<em>Fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) promotes phosphaturia and suppresses 1,25-dihydroxyvitamin D [1,25(OH)(2)D] production. PTH also promotes phosphaturia, but, in contrast, stimulates 1,25(OH)(2)D production. The relationship between FGF23 and PTH is unclear, and the acute effect of pharmacologically dosed PTH on FGF23 secretion is unknown. Twenty healthy men were infused with human PTH(1-34) [hPTH(1-34)] at 44 ng/kg/h for 24 h. Compared with baseline, FGF23, 1,25(OH)(2)D, ionized calcium (iCa), and serum N-telopeptide (NTX) increased significantly over the 18-h hPTH(1-34) infusion (p < 0.0001), whereas serum phosphate (PO(4)) transiently increased and then returned to baseline. FGF23 increased from 35 +/- 10 pg/ml at baseline to 53 +/- 20 pg/ml at 18 h (p = 0.0002); 1,25(OH)(2)D increased from 36 +/- 16 pg/ml at baseline to 80 +/- 33 pg/ml at 18 h (p < 0.0001); iCa increased from 1.23 +/- 0.03 mM at baseline to 1.46 +/- 0.05 mM at hour 18 (p < 0.0001); and NTX increased from <em>17</em> +/- 4 nM BCE at baseline to 28 +/- 8 nM BCE at peak (p < 0.0001). PO(4) was 3.3 +/- 0.6 mg/dl at baseline, transiently rose to 3.7 +/- 0.4 mg/dl at hour 6 (p = 0.016), and then returned to 3.4 +/- 0.5 mg/dl at hour 12 (p = 0.651). hPTH(1-34) infusion increases endogenous 1,25(OH)(2)D and FGF23 within 18 h in healthy men. Whereas it is possible that the rise in PO(4) contributed to the observed increase in FGF23, the increase in 1,25(OH)(2)D was more substantial and longer sustained than the change in serum phosphate. Given prior data that suggest that neither PTH nor calcium stimulate FGF23 secretion, these data support the assertion that 1,25(OH)(2)D is a potent physiologic stimulator of FGF23 secretion.

Sorafenib is effective in hepatocellular carcinoma (HCC), but patients ultimately present disease progression. Molecular mechanisms underlying acquired resistance are still unknown. Herein, we characterise the role of tumour-initiating cells (T-ICs) and signalling pathways involved in sorafenib resistance.

HCC xenograft mice treated with sorafenib (n=22) were explored for responsiveness (n=5) and acquired resistance (n=<em>17</em>). Mechanism of acquired resistance were assessed by: (1) role of T-ICs by in vitro sphere formation and in vivo tumourigenesis assays using NOD/SCID mice, (2) activation of alternative signalling pathways and (3) efficacy of anti-FGF and anti-IGF drugs in experimental models. Gene expression (microarray, quantitative real-time PCR (qRT-PCR)) and protein analyses (immunohistochemistry, western blot) were conducted. A novel gene signature of sorafenib resistance was generated and tested in two independent cohorts.

Sorafenib-acquired resistant tumours showed significant enrichment of T-ICs (164 cells needed to create a tumour) versus sorafenib-sensitive tumours (13 400 cells) and non-treated tumours (1292 cells), p<0.001. Tumours with sorafenib-acquired resistance were enriched with insulin-like growth factor (IGF) and fibroblast growth factor (FGF) signalling cascades (false discovery rate (FDR)<0.05). In vitro, cells derived from sorafenib-acquired resistant tumours and two sorafenib-resistant HCC cell lines were responsive to IGF or FGF inhibition. In vivo, FGF blockade delayed tumour growth and improved survival in sorafenib-resistant tumours. A sorafenib-resistance <em>17</em>5 gene signature was characterised by enrichment of progenitor cell features, aggressive tumorous traits and predicted poor survival in two cohorts (n=442 patients with HCC).

Acquired resistance to sorafenib is driven by T-ICs with enrichment of progenitor markers and activation of IGF and FGF signalling. Inhibition of these pathways would benefit a subset of patients after sorafenib progression.

