The highly ordered process of wound healing involves the coordinated regulation of cell proliferation and migration and tissue remodeling, predominantly by polypeptide <em>growth</em> <em>factors</em>. Consequently, the slowing of wound healing that occurs in the aged may be related to changes in the activity of these various regulatory <em>factors</em>. To gain additional insight into these issues, we quantified the absolute copy numbers of mRNAs encoding all the <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs), their receptors (FGFRs) and two other <em>growth</em> <em>factors</em> in the dorsal skin of young and aged mice during the healing of full-thickness skin excisional wounds. In young adult mice (8 weeks old), FGF7, FGF10 and FGF22 mRNAs were all strongly expressed in healthy skin, and levels of FGF7 and 10 but not 22 increased 2- to 3.5-fold over differing time courses after wounding. The levels of FGF9, <em>16</em>, 18 and especially 23 mRNAs were moderate or low in healthy skin but increased 2- to 33-fold after wounding. Among the four FGFRs, expression of only FGFR1 mRNA was augmented during wound healing. Expression of transforming <em>growth</em> <em>factor</em>-beta and hepatocyte <em>growth</em> <em>factor</em> was also high in healthy skin and was upregulated during healing. Notably, in aged mice (35 weeks old), where healing proceeded more slowly than in the young, both the basal and wound-induced mRNA expression of most of these genes was reduced. While these results confirm the established notion that FGFR2 IIIB ligands (FGF7 and FGF10) are important for wound healing, they also suggest that decreased expression of multiple FGF ligands contributes to the slowing of wound healing in aged mice and indicate the potential importance of further study of the involvement of FGF9, <em>16</em>, 18 and 23 in the wound healing process.

OBJECTIVE

Matrix metalloproteinases (MMPs) are involved in tissue remodelling, which is one of the important aspects of inflammatory disease. To assess the balance between the matrix degradation and production, we analysed the in situ expression of MMP-1, -3, -8 and -9, tissue inhibitor of metalloproteinases (TIMP)-1 and -2, and type I procollagen (PC-I) in inflammatory bowel disease.

RESULTS

Immunohistochemistry using frozen sections was performed in 17 patients with ulcerative colitis (UC) and <em>16</em> with Crohn's disease (CD). In both UC and CD, MMPs and TIMPs were expressed by inflammatory cells as well as by fibroblastic cells most prominently in actively inflamed areas in ulcer bases, but sparsely in intact inflamed mucosa in both UC and CD. In UC, inflamed mucosa with erosions expressed these substances focally. <em>Fibroblasts</em> also expressed PC-I. We identified that vascular smooth muscle cells of venules in ulcer bases expressed MMP-1 and -9, TIMP-1 and PC-I. These venules also expressed E-selectin, a cell adhesion molecule to facilitate the leucocyte extravasation, and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) receptor 2, consistent with their property of newly formed vessels.

CONCLUSIONS

Our results suggest that MMPs are involved in the tissue remodelling, angiogenesis and promotion of leucocyte extravasation in the actively inflamed area in the ulcer base in both UC and CD. MMP-1 expression in the mucosa may be related to the initial step of ulceration in UC. Therapeutic manipulation of extracellular matrix turnover would be an effective therapy to alleviate active inflammation and accelerate ulcer healing.

BACKGROUND

Recent studies have described relationships between iron status and fibroblast growth factor-23 (FGF23) but the possible confounding effects of inflammation on iron status have not been considered. The aims of this study were a) to consider a relationship between FGF23 and inflammation b) to identify relationships between iron status and FGF23 whilst correcting for inflammation and c) to assess the relationship between changes in FGF23 and iron status after supplementation.

METHODS

Blood samples from an iron supplementation study in children (n=79) were collected at baseline and after 3 months supplementation with iron sulphate. The children were from a rural Gambian population where rates of iron deficiency and infection/inflammation are high. This study identified cross-sectional and longitudinal relationships between FGF23, inflammation (C-reactive protein (CRP)) and iron status (ferritin, haemoglobin, and zinc protoporphyrin). CRP ≥ 5 mg/dL was used to indicate inflammation and FGF23 ≥ 125 RU/mL was considered elevated.

RESULTS

FGF23 was not significantly correlated with CRP. At baseline, all markers of iron status were significantly correlated with FGF23. Ferritin was the strongest independent inverse predictor of FGF23 in subjects with and without elevated CRP (coefficient (SE)): All subjects=-0.57 (0.12), R2=22.3%, P≤0.0001; subjects with CRP < 5 mg/dL=-0.89 (0.14), R2=38.9%, P≤0.0001. FGF23 was elevated in 28% of children at baseline and 16% post supplementation (P=0.1). Improved iron status was associated with a decrease in FGF23 concentration in univariate (ferritin =-0.41 (0.11), R2=14.1%, P=0.0004; haemoglobin=-2.22 (0.64), R2=12.5%, P=0.0008; zinc protoporphyrin=1.12 (0.26), R2=18.6%, P≤0.0001) and multivariate analysis (R2=33.1%; ferritin=-0.36 (0.10), P=0.0007, haemoglobin = -1.83 (0.61), P=0.004, zinc protoporphyrin=0.62 (0.26), P=0.02).

