BMP7-induced osteoblast differentiation requires hedgehog signaling and involves nuclear mechanisms of gene expression control.

During the morphological changes occurring in osteoblast differentiation, Sonic hedgehog (Shh) plays a crucial role. While some progress has been made in understanding this process, the epigenetic mechanisms governing the expression of Hh signaling members in response to bone morphogenetic protein 7 (BMP7) signaling in osteoblasts remain poorly understood. To delve deeper into this issue, we treated pre-osteoblasts (pObs) with 100 ng/mL of BMP7 for up to 21 days. Initially, we validated the osteogenic phenotype by confirming elevated expression of well-defined gene biomarkers, including Runx2, Osterix, Alkaline Phosphatase (Alp), and bone sialoprotein (Bsp). Simultaneously, Hh signaling-related members Sonic (Shh), Indian (Ihh), and Desert (Dhh) Hedgehog (Hh) exhibited nuanced modulation over the 21 days in vitro period. Subsequently, we evaluated epigenetic markers, and our data revealed a notable change in the CpG methylation profile, considering the methylation/hydroxymethylation ratio. CpG methylation is a reversible process regulated by DNA methyltransferases and demethylases, including Ten-eleven translocation (Tets), which also exhibited changes during the acquisition of the osteogenic phenotype. Specifically, we measured the methylation pattern of Shh-related genes and demonstrated a positive Pearson correlation for GLI Family Zinc Finger 1 (Gli1) and Patched (Ptch1). This data underscores the significance of the epigenetic machinery in modulating the BMP7-induced osteogenic phenotype by influencing the activity of Shh-related genes. In conclusion, this study highlights the positive impact of epigenetic control on the expression of genes related to hedgehog signaling during the morphogenetic changes induced by BMP7 signaling in osteoblasts.

Epigenetic changes in shear-stressed endothelial cells.

Epigenetic changes, particularly histone compaction modifications, have emerged as critical regulators in the epigenetic pathway driving endothelial cell phenotype under constant exposure to laminar forces induced by blood flow. However, the underlying epigenetic mechanisms governing endothelial cell behavior in this context remain poorly understood. To address this knowledge gap, we conducted in vitro experiments using human umbilical vein endothelial cells subjected to various tensional forces simulating pathophysiological blood flow shear stress conditions, ranging from normotensive to hypertensive forces. Our study uncovers a noteworthy observation wherein endothelial cells exposed to high shear stress demonstrate a decrease in the epigenetic marks H3K4ac and H3K27ac, accompanied by significant alterations in the levels of HDAC (histone deacetylase) proteins. Moreover, we demonstrate a negative regulatory effect of increased shear stress on HOXA13 gene expression and a concomitant increase in the expression of the long noncoding RNA, HOTTIP, suggesting a direct association with the suppression of HOXA13. Collectively, these findings represent the first evidence of the role of histone-related epigenetic modifications in modulating chromatin compaction during mechanosignaling of endothelial cells in response to elevated shear stress forces. Additionally, our results highlight the importance of understanding the physiological role of HOXA13 in vascular biology and hypertensive patients, emphasizing the potential for developing small molecules to modulate its activity. These findings warrant further preclinical investigations and open new avenues for therapeutic interventions targeting epigenetic mechanisms in hypertensive conditions.

JARID1B represses the osteogenic potential of human periodontal ligament mesenchymal cells.

BACKGROUND: Here, we evaluated whether the histone lysine demethylase 5B (JARID1B), is involved in osteogenic phenotype commitment of periodontal ligament cells (PDLCs), by considering their heterogeneity for osteoblast differentiation. MATERIALS AND METHODS: Epigenetic, transcriptional, and protein levels of a gene set, involved in the osteogenesis, were investigated by performing genome-wide DNA (hydroxy)methylation, mRNA expression, and western blotting analysis at basal (without osteogenic induction), and at the 3rd and 10th days of osteogenic stimulus, in vitro, using PDLCs with low (l) and high (h) osteogenic potential as biological models. RESULTS: h-PDLCs showed reduced levels of JARID1B, compared to l-PDLCs, with significant inversely proportional correlations between RUNX2 and RUNX2/p57. Epigenetically, a significant reduction in the global H3K4me3 content was observed only in h-PDLCs. Immunoblotting data reveal a significant reduction in the global H3K4me3 content, at 3 days of induction only in h-PDLCs, while an increase in the global H3K4me3 content was observed at 10 days for both PDLCs. Additionally, positive correlations were found between global H3K4me3 levels and JARID1B gene expression. CONCLUSIONS: Altogether, our results show the crucial role of JARID1B in repressing PDLCs osteogenic phenotype and this claims to pre-clinical protocols proposing JARID1B as a potential therapeutic target.

