Análise microscópica do osso bovino inorgânico microgranular implantado em subcutâneo de ratos

The tissue response to porous bovine anorganic bone implanted in rat connective tissue was evaluated by subjective light microscopy analysis. Forty rats were divided into two groups: control (empty collagen capsules) and test (collagen capsule filled with 0.1g biomaterial) and killed 10, 20, 30 and 60 days after implantation. At 10 days, intense chronic inflammatory infiltrate consisting mainly of macrophages and inflammatory multinucleated giant cells (IMGC) was observed. Neutrophils, plasma cells and lymphocytes were present in discrete amounts and slowly disappeared along the repair process. Porosity of the material was filled by reaction connective tissue exhibiting IMGC. The fibrosis was more intense after 60 days and clearly higher than the control group. Thus, the material did not cause any severe adverse reactions and did not stimulate the immune system. Based on the results it could be concluded that deproteinized bovine cancelous bone was well tolerated by rat connective tissue.A resposta tecidual ao osso inorgânico bovino medular implantado em subcutâneo de rato foi avaliada por análise subjetiva através de microscopia de luz. Quarenta ratos foram divididos em 2 grupos: controle (cápsulas vazias de colágeno) e teste (cápsulas de colágeno contendo 0,1g do biomaterial) e mortos 10, 20, 30 e 60 dias após a implantação. Histologicamente, aos 10 dias, observou-se infiltrado inflamatório crônico composto por macrófagos e Células Gigantes Multinucleadas Inflamatórias (IMGC). Neutrófilos, plasmócitos e linfócitos estavam presentes de maneira discreta, desaparecendo durante o processo de reparo tecidual. A porosidade do material foi preenchida pelo tecido conjuntivo reacional mostrando as IMGC. A fibrose foi mais intensa aos 60 dias e evidentemente superior ao grupo controle. Entretanto, o material não causou reações adversas severas, não estimulando a resposta imunológica. Baseado nos resultados encontrados, concluímos que o osso inorgânico bovino medular foi bem tolerado pelo tecido conjuntivo de rato

Updating the role of matrix metalloproteinases in mineralized tissue and related diseases

Bone development and healing processes involve a complex cascade of biological events requiring well-orchestrated synergism with bone cells, growth factors, and other trophic signaling molecules and cellular structures. Beyond health processes, MMPs play several key roles in the installation of heart and blood vessel related diseases and cancer, ranging from accelerating metastatic cells to ectopic vascular mineralization by smooth muscle cells in complementary manner. The tissue inhibitors of MMPs (TIMPs) have an important role in controlling proteolysis. Paired with the post-transcriptional efficiency of specific miRNAs, they modulate MMP performance. If druggable, these molecules are suggested to be a platform for development of “smart” medications and further clinical trials. Thus, considering the pleiotropic effect of MMPs on mammals, the purpose of this review is to update the role of those multifaceted proteases in mineralized tissues in health, such as bone, and pathophysiological disorders, such as ectopic vascular calcification and cancer

Violacein Induces Death of Resistant Leukaemia Cells via Kinome Reprogramming, Endoplasmic Reticulum Stress and Golgi Apparatus Collapse

It is now generally recognised that different modes of programmed cell death (PCD) are intimately linked to the cancerous process. However, the mechanism of PCD involved in cancer chemoprevention is much less clear and may be different between types of chemopreventive agents and tumour cell types involved. Therefore, from a pharmacological view, it is crucial during the earlier steps of drug development to define the cellular specificity of the candidate as well as its capacity to bypass dysfunctional tumoral signalling pathways providing insensitivity to death stimuli. Studying the cytotoxic effects of violacein, an antibiotic dihydro-indolone synthesised by an Amazon river Chromobacterium, we observed that death induced in CD34(+)/c-Kit(+)/P-glycoprotein(+)/MRP1(+) TF1 leukaemia progenitor cells is not mediated by apoptosis and/or autophagy, since biomarkers of both types of cell death were not significantly affected by this compound. To clarify the working mechanism of violacein, we performed kinome profiling using peptide arrays to yield comprehensive descriptions of cellular kinase activities. Pro-death activity of violacein is actually carried out by inhibition of calpain and DAPK1 and activation of PKA, AKT and PDK, followed by structural changes caused by endoplasmic reticulum stress and Golgi apparatus collapse, leading to cellular demise. Our results demonstrate that violacein induces kinome reprogramming, overcoming death signaling dysfunctions of intrinsically resistant human leukaemia cells.TopInstitute pharma (The Netherlands)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Dutch Cancer SocietyErasmus MC Univ Med Ctr, Dept Gastroenterol & Hepatol, Rotterdam, NetherlandsUniv Amsterdam, Acad Med Ctr, Ctr Expt & Mol Med, NL-1105 AZ Amsterdam, NetherlandsUniv Estadual Campinas, Brazil UNICAMP, Dept Biochem, Inst Biol, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Biochem, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Cell Biol, São Paulo, BrazilUniv Grande Rio UNIGRANRIO, Heath Sci Sch, Multidisciplinary Lab Dent Res, Rio de Janeiro, BrazilNatl Inst Metrol Qual & Technol Inmetro, Biotechnol Lab, Bioengn Sect, Rio de Janeiro, BrazilUniv Campinas UNICAMP, Inst Chem, Biol Chem Lab, Rio de Janeiro, BrazilUniv Groningen, Univ Med Ctr Groningen, Dept Pediat Oncol, Beatrix Childrens Hosp, Groningen, NetherlandsFed Univ São Paulo UNIFESP, Dept Biochem, São Paulo, BrazilFed Univ São Paulo UNIFESP, Dept Cell Biol, São Paulo, BrazilDutch Cancer Society: EMCR 2010-4737Web of Scienc

