The mechanism of peritoneal fibrosis in patients on continuous ambulatory peritoneal dialysis is poorly understood. The production of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) by human peritoneal mesothelial cells cultured in high glucose medium was investigated, and the behavior of peritoneal <em>fibroblasts</em>, as well as the inhibitory effect of prednisolone, was assessed. Reverse transcriptase-PCR and immunocytochemistry showed the expression of glucocorticoid receptors in mesothelial cells. The semiquantitative reverse transcriptase-PCR showed that high glucose medium (4.0%) increased bFGF mRNA by 2.5-fold relative to control medium (0.1% glucose), with 83% suppression of the increase by 1 microM prednisolone. The bFGF protein level in culture supernatant was also increased by 1.5-fold in high glucose medium, with this change showing 45% suppression by 1 microM prednisolone. These effects of prednisolone were prevented by a glucocorticoid receptor antagonist (RU486) in a concentration-dependent manner. The proliferation of peritoneal <em>fibroblasts</em> was increased 1.9-fold by the supernatant of mesothelial cells cultured in high glucose medium, with 85% suppression by 1 microM prednisolone and suppression to <em>16</em>% below basal proliferation by an anti-bFGF neutralizing antibody (10 microg/ml), whereas proliferation showed a concentration-dependent increase on addition of an anti-transforming <em>growth</em> <em>factor</em> beta-neutralizing antibody. Recombinant bFGF (50 to 1000 pg/ml) likewise caused a concentration-dependent increase of peritoneal <em>fibroblast</em> proliferation and fibronectin release by these cells was also increased (at 50 to 5000 pg/ml). These results suggest the potential importance of bFGF for initiation of peritoneal fibrosis and the possible efficacy of glucocorticoids for preventing such fibrosis in patients receiving peritoneal dialysis.

OBJECTIVE

Recent studies showed that Fas ligand (FasL) induced apoptosis in tumor cells and suppressed the immune response in several types of tumors. However, the toxicity of FasL limited further administration. This study delivered FasL in prostate cancer cells using an improved prostate-restricted replicative adenovirus (PRRA), thereby improving the antitumor effect while decreasing systemic toxicity.

METHODS

We designed a FasL-armed PRRA, called AdIU3, by placing adenoviral E1a and E4 genes, FasL cDNA, and E1b gene under the control of two individual PSES enhancers. Tissue-specific viral replication and FasL expression were analyzed, and the tumor killing effect of AdIU3 was investigated both in vitro and in vivo using androgen-independent CWR22rv s.c. models via local administration and bone models via systemic administration. The safety of systemic administration of AdIU3 was evaluated. AdCMVFasL, in which FasL was controlled by a universal cytomegalovirus (CMV) promoter, was used as a control.

RESULTS

AdIU3 enhanced FasL expression in prostate-specific antigen (PSA)/prostate-specific membrane antigen (PSMA)-positive cells but not in PSA/PMSA-negative cells. It induced apoptosis and killed PSA/PMSA-positive prostate cancer cells but spared normal human <em>fibroblasts</em>, hepatocytes, and negative cells. The increase in killing activity was confirmed to result in part from a bystander killing effect. Furthermore, AdIU3 was more effective than a plain PRRA in inhibiting the <em>growth</em> of androgen-independent prostate cancer xenografts and bone tumor formation. Importantly, systemic administration of AdIU3 resulted in undetectable toxicity, whereas the same doses of AdCMVFasL killed all mice due to multiviscera failure in <em>16</em> h.

CONCLUSIONS

AdIU3 decreased the toxicity of FasL by controlling its expression with PSES, with greatly enhanced prostate cancer antitumor efficacy. The results suggested that toxic antitumor factors can be delivered safely by a PRRA.

BACKGROUND

To determine the correlation between clinical activity scores (CAS) of thyroid-associated ophthalmology (TAO) patients and their locally produced and/or systemically circulating insulin-like growth factor-1 (IGF-1), and to assess the possible pathogenic role of IGF-1 in TAO.

METHODS

Eighteen patients with TAO, and 16 age- and gender-matched controls were included in the present study. Among them, orbital tissue surgically collected from five TAO patients and five healthy controls was used for orbital fibroblasts (OFs) culture and in vitro study. Total and free IGF-1 in serum levels were determined by an ELISA kit for all the participants in this study. The IGF-1 concentration in culture media of OFs was determined using a noncompetitive time-resolved radioimmunoassay kit. The effect of octreotide (OCT), a somatostatin analog, on proliferation of OFs was assessed using the MTT assay. IGF-1 mRNA levels were measured by real-time polymerase chain reaction (PCR).

RESULTS

Cultured OFs from both TAO patients and normal donors secreted IGF-1, and the secretion continued over a 72 hour period in vitro. IGF-1 secretion by OFs was elevated in the TAO group. Both the elevated secretion of IGF-1 and proliferation of OFs from TAO patients could be inhibited by OCT. Result of quantitative PCR showed that IGF-1 mRNA expression by OFs in TAO patients was up-regulated more than 2-fold compared with normal controls (P < 0.05), and this up-regulation was prevented by OCT treatment. Total and free serum IGF-1 levels in TAO patients were similar to those of normal controls. However, the IGF-1 level in cultured medium of OFs from TAO patients, but not serum levels of IGF-1, was positively correlated with CAS (r = 0.97, P = 0.017).

