Transforming <em>growth</em> <em>factors</em> beta are multifunctional proteins and regulators of cell proliferation and differentiation. Transforming <em>growth</em> <em>factor</em>-beta s have the capacity to rescue adult neurons from ischemia- and glutamate-induced cell death and are prominent in the embryonic and adult brain including striatum and substantia nigra. In the present study we show that transforming <em>growth</em> <em>factors</em>-beta 1, -2, and -3 promote, in a dose-dependent fashion, in vitro survival of tyrosine hydroxylase-immunoreactive dopaminergic neurons isolated from the embryonic rat mesencephalon floor. The magnitude of the effect, which was half-maximal at a concentration of <em>20</em> pM, was identical for all three transforming <em>growth</em> <em>factor</em>-isoforms and matched that of <em>fibroblast</em> <em>growth</em> <em>factor</em>-2. Unlike <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, however, transforming <em>growth</em> <em>factor</em>-beta s did not increase numbers of astroglial cells visualized by using antibodies to glial fibrillary acidic protein, and had no effect on cell proliferation monitored by incorporation of BrdUrd. Transforming <em>growth</em> <em>factor</em>-beta s were significantly more potent than <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 in protecting dopaminergic neurons against N-methyl-4-phenylpyridinium ion toxicity. RT-PCR analysis indicated that the effect of transforming <em>growth</em> <em>factor</em>-beta s is not mediated by glial cell-derived neurotrophic <em>factor</em>, which was not detectable in cultures at various time points. On the other hand transforming <em>growth</em> <em>factor</em>-beta 2 mRNA could be detected in freshly isolated and cultured mesencephalic cells, and its immunoreactivity has also been demonstrated in the embryonic day 14 mesencephalon floor. We conclude that transforming <em>growth</em> <em>factor</em>-beta has trophic and protective effects on developing dopaminergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

We tested the hypothesis that intravenous administration of basic fibroblast growth factor (bFGF) during 4 hours of permanent focal ischemia would affect acute brain injury.

METHODS

Halothane-anesthetized cats underwent left middle cerebral artery (MCA) occlusion for 4 hours. Control cats received diluent (n = 14). Experimental cats were treated with bFGF at a rate of 5 (n = 13), 50 (n = 13), or 250 microg/kg per hour (n = 9) intravenously beginning 60 minutes after initiation of ischemia and continuing until the end of the protocol.

RESULTS

As measured by the microsphere method, blood flow to ipsilateral caudate nucleus and ipsilateral inferior temporal cortex was decreased similarly during ischemia, before drug administration, in all groups. Likewise, there was no difference in blood flow to ipsilateral caudate nucleus or inferior temporal cortex as a result of bFGF administration during MCA occlusion. Triphenyltetrazolium-determined injury volume of the ipsilateral cerebral cortex (control, 40+/-7%; bFGF 5 microg/kg per hour, 22+/-5%; bFGF 50 microg/kg per hour, 26+/-7%; bFGF 255 microg/kg per hour, 23+/-6% of ipsilateral cerebral cortex; mean+/-SEM) was less in cats treated with bFGF. There was no difference among groups in injury volume to caudate nucleus (control, 29+/-8%; bFGF 5 microg/kg per hour, 29+/-8%; bFGF 50 microg/kg per hour, 21+/-7%; bFGF 250 microg/kg per hour, 32+/-7% of ipsilateral caudate nucleus). Somatosensory evoked potential amplitude decreased similarly (to <20% of baseline amplitude in all groups) during MCA occlusion and was not altered by bFGF administration. CONCLUSIONS; These data indicate that systemic administration of bFGF ameliorates acute injury in the cerebral cortex without increasing blood flow during focal ischemia in cats. Because bFGF afforded protection when administered after the onset of ischemia, bFGF may provide its beneficial effect by limiting progression of injury in ischemic border regions.

BACKGROUND

Full recovery is always achieved after caerulein induced pancreatitis. Cyclosporin stimulates transforming growth factor beta (TGF-beta) and may interfere with pancreatic regeneration.

OBJECTIVE

To investigate the effects of cyclosporin after caerulein induced pancreatitis or after caerulein injury.

METHODS

Protocol A: rats received cyclosporin daily (20 mg/kg) and caerulein pancreatitis was induced on days 2 and 8. Protocol B: six courses of caerulein pancreatitis were induced at weekly intervals. Cyclosporin was administered on induction and the day before. Rats recovered for two weeks before being killed. Control groups received saline, cyclosporin, or caerulein alone.

RESULTS

Protocol A: plasma TGF-beta1 and tissue collagenase rose after pancreatitis but decreased towards baseline values on day 15, matching a low collagen content. Morphology disclosed minimal inflammatory infiltration and some interstitial cells immunoreactive for smooth muscle alpha-actin (SMA). TGF-beta1 increased, and remained high in cyclosporin treated groups (cyclosporin alone and cyclosporin plus caerulein). Rats treated with cyclosporin and caerulein showed severe pancreatic weight reduction, abundant inflammatory infiltrates, increased SMA immunoreactive interstitial cells, high collagen content, and delayed collagenase response. No SMA immunoreactive cells were detected in normal rats. Cyclosporin alone also increased SMA immunoreactive cells, despite the absence of inflammatory infiltration and fairly conserved pancreatic structure. Protocol B: the combined pulse treatment induced appreciable collagen deposition and resulted in a smaller pancreas than controls. Morphological examination showed atrophy, fibrosis, fibroblast proliferation, and mononuclear infiltrates.

