We have shown previously that pp60c-src is a substrate for protein kinase C in vivo and that the target of protein kinase C phosphorylation in mammalian pp60c-src is serine 12. We now demonstrate that in addition to tumor promoters, all activators of phosphatidylinositol turnover that we have tested in <em>fibroblasts</em> (platelet-derived <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em>, serum, vasopressin, sodium orthovanadate, and prostaglandin F2 alpha) lead to the phosphorylation of pp60c-src at serine 12. In addition to stimulating serine 12 phosphorylation in pp60c-src, platelet-derived <em>growth</em> <em>factor</em> treatment of quiescent <em>fibroblasts</em> induces phosphorylation of one or two additional serine residues and one tyrosine residue within the N-terminal <em>16</em> kilodaltons of the enzyme and activates its immune complex protein-tyrosine kinase activity.

To investigate E7-dependent biochemical changes which are involved in cellular transformation, we analyzed the influence of human papillomavirus type <em>16</em> (HPV-<em>16</em>) E7 on the expression of cell cycle regulatory proteins. Expression of E7 in established rodent <em>fibroblasts</em> (NIH 3T3), which was shown to be sufficient for transformation of these cells, leads to constitutive expression of the cyclin E and cyclin A genes in the absence of external <em>growth</em> <em>factors</em>. Surprisingly, expression of the cyclin D1 gene, which encodes a major regulator of G1 progression, is unaltered in E7-transformed cells. In transient transfection experiments, the cyclin A gene promoter is activated by E7 via an E2F binding site. In 14/2 cells, which were used as a model system to analyze the role of HPV-<em>16</em> E7 in the transformation of primary cells, we observed rapid E7-dependent activation of cyclin E gene expression, which can be uncoupled from activation of the cyclin A gene, since the latter requires additional protein synthesis. E7-driven induction of cyclin E and cyclin A gene expression was accompanied by an increase in the associated kinase activities. Two domains of the E7 oncoprotein, which are designated cd1 and cd2, are essential for transformation of rodent <em>fibroblasts</em>. It is shown here that <em>growth</em> <em>factor</em>-independent expression of the cyclin E gene requires cd2 but not cd1, while activation of cyclin A gene expression requires cd1 function in addition to that of cd2. These data suggest that cyclin A gene expression is controlled by two distinct negative signals, one of which also restricts expression of the cyclin E gene. The ability of E7 to separately override each of these inhibitory signals, via cd1 and cd2, cosegregates with its ability to fully transform rodent <em>fibroblasts</em>. Unlike serum <em>growth</em> <em>factors</em>, E7 induces S-phase entry without activating cyclin D1 gene expression, in keeping with the finding that cyclin D1 function is not required in cells transformed by DNA tumor viruses.

Human papillomavirus type <em>16</em> (HPV-<em>16</em>) and HPV-18 are often detected in cervical carcinomas, while HPV-6, although frequently present in benign genital lesions, is only rarely present in cancers of the cervix. Therefore, infections with HPV-<em>16</em> and HPV-18 are considered high risk and infection with HPV-6 is considered low risk. We found, by using a heterologous promoter system, that expression of the E7 transforming protein differs between high- and low-risk HPVs. In high-risk HPV-<em>16</em>, E7 is expressed from constructs containing the complete upstream E6 open reading frame. In contrast, HPV-6 E7 was efficiently translated only when E6 was deleted. By using clones in which the coding regions of HPV-6, HPV-<em>16</em>, and HPV-18 E7s were preceded by identical leader sequences, we found that the ability of the E7 gene products to induce anchorage-independent <em>growth</em> in rodent <em>fibroblasts</em> correlated directly with the oncogenic association of the HPV types. By using an immortalization assay of normal human keratinocytes that requires complementation of E6 and E7, we found that both E6 and E7 of HPV-18 could complement the corresponding gene from HPV-<em>16</em>. However, neither E6 nor E7 from HPV-6 was able to substitute for the corresponding gene of HPV-<em>16</em> or HPV-18. Our results suggest that multiple <em>factors</em>, including lower intrinsic biological activity of E6 and E7 and differences in the regulation of their expression, account for the low activity of HPV-6, in comparison with HPV-<em>16</em> and HPV-18, in in vitro assays. These same <em>factors</em> may, in part, account for the apparent difference in oncogenic potential between these viruses.

OBJECTIVE

Inflammatory breast cancer is a distinct and aggressive form of locally advanced breast cancer with unique clinical and pathological features. Recently, histologic evidence of intense angiogenesis was found in inflammatory breast cancer specimens. The aim of this study was to confirm the angiogenic phenotype of inflammatory breast cancer and to investigate its potential to induce lymphangiogenesis.

