<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-1 binding to cell surface receptors stimulates an intracellular signaling pathway that ultimately promotes the transcriptional activation of specific genes. We have used a mRNA differential display method to identify FGF-1-inducible genes in mouse NIH 3T3 <em>fibroblasts</em>. Here, we report that one of these genes, FGF-regulated (FR)-<em>19</em>, is predicted to encode a member of the transcriptional enhancer <em>factor</em> (TEF)-1 family of structurally related DNA-binding proteins. Specifically, the deduced FR-<em>19</em> amino acid sequence has approximately89, 77, and 68% overall identity to chicken TEF-1A, mouse TEF-1, and mouse embryonic TEA domain-containing <em>factor</em>, respectively. Gel mobility shift experiments indicate that FR-<em>19</em>, like TEF-1, can bind the GT-IIC motif found in the SV40 enhancer. The FR-<em>19</em> gene maps in the distal region of mouse chromosome 6, and analysis of several FR-<em>19</em> cDNA clones indicates that at least two FR-<em>19</em> isoforms may be expressed from this locus. FGF-1 induction of FR-<em>19</em> mRNA expression in mouse <em>fibroblasts</em> is first detectable at 4 h after FGF-1 addition and is dependent on de novo RNA and protein synthesis. FGF-2, calf serum, platelet-derived <em>growth</em> <em>factor</em>-BB, and phorbol 12-myristate 13-acetate can also induce FR-<em>19</em> mRNA levels. We have also found that FR-<em>19</em> mRNA expression increases during mouse C2C12 myoblast differentiation in vitro. The FR-<em>19</em> gene is expressed in vivo in a tissue-specific manner, with a relatively high level detected in lung. These results indicate that increased expression of a TEF-1-related protein may be important for both mitogen-stimulated <em>fibroblast</em> proliferation and skeletal muscle cell differentiation.

Germline mutations of the gene encoding human <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) have been shown to be responsible for several related autosomal dominant forms of syndromic craniosynostosis and short limb dwarfism. Somatic activating mutations of FGFR3 were recently reported to occur in three of 12 (25%) uterine cervical carcinomas and nine of 26 (35%) bladder carcinomas, suggesting that constitutive activation of FGFR3 may be an important mechanism underlying the development and/or progression of these common epithelial malignancies. In order to investigate further a possible role for FGFR3 mutations in cervical carcinogenesis, we performed sequence-based mutational analysis of FGFR3 in 51 primary cervical carcinomas and seven cervical carcinoma-derived cell lines. The regions analysed (exons 7, 10, 13, 15, and <em>19</em>) encompassed all previously described FGFR3 mutations. A single nucleotide substitution at codon 249, predicting a serine to cysteine amino acid substitution (S249C) in the FGFR3 extracellular domain, was identified in one primary tumor. Only wild type FGFR3 alleles were identified in the remaining tumors and cell lines. The S249C mutation is the only FGFR3 mutation described to date in cervical carcinomas. These findings suggest that while activating mutations of FGFR3 occur in cervical cancer, they may not be as common as initially reported.

Alveolar epithelial cell (AEC) migration mediated by matrix metalloproteinases (MMPs) is required for lung development and repair after injury such as hyperoxia. Of specific interest in lung remodeling are the gelatinases, which are upregulated in AEC after hyperoxia. We correlated migration and gelatinase production in AEC cultured from fetal, adult, and hyperoxic rats. Fetal AEC (<em>19</em>-20 days) had higher MMP-2 and MMP-9 gelatinase expression than adult AEC, with fivefold higher MMP-9 activity, and were migratory through gelatin, responding to epidermal <em>growth</em> <em>factor</em>, keratinocyte <em>growth</em> <em>factor</em>, and <em>fibroblast</em> <em>growth</em> <em>factor</em>-10. MMP-2 and MMP-9 expression and migratory activity could be detected from the time of plating. In contrast, adult AEC migrated and expressed MMP-2 and MMP-9 proteins only after 48 h of culture. AEC from hyperoxic rats were significantly more migratory through gelatin than control adult AEC, with significantly higher MMP-9 activity. Inhibition of MMPs with doxycycline reduced the migration of AEC from hyperoxic rats to the level of control adult AEC. Fibronectin-cultured "hyperoxic" AEC acquired a temporary capacity for migration similar to the A549 lung cancer cell line, which is both highly migratory and invasive and is derived from the AEC type 2 lineage. These data suggest that MMP activity is associated with a migratory phenotype in fetal, hyperoxic, and transformed AEC in vitro, and we speculate that MMPs may play a key mechanistic role in AEC migration in vivo during lung development and repair.

