Acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) belong to a family of mitogenic polypeptides that are involved in cellular proliferation and differentiation. In this study we investigated the potential role of aFGF and bFGF in chronic pancreatitis (CP), a fibrotic condition associated with acinar cell dedifferentiation and atrophy, and <em>fibroblast</em>ic proliferation. By immunohistochemistry, aFGF and bFGF were abundant in pancreatic ductal and acinar cells in pancreatic tissues from CP patients. Immunoblotting with the same highly specific monoclonal antibodies demonstrated a marked increase in aFGF and bFGF in pancreatic homogenates from CP patients by comparison with the normal pancreas. Northern blot analysis indicated that, by comparison with normal controls, <em>16</em> of 21 CP tissues exhibited a 14-fold increase in aFGF mRNA levels, and 19 of 21 CP tissues exhibited a 15-fold increase in bFGF mRNA levels. In situ hybridization confirmed that this overexpression occurred in ductal and acinar cells, and indicated that both mRNA moieties colocalized with their respective proteins. These findings suggest that aFGF and bFGF may either be involved in the pathobiological mechanisms that occur in CP, or that their overexpression may be the consequence of other perturbations that occur in this disorder.

The int-2 gene, a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) super-gene family, has previously been shown to be amplified in <em>16</em>% of the 110 human breast tumors examined. In order to characterize the amplification unit containing the int-2 gene (chromosome 11q13), the same panel of breast tumors was screened for possible amplifications of other markers mapping between 11q11 and 11q24. Out of the eight additional genes analysed, simultaneous amplification of bcl-1 (11q13, a locus involved in hematopoietic malignancies) and hst (11q13, another member of the FGF family) was observed in 17/18 tumors with increased copy number of the int-2 gene. A single breast tumor showed amplification of int-2 oncogene only. Neither the bcl-1 nor the hst locus was individually amplified in any of the tumor DNAs examined.

Low-carbohydrate/high-fat diets (LC-HFDs) in rodent models have been implicated with both weight loss and as a therapeutic approach to treat neurological diseases. LC-HFDs are known to induce ketosis; however, systematic studies analyzing the impact of the macronutrient composition on ketosis induction and weight loss success are lacking. Male Wistar rats were pair-fed for 4 wk either a standard chow diet or one of three different LC-HFDs, which only differed in the relative abundance of fat and protein (percentages of fat/protein in dry matter: LC-75/10; LC-65/20; LC-55/30). We subsequently measured body composition by nuclear magnetic resonance (NMR), analyzed blood chemistry and urine acetone content, evaluated gene expression changes of key ketogenic and gluconeogenic genes, and measured energy expenditure (EE) and locomotor activity (LA) during the first 4 days and after 3 wk on the respective diets. Compared with chow, rats fed with LC-75/10, LC-65/20, and LC-55/30 gained significantly less body weight. Reductions in body weight were mainly due to lower lean body mass and paralleled by significantly increased fat mass. Levels of β-hydroxybutyate were significantly elevated feeding LC-75/10 and LC-65/20 but decreased in parallel to reductions in dietary fat. Acetone was about <em>16</em>-fold higher with LC-75/10 only (P < 0.001). In contrast, rats fed with LC-55/30 were not ketotic. Serum <em>fibroblast</em> <em>growth</em> <em>factor</em>-21, hepatic mRNA expression of hydroxymethylglutaryl-CoA-lyase, peroxisome proliferator-activated receptor-γ coactivator-1α, and peroxisome proliferator-activated receptor-γ coactivator-1β were increased with LC-75/10 only. Expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase was downregulated by 50-70% in LC-HF groups. Furthermore, EE and LA were significantly decreased in all groups fed with LC-HFDs after 3 wk on the diets. In rats, the absence of dietary carbohydrates per se does not induce ketosis. LC-HFDs must be high in fat, but also low in protein contents to be clearly ketogenic. Independent of the macronutrient composition, LC-HFD-induced weight loss is not due to increased EE and LA.

