Cellular senescence, a stress-induced irreversible growth arrest often characterized by expression of p16(Ink4a) (encoded by the Ink4a/Arf locus, also known as Cdkn2a) and a distinctive secretory phenotype, prevents the proliferation of preneoplastic cells and has beneficial roles in tissue remodelling during embryogenesis and wound healing. Senescent cells accumulate in various tissues and organs over time, and have been speculated to have a role in ageing. To explore the physiological relevance and consequences of naturally occurring senescent cells, here we use a previously established transgene, INK-ATTAC, to induce apoptosis in p16(Ink4a)-expressing cells of wild-type mice by injection of AP20187 twice a week starting at one year of age. We show that compared to vehicle alone, AP20187 treatment extended median lifespan in both male and female mice of two distinct genetic backgrounds. The clearance of p16(Ink4a)-positive cells delayed tumorigenesis and attenuated age-related deterioration of several organs without apparent side effects, including kidney, heart and fat, where clearance preserved the functionality of glomeruli, cardio-protective KATP channels and adipocytes, respectively. Thus, p16(Ink4a)-positive cells that accumulate during adulthood negatively influence lifespan and promote age-dependent changes in several organs, and their therapeutic removal may be an attractive approach to extend healthy lifespan.

Inhibition of angiogenesis is a promising therapeutic strategy against cancer. In this study, we reported that ZLM-7, a combretastain A-4 (CA-4) derivative, exhibited anti-angiogenic activity in vitro and in vivo. In vitro, ZLM-7 induced microtubule cytoskeletal disassembly. It decreased VEGF-induced proliferation, migration, invasion and tube formation in endothelial cells, which are critical steps in angiogenesis. In vivo, ZLM-7 significantly inhibited neovascularization in a chicken chorioallantoic membrane (CAM) model and reduced the microvessel density in tumor tissues of MCF-7 xenograft mouse model. ZLM-7 also displayed comparable antiangiogenic and anti-tumor activities associated with the lead compound CA-4, but exhibited lower toxicity compared with CA-4. The anti-angiogenic effect of ZLM-7 was exerted via blockade of VEGF/VEGFR-2 signaling. ZLM-7 treatment suppressed the expression and secretion of VEGF in endothelial cells and MCF-7 cells under hypoxia. Further, ZLM-7 suppressed the VEGF-induced phosphorylation of VEGFR-2 and its downstream signaling mediators including activated AKT, MEK and ERK in endothelial cells. Overall, these results demonstrate that ZLM-7 exhibits anti-angiogenic activities by impairing endothelial cell function and blocking VEGF/VEGFR-2 signaling, suggesting that ZLM-7 might be a potential angiogenesis inhibitor.

Lineage differentiation of bone marrow mesenchymal stem cells (BMMSCs) is the key to bone-fat reciprocity in bone marrow. To date, the regulators of BMMSC lineage switching have all been identified to be transcription factors, and researchers have not determined whether other genes control this process. This study aims to reveal a previously unknown role of tissue-nonspecific alkaline phosphatase (TNSALP) in controlling BMMSC lineage selection. Methods: We compared the characteristics of cultured BMMSCs from patients with hypophosphatasia (HPP), which is caused by mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene, and an ALPL knockout (ko) mouse model. We performed ALPL downregulation and overexpression experiments to investigate the regulatory role of ALPL in BMMSC lineage switching. Using the PathScan array, coimmunoprecipitation experiments and pathway-guided small molecule treatments, we explored the possible mechanism underlying the regulatory effects of ALPL on cell differentiation and evaluated its therapeutic effect on ALPL ko mice. Results: BMMSCs from both patients with HPP and ALPL ko mice exhibited defective lineage differentiation, including a decrease in osteogenic differentiation and a parallel increase in adipogenic differentiation. Mechanistically, TNSALP directly interacted with LRP6 and regulated the phosphorylation of GSK3β, subsequently resulting in lineage switching of BMMSCs. Re-phosphorylation of GSK3β induced by LiCl treatment restored differentiation of BMMSCs and attenuated skeletal deformities in Alpl+/- mice. Conclusion: Based on our findings, TNSALP acts as a signal regulator to control lineage switching of BMMSCs by regulating the LRP6/GSK3β cascade.

