Lysophosphatidic acid (LPA) is the prototypic G-protein-coupled receptor agonist that activates the Ras-mitogen-activated protein (MAP) kinase cascade through pertussis toxin (PTX)-sensitive Gi and enhanced tyrosine kinase activity. We recently detected a 100 kDa protein (p100) that binds to the C-terminal SH3 domain of <em>growth</em>-<em>factor</em>-receptor-bound protein 2 (Grb2) and becomes tyrosine phosphorylated in a PTX-sensitive manner in LPA-treated Rat-1 cells [Kranenburg, Verlaan, Hordijk and Moolenaar (1997) EMBO J. <em>16</em>, 3097-3105]. Through glutathione S-transferase-Grb2 affinity purification and microsequencing, we have now identified p100 as dynamin-II, a GTPase that regulates clathrin-mediated endocytosis. We show that in Rat-1 cells, Grb2-bound dynamin-II is rapidly tyrosine phosphorylated in response to LPA in a PTX-sensitive manner. Thus, tyrosine phosphorylation of Grb2-bound dynamin-II may be a critical event in Gi-mediated activation of the Ras-MAP kinase cascade in <em>fibroblasts</em>.

MicroRNAs (miRNAs) are well-known candidates for modulating the dysregulated signaling pathways during fibrosis. In this study, we investigated the expression pattern of <em>16</em> miRNAs, which have previously been confirmed or predicted to target genes involved in extracellular matrix (ECM) homeostasis. Primary culture of dermal <em>fibroblasts</em> was obtained from skin biopsies of diffused cutaneous SSc (dcSSc) patients and healthy controls. Expression of let-7a, miR-1, miR-15a, miR-17, miR-19a, miR-20a, miR-21, miR-27b, miR-26a, miR-29a, miR-29b, miR29c, miR-141, miR-125a-5p, miR-193a-3p, and miR-200a were quantified by Real-time PCR. Functional analysis of microRNAs was performed using synthetic oligonucleotides. To further confirm the pro- or anti-fibrotic effects of miRNAs, normal <em>fibroblasts</em> were treated with 10 ng/mL of transforming <em>growth</em> <em>factor</em> (TGF)-β to generate an <i>in vitro</i> model of dermal fibrosis. miR-21 and miR-29a were upregulated and downregulated, respectively, in both dcSSc and TGF-β-treated <em>fibroblasts</em>. We observed that restoration of miR-29a expression or blockade of miR-21 function negatively affected collagen production. COL1A1 expression in SSc <em>fibroblasts</em> is more sensitive to changes of miR-29a and miR-21 expression in compare to normal <em>fibroblasts</em>. miR-29a alone was effective to decrease TGF-β-induced collagen production in dermal <em>fibroblasts</em>. miR-21 and TGF-β had synergistic effects on induction of collagen production. However, neither miR-21 nor miR-29a affected alpha smooth muscle actin (α-SMA) expression in the presence or absence of TGF-β in dermal <em>fibroblasts</em>. miR-21 and miR-29a as pro- and anti-fibrotic miRNAs modulate collagen production in an opposing manner. Focusing on miR-21 and miR-29s as therapeutic targets would be effective in patients with SSc or other fibrotic diseases which show aberrant expression of collagen expression.

UNASSIGNED

To study the effect of duodenal-jejunal omega switch (DJOS) in combination with different dietary patterns on the retinol-binding protein (RBP4), fetuin-A, and fibroblast growth factor 21 (FGF21) plasma levels and their hepatic gene expressions in rats.

UNASSIGNED

A high-fat diet (HF) was given to 28 rats and 28 more were fed with a control diet (CD) for 2 months. After that, half of each group underwent either DJOS or SHAM surgery. For the next 2 months, half of the animals in each operation group were kept on the same diet as before and half of them had the diet changed. After 16 weeks of the experiment RBP4, fetuin-A, and FGF21 plasma levels as well as liver Rbp4, Ahsg, and Fgf21 gene expressions were measured.

UNASSIGNED

DJOS had a reductive impact on plasma levels of RBP4, fetuin-A, and FGF21 and Rbp4, Ahsg, and Fgf21 relative gene expression in the liver when compared to SHAM. The HF/HF group expressed significantly higher RBP4 and fetuin-A plasma levels in comparison to the control. The HF diet used before and/or after surgery led to upregulation of Rbp4, Ahsg, and Fgf21 relative gene expression. The lowest levels of analyzed parameters were observed in the CD/CD group.

UNASSIGNED

The efficiency of DJOS surgery, measured by hepatokines' plasma levels and their gene expressions in the liver, depends on the type of diet applied before and after surgery. Manipulation of dietary patterns can lead to marked improvements in metabolic profile after DJOS surgery.

