OBJECTIVE

Thyroid hormones have important roles in normal <em>growth</em> and skeletal muscle development. IGF-I is one of the most important <em>growth</em> <em>factors</em> and is needed for the proliferation and development of thyroid cells. It stimulates <em>fibroblasts</em>, follicular and endothelia cells in thyroid gland. It has been shown that thyroid hormones play an important role in the regulation of IGF-I and IGFBP-3. In this study we proposed that IGF-I (CA)(<em>19</em>) and IGFBP-3-202 A/C gene polymorphism may affect thyroid functions. For this purpose, frequency of IGF-I (CA)(<em>19</em>) and IGFBP-3-202 A/C gene polymorphism in hypo- and hyperthyroid patients and possible role of these polymorphism in thyroid functions were investigated.

METHODS

This study was performed on 37 volunteer hyperthyroid and 76 hypothyroid patients as well as with 50 healthy subjects as controls. DNA isolation was applied in peripheral blood samples obtained from patients and controls. Required areas were amplified with PCR by using proper primers belonging to these gene areas from the isolated DNA samples. The products were evaluated with visualization by UV gel documentation system.

RESULTS

Frequency of IGF-I (CA)(<em>19</em>) gene polymorphism among hypothyroidism patients, hyperthyroidism patients and controls were statistically significant (chi(2) = 11.55, df = 4, p = 0.021). Genotypic variations between hyper- and hypothyroid patients were significant (chi(2) = 11.39, df = 2, p = 0.003), whereas there was no difference in IGF-I (CA)(<em>19</em>) gene polymorphism between the patients and controls. Differences in the IGFBP-3-202 A/C gene polymorphism between controls and hypo- as well as hyperthyroid patients were not significant. But IGFBP-3-202 A/C gene polymorphism genotype frequencies showed a significant difference between hypo- and hyperthyroid patients (chi(2) = 6.24, df = 2, p = 0.044).

CONCLUSIONS

These findings suggests that IGF-I (CA)(<em>19</em>) and IGFBP-3-202 A/C gene polymorphisms may be a risk factor for hypothyroidism.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR) pathway has been implicated both as an escape mechanism from anti-angiogenic therapy and as a driver oncogene in different tumor types. Lucitanib is a small molecule inhibitor of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) receptors 1 to 3 (VEGFR1 to 3), platelet derived <em>growth</em> <em>factor</em> α/β (PDGFRα/β) and FGFR1-3 tyrosine kinases and has demonstrated activity in a phase I/II clinical study, with objective RECIST responses in breast cancer patients with FGFR1 or FGF3/4/<em>19</em> gene amplification, as well as in patients anticipated to benefit from anti-angiogenic agents. We report here the in vitro and in vivo antitumor activity of lucitanib in experimental models with or without FGFR1/2 amplification or mutations. In cell assays, lucitanib potently inhibited the <em>growth</em> of tumor cell lines with amplified FGFR1 or mutated/amplified FGFR2. In all xenograft models studied, lucitanib demonstrated marked tumor <em>growth</em> inhibition due to potent inhibition of angiogenesis. Notably, in two lung cancer models with FGFR1 amplification, the antitumor efficacy was higher, suggesting that the simultaneous inhibition of VEGF and FGF receptors in FGFR1 dependent tumors can be therapeutically advantageous. Similar antitumor activity was observed in FGFR2 wild-type and amplified or mutated xenograft models. Pharmacokinetic studies showed lucitanib plasma concentrations in the micro/sub-micromolar range demonstrated drug accumulation following repeated lucitanib administration.

BACKGROUND

Jansen's metaphyseal chondrodysplasia (JMC) is a rare autosomal dominant disorder caused by activating mutations in the PTH 1 receptor (PTH1R; PTH/PTHrP receptor), leading to chronic hypercalcemia and hypercalciuria. Hypophosphatemia is also a hallmark of JMC, and recently, increased fibroblast growth factor 23 (FGF23) levels have been reported in this syndrome. Hypercalcemia has been associated with increased cardiovascular risk; however, cardiovascular disease has not been extensively investigated in JMC patients.

OBJECTIVE

The aim of the study was to describe the long-term follow-up of a JMC patient with regard to the management of hypercalciuria, the evaluation of FGF23 levels under bisphosphonate treatment, and the investigation of cardiovascular repercussion of chronic hypercalcemia.

RESULTS

The diagnosis of JCM was confirmed by molecular analysis (p.H223R mutation in PTH1R). The patient was followed from 5 to 27 yr of age. Asymptomatic nephrolithiasis was diagnosed at 18 yr of age, prompting pharmacological management of hypercalciuria. Treatment with alendronate reduced hypercalciuria; however, normocalciuria was only obtained with the association of thiazide diuretic. Serum FGF23 levels, measured under alendronate treatment, were repeatedly within the normal range. Subclinical cardiovascular disease was investigated when the patient was 26 yr old, after 19 yr of sustained mild hypercalcemia; carotid and vertebral artery ultrasonography was normal, as well as coronary computed tomography angiography (calcium score = 0).

CONCLUSIONS

The long-term follow-up of our JMC patient has provided insight on therapeutic strategies to control hypercalciuria, on the potential effects of alendronate on FGF23 levels, and on the lack of detectable cardiovascular disease at young adulthood after prolonged exposure to hypercalcemia.

