The effects of fluoride (20 mumol/L) and bovine bone extract (17 micrograms/ml) were determined on cultures of human bone cells, embryonic chick bone cells, and human skin <em>fibroblasts</em>. The incorporation of [3H]thymidine into DNA was measured <em>16</em> hours after the addition of <em>factors</em>. After three to five days treatment, Triton X-100 extracts of the cells were assayed for acid phosphatase activity, in the presence and absence of tartrate, and for alkaline phosphatase activity. Fluoride stimulated [3H]thymidine incorporation and specific activity of alkaline phosphatase in human bone cells and chick bone cells but not in human skin cells. Fluoride also stimulated the cell population doubling rate of the human bone cells with an optimum of approximately 20 mumol/L. Bovine bone extract stimulated thymidine uptake into DNA several-fold and decreased alkaline phosphatase activity in all three types of cultured cells. The specific activity of tartrate-resistant acid phosphatase was increased in bone cells but not in skin <em>fibroblasts</em>. These results suggest that fluoride specifically stimulates the proliferation and differentiation of osteoblasts, while the <em>growth</em> <em>factors</em> in bovine bone extract primarily stimulate proliferation of bone cells. Cultures of human bone cells respond similarly to chick calvarial cells when treated with fluoride or bovine bone extract.

BACKGROUND

In previous experiments, we showed that heparin oligosaccharides inhibit the angiogenic cytokine fibroblast growth factor-2. Here, we present the first in vivo study of size-fractionated heparin oligosaccharides in four models of angiogenesis that are progressively less dependent on fibroblast growth factor-2.

METHODS

Heparin oligosaccharides were prepared using size-exclusion gel filtration chromatography and characterized through depolymerization and strong anion exchange high-performance liquid chromatography. Size-defined oligosaccharides (20 mg/kg/d) were given to mice bearing s.c. sponges that were injected with fibroblast growth factor-2 (100 ng/d). After 14 days, octasaccharides and decasaccharides reduced the microvessel density to levels below control. In a second experiment, HEC-FGF2 human endometrial cancer cells that overexpress fibroblast growth factor-2 were implanted in a hollow fiber placed s.c. in vivo. Oligosaccharides were given at 20 mg/kg/d for 2 weeks and the data again showed that octasaccharides significantly reduced microvessel density around the fiber (P = 0.03). In a more complex model, where angiogenesis was induced by a broad spectrum of growth factors, including vascular endothelial growth factor, we implanted H460 lung carcinoma cells in hollow fibers and treated the animals with oligosaccharides at 20 mg/kg/d over 3 weeks. Octasaccharides reduced the microvessel density to that of control. Preliminary investigation of 6-O-desulfated heparins showed that these also had antiangiogenic activity.

RESULTS

Finally, we examined the inhibitory potential of hexasaccharides and octasaccharides given at 20 mg/kg/d and these inhibited the growth of H460 lung carcinoma in vivo. At clinically attainable concentrations, significant anticoagulation (activated partial thromboplastin time, anti-factor Xa, and anti-factor IIa) was not observed in vitro unless species containing>> or =16 saccharide residues were investigated.

CONCLUSIONS

Thus, our preclinical data show that heparin octasaccharides represent novel antiangiogenic compounds that can be given without the anticoagulant effects of low molecular weight heparin.

OBJECTIVE

To determine the gene-expression profile in dermal fibroblasts from type 1 tight-skin (Tsk1) mice, and to examine the expression and potential fibrotic activity of monocyte chemoattractant protein 3 (MCP-3) in Tsk1 mouse and human systemic sclerosis (SSc) skin.

METHODS

Complementary DNA microarrays (Atlas 1.2) were used to compare Tsk1 fibroblasts with non-Tsk1 littermate cells at 10 days, 6 weeks, and 12 weeks of age. Expression of MCP-3 protein was assessed by Western blotting of fibroblast culture supernatants, and localized in the mouse and human skin biopsy samples by immunohistochemistry. Activation of collagen reporter genes by MCP-3 was explored in transgenic mouse fibroblasts and by transient transfection assays.

RESULTS

MCP-3 was highly overexpressed by neonatal Tsk1 fibroblasts and by fibroblasts cultured from the lesional skin of patients with early-stage diffuse cutaneous SSc. Immunolocalization confirmed increased expression of MCP-3 in the dermis of 4 of 5 Tsk1 skin samples and 14 of 28 lesional SSc skin samples, compared with that in matched healthy mice (n = 5) and human controls (n = 11). Proalpha2(I) collagen promoter-reporter gene constructs were activated by MCP-3 in transgenic mice and by transient transfection assays. This response was maximal between 16 and 24 hours of culture and mediated via sequences within the proximal promoter. The effects of MCP-3 could be diminished by a neutralizing antibody to transforming growth factor beta.

CONCLUSIONS

We demonstrate, for the first time, overexpression of MCP-3 in early-stage SSc and in Tsk1 skin, and suggest a novel role for this protein as a fibrotic mediator activating extracellular matrix gene expression in addition to promoting leukocyte trafficking. This chemokine may be an important early member of the cytokine cascade driving the pathogenesis of SSc.

