The aim of the present study was to investigate whether biomarkers improve the prediction of recurrence-free, disease-specific, and overall survival in patients with clinically localized prostate cancer. A tissue microarray was constructed from prostate specimens of 278 patients who underwent open radical retropubic prostatectomy for clinically localized prostate cancer. For immunohistochemical studies, antibodies were used against matrix metalloproteinase (MMP)-2, MMP-3, MMP-7, MMP-9, MMP-13, and MMP-<em>19</em>, as well as against vascular endothelial <em>growth</em> <em>factor</em>, hypoxia-induced <em>factor</em> 1α, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and cluster of differentiation 31. Univariate and multivariable analyses were performed to evaluate the potential predictors of overall, disease-specific, and recurrence-free survival. In univariate analysis of patients with clinically organ-confined prostate cancer, only higher expression levels of MMP-9 (hazard ratio [0.6], 95% CI 0.45-0.8) had a protective effect in terms of overall survival. This positive effect of high MMP-9 expression was also observed for recurrence-free (HR 0.88, 95% CI 0.78-0.99) and disease-specific survival (HR 0.5, 95% CI 0.36-0.73). In multivariable analysis, none of these potential markers was found to be an independent prognostic <em>factor</em> of survival. Of all MMPs and angiogenic <em>factors</em> tested, MMP-9 expression has the potential as a prognostic marker in patients undergoing radical prostatectomy for clinically organ-confined cases of prostate cancer.

We examined the expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) and FGF receptor by immunohistochemistry in 32 human pancreatic ductal adenocarcinomas. Mild to marked basic FGF immunoreactivity was noted in <em>19</em> (59.4%) of the 32 tumours examined, and 30 (93.3%) of the tumours exhibited a cytoplasmic staining pattern against FGF receptor. The tumours were divided into two groups according to the proportion of positively stained tumour cells: a low expression group (positive cells < 25%) and a high expression group (positive cells>> or = 25%). No statistically significant difference in tumour size, differentiation, metastases or stage was found between the low and high basic FGF expression groups. However, a significant correlation was found between FGF receptor expression level and the presence of retroperitoneal invasion, lymph node metastasis, and tumour stage. In addition, low FGF receptor expression was significantly associated with a longer post-operative survival as compared with high FGF receptor expression, whereas there was no significant difference in post-operative survival between the low and high basic FGF expression groups. Increased expression of FGF receptor is correlated with the extent of malignancy and post-operative survival in human pancreatic ductal adenocarcinomas. Thus, overexpression of FGF receptor may prove to be a more useful prognostic marker than basic FGF expression level in pancreatic cancer patients.

BACKGROUND

Bronchiolitis obliterans syndrome (BOS), chronic lung allograft rejection, remains an impediment for the function of the transplanted organ. In this study, we defined the role of the microRNA (miRNA) miR-144 in fibroproliferation leading to BOS.

METHODS

Biopsy specimens were obtained from 20 lung transplant recipients with BOS((+)) and <em>19</em> without BOS((-)). Expression of miR-144 and its target, transforming <em>growth</em> <em>factor</em>-β (TGF-β)-induced <em>factor</em> homeobox 1(TGIF1), were analyzed by real-time polymerase chain reaction and Western blot. Overexpression of miR-144 and luciferase reporter genes were performed to elucidate miRNA-target interactions. The function of miR-144 was evaluated by transfecting <em>fibroblasts</em> and determining the response to TGF-β by analyzing Sma- and Mad-related family (Smads), <em>fibroblast</em> <em>growth</em> <em>factor</em>, TGF-β, and vascular endothelial <em>growth</em> <em>factor</em>. Smooth muscle actin-α-positive stress fibers and F-actin filaments in lung <em>fibroblasts</em> were analyzed by immunofluorescence.

RESULTS

Analysis of miR-144 in the biopsy specimens demonstrated 4.1 ± 0.8-fold increases in BOS(+) compared with BOS(-) patients, with a significant reduction in TGIF1 (3.6 ± 1.2-fold), a corepressor of Smads. In vitro transfection confirmed that over-expression of miR-144 results in a reduction in TGIF1 and an increase in SMAD2, SMAD4, fibroblast growth factor-6, TGF-β, and vascular endothelial growth factor. Increasing miR-144 by transfecting, increased smooth muscle actin-α and fibronectin, and knockdown of miR-144 diminished fibrogenesis in MRC-5 fibroblasts.

CONCLUSIONS

miR-144 is a critical regulator of the TGF-β signaling cascade and is over-expressed in lungs with BOS. Therefore, miR-144 is a potential target toward preventing fibrosis leading to BOS after lung transplant.

