OBJECTIVE

Gliomas account for most primary brain tumors in adults, and survival correlates with the grade and vascularity of the tumor. The degree of tumor-related angiogenesis seems to be a significant predictor of tumor progression, recurrence, and metastatic spread in a variety of malignant diseases, including brain tumors. Our study's objective was to quantify the levels of two angiogenic factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), in the cerebrospinal fluid (CSF) and serum of patients with gliomas and to correlate these levels with tumor grade, vascularity, and overall survival.

METHODS

Twenty-six patients with the diagnosis of cerebral glioma (19 high-grade, 7 low-grade) comprised the study group. Ten patients with communicating hydrocephalus served as controls. Levels of VEGF and bFGF in the CSF and serum were determined using enzyme-linked immunosorbent assay analysis. Tumor vascularity was graded qualitatively using immunohistochemical staining for CD34. Nonparametric statistical techniques were used for data analysis.

RESULTS

Median levels of bFGF and VEGF in the CSF were significantly higher in patients with high-grade glioma as compared with patients with low-grade glioma or hydrocephalus (bFGF levels, 52, 26, and 24 ng/ml, respectively, P < 0.0001; VEGF levels, 17.6, 7.2, and 8.3 ng/ml, respectively, P < 0.005). A significant correlation was found comparing CSF levels of bFGF with levels of VEGF (P < 0.001). The levels of the angiogenic factors in the CSF correlated with the degree of tumor vascularity and were adversely associated with patient survival. Serum levels of the angiogenic factors showed no correlation to tumor grade, vascularity, or survival.

CONCLUSIONS

Our data suggest that CSF levels of bFGF and VEGF may serve as an additional marker for tumor grading and vascularity and may help predict survival.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> ligands and receptors (FGF and FGFR) play critical roles in tumorigenesis, and several drugs have been developed to target them. We report the biologic correlates of FGF/FGFR abnormalities in diverse malignancies. The medical records of patients with cancers that underwent targeted next generation sequencing (182 or 236 cancer-related genes) were reviewed. The following FGF/FGFR genes were tested: FGF3, 4, 6, 7, 10, 12, 14, <em>19</em>, 23 and FGFR1, 2, 3, and 4. Of 391 patients, 56 (14.3%) had aberrant FGF (N = 38, all amplifications) and/or FGFR (N = 22 including 5 mutations and one FGFR3-TACC3 fusion). FGF/FGFR aberrations were most frequent in breast cancers (26/81, 32.1%, p = 0.0003). In multivariate analysis, FGF/FGFR abnormalities were independently associated with CCND1/2, RICTOR, ZNF703, RPTOR, AKT2, and CDK8 alterations (all P < 0.02), as well as with an increased median number of alterations (P < 0.0001). FGF3, FGF4, FGF<em>19</em> and CCND1 were co-amplified in 22 of 391 patients (5.6%, P < 0.0001), most likely because they co-localize on the same chromosomal region (11q13). There was no significant difference in time to metastasis or overall survival when comparing patients harboring FGF/FGFR alterations versus those not. Overall, FGF/FGFR was one of the most frequently aberrant pathways in our population comprising patients with diverse malignancies. These aberrations frequently co-exist with anomalies in a variety of other genes, suggesting that tailored combination therapy may be necessary in these patients.

Glucocorticoids have been shown to accelerate fetal lung type II cell maturation, and this effect appears, in part, to be mediated via <em>fibroblasts</em>. To identify glucocorticoid induced genes in fetal lung <em>fibroblasts</em>, we screened a cDNA library from cortisol-treated fetal lung <em>fibroblasts</em> with a subtracted cDNA probe which was enriched for sequences specific for cortisol-treated fetal lung <em>fibroblasts</em>. Fifty-seven clones were isolated from the cDNA library. One cDNA represented approximately 30% of the 57 clones. Analysis of DNA sequence homology suggested that this cDNA encodes the rat transforming <em>growth</em> <em>factor</em>-beta 3 (TGF beta 3). We found that TGF beta 3 mRNA was expressed in fetal lung <em>fibroblasts</em> but not epithelial cells. Expression of message in fetal lung <em>fibroblasts</em> was developmentally regulated. TGF beta 3 mRNA levels were low during the pseudoglandular stage (day 18), peaked during the early canalicular stage of lung development (day <em>19</em>), then fell again at days 20 and 21 (term = 22 days). Exposure of fetal lung <em>fibroblasts</em> to cortisol increased TGF beta 3 mRNA expression in a time- and dose-dependent manner. Maternal administration of dexamethasone also enhanced mRNA expression of TGF beta 3 in fetal lung <em>fibroblasts</em>. These data suggest that glucocorticoids may mediate their stimulatory effect on lung maturation by inducing TGF beta 3 expression in fetal lung <em>fibroblasts</em>.

