Catecholamine stimulation of alpha1-adrenoceptors exerts <em>growth</em> <em>factor</em>-like activity, mediated by generation of reactive oxygen species, on arterial smooth muscle cells and adventitial <em>fibroblasts</em> and contributes to hypertrophy and hyperplasia in models of vascular injury and disease. Adrenergic trophic activity also contributes to flow-mediated positive arterial remodeling by augmenting proliferation and leukocyte accumulation. To further examine this concept, we studied whether catecholamines contribute to collateral <em>growth</em> and angiogenesis in hindlimb insufficiency. Support for this hypothesis includes the above-mentioned studies, evidence that ischemia augments norepinephrine release from sympathetic nerves, and proposed involvement of reactive oxygen species in angiogenesis and collateral <em>growth</em>. Mice deficient in catecholamine synthesis [by gene deletion of dopamine beta-hydroxylase (DBH-/-)] were studied. At 3 wk after femoral artery ligation, increases in adductor muscle perfusion were similar in DBH-/- and wild-type mice, whereas recovery of plantar perfusion and calf microsphere flow were attenuated, although not significantly. Preexisting collaterals in adductor of wild-type mice showed increases in lumen diameter (60%) and medial and adventitial thickness (57 and 119%, P < 0.05 here and below). Lumen diameter increased similarly in DBH-/- mice (52%); however, increases in medial and adventitial thicknesses were reduced (30 and 65%). Leukocyte accumulation in the adventitia/periadventitia of collaterals was 39% less in DBH-/- mice. Increased density of alpha-smooth muscle actin-positive vessels in wild-type adductor (45%) was inhibited in DBH-/- mice (2%). Although both groups experienced similar atrophy in the gastrocnemius (approximately 22%), the increase in capillary-to-muscle fiber ratio in wild-type mice (<em>21</em>%) was inhibited in DBH-/- mice (7%). These data suggest that catecholamines may contribute to collateral <em>growth</em> and angiogenesis in tissue ischemia.

Mesenchymal stem cells (MSCs) are adult multipotent cells found in bone marrow, adipose tissue, and other adult tissues. MSCs improve regeneration of injured tissues in vivo, but the mechanisms remain unclear. Typically, MSCs are cultured under ambient or normoxic conditions (<em>21</em>% O2 ). However, the physiological niches of MSCs have much lower oxygen tension. When used as a therapeutic tool to repair tissue injuries, MSCs cultured in standard conditions must adapt from <em>21</em>% O2 in culture to <1% O2 in ischemic tissue. We have examined the effects of hypoxia preconditioning (1% O2 ) in human adipose derived mesenchymal stem cells (AD-MSCs) to discover the conditions that best enhance their tissue regenerative potential. We demonstrate that AD-MSCs respond positively to hypoxia compared with normoxia preconditioning, show decreased apoptosis even in severe microenvironmental conditions (such as a low-serum medium), and an increased expression of the angiogenic <em>factors</em>, vascular endothelial <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. Human umbilical vein endothelial cells have higher vitality and lower apoptosis when cultured in medium taken from hypoxia-preconditioned AD-MSCs, as well as significantly increased capillary-like structures with this medium on Matrigel. The data suggest that hypoxia preconditioned AD-MSCs can improve tissue regeneration.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em>, a hormone primarily secreted by liver, has recently been shown to have beneficial effects on glucose and lipid metabolism and insulin sensitivity in animal models. This study investigated the association of serum FGF<em>21</em> levels with insulin secretion and sensitivity, as well as circulating parameters of lipid metabolism and hepatic enzymes in Chinese subjects.

METHODS

Serum FGF<em>21</em> levels were determined by ELISA in 134 normal glucose tolerance (NGT), 101 isolated-impaired fasting glucose, and 118 isolated-impaired glucose tolerance (I-IGT) Chinese subjects, and their association with parameters of adiposity, glucose, and lipid profiles, and levels of liver injury markers was studied. In a subgroup of this study, the hyperglycemic clamp technique was performed in 31 NGT, 17 isolated-impaired fasting glucose, and 15 I-IGT subjects to measure insulin secretion and sensitivity to test the associations with serum FGF<em>21</em>.

RESULTS

The serum FGF<em>21</em> levels in I-IGT were significantly higher than NGT subjects [164.6 pg/ml (89.7, 261.0) vs. 111.8 pg/ml (58.0, 198.9); P < 0.05], and correlated positively with several parameters of adiposity. Multiple stepwise regression analysis showed an independent association of serum FGF<em>21</em> with serum triglycerides, total cholesterol, and gamma-glutamyltransferase (all P < 0.05). However, FGF<em>21</em> did not correlate with insulin secretion and sensitivity, as measured by hyperglycemic clamp and a 75-g oral glucose tolerance test.

