Special AT‐rich sequence‐binding protein 2 (Satb2) synergizes with Bmp9 and is essential for osteo/odontogenic differentiation of mouse incisor mesenchymal stem cells

ObjectivesMouse incisor mesenchymal stem cells (MSCs) have self‐renewal ability and osteo/odontogenic differentiation potential. However, the mechanism controlling the continuous self‐renewal and osteo/odontogenic differentiation of mouse incisor MSCs remains unclear. Special AT‐rich sequence‐binding protein 2 (SATB2) positively regulates craniofacial patterning, bone development and regeneration, whereas SATB2 deletion or mutation leads to craniomaxillofacial dysplasia and delayed tooth and root development, similar to bone morphogenetic protein (BMP) loss‐of‐function phenotypes. However, the detailed mechanism underlying the SATB2 role in odontogenic MSCs is poorly understood. The aim of this study was to investigate whether SATB2 can regulate self‐renewal and osteo/odontogenic differentiation of odontogenic MSCs.Materials and methods Satb2 expression was detected in the rapidly renewing mouse incisor mesenchyme by immunofluorescence staining, quantitative RT‐PCR and Western blot analysis. Ad‐Satb2 and Ad‐siSatb2 were constructed to evaluate the effect of Satb2 on odontogenic MSCs self‐renewal and osteo/odontogenic differentiation properties and the potential role of Satb2 with the osteogenic factor bone morphogenetic protein 9 (Bmp 9) in vitro and in vivo.Results Satb2 was found to be expressed in mesenchymal cells and pre‐odontoblasts/odontoblasts. We further discovered that Satb2 effectively enhances mouse incisor MSCs self‐renewal. Satb2 acted synergistically with the potent osteogenic factor Bmp9 in inducing osteo/odontogenic differentiation of mouse incisor MSCs in vitro and in vivo.Conclusions Satb2 promotes self‐renewal and osteo/odontogenic differentiation of mouse incisor MSCs. Thus, Satb2 can cooperate with Bmp9 as a new efficacious bio‐factor for osteogenic regeneration and tooth engineering.     

Hypertrophic Preconditioning Attenuates Myocardial Ischaemia‐Reperfusion Injury by Modulating SIRT3‐SOD2‐mROS‐Dependent Autophagy

BackgroundIschaemic preconditioning elicited by brief periods of coronary occlusion and reperfusion protects the heart from a subsequent prolonged ischaemic insult. Here, we test the hypothesis that short‐term non‐ischaemic stimulation of hypertrophy renders the heart resistant to subsequent ischaemic injury.Methods and ResultsTransient transverse aortic constriction (TAC) was performed for 3 days in mice and then withdrawn for 4 days by aortic debanding, followed by subsequent exposure to myocardial ischaemia‐reperfusion (I/R) injury. Following I/R injury, myocardial infarct size and apoptosis were significantly decreased, and cardiac dysfunction was markedly improved in the TAC preconditioning group compared with the control group. Mechanistically, TAC preconditioning markedly suppressed I/R‐induced autophagy and preserved autophagic flux by deacetylating SOD2 via a SIRT3‐dependent mechanism. Moreover, treatment with an adenovirus encoding SIRT3 partially mimicked the effects of hypertrophic preconditioning, whereas genetic ablation of SIRT3 in mice blocked the cardioprotective effects of hypertrophic preconditioning. Furthermore, in vivo lentiviral‐mediated knockdown of Beclin 1 in the myocardium ameliorated the I/R‐induced impairment of autophagic flux and was associated with a reduction in cell death, whereas treatment with a lentivirus encoding Beclin 1 abolished the cardioprotective effect of TAC preconditioning.ConclusionsThe present study identifies TAC preconditioning as a novel strategy for induction of an endogenous self‐defensive and cardioprotective mechanism against cardiac injury. Specifically, TAC preconditioning reduced myocardial autophagic cell death in a SIRT3/SOD2 pathway‐dependent manner.     