Production of insulin-like <em>growth</em> <em>factor</em> I/somatomedin C (IGF-I) by <em>19</em>-day-gestation fetal rat lung <em>fibroblasts</em> was studied, and a paracrine (local production and action) mitogenic activity of this <em>growth</em> <em>factor</em> was explored. Specific IGF-I mRNAs were demonstrated in these cells, consistent with production of IGF-I. Using an IGF-I monoclonal antibody, IGF-I-like material was isolated from fetal lung <em>fibroblast</em> conditioned medium (FCM) and separated by molecular weight. Several molecular weight species were identified, including an 8,000 to 10,000 molecular weight species, a weight similar to purified IGF-I. IGF-I binding proteins elaborated by these cells were also demonstrated. The possibility of a paracrine mitogenic activity of the fetal lung <em>fibroblast</em>-produced IGF-I in cultures was suggested by demonstrating a reduction in DNA synthesis in cultures incubated with either the IGF-I monoclonal antibody or an IGF-I receptor antibody. These findings indicate that <em>19</em>-day-gestation fetal rat lung <em>fibroblasts</em> produce IGF-I that acts in a paracrine fashion and suggest that this <em>growth</em> <em>factor</em> is biologically active during fetal lung development.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a potent endothelial cell mitogen that does not normally circulate. Yet plasma bFGF-like bioactivity was increased in association with persistent microalbuminuria and retinopathy in adult type 2 diabetes mellitus. In the present study, we tested whether plasma bFGF immunoreactivity (IR) could predict the need for laser treatment of diabetic retinopathy in a baseline subset of advanced type 2 diabetes mellitus from the Veterans Affairs Diabetes Trial (mean: age, 59 years; diabetes duration, 11 years; baseline glycosylated hemoglobin, 9.5%). Plasma bFGF-IR was determined with a sensitive and specific 2-site enzyme-linked immunoassay in 172 patients at the baseline visit. Results were dichotomized at 4.5 pg/mL, the upper limit in healthy men. There was an unexpected significant association between low baseline plasma bFGF-IR level and the interim (4 years) need for laser treatment. First laser treatment was significantly more likely to be required in patients with low compared with high baseline bFGF (<em>19</em>% vs 6%, P = .03 for the difference). After adjusting for clinical risk <em>factors</em>, low vs high bFGF (hazard ratio [HR], 5.01; P = .012), duration of diabetes (HR, 1.05; P = .050), and low-density lipoprotein cholesterol concentration (HR, 0.98; P = .027) were all significantly associated with time to first laser occurrence. These and our prior results suggest that low plasma bFGF-IR may be a marker for the presence of anti-endothelial cell autoantibodies that may contribute to the need for laser photocoagulation treatment in adult men with advanced type 2 diabetes mellitus.

Progressive pulmonary fibrosis is a devastating consequence of many acute and chronic insults to the lung. Lung injury leads to alveolar epithelial cell (AEC) death, destruction of the basement membrane, and activation of transforming <em>growth</em> <em>factor</em>-β (TGF-β). There is subsequent resolution of the injury and a coordinated and concurrent initiation of fibrosis. Both of these processes may involve activation of similar intracellular signaling pathways regulated in part by dynamic changes to the extracellular matrix. Matrix signaling can augment the profibrotic <em>fibroblast</em> response to TGF-β. However, similar matrix/integrin signaling pathways may also be involved in the inhibition of ongoing TGF-β-induced AEC apoptosis. Focal adhesion kinase (FAK) is an integrin-associated signaling molecule expressed by many cell types. We used mice with AEC-specific FAK deletion to isolate the epithelial aspect of integrin signaling in the bleomycin model of lung injury and fibrosis. Mice with AEC-specific deletion of FAK did not exhibit spontaneous lung injury but did have significantly greater terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling-positive cells (18.6 vs. 7.1) per ×200 field, greater bronchoalveolar lavage protein (3.2 vs. 1.8 mg/ml), and significantly greater death (77 vs. <em>19</em>%) after bleomycin injury compared with littermate control mice. Within primary AECs, activated FAK directly associates with caspase-8 and inhibits activation of the caspase cascade resulting in less apoptosis in response to TGF-β. Our studies support a model in which dynamic changes to the extracellular matrix after injury promote <em>fibroblast</em> activation and inhibition of epithelial cell apoptosis in response to TGF-β through FAK activation potentially complicating attempts to nonspecifically target this pathway for antifibrotic therapy.

To compare the levels of cytokines and growth factor in aqueous humor of the patients with chronic primary angle closure glaucoma (PACG) and cataract.

Aqueous humor samples were collected from 19 chronic PACG patients and compared with 14 nonglaucomatous controls presenting for cataract surgery. The levels of 27 cytokines and growth factors were measured in the aqueous samples using multiplex bead immunoassay and compared across groups.

