The structural gene for human keratinocyte <em>growth</em> <em>factor</em> (KGF), a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family, consists of three coding exons and two introns typical of other <em>fibroblast</em> <em>growth</em> <em>factor</em> loci. A portion of the KGF gene, located on chromosome <em>15</em>, is amplified to approximately 16 copies in the human genome, and these highly related copies (which consist of exon 2, exon 3, the intron between them, and a 3' noncoding segment of the KGF transcript) are dispersed to multiple human chromosomes. The KGF-like sequences are transcriptionally active, differentially regulated in various tissues, and composed of three distinct classes of coding sequences that are 5% divergent from each other and from the authentic KGF sequence. Multiple copies of KGF-like genes were also discovered in the genomic DNAs of chimpanzee and gorilla but were not found in lesser apes (gibbon), Old World monkeys (African green monkey and macaques), mice, or chickens. The pattern of evolutionary occurrence suggests that a primordial KGF gene was amplified and chromosomally dispersed subsequent to the divergence of orangutan from African apes but before the trichotomous divergence of human, chimpanzee, and gorilla 5-8 million years ago. The appearance of a transcriptionally active and chromosomally dispersed multigene KGF family may have implications in the evolution of the great apes and humans.

A specific receptor for insulin-like <em>growth</em> <em>factor</em> I (IGF-I) has been demonstrated in cultured fetal rat osteoblast-like bone cells. Specific binding of [125I]IGF-I to bone cells incubated at <em>15</em> C reached a steady state by 5 h. Half-maximal inhibition of [125I]IGF-I binding by unlabeled IGF-I was observed at 7 ng/ml. Multiplication-stimulating activity, insulin, and proinsulin were less effective than unlabeled IGF-I in competing for receptor occupancy. Scatchard analysis showed a curvilinear plot, with a Ka similar to that observed in human <em>fibroblasts</em>. Incubation of cell monolayers with glucocorticoids resulted in a concentration-dependent increase in [125I]IGF-I binding. This increase in [125I]IGF-I binding was dependent on cell density. After a 2-day exposure to dexamethasone, no increase in binding was observed in sparsely plated cells; however, an increase in binding was observed after 3 days in culture (log phase) and was maximal by 5 days (peak log phase). These data indicate that rat bone cells possess a specific receptor for IGF-I with binding characteristics similar to those reported in human <em>fibroblasts</em>, and that IGF-I receptor concentrations are increased by exposure to glucocorticoids. A role for glucocorticoids and IGF-I in rat bone proliferation is suggested by these findings.

The aim of the study was to measure concentrations of cytokines in wound fluid from donor sites. A film dressing was applied to the donor sites (mean size 10 x <em>15</em> cm) immediately after a split skin graft had been taken. Five days later the film was punctured with a needle and 2-3 ml of the fluid accumulated under the film dressing was withdrawn into a syringe. The fluid was snap frozen and stored immediately at -70 degrees C. The fluid was examined for epidermal <em>growth</em> <em>factor</em> (EGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), transforming <em>growth</em> <em>factor</em> (TGF) alpha, TGF beta, interleukin (IL)-1 alpha and IL-1 beta. The results showed that the fluid accumulated under the film dressing contained <em>growth</em> <em>factors</em> and cytokines that are thought to promote healing. The fluid was rich in TGF alpha but contained no EGF or bFGF, which indicates that TGF alpha plays a major part for promoting local wound healing.

We tested the hypothesis that aged animals are as responsive as the young adult animals in expanding collateral vasculature under a similar treatment of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). Two age groups of male Fischer 344 rats (11 mo old; n = 32, 23 mo old; n = 43) weighing approximately 385 g were subdivided into normal, acute ligation [femoral artery (FA) ligated 3 days before blood flow (BF) measurement] or ligated groups for 16 days and received recombinant human bFGF intra-arterial infusion at doses of 0, 0.5, 5, and 50 microg x kg(-1) x day(-1). BF was determined with (85)Sr- and (141)Ce-labeled microspheres during treadmill running at <em>15</em> and 20 m/min at <em>15</em>% grade. Blood pressure (BP) values were approximately 149 and approximately 163 mmHg (p < 0.05); heart rates were approximately 496 and approximately 512 beats/min in the aged and young adult groups during running, respectively. Maximal collateral BF values were confirmed by no additional BF increase in the calf muscle at the higher speed. Ligation of the FA for 3 days reduced the BF reserve to the calf muscle by approximately 90%. Calf muscle BF was modestly greater (10 ml x min(-1) x 100 g(-1)) by 16 days in the carrier group. bFGF infusion expanded collateral BF in a dose-dependent manner with an increase of 33 and 42 ml x min(-1) x 100 g(-1) (P < 0.001) in the 5 and 50 microg x kg(-1) x day(-1) bFGF groups, respectively. Aged animals showed similar BF improvements as observed with the adult groups in response to ligation surgery and bFGF treatment. Our data indicate that the aged rats (approximately 23 mo old) remain responsive to exogenous bFGF induced in developing collateral-dependent BF as the young adult (approximately 11 mo old) controls. This suggests that the influence of bFGF in expanding collateral BF should not be preempted in the aged group, the population most affected by peripheral arterial insufficiency.