Patients with primary biliary cholangitis (PBC) who had an inadequate response to ursodiol have few treatment options. Alkaline phosphatase (ALP) and bilirubin levels correlate with the risk of liver transplant or death in PBC patients. <em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>19</em> is a hormone that acts directly in the liver to regulate bile acid synthesis. We evaluated NGM282, an engineered analogue of FGF<em>19</em>, for the treatment of PBC. In this 28-day, double-blind, placebo-controlled phase 2 trial, 45 PBC patients who had an inadequate response to ursodiol were randomly assigned 1:1:1 to receive subcutaneous daily doses of either NGM282 at 0.3 mg (n = 14), 3 mg (n = 16), or placebo (n = 15). The primary endpoint was a change in ALP from baseline after 28 days of treatment. At day 28, ALP was significantly reduced with NGM282 treatment at both 0.3 mg (least-squares mean -51.0 IU/L [standard error (SE) 15.4]) and 3 mg (-66.0 IU/L [SE 16.0]) versus placebo (3.3 IU/L [SE 14.8]), with least-squares mean differences of -54.3 IU/L (95% confidence interval -104.2 to -4.5; P = 0.0149) and -69.3 IU/L (95% confidence interval -120.5 to -18.3; P = 0.0030), respectively. Fifty percent (7 of 14) of patients receiving NGM282 0.3 mg and 46% (6 of 13) of those receiving NGM282 3mg achieved 15% or greater reduction in ALP levels from baseline, compared with 7% (1 of 15) of patients receiving placebo. NGM282 also significantly reduced serum concentrations of transaminases and immunoglobulins. Most adverse events were grade 1 (mild) to grade 2 (moderate) in severity, with gastrointestinal disorders more frequent in the NGM282 treatment groups. No worsening of pruritus was observed with NGM282 treatment. Conclusion: NGM282 administered for 28 days resulted in significant improvements in ALP and transaminase levels compared with placebo, with an acceptable safety profile in patients with PBC. (Hepatology Communications 2018; 00:000-000).

Elevated plasma fibroblast growth factor 23 (FGF23) is a prognostic marker in chronic kidney disease. Recently, FGF23 was reported to also be a predictive factor in chronic congestive heart failure (HF). To date however, plasma levels in acute decompensated HF (ADHF) have not been reported and myocardial production and distribution of FGF23 in HF is poorly defined. We aimed to determine plasma levels and myocardial production of FGF23 in ADHF.

Plasma FGF23, N-terminal pro B-type natriuretic peptide (NT-proBNP) and estimated glomerular filtration rate (eGFR) were assessed in 21 ADHF patients and 19 controls. Myocardial gene expression and distribution of FGF23 was determined on left ventricular samples from HF patients and normal controls.

Plasma FGF23 was markedly higher in ADHF patients compared with controls (1498 ± 1238 versus 66 ± 27 RU/mL, P < 0.0001). There were no correlations between FGF23 and eGFR, NT-proBNP, ejection fraction or age. ADHF subjects with eGFR >60 mL/min/1.73 m(2) had FGF23 levels of 1526 ± 1601 RU/mL versus 55 ± 20 RU/mL in controls (P = 0.007). Quantified myocardial FGF23 gene expression was similar between HF patients and controls. Myocardial FGF23 immunostaining was similar between HF patients and controls, with equal distribution throughout cardiomyocytes.

Patients with ADHF had markedly elevated plasma FGF23 levels. Myocardial FGF23 gene expression was present in HF at a similar level as normal controls, and immunohistochemistry showed similar cellular distribution of FGF23 in HF and controls, suggesting that the myocardium does not contribute to the elevated circulating FGF23 in HF.

Platelet-derived <em>growth</em> <em>factor</em> (PDGF) is produced by a variety of normal and tumor cells in vitro. We have developed an enzyme-linked immunosorbent assay for the detection of the B-chain of PDGF. This assay can reliably detect 0.1 ng/ml of homodimeric recombinant PDGF B-chain and does not cross-react with recombinant PDGF-AA, epidermal <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, or transforming <em>growth</em> <em>factor</em>-beta. Citrated plasma from 72 control individuals had a PDGF B-chain (PDGF-B) level of 0.32 +/- 0.14 ng/ml (mean +/- SD) with a range of 0.10-0.69 ng/ml. The plasma platelet <em>factor</em> 4 (PF4) level was 97 +/- 70 ng/ml, with a range of 34-363 ng/ml. Citrated plasma was obtained from 131 cancer patients, and plasma PDGF-B was elevated in <em>19</em> (15%) of the patients. Both PDGF-B and PF4 were elevated in 14 (11%) of these patients, consistent with a platelet source of PDGF-B. In 5 patients (4%), however, PDGF-B was elevated and PF4 was not elevated compared to the control group. This last group of patients may have a tumor-derived source of PDGF-B which could be important in autocrine or paracrine <em>growth</em> stimulation of the tumor cells.