Significantly higher levels of interleukin (IL)-8 (p < 0.001), eotaxin (p < 0.001), interferon gamma-induced protein (IP)-10 (p < 0.001) and macrophage inflammatory protein-1-beta (MIP-1β; p < 0.001) were observed in aqueous of chronic PACG patients compared to controls. In comparison to controls, significantly lower levels of IL-9 (p = 0.001), IL-17 (p < 0.001), tumor necrosis factor-alpha (TNF-α; p < 0.001), granulocyte-macrophage colony-stimulating factor (GM-CSF; p < 0.001), and IL-5 (p = 0.001) were observed in chronic PACG eyes. All other assayed cytokines-IL-1β, interleukin-1 receptor antagonist (IL-1rα), IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, fibroblast growth factor-basic (FGF-basic), granulocyte colony-stimulating factor (G-CSF), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1-alpha (MIP-1α), and vascular endothelial growth factor (VEGF) -showed no significant difference between the groups.

These results suggest that the aqueous cytokine levels of chronic PACG eyes differ significantly from nonglaucomatous eyes. This is the first study reporting significantly increased levels of eotaxin, MIP-1β, and IP-10 and lower levels of TNF-α, IL-5, IL-9, IL-17, and GM-CSF in chronic PACG patients, suggesting a plausible role of these inflammatory cytokines in its pathogenesis.

Critical regulation of bile acid (BA) pool size and composition occurs via an intensive molecular crosstalk between the liver and gut, orchestrated by the combined actions of the nuclear Farnesoid X receptor (FXR) and the enterokine <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) with the final aim of reducing hepatic BA synthesis in a negative feedback fashion. Disruption of BA homeostasis with increased hepatic BA toxic levels leads to higher incidence of hepatocellular carcinoma (HCC). While native FGF<em>19</em> has anti-cholestatic and anti-fibrotic activity in the liver, it retains peculiar pro-tumorigenic actions. Thus, novel analogues have been generated to avoid tumorigenic capacity and maintain BA metabolic action. Here, using BA related Abcb4-/- and Fxr-/- mouse models of spontaneous hepatic fibrosis and HCC, we explored the role of a novel engineered variant of FGF<em>19</em> protein, called FGF<em>19</em>-M52, which fully retains BA regulatory activity but is devoid of the pro-tumoral activity. Expression of the BA synthesis rate-limiting enzyme Cyp7a1 is reduced in FGF<em>19</em>-M52-treated mice compared to the GFP-treated control group with consequent reduction of BA pool and hepatic concentration. Treatment with the non-tumorigenic FGF<em>19</em>-M52 strongly protects Abcb4-/- and Fxr-/- mice from spontaneous hepatic fibrosis, cellular proliferation and HCC formation in terms of tumor number and size, with significant reduction of biochemical parameters of liver damage and reduced expression of several genes driving the proliferative and inflammatory hepatic scenario. Our data bona fide suggest the therapeutic potential of targeting the FXR-FGF<em>19</em> axis to reduce hepatic BA synthesis in the control of BA-associated risk of fibrosis and hepatocarcinoma development.

It has been proposed that bile acid suppression of CYP7A1 gene expression is mediated through a gut-liver signaling pathway <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)15/<em>19</em>-<em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 which is initiated by activation of farnesoid X receptor in the ileum but not in the liver. This study evaluated whether FGF15/<em>19</em> protein levels in the portal blood reflected changes in FGF15/<em>19</em> mRNA in the ileum. Studies were conducted in Sprague Dawley rats and New Zealand white rabbits fed regular chow (controls), supplemented with cholesterol (Ch) or cholic acid (CA). After feeding CA, ileal FGF15 mRNA increased 8.5-fold in rats and FGF<em>19</em> rose 16-fold in rabbits associated with 62 and 75% reduction of CYP7A1 mRNA, respectively. Neither FGF15 nor FGF<em>19</em> protein levels changed in the portal blood to correspond with the marked increase of FGF15/<em>19</em> mRNA levels in the ileum or inhibited CYP7A1 expression in the liver. Further, in Ch-fed rats, CYP7A1 mRNA increased 1.9-fold (P < 0.001) although FGF15 mRNA levels in the ileum and portal blood FGF15 protein levels were not decreased. In Ch-fed rabbits, although FGF<em>19</em> mRNA levels in the ileum and liver did not increase significantly, CYP7A1 mRNA declined 49% (P < 0.05). We were unable to find corresponding changes of FGF15/<em>19</em> protein levels in the portal blood in rats and rabbits where the mRNA levels of FGF15/<em>19</em> in the ileum and CYP7A1 in the liver change significantly.

A number of cytokines and <em>growth</em> <em>factors</em> are known to modulate proliferation and differentiation of human endometrium. In this study, the expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF), basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), and VEGF receptors, fms-like tyrosine kinase (Flt1) and kinase insert domain-containing region (KDR), and bFGF receptor 1 (Flg) were examined in the endometrium of rhesus monkey on Day 5, 10, 16, 20, 25 of menstrual cycle and on Day <em>19</em> of early pregnancy. Western blot analysis showed the specificity of the anti-human antibodies with the monkey tissue. The expression of mRNA and protein of VEGF was correlated with that of its receptor KDR, which was detected in epithelial, vascular, and myometrial cells. The localization of bFGF and its receptor Flg was similar to that of VEGF, except that the Flg was absent in the endothelial cells. Strong expression of VEGF and bFGF in the glandular epithelial cells was observed in the proliferative phase, declined in the secretory phase during the cycle. Stronger staining of these <em>factors</em> was also observed in the decidual cells of the pregnant uterus, as compared with the stromal cells of cycling uterus. No expression of Flt1 was detected in the tissue examined in this study. These data suggest that VEGF, bFGF, and their receptors play important roles in epithelial and stromal development, angiogenesis, and blood vessel function in the endometrium during the menstrual cycle and early pregnancy of the rhesus monkey.

