Stimulation of MMP-11 (stromelysin-3) expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

Background Matrix metalloproteinases (MMPs) are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14) and stromelysin-3 (MMP-11) are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs) were: a) treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b) grown on collagens I, IV and V; c) treated with fibronectin, con-A and matrigel; and d) co-cultured with a range of HBC (human breast cancer) cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

Evaluation of the Effects of Endodontic Materials on Fibroblast Viability and Cytokine Production

Introduction: Recently, a new sealer composed of Portland cement named Endo-CPM-Sealer was developed. The aim of this study was to investigate the effects of Endo-CPM-Sealer (EGEO SRL, Buenos Aires, Argentina), Sealapex (Sybron Endo, Glendora, CA), and Angelus MTA (Angelus, Londrina, Brazil) on cell viability and cytokine (interleukin [IL]-1 beta and IL-6) production by mouse fibroblasts. Methods: Millipore culture plate inserts with polyethylene tubes filled with materials were placed into 24-well cell culture plates with mouse fibroblasts. Cells cultured with only empty polyethylene tubes were used as the control. After 24 hours, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was used to evaluate the cell viability. For cytokine assay, mouse fibroblasts were incubated in 24-well flat-bottom plates with set material disks at the bottom. Cells cultured without the material disks served as the negative control. After 24 hours of incubation, culture media were collected for cytokine evaluation by using an enzyme-linked immunosorbent assay. The data were statistically analyzed by analysis of variance and Bonferroni correction. Results: Endo-CPM-Sealer, Sealapex, and Angelus MTA did not inhibit the cell viability. All materials induced IL-6 releasing, but the amount was not statistically significant compared with the control group. Angelus MTA induced IL-1 beta releasing significantly more than the control. Conclusions: All materials were not considered cytotoxic in fibroblast culture. (J Endod 2009;35:1577-1579)

Relationship between donor age and the replicative lifespan of human cells in culture: A reevaluation

Normal human diploid fibroblasts have a finite replicative lifespan in vitro, which has been postulated to be a cellular manifestation of aging in vivo. Several studies have shown an inverse relationship between donor age and fibroblast culture replicative lifespan; however, in all cases, the correlation was weak, and, with few exceptions, the health status of the donors was unknown. We have determined the replicative lifespans of 124 skin fibroblast cell lines established from donors of different ages as part of the Baltimore Longitudinal Study of Aging. All of the donors were medically examined and were declared “healthy,” according to Baltimore Longitudinal Study of Aging protocols, at the time the biopsies were taken. Both long- and short-lived cell lines were observed in all age groups, but no significant correlation between the proliferative potential of the cell lines and donor age was found. A comparison of multiple cell lines established from the same donors at different ages also failed to reveal any significant trends between proliferative potential and donor age. The rate of [3H]thymidine incorporation and the initial rates of growth during the first few subcultivations were examined in a subset of cell lines and were found to be significantly greater in fetal lines than in postnatal lines. Cell lines established from adults did not vary significantly either in initial growth rate or in [3H]thymidine incorporation. These results clearly indicate that, if health status and biopsy conditions are controlled, the replicative lifespan of fibroblasts in culture does not correlate with donor age.

A SIMPLE AND FAST PROCEDURE TO GROW BAT FIBROBLASTS FROM LUNG BIOPSIES FOR CYTOGENETIC STUDIES

A fast, simple, and inexpensive procedure to establish fibroblast culture from bat lungs is presented. Explants plated following mechanical disaggregation provide good quality preparations for cytogenetics studies in about one week. Cultures established with this procedure may also be used for other biological studies.

Fator de crescimento derivado de plaquetas humanas obtido por uma metodologia rápida e de baixo custo

Human platelet-derived growth factor (PDGF) was purified from lysates of clinically outdated human platelets by ionic exchange chromatography in CM-Sepharose. The eluated fraction was submitted to the Immunoblot/Slot Blot assay using anti-PDGF-AA and anti-PDGF-BB polyclonal antibodies and was evaluated as to its biological activity through the test of [H 3]-thymidine incorporation in NIH/3T3 cell line fibroblasts in culture. The Immunoblot/Slot Blot assay using anti-PDGF-AA and anti-PDGF-BB antibodies proved the presence of the PDGF in chromatographic cationic fraction. The comparison of biological activities between fiblobrast stimulation assay using recombinant PDGF-AB and partially purified PDGF was demonstrated in 165.796 and 157.567 cpm, respectively. This result, proved the potent mitogenic effect of partially purified PDGF and consequently their evidence about the wound healing activity.