Seven clones encoding interferon response element binding <em>factors</em> have been isolated from a mouse <em>fibroblast</em> lambda gt11 cDNA library by using a 32P end-labeled tandem trimer of the mouse (2'-5')oligoadenylate synthetase gene interferon response element as a probe. Clone <em>16</em> shares strong similarity (95%) at both DNA and amino acid level with YB-1, a human major histocompatibility complex class II Y-box DNA-binding protein, and with dbpB, a human epidermal <em>growth</em> <em>factor</em> receptor gene enhancer region binding protein. The product of the gene represented by clone <em>16</em> may represent a <em>factor</em> that regulates multiple genes by binding to a variety of 5' regulatory elements. Clone 25 is a 2407-base-pair-long cDNA and contains a putative 311-amino acid open reading frame corresponding to an estimated mass of 35.5 kDa. This putative protein, designated as interferon response element binding <em>factor</em> 1 (IREBF-1), contains an acidic domain, three heptad repeat leucine arrays, and a region that shares similarity with the yeast transcriptional <em>factor</em> GAL4 DNA-binding domain. Furthermore, the C terminus of IREBF-1 shows an unusual amphipathic property: within a 79-amino acid range, one side of the alpha-helical region contains a preponderance of hydrophobic amino acids and the other side contains hydrophilic amino acids. This type of structure provides a strong hydrophobic force for protein-protein interaction.

Human skin <em>fibroblasts</em> are readily accessible cells for propagation in culture without transformation that can serve for direct pathophysiology studies in subjects with inherited diseases. We thus examined by quantitative fluorescent cDNA microarray analysis the effect of thyroid hormone (TH) on the expression of more than 15,000 genes in <em>fibroblasts</em> of two normal individuals. <em>Fibroblasts</em> from two subjects with resistance to thyroid hormone (RTH) due to mutations in the TH receptor-beta gene were used to confirm the specificity of the hormonal effect by the ability to discriminate between normal cells and cells with a defect in TH action. Microarray analysis identified 148 genes induced by 1.4-fold or more and five genes repressed to 0.7 or less 24 h after treatment with 2 x 10(-9) M T(3). Taking into account duplicate genes, these represented 91 up-regulated and five down-regulated genes, respectively. Confirmation by real-time PCR was obtained in eight of 10 induced and two of three repressed genes that were tested. Further evidence for T(3)-specific induction was provided by a graded dose response absent in <em>fibroblasts</em> from the patients with RTH. The following genes not previously known to be induced by TH were identified and validated: aldo-keto reductase family 1 C1-3, collagen type VI alpha 3, member RAS oncogene family brain antigen RAB3B, platelet phosphofructokinase, hypoxia-inducible <em>factor</em>-1 alpha, and enolase 1 alpha. These genes as well as three known to be TH regulated in other species and found in this study also in human cells (glucose transporter 1, solute carrier family <em>16</em> member 3, and basic transcription element-binding protein 1) have a variety of regulatory functions in development and metabolism. TH seems to induce these genes by initiating either genomic or nongenomic mechanisms. Surprisingly, TH-mediated down-regulation of <em>fibroblast</em> <em>growth</em> <em>factor</em> 7 and alcohol dehydrogenase 1B persisted in <em>fibroblasts</em> from patients with RTH. This first systematic study of TH-mediated gene expression in normal human cells identifies several new TH-responsive genes and demonstrates that skin <em>fibroblasts</em> are suitable for the study of TH action in health and disease.

OBJECTIVE

We previously reported that the level of basic fibroblast growth factor (bFGF) is high in cerebrospinal fluid (CSF) taken from patients with moyamoya disease. The present study investigated the levels of other angiogenic growth factors in the CSF of moyamoya patients and the clinical significance of bFGF in moyamoya disease.

METHODS

The levels of bFGF, interleukin-8, platelet-derived growth factor, transforming growth factor-beta, endothelial growth factor, and vascular endothelial cell growth factor in CSF, taken from 38 patients with moyamoya disease and 16 patients with atherosclerotic occlusive disease (control group), were measured by an enzyme-linked immunosorbent assay. We analyzed the correlation between the level of bFGF and the clinical factors of age, onset pattern, development of neovascularization, and cerebral circulation.

RESULTS

The CSF of moyamoya patients contained a high concentration of bFGF to a significant (P < .05) extent. The bFGF level was apparently elevated in the patients in whom neovascularization from indirect revascularization, such as encephaloduroarteriosynangiosis, was well developed (P < .01). A linear correlation between the values of bFGF and cerebral vascular response to acetazolamide (r = .7; P < .05) was revealed. The other angiogenic factors were not significantly high compared with the control group.

CONCLUSIONS

The elevation of bFGF in moyamoya disease seems to be specific and is not related simply to cerebral ischemia. Clinically, the bFGF level is a useful indicator to predict the efficacy of indirect revascularization after surgery.

