OBJECTIVE

There is increasing evidence to suggest that various growth factors play a crucial role in fetal lung growth and morphogenesis. An array of peptide growth factors regulate cell proliferation, differentiation, and various other cell functions in the developing lung. The aim of this study was to investigate the effect of antenatal glucocorticoids administration on gene expression of basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta1 in nitrofen-induced congenital diaphragmatic hernia (CDH) in rats.

METHODS

A CDH model was induced in pregnant rats after administration of nitrofen. Dexamethasone (Dex; 0.25 mg/kg) was given intraperitoneally on day 18.5 and 19.5 of gestation (term, day 22). Cesarean section was performed on day 21 of gestation. mRNA was extracted from left lung and reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expression of each growth factors. Relative levels of mRNA were expressed as a ratio of the band density divided by that of beta-actin, a housekeeping gene known to be expressed at a constant level.

RESULTS

Relative mRNA levels of bFGF and TGF-beta1 were decreased significantly in CDH lung compared with controls. Antenatal Dex treatment up-regulated gene expression of bFGF, PDGF, and TGF-beta1 in the hypoplastic CDH lung.

CONCLUSIONS

The authors' findings suggest that decreased gene expression of bFGF, PDGF, and TGF-beta1 in the CDH lung may suppress lung growth and development. Increased gene expression of bFGF, PDGF, and TGF-beta1 in Dex-treated lung suggests that antenatal glucocorticoid administration may accelerate fetal lung growth by up-regulating these growth factors.

Newborn infants with congenital diaphragmatic hernia (CDH) still have high mortality. Recently, the possible role of a cardiac maldevelopment in the high mortality has been suggested. Human and animal studies have demonstrated that heart weight is significantly reduced in the presence of CDH. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and platelet-derived <em>growth</em> <em>factor</em> (PDGF) are pleiotropic regulatory peptides that are expressed in myocardium in precise developmental and spatial programs. PDGF and bFGF both stimulate cardiac <em>growth</em> by inducing cell proliferation and stimulating the synthesis of extracellular matrix. The aim of this study was to investigate the presence of heart hypoplasia in nitrofen-induced CDH in rats and the role of specific tissue <em>growth</em> <em>factors</em> (bFGF and PDGF) in its genesis. CDH was induced in pregnant rats following administration of 100 mg nitrofen on day 9.5 of gestation (term <em>22</em> days). In control animals the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The fetuses were divided in two groups: normal controls (n = 8) and nitrofen-induced CDH (n = 8). Total RNA, DNA, and soluble proteins were extracted from the heart in each group and measured. mRNA was extracted from total RNA and a reverse transcription-polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expression of bFGF and PDGF. The heart/body weight ratio (HBWR) and DNA content were significantly decreased (P < 0.01) in CDH animals compared to controls. RNA and protein content were also reduced in CDH. The expression of bFGF and PDGF mRNA was significantly reduced in the CDH group compared to controls (P < 0.01). The decreased HBWR, DNA, RNA, and protein content in the CDH heart indicates that the heart is hypoplastic in nitrofen-induced left CDH. The downregulation of bFGF and PDGF gene expression in the CDH heart suggests that these regulating peptides may play an important role in the genesis of cardiac hypoplasia in CDH.

BACKGROUND

Hyperinsulinaemia has been implicated in the pathogenesis of laminitis; however, laminar cell types responding to insulin remain poorly characterised.

OBJECTIVE

To identify laminar cell types expressing insulin receptor (IRc) and/or insulin-like growth factor-1 receptor (IGF-1R); and to evaluate the effect of dietary nonstructural carbohydrate (NSC) on their expression.

METHODS

Mixed-breed ponies (n = 22) received a conditioning hay chop diet (NSC ∼6%); following acclimation, ponies were stratified into lean (n = 11, body condition score [BCS]≤4) or obese (n = 11, BCS ≥7) groups and each group further stratified to remain on the low NSC diet (n = 5 each for obese and lean) or receive a high NSC diet (total diet ∼42% NSC; n = 6 each for obese and lean) for 7 days. Laminar samples were collected at the end of the feeding protocol and stained immunohistochemically for IRc and IGF-1R. The number of IRc(+) cells was quantified; distribution of IGF-1R was qualitatively described. Laminar IRc content was assessed via immunoblotting.

RESULTS

The number of IRc(+) cells was greater in the laminae of high NSC ponies than low NSC ponies (P = 0.001); there was a positive correlation between the change in serum insulin concentration and number of IRc(+) cells (r(2) = 0.74; P<0.0001). No epithelial IRc(+) cells were observed; IRc(+) cells were absent from the deep dermis. Analysis of serial sections identified IRc(+) cells as endothelial cells. The distribution of IGF-1R was more extensive than that of IRc, with signal in vascular elements, epithelial cells and fibroblasts.

CONCLUSIONS

Increased dietary NSC results in increased laminar endothelial IRc expression. Laminar keratinocytes do not express IRc, suggesting that insulin signalling in laminar epithelial cells must be mediated through other receptors (such as IGF-1R).

