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Caveolin‐1 (Cav‐1) is an integral membrane protein present in all organelles, responsible for regulating and integrating multiple signals as a platform. Mitochondria are extremely adaptable to external cues in chronic liver diseases, and expression of Cav‐1 may affect mitochondrial flexibility in hepatic stellate cells (HSCs) activation. We previously demonstrated that exogenous expression of Cav‐1 was sufficient to increase some classical markers of activation in HSCs. Here, we aimed to evaluate the influence of exogenous expression and knockdown of Cav‐1 on regulating the mitochondrial plasticity, metabolism, endoplasmic reticulum (ER)‐mitochondria distance, and lysosomal activity in HSCs. To characterize the mitochondrial, lysosomal morphology, and ER‐mitochondria distance, we perform transmission electron microscope analysis. We accessed mitochondria and lysosomal networks and functions through a confocal microscope and flow cytometry. The expression of mitochondrial machinery fusion/fission genes was examined by real‐time polymerase chain reaction. Total and mitochondrial cholesterol content was measured using Amplex Red. To define energy metabolism, we used the Oroboros system in the cells. We report that GRX cells with exogenous expression or knockdown of Cav‐1 changed mitochondrial morphometric parameters, OXPHOS metabolism, ER‐mitochondria distance, lysosomal activity, and may change the activation state of HSC. This study highlights that Cav‐1 may modulate mitochondrial function and structural reorganization in HSC activation, being a potential candidate marker for chronic liver diseases and a molecular target for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Background. Stem cells from adipose tissue (ADSCs) and platelet-rich plasma (PRP) are innovative modalities that arise due to their regenerative potential. Objective. The aim of this study was to characterize possible histological changes induced by PRP and ADSC therapies in photoaged skin. Methods. A prospective randomized study involving 20 healthy individuals, showing skin aging. They underwent two therapeutic protocols (protocol 1: PRP; protocol 2: ADSCs). Biopsies were obtained before and after treatment (4 months). Results. PRP protocol showed unwanted changes in the reticular dermis, mainly due to the deposition of a horizontal layer of collagen (fibrosis) and elastic fibers tightly linked. Structural analyses revealed infiltration of mononuclear cells and depot of fibrotic material in the reticular dermis. The ADSC protocol leads to neoelastogenesis with increase of tropoelastin and fibrillin. There was an improvement of solar elastosis inducing an increment of macrophage polarization and matrix proteinases. These last effects are probably related to the increase of elastinolysis and the remodeling of the dermis. Conclusions. The PRP promoted an inflammatory process with an increase of reticular dermis thickness with a fibrotic aspect. On the other hand, ADSC therapy is a promising modality with an important antiaging effect on photoaged human skin. [ABSTRACT FROM AUTHOR]

Internalization of hydroxyapatite nanoparticles in SAOS-2 osteoblasts for 2 and 24 h was investigated in vitro using 5 and 50 µg/mL nanoparticles in culture medium. No cytotoxic effects were observed in a PrestoBlue viability assay. Focused ion beam-scanning electron microscopy and transmission electron microscopy were used to study nanoparticle trafficking inside cells and to characterize the physicochemical properties of the remodeled nanoparticles. Nanoparticles were actively internalized by cells and maintained in intracellular membrane-bound compartments. Dissolution of hydroxyapatite nanoparticles was observed inside phagolysosome in all samples. After 24 h of internalization in cell culture assays, reprecipitation of calcium phosphate minerals was observed in membrane-bound compartments in 5 and 50 µg/mL samples. Compared to the original nanoparticles, the reprecipitated calcium phosphate phase presented a different morphology, structure, and chemical composition. Two sample preparation methods were used and confirmed that reprecipitation of the calcium phosphate crystallites occurred in the intracellular environment and not during electron microscopy sample preparation. Reprecipitation of calcium phosphate prevented the release of large amounts of calcium and phosphate ions inside the cells. This phenomenon may be linked to physiological processes in the cell that control the concentration and trafficking of intracellular calcium ions, which are highly controlled by cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 428-439, 2018. [ABSTRACT FROM AUTHOR]

