NGM282, an engineered <em>fibroblast</em> <em>growth</em> <em>factor</em> 19 analogue, rapidly and significantly reduced liver fat content in a multicenter, randomized, double-blind, placebo-controlled study in patients with biopsy-confirmed nonalcoholic steatohepatitis (NASH). However, it is unclear whether these changes would be accompanied by histological improvement. In this open-label study, we assessed the histological efficacy of NGM282 in patients with biopsy-confirmed nonalcoholic steatohepatitis. Paired liver biopsies from 43 patients who received subcutaneous NGM282 (1 mg, n = 24; 3 mg, n = 19) once daily for 12 weeks were evaluated blinded to time point, subject, and clinical information. At week 12, NGM282 significantly reduced nonalcoholic fatty liver disease activity score (NAS; -1.9; 95% confidence interval, -2.6 to -1.2; P < 0.001 in the 1 mg group; -2.2, -3.1 to -1.3; P < 0.001 in the 3 mg group) and fibrosis (-0.5; -0.9 to 0; P = 0.035 in the 3 mg group) scores. Overall, 50% and 63% of the patients receiving NGM282 1 mg or 3 mg, respectively, improved NAS by 2 or more points without fibrosis worsening. Of the patients receiving NGM282 1 mg or 3 mg, 25% and 42%, respectively, improved liver fibrosis by one stage or more without worsening of steatohepatitis. Treatment with NGM282 led to relative reductions in liver fat content (-58% and -67% in the 1 mg and 3 mg groups, respectively), corrected T1 (cT1; -8% and -9%), alanine aminotransferase (ALT) (-67% and -60%), aspartate aminotransferase (-57% and -52%), and fibrogenesis biomarkers neoepitope-specific N-terminal propeptide of type III collagen (Pro-C3; -<em>22</em>% and -33%) and enhanced liver fibrosis score (ELF; -3% and -6%) at week 12. Greater reductions in Pro-C3, ELF, and cT1, but not in liver fat content, 7alpha-hydroxy-4-cholesten-3-one, or ALT, were observed in histological responders than in nonresponders. Conclusion: In this open-label study, NGM282 improved the histological features of NASH in 12 weeks with significant reductions in NAS and fibrosis scores, accompanied by improvements in noninvasive imaging and serum markers.

BACKGROUND

Trafermin (basic fibroblast growth factor) has been shown to reduce infarct volume in acute ischemic stroke models, and to promote functional recovery and new synapse formation when given to animals with completed cerebral infarction. A previous study in acute stroke patients suggested that trafermin was safe and well tolerated when given over a 3-hour period over a wide dose range.

RESULTS

Double-blind, parallel group, placebo-controlled trial of a single 24-hour intravenous infusion of trafermin. Patients having onset of stroke symptoms within 6 h and a baseline score of>>/=7 on the NIH Stroke Scale >>/=2 motor) were randomized to receive 5 or 10 mg of trafermin or placebo intravenously infused over 24 h. The primary efficacy outcome was a categorized combination of the Barthel and Rankin scales assessed at 90 days. A total of 286 patients had been enrolled at 55 sites in 11 countries when the sponsor directed that enrollment be stopped because an interim analysis of efficacy data predicted too small a chance of demonstrating a statistically significant benefit after recruitment of the planned 900 patients. The 5-mg group showed a slight but nonsignificant advantage over placebo (OR 1.2, 95% CI 0.72-2.00, p = 0.48); the 10-mg group showed a nonsignificant disadvantage (OR 0.74, 95% CI 0.44-1.22, p = 0.24). Mortality rates at 90 days were 17% in the 5-mg group, 24% in the 10-mg group and 18% in the placebo group. Treatment with trafermin was associated with an increased leukocytosis and a decrease in blood pressure: mean decrease in systolic blood pressure from baseline was 19 mm Hg in the 5-mg group, 22 mm Hg in the 10-mg group and 8 mm Hg in the placebo group. In a post hoc subgroup analysis, patients in the 5-mg group treated more than 5 h after the onset of symptoms showed an apparent advantage over placebo (OR 2.1, 95% CI 1.00-4.41, p = 0.044; after age adjustment: OR 1.9, 95% CI 0.91-4.13, p = 0.08).

CONCLUSIONS

With the proper treatment regimen, trafermin can likely be given safely to stroke patients. The 5-mg dose showed a trend toward a treatment advantage. The ideal time window for this agent may exceed 5 h. This may open new avenues for acute stroke therapy, aiming at enhancing recovery mechanisms rather than immediate neuroprotection.