Non-coding RNAs repressive role in post-transcriptional processing of RUNX2 during the acquisition of the osteogenic phenotype of periodontal ligament mesenchymal stem cells.

Mesenchymal stem cells are candidates for therapeutic strategies in periodontal repair due to their osteogenic potential. In this study, we identified epigenetic markers during osteogenic differentiation, taking advantage of the individual pattern of mesenchymal cells of the periodontal ligament with high (h-PDLCs) and low (l-PDLCs) osteogenic capacity. We found that the involvement of non-coding RNAs in the regulation of the RUNX2 gene is strongly associated with high osteogenic potential. Moreover, we evaluated miRs and genes that encode enzymes to process miRs and their biogenesis. Our data show the high expression of the XPO5 gene, and miRs 7 and 22 observed in the l-PDLCs might be involved in acquiring osteogenic potential, suppressing RUNX2 gene expression. Further, an inversely proportional correlation between lncRNAs (HOTAIR and HOTTIP) and RUNX2 gene expression was observed in both l- and h-PDLCs, and it was also related to the distinct osteogenic phenotypes. Thus, our results indicate the low expression of XPO5 in h-PDLC might be the limiting point for blocking the miRs biogenesis, allowing the high gene expression of RUNX2. In accordance, the low expression of miRs, HOTAIR, and HOTTIP could be a prerequisite for increased osteogenic potential in h-PDLCs. These results will help us to better understand the underlying mechanisms of osteogenesis, considering the heterogeneity in the osteogenic potential of PDLCs that might be related to a distinct transcriptional profile of lncRNAs and the biogenesis machinery.

LncRNA HOTAIR is a novel endothelial mechanosensitive gene.

To better address whether the long noncoding RNAs (lncRNAs) HOTAIR and HOTTIP are mechanosensitive genes, they were investigated in differentially challenged endothelial cells with respect to a circuit of tensional forces, considering the performance of both arterial and venous endothelial cells. We subjected arterial- and venous-obtained endothelial cells to a circuit of tensional forces within a shear stress model in vitro. Real-time quantitative polymerase chain reaction analysis indicated that microRNA (miRNA)-related processing machinery is significantly required in shear stressed arterial endothelial cell metabolism, which orchestrates miRNA (small noncoding RNA) involvement, and their involvement suggests lncRNA involvement. Of lncRNAs HOTAIR and HOTTIP, only HOTAIR was mechanosensitive considering both arterial and venous endothelial cells, presenting a positive correlation between methylation signature and gene expression. Thereafter, using bioinformatics tools, lncRNA HOTAIR was predicted to modulate miRNA185, miRNA-21, and miRNA23b downregulation. We compared the values of gene expression with a Pearson's correlation test, and expected correlations were observed for miRNA185 (r = 0.8664), miRNA-21 (r = 0.8605), and miRNA23b (0.9128). Taken together, these findings clearly show that lncRNA HOTAIR responds to the shear stress and emerges as a novel mechanosensitive gene in endothelial cells. Altogether, this understanding of mechanosensitive transcriptional and posttranscriptional control involving HOTAIR can also lead to new forms of therapeutic intervention for various diseases, as well as new strategies for tissue engineering and regenerative medicine.

Wortmannin targeting phosphatidylinositol 3-kinase suppresses angiogenic factors in shear-stressed endothelial cells.

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.

HOXA9 mediates and marks premalignant compartment size expansion in colonic adenomas.

