The development of postoperative intraperitoneal adhesions continues to be a major concern for surgeons. The purpose of this study was to establish a postoperative adhesion model in rats, and to assess the effectiveness of tranilast (N-(3',4'-dimethoxycinnamoyl)anthranilic acid) in preventing postoperative adhesion formation. The adhesion model was established in 12 male Donryu rats. This involved two essential <em>factors</em>, drying and bleeding. Another <em>22</em> male Donryu rats were used to study the prevention of intraperitoneal adhesions. Tranilast was administered orally pre- and postoperatively. Adhesion strength was evaluated by grading, and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and transforming <em>growth</em> <em>factor</em>-beta-1 (TGF-beta1) concentration were measured. Postoperative intraperitoneal adhesions were seen in all rats, but the adhesions in the tranilast group were significantly less severe than those in the control group. Serum bFGF and TGF-beta1 levels in the tranilast group were lower at the time of surgery than those in the control group, and bFGF levels were lower at the endpoint of this study in the tranilast group than in the control group. The TGF-beta1 levels at the end-point did not differ between the two groups. These findings demonstrated that tranilast significantly reduced postoperative intraperitoneal adhesion formation.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 23 (FGF23) is a potent regulator of Pi and 1,25-(OH)(2)D homeostasis. Early postpartum infants show intriguing changes in serum levels of Ca, Pi, PTH and 1,25-(OH)(2)D. However, the role of FGF23 in the early neonatal mineral metabolism has not been clarified. In order to evaluate the significance of FGF23 during the early postpartum period, we examined the circulating FGF23 levels using an intact FGF23 ELISA and a C-terminal FGF23 ELISA either in <em>22</em> umbilical cord blood samples (the cord blood) or in <em>22</em> term infants at 5days of life (the 5-day-old infant). We also compared these ranges with those of 11 healthy adults. Data were expressed as mean+/-SD, and analyzed by two-way ANOVA, followed by the Tukey's test. C-terminal FGF23 in the cord blood, the 5-day-old infants and the healthy adults were 73.3+/-<em>22</em>.4, 81.0+/-28.2 and 39.0+/-7.8 RU/ml, respectively. Intact FGF23 in the cord blood, the 5-day-old infants and the healthy adults were 3.9+/-1.6, 21.8+/-17.6, and 27.6+/-7.3 pg/ml, respectively. Immunoprecipitation assays using anti-FGF23 antibodies demonstrated that the intact 32 kDa FGF23 was low and the fragmented FGF23 of 18kDa was abundant in the cord blood compared with those in the healthy adults. In conclusion, our observations indicated that the intact FGF23/C-terminal FGF23 ratio was very low due to the fragmentation of FGF23 during the early postpartum period and might have a considerable contribution to the Pi homeostasis in the healthy term infants.

The fgf-2 gene encodes low molecular weight (LMW, 18 kDa) and high molecular weight (HMW, <em>22</em>-24 kDa) forms that originate from alternative translation of a single mRNA and exhibit diverse biological functions. HMW <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) inhibits cell migration and induces cell transformation or <em>growth</em> arrest in a cell type- and dose-dependent fashion. Conversely, LMW FGF-2 upregulates both cell proliferation and migration in most cell types. Although transcriptional and translational regulation of HMW and LMW FGF-2 has been extensively investigated, little is known about post-translational control of their relative expression. Here we report that thrombin, a key coagulation <em>factor</em> and inflammatory mediator, cleaves HMW FGF-2 into an LMW FGF-2-like form that stimulates endothelial cell migration and proliferation. The effect of thrombin on these cell functions requires HMW FGF-2 cleavage. This post-translational control mechanism adds a novel level of complexity to the regulation of FGF-2, and links the activities of thrombin and FGF-2 in patho-physiological processes in which both molecules are expressed.

Overexpression of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors (FGFRs) has been observed in many types of human tumors; however, the regulatory mechanism of human FGFR expression is still largely unknown. In the present study, we first identified the transcriptional initiation site in the human FGFR 1 gene by 5'-RACE. Furthermore, we show that the expression of human FGFR 1 is regulated by E2F-1. Characterization of the human FGFR 1 promoter demonstrated that two non-consensus E2F binding sequences at positions +4 to +<em>22</em> and +25 to +43 relative to our identified transcriptional initiation site in the human FGFR 1 gene were critical for E2F-1-mediated transactivation of human FGFR 1 promoter. Mutations of these sites completely abolished the response of human FGFR 1 promoter to E2F-1 as well as E2F-1 binding in electrophoretic mobility-shift assays. Furthermore, chromatin immunoprecipitation assay showed that E2F-1 was able to bind in vivo to the human FGFR 1 promoter. Moreover, human FGFR 1 protein expression was up-regulated by the overexpression of E2F-1, but down-regulated by the overexpression of pRB in situ, suggesting that the expression of human FGFR 1 is regulated by the pRB/E2F pathway. Because disruption of the pRB/E2F pathway is frequently observed in tumor cells, our findings provide valuable information for studying the role of FGFR 1 in tumor progression.