Besides the well-established role of mast cells in allergic reactions as an important source of vasoactive and proinflammatory products, they have been related to tissue fibrosis and remodelling processes. In a heart failure (HF) animal model, mast cells were shown to synthesize transforming growth factor beta1 and basic fibroblast growth factor in myocardial tissue and were localized to an area with fibrosis. Our objective was to quantify mast cell density in left ventricles from patients with congestive heart failure who were candidates for transplantation and to analyze whether they showed a correlation with the fibrosis level of the same area.

METHODS

We obtained myocardial biopsies from 20 patients with end-stage HF secondary to idiopathic dilated cardiomyopathy (iDCM) undergoing heart transplantation and 15 controls (donors without cardiopathy). Mast cells were detected by immunohistochemistry with a human mast cell chymase antibody and fibrosis levels measured with picrosirius red staining of collagen fibrils with later quantification by morphometry.

RESULTS

The patients mean age was 51 +/- 3 years. Fibrosis levels in the myocardial sections from patients with congestive HF was three-fold higher than those in control myocardium (12.41 +/- 1.7% vs 3.98 +/- 0.63%, P < .001). Mast cell density correlated with the collagen fraction and could be fitted to a linear regression curve: collagen fraction = 0.78 + 0.05 mast cell density (n = 33, P < .005, R2 = 0.28).

CONCLUSIONS

The elevated collagen fraction present in failing hearts may be the cause of increased stiffness and loss of elasticity that is detected in patients with end-stage HF. Due to the mast cells capacity to synthesize vasoactive and fibrogenic products and the correlation between their density and fibrosis levels, they probably play a role in the ventricular remodelling in HF.

Biochemical studies have shown that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF or FGF-2) is internalized by two pathways, after binding to either FGF tyrosine kinase receptors or to heparan sulfate proteoglycans (HSPG). To get insights on the HSPG-mediated pathway, we have examined by electron microscopy the intracellular route of bFGF-HRP, a monovalent conjugate of bFGF and horseradish peroxidase which was found to bind to HSPG only and was detectable by electron microscopy. bFGF-HRP association to adult bovine aortic endothelial (ABAE) cells or baby hamster kidney (BHK) cells was inhibited by a high molar excess of native bFGF, a 2 M NaCl wash at neutral pH, heparin and heparan sulfate, but not by chondroitin 4-sulfate or chondroitin 6-sulfate. bFGF-HRP was not able to displace [125I]bFGF from its high-affinity binding sites, and the dissociation constant of its binding to ABAE cells was estimated at 3 nM. Time-course experiments were performed to follow bFGF-HRP endocytosis in ABAE cells. bFGF-HRP was found to enter the cell after binding to the plasma membrane or extracellular matrix. On the cell surface, the probe accumulated in noncoated flask-shaped invaginations and in caveolae rather than in clathrin-coated pits. Immediately after endocytosis, bFGF-HRP was detected in pleiomorphic tubulovesicular and tubulocisternal early endosomes. Multivesicular bodies contained diaminobenzidine (DAB) precipitate after 5 to <em>15</em> min, but lysosomes were not labeled before 1 h, indicating a delayed transfer from late endosomes to lysosomes. Labeling was never detected in the nucleus, even after intensification of the DAB reaction product by silver-gold enhancement. Similar endocytic pathways and intracellular locations were observed in other endothelial and non-endothelial cell types. These results suggest that bFGF associated to HSPG can enter the cell via several pathways and follows mainly a degradative route.

OBJECTIVE

Activating point mutations and protein overexpression of fibroblast growth factor receptors (FGFRs), especially FGFR3, are frequent events in bladder cancer. Little is known about gene amplifications, therefore we characterized amplification of FGFR1-3 by fluorescence in-situ hybridization (FISH).

RESULTS

Tumours of 153 patients (n = 65 pTa low-grade, n = 15 pTa high-grade, n = 37 pT1, n = 20 pT2, n = 10 pT3, n = 6 pT4) were analysed by FISH for FGFR1-3 copy numbers and screened for FGFR3 mutations and immunohistochemical protein expression. Amplifications of FGFR1 were found in 1.6% (two of 122), FGFR2 in 0.8% (one of 121) and FGFR3 in 3.4% (five of 145). All amplifications were high-level amplifications, not overlapping with polysomy. Amplifications were found in papillary/papillary-invasive tumour parts, and predominantly in tumours with enhanced Ki67 index (>10%), aberrant CK20 expression, and low p53 expression. All FGFR3-amplified samples showed concomitant FGFR3 mutations and FGFR3 protein overexpression. FGFR amplifications were not associated significantly with gender, age, grade or stage in statistical analyses.

