OBJECTIVE

Cholesterol and lipoprotein metabolism display pronounced gender differences. Premenopausal women have lower LDL and higher HDL cholesterol, whereas men display higher synthetic rates of bile acids and cholesterol. The effects of the administration of exogenous hormones to humans and animals indicate that these gender differences can often be explained by estrogens. We evaluated how increased levels of endogenous estrogens modulate cholesterol and lipoprotein metabolism in women.

RESULTS

We studied healthy women during initiation of in vitro fertilization using blood samples obtained when endogenous estrogens were low and high. Cholesterol in VLDL and LDL, but not in HDL, was reduced 20% when estrogens were high. Apolipoprotein B levels decreased 13%. Apolipoprotein A-I and triglyceride levels increased 8% and 37%, respectively, whereas lipoprotein(a) levels were unchanged. Circulating PCSK9, a suppressor of LDL receptors, was reduced 14% when estrogens were high. Serum markers of bile acid and cholesterol synthesis were unaltered. <em>Growth</em> hormone levels increased 3-fold when estrogens were high, whereas insulin-like <em>growth</em> <em>factor</em>-1 and <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> concentrations were unaltered.

CONCLUSIONS

In women, Apolipoprotein B-containing particles and circulating PCSK9 are reduced when endogenous estrogens are high, indicating that endogenous estrogens induce hepatic LDL receptors partly through a posttranscriptional mechanism. However, estrogens do not stimulate bile acid or cholesterol synthesis.

It has been shown that replication of the Japanese encephalitis virus (JEV) can trigger infected cells to undergo apoptosis. In the present study, it is further demonstrated that replication-incompetent virions of JEV, obtained by short-wavelength ultraviolet (UV) irradiation, could also induce host-cell death. It was found that UV-inactivated JEV (UV-JEV) caused cell death in neuronal cells such as mouse neuroblastoma N18 and human neuronal NT-2 cells, but not in non-neuronal baby hamster kidney BHK-<em>21</em> <em>fibroblast</em> or human cervical HeLa cells. Only actively <em>growing</em>, but not <em>growth</em>-arrested, cells were susceptible to the cytotoxic effects of UV-JEV. Killing of UV-JEV-infected N18 cells could be antagonized by co-infection with live, infectious JEV, suggesting that virions of UV-JEV might engage an as-yet-unidentified receptor-mediated death-signalling pathway. Characteristically, mitochondrial alterations were evident in UV-JEV-infected N18 cells, as revealed by electron microscopy and a loss of membrane potential. N18 cells infected by UV-JEV induced generation of reactive oxygen species (ROS) as well as the activation of nuclear <em>factor</em> kappa B (NF-kappaB), and the addition of anti-oxidants or specific NF-kappaB inhibitors to the media greatly reduced the cytotoxicity of UV-JEV. Together, the results presented here suggest that replication-incompetent UV-JEV damages actively <em>growing</em> neuronal cells through a ROS-mediated pathway.

Cerebral ischemia disrupts the neurovascular unit, involving death of neuronal, glial, and endothelial cells (ECs) in the core and penumbra regions. Whereas the neuroprotective effect of recombinant human erythropoietin (rhEPO) has been widely investigated, its effects on ECs remain elusive. We now report the effects of rhEPO treatment on EC death and neurovasculature repair following a focal ischemic stroke in postnatal day 7 neonatal rats. rhEPO (5000 U/kg i.p.) was administered 60 min after ischemia and for the next 3 days. Western blot analysis revealed increased expression of neurovascular remodeling proteins, including Tie-1, angiopoietin-2, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> in rhEPO-treated pups. rhEPO treatment significantly reduced EC death in the ischemic penumbra region 12 to 72 h after ischemia examined by immunostaining of terminal deoxynucleotidyl transferase dUTP nick-end labeling and EC marker glucose transporter-1 (GLUT-1). Treatment with rhEPO increased proliferation of ECs and neuronal cells, revealed by costaining of 5-bromo-2'-deoxyuridine with GLUT-1 or with the neuronal marker protein (NeuN) 7 to <em>21</em> days after stroke. Specifically, rhEPO increased number of NeuN-positive cells in close proximity to proliferating microvessels. These results suggest for the first time that, in addition to its protection on neural cells, EPO protects ECs and promotes the neurovascular unit repair, which may contribute to its therapeutic benefits after neonatal ischemic stroke.

BACKGROUND

MicroRNAs (miRNAs) and their role during tumor development have been studied in great detail during the last decade, albeit their expression pattern and regulation during normal development are however not so well established. Previous studies have shown that miRNAs are differentially expressed in solid human tumors. Platelet-derived <em>growth</em> <em>factor</em> (PDGF) signaling is known to be involved in normal development of the brain as well as in malignant primary brain tumors, gliomas, but the complete mechanism is still lacking. We decided to investigate the expression of the oncogenic miR-<em>21</em> during normal mouse development and glioma, focusing on PDGF signaling as a potential regulator of miR-<em>21</em>.

METHODS

We generated mouse glioma using the RCAS/tv-a system for driving PDGF-BB expression in a cell-specific manner. Expression of miR-<em>21</em> in mouse cell cultures and mouse brain were assessed using Northern blot analysis and in situ hybridization. Immunohistochemistry and Western blot analysis were used to investigate SOX2 expression. LNA-modified siRNA was used for irreversible depletion of miR-<em>21</em>. For inhibition of PDGF signaling Gleevec (imatinib mesylate), Rapamycin and U0126, as well as siRNA were used. Statistical significance was calculated using double-sided unpaired Student's t-test.

