In chronic kidney disease, vascular calcification, renal osteodystrophy, and phosphate contribute substantially to cardiovascular risk and are components of CKD-mineral and bone disorder (CKD-MBD). The cause of this syndrome is unknown. Additionally, no therapy addresses cardiovascular risk in CKD. In its inception, CKD-MBD is characterized by osteodystrophy, vascular calcification, and stimulation of osteocyte secretion. We tested the hypothesis that increased production of circulating <em>factors</em> by diseased kidneys causes the CKD-MBD in diabetic mice subjected to renal injury to induce stage 2 CKD (CKD-2 mice). Compared with non-CKD diabetic controls, CKD-2 mice showed increased renal production of Wnt inhibitor family members and higher levels of circulating Dickkopf-1 (Dkk1), sclerostin, and secreted klotho. Neutralization of Dkk1 in CKD-2 mice by administration of a monoclonal antibody after renal injury stimulated bone formation rates, corrected the osteodystrophy, and prevented CKD-stimulated vascular calcification. Mechanistically, neutralization of Dkk1 suppressed aortic expression of the osteoblastic transcription <em>factor</em> Runx2, increased expression of vascular smooth muscle protein <em>22</em>-α, and restored aortic expression of klotho. Neutralization of Dkk1 did not affect the elevated plasma levels of osteocytic <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 but decreased the elevated levels of sclerostin. Phosphate binder therapy restored plasma <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 levels but had no effect on vascular calcification or osteodystrophy. The combination of the Dkk1 antibody and phosphate binder therapy completely treated the CKD-MBD. These results show that circulating Wnt inhibitors are involved in the pathogenesis of CKD-MBD and that the combination of Dkk1 neutralization and phosphate binding may have therapeutic potential for this disorder.

Uncertainty exists regarding the physiologically relevant <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) receptor (FGFR) for FGF23 in the kidney and the precise tubular segments that are targeted by FGF23. Current data suggest that FGF23 targets the FGFR1c-Klotho complex to coordinately regulate phosphate transport and 1,25-dihydroxyvitamin D [1,25(OH)(2)D] production in the proximal tubule. In studies using the Hyp mouse model, which displays FGF23-mediated hypophosphatemia and aberrant vitamin D, deletion of Fgfr3 or Fgfr4 alone failed to correct the Hyp phenotype. To determine whether FGFR1 is sufficient to mediate the renal effects of FGF23, we deleted Fgfr3 and Fgfr4 in Hyp mice, leaving intact the FGFR1 pathway by transferring compound Fgfr3/Fgfr4-null mice on the Hyp background to create wild-type (WT), Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice. We found that deletion of Fgfr3 and Fgfr4 in Fgfr3(-/-)/Fgfr4(-/-) and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice induced an increase in 1,25(OH)(2)D. In Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, it partially corrected the hypophosphatemia (P(i) = 9.4 ± 0.9, 6.1 ± 0.2, 9.1 ± 0.4, and 8.0 ± 0.5 mg/dl in WT, Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, respectively), increased Na-phosphate cotransporter Napi2a and Napi2c and Klotho mRNA expression in the kidney, and markedly increased serum FGF23 levels (107 ± 20, 3,680 ± 284, 167 ± <em>22</em>, and 18,492 ± 1,547 pg/ml in WT, Hyp, Fgfr3(-/-)/Fgfr4(-/-), and Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice, respectively), consistent with a compensatory response to the induction of end-organ resistance. Fgfr1 expression was unchanged in Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice and was not sufficient to transduce the full effects of FGF23 in Hyp/Fgfr3(-/-)/Fgfr4(-/-) mice. These studies suggest that FGFR1, FGFR3, and FGFR4 act in concert to mediate FGF23 effects on the kidney and that loss of FGFR function leads to feedback stimulation of Fgf23 expression in bone.

