FOXO1 regulates wound-healing responses in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Forkhead box-O 1 (FOXO1) is a transcription factor actively involved in oral wound healing at the epithelial barrier. However, less is known regarding the role of FOXO1 during the tissue repair response in the connective tissue compartment. This study explored the involvement of FOXO1 in the modulation of fibroblast activity related to wound healing. METHODS: Primary cultures of human gingival fibroblasts were obtained from four healthy young donors. Myofibroblastic differentiation, collagen gel contraction, cell migration, cell spreading, and integrin activation were evaluated in the presence or absence of a FOXO1 inhibitor (AS1842856). Variations in mRNA and proteins of interest were evaluated through qRT-PCR and western blot, respectively. Distribution of actin, α-smooth muscle actin, and ß1 integrin was evaluated using immunofluorescence. FOXO1 and TGF-ß1 expression in gingival wound healing was assessed by immunohistochemistry in gingival wounds performed in C57BL/6 mice. Images were analyzed using ImageJ/Fiji. ANOVA or Kruskal-Wallis test followed by Tukey's or Dunn's post-hoc test was performed. All data are expressed as mean ± SD. p < .05 was considered statistically significant. RESULTS: FOXO1 inhibition caused a decrease in the expression of the myofibroblastic marker α-SMA along with a reduction in fibronectin, type I collagen, TGF-ß1, and ß1 integrin mRNA level. The FOXO1 inhibitor also caused decreases in cell migration, cell spreading, collagen gel contraction, and ß1 integrin activation. FOXO1 and TGF-ß1 were prominently expressed in gingival wounds in fibroblastic cells located at the wound bed. CONCLUSION: The present study indicates that FOXO1 plays an important role in the modulation of several wound-healing functions in gingival fibroblast. Moreover, our findings reveal an important regulatory role for FOXO1 on the differentiation of gingival myofibroblasts, the regulation of cell migration, and collagen contraction, all these functions being critical during tissue repair and fibrosis.

Smoking habits do not affect biological responses induced by leucocyte and platelet-rich fibrin in periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Leucocyte- and platelet-rich fibrin has been developed to stimulate wound healing response. However, it is currently unknown whether smoking affects the biological responses elicited by leucocyte- and platelet-rich fibrin on periodontal ligament-derived mesenchymal stromal cells. This study analyzes the kinetics of biomolecule release from leucocyte- and platelet-rich fibrin derived from smokers and nonsmokers and their effect on periodontal ligament cell proliferation and migration as essential biological activities during wound healing. METHODS: Biomolecules present in leucocyte- and platelet-rich fibrin exudates and conditioned media collected from smokers and nonsmokers were analyzed by Luminex arrays. Periodontal ligament-derived mesenchymal stromal cell obtained from one nonsmoker were treated with leucocyte- and platelet-rich fibrin exudates or leucocyte- and platelet-rich fibrin conditioned media derived from both smokers and nonsmokers. The parameters evaluated included cell proliferation, determined by Ki67 immunostaining and migration assessed using transwell assays. Also, cells were treated with nicotine in the presence of fetal bovine serum 10% or leucocyte- and platelet-rich fibrin conditioned media. RESULTS: A similar biomolecular profile was detected in leucocyte- and platelet-rich fibrin exudates and leucocyte- and platelet-rich fibrin conditioned media from smokers and nonsmokers, stimulating (periodontal ligament-derived mesenchymal stromal cell) proliferation, and migration to a comparable degree. Nicotine reduced cell proliferation and migration of periodontal cells; however, this effect was recovered in the presence of leucocyte- and platelet-rich fibrin conditioned media. CONCLUSION: Leucocyte- and platelet-rich fibrin derived from smokers could be an autologous source of biomolecules to stimulate cell biological activities involved in wound healing in smokers who have difficulties in ceasing this habit. Clinical trials are required to evaluate the impact of leucocyte- and platelet-rich fibrin on healing responses in smokers.