We have previously reported on a case-series of children (n=46) with suspected calcium-deficiency rickets who presented in The Gambia with rickets-like bone deformities. Biochemical analyses discounted vitamin D-deficiency as an aetiological <em>factor</em> but indicated a perturbation of Ca-P metabolism involving low plasma phosphate and high circulating <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF23) concentrations. A follow-up study was conducted 5 years after presentation to investigate possible associated <em>factors</em> and characterise recovery. 35 children were investigated at follow-up (RFU). Clinical assessment of bone deformities, overnight fasted 2 h urine and blood samples, 2-day weighed dietary records and 24 h urine collections were obtained. Age- and season-matched data from children from the local community (LC) were used to calculate standard deviation scores (SDS) for RFU children. None of the RFU children had radiological signs of active rickets. However, over half had residual leg deformities consistent with rickets. Dietary Ca intake (SDS-Ca=-0.52 (0.98) p=0.04), dietary Ca/P ratio (SDS-Ca/P=-0.80 (0.82) p=0.0008) and TmP:GFR (SDS-TmP:GFR=-0.48 (0.81) p=0.04) were significantly lower in RFU children compared with LC children and circulating FGF23 concentration was elevated in <em>19</em>% of RFU children. Furthermore an inverse relationship was seen between haemoglobin and FGF23 (R(2)=25.8, p=0.004). This study has shown differences in biochemical and dietary profiles between Gambian children with a history of rickets-like bone deformities and children from the local community. This study provided evidence in support of the calcium deficiency hypothesis leading to urinary phosphate wasting and rickets and identified glomerular filtration rate and iron status as possible modulators of FGF23 metabolic pathways.

BACKGROUND

Peripheral vasculature undergoes extensive vascular remodeling in the hypertensive state. Regulation of extracellular matrix turnover by the matrix metalloproteinase (MMP) system is an important step in the vascular remodeling process. However, the expression pattern of the vascular MMP system in human hypertension remained unknown.

RESULTS

Internal mammary artery specimens were obtained from normotensive (n = 13) and hypertensive (n = <em>19</em>) patients undergoing coronary artery bypass grafting surgery. Zymographic analysis indicated a threefold decrease in total gelatinolytic activity of MMP-2 and MMP-9 in hypertension. MMP-1 activity was also decreased by fourfold without a significant change in protein levels. Tissue levels of extracellular matrix inducer protein (EMMPRIN), MMP activator protein (MT1-MMP), MMP-1, MMP-2, and MMP-9, as well as tissue inhibitors of MMPs (TIMP-1 and TIMP-2) were assessed by immunoblotting and yielded a significant decrease in MMP-9, EMMPRIN, and MT1-MMP levels in hypertension. In addition, measurement of plasma markers of collagen synthesis (procollagen type I amino-terminal propeptide [PINP]) and collagen degradation (carboxy-terminal telopeptide of collagen type I [ICTP]) indicated no difference in PINP levels but suppressed degradation of collagen in hypertension. Evaluation of profibrotic <em>growth</em> <em>factors</em> demonstrated higher levels of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 in tissue preparations from hypertensive patients but no difference in transforming <em>growth</em> <em>factor</em>-beta1 levels.

CONCLUSIONS

These findings demonstrate that not only MMP-1 and MMP-9, but MMP inducer and activator proteins are also downregulated in the hypertensive state. Augmented FGF-2 levels may contribute to parallel decreases in MMP activity and MMP induction system resulting in enhanced collagen deposition in hypertension.

BACKGROUND

Pulmonary pleomorphic carcinoma (PPC) is a rare type of lung cancer characterized by the poor response to conventional chemotherapy and subsequent disappointing outcomes. Therefore, it is paramount to delineate the molecular characteristics of this disease entity.

METHODS

In this study, we retrospectively examined the surgical specimens of 61 patients who underwent lung surgery. Mutational or gene amplification statuses of epidermal growth factor receptor (EGFR), k-ras, c-kit, c-met, and fibroblast growth factor receptor (FGFR) were examined using genomic DNA sequencing, real-time PCR and/or fluorescence in situ hybridization (FISH).

RESULTS

The median age was 61 years, and 50 patients were men and 11 were women. In the histologic review of epithelial component, adenocarcinoma were in 44 cases (72%), squamous cell carcinoma in 15 (25%) and large cell carcinoma in 2 patients (3%). Overall, 30 cases (49%) had any molecular alterations. Nine patients (15%) possessed EGFR deletion in exon 19 (n = 8) or L858R mutations in exon 21 (n = 1), while 3 other cases having atypical EGFR mutations. Six patients (9.8%) had k-ras mutations in exon 12, and 3 had c-kit mutations. High gene copy number of c-met was found in 11 patients (18.0%) and that of FGFR was in 6 patients (9.8%). No significant relationships were identified among the occurrence and type of mutations and patient survival or any other clinicopathological variables.

CONCLUSIONS

Given the diverse repertoire of mutational profiles observed in PPC samples, clinical trials based on accurate cancer-genotyping should be considered as a legitimate treatment scheme for this rare disease entity in the future.