BACKGROUND

The cytokine, interleukin-18 (IL-18), was originally identified as an interferon-γ-inducing proinflammatory factor; however, there is increasing evidence suggesting that it has non-immunological effects on physiological functions. We have previously investigated the potential pathophysiological relationship between IL-18 and dyslipidemia, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis, which were mediated by lipid energy imbalance. Therefore, herein we focused on brown adipocytes (BAs) and brown adipose tissue (BAT) related to energy consumption as non-shivering thermogenesis.

METHODS

Il18-/- male mice were generated on the C57Bl/6 background, and littermate C57Bl/6 Il18+/+ male mice were used as controls. To reveal the direct effect of IL-18, primary cell cultures derived from both mice were established. Moreover, for molecular analysis, microarray, quantitative reverse transcription PCR and western blotting were performed using 6 and 12 weeks old mice. To evaluate the short- and long-term effects of IL-18 on BAT, recombinant IL-18 was administered for 2 and 12 weeks, respectively.

RESULTS

Compared with Il18+/+ mice, BAT of Il18-/- mice showed earlier differentiation and lipid accumulation. To examine the direct effect of IL-18 on BAT, BA cell cultures were established. Myogenic factor 5-expressing adipose precursor cells were extracted from Il18+/+ and Il18-/- mice. PR domain containing 16 (PRDM16), a differentiation inducer, was strongly expressed in Il18-/- BAs, and uncoupling protein 1, a thermogenic and differentiation marker, was upregulated, resulting in the promotion of BA differentiation. Moreover, PRDM16-dependent and independent molecules related to BAT function, such as fibroblast growth factor 21, were activated. These findings were confirmed by comparing Il18+/+ and Il18-/- mice at 6 and 12 weeks of age. Additional analyses of the molecular mechanisms influencing the 'Quantity of adipocytes' identified three associated genes, apolipoprotein C3 (Apoc3), insulin-induced gene 1 (Insig1) and vitamin D (1,25-dihydroxyvitamin D3) receptor (Vdr). Intravenous administration of IL-18 not only significantly improved the expression of some of these genes, but it also significantly decreased the adipocytes' size.

CONCLUSIONS

This study demonstrated the critical function of IL-18 in differentiation and lipid metabolism in BAs. Furthermore, IL-18 may contribute to novel treatments by improving the energy imbalance.

Amplification of <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor2 (FGFR2) has been regarded as a druggable target in gastric cancer (GC). Despite known potential of AZD4547, a selective inhibitor of FGFR 1-3, to suppress tumorigenic effects of activated FGFR2, resistance to the targeted agent has been an unresolved issue. This study was performed to elucidate the mechanism of AZD4547 resistance in GC cells. SNU-<em>16</em> cells were used to establish an AZD4547-resistant GC cell line, SNU-<em>16</em>R. Elevated phosphorylation of EphB3 was confirmed using the Human Phospho-Receptor Tyrosine Kinase Array kit. A tyrosine kinase inhibitor (TKI) of EphB3 was used to investigate the effects of suppressed EphB3 activity in the SNU-<em>16</em>R cell line. SNU-<em>16</em>R cells exhibited upregulated phosphorylation of EphB3. Treatment of SNU-<em>16</em>R cells with the EphB3 TKI resulted in induction of apoptosis, decreased cellular viability, and cell cycle arrest at sub-G1 phase. SNU-<em>16</em>R cells expressed upregulated levels of N-cadherin, vimentin, Snail, matrix metalloproteinase 2 (MMP-2), and MMP-9, and reduced levels of E-cadherin, characteristic of epithelial to mesenchymal transition (EMT). Matrigel invasion assay also demonstrated the increased invasiveness of SNU-<em>16</em>R cells. EphB3 TKI treatment inhibited EMT of SNU-<em>16</em>R cells. Activation of mammalian target of rapamycin (mTOR) through the Ras-ERK1/2 pathway was suggested as the signal transduction mechanism downstream EphB3 by showing enhanced phosphorylation of Raf-1, MEK1/2, ERK1/2, mTOR and its downstream substrates in SNU-<em>16</em>R cells. As expected, EphB3 TKI decreased phosphorylation of these proteins. Our data suggest phosphorylation of mTOR through signaling by EphB3 is a potential mechanism of AZD4547 resistance in GC cells.