Degenerated intervertebral disc has lost its normal architecture, and there are changes both in the nuclear and annular parts of the disc. Changes in cell shape, especially in the annulus fibrosus, have been reported. During degeneration the cells become more rounded, chondrocyte-like, whereas in the normal condition annular cells are more spindle shaped. These chondrocyte-like cells, often forming clusters, affect extracellular matrix turnover. In previous studies transforming <em>growth</em> <em>factor</em> beta (TGFbeta) -1 and -2, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and platelet-derived <em>growth</em> <em>factor</em> (PDGF) have been highlighted in herniated intervertebral disc tissue. In the present study the same <em>growth</em> <em>factors</em> are analysed immunohistochemically in degenerated intervertebral disc tissue. Disc material was obtained from <em>16</em> discs operated for painful degenerative disc disease. Discs were classified according to the Dallas Discogram Description. Different disc regions were analysed in parallel. As normal control disc tissue material from eight organ donors was used. Polyclonal antibodies against different <em>growth</em> <em>factors</em> and TGFbeta receptor type II were used, and the immunoreaction was detected by the avidin biotin complex method. All studied degenerated discs showed immunoreactivity for TGFbeta receptor type II and bFGF. Fifteen of <em>16</em> discs were immunopositive for TGFbeta-1 and -2, respectively, and none showed immunoreaction for PDGF. Immunopositivity was located in blood vessels and in disc cells. In the nucleus pulposus the immunoreaction was located almost exclusively in chondrocyte-like disc cells, whereas in the annular region this reaction was either in chondrocyte-like disc cells, often forming clusters, or in <em>fibroblast</em>-like disc cells. Chondrocyte-like disc cells were especially prevalent in the posterior disrupted area. In the anterior area of the annulus fibrosus the distribution was more even between these two cell types. bFGF was expressed in the anterior annulus fibrosus more often in chondrocyte-like disc cells than in <em>fibroblast</em>-like disc cells. Control discs showed cellular immunopositivity for only TGFbeta-1 and -2 and TGFbeta receptor type II . We suggest that <em>growth</em> <em>factors</em> create a cascade in intervertebral disc tissue, where they act and participate in cellular remodelling from the normal resting stage via disc degeneration to disc herniation.

The <em>16</em> kDa N-terminal fragment of prolactin (<em>16</em>K-prolactin) is a potent antiangiogenic <em>factor</em>. Here, we demonstrate that matrix metalloproteases (MMPs) produced and secreted by chondrocytes generate biologically functional <em>16</em>K-prolactin from full-length prolactin. When incubated with human prolactin at neutral pH, chondrocyte extracts and conditioned medium, as well as chondrocytes in culture, cleaved the Ser155-Leu156 peptide bond in prolactin, yielding - upon reduction of intramolecular disulfide bonds - a <em>16</em> kDa N-terminal fragment. This <em>16</em>K-prolactin inhibited basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-induced endothelial cell proliferation in vitro. The Ser155-Leu156 site is highly conserved, and both human and rat prolactin were cleaved at this site by chondrocytes from either species. Conversion of prolactin to <em>16</em>K-prolactin by chondrocyte lysates was completely abolished by the MMP inhibitors EDTA, GM6001 or 1,10-phenanthroline. Purified MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 cleaved human prolactin at Gln157, one residue downstream from the chondrocyte protease cleavage site, with the following relative potency: MMP-8>> MMP-13>> MMP-3>> MMP-1= MMP-2>> MMP-9. Finally, chondrocytes expressed prolactin mRNA (as revealed by RT-PCR) and they contained and released antiangiogenic N-terminal <em>16</em> kDa prolactin (detected by western blot and endothelial cell proliferation). These results suggest that several matrix metalloproteases in cartilage generate antiangiogenic <em>16</em>K-prolactin from systemically derived or locally produced prolactin.

OBJECTIVE

High levels of fibroblast growth factor 23 are associated with mortality, CKD progression, and calcification in CKD patients. The aim of this pilot study is to assess whether a very-low-protein diet (0.3 g/kg per day) with a consequent low intake of phosphorus would reduce fibroblast growth factor 23 compared with a low-protein diet (0.6 g/kg per day) in CKD patients not yet on dialysis.

METHODS

A prospective, randomized, controlled crossover study was performed in which 32 patients were randomized into two groups. Group A (16 patients) received a very-low-protein diet (0.3 g/kg body wt per day) supplemented with ketoanalogues during the first week and a low-protein diet during the second week, and group B (16 patients) received a low-protein diet during the first week and a very-low-protein diet during the second week. Fibroblast growth factor 23, seric, and urinary phosphate levels were measured at baseline and the end of each study period.

RESULTS

After only 1 week of the very-low-protein diet, reductions in fibroblast growth factor 23 levels (33.5%), serum phosphate (12%), and urinary phosphate (34%) with the very-low-protein diet compared with the low-protein diet were observed. Serum and urinary phosphate levels and protein intake were significant determinants of fibroblast growth factor 23 (95% confidence interval=1.04-1.19, 1.12-1.37, and 1.51-2.23, respectively).

CONCLUSIONS

A very-low-protein diet supplemented with ketoanalogues reduced fibroblast growth factor 23 levels in CKD patients not yet on dialysis.

