Hyaluronidases are enzymes that degrade hyaluronan an important constituent of the extracellular matrix. They have been used as a spreading agent, improving the absorption of drugs and facilitating the subcutaneous infusion of fluids. Here, we investigated the influence of bovine testes hyaluronidase (HYAL) during cutaneous wound healing in in vitro and in vivo assays. We demonstrated in the wound scratch assay that HYAL increased the migration and proliferation of <em>fibroblasts</em> in vitro at low concentration, e.g. 0.1 U HYAL enhanced the cell number by 20%. HYAL presented faster and higher reepithelialization in in vivo full-thickness excisional wounds generated on adult Wistar rats back skin already in the early phase at 2nd day post operatory compared to vehicle-control group. Wound closured area observed in the 16 U and 32 U HYAL treated rats reached 38% and 46% compared to <em>19</em>% in the controls, respectively. Histological and biochemical analyses supported the clinical observations and showed that HYAL treated wounds exhibited increased granulation tissue, diminished edema formation and regulated the inflammatory response by modulating the release of pro and anti-inflammatory cytokines, <em>growth</em> <em>factor</em> and eicosanoids mediators. Moreover, HYAL increased gene expression of peroxisome proliferator-activated receptors (PPAR) γ and PPAR β/δ, the collagen content in the early stages of healing processes as well as angiogenesis. Altogether these data revealed that HYAL accelerates wound healing processes and might be beneficial for treating wound disorders.

OBJECTIVE

Ficlatuzumab can be used to treat head and neck squamous cell carcinoma (HNSCC) by inhibiting c-Met receptor-mediated cell proliferation, migration, and invasion.

OBJECTIVE

To understand the effect of ficlatuzumab on HNSCC proliferation, migration, and invasion.

METHODS

The effects of ficlatuzumab on HNSCC proliferation, invasion, and migration were tested. Mitigation of c-Met and downstream signaling was assessed by immunoblotting. The tumor microenvironment has emerged as an important <em>factor</em> in HNSCC tumor progression. The most abundant stromal cells in HNSCC tumor microenvironment are tumor-associated <em>fibroblasts</em> (TAFs). We previously reported that TAFs facilitate HNSCC <em>growth</em> and metastasis. Furthermore, activation of the c-Met tyrosine kinase receptor by TAF-secreted hepatocyte <em>growth</em> <em>factor</em> (HGF) facilitates tumor invasion. Ficlatuzumab is a humanized monoclonal antibody that sequesters HGF, preventing it from binding to and activating c-Met. We hypothesized that targeting the c-Met pathway with ficlatuzumab will mitigate TAF-mediated HNSCC proliferation, migration, and invasion. Representative HNSCC cell lines HN5, UM-SCC-1, and OSC-<em>19</em> were used in these studies.

UNASSIGNED

The HNSCC cell lines were treated with ficlatuzumab, 0 to 100 µg/mL, for 24 to 72 hours.

METHODS

Ficlatuzumab inhibited HNSCC progression through c-Met and mitogen-activated protein kinase (MAPK) signaling pathway.

RESULTS

Ficlatuzumab significantly reduced TAF-facilitated HNSCC cell proliferation (HN5, P < .001; UM-SCC-1, P < .001), migration (HN5, P = .002; UM-SCC-1, P = .01; and OSC-<em>19</em>, P = .04), and invasion (HN5, P = .047; UM-SCC-1, P = .03; and OSC-<em>19</em>, P = .04) through a 3-dimensional peptide-based hydrogel (PGmatrix). In addition, ficlatuzumab also inhibited the phosphorylation of c-Met at Tyr1234/1235 and p44/42 MAPK in HNSCC cells exposed to recombinant HGF.

CONCLUSIONS

We demonstrate that neutralizing TAF-derived HGF with ficlatuzumab effectively mitigates c-Met signaling and decreases HNSCC proliferation, migration, and invasion. Thus, ficlatuzumab effectively mitigates stromal influences on HNSCC progression.

BACKGROUND

Lactate dehydrogenase A (LDHA) and Pyruvate Kinase M2 (PKM2) are important enzymes of glycolysis. Both of them can be phosphorylated and therefore regulated by Fibroblast growth factor receptor 1 (FGFR1). While phosphorylation of LDHA at tyrosine10 leads to tetramerization and activation, phosphorylation of PKM2 at tyrosine105 promotes dimerization and inactivation. Dimeric PKM2 is found in the nucleus and regulates gene transcription. Up-regulation and phosphorylation of LDHA and PKM2 contribute to faster proliferation under hypoxic conditions and promote the Warburg effect.

METHODS

Using western blot and SYBR Green Real time PCR we investigated 77 thyroid tissues including 19 goiter tissues, 11 follicular adenomas, 16 follicular carcinomas, 15 papillary thyroid carcinomas, and 16 undifferentiated thyroid carcinomas for total expression of PKM2, LDHA and FGFR1. Additionally, phosphorylation status of PKM2 and LDHA was analysed. Inhibition of FGFR was performed on FTC133 cells with SU-5402 and Dovitinib.