Cytokine profiles and the dynamic of gingivitis development in humans.

AIM: To investigate the relationship between cytokine profiles and "fast" and "slow" patterns of gingival inflammation development. MATERIALS AND METHODS: Forty-two adults participated in an experimental gingivitis study, comprising a 2-week hygiene phase (clinical examination and professional cleaning); a 3-week induction phase (absence of oral hygiene); and a 2-week resolution phase (re-establishment of oral hygiene). Plaque and gingival inflammation scores were assessed. Interferon-gamma (IFN-γ), interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and tumour necrosis factor-alpha (TNF-α) from gingival crevicular fluid were collected and measured by multiplex ELISA. Group-based-trajectory-modelling (GBTM) was used to model cytokine profiles over the induction phase. The effect of gingival inflammation on cytokine levels over time was estimated with mixed-effects modelling. RESULTS: GBTM analysis revealed two cytokine profiles, "non-organized response" (IL-4, IL-6, IL-8, IL-12, and IL-13) and "organized response" (IL-2, IL-10, and TNF-α). Among the "slow" responders, neither cytokine profile was associated with gingivitis. In contrast, a "fast" response was associated with a higher "non-organized response" factor (coef. 0.14) and a lower "organized response" factor (coef. -0.03). CONCLUSION: A "fast" gingivitis development was associated with a higher "non-organized response" and a lower "organized response", which may elucidate the role of individual variability in gingivitis susceptibility.

Uncoupled inflammatory, proliferative, and cytoskeletal responses in senescent human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Aging is characterized by a decline in tissue structure and function that may be explained by the development of cellular senescence. However, the acquisition of specific phenotypic responses in senescent gingival fibroblasts is still poorly understood. Here, we have analyzed whether proliferation of primary cultures of human gingival fibroblasts may affect different cell functions relevant to cellular senescence and tissue deterioration. METHODS: Human gingival fibroblasts from five young donors were expanded until cellular senescence was achieved. Cellular senescence was evaluated by determining modifications in cell size, cell proliferation, p16 and p21 mRNA levels, H2Ax phosphorylation, cell viability, and senescence-associated beta-galactosidase staining. Inflammation was evaluated by analyzing the secretion of cytokines and nuclear translocation of NF-κB. Collagen remodeling was evaluated using a collagen gel contraction assay. Immunofluorescence and confocal microscopy were used to determine changes in the localization of the cytoskeletal proteins. Data analysis was performed to identify changes between cultures of the same donor at early and late passages using the paired sample t test or the Wilcoxon matched-pairs signed-rank test. RESULTS: Late passage cells showed changes compatible with cellular senescence that included increased cell size, reduced cell proliferation, staining for SA-beta gal, phosphorylated H2Ax, and increased p16 and p21 mRNA levels. Late passage cells showed a decrease in collagen contraction and reduced co-localization between the cytoskeletal proteins actin and vinculin. Importantly, late passage cells neither demonstrated changes in the secretion of inflammatory cytokines nor NF-κB activation. CONCLUSION: Our results imply that cytoskeletal changes and inhibition of cell proliferation represent early modifications in the structure and function of senescent gingival fibroblasts that are not coupled with the acquisition of an inflammatory phenotype. Further studies are needed to clarify the impact of different senescence stages during aging of the periodontium.