OBJECTIVE

Mechanisms by which <em>fibroblast</em> networks between stromal lamellae are laid in the corneal stroma are far from clear. We have investigated the role of vascular endothelial <em>growth</em> <em>factor</em> receptors (VEGFRs) by in vitro studies in the human corneal network formation obtained from donors whose ages ranged from <em>19</em> to 89 years.

METHODS

Corneal fibroblasts were prepared from cornea donations. The functional properties of these cells to form networks were analyzed using a semi solid matrix (substratum) of Matrigel. The presence of VEGF receptor-1 (VEGFR-1) and the functionality in these fibroblasts were investigated using immunofluorescence, molecular analysis (gene microarray, reverse transcription polymerase chain reaction [RT-PCR] and VEGFR siRNA transfections), and cell culture.

RESULTS

Corneal fibroblasts from 61 donors were classified into two groups according to whether they formed (82%) a reticulum on Matrigel or not (18%). By RT-PCR and immunofluorescence analysis, we showed that corneal fibroblasts expressed VEGFR-1 (mRNA and protein). Further, cell culture analysis revealed that only the network (reticulum) forming corneal fibroblast expressed VEGFR-1 in contrast to non network-forming fibroblasts. Use of inhibitors such as VEGFR-1 siRNA transfection or neutralizing antibody (Avastin) indicated that VEGFR-1 was essential to the formation of the corneal network in vitro.

CONCLUSIONS

The cell reticulum formation seemed to be directly related to the expression of VEGFR-1 in the corneal fibroblast, and this expression decreased with age. The decrease in VEGFR-1 expression is probably related to the diminution of autocrine functions, which may alter the overall tissular homeostasis. This may culminate in the gradual development of poor vision, which is observed in certain pathologies and in aging individuals.

Human osteoclasts are well characterized multinucleated cells whose function is the directed resorption of normal bone (NB). Osteoclastic bone destruction accompanies lytic solid tumors and myeloma as well as Paget's disease (PD) of bone and giant cell tumors of bone (GCTB). The mechanism of this stimulation of osteoclastic bone resorption is unknown. This study was designed to detect cytokines present in the multinucleated cells of PD and GCTB in order to determine whether cytokine abnormalities exist to account for bone lysis. Nine cytokines, representing the functions of bone resorption, angiogenesis, tumor necrosis, bone cell proliferation, and osteoblast-osteoclast coupling, were examined by immunohistochemistry using tissue samples from 15 NB, 17 PD, and <em>19</em> GCTB patients. Standard nonparametric statistical analysis showed a significant increase (P < 0.01 to 0.05) in immunostaining between osteoclasts of PD and NB for interleukin-6 (Il-6), tumor necrosis <em>factor</em> beta (TNFbeta), epidermal <em>growth</em> <em>factor</em> (EGF), platelet derived <em>growth</em> <em>factor</em> (PDGF), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). There was a statistically significant decrease in immunostaining of giant cells of GCTB as compared with NB for transforming <em>growth</em> <em>factor</em> beta (TGFbeta), but no other differences from normal osteoclasts. The increase in staining of PD osteoclasts over the giant cells of GCTB was significant (P < 0.01) for Il-6, TNFbeta, PDGF, bFGF and insulin <em>growth</em> <em>factor</em>-1 (IGF-1), and (P < 0. 05) for Il-1 and EGF. It was concluded that marked cytokine differences exist in vivo between osteoclasts of NB and PD lesions consistent with stimulated resorption. Alternatively, "osteoclastoma" cells in the center of the tumor did not overexpress the cytokines associated with bone lysis, suggesting some other mechanism for stimulated resorption.

The regulation of DNA synthesis in <em>19</em> day rat fetal lung epithelial (alveolar type II) and mesenchymal (<em>fibroblast</em>) cells by protein <em>growth</em> <em>factors</em> has been studied. In each case a single <em>growth</em> <em>factor</em> is capable of stimulating 3H-thymidine incorporation into DNA: platelet-derived <em>growth</em> <em>factor</em> in the case of the alveolar type II cell and epidermal <em>growth</em> <em>factor</em> in the case of the fetal lung <em>fibroblast</em>. We hypothesize that these results indicate that the type II cell endogenously produces progression activities (i.e., epidermal <em>growth</em> <em>factor</em>-like and somatomedin-like activity) while the <em>fibroblast</em> produces competence (i.e., platelet-derived <em>growth</em> <em>factor</em>-like) and progression (i.e., somatomedin-like activity). The latter is in keeping with previous observations with skin <em>fibroblasts</em>. To test the above hypothesis, the effect of fetal lung <em>fibroblast</em>-derived conditioned media upon the <em>growth</em> of fetal alveolar type II cells has been determined. The results indicate that, indeed, such media contain competence activity for this cell type. The mitogenic activity was further characterized as heat-sensitive, trypsin-sensitive, and has an apparent molecular weight of 30,000 Daltons. It is not synthesized by <em>19</em> day fetal liver, kidney or skin <em>fibroblasts</em> and its synthesis is higher in lung <em>fibroblasts</em> isolated from <em>19</em> day fetuses as compared to those isolated on day 16 or day 22.

