We have purified acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> (c-aFGF, c-bFGF) from 11 day-old chick embryo brain, retina and vitreous by heparin-Sepharose chromatography and reverse phase HPLC. The analysis of their biological activity as well as their molecular weight indicates that they were analogous to basic or acidic human and bovine FGF. The ratio of c-aFGF to c-bFGF activity depended of the tissue. In brain c-aFGF represented 66% of the total mitogenic activity retained on the heparin-sepharose column and c-bFGF 34% while retina contained 16% of c-aFGF and 84% of c-bFGF; vitreous 78% of c-aFGF and <em>22</em>% of c-bFGF. Like human aFGF, Heparin stimulated purified c-aFGF mitogenic activity in the absence of serum but inhibited the activity of the retina acid soluble extract, in the presence of foetal calf serum (FCS). Thus, chick embryo and adult human acidic and basic FGF respectively share the same biochemical properties. Since there are no blood vessels in chick retina or vitreous, their presence in these tissues suggests that angiogenesis is not the only role of these <em>growth</em> <em>factors</em>.

Rat-1 <em>fibroblasts</em> were used to study the role of the sustained activation of extracellular signal-regulated kinase 1 (ERK1) in lysophosphatidic acid (LPA)-stimulated mitogenic signalling. Mitogenic doses of LPA, like serum, stimulated biphasic, sustained, ERK activation that persisted towards the G1/S boundary. The EC50 for LPA-stimulated ERK activation after 10 min, the time of peak response, was 2 orders of magnitude to the left of that for the sustained response after 3 h or that for DNA synthesis after <em>22</em> h, with the result that non-mitogenic doses stimulated a maximal peak response but no second phase. To complement these studies, we examined the role of different signal pathways in regulating the sustained and acute phases of ERK activation using defined biochemical inhibitors and mimetics. Activation of protein kinase C and Ca2+ fluxes played a minor and transient role in regulation of ERK1 activity by LPA in Rat-1 cells. Sustained ERK1 activation stimulated by LPA was completely inhibited by pertussis toxin, whereas the early peak response was only partly affected; this is correlated with the specific inhibition of LPA-stimulated DNA synthesis by pertussis toxin. The selective tyrosine kinase inhibitor herbimycin A completely inhibited sustained ERK1 activation by LPA but, again, the early phase of the response was only partially inhibited. In addition, low doses of staurosporine inhibited ERK1 activation by LPA. The effects of herbimycin A and staurosporine were selective for the response to LPA but did not affect that to epidermal <em>growth</em> <em>factor</em>. The results suggest a strong correlation between sustained ERK1 activation and DNA synthesis in LPA-stimulated Rat-1 cells. Furthermore, the two discrete phases of ERK activation by LPA are regulated by a combination of at least two different signalling pathways; the sustained activation of ERK1 in Rat-1 cells proceeds via a G1- or Gzero-mediated pathway which may also involve a tyrosine kinase.

The E5 protein of bovine papillomavirus is a 44-amino acid, Golgi-resident, type II transmembrane protein that efficiently transforms immortalized mouse <em>fibroblasts</em>. The transmembrane (TM) domain of E5 is not only critical for biological activity, it also regulates interactions with cellular targets including the platelet derived <em>growth</em> <em>factor</em> receptor (PDGF-R) and the 16-kDa subunit of the vacuolar proton ATPase (V-ATPase). In order to define the specific TM amino acids essential for E5 biological and biochemical activity, we performed scanning alanine mutagenesis on 25 of the 30 potential TM residues and genetically mapped discrete alpha-helical domains which separately regulated the ability of E5 to bind PDGF-R, activate PDGF-R, and to form oligomers. Alanine substitutions at positions 17, 21, and 24 (which lie on the same helical face) greatly inhibited E5 association with the PDGF-R, suggesting that this region comprises the receptor binding site. PDGF-R activation also mapped to a specific but broader domain in E5; mutant proteins with alanines on one helical face (positions 8, 9, 11, 16, 19, <em>22</em>, and 23) continued to induce PDGF-R tyrosine phosphorylation, whereas mutant proteins with alanines on the opposite helical face (positions 7, 10, 13, 17, 18, 21, 24, and 25) did not, indicating that the latter helical face was critical for mediating receptor transphosphorylation. Interestingly, these "activation-defective" mutants segregated into two classes: 1) those that were unable to form dimers but that could still form higher order oligomers and transform cells, and 2) those that were defective for PDGF-R binding and were transformation-incompetent. These findings suggest that the ability of E5 to dimerize and to bind PDGF-R is important for receptor activation. However, since several transformation-competent E5 mutants were defective for binding and/or activating PDGF-R, it is apparent that E5 must have additional activities to mediate cell transformation. Finally, alanine substitutions also defined two separate helical faces critical for E5/E5 interactions (homodimer formation). Thus, our data identify distinct E5 helical faces that regulate homologous and heterologous intramembrane interactions and define two new classes of biologically active TM mutants.

