Objectives. Human breast milk contains a heterogeneous population of cells that have the potential to provide a noninvasive source of cells for cell therapy in many neurodegenerative diseases without any ethical concern. The objectives of this study were to differentiate the breast milk-derived stem cells (BMDSC) toward neural stem cells and then into the neurons and neuroglia. Materials and Methods. To do this, the BMDSC were isolated from human breast milk and cultured in Dulbecco's modified Eagle medium/F12 (DMEM/F12) containing <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF). The cells were then characterized by evaluation of the embryonic and stem cell markers. Then, the cells were exposed to culture medium containing 1% B27 and 2% N2 for 7-10 days followed by medium supplemented with B27, N2, bFGF 10 µg/mL, and endothelial <em>growth</em> <em>factor</em> (EGF) <em>20</em> µg/mL. Then, the sphere-forming assay was performed. The spheres were then differentiated into three neural lineages by withdrawing <em>growth</em> <em>factor</em> in the presence of 5% FBS (fetal bovine serum). The immunofluorescence was done for β-tubulin III, O4, and GFAP (glial fibrillary acidic protein). Results. The results indicated that the cells expressed both embryonic and mesenchymal stem cell (MSC) markers. They also showed neurospheres formation that was nestin-positive. The cells were also differentiated into all three neural lineages. Conclusion. The BMDSC can behave in the same way with neural stem cells. They were differentiated into oligodendrocytes, and astrocytes as well as neurons.

BACKGROUND

Hyperphosphatemic Familial Tumoral Calcinosis (HFTC) and Hyperphosphatemic Hyperostosis Syndrome (HHS) are associated with autosomal recessive mutations in three different genes, FGF23, GALNT3 and KL, leading to reduced levels of fibroblast growth factor 23 (FGF23) and subsequent clinical effects.

RESULTS

We describe a consanguineous family with two affected siblings with HFTC and HHS caused by a novel homozygous G-to T substitution in exon 3 of GALNT3 (c.767 G>> T; p.Gly256Val), demonstrating great phenotypic variation and long asymptomatic intervals. Calcific tumors appeared at 14 years of age in the male, and the female displayed episodic diaphysitis from age 9 years. Symptoms of eye involvement were present in both from childhood, and progressed into band keratopathy in the female. Abnormal dental roots and tooth loss, as well as myalgia were present in both from their mid-twenties, while the female also had calcifications in the placenta, the iliac vessels and thyroid cartilage. New calcific tumors appeared more than 20 years after the initial episodes, delaying diagnosis and treatment until the ages of 37 and 50 years, respectively. Both siblings had elevated serum phosphate levels, inappropriately elevated tubular maximum phosphate reabsorption per unit glomerular filtration rate (TmP/GFR), reduced levels of intact FGF23 and increased levels of c-terminal FGF23. Review of all 54 previously published cases of GALNT3, FGF23, and KL associated HFTC and HHS demonstrated that more subjects than previously recognized have a combined phenotype.

CONCLUSIONS

We have described HFTC and HHS in a consanguineous Caucasian family with a novel GALNT3 mutation, demonstrating new phenotypic features and significant variability in the natural course of the disease. A review of the literature, show that more subjects than previously recognized have a combined phenotype of HFTC and HHS. HHS and HFTC are two distinct phenotypes in a spectrum of GALNT3 mutation related calcification disorders, where the additional factors determining the phenotypic expression, are yet to be clarified.

Pediatric burn wounds can be problematic because an accurate evaluation is difficult as the result of anatomically immature vasculature or immobilization failure, especially in patients with second-degree burns, and because the burn surface areas and the burn depth tend to worsen over the course of time. Delayed wound healing results in unsightly scarring, such as hypertrophic scars, which are problematic both esthetically and functionally. Among cytokines and <em>growth</em> <em>factors</em>, basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) is clinically proven, having demonstrated accelerated acute and chronic wound healing. Accelerated wound healing may lead to improved scarring. To elucidate the effects of bFGF on second-degree pediatric burn wounds, a comparative study was performed. A total of <em>20</em> pediatric patients ranging from 8 month to 3 years (average 1 year, 3 months +/- 6 months) who suffered from the burns by various causes were divided into two groups, conventional (n = 10) and treatment with bFGF (n = 10). A moisture meter, used to objectively measure the stratum corneum and epithelial-mesenchymal functions, was used to assess scars at least 1 year after wound healing. Clinical evaluation of pigmentation, pliability, height, and vascularity demonstrated significant differences between conventional and bFGF-treated scars (1.7 +/- 0.55 vs 0.7 +/- 0.58, 2.4 +/- 0.82 vs 1.1 +/- 0.69, 1.8 +/- 0.66 vs 0.5 +/- 0.57, 1.9 +/- 0.63 vs 0.8 +/- 0.68; conventional vs bFGF-treated, pigmentation, pliability, height, and vascularity, respectively, P < .01). The effective contact coefficient was significantly greater in conventional wounds than bFGF-treated wounds (14.6 +/- 1.68 % vs 8.7 +/- 2.82 %; conventional vs bFGF, P < .01) and bFGF-treated wounds demonstrated significantly less transepidermal water loss values than conventional treatment (8.3 +/- 1.90 g/m/h vs 5.7 +/- 1.85 g/m/hr; conventional vs bFGF, P < .01). Pediatric burn patients treated with bFGF showed less damaging function of the stratum corneum after healing both in clinical assessment and moisture meter analysis.