Alternativas para o tratamento da esquistossomose: Caracterizacao fisico-quimica do complexo de inclusao entre praziquantel e hidroxipropil-β-ciclodextrina

Praziquantel (PZQ) is the drug of choice commonly used for the treatment of shistosomiasis. However, it has low aqueous solubility, which could limit its bioavailability in the body. To circumvent these features, an inclusion complex with hydroxypropyl-beta- cyclodextrin (HP-β-CD) was prepared. Thus, the objective of this work was to prepare and characterize the PZQ/HP-β-CD inclusion complex. Morphological, spectroscopic, and calorimetric analysis showed the first signs of the guest/host interaction. The complexation kinetic analysis was used to determine the kinetic constant and, besides that, it was possible to establish the time consumed to reach equilibrium. Using the solubility isotherm, it was observed that the interaction with HP-β-CD increased 2.4 fold the aqueous solubility of plain PZQ. In vitro cytotoxicity tests, using fibroblast cells, evidenced no toxicity for these cells at the concentrations tested. These results demonstrated that there is a potential use of PZQ in formulations with HP-β-CD.

In vitro wound healing improvement by low-level laser therapy application in cultured gingival fibroblasts

The aim of this study was to determine adequate energy doses using specific parameters of LLLT to produce biostimulatory effects on human gingival fibroblast culture. Cells (3 10 4 cells/cm 2) were seeded on 24-well acrylic plates using plain DMEM supplemented with 10 fetal bovine serum. After 48-hour incubation with 5 CO2 at 37C, cells were irradiated with a InGaAsP diode laser prototype (LASERTable; 780 3 nm; 40mW) with energy doses of 0.5, 1.5, 3, 5, and 7J/cm 2. Cells were irradiated every 24h totalizing 3 applications. Twenty-four hours after the last irradiation, cell metabolism was evaluated by the MTT assay and the two most effective doses (0.5 and 3J/cm 2) were selected to evaluate the cell number (trypan blue assay) and the cell migration capacity (wound healing assay; transwell migration assay). Data were analyzed by the Kruskal-Wallis and Mann-Whitney nonparametric tests with statistical significance of 5. Irradiation of the fibroblasts with 0.5 and 3J/cm 2 resulted in significant increase in cell metabolism compared with the nonrradiated group (P 0.05). Both energy doses promoted significant increase in the cell number as well as in cell migration (P 0.05). These results demonstrate that, under the tested conditions, LLLT promoted biostimulation of fibroblasts in vitro. Copyright © 2012 Fernanda G. Basso et al.

Topical application of the lectin Artin M accelerates wound healing in rat oral mucosa by enhancing TGF-β and VEGF production

The lectin Artin M has been shown to accelerate the wound-healing process. The aims of this study were to evaluate the effects of Artin M on wound healing in the palatal mucosa of rats and to investigate the effects of Artin M on transforming growth factor beta (TGF-β) and vascular endothelial growth factor (VEGF) secretion by rat gingival fibroblasts. A surgical wound was created on the palatal mucosa of 72 rats divided into three groups according to treatment: C - Control (nontreated), A - Artin M gel, and V - Vehicle. Eight animals per group were sacrificed at 3, 5, and 7 days postsurgery for histology, immunohistochemistry and determination of the levels of cytokines, and growth factors. Gingival fibroblasts were incubated with 2.5 μg/mL of Artin M for 24, 48, and 72 hours. The expression of VEGF and TGF-β was determined by enzyme-linked immunosorbent assay. Histologically, at day 7, the Artin M group showed earlier reepithelialization, milder inflammatory infiltration, and increased collagen fiber formation, resulting in faster maturation of granular tissue than in the other groups (p < 0.05). Artin M-induced cell proliferation in vivo and promoted a greater expression of TGF-β and VEGF in both experiments (p < 0.05). Artin M was effective in healing oral mucosa wounds in rats and was associated with increased TGF-β and VEGF release, cell proliferation, reepithelialization, and collagen deposition and arrangement of fibers. © 2013 by the Wound Healing Society.