CONCLUSIONS

Serum levels of FGF<em>21</em> are closely related to adiposity, lipid metabolism, and biomarkers of liver injury but not insulin secretion and sensitivity in humans.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em> and <em>growth</em> hormone (GH) are metabolic hormones that play important roles in regulating glucose and lipid metabolism. Both hormones are induced in response to fasting and exert their actions on adipocytes to regulate lipolysis. However, the molecular interaction between these two hormones remains unclear. Here we demonstrate the existence of a feedback loop between GH and FGF<em>21</em> on the regulation of lipolysis in adipocytes. A single bolus injection of GH into C57 mice acutely increases both mRNA and protein expression of FGF<em>21</em> in the liver, thereby leading to a marked elevation of serum FGF<em>21</em> concentrations. Such a stimulatory effect of GH on hepatic FGF<em>21</em> production is abrogated by pretreatment of mice with the lipolysis inhibitor niacin. Direct incubation of either liver explants or human HepG2 hepatocytes with GH has no effect on FGF<em>21</em> expression. On the other hand, FGF<em>21</em> production in HepG2 cells is significantly induced by incubation with the conditioned medium harvested from GH-treated adipose tissue explants, which contains high concentrations of free fatty acids (FFA). Further analysis shows that FFA released by GH-induced lipolysis stimulates hepatic FGF<em>21</em> expression by activation of the transcription <em>factor</em> PPARα. In FGF<em>21</em>-null mice, both the magnitude and duration of GH-induced lipolysis are significantly higher than those in their wild type littermates. Taken together, these findings suggest that GH-induced hepatic FGF<em>21</em> production is mediated by FFA released from adipose tissues, and elevated FGF<em>21</em> in turn acts as a negative feedback signal to terminate GH-stimulated lipolysis in adipocytes.

Chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus at chromosome 14q32 represent a common mechanism of oncogene activation in lymphoid malignancies. In multiple myeloma (MM), the most consistent chromosomal abnormality is the 14q+ marker, which originates in one third of cases through a t(11; 14)(q13; q32) chromosomal translocation; in the remaining cases, the identity of the partner chromosomes has not been well established. We used a Southern blot approach based on the linkage analysis of the joining (J) and the constant (C) mu, alpha, and gamma regions to detect cases bearing IGH switch-mediated chromosomal translocations. We evaluated DNA of 88 nonkaryotyped patients with MM (78 cases) or plasma cell leukemia (PCL) (10 cases) and found the presence of "illegitimate" rearranged IGH fragments (no comigration between the J and C regions) in <em>21</em> cases. To confirm this analysis, we cloned the illegitimate rearranged fragments from three samples, and the molecular and fluorescent in situ hybridization (FISH) analyses indicated the presence of chromosomal translocations juxtaposing a switch IGH region to sequences from chromosomes 11q13 (one PCL case) or 4p16.3 (two MM cases). Interestingly, the breakpoints on 4p16.3 occurred about 14 kb apart in a genomic region located approximately 50 kb centromeric to the <em>fibroblast</em> <em>growth</em>-<em>factor</em> receptor 3 (FGFR3) gene. Moreover, Southern blot analysis using 4p16.3 genomic probes detected a rearrangement in an additional MM tumor. FISH analysis of the MM-derived KMS-11 cell line, reported to be associated with a t(4; 14)(p16.3; q32), showed that the FGFR3 gene was translocated on 14q32. High levels of FGFR3 mRNA expression were observed in the cloned MM tumors and KMS-11 cell line, but not in the cases that were apparently negative for this lesion. Furthermore, a point mutation at codon 373 in the transmembrane domain of the FGFR3 gene resulting in an amino acid substitution (Tyr ->> Cys) was detected in the KMS-11 cell line. These findings indicate that the t(4; 14)(p16.3; q32) represents a novel, recurrent chromosomal translocation in MM, and suggest that the FGFR3 gene may be the target of this abnormality and thus contribute to tumorigenesis in MM.

Endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR) and represents a critical mechanism that underlies metabolic dysfunctions. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), a hormone that is predominantly secreted by the liver, exerts a broad range of effects upon the metabolism of carbohydrates and lipids. Although increased circulating levels of FGF<em>21</em> have been documented in animal models and human subjects with obesity and nonalcoholic fatty liver disease, the functional interconnections between metabolic ER stress and FGF<em>21</em> are incompletely understood. Here, we report that increased ER stress along with the simultaneous elevation of FGF<em>21</em> expression were associated with the occurrence of nonalcoholic fatty liver disease both in diet-induced obese mice and human patients. Intraperitoneal administration of the ER stressor tunicamycin in mice resulted in hepatic steatosis, accompanied by activation of the three canonical UPR branches and increased the expression of FGF<em>21</em>. Furthermore, the IRE1α-XBP1 pathway of the UPR could directly activate the transcriptional expression of Fgf<em>21</em>. Administration of recombinant FGF<em>21</em> in mice alleviated tunicamycin-induced liver steatosis, in parallel with reduced eIF2α-ATF4-CHOP signaling. Taken together, these results suggest that FGF<em>21</em> is an integral physiological component of the cellular UPR program, which exerts beneficial feedback effects upon lipid metabolism through counteracting ER stress.