Fibrous dysplasia (FD) patients sometimes suffer from concomitant hypophosphatemic rickets/osteomalacia, resulting from renal phosphate wasting. It was recently reported that FD tissue in the patients with McCune-Albright syndrome (MAS) expressed <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF-23), which is now known to be as a pathogenic phosphaturic <em>factor</em> in patients with oncogenic osteomalacia and X-linked hypophosphatemic rickets. Since it remains controversial whether serum phosphate levels are influenced by FGF23 expressions in FD tissue, isolated FD patients without MAS syndrome were examined for the relationship between FGF23 expressions, circulating levels of FGF-23 and phosphate to negate the effects of MAS-associated endocrine abnormalities on serum phosphate. Eighteen paraffin embedded FD tissues and 2 frozen tissues were obtained for the study. Sixteen of 18 isolated FD tissues were successfully analyzed GNAS gene, which exhibited activated mutations observed in MAS. Eight of <em>16</em> FD tissues, which exhibited GNAS mutations, revealed positive staining for FGF-23. These evidence indicate that postzygotic activated mutations of GNAS is necessary for the FD tissue formation by mosaic distribution of mutated osteogenic cell lineage, but is not sufficient to elevate FGF23 expression causing generalized osteomalacia with severe renal phosphate wasting. The expression level of FGF23 in isolated FD tissue with hypophosphatemic osteomalacia determined by real-time PCR was abundant close to the levels in OOM tumors. Osteoblasts/osteocytes in woven bone were predominant source of circulating FGF-23 in FD tissues by immunohistochemistry. A negative correlation of the intensity of FGF-23 staining with serum inorganic phosphate levels indicated that the expression of FGF23 in focal FD tissues could be a prominent determinant of serum phosphate levels in isolated FD patient. These data provide novel insights into the regulatory mechanism of serum inorganic phosphate levels in isolated FD patients and extend the notion that FGF-23 originating from FD tissue may cause hypophosphatemia not only in isolated FD patients but also in the patients with MAS syndrome.

Hyperplasia of airway smooth muscle cells is present in the airways of asthmatic patients and may contribute to the development of the bronchial hyperresponsiveness that occurs in these patients. Because tryptase is an abundant component of mast cell granules and has demonstrated <em>growth</em>-stimulatory effects in other mesenchymal cells (J. Clin. Invest. 1991; 88:493-499), the goal of our study was to determine whether tryptase is a mitogen for airway smooth muscle cells. The mitogenic effects of tryptase were tested in passages 1 through 5 of dog tracheal smooth muscle cells, either by counting smooth muscle cells or by monitoring uptake of bromodeoxyuridine (BrdU) into cellular DNA during S-phase. With respect to its efficacy, at a near maximal concentration (4 nM), tryptase increased cell numbers 2.1 +/- 0.2- or 2.8 +/- 0.6-fold above controls after 2 or 4 days, respectively, and these increases were approximately the same as those induced by platelet-derived <em>growth</em> <em>factor</em> (50 ng/ml) or 10% calf serum. With respect to potency, tryptase caused concentration-dependent increases in BrdU uptake, as detected in an enzyme-linked immunosorbent assay or by counting BrdU-labeled nuclei, with an EC50 of 2 nM. Pretreatment of tryptase with diisopropylfluorophosphate, to reduce markedly its catalytic as a activity as a proteinase, attenuated its <em>growth</em>-stimulated effects by 58 +/- <em>16</em>%. Tryptase-induced mitogenesis was not a nonspecific effect of all serine proteinases, because thrombin, another proteinase with mitogenicity for <em>fibroblasts</em>, stimulated neither increases in cell counts nor BrdU uptake in our cells. We conclude that tryptase is a potent mitogen for airway smooth muscle cells in culture.

Previously, we have shown that prostaglandins are necessary, but not sufficient, for the stimulation of mitogenesis in BALB/c 3T3 <em>fibroblasts</em> by epidermal <em>growth</em> <em>factor</em> (EGF) (Nolan, R. D., Danilowicz, R. M., and Eling, T. E. (1988) Mol. Pharmacol. 33, 650-656). The purpose of this work was to extend these findings to another potent mitogen, platelet-derived <em>growth</em> <em>factor</em> (PDGF), and to determine if metabolism of arachidonic acid to prostaglandins is necessary for stimulation of expression of the protooncogene c-myc by EGF, which is an early event in the mitogenic cascade. In BALB/c 3T3 cells grown to about 70% confluence and deprived of serum for <em>16</em>-24 h, PDGF stimulated [3H]thymidine uptake into DNA significantly in a concentration-dependent manner, but did not increase production of prostaglandin E2 (PGE2). The addition of indomethacin, a prostaglandin H synthase inhibitor, or nordihydroguaiaretic acid, a lipoxygenase inhibitor, did not affect PDGF-stimulated thymidine uptake into DNA. In addition, PGE2 enhanced EGF-dependent, but not PDGF-dependent, mitogenesis. Taken together, the data support the hypothesis that prostaglandins are not involved in PDGF-dependent mitogenesis. In contrast, indomethacin (10(-6) M) and nordihydroguaiaretic acid (10(-6) M) inhibited EGF-stimulated thymidine uptake and c-myc expression by approximately 50%. Addition of PGG2 (10(-7) to 10(-5) M) in the presence of indomethacin and EGF restored the ability of EGF to elevate c-myc RNA levels and DNA synthesis. When PGF2 alpha (10(-8) to 10(-5) M) was added in the presence of EGF, c-myc RNA levels and thymidine incorporation were elevated up to 5-6-fold above levels observed with EGF alone. These data support the hypothesis that metabolism of arachidonic acid to prostaglandins is necessary for stimulation of c-myc expression by EGF in BALB/c 3T3 cells.

