Role of hyaluronan in human adipogenesis : evidence from in-vitro and in-vivo studies

Hyaluronan (HA), an extra-cellular matrix glycosaminoglycan, may play a role in mesenchymal stem cell differentiation to fat but results using murine models and cell lines are conflicting. Our previous data, illustrating decreased HA production during human adipogenesis, suggested an inhibitory role. We have investigated the role of HA in adipogenesis and fat accumulation using human primary subcutaneous preadipocyte/fibroblasts (PFs, n = 12) and subjects of varying body mass index (BMI). The impact of HA on peroxisome proliferator-activated receptor gamma (PPARγ) expression was analysed following siRNA knockdown or HA synthase (HAS)1 and HAS2 overexpression. PFs were cultured in complete or adipogenic medium (ADM) with/without 4-methylumbelliferone (4-MU = HA synthesis inhibitor). Adipogenesis was evaluated using oil red O (ORO), counting adipogenic foci, and measurement of a terminal differentiation marker. Modulating HA production by HAS2 knockdown or overexpression increased (16%, p < 0.04) or decreased (30%, p = 0.01) PPARγ transcripts respectively. The inhibition of HA by 4-MU significantly enhanced ADM-induced adipogenesis with 1.52 ± 0.18- (ORO), 4.09 ± 0.63- (foci) and 2.6 ± 0.21-(marker)-fold increases compared with the controls, also increased PPARγ protein expression (40%, (p < 0.04)). In human subjects, circulating HA correlated negatively with BMI and triglycerides (r = −0.396 (p = 0.002), r = −0.269 (p = 0.038), respectively), confirming an inhibitory role of HA in human adipogenesis. Thus, enhancing HA action may provide a therapeutic target in obesity

Hyaluronan in the Cancer Cells Microenvironment

The presence of the glycosaminoglycan hyaluronan in the extracellular matrix of tissues is the result of the cooperative synthesis of several resident cells, that is, macrophages and tumor and stromal cells. Any change in hyaluronan concentration or dimension leads to a modification in stiffness and cellular response through receptors on the plasma membrane. Hyaluronan has an effect on all cancer cell behaviors, such as evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and metastasis. It is noteworthy that hyaluronan metabolism can be dramatically altered by growth factors and matrikines during inflammation, as well as by the metabolic homeostasis of cells. The regulation of HA deposition and its dimensions are pivotal for tumor progression and cancer patient prognosis. Nevertheless, because of all the factors involved, modulating hyaluronan metabolism could be tough. Several commercial drugs have already been described as potential or effective modulators; however, deeper investigations are needed to study their possible side effects. Moreover, other matrix molecules could be identified and targeted as upstream regulators of synthetic or degrading enzymes. Finally, co-cultures of cancer, fibroblasts, and immune cells could reveal potential new targets among secreted factors

Treatment of bone defects with 1% hyaluronic acid gel in normal and diabetic animals