Functional recovery following facial nerve injury is poor. Adjacent neuromuscular junctions (NMJs) are "bridged" by terminal Schwann cells and numerous regenerating axonal sprouts. We have recently shown that manual stimulation (MS) restores whisking function and reduces polyinnervation of NMJs. Furthermore, MS requires both insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1) and brain-derived neurotrophic <em>factor</em> (BDNF). Here, we investigated whether <em>fibroblast</em> <em>growth</em> <em>factor</em>-2 (FGF-2) was also required for the beneficial effects of MS. Following transection and suture of the facial nerve (facial-facial anastomisis, FFA) in homozygous mice lacking FGF-2 (FGF-2(-/-)), vibrissal motor performance and the percentage of poly-innervated NMJ were quantified. In intact FGF-2(-/-) mice and their wildtype (WT) counterparts, there were no differences in amplitude of vibrissal whisking (about 50°) or in the percentage of polyinnervated NMJ (0%). After 2 months FFA and handling alone (i.e. no MS), the amplitude of vibrissal whisking in WT-mice decreased to <em>22</em>±3°. In the FGF-2(-/-) mice, the amplitude was reduced further to 15±4°, that is, function was significantly poorer. Functional deficits were mirrored by increased polyinnervation of NMJ in WT mice (40.33±2.16%) with polyinnervation being increased further in FGF-2(-/-) mice (50.33±4.33%). However, regardless of the genotype, MS increased vibrissal whisking amplitude (WT: 33.9°±7.7; FGF-2(-/-): 33.4°±8.1) and concomitantly reduced polyinnervation (WT: 33.9%±7.7; FGF-2(-/-): 33.4%±8.1) to a similar extent. We conclude that, whereas lack of FGF-2 leads to poor functional recovery and target reinnervation, MS can nevertheless confer some functional benefit in its absence.

Insulin has been approved for inhaled application, but safety concerns remain, because of un-physiologically high insulin concentrations in the lung. Since insulin may act as <em>growth</em> <em>factor</em>, possible proliferative effects of insulin, insulin analogues and insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1) on human lung <em>fibroblasts</em> were studied. As measure of proliferation [(3)H]-thymidine incorporation was studied in HEL-299, MRC-5, IMR-90 and primary human lung <em>fibroblasts</em>. In all cells, mRNA encoding IGF-1 receptors and two variants of insulin receptors was detected. Insulin and IGF-1 stimulated [(3)H]-thymidine incorporation in all cells. Comparison of the concentration-dependent effects in HEL-299 cells showed that IGF-1 and insulin glargine were more potent (EC(50), 3 and 6 nM) and more effective (maximum increase, by 135-150%) than insulin and insulin detemir (EC(50), <em>22</em> and 110 nM; maximum increase: by 80%). Proliferative effects of IGF-1 and insulin were inhibited to the same extent by an antibody (1H7) directed against the IGF-1 receptor α-subunit. Insulin-induced stimulation of [(3)H]-thymidine incorporation was reduced by 83% after siRNA-mediated down-regulation of IGF-1 receptor by about 75%, but not affected by a similar down-regulation of the insulin receptor. Insulin and IGF-1 caused rapid up-regulation of the early genes FOS, EGR-1 and EGR-2 as well as of the gene coding for IGF-1. In conclusion, in human lung <em>fibroblasts</em> insulin exerts marked proliferative effects and the pharmacological profile of this response as well as specific receptor knock-down experiments suggest mediation via IGF-1 receptors. The risk of unwanted structural lung alterations by long-term inhalative application of insulin should be considered.

BACKGROUND

Early- to mid-gestational fetal mammalian skin wounds heal rapidly and without scarring. Keratinocytes (KCs) have been found to exert important effects on the regulation of fibroblasts. There may be significant differences of gestational fetal KCs at different ages. The advantages in early- to mid-gestational fetal KCs could lead to fetal scarless wound healing.

METHODS

KCs from six human fetal skin samples were divided into two groups: a mid-gestation group (less than 28 weeks of gestational age) and a late-gestation group (more than 28 weeks of gestational age). RNA extracted from KCs was used to prepare a library of small RNAs for next-generation sequencing (NGS). To uncover potential novel microRNA (miRNAs), the mirTools 2.0 web server was used to identify candidate novel human miRNAs from the NGS data. Other bioinformatical analyses were used to further validate the novel miRNAs. The expression levels of the miRNAs were further confirmed by real-time quantitative RT-PCR.

RESULTS

A total of 61.59 million reads were mapped to 1,170 known human miRNAs in miRBase. Among a total of 202 potential novel miRNAs uncovered, 106 candidates have a higher probability of being novel human miRNAs. A total of 110 miRNAs, including 22 novel miRNA candidates, were significantly differently expressed between mid- and late-gestational fetal KCs. Thirty-three differentially expressed miRNAs and miR-34 family members are correlated with the transforming growth factor-β (TGF-β) pathway.