CONCLUSIONS

Manipulation of signalling downstream of IRc and IGF-1R may aid in treatment and prevention of laminitis associated with hyperinsulinaemia.

The in vitro effects of asbestos fibers on the <em>growth</em> and viability of CHO cells, an epithelioid cell line derived from Chinese hamster ovary, and on K-<em>22</em> cells, an epithelial cell line derived from rat liver, have been studied. Relatively low concentrations of asbestos (10 micrograms/ml) were quite cytotoxic to both cell types. A sample of chrysotile asbestos was more toxic than samples of crocidolite or amosite. The toxic <em>factor</em> could not be extracted from the asbestos and toxicity occurred only if there was physical contact between the fibers and the cells. Although the phorbol ester class of tumor promoters induces the synthesis of plasminogen activator in various cell cultures, asbestos did not induce this protease in epithelioid or <em>fibroblast</em> cell cultures. The results obtained are compared to previous in vitro results obtained with <em>fibroblast</em> or macrophage cultures and are discussed in terms of their possible relevance to asbestos-induced fibrosis and tumorigenesis.

<em>Growth</em> of the mouse mammary epithelial cell line designated COMMA-D has been studied in serum-free medium (SFM) formulated with Ham's F12 and Dulbecco's modified Eagle's medium (1/1) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2 mM glutamine, gentamicin (50 micrograms/ml; basal medium) and supplemented with insulin (10 micrograms/ml), transferrin (10 micrograms/ml), selenous acid (10 ng/ml), epidermal <em>growth</em> <em>factor</em> (20 ng/ml; EGF), 10 nM 3,5,3'-triiodothyronine, 50 microM ethanolamine, 1.0 nM 17 beta-estradiol, 65 microM glutathione, and ovalbumin (100 micrograms/ml). COMMA-D cells were able to undergo serial passage and continued to exhibit dome formation after 20 passages in SFM. Cells seeded at low density in SFM underwent four population doublings at low passage number in 1 week compared to six doublings for cells grown in medium containing insulin, transferrin, selenium, EGF, and 1% fetal bovine serum. After many passages in SFM, the <em>growth</em> rates of cells were similar to those in serum-supplemented medium used for stock culture. Deletion of insulin or EGF from SFM resulted in cell <em>growth</em> similar to that of cells seeded in basal medium alone. When cells were seeded in basal medium without added supplements, addition of insulin or EGF resulted in 29 and <em>22</em>%, respectively, of the number of cells grown in SFM for 5 days. However, when insulin and EGF were combined in basal medium, the cell number at 5 days was 83% of that in SFM. When insulin was deleted from SFM, COMMA-D cells became responsive to insulin-like <em>growth</em> <em>factors</em> I and II. The <em>growth</em>-promoting characteristics of EGF and transforming <em>growth</em> <em>factor</em> alpha were compared in SFM and were not distinguishable, showing identical dose-response curves. When incorporation of [3H]thymidine was used as an assay of cell <em>growth</em>, saturating levels of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (20 ng/ml) showed a stimulation 1.35 times greater than EGF (20 ng/ml). When EGF and <em>fibroblast</em> <em>growth</em> <em>factor</em> were combined, the stimulation was 1.75 times greater than EGF alone suggesting that COMMA-D cells are responsive to multiple classes of <em>growth</em> <em>factors</em>. COMMA-D cells seeded in basal medium supplemented with insulin, transferrin, and selenous acid have been used to detect mitogenic activity present in extracts of hypothalamus, uterus, and pituitary. The results show that COMMA-D cells can be grown long term in a hormonally defined serum-free medium and that maximal mitogenic effects were seen only with the addition of two or more <em>growth</em> <em>factors</em>.

Inner hair cell (IHC) ribbon synapses of cochlea play important role in transmitting sound signal into auditory nerve and are sensitive to ototoxicity. However, ototoxic damage of ribbon synapses is not understood clearly. Roles of <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>22</em> (FGF<em>22</em>) on synapse formation were explored under gentamycin ototoxicity. 6-week-old mice were injected intraperitoneally once daily with 50-150 mg/kg gentamicin for 10 days. Immunostaining with anti- GluR2&3/CtBP2 was used to estimate the number of ribbon synapses in the cochlea. Expression of FGF<em>22</em> and myocyte enhancer <em>factor</em> 2D (MEF2D) was assayed with RT-PCR. Expression and localization of FGF<em>22</em> protein were visualized with anti-FGF<em>22</em> immunostaining. Hearing thresholds were assessed using auditory brainstem responses. Gentamicin administration caused reduction in ribbon synapse number and hearing impairment without effect on hair cells in CBA/J mouse model. Immunohistochemistry showed that FGF<em>22</em> protein was expressed in IHCs, but not OHCs of cochlea. Gentamycin attenuated expression of FGF<em>22</em> but enhanced expression of MEF2D. Cochlear infusion of recombinant FGF<em>22</em> inhibited expression of MEF2D, preserved ribbon synapses, and restored hearing function impaired by gentamycin. FGF<em>22</em> restores hearing loss through maintaining ribbon synapse number, likely via inhibition of MEF2D. Activating FGF<em>22</em> might provide the conceptual basis for the therapeutic strategies.