Hyaluronate supports hESC‐cardiomyocyte cell therapy for cardiac regeneration after acute myocardial infarction

IntroductionEnormous progress has been made in cardiac regeneration using human embryonic stem cell‐derived cardiomyocyte (hESC‐CM) grafts in pre‐clinical trials. However, the rate of cell survival has remained very low due to anoikis after transplantation into the heart as single cells. Numerous solutions have been proposed to improve cell survival, and one of these strategies is to co‐transplant biocompatible materials or hydrogels with the hESC‐CMs.MethodsIn our study, we screened various combinations of biomaterials that could promote anoikis resistance and improve hESC‐CM survival upon co‐transplantation and promote cardiac functional recovery. We injected different combinations of Matrigel, alginate and hyaluronate with hESC‐CM suspensions into the myocardium of rat models with myocardial infarction (MI).ResultsOur results showed that the group treated with a combination of hyaluronate and hESC‐CMs had the lowest arrhythmia rates when stimulated with programmed electrical stimulation. While all three combinations of hydrogel‐hESC‐CM treatments improved rat cardiac function compared with the saline control group, the combination with hyaluronate most significantly reduced pathological changes from left ventricular remodelling and improved both left ventricular function and left ventricular ejection fraction by 28 days post‐infarction.ConclusionHence, we concluded that hyaluronate‐hESC‐CM is a superior combination therapy for promoting cardiac regeneration after myocardial infarction.     

Controlled X‐chromosome dynamics defines meiotic potential of female mouse in vitro germ cells

The mammalian germline is characterized by extensive epigenetic reprogramming during its development into functional eggs and sperm. Specifically, the epigenome requires resetting before parental marks can be established and transmitted to the next generation. In the female germline, X‐chromosome inactivation and reactivation are among the most prominent epigenetic reprogramming events, yet very little is known about their kinetics and biological function. Here, we investigate X‐inactivation and reactivation dynamics using a tailor‐made in vitro system of primordial germ cell‐like cell (PGCLC) differentiation from mouse embryonic stem cells. We find that X‐inactivation in PGCLCs in vitro and in germ cell‐competent epiblast cells in vivo is moderate compared to somatic cells, and frequently characterized by escaping genes. X‐inactivation is followed by step‐wise X‐reactivation, which is mostly completed during meiotic prophase I. Furthermore, we find that PGCLCs which fail to undergo X‐inactivation or reactivate too rapidly display impaired meiotic potential. Thus, our data reveal fine‐tuned X‐chromosome remodelling as a critical feature of female germ cell development towards meiosis and oogenesis.     

Decreased p53 is associated with a decline in asymmetric stem cell self‐renewal in aged human epidermis

With age, the epidermis becomes hypoplastic and hypoproliferative. Hypoproliferation due to aging has been associated with decreased stem cell (SC) self‐renewal in multiple murine tissues. The fate of SC self‐renewal divisions can be asymmetric (one SC, one committed progenitor) or symmetric (two SCs). Increased asymmetric SC self‐renewal has been observed in inflammatory‐mediated hyperproliferation, while increased symmetric SC self‐renewal has been observed in cancers. We analyzed SC self‐renewal divisions in aging human epidermis to better understand the role of SCs in the hypoproliferation of aging. In human subjects, neonatal to 78 years, there was an age‐dependent decrease in epidermal basal layer divisions. The balance of SC self‐renewal shifted toward symmetric SC self‐renewal, with a decline in asymmetric SC self‐renewal. Asymmetric SC divisions maintain epidermal stratification, and this decrease may contribute to the hypoplasia of aging skin. P53 decreases in multiple tissues with age, and p53 has been shown to promote asymmetric SC self‐renewal. Fewer aged than adult ALDH+CD44+ keratinocyte SCs exhibited p53 expression and activity and Nutlin‐3 (a p53 activator) returned p53 activity as well as asymmetric SC self‐renewal divisions to adult levels. Nutlin‐3 increased Notch signaling (NICD, Hes1) and DAPT inhibition of Notch activation prevented Nutlin‐3 (p53)‐induced asymmetric SC self‐renewal divisions in aged keratinocytes. These studies indicate a role for p53 in the decreased asymmetric SC divisions with age and suggest that in aged keratinocytes, Notch is required for p53‐induced asymmetric SC divisions.     