Orientador: Antonio Wilson SallumTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de PiracicabaResumo: O ácido hialurônico (HA) é um importante componente da matriz extracelular e desempenha importante papel na cicatrização. Além de sua função durante o processo cicatricial ele também atua na homeostasia dos tecidos e no reparo ósseo. Devido as suas características propomos este estudo com o objetivo de avaliar o efeito do tratamento de defeitos ósseos com ácido hialurônico em ratos normais e diabéticos. Para este trabalho foram utilizados 64 ratos Wistar machos, sendo que 32 destes animais tiveram um quadro de diabetes induzida por meio de injeção única intraperitoneal de estreptozotocina (STZ) (60mg/Kg). Os animais somente foram considerados diabéticos quando o nível de glicose no sangue ultrapassasse 250mg/dL. Dois defeitos de tamanho crítico, de 5mm de diâmetro, foram confeccionados na calota dos animais e os tratamentos 1) gel de HA a 1%; 2) gel de HA a 1% associado a esponja de colágeno absorvível (ACS); 3) ACS; 4) controle (coágulo sanguíneo) foram aleatoreamente distribuídos entre os defeitos. Após 60 dias do procedimento cirúrgico os animais foram sacrificados e os especimes passaram por processamento histológico para posterior avaliação histométrica. Análise histométrica foi realizada tomando duas medidas lineares nos defeitos: tamanho inicial e tamanho final do defeito. A quantidade de preenchimento do defeito foi obtida pela diferença entre os tamanhos inicial e final do defeito e os dados obtidos analisados estatisticamente. Para análise dos dados foram utilizados o teste t de Student para comparação entres os pesos inicial e final dos animais. O teste ANOVA comparou os níveis glicêmicos dos animais diabéticos antes e após a administração de STZ e no sacrifício, e o teste de Tukey foi utilizado para detectar as diferenças significativas. Para comparação dos tratamentos o teste ANOVA one-way foi usado com o teste de Bonferroni post hoc. O nível de significânica foi estabelecido a 5%. Os animais apresentaram ganho de peso significativo (p0.05). Dentro dos limites deste estudos pode-se concluir que o HA pode ser utilizado como adjunto no tratamento de defeitos ósseos.Abstract: Hyaluronic acid (HA) is an important component of extracellular matrix and has an important role in wound healing. Besides its role in wound healing it also participates in tissue hemostasis e bone repair. Due to these characteristics this study was suggested with the aim of evaluating the effects of HA treatment of bone defects in normal and diabetic animals. Sixty-four male Wistar rats were used in this study, and 32 of these animals underwent diabetes induction by a single intraperitoneal injection of streptozotocin (STZ) (60mg/Kg). The animals were considered diabetic only if their glucose levels were higher that 250mg/dL. Two 5mm round defects were created in the calvaria of the animals and four treatments were randomly distributed: 1) 1% HA gel, 2) 1% HA gel soaked absorbable collagen sponge (ACS), 3) ACS and 4) control (blood clot). Sixty days post-surgery the animals were sacrificed and the specimens processed for histometric analysis. Histometric analyses were performed and 2 measurements were taken: initial and final sizes of the defect. The amount of bone fill was calculated as the difference between the initial and final sizes of the defects and the data statistically analyzed. For statistical analysis Student t test was used to compare initial and final body weights. One-way ANOVA test compared glucose levels between diabetic animals before and after STZ injections and at sacrifice, Tukey test was used to identify significant differences. Comparisons between treatments were performed by one-way ANOVA and Bonferroni post hoc. Significance level was set at 5%. The animals had a significant increase in body weight (p0.05), though. Within the limits of this study it can be concluded that HA can be used as an adjunct in the treatment of bone defects.DoutoradoPeriodontiaDoutor em Clínica Odontológic

Inflammatory Cascades in the Pathogenesis of Multiple Sclerosis Lesions

Multiple sclerosis (MS) is a disease of the human central nervous system (CNS) characterised by inflammation and demyelination. Initially the MS lesion has a distinct histopathological picture with myelin-positive microglia in the midst of apparently intact myelin but minimal perivascular inflammation. Inflammatory mediators produced by these activated microglia may precipitate the infiltration of mononuclear cells and the overt myelin loss seen in actively demyelinating MS lesions. Nuclear factor-κB (NF-κB) is a transcriptional regulator of proteolytic enzymes, adhesion molecules and inflammatory cytokines which rapidly translates extracellular signals into protein synthesis. The immunocytochemical detection of the transcriptionally active form of NF-κB, but not the inhibitory protein IκBα, in the nuclei of microglia in normal human CNS white matter indicates the capability of microglia to respond rapidly to pathological stimuli in the CNS. Activation of NF-κB in MS plaques, evident from the nuclear localisation of the NF-κB subunits RelA, c-Rel and p50 in macrophages, may propagate inflammatory demyelination through upregulation of NF-κB-controlled macrophage genes for inflammatory mediators. In demyelinating disease the plasmin-matrix metalloprotease (MMP) enzymatic cascade promotes blood-brain barrier (BBB) damage, generation of encephalitogenic myelin peptides and activation of pro-inflammatory cytokines. Constitutive expression of MMPs 1, 2, 3 and 9 in glial cells in normal control white matter was demonstrated by immunocytochemistry. However, the lack of tissue (t-PA) and urokinase (u-PA) plasminogen activators in glial cells and the absence of caseinolytic activity as shown by in situ zymography emphasises the latent nature of the plasmin-MMP cascade in normal CNS tissue. In contrast, the co-localisation of t-PA and u-PA, rate-limiting serine-proteases, and MMPs in macrophages and astrocytes in active MS lesions forms the basis of a functional enzymatic cascade. Furthermore, increased amounts and activity of u-PA and MMP-9 in homogenates of active MS plaques coupled with the presence of caseinolytic activity in foamy macrophages implicates these cells as the major source of MMPs, which cause proteolytic damage in MS. Insulin-like growth factors (IGFs) play an important role in development and myelination in the CNS but can also stimulate phagocytosis and production of inflammatory mediators by macrophages. In active MS lesions binding of IGF-II to the IGF receptor on foamy macrophages may induce mitogenic responses and invasiveness of macrophages which can be further enhanced by MMP-mediated proteolytic removal of inhibitory IGF-binding proteins. Similarly, the potent mitogens IGF-I and insulin may stimulate astrocytosis and gliosis. In contrast, oligodendrocytes in normal-appearing white matter do not express IGFs or IGF-I receptor which implies that the oligodendrocyte response to these remyelinating growth factors is impaired. Therefore, the prevailing role of IGFs in MS lesions may be in line with pro-inflammatory mediators promoting macrophage and astrocyte responses to tissue damage. In conclusion, NF-κB activation in microglia and macrophages upregulates the production of PAs and inflammatory cytokines which trigger the plasmin-MMP cascade, leading to BBB damage and enhanced inflammatory cell migration and demyelination in white matter. Influx of IGFs through the damaged BBB and their increased local production may promote myelin phagocytosis and reactive astrocytosis. In turn IGF-mediated upregulation of PAs in glial cells could provide a feedback amplification of the MMP cascade. Therefore, the findings from these studies bring together three systems of mediators, NF-κB, MMPS and IGFs, into a hypothetical model for the propagation of demyelination in MS lesions