BACKGROUND

Transforming growth factor-β1 (TGF-β1) is a potent regulator of cell growth and differentiation. TGF-β1 has been shown to be a key player in tissue remodeling processes in a number of disease states by inducing expression of extracellular matrix proteins. In this study a quantitative proteomic analysis was undertaken to investigate if TGF-β1 contributes to tissue remodeling by mediating mRNA splicing and production of alternative isoforms of proteins.

RESULTS

The expression of proteins involved in mRNA splicing from TGF-β1-stimulated lung fibroblasts was compared to non-stimulated cells by employing isotope coded affinity tag (ICATTM) reagent labeling and tandem mass spectrometry. A total of 1733 proteins were identified and quantified with a relative standard deviation of 11% +/- 8 from enriched nuclear fractions. Seventy-six of these proteins were associated with mRNA splicing, including 22 proteins involved in splice site selection. In addition, TGF-β1 was observed to alter the relative expression of splicing proteins that may be important for alternative splicing of fibronectin. Specifically, TGF-β1 significantly induced expression of SRp20, and reduced the expression of SRp30C, which has been suggested to be a prerequisite for generation of alternatively spliced fibronectin. The induction of SRp20 was further confirmed by western blot and immunofluorescence.

CONCLUSIONS

The results show that TGF-β1 induces the expression of proteins involved in mRNA splicing and RNA processing in human lung fibroblasts. This may have an impact on the production of alternative isoforms of matrix proteins and can therefore be an important factor in tissue remodeling and disease progression.

OBJECTIVE

We studied <em>22</em> dogs to examine the effect of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) alone, in comparison with omental or muscular wrapping on airway healing in a tracheal autotransplantation model.

BACKGROUND

Basic fibroblast growth factor is one of the most potent promoters of angiogenesis and has an ability to enhance blood supply to the ischemic airway. Topical administration of a fibrin glue enriched with 5 microg/cm2 bFGF, determined as a proportion of surface area of the tracheal grafts, improved revascularization of orthotopic canine tracheal autografts in a previous study.

METHODS

All animals received orthotopic tracheal transplantation using 6-ring autografts that occupied a distal part of the thoracic trachea. Twenty-two animals were classified randomly into the following four groups: no treatment (Group G1, n = 4), muscular wrapping (Group G2, n = 4), omental wrapping (Group G3, n = 4), and topical administration of fibrin glue enriched with 5 microg/cm2 bFGF (Group G4, n = 10). Autografts were harvested 60 days after transplantation and assessed by the percent patency and histology.

RESULTS

Devascularized tracheal autografts could not maintain their structural integrity without other treatments (Group G1). In contrast, more than half of all autografts receiving treatments remained viable, as demonstrated by gross and histologic findings (Groups G2, G3, and G4). Treatments with bFGF and omentum showed significantly better graft viability than no treatment. However, there was no statistical difference in the viability of tracheal autografts among the three treatment groups. In terms of the time performance ratio, bFGF was the best treatment for the devascularized autografts.

CONCLUSIONS

Topical administration of bFGF was superior to the omental or muscular wrapping in terms of the time performance ratio. Clinical trials will be necessary to determine whether these findings are applicable to humans.

Purinergic signaling mediates many cellular processes, including embryonic development and regulation of endocrine signaling. The ADP P2Y13 receptor is known to regulate bone and stem cells activities, although relatively little is known about its role in bone development. In this study we demonstrate, using contemporary techniques, that deletion of the P2Y13 receptor results in an age-dependent skeletal phenotype that is governed by changes in phosphate metabolism and hormone levels. Neonatal and postnatal (2 wk) P2Y13 receptor-knockout (KO) mice were indistinguishable from their wild-type (WT) littermate controls. A clear bone phenotype was observed in young (4-wk-old) KO mice compared WT controls, with 14% more trabecular bone, 35% more osteoblasts, 73% fewer osteoclasts, and a 17% thicker <em>growth</em> plate. Mature (>10 wk of age) KO mice showed the opposite bone phenotype, with 14% less trabecular bone, <em>22</em>% fewer osteoblasts, and 10% thinner <em>growth</em> plate. This age-dependent phenotype correlated with serum <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF-23) and phosphorus levels that were 65 and 16% higher, respectively, in young KO mice but remained unchanged in mature mice. These findings provide novel insights for the role of the P2Y13 receptor in skeletal development via coordination with hormonal regulators of phosphate homeostasis.