CONCLUSIONS

Local production of IGF-1 by cultured OFs may be positively correlated with CAS, whereas systemically circulating IGF-1 may remain unchanged in TAO patients. Thus, locally produced IGF-1 may develop a role in the pathogenesis of TAO in an autocrine or paracrine fashion. The inhibitory effect of OCT on proliferating and IGF-1 mRNA levels of cultured OFs from TAO patients may be used as the mechanistic explanation for somatostatin analog as a valuable option in the treatment of TAO.

OBJECTIVE

Transforming growth factor beta-1 (TGF-beta1) is a known fibrogenic factor with immunosuppressive properties. We wanted to determine the effect of stimulation with TGF-beta1 on nasal polyp-derived fibroblasts and assess the role this molecule would have in polyp formation and growth.

METHODS

Nasal-polyp derived fibroblasts were cultured with or without TGF-beta1, and proliferation and cytokine secretion were measured.

METHODS

Fibroblasts were isolated from nasal polyps following endoscopic surgery. Cells were plated and grown until confluent, after which they were split and used in assays. Cells were stimulated with TGF- beta1 and mRNA collected after 16 hours, supernatants after 72 hours, and proliferation measured after 96 hours of culture.

RESULTS

TGF-beta1 significantly (P < .02) increased proliferation of nasal-polyp derived fibroblasts. We examined the expression of inflammatory cytokines and found that TGF-beta1 decreased expression of CCL2 (MCP-1), CCL5 (RANTES), CCL11 (eotaxin), granulocyte-colony stimulating factor (G-CSF), and GM-CSF (P < .05). In contrast, incubation with TGF-beta1 increased fibronectin, procollagen, vascular endothelial growth factor (VEGF), and TGF-beta2 protein production (P < .05). For select samples, we confirmed that the increased protein production was due to increased mRNA expression.

CONCLUSIONS

These studies suggest that TGF-beta1 expression in polyp tissue can have dual effects. One role is to act as an anti-inflammatory agent shown by the ability to inhibit pro-inflammatory mRNA and protein production. At the same time, TGF-beta1 expression leads to increases in factors involved in fibrosis and angiogenesis, promoting remodeling and cell growth.

BACKGROUND

This research elucidates the question of whether common and widespread dental procedures (DP) like root filling (RF) and the removal of wisdom teeth (WT) contribute to chronic inflammation in the jawbone. Dentists, in carrying out these DP, can set off defective wound healing in the jawbone in ignorance of its connection to inflammatory mediators and the possibility of it being a hidden cause of chronic systemic diseases (SYD).

METHODS

We examined samples of the jawbone for seven cytokines by multiplex analysis in three groups of jawbone areas. In order to clarify systemic interrelations, specimens from <em>16</em> patients were analyzed in areas of former surgery in the retromolar wisdom tooth area; specimens from <em>16</em> patients were analyzed in the jawbone, apically of teeth with RF; and specimens from 19 patients were of the healthy jawbone. Each of the retromolar and the apical jawbone samples showed clinically fatty degenerated and osteonecrotic medullary changes.

RESULTS

All fatty necrotic and osteolytic jawbone (FDOJ) samples showed regulated on activation, normal T-cell expressed and secreted (RANTES) and fibroblast growth factor (FGF)-2 as the only extremely overexpressed cytokines. FDOJ cohorts showed a 30-fold mean overexpression of RANTES and a 20-fold overexpressed level of FGF-2 when compared to healthy controls.

CONCLUSIONS

As RANTES is discussed in the literature as a possible contributor to inflammatory diseases, and though it might have oncogenic effects, we hypothesize that FDOJ in areas of improper and incomplete wound healing in the jawbone might act as hyperactivated signaling pathways, while serving as an unknown source of "silent inflammation". Because of the wide range of RANTES in immune diseases, treating FDOJ can cover many potential prediction or prognosis of individual outcomes.

Intravenous administration of human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> up to 100 micrograms/kg/day to Sprague-Dawley rats caused changes in the kidneys that included enlargement, vacuolation, and karyomegaly of podocytes in glomeruli, dilatation and cast formation in tubules, thickening of the media in the lobular arteries, and hyperplasia of the epithelium of the papilla and collecting ducts. In cynomolgus monkeys there was hyperplasia of the parietal epithelium of Bowman's capsule in the glomeruli, tubular dilatation, and minimal arteriopathy. These changes were only seen at 100 micrograms/kg/day. The development and eventual recovery over time were investigated in a sequence of sacrifices. In monkeys the first changes were seen after 7 days of treatment, but in rats only after <em>16</em> days. In both species the changes had partially resolved after 30 days of recovery and were considered to return to normal after 60 days without treatment. The morphological changes were accompanied by functional alterations that included proteinuria and raised blood urea. Changes that occurred in other tissues including bone, red blood cells, adrenals, ovaries, liver, gall bladder, spleen, mesenteric lymph nodes, thymus, aorta, salivary glands, and injection site are not described in this paper.

