Modifications on shear stress‐based mechanical forces are associated with pathophysiological susceptibility and their effect on endothelial cells (EC) needs to be better addressed looking for ...comprehending the cellular and molecular mechanisms. This prompted us to better evaluate the effects of shear stress in human primary venous EC obtained from the umbilical cord, using an in vitro model to mimic the laminar blood flow, reaching an intensity 1–4 Pa. First, our data shows there is a significant up‐expression of phosphatidylinositol 3‐kinase (PI3K) in shear‐stressed cells culminating downstream with an up‐phosphorylation of AKT and up‐expression of MAPK‐ERK, concomitant to a dynamic cytoskeleton rearrangement upon integrin subunits (α4 and ß 3) requirements. Importantly, the results show there is significant involvement of nitric oxide synthase (eNOS), nNOS, and vascular endothelial growth factors receptor 2 (VEGFR2) in shear‐stressed EC, while cell cycle‐related events seem to being changed. Additionally, although diminution of 5‐hydroxymethylcytosine in shear‐stressed EC, suggesting a global repression of genes transcription, the promoters of PI3K and eNOS genes were significantly hydroxymethylated corroborating with their respective transcriptional profiles. Finally, to better address, the pivotal role of PI3K in shear‐stressed EC we have revisited these biological issues by wortmannin targeting PI3K signaling and the data shows a dependency of PI3K signaling in controlling the expression of VGFR1, VGFR2, VEGF, and eNOS, once these genes were significantly suppressed in the presence of the inhibitor, as well as transcripts from Ki67 and CDK2 genes. Finally, our data still shows a coupling between PI3K and the epigenetic landscape of shear‐stressed cells, once wortmannin promotes a significant suppression of ten‐11 translocation 1 (TET1), TET2, and TET3 genes, evidencing that PI3K signaling is a necessary upstream pathway to modulate TET‐related genes. In this study we determined the major mechanotransduction pathway by which blood flow driven shear stress activates PI3K which plays a pivotal role on guaranteeing endothelial cell phenotype and vascular homeostasis, opening novel perspectives to understand the molecular basis of pathophysiological disorders related with the vascular system.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Obesity is a worldwide health problem and is directly associated with insulin resistance and type 2 diabetes. The liver is an important organ for the control of healthy glycemic levels, since insulin ...resistance in this organ reduces phosphorylation of forkhead box protein 1 (FOXO1) protein, leading to higher hepatic glucose production (HGP) and fasting hyperglycemia. Aerobic physical training is known as an important strategy in increasing the insulin action in the liver by increasing FOXO1 phosphorylation and reducing gluconeogenesis. However, little is known about the effects of strength training in this context. This study aimed to investigate the effects of short‐term strength training on hepatic insulin sensitivity and glycogen synthase kinase‐3β (GSK3β) and FOXO1 phosphorylation in obese (OB) mice. To achieve this goal, OB Swiss mice performed the strength training protocol (one daily session for 15 days). Short‐term strength training increased the phosphorylation of protein kinase B and GSK3β in the liver after insulin stimulus and improved the control of HGP during the pyruvate tolerance test. On the other hand, sedentary OB animals reduced FOXO1 phosphorylation and increased the levels of nuclear FOXO1 in the liver, increasing the phosphoenolpyruvate carboxykinase (PEPCK) and glucose‐6‐phosphatase (G6Pase) content. The bioinformatics analysis also showed positive correlations between hepatic FOXO1 levels and gluconeogenic genes, reinforcing our findings. However, strength‐trained animals reverted to this scenario, regardless of body adiposity changes. In conclusion, short‐term strength training is an efficient strategy to enhance the insulin action in the liver of OB mice, contributing to glycemic control by reducing the activity of hepatic FOXO1 and lowering PEPCK and G6Pase contents.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
It is known that cellular events underlying the processes of bone maintenance, remodeling, and repair have their basis in the embryonic production of bone. Shh signaling is widely described ...developing important morphogenetic control in bone by modifying the activity of osteoblast. Furthermore, identifying whether it is associated with the modulation of nuclear control is very important to be the basis for further applications. Experimentally, osteoblasts were exposed with cyclopamine (CICLOP) considering up to 1 day and 7 days, here considered an acute and chronic responses respectively. Firstly, we have validated the osteogenic model in vitro by exposing the osteoblasts to classical differentiating solution up to 7 days to allow the analysis of alkaline phosphatase and mineralization. Conversely, our data shows that differentiating osteoblasts present higher activity of inflammasome-related genes, while Shh signaling members were lower, suggesting a negative feedback between them. Thereafter, to better know about the role of Shh signaling on this manner, functional assays using CICLOP (5 μM) were performed and the data validates the previously hypothesis that Shh represses inflammasome related genes activities. Altogether, our data supports the anti-inflammatory effect of Shh signaling by suppressing Tnfα, Tgfβ and inflammasome related genes during osteoblast differentiation, and this comprehension might support the understanding the molecular and cellular mechanisms related in bone regeneration by reporting molecular-related osteoblast differentiation.