Smooth muscle myosin light chain kinase (MLCK) is a multifunctional molecule composed of an N-terminal actin binding domain, a central kinase domain, and C-terminal calmodulin- and myosin-binding domains. We previously cloned and characterized a novel MLCK isoform from endothelial cells (EC MLCK) consisting of 1,914 amino acids displaying a higher molecular weight (210 kDa) and a novel-amino-terminal stretch of 922 amino acids not shared by the smooth muscle isoform (smMLCK, 150 kDa). To further define the role of specific EC MLCK motifs in endothelial and non-muscle cells, we constructed two epitope-tagged EC MLCK deletion mutants in mammalian expression vectors lacking either the C-terminal auto-inhibitory and calmodulin-binding domain (EC MLCK1745) or the ATP-binding site (EC MLCKATPdel). Expression of EC MLCK1745 in CV1 <em>fibroblasts</em> showed increased basal actin stress fiber formation, which was markedly enhanced after tumor necrosis <em>factor</em> (TNF-alpha) or thrombin treatment. Distribution of EC MLCK1745 was largely confined to stress fibers, cortical actin filaments, and focal adhesion contacts, and co-localized with myosin light chains (MLCs) diphosphorylated on Ser(<em>19</em>) and Thr(18). In contrast, immunofluorescence staining demonstrated that EC MLCKATPdel abolished thrombin- and TNFalpha-induced stress fiber formation and MLC phosphorylation, suggesting this kinase-dead mutant functions as a dominant-negative MLCK construct, thereby confirming the role of EC MLCK in stress fiber formation. Finally, we compared the serum-stimulated <em>growth</em> rate of mutant MLCK-transfected <em>fibroblasts</em> to sham controls, and found EC MLCK1745 to augment thymidine incorporation whereas EC MLCKATPdel reduced CV1 <em>growth</em> rates. These data demonstrate the necessary role for MLCK in driving the contractile apparatus via MLC phosphorylation, which can alter <em>fibroblast</em> <em>growth</em> and contractility.

The 3T3-L1 cell line is a preadipocyte cell line derived from the Swiss 3T3 mouse <em>fibroblast</em> cell line. We have compared the effect of 3T3-L1 conditioned medium (3T3-L1 CM) and Swiss 3T3 conditioned medium (3T3 CM) on the <em>growth</em> of normal mouse mammary cells (NMMG) and the human MCF-7 breast carcinoma cell line. 3T3 CM increased the <em>growth</em> of both NMMG and MCF-7 cells by <em>19</em> +/- 2% (SD) and 24 +/- 3%, respectively, and increased thymidine incorporation by 74 +/- 4% and 104 +/- 8%, respectively. Conditioned medium from 3T3-L1 cells stimulated the <em>growth</em> of NMMG cells by 64 +/- 2%; in contrast, 3T3-L1 CM inhibited the <em>growth</em> of MCF-7 cells by 36 +/- 1%. In parallel with these <em>growth</em> studies, thymidine incorporation increased by 20 +/- 4% in NMMG cells and decreased by 72 +/- 5% in the MCF-7 cells. Moreover, a similar effect was also noted in NCI H630 colon cancer cells, where 3T3-L1 CM produced a 58 +/- 4% decrease in <em>growth</em> and a 82 +/- 6% decrease in thymidine incorporation. Heating the 3T3-L1 CM at 100 degrees C for 30 min destroyed all inhibitory activity. Several known inhibitory <em>growth</em> <em>factors</em> (<em>fibroblast</em> <em>growth</em> <em>factor</em>, 20 ng/ml; interleukin 6, 1000 units/ml; tumor necrosis <em>factor</em> alpha, 15 ng/ml; transforming <em>growth</em> <em>factor</em> beta, 1 ng/ml) were tested for activity in the MCF-7 cells. Tumor necrosis <em>factor</em> alpha and transforming <em>growth</em> <em>factor</em> beta produced a 97% and 67% inhibition of thymidine uptake, respectively, whereas interleukin 6 and <em>fibroblast</em> <em>growth</em> <em>factor</em> had no effect. Neither transforming <em>growth</em> <em>factor</em> beta nor tumor necrosis <em>factor</em> alpha activity was detectable in 3T3-L1 CM using an enzyme-linked immunosorbent assay. High-performance liquid chromatography fractionation of the 3T3-L1 CM revealed that the inhibitory activity eluted at a molecular weight of 67,000; moreover, silver staining of these eluates on a denaturing polyacrylamide gel revealed that M(r) 69,000 peptide was the predominant protein band in the inhibitory fractions. Thus 3T3-L1 CM stimulates the <em>growth</em> of normal breast epithelial cells and inhibits the <em>growth</em> of MCF-7 breast cancer cells. This inhibitory activity appears to be due to a protein secreted by 3T3-L1 preadipocytes.

OBJECTIVE

We have developed a technique for biologic coronary artery bypass grafting, which is a revival of a classic concept with modern biotechnology.