BACKGROUND

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity. It acts as a key downstream target of both peroxisome proliferator-activated receptor α and γ, the agonists of which have been used for lipid lowering and insulin sensitization, respectively. However, the role of FGF<em>21</em> in the cardiovascular system remains elusive.

RESULTS

The roles of FGF<em>21</em> in atherosclerosis were investigated by evaluating the impact of FGF<em>21</em> deficiency and replenishment with recombinant FGF<em>21</em> in apolipoprotein E(-/-) mice. FGF<em>21</em> deficiency causes a marked exacerbation of atherosclerotic plaque formation and premature death in apolipoprotein E(-/-) mice, which is accompanied by hypoadiponectinemia and severe hypercholesterolemia. Replenishment of FGF<em>21</em> protects against atherosclerosis in apolipoprotein E(-/-)mice via 2 independent mechanisms, inducing the adipocyte production of adiponectin, which in turn acts on the blood vessels to inhibit neointima formation and macrophage inflammation, and suppressing the hepatic expression of the transcription <em>factor</em> sterol regulatory element-binding protein-2, thereby leading to reduced cholesterol synthesis and attenuation of hypercholesterolemia. Chronic treatment with adiponectin partially reverses atherosclerosis without obvious effects on hypercholesterolemia in FGF<em>21</em>-deficient apolipoprotein E(-/-) mice. By contrast, the cholesterol-lowering effects of FGF<em>21</em> are abrogated by hepatic expression of sterol regulatory element-binding protein-2.

CONCLUSIONS

FGF<em>21</em> protects against atherosclerosis via fine tuning the multiorgan crosstalk among liver, adipose tissue, and blood vessels.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>), a hormone with short half-life, has consistently shown strong pharmacological efficacy. We first assessed the efficacy of murine recombinant FGF<em>21</em> in C57BL6 lean mice for 5 wk. We then generated a long-acting FGF<em>21</em> molecule by fusing a Fc to a variant of human recombinant FGF<em>21</em> (hrFGF<em>21</em>) that contained two engineered mutations [L98R, P171G; Fc-FGF<em>21</em>(RG)] and tested it in C57BL6 diet-induced obese mice and obese rhesus monkeys. We compared its metabolic properties with those of the hrFGF<em>21</em>. Groups of diet-induced obese mice were treated for 36 d with different doses of hrFGF<em>21</em> (01, 0.3, and 1 mg/kg twice daily) and with Fc-FGF<em>21</em>(RG) (2.3 mg/kg, every 5 d). Body weight, glucose, insulin, cholesterol, and triglyceride levels were decreased after treatment with either compound. A glucose tolerance test (GTT) was also improved. Obese rhesus monkeys were treated with hrFGF<em>21</em> (once a day) and Fc-FGF<em>21</em>(RG) (once a week) in a dose-escalation fashion. Doses started at 0.1 and 0.3 mg/kg and ended at 3 and 5 mg/kg for hrFGF<em>21</em> and Fc-FGF<em>21</em>(RG), respectively. Doses were escalated every 2 wk, and animals were followed up for a washout period of 3 wk. Body weight, glucose, insulin, cholesterol, and triglyceride levels and the GTT profile were decreased to a greater extent with Fc-FGF<em>21</em>(RG) than with hrFGF<em>21</em>. The PK-PD relationship of Fc-FGF<em>21</em>(RG) exposure and triglyceride reduction was also conducted with a maximum response model. In conclusion, in more than one species, Fc-FGF<em>21</em>(RG) chronically administered once a week showed similar or greater efficacy than hrFGF<em>21</em> administered daily.

BACKGROUND

Fibroblast growth factor 19 (FGF19) and FGF21 are novel metabolic regulators that improve insulin sensitivity and decrease adiposity in mice. However, little is known about the nutritional regulation of these factors in humans.

OBJECTIVE

The objective of this study was to measure plasma FGF19 and FGF21 levels in patients with anorexia nervosa (AN) and to explore its relationship with anthropometric and endocrine parameters.

METHODS

This was a single-center cross-sectional study.

METHODS

The study was performed in a university hospital.

METHODS

Seventeen untreated women with a restrictive type of AN and 17 healthy women (control group) were included.

METHODS

Fasting plasma FGF19 and FGF21, serum insulin, leptin, soluble leptin receptor, adiponectin, resistin, and C-reactive protein were the main outcome measures.