CONCLUSIONS

Iron status rather than inflammation is a negative predictor of plasma FGF23 concentration. Improvements in iron status following iron supplementation are associated with a significant decrease in FGF23 concentration.

OBJECTIVE

The involvement of cytokines in the aqueous humour is important in the development and progression of diabetic macular oedema (DMO) and macular oedema (MO) due to branch retinal vein occlusion (BRVO-MO). In this study, the concentrations of cytokines in the aqueous humour of eyes with DMO and BRVO-MO were measured and compared.

METHODS

Prospective, observational case series. Aqueous samples were obtained from 18 eyes with DMO (DMO group), 12 eyes with BRVO-MO (BRVO-MO group), and <em>16</em> normal eyes (control group). The aqueous levels of cytokines, including interleukin (IL)-2, IL-5, IL-6, IL-8, IL-12p70, IL-13, monocytochemotactic protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), platelet-derived <em>growth</em> <em>factor</em>-AA (PDGF)-AA, transforming <em>growth</em> <em>factor</em>-α (TGF-α), interferon-γ (IFN-γ), epidermal <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF), were measured.

RESULTS

The aqueous levels of IL-8, MCP-1, PDGF-AA and VEGF were higher, and IL-13 was lower in the DMO group compared with the control group. The aqueous levels of IL-8 and VEGF were higher in the BRVO-MO group than in the control group. Compared with the IL-6 and MCP-1 levels in the BRVO-MO group, those levels were significantly higher in the DMO group. Correlation analyses revealed that IL-8 was positively and IFN-γ was negatively correlated with the severity of MO in the DMO group. In the BRVO-MO group, IL-8 and was positively correlated with severity of MO and retinal ischaemia.

CONCLUSIONS

DMO and BRVO-MO may differ in terms of pathogenesis because the cytokine concentrations in the aqueous humour differ. The results suggest that the inflammatory reaction may be more activated in DMO than in BRVO-MO, and ischaemic insult may play a central role in the development of BRVO-MO.

In vertebrates, a number of <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) have been shown to play important roles in developing embryos and adult organisms. However, the molecular relationships of the vertebrate FGFs are not yet completely understood, partly due to the divergence of their amino acid sequences. To solve this problem, we have identified six FGF genes in a basal chordate, the ascidian Ciona intestinalis. A phylogenetic analysis confidently assigned two of them to vertebrate FGF8/17/18 and FGF11/12/13/14, respectively. Based on the presence of the conserved domains within or outside of the FGF domains, we speculate that three of the other genes are orthologous to vertebrate FGF3/7/10/22, FGF4/5/6 and FGF9/<em>16</em>/20, respectively, although we cannot assign the sixth member to any of the vertebrate FGFs. A survey of the raw whole genome shotgun sequences of C. intestinalis demonstrated the presence of no FGF genes other than the six genes in the genome. The identification of these six FGF genes in the basal chordate gave us an insight into the diversification of specific subfamilies of vertebrate FGFs.

OBJECTIVE

Effective systemic therapy options for carcinoid tumors are lacking. We conducted in vitro studies and a phase II clinical trial to explore the activity of imatinib in carcinoid tumors.

METHODS

Cells of the human bronchial carcinoid cell line NCI-H727 and the human pancreatic carcinoid cell line BON-1 were treated with increasing concentrations of imatinib using standard procedures to assess in vitro growth-inhibitory activity. A clinical trial using a two-stage phase II design to assess the response rate and safety profile of imatinib at a dose of 400 mg given twice daily in patients with advanced carcinoid tumors was completed.

RESULTS

In both cell lines, there was a dose- and time-dependent cytotoxic effect. The clinical trial enrolled 27 evaluable patients. Median duration on trial was 16 weeks. One patient had a partial response, 17 had stable disease, and 9 had progressive disease by the Response Evaluation Criteria in Solid Tumors criteria. Median progression-free survival time was 24 weeks. Median overall survival is 36 months. Seven patients who achieved a biochemical response had a superior progression-free survival time compared with patients without biochemical response (115 weeks compared with 24 weeks; P = 0.003). An increase in plasma basic fibroblast growth factor was associated with a shorter progression-free survival duration (P = 0.02).

CONCLUSIONS

Our data suggest that imatinib is active in vitro and has a modest clinical activity in carcinoid patients. Changes in tumor markers may help select patients who are likely to benefit from therapy.