RESULTS

All examined thyroid cancer subtypes overexpressed PKM2 as compared to goiter. LDHA was overexpressed in follicular and papillary thyroid cancer as compared to goiter. Elevated phosphorylation of LDHA and PKM2 was detectable in all analysed cancer subtypes. The highest relative phosphorylation levels of PKM2 and LDHA compared to overall expression were found in undifferentiated thyroid cancer. Inhibition of FGFR led to significantly decreased phosphorylation levels of PKM2 and LDHA.

CONCLUSIONS

Our data shows that overexpression and increased phosphorylation of PKM2 and LHDA is a common finding in thyroid malignancies. Phospho-PKM2 and Phospho-LDHA could be valuable tumour markers for thyroglobulin negative thyroid cancer.

A new culture medium, NCTC 168, has been designed for human skin epithelial cells. This medium formulation was developed, by combining and testing at various concentrations, components of media NCTC 135 and 163, since a 1:1 mixture of these two media with 10% horse serum supplement was found to promote epithelial cell out<em>growth</em> from human skin explants. The buffer system in NCTC 168 maintains the pH of the medium between 7.0 and 7.2. In contrast to other media tested, NCTC 168 with 10% horse serum is capable of initiating and sustaining larger epithelial cell out<em>growth</em>s. Explants in serum-supplemented NCTC 168 in the absence of feeder cells reproducibly yield confluent epithelial cell sheets apparently free of <em>fibroblasts</em> after only <em>19</em> to 28 days as compared with 5 weeks or longer for the other media tested. NCTC 168 also supports passage of human epithelial cells to the sixth subculture generation without feeder cells. Electron microscopy has shown the presence of desmosomes and tonofilaments in the passaged cells indicating the epithelial nature of the cells. The addition of epithelial <em>growth</em> <em>factor</em>, hydrocortisone and insulin at 5 ng per ml, 4 microgram per ml and 5 microgram per ml, respectively, did not appreciably enhance the <em>growth</em> of the epithelial cells.

Germ-cell transplantation is a powerful tool for studying gametogenesis in many species. We previously showed that spermatogonia transplanted into the peritoneal cavity of trout hatchlings were able to colonize recipient gonads, and produced fully functional sperm and eggs in synchrony with the germ cells of the recipient. An in vitro-culture system enabling spermatogonia to expand, when combined with transplantation, would be valuable in both basic and applied biology. To this end, we optimized culture conditions for type A spermatogonia in the present study using immature rainbow trout at 8-10 month of age. Spermatogonial survival and mitotic activity were improved during culture in Leibovitz's L-15 medium (pH 7.8) supplemented with 10% fetal bovine serum at 10 degrees C compared with culture under standard conditions for salmonids (Hank's MEM (pH 7.3) supplemented with 25 mM HEPES and 5% FBS, and culture at 20 degrees C). Elimination of testicular somatic cells promoted spermatogonial mitotic activity. In addition, insulin, trout embryonic extract, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> promoted the mitosis of purified spermatogonia in an additive manner. Mitotic activity increased nearly sevenfold over <em>19</em> days of culture compared with <em>growth</em> <em>factor</em>-free conditions and was maintained for >1 month. Furthermore, the cultured spermatogonia could colonize and proliferate in recipient gonads following transplantation. This study represents the first step towards establishing a cell line that can be transplanted for use in surrogate broodstock technology and cell-mediated gene-transfer systems.

OBJECTIVE

Lipoprotein(a) is a highly atherogenic lipoprotein, whose metabolism is poorly understood. Currently no safe drugs exists that lower elevated plasma lipoprotein(a) concentrations. We therefore focused on molecular mechanisms that influence apolipoprotein(a) (APOA) biosynthesis.

RESULTS

Transgenic human APOA mice (tg-APO mice) were injected with 1 mg/kg of recombinant human <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>). This led to a significant reduction of plasma APOA and hepatic expression of APOA. Incubation of primary hepatocytes of tg-APOA mice with FGF<em>19</em> induced ERK1/2 phosphorylation and, in turn, downregulated APOA expression. Repression of APOA by FGF<em>19</em> was abrogated by specific ERK1/2 phosphorylation inhibitors. The FGF<em>19</em> effect on APOA was attenuated by transfection of primary hepatocytes with siRNA against the FGF<em>19</em> receptor 4 (FGFR4). Using promoter reporter assays, mutation analysis, gel shift, and chromatin immune-precipitation assays, an Ets-1 binding element was identified at -1630/-1615bp region in the human APOA promoter. This element functions as an Elk-1 binding site that mediates repression of APOA transcription by FGF<em>19</em>.

