Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial <em>growth</em> <em>factor</em> and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, induced up to approximately <em>16</em>-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis <em>factor</em> alpha, and interleukin-1 did not induce survivin expression in endothelial cells. Formation of three-dimensional vascular tubes in vitro was associated with strong induction of survivin in endothelial cells, as compared with two-dimensional cultures. By immunohistochemistry, survivin was minimally expressed in endothelium of nonproliferating capillaries of normal skin, whereas it became massively up-regulated in newly formed blood vessels of granulation tissue in vivo. Recombinant expression of green fluorescent protein survivin in endothelial cells reduced caspase-3 activity and counteracted apoptosis induced by tumor necrosis <em>factor</em> alpha/cycloheximide. These findings identify survivin as a novel <em>growth</em> <em>factor</em>-inducible protective gene expressed by endothelial cells during angiogenesis. Therapeutic manipulation of survivin expression and function in endothelium may influence compensatory or pathological (tumor) angiogenesis.

OBJECTIVE

FGFR gene aberrations are associated with tumor growth and survival. We explored the role of FGFR2 amplification in gastric cancer and the therapeutic potential of AZD4547, a potent and selective ATP-competitive receptor tyrosine kinase inhibitor of fibroblast growth factor receptor (FGFR)1-3, in patients with FGFR2-amplified gastric cancer.

METHODS

Array-comparative genomic hybridization and FISH were used to identify FGFR2 amplification in gastric cancer patient tumor samples. The effects of FGFR2 modulation were investigated in gastric cancer cells with FGFR2 amplification and in patient-derived gastric cancer xenograft (PDGCX) models using two approaches: inhibition with AZD4547 and short hairpin RNA (shRNA) knockdown of FGFR2.

RESULTS

Amplification of the FGFR2 gene was identified in a subset of Chinese and Caucasian patients with gastric cancer. Gastric cancer cell lines SNU-16 and KATOIII, carrying the amplified FGFR2 gene, were extremely sensitive to AZD4547 in vitro with GI50 values of 3 and 5 nmol/L, respectively. AZD4547 effectively inhibited phosphorylation of FGFR2 and its downstream signaling molecules and induced apoptosis in SNU-16 cells. Furthermore, inhibition of FGFR2 signaling by AZD4547 resulted in significant dose-dependent tumor growth inhibition in FGFR2-amplified xenograft (SNU-16) and PDGCX models (SGC083) but not in nonamplified models. shRNA knockdown of FGFR2 similarly inhibited tumor growth in vitro and in vivo. Finally, compared with monotherapy, we showed enhancement of in vivo antitumor efficacy using AZD4547 in combination with chemotherapeutic agents.

CONCLUSIONS

FGFR2 pathway activation is required for driving growth and survival of gastric cancer carrying FGFR2 gene amplification both in vitro and in vivo. Our data support therapeutic intervention with FGFR inhibitors, such as AZD4547, in patients with gastric cancer carrying FGFR2 gene amplification.

OBJECTIVE

The lung is the major dose-limiting organ for radiotherapy of cancer in the thoracic region. The pathogenesis of radiation-induced lung injury at the molecular level is still unclear. Immediate cellular damage after irradiation is supposed to result in cytokine-mediated multicellular interactions with induction and progression of fibrotic tissue reactions. The purpose of this investigation was to evaluate the acute and long-term effects of radiation on the gene expression of transforming growth factor beta (TGF-beta) in a model of lung injury using fibrosis-sensitive C57BL/6 mice.

METHODS

The thoraces of C57BL/6 mice were irradiated with 6 and 12 Gy, respectively. Treated and sham-irradiated control mice were sacrificed at times corresponding to the latent period (1, 3, 6, 12, 24, 48, 72 hours and 1 week postirradiation), the pneumonic phase (2, 4, 8, and 16 weeks postirradiation), and the beginning of the fibrotic phase (24 weeks postirradiation). The lung tissue from three different mice per dosage and time point was analyzed by a combination of polymerase chain reaction (PCR), immunohistochemistry, and light microscopy. The mRNA expression of TGF-beta was quantified by competitive reverse transcriptase/polymerase chain reaction (RT-PCR); the cellular origin of the TGF-beta protein was identified by immunohistochemical staining (alkaline phosphatase-anti-alkaline phosphatase [APAAP]). The cytokine expression on mRNA and protein level was correlated with the histopathological alterations.

RESULTS

Following thoracic irradiation with a single dose of 12 Gy, radiation-induced TGF-beta release in lung tissue was appreciable already within the first hours (1, 3, and 6 hours postirradiation) and reached a significant increase after 12 hours; subsequently (48 hours, 72 hours, and 1 week postirradiation) the TGF-beta expression declined to basal levels. At the beginning of the pneumonic phase, irradiation-mediated stimulation of TGF-beta release reached maximal values at 2 and 4 weeks. The elevated levels of TGF-beta mRNA during the latent phase have been found to correlate with immunohistochemical staining of alveolar macrophages. The most striking increase in TGF-beta immunoreactivity was seen during the acute phase of pneumonitis. Throughout this observation period, type II pneumocytes and fibroblasts (apart from inflammatory cells) served as important sources of TGF-beta expression. Increased TGF-beta expression was detected prominently in regions of histopathologic radiation injury. After exposure to a single radiation dose of 6 Gy, the lung tissue revealed only a minor radiation-mediated TGF-beta mRNA response. The modest upregulation ranged from 6 hours to 48 hours after irradiation. Corresponding to the only minor histopathologic changes after thoracic irradiation with 6 Gy, measurement of TGF-beta mRNA levels during the later time points revealed no significant alterations in comparison to untreated control mice.

