Vascularization plays a key role in processes such as wound healing and tissue engineering. Non-thermal plasma, which primarily produces reactive oxygen species (ROS), has recently emerged as an efficient tool in medical applications including blood coagulation, sterilization and malignant cell apoptosis. Liquids and porcine aortic endothelial cells were treated with a non-thermal dielectric barrier discharge plasma in vitro. Plasma treatment of phosphate-buffered saline (PBS) and serum-free medium increased ROS concentration in a dose-dependent manner, with a higher concentration observed in serum-free medium compared with PBS. Species concentration inside cells peaked 1 h after treatment, followed by a decrease 3 h post treatment. Endothelial cells treated with a plasma dose of 4.2 J cm(-2) had 1.7 times more cells than untreated samples 5 days after plasma treatment. The 4.2 J cm(-2) plasma dose increased two-dimensional migration distance by 40 per cent compared with untreated control, while the number of cells that migrated through a three-dimensional collagen gel increased by <em>15</em> per cent. Tube formation was also enhanced by plasma treatment, with tube lengths in plasma-treated samples measuring 2.6 times longer than control samples. A <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) neutralizing antibody and ROS scavengers abrogated these angiogenic effects. These data indicate that plasma enhanced proliferation, migration and tube formation is due to FGF-2 release induced by plasma-produced ROS. Non-thermal plasma may be used as a potential tool for applying ROS in precise doses to enhance vascularization.

There is great interest in genetic modification of bone marrow-derived mesenchymal stem cells (MSC), not only for research purposes but also for use in (autologous) patient-derived-patient-used transplantations. A major drawback of bulk methods for genetic modifications of (stem) cells, like bulk-electroporation, is its limited yield of DNA transfection (typically then 10%). This is even more limited when cells are present at very low numbers, as is the case for stem cells. Here we present an alternative technology to transfect cells with high efficiency (>75%), based on single cell electroporation in a microfluidic device. In a first experiment we show that we can successfully transport propidium iodide (PI) into single mouse myoblastic C2C12 cells. Subsequently, we show the use of this microfluidic device to perform successful electroporation of single mouse myoblastic C2C12 cells and single human MSC with vector DNA encoding a green fluorescent-erk1 fusion protein (EGFP-ERK1 (MAPK3)). Finally, we performed electroporation in combination with live imaging of protein expression and dynamics in response to extracellular stimuli, by <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2). We observed nuclear translocation of EGFP-ERK1 in both cell types within <em>15</em> min after FGF-2 stimulation. Due to the successful and promising results, we predict that microfluidic devices can be used for highly efficient small-scale 'genetic modification' of cells, and biological experimentation, offering possibilities to study cellular processes at the single cell level. Future applications might be small-scale production of cells for therapeutic application under controlled conditions.

OBJECTIVE

Fibroblast growth factor (FGF)-21, and possibly FGF19, protect against type 2 diabetes mellitus (T2DM) and obesity in rodents. We investigated the circulating levels of FGF21 and FGF19 in obese patients with varying degrees of abnormal glucose homeostasis, and we determined gene expression for FGF receptors (FGFR1-4) and the co-receptor β-Klotho, in liver and adipose tissues.

METHODS

We analyzed 35 lean healthy (71% men) and 61 obese patients (49% men, median body mass index (BMI): 40.5 kg m(-2), interquartile range: 34.7-46.2). Among obese patients, 36 were normoglycemic, 15 showed impaired glucose tolerance and 10 had T2DM. Biopsies from liver and visceral and subcutaneous fat from a subset of obese patients and controls were analyzed. FGF19 and FGF21 levels were measured using enzyme-linked immunosorbent assay, and tissue mRNA and protein levels by reverse transcription-polymerase chain reaction and immunoblotting.

RESULTS

FGF21 serum levels were significantly increased in obese patients compared with controls (P<0.001), whereas FGF19 levels were decreased (P < 0.001). FGF21 levels were positively correlated with homeostasis model assessment of insulin resistance (P = 0.0002, r = 0.37) and insulin (P = 0.001, r = 0.32), whereas FGF19 levels were negatively correlated (P = 0.007, r = -0.27; P=0.003, r = -0.28; respectively). After adjusting for BMI, the correlations of FGF21 and FGF19 levels with indicators of abnormal glucose homeostasis were not significant. In obese patients, the hepatic expression of FGF21 was increased. (P = 0.04). β-Klotho transcript levels in visceral fat (P = 0.002) and β-Klotho protein levels in subcutaneous (P = 0.03) and visceral fat (P = 0.04) were significantly reduced in obese patients, whereas hepatic levels for β-Klotho (P = 0.03), FGFR1 (P = 0.04) and FGFR3 (P = 0.001) transcripts were significantly increased.