CONCLUSIONS

These findings provide mechanistic insights into the transcriptional regulation of human APOA by FGF<em>19</em>. Further studies in the human system are required to substantiate our findings and to design therapeutics for hyper lipoprotein(a).

The sulfonic acid polymers poly(2-acrylamido-2-methyl-1-propanesulfonic acid) (PAMPS), poly(4-styrenesulfonic acid) (PSS), and poly(anetholesulfonic acid) (PAS) proved to be highly potent inhibitors of angiogenesis in the chick chorioallantoic membrane (CAM) assay. PAMPS was found to achieve a dose-dependent inhibition of microvessel formation in the CAM assay ranging from 57 +/- 16% inhibition at 10 micrograms/disc to 72 +/- 15% at 150 micrograms/ disc. Also, PSS and PAS caused a strong inhibition of angiogenesis (55 +/- <em>19</em>% and 48 +/- 16%, respectively, at 50 micrograms/disc), whereas poly(vinylsulfonic acid) (PVS) was found to be inactive at this dose. The compounds proved to be nontoxic for the developing chick embryo at these doses. Suramin, which was included as a reference compound, caused only a slight inhibition of vascular density, at a dose of 150 micrograms/disc, whereas pentosan polysulfate (PPS) was found to be toxic. PAMPS, PAS, and PSS, but not PVS, inhibited microvessel formation in the rat aorta-ring assay. In addition, the increased [3H-methyl]dThd uptake in endothelial cells in vitro upon stimulation with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was inhibited by PAMPS, PAS, and PSS at 20 micrograms/ml. A strong correlation (r = 0.95) was found between the antiangiogenic effect of the sulfonic acid polymers in the CAM assay and their inhibition of the bFGF-induced mitogenic response, indicating that bFGF is the target for these sulfonic acid polymers. These results suggest that sulfonic acid polymers, and in particular PAMPS, may be considered as specific, nontoxic angiogenesis inhibitors.

Our objective was to delineate the temporal sequence of mitogenic activity in myocardial interstitial fluid (IF) during enhancement of collateral <em>growth</em>. Collateral development in chronically instrumented dogs was induced by eight 2-min coronary occlusions/day for 21 days. Collateralization was assessed by measurement of blood flow in the region distal to a total coronary occlusion. Myocardial IF was obtained periodically from an intramyocardial catheter, and mitogenic activity was assessed by proliferative response of cultured endothelial cells (EC) and vascular smooth muscle cells (VSMC) to the IF. Three experiments were conducted to test that the mitogenic activity is induced by protein <em>growth</em> <em>factors</em>: 1) protein digestion of the myocardial IF with Pronase-coupled latex beads; 2) heat inactivation (boiling) of the IF; and 3) neutralization of the mitogenic activity with antibodies for basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF). Blood flow was reconstituted to baseline levels during occlusion after 3 wk of repetitive coronary occlusions. After initiation of occlusion the mitogenic activity of the myocardial IF on VSMC and EC increased up to days 12-14 and was reduced on days <em>19</em>-23. Pronase treatment and heat inactivation blocked the mitogenic effect. Treatment with antibodies for bFGF and VEGF neutralized the proliferative response to the myocardial IF at specific times. bFGF antibody inhibited the mitogenic effect significantly on days 12-14. VEGF antibody neutralized the mitogenicity of the myocardial IF on day 7, days 12 and 13, and days <em>19</em> and 20 significantly. We conclude that myocardial IF harvested from ischemic myocardium is highly mitogenic up to 2 wk after initiation of repetitive coronary occlusions. After 3 wk of ischemia, the degree of mitogenic activity for VSMC and EC was decreased from peak levels. The antibodies could not neutralize the mitogenic effect of the myocardial IF during this time period. These results suggest that mitogens are expressed during various stages of collateral development in a time-dependent manner, that the mitogens are proteinaceous in nature, and that bFGF and VEGF are released into the myocardial IF.

Monitoring bile acids as signal molecules in combination with a bile acid synthesis marker and the FXR regulator <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>), this study addresses significant postprandial changes. The efficacy of the different pathways to regulate bile acid synthesis through short heterodimer partner (SHP) dependent FXR modulation in liver, and SHP independent activation via FGF<em>19</em> is demonstrated. Characteristic changes of the bile acid profile during an oral glucose tolerance test (oGTT) were investigated in 73 individuals. 15 bile acid species including conjugated and unconjugated forms were quantitatively determined with LC-MS/MS in serum samples collected at three time points during the oGTT. All conjugated bile acid species showed the same time course, a significant increase at 60 min after the glucose intake and an incline at 120 min. In contrast, a consistent decline of all unconjugated bile acids was monitored. 7α-Hydroxy-4-cholesten-3-one, an early bile acid synthesis marker, showed an inverse response with a significant decrease at 60 min which proves the efficient and rapid downregulation of CYP7A1 via FXR activation through bile acid signaling. Significantly higher levels of FGF<em>19</em> were observed 120 min after glucose intake and 60 min after bile acid excursion.