RESULTS

Plasma FGF19 levels did not significantly differ between the groups studied, whereas plasma FGF21 levels were significantly reduced in AN relative to the control group. Plasma FGF21 positively correlated with body mass index and serum leptin and insulin and was inversely related to serum adiponectin in both groups. In contrast, plasma FGF19 was not related to any of parameters studied. Partial realimentation significantly reduced plasma FGF21 levels in AN.

CONCLUSIONS

Circulating levels of FGF21 but not FGF19 are strongly related to body weight and serum levels of leptin, adiponectin, and insulin in both anorectic and normal-weight women. We suggest that reduced plasma FGF21 levels could be involved in the pathophysiology of AN or in a complex adaptive response to this disease.

OBJECTIVE

To investigate the possible role of transforming growth factor beta (TGF beta) in lens development and growth, the authors studied the influence of TGF beta, alone and in combination with fibroblast growth factor (FGF), on lens epithelial explants.

METHODS

Lens explants were prepared from both postnatal and adult rats, and changes during 5 days of culture with growth factor(s) were monitored by light and electron microscopy, immunolocalization of laminin, heparan sulfate proteoglycan and fiber-specific crystallins, and crystallin enzyme-linked immunosorbent assays.

RESULTS

TGF beta induced cells in explants to undergo an extensive and rapid elongation with features that distinguished it from FGF-induced fiber differentiation. TGF beta also induced accumulation of extracellular matrix, capsule wrinkling, cell death by apoptosis, and distinctive arrangements of cells. Standard explants from 10-day-old rats responded to TGF beta only in the presence of FGF. Comparable explants from adult rats or from 21-day-old rats (cultured on a laminin substratum) responded readily to TGF beta whether or not FGF was present.

CONCLUSIONS

First, these results suggest a role for TGF beta in regulating normal processes in lens cells such as the production of extracellular matrix and capsule formation. Second, because many of the changes induced by TGF beta resembled changes reported to occur during the formation of various kinds of subcapsular cataracts, the results suggest that detailed studies of factors that influence the ability of lens cells to respond to TGF beta and the bioavailability of TGF beta in the ocular media may provide important insights into the etiology of some forms of cataract.

Mesenchymal stem cell (MSC) transplantation is a promising therapy to regenerate the brain after an ischemic event. We investigated the possibility to use the nasal route as a noninvasive method to repair the neonatal damaged brain. Nine-day-old mice underwent cerebral hypoxia-ischemia (HI), and MSCs were transplanted intranasally 10 d after HI. At 28 d after HI, MSCs were still present in the affected hemisphere but had not differentiated into cerebral cell types. Intranasal MSC treatment significantly improved sensorimotor function in the cylinder rearing test at <em>21</em> and 28 d after HI. Furthermore, intranasal MSC treatment decreased gray and white matter area loss when determined 28 d after HI by 34 and 37%, respectively. MSC cultured in vitro with brain extracts obtained 10 d after HI, responded to the ischemic brain by up-regulation of several <em>growth</em> <em>factors</em>, including <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 and nerve <em>growth</em> <em>factor</em> in comparison with brain extracts of sham-operated controls. In conclusion, MSC can reliably be delivered to the brain via the nasal route to induce functional recovery and a reduction in brain lesion size. We propose that MSC function by stimulating endogenous cerebral repair by adapting their secretion profile to the ischemic brain leading to up-regulation of repair promoting <em>factors</em>.

BACKGROUND

Adult endothelial progenitor cells (EPC) may be a useful source for engineering the endothelialization of vascular grafts. However, the optimal factors that promote differentiation of EPCs into endothelium remain to be elucidated. The goal of this current report was to determine which extracellular matrix (ECM) protein might modulate or enhance the effects of EPCs on differentiation into mature endothelium.

METHODS

Human EPCs (CD34(+) cells) were cultured in ECM-coated six-well plates in MCDB-131 medium containing vascular endothelial growth factor (VEGF), insulin-like growth factor-1, and basic fibroblast growth factor. After 21 days, differentiated endothelial colonies were confirmed by immunofluorescence for von Willebrand factor (vWF) and vascular-endothelial (VE)-cadherin and mRNA expression of the endothelial markers Flk-1, vWF, and VE-cadherin. Cell migration toward the VEGF-matrix protein combinations was also measured.

RESULTS

As judged by positive staining for endothelial markers vWF and VE-cadherin, the combination of VEGF with fibronectin (FN) produced significantly more endothelial colonies (P <.05) than did collagens I or IV or vitronectin. Defined fragments of FN did not enhance VEGF-mediated effects. Fibrinogen produced intermediate stimulation of differentiation. FN also enhanced VEGF-mediated CD34(+) cell migration. Blockade of alpha5beta1, but not alphavbeta3 or alphavbeta5, inhibited both VEGF-mediated CD34(+) cell differentiation and migration.