We investigated potential mechanisms by which lymphocytes infiltrating rheumatoid synovium become immunosuppressed. In 20 of <em>22</em> synovial fluids and 12 of 13 synovial tissue culture supernatants, no IL-1 bioactivity could be detected in the thymocyte proliferation assay. These same preparations could, however, support proliferation of <em>fibroblast</em> monolayers, consistent with the presence of IL-1 and/or other <em>fibroblast</em> <em>growth</em> <em>factors</em>. Addition of either rheumatoid synovial fluids or synovial culture supernatants to exogenous IL-1 in the IL-1 bioassay caused marked inhibition of the assay indicative of an IL-1 inhibitor. This inhibition of IL-1 could be reversed by treating the effusions or supernatants with a neutralizing antibody to transforming <em>growth</em> <em>factor</em>-beta (TGF-beta). Furthermore, monocyte-macrophages isolated from rheumatoid synovial fluid constitutively released both latent and active TGF-beta in culture at levels sufficient to completely block the biologic activity of 100 U/ml IL-1. The production of substantial levels of TGF-beta by synovial macrophages, as well as the apparent ability of these inflammatory macrophages to activate latent TGF-beta, implicates TGF-beta not only as an important inhibitor of IL-1-induced lymphocyte proliferation, but also as a key cytokine in promoting synovial <em>fibroblast</em> hyperplasia and pathology.

BACKGROUND

Thalidomide is a putative antiangiogenesis agent with activity in several hematologic malignancies.

METHODS

Forty-four patients who had myelofibrosis with myeloid metaplasia received treatment with thalidomide in a Phase II clinical trial at a dose of 200 mg daily with escalation by 200 mg weekly until the best tolerated dose (maximum, 800 mg) was reached.

RESULTS

Seventeen of 41 evaluable patients (41%) who received treatment for at least 15 days had a response. A complete response (without reversal of bone marrow fibrosis) was achieved in 4 patients (10%), a partial response was achieved in 4 patients (10%), and hematologic improvements in anemia, thrombopenia, and/or splenomegaly were observed in 9 patients (21%). Improvements in anemia occurred in 7 of 35 patients (20%) with hemoglobin levels <10.0 g/dL, and improvements in thrombopenia occurred in 5 of 24 patients (21%) with platelet counts <100 x 10(9)/L. Five of 24 patients (21%) became transfusion-independent. Major or minor regression of splenomegaly was noted in 9 of 29 evaluable patients (31%), and complete regression was noted in 5 patients. Responders had a lower baseline median vascular endothelial <em>growth</em> <em>factor</em> levels (77.9 pg/mL vs. 97.7 pg/mL; P <.01) and higher median basis <em>fibroblast</em> <em>growth</em> <em>factor</em> levels (60.8 pg/mL vs. 37.4 pg/mL; P <.01) compared with nonresponders. Nine patients (<em>22</em>%) had deterioration that was attributed to thalidomide (resolved after withdrawal) with either progressive cytopenias or excessive proliferation. Two patients developed Grade 3 neutropenia with recovery and resumed therapy with dose reductions, and both later achieved a complete response. Dose-related toxicities included fatigue (50%), constipation (48%), rash or pruritus (37%), sedation (35%), peripheral edema (29%), tremors (23%), peripheral neuropathy (<em>22</em>%), and orthostasis (16%).

CONCLUSIONS

Thalidomide warrants further evaluation in patients with MMM, particularly in combination regimens, along with the investigation of newer analogs.

Recently we have demonstrated that the release of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) from a biodegradable gelatin hydrogel carrier depends on the degradation of hydrogel in vivo. The purpose of our study was to assess whether bFGF-incorporating gelatin hydrogels induce myocardial angiogenesis and improve left ventricular function in the infarcted myocardium of rats. Studies were conducted in <em>22</em> Lewis rats after a 4-week ligation of the proximal left anterior descending coronary artery. The rats were randomized into the following two groups: the control group (n = 11) had an intramyocardial injection of saline alone, and the FGF group (n = 11) had gelatin hydrogel microspheres containing 100 microg of bFGF injected into the border zone of the infarct area after the repeat left thoracotomy. For visualization of the regional myocardial blood flow in the rat heart, (201)Tl images were taken just before and 4 weeks after the treatment using a 4-head single photon emission computed tomography scanner with pinhole collimators. Left ventricular function was also assessed with echocardiography and a micromanometer-tipped catheter. Finally, the extent of myocardial angiogenesis was evaluated quantitatively in the postmortem analysis. The (201)Tl defect score in the control group remained unchanged before and after the treatment, whereas it decreased significantly in the FGF group. Both regional and global left ventricular function was significantly better in the FGF group compared with the control group. The vascular density in the border zone of the infarct in the FGF group was significantly higher than that in the control group. In conclusion, intramyocardial injection of bFGF-impregnated gelatin hydrogels induces functionally significant angiogenesis and improves left ventricular systolic and diastolic function in the infarcted myocardium of rats.

