OBJECTIVE

To study the effects of transforming growth factor-β1 (TGF-β1) on the gene expression of connective tissue growth factor (CTGF) in cultured lung fibroblasts of embryonic rats in vitro.

METHODS

Wistar rats of embryonic 19 days were used for primary culture of lung fibroblasts (LFs). The cells in the experimental group were treated by different concentrations (1, 5 or 10 ng/mL) and different durations (12, 24 or 48 hrs) of TGF-β1 to stimulate the LFs. The cells in the control group were cultured in serum-free medium. RT-PCR method was applied to detect CTGF mRNA expression in LFs.

RESULTS

Compared with the control group, the levels of CTGF mRNA in LFs in the experimental group increased significantly (P<0.05). CTGF mRNA expression gradually increased with increasing concentration and duration of TGF-β1 treatment (P<0.05).

CONCLUSIONS

TGF-β1 can stimulate CTGF gene expression in LFs and increase CTGF gene expression in a dose-and time-dependent manner.

A previous publication (Leith et al., <em>19</em>92) showed that administration of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-2, 0.25 mg kg-1, q.i.d. x 7) to mice bearing xenografted DLD-2 human colon cancers would increase treated tumour <em>growth</em> rates as compared to control neoplasms. Additionally, at the end of the 7 day treatment period, clonogenic excision assays showed that the percentage of hypoxic cells in tumours from mice receiving FGF-2 administration was significantly decreased as compared to control neoplasms (from about 42 to about <em>19</em>%). The present study was undertaken to better define the kinetics of changes in hypoxic percentages as a function of tumour volume and FGF-2 treatment. In sham-injected control tumours, the hypoxic percentage increased from about 14% at day 15 postimplantation, (i.e. when sham- or FGF-2 injections were begun) to about 42% by day 22, and to about 75% at 29 days postimplantation (respective average volumes 220, 910, and 2810 mm3). In contrast, the hypoxic percentages in mice treated with FGF-2 remained at the levels seen in control mice on day 15, not only throughout the 7 day FGF-2 treatment schedule, but for at least 1 week after the cessation of <em>growth</em> <em>factor</em> administration. The hypoxic percentage was 16% on day 29 postimplantation, even though average tumour volumes were about 4325 mm3. These data show that the effect of FGF-2 administration on tumour <em>growth</em> rate and hypoxic percentages in xenografted DLD-2 neoplasms is rapid, and continues for some period of time even after administration is ended. Studies of tumour perfusion with injected 86RbCl at equivalent tumour volumes of about 1800 mm3 indicated that the percentage of cardiac output to FGF-2 treated tumours was 33% greater than in sham-injected control neoplasms.

BACKGROUND

Basic fibroblast growth factor (FGF-2), a potent angiogenic peptide, is known to be present in gonadotropes of the anterior pituitary parenchyma of rats and mice, and has been isolated from endothelial cells of many organs. Its localization within endothelial cells has not been determined, nor the mechanisms by which it might be released from endothelial cells during normal organogenesis.

METHODS

Localization of FGF within endothelial cells of the anterior pituitary was accomplished by immunocytochemistry and studied by light- and electron microscopy. Capillaries within the anterior pituitary were studied in fetal rats from day 15 to term, and in adult rats.

RESULTS

At the onset stages of vascularization (15-18 days fetal), the cytoplasm of the endothelial cells of many of the invading, immature capillaries (thick-walled with few or no fenestrations) was intensely immunopositive for FGF. Immunoprecipitate-filled blebs and slender cytoplasmic processes projected from the endothelial cells into the presumptive pericapillary space and toward the parenchymal cells. As gestation progressed (19-20 day fetal), and an increasing number of capillaries acquired the features characteristic of capillaries in the anterior pituitary of adult animals, i.e., thin-walled and fenestrated, there were fewer capillaries demonstrating immunopositivity for FGF. Foci of released FGF, i.e., extracellular, were occasionally evident within the presumptive pericapillary spaces throughout gestation. By comparison, capillaries of the anterior pituitary of adult rats did not contain immunostainable FGF in their cytoplasm, nor were any blebs and/or processes filled with immunoprecipitate evident. However capillaries did reveal an immunopositive enhancement of their lumenal and ablumenal surfaces.

