Effects of vitamin e on the epithelial-mesenchymal transition in testicular development exposed to valproic acid / Efectos de la vitamina e en la interacción epitelio-mesenquimático en el desarrollo testicular expuesto a ácido valproico

SUMMARY: In testicular differentiation, somatic cells must adopt a specific destiny towards sustentacular, peritubular and interstitial cells, being fundamental for the morphogenesis of seminiferous tubules, mediated by morphogens such as Desert Hedgehog (DHH), insulin-like growth factor-1 (IGF-1) and fibroblastic growth factor 2 (FGF-2). Its alteration could be related to failures in the development mechanisms, such as those caused by valproic acid (VPA), which can be reversed with vitamin E (VE). The objective of the study was to evaluate the epithelial-mesenchymal transition (EMT) in the testicular development of mice exposed to VPA and VE. 12 groups of pregnant female mice were formed that were separated by days post-coital (dpc) at 12.5 dpc, 17.5 dpc and 6 weeks postnatal, each one subdivided into 4 groups of 5 pregnant women each. Subgroups received different treatments from the beginning to the end of gestation orally: 600 mg/kg of VPA, 600 mg/kg of VPA and 200 IU of VE, 200 IU of VE and the control group 0.3 mL of 0.9% physiological solution. Immunohistochemistry was performed for the detection of DHH, IGF-1 and FGF-2. Immunolocalization of DHH was observed in all stages, with more evident significant differences in integrated optical density (IOD) and percentage of immunoreaction area at 6 weeks postnatal, being lower in the VPA group. In IGF-1, lower intensity and distribution of immunostaining was observed in the fetal and pubertal stages in the VPA groups, a similar situation with FGF-2, but only evident at 17.5 dpc, with significant differences. These results demonstrate that VPA can alter EMT between somatic cells in testicular development, with VE being an agent capable of attenuating this process.

RESUMEN: En la diferenciación testicular, es necesario que las células somáticas adopten un destino específico hacia células sustentaculares, peritubulares e intersticiales, siendo fundamental para la morfogénesis de los túbulos seminíferos, mediado por morfógenos como Desert Hedgehog (DHH), Factor de Crecimiento Fibroblástico 2 (FGF-2) y Factor de Crecimiento símil a Insulina (IGF-1). Su alteración se podría relacionar a fallas en los mecanismos de desarrollo, como los que ocasiona el ácido valproico (VPA), los cuales pueden ser revertidos con la vitamina E (VE). El objetivo de estudio fue evaluar la transición epitelio-mesenquimática (EMT) en el desarrollo testicular de ratones expuestos a VPA y VE. Se conformaron 12 grupos de ratones hembra gestantes que se separaron por días post-coital (dpc) a los 12.5 dpc, 17.5 dpc y 6 semanas post-natal, cada uno subdividido en 4 grupos de 5 gestantes cada uno. Cada subgrupo recibió diferentes tratamientos desde el inicio hasta el término de la gestación vía oral: 600 mg/kg de VPA, 600 mg/kg de VPA y 200 UI de VE, 200 UI de VE y el grupo control 0,3 mL de solución fisiológica 0,9%. Se realizó técnica inmunohistoquímica para la detección de DHH, IGF-1 y FGF-2. Se observó la inmunolocalización de DHH en todos los estadios, con diferencias significativas más evidentes en la densidad óptica integrada (IOD) y porcentaje de área de inmunoreacción a las 6 semanas post-natal, siendo menor en el grupo VPA. En IGF-1, se observó en la etapa fetal y puberal menor intensidad y distribución de la marcación en los grupos VPA, situación similar con la inmunomarcación de FGF-2, pero sólo evidenciándose a los 17.5 dpc, con diferencias significativas. Estos resultados demuestran que el VPA puede alterar la EMT entre las células somáticas en el desarrollo testicular, siendo la VE un agente capaz de atenuar este proceso.

Functional analysis of human fibrocytes derived from monocytes reveals their profibrotic phenotype through paracrine effects.