CONCLUSIONS

Obesity is characterized by reciprocal alterations in FGF19 (decrease) and FGF21 (increase) levels. Although worsened in diabetic obese patients, obesity itself appears as the predominant determinant of the abnormalities in FGF21 and FGF19 levels. Opposite changes in β-Klotho expression in fat and liver indicate potential tissue-specific alterations in the responsiveness to endocrine FGFs in obesity.

The pneumoconioses, interstitial lung disorders resulting from the inhalation of inorganic dusts, are associated with chronic inflammatory processes in the lower respiratory tract. To characterize these inflammatory processes in relation to the pathogenesis of these disorders, we studied 39 nonsmoking individuals with long-term occupational exposures to inorganic dust and functional evidence of interstitial disease (asbestosis, n = 18; coal workers' pneumoconiosis, n = <em>15</em>; silicosis, n = 6). In all 3 disorders, the inflammation was dominated by alveolar macrophages. Because a common feature of these interstitial lung diseases is concurrent injury and fibrosis of alveolar walls, we assessed whether these alveolar macrophages were spontaneously releasing mediators capable of giving rise to these changes. Alveolar macrophages from the study population were spontaneously releasing increased amounts of superoxide anion and hydrogen peroxide (both p less than 0.01 compared to normals), oxidants capable of injuring lung parenchymal cells. The alveolar macrophages were also spontaneously releasing significantly increased amounts of fibronectin and alveolar macrophage-derived <em>growth</em> <em>factor</em> (both p less than 0.01 compared to normals), mediators that act synergistically to signal <em>fibroblast</em> replication. Taken together, these findings define a major role for the alveolar macrophage in mediating the alveolar wall injury and fibrosis that characterize the common pneumoconioses and suggest that the alveolar macrophage is an important "target" for developing strategies designed to prevent loss of lung function in these individuals.

Angiotensin II (angiotensin) and transforming <em>growth</em> <em>factor</em> (TGF)-beta(1) play an important role in cardiac fibrosis. We examined Smad proteins in 8-wk post-myocardial infarction (MI) rat hearts. AT(1) blockade (losartan) attenuated the activation of TGF-beta(1) in target tissues. Losartan administration (8 wk, <em>15</em> mg. kg(-1). day(-1)) normalized total Smad 2 overexpression in infarct scar and remnant heart tissue and normalized Smad 4 in infarct scar. Phosphorylated Smad 2 (P-Smad 2) staining decreased in cytosol from failing heart vs. the control, which was normalized by losartan, suggesting augmented P-Smad 2 movement into nuclei in untreated failing hearts. Using adult primary rat <em>fibroblasts</em> treated with angiotensin (10(-6) M), we noted rapid translocation (<em>15</em> min) of P-Smad 2 into the nuclei from the cytosol. Nuclear P-Smad 2 protein level increased with angiotensin treatment, which was blocked by losartan. We conclude that angiotensin may influence total Smad 2 and 4 expression in post-MI heart failure and that angiotensin treatment is associated with rapid P-Smad 2 nuclear translocation in isolated <em>fibroblasts</em>. This study suggests that cross talk between angiotensin and Smad signaling is associated with fibrotic events in post-MI hearts.

Although the roles of Jak-Stat pathways in type I and II interferon (IFN)-dependent transcriptional regulation are well established, the precise mechanisms of mRNA translation for IFN-sensitive genes remain to be defined. We examined the effects of IFNs on the phosphorylation/activation of eukaryotic translation initiation <em>factor</em> 4B (eIF4B). Our data show that eIF4B is phosphorylated on Ser422 during treatment of sensitive cells with alpha IFN (IFN-alpha) or IFN-gamma. Such phosphorylation is regulated, in a cell type-specific manner, by either the p70 S6 kinase (S6K) or the p90 ribosomal protein S6K (RSK) and results in enhanced interaction of the protein with eIF3A (p170/eIF3A) and increased associated ATPase activity. Our data also demonstrate that IFN-inducible eIF4B activity and IFN-stimulated gene <em>15</em> protein (ISG<em>15</em>) or IFN-gamma-inducible chemokine CXCL-10 protein expression are diminished in S6k1/S6k2 double-knockout mouse embryonic <em>fibroblasts</em>. In addition, IFN-alpha-inducible ISG<em>15</em> protein expression is blocked by eIF4B or eIF3A knockdown, establishing a requirement for these proteins in mRNA translation/protein expression by IFNs. Importantly, the generation of IFN-dependent <em>growth</em> inhibitory effects on primitive leukemic progenitors is dependent on activation of the S6K/eIF4B or RSK/eIF4B pathway. Taken together, our findings establish critical roles for S6K and RSK in the induction of IFN-dependent biological effects and define a key regulatory role for eIF4B as a common mediator and integrator of IFN-generated signals from these kinases.