The <em>growth</em> and development of hair follicles is influenced by a number of different <em>growth</em> <em>factors</em> and cytokines, particularly members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family. Keratinocyte <em>growth</em> <em>factor</em> (KGF or FGF-7) is a recently identified 28-kd member of the FGF family that induces proliferation of a wide variety of epithelial cells, including keratinocytes within the epidermis and dermal adnexa. Because KGF induces marked proliferation of keratinocytes, and both KGF and KGF receptor (KGFR) mRNA are expressed at high levels in skin, we sought to localize KGF and KGFR in skin by in situ hybridization. KGFR mRNA was relatively strongly expressed by keratinocytes in the basilar epidermis as well as throughout developing hair follicles of rat embryos and neonates. KGF mRNA was expressed at lower levels than was KGFR but could be localized to follicular dermal papillae in rat embryos and neonates. These results prompted us to investigate the effects of KGF on hair follicles in two distinct murine models of alopecia. In the first model, recombinant KGF (rKGF) induced dose-dependent hair <em>growth</em> over most of the body in nu/nu athymic nude mice when administered intraperitoneally or subcutaneously over <em>17</em> to 18 days. When administered subcutaneously, rKGF induced the most extensive hair <em>growth</em> at the sites of injection. Histologically, rKGF induced marked follicular and sebaceous gland hypertrophy, a normalization of the nu/nu follicular keratinization defect, and an increase in follicular keratinocyte proliferation as assessed by bromodeoxyuridine labeling. In the second model, a neonatal rat model of cytosine arabinoside chemotherapy-induced alopecia in which interleukin-1, epidermal <em>growth</em> <em>factor</em>, and acidic FGF have all demonstrated some degree of alopecia cytoprotection, rKGF induced a dose-dependent cytoprotective effect, abrogating as much as 50% of the alopecia in this model when administered beginning 1 day before the onset of chemotherapy. Taken together, these data suggest that KGF is an important endogenous mediator of normal hair follicle <em>growth</em>, development, and differentiation.

An immunohistochemical study combined with morphometry was carried out to examine the time-dependent expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) using 53 human skin wounds with different wound ages (groups I: 0-12 h, II: 1-4 days, III: 7-14 days and IV: <em>17</em>-21 days). In the human wound specimens aged 4-12 h, neutrophils recruited at the wound showed no positive signals for VEGF. With an increase in wound ages of>> or =7 days, granulation tissue and angiogenesis were observed, with the migration of macrophages and <em>fibroblasts</em> of which the cytoplasm expressed VEGF-positive reactions. Morphometrically, the average VEGF-positive ratio was highest in group III, followed by that of group IV. In groups III and IV, 13 out of 26 wound samples had VEGF-positive ratios of more than 50%. However, all of the wound samples in groups I and II showed VEGF-positive ratios of less than 50%. With regard to the practical applicability and forensic validity, these observations suggest that a VEGF-positive ratio of more than 50% possibly indicates a wound age of 7 days or more.

Grb10 is a protein that binds to the intracellular domains of activated tyrosine kinase receptors, including insulin-like <em>growth</em> <em>factor</em> (IGF-I) and insulin receptors. This occurs through the interaction of two C-terminal Grb10 motifs (BPS and Src homology domains) with receptor phosphotyrosine residues. Published data from transfection/overexpression studies support both positive and negative regulatory effects of Grb10, thus leaving its physiological role unclear. Because Grb10 has the structure of an adapter protein, the objective of this study was to determine whether Grb10 links other proteins to IGF-I receptors and thus modulates IGF-I signaling. Using yeast two-hybrid screening, the N terminus of Grb10 was shown to interact with two novel proteins, designated GIGYF1 (Grb10 interacting GYF protein 1) and GIGYF2. Mutation analysis indicates that a <em>17</em>-amino acid sequence in GIGYF1 and GIGYF2, homologous to the GYF domain described previously, binds to tandem proline-rich regions in the N terminus of Grb10. In IGF-I receptor-expressing R+ <em>fibroblasts</em>, there is detectable binding of a Myc-tagged fragment of GIGYF1 to Grb10 in the basal state. Stimulation with IGF-I results in increased binding of GIGYF1 to Grb10 and transient binding of both Grb10 and GIGYF1 to IGF-I receptors, presumably via the adapter function of Grb10. At later time points, GIGYF1 dissociates, but Grb10 remains linked to IGF-I receptors. Overexpression of the Grb10 binding fragment of GIGYF1 in R+ cells results in a significant increase in IGF-I-stimulated receptor tyrosine phosphorylation. In conclusion, we have identified two members of a novel protein family, which become transiently linked to IGF-I receptors by the Grb10 adapter protein following IGF-I stimulation. Grb10 and GIGYFs may act cooperatively to regulate receptor signaling.

