Psoralen synergizes with exosome-loaded SPC25 to alleviate senescence of nucleus pulposus cells in intervertebral disc degeneration.

OBJECTIVE: To explore the mechanism of psoralen synergized with exosomes (exos)-loaded SPC25 on nucleus pulposus (NP) cell senescence in intervertebral disc degeneration (IVDD). METHODS: IVDD cellular models were established on NP cells by tert-butyl hydroperoxide (TBHP) induction, followed by the treatment of psoralen or/and exos from adipose-derived stem cells (ADSCs) transfected with SPC25 overexpression vector (ADSCs-oe-SPC25-Exos). The viability, cell cycle, apoptosis, and senescence of NP cells were examined, accompanied by the expression measurement of aggrecan, COL2A1, Bcl-2, Bax, CDK2, p16, and p21. RESULTS: After TBHP-induced NP cells were treated with psoralen or ADSCs-oe-SPC25-Exos, cell proliferation and the expression of aggrecan, COL2A1, Bcl-2, and CDK2 were promoted; however, the expression of Bax, p16, p21, and inflammatory factors was decreased, and cell senescence, cycle arrest, and apoptosis were inhibited. Of note, psoralen combined with ADSCs-oe-SPC25-Exos further decelerated NP cell senescence and cycle arrest compared to psoralen or ADSCs-oe-SPC25-Exos alone. CONCLUSION: Combined treatment of psoralen and ADSCs-oe-SPC25-Exos exerted an alleviating effect on NP cell senescence, which may provide an insightful idea for IVDD treatment.

Ischemia reperfusion myocardium injuries in type 2 diabetic rats: Effects of ketamine and insulin on LC3-II and mTOR expression.

Objectives: Myocardiopathy occurs in ischemia-induced injury caused by dysregulation of autophagy of cardiac tissues. The present report evaluates the protective effect of ketamine and insulin against myocardial injury in type 2 diabetic rats (T2DM).Methods: The effects of ketamine and its combination with insulin on biochemical parameters and inflammatory cytokines in the serum of I/R-induced myocardial injury in T2DM rats were evaluated. The parameters of reactive oxygen species and the expression of autophagosome signaling pathway proteins were also determined. Using transmission electron microscopy, we investigated autophagosomes. Western blots were used to detect autophagy-associated signaling pathways. Myocardial function was determined by echocardiography and histopathological changes in myocardial tissues were also determined in I/R-induced myocardial injury in type 2 diabetic rats.Results: There was a significant reduction in glucose, AST, LDH, and CK-MB levels and cytokines (IL-1ß, IL-6, and TNF-α) in serum of the ketamine (p < .05) and ketamine + insulin (p < .01) groups than in the diabetic + I/R. MDA and ROS levels were reduced with a substantial (p < .05) increase in GSH levels through improved cardiac function in the ketamine (p < .05) and ketamine + insulin (p < .01) groups than the diabetic + I/R group. There was an increase in mature autophagosomes in diabetic+I/R+Kt+In compared to diabetic+I/R+Kt alone in infarction and marginal zones. It should be noted that the significant increase (p < .01) in protein levels of the autophagy-associated intracellular signaling pathways AMPK and mTOR, as well as an increase in LC3-II and BECLIN-1, suggests that ketamine combined with insulin-activated autophagy-associated intracellular signaling AMPK and mTOR.Conclusion: The findings of the study suggest that ketamine combined with insulin administration remarkably protects I/R-induced myocardial injury in rats with T2DM by reducing the dysregulation of autophagy.

Transcription Factor E2F8 Promotes Cisplatin Resistance in Hepatocellular Carcinoma by Regulating DNA Damage via NUSAP1.

DNA damage repair has been the key mechanism of cisplatin resistance in hepatocellular carcinoma (HCC). The present study elucidated the molecular mechanism by which nucleolar and spindle-associated protein 1 (NUSAP1) influenced cisplatin tolerance in HCC by regulating DNA damage. First, high mRNA expression of E2F8 and NUSAP1 in HCC was detected by real-time quantitative PCR in cells and tumor tissue. The interaction between E2F8 and NUSAP1 was confirmed by chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays that E2F8 bound to the promoter region of NUSAP1 and regulated its transcriptional activity. The effects of the E2F8/NUSAP1 axis on cell viability, cell cycle, DNA damage protein γ-H2AX, and cisplatin resistance were investigated by CCK-8, flow cytometry, comet detection, and western blot. The results showed that NUSAP1 knockdown blocked the cell cycle in G0/G1 phase, promoted cisplatin-induced DNA damage, and enhanced cisplatin sensitivity in HCC. Overexpressed E2F8 promoted cell cycle arrest by silencing NUSAP1 in HCC, and promoting DNA damage as well as cisplatin sensitivity. In conclusion, our results suggested that E2F8 enhanced the chemoresistance of HCC cells to cisplatin by activating NUSAP1 to inhibit DNA damage, which provides a basis for describing new therapeutic targets that effectively exacerbate DNA damage and improve the chemical sensitivity of HCC to cisplatin.