The mechanism of <em>growth</em> <em>factor</em> protection against metabolic/excitotoxic insults was examined. The time course of changes in ATP levels, mitochondrial transmembrane potential, intracellular free calcium levels ([Ca2+]i), and cell survival resulting from glucose deprivation were assessed in cultured hippocampal neurons. ATP levels were significantly reduced within 1 h of the onset of glucose deprivation and reached less than 20% of control levels by 12 h. Mitochondrial transmembrane potential (assessed by rhodamine 123 accumulation in mitochondria) declined progressively between 4 and 20 h following the onset of glucose deprivation. The [Ca2+]i was reduced during the first 1 h of glucose deprivation, gradually rose through 12 h, and then rose rapidly and was elevated five- to sevenfold after <em>16</em> h. The [Ca2+]i did not increase, and mitochondrial dysfunction and cell damage were prevented, in hypoglycemic neurons incubated in Ca(2+)-deficient medium. Elevation of [Ca2+]i by exposure of neurons to glutamate caused loss of rhodamine 123 fluorescence and structural damage to mitochondria. Mitochondrial function could be restored and cell survival maintained by addition of glucose prior to the late elevation of [Ca2+]i. Nerve <em>growth</em> <em>factor</em> (NGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and insulin-like <em>growth</em> <em>factor</em> II (IGF-II) prevented loss of both [Ca2+]i homeostasis and mitochondrial transmembrane potential, and protected hippocampal neurons against hypoglycemic injury, but did not prevent the hypoglycemia-induced reduction in ATP levels. NaCN and 2,4-dinitrophenol (DNP) caused a large elevation of [Ca2+]i, mitochondrial dysfunction, and cell death. NGF, bFGF, and IGF-II each significantly reduced the adverse effects of NaCN and DNP on [Ca2+]i, mitochondrial function, and cell survival. Loss of [Ca2+]i homeostasis may be a critical event leading to mitochondrial damage and cell death resulting from energy failure. Preventing loss of [Ca2+]i homeostasis may be a general mechanism for the neuroprotective action of <em>growth</em> <em>factors</em>.

Graves' disease (GD) is an autoimmune process involving the thyroid and connective tissues in the orbit and pretibial skin. Activating anti-thyrotropin receptor Abs are responsible for hyperthyroidism in GD. However, neither these autoAbs nor the receptor they are directed against have been convincingly implicated in the connective tissue manifestations. Insulin-like <em>growth</em> <em>factor</em>-1 receptor (IGF-1R)-bearing <em>fibroblasts</em> overpopulate connective tissues in GD and when ligated with IgGs from these patients, express the T cell chemoattractants, IL-<em>16</em>, and RANTES. Disproportionately large fractions of peripheral blood T cells also express IGF-1R in patients with GD and may account, at least in part, for expansion of IGF-1R(+) memory T cells. We now report a similarly skewed B cell population exhibiting the IGF-1R(+) phenotype from the blood, orbit, and bone marrow of patients with GD. This expression profile exhibits durability in culture and is maintained or increased with CpG activation. Moreover, IGF-1R(+) B cells produce pathogenic Abs against the thyrotropin receptor. In lymphocytes from patients with GD, IGF-1 enhanced IgG production (p < 0.05) and increased B cell expansion (p < 0.02) in vitro while those from control donors failed to respond. These findings suggest a potentially important role for IGF-1R display by B lymphocytes in patients with GD in supporting their expansion and abnormal Ig production.

Systemic sclerosis (SSc) is a disorder of systemic and dermal fibrosis of uncertain etiology. Recently, we found that SSc epidermis is abnormal, taking on an activated phenotype observed during wound healing and tissue repair. As epithelial-<em>fibroblast</em> interactions are important during wound repair and in fibrosis in general, we investigated further the phenotype of the SSc epidermis, and tested whether the SSc epidermis provides a pro-fibrotic stimulus to <em>fibroblasts</em>. In this study we show that in SSc epidermis keratinocyte maturation is delayed, and wound-associated keratins 6 and <em>16</em> are induced, in both involved and clinically uninvolved skin. Phosphorylation array analysis revealed induction of stress-induced mitogen-activated protein kinase signaling and mesenchymal feedback through hepatocyte <em>growth</em> <em>factor</em>/c-Met in SSc epidermis. SSc epidermal cells maintained with normal <em>fibroblasts</em> in three-dimensional co-culture were found to stimulate <em>fibroblasts</em>, leading to contractility and connective tissue <em>growth</em> <em>factor</em> expression. These effects depend on elevation of IL-1alpha by the epidermal cells and induction of endothelin-1 and transforming <em>growth</em> <em>factor</em>-beta in <em>fibroblasts</em>. Antagonism of endogenous IL-1alpha using IL-1 receptor antagonist blocked gel contraction by SSc epidermis. We propose that in SSc, epidermal cells are in a persistently activated state and are able to promote dermal fibrosis. These findings are important because biologic therapies could target epithelial-<em>fibroblast</em> interactions in the disease.