The transformation of normal colonic epithelium to colorectal cancer (CRC) involves a relatively ordered progression, and understanding the molecular alterations involved may aid rational design of strategies aimed at preventing or counteracting disease. Homeobox A9 (HOXA9) is an oncogene in leukemia and has been implicated in CRC pathology, although its role in disease etiology remains obscure at best. We observe that HOXA9 expression is increased in colonic adenomas compared with location-matched healthy colon epithelium. Its forced expression results in dramatic genetic and signaling changes, with increased expression of growth factors IGF1 and FLT3, super-activity of the AKT survival pathway and a concomitant increase in compartment size. Furthermore, a reduced mRNA expression of the epithelial to mesenchymal transition marker N-cadherin as well as reduced activity of the actin cytoskeletal mediator PAK was seen, which is in apparent agreement with an observed reduced migratory response in HOXA9-overexpressing cells. Thus, HOXA9 appears closely linked with adenoma growth while impairing migration and metastasis and hence is both a marker and driver of premalignant polyp growth. Colonic polyps grow but remain premalignant for up to decades. Here, we show that HOXA9 drives growth in premalignant polyps, but simultaneously prevents further transformation.

Laminar shear stress-provoked cytoskeletal changes are mediated by epigenetic reprogramming of TIMP1 in human primary smooth muscle cells.

Whereas endothelial responses to shear stress are well-characterized, the cell physiological effects of shear stress in smooth muscle cells (SMCs) remain largely obscure. As SMCs are directly challenged by shear stress after endothelial denuding injury following procedures such as angioplasty or endarterectomy, characterization of these responses represents an important scientific question. Hence we decided to contrast cytoskeletal reorganization, epigenetic reprogramming, signaling transduction, and changes in miRNA (miRs) profiles in primary human aortic smooth muscle cells (AoSMCs) between unstressed cells and cells exposed to shear stress. We observed that shear stress-provoked reorganization of the actin cytoskeleton in an apparently Cofilin-dependent fashion and which related to altered integrin signaling, apparently caused by remodeling of the extracellular matrix. The latter appeared a downstream effect of increased expression of matrix metalloproteinases and downregulation of tissue metalloproteinase inhibitor 1 (TIMP1) protein levels. In turn, these effects related to shear stress-provoked changes in expression and nuclear localization of the epigenetic regulators demethylases TET1, TET2, DNMT1, DNMT3A and DNMT3B, HDAC6, and SIRT1. Accordingly, TIMP1 promotor CpG hypomethylation was a prominent effect, and resulted in a significant increase in TIMP1 transcription, which may also have related increased expression of miRs involved in modulating TIMP1 translation. Thus epigenetic-reprogramming of TIMP1 emerges as critical element in smooth muscle responses to mechanical signals and as epigenetic machinery is amendable to pharmacological manipulation, this pathway may have important clinical consequences.

Apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) contributes to osteoblast differentiation and osteogenesis.

The role of apoptosis-associated speck-like protein containing a caspase-1 recruitment domain (ASC) in bone healing remains to be understood. To address this issue, we investigated the requirement of inflammasome-related genes in response to bone morphogenetic protein 7 (BMP7)-induced osteoblast differentiation in vitro. To validate the importance of ASC on osteogenesis, we subjected wild-type (WT) and ASC knockout C57BL/6 mice (ASC KO) to tibia defect to evaluate the bone healing process (up to 28 days). Our in vitro data showed that there is an involvement of ASC during BMP7-induced osteoblast differentiation, concomitant to osteogenic biomarker expression. Indeed, primary osteogenic cells from ASC KO presented a lower osteogenic profile than those obtained from WT mice. To validate this hypothesis, we evaluated the bone healing process of tibia defects on both WT and ASC KO mice genotypes and the ASC KO mice were not able to fully heal tibia defects up to 28 days, whereas WT tibia defects presented a higher bone de novo volume at this stage, evidencing ASC as an important molecule during osteogenic phenotype. In addition, we have shown a higher involvement of runt-related transcription factor 2 in WT sections during bone repair, as well as circulating bone alkaline phosphatase isoform when both were compared with ASC KO mice behavior. Altogether, our results showed for the first time the involvement of inflammasome during osteoblast differentiation and osteogenesis, which opens new avenues to understand the pathways involved in bone healing.

On the Location of Fog Nodes in Fog-Cloud Infrastructures.

In the fog computing paradigm, fog nodes are placed on the network edge to meet end-user demands with low latency, providing the possibility of new applications. Although the role of the cloud remains unchanged, a new network infrastructure for fog nodes must be created. The design of such an infrastructure must consider user mobility, which causes variations in workload demand over time in different regions. Properly deciding on the location of fog nodes is important to reduce the costs associated with their deployment and maintenance. To meet these demands, this paper discusses the problem of locating fog nodes and proposes a solution which considers time-varying demands, with two classes of workload in terms of latency. The solution was modeled as a mixed-integer linear programming formulation with multiple criteria. An evaluation with real data showed that an improvement in end-user service can be obtained in conjunction with the minimization of the costs by deploying fewer servers in the infrastructure. Furthermore, results show that costs can be further reduced if a limited blocking of requests is tolerated.

