Five human PRL-secreting pituitary tumors were tested for the presence of DNA-transforming sequences. After calcium phosphate transfection to NIH-3T3 mouse <em>fibroblast</em> cells, DNA samples derived from two prolactinomas induced foci of morphologically transformed cells which subsequently grew in soft agar. After retransfection of transformant DNA, resulting secondary transformants elicited rapidly <em>growing</em> solid tumors in nude mice. Southern analysis of transformant DNA revealed the integration of Alu-positive human DNA sequences into the mouse <em>fibroblast</em> NIH-3T3 cells, as judged by hybridization to a Blur-8 probe. The Alu signal became increasingly more difficult to detect with the multiple passaging (greater than <em>20</em>) of transformant cells in culture. Alu polymerase chain reaction (PCR) was, therefore, used to selectively amplify human DNA sequences from the NIH-3T3 rodent background. PCR using a human Alu-specific primer resulted in amplification of an Alu-containing DNA region within these transformants. The transformant DNA did not hybridize to human genomic probes for genes known to evoke focus formation in this assay, including H-ras, K-ras, N-ras, trk, ret, ros, or met. Further identification of the Alu-containing region revealed that it contained sequences from the human hst gene, a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family. The presence of human hst was demonstrated by strong hybridization to a 40-mer oligonucleotide probe to the second exon of hst, by amplification of this region with human hst-nested amplimers within the first and second introns, and finally by direct sequencing.(ABSTRACT TRUNCATED AT 250 WORDS)

Progenitor cells that endure in different regions of the CNS after the initial neurogenesis can be expanded in culture and used as a source of donor tissue for grafting in neurodegenerative diseases. However, the proliferation and differentiation characteristics of residual neural progenitor cells from distinct regions of the CNS are mostly unknown. This study elucidated the characteristics of progenitor cells that endure in the CA3 region of the hippocampus after neurogenesis, by in vitro analyses of cells that are responsive to epidermal <em>growth</em> <em>factor</em> (EGF) or <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) in the embryonic day 19 (E19) rat hippocampus. Isolated cells from the E19 CA3 region formed neurospheres in the presence of either EGF or FGF-2, but the yield of neurospheres was greater with FGF-2 exposure, Differentiation cultures revealed a greater yield of neurons from FGF-2 neurospheres (60%) than from EGF neurospheres (35%). Exposure to brain-derived neurotrophic <em>factor</em> (BDNF) enhanced the yield of neurons from EGF neurospheres but had no consequence on FGF-2 neurospheres. A large number of neurons from EGF/FGF-2 neurospheres demonstrated clearly palpable morphological features of CA3 pyramidal neurons and lacked gamma-aminobutyric acid (GABA) expression. However, a fraction of neurons (17-<em>20</em>%) from EGF/FGF-2 neurospheres expressed GABA, and exposure to BDNF increased the number of GABAergic neurons (30%) from EGF neurospheres. Neurons from EGF/FGF-2 neurospheres also contained smaller populations of calbindin- and calretinin-positive interneuron-like cells. Thus, progenitor cells responsive to FGF-2 are prevalent in the CA3 region of the E19 rat hippocampus and give rise to a greater number of neurons than progenitor cells responsive to EGF. However, both FGF-2- and EGF-responsive progenitor cells from E19 CA3 region are capable of giving rise to CA3 field-specific phenotypic neurons. These results imply that progenitor cells that persist in the hippocampus after neurogenesis remain regionally restricted and hence retain their ability to give rise to region-specific phenotypic neurons even after isolation and expansion in vitro.