Previous studies have established the link between aberrant microRNA (miRNA) expression and hypoxia in various neoplasms. However, how these hypoxia-related miRNAs modulate tumor progression is still unclear. Therefore, the patterns of miRNA in colorectal carcinoma cell lines in response to hypoxia or not were first screened and the hypoxia-induced repression of the miR-15-<em>16</em> cluster was confirmed. Then, this repression was found to be associated with high tumor stage and poor prognosis in colorectal carcinoma and is shown to promote tumor angiogenesis and metastasis by the loss of restriction of its target gene, <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF2). Moreover, the general and alterative promoters of the miR-15-<em>16</em> host (deleted in lymphocytic leukemia 2, DLEU2) were mapped, and three c-Myc/Max binding sites in response to the hypoxia-induced repression of miR-15-<em>16</em> were further identified. Finally, an enhanced stability of c-Myc/Max heterodimer promoted by increased hypoxia-inducible <em>factor</em>-2α (HIF-2α) was validated, and we also verified that the enhancement contributed to the hypoxia-induced repression of miR-15-<em>16</em>. In brief, the c-Myc-mediated transcriptional repression of miR-15-<em>16</em> in hypoxia is induced by increased HIF-2α and promoted tumor angiogenesis and hematogenous metastasis by the further loss of post-transcriptional inhibition of FGF2. Our study provides a better understanding of the coping mechanisms in response to tumor hypoxia and may be helpful in developing an effective prognostic marker or treatment target against solid tumors.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> (FGF)/FGF receptor (FGFR) signaling plays a crucial role in mesoderm formation and patterning. Heartless mutant studies in Drosophila suggest that FGFR1, among the different FGFRs, may play a role in cardiogenesis. However, fgfr1-/- mice die during gastrulation before heart formation. To establish the contribution of FGFR1 in cardiac development, we investigated the capacity of murine fgfr1+/- and fgfr1-/- embryonic stem (ES) cells to differentiate to cardiomyocytes in vitro. Clusters of pulsating cardiomyocytes were observed in >90% of 3-dimensional embryoid bodies (EBs) originated from fgfr1+/- ES cells at day 9 to 10 of differentiation. In contrast, 10% or less of fgfr1-/- EBs showed beating foci at day <em>16</em>. Accordingly, fgfr1-/- EBs were characterized by impaired expression of early cardiac transcription <em>factors</em> Nkx2.5 and d-Hand and of late structural cardiac genes myosin heavy chain (MHC)-alpha, MHC-beta, and ventricular myosin light chain. Homozygous fgfr1 mutation resulted also in alterations of the expression of mesoderm-related early genes, including nodal, BMP2, BMP4, T(bra), and sonic hedgehog. Nevertheless, fgfr1+/- and fgfr1-/- EBs similarly express cardiogenic precursor, endothelial, hematopoietic, and skeletal muscle markers, indicating that fgfr1-null mutation exerts a selective effect on cardiomyocyte development in differentiating ES cells. Accordingly, inhibitors of FGFR signaling, including the FGFR1 tyrosine kinase inhibitor SU 5402, the MEK1/2 inhibitor U0126, and the protein kinase C inhibitor GF109 all prevented cardiomyocyte differentiation in fgfr1+/- EBs without affecting the expression of the hematopoietic/endothelial marker flk-1. In conclusion, the data point to a nonredundant role for FGFR1-mediated signaling in cardiomyocyte development.

Angiogenic <em>growth</em> <em>factors</em> could prove to be useful in managing peripheral arterial insufficiency. The present study was designed to evaluate the dose response of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), the efficacy of critical routes and dosing regimens, and the specificity of action in rats with peripheral arterial insufficiency. Bilateral ligation of femoral arteries greatly reduces blood flow capacity to the calf muscles but does not impair resting flow needs. Collateral blood flow to calf muscles was determined <em>16</em> days postocclusion, during treadmill running, with (85)Sr and (141)Ce microspheres, in blinded-randomized trials that included intra-arterial and intravenous infusions and subcutaneous injections of recombinant human bFGF. Peak blood flow of 75-80 ml. min(-1). 100 g(-1) for calf muscle was observed at a bFGF dose of 5 microg. kg(-1). day(-1) (ia for 14 days) compared with 50 ml. min(-1). 100 g(-1) for vehicle groups. Similar increases in collateral blood flow were observed with short-term or prolonged and continuous or intermittent delivery of bFGF by any route. Collateral blood flows were similar in corresponding muscles across both limbs. Vascular remodeling induced by bFGF required attendant vascular occlusion, inasmuch as vessels in the normal nonoccluded vascular tree were unresponsive to circulating bFGF. Improvement in collateral blood flow with exogenous bFGF is robust, amenable to short-term administration, and requires vascular occlusion to be effective.

Connective tissue <em>growth</em> <em>factor</em> (CTGF) is a <em>growth</em> and chemotactic <em>factor</em> for <em>fibroblasts</em> encoded by an immediate early gene that is transcriptionally activated by transforming <em>growth</em> <em>factor</em> ss. Although the primary translational product of the pig CTGF gene is predicted to be of approximate Mr 38 000, pig uterine luminal flushings (ULF) contained 10- to 20-kDa CTGF proteins that were heparin-binding and mitogenic, whereas 38-kDa CTGF was not apparent. The N-termini of two microheterogeneous forms of <em>16</em>-kDa CTGF, as well as 18-kDa and 20-kDa forms of CTGF, commenced at, respectively, Cys199, Ala197, Asp186, and Asp186 and did not correspond to intron-exon boundaries in the CTGF gene. Northern blotting revealed a single porcine (p) CTGF transcript of 2.4 kilobases in endometrium from Day 10 to <em>16</em> cycling or pregnant pigs. Ten- to twenty-kilodalton pCTGF proteins in ULF were stable for 48 h at 37 degreesC whereas native 38-kDa pCTGF was degraded within 10 min under the same conditions. CTGF-degrading activity in pig ULF was heat-sensitive and concentration- and time-dependent. Ten- to twenty-kilodalton CTGF levels in ULF peaked on Day <em>16</em> of the cycle and on Day 12 of pregnancy and were highly correlated with the levels of proteolytic activity for 38-kDa CTGF. Collectively these data suggest that bioactive 10- to 20-kDa CTGF proteins are generated in utero through limited proteolysis of the 38-kDa CTGF primary translational product.