CONCLUSIONS

During vascularization of the anterior pituitary, FGF within the cytoplasm of endothelial cells is released from blebs and/or processes of endothelial cells, and after the capillary bed is stabilized postnatally, these characteristics of vascularization are absent.

OBJECTIVE

To explore the in vitro methods of isolation and culture of human fetal epidermal stem cells (HFESCs) and the feasibility of the cultured cells as the target cells for gene transfection.

METHODS

The HFESCs were isolated by means of type IV collagen rapid adhering method. The culture medium for HFESCs was prepared according to that for human fetal <em>fibroblasts</em>. The cultured cells were identified by immunohistochemistry staining of keratin-<em>19</em> and integrin-beta1, cell cycle analysis and clone forming rate determination. Then the cultured cells were gene transfected in vitro by liposome mediating method in which eukaryon expression vector pcDNA3.1/VEGF165 containing vascular endothelial <em>growth</em> <em>factor</em> 165 (VEGF165) were transfected into cultured cells, or by virus vector mediating method in which recombinant adenovirus accompanied vector (raav) containing green fluorescent protein (GFP) (raav/GFP) were transfected into the cultured cells, respectively. The results of in vitro gene transfection of HFESCs were observed by immunohistochemisty staining and fluorescence microscope.

RESULTS

HFESCs grew well and formed large clones with higher cloning efficiency and higher ratio of G1 cells than keratinocytes. The cultured cells were strongly positive with immunohistochemistry staining of keratin-<em>19</em> and integrin-beta1. After being gene-transfected by pcDNA3.1/VEGF165, the VEGF165 of HFESCs showed positive immunohistochemistry staining property, while the HFESCs transfected by raav/GFP exhibited strong fluorescence.

CONCLUSIONS

HFESCs could be isolated and cultured in vitro by means of rapid adherence to type IV collagen. It seemed feasible that HFESCs were gene transfected with liposome or adeno-associated virus as the vector.

Tubular damage and loss associated with interstitial inflammation and fibrosis may be the most important determinants in chronic renal allograft rejection. To elucidate potential pathophysiologic mechanisms associated with tubulointerstitial lesions, we examined the expression of a fibrogenic cytokine, acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF-1) and its high-affinity receptors, in both relevant renal transplant controls (n=5) and tissue from patients (n=<em>19</em>) who underwent nephrectomy after graft loss, secondary to chronic rejection. In situ hybridization and immunohistochemical analyses demonstrated minimal expression of FGF-1 mRNA and protein in the tubulointerstitial compartment of the normal human kidney. In contrast, tubulointerstitial lesions in kidney allografts experiencing chronic rejection demonstrated the exaggerated appearance of both FGF-1 protein and mRNA in resident inflammatory and tubular epithelial cells. Patterns of staining were consistent throughout tubular compartments and did not appear to be localized to any particular region. The tubulointerstitium in kidneys with findings of chronic rejection also exhibited increased immunodetection of proliferating cell nuclear antigen in the tubular epithelium, inflammatory cell infiltrate, and neovascular structures. The enhanced appearance of FGF-1 and readily detectable <em>fibroblast</em> <em>growth</em> <em>factor</em> receptors suggests that this polypeptide mitogen may serve as an important mediator of <em>growth</em> and repair responses, associated with development of angiogenesis and tubulointerstitial lesions during chronic rejection of human renal allografts.

OBJECTIVE

Fibroblast growth factor-2 (FGF-2) is a multifunctional protein which plays a role in smooth muscle cell growth, wound healing, tissue repair and angiogenesis. FGF-2 is also released by mechanically wounded cells. Herein, the importance of FGF-2 release from periannular tissue in the mechanism of pannus formation in obstructed mechanical prostheses was investigated.

METHODS

Between January 1993 and December 2002, 35 patients with an obstructed bileaflet prosthetic mitral valve were classified according to the nature of obstruction as either thrombus or pannus. Data were related to patient age and gender, prosthesis model and size, intraoperative and pathology findings, and interval between implant and thrombosis. FGF-2 release was monitored immunohistochemically in all cases.

