Severe neonatal Marfan syndrome has features of the Marfan syndrome and congenital contractural arachnodactyly present at birth, along with unique features such as loose, redundant skin and pulmonary emphysema. Since the Marfan syndrome and congenital contractural arachnodactyly are due to mutations in different genes, it has been uncertain whether neonatal Marfan syndrome is due to mutations in the fibrillin gene on chromosome <em>15</em> or in another gene. We studied an infant with severe neonatal Marfan syndrome. Dermal <em>fibroblasts</em> were metabolically labeled and found to secret fibrillin inefficiently when compared with control cells. Reverse transcription and amplification of the proband's <em>fibroblast</em> RNA was used to identify a 3-bp insertion between nucleotides 480-481 or 481-482 of the fibrillin cDNA. The insertion maintains the reading frame of the protein and inserts a cysteine between amino acids 160 and 161 in an epidermal <em>growth</em>-<em>factor</em>-like motif of fibrillin. This 3-bp insertion was not found in the fibrillin gene in 70 unrelated, unaffected individuals and 11 unrelated individuals with the Marfan syndrome. We conclude that neonatal Marfan syndrome is the result of mutations in the fibrillin gene on chromosome <em>15</em> and is part of the Marfan syndrome spectrum.

A novel family of RGD-containing molecules (Tablysin-<em>15</em>) has been molecularly characterised from the salivary gland of the haematophagous horsefly Tabanus yao. Tablysin-<em>15</em> does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-<em>15</em> exhibits high affinity binding for platelet αIIbβ3 and endothelial cell αVβ3 integrins, but not for α5β1 or α2β1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~1 nM) and marginally to fibronectin (IC50 ~1 μM), but not to collagen. It also inhibits <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-<em>15</em> inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilised fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbβ3 binds to Tablysin-<em>15</em>. Moreover, immobilised Tablysin-<em>15</em> supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbβ3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-<em>15</em> dose-dependently attenuates thrombus formation to collagen under flow. Consistent with these findings, Tablysin-<em>15</em> displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbβ3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-<em>15</em> represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.

UNASSIGNED

Approximately 5%-10% of gastric cancers have a fibroblast growth factor receptor-2 (FGFR2) gene amplification. AZD4547 is a selective FGFR-1, 2, 3 tyrosine kinase inhibitor with potent preclinical activity in FGFR2 amplified gastric adenocarcinoma SNU16 and SGC083 xenograft models. The randomized phase II SHINE study (NCT01457846) investigated whether AZD4547 improves clinical outcome versus paclitaxel as second-line treatment in patients with advanced gastric adenocarcinoma displaying FGFR2 polysomy or gene amplification detected by fluorescence in situ hybridization.

UNASSIGNED

Patients were randomized 3:2 (FGFR2 gene amplification) or 1:1 (FGFR2 polysomy) to AZD4547 or paclitaxel. Patients received AZD4547 80 mg twice daily, orally, on a 2 weeks on/1 week off schedule of a 21-day cycle or intravenous paclitaxel 80 mg/m2 administered weekly on days 1, 8, and 15 of a 28-day cycle. The primary end point was progression-free survival (PFS). Safety outcomes were assessed and an exploratory biomarker analysis was undertaken.

UNASSIGNED

Of 71 patients randomized (AZD4547 n = 41, paclitaxel n = 30), 67 received study treatment (AZD4547 n = 40, paclitaxel n = 27). Among all randomized patients, median PFS was 1.8 months with AZD4547 and 3.5 months with paclitaxel (one-sided P = 0.9581); median follow-up duration for PFS was 1.77 and 2.12 months, respectively. The incidence of adverse events was similar in both treatment arms. Exploratory biomarker analyses revealed marked intratumor heterogeneity of FGFR2 amplification and poor concordance between amplification/polysomy and FGFR2 mRNA expression.

UNASSIGNED

AZD4547 did not significantly improve PFS versus paclitaxel in gastric cancer FGFR2 amplification/polysomy patients. Considerable intratumor heterogeneity for FGFR2 gene amplification and poor concordance between FGFR2 amplification/polysomy and FGFR2 expression indicates the need for alternative predictive biomarker testing. AZD4547 was generally well tolerated.

