Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic (b) <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2/bFGF) are involved in vascular development and angiogenesis. Pulmonary artery smooth muscle cells express VEGF and FGF-2 and are subjected to mechanical forces during pulsatile blood flow. The effect of stretch on <em>growth</em> <em>factor</em> expression in these cells is not well characterized. We investigated the effect of cyclic stretch on the expression of VEGF and FGF-2 in ovine pulmonary artery smooth muscle cells. Primary confluent cells from 6-wk-old lambs were cultured on flexible silicon membranes and subjected to cyclic biaxial stretch (1 Hz; 5-25% stretch; 4-48 h). Nonstretched cells served as controls. Expression of VEGF and FGF-2 was determined by Northern blot analysis. Cyclic stretch induced expression of both VEGF and FGF-2 mRNA in a time- and amplitude-dependent manner. Maximum expression was found at 24 h and <em>15</em>% stretch (VEGF: 1.8-fold; FGF-2: 1.9-fold). These results demonstrate that mechanical stretch regulates VEGF and FGF-2 gene expression, which could play a role in pulmonary vascular development or in postnatal pulmonary artery function or disease.

The endometrium is the only human tissue to undergo cyclic breakdown and regeneration. This physiological alternation renders it an advantageous system for studying tissue remodelling. Our previous observations indicate that menstrual endometrial breakdown is initiated by matrix metalloproteinases (MMPs), which are controlled overall by ovarian steroids but are also locally regulated by cytokines. We have therefore compared the effect of several endometrial cytokines on the gene expression of eight MMPs and their tissue inhibitors (TIMP)-1, -2 and -3, in primary cultures of human endometrial <em>fibroblasts</em>. Three categories of gene expression were identified: (a) MMP-13, -<em>15</em> and -16 mRNAs were not detected despite stimulation by various cytokines; (b) MMP-2 and -14 as well as TIMP-1, -2 and -3 mRNAs were constitutively expressed but not markedly affected by the six cytokines tested; (c) mRNAs for MMP-1, -9 and -11 were selectively induced by specific cytokines: insulin-like <em>growth</em> <em>factor</em>-II, epidermal <em>growth</em> <em>factor</em> (EGF), platelet derived <em>growth</em> <em>factor</em> (PDGF)-BB and interleukin (IL)-6 stimulated MMP-11 expression; MMP-1 was induced by EGF, PDGF-BB, tumour necrosis <em>factor</em> (TNF)alpha and IL-1alpha, which also exerted additive effects. In contrast with MMP-1 and MMP-11 gene expression, which was sustained for 48 h, MMP-9 mRNA was quickly induced by TNFalpha, but disappeared within 12 h despite continuing stimulation. These results show that several cytokines are able to induce the selective expression of MMPs in cultured human endometrial <em>fibroblasts</em> and are thus good candidates for involvement in local triggering of menstrual tissue breakdown.

We have analysed DNA from 183 primary breast cancers for amplification or rearrangement of a number of cellular proto-oncogenes, focusing primarily on a cluster of markers on the long arm of chromosome 11. Two of these oncogenes, INT2 and HST1, both of which encode members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family, are implicated in the generation of virally induced mammary tumours in mice. Here we confirm earlier reports that the q13 region of chromosome 11, in which these genes are tandemly linked, is modestly amplified in approximately <em>15</em>% of primary human breast cancers. This amplification is confined, with one exception, to cases in which the oestrogen receptor (ER) levels are in excess of 20 fmol/mg protein (P = 0.001). However, DNA amplification does not usually result in detectable expression of either the INT2 or HST1 gene. The data imply that some other gene in the vicinity must contribute to the development of a subset of ER-positive tumours and that assessing the amplification of this region of DNA may be of value in defining a separate category of ER-positive tumour.

We prepared a 6-O-desulfated (DS-) heparin (Hep) hydrogel as an excellent carrier for the controlled release of Hep-binding <em>growth</em> <em>factors</em>, such as <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2. This material, which is partially derived from photoreactive groups, such as cinnamate, is easily crosslinked upon ultraviolet light (UV)-irradiation, resulting in a water-insoluble, viscous, and injectable hydrogel. In the present study, we examined the capacity of 6-O-DS-Hep hydrogel to immobilize FGF-2, as well as the controlled release of FGF-2 molecules from this hydrogel in vitro and in vivo. Only 10% of FGF-2 was gradually released from the FGF-2-containing 6-O-DS-Hep hydrogel (photocrosslinked 6-O-DS-Hep (4%; w/w) hydrogel containing 50 microg/mL FGF-2) into PBS (phosphate-buffered saline) within first 7 days. The 6-O-DS-Hep hydrogel in vitro maintained the original form through 1 weeks incubation in PBS, but it was gradually fragmented and could not maintain the original form by 2-3 week-washing. When the FGF-2-containing 6-O-DS-Hep hydrogel was subcutaneously injected into the back of rats, significant neovascularization and fibrous tissue formation were induced near the injected site from day 3 after the injection. And, the hydrogel had been biodegraded and completely disappeared from the injected sites in vivo within about <em>15</em>-20 days after the injection. These findings indicate a controlled release of biologically active FGF-2 molecules together with fragmentation and biodegradation of 6-O-DS-Hep hydrogel and the subsequent induction of neovascularization in vivo.