Cancer-associated <em>fibroblasts</em> (CAFs), represent a pivotal compartment of solid cancers (desmoplasia), and are causatively implicated in cancer development and progression. CAFs are recruited by <em>growth</em> <em>factors</em> secreted by cancer cells and they present a myofibroblastic phenotype, similar to the one obtained by resident <em>fibroblasts</em> during wound healing. Paracrine signaling between cancer cells and CAFs results in a unique protein expression profile in areas of desmoplastic reaction, which is speculated to drive metastasis. In an attempt to decipher large-scale proteomic profiles of the cancer invasive margins, we developed an in vitro coculture model system, based on tumor-host cell interactions between colon cancer cells and CAFs. Proteomic analysis of conditioned media derived from these cocultures coupled to mass spectrometry and bioinformatic analysis was performed to uncover myofibroblastic signatures of the cancer invasion front. Our analysis resulted in the identification and generation of a desmoplastic protein dataset (DPD), consisting of 152 candidate proteins of desmoplasia. By using monoculture exclusion datasets, a secretome algorithm and gene-expression meta-analysis in DPD, we specified a <em>22</em>-protein "myofibroblastic signature" with putative importance in the regulation of colorectal cancer metastasis. Of these proteins, we investigated collagen type XII by immunohistochemistry, a fibril-associated collagen with interrupted triple helices (FACIT), whose expression has not been reported in desmoplastic lesions in any type of cancer. Collagen type XII was highly expressed in desmoplastic stroma by and around alpha-smooth muscle actin (α-SMA) positive CAFs, as well as in cancer cells lining the invasion front, in a small cohort of colon cancer patients. Other stromal markers, such as collagen type III, were also expressed in stromal collagen, but not in cancer cells. In a complementary fashion, gene expression meta-analysis revealed that COL12A1 is also an upregulated gene in colorectal cancer. Our proteomic analysis identified previously documented markers of tumor invasion fronts and our DPD could serve as a pool for future investigation of the tumor microenvironment. Collagen type XII is a novel candidate marker of myo<em>fibroblasts</em>, and/or cancer cells undergoing dedifferentiation.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) is a heparin-binding <em>growth</em> <em>factor</em> which occurs in several isoforms resulting from alternative initiations of translation: an 18 kD cytoplasmic isoform and four larger molecular weight nuclear isoforms (<em>22</em>, <em>22</em>.5, 24 and 34 kD). FGF-2 has pleiotropic roles in many cell types and tissues; it is a motogenic, angiogenic and survival <em>factor</em> which is involved in cell migration, cell differentiation and in a variety of developmental processes. Although devoid of signal peptide, it could be secreted. It acts mainly through a paracrine/autocrine mechanism involving high affinity transmembrane receptors and heparan sulfate proteoglycan low affinity receptors, but also through still unknown intracrine process(es) on intracellular targets. FGF-2 has many biological functions which are probably isoform-specific. Nevertheless, FGF-2 is not essential for embryonic development as knock-out mice for the <em>growth</em> <em>factor</em> are viable and fertile although they exhibit abnormalities in neuronal differentiation. Use of FGF-2 as therapeutic agent for the treatment of ischemic cardiovascular disease is promising and clinical trials are in progress.

Previous studies have described a <em>22</em> kD IL-1 inhibitor in the supernatant of human monocytes cultured on adherent immune complexes (J. Immunol. 134:3868, 1985). The studies reported herein further detail the conditions of production and biological properties of this IL-1 inhibitor. The inhibitor was produced by human monocytes cultured on adherent human IgG with maximal production between 8 and 24 hr. The IL-1 inhibitor was not performed in the cells but required transcription and new protein synthesis. The inhibitor blocked IL-1 augmentation of PHA-induced murine thymocyte proliferation but not IL-2-induced stimulation of CTLL or HT-2 cell lines. In addition, the inhibitor blocked IL-1-stimulated collagenase production from rabbit articular chondrocytes and IL-1-induced PGE2 production from human <em>fibroblasts</em> and synovial cells. The IL-1 inhibitor was not transforming <em>growth</em> <em>factor</em> beta (TGF beta) as determined by: the failure of anti-TGF beta antibodies to reduce IL-1 inhibitory activity, the separation of TGF beta from the IL-1 inhibitor by ion exchange chromatography, and the failure of TGF beta to inhibit IL-1-induced PGE2 production from synovial cells. IL-1 and the inhibitor showed no immunological cross-reactivity by Western blot analysis. The inhibitor specifically blocked binding of IL-1 to its receptor on the murine thymoma cell line EL4-6.1. These results indicate that a specific inhibitor of IL-1-induced immune and inflammatory cell responses is produced by monocytes cultured on adherent immune complexes or adherent IgG. This IL-1 inhibitor may be of importance in modulating the effects of IL-1 in the monocyte microenvironment.

To learn more about the relationship between extracellular matrix organization, cell shape, and cell <em>growth</em> control, we studied DNA synthesis by <em>fibroblasts</em> in collagen gels that were either attached to culture dishes or floating in culture medium during gel contraction. After 4 days of contraction, the collagen density (initially 1.5 mg/ml) reached <em>22</em> mg/ml in attached gels and 55 mg/ml in floating gels. After contraction, attached collagen gels were well organized; collagen fibrils were aligned in the plane of cell spreading; and <em>fibroblasts</em> had an elongated, bipolar morphology. Floating collagen gels, however, were unorganized; collagen fibrils were arranged randomly; and <em>fibroblasts</em> had a stellate morphology. DNA synthesis by <em>fibroblasts</em> in contracted collagen gels was suppressed if the gels were floating in medium but not if the gels were attached, and inhibition was independent of the extent of gel contraction. Therefore, <em>growth</em> of <em>fibroblasts</em> in contracted collagen gels could be regulated by differences in extracellular matrix organization and cell shape independently of extracellular matrix density. We also compared the responses of <em>fibroblasts</em> in contracted collagen gels and monolayer culture to peptide <em>growth</em> <em>factors</em> including <em>fibroblast</em> <em>growth</em> <em>factor</em>, platelet-derived <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-beta, and interleukin 1. Cells in floating collagen gels were generally unresponsive to any of the <em>growth</em> <em>factors</em>. Cells in attached collagen gels and monolayer culture were affected similarly by <em>fibroblast</em> <em>growth</em> <em>factor</em> but not by the others. Our results indicate that extracellular matrix organization influenced not only cell <em>growth</em>, but also <em>fibroblast</em> responsiveness to peptide <em>growth</em> <em>factors</em>.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a member of the heparin-binding <em>growth</em> <em>factor</em> (HBGF) family that includes at least seven species. These proteins are potent regulators of a number of cellular processes, including cell division and angiogenesis. Multiple forms of bFGF exist differing only in the length of their NH2-terminal extensions. These species of bFGF also have unique subcellular distributions. The smallest form (18 kD) occurs predominantly in the cytosol, while the higher molecular weight forms (<em>22</em>, <em>22</em>.5, 24 kD) are associated with the nucleus and ribosomes. Here we report that the nuclear localization of the higher molecular weight forms of bFGF derives specifically from the amino acid sequences within the NH2-terminal extension. This has been demonstrated by constructing a chimeric protein containing the NH2-terminal extension of the highest molecular weight form of bFGF fused to beta-galactosidase (beta-gal). After transfection in a transient expression system, the chimeric protein accumulated in the nuclei of transfected cells, while the wild-type beta-gal was found predominantly in the cytoplasm.