CONCLUSIONS

This study demonstrates an acute and long-lasting increase in the expression of TGF-beta in lung tissue following thoracic irradiation with 12 Gy. The predominant localization of TGF-beta in areas of inflammatory cell infiltrates and fibrosis suggests involvement of this cytokine in the pathogenesis of radiation-induced pulmonal fibrosis. Further studies should be performed to explore the role of other cytokines in the development of radiation injury. An improved understanding of the underlying mechanisms of pulmonary fibrosis may eventually lead to modulatory intervention at the molecular level to modify the fibrotic process.

P450 oxidoreductase (POR) is the obligatory flavoprotein intermediate that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 enzymes. Although mouse Por gene ablation causes embryonic lethality, POR missense mutations cause disordered steroidogenesis, ambiguous genitalia, and Antley-Bixler syndrome (ABS), which has also been attributed to <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 2 (FGFR2) mutations. We sequenced the POR gene and FGFR2 exons 8 and 10 in 32 individuals with ABS and/or hormonal findings that suggested POR deficiency. POR and FGFR2 mutations segregated completely. Fifteen patients carried POR mutations on both alleles, 4 carried mutations on only one allele, 10 carried FGFR2 or FGFR3 mutations, and 3 patients carried no mutations. The 34 affected POR alleles included 10 with A287P (all from whites) and 7 with R457H (four Japanese, one African, two whites); 17 of the 34 alleles carried <em>16</em> "private" mutations, including 9 missense and 7 frameshift mutations. These 11 missense mutations, plus 10 others found in databases or reported elsewhere, were recreated by site-directed mutagenesis and were assessed by four assays: reduction of cytochrome c, oxidation of NADPH, support of 17alpha-hydroxylase activity, and support of 17,20 lyase using human P450c17. Assays that were based on cytochrome c, which is not a physiologic substrate for POR, correlated poorly with clinical phenotype, but assays that were based on POR's support of catalysis by P450c17--the enzyme most closely associated with the hormonal phenotype--provided an excellent genotype/phenotype correlation. Our large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and disordered steroidogenesis should be recognized as having a distinct new disease: POR deficiency.

BACKGROUND

Human umbilical mesenchymal stem cells (HUMSCs) isolated from Wharton's jelly of the umbilical cord can be easily obtained and processed compared with embryonic or bone marrow stem cells. These cells may be a valuable source in the repair of spinal cord injury.

RESULTS

We examine the effects of HUMSC transplantation after complete spinal cord transection in rats. Approximately 5x10(5) HUMSCs were transplanted into the lesion site. Three groups of rats were implanted with either untreated HUMSCs (referred to as the stem cell group), or HUMSCs treated with neuronal conditioned medium (NCM) for either three days or six days (referred to as NCM-3 and NCM-6 days, respectively). The control group received no HUMSCs in the transected spinal cord. Three weeks after transplantation, significant improvements in locomotion were observed in all the three groups receiving HUMSCs (stem cell, NCM-3 and NCM-6 days groups). This recovery was accompanied by increased numbers of regenerated axons in the corticospinal tract and neurofilament-positive fibers around the lesion site. There were fewer microglia and reactive astrocytes in both the rostral and caudal stumps of the spinal cord in the stem cell group than in the control group. Transplanted HUMSCs survived for <em>16</em> weeks and produced large amounts of human neutrophil-activating protein-2, neurotrophin-3, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, glucocorticoid induced tumor necrosis <em>factor</em> receptor, and vascular endothelial <em>growth</em> <em>factor</em> receptor 3 in the host spinal cord, which may help spinal cord repair.

CONCLUSIONS

Transplantation of HUMSCs is beneficial to wound healing after spinal cord injury in rats.

p38 is a member of the mitogen-activated protein (MAP) kinase superfamily activated by stress signals and implicated in cellular processes involving inflammation and apoptosis. Unlike the extracellular signal-regulated kinases (p42 and p44 MAP kinases), which are stimulated by insulin in many cell types, p38 activity is inhibited by insulin in postmitotic fetal neurons for which insulin is a potent survival <em>factor</em> (Heidenreich, K. A., and Kummer, J. L. (1996) J. Biol. Chem. 271, 9891-9894). These data suggested that insulin's effects on neuronal survival are mediated by inhibition of a p38-mediated apoptotic pathway. To better understand the relationship between p38 activity and cell survival, we induced apoptosis in two cell lines and examined the ability of insulin or a specific p38 inhibitor (a pyridinyl imidazole compound PD<em>16</em>93<em>16</em>) to block p38 activity and cell death. In Rat-1 <em>fibroblasts</em> grown in the presence of serum, p38 activity was undetectable by immune complex assays, and the number of apoptotic cells was very low (<0.5%). After the removal of serum for <em>16</em> h, p38 activity was markedly elevated, and apoptosis increased by 14-15-fold. Insulin (50 ng/ml) inhibited p38 activity by approximately 70% and blocked apoptosis by at least 80%. PD<em>16</em>93<em>16</em> also blocked p38 enzyme activity and apoptosis by approximately 80%. Similar results were obtained in differentiated PC12 cells that were deprived of nerve <em>growth</em> <em>factor</em> (NGF) for <em>16</em> h. In the presence of NGF, p38 activity and the number of apoptotic cells was very low (approximately 1.0%). After NGF withdrawal, p38 activity was selectively elevated and apoptosis increased to 15%. Both insulin and PD<em>16</em>93<em>16</em> markedly blocked the increase in p38 activity and apoptosis. The MAP kinase kinase inhibitor, PD98059, had no effect on apoptosis in Rat-1 <em>fibroblasts</em> and only partially blocked apoptosis in PC12 cells. PD98059 did not influence insulin's ability to block apoptosis, indicating that the extracellular signal-regulated kinase pathway does not mediate insulin's survival effects. These data further support the role of p38 in cellular apoptosis and support the hypothesis that insulin promotes cell survival, at least in part, by inhibiting the p38 pathway.