Fibrocytes, which are bone marrow-derived collagen-producing cells, were reported to play a role in the pathogenesis of pulmonary fibrosis. However, their function in pulmonary fibrosis is unclear. We analyzed their function compared with that of monocytes and localization in fibrotic tissues in patients with idiopathic pulmonary fibrosis (IPF). We compared the gene expression profile of monocyte-derived fibrocytes with that of monocytes by microarray analysis. Proliferation and differentiation into myofibroblasts were examined by 3H-thymidine incorporation assay and Western blotting. We measured the level of growth factors in the culture supernatant of fibrocytes by ELISA. The localization of fibrocytes in lung tissues of patients with IPF was determined by immunofluorescence staining. Compared with monocytes, fibrocytes had higher expression of extracellular matrix- and growth factor-encoding genes, including PDGF-B, FGF-2 and VEGF-B. Although fibrocytes did not proliferate in response to PDGF, co-culture of fibrocytes stimulated the growth of lung fibroblasts through the production of PDGF-BB. In the lung of IPF patients, CD45+Collagen-I+FSP-1+ cells, which have a similar phenotype to fibrocytes, were detected and co-stained with anti-PDGF antibody. This study suggested that fibrocytes function in pulmonary fibrosis partly by producing PDGF in the lungs of IPF patients. J. Med. Invest. 67 : 102-112, February, 2020.

Angiomatoid Spitz nevus with surrounding pagetoid melanocytic proliferation on the sole of the foot: An unusual case report with immunohistochemical studies for angiogenic factors.

Angiomatoid Spitz nevus (ASN) is a rare histological variant of Spitz nevus (SN) that is characterized by prominent blood vessel proliferation around the intradermal melanocytes of SN. In contrast, SN may have pagetoid components, which are characterized by epidermal proliferation of single melanocytes. However, cases of ASN with predominant pagetoid melanocytic proliferation in the epidermis have not been reported. Here, we report a case of ASN with surrounding pagetoid melanocytic proliferation without formation of tumor nests in the epidermis in the plantar region. A 12-year-old girl presented with a bright red nodule surrounded by a brown macule on the sole of her right foot. Histologically, the nodule showed tumor nests in the dermis, composed of spindle or epithelioid melanocytes containing abundant cytoplasm and large nuclei. Around the nests, numerous blood vessels were seen. In the overlying epidermis of the nodule, numerous eosinophilic Kamino bodies were found along the dermal-epidermal interface. In the macule, proliferation of oval melanocytes was present as single-cell units in the epidermis. Theses melanocytes had abundant cytoplasm with large nuclei, which were larger than those of the surrounding keratinocytes. From these findings, a diagnosis of ASN with surrounding pagetoid melanocytic proliferation was made. Vascular endothelial growth factor and fibroblast growth factor 2 were strongly expressed in the melanocytes as well as in the endothelial cells in our case. Therefore, angiogenic factors produced by the melanocytes of SN might have played important roles in the surrounding angiogenesis of this case.

Effect of a novel bioceramic root canal sealer on the angiogenesis-enhancing potential of assorted human odontogenic stem cells compared with principal tricalcium silicate-based cements.

OBJECTIVE: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). METHODOLOGY: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. RESULTS: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). CONCLUSION: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.

Effect of a novel bioceramic root canal sealer on the angiogenesis-enhancing potential of assorted human odontogenic stem cells compared with principal tricalcium silicate-based cements

Abstract Objective: This study evaluated the angiogenesis-enhancing potential of a tricalcium silicate-based mineral trioxide aggregate (ProRoot MTA), Biodentine, and a novel bioceramic root canal sealer (Well-Root ST) in human dental pulp stem cells (hDPSCs), human periodontal ligament stem cells (hPLSCs), and human tooth germ stem cells (hTGSCs). Methodology: Dulbecco's modified Eagle's medium was conditioned for 24 h by exposure to ProRoot MTA, Biodentine, or Well-Root ST specimens (prepared according to the manufacturers' instructions). The cells were cultured in these conditioned media and their viability was assessed with 3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxy-methoxy-phenyl)-2-(4-sulfo-phenyl)-2H tetrazolium (MTS) on days 1, 3, 7, 10, and 14. Angiogenic growth factors [platelet-derived growth factor (PDGF), basic fibroblast growth factor (FGF-2), and vascular endothelial growth factor (VEGF)] were assayed by sandwich enzyme-linked immunosorbent assay (ELISA) on days 1, 7, and 14. Human umbilical vein endothelial cell (HUVEC) migration assays were used to evaluate the vascular effects of the tested materials at 6-8 h. Statistical analyses included Kruskal-Wallis, Mann-Whitney U, and Friedman and Wilcoxon signed rank tests. Results: None of tricalcium silicate-based materials were cytotoxic and all induced a similar release of angiogenic growth factors (PDGF, FGF-2, and VEGF) (p>0.05). The best cell viability was observed for hDPSCs (p<0.05) with all tricalcium silicate-based materials at day 14. Tube formation by HUVECs showed a significant increase with all tested materials (p<0.05). Conclusion: The tricalcium silicate-based materials showed potential for angiogenic stimulation of all stem cell types and significantly enhanced tube formation by HUVECs.