CONCLUSIONS

FGFR amplifications are rare events in bladder cancer, with FGFR3 amplification being the most prevalent (3.4% of cases). Concomitant FGFR3 mutations and protein overexpression indicate that FGFR3-mediated signalling in these tumours would probably be highly active. This patient subgroup may be particularly suited to FGFR-targeted pharmacotherapy.

1alpha,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), a natural ligand of the vitamin D receptor (VDR), was found to increase the rat ileal Asbt and bile acid absorption. The effects of VDR, whose expression is low in liver, on hepatic transporters and enzymes are unknown. Protein and mRNA levels of target genes in the small intestine, colon and liver after intraperitoneal dosing of 1,25(OH)(2)D(3) (0-2.56 nmol/kg/day for 4 days) to the rat were determined by Western blotting and qPCR, respectively. The 1,25(OH)(2)D(3) treatment increased total Cyp3a protein and Cyp3a1 mRNA expressions in the proximal small intestine, and the short heterodimer partner (SHP), the <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> (FGF<em>15</em>), organic solute transporter (Ostalpha and Ostbeta) mRNA and Asbt protein expressions in the ileum. About 50% higher portal bile acid concentration (65.1+/-14.9 vs 41.9+/-7.8 microm, p<0.05) and elevated expressions of the hepatic farnesoid X receptor (FXR) and SHP mRNA resulted with 1,25(OH)(2)D(3) treatment. Increased Bsep and Ostalpha mRNA expressions in liver and a>50% reduction in the Cyp7a1 protein level (p<0.05) and cholesterol metabolism in rat liver microsomes (p=0.002), likely consequences of the bile acid-FXR-SHP cascade and activation of the signaling pathway for Cyp7a1 inhibition by FGF<em>15</em>, were found. Increased hepatic multidrug resistance-associated protein (Mrp3) and multidrug resistance protein 1a (Mdr1a) mRNA and P-gp protein were also observed. It was concluded that the changes in hepatic transporters and enzymes in the rat were indirect, secondary effects of the liver FXR-SHP cascade due to increased intestinal absorption of bile acids and elevated levels of FGF<em>15</em>, events that led to the activation of FXR.

Effective local delivery to the heart remains an obstacle to successful therapeutic application of a number of drugs and biological agents. This study was designed to study and optimize the delivery characteristics of transendocardial intramyocardial (IM) administration, determine myocardial deposition and retention over time, and compare it to transepicardial IM injection. Thirty-nine pigs were used for the study (<em>15</em> for catheter optimization, <em>15</em> for transendocardial IM delivery, and 9 for transepicardial IM delivery). (125)I-<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2) (25 microCi) was used as the prototype molecule. Tissue and myocardial distribution was determined at 1 and 24 h and 7 days. Using 1-h (125)I-FGF2 myocardial deposition as a parameter for delivery efficiency, the optimal needle length and delivery volume for transendocardial based delivery were determined to be 6 mm and 0.1 ml, respectively. Using these parameters for endocardial delivery, (125)I-FGF2 cardiac activity was 18.01 +/- 3.84% of delivered activity at 1 h, 11.65 +/- 5.17% at 24 h, and 2.32 +/- 0.87% at 7 days in ischemic animals. Studies in nonischemic animals produced similar results. For transepicardial delivery, (125)I-FGF2 cardiac-specific activity was 23.14 +/- 12.67% for the 6-mm needle, declining to 12.32 +/- 8.50% at 24 h, and did not significantly differ from values obtained following transendocardial delivery. Thus, optimized transendocardial intramyocardial delivery using Biosense guidance results in efficient delivery of FGF2 to the target myocardium that is comparable with transepicardial delivery, both providing markedly higher myocardial deposition and retention and lower systemic recirculation of FGF2 than intracoronary, intrapericardial, or intravenous delivery. However, myocardial distribution is limited to injection sites.