RESULTS

We identified miR-<em>21</em> to be highly expressed during embryonic and newborn brain development followed by a gradual decrease until undetectable at postnatal day 7 (P7), this pattern correlated with SOX2 expression. Furthermore, miR-<em>21</em> and SOX2 showed up-regulation and overlapping expression pattern in RCAS/tv-a generated mouse brain tumor specimens. Upon irreversible depletion of miR-<em>21</em> the expression of SOX2 was strongly diminished in both mouse primary glioma cultures and human glioma cell lines. Interestingly, in normal fibroblasts the expression of miR-<em>21</em> was induced by PDGF-BB, and inhibition of PDGF signaling in mouse glioma primary cultures resulted in suppression of miR-<em>21</em> suggesting that miR-<em>21</em> is indeed regulated by PDGF signaling.

CONCLUSIONS

Our data show that miR-<em>21</em> and SOX2 are tightly regulated already during embryogenesis and define a distinct population with putative tumor cell of origin characteristics. Furthermore, we believe that miR-<em>21</em> is a mediator of PDGF-driven brain tumors, which suggests miR-<em>21</em> as a promising target for treatment of glioma.

In many mammals, lactation success depends on substantial use of lipid reserves and requires integrated metabolic activities between white adipose tissue (WAT) and liver. Mechanisms responsible for this integration in lactation are poorly understood, but data collected in other conditions of elevated lipid use suggest a role for <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em> (FGF<em>21</em>). To address this possibility in the context of lactation, we studied high-yielding dairy cows during the transition from late pregnancy (LP) to early lactation (EL). Plasma FGF<em>21</em> was nearly undetectable in LP, peaked on the day of parturition, and then stabilized at lower, chronically elevated concentrations during the energy deficit of EL. Plasma FGF<em>21</em> was similarly increased in the absence of parturition when an energy-deficit state was induced by feed restricting late-lactating dairy cows, implicating energy insufficiency as a cause of chronically elevated FGF<em>21</em> in EL. Gene expression studies showed that liver was a major source of plasma FGF<em>21</em> in EL with little or no contribution by WAT, skeletal muscle, and mammary gland. Meaningful expression of the FGF<em>21</em> coreceptor β-Klotho was restricted to liver and WAT in a survey of 15 tissues that included the mammary gland. Expression of β-Klotho and its subset of interacting FGF receptors was modestly affected by the transition from LP to EL in liver but not in WAT. Overall, these data suggest a model whereby liver-derived FGF<em>21</em> regulates the use of lipid reserves during lactation via focal actions on liver and WAT.

We have isolated a strongly mitogenic, type beta transforming <em>growth</em> <em>factor</em> (beta TGF) released by Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells that induces phenotypic transformation of normal NRK cells when they are concomitantly stimulated by analogues of epidermal <em>growth</em> <em>factor</em> (EGF). Molecule filtration chromatography separates beta TGF from an EGF-like TGF (eTGF) which is also present in acid extracts from medium conditioned by FeSV-Fre cells (J. Massagué, (1983) J. Biol. Chem. 258, 13606-13613). Final purification of beta TGF is achieved by reverse phase high pressure liquid chromatography (HPLC) on octadecyl support, molecular filtration HPLC, and nonreducing dodecyl sulfate-polyacrylamide gel electrophoresis steps, yielding a 300,000-fold purified polypeptide with a final recovery of <em>21</em>%. The purified rat beta TGF consists of two Mr = 11,000-12,000 polypeptide chains disulfide-linked as a Mr = 23,000 dimer. Induction of anchorage-independent proliferation of NRK cells by rat beta TGF depends on the simultaneous presence of eTGF or EGF. In the presence of a saturating (300 pM) concentration of either rat eTGF or mouse EGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with 4-6 pM rat beta TGF. In the presence of a saturating (20 pM) concentration of rat beta TGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with either rat eTGF or mouse EGF at a 50-70 pM concentration. Rat beta TGF is also able to induce DNA synthesis and cell proliferation on <em>growth</em>-arrested NRK, human lung, and Swiss mouse 3T3 <em>fibroblast</em> monolayers, this effect being half-maximal at 2-3 pM beta TGF for NRK cells. These results identify eTGF and beta TGF as the two synergistically acting <em>factors</em> responsible for the transforming action of culture fluids from FeSV-Fre cells.