<b>Context:</b><i>Cinnamomum verum</i> J. Presl. (Lauraceae) has a high number of polyphenols with insulin-like activity, increases glucose utilization in animal muscle, and might be beneficial for diabetic patients.<b>Objective:</b> This study evaluated the effectiveness of an ointment prepared from <i>Cinnamomum verum</i> hydroethanolic extract on wound healing in diabetic mice.<b>Materials and methods:</b> A total of 54 male BALB/c mice were divided into three groups: (1) diabetic non-treated group mice that were treated with soft yellow paraffin, (2 and 3) mice that were treated with 5 and 10% <i>C. verum</i>. Two circular full-thickness excisional wounds were created in each mouse, and the trial lasted for <em>16</em> d following induction of the wound. Further evaluation was made on the wound contraction ratio, histopathology parameters and mRNA levels of cyclin D1, insulin-like <em>growth</em> <em>factor</em> 1 (IGF-1), glucose transporter-1 (GLUT-1), total antioxidant capacity, and malondialdehyde of granulation tissue contents. HPLC apparatus was utilized to identify the compounds.<b>Results:</b> The HPLC data for cinnamon hydroethanolic extract identified cinnamaldehyde (11.26%) and 2-hydroxyl cinnamaldehyde (6.7%) as the major components. A significant increase was observed in wound contraction ratio, <em>fibroblast</em> proliferation, collagen deposition, re-epithelialization and keratin biosynthesis in the <i>C. verum</i>-treated groups in comparison to the diabetic non-treated group (<i>p</i> < 0.05). The expression level of cyclin D1, IGF1, GLUT 1 and antioxidant capacity increased in the <i>C. verum</i>-treated groups in comparison to the diabetic non-treated group (<i>p</i> < 0.05).<b>Conclusions:</b> Topical administration of <i>C. verum</i> accelerated wound healing and can possibly be employed in treating the wounds of diabetic patients.

Preclinical and clinical data suggest that <em>fibroblast</em> <em>growth</em> <em>factor</em> 21 (FGF21) is anti-fibrotic, improves metabolic status and has potential to treat non-alcoholic steatohepatitis (NASH). We assessed the safety and efficacy of efruxifermin, a long-acting Fc-FGF21 fusion protein, for the treatment of NASH. BALANCED was a randomized, placebo-controlled study in patients with NASH conducted at 27 centers in the United States (ClinicalTrials.gov <a href="http://clinicaltrials.gov/show/NCT03976401" title="See in ClinicalTrials.gov">NCT03976401</a> ). Eighty patients, stratified by hepatic fat fraction (HFF) and fibrosis stage, were randomized using a centrally administered minimization algorithm 1:1:1:1 to receive placebo (n = 21) or efruxifermin 28 mg (n = 19), efruxifermin 50 mg (n = 20) or efruxifermin 70 mg (n = 20) via weekly subcutaneous injection for <em>16</em> weeks. The primary endpoint-absolute change from baseline in HFF measured as magnetic resonance imaging-proton density fat fraction at week 12-was met. For the full analysis set, the least squares mean absolute changes (one-sided 97.5% confidence interval) from baseline in HFF were -12.3% (-infinity (-inf), -10.3), -13.4% (-inf, -11.4) and -14.1% (-inf, -12.1) in the 28-, 50- and 70-mg groups, respectively, versus 0.3% (-inf, 1.6) in the placebo group, with statistically significant differences between efruxifermin groups and placebo (P < 0.0001 each). Overall, 70 of 79 patients who received the study drug (89%) experienced at least one treatment-emergent adverse event (TEAE), with the majority grade 1-2 (64 (81%)), five (6%) grade 3 and one grade 4. The most commonly reported drug-related TEAEs were grade 1-2 gastrointestinal (36 (46%)). Treatment with efruxifermin significantly reduced HFF in patients with F1-F3 stage NASH, with an acceptable safety profile.

<em>Fibroblast</em> <em>growth</em> <em>factors</em> (FGF), hepatocyte <em>growth</em> <em>factor</em> (HGF) and their receptors, FGFR and c-Met, are essential components of the regulatory networks between the epithelium and mesenchyme in embryonic lung, but their respective roles in tumour <em>growth</em> are not clear. We performed allelotyping at loci containing the candidate genes FGFR-1-2-3-4, FGF-1-2-7-10, c-Met and HGF in 36 non-small cell lung cancer (NSCLC) (20 squamous-cell carcinomas (SQC) and <em>16</em> adenocarcinomas (ADC)), by surrounding each locus with two microsatellites (MS), as close as possible to the genes of interest. Unexpectedly, SQC and ADC were frequently altered at all of these loci, and SQC showed more simultaneously altered loci. In ADC, alterations at the 15q13-22 locus (FGF7 candidate gene) were significantly more frequent. Thus, these loci showed different patterns of molecular alterations between SQC and ADC. Finally, alterations at loci containing FGFR and HGF candidate genes were inversely correlated to the lymph node status in SQC and ADC, respectively.

BACKGROUND

Captopril, an angiotensin-converting enzyme inhibitor, used for treating hypertension and heart failure, inhibits angiogenesis in the corneas of rats in response to basic fibroblast growth factor, slows the growth of experimental tumors in rats, and leads to the regression of Kaposi's sarcoma. Because angiogenesis is key to wound healing, we hypothesized that captopril would impair wound healing. We hypothesized also that because local application at operation of Staphylococcus aureus peptidoglycan (SaPG) increases angiogenesis and accelerates wound healing in rats, SaPG would prevent or ameliorate the postulated captopril-impaired wound healing.