OBJECTIVE

Rheumatoid arthritis (RA) is characterized by leukocyte recruitment and angiogenesis. We investigated the effects of sulfasalazine (SSZ) and its metabolites, sulfapyridine (SP) and 5-aminosalicylic acid (5-ASA), on components of angiogenesis, namely, endothelial cell (EC) chemotaxis and proliferation, as well as on EC chemokine and soluble adhesion molecule expression.

METHODS

SSZ, SP, and 5-ASA were assayed for their effects on basic fibroblast growth factor (bFGF)-induced human dermal microvascular endothelial cell (HMVEC) chemotaxis and proliferation. EC were plated on Matrigel to assess the effect of SSZ on EC tube formation. Enzyme-linked immunosorbent assays were performed to determine changes in HMVEC production of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), growth-related oncogene alpha (GROalpha), epithelial neutrophil-activating peptide 78 (ENA-78), soluble E-selectin (sE-selectin), and soluble intercellular adhesion molecule 1 (sICAM-1) upon treatment with SSZ or its metabolites.

RESULTS

HMVEC incubated with SSZ or SP exhibited reduced bFGF-induced chemotaxis (59%, [n = 7] and 22%, [n = 3], respectively) (P<0.05). SSZ and SP decreased basal HMVEC proliferation, while 5-ASA increased proliferation (P<0.05; [n = 5]). SSZ decreased bFGF-induced HMVEC proliferation (P<0.05 [n = 5]). SSZ inhibited phorbol 12-myristate 13-acetate-induced HMVEC tube formation (P<0.05; [minimum n = 5]). Tumor necrosis factor alpha-stimulated HMVEC shedding of sICAM-1 was reduced by incubation with either SSZ (19%) or 5-ASA (23%) (P<0.05; [n = 6]). SP inhibited cytokine-stimulated HMVEC expression of IL-8 and MCP-1 (P<0.05; [n = 4]). Neither SSZ nor its metabolites had any effect on HMVEC production of sE-selectin, GROalpha, or ENA-78.

CONCLUSIONS

These results demonstrate that SSZ and its metabolite SP may affect the pathogenesis of RA by inhibiting EC chemotaxis, proliferation, tube formation, and expression of sICAM-1, IL-8, and MCP-1.

Progenitor cells that endure in different regions of the CNS after the initial neurogenesis can be expanded in culture and used as a source of donor tissue for grafting in neurodegenerative diseases. However, the proliferation and differentiation characteristics of residual neural progenitor cells from distinct regions of the CNS are mostly unknown. This study elucidated the characteristics of progenitor cells that endure in the CA3 region of the hippocampus after neurogenesis, by in vitro analyses of cells that are responsive to epidermal <em>growth</em> <em>factor</em> (EGF) or <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) in the embryonic day <em>19</em> (E<em>19</em>) rat hippocampus. Isolated cells from the E<em>19</em> CA3 region formed neurospheres in the presence of either EGF or FGF-2, but the yield of neurospheres was greater with FGF-2 exposure, Differentiation cultures revealed a greater yield of neurons from FGF-2 neurospheres (60%) than from EGF neurospheres (35%). Exposure to brain-derived neurotrophic <em>factor</em> (BDNF) enhanced the yield of neurons from EGF neurospheres but had no consequence on FGF-2 neurospheres. A large number of neurons from EGF/FGF-2 neurospheres demonstrated clearly palpable morphological features of CA3 pyramidal neurons and lacked gamma-aminobutyric acid (GABA) expression. However, a fraction of neurons (17-20%) from EGF/FGF-2 neurospheres expressed GABA, and exposure to BDNF increased the number of GABAergic neurons (30%) from EGF neurospheres. Neurons from EGF/FGF-2 neurospheres also contained smaller populations of calbindin- and calretinin-positive interneuron-like cells. Thus, progenitor cells responsive to FGF-2 are prevalent in the CA3 region of the E<em>19</em> rat hippocampus and give rise to a greater number of neurons than progenitor cells responsive to EGF. However, both FGF-2- and EGF-responsive progenitor cells from E<em>19</em> CA3 region are capable of giving rise to CA3 field-specific phenotypic neurons. These results imply that progenitor cells that persist in the hippocampus after neurogenesis remain regionally restricted and hence retain their ability to give rise to region-specific phenotypic neurons even after isolation and expansion in vitro.

Some primary tumors are capable of suppressing the <em>growth</em> of their metastases by presumably generating antiangiogenic <em>factors</em> such as angiostatin. We hypothesized that the amount of inhibitor(s) released by a tumor increases with tumor <em>growth</em>. We tested this hypothesis by evaluating the relationship between the size of a primary tumor and its ability to inhibit angiogenesis at a secondary site. Furthermore, we characterized the effects of the primary tumor on physiological properties of newly formed vessels at the secondary site. Angiogenesis and physiological properties were measured using intravital microscopy of angiogenic vessels in the gels containing basic <em>fibroblast</em> <em>growth</em> <em>factor</em> placed into cranial windows of immunodeficient mice bearing human prostatic carcinoma (PC-3) in their flank. The PC-3 tumor inhibited angiogenesis in the gels, and surgical resection of tumor reversed this inhibition. The inhibition of angiogenesis 20 days after gel implantation (range, 0-83%) correlated positively (r = 0.625; P < 0.008) with the tumor size on the day of gel implantation (range, <em>19</em>-980 mm3). The primary tumor also suppressed leukocyte-adhesion in angiogenic vessels, thus helping them evade the immune recognition. These results provide an additional rationale for combining antiangiogenic treatment with local therapies.