OBJECTIVE

To analyse patterns of gene expression for peptide regulatory factors in patients with Dupuytren's contracture.

METHODS

Tissue samples (palmer fascia) from 12 patients with Dupuytren's contracture and 12 controls were studied using the reverse transcription/polymerase chain reaction (RT/PCR) technique.

RESULTS

Tissue from patients with Dupuytren's contracture expressed a higher percentage of peptide regulatory factors than that of controls: interleukin-1 alpha (83% v 16%; p < 0.01); interleukin-1 beta (66% v 8%; p < 0.01); transforming growth factor beta (75% v 25%; p < 0.02); and basic fibroblast growth factor (66% v 25%; p < 0.05). Platelet derived growth factors alpha and beta were also expressed more commonly (66% v 33% and 25% v 16%, respectively), but these differences were not significant.

CONCLUSIONS

The increased prevalence of expression for the above mRNAs in Dupuytren's tissue is relevant as interleukin-1, basic fibroblast growth factor, and transforming growth factor beta stimulate the growth of fibroblasts and transforming growth factor beta also enhances production of collagen and other extracellular matrix proteins. Excessive local release of these peptide regulatory factors may have an important role in the pathogenesis of Dupuytren's contracture.

The DBA/2FG-pcy mouse has a form of slowly progressive kidney disease that appears similar in many respects to that seen in the autosomal dominant form of human polycystic kidney disease. The aim of this study was to examine the mRNA expression of <em>growth</em>-related proteins in kidney obtained from DBA/2FG-pcy mice and control DBA/2 mice at 8, <em>16</em>, and 30 wk of age. The mRNA levels encoding for proliferating cell nuclear antigen (PCNA), transforming <em>growth</em> <em>factor</em> (TGF)-beta, platelet-derived <em>growth</em> <em>factor</em> (PDGF)-A and PDGF-B chains, insulin-like <em>growth</em> <em>factor</em> (IGF)-I, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) were increased with the progression of cystic lesions in the kidneys of DBA/2FG-pcy mice. At 30 wk of age, mRNA levels of PCNA, TGF-beta, PDGF-A and PDGF-B chains, IGF-I, and bFGF were increased 5.4-fold, 4.8-fold, 4.4-fold, 3.8-fold, 3.7-fold, and 4.6-fold, respectively, compared with those of control DBA/2 mice. In contrast, mRNA levels for epidermal <em>growth</em> <em>factor</em> in kidney of DBA/2FG-pcy mice decreased with age as compared with those of DBA/2 mice. These results suggest that decreased epidermal <em>growth</em> <em>factor</em> mRNA expression and increased expression of PCNA, TGF-beta, PDGF-A and PDGF-B chains, IGF-I, and bFGF mRNA may contribute to the progression of cystic lesions in DBA/2FG-pcy mice.

