed cumulus expansion in both EGF- and FSH-treated CEO, but did not prevent GVB stimulated by either hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in endogenous levels of the angiogenic proteins basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) were assessed in rabbit hindlimb muscles subjected to 1, 5, or <em>21</em> days of ischemia. In the glycolytic [tibialis anterior (TA)] and the oxidative [soleus (SOL)] muscles from the ischemic and contralateral (control) hindlimb, bFGF and VEGF protein expression was determined by ELISA and immunoblot analysis. Total VEGF protein expression was greater in oxidative than in glycolytic muscles after 5 days of hindlimb ischemia. In SOL muscle, total VEGF detected by ELISA in ischemic limbs was increased to 137, 300, and 220% of control at 1, 5, and <em>21</em> days, respectively. However, in TA, total VEGF expression by ELISA was increased only at 1 and 5 days of ischemia to 140 and 134% of control, respectively. By immunoblotting, the expression of the 165-amino acid isoform (VEGF(165)) was initially decreased to 55% of control in ischemic SOL at 1 day but was increased to 250% of control at day 5 and remained at 155% at day <em>21</em>. In TA, VEGF(165) was increased to 260% of control at 1 day of ischemia but only to 150% of control by day 5. The only significant change in bFGF expression in either the oxidative or glycolytic muscles was a small increase (129% of control) at <em>21</em> days in SOL. This study demonstrates that the magnitude and direction of change in VEGF protein expression depend on VEGF subtype, muscle fiber type, and duration of ischemia. These findings suggest that strategies in therapeutic angiogenesis may need to differ depending on muscle fiber type.

Ischemic heart disease is one of the leading causes of death. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a circulating <em>factor</em> with an anti-diabetic property. Skeletal muscle is an important source of FGF<em>21</em> production. Here, we investigated whether skeletal muscle-derived FGF<em>21</em> modulates cardiac remodeling in a murine model of myocardial infarction. Myocardial infarction was produced in C57BL/6J wild-type (WT) mice by the permanent ligation of the left anterior descending coronary artery (LAD). Adenoviral vectors expressing FGF<em>21</em> (Ad-FGF<em>21</em>) or control β-galactosidase were intramuscularly injected into mice at 3 days before permanent LAD ligation. Intramuscular injection of Ad-FGF<em>21</em> increased plasma FGF<em>21</em> levels in WT mice compared with control. Treatment of WT mice with Ad-FGF<em>21</em> led to improvement of left ventricular systolic dysfunction and dilatation at 2 weeks after LAD ligation. Ad-FGF<em>21</em> administration to WT mice also led to enhancement of capillary density in the infarct border zone, and reduction of myocyte apoptosis in the remote zone, which were accompanied by decreased expression of pro-inflammatory cytokines. Furthermore, treatment of WT mice with Ad-FGF<em>21</em> increased plasma levels of adiponectin, which is a cardioprotective adipokine. The beneficial effects of Ad-FGF<em>21</em> on cardiac dysfunction and inflammatory response after myocardial infarction were diminished in adiponectin-knockout mice. These data suggest that muscle-derived FGF<em>21</em> ameliorates adverse cardiac remodeling after myocardial infarction, at least in part, through an adiponectin-dependent mechanism.