Normal, viral transformed and tumor-derived cells grown in tissue culture and representing different species were tested for their ability to produce an extracellular tumor angiogenesis <em>factor</em> (TAF). TAF was assayed by measuring the host-mediated vascular response of the chorioallantoic membrane to TAF preparations. All of the viral transformed and tumor-derived cells tested, including SVT2, SVW126, Welker 256 rat carcinoma, B-<em>16</em> mouse melanoma, human glioblastoma, and human meningioma cells, produced TAF. The potency of the TAF preparations, as measured by the number of cells needed to induce a positive vascular response on the chorioallantoic membrane, varied from cell line to cell line. The most potent cells tested were the glioblastoma and maningioma brain tumor cells. Since these brain tumors are found to be the most highly vascularized tumors in vivo, it was concluded that a correlation exists between the vascularity of a tumor in vivo and the potency of TAF in vitro. There was no detectable angiogenesis activity induced by density-inhibited BALB/c primary mouse embryo or early-passage human skin <em>fibroblasts</em>, even when relatively large numbers of cells were used to make a sample. However, density-inhibited BALB/c 3T3 aan W138 human embryonic lung <em>fibroblasts</em>, two cell lines widely regarded as demonstrating "normal" <em>growth</em> behavior in culture, produced TAF. From these and other observations, it was suggested that BALB/c 3T3 and W138 are not fully "normal" cells. Furthermore, it was suggested that the production of TAF is an early event in the cell transformation process that precedes the loss of density inhibition of <em>growth</em> in vitro.

OBJECTIVE

To correlate levels of angiogenic growth factors with Doppler ultrasound parameters in pregnancies complicated by pre-eclampsia and intrauterine growth restriction (IUGR).

METHODS

In 16 women with pre-eclampsia and 15 women with isolated IUGR, pulsatility indices (PI) in the umbilical and uterine arteries were measured by Doppler ultrasonography. At delivery, maternal and fetal blood (umbilical vein and artery separately) was sampled and angiogenic growth factors measured by means of enzyme linked immunosorbent assay (ELISA).

RESULTS

Umbilical artery PI was significantly higher in women with IUGR than in those with pre-eclampsia, whereas uterine artery PI was not statistically significantly different. Maternal soluble fms-like tyrosine kinase-1 (sFlt-1) levels were higher in women with pre-eclampsia than in those with IUGR (P < 0.0001). Umbilical vein basic fibroblast growth factor (bFGF) levels were lower in women with pre-eclampsia than in those with IUGR (P < 0.05). Placental growth factor (PlGF) levels in the umbilical vein were below the detection limit in nearly all samples of IUGR fetuses and lower than in those with pre-eclampsia (P < 0.001). Maternal PlGF levels were inversely correlated with PI values of both vessels. In the umbilical vein sFlt-1 was positively and soluble kinase insert domain receptor (sKDR) negatively correlated with umbilical artery PI. No correlation could be found in the serum of the umbilical artery for all growth factors and for vascular endothelial growth factor (VEGF) in all compartments.

CONCLUSIONS

The correlations between maternal and fetal angiogenic growth factor serum levels and Doppler ultrasound indices of uterine and umbilical arteries in pre-eclampsia and IUGR reflect the severity of the disorders especially for the fetus. A combination of both measurements may be useful in future screening for early prediction of pregnancy complications. Published by John Wiley & Sons, Ltd.

BACKGROUND

Recent studies show associations between inorganic phosphate and risk of heart failure in the general population as well as between fibroblast growth factor 23 (FGF-23) and outcome in coronary heart disease. This study was carried out to assess whether circulating levels of inorganic phosphate and FGF-23, a new central hormone in mineral bone metabolism, predict outcome in systolic heart failure.

METHODS

Ninety-nine consecutive outpatients with systolic heart failure were enrolled. Mean (SD) age was 61 years (11), mean left ventricular ejection fraction (LVEF) was 33% (10), 82 patients were men, median estimated creatinine clearance was 83 mL/min (Q(1) -Q(3) 58-106), median NTproBNP level was 803 pg/mL (Q(1) -Q(3) 404-2757), median inorganic phosphate was 1·12 mM (Q(1) -Q(3) 1·02-1·22), median FGF-23 was 39·02 pg/mL (Q(1) -Q(3) 32·45-55·86) and median follow-up was 35 months. Associations between inorganic phosphate, FGF-23 and endpoints were assessed using Cox regression analyses.

RESULTS

Inorganic phosphate and FGF-23 levels were significantly higher (P < 0·001 and P = 0·009) in patients reaching the combined endpoint of cardiac hospitalization or death. FGF-23 (ln) predicted all-cause mortality (hazard ratio (HR) 5·042, P = 0·032) in a model adjusted for age, gender, estimated creatinine clearance, LVEF, New York Heart Association (NYHA) stage and NTproBNP level. Inorganic phosphate predicted heart failure hospitalization (HR 26·944, P = 0·021), cardiac hospitalization (HR 16·016, P = 0·017) and the combined endpoint (HR 13·294, P = 0·015) in models adjusted for the same co-variables.