The effect of a hyaluronic acid nasal pack, and insulin-like growth factor 1, on mucosal healing after endoscopic

Thesis (M.S.)--University of Adelaide, Dept. of Surgery, 200

Role of macrophage colony stimulating factor-1 (CSF-1) in postnatal growth

Colony-Stimulating Factor (CSF-1) is required for the proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Mice with a mutation in their CSF-1 gene demonstrate abnormal development in many organ systems and severe growth retardation. These defects can be corrected by administration of rh- CSF-1, and when similarly administered to wild-type mice, can increase organ and body weight, thus highlighting the importance of CSF-1 in postnatal growth. CSF-1 is known to be elevated in the circulation in the immediate postnatal period of both mice and humans. It remains to be seen whether CSF-1 deficiency underlies important clinical issues such as low birth weight, and whether there are any functionally important variations in expression or biology of CSF-1, or the alternative CSF-1R ligand IL-34 that contributes to variation in somatic growth between individuals. This thesis aimed to use the pig as a model for human innate immunity and disease based upon recent publications that highlighted the similarities in their immune systems. To investigate the effects of CSF-1 on postnatal growth the first aim was to characterise the CSF-1R system in pigs and produce reagents. Biologically active porcine CSF-1 and IL-34 were produced along with expression of full length functional porcine CSF-1R and production of anti-CSF-1R antibodies. A bioassay was developed and optimised to assess the biological activity of these proteins. The cross-species reactivity of a range of species CSF-1 and IL-34 proteins was investigated in-vitro using the bioassay and cell culture systems. Recombinant CSF-1 is known to have a short half-life. Since conjugation of proteins to the Fc region of immunoglobulins has been used extensively to improve circulating half-life; a porcine Fc CSF-1 fusion protein was generated by commercial partners, Pfizer Animal Health. The conjugated and un-conjugated CSF-1 proteins had identical activity in cell line and primary cell assays in-vitro. The in-vivo activity of porcine Fc CSF-1 was tested initially in the Csf1r-EGFP+ mouse reporter line and C57BL/6 mice. The Fc CSF-1 protein was more active than the native protein in promoting increased monocyte and tissue macrophage numbers, increasing body weight and inducing hepatosplenomegaly. Hepatic growth was associated with extensive macrophage infiltration and hepatocyte proliferation, identified by gene expression profiling as well as immunohistochemistry. Fc CSF-1 was then tested in neonatal pigs. They were found to have an immature immune system that develops with age. No postnatal surge of CSF-1 was detected. Fc CSF-1 administration increased blood monocyte and neutrophil numbers confirming that CSF-1 is not saturating at this time. Nevertheless, no influence on postnatal growth rate was identified. This is discussed in terms of the differences in placental architecture in the pig compared to human and mouse. This thesis demonstrates the effectiveness of porcine Fc CSF-1 in both mice and porcine and highlights the important role that CSF-1 and macrophages play in liver homeostasis. Fc CSF-1 is identified as candidate therapeutic agent in humans and companion animals for tissue regeneration, and a tool for the study of the role of macrophages in physiology and pathology