Nitric oxide promotes cell-matrix adhesion of endothelial progenitor cells under hypoxia condition via ITGA5 CpG promoter demethylation.

Hypoxia or low oxygen tension causes changes in the structure and functional phenotype of the endothelial progenitor cells (EPCs). EPCs are found to be involved in angiogenesis and vascular repair. However, EPC's role in cell-matrix adhesion under hypoxia conditions is not clearly established. Nitric oxide (NO) exerts a wide range of biological functions, especially in regulating the mobilization and vascular repair of EPCs. In contrast, the link between NO and its role in cell-matrix deadhesion under hypoxia is not studied yet. Here, we investigated the protective role of NO in hypoxia-induced cell-matrix deadhesion of EPCs through an epigenetic mechanism. The EPCs were exposed to 2% hypoxia in the presence or absence of 10 µM Spermine NONOate (NO donor). The result demonstrates that hypoxia exposure intensified mitochondrial oxidative damage and energy defects. Using miScript miRNA qPCR array-based screening, the study found miR-148 as a novel target of hypoxia-induced DNMT1 activation. Mechanistically, the study discovered that hypoxia suppressed miR-148 levels and stimulated EPCs cell-matrix deadhesion via increasing DNMT1 mediated Integrin alpha-5 (ITGA5) CpG promoter hypermethylation. Treatment with a mitochondria-targeted antioxidant, MitoTEMPO, or epigenetic DNMT inhibitor, 5'-azacitidine, or miR-148 overexpression in hypoxic EPCs culture, prevented the cell-matrix deadhesion compared to hypoxic EPCs. Further, treatment of spNO or transient expression of eNOS-GFP attenuated hypoxia-induced cell-matrix deadhesion via inhibition of ITGA5 CpG island promoter methylation. In conclusion, the study provides evidence that NO is essential for cell-matrix adhesion of EPCs by epigenetically mitigating ITGA5 CpG promoter hypermethylation under hypoxia conditions. This finding uncovers the previously undefined mechanism of NO-mediated diminution of hypoxia-induced cell-matrix deadhesion and dysfunction induced by low oxygen tension.

Diabetic Covid-19 severity: Impaired glucose tolerance and pathologic bone loss.

Diabetes mellitus (DM), hypertension, and cardiovascular diseases (CVDs) are the leading chronic comorbidities that enhance the severity and mortality of COVID-19 cases. However, SARS-CoV-2 mediated deregulation of diabetes pathophysiology and comorbidity that links the skeletal bone loss remain unclear. We used both streptozocin-induced type 2 diabetes (T2DM) mouse and hACE2 transgenic mouse to enable SARS-CoV-2-receptor binding domain (RBD) mediated abnormal glucose metabolism and bone loss phenotype in mice. The data demonstrate that SARS-CoV-2-RBD treatment in pre-existing diabetes conditions in hACE2 (T2DM + RBD) mice results in the aggravated osteoblast inflammation and downregulation of Glucose transporter 4 (Glut4) expression via upregulation of miR-294-3p expression. The data also found increased fasting blood glucose and reduced insulin sensitivity in the T2DM + RBD condition compared to the T2DM condition. Femoral trabecular bone mass loss and bone mechanical quality were further reduced in T2DM + RBD mice. Mechanistically, silencing of miR-294 function improved Glut4 expression, glucose metabolism, and bone formation in T2DM + RBD + anti-miR-294 mice. These data uncover the previously undefined role of SARS-CoV-2-RBD treatment mediated complex pathological symptoms of diabetic COVID-19 mice with abnormal bone metabolism via a miRNA-294/Glut4 axis. Therefore, this work would provide a better understanding of the interplay between diabetes and SARS-CoV-2 infection.

Temporal dynamics of nitric oxide wave in early vasculogenesis.

Endothelium-derived nitric oxide (NO) is a mediator of angiogenesis. However, NO-mediated regulation of vasculogenesis remains largely unknown. In the present study, we show that the inhibition of NO significantly attenuated endothelial migration, ring formation, and tube formation. The contribution of nitric oxide synthase (NOS) enzymes during early vasculogenesis was assessed by evaluating endothelial NOS (eNOS) and inducible NOS (iNOS) mRNA expression during HH10-HH13 stages of chick embryo development. iNOS but not eNOS was expressed at HH12 and HH13 stages. We hypothesized that vasculogenic events are controlled by NOS-independent reduction of nitrite to NO under hypoxia during the very early phases of development. Semi-quantitative polymerase chain reaction analysis of hypoxia-inducible factor-1α (HIF-1α) showed higher expression at HH10 stage, after which a decrease was observed. This observation was in correlation with the nitrite reductase (NR) activity at HH10 stage. We observed a sodium nitrite-induced increase in NO levels at HH10, reaching a gradual decrease at HH13. The possible involvement of a HIF/NF-κB/iNOS signaling pathway in the process of early vasculogenesis is suggested by the inverse relationship observed between nitrite reduction and NOS activation between HH10 and HH13 stages. Further, we detected that NR-mediated NO production was inhibited by several NR inhibitors at the HH10 stage, whereas the inhibitors eventually became less effective at later stages. These findings suggest that the temporal dynamics of the NO source switches from NR to NOS in the extraembryonic area vasculosa, where both nitrite reduction and NOS activity are defined by hypoxia.