Explants of <em>19</em>- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like <em>growth</em> <em>factor</em>) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo <em>fibroblasts</em> and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) is the human ortholog of mouse FGF15, and both proteins function as an endocrine signal to regulate various liver functions. FGF15/FGF<em>19</em> protein contains two disulfide bonds. It is unfavorable to form disulfide bonds in Escherichia coli (E. coli) cytoplasm because of the bacterial cytoplasmic reducing environment. Modification of the cytoplasmic reducing environment and/or co-expression of protein chaperones are common strategies to express disulfide bond containing proteins in E. coli. In the current study, we report a method to produce soluble FGF15/FGF<em>19</em> protein in cytoplasm of E. coli. Several commercial available strains with the disruption of thiol-redox pathways, and/or co-expression of redoxase or refolding chaperones were used to develop this novel method for expression of FGF15/FGF<em>19</em> in E. coli. Mutation of the thiol-disulfide bond reducing pathway in E. coli or N-terminal fusion of thioredox (TRX) alone is not enough to support disulfide bond formation in FGF15/<em>19</em> proteins. However, TRX fusion protein improved FGF<em>19</em> solubility in strains of thiol-redox system mutants. In addition, DsbC co-expressed in thiol-redox system mutants alone improved and further enhanced FGF<em>19</em> solubility with combination of TRX fusion tag. The soluble FGF<em>19</em> proteins were easily purified through Ni-NTA affinity chromatography and anion exchange chromatography, and the purified protein maintained its biological activities, confirmed by suppressing hepatic Cyp7a1 gene transcription in mice and by activating ERK1/2 signaling pathway in HepG2 cells. In contrast, soluble FGF15 protein in cytoplasm remained very low using these strategies. In summary, we have successfully developed a method to express functional FGF<em>19</em> protein in prokaryotic cells, and this strategy may be adapted for the expression of other disulfide-containing proteins.

Treatment of quiescent rat aortic smooth muscle cells with either alpha-thrombin or a thrombin receptor-derived agonist peptide (SFLLRNP) resulted in pronounced increases in [3H]thymidine incorporation that were concentration dependent and reached a maximum of approximately 15-fold above serum-starved controls. However, in contrast to FBS, PDGF-BB, or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), that initiated DNA synthesis promptly after 16-<em>19</em> h, thymidine incorporation in response to thrombin was delayed by an additional 3-6 h. Delayed mitogenesis correlated with the appearance of a potent mitogenic activity in conditioned media samples obtained from thrombin-stimulated rat aortic smooth muscle cells, as assayed using Swiss 3T3 <em>fibroblasts</em>. This activity was not inhibited by neutralizing antibodies directed against PDGF or bFGF. Furthermore, in the Swiss 3T3 cells, simple addition of either alpha-thrombin or SFLLRNP failed to elicit a significant mitogenic response. In signal transduction studies, both thrombin and SFLLRNP treatment led to rapid tyrosine phosphorylation of proteins with apparent molecular masses of 42, 44, 75, 120, and <em>19</em>0 kD, respectively, as assessed by antiphosphotyrosine immunoblotting. The overall pattern of protein tyrosine phosphorylation was distinct from that observed after PDGF-BB addition. Activation of Raf-1 and the mitogen-activated protein (MAP) kinases p44mapk and p42mapk was also observed. However, the time course and duration of Raf-1/MAP kinase activation after thrombin stimulation were similar to those elicited by PDGF-BB. Taken together, our results indicate that thrombin-stimulated vascular smooth muscle proliferation is delayed and requires the de novo expression of one or more autocrine mitogens. In addition, the rapid induction of discrete intracellular signaling mechanisms by thrombin, including the Raf-1/MAP kinase pathway, appears to be insufficient alone to promote vascular smooth muscle cell mitogenesis.