Signaling by <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) and their receptors has been previously implicated in control of cell proliferation, differentiation and migration. Here we report a novel role for signaling by the EGL-<em>15</em> FGFR of Caenorhabditis elegans in controlling protein degradation in differentiated muscle. Activation of EGL-<em>15</em>, by means of a reduction of function mutation (clr-1) affecting an inhibitory phosphatase, triggers protein degradation in adult muscle cells using a pre-existing proteolytic system. This activation is not suppressed by mutation in either of the known genes encoding FGF ligands (egl-17 or let-756) but is well suppressed when both are mutated, indicating that either ligand is sufficient and at least one is necessary for FGFR activation. Activity of the Ras pathway through mitogen-activated protein kinase (MAPK) is required to trigger protein degradation. This is the first report that degradation of intracellular protein can be triggered by a <em>growth</em> <em>factor</em> receptor using an identified signal transduction pathway. The data raise the possibility that FGF-triggered proteolysis may be relevant to muscle remodeling or dedifferentiation.

The tyrosine kinase Src has been implicated in the process of osteoclast-mediated bone resorption. Here, we describe a novel class of Src inhibitors, substituted 5,7-diphenyl-pyrrolo[2,3-d]pyrimidines, and characterize one of them, CGP77675, in vitro and in models of bone resorption in vivo. In vitro, CGP77675 inhibited phosphorylation of peptide substrates and autophosphorylation of purified Src (concentration producing half-maximal inhibition [IC50] values 5-20 and 40 nmol/L, respectively). The compound was selective toward other protein kinases: the Src IC50 value was lower than those for Cdc2 (>500-fold), epidermal <em>growth</em> <em>factor</em> (EGF) receptor (7.5-fold), and vascular endothelial <em>growth</em> <em>factor</em> receptor (>50-fold), and for v-Abl (<em>15</em>-fold) and focal adhesion kinase (Fak) (>25-fold). The Src kinase family members Lck and Yes were inhibited with IC50 values 20-fold higher than or equal to Src. To measure the inhibition of cellular Src activity, we identified the major tyrosine-phosphorylated proteins in an Src-overexpressing cell line IC8.1 as Src, Fak, and paxillin. CGP77675 potently inhibited tyrosine phosphorylation of the Src substrates Fak and paxillin, but had much less effect on Src (IC50 values 0.3, 0.5, and 5.7 micromol/L). The phosphorylation of Src in IC8.1 cells reflected phosphorylation of the negative regulatory tyrosine 527 (Y527); thus, the inhibitor was selective against the Y527 C-terminal Src kinase Csk. In osteoblastic MC3T3-E1 cells, CGP77675 inhibited signaling induced by PDGF at the receptor level, but not signaling by EGF, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>-1, and phorbol 12-myristate 13-acetate. The effect of CGP77675 on bone resorption was evaluated in vitro and in vivo. The parathyroid hormone-induced bone resorption in rat fetal long bone cultures was inhibited with an IC50 of 0.8 micromol/L. CGP77675 dose-dependently reduced the hypercalcemia induced in mice by interleukin-1beta and partly prevented bone loss and microarchitectural changes in young ovariectomized rats, showing that the protective effect on bone was exerted via the inhibition of bone resorption. Thus, specific Src family kinase inhibitors may be useful for the treatment of diseases associated with elevated bone loss.

To understand the mechanisms underlying <em>15</em>(S)-hydroxyeicosatetraenoic acid [<em>15</em>(S)-HETE]-induced angiogenesis, we studied the role of Egr-1. <em>15</em>(S)-HETE induced Egr-1 expression in a time-dependent manner in human dermal microvascular endothelial cells (HDMVECs). Blockade of Egr-1 via forced expression of its dominant-negative mutant attenuated <em>15</em>(S)-HETE-induced HDMVEC migration and tube formation as well as Matrigel plug angiogenesis. <em>15</em>(S)-HETE-induced Egr-1 expression requires Src activation. In addition, adenovirus-mediated expression of dominant-negative mutant of Src blocked <em>15</em>(S)-HETE's effects on migration and tube formation of HDMVECs and Matrigel plug angiogenesis. <em>15</em>(S)-HETE induced <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) expression rapidly via Src-mediated production of Egr-1. Cloning and mutational analysis of FGF-2 promoter revealed that Egr-1 binding site proximal to transcription start site is required for <em>15</em>(S)-HETE-induced FGF-2 expression. Neutralizing antibody-mediated suppression of FGF-2 function also attenuated the effects of <em>15</em>(S)-HETE on HDMVEC migration and tube formation as well as Matrigel plug angiogenesis. Furthermore, in contrast to wild-type mice, 12/<em>15</em>-LOX(-/-) mice exhibited decreased Matrigel plug angiogenesis in response to AA, which was rescued by <em>15</em>(S)-HETE. On the basis of these observations, we conclude that <em>15</em>(S)-HETE-induced angiogenesis requires Src-mediated Egr-1-dependent rapid induction of FGF-2. These findings may suggest that <em>15</em>(S)-HETE could be a potential endogenous regulator of pathologic angiogenesis associated with atherosclerosis and restenosis.