RESULTS

Thrombus formation was found in 19 patients, and pannus formation in 16. Patients were reoperated on after 3.10 +/- 0.7 years in the thrombus group, and after 6.3 +/- 0.46 years in the pannus group (p = 0.04). A foreign body reaction was found 78.9% of thrombus patients and 81.2% of pannus patients (p = 0.602), chronic inflammation in 31.5% and 50%, respectively (p = 0.317), and FGF-2 release in 78.9% and 87.5%, respectively (p = 0.582).

CONCLUSIONS

As FGF-2 release was similar in both patient groups, the duration of FGF-2 release from injured periannnular tissue was considered to form part of the chronic healing process, and was not attributed to mitral valve obstruction by pannus formation.

<AbstractText>Sensory neuropathies (SNs) are often classified as idiopathic even if immunological mechanisms can be suspected. Antibodies against the intracellular domain of the <em>fibroblast</em> <em>growth</em> <em>factor</em> receptor 3 (FGFR3) possibly identify a subgroup of SN affecting mostly the dorsal root ganglion (DRG). The aim of this study was to identify the frequency of anti-FGFR3 antibodies and the associated clinical pattern in a large cohort of patients with SN.</AbstractText><AbstractText>A prospective, multicentric, European and Brazilian study included adults with pure SN. Serum anti-FGRF3 antibodies were analysed by ELISA. Detailed clinical and paraclinical data were collected for each anti-FGFR3-positive patient and as control for anti-FGFR3-negative patients from the same centres ('center-matched').</AbstractText><AbstractText>Sixty-five patients out of 426 (15%) had anti-FGFR3 antibodies, which were the only identified autoimmune markers in 43 patients (66%). The neuropathy was non-length dependent in 89% and classified as sensory neuronopathy in 64%, non-length-dependent small fibre neuropathy in 17% and other neuropathy in <em>19</em>%. Specific clinical features occurred after 5-6 years of evolution including frequent paresthesia, predominant clinical and electrophysiological involvement of the lower limbs, and a less frequent mixed large and small fibre involvement. Brazilians had a higher frequency of anti-FGFR3 antibodies than Europeans (36% vs 13%, p<0.001), and a more frequent asymmetrical distribution of symptoms (OR 169, 95% CI 3.4 to 8424).</AbstractText><AbstractText>Anti-FGFR3 antibodies occur in a subgroup of SN probably predominantly affecting the DRG. Differences between Europeans and Brazilians could suggest involvement of genetic or environmental <em>factors</em>.</AbstractText>

Abnormal fatty acid (FA) metabolism contributes to diabetes and cardiovascular disease. The FA receptor CD36 has been linked to risk of metabolic syndrome. In rodents CD36 regulates various aspects of fat metabolism, but whether it has similar actions in humans is unknown. We examined the impact of a coding single-nucleotide polymorphism in CD36 on postprandial hormone and bile acid (BA) responses.

To examine whether the minor allele (G) of coding CD36 variant rs321<em>19</em>38 (G/T), which reduces CD36 level by ∼50%, influences hormonal responses to a high-fat meal (HFM).

Obese African American (AA) women carriers of the G allele of rs321<em>19</em>38 (G/T) and weight-matched noncarriers (T/T) were studied before and after a HFM.

Two-center study.

Obese AA women.

HFM.

Early preabsorptive responses (10 minutes) and extended excursions in plasma hormones [C-peptide, insulin, incretins, ghrelin <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)<em>19</em>, FGF21], BAs, and serum lipoproteins (chylomicrons, very-low-density lipoprotein) were determined.

At fasting, G-allele carriers had significantly reduced cholesterol and glycodeoxycholic acid and consistent but nonsignificant reductions of serum lipoproteins. Levels of GLP-1 and pancreatic polypeptide (PP) were reduced 60% to 70% and those of total BAs were 1.8-fold higher. After the meal, G-allele carriers displayed attenuated early (-10 to 10 minute) responses in insulin, C-peptide, GLP-1, gastric inhibitory peptide, and PP. BAs exhibited divergent trends in G allele carriers vs noncarriers concomitant with differential FGF<em>19</em> responses.

CD36 plays an important role in the preabsorptive hormone and BA responses that coordinate brain and gut regulation of energy metabolism.