Genome-wide association studies (GWAS) and candidate gene analyses have led to the discovery of several dozen genetic polymorphisms associated with breast cancer susceptibility, many of which are considered well-established risk <em>factors</em> for the disease. Despite attempts to replicate these same variant-disease associations in African Americans, the evaluable populations are often too small to produce precise or consistent results. We estimated the associations between 83 previously identified single nucleotide polymorphisms (SNPs) and breast cancer among Carolina Breast Cancer Study (<em>19</em>93-2001) participants using maximum likelihood, Bayesian, and hierarchical methods. The selected SNPs were previous GWAS hits (n = 22), near-hits (n = <em>19</em>), otherwise well-established risk loci (n = 5), or located in the same genes as selected variants (n = 37). We successfully replicated 18 GWAS-identified SNPs in whites (n = 2,352) and 10 in African Americans (n = 1,447). SNPs in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 gene (FGFR2) and the TOC high mobility group box family member 3 gene (TOX3) were strongly associated with breast cancer in both races. SNPs in the mitochondrial ribosomal protein S30 gene (MRPS30), mitogen-activated protein kinase kinase kinase 1 gene (MAP3K1), zinc finger, MIZ-type containing 1 gene (ZMIZ1), and H<em>19</em>, imprinted maternally expressed transcript gene (H<em>19</em>) were associated with breast cancer in whites, and SNPs in the estrogen receptor 1 gene (ESR1) and H<em>19</em> gene were associated with breast cancer in African Americans. We provide precise and well-informed race-stratified odds ratios for key breast cancer-related SNPs. Our results demonstrate the utility of Bayesian methods in genetic epidemiology and provide support for their application in small, etiologically driven investigations.

This report describes the profiling of proteins in a sample prepared by laser capture microdissection (LCM) from a breast cancer cell line (SKBR-3). This experimental approach serves as a model system for proteomic studies on selected tissue samples and for studies of specific cell types. The captured cells were isolated in a dehydrated and reduced state and solubilized with a denaturing buffer. After dilution the protein mixture was digested with trypsin and the resulting peptide mixture was fractionated by reversed phase HPLC (RPLC) and analyzed on an ion trap mass spectrometer. A key part of this study is the combination of the LCM process with an extraction/digestion procedure that allowed effective solubilization of a significant part of the cellular sample in a single step. The identity of the peptides was determined by tandem mass spectrometry measurements in which the resulting spectra were compared with genomic and proteomic databases and protein identifications were made. While only peptides with a high probability assignment were used, the interpretation of mass spectral fragmentation patterns were also confirmed by manual interpretation of the spectra. Also, for the more abundant proteins the initial protein assignment from the best match peptide was strengthened by the observation of additional confirmatory peptide identifications. Another selection criteria was correlation of the mass spectrometric studies with clinical and genomic studies of potential cancer markers in tumor samples. This proteomic study allowed identification of the following proteins: human receptor protein kinase HER-2 or ERBB-2 and related kinases HER-3 and HER-4, the gene products from breast cancer type I and II susceptibility genes and cytoskeletal components such as cytokeratins 8, 18 and <em>19</em>. Other proteins include <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor variants (FGFR-2&4) and T-lymphoma invasion and metastasis inducing protein 1 (TIAM1). In addition several nonreceptor protein kinases YES, FAK and JAK-1 and 3 were identified. Since the study was performed on a limited number of cells (approximately 10,000) it raises the possibility of such studies being performed on individual patient samples prepared by needle biopsy.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) is a developmentally regulated transmembrane protein. Three other FGFRs (1, 2, and 4) in conjunction with FGFR3 are part of the receptor tyrosine kinase super-family. Mutations in three of these genes (FGFR1, 2, and 3) have been determined to be the cause of human <em>growth</em> and developmental disorders. We have characterized a 22-kb DNA fragment containing the human FGFR3 gene and determined 11 kb of its nucleotide sequence. The gene consists of <em>19</em> exons and 18 introns spanning 16.5 kb, and the boundaries between exons and introns follow the GT/AG rule. The translation initiation and termination sites are located in exon 2 and exon <em>19</em> respectively. The sequence of the 5'-flanking region (1.5 kb) lacks the typical TATA or CAAT boxes. However, several putative binding sites for transcription <em>factors</em> SP1, AP2, Krox 24, IgHC.4, and Zeste are present. The 0.77-kb region from position -889 (5'-flanking region) to -1<em>19</em> (intron 1) contains a CpG island. A comparative sequence analysis of the human and mouse FGFR3 genes indicates that the overall genomic structure and organization of the human gene are nearly identical to those of its mouse counterpart. Furthermore, there is a striking similarity in the promoter regions of both genes, and several of the putative transcription <em>factor</em>-binding sites are conserved across species, suggesting a definitive role of these <em>factors</em> in the transcriptional regulation of these genes.