OBJECTIVE

The purpose of this study was to simultaneously monitor the transcriptional levels of 12 endothelial growth factor genes in response to alterations in wall shear stress (WSS) under conditions relevant to the development of intimal hyperplasia, a major cause of arterial bypass graft failure.

METHODS

Human umbilical vein endothelial cells were preconditioned in vitro under steady flow (WSS, 15 dynes/cm(2)) for 24 hours before being subjected to WSS at 25 (Delta = +10), 15 (Delta = 0), 5 (Delta = -10), 2.5 (Delta = -12.5), and 0 (Delta = -15) dynes/cm(2) or low magnitude WSS reversal (-2.5 dynes/cm(2)) for 6 hours. A focused complementary DNA array was used to simultaneously measure messenger RNA expression levels for END1, endothelial nitric oxide synthase (NOS3), platelet-derived growth factor A, platelet-derived growth factor B (PDGFB), acidic fibroblast growth factor, basic fibroblast growth factor, transforming growth factor-alpha, transforming growth factor-beta, vascular endothelial growth factor, insulin-like growth factor-1, epidermal growth factor, and angiotensin converting enzyme.

RESULTS

Preconditioning significantly (P <.05) increased the fold expression of NOS3 (4.1 +/- 1.4), basic fibroblast growth factor (3.90 +/- 1.16), vascular endothelial growth factor (3.39 +/- 1.04), and insulin-like growth factor-1 (2.8 +/- 0.7) but decreased END1 (0.47 +/- 0.05) and PDGFB (0.70 +/- 0.04) messenger RNA expression levels relative to no-flow controls, an effect that was sustained on removal from flow for 6 hours. Notably, the ratio of END1/NOS3 expression was diminished (0.11 +/- 0.03) relative to that of cells maintained in static culture. Although few differences in gene expression from baseline (15 dynes/cm(2)) were measured in cells exposed to either constant (Delta = 0) or step decreases (Delta = -10, -12.5, or -15 dynes/cm(2)) in WSS, marked changes were seen in the group exposed to a step increase in WSS (Delta = +10) or to WSS reversal. Low magnitude retrograde WSS evoked significant (P <.05) transcriptional changes in multiple genes, including elevated END1 (4.1 +/- 0.5), platelet-derived growth factor A (1.5 +/- 0.2), PDGFB (2.3 +/- 0.3), and transforming growth factor-beta (1.5 +/- 0.2) levels, but depressed NOS3 (0.60 +/- 0.17) levels, and a marked increase in END1/NOS3 (6.7 +/- 1.6) when compared with equal magnitude antegrade WSS (2.5 dynes/cm(2)).

CONCLUSIONS

These results support the implementation of a preconditioning phase for in vitro WSS studies to establish a physiologic baseline. Our findings complement previous macroscale findings and are consistent with a cellular mechanism involving increased END1 and PDGFB levels, but decreased NOS3 levels, leading to intimal hyperplasia at regions of low magnitude reversing WSS.

Recently, we characterized <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 amplification as a target for a rational therapy in lung squamous cell carcinoma. Patients harboring this genetic event are currently eligible for treatment with anti<em>fibroblast</em> <em>growth</em> <em>factor</em> receptor small-molecule inhibitors in phase I clinical trials. This has the potential to significantly improve standard therapy for lung squamous cell carcinoma patients. The aim of this study was to elucidate whether <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 amplification is also a common genetic event in head and neck squamous cell carcinoma. For this purpose, we assembled a cohort of 555 patients, including 264 with metastatic disease and 147 with recurrent disease. Formalin-fixed, paraffin-embedded material of primary tumors, metastases and recurrences were assessed for <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 copy number status using fluorescence in situ hybridization. Human papilloma virus status was detected by p16 immunohistochemistry staining and PCR-ELISA. Molecular parameters were correlated with each other and with clinicopathological data. We found <em>15</em>% of primary head and neck squamous cell carcinoma to display a <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 amplification. In nearly all cases, metastatic and recurrent tumor samples shared the same amplification status as the corresponding primary tumor. <em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 amplification was associated with nicotine and alcohol consumption, but was mutually exclusive with human papilloma virus infection. Amplification of the gene was associated with parameters of worse outcome. Our data identify <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 amplification as a frequent event in primary and metastatic head and neck squamous cell carcinoma and represents a potential biomarker for more aggressive disease. <em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 1-amplified tumors could potentially comprise a subset of head and neck squamous cell carcinoma against which targeted small-molecule inhibitors hold therapeutic efficacy.