METHODS

Real-time quantitative reverse transcriptase-PCR was used to measure levels of mRNA of tumor angiogenesis and lymphangiogenesis-related <em>factors</em> [vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-A, VEGF-C, VEGF-D, Flt-1, KDR, Flt-4, Ang-1, Ang-2, Tie-1, Tie-2, cyclooxygenase-2, <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2), Egr-1, Prox-1, and LYVE-1] in tumor specimens of <em>16</em> inflammatory breast cancer and 20 noninflammatory breast cancer patients. Tissue microarray technology and immunohistochemistry were used to study differential protein expression of some of the angiogenic <em>factors</em> in inflammatory breast cancer and noninflammatory breast cancer. Active lymphangiogenesis was further assessed by measuring lymphatic endothelial cell proliferation.

RESULTS

Inflammatory breast cancer specimens had significantly higher mRNA expression levels than noninflammatory breast cancer specimens of the following genes: KDR (P = 0.033), Ang-1, (P = 0.0001), Tie-1 (P = 0.001), Tie-2 (P = 0.001), FGF-2 (P = 0.002), VEGF-C (P = 0.001), VEGF-D (P = 0.012), Flt-4 (P = 0.001), Prox-1 (P = 0.005), and LYVE-1 (P = 0.013). High mRNA levels of FGF-2 and cyclooxygenase-2 corresponded to increased protein expression by immunohistochemistry. Inflammatory breast cancer specimens contained significantly higher fractions of proliferating lymphatic endothelial cells than noninflammatory breast cancer specimens (P = 0.033).

CONCLUSIONS

Using real-time quantitative reverse transcriptase-PCR and immunohistochemistry, we confirmed the intense angiogenic activity in inflammatory breast cancer and demonstrated the presence of active lymphangiogenesis in inflammatory breast cancer. This may help explain the high metastatic potential of inflammatory breast cancer by lymphatic and hematogenous route. Both pathways are potential targets for the treatment of inflammatory breast cancer.

This study evaluated the efficacy and safety of intramuscular administration of NV1FGF, a plasmid-based angiogenic gene delivery system for local expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> 1 (FGF-1), versus placebo, in patients with critical limb ischemia (CLI). In a double-blind, randomized, placebo-controlled, European, multinational study, 125 patients in whom revascularization was not considered to be a suitable option, presenting with nonhealing ulcer(s), were randomized to receive eight intramuscular injections of placebo or 2.5 ml of NV1FGF at 0.2 mg/ml on days 1, 15, 30, and 45 (total <em>16</em> mg: 4 x 4 mg). The primary end point was occurrence of complete healing of at least one ulcer in the treated limb at week 25. Secondary end points included ankle brachial index (ABI), amputation, and death. There were 107 patients eligible for evaluation. Improvements in ulcer healing were similar for use of NV1FGF (19.6%) and placebo (14.3%; P = 0.514). However, the use of NV1FGF significantly reduced (by twofold) the risk of all amputations [hazard ratio (HR) 0.498; P = 0.015] and major amputations (HR 0.371; P = 0.015). Furthermore, there was a trend for reduced risk of death with the use of NV1FGF (HR 0.460; P = 0.105). The adverse event incidence was high, and similar between the groups. In patients with CLI, plasmid-based NV1FGF gene transfer was well tolerated, and resulted in a significantly reduced risk of major amputation when compared with placebo.

BACKGROUND

Amplification of fibroblast growth factor receptor 1 (FGFR1) has been reported in squamous cell lung carcinoma and may be a molecular target for therapy. Little is known, however, about the clinical and demographic correlates of FGFR1 amplification.

METHODS

The study is an Institutional Review Board approved retrospective analysis of 226 patients with squamous cell lung cancer seen at the Massachusetts General Hospital from 2005 to 2011. Clinical and demographic characteristics of all patients were obtained, as well as treatment details including surgery, radiation, and chemotherapy, and overall survival. fluorescence in situ hybridization was performed for FGFR1 on formalin-fixed paraffin-embedded tumor tissue. Clinical genotyping results were also reviewed where available.

RESULTS

Thirty-seven of 226 patients (16%) with squamous cell lung cancer were found positive for amplification using a definition of amplification of a gene to copy number control ratio of 2.2 or higher. FGFR1 amplification status was not associated with age, sex, stage, histologic subtype within squamous cell, smoking history, or pack-years of smoking. We found no significant difference in overall survival by FGFR1 amplification status as a whole; in the advanced stage subset, our findings are inconclusive because of the small sample size.