Polarizing receptor activation dissociates fibroblast growth factor 2 mediated inhibition of myelination from its neuroprotective potential.

Fibroblast growth factor (FGF) signaling contributes to failure of remyelination in multiple sclerosis, but targeting this therapeutically is complicated by its functional pleiotropy. We now identify FGF2 as a factor up-regulated by astrocytes in active inflammatory lesions that disrupts myelination via FGF receptor 2 (FGFR2) mediated activation of Wingless (Wnt) signaling; pharmacological inhibition of Wnt being sufficient to abrogate inhibition of myelination by FGF2 in tissue culture. Using a novel FGFR1-selective agonist (F2 V2) generated by deleting the N-terminal 26 amino acids of FGF2 we demonstrate polarizing signal transduction to favor FGFR1 abrogates FGF mediated inhibition of myelination but retains its ability to induce expression of pro-myelinating and immunomodulatory factors that include Cd93, Lif, Il11, Hbegf, Cxcl1 and Timp1. Our data provide new insights into the mechanistic basis of remyelination failure in MS and identify selective activation of FGFR1 as a novel strategy to induce a neuroprotective signaling environment in multiple sclerosis and other neurological diseases.

Correlations of insulin resistance and HbA1c with cytokines IGF-1, bFGF and IL-6 in the aqueous humor of patients with diabetic cataract.

OBJECTIVE: The study aimed to investigate the correlations of insulin resistance and hemoglobin A1c (HbA1c) with cytokines [insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF) and interleukin-6 (IL-6)] in the aqueous humor of patients with diabetic cataract. PATIENTS AND METHODS: 59 patients with diabetic cataract and 58 patients with simple cataract treated in Jining No. 1 People´s Hospital (Jining, China) from January 2017 to February 2018, were selected randomly. The levels of homeostasis model assessment of insulin resistance (HOMA-IR) and HbAlc, as well as IGF-1, bFGF and IL-6 in the aqueous humor were compared between the two groups. The correlations of HOMA-IR and HbAlc with IGF-1, bFGF and IL-6 were analyzed. In control group, the levels of HOMA-IR and HbAlc, as well as IGF-1, bFGF and IL-6 in the aqueous humor were significantly lower than those in observation group (p<0.05). RESULTS: Compared with the group with HbAlc ≤ 7%, the groups with HbAlc ≥ 9% and 7%

Imaging of growth factors on a human tooth root canal by surface-enhanced Raman spectroscopy.

Endodontic treatment of immature permanent teeth with necrotic pulp poses several clinical challenges and is one of the most demanding interventions in endodontics. Recently, with new discoveries in the field of tissue engineering, novel treatment protocols have been established. The most promising treatment modality is revascularization, whose integral part is the exposure of collagen matrix and embedded growth factors. However, optimization of the treatment protocol requires a development of analytical procedures able to analyze growth factors directly on the sample surface. In this work, method based on surface-enhanced Raman spectroscopy (SERS) was developed to investigate the influence of the time of the medical treatment using EDTA on exposure and accessibility of the growth factors, namely TGF-ß1, BMP-2, and bFGF on the dentine surface. The nanotags, which consist of magnetic Fe3O4@Ag nanocomposite covalently functionalized by tagged antibodies (anti-TGF-ß1-Cy3, anti-BMP-2-Cy5, and anti-bFGF-Cy7), were employed as a SERS substrate. Each antibody was coupled with a unique label allowing us to perform a parallel analysis of all three growth factors within one analytical run. Developed methodology presents an interesting alternative to a fluorescence microscopy and in contrary allows evaluating a chemical composition and thus minimizing possible false-positive results. Graphical abstract.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells.

We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

Evaluation of prophylactic and therapeutic effects of sildenafil on acute radiation proctitis in rats.