Insulin exerts many of its metabolic actions via the canonical phosphatidylinositide 3 kinase (PI3K)/Akt pathway, leading to phosphorylation and 14-3-3 binding of key metabolic targets. We previously identified a GTPase-activating protein (GAP) for Rac1 called RhoGAP22 as an insulin-responsive 14-3-3 binding protein. Insulin increased 14-3-3 binding to RhoGAP22 fourfold, and this effect was PI3K dependent. We identified two insulin-responsive 14-3-3 binding sites (pSer(<em>16</em>) and pSer(395)) within RhoGAP22, and mutagenesis studies revealed a complex interplay between the phosphorylation at these two sites. Mutating Ser(<em>16</em>) to alanine blocked 14-3-3 binding to RhoGAP22 in vivo, and phosphorylation at Ser(<em>16</em>) was mediated by the kinase Akt. Overexpression of a mutant RhoGAP22 that was unable to bind 14-3-3 reduced cell motility in NIH-3T3 <em>fibroblasts</em>, and this effect was dependent on a functional GAP domain. Mutation of the catalytic arginine of the GAP domain of RhoGAP22 potentiated <em>growth</em> <em>factor</em>-stimulated Rac1 GTP loading. We propose that insulin and possibly <em>growth</em> <em>factors</em> such as platelet-derived <em>growth</em> <em>factor</em> may play a novel role in regulating cell migration and motility via the Akt-dependent phosphorylation of RhoGAP22, leading to modulation of Rac1 activity.

This study was undertaken to investigate the potential of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) to delay photoreceptor cell loss in retinas of Fischer 344 rats which exhibit an age-related peripheral retinopathy. Eight male <em>16</em>-month-old Fischer 344 rats were injected intravitreally with 2.0 micrograms bFGF in the right eye, while the vehicle was injected into the left eye and all rats were killed at two months post-injection. Eight other Fischer rats were injected with either bFGF or vehicle at <em>16</em> and again at 18 months, then killed two months later. After enucleation, eyes were processed for light and electron microscopic and morphometric analyses. The distance from the ora serrata to the point where the outer nuclear layer (ONL) in the superior retina was two cells in thickness, called the die-back zone, that represents the extent of the peripheral retinopathy, was compared in bFGF- and vehicle-injected Fischer rats. These measurements revealed that the die-back zone in retinas of eyes injected twice with bFGF was significantly reduced (P < 0.01) when compared to this zone in retinas of age-matched rats that received vehicle injections. However, there was no significant difference (P>> 0.05) in this degeneration zone in retinas of 18-month-old rats that received a single-bFGF injection when compared to retinas of respective vehicle-injected rats. In addition, the ONL at 2 and 3 mm from the ora serrata in the superior retina of bFGF-injected 20-month-old Fischer rats was significantly thicker than corresponding regions in retinas of vehicle-control rats. Furthermore, the photoreceptor cells in the superior retina of 20-month-old rats which received two injections of bFGF had normal-appearing inner and outer segments, while the few photoreceptors had short inner and outer segments in vehicle-injected retinas of Fisher rats. These findings reveal that injections of bFGF into eyes of Fischer 344 rats significantly delays the progress of photoreceptor cell degeneration suggesting that exogenous bFGF may act as a survival-promoting <em>factor</em> in these aged retinas.

Alcoholic liver disease (ALD) develops in a subset of heavy drinkers (HDs). The goals of our study were to (1) characterize the global serum metabolomic changes in well-characterized cohorts of controls (Cs), HDs, and those with alcoholic cirrhosis (AC); (2) identify metabolomic signatures as potential diagnostic markers, and (3) determine the trajectory of serum metabolites in response to alcohol abstinence. Serum metabolic profiling was performed in 22 Cs, 147 HDs, and 33 patients with AC using ultraperformance liquid chromatography-tandem mass spectrometry. Hepatic gene expression was conducted in Cs (n = <em>16</em>) and those with AC (n = 32). We found progressive changes in the quantities of metabolites from heavy drinking to AC. Taurine-conjugated bile acids (taurocholic acid [TCA], 127-fold; taurochenodeoxycholic acid [TCDCA], 131-fold; and tauroursodeoxycholic acid, 56-fold) showed more striking elevations than glycine-conjugated forms (glycocholic acid [GCA], 22-fold; glycochenodeoxycholic acid [GCDCA], 22-fold; and glycoursodeoxycholic acid [GUDCA], 11-fold). This was associated with increased liver cytochrome P450, family 7, subfamily B, member 1 and taurine content (more substrates); the latter was due to dysregulation of homocysteine metabolism. Increased levels of GCDCA, TCDCA, GCA, and TCA positively correlated with disease progression from Child-Pugh A to C and Model for End-Stage Liver Disease scores, whereas GCDCA, GCA, and GUDCA were better predictors of alcohol abstinence. The levels of glucagon-like peptide 1 (GLP-1) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 21 but not FGF19 were increased in HDs, and all three were further increased in those with AC. <i>Conclusion:</i> Serum taurine/glycine-conjugated bile acids could serve as noninvasive markers to predict the severity of AC, whereas GLP-1 and FGF21 may indicate a progression from heavy drinking to AC.