The fibroproliferative phase of acute respiratory distress syndrome (ARDS) has traditionally been regarded as a late event but recent studies that suggest increased lung collagen turnover within 24 h of diagnosis challenge this view. We hypothesized that fibroproliferation is initiated early in ARDS, characterized by the presence of <em>fibroblast</em> <em>growth</em> <em>factor</em> activity in the lung and would relate to clinical outcome. Patients fulfilling American/European Consensus Committee criteria for ARDS and control patients ventilated for non-ARDS respiratory failure underwent bronchoalveolar lavage (BAL) and serum sampling within 24 h of diagnosis and again at 7 d. The ability of BAL fluid (BALF) to stimulate human lung <em>fibroblast</em> proliferation in vitro was examined in relation to concentrations of N-terminal peptide for type III procollagen (N-PCP-III) in BALF/serum and clinical indices. At 24 h, ARDS lavage fluid demonstrated potent mitogenic activity with a median value equivalent to 70% (range 31-<em>16</em>4) of the response to serum, and was significantly higher than control lavage (32% of serum response, range 11-42; p < 0.05). At 24 h, serum N-PCP-III concentrations were elevated in the ARDS group compared with control patients (2.8 U/ml; range 0.6-14.8 versus 1.1 U/ml; range 0.4-3.7, p < 0.0001) as were BALF N-PCP-III concentrations (2.9 U/ml; range 0. 6-11.4 versus 0.46 U/ ml; range 0.00-1.63, p < 0.01). In addition, BALF N-PCP-III concentrations at 24 h were significantly elevated in nonsurvivors of ARDS compared with survivors (p < 0.05). At 7 d, the mitogenic activity remained elevated in the ARDS group compared with control (p < 0.05) and was also significantly higher in ARDS nonsurvivors compared with survivors (67%; range 45-120 versus 31%; range <em>16</em>-64, p < 0.05). These data are consistent with the hypothesis that fibroproliferation is an early response to lung injury and an important therapeutic target.

Intercellular adhesion molecule 1 (ICAM-1) is involved in the recirculation of blood leukocytes and, presumably, in the infiltration of cytolytic effector leukocytes into tumors. The present report describes a down-regulated expression of vascular ICAM-1 on tumor-infiltrating endothelial cells (EC) in renal cell carcinoma. This finding was obtained by flow cytometric analysis of tumor EC compared to EC obtained from healthy tissue. Since <em>growth</em> of solid tumors is dependent on the formation of new blood vessels (angiogenesis), we hypothesized that angiogenic <em>factors</em> are responsible for the down-regulation of ICAM-1. This hypothesis was investigated in vitro using human umbilical vein- and dermis-derived EC. Using flow cytometry, we found a biphasic regulation of ICAM-1 during stimulation of cultured EC with the angiogenic agent basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). Although <em>16</em>-24 h after activation a marked up-regulation of ICAM-1 was observed, expression was significantly decreased after 48h. The longevity of this down-regulation was at least 7 days. Northern blot analysis revealed down-regulation of the steady-state mRNA level of the gene. ICAM-2 showed similar results of intial up- and later down-regulation. Functional relevance for the changes in ICAM-1 expression was demonstrated by a corresponding biphasic regulation of EC-leukocyte adhesion after EC activation by bFGF. The described effects are specific for bFGF since other angiogenic <em>factors</em> (such as vascular endothelial <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em> beta, and interleukin 8) did not affect adhesion molecule expression. Subsequent experiments showed that angiogenic <em>factors</em> decrease the sensitivity of EC to activation with tumor necrosis <em>factor</em>-alpha in regard to adhesion molecule expression. The present results reveal a tumor-derived escape mechanism from cytolytic effector leukocytes by down-regulation of vascular adhesion molecules in vivo and in vitro and decreased responsiveness to proinflammatory cytokines.

Graves' disease (GD) is associated with T cell infiltration, but the mechanism for lymphocyte trafficking has remained uncertain. We reported previously that <em>fibroblasts</em> from patients with GD express IL-<em>16</em>, a CD4-specific chemoattractant, and RANTES, a C-C chemokine, in response to GD-specific IgG (GD-IgG). We unexpectedly found that these responses result from a functional interaction between GD-IgG and the insulin-like <em>growth</em> <em>factor</em> (IGF)-I receptor (IGF-IR). IGF-I and the IGF-IR-specific IGF-I analog, des(1-3), mimic the effects of GD-IgG. Neither GD-IgG nor IGF-I activates chemoattractant expression in control <em>fibroblasts</em> from donors without GD. Interrupting IGF-IR function with specific receptor-blocking Abs or by transiently transfecting <em>fibroblasts</em> with a dominant negative mutant IGF-IR completely attenuates signaling provoked by GD-IgG. Moreover, GD-IgG displaces specific (125)I-labeled IGF-I binding to <em>fibroblasts</em> and attenuates IGF-IR detection by flow cytometry. These findings identify a novel disease mechanism involving a functional GD-IgG/IGF-IR bridge, which potentially explains T cell infiltration in GD. Interrupting this pathway may constitute a specific therapeutic strategy.