The biosynthesis of type V collagen and its regulation were studied using diploid human gingival <em>fibroblasts</em>. Cells were metabolically labeled with radioactive amino acids and labeled proteins were subjected to limited pepsin digestion, DEAE-cellulose chromatography at <em>15</em> degrees C, and polyacrylamide gel electrophoresis. Proteins eluted from DEAE-cellulose columns by 0.25 M NaCl contained a collagen species which was resistant to mammalian collagenase and had alpha chains with hydroxylysine/lysine ratios and CNBr peptide patterns similar to alpha 1(V) and alpha 2(V). Procollagen(V) fractions obtained by DEAE-cellulose chromatography and immunoprecipitates of type V collagen antibody contained polypeptides with Mr = 239,000, 219,000, 198,000, 174,000, <em>15</em>7,000, and 132,000. By comparing the CNBr peptide maps of these proteins with those of standard alpha 1(V) and alpha 2(V) chains, the first three polypeptides were shown to be related to alpha 1(V) and the others to alpha 2(V). It was concluded that the gingival <em>fibroblasts</em> synthesize type V collagen, that the pro alpha 1(V) and the pro alpha 2(V) chains have Mr = 239,000 and 174,000, respectively, and that the alpha 1(V) and alpha 2(V) chains laid in the form of fibrils have Mr = 198,000 and 132,000, respectively. A detectable amount of type V collagen was synthesized only at high cell density, and it was associated with the cell layer. The amount and proportion of type V synthesized were increased when the cells were labeled in the presence of serum, and the increase was accompanied by a decrease in type III. This effect was dependent on serum concentration. Serum obtained from platelet-poor plasma failed to elicit this effect, and it was restored by the addition of platelet-derived <em>growth</em> <em>factor</em>. Platelet-derived <em>growth</em> <em>factor</em> was effective in medium with and without platelet-poor serum. Thus, it appears that platelet-derived <em>growth</em> <em>factor</em> may be an important regulatory <em>factor</em> in the synthesis of types V and III collagens.

One response of BALB/c 3T3 cells to epidermal <em>growth</em> <em>factor</em> (EGF) is the release and subsequent metabolism of arachidonic acid. Prostaglandins generated from EGF treatment appear to play a role in the mitogenic signal. Lipoxygenase inhibitors (nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid) were previously shown to be very effective in blocking EGF-stimulated DNA synthesis; however, only low levels of lipoxygenase-derived arachidonate metabolites were detected. In an extension of these investigations, we have now found that EGF stimulates lipoxygenase metabolites of linoleic acid in BALB/c 3T3 <em>fibroblasts</em>. In the presence of EGF (10 ng/ml), the cells converted 10-<em>15</em>% of exogenous linoleic acid (10 microM) to hydroxy fatty acids that were isolated on reverse phase high performance liquid chromatography. No linoleate metabolites were detected in the absence of EGF. The isolated compounds were characterized further by straight phase high performance liquid chromatography, UV spectroscopy, and gas chromatography-mass spectrometry analyses, and they were identified as 13-hydroxyoctadecadienoic acid and 9-hydroxyoctadecadienoic acid. The hydroxy metabolites and their hydroperoxy precursors produced a 2- to 4-fold potentiation of EGF-stimulated [3H]thymidine incorporation in BALB/c 3T3 cells. These linoleate derivatives stimulated DNA synthesis at concentration ranges of 10(-8) to 10(-6) M. Thus, linoleic acid metabolism might be an important element in the EGF-regulated cascade of biochemical events leading to <em>fibroblast</em> mitogenesis.

OBJECTIVE

Patients with cystic fibrosis (CF) have poorly defined defects in biliary function. We evaluated the effects of cystic fibrosis transmembrane conductance regulator (CFTR) deficiency on the enterohepatic disposition of bile acids (BAs).

METHODS

Bile secretion and BA homeostasis were investigated in Cftr(tm1Unc) (Cftr-/-) and CftrΔF508 (ΔF508) mice.

RESULTS

Cftr-/- and ΔF508 mice did not grow to normal size, but did not have liver abnormalities. The gallbladders of Cftr-/- mice were enlarged and had defects in emptying, based on (99m)technetium-mebrofenin scintigraphy or post-prandial variations in gallbladder volume; gallbladder contraction in response to cholecystokinin-8 was normal. Cftr-/- mice had abnormal gallbladder bile and duodenal acidity, and overexpressed the vasoactive intestinal peptide-a myorelaxant <em>factor</em> for the gallbladder. The BA pool was larger in Cftr-/- than wild-type mice, although there were no differences in fecal loss of BAs. Amounts of secondary BAs in portal blood, liver, and bile of Cftr-/- mice were much lower than normal. Expression of genes that are induced by BAs, including <em>fibroblast</em> <em>growth</em> <em>factor</em>-<em>15</em> and BA transporters, was lower in the ileum but higher in the gallbladders of Cftr-/- mice, compared with wild-type mice, whereas enzymes that synthesize BA were down-regulated in livers of Cftr-/- mice. This indicates that BAs underwent a cholecystohepatic shunt, which was confirmed using cholyl-(Ne-NBD)-lysine as a tracer. In Cftr-/- mice, cholecystectomy reversed most changes in gene expression and partially restored circulating levels of secondary BAs. The ΔF508 mice overexpressed vasoactive intestinal peptide and had defects in gallbladder emptying and in levels of secondary BAs, but these features were less severe than in Cftr-/- mice.