MiR-145 inhibits the differentiation and proliferation of bone marrow stromal mesenchymal stem cells by GABARAPL1 in steroid-induced femoral head necrosis.

Steroid-induced osteonecrosis of femoral head (SANFH) involves impaired differentiation of bone marrow mesenchymal stem cells (BMSC), the mechanism of which is regulated by multiple microRNAs. Studies have shown that miR-145 is a key regulatory molecule of BMSC cells, but its mechanism in steroid-induced femur head necrosis remains unclear. The present study mainly explored the specific mechanism of miR-145 involved in SANFH. In this study dexamethasone, a typical glucocorticoid, was used to induce osteogenic differentiation of BMSC cells. Western blot, qPCR, CCK8 and flow cytometry were used to investigate the effects of miR-145 on the proliferation and differentiation of BMSC. The relationship between miR-145 and GABA Type A Receptor Associated Protein Like 1(GABARAPL1) was identified using dual luciferase reports and the effects of the two molecules on BMSC were investigated in vitro. The results showed that miR-145 was up-regulated in SANFH patients, while GABARAPL1 was down-regulated. Inhibition of miR-145 can improve apoptosis and promote proliferation and activation of BMSC. GABARAPL1 is a downstream target gene of miR-145 and is negatively regulated by miR-145. In conclusion, miR-145 regulates the proliferation and differentiation of glucocorticoid-induced BMSC cells through GABARAPL1 and pharmacologically inhibit targeting miR-145 may provide new aspect for the treatment of SANFH.

Adiponectin Protects Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury by Suppressing Autophagy.

Adiponectin is a cytokine produced by adipocytes and acts as a potential cardioprotective agent and plays an important role in myocardial ischemia/reperfusion injury. In a myocardial hypoxia/reoxygenation model using neonatal rat ventricular myocytes, we investigated the contribution of adiponectin-mediated autophagy to its cardioprotective effects. Cardiomyocytes were exposed to hypoxia/reoxygenation pretreated with or without adiponectin in the presence of absence of rapamycin. Cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Western blotting assay was used to determine the expression levels of microtubule-associated proteins 1A/1B light chain 3B (LC3B), adenosine monophosphate-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), p62/sequestosome 1, unc-51 like autophagy activating kinase 1 (ULK1), and Beclin-1. Autophagosome formation was detected by monodansylcadaverine staining. We found that hypoxia induced a time dependent decline in cardiomyocyte viability, and increase in autophagy and reoxygenation further augmented hypoxia-induced autophagy induction and consequently reduced cell viability. Adiponectin treatment alleviated hypoxia/reoxygenation-induced cellular damage and autophagy in cardiomyocytes. Adiponectin treatment also attenuated hypoxia/reoxygenation-promoted cardiomyocyte autophagy even in the presence of another autophagy stimulator rapamycin in part by inhibiting vacuolar hydron-adenosine triphosphatase. Additionally, autophagy suppression by adiponectin during hypoxia/reoxygenation was associated with the attenuated phosphorylation of AMPK and ULK1, augmented phosphorylation of mTOR, and the reduced protein expression levels of Beclin-1 in cardiomyocytes. Taken together, these results suggest that adiponectin protects ischemia/reperfusion-induced cardiomyocytes by suppressing autophagy in part through AMPK/mTOR/ULK1/Beclin-1 signaling pathway.

[Effect of heat-reinforcing needling on expression of serum inflammatory factors and autophagy of knee joint synovial tissue in rheumatoid arthritis rabbits with cold syndrome].