The immortal human keratinocyte line HaCaT is frequently used as a paradigm for skin keratinocytes in vitro because of its highly preserved differentiation capacity. HaCaT cells form a nearly regular epidermal architecture when transplanted onto subcutaneous tissue of athymic mice. In order to analyze further their differentiation capacity in vitro, HaCaT cells were studied in organotypic cocultures on top of collagen gels containing human dermal <em>fibroblasts</em>. Within 1 wk HaCaT cells formed a still dysplastic epithelium, the thickness of which correlated with the number of <em>fibroblasts</em> in the collagen gel. With further culture time of up to 3 wk a remarkably well structured and differentiated squamous epithelium developed. After 1 wk, keratins 10 and <em>16</em>, involucrin, and transglutaminase I were expressed in suprabasal layers, whereas filaggrin, keratin 2e, and loricrin appeared after 2-3 wk. Within this time, a nearly complete basement membrane had formed including hemidesmosomes and anchoring fibrils. Epithelial cell proliferation became restricted to the basal layer after 2 and 3 wk. Using the TdT-mediated dUTP nick end labeling assay, fragmentation of DNA was detectable in nuclei of the parakeratotic stratum corneum. Ultrastructurally, many features of keratinization accumulated after 2 and 3 wk, though an orthokeratotic keratinization was not achieved, in contrast to HaCaT transplants. This differentiation deficiency - as compared with normal keratinocytes -- might be due to a lack of paracrine <em>factors</em> important for keratinocyte differentiation or to a reduced sensitivity of these cells. Nevertheless, this high degree of differentiation under organotypic conditions qualifies this cell line as an appropriate model for elucidation of the molecular mechanisms regulating keratinocyte <em>growth</em> and differentiation and for use in pharmacotoxicology.

The transforming <em>growth</em> <em>factor</em> (TGF)-beta/Smad3 signaling pathway is considered a central mediator of pathological organ fibrosis; however, contribution of Smad2/3-independent TGF-beta signaling has not been fully explored. The present study utilized previously a described model of scleroderma (SSc) fibrosis based on forced expression of the TGF-betaRI (ALK5) (Pannu, J., Gardner, H., Shearstone, J. R., Smith, E., and Trojanowska, M. (2006) Arthritis Rheum. 54, 3011-3021). This study was aimed at determining the molecular mechanisms underlying the profibrotic program in this model. We demonstrate that the TGF-betaRI-dependent up-regulation of collagen and CCN2 (CTGF) does not involve Smad2/3 activation but is mediated by ALK1/Smad1 and ERK1/2 pathways. The following findings support this conclusion: (i) Smad2 and -3 were not phosphorylated in response to TGF-betaRI, (ii) a TGF-betaRI mutant defective in Smad2/3 activation, ALK5(3A), potently stimulated collagen production, (iii) elevation of TGF-betaRI triggered sustained association of ALK5 with ALK1 and high levels of Smad1 phosphorylation, (iv) blockade of Smad1 via small interfering RNA abrogated collagen and CCN2 up-regulation in this model, (v) elevated TGF-betaRI led to a prolonged activation of ERK1/2, (vi) the pharmacologic inhibitor of ERK1/2 inhibited Smad1 phosphorylation and abrogated profibrotic effects of elevated TGFbeta-RI. Additional experiments demonstrated that a GC-rich response element located -6 to -<em>16</em> (upstream of the transcription start site) in the CCN2 promoter mediated Smad1-dependent increased promoter activity in this model. This element was shown previously to mediate up-regulation of the CCN2 promoter in SSc <em>fibroblasts</em>. In conclusion, this study defines a novel ALK1/Smad1- and ERK1/2-dependent, Smad3-independent mode of TGF-beta signaling that may operate during chronic stages of fibrosis in SSc.

OBJECTIVE

Fibroblast growth factor receptor 3 (FGFR3) mutations were recently found at a high frequency in well-differentiated urothelial cell carcinoma (UCC). We investigated the relationship between FGFR3 status and three molecular markers (MIB-1, P53, and P27kip1) associated with worse prognosis and determined the reproducibility of pathologic grade and molecular variables.

METHODS

In this multicenter study, we included 286 patients with primary (first diagnosis) UCC. The histologic slides were reviewed. FGFR3 status was examined by polymerase chain reaction-single strand conformation polymorphism and sequencing. Expression levels of MIB-1, P53, and P27kip1 were determined by immunohistochemistry. Mean follow-up was 5.5 years (range, 0.4 to 18.4 years).

RESULTS

FGFR3 mutations were detected in 172 (60%) of 286 UCCs. Grade 1 tumors had an FGFR3 mutation in 88% of patient samples and grade 3 tumors in 16% of patient samples. Conversely, aberrant expression patterns of MIB-1, P53, and P27kip1 were seen in 5%, 2%, and 3% of grade 1 tumors and in 85%, 60%, and 56% of grade 3 tumors, respectively. In multivariate analysis with recurrence rate, progression, and disease-specific survival as end points, the combination of FGFR3 and MIB-1 proved independently significant in all three cases. By using these two molecular markers, three molecular grades (mGs) could be identified: mG1 (mutation; normal expression), favorable prognosis; mG2 (two remaining combinations), intermediate prognosis; and mG3 (no mutation; high expression), poor prognosis. The molecular variables were more reproducible than pathologic grade (85% to 100% v 47% to 61%).