Galectin-8 mediates fibrogenesis induced by cyclosporine in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: During cyclosporine-induced gingival overgrowth, the homeostatic balance of gingival connective tissue is disrupted leading to fibrosis. Galectins are glycan-binding proteins that can modulate a variety of cellular processes including fibrosis in several organs. Here, we study the role of galectin-8 (Gal-8) in the response of gingival connective tissue cells to cyclosporine. METHODS: We used human gingival fibroblasts and mouse NIH3T3 cells treated with recombinant Gal-8 and/or cyclosporine for analyzing specific mRNA and protein levels through immunoblot, real-time polymerase chain reaction, ELISA and immunofluorescence, pull-down with Gal-8-Sepharose for Gal-8-to-cell surface glycoprotein interactions, short hairpin RNA for Gal-8 silencing and Student's t test and ANOVA for statistical analysis. RESULTS: Galectin-8 stimulated type I collagen and fibronectin protein levels and potentiated CTGF protein levels in TGF-ß1-stimulated human gingival fibroblasts. Gal-8 interacted with α5ß1-integrin and type II TGF-ß receptor. Gal-8 stimulated fibronectin protein and mRNA levels, and this response was dependent on FAK activity but not Smad2/3 signaling. Cyclosporine and tumor necrosis factor alpha (TNF-α) increased Gal-8 protein levels. Finally, silencing of galectin-8 in NIH3T3 cells abolished cyclosporine-induced fibronectin protein levels. CONCLUSION: Taken together, these results reveal for the first time Gal-8 as a fibrogenic stimulus exerted through ß1-integrin/FAK pathways in human gingival fibroblasts, which can be triggered by cyclosporine. Further studies should explore the involvement of Gal-8 in human gingival tissues and its role in drug-induced gingival overgrowth.

Diabetes alters the involvement of myofibroblasts during periodontal wound healing.

OBJECTIVES: Myofibroblasts constitute a specific cell phenotype involved in connective tissue healing. Diabetes alters the wound healing response. However, it is not clear whether diabetes modifies the involvement of myofibroblasts in periodontal wounds. MATERIALS AND METHODS: Type I diabetes was induced in rats through streptozotocin injection, and periodontal wounds were performed. Wound healing was evaluated histologically at 2, 5, 7, and 15 days by measuring epithelial migration, neutrophil infiltration, and collagen and biofilm formation. Distribution of myofibroblasts was evaluated through immunofluorescence for α-smooth muscle actin. Data analyses were performed using the Shapiro-Wilk, ANOVA, or Kruskal-Wallis tests. RESULTS: Diabetic wounds were characterized by delayed epithelial closure, increased neutrophil infiltration, biofilm formation, and reduced collagen formation. Quantification of the myofibroblasts showed a significant reduction at 5 and 7 days in wounds of diabetic rats and an increase at 15 days when compared to wounds of non-diabetic rats. CONCLUSIONS: Diabetic wound healing was associated with decreased epithelial and connective tissue healing, increased levels of inflammation, and biofilm formation. Myofibroblast differentiation was delayed in diabetic periodontal wounds at early time points. However, myofibroblasts persisted at later time points of healing. The present study suggests that diabetes alters the involvement of myofibroblasts during periodontal wound healing.

Comparative effect of platelet-rich plasma, platelet-poor plasma, and fetal bovine serum on the proliferative response of periodontal ligament cell subpopulations.

OBJECTIVES: Cell-based therapies involve the need to expand cell cultures ex vivo for their subsequent implantation in an autologous manner. An important limitation regarding this technology is the use of fetal bovine serum (FBS) that has critical safety limitations. Platelet-derived fractions represent an autologous source of growth factors that may be used for the expansion of these cell cultures. Periodontal ligament (PDL) cells comprise a heterogeneous cell population that may not necessarily respond in a uniform manner to proliferative stimuli. The aim of this study was to evaluate the ability of two platelet-derived fractions, platelet-rich plasma (PRP) and platelet-poor plasma (PPP) and FBS on the proliferative response of different subpopulations of PDL cell cultures. MATERIALS AND METHODS: PDL cells were characterized and then exposed to PRP, PPP, or FBS during 2, 5, or 14 days to analyze cell proliferation and clonogenic capability. Cell proliferation was evaluated through immunofluorescence for Ki67 and by tracing carboxyfluorescein diacetate succinimidyl ester (CFSE) dye in combination with mesenchymal stem cell markers using flow cytometry. RESULTS: Both PRP and PPP stimulated PDL cell proliferation and their clonogenic ability. We found a significant increase of CD73- and CD90-positive cells after PRP or PPP treatment, compared to FBS. Otherwise, no differences were found regarding the response of CD146-or CD105-positive cells when stimulated with PRP, PPP, or FBS. CONCLUSION: PRP and PPP can stimulate the proliferation and clonogenicity of PDL cell populations including cells positive for CD90 and CD73 markers. CLINICAL RELEVANCE: These findings may have implications for future therapies aiming to stimulate periodontal regeneration using autologous growth factors.

