Vascular smooth muscle cell-derived exosomes promote osteoblast-to-osteocyte transition via ß-catenin signaling.

Blood vessel growth and osteogenesis in the skeletal system are coupled; however, fundamental aspects of vascular function in osteoblast-to-osteocyte transition remain unclear. Our study demonstrates that vascular smooth muscle cells (VSMCs), but not endothelial cells, are sufficient to drive bone marrow mesenchymal stromal cell-derived osteoblast-to-osteocyte transition via ß-catenin signaling and exosome-mediated communication. We found that VSMC-derived exosomes are loaded with transcripts encoding proteins associated with the osteocyte phenotype and members of the WNT/ß-catenin signaling pathway. In contrast, endothelial cell-derived exosomes facilitated mature osteoblast differentiation by reprogramming the TGFB1 gene family and osteogenic transcription factors osterix (SP7) and RUNX2. Notably, VSMCs express significant levels of tetraspanins (CD9, CD63, and CD81) and drive the intracellular trafficking of exosomes with a lower membrane zeta potential than those from other cells. Additionally, the high ATP content within these exosomes supports mineralization mechanisms, as ATP is a substrate for alkaline phosphatase. Osteocyte function was further validated by RNA sequencing, revealing activity in genes related to intermittent mineralization and sonic hedgehog signaling, alongside a significant increase in TNFSF11 levels. Our findings unveil a novel role of VSMCs in promoting osteoblast-to-osteocyte transition, thus offering new insights into bone biology and homeostasis, as well as in bone-related diseases. Clinically, these insights could pave the way for innovative therapeutic strategies targeting VSMC-derived exosome pathways to treat bone-related disorders such as osteoporosis. By manipulating these signaling pathways, it may be possible to enhance bone regeneration and improve skeletal health in patients with compromised bone structure and function.

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.

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.

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.

Genome-wide DNA (hydroxy) methylation reveals the individual epigenetic landscape importance on osteogenic phenotype acquisition in periodontal ligament cells.

BACKGROUND: Mesenchymal cells' biology has been an important investigative tool to maximize bone regeneration through tissue engineering. Here we used mesenchymal cells from periodontal ligament (PDLCs) with high (h-) and low (l-) osteogenic potential, isolated from different donors, to investigate the impact of the individual epigenetic and transcriptional profiles on the osteogenic potential. METHODS: Genome-wide and gene-specific DNA (hydroxy) methylation, mRNA expression and immunofluorescence analysis were carried out in h- and l-PDLCs at DMEM (non-induced to osteogenesis) and OM (induced-3rd and 10th days of osteogenic differentiation) groups in vitro. RESULTS: Genome-wide results showed distinct epigenetic profile among PDLCs with most of the differences on 10th day of OM; DMEMs showed higher concentrations (xOM) of differentially methylated probes in gene body, intronic and open sea (3rd day), increasing this concentration in TSS200 and island regions, at 10 days. At basal levels, h- and l-PDLCs showed different transcriptional profiles; l-PDLCs demonstrated higher levels of NANOG/OCT4/SOX2, BAPX1, DNMT3A, TET1/3, and lower levels of RUNX2 transcripts, confirmed by NANOG/OCT4 and RUNX2 immunofluorescence. After osteogenic induction, the distinct transcriptional profile of multipotentiality genes was maintained among PDLCs. In l-PDLCs, the anti-correlation between DNA methylation and gene expression in RUNX2 and NANOG indicates methylation could play a role in modulating both transcripts. CONCLUSIONS: Epigenetic and transcriptional distinct profiles detected at basal levels among PDLCs were maintained after osteogenic induction. We cannot discard the existence of a complex that represses osteogenesis, suggesting the individual donors' characteristics have significant impact on the osteogenic phenotype acquisition.

HOXA13 in etiology and oncogenic potential of Barrett's esophagus.

Barrett's esophagus in gastrointestinal reflux patients constitutes a columnar epithelium with distal characteristics, prone to progress to esophageal adenocarcinoma. HOX genes are known mediators of position-dependent morphology. Here we show HOX collinearity in the adult gut while Barrett's esophagus shows high HOXA13 expression in stem cells and their progeny. HOXA13 overexpression appears sufficient to explain both the phenotype (through downregulation of the epidermal differentiation complex) and the oncogenic potential of Barrett's esophagus. Intriguingly, employing a mouse model that contains a reporter coupled to the HOXA13 promotor we identify single HOXA13-positive cells distally from the physiological esophagus, which is mirrored in human physiology, but increased in Barrett's esophagus. Additionally, we observe that HOXA13 expression confers a competitive advantage to cells. We thus propose that Barrett's esophagus and associated esophageal adenocarcinoma is the consequence of expansion of this gastro-esophageal HOXA13-expressing compartment following epithelial injury.