Skin is a major source of secretion of the neurotrophic <em>factors</em> nerve <em>growth</em> <em>factor</em> (NGF), brain-derived neurotrophic <em>factor</em> (BDNF), neurotrophin-3 (NT-3), and glial-derived neurotrophic <em>factor</em> (GDNF) controlling cutaneous sensory innervation. Beside their neuronal contribution, we hypothesized that neurotrophic <em>factors</em> also modulate the cutaneous microvascular network. First, we showed that NGF, BDNF, NT-3, and GDNF were all expressed in the epidermis, while only NGF and NT-3 were expressed by cultured <em>fibroblasts</em>, and BDNF by human endothelial cells. We demonstrated that these peptides are highly potent angiogenic <em>factors</em> using a human tissue-engineered angiogenesis model. A 40% to 80% increase in the number of capillary-like tubes was observed after the addition of 10 ng/mL of NGF, 0.1 ng/mL of BDNF, <em>15</em> ng/mL of NT-3, and 50 ng/mL of GDNF. This is the first characterization of the direct angiogenic effect of NT-3 and GDNF. This angiogenic effect was mediated directly through binding with the neurotrophic <em>factor</em> receptors tropomyosin-receptor kinase A (TrkA), TrkB, GFRα-1 and c-ret that were all expressed by human endothelial cells, while this effect was blocked by addition of the Trk inhibitor K252a. Thus, if NGF, BDNF, NT-3, and GDNF may only moderately regulate the microvascular network in normal skin, they might have the potential to greatly increase angiogenesis in pathological situations.

OBJECTIVE

Myofibroblasts express alpha-smooth muscle actin (alphaSMA), are important in tissue repair, and are present in the early phase of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). We wished to determine whether bronchoalveolar lavage fluid (BALF) from ALI/ARDS patients can induce myofibroblast differentiation and if this induction is associated with outcome.

METHODS

A single-center cohort study enrolling patients between 2002 and 2005.

METHODS

Medical intensive care unit of a tertiary care medical center.

METHODS

Adult patients meeting the American-European Consensus Conference definition of ALI/ARDS.

METHODS

BALF was collected from ALI/ARDS patients within 48 hrs of intubation and incubated with normal human lung fibroblasts in vitro, and alphaSMA expression was assessed by reverse transcription polymerase chain reaction. BALF was also collected and tested from negative control patients. ALI/ARDS patients were followed for 28 days or death.

RESULTS

Thirty-one lung injury and 11 negative control patients were enrolled from 2002 to 2005. ALI/ ARDS BALF demonstrated potent alphaSMA induction with a mean value 92% greater than negative controls (34.5% +/- 7.6% vs. 18% +/- 2.4% of maximal transforming growth factor [TGF]-beta1 [5 ng/mL], p < .02). The specific TGF-beta1 receptor inhibitor SB431542 reduced ALI/ARDS BALF-stimulated alphaSMA induction by 52% (p < .005). There was no correlation between ALI/ARDS BALF-induced alphaSMA and procollagen 3 induction (r = -.08, p = .66). The odds ratio for survival was 6.75 (1.15-39.80) times higher for ALI/ARDS patients with alphaSMA induction between 15% and 75% of maximal TGF-beta1 induction (5 ng/mL) than outside this range.

CONCLUSIONS

ALI/ARDS BALF-induced myofibroblast differentiation is partially attributable to TGF-beta1. Procollagen 3 and alphaSMA are regulated by distinct mechanisms in ALI/ARDS and there may be an optimal level of myofibroblast induction that is associated with better outcome.

Keratinocyte <em>growth</em> <em>factor</em> (KGF) is a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family. Portions of the gene encoding KGF were amplified during primate evolution and are present in multiple nonprocessed copies in the human genome. Nucleotide analysis of a representative sampling of these KGF-like sequences indicated that they were at least 95% identical to corresponding regions of the KGF gene. To localize these sequences to specific chromosomal sites in human and higher primates, we used fluorescence in situ hybridization. In human, using a cosmid probe encoding KGF exon 1, we assigned the location of the KGF gene to chromosome <em>15</em>q<em>15</em>-21.1. In addition, copies of KGF-like sequences hybridizing only with a cosmid probe encoding exons 2 and 3 were localized to dispersed sites on chromosome 2q21, 9p11, 9q12-13, 18p11, 18q11, 21q11, and 21q21.1. The distribution of KGF-like sequences suggests a role for alphoid DNA in their amplification and dispersion. In chimpanzee, KGF-like sequences were observed at five chromosomal sites, which were each homologous to sites in human, while in gorilla, a subset of four of these homologous sites was identified; in orangutan two sites were identified, while gibbon exhibited only a single site. The chromosomal localization of KGF sequences in human and great ape genomes indicates that amplification and dispersion occurred in multiple discrete steps, with initial KGF gene duplication and dispersion taking place in gibbon and involving loci corresponding to human chromosomes <em>15</em> and 21. These findings support the concept of a closer evolutionary relationship of human and chimpanzee and a possible selective pressure for such dispersion during the evolution of higher primates.