OBJECTIVE

Central administration of ligands for <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs) such as <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>19</em> (FGF<em>19</em>) and FGF21 exert glucose-lowering effects in rodent models of obesity and type 2 diabetes (T2D). Conversely, intracerebroventricular (icv) administration of the non-selective FGFR inhibitor (FGFRi) PD173074 causes glucose intolerance, implying a physiological role for neuronal FGFR signaling in glucose homeostasis. The current studies were undertaken to identify neuroendocrine mechanisms underlying the glucose intolerance induced by pharmacological blockade of central FGFRs.

METHODS

Overnight fasted, lean, male, Long-Evans rats received icv injections of either PD173074 or vehicle (Veh) followed 30 min later by performance of a frequently sampled intravenous glucose tolerance test (FSIGT). Minimal model analysis of glucose and insulin data from the FSIGT was performed to estimate insulin-dependent and insulin-independent components of glucose disposal. Plasma levels of lactate, glucagon, corticosterone, non-esterified free fatty acids (NEFA) and catecholamines were measured before and after intravenous (iv) glucose injection.

RESULTS

Within 20 min of icv PD173074 injection (prior to the FSIGT), plasma levels of lactate, norepinephrine and epinephrine increased markedly, and each returned to baseline rapidly (within 8 min) following the iv glucose bolus. In contrast, plasma glucagon levels were not altered by icv FGFRi at either time point. Consistent with a previous report, glucose tolerance was impaired following icv PD173074 compared to Veh injection and, based on minimal model analysis of FSIGT data, this effect was attributable to reductions of both insulin secretion and the basal insulin effect (BIE), consistent with the inhibitory effect of catecholamines on pancreatic β-cell secretion. By comparison, there were no changes in glucose effectiveness at zero insulin (GEZI) or the insulin sensitivity index (SI). To determine if iv glucose (given during the FSIGT) contributed to the rapid resolution of the sympathoadrenal response induced by icv FGFRi, we performed an additional study comparing groups that received iv saline or iv glucose 30 min after icv FGFRi. Our finding that elevated plasma catecholamine levels returned rapidly to baseline irrespective of whether rats subsequently received an iv bolus of saline or glucose indicates that the rapid reversal of sympathoadrenal activation following icv FGFRi was unrelated to the subsequent glucose bolus.

CONCLUSIONS

The effect of acute inhibition of central FGFR signaling to impair glucose tolerance likely involves a stress response associated with pronounced, but transient, sympathoadrenal activation and an associated reduction of insulin secretion. Whether this effect is a true consequence of FGFR blockade or involves an off-target effect of the FGFR inhibitor requires additional study.

The influence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), a central nervous system (CNS)-derived molecule, on survival of trunk neural crest cells was investigated. As previously shown (C. Kalcheim and N. M. Le Douarin, <em>19</em>86, Dev. Biol. 116, 451-466), the interposition of untreated silastic membranes between neural tube and neural crest cells of the dorsal root ganglion (DRG) anlage led to selective death of neural crest cells that remained distally located with respect to the implants. Membranes were then treated with laminin and bFGF (100 ng/ml) and implanted. Under these conditions, rescued cells were observed for over 30 hr after grafting in 15 of <em>19</em> embryos. In contrast, no surviving cells could be found in any of 10 control embryos implanted with laminin-treated silastic membranes. We have also investigated the effects of bFGF on survival of identified subpopulations of trunk neural crest cells cultured with somite cells in a serum-free, chemically defined medium. bFGF promoted a dose-dependent increase in the number of HNK-1-positive nonneuronal cells in 1- to 4-day-old cultures (1.8- to 8.2-fold over controls using FGF at concentrations of 10 pg/ml to 1 ng/ml, respectively). FGF had no mitogenic effect on the neural crest-derived nonneuronal cells since the number of HNK-1-immunoreactive nonneuronal cells having incorporated [3H]thymidine into their nuclei remained unchanged in control as compared to treated cultures. However, the same concentrations of FGF were found to stimulate the incorporation of [3H]thymidine into acid-insoluble material in somite cultures devoid of neural crest. Moreover, bFGF significantly enhanced survival of nonneuronal cells in pure neural crest cultures established from neural crest clusters, thus demonstrating a direct effect of bFGF on survival and/or differentiation of neural crest-derived nonneuronal cells. These data support the hypothesis that CNS-derived molecules influence early development of selective subsets of neural crest cells developing into sensory ganglia.