We have used the technique of DNA-excess filter hybridization to measure directly the content and metabolism of the mRNA for dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). The studies were conducted with a methotrexate-resistant derivative of mouse 3T6 <em>fibroblasts</em> (M50L3) that overproduces the enzyme and its mRNA by a <em>factor</em> of 300 but regulates the level of the enzyme during the cell cycle in the same manner as normal 3T6 cells. We found that, when resting (G0) M50L3 cells were serum-stimulated to reenter the cell cycle, the 10-fold increase in the rate of synthesis of DHFR that occurs at the beginning of S phase was the result of a corresponding increase in DHFR mRNA content. In pulse-labeling experiments, we found that there was a similar increase in the rate of production of the mRNA just prior to S phase. However, the half-life of the mRNA was the same (7.5 hr) in resting and exponentially <em>growing</em> cells. Therefore, the increase in DHFR mRNA content was due to an increase in the rate of production rather than an increase in the stability of the message. The delay between addition of [3H]-uridine to the culture medium and the emergence of DHFR mRNA from the nucleus was <em>15</em>-20 min for both resting and <em>growing</em> M50L3 cells. A similar delay was observed for total mRNA. Therefore, the time required for the processing of newly synthesized DHFR heterogeneous nuclear RNA into DHFR mRNA is about the same as that for the average mRNA.

The objective of the study was to investigate whether the response profile of the <em>growth</em> <em>factor</em> of human tendon <em>fibroblasts</em> could be beneficially influenced through the application of mechanical stretch. It was considered that this would elucidate structural and functional problems, often seen after tendon and ligament healing. The secretion pattern of transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), platelet-derived <em>growth</em> <em>factor</em> (PDGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was determined in mechanically stretched <em>fibroblasts</em> and compared to non-stretched controls. Human tendon <em>fibroblasts</em> were experimentally stretched for <em>15</em> and 60 mm at a frequency of 1 Hz and an amplitude of 5%. The secretion of TGF-beta PDGF and bFGF was measured by enzyme-linked immuno-sorbent assay. All the <em>growth</em> <em>factors</em> investigated were indeed secreted by human tendon <em>fibroblasts</em> both in stretched cells and controls. Mechanical stretch increased the secretion pattern of the <em>growth</em> <em>factors</em>. The increased concentrations of TGF-beta bFGF and PDGF after cyclical mechanical stretching may have a positive influence on tendon and ligament healing through stimulation of cell proliferation, differentiation and matrix formation.

Diabetic skin ulcers are difficult to heal spontaneously due to the reduced levels and activity of endogenous <em>growth</em> <em>factors</em>. Recombinant human vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) are known to stimulate cell proliferation and accelerate wound healing. Direct delivery of VEGF and bFGF at the wound site in a sustained and controllable way without loss of bioactivity would enhance their biological effects. The aim of this study was to develop a poly(ether)urethane-polydimethylsiloxane/fibrin-based scaffold containing poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with VEGF and bFGF (scaffold/GF-loaded NPs) and to evaluate its wound healing properties in genetically diabetic mice (db/db). The scaffold application on full-thickness dorsal skin wounds significantly accelerated wound closure at day <em>15</em> compared to scaffolds without <em>growth</em> <em>factors</em> (control scaffold) or containing unloaded PLGA nanoparticles (scaffold/unloaded NPs). However, the closure rate was similar to that observed in mice treated with scaffolds containing free VEGF and bFGF (scaffold/GFs). Both scaffolds containing <em>growth</em> <em>factors</em> induced complete re-epithelialization, with enhanced granulation tissue formation/maturity and collagen deposition compared to the other groups, as revealed by histological analysis. The ability of the scaffold/GF-loaded NPs to promote wound healing in a diabetic mouse model suggests its potential use as a dressing in patients with diabetic foot ulcers.

OBJECTIVE

To identify changes in cellular signaling pathways and AP-1 expression in retina and retinal pigmented epithelium (RPE) after experimental retinal detachment (RD).

METHODS

Cat and rabbit neural retinas were separated from the RPE in vivo for 5 minutes to 28 days. Tissues were removed and processed for Western blotting, immunohistochemistry, in situ hybridization, and immunoprecipitation experiments.