Heparan sulfate proteoglycans (HSPGs) are essential to respiratory morphogenesis in species as diverse as Drosophila and mice; they play a role in the regulation of numerous HS-binding <em>growth</em> <em>factors</em>, e.g. <em>fibroblast</em> <em>growth</em> <em>factors</em>. Moreover, an HS analogue, heparin, modulates lung <em>growth</em> in vitro. However, it has been difficult to assess the roles of specific HS structures in lung development due to technical barriers to their spatial localisation. Lungs from Sprague-Dawley rats were harvested between E<em>15</em>.5 and E19.5 and immediately fixed in 4 % (w/v) paraformaldehyde (in 0.1 M phosphate-buffered saline (PBS), pH 7.4). Lungs were washed in PBS, cryoprotected with 20% (w/v) sucrose (in PBS), gelatin embedded [7.5% (w/v) gelatin, <em>15</em>% (w/v) sucrose in PBS], before being covered in Cryo-M-Bed (Bright, Huntingdon, UK) and snap frozen at -40 degrees C. Cryosections were cut at 8 microm and stained with the HSPG core protein specific antibody 3G10 and a HS 'phage display antibody, EW4G2V. 3G10 and EW4G2V immunohistochemistry highlighted the presence of specific HS structures in lungs at all gestational ages examined. 3G10 strongly labelled airway basement membranes and the surrounding mesenchyme and showed weak staining of airway epithelial cells. EW4G2V, however, was far more selective, labelling the airway basement membranes only. Mesenchymal and epithelial cells did not appear to possess the HS epitope recognised by EW4G2V at these gestational ages. Novel 'phage display antibodies allow the spatial distribution of tissue HS to be analysed, and demonstrate in situ that distinct cellular compartments of a tissue possess different HS structures, possibly on the same proteoglycan core protein. These probes offer a new opportunity to determine the role of HS in the pathogenesis of congenital defects such as congenital diaphragmatic hernia (CDH), where lung development is aberrant, and the resulting pulmonary hypoplasia and hypertension are a primary cause of mortality.

Connective tissue <em>growth</em> <em>factor</em> (CTGF) is a recently described heparin-binding mitogen for <em>fibroblasts</em> and smooth muscle cells. The aim of this study was to investigate the production of CTGF by human uterine tissues using immunohistochemical and Northern blotting analyses. For immunohistochemistry, formalin-fixed human proliferative (n = 5), early secretory (n = 5; days <em>15</em>-19), mid-secretory (n = 5; days 20-23), late secretory (n = 5; days 24-28) endometrial, and decidual (n = 5) tissues were stained using a highly specific affinity-purified polyclonal antibody raised against residues 81-94 of human CTGF. Myometrial (n = 5) and leiomyoma (n = 5) tissues were also used for CTGF immunochemistry. In proliferative endometrium, epithelial and vascular endothelial cells showed strong CTGF immunoreactivity, whereas stromal cells were negative or only weakly positive for the CTGF protein. Throughout the entire secretory stage, CTGF was detected in epithelial and endothelial cells of endometrium. Stromal cells showed strong immunoreactivity to CTGF only in oedematous areas for early and mid-secretory endometrium, and in decidualized regions of late secretory endometrium. During pregnancy, the decidual, epithelial and endothelial cells of the endometrium were all immunoreactive to CTGF. In myometrial and leiomyoma samples, CTGF immunoreactivity was found only in the endothelial cells. Northern blotting of mRNA from normal uterus (n = 2) or leiomyoma (n = 6) using a 320 bp human CTGF cDNA probe revealed a single 2.4 kb transcript. This study is the first to demonstrate CTGF gene expression and localization of its encoded protein in human uterine tissues. The cell- and cycle-specific localization of CTGF support a role for this molecule in regulating aspects of uterine cell <em>growth</em>, migration, and/or matrix production during the menstrual cycle and pregnancy.

The transplantation of <em>fibroblasts</em>, genetically modified to secrete neurotrophin-3 (NT-3) and/or brain-derived neurotrophic <em>factor</em> (BDNF), into spinal cord-injured rats increases the production of new oligodendrocytes and myelination (McTigue et al. [1998] J. Neurosci. 18:5354-5365). This experiment did not fully resolve whether the effect was exerted on oligodendrocyte precursors or on oligodendrocytes, or whether there was stimulation of both proliferation and differentiation of the oligodendrocyte lineage cells. To clarify the effects of NT-3 and BDNF, adult rat spinal cord was dissociated to produce cultures in which both oligodendrocyte precursors (O1(-)O4(+)) and oligodendrocytes (O1(+)) were present. Thymidine labeling of cells was determined in the presence and absence of added NT-3 and/or BDNF. In addition, the effect of these neurotrophins on myelination was determined by treating purified adult O1(+) oligodendrocyte/embryonic dorsal root ganglion (DRG) neuron cocultures with neurotrophins, only during the myelination period. O1(+) oligodendrocyte proliferation was not stimulated by NT-3 or BDNF; however, the proliferation of O1(-)O4(+) cells was increased in NT-3-treated cultures to a labeling index (LI: 24 hr) of <em>15</em>-20%. This effect was observed at 5 but not at 10 days in vitro. In comparison, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) induced the proliferation of both O1(+) oligodendrocytes (LI approximately 60%) and O1(-)O4(+) cells (LI approximately 75%). The amount of myelin formed in purified O1(+) oligodendrocyte/DRG neuron cocultures was significantly increased in NT-3-treated cultures compared to untreated cultures. These results indicate that NT-3 is weakly but transiently mitogenic for adult-derived oligodendrocyte precursors and support the suggestion that NT-3 promotes the maturation of O1(+) oligodendrocytes into myelin-forming cells.