Modulation of Mammary Stromal Cell Lactate Dynamics by Ambient Glucose and Epithelial Factors.

Hyperglycemia is a risk factor for a variety of human cancers. Increased access to glucose and that tumor metabolize glucose by a glycolytic process even in the presence of oxygen (Warburg effect), provide a framework to analyze a particular set of metabolic adaptation mechanisms that may explain this phenomenon. In the present work, using a mammary stromal cell line derived from healthy tissue that was subjected to a long-term culture in low (5 mM) or high (25 mM) glucose, we analyzed kinetic parameters of lactate transport using a FRET biosensor. Our results indicate that the glucose pre-culture and soluble epithelial factors constitute a stimulus for lactate stromal production, factors that also modify the kinetic parameters and the monocarboxylate transporters expression in stromal cells. We also observed a vectorial flux of lactate from stroma to epithelial cells in a co-culture setting and found that the uptake of lactate by epithelial cells correlates with the degree of malignancy. Glucose preconditioning of the stromal cell stimulated epithelial motility. Our findings suggest that lactate generated by stromal cells in the high glucose condition stimulate epithelial migration. Overall, our results support the notion that glucose not only provides a substrate for tumor nutrition but also behaves as a signal promoting malignancy. J. Cell. Physiol. 232: 136-144, 2017. © 2016 Wiley Periodicals, Inc.

Glucose Promotes a Pro-Oxidant and Pro-Inflammatory Stromal Microenvironment Which Favors Motile Properties in Breast Tumor Cells.

Chronic inflammation and metabolic reprogramming have been proposed as hallmarks of cancer development. Currently, many of the functional clues between these two phenomena are studied under the integrative view of functional stroma-epithelia interaction. It has been proposed that stromal cells, due to their abundance and avidity for glucose, are able to modify the metabolic behavior of an entire solid tumor. In the present study, using a mammary stromal cell line derived from healthy tissue subjected to long-term culture in low (5 mM) or high (25 mM) glucose, we found that the hyperglycemic condition favors the establishment of a pro-inflammatory and pro-oxidant environment characterized by the induction of the COX-2/PGE2 axis. In this condition, epithelial migration was stimulated. Moreover, we also found that stromal-derived PGE2, acting as a stimulator of IL-1 epithelial expression was one of the factors that promote the acquisition of motile properties by epithelial cells and the maintenance of a COX-2/PGE2-dependent inflammatory condition. Overall, our work provides experimental evidence that glucose stimulates a tumor inflammatory environment that, as a result of a functional cross-talk between stroma and epithelia, may be responsible for tumor progression. J. Cell. Biochem. 118: 994-1002, 2017. © 2016 Wiley Periodicals, Inc.

Ageing, dental caries and periodontal diseases.

AIM: To review the burden of caries and periodontitis in the elderly, changes with age that can explain this burden, and the vulnerability to disease of elderly populations. METHODS: An assessment of surveys in two populations was conducted. Indicators for caries were identified by updating a systematic review. Secular trends for smoking and type 2 diabetes were discussed. RESULTS: Changes in the susceptibility to periodontitis with age may be explained by exposure to pro-inflammatory conditions and changes in the healing capacity of cells and tissues. Due to accumulated periodontal destruction, the number of surfaces at risk for caries increases. The sequels of restorative treatment contribute to an increased susceptibility for caries development. Population-based surveys in the United States and Germany demonstrate a high caries experience among elderly people. A comparison of surveys demonstrates a relative improvement of periodontal health among elderly during the last few decades. Nevertheless, prevalence estimates for periodontitis remain high. Risk indicators for root caries include caries experience, the number of surfaces at risk and poor oral hygiene. Secular trends of main risk factors for periodontitis and their likely influence on the future periodontitis burden in the elderly are discussed. CONCLUSION: Caries and periodontitis burden in the elderly remain high.