The thyroid hormone triiodothyronine (T3) activates thermogenesis by uncoupling electron transport from ATP synthesis in brown adipose tissue (BAT) mitochondria. Although T3 can induce thermogenesis by sympathetic innervation, little is known about its cell autonomous effects on BAT mitochondria. We thus examined effects of T3 on mitochondrial activity, autophagy, and metabolism in primary brown adipocytes and BAT and found that T3 increased fatty acid oxidation and mitochondrial respiration as well as autophagic flux, mitophagy, and mitochondrial biogenesis. Interestingly, there was no significant induction of intracellular reactive oxygen species (ROS) despite high mitochondrial respiration and UCP1 induction by T3. However, when cells were treated with Atg5 siRNA to block autophagy, induction of mitochondrial respiration by T3 decreased, and was accompanied by ROS accumulation, demonstrating a critical role for autophagic mitochondrial turnover. We next generated an Atg5 conditional knockout mouse model (Atg5 cKO) by injecting Ucp1 promoter-driven Cre-expressing adenovirus into Atg5Flox/Flox mice to examine effects of BAT-specific autophagy on thermogenesis in vivo. Hyperthyroid Atg5 cKO mice exhibited lower body temperature than hyperthyroid or euthyroid control mice. Metabolomic analysis showed that T3 increased short and long chain acylcarnitines in BAT, consistent with increased β-oxidation. T3 also decreased amino acid levels, and in conjunction with SIRT1 activation, decreased MTOR activity to stimulate autophagy. In summary, T3 has direct effects on mitochondrial autophagy, activity, and turnover in BAT that are essential for thermogenesis. Stimulation of BAT activity by thyroid hormone or its analogs may represent a potential therapeutic strategy for obesity and metabolic diseases. Abbreviations: ACACA: acetyl-Coenzyme A carboxylase alpha; AMPK: AMP-activated protein kinase; Acsl1: acyl-CoA synthetase long-chain family member 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATP: adenosine triphosphate; BAT: brown adipose tissue; cKO: conditional knockout; COX4I1: cytochrome c oxidase subunit 4I1; Cpt1b: carnitine palmitoyltransferase 1b, muscle; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DIO2: deiodinase, iodothyronine, type 2; DMEM: Dulbecco's modified Eagle's medium; EIF4EBP1: eukaryotic translation initiation <em>factor</em> 4E binding protein 1; Fabp4: fatty acid binding protein 4, adipocyte; FBS: fetal bovine serum; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FGF: <em>fibroblast</em> <em>growth</em> <em>factor</em>; FOXO1: forkhead box O1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; Gpx1: glutathione peroxidase 1; Lipe: lipase, hormone sensitive; MAP1LC3B: microtubule-associated protein 1 light chain 3; mRNA: messenger RNA; MTORC1: mechanistic target of rapamycin kinase complex 1; NAD: nicotinamide adenine dinucleotide; Nrf1: nuclear respiratory <em>factor</em> 1; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; Pnpla2: patatin-like phospholipase domain containing 2; Prdm<em>16</em>: PR domain containing <em>16</em>; PRKA: protein kinase, AMP-activated; RPS6KB: ribosomal protein S6 kinase; RFP: red fluorescent protein; ROS: reactive oxygen species; SD: standard deviation; SEM: standard error of the mean; siRNA: small interfering RNA; SIRT1: sirtuin 1; Sod1: superoxide dismutase 1, soluble; Sod2: superoxide dismutase 2, mitochondrial; SQSTM1: sequestosome 1; T3: 3,5,3'-triiodothyronine; TFEB: transcription <em>factor</em> EB; TOMM20: translocase of outer mitochondrial membrane 20; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); ULK1: unc-51 like kinase 1; VDAC1: voltage-dependent anion channel 1; WAT: white adipose tissue.

Graves' disease when fully expressed affects the thyroid gland and connective tissues of the orbit and pretibium. While the glandular disease is relatively well-characterized, the pathogenesis of the orbital and dermal components remains enigmatic. In the following article, we review some of the evidence suggesting that <em>fibroblast</em> activation in Graves' disease might play an integral role in the tissue remodeling associated with ophthalmopathy. The thyrotropin receptor (TSHR) is expressed at low levels in several connective tissue depots and by their derivative <em>fibroblasts</em>, including those from the orbit. Little direct evidence currently links extra-thyroidal TSHR expression with Graves' disease. Very recent observations now implicate the insulin-like <em>growth</em> <em>factor</em>-1 receptor (IGF-1R) as a <em>fibroblast</em> activating antigen. When immunoglobulins from patients with the disease, with or without clinical ophthalmopathy, bind IGF-1R on the surface of <em>fibroblasts</em>, the receptor becomes activated and upregulates the expression of two T lymphocyte chemoattractants, IL-<em>16</em> and RANTES. Thus, IGF-1R may represent a second self-antigen with a pathogenic role in extra-thyroidal Graves' disease.