Dysregulated signaling contributes to altered cellular <em>growth</em>, motility, and survival during cancer progression. We have evaluated the ability of several <em>factors</em> to stimulate migration in WM1341D, a cell line derived from an invasive human vertical <em>growth</em> phase melanoma. Basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, hepatocyte <em>growth</em> <em>factor</em>, interleukin-8, and CCL27 each slightly increased migration. Insulin-like <em>growth</em> <em>factor</em> I (IGF-I), however, stimulated a <em>15</em>-fold increase in migration. This response required the IGF-I receptor, which activates phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways. Both pathways have been implicated in migration in a variety of cell types, but the signaling required for IGF-I-induced melanoma cell migration is not well defined. IGF-I-stimulated activation of MAPK/ERK signaling in WM1341D cells was inhibited by U0126, but a 33-fold higher dose of U0126 was needed to inhibit IGF-I-stimulated cellular migration. In contrast, similar concentrations of either wortmannin or LY294002 were required to inhibit both IGF-I-induced PI3K activation and migration. These results indicate that IGF-I-stimulated migration of WM1341D cells requires PI3K activation but is independent of MAPK/ERK signaling. Determining the contributions of IGF-I signaling pathways to migration will help us to understand melanoma progression and may lead to new therapeutic targets of this highly metastatic cancer.

The role of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in the proliferation and differentiation of rat bone marrow cells in culture was studied. bFGF stimulated [3H]thymidine incorporation into these cells by 4-fold at a concentration of 0.3 ng/ml and half-maximal effect was observed at a concentration of <em>15</em> pg/ml. In addition to its mitogenic effect, bFGF stimulated alkaline phosphatase activity by 3.6-fold. Continuous treatment with bFGF (for 21 days) resulted in a 6.3-fold increase in the culture dish surface area covered by bone-like mineralized tissue. Maximal bone-like tissue formation was observed in the presence of 3 ng/ml bFGF with half-maximal effect at a concentration of 0.3 ng/ml. These results indicate the possible role of bFGF in the proliferation of osteogenic rat bone marrow cells and their differentiation into cells of osteoblast-like phenotype.

Extracellular short fibulins, fibulin-3, -4, and -5, are components of the elastic fiber/microfibril system and are implicated in the formation and homeostasis of elastic tissues. In this study, we report new structural and functional properties of the short fibulins. Full-length human short fibulins were recombinantly expressed in human embryonic kidney cells and purified by immobilized metal ion affinity chromatography. All three fibulins showed various levels of degradation after the purification procedure. N-terminal sequencing revealed that all three fibulins are highly susceptible to proteolysis within the N-terminal linker region of the first calcium-binding epidermal <em>growth</em> <em>factor</em> domain. Proteolytic susceptibility of the linker correlated with its length. Exposure of these fibulins to matrix metalloproteinase (MMP)-1, -2, -3, -7, -9, and -12 resulted in similar proteolytic fragments with MMP-7 and -12 being the most potent proteases. Fibulin-3 proteolysis was almost completely inhibited in cell culture by the addition of 25 μm doxycycline (a broad spectrum MMP inhibitor). Reducible fibulin-4 dimerization and multimerization were consistently observed by SDS-PAGE, Western blotting, and mass spectrometry. Atomic force microscopy identified monomers, dimers, and multimers in purified fibulin-4 preparations with sizes of ∼10-<em>15</em>, ∼20-25, and ∼30-50 nm, respectively. All short fibulins strongly adhered to human <em>fibroblasts</em> and smooth muscle cells. Although only fibulin-5 has an RGD integrin binding site, all short fibulins adhere at a similar level to the respective cells. Solid phase binding assays detected strong calcium-dependent binding of the short fibulins to immobilized heparin, suggesting that these fibulins may bind cell surface-located heparan sulfate.