CONCLUSIONS

Cyclosporin greatly distorts pancreatic repair, transforming caerulein induced pancreatitis into a fibrotic chronic-like disease. The mechanism involves TGF-beta, myofibroblasts, and defective collagenase activation.

Kallmann syndrome (KS) describes the association of isolated hypogonadotropic hypogonadism with hypo/anosmia. A few KS patients may reverse hypogonadism after testosterone withdrawal, a variant known as reversible KS. Herein, we describe the first mutation in KAL1 in a patient with reversible KS and review the literature. The proband was first seen at 22 years complaining of anosmia and lack of puberty. His brother had puberty at 30 years and a maternal granduncle had anosmia and delayed puberty. On physical examination, he was P(2)G(1), testes were 3 ml and bone age was 14 years. During <em>20</em> years of irregular testosterone replacement, he developed secondary sexual characteristics and testicular enlargement. At the age of 41 years, after stopping testosterone replacement for 5 months, his testes were 15 ml, serum testosterone, LH, and FSH responses to GnRH were normal, and his wife was pregnant. The molecular study revealed a cytosine insertion in exon 2 of KAL1, generating a frameshift at codon 75 and a premature stop at codon 85. The expected gene product is a truncated peptide with 85 of the 680 [corrected] amino acids present in the wild-type protein. Fourteen cases of reversible KS have been described but the genotype was only studied in a single case showing a heterozygous <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor type 1 (FGFR1) mutation. Considering the low prevalence of mutations in KAL1 or FGFR1 in KS, it is possible that these genotypes are more prevalent in reversible KS than in other KS patients, but additional studies are necessary to confirm this hypothesis.

Craniosynostosis is a common disorder with an unknown etiology. Recent genetic mapping studies have demonstrated a strong linkage between several familial craniosynostotic syndromes and mutations in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGF-R1) and 2 (FGF-R2). The purpose of this experiment was to investigate by immunohistochemistry the protein production of these receptors as well as of their most prevalent ligand, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), before, during, and after sutural fusion in rat cranial sutures. The posterior frontal (normally fuses between postnatal days 12 and 22) and sagittal (remains patent) sutures of embryonic day <em>20</em> and neonatal days 6, 12, 17, 22, and 62 (n = 3 per group) were harvested, fixed, and decalcified. Five-micrometer sections were stained with polyclonal antibodies against bFGF, FGF-R1, and FGF-R2, and patterns of immunohistochemical staining were assessed by independent reviewers. Our results indicate that increased bFGF production correlates temporally with suture fusion, with increased staining of the dura underneath the fusing suture prior to fusion followed by increased staining within osteoblasts and sutural cells during fusion. FGF-R1 and, to a lesser extent FGF-R2 immunostaining revealed a different pattern of localization with increased immunostaining within the patent sagittal suture at these time points. These results implicate bFGF in the regulation of sutural fusion and may imply autoregulatory mechanisms in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor expression.

Recently, a unique Pro250Arg point mutation in <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) was reported in 61 individuals with coronal craniosynostosis from <em>20</em> unrelated families [Muenke et al. (1997): Am J Hum Genet 60:555-564]. The discovery of this apparently common mutation has resulted in the definition of a recognizable syndrome, through analysis of subtle clinical findings in families who were previously thought to have a variety of other craniosynostosis syndromes. Previous diagnoses in some of these families have included Jackson-Weiss, Saethre-Chotzen, and Pfeiffer syndromes, as well as Adelaide-type craniosynostosis and brachydactyly-craniosynostosis syndrome [Adès et al. (1994): Am J Med Genet 51:121-130; von Gernet et al. (1996): Am J Med Genet 63:177-184; Reardon et al. (1997): J Med Genet 34:632-636; Bellus et al. (1996): Nat Genet 14:174-176; Hollaway et al. (1995): Hum Mol Genet 4:681-683; Glass et al. (1994): Clin Dysmorphol 3:215-223]. There appears to be a need to further delineate the phenotype associated with this common mutation in FGFR3. We compare the clinical characteristics of previously reported cases of this unique Pro250Arg mutation with those of two additional families and suggest that this syndrome with a unique mutational basis be designated coronal craniosynostosis with brachydactyly and carpal/tarsal coalition due to Pro250Arg mutation in FGFR3 gene, to emphasize the distinctive findings which may be present even in the absence of coronal craniosynostosis.

Ascidian larvae develop mesenchyme cells in their trunk. A <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF9/16/<em>20</em>) is essential and sufficient for induction of the mesenchyme in Ciona savignyi. We have identified two basic helix-loop-helix (bHLH) genes named Twist-like1 and Twist-like2 as downstream <em>factors</em> of this FGF. These two genes are phylogenetically closely related to each other, and were expressed specifically in the mesenchymal cells after the 110-cell stage. Gene-knockdown experiments using a specific morpholino oligonucleotide demonstrated that Twist-like1 plays an essential role in determination of the mesenchyme and that Twist-like2 is a downstream <em>factor</em> of Twist-like1. In addition, both overexpression and misexpression of Twist-like1 converts non-mesenchymal cells to mesenchymal cells. We also demonstrate that the upstream regulatory mechanisms of Twist-like1 are different between B-line mesenchymal cells and the A-line mesenchymal cells called 'trunk lateral cells'. FGF9/16/<em>20</em> is required for the expression of Twist-like1 in B-line mesenchymal precursor cells, whereas FGF, FoxD and another novel bHLH <em>factor</em> called NoTrlc are required for Twist-like1 to be expressed in the A-line mesenchymal precursor cells. Therefore, two different but partially overlapping mechanisms are required for the expression of Twist-like1 in the mesenchymal precursors, which triggers the differentiation of the mesenchyme in Ciona embryos.