BACKGROUND

Triple-negative breast cancer (TNBC) makes up about 10 - <em>20</em>% of all breast cancers and the lack of hormone receptors and human epidermal <em>growth</em> <em>factor</em> receptor-2/Neu expression is responsible for poor prognosis, no targeted therapies and trouble in the clinical management. Tumor heterogeneity, also within the same tumor, is a major cause for this difficulty. Based on the introduction of new biological drugs against different kinds of tumor, many efforts have been made for classification of genetic alterations present in TNBC, leading to the identification of several oncogenes and tumor suppressor genes involved in breast cancer carcinogenesis.

METHODS

In this review we investigated the molecular alteration present in TNBC which could lead to the creation of new targeted therapies in the future, with the aim to counteract this disease in the most effective way.

CONCLUSIONS

In this context some hormone receptors like G-protein-coupled receptor 30 and androgen receptors may be a fascinating area to investigate; also, angiogenesis, represented not only by the classical VEGF/VEGFR relationship, but also by other molecules, like semaphorins, fibroblast growth factor and heparin-binding-EGF-like, is a mechanism in which new developments are expected. In this perspective, one technique that may show promise is the gene therapy; in particular the gene transfer could correct abnormal genetic function in cancer cells.

OBJECTIVE

To determine the protective effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo.

METHODS

In Experiment I, cultured spiral ganglion neurons (SGNs) prepared from postnatal Day 3 mice were exposed to 20 mM glutamate for 2 h before the culture medium was replaced with fresh medium containing 0, 25, 50 or 100 ng/ml bFGF. Fourteen days later, all cultures were fixed with 4% paraformaldehyde and stained with 1% toluidine blue. The number of surviving SGNs was counted and the length of the neurites of the SGNs was measured. In Experiment II, in vivo studies were carried out with guinea pigs in which bFGF or normal saline was injected intramuscularly to assess possible protective effects of bFGF on cochlear hair cells and to accelerate the recovery of the auditory brainstem response (ABR). The ABRs were measured before, immediately after and 2 and 4 weeks after exposure to noise.

RESULTS

Exposure to 20 mM glutamate for 2 h resulted in an inhibition of neurite outgrowth of SGNs and an increase in cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and an increase in the number of surviving SGNs. In Experiment II, significant (p < 0.05) differences in ABR thresholds were observed between the groups injected with bFGF and saline (t = 2.689) at 4 weeks after noise exposure. Cochleae were removed and hair cell loss analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 240 and 2160 inner hair cells in the groups injected with bFGF and saline, respectively. Similarly, more outer hair cells were lost in the normal saline injection group (99,291) than in the group treated with bFGF (70,377).

CONCLUSIONS

Our results demonstrate that bFGF protects SGNs against glutamate neurotoxicity in vitro. In addition, treatment with bFGF protects hair cells from acoustic trauma.

<em>Fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2) and its main receptor FGFR1 have been shown to promote hepatic stellate cell (HSC) activation and proliferation. However, scant information is available on the anti-fibrogenic activity of FGFR1 inhibitors. The aim of this study was to assess the impact of a selective FGFR1 tyrosine kinase inhibitor NP603 on HSC proliferation and hepatic fibrosis. We demonstrated that rat primary HSCs secreted significant amounts of FGF-2, and its tyrosine phosphorylation of FGFR1 was attenuated by NP603. NP603 inhibited HSC activaton by measuring the expression of α-smooth muscle actin (α-SMA) and the production of type I collagen using ELISA. Furthermore, NP603 (25 μM) in vitro strongly suppressed HSC <em>growth</em> induced by FGF-2 (10 ng/ml) and FCS. This effect correlated with the suppression of extracellular-regulated kinase (ERK) activity and its downstream targets cyclin D1 and p21. In addition, PO NP603 (<em>20</em> mg·kg(-1)·day(-1)) administration significantly decreased hepatic collagen deposition and α-SMA expression in CCl(4)-treated rats. Collectively, these studies suggest that selective blocking of the FGFR1-mediated pathway could be a promising therapeutic approach for the treatment of hepatic fibrosis.