Accelerated rehabilitation after tendon and ligament injuries is widely accepted to avoid adverse effects of immobilization. However, progressive rehabilitation may also lead to an excessive inflammatory soft tissue response. To investigate the amount of loading necessary to accelerate the healing process without causing damage to the healing tissue, we experimentally stretched human tendon <em>fibroblasts</em> of healthy tendons <em>15</em> and 60 min with 1 Hz and an elongation of 5% and measured the secretion of interleukin 6 (IL-6), tumor necrosis <em>factor</em> alpha (TNF-alpha), transforming <em>growth</em> <em>factor</em> beta1 (TGF-beta1), platelet-derived <em>growth</em> <em>factor</em> (PDGF), and <em>fibroblast</em> <em>growth</em> <em>factor</em> basic (bFGF). Secretion of IL-6 was significantly induced by <em>15</em> min of cyclic biaxial mechanical stretching after 4 and 8 h observation time and by 60 min stretching and 2 h observation time. The <em>growth</em> <em>factors</em> TGF-beta1, bFGF, and PDGF were secreted by human tendon <em>fibroblasts</em> both in stretched cells and controls; however, no increases were related to mechanical stretching. There was no measurable secretion of TNF-alpha in human tendon <em>fibroblasts</em>. These findings suggest that the inflammatory reaction often seen during physiotherapy after tendon and ligament injuries is caused in part by secretion of IL-6 from the stretched human tendon <em>fibroblasts</em>. IL-6 may cause exaggerated proliferation of <em>fibroblasts</em> and synovial cells as seen in rheumatoid arthritis and arthrofibrosis. However, physiological proliferative reactions leading to repair of injured tissue are also possible. IL-6 measured in the synovial fluid may be an important predictor for monitoring and improving therapeutic strategies in terms of tendon/ligament healing.

Radiation-induced pulmonary fibrosis is a common disease and has a poor prognosis owing to the progressive breakdown of gas exchange regions in the lung. Recently, a novel strategy of administering mesenchymal stem cells for pulmonary fibrosis has achieved high therapeutic efficacy. In the present study, we attempted to use human adipose tissue-derived mesenchymal stem cells to prevent disease in Sprague-Dawley rats that received semi-thoracic irradiation (<em>15</em> Gy). To investigate the specific roles of mesenchymal stem cells in ameliorating radiation-induced pulmonary fibrosis, we treated control groups of irradiated rats with human skin <em>fibroblasts</em> or phosphate-buffered saline. After mesenchymal stem cells were infused, host secretions of hepatocyte <em>growth</em> <em>factor</em> (HGF) and prostaglandin E2 (PGE2) were elevated compared with those of the controls. In contrast, tumour necrosis <em>factor</em>-alpha (TNF-α) and transforming <em>growth</em> <em>factor</em>-beta1 (TGF-β1) levels were decreased after infusion of mesenchymal stem cells. Consequently, the architecture of the irradiated lungs was preserved without marked activation of <em>fibroblasts</em> or collagen deposition within the injured sites. Moreover, mesenchymal stem cells were able to prevent the irradiated type II alveolar epithelial cells from undergoing epithelial-mesenchymal transition. Collectively, these data confirmed that mesenchymal stem cells have the potential to limit pulmonary fibrosis after exposure to ionising irradiation.

Well-differentiated liposarcoma (WDLS) is one of the most common malignant mesenchymal tumors and dedifferentiated liposarcoma (DDLS) is a malignant tumor consisting of both WDLS and a transformed nonlipogenic sarcomatous component. Cytogenetically, WDLS is characterized by the presence of ring or giant rod chromosomes containing several amplified genes, including MDM2, TSPAN31, CDK4, and others mainly derived from chromosome bands 12q13-<em>15</em>. However, the 12q13-<em>15</em> amplicon is large and discontinuous. The focus of this study was to identify novel critical genes that are consistently amplified in primary (nonrecurrent) WDLS and with potential relevance for future targeted therapy. Using a high-resolution (5.0 kb) "single nucleotide polymorphism"/copy number variation microarray to screen the whole genome in a series of primary WDLS, two consistently amplified areas were found on chromosome 12: one region containing the MDM2 and CPM genes, and another region containing the FRS2 gene. Based on these findings, we further validated FRS2 amplification in both WDLS and DDLS. Fluorescence in situ hybridization confirmed FRS2 amplification in all WDLS and DDLS tested (n = 57). Real time PCR showed FRS2 mRNA transcriptional upregulation in WDLS (n = 19) and DDLS (n = 13) but not in lipoma (n = 5) and normal fat (n = 9). Immunoblotting revealed high expression levels of phospho-FRS2 at Y436 and slightly overexpression of total FRS2 protein in liposarcoma but not in normal fat or preadipocytes. Considering the critical role of FRS2 in mediating <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor signaling, our findings indicate that FRS2 signaling should be further investigated as a potential therapeutic target for liposarcoma.