Hydrogen sulfide prevents ethanol-induced ZO-1 CpG promoter hypermethylation-dependent vascular permeability via miR-218/DNMT3a axis.

Ethanol (ET) causes cerebrovascular dysfunction by altering homocysteine (Hcy) metabolism and by causing oxidative stress. However, there are no strategies to prevent ET-induced epigenetic deregulation of tight junction protein (hyper-methylation) and endothelial cell permeability to date. Hydrogen sulfide (H2 S) has an antioxidative, antiapoptotic, and anti-inflammatory effect. Here, we investigated the protective role of H2 S in ET-induced endothelial permeability through epigenetic changes in mouse brain endothelial cells (bEnd3). The bEnd3 cells were exposed to 50 mM ET treatment in the presence or absence of 50 µM NaHS (H2 S donor). The result demonstrates that ET-induced cellular toxicity increased intracellular Hcy levels, which further intensified mitochondrial dysfunction and energy defects. Using miScript microRNA (miRNA) polymerase chain reaction array-based screening, we identified a particular miRNA, miR-218, as a novel target of ET-induced DNA methyltransferase-3a (DNMT3a) activation. miR-218 influences CpG island methylation of the zonula occludens 1 (ZO-1) promoter in the endothelial cells. We discovered that ET suppressed miR-218 levels and induced endothelial permeability via DNMT3a-mediated ZO-1 hyper-methylation. Treatment with mito-TEMPO (mitochondria-targeted antioxidant), 5'-azacitidine (DNMT inhibitor), or miR-218 overexpression was shown to protect endothelial cells against ET-induced permeability. Also, bEnd3 cells pretreated with NaHS attenuated ET-induced vascular permeability and prevented CpG island methylation at the promoter. In conclusion, our data provide evidence that H2 S treatment protects vascular integrity from ET-induced stress by mitigating CpG (ZO-1 promoter) DNA hyper-methylation. This finding uncovers a new mechanistic understanding of NaHS/H2 S, that may have therapeutic potential in preventing or diminishing ET-induced brain vascular permeability and dysfunction induced by alcoholism.

Allyl sulfide promotes osteoblast differentiation and bone density via reducing mitochondrial DNA release mediated Kdm6b/H3K27me3 epigenetic mechanism.

Age-associated bone loss or osteoporosis is a common clinical manifestation during aging (AG). The mechanism underlying age-associated osteoblast dysfunction induced by oxidative damage in the mitochondria and loss of bone density remains elusive. Here, we demonstrated the effect of allyl sulfide (AS), a natural organosulfur compound, on mitochondrial (mt) function in bone marrow-derived mesenchymal stem cells (BMMSCs) and bone density in AG mice. The data demonstrate that AS treatment in AG mice promotes BMMSCs differentiation and mineralization via inhibition of mitochondrial oxidative damage. The data also indicate that AG related mito-damage was associated with reduced mitochondrial biogenesis and oxidative phosphorylation, and release of a greater concentration of mtDNA. Furthermore, the data showed that mtDNA caused histone H3K27 demethylase inhibition, KDM6B, and subsequent inflammation by unbalancing mitochondrial redox homeostasis. KDM6B overexpression in AG BMMSCs or AS administration in AG mice restores osteogenesis and bone density in vitro and in vivo. Mechanistically, AS or the mitochondrial-specific antioxidant Mito-TEMPO increased KDM6B expression and upregulated the expression of Runx2 in BMMSCs, probably via epigenetic inhibition of H3K27me3 methylation at the promoter. These data uncover the previously undefined role of AS mediated prevention of mtDNA release, promoting osteogenesis and bone density via an epigenetic mechanism. Therefore, AS could be a potential drug target for the treatment of aging-associated osteoporosis.

Nitric oxide restores peripheral blood mononuclear cell adhesion against hypoxia via NO-cGMP signalling.