METHODS

In each experiment, rats were divided randomly into two groups: one drinking tap water, and the other, tap water containing 0.5 mg captopril/ml. All ate chow and drank ad libitum, pre-operatively (4-12 days) and postoperatively (7 days). In experiments 1 and 2, bilateral paravertebral 5.5-cm skin incisions were made aseptically (intraperitoneal sodium pentobarbital), and closed with interrupted No. 35 stainless-steel sutures. On one side, the wound was immediately inoculated with 157 microliter pyrogen-free isotonic saline and on the other side the wound was inoculated with 157 microliter saline containing 4.7 mg SaPG (860 microgram SaPG/cm incision). In the third experiment, polyvinyl alcohol (PVA) sponges (16-17 mg dry wt each) containing either 50 microliter saline or 0.5 mg SaPG in 50 microliter saline were implanted subcutaneously, two on each side, via 1-cm incisions closed with a single suture. In the fourth experiment, 5.5-cm bilateral skin incisions and subcutaneous implantation of PVA sponges were done as described but all sites were instilled with saline only. All rats were euthanized (CO(2) asphyxia) 7 days postoperatively.

RESULTS

Wound breaking strength (WBS) of the saline-treated incisions was significantly higher (P < 0.001) in captopril-treated rats than in controls (172 +/- 13 g vs 105 +/- 6 g) in experiment 1 and higher, but not significantly in captopril-treated rats in experiment 2 (153 +/- 8 g vs 114 +/- 6 g) (PNS). SaPG inoculation of the incisions increased WBS significantly in both control and captopril-treated rats: 187 +/- 11 g vs 105 +/- 6 g (P < 0.001) and 283 +/- 16 g vs 172 +/- 13 g (P < 0.001), respectively, in experiment 1, and 217 +/- 13 g vs 114 +/- 6 g (P < 0.0001) (controls) and 266 +/- 17 g vs 153 +/- 8 g (captopril-treated rats) (P < 0.0001) in experiment 2. In experiment 3, subcutaneous PVA saline-inoculated sponge reparative tissue hydroxyproline (OHP) content was similar in control and captopril-treated rats, and SaPG inoculation increased reparative tissue OHP significantly in both groups: 2458 +/- 218 microgram/100 mg dry sponge vs 3869 +/- 230 microgram/100 mg (P < 0.001) (controls) and 2489 +/- 166 microgram/100 mg vs 4176 +/- 418 microgram/100 mg (P < 0.001) (captopril-treated rats). Histologically, angiogenesis and reparative tissue collagen were similar in control and captopril-treated rats, in both saline-inoculated and SaPG-inoculated sponges. In experiment 4 (all incisions and subcutaneous PVA sponges were saline-inoculated), there was no significant difference in WBS between control and captopril-treated rats (107 +/- 6 g vs 96 +/- 5 g, NS). PVA sponge reparative tissue OHP was significantly higher in captopril-treated rats: 3698 +/- 170 microgram/100 mg dry sponge vs 2534 +/- 100 microgram/100 mg (P < 0.0001).

CONCLUSIONS

Unexpectedly, in four experiments, captopril did not inhibit WBS or PVA sponge reparative tissue angiogenesis or collagen accumulation; in fact, WBS was increased significantly in one of three experiments, and PVA sponge reparative tissue OHP was increased significantly in one of two experiments. Also, captopril did not interfere with the wound healing-accelerating effect of SaPG.

<em>Fibroblast</em> <em>growth</em> <em>factor</em>-2 (Fgf-2 or basic Fgf) is known to promote the survival, proliferation, and differentiation of neural precursor cells. We have examined and compared the effects of Fgf-2 with those of Fgf-1, -4, -6, -7, -9, and -10, as well as three isoforms of Fgf-8 (-8a, -8b, and -8c), on the fate of cultured embryonic day 15 (E15) rat cortical cells. Clonal analysis, using retroviral tagging, shows that only Fgf-2, -4, and -8b can efficiently promote the survival of cortical precursor cells, the majority of which give rise to neurons. Surprisingly, and in contrast to other Fgfs, Fgf-8b also promotes astroglial differentiation of a subpopulation of these cells, which would otherwise appear to yield neurons. We also show that E15 cortical cells initially express the IIIc isoforms of Fgf-receptors (R-1,-2, and -3 but within <em>16</em> h of culturing they down regulate FgfR2-IIIc. These studies demonstrate that cortical precursor cells respond to Fgf stimulation in different ways depending on the ligand and by inference the Fgf receptors activated.