BACKGROUND

Chronic injury to the liver, such as viral hepatitis, alcoholism, non-alcoholic fatty liver disease (NAFLD) or nonalcoholic steatohepatitis (NASH), promotes extracellular matrix deposition and organ scarring, termed hepatic fibrosis. Fibrosis might progress to cirrhosis and predisposes to hepatocellular carcinoma (HCC), but is also associated with extrahepatic morbidity and mortality in NAFLD/NASH. The improved understanding of pathogenic mechanisms underlying chronic inflammation and fibrogenesis in the liver prompted recent advances in antifibrotic therapies. Areas covered: We review recent advances in antifibrotic therapy, of which most are currently tested in clinical trials for NAFLD or NASH. This explains the manifold metabolic pathways as antifibrotic targets, including farnesoid X receptor (FXR) agonism (obeticholic acid, nonsteroidal FXR agonists), acetyl-CoA carboxylase inhibition, peroxisome proliferator-activator receptor agonism (elafibranor, lanifibranor, saroglitazar), and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-21 or FGF-<em>19</em> activation. Other antifibrotic drug candidates target cell death or inflammation, such as caspase (emricasan) or ASK1 inhibitors (selonsertib), galectin-3 inhibitors and reducing inflammatory macrophage recruitment by blocking chemokine receptors CCR2/CCR5 (cenicriviroc). Expert commentary: The tremendous advances in translational and clinical research fuels the hope for efficacious antifibrotic therapies within the next 5 years. Very likely, a combination of etiology-specific, metabolic, anti-inflammatory, and direct antifibrotic interventions will be most effective.

BACKGROUND

In the current study, the authors present a comprehensive genomic profile (CGP)-based study of advanced urothelial carcinoma (UC) designed to detect clinically relevant genomic alterations (CRGAs).

METHODS

DNA was extracted from 40 µm of formalin-fixed, paraffin-embedded sections from 295 consecutive cases of recurrent/metastatic UC. CGP was performed on hybridization-captured, adaptor ligation-based libraries to a mean coverage depth of 688X for all coding exons of 236 cancer-related genes plus 47 introns from <em>19</em> genes frequently rearranged in cancer, using process-matched normal control samples as a reference. CRGAs were defined as GAs linked to drugs on the market or currently under evaluation in mechanism-driven clinical trials.

RESULTS

All 295 patients assessed were classified with high-grade (International Society of Urological Pathology classification) and advanced stage (stage III/IV American Joint Committee on Cancer) disease, and 294 of 295 patients (99.7%) had at least 1 GA on CGP with a mean of 6.4 GAs per UC (61% substitutions/insertions/deletions, 37% copy number alterations, and 2% fusions). Furthermore, 275 patients (93%) had at least 1 CRGA involving 75 individual genes with a mean of 2.6 CRGAs per UC. The most common CRGAs involved cyclin-dependent kinase inhibitor 2A (CDKN2A) (34%), fibroblast growth factor receptor 3 (FGFR3) (21%), phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) (20%), and ERBB2 (17%). FGFR3 GAs were diverse types and included 10% fusions. ERBB2 GAs were equally divided between amplifications and substitutions. ERBB2 substitutions were predominantly within the extracellular domain and were highly enriched in patients with micropapillary UC (38% of 32 cases vs 5% of 263 nonmicropapillary UC cases; P<.0001).

CONCLUSIONS

Using a CGP assay capable of detecting all classes of GA simultaneously, an extraordinarily high frequency of CRGA was identified in a large series of patients with advanced UC. Cancer 2016;122:702-711. © 2015 American Cancer Society.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) have been known to be potent stimulators of vascular endothelial cell proliferation and angiogenesis. Recent experimental evidence indicates that basic FGF (bFGF) is also involved in modulation of arterial pressure. In this study, we investigated the effects of acidic FGF (aFGF) and bFGF on muscle microcirculation using isolated arterioles and intact cremaster muscles of the at. In isolated microvessels, aFGF and bFGF (10(-12)-10(-8) M) significantly increased arteriolar diameter in a dose-dependent and time-dependent manner. This effect was abolished during inhibition of nitric oxide synthesis by NG-monomethyl-L-arginine (L-NMMA, 10(-4) M) but was not affected by indomethacin (10(-4) M), an inhibitor of the cyclooxygenase pathway of arachidonic acid metabolism. The vasodilation induced by FGFs was not observed in endothelium-denuded vessels. Furthermore, we studied microvascular hemodynamics in response to the <em>growth</em> <em>factors</em> in the cremaster muscle using intravital microscopy. Both aFGF and bFGF dilated arterioles of the intact cremaster muscle in a pattern similar to that observed in the isolated arterioles. At a concentration of 10(-10) M, aFGF caused a <em>19</em>% increase in vessel diameter and 56% increase in blood flow. Administration of L-NMMA blocked by FGF-induced vasodilation and hyperemia. These results suggest that FGFs modulate blood flow in the skeletal muscle by acting on the endothelium of arterioles. The signaling mechanism of FGF-induced vasodilation involves the synthesis of nitric oxide by arteriolar endothelium.