Research on growth factors in periodontology.

Growth factors play critical roles in periodontal repair through the regulation of cell behavior. Many of the cell responses regulated by these proteins include cell adhesion, migration, proliferation and differentiation. Periodontal regeneration involves an organized response of different cells, tissues and growth factors implicated in the coordination of these events. However, periodontal tissue reconstruction is an extremely difficult task. Multiple studies have been performed to understand the specific role of growth factors in periodontal wound healing. In the present review we analyze the evidence that supports the roles of growth factors in periodontal wound healing and regeneration.

Soluble MMP-14 produced by bone marrow-derived stromal cells sheds epithelial endoglin modulating the migratory properties of human breast cancer cells.

It has been proposed that epithelial cells can acquire invasive properties through exposure to paracrine signals originated from mesenchymal cells within the tumor microenvironment. Transforming growth factor-ß (TGF-ß) has been revealed as an active factor that mediates the epithelial-stroma cross-talk that facilitates cell invasion and metastasis. TGF-ß signaling is modulated by the coreceptor Endoglin (Eng), which shows a tumor suppressor activity in epithelial cells and regulates the ALK1-Smad1,5,8 as well as the ALK5-Smad2,3 signaling pathways. In the current work, we present evidence showing that cell surface Eng abundance in epithelial MCF-7 breast cancer cells is inversely related with cell motility. Shedding of Eng in MCF-7 cell surface by soluble matrix metalloproteinase-14 (MMP-14) derived from the HS-5 bone-marrow-derived cell line induces a motile epithelial phenotype. On the other hand, restoration of full-length Eng expression blocks the stromal stimulus on migration. Processing of surface Eng by stromal factors was demonstrated by biotin-neutravidin labeling of cell surface proteins and this processing generated a shift in TGF-ß signaling through the activation of Smad2,3 pathway. Stromal MMP-14 abundance was stimulated by TGF-ß secreted by MCF-7 cells acting in a paracrine manner. In turn, the stromal proteolytic activity of soluble MMP-14, by inducing Eng shedding, promoted malignant progression. From these data, and due to the capacity of TGF-ß to regulate malignancy in epithelial cancer, we propose that stromal-dependent epithelial Eng shedding constitutes a putative mechanism that exerts an environmental control of cell malignancy.

c-Jun N terminal kinase modulates NOX-4 derived ROS production and myofibroblasts differentiation in human breast stromal cells.

BACKGROUND: Hard consistency, developed under the influence of tumor cell factors, is a characteristic feature of a breast tumor. Activation of resident fibroblasts leading to a myofibroblast phenotype is the principal feature that orchestrates this fibrotic process. The aim of this study was to assess the effects induced by TGF-ß1, a growth factor abundantly present in tumor microenvironment, on the molecular mechanisms that mediate myofibroblastic differentiation of normal human mammary fibroblasts. METHODS: We used an immortalized fibroblastic cell line derived from normal mammary tissue (RMF-EG cells) to study the effect of TGF-ß1 in the expression of α-SMA and CTGF as markers of myofibroblastic differentiation. The influence of redox status and JNK activity on TGF-ß1-induced transcriptional activity was measured by a luciferase reporter assay. We also used a shRNA approach to evaluate the influence of NOX4 in myofibroblastic differentiation. RESULTS: TGF-ß1 stimulates the expression of myofibroblast markers α-SMA and CTGF. Using a NOX inhibitor (DPI) and cells expressing a shRNA for NOX4, we demonstrated that TGF-ß1 promotes an oxidative environment that favors myofibroblastic differentiation. We also found that activation of c-Jun N-terminal kinase is required for TGF-ß1-dependent expression of CTGF, NOX4 and α-SMA. CONCLUSIONS: Human mammary stromal fibrosis, evaluated by the expression of early and late markers as CTGF and α-SMA, depends on the activation of JNK signaling pathway. Our results show that JNK activation is an early event that precedes the increase in ROS levels leading to myofibroblastic differentiation and tumor fibrosis, suggesting that inhibition of JNK may be used a method to interrupt the development of tumor desmoplasia.