We report here that the organotypic co-culture (OCC) system allows for significant preservation of the tissue-specific phenotype of human gingival keratinocytes (IHGK) immortalized with the E6/E7 gene of the human papillomavirus type <em>16</em> (HPV<em>16</em>). The approach adopted is based on the OCC system facilitating spatially separated cell <em>growth</em> and cell-to-cell interactions via diffusible <em>growth</em> <em>factors</em>. Generally, IHGK reveal transcription of the HPV<em>16</em> E6/E7 gene at rising passages. Fluorescence in situ hybridization performed for chromosomes 1, 8, 10, and 18 demonstrates that disomic fractions differ between the tested chromosomes but otherwise remain fairly constant. Monosomies of chromosome 18 are more prominent in late passages 81 and 83, while polysomies of chromosome 10 and 18 are detected in early passages 25 and 27. In comparison with corresponding monolayer cultures (MCs), IHGK in OCCs form stratified epithelia, proliferate, and express gingival-specific gene products in vitro. Moreover, mRNA gene transcription for <em>growth</em> <em>factors</em> interleukin 1beta, granulocyte-macrophage colony stimulating <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em> 7, and EGF in OCCs is different from that in MCs. When grafted onto nude mice, IHGK develop hyperplastic, differentiated surface epithelia devoid of malignant <em>growth</em>. We are not aware of any other OCC system comprising of IHGK, which allows for site-specific expression of gingival epithelial markers. This substantiates reconstitution of a gingival epithelial phenotype in vitro.

In order to investigate whether chronic duodenal ulcer disease is a consequence of disturbed mucosal turnover and <em>growth</em> <em>factor</em> expression, we studied <em>16</em> patients with duodenal ulcers before, during, and after endoscopic healing with lansoprazole or sucralfate. Before treatment, gastric fundal and antral mucosal protein turnover rates were higher in patients than controls, without parallel increases in <em>growth</em> <em>factors</em>. Both forms of therapy produced similar changes, with overall increases in duodenal mucosal turnover and transforming <em>growth</em> <em>factor</em>-alpha (TGF-alpha) and epidermal <em>growth</em> <em>factor</em> receptor (EGF-r) levels. Measurements after healing showed persistent elevations of mucosal turnover in the antrum and duodenum and depressions of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in gastric fundal and duodenal mucosa. We conclude that mucosal turnover is abnormally high in patients with chronic duodenal ulcer disease and is not easily explained by <em>growth</em> <em>factor</em> changes. The failure of lansoprazole and sucralfate to normalize rates, despite endoscopic healing, may explain the high ulcer relapse rates in non-HP-eradicated patients.

OBJECTIVE

Hypercholesterolemia causes erectile dysfunction that is associated with abnormalities in vascular smooth muscle and endothelial cells. We determined the effects of basic fibroblast growth factor (bFGF) on corporeal tissue in hypercholesterolemic rabbits.

METHODS

A total of 16 New Zealand White rabbits were fed a 1% cholesterol diet for 6 weeks and were randomly divided into 3 groups. Group 1 (5 rabbits) received 2.5 microg recombinant bFGF intravenously once and again 3 weeks later. Group 2 (6 rabbits) received 2.5 microg bFGF intravenously once and placebo 3 weeks later. Group 3 (5 rabbits) received placebo intravenously each time. Rabbits were continuously fed a 1% cholesterol diet and sacrificed 3 weeks after the last treatment. Smooth muscle, endothelial cell and collagen content were assessed by immunohistochemistry and histochemical staining of corporeal tissue. Vascular endothelial growth factor (VEGF) protein and mRNA expression were assessed by enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction.

RESULTS

Corporeal smooth muscle content was greater in groups 1 and 2 (35.24% +/- 4.25% and 24.79% +/- 3.39%, p <0.01) vs group 3 (19.68% +/- 2.94%, vs groups 1 and 2 p <0.001 and <0.05, respectively). Endothelial cell and collagen content were similar among the groups. VEGF protein was increased in group 1 vs group 2 (97.90 +/- 26.00 vs 57.03 +/- 14.99 pg/ml, p <0.01) and vs group 3 (39.93 +/- 15.08, p <0.01). There was no statistical difference between groups 2 and 3. VEGF mRNA expression was similar among the groups.

CONCLUSIONS

Systemic bFGF increases smooth muscle content and VEGF protein in hypercholesterolemic rabbit corporeal tissue.

Although a number of ablative-laser techniques based on CO(2) and Er: YAG laser devices have been successfully developed and used in the clinical setting, the bio-molecular processes influencing wound healing after exposure to laser energy are not well elucidated. In this study, we aim to assess the impact of the mechanism of injury on the secretion of transforming <em>growth</em> <em>factor</em> beta1 (TGF-beta1) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in various stages of wound healing, in wounds created with a CO(2) laser and scalpel. Ten Wistar rats were used to determine the levels of <em>growth</em> <em>factor</em> proteins TGF-beta1 and bFGF after CO(2) laser- and scalpel-induced skin injury. Tissue was excised on day 0 for untreated skin (control sites), and on days 1, 10, 30, and 90 following laser and scalpel surgery. Specimens were processed for histopathological analysis and for determining the concentration of <em>growth</em> <em>factors</em> by a Western blot technique. The concentration of TGF-beta1 increased markedly, at day 1 postinjury, from a baseline of 130+/-<em>16</em> mm(2) (mean surface area of blotted-protein lanes) to 261+/-23 mm(2) and 394+/-22 mm(2) for laser-inflicted injury and scalpel wounds, respectively; the latter values were found to differ significantly (p<0.001). The concentration of b-FGF on day 10 postinjury differed significantly (p<0.001) between the laser sites (553+/-45 mm(2)) and the corresponding scalpel sites (418+/-41 mm(2)). Laser energy alters local tissue secretion of TGF-beta1 and bFGF of skin injuries created with the CO(2) laser compared with wounds created with a scalpel. These differences might have an impact on various aspects of wound healing of skin injuries created by a laser.