We have previously utilized a combination of high throughput sequencing and genome-wide microarray profiling analyses to identify novel cell-surface proteins expressed in human umbilical vein endothelial cells. One gene identified by this approach encodes a type I transmembrane receptor that shares sequence homology with the intracellular domain of members of the interleukin-<em>17</em> (IL-<em>17</em>) receptor family. Real-time quantitative PCR and Northern analyses revealed that this gene is highly expressed in human umbilical vein endothelial cells and in several highly vascularized tissues such as kidney, colon, skeletal muscle, heart, and small intestine. In addition, we also found that it is also highly expressed in the ductal epithelial cells of human salivary glands, seminal vesicles, and the collecting tubules of the kidney by in situ hybridization. This putative receptor, which we have termed human SEF (hSEF), is also expressed in a variety of breast cancer tissues. In co-immunoprecipitation assays, this receptor is capable of forming homomeric complexes and can interact with <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) receptor 1. Overexpression of this receptor inhibits FGF induction of an FGF-responsive reporter gene in human 293T cells. This appears to occur as a result of specific inhibition of p42/p44 ERK in the absence of upstream MEK inhibition. This inhibitory effect is dependent upon a functional intracellular domain since deletion mutants missing the IL-<em>17</em> receptor-like domain lack this inhibitory effect. These findings are consistent with the recent discovery of the zebrafish homologue, Sef (similar expression to fgf genes), which specifically antagonizes FGF signaling when ectopically expressed in zebrafish or Xenopus laevis embryos. Based on sequence and functional similarities, this novel IL-<em>17</em> receptor homologue represents a potential human SEF and is likely to play critical roles in endothelial or epithelial functions such as proliferation, migration, and angiogenesis.

OBJECTIVE

The purpose of this study was to investigate the antiangiogenic properties of <em>17</em>-(dimethylaminoethylamino)-<em>17</em>-demethoxygeldanamycin (<em>17</em>-DMAG; NSC707545), a water-soluble benzoquinone ansamycin.

METHODS

The activity of <em>17</em>-DMAG, in vivo, was evaluated for inhibition of fibroblast growth factor (FGF)-2-induced angiogenesis in s.c. implanted Matrigel in mice. In vitro, the activity of <em>17</em>-DMAG on endothelial cells (human umbilical vein endothelial cells; HUVEC) was tested in FGF-2; and vascular endothelial growth factor (VEGF)-induced proliferation and apoptosis, motility, and extracellular matrix invasion; and on the alignment of capillary like structures in Matrigel. The protein level of heat shock protein (Hsp)90 and client proteins was examined by Western blot in FGF-2 and VEGF-stimulated HUVEC.

RESULTS

Daily oral administration of <em>17</em>-DMAG affected the angiogenic response in Matrigel in a dose-dependent manner. The hemoglobin content in the Matrigel implants was significantly inhibited, and the histological analysis confirmed a decrease of CD31(+) endothelial cells and of structures organized in cord and erythrocyte-containing vessels. In vitro, the compound inhibited dose-dependently the migration and the extracellular matrix-invasiveness of HUVEC and their capacity to form capillary like structures in Matrigel. <em>17</em>-DMAG treatment also inhibited FGF-2 and VEGF-induced HUVEC proliferation and resulted in apoptosis. Accordingly, the expression of Hsp90 direct client proteins (pAkt and c-Raf-1) or their downstream substrates including pERK was also affected. <em>17</em>-DMAG consistently increased the expression of Hsp70. Throughout the study similar results were obtained with <em>17</em>-allylamino-<em>17</em>-demethoxygeldanamycin (<em>17</em>-AAG; NSC330507), the analog compound currently undergoing clinical trials.

CONCLUSIONS

We show that the Hsp90 targeting agents <em>17</em>-DMAG and <em>17</em>-AAG inhibit angiogenesis. The strong effects on endothelial cell functions, in vitro, indicate that the antiangiogenic activity of <em>17</em>-DMAG/<em>17</em>-AAG could also be due to a direct effect on endothelial cells. The oral bioavailability of <em>17</em>-DMAG might be of advantage in investigating the potential of this compound in clinical trials with antiangiogenic as well as antiproliferative endpoints.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs) have been implicated in various forms of human hyperproliferative disorders such as cancers of the cervix and bladder. We investigated the expression pattern of FGFR4 and the clinical significance of the recently identified Gly/Arg polymorphism (388) in head and neck squamous cell carcinomas (HNSCCs) of the oral cavity and the oropharynx. Sections from 104 paraffin-embedded tumors were analyzed by a restriction fragment length polymorphism-based method to determine the FGFR4 genotypes. Protein expression was investigated immunohistochemically and graded into a low, intermediate, or high degree of staining. FGFR4 expression was scored as high in <em>17</em>, as intermediate in 59 and as low in 28 cases. The FGFR4 Arg388 allele was found in 59 tumors, 46 of them having heterozygous and 13 homozygous genotypes. High expression of the FGFR4 Arg388 allele was significantly associated with reduced overall survival (p = 0.032) and with an advanced tumor stage (p = 0.023), whereas expression of the FGFR4 Gly388 had no impact on disease progression. Our findings indicate that high expression of FGFR4 in connection with the Arg388 allele is associated with poor clinical outcome and support the significance of FGFR4 as a diagnostic marker and a target for therapeutic intervention in human HNSCC.

