Originally identified as <em>factors</em> affecting Drosophila embryogenesis, the Hedgehog (Hh) pathway is one of the primary signaling systems that specify patterns of cell <em>growth</em> and differentiation during vertebrate development. Mutations in various components of this pathway frequently occur in tumors originated from the skin, cerebellum, and skeletal muscle, and abnormal pathway activity is associated with a subset of lung, digestive tract, pancreatic, and prostate cancers. Because of these potent biological activities, this pathway is negatively regulated at multiple levels to ensure appropriate signaling responses. Suppressor of fused (Sufu) is one such negative regulator of Hh signaling. Although not essential in Drosophila, Sufu is absolutely required for mouse embryonic development. Mutations of Sufu are associated with a childhood brain tumor in human and an increased susceptibility to the same type of cancer in the TP53 null background in mice, and RNAi-mediated silencing of Sufu is sufficient to activate the Hh signaling in cultured <em>fibroblasts</em>. All these data point to a central role of Sufu in controlling the vertebrate Hh signaling pathway; however, for years what exactly Sufu does in the Hh pathway and what controls its activity remains a deep mystery. This chapter will go over all studies curated in the PubMed database with Sufu as a main subject during the past <em>17</em> years, and attempt to provide a balanced view on Sufu gene and protein structure, activities in Drosophila as well as mammalian development, and its involvement in cancer.

Epithelial-mesenchymal interactions play an important role in controlling epidermal morphogenesis and homeostasis but little is known about the mechanisms of these interactions. To examine whether diffusible <em>factors</em> produced by <em>fibroblasts</em> and/or keratinocytes support epidermal morphogenesis and basement membrane formation, organotypic keratinocyte monocultures were established in media collected either from organotypic <em>fibroblast</em> or keratinocyte-monocultures or from keratinocyte-<em>fibroblast</em> cocultures, and the expression of keratin 10, 16, and <em>17</em> and basement membrane components (types IV and VII collagen, laminin 5, nidogen, BP 180, LAD-1) were examined. We found that diffusible <em>factors</em> released by keratinocytes were not sufficient to support the establishment of normalized epidermal phenotype and deposition of basement membrane components in contrast to <em>fibroblast</em>- or keratinocyte/<em>fibroblast</em>-derived <em>factors</em>. Keratinocytes appear to affect the spectrum of secreted soluble <em>factors</em>, as keratinocyte/<em>fibroblast</em>-derived <em>factors</em> were more effective to accomplish continuous linear deposition of laminin 5 and of nidogen. The finding that released amounts of keratinocyte <em>growth</em> <em>factor</em> and granulocyte macrophage colony stimulating <em>factor</em> were not sufficient to fully support epidermal morphogenesis and deposition of basement membrane components is suggestive for the involvement of other released diffusible <em>factors</em>. Generation of organotypic keratinocyte monocultures in the presence of <em>fibroblast</em>- or keratinocyte/<em>fibroblast</em>-derived soluble <em>factors</em> resulted in enhanced expression of keratins K16 and K<em>17</em> and the absence of type IV collagen. This observation indicates that next to paracrine acting <em>factors</em>, epidermal homeostasis is controlled by mutual keratinocyte-<em>fibroblast</em> interaction.

A high-throughput small-molecule screen was conducted to identify inhibitors of epithelial-mesenchymal transition (EMT) that could be used as tool compounds to test the importance of EMT signaling in vivo during fibrogenesis. Transforming <em>growth</em> <em>factor</em> (TGF)-β1-induced fibronectin expression and E-cadherin repression in A549 cells were used as 48-hour endpoints in a cell-based imaging screen. Compounds that directly blocked Smad2/3 phosphorylation were excluded. From 2,100 bioactive compounds, methacycline was identified as an inhibitor of A549 EMT with the half maximal inhibitory concentration (IC50) of roughly 5 μM. In vitro, methacycline inhibited TGF-β1-induced α-smooth muscle actin, Snail1, and collagen I of primary alveolar epithelial cells . Methacycline inhibited TGF-β1-induced non-Smad pathways, including c-Jun N-terminal kinase, p38, and Akt activation, but not Smad or β-catenin transcriptional activity. Methacycline had no effect on baseline c-Jun N-terminal kinase, p38, or Akt activities or lung <em>fibroblast</em> responses to TGF-β1. In vivo, 100 mg/kg intraperitoneal methacycline delivered daily beginning 10 days after intratracheal bleomycin improved survival at Day <em>17</em> (P < 0.01). Bleomycin-induced canonical EMT markers, Snail1, Twist1, collagen I, as well as fibronectin protein and mRNA, were attenuated by methacycline (Day <em>17</em>). Methacycline did not attenuate inflammatory cell accumulation or alter TGF-β1-responsive genes in alveolar macrophages. These studies identify a novel inhibitor of EMT as a potent suppressor of fibrogenesis, further supporting the concept that EMT signaling is important to lung fibrosis. The findings also provide support for testing the impact of methacycline or doxycycline, an active analog, on progression of human pulmonary fibrosis.