Pemigatinib (PEMAZYRE™), a small molecule inhibitor of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) 1, FGFR2 and FGFR3, received accelerated approval in April 2020 in the USA for the treatment of adults with previously treated, unresectable, locally advanced or metastatic cholangiocarcinoma and a FGFR2 fusion or other rearrangement, as detected by a US FDA-approved test. Developed by Incyte Corporation, it is the first targeted treatment for cholangiocarcinoma in the USA. The recommended dosage of pemigatinib is 13.5 mg once daily, administered orally with or without food, on days 1-14 of a <em>21</em>-day cycle until disease progression or unacceptable toxicity. Pemigatinib received orphan designation for the treatment of myeloid/lymphoid neoplasms with eosinophilia and rearrangement of PDGFRA, PDGFRB or FGFR1, or with PCM1-JAK2 in August 2019 in the USA. A regulatory assessment for pemigatinib as a treatment for adults with locally advanced or metastatic cholangiocarcinoma and a FGFR2 fusion or rearrangement that is relapsed or refractory after ≥ 1 line of systemic therapy is underway in the EU. Pemigatinib is also undergoing clinical development in various countries worldwide for use in several other FGFR-driven malignancies (e.g. solid tumour, urothelial carcinoma). This article summarizes the milestones in the development of pemigatinib leading to this first approval for the treatment of adults with previously treated, unresectable, locally advanced or metastatic cholangiocarcinoma and a FGFR2 fusion or other rearrangement, as detected by a US FDA-approved test.

Left ventricular (LV) pressure overload is a major cause of heart failure. Transforming <em>growth</em> <em>factors</em>-β (TGF-βs) promote LV remodeling under biomechanical stress. BAMBI (BMP and activin membrane-bound inhibitor) is a pseudoreceptor that negatively modulates TGF-β signaling. The present study tests the hypothesis that BAMBI plays a protective role during the adverse LV remodeling under pressure overload. The subjects of the study were BAMBI knockout mice (BAMBI(-/-)) undergoing transverse aortic constriction (TAC) and patients with severe aortic stenosis (AS). We examined LV gene and protein expression of remodeling-related elements, histological fibrosis, and heart morphology and function. LV expression of BAMBI was increased in AS patients and TAC-mice and correlated directly with TGF-β. BAMBI deletion led to a gain of myocardial TGF-β signaling through canonical (Smads) and non-canonical (TAK1-p38 and TAK1-JNK) pathways. As a consequence, the remodeling response to pressure overload in BAMBI(-/-) mice was exacerbated in terms of hypertrophy, chamber dilation, deterioration of long-axis LV systolic function and diastolic dysfunction. Functional remodeling associated transcriptional activation of fibrosis-related TGF-β targets, up-regulation of the profibrotic micro-RNA-<em>21</em>, histological fibrosis and increased metalloproteinase-2 activity. Histological remodeling in BAMBI(-/-) mice involved TGF-βs. BAMBI deletion in primary cardiac <em>fibroblasts</em> exacerbated TGF-β-induced profibrotic responses while BAMBI overexpression in NIH-3T3 <em>fibroblasts</em> attenuated them. Our findings identify BAMBI as a critical negative modulator of myocardial remodeling under pressure overload. We suggest that BAMBI is involved in negative feedback loops that restrain the TGF-β remodeling signals to protect the pressure-overloaded myocardium from uncontrolled extracellular matrix deposition in humans and mice.

Two models of wound repair compared the effect of defined, recombinant <em>growth</em> <em>factors</em> on the rate of wound repair in both normal and streptozotocin-induced diabetic rats: subcutaneous implantation of polyvinyl alcohol sponges and incisional wounding. Transverse incisional wounds were made on the dorsal surface of rats and closed with steel sutures. Three days postwounding the rats received a single injection of either transforming <em>growth</em> <em>factor</em>-beta or vehicle alone directly into the wound site. Animals were sacrificed 7, 14, and <em>21</em> days postwounding, and fresh and formalin-fixed wound tensile strength were measured. Diabetic rats had expected defects in wound repair, including decreased granulation tissue and reduced amounts of collagen, protein, and DNA. Fresh tensile strength of the diabetic incisions was 53% of normal on Day 7 (p < or = .01) and 29% of normal on Day <em>21</em>. Fixed tensile strength was 41% of normal on Day 7 (p < or = .01) and fell to 78% of normal by Day <em>21</em> (p < or = .01), suggesting that collagen concentrations of diabetic wounds increased towards normal but did not undergo maturation. TGF beta produced a moderate increase in tensile strength of fresh and fixed wounds of diabetic rats, but not to the levels of wounds in untreated normal rats. Sponges fill with granulation tissue, their reproducible rate of organization being measured by histological and biochemical methods. A single injection into sponges 3 days postimplantation of basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-beta, or vehicle only, was evaluated at 7 and 9 days postimplantation. In the sponge model, bFGF and TGF beta were each able to induce significant increases in the accumulation of granulation tissue in both diabetic and normal rats. TGF beta increased the collagen content of sponges by 136% in sponges from diabetic animals (p < or = .001), thereby raising the collagen content to that of normal control wounds, while stimulating a 49% (p < or = .02) increase in sponges from normal animals on Day 9. By contrast, the response to bFGF was predominantly an increase in the protein and DNA content of the sponges. These results emphasize the differential effects of the two cytokines in accelerating healing under conditions of defective wound repair.