CONCLUSIONS

Cftr-/- and CftrΔF508 mice have defects in gallbladder emptying that disrupt enterohepatic circulation of BAs. These defects create a shunt pathway that restricts the amount of toxic secondary BAs that enter the liver.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) has been shown to enhance the survival and neurite extension of various types of neurons including spinal ganglion neurons. In addition, endogenous FGF-2 and FGF receptors are upregulated following peripheral nerve lesion in ganglia and at the lesion site. FGF-2 protein is expressed in different isoforms (18 kDa, 21 kDa, 23 kDa) and differentially regulated after nerve injury. In the rat we analyzed the regenerative capacity of the high molecular weight (HMW) FGF-2 isoforms (21/23 kDa) to support the regeneration of the axotomized adult sciatic nerve across long gaps. The nerve stumps were inserted into the opposite ends of a silicone chamber resulting in an interstump gap of <em>15</em> mm. Silicone tubes were filled with Matrigel or a mixture of Schwann cells (SC) and Matrigel. SC were prepared from newborn rats and transfected to overexpress HMW FGF-2. Four weeks after the operation procedure, channels were analyzed with regard to tissue cables bridging both nerve stumps and myelinated axons distal to the original proximal nerve stump. Peripheral nerves interposed with HMW Schwann cells displayed significantly enhanced nerve regeneration, with the greatest number of tissue cables containing myelinated axons and the highest number of myelinated axons. These results suggest that a cellular substrate together with a source of a trophic <em>factor</em> could be a promising tool to promote nerve regeneration and, therefore, become useful also for a clinical approach to repair long gaps.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (Fgf<em>15</em>) is a gene regulated by the expression of Otx2 in developing mouse brain (Proc Natl Acad Sci USA 97 (2000) 14388). Otx2 gene codes for a transcription <em>factor</em> and is fundamental for the regionalisation and development of the anterior neural plate and cephalic region of the vertebrate embryo (Development 124 (1997) 3639). In addition, the thalamic expression of Fgf<em>15</em> has been recently reported under the control of Shh signalling gene, expressed in the diencephalic basal plate (Development 129 (2002) 4807). In the present work, we have analysed Fgf<em>15</em> expression pattern during mouse neural development. Fgf<em>15</em> appeared early in the developing neural epithelium, in domains where Fgf8 gene is also expressed and, at later stages, in specific groups of neural cells. Fgf8 is an important signalling protein with demonstrated morphogenetic activity in several embryonic regions. Fgf<em>15</em> expression is localized, like Fgf8, in secondary neural tube organizers: the isthmic organizer (IsO) and the anterior neural ridge (ANR).

Postprandial hyperglycemia is implicated as a risk <em>factor</em> predisposing to vascular complications. This study was designed to assess recurrent short-term increases in glucose on markers of renal fibrogenesis. Human renal cortical <em>fibroblasts</em> were exposed to fluctuating short-term (2 h) increases to <em>15</em> mM d-glucose, three times a day over 72 h, on a background of 5 mM d-glucose. To determine whether observed changes were due to fluctuating osmolality, identical experiments were undertaken with cells exposed to l-glucose. Parallel experiments were performed in cells exposed to 5 mM d-glucose and constant exposure to either <em>15</em> or 7.5 mM d-glucose. Fluctuating d-glucose increased extracellular matrix, as measured by proline incorporation (P < 0.05), collagen IV (P < 0.005), and fibronectin production (P < 0.001), in association with increased tissue inhibitor of matrix metalloproteinase (MMP) (P < 0.05). Sustained exposure to <em>15</em> mM d-glucose increased fibronectin (P < 0.001), in association with increased MMP-2 (P = 0.01) and MMP-9 activity (P < 0.05), suggestive of a protective effect on collagen matrix accumulation. Transforming <em>growth</em> <em>factor</em>-beta(1) (TGF-beta(1)) mRNA was increased after short-term (90 min) exposure to <em>15</em> mM glucose (P < 0.05) and after 24-h exposure to 7.5 mM ? (P < 0.05). Normalization of TGF-beta(1) secretion occurred within 48 h of constant exposure to an elevated glucose. Fluctuating l-glucose also induced TGF-beta(1) mRNA and a profibrotic profile, however, to a lesser extent than observed with exposure to fluctuating d-glucose. The results suggest that exposure to fluctuating glucose concentrations increases renal interstitial fibrosis compared with stable elevations in d-glucose. The effects are, in part, due to the inherent osmotic changes.

BACKGROUND

Avastin Biomarkers In lunG And 3D Innovative anaLysis (ABIGAIL), which is a phase II, open-label, randomized study, investigated correlations between biomarkers and best overall response to bevacizumab plus platinum-doublet chemotherapy for patients with advanced/recurrent non-small-cell lung cancer.