CONCLUSIONS

The results of this study demonstrate the independent prognostic value of inorganic phosphate and FGF-23 in heart failure even in the context of established risk markers.

Oversulfated chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chains were purified from the notochord of hagfish. The chains (previously named CS-H for hagfish) have an average molecular mass of 18 kDa. Composition analysis using various chondroitinases demonstrated a variety of D-glucuronic acid (GlcUA)- and L-iduronic acid (IdoUA)-containing disaccharides variably sulfated with a higher proportion of GlcUA/IdoUA-GalNAc 4,6-O-disulfate, revealing complex CS/DS hybrid features. The hybrid chains showed neurite out<em>growth</em>-promoting activity of an axonic nature, which resembled the activity of squid cartilage CS-E and which was abolished fully by chondroitinase ABC digestion and partially by chondroitinase AC-I or B digestion, suggesting the involvement of both GlcUA and IdoUA in neuritogenic activity. Purified CS-H exhibited interactions in a BIAcore system with various heparin-binding proteins and neurotrophic <em>factors</em> (viz. <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, -10, -<em>16</em>, and -18; midkine; pleiotrophin; heparin-binding epidermal <em>growth</em> <em>factor</em>-like <em>growth</em> <em>factor</em>; vascular endothelial <em>growth</em> <em>factor</em>; brain-derived neurotrophic <em>factor</em>; and glial cell line-derived neurotrophic <em>factor</em>), most of which are expressed in the brain, although <em>fibroblast</em> <em>growth</em> <em>factor</em>-1 and ciliary neurotrophic <em>factor</em> showed no binding. Kinetic analysis revealed high affinity binding of these <em>growth</em> <em>factors</em> and, for the first time, of the neurotrophic <em>factors</em>. Competitive inhibition revealed the involvement of both IdoUA and GlcUA in the binding of these <em>growth</em> <em>factors</em>, suggesting the importance of the hybrid nature of CS-H for the efficient binding of these <em>growth</em> <em>factors</em>. These findings, together with those from the recent analysis of brain CS/DS chains from neonatal mouse and embryonic pig (Bao, X., Nishimura, S., Mikami, T., Yamada, S., Itoh, N., and Sugahara, K. (2004) J. Biol. Chem. 279, 9765-9776), suggest physiological roles of the hybrid chains in the development of the brain.

Recently, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) has been found to increase trabecular bone mass and connectivity in the proximal tibial metaphyses (PTM) in osteopenic rats. The purpose of this study was to determine the bone anabolic effects of bFGF in the lumbar vertebral body (LVB), a less loaded skeletal site with a lower rate of bone turnover than the PTM. Six-month old female Sprague-Dawley rats were ovariectomized (OVX) or sham-operated and untreated for 8 weeks to induce osteopenia. Then group 1 (sham) and group 2 (OVX) were treated subcutaneously (s.c.) with vehicle, and OVXed groups 3 and 4 were treated s.c. with PTH [hPTH (1-34) at 40 microg/kg, 5x/week] and bFGF (1 mg/kg, 5x/week), respectively, for 8 weeks. At sacrifice, the fifth LVB was removed, subjected to micro-CT for determination of trabecular bone structure and then processed for histomorphometry to assess bone turnover. The sixth LVB was used for mechanical compression testing (MTS, Bionix 858). The data were analyzed with the Kruskal-Wallis test followed by post-hoc testing as needed. After <em>16</em> weeks of estrogen deficiency, there were significant reductions in vertebral trabecular bone volume and trabecular thickness. Treatment with either bFGF or hPTH (1-34) increased BV/TV in OVX animals. Human PTH (1-34)-treated animals had significant increases in trabecular (48%) and cortical thickness (30%) and bone strength [maximum load (53%) and work to failure (175%)] compared to OVX + Vehicle animals. Treatment of osteopenic rats with bFGF increased bone volume (15%), trabecular thickness (13%), maximum load (45%) and work to failure (140%) compared to OVX + Vehicle animals (all P <0.05). Basic FGF increased trabecular bone volume in the lumbar vertebral body of osteopenic rats by restoring trabecular number, thickness and connectivity density. Also, bFGF improved bone mechanical properties (maximum force and work to failure) compared to the OVX + Vehicle group. Therefore, increasing the number, thickness and connections of the trabeculae contributes to increased bone strength in this small animal model of osteoporosis.