OBJECTIVE

Hyperostosis-hyperphosphataemia syndrome (HHS) is a rare hereditary disorder characterized by hyperphosphataemia, inappropriately normal or elevated 1,25-dihydroxyvitamin D(3) and localized painful cortical hyperostosis. HHS was shown to be caused by inactivating mutations in GALNT3, encoding UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 3 (GalNAc-transferase; GALNT3). Herein, we sought to identify the genetic cause of hyperphosphataemia and tibial hyperostosis in a <em>19</em>-year-old girl of Colombian origin.

METHODS

Genomic DNA was extracted and sequencing analysis of the GALNT3 and fibroblast growth factor 23 (FGF23) genes performed. Serum levels of intact and C-terminal FGF23 were measured using two different ELISA methods.

RESULTS

Mutational analysis identified a novel homozygous missense mutation in exon 6 of GALNT3 (1584 G>A), leading to an amino acid shift from Arg to His at residue 438 (R438H). The mutation was not found in over 200 control alleles or in any single nucleotide polymorphism databases. The R438 residue is highly conserved throughout species and in all known GalNAc-transferase family members. Modelling predicted the substitution deleterious for protein structure. Importantly, the phosphaturic factor FGF23 was differentially processed, as reflected by low intact (15 pg/ml) but high C-terminal (839 RU/ml) serum FGF23 levels.

CONCLUSIONS

We report on the first missense mutation in GALNT3 giving rise to HHS, since previous GALNT3 mutations in HHS caused aberrant splicing or premature truncation of the protein. The R438H substitution likely abrogates GALNT3 activity, in turn causing enhanced FGF23 degradation and subsequent hyperostosis/hyperphosphataemia.

The effect of PTH on chondrocyte proliferation as a function of cartilage age was examined. PTH[1-34] induced a 12- to 15-fold increase in the efficiency of colony formation in soft agar by chondrocytes from embryonic 13- to <em>19</em>-d-old chickens and fetal 25-d-old rabbits with a 10-fold increase in their DNA content. It also caused a 2.5-fold increase in [3H]thymidine incorporation into DNA in fetal 25-d-old rabbit chondrocytes. No mitogenic responses to PTH were observed, however, in postnatal 7- to 21-d-old chick chondrocytes or postnatal 21-d-old rabbit chondrocytes. This age dependency was observed only with PTH: <em>fibroblast</em> <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>, and insulin stimulated chondrocyte proliferation irrespective of cartilage age. The absence of a mitogenic effect in postnatal chondrocytes was not due to a decrease in number or a reduction in affinity of receptors for PTH. PTH also increased [35S]sulfate incorporation into proteoglycans and the cyclic AMP level in fetal and postnatal chondrocytes, but at 100-fold higher concentrations (10(-8)-10(-7) M) than those (10(-10)-10(-9) M) required for the stimulation of cell division. These results suggest that PTH is a potent mitogen for embryonic chondrocytes, and that its mitogenic effect disappears selectively after birth.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) 4 possesses high affinity to acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs). The authors focused on FGFR 4 expression in astrocytoma because the FGF expression increases as the tumor malignancy progresses. Forty-one astrocytoma specimens were examined by immunohistochemistry and polymerase chain reaction-Southern blot. FGFR 4 was negative in all seven Grade II astrocytomas by immunohistochemistry, while positive in four among 15 Grade III and in 13 among <em>19</em> Grade IV astrocytomas. The median survival time of Grade III astrocytoma patients was 22.3 months in FGFR 4 negative group and 14.5 months in positive group (p < 0.05). Those of Grade IV patients were 14.2 months in FGFR 4 negative group and 11.9 months in positive group (p>> 0.05, not significant). However, FGFR 4 mRNA was detected in all specimens suggesting activated translation system of FGFR 4 in progression of the tumor malignancy. Histologically diagnosed Grade III astrocytoma patients can be divided into two groups; one with median survival time close to those with Grade II astrocytoma patients, and the other similar to that of glioblastoma patients. The authors concluded that FGFR 4 must be an important <em>factor</em> which predicts short survival Grade III astrocytoma patients, who require strict adjuvant therapy in accordance with glioblastoma.