Glioblastoma (GBM) is a lethal cancer of the central nervous system with a median survival rate of 15 months with treatment. Thus, there is a critical need to develop novel therapies for GBM. Immunotherapy is emerging as a promising therapeutic strategy. However, current therapies for GBM, in particular anti-angiogenic therapies that block vascular endothelial growth factor (VEGF), may have undefined consequences on the efficacy of immunotherapy. While this treatment is primarily prescribed to reduce tumor vascularization, multiple immune cell types also express VEGF receptors, including the most potent antigen-presenting cell, the dendritic cell (DC). Therefore, we assessed the role of anti-VEGF therapy in modifying DC function. We found that VEGF blockade results in a more mature DC phenotype in the brain, as demonstrated by an increase in the expression of the co-stimulatory molecules B7-1, B7-2, and MHC II. Furthermore, we observed reduced levels of the exhaustion markers PD-1 and Tim-3 on brain-infiltrating CD8 T cells, indicating improved functionality. Thus, anti-angiogenic therapy has the potential to be used in conjunction with and enhance immunotherapy for GBM.

[Purpose] An estimation model of the knee and ankle joint angles during the extension phase was proposed in the previous study. However, it had limited use because of the fixed initial lower limb angle before standing up. This study aimed to propose a new estimation model of the initial lower limb angle to improve the angle estimation during extension phase. [Subjects and Methods] Seven healthy male volunteers were enrolled. The new estimation model approximated the initial lower limb angle using a force sensor plate that measured the plantar pressure of the subjects. The estimated angle and force were compared to those obtained by a motion capture system and force plate. [Results] The new estimation model of initial lower limb angle showed no significant difference compared with the true values obtained by motion capture, except for the subject who had a greater foot-pressure measurement error compared with the force plate measurement, with maximum errors of 5.98° and 6.31°, respectively. [Conclusion] The proposed model in this study can estimate the initial lower limb angle before standing and can be applied to the angle estimation model during the extension phase of the standing-up movement.

Mice overexpressing the mitotic checkpoint kinase gene BubR1 live longer, whereas mice hypomorphic for BubR1 (BubR1(H/H)) live shorter and show signs of accelerated aging. As wild-type mice age, BubR1 levels decline in many tissues, a process that is proposed to underlie normal aging and age-related diseases. Understanding why BubR1 declines with age and how to slow this process is therefore of considerable interest. The sirtuins (SIRT1-7) are a family of NAD(+)-dependent deacetylases that can delay age-related diseases. Here, we show that the loss of BubR1 levels with age is due to a decline in NAD(+) and the ability of SIRT2 to maintain lysine-668 of BubR1 in a deacetylated state, which is counteracted by the acetyltransferase CBP. Overexpression of SIRT2 or treatment of mice with the NAD(+) precursor nicotinamide mononucleotide (NMN) increases BubR1 abundance in vivo. Overexpression of SIRT2 in BubR1(H/H) animals increases median lifespan, with a greater effect in male mice. Together, these data indicate that further exploration of the potential of SIRT2 and NAD(+) to delay diseases of aging in mammals is warranted.

The aim of the present study was to investigate the periodontitis-associated changes in the number, proliferation and differentiation potential of human periodontal ligament stem cells (PDLSCs). Cultures of human periodontal ligament cells (PDLCs) were established from healthy donors and donors with periodontitis. The numbers of stem cell were characterized using flow cytometry. PDLSCs were isolated from the PDLCs by immunomagnetic bead selection. Colony‑forming abilities, osteogenic and adipogenic potential, gene expression of cementoblast phenotype, alkaline phosphatase activity and in vivo differentiation capacities were then evaluated. Periodontitis caused an increase in the proliferation of PDLSCs and a decrease in the commitment to the osteoblast lineage. This is reflected by changes in the expression of osteoblast markers. When transplanted into immunocompromised mice, PDLSCs from the healthy donors exhibited the capacity to produce cementum PDL‑like structures, whereas, the inflammatory PDLSCs transplants predominantly formed connective tissues. In conclusion, the data from the present study suggest that periodontitis affects the proliferation and differentiation potential of human PDLSCs in vitro and in vivo.