OBJECTIVE

Recent data indicate that oxidized low-density lipoprotein (ox-LDL) has several proatherogenic effects, e.g. induction of macrophage chemoattractants, adhesion molecules, cytokines, type-1 plasminogen activator inhibitor and platelet-derived <em>growth</em> <em>factor</em> A-chain by smooth muscle cells. Therefore, ox-LDL has been utilized as a marker of oxidative modification of proteins in atherosclerosis. Because heart valves consist of smooth muscle cells, <em>fibroblasts</em> and endothelial cells, and because valvular disease and coronary atherosclerosis could result from similar biological processes, we investigated ox-LDL accumulation in isolated aortic and pulmonary valves and coronary arteries from patients with angiographically proven coronary heart disease (CHD, n = <em>19</em>), patients with idiopathic congestive heart failure (IDCM = idiopathic dilated cardiomyopathy, n = 20), and transplant donors.

METHODS

Masson-Goldner staining and immunohistochemistry utilizing anti ox-LDL and CD68 were performed on paraffin sections of freshly isolated semilunar valves. Data were analyzed by digital image planimetry and by visual scoring of staining intensity.

RESULTS

Ox-LDL immunoreactivity was identified in the vascular aspect of the attachment line, in the deep valve stroma, and in the ventricular and vascular endothelium of the semilunar valves, colocalizing with macrophages. Valvular ox-LDL area was significantly increased in CHD-patients (P < 0.03) and IDCM-patients (P < 0.04) compared with controls. More ox-LDL was accumulating in the pulmonary valves than in the aortic valves (P = 0.04) as assessed by area and staining intensity. Valvular ox-LDL area in pulmonary valve and aortic valve was significantly correlated with ox-LDL accumulation in the intimal layer (P < 0.001) and medial layer (P < 0.001) of coronary arteries from the same patients.

CONCLUSIONS

The data suggest that the biological process leading to ox-LDL accumulation in coronary atherosclerosis also involves heart valves. Therefore, accumulation of the oxidative stress marker ox-LDL in heart valves illustrates atherosclerosis as an additional mechanisms accelerating valvular degeneration in these patients.

The FGL peptide is a neural cell adhesion molecule (NCAM) mimetic comprising a 15-amino-acid-long sequence of the FG loop region of the second fibronectin type III module of NCAM. It corresponds to the binding site of NCAM for the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1. FGL improves cognitive function through enhancement of synaptic function. We examined the effect of FGL on synaptic and dendritic structure in the brains of aged (22-month-old) rats that were injected subcutaneously (8 mg/kg) at 2-day intervals until <em>19</em> days after the start of the experiment. Animals were perfused with fixative, brains removed and coronal sections cut at 50 microm. The hippocampal volume was measured, tissue embedded and ultrathin sections viewed in a JEOL 1010 electron microscope. Analyses were made of synaptic and dendritic parameters following three-dimensional reconstruction via images from a series of approximately 100 serial ultrathin sections. FGL affected neither hippocampal volume nor spine or synaptic density in the middle molecular layer of the dentate gyrus. However, it increased the ratio of mushroom to thin spines, number of multivesicular bodies and also increased the frequency of appearance of coated pits. Three-dimensional analysis showed a significant decrease in both post-synaptic density and apposition zone curvature of mushroom spines following FGL treatment, whereas for thin spines the convexity of the apposition zone increased. These data indicate that FGL induces large changes in the fine structure of synapses and dendritic spines in hippocampus of aged rats, complementing data showing its effect on cognitive processes.

The forkhead C1 (FOXC1) transcription <em>factor</em> is involved in the development and regulation of several organs, including the eye, where FOXC1 alterations cause iris, trabecular meshwork and corneal anomalies. Using nickel agarose chromatin enrichment with human anterior segment cells, we previously identified the <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) locus as a gene potentially regulated by FOXC1. Here, we demonstrate that FGF<em>19</em> is a direct target of FOXC1 in the eye. FOXC1 positively regulates FGF<em>19</em> expression in corneal and periocular mesenchymal cells in cell culture and in zebrafish embryos. Through the FGFR4 tyrosine kinase, FGF<em>19</em> promotes MAPK phosphorylation in the developing and mature cornea. During development, loss of either FOXC1 or FGF<em>19</em> results in complementary, but distinct, anterior segment dysgeneses. This study reveals an important role for FOXC1 in the direct regulation of the FGF<em>19</em>-FGFR4-MAPK pathway to promote both the development and maintenance of anterior segment structures within the eye.