Cathepsin B (CTSB) is a proteolytic enzyme potentially modulating angiogenic processes and extracellular matrix remodeling. While matrix metalloproteinases are shown to be implicated in tissue fibrosis and vasculopathy associated with systemic sclerosis (SSc), the role of cathepsins in this disease has not been well studied. The aim of this study is to evaluate the roles of CTSB in SSc. Serum pro-CTSB levels were determined by enzyme-linked immunosorbent assay in 55 SSc patients and <em>19</em> normal controls. Since the deficiency of transcription <em>factor</em> Fli1 in endothelial cells is potentially associated with the development of SSc vasculopathy, cutaneous CTSB expression was evaluated by immunostaining in Fli1(+/-) and wild type mice as well as in SSc and control subjects. The effects of Fli1 gene silencing and transforming <em>growth</em> <em>factor</em>-β (TGF-β) on CTSB expression were determined by real-time PCR in human dermal microvascular endothelial cells (HDMECs) and dermal <em>fibroblasts</em>, respectively. Serum pro-CTSB levels were significantly higher in limited cutaneous SSc (lcSSc) and late-stage diffuse cutaneous SSc (dcSSc) patients than in healthy controls. In dcSSc, patients with increased serum pro-CTSB levels showed a significantly higher frequency of digital ulcers than those with normal levels. CTSB expression in dermal blood vessels was increased in Fli1(+/-) mice compared with wild type mice and in SSc patients compared with healthy controls. Consistently, Fli1 gene silencing increased CTSB expression in HDMECs. In cultured dermal <em>fibroblasts</em> from early dcSSc, CTSB expression was decreased compared with normal <em>fibroblasts</em> and significantly reversed by TGF-β1 antisense oligonucleotide. In conclusion, up-regulation of endothelial CTSB due to Fli1 deficiency may contribute to the development of SSc vasculopathy, especially digital ulcers, while reduced expression of CTSB in lesional dermal <em>fibroblasts</em> is likely to be associated with skin sclerosis in early dcSSc.

OBJECTIVE

Vitronectin-receptor-type integrins (alpha(v) beta(3) and alpha(v) beta(5)) are thought to be involved in the selective ablation of tumorigenic and other pathologic angiogenesis. Specifically, it has been shown that ligation inhibition of the alpha(v)-type integrins with cyclic penta-peptid peptide inhibits proliferative retinopathy by almost 80 % in a hypoxia-induced mouse model.

METHODS

On the basis of growth factor and integrin expression dynamics in this model, secondary intervention approaches with cyclic RGDfV peptide were investigated.

RESULTS

alpha(v)-integrin expression started immediately after induction of hypoxia (at postnatal day 12, p12) and persisted only during the initial period of neovascularization (until day p14). Vascular endothelial growth factor (VEGF) expression started at high values immediately after return of the mice into room air, and dropped rapidly to low values beyond day 13. In contrast, basic fibroblast growth factor (bFGF) was predominantly expressed during the phase of maximum angiogenesis which was noted between day p17 and 19. Based on these findings, cyclic penta peptide was administered subcutaneously at varying doses (2-20 microg/kg/day) for 5 days beginning either at day p14 (early intervention) or at day p17 (late intervention). Early secondary intervention showed a dose-dependent reduction of new vessels with maximum inhibition of 57 % (control 68.08 +/- 3.21 nuclei/section compared with RGDfV-treated 29.35 +/- 2.39 nuclei/section; p < 0.0001), whereas late secondary intervention had no effect.

CONCLUSIONS

These data indicate that angiogenesis-related alpha(v)-integrin expression is VEGF- rather than bFGF-dependent, and the efficacy of cyclic penta-peptid (RGDfV)-treatment in proliferative retinopathy is only effective as long as the alpha(v)-integrin target is prominently expressed.

The tumor necrosis <em>factor</em> superfamily (TNFSF) of cytokines comprises <em>19</em> ligands and 28 receptors (1). Ligand-mediated activation of TNFSF receptors plays a role in the pathogenesis of multiple central nervous system (CNS) diseases such as multiple sclerosis (2) and cerebral ischemia (3). Tumor necrosis <em>factor</em>-like weak inducer of apoptosis (TWEAK) is a member of the TNFSF (4) that binds a small cell surface receptor known as <em>fibroblast</em> <em>growth</em> <em>factor</em>-inducible 14 (Fn14) (5-7). There is a <em>growing</em> body of evidence indicating that the interaction between TWEAK and Fn14 plays a role on cell death, regulation of the permeability of the neurovascular unit (NVU) and development of an inflammatory response in the CNS under physiological and pathological conditions (8, 9). Accordingly, here we will review the information available to this date on the role of this cytokine and its receptor in the CNS.

OBJECTIVE

The pathogenesis of intestinal failure (IF) associated liver disease (IFALD) is uncertain, we therefore investigated the role of FGF19 and pro-inflammatory cytokines has on this disease state.