Nano hydroxyapatite-blasted titanium surface affects pre-osteoblast morphology by modulating critical intracellular pathways.

Although, intracellular signaling pathways are proposed to predict the quality of cell-surface relationship, this study addressed pre-osteoblast behavior in response to nano hydroxyapatite (HA)-blasted titanium (Ti) surface by exploring critical intracellular pathways and pre-osteoblast morphological change. Physicochemical properties were evaluated by atomic force microscopy (AFM) and wettability considering water contact angle of three differently texturized Ti surfaces: Machined (Mac), Dual acid-etching (DAE), and nano hydroxyapatite-blasted (nHA). The results revealed critical differences in surface topography, impacting the water contact angle and later the osteoblast performance. In order to evaluate the effect of those topographical characteristics on biological responses, we have seeded pre-osteoblast cells on the Ti discs for up to 4 h and subjected the cultures to biological analysis. First, we have observed pre-osteoblasts morphological changes resulting from the interaction with the Ti texturized surfaces whereas the cells cultured on nHA presented a more advanced spreading process when compared with the cells cultured on the other surfaces. These results argued us for analyzing the molecular machinery and thus, we have shown that nHA promoted a lower Bax/Bcl2 ratio, suggesting an interesting anti-apoptotic effect, maybe explained by the fact that HA is a natural element present in bone composition. Thereafter, we investigated the potential effect of those surfaces on promoting pre-osteoblast adhesion and survival signaling by performing crystal violet and immunoblotting approaches, respectively. Our results showed that nHA promoted a higher pre-osteoblast adhesion supported by up-modulating FAK and Src activations, both signaling transducers involved during eukaryotic cell adhesion. Also, we have shown Ras-Erk stimulation by the all evaluated surfaces. Finally, we showed that all Ti-texturing surfaces were able to promote osteoblast differentiation up to 10 days, when alkaline phosphatase (ALP) activity and osteogenic transcription factors were up-modulated. Altogether, our results showed for the first time that nano hydroxyapatite-blasted titanium surface promotes crucial intracellular signaling network responsible for cell adapting on the Ti-surface.Biotechnol. Bioeng. 2017;114: 1888-1898. © 2017 Wiley Periodicals, Inc.

Fibroblast contributes for osteoblastic phenotype in a MAPK-ERK and sonic hedgehog signaling-independent manner.

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.

DNA methylation analysis of SOCS1, SOCS3, and LINE-1 in microdissected gingival tissue.

OBJECTIVES: DNA methylation plays a critical role in the regulation of the transcription of the suppressors of cytokine signaling (SOCS) 1 and SOCS3, which are modulators in the inflammation. We hypothesized that the methylation status of SOCS1, SOCS3, and long interspersed nuclear element (LINE)-1 in gingival tissues previously inflamed would be similar to that found in gingival tissues without clinical inflammation in the period studied. MATERIALS AND METHODS: Laser capture microdissection was performed to isolate epithelial and connective gingival tissues. The groups were comprised by ten patients without history of periodontitis and absence of clinical signs of inflammation in the gingiva during the study (healthy group) and ten patients with history of periodontitis, presenting inflammation in the gingival tissue at the first examination of the study (controlled chronic periodontitis group). The gingival biopsies from the controlled chronic periodontitis group were collected after controlling the inflammation. DNA methylation patterns were analyzed using methylation-specific high-resolution melting and combined bisulfite restriction analysis. RESULTS: DNA methylation levels for SOCS1 and SOCS3 did not differ between groups or tissues; likewise, no differences were observed in total LINE-1 methylation or at specific loci. CONCLUSION: At 3 months following control of inflammation in gingival tissues, the methylation profile of SOCS1, SOCS3, and LINE-1 is similar between connective and epithelial tissues from patients that were previously affected or not by chronic inflammation. CLINICAL RELEVANCE: Clinical results of a successful treatment are observed after inflammation control and the molecular findings illustrate local and general methylation patterns in recovering tissues toward health conditions and might help to understand events that are occurring in oral cells.