Activation of the Low Molecular Weight Protein Tyrosine Phosphatase in Keratinocytes Exposed to Hyperosmotic Stress

Herein, we provide new contribution to the mechanisms involved in keratinocytes response to hyperosmotic shock showing, for the first time, the participation of Low Molecular Weight Protein Tyrosine Phosphatase (LMWPTP) activity in this event. We reported that sorbitol-induced osmotic stress mediates alterations in the phosphorylation of pivotal cytoskeletal proteins, particularly Src and cofilin. Furthermore, an increase in the expression of the phosphorylated form of LMWPTP, which was followed by an augment in its catalytic activity, was observed. of particular importance, these responses occurred in an intracellular milieu characterized by elevated levels of reduced glutathione (GSH) and increased expression of the antioxidant enzymes glutathione peroxidase and glutathione reductase. Altogether, our results suggest that hyperosmostic stress provides a favorable cellular environment to the activation of LMWPTP, which is associated with increased expression of antioxidant enzymes, high levels of GSH and inhibition of Src kinase. Finally, the real contribution of LMWPTP in the hyperosmotic stress response of keratinocytes was demonstrated through analysis of the effects of ACP1 gene knockdown in stressed and non-stressed cells. LMWPTP knockdown attenuates the effects of sorbitol induced-stress in HaCaT cells, mainly in the status of Src kinase, Rac and STAT5 phosphorylation and activity. These results describe for the first time the participation of LMWPTP in the dynamics of cytoskeleton rearrangement during exposure of human keratinocytes to hyperosmotic shock, which may contribute to cell death.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Estadual Campinas, Dept Bioquim, Inst Biol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, SP, BrazilUniv Estadual Paulista, Dept Quim & Bioquim, IBB, São Paulo, BrazilUniv São Paulo, Dept Genet & Biol Evolut, São Paulo, SP, BrazilUniv Fed ABC, Ctr Ciencias Nat & Humanas, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Bioquim, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Ciencias Biol, São Paulo, SP, BrazilFAPESP: 2006/07315-3CNPq: PQ-2Web of Scienc

Human mesenchymal stromal cells in adhesion to cell-derived extracellular matrix and titanium: Comparative kinome profile analysis

The extracellular matrix (ECM) physically supports cells and influences stem cell behaviour, modulating kinase-mediated signalling cascades. Cell-derived ECMs have emerged in bone regeneration as they reproduce physiological tissue-architecture and ameliorate mesenchymal stromal cell (MSC) properties. Titanium scaffolds show good mechanical properties, facilitate cell adhesion, and have been routinely used for bone tissue engineering (BTE). We analyzed the kinomic signature of human MSCs in adhesion to an osteopromotive osteoblast-derived ECM, and compared it to MSCs on titanium. PamChip kinase-array analysis revealed 63 phosphorylated peptides on ECM and 59 on titanium, with MSCs on ECM exhibiting significantly higher kinase activity than on titanium. MSCs on the two substrates showed overlapping kinome profiles, with activation of similar signalling pathways (FAK, ERK, and PI3K signalling). Inhibition of PI3K signalling in cells significantly reduced adhesion to ECM and increased the number of nonadherent cells on both substrates. In summary, this study comprehensively characterized the kinase activity in MSCs on cell-derived ECM and titanium, highlighting the role of PI3K signalling in kinomic changes regulating osteoblast viability and adhesion. Kinome profile analysis represents a powerful tool to select pathways to better understand cell behaviour. Osteoblast-derived ECM could be further investigated as titanium scaffold-coating to improve BTE