METHODS

Serum FGF19, IL-6 and, TNF-α were measured in 52 IF patients at median age 6.0 years (IQR 2.2-13) after 10 months (4.1-39) on parenteral nutrition (PN). Thirty-nine patients underwent liver biopsies.

RESULTS

In IF patients, FGF19 concentrations were lower and those of IL-6 and TNF-α higher compared to healthy matched controls (p ⩽ 0.001 for all). FGF19 concentrations were further decreased in patients without a remaining ileum [37 pg/ml (IQR 30-68) vs. 74 (35-135) p=0.028], and correlated with remaining ileum length (r = 0.333, p = 0.018) and markers of cholesterol synthesis (r = -0.552 to -0.643, p < 0.001). Patients with histological portal inflammation [30 pg/ml (28-45) vs. 48 (33-100), p = 0.019] or fibrosis [35 pg/ml (30-66) vs. 99 (38-163), p = 0.013] had lower serum FGF19 concentrations than others. FGF19 negatively correlated with portal inflammation grade (r = -0.442, p = 0.005), serum TNF-α (r = -0.318, p = 0.025), METAVIR fibrosis stage (r = -0.441, p = 0.005) and APRI (r = -0.328, p = 0.028). IL-6 was higher during PN [6 pg/ml (2-31)] than after weaning off PN [2 pg/ml (1-5), p = 0.009], correlated weakly with cholestasis grade (r = 0.328, p = 0.044), and tended to associate with histological cholestasis [n = 5, 5 pg/ml (5-267) vs. n=34, 2 pg/ml (1-7), p = 0.058].

CONCLUSIONS

In pediatric onset of IF, total or partial loss of ileum decreases serum FGF19 concentration corresponding to hepatic inflammation and fibrosis, along with increased cholesterol synthesis. In contrast, serum IL-6 increases during PN and may associate with concurrent cholestasis. These data suggests that FGF19 may contribute to the pathogenesis of IFALD.

Micro RNAs (miRNAs) are small non-coding RNAs which are <em>19</em>-24 nucleotides in length. MiRNAs play a vital role in the whole process of tumour development, but how they influence the tumourigenecity of epithelial ovarian cancer (EOC)cells is rarely researched. In our study, it was verified that miR-628-5p decreased the stem like cell percentage of EOC cells by inducing their apoptosis. The animal experiments showed that miR-628-5p decreased the tumourigenecity of EOC cells. Besides, we found miR-628-5p targeted at and down-regulated the expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2). FGFR2 expressed higher in ovarian cancer tissues and was correlated with worse prognosis. Our findings indicated that miR-628-5pplays an important role in ovarian cancer stem cell driven tumorigenesis.

Myelomeningocele (MMC) is the most severe form of spina bifida, one of the most common congenital anomalies. Although open fetal surgical repair of the MMC defect has been shown to result in improved outcomes, a less invasive approach applicable earlier in gestation than the current open surgical approach between <em>19</em> and 26 weeks of gestation is desirable for further improvement of neurological symptoms, as well as reduction of maternal and fetal risks. We previously reported the therapeutic potential of a scaffold-based tissue engineering approach in a fetal rat MMC model. The objective of this study was to confirm the long-term efficacy of this approach in the surgically created fetal sheep MMC model. Gelatin-based or gelatin/collagen hybrid sponges were prepared with and without basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) incorporation. The defect was covered by a sponge and secured by a supporting sheet with adhesive at 100 days of gestation or the gelatin/collagen hybrid with bFGF was secured with adhesive without the sheet. Although sheets were found detached at term (140 days' gestation), both gelatin-based and gelatin/collagen hybrid sponges had integrated within the newly formed granulation tissue, resulting in complete coverage of the MMC defect. The release of bFGF from sponges resulted in enhanced formation of granulation tissue and epithelialization. There was also evidence of improved preservation of the spinal cord with less associated damage on histological analysis and reversal of hindbrain herniation. These experiments provide important proof-of-principle evidence of the efficacy of scaffold-based tissue engineered coverage for the prenatal treatment of MMC.

OBJECTIVE

To detect new predictive markers from the prostate cancer tissue, to study the expression by cultured cancer-associated fibroblasts (CAFs) of stromal factors implicated in prostate carcinogenesis, and to compare their expressions in localized, metastatic, castration-sensitive (CSCP), castration-resistant prostate tumors (CRCP) as well as in fibroblasts from benign prostatic hyperplasia (BPH).