Adora2A downregulation promotes caffeine neuroprotective effect against LPS-induced neuroinflammation in the hippocampus.

Caffeine has been extensively studied in the context of CNS pathologies as many researchers have shown that consuming it reduces pro-inflammatory biomarkers, potentially delaying the progression of neurodegenerative pathologies. Several lines of evidence suggest that adenosine receptors, especially A1 and A2A receptors, are the main targets of its neuroprotective action. We found that caffeine pretreatment 15 min before LPS administration reduced the expression of Il1b in the hippocampus and striatum. The harmful modulation of caffeine-induced inflammatory response involved the downregulation of the expression of A2A receptors, especially in the hippocampus. Caffeine treatment alone promoted the downregulation of the adenosinergic receptor Adora2A; however, this promotion effect was reversed by LPS. Although administering caffeine increased the expression of the enzymes DNA methyltransferases 1 and 3A and decreased the expression of the demethylase enzyme Tet1, this effect was reversed by LPS in the hippocampus of mice that were administered Caffeine + LPS, relative to the basal condition; no significant differences were observed in the methylation status of the promoter regions of adenosine receptors. Finally, the bioinformatics analysis of the expanded network demonstrated the following results: the Adora2B gene connects the extended networks of the adenosine receptors Adora1 and Adora2A; the Mapk3 and Esr1 genes connect the extended Adora1 network; the Mapk4 and Arrb2 genes connect the extended Adora2A network with the extended network of the proinflammatory cytokine Il1ß. These results indicated that the anti-inflammatory effects of acute caffeine administration in the hippocampus may be mediated by a complex network of interdependencies between the Adora2B and Adora2A genes.

Analyzes In Silico Indicate the lncRNAs MIR31HG and LINC00939 as Possible Epigenetic Inhibitors of the Osteogenic Differentiation in PDLCs.

Chromatin conformation, DNA methylation pattern, transcriptional profile, and non-coding RNAs (ncRNAs) interactions constitute an epigenetic pattern that influences the cellular phenotypic commitment and impacts the clinical outcomes in regenerative therapies. Here, we investigated the epigenetic landscape of the SP7 transcriptor factor (SP7) and Distal-Less Homeobox 4 (DLX4) osteoblastic transcription factors (TFs), in human periodontal ligament mesenchymal cells (PDLCs) with low (l-PDLCs) and high (h-PDLCs) osteogenic potential. Chromatin accessibility (ATAC-seq), genome DNA methylation (Methylome), and RNA sequencing (RNA-seq) assays were performed in l- and h-PDLCs, cultured at 10 days in non-induced (DMEM) and osteogenic (OM) medium in vitro. Data were processed in HOMER, Genome Studio, and edgeR programs, and metadata was analyzed by online bioinformatics tools and in R and Python environments. ATAC-seq analyses showed the TFs genomic regions are more accessible in l-PDLCs than in h-PDLCs. In Methylome analyses, the TFs presented similar average methylation intensities (AMIs), without differently methylated probes (DMPs) between l- and h-PDLCs; in addition, there were no differences in the expression profiles of TFs signaling pathways. Interestingly, we identified the long non-coding RNAs (lncRNAs), MIR31HG and LINC00939, as upregulated in l-PDLCs, in both DMEM and OM. In the following analysis, the web-based prediction tool LncRRIsearch predicted RNA:RNA base-pairing interactions between SP7, DLX4, MIR31HG, and LINC00939 transcripts. The machine learning program TriplexFPP predicted DNA:RNA triplex-forming potential for the SP7 DNA site and for one of the LINC00939 transcripts (ENST00000502479). PCR data confirmed the upregulation of MIR31HG and LINC00939 transcripts in l-PDLCs (× h-PDLCs) in both DMEM and OM (p < 0.05); conversely, SP7 and DLX4 were downregulated, confirming those results observed in the RNA-Seq analysis. Together, these results indicate the lncRNAs MIR31HG and LINC00939 as possible epigenetic inhibitors of the osteogenic differentiation in PDLCs by (post)transcriptional and translational repression of the SP7 and DLX4 TFs.

MicroRNA biogenesis machinery activation and lncRNA and REST overexpression as neuroprotective responses to fight inflammation in the hippocampus.