Insulin-like <em>growth</em> <em>factor</em> binding protein-3 (IGFBP-3) purified from bovine serum shares <em>19</em> of 25 amino-terminal amino acid residues with IGFBP-3 purified from human, rat, and porcine sources. A newly characterized bovine <em>fibroblast</em> model was used to investigate the biological effects of purified bovine IGFBP-3 (bIGFBP-3). Coincubation of insulin-like <em>growth</em> <em>factor</em> I (IGF-I) with increasing concentrations of bIGFBP-3 produced a dose-dependent inhibition of IGF-I-stimulated [3H]aminoisobutyric acid (AIB) uptake in cultured bovine <em>fibroblasts</em>. Inhibition was complete at equimolar concentrations of IGF-I and bIGFBP-3. Inhibition of IGF-I-stimulated [3H]AIB uptake paralleled the ability of bIGFBP-3 to prevent [125I]IGF-I cell surface binding. In contrast, preincubation with bIGFBP-3 resulted in a dose-dependent enhancement of IGF-I-stimulated [3H]AIB uptake; a 32-86% increase in IGF-I bioactivity was seen after a 24 h preexposure to 10 nM bIGFBP-3, and a 2- to 6-fold potentiation was seen after a 72 h preincubation. Preincubation with bIGFBP-3 increased both the sensitivity and maximal responsiveness of the cells to IGF-I. The potentiating effects of bIGFBP-3 were associated with increased [125I]IGF-I binding to cultured bovine <em>fibroblasts</em>. Affinity cross-linking experiments indicated that the increase in IGF-I binding was due to increased membrane-associated bIGFBP-3 rather than to a bIGFBP-3-induced increase in type I IGF receptors. bIGFBP-3 had no effect on insulin stimulation of [3H]AIB uptake under either experimental condition. These data suggest that soluble bIGFBP-3 inhibits IGF-I action by sequestering and preventing IGF-I receptor binding, whereas surface-associated bIGFBP-3 enhances the <em>growth</em>-promoting effects of IGF-I in bovine <em>fibroblasts</em>. We propose that IGFBP-3 serves a dual function in modulating IGF action in vivo.

Platelet-derived <em>growth</em> <em>factors</em> (PDGF) are mitogenic polypeptides that are involved in cellular proliferation and tissue repair. The expression of PDGFs and type beta PDGF receptor was examined in the normal human pancreas and in chronic pancreatitis, a fibrotic disease associated with fibroblastic proliferation, atrophy, and acinar cell dedifferentiation. In the normal human pancreas, PDGF A chain mRNA levels were relatively abundant, whereas PDGF B chain mRNA levels were not detected, and type beta PDGF receptor mRNA transcripts were present at low levels. In the normal pancreas, PDGF immunoreactivity was present in islet cells, whereas type beta PDGF receptor immunoreactivity was present in acinar cells. In chronic pancreatitis, PDGF A chain mRNA transcripts were also abundant, and 11 of <em>19</em> samples exhibited the PDGF B chain mRNA transcript. In addition, there was a significant increase in the mRNA levels of type beta PDGF receptor in the pancreatitis samples by comparison with the normal pancreas (P < 0.001). In chronic pancreatitis tissues, PDGF and type beta PDGF receptor immunoreactivity were present in acinar, ductal, islet, and endothelial cells, <em>fibroblasts</em>, and leukocytes. The concomitant overexpression of PDGFs and of the type beta PDGF receptor points to the existence of autocrine and paracrine PDGF-dependent loops in human chronic pancreatitis.

We cloned a novel matrix metalloproteinase (MMP) called CMMP from cultured primary chicken embryo <em>fibroblasts</em>. The cDNA-derived CMMP sequence contains 472 amino acids including a putative <em>19</em>-residue signal peptide and a unique cysteine in the catalytic domain, an insertion in a sequence motif that binds the structural (noncatalytic) zinc of MMPs. Strikingly, a homologously inserted cysteine is also found in Xenopus XMMP and human MMP<em>19</em>, two recently cloned novel members of the MMP family. Phylogenetic analysis suggest that XMMP and MMP<em>19</em> represent founding members of the MMP family, whereas CMMP is related to collagenase MMPs. Bacterially produced recombinant CMMP (without the amino-terminal inhibition domain), which was autoproteolyzed at the carboxyl-terminal domain, digested casein and gelatin. As shown by Northern blotting, CMMP mRNA of 1.8 kilobase pairs was constitutively expressed in cultured primary chicken embryo <em>fibroblasts</em> and up-regulated by tumor necrosis <em>factor</em>-alpha and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, but it was not regulated by interleukin-1, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, or retinoic acid. CMMP mRNA of 1.8 kb was also detected in the head and body of 8-day-old chicken embryos and dramatically up-regulated in 9-day-old embryos.