Bone marrow stromal cells (BMSCs) are considered a promising tool for bone bioengineering. However, the mechanisms controlling osteoblastic commitment are still unclear. Osteogenic differentiation of BMSCs requires the activation of β-catenin signaling, classically known to be regulated by the canonical Wnt pathway. However, BMSCs treatment with canonical Wnts in vitro does not always result in osteogenic differentiation and evidence indicates that a more complex signaling pathway, involving cadherins, would be required to induce β-catenin signaling in these cells. Here we showed that Wnt3a alone did not induce TCF activation in BMSCs, maintaining the cells at a proliferative state. On the other hand, we verified that, upon BMSCs osteoinduction with dexamethasone, cadherins were cleaved by the PS1/γ-secretase complex at the plasma membrane, and this event was associated with an enhanced β-catenin translocation to the nucleus and signaling. When PS1/γ-secretase activity was inhibited, the osteogenic process was impaired. Altogether, we provide evidence that PS1/γ-secretase-mediated cadherin cleavage has as an important role in controlling β-catenin signaling during the onset of BMSCs osteogenic differentiation, as part of a complex signaling pathway responsible for cell fate decision. A comprehensive map of these pathways might contribute to the development of strategies to improve bone repair. [ABSTRACT FROM AUTHOR]

Recurrent chronic leg ulcers are among the most severe vasculopathic complications of sickle cell disease ( SCD). Their treatment remains a challenge. Stem cell therapy with bone marrow mononuclear cells ( BMMC) is a promising new therapeutic option for other forms of chronic ulcers. This prospective pilot study was performed to evaluate safety and feasibility of BMMC implantation in patients with SCD and chronic leg ulcers ( SCLU). Ulcer closure, recurrence and local pain were evaluated. BMMC were successfully administered to 23 SCLU patients and no serious adverse events occurred. During the 6-month follow-up period, 91·3% of patients had improved ulcer pain compared with baseline and 29·2% of the treated ulcers achieved total healing. The frequency of progenitor stem cells ( CD34 CD45low and fibroblast colony-forming units) in BMMC was found to be significantly reduced in SCLU patients and compared to SCD patients without ulcers ( P < 0·004 and P < 0·01, respectively). No relationship was observed between treatment outcome and the number of implanted BM progenitor stem cells. In conclusion, BMMC implantation is a feasible and safe procedure, showing favourable outcomes for the treatment of SCLU, and encouraging further controlled clinical trials. [ABSTRACT FROM AUTHOR]

The presence of phenolic compounds in fruit- and vegetable-rich diets has attracted researchers' attention due to their health-promoting effects. The objective of this study was to evaluate the effects of purple pitanga ( Eugenia uniflora L.) extract on cell proliferation, viability, mitochondrial membrane potential, cell death and cell cycle in murine activated hepatic stellate cells (GRX). Cell viability by 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was significantly decreased on cells treated with 50 and 100 µg ml−1 of purple pitanga extract for 48 and 72 h, and the percentage of dead cell stained with 7-amino-actinomycin D was significantly higher in treated cells. The reduction of cell proliferation was dose dependent, and we also observed alterations on cell cycle progression. At all times studied, GRX cells treated with 50 and 100 µg ml−1 of purple pitanga showed a significant reduction in cellular mitochondrial content as well as a decrease in mitochondrial membrane potential. Furthermore, our results indicated that purple pitanga extract induces early and late apoptosis/necrosis and necrotic death in GRX cells. This is the first report describing the antiproliferative, cytotoxic and apoptotic activity for E. uniflora fruits in hepatic stellate cells. The present study provides a foundation for the prevention and treatment of liver fibrosis, and more studies will be carried to elucidate this effect. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The objective of our study was to investigate chondrogenesis potential of human adipose-derived mesenchymal stromal cells ( MSCs), using as a positive control a human source of cartilage-derived progenitor cells ( PCs). This source of PCs was recently described by our group and dwells on the surface of nasoseptal cartilage. Histological analysis using Safranin O staining and immunofluorescence for actin filaments and collagen type II was performed on three-dimensional ( 3D) pellet cultures. Cartilage PCs and adipose MSCs showed similarities in monolayer culture related to cell morphology and proliferation. Our 3D pellet cultures substantially reduced the actin stress and after 21 days under chondrogenic medium, we observed an increase in the pellet diameter for cartilage PCs (7.4%) and adipose MSCs (21.2%). Adipose-derived MSCs responded to chondrogenic stimulus, as seen by positive areas for collagen type II, but they were not able to recreate a mature extracellular matrix. Using semi-quantitative analysis, we observed a majority of Safranin O areas rising from blue (no stain) to orange (moderate staining) and no changes in fibroblastic morphology ( P < 0.0001). For cartilage PCs, chondrogenic induction is responsible for morphological changes and a high percentage of matrix area/number of cells ( P ≤ 0.0001), evaluated by computerized histomorphometry. Morphological analyses reveal that adipose-derived MSCs were not able to recreate a bioengineered cartilage. The cost of culture was reduced, as the cartilage PCs under growth-factor free medium exhibit a high score for cartilage formation compared with the induced adipose mesenchymal stromal cells ( P = 0.0021). Using a pellet 3D culture, our cartilage PCs were able to produce a cartilage tissue in vitro, leading to the future development of bioengineered products. [ABSTRACT FROM AUTHOR]