CONCLUSIONS

Taken together, our results provide compelling evidence supporting the existence of 106 novel miRNAs and the dynamic expression of miRNAs that extensively targets the TGF-β pathway at different gestational ages in fetal KCs. MiRNAs showing altered expression at different gestational ages in fetal KCs may contribute to scarless wound healing in early- to mid-gestational fetal KCs, and thus may be new targets for potential scar prevention and reduction therapies.

Experimental evidence suggests that the fibroblastic proliferation often associated with the myeloproliferative disorders is not part of the neoplastic process, but is secondary to an unknown stimulus. This stimulus may be a <em>factor</em> derived from platelets which promotes the proliferation of <em>fibroblasts</em> in vitro (PDGF). Platelet-derived <em>growth</em> <em>factor</em> is localized to platelet alpha-granules together with PF4 and beta-TG. As an indicator of alpha-granule release, we have measured PF4 levels in plasma, platelets and urine in 46 normal subjects and 49 patients with myeloproliferative disorders, secondary thrombocytosis and miscellaneous malignancies. All 11 patients with elevated urinary PF4 excretion exhibited myelofibrosis, whereas 11 of <em>22</em> patients with documented myelofibrosis had urinary PF4 excretion in the normal range. No correlation was seen between marrow fibrosis and plasma levels or the platelet content of PF4. The data are consistent with the possibility that release of mitogen(s) from platelet or megakaryocyte alpha-granules in some patients with myeloproliferative disorders is pathogenetically related to the development of marrow fibrosis.

Recent reports have suggested that basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) could play a permissive role in hematopoiesis, in combination with specific colony-stimulating <em>factors</em>. We investigated the expression of bFGF and FGF-receptors (FGF-Rs) in leukemic cell lines of various hematopoietic lineages. Three protein isoforms of bFGF of approximately 18, <em>22</em> and 24 kDa were detected in the myeloid cell line K562, but not in myelomonocytic or lymphoid (T or B) cell lines. In vitro-induced differentiation of K562 cells did not change the pattern of expression of the different bFGF isoforms. Accordingly, the mRNA of bFGF was found expressed in K562, but not in other leukemic lines tested, as assayed by reverse transcript amplification (RT-PCR). Using the same technique, we searched for the presence of high affinity FGF-Rs on these cells: in eight out of ten cell lines tested, mRNA for at least one FGF-R among FGF-R1, FGF-R3 or FGF-R4 was expressed, whereas FGF-R2 was never detected. We found that two cell lines were responsive to bFGF in different biological assays: (i) in K562 myeloid cells induced to differentiate by hemin, preincubation with bFGF and heparin increased cell viability and decreased hemin-induced DNA fragmentation, without affecting erythroid differentiation; and (ii) in U937 monocytic cells, the production of plasminogen activator was increased by bFGF or aFGF in combination with heparin. Binding experiments with 125I-bFGF (up to 200 pM) in the presence of heparin revealed high affinity receptors on the K562 and U937 cell lines (1177 +/- 440 and 392 +/- 184 sites/cell, Kd = 61.7 +/- 8.6 and 43.1 +/- 13.5 pM, respectively). Thus our results strongly suggest that cells of hematopoietic origin could express functional FGF-receptors.

Cultured endothelial cells have been shown to possess two mechanisms of intercellular adhesion: Ca2(+)-dependent and Ca2(+)-independent. We report here that <em>growth</em> of bovine aortic endothelial cells (BAEC) in complete medium containing purified basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF, 6 ng/ml) results in loss of Ca2(+)-dependent intercellular adhesion. In the presence of heparin (90 micrograms/ml), this effect is reproduced upon treatment with acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF, 6 ng/ml) or endothelial cell <em>growth</em> supplement (ECGS, 100 micrograms/ml), in both human umbilical vein endothelial cells (HUVEC) and BAEC. Treatment at these doses with aFGF in the absence of heparin or with heparin alone is without significant effect. Loss of Ca2(+)-dependent adhesion following treatment of cells with heparin-binding <em>growth</em> <em>factors</em> (HBGFs) is prevented by pre-treatment of cell layers with cycloheximide. The Ca2(+)-independent adhesion mechanism is unaffected by HBGF treatment. Exposure of endothelial cells to HBGFs, moreover, prevents the eventual establishment of quiescence in <em>growing</em> cultures and restimulates replication in confluent cultures that have reached a final density-inhibited state. Addition of bFGF alone or aFGF + heparin at these doses results in a 4-fold increase in DNA synthesis over untreated control cultures at saturation density as reflected by thymidine index. A single addition of bFGF (6 ng/ml) to untreated quiescent confluent BAEC monolayers results in an increase in 3H-TdR incorporation reaching a peak at <em>22</em> hours with a parallel loss of Ca2(+)-dependent adhesiveness. Fluorescent staining with rhodamine-phalloidin demonstrates an altered distribution of polymerized F-actin in the bFGF-treated monolayers, marked by disruption of the dense peripheral microfilament bands retained by untreated confluent monolayers. Together, these results indicate that the mitogenic effect of HBGFs in cultured endothelial cells is associated with a "morphogenic" set of responses, perhaps dependent on breakdown of calcium-dependent cell-cell contacts.