alpha 2-Macroglobulin (alpha 2M) binds numerous cytokines; however, since binding affinities have not been determined, it is difficult to compare various alpha 2M-cytokine interactions or predict whether alpha 2M-cytokine complexes will form in the presence of other cytokine-binding macromolecules. In this investigation, we used a novel method to demonstrate that transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1), TGF-beta 2, nerve <em>growth</em> <em>factor</em>-beta (NGF-beta), platelet derived <em>growth</em> <em>factor</em>-BB (PDGF-BB), tumor necrosis <em>factor</em>-alpha (TNF-alpha), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) reversibly associate with alpha 2M-methylamine to form noncovalent complexes. Apparent equilibrium was achieved in less than <em>15</em> min. Noncovalent alpha 2M-cytokine complexes were converted into covalent complexes; however, this occurred slowly. Therefore, a rapid equilibrium assumption was applied and equilibrium dissociation constants were determined using a single binding site model. KD values for the binding of cytokines to alpha 2M-methylamine varied by 2 orders of magnitude. The rank order of affinity was TGF-beta 2 (13 +/- 2 nM)>> TGF-beta 1, NGF-beta>> PDGF-BB>> or = bFGF>> TNF-alpha. Native alpha 2M bound TGF-beta 1, TGF-beta 2, NGF-beta, PDGF-BB, and TNF-alpha. Interferon-gamma did not bind to native alpha 2M or alpha 2M-methylamine. Each cytokine bound native alpha 2M with lower affinity than alpha 2M-methylamine except for TGF-beta 2 which bound both forms with equal affinity. In non-equilibrium systems, alpha 2M-methylamine appeared to bind more TGF-beta 2 due to the more rapid dissociation of TGF-beta 2-native alpha 2M complex. The classification of alpha 2M-cytokine complexes according to binding affinity should predict which complexes are most likely to form in cell culture and under various conditions in vivo.

BACKGROUND

Therapeutic angiogenesis is a promising approach for the treatment of cardiovascular diseases, including myocardial infarction and chronic heart failure. We aimed to improve proangiogenic therapies by identifying novel arteriogenic growth factor combinations, developing injectable delivery systems for spatiotemporally controlled growth factor release, and evaluating functional consequences of targeted intramyocardial growth factor delivery in chronic heart failure.

RESULTS

First, we observed that fibroblast growth factor and hepatocyte growth factor synergistically stimulate vascular cell migration and proliferation in vitro. Using 2 in vivo angiogenesis assays (n=5 mice per group), we found that the growth factor combination results in a more potent and durable angiogenic response than either growth factor used alone. Furthermore, we determined that the molecular mechanisms involve potentiation of Akt and mitogen-activated protein kinase signal transduction pathways, as well as upregulation of angiogenic growth factor receptors. Next, we developed crosslinked albumin-alginate microcapsules that sequentially release fibroblast growth factor-2 and hepatocyte growth factor. Finally, in a rat model of chronic heart failure induced by coronary ligation (n=14 to 15 rats per group), we found that intramyocardial slow release of fibroblast growth factor-2 with hepatocyte growth factor potently stimulates angiogenesis and arteriogenesis and prevents cardiac hypertrophy and fibrosis, as determined by immunohistochemistry, leading to improved cardiac perfusion after 3 months, as shown by magnetic resonance imaging. These multiple beneficial effects resulted in reduced adverse cardiac remodeling and improved left ventricular function, as revealed by echocardiography.

CONCLUSIONS

Our data showing the selective advantage of using fibroblast growth factor-2 together with hepatocyte growth factor suggest that this growth factor combination may constitute an efficient novel treatment for chronic heart failure.

T cell differentiation in the thymus is dependent upon signals from thymic stromal cells. Most studies into the nature of these signals have focused only on the support provided by the thymic epithelium, but there is an emerging view that other stromal cells such as mesenchymal <em>fibroblasts</em> may also be involved. Study of the latter has been hindered by a lack of appropriate markers, particularly those allowing their isolation. In this study, we describe a new surface marker of thymic stroma, MTS-<em>15</em>, and demonstrate its specificity for <em>fibroblasts</em> and a subset of endothelial cells. Coculture experiments showed that the determinant could be transferred between cells. Extensive biochemical analysis demonstrated that the Ag bound by MTS-<em>15</em> was the glycosphingolipid Forssman determinant, consistent with the distribution observed. Transcriptional analysis of purified MTS-<em>15</em>(+) thymic <em>fibroblasts</em> revealed a unique expression profile for a number of chemokines and <em>growth</em> <em>factors</em> important to thymocyte and epithelial cell development. In a model of cyclophosphamide-induced thymic involution and regeneration, <em>fibroblasts</em> were found to expand extensively and express <em>growth</em> <em>factors</em> important to epithelial proliferation and increased T cell production just before thymic regeneration. Overall, this study identifies a useful marker of thymic <em>fibroblasts</em> and highlights this subpopulation as a key player in thymic function by virtue of their support of both thymocytes and epithelial cells.