Hypoxia is the most detrimental threat to humans residing at high altitudes, affecting multifaceted cellular responses that are crucial for normal homeostasis. Inhalation of nitric oxide has been successfully implemented to combat the hypoxia effect in the high altitude patients. We hypothesize that nitric oxide (NO) restores the peripheral blood mononuclear cell-matrix deadhesion during hypoxia. In the present study, we investigate the cellular action of exogenous NO in the hypoxia-mediated diminution of cell-matrix adhesion of PBMNC and NO bioavailability in vitro. The result showed that NO level and cell-matrix adhesion of PBMNC were significantly reduced in hypoxia as compared with normoxia, as assessed by the DAF-FM and cell adhesion assay, respectively. In contrast, cellular oxidative damage response was indeed upregulated in hypoxic PBMNC. Further, gene expression analysis revealed that mRNA transcripts of cell adhesion molecules (Integrin α5 and ß1) and eNOS expressions were significantly downregulated. The mechanistic study revealed that administration of NO and 8-Br-cGMP and overexpression of eNOS-GFP restored the basal NO level and recovers cell-matrix adhesion in PBMNC via cGMP-dependent protein kinase I (PKG I) signalling. In conclusion, NO-cGMP/PKG signalling may constitute a novel target to recover high altitude-afflicted cellular deadhesion. SIGNIFICANCE OF THIS STUDY: Cellular adhesion is a complex multistep process. The ability of cells to adhere to extracellular matrix is an essential physiological process for normal homeostasis and function. Hypoxia exposure in the PBMNC culture has been proposed to induce oxidative damage and cellular deadhesion and is generally believed to be the key factor in the reduction of NO bioavailability. In the present study, we demonstrated that NO donor or overexpression of eNOS-GFP has a protective effect against hypoxia-induced cellular deadhesion and greatly improves the redox balance by inhibiting the oxidative stress. Furthermore, this protective effect of NO is mediated by the NO-cGMP/PKG signal pathway, which may provide a potential strategy against hypoxia.

Hydrogen sulfide attenuates homocysteine-induced osteoblast dysfunction by inhibiting mitochondrial toxicity.

Homocysteine (Hcy) is detrimental to bone health in a mouse model of diet-induced hyperhomocysteinemia (HHcy). However, little is known about Hcy-mediated osteoblast dysfunction via mitochondrial oxidative damage. Hydrogen sulfide (H2 S) has potent antioxidant, anti-inflammatory, and antiapoptotic effects. In this study, we hypothesized that the H2 S mediated recovery of osteoblast dysfunction by maintaining mitochondrial biogenesis in Hcy-treated osteoblast cultures in vitro. MC3T3-E1 osteoblastic cells were exposed to Hcy treatment in the presence or absence of an H2 S donor (NaHS). Cell viability, osteogenic differentiation, reactive oxygen species (ROS) production were determined. Mitochondrial DNA copy number, adenosine triphosphate (ATP) production, and oxygen consumption were also measured. Our results demonstrated that administration of Hcy increases the intracellular Hcy level and decreases intracellular H2 S level and expression of the cystathionine ß-synthase/Cystathionine γ-lyase system, thereby inhibiting osteogenic differentiation. Pretreatment with NaHS attenuated Hcy-induced mitochondrial toxicity (production of total ROS and mito-ROS, ratio of mitochondrial fission (DRP-1)/fusion (Mfn-2)) and restored ATP production and mitochondrial DNA copy numbers as well as oxygen consumption in the osteoblast as compared with the control, indicating its protective effects against Hcy-induced mitochondrial toxicity. In addition, NaHS also decreased the release of cytochrome c from the mitochondria to the cytosol, which induces cell apoptosis. Finally, flow cytometry confirmed that NaHS can rescue cells from apoptosis induced by Hcy. Our studies strongly suggest that NaHS has beneficial effects on mitochondrial toxicity, and could be developed as a potential therapeutic agent against HHcy-induced mitochondrial dysfunction in cultured osteoblasts in vitro.

Hyperhomocysteinemia induced endothelial progenitor cells dysfunction through hyper-methylation of CBS promoter.