Avaliação da atividade da unidade epidermo-melânica e do dano dérmico no melasma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Post-receptor IGF1 insensitivity restricted to the MAPK pathway in a Silver-Russell syndrome patient with hypomethylation at the imprinting control region on chromosome 11

Background: Hypomethylation of the paternal imprinting center region 1 (ICR1) is the most frequent molecular cause of Silver-Russell syndrome (SRS). Clinical evidence suggests that patients with this epimutation have mild IGF1 insensitivity. Objective: To assess in vitro IGF1 action in fibroblast culture from a patient with SRS and IGF1 insensitivity. Methods: Fibroblast cultures from one patient with SRS due to ICR1 demethylation and controls were established. The SRS patient has severe growth failure, elevated IGF1 level, and poor growth rate during human recombinant GH treatment. IGF1 action was assessed by cell proliferation, AKT, and p42/44-MAPK phosphorylation. Gene expression was determined by real-time PCR. Results: Despite normal IGF1R sequence and expression, fibroblast proliferation induced by IGF1 was 50% lower in SRS fibroblasts in comparison with controls. IGF1 and insulin promoted a p42/44-MAPK activation in SRS fibroblasts 40 and 36%, respectively, lower than that in control fibroblasts. On the other hand, p42/44-MAPK activation induced by EGF stimulation was only slightly reduced (75% in SRS fibroblasts in comparison with control), suggesting a general impairment in MAPK pathway with a greater impairment of the stimulation induced by insulin and IGF1 than by EGF. A PCR array analysis disclosed a defect in MAPK pathway characterized by an increase in DUSP4 and MEF2C gene expressions in patient fibroblasts. Conclusion: A post-receptor IGF1 insensitivity was characterized in one patient with SRS and ICR1 hypomethylation. Although based on one unique severely affected patient, these results raise an intriguing mechanism to explain the postnatal growth impairment observed in SRS patients that needs confirmation in larger cohorts.

In vitro cytotoxicity of white MTA, MTA Fillapex® and Portland cement on human periodontal ligament fibroblasts

The aim of this study was to compare the in vitro cytotoxicity of white mineral trioxide aggregate (MTA), MTA Fillapex® and Portland cement (PC) on human cultured periodontal ligament fibroblasts. Periodontal ligament fibroblast culture was established and the cells were used for cytotoxic tests after the fourth passage. Cell density was set at 1.25 X10 4 cells/well in 96-well plates. Endodontic material extracts were prepared by placing sealer/cement specimens (5X3mm) in 1mL of culture medium for 72 h. The extracts were then serially two-fold diluted and inserted into the cell-seeded wells for 24, 48 and 72 h. MTT assay was employed for analysis of cell viability. Cell supernatants were tested for nitric oxide using the Griess reagent system. MTA presented cytotoxic effect in undiluted extracts at 24 and 72 h. MTA Fillapex® presented the highest cytotoxic levels with important cell viability reduction for pure extracts and at ½ and ¼ dilutions. In this study, PC did not induce alterations in fibroblast viability. Nitric oxide was detected in extract-treated cell supernatants and also in the extracts only, suggesting presence of nitrite in the soluble content of the tested materials. In the present study, MTA Fillapex displayed the highest cytotoxic effect on periodontal ligament fibroblasts followed by white MTA and PC.

Alanine-rich amphiphilic peptide containing the RGD cell adhesion motif: a coating material for human fibroblast attachment and culture

We studied the self-assembly of peptide A6RGD (A: alanine, R: arginine, G: glycine, D: aspartic acid) in water, and the use of A6RGD substrates as coatings to promote the attachment of human cornea stromal fibroblasts (hCSFs). The self-assembled motif of A6RGD was shown to depend on the peptide concentration in water, where both vesicle and fibril formation were observed. Oligomers were detected for 0.7 wt% A6RGD, which evolved into short peptide fibres at 1.0 wt% A6RGD, while a co-existence of vesicles and long peptide fibres was revealed for 2–15 wt% A6RGD. A6RGD vesicle walls were shown to have a multilayer structure built out of highly interdigitated A6 units, while A6RGD fibres were based on β-sheet assemblies. Changes in the self-assembly motif with concentration were reflected in the cell culture assay results. Films dried from 0.1–1.0 wt% A6RGD solutions allowed hCSFs to attach and significantly enhanced cell proliferation relative to the control. In contrast, films dried from 2.5 wt% A6RGD solutions were toxic to hCSFs.