Psychological stress emerges to be a common health burden in the current society for its highly related risk of mental and physical disease outcomes. However, how the quickly-adaptive stress response process connects to the long-observed organismal alterations still remains unclear. Here, we investigated the profile of circulatory extracellular vesicles (EVs) after acute stress (AS) of restraint mice by phenotypic and proteomic analyses. We surprisingly discovered that AS-EVs demonstrated significant changes in size distribution and plasma concentration compared to control group (CN) EVs. AS-EVs were further characterized by various differentially expressed proteins (DEPs) closely associated with biological, metabolic and immune regulations and were functionally important in potentially underlying multiple diseases. Notably, we first identified the lipid raft protein Stomatin as an essential biomarker expressed on the surface of AS-EVs. These findings collectively reveal that EVs are a significant function-related liquid biopsy indicator that mediate circulation alterations impinged by psychological stress, while also supporting the idea that psychological stress-associated EV-stomatin can be used as a biomarker for potentially predicting acute stress responses and monitoring psychological status. Our study will pave an avenue for implementing routine plasma EV-based theranostics in the clinic.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by articular destruction and functional loss. Methotrexate (MTX) is effective in RA treatment. However, MTX induces several adverse events and 20%-30% of patients do not respond to MTX. Thus, it is urgent to enhance the therapeutic effects and reduce the side effects of MTX. Recent studies showed that mesenchymal stem cells (MSCs) were participants in anti-inflammation, immunoregulation, and tissue regeneration. However, whether the combined application of MSCs and MTX promotes the therapeutic effects and reduces the side effects of MTX has not been studied. In this study, we used bovine type II collagen to induce rheumatoid arthritis in mice (collagen-induced arthritis, CIA). Then, CIA mice were subjected to MTX or MSC treatment, or both. The therapeutic effect and adverse events of different treatments on RA were evaluated with micro-CT, HE staining, and immunohistochemistry in vivo. Apoptosis and proliferation of MODE-K cells were measured after treated with MTX or/and cocultured with UCs. To test M2 polarization, Raw264.7 macrophages were stimulated by MTX with different concentrations or cocultured with UCs. We found that the combined application of MSCs and MTX increased the therapeutic effects on RA, as evidenced by decreased arthritis score, inflammatory responses, and mortality. Moreover, in this combination remedy, MTX prefers to suppress inflammation by facilitating macrophage polarization to M2 type while UCs prefer to eliminate gastrointestinal side effects of MTX via mitigating the apoptosis of intestinal epithelial cells. Thus, a combination of MTX and UCs is a promising strategy for RA treatment.

The mammalian liver's regenerative ability has led researchers to engineer animals as incubators for expansion of human hepatocytes. The expansion properties of human hepatocytes in immunodeficient mice are well known. However, little has been reported about larger animals that are more scalable and practical for clinical purposes. Therefore, we engineered immunodeficient swine to support expansion of human hepatocytes and identify barriers to their clinical application. Immunodeficient swine were engineered by knockout of the recombinase-activating gene 2 (RAG2) and fumarylacetoacetate hydrolase (FAH). Immature human hepatocytes (ihHCs) were injected into fetal swine by intrauterine cell transplantation (IUCT) at day 40 of gestation. Human albumin was measured as a marker of engraftment. Cytotoxicity against ihHCs was measured in transplanted piglets and control swine. We initially detected higher levels of human albumin in cord blood of newborn FAH/RAG2-deficient (FR) pigs compared with immunocompetent controls (196.26 ng/dL vs. 39.29 ng/dL, p = 0.008), indicating successful engraftment of ihHCs after IUCT and adaptive immunity in the fetus. Although rare hepatocytes staining positive for human albumin were observed, levels of human albumin did not rise after birth, but declined, suggesting rejection of xenografted ihHCs. Cytotoxicity against ihHCs increased after birth by 3.8% (95% CI: [2.1%-5.4%], p < 0.001) and inversely correlated with declining levels of human albumin (p = 2.1 × 10-5, R2 = 0.17). Circulating numbers of T cells and B cells were negligible in FR pigs. However, circulating natural killer (NK) cells exerted cytotoxicity against ihHCs. NK cell activity was lower in immunodeficient piglets after IUCT than in naive controls (30.4% vs. 40.1%, p = 0.011, 95% CI for difference [2.7%-16.7%]). In conclusion, ihHCs were successfully engrafted in FR swine after IUCT. NK cells were a significant barrier to expansion of hepatocytes. New approaches are needed to overcome this hurdle and allow large-scale expansion of human hepatocytes in immunodeficient swine. Impact statement There is currently a need for robust expansion of human hepatocytes. We describe an immunodeficient swine model into which we engrafted immature human hepatocytes (ihHCs). We identified the mechanism of the eventual graft rejection by the intact NK cell population, which has not been previously shown to have a significant role in xenograft rejection. By both improving engraftment and reducing NK cell-mediated cytotoxicity toward the graft through intrauterine cell transfer, we confirmed the presence of residual adaptive immunity in this model of immunodeficiency and the ability to induce hyposensitization in the NK cell population by taking advantage of the fetal microenvironment.