OBJECTIVE

The pathological accumulation of extracellular matrix is a characteristic feature of diabetic cardiomyopathy that is directly related to a loss of function. Tranilast (n-[3,4-anthranilic acid), used for the treatment of fibrotic skin diseases, has also been shown to inhibit transforming growth factor-beta (TGF-beta)-induced matrix production in kidney epithelial cells.

METHODS

To investigate the effects of tranilast in the diabetic heart, we examined its effects in cultured cardiac fibroblasts and then assessed its effects in (mRen-2)27 diabetic rats with established disease (8 weeks after streptozotocin).

RESULTS

In vitro studies demonstrated a 58% reduction in TGF-beta1-induced 3[H]-hydroxyproline incorporation with tranilast 30 microM (p<0.01). At 16 weeks, diabetes in the Ren-2 rat was associated with increased cardiac fibrosis and evidence of TGF-beta1 activation, as measured by the abundance of phosphorylated Smad2. Despite persistent hyperglycaemia and hypertension, tranilast attenuated cardiac fibrosis by 37% (p<0.05) in association with reduction in phospho-Smad2 (p<0.01).

CONCLUSIONS

These findings indicate that tranilast has antifibrotic actions in the Ren-2 model of experimental diabetic cardiac disease by mechanisms that might attributable to reduced TGF-beta activity.

The molecular weight of rat basic <em>fibroblast</em> <em>growth</em> <em>factor</em> is predicted to be 18 kDa when the amino acid sequence is read from the single AUG initiation codon found in the cDNA. DNA sequencing upstream of this AUG codon indicated, however, that there was an extended open reading frame. In vitro translation of the rat cDNA for basic FGF gave three proteins of 18.0, 21.5, and 22.0 kDa in equal abundance. The same proteins were produced in vivo by COS cells transfected with the rat cDNA. Deletion of 81 base pairs from the reading frame upstream of the AUG codon resulted in the expression of only one protein observed at 18.0 kDa. These results indicated that the 22.0 and 21.5 kDa forms of rat basic FGF were formed when translation initiates at the alternative upstream non-AUG codons. Rat cell lines and tissues were found to express all three forms of basic FGF protein. The cDNA was used to analyze the subcellular distribution of the different forms of rat basic FGF. Subcellular fractionation and immunofluorescence of transfected COS cells showed that all three forms of the protein localized preferentially in the nucleus. Expression of a truncated cDNA from which 81 base pairs (27 amino acids) of the upstream reading frame had been deleted, showed localization of the smaller form of bFGF alone in the nucleus. These results demonstrated that although the amino acids that were deleted from the N-terminus of rat basic <em>fibroblast</em> <em>growth</em> <em>factor</em> have a sequence characteristic of nuclear localization motifs, they are not obligatory for the transport of the <em>growth</em> <em>factor</em> into the nucleus. Nuclear extracts taken from transfected cells also contained two smaller proteins of <em>16</em> and 12 kDa that were detected by Western blot analysis. It is possible that these are proteolytic products of bFGF.

The <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family consists of seven members whose activities are thought to be mediated by multiple receptors. Here we describe the cDNA cloning, expression, and characterization of a cysteine-rich FGF receptor (CFR) that is distinct from previously identified FGF receptors. The deduced amino acid sequence for CFR suggests that it is an integral membrane protein containing a large extracellular domain comprising <em>16</em> cysteine-rich repeated units and an intracellular domain of 13 amino acids. No reported sequences exhibit significant homologies to either the repeated extracellular motif or to the entire CFR amino acid sequence. Several CFR transcripts are present in embryonic chick tissue, suggesting that CFR undergoes alternate mRNA splicing or that related genes are present. Chinese hamster ovary cells transfected with the CFR cDNA express a 150-kDa polypeptide that binds FGF-1, FGF-2, and FGF-4 but does not bind several non-FGF family members. The high degree of evolutionary conservation among vertebrate CFRs and its ability to bind three different FGFs with high affinity suggest that this unique receptor plays an important role in FGF biology.