1. Currently, there is no satis<em>factor</em>y treatment for pulmonary fibrosis. Emodin, a component in Chinese herbs, has been shown to have an antifibrotic effect on pancreatic fibrosis and liver fibrosis. In the present study, we tested the hypothesis that emodin may attenuate the development of pulmonary fibrosis. 2. Mice were randomly divided into five groups (n = 16 in each). One group was a control group; the remaining four groups were treated with intratracheal instillation of 3 mg/kg bleomycin (BLM). The following day, emodin (5, 10 or <em>20</em> mg/kg per day, p.o.) treatment was started for three of the BLM-treated groups and was continued for 21 days. The fourth BLM-treated group (and the control group) received daily 0.5% sodium carboxymethyl cellulose (placebo) by gavage over the same period. 3. Bleomycin challenge provoked severe pulmonary fibrosis, with marked increases in fibrosis fraction, hydroxyproline content and myeloperoxidase activity in lung tissue. Emodin treatment (10 and <em>20</em> mg/kg per day, p.o.) attenuated all these biochemical indices, as well as histopathological alterations induced by BLM. Furthermore, in mice injected with BLM, elevated levels of transforming <em>growth</em> <em>factor</em>-beta1, interleukin (IL)-4 and IL-13 were found in bronchoalveolar lavage fluid. These increases were significantly inhibited by 10 and <em>20</em> mg/kg per day emodin. 4. In cell culture, exposure of cells to 6.25, 12.5, 25 or 50 micromol/L emodin for 24 h decreased <em>fibroblast</em> proliferation. Treatment of cells with the same concentrations of emodin for 72 h decreased collagen production by <em>fibroblasts</em>. In addition, emodin (6.25, 12.5, 25 or 50 micromol/L) inhibited the steady state expression of alpha1 (I) procollagen and alpha2 (I) procollagen mRNA in a dose-dependent manner. 5. The results of the present study suggest that emodin may be effective in the treatment of pulmonary fibrosis.

Expression of early <em>growth</em> response protein (Egr)-1, a protein of the Egr family of zinc finger transcription <em>factors</em>, is stimulated in glucose-treated pancreatic β-cells and insulinoma cells. The purpose of this study was to elucidate the role of Egr transcription <em>factors</em> in pancreatic β-cells in vivo. To overcome the problem associated with redundancy of functions between Egr proteins, conditional transgenic mice were generated expressing a dominant-negative mutant of Egr-1 in pancreatic β-cells. The Egr-1 mutant interferes with DNA binding of all Egr proteins and thus impairs the biological functions of the entire Egr family. Expression of the Egr-1 mutant reduced expression of TGFβ and basic <em>fibroblast</em> <em>growth</em> <em>factor</em>, known target genes of Egr-1, whereas the expression of Egr-1, Egr-3, Ets-like gene-1 (Elk-1), and specificity protein-3 was not changed in the presence of the Egr-1 mutant. Expression of the homeobox protein pancreas duodenum homeobox-1, a major regulator of insulin biosynthesis, was reduced in islets expressing the Egr-1 mutant. Accordingly, insulin mRNA and protein levels were reduced by 75 or 25%, respectively, whereas expression of glucagon and somatostatin was not altered after expression of the Egr-1 mutant in β-cells. Glucose tolerance tests revealed that transgenic mice expressing the Egr-1 mutant in pancreatic β-cells displayed impaired glucose tolerance. In addition, increased caspase-3/7 activity was detected as a result of transgene expression, leading to a <em>20</em>% decrease of the size of the islets. These results show that Egr proteins play an important role in controlling insulin biosynthesis, glucose homeostasis, and islet size of pancreatic β-cells in vivo.

OBJECTIVE

Fibroblast growth factor 21 (FGF21), an endocrine factor predominantly secreted from liver, possesses multiple beneficial effect on energy metabolism and insulin sensitivity in animals. This study aimed to investigate the acute change of serum FGF21 in response to glucose challenge in humans.

METHODS

A 75-g oral glucose tolerance test was performed among 20 healthy subjects, 18 with impaired glucose tolerance (IGT) and 21 with type 2 diabetes mellitus (T2DM). Blood samples were collected for measurement of FGF21 and other biochemical parameters. The associations of FGF21 with insulin and other metabolic parameters were analyzed.

RESULTS

Fasting serum FGF21 levels increased progressively from healthy, IGT to T2DM subjects (P < 0.05 for global trend). After oral glucose administration, the serum FGF21 level showed a similar biphasic change in all three groups. It declined to a nadir level at 60 min and then increased gradually to its peak level at 180 min. FGF21 levels at different time points of oral glucose tolerance test negatively correlated with glucose levels in all subjects, and the fold change of serum FGF21 at different time points (compared with the basal level) were inversely associated with fold changes of insulin (P = 0.012) and C-peptide (P = 0.043) levels in healthy subjects but not in IGT and T2DM patients.

CONCLUSIONS

The dynamic change of circulating FGF21 was associated with alterations in insulin levels in response to glucose challenge in humans. These findings support the role of FGF21 as a potential regulator of insulin secretion and glucose metabolism in humans.