Epidermal <em>growth</em> <em>factor</em> (EGF) augments late fetal lung maturation by advancing the ontogeny of fetal lung development and by stimulating surfactant synthesis. Previous studies have indicated that fibroblastalveolar epithelial cell communications mediate surfactant synthesis in the fetal lung and EGF acts through such a mechanism. We investigated the hypothesis that is differential activity and expression of the epidermal <em>growth</em> <em>factor</em> receptor (EGF-R) in fetal lung <em>fibroblasts</em> during the canalicular stage of lung development mediates EGF effects. To test this hypothesis, we examined fetal rat lung <em>fibroblasts</em> (FLFs) and type II cells of late gestation (canalicular and saccular stages; 17-<em>22</em> days) by EGF-R binding techniques, SDS-PAGE, and Western blot analysis. Specific EGF binding increased 181% in day 18 female FLFs, with male FLFs exhibiting a similar increase on day 19. In contrast, specific EGF binding was low in type II cells, did not increase during late gestation, and there were no sex-specific differences. SDS-PAGE and Western blot analysis revealed a predominant 170-kDa EGF-R band in <em>fibroblasts</em> that increased with gestation (peak = 19 days), and was stronger in females. Immunoprecipitation of EGF-treated cells demonstrated the tyrosine kinase activity of the identified receptor. In contrast, type II cells showed minimal signal that did not increase until day 21 of gestation. We also examined whole fetal lung sections by immunohistochemistry to determine cell-specific expression of the EGF-R in vivo. Immunohistochemistry revealed specific EGF-R staining in columnar and cuboidal epithelia of small conducting airways and in mesenchyme of epithelial-mesenchymal borders (including subepithelial mesenchyme). In contrast, alveolar epithelia showed minimal staining, while subalveolar mesenchyme EGF-R staining peaked at day 19 of gestation. We conclude that cell-specific and sex-specific differences in EGF-R binding and EGF-R immunolocalization appears in the fetal lung at a developmental stage that is critical for alveolar epithelial cell differentiation. The results suggest a role for EGF-R activation in late fetal alveolar epithelial cell maturation, which is mediated through mesenchymal-epithelial cell communication.

Left ventricular (LV) hypertrophy and one of its inducers, the <em>fibroblast</em> <em>growth</em> <em>factor</em>-23 (FGF-23) were found to be associated with unfavourable outcome in end-stage renal disease (ESRD) patients. We sought to investigate the influence of hemodialysis (HD), increased LV mass and FGF-23 on LV mechanics using three-dimensional (3D) speckle tracking echocardiography. Forty-four ESRD patients on maintenance HD were examined just before and immediately after HD, and were compared to 44 normal controls (NC). Transthoracic 3D recordings were obtained using multi-beat reconstruction from 6 consecutive cardiac cycles. LV mass index (LVMi) was evaluated and 3D speckle tracking analysis was performed to calculate global longitudinal (GLS), circumferential (GCS), area (GAS) and radial (GRS) peak systolic strain. Serum FGF-23 levels were also measured. Strain values improved in all directions after HD [pre- vs. post-HD; GLS: -20(3) vs. -21(6), GCS: -20(4) vs. -<em>22</em>(7), GAS: -33(5) vs. -35(10), GRS: 50(12) vs. 53.5(20) %, all p < 0.01]. LVMi was remarkably increased in our patients [ESRD vs. NC; 136(46) vs. 71(8) g/m(2), p < 0.001]. Elevated FGF-23 levels were associated with increased LV mass (ρ = 0.581, p < 0.001). LVMi was inversely related to pre-HD GCS (ρ = 0.626, p < 0.001) and post-HD GCS (ρ = 0.761, p < 0.001), GAS (ρ = 0.534, p < 0.05) and GRS (ρ = -0.639, p < 0.01). Serum FGF-23 levels correlated with post-HD GAS (ρ = 0.513, p < 0.01) and GRS (ρ = -0.512, p < 0.05). HD treatment results in immediate improvement in all strain directions. Besides inducing LV hypertrophy, FGF-23 may play a role in the deterioration of LV mechanics in patients with ESRD.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF2) is synthesized as four isoforms with molecular weights of 24, <em>22</em>.5, <em>22</em>, and 18 kDa, with each of the three higher molecular weight forms (hmwFGF2) produced by the initiation of translation at one of three upstream CUG codons. We have shown that bovine arterial endothelial cells export the high molecular weight forms of FGF2 (hmwFGF2) in a 17beta-estradiol-dependent manner (Piotrowicz et al., 1997, J Biol Chem 272:7042-7047). To determine whether the hmwFGF2 forms affected cell behavior after release, we evaluated the effect of recombinant hmwFGF2 on the <em>growth</em> and migration of endothelial cells and mammary carcinoma cells (MCF-7). Treatment with the recombinant protein resulted in the inhibition of endothelial cell migration by 45% and MCF-7 cell migration by 70%. HmwFGF2-dependent inhibition was observed when endothelial cell migration was stimulated by 18 kDa FGF2 or vascular endothelial <em>growth</em>, and MCF cell migration was stimulated with insulin-like <em>growth</em> <em>factor</em>. In each case, inclusion of an antibody against the 55 amino acid amino terminal end of 24 kDa FGF2 abrogated the inhibition of migration, while antibodies to the 18 kDa FGF2 domain had no effect. When endothelial cells were cultured under conditions which promoted export of hmwFGF2, a 40% decrease in motility was observed which was reversed by the antibodies to the 24 kDa FGF2. Thus, both recombinant and endogenously produced hmw-FGF2 are capable of inhibiting migration. In contrast to the ubiquitous effect on migration, hmwFGF2 had no effect on endothelial cell <em>growth</em> but stimulated MCF-7 <em>growth</em> equally as well as the 18 kDa FGF2 (threefold). Antibodies to the 18 kDa domain of 24 kDa FGF2 blocked the <em>growth</em>-promoting activity of hmwFGF2, but those to the amino terminal end were ineffective. These data suggest that hmwFGF2 has dual activities, an inhibitory effect on cell migration and a <em>growth</em>-stimulating effect. The two activities can be localized to different parts of hmwFGF2: inhibitory activity to the amino terminal 55 amino acids (which are absent from the 18 kDa FGF2) and <em>growth</em>-promoting activity to the 18 kDa domain. Therefore, the ratio of hmwFGF2 and 18 kDa FGF2 in the extracellular space may provide a mechanism of control for angiogenesis and mammary tumor development.