Angiotensin II (Ang II) stimulation has been shown to regulate proliferation of skin <em>fibroblasts</em> and the production of extracellular matrix, which are very important processes in skin wound healing and fibrosis; however, there is little knowledge about the mechanisms involved in this process. We investigated the molecular aspects of this system with regards to Ang II in human dermal <em>fibroblasts</em> (HDF) and its potential role in fibrosis. <em>Fibroblasts</em> derived from human skin were subjected to examine differential relative gene and protein expression after transfection with specific reporter expression vectors and Ang II in vitro. In <em>growth</em>-arrested HDFs, Ang II treatment for <em>20</em> min caused acute activation of Smad2 phosphorylation, Smad overexpression and Smad-dependent gene transcription. The angiotensin type 1 (AT1) antagonist losartan diminished Ang II-induced Smad activation. The blockade of endogenous transforming <em>growth</em> <em>factor</em>-beta1 did modify the activation of Smad caused by Ang II. The p38 mitogen-activated protein kinase (MAPK) inhibitor SB<em>20</em>3580 diminished Ang II-induced Smad2 phosphorylation. Transient transfection with Smad7, which interferes with receptor-mediated activation of Smad2, diminished Ang II-induced connective tissue <em>growth</em> <em>factor</em> promoter activation, gene and protein expression and fibronectin, type I procollagen and type III procollagen overexpression, showing that Smad activation is involved in Ang II-induced dermal fibrosis. Our results show that Ang II activation of Smad2 occurs via the AT1 receptor, but not the AT2 receptor. Activation of Smad2 required p38 MAPK but not p42/p44 MAPK or the epidermal <em>growth</em> <em>factor</em> receptor.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>20</em> (FGF<em>20</em>) and monoamine oxidase B (MAOB) genes are associated with Parkinson Disease (PD) risk and both are in the dopamine bio-pathway. Therefore, we investigated the joint effect between polymorphisms in the FGF<em>20</em> and MAOB genes for evidence of interaction contributing to PD risk. Fourteen polymorphisms (eight for FGF<em>20</em>, six for MAOB) were genotyped in 736 families and analyzed using conditional logistic regression (CLR). Significant two-locus interactions were found in females between the polymorphisms rs1721100 of FGF<em>20</em> and rs1799836 of MAOB, and between the polymorphisms rs1721082 of FGF<em>20</em> and rs1799836 of MAOB. The risk alleles for each SNP identified from CLR, rs1721100 C, rs1721082 T and rs1799836 A, are consistent with previous reports. Using indicator variables for the SNP genotypes, rs1721100 GC with rs1799836 AA showed significant interaction (P = 0.021), compared with the reference group rs1721100 GG with rs1799836 GG. Using an allele-dose model for the risk alleles, rs1721100 and rs1799836 showed significant interaction (P = 0.019). We found similar interaction results between rs1721082 and rs1799836. In conclusion, variants in FGF<em>20</em> and MAOB show evidence of statistical interactions, which emphasizes the importance of considering them jointly in genetic analysis of PD and illustrates potential patterns of biological interaction contributing to PD risk.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) stimulated the sustained proliferation of mouse epidermal melanoblasts derived from epidermal cell suspensions in a serum-free medium supplemented with dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP). The melanoblasts could be subcultured in the serum-free medium supplemented with the two <em>factors</em> in the presence of keratinocytes, but not in the absence of keratinocytes. In these conditions, some melanoblasts proliferated without differentiating for more than <em>20</em> days including a subculture. This is the first report of a successful culture of melanoblasts from mammalian skin. This culture system is expected to clarify further markers for melanoblasts and requirements for their proliferation and differentiation.

Several proteins involved in transmembrane signaling have been shown previously to be modified covalently by long-chain fatty acids. Using the BC3H1 cell line, which contains a broad array of fatty acylated proteins, we have examined the possibility that acylation of certain proteins is modulated in response to mitogenic stimulation. In the present study, we describe a 64-kDa palmitoylated protein, referred to as p64, that is deacylated following stimulation of quiescent cells with fetal bovine serum, <em>fibroblast</em> <em>growth</em> <em>factor</em>, and phorbol dibutyrate. Western blot analysis of membrane and soluble fractions using a polyclonal antibody against p64 revealed that approximately 70% of p64 in unstimulated cells is present in the cytosol in a non-acylated form, whereas palmitoylated p64 is found exclusively in the membrane fraction. Extraction of membranes with 0.5 M sodium chloride, 0.2 M sodium pyrophosphate, or 0.2 M sodium carbonate failed to release p64, suggesting that the acylated form of this protein is tightly associated with membranes. Pulse labeling of proteins in quiescent cells with [3H] palmitate and subsequent chasing in medium containing <em>20</em>% fetal bovine serum, <em>fibroblast</em> <em>growth</em> <em>factor</em>, or phorbol dibutyrate revealed that the fatty acid associated with p64 undergoes mitogen-stimulated turnover, whereas turnover of fatty-acid on other acylated proteins is not observed. Palmitate is the predominant fatty acid associated with p64; however, small amounts of covalent myristate are also detected. Both fatty acids are attached post-translationally to p64 through a hydroxylamine-sensitive linkage, suggesting that acylation of this protein is catalyzed by a palmitoyl transferase with relaxed specificity for fatty acid substrates. Together, these results suggest that palmitoylation may participate in the association of p64 with the plasma membrane and that mitogen-dependent deacylation might alter interactions between this protein and other membrane components.