An increase of bile acids (BAs), <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>), and glucagon-like peptide 1 (GLP-1) has been implicated in metabolic improvements after Roux-en-Y gastric bypass and vertical sleeve gastrectomy. However, data are still conflicting regarding their role after laparoscopic adjustable gastric banding (LAGB).

To assess the fasting BA, FGF<em>19</em>, and GLP-1 concentrations in plasma before and after LAGB and to test for correlations with immunometabolic parameters. Furthermore, hepatic farnesoid X receptor (FXR) expression and regulation of FXR-dependent genes were analyzed.

Observational study at the University Hospital Innsbruck.

Twenty obese patients.

Fasting plasma samples were taken before, 3, 6, and 12 months after LAGB. Liver biopsies were obtained at surgery and after 6 months postoperatively.

BA profiles, GLP-1 and FGF<em>19</em> levels, hepatic FXR expression and regulation of FXR target genes were determined.

Total, conjugated, and secondary BAs transiently increased 3 months after LAGB (P < 0.01). Only one BA, glycolithocholic acid sulfate, remained significantly elevated throughout the whole follow-up period (P < 0.05). GLP-1 had increased transiently 3 months after surgery (P < 0.01), whereas FGF<em>19</em> levels increased continuously (P < 0.05). Insulin, homeostasis model assessment index, C-reactive protein, FGF<em>19</em>, and GLP-1 correlated positively with different BAs. No differences were seen in hepatic FXR expression and FXR-regulated genes.

Our study results, not only identified LAGB-induced changes in BAs and BA-induced hormones, but also revealed associations between changes in BA profile with GLP-1 and FGF<em>19</em>.

OBJECTIVE

Vitamin D-deficient rickets (DR) has recently re-emerged among developed countries. Vitamin D deficiency can influence biochemical results of patients with fibroblast growth factor 23 (FGF23)-related hereditary hypophosphatemic rickets (HR), making differential diagnosis difficult. In the present study we evaluated the utility of serum FGF23 levels in the diagnosis of DR and during its treatment.

METHODS

The study group comprised 24 children with DR and 8 children with HR. Serum FGF23 levels and bone metabolism-related measurements were assessed.

RESULTS

Serum FGF23 levels in patients with DR were less than 19 pg/ml, while those in patients with HR were more than 57 pg/ml. There were significant differences in serum levels of calcium, phosphate, parathyroid hormone, and 1,25-dihydroxyvitamin D, as well as tubular maximum phosphate reabsorption per glomerular filtration rate between patients with DR and HR, but these values were not fully mutually exclusive. In addition, serum FGF23 and phosphate levels were increased following treatment.

CONCLUSIONS

Serum FGF23 level is the most critical biochemical marker for distinguishing DR from HR and might be a good indicator of biochemical response to the intervention. Serum FGF23 levels show utility for the diagnosis of DR and in the assessment of its response to treatment.

The corneae of 17- to <em>19</em>-day-old chick embryos were dissected and grafted on the chorioallantoic membrane (CAM) of 10- to 14-day-old embryos with reincubation periods of 3-9 days. After fixation, serial semithin or paraffin sections were made. Furthermore, two <em>growth</em> <em>factors</em> were applied together with the corneae. The 165-amino-acid vascular endothelial <em>growth</em> <em>factor</em> (VEGF165) or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was either pipetted onto the corneae, or pieces of shell membrane were soaked in a <em>factor</em> solution, dried and inserted into an incision in the corneae. After a reincubation of 4-6 days, serial paraffin sections were made. The controls show that the viability of the grafts decreases with increasing age of the host CAM. Furthermore, with prolonged reincubation, the viable corneae become more and more spherical. Due to this movement, necrotic tissue of the CAM is shifted into the center of the sphere. After 9 days, blood vessels can be seen <em>growing</em> in the direction of the necrosis. bFGF pipetted onto the grafts induces marked proliferation of the stroma of the CAM beneath the corneae. Additionally, bFGF carriers inserted into the corneae induce fibrocyte in<em>growth</em> in the grafts together with a few blood vessels. VEGF165 specifically induces vascular <em>growth</em> in the CAM beneath the cornea but did not induce blood vessel <em>growth</em> into the grafts. The pros and cons of the method are discussed.