METHODS

Patients received bevacizumab (7.5 or <em>15</em> mg/kg, 3-weekly until disease progression/unacceptable toxicity) plus carboplatin/gemcitabine or carboplatin/paclitaxel (maximum six cycles). Plasma samples (baseline/throughout treatment) were analyzed for vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-A (baseline only), VEGF receptors (VEGFR-1/VEGFR-2), basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, E-selectin, intercellular adhesion molecule-1, and placental <em>growth</em> <em>factor</em> (baseline only). Tumor samples (primary specimen) were analyzed for VEGF-A, VEGFR-1/VEGFR-2, neuropilin (NRP), and CD31. Response was evaluated at baseline and every 6 weeks (Response Evaluation Criteria in Solid Tumors).

RESULTS

Patients were randomized to receive chemotherapy plus 7.5 mg/kg (n =<em>15</em>4) or <em>15</em> mg/kg (n =149) bevacizumab. For the primary analysis, none of the baseline plasma biomarkers correlated with best overall response. Exploratory analyses showed that low VEGF-A levels were associated with longer progression-free survival (7.4 versus 6.1 months; hazard ratio, 1.57; 95% confidence intervals, 1.17 to 2.09; p = 0.002) and overall survival (19.8 versus 11.1 months; hazard ratio, 1.57; 95% confidence interval, 1.<em>15</em>-2.13; p = 0.004) compared with these in high baseline plasma VEGF-A levels. No plasma biomarkers changed significantly over time. No significant correlations were observed between tumor biomarkers and clinical outcomes. No new safety signals were observed.

CONCLUSIONS

Baseline and/or dynamic changes in plasma basic fibroblast growth factor, E-selectin, intercellular adhesion molecule-1, placental growth factor, VEGFR-1 and VEGFR-2, and tumor biomarkers did not correlate statistically with treatment outcomes for bevacizumab plus chemotherapy. Only baseline plasma VEGF-A was significantly correlated with progression-free survival/overall survival.

Young (4 month) and aged (<em>15</em>-18 months) mice were given intranasal saline or γ--herpesvirus-68 infection. After 21 days, aged, but not young mice, showed significant increases in collagen content and fibrosis. There were no differences in viral clearance or inflammatory cells (including fibrocytes) between infected aged and young mice. Enzyme-linked immunosorbent assays showed increased transforming <em>growth</em> <em>factor</em>-β in whole lung homogenates of infected aged mice compared with young mice. When <em>fibroblasts</em> from aged and young mice were infected in vitro, aged, but not young, <em>fibroblasts</em> upregulate alpha-smooth muscle actin and collagen I protein. Infection with virus in vivo also demonstrates increased alpha-smooth muscle actin and collagen I protein and collagen I, collagen III, and fibronectin messenger RNA in aged <em>fibroblasts</em>. Furthermore, evaluation revealed that aged <em>fibroblasts</em> at baseline have increased transforming <em>growth</em> <em>factor</em>-β receptor 1 and 2 levels compared with young <em>fibroblasts</em> and are resistant to apoptosis. Increased responsiveness to transforming <em>growth</em> <em>factor</em>-β was verified by increased collagen III and fibronectin messenger RNA after treatment in vitro with transforming <em>growth</em> <em>factor</em>-β.

COX-1 and COX-2 are two cyclooxygenase enzymes responsible for prostanoid production. COX-2 is expressed in inflammatory cells and <em>fibroblasts</em> of the gastric mucosa, and through the production of various <em>growth</em> <em>factors</em> including hepatocyte <em>growth</em> <em>factor</em> (HGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF), plays a key role in the tissue repair process. Aspirin induces and acetylates COX-2 to produce <em>15</em>-(R)-epi-lipoxinA4, an anti-inflammatory mediator thought to protect the gastric mucosa against aspirin-induced injury. Recently, three different PGE synthases have been identified, that convert COX-2 metabolites into PGE2. mPGE synthase (mPGES)-1 has been shown to be inducible, and to colocalize with COX-2 in <em>fibroblasts</em> and macrophages infiltrating the gastric ulcer bed. cPGES and mPGES-2 have been found expressed in normal gastric mucosa, with no change in expression levels seen in gastritis or gastric ulcer tissue. Finally, this review discusses the role of these enzymes in the pathophysiology of the gastric mucosa, as well as the biologcal significance of their inhibition.

OBJECTIVE

The purpose of this study was to characterize cell cultures and xenografts derived from patients with ovarian cancer.

METHODS

Ninety specimens from 67 patients were plated in RPMI 1640 or inoculated in nude mice. Growth characteristics of cell cultures and xenografts were determined. Expression of receptors for estrogen, progesterone, androgen, epithelial growth factor, fibroblast growth factor, HER-2/erbB-2/c-neu proto-oncogene, and the P53 expression were characterized by immunocytochemistry in 28 cell cultures.