Crouzon syndrome and Pfeiffer syndrome are both autosomal dominant craniosynostotic disorders that can be caused by mutations in the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) gene. To determine the parental origin of these FGFR2 mutations, the amplification refractory mutation system (ARMS) was used. ARMS PCR primers were developed to recognize polymorphisms that could distinguish maternal and paternal alleles. A total of 4,374 bases between introns IIIa and 11 of the FGFR2 gene were sequenced and were assayed by heteroduplex analysis, to identify polymorphisms. Two polymorphisms (1333TA/TATA and 2710 C/T) were found and were used with two previously described polymorphisms, to screen a total of 41 families. Twenty-two of these families were shown to be informative (11 for Crouzon syndrome and 11 for Pfeiffer syndrome). Eleven different mutations in the <em>22</em> families were detected by either restriction digest or allele-specific oligonucleotide hybridization of ARMS PCR products. We molecularly proved the origin of these different mutations to be paternal for all informative cases analyzed (P=2. 4x10-7; 95% confidence limits 87%-100%). Advanced paternal age was noted for the fathers of patients with Crouzon syndrome or Pfeiffer syndrome, compared with the fathers of control individuals (34. 50+/-7.65 years vs. 30.45+/-1.28 years, P<.01). Our data on advanced paternal age corroborates and extends previous clinical evidence based on statistical analyses as well as additional reports of advanced paternal age associated with paternal origin of three sporadic mutations causing Apert syndrome (FGFR2) and achondroplasia (FGFR3). Our results suggest that older men either have accumulated or are more susceptible to a variety of germline mutations.

The EFFECT-II study aimed to investigate the effects of dapagliflozin and omega-3 (n-3) carboxylic acids (OM-3CA), individually or combined, on liver fat content in individuals with type 2 diabetes and non-alcoholic fatty liver disease (NAFLD).

This randomised placebo-controlled double-blind parallel-group study was performed at five clinical research centres at university hospitals in Sweden. 84 participants with type 2 diabetes and NAFLD were randomly assigned 1:1:1:1 to four treatments by a centralised randomisation system, and all participants as well as investigators and staff involved in the study conduct and analyses were blinded to treatments. Each group received oral doses of one of the following: 10 mg dapagliflozin (n = 21), 4 g OM-3CA (n = 20), a combination of both (n = <em>22</em>) or placebo (n = 21). The primary endpoint was liver fat content assessed by MRI (proton density fat fraction [PDFF]) and, in addition, total liver volume and markers of glucose and lipid metabolism as well as of hepatocyte injury and oxidative stress were assessed at baseline and after 12 weeks of treatment (completion of the trial).

Participants had a mean age of 65.5 years (SD 5.9), BMI 31.2 kg/m2 (3.5) and liver PDFF 18% (9.3). All active treatments significantly reduced liver PDFF from baseline, relative changes: OM-3CA, -15%; dapagliflozin, -13%; OM-3CA + dapagliflozin, -21%. Only the combination treatment reduced liver PDFF (p = 0.046) and total liver fat volume (relative change, -24%, p = 0.037) in comparison with placebo. There was an interaction between the PNPLA3 I148M polymorphism and change in liver PDFF in the active treatment groups (p = 0.03). Dapagliflozin monotherapy, but not the combination with OM-3CA, reduced the levels of hepatocyte injury biomarkers, including alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase (γ-GT), cytokeratin (CK) 18-M30 and CK 18-M65 and plasma fibroblast growth factor 21 (FGF21). Changes in γ-GT correlated with changes in liver PDFF (ρ = 0.53, p = 0.02). Dapagliflozin alone and in combination with OM-3CA improved glucose control and reduced body weight and abdominal fat volumes. Fatty acid oxidative stress biomarkers were not affected by treatments. There were no new or unexpected adverse events compared with previous studies with these treatments.

Combined treatment with dapagliflozin and OM-3CA significantly reduced liver fat content. Dapagliflozin monotherapy reduced all measured hepatocyte injury biomarkers and FGF21, suggesting a disease-modifying effect in NAFLD.

ClinicalTrials.gov NCT0<em>22</em>79407 FUNDING: The study was funded by AstraZeneca.