Collagen VII, the major component of cutaneous anchoring fibrils is expressed at a low level by normal human keratinocytes and <em>fibroblasts</em> in vitro. In cocultures of these two cell types, signals from <em>fibroblasts</em> enhance expression of collagen VII by keratinocytes and vice versa. In this study, the effects of a possible mediator of such a stimulation, transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), were investigated. Its effect on the expression and deposition of the highly insoluble collagen VII was assessed in a semiquantitative manner by a newly developed enzyme-linked immunoassay which is based on immunoblotting. In keratinocyte monocultures, 0.5-<em>20</em> ng/ml of TGF-beta 2 induced a dose-dependent stimulation of collagen VII expression as measured per microgram of DNA. The maximal enhancement was about sevenfold compared to controls. The effect of TGF-beta 2 was observed already after 12 h, with a steady increase at least up to 3 d. As previous studies have implicated, untreated cocultures of keratinocytes and <em>fibroblasts</em> exhibited a higher basic level of collagen VII expression, which could be further stimulated about twofold by TGF-beta 2. <em>Fibroblasts</em> alone synthesized very minor quantities of collagen VII and could be only weakly stimulated by TGF-beta 2. This <em>growth</em> <em>factor</em> seems a specific enhancer of collagen VII since the expression of laminin, collagen IV, as well as total protein was increased to a much lesser extent. Our data suggest that TGF-beta may be an important mediator of epithelial-mesenchymal interactions and may regulate the synthesis of the anchoring fibrils at the skin basement membrane zone.

The present study was undertaken to further define the role of alveolar macrophages (AM) in the pulmonary response to crocidolite fibers. Briefly, groups of 4 male F344 rats were intratracheally instilled with saline or saline suspensions of crocidolite at 2 or <em>20</em> mg/kg body weight. Animals were sacrificed 3, 7, 14, and 28 d after exposure and the lung response was characterized by analysis of bronchoalveolar lavage fluid (BALF) for markers of lung injury and inflammation. AM obtained in BALF were cultured and their production of the pro-inflammatory cytokines, tumor necrosis <em>factor</em> alpha (TNF alpha), and interleukin-1 (IL-1) were characterized along with fibronectin, a protein known to stimulate <em>fibroblast</em> migration and proliferation. Lung hydroxyproline content was determined 28 d after exposure and lung histopathology was characterized on d 28 and 90 after exposure. Crocidolite instillation resulted in transient dose-related pulmonary inflammation as evidenced by increased numbers of BALF neutrophils at the low dose and neutrophils, macrophages, and lymphocytes at the high dose. Cytotoxicity and increased permeability were demonstrated by increased levels of BALF lactate dehydrogenase (LDH) and total protein, respectively. AM TNF alpha and IL-1 production were increased only at the high crocidolite dose. This cytokine response was greatest at d 3 and decreased thereafter. AM TNF alpha and IL-1 release were positively correlated with the increased BALF neutrophils. In contrast to TNF alpha and IL-1, AM fibronectin release was increased at both the low and high doses, with the magnitude of response increasing over time. Consistent with previous acute asbestos inhalation studies, histopathology revealed inflammation localized at the level of the terminal bronchioles and alveolar ducts. Fibrosis was demonstrated at both doses by increased trichrome staining of lung tissue sections. Only the high dose resulted in a detectable increase in lung hydroxyproline. Given the bioactivities of TNF alpha, IL-1, and fibronectin, their increased production after crocidolite exposure indicates they contribute to the pulmonary inflammation and fibrosis occurring with this mineral fiber. In addition, the correlation of increased AM TNF alpha and IL-1 production with increased BALF neutrophils supports a role for these cytokines in crocidolite-induced inflammatory cell recruitment. Lastly, association of a persistent increase in AM fibronectin production with an eventual increase in lung collagen deposition extends the <em>growing</em> database indicating this response is a predictive marker of pulmonary fibrosis.