We have examined the possible role of transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) in metastatic malignancy by analyzing the production and activation of TGF-beta 1 and -beta 2 and the regulation of TGF-beta-responsive genes in oncogene-transformed metastatic fibrosarcomas. All transformed lines derived from either 10T1/2 or NIH 3T3 by either H-ras or protein-kinase encoding oncogenes produced more TGF-beta than parental cells. However, only highly metastatic fibrosarcomas secreted activated TGF-beta at rates that were greater than parental <em>fibroblasts</em>. Immunohistochemical staining for TGF-beta 1 showed widespread intra- and extracellular distribution in metastatic lung nodules and adjacent tissue. Cells isolated from tumors successfully metastasizing to the lung had TGF-beta 1 mRNA levels which were increased <em>19</em>-fold over in vitro controls. Despite the greatly enhanced rate of secretion of activated TGF-beta, metastatic cells exhibited markedly altered responses of TGF-beta 1 and TGF-beta 2, being unable to either increase collagen secretion or enhance collagen alpha 2(1) or TGF-beta 1 mRNA levels. This lack of response was not due to either altered TGF-beta receptor affinity or numbers. Metastatic progression was, therefore, associated with an increase in the secretion of activated TGF-beta 1 and a loss of the ability to deregulate TGF-beta-responsive genes.

The objective of this study was to measure plasma <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 and <em>19</em> (FGF21 and FGF<em>19</em>) levels in patients with Cushing's syndrome (CS) and to compare it with those of lean control subjects (C) and patients with obesity (OB). Fourteen untreated patients with CS, <em>19</em> patients with OB and 36 controls were included in the study. Plasma FGF21 and FGF<em>19</em> levels were measured by ELISA kits, other hormonal and biochemical parameters were measured by standard laboratory methods. Plasma FGF<em>19</em> did not significantly differ among the studied groups. Plasma FGF21 levels were significantly higher in both CS and OB groups relative to C group but they did not differ between CS and OB groups. In a combined population of all three groups FGF21 levels positively correlated with BMI, waist circumference and percentage of total and truncal fat mass. Less prominent inverse relationship with these parameters was found for FGF<em>19</em>. Neither FGF21 nor FGF<em>19</em> were significantly related to cortisol concentrations. Increased FGF21 concentrations in both patients with CS and OB relative to lean subjects suggest that excessive body fat and/or related metabolic abnormalities rather than direct effects of cortisol are responsible. In contrast neither obesity nor hypercortisolism significantly affected FGF<em>19</em> concentrations.

OBJECTIVE

The aim of this study was to develop high-throughput assays for the analysis of major chondrocyte functions that are important in osteoarthritis (OA) pathogenesis and methods for high-level gene expression and analysis in primary human chondrocytes.

METHODS

In the first approach, complementary DNA (cDNA) libraries were constructed from OA cartilage RNA and full-length clones were selected. These cDNAs were transferred into a retroviral vector using Gateway Technology. Full-length clones were over-expressed in human articular chondrocytes (HAC) by retroviral-mediated gene transfer. The induction of OA-associated markers, including aggrecanase-1 (Agg-1), matrix metalloproteinase-13 (MMP-13), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), collagen IIA and collagen X was measured by quantitative real-time polymerase chain reaction (QPCR). Induction of a marker gene was verified by independent isolation of 2-3 clones per gene, re-transfection followed by QPCR as well as nucleotide sequencing. In the second approach, whole cDNA libraries were transduced into chondrocytes and screened for chondrocyte cluster formation in three-dimensional agarose cultures.

RESULTS

Using green fluorescent protein (eGFP) as a marker gene, it was shown that the retroviral method has a transduction efficiency of >90%. A total of 40 verified hits were identified in the QPCR screen. The first set of <em>19</em> hits coordinately induced iNOS, COX-2, Agg-1 and MMP-13. The most potent of these genes were the tyrosine kinases Axl and Tyro-3, receptor interacting kinase-2 (RIPK2), tumor necrosis <em>factor</em> receptor 1A (TNFR1A), <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) and its receptor FGFR, MUS81 endonuclease and Sentrin/SUMO-specific protease 3. The second set of seven hits induced both Agg-1 and MMP-13 but none of the other markers. Five of these seven genes regulate the phosphoinositide-3-kinase pathway. The most potently induced OA marker was iNOS. This marker was induced 20-500 fold by seven genes. Collagen IIA was also induced by seven genes, the most potent being transforming <em>growth</em> <em>factor</em> beta (TGFbeta)-stimulated protein TSC22, vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and splicing <em>factor</em> 3a. This screening assay did not identify inducers of collagen X. The second chondrocyte cluster formation screen identified 14 verified hits. Most of the genes inducing cluster formation were kinases. Additional genes had not been previously known to regulate chondrocyte cluster formation or any other chondrocyte function.