Hepatocyte <em>growth</em> <em>factor</em> (HGF) is a potent mitogen for adult rat hepatocytes in primary culture. HGF stimulates <em>growth</em> and DNA synthesis of normal human epidermal melanocytes in culture. The maximal stimulation of DNA synthesis by 4.0-fold occurred with 10 ng/ml HGF. This stimulatory effect was additive with both acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em>, while it was inhibited by transforming <em>growth</em> <em>factor</em>-beta 1. Melanocytes expressed a single class of specific, high-affinity receptors for HGF with a Kd of <em>22</em> pM and approximately 120 receptors/cell. Thus, HGF is a potent mitogen for normal human epidermal melanocytes.

In this study, we examined the impact of matrix metalloproteinases (MMP) on epithelialization, granulation tissue development, wound contraction, and alpha-smooth muscle actin (ASMA) expression during cutaneous wound repair through systemic administration of the synthetic broad-spectrum MMP inhibitor GM 6001 (N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide). Four full-thickness excisional wounds (50 mm2) on the back of <em>22</em> young female Sprague-Dawley rats, 12 treated with GM 6001 100 mg/kg and 10 with vehicle, were allowed to heal by secondary intention. GM 6001-treated wounds were minimally resurfaced with neoepithelium, despite unaltered keratinocyte proliferation in wound edges, whereas control wounds were completely covered with 3-7 cell layers of parakeratinized epithelium on post-wounding day 7. Hydroxyproline concentration, a marker of collagen, and cell proliferation in granulation tissue did not differ significantly between GM 6001-treated and control groups. Impaired wound contraction (P < 0.01) was associated with a dramatic reduction of ASMA-positive myo<em>fibroblasts</em> in granulation tissue of GM 6001 wounds. This was not due to GM6001 blocking transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1)-induced myofibroblast differentiation since GM 6001 did not inhibit TGF-beta1-induced ASMA expression and force generation in cultured rat dermal <em>fibroblasts</em>. The profound impairment of skin repair by the nonselective MMP inhibitor GM 6001 suggests that keratinocyte resurfacing, wound contraction, and granulation tissue organization are highly MMP-dependent processes.

BACKGROUND

Autosomal dominant cerebellar ataxias are a clinical and genetically heterogeneous group of progressive neurodegenerative diseases, at present associated with <em>22</em> loci (spinocerebellar ataxia [SCA] 1-SCA8, SCA10-SCA19, SCA21, SCA<em>22</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em> 14 [FGF14]-SCA, and dentatorubral-pallidoluysian atrophy [DRPLA]). The relevant gene has been identified in 12 cases (SCA1-3, SCA6-8, SCA10, SCA12, FGF14, and DRPLA), and in all but the recently identified SCA14, SCA17, PRKCG and FGF14 genes, the defect consists of the expansion of a short nucleotide repeat.

OBJECTIVE

To investigate the relative prevalence of SCA1-3, SCA6-8, SCA10, SCA12, and SCA17 gene expansions in Italian families with hereditary ataxia, specifically to verify the occurrence of SCA10, SCA12, and SCA17 in Italy; and to analyze samples from probands with negative test results at the initial screening by means of the repeat expansion detection technique to identify CAG/CTG expansions in novel loci.Patients Two hundred twenty-five unrelated Italian index cases with hereditary ataxia, most (n = 183) of whom presented with a clear dominantly transmitted trait.

RESULTS

We found that SCA1 and SCA2 gene mutations accounted for most cases (21% and 24%, respectively). We found SCA3, SCA6, SCA7, SCA8, and SCA17 to be very rare (approximately 1% each), and no case of SCA10 or SCA12 was identified. Half of the index cases (113/<em>22</em>5) were negative for expansions in the known SCA genes. Repeat expansion detection analysis performed on 111 of these cases showed a CAG/CTG repeat expansion of at least 50 triplets in <em>22</em> (20%). Twenty-one of <em>22</em> expansions could be attributed to length variation at 2 polymorphic loci (expanded repeat domain CAG/CTG 1 [ERDA1] or CTG repeat on chromosome 18q21.1 [CTG18.1]). In 1 patient, the expansion was assigned to the DRPLA gene.

CONCLUSIONS

The distribution of SCA1-3 and SCA6-7 gene mutations is peculiar in Italy. We found a relatively high frequency of SCA1 and SCA2 gene expansions; SCA3, SCA6, and SCA7 mutations were rare, compared with other European countries. No SCA10 or SCA12 and only a few SCA8 (2/<em>22</em>5) and SCA17 (2/<em>22</em>5) families were detected. In patients negative for defects in known SCA genes, repeat expansion detection data strongly suggest that, at least in our population, CAG/CTG expansions in novel genes should be considered an unlikely cause of the SCA phenotype.