Interleukin-<em>17</em> (IL-<em>17</em>) and IL-25 signaling induce the expression of genes encoding inflammatory <em>factors</em> and are implicated in the pathology of various inflammatory diseases. Nuclear <em>factor</em> κB (NF-κB) activator 1 (Act1) is an adaptor protein and E3 ubiquitin ligase that is critical for signaling by either IL-<em>17</em> or IL-25, and it is recruited to their receptors (IL-<em>17</em>R and IL-25R) through heterotypic interactions between the SEFIR [SEF (similar expression to <em>fibroblast</em> <em>growth</em> <em>factor</em> genes) and IL-<em>17</em>R] domain of Act1 and that of the receptor. SEFIR domains have structural similarity with the Toll-IL-1 receptor (TIR) domains of Toll-like receptors and IL-1R. Whereas the BB' loop of TIR is required for TIR-TIR interactions, we found that deletion of the BB' loop from Act1 or IL-<em>17</em>RA (a common subunit of both IL-<em>17</em>R and IL-25R) did not affect Act1-IL-<em>17</em>RA interactions; rather, deletion of the CC' loop from Act1 or IL-<em>17</em>RA abolished the interaction between both proteins. Surface plasmon resonance measurements showed that a peptide corresponding to the CC' loop of Act1 bound directly to IL-<em>17</em>RA. A cell-permeable decoy peptide based on the CC' loop sequence inhibited IL-<em>17</em>- or IL-25-mediated signaling in vitro, as well as IL-<em>17</em>- and IL-25-induced pulmonary inflammation in mice. Together, these findings provide the molecular basis for the specificity of SEFIR-SEFIR versus TIR-TIR domain interactions and consequent signaling. Moreover, we suggest that the CC' loop motif of SEFIR domains is a promising target for therapeutic strategies against inflammatory diseases associated with IL-<em>17</em> or IL-25 signaling.

Bevacizumab is a humanized recombinant monoclonal antibody that neutralizes vascular endothelial <em>growth</em> <em>factor</em>, an agent with proangiogenic effects in melanoma. Interferon alpha (IFN-α) has antiangiogenic properties through its ability to downregulate basic-<em>fibroblast</em> <em>growth</em> <em>factor</em> levels. We hypothesized that the coadministration of these agents would lead to tumor regression. Patients with metastatic melanoma received bevacizumab 15 mg/kg intravenously on day 1 of the 2-week cycle. IFN-α was administered thrice weekly at 5 MU/m subcutaneously during cycle 1 and was increased to 10 MU/m during cycle 2. Patients were restaged every 6 cycles. Patients with stable disease or a response continued with therapy. Baseline serum vascular endothelial <em>growth</em> <em>factor</em> and <em>fibroblast</em> <em>growth</em> <em>factor</em> were measured. Twenty-five patients were accrued. Mean age was 58.4 years. Eleven patients required IFN-α dose reductions due to toxicity. Common grade 3 toxicities associated with IFN-α included fatigue and myalgia. Bevacizumab administration was associated with grade 2-3 proteinuria in 6 patients. Grade 4 adverse events were pulmonary embolus (1), myocardial infarction (1), and stroke (1). Six patients had a partial response, and 5 patients exhibited stable disease that lasted more than 24 weeks (range: 30 to 122 wk). Median progression-free survival and overall survival were 4.8 and <em>17</em> months, respectively. Significantly lower <em>fibroblast</em> <em>growth</em> <em>factor</em> levels were observed in patients with a partial response compared to those with stable or progressive disease (P=0.040). Administration of bevacizumab with IFN led to a clinical response in 24% of patients with stage IV melanoma and stabilization of disease in another 20% of patients. This regimen has activity in advanced melanoma.

In vertebrates, <em>Fibroblast</em> <em>Growth</em> <em>Factors</em> (FGFs) and their receptors are involved in various developmental and pathological processes, including neoplasia. The number of FGFs and their large range of activities have made the understanding of their precise functions difficult. Investigating their biology in other species might be enlightening. A sequence encoding a putative protein presenting 30-40% identity with the conserved core of vertebrate FGFs has been identified by the C. elegans sequencing consortium. We show here that this gene is transcribed and encodes a putative protein of 425 amino acids (aa). The gene is expressed at all stages of development beyond late embryogenesis, peaking at the larval stages. Loss-of-function mutants of the let-756 gene are rescued by the wild type fgf gene in germline transformation experiments. Two partial loss-of-function alleles, s2613 and s2809, have a mutation that replaces aa 3<em>17</em> by a stop. The truncated protein retains the FGF core but lacks a C-termins portion. These worms are small and develop slowly into clear and scrawny, yet viable and fertile adults. A third allele, s2887, is inactivated by an inversion that disrupts the first exon. It causes a developmental arrest early in the larval stages. Thus, in contrast to the other nematode fgf gene egl-<em>17</em>, let-756/fgf is essential for worm development.

Neovascularization of the atherosclerotic plaque is responsible for its weakening and consequently for the complications of vascular disease. Macrophages are a source of <em>growth</em> <em>factors</em> that can modulate angiogenesis. In this study, we analyzed the effect of oncostatin M (OSM) on angiogenesis, as it could be involved in the development of atherosclerosis. The effect of OSM was compared with those of leukemia inhibitory <em>factor</em> (LIF) and interleukin-6 (IL-6). On human dermal microvasculature endothelial cells (HMEC-1s), OSM (22.5 to 112.5 pmol/L) induced a dose-dependent increase in cell proliferation greater than that induced by the classic angiogenic <em>factors</em> vascular endothelial <em>growth</em> <em>factor</em> (VEGF; 543 pmol/L) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF; 1.1 nmol/L). LIF (19 to 475 pmol/L) induced only a 30% increase in cell proliferation, and IL-6 had no effect. Furthermore, in a modified Boyden-chamber model, OSM, LIF, and IL-6 were chemoattractant for HMEC-1s. In a tridimensional gel of fibrin, OSM increased tube formation and tube length, which were already noticeable by day 3. LIF and IL-6 induced a weaker effect that was only obvious by day 10. The angiogenic effect of OSM was also demonstrated in vivo in a rabbit corneal model: OSM was more potent than LIF, the length of the neovessels being longer with OSM than with LIF, whereas IL-6 was without effect. We tested <em>factors</em> that could be involved in the proliferative effect of OSM on HMEC-1s. OSM induced only a slight increase in the urokinase receptor and a 60% increase in VEGF secretion, whereas it does not modify IL-8 secretion or bFGF levels. The effect of OSM seems to depend on endothelial cell origin and cell species: OSM (up to 112.5 pmol/L) did not induce human umbilical vein endothelial cell proliferation and even had a small inhibitory effect (<em>17</em>%) on calf pulmonary artery endothelial cells. In conclusion, OSM induces an angiogenic effect on capillary endothelial cells, which could be, at least in part, implicated in pathological processes such as atherosclerosis or tumor <em>growth</em>.