Hematopoietic stem cell stretches and moves in its bone marrow niche.

Hematopoietic stem cells are the most illustrious inhabitants of the bone marrow. Direct visualization of endogenous hematopoietic stem cells in this niche is essential to study their functions. Until recently this was not possible in live animals. Recent studies, using state-of-the-art technologies, including sophisticated in vivo inducible genetic approaches in combination with two-photon laser scanning microscopy, allow the follow-up of endogenous hematopoietic stem cells' behavior in their habitat. Strikingly, the new findings reveal that quiescent hematopoietic stem cells are more mobile than previously thought, and link their retained steady state within the niche to a mobile behavior. The arising knowledge from this research will be critical for the therapy of several hematological diseases. Here, we review recent progress in our understanding of hematopoietic stem cell biology in their niches.

DNMT1 Inhibitor Restores RUNX2 Expression and Mineralization in Periodontal Ligament Cells.

Periodontal ligament cells (PDLCs) have well documented osteogenic potential; however, this commitment can be highly heterogenous, limiting their applications in tissue regeneration. In this study, we use PDLC populations characterized by high and low osteogenic potential (h-PDLCs and l-PDLCs, respectively) to identify possible sources of such heterogeneity and to investigate whether the osteogenic differentiation can be enhanced by epigenetic modulation. In h-PDLCs, low basal expression levels of pluripotency markers (NANOG, OCT4), DNA methyltransferases (DNMT1, DNMT3B), and enzymes involved in active DNA demethylation (TET1, TET3) were prerequisite to high osteogenic potential. Furthermore, these genes were downregulated upon early osteogenesis, possibly allowing for the increase in expression of the key osteogenic transcription factors, Runt-related transcription factor 2 (RUNX2) and SP7, and ultimately, mineral nodule formation. l-PDLCs appeared locked in the multipotent state and this was further enhanced upon early osteogenic stimulation, correlating with low RUNX2 expression and impaired mineralization. Further upregulation of DNMTs was also evident, while pretreatment with RG108, the DNMTs' inhibitor, enhanced the osteogenic program in l-PDLCs through downregulation of DNMTs, increased RUNX2 expression and nuclear localization, accelerated expression of osteogenic markers, and increased mineralization. These findings point toward the role of DNMTs and Ten Eleven Translocations (TETs) in osteogenic commitment and support application of epigenetic approaches to modulate biomineralization in PDLCs.

Sensory nerves in the spotlight of the stem cell niche.

Niches are specialized tissue microenvironments that control stem cells functioning. The bone marrow mesenchymal stem cell niche defines a location within the marrow in which mesenchymal stem cells are retained and produce new cells throughout life. Deciphering the signaling mechanisms by which the niche regulates stem cell fate will facilitate the use of these cells for therapy. Recent studies, by using state-of-the-art methodologies, including sophisticated in vivo inducible genetic techniques, such as lineage-tracing Cre/loxP mediated systems, in combination with pharmacological inhibition, provide evidence that sensory neuron is an important component of the bone marrow mesenchymal stem cell niche. Strikingly, knockout of a specific receptor in sensory neurons blocked stem cell function in the bone marrow. The knowledge arising from these discoveries will be crucial for stem cell manipulation in the future. Here, we review recent progress in our understanding of sensory nerves biology in the stem cell niche.

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.

Characterization of the Renin-Angiotensin System in Aged Cavernosal Tissue and its Role in Penile Fibrosis.