PURPOSE: To investigate the prophylactic and therapeutical effects of sildenafil in a model of acute radiation proctitis (ARP). METHODS: All experimental procedures of this study was examined by histopathological, immunohistochemical and transmission electron microscopic analysis. RESULTS: Our histopathological evaluations indicated significant increases in lesion severity, cryptic apsis, cryptitis, cryptic distortion, reactive atypia and infiltration depth of the control (proctitis) group. While the prophylaxis group and the treatment group had significantly lower scores. High-dose group showed similar results as prophylaxis group. Histopathological findings of the prophylaxis group was more significant than the treatment group. Immunoreactivities of IL-1ß, FGF-2, TNF- α and HIF-1α increased in the control group especially in the epithelial and cryptic regions. On the contrary, sildenafil application caused significant decreases of inflammatory markers in all treatment groups, specifically better results in the prophylaxis group. CONCLUSION: The sildenafil has anti-inflammatory effects on ARP, as well as protective effects against ARP and the protective effect of sildenafil surpasses its therapeutic effect histopathologically.

Evaluation of prophylactic and therapeutic effects of sildenafil on acute radiation proctitis in rats

Abstract Purpose: To investigate the prophylactic and therapeutical effects of sildenafil in a model of acute radiation proctitis (ARP). Methods: All experimental procedures of this study was examined by histopathological, immunohistochemical and transmission electron microscopic analysis. Results: Our histopathological evaluations indicated significant increases in lesion severity, cryptic apsis, cryptitis, cryptic distortion, reactive atypia and infiltration depth of the control (proctitis) group. While the prophylaxis group and the treatment group had significantly lower scores. High-dose group showed similar results as prophylaxis group. Histopathological findings of the prophylaxis group was more significant than the treatment group. Immunoreactivities of IL-1β, FGF-2, TNF- α and HIF-1α increased in the control group especially in the epithelial and cryptic regions. On the contrary, sildenafil application caused significant decreases of inflammatory markers in all treatment groups, specifically better results in the prophylaxis group. Conclusion: The sildenafil has anti-inflammatory effects on ARP, as well as protective effects against ARP and the protective effect of sildenafil surpasses its therapeutic effect histopathologically.

Activation of AMPK by simvastatin inhibited breast tumor angiogenesis via impeding HIF-1α-induced pro-angiogenic factor.

Substantial data from preclinical studies have revealed the biphasic effects of statins on cardiovascular angiogenesis. Although some have reported the anti-angiogenic potential of statins in malignant tumors, the underlying mechanism remains poorly understood. The aim of this study is to elucidate the mechanism by which simvastatin, a member of the statin family, inhibits tumor angiogenesis. Simvastatin significantly suppressed tumor cell-conditioned medium-induced angiogenic promotion in vitro, and resulted in dose-dependent anti-angiogenesis in vivo. Further genetic silencing of hypoxia-inducible factor-1α (HIF-1α) reduced vascular endothelial growth factor and fibroblast growth factor-2 expressions in 4T1 cells and correspondingly ameliorated HUVEC proliferation facilitated by tumor cell-conditioned medium. Additionally, simvastatin induced angiogenic inhibition through a mechanism of post-transcriptional downregulation of HIF-1α by increasing the phosphorylation level of AMP kinase. These results were further validated by the fact that 5-aminoimidazole-4-carboxamide ribonucleotide reduced HIF-1α protein levels and ameliorated the angiogenic ability of endothelial cells in vitro and in vivo. Critically, inhibition of AMPK phosphorylation by compound C almost completely abrogated simvastatin-induced anti-angiogenesis, which was accompanied by the reduction of protein levels of HIF-1α and its downstream pro-angiogenic factors. These findings reveal the mechanism by which simvastatin induces tumor anti-angiogenesis, and therefore identifies the target that explains the beneficial effects of statins on malignant tumors.

Comparison of primary human gingival fibroblasts from an older and a young donor on the evaluation of cytotoxicity of denture adhesives.