In various invasive human tumors, c-ets-1 mRNA was found to be selectively expressed in stromal <em>fibroblasts</em>. We have now investigated the possibility that soluble <em>factors</em> could regulate c-ets-1 expression in cultured human <em>fibroblasts</em>. We show that both conditioned media from tumor cell lines and a number of characterized cytokines and <em>growth</em> <em>factors</em> were able to induce c-ets-1 expression. TNF alpha and IL-1 alpha were the most potent c-ets-1 stimulators, inducing rapid (within 1 h) and long-lasting (<em>19</em> h) increases of c-ets-1 mRNA and protein expression. In contrast, bFGF, EGF, and PDGF were mainly delayed stimulators, with maximal stimulation being detected by <em>19</em> h. In addition, these <em>growth</em> <em>factors</em> potentiated the rapid induction of c-ets-1 by TNF alpha. While all these <em>factors</em> were able to stimulate c-ets-1 expression, TGF beta was found to be ineffective. Using inhibitors of transcription and translation, we also found that increase of c-ets-1 mRNA by TNF alpha resulted from new transcription rather than from stabilization and did not require new protein synthesis. These results demonstrated that c-ets-1 is a new nuclear target for several <em>factors</em> and behaves as an early-response gene for TNF alpha.

The purpose of this review is to highlight clinical research in osteoarthritis (OA). A literature search was conducted using PubMed (http://www.ncbi.nlm.nih.gov/pubmed/) with the search terms "osteoarthritis [All Fields] AND treatment [All Fields]" and the following limits activated: humans, English language, all adult <em>19</em>+ years, published between April 1, 2014 and April 1, 2015. A second literature search was then conducted with the search terms "osteoarthritis [All Fields] AND epidemiology [All Fields]", with the same limits. Reports of surgical outcome, case series, surgical technique, tissue sample or culture studies, trial protocols, and pilot studies were excluded. Of 1523, 150 were considered relevant. Among epidemiologic and observational clinical studies, themes included physical activity, early knee OA, and confidence/instability/falls. Symptom outcomes of pharmacologic treatments were reported for methotrexate, adalimumab, anti-nerve <em>growth</em> <em>factor</em> monoclonal antibodies, strontium ranelate, bisphosphonates, glucosamine, and chondroitin sulfate, and structural outcomes of pharmacologic treatments for strontium ranelate, recombinant human <em>fibroblast</em> <em>growth</em> <em>factor</em> 18, and glucosamine and chondroitin sulfate. Symptom outcomes of non-pharmacologic interventions were reported for: neuromuscular exercise, quadriceps strengthening, weight reduction and maintenance, TENS, therapeutic ultrasound, stepped care strategies, cognitive behavior therapy for sleep disturbance, acupuncture, gait modification, booster physical therapy, a web-based therapeutic exercise resource center for knee OA; hip physical therapy for hip OA; and joint protection and hand exercises for hand OA. Structure outcomes of non-pharmacologic interventions were reported for patellofemoral bracing.

Chronic idiopathic constipation is highly prevalent among adults. Bile acids (BAs) and the enterohepatic BA circulation modulate colonic secretion and motility that affect transit. BAs in the colon have a dual action as osmotic and stimulant agents. Newer agents, such as elobixibat (A3309), an inhibitor of the ileal BA transporter, have the potential to improve significantly the management of chronic constipation, with minimal adverse effects. Elobixibat modulates the enterohepatic BA circulation, enhancing the delivery of BAs to the colon where they induce secretory and motor effects. Secondary effects of the inhibition of BA absorption are reduced activation of the farnesoid X receptor, decreased secretion of <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>19</em> into the portal circulation, and increased BA synthesis. This review focuses on the role of BAs, the enterohepatic BA circulation, and an ileal BA transporter inhibitor (elobixibat) in chronic constipation.

Multipotential progenitor cells grown from central nervous system (CNS) tissues in defined media supplemented with epidermal <em>growth</em> <em>factor</em> (EGF), when attached to a suitable substratum, differentiate to express neural and glial histochemical markers and morphologies. To assess the functional characteristics of such cells, expression of voltage-gated Na+ and K+ currents (INa, IK) was studied by whole-cell patch clamp methods in progenitors raised from postnatal rat forebrain. Undifferentiated cells were acutely dissociated from proliferative "spheres," and differentiated cells were studied 1-25 days after plating spheres onto polylysine/laminin-treated coverslips. INa and IK were detected together in 58%, INa alone in 11%, and IK alone in <em>19</em>% of differentiated cells recorded with K(+)-containing pipettes. With internal Cs+ (to isolate INa), INa up to 45 pA/pF was observed in some cells within 1 day after plating. I Na ranged up to 150 pA/pF subsequently. Overall, 84% of cells expressed I Na, with an average of 38 pA/pF. INa had fast kinetics, as in neurons, but steadystate inactivation curves were strongly negative, resembling those of glial INa. Inward tail currents sensitive to [K+]out were observed upon repolarization after the 10-ms test pulse with internal Cs+, indicating the expression of K+ channels in 82% of cells. In contrast to the substantial currents observed in differentiating cells, little or no INa or Ik-tail currents were detected in recordings from cells acutely dissociated from spheres. Thus, in the presence of EGF, ionic currents develop early during differentiation induced by attachment to an appropriate substratum. Cells switched from EGF to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) when plated onto coverslips showed greatly reduced proliferation and developed less neuron-like morphologies than cells plated in the presence of EGF. INa was observed in only 53% of bFGF-treated cells, with an average of 9 pA/pF. Thus, in contrast to reports that bFGF promotes neuronal differentiation in some CNS progenitor populations, our EGF-generated postnatal rat CNS progenitors do not develop neuronal characteristics when switched to medium containing bFGF. Thus, differentiated CNS progenitors can express a mix of neuronal and glial molecular, morphological, and electrophysiological properties that can be modified by culture conditions.