CONCLUSIONS

FGFR1 amplification was found in 16% of a clinical cohort of squamous cell lung cancer patients. The lack of any specific clinicodemographic features that correlates with FGFR1 amplification suggests that all squamous cell patients should be tested for this genomic change.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) binds to cell surface receptor (CSR) proteins and to heparan sulfate proteoglycans (HSPG). On the basis of equilibrium dissociation constants (Kd), the CSR has been considered a "high-affinity" binding site and HSPG a "low-affinity" site. We measured the apparent individual on and off rate constants (kon and koff) for bFGF binding to these two sites on intact cells and to each class of binding site in the absence of the other. While the kon's for CSR and HSPG on intact cells were not statistically different (konC = 2.27 x 10(8) M-1 min-1; konH = 0.90 x 10(8) M-1 min-1), the koff for the HSPG was 22.7-fold greater than that for the CSR (koffC = 0.003 min-1; koffH = 0.68 min-1). Thus, the difference in Kd's appears to result from the faster rate at which bFGF is released from the HSPG sites compared to the CSR. The kon's for isolated CSR and HSPG, and the koff for isolated HSPG, did not differ significantly from those for intact cells konC = 2.50 x 10(8) M-1 min-1; konH = 0.92 x 10(8) M-1 min-1; koffH = 0.095 min-1). However, the off rate for isolated CSR (koffC = 0.048 min-1) was statistically indistinguishable from the off rate for HSPG and <em>16</em>-fold greater than the off rate for CSR on intact cells. The "high-affinity" binding of bFGF to intact cells probably refers only to a complex of bFGF with both CSR and HSPG, and not to the CSR alone.

The purpose of this study was to identify microRNAs (miRNAs) involved in the pathology of colorectal cancer (CRC) liver metastasis and investigate their underlying mechanisms. A total of 39 miRNAs were identified to be differentially expressed between <em>16</em> primary CRC tissues with liver metastases and <em>16</em> CRC tissues without liver metastases from 32 patients by Affymetric miRNA microarrays. A panel of eight miRNAs were confirmed to be significantly and differentially expressed between CRC tissues with and without liver metastases through quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis in the 32 patients. In a validated cohort of 99 CRC patients (44 with and 55 without liver metastases), only miR-214 was validated to be significantly down-regulated in CRC with liver metastases, which was associated with an unfavorable prognosis. Ectopic expression of miR-214 suppressed proliferation, migration, and invasion in vitro, tumor <em>growth</em> and liver metastasis in an in vivo xenograft mouse model, whereas miR-214 knockdown promoted proliferation, migration, and invasion in CRC cell lines. Further studies indicated that <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1) was a potential target of miR-214. Restoring miR-214 expression in CRC cells decreased endogenous FGFR1 messenger RNA (mRNA) and protein levels. FGFR1 knockdown mimicked the tumor suppressive effect of miR-214 on CRC cells, while reintroduction of FGFR1 abolished the tumor suppressive effect of miR-214 on CRC cells. Moreover, miR-214 expression levels were inversely correlated with FGFR1 in CRC patients.

CONCLUSIONS

Down-regulation of miR-214 expression was correlated with increased FGFR1 expression levels, which may contribute to increased CRC liver metastasis. miR-214 may serve as a potential marker to predict survival, and the miR-214-FGFR1 axis may be a therapeutic target in CRC patients.

We examined the role of vascular function and inflammation in the development and failure to heal diabetic foot ulcers (DFUs). We followed 104 diabetic patients for a period of 18.4 ± 10.8 months. At the beginning of the study, we evaluated vascular reactivity and serum inflammatory cytokines and <em>growth</em> <em>factors</em>. DFUs developed in 30 (29%) patients. DFU patients had more severe neuropathy, higher white blood cell count, and lower endothelium-dependent and -independent vasodilation in the macrocirculation. Complete ulcer healing was achieved in <em>16</em> (53%) patients, whereas 13 (47%) patients did not heal. There were no differences in the above parameters between the two groups, but patients whose ulcers failed to heal had higher tumor necrosis <em>factor</em>-α, monocyte chemoattractant protein-1, matrix metallopeptidase 9 (MMP-9), and <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 serum levels when compared with those who healed. Skin biopsy analysis showed that compared with control subjects, diabetic patients had increased immune cell infiltration, expression of MMP-9, and protein tyrosine phosphatase-1B (PTP1B), which negatively regulates the signaling of insulin, leptin, and <em>growth</em> <em>factors</em>. We conclude that increased inflammation, expression of MMP-9, PTP1B, and aberrant <em>growth</em> <em>factor</em> levels are the main <em>factors</em> associated with failure to heal DFUs. Targeting these <em>factors</em> may prove helpful in the management of DFUs.