METHODS

Acute myocardial infarction was created by ligating the major branch of the circumflex artery in rabbits. Animals were divided into four groups: a nontreated group (group N), a group in which omentum was used to wrap the infarcted area (group G), a group in which a gelatin hydrogel sheet incorporating 100 microg basic fibroblast growth factor was placed over the infarcted area (group F), and a group in which the infarcted area was similarly treated with basic fibroblast growth factor followed by omental wrapping (group FG). Cardiac function was subsequently assessed by echocardiography. Postmortem angiography through the gastroepiploic artery was done in groups G and FG. Infarct size and arteriolar density were evaluated.

RESULTS

Group FG showed a better fractional area change than did the other groups (group N P <.001, group G P =.002, group F P <.001). Angiography revealed that communication from the gastroepiploic artery to the coronary artery was created through a rich bed of neovascularization in all 7 animals of group FG, whereas poor collaterals were recognized in only 2 of 7 animals in group G. Infarct size was reduced to a greater extent in group FG than in groups F, G, and N (10% +/- 3%, 16% +/- 5%, 19% +/- 7%, 23% +/- 2%, respectively, group F P =.04, groups G and N P <.01). The number of arterioles 20 to 100 microm in diameter was increased to a greater extent in group FG than in groups F, G, and N (23 +/- 5 arterioles/mm(2), 14 +/- 3 arterioles/mm(2), 10 +/- 1 arterioles/mm(2), 4 +/- 2 arterioles/mm(2), respectively), with the differences being significant.

CONCLUSIONS

These results show that bypass from the gastroepiploic artery to coronary arteries can be achieved without surgical anastomosis through slow release of basic fibroblast growth factor in this rabbit acute myocardial infarction model. This new revascularization concept, biologic coronary artery bypass grafting, could be applicable for revascularizing many tiny coronary vessels in patients who are difficult to treat with conventional surgery or catheter intervention.

It was investigated whether cryopreserved rat liver epithelial cells (RLEC) from biliary origin are capable of undergoing hepatic differentiation upon sequential exposure to liver-specific <em>factors</em> (<em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-4, hepatocyte <em>growth</em> <em>factor</em> (HGF), insulin-transferrin-sodium-selenite (ITS), dexamethasone (Dex) and oncostatin M (OSM)), reflecting liver embryogenesis in vivo. As differentiation progressed, cells acquired a hepatic morphology (polygonal-to-cuboidal shaped, binucleated cells), corresponding well with the phenotypic changes observed. Biliary cytokeratin (CK)<em>19</em> and connexin (Cx)43-expression both gradually decreased; CK<em>19</em>-expression disappeared even completely. In contrast, hepatic CK18-expression persisted throughout the culture time. Hepatocyte nuclear <em>factor</em> (HNF)3beta, alpha-foetoprotein (AFP), transthyretin (TTR), HNF4, albumin (ALB), HNF1alpha, multidrug resistance protein (MRP)2 and Cx32 were expressed at specific stages during RLEC-differentiation, thereby showing a progressive hepatic maturation. Indeed, immature AFP and mature ALB were sequentially expressed, in line with the in vivo liver embryogenesis. Expression of the early and mid-late 'liver-enriched' transcription <em>factors</em> (LETF) HNF3beta and HNF4 declined and translocated to the cytosol, respectively, while the late LETF HNF1alpha underwent a nuclear upregulation. In conclusion, RLEC are bipotent cells, capable of differentiation into immature hepatocytes in a hepatic-stimulating micro-environment. The robustness of the sequential conditions, developed before for hepatic 'transdifferentiation' of rat bone marrow stem cells (rBMSC), was hereby confirmed.

OBJECTIVE

To investigate the expression and function of fibroblast growth factor-inducible 14 (Fn14) in human retinal pigment epithelial cells.

METHODS

A human retinal pigment epithelial cell line (RPE cells: ARPE-19) was used. Expression of Fn14 protein was assessed by flow cytometry. An antibody array and ELISA were used to detect chemokines and cytokines in the supernatant of RPE cells cultured with or without stimulation by TWEAK and/or TGF-beta(1). To explore the mechanism by which TWEAK stimulates RPE cells, we investigated phosphorylation of MAP kinase in TWEAK-stimulated cells. We also investigated whether TWEAK induced the migration of RPE cells by performing an in vitro wound assay.

RESULTS

RPE cells showed constitutive surface expression of Fn14 protein. FGF, VEGF, and TGF-beta(1) did not induce Fn14 expression by RPE cells. TWEAK increased the production of IL-8 and MCP-1 by RPE cells via Fn14, and TGF-beta(1) augmented TWEAK-induced production of these chemokines. TWEAK induced the phosphorylation of MAP kinase in RPE cells and promoted the migration of these cells via MAP kinase.

CONCLUSIONS

TWEAK/Fn14 interaction may have proinflammatory effects in RPE cells.

OBJECTIVE

Elevated serum phosphate concentrations are established risk factors for cardiovascular disease and mortality in chronic kidney disease (CKD). Independent associations of other indices of phosphorus metabolism, such as phosphorus intake, urinary phosphate excretion, or hormones that regulate these systems, like fibroblast growth factor 23 (FGF23), with markers of cardiovascular disease in CKD, have been studied in less detail.

METHODS

Cross-sectional study.

METHODS

Seventy-four adult CKD patients with mean creatinine clearance of 51 ± 19 mL/minute.

RESULTS

Augmentation index (AI)--a surrogate marker of arterial stiffness.