Initiation of translation from alternate codons in the same mRNA results in multiple forms of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). High molecular weight species of bFGF make use of leucine translation initiation sites located upstream of the methionine residue used to produce the 18 kiloDalton (kDa) form. Although the addition of exogenous 18 kDa bFGF is known to stimulate DNA synthesis and proliferation of several cell types including embryonic chicken cardiac myocytes, little is known about the role of high molecular weight forms of bFGF. We modified the rat bFGF cDNA to yield high (<em>22</em>/21.5 kDa) or low (18 kDa) molecular weight species of bFGF. Expression of <em>22</em>/21.5 kDa or 18 kDa bFGF in transfected embryonic chicken ventricular myocyte cultures was confirmed by protein blotting. Expression of both high and low molecular weight species of bFGF was associated with (i) a three-fold increase in overall thymidine incorporation as well as cardiomyocyte labelling index (fraction of cardiomyocyte nuclei incorporating tritiated thymidine); (ii) a two- to three-fold increase in cell number; (iii) an eight-fold increase in protein synthesis; and (iv) a three-fold decrease in myosin accumulation. Subcellular localization of bFGF in the transfected myocyte cultures was also assessed by immunofluorescence microscopy. Over-expression of cDNAs yielding high molecular weight bFGF resulted in predominantly nuclear bFGF staining. By contrast, both cytoplasmic and nuclear staining were observed following over-expression of 18 kDa bFGF. Over-expression of <em>22</em>/21.5 kDa bFGF was associated with the formation of multiple DNA-containing "clumps" resembling condensed chromatin in cardiac myocyte nuclei. These DNA "clumps" were not observed in cardiac myocyte cultures over-expressing 18 kDa bFGF. These data indicate that over-expression of high as well as low molecular weight forms of bFGF can stimulate cardiac myocyte proliferative potential and decrease myosin accumulation. However, these forms possess distinct subcellular localizations and can have different biological functions in the nucleus.

BACKGROUND

Hepatocellular carcinoma (HCC) is a preventable disease rather than a curable one, since there is no well-documented effective treatment modality until now, making the molecular study of this disease mandatory.

RESULTS

We studied gene expression profile of 17 Egyptian HCC patients associated with HCV genotype-4 infection by c-DNA microarray. Out of the 15,660 studied genes, 446 were differentially expressed; 180 of them were up regulated and 134 were down regulated. Seventeen genes out of the 180 up-regulated genes are involved in 28 different pathways. Protein phosphatase 3 (PPP3R1) is involved in 10 different pathways followed by <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1), Cas-Br-M ecotropic retroviral transforming sequence b (CBLB), spleen tyrosine kinase (SYK) involved in three pathways; bone morphogenetic protein 8a (BMP8A), laminin alpha 3 (LAMA3), cell division cycle 23 (CDC23) involved in 2 pathways and NOTCH4 which regulate Notch signaling pathway. On the other hand, 25 out of the 134 down-regulated genes are involved in 20 different pathways. Integrin alpha V alpha polypeptide antigen CD51 (ITGVA) is involved in 4 pathways followed by lymphotoxin alpha (TNF superfamily, member 1) (LTA) involved in 3 pathways and alpha-2-macroglobulin (A2M), phosphorylase kinase alpha 2-liver (PHKA2) and MAGI1 membrane associated guanylate kinase 1 (MAGI1) involved in 2 pathways. In addition, <em>22</em> genes showed significantly differential expression between HCC cases with cirrhosis and without cirrhosis. Confirmation analysis was performed on subsets of these genes by RT-PCR, including some up-regulated genes such as CDK4, Bax, NOTCH4 and some down-regulated genes such as ISGF3G, TNF, and VISA.

CONCLUSIONS

This is the first preliminary study on gene expression profile in Egyptian HCC patients associated with HCV-Genotype-4 using the cDNA microarray. The identified genes could provide a new gate for prognostic and diagnostic markers for HCC associated with HCV. They could also be used to identify candidate genes for molecular target therapy.