Postnatal cardiac remodeling is characterized by a marked decrease in the insulin-like <em>growth</em> <em>factor</em> 1 (IGF1) and IGF1 receptor (IGF1R) expression. The underlying mechanism remains unexplored. This study examined the role of microRNAs in postnatal cardiac remodeling. By expression profiling, we observed a 10-fold increase in miR-378 expression in 1-week-old neonatal mouse hearts compared with <em>16</em>-day-old fetal hearts. There was also a 4-6-fold induction in expression of miR-378 in older (10 months) compared with younger (1 month) hearts. Interestingly, tissue distribution analysis identified miR-378 to be highly abundant in heart and skeletal muscles. In the heart, specific expression was observed in cardiac myocytes, which was inducible by a variety of stressors. Overexpression of miR-378 enhanced apoptosis of cardiomyocytes by direct targeting of IGF1R and reduced signaling in Akt cascade. The inhibition of miR-378 by its anti-miR protected cardiomyocytes against H(2)O(2) and hypoxia reoxygenation-induced cell death by promoting IGF1R expression and downstream Akt signaling cascade. Additionally, our data show that miR-378 expression is inhibited by IGF1 in cardiomyocytes. In tissues such as <em>fibroblasts</em> and fetal hearts, where IGF1 levels are high, we found either absent or significantly low miR-378 levels, suggesting an inverse relationship between these two <em>factors</em>. Our study identifies miR-378 as a new cardioabundant microRNA that targets IGF1R. We also demonstrate the existence of a negative feedback loop between miR-378, IGF1R, and IGF1 that is associated with postnatal cardiac remodeling and with the regulation of cardiomyocyte survival during stress.

METHODS

We collected the specimens of lumbar intervertebral disc (i.e., the symptomatic degenerative disc) from patients with discogenic low back pain to study the histopathologic features and growth factor expressions.

OBJECTIVE

To study the pathogenesis of disc degeneration, meanwhile discriminating between common disc degeneration (aging disc) (i.e., black asymptomatic disc, not clinically relevant) and painful disc degeneration (i.e., symptomatic disc, clinically relevant).

BACKGROUND

The pathogenesis of intervertebral disc degeneration is poorly understood, mainly because of the difficulty to establish the experimental model with good reproducibility. Recently, the popularity of spinal fusion leads to more opportunities to obtain disc specimens, which could be applied to explore the pathogenesis of disc degeneration with modern biologic techniques.

METHODS

There were 21 specimens of lumbar intervertebral discs from 15 patients with discogenic low back pain during posterior lumbar interbody fusion, 16 aging discs from patients without low back pain, and 10 normal discs as control collected for the study of their histopathologic features, as well as the expressions of basic fibroblast growth factor (bFGF) and its receptor (Flg), transforming growth factor-beta1 (TGF-beta1) and its receptor (TGF-betaRI) by immunohistochemistry. The distribution of macrophages and mast cells was also noted. Proliferating cell nuclear antigen was assessed to evaluate proliferating activities of disc cells.

RESULTS

The distinct histologic characteristic of the disc from the patient with discogenic low back pain was the ingrowth of vascularized granulation tissue along torn fissures, extending from the external layer of the anulus fibrosus into the nucleus pulposus. The immunohistochemical staining showed that there were strong expressions of bFGF and TGF-beta1 and their receptors, as well as a strong expression of proliferating cell nuclear antigen in the zones of granulation tissue in the painful discs. However, there were only weak expressions in the nongranulation tissue zones in the painful discs and aging discs, and no expression in the control discs. In addition, abundant macrophages and mast cells were found in the granulation tissue zones of painful discs but absent in the nongranulation tissue zones of painful discs or aging discs and the normal control discs.

CONCLUSIONS

The findings indicated that degeneration of the painful disc might originate from the injury and subsequent repair of anulus fibrosus. Growth factors, such as bFGF and TGF-beta1, macrophages and mast cells might play a key role in the repair of the injured anulus fibrosus and subsequent disc degeneration.

Bovine capillary endothelial (BCE) cells were incubated at 4 degrees C with 5 ng/ml 125I-basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) to equilibrate 125I-bFGF with high affinity cell surface receptors and low affinity matrix binding sites. 67% of the added 125I-bFGF bound to the matrix and 7% bound to receptors. The fate of bound bFGF was followed after cells were incubated in bFGF-free medium and were shifted to 37 degrees C to restore cell metabolism. 125I-bFGF bound to receptors decreased rapidly while the amount of 125I-bFGF bound to matrix was reduced more slowly. The rapid decrease in receptor-bound 125I-bFGF appeared to be due to a down-regulation of bFGF receptors; cells that had been treated for 5 h with bFGF had 60% fewer high affinity receptors than untreated cells. Despite the initial high level of 125I-bFGF binding to matrix, most of this 125I-bFGF was mobilized and metabolized by the cells. 125I-bFGF was internalized by the cells at 37 degrees C, leading to a constant accumulation of 125I-bFGF within the cell. Internalized bFGF was rapidly cleaved from an 18-kD form to a <em>16</em>-kD form. The <em>16</em>-kD form was more slowly degraded with a half-life of approximately 8 h. Degradation of internalized 125I-bFGF was inhibited by chloroquine, suggesting that the digestion occurred in a lysosomal compartment. The role of matrix binding sites in the internalization process was investigated. Binding to matrix sites seemed not to be directly involved in the internalization process, since addition of heparin at a concentration that blocked 95% of the binding to matrix had no effect on the initial rate of internalization of bFGF. BCE cells also released a substance that competed for the binding of bFGF to matrix but not to receptors. This substance bound to DEAE-cellulose and was sensitive to heparinase treatment, suggesting that it was a heparinlike molecule. Thus, heparinlike molecules produced by BCE cells can modulate the cellular interaction with bFGF. Matrix-associated heparinlike molecules bind bFGF which can later be metabolized by the cell, and secreted heparinlike molecules release bFGF from matrices.