Bone marrow (BM)-derived endothelial progenitor cells (EPCs) are the key players in angiogenesis and vascular function. Cystathionine-ß-synthase (CBS), an H2S-generating enzyme in methionine metabolism, regulates the function of these EPCs. This study aims to examine whether CBS hyper-methylation contributes to the bone marrow endothelial progenitor cell (BM-EPCs) function and subsequent bone blood flow in mice fed with a high methionine diet (HMD). Bone marrow (BM) cells were collected from HMD and control mice, differentiated into BM-EPCs, and were characterized by acLDL-DiI labeling. CBS mRNA expression was analyzed by real-time PCR, and the global methylation status and methylation of the CBS promoter were detected by nuclear 5-mC assay and methylation-specific PCR (qMSP) respectively. The result reveals that CBS promoter in BM-EPCs from HMD mice was hyper-methylated and the methylation level was, indeed, negatively correlated with CBS mRNA and angiogenic function of BM-EPCs. In addition, global methylation (5-mC) and DNA methyltransferase-1 (DNMT1) expression were increased in HMD condition. In vitro study also shows that HMD induced hyperhomocysteinemia (HHcy) impaired both adhesion and angiogenesis properties of BM-EPCs, accompanied by higher methylation level of CBS promoter that compared to control. Furthermore, bone blood flow was found to be decreased in HMD mice as compared to wild-type mice. To dissect the epigenetic mechanism, we also administrated DNMT inhibitor, 5-azacytidine (5-Aza) to HMD mice. The administration of 5-Aza in HMD mice restored the CBS expression, EPC mediated angiogenesis and blood flow by reducing abnormal DNA hyper-methylation. In conclusion, HHcy dismantles BM-EPCs function and bone blood flow through the hyper-methylation of the CBS promoter in HMD fed mice.

Hydrogen sulfide alleviates hyperhomocysteinemia-mediated skeletal muscle atrophy via mitigation of oxidative and endoplasmic reticulum stress injury.

Although hyperhomocysteinemia (HHcy) occurs because of the deficiency in cystathionine-ß-synthase (CBS) causing skeletal muscle dysfunction, it is still unclear whether this effect is mediated through oxidative stress, endoplasmic reticulum (ER) stress, or both. Nevertheless, there is no treatment option available to improve HHcy-mediated muscle injury. Hydrogen sulfide (H2S) is an antioxidant compound, and patients with CBS mutation do not produce H2S. In this study, we hypothesized that H2S mitigates HHcy-induced redox imbalance/ER stress during skeletal muscle atrophy via JNK phosphorylation. We used CBS+/- mice to study HHcy-mediated muscle atrophy, and treated them with sodium hydrogen sulfide (NaHS; an H2S donor). Proteins and mRNAs were examined by Western blots and quantitative PCR. Proinflammatory cytokines were also measured. Muscle mass and strength were studied via fatigue susceptibility test. Our data revealed that HHcy was detrimental to skeletal mass, particularly gastrocnemius and quadriceps muscle weight. We noticed that oxidative stress was reversed by NaHS in homocysteine (Hcy)-treated C2C12 cells. Interestingly, ER stress markers (GRP78, ATF6, pIRE1α, and pJNK) were elevated in vivo and in vitro, and NaHS mitigated these effects. Additionally, we observed that JNK phosphorylation was upregulated in C2C12 after Hcy treatment, but NaHS could not reduce this effect. Furthermore, inflammatory cytokines IL-6 and TNF-α were higher in plasma from CBS as compared with wild-type mice. FOXO1-mediated Atrogin-1 and MuRF-1 upregulation were attenuated by NaHS. Functional studies revealed that NaHS administration improved muscle fatigability in CBS+/- mice. In conclusion, our work provides evidence that NaHS is beneficial in mitigating HHcy-mediated skeletal injury incited by oxidative/ER stress responses.

Tetrahydrocurcumin ameliorates homocysteine-mediated mitochondrial remodeling in brain endothelial cells.

Homocysteine (Hcy) causes endothelial dysfunction by inducing oxidative stress in most neurodegenerative disorders. This dysfunction is highly correlated with mitochondrial dynamics such as fusion and fission. However, there are no strategies to prevent Hcy-induced mitochondrial remodeling. Tetrahydrocurcumin (THC) is an anti-inflammatory and anti-oxidant compound. We hypothesized that THC may ameliorates Hcy-induced mitochondria remodeling in mouse brain endothelial cells (bEnd3) cells. bEnd3 cells were exposed to Hcy treatment in the presence or absence of THC. Cell viability and autophagic cell death were measured with MTT and MDC staining assay. Reactive oxygen species (ROS) production was determined using DCFH-DA staining by confocal microscopy. Autophagy flux was assessed using a conventional GFP-microtubule-associated protein 1 light chain 3 (LC3) dot assay. Interaction of phagophore marker LC-3 with mitochondrial receptor NIX was observed by confocal imaging. Mitochondrial fusion and fission were evaluated by western blot and RT-PCR. Our results demonstrated that Hcy resulted in cell toxicity in a dose-dependent manner and supplementation of THC prevented the detrimental effects of Hcy on cell survival. Furthermore, Hcy also upregulated fission marker (DRP-1), fusion marker (Mfn2), and autophagy marker (LC-3). Finally, we observed that Hcy activated mitochondrial specific phagophore marker (LC-3) and co-localized with the mitochondrial receptor NIX, as viewed by confocal microscopy. Pretreatment of bEnd3 with THC (15 µM) ameliorated Hcy-induced oxidative damage, mitochondrial fission/fusion, and mitophagy. Our studies strongly suggest that THC has beneficial effects on mitochondrial remodeling and could be developed as a potential therapeutic agent against hyperhomocysteinemia (HHcy) induced mitochondrial dysfunction.