In order to obtain greater insights into the molecular mechanisms accompanying hormonal aging the effects of <em>growth</em> hormone (GH), insulin-like <em>growth</em> <em>factor</em>-I (IGF-I), 17beta-estradiol, progesterone and dehydroepiandrosterone were tested as single agents in concentrations corresponding to <em>20</em>- and 60-year-old females on human SZ95 sebocytes and <em>fibroblasts</em>. Cell proliferation and viability were measured by 4-methylumbelliferyl heptanoate and lactate dehydrogenase microassays, respectively, whereas lipid accumulation was documented via nile red microassay and fluorescence microscopy. mRNA and protein expression were evaluated via real-time RT-PCR and Western blotting or ELISA, accordingly. Our results depict the importance of IGF-I for lipid synthesis in SZ95 sebocyte and demonstrate the lack of 17beta-estradiol, dehydroepiandrosterone and progesterone activity on lipid synthesis and SZ95 sebocyte proliferation suggesting that the action of these hormones in vivo may be implemented through indirect pathways. <em>Fibroblast</em> showed to be more susceptible to 17beta-estradiol treatment, while IGF-I could significantly stimulate <em>fibroblast</em> proliferation in a dose-dependent manner. Furthermore, an interplay between the 17beta-estradiol and IGF-I signaling pathway was documented in both cell types. In conclusion, IGF-I is a key regulator of human skin aging and declining IGF-I levels with age may play a significant role in the reduction of skin surface lipids and thickness.

The distribution of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) at the ultrastructural level in the brain of young (15- and <em>20</em>-day-old) and adult (3-month-old) rats was investigated by immunocytochemistry. Strong staining was observed in most neurons of the cortex of young rat brain. In the same brain area of adult rat many neurons were also stained intensely, while others were negative. Neurons in the other parts of the brain and especially in the adult rat, were generally more weakly stained. The reaction product was located in the cytoplasm of the neuronal cell bodies and their processes. Astrocytes, oligodendrocytes, microglial cells, meningeal cells, choroïd epithelial cells, ependymal cells and capillary endothelial cells showed no staining.

Multiple neurotrophic <em>factors</em> play a role in proliferation, differentiation and survival in the ol<em>factor</em>y epithelium (OE); however, the signaling cascade has not been fully elucidated. We tested the hypotheses that ATP induces the synthesis and secretion of two neurotrophic <em>factors</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) and transforming <em>growth</em> <em>factor</em> alpha (TGFα), and that these neurotrophic <em>factors</em> have a role in inducing proliferation. Protein levels of FGF2 and TGFα were increased <em>20</em> h post-intranasal instillation of ATP compared to vehicle control in adult Swiss Webster mice. Pre-intranasal treatment with purinergic receptor antagonist pyridoxal-phosphate-6-azophenyl-<em>20</em>,40-disulfonic acid (PPADS) significantly blocked this ATP-induced increase, indicating that upregulation of FGF2 and TGFα expression is mediated by purinergic receptor activation. However, in neonatal mouse, intranasal instillation of ATP significantly increased the protein levels of FGF2, but not TGFα. Likewise, ATP evoked the secretion of FGF2, but not TGFα, from neonatal mouse ol<em>factor</em>y epithelial slices and PPADS significantly blocked ATP-evoked FGF2 release. To determine the role of FGF2 and TGFα in inducing proliferation, 5-bromo-2-deoxyuridine (BrdU) incorporation was examined in adult ol<em>factor</em>y epithelium. Intranasal treatment with FGF receptor inhibitor PD173074 or epidermal <em>growth</em> <em>factor</em> receptor inhibitor AG1478 following ATP instillation significantly blocked ATP-induced BrdU incorporation. Collectively, these data demonstrate that ATP induces proliferation in adult mouse ol<em>factor</em>y epithelium by promoting FGF2 and TGFα synthesis and activation of their receptors. These data suggest that different mechanisms regulate neurogenesis in neonatal and adult OE, and FGF2 and TGFα may have different roles throughout development.

OBJECTIVE

Epigallocatechin-gallate (EGCG) claims a plethora of health benefits including protection against neoplastic diseases. Meanwhile, heparan-sulfate proteoglycans (HSPGs) have defensive role against tumour cell invasion. Therefore, the chemopreventive and hepatoprotective effects of EGCG were studied in hepatocellular carcinoma (HCC) in vivo and in vitro and compared with strong water soluble antioxidant, sodium ascorbate.