CONCLUSIONS

VEGF and FN together significantly promote the migration and differentiation of CD34(+) cells. This synergism is specific to FN and the alpha5beta1 integrin. Combinations of VEGF and FN may be useful in promoting differentiation of circulating endothelial progenitors into endothelial cells for tissue engineering. Clinical relevance Treatment of injured or diseased tissues with adult stem cells is a promising approach. In particular, bone marrow derived circulating endothelial progenitors (CEP's) have been shown to differentiate into endothelial cells in vitro and promote tissue revascularization of ischemic limbs and myocardium in vivo. Because of the relative ease of obtaining CEP's and as well as its high proliferative rate, CEP's may have clinical potential for endothelialization of prosthetic vascular grafts and revascularization of injured myocardium. However, there is a need to better understand the molecular pathways involved in the proliferation and differentiation of CEP's to take full advantage of its clinical potential.

Duchenne muscular dystrophy (DMD) is the most common inherited neuromuscular disease, and is characterized by the lack of dystrophin, muscle wasting, increased transforming <em>growth</em> <em>factor</em> (TGF)-β Smad-dependent signalling and fibrosis. Acting via the Mas receptor, angiotensin-1-7 [Ang-(1-7)], is part of the renin-angiotensin system, with the opposite effect to that of angiotensin II. We hypothesized that the Ang-(1-7)/Mas receptor axis might protect chronically damaged tissues as in skeletal muscle of the DMD mouse model mdx. Infusion or oral administration of Ang-(1-7) in mdx mice normalized skeletal muscle architecture, decreased local fibrosis and improved muscle function in vitro and in vivo. These positive effects were mediated by the inhibition of TGF-β Smad signalling, which in turn led to reduction of the pro-fibrotic microRNA miR-<em>21</em> concomitant with a reduction in the number of TCF4 expressing <em>fibroblasts</em>. Mdx mice infused with Mas antagonist (A-779) and mdx deficient for the Mas receptor showed highly deteriorated muscular architecture, increased fibrosis and TGF-β signalling with diminished muscle strength. These results suggest that this novel compound Ang-(1-7) might be used to improve quality of life and delay death in individuals with DMD and this drug should be investigated in further pre-clinical trials.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has multiple metabolic actions, including the induction of browning in white adipose tissue. Although FGF<em>21</em> stimulated browning results from a direct interaction between FGF<em>21</em> and the adipocyte, browning is typically associated with activation of the sympathetic nervous system through cold exposure. We tested the hypothesis that FGF<em>21</em> can act via the brain, to increase sympathetic activity and induce browning, independent of cell-autonomous actions. We administered FGF<em>21</em> into the central nervous system via lateral ventricle infusion into male mice and found that the central treatment increased norepinephrine turnover in target tissues that include the inguinal white adipose tissue and brown adipose tissue. Central FGF<em>21</em> stimulated browning as assessed by histology, expression of uncoupling protein 1, and the induction of gene expression associated with browning. These effects were markedly attenuated when mice were treated with a β-blocker. Additionally, neither centrally nor peripherally administered FGF<em>21</em> initiated browning in mice lacking β-adrenoceptors, demonstrating that an intact adrenergic system is necessary for FGF<em>21</em> action. These data indicate that FGF<em>21</em> can signal in the brain to activate the sympathetic nervous system and induce adipose tissue thermogenesis.

Our objective was to establish the role of <em>fibroblasts</em> in medial vascular calcification, a pathological process known to be associated with elastin degradation and remodeling. Rat dermal <em>fibroblasts</em> were treated in vitro with elastin degradation products and transforming <em>growth</em> <em>factor</em> (TGF)-beta1, <em>factors</em> usually present in deteriorated matrix environments. Cellular changes were monitored at the gene and protein level by reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and von Kossa staining for calcium deposits. By <em>21</em> days, multicellular calcified nodules were formed in the presence of elastin degradation products and TGF-beta1 separately and to a significantly greater extent when used together. Before mineralization, cells expressed alpha-smooth muscle actin and large amounts of collagen type I and matrix metalloproteinase-2, characteristic features of myo<em>fibroblasts</em>, key elements in tissue remodeling and repair. Stimulated cells expressed increased levels of core-binding <em>factor</em> alpha1, osteocalcin, alkaline phosphatase, and osteoprotegerin, representative bone-regulating proteins. For most proteins analyzed, TGF-beta1 synergistically amplified responses of <em>fibroblasts</em> to elastin degradation products. In conclusion, elastin degradation products and TGF-beta1 promote myofibroblastic and osteogenic differentiation in <em>fibroblasts</em>. These results support the idea that elastin-related calcification involves dynamic remodeling events and suggest the possibility of a defective tissue repair process.