Homocysteine as a Pathological Biomarker for Bone Disease.

In the last few decades, perturbation in methyl-group and homocysteine (Hcy) balance have emerged as independent risk factors in a number of pathological conditions including neurodegenerative disease, cardiovascular dysfunction, cancer development, autoimmune disease, and kidney disease. Recent studies report Hcy to be a newly recognized risk factor for osteoporosis. Elevated Hcy levels are known to modulate osteoclastgenesis by causing detrimental effects on bone via oxidative stress induced metalloproteinase-mediated extracellular matrix degradation and decrease in bone blood flow. Evidence from previous studies also suggests that the decreased chondrocytes mediated bone mineralization in chick limb-bud mesenchymal cells and during the gestational period of ossification in rat model. However, Hcy imbalance and its role in bone loss, regression in vascular invasion, and osteoporosis, are not clearly understood. More investigations are required to explore the complex interplay between Hcy imbalance and onset of bone disease progression. This article reviews the current body of knowledge on regulation of Hcy mediated oxidative stress and its role in bone remodeling, vascular blood flow and progression of bone disease. J. Cell. Physiol. 232: 2704-2709, 2017. © 2016 Wiley Periodicals, Inc.

Hypermethylation: Causes and Consequences in Skeletal Muscle Myopathy.

A detrimental consequence of hypermethylation is hyperhomocysteinemia (HHcy), that causes oxidative stress, inflammation, and matrix degradation, which leads to multi-pathology in different organs. Although, it is well known that hypermethylation leads to overall gene silencing and hypomethylation leads to overall gene activation, the role of such process in skeletal muscle dysfunction during HHcy condition is unclear. In this study, we emphasized the multiple mechanisms including epigenetic alteration by which HHcy causes skeletal muscle myopathy. This review also highlights possible role of methylation, histone modification, and RNA interference in skeletal muscle dysfunction during HHcy condition and potential therapeutic molecules, putative challenges, and methodologies to deal with HHcy mediated skeletal muscle dysfunction. We also highlighted that B vitamins (mainly B12 and B6), with folic acid supplementation, could be useful as an adjuvant therapy to reverse these consequences associated with this HHcy conditions in skeletal muscle. However, we would recommend to further study involving long-term trials could help to assess efficacy of the use of these therapeutic agents. J. Cell. Biochem. 118: 2108-2117, 2017. © 2017 Wiley Periodicals, Inc.

Breast cancer drugs dampen vascular functions by interfering with nitric oxide signaling in endothelium.

Widely used chemotherapeutic breast cancer drugs such as Tamoxifen citrate (TC), Capecitabine (CP) and Epirubicin (EP) are known to cause various cardiovascular side-effects among long term cancer survivors. Vascular modulation warrants nitric oxide (NO) signal transduction, which targets the vascular endothelium. We hypothesize that TC, CP and EP interference with the nitric oxide downstream signaling specifically, could lead to cardiovascular dysfunctions. The results demonstrate that while all three drugs attenuate NO and cyclic guanosine mono-phosphate (cGMP) production in endothelial cells, they caused elevated levels of NO in the plasma and RBC. However, PBMC and platelets did not show any significant changes under treatment. This implies that the drug effects are specific to the endothelium. Altered eNOS and phosphorylated eNOS (Ser-1177) localization patterns in endothelial cells were observed following drug treatments. Similarly, the expression of phosphorylated eNOS (Ser-1177) protein was decreased under the treatment of drugs. Altered actin polymerization was also observed following drug treatment, while addition of SpNO and 8Br-cGMP reversed this effect. Incubation with the drugs decreased endothelial cell migration whereas addition of YC-1, SC and 8Br-cGMP recovered the effect. Additionally molecular docking studies showed that all three drugs exhibited a strong binding affinity with the catalytic domain of human sGC. In conclusion, results indicate that TC, CP and EP cause endothelial dysfunctions via the NO-sGC-cGMP pathway and these effects could be recovered using pharmaceutical agonists of NO signaling pathway. Further, the study proposes a combination therapy of chemotherapeutic drugs and cGMP analogs, which would confer protection against chemotherapy mediated vascular dysfunctions in cancer patients.