Despite numerous studies on the effects of gonadotropins on ovarian cells in tissue culture, the <em>factors</em> controlling the proliferation of granulosa cells in vitro remain unknown. We have examined the effect of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) and epidermal <em>growth</em> <em>factor</em> (EGF) on granulosa cell proliferation in vitro in an attempt to clarify their possible roles in the control of ovarian development. FGF and EGF both stimulate DNA synthesis in resting populations of granulosa cells. The half-maximal response forthis effect with FGF was observed at 4 X 10(-11)M and with EGF at 1.5 X 10(-13)M. Autoradiography demonstrated that the whole cell population initiated DNA synthesis in the presence of either EGF or FGF, thus precluding an additive effect of the two mitogens. When cells were maintained at low density (100 cells/cm2) in the presence of low serum (1%) they divided with a doubling time of 48-72 h, but addition of either EGF or FGF accelerated their proliferation. The doubling time observed in the presence of FGF was <em>16</em> h versus 20 h with EGF and the final cell density reached in the presence of EGF or FGF was 20 times that of cells maintained in the presence of 1% calf serum alone. In the presence of 10% serum, granulosa cells had a doubling time of 24 h and the final density reached was similar to that observed in 1% serum with EGF and FGF. Addition of EGF or FGF to 10% serum resulted in a final density 3 to 4-fold higher than that observed with 10% serum alone. The ultrastructure of the granulosa cells grown in the presence of EGF or FGF was similar to that of cells maintained in the absence of added mitogens. The only marked difference was that cells grown in the presence of FGF or EGF had a high lipid granule content while cells grown in their absence had a low lipid granule content. The effect of various concentrations of FGF and EGF on the proliferation of granulosa cells has been analyzed. The minimal effective dose of EGF was 3 X 10(-14)M and saturation was observed at 3 X 10(-11)M, with a half-maximal response at 6 X 10(-13)M. With FGF the minimal dose stimulating proliferation was 1.5 X 10(-12)M and saturation was achieved at 1.5 X 10(-10)M, with a half-maximal response at 3 X 10(-11)M. Our results show that EGF and FGF are the most potent mitogens ever observed and are mitogenic for granulosa cells at 300 to 3000-fold lower concentrations than for other cell types which have been studied, such as <em>fibroblasts</em> or lens epithelial cells.

<em>Factor</em> H (FH) of the complement system acts as a regulatory co<em>factor</em> for the <em>factor</em> I-mediated cleavage of C3b and binds to polyanionic substrates. FH is composed of 20 short consensus/complement repeat (SCR) domains. A set of 12 missense mutations in the C-terminal domains between SCR-<em>16</em> to SCR-20 is associated with haemolytic uraemic syndrome. Recent structural models for intact FH permit the molecular interpretation of these amino acid substitutions. As all nine SCR-20 substitutions correspond to normal amounts of FH in plasma, and were localised in mostly surface-exposed positions, these are inferred to lead to a functional defect in FH. The nine substitutions occur in the same spatial region of SCR-20. As this surface coincides with conserved basic residues in the C-terminal SCR-20 domain, the substitutions provide direct evidence for a polyanionic binding surface. The positions of these conserved basic residues coincide with those of heparin-binding residues in the crystal structure of the acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>-heparin complex. A tenth substitution and another conserved basic residue in SCR-19 are proximate to this binding site. As the remaining FH substitutions could also be correlated with their proximity to conserved basic residues, haemolytic uraemic syndrome may result from a failure of FH to interact with polyanions at cell surfaces in the kidney.

Recent evidence suggests that <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) is a primary mesoderm inducer in Xenopus development. We have isolated a full-length cDNA clone for the Xenopus FGF receptor. Like other FGF receptors, the Xenopus homolog is a membrane-spanning protein with a split intracellular tyrosine kinase domain. The Xenopus FGF receptor mRNA is present as a maternal message whose levels are constant through early development. There is no specific regional localization of the transcript by analysis of FGF receptor mRNA levels in microdissected embryonic tissue. In isolated animal-pole blastomeres, FGF receptor mRNA declines over <em>16</em> hr in culture and this loss can be prevented by incubation with FGF or activin. Despite the presence of the FGF receptor mRNA in the oocyte, oocytes in culture do not respond to added FGF. However, injection of exogenous Xenopus FGF receptor transcripts into oocytes does generate a functional response to FGF. Our data suggest that posttranscriptional response to FGF. Our data suggest that posttranscriptional mechanisms regulate the FGF receptor in the oocyte and early embryo and further suggest that mesoderm-inducing <em>factors</em> influence receptor mRNA levels during the time of early tissue formation.

The minimal signalling unit for tyrosine kinase receptors is two protomers dimerized by one or more ligands. However, it is clear that maximal signalling requires the formation of larger complexes of many receptors at discrete foci on the cell surface. The biological interactions that lead to this are likely to be diverse and have system specific components. In the present study, we demonstrate that, in the FGF (<em>fibroblast</em> <em>growth</em> <em>factor</em>)-FGFR (FGF receptor) system, multimers of the minimal complex composed of two FGF1 and two FGFR2 protomers can form on a single chain of the co-receptor heparin. Using size-exclusion chromatography, we show that two complexes can form on heparin chains as small as <em>16</em> saccharide units. We also show by MS that discrete complexes containing exactly two copies of the minimal signalling unit are formed. However, the doublet of complexes appears to be less co-operative than the formation of the 2:2:1 FGF1:FGFR2:heparin complex, suggesting that this mechanism is one of a number of weaker interactions that might be involved in the formation of a focal complex on the cell surface.