RESULTS

Although serum phosphate varied little across quartiles of creatinine clearance, average daily phosphorus intake and 24-hour urinary phosphate excretion decreased from highest to lowest quartile (by 31% and 60%, respectively, P for trend <.05). FGF23 was associated with serum phosphate (r = 0.24, P = .03) and creatinine clearance (r = -0.4, P = .001), but not with dietary phosphorus or 24-hour urinary phosphate excretion (P>> .05 for both). Older age, higher systolic blood pressure, female gender, and black race were independently associated with increased AI. In contrast, there were no associations of serum phosphate, dietary phosphorus intake, urinary phosphate excretion, or FGF23 with AI in multivariate-adjusted models.

CONCLUSIONS

In this sample of patients with CKD, established risk factors for arterial stiffness, but not mediators of phosphorus metabolism, were associated with increased AI. In addition, there were no significant associations between FGF23 and dietary phosphorus or urinary phosphate excretion. Future studies are needed to determine the main factors associated with elevations in FGF23 in CKD and to further assess the association of disordered phosphorus metabolism with subclinical markers of vascular disease.

OBJECTIVE

This study investigated the metabolism and excretion of dovitinib (TKI258), a tyrosine kinase inhibitor that inhibits fibroblast, vascular endothelial, and platelet-derived growth factor receptors, in patients with advanced solid tumors.

METHODS

Four patients (cohort 1) received a single 500 mg oral dose of (14)C-dovitinib, followed by the collection of blood, urine, and feces for ≤10 days. Radioactivity concentrations were measured by liquid scintillation counting and plasma concentrations of dovitinib by liquid chromatography-tandem mass spectrometry. Both techniques were applied for metabolite profiling and identification. A continuous-dosing extension phase (nonlabeled dovitinib 400 mg daily) was conducted with the 3 patients from cohort 1 and 9 additional patients from cohort 2.

RESULTS

The majority of radioactivity was recovered in feces (mean 61 %; range 52-69 %), as compared with urine (mean 16 %; range 13-21 %). Only 6-19 % of the radioactivity was recovered in feces as unchanged dovitinib, suggesting high oral absorption. (14)C-dovitinib was eliminated predominantly via oxidative metabolism, with prominent primary biotransformations including hydroxylation on the fluorobenzyl ring and N-oxidation and carbon oxidation on the methylpiperazine moiety. Dovitinib was the most prominent radioactive component in plasma. The high apparent volume of distribution (2,160 L) may indicate that dovitinib distributes extensively to tissues. Adverse events were predominantly mild to moderate, and most common events included nausea, vomiting, constipation, diarrhea, and fatigue.

CONCLUSIONS

Dovitinib was well absorbed, extensively distributed, and eliminated mainly by oxidative metabolism, followed by excretion, predominantly in feces. The adverse events were as expected for this class of drug.

OBJECTIVE

To investigate the efficacy of basic fibroblast growth factor (bFGF) combined with topical oxygen therapy for deep II degree burn wounds, by comparing the effects of bFGF combined with topical oxygen therapy and bFGF with routine therapy.

METHODS

From February 2004 to July 2009, 85 patients with deep II degree burn wounds (117 wounds) were enrolled and divided into 4 groups randomly according to different treatments. There was no significant difference in sex, age, disease course, wound size, and wound treatment size among 4 groups (P>> 0.05). In group A, 18 patients (28 wounds) were treated routinely; in group B, 23 patients (30 wounds) were treated with routine methods and topical oxygen therapy; in group C, 19 patients (25 wounds) were treated with routine methods and bFGF therapy; and in group D, 25 patients (34 wounds) were treated with routine methods and bFGF/topical oxygen therapy. Topical oxygen therapy was administered to the wound for 90 minutes per day for 3 weeks. The bFGF therapy was applied everyday (150 U/cm2) for 3 weeks.

RESULTS

All cases were followed up 6-12 months (9 months on average). The wound healing times in groups A, B, C, and D were (27.3 +/- 6.6), (24.2 +/- 5.8), (22.2 +/- 6.8), and (18.2 +/- 4.8) days, respectively; showing significant difference between group A and group D (P < 0.05). The wound healing rates in groups A, B, C, and D were 67.8% +/- 12.1%, 85.1% +/- 7.5%, 89.2% +/- 8.3%, and 96.1% +/- 5.6%, respectively; showing significant differences between group A and groups B, C, D (P < 0.05). The therapic effective rates in groups A, B, C, and D were 75%, 90%, 92%, and 100%, respectively; showing significant difference between group A and group D (P < 0.05). The Vancouver scar scale scoring of group D 6 months after treatment was better than that of group A (P < 0.05).

CONCLUSIONS

The bFGF combined with topical oxygen therapy can enhance deep II degree burn wound healing. Furthermore, the therapy method is simple and convenient.