The study demonstrates that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) serves as an inducer of radiation damage repair in bovine aortic endothelial cells (BAEC). Radiation dose-survival curves were generated with plateau-phase BAEC using culture dishes precoated with HR9-bFGF/extracellular matrix (ECM) for the postradiation colony formation assay. This natural basement membrane-like ECM is enriched with ECM-bound bFGF. Under these conditions the cells exhibited increased repair of radiation damage as compared to cells plated on top of the bFGF-free isotype of this extracellular matrix (the HR9/ECM). While the slopes of the curves did not differ significantly (Do 107 +/- 6.8 cGy on the HR9/ECM, compared to 112 +/- 1.3 cGy on the HR9-bFGF/ECM), there was a nearly complete elimination of the threshold shoulder in the curves generated on the bFGF-free HR9/ECM (Dq 29 +/- 19 cGy, compared to 174 +/- <em>22</em> cGy on the HR9-bFGF/ECM; P less than 0.05). Delayed plating experiments, in which the cells were irradiated under bFGF-free conditions (while adherent as contact-inhibited monolayers to the HR9/ECM in bFGF-free medium) and maintained after irradiation in the same culture for various periods of time, showed that the cells performed repair of potentially lethal damage (PLDR) and restored clonogenic ability, with a 24 h to immediate postradiation recovery ratio of 3.27. This expression of PLDR was inhibited by neutralizing monoclonal antibodies against bFGF, indicating that the irradiated cells secreted bFGF into their conditioned medium. Northern blot hybridization showed a 5.6-fold increase of the 3.7-kilobase species and a 4.7-fold increase of the 7.0-kilobase species of the bFGF-specific mRNA within 6 h after delivery of a single dose of 400 cGy. The data suggest that radiation induces a complete cycle of an autoregulated damage-repair pathway in BAEC, initiated by radiation-induced damage to cellular DNA and followed by stimulation of bFGF synthesis and its secretion into the medium. The newly synthesized bFGF stimulates the PLDR pathway, acting via an extracellular autocrine loop (inhibitable by specific anti-bFGF antibodies), leading to recovery of cells from radiation lesions and restoration of their clonogenic capacity.

Indomethacin delays healing of experimental gastric ulcers. We investigated whether inhibition of gastric acid secretion by omeprazole or stimulation of angiogenesis by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) may reverse this delay. Rats with gastric ulcers induced by cryoprobe were treated subcutaneously with either placebo, indomethacin (2 x 0.5 mg/kg), bFGF (2 x 100 micrograms/kg), omeprazole (1 x 40 mumol/kg), indomethacin plus omeprazole, or indomethacin plus bFGF given daily for 8, 10, 15, and <em>22</em> days. Ulcer size, epithelial cell proliferation, angiogenesis, and maturation of granulation tissue were sequentially quantified. Omeprazole significantly accelerated ulcer healing in an early phase (days 3-8). In contrast, bFGF accelerated healing in a late phase (days 10-15). Indomethacin significantly delayed ulcer healing in late phase and decreased prostaglandin generation, cell proliferation, angiogenesis, and maturation of granulation tissue. Despite stimulation of angiogenesis, bFGF did not reverse indomethacin-induced delay in ulcer healing. In contrast, omeprazole reversed indomethacin-induced effects on angiogenesis, cell proliferation, maturation of granulation tissue, and ulcer healing rate.

Although pituitary tumors arise as benign monoclonal neoplasms, genetic alterations have not readily been identified in these adenomas. We studied restriction fragment abnormalities involving the GH gene locus, and mutations in the p53 and H-, K- and N-ras genes in <em>22</em> human GH cell adenomas. Twenty two nonsecretory adenomas were also examined for p53 and ras gene mutations. Seven prolactinoma DNA samples were tested for deletions in the multiple endocrine neoplasia-1 (MEN-1) locus, as well as for rearrangements in the hst gene, a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family. Pituitary adenoma tissue and lymphocytes were obtained from patients at the time of transsphenoidal surgery. In DNA from GH-cell adenomas, identical GH restriction patterns were detected in both pituitary and lymphocyte DNA in all patients and in one patient with a mixed GH-TSH cell adenoma. Using polymerase chain reaction (PCR)-single stranded conformation polymorphism analysis, no mutations were detected in exons 5, 6, 7, and 8 of the p53 gene in GH cell adenomas nor in <em>22</em> nonsecretory adenomas. Codons 12/13 and 61 of H-ras, K-ras, and N-ras genes were also intact in GH cell adenomas and in nonsecretory adenomas. Site-specific probes for chromosome 11q13 including PYGM, D11S146, and INT2 were used in 7 sporadic PRL-secreting adenomas to detect deletions of the MEN-1 locus on chromosome 11. One patient was identified with a loss of 11p, and the remaining 6 patients did not demonstrate loss of heterozygosity in the pituitary 11q13 locus, compared to lymphocyte DNA. None of these patients, demonstrated hst gene rearrangements which also maps to this locus. These results show that p53 and ras gene mutations are not common events in the pathogenesis of acromegaly and nonsecretory tumors. Although hst gene rearrangements and deletions of 11q13 are not associated with sporadic PRL-cell adenoma formation, a single patient was detected with a partial loss of chromosome 11, including the putative MEN-1 site.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs) exist as a gene family of 4 membrane bound receptor tyrosine kinases (FGFR1-4) that mediate signals of at least <em>22</em> <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF1-<em>22</em>). FGFs/FGFRs play important roles in multiple biological processes, including mesoderm induction and patterning, cell <em>growth</em> and migration, organ formation and bone <em>growth</em>. Furthermore, it has been shown that missense mutations of FGFR1-3 in human result in, at least, 14 congential bone diseases that are broadly classified into two groups: chondrodysplasia syndromes and craniosynostosis syndromes. The chondrodysplasia affects primarily the skeleton formed through endochondral ossification, resulting short-limbed dwarfisms, while the craniosynostosis affects mainly bones formed through intramembraneous ossification, leading to premature fusion of the craniofacial sutures. Using gene targeting, mouse models mimicking some of these human diseases have been created. Analysis of these mutant mice revealed essential functions of FGFs/FGFRs in skeletal development and maintenance. These models may be beneficial in future studies aimed at developing novel therapeutic strategies for FGFR-related skeletal dysplasias. In this review, we discuss the results of recent studies on FGF receptors to illustrate mechanisms through which the abnormally activated FGF/FGFR signaling results in these diseases.