Denture adhesives (DA) improve the retention and stability of ill-fitting dentures, especially for older adults. These materials should be biocompatible, i.e., they cannot cause undesired biological responses and be non-cytotoxic to oral tissues. However, in vitro testing of DA biocompatibility employing primary cell culture may possibly be affected by other factors, such as the donor age. OBJECTIVE: To compare the cytotoxicity of three different denture adhesives when assessed in primary gingival fibroblasts from a young donor or from an older donor, as well as the release of the basic fibroblast growth factor (bFGF), and the inflammatory response marker interleukin-6 (IL-6). MATERIAL AND METHODS: Gingival fibroblasts isolated from a 30- and a 62-year-old donor were assayed for proliferation (1-7 days) and sensitivity to latex (positive control). Fibroblasts were indirectly exposed to Corega Ultra (cream), Corega powder and Fixodent Original for a 24 h period and assayed by XTT and Crystal Violet tests. The release of IL-6 and bFGF by exposed cells was determined by ELISA. RESULTS: While cells from the young donor presented higher cell growth after 7 days, the sensitivity to increasing concentrations of latex extracts was very similar between young and older cells. Both XTT and CVDE detected no difference between the DA and the control group. All materials induced higher levels of IL-6 and bFGF compared to control. Cells from the older donor exposed to Corega Ultra released lower levels of cytokine and growth factor. CONCLUSIONS: All materials were considered non-cytotoxic, but affected cytokine and growth factor release. The biological differences found between fibroblasts from both donors could be due to individual or age-related factors. The authors suggest the use of cells from older donors on studies of dental products aimed at older patients, to better simulate their physiological response.

Mecanismos moleculares do efeito citotóxico de FGF2 em células transformadas por RAS / Molecular mechanisms of the cytotoxic effect of FGF2 in rastransformed cells

O FGF2 (Fibroblast Growth Factor 2) é um clássico fator peptídico de crescimento que ativa vias intracelulares de sinalização molecular promovendo a transição G0 → G1 e o comprometimento com o ciclo celular. Não surpreendentemente, seus papéis pró-tumoral e angiogênico estão bem caracterizados e estabelecidos na literatura. No entanto, um crescente corpo de evidências tem indicado que o FGF2 também pode exercer efeitos anti-tumorais in vitro e in vivo, em modelos murinos e também humanos. Neste contexto, nosso grupo publicou em 2008 que o FGF2 exerce um efeito antiproliferativo seletivo em células murinas malignas dependentes de alta atividade de K-Ras e H-Ras. Os genes ras compõem a família de oncogenes mais frequentemente mutada em tumores malignos humanos, alcançando aproximadamente 30% de todos os casos. O desenvolvimento de terapias contra tumores dependentes de Ras fracassou, apesar dos intensos esforços e investimentos desde a descoberta em 1982 de suas mutações ativadoras em múltiplos cânceres. O objetivo deste trabalho foi desvendar os mecanismos moleculares pelo quais o FGF2 inibe irreversivelmente a proliferação de células malignas dependentes da atividade de Ras, empregando como modelos experimentais a linhagem murina Y1 de células adrenocorticais, e 4 linhagens humanas derivadas de sarcomas de Ewing. Identificamos que o efeito citotóxico do FGF2 não se processa por um mecanismo novo e independente das viasproliferativas classicamente ativadas por fatores peptídicos de crescimento. Ao contrário, seu efeito tóxico é resultado de sinalização mitogênica exagerada decorrente de estimulação sustentada por FGF2. A ativação da via de MAPK, principal sinalização mitogênica intracelular, a níveis elevados e sustentados provoca estresse mitogênico, que se propaga para a fase S na forma de estresse replicativo. Nesta situação, a célula passa a depender exageradamente da sinalização protetora de ATR, de modo que a combinação de estimulação com FGF2 e inibição de ATR foi altamente letal para as células malignas dependentes de Ras empregadas neste trabalho. Também analisamos as bases moleculares de resistência a FGF2 exibida por células Y1 anteriormente selecionadas para resistir ao efeito tóxico do FGF2 (Y1FRs). Descobrimos que a pressão seletiva do FGF2 não teve efeito na expressão de seus receptores, mas provocou a eliminação de um dos dois cromossomos que portam a amplificação gênica de ras nesta linhagem, enquanto o segundo cromossomo foi mantido por ser a única fonte de genes ribossomais ativos. Suas cópias de ras, no entanto, mostraram-se transcricionalmente silenciadas. Além disso, as sublinhagens Y1FRs não expressam o principal RasGEF, GRP4, encontrado nas células parentais Y1, o que pode ter influenciado o surgimento do fenótipo resistente ao FGF2. As linhagens resistentes mostraram grande redução no número de cromossomos e aumento da frequência de fusões entre cromossomos não homólogos em relação às células parentais