In the course of experiments to identify and characterize the <em>factors</em> that function in bovine conceptuses during peri-attachment periods, various transcripts related to the epithelial-mesenchymal transition (EMT) were found. In this study, RNA was extracted from different sets of days <em>17</em>, 20, and 22 (day 0=day of estrous) bovine conceptuses and subjected to real-time PCR analysis as well as Western blotting, from which abundances of N-cadherin (CDH2), vimentin, matrix metalloproteinase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase) (MMP2), and matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) (MMP9) mRNAs were determined on day 22, concurrent with (CDH1) mRNA and protein downregulation. Transcription <em>factors</em> in EMT processes were then analyzed and changes in snail homolog 2 (Drosophila) (SNAI), zinc finger E-box binding homeobox 1 (ZEB1), zinc finger E-box binding homeobox 2 (ZEB2), twist homolog 1 (Drosophila) (TWIST1), twist homolog 2 (Drosophila) (TWIST2), and Kruppel-like <em>factor</em> 8 (KLF8) transcripts were found in day 22 conceptuses, while confirming SNAI2 expression by Western blotting. Immunohistochemical analysis revealed that the day 22 trophectoderm expressed the mesenchymal markers N-cadherin and vimentin as well as the epithelial marker cytokeratin. In attempts to identify the molecular mechanisms by which the trophectoderm expressed EMT-related genes, <em>growth</em> <em>factor</em> receptors associated with EMT were analyzed. Upregulation of the <em>growth</em> <em>factor</em> receptor transcripts, <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1), platelet-derived <em>growth</em> <em>factor</em> receptor, alpha polypeptide (PDGFRA), platelet-derived <em>growth</em> <em>factor</em> receptor, beta polypeptide (PDGFRB), and transforming <em>growth</em> <em>factor</em>, beta receptor II (70/80 kDa) (TGFBR2) mRNAs, was found on day 22. The analysis was extended to determine the integrin (ITG) transcripts and found high levels of integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor) (ITGA4), integrin, alpha 8 (ITGA8), integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61) (ITGB3), and integrin, beta 5 (ITGB5) mRNAs on day 22. These observations indicate that after the conceptus-endometrium attachment, EMT-related transcripts as well as the epithelial marker cytokeratin were present in the bovine trophectoderm and suggest that the implantation process for noninvasive trophoblasts requires not only extracellular matrix expression but also partial EMT.

Pulmonary fibrosis is a progressive inflammatory disease with high mortality and limited therapeutic options. Previous genetic and immunologic investigations suggest common intersections between idiopathic pulmonary fibrosis (IPF), sarcoidosis, and murine models of pulmonary fibrosis. To identify immune responses that precede collagen deposition, we conducted molecular, immunohistochemical, and flow cytometric analysis of human and murine specimens. Immunohistochemistry revealed programmed cell death-1 (PD-1) up-regulation on IPF lymphocytes. PD-1+CD4+ T cells with reduced proliferative capacity and increased transforming <em>growth</em> <em>factor</em>-β (TGF-β)/interleukin-<em>17</em>A (IL-<em>17</em>A) expression were detected in IPF, sarcoidosis, and bleomycin CD4+ T cells. PD-1+ T helper <em>17</em> cells are the predominant CD4+ T cell subset expressing TGF-β. Coculture of PD-1+CD4+ T cells with human lung <em>fibroblasts</em> induced collagen-1 production. Strikingly, ex vivo PD-1 pathway blockade resulted in reductions in TGF-β and IL-<em>17</em>A expression from CD4+ T cells, with concomitant declines in collagen-1 production from <em>fibroblasts</em>. Molecular analysis demonstrated PD-1 regulation of the transcription <em>factor</em> STAT3 (signal transducer and activator of transcription 3). Chemical blockade of STAT3, using the inhibitor STATTIC, inhibited collagen-1 production. Both bleomycin administration to PD-1 null mice or use of antibody against programmed cell death ligand 1 (PD-L1) demonstrated significantly reduced fibrosis compared to controls. This work identifies a critical, previously unrecognized role for PD-1+CD4+ T cells in pulmonary fibrosis, supporting the use of readily available therapeutics that directly address interstitial lung disease pathophysiology.