Four members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) family of tyrosine kinases transduce signals of a diverse group of more than 23 <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) ligands. Each prototypic receptor is composed of three immunoglobulin-like extracellular domains, two of which are involved in ligand binding. Alternative RNA splicing of one of two exons results in two different forms of the second half of the third immunoglobulin-like domain, the IIIb or IIIc isoforms. The contribution of each receptor and their isoforms in tumorigenesis remains unknown. In the pituitary, FGFR2 is expressed primarily as the IIIb isoform in normal adenohypophysial cells. In contrast, FGFR2 is significantly down-regulated in mouse corticotroph AtT20 tumor cells where the 5' promoter is methylated. Treatment of AtT20 cells with 5'-azacytidine resulted in FGFR2 re-expression, mainly as the FGFR2-IIIb isoform. Chromatin immunoprecipitation revealed evidence of histone methylation, but not of deacetylation, in the silencing of FGFR2 in AtT20 cells. Exposure of these cells to the cognate FGFR2-IIIb ligand FGF-7 resulted in diminished Rb phosphorylation and accumulation of p21 and p27, indicating diminished cell cycle progression. Examination of primary human pituitary adenomas revealed FGFR2 down-regulation in 52% (11 of 21) of samples and FGFR2 promoter DNA methylation in 45% (10 of <em>22</em>) of samples. These data highlight the contribution from DNA and histone methylation as epigenetic mechanisms responsible for FGFR2 silencing in pituitary neoplasia.

Numerous heparin-binding <em>growth</em> <em>factors</em> active in different types of cells have recently been shown to belong to the family of <em>fibroblast</em> <em>growth</em> <em>factors</em>. Because these <em>factors</em> are active in some types of epithelial cells, we tested the activity of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) from bovine brain in human keratinocyte cultures. bFGF stimulated thymidine incorporation and cellular proliferation in these cultures with half-maximal activity at approximately 60 pg/ml (4 X 10(-12) M). Stimulation of thymidine incorporation was associated with increased nuclear labeling after <em>22</em> h in the presence of bFGF under the same conditions used in the thymidine incorporation assay. bFGF was nearly as effective as epidermal <em>growth</em> <em>factor</em> (EGF) in stimulating keratinocyte <em>growth</em> and substantially less effective than crude placental extract, and was not additive with EGF in stimulating thymidine incorporation or proliferation of cells. The findings indicate that bFGF is a potent <em>growth</em> <em>factor</em> for keratinocytes.

We have analysed expression of the first 7 members of the family of heparin-binding <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGFs) and their 4 high-affinity receptors (FGFRs) in human pancreatic carcinoma cell lines, both at the mRNA and protein levels. In cell lines expressing FGFRs, 2 typical patterns were observed: (i) expression of FGFR-I, -3 or -4 along with the expression of at least one FGF; (ii) co-expression of FGFR-3 and FGFR-4 in the absence of FGF expression. Using RT-PCR, transcripts representing multiple isoforms of both extracellular and intracellular domains of FGFR-I were detected in the cell line PT45. A novel extracellular domain variant of FGFR-I was predicted to encode the first immunoglobulin loop in a potentially secreted form. Protein expression of the splice variants of FGFR-I was confirmed by immunoprecipitation with specific antibodies in radiolabelled ligand cross-linking experiments. The type I carboxyl end and the alpha subtype extracellular domain were detected in the PANC-I cell line, while the type I carboxyl terminus and the gamma subtype extracellular domain were expressed in the PT45 cell line. Expression of FGF-2 in PT45 was also detected by immunoprecipitation using 3 different anti-FGF-2 antibodies. Apart from the 18-kDa product, higher molecular weight isoforms, namely <em>22</em>- and 23-kDa isoforms, were expressed. In an assay of anchorage-independent <em>growth</em>, exogenous FGF-2 stimulated a maximum 15-fold and 10-fold increase in colony formation by the cell lines MIA PACA-2 and PANC-I respectively. Treatment of monolayer cultures of the same cell lines did not promote <em>growth</em>. However, a specific neutralising antibody against FGF-2 reduced cell proliferation of MIA PACA-2 cells by 50%.