Oncostatin M (OSM), a pleiotropic cytokine originally isolated from supernatants of the U937 histiocytic lymphoma cell line, has been shown to have regulatory effects on a wide variety of cultured and tumor cells. We investigated the effects of OSM on basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) gene expression in bovine arterial endothelial (BAE) cells. Levels of bFGF mRNA transcripts were low in uninduced BAE cells, were maximal at 8 hours of exposure to OSM, and returned to control levels by 24 hours. Induction of bFGF mRNA transcripts by OSM was dose-dependent. Nuclear transcriptional run-on analysis demonstrated that exposure of BAE cells to OSM stimulated bFGF gene transcription. OSM treatment of BAE cells enhanced the synthesis of bFGF protein as determined by ELISA assays. Immunocytochemistry studies demonstrated the presence of low levels of bFGF protein within the cytoplasm in uninduced cells. After stimulation for 8 hours with OSM there was significant staining for bFGF in the cytoplasm. However, 24 hours after exposure to OSM, bFGF antigen was located only within the nuclei. Western blot analysis demonstrated that OSM stimulated predominantly the synthesis of a <em>22</em> kDa form of bFGF. In addition, OSM stimulated endothelial cell proliferation and migration as well as acquisition of a spindle shape. Phosphorothioate antisense oligonucleotide directed against bFGF inhibited OSM induced BAE cell proliferation and spindle shape formation but had only a minimal effect on migration. The levels of the <em>22</em> kDa form of bFGF were reduced by antisense treatment indicating that OSM induced proliferation and morphology change is likely to be regulated by intracellular bFGF. Our studies suggest that OSM released at sites of vascular injury could stimulate angiogenesis by inducing bFGF synthesis, endothelial cell proliferation and migration.

BACKGROUND

SMAD proteins, intracellular mediators of the transforming growth factor (TGF)-beta pathway, function within two axes, the SMAD1/5/8 and SMAD2/3, connected to TGF-beta and bone morphogenetic protein (BMP) ligands. The SMAD proteins of these two axes dimerize with SMAD4 and translocate to the nucleus. SMAD signaling is characterized by a dichotomic functioning, with tumor-suppressive functions and with loss of normal growth inhibitory responses, depending on the carcinogenesis stage. SMAD proteins also have pro-tumor effects including abnormal extracellular matrix production. Among tumors, pancreatic cancers harbor SMAD4 inactivation the most frequently and the SMAD proteins are considered to be key factors in pancreatic carcinogenesis.

METHODS

Our aims were to study the expression patterns of the different types of SMAD proteins in pancreatic ductal adenocarcinomas treated by surgical resection (without neoadjuvant treatment) and their correlations with morphological and clinical characteristics. We examined the immunohistochemical expression of SMAD4, SMAD1/5/8, and SMAD2/3 in 99 pancreatic ductal adenocarcinomas. Antibodies directed against the activated, phosphorylated forms of proteins were used when appropriate (SMAD1/5/8, SMAD2/3). Protein expression in the epithelial tumor cells and in stromal fibroblasts was analyzed with regard to morphological and clinical data.

RESULTS

Epithelial tumor cells showed SMAD1/5/8, SMAD2/3, and, SMAD4 expression in 13, 93, and 45 tumors, respectively, and stromal fibroblast expression in 5, 11, and 22 tumors, respectively. Epithelial SMAD4 was associated with a low, T1 or T2, TNM stage, and with the presence of an abundant stroma (p = 0.05 and <0.01, respectively). Activated stromal fibroblast SMAD2/3 expression was correlated with the presence of a fibrotic focus (p = 0.01), whereas fibroblast SMAD4 was related to a tendency for shorter postsurgical overall survival (p = 0.07). The relationship of stromal, fibroblast SMAD4 to a worse outcome attained statistical significance in the group of patients with T1 and with N1 stage tumors (p < 0.01 and p = 0.04, respectively).

CONCLUSIONS

In pancreatic ductal adenocarcinomas, SMAD protein expression in epithelial tumor cells or in stromal fibroblasts was related to stromal features and to a shorter postsurgical overall survival. Our results point out that the SMAD proteins play a role in the microenvironment of this highly fibrotic tumor type.