METHODS

HCC was induced in SD rats by thioacetamide (<em>20</em>0 mg/Kg). Some rats were treated with EGCG (<em>20</em> mg/Kg) or sodium ascorbate (100 mg/Kg). Liver impairment was assessed by measuring serum α-fetoprotein and investigating liver sections stained with H/E. Hepatic HSPGs, syndecan-1 and matrix metalloproteinase-9 (MMP-9) were measured by ELISA. Gene expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-2 was measured. Cell death was assessed by caspase-3 activity. In addition, all markers were measured in human hepatocellular carcinoma cell line (HepG2).

RESULTS

EGCG increased the animal survival and decreased both α-fetoprotein and HepG2 viability. In addition, EGCG ameliorated fibrosis and massive hepatic tissue breakdown. EGCG restored HSPGs and reduced expression of MMP-9, syndecan-1 and FGF-2 in-vivo and in-vitro. Sodium ascorbate showed significantly lower results than EGCG.

CONCLUSIONS

Besides antioxidant activity, other mechanisms are involved in the chemopreventive and hepatoprotective effects of EGCG including restoration of HSPGs receptors and inhibition of vascular invasion.

<em>Fibroblast</em> contraction of collagen gels is regarded as a model of wound contraction. Transforming <em>growth</em> <em>factor</em> (TGF)-beta added to such gels can augment contraction consistent with its suggested role as a mediator of fibrotic repair. Since <em>fibroblasts</em> isolated from fibrotic tissues have been suggested to express a "fibrotic phenotype," we hypothesized that TGF-beta exposure may lead to a persistent increase in <em>fibroblasts</em>' contractility. To evaluate this question, confluent human fetal lung <em>fibroblasts</em> were treated with serum-free Dulbecco modified Eagle medium (DMEM), with or without 100 pM [corrected] TGF-beta1, TGF-beta2, or TGF-beta3 for 48 h. <em>Fibroblasts</em> were then trypsinized and cast into gels composed of native type I collagen isolated from rat tail tendons. After <em>20</em> min for gelation, the gels were released and maintained in serum-free DMEM. TGF-beta-pretreated <em>fibroblasts</em> caused significantly more rapid gel contraction (52.5+/-0.6, 50.9+/-0.2, and 50.3+/-0.5% by TGF-beta1, -beta2, and -beta3 pretreated <em>fibroblasts</em>, respectively) than control <em>fibroblasts</em> (74.0+/-0.3%, P < 0.01). This effect is concentration dependent (50-<em>20</em>0 nM), and all three isoforms had equal activity. The effect of TGF-beta1, however, persisted for only a short period of time following the removal of TGF-beta, and was lost with sequential passage. These observations suggest that the persistent increase in collagen-gel contractility, mediated by <em>fibroblasts</em> from fibrotic tissues, would not appear to be solely due to previous exposure of these cells to TGF-beta.

The conditioned medium from the HT-29 human colonic adenocarcinoma cell line contains a potent mitogenic activity that can markedly stimulate the proliferation of both rat and human <em>fibroblasts</em> in the absence of serum. Fractionation of conditioned medium on Bio-Gel P-100 shows that HT-29 cells simultaneously produce 2 different types of endogenous <em>growth</em> <em>factors</em>. The first one (molecular mass of 35, 8 and 5.5 kDa) exhibits an IGF-I competing activity which is positively correlated to mitogenic activity. This mitogen is recognized by anti-IGF-I antibodies but is resistant to reducing agents. It is distinct from IGF-II, insulin and PDGF. The second one (molecular mass of 40- and <em>20</em>-kDa) is able to displace EGF binding to its receptor. This <em>factor</em> is immunologically recognized by anti-EGF antibodies but with a lower affinity as compared to EGF. This suggests that this endogenous HT-29-<em>growth</em> <em>factor</em> is related to but distinct from native EGF. Although more active in radioreceptor assay than in radioimmunoassay, the EGF-competing <em>factor</em> is distinct from TGF alpha or beta since it is unable to induce anchorage-independent <em>growth</em> of NRK or FR3T3 target cells in the presence or absence of exogenous EGF. Moreover, free functional EGF receptors are available at the HT-29 cell surface.