Iron-sulfur (Fe-S) clusters are ancient enzyme co<em>factor</em>s found in virtually all life forms. We evaluated the physiological effects of chronic Fe-S cluster deficiency in human skeletal muscle, a tissue that relies heavily on Fe-S cluster-mediated aerobic energy metabolism. Despite greatly decreased oxidative capacity, muscle tissue from patients deficient in the Fe-S cluster scaffold protein ISCU showed a predominance of type I oxidative muscle fibers and higher capillary density, enhanced expression of transcriptional co-activator PGC-1α and increased mitochondrial fatty acid oxidation genes. These Fe-S cluster-deficient muscles showed a dramatic up-regulation of the ketogenic enzyme HMGCS2 and the secreted protein FGF<em>21</em> (<em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>). Enhanced muscle FGF<em>21</em> expression was reflected by elevated circulating FGF<em>21</em> levels in the patients, and robust FGF<em>21</em> secretion could be recapitulated by respiratory chain inhibition in cultured myotubes. Our findings reveal that mitochondrial energy starvation elicits a coordinated response in Fe-S-deficient skeletal muscle that is reflected systemically by increased plasma FGF<em>21</em> levels.

OBJECTIVE

High intraglomerular pressure causes renal inflammation in experimental models of diabetes. Our objective was to determine whether renal hyperfiltration, a surrogate for intraglomerular hypertension, is associated with increased excretion of urinary cytokines/chemokines in patients with type 1 diabetes mellitus.

METHODS

Blood pressure, renal haemodynamic function (inulin and para-aminohippurate clearances for glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), respectively) and urine samples were obtained during clamped euglycaemia in individuals with type 1 diabetes with either hyperfiltration (GFR determined using inulin [GFRINULIN] ≥ 135 ml min⁻¹ 1.73 m⁻², n = 28) or normofiltration (n = <em>21</em>) and healthy control individuals (n = 18).

RESULTS

Baseline clinical characteristics, dietary sodium and protein intake and blood pressure levels were similar in the diabetic and healthy control groups. In addition, HbA1c levels were similar in the two diabetic groups. As expected baseline GFR was higher in hyperfilterers than either normofiltering diabetic patients or healthy control patients (165 ± 9 vs 113 ± 2 and 116 ± 4 ml min⁻¹ 1.73 m⁻², respectively, p < 0.01). ERPF and renal blood flow were also comparatively higher and renal vascular resistance was lower in hyperfiltering patients (p < 0.01). Hyperfiltering diabetic patients had higher excretion rates for eotaxin, IFNα2, macrophage-derived chemokine, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB and granulocyte-macrophage colony-stimulating factor (p ≤ 0.01). Urinary monocyte chemoattractant protein (MCP)-1 and RANTES (regulated on activation, normal T expressed and secreted) excretion was also higher in hyperfiltering vs normofiltering diabetic individuals (p < 0.01) and fibroblast growth factor-2, MCP-3 and CD40K excretion was elevated in hyperfiltering diabetic individuals vs healthy controls (p < 0.01).