An angiogenic <em>growth</em> <em>factor</em> present in bovine corpus luteum (CL) has been purified to apparent homogeneity by a combination of differential salt precipitation, ion exchange chromatography, and heparin-Sepharose chromatography. It is a single chain polypeptide with an apparent mol wt of 15,000 and an amino acid composition similar to that previously reported for pituitary and brain <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF). Sequence analysis of the first 17 residues of the CL-derived <em>growth</em> <em>factor</em> identified the sequence; His-Phe-Lys-Asp-Pro-Lys-Arg-Leu-Tyr-X-Lys-Asn-Gly-Gly-X-Phe-Leu. This sequence is identical to residues <em>16</em>-33 of bovine pituitary and brain FGF, indicating that the CL-derived <em>growth</em> <em>factor</em> is an amino-terminally truncated form of FGF and is otherwise similar, if not identical, to FGF. The biological activity of CL FGF is indistinguishable from that of pituitary or brain FGF. It is highly active in triggering the proliferation of cultured bovine vascular endothelial cells derived either from large vessels (aortic arch) or from corpus luteum and adrenal cortex capillaries (half-maximal stimulation at 20-40 pg/ml and saturation at 400-600 pg/ml). In vivo implants containing 50 ng to 1 microgram CL-derived <em>growth</em> <em>factor</em> stimulate neovascularization in the chorioallantoic membrane of the chick embryo. In addition to being mitogenic for vascular endothelial cells, CL FGF also stimulates the proliferation of a wide variety of mesoderm- and neuroectoderm-derived cells, including vascular smooth muscle cells, granulosa and adrenal cortex cells, rabbit costal chondrocytes, and corneal endothelial cells.

Tumor-induced osteomalacia (TIO) is a rare disorder of phosphate wasting due to <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF23)-secreting tumors that are often difficult to locate. We present a systematic approach to tumor localization and postoperative biochemical changes in 31 subjects with TIO. All had failed either initial localization, or relocalization (in case of recurrence or metastases) at outside institutions. Functional imaging with ¹¹¹Indium-octreotide with single photon emission computed tomography (octreo-SPECT or SPECT/CT), and ¹⁸fluorodeoxyglucose positron emission tomography/CT (FDG-PET/CT) were performed, followed by anatomic imaging (CT, MRI). Selective venous sampling (VS) was performed when multiple suspicious lesions were identified or high surgical risk was a concern. Tumors were localized in 20 of 31 subjects (64.5%). Nineteen of 20 subjects underwent octreo-SPECT imaging, and <em>16</em> of 20 FDG-PET/CT imaging. Eighteen of 19 (95%) were positive on octreo-SPECT, and 14 of <em>16</em> (88%) on FDG-PET/CT. Twelve of 20 subjects underwent VS; 10 of 12 (83%) were positive. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were as follows: sensitivity = 0.95, specificity = 0.64, PPV = 0.82, and NPV = 0.88 for octreo-SPECT; sensitivity = 0.88, specificity = 0.36, PPV = 0.62, and NPV = 0.50 for FDG-PET/CT. Fifteen subjects had their tumor resected at our institution, and were disease-free at last follow-up. Serum phosphorus returned to normal in all subjects within 1 to 5 days. In 10 subjects who were followed for at least 7 days postoperatively, intact FGF23 (iFGF23) decreased to near undetectable within hours and returned to the normal range within 5 days. C-terminal FGF23 (cFGF23) decreased immediately but remained elevated, yielding a markedly elevated cFGF23/iFGF23 ratio. Serum 1,25-dihydroxyvitamin D₃ (1,25D) rose and exceeded the normal range. In this systematic approach to tumor localization in TIO, octreo-SPECT was more sensitive and specific, but in many cases FDG-PET/CT was complementary. VS can discriminate between multiple suspicious lesions and increase certainty prior to surgery. Sustained elevations in cFGF23 and 1,25D were observed, suggesting novel regulation of FGF23 processing and 1,25D generation.