The stimulation of DNA synthesis by epidermal <em>growth</em> <em>factor</em>, insulin, and serum is inhibited by a variety of alkylamines when present for the duration of the stimulatory preincubation (<em>20</em>-24 hr). These results contradict an earlier report [Maxfield, F. R., Davies, P. J. A., Klempner, L., Willingham, M. C. & Pastan, I. (1979) Proc. Natl. Acad. Sci. USA 76, 5731-5735] and can be explained by differences in incubation conditions. The most straightforward interpretation of our results is that the mitogenic activities of <em>growth</em> <em>factors</em> are blocked by agents that inhibit the intracellular processing of hormone-receptor complexes. Therefore, the continued internalization and degradation of <em>growth</em> <em>factors</em> or their receptors within cells may play an important role in inducing mitogenesis in cultured human <em>fibroblasts</em> and may explain the prolonged requirement for epidermal <em>growth</em> <em>factor</em> in the culture medium (8 hr) to elicit a mitogenic response. We also found that bacitracin, a potent inhibitor of the enzyme transglutaminase, neither prevents receptor internalization or degradation in human <em>fibroblasts</em> nor inhibits the mitogenic activity of epidermal <em>growth</em> <em>factor</em>. These results suggest that transglutaminase activity may not be relevant to the mechanisms of <em>growth</em>-<em>factor</em>-induced receptor internalization or mitogenesis.

OBJECTIVE

To evaluate quantitative functional ultrasonography (US) in a murine gel model by using microbubble destruction kinetics to determine whether parametric indices provided with US could help assess angiogenesis.

METHODS

Institutional Animal Subjects Committee approved experiments and procedures. In 36 normal mice, two 0.4-mL gel implants were placed subcutaneously on either side of spine. One implant contained 0.5, 1.0, or 1.5 microg human basic fibroblast growth factor (bFGF) per milliliter of gel. Functional US quantitative analysis of angiogenesis with microbubble contrast agent was performed on days 3, 6, 9, and 12; histologic data were collected. Time-intensity curve of implant was fitted to mathematic decay model to calculate fractional blood volume and fraction of blood replaced per unit of time. Microvascular density (MVD) and percentage of microvascular area (MVA) were measured after anti-CD31 staining. Spearman rank order correlation was used in analyses.

RESULTS

bFGF-containing implants induced MVD of eight, 35, 42, and 42 vessels per square millimeter on days 3, 6, 9, and 12, respectively; in controls, MVD was four vessels/mm2 (P<.05 on days 6, 9, and 12). bFGF-containing implants induced percentage MVA of 2%, 5%, 20%, and 27%, respectively; in controls, it was 0.5% (P<.05). Maximum enhancement was significantly increased in bFGF implants (23.3 gray level+/-14.1 [standard deviation]) compared with controls (11.0+/-5.5, P<.001). Implants containing bFGF showed poor correlations between fractional blood volume and MVD (r2=0.42) or percentage MVA (r2=0.51) at US. There was no correlation between microbubble velocity and MVD (r2<0.05) or percentage MVA (r2<0.13).

CONCLUSIONS

Functional US perfusion parameters do not correlate with current histologic indices for quantifying angiogenesis. MVD, as a histologic quantitative measurement of angiogenesis, may not be an appropriate standard for contrast-enhanced imaging that relies on perfused neovessels.

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

OBJECTIVE

Fibroblast growth factor receptors (FGFRs)-1, -2, and -3 are expressed in the developing brain and may participate in CNS neoplasia. Fibroblast growth receptor-3 has not been demonstrated in the human CNS or its tumors. Nonetheless, it has been implicated in the pathogenesis of several other forms of neoplasia.

METHODS

Twenty-four human meningiomas were evaluated using Western blot analysis for expression of FGFR3, its ligand acidic FGF, and concomitant phosphorylation/activation of p44/42 mitogen-activated protein kinase (MAPK), Akt, and STAT3. Mutations in exons 7 and 10 of the FGFR3 gene were analyzed by polymerase chain reaction in 10 meningiomas. Primary meningioma cells cultured from 10 human meningiomas were also treated with acidic FGF and evaluated for cell proliferation or activation/phosphorylation of p44/42 MAPK, Akt, and STAT3.

RESULTS

Immunoblotting demonstrated the presence of FGFR3 in 12 (71%) of 17 primarily fibroblastic and transitional WHO Grade I meningiomas. The FGFR3 was detected in 4 (80%) of 5 WHO Grade II, and 2 of 2 Grade III tumors. Acidic FGF was detected in 3 (18%) of 17 Grade I, 1 (20%) of 5 Grade II, and 1 (50%) of 2 Grade III meningiomas. In WHO Grade I meningiomas, 3 of 6 tumors with no detectable FGFR3 had no detectable p-STAT3. In WHO Grades II and III meningiomas, FGFR3 expression was associated with p-STAT3, p-Akt, and p-p44/42 MAPK expression. No mutations were demonstrated in exons 7 or 10 by polymerase chain reaction in any meningioma. Treatment with acidic FGF, a ligand for FGFR3, stimulated meningioma cell proliferation and activation of Akt and STAT3 in primary meningioma cell cultures.