Major features of a long-standing inflammation in the kidney are vascular proliferation, glomerulosclerosis, interstitial fibrosis and tubular atrophy, leading to a gradual deterioration of the renal function. In this study we have investigated the expression of B-type receptors for platelet-derived <em>growth</em> <em>factor</em> (PDGF) in frozen sections from normal and inflamed kidneys. Immunohistochemical techniques, employing two monoclonal antibodies specific for PDGF B-type receptors, were used. The specimens investigated were <em>15</em> kidneys removed by transplantectomy because of chronic rejection, 20 cases of glomerulonephritis with crescent formation, mesangial proliferation or non-proliferative glomerulonephritis, and six normal kidneys. In parallel we characterized cellular infiltrates and class II transplantation antigen expression in the inflamed kidneys. An enhanced PDGF receptor expression was found on intimal cells and on smooth muscle cells of the proliferating vessels, on glomerular cells in glomeruli with mesangial proliferation, and on <em>fibroblast</em>-like cells in the proximity of clusters of infiltrating macrophages and T-lymphocytes of the interstitial tissue. Induction of PDGF receptor expression may render cells responsive to stimulation by PDGF, released from PDGF-producing cells, such as activated macrophages and from platelets. Our data suggest that PDGF is involved in the proliferation of mesenchymal cells that is seen in rejected kidney transplants and glomerulonephritis.

Human neural precursor cell cultures (neurospheres) were established from fetal brain tissues of <em>15</em>-20 gestation weeks and propagated for over a year in the presence of epidermal <em>growth</em> <em>factor</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and leukemia inhibitory <em>factor</em>. Neurospheres were differentiated without the presence of above <em>growth</em> <em>factors</em> to follow the development of oligodendroglia. Oligodendroglial progenitors, identified by their bipolar morphology and expression of platelet-derived <em>growth</em> <em>factor</em> receptor-alpha (PDGFRalpha), emerged from spheres as early as 1 DIV; O4+ cells with bipolar to multipolar processes were observed at 3 DIV whereas O1+ multiprocess-bearing oligodendroglia did not appear until 5-7 DIV. They further differentiated to myelin basic protein-expressing oligodendrocytes after 2-3 weeks in culture. Thus, human oligodendroglial maturation in vitro follows the same pathway as rat cells but takes twice as long as their rodent counterparts. Bromodeoxyuridine incorporation indicated that PDGFRalpha-expressing cells but not O4+ oligodendroglia proliferated. More oligodendroglial progenitors incorporated BrdU and more O4+ cells survived when they were in contact with neurons and astrocytes than when they developed beyond the astrocyte layer. In addition, oligodendroglia on astrocytes had a complex process branching whereas those <em>growing</em> beyond astrocyte layer often formed membrane sheaths. Thus the survival, proliferation and maturation of oligodendroglia are influenced by other cell types.

The relative contributions of type I and type II insulinlike <em>growth</em> <em>factor</em> (IGF) receptors and IGF carrier proteins to the binding of IGF-I tracer to cultured human <em>fibroblasts</em> were determined in competitive binding experiments that used unlabeled insulin and synthetic insulin-IGF-I hybrid molecules containing the A chain of insulin and the B domain of IGF-I. Whereas insulin binds only to type I IGF receptors, the B-IGF-I hybrids bind to type I receptors and IGF carrier proteins but not to type II receptors. In suspended human <em>fibroblasts</em>, IGF-I tracer binds predominantly to type I IGF receptors (inhibition by IGF-I much greater than insulin greater than B-IGF-I hybrid molecules). By contrast, in <em>fibroblast</em> monolayers, IGF-I binding was minimally inhibited by insulin or hybrid molecules, suggesting predominant binding to the type II IGF receptor. The type I receptor appears to be masked on <em>fibroblast</em> monolayers, and to require suspension or detergent solubilization of the cells to be demonstrated. In the course of the monolayers binding experiments, we noted that low concentrations of unlabeled IGF-I (5-10 ng/ml) or B-IGF-I hybrids (100 ng/ml) paradoxically increased IGF-I tracer binding up to twofold. We postulated that during the binding incubation (5 h, <em>15</em> degrees C), IGF-I tracer partitioned between binding sites on the cell surface and IGF carrier proteins released to the incubation media. Preferential occupancy of binding sites in the media by unlabeled ligand increased the tracer available to bind to the cells. In support of this hypothesis, carrier proteins were demonstrated in the media at the end of the binding incubation with <em>fibroblast</em> monolayers, and the concentration of unsaturated binding sites in the media correlated inversely with tracer binding to the cells. Thus carrier proteins released to the media during the binding incubation modulate the binding of IGF-I tracer to cell receptors, suggesting that the carrier proteins may play an important role in regulating cellular responsiveness to the IGFs.