Effects of platelet-rich and -poor plasma on the reparative response of gingival fibroblasts.

OBJECTIVES: Although platelet-rich plasma (PRP) has been proposed as a therapeutic tool to enhance wound repair, the cellular and molecular mechanisms stimulated by this agent are still not completely understood. The present study was designed to characterize the effects of PRP and platelet-poor plasma (PPP) supernatants on cell responses involved in gingival tissue repair. METHODS: We studied the response of human gingival fibroblasts (HGF) to PRP and PPP fractions on: matrix contraction, cell migration, myofibroblastic differentiation, production of matrix components and proteolytic enzymes. PRP and PPP were obtained from donors using a commercial kit. Matrix contraction was evaluated by means of collagen lattices in the presence of matrix metalloproteinase (MMP) and actin polymerization inhibitors. The production of matrix molecules and proteinases was assessed through Western-blot. RhoA activity was evaluated through a pull-down assay. Actin distribution and focal adhesions were assessed through immunofluorescence. Transforming growth factor-beta (TGF-ß) was quantified through ELISA. RESULTS: Both PRP and PPP stimulated human gingival fibroblasts-populated collagen gel contraction and Ilomastat and cytochalasin D inhibited this response. PRP and PPP also stimulated MT1-MMP and TIMP-2 production, RhoA activation and actin cytoskeleton remodeling, cell migration/invasion and myofibroblastic differentiation. TGF-ß1 was found at significantly higher concentrations in PRP than in PPP. CONCLUSIONS: Both PRP and PPP promote wound tissue remodeling and contraction through the stimulation of actin remodeling, the activity of MMPs, promotion of cell migration, and myofibroblastic differentiation. The similar biological responses induced by PRP and PPP suggest that both platelet-derived fractions may exert a positive effect on gingival repair.

The Dynamic Interaction between Extracellular Matrix Remodeling and Breast Tumor Progression.

Desmoplastic tumors correspond to a unique tissue structure characterized by the abnormal deposition of extracellular matrix. Breast tumors are a typical example of this type of lesion, a property that allows its palpation and early detection. Fibrillar type I collagen is a major component of tumor desmoplasia and its accumulation is causally linked to tumor cell survival and metastasis. For many years, the desmoplastic phenomenon was considered to be a reaction and response of the host tissue against tumor cells and, accordingly, designated as "desmoplastic reaction". This notion has been challenged in the last decades when desmoplastic tissue was detected in breast tissue in the absence of tumor. This finding suggests that desmoplasia is a preexisting condition that stimulates the development of a malignant phenotype. With this perspective, in the present review, we analyze the role of extracellular matrix remodeling in the development of the desmoplastic response. Importantly, during the discussion, we also analyze the impact of obesity and cell metabolism as critical drivers of tissue remodeling during the development of desmoplasia. New knowledge derived from the dynamic remodeling of the extracellular matrix may lead to novel targets of interest for early diagnosis or therapy in the context of breast tumors.

Matrix Stiffness Modulates Metabolic Interaction between Human Stromal and Breast Cancer Cells to Stimulate Epithelial Motility.