MST1/2 inhibitor XMU‐MP‐1 alleviates the injury induced by ionizing radiation in haematopoietic and intestinal system

The Hippo signalling pathway has been considered as potential therapeutic target in self‐renewal and differentiation of stem and progenitor cells. Thus, mammalian Ste20‐like kinase 1/2 (MST1/2) as the core serine‐threonine kinases in the Hippo signalling pathway has been investigated for its role in immunological disease. However, little information of MST1/2 function in bone marrow suppression induced by ionizing radiation was fully investigated. Here, we reported that MST1/2 inhibitor XMU‐MP‐1 could rescue the impaired haematopoietic stem cells (HSCs) and progenitor cells (HPCs) function under oxidative stress condition. Also, XMU‐MP‐1 pretreatment markedly alleviated the small intestinal system injury caused by the total body irradiation 9 Gy and extended the average survival days of the mice exposed to the lethal dose radiation. Therefore, irradiation exposure causes the serious pathological changes of haematopoietic and intestinal system, and XMU‐MP‐1 could prevent the ROS production, the haematopoietic cells impairment and the intestinal injury. These detrimental effects may be associated with regulating NOX/ROS/P38MARK pathway by MST1/2.     

New insights into the central sympathetic hyperactivity post‐myocardial infarction: Roles of METTL3‐mediated m6A methylation

Ventricular arrhythmias (VAs) triggers by sympathetic nerve hyperactivity contribute to sudden cardiac death in myocardial infarction (MI) patients. Microglia‐mediated inflammation in the paraventricular nucleus (PVN) is involved in sympathetic hyperactivity after MI. N6‐methyladenosine (m⁶A), the most prevalent mRNA and epigenetic modification, is critical for mediating cell inflammation. We aimed to explore whether METTL3‐mediated m⁶A modification is involved in microglia‐mediated sympathetic hyperactivity after MI in the PVN. MI model was established by left coronary artery ligation. METTL3‐mediated m⁶A modification was markedly increased in the PVN at 3 days after MI, and METTL3 was primarily located in microglia by immunofluorescence. RNA‐seq, MeRIP‐seq, MeRIP‐qPCR, immunohistochemistry, ELISA, heart rate variability measurements, renal sympathetic nerve activity recording and programmed electrical stimulation confirmed that the elevated toll‐like receptor 4 (TLR4) expression by m⁶A modification on TLR4 mRNA 3''‐UTR region combined with activated NF‐κB signalling led to the overwhelming production of pro‐inflammatory cytokines IL‐1β and TNF‐α in the PVN, thus inducing the sympathetic hyperactivity and increasing the incidence of VAs post‐MI. Targeting METTL3 attenuated the inflammatory response and sympathetic hyperactivity and reduced the incidence of VAs post‐MI.     

PINK1‐mediated mitophagy maintains pluripotency through optineurin

ObjectivesDysfunction of autophagy results in accumulation of depolarized mitochondria and breakdown of self‐renewal and pluripotency in ESCs. However, the regulators that control how mitochondria are degraded by autophagy for pluripotency regulation remains largely unknown. This study aims to dissect the molecular mechanisms that regulate mitochondrial homeostasis for pluripotency regulation in mouse ESCs.Materials and methods Parkin^+/+ and parkin ^−/− ESCs were established from E3.5 blastocysts of parkin^+/− x parkin^+/− mating mice. The pink1 ^−/−, optn ^−/− and ndp52 ^−/− ESCs were generated by CRISPR‐Cas9. shRNAs were used for function loss assay of target genes. Mito‐Keima, ROS and ATP detection were used to investigate the mitophagy and mitochondrial function. Western blot, Q‐PCR, AP staining and teratoma formation assay were performed to evaluate the PSC stemness.ResultsPINK1 or OPTN depletion impairs the degradation of dysfunctional mitochondria during reprogramming, and reduces the reprogramming efficiency and quality. In ESCs, PINK1 or OPTN deficiency leads to accumulation of dysfunctional mitochondria and compromised pluripotency. The defective mitochondrial homeostasis and pluripotency in pink1 ^−/− ESCs can be compensated by gain expression of phosphomimetic Ubiquitin (Ub‐S65D) together with WT or a constitutively active phosphomimetic OPTN mutant (S187D, S476D, S517D), rather than constitutively inactive OPTN (S187A, S476A, S517A) or a Ub‐binding dead OPTN mutant (D477N).ConclusionsThe mitophagy receptor OPTN guards ESC mitochondrial homeostasis and pluripotency by scavenging damaged mitochondria through TBK1‐activated OPTN binding of PINK1‐phosphorylated Ubiquitin.     