OBJECTIVE

To clarify whether synovial cell proliferation indicates an imbalance in production between angiogenic growth factors and angiogenesis inhibitors in rheumatoid arthritis (RA), we investigated the production of basic fibroblast growth factor (b-FGF) and vascular endothelial growth factor (VEGF) as representative angiogenic growth factors and endostatin as a representative angiogenesis inhibitor.

METHODS

The b-FGF, VEGF, and endostatin levels in 90 samples of peripheral blood (PB) and 15 samples of joint fluid obtained from patients with RA and 30 samples of PB and 10 samples of joint fluid from patients without RA, including 20 patients with inflammatory arthritis without purulent arthritis, and 10 patients with osteoarthritis were measured by ELISA. VEGF and endostatin levels in blood samples from 22 patients with RA were measured at 2 points: before and 4 or 5 months after the commencement of medication.

RESULTS

The b-FGF and VEGF levels in the PB and joint fluid samples from patients with RA were markedly elevated compared to samples from patients without RA. In contrast, endostatin levels in PB and joint fluid samples from patients with RA were almost the same as in the samples from patients without RA. VEGF levels in blood samples obtained 4 or 5 months after the commencement of medication (combination of prednisolone 5 mg/day and disease modifying antirheumatic drugs: either bucillamine 100 mg/day or salazosulfapyridine 1,000 mg/day) were significantly decreased from 27.1 +/- 8.5 pg/ml in samples obtained before commencement of medication to 18.1 +/- 16.2 pg/ml. Endostatin levels in the corresponding samples were significantly increased, from 31.5 +/- 7.0 to 57.1 +/- 22.8 ng/ml [correction].

CONCLUSIONS

Our results reveal significant differences in b-FGF and VEGF levels in PB and joint fluid samples, but no difference in endostatin levels, between patients with RA and those without RA, suggesting that angiogenesis in RA occurs as a result of an imbalance in production between angiogenic growth factors and angiogenesis inhibitors.

Bovine brain and pituitary <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGF) have been compared with regard to their chemical and biological properties. Pituitary and one preparation of brain FGF (Prep A) contain a basic mitogenic activity, which migrates to the same position on electrophoresis in acid pH gels as detected by incorporation of [methyl-3H]-thymidine into BALB/c 3T3 cells. In contrast, another preparation of brain FGF (Prep B) contains two mitogens, one (20-30%) indistinguishable from the basic components in pituitary and brain (Prep A) FGF preparations and an acidic activity (70-80%), pl 5-6, that migrates more slowly on acid gels, corresponding to the acidic component of brain FGF described previously (Thomas, K. A., M. C. Riley, S. K. Lemmon, N. C. Baglan, and R. A. Bradshaw. 1980. J. Biol. Chem. 255:5517-5520.) In agreement with that report, none of the mitogens comigrates with fragments of myelin basic protein. Pituitary FGF was virtually inactive, brain (Prep A) FGF had a small amount of activity, and brain (Prep B) FGF was highly potent (50% maximal stimulation at <em>15</em>-30 ng/ml) in stimulating the <em>growth</em> of human umbilical vein endothelial (HUVE) cells. The acidic component of brain FGF, which is much more unstable at pH 8.5 than the basic one, can be protected by reducing agents, whereas the basic constituent of brain FGF as well as pituitary FGF is unaffected by reducing conditions. Thus, brain FGF preparations may contain two distinct mitogenic activities, one that is acidic and contains HUVE cell activity, and a basic mitogen that is similar to and may be identical with pituitary FGF.