OBJECTIVE: To observe the effect of heat-reinforcing needling on the expression of serum inflammatory factors and autophagy of knee synovial tissue in rheumatoid arthritis (RA) rabbits with cold syndrome, so as to explore its mechanism of anti-inflammatory in the treatment of RA. METHODS: Fifty rabbits were randomly divided into normal, model, heat-reinforcing needling, inhibitor and agonist groups (n=10 rabbits in each group). The model of RA with cold syndrome was established by Freund's adjuvant and ovalbumin mixed solution injection combined with freezing and wind-cold dampness method. Heat-reinforcing needling was applied at "Zusanli" (ST36) for 30 min, once a day for 14 days. Rabbits of the inhibitor and agonist groups were given intraperitoneally injected with autophagy inhibitor 3-methyladenine (3-MA) or autophagy agonist rapamycin, once every 2 days for 7 days. The knee circumference and skin temperature of the rabbits in each group were measured. Color doppler ultrasonography was applied to examine the synovial membrane, joint effusion and blood flow signals in the knee joints of the rabbits in each group. Serum tumor necrosis factor (TNF) -α, interleukin (IL)-1ß, IL-6 and C-creactive protein (CRP) were detected by ELISA. Transmission electron microscopy was applied to observe the ultrastructure and autophagosomes of synovial cells. The protein expressions of autophagy-related protein Atg5, serine/threonine protein kinase-dysregulated 51-like kinase 1 (ULK1), microtubule-associated protein light chain 3B (LC3B), and Beclin-1 were detected by Western blot. Fluorescence quantitative PCR was used to detect the mRNA expressions of NOD-like receptor 3 (NLRP3) and nuclear factor-κB (NF-κB). RESULTS: Compared with the normal group, the circumference of the knee joint was increased (P<0.01), the skin temperature was decreased (P<0.01), the knee joint synovium was thickened and the blood flow signal was abundant, the contents of serum TNF-α, IL-1ß, IL-6, and CRP were increased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3BⅡ/LC3BⅠof synovial tissue were significantly decreased (P<0.01), the mRNA expressions of NLRP3 and NF-κB were increased (P<0.01) in the model group. In comparison with the model and inhibitor groups, the circumference of the knee joint was decreased (P<0.01), whlie the skin temperature was increased (P<0.01), the synovial membrane became thinner and the blood flow signal was wea-kened, the contents of TNF-α, IL-1ß, IL-6 and CRP were decreased (P<0.01), the protein expressions of Atg5, ULK1, Beclin-1 and LC3B Ⅱ/LC3B Ⅰ were increased (P<0.01), and the mRNA expressions of NLRP3 and NF-κB were decreased (P<0.01) in the heat-reinforcing needling and agonist groups. CONCLUSION: Heat-reinforcing needling can alleviate the inflammatory response of the knee joint synovium in RA rabbits with cold syndrome, which may be related to its function in enhancing the autophagy activity of synovial cells and inhibiting the synthesis and release of inflammatory factors TNF-α, IL-1ß, IL-6 and CRP.

Ginseng volatile oil prolongs the lifespan and healthspan of Caenorhabditis elegans.

Ginseng volatile oil (GVO) is one of the main components of ginseng and has antibacterial and anti-inflammatory properties. In this study, gas chromatography-mass spectrometry (GC-MS) was applied to characterize GVO chemical composition, and 73 volatile components were detected from GVO. Caenorhabditis elegans was used as animal model to further elucidate the antioxidant and anti-aging effects of GVO in vivo. The results suggested that GVO significantly prolonged the lifespan of C. elegans and promoted its health without damaging its reproductive capacity. In addition, GVO increased the antioxidant capacity and survival rate of nematodes after heat shock. Transcriptional sequencing showed that autophagy-related genes atg-4.2, atg-7, lgg-2, and cyd-1 were up-regulated, and superoxide dismutase 1 (sod-1) expression was increased after GVO pretreatment. Considering the role of autophagy and antioxidant in aging, the expression of autophagy substrate P62 protein in BC12921 strain was analyzed and found to decrease by more than 50.00% after treatment with GVO. In addition, the lifespan of SOD-1 mutant nematodes was not significantly different from that of the control group. SOD activity and autophagy were activated, which is a clear expression of hormesis. All these results suggest that GVO prolongs the lifespan and healthspan of C. elegans, and its biological functions may be related to hormesis.

The preventive effect of loganin on oxidative stress-induced cellular damage in human keratinocyte HaCaT cells.

Loganin is a type of iridoid glycosides isolated from Corni fructus and is known to have various pharmacological properties, but studies on its antioxidant activity are still lacking. Therefore, in this study, the preventive effect of loganin on oxidative stress-mediated cellular damage in human keratinocyte HaCaT cells was investigated. Our results show that loganin pretreatment in a non-toxic concentration range significantly improved cell survival in hydrogen peroxide (H2O2)-treated HaCaT cells, which was associated with inhibition of cell cycle arrest at the G2/M phase and induction of apoptosis. H2O2-induced DNA damage and reactive oxygen species (ROS) generation were also greatly reduced in the presence of loganin. Moreover, H2O2 treatment enhanced the cytoplasmic release of cytochrome c, upregulation of the Bax/Bcl-2 ratio and degradation of cleavage of poly (ADP-ribose) polymerase, whereas loganin remarkably suppressed these changes. In addition, loganin obviously attenuated H2O2-induced autophagy while inhibiting the increased accumulation of autophagosome proteins, including as microtubule-associated protein 1 light chain 3-II and Beclin-1, and p62, an autophagy substrate protein, in H2O2-treated cells. In conclusion, our current results suggests that loganin could protect HaCaT keratinocytes from H2O2-induced cellular injury by inhibiting mitochondrial dysfunction, autophagy and apoptosis. This finding indicates the applicability of loganin in the prevention and treatment of skin diseases caused by oxidative damage.