HIV-1-infected patients develop a generalized vasculopathy that is clinically most evident as Kaposi's sarcoma (KS), a multifocally appearing endothelial cell-derived tumor. <em>Fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2) is a potent autocrine and paracrine mitogen of endothelial cells and has been implicated in the cell proliferation and angiogenesis observed in KS. Here we determined by ELISA the FGF-2 serum concentrations in different clinical groups of HIV-1-infected patients. AIDS-KS patients (n = 53) and HIV-1-infected patients without KS (n = 39) revealed significantly increased FGF-2 serum concentrations (median, 4.5 and 4.6 pg/ml, respectively), as compared with the healthy control group (n = <em>22</em>; median, 2.2 pg/ml; p < 0.01). FGF-2 concentrations were highest in untreated HIV-1-infected patients (median, 8.6 pg/ml) and were significantly decreased in patients undergoing antiretroviral therapy (AZT-median, 4.5 pg/ml; HAART-median, 2.5 pg/ml; p < 0.01). In addition, FGF-2 serum concentrations above 5.2 pg/ml were associated with a statistically significant higher risk of death in HIV-1-infected patients. Multivariate analysis showed that this effect is independent of CD4 levels, localization of KS (cutaneous or visceral), AIDS-defining opportunistic diseases, and therapy. Circulating FGF-2 may contribute to AIDS-associated vasculopathy and may be a sensitive and easily accessible surrogate marker to determine the survival time of HIV-1-infected patients and the efficacy of antiretroviral therapy.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) is a key regulator in glucose and lipid metabolism and its plasma levels have been shown to be increased not only in humans in different situations such as type 2 diabetes, obesity, and nonalcoholic fatty liver disease but also in animal models of sepsis and pancreatitis. FGF21 is considered as a pharmacological candidate in conditions associated with insulin resistance. The aim of this study was to compare FGF21 plasma levels in patients with sepsis, in patients with systemic inflammatory response syndrome (SIRS), and in healthy controls. We measured FGF21 plasma concentrations in <em>22</em> patients with established sepsis, in 11 with SIRS, and in 12 healthy volunteers. Here, we show that FGF21 levels were significantly higher in plasma obtained from patients with sepsis and SIRS in comparison with healthy controls. Also, FGF21 levels were significantly higher in patients with sepsis than in those with noninfectious SIRS. FGF21 plasma levels measured at study entry correlated positively with the APACHE II score, but not with procalcitonin levels, nor with C-reactive protein, classical markers of sepsis. Plasma concentrations of FGF21 peaked near the onset of shock and rapidly decreased with clinical improvement. Taken together, these results indicate that circulating levels of FGF21 are increased in patients presenting with sepsis and SIRS, and suggest a role for FGF21 in inflammation. Further studies are needed to explore the potential role of FGF21 in sepsis as a potential therapeutic target.

OBJECTIVE

Newborns with congenital diaphragmatic hernia (CDH) still have a high mortality rate, which has been attributed to pulmonary hypoplasia and pulmonary hypertension. Fibroblast growth factors (FGFs) are essential components of the gene network that regulates lung development. Recent studies suggest that the new member of FGF family, FGF-10, plays a fundamental role in branching morphogenesis and is essential for lung formation. FGF-10-deficient mice exhibit complete absence of lungs. FGF-7 promotes epithelial proliferation and expansion leading to the formation of cystlike structures. The aim of this study was to determine the gene level expression of FGF-10 and FGF-7 in the lung of nitrofen-induced CDH.

METHODS

Congenital diaphragmatic hernia (CDH) was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, 22 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 into 3 groups: normal controls (n = 16), nitrofen induced without CDH (n = 16), and nitrofen-induced CDH (n = 16). Total RNA and DNA were extracted from the lung in each group and measured. mRNA was extracted from total RNA. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate mRNA expressions of FGF-10 and FGF-7. Levels of mRNA were expressed as a ratio of the band density divided by that of beta-actin, a house-keeping gene.

RESULTS

FGF-10 mRNA expression was decreased significantly in CDH lung (2.914 +/- 0.320) compared with controls (4.062 +/- 0.307; P <.05) and nitrofen induced without CDH lung (3.923 +/- 0.250; P <.01). FGF-7 mRNA expression was decreased significantly in CDH lung (0.777 +/- 0.097) compared with controls (1.028 +/- 0.093; P <.01).

CONCLUSIONS

Decreased gene expression of FGF-10 and FGF-7 in the hypoplastic lung suggests that pulmonary hypoplasia in nitrofen-induced CDH rat may be caused by reduced synthesis of FGF-10 and FGF-7 during lung morphogenesis.