CONCLUSIONS

The FGFR3 mutation represents the favorable molecular pathway of UCC. Molecular grading provides a new, simple, and highly reproducible tool for clinical decision making in UCC patients.

The nontransmembrane protein tyrosine phosphatase SHP-2 plays a critical role in <em>growth</em> <em>factor</em> and cytokine signaling pathways. Previous studies revealed that a fraction of SHP-2 moves to focal contacts upon integrin engagement and that SHP-2 binds to SHP substrate 1 (SHPS-1)/SIRP-1alpha, a transmembrane glycoprotein with adhesion molecule characteristics (Y. Fujioka et al., Mol. Cell. Biol. <em>16</em>:6887-6899, 1996; M. Tsuda et al., J. Biol. Chem. 273:13223-13229). Therefore, we asked whether SHP2-SHPS-1 complexes participate in integrin signaling. SHPS-1 tyrosyl phosphorylation increased upon plating of murine <em>fibroblasts</em> onto specific extracellular matrices. Both in vitro and in vivo studies indicate that SHPS-1 tyrosyl phosphorylation is catalyzed by Src family protein tyrosine kinases (PTKs). Overexpression of SHPS-1 in 293 cells potentiated integrin-induced mitogen-activated protein kinase (MAPK) activation, and potentiation required functional SHP-2. To further explore the role of SHP-2 in integrin signaling, we analyzed the responses of SHP-2 exon 3(-/-) and wild-type cell lines to being plated on fibronectin. Integrin-induced activation of Src family PTKs, tyrosyl phosphorylation of several focal adhesion proteins, MAPK activation, and the ability to spread on fibronectin were defective in SHP-2 mutant <em>fibroblasts</em> but were restored upon SHP-2 expression. Our data suggest a positive-feedback model in which, upon integrin engagement, basal levels of c-Src activity catalyze the tyrosyl phosphorylation of SHPS-1, thereby recruiting SHP-2 to the plasma membrane, where, perhaps by further activating Src PTKs, SHP-2 transduces positive signals for downstream events such as MAPK activation and cell shape changes.

OBJECTIVE

To characterize the molecular response of adult human articular cartilage to acute mechanical injury.

METHODS

An established ex vivo model was used to compare gene expression of adult human articular cartilage explants 24 hours after mechanical injury with that of uninjured controls by microarray analysis of gene expression. Confirmation for selected genes was obtained by real-time polymerase chain reaction and immunohistochemical analysis. Expression of selected genes was also investigated in preserved and osteoarthritic (OA) cartilage.

RESULTS

Six hundred ninety genes were significantly regulated at least 2-fold following mechanical injury. They included genes previously reported to be differentially expressed in OA versus normal cartilage or having allelic variants genetically linked to OA. Significant functional clusters included genes associated with wound healing, developmental processes, and skeletal development. The transforming <em>growth</em> <em>factor</em> beta, <em>fibroblast</em> <em>growth</em> <em>factor</em>, and Wnt pathways were modulated. A systematic analysis of the Wnt signaling pathway revealed up-regulation of Wnt-<em>16</em>, down-regulation of FRZB, up-regulation of Wnt target genes, and nuclear localization of beta-catenin in injured cartilage. In addition, in OA, Wnt-<em>16</em> and beta-catenin were barely detectable in preserved cartilage areas, but were dramatically up-regulated in areas of the same joint with moderate to severe OA damage.

CONCLUSIONS

Our findings indicate that mechanical injury to adult human articular cartilage results in the activation of a signaling response, with reactivation of morphogenetic pathways. Therapeutic targeting of such pathways may improve current protocols of joint surface defect repair and/or prevent the evolution of such lesions into posttraumatic OA.

OBJECTIVE

The concept of neovascularization in response to tissue ischemia has been extended by the finding of postnatal vasculogenesis initiated by endothelial progenitor cells (EPCs). The aim of this study was to analyze whether a maximal stress test in patients with coronary artery disease (CAD) increases the number of circulating EPCs.

RESULTS

Blood concentration of EPCs was analyzed by FACS and cell culture assay in CAD patients with (n=<em>16</em>) or without (n=12) exercise-induced myocardial ischemia and in healthy subjects (n=11) for up to 144 hours after maximal stress test. Plasma concentrations of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, tumor necrosis <em>factor</em>-alpha, and granulocyte macrophage-colony stimulating <em>factor</em> were determined by ELISA. EPCs increased significantly in ischemic patients, with a maximum after 24 to 48 hours (cell culture: 3.3+/-0.5-fold increase; FACS: 3.1+/-0.6-fold increase) and returned to baseline within 72 hour. In nonischemic patients and healthy subjects, no EPC increase was detectable. VEGF levels in ischemic patients increased significantly after 2 to 6 hours (maximum after 2 hours; 4.0+/-1.1-fold increase) and no change was observed in nonischemic patients and healthy subjects; DeltaVEGF and DeltaEPC correlated significantly (r=0.66).