RESULTS

An ordered sequence of events occurs after RD: (1) <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) receptor 1 (FGFR1, flg) is phosphorylated in the retina within <em>15</em> minutes and dephosphorylated 2 hours after RD; (2) The extracellular signal-regulated kinase (ERK) is phosphorylated in both Müller and RPE cells within <em>15</em> minutes and remains so for several days; (3) De novo expression of c-fos mRNA coincides with increased c-Fos and c-Jun immunoreactivity in both Müller and RPE cells; (4) CREB is phosphorylated in a subpopulation of photoreceptors; and (5) STAT3 and NF-kappaB are activated in inner nuclear layer cells by 1 day of RD.

CONCLUSIONS

These data suggest that nonneuronal cells (RPE and Müller cells) respond to RD very rapidly by stimulating ERK signaling and AP-1 transcription factor expression. Furthermore, these data suggest that basic fibroblast growth factor (FGF-2, bFGF) is involved in initiating the retina's earliest responses to RD. The events described here precede changes in gene expression and morphology that can have serious effects on visual outcome in humans treated for retinal detachment or other retinal injuries.

OBJECTIVE

To determine the vitreous levels of 27 types of cytokines in eyes with retinopathy of prematurity (ROP).

METHODS

Retrospective case-control study.

METHODS

Twenty-seven eyes of 19 infants with stage 4 ROP were studied. Six eyes of 5 patients with congenital cataract who underwent lensectomy were used as controls.

METHODS

The ROP eyes were divided into 2 groups according to vascular activity: 12 eyes with vascularly active ROP and <em>15</em> eyes with vascularly inactive ROP. Undiluted vitreous samples were collected, and the vitreous concentrations of 27 types of cytokines were determined by a multiplex bead analysis system: interleukin (IL)-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-<em>15</em>, IL-17, Eotaxin, <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) basic, granulocyte-colony stimulating <em>factor</em> (G-CSF), granulocyte macrophage colony-stimulating <em>factor</em> (GM-CSF), interferon-r, interferon-gamma-inducible protein (IP)-10, monocyte chemoattractant protein 1, macrophage inflammatory protein (MIP)-1a, MIP-1b, platelet-derived <em>growth</em> <em>factor</em> bb, regulated on activation, normal T cell expressed and secreted (RANTES), tumor necrosis <em>factor</em> alpha, and vascular endothelial <em>growth</em> <em>factor</em> (VEGF).

METHODS

The vitreous levels of the 27 types of cytokines and a comparison of the levels in the 3 groups.

RESULTS

The postmenstrual age at vitrectomy was significantly younger in the vascularly active ROP eyes than in vascularly inactive ROP eyes. The cytokines that had significantly different vitreous levels among the 3 groups were: IL-6, IL-7, IL-10, IL-<em>15</em>, Eotaxin, FGF basic, G-CSF, GM-CSF, IP-10, RANTES, and VEGF (P<0.05). The vitreous levels of IL-6, IL-7, IL-<em>15</em>, Eotaxin, G-CSF, IP-10, and RANTES were significantly higher (P<0.05) in both vascularly active and inactive ROP eyes than in control eyes, whereas the vitreous level of VEGF was significantly higher (P<0.05) only in vascularly active ROP eyes than in control eyes. There was a significantly negative correlation (r = -0.382; P = 0.0495) between the VEGF level and the postmenstrual age at vitrectomy.

CONCLUSIONS

These results indicate that, although cytokines other than VEGF may be involved in the pathologic changes in eyes with ROP, VEGF is likely to have the strongest correlation with the vascular activity in ROP eyes among these cytokines.