Interleukin-<em>15</em> (IL-<em>15</em>) is a recently discovered cytokine produced by a wide range of different cell types including <em>fibroblasts</em>, keratinocytes, endothelial cells, and macrophages in response to lipopolysaccharide or microbial infection. This suggests that IL-<em>15</em> may play a crucial role in the activation of phagocytic cells against pathogens. We studied polymorphonuclear leukocyte (PMN) activation by IL-<em>15</em>, evaluated as enhancement of PMN anti-Candida activity as well as IL-8 production, following stimulation with the cytokine. The PMN response to IL-<em>15</em> depends on binding to the IL-<em>15</em> receptor. Our experiments show that binding of a biotinylated human IL-<em>15</em>-immunoglobulin G2b IgG2b fusion protein was competed by the addition of human recombinant IL-<em>15</em> (rIL-<em>15</em>) or of human rIL-2, suggesting that IL-<em>15</em> binding to PMN might involve the IL-2Rbeta and IL-2Rgamma chains, which have been shown to be constitutively expressed by PMN. In addition, we show by reverse transcription-PCR and by flow cytometry with a specific anti-IL-<em>15</em>Ralpha chain monoclonal antibody that PMN express the IL-<em>15</em>Ralpha chain at the mRNA and protein levels. Incubation with IL-<em>15</em> activated PMN to secrete the chemotactic <em>factor</em> IL-8, and the amount secreted was increased by costimulation with heat-inactivated Candida albicans. In addition, IL-<em>15</em> primed the metabolic burst of PMN in response to formyl-methionyl-leucyl-phenylalanine but was not sufficient to trigger the respiratory burst or to increase the production of superoxide in PMN exposed to C. albicans. IL-<em>15</em> also increased the ability of PMN to phagocytose heat-killed C. albicans organisms in a dose-dependent manner, without opsonization by antibodies or complement-derived products. In the same concentration range, IL-<em>15</em> was as effective as gamma interferon (IFN-gamma) and IL-2 in increasing the C. albicans <em>growth</em>-inhibitory activity of PMN. Taken together, these results suggest that IL-<em>15</em> is a potent stimulant of both proinflammatory and antifungal activities of PMN, activating several antimicrobial functions of PMN involved in the cellular response against C. albicans.

OBJECTIVE

Thyroid eye disease (TED) patients are classified as type I (predominantly fat compartment enlargement) or type II (predominantly extraocular muscle enlargement) based on orbital imaging. Orbital fibroblasts (OFs) can be driven to proliferate or differentiate into adipocytes in vitro. We tested the hypothesis that type I OFs undergo more adipogenesis than type II OFs, whereas type II OFs proliferate more than type I OFs. We also examined the effect of cyclooxygenase (COX) inhibitors on OF adipogenesis and proliferation.

METHODS

Type I, type II, and non-TED OFs were treated with transforming growth factor-beta (TGFβ) to induce proliferation and with 15-deoxy-Δ(-12,14)-prostaglandin J2 (15d-PGJ2) to induce adipogenesis. Proliferation was measured using the [(3)H]thymidine assay, and adipogenesis was measured using the AdipoRed assay, Oil Red O staining, and flow cytometry. The effect of COX inhibition on adipogenesis and proliferation was also studied.

RESULTS

Type II OFs incorporated 1.7-fold more [(3)H]thymidine than type I OFs (P < 0.05). Type I OFs accumulated 4.8-fold more lipid than type II OFs (P < 0.05) and 12.6-fold more lipid than non-TED OFs (P < 0.05). Oil Red O staining and flow cytometry also demonstrated increased adipogenesis in type I OFs compared to type II and non-TED OFs. Cyclooxygenase inhibition significantly decreased proliferation and adipogenesis in type II OFs, but not type I OFs.

CONCLUSIONS

We have demonstrated that OFs from TED patients have heterogeneous responses to proproliferative and proadipogenic stimulators in vitro in a manner that corresponds to their different clinical manifestations. Furthermore, we demonstrated a differential effect of COX inhibitors on type I and type II OF proliferation and adipogenesis.

Chronic granulating wounds were established in rats by excising burns inoculated with Escherichia coli. Recombinant human basic <em>fibroblast</em> <em>growth</em> <em>factor</em> was applied at dosages of 1, 10, and 100 micrograms/cm2 to the wounds of three groups of 20 animals on days 5, 9, 12, <em>15</em>, and 18 after injury. The rate of wound closure was compared with that of similarly wounded animals treated with saline vehicle alone. High levels of bacteria caused significant retardation of wound contraction. The addition of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> at the 100 micrograms/cm2 dosage level markedly improved the rate of wound closure whereas inert vehicles applied alone were ineffective. Since bacterial counts did not decrease in the basic <em>fibroblast</em> <em>growth</em> <em>factor</em> treated wounds, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> was not inherently bactericidal. Histologic examination of the wounds treated with basic <em>fibroblast</em> <em>growth</em> <em>factor</em> showed increased cellularity with increased numbers of <em>fibroblasts</em> and round cells. These results suggest basic <em>fibroblast</em> <em>growth</em> <em>factor</em> can overcome the defect in healing created by bacterial infection, and this peptide may have efficacy in the management of the contaminated wound.