The sleeping brain differs from the waking brain in its electrophysiological and molecular properties, including the expression of <em>growth</em> <em>factors</em> and immediate early genes (IEG). Sleep architecture and homeostatic regulation of sleep in neonates is distinct from that of adults. Hence, the present study addressed the question whether the unique homeostatic response to sleep deprivation in neonates is reflected in mRNA expression of the IEG cFos, brain-derived nerve <em>growth</em> <em>factor</em> (BDNF), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2) in the cortex. As sleep deprivation is stressful to developing rats, we also investigated whether the increased levels of corticosterone would affect the expression of <em>growth</em> <em>factors</em> in the hippocampus, known to be sensitive to glucocorticoid levels. At postnatal days <em>16</em>, 20, and 24, rats were subjected to sleep deprivation, maternal separation without sleep deprivation, sleep deprivation with 2 h recovery sleep, or no intervention. mRNA expression was quantified in the cortex and hippocampus. cFos was increased after sleep deprivation and was similar to control level after 2 h recovery sleep irrespective of age or brain region. BDNF was increased by sleep deprivation in the cortex at P20 and P24 and only at P24 in the hippocampus. FGF2 increased during recovery sleep at all ages in both brain regions. We conclude that cortical BDNF expression reflects the onset of adult sleep-homeostatic response, whereas the profile of expression of both <em>growth</em> <em>factors</em> suggests a trophic effect of mild sleep deprivation.

BACKGROUND

Fibroblast growth factor 23 (FGF23) increases renal phosphate excretion and decreases the formation of 1,25 dihydroxyvitamin D. In patients with chronic kidney disease, plasma FGF23 levels are markedly elevated by unknown mechanisms. We explored the changes in FGF23 during treatment of secondary hyperparathyroidism (SHPT) with alfacalcidol or paricalcitol in haemodialysis patients.

METHODS

Intravenous alfacalcidol and paricalcitol were compared in haemodialysis patients with SHPT in a randomized 2 × 16-week cross-over study, with 6 weeks washout period preceding treatment and between treatment periods. In 57 of the enrolled patients, blood samples were frozen before and after each treatment period and available for measurement of FGF23.

RESULTS

Treatment with both analogues increased FGF23 (P < 0.05 in all treatment periods). The magnitude of increase was similar for alfacalcidol and paricalcitol (Period 1: 223 versus 314%; P = 0.384 and Period 2: 174 versus 227%; P = 0.510) and the levels returned to pre-treatment levels during the washout period. Independent predictors of rise in FGF23 were baseline levels of FGF23 (P < 0.01), changes in ionized calcium (P < 0.01) and phosphate (P < 0.01) and cumulative dose of vitamin D analogues (P = 0.024).

CONCLUSIONS

Alfacalcidol and paricalcitol increase the plasma levels of FGF23 equally and substantially in haemodialysis patients.

OBJECTIVE

Fibroblast growth factor (FGF21) is a potent regulator of glucose and lipid metabolism. In rodents, the hepatic expression of FGF21 is controlled by fasting and a circadian regulation, but the physiological role and regulation of FGF21 in humans is not well established. Therefore, the objective of this study was to elucidate the 24-h profiling of plasma FGF21 during a 72-h fast.

METHODS

After an initial 12-h overnight fast, plasma levels of FGF21 together with circulating levels of glucose, insulin, glucagon and free fatty acids (FFA) were measured every 6 h during a 72-h fast in healthy female volunteers (n = 14).

RESULTS

During the fast, plasma glucose and serum insulin gradually decreased whilst glucagon and FFA levels increased. Mean 24-h plasma FGF21 levels did not increase during the 72-h fast, but, despite large inter-individually variations, a significant circadian rhythm was observed. Thus, during the first day, plasma FGF21 increased from 125 pg/ml (16-142) at 8.30 a.m to peak levels of 224 pg/ml (88-326) at 2.30 a.m (P = 0·0215), the second day from 60 pg/ml (7-74) at 8.30 a.m to 268 pg/ml (103-410) at 2.30 a.m. (P = 0·0010) and the third day from 94 pg/ml (9-146) to 151 pg/ml (87-207) at 2.30 a.m. (P = 0·0049). Cortisol also showed circadian regulation and peaked at 8.30 a.m every day, 6 h after the observed FGF21 peak.

CONCLUSIONS

Plasma FGF21 follows a circadian rhythm during a 72-h fast in healthy female subjects. The circadian regulation has a stronger impact on plasma FGF21 than the fasting status over 72-h period.