Direct cell to cell contact has been suggested as the means whereby <em>fibroblast</em> <em>factors</em> influence epithelial cell differentiation in the developing lung. To obtain further evidence for this hypothesis, the role of epithelial-mesenchymal cell contacts is now examined when lung development is accelerated after steroid injection. Male and female rat fetuses were studied from day 17 to day <em>22</em> of gestation. The fetuses were from mothers injected with 2 mg/kg of dexamethasone at 2 days before death and [3H]thymidine at 1 hour before death. Steroid injected during the rapid <em>growth</em> phase reduced total DNA in fetal lung, and from autoradiographs, the effect was most noticeable in epithelial cells. Differentiation began sooner after steroid; more epithelial cells contained lamellar bodies and disaturated phosphatidylcholine levels were higher. From quantitative ultrastructural studies, these changes in phospholipid correlated with an increased frequency of epithelial-interstitial cell contacts. Steroid treatment did not abolish the sex-related difference seen in controls; both males and females showed increases in cell to cell contacts and in lipid synthesis in the lung. The results demonstrate that acceleration of epithelial cell maturation with increased surfactant synthesis is associated with increased epithelial-interstitial cell contacts. This supports the concept that regulatory messages from the fetal <em>fibroblast</em> are passed directly to specific epithelial cells to initiate surfactant synthesis.

Undifferentiated fetal rat lung epithelial cells were isolated on gestational days 15 or 17 (term <em>22</em> days) and cultured in a defined medium. On plastic, most of the cells developed structurally abnormal lamellar bodies. On a basement membrane matrix (BMM), they sequentially accumulated glycogen and formed typical lamellar bodies. Biochemical analysis of the latter indicated that they had a phospholipid composition typical of surfactant for cells on BMM but not on plastic and that surfactant protein A appeared on BMM only. Progressing maturation from day 1 to day 6 in culture was demonstrated for 17-day cells on BMM by a sevenfold increase of labeled precursor incorporation into surfactant phospholipids. Exposure to medium conditioned by 21-day fetal <em>fibroblasts</em> enhanced incorporation already after a 1-day culture. The antisteroid RU 486 had no effect on differentiation, whereas transforming <em>growth</em> <em>factor</em>-beta, a <em>factor</em> produced by lung mesenchyme at early fetal stages, inhibited it markedly. Alveolar epithelial type II cells appear to be committed early, but their maturational process would be prevented until a definite gestational stage.