The aims of this study were to investigate the expression of transforming <em>growth</em> <em>factor</em>-β1 (TGF-β1) and α-smooth muscle actin (α-SMA) in surgical resection specimens from nonsmall cell lung cancer (NSCLC) and to evaluate the prognostic significance of this gene expression in stromal <em>fibroblasts</em> for patients with clinical stage I-IIIA NSCLC. The immunohistochemical expression of TGF-β1 and α-SMA was evaluated in 78 formalin-fixed paraffin-embedded tumor specimens from clinical stage I-IIIA NSCLC. Correlations between this gene expression and the clinicopathologic characteristics were determined by chi-square test. The prognostic impact of this gene expression in stromal <em>fibroblasts</em> with regard to overall survival (OS) was determined by Kaplan-Meier and Cox hazard proportional model. The percentages of high TGF-β1 expression in stromal <em>fibroblasts</em> and cancer cells were 19.2 % (15/78) and 35.9 % (28/78), respectively. There were 28.2 % (<em>22</em>/78) of patients with high α-SMA expression in stromal <em>fibroblasts</em>. The analysis revealed a significant positive association between TGF-β1 expression in stromal <em>fibroblasts</em> and in cancer cells (χ (2) = 4.86, p = 0.03). No significant association was found between TGF-β1 in cancer cells and α-SMA expression in stromal <em>fibroblasts</em> (χ (2) = 0.978, p = 0.326). The 3-year OS rates with low and high TGF-β1 expression in stromal <em>fibroblasts</em> were 52.4 and 26.7 %, respectively (χ (2) = 5.42, p = 0.019). The 3-year OS rates with low and high α-SMA expression in stromal <em>fibroblasts</em> were 53.9 and 31.0 %, respectively (χ (2) =5.01, p=0.025). The multivariate analysis revealed that clinical stage and TGF-β1 and α-SMA expression levels in stromal <em>fibroblasts</em> were identified as independent predictive <em>factors</em> of OS. The results suggest that the expression level of TGF-β1 and α-SMA in stromal <em>fibroblasts</em> may have prognostic significance in patients with clinical stage I-IIIA NSCLC after curative resection.

The monogenetic disease Spinal Muscular Atrophy (SMA) is characterized by a progressive loss of motoneurons leading to muscle weakness and atrophy due to severe reduction of the Survival of Motoneuron (SMN) protein. Several models of SMA show deficits in neurite outgrowth and maintenance of neuromuscular junction (NMJ) structure. Survival of motoneurons, axonal outgrowth and formation of NMJ is controlled by neurotrophic <em>factors</em> such as the <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> (FGF) system. Besides their classical role as extracellular ligands, some FGFs exert also intracellular functions controlling neuronal differentiation. We have previously shown that intracellular FGF-2 binds to SMN and regulates the number of a subtype of nuclear bodies which are reduced in SMA patients. In the light of these findings, we systematically analyzed the FGF-system comprising five canonical receptors and <em>22</em> ligands in a severe mouse model of SMA. In this study, we demonstrate widespread alterations of the FGF-system in both muscle and spinal cord. Importantly, FGF-receptor 1 is upregulated in spinal cord at a pre-symptomatic stage as well as in a mouse motoneuron-like cell-line NSC34 based model of SMA. Consistent with that, phosphorylations of FGFR-downstream targets Akt and ERK are increased. Moreover, ERK hyper-phosphorylation is functionally linked to FGFR-1 as revealed by receptor inhibition experiments. Our study shows that the FGF system is dysregulated at an early stage in SMA and may contribute to the SMA pathogenesis.

Bone is a metabolically active organ that undergoes continuous remodeling throughout life. However, many complex skeletal defects such as large traumatic bone defects or extensive bone loss after tumor resection may cause failure of bone healing. Effective therapies for these conditions typically employ combinations of cells, scaffolds, and bioactive <em>factors</em>. In this review, we pay attention to one of the three <em>factors</em> required for regeneration of bone, bioactive <em>factors</em>, especially the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family. This family is composed of <em>22</em> members and associated with various biological functions including skeletal formation. Based on the phenotypes of genetically modified mice and spatio-temporal expression levels during bone fracture healing, FGF2, FGF9, and FGF18 are regarded as possible candidates useful for bone regeneration. The role of these candidate FGFs in bone regeneration is also discussed in this review.

Neurons in the brain must establish a balanced network of excitatory and inhibitory synapses during development for the brain to function properly. An imbalance between these synapses underlies various neurological and psychiatric disorders. The formation of excitatory and inhibitory synapses requires precise molecular control. In the hippocampus, the structure crucial for learning and memory, <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>22</em> (FGF<em>22</em>) and FGF7 specifically promote excitatory or inhibitory synapse formation, respectively. Knockout of either Fgf gene leads to excitatory-inhibitory imbalance in the mouse hippocampus and manifests in an altered susceptibility to epileptic seizures, underscoring the importance of FGF-dependent synapse formation. However, the receptors and signaling mechanisms by which FGF<em>22</em> and FGF7 induce excitatory and inhibitory synapse differentiation are unknown. Here, we show that distinct sets of overlapping FGF receptors (FGFRs), FGFR2b and FGFR1b, mediate excitatory or inhibitory presynaptic differentiation in response to FGF<em>22</em> and FGF7. Excitatory presynaptic differentiation is impaired in Fgfr2b and Fgfr1b mutant mice; however, inhibitory presynaptic defects are only found in Fgfr2b mutants. FGFR2b and FGFR1b are required for an excitatory presynaptic response to FGF<em>22</em>, whereas only FGFR2b is required for an inhibitory presynaptic response to FGF7. We further find that FGFRs are required in the presynaptic neuron to respond to FGF<em>22</em>, and that FRS2 and PI3K, but not PLCγ, mediate FGF<em>22</em>-dependent presynaptic differentiation. Our results reveal the specific receptors and signaling pathways that mediate FGF-dependent presynaptic differentiation, and thereby provide a mechanistic understanding of precise excitatory and inhibitory synapse formation in the mammalian brain.