Since its identification in 2000, the interest of scientists in the hepatokine <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>21</em> has tremendously grown, and still remains high, due to a wealth of very robust data documenting this <em>factor</em>'s favorable effects on glucose and lipid metabolism in mice. For more than ten years now, intense in vivo and ex vivo experimentation addressed the physiological functions of FGF<em>21</em> in humans as well as its pathophysiological role and pharmacological effects in human metabolic disease. This work produced a comprehensive collection of data revealing overlaps in FGF<em>21</em> expression and function but also significant differences between mice and humans that have to be considered before translation from bench to bedside can be successful. This review summarizes what is known about FGF<em>21</em> in mice and humans with a special focus on this <em>factor</em>'s role in glucose and lipid metabolism and in metabolic diseases, such as obesity and type 2 diabetes mellitus. We highlight the discrepancies between mice and humans and try to decipher their underlying reasons.

Antibodies targeting PD-1 or its ligand 1 PD-L1 such as atezolizumab, have great efficacy in a proportion of metastatic urothelial cancers^1,2. Biomarkers may facilitate identification of these responding tumors³. Neoadjuvant use of these agents is associated with pathological complete response in a spectrum of tumors, including urothelial cancer^4-7. Sequential tissue sampling from these studies allowed for detailed on-treatment biomarker analysis. Here, we present a single-arm phase 2 study, investigating two cycles of atezolizumab before cystectomy in 95 patients with muscle-invasive urothelial cancer (ClinicalTrials.gov identifier: NCT02662309 ). Pathological complete response was the primary endpoint. Secondary endpoints focused on safety, relapse-free survival and biomarker analysis. The pathological complete response rate was 31% (95% confidence interval: <em>21</em>-41%), achieving the primary efficacy endpoint. Baseline biomarkers showed that the presence of preexisting activated T cells was more prominent than expected and correlated with outcome. Other established biomarkers, such as tumor mutational burden, did not predict outcome, differentiating this from the metastatic setting. Dynamic changes to gene expression signatures and protein biomarkers occurred with therapy, whereas changes in DNA alterations with treatment were uncommon. Responding tumors showed predominant expression of genes related to tissue repair after treatment, making tumor biomarker interpretation challenging in this group. Stromal <em>factors</em> such as transforming <em>growth</em> <em>factor</em>-β and <em>fibroblast</em> activation protein were linked to resistance, as was high expression of cell cycle gene signatures after treatment.

BACKGROUND

Fibroblast growth factor (FGF) 23 is a recently identified circulating factor that regulates phosphate (Pi) metabolism. Since the derangement of Pi control is an important risk factor for vascular calcification, we investigated the importance of plasma FGF-23 in the development of vascular calcification in the aorta and peripheral artery in hemodialysis patients with and without diabetes mellitus (DM).

METHODS

Male hemodialysis patients with DM (n=32) and without DM (n=56) were examined. Plasma samples were obtained before the start of dialysis sessions, and the FGF-23 levels were determined by enzyme-linked immunosorbent assay. Roentgenography of the aorta and hand artery was performed, and visible vascular calcification was evaluated by one examiner, who was blinded to the patient characteristics.

RESULTS

In the 56 non-DM hemodialysis patients, vascular calcification was found in the hand artery in 5 patients (8.9%) and in the aorta in 23 patients (41.1%). These levels were significantly lower (p<0.05) than in the 32 DM patients, of whom, 19 (59.4%) and 21 (65.6%) had vascular calcification of the hand artery and aorta, respectively. Multiple regression analyses performed separately in the non-DM and DM patients showed that the plasma FGF-23 level, CaxPi product, and body weight are independent factors significantly associated with hand-artery calcification and that diastolic blood pressure is associated with aorta calcification in non-DM patients. In DM patients, the plasma FGF-23 level and hemodialysis duration emerged as independent factors associated with hand-artery calcification and diastolic blood pressure was associated with aorta calcification. The independent association of the plasma FGF-23 level with hand-artery calcification was retained in both non-DM and DM patients when adjusted for the CaxPi product.

CONCLUSIONS

Our findings show that the plasma FGF-23 level is an independent factor negatively associated with peripheral vascular calcification in the hand artery, but not in the aorta, in both male non-DM and DM hemodialysis patients, even when adjusted for the CaxPi product. This study raises the possibility that the plasma FGF-23 level may provide a reliable marker for Moenckeberg's medial calcification in male hemodialysis patients, independent of its regulatory effect on Pi metabolism.