During development, bone morphogenetic proteins (BMPs) induce the differentiation of mesenchymal progenitor cells to enter into the osteoblastic lineage, and BMPs enhance osteoblastic function. BMPs and noggin, a specific binding protein that blocks BMP actions, are expressed by osteoblastic cells but there is limited information about regulation of BMP synthesis in skeletal cells. We tested for the expression and regulation of BMP-4 in cultures of osteoblast-enriched cells from <em>22</em>-day fetal rat calvariae (Ob cells). BMP-4 caused a short-lived increase in BMP-4 mRNA followed by a marked inhibition of BMP-4 expression. The stimulatory effect was transcriptional, as determined by nuclear run-on assays, whereas the inhibitory effect was transcriptional and posttranscriptional, because longer BMP-4 exposure decreased its rate of transcription and shortened the half-life of BMP-4 mRNA in transcriptionally arrested Ob cells. BMP-2 and BMP-6 also inhibited BMP-4 mRNA levels. Transforming <em>growth</em> <em>factor</em> beta1 increased, whereas <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, platelet-derived <em>growth</em> <em>factor</em> BB, and insulin-like <em>growth</em> <em>factor</em> I decreased BMP-4 mRNA in Ob cells. BMP-2 also was expressed by Ob cells and it was downregulated by BMP-2, BMP-4, and BMP-6. Noggin increased BMP-4 transcripts, suggesting autocrine control of BMP-4 expression. In conclusion, BMP-4 inhibits its own expression in Ob cells, a mechanism to limit BMP availability to osteoblasts.

We studied the role of angiogenesis in patients with medullary type poorly differentiated adenocarcinoma (MTPDA) of the stomach. Immunohistochemical analyses were conducted using antibodies against <em>factor</em> VIII (endothelial cells), vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and its receptors (KDR andflt-1), and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and its receptors (bek andflg). Archival specimens of MTPDA (n=<em>22</em>) and non-MTPDA (n=47) were studied. The expression of VEGF and bFGF, the vessel count, and positivity of KDR on endothelium were all significantly higher in MTPDA than in non-MTPDA. The vessel count correlated with the VEGF expression in MTPDA. The vessel count and VEGF expression increased with the increasing stage of disease in MTPDA but not in non-MTPDA. The expression of bFGF and its receptors did not correlate with the vessel count and stage of disease in either type. These findings thus suggest that the biological behavior of medullary type poorly differentiated adenocarcinoma of the stomach is angiogenesis-dependent. The correlation of the VEGF expression and its endothelial receptors with the vessel count and the stage of disease thus suggests that VEGF is a <em>factor</em> responsible for the induction of angiogenesis in this type.

Broad cell tropism contributes to the pathogenesis of human cytomegalovirus (HCMV), but the extent to which cell type influences HCMV gene expression is unclear. A bespoke HCMV DNA microarray was used to monitor the transcriptome activity of the low passage Merlin strain of HCMV at 12, 24, 48 and 72 h post-infection, during a single round of replication in human fetal foreskin <em>fibroblast</em> cells (HFFF-2s), human retinal pigmented epithelial cells (RPE-1s) and human astrocytoma cells (U373MGs). In order to correlate transcriptome activity with concurrent biological responses, viral cytopathic effect, <em>growth</em> kinetics and genomic loads were examined in the three cell types. The temporal expression pattern of viral genes was broadly similar in HFFF-2s and RPE-1s, but dramatically different in U373MGs. Of the 165 known HCMV protein-coding genes, 41 and 48 were differentially regulated in RPE-1s and U373MGs, respectively, compared with HFFF-2s, and <em>22</em> of these were differentially regulated in both RPE-1s and U373MGs. In RPE-1s, all differentially regulated genes were downregulated, but, in U373MGs, some were down- and others upregulated. Differentially regulated genes were identified among the immediate-early, early, early late and true-late viral gene classes. Grouping of downregulated genes according to function at landmark stages of the replication cycle led to the identification of potential bottleneck stages (genome replication, virion assembly, and virion maturation and release) that may account for cell type-dependent viral <em>growth</em> kinetics. The possibility that cell type-specific differences in expressed cellular <em>factors</em> are responsible for modulation of viral gene expression is discussed.

Heparanase is an endoglycosidase that degrades heparan sulfate (HS) in the extracellular matrix (ECM) and cell surfaces, and fulfills a significant role in cancer metastasis and angiogenesis. We evaluated the expression of heparanase and its possible association with the expression of angiogenic molecules in malignant mesothelioma (MM), and analyzed whether expression of these proteins is site-related (pleural vs peritoneal MM, solid lesions vs effusions). Sections from 80 MM (56 biopsies, 24 effusions) were analyzed for heparanase protein expression using immunohistochemistry (IHC). Sixty MM were of pleural origin, and 20 were peritoneal. Effusion specimens consisted of 6 peritoneal and 18 pleural effusions, while biopsies consisted of 14 peritoneal and 42 pleural lesions. Fifty-four specimens were additionally evaluated for expression of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF), interleukin-8 (IL-8) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) proteins using IHC. Microvessel density (MVD) was studied in 28 biopsies using an anti-CD31 antibody. mRNA expression of heparanase (HPSE-1), VEGF and the VEGF receptor KDR was analyzed in 23 effusions using RT-PCR. Heparanase protein expression was seen in 69/80 (86%) tumors. Of these, 35 showed combined membrane and cytoplasmic expression, 30 cytoplasmic expression, and four exclusively membrane expression. Both total (P = 0.001) and cytoplasmic (P = 0.002) expression was significantly higher in solid tumors compared to effusions. Protein expression of VEGF, IL-8 and bFGF was seen in 21/54 (39%), <em>22</em>/54 (41%) and 44/54 (81%) specimens, respectively. Protein expression of bFGF was significantly higher in solid tumors (P < 0.001) and correlated with heparanase expression (P = 0.005). HPSE-1 and VEGF mRNA expression was detected in all 23 effusions using RT-PCR, while KDR mRNA was found in 12/23 MM. KDR mRNA expression correlated with that of both HPSE-1 (P = 0.005) and VEGF (P = 0.001). Our results document frequent expression of heparanase in MM, in agreement with the biological aggressiveness of this tumor. The co-expression of heparanase with bFGF is in agreement with the role of the former in releasing bFGF from the ECM. The concomitant reduction in protein expression of both molecules in effusions as compared to solid tumors, supports the hypothesis of a reduced need for pro-angiogenic stimuli in effusions, and may aid in defining tumor progression in this setting.