OBJECTIVE

The initiation, progression, and maintenance of pancreatic ductal adenocarcinoma (PDAC) results from the interplay of genetic and epigenetic events. While the genetic alterations of PDAC have been well characterized, epigenetic pathways regulating PDAC remain, for the most part, elusive. The goal of this study was to identify novel epigenetic regulators contributing to the biology of PDAC.

METHODS

In vivo pooled shRNA screens targeting 118 epigenetic proteins were performed in two orthotopic PDAC xenograft models. Candidate genes were characterized in 19 human PDAC cell lines, heterotopic xenograft tumor models, and a genetically engineered mouse (GEM) model of PDAC. Gene expression, IHC, and immunoprecipitation experiments were performed to analyze the pathways by which candidate genes contribute to PDAC.

RESULTS

In vivo shRNA screens identified BRD2 and BRD3, members of the BET family of chromatin adaptors, as key regulators of PDAC tumor growth. Pharmacologic inhibition of BET bromodomains enhanced survival in a PDAC GEM model and inhibited growth of human-derived xenograft tumors. BET proteins contribute to PDAC cell growth through direct interaction with members of the GLI family of transcription factors and modulating their activity. Within cancer cells, BET bromodomain inhibition results in downregulation of SHH, a key mediator of the tumor microenvironment and canonical activator of GLI. Consistent with this, inhibition of BET bromodomains decreases cancer-associated fibroblast content of tumors in both GEM and xenograft tumor models.

CONCLUSIONS

Therapeutic inhibition of BET proteins offers a novel mechanism to target both the neoplastic and stromal components of PDAC. Clin Cancer Res; 22(16); 4259-70. ©2016 AACR.

BACKGROUND

Leptin, apart from the regulation of food intake, has been implicated in hematopoiesis, the immune response and angiogenesis. Leptin has been found to be decreased in various hematological malignancies. In the present study leptin was measured in multiple myeloma (MM) patients before and after treatment and correlated with other angiogenic molecules and markers of disease activity.

METHODS

Serum leptin, vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (b-FGF), interleukin-1 beta (IL-1beta), beta 2 microglobulin (beta2M) and C-reactive protein (CRP) were measured in 62 newly diagnosed MM patients, <em>22</em> of whom obtaining disease stabilization after treatment. The same parameters were measured in 20 healthy controls. Disease stage was defined according to the Durie-Salmon criteria.

RESULTS

Leptin, VEGF, b-FGF, IL-1beta, and beta2M were significantly higher in newly diagnosed MM patients than in controls (p<0.05). VEGF, b-FGF, IL-1beta, beta2M, CRP but not leptin increased with advancing stage of disease (p<0.01). All parameters decreased significantly following treatment (p<0.001). Although IL-1beta correlated positively with VEGF, beta2M, b-FGF and CRP, leptin did not correlate with any of the measured parameters.

CONCLUSIONS

Leptin serum levels do not reflect disease severity in MM. However, there seems to be a decrease in leptin following treatment, which may be associated with an alteration in the metabolic state or the chemokine milieu.

The present study evaluated the effect of epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, on irradiation-induced pulmonary fibrosis and elucidated its mechanism of action. A rat model of irradiation-induced pulmonary fibrosis was generated using a (60)Co irradiator and a dose of <em>22</em> Gy. Rats were intraperitoneally injected with EGCG (25 mg/kg) or dexamethasone (DEX; 5 mg/kg) daily for 30 days. Mortality rates and lung index values were calculated. The severity of fibrosis was evaluated by assaying the hydroxyproline (Hyp) contents of pulmonary and lung tissue sections post-irradiation. Alveolitis and fibrosis scores were obtained from semi-quantitative analyses of hematoxylin and eosin (H&E) and Masson's trichrome lung section staining, respectively. The serum levels of transforming <em>growth</em> <em>factor</em> β1 (TGF-β1), interleukin (IL)-6, IL-10, and tumor necrosis <em>factor</em>-α (TNF-α) were also measured. Surfactant protein-B (SPB) and α-SMA expression patterns were evaluated using immunohistochemistry, and the protein levels of nuclear transcription <em>factor</em> NF-E2-related <em>factor</em> 2 (Nrf-2) and its associated antioxidant enzymes heme oxygenase-1 enzyme (HO-1) and

UNASSIGNED

quinone oxidoreductase-1 (NQO-1) were examined via western blot analysis. Treatment with EGCG, but not DEX, reduced mortality rates and lung index scores, improved histological changes in the lung, reduced collagen depositions, reduced MDA content, enhanced SOD activity, inhibited (myo)fibroblast proliferation, protected alveolar epithelial type II (AE2) cells, and regulated serum levels of TGF-β1, IL-6, IL-10, and TNF-α. Treatment with EGCG, but not DEX, activated Nrf-2 and its downstream antioxidant enzymes HO-1 and NQO-1. Taken together, these results showed that EGCG treatment significantly inhibits irradiation-induced pulmonary fibrosis. Furthermore, the results suggested promising clinical EGCG therapies to treat this disorder.