CONCLUSIONS

These findings suggest that FGFR3 and acidic FGF are expressed in adult human leptomeninges as well as WHO Grades I and II meningiomas. Fibroblast growth factor receptor-3 activation stimulates meningioma cell proliferation by activation of the phosphoinositide 3 kinase-Akt-PRAS40-mTOR and STAT3 pathways.

A recent report suggested that platelet-derived <em>growth</em> <em>factor</em> (PDGF) activates nuclear <em>factor</em>-kappa B (NF-kappa B) by phosphorylation of the protein kinase Akt [Romashkova and Makarov, Nature 401 (1999) 86-90]. The present study investigates the role of Akt in the activation of NF-kappa B by tumor necrosis <em>factor</em>-alpha (TNF alpha, 10 ng/ml) and PDGF-BB (<em>20</em> ng/ml) in human vascular smooth muscle cells (SMC), skin and foreskin <em>fibroblasts</em>. TNF alpha stimulated serine phosphorylation and degradation of the inhibitory protein I kappa B alpha and strongly induced nuclear NF-kappa B translocation and binding activity. PDGF did not induce serine phosphorylation or degradation of I kappa B alpha and did not enhance binding activity of NF-kappa B. In contrast, stimulation with PDGF resulted in a marked phosphorylation of Akt, but no Akt phosphorylation occurred after stimulation with TNF alpha. These data suggest that Akt phosphorylation is not involved in NF-kappa B activation in human SMC and <em>fibroblasts</em>.

The effects of heparin and other glycosaminoglycans (GAGs) on the mitogenicity and stability of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) were studied. The mitogenic activity of aFGF was assayed utilizing cultured adult human endothelial cells (AHECs) isolated from iliac arteries and veins as target cells. In most experiments, aFGF purified from bovine brain was employed; in some experiments recombinant bovine aFGF was used and qualitatively similar results were obtained. In the presence of heparin, bovine aFGF at doses between 0.5 and 1.0 ng/ml (30-60 pM) elicited half the maximum AHEC <em>growth</em> over a 4-day period depending on the cell line tested; in the absence of heparin, significant <em>growth</em> was not observed at aFGF concentrations less than 10-<em>20</em> ng/ml. This effect of heparin was dose-dependent over the range 0.1-10 micrograms/ml (half-maximum dose, 2 micrograms/ml). The mitogenic activity of bovine aFGF for AHECs decreased by 50% after preincubation in culture medium without cells at 37 degrees C for 2 1/2 to 3 hours. In contrast, the mitogenic activity of bovine aFGF preincubated in the presence of heparin-containing culture medium without cells was dramatically stabilized (half-life 24-29 hours). These effects also were observed in serum-free medium. Several GAGs structurally related to heparin such as chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and hyaluronic acid neither potentiated nor stabilized aFGF mitogenic activity. However, heparan sulfate from bovine lung was found to be nearly as active as heparin in both these effects. These data suggest that the binding and stabilization of mitogens by extracellular and tissue-associated heparan sulfates might play important roles in the regulation of AHEC <em>growth</em>.

HST1 (or HSTF1 in human gene nomenclature) transforming gene encodes a novel heparin-binding <em>growth</em> <em>factor</em> which has 40-50% homology with <em>fibroblast</em> <em>growth</em> <em>factors</em> and mouse Int-2 protein. Expression of mouse Hst1 or Int-2 is rare in adult tissues, but both of them are transcribed in embryos. We found that mouse Hst1 and Int-2, like their human counterparts, were located close to each other on the genome: the distance was less than <em>20</em> kbp. Hst1 was expressed in an undifferentiated mouse teratocarcinoma cell line, F9. Upon induction of differentiation of F9 cells, the amount of Hst1 transcript was markedly decreased, while that of Int-2 transcripts increased concomitantly.

BACKGROUND

Systemic therapy with cyclosporin A, phenytoin, and nifedipine modulates cytokine levels in human gingival tissues. Functional relationships between altered cytokine levels and gingival extracellular matrix production are partially characterized. The present study investigates in cultured human gingival fibroblasts the regulation of lysyl oxidase, alpha-1 type I collagen, and elastin by selected cytokines that are elevated in drug-induced gingival overgrowth tissues.