Introduction: Muscle sympathetic nerve activity (MSNA) may play a role in insulin resistance in obesity. However, the direction and nature of the relationship between MSNA and insulin resistance in obesity remain unclear. We hypothesized that resting MSNA would correlate inversely with both muscle and liver insulin sensitivity and that it would be higher in insulin-resistant vs. insulin-sensitive subjects. Materials and methods: Forty-five non-diabetic obese subjects were studied. As no significant relationships were found in women, the data presented in on 22 men aged 48 ± 12 years. Two-step (15 and 80 mU/m2/min) hyperinsulinaemic-euglycaemic clamps were performed using deuterated glucose to determine liver and muscle insulin sensitivity. Clinical and metabolic parameters were assessed. MSNA was measured via a microelectrode inserted percutaneously into the common peroneal nerve. Results: MSNA burst frequency correlated inversely with liver insulin sensitivity (r = -0.53, P = 0.02) and positively with the hepatokines C-reactive protein (CRP) and <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF)-<em>19</em> (r = 0.57, P = 0.006, and r = -0.47, P = 0.03, respectively). MSNA burst frequency was lower in Liversen compared to Liverres (27 ± 5 vs. 38 ± 2 bursts per minute; P = 0.03). Muscle insulin sensitivity was unrelated to MSNA. Discussion: Sympathetic neural activation is related to liver insulin sensitivity and circulating hepatokines CRP and FGF-<em>19</em> in non-diabetic obese men. These results suggest a potential hepato-endocrine-autonomic axis. Future studies are needed to clarify the influence of MSNA on liver insulin sensitivity in men.

To investigate the role of bile acids (BA) in the pathogenesis of diet-induced non-alcoholic steatohepatitis (NASH), we fed a "Western-style diet" (HFF) enriched with fructose, cholesterol and saturated fat for 10 weeks to juvenile Iberian pigs. We also supplemented probiotics with <i>in vitro</i> BA deconjugating activity to evaluate their potential therapeutic effect in NASH. Liver lipid and function, cytokines and hormones were analyzed using commercially available kits. Metabolites, BAs, and fatty acids were measured by liquid chromatography-mass spectrometry. Histology and gene and protein expression analyses were performed using standard protocols. HFF-fed pigs developed NASH, cholestasis and impaired enterohepatic Farnesoid-X receptor (FXR)-<em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> (FGF<em>19</em>) signaling, in the absence of obesity and insulin resistance. Choline depletion in HFF livers was associated with decreased lipoprotein and cholesterol in serum, and an increase of choline-containing phospholipids in colon contents and trimethylamine-N-oxide in the liver. Additionally, gut dysbiosis and hyperplasia increased with the severity of NASH, and were correlated with increased colonic levels of choline metabolites and secondary BAs. Supplementation of probiotics in the HFF diet enhanced NASH, inhibited hepatic autophagy, increased excretion of taurine and choline, and decreased gut microbial diversity. In conclusion, dysregulation of BA homeostasis was associated with injury and choline depletion in the liver, as well as increased biliary secretion, gut metabolism and excretion of choline-based phospholipids. Choline depletion limited lipoprotein synthesis resulting in hepatic steatosis, while secondary BAs and choline-containing phospholipids in colon may have promoted dysbiosis, hyperplasia and trimethylamine synthesis, causing further damage to the liver.