Inherited cutis laxa is a connective tissue disorder characterized by loose skin and variable internal organ involvement, resulting from paucity of elastic fibers. Elsewhere, frameshift mutations in the elastin gene have been reported in three families with autosomal dominant inheritance, and a family with autosomal recessive cutis laxa was recently reported to have a homozygous missense mutation in the fibulin-5 gene. In the present study, we analyzed the gene expression of elastin and fibulins 1-5 in <em>fibroblasts</em> from five patients with cutis laxa. One patient was found to express both normal (2.2 kb) and mutant (2.7 kb) fibulin-5 mRNA transcripts. The larger transcript contains an internal duplication of 483 nucleotides, which resulted in the synthesis and secretion of a mutant fibulin-5 protein with four additional tandem calcium-binding epidermal <em>growth</em> <em>factor</em>-like motifs. The mutation arose from a <em>22</em>-kb tandem gene duplication, encompassing the sequence from intron 4 to exon 9. No fibulin-5 or elastin mutations were detected in the other patients. The results demonstrate that a heterozygous mutation in fibulin-5 can cause cutis laxa and also suggest that fibulin-5 and elastin gene mutations are not the exclusive cause of the disease.

OBJECTIVE

We analyzed the correlations between pretreatment serum levels of 11 cytokines and soluble cytokine receptors (interleukin 6 (IL-6); interleukin 8 (IL-8); interleukin 10 (IL-10); vascular endothelial growth factor (VEGF); basic fibroblast growth factor (bFGF); macrophage colony-stimulating factor (M-CSF); granulocyte colony-stimulating factor (G-CSF); interleukin 1 receptor antagonist (IL-1ra); sIL-2Ralpha; tumor necrosis factor receptor I (TNF RI), and TNF RII) with clinico-pathological features and survival of patients with bone sarcomas.

METHODS

Altogether, 72 patients with bone sarcomas without distant metastases before treatment (26 osteosarcomas-36%, 23 chondrosarcomas-32%, 13 Ewing's sarcomas/PNET-18%, 10 giant-cell tumors-14%), 22 patients with benign non-inflammatory bone tumors and 50 age-matched healthy controls were included into this prospective study.

RESULTS

Median serum levels of 9/11 cytokines, with the exception of sIL-2Ralpha and G-CSF, were significantly higher in sarcoma patients than in controls. Median serum levels of IL-6, IL-8, IL-1ra, TNF RI, and M-CSF were significantly higher in patients with bone sarcoma as compared to patients with benign bone tumors. In 45.9% of sarcoma patients, six or more cytokines and cytokine receptors, including those that are involved in bone destruction (e.g., IL-6 and IL-8) and bone formation (e.g., IL-1ra and TNFRI and TNFRII), were elevated in parallel. Serum levels of IL-6, IL-8, TNF RI, TNF RII, and VEGF correlated significantly with tumor size (<10 cm vs.>>or=10 cm in diameter) and serum levels of IL-6, IL-8, TNF RI, and IL-1ra correlated significantly with local tumor extent (E2/4 vs. E5/6 according to the classification proposed by Spanier et al. 46). Moreover, serum levels of IL-1ra and IL-6 were significantly higher in patients with small tumors (<5 cm in diameter) infiltrating structures adjacent to the periosteum (E5/6) than in large tumors (>10 cm in diameter) but confined to the bone and periosteum (E < 4). The lowest median serum levels of 8/11 cytokines/cytokine receptors were found in patients with giant-cell tumors. In an univariate analysis, increased serum levels of IL-1ra, IL-6, IL-8, IL-10, sIL-2Ralpha, M-CSF, TNF RI, and TNF RII, the number of cytokines elevated, higher tumor grade, larger tumor size, greater local extent (E) and patients' age >35 years correlated with poor overall survival (OS) (P < 0.05). Similarly, high serum levels of IL-1ra, IL-6, TNF RI and TNF RII, tumor grade, tumor size, and tumor local extent (E) (P < 0.05) affected disease free survival (DFS) in univariate analysis. Multivariate analysis using Cox's proportional hazards model showed that high serum levels of IL-1ra (P = 0.039) and TNF RI (P = 0.048), the number of serum cytokines above normal cut-off values (0-1 vs. 2-5 vs.>>or=6; P = 0.029), greater tumor local extent E (E2/4 vs. E5/6; P = 0.02) correlated significantly with shorter OS. Only E was found as an independent prognostic factor for DFS (P = 0.04).

CONCLUSIONS

These findings indicate that cytokines and soluble cytokine receptors, both physiologically involved in bone destruction and bone formation, have an essential role in the progression of malignant bone tumors.

BACKGROUND

Biliary tract cancers (BTCs) typically present at an advanced stage, and systemic chemotherapy is often of limited benefit.

METHODS

Hybrid capture-based comprehensive genomic profiling (CGP) was performed for 412 intrahepatic cholangiocarcinomas (IHCCAs), 57 extrahepatic cholangiocarcinomas (EHCCAs), and 85 gallbladder carcinomas (GBCAs). The mutational profile was correlated with the clinical outcome of standard and experimental therapies for 321 patients. Clinical variables, detected mutations, and administered therapies were correlated with overall survival (OS) in a Cox regression model.