Treatment of rabbit corneal wounds with topical corticosteroid retards both epithelial regeneration and healing of penetrating stromal wounds. Currently, no clinical agent is available which accelerates the rate of stromal wound healing. Epidermal <em>growth</em> <em>factor</em> (EGF, 0.5 mg ml-1), <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF, <em>20</em> micrograms ml-1), and insulin (0.5 mg ml-1) were tested for their ability to accelerate healing of totally penetrating wounds in rabbit corneas when the hormones were administered alone or in combination with dexamethasone (1 mg ml-1). After 5 days of treatment with eye drops, the tensile strengths of corneal wounds treated with EGF (54 +/- 4 g mm-1) or treated with EGF and dexamethasone (32 +/- 9 g mm-1) were significantly higher than the tensile strengths of corneal wounds treated with only saline vehicle (3 +/- 1 g mm-1) or dexamethasone (1 +/ 0 g mm-1) (P less than 0.001). The combination of dexamethasone with EGF significantly (P less than 0.025) reduced the strength of corneal wounds compared to treatment with EGF alone. Similarly, the tensile strength of corneal wounds after 5 days of insulin treatment alone (28 +/- 8 g mm-1) or in combination with dexamethasone (25 +/- 7 g mm-1) was significantly increased compared with saline- or dexamethasone-treated corneas (P less than 0.001). In the absence of dexamethasone, EGF increased the tensile strength of corneal wounds significantly better than insulin (P less than 0.01). However, when EGF or insulin were given in combination with dexamethasone there was no significant difference between the tensile strength produced by the peptide hormones. In comparison to the tensile strength of corneal wounds treated by EGF or insulin, treatment with FGF alone (5 +/- 4 g mm-1) or in combination with dexamethasone (2 +/- 1 g mm-1) produced poor wound healing. The in vitro actions of EGF or FGF alone or in combination with dexamethasone were tested for ability to stimulate [3H]-thymidine incorporation into pure cultures of human corneal <em>fibroblasts</em> (HCF) in defined culture medium. EGF (5 mM) or FGF (100 ng ml-1) alone stimulated [3H]-thymidine incorporation approximately 2.5-fold compared to control cultures, whereas in combination with dexamethasone (10 nM), the stimulatory action of FGF, but not EGF, was abolished. Dose-response curves indicated that HCF in culture were very sensitive to EGF, insulin, and FGF with maximum stimulation of [3H]-thymidine incorporation occurring at approximately 1 nM for EGF and insulin and at 100 micrograms ml-1 for FGF. Binding of 125I-EGF to HCF reached maximum after 2 hr at 37 degrees C and was specific, saturable, and of high affinity (half saturation at 1 nM). (ABSTRACT TRUNCATED AT 400 WORDS)

Enhanced proliferation of <em>fibroblasts</em> is a primary characteristic of lung fibrosis. Macrophage-secreted platelet-derived <em>growth</em> <em>factor</em> (PDGF) is a potent mitogen and chemoattractant for lung <em>fibroblasts</em>. The magnitude of the <em>fibroblast</em> PDGF response is dependent on the number of PDGF receptor alpha (PDGF-R alpha) relative to PDGF-R beta at the cell surface. We recently reported that upregulation of the PDGF-R alpha subtype by interleukin (IL)-1beta results in enhanced lung <em>fibroblast</em> proliferation in response to PDGF-AA, PDGF-AB, and PDGF-BB whereas transforming <em>growth</em> <em>factor</em> (TGF)-beta1 has the opposite effect. Both IL-1beta and TGF-beta1 are produced by particle-activated macrophages in vivo and in vitro. We studied the net effect of macrophage conditioned medium (MOCM), which contains both IL-1beta and TGF-beta1, on the expression of the lung <em>fibroblast</em> PDGF receptor system. MOCM obtained from unstimulated, titanium dioxide (TiO2)-, chrysotile asbestos-, or residual oil fly ash (ROFA)-exposed macrophages in vitro increased [125I]PDGF-AA binding 3-, 6-, 6-, and <em>20</em>-fold, respectively. These increases correlated with increased PDGF-R alpha mRNA and protein expression as shown by northern and western assays. PDGF-AB and -BB-stimulated [3H]thymidine incorporation by <em>fibroblasts</em> was enhanced 5-, 5-, 10-, and <em>20</em>-fold by pretreatment with MOCM from unstimulated, TiO2-, asbestos-, and ROFA-exposed macrophages, respectively. [125I]PDGF-AA binding experiments using the IL-1 receptor antagonist blocked the upregulatory effect of all MOCM samples. Latent TGF-beta1 present in MOCM was activated by acid treatment, inhibiting upregulation by approximately 60%, a result similar to experiments with IL-1beta and TGF-beta1 mixtures. Treatment with a TGF-beta neutralizing antibody restored full upregulatory activity to acidified MOCM. Thus activated macrophages increase lung <em>fibroblast</em> PDGF-R alpha primarily due to the secretion of IL-1beta. Intratracheal instillation of ROFA particles in rats induced a 2-fold increase in total lung PDGF-R alpha mRNA in vivo. These findings support the idea that macrophage-derived IL-1beta plays a key role in the initiation of a fibrotic response by increasing <em>fibroblast</em> PDGF-R alpha expression, thereby dramatically potentiating the mitogenic response to PDGF.

BACKGROUND

Basic fibroblast growth factor (bFGF) stimulates neoangiogenesis. Incorporation into biodegradable gelatin hydrogels provides the sustained release of bFGF. The effects of intramyocardial injections of slow-release bFGF on neoangiogenesis in a rat model of infarction were investigated.

RESULTS

Myocardial infarction was induced in rats using coronary artery ligation. A total of 124 rats received an intramyocardial injection of 20 microg of bFGF, the same amount of bFGF incorporated into gelatin hydrogel (bFGF + gel), gelatin hydrogel (gel) or saline. Ventricular function was evaluated by echocardiography 2 or 4 weeks later. Morphometric and histological analyses were used to evaluate infarct size, vascular density and myocardial apoptosis. Capillary density in the infarct border zone was higher in the bFGF and bFGF + gel groups than in the saline and gel groups at 4 weeks (p<0.001). Arteriolar density was higher in the bFGF + gel group than in the other 3 groups (p<0.05). The bFGF and bFGF + gel groups contained fewer apoptotic cardiomyocytes in the border zone than the saline and gel groups (p<0.01). The bFGF+gel group had thicker (p<0.05) and less expanded infarcts (p<0.01) compared with the saline group at 4 weeks.