RESULTS

Forty-nine percent of samples were cultured successfully in vitro. Ascitic and pleural effusion specimens were more likely to produce a cell culture or a xenograft than solid tissue specimens (P < 0.005). All of the cell cultures had an epithelial morphology, and 89% were aneuploid with a mean DNA index of 1.6 (range, 0.9-3.0). Of 54 and 61 specimens inoculated into nude mice i.p. and s.c., 15 (28%) and 18 (30%) produced a xenograft, respectively, with two-thirds of these xenografts being reproducibly tumorigenic. The median time to first passage was 21 weeks for cell cultures and 8-12 weeks for xenografts. Expression of epithelial growth factor receptor, HER-2/erbB-2/c-neu proto-oncogene, fibroblast growth factor receptor, estrogen, progesterone, and androgen was seen in 24, 21, 31, 17, 43, and 18%, respectively. P53 was overexpressed in 62% of cell cultures analyzed.

CONCLUSIONS

Ovarian cancer cells collected from effusions are easier to grow in vitro than in vivo. The only characteristic that may be associated with tumorigenicity was abnormal P53 expression. This panel of ovarian cancer materials provides useful models for biological or therapeutical studies.

Sample preparation for proteomic analysis involves precipitation of protein using 2,2,2-trichloroacetic acid (TCA). In this study, we examine the mechanism of the TCA-induced protein precipitation reaction. TCA-induced protein precipitation curves are U-shaped and the shape of the curve is observed to be independent of the physicochemical properties of proteins. TCA is significantly less effective in precipitating unfolded states of proteins. Results of the 1-anilino-8-napthalene sulfonate (ANS) and size-exclusion chromatography, obtained using acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF), show that a stable "molten globule-like" partially structured intermediate accumulates maximally in 5% (w/v) of trichloroacetate. Urea-induced unfolding and limited proteolytic digestion data reveal that the partially structured intermediate is significantly less stable than the native conformation. (1)H-(<em>15</em>)N chemical shift perturbation data obtained using NMR spectroscopy indicate that interactions stabilizing the beta-strands at the N- and C- terminal ends (of aFGF) are disrupted in the trichloroacetate-induced "MG-like" state. The results of the study clearly demonstrate that TCA-induced protein precipitation occurs due to the reversible association of the "MG-like" partially structured intermediate state(s). In our opinion, the findings of this study provide useful clues toward development of efficient protocols for the isolation and analysis of the entire proteome.

BACKGROUND

Brain tumour stem cells (BTSCs) are a small population of cancer cells that exhibit self-renewal, multi-drug resistance, and recurrence properties. We have shown earlier that peroxisome proliferator-activated receptor gamma (PPARγ) agonists inhibit the expansion of BTSCs in T98G and U87MG glioma. In this study, we analysed the influence of PPARγ agonists on the expression of stemness and differentiation genes in BTSCs.

METHODS

The BTSCs were isolated from T98G and DB29 glioma cells, and cultured in neurobasal medium with epidermal growth factor+basic fibroblast growth factor. Proliferation was measured by WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2 H-5-tetrazolio]-1,3-benzene disulphonate) and 3H thymidine uptake assays, and gene expression was analysed by quantitative reverse--transcription PCR and Taqman array. The expression of CD133, SRY box 2, and nanog homeobox (Nanog) was also evaluated by western blotting, immunostaining, and flow cytometry.

RESULTS

We found that PPARγ agonists, ciglitazone and 15-deoxy-Δ(12,14)-ProstaglandinJ(2), inhibited cell viability and proliferation of T98G- and DB29-BTSCs. The PPARγ agonists reduced the expansion of CD133(+) BTSCs and altered the expression of stemness and differentiation genes. They also inhibited Sox2 while enhancing Nanog expression in BTSCs.

CONCLUSIONS

These findings highlight that PPARγ agonists inhibit BTSC proliferation in association with altered expression of Sox2, Nanog, and other stemness genes. Therefore, targeting stemness genes in BTSCs could be a novel strategy in the treatment of glioblastoma.

OBJECTIVE

The purpose of this study was to identify a mediator produced by human colon cancer cells that is responsible for the induction of hyperplasia in the adjacent mucosa.

METHODS

Seventy human colon cancer surgical specimens were immunostained to determine the presence of cytokines that can induce hyperplasia in the adjacent mucosal. Human colon cancer cells with low and high metastatic potential were implanted into the cecal wall of nude mice. The resulting lesions were studied by immunohistochemistry to detect possible mediators of mucosal hyperplasia.