The present study demonstrated that the vessel number in bone marrow biopsies from acute myeloid leukaemia (AML) patients (n = 23) was significantly increased at diagnosis compared with normal bone marrow (P = 0.019) and was restored to normal levels after achieving complete remission (P = 0.03). The in vitro angiogenic potential of culture supernatant of AML cells was assessed using endothelial cell (EC) migration and proliferation assays. Increased EC migration and EC proliferation was induced in 7/20 and 19/20 AML supernatents respectively. The degree of in vivo neovascularization did not correlate with the ability of AML cells to stimulate in vitro endothelial cell migration and/or proliferation. This might be in part a result of the heterogeneous pattern of angiogenic <em>factors</em> produced by AML cells. The expression of different angiogenic <em>factors</em> was studied using reverse transcription polymerase chain reaction. Cells from <em>17</em>/20 AML patients showed wide variation in spontaneous vascular endothelial <em>growth</em> <em>factor</em> (VEGF) expression, 4/19 expressed varied spontaneous blastic <em>fibroblast</em> <em>growth</em> <em>factor</em> mRNA levels and all patient samples showed spontaneous interleukin 8 mRNA expression. All AML samples expressed matrix metalloproteinase (MMP)-2 and/or MMP-9. VEGF mRNA expression correlated well with protein level (P = 0.006). A correlation was found between the degree of VEGF expression and neoangiogenesis (correlation coefficient = 0.448, P = 0.05). These results suggest that malignant cell proliferation, angiogenesis and VEGF expression are linked in AML and might contribute to the <em>growth</em> advantage of the malignant counterpart as a result of the paracrine production of <em>growth</em> <em>factors</em> produced by the surrounding endothelial cells.

A highly reproducible and technically straightforward technique for the isolation and long-term culture of normal human endometrial epithelial cells is described. The essential conditions for long-term culture are that the cells be seeded onto a gelatin matrix and that 'endothelial cell <em>growth</em> supplement' be present in the culture medium. Normal endometrial epithelial cells express cytokeratins and oestrogen receptors. They may be passaged five to six times without change in properties. <em>Growth</em> of normal endometrial epithelial cells was stimulated by <em>17</em>-beta-oestradiol and epidermal <em>growth</em> <em>factor</em>. Expression of the mRNA coding for seven polypeptide angiogenic <em>factors</em>, by normal endometrial epithelial, stromal and three endometrial carcinoma lines, was examined. The endometrial epithelial and stromal cells express mRNA for the polypeptide angiogenic <em>factors</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, vascular endothelial cell <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-beta 1 and pleiotrophin, as well as the cytokine midkine. Expression of the mRNA for both vascular endothelial <em>growth</em> <em>factor</em> and midkine by normal endometrial epithelial cells showed a 2-fold increase on treatment with a physiological dose of <em>17</em>-beta-oestradiol (10(-10) M) while, in contrast, the mRNA of transforming <em>growth</em> <em>factor</em>-beta 1 decreased 4-fold on treatment with <em>17</em>-beta-oestradiol (10(-10) M) and was abolished by exposure to progesterone (5 x 10(-9) M). Expression of the mRNAs for angiogenic polypeptides by the endometrial carcinoma lines was more restricted.

Recombinant expression of either the alpha or beta platelet-derived <em>growth</em> <em>factor</em> (PDGF) receptors in 32D hematopoietic cells allows efficient coupling of PDGF with mitogenic and chemotactic signaling pathways inherently expressed by those cells. PDGF-BB stimulation of 32D-alpha R or beta R cells results in anti-P-Tyr recovery of cellular proteins possessing similar as well as distinct phosphotyrosine signals. Comparison of the ability of each receptor to couple with known second messengers revealed that both receptors associated with and/or tyrosine phosphorylated phospholipase C-gamma (PLC gamma) and phosphatidylinositol 3-kinase (p85) with similar stoichiometry. However, the beta platelet-derived <em>growth</em> <em>factor</em> receptor (PDGFR) was significantly more efficient at in vivo tyrosine phosphorylation of GTPase-activating protein (GAP). Similar differences in binding affinity for GAP were observed in NIH/3T3 cells which express both receptors. To quantitate the affinities of each receptor for GAP or PLC gamma, we utilized baculovirus-expressed alpha and beta PDGFRs purified by anti-P-Tyr affinity chromatography. Exposure of immunoblots containing bacterially expressed GAP or PLC gamma to activated alpha or beta PDGF receptors led to a comparable high affinity binding of each receptor to PLC gamma, while the beta PDGFR showed a 5-fold higher binding affinity for GAP. In an effort to correlate differences in their substrate specificities with biological properties of the receptors, we compare their abilities to enhance PDGF-A transforming function in NIH/3T3 cells. Cotransfection of PDGF-A with the alpha PDGFR increased PDGF-A transforming activity by approximately 2-fold. However, cotransfection with a chimeric receptor with the catalytic domain of the beta PDGFR but possessing alpha PDGFR ligand binding properties resulted in <em>17</em>-fold enhancement of PDGF-A transformation. These findings argue that differences in alpha and beta PDGF receptor substrate specificity in NIH/3T3 <em>fibroblasts</em> correlate with greater transforming activity mediated by the beta PDGFR.