Erythropoietin (EPO), clinically used as a hematopoietic drug, has received much attention due to its nonhematopoietic effects. EPO reportedly has beneficial effects on obesity and diabetes mellitus. We investigated whether interscapular brown adipose tissue (iBAT: main part of classical BAT) could play a role in EPO's anti-obesity and anti-diabetic effects in diet-induced obese mice. Four-week-old male C57BL/6J mice were fed a high-fat diet (HFD-Con), and half were additionally given an intraperitoneal injection of recombinant human EPO (200 IU/kg) (HFD-EPO) thrice a week for four weeks. At 8 weeks, EPO-injected mice showed significantly reduced body weight with reduced epididymal and subcutaneous white fat mass and unchanged caloric intake and locomotor activity. HOMA-IR (insulin resistance index) and glucose levels during intraperitoneal glucose tolerance test (IPGTT) were significantly lower in HFD-EPO mice than in HFD-Con mice. EPO-injected mice also showed increased oxygen consumption, indicative of metabolic rate, and skin temperature around iBAT tissue masses. EPO significantly upregulated the PRD1-BF1-RIZ1 homologous domain containing <em>16</em> (PRDM<em>16</em>), a transcriptional <em>factor</em> with a crucial role in brown adipocyte differentiation. EPO significantly increased phosphorylated signal transducer and activator of transcription 3 (STAT3), which is downstream of erythropoietin receptor (EpoR) and known to stabilize PRDM<em>16</em>. EPO's suppression of myocyte enhancer <em>factor</em> 2c (Mef2c) and microRNA-133a (miR-133a) via β3-adrenergic receptor caused PRDM<em>16</em> upregulation. EPO-mediated enhancement of EpoR/STAT3 and β-adrenergic receptor/Mef2c/miR-133 pathways dramatically increases total uncoupling protein 1 (UCP1), an essential enzyme for BAT thermogenesis. Furthermore, EPO activated BAT's endocrine functions. EPO facilitated <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) production and excretion in iBAT, associated with reduction of liver gluconeogenesis-related genes. Thus, EPO's improvement of obesity and glucose homeostasis can be attributed to increased iBAT thermogenic capacity and activation of BAT's endocrine functions.

OBJECTIVE

Progranulin is a growth factor that is active in wound repair and is an antagonist of tumor necrosis factor (TNF) receptors, regulating fibroblast activation, angiogenesis, and inflammation. Because long-standing activation of gene programs related to wound healing is a hallmark of systemic sclerosis (SSc), we sought to investigate the role of progranulin in SSc.

METHODS

Progranulin expression levels in human and murine skin samples were determined by immunohistochemical analysis and quantitative reverse transcription-polymerase chain reaction. The role of progranulin in fibroblast activation was examined using a gene-silencing technique. Progranulin levels in serum obtained from 60 patients with SSc and 16 healthy control subjects were determined by enzyme-linked immunosorbent assay.

RESULTS

Progranulin expression was increased in SSc dermal fibroblasts compared with normal dermal fibroblasts, both in vivo and in vitro. Transcription factor Fli-1, a deficiency of which is involved in the activation of SSc dermal fibroblasts, served as a potent repressor of the progranulin gene, and Fli-1(+/-) mice and bleomycin-treated wild-type mice exhibited up-regulated expression of progranulin in dermal fibroblasts. SSc dermal fibroblasts were resistant to the antifibrotic effect of TNF, but this resistance was reversed by gene silencing of progranulin. Serum progranulin levels were elevated in patients with early diffuse cutaneous SSc (dcSSc), especially in those with inflammatory skin symptoms, and were positively correlated with the C-reactive protein level.

CONCLUSIONS

Progranulin overproduction due to Fli-1 deficiency may contribute to the constitutive activation of SSc dermal fibroblasts by antagonizing the antifibrotic effect of TNF. Progranulin may also be involved in the inflammatory process associated with progressive skin sclerosis in early dcSSc.