Ischemic airway complications after lung transplantation remain a significant problem despite the use of bronchial omentopexy. Clinical observations suggest that enhancement of vascular in<em>growth</em> could possibly increase the efficacy of a bronchial omental flap. This study was therefore designed to investigate whether basic <em>fibroblast</em> <em>growth</em> <em>factor</em> can enhance blood supply of an ischemic airway by acceleration of vascular in<em>growth</em> in a rabbit autotransplant model. Segments of the trachea were harvested and transplanted into a subcutaneous pouch. The animals were randomly assigned to one of four groups: group I, no omentopexy; group II, omentopexy; group III, omentopexy and fibrin glue; or group IV, omentopexy and fibrin glue enriched with 2.5 micrograms basic <em>fibroblast</em> <em>growth</em> <em>factor</em>. After 14 days the animals were sacrificed. The extent of perfusion was investigated by means of radioactive microspheres. The morphology of the tracheal segments was investigated in a blinded fashion macroscopically, by means of light microscopy, and by means of scanning electron microscopy. The radioactivity measurements revealed a significantly increased perfusion of group IV (77% +/- 42%) as compared with groups I (17% +/- 13%) and III (20% +/- <em>16</em>%). By macroscopic and light microscopic assessment, the epithelial integrity of group IV was significantly improved compared with groups I and II. At electron microscopy the integrity of group IV was significantly superior to all remaining groups. We conclude that a deposit of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> and fibrin glue appears to increase revascularization of an ischemic airway from omentum and thus results in improved epithelial preservation of a tracheal autograft.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>16</em> (FGF-<em>16</em>) was originally cloned from rat heart. Subsequent investigation of mouse FGF-<em>16</em>, including generation of null mice, revealed a specific pattern of expression in the endocardium and epicardium, and role for FGF-<em>16</em> during embryonic heart development. FGF-<em>16</em> is expressed mainly in brown adipose tissue during rat embryonic development, but is expressed mainly in the murine heart after birth. There is also an apparent switch from limited endocardial and epicardial expression in the embryo to the myocardium in the perinatal period. The FGF-<em>16</em> gene and its location on the X chromosome are conserved between human and murine species, and no other member of the FGF family shows this pattern of spatial and temporal expression. The human and murine FGF-<em>16</em> gene promoter regions also share an equivalent location for TATA sequences, as well as adjacent putative binding sites for transcription <em>factors</em> linked to cardiac expression and response to stress. Recent evidence has implicated nonsense mutation of FGF-<em>16</em> with increased cardiovascular risk, and FGF-<em>16</em> supplementation with cardioprotection. Here we review the important role of FGF-<em>16</em> in embryonic heart development, its gene regulation, and evidence for FGF-<em>16</em> as an endogenous and exogenous cardiac-specific and protective <em>factor</em> in the postnatal heart. Moreover, given the conservation of the FGF-<em>16</em> gene and its chromosomal location between species, the question of support for a cardiac role in the human population is also considered.

Cell-to-cell interactions between human mesenchymal stem cells and potential adjacent cells such as endothelial cells, dermal <em>fibroblasts</em>, and epidermal keratinocytes was investigated. A modified dual Boyden chamber assay using 8-microm pores revealed a more powerful chemotactic cell migration of human mesenchymal stem cells toward human epidermal keratinocytes than other cells, such as umbilical artery endothelial cells and dermal <em>fibroblasts</em>, during <em>16</em> hours of incubation (336.2+/-52.33, 36.0+/-11.20, and 62.7+/-18.<em>16</em>, cells/field, respectively, p<0.01; comparison between endothelial cells and keratinocytes, and <em>fibroblasts</em> and keratinocytes). Scanning electron microscopy showed human mesenchymal stem cell migration through the pores, with endothelial cells, <em>fibroblasts</em>, or keratinocytes in the lower chambers. Mesenchymal stem cell ultrastructural changes occurred, including a larger euchromatin nucleus, when the cells were placed in medium containing 10 percent fetal bovine serum, whereas basic <em>fibroblast</em> <em>growth</em> <em>factor</em> maintained the immature cell morphology for 4 days. Monolayer coculture also showed human mesenchymal stem cell changes in ultrastructural morphology in the vicinity of the epidermal keratinocytes. These data suggest that human mesenchymal stem cells may interact with human epidermal keratinocytes to accelerate wound healing and coverage.