CONCLUSIONS

The methods developed in this study can be applied to screen for genes capable of inducing an OA-like phenotype in chondrocytes on a genome-wide scale and identify novel mediators of OA pathogenesis. Thus, coordinated functional genomic approaches can be used to delineate key genes and pathways activated in complex human diseases such as OA.

We studied the in vitro secretion of macrophage-derived <em>growth</em> <em>factor</em> (MDGF) activity by peritoneal macrophages from fertile and infertile women. Peritoneal fluid was obtained from 55 women undergoing laparoscopy for evaluation and treatment of infertility or for tubal sterilization. Isolated macrophages were plated in tissue culture wells and incubated in Dulbecco's Modified Eagle's Medium plus 0.2% lactalbumin hydrolyzate at 37 C for 24 h. Medium MDGF activity was assayed by determining the ability of medium to stimulate [3H] thymidine incorporation in BALB-c 3T3 <em>fibroblasts</em>. Macrophages from 23 women released significant MDGF activity in vitro; the release was linear for up to 72 h. Among the 55 women, macrophages from 10 of 36 (28%) women with normal pelvic anatomy or tubal occlusion/pelvic adhesions released significant MDGF activity. In contrast, macrophages from 13 of <em>19</em> (68%) women with endometriosis, a significantly higher proportion (P less than 0.02), released MDGF. The finding that endometriosis is associated with in vivo primed peritoneal macrophages that produce MDGF in vitro may help to explain the proliferation or maintenance of endometrial tissue in the peritoneal cavity.

The assignments of the 1H, 15N, 13CO and 13C resonances of recombinant human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2), a protein comprising of 154 residues and with a molecular mass of 17.2 kDa, is presented based on a series of three-dimensional triple-resonance heteronuclear NMR experiments. These studies employ uniformly labeled 15N- and 15N-/13C-labeled FGF-2 with an isotope incorporation>> 95% for the protein expressed in E. coli. The sequence-specific backbone assignments were based primarily on the interresidue correlation of C alpha, C beta and H alpha to the backbone amide 1H and 15N of the next residue in the CBCA(CO)NH and HBHA(CO)NH experiments and the intraresidue correlation of C alpha, C beta and H alpha to the backbone amide 1H and 15N in the CBCANH and HNHA experiments. In addition, C alpha and C beta chemical shift assignments were used to determine amino acid types. Sequential assignments were verified from carbonyl correlations observed in the HNCO and HCACO experiments and C alpha correlations from the HNCA experiment. Aliphatic side-chain spin systems were assigned primarily from H(CCO)NH and C(CO)NH experiments that correlate all the aliphatic 1H and 13C resonances of a given residue with the amide resonance of the next residue. Additional side-chain assignments were made from HCCH-COSY and HCCH-TOCSY experiments. The secondary structure of FGF-2 is based on NOE data involving the NH, H alpha and H beta protons as well as 3JHNH alpha coupling constants, amide exchange and 13C alpha and 13C beta secondary chemical shifts. It is shown that FGF-2 consists of 11 well-defined antiparallel beta-sheets (residues 30-34, 39-44, 48-53, 62-67, 71-76, 81-85, 91-94, 103-108, 113-118, 123-125 and 148-152) and a helix-like structure (residues 131-136), which are connected primarily by tight turns. This structure differs from the refined X-ray crystal structures of FGF-2, where residues 131-136 were defined as beta-strand XI. The discovery of the helix-like region in the primary heparin-binding site (residues 128-138) instead of the beta-strand conformation described in the X-ray structures may have important implications in understanding the nature of heparin-FGF-2 interactions. In addition, two distinct conformations exist in solution for the N-terminal residues 9-28. This is consistent with the X-ray structures of FGF-2, where the first 17-<em>19</em> residues were ill defined.

Bile acids regulate lipid and carbohydrate metabolism by interaction with membrane or intracellular proteins including the nuclear farnesoid X receptor (FXR). Postprandial activation of ileal FXR leads to secretion of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF-<em>19</em>), a gut hormone that may be implicated in postprandial glucose metabolism.

To describe postprandial plasma concentrations of 12 individual bile acids and FGF-<em>19</em> in patients with type 2 diabetes (T2D) and healthy controls.

Descriptive study, performed at the Center for Diabetes Research, Gentofte Hospital, Hellerup, Denmark.

Fifteen patients with T2D and 15 healthy matched controls with normal glucose tolerance.

A 75-g oral glucose tolerance test and three isocaloric and isovolemic liquid meals with low, medium, and high fat content, respectively.

Bile acid and FGF-<em>19</em> concentrations.