METHODS

The genomic expression of 20 stroma-derived factors, including the androgen receptor (AR), growth factors (FGF2, FGF7, FGF10, HGF, TGFβ, PDGFB), protein implicated in invasion (MMP-2, MMP-9 and MMP-11), inflammation (IL-6, IL-17, STAT-3 and NFκB), stroma/epithelium interaction (CDH11, FAP, CXCL12 and CXCL14) and chaperones (HPA1A and HSF1), was evaluated in cultured fibroblasts both from BHP and prostate carcinomas (PCa). After isolation and culture of fibroblasts by biopsy specimens, RNA was isolated and genomic studies performed.

RESULTS

Finally, 5 BPH and 37 PCa specimens were selected: clinically localized (19), metastatic (5), CSCP (7) and CRPC (6). Interleukin-17 receptor (IL-17RB) was highly expressed in CAFs compared with fibroblasts from BPH. However, metalloproteinase-2 and chemokine ligand 14 (CXCL14) were expressed at higher levels by fibroblasts from BPH. The fibroblastic growth factor-7 was highly expressed by CAFs from localized tumors, but metalloproteinase-11 in metastatic tumors. MMP-11, androgen receptor (AR) and heat-shock-70kda-protein-1A (HSPA1A) expressions were significantly higher in CAFs from CRPC.

CONCLUSIONS

These results demonstrate a CAFs heterogeneity among prostate carcinomas with regard to some molecular profile expressions that may be relevant in tumor development (IL-17RB), progression (MMP-11) and castration resistance (AR, MMP-11 and HSPA1A).

BACKGROUND

Human <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>), its receptor (FGFR4) and EpCAM play an important role in cell proliferation, differentiation, motility, and overexpression have been linked to hepatocellular carcinoma (HCC). The aim of this study was to evaluate the FGF<em>19</em> signals responsible for the progression of HCC arising from fatty liver.

RESULTS

FGF<em>19</em> level was significantly increased in the HCC patients' serum compared to non-HCC controls. The IHC results demonstrated significant increases of protein expressions of FGF<em>19</em>, FGFR4 and EpCAM in specimens with fatty liver, NASH, cirrhosis, and HCC compared to healthy liver tissue. There was a significant positive correlation between the protein expressions (FGF<em>19</em>, FGFR4, and EpCAM) and histopathologic changes from FL to HCC. Furthermore, FGF<em>19</em> was positively correlated with FGFR4 and with EpCAM.

METHODS

FGF<em>19</em> protein levels in serum and tissues were determined by ELISA assay. The FGFR4, and EpCAM expression and tissue distribution were further evaluated by immunohistochemical staining in tissue array samples. FGF<em>19</em>, FGFR4 and EpCAM expressions between the different histologic stages of fatty liver steatohepatitis-cirrhosis-HCC carcinogenesis sequence were compared to healthy hepatic tissue.

CONCLUSIONS

Overexpression of FGF<em>19</em>/FGFR4 significantly correlated with EpCAM as a marker of hepatic cancer stem cells within the fatty liver-steatosis-cirrhosis-HCC sequence.

CONCLUSIONS

This is the first study to elucidate FGF<em>19</em>/FGFR4 signaling in favor of HCC cells developing as indicated by increased EpCAM within the carcinogenesis sequence from fatty liver to hepatocellular carcinoma. Our study has the potential to yield novel and cost effective screening strategies for HCC patients.

β-Tricalcium phosphate (β-TCP) is an osteoconductive material. For this research we have combined it with a low degradation calcium silicate (CS) to enhance its bioactive and osteostimulative properties. To check its effectiveness, a series of β-TCP/CS composites with different ratios were prepared to make new bioactive and biodegradable biocomposites for bone repair. Formation of bone-like apatite, the diametral tensile strength, and weight loss of composites were considered before and after immersion in simulated body fluid (SBF). In addition, we also examined the effects of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) released from β-TCP/CS composites and in vitro human dental pulp cell (hDPC) and studied its behavior. The results showed that the apatite deposition ability of the β-TCP/CS composites was enhanced as the CS content was increased. For composites with more than 50% CS contents, the samples were completely covered by a dense bone-like apatite layer. At the end of the immersion point, weight losses of <em>19</em>%, 24%, 33%, 42%, and 51% were observed for the composites containing 0%, 30%, 50%, 70% and 100% β-TCP cements, respectively. In vitro cell experiments show that the CS-rich composites promote human dental pulp cell (hDPC) proliferation and differentiation. However, when the CS quantity in the composite is less than 70%, the amount of cells and osteogenesis protein of hDPCs was stimulated by FGF-2 released from β-TCP/CS composites. The combination of FGF-2 in degradation of β-TCP and osteogenesis of CS gives a strong reason to believe that these calcium-based composite cements may prove to be promising bone repair materials.