•Osteoblast under differentiation stimulus requires inflammasome-related gene activation.•Shh signaling contributes with the repression of TGFβ gene activation in osteoblasts.•Cyclopamine targeting hedgehog modulates nuclear control of the osteoblast activity.•Differentiating osteoblasts present higher activity of inflammasome-related genes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The biological response to zirconia (ZrO
2
) is not completely understood, which prompted us to address its effect on pre-osteoblastic cells in both direct and indirect manner. Our results showed ...that zirconia triggers important intracellular signaling mainly by governing survival signals which leads to cell adhesion and proliferation by modulating signaling cascade responsible for dynamic cytoskeleton rearrangement, as observed by fluorescence microscopy. The phosphorylations of Focal Adhesion Kinase (FAK) and Rac1 decreased in response to ZrO
2
enriched medium. This corroborates the result of the crystal violet assay, which indicated a significant decrease of pre-osteoblast adhesion in responding to ZrO
2
enriched medium. However, we credit this decrease on pre-osteoblast adhesion to the need to govern intracellular repertory of intracellular pathways involved with cell cycle progression, because we found a significant up-phosphorylation of Mitogen-Activated Protein Kinase (MAPK)-p38 and Cyclin-dependent kinase 2 (CDK2), while p15 (a cell cycle suppressor) decreased. Importantly, Protein phosphatase 2 A (PP2A) activity decreased, guaranteeing the significant up-phosphorylation of MAPK -p38 in response to ZrO
2
enriched medium. Complementarily, there was a regulation of Matrix Metalloproteinases (MMPs) in response to Zirconia and this remodeling could affect cell phenotype by interfering on cell anchorage. Altogether, our results show a repertory of signaling molecules, which suggests that ECM remodel as a pre-requisite to pre-osteoblast phenotype by affecting their anchoring in responding to zirconia.
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FZAB, GEOZS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
We hypothesized that a crosstalk between osteoblast and fibroblast (FB) exists, which contributes to bone as a dynamic tissue. Cell-free supernatants were harvested from fibroblast cultures and later ...subject pre-osteoblasts to investigate there capacity to modulate cell viability and differentiation mechanisms, reporting the possible involvement of Shh signaling as a paracrine mechanism. By exploring immunoblotting technology, we have shown that FB-released factors interfere with osteoblast metabolism by up-regulating the phosphorylation of FAK and Rac-1 proteins at the early stage and later contribute to osteoblast differentiation by up-modulating alkaline phosphatase (ALP) and in vitro mineralization. We also found that Shh signaling was not required during osteoblastic differentiation promoted by the FB-released factors as well as MAPK-ERK phosphorylation, while pre-osteoblast cultures subjected to osteogenic medium (O.M.) require downstream transducers of Shh, such as Patched and Gli-1, and MAPK-ERK. Altogether, our results indicate for the first time a possible mechanism involved in the crosstalk between fibroblasts and osteoblasts, as it was possible to observe trophic factors released by fibroblasts interfering decisively in osteoblast metabolism in a Shh-independent manner. This study collaborates the body of work that indicates paracrine signaling molecules participate in the crosstalk among bone-resident cells and explains, at least partially, the biological mechanisms responsible for bone tissue dynamism, opening new avenues to understand etiologies of bone diseases.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Titanium is widely used for biomedical applications, but little information is being delivered regarding the cellular/molecular mechanisms explaining their efficacy, mainly considering the effects of ...the Ti-released trace elements on pre-osteoblasts. We addressed this issue by investigating decisive intracellular signal transduction able to modulate cytoskeleton rearrangement, proliferative phenotype and extracellular matrix (ECM) remodeling. We considered titanium grades IV and V, submitted or not to dual acid-etching (w/DAE or wo/DAE, respectively). Our results showed there is no cytotoxicity, preserving AKT involvement. Additionally, Ti-enriched medium promoted a diminution of the downstream signaling upon integrin activation (phosphorylating Rac1 and cofilin), guaranteeing a dynamic cytoskeleton rearrangement. Moreover, the low profile of ECM remodeling obtained in response to trace molecules released by Ti-based devices seems contributing to the osteoblast performance in mediating extracellular support to cell anchorage. This hypothesis was validated by the up-expression of ß1-integrin, src and Focal adhesion kinase (fak) genes, mainly in response to titanium grade V. Proliferative phenotype showed an unbalance between cyclin-dependent kinases (CDKs) and p15INK4b/p21Cip1. In conjunction, we showed for the first time that trace elements from Ti-based biomedical devices provoke important modulation of the osteoblast biology, driving cell anchoring, viability, and proliferative phenotype. Certainly, these biological outcomes compromise implant osseointegration.