Estudo da imunoexpressão de RANKL e OPG, do índice angiogênico (CD34) e da presença de miofibroblastos (α-SMA) em ceratocistos odontogênicos isolados e associados à síndrome de Gorlin

The odontogenic keratocysts are distinguished from other odontogenic cystic lesions by their potentially aggressive clinical behavior and association, in some cases, with Gorlin syndrome. Studies have suggested that syndrome keratocysts, in comparison with sporadic lesions, have higher growth and infiltration capacity and higher recurrence tendency. The aim of this study was to analyze, by means of immunohistochemistry, the expressions of receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG), the angiogenic index (CD34) and the presence of myofibroblasts (α-SMA) in primary and recurrent sporadic keratocysts and in keratocysts associated with Gorlin syndrome. The sample was composed by 30 sporadic keratocysts (22 primary and 8 recurrent) and 22 syndrome keratocysts. In the epithelium and in the fibrous capsule of the lesions, the immunoexpression of RANKL and OPG was evaluated by determination of the percentage of positive cells, according to the following scores: 0 (less than 10% of positive cells), 1 (11% - 50% of positive cells), 2 (51% - 75% of positive cells) and 3 (more than 76% of positive cells). In addition, cases were classified according to the RANKL score/ OPG score ratio, as follows: RANKL > OPG, RANKL < OPG, and RANKL = OPG. The angiogenic index was analyzed by counting the microvessels immunoreactive to anti-CD34 antibody in 5 fields (200). The analysis of myofibroblasts was performed by counting the cells immunoreactive to anti-α-SMA antibody in 10 fields (400). The analysis of the expressions of RANKL and OPG in the epithelial lining and in the fibrous capsule did not reveal significant differences between groups (p > 0.05). Regarding the RANKL/ OPG ratio in the epithelial lining, most sporadic primary (54.5%) and syndrome lesions (59.1%) showed RANKL < OPG ratio and RANKL = OPG ratio, respectively (p > 0.05). With respect to the RANKL/ OPG ratio in the fibrous capsule, the majority of sporadic primary (81.8%) and sporadic recurrent lesions (75.0%) and most syndrome lesions (45.5%) showed RANKL = OPG ratio (p > 0.05). The mean number of microvessels was 69.2 in sporadic primary lesions, 67.6 in recurrent lesions, and 71.6 in syndrome lesions, with no significant differences between groups (p > 0.05). The mean number of myofibroblasts was 34.4 in sporadic primary lesions, 29.3 in recurrent lesions, and 33.7 in syndrome lesions, with no significant differences between groups (p > 0.05). In conclusion, the results of the present study suggest that the differences in the biological behavior between sporadic keratocysts and keratocysts associated with Gorlin syndrome may not be related to the expressions of RANKL and OPG, the RANKL/ OPG ratio, the angiogenic index or the number of myofibroblasts in these lesions

Common chromosomal imbalances and stemness-related protein expression markers in endometriotic lesions from different anatomical sites: the potential role of stem cells

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

CD34 EXPRESSION IN STROMAL TUMORS OF THE GASTROINTESTINAL-TRACT

Gastrointestinal stromal tumors (GISTs) are mesenchymal tumors that may exhibit varied morphologic appearances (spindle, epithelioid) and biologic potentials. Given the continuing controversy regarding the type of cell differentiation present in these tumors (muscle versus nerve sheath versus null), we evaluated a set of GISTs, most of which had been previously examined for the presence of smooth muscle differentiation, for expression of CD34, a 115 kDa cell-surface progenitor cell marker also recently identified in a subset of mesenchymal tumors. Using antibody My 10 in deparaffinized, formalin-fixed tissue after pretreatment with microwave energy, we found that 46 of 57, or 81%, of GISTs were CD34+; this fraction of CD34+ tumors exceeded the fraction of these same GISTs found to show muscle actin (72%) expression. In addition, a consistently higher fraction of the tumor cell population was CD34+ than was muscle actin positive. These findings suggest that CD34 is a very sensitive marker for the identification of GISTs. CD34 is normally expressed by endothelial as well as perivascular cells, perhaps related to, but distinct from, vascular smooth muscle cells. While the nature of these latter cells is uncertain, the expression of CD34 in such a large fraction of GISTs may provide evidence of a unique differentiation pathway in these tumors.