miRNA-15/IL-10Rα axis promotes Kabasura Kudineer (Indian traditional Siddha formulation) induced immunomodulation by suppressing oxidative stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Kabasura Kudineer (KK), the traditional Indian medicine of Siddha, effectively manages common respiratory symptoms such as flu, cold, and fever. However, there is no evidence of the immunomodulatory capacity of KK in the cultured Jurkat T-lymphocytes under the LPS insult studied. AIM OF THE STUDY: Assess the effect of the traditional Indian medicine of Siddha, Kabasura Kudineer (KK) on immunomodulation by suppressing oxidative damage in cultured Jurkat T cells in vitro. The miRNA activity on anti-inflammatory gene receptors and cellular nitric oxide levels also was studied. MATERIALS AND METHODS: Jurkat T cells were exposed to LPS treatment in the presence or absence of KK. Cell viability and nitric oxide (NO) were measured with MTT and Griess assay. Cellular antioxidant systems (glutathione and SOD) were determined using glutathione and SOD assay. Lipid peroxidation was measured using an MDA assay. MiRNA-15a-5p expression was performed using microRNA qPCR Assays. Both inflammatory and anti-inflammatory genes (IL-6, IL-1, IL-10, IL-13) were performed using a qPCR and ELISA assay. RESULTS: The data showed that reduced cell proliferation and exaggerated NO production was observed in LPS treated condition compared to the control condition. Further, LPS treatment increased lipid peroxidation and reduced antioxidant enzyme activities (SOD and glutathione) in cultured Jurkat T cells. However, treatment with KK or N-acetyl cysteine (NAC; antioxidant) treatment mitigates the above effect. Mechanistically, LPS-induced oxidative stress upregulated miR- 15-5p expression and suppressed IL-10 Receptor alpha (IL-10Rα) by binding to its 3'-UTR region. The deregulated expression of IL-10Rα expression leads to increased IL-6 and IL-1ß expression in LPS-induced Jurkat T cells; however, treatment with KK or NAC reversed the above effects. CONCLUSION: Collectively, our study revealed the previously undefined mechanistic role of Kabasura Kudineer (KK) that alleviates the LPS-induced oxidative damage associated with inflammation by inhibiting the miRNA-15-5p/IL-10Rα axis.

Exercise-Linked Skeletal Irisin Ameliorates Diabetes-Associated Osteoporosis by Inhibiting the Oxidative Damage-Dependent miR-150-FNDC5/Pyroptosis Axis.

Recent evidence suggests that physical exercise (EX) promotes skeletal development. However, the impact of EX on the progression of bone loss and deterioration of mechanical strength in mice with type 2 diabetic mellitus (T2DM) remains unexplored. In the current study, we investigated the effect of EX on bone mass and mechanical quality using a diabetic mouse model. The T2DM mouse model was established with a high-fat diet with two streptozotocin injections (50 mg/kg/body wt) in C57BL/6 female mice. The diabetic mice underwent treadmill exercises (5 days/week at 7-11 m/min for 60 min/day) for 8 weeks. The data showed that diabetes upregulated miR-150 expression through oxidative stress and suppressed FNDC5/Irisin by binding to its 3'-untranslated region. The decreased level of irisin further triggers the pyroptosis response in diabetic bone tissue. EX or N-acetyl cysteine or anti-miRNA-150 transfection in T2DM mice restored FNDC5/Irisin expression and bone formation. Furthermore, EX or recombinant irisin administration prevented T2DM-Induced hyperglycemia and improved glucose intolerance in diabetic mice. Furthermore, osteoblastic knockdown of Nlrp3 silencing (si-Nlrp3) or pyroptosis inhibitor (Ac-YVADCMK [AYC]) treatment restores bone mineralization in diabetic mice. Micro-computed tomography scans and mechanical testing revealed that trabecular bone microarchitecture and bone mechanical properties were improved after EX in diabetic mice. Irisin, either induced by skeleton or daily EX or directly administered, prevents bone loss by mitigating inflammasome-associated pyroptosis signaling in diabetic mice. This study demonstrates that EX-induced skeletal irisin ameliorates diabetes-associated glucose intolerance and bone loss and possibly provides a mechanism of its effects on metabolic osteoporosis.