In rat embryos, <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>16</em> is predominantly expressed in brown adipose tissue. To elucidate the role of FGF-<em>16</em>, we examined the expression of FGF-<em>16</em> mRNA in rat embryonic brown adipose tissue at different developmental stages by Northern blotting analysis and in situ hybridization. FGF-<em>16</em> mRNA was expressed abundantly in brown adipose tissue during embryonic day 17. 5, embryonic days 17.5-19.5, and thereafter at lower levels into the neonatal period. The expression profile of FGF-<em>16</em> mRNA well corresponds to the proliferative profile of embryonic brown adipose tissue reported. We also examined the mitogenic activity of recombinant rat FGF-<em>16</em> for primary brown adipocytes prepared from rat embryonic brown adipose tissue. FGF-<em>16</em> showed significant mitogenic activity for primary brown adipocytes. The mitogenic activity was found to be exerted by binding and activating FGF receptor-4 in the brown adipose tissue. As a great induction of proliferation of rat brown adipose tissue during cold acclimation was reported, we also examined the expression of FGF-<em>16</em> mRNA in the brown adipose tissue during cold acclimation by Northern blotting analysis. The expression of FGF-<em>16</em> mRNA was not increased, but rather decreased. The expression profile of FGF-<em>16</em> mRNA and the mitogenic activity of FGF-<em>16</em> reported here indicate that FGF-<em>16</em> is a unique <em>growth</em> <em>factor</em> involved in proliferation of embryonic brown adipose tissue.

A number of <em>growth</em> <em>factors</em> have been implicated as stimuli of thyroid cell proliferation; overexpression of these <em>growth</em> <em>factors</em> and/or their receptors may play a role in the <em>growth</em> of thyroid tumors. To determine if immunohistochemical detection of <em>growth</em> <em>factors</em> and/or their receptors correlates with morphological alterations in proliferative lesions of thyroid, we examined the localization of epidermal <em>growth</em> <em>factor</em> (EGF), transforming <em>growth</em> <em>factor</em>-alpha (TGF-alpha) and their common receptor, EGF-receptor (EGF-R), insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1), IGF-1-receptor (IGF-R) and IGF binding proteins (IGFBP)-1, -2, -3, and -4, nerve <em>growth</em> <em>factor</em> (NGF), and its receptor NGF-receptor (NGF-R), transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), in normal thyroid tissue and various thyroid tumors. We applied the streptavidin-biotin technique to formalin-fixed, paraffin-embedded tissues. We studied 8-<em>16</em> different cases of each of the following: normal human thyroid, multinodular hyperplasia, follicular adenoma, papillary carcinoma, follicular carcinoma, medullary carcinoma, and anaplastic carcinoma. EGF, TGF-alpha, and their receptor EGF-R were widely expressed in normal thyroid and in all the thyroid lesions examined. IGF-1 and IGFBP-1 were diffusely present in all different thyroid tissues as well. There was no difference in staining intensity or distribution that correlated with the pathological process. IGFBP-4 seemed to have a variable expression. IGFBP-2 and -3 were detected only in medullary carcinomas.(ABSTRACT TRUNCATED AT 250 WORDS)

The identification of cancer-associated <em>fibroblast</em> (CAF)-derived proteins that mediate interactions between the tumor stroma and cancer cells is a crucial step toward the discovery of new molecular targets for therapy or molecular signatures that improve tumor classification and predict clinical outcome. CAF are α-smooth muscle actin positive, representing a myo<em>fibroblast</em> phenotype that may differentiate from multiple precursor cells, including bone marrow-derived mesenchymal stem cells (MSC). Transforming <em>growth</em> <em>factor</em>-β1 (TGF-β1) is a crucial inducer of α-smooth muscle actin positive CAFs. In this study, we aimed to identify CAF-derived regulators of colon cancer progression by performing a high-throughput differential secretome profiling between CAF compared to noncancer-activated bone marrow-derived MSC. In addition, we explored the effect of TGF-β1 on the secretion of proteins by bone marrow-derived MSC in comparison with the protein secretion profile of CAF. TGF-β1 induced de novo secretion of 84 proteins in MSC, of which <em>16</em> proteins, including stromal-derived <em>factor</em>-1α and Rantes, were also present in CAF secretome. Immunohistochemistry further validated the expression of selected candidates such as tenascin C, fibronectin ED-A domain and stromal-derived <em>factor</em>-1 in clinical colon cancer specimens. In conclusion, this differential secretome approach enabled us to identify a series of candidate biomarkers for colon cancer that are associated with a CAF-specific phenotype.