CONCLUSIONS

Patients with symptomatic CAD respond to a single episode of exercise-induced myocardial ischemia with a time-dependent increase in circulating EPCs. This increase may be related to and preceded by an increase in plasma VEGF.

BACKGROUND

An unmet medical need exists for patients with metastatic renal cell carcinoma who have progressed on VEGF-targeted and mTOR-inhibitor therapies. Fibroblast growth factor (FGF) pathway activation has been proposed as a mechanism of escape from VEGF-targeted therapies. Dovitinib is an oral tyrosine-kinase inhibitor that inhibits VEGF and FGF receptors. We therefore compared dovitinib with sorafenib as third-line targeted therapies in patients with metastatic renal cell carcinoma.

METHODS

In this multicentre phase 3 study, patients with clear cell metastatic renal cell carcinoma who received one previous VEGF-targeted therapy and one previous mTOR inhibitor were randomly assigned through an interactive voice and web response system to receive open-label dovitinib (500 mg orally according to a 5-days-on and 2-days-off schedule) or sorafenib (400 mg orally twice daily) in a 1:1 ratio. Randomisation was stratified by risk group and region. The primary endpoint was progression-free survival (PFS) assessed by masked central review. Efficacy was assessed in all patients who were randomly assigned and safety was assessed in patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT01223027.

RESULTS

284 patients were randomly assigned to the dovitinib group and 286 to the sorafenib group. Median follow-up was 11·3 months (IQR 7·9-14·6). Median PFS was 3·7 months (95% CI 3·5-3·9) in the dovitinib group and 3·6 months (3·5-3·7) in the sorafenib group (hazard ratio 0·86, 95% CI 0·72-1·04; one-sided p=0·063). 280 patients in the dovitinib group and 284 in the sorafenib group received at least one dose of study drug. Common grade 3 or 4 adverse events included hypertriglyceridaemia (38 [14%]), fatigue (28 [10%]), hypertension (22 [8%]), and diarrhoea (20 [7%]) in the dovitinib group, and hypertension (47 [17%]), fatigue (24 [8%]), dyspnoea (21 [7%]), and palmar-plantar erythrodysaesthesia (18 [6%]) in the sorafenib group. The most common serious adverse event was dyspnoea (16 [6%] and 15 [5%] in the dovitinib and sorafenib groups, respectively).

CONCLUSIONS

Dovitinib showed activity, but this was no better than that of sorafenib in patients with renal cell carcinoma who had progressed on previous VEGF-targeted therapies and mTOR inhibitors. This trial provides reference outcome data for future studies of targeted inhibitors in the third-line setting.

BACKGROUND

Novartis Pharmaceuticals Corporation.

BACKGROUND

High levels of fibroblast growth factor 23 (FGF23) are associated with mortality and progression of chronic kidney disease (CKD). Reducing dietary phosphorus intake lowers FGF23 secretion in healthly individuals, but there is little data on its effects in patients with pre-dialysis CKD.

METHODS

Using a 2×2 factorial design, we randomly assigned 16 normophosphataemic CKD stage 3-4 patients to receive a 2-week treatment with either lanthanum carbonate 1000 mg three times daily or placebo, and to ingest a tightly controlled diet containing 750 or 1500 mg of dietary phosphorus daily. We analysed serial measurements of FGF23, parathyroid hormone, serum phosphate and calcium, and 24-h urinary phosphate and calcium excretion using repeated-measures analyses.

RESULTS

Compared with the 1500-mg phosphorus diet, patients assigned to the 750-mg diet had greater reduction in 24-h urinary phosphate excretion (66% vs. 29%; P<0.0001). Lanthanum-treated patients experienced a significant reduction in 24-h urinary phosphate excretion compared with baseline (64%; P<0.0001), but the difference compared with placebo did not reach significance (64% vs. 31%). Despite the significant reductions in 24-h urinary phosphate excretion, no group demonstrated a significant reduction in FGF23 levels; FGF23 levels actually increased significantly in the 1500-mg diet plus placebo group, suggesting dietary phosphorus loading.

CONCLUSIONS

Although dietary phosphorus restriction and lanthanum lowered urinary phosphate excretion consistent with a rapid decrease in phosphorus absorption, inducing a reduction in FGF23 levels in CKD patients may require interventions with a longer duration than in healthy volunteers.

Previous crystallographic analyses of the Kunitz inhibitors from soybean. Erythrina caffra and wheat, the interleukins-1 beta and 1 alpha and the acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> have shown that they contain a most unusual fold. It is formed by six two-stranded hairpins. Three of these form a barrel structure and the other three are in a triangular array that caps the barrel. The arrangement of the secondary structures gives the molecules a pseudo 3-fold axis. Although the different proteins have very similar structures, many of their sequences have no significant similarities overall. The structural determinants of this fold are described and discussed in this paper. The barrels in the different proteins have the same geometrical features: six strands tilted at 56 degrees to the barrel axis; a barrel diameter of <em>16</em> A, and the beta-sheet hydrogen bonded so that it is staggered with a shear number of 12. These features fit McLachlan's equations for ideal barrels formed by beta-sheets. The wide diameter of the barrels is filled by layers of residues that, while not identical in the different proteins, are, in almost all cases, large. The structure of the triangular array of hairpins is determined by the coiling of the strands and the packing of hairpin residues against each other and against residues from the interior of the barrel. The major sequence requirements of this fold are large or medium hydrophobic residues at 18 buried sites. In the different structures the total volume of these residues is 3000 (+/- 120) A. The polyhedron model of protein architecture is used to demonstrate that the main, and in particular the symmetrical, features of this fold arise from the ideal and equal packing of six hairpins, modified only slightly to form hydrogen bonds between the hairpins.