Postprandial total bile acid concentrations increased with increasing meal fat content (P < .05), peaked after 1-2 hours, and were higher in T2D patients vs controls (oral glucose tolerance test, low and medium fat meals, P < .05; high fat meal, P = .30). Differences reflected mainly unconjugated and glycine-conjugated forms of deoxycholic acid (DCA) and to a lesser extent cholic acid (CA) and ursodeoxycholic acid (UDCA), whereas chenodeoxycholic acid (CDCA) concentrations were comparable in the two groups. FGF-<em>19</em> concentrations tended to be lower in T2D patients vs controls, but differences were not statistically significant due to considerable variation.

Postprandial plasma patterns of bile acids with FXR agonistic properties (CDCA, DCA, and CA) and FXR antagonistic properties (UDCA) in T2D patients support the notion of a "T2D-bile acid-FGF-<em>19</em>" phenotype with possible pathophysiological implications.

Reduced plasma LDL-cholesterol is a hallmark of hyperthyroidism and is caused by transcriptional stimulation of LDL receptors in the liver. Here, we investigated whether thyroid hormone (TH) actions involve other mechanisms that may also account for the reduction in LDL-cholesterol, including effects on proprotein convertase subtilisin/kexin type 9 (PCSK9) and bile acid synthesis. Twenty hyperthyroid patients were studied before and after clinical normalization, and the responses to hyperthyroidism were compared with those in 14 healthy individuals after 14 days of treatment with the liver-selective TH analog eprotirome. Both hyperthyroidism and eprotirome treatment reduced circulating PCSK9, lipoprotein cholesterol, apoB and AI, and lipoprotein(a), while cholesterol synthesis was stable. Hyperthyroidism, but not eprotirome treatment, markedly increased bile acid synthesis and reduced <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>19</em> and dietary cholesterol absorption. Eprotirome treatment, but not hyperthyroidism, reduced plasma triglycerides. Neither hyperthyroidism nor eprotirome treatment altered insulin, glucose, or FGF21 levels. TH reduces circulating PSCK9, thereby likely contributing to lower plasma LDL-cholesterol in hyperthyroidism. TH also stimulates bile acid synthesis, although this response is not critical for its LDL-lowering effect.

Based on the K8/JTS-1-mediated transfection technique, we developed an in vivo protocol for an efficient transfer of plasmid DNA to ocular cells. As determined with condensed plasmids containing reporter genes for either beta-galactosidase (pcDNA-lacZ) or enhanced green fluorescent protein (pREP-EGFP), the immortalized human retinal epithelial cells RPE D407 and human embryonic kidney 293 cells can be transfected with typical efficiencies of 11 and <em>19</em>%, respectively. Unlike 293 cells, RPE D407 cells had a reduced viability on transfection with both plasmids. In vivo, subretinal injections of DNA-K8/JTS-1 complexes revealed reporter gene expression in choroidal and RPE cells of normal pink-eyed Royal College of Surgeons (RCS) rats. The validity of this transfection technique in terms of retinal cell survival in RCS rats was then examined by using pREP-hFGF2 plasmid, which encodes the human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> isoforms (hFGF2). Subretinal injection of pREP-hFGF2-K8/JTS-1 complexes into 3-week-old dystrophic RCS rat eyes reveals a delayed photoreceptor cell degeneration 60 days postinjection. In this case, the average analyzed field points with delayed cell dystrophy represent 14 to 17% of the retinal surface as compared with 2.6 and 4% in pREP5beta and vehicle-injected eyes, respectively. Peptide-mediated in oculo transfection thus appears to be a promising technique for the treatment of retinal cell and photoreceptor degenerations.

Outcomes for patients with advanced hepatocellular carcinoma (HCC) remain poor despite recent progress in drug development. Emerging data implicate FGF19 as a potential HCC driver, suggesting its receptor, FGFR4, as a novel therapeutic target. We evaluated fisogatinib (BLU-554), a highly potent and selective oral FGFR4 inhibitor, in a phase I dose-escalation/dose-expansion study in advanced HCC using FGF19 expression by immunohistochemistry as a biomarker for pathway activation. For dose escalation, 25 patients received 140 to 900 mg fisogatinib once daily; the maximum-tolerated dose (600 mg once daily) was expanded in 81 patients. fisogatinib was well tolerated; most adverse events were manageable, grade 1/2 gastrointestinal events, primarily diarrhea, nausea, and vomiting. Across doses, the overall response rate was 17% in FGF19-positive patients (median duration of response: 5.3 months [95% CI, 3.7-not reached]) and 0% in FGF19-negative patients. These results validate FGFR4 as a targetable driver in FGF19-positive advanced HCC.

OBJECTIVE

To study the effect of the toxic secondary bile acid lithocholic acid (LCA) on the expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) in intestinal cells and to characterize the pregnane-X-receptor (PXR) response of the FGF<em>19</em> promoter region.