OBJECTIVE

To clarify the role of pentraxin 3 (PTX3), a multifunctional pattern recognition protein that can suppress fibroblast growth factor 2 (FGF-2), in systemic sclerosis (SSc)-related vasculopathy.

METHODS

We assessed 171 SSc patients and 19 age- and sex-matched healthy control subjects. Circulating PTX3 and FGF-2 levels were measured by enzyme immunoassay, and CD34+CD133+CD309+ endothelial progenitor cells (EPCs) were counted by flow cytometry. Correlations between PTX3 and FGF-2 and the presence or future development of vascular manifestations, including digital ulcers and pulmonary arterial hypertension (PAH), were identified by univariate and multivariate analysis. The effect of PTX3 on EPC differentiation was evaluated in proangiogenic cultures of mouse bone marrow cells in combination with colony formation assay.

RESULTS

Circulating PTX3 and FGF-2 levels were significantly higher in SSc patients than in healthy control subjects. PTX3 was elevated in SSc patients who had digital ulcers or PAH, while FGF-2 was reduced in SSc patients with PAH. Multivariate analysis identified elevated PTX3 as an independent parameter associated with the presence of digital ulcers and PAH, and PTX3 levels were a useful predictor of future occurrences of digital ulcers. Reduced FGF-2 was independently associated with the presence of PAH. EPC counts were significantly lower in patients with digital ulcers or PAH and correlated negatively with circulating PTX3 concentrations. Finally, PTX3 inhibited EPC differentiation in vitro.

CONCLUSIONS

In SSc patients, exposure to high concentrations of PTX3 may suppress EPC-mediated vasculogenesis and promote vascular manifestations such as digital ulcers and PAH.

Hepatocellular carcinoma (HCC) is a lethal disease with limited therapeutic options and a particularly poor prognosis. Aberrant <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) signaling through <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 (FGFR4) has been identified as an oncogenic driver for a subset of patients with HCC. FGFR4 is therefore a promising target for the treatment of HCC harboring aberrant FGF<em>19</em>-FGFR4 signaling, and several FGFR4 inhibitors have advanced to clinical trial. In this review, we summarize the latest developments in FGFR4 inhibitors, including the known pharmacophores, their binding mode, selectivity, and clinical implications, as well as the optimization strategy of introducing an acrylamide into a known pan-FGFR inhibitor targeting Cys552 of FGFR4 to provide selective covalent FGFR4 inhibitors.

Bile acids (BAs) are signaling molecules controlling energy homeostasis that can be both toxic and protective for the liver. BA alterations have been reported in obesity, insulin resistance (IR), and nonalcoholic steatohepatitis (NASH). However, whether BA alterations contribute to NASH independently of the metabolic status is unclear.

To assess BA alterations associated with NASH independently of body mass index and IR.

Patients visiting the obesity clinic of the Antwerp University Hospital (a tertiary referral facility) were recruited from 2006 to 2014.

Obese patients with biopsy-proven NASH (n = 32) and healthy livers (n = 26) were matched on body mass index and homeostasis model assessment of IR.

Transcriptomic analyses were performed on liver biopsies. Plasma concentrations of 21 BA species and 7α-hydroxy-4-cholesten-3-one, a marker of BA synthesis, were determined by liquid chromatography-tandem mass spectrometry. Plasma <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> was measured by enzyme-linked immunosorbent assay.

Plasma BA concentrations did not correlate with any hepatic lesions, whereas, as previously reported, primary BA strongly correlated with IR. Transcriptomic analyses showed unaltered hepatic BA metabolism in NASH patients. In line, plasma 7α-hydroxy-4-cholesten-3-one was unchanged in NASH. Moreover, no sign of hepatic BA accumulation or activation of BA receptors-farnesoid X, pregnane X, and vitamin D receptors-was found. Finally, plasma <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em>, secondary-to-primary BA, and free-to-conjugated BA ratios were similar, suggesting unaltered intestinal BA metabolism and signaling.

In obese patients, BA alterations are related to the metabolic phenotype associated with NASH, especially IR, but not liver necroinflammation.

The hemodialysis (HD) procedure induces an inflammatory response potentially contributing to cardiovascular disease. Here we investigated the acute impact of HD on circulating biomarkers. Circulating biomarkers (small solutes, middle molecular-sized peptides, and proteins) related to inflammation, oxidative stress, and vascular calcification (VC) were measured before and after a single session of HD in 45 clinically stable patients. Concentrations were corrected for ultrafiltration-induced hemoconcentration. Among vascular calcification-related biomarkers, osteoprotegerin and fetuin-A remained unchanged while <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF23) decreased by -<em>19</em>%. Changes of FGF23 and changes of phosphate correlated (ρ = 0.61, P < 0.001). While C-reactive protein did not change, interleukin-6 (IL-6) increased by 14% and pentraxin 3 (PTX3) increased by 45%. IL-6 and PTX3 appear to be valid biomarkers of the intradialytic inflammatory response. VC-related markers were in general not affected by the single HD session; however, the observed correlation between acute changes of FGF-23 and phosphate during HD warrants further studies.