Signal transduction activated during differentiation of osteoblsts and osteocytes

Orientadores: Carmen Verissima Ferreira, Jose Mauro Granjeiro, Maikel PeppelenboschTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Este trabalho teve como principal objetivo investigar os mecanismos de transdução de sinal disparados durante a diferenciação de células ósseas. Desta forma, vários aspectos moleculares desse processo foram analisados. A modulação da Src kinase pela proteína tirosina fosfatase de baixo peso molecular (LMWPTP) é essencial para a diferenciação dos pré-osteoblastos induzida pelo ácido ascórbico/glicerofosfato. Outro enfoque dado nesse trabalho foi a avaliação, sob o aspecto molecular e morfológico, da diferenciação de pré-osteoblastos em ¿osteocyte-like cells¿, processo esse induzido quando o Matrigel foi utilizado como substrato. De forma inédita demonstramos que nessa condição as células produziram a proteína sonic hedgehog (Shh), a qual também foi essencial para o processo de diferenciação. A análise do perfil quinômico dessas células apontou uma prevalência das quinases envolvidas com a comunicação celular. Este fato é coerente com a super-expressão de conexina 43 observada. Além disso, observamos que RECK e TIMP-1 modulam a atividade do rearranjo da matriz extracelular durante a diferenciação de osteoblastos, bem como o requerimento das proteínas PP2A e p38 MAPK durante a adesão de osteoblastos. Adicionalmente observamos uma modulação refinada das PTPs (LMWPTP, SHP2 e PTPa) bem como do status redox celular. Os resultados em conjunto demonstram que o estudo de transdução de sinal pode fornecer informações importantes para o entendimento do funcionamento celular bem como definir alvos moleculares que podem servir como ferramentas para diferentes aplicaçõesAbstract: The main goal of this work was to investigate the signal transduction pathways triggered during the bone cells differentiation. In this way, several molecular aspects of this process were analyzed. Src kinase modulation by the low molecular weight protein tyrosine phosphatase (LMWPTP) was essential for the occurrence of the differentiation induced by ascorbic acid and glycerophosphate. We also evaluated, under molecular and morphological patterns, the differentiation of pre-osteoblasts into osteocyte-like cells induced by 3D scaffold (matrigel as substrate). Interestingly, under this condition, the cells produced sonic hedgehog (Shh), which also was essential for stimulating the differentiation signaling pathway. Kinomic profiling of these cells revealed a prevalence of kinases involved in cellular communication, which is in agreement with the overexpression of connexin 43 observed. Besides we observed that RECK and TIMP-1 modulated the extracellular matrix rearrangement and that PP2A and p38 MAPK are required for osteoblasts adhesion. In addition, we observed that during pre-osteoblasts differentiation both PTPs and cellular redox are tightly regulated. Our findings demonstrated that the signal transduction evaluation can provide important information for understanding the cell biology as well as defining molecular targets that can be useful for different applicationsDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Expanding the role of Src and protein-tyrosine phosphatases balance in modulating osteoblast metabolism : lessons from mice

6 p. : il.The widespread nature of protein phosphorylation/dephosphorylation underscores its key role in cell signaling metabolism, growth and differentiation. Tyrosine phosphorylation of cytoplasmic proteins is a critical event in the regulation of intracellular signaling pathways activated by external stimuli. An adequate balance in protein phosphorylation is a major factor in the regulation of osteoclast and osteoblast activities involved in bone metabolism. However, although phosphorylation is widely recognized as an important regulatory pathway in skeletal development and maintenance, the mechanisms involved are not fully understood. Among the putative protein-tyrosine kinases (ptk) and protein-tyrosine phosphatases (ptp) involved in this phenomenon there is increasing evidence that Src and low molecular weight-ptps play a central role in a range of osteoblast activities, from adhesion to differentiation. A role for Src in bone metabolism was first demonstrated in Src-deficient mice and has since been confirmed using low molecular weight Src inhibitors in animal models of osteoporosis. Several studies have shown that Src is important for cellular proliferation, adhesion and motility. In contrast, few studies have assessed the importance of the ptk/ptp balance in driving osteoblast metabolism. In this review, we summarize our current knowledge of the functional importance of the ptk/ptp balance in osteoblast metabolism, and highlight directions for future research that should improve our understanding of these critical signaling molecules