It is reported that osteoporosis commonly occurs among patients with rheumatoid arthritis (RA), whereas the association between osteoporosis and osteoarthritis (OA) remains controversial. Our aim in this study was to investigate the association between BMD, as a marker of osteoporosis, and OA and RA among adults 20-59 years of age, using a population-based sample from the National Health and Nutrition Examination Survey (NHANES).

Osteoarthritis (OA) is a chronic degenerative joint disease accompanied by an elevated macrophage proinflammatory phenotype, which is triggered by persistent pathologically elevated calcium ion levels in mitochondria. However, existing pharmacological compounds targeting the inhibition of mitochondrial calcium ion (m[Ca2+]) influx are currently limited in terms of plasma membrane permeability and low specificity for ion channels and transporters. In the present study, we synthesized mesoporous silica nanoparticle-amidated (MSN)-ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA)/triphenylphosphine (TPP)-polyethylene glycol (PEG) [METP] nanoparticles (NPs), which specifically target mitochondria and block excess calcium ion influx.

The role of bone marrow-derived mesenchymal stem cells(BMSCs)in the pathogenesis and therapy of osteoporosis has drawn increasing attention in recent years. In the development of osteoporosis, it has been demonstrated that many changes occurred in the behavior of BMSCs. For example, the biological system of FasL pathways mediated differentiation of ERK and GSK-3β-catenin pathway was damaged. Here we found that 0.35 mg/L Licochalcone A (L-A) had a strong effect in increasing the osteogenic differentiation and mineralization of BMSCs both in vivo and in vitro by up-regulating FasL and further playing a role in regulating the ERK and GSK-3β-catenin systems. It has also demonstrated that the administration of L-A could restore the biological function of the damaged BMSCs differentiation by recovering or protecting bone mass in a disease state through activating the endosteal bone formation and partially inhibiting bone resorption in acute estrogen deficiency model. Results of our study suggested that careful titration of MSC was response to L-A and up-regulated FasL pathways mediating differentiation of ERK and GSK-3β-catenin biological systems under disease state in vivo, restore the impaired function, is one of the ways of L-A relieve or treatment osteoporosis.

Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C), initiates sister-chromatid separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome missegregation and formation of aneuploid gametes.

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome-microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element-binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.

High expression of the protein kinase Bub1 has been observed in a variety of human tumors and often correlates with poor clinical prognosis, but its molecular and cellular consequences and role in tumorigenesis are unknown. Here, we demonstrate that overexpression of Bub1 in mice leads to near-diploid aneuploidies and tumor formation. We found that chromosome misalignment and lagging are the primary mitotic errors responsible for the observed aneuploidization. High Bub1 levels resulted in aberrant Bub1 kinase activity and hyperactivation of Aurora B kinase. When Aurora B activity is suppressed, pharmacologically or via BubR1 overexpression, chromosome segregation errors caused by Bub1 overexpression are largely corrected. Importantly, Bub1 transgenic mice overexpressing Bub1 developed various kinds of spontaneous tumors and showed accelerated Myc-induced lymphomagenesis. Our results establish that Bub1 has oncogenic properties and suggest that Aurora B is a critical target through which overexpressed Bub1 drives aneuploidization and tumorigenesis.