METHODS

Normal human gingival fibroblasts were cultured and then treated with selected cytokines: interleukin (IL)-1beta, IL-6, platelet-derived growth factor (PDGF)-BB, and basic fibroblast growth factor (bFGF or FGF-2). Cells were harvested at intervals, and changes in lysyl oxidase enzyme activity, and in mRNA levels of lysyl oxidase, alpha-1 type I collagen, and elastin were determined.

RESULTS

bFGF reproducibly and significantly decreased human gingival fibroblast lysyl oxidase and alpha-1 type I collagen mRNA levels in a dose- and time-dependent manner; 1 nM bFGF reduced lysyl oxidase and collagen mRNA levels to 53% and to less than 10% of control after 48 hours of treatment. Interestingly, bFGF downregulated lysyl oxidase enzyme activity by 10% to 20%. IL-1, IL-6, and PDGF-BB did not significantly regulate lysyl oxidase enzyme activity, or alpha-1 type I collagen, elastin, and lysyl oxidase mRNA levels under the conditions tested.

CONCLUSIONS

Previous studies have shown that modulated levels of bFGF occur in gingiva as a result of certain pharmacologic therapies. The present study suggests that modulated levels of bFGF likely influence gingival connective tissue metabolism.

OBJECTIVE

We recently identified a novel member of the human <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family of signaling molecules, designated FGF-<em>20</em>. In the present study, we examined the activity of this protein in 2 animal models of acute intestinal inflammation and in mechanistic studies in vitro.

METHODS

In vivo experiments consisted of a murine dextran sulfate sodium (DSS) model of colitis and a rat indomethacin model of small intestinal ulceration/inflammation. Cell growth, restitution, gene expression (cyclooxygenase-2 [COX-2] and intestinal trefoil factor [ITF]), and prostaglandin E2 (PGE2) levels were examined in vitro.

RESULTS

In the DSS-colitis model, prophylactic administration of FGF-<em>20</em> significantly reduced the severity and extent of mucosal damage as indicated by a 55%-93% reduction in luminal blood loss, distal colonic edema, histologic inflammation, and epithelial cell loss relative to animals administered vehicle control. No toxicity was noted during administration of FGF-<em>20</em> to normal controls. In addition, therapeutic administration of FGF-<em>20</em> enhanced survival in this model. In the indomethacin-small bowel ulceration/inflammation model, administration of FGF-<em>20</em> reduced small intestinal weight gain, necrosis, inflammation, and weight loss (36%-53% relative to vehicle control). In vitro studies demonstrated that FGF-<em>20</em> stimulates <em>growth</em>, restitution, mRNA expression of COX-2 and ITF, and PGE2 levels in human intestinal epithelial cells and enhances the <em>growth</em> of human intestinal <em>fibroblast</em>s.

CONCLUSIONS

FGF-<em>20</em>, having demonstrated therapeutic activity in 2 experimental models of intestinal inflammation, represents a promising new candidate for the treatment of human inflammatory bowel disease.

We investigated the serum concentration of hepatocyte <em>growth</em> <em>factor</em> (HGF), vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and transforming <em>growth</em> <em>factor</em> beta1 (TGF-beta1) using an enzyme-linked immunosorbent assay (ELISA) in a group of 60 patients with systemic lupus erythematosus (SLE), and <em>20</em> healthy controls. We also examined the possible association between the serum concentrations of these <em>factors</em> and certain clinical, laboratory parameters and SLE activity. HGF, VEGF and TGF-beta1 were detectable in all patients with SLE, and in all normal individuals. bFGF was measurable in 70% of the patients with SLE and in 65% of the healthy controls. The HGF level was higher in active SLE (median 1,019.5pg/ml) than in inactive SLE (median 787.8 pg/ml) (p < 0.005) or in the control group (median 847.0 pg/ml) (p < 0.009). The level of VEGF in active SLE was also higher (<em>20</em>3.5 pg/ml) than in inactive disease (116.1 pg/ml) (p < 0.05) or in healthy persons (133.5 pg/ml) (p < 0.04). The levels of bFGF and TGF-beta1 were similar for both the active and inactive SLE, and the control group (p>> 0.05). We found a significant, positive correlation between the levels of HGF and bFGF (r = 0.268, p < 0.04), HGF and TGF-beta1 (r = 0.365, p < 0.005) and HGF and VEGF (r = 0.327, p < 0.02) as well as VEGF and TGF-beta1 (r = 0.543, p < 0.001). We found a positive correlation between VEGF serum levels and platelet counts (r = 0.272, p < 0.04), and the TGF-beta1 concentration and platelet count (r = 0.313; p < 0.02). There was also a positive correlation between HGF serum concentration and the SLE activity score (r = 0.435, p < 0.001), as well as between the level of VEGF and SLE activity (r = 0.252, p = 0.05). In conclusion, serum levels of the angiogenic <em>factors</em> HGF and VEGF may be relevant in SLE pathogenesis. Their concentrations seem to be markers of SLE activity.