Phototherapy stimulates metabolic processes in healing wounds. Despite worldwide interest, phototherapy is not firmly established or practiced in South Africa. This study aimed to determine which dose and wavelength would better induce healing in vitro. Diabetic-induced wounded <em>fibroblasts</em> were irradiated with 5 or <em>16</em> J/cm(2) at 632.8, 830, or 1,064 nm. Cellular morphology, viability (Trypan blue and apoptosis), and proliferation (basic <em>fibroblast</em> <em>growth</em> <em>factor</em>) were then determined. Cells irradiated with 5 J/cm(2) at 632.8 nm showed complete wound closure and an increase in viability and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) expression. Cells irradiated at 830 nm showed incomplete wound closure and an increase in bFGF expression. Cells irradiated at 1,064 nm showed incomplete closure and increased apoptosis. All cells irradiated with <em>16</em> J/cm(2) at all three wavelengths showed incomplete wound closure, increased apoptosis, and decreased bFGF expression. This study showed that diabetic-wounded cells respond in a dose- and a wavelength-dependent manner to laser light. Cells responded the best when irradiated with a fluence of 5 J/cm(2) at a wavelength of 632.8 nm.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a potent mitogenic and angiogenic <em>factor</em> that is known to regulate GH, PRL, and TSH secretion. Sequences within a bFGF gene family member have been detected in transforming DNA samples derived from human PRL-secreting tumors. Furthermore, elevated serum concentrations of bFGF have been noted in patients with multiple endocrine neoplasia-1. To further examine the significance of bFGF in sporadic human pituitary adenomas, we investigated the expression of bFGF by these tumors. Using an enzyme-linked immunoassay that recognizes all <em>16</em>-24 kilodalton molecular mass forms of bFGF, we measured circulating serum concentrations in 21 patients with sporadic pituitary adenomas; they ranged from less than 0.5-84 pg/mL and declined following surgical adenomectomy. To confirm the pituitary source of this <em>growth</em> <em>factor</em>, we determined in vitro bFGF release from 43 adenomas (10 GH, 7 PRL, 10 ACTH, 14 gonadotrope adenomas/oncocytomas, and 2 silent subtype 3 adenomas). bFGF was present with wide variability (0.75-2100 pg/24 h.10(5) cells) in conditioned culture media of all adenomas examined. The adenohypophysial source of this <em>growth</em> <em>factor</em> was further demonstrated by the reverse hemolytic plaque assay. Variable bFGF messenger RNA expression was identified by the reverse-transcription polymerase chain reaction technique in 9 functional (2 PRL, 5 GH, 2 ACTH) and 7 nonfunctional (1 oncocytoma, 2 null cell, 2 gonadotrope, 2 Silent Subtype 3) adenomas examined. bFGF levels were unaltered in vitro following hypothalamic hormone stimulation/inhibition. The lack of a bFGF signal peptide sequence and hypothalamic hormone-independence suggest that secretion of this <em>factor</em> may be independent of pituitary hormone regulation. Immunocytochemistry failed to localize bFGF in tumors that released this <em>factor</em> in vitro, suggesting that storage of this peptide does not correlate with its synthesis and release. In conclusion, the heterogenous expression of bFGF suggests that it may play a specific and selective role in the tumorigenic process of some pituitary adenomas.

We have determined that Strong's Luxoid (lstJ) [corrected] mice have a <em>16</em> bp deletion in the homeobox region of the Alx-4 gene. This deletion, which leads to a frame shift and a truncation of the Alx-4 protein, could cause the polydactyly phenotype observed in lstJ [corrected] mice. We have cloned the chick homologue of Alx-4 and investigated its expression during limb outgrowth. Chick Alx-4 displays an expression pattern complementary to that of shh, a mediator of polarizing activity in the limb bud. Local application of Sonic hedgehog (Shh) and <em>Fibroblast</em> <em>Growth</em> <em>Factor</em> (FGF), in addition to ectodermal apical ridge removal experiments suggest the existence of a negative feedback loop between Alx-4 and Shh during limb outgrowth. Analysis of polydactylous mutants indicate that the interaction between Alx-4 and Shh is independent of Gli3, a negative regulator of Shh in the limb. Our data suggest the existence of a negative feedback loop between Alx-4 and Shh during vertebrate limb outgrowth.

To identify the clinical relevance of cytokines involved in the development of lung fibrosis observed in patients with coal workers' pneumoconiosis (CWP), we investigated the BAL fluid contents and AM secretions of three mediators that modulate <em>fibroblast</em> <em>growth</em>: platelet-derived <em>growth</em> <em>factor</em> (PDGF), Type I insulin-like <em>growth</em> <em>factor</em> (IGF-I), and transforming <em>growth</em> <em>factor</em> Type beta (TGF-beta). Our study population consisted of 25 patients with CWP (<em>16</em> simple pneumoconiosis, SP, 9 progressive massive fibrosis, PMF, 9 control subjects, and 6 patients with idiopathic pulmonary fibrosis (IPF). The fibrotic potency of AM supernatants was also tested for their ability to promote the <em>growth</em> of a human lung <em>fibroblast</em> cell line appreciated by [3H]-thymidine incorporation. PDGF and IGF-I concentrations were increased in BAL fluids of patients with PMF compared with SP and control subjects, whereas TGF-beta concentration was significantly higher in BAL fluid of patients with SP compared with PMF and control subjects. PDGF, IGF-I, and TGF-beta concentrations in AM supernatants followed the same profile observed in BAL fluids, suggesting that AM is one of the main cell sources of PDGF, IGF-I, and TGF-beta in the lung of pneumoconiotic patients. After treatment by acidification, which activated the latent form of TGF-beta, AM from patients with SP induced an inhibition of [3H]-thymidine incorporation and <em>fibroblast</em> <em>growth</em> was restored after neutralization of TGF-beta by specific antibodies. In contrast, AM supernatants from patients with PMF and IPF promoted the proliferation of <em>fibroblasts</em> and treatment by acidification did not modify this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to understand the regulation of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) gene expression, we have cloned and characterized the human bFGF gene and its regulatory elements. Using restriction endonuclease digestion, we have mapped the entire gene and sequenced all intron/exon boundaries to confirm authenticity and to determine organization. The data show that intron 1 is at least <em>16</em> kb long while intron 2 is <em>16</em> kb long. The human bFGF gene, including its three exons, is therefore at least 36 kb long. There are five GC boxes which may represent SP-1 binding sites and one potential AP-1 binding site within the core promoter region. Primer extension analysis indicates the presence of one bFGF-RNA transcription start site. We used a standard bacterial CAT gene expression system to identify the DNA sequence containing the functional bFGF gene promoter. Deletion analysis suggests the presence of two negative regulatory elements; one in the non-transcribed 5'-promoter region and the other within transcribed (but non-translated) sequences 3' of the promoter core.