METHODS

The intestinal cell line LS174T was stimulated with various concentrations of chenodeoxy-cholic acid and lithocholic acid for several time points. FGF<em>19</em> mRNA levels were determined with quantitative realtime RT-PCR. FGF<em>19</em> deletion promoter constructs were generated and the LCA response was analzyed in reporter assays. Co-transfections with PXR and RXR were carried out to study FGF<em>19</em> regulation by these <em>factor</em>s.

RESULTS

LCA and CDCA strongly up-regulate FGF<em>19</em> mRNA expression in LS174T cells in a time and dose dependent manner. Using reporter gene assays with several deletion constructs we found that the LCA responsive element in the human FGF<em>19</em> promoter maps to the proximal regulatory region containing two potential binding sites for PXR. Overexpression of PXR and its dimerization partner retinoid X receptor (RXR) and stimulation with LCA or the potent PXR ligand rifampicin leads to a significant induction of FGF<em>19</em> promoter activity in intestinal cells.

CONCLUSIONS

LCA induced feedback inhibition of bile acid synthesis in the liver is likely to be regulated by PXR inducing intestinal FGF<em>19</em> expression.

The ability of the <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> (FGF) <em>19</em> subfamily to signal in an endocrine fashion sets this subfamily apart from the remaining five FGF subfamilies known for their paracrine functions during embryonic development. Compared to the members of paracrine FGF subfamiles, the three members of the FGF<em>19</em> subfamily, namely FGF<em>19</em>, FGF21 and FGF23, have poor affinity for heparan sulfate (HS) and therefore can diffuse freely in the HS-rich extracellular matrix to enter into the bloodstream. In further contrast to paracrine FGFs, FGF<em>19</em> subfamily members have unusually poor affinity for their cognate FGF receptors (FGFRs) and therefore cannot bind and activate them in a solely HS-dependent fashion. As a result, the FGF<em>19</em> subfamily requires α/βklotho coreceptor proteins in order to bind, dimerize and activate their cognate FGFRs. This klotho-dependency also determines the tissue specificity of endocrine FGFs. Recent structural and biochemical studies have begun to shed light onto the molecular basis for the klotho-dependent endocrine mode of action of the FGF<em>19</em> subfamily. Crystal structures of FGF<em>19</em> and FGF23 show that the topology of the HS binding site (HBS) of FGF<em>19</em> subfamily members deviates drastically from the common topology adopted by the paracrine FGFs. The distinct topologies of the HBS of FGF<em>19</em> and FGF23 prevent HS from direct hydrogen bonding with the backbone atoms of the HBS of these ligands and accordingly decrease the HS binding affinity of this subfamily. Recent biochemical data reveal that the ?klotho ectodomain binds avidly to the ectodomain of FGFR1c, the main cognate FGFR of FGF23, creating a de novo high affinity binding site for the C-terminal tail of FGF23. The isolated FGF23 C-terminus can be used to effectively inhibit the formation of the FGF23-FGFR1c-αklotho complex and alleviate hypophosphatemia in renal phosphate disorders due to elevated levels of FGF23.

BACKGROUND

The <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) has been implicated in recent studies as a potential regulator of glucose and lipid metabolism, which may lead to atherosclerosis. Here, we investigated the association of FGF<em>19</em> with the presence and severity of coronary artery disease (CAD) in a Chinese population.

METHODS

A total of 315 patients with suspected or established CAD, including 205 males and 110 postmenopausal females, were enrolled and assessed by coronary angiography. CAD severity was determined by the Gensini score. Serum FGF<em>19</em> was measured by quantitative sandwich ELISA.

RESULTS

FGF<em>19</em> levels were not significantly different between male and female patients (median [interquartile range], 143.40 [87.96-250.80] vs. 141.60 [87.13-226.32] pg/mL, P = 0.773). CAD patients had lower levels of FGF<em>19</em> than those without CAD (128.20 [80.62-226.58] vs. 188.00 [105.10-284.70] pg/mL, P = 0.007). FGF<em>19</em> was negatively correlated with 2hPG (r = -0.150, P = 0.008), FINS (r = -0.169, P = 0.004), HOMA-IR (r = -0.171, P = 0.004), and the Gensini score (r = -0.141, P = 0.012), but positively correlated with HDL-c (r = 0.116, P = 0.041) and adiponectin (r = 0.128, P = 0.024). Moreover, FGF<em>19</em> was found to be independently correlated with 2hPG (β = -0.146, P = 0.022) and adiponectin (β = 0.154, P = 0.016). After adjusting for other CAD risk <em>factor</em>s, FGF<em>19</em> was demonstrated to be an independent <em>factor</em> for Gensini score (β = -0.140, P = 0.0<em>19</em>) and the presence of CAD (β = -1.248, P = 0.036).

CONCLUSIONS

Serum FGF<em>19</em> is associated with the presence and severity of CAD in a Chinese population.