Tissue-specific alternative splicing in the second half of Ig-like domain 3 (D3) of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors 1-3 (FGFR1 to -3) generates epithelial FGFR1b-FGFR3b and mesenchymal FGFR1c-FGFR3c splice isoforms. This splicing event establishes a selectivity filter to restrict the ligand binding specificity of FGFRb and FGFRc isoforms to mesenchymally and epithelially derived <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs), respectively. FGF1 is termed the "universal FGFR ligand" because it overrides this specificity barrier. To elucidate the molecular basis for FGF1 cross-reactivity with the "b" and "c" splice isoforms of FGFRs, we determined the first crystal structure of FGF1 in complex with an FGFRb isoform, FGFR2b, at 2.1 Å resolution. Comparison of the FGF1-FGFR2b structure with the three previously published FGF1-FGFRc structures reveals that plasticity in the interactions of the N-terminal region of FGF1 with FGFR D3 is the main determinant of FGF1 cross-reactivity with both isoforms of FGFRs. In support of our structural data, we demonstrate that substitution of three N-terminal residues (Gly-<em>19</em>, His-25, and Phe-26) of FGF2 (a ligand that does not bind FGFR2b) for the corresponding residues of FGF1 (Phe-16, Asn-22, and Tyr-23) enables the FGF2 triple mutant to bind and activate FGFR2b. These findings taken together with our previous structural data on receptor binding specificity of FGF2, FGF8, and FGF10 conclusively show that sequence divergence at the N termini of FGFs is the primary regulator of the receptor binding specificity and promiscuity of FGFs.

OBJECTIVE

The aim of this study was to assess the roles of plasma cytokines in diabetic retinopathy (DR) and their relationship with the severity of DR.

METHODS

This study included 59 diabetic patients and <em>19</em> non-diabetic controls. The plasma concentrations of endothelial <em>growth</em> <em>factor</em> (EGF), eotaxin, <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2), Flt-3 ligand (Flt-3L), fractalkine, granulocyte colony-stimulating <em>factor</em> (G-CSF), granulocyte-macrophage colony-stimulating <em>factor</em> (GM-CSF), <em>growth</em>-related oncogene (GRO), interferon (IFN)-α2, IFN-γ, interleukin (IL)-1α, IL-1β, IL-1Ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IFN-inducible protein-10 (IP-10), monocyte chemoattractant protein (MCP)-1, MCP-3, macrophage-derived cytokine (MDC), macrophage inflammatory protein (MIP)-1α, MIP-1β, sCD40L, sIL-2Rα, transforming <em>growth</em> <em>factor</em> (TGF)-α, tumor necrosis <em>factor</em> (TNF)- α, TNF-β, and VEGF were measured with Luminex multiplex bead immunoassay. The levels of these cytokines were investigated according to the DR stage.

RESULTS

The plasma level of ten cytokines-MCP-1, IL-6, IL-7, IL-9, IL-13, IL-15, IL-17, sCD40L, sIL-2Rα and TNF-β-increased significantly in the diabetic group compared to the controls. The Flt-3L, IL-1Ra, IL-3, IL-5, and IL-12 (p40) levels were lower in the diabetic group than in the control group. The TNF-α plasma level was significantly elevated in patients with proliferative diabetic retinopathy (PDR) compared with the levels in patients with non-proliferative diabetic retinopathy (NPDR) and patients with no apparent diabetic retinopathy (NDR).

CONCLUSIONS

TNF-α might be involved in the progression of DR, especially in the pathogenesis of PDR. TNF-α is a potential cytokine for the prognosis of DR and might act as a therapeutic target in DR.

The aim of the present study was to identify differentially expressed genes and their related biological pathways in the secretory phase endometrium from patients with recurrent miscarriage (RM) and fertile subjects. Endometrial samples from RM and fertile patients were analyzed using the Affymetrix GeneChip® ST Array. The bioinformatic analysis using the Partek Genomic Suite revealed 346 genes (175 up-regulated and 171 down-regulated) that were differentially expressed in the endometrium of RM patients compared to the fertile subjects (fold change ≥1.5, p<0.005). Validation step using quantitative real-time polymerase chain reaction (qPCR) confirmed a similar expression pattern of four exemplary genes: one up-regulated gene (<em>fibroblast</em> <em>growth</em> <em>factor</em> 9, FGF9) and three down-regulated genes: integrin β3 (ITGB3), colony stimulating <em>factor</em> 1 (CSF1) and matrix-metalloproteinases <em>19</em> (MMP<em>19</em>). The Gene Set Enrichment Analysis (GSEA) and the Pathway Studio software have found 101 signaling pathways (p<0.05) associated with the affected genes including the FGFR3 /signal transducer and activator of transcription (STAT) pathway and the CSF1R/STAT pathway. Cell adhesion, cell differentiation and angiogenesis were among biological processes indicated by this system. In conclusion, microarray technique is a useful tool to study gene expression in the secretory phase-endometrium of RM patients. The differences in endometrial gene expressions between healthy and RM subjects contribute to an increase in our knowledge on molecular mechanisms of RM development and may improve the outcome of pregnancies in high-risk women with RM.