OBJECTIVE

Vascular endothelial growth factor (VEGF) causes widespread retinal vascular dilation, produces breakdown of the blood-retinal barrier, and is implicated in ocular neovascularization (NV). Basic fibroblast growth factor (bFGF) also has been implicated in the production of ocular NV. This study was performed to investigate the ability of simultaneous sustained intravitreal release of both VEGF and bFGF to induce robust retinal NV in the rabbit.

METHODS

Intravitreal implantation of sustained-release Hydron polymeric pellets containing both 20 microg of VEGF and 20 microg of bFGF was performed on adult male Dutch belted rabbits. In other animals either 20 microg or 50 microg bFGF-containing pellets was implanted intravitreally; also, either 20 microg VEGF or 50 microg VEGF-containing pellets was implanted. Control rabbits received either blank polymeric pellets or a pellet containing 30 microg bovine serum albumin. Eyes were examined by indirect ophthalmoscopy after surgery at 24 hrs, 48 hrs, 4 days, 7 days, 14 days, 21 days, and 28 days. Findings were documented by color fundus photography and fluorescein angiography (FA). Eyes were enucleated and prepared for histologic analysis at 28 days following intravitreal implantation of the VEGF/bFGF-containing pellets.

RESULTS

In all eyes implanted with VEGF/bFGF pellets, dilation and tortuosity of existing blood vessels were observed within 48 hrs after pellet implantation. The progression of retinal vascular changes was rapid and occurred over the entire optic disk and medullary rays between 4 and 7 days. Hemorrhage occurred as early as 14 days after VEGF/bFGF pellet implantation. In eyes with massive hemorrhage, total traction retinal detachment developed after the second week. The presence of abnormal tissues at the vitreo-retinal interface within 28 days was demonstrated by light microscopy while FA showed profuse leakage of dye from anomalous vessels within the first week. Neither bFGF-exposed eyes nor control eyes showed any vascular changes. Eyes that received only VEGF-containing pellets exhibited tortuosity of existing vessels, but neither hemorrhaging nor retinal detachment occurred.

CONCLUSIONS

These results demonstrate that retinal vascular changes leading to hemorrhaging is produced rapidly in the rabbit by simultaneous intravitreal release of both VEGF and bFGF. Understanding how these growth factors induce retinal NV may suggest novel therapeutic treatment strategies.

Conditioned media (CM) harvested from bovine smooth muscle cells (SMCs) of aortic media cultured under hypoxic conditions remarkably enhanced angiogenesis in vitro, that is, the tube formation of bovine capillary endothelial cells (BCEs) cultured on type I collagen gels. The extent of in vitro angiogenesis was assessed by the total length of tube structures formed by BCEs per area measured quantitatively with an image analyzer. The tube formation in CM obtained from the cultivation of SMCs at 1% O2 for 24 hours was enhanced by about 1.5 times and 3.4 times as compared with those at 5% O2 and <em>20</em>% O2, respectively. This tube-forming activity was abrogated by the pretreatment of CM with anti-transforming <em>growth</em> <em>factor</em> (TGF)-beta 1 IgG, but not by anti-basic <em>fibroblast</em> <em>growth</em> <em>factor</em> IgG. The SMC-CM obtained from hypoxic cultivation (1% O2 for 24 hours) inhibited [3H] thymidine incorporation by BCEs, SMCs, and <em>fibroblasts</em> more than about <em>20</em>% of control. Anti-TGF-beta 1 IgG thus significantly reduced the inhibitory effect of hypoxic SMC-CM on DNA synthesis of these cells. These results suggest that SMCs in a hypoxic state release active in vitro angiogenic <em>factors</em> into CM, and active TGF-beta 1 is closely related to the in vitro angiogenic enhancement of media conditioned by SMCs cultured in a hypoxic state.