Addition of serum to quiescent cultures of Swiss 3T3 cells and mouse embryo <em>fibroblasts</em> causes a rapid increase in the phosphorylation of an 80 000 mol. wt. cellular protein (termed 80 K). The effect is dose- and time-dependent; enhancement in 80 K phosphorylation can be detected as early as 10-<em>15</em> s after adding serum. In contrast, platelet-derived <em>growth</em> <em>factor</em> elicits the response after a lag of 1.5 min suggesting that this <em>growth</em> <em>factor</em> does not mediate the response to serum. Recently a rapid increase in the phosphorylation of an 80 K cellular protein following treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact <em>fibroblasts</em>. The 80 K phosphoproteins generated in response to serum and to phorbol dibutyrate (PBt2) co-migrated in one- and two-dimensional PAGE and produced identical phosphopeptide fragments when subjected to partial digestion with Staphyloccocus aureus V8 protease. These observations suggest that the same 80 K protein is generated in response to serum and PBt2. We conclude that activation of protein kinase C in intact cells is one of the earliest effects elicited by serum in quiescent <em>fibroblasts</em>.

Bile acids play a critical role in liver injury and regeneration, but their role in acetaminophen (APAP)-induced liver injury is not known. We tested the effect of bile acid modulation on APAP hepatotoxicity using C57BL/6 mice, which were fed a normal diet, a 2% cholestyramine (CSA)-containing diet for bile acid depletion, or a 0.2% cholic acid (CA)-containing diet for 1 week before treatment with 400 mg/kg APAP. CSA-mediated bile acid depletion resulted in significantly higher liver injury and delayed regeneration after APAP treatment. In contrast, 0.2% CA supplementation in the diet resulted in a moderate delay in progression of liver injury and significantly higher liver regeneration after APAP treatment. Either CSA-mediated bile acid depletion or CA supplementation did not affect hepatic CYP2E1 levels or glutathione depletion after APAP treatment. CSA-fed mice exhibited significantly higher activation of c-Jun N-terminal protein kinases and a significant decrease in intestinal <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> mRNA after APAP treatment. In contrast, mice fed a 0.2% CA diet had significantly lower c-Jun N-terminal protein kinase activation and 12-fold higher <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>15</em> mRNA in the intestines. Liver regeneration after APAP treatment was significantly faster in CA diet-fed mice after APAP administration secondary to rapid cyclin D1 induction. Taken together, these data indicate that bile acids play a critical role in both initiation and recovery of APAP-induced liver injury.

BACKGROUND

Collateral growth is induced by chemical signals from the ischemic myocardium. We hypothesized that angiogenic growth factors are produced by cardiac tissue; they are diffusible, more concentrated in pericardial fluids, and are increased by myocardial ischemia.

RESULTS

With the use of an enzyme-linked immunosorbent assay, we measured the concentrations of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in pericardial fluids of 12 patients with unstable angina (group 1) and of 8 patients with nonischemic heart diseases (group 2). The levels of protein in pericardial fluids were quite comparable between the two groups (34 +/- 2 versus 32 +/- 4 mg/mL). The concentration of bFGF in pericardial fluids in group 1 was 2036 +/- 357 pg/mL, significantly (P < .001) higher than the 289 +/- 72 pg/mL in group 2. The amount of bFGF per milligram of protein was also significantly (P < .05) higher in group 1 than in group 2 (67 +/- 15 versus 12 +/- 4 pg/mg). The concentration of VEGF in pericandial fluids tended to be higher in group 1, but the difference was statistically insignificant (39 +/- 7 versus 22 +/- 6 pg/mL). The amount of VEGF per milligram of protein was 1.2 +/- 0.3 pg/mg in group 1, similar to the 0.8 +/- 0.4 pg/mg in group 2.

CONCLUSIONS

This finding provides new evidence that bFGF plays an important role in mediating collateral growth in humans.

Native human IL-1 beta and IL-1 alpha stimulated prostaglandin E2 secretion by human embryonic lung <em>fibroblasts</em> at half-maximal concentrations of 3 +/- 1.2 pM (+/- SEM) and 10 +/- 2.3 pM, respectively. In contrast to the 20-50-fold lower affinities previously found for IL-1-R on 3T3 cells as well as murine and human lymphoblastoid lines, monoiodo 125I-IL-1 beta bound to normal human <em>fibroblasts</em> with a Kd of 8.4 +/- 4.1 pM in direct binding experiments, and with a Ki of 11.2 +/- 2.8 pM in competitive binding experiments. IL-1 alpha bound to the receptor identified by 125I-IL-1 beta with a Ki of 50 +/- 18 pM. The receptor exhibited homogeneous affinity for IL-1 beta or IL-1 alpha. The receptor did not recognize IL-2, IFN-gamma, tumor necrosis <em>factor</em> alpha, a functionally related monokine, or bovine acidic <em>fibroblast</em> <em>growth</em> <em>factor</em>, a structurally related mediator. Comparison of the biological response curves and binding curves obtained for IL-1 alpha and IL-1 beta showed that they were parallel and that 10-<em>15</em>% occupancy of the estimated 3,000 sites by either species of IL-1 was sufficient to give half-maximal stimulation of prostaglandin E2 secretion. Thus, the amount of apparent signal amplification observed on <em>fibroblasts</em> was considerably lower than the 100-100,000 fold amplification previously reported for lymphoid lines. Crosslinking experiments revealed a major band with a corrected molecular mass of approximately 80 kD and a minor band of approximately 200 kD. Labeling of these bands was blocked by IL-1 beta and IL-1 alpha but not by IL-2, IFN-gamma, or tumor necrosis <em>factor</em> alpha. These results demonstrate that normal human embryonic lung <em>fibroblasts</em> bear IL-1-R of sufficiently high affinity to mediate their biological responsiveness to low picomolar concentrations of IL-1 beta and IL-1 alpha and are consistent with the existence of a single receptor mediating the biological properties of both human IL-1 species.