Metabolic diseases like diabetes and obesity are major risk <em>factors</em> for breast cancer. Aberrant expression of metabolic effectors such as <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) could be therefore associated with the disease. The expression of FGF<em>19</em> was examined in <em>19</em>3 archival breast tumor samples by immunohistochemistry and evaluated semi-quantitatively by determining the staining index and correlating it with clinicopathological parameters using Fisher's exact test. The correlation between FGF<em>19</em> expression and 5-year disease-specific survival rate was determined using the univariate Kaplan-Meier analysis. The prognostic value of FGF<em>19</em> expression was evaluated using the multivariate Cox regression analysis. Of the <em>19</em>3 tumors analyzed, 40% were classified with low FGF<em>19</em> expression, whereas 60% were categorized as tumors with high FGF<em>19</em> expression. There was a highly significant correlation between high FGF<em>19</em> expression and patients' age (p = 0.008) as well as 5-year disease-specific survival (p = 0.001). However, FGF<em>19</em> expression did not show any significant correlations with other clinicopathological parameters, including hormonal status, tumor grade, tumor size, or lymph node status. Univariate Kaplan-Meier log rank analysis showed that patients with high FGF<em>19</em> expression exhibited a significantly shorter disease-specific 5-year survival (p = 0.007). This effect was exacerbated by lymph node metastasis (p = 0.001), negative estrogen receptor (ER) status (p = 0.002), or old age (p = 0.013). Multivariate analysis showed that high FGF<em>19</em> expression could be an independent prognostic marker of disease-specific survival in breast cancer patients (p = 0.030). Quantification of FGF<em>19</em> expression appears to provide valuable prognostic information in breast cancer, particularly in older patients with lymph node metastasis and negative ER status.

Routine treatment of burns with cultured skin substitutes (CSS) has been limited by poor engraftment and by scarring. Hypothetically, topical application of essential nutrients and/or <em>growth</em> <em>factors</em> may support epithelial survival temporarily during graft vascularization. CSS, composed of human epidermal keratinocytes and dermal <em>fibroblasts</em> attached to collagen-glycosaminoglycan substrates, were incubated for <em>19</em> d in media optimized for keratinocytes. CSS, human xenografts, murine autografts, or no grafts were applied orthotopically to full-thickness skin wounds (2 x 2 cm) in athymic mice. Wounds were irrigated for 14 d with 1 ml/d modified cell culture medium or with saline containing epidermal <em>growth</em> <em>factor</em>, or were treated with dry dressings. After 6 weeks, treated sites were scored for percentage original wound area (mean +/- SEM) and percentage HLA-ABC-positive healed wounds [(number positive/n) x 100], and tested for significance (analysis of variance, p < 0.0001; Tukey test, p < 0.05). The data showed that CSS irrigated with nutrient medium were not statistically different in wound area (67.8 +/- 5.1%) from murine autografts (63.3 +/- 2.9%) but were statistically larger than human xenograft, no graft, or CSS treated with saline irrigation or dry dressings. HLA-ABC expression was 100% in CSS with nutrient irrigation, 86% in CSS with saline irrigation, 83% in CSS without irrigation, and 75% in xenografts with nutrient irrigation. These findings suggest that availability of essential nutrients supports keratinocyte viability during graft vascularization of CSS.

OBJECTIVE

The purpose of this study is to demonstrate the effect of culture density on the steady state mRNA levels of fibroblast growth factor-2 (FGF-2) when retinal pigment epithelial (RPE) cells are subjected to oxidative stress in vitro.

METHODS

Subconfluent and confluent cultures of the established RPE cell line ARPE-19, were treated with increasing concentrations of tert-butyl hydroperoxide (tBH) or hydrogen peroxide (H(2)O(2)). Cell viability was measured using the WST-1 assay, and intracellular reactive oxygen intermediate (ROI) production was quantified by dichlorofluoroscein (DCF) fluorescence. Steady state changes in heme oxygenase-1 (HO-1) and FGF-2 mRNAs were measured by Northern blot analysis.