DNA synthesis in prolyl 4-hydroxylase (PH; EC 1.14.11.2) positive <em>fibroblasts</em> in situ in synovial tissue was studied using an autoradiography-avidin-biotin-peroxidase complex (ABC) double labeling. <em>Fibroblasts</em> in monolayer culture and in situ in synovial tissue were PH positive, whereas freshly isolated peripheral blood lymphocytes, monocytes, dendritic cells and granulocytes were PH negative. In rheumatoid arthritis (RA) 37 +/- 3 (22-56)% of all DNA synthesizing cells in situ were PH containing <em>fibroblasts</em>, whereas all DNA synthesizing cells in patients with meniscus lesion were PH positive. In both conditions, more than half of the self-replicating <em>fibroblasts</em> were located in the lining cell layer. This is probably not an artifact caused by insufficient penetration of 3H-thymidine because most of the DNA synthesizing lymphocytes were deep down in the synovial stroma. In RA 51 +/- 8 (<em>17</em>-88) PH positive <em>fibroblasts</em> in the S phase of the cell cycle were observed/3 mm2 synovial tissue, whereas the corresponding figure in meniscus patients was only 1 +/- 1 (0-5) (p less than 0.01). This suggests that the local <em>fibroblasts</em> in RA are activated, probably as a result of various <em>fibroblast</em> <em>growth</em> <em>factors</em> produced locally as a result of the inflammatory synovitis. In RA however, less than 1% of all local <em>fibroblasts</em> were self-replicating in situ, whereas labeling indices over 5% were not uncommon in RA synovial <em>fibroblast</em> cultures. This finding suggests that uncontrolled <em>fibroblast</em> proliferation is regulated in vivo by negative feedback mechanisms.

Compared to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), a widely distributed, broad spectrum mitogen and mesoderm inducer, acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) is reported to have an essentially neural distribution and to be undetectable in the early embryo. In the present investigation, we used immunoblotting and immunochemistry to assess the cellular and tissue distributions of aFGF and bFGF in 11-20-d rat embryos. Immunoblotting of crude and heparin-bound embryo extracts revealed faint bands at the expected <em>17</em>-18-kD and predominant bands at an apparent molecular mass of 26 to 28-kD (despite reducing conditions) using multiple specific antibodies for aFGF and bFGF. Pretreatment with 8 M urea yielded 18-20-kD aFGF and bFGF and some 24-26-kD bFGF. Immunoreactivity for both aFGF and bFGF was positive and similar in the cytoplasm, nuclei, and extracellular matrix of cells of neuroectodermal and mesodermal origin, while it was negative in endoderm-derived cells. The distribution of immunoreactive aFGF and bFGF also showed changes during development that were associated with the process of cellular and tissue differentiation. For example, intensity and extent of immunoreactivity for both peptides progressively increased in the middle layer of the spinal cord with increasing differentiation of the neural cells. The immunostaining patterns were very similar for aFGF and bFGF for each organ and at each stage. In conclusion, high molecular mass forms of immunoreactive aFGF and bFGF are present in the rat embryo. Acidic FGF and bFGF are both widely distributed in tissues of neuroectodermal and mesodermal origin, and their distribution was very similar.

We isolated the cDNA encoding a novel member (216 amino acids) of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family from rat embryos. As this protein is the <em>17</em>th documented member of the FGF family, we tentatively termed it FGF-<em>17</em>. We have also determined the structures of mouse and human FGF-<em>17</em> with high amino acid identity (100 and 98.6%) to rat FGF-<em>17</em>, respectively. Among FGF family members, FGF-<em>17</em> is most similar (53.7% amino acid identity) to FGF-8. FGF-<em>17</em> has a typical signal sequence at its amino terminus. As expected, recombinant rat FGF-<em>17</em> was efficiently secreted by High Five insect cells infected with recombinant baculovirus containing the cDNA indicating that FGF-<em>17</em> is a secreted protein. FGF-<em>17</em> mRNA of approximately 2.1 kb was detected in rat embryos at E14.5, but not at E10.5 and E19.5 by Northern analysis. The mRNA was found to be preferentially expressed in the neuroepithelia of the isthmus and septum of the rat embryonic brain at E14.5 by in situ hybridization. The present results indicate that FGF-<em>17</em> might be a novel secreted signaling molecule in the induction and patterning of the embryonic brain.

We recently reported that acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> (aFGF and bFGF) confer a broad-spectrum chemoresistance in solid tumors, and that inhibitors of these proteins enhanced the antitumor activity of several anticancer drugs. The present study investigated the effect of FGF inhibitors on doxorubicin activity in human prostate PC3 tumors. In in vitro studies, conditioned medium (CM) obtained from histocultures of rat MAT-LyLu lung metastases and different combinations of recombinant FGF induced a 7- to 15-fold doxorubicin resistance. Suramin had no effect on the doxorubicin activity in the absence of CM or FGF, but reversed the CM- and FGF-induced resistance by>> or =90% at concentrations that had no cytotoxicity (i.e., 1-<em>17</em> microM suramin). In the in vivo study, immunodeficient mice bearing well established, subcutaneous PC3 tumors (approximately 100 mg in size) were treated intravenously with doxorubicin (5 mg/kg) and suramin (10 mg/kg), administered twice weekly for 3 weeks. The suramin dose, selected to yield plasma concentration of below 50 microM, had neither antitumor activity nor toxicity. Doxorubicin alone reduced tumor <em>growth</em> rate by approximately 60%, reduced the density of nonapoptotic tumor cells by approximately 60%, enhanced the apoptotic cell fraction by 4-fold, and reduced the body weight by approximately 15% (p < 0.05 compared with control). Addition of suramin to doxorubicin therapy did not increase weight loss but significantly enhanced the antitumor effect, resulting in complete inhibition of tumor <em>growth</em>, an additional 3-fold reduction in the density of nonapoptotic tumor cells, and an additional 2-fold enhancement of the apoptotic tumor cell fraction (p < 0.05 compared with all other groups). These data indicate significant enhancement of the effectiveness of doxorubicin in prostate tumors by nontoxic and subtherapeutic doses of suramin.