The brain acid-soluble protein BASP1 (CAP-23, NAP-<em>22</em>) belongs to the family of <em>growth</em>-associated proteins, which also includes GAP-43, a protein recently shown to regulate neural cell adhesion molecule (NCAM)-mediated neurite out<em>growth</em>. Here, the effects of BASP1 overexpression were investigated in PC12E2 cells and primary hippocampal neurons. BASP1 overexpression stimulated neurite out<em>growth</em> in both cell types. The effects of BASP1 and trans-homophilic NCAM interactions were additive, and BASP1-induced neurite out<em>growth</em> was not inhibited by ectopic expression of cytoplasmic NCAM domains. Furthermore, inhibition of signaling via the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor, Src-family nonreceptor tyrosine kinases, protein kinase C, or GSK3beta, and expression of constructs of the cytoskeletal proteins spectrin and tau inhibited NCAM- but not BASP1-induced neurite out<em>growth</em>. Expression of BASP1 mutated at the serine-5 phosphorylation site stimulated neurite out<em>growth</em> to a degree comparable to that observed in response to overexpression of wild-type BASP1, whereas expression of BASP1 mutated at the myristoylation site at glycine-1 completely abrogated the stimulatory effects of the protein on neurite out<em>growth</em>. Finally, coexpression experiments with dominant negative and wild-type versions of GAP-43 and BASP1 demonstrated that the two proteins could substitute for each other with respect to induction of NCAM-independent neurite out<em>growth</em>, whereas BASP1 was unable to replace the stimulatory effect of GAP-43 on NCAM-mediated neurite out<em>growth</em>. These observations demonstrate that BASP1 and GAP-43 have overlapping, but not identical, functions in relation to neurite out<em>growth</em> and indicate that the main function of BASP1 is to regulate the organization and morphology of the plasma membrane.

BACKGROUND

The involvement of the cyclooxygenases (COX), in particular COX-2, is well documented for many tumours, e.g. colon, breast and prostate cancer, by both experimental and clinical studies. There are epidemiological data from subjects using NSAIDs, and experimental evidence supporting the hypothesis of prostaglandins (PGs) as regulators of tumourigenesis in the ovary. One of the end products of PG-synthesis, PGE2, regulates several key-processes, which are characteristic for tumour <em>growth</em>, e.g. angiogenesis, proliferation and apoptosisis. The present study investigated the pathway for PGE2-synthesis and signalling in ovarian tumourigenesis by analysing specimen from normal ovaries (n = 18), benign (B) (n = 8), borderline type (BL) (n = 6) and malignant tumours (AC) (n = <em>22</em>). The expression and cell-specific localization of COX-1, COX-2, microsomal prostaglandin E synthase-1 (mPGES-1) and two of the receptors for PGE2, EP1 and EP2, were examined by immunoblotting (IB) and immunohistochemistry (IHC).

RESULTS

The results are in line with earlier studies demonstrating an increase of COX-2 in AC compared to the normal ovary, B and BL tumours. Increased expressions were also observed for COX-1, mPGES-1 and EP-1 which all were significantly (p < 0.05) augmented in less differentiated AC (grades: moderately-, poorly- and undifferentiated). The increase of COX-2 was also correlated to stage (FIGO classification) with significant elevations in stages II and III. EP1 was increased in stage III while no significant alterations were demonstrated for COX-1, mPGES-1 or EP2 for stage. IHC revealed staining of the tumour cells, but also increase of COX-1, COX-2, mPGES-1 and EP1-2 in the stromal compartment of AC (grades: moderately-, poorly- and undifferentiated). This observation suggests interactions between tumour cells and stromal cells (fibroblasts, immune cells), e.g. paracrine signalling mediated by growth factors, cytokines and possibly PGs.

CONCLUSIONS

The increases of COX-1, COX-2, mPGES-1 and EP1-2 in epithelial ovarian cancer, supports the hypothesis that PGE2-synthesis and signalling are of importance for malignant transformation and progression. The observed augmentations of COX-1, COX-2 and mPGES-1 have implications for future therapeutic strategies.

OBJECTIVE

We measured the serum concentration of a panel of inflammatory cytokines and evaluated their association with circulating proangiogenic biomarkers and with outcome in patients with pancreatic ductal adenocarcinoma (PDAC).

METHODS

We collected serum samples from 36 patients with PDAC, 9 patients with chronic pancreatitis, and <em>22</em> healthy volunteers as a control. Inflammatory cytokines and proangiogenic biomarkers were measured using the multianalyte xMAP array and carcinoembryonic antigen (CEA) and carbohydrate 19-9 by immunoassay.

RESULTS

Patients with PDAC had higher circulating levels of interleukin 6 (IL-6) than those of patients with pancreatitis or healthy individuals and higher levels of IL-10 and tumor necrosis factor α (TNF-α) compared with those of healthy individuals. In patients with PDAC, circulating IL-6, TNF-α, IL-1β, and IL-10 correlated with serum concentrations of vascular endothelial growth factor and basic fibroblast growth factor; circulating IL-6, IL-1β, and TNF-α correlated with carbohydrate 19-9; and IL-8, IL-10, and TNF-α correlated with CEA levels. Circulating IL-8, TNF-α, and CEA; tumor stage; and lymph node metastases were associated with a poor outcome.