Gingival <em>fibroblasts</em> actively accumulate tetracyclines, thereby enhancing their redistribution from blood to gingiva. Since <em>growth</em> <em>factors</em> and pro-inflammatory cytokines regulate many <em>fibroblast</em> activities, they could potentially enhance <em>fibroblast</em> minocycline accumulation. To test this hypothesis, we treated gingival <em>fibroblast</em> monolayers for 1 or 6 hours with platelet-derived <em>growth</em> <em>factor</em>-BB (PDGF), <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF), transforming <em>growth</em> <em>factor</em>-beta1 (TGF), or tumor necrosis <em>factor</em>-alpha (TNF). Minocycline uptake was assayed at 37 degrees by a fluorescence method. All 4 <em>factors</em> significantly enhanced minocycline uptake (P < or = 0.008, ANOVA), primarily by increasing the affinity of transport. Treatment for 6 hours with 10 ng/mL FGF, PDGF, TGF, or TNF enhanced <em>fibroblast</em> minocycline uptake by <em>19</em>% to 25%. Phorbol myristate acetate enhanced <em>fibroblast</em> minocycline uptake by 28%, suggesting that protein kinase C plays a role in up-regulating transport. These effects on transport provide a mechanism by which systemic tetracyclines could be preferentially distributed to gingival wound or inflammatory sites.

The development of the fetal lung is regulated by <em>fibroblast</em>-type-II cell communications which involve <em>fibroblast</em> pneumonocyte <em>factor</em> (FPF). FPF production is positively regulated by glucocorticoids and negatively regulated by dihydrotestosterone (DHT) and transforming <em>growth</em>-<em>factor</em> beta (TGF-beta). We studied whether DHT or TGF-beta affected other steps in the process of lung maturation, by studying how the developing lung in organ culture would respond to exogenously supplied FPF after DHT or TGF-beta exposure. Fetal rabbit (day <em>19</em> of gestation) lung organ cultures were prepared and cultured in the presence of cortisol, DHT or TGF-beta. After seven days, the media were replaced with serum-free medium containing either cortisol or FPF conditioned medium. The incorporation of [14C]glycerol into surfactant lamellar body DSPC was studied over 24 h as the index of surfactant synthesis. Results were compared to simultaneous control cultures. Treatment had no significant effect on tissue protein concentration or on the efficiency of lamellar body recovery. Cortisol stimulated baseline incorporation of glycerol into DSPC. This was inhibited by DHT, such that DHT plus cortisol treatment was no different from untreated controls. FPF stimulated the incorporation of glycerol into DSPC, and did so even after culture treatment with DHT. Cultures treated with TGF-beta exhibited glycerol incorporation similar to untreated controls. After TGF-beta exposure, FPF did not stimulate glycerol incorporation into DSPC. We conclude that DHT interferes with progression of lung development by delaying the appearance of FPF production by the <em>fibroblast</em>. TGF-beta, on the other hand, inhibits other elements of lung maturation besides FPF production. We speculate that TGF-beta interferes with type-II cell development such that the cell cannot respond to FPF.

Keloid disease (KD) is a fibroproliferative disorder of unknown etiology. Current use of corticosteroid injection is partially beneficial with 80% recurrence rate. Additionally, the efficacy of different steroids, alone or in combination as opposed to monotherapy, in treating KD remains unclear. Here, we compared the single and combined efficacy of glucocorticoids-dexamethasone (Dex), triamcinolone (TAC), and methylprednisolone (Medrol)-on primary keloid <em>fibroblasts</em> (KFs) (n = 27) and normal skin (n = <em>19</em>) <em>fibroblasts</em> at cellular, protein, and messenger RNA levels in vitro. Our results demonstrated that cytotoxicity to steroids was dose dependent. Cell spreading, attachment, and proliferation were significantly (p < 0.05) reduced by Medrol and TAC. Migration and invasion properties of KF were inhibited significantly (p < 0.05) by Medrol and TAC compared with Dex. At both protein and messenger RNA levels, keloid-associated fibrotic markers were significantly (p < 0.05) decreased by Medrol and TAC compared with Dex. However, vascular endothelial <em>growth</em> <em>factor</em> expression was significantly (p = 0.01) decreased by Dex compared with TAC and Medrol. Medrol and TAC caused significant (p < 0.04) apoptosis, whereas Dex inhibited the UV-induced apoptosis and up-regulated survivin. Blocking of glucocorticoid receptor by RU486 inhibited cytoprotective property of Dex and apoptotic properties of TAC and Medrol. Double treatment with Dex + TAC and Dex + Medrol significantly (p < 0.05) induced apoptosis. In conclusion, this is the first study to report the efficacy of three well-known steroids on KF and suggest that combination may be superior than using a single steroid in treating KD.