Irradiation of cells with short-wavelength ultraviolet light (UVC) changes the program of gene expression, in part within less than <em>15</em> min. As one of the immediate-early genes in response to UV, expression of the oncogene c-fos is upregulated. This immediate induction is regulated at the transcriptional level and is transient in character, due to the autocatalyzed shutoff of transcription and the rapid turnover of c-fos mRNA. In an experiment analyzing the kinetics of c-fos mRNA expression in murine <em>fibroblasts</em> irradiated with UVC, we found that, in addition to the initial transient induction, c-fos mRNA accumulated in a second wave starting at 4 to 5 h after irradiation, reaching a maximum at 8 h, and persisting for several more hours. It was accompanied by an increase in Fos protein synthesis. The second peak of c-fos RNA was caused by an UV dose-dependent increase in mRNA half-life from about 10 to 60 min. With similar kinetics, the mRNAs of other UV target genes (i.e., the Kin17 gene, c-jun, IkappaB, and c-myc) were stabilized (e.g., Kin17 RNA from 80 min to more than 8 h). The delayed response was not due to autocrine cytokine secretion with subsequent autostimulation of the secreting cells or to UV-induced <em>growth</em> <em>factor</em> receptor activation. Cells unable to repair UVC-induced DNA damage responded to lower doses of UVC with an even greater accumulation of c-fos and Kin17 mRNAs than repair-proficient wild-type cells, suggesting that a process in which a repair protein is involved regulates mRNA stability. Although resembling the induction of p53, a DNA damage-dependent increase in p53 was not a necessary intermediate in the stabilization reaction, since cells derived from p53 knockout mice showed the same pattern of c-fos and Kin17 mRNA accumulation as wild-type cells. The data indicate that the signal flow induced by UV radiation addresses not only protein stability (p53) and transcription but also RNA stability, a hitherto-unrecognized level of UV-induced regulation.

Deletions and amplifications are frequent alterations of the short arm of chromosome 8 associated with various types of cancers, including breast cancers. This indicates the likely presence of tumor suppressor genes and oncogenes. In the present study, we have used the expressed sequence tag (EST) map of 8p11-21 to assemble a set of available cDNAs representing genes from this region. DNA arrays were prepared for expression analysis and search for genes potentially involved in breast cancer. Underexpresion in tumoral breast cells (versus normal breast) was observed for <em>15</em> transcripts. Among these, the Frizzled-related gene FRP1/FRZB, was turned off in 78% of breast carcinomas, suggesting that the lack of its product may be associated with malignant transformation. Overexpression in tumoral breast cells was observed for 13 genes. The FGFR1 gene, that encodes a tyrosine kinase receptor for members of the <em>fibroblast</em> <em>growth</em> <em>factor</em> family, was identified as a good candidate for one amplification unit. Taken together, our results demonstrate that such a strategy can rapidly identify genes with an altered pattern of expression and provide candidate genes for malignancies.

Wiring of the nervous system requires that axons navigate to their targets and maintain their correct positions in axon fascicles after termination of axon out<em>growth</em>. We show here that the C. elegans <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR), EGL-<em>15</em>, affects both processes in fundamentally distinct manners. FGF-dependent activation of the EGL-<em>15</em> tyrosine kinase and subsequently the GTPase LET-60/ras is required within epidermal cells, the substratum for most outgrowing axon, for appropriate out<em>growth</em> of specific axon classes to their target area. In contrast, genetic elimination of the FGFR isoform EGL-<em>15</em>(5A), defined by the inclusion of an alternative extracellular interimmunoglobulin domain, has no consequence for axon out<em>growth</em> but leads to a failure to postembryonically maintain axon position within defined axon fascicles. An engineered, secreted form of EGL-<em>15</em>(5A) containing only its ectodomain is sufficient for maintenance of axon position, thus providing novel insights into receptor tyrosine kinase function and the process of maintaining axon position.

Tumor-induced osteomalacia (TIO) is characterized by renal phosphate wasting, hypophosphatemia, and aberrant vitamin D(3) metabolism and is caused by <em>fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF-23)-producing mesenchymal tumors, which are often difficult to locate. We investigated the utility of selective venous sampling in tumor localization. The primary endpoint was identification of the FGF-23 concentration ratio between the venous drainage of the tumor bed and the general circulation that was diagnostic of the location of an FGF-23-secreting tumor. Fourteen subjects underwent <em>15</em> sampling procedures after functional and anatomic imaging studies. Subjects fit into three imaging categories: no suspicious site, multiple sites, and single site (positive controls). FGF-23 levels were measured by ELISA. Suspicious tumors were resected for diagnosis, confirmation, and cure. In subjects with a positive venous sampling study and subsequent cure, a minimum ratio of 1.6 was diagnostic. In 7 of 14 subjects there was suggestive imaging, a diagnostic ratio, and an associated TIO tumor (true positive). Four of these required complicated resection procedures. In 4 of 14 subjects with no suspicious site on imaging studies, an FGF-23 diagnostic ratio was not detected (true negative). Biopsy or resection of a single lesion in 2 of 14 subjects with a diagnostic ratio failed to identify a TIO tumor (false positive). A diagnostic FGF-23 ratio was absent in 1 of 14 subjects whose tumor was a single highly suspicious lesion on imaging studies (false negative). These data yield a sensitivity of 0.87 [95% confidence interval (CI) 0.47-0.99] and a specificity of 0.71 (95% CI 0.29-0.96). Selective venous sampling for FGF-23 was particularly useful in subjects with multiple suspicious sites or an anatomically challenging planned resection but not in the absence of a suspicious lesion on imaging studies.