The role of heparin and heparan sulfate in the binding and signaling of <em>fibroblast</em> <em>growth</em> <em>factors</em> (FGFs) has been subject to intense investigation, but the studies have largely been confined to two species (FGF1 and FGF2) of the family with approximately <em>20</em> members. We have investigated the structural requirements for heparin/heparan sulfate in binding and activation of FGF8 (splice variant b). We present evidence that the minimal FGF8b-binding saccharide domain encompasses 5-7 monosaccharide units. The N-, 2-O-, and 6-O-sulfate substituents of heparin/heparan sulfate (HS) are all involved in the interaction, preferentially in the form of trisulfated IdoUA(2-OSO(3))-GlcNSO(3)(6-OSO(3)) disaccharide constituents. These structural characteristics resemble those described earlier for FGF1. By contrast, the saccharide structures required for the biological activity of FGF8b differed significantly from those characteristic for FGF1 and FGF2. Experiments with cells lacking active HS indicated that extended>>/=14-mer heparin domains were needed to enhance cell proliferation and Erk phosphorylation by FGF8b, whereas in cells stimulated with FGF1 or FGF2 the corresponding responses were achieved by much shorter, 6-8-mer, oligosaccharides. Furthermore, still longer domains were needed to activate FGF8b in cells with "non-optimal" FGF receptor expression. Collectively, our data suggest that the heparin/HS structures enhancing the biological activity of FGFs were influenced by the FGF species involved as well as by the cellular composition of FGF receptors.

Although regioselective removal of 6-O-sulfate groups of heparin has been undertaken by several researchers, complete 6-O-desulfation with little side reaction has not been attained successfully. In this work, a modified method with a certain silylating reagent, N-methyl-N-(trimethylsilyl)trifluoroacetamide, has been established to produce completely 6-O-desulfated heparin with few other chemical changes. The degrees of 6-O-desulfation were estimated by means of chemical disaccharide analyses and/or (13)C NMR spectra. Although the completely 6-O-desulfated heparin lost about <em>20</em>% of 2-O-sulfate groups, any other chemical changes and depolymerization were not detected. The completely 6-O-desulfated heparin displayed strong inhibition of COS-1 cell adhesion to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF)-coated well in a dose-dependent manner, as was clarified by the competitive cell-adhesion assay. Furthermore, the completely 6-O-desulfated heparin was shown to promote in vitro A31 <em>fibroblast</em> proliferation in a dose-dependent manner in the presence of bFGF. These results suggest that signal transduction through bFGF/bFGF receptor in A31 cells occurs in the absence of 6-O-sulfate groups in heparin. The involvement of 6-O-sulfate group(s) of heparin/heparan sulfate in the promotion of bFGF mitogenic activity was reported by several groups. This discrepancy between our results and those of other groups would be due to the differences in molecular size of heparin/heparan sulfate derivatives and/or cell species used for the assay.

OBJECTIVE

To evaluate the efficacy of topically applied autologous plasma rich in growth factors (PRGF) as a treatment for persistent epithelial defects (PEDs) of the cornea.

METHODS

A series of prospective noncomparative cases.

METHODS

Twenty eyes from 18 patients with PED with various underlying etiopathologies: neurogenic, iatrogenic, associated with burning or secondary to severe dry eye. Patients were treated with a PRGF eyedrop solution. Serial photographs of the cornea were taken until epithelialization was complete. We had previously characterized the levels of a panel of growth factors (platelet-derived growth factor, epithelial growth factor, vascular endothelial growth factor, hepatocyte growth factor, fibroblast growth factor, and nerve growth factor) in the PRGF of 11 of these patients. The following variables were additionally recorded: (1) duration of PED before treatment, (2) previous treatments, (3) time for complete epithelialization, and (4) treatments required concomitantly with PRGF.

RESULTS

Epithelial defects healed in 17 of 20 cases (85%), with a mean therapeutic time of 10.9 weeks (range 2-39 weeks). Mean progression time before treatment was 26.7 weeks (range 2-104 weeks). Growth factor concentrations were platelet-derived growth factor 12645.9 +/- 1690.0 pg/mL, epithelial growth factor 468.9 +/- 97.6 pg/mL, vascular endothelial growth factor 204.5 +/- 119.4 pg/mL, hepatocyte growth factor 149.5 +/- 173.5 pg/mL, fibroblast growth factor 82.6 +/- 95.9 pg/mL, and nerve growth factor 37.7 +/- 18.6 pg/mL.

CONCLUSIONS

PRGF, when applied as eyedrops, is a highly effective therapeutic agent for the treatment of a broad etiopathological spectrum of corneal PEDs.

BACKGROUND

Functional antagonism between transforming <em>growth</em> <em>factor</em> beta (TGF-beta) and hyaluronidase has been demonstrated. For example, testicular hyaluronidase PH-<em>20</em> counteracts TGF-beta1-mediated <em>growth</em> inhibition of epithelial cells. PH-<em>20</em> sensitizes various cancer cells to tumor necrosis <em>factor</em> (TNF) cytotoxicity by upregulating proapoptotic p53 and WW domain-containing oxidoreductase (WOX1). TGF-beta1 blocks PH-<em>20</em>-increased TNF cytotoxicity. In the present study, the functional antagonism between TGF-beta1 and lysosomal hyaluronidases Hyal-1 and Hyal-2 was examined.