CONCLUSIONS

The results of this exploratory study indicate that inflammatory cytokines should be pursued as potential prognostic biomarkers as well as targets for therapy in larger studies in PDAC.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family is comprised of <em>22</em> ligands that bind and activate several FGF receptor (FGFR) isoforms. Critical roles for FGFs and FGFRs have been well-established during embryonic development. For example, the FGF10/FGFR2IIIb axis has been linked to embryonic development of both the mammary and prostate glands, which are the subject of this review. Furthermore, recent studies using novel mouse models have suggested that this pathway also participates in postnatal development in the mammary and prostate glands. These studies have provided novel insights into the mechanisms by which FGFs and FGFRs promote ductal out<em>growth</em> and branching morphogenesis. In addition to the established roles of FGFs in development, aberrant activation of the FGF pathway has been linked to tumor progression in both breast and prostate cancer. Recent studies have linked FGFR1 expression and single nucleotide polymorphisms in FGFR2 to breast cancer. Furthermore, novel pre-clinical models have demonstrated the ability of FGFRs to promote numerous aspects of breast and prostate cancer. Understanding the roles of FGFs in development will provide insights into the mechanisms by which deregulation of the FGF pathway leads to tumorigenesis, ultimately leading to the development of novel therapeutic strategies designed to target this pathway in cancer patients.

The HC11 mouse mammary epithelial cell line has proven to be a valuable in vitro model to study the roles of peptide <em>factors</em> and hormones involved in the <em>growth</em> and differentiation of mammary cells. Treatment of HC11 cells with the lactogenic hormones, dexamethasone, insulin, and PRL (DIP), leads to cellular differentiation and production of the milk protein beta-casein. We have analyzed the effects of Neu differentiation <em>factor</em> (NDF)/heregulin, a newly described activating ligand for erbB-2 and other members of the epidermal <em>growth</em> <em>factor</em> (EGF) receptor family, on cell <em>growth</em> and the expression of milk proteins in HC11 cells. In these cells, NDF induces tyrosine phosphorylation of erbB-2 and erbB-3. Both NDF and EGF stimulate HC11 cell proliferation and promote the responsiveness of HC11 cells to lactogenic hormones. NDF induces the expression of a <em>22</em>-kilodalton milk protein. This protein is up-regulated by other <em>factors</em>, including dexamethasone, EGF, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and is controlled in a manner distinct from that of beta-casein. Like EGF, NDF inhibits the DIP-induced expression of beta-casein at the level of transcription. The inhibition is due to the negative effect of NDF on the activation of mammary gland <em>factor</em> (MGF/Stat5), a member of the Stat family of transcription <em>factors</em>, which is essential for beta-casein gene expression.

OBJECTIVE

Transforming growth factor-beta1 (TGFbeta1) and fibroblast growth factor (FGF) families play a pivotal role during vascular development and in the pathogenesis of vascular disease. However, the interaction of intracellular signaling evoked by each of these growth factors is not well understood. The present study was undertaken to examine the molecular mechanisms that mediate the effects of TGFbeta1 and basic FGF (bFGF) on smooth muscle cell (SMC) gene expression.

RESULTS

TGFbeta1 induction of SMC gene expression, including smooth muscle protein 22-alpha (SM22alpha) and smooth muscle alpha-actin, was examined in the pluripotent 10T1/2 cells. Marked increase in these mRNA levels by TGFbeta1 was inhibited by c-Src-tyrosine kinase inhibitors and protein synthesis inhibitor cycloheximide. Functional studies with deletion and site-directed mutation analysis of the SM22alpha promoter demonstrated that TGFbeta1 activated the SM22alpha promoter through a CC(A/T-rich)6GG (CArG) box, which serves as a serum response factor (SRF)-binding site. TGFbeta1 increased SRF expression through an increase in transcription of the SRF gene. In the presence of bFGF, TGFbeta1 induction of SMC marker gene expression was significantly attenuated. Transient transfection assays showed that bFGF significantly suppressed induction of the SM22alpha promoter-driven luciferase activity by TGFbeta1, whereas bFGF had no effects on the TGFbeta1-mediated increase in SRF expression and SRF:DNA binding activity. Mitogen-activated protein kinase kinase-1 (MEK1) inhibitor PD98059 abrogated the bFGF-mediated suppression of TGFbeta1-induced SMC gene expression.

CONCLUSIONS

Our data suggest that bFGF-induced MEK/extracellular signal-regulated kinase signaling plays an antagonistic role in TGFbeta1-induced SMC gene expression through suppression of the SRF function. These data indicate that opposing effects of bFGF and TGFbeta1 on SMC gene expression control the phenotypic plasticity of SMCs.

Pituitary tumor-transforming gene (PTTG) is a recently characterized oncogene whose expression product contains a transcriptional activation domain at the C terminus. To understand the mechanisms involved in PTTG biological functions, we used yeast two-hybrid screening to identify proteins that interact with PTTG. This study reports the isolation and characterization of a novel PTTG-binding <em>factor</em> (PBF). PBF contains an open reading frame of 179 amino acids with a predicted molecular mass of <em>22</em> kDa. In Northern blot analyses, PBF mRNA was ubiquitously expressed in human tissues. Glutathione S-transferase pull-down and co-immunoprecipitation assays demonstrate that PBF interacts specifically with PTTG under both in vitro and in vivo conditions. The PTTG binding domain in PBF was located within the C-terminal 30-amino acid region that contain a nuclear localization signal. Immunofluorescence and subcellular fractionation studies showed that PTTG is predominantly expressed in the cytoplasm with partial nuclear localization, whereas PBF is localized both in the cytoplasm and the nucleus. The interaction between PBF and PTTG facilitated PTTG translocation from the cytoplasm to the nucleus. Furthermore, PBF is required for transcriptional activation of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> by PTTG. In summary, we have characterized a novel PTTG-binding protein that facilitates PTTG nuclear translocation and potentiates its transcriptional activation function.