Nuclear <em>factor</em> kappaB (NFkappaB) plays a pivotal role in early gene responses by promoting messenger RNA (mRNA) synthesis for various cell-adhesion molecules and inducible nitric oxide synthase. In this study, we examined whether increases in glucose concentration enhance NFkappaB expression in nuclear fractions of endothelial cells by using electrophoretic mobility shift assay. Bovine aortic endothelial cells (BAECs) were incubated in media containing 5.5-35 mM glucose. NFkappaB activity was increased as early as 1 h (peak activation at 2-4 h) after incubation with 35 mM glucose compared with 5.5 mM. Similar increases at 2 h of incubation were observed by using 25 but not <em>15</em> mM glucose. Glucose-induced NFkappaB activation was blocked by inhibiting nuclear translocation by using a peptide (SN-50) containing the nuclear-localization sequence of NFkappaB p50 linked to a membrane-permeable motif of the sequence for Kaposi <em>fibroblast</em> <em>growth</em> <em>factor</em>. Co-incubation with a selective protein kinase C (PKC) inhibitor, calphostin C, produced a concentration-dependent inhibition of glucose-induced NFkappaB activation. Thus NFkappaB activation is an early event in response to elevations in glucose, which may elicit multiple pathways contributing to the origin of hyperglycemia- or diabetes-induced endothelial cell injury.

The proper guidance of the Caenorhabditis elegans hermaphrodite sex myoblasts (SMs) requires the genes egl-<em>15</em> and egl-17. egl-<em>15</em> has been shown to encode the C. elegans orthologue of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor (FGFR). Here we clone egl-17 and show it to be a member of the <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family, one of the first functional invertebrate FGFs known. egl-17 shares homology with other FGF members, conserving the key residues required to form the distinctive tertiary structure common to FGFs. Genetic and molecular evidence demonstrates that the SM migration defect seen in egl-17 mutant animals represents complete loss of egl-17 function. While mutations in egl-17 affect only SM migration, mutations in egl-<em>15</em> can result in larval arrest, scrawny body morphology, and the ability to suppress mutations in clr-1. We propose that EGL-17 (FGF) acts as a ligand for EGL-<em>15</em> (FGFR) specifically during SM migration and that another ligand(s) activates EGL-<em>15</em> for its other functions.

BACKGROUND

Diurnal fluctuation of bile acid (BA) concentrations in the enterohepatic system of mammals has been known for a long time. Recently, BAs have been recognized as signaling molecules beyond their well-established roles in dietary lipid absorption and cholesterol homeostasis.

RESULTS

The current study depicted diurnal variations of individual BAs detected by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) in serum and livers collected from C57BL/6 mice fed a regular chow or a chow containing cholestyramine (resin). Circadian rhythms of mRNA of vital BA-related nuclear receptors, enzymes, and transporters in livers and ilea were determined in control- and resin-fed mice, as well as in farnesoid X receptor (FXR) null mice. The circadian profiles of BAs showed enhanced bacterial dehydroxylation during the fasting phase and efficient hepatic reconjugation of BAs in the fed phase. The resin removed more than 90% of BAs with β-hydroxy groups, such as muricholic acids and ursodeoxycholic acid, from serum and livers, but did not exert as significant influence on CA and CDCA in both compartments. Both resin-fed and FXR-null mouse models indicate that BAs regulate their own biosynthesis through the FXR-regulated ileal <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em>. BA flux also influences the daily mRNA levels of multiple BA transporters.

CONCLUSIONS

BA concentration and composition exhibit circadian variations in mouse liver and serum, which influences the circadian rhythms of BA metabolizing genes in liver and ileum. The diurnal variations of BAs appear to serve as a signal that coordinates daily nutrient metabolism in mammals.

The abilities of plasma and serum to support the <em>growth</em> of vascular smooth muscle cells maintained on uncoated tissue culture dishes or dishes coated with an extracellular matrix (ECM) have been compared. Vascular smooth muscle cells maintained on plastic dishes and exposed to plasma proliferate poorly; when exposed to serum they proliferate actively. Addition of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) incrases the <em>growth</em> rate of the cultures in both cases. In contrast, when vascular smooth muscle cells are maintained on an ECM, they proliferate equally well exposed to either plasma or serum. Because the cultures had an average doubling time (<em>15</em> hr) that was already at a minimum, FGF no longer had an effect on vascular smooth muscle cell proliferation. These results raise the possibility that the lack of response of vascular smooth muscle cells, as well as that of other cell types in vitro, to plasma <em>factors</em> is not an intrinsic property of the cells but is rather due to the substrate upon which the cells rest. Because cells maintained on an ECM respond to plasma <em>factors</em>, it is likely that the close contact of the cells with the ECM restores their sensitivity to physiological <em>factors</em> present in plasma.