Rheumatoid arthritis (RA) is a systemic autoimmune disease with high rates of morbidity and mortality. Previous studies proposed that the A disintegrin and metalloprotease (ADAM) family is involved in the regulation of inflammation and arthritis. Thus, the present study investigated whether ADAM10 is involved in the progression of RA. The effects of ADAM10 small interfering (si)RNA on the expression levels of tumor necrosis <em>factor</em> (TNF)‑α, interleukin (IL)‑6, IL‑8 and chemokine (C-X-C motif) ligand <em>16</em> (CXCL<em>16</em>) were determined in <em>fibroblast</em>‑like synoviocytes (FLS). In addition, the effects of ADAM10 siRNA on cell proliferation, invasion and migration in human RA‑FLS were assessed in vitro. The therapeutic efficacy and side‑effects of ADAM10 siRNA were examined in a mouse model of collagen‑induced arthritis (CIA). In vitro, ADAM10 silencing suppressed the expression of TNF‑α, IL‑6, IL‑8 and CXCL<em>16</em> in lipopolysaccharide (LPS)‑stimulated human RA‑FLS. LPS‑induced RA‑FLS proliferation, migration and invasion were significantly attenuated by ADAM10 knockdown. ADAM10 silencing inhibited the secretion of vascular endothelial <em>growth</em> <em>factor</em> A (VEGF‑A) and matrix metalloproteinase (MMP)‑3 and ‑9 from LPS‑stimulated human RA‑FLS, in addition to inhibiting the phosphoinositide 3‑kinase/AKT activation in LPS‑stimulated human RA‑FLS. In vivo, treatment with siRNA against ADAM10 for three weeks reduced the arthritis score. Serum levels of VEGF‑A, MMP‑3 and MMP‑9 were also reduced in CIA mice. These observations indicate that the inhibition of ADAM10 may be a viable therapeutic target in the amelioration of disease progression in RA by attenuating FLS proliferation, migration and invasion.

We isolated a population of proliferating cells from cultured human fetal hepatocytes of <em>16</em>-22 weeks gestational age. The cells shared a similar phenotype to that of mesenchymal stromal cells (MSCs) according to the International Society for Cellular Therapy (ISCT), including plastic adherence, antigen expression profile, and in vitro multilineage differentiation potential. Fetal liver (FL)-MSCs expressed the albumin gene, and harbored a subpopulation of CK18⁺ cells (20-40%), which defined their hepatic origin. However, when subjected to in vitro hepatic differentiation, FL-MSCs did not acquire significant liver functions. Quantitative analysis of conditioned medium (CM) collected from cultured cells revealed the presence of <em>growth</em> <em>factors</em> and chemokines with potential liver regenerative properties, the most relevant of which (concentration ≥3000 pg/ml) were SDF-1 alpha, IL-6, MCP-1, IL-8, MIP-1 beta, VEGF-A, Gro-alpha, and HGF. Culturing of FL-MSCs as spheroids significantly enhanced the secretion of HGF and bFGF (approximately 5-fold) compared with culture monolayers. Moreover, CM assessed in vitro induced capillary-like organization and migration of human umbilical vein endothelial cells (HUVECs) and <em>fibroblasts</em> as target cells. Interestingly, exosomes isolated from CM induced similar cellular responses in vitro with high efficiency and in a dose-dependent manner. FL-MSCs underwent several in vitro subcultivations, and did not stimulate allogenic T-cell proliferation thus suggesting a low immunogenicity. Furthermore, 5-year cryopreservation did not affect cell viability (approximately 90% of viable post-thawed FL-MSCs). These observations support the feasibility of a cell bank establishment for allogenic transplantation. We concluded that FL-MSCs or they secreted <em>factors</em> may be a valid alternative to hepatocyte transplantation in liver cell-based therapies.

As cancer development involves pathological vessel formation, <em>16</em> angiogenesis markers were evaluated as potential ovarian cancer (OC) biomarkers. Blood samples collected from 172 patients were divided based on histopathological result: OC (n = 38), borderline ovarian tumours (n = 6), non-malignant ovarian tumours (n = 62), healthy controls (n = 50) and <em>16</em> patients were excluded. Sixteen angiogenesis markers were measured using BioPlex Pro Human Cancer Biomarker Panel 1 immunoassay. Additionally, concentrations of cancer antigen 125 (CA125) and human epididymis protein 4 (HE4) were measured in patients with adnexal masses using electrochemiluminescence immunoassay. In the comparison between OC vs. non-OC, osteopontin achieved the highest area under the curve (AUC) of 0.79 (sensitivity 69%, specificity 78%). Multimarker models based on four to six markers (basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-FGF-basic, follistatin, hepatocyte <em>growth</em> <em>factor</em>-HGF, osteopontin, platelet-derived <em>growth</em> <em>factor</em> AB/BB-PDGF-AB/BB, leptin) demonstrated higher discriminatory ability (AUC 0.80-0.81) than a single marker (AUC 0.79). When comparing OC with benign ovarian tumours, six markers had statistically different expression (osteopontin, leptin, follistatin, PDGF-AB/BB, HGF, FGF-basic). Osteopontin was the best single angiogenesis marker (AUC 0.825, sensitivity 72%, specificity 82%). A three-marker panel consisting of osteopontin, CA125 and HE4 better discriminated the groups (AUC 0.958) than HE4 or CA125 alone (AUC 0.941 and 0.932, respectively). Osteopontin should be further investigated as a potential biomarker in OC screening and differential diagnosis of ovarian tumours. Adding osteopontin to a panel of already used biomarkers (CA125 and HE4) significantly improves differential diagnosis between malignant and benign ovarian tumours.