Histologically, desmoplastic small round cell tumor is composed of the characteristic neoplastic small round cells with divergent differentiation, and distinct desmoplastic stroma. Genetically, the tumor shows a characteristic 11;<em>22</em> translocation, involving the EWS gene on chromosome <em>22</em> and the WT1gene on chromosome 11 to produce an EWS-WT1 fusion gene which generates a chimeric protein functioning as a novel transcription <em>factor</em> that activates expression of target genes such as PDGF-A. Expression of PDGF-A, a potent <em>growth</em> <em>factor</em> for <em>fibroblasts</em>, has been detected in desmoplastic small round cell tumors and has been linked to the characteristic desmoplasia in these tumors. Bone morphogenic proteins, which are members of the TGFbeta superfamily play a complex role in regulating cell <em>growth</em> and differentiation and bone formation but have not been evaluated in desmoplastic small round cell tumors. In all, 24 desmoplastic small round cell tumors with EWS-WT1 fusion product confirmed by RT-PCR analysis were evaluated for expression of PDGF-A, PDGF-Rbeta, TGFbeta3 and bone morphogenic protein-4 by standard immunohistochemical methods with antigen retrieval on paraffin sections. Immunoreactivity was evaluated semiquantitively. Tumor-associated desmoplasia was quantified using a three-tier scale on hematoxylin- and eosin-stained sections. Desmoplastic small round cell tumors showed variable immunoreactivity with TGFbeta3 (21/24), BMP4 (14/21), PDGF-A (19/24) and PDGF-Rbeta (16/<em>22</em>). Less frequently, the stromal cells showed reactivity with TGFbeta3, PDGF-Rbeta and PDGF-A. Tumor-associated desmoplasia was prominent in eight, intermediate in seven and weak in nine cases. There was no correlation between tumor-associated desmoplasia and the markers tested except PDGF-A. In contrast to a previous study, our study showed that the level of PDGF-A expression inversely correlated with tumor-associated desmoplasia. Other targets of the EWS-WT1 transcription <em>factor</em> other than PDGF-A may be directly responsible for the prominent tumor-associated desmoplasia seen in desmoplastic small round cell tumor.

OBJECTIVE

We hypothesized that basic fibroblast growth factor (bFGF) may exert a role in carotid plaque instability by regulating the expression of matrix metalloproteinases (MMP).

METHODS

Plaques obtained from 40 consecutive patients undergoing carotid endarterectomy were preoperatively classified as soft or hard. Serum bFGF was pre- and postoperatively measured. The release of MMP-2 and MMP-9 in the blood serum, and the activity, production and expression in the carotid specimens was analyzed. Specific anti-bFGF inhibition tests were performed in vitro on human umbilical artery smooth muscle cells (HUASMC) to evaluate the role of bFGF in the activity, production and expression of MMP-2 and -9.

RESULTS

Twenty-one (53%) patients had a soft carotid plaque and 19 (48%) a hard plaque. Preoperative bFGF serum levels were higher in patients with soft plaques [soft=34 (28-39) pg/mL and hard=20 (17-22) pg/mL-p<0.001] and postoperatively returned to normal values (when compared to 10 healthy volunteers). The serum levels of MMP-2 in patients' with soft plaques were higher than those in patients' with hard plaques [soft=1222 (1190-1252) ng/mL and hard=748 (656-793)ng/mL-p<0.0001]. MMP-9 serum values were 26 (22-29) ng/mL for soft plaques and 18 (15-21) ng/mL for hard plaques (p<0.0001). We found increased activity, production and expression of MMP-2 and -9 in soft plaques compared to hard plaques (p<0.001). In vitro inhibition tests on HUASMC showed the direct influence of bFGF on the activity, production and expression of MMP-2 and -9 (p<0.001).

CONCLUSIONS

bFGF seems to exert a key role in carotid plaque instability regulating the activity, production and expression of MMP thus altering the physiologic homeostasis of the carotid plaque.