In this report, we describe the isolation from human urine of a predominant 160-kDa epidermal <em>growth</em> <em>factor</em> (EGF)-immunoreactive glycoprotein that exhibits affinity for heparin. The purification procedure involved concentration and dialysis of 20-30-liter batches of fresh urine on a high capacity ultrafiltration apparatus followed by chromatography on DEAE-Sephacel, heparin-agarose, and Sephacryl S-300. A nearly homogeneous preparation of 160-kDa protein was obtained with a yield of approximately 1 mg of 160-kDa protein from 25 liters of urine. The amino-terminal sequence of the purified 160-kDa protein, H2N-SAPQHXSXPEGTXA-, matched residues <em>21</em>-34 of the predicted sequence of human prepro-EGF and established that the 160 kDa protein (pro-EGF) is a product of the prepro-EGF gene. Characterization of the carboxyl terminus of the purified protein by digestion with carboxypeptidase B and by immunoblotting with antisera against synthetic carboxyl-terminal and juxtatransmembrane peptides of prepro-EGF indicated that the carboxyl terminus has been truncated at an arginine residue that corresponds, most likely, to the carboxyl-terminal arginine of the EGF moiety. The intact 160-kDa pro-EGF is biologically active as evidenced by its specific binding to the EGF receptor and activation of the EGF receptor tyrosine kinase in A-431 cell membranes. Purified pro-EGF competitively inhibited the binding of 125I-EGF to human <em>fibroblasts</em>, and it stimulated the proliferation of these cells in culture. When immobilized onto culture dishes, the heparin-binding pro-EGF appeared to function both as an adhesion molecule and as a <em>growth</em> <em>factor</em> for serum-free mouse embryo cells.

BACKGROUND

High fibroblast growth factor-23 (FGF-23) levels are associated with adverse outcomes. We studied the responsiveness of FGF-23 and mineral metabolism to altered dietary phosphate intake in chronic kidney disease (CKD) and healthy control patients.

METHODS

Thirty patients were enrolled: 18 normophosphatemic CKD subjects and 12 healthy controls. The study duration was 21 days with three 7-day dietary interventions; a high phosphate (HP, 2000 mg/day), low phosphate (750 mg/day) and low phosphate plus phosphate binder (aluminum hydroxide, 500 mg thrice daily with meals), with comparable macronutrient content, administered in random sequence. Baseline and weekly fasting morning measurements of FGF-23, serum phosphate (sPO(4)), 1,25-hydroxyvitamin D (1,25 D) and 24-h urinary calcium (uCa) and phosphate (uPO(4)) were collected.

RESULTS

FGF-23 levels were higher in subjects versus controls (72 pg/mL versus 30 pg/mL) at baseline, while sPO(4) remained in the normal range throughout the study. The absolute changes of uPO(4) and uCa for CKD and controls vary according to diet. The absolute changes of FGF-23 and sPO(4) suggest that the effect of the diets might also depend on the CKD status (P-values interaction effect = 0.08 and 0.07, respectively); nonetheless, these changes are evident as a function of dietary interventions, irrespective of CKD status (P-values diet effect = 0.006 and <0.001, respectively).

CONCLUSIONS

FGF-23 levels appear to be responsive to changes in diet in both CKD patients and controls. Further studies are required to determine whether lowering dietary phosphate and thus FGF-23 levels are of long-term benefit in CKD patients, irrespective of sPO(4) levels.

OBJECTIVE

Obesity is a global health crisis with detrimental effects on all organ systems leading to worsening disease state and rising costs of care. Persons with obesity failing lifestyle therapies need to be escalated to appropriate pharmacological treatment modalities, medical devices, and/or bariatric surgery if criteria are met and more aggressive intervention is needed. The progression of severe obesity in the patient population coupled with related co-morbidities necessitates the development of novel therapies for the treatment of obesity. This development is preceded by increased understanding of the underpinnings of energy regulation and neurohormonal pathways involved in energy homeostasis.

RESULTS

Though there are approved anti-obesity drugs available in the USA, newer drugs are now in the pipeline for development given the urgent need. This review focuses on anti-obesity drugs in the pipeline including centrally acting agents (setmelanotide, neuropeptide Y antagonist [velneperit], zonisamide-bupropion [Empatic], cannabinoid type-1 receptor blockers), gut hormones and incretin targets (new glucagon-like-peptide-1 [GLP-1] analogues [semaglutide and oral equivalents], amylin mimetics [davalintide, dual amylin and calcitonin receptor agonists], dual action GLP-1/glucagon receptor agonists [oxyntomodulin], triple agonists [tri-agonist 1706], peptide YY, leptin analogues [combination pramlintide-metreleptin]), and other novel targets (methionine aminopeptidase 2 inhibitor [beloranib], lipase inhibitor [cetilistat], triple monoamine reuptake inhibitor [tesofensine], <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>), including anti-obesity vaccines (ghrelin, somatostatin, adenovirus36). With these new drugs in development, anti-obesity therapeutics have potential to vastly expand allowing better treatment options and personalized approach to obesity care.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is an important endocrine metabolic regulator expressed in multiple tissues including liver and adipose tissue. Although highest levels of expression are in pancreas, little is known about the function of FGF<em>21</em> in this tissue. In order to understand the physiology of FGF<em>21</em> in the pancreas, we analyzed its expression and regulation in both acinar and islet tissues. We found that acinar tissue express 20-fold higher levels than that observed in islets. We also observed that pancreatic FGF<em>21</em> is nutritionally regulated; a marked reduction in FGF<em>21</em> expression was noted with fasting while obesity is associated with 3-4 fold higher expression. Acinar and islet cells are targets of FGF<em>21</em>, which when systemically administered, leads to phosphorylation of the downstream target ERK 1/2 in about half of acinar cells and a small subset of islet cells. Chronic, systemic FGF<em>21</em> infusion down-regulates its own expression in the pancreas. Mice lacking FGF<em>21</em> develop significant islet hyperplasia and periductal lymphocytic inflammation when fed with a high fat obesogenic diet. Inflammatory infiltrates consist of TCRb+ Thy1+ T lymphocytes with increased levels of Foxp3+ regulatory T cells. Increased levels of inflammatory cells were coupled with elevated expression of cytokines such as TNFα, IFNγ and IL1β. We conclude that FGF<em>21</em> acts to limit islet hyperplasia and may also prevent pancreatic inflammation.