OBJECTIVE

Aortic stenosis (AS) is characterized by extensive remodelling of the valves, including infiltration of inflammatory cells, extracellular matrix degradation, and fibrosis. The molecular mechanisms behind this adverse remodelling have remained obscure. In this article, we study whether cathepsin G, an angiotensin II (Ang II)-forming elastolytic enzyme, contributes to progression of AS.

RESULTS

Stenotic aortic valves (n = 86) and control valves (n = <em>17</em>) were analysed for cathepsin G, transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1), and collagens I and III with RT-PCR and immunohistochemistry. Valvular collagen/elastin ratio was quantified by histochemistry. In stenotic valves, cathepsin G was present in mast cells and showed increased expression (P < 0.001), which correlated positively (P < 0.001) with the expression levels of TGF-beta1 and collagens I and III. TGF-beta1 was also present in mast cell-rich areas and cathepsin G induced losartan-sensitive TGF-beta1 expression in cultured <em>fibroblasts</em>. Collagen/elastin ratio was increased in stenotic valves (P < 0.001) and correlated positively with smoking (P = 0.02). Nicotine in cigarette smoke activated mast cells and induced TGF-beta1 expression in cultured <em>fibroblasts</em>. Fragmented elastin was observed in stenotic valves containing activated cathepsin G-secreting mast cells and in normal valves treated with cathepsin G.

CONCLUSIONS

In stenotic aortic valves, mast cell-derived cathepsin G may cause adverse valve remodelling and AS progression.

2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol-<em>17</em>beta and the oral contraceptive agent <em>17</em>-ethylestradiol. 2-ME was recently reported to inhibit endothelial cell proliferation. The current study was undertaken to explore the mechanism of 2-ME effects on endothelial cells, especially whether 2-ME induces apoptosis, a prime mechanism in tissue remodeling and angiogenesis. Cultured bovine pulmonary artery endothelial cells (BPAEC) exposed to 2-ME showed morphological (including ultrastructural) features characteristic of apoptosis: cell shrinkage, cytoplasmic and nuclear condensation, and cell blebbing. 2-ME-induced apoptosis in BPAEC was a time- and concentration-dependent process (EC50 = 0.45 +/- 0.09 microM, n = 8). Nucleosomal DNA fragmentation in BPAEC treated with 2-ME was identified by agarose gel electrophoresis (DNA ladder) as well as in situ nick end labeling. Under the same experimental conditions, estradiol-<em>17</em>beta and two of its other metabolites, estriol and 2-methoxyestriol (< or =10 microM), did not have an apoptotic effect on BPAEC. 2-ME activated stress-activated protein kinase (SAPK)/c-Jun amino-terminal protein kinase in BPAEC in a concentration-dependent manner. The activity of SAPK was increased by <em>17</em>0 +/- 27% and 314 +/- 22% over the basal level in the presence of 0.4 and 2 microM 2-ME (n = 3-6), respectively. The activation of SAPK was detected at 10 min, peaked at 20 min, and returned to basal levels at 60 min after exposure to 2-ME. Inhibition of SAPK/c-Jun amino-terminal protein kinase activation by basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>, or forskolin reduced 2-ME-induced apoptosis. Immunohistochemical analysis of BPAEC indicated that 2-ME up-regulated expression of both Fas and Bcl-2. In addition, 2-ME inhibited BPAEC migration (IC50 = 0.71 +/- 0.11 microM, n = 4) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>-induced angiogenesis in the chick chorioallantoic membrane model. Taken together, these results suggest that promotion of endothelial cell apoptosis, thereby inhibiting endothelial cell proliferation and migration, may be a major mechanism by which 2-ME inhibits angiogenesis.

Four principal cell types involved in the pathophysiologic response of the vessel wall--endothelial cells, smooth muscle cells, platelets, and monocyte/macrophages--secrete platelet-derived <em>growth</em> <em>factor</em>-like (PDGF-like) mitogenic activities. Extensive structural data on these activities exist only for the mitogen produced by platelets, which is a 30-kd dimeric protein composed of structurally related A and B polypeptide chains encoded by different genes. It was previously demonstrated that normal cultured endothelial cells transcribe mRNA encoding the B chain of PDGF from the c-sis gene. Here several new structural features of the mitogen produced by cultured vascular endothelial cells are shown. Hybridization analysis of RNA from normal cultured human umbilical vein endothelial (HUVE) cells revealed that they contain three PDGF A chain transcript species. These RNA species comigrated with and appeared to have the same relative abundance as the three RNA species previously identified in RNA from two human tumor cell lines. A chain transcripts were not identified in RNA from a strain of bovine aortic endothelial cells or in human dermal <em>fibroblasts</em>. The A chain transcripts in HUVE had the same relative abundance as the B chain transcripts. Immunoprecipitation of metabolically labeled endothelial conditioned medium with anti-PDGF antiserum revealed a 31-kd species which was split by reduction and alkylation into two species of 16.5 and <em>17</em> kd. Thus, endothelial cells secrete a dimeric mitogen antigenically related to PDGF, with a structure identical to previously isolated PDGF A-chain homodimer. These findings are consistent with the possibility that secretion of PDGF by human endothelial cells may be regulated independently of B-chain expression.