Clear cell renal cell carcinoma (ccRCC) is the most common renal cell carcinoma subtype, and metastatic ccRCC is associated with 5-year survival rates of 10% to 20%. Genetically, ccRCC originates from sequential losses of multiple tumor suppressor genes. Remarkably, chromosome 3p loss occurs in more than 90% of sporadic ccRCCs. This results in concurrent one-copy loss of four tumor suppressor genes that are also mutated individually at high frequency in ccRCC (ie, VHL, 80%; PBRM1, 29% to 46%; BAP1, 6% to <em>19</em>%; and SETD2, 8% to 30%). Pathogenically, 3p loss probably represents the first genetic event that occurs in sporadic ccRCC and the second genetic event in VHL-mutated hereditary ccRCC. VHL constitutes the substrate recognition module of the VCB-Cul2 E3 ligase that degrades HIF1/2α, whereas PBRM1, BAP1, and SETD2 are epigenetic modulators that regulate gene transcription. Because 3p loss and VHL inactivation are nearly universal truncal events in ccRCC, the resulting HIF1/2 signaling overdrive and accompanied tumor hypervascularization probably underlie the therapeutic benefits observed with vascular endothelial <em>growth</em> <em>factor</em> receptor inhibitors, including sorafenib, sunitinib, pazopanib, axitinib, bevacizumab, cabozantinib, and lenvatinib. Furthermore, recent marked advances in ccRCC genomics, transcriptomics, proteomics, metabolomics, molecular mechanisms, mouse models, prognostic and predictive biomarkers, and clinical trials have rendered invaluable translational insights concerning precision kidney cancer therapeutics. With an armamentarium encompassing 13 drugs that exploit seven unique therapeutic mechanisms (ie, cytokines, vascular endothelial <em>growth</em> <em>factor</em> receptor, mTORC1, cMET/AXL, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor, programmed cell death-1 and programmed death-ligand 1, and cytotoxic T-cell lymphocyte associated-4) to treat metastatic renal cell carcinoma, one of the imminent clinical questions concerning care of patients with metastatic ccRCC is how a personalized treatment strategy, through rationally combining and sequencing different therapeutic modalities, can be formulated to offer the best clinical outcome for individual patients. Here, we attempt to integrate recent discoveries of immediate translational impacts and discuss future translational challenges and opportunities.

The Na+ -taurocholate cotransporting polypeptide (NTCP/SLC10A1) is believed to be pivotal for hepatic uptake of conjugated bile acids. However, plasma bile acid levels are normal in a subset of NTCP knockout mice and in mice treated with myrcludex B, a specific NTCP inhibitor. Here, we elucidated which transport proteins mediate the hepatic uptake of conjugated bile acids and demonstrated intestinal sensing of elevated bile acid levels in plasma in mice. Mice or healthy volunteers were treated with myrcludex B. Hepatic bile acid uptake kinetics were determined in wild-type (WT), organic anion transporting polypeptide (OATP) knockout mice (lacking Slco1a/1b isoforms), and human OATP1B1-transgenic mice. Effects of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) on hepatic transporter mRNA levels were assessed in rat hepatoma cells and in mice by peptide injection or adeno-associated virus-mediated overexpression. NTCP inhibition using myrcludex B had only moderate effects on bile acid kinetics in WT mice, but completely inhibited active transport of conjugated bile acid species in OATP knockout mice. Cholesterol 7α-hydroxylase Cyp7a1 expression was strongly down-regulated upon prolonged inhibition of hepatic uptake of conjugated bile acids. Fgf15 (mouse counterpart of FGF<em>19</em>) expression was induced in hypercholanemic OATP and NTCP knockout mice, as well as in myrcludex B-treated cholestatic mice, whereas plasma FGF<em>19</em> was not induced in humans treated with myrcludex B. Fgf15/FGF<em>19</em> expression was induced in polarized human enterocyte-models and mouse organoids by basolateral incubation with a high concentration (1 mM) of conjugated bile acids.

NTCP and OATPs contribute to hepatic uptake of conjugated bile acids in mice, whereas the predominant uptake in humans is NTCP mediated. Enterocytes sense highly elevated levels of (conjugated) bile acids in the systemic circulation to induce FGF15/<em>19</em>, which modulates hepatic bile acid synthesis and uptake. (Hepatology 2017;66:1631-1643).

OBJECTIVE

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>), a hormone secreted from the small intestine, has recently been shown to stimulate glycogen synthesis and inhibit gluconeogenesis through insulin-independent pathways. This study investigated the change of FGF<em>19</em> in prediabetes and newly diagnosed type 2 diabetes mellitus (T2DM) and explored the association of serum FGF<em>19</em> levels with parameters of glucose metabolism in Chinese subjects.

METHODS

Fasting serum FGF<em>19</em> levels were determined by ELISA in 81 normal glucose tolerance (NGT), 91 impaired fasting glucose (IFG), 93 impaired glucose tolerance (IGT), and 104 newly diagnosed T2DM subjects, and their association with parameters of glucose metabolism was studied. An ordinal logistic regression analysis was performed in subjects with NGT, IFG, and T2DM. Serum FGF<em>19</em> levels at 2 h after a 75-g oral glucose tolerance test in the different glucose tolerance categories were studied in a subgroup.

