Role of gut microbe-derived metabolites in cardiometabolic diseases: Systems based approach.

BACKGROUND: The gut microbiome influences host physiology and cardiometabolic diseases by interacting directly with intestinal cells or by producing molecules that enter the host circulation. Given the large number of microbial species present in the gut and the numerous factors that influence gut bacterial composition, it has been challenging to understand the underlying biological mechanisms that modulate risk of cardiometabolic disease. SCOPE OF THE REVIEW: Here we discuss a systems-based approach that involves simultaneously examining individuals in populations for gut microbiome composition, molecular traits using "omics" technologies, such as circulating metabolites quantified by mass spectrometry, and clinical traits. We summarize findings from landmark studies using this approach and discuss future applications. MAJOR CONCLUSIONS: Population-based integrative approaches have identified a large number of microbe-derived or microbe-modified metabolites that are associated with cardiometabolic traits. The knowledge gained from these studies provide new opportunities for understanding the mechanisms involved in gut microbiome-host interactions and may have potentially important implications for developing novel therapeutic approaches.

Potencial Rol de la Periodontitis en la Severidad de COVID-19. Revisión

RESUMEN: Actualmente la asociación entre periodontitis y enfermedades o condiciones sistémicas es ampliamente reconocida. Sin embargo, el rol de la periodontitis en la severidad de COVID-19, se comienza a estudiar. El objetivo de esta revisión es relacionar la presencia de periodontitis como factor asociado en casos más severos de COVID-19. Se aborda el posible rol de la periodontitis y su conexión a través de las citoquinas y otros mecanismos asociados como la senescencia celular y la NETosis, que podrían explicar su influencia en el desarrollo y severidad de COVID-19. Además, se abordan aspectos complementarios como el rol de la higiene oral y mantención de la salud oral como medidas adicionales de prevención.

ABSTRACT: Currently, the association between periodontitis and systemic diseases is widely recognized. However, a potential role of periodontal inflammation on the severity of COVID-19 has recently been studied. The goal of this review is to highlight different mechanisms by which the presence of periodontitis could contribute to the increased severity of COVID-19. These mechanisms include the secretion of proinflammatory cytokines, accelerated cellular senescence, and NETosis. We also emphasize the importance of oral hygiene and maintenance of oral health as additional measures to avoid COVID-19 dissemination.

Severe COVID-19 Lung Infection in Older People and Periodontitis.

Periodontal bacteria dissemination into the lower respiratory tract may create favorable conditions for severe COVID-19 lung infection. Once lung tissues are colonized, cells that survive persistent bacterial infection can undergo permanent damage and accelerated cellular senescence. Consequently, several morphological and functional features of senescent lung cells facilitate SARS-CoV-2 replication. The higher risk for severe SARS-CoV-2 infection, the virus that causes COVID-19, and death in older patients has generated the question whether basic aging mechanisms could be implicated in such susceptibility. Mounting evidence indicates that cellular senescence, a manifestation of aging at the cellular level, contributes to the development of age-related lung pathologies and facilitates respiratory infections. Apparently, a relationship between life-threatening COVID-19 lung infection and pre-existing periodontal disease seems improbable. However, periodontal pathogens can be inoculated during endotracheal intubation and/or aspirated into the lower respiratory tract. This review focuses on how the dissemination of periodontal bacteria into the lungs could aggravate age-related senescent cell accumulation and facilitate more efficient SARS-CoV-2 cell attachment and replication. We also consider how periodontal bacteria-induced premature senescence could influence the course of COVID-19 lung infection. Finally, we highlight the role of saliva as a reservoir for both pathogenic bacteria and SARS-CoV-2. Therefore, the identification of active severe periodontitis can be an opportune and valid clinical parameter for risk stratification of old patients with COVID-19.

Senescent cells exacerbate chronic inflammation and contribute to periodontal disease progression in old mice.