BACKGROUND

Von Hippel-Lindau (VHL) disease induces vascular neoplasms in multiple organs. We evaluated the safety and efficacy of sunitinib in VHL patients and examined the expression of candidate receptors in archived tissue.

METHODS

Patients with VHL were given four cycles of 50 mg sunitinib daily for 28 days, followed by 14 days off. Primary end point was toxicity. Modified RECIST were used for efficacy assessment. We evaluated 20 archival renal cell carcinomas (RCCs) and 20 hemangioblastomas (HBs) for biomarker expression levels using laser-scanning cytometry (LSC).

RESULTS

Fifteen patients were treated. Grade 3 toxicity included fatigue in five patients. Dose reductions were needed in 10 patients. Eighteen RCC and <em>21</em> HB lesions were evaluable. Six of the RCCs (33%) responded partially, versus none of the HBs (P = 0.014). LSC revealed that mean levels of phosphorylated vascular endothelial <em>growth</em> <em>factor</em> receptor-2 were lower in HB than in RCC endothelium (P = 0.003) and mean phosphorylated <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor substrate-2 (pFRS2) levels were higher in HB (P = 0.003).

CONCLUSIONS

Sunitinib treatment in VHL patients showed acceptable toxicity. Significant response was observed in RCC but not in HB. Greater expression of pFRS2 in HB tissue than in RCC raises the hypothesis that treatment with fibroblast growth factor pathway-blocking agents may benefit patients with HB.

Tyrosine kinase receptors lead to rapid activation of phosphatidylinositol 3-kinase (PI3 kinase) and the subsequent formation of phosphatidylinositides (PtdIns) 3,4-P2 and PtdIns 3,4, 5-P3, which are thought to be involved in signaling for glucose transporter GLUT4 translocation, cytoskeletal rearrangement, and DNA synthesis. However, the specific role of each of these PtdIns in insulin and <em>growth</em> <em>factor</em> signaling is still mainly unknown. Therefore, we assessed, in the current study, the effect of SH2-containing inositol phosphatase (SHIP) expression on these biological effects. SHIP is a 5' phosphatase that decreases the intracellular levels of PtdIns 3,4,5-P3. Expression of SHIP after nuclear microinjection in 3T3-L1 adipocytes inhibited insulin-induced GLUT4 translocation by 100 +/- <em>21</em>% (mean +/- the standard error) at submaximal (3 ng/ml) and 64 +/- 5% at maximal (10 ng/ml) insulin concentrations (P < 0.05 and P < 0.001, respectively). A catalytically inactive mutant of SHIP had no effect on insulin-induced GLUT4 translocation. Furthermore, SHIP also abolished GLUT4 translocation induced by a membrane-targeted catalytic subunit of PI3 kinase. In addition, insulin-, insulin-like <em>growth</em> <em>factor</em> I (IGF-I)-, and platelet-derived <em>growth</em> <em>factor</em>-induced cytoskeletal rearrangement, i.e., membrane ruffling, was significantly inhibited (78 +/- 10, 64 +/- 3, and 62 +/- 5%, respectively; P < 0.05 for all) in 3T3-L1 adipocytes. In a rat <em>fibroblast</em> cell line overexpressing the human insulin receptor (HIRc-B), SHIP inhibited membrane ruffling induced by insulin and IGF-I by 76 +/- 3% (P < 0.001) and 68 +/- 5% (P < 0.005), respectively. However, <em>growth</em> <em>factor</em>-induced stress fiber breakdown was not affected by SHIP expression. Finally, SHIP decreased significantly <em>growth</em> <em>factor</em>-induced mitogen-activated protein kinase activation and DNA synthesis. Expression of the catalytically inactive mutant had no effect on these cellular responses. In summary, our results show that expression of SHIP inhibits insulin-induced GLUT4 translocation, <em>growth</em> <em>factor</em>-induced membrane ruffling, and DNA synthesis, indicating that PtdIns 3,4,5-P3 is the key phospholipid product mediating these biological actions.