RESULTS

The most frequent genetic aberrations (GAs) observed were tumor protein 53 (TP53; 27%), cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B; 27%), KRAS (<em>22</em>%), AT-rich interactive domain-containing protein 1A (ARID1A; 18%), and isocitrate dehydrogenase 1 (IDH1; 16%) in IHCCA; KRAS (42%), TP53 (40%), CDKN2A/B (17%), and SMAD4 (21%) in EHCCA; and TP53 (59%), CDKN2A/B (19%), ARID1A (13%), and ERBB2 (16%) in GBCA. <em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR; 11%) and IDH mutations (20%) were mostly limited to IHCCA but appeared to be mutually exclusive. In the IHCCA group, TP53 and KRAS mutations were associated significantly with poor OS, whereas FGFR2 mutations were associated with improved OS (P = .001), a younger age at onset, and female sex. IDH1/2 mutations were not prognostic. In a multivariate model, the effects of TP53 and FGFR GAs remained significant (P < .05). Patients with FGFR GAs had superior OS with FGFR-targeted therapy versus standard regimens (P = .006). Targeted therapy in IHCCA was associated with a numerical OS improvement (P = .07).

CONCLUSIONS

This is the largest clinically annotated data set of BTC cases with CGP and indicates the potential of CGP for improving outcomes. CGP should be strongly considered in the management of BTC patients. Cancer 2016;1<em>22</em>:3838-3847. © 2016 American Cancer Society.

OBJECTIVE

To examine the expression of the thioredoxin (TRX)-thioredoxin reductase (TR) system in patients with rheumatoid arthritis (RA) and patients with other rheumatic diseases.

METHODS

Levels of TRX in plasma and synovial fluid (SF) were measured using enzyme-linked immunosorbent assay. Cellular distribution of TRX was determined by flow cytometry and histochemistry. Cellular expression of TR was studied by in situ messenger RNA (mRNA) hybridization. The effect of oxidative stress and tumor necrosis factor alpha (TNF alpha) on TRX expression by cultured rheumatoid fibroblast-like synoviocytes was studied.

RESULTS

Significantly increased TRX levels were found in the SF from 22 patients with RA, when compared with plasma levels in the same patients (P < 0.001) and compared with SF TRX levels in 15 patients with osteoarthritis (P < 0.001), 13 patients with gout (P < 0.05), and 9 patients with reactive arthritis (P < 0.0001). The presence of TRX could be demonstrated within the SF-derived mononuclear cells and synovial tissue (ST) of RA patients. Concordantly, expression of TR mRNA was observed in the ST of these patients. Stimulation of synovial fibroblast-like synoviocytes with either H2O2 or TNF alpha induced an increase in the production of TRX.

CONCLUSIONS

The data demonstrate significantly increased concentrations of TRX in the SF and ST of RA patients when compared with the levels in patients with other joint diseases. Evidence is presented that the local environment in the rheumatic joint contributes to increased TRX production. Based on its growth-promoting and cytokine-like properties, it is proposed that increased expression of TRX contributes to the disease activity in RA.

To study possible functional differences of the 18-kD and high molecular weight forms of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), we have examined the effect of endogenous production of different bFGF forms on the phenotype of NIH 3T3 cells. Cells transfected with cDNAs coding for either 18-kD bFGF (18-kD bFGF) or all four molecular forms (18, <em>22</em>, <em>22</em>.5, 24 kD; wild type [WT] bFGF) exhibit increased migration and decreased FGF receptor number compared to parental cells. However, migration and FGF receptor number of cells transfected with a cDNA coding only for high molecular weight bFGF (<em>22</em>, <em>22</em>.5, and 24 kD; HMW bFGF) were similar to that of parental cells transfected with vector alone. Cells expressing HMW, 18 kD, or WT bFGF grew to high saturation densities in 10% serum. However, only cells expressing HMW or WT bFGF grew in low serum. Cell surface or metabolic labeling of the different cell types followed by immunoprecipitation with anti-bFGF antibody showed primarily cell surface-associated 18-kD bFGF. In addition, when cells expressing exclusively HMW bFGF were transfected with a cDNA coding for 18-kD bFGF, migration was increased, bFGF receptors were down-regulated, and 18-kD bFGF was found on the cell surface. Cells expressing 18-kD bFGF transfected with a cDNA encoding FGF receptor-2 lacking the COOH-terminal domain (dominant negative bFGF receptor) exhibited a flat morphology and decreases in migration and saturation density. Cells expressing HMW bFGF transfected with the dominant negative bFGF receptor continued to grow to a high saturation density, proliferated in low serum, and exhibited no morphological changes. These results indicate that increased cell migration and FGF receptor down-regulation are mediated by the extracellular interaction of 18-kD bFGF with its cell surface receptor. <em>Growth</em> in low serum may be stimulated by the intracellular action of HMW bFGF through mechanisms independent of the presence of a cell surface receptor. Thus, the different molecular forms of bFGF may act through distinct but convergent pathways.