Hippocampal damage after status epilepticus (SE) leads to multiple epileptogenic changes, which lead to chronic temporal lobe epilepsy (TLE). Morbidities such as spontaneous recurrent seizures (SRS) and memory and mood impairments are seen in a significant fraction of SE survivors despite the administration of antiepileptic drugs after SE. We examined the efficacy of bilateral intra-hippocampal grafting of neural stem/progenitor cells (NSCs) derived from the embryonic day <em>19</em> rat hippocampi, six days after SE for restraining SE-induced SRS, memory, and mood impairments in the chronic phase. Grafting of NSCs curtailed the progression of SRS at 3-5 months post-SE and reduced the frequency and severity of SRS activity when examined at eight months post-SE. Reduced SRS activity was also associated with improved memory function. Graft-derived cells migrated into different hippocampal cell layers, differentiated into GABA-ergic interneurons, astrocytes, and oligodendrocytes. Significant percentages of graft-derived cells also expressed beneficial neurotrophic <em>factors</em> such as the <em>fibroblast</em> <em>growth</em> <em>factor</em>-2, brain-derived neurotrophic <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>-1 and glial cell line-derived neurotrophic <em>factor</em>. NSC grafting protected neuropeptide Y- and parvalbumin-positive host interneurons, diminished the abnormal migration of newly born neurons, and rescued the reelin+ interneurons in the dentate gyrus. Besides, grafting led to the maintenance of a higher level of normal neurogenesis in the chronic phase after SE and diminished aberrant mossy fiber sprouting in the dentate gyrus. Thus, intrahippocampal grafting of hippocampal NSCs shortly after SE considerably curbed the progression of epileptogenic processes and SRS, which eventually resulted in less severe chronic epilepsy devoid of significant cognitive and mood impairments.

Keywords: EEG; cell transplantation; cognitive dysfunction; depression; hippocampal NSCs; memory; neural stem cells; neuroprotection; stem cell grafts; temporal lobe epilepsy.

Nintedanib is a synthetic orally active tyrosine kinase inhibitor, whose main action is to inhibit the receptors of the platelet-derived <em>growth</em> <em>factor</em>, <em>fibroblast</em> <em>growth</em> <em>factor</em> and vascular endothelial <em>growth</em> <em>factor</em> families. The drug also affects other kinases, including Src, Flt-3, LCK, LYN. Nintedanib is used in the treatment of idiopathic pulmonary fibrosis, chronic fibrosing interstitial lung diseases and lung cancer. The mechanism of action suggests that nintedanib should be considered one of the potential agents for inhibiting and revising the fibrosis process related to COVID-<em>19</em> infections. Due to the known induction of coagulation pathways during COVID-<em>19</em> infections, possible interaction between nintedanib and anticoagulant seems to be an extremely important issue. In theory, nintedanib could increase the bleeding risk, thrombosis and lead to thrombocytopenia. The data from clinical trials on the concomitant use of nintedanib and antithrombotic agents is very limited as this patient group was within the standard exclusion criteria. Nintedanib is an important therapeutic option, despite its interaction with anticoagulants. If anticoagulant therapy is necessary, the more effective and safer option is the concomitant administration of DOACs and nintedanib, especially when drug-monitored therapy will be used in patients at high risk of bleeding complications.

Keywords: direct oral anticoagulants; idiopathic pulmonary fibrosis; nintedanib.

Canine perivascular wall tumors (PWTs) are a group of subcutaneous soft tissue sarcomas developing from vascular mural cells. Mural cells are involved in angiogenesis through a complex crosstalk with endothelial cells mediated by several <em>growth</em> <em>factors</em> and their receptors. The evaluation of their expression may have relevance since they may represent a therapeutic target in the control of canine PWTs. The expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF) and receptors VEGFR-I/II, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) and receptor Flg, platelet-derived <em>growth</em> <em>factor</em> B (PDGFB) and receptor PDGFRβ, transforming <em>growth</em> <em>factor</em> β1 (TGFβ1) and receptors TGFβR-I/II, and cyclooxygenase 2 (COX2) was evaluated on frozen sections of 40 PWTs by immunohistochemistry and semiquantitatively scored to identify their potential role in PWT development. Statistical analysis was performed to analyze possible correlations between Ki67 labeling index and the expression of each molecule. Proteins of the VEGF-, PDGFB-, and bFGF-mediated pathways were highly expressed in 27 (67.5%), 30 (75%), and <em>19</em> (47.5%) of 40 PWTs, respectively. Proteins of the TGFβ1- and COX2-mediated pathways were highly expressed in 4 (10%) and 14 (35%) of 40 cases. Statistical analysis identified an association between VEGF and VEGFR-I/II (P = .015 and .003, respectively), bFGF and Flg (P = .038), bFGF and PDGFRβ (P = .003), and between TGFβ1 and COX2 (P = .006). These findings were consistent with the mechanisms that have been reported to play a role in angiogenesis and in tumor development. No association with Ki67 labeling index was found. VEGF-, PDGFB-, and bFGF-mediated pathways seem to have a key role in PWT development and <em>growth</em>. Blockade of tyrosine kinase receptors after surgery could represent a promising therapy with the aim to reduce the PWT relapse rate and prolong the time to relapse.