FGF2 (Fibroblast Growth Factor 2) is a classic peptide growth factor that activates intracellular molecular signaling pathways promoting the G0 → G1 transition and cell cycle commitment. Not surprisingly, its pro-tumor and angiogenic roles are well characterized and established in the literature. However, a growing body of evidence has indicated that FGF2 may also exert anti-tumor effects in vitro and in vivo in murine and human models. In this context, our group reported in 2008 that FGF2 exerts a selective antiproliferative effect in murine cells dependent on high activity of K-Ras and H-Ras. Ras genes make up the most frequently mutated oncogene family in human malignant tumors, reaching approximately 30% of all cases. The development of therapies against Ras-dependent tumors has failed despite intense efforts and investments since the discovery in 1982 of its activating mutations in multiple cancers. The objective of this work was to uncover the molecular mechanisms by which FGF2 irreversibly inhibits the proliferation of malignant cells dependent on Ras activity, using as experimental models the Y1 murine lineage of adrenocortical malignant cells and 4 human lineages derived from Ewing sarcomas. We showed that the cytotoxic effect of FGF2 did not involve novel cell cycle regulatory pathways; instead, this cytotoxic effect is a result of sustainedhyper mitogenic stimulation by FGF2. Activation of the KRas/MAPK pathway, the major intracellular mitogenic signaling, at high and sustained levels provokes mitogenic stress, which is propagated to S phase as replicative stress. In this situation, the cell dependence on the ATR protective signaling is enhanced, so that the combination of stimulation with FGF2 and inhibition of ATR was highly lethal for the Ras dependent malignant cells employed in this work. We also analyzed the molecular basis of FGF2 resistance exhibited by Y1 cells previously selected for resistance to FGF2. We found that the selective pressure of FGF2 had no effect on the expression of its receptors but promoted the elimination of one of the two marker chromosomes that carry the K-ras amplified copies, while the second chromosome was maintained because it is the only source of active ribosomal genes; however, its K-ras amplified copies were transcriptionally silenced. In addition, the Y1FRs sublines did not express the main RasGEF, GRP4, found in the parental Y1 cells, which might have played a role in the emergence of the FGF2-resistant phenotype. The resistant Y1FRs sublines showed a large reduction in chromosome numbers and increased frequency of fusions between non-homologous chromosomes in relation to parental cells

Comparison of primary human gingival fibroblasts from an older and a young donor on the evaluation of cytotoxicity of denture adhesives

Abstract Denture adhesives (DA) improve the retention and stability of ill-fitting dentures, especially for older adults. These materials should be biocompatible, i.e., they cannot cause undesired biological responses and be non-cytotoxic to oral tissues. However, in vitro testing of DA biocompatibility employing primary cell culture may possibly be affected by other factors, such as the donor age. Objective To compare the cytotoxicity of three different denture adhesives when assessed in primary gingival fibroblasts from a young donor or from an older donor, as well as the release of the basic fibroblast growth factor (bFGF), and the inflammatory response marker interleukin-6 (IL-6). Material and Methods Gingival fibroblasts isolated from a 30- and a 62-year-old donor were assayed for proliferation (1-7 days) and sensitivity to latex (positive control). Fibroblasts were indirectly exposed to Corega Ultra (cream), Corega powder and Fixodent Original for a 24 h period and assayed by XTT and Crystal Violet tests. The release of IL-6 and bFGF by exposed cells was determined by ELISA. Results While cells from the young donor presented higher cell growth after 7 days, the sensitivity to increasing concentrations of latex extracts was very similar between young and older cells. Both XTT and CVDE detected no difference between the DA and the control group. All materials induced higher levels of IL-6 and bFGF compared to control. Cells from the older donor exposed to Corega Ultra released lower levels of cytokine and growth factor. Conclusions All materials were considered non-cytotoxic, but affected cytokine and growth factor release. The biological differences found between fibroblasts from both donors could be due to individual or age-related factors. The authors suggest the use of cells from older donors on studies of dental products aimed at older patients, to better simulate their physiological response.