In the present study, the ability of tumor necrosis <em>factor</em>-alpha (TNF) to stimulate hexose transport in quiescent 3T3-L1 <em>fibroblasts</em> has been examined. Activation of transport occurred in a dose- and time-dependent manner, with maximal stimulation (6-8-fold) observed <em>16</em> h after exposure to 2.5 nM TNF. Early activation of hexose transport by TNF (2-fold within 30 min) was associated with increased plasma membrane immunoreactive glucose transporters. Prolonged exposure to TNF (<em>16</em> h) resulted in a 2-fold increase in glucose transporter content of both plasma and inner membrane compartments. The magnitude of increased glucose transport (6-8-fold) was greater than the increased content of plasma membrane glucose transporters (2-fold), suggesting that the TNF-treatment altered the intrinsic activity of the glucose transporters. Increased transcription of the glucose transporter (GLUT-1) gene, as well as several immediate-early genes (c-fos, c-jun, jun-B, and beta-actin) was observed within 15 min of exposure to TNF. Transcriptional activation of immediate-early genes was tightly coupled to subsequent accumulation of their respective mRNAs. However, increased GLUT-1 mRNA (8 h after TNF treatment) was due to an apparent 3-fold increase in the stability of this message and not to increased transcription. The time course of TNF-induced hexose transport occurred concomitant with a 6-fold increase in total RNA synthesis which preceded a 3-fold increase in protein synthesis. Moreover, TNF induced cell-cycle progression through S-phase, as measured by aphidicolin-sensitive thymidine uptake. Phorbol myristate acetate also stimulated hexose transport as well as expression of the GLUT-1 gene and several immediate-early genes in quiescent 3T3-L1 cells. TNF-induced immediate-early gene expression was intact in PMA-pretreated cells (with the exception of GLUT-1 and beta-actin genes where the response was muted), suggesting the involvement of multiple pathways in TNF signal transduction. Our results indicate that TNF initiates mitogenic events in quiescent 3T3-L1 <em>fibroblasts</em> reminiscent of serum-derived <em>growth</em> <em>factors</em>.

OBJECTIVE

To study the role of some soluble factors in the process of angiogenesis that accompanies multiple myeloma (MM).

METHODS

The concentrations of three well-known angiogenic peptides, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) were evaluated by an ELISA method. All of these factors were measured in the plasma obtained from peripheral blood (PB) and bone marrow (BM) aspirates of 34 patients affected by plasma cell disorders. This series included one patient with a solitary extramedullary plasmacytoma, 17 patients with MM at diagnosis, and 16 with previously treated MM.

RESULTS

In all the patients, the concentration of each angiogenic factor was higher in bone marrow than in peripheral blood. Mean values of the three angiogenic factors in BM or in PB were lower in stage I than stage II-III. One patient with extramedullary solitary myeloma had high levels of VEGF and bFGF but this increase was not found in the other 6 patients with extramedullary disease when compared with patients without extramedullary disease. VEGF and bFGF did not correlate with each other while HGF showed a weak correlation with VEGF and a stronger one with bFGF. Moreover, VEGF correlated with features of disease activity, such as C-reactive protein, and 2-microglobulin, while both bFGF and HGF showed an inverse correlation with albumin level. No correlation was found between VEGF, bFGF and HGF levels and age, M protein level, osteolytic lesions, or percentage of BM plasma cells. Since angiogenic factors may be released by normal cells in response to hypoxia, we also evaluated erythropoietin (EPO) levels (which correlate with the hypoxic stimulus) both in PB and BM plasma of these patients but none of the measured angiogenic factors correlated with EPO levels. Interpretation and Conclusions. Several soluble factors may play a role in the angiogenic activity described in MM but their contribution to the progression of disease may be different. The finding of higher levels of these factors in BM than in PB might indicate that the bone marrow environment is their major source. Concentrations of angiogenic factors parallel the activity of disease and are independent of the hypoxic stimulus.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a secreted multifunctional cytokine and a potent stimulator of angiogenesis. We measured bFGF concentrations from serum samples taken from 103 patients with small cell lung cancer at the time of diagnosis. Serum concentration of bFGF (S-bFGF) ranged from undetectable to 54 pg/ml (median, 6 pg/ml). S-bFGF was not associated with age, sex, performance status, or stage. A high pretreatment S-bFGF was associated with poor overall survival. The 1- and 2-year survival rates of the patients within the highest quartile of S-bFGF >>or=17 pg/ml) were only 26% and 11%, respectively, in contrast to the 49% and 20% 1- and 2-year survival rates of those patients with S-bFGF < 17 pg/ml (P = 0.013). The 1- and 2-year survival rates of the patients with extensive-stage disease were 33% and 10%, respectively (P = 0.0091). Interestingly, S-bFGF provided additional prognostic information to the stage because the 1- and 2-year survival rates of patients with extensive-stage disease and a high S-bFGF >>or=17 pg/ml) were as low as <em>16</em>% and 5%, respectively (P = 0.0026). Similarly, in the multivariate model of survival analysis, patients with both extensive-stage disease and a high S-bFGF >>or=17 pg/ml) were found to have a particularly poor prognosis (relative risk of death, 2.1; 95% confidence interval, 1.2-3.6; P = 0.0057). We conclude that a high S-bFGF at diagnosis is associated with poor outcome in small cell lung cancer, possibly reflecting active angiogenesis and rapid tumor <em>growth</em>, and may complement prognostic information obtained by staging.