In mammalian organs under normoxic conditions, O2 concentration ranges from 12% to <0.5%, with O2 approximately 14% in arterial blood and <10% in the myocardium. During mild hypoxia, myocardial O2 drops to approximately 1% to 3% or lower. In response to chronic moderate hypoxia, cells adjust their normoxia set point such that reoxygenation-dependent relative elevation of PO2 results in perceived hyperoxia. We hypothesized that O2, even in marginal relative excess of the PO2 to which cardiac cells are adjusted, results in activation of specific signal transduction pathways that alter the phenotype and function of these cells. To test this hypothesis, cardiac <em>fibroblasts</em> (CFs) isolated from adult murine ventricle were cultured in 10% or <em>21</em>% O2 (hyperoxia relative to the PO2 to which cells are adjusted in vivo) and were compared with those cultured in 3% O2 (mild hypoxia). Compared with cells cultured in 3% O2, cells that were cultured in 10% or <em>21</em>% O2 demonstrated remarkable reversible G2/M arrest and a phenotype indicative of differentiation to myo<em>fibroblasts</em>. These effects were independent of NADPH oxidase function. CFs exposed to high O2 exhibited higher levels of reactive oxygen species production. The molecular signature response to perceived hyperoxia included (1) induction of p<em>21</em>, cyclin D1, cyclin D2, cyclin G1, Fos-related antigen-2, and transforming <em>growth</em> <em>factor</em>-beta1, (2) lowered telomerase activity, and (3) activation of transforming <em>growth</em> <em>factor</em>-beta1 and p38 mitogen-activated protein kinase. CFs deficient in p<em>21</em> were resistant to such O2 sensitivity. This study raises the vital broad-based issue of controlling ambient O2 during the culture of primary cells isolated from organs.

To understand the physiological significance of the reduction in fasting insulin produced by dietary methionine restriction (MR), hyperinsulinemic-euglycemic clamps were used to examine the effect of the diet on overall and tissue-specific insulin sensitivity in mice. The steady-state glucose infusion rate was threefold higher in the MR group and consistent with the 2.5- to threefold increase in 2-deoxyglucose uptake in skeletal muscle, heart, and white adipose tissue. Dietary MR enhanced suppression of hepatic glucose production by insulin, enhanced insulin-dependent Akt phosphorylation in the liver, and increased hepatic expression and circulating <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF-<em>21</em>) by fourfold. Limitation of media methionine recapitulated amplification of Akt phosphorylation by insulin in HepG2 cells but not in 3T3-L1 adipocytes or C2C12 myotubes. Amplification of insulin signaling in HepG2 cells by MR was associated with reduced glutathione, where it functions as a co<em>factor</em> for phosphatase and tensin homolog. In contrast, FGF-<em>21</em>, but not restricting media methionine, enhanced insulin-dependent Akt phosphorylation in 3T3-L1 adipocytes. These findings provide a potential mechanism for the diet-induced increase in insulin sensitivity among tissues that involves a direct effect of methionine in liver and an indirect effect in adipose tissue through MR-dependent increases in hepatic transcription and release of FGF-<em>21</em>.

Previous studies have shown that starvation or consumption of a high fat, low carbohydrate (HF-LC) ketogenic diet induces hepatic <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) gene expression in part by activating the peroxisome proliferator-activated receptor-α (PPARα). Using primary hepatocyte cultures to screen for endogenous signals that mediate the nutritional regulation of FGF<em>21</em> expression, we identified two sources of PPARα activators (i.e. nonesterified unsaturated fatty acids and chylomicron remnants) that induced FGF<em>21</em> gene expression. In addition, we discovered that natural (i.e. bile acids) and synthetic (i.e. GW4064) activators of the farnesoid X receptor (FXR) increased FGF<em>21</em> gene expression and secretion. The effects of bile acids were additive with the effects of nonesterified unsaturated fatty acids in regulating FGF<em>21</em> expression. FXR activation of FGF<em>21</em> gene transcription was mediated by an FXR/retinoid X receptor binding site in the 5'-flanking region of the FGF<em>21</em> gene. FGF19, a gut hormone whose expression and secretion is induced by intestinal bile acids, also increased hepatic FGF<em>21</em> secretion. Deletion of FXR in mice suppressed the ability of an HF-LC ketogenic diet to induce hepatic FGF<em>21</em> gene expression. The results of this study identify FXR as a new signaling pathway activating FGF<em>21</em> expression and provide evidence that FXR activators work in combination with PPARα activators to mediate the stimulatory effect of an HF-LC ketogenic diet on FGF<em>21</em> expression. We propose that the enhanced enterohepatic flux of bile acids during HF-LC consumption leads to activation of hepatic FXR and FGF19 signaling activity and an increase in FGF<em>21</em> gene expression and secretion.