Both mechanical and humoral triggers have been put forward to explain the hypertrophic response of the challenged cardiomyocyte. The aim of the present study was to investigate whether cyclic equibiaxial stretch is a direct stimulus for isolated adult rabbit cardiomyocytes to develop hypertrophy and to explore the potential involvement of the autocrine/paracrine <em>factors</em> ANG II, transforming <em>growth</em> <em>factor</em> (TGF)-beta(1), and IGF-I in this process. Isolated cardiomyocytes were exposed to 10% cyclic equibiaxial stretch (1 Hz) for up to 48 h or treated with ANG II (100 nM), TGF-beta(1) (5 ng/ml), IGF-I (100 ng/ml), ANG II type 1 (AT(1)) receptor blockers, or conditioned medium of stretched <em>fibroblasts</em>. Cyclic stretch significantly increased cell surface area (+3.1%), protein synthesis (+<em>21</em>%), and brain natriuretic peptide (BNP) mRNA expression (6-fold) in cardiomyocytes. TGF-beta(1) expression increased (+42%) transiently at 4 h, whereas cardiomyocyte IGF-I expression was not detectable under all experimental conditions. The AT(1) receptor blockers candesartan and irbesartan (100 nM) did not prevent the stretch-induced hypertrophic response. Direct exposure to ANG II, TGF-beta(1), or IGF-I did not enhance cardiomyocyte BNP expression. In cardiac <em>fibroblasts</em>, stretch elicited a significant approximately twofold increase in TGF-beta(1) and IGF-I expression. Conditioned medium of stretched <em>fibroblasts</em> increased BNP expression in cardiomyocytes ( approximately 2-fold, P = 0.07). This study clearly indicates that cyclic stretch is a strong, direct trigger to induce hypertrophy in fully differentiated rabbit cardiomyocytes. The present findings do not support the notion that stretch-mediated hypertrophy of adult rabbit cardiomyocytes involves autocrine/paracrine actions of ANG II, TGF-beta(1), or IGF-I.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) is a novel metabolic regulator produced primarily by the liver that exerts potent antidiabetic and lipid-lowering effects in animal models of obesity and type 2 diabetes mellitus. This hormone contributes to body weight regulation and is strongly involved in the response to nutritional deprivation and ketogenic state in mice. The principal sites of metabolic actions of FGF<em>21</em> are adipose tissue, liver and pancreas. Experimental studies have shown marked improvements in diabetes compensation and dyslipidemia after FGF<em>21</em> administration in diabetic mice and primates. Positive metabolic actions of FGF<em>21</em> without the presence of apparent side effects make this <em>factor</em> a hot candidate to treat type 2 diabetes and accompanying metabolic diseases. The aim of this review is to summarize the current knowledge about the metabolic effects of FGF<em>21</em> including some preliminary data on changes of its levels in humans with a special emphasis on its therapeutic potential in type 2 diabetes mellitus.

The obesity pandemic has spurred a need for novel therapies to prevent and treat metabolic complications. The recent rediscovery of brown adipose tissue (BAT) in humans made this tissue a possible therapeutic target, due to its potentially substantial contributions to energy homeostasis. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has been identified as a facilitator of cold-induced thermogenesis in humans. Furthermore, pre-clinical studies revealed that FGF<em>21</em> administration leads to improvement in the metabolic consequences of obesity, such as dyslipidemia and type 2 diabetes. Here we studied plasma FGF<em>21</em> levels in two cohorts of human subjects, in whom BAT activity was determined using an individualized cooling protocol by [(18)F]FDG-PET/CT scan. Importantly, we found that circulating FGF<em>21</em> levels correlated with BAT activity during acute cold exposure in male subjects. In addition, FGF<em>21</em> levels were related to the change in core temperature upon acute cold exposure, indicating a role for FGF<em>21</em> in maintaining normothermia, possibly via activation of BAT. Furthermore, cold acclimation increased BAT activity in parallel with increased FGF<em>21</em> levels. In conclusion, our results demonstrate that FGF<em>21</em> levels in humans are related to BAT activity, suggesting that FGF<em>21</em> may represent a novel mechanism via which BAT activity in humans may be enhanced.

Hepatocyte <em>growth</em> <em>factor</em> (HGF) and macrophage-stimulating protein (MSP) are structurally related molecules that stimulate epithelial cell proliferation and migration. MSP also acts directly as a chemoattractant for resident macrophages. These activities are integral to the wound repair processes of inflammation, epithelialization and tissue remodelling. To begin to examine the involvement of HGF and MSP in healing of cutaneous wounds we have mapped the temporal expression of these two molecules and their receptors, MET and RON respectively, in adult rat excisional wounds. Four 2x2-cm full-thickness excisional wounds were created on the dorsum of 18 rats, and biopsies were taken through the wounds at 3, 5, 7, 14, <em>21</em>, and 28 days postwounding. These biopsies were analyzed using immunofluorescent staining and in situ hybridization (ISH). The number of cells staining positively for HGF and MET significantly increased in response to wounding. HGF staining and mRNA peaked at 7 days postwounding whereas MET was upregulated earlier, peaking after 3 days. Both HGF and MET protein were observed in <em>fibroblasts</em> of the dermis and in the newly forming granulation tissue. ISH studies also revealed that <em>fibroblasts</em> at the wound edges and within the newly forming granulation tissue also expressed HGF and c-met mRNA. Immunofluorescent staining revealed both MSP and RON within the wound, with maximum staining occurring between 7 and <em>21</em> days for both the ligand and receptor. In addition, MSP co-localized with a small subset of ED1-positive cells (monocytes). In contrast, ED2-positive cells (macrophages) did not co-localize with MSP. Thus, increased expression of HGF, MSP and their receptors MET and RON respectively was observed in response to wounding. Furthermore, MSP co-localization with a subset of monocytes may confirm a role for MSP in the activation of mature macrophages, which may be important in tissue remodelling.