OBJECTIVE

Preparations rich in growth factors (PRGF) release them plus bioactive proteins at localized sites, with the aim of triggering healing and regenerative processes. The prevailing paradigm suggests that their influence on proliferation, angiogenesis and the extracellular matrix synthesis is minimal. However, variations in their composition and impact on different cell phenotypes have not been examined.

METHODS

Sixteen fibroblast cultures obtained from three different anatomical sites (skin, synovium and tendon) of 16 donors were exposed to the molecular pool released from PRGF scaffolds, with increasing amounts of platelets. We evaluated cell proliferation, secretion of angiogenic growth factors (VEGF and HGF), synthesis of type I collagen and hyaluronic acid (HA), considering platelet dose and anatomical origin of the cells. Activity of transforming growth factor-beta (TGF-beta) in type I procollagen and HA synthesis was examined by adding exogenous TGF-beta to plasma preparations.

RESULTS

All plasma preparations induced a significant proliferative response compared to non-stimulated cells (P < 0.05). Maximum proliferation rate was obtained with PRGF with 2-fold or 4-fold platelet concentration. Exposure to PRGF stimulated VEGF synthesis exclusively in tendon cells (P < 0.05), which also exhibited a different pattern of HGF production (P < 0.05). PRGF enhanced HA synthesis (P < 0.05), but did not alter collagen I production. Platelet-secreted TGF-beta may be involved in HA, but not in type I procollagen synthesis.

CONCLUSIONS

Optimizing composition and use of platelet-rich products is crucial to enhancing the therapeutic potential of this technology. Our data show that the biological effects of PRGF may depend on concentration of platelets and on the anatomical source of the cells.

Chromosomal aberrations are known to have an impact on the initiation and progression of oral squamous cell carcinoma (OSCC), but individual genes involved in OSCC pathogenesis are poorly described. To elucidate the molecular events underlying oral carcinogenesis, a set of primary OSCC were screened for distinct genetic imbalances by means of array-based comparative genomic hybridisation. For this, a DNA array was used containing 812 genomic targets including oncogenes, tumour-suppressor genes and chromosomal regions frequently altered in human neoplasms. The most frequent aberrations were amplification of MYC, EGFR, CCND1 and PIK3CA, whereas deletions affected TRAILR1 and ATM. Furthermore, a distinct high-level amplification of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 (FGFR1) locus was detected in two cases. Detailed FISH analysis on OSCC tissue microarray sections revealed amplification prevalence for FGFR1 of 17.4% (<em>16</em>/92). Furthermore, FGFR1 protein analysis by immunohistochemistry on a TMA containing 178 OSCC found a high FGFR1 expression in tumours of early t-stadium and UICC stage (T1/2 vs. T3/4: p=0.002; SI-II vs. S III-IV: p=0.048). Our results indicate that an increase in FGFR1 expression contributes to oral carcinogenesis at an early stage of development.

Uterine <em>growth</em> <em>factors</em> are potential effector molecules in embryo <em>growth</em> signaling pathways. Pig uterine luminal flushings contained a heparin-binding <em>growth</em> <em>factor</em> (HBGF) that required 0.8 M NaCl for elution from heparin columns and was termed HBGF-0.8. This <em>factor</em>, which was heat- and acid-labile and of Mr 10,000 as assessed by gel filtration, stimulated DNA synthesis in <em>fibroblasts</em> and smooth muscle cells but not endothelial cells. Two forms of HBGF-0.8, termed HBGF-0.8-P1 and HBGF-0.8-P2, exhibited differential heparin-binding properties. SDS-polyacrylamide gel electrophoresis showed that each form of HBGF-0.8 migrated with an apparent Mr of 10, 000 under reducing conditions. Amino acid sequencing revealed the N-terminal sequence EENIKKGKKXIRTPKI for HBGF-0.8-P1 and ENIKKGKKXIRT for HBGF-0.8-P2. These sequences corresponded, respectively, to residues 247-262 and 248-259 of the 349-residue predicted primary translation product of porcine connective tissue <em>growth</em> <em>factor</em> (pCTGF). 10-kDa CTGF-mediated <em>fibroblast</em> DNA synthesis was modulated by exogenous heparin, and CTGF-immunoreactive proteins of 10, <em>16</em>, and 20 kDa were present in unfractionated uterine luminal flushings. These data reveal the identity of a novel <em>growth</em> <em>factor</em> in uterine fluids as a highly truncated form of CTGF and show that the N-terminal two-thirds of the CTGF primary translation product is not required for mitogenic activity or heparin binding.