Epidermal <em>growth</em> <em>factor</em> (EGF) and transforming <em>growth</em> <em>factor</em>-alpha (TGF-alpha) promote the differentiation and proliferation of epithelia as well as the proliferation and chemotaxis of <em>fibroblasts</em>. Additionally, EGF promotes wound healing in tissues composed largely of epithelial cells and <em>fibroblasts</em>. We hypothesized that EGF and TGF-alpha regulate the differentiation and proliferation of the epithelial lining and the migration and proliferation of <em>fibroblasts</em> in the subepithelial space of the middle ear mucosa in children with otitis media. As an initial test of this hypothesis, EGF and TGF-alpha concentrations were measured in 82 middle ear effusions of children undergoing tympanostomy tube placement. EGF was present in 45% of these effusions, and TGF-alpha was present in 6%. The mean concentration +/- SEM values for EGF and TGF-alpha were <em>19</em>+/-7.6 and 3.7+/-7.9 pg/mL, respectively. In addition, neutrophils, macrophages, and lymphocytes in middle ear effusions stained for EGF by immunocytochemistry. We conclude that <em>growth</em> <em>factors</em> are frequently present in middle ear effusions of children with otitis media.

We have discovered two new exons in the mouse <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2 or bFGF) gene that can be alternatively spliced to the second coding exon of the gene. The newly identified exons 1b and 1c are located at, respectively, approximately <em>19</em> and 32 kb downstream of the canonical exon 1a. Using RT-PCR analysis, mRNAs containing exon 1c and canonical exons 2 and 3 were identified in embryonic limb, placenta, face, carcass and ocular tissues. A 3.7-kb transcript present in placenta and embryonic limb hybridizes with an exon 1c-derived probe in Northern blot analysis. Alternative splicing of exon 1c to exon 2 creates a transcript for which the predicted alternative FGF-2 (altFGF-2) polypeptide contains a novel N-terminal domain. Our data indicate that in mouse embryos multiple novel mRNA variants are transcribed from the FGF-2 locus using alternative splicing. These data suggest that proteins arising from these alternative transcripts may play a role in mouse embryogenesis.

We hypothesized that constitutive formation of reactive oxygen species by respiratory cells is a barrier to gene transfer when liposome-DNA complexes are used, by contributing to rapid degradation of plasmid DNA. When plasmid DNA is complexed to liposomes it is protected against H(2)O(2)-mediated degradation but not that mediated by the hydroxyl radical. Treatment of distal rat fetal lung epithelial cells (RFL(<em>19</em>)Ep) with the vitamin E analog Trolox (50 microM) reduced intracellular plasmid degradation. Both Trolox (50 microM) and an iron chelator, phenanthroline (0.1 microM), significantly increased transgene expression in RFL(<em>19</em>)Ep approximately twofold, consistent with a hydroxyl radical-mediated inhibition of transgene expression. When basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF; 20 ng/ml), a <em>growth</em> <em>factor</em> with antioxidant properties, was included within liposomes, we observed a significantly greater enhancement of RFL(<em>19</em>)Ep transgene expression (approximately 2-fold) over that seen with Trolox or phenanthroline. Inclusion of bFGF within liposomes also significantly enhanced (approximately 4-fold) transgene expression in mice following intratracheal instillation.