CONCLUSIONS

Renal hyperfiltration is associated with increased urinary excretion of inflammatory cytokines/chemokines in patients with uncomplicated type 1 diabetes.

We developed an experimental approach with genetically engineered and encapsulated mouse NIH 3T3 <em>fibroblasts</em> to delay the progressive degeneration of photoreceptor cells in dark-eyed Royal College of Surgeons rats. These xenogeneic <em>fibroblasts</em> can survive in 1. 5-mm-long microcapsules made of the biocompatible polymer AN69 for at least 90 days under in vitro and in vivo conditions because of their stable transfection with the gene for the 18-kDa form of the human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (hFGF-2). Furthermore, when transferred surgically into the vitreous cavity of <em>21</em>-day-old Royal College of Surgeons rats, the microencapsulated hFGF-2-secreting <em>fibroblasts</em> provoked a local delay of photoreceptor cell degeneration, as seen at 45 days and 90 days after transplantation. This effect was limited to 2.08 mm2 (45 days) and 0.95 mm2 (90 days) of the retinal surface. In both untreated eyes and control globes with encapsulated hFGF-2-deficient <em>fibroblasts</em>, the rescued area (of at most 0.08 mm2) was significantly smaller at both time points. Although, in a few ocular globes, surgical trauma induced a reorganization of the retinal cytoarchitecture, neither microcapsule rejection nor hFGF-2-mediated tumor formation were detected in any treated eyes. These findings indicate that encapsulated <em>fibroblasts</em> secreting hFGF-2 or perhaps other agents can be applied as potential therapeutic tools to treat retinal dystrophies.

Injuries to avascular regions of menisci do not heal and result in significant discomfort to patients. Current treatments, such as partial meniscectomy, alleviate these symptoms in the short term but lead to premature osteoarthritis as a result of compromised stability and changes in knee biomechanics. Thus, tissue engineering of the meniscus may provide an alternative treatment modality to overcome this problem. In this experiment, a scaffold-based tissue-engineering approach was utilized to regenerate the meniscus. Meniscus cells were cultured on poly-L-lactic acid scaffolds in normoxic (approximately <em>21</em>% oxygen) or hypoxic (approximately 2% oxygen) conditions in the presence or absence of the <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). At t = 4 weeks, histological sections of constructs showed presence of collagen and glycosaminoglycan (GAG) in all groups. Immunohistochemical staining showed the presence of collagen I in all groups and collagen II in groups cultured under hypoxic conditions. bFGF in the culture medium significantly increased cell number/construct by 25%, regardless of culture conditions. For GAG/construct, synergistic increases were observed in constructs cultured in hypoxic conditions and bFGF (two-fold) when compared to constructs cultured in normoxic conditions. Compressive tests showed synergistic increases in the relaxation modulus and coefficient of viscosity and additive increases in the instantaneous modulus for constructs cultured under hypoxic conditions and bFGF, when compared to constructs cultured under normoxic conditions. Overall, these results demonstrate that bFGF and hypoxia can significantly enhance the ability of meniscus cells to produce GAGs and improve the compressive properties of tissue-engineered meniscus constructs in vitro.

OBJECTIVE

We hypothesised that serum <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>), a novel adipokine with postulated insulin-sensitizing effects, may be predictive of cardiovascular (CV) events in patients with type 2 diabetes (DM2) at a relatively short-term follow-up.

METHODS

Serum FGF-<em>21</em> levels were assessed in 87 DM2 patients, aged 57-66 years, with the median duration of diabetes of 10 years, who were referred to the Department of Endocrinology for routine annual metabolic assessment. During a follow-up of 24 months, overall mortality, CV mortality and CV nonfatal events were registered. Cox proportional hazards regression assessed adjusted differences in CV morbidity and mortality risk.

