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.

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.