To examine which <em>growth</em> <em>factors</em> correlate with neovascularization in human brain tumors, the mRNA levels of transforming <em>growth</em> <em>factor</em> alpha, transforming <em>growth</em> <em>factor</em> beta, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) genes were determined by a Northern blot analysis in surgically obtained human gliomas and meningiomas. The vascular development was determined by counting the number of microvessels which were immunostained with von Willebrand <em>factor</em>. We normalized the <em>growth</em> <em>factor</em> mRNA levels versus the glyceraldehyde phosphate dehydrogenase mRNA level. In the 17 gliomas and <em>16</em> meningiomas examined, the mRNA of transforming <em>growth</em> <em>factors</em> alpha and beta, basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, and VEGF were expressed at various levels. Among those 4 <em>growth</em> <em>factors</em>, the mRNA levels of VEGF, but not those of transforming <em>growth</em> <em>factors</em> alpha and beta and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, correlated significantly with vascularity in both gliomas (correlation coefficient r = 0.499; P < 0.05) and meningiomas (correlation coefficient r = 0.779; P < 0.001). These findings thus suggest that VEGF may be a positive <em>factor</em> in tumor angiogenesis in both human gliomas and meningiomas.

Syndecan-1 is a ubiquitous and multifunctional extracellular matrix proteoglycan,which mediates basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) binding and activity. Shedding of syndecan-1 ectodomain from the plasma membrane is highly regulated. We evaluated the influence of soluble syndecan-1 and serum bFGF determined by ELISA on outcome in 184 lung cancer patients (non-small cell lung cancer, n = 138; small cell lung cancer, n = 46). Serum syndecan-1 and bFGF levels were determined from sera taken before treatment. The median follow-up of the patients alive (n = 21) was 8.1 years (range, 6.6-8.9 years). High serum syndecan-1 and bFGF levels tended to occur in the same patients (P = 0.044). When the serum values corresponding to the highest tertile were used as the cutoff value, the median survival time of the patients with a high serum syndecan-1 level (>59 ng/ml) was 4 months [95% confidence interval (CI), 3-6 months] as compared with 11 months (9-<em>16</em> months) among those with lower serum levels (P = 0.0001), and the median survival time of the patients with a high bFGF level (>3.4 pg/ml) was 5 months (3-8 months) versus 11 months (8-14 months) in those with a lower level (P = 0.023). In general, the prognostic influence of both <em>factors</em> was independent of the histological subtype. Both serum syndecan-1 level (relative risk, 1.8; 95% CI, 1.1-3.1) and serum bFGF level (relative risk, 1.6; 95% CI, 1.0-2.7) had independent influence on survival in a multivariate survival analysis in non-small cell lung cancer. We conclude that high serum syndecan-1 and bFGF levels at diagnosis are associated with poor outcome in lung cancer.

OBJECTIVE

Nonalcoholic fatty liver disease is a common consequence of human and rodent obesity. Disruptions in lipid metabolism lead to accumulation of triglycerides and fatty acids, which can promote inflammation and fibrosis and lead to nonalcoholic steatohepatitis. Circulating levels of fibroblast growth factor (FGF)21 increase in patients with nonalcoholic fatty liver disease or nonalcoholic steatohepatitis; therefore, we assessed the role of FGF21 in the progression of murine fatty liver disease, independent of obesity, caused by methionine and choline deficiency.

METHODS

C57BL/6 wild-type and FGF21-knockout (FGF21-KO) mice were placed on methionine- and choline-deficient (MCD), high-fat, or control diets for 8-16 weeks. Mice were weighed, and serum and liver tissues were collected and analyzed for histology, levels of malondialdehyde and liver enzymes, gene expression, and lipid content.

RESULTS

The MCD diet increased hepatic levels of FGF21 messenger RNA more than 50-fold and serum levels 16-fold, compared with the control diet. FGF21-KO mice had more severe steatosis, fibrosis, inflammation, and peroxidative damage than wild-type C57BL/6 mice. FGF21-KO mice had reduced hepatic fatty acid activation and β-oxidation, resulting in increased levels of free fatty acid. FGF21-KO mice given continuous subcutaneous infusions of FGF21 for 4 weeks while on an MCD diet had reduced steatosis and peroxidative damage, compared with mice not receiving FGF21. The expression of genes that regulate inflammation and fibrosis were reduced in FGF21-KO mice given FGF21, similar to those of wild-type mice.

CONCLUSIONS

FGF21 regulates fatty acid activation and oxidation in livers of mice. In the absence of FGF21, accumulation of inactivated fatty acids results in lipotoxic damage and increased steatosis.