Brain Long non-coding RNA (lncRNA) and microRNAs (miRs) play essential roles in the regulation of several important biological processes, including neuronal activity, cognitive processes, neurogenesis, angiogenesis, and neuroinflammation. In this context, the transcriptional repressor, RE1 silencing transcription factor (Rest), acts regulating the expression of neuronal genes as well as of lncRNAs and multiple miRNAs in the central nervous system. Nevertheless, its role in neuroinflammation was less explored. Here, we demonstrate, using an in vivo model of neuroinflammation induced by i.p. injection of LPS (0.33 mg/kg), that neuroinflammation increases gene expression of pro-inflammatory cytokines concomitant with the native and truncated forms of Rest and of non-coding RNAs. Additionally, the increased expression of enzymes Drosha ribonuclease III) (Drosha), Exportin 5 (Xpo5) and Endoribonuclease dicer (Dicer), associated with high expression of neuroprotective miRs 22 and 132 are indicative that the activation of biogenesis of miRs in the hippocampal region is a Central Nervous System (CNS) protective mechanism for the deleterious effects of neuroinflammation. Our results indicate that positive regulation of Rest gene expression in the hippocampal region by neuroinflammation correlates directly with the expression of miRs 22 and 132 and inversely with miR 335. In parallel, the confirmation of the possible alignment between the lncRNAs with miR 335 by bioinformatics corroborates with the sponge effect of Hottip and Hotair hybridizing and inhibiting the pro-inflammatory action of miR 335. This suggests the existence of a possible correlation between the activation of miR biogenesis machinery with increased expression of the transcription factor Rest, contributing to neuroprotection.

Anti-Inflammatory Effect of Caffeine on Muscle under Lipopolysaccharide-Induced Inflammation.

Evidence has shown that caffeine administration reduces pro-inflammatory biomarkers, delaying fatigue and improving endurance performance. This study examined the effects of caffeine administration on the expression of inflammatory-, adenosine receptor- (the targets of caffeine), epigenetic-, and oxidative metabolism-linked genes in the vastus lateralis muscle of mice submitted to lipopolysaccharide (LPS)-induced inflammation. We showed that caffeine pre-treatment before LPS administration reduced the expression of Il1b, Il6, and Tnfa, and increased Il10 and Il13. The negative modulation of the inflammatory response induced by caffeine involved the reduction of inflammasome components, Asc and Casp1, promoting an anti-inflammatory scenario. Caffeine treatment per se promoted the upregulation of adenosinergic receptors, Adora1 and Adora2A, an effect that was counterbalanced by LPS. Moreover, there was observed a marked Adora2A promoter hypermethylation, which could represent a compensatory response towards the increased Adora2A expression. Though caffeine administration did not alter DNA methylation patterns, the expression of DNA demethylating enzymes, Tet1 and Tet2, was increased in mice receiving Caffeine+LPS, when compared with the basal condition. Finally, caffeine administration attenuated the LPS-induced catabolic state, by rescuing basal levels of Ampk expression. Altogether, the anti-inflammatory effects of caffeine in the muscle can be mediated by modifications on the epigenetic landscape.

Epigenetic Differences Arise in Endothelial Cells Responding to Cobalt-Chromium.

Cobalt-chromium (Co-Cr)-based alloys are emerging with important characteristics for use in dentistry, but the knowledge of epigenetic mechanisms in endothelial cells has barely been achieved. In order to address this issue, we have prepared a previously Co-Cr-enriched medium to further treat endothelial cells (HUVEC) for up to 72 h. Our data show there is important involvement with epigenetic machinery. Based on the data, it is believed that methylation balance in response to Co-Cr is finely modulated by DNMTs (DNA methyltransferases) and TETs (Tet methylcytosine dioxygenases), especially DNMT3B and both TET1 and TET2. Additionally, histone compaction HDAC6 (histone deacetylase 6) seems to develop a significant effect in endothelial cells. The requirement of SIRT1 seems to have a crucial role in this scenario. SIRT1 is associated with a capacity to modulate the expression of HIF-1α in response to hypoxia microenvironments, thus presenting a protective effect. As mentioned previously, cobalt is able to prevent HIF1A degradation and maintain hypoxia-related signaling in eukaryotic cells. Together, our results show, for the first time, a descriptive study reporting the relevance of epigenetic machinery in endothelial cells responding to cobalt-chromium, and it opens new perspectives to better understand their repercussions as prerequisites for driving cell adhesion, cell cycle progression, and angiogenesis surrounding this Co-Cr-based implantable device.