Bone marrow endothelial cells (BMECs) are important components of the hematopoietic microenvironment in bone marrow, and they can secrete several types of cytokines to regulate the functions of hematopoietic stem/progenitor cells. To date, it is unknown whether BMECs undergo functional changes and lead to hematopoietic abnormalities in cases of liver cirrhosis (LC). In the present study, whole genome microarray analysis was carried out to detect differentially expressed genes in human BMECs treated for 48 h with medium supplemented with 20% pooled sera from 26 patients with LC or 10 healthy volunteers as the control group. A total of 1,106 upregulated genes and 766 downregulated genes were identified. In Gene Ontology analysis, the most significant categories of genes were revealed. A large number of the upregulated genes were involved in processes, such as cell-cell adhesion, apoptosis and cellular response to stimuli and the downregulated genes were involved in the negative regulation of secretion, angiogenesis, blood vessel development and cell <em>growth</em>. Pathway analysis revealed that the upregulated genes were either cell adhesion molecules or parts of the apoptotic signaling pathway and the downregulated genes were involved in the Wnt signaling pathway and MAPK signaling pathway. These were the pathways with the highest enrichment scores. The results of apoptosis assays revealed that the humoral inhibitors in the sera of patients with LC induced the apoptosis of BMECs, which confirmed the accuracy of bioinformatic analysis. Moreover, we screened and verified 21 differentially expressed cytokine genes [transforming <em>growth</em> <em>factor</em> (TGF)B1, tumor necrosis <em>factor</em> (TNF)B, TNF receptor superfamily, member 11b (TNFRSF11B), TNF (ligand) superfamily, member 13b (TNFSF13B), interleukin (IL)1A, IL6, IL11, IL17C, IL24, family with sequence similarity 3, member B (FAM3B), Fas ligand (FASLG), matrix metallopeptidase (MMP)3, MMP15, vitronectin (VTN), insulin-like <em>growth</em> <em>factor</em> 1 (IGF1), <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>22</em> (FGF<em>22</em>), slit homolog 2 (Drosophila) (SLIT2), thrombospondin (THBS)2, THBS3, chemokine (C-C motif) ligand 28 (CCL28) and macrophage stimulating 1 (MST1)] from 97 cytokine genes in BMECs treated with serum from patients with LC. The results from our study demonstrate that the humoral inhibitors in the sera of patients with LC induce the dysfunction and abnormal cytokine secretion by BMECs, which may be a novel mechanism responsible for hematological abnormalities in patients with LC.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is an established <em>growth</em> <em>factor</em> for melanocytes and a potent angiogenic <em>factor</em>. The expression of bFGF was investigated in 23 desmoplastic melanomas. (DM) (12 males, median age 64 years, and 11 females, median age 54 years) by immunostaining of formalin-fixed, paraffin-embedded sections with high-affinity purified antibody raised against recombinant human bFGF (Scios Nova, Inc.). The tumors were characterized by level II invasion in 1 case (5%), level IV invasion in 11 cases (48%), level V invasion in 8 cases (35%), and indeterminate in 3 cases. bFGF expression was observed in <em>22</em> of 23 tumors (95%), either immune localized to tumor cell nuclei in 17 of <em>22</em> tumors (77%), or to the cytoplasm of tumor cells in 5 of <em>22</em> tumors (23%). Also in these cases, bFGF was strongly expressed in the nuclei of vascular endothelial cells. Maximal expression was noted in the peripheral blood vessels of 20 tumors (91%) versus intratumoral vessels of 13 DM (59%). In conclusion, the expression of predominantly nuclear bFGF by tumor cells in DM suggests a role in mediating the desmoplastic phenotype. In addition, the localization of bFGF to vascular endothelium, particularly at the periphery of the tumor, may be relevant to tumor angiogenesis.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (basic FGF) is a mitogen for isolated epiphyseal <em>growth</em> plate chondrocytes. To determine whether basic FGF might function as an autocrine stimulus to longitudinal skeletal <em>growth</em> in utero, we investigated the synthesis and release of basic FGF by isolated <em>growth</em> plate chondrocytes from the ovine fetus, the expression of mRNA for a high affinity basic FGF receptor by these cells, and the contribution of endogenous basic FGF to the DNA synthetic rate of the cells in vitro. Chondrocytes were isolated from the proximal tibial <em>growth</em> plate of the lamb fetuses between 35 and 132 days' gestation using collagenase, and were cultured in monolayer before use between passages 3 and 6. Viability was confirmed over the duration of the experiments by the exclusion of trypan blue, and an absence of lactate dehydrogenase accumulation in conditioned medium. Immunocytochemistry of chondrocyte monolayers showed immunoreactive basic FGF to be present in the cytoplasm of approximately 80% of sub-confluent cells which was accompanied by pronounced nuclear staining in approximately 30% of cells. Serum-free, conditioned culture medium, extracellular matrix and chondrocyte cytoplasm contained 52 +/- 2 pM/micrograms DNA, 66 +/- 2 pM/micrograms DNA and <em>22</em> +/- 