Extracellular calcium increases fibroblast growth factor 2 gene expression via extracellular signal-regulated kinase 1/2 and protein kinase A signaling in mouse dental papilla cells

Abstract We previously reported that elevated extracellular calcium (Ca2+) levels increase bone morphogenetic protein 2 expression in human dental pulp (hDP) cells. However, it is unknown whether extracellular Ca2+ affects the expression of other growth factors such as fibroblast growth factor 2 (FGF2). Objective: The present study aimed to examine the effect of extracellular Ca2+ on FGF2 gene expression in hDP and immortalized mouse dental papilla (mDP) cells. Materials and Methods: Cells were stimulated with 10 mM CaCl2 in the presence or absence of cell signaling inhibitors. FGF2 gene expression was assessed using real-time polymerase chain reaction. The phosphorylation status of signaling molecules was examined by Western blotting. Results: Extracellular Ca2+ increased FGF2 gene expression in mDP and hDP cells. Gene expression of the calcium-sensing receptor and G protein-coupled receptor family C group 6 member A, both of which are extracellular Ca2+ sensors, was not detected. Ca2+-mediated Fgf2 expression was reduced by pretreatment with the protein kinase A (PKA) inhibitor H-89 or extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD98059 but not by pretreatment with the protein kinase C inhibitor GF-109203X or p38 inhibitor SB203580. Extracellular Ca2+ increased PKA activity and ERK1/2 phosphorylation. Ca2+-induced PKA activity decreased by pretreatment with PD98059. Conclusions: These findings indicate that elevated extracellular Ca2+ levels led to increased Fgf2 expression through ERK1/2 and PKA in mDP cells and that this mechanism may be useful for designing regenerative therapies for dentin.

Infusion of Valproic Acid Into the Renal Medulla Activates Stem Cell Population and Attenuates Salt-Sensitive Hypertension in Dahl S Rats.

BACKGROUND: Our previous study has detected a stem cell deficiency in the renal medulla in Dahl salt-sensitive (S) rats. This study determined whether infusion of valproic acid (VA), an agent known to stimulate the stem cell function, attenuated salt-sensitive hypertension in Dahl S rats. METHODS: Uninephrectomized Dahl S rats were infused with vehicle or VA (50mg/kg/d) into the renal medulla and fed with a low (LS) or high salt diet (HS). Stem cell marker and number were analyzed by immunohistochemistry, Real-time RT-PCR and Western blot. Sodium excretion and blood pressure were measured. RESULTS: VA significantly increased the mRNA and protein levels of FGF2, a stem cell niche factor, and CD133, a stem cell marker. The number of CD133+ cells was significantly increased in the renal medulla in VA-treated rats. Meanwhile, high salt-induced increases in the mRNA level of proinflammatory factors interleukin-1ß and interleukin-6 were blocked in VA-treated rats. Functionally, sodium excretion in response to the blood pressure increase and acute sodium loading was significantly enhanced, sodium retention attenuated, high salt-induced increase of blood pressure reduced in VA-treated rats. CONCLUSION: Activation of stem cell function by VA inhibits the activation of proinflammatory factors and attenuates salt-sensitive hypertension in Dahl S rats.

Platelet-Rich Plasma Powder: A New Preparation Method for the Standardization of Growth Factor Concentrations.

BACKGROUND: Platelet-rich plasma (PRP) is widely used in sports medicine. Available PRP preparations differ in white blood cell, platelet, and growth factor concentrations, making standardized research and clinical application challenging. PURPOSE: To characterize a newly standardized procedure for pooled PRP that provides defined growth factor concentrations. STUDY DESIGN: Controlled laboratory study. METHODS: A standardized growth factor preparation (lyophilized PRP powder) was prepared using 12 pooled platelet concentrates (PCs) derived from different donors via apheresis. Blood samples and commercially available PRP (SmartPrep-2) served as controls (n = 5). Baseline blood counts were analyzed. Additionally, single PCs (n = 5) were produced by standard platelet apheresis. The concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor AB (PDGF-AB), transforming growth factor ß1 (TGF-ß1), insulin-like growth factor 1 (IGF-1), interleukin (IL)-1α, IL-1ß, and IL-1 receptor agonist (IL-1RA) were analyzed by enzyme-linked immunosorbent assay, and statistical analyses were performed using descriptive statistics, mean differences, 95% CIs, and P values (analysis of variance). RESULTS: All growth factor preparation methods showed elevated concentrations of the growth factors VEGF, bFGF, PDGF-AB, and TGF-ß1 compared with those of whole blood. Large interindividual differences were found in VEGF and bFGF concentrations. Respective values (mean ± SD in pg/mL) for whole blood, SmartPrep-2, PC, and PRP powder were as follows: VEGF (574 ± 147, 528 ± 233, 1087 ± 535, and 1722), bFGF (198 ± 164, 410 ± 259, 151 ± 99, and 542), PDGF-AB (2394 ± 451, 17,846 ± 3087, 18,461 ± 4455, and 23,023), and TGF-ß1 (14,356 ± 4527, 77,533 ± 13,918, 68,582 ± 7388, and 87,495). IGF-1 was found in SmartPrep-2 (1539 ± 348 pg/mL). For PC (2266 ± 485 pg/mL), IGF-1 was measured at the same levels of whole blood (2317 ± 711 pg/mL) but was not detectable in PRP powder. IL-1α was detectable in whole blood (111 ± 35 pg/mL) and SmartPrep-2 (119 ± 44 pg/mL). CONCLUSION: Problems with PRP such as absent standardization, lack of consistency among studies, and black box dosage could be solved by using characterized PRP powder made by pooling and lyophilizing multiple PCs. The new PRP powder opens up new possibilities for PRP research as well as for the treatment of patients. CLINICAL RELEVANCE: The preparation of pooled PRP by means of lyophilization may allow physicians to apply a defined amount of growth factors by using a defined amount of PRP powder. Moreover, PRP powder as a dry substance with no need for centrifugation could become ubiquitously available, thus saving time and staff resources in clinical practice. However, before transferring the results of this basic science study to clinical application, regulatory issues have to be cleared.