Immunohistochemical studies were performed using specific polyclonal antibodies to transforming <em>growth</em> <em>factor</em> (TGF)-beta 1 and TGF-beta 2 to determine their presence and cellular localization in human ovarian tissues of various reproductive states. In the small ovarian follicles, the immunostaining for TGF-beta 1 was present in oocytes, follicle cells, and granulosa and theca cell layers. The level of immunostaining associated with granulosa and theca cell layers intensified as the size of the follicles increased. In the luteal tissue, both the small and large luteal cells immunostained for TGF-beta 1 and their intensities were similar to theca and granulosa cell layers, respectively. The patterns of immunostaining were similar in early (days 14-19), mid (days <em>22</em>-25), and late (days 26-29) luteal phases; however, the intensity was highest at mid and decreased at late luteal phase. Corpus albicans showed a very weak immunostaining for TGF-beta 1, whereas ectopic pregnancy small luteal cells immunostained relatively intensely. The ovarian stromal, luteal tissue <em>fibroblasts</em>, and arterioles endothelial and smooth muscle cells were also immunostained for TGF-beta 1. The immunostaining of the ovarian tissues for TGF-beta 2 indicated that the theca cell layers were the exclusive cells in the follicles with intense immunostaining, which increased in the larger follicles. A low immunostaining was also observed in granulosa cell of the large follicles. In the luteal tissues, only small luteal cells showed intense immunostaining for TGF-beta 2, which was similar in intensity to that in the theca cells; however, the large luteal cells showed a low level of immunostaining at midluteal phase. The small luteal cells in corpus albicans and ectopic pregnancy luteal tissues retained their immunostaining for TGF-beta 2, but with lower intensity. Endothelial and smooth muscle cells of arterioles also immunostained for TGF-beta 2, but not ovarian stromal cells. Atretic follicles showed very low or no detectable immunostaining for TGF-beta 1 or TGF-beta 2. The results of present studies show that human ovarian tissue at all the reproductive states locally produces TGF-beta 1 and TGF-beta 2, and although TGF-beta 1 is present in most major ovarian cell types, TGF-beta 2 is only produced by theca cells in the follicles and small luteal cells in luteal tissues.

OBJECTIVE

To assess fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor-B (VEGF-B) effects on flow reserve and morphological adaptation in the rabbit ischemic hind limb.

METHODS

Following bilateral femoral artery ligation, calf blood pressure (C(BP)), flow reserve, collateral artery numbers and capillary numbers were assessed. Treatment consisted of rabbit serum albumin (RSA), FGF-2, VEGF-B or FGF-2 + VEGF-B.

RESULTS

Ligation decreased C(BP); on day 14, a 48% deficit remained in the RSA group compared with a deficit of only 22% in FGF-2 and VEGF-B groups. On day 3, flow reserve was attenuated 60%, but recovered by day 14 (with no treatment effects). Collateral artery numbers increased with RSA (+28%), FGF-2 (+53%), VEGF-B (+47%) and FGF-2 + VEGF-B (+59%). Rectus femoris muscle total capillary profiles and fibers per cross-section were alike across groups. Tibialis anterior muscle cross-sectional area was lower with ligation and total capillary number was less in RSA and FGF-2 groups, providing evidence for angiogenesis with VEGF-B. Capillary/muscle fiber ratio was similar in each group.

CONCLUSIONS

FGF-2 and VEGF-B enhanced lower limb perfusion as indicated by improved C(BP) and combined treatment increased collateral artery number. Flow reserve recovery was not enhanced by cytokine treatment. VEGF-B, but not FGF-2, caused angiogenesis in the tibialis anterior muscle. Overall, VEGF-B may have advantages over FGF-2 in this setting; however, their combination may further improve arteriogenesis.