RESULTS

Fasting serum FGF<em>19</em> levels in subjects with IFG (210 pg/mL [142-327]) (median [interquartile range]) and T2DM (<em>19</em>6 pg/mL [137-280]) were significantly lower than those in NGT subjects (289 pg/mL [224-393]) (both P < 0.001). However, no significant difference in fasting FGF<em>19</em> levels was observed between IGT (246 pg/mL [138-379]) and NGT subjects. Fasting serum FGF<em>19</em> levels were negatively associated with fasting plasma glucose and independently associated with the deterioration of glucometabolic status from NGT to IFG and T2DM.

CONCLUSIONS

Fasting serum FGF<em>19</em> levels were decreased in Chinese subjects with IFG and inversely associated with fasting glucose levels.

Oval cells, putative hepatic stem cells, could potentially provide a novel solution to the severe shortage of donor livers, because of their ability to proliferate and differentiate into functional hepatocytes. We have previously demonstrated that oval cells can be induced to differentiate into cells with morphologic, phenotypic, and functional characteristics of mature hepatocytes. In this study, we have established a new model combining ethionine treatment with partial hepatectomy to activate oval cells, then developed a procedure utilizing selective enzymatic digestion and density gradient centrifugation to isolate and purify such cells from heterogeneous liver cell population. We identified oval cells by their morphological characteristics and phenotypic properties, thereby providing definitive evidence of the presence of hepatic stem-like cells in adult rat livers. Viewed by transmission electron microscopy, they were small cells with ovoid nuclei, a high nucleus/cytoplasm ratio and few organelles, including mitochondria and endoplasmic reticulum. Flow cytometric assay showed that these cells highly expressed OV-6, cytokeratin-<em>19</em> (CK-<em>19</em>) and albumin. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis displayed that the freshly isolated cells co-expressed albumin, cytokeratin-7 (CK-7) and CK-<em>19</em> mRNA, indicating that they were essentially bipotential hepatic stem-like cells. Furthermore, we set up a culture system containing <em>growth</em> <em>factors</em> and a <em>fibroblast</em> feeder layer, to provide nourishment to these cells. Thus, we were able to culture them in vitro for more than 3 months, with the number of cells doubling 100 times. Gene expressions of albumin, CK-7 and CK-<em>19</em> in the cells derived from the expanding colonies at day 95 were confirmed by RT-PCR analysis. These data suggested that the hepatic oval cells derived from adult rat livers possess a high potential to proliferate in vitro with a large increase in number, while maintaining the bipotential nature of hepatic stem cells.

The gut-derived hormone <em>fibroblast</em> <em>growth</em> <em>factor</em> 15/<em>19</em> (FGF15/<em>19</em>) is an emerging versatile regulator of various metabolic pathways. As such, FGF15/<em>19</em> has been implicated in homeostatic control of bile acid, carbohydrate and lipid metabolism in multiple target organs including the liver, adipose tissue and brain. In line with this, <em>growing</em> evidence suggests that dysregulation of FGF15/<em>19</em> contributes to a number of metabolic and bile acid-associated disorders such as fatty liver disease, Type 2 diabetes and different gastrointestinal dysfunctions. In this review we summarize the current knowledge on the organ-specific functions of FGF15/<em>19</em> and address their underlying molecular mechanisms. Moreover, recent advances in the characterization of <em>factors</em> that control the release of the hormone in the gut will be discussed and linked to the current view of how alterations of FGF15/<em>19</em> signaling may contribute to disease development. Finally, the suitability of FGF15/<em>19</em> as a potential therapeutic target will be critically reviewed.

BACKGROUND

Impaired wound healing in diabetes is related to decreased production of growth factors. Hence, gene therapy is considered as promising treatment modality. So far, efforts concentrated on single gene therapy with particular emphasis on vascular endothelial growth factor-A (VEGF-A). However, as multiple proteins are involved in this process it is rational to test new approaches. Therefore, the aim of this study was to investigate whether single AAV vector-mediated simultaneous transfer of VEGF-A and fibroblast growth factor 4 (FGF4) coding sequences will improve the wound healing over the effect of VEGF-A in diabetic (db/db) mice.

METHODS

Leptin receptor-deficient db/db mice were randomized to receive intradermal injections of PBS or AAVs carrying β-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A), FGF-4 (AAV-FGF4-IRES-GFP) or both therapeutic genes (AAV-FGF4-IRES-VEGF-A). Wound healing kinetics was analyzed until day 21 when all animals were sacrificed for biochemical and histological examination.

RESULTS

Complete wound closure in animals treated with AAV-VEGF-A was achieved earlier (day 19) than in control mice or animals injected with AAV harboring FGF4 (both on day 21). However, the fastest healing was observed in mice injected with bicistronic AAV-FGF4-IRES-VEGF-A vector (day 17). This was paralleled by significantly increased granulation tissue formation, vascularity and dermal matrix deposition. Mechanistically, as shown in vitro, FGF4 stimulated matrix metalloproteinase-9 (MMP-9) and VEGF receptor-1 expression in mouse dermal fibroblasts and when delivered in combination with VEGF-A, enhanced their migration.

CONCLUSIONS

Combined gene transfer of VEGF-A and FGF4 can improve reparative processes in the wounded skin of diabetic mice better than single agent treatment.