MAIR‐II deficiency ameliorates cardiac remodelling post‐myocardial infarction by suppressing TLR9‐mediated macrophage activation

Macrophages are fundamental components of inflammation in post‐myocardial infarction (MI) and contribute to adverse cardiac remodelling and heart failure. However, the regulatory mechanisms in macrophage activation have not been fully elucidated. Previous studies showed that myeloid‐associated immunoglobulin–like receptor II (MAIR‐II) is involved in inflammatory responses in macrophages. However, its role in MI is unknown. Thus, this study aimed to determine a novel role and mechanism of MAIR‐II in MI. We first identified that MAIR‐II–positive myeloid cells were abundant from post‐MI days 3 to 5 in infarcted hearts of C57BL/6J (WT) mice induced by permanent left coronary artery ligation. Compared to WT, MAIR‐II–deficient (Cd300c2 ^−/−) mice had longer survival, ameliorated cardiac remodelling, improved cardiac function and smaller infarct sizes. Moreover, we detected lower pro‐inflammatory cytokine and fibrotic gene expressions in Cd300c2 ^−/−‐infarcted hearts. These mice also had less infiltrating pro‐inflammatory macrophages following MI. To elucidate a novel molecular mechanism of MAIR‐II, we considered macrophage activation by Toll‐like receptor (TLR) 9–mediated inflammation. In vitro, we observed that Cd300c2 ^−/− bone marrow–derived macrophages stimulated by a TLR9 agonist expressed less pro‐inflammatory cytokines compared to WT. In conclusion, MAIR‐II may enhance inflammation via TLR9‐mediated macrophage activation in MI, leading to adverse cardiac remodelling and poor prognosis.     

Genome‐scale metabolic modeling reveals SARS‐CoV‐2‐induced metabolic changes and antiviral targets

Tremendous progress has been made to control the COVID‐19 pandemic caused by the SARS‐CoV‐2 virus. However, effective therapeutic options are still rare. Drug repurposing and combination represent practical strategies to address this urgent unmet medical need. Viruses, including coronaviruses, are known to hijack host metabolism to facilitate viral proliferation, making targeting host metabolism a promising antiviral approach. Here, we describe an integrated analysis of 12 published in vitro and human patient gene expression datasets on SARS‐CoV‐2 infection using genome‐scale metabolic modeling (GEM), revealing complicated host metabolism reprogramming during SARS‐CoV‐2 infection. We next applied the GEM‐based metabolic transformation algorithm to predict anti‐SARS‐CoV‐2 targets that counteract the virus‐induced metabolic changes. We successfully validated these targets using published drug and genetic screen data and by performing an siRNA assay in Caco‐2 cells. Further generating and analyzing RNA‐sequencing data of remdesivir‐treated Vero E6 cell samples, we predicted metabolic targets acting in combination with remdesivir, an approved anti‐SARS‐CoV‐2 drug. Our study provides clinical data‐supported candidate anti‐SARS‐CoV‐2 targets for future evaluation, demonstrating host metabolism targeting as a promising antiviral strategy.     