RESULTS

Confluent cultures of ARPE-19 cells were less susceptible than subconfluent cultures to the toxic effects of the chemical oxidants. The intracellular reactive oxygen intermediate production was higher in subconfluent than confluent cultures with increasing tBH concentration. At nontoxic concentrations of tBH and H(2)O(2), a dose dependent increase in FGF-2 expression was seen as a function of culture density. FGF-2 mRNA expression was induced after tBH treatment in subconfluent, but not confluent cells. On the other hand, FGF-2 mRNA induction was observed after H(2)O( 2) treatment in confluent, but not subconfluent cultures. In contrast, no density dependent induction of HO-1 mRNA was seen after treatment with either tBH or H(2)O(2).

CONCLUSIONS

These results suggest that care should be taken to control for cell density in similar types of in vitro experiments.

A protein with a molecular mass of <em>19</em> kDa has been purified to homogeneity from 11-day-old chick embryos using a procedure involving chromatography on heparin - Sepharose, immunoaffinity resin and C4 reversed-phase. Indirect immunofluorescence studies, using polyclonal and monoclonal antibodies raised against this protein, indicate that it is essentially localized within the basement membranes in early embryonic tissues. After the 18th day of embryonic life and in post-hatched chicken, this protein could only be detected in some eye basement membranes. It appears to be bound to heparan sulfate chains of the proteoglycan present in these structures. Thus, the protein exhibits similar properties to those previously described for <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF), such as heparin affinity, molecular mass and localization in the basement membranes. In contrast, this protein is present in much larger amounts than FGFs, at least in 11-day-old embryos. Furthermore, the first 17 amino acid residues of the N-terminal sequence show that it does not strictly correspond to any previously described protein.

Adults with diabetes (DM) and chronic kidney disease (CKD) are at risk for vitamin D (vitD) insufficiency, suboptimal bone health and reduced quality of life (QoL) due to limited sunlight exposure, poor vitD intake and CKD.

This open-labeled, randomized clinical trial, compared the impact of daily (2000 IU/D) verses monthly (40,000 IU/month) vitD3 supplementation over six months on markers of vitD status, bone health and QoL in adults with DM and CKD (stages: 1-4).

Participants (18-80 years) were randomized to daily (n = 60) or monthly (n = 60) vitD3 for six months. Primary outcomes included: vitD status (25-hydroxyvitD [25(OH)D], 1, 25-dihydroxyvitD [1,25(OH)2D], bone health (bone mineral density [BMD] and serum concentrations of bone-specific alkaline phosphatase [BSAP], osteocalcin [OC], N-telopeptide-type 1-collagen [NTx]) and Fibroblast Growth Factor-23 (FGF-23). Secondary outcomes included QoL (Short Form-36 questionnaire).

Adherence by dose allocation over six months was 95.0 ± 5.7% (daily) and 94.1 ± 4.1% (monthly), respectively (p = 0.44); resulting in an overall median [95% CI] increase in serum 25(OH)D of 19 (12-26) nmol/L (p < 0.001). Serum 25(OH)D increased at three (p < 0.001) and six months (p < 0.001) in the daily and monthly groups, respectively. No significant differences over six months between groups were observed in serum concentrations of 1,25(OH)2D, FGF-23, OC and NTx, BMD and QoL measures (p>> 0.05). Serum 25(OH)D ≥ 75 nmol/L was associated with significant reductions in BSAP (p = 0.01) and improved physical functioning vs those with concentrations < 75 nmol/L (62.5 ± 26.8 vs 52.7 ± 26.3; p = 0.03) in the monthly and daily groups, respectively.

Daily (2000 IU/D) and monthly (40,000 IU/month) vitD3 supplementation for six months in adults with DM and CKD was safe, and resulted in equivalent adherence and improvements in overall vitD status, but only modest changes in markers of bone health and QoL.

BACKGROUND

Diabetes and associated metabolic conditions have reached pandemic proportions worldwide and there is a clear unmet medical need for new therapies that are both effective and safe. FGF19 is a distinctive member of the FGF family that functions as an endocrine hormone.

METHODS

An up-to-date report on the exciting findings related to the involvement of FGF19 in the regulation of glucose, bile acid metabolism and energy expenditure. The role of FGF receptors in these different activities. The therapeutic potential of FGF19 and the engineering opportunities for removing undesirable mitogenic activity.

CONCLUSIONS

The ability of FGF19 to regulate bile acid homeostasis, gallbladder filling and tumor development and its potent ability to normalize glucose, lipid and energy homeostasis have made it a potential therapeutic target for the treatment of patients with gallstones, cancer and metabolic diseases, among others. Its potential utility as a novel therapeutic for both type 1 and type 2 diabetes is of particular interest. The ability to separate the undesired mitogenic activity from its potent metabolic activities has opened new opportunities for the development of potential therapeutic molecules based on FGF19 in treating various conditions associated with metabolic syndrome.