RESULTS

Patients stratified according to serum FGF-<em>21</em> levels ≤ and>> the median value of 240.7 pg/mL showed no significant differences at baseline in gender distribution, diabetes duration, insulin therapy, BMI, biochemical profiles and previous CV events. At 24-month follow-up, <em>21</em> (24.1%) patients experienced a nonfatal CV event. A significantly (P=0.0013) higher incidence of the combined end point of CV morbidity and mortality was observed in the FGF-<em>21</em>)240.7 pg/mL group. In the multivariate Cox proportional hazards regression model, the presence of FGF-<em>21</em>)the median value was associated with a significant increase in the risk of the combined end point of CV morbidity and mortality (HR: 4.7, 95% CI 1.67-13.24).

CONCLUSIONS

The obtained results support the prognostic value of FGF-<em>21</em> in DM2 and may provide a useful tool for stratification of CV prognosis in DM2 patients.

It has been demonstrated that circulating FGF21 levels are elevated in the serum and synovial fluid of patients with rheumatoid arthritis (RA). The aim of this study is to investigate efficacy of FGF21 for treatment of RA and the molecular mechanisms of the therapeutic effect on collagen-induced arthritis (CIA). Mice with CIA were subcutaneously administered with FGF21 (5, 2 or 1mg·kg(-1)·d(-1)), IL-1β antibody (5mg·kg(-1)·d(-1)), IL-17A antibody (5mg·kg(-1)·d(-1)) and dexamethasone (DEX) (1mg·kg(-1)·d(-1)), respectively. The effects of treatment were determined by arthritis severity score, histological damage and cytokine production. The activation of NF-κB was analyzed by Western blotting. We also detected the levels of oxidative stress parameters. Our results showed that FGF21 had beneficial effects on clinical symptom and histological lesion of CIA mice. Similar to antibody and DEX, FGF21 treatment alleviated the severity of arthritis by reducing humoral and cellular immune responses and down-regulating the expression of pro-inflammatory cytokines. FGF21 treatment also reduced the expression of TNF-α, IL-1β, IL-6, IFN-γ and MMP-3 and increased level of IL-10 in the spleen tissue or the plasma of CIA mice in a dose-dependent manner. Furthermore, FGF21 inhibited IκBα degradation and NF-κB p65 nuclear translocation and induced significant changes of oxidative stress parameters (MDA, SOD, CAT, GSH-PX and GSH) in the plasma. FGF21 exerts therapeutic efficacy for RA through antioxidant reaction and inhibiting NF-κB inflammatory pathway. This study provides evidence that FGF21 may be a promising therapeutic agent for RA patients.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has been identified as a novel metabolic regulator. This cross-sectional study was performed to clarify how serum FGF<em>21</em> levels were associated with clinical parameters in Japanese subjects with type 2 diabetes (n=139). Anthropometric and blood biochemical parameters, uses of drugs for diabetes, hypertension and dyslipidemia were examined regarding associations with fasting serum FGF<em>21</em> concentrations. FGF<em>21</em> levels were 6-times higher in those subjects taking fibrates. However, a use of thiazolidinediones did not affect serum FGF<em>21</em> levels while it induced higher serum adiponectin levels. In univariate analyses, FGF<em>21</em> levels showed associations with a use of fibrates, triglyceride levels, creatinine levels, waist circumference, and BMI. Multiple regression analyses adjusted for age, gender and BMI showed that a use of fibrates, triglyceride levels and creatinine levels were strong contributors to serum FGF<em>21</em> levels. In contrast, a use of thiazolidinediones, HDL-cholesterol levels and fasting insulin levels were strong contributors to serum adiponectin levels. This study revealed that serum FGF<em>21</em> levels were biochemical indicators correlating to a set of essential metabolic parameters, which was distinct from that correlating to serum adiponectin levels in subjects with type 2 diabetes.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a potent endocrine regulator with physiological effects on glucose and lipid metabolism and thus garners much attention for its translational potential for the management of obesity and related metabolic syndromes. FGF<em>21</em> is mainly expressed in several metabolically active tissue organs, such as the liver, adipose tissue, skeletal muscle, and pancreas, with profound effects and therapeutic relevance. Emerging experimental and clinical data point to the demonstrated metabolic benefits of FGF<em>21</em>, which include, but are not limited to, weight loss, glucose and lipid metabolism, and insulin sensitivity. In addition, FGF<em>21</em> also acts directly through its coreceptor β-klotho in the brain to alter light-dark cycle activity. In this review, we critically appraise current advances in understanding the physiological actions of FGF<em>21</em> and its role as a biomarker of various metabolic diseases, especially type 2 diabetes mellitus. We also discuss the potentially exciting role of FGF<em>21</em> in improving our health and prolonging our life span. This information will provide a fuller understanding for further research into FGF<em>21</em>, as well as providing a scientific basis for potentially establishing health care guidelines for this promising molecule.