The single copy gene for human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has been shown to encode not one but multiple proteins of 24, 23, <em>22</em> and 18 kD. Although bioactivities of the 18 kD protein are currently used to define bFGF gene function, it is not yet known if the three larger proteins have these same bioactivities or whether they will serve to define new bFGF gene functions. In this report we present a comparative study describing the de novo synthesis, transport, processing and intracellular location of individual bFGF isoforms. Data from cDNA mutagenesis and COS cell expression experiments show that individual isoforms are differentially localized to either the cell surface or to the nucleus. The 24, 23 and <em>22</em> kD proteins (CUG-mediated initiation) exclusively localize in the nucleus while the 18 kD protein (AUG-mediated initiation) is preferentially exported onto the cell surface, but is not released into the surrounding culture medium. Specific CUG or AUG translation initiation codons are necessary and sufficient for the synthesis of each isoform examined and thereby, indirectly, mediate differential localization. Since bFGF does not contain the characteristic signals predicted for cell surface or nuclear targeting, our continuing studies will either unmask its functionally equivalent domain(s) or will identify the requisite participation of yet unknown cellular components.

Four <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFR1-4) constitute a family of transmembrane tyrosine kinases that serve as high affinity receptors for at least <em>22</em> FGF ligands. Gene targeting in mice has yielded valuable insights into the functions of this important gene family in multiple biological processes. These include mesoderm induction and patterning; cell <em>growth</em>, migration, and differentiation; organ formation and maintenance; neuronal differentiation and survival; wound healing; and malignant transformation. Furthermore, discoveries that mutations in three of the four receptors result in more than a dozen human congenital diseases highlight the importance of these genes in skeletal development. In this review, we will discuss recent progress on the roles of FGF receptors in mammalian development and congenital diseases, with an emphasis on signal transduction pathways.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), a polypeptide with regulatory activity on cell <em>growth</em> and differentiation, lacks a conventional secretory signal sequence, and its mechanism of release from cells remains unclear. We characterized the role of extracellular vesicle shedding in FGF-2 release. Viable cells released membrane vesicles in the presence of serum. However, in serum-free medium vesicle shedding was dramatically down-regulated, and the cells did not release FGF-2 activity into their conditioned medium. Addition of serum to serum-starved cells rapidly induced intracellular FGF-2 clustering under the plasma membrane and into granules that colocalized with patches of the cell membrane with typical features of shed vesicle membranes. Shed vesicles carried three FGF-2 isoforms (18, <em>22</em>, 24 kDa). Addition of vesicles to endothelial cells stimulated chemotaxis and urokinase plasminogen activator production, which were blocked by anti-FGF-2 antibodies. Treatment of intact vesicles with 2.0 m NaCl or heparinase, which release FGF-2 from membrane-bound proteoglycans, did not abolish their stimulatory effect on endothelial cells, indicating that FGF-2 is carried inside vesicles. The comparison of the stimulatory effects of shed vesicles and vesicle-free conditioned medium showed that vesicles represent a major reservoir of FGF-2. Thus, FGF-2 can be released from cells through vesicle shedding.

Sorafenib is effective in hepatocellular carcinoma (HCC), but patients ultimately present disease progression. Molecular mechanisms underlying acquired resistance are still unknown. Herein, we characterise the role of tumour-initiating cells (T-ICs) and signalling pathways involved in sorafenib resistance.

HCC xenograft mice treated with sorafenib (n=<em>22</em>) were explored for responsiveness (n=5) and acquired resistance (n=17). Mechanism of acquired resistance were assessed by: (1) role of T-ICs by in vitro sphere formation and in vivo tumourigenesis assays using NOD/SCID mice, (2) activation of alternative signalling pathways and (3) efficacy of anti-FGF and anti-IGF drugs in experimental models. Gene expression (microarray, quantitative real-time PCR (qRT-PCR)) and protein analyses (immunohistochemistry, western blot) were conducted. A novel gene signature of sorafenib resistance was generated and tested in two independent cohorts.

Sorafenib-acquired resistant tumours showed significant enrichment of T-ICs (164 cells needed to create a tumour) versus sorafenib-sensitive tumours (13 400 cells) and non-treated tumours (1292 cells), p<0.001. Tumours with sorafenib-acquired resistance were enriched with insulin-like growth factor (IGF) and fibroblast growth factor (FGF) signalling cascades (false discovery rate (FDR)<0.05). In vitro, cells derived from sorafenib-acquired resistant tumours and two sorafenib-resistant HCC cell lines were responsive to IGF or FGF inhibition. In vivo, FGF blockade delayed tumour growth and improved survival in sorafenib-resistant tumours. A sorafenib-resistance 175 gene signature was characterised by enrichment of progenitor cell features, aggressive tumorous traits and predicted poor survival in two cohorts (n=442 patients with HCC).

Acquired resistance to sorafenib is driven by T-ICs with enrichment of progenitor markers and activation of IGF and FGF signalling. Inhibition of these pathways would benefit a subset of patients after sorafenib progression.

We have determined the relationship between the binding sites for acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) and basic FGF (bFGF) in heparan sulfate (HS) prepared from a panel of mammary cell lines and the ability of the HS to activate aFGF and bFGF in DNA synthesis assays. The ka of the HS for aFGF fell into three groups, whereas the kd (0.0015-0.016 s-1) and the Kd (0.4-8.6 microM) formed a continuum. bFGF possessed a high affinity binding site (Kd <em>22</em>-30 nM) with a fast ka (320,000-550,000 M-1 s-1), termed "fast/high," and a lower affinity site (Kd 47-320 nM) with a slower ka (35,000-150,000 M-1 s-1), termed "slow/low." Most of the species of HS possessed the latter binding site, which was able to activate bFGF in HS-deficient <em>fibroblasts</em>. However, the HS from the culture medium of the mammary <em>fibroblasts</em> and the myoepithelial-like cells possessed both a fast/high and a slow/low binding site and could not activate bFGF, although it could potentiate the <em>growth</em>-stimulatory activity of aFGF. Treatment of the HS possessing two binding sites for bFGF with heparitinase 1 released oligosaccharides that were able to restore the activity of bFGF in HS-deficient <em>fibroblasts</em>.