The optimal cryopreservation cell concentration during the peripheral blood stem cell (PBSC) collection is a controversial topic. We evaluated the influence of cryopreservation concentration on the recovery of hematopoietic progenitor cells and the kinetics of hematopoietic recovery of autologous stem cell transplant patients. In this retrospective study, we compared two different cryopreservation protocols: 1 × 10 cells/mL (Protocol A) and 2 × 10 cells/mL (Protocol B). A total of 419 PBSCs were analyzed with regard to the number of viable cells and colony-forming units-granulocytes-monocytes (CFU-GM) progenitors. The hematopoietic recovery of 275 patients who received PBSCs cryopreserved at a dose of 1 × 10 cells/mL (Group A) and 2 × 10 cells/mL (Group B) were compared. There were no significant differences in recovery of viable cells between Protocol A and Protocol B. The median of recovery of CFU-GM progenitors was significantly higher in Protocol B (41.2 vs. 57.3, p < 0.01). The median times to neutrophil recovery (≥500 × 10/L) and platelet (PLT) recovery (≥20 × 10/L) in Groups A and B were 11 days versus 11 days and 12 days versus 12 days, respectively. However, by Kaplan and Meier analyses, Group B recovered neutrophils with a little delay (p = 0.01). No difference was observed with regard to time to PLT recovery. On multivariate analysis, we found that the number of CD34+ cells and CFU-GM progenitors had a significant influence on hematopoietic recovery. Cryopreservation of PBSCs at a dose of 2 × 10 cells/mL did not affect the recovery rate of viable cells or the hematopoietic recovery of autologous stem cell transplant patients. [ABSTRACT FROM AUTHOR]