Mechanisms regulating subcutaneous adipose tissue (SAT) insulin sensitivity and gene network expression during the peripartal period were evaluated in cows fed to meet or exceed prepartal energy requirements. Holstein cows were dried off at -50 d relative to expected parturition and fed a controlled-energy diet [CON; net energy for lactation=1.24 Mcal/kg of dry matter (DM); 36% of DM as wheat straw] until -<em>21</em> d. Cows were then randomly assigned (n=7/diet) to either the same CON diet or a moderate-energy close-up diet (OVE; net energy for lactation=1.47 Mcal/kg of DM) until parturition. Biopsies of SAT were harvested at -10, 7, and <em>21</em> d for mRNA expression of 48 genes associated with insulin signaling, adipogenesis, and lipolysis. In vitro basal and insulin-stimulated insulin receptor substrate 1 tyrosine phosphorylation (IRS1-PY) was assessed at -10 and 7 d. The OVE led to more positive energy balance and greater serum insulin concentration prepartum. Compared with CON, OVE led to a more drastic increase in serum NEFA and also greater overall serum BHBA postcalving, both of which were associated with greater hepatic total lipid and triacylglycerol concentration. Close-up OVE did not improve any aspect of performance. In prepartal SAT, insulin-stimulated IRS1-PY was greater in OVE than in CON. However, IRS1-PY, serum insulin, and GLUT4 expression decreased postpartum regardless of prepartal treatment, suggesting a more severe state of insulin resistance. The expression of all genes encoding adipogenic regulators (PPARG and ZFP423), most lipogenic enzymes/inducers (FASN, SCD, DGAT2, and INSIG1), and basal-lipolysis regulators (ATGL and ABDH5) was greater at -10 d in OVE than in CON. Whereas adipogenic and basal lipolysis regulator expression remained greater in cows fed OVE by 7 d postpartum, expression of all lipogenic enzymes decreased regardless of diet. Despite those responses, the approximately 3-fold increase in expression of IRS1 and ZFP423 between 7 and <em>21</em> d suggested that insulin responsiveness and adipogenic capacity of SAT were partially restored. Expression of the preadipocyte marker DLK1, adiponutrin (PNPLA3), and <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) was undetectable. Results suggested that close-up energy overfeeding did not exacerbate insulin resistance in SAT. Signs of restored insulin responsiveness (upregulation of IRS1, INSIG2, SREBF1, and ZFP423) were apparent as early as 3 wk postpartum. Thus, identifying specific nutrients capable of activating PPARγ after calving in AT might help accelerate its replenishment. A regulatory network encompassing the genes and physiological measurements obtained is proposed.

BACKGROUND

Emerging as an important metabolic regulator, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) has gained considerable interest in diabetes and obesity research. The circulating FGF<em>21</em> concentration is fairly low in normal individuals, but elevated hormone levels may associate with obesity. The determining <em>factors</em> of FGF<em>21</em> levels in humans are not clear.

OBJECTIVE

Our objective was to study the influence of genetic and acquired components to serum FGF<em>21</em> variability in healthy young adult twins.

METHODS

Fasting serum FGF<em>21</em>, lipids, body fat, and oral glucose tolerance test were investigated in 46 monozygotic (MZ) and 75 dizygotic twin pairs aged 22.8-33.1 yr. Subcutaneous, intraabdominal, and liver fat content were measured by magnetic resonance imaging/spectroscopy in a subsample of 24 MZ pairs.

RESULTS

Genetic <em>factors</em> contributed moderately (heritability 40%) to circulating serum FGF<em>21</em> levels. Subjects with high FGF<em>21</em> concentrations (≥ 250 pg/ml, n = 30) had higher fasting triglycerides, insulin, homeostasis model assessment index, and area under the curve glucose and lower high-density lipoprotein cholesterol but similar measures of overall adiposity (body mass index, body fat percent) than subjects with lower FGF<em>21</em> (<100 pg/ml, n = 148). Importantly, in the MZ subsample, higher liver fat but not sc or intraabdominal fat content was found in subjects with high FGF<em>21</em>. Furthermore, in analyses controlling for genetic/familial effects in twin pairs, within-pair differences in liver fat (MZ) and triglycerides (dizygotic pairs) were the major acquired <em>factors</em> that correlated with differences in FGF<em>21</em> concentrations.

CONCLUSIONS

Genetic <em>factors</em> influence serum FGF<em>21</em> levels. Of the acquired components, high liver fat and triglycerides rather than overall adiposity associate with high FGF<em>21</em> levels.

Left ventricular (LV) remodeling is the main reason for the development of progressive cardiac dysfunction after myocardial infarction (MI). This study investigated whether stimulation of the angiotensin type 2 receptor is able to ameliorate post-MI cardiac remodeling and what the underlying mechanisms may be. MI was induced in Wistar rats by permanent ligation of the left coronary artery. Treatment with the angiotensin type 2 receptor agonist compound <em>21</em> (0.03 mg/kg) was started 6 hours post-MI and continued for 6 weeks. Hemodynamic parameters were measured by echocardiography and intracardiac catheter. Effects on proteolysis were studied in heart tissue and primary cardiac <em>fibroblasts</em>. Compound <em>21</em> significantly improved systolic and diastolic functions, resulting in improved ejection fraction (71.2±4.7% versus 53.4±7.0%; P<0.001), fractional shortening (P<0.05), LV internal dimension in systole (P<0.05), LV end-diastolic pressure (16.9±1.2 versus 22.1±1.4 mm Hg; P<0.05), ratio of early (E) to late (A) ventricular filling velocities, and maximum and minimum rate of LV pressure rise (P<0.05). Compound <em>21</em> improved arterial stiffness parameters and reduced collagen content in peri-infarct myocardium. Tissue inhibitor of matrix metalloproteinase 1 was strongly upregulated, whereas matrix metalloproteinases 2 and 9 and transforming <em>growth</em> <em>factor</em> β1 were diminished in LV of treated animals. In cardiac <em>fibroblasts</em>, compound <em>21</em> initially induced tissue inhibitor of matrix metalloproteinase 1 expression followed by attenuated matrix metalloproteinase 9 and transforming <em>growth</em> <em>factor</em> β1 secretion. In conclusion, angiotensin type 2 receptor stimulation improves cardiac function and prevents cardiac remodeling in the late stage after MI, suggesting that angiotensin type 2 receptor agonists may be considered a future pharmacological approach for the improvement of post-MI cardiac dysfunction.