The aim of the present study was to compare healing obtained with biomembranes with the natural healing process (sham) using biochemical and immunohistological assays. C57BL/6 mice were divided into 4 groups of <em>15</em> mice each and received different subcutaneous implants: natural latex biomembrane (NLB), denatured latex (DL), expanded polytetrafluorethylene (ePTFE), or sham. On the 2nd, 7th, and 14th days post-treatment, 5 mice per group were sacrificed and biopsied for the following measurements: oxidative stress based on malondialdehyde (MDA), myeloperoxidase (MPO) and hydrogen peroxide by the method of ferrous oxidation-xylenol orange (FOX), as well as glutathione and total proteins; histological evaluation to enumerate inflammatory cells, <em>fibroblasts</em>, blood vessels, and collagen, and immunohistochemical staining for inducible nitric oxide synthase, interleukin-1β, vascular endothelial <em>growth</em> <em>factor</em> (VEGF), and transforming <em>growth</em> <em>factor</em>-β1 (TGF-β1). On day 2 post-treatment, NLB stimulated a dense inflammatory infiltrate mainly consisting of polymorphonuclear cells, as indicated by increased MPO (P < 0.05), but oxidative stress due to MDA was not observed until the 7th day (P < 0.05). The number of blood vessels was greater in NLB (P < 0.05) and DL (P < 0.05) mice compared to sham animals on day 14. NLB induced fibroplasia by day 14 (P < 0.05) with low expression of TGF-β1 and collagenesis. Thus, NLB significantly induced the inflammatory phase of healing mediated by oxidative stress, which appeared to influence the subsequent phases such as angiogenesis (with low expression of VEGF) and fibroplasia (independent of TGF-β1) without influencing collagenesis.

Heparin and heparan sulfate (HS) are structurally diverse glycosaminoglycans (GAG) that are known to interact, via unique structural motifs, with a wide range of functionally distinct proteins and modulate their biological activity. To define the GAG motifs that interact with proteins, we assessed the ability of <em>15</em> totally synthetic HS mimetics to interact with 10 functionally diverse proteins that bind heparin/HS. The HS mimetics consisted of cyclitol-based pseudo-sugars coupled by linkers of variable chain length, flexibility, orientation, and hydrophobicity, with variations in sulfation also being introduced into some molecules. Three of the proteins tested, namely hepatocyte <em>growth</em> <em>factor</em>, eotaxin, and elastase, failed to interact with any of the sulfated linked cyclitols. In contrast, each of the remaining seven proteins tested exhibited a unique reactivity pattern with the panel of HS mimetics, with tetrameric cyclitols linked by different length alkyl chains being particularly informative. Thus, compounds with short alkyl spacers (2-3 carbon atoms) effectively blocked the interaction of <em>fibroblast</em> <em>growth</em> <em>factor</em>-1 (FGF-1) and lipoprotein lipase with heparin/HS, whereas longer chain spacers (7-10 carbon atoms) were required for optimal inhibition of FGF-2 and vascular endothelial <em>growth</em> <em>factor</em> binding. This effect was most pronounced with the chemokine, interleukin-8, where alkyl-linked tetrameric cyclitols were essentially inactive unless a spacer of >7 carbon atoms was used. The heparin-inhibitable enzymes heparanase and cathepsin G also displayed characteristic inhibition patterns, cathepsin G interacting promiscuously with most of the sulfated cyclitols but heparanase activity being inhibited most effectively by HS mimetics that structurally resemble a sulfated pentasaccharide. These data indicate that a simple panel of HS mimetics can be used to probe the HS binding specificity of proteins, with the position of anionic groups in the HS mimetics being critical.

The effect of culture conditions on calf dorsal aorta endothelial cells was studied. Population doubling time varied as a function of the cell seeding density, <em>growth</em> Medium, serum supplement, and concentration of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). The shortest population doubling time was found for cells (population doubling level 0--30) grown in Eagle's Minimal Essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 100 ng/ml FGF. The stimulatory effect of FGF on bovine endothelial cell proliferation was dependent on cell inoculation density. FGF significantly increased cell division rate at cell inocula less than 1 x 10(4) cells/cm2 but not at higher densities. The population doubling time and cell size increased as the mass culture population doubling level increased. The replicative lifespans of bovine endothelial cells grown in medium supplemented with 20% FBS were 10--<em>15</em>% greater than parallel cultures supplemented with 10% FBS. Cultures grown in medium supplemented with 10% FBS and 50 ng/ml FGF showed a 50% increase in replicative lifespan compared to cultures grown in medium supplemented with 10% FBS alone. When FGF was used the increase in the number of doublings was a function of the length of time the cells were grown in the presence of FGF. This report extends comparable observations on the in vitro aging of human diploid <em>fibroblasts</em> to bovine endothelial cells.