Neuronatin was recently cloned from neonatal rat brain (Biochem, Biophys. Res. Commun., 201 (1994) 1<em>22</em>7-1234). In subsequent studies, we noted neuronatin mRNA was brain-specific and that there were two alternatively spliced forms, alpha and beta (Brain Res., 690 (1995) 92-98). Furthermore, on sequencing the human neuronatin gene, it was determined that the alpha-form was encoded by three exons, and the beta-form was encoded by the first and third exons only (Genomics, 33 (1996) 292-297). The middle exon was spliced out in the beta-form. The human neuronatin gene is located in single copy of chromosome 20q 11.2-12 (Brain Res., 723 (1996) 8-<em>22</em>). These studies called for an understanding of the function of this gene. Therefore, we studied the expression of neuronatin in PC12 cells, an established model of neuronal <em>growth</em> and differentiation. Neuronatin mRNA expression was found to be abundant in undifferentiated PC12 cells. Treatment with nerve <em>growth</em> <em>factor</em> (NGF), resulting in neuronal differentiation, was associated with a downregulation of neuronatin mRNA expression. Removal of NGF was associated with a return of neuronatin mRNA levels towards baseline. These effects appear to be specific for NGF as they were not seen with transforming <em>growth</em> <em>factor</em>, epidermal <em>growth</em> <em>factor</em>, 12-O-tetradecanoylphorbol-13-acetate or dexamethasone. Although, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> also reduced neuronatin mRNA levels, the effect was less pronounced than with NGF. The NGF-induced decreased in neuronatin mRNA occurred even in the presence of protein and RNA syntheses inhibitors. Of the two spliced forms, only the alpha-form was expressed in PC12 cells. In conclusion, we report the presence of neuronatin mRNA in PC12 cells, and that NGF downregulates its expressions. These findings provide a basis for investigating the role of neuronatin in neuronal <em>growth</em> and differentiation.

OBJECTIVE

Vascular endothelial growth factor and basic fibroblast growth factor are potent stimulators of angiogenesis. Children with cyanotic congenital heart disease often experience the development of widespread formation of collateral blood vessels, which may represent a form of abnormal angiogenesis. We undertook the present study to determine whether children with cyanotic congenital heart disease have elevated serum levels of vascular endothelial growth factor and basic fibroblast growth factor.

METHODS

Serum was obtained from 22 children with cyanotic congenital heart disease and 19 children with acyanotic heart disease during cardiac catheterization. Samples were taken from the superior vena cava, inferior vena cava, and a systemic artery. Vascular endothelial growth factor and basic fibroblast growth factor levels were measured in the serum from each of these sites by enzyme-linked immunosorbent assay.

RESULTS

Vascular endothelial growth factor was significantly elevated in the superior vena cava (P =.04) and systemic artery (P =.02) but not in the inferior vena cava (P =.2) of children with cyanotic congenital heart disease compared to children with acyanotic heart disease. The mean vascular endothelial growth factor level, determined by averaging the means of all 3 sites, was also significantly elevated (P =.03). Basic fibroblast growth factor was only significantly elevated in the systemic artery (P =.02).

CONCLUSIONS

Children with cyanotic congenital heart disease have elevated systemic levels of vascular endothelial growth factor. These findings suggest that the widespread formation of collateral vessels in these children may be mediated by vascular endothelial growth factor.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family consists of <em>22</em> widely expressed regulatory polypeptides and controls a broad spectrum of cellular processes. Accumulating data show that FGF9 plays important roles both in embryogenesis and in adult tissue homeostasis. Ablation of Fgf9 alleles leads to lethality at the neonatal stage mainly due to malformations of the lung, as well as causing male-to-female sex reversal. To circumvent the neonatal lethality resulting from disruption of the Fgf9 gene, which hinders further characterization of the role of FGF9 in adult tissue function and homeostasis, we generated an Fgf9 conditional null allele for spatiotemporal- and tissue-specific disruption of Fgf9. Using gene targeting in mouse embryonic stem (ES) cells, we introduced two loxP sites flanking exon 1 in the Fgf9 allele, which encodes 93 amino acid residues at the N-terminal of FGF9. Our results indicate that the Fgf9 conditional null allele is a true conditional null that encodes wildtype activity and reverts to a null allele after recombination mediated by the Cre recombinase.

A serum-free defined medium has been formulated that supports proliferation and morphologic differentiation of U-251 MGsp human and C6-2BD rat glioma cells. This defined medium consists of a basal medium supplemented with transferrin, <em>fibroblast</em> <em>growth</em> <em>factor</em>, hydrocortisone, selenium, biotin, and fibronectin (G2 medium). When U-251 cells were plated in G2 medium on poly-D-lysine precoated dishes, their <em>growth</em> rate was 77% and final cell density was 82% of serum-grown counterparts. The <em>growth</em> rate of C6 cells in G2 medium was 67% compared to cells cultured in serum supplemented medium. Although G2 medium supported the <em>growth</em> of human and rat glioma cells, LA-N-1 human neuroblastoma and WI-38 human <em>fibroblast</em> cells showed no increase in cell number when grown in G2 medium compared to basal medium. A similar formulation (G3 medium), lacking <em>fibroblast</em> <em>growth</em> <em>factor</em> and hydrocortisone, supported the proliferation of RN-<em>22</em> rat schwannoma cells. Morphologic differences were observed between cells grown in the presence of serum and in defined media. All three glial cell lines changed from a flattened shape in serum supplemented medium to a more spherical appearance in defined medium. In addition, both U-251 and C6 cells developed numerous processes, some reaching several cell diameters in length. These defined media will facilitate studies of the <em>growth</em> and differentiation of glial-derived cells.