Mechanisms of autophagy and mitophagy in skeletal development, diseases and therapeutics.

Autophagy is a highly evolutionarily conserved process in the eukaryotic cellular system by which dysfunctional organelles are selectively degraded through a series of processes of lysosomal activity and then returned to the cytoplasm for reuse. All cells require this process to maintain cellular homeostasis and promote cell survival during stress responses such as deprivation and hypoxia. Osteoblasts and osteoclasts are two cellular phenotypes in the bone that mediate bone homeostasis. However, an imbalance between osteoblastic bone formation and osteoclastic bone resorption contributes to the onset of bone diseases. Recent studies suggest that autophagy, mitophagy, and selective mitochondrial autophagy may play an essential role in regulating osteoblast differentiation and osteoclast maturation. Autophagic activity dysregulation alters the equilibrium between osteoblastic bone creation and osteoclastic bone resorption, allowing bone disorders like osteoporosis to develop more easily. The current review emphasizes the role of autophagy and mitophagy and their related molecular mechanisms in bone metabolic disorders. In the current review, we emphasize the role of autophagy and mitophagy as well as their related molecular mechanism in bone metabolic disorders. Furthermore, we will discuss autophagy as a target for the treatment of metabolic bone disease and future application in therapeutic translational research.

Garlic exosome-like nanoparticles reverse high-fat diet induced obesity via the gut/brain axis.

Background: Obesity is becoming a global epidemic and reversing the pathological processes underlying obesity and metabolic co-morbidities is challenging. Obesity induced chronic inflammation including brain inflammation is a hallmark of obesity via the gut-brain axis. The objective of this study was to develop garlic exosome-like nanoparticles (GaELNs) that inhibit systemic as well as brain inflammatory activity and reverse a HFD induced obesity in mice. Methods: GELNs were isolated and administrated orally into HFD fed mice. GaELNs were fluorescent labeled for monitoring their in vivo trafficking route after oral administration and quantified the number particles in several tissues. The brain inflammation was determined by measuring inflammatory cytokines by ELISA and real-time PCR. Mitochondrial membrane permeability of microglial cells was determined using JC-10 fluorescence dye. The in vivo apoptotic cell death was quantified by TUNEL assay. The brain metabolites were identified and quantified by LC-MS analysis. Memory function of the mice was determined by several memory functional analysis. The effect of GaELNs on glucose and insulin response of the mice was determined by glucose and insulin tolerance tests. c-Myc localization and interaction with BASP1 and calmodulin was determined by confocal microscopy. Results: Our results show that GaELNs is preferentially taken up microglial cells and inhibits the brain inflammation in HFD mice. GaELN phosphatidic acid (PA) (36:4) is required for the uptake of GaELNs via interaction with microglial BASP1. Formation of the GaELNs/BASP1 complex is required for inhibition of c-Myc mediated expression of STING. GaELN PA binds to BASP1, leading to inhibition of c-Myc expression and activity through competitively binding to CaM with c-Myc transcription factor. Inhibition of STING activity leads to reducing the expression of an array of inflammatory cytokines including IFN-γ and TNF-α. IFN-γ induces the expression of IDO1, which in turn the metabolites generated as IDO1 dependent manner activate the AHR pathway that contributes to developing obesity. The metabolites derived from the GaELNs treated microglial cells promote neuronal differentiation and inhibit mitochondrial mediated neuronal cell death. GaELNs treated HFD mice showed improved memory function and increased glucose tolerance and insulin sensitivity in these mice. Conclusion: Collectively, these results demonstrate how nanoparticles from a healthy diet can inhibit unhealthy high-fat diet induced brain inflammation and reveal a link between brain microglia/diet to brain inflammatory disease outcomes via diet-derived exosome-like nanoparticles.

Exosomal lncRNA-H19 promotes osteogenesis and angiogenesis through mediating Angpt1/Tie2-NO signaling in CBS-heterozygous mice.