OBJECTIVE

To examine the potential role of the angiogenic growth factor angiopoietin-1 (Ang-1) in inflammatory arthritis.

METHODS

Eighteen synovial tissue samples were obtained from 17 patients with a clinical diagnosis of rheumatoid arthritis (RA) and compared with six synovial tissue samples from six patients with osteoarthritis (OA). Ang-1 expression in synovial tissues was determined by immunohistochemistry and in situ hybridisation. Ang-1 mRNA and protein expression were also examined by northern blot analysis and enzyme linked immunosorbent assay (ELISA) in cultured synovial fibroblasts and human umbilical vein endothelial cells (HUVECs) before and after treatment with tumour necrosis factor (TNF)alpha.

RESULTS

Ang-1 protein expression was detected by immunohistochemistry in 16/18 RA synovial tissue samples. Ang-1 protein was frequently observed in the synovial lining layer and in cells within the sublining synovial tissue, in both perivascular areas and in areas remote from vessels. In contrast, Ang-1 was only weakly detected in these sites in OA samples. Ang-1 mRNA and protein were also expressed in cultured synovial fibroblasts derived from patients with RA. In addition, induction of Ang-1 mRNA and protein was observed by northern blot analysis and ELISA after stimulation of RA synovial fibroblasts, but not HUVECs, with the proinflammatory cytokine TNF alpha.

CONCLUSIONS

Ang-1 mRNA and protein are expressed in the synovium of patients with RA, and are up regulated in synovial fibroblasts by TNF alpha. Ang-1 may therefore be an important regulator of angiogenesis in inflammatory arthritis.

Overexpression of epidermal <em>growth</em> <em>factor</em> receptor (EGFR) is correlated with loss of estrogen receptor and poor prognosis in breast cancer. To investigate this phenomenon, we transfected a cytomegalovirus expression vector directing the expression of EGFR into estrogen receptor-positive MCF-7 breast cancer cells and into a clone of MCF-7 cells previously transfected with transforming <em>growth</em> <em>factor</em> alpha. Cells arising from single clones or pooled polyclonal populations maintained in charcoal-stripped calf serum, a medium devoid of estrogen, overexpressed EGFR. Switching these cells to a medium containing fetal calf serum or charcoal-stripped calf serum plus <em>17</em> beta-estradiol resulted in the emergence of a population expressing low EGFR levels. Loss of expression was not a consequence of nonspecific repression of the cytomegalovirus promoter, because expression of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-4 complementary DNA in a similar vector was not lost in fetal calf serum. While loss of EGFR overexpression in fetal calf serum was seen at both the protein and mRNA levels, Southern blotting shows that this was not due to loss of the transfected gene. Subclones of a cell population with low EGFR expression were capable of increasing expression upon estrogen withdrawal, demonstrating that the changes in EGFR expression were reversible and suggesting a <em>growth</em> advantage conferred by EGFR overexpression under these restrictive <em>growth</em> conditions. Overexpression of EGFR did not result in loss of ER expression. These results suggest a role for overexpression of EGFR in the <em>growth</em> of estrogen receptor-positive breast cancer cells in the absence of estrogen.

Extracellular signal-regulated kinase (ERK) is an important intermediate in signal transduction pathways that are initiated by many types of cell surface receptors. It is thought to play a pivotal role in integrating and transmitting transmembrane signals required for <em>growth</em> and differentiation. Constitutive activation of ERK in <em>fibroblasts</em> elicits oncogenic transformation, and recently, constitutive activation of ERK has been observed in some human malignancies, including acute leukemia. However, mechanisms underlying constitutive activation of ERK have not been well characterized. In this study, we examined the activation of ERK in 79 human acute leukemia samples and attempted to find <em>factors</em> contributing to constitutive ERK activation. First, we showed that ERK and MEK were constitutively activated in acute leukemias by in vitro kinase assay and immunoblot analysis. However, in only one half of the studied samples, the pattern of ERK activation was similar to that of MEK activation. Next, by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analysis, we showed hyperexpression of ERK in a majority of acute leukemias. In <em>17</em> of 26 cases (65.4%) analyzed by immunoblot, the pattern of ERK expression was similar to that of ERK activation. The fact of constitutive activation of ERK in acute leukemias suggested to us the possibility of an abnormal downregulation mechanism of ERK. Therefore, we examined PAC1, a specific ERK phosphatase predominantly expressed in hematopoietic tissue and known to be upregulated at the transcription level in response to ERK activation. Interestingly, in our study, PAC1 gene expression in acute leukemias showing constitutive ERK activation was significantly lower than that in unstimulated, normal bone marrow (BM) samples showing minimal or no ERK activation (P =.002). Also, a significant correlation was observed between PAC1 downregulation and phosphorylation of ERK in acute leukemias (P =.002). Finally, by further analysis of 26 cases, we showed that a complementary role of MEK activation, ERK hyperexpression, and PAC1 downregulation could contribute to determining the constitutive activation of ERK in acute leukemia. Our results suggest that ERK is constitutively activated in a majority of acute leukemias, and in addition to the activation of MEK, the hyperexpression of ERK and downregulation of PAC1 also contribute to constitutive ERK activation in acute leukemias.