BACKGROUND

Idiopathic pulmonary fibrosis (IPF) and chronic hypersensitivity pneumonitis (HP) are diffuse parenchymal lung diseases characterized by a mixture of inflammation and fibrosis, leading to lung destruction and finally death.

OBJECTIVE

The aim of this study was to compare different pathophysiological mechanisms, such as angiogenesis, coagulation, fibrosis, tissue repair, inflammation, epithelial damage, oxidative stress, and matrix remodeling, in both disorders using bronchoalveolar lavage (BAL).

METHODS

at diagnosis, patients underwent bronchoscopy with BAL and were divided into three groups: Control (n = 10), HP (n = 11), and IPF (n = 11), based on multidisciplinary approach (clinical examination, radiology, and histology): Multiplex searchlight technology was used to analyze 25 proteins representative for different pathophysiological processes: Eotaxin, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGFb), fibronectin, hepatocyte <em>growth</em> <em>factor</em> (HGF), interleukine (IL)-8, IL-12p40, IL-<em>17</em>, IL-23, monocyte chemotactic protein (MCP-1), macrophage-derived chemokine (MDC), myeloperoxidase (MPO), matrix metalloproteinase (MMP)-8, MMP-9, active plasminogen activating inhibitor 1 (PAI-1), pulmonary activation regulated chemokine (PARC), placental <em>growth</em> <em>factor</em> (PlGF), protein-C, receptor for advanced glycation end products (RAGE), regulated on activation normal T cells expressed and secreted (RANTES), surfactant protein-C (SP-C), transforming <em>growth</em> <em>factor</em>-β1 (TGF-β1), tissue inhibitor of metalloproteinase-1 (TIMP-1), tissue <em>factor</em>, thymic stromal lymphopoietin (TSLP), and vascular endothelial <em>growth</em> <em>factor</em> (VEGF).

RESULTS

All patients suffered from decreased pulmonary function and abnormal BAL cell differential compared with control. Protein levels were increased in both IPF and HP for MMP-8 (P = 0.022), MMP-9 (P = 0.0020), MCP-1 (P = 0.0006), MDC (P = 0.0048), IL-8 (P = 0.013), MPO (P = 0.019), and protein-C (P = 0.0087), whereas VEGF was decreased (P = 0.0003) compared with control. HGF was upregulated in HP (P = 0.0089) and active PAI-1 was upregulated (P = 0.019) in IPF compared with control. Differences in expression between IPF and HP were observed for IL-12p40 (P = 0.0093) and TGF-β1 (P = 0.0045).

CONCLUSIONS

Using BAL, we demonstrated not only expected similarities but also important differences in both disorders, many related to the innate immunity. These findings provide new clues for further research in both disorders.

The periodontal ligament (PDL) that anchors the tooth root to the alveolar bone influences the lifespan of the tooth, and PDL lost through periodontitis is difficult to regenerate. The development of new PDL-regenerative therapies requires the isolation of PDL stem cells. However, their characteristics are unclear due to the absence of somatic PDL stem cell lines and because PDL is composed of heterogeneous cell populations. Recently, we succeeded in immortalizing human PDL <em>fibroblasts</em> that retained the properties of the primary cells. Therefore, we aimed to establish a human PDL-committed stem cell line and investigate the effects of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) on the osteoblastic differentiation of the cells. Here, we report the development of cell line 1-<em>17</em>, a multipotent clonal human PDL cell line that expresses the embryonic stem cell-related pluripotency genes Oct3/4 and Nanog, as well as the PDL-related molecules periostin and scleraxis. Continuous treatment of cell line 1-<em>17</em> with bFGF in osteoblastic induction medium inhibited its calcification, with down-regulated expression of FGF-Receptor 1 (FGF-R1), whereas later addition of bFGF potentiated its calcification. Furthermore, bFGF induced calcification of cell line 1-<em>17</em> when it was co-cultured with osteoblastic cells. These results suggest that cell line 1-<em>17</em> is a PDL-committed stem cell line and that bFGF exerts dualistic (i.e., promoting and inhibitory) effects on the osteoblastic differentiation of cell line 1-<em>17</em> based on its differentiation stage.

