Simone Nunes de Carvalho

OSTEOPONTIN EXPRESSION IN COCULTURE OF DIFFERENTIATING RAT FETAL SKELETAL FIBROBLASTS AND MYOBLASTS

SummarySkeletal fibroblasts in vitro can acquire myofibroblast phenotypes by the development of biochemical and morphological features, mainly the expression of alpha-smooth-muscle actin (α-SMA). Myogenic differentiation is a central event in skeletal muscle development, and has commonly been studied in vitro in the context of skeletal muscle development and regeneration. Controlling this process is a complex set of interactions between myoblasts and the extracellular matrix. Osteopontin (OPN) is an acidic, phosphorylated matrix protein that contains an Arg-Gly-Asp (RGD) cell attachment sequence and has been identified as an adhesive and migratory substrate for several cell types. The aim of this study was to investigate osteopontin expression during the differentiation of skeletal fibroblasts into myofibroblasts and during myogenesis in a coculture model. Fibroblasts and myoblasts were obtained from skeletal muscle of 18-d-old Wistar strain rat fetuses by enzymatic dissociation. At 1 and 9 d, cocultures were immunolabeled, and the cells were also separately subjected to Western blotting to analyze OPN expression. Our data using confocal microscopy showed that myoblasts displayed a strong staining for OPN and that this labeling was maintained after myotube differentiation. Conversely, during fibroblast differentiation into myofibroblasts, we observed a significant increase in OPN expression. The results obtained by immunolabeling were confirmed by Western blotting. We suggest that OPN is important mainly during early stages of myogenesis, facilitating myoblast fusion and differentiation, and that the increased expression of OPN in myofibroblasts might be related to its effects as a key cytokine regulating tissue repair and inflammation.

Progenitor cells and TNF-alpha involvement during morphological changes in pancreatic islets of obese mice

Overnutrition during pregnancy and lactation lend increasing support to the development of obesity and several chronic diseases in adulthood such as type 2 diabetes mellitus, which leads to beta-cell dysfunction and insulin resistance. In this work, we aimed to study the effects of early life overnutrition on the development of obesity, analyzing the morphological changes, expression of TNF-α, and also the stem cell marker CD133 in the pancreatic islets of young and adult mice. Overnutrition during lactation phase was used as an experimental model to induce obesity. The animals were analyzed at 28 and 150 days of age, when pancreata were collected for histological, ultrastructural and western blotting analysis. The results showed that islet hypertrophy is established in obese groups at day 28 and remained until adulthood. CD133+ cells were observed as small cells within pancreatic islets in both control and obese young mice. However, at day 150, these cells were observed only in the islet peripheries and near ducts of the obese group. Furthermore, TNF-α expression in pancreatic islets was increased in both young and adult obese groups when compared to control groups. This work shows interesting data about CD133 receptor and TNF-α roles in the pancreas during obesity development.

The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway

Leishmania amazonensis activates the NF-κB transcriptional repressor homodimer (p50/p50) and promotes nitric oxide synthase (iNOS) downregulation. We investigated the role of PI3K/Akt in p50/p50 NF-κB activation and the effect on iNOS expression in L. amazonensis infection. The increased occupancy of p50/p50 on the iNOS promoter of infected macrophages was observed and we demonstrated that both p50/p50 NF-κB induction and iNOS downregulation in infected macrophages depended on PI3K/Akt activation. Importantly, the intracellular growth of the parasite was also impaired during PI3K/Akt signalling inhibition and in macrophages knocked-down for Akt 1 expression. It was also observed that the increased nuclear levels of p50/p50 in L. amazonensis-infected macrophages were associated with reduced phosphorylation of 907 Ser p105, the precursor of p50. Corroborating these data, we demonstrated the increased levels of phospho-9 Ser GSK3β in infected macrophages, which is associated with GSK3β inhibition and, consequently, its inability to phosphorylate p105. Remarkably, we found that the levels of pPTEN 370 Ser, a negative regulator of PI3K, increased due to L. amazonensis infection. Our data support the notion that PI3K/Akt activity is sustained during the parasite infection, leading to NF-κB 105 phosphorylation and further processing to originate p50/p50 homodimers and the consequent downregulation of iNOS expression.

Central role of PAFR signalling in ExoU-induced NF-κB activation.