OBJECTIVE

Circulating <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em> (FGF<em>21</em>) levels are often elevated in obesity, dyslipidaemia, insulin resistance and type 2 diabetes. This study investigated the relationship of plasma FGF<em>21</em> levels with cardiovascular events in patients with type 2 diabetes.

METHODS

Plasma FGF<em>21</em> levels were measured by ELISA at baseline in 9,697 individuals with type 2 diabetes participating in the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) study. We assessed the association of FGF<em>21</em> levels with the incidence of different cardiovascular outcomes over 5 years. The primary outcome was total cardiovascular disease (CVD) events and the secondary outcomes were the four individual components: coronary heart disease events, total stroke, CVD mortality and coronary and carotid revascularisation. The tertiary outcome was hospitalisation for angina pectoris.

RESULTS

Higher baseline FGF<em>21</em> levels were associated with higher risks of all cardiovascular outcome events after adjusting for the study treatment allocation (all p < 0.01). The associations remained significant for total CVD events and for coronary and carotid revascularisation after further adjusting for confounding <em>factor</em>s, with the HR (95% CI) being 1.28 (1.10, 1.50) and 1.26 (1.01, 1.56), respectively, for the highest tertile compared with the lowest tertile (overall effect p = 0.002 and 0.007, respectively). The addition of FGF<em>21</em> levels to a model including established CVD risk <em>factor</em>s predicting total CVD events led to a non-significant increase in the C-statistic but there was a significant improvement in integrated discrimination and net reclassification.

CONCLUSIONS

Higher baseline plasma FGF<em>21</em> levels were associated with higher risk of cardiovascular events in patients with type 2 diabetes.

BACKGROUND

ISRCTN64783481.

Although the liver is generally considered the main site of production of FGF<em>21</em> (<em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>21</em>), high FGF<em>21</em> levels have been found to be associated with neuromuscular mitochondrial genetic diseases, and there are indications that the muscle may be a relevant site of FGF<em>21</em> production under conditions of muscular mitochondrial stress. In the present study, we found that expression and release of FGF<em>21</em> was associated with myogenic differentiation, and we identified MyoD as a major controller of FGF<em>21</em> gene transcription. Mimicking mitochondrial dysfunction using respiratory chain/oxidative phosphorylation inhibitors resulted in enhanced expression and release of FGF<em>21</em> by muscle cells. The increased production of reactive oxygen species, subsequent induction of p38 MAPK (mitogen-activated protein kinase) and activation of an ATF2 (activating transcription <em>factor</em> 2)-binding site at the proximal promoter region of the FGF<em>21</em> gene was found to be a major mechanism linking mitochondrial dysfunction with enhanced FGF<em>21</em> gene transcription in myogenic cells. The myogenic <em>factor</em> MyoD was required for the induction of FGF<em>21</em> gene transcription by mitochondrial dysfunction, thus explaining the preferential response of muscle cells to mitochondrial dysfunction-induced FGF<em>21</em> expression and secretion. FGF<em>21</em> release by muscle cells in response to mitochondrial alterations may represent a physiological mechanism by which the sensing of internal energetic status by muscles results in the release of FGF<em>21</em> to favour systemic metabolic adaptations.

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>21</em> has recently been introduced as a circulating adipokine which reverses insulin resistance and obesity in rodents. In this study, regulation of FGF-<em>21</em> in renal dysfunction was elucidated in both chronic kidney disease (CKD) and acute kidney dysfunction (AKD).

METHODS

Serum concentrations of total FGF-<em>21</em> were quantified by enzyme-linked immunosorbent assay in 499 patients with CKD stages 1-5 (study population 1). Furthermore, total FGF-<em>21</em> was determined before and within 30 h after unilateral nephrectomy, a model of AKD, in 32 patients (study population 2). FGF-<em>21</em> levels were correlated to anthropometric and biochemical parameters of renal function, glucose and lipid metabolism, as well as inflammation, in both studies.

RESULTS

In study population 1, median [interquartile range] circulating FGF-<em>21</em> adjusted for age, gender and body mass index was significantly different between CKD stages with highest values detectable in stage 5 (stage 1: 86·4 [132·9]; 2: 206·4 [223·1]; 3: 289·8 [409·3]; 4: 591·3 [789·0]; 5: 1918·1 [4157·0] ng/l). Furthermore, estimated glomerular filtration rate remained a strong independent and negative predictor of FGF-<em>21</em>. In study population 2, FGF-<em>21</em> increased significantly postsurgically (325·0 [984·0] ng/l) as compared to presurgical values (255·5 [243·0] ng/l). Furthermore, relative changes of FGF-<em>21</em> were independently and positively predicted by relative changes of creatinine.