The E5 oncoprotein of bovine papillomavirus type 1 is a 44 amino acid, highly hydrophobic protein that induces the stable transformation of immortalized murine <em>fibroblasts</em>, presumably through its activation of <em>growth</em> <em>factor</em> receptors. Previous studies have shown that the E5 protein complexes with the <em>16</em> kDa (<em>16</em>k) pore-forming protein of vacuolar H(+)-ATPases. This integral membrane protein is essential for the acidification and function of subcellular compartments that process <em>growth</em> <em>factor</em> receptors. Using an SV40 expression system in COS cells, we analyzed whether the E5-<em>16</em>k complexes bind additional cellular proteins, including <em>growth</em> <em>factor</em> receptors. These studies demonstrate that E5 binds to both the <em>16</em>k protein and the PDGF receptor and that this tri-component complex can be isolated with antibodies specific for each protein. Importantly, the <em>16</em>k protein bound to the PDGF receptor in the absence of E5, suggesting that E5 binds to the PDGF receptor via its interaction with the <em>16</em>k protein. An E5 mutant lacking the hydrophilic carboxyl-terminal 14 amino acids retained binding to both <em>16</em>k and the PDGF receptor, indicating that E5 binds to these proteins through its hydrophobic, membrane-associating domain. These studies reveal that hydrophobic, intramembrane interactions govern the association of E5, <em>16</em>k and the PDGF receptor, suggesting a ligand-independent mechanism for receptor activation and a potential link between receptor signal transduction pathways and membrane pore activity.

OBJECTIVE

To determine whether the heterogeneous clinical response to tumour necrosis factor (TNF)alpha blocking therapy in rheumatoid arthritis (RA) can be predicted by TNFalpha expression in the synovium before initiation of treatment.

METHODS

Prior to initiation of infliximab treatment, arthroscopic synovial tissue biopsies were obtained from 143 patients with active RA. At week 16, clinical response was evaluated using the 28-joint Disease Activity Score (DAS28). Immunohistochemistry was used to analyse the cell infiltrate as well as the expression of various cytokines, adhesion molecules and growth factors. Stained sections were evaluated by digital image analysis. Student t tests were used to compare responders (decrease in DAS28>> or =1.2) with non-responders (decrease in DAS28 <1.2) and multivariable regression was used to identify the independent predictors of clinical response.

RESULTS

Synovial tissue analysis confirmed our hypothesis that the baseline level of TNFalpha expression is a significant predictor of response to TNFalpha blocking therapy. TNFalpha expression in the intimal lining layer and synovial sublining were significantly higher in responders than in non-responders (p = 0.047 and p = 0.008, respectively). The numbers of macrophages, macrophage subsets and T cells (all able to produce TNFalpha) were also significantly higher in responders than in non-responders. The expression of interleukin (IL)1beta, IL6, IL18, IL10, E-selectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was not associated with response to anti-TNFalpha treatment.

CONCLUSIONS

The effects of TNFalpha blockade are in part dependent on synovial TNFalpha expression and infiltration by TNFalpha producing inflammatory cells. Clinical response cannot be predicted completely, indicating involvement of other as yet unknown mechanisms.

Bile acids (BAs) are essential for fat absorption and appear to modulate glucose and energy metabolism. Colesevelam, a BA sequestrant, improves glycemic control in type 2 diabetes mellitus (T2DM). We aimed to characterize the alterations in BA metabolism associated with T2DM and colesevelam treatment and to establish whether metabolic consequences of T2DM and colesevelam are related to changes in BA metabolism. Male subjects with T2DM (n = <em>16</em>) and controls (n = 12) were matched for age and body mass index. BA pool sizes and synthesis/input rates were determined before and after 2 and 8 weeks of colesevelam treatment. T2DM subjects had higher cholic acid (CA) synthesis rate, higher deoxycholic acid (DCA) input rate, and enlarged DCA pool size. Colesevelam resulted in a preferential increase in CA synthesis in both groups. CA pool size was increased whereas chenodeoxycholic acid and DCA pool sizes were decreased upon treatment. Fasting and postprandial <em>fibroblast</em> <em>growth</em> <em>factor</em> 19 (FGF19) levels did not differ between controls and diabetics, but were decreased by treatment in both groups. Colesevelam treatment reduced hemoglobin A1C by 0.7% (P < 0.01) in diabetics. Yet, no relationships between BA kinetic parameters and changes in glucose metabolism were found in T2DM or with colesevelam treatment.

CONCLUSIONS

Our results reveal significant changes in BA metabolism in T2DM, particularly affecting CA and DCA. Colesevelam treatment reduced FGF19 signaling associated with increased BA synthesis, particularly of CA, and resulted in a more hydrophilic BA pool without altering total BA pool size. However, these changes could not be related to the improved glycemic control in T2DM.