The formation of a new blood supply, angiogenesis, is an essential component of carcinogenesis and unrestricted tumor <em>growth</em>. A substance capable of inhibiting angiogenesis would be of considerable therapeutic potential in the treatment of cancer. We previously reported that the <em>16</em>-kilodalton N-terminal fragment of rat PRL (<em>16</em>K rPRL) was a potent inhibitor of capillary endothelial cell proliferation via a novel receptor. We now report that the nanomolar concentrations of recombinant human <em>16</em>K PRL inhibit basal and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>- or vascular endothelial <em>growth</em> <em>factor</em>-stimulated <em>growth</em> of bovine brain capillary endothelial cells. <em>16</em>K human (h) PRL also inhibits stimulation of human umbilical vein endothelial cell proliferation by basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. The organization of endothelial cells into capillary-like structures in type I collagen gels is also prevented by <em>16</em>K hPRL. Furthermore, in an in vivo assay, the chick embryo chorioallantoic membrane assay, <em>16</em>K hPRL as well as <em>16</em>K rPRL were potent inhibitors of capillary formation. <em>16</em>K hPRL, like <em>16</em>K rPRL, maintains its biological activity as a partial PRL agonist at PRL receptors on mammary gland epithelial cells. These data demonstrate for the first time that the biological activity of <em>16</em>K rPRL is not unique and that similar fragments of hPRL are active. The antiangiogenic activity of these molecules is conserved across avian and mammalian species. That <em>16</em>K hPRL is a potent antiangiogenic <em>factor</em> in in vitro and an in vivo assay raises the exciting potential of this peptide being capable of inhibiting tumor <em>growth</em>.

OBJECTIVE

JNJ-42756493 is an orally administered pan-fibroblast growth factor receptor (FGFR) tyrosine kinase inhibitor. This first-in-human study evaluates the safety, pharmacokinetics, and pharmacodynamics and defines the recommended phase II dose (RP2D) of JNJ-42756493.

METHODS

Eligible patients with advanced solid tumors received escalating doses of JNJ-42756493 from 0.5 to 12 mg administered continuously daily or JNJ-42756493 10 or 12 mg administered intermittently (7 days on/7 days off).

RESULTS

Sixty-five patients were enrolled. The most common treatment-emergent adverse events included hyperphosphatemia (65%), asthenia (55%), dry mouth (45%), nail toxicity (35%), constipation (34%), decreased appetite (32%), and dysgeusia (31%). Twenty-seven patients (42%) experienced grade ≥ 3 treatment-emergent adverse events, and one dose-limiting toxicity of grade 3 ALT elevation was observed at 12 mg daily. Maximum-tolerated dose was not defined. Nine milligrams daily was considered as the initial RP2D; however, tolerability was improved with intermittent schedules, and 10 mg administered on a 7-days-on/7-days-off schedule was considered the final RP2D. Pharmacokinetics were linear, dose proportional, and predictable, with a half-life of 50 to 60 hours. Dose-dependent elevations in serum phosphate, a manifestation of pharmacodynamic effect, occurred in all patients starting at 4 mg daily. Among 23 response-evaluable patients with tumor FGFR pathway alterations, four confirmed responses and one unconfirmed partial response were observed in patients with glioblastoma and urothelial and endometrial cancer (all with FGFR2 or FGFR3 translocations); 16 patients had stable disease.

CONCLUSIONS

JNJ-42756493 administered at 10 mg on a 7-days-on/7-days-off schedule achieved exposures at which clinical responses were observed, demonstrated pharmacodynamic biomarker activity, and had a manageable safety profile.

We report that human breast cancer cells secrete a <em>growth</em> <em>factor</em> that is biologically and immunologically similar to platelet-derived <em>growth</em> <em>factor</em> (PDGF). Serum-free medium conditioned by estrogen-independent MDA-MB-231 or estrogen-dependent MCF-7 cells contains a mitogenic or "competence" activity that is capable of inducing incorporation of [3H]thymidine into quiescent Swiss 3T3 cells in the presence of platelet-poor plasma. In addition, the conditioned medium contains an activity that competes with 125I-labeled PDGF for binding to PDGF receptors on normal human <em>fibroblasts</em>. The secretion of PDGF-like activity by the hormone-responsive cell line MCF-7 is stimulated by 17 beta-estradiol. Like authentic PDGF, the PDGF-like activity produced by breast cancer cells is stable after acid and heat treatment (95 degrees C) and inhibited by reducing agents. The mitogenic activity comigrates with a material of approximately equal to 30 kDa on NaDodSO4/polyacrylamide gels. Immunoprecipitation with PDGF antiserum of proteins from metabolically labeled cell lysates and conditioned medium followed by analysis on nonreducing NaDodSO4/polyacrylamide gels identified proteins of 30 and 34 kDa. Upon reduction, the 30- and 34-kDa bands were converted to 15- and <em>16</em>-kDa bands suggesting that the immunoprecipitated proteins were made up of two disulfide-linked polypeptides similar to PDGF. Hybridization studies with cDNA probes for the A chain of PDGF and the B chain of PDGF/SIS identified transcripts for both PDGF chains in the MCF-7 and MDA-MB-231 cells. The data summarized above provide conclusive evidence for the synthesis and hormonally regulated secretion of a PDGF-like mitogen by breast carcinoma cells. Production of a PDGF-like <em>growth</em> <em>factor</em> by breast cancer cell lines may be important in mediating paracrine stimulation of tumor <em>growth</em>.