BACKGROUND: Coinciding with other chronic comorbidities, the prevalence of periodontal disease increases with aging. Mounting evidence has established that senescent cells accumulate at sites of age-related pathologies, where they promote "non-microbial" inflammation. We hypothesized that alveolar bone osteocytes develop senescence characteristics in old age. METHODS: Alveolar bone samples were obtained from young (6 months) and old (20 to 22 months) mice to evaluate the expression of senescence biomarkers by immunofluorescent staining. Osteocyte-enriched fractions were used to characterize the age-related senescence-associated secretory phenotype (SASP) gene expression profile. Primary alveolar bone cells were exposed to the SASP via in vitro senescent conditioned media (SCM) administration. A multiplex assay confirmed protein levels of specific cytokines. Interactions with bacterial components were evaluated by stimulating cells with lipopolysaccharide (LPS). RESULTS: Increased senescence-associated distension of satellites (SADS) and p16Ink4a mRNA expression were identified in alveolar bone osteocytes with aging. These findings were associated with increased levels of DNA damage, and activated p38 MAPK, both inducers of senescence. Furthermore, interleukin-6 (IL6), IL17, IGFBP4, and MMP13 were significantly upregulated with aging in osteocyte-enriched samples. Interestingly, SCM potentiated the LPS-induced expression of IL1α, IL1ß, and IL6. Cell migration and differentiation were also impeded by SCM. These in vitro effects were ameliorated by the p38 MAPK inhibitor SB202190. CONCLUSIONS: Accumulation of senescent osteocytes contributes to deterioration of the periodontal environment by exacerbating chronic inflammation and reducing regeneration in old age. Cellular senescence is a cell-intrinsic response to DNA damage, and a host-related mechanism associated with aging that could potentiate inflammation induced by bacterial components.

Periodontal Disease and Senescent Cells: New Players for an Old Oral Health Problem?

The recent identification of senescent cells in periodontal tissues has the potential to provide new insights into the underlying mechanisms of periodontal disease etiology. DNA damage-driven senescence is perhaps one of the most underappreciated delayed consequences of persistent Gram-negative bacterial infection and inflammation. Although the host immune response rapidly protects against bacterial invasion, oxidative stress generated during inflammation can indirectly deteriorate periodontal tissues through the damage to vital cell macromolecules, including DNA. What happens to those healthy cells that reside in this harmful environment? Emerging evidence indicates that cells that survive irreparable genomic damage undergo cellular senescence, a crucial intermediate mechanism connecting DNA damage and the immune response. In this review, we hypothesize that sustained Gram-negative bacterial challenge, chronic inflammation itself, and the constant renewal of damaged tissues create a permissive environment for the abnormal accumulation of senescent cells. Based on emerging data we propose a model in which the dysfunctional presence of senescent cells may aggravate the initial immune reaction against pathogens. Further understanding of the role of senescent cells in periodontal disease pathogenesis may have clinical implications by providing more sophisticated therapeutic strategies to combat tissue destruction.

LPS-induced premature osteocyte senescence: Implications in inflammatory alveolar bone loss and periodontal disease pathogenesis.

Cellular senescence is associated with inflammation and extracellular matrix tissue remodeling through the secretion of proteins termed the senescence-associated secretory phenotype (SASP). Although osteocyte senescence in older individuals in the skeleton is well recognized, whether young alveolar osteocytes can also become senescent is unknown. This is potentially important in the context of periodontal disease, which is an inflammatory condition caused by a gradual change from symbiotic to pathogenic oral microflora that can lead to tooth loss. Our aim was to identify whether senescent osteocytes accumulate in young alveolar bone and whether bacterial-derived lipopolysaccharide (LPS) can influence cellular senescence in alveolar bone. An osteocyte-enriched cell population isolated from alveolar bone expressed increased levels of the known senescence marker p16Ink4a, as well as select SASP markers known to be implicated alveolar bone resorption (Icam1, Il6, Il17, Mmp13 and Tnfα), compared to ramus control cells. Increased senescence of alveolar bone osteocytes was also observed in vivo using the senescence-associated distension of satellites (SADS) assay and increased γH2AX, a marker of DNA damage associated with senescent cells. To approximate a bacterial infection in vitro, alveolar osteocytes were treated with LPS. We found increased expression of various senescence and SASP markers, increased γH2AX staining, increased SA-ß-Gal activity and the redistribution of F-actin leading to a larger and flattened cell morphology, all hallmarks of cellular senescence. In conclusion, our data suggests a model whereby bacterial-derived LPS stimulates premature alveolar osteocyte senescence, which in combination with the resultant SASP, could potentially contribute to the onset of alveolar bone loss.

Calcium mimics the chemotactic effect of conditioned media and is an effective inducer of bone regeneration.