Breast tumors belong to the type of desmoplastic lesion in which a stiffer tissue structure is a determinant of breast cancer progression and constitutes a risk factor for breast cancer development. It has been proposed that cancer-associated stromal cells (responsible for this fibrotic phenomenon) are able to metabolize glucose via lactate production, which supports the catabolic metabolism of cancer cells. The aim of this work was to investigate the possible functional link between these two processes. To measure the effect of matrix rigidity on metabolic determinations, we used compliant elastic polyacrylamide gels as a substrate material, to which matrix molecules were covalently linked. We evaluated metabolite transport in stromal cells using two different FRET (Fluorescence Resonance Energy Transfer) nanosensors specific for glucose and lactate. Cell migration/invasion was evaluated using Transwell devices. We show that increased stiffness stimulates lactate production and glucose uptake by mammary fibroblasts. This response was correlated with the expression of stromal glucose transporter Glut1 and monocarboxylate transporters MCT4. Moreover, mammary stromal cells cultured on stiff matrices generated soluble factors that stimulated epithelial breast migration in a stiffness-dependent manner. Using a normal breast stromal cell line, we found that a stiffer extracellular matrix favors the acquisition mechanistical properties that promote metabolic reprograming and also constitute a stimulus for epithelial motility. This new knowledge will help us to better understand the complex relationship between fibrosis, metabolic reprogramming, and cancer malignancy.

Soluble factors derived from tumor mammary cell lines induce a stromal mammary adipose reversion in human and mice adipose cells. Possible role of TGF-beta1 and TNF-alpha.

In carcinomas such as those of breast, pancreas, stomach, and colon, cancer cells support the expansion of molecular and cellular stroma in a phenomenon termed desmoplasia, which is characterized by a strong fibrotic response. In the case of breast tissue, in which stroma is mainly a fatty tissue, this response presumably occurs at the expense of the adipose cells, the most abundant stromal phenotype, generating a tumoral fibrous structure rich in fibroblast-like cells. In this study, we aimed to determine the cellular mechanisms by which factors present in the media conditioned by MDA-MB-231 and MCF-7 human breast cancer cell lines induce a reversion of adipose cells to a fibroblastic phenotype. We demonstrated that soluble factors generated by these cell lines stimulated the reversion of mammary adipose phenotype evaluated as intracellular lipid content and expression of C/EBP alpha and PPAR gamma. We also demonstrated that exogenous TGF-beta 1 and TNF-alpha exerts a similar function. The participation of both growth factors, components of media conditioned by tumoral mammary cells, on the expression and nuclear translocation of C/EBP alpha and PPAR gamma was tested in 3T3-L1 cells by interfering with the inhibitory effects of media with agents that block the TGF-beta 1 and TNF-alpha activity. These results allow us to postulate that TGF-beta 1 and TNF-alpha present in this media are in part responsible for this phenotypic reversion.

Cetylpyridinium chloride blocks herpes simplex virus replication in gingival fibroblasts.

Infections with herpes simplex viruses are lifelong and highly prevalent worldwide. Individuals with clinical symptoms elicited by HSVs may suffer from occasional or recurrent herpetic lesions in the orofacial and genital areas. Despite the existence of nucleoside analogues that interfere with HSV replication, such as acyclovir, these drugs are somewhat ineffective in treating skin lesions as topical formulations only reduce in one or few days the duration of the herpetic ulcers. Cetylpyridinium chloride (CPC) is a quaternary ammonium compound present in numerous hygiene products, such as mouthwashes, deodorants, aphtae-treating formulations and oral tablets as an anti-septic to limit bacterial growth. Some reports indicate that CPC can also modulate host signaling pathways, namely NF-κB signaling. Because HSV infection is modulated by NF-κB, we sought to assess whether CPC has antiviral effects against HSVs. Using wild-type HSV-1 and HSV-2, as well as viruses that are acyclovir-resistant or encode GFP reporter genes, we assessed the antiviral capacity of CPC in epithelial cells and human gingival fibroblasts expanded from the oral cavity and its mechanism of action. We found that a short, 10-min exposure to CPC added after HSV entry into the cells, significantly limited viral replication in both cell types by impairing viral gene expression. Interestingly, our results suggest that CPC blocks HSV replication by interfering with the translocation of NF-κB into the nucleus of HSV-infected cells. Taken together, these findings suggest that formulations containing CPC may help limit HSV replication in infected tissues and consequently reduce viral shedding.