3 pM/micrograms DNA basic FGF, respectively (mean +/- S.E.M., n = 8 fetuses), for cells obtained from animals of 35-40 days' gestation when assessed by radioimmunoassay. Chondrocyte-conditioned medium increased endothelial cell proliferation in vitro (a specific bio-assay for basic FGF and related peptides); and the mitogenic activity was removed from conditioned medium by incubation with heparin-Sepharose demonstrating that this was due to heparin-binding protein(s). Western blot analysis of conditioned medium using a specific basic FGF antibody revealed a single immunoreactive protein of approximately 18 kDa molecular size. The appearance of radiommunoassayable basic FGF in conditioned medium, extracellular matrix, and chondrocyte cytoplasm observed during culture was blocked by co-incubation with cycloheximide. The levels of immunoreactive basic FGF present in each compartment decreased with gestational age as did basal DNA synthetic rate assessed by the incorporation of [3H] thymidine. Incubation of chondrocytes with transforming <em>growth</em> <em>factor</em> beta, resulted in a significant increase while exposure to insulin-like <em>growth</em> <em>factors</em> or insulin caused a decrease, in the content and release of basic FGF. Basic FGF presence was unaltered when medium was supplemented with varying amounts of glucose (2.7-16.7 mM). In situ hybridization on cell monolayers using a cRNA probe encoding the high affinity flg receptor for FGFs showed an abundant expression of mRNA for the receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromaffin granules, the secretory organelles of the neuron-like adrenal medullary chromaffin cells, have previously been shown to store and liberate neurotrophic activities that support in vitro survival of several neuron populations including those innervating the adrenal medulla. Molecules resembling <em>fibroblast</em> <em>growth</em> <em>factor</em> and ciliary neurotrophic <em>factor</em> have been identified among these activities. Since chromaffin granules store a variety of neuropeptides and many neuropeptides can have pleiotropic effects on neuronal <em>growth</em> and maintenance we have tested 24 different neuropeptides for their capacities to promote survival of embryonic chick ciliary, dorsal root and sympathetic ganglionic neurons. Peptides tested included several derivatives of proenkephalin (Leu- and met-enkephalin, fragments BAM <em>22</em>, B, F and E), somatostatin, substance P, neuropeptide Y, neurotensin, VIP, bombesin, secretin, pancreastatin, dynorphin B, dynorphin 1-13, beta-endorphin, alpha-, beta-, and gamma-MSH. Control cultures received saturating concentrations of ciliary neurotrophic or nerve <em>growth</em> <em>factor</em> (CNTF; NGF), or no trophic supplements. At 1 x 10(-5) M leu- and met-enkephalin as well as somatostatin supported sympathetic neurons to the same extent as NGF. At the same concentrations, leu-enkephalin, the proenkephalin fragments BAM <em>22</em> and E, and somatostatin maintained about half of the dorsal root ganglionic neurons supported by NGF, but were not effective on ciliary neurons. VIP promoted the survival of approximately 50% of the ciliary and embryonic day 10 dorsal root ganglionic neurons as compared to saturating amounts of CNTF, but required the presence of non-neuronal cells in the cultures to be effective. Neurotensin (1 x 10(-5) M had a small effect on ciliary neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Excessive ultraviolet B (UVB) irradiation causes apoptotic cell death or induction of tumors in skin. Melatonin is a promising antioxidant and direct radical scavenger. Recently, it was reported that melatonin increases the survival of ultraviolet-B (UVB)-irradiated HaCaT keratinocyte cell lines. However, the precise molecular mechanisms underlying protective effect of melatonin on UVB damage are largely unknown. In this study, to gain more insight into the molecular mechanisms involved in melatonin-induced cell survival on UVB-irradiated HaCaT keratinocytes, we performed cDNA microarray analysis. HaCaT keratinocytes were incubated without or with melatonin at 100 nm for 30 min prior to UVB irradiation at 100 mJ/cm(2), and total RNA was isolated. Our data showed that the expression of apoptosis regulator genes (apoptosis related protein-3, apoptotic chromatin condensation inducer in the nucleus), cancer related genes (tumor suppressor deleted in oral cancer-related 1), cell cycle regulator (cyclin-dependent kinase 2 interacting protein), enzymes (glutathione peroxidase 1, ubiquitin-conjugating enzyme E2M), and signal transducer genes [<em>fibroblast</em> <em>growth</em> <em>factor</em> (acidic) intracellular binding protein, transforming <em>growth</em> <em>factor</em> beta-stimulated protein TSC-<em>22</em>] were decreased by melatonin treatment in the UVB-irradiated HaCaT keratinocyte cell lines, compared to that of UVB-irradiated HaCaT cells without melatonin. Thus, findings of the present study demonstrate that melatonin modulates the expression of apoptosis related genes in UVB-irradiated HaCaT cells, resulting in increasing cell survival, thereby suggesting that melatonin may be used as a promising sunscreen substance to reduce cell death of keratinocytes after excessive UVB irradiation.