Background Vascular endothelial growth factor (VEGF) has mostly been tested to treat ischemic diseases, although the outcomes obtained are not satisfactory. Our hypothesis is that the local transient expression of VEGF and stem cell mobilizer granulocyte colony-stimulating factor (G-CSF) genes in ischemic limbs can complement their activities and be more efficient for limb recovery. Methods Limb ischemia was surgically induced in mice and 50 µg of VEGF and/or G-CSF genes were locally transferred by electroporation. After 3-4 weeks, evidence of necrosis by visual inspection, capillary density, muscle mass, muscle force and hematopoietic cell mobilization were evaluated. Results After 4 weeks, 70% and 90% of the animals of the ischemic group (IG) and VEGF-treated group (VG), respectively, presented limb necrosis, in contrast to only 10% observed in the group of mice treated with both VEGF and G-CSF genes (VGG). Recovery of muscle mass and muscle force was higher than 60% in the VGG compared to the non-ischemic group. The mobilization of Sca1+ cells and neutrophils was also higher in the VGG, which may explain the lower level of necrosis observed in this group (22%, in contrast to 70% in the IG). Capillary density and degree of fibrosis were determined in weeks 3 and 4, and also showed a clear benefit as a result of the use of the G-CSF and VEGF genes together. Conclusions Gene therapy using VEGF and G-CSF demonstrated a synergistic effect promoting vessel and tissue repair in mouse hind limb ischemia. Copyright © 2010 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background Granulocyte-colony-stimulating factor (GM-CSF) is a pleiotropic factor for hematopoiesis that stimulates myeloblasts, monoblasts and mobilization of bone marrow stem cells. Therefore, the GM-CSF gene is a potential candidate for vessel formation and tissue remodeling in the treatment of ischemic diseases. Methods A new mouse limb ischemia was established by surgery and gene transfer was performed by injection of 100 µg of a plasmid carrying GM-CSF. Muscle force and weight, histology, capillary density, circulating stem cells and monocytes were determined after 3-4 weeks. Results More than 60% of nontreated ischemic animals showed gangrene below the heel after 4 weeks, whereas the GM-CSF gene-treated animals showed only darkening of nails or toes. These animals demonstrated a full recovery of the affected muscles in terms of weight, force and muscle fiber structure, but the muscles of nontreated ischemic animals lost approximately 50% weight, 86% force and their regular structure. When the GM-CSF gene was injected into the contralateral limb, only partial loss was observed, demonstrating a distant effect of GM-CSF. The capillary density in the GM-CSF-treated group was 52% higher in relation to the nontreated group. Blood analysis by flow cytometry showed that the GM-CSF-treated group had 10-20% higher levels of circulating monocytes and Sca-1+. Conclusions We conclude that the direct administration of GM-CSF gene in limb ischemia had a strong therapeutic effect because it promoted the recovery of muscle mass, force and structure by mobilizing therapeutic cells and augmenting the number of vessels. Copyright © 2009 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background/Aims: Pre-adipocyte differentiation into adipocyte is a terminal differentiation process triggered by a cascade of transcription factors. Conversely, hepatic stellate cells (HSC) can switch between lipid storing and the myofibroblast phenotype in association with liver fibrotic processes. Here, adipogenic/lipogenic-related transcription factors and downstream-regulated genes were evaluated in a murine HSC cell line. GRX-HSC cells are transitional myofibroblasts that differentiate into lipocytes following retinol or indomethacin treatment. Methods/Results: Specific mRNAs were quantified by a real-time polymerase chain reaction after 24 h or 7 days of cell culture with indomethacin or retinol. Proliferator-activated receptorγ and Pex16 transcripts were increased either by retinol or indomethacin. Retinol induced a minor increase in C/enhancer binding proteinα transcripts, while only indomethacin increased adipsin transcripts. Conclusions: Our results showed that the myofibroblast to lipocyte phenotype switch follows partially different transcriptional pathways, according to the effector. Retinol induces lipid synthesis and storage without affecting characteristic adipocytic genes, while indomethacin treatment restores the lipocytic phenotype with increased adipisin expression. [ABSTRACT FROM AUTHOR]

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Although some studies have shown a possible modulation of the stroma on the hormonal secretion, it is not clear as to what are the requirements for these cellular interactions. In the present work, a homogeneous and continuous lineage of rat adenohypophysis stromal cells (APS9 cells) obtained from rat adenohypophysis primary culture was established. Using immunocytochemical methods and electron microscopy, we have characterised APS9 cells as elongated fibroblastoid-like cells with intercellular contacts, expressing alpha-smooth muscle actin, type IV collagen and laminin. By biochemical procedures, higher amounts of chondroitin sulphate and heparan sulphate were found in the pericellular and extracellular compartments of APS9 cell culture. In order to evaluate the possible effects of APS9 cell on GH(3)B(6) prolactin-secreting cell survival and/or proliferation, we established co-culture and proliferation assays. When GH(3)B(6) cells were cultivated on APS9 cell substrate, they displayed an organisation of many cellular cords strongly attached and covering all the stromal cell area, establishing punctual interactions or extensive surface associations between adjacent cells. Prolactin immunoreactivity appeared to be more scattered throughout the cytoplasm and accumulated in its periphery. When plated on glass coverslips, on newborn rat skin fibroblasts, on murine haematopoietic bone marrow stroma cell line or on murine foetal liver stroma cell line, GH(3)B(6) cells changed their organisation and presented a decrease in cell number and adherence to the substrate. Our results showed that the APS9 cell/GH(3)B(6) cell interactions favour cell growth and probably PRL secretion, and raises questions about the specificity of different organs and/or animal species stromas on the hormone secretion.