To investigate the clinical significance of circulating angiogenic <em>factors</em>, especially in association with early relapse of osteosarcoma, we quantified pre-therapeutic levels of vascular endothelial <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and placenta <em>growth</em> <em>factor</em> in the sera of <em>16</em> patients with osteosarcoma using an enzyme-linked immunosorbent assay. After a 1-year follow-up, the serum level of angiogenic <em>factors</em> was analysed with respect to microvessel density of the biopsy specimen and clinical disease relapse. The serum vascular endothelial <em>growth</em> <em>factor</em> levels were positively correlated with the microvessel density with statistical significance (P=0.004; Spearman rank correlation) and also significantly higher in seven patients who developed pulmonary metastasis than the remaining nine patients without detectable disease relapse (P=0.0009; The Mann-Whitney U-test). In contrast, the serum levels of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> or placenta <em>growth</em> <em>factor</em> failed to show significant correlation with the microvessel density or relapse of the disease. Although there was no significant correlation between serum vascular endothelial <em>growth</em> <em>factor</em> levels and the tumour volume, the serum vascular endothelial <em>growth</em> <em>factor</em> levels were significantly higher in patients with a vascular endothelial <em>growth</em> <em>factor</em>-positive tumour than those with a vascular endothelial <em>growth</em> <em>factor</em>-negative tumour. These findings suggest that the pre-therapeutic serum vascular endothelial <em>growth</em> <em>factor</em> level reflects the angiogenic property of primary tumour and may have a predictive value on early disease relapse of osteosarcoma.

BACKGROUND

We previously identified amplification of the fibroblast growth factor receptor 1 gene (FGFR1) as a potential therapeutic target for small-molecule inhibitor therapy in squamous cell lung cancer (L-SCC). Currently, clinical phase I trials are underway to examine whether patients with FGFR1-amplified L-SCC benefit from a targeted therapy approach using small-molecule inhibitors. Because most patients with lung cancer present with metastatic disease, we investigated whether lymph node metastases in L-SCC share the FGFR1 amplification status of their corresponding primary tumor.

METHODS

The study cohort consisted of 72 patients with L-SCC, 39 with regional lymph node metastases. Tissue microarrays were constructed from formalin-fixed, paraffin-embedded tissue of the primary tumors and, where present, of the corresponding lymph node metastasis. A biotin-labeled target probe spanning the FGFR1 locus (8p11.22-23) was used to determine the FGFR1 amplification status by fluorescence in situ hybridization.

RESULTS

FGFR1 amplification was detected in 16% (12 of 72) of all primary L-SCCs. In metastatic tumors, 18% (seven of 39) of the lymph node metastases displayed FGFR1 amplification with an exact correlation of FGFR1 amplification status between tumor and metastatic tissue.

CONCLUSIONS

FGFR1 amplification is a common genetic event occurring at a frequency of 16% in L-SCCs. Moreover, lymph node metastases derived from FGFR1-amplified L-SCCs also exhibit FGFR1 amplification. Therefore, we suggest that the FGFR1 amplification is a clonal event in tumor progression. Beyond this biologically relevant observation, the findings carry potential therapeutic implications in that small-molecule inhibitors may be applicable to the treatment of a subset of patients with metastatic L-SCC.

Ornithine decarboxylase activity was assessed in serum-deprived quiescent NIH-3T3 murine <em>fibroblasts</em> after exposure to a variety of <em>growth</em>-promoting <em>factors</em>. Ornithine decarboxylase activity increased after treatment with phorbol 12-myristate 13-acetate (PMA), fetal calf serum, bovine pituitary <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), platelet-derived <em>growth</em> <em>factor</em> (PDGF), and the synthetic diacyglycerol sn-1,2-dioctanolyglycerol but not after treatment with epidermal <em>growth</em> <em>factor</em>, insulin, 4 alpha-phorbol 12,13-didecanoate, sn-1,2-dibutyrylglycerol, or the calcium ionophore A23187. Activity peaked at 3-4 h and returned to basal levels after 8 h. To determine the importance of protein kinase C in this increase, cells were pretreated with PMA for <em>16</em> h to make the cells effectively deficient in protein kinase C; this deficiency was documented by direct measurement of enzyme activity and immunoreactivity. The ornithine decarboxylase response to each mitogen was then compared in cells pretreated with PMA or control conditions. PMA pretreatment abolished the increase in ornithine decarboxylase activity due to additional PMA and decreased but did not eliminate the ability of serum, FGF, and PDGF to cause increases in ornithine decarboxylase activity. Similarly, pretreatment with PMA abolished the ability of additional PMA to increase ornithine decarboxylase mRNA levels but did not prevent the increases in these mRNA levels caused by FGF or serum. These data suggest that the increases in ornithine decarboxylase activity and mRNA levels that occur in quiescent <em>fibroblasts</em> in response to serum, FGF, or PDGF are due to activation of at least two separate pathways, one involving protein kinase C and the other independent of protein kinase C.