Specific interactions between <em>fibroblast</em> <em>growth</em> <em>factors</em> (Fgf1-<em>22</em>) and their tyrosine kinase receptors (FgfR1-4) activate different signalling pathways that are responsible for the biological processes in which Fgf signalling is implicated during embryonic development. In the chick, several Fgf ligands (Fgf2, 4, 8, 9, 10, 12, 13 and 18) and the four FgfRs (FgfR 1, 2, 3 and 4) have been reported to be expressed in the developing limb. The precise spatial and temporal expression of these transcripts is important to guide the limb bud to develop into a wing/leg. In this paper, we present a detailed and systematic analysis of the expression patterns of FgfR1, 2, 3 and 4 throughout chick wing development, by in situ hybridisation on whole mounts and sections. Moreover, we characterize for the first time the different isoforms of FGFR1-3 by analysing their differential expression in limb ectoderm and mesodermal tissues, using RT-PCR and in situ hybridisation on sections. Finally, isoform-specific sequences for FgfR1IIIb, FgfR1IIIc, FgfR3IIIb and FgfR3IIIc were determined and deposited in GenBank with the following accession numbers: GU053725, GU065444, GU053726, GU065445, respectively.

We evaluated the effect of a "tailor-made" chemo-gene therapy in scirrhous gastric cancer (SGC)-bearing nude mice. For this tailor-made approach, we first selected gefitinib (epidermal <em>growth</em> <em>factor</em> receptor-tyrosine kinase inhibitor)-sensitive SGC cell lines, and 5/8 cell lines demonstrated various degrees of gefitinib-sensitivity. In the highly gefitinib-sensitive NUGC-4, the biological response to NK4 (HGF antagonist/angiogenesis inhibitor) was examined. Subsequently, the composition of an NK4-expressing ternary complex (cationic lipid/nucleic acid/HMG-1, 2 protein) was optimized for maximum transfection activity in NUGC-4. Finally, mice were peritoneally coinoculated with NUGC-4 and scirrhous-associated gastric <em>fibroblasts</em>, NF<em>22</em>, on day 0. Animal models were orally administrated gefitinib (50 mg/kg/day, on days 7-28), and peritoneally NK4-expressing ternary complex (on days 14, 21 and 28). NK4-expression suppressed the gefitinib-resistance induced by the interaction between <em>fibroblasts</em> and SGC, and eventually, this tailor-made combination synergistically decelerated the disease progression by inhibiting proliferative, angiogenic and antiapoptotic effects in tumor tissues. On day 28, both the hemoglobin concentration (g/dl) (control (n = 8), 11.9; treated (n = 8), 17.3; p = 0.0014) and the numbers of mice in good condition (control, 2; treated, 8; p = 0.0012) were significantly greater, and the abdominal girth (mm) (control, 81.1; treated, 70.3; p = 0.0036) was significantly reduced. The median points of bloody ascite-free survival time (days) (control, <em>22</em>; treated, 44; p < 0.0001) and time to euthanasia (days) (control, 36.5; treated, 56; p < 0.0001) were also significantly prolonged. This combination is a potentially useful approach to the treatment of peritoneal gefitinib-sensitive SGC dissemination.

TSC-<em>22</em> (transforming <em>growth</em> <em>factor</em>-beta-stimulated clone <em>22</em>) belongs to a family of leucine zipper transcription <em>factors</em> that includes sequences from invertebrates and vertebrates. The single Drosophila family member, encoded by the bunched gene, serves to integrate opposing bone morphogenic protein (BMP) and epidermal <em>growth</em> <em>factor</em> (EGF) signals during oogenesis. Similarly, mammalian TSC-<em>22</em> expression is regulated by several families of secreted signaling molecules in cultured cells. Here, we show that chick TSC-<em>22</em> is dynamically expressed in the condensing feather bud, as well as in many tissues of the chick embryo. BMP-2/4, previously shown to inhibit bud development, repress TSC-<em>22</em> expression during feather bud formation in vivo. Noggin, a BMP antagonist, promotes TSC-<em>22</em> expression. EGF, TGF-alpha, and <em>fibroblast</em> <em>growth</em> <em>factor</em> all promote both feather bud development and TSC-<em>22</em> expression; each can promote ectopic feather buds that are regularly spaced between existing feather buds. Thus, TSC-<em>22</em> is a candidate to integrate small imbalances in receptor tyrosine kinase and BMP signaling during feather tract development to generate stable and reproducible morphogenetic responses.

The urinary concentration of calmodulin and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was determined in a total of 53 patients with various chronic myeloproliferative disorders (CMPD), including <em>22</em> patients with idiopathic myelofibrosis (IMF). Calmodulin excretion was significantly elevated in IMF (0.29 +/- 0.04 microgram/mmol creatinine) (P < 0.001), when compared to polycythaemia vera (PV) (0.14 +/- 0.02), essential thrombocythaemia (ET) (0.13 +/- 0.04), chronic myeloid leukaemia (CML) (0.16 +/- 0.02), unclassified myeloproliferative disorders (UMPD) (0.11 +/- 0.02) and age-matched controls (0.1 +/- 0.02) (P < 0.001). In contrast, bFGF was slightly elevated in all CMPD conditions when compared to age-matched controls. A neutralizing antibody to calmodulin was demonstrated to significantly influence the in vitro proliferation of normal human <em>fibroblasts</em>, an effect dependent on both cell density and the presence of fetal calf serum (FCS). Essentially, the antibody reduced FCS-induced proliferation of low-density <em>fibroblasts</em> but had little or no inhibitory effect on high-density <em>fibroblasts</em> in the absence of FCS. In addition, extracellular calmodulin was shown not to interact with known <em>fibroblast</em> mitogens, namely, IFG-1, EGF, bDGF and PDGF. We conclude that extracellular calmodulin should be considered, in addition to PDGF, TFG-beta and EGF, as a potential mitogen involved in the stromal reaction of idiopathic myelofibrosis.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 is an abundant astroglial cytokine. We have previously shown that FGF-2 downregulates gap junctions in primary astroglial cultures (B. Reuss et al., 1998, Glia <em>22</em>, 19-30). We demonstrate now that FGF-2 induces astroglial dopamine (DA) sensitivity and D1 dopamine-receptor (D1DR) antigen and message in cortical and striatal astroglial cultures. On the functional level 10 micromol/L DA triggered transient increases in astroglial [Ca(2+)](i). In gap-junction-coupled cells, no FGF-2-dependent changes in proportions of DA-responsive cells were observable. However, uncoupling with octanol or 18alpha-glycirrhetinic acid isolated the smaller population of astrocytes intrinsically sensitive to DA which was significantly increased by FGF-2 in cortical and striatal cultures. Administration of DR-specific substances revealed that FGF-2 upregulated D1DR. These results indicate that downregulation of astroglial gap junctions by FGF-2 is accompanied by an upregulation of D1DR and DA sensitivity, adding a new aspect to the role of FGF-2 in the regulation of brain functions.