Adipose tissue-derived signals potentially link obesity and adipose tissue dysfunction with metabolic and cardiovascular diseases. Although some adipocytokines have been closely related to metabolic and cardiovascular traits, it is unknown which adipocytokine or adipocytokine clusters serve as meaningful markers of metabolic syndrome (MS) components. Therefore, this study investigated the associations of 12 adipocytokines with components of the MS to identify the most relevant cytokines potentially related to specific metabolic profiles.

Twelve cytokines [adiponectin, adipocyte fatty acid-binding protein (AFABP), angiopoietin-related <em>growth</em> <em>factor</em>, chemerin, <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>19</em>, FGF21, FGF23, insulin-like <em>growth</em> <em>factor</em>-1, interleukin 10, irisin, progranulin, and vaspin] were quantified in a cross-sectional cohort of 1046 subjects. Hypothesis-free cluster analysis, multivariate regression analyses with parameters of the MS, and discriminant analysis were performed to assess associations and the relative importance of each cytokine for reflecting MS and its components.

Among the studied adipocytokines, adiponectin, AFABP, chemerin, and FGF21 showed the strongest associations with MS and several MS components in discriminant analyses and multiple regression models. For certain metabolic components, these adipocytokines were better discriminators than routine metabolic markers. Other cytokines investigated in the present cohort are less able to distinguish between metabolically healthy and unhealthy subjects.

Adiponectin, AFABP, chemerin, and FGF21 showed the strongest associations with MS components in a general population, suggesting that adverse adipose tissue function is a major contributor to these metabolic abnormalities. Future prospective studies should address the question whether these adipocytokines can predict the development of metabolic disease states.

UNASSIGNED

To evaluate the expression of <em>19</em> angiogenic biomarkers in the aqueous humor before and after intravitreal bevacizumab injection (IVB) in eyes with neovascular age-related macular degeneration (AMD).

UNASSIGNED

Prospective, noncomparative, interventional case series.

UNASSIGNED

Twenty-three eyes of 23 treatment-naïve patients with choroidal neovascularization (CNV) secondary to neovascular AMD.

UNASSIGNED

Eyes were diagnosed with CNV secondary to neovascular AMD and were treated with 3 monthly IVBs. Aqueous humor samples were obtained by anterior chamber paracentesis at baseline and immediately before each intravitreal bevacizumab injection.

UNASSIGNED

Aqueous humor levels of <em>19</em> angiogenic biomarkers (angiopoietin 2, bone morphogenetic protein 9 [BMP-9], epidermal growth factor [EGF], endoglin, endothelin 1, fibroblast growth factor [FGF]-1 and FGF-2, follistatin, granulocyte colony-stimulating factor [GCSF], heparin-binding EGF-like growth factor [HB-EGF], hepatocyte growth factor [HGF], interleukin 8, leptin, placental growth factor [PLGF], vascular endothelial growth factor [VEGF]-A, VEGF-C, VEGF-D, and tissue inhibitor of metalloproteinases [TIMP]-1 and TIMP-2) were measured. Best-corrected visual acuity (BCVA), spectral-domain OCT parameters, and intraocular pressure also were evaluated.

UNASSIGNED

Baseline aqueous VEGF-A expression was elevated in all study eyes before treatment initiation. A statistically significant decrease of VEGF-A was observed at the 1- and 2-month follow-ups. A statistically significant increased concentration was observed in 7 biomarkers: VEGF-C, angiopoietin 2, endothelin 1, follistatin, HB-EGF, HGF, and interleukin 8. The other 11 study biomarker levels (VEGF-D, BMP-9, EGF, endoglin, FGF-1, FGF-2, GCSF, leptin, PLGF, TIMP-1, and TIMP-2) did not show any significant difference during follow-up. The BCVA statistically improved significantly at 2 months. Spectral-domain OCT parameters improved significantly at all follow-ups. Mean intraocular pressure values were not statistically different during the study period.

UNASSIGNED

Despite a decrease in VEGF-A, the aqueous levels of VEGF-C, angiopoietin 2, endothelin 1, follistatin, HB-EGF, HGF, and interleukin 8 increased significantly after intravitreal injection of bevacizumab. These upregulated angiogenic biomarkers may represent new therapeutic targets in exudative AMD.