ExoU is an important virulence factor in acute Pseudomonas aeruginosa infections. Here, we unveiled the mechanisms of ExoU‐driven NF‐κB activation by using human airway cells and mice infected with P. aeruginosa strains. Several approaches showed that PAFR was crucially implicated in the activation of the canonical NF‐κB pathway. Confocal microscopy of lungs from infected mice revealed that PAFR‐dependent NF‐κB activation occurred mainly in respiratory epithelial cells, and reduced p65 nuclear translocation was detected in mice PAFR−/− or treated with the PAFR antagonist WEB 2086. Several evidences showed that ExoU‐induced NF‐κB activation regulated PAFR expression. First, ExoU increased p65 occupation of PAFR promoter, as assessed by ChIP. Second, luciferase assays in cultures transfected with different plasmid constructs revealed that ExoU promoted p65 binding to the three κB sites in PAFR promoter. Third, treatment of cell cultures with the NF‐κB inhibitor Bay 11–7082, or transfection with IκBα negative‐dominant, significantly decreased PAFR mRNA. Finally, reduction in PAFR expression was observed in mice treated with Bay 11–7082 or WEB 2086 prior to infection. Together, our data demonstrate that ExoU activates NF‐κB by PAFR signalling, which in turns enhances PAFR expression, highlighting an important mechanism of amplification of response to this P. aeruginosa toxin.

Impaired mitochondrial function and reduced viability in bone marrow cells of obese mice

Bone marrow cells (BMCs) are the main type of cells used for transplantation therapies. Obesity, a major world health problem, has been demonstrated to affect various tissues, including bone marrow. This could compromise the success of such therapies. One of the main mechanisms underlying the pathogenesis of obesity is mitochondrial dysfunction, and recent data have suggested an important role for mitochondrial metabolism in the regulation of stem cell proliferation and differentiation. Since the potential use of BMCs for clinical therapies depends on their viability and capacity to proliferate and/or differentiate properly, the analysis of mitochondrial function and cell viability could be important approaches for evaluating BMC quality in the context of obesity. We therefore compared BMCs from a control group (CG) and an obese group (OG) of mice and evaluated their mitochondrial function, proliferation capacity, apoptosis, and levels of proteins involved in energy metabolism. BMCs from OG had increased apoptosis and decreased proliferation rates compared with CG. Mitochondrial respiratory capacity, biogenesis, and the coupling between oxidative phosphorylation and ATP synthesis were significantly decreased in OG compared with CG, in correlation with increased levels of uncoupling protein 2 and reduced peroxisome proliferator-activated receptor-coactivator 1α content. OG also had decreased amounts of the glucose transporter GLUT-1 and insulin receptor (IRβ). Thus, Western-diet-induced obesity leads to mitochondrial dysfunction and reduced proliferative capacity in BMCs, changes that, in turn, might compromise the success of therapies utilizing these cells.

Early Proliferation of Bone Marrow Mononuclear Cells on Collagen Membrane, Bone Graft and Tooth Cementum

El objetivo ultimo de la terapia periodontal es reparar el dano tejidos periodontales de soporte, permitiendo la regeneracion del ligamento periodontal. Sin embargo, la respuesta de la celula, la matriz de apoyo y las moleculas bioactivas aun no han sido bien establecidas. Celulas mononucleares de la medula osea se extrajeron del femur y fibula de rata, y fueron cultivadas sobre un reticulado de membrana de colageno, de injerto de hueso o de un diente molar para comparar la adhesion celular y la proliferacion temprana sobre estos materiales. La adhesion celular fue cuantificada por microscopia de luz a las 24, 48 y 72h, y la proliferacion celular fue observada bajo MEB despues de 72h. Despues de 24 horas, el numero de celulas sobre el injerto de hueso fue similar a la del control y mas del doble en comparacion con la membrana de colageno (q=7,473 p<0,001) y 1,75 veces mayor que con el cemento dental (q=5,613 p<0,01). Sin embargo, el numero de celulas cerca del injerto oseo disminuyo el segundo dia en comparacion con el control. El examen al MEB revelo una disminucion significativa en el numero de celulas que se unen y proliferan sobre los dientes y el injerto oseo en comparacion con la membrana. Los resultados mostraron que las celulas mesenquimales de la medula osea tienen un gran potencial para colonizar la membrana de colageno.

Laminin expression during bone marrow mononuclear cell transplantation in hepatectomized rats.

The adult bone marrow retains two populations of stem cells with emerging importance for the treatment of diverse liver diseases: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). However, the mechanisms that control liver regeneration after bone marrow cell transplantation are still controversial. Liver regeneration after partial hepatectomy is a complex process that requires the proliferation of all hepatic cells. Growth factors, cytokines and extracellular matrix molecules are key elements in this process. Laminins are a family of extracellular matrix proteins with adhesive and chemotactic functions, expressed in the portal and centrolobular veins of the normal liver. The aim of this study was to investigate laminin expression during liver regeneration induced by partial hepatectomy followed by bone marrow mononuclear cell (BMMNC) transplantation. Rat BMMNCs were isolated by Ficoll‐gradient centrifugation, stained with DAPI and injected into recently hepatectomyzed rats via the portal vein. Liver sections obtained 15 min, 1 day and 3 days after the surgery were immunolabeled with anti‐rat CD34 and/or laminin primary antibodies and observed under a laser scanning confocal microscope. Results showed that 15 min after partial hepatectomy, a transplanted CD34+ HSC was found in contact with laminin, which was localized in the portal and centrolobular veins of rat livers. Furthermore, 1 and 3 days after hepatectomy, transplanted BMMNCs were found in the hepatic sinusoids expressing laminin. These results strongly suggest that laminin might be an important extracellular matrix component for bone marrow cell attachment and migration in the injured liver.