Release of Growth Factors into Root Canal by Irrigations in Regenerative Endodontics.

INTRODUCTION: The aim of this study was to investigate the release of growth factors into root canal space after the irrigation procedure of regenerative endodontic procedure. METHODS: Sixty standardized root segments were prepared from extracted single-root teeth. Nail varnish was applied to all surfaces except the root canal surface. Root segments were irrigated with 1.5% NaOCl + 17% EDTA, 2.5% NaOCl + 17% EDTA, 17% EDTA, or deionized water. The profile of growth factors that were released after irrigation was studied by growth factor array. Enzyme-linked immunosorbent assay was used to validate the release of transforming growth factor (TGF)-ß1 and basic fibroblast growth factor (bFGF) at 4 hours, 1 day, and 3 days after irrigation. The final concentrations were calculated on the basis of the root canal volume measured by cone-beam computed tomography. Dental pulp stem cell migration on growth factors released from root segments was measured by using Transwell assay. RESULTS: Total of 11 of 41 growth factors were detected by growth factors array. Enzyme-linked immunosorbent assay showed that TGF-ß1 was released in all irrigation groups. Compared with the group with 17% EDTA (6.92 ± 4.49 ng/mL), the groups with 1.5% NaOCl + 17% EDTA and 2.5% NaOCl + 17% EDTA had significantly higher release of TGF-ß1 (69.04 ± 30.41 ng/mL and 59.26 ± 3.37 ng/mL, respectively), with a peak release at day 1. The release of bFGF was detected at a low level in all groups (0 ng/mL to 0.43 ± 0.22 ng/mL). Migration assay showed the growth factors released from root segments induced dental pulp stem cell migration. CONCLUSIONS: The root segment model in present study simulated clinical scenario and indicated that the current irrigation protocol released a significant amount of TGF-ß1 but not bFGF. The growth factors released into root canal space induced dental pulp stem cell migration.

Novel Bioluminescent Binding Assays for Ligand-Receptor Interaction Studies of the Fibroblast Growth Factor Family.

We recently developed novel bioluminescent binding assays for several protein/peptide hormones to study their interactions with receptors using the so far brightest NanoLuc reporter. To validate the novel bioluminescent binding assay using a variety of protein/peptide hormones, in the present work we applied it to the fibroblast growth factor (FGF) family using the prototype member FGF2 as an example. A fully active recombinant FGF2 retaining a unique exposed cysteine (Cys) residue was chemically conjugated with an engineered NanoLuc carrying a unique exposed Cys residue at the C-terminus via formation of an intermolecular disulfide linkage. The NanoLuc-conjugated FGF2 (FGF2-Luc) retained high binding affinity to the overexpressed FGFR1 and the endogenous FGF receptor with the calculated dissociation constants of 161 ± 21 pM (n = 3) and 25 ± 4 pM (n = 3), respectively. In competition binding assays using FGF2-Luc as a tracer, receptor-binding potencies of wild-type or mutant FGF2s were accurately quantified. Thus, FGF2-Luc represents a novel non-radioactive tracer for the quantitative measurement of ligand-receptor interactions in the FGF family. These data suggest that the novel bioluminescent binding assay can be applied to a variety of protein/peptide hormones for ligand-receptor interaction studies.