Dermatofibrosarcoma protuberans (DFSP) and its juvenile form, giant-cell fibroblastoma (GCF), are uncommon infiltrative tumors of the dermis, which present unique cytogenetic features, such as the reciprocal translocation t(17;22) or, more commonly, supernumerary ring chromosomes containing sequences from chromosomes 17 and 22. We have recently shown that these aberrations are cytogenetic manifestations of gene fusions between the platelet-derived <em>growth</em> <em>factor</em> B-chain gene (PDGFB), the cellular equivalent of the v-sis oncogene, and the collagen type 1 alpha 1 gene (COL1A1), the major protein constituent of the extracellular matrix in connective tissue of skin. We now report characterization of COL1A1/PDGFB chimeric genes at the RNA and DNA sequence levels in a series of DFSPs and GCFs. All <em>16</em> tumors studied contained the COL1A1/PDGFB gene. The location of breakpoints within COL1A1 varied greatly, but was always limited to the region encoding the alpha-helical domain. The PDGFB segment of the chimeric transcript always starts with exon 2, placing PDGFB under the control of the COL1A1 promoter and removing all known elements negatively controlling PDGFB transcription and translation. Production of these aberrant transcripts in <em>fibroblasts</em>, the suspected cell of origin of DFSP/GCF, likely causes autocrine stimulation and cell proliferation. No specific function has yet been assigned to exon 2 of PDGFB, and this exon does not encode for the mature <em>growth</em> <em>factor</em>. Its retention in all chimeric COL1A1/PDGFB genes suggests that it is important for the normal processing of the PDGFB polypeptide.

Limited proteolysis of insulin-like <em>growth</em> <em>factor</em> binding protein-3 (IGFBP-3) is increasingly becoming recognized as an essential mechanism in the regulation of insulin-like <em>growth</em> <em>factor</em> (IGF) bioavailability, both in the bloodstream and at cellular level. Plasmin generated on contact with various cell types provokes proteolytic cleavages that are similar to those induced in vivo by (as yet unidentified) IGFBP-3 proteases. Experimental conditions were determined to achieve plasmin-induced limited proteolysis of recombinant human nonglycosylated IGFBP-3. Two major fragments of 22/25 kilodaltons (kDa) and one of <em>16</em> kDa were identified by Western immunoblotting and isolated by reverse-phase chromatography. The 22/25-kDa fragments correspond to the major approximately 30-kDa glycosylated fragment of IGFBP-3 in serum and the <em>16</em>-kDa fragment, to one of the same size, that is nonglycosylated. Western ligand blot analysis, affinity cross-linking, and competitive binding experiments using radiolabeled IGF and unlabeled IGF-I or -II showed that in the high performance liquid chromatography eluate containing the <em>16</em>-kDa fragment, all affinity for IGFs had been lost, whereas the affinity of the 22/25-kDa fragments was considerably reduced. Scatchard analysis of the data indicated a 20-fold loss of affinity for IGF-II and an 50-fold loss for IGF-I compared with that of recombinant human IGFBP-3. In a chick embryo <em>fibroblast</em> assay in which DNA synthesis was stimulated both by IGF-I and by insulin (at 100-fold concentrations, so that interaction with the Type 1 IGF receptor would occur), IGFBP-3 was found to inhibit IGF-I-induced stimulation almost totally. It had no effect on stimulation by insulin, which has no affinity for the IGFBPs. With the 22/25-kDa fragments, barely 50% inhibition of IGF-I stimulation was achieved and no inhibition of insulin stimulation. Unexpectedly, with the fraction containing the <em>16</em>-kDa fragment (despite the total lack of affinity for IGF-I), IGF-I-induced stimulation was inhibited to nearly the same extent as with intact IGFBP-3. In addition, insulin-induced stimulation was inhibited with similar potency. IGFBP-3 proteolysis therefore generates two types of fragment with different activities. One has weak affinity for IGF-I and is only a weak antagonist of IGF action. The other lacks affinity for the IGFs, but nevertheless inhibits IGF-stimulated mitogenesis, thus acting by a mechanism that is independent of the IGFs.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) is a potent endothelial cell mitogen that has been proposed to play a role in proliferative diabetic retinopathy and other neovascular processes. Our understanding of the in vivo role of basic FGF in the pathogenesis of these disorders is limited. We studied the immunolocalization of basic FGF in <em>16</em> clinical cases of diabetic retinopathy to determine whether the normal retinal distribution of basic FGF changed during the development of diabetic retinopathy and correlated with the onset of retinal neovascularization. By using monoclonal and affinity-purified polyclonal antibodies against basic FGF and heparan sulfate proteoglycan (HSPG), we found that basic FGF colocalized with HSPG to vascular basement membranes. As the basement membranes thickened during the progression of diabetic retinopathy, the intraretinal stores of immunoreactive basic FGF and HSPG expanded. With the development of neovascularization, the colocalization of basic FGF and HSPG changed; HSPG localized to basement membranes, while basic FGF localized intracellularly, with only minimal basement membrane immunoreactivity. Incubations of the neovascular fronds with exogenous basic FGF demonstrated multiple HSPG glycosaminoglycan-binding sites for basic FGF, indicating that basic FGF had not been released from the matrix of neovascular fronds by heparitanase digestion.