BACKGROUND: The renin-angiotensin system (RAS) plays an important role in erectile function. The RAS contains 2 major axes: one deleterious, composed of ACE-Ang II-AT1 receptor, and another protective, composed of ACE2-Ang-(1-7)-Mas receptor. While aging is a well-known cause for development of male sexual disorders, little is known about local regulation of the RAS in age-related erectile dysfunction (ED). AIM: The present study aimed to assess regulation of the RAS in aging-associated ED rat model and evaluate possible options for disease management through pharmacological modulation of the RAS. METHODS: Penile tissues were harvested from 3-, 12-, and 24-month-old Wistar rats. Local expression of major RAS components and ED markers was measured by RT-PCR. Protein expression of RAS components was assessed by western blot. Collagen deposition was measured by Sirius Red and immunohistochemical staining. Evaluation of collagen content was also performed in penile sections of Mas-knockout mice by Sirius Red and Masson's trichrome stainings. Finally, the effect of Ang-(1-7) pretreatment on TGF-ß-induced myofibroblast activation was studied in primary cavernosal and immortalized fibroblasts. OUTCOMES: Experimental results highlighted the essential role of the RAS in modulation of cavernosal fibrosis. RESULTS: The present study demonstrates local expression of angiotensinogen mRNA alongside with major RAS components, which suggests local autonomous functioning of the RAS within penile tissue. Gene expression analysis revealed strong positive correlation between ACE-Ang II-AT1 axis with markers for inflammation and fibrosis. While corpus cavernosum from 24-month-old rats was characterized by increased collagen deposition, protein expression of ACE, AT1, and Mas was shown to be upregulated in the penile tissue of this group. At the same time, penile sections from Mas-knockout mice (FVB/N background) were also shown to have increased collagen deposition. Finally, it was demonstrated that Ang-(1-7) treatment of primary cavernosal and immortalized fibroblasts was able to alleviate TGF-ß-induced fibroblast-to-myofibroblast transition. CLINICAL TRANSLATION: The present study suggests Ang-(1-7) treatment as a possible strategy for pharmacological management of fibrosis-associated ED in aging. STRENGTHS & LIMITATIONS: The link between the RAS and penile fibrosis, indicated by a holistic screening of different ED markers, was confirmed by in vivo and in vitro data. However, results, presented in the manuscript, need to be further reinforced by human data. Important to note, the main goal of the study was to characterize RAS regulation in aging condition rather than state any causal relationships. CONCLUSION: Present study characterizes RAS regulation in aging-associated ED and indicates its important role in cavernosal fibrosis. Bragina ME, Costa-Fraga F, Sturny M, et al. Characterization of the Renin-Angiotensin System in Aged Cavernosal Tissue and its Role in Penile Fibrosis. J Sex Med 2020;17:2129-2140.

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.

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.

HOXA cluster gene expression during osteoblast differentiation involves epigenetic control.

The HOXA gene cluster is generally recognized as a pivotal mediator of positional identity in the skeletal system, expression of different orthologues conferring alternative locational phenotype of the vertebrate bone. Strikingly, however, the molecular mechanisms that regulate orthologue-specific expression of different HOXA cluster members in gestating osteoblasts remain largely obscure, but in analogy to the processes observed in acute lymphatic leukemia it is assumed that alternative methylation of HOXA promoter regions drives position specific expression patterns. In an effort to understand HOXA cluster gene expression in osteogenesis we characterize both expression and the epigenetic landscape of the HOXA gene cluster during in vitro osteoblast formation from mesenchymal precursors. We observe that osteoblast formation per se provokes strong upregulation of HOXA gene cluster expression, in particular of midcluster genes, and paradoxal downregulation of HOXA7 and HOXA10. These differences in expression appear related to promoter methylation. LnRNAs HOTAIR and HOTTIP, known to modulate HOXA expression, are also regulated by their promoter methylation processing, but do not correlate with HOXA cluster expression profile. We thus conclude that HOXA expression is profoundly regulated during osteoblast differentiation through canonical methylation-dependent mechanisms but not through the flanking lnRNAs.

RG108 increases NANOG and OCT4 in bone marrow-derived mesenchymal cells through global changes in DNA modifications and epigenetic activation.

Human bone marrow-derived mesenchymal stem cells (hBMSCs) are important for tissue regeneration but their epigenetic regulation is not well understood. Here we investigate the ability of a non-nucleoside DNA methylation inhibitor, RG108 to induce epigenetic changes at both global and gene-specific levels in order to enhance mesenchymal cell markers, in hBMSCs. hBMSCs were treated with complete culture medium, 50 µM RG108 and DMSO for three days and subjected to viability and apoptosis assays, global and gene-specific methylation/hydroxymethylation, transcript levels' analysis of epigenetic machinery enzymes and multipotency markers, protein activities of DNMTs and TETs, immunofluorescence staining and western blot analysis for NANOG and OCT4 and flow cytometry for CD105. The RG108, when used at 50 µM, did not affect the viability, apoptosis and proliferation rates of hBMSCs or hydroxymethylation global levels while leading to 75% decrease in DNMTs activity and 42% loss of global DNA methylation levels. In addition, DNMT1 was significantly downregulated while TET1 was upregulated, potentially contributing to the substantial loss of methylation observed. Most importantly, the mesenchymal cell markers CD105, NANOG and OCT4 were upregulated being NANOG and OCT4 epigenetically modulated by RG108, at their gene promoters. We propose that RG108 could be used for epigenetic modulation, promoting epigenetic activation of NANOG and OCT4, without affecting the viability of hBMSCs. DMSO can be considered a modulator of epigenetic machinery enzymes, although with milder effect compared to RG108.