Cell‐extracellular matrix interactions in the fluidic phase direct the topology and polarity of self‐organized epithelial structures

IntroductionIn vivo, cells are surrounded by extracellular matrix (ECM). To build organs from single cells, it is generally believed that ECM serves as scaffolds to coordinate cell positioning and differentiation. Nevertheless, how cells utilize cell‐ECM interactions for the spatiotemporal coordination to different ECM at the tissue scale is not fully understood.MethodsHere, using in vitro assay with engineered MDCK cells expressing H2B‐mCherry (nucleus) and gp135/Podocalyxin‐GFP (apical marker), we show in multi‐dimensions that such coordination for epithelial morphogenesis can be determined by cell‐soluble ECM interaction in the fluidic phase.ResultsThe coordination depends on the native topology of ECM components such as sheet‐like basement membrane (BM) and type I collagen (COL) fibres: scaffold formed by BM (COL) facilitates a close‐ended (open‐ended) coordination that leads to the formation of lobular (tubular) epithelium. Further, cells form apicobasal polarity throughout the entire lobule/tubule without a complete coverage of ECM at the basal side, and time‐lapse two‐photon scanning imaging reveals the polarization occurring early and maintained through the lobular expansion. During polarization, gp135‐GFP was converged to the apical surface collectively in the lobular/tubular structures, suggesting possible intercellular communications. Under suspension culture, the polarization was impaired with multi‐lumen formation in the tubules, implying the importance of ECM biomechanical microenvironment.ConclusionOur results suggest a biophysical mechanism for cells to form polarity and coordinate positioning at tissue scale, and in engineering epithelium through cell‐soluble ECM interaction and self‐assembly.     

Oral intake of Lactobacillus plantarum L‐14 extract alleviates TLR2‐ and AMPK‐mediated obesity‐associated disorders in high‐fat‐diet‐induced obese C57BL/6J mice

ObjectivesWhether periodic oral intake of postbiotics positively affects weight regulation and prevents obesity‐associated diseases in vivo is unclear. This study evaluated the action mechanism of Lactobacillus plantarum L‐14 (KTCT13497BP) extract and the effects of its periodic oral intake in a high‐fat‐diet (HFD) mouse model.Materials and methodsMouse pre‐adipocyte 3T3‐L1 cells and human bone marrow mesenchymal stem cells (hBM‐MSC) were treated with L‐14 extract every 2 days during adipogenic differentiation, and the mechanism underlying anti‐adipogenic effects was analysed at cellular and molecular levels. L‐14 extract was orally administrated to HFD‐feeding C57BL/6J mice every 2 days for 7 weeks. White adipose tissue was collected and weighed, and liver and blood serum were analysed. The anti‐adipogenic mechanism of exopolysaccharide (EPS) isolated from L‐14 extract was also analysed using Toll‐like receptor 2 (TLR2) inhibitor C29.ResultsL‐14 extract inhibited 3T3‐L1 and hBM‐MSC differentiation into mature adipocytes by upregulating AMPK signalling pathway in the early stage of adipogenic differentiation. The weight of the HFD + L‐14 group (31.51 ± 1.96 g) was significantly different from that of the HFD group (35.14 ± 3.18 g). L‐14 extract also significantly decreased the serum triacylglycerol/high‐density lipoprotein cholesterol ratio (an insulin resistance marker) and steatohepatitis. In addition, EPS activated the AMPK signalling pathway by interacting with TLR2, consequently inhibiting adipogenesis.ConclusionsEPS from L‐14 extract inhibits adipogenesis via TLR2 and AMPK signalling pathways, and oral intake of L‐14 extract improves obesity and obesity‐associated diseases in vivo. Therefore, EPS can be used to prevent and treat obesity and metabolic disorders.     

Mitochondrial derived peptide MOTS‐c prevents the development of heart failure under pressure overload conditions in mice

MOTS‐c, a mitochondrial‐derived peptide (MDP), has been shown to have multiple biological activities such as antioxidation, anti‐inflammation, and anti‐apoptosis properties. In the present study, we aimed at evaluating the therapeutic effect of MOTS‐c peptide in an animal model of heart failure. The heart failure mouse model was made by transverse aortic constriction (TAC) operations. The MOTS‐c peptide was administrated subcutaneously by using an osmotic pump. At the end of the animal experiment, cardiac function was evaluated by echocardiography, and heart tissues were subjected to histological and molecular analysis. In vitro cultured H9C2 cells were used to test the effects of MOTS‐c overexpression on cell death in response to H₂O₂ stimulation. Our study showed that MOTS‐c peptide attenuated TAC‐induced cardiac dysfunction and remodelling. In addition, the MOTS‐c peptide reduced the inflammatory response and upregulated the antioxidant capacity, coupled with the activation of the AMPK pathway in the heart of the TAC mouse model. In in vitro cultured cardiac cells, overexpression of MOTS‐c was shown to activate the AMPK pathway and protect cell apoptosis in response to H₂O₂ stimulation. Taken together, our study suggested that MOTS‐c peptides may have therapeutic potential in treating HF.     