RESULTS

Immunostaining of 70 colon cancer specimens from 70 patients suggested that mucosal hyperplasia and distant metastasis were associated with the expression of interleukin (IL)-<em>15</em> and, to a lesser extent, transforming <em>growth</em> <em>factor</em> alpha. The production of IL-<em>15</em> by colon cancer cells was not associated with the infiltration of natural killer cells into the tumors. Cecal tumors produced in nude mice by human colon cancer cells with low and high metastatic potential (KM12C and KM12SM cells, respectively) expressed similar levels of transforming <em>growth</em> <em>factor</em> alpha, and expression of IL-<em>15</em> was detected only in the metastatic KM12SM cells and was associated with hyperplasia of the surrounding mucosa. The expression of the IL-<em>15</em> receptor in rat intestinal epithelial cells (IEC6 cells) was confirmed by immunoblotting with antibodies against IL-<em>15</em> receptor alpha and IL-2 receptor beta and gamma subunits and by a binding assay using (125)I-labeled IL-<em>15</em> (K(d) = 0.011 nM). IL-<em>15</em> stimulated proliferation of the IEC6 cells, even under serum starvation. Treatment of IEC6 cells with IL-<em>15</em> decreased doxorubicin-mediated cytotoxicity. In IEC6 cells treated with either IL-<em>15</em>- or KM12SM-conditioned medium, immunoblotting revealed a decrease in the production of p21Waf1, Bax, and Bak and an increase in the production of cyclin E, proliferating cell nuclear antigen, the phosphorylated active form of AKT, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and vascular endothelial <em>growth</em> <em>factor</em>, changes associated with cell <em>growth</em>, survival, and induction of angiogenesis.

CONCLUSIONS

These data indicate that IL-<em>15</em> produced by metastatic colon carcinoma cells can induce hyperplasia in the mucosa adjacent to colon cancer, thus contributing to angiogenesis and progression of the disease.

BACKGROUND

Adrenocortical cancer (ACC) is a rare cancer with poor prognosis and scant treatment options. In ACC, no personalized approach has emerged but no extensive molecular screening has been performed to date.

OBJECTIVE

The objective of the study was to evaluate the presence of a large number of potentially targetable molecular events in a large cohort of advanced ACC.

METHODS

We used hot spot gene sequencing (Ion Torrent, 40 patients) and comparative genomic hybridization (CGH; 28 patients; a subset of the entire cohort) in adult stage III-IV ACC samples to screen for mutations and copy number abnormalities of potential interest for therapeutic use in 46 and 130 genes, respectively.

RESULTS

At least one copy number alteration or mutation was found in 19 patients (47.5%). The most frequent mutations were detected on TP53, ATM, and CTNNB1 [6 of 40 (<em>15</em>%), 5 of 40 (12.5%), and 4 of 40 (10%), respectively]. The most frequent copy number alterations identified were: amplification of the CDK4 oncogene (5 of 28; 17.9%) and deletion of the CDKN2A (4 of 28; 14.3%) and CDKN2B (3 of 28; 10.7%) tumor suppressor genes. Amplifications of FGFR1, FGF9, or FRS2 were discovered in three subjects (10.7%). Associated alterations were: deletions of CDKN2A, CDKN2B with ATM mutations, and TP53 mutations with CTNNB1 mutations.

CONCLUSIONS

No simple targetable molecular event emerged. Drugs targeting the cell cycle could be the most relevant new therapeutic approach for patients with advanced ACC. Inhibitors of the fibroblast growth factor receptor pathway could also be a therapeutic option in a subset of patients, whereas other targeted therapies should be considered on a case-by-case basis.

BACKGROUND

Persistent upregulation of signaling by cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK) through its receptor fibroblast growth factor-inducible molecule-14 (Fn14) promotes chronic inflammation and tissue destruction.

OBJECTIVE

The aim of this study was to explore the safety and tolerability of the TWEAK-blocking monoclonal antibody BIIB023 and determine its pharmacokinetics and effects on TWEAK pathway pharmacodynamic markers in rheumatoid arthritis (RA).

METHODS

Phase I, first-in-human, 2-part, multicenter, double-blind, dose-escalation study. Patients were randomized to a single dose of BIIB023 (0.03-20 mg/kg) (n = 38) or placebo (n = 15) as an add-on to methotrexate. Three open-label cohorts of RA patients taking background disease-modifying antirheumatic drugs and stable tumor necrosis factor (TNF) inhibitor therapy (n = 12) received a single-dose of BIIB023 of 2, 10, or 20 mg/kg and were assessed over 70 days.

RESULTS

The incidence of treatment-emergent adverse events for the BIIB023 monotherapy cohorts and open-label cohorts of BIIB023 as add-on therapy to TNF inhibitors compared with placebo were 47% and 50% versus 33%, respectively. Serum exposure to BIIB023 increased in a dose-dependent manner from 0.03 to 20 mg/kg, but not in direct proportion to dose level. After administration, the time course of BIIB023 serum concentration was multiphasic and showed expedited elimination when levels decreased to < 10 µg/mL. Serum-soluble TWEAK levels were suppressed at all dose levels by 6 hours post-dose and recovered to baseline between days 7 and 28. A trend toward downward modulation of serum biomarkers of inflammatory response was suggested in monocyte chemoattractant protein 1, inducible protein 10, macrophage inflammatory protein 1β, and tissue inhibitor of metalloproteinase 1 in the BIIB023 group versus placebo.

CONCLUSIONS

Single-dose BIIB023 showed a favorable safety and tolerability profile in RA. Suppression of serum-soluble TWEAK for ≤ 28 days was observed and downward trends in serum biomarkers suggested.