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.

Age-related changes in vascular responses to angiotensin-(1-7) in female mice.

The vasodilatory effect of angiotensin-(1-7) seems to vary between sexes, and estradiol (E2) can modulate the magnitude of the Ang-(1-7) vasodilatory response in female rats. However, there are few studies addressing the influence of sex on the age-related vasodilatory effect of Ang-(1-7). Here, we evaluated the vasodilatory response to Ang-(1-7) on vascular ageing. Ang-(1-7) dose-response curves were determined in mice aortic rings from males (old and young) and females (E2 treated/non-treated old and young) mounted in an isolated organ chamber. Abdominal aortic rings were used for protein expression analysis and determination of reactive oxygen species (ROS) and nitric oxide (NO) production. Our results showed that the Ang-(1-7) vasodilatory effect was absent in aorta from old females, contrasting with a full response in vessels from young females. The Ang-(1-7) vasodilatory effect was restored by E2 replacement in old females. A robust increase in Mas receptor, SOD2, NRF-2 and NOX2 expression was observed in aorta from old females, which was normalized by E2. This effect of E2 was also associated with lower production of ROS and normal levels of NO. In conclusion, our data demonstrated that pathways involved in the Ang-(1-7) vasodilatory response in female mice is affected by hormonal changes in ageing and rescued by E2.

Oxidative stress and mitochondrial adaptive shift during pituitary tumoral growth.

The cellular transformation of normal functional cells to neoplastic ones implies alterations in the cellular metabolism and mitochondrial function in order to provide the bioenergetics and growth requirements for tumour growth progression. Currently, the mitochondrial physiology and dynamic shift during pituitary tumour development are not well understood. Pituitary tumours present endocrine neoplastic benign growth which, in previous reports, we had shown that in addition to increased proliferation, these tumours were also characterized by cellular senescence signs with no indication of apoptosis. Here, we show clear evidence of oxidative stress in pituitary cells, accompanied by bigger and round mitochondria during tumour development, associated with augmented biogenesis and an increased fusion process. An activation of the Nrf2 stress response pathway together with the attenuation of the oxidative damage signs occurring during tumour development were also observed which will probably provide survival advantages to the pituitary cells. These neoplasms also presented a progressive increase in lactate production, suggesting a metabolic shift towards glycolysis metabolism. These findings might imply an oxidative stress state that could impact on the pathogenesis of pituitary tumours. These data may also reflect that pituitary cells can modulate their metabolism to adapt to different energy requirements and signalling events in a pathophysiological situation to obtain protection from damage and enhance their survival chances. Thus, we suggest that mitochondria function, oxidative stress or damage might play a critical role in pituitary tumour progression.

Apelin-13 treatment enhances the stability of atherosclerotic plaques.

BACKGROUND: Apelin is an endogenous peptidergic system which modulates cardiovascular function. Recent studies pointed out a fundamental contribution of apelin on atherosclerosis development; however, such reports revealed contradictory data, and to date, it is difficult to accurately define a beneficial or deleterious role. To better understand apelin function on atherosclerosis, we aimed to investigate apelin-13 treatment effects on atherosclerotic plaques composition. DESIGN: Apolipoprotein E gene-deleted mice were fed on Western-type diet for 11 weeks. Atherosclerotic plaque formation was induced in the carotid artery by a shear stress modifier device, which exposes the same vessel to distinct patterns of shear stress enabling the formation of plaques with different composition. Mice were treated with apelin-13 (2 mg kg-1 day-1 ) or vehicle for the last 3 weeks. RESULTS: Apelin-13 treatment did not alter the lipid content of low shear stress- and oscillatory shear stress-induced plaques in the carotid. However, apelin-13 greatly ameliorated plaque stability by increasing intraplaque collagen content and reducing MMP-9 expression. Furthermore, apelin-13 decreased the infiltration of inflammatory cells (neutrophil and macrophage) and intraplaque reactive oxygen species content. Interestingly, apelin-13 treatment reduced total cholesterol, LDL levels and free fatty acid serum levels, while HDL, triglycerides serum levels were not significantly changed. CONCLUSIONS: Apelin-13 treatment for 3 weeks did not alter the lesion size, but it significantly enhanced the stable phenotype of atherosclerotic plaques and improved serum lipid profile. These results indicate that activation of apelin system decreases plaque vulnerability.