CONCLUSIONS

Incorporation of bFGF in gelatin hydrogels enhanced the effects of bFGF on arteriogenesis, ventricular remodeling and cardiac function.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) exerts angiogenic and mitogenic properties in human tissue. Since changes in ion currents modulate essential Ca2+-dependent intracellular pathways in endothelial cells, we have investigated a possible contribution of Ca2+-activated K+ channels (BKCa) on bFGF-induced endothelial cell proliferation. The patch-clamp technique was used to identify BKCa and to study their modulation by bFGF in cultured endothelial cells of human umbilical cord veins (HUVEC). Cell counts of HUVEC were carried out on different days to analyze bFGF-induced cell proliferation and its influence by the specific BKCa blocker iberiotoxin (IBX). Using single-channel recordings, we found characteristic BKCa with a single-channel slope conductance of 170.3 +/- 2.1 pS (n = 7), half-maximal activation at internal pCa = 5.7 (n = 5; test potential: 80 mV), and dose-dependent block by IBX (25-100 nmol/l). In cell-attached patches bFGF (50 ng/ml) caused a significant increase in the open-state probability (NPo) after 6 min at test potentials of 80 and 100 mV (n = 28; p < 0.001), respectively, which lasted up to 30 min. After preincubation with pertussis toxin (100 ng/ml; 4 h) bFGF superfusion did not cause a significant increase in BKCa activity until 25 min had passed (n = <em>20</em>; p < 0.01). Addition of 100 nmol/l IBX to the pipette solution caused a total block of BKCa within 2 min in cell-attached patches, whereas bFGF (50 ng/ml) was not able to activate BKCa. When incubated with IBX (25-100 nmol/l) every 2 days, bFGF-induced proliferation of HUVEC was significantly decreased by 50 (-41%) and 100 nmol/l (-50%) IBX (n = 5; p < 0.001) after 7 days. We conclude that activation of BKCa by bFGF may play an important role in bFGF-induced proliferation of human endothelial cells and thus might be important in the process of angiogenesis and vascular remodelling.

Despite the improvements in the human embryonic stem cell (hESC) culture systems, very similar conditions to those used to maintain hESCs on mouse feeders are broadly applied to culture methods based on human feeders. Indeed, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), a master hESC-sustaining <em>factor</em>, is still added in nearly all medium formulations for hESC propagation. Human foreskin <em>fibroblasts</em> (HFFs) and mesenchymal stem cells (MSCs) used as feeders have recently been reported to support hESC <em>growth</em> without exogenous bFGF. However, whether hESCs may be maintained undifferentiated without exogenous bFGF using media conditioned (CM) by human feeders remains elusive. We hypothesize that HFFs and hMSCs are likely to be functionally different and therefore the mechanisms by which HFF-CM and MSC-CM support undifferentiated <em>growth</em> of hESCs may differ. We have thus determined whether HFF-CM and/or MSC-CM sustain feeder-free undifferentiated <em>growth</em> of hESC without exogenous supplementation of bFGF. We report that hMSCs synthesize higher levels of endogenous bFGF than HFFs. Accordingly and in contrast to HFF-CM, MSC-CM produced without the addition of exogenous bFGF supports hESC pluripotency and culture homeostasis beyond <em>20</em> passages without the need of bFGF supplementation. hESCs maintained without exogenous bFGF in MSC-CM retained hESC morphology and expression of pluripotency surface markers and transcription <em>factors</em>, formed teratomas, and showed spontaneous and lineage-directed in vitro differentiation capacity. Our data indicate that MSC-CM, but not HFF-CM, provides microenvironment cues supporting feeder-free long-term maintenance of pluripotent hESCs and obviates the requirement of exogenous bFGF at any time.

OBJECTIVE

Fibroblast growth factors (FGFs) are mitogenic polypeptides that signal via FGF receptors (FGFRs). Pancreatic ductal adenocarcinomas (PDACs) overexpress multiple FGFs, implying a potential for growth modulation. In this study we investigated the importance of the IIIc splice variant of FGFR-1 (FGFR-1 IIIc) in PDAC.

METHODS

Expression of FGFR-1 IIIc was determined by a ribonuclease protection assay in pancreatic cancer cell lines and in tissues. In situ hybridization was used to localize FGFR-1 IIIc messenger RNA (mRNA) in pancreatic tissues. A cDNA encoding FGFR-1 IIIc was stably transfected into the well-differentiated TAKA-1 pancreatic ductal cell line that is not responsive to FGF5 and does not express FGFR-1.

RESULTS

FGFR-1 IIIc was expressed in 5 of 7 pancreatic cancer cell lines and in the majority of the cancer cells in 4 of 7 PDAC samples. In vitro, TAKA-1 cells stably transfected with FGFR-1 IIIc exhibited increased basal growth; enhanced basal tyrosine phosphorylation of FGFR substrate-2 (FRS2), Shc, and phospholipase Cgamma; and increased activation of mitogen-activated protein kinase (MAPK). PD98059, an inhibitor of MAPK, suppressed the basal growth of parental and transfected clones, but the effect was more marked in clones expressing FGFR-1 IIIc. In vivo, tumor formation in nude mice was dramatically enhanced with FGFR-1 IIIc transfected (20 of 20) in comparison with sham transfected (0 of 10) cells.