The role of angiogenesis and lymphangiogenesis in thyroid cancer pathogenesis has not been elucidated. Patterns for tumour behaviour and metastasic spread vary according to tumour type and whether differences in the angiogenic or lymphangiogenic phenotype influence the route for tumour metastases or determine a more aggressive behaviour has not been fully explored. The angiogenic and lymphangiogenic phenotypes of a large cohort of thyroid proliferative lesions (n=191) were studied. Using immunohistochemistry for CD34, lymphatic vessel endothelial receptor-1 (LYVE-1) (specific markers for vascular and lymphatic endothelium respectively), vascular endothelial <em>growth</em> <em>factor</em> (VEGF-A), VEGF-C and <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), this study analyses microvascular density (MVD), lymphatic vascular density (LVD), and expression of angiogenic and lymphangiogenic <em>factors</em> in normal thyroid (NT; n=19), multinodular goitre (n=25), toxic multinodular goitre (n=8), Graves' hyperplasia (n=<em>22</em>), follicular adenoma (n=54), papillary carcinoma (PC; n=27), incidental papillary microcarcinoma (PMC; n=8), follicular carcinoma (FC; n=20) and medullary carcinoma (MC; n=8). MVD was decreased in proliferative lesions, benign and malignant, compared with NT (P<0.0001). In contrast, VEGF-A expression was increased in thyroid carcinomas (PC, FC and MC) when compared with PMC, benign lesions and NT (P<0.0001). LVD was higher in PC and PMC (P=0.001), and VEGF-C expression was increased in PC (P<0.0001). Despite higher LVD and increased expression of VEGF-A and VEGF-C in thyroid cancers, these markers were not related to poor prognosis in terms of tumour size, multifocality and/or presence of lymphatic or distant metastases. In conclusion, angiogenesis is reduced in thyroid proliferative lesions compared with NT tissue. However, VEGF-A expression is upregulated in thyroid cancers. Lymphangiogenesis and VEGF-C expression are increased in thyroid tumours prone to lymphatic metastases. This may be an important mechanism underlying the differences in metastatic behaviour between papillary and follicular thyroid cancer.

BACKGROUND

In X-linked hypophosphatemia (XLH), elevated fibroblast growth factor 23 (FGF23) decreases the renal tubular maximum reabsorption rate of phosphate/glomerular filtration rate (TmP/GFR) and serum inorganic phosphorus (Pi), resulting in rickets and/or osteomalacia.

OBJECTIVE

The objective was to test the hypothesis that monthly KRN23 (anti-FGF23 antibody) would safely improve serum Pi in adults with XLH.

METHODS

Two sequential open-label phase 1/2 studies were done.

METHODS

Six academic medical centers were used.

METHODS

Twenty-eight adults with XLH participated in a 4-month dose-escalation study (0.05-0.6 mg/kg); 22 entered a 12-month extension study (0.1-1 mg/kg).

METHODS

KRN23 was injected sc every 28 days.

METHODS

The main outcome measure was the proportion of subjects attaining normal serum Pi and safety.

RESULTS

At baseline, mean TmP/GFR, serum Pi, and 1,25-dihydroxyvitamin D [1,25(OH)2D] were 1.6 ± 0.4 mg/dL, 1.9 ± 0.3 mg/dL, and 36.6 ± 14.3 pg/mL, respectively. During dose escalation, TmP/GFR, Pi, and 1,25(OH)2D increased, peaking at 7 days for TmP/GFR and Pi and at 3-7 days for 1,25(OH)2D, remaining above (TmP/GFR, Pi) or near [1,25(OH)2D] pre-dose levels at trough. After each of the four escalating doses, peak Pi was between 2.5 and 4.5 mg/dL in 14.8, 37.0, 74.1, and 88.5% of subjects, respectively. During the 12-month extension, peak Pi was in the normal range for 57.9-85.0% of subjects, and ≥25% maintained trough Pi levels within the normal range. Serum Pi did not exceed 4.5 mg/dL in any subject. Although 1,25(OH)2D levels increased transiently, mean serum and urinary calcium remained normal. KRN23 treatment increased biomarkers of skeletal turnover and had a favorable safety profile.

CONCLUSIONS

Monthly KRN23 significantly increased serum Pi, TmP/GFR, and 1,25(OH)2D in all subjects. KRN23 has potential for effectively treating XLH.