Inhibition of the vascular endothelial <em>growth</em> <em>factor</em> VEGF-VEGF receptor (VEGF-R) kinase axes in the tumor angiogenic cascade is a promising therapeutic strategy in oncology. CEP-7055 is the fully synthetic orally active N,N-dimethyl glycine ester of CEP-5214, a C3-(isopropylmethoxy) fused pyrrolocarbazole with potent pan-VEGF-R kinase inhibitory activity. CEP-5214 demonstrates IC(50) values of 18 nM, 12 nM, and <em>17</em> nM against human VEGF-R2/KDR kinase, VEGF-R1/FLT-1 kinase, and VEGF-R3/FLT-4 kinase, respectively, in biochemical kinase assays. CEP-5214 inhibited VEGF-stimulated VEGF-R2/KDR autophosphorylation in human umbilical vein endothelial cells (HUVECs) with an IC (50) of approximately 10 nM and demonstrated an equivalent inhibition of murine FLK-1 autophosphorylation in transformed SVR endothelial cells. Evaluation of the antiangiogenic activity of CEP-5214 revealed a dose-related inhibition of microvessel <em>growth</em> ex vivo in rat aortic ring explant cultures and in vitro on HUVEC capillary-tube formation on Matrigel at low nanomolar concentrations. The antiangiogenic activity of CEP-5214 in these bioassays was observed in the absence of apparent cytotoxicity. Single-dose p.o. or s.c. administration of CEP-7055 or CEP-5214 to CD-1 mice at 23.8 mg/kg/dose b.i.d. resulted in a reversible inhibition of VEGF-R2/FLK-1 phosphorylation in murine lung tissues. Administration p.o. of CEP-7055 at 2.57 to 23.8 mg/kg/dose b.i.d. resulted in dose-related reductions in neovascularization in vivo in porcine aortic endothelial cell (PAEC)-VEGF/basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-Matrigel implants in nude mice (maximum, 82% inhibition), significant reductions in granuloma formation (30%) and granuloma vascularity (42%) in a murine chronic inflammation-induced angiogenesis model, and significant and sustained (6 h) inhibition of VEGF-induced plasma extravasation in rats, with an ED(50) of 20 mg/kg/dose. Chronic p.o. administration of CEP-7055 at doses of 11.9 to 23.8 mg/kg/dose b.i.d. resulted in significant inhibition (50-90% maximum inhibition relative to controls) in the <em>growth</em> of a variety of established murine and human s.c. tumor xenografts in nude mice, including A375 melanomas, U251MG and U87MG glioblastomas, CALU-6 lung carcinoma, ASPC-1 pancreatic carcinoma, HT-29 and HCT-116 colon carcinomas, MCF-7 breast carcinomas, and SVR angiosarcomas. Significant antitumor efficacy was observed similarly against orthotopically implanted LNCaP human prostate carcinomas in male nude mice and orthotopically implanted renal carcinoma (RENCA) tumors in BALB/c mice, in terms of a significant reduction in the metastatic score and the extent of pulmonary metastases. These antitumor responses were associated with marked increases in tumor apoptosis, and significant reductions in intratumoral microvessel density (CD34 and <em>Factor</em> VIII staining) of 22-38% relative to controls depending on the specific tumor xenograft. The antitumor efficacy of chronic CEP-7055 administration was independent of initial tumor volume (in the ASPC-1 pancreatic carcinoma model) and reversible on withdrawal of treatment. Chronic p.o. administration of CEP-7055 in preclinical efficacy studies for periods of up to 65 days was well tolerated with no apparent toxicity or significant morbidity. Orally administered CEP-7055 has entered Phase I clinical trials in cancer patients.

Tamoxifen treatment of estrogen-dependent breast cancer ultimately loses its effectiveness due to the development of resistance. From a functional screen for identifying genes responsible for tamoxifen resistance in human ZR-75-1 breast cancer cells, <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>17</em> was recovered. The aim of this exploratory study was to assess the predictive value of FGF<em>17</em> and the receptors FGFR1-4 for the type of response to tamoxifen treatment (clinical benefit) and the duration of progression-free survival (PFS) in patients with recurrent breast cancer. mRNA levels of FGF<em>17</em> and FGFR1-4 were quantified by real-time reverse transcriptase PCR in 285 estrogen receptor-positive breast carcinomas with clinical follow-up. All patients had recurrent disease and were treated with tamoxifen as first-line systemic therapy for local or distant relapse. FGF<em>17</em> and FGFR1-3 mRNA levels had no significant predictive value for this group of patients. However, high FGFR4 mRNA levels analyzed as a continuous log-transformed variable predicted poor clinical benefit (odds ratio=1.22; P=0.009) and shorter PFS (hazard ratio=1.18; P<0.001). In addition, in multivariable analysis, the predictive value of FGFR4 was independent from the traditional predictive <em>factors</em>. Our analyses show that FGFR4 may play a role in the biological response of the tumor to tamoxifen treatment. In addition, as altered expression of FGF<em>17</em> causes tamoxifen resistance in vitro, the FGF signaling pathway could be a valuable target in the treatment of breast cancer patients resistant to endocrine treatment.

Obesity is a chronic inflammation with increased serum levels of insulin, insulin-like <em>growth</em> <em>factor</em> 1 (IGF1), and interleukin-<em>17</em> (IL-<em>17</em>). The objective of this study was to test a hypothesis that insulin and IGF1 enhance IL-<em>17</em>-induced expression of inflammatory chemokines/cytokines through a glycogen synthase kinase 3β (GSK3B)-dependent mechanism, which can be inhibited by melatonin. We found that insulin/IGF1 and lithium chloride enhanced IL-<em>17</em>-induced expression of C-X-C motif ligand 1 (Cxcl1) and C-C motif ligand 20 (Ccl20) in the Gsk3b(+/+) , but not in Gsk3b(-/-) mouse embryonic <em>fibroblast</em> (MEF) cells. IL-<em>17</em> induced higher levels of Cxcl1 and Ccl20 in the Gsk3b(-/-) MEF cells, compared with the Gsk3b(+/+) MEF cells. Insulin and IGF1 activated Akt to phosphorylate GSK3B at serine 9, thus inhibiting GSK3B activity. Melatonin inhibited Akt activation, thus decreasing P-GSK3B at serine 9 (i.e., increasing GSK3B activity) and subsequently inhibiting expression of Cxcl1 and Ccl20 that was induced either by IL-<em>17</em> alone or by a combination of insulin and IL-<em>17</em>. Melatonin's inhibitory effects were only observed in the Gsk3b(+/+) , but in not Gsk3b(-/-) MEF cells. Melatonin also inhibited expression of Cxcl1, Ccl20, and Il-6 that was induced by a combination of insulin and IL-<em>17</em> in the mouse prostatic tissues. Further, nighttime human blood, which contained high physiologic levels of melatonin, decreased expression of Cxcl1, Ccl20, and Il-6 in the PC3 human prostate cancer xenograft tumors. Our data support our hypothesis and suggest that melatonin may be used to dampen IL-<em>17</em>-mediated inflammation that is enhanced by the increased levels of insulin and IGF1 in obesity.