BACKGROUND: After bone resorption, ions and degraded organic components are co-released into the extracellular space. Ions and growth factors, although different in their biological nature, induce a common and coordinated chemotactic effect. Conditioned media has been used successfully in bone regeneration by promoting endogenous cell recruitment. Likewise, calcium alone act as a paracrine chemotactic signal, inducing the host's undifferentiated progenitor cell infiltration into the implanted biomaterials. The aim of the present study was to compare the chemotactic effect of calcium and conditioned media in primary calvarial cells. METHODS: The chemotactic cell response was evaluated in vitro using an agarose spot and a wound healing assay. In addition, we used a calvarial bone explant model ex-vivo. The healing potential was also tested through an in vivo model, a critical-size calvarial bone defect in mice. For the in vivo experiment, cell-free calcium-containing or conditioned media-containing scaffolds were implanted, and MSC's seeded scaffolds were used as positive control. After seven weeks post-implantation, samples were retrieved, and bone regeneration was evaluated by µCT and histological analysis. Osteogenic gene expression was evaluated by qPCR. RESULTS: We found that chemotactic cell migration in response to either calcium or conditioned media was equivalent in vitro and ex vivo. Accordingly, µCT analysis showed that bone regeneration induced by the MSC's seeded scaffolds was similar to that obtained with cell-free calcium or conditioned media-containing scaffolds. Pre-treatment with SB202190, a highly selective p38 inhibitor, abrogated the chemotactic effect induced by conditioned media. In contrast, p38 activity was not essential for the calcium-induced chemotaxis. Moreover, BAPTA-AM treatment, a cytosolic calcium chelator, decreased the chemotactic effect and the expression of key osteogenic genes induced by calcium or conditioned media. CONCLUSION: We show that calcium ions alone not only mimic the conditioned media chemotactic effect, but also induce an osteogenic effect similar to that produced by transplanted MSC's in vivo. Furthermore, the chemotactic effect induced by conditioned media is calcium and p38 dependent. The rise in cytosolic calcium might integrate the different signaling pathways triggered by conditioned media and extracellular Ca2+. This calcium-driven in situ bone regeneration is a promising and convenient alternative to promote endogenous cell recruitment into the injured bone site. This pre-clinical cell-free and growth factor-free approach might avoid the disadvantages of the ex vivo cell manipulation.

miR-219a-5p Regulates Rorß During Osteoblast Differentiation and in Age-related Bone Loss.

Developing novel approaches to treat skeletal disorders requires an understanding of how critical molecular factors regulate osteoblast differentiation and bone remodeling. We have reported that (1) retinoic acid receptor-related orphan receptor beta (Rorß) is upregulated in bone samples isolated from aged mice and humans in vivo; (2) Rorß expression is inhibited during osteoblastic differentiation in vitro; and (3) genetic deletion of Rorß in mice results in preservation of bone mass during aging. These data establish that Rorß inhibits osteogenesis and that strict control of Rorß expression is essential for bone homeostasis. Because microRNAs (miRNAs) are known to play important roles in the regulation of gene expression in bone, we explored whether a predicted subset of nine miRNAs regulates Rorß expression during both osteoblast differentiation and aging. Mouse osteoblastic cells were differentiated in vitro and assayed for Rorß and miRNA expression. As Rorß levels declined with differentiation, the expression of many of these miRNAs, including miR-219a-5p, was increased. We further demonstrated that miR-219a-5p was decreased in bone samples from old (24-month) mice, as compared with young (6-month) mice, concomitant with increased Rorß expression. Importantly, we also found that miR-219a-5p expression was decreased in aged human bone biopsies compared with young controls, demonstrating that this phenomenon also occurs in aging bone in humans. Inhibition of miR-219a-5p in mouse calvarial osteoblasts led to increased Rorß expression and decreased alkaline phosphatase expression and activity, whereas a miR-219a-5p mimic decreased Rorß expression and increased osteogenic activity. Finally, we demonstrated that miR-219a-5p physically interacts with Rorß mRNA in osteoblasts, defining Rorß as a true molecular target of miR-219a-5p. Overall, our findings demonstrate that miR-219a-5p is involved in the regulation of Rorß in both mouse and human bone. © 2018 American Society for Bone and Mineral Research.

Calcium-containing scaffolds induce bone regeneration by regulating mesenchymal stem cell differentiation and migration.