Numerous in vitro studies indicate that <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) play a role in both the development and maintenance of oligodendrocytes. Addition of FGF to mature oligodendrocytes in culture was reported to downregulate the expression of genes encoding proteins of the myelin sheath and to induce a loss of myelin compaction. In this study, a model was developed to functionally block FGF signaling in oligodendrocytes in vivo, by generating transgenic mice expressing a dominant-negative FGF receptor (FGFR1), under the control of the myelin basic protein (MBP) promoter. To demonstrate the effectiveness of this model, truncated FGFR1 was first overexpressed in an FGF-responsive cell line in vitro. It was confirmed that FGF-signalling was blocked in these cells. Subsequently, five independent transgenic lines ("MBP-FRD") were generated. Three lines expressing the highest level of the transgene were further studied. Initial investigation by Western blot and light microscopic analyses revealed no apparent alterations in myelination of the MBP-FRD mouse brains. However, ultrastructural analysis of myelinated optic nerve fibres from two independent MBP-FRD lines revealed a significant increase in myelin thickness as a function of fibre diameter for both transgenic lines (13% and <em>16</em>% increase). This increase in myelin thickness was not accompanied by alterations in myelin compaction. These results support the idea that FGF signaling in oligodendrocytes plays a role in the modulation of axon myelination in vivo.

Acidic and basic <em>fibroblast</em> <em>growth</em> <em>factors</em> (aFGF and bFGF, respectively) are expressed in high levels in adult central nervous system (CNS). We report the time course of developmental appearance and distribution of these <em>factors</em> and of two FGF receptors, FGFR-1 and FGFR-2, in the CNS of rats ranging in age from embryonic day <em>16</em> to adult. Immunohistochemical analysis showed that sensory neurons in the midbrain were the first cells to contain detectable aFGF immunoreactivity at embryonic day 18. The next cell group to contain aFGF were motor neurons, which were found to be aFGF-positive at the day of birth. A number of other subcortical neuronal populations were observed to contain aFGF immunoreactivity after postnatal day 7. Adult levels and distribution patterns of aFGF were reached in all CNS areas by postnatal day 28. Basic FGF immunoreactivity was observed at postnatal day 0 in neurons in the CA2 subfield of hippocampus. Astrocytes contained detectable bFGF immunoreactivity, starting at postnatal day 7. Adult levels and patterns of distribution of bFGF were reached in all CNS areas by postnatal day 28. These immunohistochemical observations were confirmed by using bioassay and Western blot techniques. FGFR-1 and FGFR-2 mRNA were expressed in significant levels in all CNS areas at all time points analyzed. The observation that aFGF and bFGF appear in specific and distinct cellular populations at relatively late developmental times suggests that these FGFs may be involved in specific mechanisms of CNS maturation, maintenance, and repair.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) plays an important role in tumor angiogenesis. Several studies have reported that bFGF may influence cell apoptosis through different signaling pathways. The aim of the present investigation was to study the effect of bFGF on the activities of protein kinase B (PKB)/survivin and cell apoptosis in hepatocellular carcinoma cells (Bel-7402). We treated Bel-7402 cells with bFGF and wortmannin [phosphatidylinositol 3-kinase (PI3K)-specific inhibitor] separately to observe the expression of PKB and survivin detected with RT-PCR and western blotting. The cell cycle and apoptosis were assayed with flow cytometry. We found a significant increase in PKB expression in the group treated with 25 ng/ml bFGF for 10 min (P<0.05), and this effect was significantly inhibited by pretreatment with wortmannin (200 nM) for 1 h. After treatment with 10 ng/ml bFGF, the expression of survivin mRNA in Bel-7402 cells increased significantly, and reached the peak at <em>16</em> h (P<0.05); however, this effect could be significantly inhibited by pretreatment with wortmannin (200 mM) in a time-dependent manner. Following incubation with 25 ng/ml bFGF for 10 min, the apoptosis rate and M phase were significantly decreased and S phase cells increased compared with the wortmannin (200 nM)-treated group. When this group was pretreated with wortmannin (200 nM) for 1 h, the apoptosis rate and S phase were significantly increased, M phase cells decreased. The results revealed that wortmannin could induce high apoptosis rates in hepatocellular carcinoma cells, and bFGF could inhibit the cell apoptosis induced by wortmannin. These findings indicate that bFGF could rapidly activate the PKB activities, enhance the expression of survivin and the proliferation of hepatocellular carcinoma cells via the PI3K pathway, thus it may serve as a novel molecule for early targeting therapy of hepatocellular carcinoma.