OBJECTIVE

To examine the expression of the thioredoxin (TRX)-thioredoxin reductase (TR) system in patients with rheumatoid arthritis (RA) and patients with other rheumatic diseases.

METHODS

Levels of TRX in plasma and synovial fluid (SF) were measured using enzyme-linked immunosorbent assay. Cellular distribution of TRX was determined by flow cytometry and histochemistry. Cellular expression of TR was studied by in situ messenger RNA (mRNA) hybridization. The effect of oxidative stress and tumor necrosis factor alpha (TNF alpha) on TRX expression by cultured rheumatoid fibroblast-like synoviocytes was studied.

RESULTS

Significantly increased TRX levels were found in the SF from 22 patients with RA, when compared with plasma levels in the same patients (P < 0.001) and compared with SF TRX levels in 15 patients with osteoarthritis (P < 0.001), 13 patients with gout (P < 0.05), and 9 patients with reactive arthritis (P < 0.0001). The presence of TRX could be demonstrated within the SF-derived mononuclear cells and synovial tissue (ST) of RA patients. Concordantly, expression of TR mRNA was observed in the ST of these patients. Stimulation of synovial fibroblast-like synoviocytes with either H2O2 or TNF alpha induced an increase in the production of TRX.

CONCLUSIONS

The data demonstrate significantly increased concentrations of TRX in the SF and ST of RA patients when compared with the levels in patients with other joint diseases. Evidence is presented that the local environment in the rheumatic joint contributes to increased TRX production. Based on its growth-promoting and cytokine-like properties, it is proposed that increased expression of TRX contributes to the disease activity in RA.

OBJECTIVE

We evaluated adeno-associated virus (AAV)-mediated gene transfer of basic fibroblast growth factor (FGF-2) as a therapy for photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa.

METHODS

Recombinant adeno-associated virus vector (rAAV) incorporating a constitutive cytomegalovirus (CMV) promoter was used to transfer the bovine FGF-2 gene to photoreceptors. AAV was administered by subretinal injection to transgenic rats (TgN S334ter-4) at postnatal day 15 (P15). Control eyes were uninjected, injected with PBS, or AAV-LacZ. Eyes were examined by histopathology, morphometric analysis, and electroretinography at P60.

RESULTS

Expression of recombinant FGF-2 slowed the rate of photoreceptor degeneration. Morphologic studies demonstrated significantly more photoreceptors surviving in eyes injected with AAV-FGF-2 than in controls. Insignificant rescue effects were seen in retinas injected with buffer only. No significant inflammatory response or neovascularization was detected. Electroretinographic (ERG) responses of eyes injected with AAV-FGF-2 were increased compared with uninjected eyes; however, these amplitudes were not significantly larger than eyes receiving an AAV-LacZ control vector.

CONCLUSIONS

Transduction of retinal cells with AAV-FGF-2 reduces the rate of photoreceptor degeneration in an S334ter-4 animal model. Despite the lack of significantly increased ERG amplitudes from eyes expressing FGF-2, a greater number of surviving photoreceptors was demonstrated. Delivery of FGF-2 using recombinant AAV has potential as a therapy for retinal degeneration.

Mechanisms accounting for the development of proliferative vitreoretinopathy (PVR) in patients with rhegmatogenous retinal detachment remain poorly understood. In a previous study, we found the presence of various <em>growth</em> <em>factors</em> in preretinal membranes that were surgically removed from patients with PVR. The present immunohistological study was undertaken in intravitreal and subretinal fluid cells from patients suffering from PVR in various stages of development, in order to seek the presence of 4 <em>growth</em>-promoting <em>factors</em> for retinal pigment epithelial cells: acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), epidermal <em>growth</em> <em>factor</em> (EGF), insulin-like <em>growth</em> <em>factor</em> type I (IGF-I) and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta). Results were quite similar in vitreous and subretinal fluid. Acidic FGF was found in all vitreous and subretinal specimens, in 30-100% of the examined cells. Immunoreactivity for EGF could be found in 53% of intravitreal cells and 69% of subretinal fluid cells. Positive cells were seen in all vitreous specimens and in all but 1 of the subretinal fluid specimens. IGF-I-containing cells were present in 13 of <em>15</em> vitreous specimens and in 18 of 20 subretinal fluid samples (mean percentages of reactivity in positive specimens 70% and 78%, respectively). In contrast, TGF-beta 1 reactivity was found in only 8 of <em>15</em> vitreous specimens and in 11 of 20 subretinal samples. Mean percentages of reactive cells were 30% and 50%, respectively. These results suggest that several <em>growth</em> <em>factors</em> could be involved in the proliferation and migration of retinal pigment epithelial cells during the course of PVR.(ABSTRACT TRUNCATED AT 250 WORDS)