The anaphase-promoting complex/cyclosome (APC/C) E3 ubiquitin ligase functions with the E2 ubiquitin-conjugating enzyme UbcH10 in the orderly progression through mitosis by marking key mitotic regulators for destruction by the 26-S proteasome. UbcH10 is overexpressed in many human cancer types and is associated with tumor progression. However, whether UbcH10 overexpression causes tumor formation is unknown. To address this central question and to define the molecular and cellular consequences of UbcH10 overexpression, we generated a series of transgenic mice in which UbcH10 was overexpressed in graded fashion. In this study, we show that UbcH10 overexpression leads to precocious degradation of cyclin B by the APC/C, supernumerary centrioles, lagging chromosomes, and aneuploidy. Importantly, we find that UbcH10 transgenic mice are prone to carcinogen-induced lung tumors and a broad spectrum of spontaneous tumors. Our results identify UbcH10 as a prominent protooncogene that causes whole chromosome instability and tumor formation over a wide gradient of overexpression levels.

Mosaic Variegated Aneuploidy (MVA) syndrome is a rare autosomal recessive disorder characterized by inaccurate chromosome segregation and high rates of near-diploid aneuploidy. Children with MVA syndrome die at an early age, are cancer prone, and have progeroid features like facial dysmorphisms, short stature, and cataracts. The majority of MVA cases are linked to mutations in BUBR1, a mitotic checkpoint gene required for proper chromosome segregation. Affected patients either have bi-allelic BUBR1 mutations, with one allele harboring a missense mutation and the other a nonsense mutation, or mono-allelic BUBR1 mutations combined with allelic variants that yield low amounts of wild-type BubR1 protein. Parents of MVA patients that carry single allele mutations have mild mitotic defects, but whether they are at risk for any of the pathologies associated with MVA syndrome is unknown. To address this, we engineered a mouse model for the nonsense mutation 2211insGTTA (referred to as GTTA) found in MVA patients with bi-allelic BUBR1 mutations. Here we report that both the median and maximum lifespans of the resulting BubR1(+/GTTA) mice are significantly reduced. Furthermore, BubR1(+/GTTA) mice develop several aging-related phenotypes at an accelerated rate, including cataract formation, lordokyphosis, skeletal muscle wasting, impaired exercise ability, and fat loss. BubR1(+/GTTA) mice develop mild aneuploidies and show enhanced growth of carcinogen-induced tumors. Collectively, these data demonstrate that the BUBR1 GTTA mutation compromises longevity and healthspan, raising the interesting possibility that mono-allelic changes in BUBR1 might contribute to differences in aging rates in the general population.

The mitotic checkpoint protein Bub1 is essential for embryogenesis and survival of proliferating cells, and bidirectional deviations from its normal level of expression cause chromosome missegregation, aneuploidy, and cancer predisposition in mice. To provide insight into the physiological significance of this critical mitotic regulator at a modular level, we generated Bub1 mutant mice that lack kinase activity using a knockin gene-targeting approach that preserves normal protein abundance. In this paper, we uncover that Bub1 kinase activity integrates attachment error correction and mitotic checkpoint signaling by controlling the localization and activity of Aurora B kinase through phosphorylation of histone H2A at threonine 121. Strikingly, despite substantial chromosome segregation errors and aneuploidization, mice deficient for Bub1 kinase activity do not exhibit increased susceptibility to spontaneous or carcinogen-induced tumorigenesis. These findings provide a unique example of a modular mitotic activity orchestrating two distinct networks that safeguard against whole chromosome instability and reveal the differential importance of distinct aneuploidy-causing Bub1 defects in tumor suppression.

BubR1 is a key component of the spindle assembly checkpoint (SAC). Mutations that reduce BubR1 abundance cause aneuploidization and tumorigenesis in humans and mice, whereas BubR1 overexpression protects against these. However, how supranormal BubR1 expression exerts these beneficial physiological impacts is poorly understood. Here, we used Bub1b mutant transgenic mice to explore the role of the amino-terminal (BubR1(N)) and internal (BubR1(I)) Cdc20-binding domains of BubR1 in preventing aneuploidy and safeguarding against cancer. BubR1(N) was necessary, but not sufficient to protect against aneuploidy and cancer. In contrast, BubR1 lacking the internal Cdc20-binding domain provided protection against both, which coincided with improved microtubule-kinetochore attachment error correction and SAC activity. Maximal SAC reinforcement occurred when both the Phe- and D-box of BubR1(I) were disrupted. Thus, while under- or overexpression of most mitotic regulators impairs chromosome segregation fidelity, certain manipulations of BubR1 can positively impact this process and therefore be therapeutically exploited.