At least part of the mechanism underlying fetal development appears to be the production of a number of <em>growth</em> <em>factors</em> considered important in the process of tumour formation. Using immunocytochemistry, we have investigated the temporal and spatial pattern of expression of some of the important <em>growth</em> <em>factors</em>, by the fetus. We describe here the cellular localization of transforming <em>growth</em> <em>factor</em> beta 3 (TGF-beta 3), platelet derived <em>growth</em> <em>factor</em> (PDGF) and its receptor (PDGF-R), TGF-alpha and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) in the fetal rat from day 13 to 21 of gestation. Using antisera raised against an N-terminal portion of TGF-beta 3, immunoreactivity peaked around day <em>16</em> and was seen predominantly within epithelial cells. However, using antisera raised against the C-terminal of this molecule immunoreactivity was seen exclusively within the extracellular matrix underlying adjacent epithelia, and was maintained up until day 21 of gestation. Strong expression of TGF-alpha was seen in cells of most organs throughout the gestation period studied. Immunoreactivity for bFGF, PDGF and PDGF-R peaked around day 18 in both epithelial and mesenchymal cells of all major organ systems and then declined by day 21. These data suggest distinct roles for each <em>factor</em> during embryogenesis and tumorigenesis.

The Marfan syndrome, an autosomal dominant heritable disorder of connective tissue, is caused by mutations in the gene for fibrillin-1, FBN1. A novel FBN1 mutation was identified using temperature-gradient gel electrophoresis of a reverse-transcribed polymerase chain reaction product spanning exons 14 to <em>16</em>. The mutation, G1760A, is predicted to result in the amino acid substitution C587Y and thus to disrupt one of the disulfide bonds of the calcium-binding epidermal <em>growth</em> <em>factor</em>-like module encoded by exon 14. C587Y was found to be a de novo mutation in a relatively mildly affected 15-year-old girl whose clinical phenotype was characterized mainly by ectopia lentis and thoracic scoliosis. Metabolic labeling of cultured dermal <em>fibroblasts</em> from the affected patient demonstrated delayed secretion of fibrillin with normal synthesis and no decrease in incorporation into the extracellular matrix compartment. Fibrillin immunostaining of confluent dermal <em>fibroblast</em> cultures revealed no visible difference between the patient's cells and control cells. Characterization of many different FBN1 mutations from different regions of the gene may provide a better understanding of clinical and biochemical genotype-phenotype relationships.

OBJECTIVE

The <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) family plays an important role in cardiac <em>growth</em> and development. However, only FGF-<em>16</em> RNA levels are reported to increase during the perinatal period and to be expressed preferentially in the myocardium, suggesting control at the transcriptional level and a role for FGF-<em>16</em> in the postnatal heart. Beyond the identification of two TATA-like elements (TATA1 and TATA2) in the mouse FGF-<em>16</em> promoter region and the preferential cardiac activity of TATA2, there is no report of Fgf-<em>16</em> gene regulation. Assessment of promoter sequences, however, reveals putative nuclear <em>factor</em>-kappaB (NF-kappaB) elements, suggesting that Fgf-<em>16</em> is regulated via NF-kappaB activation and thereby implicated in a number of cardiac events. Thus, the Fgf-<em>16</em> gene was investigated as a target for NF-kappaB activation in cardiac cells.

RESULTS

Assessments of Fgf-<em>16</em> promoter activity were made using truncated and transfected hybrid genes with NF-kappaB inhibitors and/or beta-adrenergic stimulation via isoproterenol (IsP) treatment (a known NF-kappaB activator) in culture, and on endogenous mouse and human Fgf-<em>16</em> genes in situ. The mouse Fgf-<em>16</em> promoter region was stimulated in response to IsP treatment, but this response was lost with NF-kappaB inhibitor pretreatment. Deletion analysis revealed IsP responsiveness linked to sequences between TATA2 and TATA1 and, more specifically, a NF-kappaB element upstream and adjacent to TATA1 that associates with NF-kappaB p50/p65 subunits in chromatin. Finally, TATA1 and the proximal NF-kappaB element are conserved in the human genome and responsive to IsP.

CONCLUSIONS

The mouse and human Fgf-<em>16</em> gene is a target for NF-kappaB activation in the postnatal heart.

Radiation-induced hair loss is a clinically important, but under-researched topic. The aim of the study was to develop an in vivo assay system for radiation-induced apoptosis in hair follicles to promote hair research and exploit new radioprotectors. BALB/c mice received total body irradiation (TBI) with gamma-rays at doses in the range from 8 to <em>16</em> Gy at 6 days after depilation. Pathological changes were detected progressively in the hair follicles over the time course after TBI and the dystrophy was evaluated on the basis of stage-specific parameters reported previously, which were found to be well-suited for classification of the radiation-induced hair follicle dystrophy. As a result, regression from anagen to catagen was determined in these follicles after irradiation. In addition, radiation-induced apoptosis was a good early dystrophic parameter. In this system, it was found that <em>fibroblast</em> <em>growth</em> <em>factor</em>-1 effectively prevented hair follicle apoptosis in mice.