The microRNAs (miRNAs) are recently discovered short, noncoding RNAs, that regulate gene expression in metazoans. We have cloned short RNAs from chicken embryos and identified five new chicken miRNA genes. Genome analysis identified <em>17</em> new chicken miRNA genes based on sequence homology to previously characterized mouse miRNAs. Developmental Northern blots of chick embryos showed increased accumulation of most miRNAs analyzed from 1.5 days to 5 days except, the stem cell-specific mir-302, which was expressed at high levels at early stages and then declined. In situ analysis of mature miRNAs revealed the restricted expression of mir-124 in the central nervous system and of mir-206 in developing somites, in particular the developing myotome. In addition, we investigated how miR-206 expression is controlled during somite development using bead implants. These experiments demonstrate that <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) -mediated signaling negatively regulates the initiation of mir-206 gene expression. This may be mediated through the effects of FGF on somite differentiation. These data provide the first demonstration that developmental signaling pathways affect miRNA expression. Thus far, miRNAs have not been studied extensively in chicken embryos, and our results show that this system can complement other model organisms to investigate the regulation of many other miRNAs.

Rheumatoid arthritis (RA) is characterized by infiltrations of inflammatory cells accompanied by neovascularization in the joint. We hypothesized that cell activation via the toll-like receptor (TLR) may be involved in the induction of angiogenic molecules, which are relevant to the pathogenesis of RA. RA <em>fibroblast</em> like synoviocytes (FLS) were stimulated with TLR-2 ligand bacterial peptidoglycan (PGN), TLR-4 ligand lipopolysaccharide (LPS) and various cytokines. Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and IL-8 were measured by ELISA in culture supernatants; mRNA levels were assessed by RT-PCR and real time PCR. The levels of TLR-2, VEGF and IL-8 were analyzed by dual immunohistochemistry in RA synovium and compared with osteoarthritis (OA). Regulation of MyD88, IRAK4, IRAK1, IRAK-M and TRAF-6 mRNA expression levels by PGN were analyzed by RT-PCR. Phosphorylation of I kappa B alpha was evaluated by western blotting. Levels of VEGF and IL-8 were upregulated in culture supernatants of RA FLS stimulated with PGN, similar to the levels of IL-1beta and IL-<em>17</em> stimulation. Neutralization of TLR-2 with a blocking monoclonal antibody significantly reduced both VEGF and IL-8 levels (P<0.05), which reflected the functional relevance of TLR-2 activation to the induction of VEGF and IL-8 production. Downstream intracellular signaling following TLR-2 stimulation involved MyD88-IRAK-4-TRAF-6 pathways, resulting in NF-kappaB activation. Thus, TLR-2 activation in RA FLS by microbial constituents could be involved in the induction of VEGF and IL-8 and thereby promote inflammation either directly or via angiogenesis. This possibly contributes to the perpetuation of synovitis in patients with RA.

Responses to G protein-coupled receptor stimulation may be mediated by paracrine <em>factors</em>. We have developed a coculture system to study paracrine regulation of migration of gastric epithelial (AGS) cells after stimulation of gastrin-CCK(B) receptors. In cells expressing this receptor, G-<em>17</em> stimulated migration by activation of protein kinase C. However, G-<em>17</em> also stimulated the migration of cells expressing green fluorescent protein, but not the receptor, when they were cocultured with receptor-expressing cells consistent with activation of paracrine signals. The use of various pharmacological inhibitors indicated that gastrin stimulated migration via activation of the EGF receptor (EGR-R), the erbB-2 receptor tyrosine kinase, and the MAP kinase pathway. However, gastrin also released <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-1, and migration was inhibited by the FGF receptor tyrosine kinase inhibitor SU-5402. Flow cytometry indicated that in both cell types, gastrin increased MAP kinase via activation of EGF-R but not FGF-R1 or erbB-2. We conclude that gastrin-CCK(B) receptors stimulate epithelial cell migration partly via paracrine mechanisms; transactivation of EGF-R is only one component of the paracrine pathway.