The effects of nerve <em>growth</em> <em>factor</em> (NGF) on induction of Na+,K+-ATPase were examined in a rat pheochromocytoma cell line, PC12h. Na+,K+-ATPase activity in a crude particulate fraction from the cells increased from 0.37 +/- 0.02 (n = <em>19</em>) to 0.55 +/- 0.02 (n = 20) (means +/- SEM, mumol Pi/min/mg of protein) when cultured with NGF for 5-11 days. The increase caused by NGF was prevented by addition of specific anti-NGF antibodies. Epidermal <em>growth</em> <em>factor</em> and insulin had only a small effect on induction of Na+,K+-ATPase. A concentration of basic <em>fibroblast</em> <em>growth</em> <em>factor</em> three times higher than that of NGF showed a similar potency to NGF. The molecular form of the enzyme was judged as only the alpha form in both the untreated and the NGF-treated cells by a simple pattern of low-affinity interaction with cardiotonic steroids: inhibition of enzyme activity by strophanthidin (Ki approximately 1 mM) and inhibition of Rb+ uptake by ouabain (Ki approximately 100 microM). As a consequence, during differentiation of PC12h cells to neuron-like cells, NGF increases the alpha form of Na+,K+-ATPase, but does not induce the alpha(+) form of the enzyme, which has a high sensitivity for cardiotonic steroid and is a characteristic form in neurons.

OBJECTIVE

Bile acid biosynthesis is strictly regulated under physiological conditions. The expression of <em>fibroblast</em> <em>growth</em> <em>factor</em> (FGF) <em>19</em> is induced when bile acids bind to the farnesoid X receptor in the intestinal epithelium. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> is then transported by the portal flow, causing transcriptional inhibition of cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1), a key enzyme in bile acid biosynthesis, through the extracellular signal-regulated kinase (ERK) pathway. However, the regulatory mechanisms of these signaling pathways in hepatocytes under chronic cholestasis remain unclear. We investigated the regulation of these signaling pathways in patients with biliary atresia (BA).

METHODS

We analyzed the regulation of molecules in these signaling pathways using liver and serum samples from eight BA children and four non-cholestatic disease controls.

RESULTS

CYP7A1 mRNA expression was not inhibited in BA microdissected hepatocyte-enriched tissue (HET) despite high serum bile acid concentrations. The FGF<em>19</em> protein was synthesized in BA HET, and its serum concentration was elevated. <em>Fibroblast</em> <em>growth</em> <em>factor</em> receptor 4 was phosphorylated in BA livers. However, ERK phosphorylation was significantly reduced. We examined SPRY2 expression to determine how the ERK pathway was inactivated downstream of the FGF receptor; the expression was significantly increased in BA HET.

CONCLUSIONS

This is the first study to measure the CYP7A1 mRNA levels in human BA HET. <em>Fibroblast</em> <em>growth</em> <em>factor</em> <em>19</em> was increased in BA hepatocytes. By focusing on its regulation in hepatocytes, we showed that the FGF<em>19</em> pathway did not suppress bile acid synthesis, probably due to an altered mechanism involving upregulated SPRY2 in BA patients.

Angiogenesis plays a pivotal role in the progression and metastasis of hepatocellular carcinoma (HCC). However, the expression of a wide range of angiogenic <em>factors</em> remains obscure in HCC. The purpose of the present study was to determine the expression of various angiogenic <em>factors</em> related to hepatocarcinogenesis. We examined the expression of <em>19</em> angiogenic <em>factors</em> using antibody arrays in human tissues of various liver diseases, including HCC. We also studied the expression of <em>19</em> angiogenic <em>factors</em> in the human HCC cell lines PLC/PRF/5, Hep 3B, HuH7, HLE, HLF and Li-7 and the normal hepatocyte cell line ACBRI3716. In human tissues, although the expression of acidic <em>fibroblast</em> <em>growth</em> <em>factor</em> (aFGF) was found to increase from normal liver to chronic hepatitis, its expression remained unchanged in the transition from chronic hepatitis to HCC. Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) was elevated in liver cirrhosis, but the amounts remained unchanged in the transition from liver cirrhosis to HCC. In contrast, either interleukin-8 (IL-8) or basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) was upregulated in HCC. In the HCC cell lines PLC/PRF/5, Hep 3B and HuH-7, the expression of IL-8 was elevated. Although IL-8 was not elevated, bFGF was upregulated in the other HCC cell lines HLE, HLF and Li-7. Thus, either IL-8 or bFGF was upregulated in HCC cell lines and in HCC tissue samples. These data suggest that the upregulation of either IL-8 or bFGF is closely related to the transition from liver cirrhosis into HCC. Therefore, the analysis of the expression of these cytokines using protein arrays may identify novel therapies for individual patients with HCC.