Immunophenotypic, immunocytochemistry, ultrastructural, and cytogenetic characterization of mesenchymal stem cells from equine bone marrow

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium-rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n=64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. © 2013 Wiley Periodicals, Inc.

Immunohistochemical investigation of the angiogenic proteins VEGF, HIF-1α and CD34 in invasive ductal carcinoma of the breast

The expression of prognostic markers in cancer has become important in diagnostic routine and research. A high mitotic rate compromises the individual cell access to oxygen and nutrients, due to reduced blood supply. Cells undertake adaptive measures such as vascular endothelial growth factor (VEGF), expressed under the control of hypoxia-inducible factor-1α (HIF-1α). CD34 is an endothelial marker which can show the presence and distribution of blood vessels. This study evaluated the presence and relative expression of VEGF, HIF-1α and CD34 using immunohistochemistry of 60 breast tumors and double staining, correlating the findings with clinical and pathological variables. High VEGF expression was correlated with low cell proliferation, lymph node-negative, smaller tumor size and patients not receiving hormone therapy. High HIF-1α expression predominated in younger (<50-year) patients, subjected to neo-adjuvant therapy and in p53-negative tumors. Absence of metastasis, radiotherapy or hormone treatment, and estrogen receptor (ER)-positive tumors showed high CD34 immunoreactivity. We suggest that the angiogenic factors VEGF, HIF-1α and CD34 are important in breast cancer progression and their abundance in breast tumors has prognostic and predictive value. Crown Copyright © 2013.

Efeito do fator estimulante de colônia de granulócitos recombinante humano (rhG-CSF) sobre o número de leucócitos, plaquetas e sobre a mobilização de células-tronco hematopoéticas CD34+ para o sangue periférico de cães sadios

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A new fibrin sealant as a three-dimensional scaffold candidate for mesenchymal stem cells

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

Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Bone marrow mesenchymal stem cells stimulated by bFGF up-regulated protein expression in comparison with periodontal fibroblasts in vitro

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

Obtenção e caracterização de populações de células-tronco CD200 e CD34 positivas da pele na espécie canina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Obtenção e caracterização de populações de células-tronco CD200 e CD34 positivas da pele na espécie canina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Antiproliferative Effects of Fluoxetine on Colon Cancer Cells and in a Colonic Carcinogen Mouse Model

The antidepressant fluoxetine has been under discussion because of its potential influence on cancer risk. It was found to inhibit the development of carcinogen-induced preneoplastic lesions in colon tissue, but the mechanisms of action are not well understood. Therefore, we investigated anti-proliferative effects, and used HT29 colon tumor cells in vitro, as well as C57BL/6 mice exposed to intra-rectal treatment with the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) as models. Fluoxetine increased the percentage of HT29 cells in the G(0)/G(1) phase of cell-cycle, and the expression of p27 protein. This was not related to an induction of apoptosis, reactive oxygen species or DNA damage. In vivo, fluoxetine reduced the development of MNNG-induced dysplasia and vascularization-related dysplasia in colon tissue, which was analyzed by histopathological techniques. An anti-proliferative potential of fluoxetine was observed in epithelial and stromal areas. It was accompanied by a reduction of VEGF expression and of the number of cells with angiogenic potential, such as CD133, CD34, and CD31-positive cell clusters. Taken together, our findings suggest that fluoxetine treatment targets steps of early colon carcinogenesis. This confirms its protective potential, explaining at least partially the lower colon cancer risk under antidepressant therapy.