Kcnh2 mediates FAK/AKT‐FOXO3A pathway to attenuate sepsis‐induced cardiac dysfunction

ObjectivesMyocardial dysfunction is a significant manifestation in sepsis, which results in high mortality. Even Kcnh2 has been hinted to associate with the pathological process, its involved signalling is still elusive.Materials and methodsThe caecal ligation puncture (CLP) surgery or lipopolysaccharide (LPS) injection was performed to induce septic cardiac dysfunction. Western blotting was used to determine KCNH2 expression. Cardiac function was examined by echocardiography 6 hours after CLP and LPS injection in Kcnh2 knockout (Kcnh2^+/‐) and NS1643 injection rats (n ≥ 6/group). Survival was monitored following CLP‐induced sepsis (n ≥ 8/group).ResultsSepsis could downregulate KCNH2 level in the rat heart, as well as in LPS‐stimulated cardiomyocytes but not cardiac fibroblast. Defect of Kcnh2 (Kcnh2^+/‐) significantly aggravated septic cardiac dysfunction, exacerbated tissue damage and increased apoptosis under LPS challenge. Fractional shortening and ejection fraction values were significantly decreased in Kcnh2^+/‐ group than Kcnh2^+/+ group. Survival outcome in Kcnh2^+/‐ septic rats was markedly deteriorated, compared with Kcnh2^+/+ rats. Activated Kcnh2 with NS1643, however, resulted in opposite effects. Lack of Kcnh2 caused inhibition of FAK/AKT signalling, reflecting in an upregulation for FOXO3A and its downstream targets, which eventually induced cardiomyocyte apoptosis and heart tissue damage. Either activation of AKT by activator or knockdown of FOXO3A with si‐RNA remarkably attenuated the pathological manifestations that Kcnh2 defect mediated.ConclusionKcnh2 plays a protection role in sepsis‐induced cardiac dysfunction (SCID) via regulating FAK/AKT‐FOXO3A to block LPS‐induced myocardium apoptosis, indicating a potential effect of the potassium channels in pathophysiology of SCID.     

Calhex231 ameliorates myocardial fibrosis post myocardial infarction in rats through the autophagy‐NLRP3 inflammasome pathway in macrophages

The calcium‐sensing receptor (CaSR) is involved in the pathophysiology of many cardiovascular diseases, including myocardial infarction (MI) and hypertension. The role of Calhex231, a specific inhibitor of CaSR, in myocardial fibrosis following MI is still unclear. Using Wistar rats, we investigated whether Calhex231 ameliorates myocardial fibrosis through the autophagy‐NLRP3 inflammasome pathway in macrophages post myocardial infarction (MI). The rats were randomly divided into sham, MI and MI + Calhex231 groups. Compared with the sham rats, the MI rats consistently developed severe cardiac function, myocardial fibrosis and infiltration of inflammatory cells including macrophages. Moreover, inflammatory pathway including activation of NLRP3 inflammasome, IL‐1β and autophagy was significantly up‐regulated in myocardial tissue, infiltrated cardiac macrophages and peritoneal macrophages of the MI rats. These impacts were reversed by Calhex231. In vitro, studies revealed that calindol and rapamycin exacerbated MI‐induced autophagy and NLRP3 inflammasome activation in peritoneal macrophages. Calhex231 and 3‐Methyladenine (a specific inhibitor of autophagy) attenuated both autophagy and NLRP3 inflammasome activation; however, the caspase‐1 inhibitor Z‐YVAD‐FMK did not. Our study indicated that Calhex231 improved cardiac function and ameliorated myocardial fibrosis post MI, likely via the inhibition of autophagy‐mediated NLRP3 inflammasome activation; this provides a new therapeutic target for ventricular remodelling‐related cardiovascular diseases.