Hepatocellular carcinoma is an aggressive cancer with poor prognosis. <em>Fibroblast</em> <em>growth</em> <em>factor</em> 19, a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family, is a ligand for <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4. Moreover, it plays a crucial role in the progression of hepatocellular carcinoma. ASP5878 is a novel inhibitor of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors 1, 2, 3, and 4 that is under development. It inhibits <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 kinase activity with an IC50 of 3.5 nmol/L. ASP5878 potently suppressed the <em>growth</em> of the <em>fibroblast</em> <em>growth</em> <em>factor</em> 19-expressing hepatocellular carcinoma cell lines Hep3B2.1-7, HuH-7, and JHH-7. In the Hep3B2.1-7 cell line, ASP5878 inhibited the phosphorylation of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 and its downstream signaling molecules as well as induced apoptosis. Oral administration of ASP5878 at 3 mg/kg induced sustained tumor regression in a subcutaneous xenograft mouse model using Hep3B2.1-7. In HuH-7, an orthotopic xenograft mouse model, ASP5878 induced complete tumor regression and dramatically extended the survival of the mice. These results suggest that ASP5878 is a potentially effective therapeutic agent for hepatocellular carcinoma patients with tumors expressing <em>fibroblast</em> <em>growth</em> <em>factor</em> 19. Mol Cancer Ther; <em>16</em>(1); 68-75. ©20<em>16</em> AACR.

Idiopathic myelofibrosis (IMF) and secondary myelofibrosis (MF) are characterized by bone marrow (BM) fibrosis, neoangiogenesis, and increased extracellular matrix (ECM) proteins. These characteristics may be partially attributed to transforming <em>growth</em> <em>factor</em> beta (TGF-beta), a cytokine produced by monocytes. In myelofibrosis, monocytes are increased and activated with concomitant up-regulation of intracytoplasmic TGF-beta. We have therefore determined systemic TGF-beta in patients with either BM fibrosis: IMF, n = 18; MF, n = <em>16</em>; or without BM fibrosis: hematologic disorders with normal platelets (n = 31); high platelets (n = 9); or normal controls (n = 27). Compared with nonfibrosis sera, there was significant TGF-beta elevation in BM fibrosis sera (P < 0.0001). Most (>80%) of the TGF-beta is active and belongs to the-beta1 isoform. In situ hybridization and immunohistochemical analyses in BM biopsy sections showed a marked increase in TGF-beta1 only in patients with fibrosis. Moreover, TGF-beta protein was detected mainly in myelomonocytic-like predominant areas. To determine if another functionally similar cytokine, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), may be important to BM fibrosis, we quantitated sera levels and found elevation in 57% compared with 100% elevation for TGF-beta. The data indicate that irrespective of etiology, systemic TGF-beta is elevated in patients with BM fibrosis. TGF-beta likely plays an important role in the development of BM fibrosis. The study also provides a significant parameter for early therapeutic intervention in BM fibrosis.

The beneficial effects of <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> 21 (FGF21) on metabolic function and neuroprotection have been shown in earlier research. We have previously shown that the Dipeptidyl Peptidase 4 inhibitor, vildagliptin, also led to improved insulin sensitivity and brain function in the obese-insulin resistant condition. However, the comparative efficacy on the improvement of metabolic function and neuroprotection between FGF21 and vildagliptin in the obese-insulin resistant condition has never been investigated. Twenty-four male Wistar rats were divided into two groups, and received either a normal diet (ND, n=6) or a high fat diet (HFD, n=18) for <em>16</em> weeks. At week 13, the HFD-fed rats were divided into three subgroups (n=6/subgroup) to receive either a vehicle, recombinant human FGF21 (0.1mg/kg/day) or vildagliptin (3mg/kg/day), for four weeks. ND-fed rats were given a vehicle for four weeks. The metabolic parameters and brain function were subsequently investigated. The results demonstrated that the rats fed on HFD had obese-insulin resistance, increased systemic inflammation, brain mitochondrial dysfunction, increased brain apoptosis, impaired hippocampal plasticity, and demonstrated cognitive decline. FGF21 and vildagliptin effectively attenuated peripheral insulin resistance, brain mitochondrial dysfunction, brain apoptosis and cognitive decline. However, only FGF21 treatment led to significantly reduced body weight gain, visceral fat, systemic inflammation, improved hippocampal synaptic plasticity, enhanced FGF21 mediated signaling in the brain leading to prevention of early cognitive decline. These findings suggest that FGF21 exerts greater efficacy than vildagliptin in restoring metabolic function as well as brain function in cases of obese-insulin resistant rats.