RESULTS

Murine L929 fibroblasts were engineered to stably express green-fluorescent protein (GFP)-tagged hyaluronidase (GFP-Hyal-1 or GFP-Hyal-2) or GFP alone. Compared to control cells, Hyal-2-expressing cells had a significantly increased sensitivity to TNF cytotoxicity (approximately 60-110% increase), while Hyal-1-expressing cells were less sensitive to TNF (approximately <em>20</em>-90% increase). TNF activated NF-kappaB, along with IkappaBalpha degradation, occurred at <em>20</em> to 60 min in Hyal-2 cells post stimulation, but at the <em>20</em> min time point in both control and Hyal-1 cells. Hyal-2 cells, but not Hyal-1 and control cells, constitutively expressed WOX1, and transiently expressed Hyal-2 enhanced WOX1-mediated cell death. Unlike PH-<em>20</em>, Hyal-1 and Hyal-2 did not induce p53 expression. Hyal-2 translocated from the lysosome to the mitochondria during staurosporine-mediated apoptosis, suggesting that Hyal-2 may damage mitochondria. Finally, Hyal-1 and Hyal-2 blocked TGF-beta1-enhanced L929 cell <em>growth</em>. In contrast, TGF-beta1 inhibited Hyal-1- and Hyal-2-increased TNF cytotoxicity in L929 cells by 30-50%.

CONCLUSIONS

TGF-beta1 limits the ability of Hyal-2 to induce TNF cytotoxicity in L929 cells. Hyal-2-increased TNF cytotoxicity in L929 cells appears to be correlated with upregulation of WOX1, a prolonged NF-kappaB activation, and Hyal-2 translocation to the mitochondria during apoptosis.

BACKGROUND

Lower leg ischemia, myopathy, and limb dysfunction are distinguishing features of peripheral artery disease (PAD). The myopathy of PAD is characterized by myofiber degeneration in association with extracellular matrix expansion, and increased expression of transforming growth factor-beta 1 (TGF-β1; a pro-fibrotic cytokine). In this study, we evaluated cellular expression of TGF-β1 in gastrocnemius of control (CTRL) and PAD patients and its relationship to deposited collagen, fibroblast accumulation and limb hemodynamics.

METHODS

Gastrocnemius biopsies were collected from PAD patients with claudication (PAD-II; N = 25) and tissue loss (PAD-IV; N = 20) and from CTRL patients (N = 20). TGF-β1 in slide-mounted specimens was labeled with fluorescent antibodies and analyzed by quantitative wide-field, fluorescence microscopy. We evaluated co-localization of TGF-β1 with vascular smooth muscle cells (SMC) (high molecular weight caldesmon), fibroblasts (TE-7 antigen), macrophages (CD163), T cells (CD3) and endothelial cells (CD31). Collagen was stained with Masson Trichrome and collagen density was determined by quantitative bright-field microscopy with multi-spectral imaging.

RESULTS

Collagen density increased from CTRL to PAD-II to PAD-IV specimens (all differences p < 0.05) and was prominent around microvessels. TGF-β1 expression increased with advancing disease (all differences p < 0.05), correlated with collagen density across all specimens (r = 0.864; p < 0.001), associated with fibroblast accumulation, and was observed exclusively in SMC. TGF-β1 expression inversely correlated with ankle-brachial index across PAD patients (r = -0.698; p < 0.001).

CONCLUSIONS

Our findings support a progressive fibrosis in the gastrocnemius of PAD patients that is caused by elevated TGF-β1 production in the SMC of microvessels in response to tissue hypoxia.

Thirty percent of patients undergoing percutaneous transluminal coronary angioplasty develop recurrent disease within a year. This is usually due to the rapid accumulation of intimal smooth muscle cells and extracellular matrix, which causes luminal narrowing, and is probably orchestrated by several mitogenic and chemotactic <em>factors</em>, of which platelet-derived <em>growth</em> <em>factor</em> (PDGF) and basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) appear to be particularly important. We have investigated the effects of administering a combination of neutralizing antibodies directed against PDGF-BB and bFGF on neo-intima development following balloon catheter injury in the rat carotid artery. Purified sheep anti-PDGF-BB and anti-bFGF immunoglobulins (IgGs) were administered singly and in combination prior to mechanical injury and daily until sacrifice, 8 days later. Plasma titres of exogenous anti-PDGF-BB and anti-bFGF were maintained at levels 10-<em>20</em>-fold higher than those required to neutralise the mitogenic and chemotactic effects of <em>20</em> ng/ml of PDGF-BB, or 10 ng/ml bFGF in vitro. Used singly, anti-PDGF IgG treatment was associated with a 47% reduction in intimal thickness and a 59% reduction in intimal:medial area ratio; anti-bFGF IgG administration caused a 53% reduction in intimal thickness, and a 50% reduction in intimal:medial area ratio. Treatment with a combination of these antibodies resulted in a 83.8% reduction in intimal thickness (P < 0.05), and a 91% reduction in intimal:medial area ratio (P < 0.01). The latter treatment was also associated with a significantly higher intimal cell density (14.2 +/- 1.6 x 10(3) nuclei/mm2) compared to animals receiving non-immune IgG (7.8 +/- 0.8 x 10(3) nuclei/mm2; P < 0.025), although intimal and medial cell proliferation indices were not significantly different between the groups (P>> 0.05). Our results suggest that in this particular model, PDGF-BB and bFGF are the major <em>factors</em> controlling neointimal hyperplasia, and that these <em>growth</em> <em>factors</em> are operating principally via an effect on smooth muscle cell migration and extracellular matrix protein accumulation.

Basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF) delivery to the brain of animals appears to be an emerging potential therapeutic approach to neurodegenerative diseases, such as Alzheimer's disease (AD). The intranasal route of administration could provide an alternative to intracerebroventricular infusion. A nasal spray of bFGF had been developed previously and the objective of the present study was to investigate whether bFGF nasal spray could enhance brain uptake of bFGF and ameliorate memory impairment induced by co-injection of β-amyloid(25-35) and ibotenic acid into bilateral hippocampus of rats. The results of brain uptake study showed that the AUC(0-12h) of bFGF nasal spray in ol<em>factor</em>y bulb, cerebrum, cerebellum and hippocampus was respectively 2.47, 2.38, 2.56 and 2.19 times that of intravenous bFGF solution, and 1.11, 1.95, 1.40 and 1.93 times that of intranasal bFGF solution, indicating that intranasal administration of bFGF nasal spray was an effective means of delivering bFGF to the brain, especially to cerebrum and hippocampus. In Morris water maze tasks, intravenous administration of bFGF solution at high dose (40 μg/kg) showed little improvement on spatial memory impairment. In contrast, bFGF solution of the same dose following intranasal administration could significantly ameliorate spatial memory impairment. bFGF nasal spray obviously improved spatial memory impairment even at a dose half (<em>20</em> μg/kg) of bFGF solution, recovered their acetylcholinesterase and choline acetyltransferase activity to the sham control level, and alleviated neuronal degeneration in rat hippocampus, indicating neuroprotective effects on the central nerve system. In a word, bFGF nasal spray may be a new formulation of great potential for treating AD.

Produced by senescent cells, the senescence-associated secretory phenotype (SASP) is a potential driver of age-related dysfunction. We tested whether circulating concentrations of SASP proteins reflect age and medical risk in humans. We first screened senescent endothelial cells, <em>fibroblasts</em>, preadipocytes, epithelial cells, and myoblasts to identify candidates for human profiling. We then tested associations between circulating SASP proteins and clinical data from individuals throughout the life span and older adults undergoing surgery for prevalent but distinct age-related diseases. A community-based sample of people aged <em>20</em>-90 years (retrospective cross-sectional) was studied to test associations between circulating SASP <em>factors</em> and chronological age. A subset of this cohort aged 60-90 years and separate cohorts of older adults undergoing surgery for severe aortic stenosis (prospective longitudinal) or ovarian cancer (prospective case-control) were studied to assess relationships between circulating concentrations of SASP proteins and biological age (determined by the accumulation of age-related health deficits) and/or postsurgical outcomes. We showed that SASP proteins were positively associated with age, frailty, and adverse postsurgery outcomes. A panel of 7 SASP <em>factors</em> composed of <em>growth</em> differentiation <em>factor</em> 15 (GDF15), TNF receptor superfamily member 6 (FAS), osteopontin (OPN), TNF receptor 1 (TNFR1), ACTIVIN A, chemokine (C-C motif) ligand 3 (CCL3), and IL-15 predicted adverse events markedly better than a single SASP protein or age. Our findings suggest that the circulating SASP may serve as a clinically useful candidate biomarker of age-related health and a powerful tool for interventional human studies.

Keywords: Aging; Cellular senescence.

Eicosapentaenoic acid (EPA; <em>20</em>:5, n-3) can restrain tumor <em>growth</em> and metastasis in vivo; however, the mechanism of its antitumor effect is still not fully understood. Angiogenesis is a crucial process for tumor <em>growth</em> and metastasis and inhibition of tumor angiogenesis can suppress tumor <em>growth</em> and metastasis in vivo. Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) is an important angiogenic <em>factor</em>. In this study, we investigated the mechanisms of the inhibitory effect of EPA on VEGF-induced proliferation of bovine carotid artery endothelial (BAE) cells. BAE cells, treated with 0-5 microg/ml EPA for 48 h, displayed a dose-dependent suppression to VEGF (0.2 nM)-induced proliferation. Similar inhibitory effect was not found in BAE cells treated with arachidonic acid (AA; <em>20</em>:4, n-6), or docasahexaenoic acid (DHA; 22:5, n-3). In contrast to its effect on VEGF-induced proliferation, EPA had no inhibition to basic <em>fibroblast</em> <em>growth</em> <em>factor</em> (bFGF, 0.2 nM)-induced proliferation in BAE cells. Both VEGF and bFGF activated mitogen-activated protein (MAP) kinase in BAE cells; however, EPA selectively inhibited VEGF-induced, but not bFGF-induced activation of MAP kinase. Flk-1 expression was inhibited dose-dependently in EPA-treated cells, whereas Flt-1 expression was increased in EPA treated cells. This in vitro inhibitory effect by EPA on Flk-1 receptor expression provides indirect evidence that one of the mechanisms of EPA for antitumor action in vivo maybe related to its antiangiogenic action.