Titanium-Enriched Medium Promotes Environment-Induced Epigenetic Machinery Changes in Human Endothelial Cells.

It is important to understand whether endothelial cells are epigenetically affected by titanium-enriched media when angiogenesis is required during bone development and it is expected to be recapitulated during osseointegration of biomaterials. To better address this issue, titanium-enriched medium was obtained from incubation of titanium discs for up to 24 h as recommended by ISO 10993-5:2016, and further used to expose human umbilical vein endothelial cells (HUVECs) for up to 72 h, when the samples were properly harvested to allow molecular analysis and epigenetics. In general, our data show an important repertoire of epigenetic players in endothelial cells responding to titanium, reinforcing protein related to the metabolism of acetyl and methyl groups, as follows: Histone deacetylases (HDACs) and NAD-dependent deacetylase sirtuin-1 (Sirt1), DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) methylcytosine dioxygenases, which in conjunction culminate in driving chromatin condensation and the methylation profile of DNA strands, respectively. Taking our data into consideration, HDAC6 emerges as important player of this environment-induced epigenetic mechanism in endothelial cells, while Sirt1 is required in response to stimulation of reactive oxygen species (ROS) production, as its modulation is relevant to vasculature surrounding implanted devices. Collectively, all these findings support the hypothesis that titanium keeps the surrounding microenvironment dynamically active and so affects the performance of endothelial cells by modulating epigenetics. Specifically, this study shows the relevance of HDAC6 as a player in this process, possibly correlated with the cytoskeleton rearrangement of those cells. Furthermore, as those enzymes are druggable, it opens new perspectives to consider the use of small molecules to modulate their activities as a biotechnological tool in order to improve angiogenesis and accelerate bone growth with benefits of a fast recovery time for patients.

Hyperglycemic microenvironment compromises the homeostasis of communication between the bone-brain axis by the epigenetic repression of the osteocalcin receptor, Gpr158 in the hippocampus.

Diabetes mellitus (DM) is a chronic metabolic disease, mainly characterized by increased blood glucose and insulin dysfunction. In response to the persistent systemic hyperglycemic state, numerous metabolic and physiological complications have already been well characterized. However, its relationship to bone fragility, cognitive deficits and increased risk of dementia still needs to be better understood. The impact of chronic hyperglycemia on bone physiology and architecture was assessed in a model of chronic hyperglycemia induced by a single intraperitoneal administration of streptozotocin (STZ; 55 mg/kg) in Wistar rats. In addition, the bone-to-brain communication was investigated by analyzing the gene expression and methylation status of genes that encode the main osteokines released by the bone [Fgf23 (fibroblast growth factor 23), Bglap (bone gamma-carboxyglutamate protein) and Lcn2 (lipocalin 2) and their receptors in both, the bone and the brain [Fgfr1 (fibroblast growth factor receptor 1), Gpr6A (G-protein coupled receptor family C group 6 member A), Gpr158 (G protein-coupled receptor 158) and Slc22a17 (Solute carrier family 22 member 17)]. It was observed that chronic hyperglycemia negatively impacted on bone biology and compromised the balance of the bone-brain endocrine axis. Ultrastructural disorganization was accompanied by global DNA hypomethylation and changes in gene expression of DNA-modifying enzymes that were accompanied by changes in the methylation status of the osteokine promoter region Bglap and Lcn2 (lipocalin 2) in the femur. Additionally, the chronic hyperglycemic state was accompanied by modulation of gene expression of the osteokines Fgf23 (fibroblast growth factor 23), Bglap (bone gamma-carboxyglutamate protein) and Lcn2 (lipocalin 2) in the different brain regions. However, transcriptional regulation mediated by DNA methylation was observed only for the osteokine receptors, Fgfr1(fibroblast growth factor receptor 1) in the striatum and Gpr158 (G protein-coupled receptor 158) in the hippocampus. This is a pioneer study demonstrating that the chronic hyperglycemic state compromises the crosstalk between bone tissue and the brain, mainly affecting the hippocampus, through transcriptional silencing of the Bglap receptor by hypermethylation of Gpr158 gene.