Celastrol (CSL) is a naturally occurring triterpenoid acid that exhibits anticancer activity, and in KU7 and 253JB-V bladder cells, CSL induced apoptosis, inhibited <em>growth</em>, colony formation and migration and CSL decreased bladder tumor <em>growth</em> in vivo. CSL also decreased expression of specificity protein (Sp) transcription <em>factors</em> Sp1, Sp3 and Sp4 and several Sp-regulated genes/proteins including vascular endothelial <em>growth</em> <em>factor</em>, survivin and cyclin D1 and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor-3, a potential drug target for bladder cancer therapy, has now been characterized as an Sp-regulated gene downregulated by CSL. The mechanism of Sp downregulation by CSL was cell context-dependent due to activation of proteosome-dependent (KU7) and -independent (253JB-V) pathways. In 253JB-V cells, CSL induced reactive oxygen species (ROS) and inhibitors of ROS blocked CSL-induced <em>growth</em> inhibition and repression of Sp1, Sp3 and Sp4. This response was due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of miR-27a and miR-20a/<em>17</em>-5p, respectively, which regulate expression of these transcriptional repressors. Thus, the anticancer activity of CSL in 253JB-V cells is due to induction of ROS and ROS-mediated induction of Sp repressors (ZBTB4/ZBTB10) through downregulation of miR-27a and miR-20a/<em>17</em>-5p.

BACKGROUND

There is an ongoing controversy on the benefits of treatment protocols, including dental lasers and photodynamic therapy (PDT). The purpose of this study is to compare the local biologic effects of PDT, diode soft laser (DSL) therapy, and conventional deep scaling and root planing (SRP) in residual pockets.

METHODS

Thirty-two individuals were included based on a history of previous treatment for periodontitis and the persistence of sites with probing depths >4 mm and bleeding on probing. Residual pockets were debrided with an ultrasonic device and then randomly assigned either to PDT, DSL, or SRP. Gingival crevicular fluid was collected before treatment, after 14 days, and at 2 and 6 months. Levels of 13 cytokines and nine acute-phase proteins were measured using a bead-based multiplexing analysis system.

RESULTS

Treatment with PDT, DSL, or SRP led to significant changes in several cytokines and acute-phase proteins: Compared with baseline, levels of interleukin-<em>17</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, granulocyte colony-stimulating <em>factor</em>, granulocyte macrophage colony-stimulating <em>factor</em>, and macrophage inflammatory protein 1-α were lower 14 days and 2 months after treatment. Except for granulocyte colony-stimulating <em>factor</em>, these differences remained significant throughout the observation period. The levels of five acute-phase proteins (α-2 macroglobulin, haptoglobin, serum amyloid P, procalcitonin, and tissue plasminogen activator) were significantly higher at 6 months than at baseline. No significant differences were observed among the three treatment modalities at any time point for any biochemical parameter.

CONCLUSIONS

Levels of several cytokines and acute-phase proteins significantly changed after treatment regardless of treatment modality. There was no evidence for a specific DSL- or PDT-enhanced expression of inflammatory mediators.

Active angiogenesis, together with an up-regulation of angiogenic <em>factors</em>, is evident in the synovium of both rheumatoid arthritis (RA) and osteoarthritis (OA). The present study assessed, by immunohistochemistry, the microvessel density in the synovium of these arthritides and in normal controls, in relation to the expression of the angiogenic <em>factors</em> vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and platelet-derived endothelial cell <em>growth</em> <em>factor</em> (PD-ECGF) and the apoptosis-related proteins bcl-2 and p53. More importantly, using the novel 11B5 MAb, the activated "VEGF/flk-1(KDR)-receptor" microvessel density was assessed. VEGF expression in <em>fibroblasts</em> was diffuse in both RA and OA. Diffuse PD-ECGF expression of <em>fibroblasts</em> was noted in all cases of RA, while <em>fibroblast</em> reactivity was focal in the OA material. The standard microvessel density (sMVD), as assessed with the anti-CD31 monoclonal antibody (MAb), was higher in RA (64+/-12) and in OA (65+/-16) than in normal tissues (52+/-8; p=0.008 and 0.0004, respectively). The activated microvessel density (aMVD), assessed with the 11B5 MAb, was significantly higher in RA (29+/-10) than in OA (<em>17</em>+/-4; p<0.0001) and than in normal tissues (14+/-2; p<0.0001). The "activation ratio" (aMVD/sMVD) was statistically higher in RA (0.46+/-0.<em>17</em>) than in OA and normal synovial tissues, the latter two having a similar ratio (0.28+/-0.08 and 0.26+/-0.03, respectively). Cytoplasmic bcl-2 expression was frequent in the synovial cells of OA, but rare in RA. Nuclear p53 protein accumulation was never observed. It is suggested that the angiogenic pathway VEGF/flk-1(KDR) may play an important role in the pathogenesis of RA and OA. Thus, failure of VEGF/flk-1(KDR) activation, in the presence of increased VEGF expression, may indicate a synovium with an impaired capacity to establish a viable vasculature, consistent with the degenerative nature of OA. On the other hand, the activated angiogenesis in RA shows a functional, still pathologically up-regulated VEGF/flk-1(KDR) pathway. Whether restoration of an impaired VEGF/flk-1(KDR) pathway in OA, or inhibition of this in RA, would prove of therapeutic importance requires further investigation.