Cortical human brain tissue was obtained from 11 craniotomies for intractable epilepsy or tumor resection. Neuregen transport medium preserved viability at 4 degrees C during transfer to the culture laboratory. Cells were isolated and cultured by methods previously developed for adult rat neurons (Brewer GJ. Isolation and culture of adult rat hippocampal neurons. J. Neurosci. Meth. 1997:71:143-55). In about 40% of the cases, cultures regenerated with a majority of neuron-like cells that stained for neurofilament and not GFAP. After 3 weeks of culture from a 70 year old meningioma case, synapse-like structures were revealed by electron microscopy. Trophic support from basic human recombinant <em>fibroblast</em> <em>growth</em> <em>factor</em> was synergistically improved with the steroid hormone dehydroepiandrosterone 3-sulfate. Another 40% of the cases resulted in cultures that were predominantly GFAP positive astroglia. The remaining <em>20</em>% of the cases did not regenerate cells with neuron-like or glial processes. Three postmortem cases did not regenerate neurites. These methods may aid development of human culture models of epilepsy as well as human pharmacology, toxicology and development of improved methods for brain grafts.

Parkinson disease (PD) is a neurodegenerative disorder characterized by dopaminergic neurons affected by inflammatory processes. Post-mortem analyses of brain and cerebrospinal fluid from PD patients show the accumulation of proinflammatory cytokines, confirming an ongoing neuroinflammation in the affected brain regions. These inflammatory mediators may activate transcription <em>factors</em>-notably nuclear <em>factor</em> κB, Ying-Yang 1 (YY1), <em>fibroblast</em> <em>growth</em> <em>factor</em> <em>20</em> (FGF<em>20</em>), and mammalian target of rapamycin (mTOR)-which then regulate downstream signaling pathways that in turn promote death of dopaminergic neurons through death domain-containing receptors. Dopaminergic neurons are vulnerable to oxidative stress and inflammatory attack. An increased level of inducible nitric oxide synthase observed in the substantia nigra and striatum of PD patients suggests that both cytokine-and chemokine-induced toxicity and inflammation lead to oxidative stress that contributes to degeneration of dopaminergic neurons and to disease progression. Lipopolysaccharide activation of microglia in the proximity of dopaminergic neurons in the substantia nigra causes their degeneration, and this appears to be a selective vulnerability of dopaminergic neurons to inflammation. In this review, we will look at the role of various transcription <em>factors</em> and signaling pathways in the development of PD.

Exosomes are nano-scale and closed membrane vesicles which are promising for therapeutic applications due to exosome-enclosed therapeutic molecules such as DNA, small RNAs, proteins and lipids. Recently, it has been demonstrated that mesenchymal stem cell (MSC)-derived exosomes have capacity to regulate many biological events associated with wound healing process, such as cell proliferation, cell migration and blood vessel formation. This study investigated the regenerative potentials for cutaneous tissue, in regard to <em>growth</em> <em>factors</em> associated with wound healing and skin cell proliferation and migration, by exosomes released from primary MSCs originated from bone marrow (BM), adipose tissue (AD), and umbilical cord (UC) under serum- and xeno-free condition. We found crucial wound healing-mediated <em>growth</em> <em>factors</em>, such as vascular endothelial <em>growth</em> <em>factor</em> A (VEGF-A), <em>fibroblast</em> <em>growth</em> <em>factor</em> 2 (FGF-2), hepatocyte <em>growth</em> <em>factor</em> (HGF), and platelet-derived <em>growth</em> <em>factor</em> BB (PDGF-BB) in exosomes derived from all three MSC sources. However, expression levels of these <em>growth</em> <em>factors</em> in exosomes were influenced by MSC origins, especially transforming <em>growth</em> <em>factor</em> beta (TGF-β) was only detected in UCMSC-derived exosomes. All exosomes released by three MSCs sources induced keratinocyte and <em>fibroblast</em> proliferation and migration; and, the induction of cell migration is a dependent manner with the higher dose of exosomes was used (<em>20</em> μg), the faster migration rate was observed. Additionally, the influences of exosomes on cell proliferation and migration was associated with exosome origins and also target cells of exosomes that the greatest induction of primary dermal <em>fibroblasts</em> belongs to BMMSC-derived exosomes and keratinocytes belongs to UCMSC-derived exosomes. Data from this study indicated that BMMSCs and UCMSCs under clinical condition secreted exosomes are promising to develop into therapeutic products for wound healing treatment.

Keywords: ADMSC-derived exosomes; BMMSC-derived exosomes; UCMSC-derived exosomes; exosomes; growth factors; mesenchymal stem cells; wound healing.