CONCLUSIONS

Our data indicate that FGFR-1 IIIc is expressed in human pancreatic cancer cells, promotes mitogenic signaling via the FRS2-MAPK pathway, and has the potential to enhance pancreatic ductal cell transformation.

An extract of 21-day rainbow trout embryos stimulated <em>growth</em> of several piscine cell lines in the absence of added serum. Established lines from trout (RTG-2 and STE-137), salmon (CHSE-214), carp (EPC), and goldfish (CAR) and early-passage cells initiated from trout embryos grew in serum-free medium containing the embryo extract. In addition the extract was sufficient for maintaining long-term cultures of CHSE-214 cells for several months through a minimum of <em>20</em> passages (approximately 50 population doublings) in the absence of serum. Optimal response was achieved with 100 micrograms of extract protein per ml, but a significant <em>growth</em>-promoting effect was observed with as little as 2.5 micrograms/ml. The activity was nondialyzable, protease-sensitive, and stable in <em>20</em>0 mM acetic acid. The level of mitogenic response induced by the extract could not be duplicated with purified mammalian <em>growth</em> <em>factors</em> added individually or in combination, and the extract did not stimulate DNA synthesis in quiescent mouse <em>fibroblasts</em>. These results suggest that trout embryo extract may contain a novel <em>growth</em>-promoting activity for fish cells.

BACKGROUND

In adults with heart failure, elevated levels of fibroblast growth factor 23 (FGF23) are associated with mortality. Data on FGF23 levels in pediatric heart failure are lacking.

METHODS

We conducted a cross-sectional study of 17 healthy children (mean age 13 years) and 20 pediatric patients with heart failure (mean age 12 years) who underwent echocardiography and for whom the following measurements were taken: plasma FGF23 and parathyroid hormone (PTH) and serum phosphate, creatinine and N-terminal prohormone brain natriuretic peptide (NT-proBNP). Symptom severity was assessed with the New York Heart Association and the Ross classification systems.

RESULTS

Of the 20 patients, 11 had dilated cardiomyopathy, four had congenital heart disease, three had hypertrophic cardiomyopathy, one had a failing heart transplant and one had pulmonary hypertension. Mean phosphate levels in these patients were within the reported reference range for healthy children. Median PTH levels were in the normal range in patients and controls. The median FGF23 level was higher in patients versus controls (110.9 vs. 66.4 RU/ml; P = 0.03) and higher in patients on diuretics versus other patients (222.4 vs. 82.1 RU/ml; P = 0.01). Levels of FGF23 and NT-proBNP were directly correlated (r = 0.47, P = 0.04), and patients with greater physical functional impairment had higher FGF23 levels (142.5 in those with moderate-severe limitation vs. 92.8 RU/ml in those with no limitation; P = 0.05). Among patients with dilated cardiomyopathy, higher FGF23 levels were associated with a greater left ventricular end-diastolic diameter (r = 0.63, P = 0.04).

CONCLUSIONS

FGF23 levels are elevated in children with heart failure and are associated with diuretic use, severity of heart failure and left ventricular dilation.

BACKGROUND

There is growing evidence to support an important role for vitamin D and related hormones, parathyroid hormone and fibroblast growth factor 23 (FGF23), on cardiac remodeling in chronic kidney disease. Our objective was to determine the relationships between vitamin D and cardiac remodeling in chronic kidney disease and the effects of parathyroid hormone and FGF23 on these associations.

RESULTS

In 1431 participants from the Chronic Renal Insufficiency Cohort study, we measured 25-hydroxyvitamin D (25(OH)D), 1,25-dihydroxyvitamin D (1,25(OH)2D), FGF23, and parathyroid hormone and performed quantitative echocardiography. Using linear regression methods, we determined significant negative interactions between 25(OH)D and FGF23 on left ventricular (LV) mass (P=0.016), end-diastolic volume (P=0.029), and end-systolic volumes (P=0.021). In participants with an FGF23 level greater than the median of 123.5 RU/mL, each doubling of 25(OH)D was associated with a 2.5% (95% confidence interval, -4.8, -0.2) lower LV mass. This association was less pronounced with FGF23 levels less than the median (0.4%; 95% confidence interval, -1.9, 2.7). Conversely, in participants with deficient 25(OH)D levels <20 ng/mL, each doubling of FGF23 was associated with a 3.4% (95% confidence interval, 1.2, 5.6) greater LV mass compared with only a 1.6% (95% confidence interval, -0.2, 3.5) difference in participants with sufficient 25(OH)D. Similar findings were observed with 25(OH)D and volumes (P<0.05), and 1,25(OH)2D and LV mass and volumes (P<0.005). There was no effect modification by parathyroid hormone.

CONCLUSIONS

We identified significant interactions among 25(OH)D, 1,25(OH)2D, and FGF23 on cardiac remodeling. Increased LV mass and cavity dilatation were observed with low 25(OH)D and high FGF23. Our findings suggest that consideration of both hormones is crucial to understanding the role of either in cardiac remodeling, and may have important therapeutic implications.