Endothelial cell seeding, a promising method to improve the performance of small-diameter vascular grafts, requires a suitable substrate, e.g. crosslinked collagen. In addition, the <em>growth</em> of seeded endothelial cells can be improved by local release of a heparin-binding protein, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). In this study, the influence of immobilization of heparin to collagen, crosslinked using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) in combination with N-hydroxysuccinimide (NHS), on the binding and release of bFGF was determined. Heparin was immobilized also using EDC and NHS. Furthermore, the effects of the release of bFGF from (heparinized) EDC/NHS-crosslinked collagen on the proliferation of seeded endothelial cells was studied in vitro. Immobilization of increasing amounts of heparin to EDC/NHS-crosslinked collagen (containing 14 free epsilon-amino groups per 1000 amino acid residues, E/N14C) resulted in binding of increasing amounts of bFGF to the material. Maximal bFGF binding was observed for E/N14C containing 20-30 mg heparin immobilized per gram of collagen which was obtained using a molar ratio of EDC to heparin-carboxylic acid groups of 0.4 for heparin immobilization (E/N14C-H(0.4)). Up to concentrations of 320 ng bFGF/ml, 10% of the added bFGF bound to E/N14C, while binding of bFGF to E/N14C-H(0.4) was <em>22</em>%. The initial release rate of bFGF bound to E/N14C was much higher compared to bFGF bound to E/N14C-H(0.4): respectively, 30 vs. 2% in the first 6 h. After 10 days, the bFGF release from E/N14C and E/N14C-H(0.4) amounted to 83 vs. 42%, respectively. Binding of increasing amounts of bFGF resulted in increased <em>growth</em> of human umbilical vein endothelial cells (HUVECs) seeded on both E/N14C and E/N14C-H(0.4). Nevertheless, after 6 and 10 days of proliferation cell numbers on E/N14C-H(0.4) where higher than cell numbers on E/N14C, irrespective of the bFGF concentration used for loading of the matrix. It is concluded that heparinized, EDC/NHS-crosslinked collagen is a good synthetic vascular graft coating for in vivo endothelial cell seeding.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF) signaling is essential for endochondral bone formation. Mutations cause skeletal dysplasias including achondroplasia, the most common human skeletal dysplasia. Most previous work in this area has focused on embryonic chondrogenesis. To explore the role of FGF signaling in the postnatal <em>growth</em> plate, we quantitated expression of FGFs and FGF receptors (FGFRs) and examined both their spatial and temporal regulation. Toward this aim, rat proximal tibial <em>growth</em> plates and surrounding tissues were microdissected, and specific mRNAs were quantitated by real-time RT-PCR. To assess the FGF system without bias, we first screened for expression of all known FGFs and major FGFR isoforms. Perichondrium expressed FGFs 1, 2, 6, 7, 9, and 18 and, at lower levels, FGFs 21 and <em>22</em>. <em>Growth</em> plate expressed FGFs 2, 7, 18, and <em>22</em>. Perichondrial expression was generally greater than <em>growth</em> plate expression, supporting the concept that perichondrial FGFs regulate <em>growth</em> plate chondrogenesis. Nevertheless, FGFs synthesized by <em>growth</em> plate chondrocytes may be physiologically important because of their proximity to target receptors. In <em>growth</em> plate, we found expression of FGFRs 1, 2, and 3, primarily, but not exclusively, the c isoforms. FGFRs 1 and 3, thought to negatively regulate chondrogenesis, were expressed at greater levels and at later stages of chondrocyte differentiation, with FGFR1 upregulated in the hypertrophic zone and FGFR3 upregulated in both proliferative and hypertrophic zones. In contrast, FGFRs 2 and 4, putative positive regulators, were expressed at earlier stages of differentiation, with FGFR2 upregulated in the resting zone and FGFR4 in the resting and proliferative zones. FGFRL1, a presumed decoy receptor, was expressed in the resting zone. With increasing age and decreasing <em>growth</em> velocity, FGFR2 and 4 expression was downregulated in proliferative zone. Perichondrial FGF1, FGF7, FGF18, and FGF<em>22</em> were upregulated. In summary, we have analyzed the expression of all known FGFs and FGFRs in the postnatal <em>growth</em> plate using a method that is quantitative and highly sensitive. This approach identified ligands and receptors not previously known to be expressed in <em>growth</em> plate and revealed a complex pattern of spatial regulation of FGFs and FGFRs in the different zones of the <em>growth</em> plate. We also found temporal changes in FGF and FGFR expression which may contribute to <em>growth</em> plate senescence and thus help determine the size of the adult skeleton.