Utero-placental <em>growth</em> and vascular development are critical for pregnancy establishment that may be altered by various <em>factors</em> including assisted reproductive technologies (ART), nutrition, or others, leading to compromised pregnancy. We hypothesized that placental vascularization and expression of angiogenic <em>factors</em> are altered early in pregnancies after transfer of embryos created using selected ART methods. Pregnancies were achieved through natural mating (NAT), or transfer of embryos from NAT (NAT-ET), or IVF or in vitro activation (IVA). Placental tissues were collected on day <em>22</em> of pregnancy. In maternal caruncles (CAR), vascular cell proliferation was less (P<0.05) for IVA than other groups. Compared with NAT, density of blood vessels was less (P<0.05) for IVF and IVA in fetal membranes (FM) and for NAT-ET, IVF, and IVA in CAR. In FM, mRNA expression was decreased (P<0.01-0.08) in NAT-ET, IVF, and IVA compared with NAT for vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and its receptor FLT1, placental <em>growth</em> <em>factor</em> (PGF), neuropilin 1 (NP1) and NP2, angiopoietin 1 (ANGPT1) and ANGPT2, endothelial nitric oxide synthase 3 (NOS3), hypoxia-inducible <em>factor</em> 1A (HIF1A), <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF2), and its receptor FGFR2. In CAR, mRNA expression was decreased (P<0.01-0.05) in NAT-ET, IVF, and IVA compared with NAT for VEGF, FLT1, PGF, ANGPT1, and TEK. Decreased mRNA expression for 12 of 14 angiogenic <em>factors</em> across FM and CAR in NAT-ET, IVF, and IVA pregnancies was associated with reduced placental vascular development, which would lead to poor placental function and compromised fetal and placental <em>growth</em> and development.

The frequently detected t(4;14)(p16.3;q32) translocation in multiple myeloma (MM) results in a dysregulation of two potential oncogenes: multiple myeloma SET domain (MMSET) and <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3). As the expression of FGFR3 is undetectable in 30% of the t(4;14)+ MM patients, MMSET has been suggested to play an important role in the malignant transformation associated with the t(4;14) translocation. Screening with a real-time polymerase chain reaction (PCR) found complex expression patterns of the MMSET transcripts in fluorescence-activated cell sorted (FACS)-purified plasma cells (PCs) from 15 t(4;14)+ MM patients. In addition, potential target genes of MMSET type I and II were identified, using microarray analyses of MMSET transfected cell lines. Subsequently, the expression of potential target genes was verified by real-time PCR in FACS-purified PCs from 15 t(4;14)+ and <em>22</em> t(4;14)- MM patients. We suggest that the inhibitor of differentiation 1 (ID-1) is a target gene of MMSET, based on its upregulation in MMSET transfected cell lines and a significant association between the t(4;14) translocation and ID-1 expression in MM patients (P = 0.002). As high levels of ID-1 are associated with cancer, our findings indicate that MMSET promotes oncogenic transformation in t(4;14)+ MM patients by transcriptional activation of ID-1 expression.

OBJECTIVE

The use of growth factors to achieve closure of perforated tympanic membranes (TMs) has recently become popular. However, preoperative factors affecting treatment outcomes have seldom been discussed.

OBJECTIVE

To evaluate preoperative factors contributing to the success or failure of healing of perforated TMs.

METHODS

Retrospective cohort study of 153 patients (48 males and 105 females) in whom the duration of perforation was longer than 6 months prior to treatment and who were observed for at least 1 year after treatment between July 2009 and June 2012. Eight factors considered likely to affect the outcome of perforation closure were statistically evaluated using multivariate logistic regression analysis.

METHODS

Each perforated TM was filled with a synthetic graft material (atelocollagen sponge and silicone membrane) containing human basic fibroblast growth factor to promote wound healing after TM perforation closure.

METHODS

Complete closure vs residual perforation.

RESULTS

After 1 year of follow-up, 101 patients (66.0%) achieved complete closure, 30 patients (19.6%) had residual pinhole perforations (<1 mm diameter), and 22 patients (14.4%) had larger residual perforations. Multivariate logistic regression analysis adjusted for each explanatory variable identified a TM without calcification (odds ratio [OR], 2.68 [95% CI, 1.17-6.15]; P = .03) and a perforation not involving the tympanic annulus (odds ratio, 2.75 [95% CI, 1.09-6.94]; P = .04) as significant. Insignificant factors included perforation margin identified on microscopy (OR, 0.24 [95% CI, 0.99-6.27]; P < .001), perforation margin without epithelial migration (OR, 7.27 [95% CI, 0.66-80.49]; P = .11), absence of preoperative otorrhea (P = .38), no previous ear operations (P = .82), perforation size (P = .14), and patient age (P = .26).

CONCLUSIONS

Tympanic membrane regeneration therapy can be applied to all patients, except those with cholesteatoma or malignant neoplasm. However, patients with severe calcification of the TM and those with marginal perforations close to the fibrous annulus should be treated more prudently to achieve perforation closure.