OBJECTIVE

Identification of isolated congenital heart block (CHB) predicts, with near certainty, the presence of maternal anti-SSA/Ro antibodies; however, the 2% incidence of CHB in first offspring of anti-SSA/Ro+ mothers, <em>20</em>% recurrence in subsequent pregnancies, and discordance in identical twins suggest that an environmental <em>factor</em> amplifies the effect of the antibody. Accordingly, this study was carried out to explore the hypothesis that hypoxia potentiates a profibrosing phenotype of the fetal cardiac <em>fibroblast</em>.

METHODS

Evidence of an effect of hypoxia was sought by immunohistologic evaluation of CHB-affected fetal heart tissue and by determination of erythropoietin levels in cord blood. The in vitro effect of hypoxia on gene expression and phenotype in fibroblasts derived from fetal hearts and lungs was investigated by Affymetrix arrays, quantitative polymerase chain reaction (PCR), immunofluorescence, and immunoblotting.

RESULTS

In vivo hypoxic exposure was supported by the prominent intracellular fibroblast expression of hypoxia-inducible factor 1alpha in conduction tissue from 2 fetuses in whom CHB led to death. The possibility that hypoxia was sustained was suggested by significantly elevated erythropoietin levels in cord blood from CHB-affected, as compared with unaffected, anti-SSA/Ro-exposed neonates. In vitro exposure of cardiac fibroblasts to hypoxia resulted in transdifferentiation to myofibroblasts (a scarring phenotype), as demonstrated on immunoblots and immunofluorescence by increased expression of smooth muscle actin (SMA), an effect not seen in lung fibroblasts. Hypoxia-exposed cardiac fibroblasts expressed adrenomedullin at 4-fold increased levels, as determined by Affymetrix array, quantitative PCR, and immunofluorescence, thus focusing attention on cAMP as a modulator of fibrosis. MDL12,330A, an adenylate cyclase inhibitor that lowers the levels of cAMP, increased expression of fibrosis-related proteins (mammalian target of rapamycin, SMA, plasminogen activator inhibitor type 1, and type I collagen), while the cAMP activator forskolin attenuated transforming growth factor beta-elicited fibrosing end points in the cardiac fibroblasts.

CONCLUSIONS

These findings provide evidence that hypoxia may amplify the injurious effects of anti-SSA/Ro antibodies. Modulation of cAMP may be a key component in the scarring phenotype. Further assessment of the susceptibility of cardiac fibroblasts to cAMP modulation offers a new research direction in CHB.

Placental blood flow, nitric-oxide (NO) levels, and endothelial NO synthase (eNOS) expression increase during human and ovine pregnancy. Shear stress stimulates NO production and eNOS expression in ovine fetoplacental artery endothelial (OFPAE) cells. Because eNOS is the rate-limiting enzyme essential for NO synthesis, its activity and expression are both closely regulated. We investigated signaling mechanisms underlying pulsatile shear stress-induced increases in eNOS phosphorylation and protein expression by OFPAE cells. The OFPAE cells were cultured at 3 dynes/cm2 shear stress, then exposed to 15 dynes/cm2 shear stress. Western blot analysis for phosphorylated ERK1/2, Akt, p38 mitogen activated protein kinase (MAPK), and eNOS showed that shear stress rapidly increased phosphorylation of ERK1/2 and Akt but not of p38 MAPK. Phosphorylation of eNOS Ser1177 under shear stress was elevated by <em>20</em> min, a response that was blocked by the phosphatidyl inositol-3-kinase (PI-3K)-inhibitors wortmannin and LY294002 but not by the mitogen activated protein kinase kinase (MEK)-inhibitor UO126. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) enhanced eNOS protein levels in static culture via a MEK-mediated mechanism, but it could not further augment the elevated eNOS protein levels otherwise induced by the 15 dynes/cm2 shear stress. Blockade of either signaling pathway changed the shear stress-induced increase in eNOS protein levels. In conclusion, shear stress induced rapid eNOS phosphorylation on Ser1177 in OFPAE cells through a PI-3K-dependent pathway. The bFGF-induced rise in eNOS protein levels in static culture was much less than those observed under flow and was blocked by inhibition of MEK. Prolonged shear stress-stimulated increases in eNOS protein were not affected by inhibition of MEK- or PI-3K-mediated pathways.