Previous animal data showed that platelets contain <em>growth</em> <em>factors</em> that stimulate capillary endothelial migration (angiogenesis), <em>fibroblast</em> proliferation and migration, and collagen synthesis. This study utilized autologous platelet-derived wound healing <em>factors</em> (PDWHF) to treat 49 patients with chronic nonhealing cutaneous ulcers. Patients were classified on the basis of 20 clinical and wound status parameters to generate a wound severity index. Forty-nine patients--58% diabetic (20% with renal transplants); <em>16</em>% with trauma, vasculitis, etc.; 14% with decubitus ulcers; and 6% each with venous stasis or arterial insufficiency--with a total of 95 wounds had received conventional wound care for an average of 198 weeks (range: 1-1820 weeks). After informed consent was obtained, patients received autologous PDWHF. Mean 100% healing time for all patients was 10.6 weeks. There was no abnormal tissue formation, keloid, or hypertrophic scarring. A multivariant analysis showed a direct correlation to 100% healing with initial wound size and the initiation of PDWHF therapy. This is the first clinical demonstration that locally acting <em>growth</em> <em>factors</em> promote healing of chronic cutaneous ulcers.

Targeted gene studies have demonstrated the importance of insulin-like <em>growth</em> <em>factor</em>-I (IGF-I) for osteoblast (OB) differentiation and the acquisition of peak bone mineral density (BMD). The skeletal response to allelic differences in IGF-I expression can also be measured in vivo, using congenic mice. We created a congenic strain with reduced (approximately 20%) circulating IGF-I (C3H.B6-6T [6T]) by backcrossing a small genomic region (30 cM) of Chromosome 6 (Chr6) from C3H/HeJ (C3H) onto a C57Bl/6J (B6) background. 6T female mice have lower serum IGF-I (P<0.001 vs. B6) but similar <em>growth</em> hormone (GH) and serum IGF binding protein (IGFBP) concentrations as B6. At <em>16</em> weeks of age, congenics have greater body fat (P<0.02 vs. B6) despite less total body weight, and exhibit smaller femoral cross-sectional size (P=0.001), reduced cortical thickness (P<0.001) and lower trabecular BV/TV (P<0.05) than B6. 6T mice also have suppressed serum leptin (P<0.01), but compared to B6 have similar markers of bone resorption (i.e., urine CTx and serum TRAP 5B). At 8 weeks of age, skeletal IGF-I mRNA from long bones was reduced by 40% (P<0.05) as were liver mRNA transcripts (i.e., 50%, P<0.01). Osteoblast progenitors from the bone marrow of 6T mice formed less colony forming unit <em>fibroblasts</em> by crystal violet staining than B6 (P<0.007) and had significantly reduced alkaline phosphatase-positive colonies than B6(P<0.0001). In addition, staining of bone marrow with oil red O revealed greater numbers of adipocytes in 6T than B6. Several candidate genes in the Chr6 QTL were excluded by lack of strain-related expression differences in bone, but genes positively regulating adipocyte differentiation including Alox 5 and PPAR-gamma require further study as either "pathway" or candidate genes. In summary, allelic differences in a QTL on Chr6 result in altered IGF-I gene expression, changes in OB lineage allocation, and reduced peak bone mass. Congenic mice are useful models not only for mapping genes related to bone mass but also for elucidating the biology underlying various skeletal phenotypes associated with more subtle manipulation of the mouse genome.

BACKGROUND

Fibroblast growth factor 23 (FGF23) is a phosphaturic factor and a suppressor of 1alpha-hydroxylase activity in the kidney. Although its importance in chronic kidney disease (CKD) has been demonstrated in adults, there is little information in pediatric patients.

OBJECTIVE

The aims of this study were: 1) to determine reference values for FGF23 serum levels according to glomerular filtration rate (GFR) (measured by the reference standard, inulin clearance), gender, and age; and 2) to evaluate the effects of different etiologies and treatments on FGF23 serum levels in a prospective single-center cohort of 227 CKD children (119 boys).

RESULTS

Age, body weight, height, and GFR (mean +/- sd) values were: 11.3 +/- 4.1 yr, 37 +/- 16 kg, 140 +/- 20 cm, and 98 +/- 34 ml/min per 1.73 m(2), respectively. Calcium, phosphate, PTH, 25 hydroxyvitamin D, 1,25 dihydroxyvitamin D, C-terminal FGF23, and intact FGF23 (mean +/- sd) levels were: 2.43 +/- 0.11 mmol/liter, 1.41 +/- 0.22 mmol/liter, 41 +/- 23 pg/ml, 24 +/- 10 ng/ml, 152 +/- 72 pmol/liter, 76 +/- 134 relative units/ml, and 44 +/- 37 pg/ml, respectively. There was a wide range of FGF23 serum levels, but FGF23 levels increased when GFR decreased. FGF23 serum levels were not modified by gender, but they increased with age. In univariate analysis, corticosteroid therapy seemed to be associated with increased FGF23 serum levels. A multivariate linear regression analysis found a significant impact of GFR, body mass index, and solid organ transplantation on FGF23 serum levels.

CONCLUSIONS

Age, GFR, body mass index, and solid organ transplantation seem to influence FGF23 serum levels in a pediatric population. The impact of corticosteroids on FGF23 metabolism should be further investigated; further longitudinal studies will also help to better define the prognostic impact of FGF23 serum levels in pediatric CKD in terms of disease progression, cardiovascular morbidities, and bone disabilities.