In 1993 miRNAs were discovered during a research on Caenorhabditis elegans conducted by Victor Ambros and Gary Ruvkun. The gene lin-4 that played important role in development in C. elgans was observed not encoding any protein but a very small RNA molecule of just <em>22</em> nucleotides. Main objective of this review is to highlight the significance of miRNAs in regulating the expression of many genes, which are either directly or indirectly involved in many diseases. One of the major causes of illness and death in developed countries of the world is cardiovascular disease. Some of the miRNAs have certain role to play in heart that are not specified for heart. So miRNAs have been found to be in other tissues like <em>fibroblasts</em>, endothelial cells and smooth muscle cells that are part of physiological study of cardiovascular system. Adult heart has limited capacity of regeneration therefore lost cardiomyocytes due to myocardial ischemia or infarction can result in low performance of heart. miRNAs have been shown to play a role in apoptotic regulation of cardiomyocytes in vivo. Many studies have shown that miR146a and 155 are up regulated in peripheral blood mononuclear cells, synovial <em>fibroblasts</em>, synovial fluid and Th-17 cells from rheumatoid arthritis patients as compared to healthy persons. Several types of miRNAs are playing important roles in type 1 diabetes mellitus including miR-375 and miR-375 with intolerance to glucose and decreased beta cells account due to impaired proliferation. Up regulation of miR-125a in WAT of type 2 Diabetes mellitus have been observed. miRNAs have proved to be the important regulators of cytokines and <em>growth</em> <em>factor</em> expression. Thus, suggested as a good biomarker and target of therapy. miRNA profiling techniques have revealed the role of miRNAs in Multiple sclerosis.

The causes of the increased cardiovascular risk associated with kidney diseases partly reside in the chronic kidney disease-mineral bone disorder (CKD-MBD) syndrome. Three cardiovascular risk <em>factors</em> [hyperphosphatemia, vascular calcification, and elevated <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23)] levels have been discovered within the CKD-MBD over the last decades. In addition, sclerostin is recently presented as a new bone and vascular disease biomarker. This <em>22</em>-kDa glycoprotein, secreted mainly by osteocytes, is a soluble inhibitor of the canonical Wnt pathway that has a pivotal role in bone biology and turnover. CKD patients are reported with higher levels of sclerostin, and levels decrease during dialysis. Sclerostin is associated with vascular calcification and CV risk in CKD, although data are still controversial. The question whether serum sclerostin has protective or deleterious role in CKD-MBD pathophysiology, and therefore in cardiovascular risk and overall mortality, is still open and needs to be answered. The standardization of assays and the establishment of a clear cut-off values when sclerostin starts to switch from physiological to pathophysiological role have to be another important step. Further research is needed also to define its relationship with other CKD-MBD biomarkers for future diagnostic and therapeutic strategies.

In the present study, we investigated the potential anti-angiogenic mechanism and anti-tumour activity of beta-eudesmol using in vitro and in vivo experimental models. Proliferation of human umbilical vein endothelial cells (HUVEC) stimulated with vascular endothelial <em>growth</em> <em>factor</em> (VEGF, 30 ng/ml) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF, 30 ng/ml) was significantly inhibited by beta-eudesmol (50-100 microM). Beta-eudesmol (100 microM) also blocked the phosphorylation of cAMP response element binding protein (CREB) induced by VEGF (30 ng/ml) in HUVEC. Beta-eudesmol (10-100 microM) inhibited proliferation of HeLa, SGC-7901, and BEL-7402 tumour cells in a time- and dose-dependent manner. Moreover, beta-eudesmol treatment (2.5-5 mg/kg) significantly inhibited <em>growth</em> of H(<em>22</em>) and S(180) mouse tumour in vivo. These results indicated that beta-eudesmol inhibited angiogenesis by suppressing CREB activation in <em>growth</em> <em>factor</em> signalling pathway. This is the first study to demonstrate that beta-eudesmol is an inhibitor of tumour <em>growth</em>.