Rat extensor digitorum longus muscles were overloaded by stretch after removal of the synergist tibialis anterior muscle to determine the relationship between capillary <em>growth</em>, muscle blood flow, and presence of <em>growth</em> <em>factors</em>. After 2 wk, sarcomere length increased from 2.4 to 2.9 micrometers. Capillary-to-fiber ratio, estimated from alkaline phosphatase-stained frozen sections, was increased by 33% (P < 0.0001) and 60% (P < 0.01), compared with control muscles (1.44 +/- 0.06) after 2 and 8 wk, respectively. At 2 wk, the increased capillary-to-fiber ratio was not associated with any changes in mRNA for basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2) or its protein distribution. FGF-2 immunoreactivity was present in nerves and large blood vessels but was negative in capillaries, whereas the activity of low-molecular endothelial-cell-stimulating angiogenic <em>factor</em> (ESAF) was 50% higher in stretched muscles. Muscle blood flows measured by radiolabeled microspheres during contractions were not significantly different after 2 or 8 wk (132 +/- 37 and 177 +/- <em>22</em> ml. min-1. 100 g-1, respectively) from weight-matched controls (156 +/- 12 and 150 +/- 10 ml. min-1. 100 g-1, respectively). Resistance to fatigue during 5-min isometric contractions (final/peak tension x 100) was similar in 2-wk overloaded and contralateral muscles (85 vs. 80%) and enhanced after 8 wk to 92%, compared with 77% in contralateral muscles and 67% in controls. We conclude that increased blood flow cannot be responsible for initiating expansion of the capillary bed, nor does it explain the reduced fatigue within overloaded muscles. However, stretch can present a mechanical stimulus to capillary <em>growth</em>, acting either directly on the capillary abluminal surface or by upregulating ESAF, but not FGF-2, in the extracellular matrix.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has been shown to promote wound healing. The present trial evaluated the clinical efficacy of bFGF for diabetic ulcer, a type of refractory skin ulcer, and the dose-response relationship. This was designed as a randomized, double-blind, dose-ranging, placebo-controlled trial. A total of 150 patients with non-ischaemic diabetic ulcers measuring 900 mm2 or less were randomized into a placebo group (n = 51), a 0.001% bFGF group (n = 49) and a 0.01% bFGF group (n = 50), and 148 of these patients received treatment for 8 weeks or less. The efficacy evaluation was carried out on 139 patients who met the protocol in this trial. The primary outcome was the percentage of patients showing 75% or greater reductions in the area of ulcer. The area of ulcer decreased by 75% or more in 57.5% (27/47), 72.3% (34/47), and 82.2% (37/45) in the placebo, 0.001% bFGF and 0.01% bFGF groups, respectively, and differences were significant between the 0.01% bFGF and placebo groups (p = 0.025). The cure rate was 46.8% (<em>22</em>/47), 57.4% (27/47), and 66.7% (30/45) in the placebo, 0.001% bFGF and 0.01% bFGF groups, respectively. The findings obtained in this trial showed wound healing accelerating effects of bFGF on diabetic ulcers.

We have shown that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) is heterogeneously expressed by human pituitary adenomas and may be implicated as a <em>growth</em> stimulus for these tumors. There are four mammalian FGF receptor (FGFR) genes encoding a complex family of transmembrane tyrosine kinases. The prototypic receptor is composed of three Ig-like extracellular ligand-binding domains, a transmembrane domain, and a cytoplasmic split tyrosine kinase. Multiple forms of cell-bound or secretable isoforms of FGFR-1, -2, and -3 can be generated by cell- and tissue-specific alternative splicing, resulting in tissue-specific FGF function. Shifts in isoform expression accompany tumor progression in some systems. We examined the normal human adenohypophysis and 40 pituitary adenomas to determine the pattern of FGFR expression by reverse transcription-PCR; all tumors were characterized clinically and morphologically. Ribonucleic acid (RNA) was extracted from frozen tumor tissue and primers were used to distinguish messenger RNA of the secretable first Ig-like domain (I) and those of the transmembrane and kinase domains (K) of each FGFR subtype. The normal pituitary-expressed mRNAs for FGFR-1 I and K, FGFR-2 I and K, FGFR-3 I and K, and FGFR-4 I but not FGFR-4 K; this represents the first report of a truncated isoform of FGFR-4, indicating possible alternative polyadenylation sites in this receptor. Only 3 tumors had the same pattern of expression of the 4 FGFRs as the normal gland. Although all tumors expressed FGFR-1 I, 1 tumor did not express FGFR-1 K, suggesting the production of only a secretable form of FGFR-1 by this tumor. Four tumors were negative for FGFR-2 I and K; 6 expressed the secretable form only, and 17 expressed FGFR-2 K but not I. All tumors expressed FGFR-3 I; 14 had secretable forms only, and no tumors expressed FGFR-3 K alone. As in the normal gland, 13 tumors expressed only the secretable I form of FGFR-4. Unlike the normal pituitary, however, <em>22</em> expressed FGFR-4 I and K, indicating a possible tumor-specific transmembrane receptor. Five tumors were negative for FGFR-4 I and K. Expression of FGFR proteins was confirmed by immunohistochemical localization of the C-terminal portion of FGFR-1, -2, -3, and -4; the results correlated with the RNA data in each case. There was no correlation between tumor type, size, or aggressiveness and the expression pattern of FGFRs. Our study suggests that pituitary adenomas have altered FGFR subtype and isoform expression, which may determine their hormonal and proliferative responses to FGFs.