Keratoacanthomas are rapidly <em>growing</em> hyperproliferative skin tumors that may clinically or histologically be difficult to distinguish from well-differentiated squamous cell cancers (SCCs). UV light, trauma, and immune suppression represent their etiological <em>factors</em>. As matrix metalloproteinases (MMPs) are implicated at all stages of tumorigenesis, we investigated the expression profile of several cancer-related MMPs to find markers that would differentiate keratoacanthomas from SCCs and shed light to the pathobiology of keratoacanthoma. Samples from 31 keratoacanthomas and 15 grade I SCCs were studied using immunohistochemistry for MMP-2, -7, -8, -9, -10, -13, and -<em>19</em> and p16 and laminin-5gamma2 chain. In situ hybridization for MMP-7, -10, and -13 was performed in a subset of tumors. Keratinocytes with atypia, presence of neovascularization, and composition of the inflammatory infiltrate were graded from hematoxylin-eosin stainings. MMP-7 was present in the epithelium of 4/31 keratoacanthomas and 9/15 SCCs, MMP-8 in 3/30 keratoacanthomas and 0/15 SCCs, but MMP-13 in 16/31 keratoacanthomas and 10/15 SCCs, and MMP-10 in 28/31 keratoacanthomas and all cancers. MMP-9 was detected in the epithelium in 5/31 keratoacanthomas and 8/15 SCCs, whereas MMP-2 was only present in <em>fibroblasts</em> in both tumors. MMP-<em>19</em> was upregulated in proliferating epithelium of keratoacanthomas as was p16. Cytoplasmic laminin-5gamma2 was particularly abundant in keratinocytes at the pushing border of MMP-13-positive keratoacanthomas. We conclude that although some MMPs (MMP-10 and -13) are abundantly expressed in keratoacanthomas, the presence of MMP-7 and -9 in their epithelial pushing border is rare and should raise suspicion of SCC. Further, the loss of MMP-<em>19</em> and p16 could aid in making the differential diagnosis between well-differentiated SCC and keratoacanthoma. Frequent expression of the transformation-specific MMP-13 in keratoacanthomas suggests that they are not benign tumors but incomplete SCCs.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 1 and 3 (FGFR1, FGFR3) impact on tissue homoeostasis, embryonic development and carcinogenesis. Murine double minute protein 4 (MDM4) and mouse double minute 2 homologue (MDM2) are regulators of p53-protein and may be the origin of an apoptosis overpowering cascade. A collective of 266 carcinomas of salivary glands were investigated for MDM2, MDM4, FGFR1 and FGFR3 aberrations by fluorescence in situ hybridization (FISH). The results were matched with clinicopathological parameters and with expression of PTEN and p53. MDM2 gene amplification (n = 9) and chromosomal aberrations (trisomy, n = 47; high polysomy, n = 7) are linked to high-grade malignancy (P < 0.001), lymph node metastasis (P = 0.001), advanced tumour size (P = 0.013) and stage (P < 0.001), gender (P = 0.002) and age (P = 0.001). MDM4 gene amplification (n = <em>19</em>) and chromosomal aberrations (trisomy, n = 34; high polysomy, n = 31) are correlated to high-grade malignancy (P < 0.001), lymph node metastasis (P = 0.008), advanced tumour size (P = 0.039), stage (P = 0.004) and loss of PTEN (P < 0.001). Only, high-grade malignancy (P < 0.001), lymph node metastasis (P = 0.036) and advanced tumour stage (P = 0.025) are associated with FGFR3 amplification (n = 1) or chromosomal aberrations (low polysomy, n = 61; high polysomy, n = 55) but not with MDM4 alterations. FGFR1 amplifications (n = 5) and chromosomal aberrations (trisomy, n = 38; high polysomy, n = 30) are associated with high-grade malignancy (P < 0.001), advanced tumour size (P = 0.026) and stage (P = 0.004), gender (P = 0.016) and age (P = 0.023). Aberrations of MDM2, MDM4, FGFR1 and FGFR3 correlate with aggressive tumour <em>growth</em> and nodal metastasis. MDM2 (P < 0.001), MDM4 (P = 0.005) and FGFR3 (P = 0.006) alterations are associated with worse overall survival of patients with salivary gland cancer.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is a potent tumor angiogenesis <em>factor</em> which lacks an amino-terminal signal sequence and does not normally circulate in serum from normal subjects. Naturally-occurring autoantibodies which mimicked basic <em>fibroblast</em> <em>growth</em> <em>factor</em> were described in serum from patients with multiple endocrine neoplasia type 1 prolactinoma or sporadic <em>growth</em>-hormone-secreting adenoma associated with increased bFGF. Since bFGF was increased in serum from a variety of cancers, we used endothelial cell proliferation assay(s) to test for bioactivity in the IgG fraction of serum from 56 patients with cancer-associated hypercalcemia, and normal or control subjects. We now report increased IgG-like endothelial cell activity in serum from a hyper prolactinemic subset (4/<em>19</em> breast cancer; 1/14 renal cancer; 0/23 lung cancer) of cancer-associated hypercalcemic subjects. Highest activity was found in serum from three breast cancer patients who suffered spinal cord compression/metastases. The activity had properties of antiidiotype bFGF antibodies including reaction with anti-human IgG antibodies, and complete neutralization by rabbit antibodies to intact bFGF. The activity in endothelial cells persisted after storage at 0-4 C for 5 yrs; and [prepared by SDS-PAGE and immunoblotting with anti-human IgG] had apparent mol wt corresponding to the heavy chains of IgG. Serum IgG-like activity from 5 of 5 breast cancer patients and 2 of 2 prostate cancer subjects tested [prepared by anti-bFGF antibody, protein-A immunoaffinity, and hydroxyapatite (HA) chromatography] yielded peak HA-adsorbed activity that eluted with 0.4 M sodium phosphate, and was neutralized 70% by antibodies to intact bFGF. Cancer sera mean peak specific activity (12.0 ng-eq bFGF/ug protein) (n = 7) significantly exceeded (P < 0.001) normal sera mean peak specific activity (0.46 ng-eq bFGF/ug protein) (n = 6) in the 0.4 M sodium phosphate eluate fraction from hydroxyapatite columns. These results imply that long-lasting, bioactive FGF-like autoantibodies may arise spontaneously (and contribute to pathophysiology) in subsets of cancer patients with osseous metastases.