Several matrix metalloproteinases (MMPs), including MMP-1, -3, and -9, mediate matrix destruction during chronic inflammatory diseases such as arthritis and atherosclerosis. MMP up-regulation by inflammatory cytokines involves interactions between several transcription <em>factors</em>, including activator protein-1 and nuclear <em>factor</em> kappaB (NF-kappaB). The upstream regulatory pathways are less well understood. We investigated the role of isoforms of protein kinase C (PKC) in basic <em>fibroblast</em> <em>growth</em> <em>factor</em>- and interleukin-1alpha-mediated MMP production from cultured rabbit aortic smooth muscle cells. A synthetic PKC inhibitor, RO318220, inhibited MMP-1, -3, and -9 production by 89 +/- 3, 75 +/- 18, and 89 +/- 9%, respectively. However, down-regulation of conventional and novel isoforms did not inhibit but rather increased MMP-9 production by 48 +/- <em>16</em>%, implicating an atypical PKC isoform. Consistent with this, PKCzeta protein levels and activity were stimulated 3.3- and 13-fold, respectively, by basic <em>fibroblast</em> <em>growth</em> <em>factor</em> plus interleukin-1alpha and antisense oligonucleotides to PKCzeta significantly decreased MMP-9 formation by 62 +/- 18% compared with scrambled sequences. Moreover, adenovirus-mediated overexpression of a dominant-negative (DN) PKCzeta reduced MMP-1, -3, and -9 production by 78 +/- 9, 76 +/- 8, and 76 +/- 5%, respectively. DN-PKCzeta inhibited NF-kappaB DNA binding but did not affect ERK1/2 activation or AP-1 binding. Antisense PKCzeta oligonucleotides and DN-PKCzeta stimulated cell proliferation by 89 +/- 14% (n = 4) and 305 +/- 74% (n = 3), respectively (both p < 0.05). Our results show that PKCzeta is essential for cytokine-induced up-regulation of MMP-1, -3, and -9, most likely by activating NF-kappaB. Selective inhibition of PKCzeta is therefore a possible strategy to inhibit MMP production in inflammatory diseases such as atherosclerosis.

Follistatin (Fst) functions to bind and neutralize the activity of members of the transforming <em>growth</em> <em>factor</em>-β superfamily. Fst has a well-established role in skeletal muscle, but we detected significant Fst expression levels in interscapular brown and subcutaneous white adipose tissue, and further investigated its role in adipocyte biology. Fst expression was induced during adipogenic differentiation of mouse brown preadipocytes and mouse embryonic <em>fibroblasts</em> (MEFs) as well as in cold-induced brown adipose tissue from mice. In differentiated MEFs from Fst KO mice, the induction of brown adipocyte proteins including uncoupling protein 1, PR domain containing <em>16</em>, and PPAR gamma coactivator-1α was attenuated, but could be rescued by treatment with recombinant FST. Furthermore, Fst enhanced thermogenic gene expression in differentiated mouse brown adipocytes and MEF cultures from both WT and Fst KO groups, suggesting that Fst produced by adipocytes may act in a paracrine manner. Our microarray gene expression profiling of WT and Fst KO MEFs during adipogenic differentiation identified several genes implicated in lipid and energy metabolism that were significantly downregulated in Fst KO MEFs. Furthermore, Fst treatment significantly increases cellular respiration in Fst-deficient cells. Our results implicate a novel role of Fst in the induction of brown adipocyte character and regulation of energy metabolism.

BACKGROUND

Data from studies in patients with nonalcoholic steatohepatitis (NASH) suggest an increased hepatic fatty acid oxidation. We have previously shown higher fasting plasma bile acid concentrations in patients with NASH. In-vivo and in-vitro studies suggest that bile acids by binding to peroxisome proliferator-activated receptor α activate fibroblast growth factor 21 (FGF21) and increase hepatic fatty acid oxidation.

METHODS

Plasma bile acid levels were quantified in healthy controls (n=38) and patients with biopsy-proven NASH (n=36). Plasma concentration of fatty acids, β-hydroxybutyrate, insulin, glucose, leptin, alanine aminotransferase, FGF21, and 8-hydroxydeoxyguanosine, a measure of oxidative stress, were measured in 16 healthy controls and 10 patients with NASH in the fasted state and in response to 3 h of infusion of intralipid. In a subgroup of these patients (n=6 each), plasma ceramide subspecies were quantified.

RESULTS

Fasting plasma bile acids, FGF21, and leptin concentrations were significantly higher in patients with NASH. In response to intralipid infusion there was an increase in plasma β-hydroxybutyrate and free fatty acid levels in both controls and NASH; however, the ratio of β-hydroxybutyrate/free fatty acid was higher in NASH (P=0.02). Plasma FGF21 concentration increased in response to intralipid in patients with NASH only (P<0.01). Plasma leptin, insulin, glucose, and alanine transferase concentrations did not change in either group after infusion of intralipid. Increase in total ceramides in response to intralipid was greater in NASH.

CONCLUSIONS

Elevated bile acids and FGF21 may be responsible for the higher hepatic fatty acid oxidation in NASH.