Quiescent cultures of chick embryo <em>fibroblasts</em> incubated with human alpha-thrombin (14-219 pM) incorporated [methyl-3H]thymidine proportional to concentration. Inactivated forms of this protease (e.g. active-site-conjugated alpha-thrombin or its hirudin complex) had no mitogenic activity and did not compete with 124I-alpha-thrombin for binding to specific plasma membrane receptors. The noncoagulant but esterolytic active forms, gamma- and nitro-alpha-thrombins, were weakly mitogenic and correspondingly competed weakly for binding. Trypsin competed equally as well as native thrombin for binding, whereas chymotrypsin, elastase, and human urokinase competed with 80-fold less affinity. Plasma, arginine-specific proteases associated with nerve or epidermal <em>growth</em> <em>factors</em>, insulin, and insulin-like <em>growth</em> <em>factors</em> did not compete for binding. These data demonstrate that (a) functional catalytic residues of the thrombin active site are necessary for mitogenic activity and for specific binding; (b) regions adjacent to the active site, i.e. the high affinity protein recognition site, appear to enhance binding; and (c) the receptor can discriminate between other proteases and binds those which are also mitogens for the avian cells. The characteristics of 125I-alpha-thrombin binding were determined, and it was found to be (i) proportional to cell number; (ii) optimal at pH 6.8; (iii) 70-90% specific; (iv) at equilibrium after 60 min of incubation at <em>22</em>-24 degrees C or 180 min at 0-4 degrees C (the rate constants for association, i.e. ka, at <em>22</em> and 4 degrees C were 18 and 1.1 x 10(7) M-1 min-1, respectively); and (v) essentially nondissociable. Nondissociable thrombin that bound during incubation at 0-4 degrees C was distributed equally between trypsin-sensitive and insensitive compartments. Thrombin associated with the former was released into the media when the cells were incubated at 0-4 degrees C with hirudin or hydroxylamine, or transferred to the insensitive compartment when incubated at <em>22</em> degrees C. Finally, confluent cultures of <em>fibroblasts</em> bind 2-3 x 10(4) 125I-alpha-thrombin molecules/cell with an apparent binding constant, i.e. Kd, of 0.7 nM (a true Kd could not be determined because of the irreversible nature of thrombin binding). The binding capacity per cell and the apparent Kd value increased proportionally to an increase in culture density.

The aim of this investigation was to study the regulation of insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) gene expression in cultured rat aortic smooth muscle cells. Near-confluent cells were deprived of serum for 24 h and then exposed to IGF-I, insulin, serum, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (basic FGF), platelet-derived <em>growth</em> <em>factor</em> (PDGF-BB; consisting of B-chain homodimer) or GH for 24 h. Levels of IGF-I mRNA were measured by solution hybridization. The level of IGF-I mRNA was markedly decreased by 10% (v/v) newborn calf serum (78 +/- 4 (S.E.M.) % decrease), 1 nmol basic FGF/l (53 +/- 8%), and 1 nmol PDGF-BB/l (40 +/- 3%) when measured after 24 h. The effect of PDGF-BB was significant after 6 h and became more marked after 24 h. GH (1 nmol/l or 0.1 mumol/l) or insulin (1 nmol/l) had no effect after 24 h, whereas IGF-I (1 nmol/l) and insulin (10 mumol/l) increased IGF-I mRNA 64 +/- 20% and 46 +/- 14% respectively. The increase caused by IGF-I was demonstrated after 3 h, and was most marked after 24 h. Using Northern blot analysis of cultured aortic smooth muscle cells, IGF-I transcripts of 7.4, 1.7 and 1.1-0.8 kilobases were observed. Exposure of the cells to 10% serum, 1 nmol basic FGF/l or 1 nmol PDGF-BB/l for 48 h increased the cell number by 104 +/- 7%, 64 +/- 3% and 61 +/- <em>22</em>% respectively, while IGF-I, insulin and GH had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of the <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble <em>growth</em> <em>factors</em>. Treatment of subconfluent astrocytes with interleukin-1beta, epidermal or platelet-derived <em>growth</em> <em>factors</em>, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, <em>22</em>-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter-luciferase constructs identified a unique -555/-513 bp <em>growth</em> <em>factor</em>-responsive element (GFRE) that confers high basal promoter activity and activation by <em>growth</em> <em>factors</em> to a downstream promoter region. It also identified a separate region (-624/-556 bp) essential for PKC and cAMP stimulation. DNA-protein binding assays indicated that novel cis-acting elements and trans-acting <em>factors</em> mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by <em>growth</em> <em>factors</em> or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and <em>growth</em> <em>factors</em> or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.