Osteogenic gene markers are epigenetically reprogrammed during contractile-to-calcifying vascular smooth muscle cell phenotype transition.

The understanding of vascular calcification-based mechanism is an urgent pending task in vascular biology and this prompted us to better address this issue by investigating whether DNA methylation mechanism might drive osteogenic marker genes modulation in primary human vascular smooth muscle cells (VSMCs) responding to calcium and phosphate levels overload up to 72 h. Firstly, our data shows this calcifying process recapitulates the molecular repertory of osteogenic biomarkers and specifically requiring RUNX2, Osterix and ALP, BSP genes activations along 72 h in vitro, and this behavior was validated here using other lineages. Conversely, both BMPs 4 and 7 were significantly overexpressed, maybe already as a mechanism in response to RUNX2 and Osterix genes activities identified earlier in response to the calcifying condition, and taken into maintain the calcifying phenotype of VSMCs. Additionally, survival signaling was maintained active and accompanied by a dynamic cytoskeleton rearrangement signaling requiring MAPK and AKT phosphorylations. Moreover, during the contractile-to-calcifying transition phenotype of VSMCs, epigenetic machinery was finely modulated, requiring the translocation of DNMT3B and TET2 into nucleus and this prompted us evaluating whether the profile of osteogenic-related gene promoters' methylation might contribute with this process. By firstly estimating 5meC/5 hmeC ratio changes, we further specifically show the significance of the epigenetic modulation of Osterix and Bone sialoprotein related gene promoters, presenting a positive correlation between the epigenetic signature of their gene promoters and transcriptional patterns. Altogether, our results show for the first time the importance of epigenetic mechanism on modulating osteogenic gene markers reprogramming during calcifying VSMCs phenotype acquisition, which might drive the genesis of vascular ectopic calcification.

Differential inflammatory landscape stimulus during titanium surfaces obtained osteogenic phenotype.

Molecular mechanism governing inflammatory scenario in response to titanium (Ti)-nanotexturing surfaces needs to be better addressed. Thus, we subjected pre-osteoblast to different Ti-texturing surfaces, as follows: machined (Mac), double acid-etching (DAE), and nanoscaled hydroxyapatite-blasted titanium surface (nHA), considering the cells chronically responding either directly (when the cells were cultured onto the surfaces) or indirectly (when the cells were challenged with the conditioned medium by the surfaces), up to 10 days. Our results showed that there is a dynamic requirement of inflammatory-related genes activation in response to nHA by up expressing IL1ß, IL6, IL10, and IL33 (direct condition) and IL6, IL10, IL18 (indirect condition). Importantly, our data show that there is inflammasome involvement, once NLRP3, ASC1, and CASP1 genes were also required. As we found a strong signal of IL10, an anti-inflammatory cytokine, we further investigated Sonic Hedgehog (Shh) signaling cascade. Surprisingly, Shh ligand and Smoothened (Smo) genes were up-modulated in response to nHA, while Patched (Ptc) was down-modulated. Finally, an interactome was built using bioinformatics reinforcing Shh signaling cascade on modulating IL10 transcripts by Src mediating this process and this prevalence of anti-inflammatory picture might explain the low profile of RANKL transcripts in response to nHA, compromising the osteoclastogenesis surrounding the implants. Taking our results into account, our data show that the inflammatory landscape promoted by nHA is strictly modulated by Shh signaling promoted anti-inflammatory pathways. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1597-1604, 2019.