The binding of human acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) to heparin has been analyzed by a variety of different approaches to better elucidate the nature of this protein/sulfated polysaccharide interaction. Static and dynamic light scattering as well as analytical ultracentrifugation analyses indicates that 14-<em>15</em> molecules of a FGF can bind to a 16-kDa heparin chain, with approximately 10 of these bound relatively uniformly to high-affinity sites. The dissociation constants of these latter sites are estimated to be approximately 50-140 nM on the basis of surface plasmon resonance experiments in which the association and dissociation rates of aFGF interaction with immobilized heparin were measured. The size of the binding site of a FGF on heparin was also determined by heparin lyase digestion of a FGF/heparin complexes followed by isolation and characterization of protected oligosaccharides. The smallest aFGF-protected oligosaccharide comigrated with delta UA2S(1-->4)-alpha-D-GlcNp2S6S(1-->4)-alpha-L-IdoAp-2S( 1-->4)-alpha-D-GlcNp2S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid and S is sulfate). Thus, aFGF appears to bind at high density (one molecule every 4-5 polysaccharide units) and with high affinity to heparin. This potentially provides a concentrated, stabilized storage form of the <em>growth</em> <em>factor</em> that can be released for receptor-mediated cellular activation in response to the proper stimuli. It is also possible that close proximity of aFGF molecules on the highly sulfated regions of heparan chains may be involved in the induction of receptor aggregation as suggested by Ornitz et al. [Ornitz, D. M., Yayon, A., Flanagan, J. G., Svahn, C. M., Levi, E., & Leder, P. (1992) Mol. Cell. Biol. 12, 240-247].

Angiogenesis has an important role in the progression of solid tumors. Therefore, we measured the blood levels (ELISA) of angiogenic <em>factors</em> [basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), hepatocyte <em>growth</em> <em>factor</em>/scatter <em>factor</em>, and vascular endothelial <em>growth</em> <em>factor</em> (VEGF)] and soluble adhesion molecules [E-selectin, intercellular adhesion molecule (ICAM-1), platelet endothelial cell adhesion molecule-1, and vascular cell adhesion molecule-1] in 76 consecutive patients with untreated renal cell carcinoma and 41 healthy controls to evaluate their prognostic value. The serum levels of bFGF, hepatocyte <em>growth</em> <em>factor</em>, and VEGF were significantly higher in patients with renal cancer than they were in healthy subjects. bFGF and VEGF values were significantly higher in patients with disseminated cancer (N+ and/or M+) than they were in those with undisseminated (M-N-) cancer: median = 27 pg/ml, range = 5-118, n = <em>15</em> versus median = 8 pg/ml, range = 1-149, n = 61 (P = 10(-4)) for bFGF; and median = 883 pg/ml, range = 200-2317, n = <em>15</em> versus median = 278 pg/ml, range = 0-1704, n = 61 (P = 0.006) for VEGF. The blood levels of ICAM-1 and vascular cell adhesion molecule-1 were significantly higher, and the levels of E-selectin and platelet endothelial cell adhesion molecule-1 were significantly lower in patients with renal cancer than they were in controls. Plasma ICAM-1 was higher in metastatic patients (M+) than they were in nonmetastatic (M-) patients: median = 687 ng/ml, range = 294-1091, n = 12 versus median = 408 ng/ml, range = 217-1375, n = 64 (P = 10(-4)). ICAM-1 and bFGF blood values were correlated with the size of the primary tumor. The interleukin 6 and tumor necrosis <em>factor</em>-alpha (TNF-alpha) values of these patients have been previously published and are included in the survival analysis. Univariate analysis showed that bFGF, ICAM-1, interleukin 6, and TNF-alpha, before treatment, were prognostic <em>factors</em>. In multivariate analysis for proportional hazard regression, only TNF-alpha was an independent prognostic indicator, with a normal plasma TNF-alpha being highly predictive for a good prognosis in patients with untreated renal cell carcinoma.