Transarterial chemoembolization (TACE) is the current standard of treatment for unresectable intermediate-stage hepatocellular carcinoma (HCC). Brivanib, a selective dual inhibitor of vascular endothelial <em>growth</em> <em>factor</em> and <em>fibroblast</em> <em>growth</em> <em>factor</em> signaling, may improve the effectiveness of TACE when given as an adjuvant to TACE. In this multinational, randomized, double-blind, placebo-controlled, phase III study, 870 patients with TACE-eligible HCC were planned to be randomly assigned (1:1) after the first TACE to receive either brivanib 800 mg or placebo orally once-daily. The primary endpoint was overall survival (OS). Secondary endpoints included time to disease progression (TTDP; a composite endpoint based on development of extrahepatic spread or vascular invasion, deterioration of liver function or performance status, or death), time to extrahepatic spread or vascular invasion (TTES/VI), rate of TACE, and safety. Time to radiographic progression (TTP) and objective response rate were exploratory endpoints. The trial was terminated after randomization of 502 patients (brivanib, 249; placebo, 253) when two other phase III studies of brivanib in advanced HCC patients failed to meet OS objectives. At termination, median follow-up was approximately <em>16</em> months. Intention-to-treat analysis showed no improvement in OS with brivanib versus placebo (median, 26.4 [95% confidence interval {CI}: 19.1 to not reached] vs. 26.1 months [19.0-30.9]; hazard ratio [HR]: 0.90 [95% CI: 0.66-1.23]; log-rank P=0.5280). Brivanib improved TTES/VI (HR, 0.64 [95% CI: 0.45-0.90]), TTP (0.61 [0.48-0.77]), and rate of TACE (0.72 [0.61-0.86]), but not TTDP (0.94 [0.72-1.22]) versus placebo. Most frequent grade 3-4 adverse events included hyponatremia (brivanib, 18% vs. placebo, 5%) and hypertension (13% vs. 3%).

CONCLUSIONS

In this study, brivanib as adjuvant therapy to TACE did not improve OS.

BACKGROUND

Tumor-induced osteomalacia (TIO) is a paraneoplastic syndrome of hypophosphatemia, decreased renal phosphate reabsorption, normal or low serum 1,25-dihydryxyvitamin-D concentration, myopathy, and osteomalacia. Fibroblast growth factor 23 (FGF23) is a phosphaturic protein overexpressed in tumors that cause TIO and is, at least partly, responsible for the manifestations of TIO.

OBJECTIVE

The objective of this study was to determine the sensitivity of FGF23 measurements in TIO.

METHODS

FGF23 concentrations were measured on stored samples with three ELISAs.

METHODS

This study was conducted at subspecialty referral centers.

METHODS

Twenty-two patients with suspected TIO, 13 with confirmed tumors, were studied.

METHODS

There were no interventions in this study.

METHODS

FGF23 concentration was the main outcome measure of this study.

RESULTS

Elevated FGF23 concentrations were detected using the Immunotopics C-terminal assay in 16 of 22 TIO patients (for a sensitivity of 73%), the Immunotopics Intact assay in five of 22 patients (sensitivity, 23%), and the Kainos Intact assay in 19 of 22 patients (sensitivity, 86%). In the 13 patients with confirmed tumors, the sensitivity was higher with all assays: 92% for the Immunotopics C-terminal assay, 38% for the Immunotopics Intact assay, and 100% for the Kainos assay.

CONCLUSIONS

The Kainos Intact assay was the most sensitive, followed by the Immunotopics C-terminal assay. The findings of normal FGF23 concentrations in some patients with TIO may indicate that FGF 23 is not responsible for the hypophosphatemia in these patients or that FGF23 secretion by some tumors is partially responsive to serum phosphate. Normal FGF23 concentrations should be interpreted in relation to the serum phosphate and 1,25-dihydryxyvitamin-D concentrations.

The potent vasoconstrictor endothelin-1 (ET-1) is at its highest concentration in the normal human ejaculate and is associated with the progression of metastatic prostate cancer. ET-1 protein expression is detected in situ in 14 of 14 primary cancers and 14 of <em>16</em> metastatic sites of human prostatic carcinoma. Exogenous ET-1 induces prostate cancer proliferation directly and enhances the mitogenic effects of insulin-like <em>growth</em> <em>factor</em> I, insulin-like <em>growth</em> <em>factor</em> II, platelet-derived <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and epidermal <em>growth</em> <em>factor</em> in serum-free conditions in vitro. The ETA-selective receptor antagonist A-127722 inhibits ET-1-stimulated <em>growth</em>, but the ETB-selective receptor antagonist BQ-788 does not. ET-3, an ETB-selective agonist, also had no effect on prostate cancer <em>growth</em>. No specific ETB-binding sites could be demonstrated in any established human prostate cancer cell line tested, and ETB mRNA, detected by reverse transcription PCR, was reduced. The predominance of ETB binding on human benign prostatic epithelial tissue is not present in metastatic prostate cancer by autoradiography. In human prostate cancer progression to metastases, ET-1 and ETA expression are retained, whereas ETB receptor expression is reduced.