<AbstractText>Global consumption of protein per capita is rising, while rates of infertility are increasing. However, a clear relationship between protein intake and reproductive health has not been demonstrated. The activation of the quiescent primordial follicles is the first step of folliculogenesis, and their activation must be tightly controlled to prevent premature exhaustion of the ovarian follicular reserve.</AbstractText><AbstractText>The primordial follicle reserve of wild-type or liver-specific ablation of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) in mice, subjected to limited or excessive protein diets or oral gavage test, were detected in vivo. Mouse ovary organ cultures were used to examine the direct role of metabolites or metabolic hormones on primordial follicle activation.</AbstractText><AbstractText>Mouse primordial follicle activation, was reduced by restricted protein intake and was accelerated by excessive protein intake, in an ovarian mTORC1 signaling-dependent manner. Furthermore, restricted or excessive protein intake resulted in an augmentation or decline of oocyte number and fertility at older age, respectively. Liver-specific ablation of FGF<em>21</em>, which resulted in a reduction of 87% in circulating FGF<em>21</em>, abrogated the preserving effect of low-protein intake on primordial follicle pool. Interestingly, FGF<em>21</em> had no direct effect on the activation of primordial follicles, but instead required an adipokine adiponectin. Moreover, AdipoRon, an oral adiponectin receptor agonist, prevented the over-activation effect of excessive protein intake on primordial follicle activation.</AbstractText><AbstractText>Dietary protein consumption controlled ovarian primordial follicle reserve and fertility, which required coordination between FGF<em>21</em> and adiponectin. FUND: Natural Science Foundation of China (Grant 31772616).</AbstractText>

The antileukemic agent asparaginase triggers the amino acid response (AAR) in the liver by activating the eukaryotic initiation <em>factor</em> 2 (eIF2) kinase general control nonderepressible 2 (GCN2). To explore the mechanism by which AAR induction is necessary to mitigate hepatic lipid accumulation and prevent liver dysfunction during continued asparaginase treatment, wild-type and Gcn2 null mice were injected once daily with asparaginase or phosphate buffered saline for up to 14 days. Asparaginase induced mRNA expression of multiple AAR genes and greatly increased circulating concentrations of the metabolic hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) independent of food intake. Loss of Gcn2 precluded mRNA expression and circulating levels of FGF<em>21</em> and blocked mRNA expression of multiple genes regulating lipid synthesis and metabolism including Fas, Ppara, Pparg, Acadm, and Scd1 in both liver and white adipose tissue. Furthermore, rates of triglyceride export and protein expression of apolipoproteinB-100 were significantly reduced in the livers of Gcn2 null mice treated with asparaginase, providing a mechanistic basis for the increase in hepatic lipid content. Loss of AAR-regulated antioxidant defenses in Gcn2 null livers was signified by reduced Gpx1 gene expression alongside increased lipid peroxidation. Substantial reductions in antithrombin III hepatic expression and activity in the blood of asparaginase-treated Gcn2 null mice indicated liver dysfunction. These results suggest that the ability of the liver to adapt to prolonged asparaginase treatment is influenced by GCN2-directed regulation of FGF<em>21</em> and oxidative defenses, which, when lost, corresponds with maladaptive effects on lipid metabolism and hemostasis.