Anti-herpetic Activity of Macrocystis pyrifera and Durvillaea antarctica Algae Extracts Against HSV-1 and HSV-2.

Herpes simplex viruses (HSVs) type 1 (HSV-1) and type 2 (HSV-2) are highly prevalent in the human population, and the infections they produce are lifelong with frequent reactivations throughout life. Both viruses produce uncomfortable and sometimes painful lesions in the orofacial and genital areas, as well as herpetic gingivostomatitis, among other clinical manifestations. At present, the most common treatments against HSVs consist of nucleoside analogs that target the viral polymerases. However, such drugs are poorly effective for treating skin lesions, as they only reduce in 1-2 days the duration of the herpetic lesions. Additionally, viral isolates resistant to these drugs can emerge in immunosuppressed individuals, and second-line drugs for such variants are frequently accompanied by adverse effects requiring medical supervision. Thus, novel or improved therapeutic drugs for treating HSV lesions are needed. Here, we assessed the potential antiviral activity of aqueous extracts obtained from two brown macroalgae, namely Macrocystis pyrifera and Durvillaea antarctica against HSVs. Both extracts showed antiviral activity against acyclovir-sensitive and acyclovir-resistant HSV-1 and HSV-2. Our analyses show that there is a significant antiviral activity associated with proteins in the extract, although other compounds also seem to contribute to inhibiting the replication cycle of these viruses. Evaluation of the algae extracts as topical formulations in an animal model of HSV-1 skin infection significantly reduced the severity of the disease more than acyclovir, as well as the duration of the herpetic lesions, when compared to mock-treated animals, with the D. antarctica extract performing best. Taken together, these findings suggest that these algae extracts may be potential phytotherapeutics against HSVs and may be useful for the treatment and reduction of common herpetic manifestations in humans.

Role of Fibroblast Populations in Periodontal Wound Healing and Tissue Remodeling.

After injury to periodontal tissues, a sequentially phased healing response is initiated that enables wound closure and partial restoration of tissue structure and function. Wound closure in periodontal tissues involves the tightly regulated coordination of resident cells in epithelial and connective tissue compartments. Multiple cell populations in these compartments synergize their metabolic activities to reestablish a mucosal seal that involves the underlying periodontal connective tissues and the attachment of these tissues to the tooth surface. The formation of an impermeable seal around the circumference of the tooth is of particular significance in oral health since colonization of tooth surfaces by pathogenic biofilms promotes inflammation, which can contribute to periodontal tissue degradation and tooth loss. The reformation of periodontal tissue structures in the healing response centrally involves fibroblasts, which synthesize and organize the collagen fibers that link alveolar bone and gingiva to the cementum covering the tooth root. The synthesis and remodeling of nascent collagen matrices are of fundamental importance for the reestablishment of a functional periodontium and are mediated by diverse, multi-functional fibroblast populations that reside within the connective tissues of gingiva and periodontal ligament. Notably, after gingival wounding, a fibroblast sub-type (myofibroblast) arises, which is centrally involved in collagen synthesis and fibrillar remodeling. While myofibroblasts are not usually seen in healthy, mature connective tissues, their formation is enhanced by wound-healing cytokines. The formation of myofibroblasts is also modulated by the stiffness of the extracellular matrix, which is mechanosensed by resident precursor cells in the gingival connective tissue microenvironment. Here, we consider the cellular origins and the factors that control the differentiation and matrix remodeling functions of periodontal fibroblasts. An improved understanding of the regulation and function of periodontal fibroblasts will be critical for the development of new therapies to optimize the restoration of periodontal structure and function after wounding.