<AbstractText>Non-alcoholic fatty liver disease (NAFLD) is associated with obesity and is considered to be an inflammatory disorder characterized by fatty acid accumulation, oxidative stress, and lipotoxicity. We have previously reported that epoxyeicosatrienoic acid-agonist (EET-A) has multiple beneficial effects on cardiac, renal and adipose tissue function while exhibiting both anti-inflammatory and anti-oxidant activities. We hypothesized that EET-A intervention would play a central role in attenuation of obesity-induced steatosis and hepatic fibrosis that leads to NAFLD.</AbstractText><AbstractText>We studied the effect of EET-A on fatty liver using db/db mice as a model of obesity. Mice were fed a high fat diet (HFD) for <em>16</em> weeks and administered EET-A twice weekly for the final 8 weeks.</AbstractText><AbstractText>db/db mice fed HFD significantly increased hepatic lipid accumulation as manifested by increases in NAS scores, hepatic fibrosis, insulin resistance, and inflammation, and decreases in mitochondrial mitofusin proteins (Mfn 1/2) and anti-obesity genes <em>Fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) and Cellular Repressor of E1A-Stimulated Genes 1 (CREG1). EET-A administration reversed the decrease in these genes and reduced liver fibrosis. Knockout of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in EET-A treated mice resulted in a reversal of the beneficial effects of EET-A administration.</AbstractText><AbstractText>EET-A intervention diminishes fatty acid accumulation, fibrosis, and NFALD associated with an increase in HO-1-PGC1α and increased insulin receptor phosphorylation. A pharmacological strategy involving EETs may offer a potential therapeutic approach in preventing fibrosis, mitochondrial dysfunction, and the development of NAFLD.</AbstractText>

UNASSIGNED

Wen Luo Yin (WLY) is a traditional Chinese formula, which has the traditional use of scattering cold pathogen, draining dampness, freeing the flow of network vessels and relieving pains. It is extensively used in the treatment of rheumatoid arthritis (RA) patients for more than 2000 years, but its actions on angiogenesis of RA have not been clarified. The present study aims to determine the anti-angiogenic activity of WLY on collagen-induced arthritis (CIA) rat model and in human fibroblast-like synoviocytes of RA (HFLS-RA) and human umbilical vein endothelial cells (HUVEC).

METHODS

For in vivo experiment, arthritis was induced by immunization with bovine II collagen in DA rats. Treatment with WLY (3.45, 6.9, 13.8 g/kg, p.o., daily), or vehicle began from day 1 to day 28 of first immunization. The arthritis score, arthritis incidence, microfocal computed tomography analysis and histopathology evaluation of inflamed joints were assessed. Angiogenesis was measured by synovial vessel density with immunohistochemistry and histomorphometric analysis in synovial membrane tissues of joints. For in vitro experiments, HFLS-RA and HUVEC were used. Assays to determine HFLS-RA migration and adhesion were performed in the presence of vascular endothelial growth factor (VEGF)165 or interleukin (IL)-1β and/or the WLY (8, 16, 32 mg/ml). Angiogenesis was assessed by measuring the migration, adhesion, and tube formation of HUVEC. Further the effect of treatment with WLY on expression levels of angiogenic activators in sera of CIA rats and in IL-1β-induced HFLS-RA were evaluated by enzyme linked immunosorbent assay.

RESULTS

WLY significantly decreased the arthritis score and arthritis incidence, and inhibited inflammation, pannus formation, cartilage and bone destruction of inflamed joints in CIA rats. More interestingly, doses of 3.45-13.8 g/kg WLY could markedly reduce the capillaries, small, medium and large vessel density in synovial membrane tissues of inflamed joints. Moreover, WLY suppressed the VEGF-induced chemotactic migration of HFLS-RA and HUVEC, and inhibited matrigel-induced cell adhesion of them. It also disrupted tube formation of HUVEC on matrigel. Furthermore, WLY significantly reduced the expression of angiogenic activators including tumor necrosis factor-α, IL-1β, IL-17, VEGF, VEGFR, angiopoietin (Ang)-1, Ang-2 and Ang-2 receptor in sera of CIA rats and/or in IL-1β-induced HFLS-RA/HUVEC.

CONCLUSIONS

Our data suggest for the first time that WLY posses the anti-angiogenic effect in RA both in vivo and in vitro by downregulating angiogenic activators.