A major complication of diabetes mellitus is the disruption of normal wound repair process, characterised by insufficient production of <em>growth</em> <em>factors</em>. A molecular genetic approach wherein resident cells synthesise and deliver the <em>growth</em> <em>factors</em> to the wound site would be a powerful therapeutic strategy to treat diabetic wounds. One such molecular approach could be the application of microRNAs (miRNAs). This study reports differential expression of miRNAs related to cell development and differentiation, during wound healing in diabetic mice. Comparison of skin tissue from normal and diabetic mice showed that 14 miRNAs were differentially expressed in diabetic skin; miR-146b and miR-<em>21</em> were the most noteworthy. Expression pattern of these miRNAs was also altered during healing of diabetic wounds. A subset of miRNAs (miR-20b, miR-10a, miR-10b, miR-96, miR-128, miR-452 and miR-541) exhibited similar basal levels in normal and diabetic skins, but displayed dysregulation during healing of diabetic wounds. Amongst the miRNAs studied, miR-<em>21</em> showed a distinct signature with increased expression in diabetic skin but decreased expression during diabetic wound healing. We analysed the role of miR-<em>21</em> in <em>fibroblast</em> migration, because migration of <em>fibroblasts</em> into the wound area is an important landmark facilitating secretion of <em>growth</em> <em>factors</em> and migration of other cell types into the wound, thus enhancing the healing process. Using gain-of and loss-of function approaches, we show that miR-<em>21</em> is involved in <em>fibroblast</em> migration. Our preliminary studies implicate an important role for miRNAs in the pathogenesis of diabetic wounds.

Gestational exposure to maternal overweight (OW) influences the risk of obesity in adult life. Male offspring from OW dams gain greater body weight and fat mass and develop insulin resistance when fed high-fat diets (45% fat). In this report, we identify molecular targets of maternal OW-induced programming at postnatal d <em>21</em> before challenge with the high-fat diet. We conducted global transcriptome profiling, gene/protein expression analyses, and characterization of downstream signaling of insulin and adiponectin pathways in conjunction with endocrine and biochemical characterization. Offspring born to OW dams displayed increased serum insulin, leptin, and resistin levels (P < 0.05) at postnatal d <em>21</em> preceding changes in body composition. A lipogenic transcriptome signature in the liver, before development of obesity, was evident in OW-dam offspring. A coordinated locus of 20 sterol regulatory element-binding protein-1-regulated target genes was induced by maternal OW. Increased nuclear levels of sterol regulatory element-binding protein-1 and recruitment to the fatty acid synthase promoter were confirmed via ELISA and chromatin immunoprecipitation analyses, respectively. Higher fatty acid synthase and acetyl coenzyme A carboxylase protein and pAKT (Thr(308)) and phospho-insulin receptor-beta were confirmed via immunoblotting. Maternal OW also attenuated AMP kinase/peroxisome proliferator-activated receptor-alpha signaling in the offspring liver, including transcriptional down-regulation of several peroxisome proliferator-activated receptor-alpha-regulated genes. Hepatic mRNA and circulating <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> levels were significantly lower in OW-dam offspring. Furthermore, serum levels of high-molecular-weight adiponectin (P < 0.05) were decreased in OW-dam offspring. Phosphorylation of hepatic AMP-kinase (Thr(172)) was significantly decreased in OW-dam offspring, along with lower AdipoR1 mRNA. Our results strongly suggest that gestational exposure to maternal obesity programs multiple aspects of energy-balance regulation in the offspring.

OBJECTIVE

The actions of growth factors during healing of injured flexor tendons are not well characterized, although information pertinent to some individual growth factors is available. We studied gene expression and protein production of a number of growth factors at several time points during the early healing period in a chicken model.

METHODS

Seventy-four long toes of 37 white Leghorn chickens were used. The flexor digitorum profundus tendons of 60 toes were surgically repaired after complete transection and were harvested for analysis 3, 5, 7, 9, 14, and 21 days after surgery. The expression of 6 growth factors was studied at 4 time points after surgery with real-time quantitative polymerase chain reactions, and production and distribution of 3 growth factors at all 6 time points were studied by immunohistochemical staining with antibodies. Fourteen tendons that had no surgery served as day 0 controls. Tendon healing status was also assessed histologically.

RESULTS

Throughout the early tendon healing period, connective tissue growth factor (CTGF) and transforming growth factor beta (TGF-beta) showed high levels of gene expression. Levels of gene expression of vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) were high or moderately high. Expression of the TGF-beta gene was upregulated after injury, whereas the basic fibroblast growth factor (bFGF) gene was downregulated at all postsurgical time points and expressed at the lowest levels among 6 growth factor genes 2 to 3 weeks after surgery. The platelet-derived growth factor B (PDGF-B) gene was also minimally expressed. Findings of immunohistochemistry corresponded to TGF-beta, bFGF, and IGF-1 gene expression.

CONCLUSIONS

In this model, up to 3 weeks after surgery, gene expression and production of TGF-beta are high and are upregulated in this healing period. However, expression of the bFGF gene and protein is low and decreases in the healing tendon. The CTGF, VEGF, and IGF-1 genes are expressed at high or moderately high levels, but PDGF-B is minimally expressed.