Pelvic organ prolapse (POP) is a global health problem, for which the pathophysiological mechanism remains to be fully elucidated. The loss of extracellular matrix protein has been considered to be the most important molecular basis facilitating the development of POP. Oxidative stress (OS) is a well‑recognized mechanism involved in fiber metabolic disorders. The present study aimed to clarify whether OS exists in the uterosacral ligament (USL) with POP, and to investigate the precise role of OS in collagen metabolism in human USL <em>fibroblasts</em> (hUSLFs). In the present study, 8‑hydroxyguanosine (8‑OHdG) and 4 hydroxynonenal (4‑HNE), as oxidative biomarkers, were examined by immunohistochemistry to evaluate oxidative injury in USL sections in POP (n=<em>20</em>) and non‑POP (n=<em>20</em>) groups. The primary cultured hUSLFs were treated with exogenous H2O2 to establish an original OS cell model, in which the expression levels of collagen, type 1, α1 (COL1A1), matrix metalloproteinase (MMP)‑2, tissue inhibitor of metalloproteinase (TIMP)‑2 and transforming <em>growth</em> <em>factor</em> (TGF)‑β1 were evaluated by western blot and reverse transcription‑quantitative polymerase chain reaction analyses. The results showed that the expression levels of 8‑OHdG and 4‑HNE in the POP group were significantly higher, compared with those in the control group. Collagen metabolism was regulated by H2O2 exposure in a concentration‑dependent manner, in which lower concentrations of H2O2 (0.1‑0.2 mM) stimulated the anabolism of COL1A1, whereas a higher concentration (0.4 mM) promoted catabolism. The expression levels of MMP‑2, TIMP‑2 and TGF‑β1 exhibited corresponding changes with the OS levels. These results suggested that OS may be involved in the pathophysiology of POP by contributing to collagen metabolic disorder in a severity‑dependent manner in hUSLFs, possibly through the regulation of MMPs, TIMPs and TGF‑β1 indirectly.

Disruption of the X-chromosome <em>fibroblast</em> <em>growth</em> <em>factor</em> 16 (Fgf-16) gene, a member of the FGF-9 subfamily with FGF-<em>20</em>, was linked with an effect on cardiac development in two independent studies. However, poor trabeculation with lethality by embryonic day (E) 11.5 was associated with only one, involving maintenance in Black Swiss (Bsw) versus C57BL/6 mice. The aim of this study was to examine the potential influence of genetic background through breeding the null mutation onto an alternate (C57BL/6) background. After three generations, 25% of Fgf-16(-/Y) mice survived to adulthood, which could be reversed by reducing the contribution of the C57BL/6 genetic background by back crossing to another strain. There was no significant difference between FGF-9 and FGF-<em>20</em> RNA levels in Fgf-16 null versus wild-type mice regardless of strain. However, FGF-8 RNA levels were reduced significantly in Bsw but not C57BL/6 mice. FGF-8 is linked to anterior heart development and like the FGF-9 subfamily is reportedly expressed at E10.5. Like FGF-16, neuregulin as well as signaling via ErbB2 and ErbB4 receptors have been linked to trabeculae formation and cardiac development around E10.5. Basal neuregulin, ErbB2, and ErbB4 as well as FGF-8, FGF-9, and FGF-16 RNA levels varied in Bsw versus C57BL/6 mice. These data are consistent with the ability of genetic background to modify the phenotype and affect embryonic survival in Fgf-16 null mice.

BACKGROUND

Melanoma escape mechanisms include immunosuppressive and angiogenic cytokine production.

OBJECTIVE

We sought to determine vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) expression by immunohistochemistry, and soluble circulating plasma levels of VEGF, bFGF, IL-10, and transforming growth factor-beta2 in patients with different stages of melanoma.

METHODS

Biopsy specimens from 42 patients with primary melanoma and 9 with cutaneous metastases were studied by immunohistochemistry. In another 46 patients with melanoma (8 stage I and II; 18, III; and 20, IV) and in 10 healthy control participants, bFGF, VEGF, IL-10, and transforming growth factor-beta2 circulating levels were analyzed.

RESULTS

bFGF was positive in 85% and VEGF in 47.5% of 42 primary melanomas. Of 10 patients with primary melanoma (Breslow depth 1.5-3 mm) 6 were VEGF positive and had metastases develop, whereas 4 were VEGF negative and had no metastases at 5 years of follow up. VEGF, bFGF, and IL-10 plasma levels in patients with stages III and IV melanoma were higher than the control group (P <.05 and P <.01, respectively). An inverse relationship was found between VEGF and IL-10. Specifically, in 7 patients with IL-10 levels higher than 10 pg/mL, VEGF levels were less than 49 pg/mL (P <.05); in 9 patients with VEGF levels higher than 100 pg/mL, IL-10 levels were less than 6.7 pg/mL (P <.01).

CONCLUSIONS

VEGF expression in 1.5- to 3.0-mm Breslow depth melanomas may be considered as an unfavorable prognostic factor. Immunosuppressive (IL-10, transforming growth factor-beta2) and proangiogenic (bFGF, VEGF) cytokines are increased in metastatic melanoma. Inverse plasma levels between IL-10 and VEGF in patients with metastatic melanoma are shown in vivo for the first time, the significance of which must be further investigated.