CONCLUSIONS

We demonstrate that circulating FGF-<em>21</em> is increased in both CKD and AKD. Our results suggest renal excretion as a major route for FGF-<em>21</em> elimination. The pathophysiological significance of these findings needs to be elucidated in more detail.

Increased cardiovascular disease in aging is partly a consequence of the vascular endothelial cell (EC) senescence and associated vascular dysfunction. In this contest, EC senescence is a pathophysiological process of structural and functional changes including dysregulation of vascular tone, increased endothelium permeability, arterial stiffness, impairment of angiogenesis and vascular repair, and a reduction of EC mitochondrial biogenesis. Dysregulation of cell cycle, oxidative stress, altered calcium signaling, hyperuricemia, and vascular inflammation have been implicated in the development and progression of EC senescence and vascular disease in aging. A number of abnormal molecular pathways are associated with these underlying pathophysiological changes including Sirtuin 1, Klotho, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>21</em>, and activation of the renin angiotensin-aldosterone system. However, the molecular mechanisms of EC senescence and associated vascular impairment in aging are not completely understood. This review provides a contemporary update on molecular mechanisms, pathophysiological events, as well functional changes in EC senescence and age-associated cardiovascular disease. This article is part of a Special Issue entitled: Genetic and epigenetic regulation of aging and longevity edited by Jun Ren & Megan Yingmei Zhang.

<AbstractText>The clinical activity of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) inhibitors seems restricted to cancers harbouring rare FGFR genetic aberrations. In preclinical studies, high tumour FGFR mRNA expression predicted response to rogaratinib, an oral pan-FGFR inhibitor. We aimed to assess the safety, maximum tolerated dose, recommended phase 2 dose, pharmacokinetics, and preliminary clinical activity of rogaratinib.</AbstractText><AbstractText>We did a phase 1 dose-escalation and dose-expansion study of rogaratinib in adults with advanced cancers at 22 sites in Germany, Switzerland, South Korea, Singapore, Spain, and France. Eligible patients were aged 18 years or older, and were ineligible for standard therapy, with an Eastern Cooperative Oncology Group performance status of 0-2, a life expectancy of at least 3 months, and at least one measurable or evaluable lesion according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. During dose escalation, rogaratinib was administered orally twice daily at 50-800 mg in continuous <em>21</em>-day cycles using a model-based dose-response analysis (continuous reassessment method). In the dose-expansion phase, all patients provided an archival formalin-fixed paraffin-embedded (FFPE) tumour biopsy or consented to a new biopsy at screening for the analysis of FGFR1-3 mRNA expression. In the dose-expansion phase, rogaratinib was given at the recommended dose for expansion to patients in four cohorts: urothelial carcinoma, head and neck squamous-cell cancer (HNSCC), non-small-cell lung cancer (NSCLC), and other solid tumour types. Primary endpoints were safety and tolerability, determination of maximum tolerated dose including dose-limiting toxicities and determination of recommended phase 2 dose, and pharmacokinetics of rogaratinib. Safety analyses were reported in all patients who received at least one dose of rogaratinib. Patients who completed cycle 1 or discontinued during cycle 1 due to an adverse event or dose-limiting toxicity were included in the evaluation of recommended phase 2 dose. Efficacy analyses were reported for all patients who received at least one dose of study drug and who had available post-baseline efficacy data. This ongoing study is registered with ClinicalTrials.gov, number NCT01976741, and is fully recruited.</AbstractText><AbstractText>Between Dec 30, 2013, and July 5, 2017, 866 patients were screened for FGFR mRNA expression, of whom 126 patients were treated (23 FGFR mRNA-unselected patients in the dose-escalation phase and 103 patients with FGFR mRNA-overexpressing tumours [52 patients with urothelial carcinoma, eight patients with HNSCC, 20 patients with NSCLC, and 23 patients with other tumour types] in the dose-expansion phase). No dose-limiting toxicities were reported and the maximum tolerated dose was not reached; 800 mg twice daily was established as the recommended phase 2 dose and was selected for the dose-expansion phase. The most common adverse events of any grade were hyperphosphataemia (in 77 [61%] of 126 patients), diarrhoea (in 65 [52%]), and decreased appetite (in 48 [38%]); and the most common grade 3-4 adverse events were fatigue (in 11 [9%] of 126 patients) and asymptomatic increased lipase (in 10 [8%]). Serious treatment-related adverse events were reported in five patients (decreased appetite and diarrhoea in one patient with urothelial carcinoma, and acute kidney injury [NSCLC], hypoglycaemia [other solid tumours], retinopathy [urothelial carcinoma], and vomiting [urothelial carcinoma] in one patient each); no treatment-related deaths occurred. Median follow-up after cessation of treatment was 32 days (IQR 25-36 days). In the expansion cohorts, 15 (15%; 95% CI 8·6-23·5) out of 100 evaluable patients achieved an objective response, with responses recorded in all four expansion cohorts (12 in the urothelial carcinoma cohort and one in each of the other three cohorts), and in ten (67%) of 15 FGFR mRNA-overexpressing tumours without apparent FGFR genetic aberration.</AbstractText><AbstractText>Rogaratinib was well tolerated and clinically active against several types of cancer. Selection by FGFR mRNA expression could be a useful additional biomarker to identify a broader patient population who could be eligible for FGFR inhibitor treatment.</AbstractText><AbstractText>Bayer AG.</AbstractText>