Common chromosomal imbalances and stemness-related protein expression markers in endometriotic lesions from different anatomical sites: the potential role of stem cells

Endometriosis is a multifactorial gynecological disease characterized by the presence of functional endometrium-like tissue in ectopic sites. Several studies have focused on elucidating the immunological, endocrine, environmental and genetic factors involved in endometriosis. However, its pathogenesis is still unclear. High-resolution comparative genomic hybridization was applied to screen for genomic imbalances in laser microdissected stromal and epithelial cells from 20 endometriotic lesions and three samples of eutopic endometrium derived from eight patients. The expression of seven stemness-related markers (CD9, CD13, CD24, CD34, CD133, CD117/c-Kit and Oct-4) in endometrial tissue samples was evaluated by immunohistochemistry. Samples of eutopic endometrium showed normal genomic profiles. In ectopic tissues, an average of 68 genomic imbalances was detected per sample. DNA losses were more frequently detected and involved mainly 3p, 5q, 7p, 9p, 11q, 16q, 18q and 19q. Many of the genomic imbalances detected were common to endometriotic stroma and epithelia and also among different endometriotic sites from the same patient. These findings suggested a clonal origin of the endometriotic cells and the putative involvement of stem cells. Positive immunostaining for CD9, CD34, c-Kit and Oct-4 markers was detected in isolated epithelial and/or stromal cells in eutopic and ectopic endometrium in the majority of cases. The presence of shared genomic alterations in stromal and epithelial cells from different anatomical sites of the same patient and the expression of stemness-related markers suggested that endometriosis arises as a clonal proliferation with the putative involvement of stem cells.

Exercise training restores the endothelial progenitor cells number and function in hypertension: implications for angiogenesis

Objectives: Aerobic exercise training has been established as an important nonpharmacological treatment for hypertension. We investigated whether the number and function of endothelial progenitor cells (EPCs) are restored after exercise training, potentially contributing to neovascularization in hypertension. Methods: Twelve-week-old male spontaneously hypertensive rats (SHRs, n = 14) and Wistar Kyoto (WKY, n = 14) rats were assigned to four groups: SHR; trained SHR (SHR-T); WKY; and trained WKY. Exercise training consisted of 10 weeks of swimming. EPC number and function, as well as the vascular endothelial growth factor (VEGF), nitrotyrosine and nitrite concentration in peripheral blood were quantified by fluorescence-activated cell sorter analysis (CD34+/Flk1+ cells), colony-forming unit assay, ELISA and nitric oxide (NO) analyzer, respectively. Soleus capillary/fiber ratio and protein expression of VEGF and endothelial NO synthase (eNOS) by western blot were assessed. Results: Exercise training was effective in reducing blood pressure in SHR-T accompanied by resting bradycardia, an increase in exercise tolerance, peak oxygen uptake (VO2) and citrate synthase activity. In response to hypertension, the amount of peripheral blood-EPC and number of colonies were decreased in comparison with control levels. In contrast, exercise training normalized the EPC levels and function in SHR-T accompanied by an increase in VEGF and NO levels. In addition, oxidative stress levels were normalized in SHR-T. Similar results were found in the number and function of bone marrow EPC. Exercise training repaired the peripheral capillary rarefaction in hypertension by a signaling pathway VEGF/eNOS-dependent in SHR-T. Moreover, improvement in EPC was significantly related to angiogenesis. Conclusion: Our data show that exercise training repairs the impairment of EPC in hypertension, which could be associated with peripheral revascularization, suggesting a mechanism for its potential therapeutic: application in vascular diseases.

Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

The dystrophin gene, located at Xp21, codifies dystrophin, which is part of a protein complex responsible for the membrane stability of muscle cells. Its absence on muscle causes Duchenne Muscular Dystrophy (DMD), a severe disorder, while a defect of muscle dystrophin causes Becker Muscular Dystrophy (DMB), a milder disease. The replacement of the defective muscle through stem cells transplantation is a possible future treatment for these patients. Our objective was to analyze the potential of CD34+ stem cells from umbilical cord blood to differentiate in muscle cells and express dystrophin, in vitro. Protein expression was analyzed by Immunofluorescence, Western Blotting (WB) and Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR). CD34+ stem cells and myoblasts from a DMD affected patient started to fuse with muscle cells immediately after co-cultures establishment. Differentiation in mature myotubes was observed after 15 days and dystrophin-positive regions were detected through Immunofluorescence analysis. However, WB or RT-PCR analysis did not detect the presence of normal dystrophin in co-cultures of CD34+ and DMD or DMB affected patients' muscle cells. In contrast, some CD34+ stem cells differentiated in dystrophin producers' muscle cells, what was observed by WB, reinforcing that this progenitor cell has the potential to originate muscle dystrophin in vitro, and not just in vivo like reported before.