BACKGROUND: Osteoinduction and subsequent bone formation rely on efficient mesenchymal stem cell (MSC) recruitment. It is also known that migration is induced by gradients of growth factors and cytokines. Degradation of Ca2+-containing biomaterials mimics the bone remodeling compartment producing a localized calcium-rich osteoinductive microenvironment. The aim of our study was to determine the effect of calcium sulfate (CaSO4) on MSC migration. In addition, to evaluate the influence of CaSO4 on MSC differentiation and the potential molecular mechanisms involved. METHODS: A circular calvarial bone defect (5 mm diameter) was created in the parietal bone of 35 Balb-C mice. We prepared and implanted a cell-free agarose/gelatin scaffold alone or in combination with different CaSO4 concentrations into the bone defects. After 7 weeks, we determined the new bone regenerated by micro-CT and histological analysis. In vitro, we evaluated the CaSO4 effects on MSC migration by both wound healing and agarose spot assays. Osteoblastic gene expression after BMP-2 and CaSO4 treatment was also evaluated by qPCR. RESULTS: CaSO4 increased MSC migration and bone formation in a concentration-dependent manner. Micro-CT analysis showed that the addition of CaSO4 significantly enhanced bone regeneration compared to the scaffold alone. The histological evaluation confirmed an increased number of endogenous cells recruited into the cell-free CaSO4-containing scaffolds. Furthermore, MSC migration in vitro and active AKT levels were attenuated when CaSO4 and BMP-2 were in combination. Addition of LY294002 and Wortmannin abrogated the CaSO4 effects on MSC migration. CONCLUSIONS: Specific CaSO4 concentrations induce bone regeneration of calvarial defects in part by acting on the host's undifferentiated MSCs and promoting their migration. Progenitor cell recruitment is followed by a gradual increment in osteoblast gene expression. Moreover, CaSO4 regulates BMP-2-induced MSC migration by differentially activating the PI3K/AKT pathway. Altogether, these results suggest that CaSO4 scaffolds could have potential applications for bone regeneration.

Extracellular calcium promotes bone formation from bone marrow mesenchymal stem cells by amplifying the effects of BMP-2 on SMAD signalling.

Understanding the molecular events that regulate osteoblast differentiation is essential for the development of effective approaches to bone regeneration. In this study, we analysed the osteoinductive properties of extracellular calcium in bone marrow-derived mesenchymal stem cell (BM-MSC) differentiation. We cultured BM-MSCs in 3D gelatin scaffolds with Ca2+ and BMP-2 as osteoinductive agents. Early and late osteogenic gene expression and bone regeneration in a calvarial critical-size defect model demonstrate that extracellular Ca2+ enhances the effects of BMP-2 on Osteocalcin, Runx2 and Osterix expression and promotes bone regeneration in vivo. Moreover, we analysed the molecular mechanisms involved and observed an antagonistic effect between Ca2+ and BMP-2 on SMAD1/5, ERK and S6K signalling after 24 hours. More importantly, a cooperative effect between Ca2+ and BMP-2 on the phosphorylation of SMAD1/5, S6, GSK3 and total levels of ß-CATENIN was observed at a later differentiation time (10 days). Furthermore, Ca2+ alone favoured the phosphorylation of SMAD1, which correlates with the induction of Bmp2 and Bmp4 gene expression. These data suggest that Ca2+ and BMP-2 cooperate and promote an autocrine/paracrine osteogenic feed-forward loop. On the whole, these results demonstrate the usefulness of calcium-based bone grafts or the addition of exogenous Ca2+ in bone tissue engineering.

Mesenchymal Stem Cells Within Gelatin/CaSO4 Scaffolds Treated Ex Vivo with Low Doses of BMP-2 and Wnt3a Increase Bone Regeneration.

The delivery of osteogenic factors is a proven therapeutic strategy to promote bone regeneration. Bone morphogenetic proteins (BMPs) constitute a family of cytokines with well-known osteogenic and bone regenerative abilities. However, clinical uses of BMPs require high doses that have been associated with complications such as osteolysis, ectopic bone formation, or hematoma formation. In the present work, we sought to improve bone tissue engineering through an approach that combines the use of bone marrow-derived mesenchymal stem cells (BMMSCs), composite scaffolds, and osteoinductive agents. We employed a composite gelatin/CaSO4 scaffold that allows for an early expansion of seeded BMMSCs, which is followed by an increased level of osteogenic differentiation after 10 days in culture. Furthermore, this scaffold enhanced bone formation by BMMSCs in a mouse model of critical-sized calvarial defect. More importantly, our results demonstrate that ex vivo pretreatment of BMMSCs with low amounts of BMP-2 (2 nM) and Wnt3a (50 ng/mL) for 24 h cooperatively increases the expression of osteogenic markers in vitro and bone regeneration in the critical-sized calvarial defect mouse model. These data provide a strong rationale for the development of an ex vivo cooperative use of BMP-2 and Wnt3a. Osteogenic factor cooperation might be applied to reduce the required amount of growth factors while obtaining higher therapeutic effects.