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.

Mechanosignaling-related angiocrine factors drive osteoblastic phenotype in response to zirconia.

BACKGROUND: The growing use of zirconia as a ceramic material in dentistry is attributed to its biocompatibility, mechanical properties, esthetic appearance, and reduced bacterial adhesion. These favorable properties make ceramic materials a viable alternative to commonly used titanium alloys. Mimicking the physiological properties of blood flow, particularly the mechanosignaling in endothelial cells (ECs), is crucial for enhancing our understanding of their role in the response to zirconia exposure. METHODS: In this study, EC cultures were subjected to shear stress while being exposed to zirconia for up to 3 days. The conditioned medium obtained from these cultures was then used to expose osteoblasts for a duration of 7 days. To investigate the effects of zirconia on osteoblasts, we examined the expression of genes associated with osteoblast differentiation, including Runx2, Osterix, bone sialoprotein, and osteocalcin genes. Additionally, we assessed the impact of mechanosignaling-related angiocrine factors on extracellular matrix (ECM) remodeling by measuring the activities of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) during the acquisition of the osteogenic phenotype, which precedes mineralization. RESULTS: Our data revealed that mechanosignaling-related angiocrine factors play a crucial role in promoting an osteoblastic phenotype in response to zirconia exposure. Specifically, exposed osteoblasts exhibited significantly higher expression levels of genes associated with osteoblast differentiation, such as Runx2, Osterix, bone sialoprotein, and osteocalcin genes. Furthermore, the activities of MMP2 and MMP9, which are involved in ECM remodeling, were modulated by mechanosignaling-related angiocrine factors. This modulation is likely an initial event preceding the mineralization phase. CONCLUSION: Based on our findings, we propose that mechanosignaling drives the release of angiocrine factors capable of modulating the osteogenic phenotype at the biointerface with zirconia. This process creates a microenvironment that promotes wound healing and osseointegration. Moreover, these results highlight the importance of considering the mechanosignaling of endothelial cells in the modulation of bone healing and osseointegration in the context of blood vessel effects. Our data provide new insights and open avenues for further investigation into the influence of mechanosignaling on bone healing and the osseointegration of dental devices.

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.

The role of triiodothyronine hormone and mechanically-stressed endothelial cell paracrine signalling synergism in gene reprogramming during hBMSC-stimulated osteogenic phenotype in vitro.

We therefore investigated whether there is synergism between triiodothyronine (T3) hormone and trophic molecules released from mechanically-stressed endothelial cells (EC-enriched medium) in osteogenic phenotype by mapping classical repertory of genes. Although there are studies reporting the efficiency of T3 hormone on bone cells, it is scarce considering their effect in conjunction with other physiologically active molecules, such as those released by the active endothelial cells. To address this issue, human bone marrow-derived mesenchymal stem cells (hBMSCs) were treated with EC-enriched medium subjected to shear-stress up to 72 h in vitro, in conjunction or not with T3 hormone. Although our results found an important synergism considering these parameters on modulating key bone-related gene markers, such as on the alkaline phosphatase (ALP) behavior (at both mRNA and protein content), contributing for osteoblast differentiation, important genes such as OSTERIX and RUNX2 were significantly down-expressed, while a over-expression of RANKL was found when the conjunction effect of T3 and endothelial paracrine signaling was considered. In addition, T3 hormone over expressed both OCT4 and NANOG genes in a DNA epigenetic-independent manner. However, we observed a dynamic reprogramming of DNMT1, DNMT3A, DNMT3B and TET1, important DNA-related epigenetic markers. Specifically, T3 hormone alone up-modulated TET2 transcripts profile. Complimentarily, expression of microRNA (miRs) processing-related genes also was modulated, as well as miR-10b, miR-22, miR-21, miR-143 and miR-145 transcriptional related profiles. Altogether, our results suggested a positive effect of mechanically-stressed endothelial cells-induced paracrine signaling on T3 hormone-obtaining osteogenic phenotype, contributing to understanding the paradoxal effect of T3 hormone on the bone physiology.