Accumulation of TIS1 and TIS11 (Lim et al.: Oncogene 1:263-270, 1987) mRNAs in secondary cultures of rat neocortical astrocytes was much greater in response to tetradecanoyl phorbol acetate (TPA) than in response to either epidermal <em>growth</em> <em>factor</em> (EGF) or <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). In contrast, EGF, FGF, and TPA were equally effective in inducing accumulation of TIS8 and TIS28/c-fos mRNAs. These data suggested that TPA and the polypeptide mitogens might induce TIS gene expression by distinct pathways. When maximally inducing concentrations of EGF and FGF were co-administered to astrocyte cultures, TIS mRNA accumulations were no greater than those observed for the individual <em>growth</em> <em>factors</em>, suggesting that EGF and FGF saturate a common, limiting step in their induction pathways. In contrast, when either EGF or FGF was presented to astrocytes in combination with maximally inducing levels of TPA, the resulting levels of accumulation of TIS mRNAs were at least as great as the sum of the levels induced by the individual mitogens. Stimulation of [3H]-thymidine incorporation demonstrated an identical pattern of interaction; EGF and FGF co-administration was no more effective than either polypeptide mitogen alone, but, when presented to astrocyte cultures along with maximally inducing concentrations of TPA, either EGF or FGF was able to increase incorporation of [3H]-thymidine. Superinduction of all the TIS genes occurred if cycloheximide (CHX) was present during TPA exposure. Once again, two distinct classes of responses of the various TIS genes occurred; superinduction of TIS1, TIS7, TIS11, and TIS28/c-fos mRNA accumulation ranged from 10- to <em>20</em>-fold, while CHX superinduction of TIS8 and TIS10 was far more modest, ranging from 2- to 3-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Activating mutations of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3), found in autosomal dominant human skeletal dysplasia, were reported to be involved in tumorigenesis and correlate with low-grade and superficial lesions of urothelial carcinoma. FGFR3 protein expression was immunohistochemically investigated in 126 cases of urothelial carcinoma of the urinary bladder to evaluate the role of this receptor in tumor behavior. p53 expression and the proliferating activity of tumor cells, assessed by Ki-67 expression, were also analyzed in parallel. Cytoplasmic and/or membrane immunostaining for FGFR3 was observed in 62 (49.2%) cases, including <em>20</em> (15.9%) cases of intense staining and 42 (33.3%) of moderate staining. p53 expression and Ki-67 labeling index (LI) were significantly correlated with high tumor grade (p=0.0093 and <0.0001, respectively) and invasion (p=0.0041 and <0.0001, respectively). Although there were two groups of interesting cases: low-grade and non-invasive tumors negative for p53 but positive for FGFR3, and high-grade and invasive tumors positive for p53 but negative for FGFR3, no statistically significant relationship was found between FGFR3 expression and tumor grade, invasion, p53 expression or Ki-67 LI. These results suggest that FGFR3 protein expression in bladder cancer is unlikely to affect tumor behavior as a unique single <em>factor</em>.

Transforming <em>growth</em> <em>factor</em> beta 1 (TGF-beta1) upregulation has been implicated in hypertrophic scars and keloids, but it is unclear if it is the cause or an effect of excessive scar formation. In this study, we overexpressed TGF-beta1 in <em>fibroblasts</em> and characterized its role. Normal human dermal <em>fibroblasts</em> were genetically modified to overexpress TGF-beta1 as the wild-type latent molecule or as a mutant constitutively active molecule. TGF-beta1 secretion was measured, as were the effects of TGF-beta1 upregulation on cell proliferation, expression of smooth muscle cell alpha actin (SMC alpha-actin) and ability to contract collagen lattices. <em>Fibroblasts</em> were implanted intradermally into athymic mice and tissue formation was analyzed over time by histology and immunostaining. Gene-modified <em>fibroblasts</em> secreted approximately <em>20</em> times the TGF-beta1 released by control cells, but only cells expressing mutant TGF-beta1 secreted it in the active form. <em>Fibroblasts</em> expressing the active TGF-beta1 gene had increased levels of SMC alpha-actin and enhanced ability to contract a collagen lattice. After intradermal injection into athymic mice, only <em>fibroblasts</em> expressing active TGF-beta1 formed "keloid-like" nodules containing collagen, which persisted longer than implants of the other cell types. We conclude that upregulation of TGF-beta1 by <em>fibroblasts</em> may be necessary, but is not sufficient for excessive scarring. Needed are other signals to activate TGF-beta1 and prolong cell persistence.

ADAMs (a disintegrin and metalloproteinases) comprise a new gene family of metalloproteinases, and may play roles in cell-cell interaction, cell migration, signal transduction, shedding of membrane-anchored proteins and degradation of extracellular matrix. We screened the mRNA expression of 10 different ADAMs with a putative metalloproteinase motif in synovial tissues from patients with rheumatoid arthritis (RA) or osteoarthritis (OA). Reverse transcription PCR and real-time quantitative PCR analyses indicated that among the ADAMs, ADAM15 mRNA was more frequently expressed in the RA samples and its expression level was significantly 3.8-fold higher in RA than in OA (p < 0.01). In situ hybridization, immunohistochemistry and immunoblotting demonstrated that ADAM15 is expressed in active and precursor forms in the synovial lining cells, endothelial cells of blood vessels and macrophage-like cells in the sublining layer of RA synovium. There was a direct correlation between ADAM15 mRNA expression levels and vascular density in the synovial tissues (r = 0.907, p < 0.001; n = <em>20</em>). ADAM15 was constitutively expressed in RA synovial <em>fibroblasts</em> and human umbilical vein endothelial cells (HUVECs), and the expression level was increased in HUVECs by treatment with vascular endothelial <em>growth</em> <em>factor</em> (VEGF)165. On the other hand, ADAM15 expression in RA synovial <em>fibroblasts</em> was enhanced with VEGF165 only if vascular endothelial <em>growth</em> <em>factor</em> receptor (VEGFR)-2 expression was induced by treatment with tumor necrosis <em>factor</em>-alpha, and the expression was blocked with SU1498, a specific inhibitor of VEGFR-2. These data demonstrate that ADAM15 is overexpressed in RA synovium and its expression is up-regulated by the action of VEGF165 through VEGFR-2, and suggest the possibility that ADAM15 is involved in angiogenesis in RA synovium.