OBJECTIVE

Adeno-associated virus-mediated gene transfer is promising in the delivery of genes to tendons because this vector stimulates few adverse tissue reactions. Basic fibroblast growth factor (bFGF) promotes collagen production in healing tendons. We transferred the exogenous bFGF gene to proliferating tenocytes by adeno-associated viral (AAV) vectors and investigated its effects on the expression of the collagen genes in an in vitro tenocyte model.

METHODS

AAV2 vectors harboring the rat bFGF gene were constructed. Tenocytes were obtained from explant cultures of rat intrasynovial tendons and were distributed into 21 culture dishes and 8 wells. Tenocytes in 7 dishes were treated with AAV2 bFGF for 3 hours and then were cultured for 10 days. Tenocytes in 14 dishes (sham vector and nontreatment controls) did not receive the transgene. Efficiency of the gene transfer was evaluated by in situ beta-galactosidase staining in 8 wells after treatment with AAV2 lacZ. Expression of the target genes was assessed by reverse-transcription polymerase chain reactions with primers specifically amplifying the target genes. Expression of bFGF and type I and III collagen genes was determined by quantitative analysis of the polymerase chain reaction products.

RESULTS

Positive beta-galactosidase staining confirmed the effectiveness of AAV2-mediated gene delivery to tenocytes. The level of expression of the bFGF gene was increased significantly after gene transfer. Levels of expression of type I and III collagen genes after transfer of the exogenous bFGF gene were increased significantly compared with those in the cells treated with sham vectors or in nontreatment controls.

CONCLUSIONS

Delivery of exogenous bFGF gene to tenocytes can increase significantly the levels of expression of the bFGF and type I and III collagen genes. AAV2 vectors provide a novel method for delivering growth factor genes to tenocytes. These findings warrant future in vivo study of the delivery of genes pertinent to tendon healing through AAV2-based gene therapy to enhance repairs of injured flexor tendons.

OBJECTIVE

Beside its beneficial effects on weight loss, ketogenic diet (KD) causes dyslipidemia, a pro-inflammatory state involved in the development of hepatic steatosis, glucose intolerance and insulin resistance, although the latter is still being debated. Additionally, KD is known to increase <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) plasma levels. However, FGF<em>21</em> cannot initiate its beneficial actions on metabolism in these conditions. We therefore hypothesized and tested in the present study that KD may impair FGF<em>21</em> signaling.

RESULTS

Using indirect calorimetry, we found that KD-fed mice exhibited higher energy expenditure than regular chow (RC)-fed mice associated with increased Ucp1 levels in white adipose tissue (WAT), along with increased plasma FGF<em>21</em> levels. We then assessed the effect of KD on FGF<em>21</em> signaling in both the liver and WAT. We found that Fgfr4 and Klb (β-klotho) were downregulated in the liver, while Fgfr1 was downregulated in WAT of KD-fed mice. Because inflammation could be one of the mechanisms linking KD to impaired FGF<em>21</em> signaling, we measured the expression levels of inflammatory markers and macrophage accumulation in WAT and liver and found an increased inflammation and macrophage accumulation in the liver, but surprisingly, a reduction of inflammation in WAT.We also showed that KD enhances lipid accumulation in the liver, which may explain hepatic inflammation and impaired Fgfr4 and Klb expression. In contrast, import of lipids from the circulation was significantly reduced in WAT of KD-fed mice, as suggested by a downregulation of Lpl and Cd36. This was further associated with reduced inflammation in WAT.

CONCLUSIONS

Altogether, these results indicate that KD could be beneficial for a given tissue but deleterious for another.