The cranial sutures are the primary sites of bone formation during skull <em>growth</em>. Morphogenesis and phenotypic maintenance of the cranial sutures are regulated by tissue interactions, especially those with the underlying dura mater. Removal of the dura mater in fetuses causes abnormal suture development and premature suture obliteration. The dura mater interacts with overlying tissues of the cranial vault by providing: (1) intercellular signals, (2) mechanical signals and (3) cells, which undergo transformation and migrate to the suture. The intercellular signaling governing suture development employs the <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs). In rats during formation of the sutures in the fetus, FGF-1 is localized mainly in the dura mater, while other FGFs are expressed in the overlying tissues. By birth, FGF-2 largely replaces FGF-1 in the dura mater. FGFs present in the calvaria bind either the IIIb or IIIc mRNA splice variants of the FGF receptors (FGFRs) 1, 2, or 3. Monoclonal antibodies to the b variant of FGFR2 were used to determine the distribution of FGFR2IIIb during suture development and its extracellular localization. FGFR2IIIb is present in association with mature osteoblasts and osteogenic precursor cells of the suture in the fetus. Ectodomains of FGFR2IIIb, the products of proteolytic cleavage of the receptors, were present throughout the extracellular matrix of sutures resisting obliteration (coronal and sagittal), but absent from the core of sutures undergoing normal fusion (posterior intrafrontal). This observation is consistent with a possible mechanism, in which truncated receptors bind FGFs, thus regulating free FGF available to nearby cells. Mechanical signaling in the calvaria results from tensional forces in the dura mater generated during rapid expansion of the neurocranium. Posterior intrafrontal sutures of rats, which fuse between days <em>16</em> and 24, were subjected to cyclical tensional forces in vitro. Significant delay in the timing of suture fusion and increases in the expression domains of FGFR1 and 2 were observed, demonstrating the sensitivity of suture patency to mechanical signals and a possible role of the FGF system in mediating such stimuli. Finally, cells of the dura mater beneath the intrafrontal and sagittal sutures were observed to undergo a morphological transformation to a dendritic morphology and migrate into the suture mesenchyme between days 10 and <em>16</em> of development. This process may participate in suture and bone morphogenesis and influence the patency of the sutures along the anterior-posterior axis.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) play important roles in embryonic development, angiogenesis, wound healing, and cell proliferation and differentiation. In search of inhibitors of FGFR1 kinase, 2.2 million compounds were docked into the ATP binding site of the protein. A co-crystal structure, which shows two alternative conformations for the nucleotide binding loop, is reported. Docking was performed on both conformations and, ultimately, 23 diverse compounds were purchased and assayed. Following hit validation, two compounds 10 and <em>16</em>, a benzylidene derivative of pseudothiohydantoin and a thienopyrimidinone derivative, respectively, were discovered that inhibit FGFR1 kinase with IC(50) values of 23 and 50 microM. Initial optimization of <em>16</em> led to the more unsaturated 40, which has significantly enhanced potency, 1.9 microM. The core structures represent new structural motifs for FGFR1 kinase inhibitors. The study also illustrates complexities associated with the choice of protein structures for docking, possible use of multiple kinase structures to seek selectivity, and hit identification.

BACKGROUND

Advanced hepatocellular carcinoma (HCC) in humans is characterized by hypervascularity. In the present study, the expressions of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2) and endostatin were analyzed in patients with chronic liver disease to clarify the effect of these major angiogenic factors.

METHODS

Serum concentrations of VEGF, FGF-2 and endostatin in 24 patients with HCC, 16 patients with liver cirrhosis (LC) and 13 healthy volunteers were measured by enzyme-linked immunosorbent assay. The expression of VEGF in 21 surgically resected HCC samples was analyzed by immunohistochemistry, and that of VEGF isoforms in 15 HCC samples was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS

Serum VEGF, FGF-2 and endostatin concentrations were significantly elevated in patients with HCC compared with healthy volunteers; but there was no significant difference between patients with HCC and those with non-HCC liver disease. Immunohistochemical analysis showed that VEGF protein was strongly expressed in both well-differentiated HCC cells and non-cancerous hepatocytes, whereas in moderately and poorly differentiated HCC the expression was stronger in the endothelial cells (EC) lining intratumor vessels than in the cancer cells. On RT-PCR for VEGF isoforms it was found that VEGF-121, VEGF-165 and VEGF-189 were expressed in all but one of the HCC samples and in all corresponding non-HCC samples.

CONCLUSIONS

The results suggest that VEGF, FGF-2, and endostatin concentrations are elevated prior to the emergence of HCC and that the distribution of VEGF changes dynamically during the development of HCC.