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Angiogenesis is a relevant mechanism to be considered for the success of bone healing, even considering endosseous implantable devices, providing adequate delivery of substances necessaries for the ...cell viability and bone de novo deposition. Within of the repertory of metal-based implantable alloys, cobalt-chromium (CoCr) has emerged with very interesting properties for biomedical applications. Additionally, we have shown that released molecules from implants devices are able to modulate cells away and because that we hypothesized these released molecules might act on endothelial cells. In order to better address this issue, we investigated the effect of Co-Cr-enriched medium on endothelial cells (HUVECs), considering a biological model subjecting those cells to shear-stress to partially mimic the physiological environment and further allow investigating intracellular pathways responsible to drive cytoskeletal rearrangement, cell viability and extracellular matrix (ECM) remodeling processes. Considering the analysis of the metalloproteinases (MMPs) activities, our data indicates an intense ECM remodeling in response to CoCr-enriched medium suggesting some role on angiogenesis once ECM remodeling is prerequisite to cell growth. This was better addressed by revealing its involvement on modifying both mRNA expression and protein levels of members of the MAPK family. Additionally, the expression of CDK4 gene was modulated within the cell response to Co-Cr-enriched medium, while the modulation in the expression of P15 and P21 indicates an important regulatory mechanism required. Overall, our results demonstrate that trace of CoCr elements triggers decisive intracellular signaling in shear-stressed endothelial cells, suggesting influence on angiogenesis-related mechanism and they bring novel insights to explain the biological activity of CoCr as it has been emerged as interesting biomedical materials within the medical and dentistry fields.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Cobalt-chromium (CoCr)-based alloys have emerged as an interesting biomaterial within biomedical field, mainly considering their biocompatibility, resistance to corrosion and absence of magnetism; ...however, its effect on cell metabolism is barely known and this prompted us better evaluating whether CoCr-enriched medium affects the metabolism of both osteoblast and endothelial cells, and also if there is a coupling between them. This is also considered here the already-known effect of Cobalt (Co) as a hypoxic element. Firstly, discs of CoCr subjecting (W) or not (Wo) to dual acid-etched (DAE) were incubated into FBS-free cell culture medium up to 24 h (37 °C). This CoCr-enriched medium was further used to treat shear-stressed endothelial cells cultures up to 72 h. Thereafter, the conditioned medium containing metabolites of shear-stressed endothelial cells in response to CoCr-enriched medium was further used to subject osteoblast's cultures, when the samples were properly harvested to allow the analysis of the molecular issues. Our data shows that CoCr-enriched medium contains 1.5 ng–2.0 ng/mL of Co, which was captured by endothelial cells and osteoblasts in about 30% in amount and it seems modulate their metabolic pathways: shear-stressed endothelial cells expressed higher profile of HIF1α, VEGF and nNOS genes, while their global profile of protein carbonylation was lower than the control cultures, suggesting lower oxidative stress commitment. Additionally, osteoblasts responding to metabolites of CoCr-challenged endothelial cells show dynamic expression of marker genes in osteogenic differentiation, with alkaline phosphatase (ALP), osteocalcin, and bone sialoprotein (BSP) genes being significantly increased. Additionally, tensional shear-stress forces decrease the stimulus for ColA1gene expression in osteoblasts responding to endothelial cells metabolites, as well as modifying the extracellular matrix remodeling related genes. Analyzing the activities of matrix metalloproteinases (MMPs), the data shows that shear-stressed endothelial cells metabolites increase the activities of both MMP9 and MMP2 in osteoblasts. Altogether, our data shows for the first time that shear-stressed endothelial metabolites responding to CoCr discs contribute to osteogenic phenotype in vitro, and this predicts an active crosstalk between angiogenesis and osteogenesis during osseointegration of CoCr alloy and bone healing, maybe guided by the Co-induced hypoxic condition.
•Metabolic molecules released by endothelial cell responding to CoCr effects osteoblast activity.•Endothelial gene biomarkers are up-expressed in HUVECs responding to CoCr.•Pivotal gene markers of osteoblast differentiation were modulated by metabolic molecules released by endothelial cell.•ECM remodeling stimulus is affected by the metabolic effects of CoCr-enriched medium on osteoblasts.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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