Bone marrow mononuclear cell transplantation improves mitochondrial bioenergetics in the liver of cholestatic rats

Mitochondrial dysfunction has been associated with liver cholestatis. Toxic bile salt accumulation leads to chronic injury with mitochondrial damage, ROS increase and apoptosis, resulting in liver dysfunction. This study aimed to analyze mitochondrial bioenergetics in rats with hepatic fibrosis induced by bile duct ligation (BDL) after BMMNC transplantation. Livers were collected from normal rats, fibrotic rats after 14 and 21 days of BDL (F14d and F21d) and rats that received BMMNC at 14 days of BDL, analyzed after 7 days. F21d demonstrated increased collagen I content and consequently decrease after BMMNC transplantation. Both F14d and F21d had significantly reduced mitochondrial oxidation capacity and increased mitochondrial uncoupling, which were restored to levels similar to those of normal group after BMMNC transplantation. In addition, F21d had a significantly increase of UCP2, and reduced PGC-1α content. However, after BMMNC transplantation both proteins returned to levels similar to normal group. Moreover, F14d had a significantly increase in 4-HNE content compared to normal group, but after BMMNC transplantation 4-HNE content significantly reduced, suggesting oxidative stress reduction. Therefore, BMMNC transplantation has a positive effect on hepatic mitochondrial bioenergetics of cholestatic rats, increasing oxidative capacity and reducing oxidative stress, which, in turn, contribute to liver function recover.

Cytokines, hepatic cell profiling and cell interactions during bone marrow cell therapy for liver fibrosis in cholestatic mice

Bone marrow cells (BMC) migrate to the injured liver after transplantation, contributing to regeneration through multiple pathways, but mechanisms involved are unclear. This work aimed to study BMC migration, characterize cytokine profile, cell populations and proliferation in mice with liver fibrosis transplanted with GFP+ BMC. Confocal microscopy analysis showed GFP+ BMC near regions expressing HGF and SDF-1 in the fibrotic liver. Impaired liver cell proliferation in fibrotic groups was restored after BMC transplantation. Regarding total cell populations, there was a significant reduction in CD68+ cells and increased Ly6G+ cells in transplanted fibrotic group. BMC contributed to the total populations of CD144, CD11b and Ly6G cells in the fibrotic liver, related to an increment of anti-fibrotic cytokines (IL-10, IL-13, IFN-γ and HGF) and reduction of pro-inflammatory cytokines (IL-17A and IL-6). Therefore, HGF and SDF-1 may represent important chemoattractants for transplanted BMC in the injured liver, where these cells can give rise to populations of extrahepatic macrophages, neutrophils and endothelial progenitor cells that can interact synergistically with other liver cells towards the modulation of an anti-fibrotic cytokine profile promoting the onset of liver regeneration.

Transplantation of bone marrow-derived MSCs improves renal function and Na + +K + -ATPase activity in rats with renovascular hypertension

Renovascular hypertension (RVH) is a progressive disease, leading to chronic kidney disease when untreated and no specific treatment is available. Therefore, development of new therapeutic modalities is imperative. RVH is triggered by renal artery stenosis and subsequent renin–angiotensin–aldosterone system activation; it can be experimentally induced by the 2 Kidneys–1 Clip (2K1C) model. This study investigates the therapeutic potential of renal subcapsular mesenchymal stem cell (MSC) infusion in 2K1C rats. Renal morphological and functional changes were analyzed, including Na++K+-ATPase activity and expression, renin angiotensin-converting enzyme (ACE) and angiotensin-II type 1 (AT1R) and type 2 (AT2R) receptors expression. 2K1C rats developed hypertension accompanied by renin upregulation (clipped kidney) and renal Na++K+-ATPase activity and expression reduction. MSC therapy decreased systolic blood pressure, renin, ACE, and AT1R, upregulated AT2R and podocin expression and restored renal Na++K+-ATPase activity and expression. In addition, MSC improved renal morphology, reduced fibrosis and TGF-β expression in the clipped kidney, decreased proteinuria and restored protein plasma levels. In conclusion, transplantation into a renal subcapsule is an efficient route and MSC is a good candidate for cell therapy, which may represent an interesting approach for chronic kidney disease treatment.