OBJECTIVE

Matrix metalloproteinases (MMPs) are involved in tumor invasion and metastasis and are overexpressed in Kaposi's sarcoma (KS) cells. The primary aim was to define the safety and toxicity of the MMP inhibitor COL-3 in patients with AIDS-related KS. Secondary aims were to evaluate tumor response, pharmacokinetics, and changes in blood levels of MMP-2, MMP-9, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF).

METHODS

COL-3 was administered orally once daily, and doses were escalated in cohorts of three to six subjects. Patients with symptomatic visceral KS or severe tumor-associated edema were excluded. Antiretroviral therapy was permitted but not required. Study end points were grade 3 or 4 toxicity or progressive KS. Serial blood specimens were obtained for pharmacokinetics and levels of MMP-2, MMP-9, VEGF, and bFGF.

RESULTS

Eighteen patients received COL-3 in dosing cohorts of 25, 50, and 70 mg/m(2)/d. Prior KS therapy was reported by 17 patients (94%). COL-3-related grade 3 or 4 adverse events were reported by six patients and included photosensitivity, rash, and headache. There was one complete response and seven partial responses, for an overall response rate of 44%, with a median response duration of 25+ weeks. The median COL-3 half-life was 39.3 hours (range, 4.1 to 251.1 hours). There was a significant difference between responders and nonresponders with respect to the change in MMP-2 serum levels from baseline to minimum value on treatment (P =.037).

CONCLUSIONS

COL-3 administered orally once daily to patients with AIDS-related KS is reasonably well tolerated. The most common adverse event was dose-related photosensitivity. Antitumor activity was noted. Further evaluation of COL-3 for the treatment of KS is warranted.

Eosinophils are multifunctional leukocytes involved in various inflammatory processes, as well as tissue remodeling and immunoregulation. During inflammation and infection, injured cells and damaged tissues release uric acid and monosodium urate (MSU) crystals as important endogenous danger signals. Uric acid is also implicated in the immunogenic effects of an authentic Th2 adjuvant, aluminum hydroxide. Eosinophils often localize at sites of Th2-type chronic inflammation; therefore, we hypothesized that eosinophils may react to endogenous danger signals. We found that human eosinophils migrate toward soluble uric acid and MSU crystals in a gradient-dependent manner. Eosinophils incubated with MSU crystals, but not those incubated with uric acid solution, produced elevated levels of IL-6 and IL-8/CXCL8. Other cytokines and chemokines, including IL-1beta, IL-10, IL-<em>17</em>, IFN-gamma, CCL2, CCL3, CCL4, TNF-alpha, G-CSF, GM-CSF, <em>fibroblast</em> <em>growth</em> <em>factor</em>, vascular endothelial <em>growth</em> <em>factor</em>, and TGF-beta, were also produced by eosinophils incubated with MSU crystals. Eosinophils exposed to MSU crystals rapidly (i.e., within 1 min of exposure) released ATP into the extracellular milieu. Importantly, this autocrine ATP was necessary for eosinophils to produce cytokines in response to MSU crystals, and P2 nucleotide receptors, in particular P2Y(2), are likely involved in this positive feedback loop. Finally, at higher concentrations, MSU crystals promoted P2R-dependent release of a granule protein (eosinophil-derived neurotoxin) and cell death. Thus, human eosinophils may respond to particulate damage-associated endogenous danger signals. These responses by eosinophils to tissue damage may explain the self-perpetuating nature of chronic inflammation in certain human diseases, such as asthma.

Thisbe (Ths) and Pyramus (Pyr), two closely related Drosophila homologues of the vertebrate <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) 8/<em>17</em>/18 subfamily, are ligands for the FGF receptor Heartless (Htl). Both ligands are required for mesoderm development, but their differential expression patterns suggest distinct functions during development. We generated single mutants and found that ths or pyr loss-of-function mutations are semi-lethal and mutants exhibit much weaker phenotypes as compared with loss of both ligands or htl. Thus, pyr and ths display partial redundancy in their requirement in embryogenesis and viability. Nevertheless, we find that pyr and ths single mutants display defects in gastrulation and mesoderm differentiation. We show that localised expression of pyr is required for normal cell protrusions and high levels of MAPK activation in migrating mesoderm cells. The results support the model that Pyr acts as an instructive cue for mesoderm migration during gastrulation. Consistent with this function, mutations in pyr affect the normal segmental number of cardioblasts. Furthermore, Pyr is essential for the specification of even-skipped-positive mesodermal precursors and Pyr and Ths are both required for the specification of a subset of somatic muscles. The results demonstrate both independent and overlapping functions of two FGF8 homologues in mesoderm morphogenesis and differentiation. We propose that the integration of Pyr and Ths function is required for robustness of Htl-dependent mesoderm spreading and differentiation, but that the functions of Pyr have become more specific, possibly representing an early stage of functional divergence after gene duplication of a common ancestor.