Rationale: Emerging evidence indicates that the growth of blood vessels and osteogenesis is tightly coordinated during bone development. However, the molecular regulators of intercellular communication in the bone microenvironment are not well studied. Therefore, we aim to investigate whether BMMSC-Exo promotes osteogenesis and angiogenesis via transporting lnc-H19 in the CBS- heterozygous mouse model. Methods: Using RT2 lncRNA PCR array screening, we identify a bone-specific, long noncoding RNA-H19 (lncRNA-H19/lnc-H19) in exosomes derived from bone marrow mesenchymal stem cells (BMMSC-Exo) during osteogenesis. Using bioinformatics analysis, we further discovered the seed sequence of miR-106a that could bind to lnc-H19. A luciferase reporter assay was performed to demonstrate the direct binding of miR-106a to the target gene angiopoietin 1 (Angpt1). We employed an immunocompromised Nude mouse model, to evaluate the effects of BMMSC-Exo on angiogenesis in vivo. Using a micro-CT scan, we monitored microstructural changes of bone in the experimental mice. Results: BMMSC-Exo possessed exosomal characteristics including exosome size, and typical markers including CD63, CD9, and TSD101. In vitro, BMMSC-Exo significantly promoted endothelial angiogenesis and osteogenesis. Mechanistic studies have shown that exosomal lnc-H19 acts as "sponges" to absorb miR-106 and regulate the expression of angiogenic factor, Angpt1 that activates lnc-H19/Tie2-NO signaling in mesenchymal and endothelial cells. Both of these effects on osteogenesis and angiogenesis are inhibited by antagonizing Tie2 signaling. Treatment of BMMSC-Exo also restored the bone formation and mechanical quality in vivo. Conclusion: These findings provide a novel insight into how the extracellular role of exosomal lnc-H19 affects osteogenesis and angiogenesis through competing endogenous RNA networks.

Probiotics Stimulate Bone Formation in Obese Mice via Histone Methylations.

Rationale: Manipulation of the gut microbiome can prevent pathologic bone loss. However, the effects of probiotics on mitochondrial epigenetic remodeling and skeletal homeostasis in the high-fat diet (HFD)-linked obesity remains to be explored. Here, we examined the impact of probiotics supplementation on mitochondrial biogenesis and bone homeostasis through the histone methylation mechanism in HFD fed obese mice. Methods: 16S rRNA gene sequencing was performed to study the microbiota composition in the gut and microbial dysbiosis in obese mouse model. High resolution (microPET/CT) imaging was performed to demonstrate the obese associated colonic inflammation. Obese-associated upregulation of target miRNA in osteoblast was investigated using a microRNA qPCR array. Osteoblastic mitochondrial mass was evaluated using confocal imaging. Overexpression of mitochondrial transcription factor (Tfam) was used to investigate the glycolysis and mitochondrial bioenergetic metabolism using Tfam-transgenic (Tg) mice fed on HFD. The bone formation and mechanical strength was evaluated by microCT analysis and three-point bending analysis. Results: High-resolution imaging (µ-CT) and mechanical testing revealed that probiotics induced a significant increase of trabecular bone volume and bone mechanical strength respectively in obese mice. Probiotics or Indole-3-propionic acid (IPA) treatment directly to obese mice, prevents gut inflammation, and improved osteoblast mineralization. Mechanistically, probiotics treatment increases mitochondrial transcription factor A (Tfam) expression in osteoblasts by promoting Kdm6b/Jmjd3 histone demethylase, which inhibits H3K27me3 epigenetic methylation at the Tfam promoter. Furthermore, Tfam-transgenic (Tg) mice, fed with HFD, did not experience obesity-linked reduction of glucose uptake, mitochondrial biogenesis and mineralization in osteoblasts. Conclusions: These results suggest that the probiotics mediated changes in the gut microbiome and its derived metabolite, IPA are potentially be a novel agent for regulating bone anabolism via the gut-bone axis.

Rebuilding Microbiome for Mitigating Traumatic Brain Injury: Importance of Restructuring the Gut-Microbiome-Brain Axis.

Traumatic brain injury (TBI) is a damage to the brain from an external force that results in temporary or permanent impairment in brain functions. Unfortunately, not many treatment options are available to TBI patients. Therefore, knowledge of the complex interplay between gut microbiome (GM) and brain health may shed novel insights as it is a rapidly expanding field of research around the world. Recent studies show that GM plays important roles in shaping neurogenerative processes such as blood-brain-barrier (BBB), myelination, neurogenesis, and microglial maturation. In addition, GM is also known to modulate many aspects of neurological behavior and cognition; however, not much is known about the role of GM in brain injuries. Since GM has been shown to improve cellular and molecular functions via mitigating TBI-induced pathologies such as BBB permeability, neuroinflammation, astroglia activation, and mitochondrial dysfunction, herein we discuss how a dysbiotic gut environment, which in fact, contributes to central nervous system (CNS) disorders during brain injury and how to potentially ward off these harmful effects. We further opine that a better understanding of GM-brain (GMB) axis could help assist in designing better treatment and management strategies in future for the patients who are faced with limited options.