There are two well-described thermogenic sites; brown adipose tissue (BAT) and skeletal muscle, which utilize distinct mechanisms of heat production. In BAT, mitochondrial metabolism is the molecular basis of heat generation, while it serves only a secondary role in supplying energy for thermogenesis in muscle. Here, we wanted to document changes in mitochondrial ultrastructure in these two tissue types based upon adaptation to mild (<em>16</em>°C) and severe (4°C) cold in mice. When reared at thermoneutrality (29°C), mitochondria in both tissues were loosely packed with irregular cristae. Interestingly, adaptation to even mild cold initiated ultrastructural remodeling of mitochondria including acquisition of more elaborate cristae structure in both thermogenic sites. The shape of mitochondria in the BAT remained mostly circular, whereas the intermyofibrilar mitochondria in the skeletal muscle became more elongated and tubular. The most dramatic remodeling of mitochondrial architecture was observed upon adaptation to severe cold. In addition, we report cold-induced alteration in levels of humoral <em>factors</em>: <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21), IL1α, peptide YY (PYY), tumor necrosis <em>factor</em> α (TNFα), and interleukin 6 (IL6) were all induced whereas both insulin and leptin were down-regulated. In summary, adaptation to cold leads to enhanced cristae formation in mitochondria in skeletal muscle as well as the BAT. Further, the present study indicates that circulating cytokines might play an important role in the synergistic recruitment of the thermogenic program including cross-talk between muscle and BAT.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 5 (FGF5) regulates hair length in humans and a variety of other animals. To investigate whether FGF5 has similar effects in sheep, we used clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) to generate loss-of-function mutations with the FGF5 gene in Chinese Merino sheep. A total of <em>16</em> lambs were identified with genetic mutations within the targeting locus: 13 lambs had biallelic modifications and three lambs had monoallelic modifications. Characterization of the modifications revealed that 13 were frameshift mutations that led to premature termination, whereas the other three were in-frame deletions. Thus, CRISPR/Cas9 efficiently generated loss-of-function mutations in the sheep FGF5 gene. We then investigated the effect of loss of FGF5 function on wool traits in 12 lambs and found that wool staple length and stretched length of genetically modified (GM) yearling sheep were significantly longer compared with that of wild-type (WT) control animals. The greasy fleece weight of GM yearling sheep was also significantly greater compared with that of WT sheep. Moreover, the mean fiber diameter in GM sheep showed no significant difference compared with WT sheep, suggesting that the increase in greasy fleece weight was likely attributed to the increase in wool length. The results of this study suggest that CRISPR/Cas9-mediated loss of FGF5 activity could promote wool <em>growth</em> and, consequently, increase wool length and yield.

Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type <em>16</em> E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN); these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent <em>growth</em> and tumorigenicity in nude mice, (sarcoma) and exhibited increased expression of genes encoding <em>growth</em> <em>factor</em> and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5), which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1), the cell surface receptor for hedgehog (Hh) signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in <em>growth</em> in U3-DT cells (transformants derived from UE6E7T-3 cells) through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional contribution of genes showed by expression profiling to the neoplastic transformation of human <em>fibroblasts</em> and human mesenchymal stem cells (hMSC).

Urachal cancer is a rare cancer that develops in the urachus. Because of its rarity, standard treatment therapies for urachal cancer are not established, and chemotherapeutic regimens for bladder cancer have been unsuccessful for patients with urachal cancer. Hence, we aim to understand a systematic molecular characterisation of urachal cancer.

We identified somatic single-nucleotide variations (SNVs)/indels and somatic copy number aberrations (SCNAs) in the 17 patients by using whole-exome sequencing (WES) and OncoScan platform (Affymetrix) as follows: tumour-normal paired sequencing (WES, n=10), tumour-only sequencing (WES, n=1; targeted deep sequencing, n=<em>16</em>), and OncoScan (n=17).

Our analyses identified 27 genes with somatic SNVs and indels, as well as six genes (APC, COL5A1, KIF26B, LRP1B, SMAD4 and TP53) that were recurrent in at least two patients. By analysing the SCNAs, we found that the extent of chromosomal amplification was highly associated with the patient's cancer stage. Interestingly, 35% (6/17) of the patients had focal DNA amplifications in fibroblast growth factor receptor family genes. The integration of somatic SNVs, indels and SCNAs revealed significant alterations in the mitogen-activated protein kinase signalling pathways.

Our genome-wide analysis of urachal cancer suggests that molecular characteristics may be important for the treatment of urachal cancer.

BACKGROUND

Fibroblast growth factors (FGFs) play important roles in multiple cancers by supporting tumor growth and angiogenesis. FP-1039 (GSK3052230) is a FGF ligand trap consisting of the extracellular domain of FGF receptor 1 (FGFR1) fused with the Fc region of IgG1. FP-1039 binds and neutralizes multiple FGFs that normally bind FGFR1. The primary objective of this phase I study was to evaluate the safety and tolerability of FP-1039.

METHODS

Eligible patients with metastatic or locally advanced solid tumors for which standard treatments were ineffective were treated with weekly doses of FP-1039 for 4 weeks, followed by 2 weeks observation.

RESULTS

Thirty-nine subjects received a mean of 6 infusions of FP-1039 at doses ranging from 0.5 to 16 mg/kg weekly, with no maximally tolerated dose identified. Grade 3 or greater treatment emergent adverse events were uncommon. Four dose-limiting toxicities were reported at doses of 0.75 mg/kg (urticaria), 1 mg/kg (intestinal perforation and neutropenia), and 16 mg/kg (muscular weakness). Drug exposure was dose proportional, and the terminal elimination half-life was 2.6-3.9 days following a single dose. Target engagement as measured by low free plasma FGF2 levels was achieved. FGF pathway dysregulation was uncommon. No objective responses were observed.

CONCLUSIONS

In nonselected cancer patients with advanced disease, treatment with FP-1039 was well tolerated and toxicities associated with small molecule drugs that inhibit FGFR tyrosine kinases, including hyperphosphatemia, were not observed. Further studies of FP-1039 in patients selected for FGF pathway dysregulation, who are most likely to benefit, are now underway.