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog (Hh), <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF), and bone morphogenetic protein (BMP) families. To better understand when and where Hh signaling influences pituitary development, we have analyzed the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants). While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, our data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. We report that Hh signaling is necessary between 10 and <em>15</em> h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. We show that the Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (<em>15</em>-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. We also use the zebrafish Hh pathway mutants to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. Our results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland.

BACKGROUND

Advanced hepatocellular carcinoma (HCC) in humans is characterized by hypervascularity. In the present study, the expressions of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2) and endostatin were analyzed in patients with chronic liver disease to clarify the effect of these major angiogenic factors.

METHODS

Serum concentrations of VEGF, FGF-2 and endostatin in 24 patients with HCC, 16 patients with liver cirrhosis (LC) and 13 healthy volunteers were measured by enzyme-linked immunosorbent assay. The expression of VEGF in 21 surgically resected HCC samples was analyzed by immunohistochemistry, and that of VEGF isoforms in 15 HCC samples was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS

Serum VEGF, FGF-2 and endostatin concentrations were significantly elevated in patients with HCC compared with healthy volunteers; but there was no significant difference between patients with HCC and those with non-HCC liver disease. Immunohistochemical analysis showed that VEGF protein was strongly expressed in both well-differentiated HCC cells and non-cancerous hepatocytes, whereas in moderately and poorly differentiated HCC the expression was stronger in the endothelial cells (EC) lining intratumor vessels than in the cancer cells. On RT-PCR for VEGF isoforms it was found that VEGF-121, VEGF-165 and VEGF-189 were expressed in all but one of the HCC samples and in all corresponding non-HCC samples.

CONCLUSIONS

The results suggest that VEGF, FGF-2, and endostatin concentrations are elevated prior to the emergence of HCC and that the distribution of VEGF changes dynamically during the development of HCC.

We have explored the plastic ability of neuronal precursors to acquire different identities by manipulating their surrounding environment. Specifically, we sought to identify potential signals involved in the specification of forebrain dopaminergic neurons. Here we describe culture conditions under which tyrosine hydroxylase (TH) expression is induced in neuronal precursors, which were derived directly from the embryonic striatum and adult subependyma (SE) of the lateral ventricle or generated from multipotent forebrain stem cells. TH was successfully induced in all of these cell types by 24 hr exposure to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2) and glial cell conditioned media (CM). The greatest magnitude of the inductive action was on embryonic striatal precursors. Although FGF2 alone induced limited TH expression in striatal cells (1.1 +/- 0.2% of neurons), these actions were potentiated 17.5-fold (19.6 +/- 1.5% of neurons) when FGF2 was coadministered with B49 glial cell line CM. Of these TH-immunoreactive cells, approximately <em>15</em>% incorporated bromodeoxyuridine (BrdU), indicating that they were newly generated, and 95% coexpressed the neurotransmitter GABA. To investigate whether precursors of the adult forebrain subependyma were competent to respond to the instructive actions of FGF2+CM, they were first labeled in vivo with a pulse of BrdU. Although none of the cells expressed TH in control, 0.2% of total cells showed TH immunoreactivity in FGF2+CM-treated cultures. Under these same conditions only, in vitro-generated precursors from epidermal <em>growth</em> <em>factor</em>-responsive stem cells exhibited TH expression in 10% of their total neuronal progeny. Regulation of neurotransmitter phenotype in forebrain neuronal precursors, by the synergistic action of FGF2 and glial-derived diffusible <em>factors</em>, may represent a first step in understanding how these cells are generated in the embryonic and adult brain and opens the prospect for their manipulation in vitro and in vivo for therapeutic use.

Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), a mediator capable of modulating a broad range of effects on the behavior of many normal cells, was found in high concentrations in the epithelial lining fluid (ELF) of the normal human lower respiratory tract. Although plasma contained small amounts of TGF-beta, the concentrations of TGF-beta in normal ELF were in the 200 to 300 pM range, more than <em>15</em>-fold higher. This ELF TGF-beta had similar physical characteristics to purified human platelet TGF-beta, competed with platelet TGF-beta for its receptor on A549 carcinoma cells, and stimulated the anchorage-independent <em>growth</em> of NRK cells in soft agar in the presence of epidermal <em>growth</em> <em>factor</em>. Furthermore, ELF TGF-beta suppressed diploid lung <em>fibroblast</em> proliferation in a dose-dependent fashion similar to platelet TGF-beta. In the context of these observations and with the known biologic properties of this molecule, TGF-beta in ELF has the potential to play a role in a variety of cellular processes in the lower respiratory tract.

BACKGROUND

We have recently shown, in an animal model, that amniotic fluid can be a source of cells for fetal tissue engineering. This study was aimed at determining whether fetal tissue constructs could also be engineered from cells normally found in human amniotic fluid.

METHODS

Cells obtained from the amniotic fluid of pregnant women at <em>15</em> to 19 weeks of gestation (n=6) were cultured in Dulbecco's Modified Eagle's medium (Sigma Chemical, St Louis, MO) containing 20% fetal bovine serum and 5 ng/mL basic <em>fibroblast</em> <em>growth</em> <em>factor</em> in a 95% humidified, 5% CO(2) chamber at 37 degrees C. A subpopulation of morphologically distinct cells was then mechanically isolated from the rest and selectively expanded. The lineage of this subpopulation of amniocytes was determined by immunofluorescent staining with antibodies against standard intermediate filaments and surface antigens. Cell proliferation rates were determined by oxidation assay. After cell expansion, colonies of amniocytes were statically and dynamically seeded onto both unwoven, 1-mm-thick polyglycolic acid polymer scaffold and acellular human dermis for 72 hours. The resulting constructs were analyzed by scanning electron microscopy.

RESULTS

Amniocytes stained positively for smooth muscle actin, vimentin, cytokeratin 18, and fibroblast surface protein, and negatively for desmin, cluster of differentiation 31, and von Willebrand's factor (Dako, Carpenteria, CA). These findings are consistent with a mesenchymal, fibroblast-myofibroblast cell lineage. Mesenchymal amniocytes could be rapidly expanded in culture, based on results of the proliferation assay. Scanning electron microscopy of amniocyte constructs revealed dense, confluent layers of cells surrounding the polymer matrices and firm cell adhesion to both PGA and Alloderm (Lifecell Corp, Branchburg, NJ) scaffolds. No evidence of cell death was observed.

CONCLUSIONS

Subpopulations of fetal mesenchymal cells can be consistently isolated from human amniotic fluid and rapidly expanded in vitro. Human mesenchymal amniocytes attach firmly to both polyglycolic acid polymer and acellular human dermis. The amniotic fluid can be a valuable and practical cell source for fetal tissue engineering.

EGL-<em>15</em> is a <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor in the nematode Caenorhabditis elegans. Components that mediate EGL-<em>15</em> signaling have been identified via mutations that confer a Clear (Clr) phenotype, indicative of hyperactivity of this pathway, or a suppressor-of-Clr (Soc) phenotype, indicative of reduced pathway activity. We have isolated a gain-of-function allele of let-60 ras that confers a Clr phenotype and implicated both let-60 ras and components of a mitogen-activated protein kinase cascade in EGL-<em>15</em> signaling by their Soc phenotype. Epistasis analysis indicates that the gene soc-1 functions in EGL-<em>15</em> signaling by acting either upstream of or independently of LET-60 RAS. soc-1 encodes a multisubstrate adaptor protein with an amino-terminal pleckstrin homology domain that is structurally similar to the DOS protein in Drosophila and mammalian GAB1. DOS is known to act with the cytoplasmic tyrosine phosphatase Corkscrew (CSW) in signaling pathways in Drosophila. Similarly, the C. elegans CSW ortholog PTP-2 was found to be involved in EGL-<em>15</em> signaling. Structure-function analysis of SOC-1 and phenotypic analysis of single and double mutants are consistent with a model in which SOC-1 and PTP-2 act together in a pathway downstream of EGL-<em>15</em> and the Src homology domain 2 (SH2)/SH3-adaptor protein SEM-5/GRB2 contributes to SOC-1-independent activities of EGL-<em>15</em>.