OBJECTIVE

The possible role of inflammatory reaction of the cerebral artery in the pathogenesis of cerebral vasospasm has been noted in recent studies. We quantitatively measured the levels of expression of genes related to inflammation in the spastic artery in a canine double-hemorrhage model.

METHODS

Twenty dogs were assigned to 4 groups: group D0, control; group D2, dogs killed 2 days after cisternal injection of blood; group D7, dogs given double cisternal injections of blood and killed 7 days after the first injection; and group D14. Angiography was performed twice: on the first day and before the animals were killed. Total RNA was extracted from the basilar artery. The expressions of interleukin (IL)-1alpha, IL-6, IL-8, IL-10, tumor necrosis factor-alpha, E-secretin, fibronectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule-1, transforming growth factor-ss, basic fibroblast growth factor, and collagen types I, III, and IV were examined with TaqMan real-time quantitative reverse transcription-polymerase chain reaction.

RESULTS

Prolonged arterial narrowing peaking on 7 day was observed. There was a significant difference in vessel caliber between D0, D2, D7, and D14 groups (P:<0.0001). There were significant differences in mRNA expression in the basilar artery for IL-1alpha, IL-6, IL-8, ICAM-1, and collagen type I between D0, D2, D7, and D14 groups (P:=0.0079, 0. 0196, 0.0040, 0.0017, and <0.0001, respectively). The average level of mRNA was highest in D7 for IL-1alpha, IL-6, IL-8, and ICAM-1 (17-, 16-, 131-, and 1.7-fold compared with those of D0, respectively) and in D14 for collagen type I (10.9-fold).

CONCLUSIONS

Increased expression of genes related to inflammation in the spastic artery suggests that inflammatory reaction of the cerebral artery is associated with sustained contraction.

In order to document a possible involvement of structural alterations of FGF (<em>Fibroblast</em> <em>Growth</em> <em>Factor</em>)-like genes in human oncogenesis, we have screened a large series of human tumors for amplification of five FGF-related genes (Basic-FGF, INT2, HST, FGF5 and FGF6). None of 37 hematopoietic neoplasms, one out of 13 melanomas (8%), three out of 43 bladder tumors (7%) and 41 out of 238 breast carcinomas (<em>17</em>%) contained amplified FGF-related sequences, namely HST and INT2. Only these two genes, both located on band q13 of chromosome 11 have been found amplified. In all cases they were co-amplified and in only one instance did amplification extend to the ETS1 locus at position 11q23. INT2 and HST RNA could be evidenced by RNA/RNA in situ hybridization in breast carcinomas. Our results indicate a correlation between RNA expression and gene amplification in the case of HST but not of INT2. Although evaluation of the clinical significance of HST amplification and expression must await long-term follow-up of the patients, we suggest that HST gene product could play a role in development and/or progression of human breast cancer.

Caveolin is a major structural component of caveolae and has been implicated in the regulation of the function of several caveolae-associated signaling molecules. Platelet-derived <em>growth</em> <em>factor</em> (PDGF) receptors and caveolin were colocalized in the same subcellular fraction after sucrose density gradient fractionation of <em>fibroblasts</em>. Additionally, we found that the PDGF receptors interacted with caveolin in NIH3T3 <em>fibroblast</em> cells. We then examined whether caveolin directly binds to PDGF receptors and inhibits kinase activity using a recombinant PDGF receptor overexpressed in insect cells and peptides derived from the scaffolding domain of caveolin subtypes. We found the peptide from caveolin-1 and -3, but not -2, inhibited the autophosphorylation of PDGF receptors in a dose-dependent manner. Similarly, caveolin-1 and -3 peptides directly bound to PDGF receptors. Mutational analysis using a series of truncated caveolin-3 peptides (20-, <em>17</em>-, 14-, and 11-mer peptides) revealed that at least <em>17</em> amino acid residues of the peptide were required to inhibit and directly bind to PDGF receptors. Thus, our findings suggest that PDGF receptors directly interact with caveolin subtypes, leading to the inhibition of kinase activity. Caveolin may be another regulating <em>factor</em> of PDGF-mediated tyrosine kinase signaling.

Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ES-like cells (E-1 and E-2) appear to maintain a normal diploid karyotype indefinitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-specific embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than <em>17</em> passages, some ES-like cells differentiated to neural precursor cells in the presence of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), epidermal <em>growth</em> <em>factor</em> and platelet-derived <em>growth</em> <em>factor</em>. We also developed a protocol that resulted in the differentiation of ES-like cells in vitro to hematopoietic and endothelial cell lineages in response to bFGF, stem cell <em>factor</em> and oncostatin M. Our observations set the stage for future developments that may allow the use of equine ES-like cells for the treatment of neurological and hematopoietic disorders.