Atrogin-1 knockdown inhibits the autophagy-lysosome system in mammalian and avian myotubes.

Atrogin-1 plays an important role in ubiquitin-proteasome proteolysis in vertebrate skeletal muscles. Recently, atrogin-1 has been shown to be involved in the autophagy-lysosome system, another proteolytic system, in the murine and fish hearts and skeletal muscles. With the aim to elucidate the effect of atrogin-1 on the autophagy-lysosome system in mammalian and avian skeletal muscles, this study has examined the effects of atrogin-1 knockdown on autophagy-lysosome-related proteins in C2C12 and chicken embryonic myotubes. Using the levels of microtubule-associated protein light chain 3 (LC3)-II protein, it was confirmed that atrogin-1 knockdown blocked the autophagic flux in both the myotubes. In addition, atrogin-1 knockdown in C2C12 myotubes significantly decreased the level of autophagy-related gene (ATG)12-ATG5 conjugate, which is supposedly necessary for the fusion of autophagosomes and lysosomes. Atrogin-1 knockdown also resulted in downregulation of forkhead box O3, a transcription factor for ATG12. These data suggest that atrogin-1 is essential for the normal autophagy-lysosome system in the striated muscles of vertebrates.

High-altitude hypoxia-induced rat alveolar cell injury by increasing autophagy.

Autophagy has been implicated in the pathogenesis of various lung diseases. This study aimed to investigate the role of autophagy in lung injury induced by high-altitude hypoxia. Wistar rats were randomized into four groups for exposure to normal altitude or high altitude for 1, 7, 14 and 21 days with no treatment or with the treatment of 1 mg/kg rapamycin or 2 mg/kg 3-methyladenine (3-MA) for consecutive 21 days respectively. In control rats, the alveolar structure was intact with regularly arranged cells. However, inflammatory cell infiltration and shrunk alveoli were observed in rats exposed to hypoxia. Rapamycin treatment led to many shrunken alveoli with a large number of red blood cells in them. In contrast, 3-MA treatment led to almost intact alveoli or only a few shrunken alveoli. Compared to the control group exposure to high-altitude hypoxia for longer periods resulted in the aggravation of the lung injury, the formation of autophagosomes with a double-membrane structure and increased levels of Beclin-1 and LC3-II in alveolar tissues. Rapamycin treatment resulted in significant increase in Beclin-1 and LC3-II levels and further aggravation of alveolar tissue damage, while 3-MA treatment led to opposite effects. In conclusion, exposure to high-altitude hypoxia can induce autophagy of alveolar cells, which may be an important mechanism of high-altitude hypoxia-induced lung injury. The inhibition of autophagy may be a promising therapy strategy for high-altitude hypoxia-induced lung injury.

Therapeutic potential of pregnenolone and pregnenolone methyl ether on depressive and CDKL5 deficiency disorders: Focus on microtubule targeting.

Pregnenolone methyl-ether (PME) is a synthetic derivative of the endogenous neuroactive steroid pregnenolone (PREG), which is an important modulator of several brain functions. In addition to being the precursor of steroids, PREG acts directly on various targets including microtubules (MTs), the functioning of which is fundamental for the development and homeostasis of nervous system. The coordination of MT dynamics is supported by a plethora of MT-associated proteins (MAPs) and by a specific MT code that is defined by the post-translational modifications of tubulin. Defects associated with MAPs or tubulin post-translational modifications are linked to different neurological pathologies including mood and neurodevelopmental disorders. In this review, we describe the beneficial effect of PME in major depressive disorders (MDDs) and in CDKL5 deficiency disorder (CDD), two pathologies that are joint by defective MT dynamics. Growing evidence indeed suggests that PME, as well as PREG, is able to positively affect the MT-binding of MAP2 and the plus-end tracking protein CLIP170 that are both found to be deregulated in the above mentioned pathologies. Furthermore, PME influences the state of MT acetylation, the deregulation of which is often associated with neurological abnormalities including MDDs. By contrast to PREG, PME is not metabolised into other downstream molecules with specific biological properties, an aspect that makes this compound more suitable for therapeutic strategies. Thus, through the analysis of MDDs and CDD, this work focuses attention on the possible use of PME for neuronal pathologies associated with MT defects.

GABARAP ameliorates IL-1ß-induced inflammatory responses and osteogenic differentiation in bone marrow-derived stromal cells by activating autophagy.

Bone mesenchymal stem cells (BMSCs) are the most commonly investigated progenitor cells in bone defect repair and osteoarthritis subchondral bone regeneration; however, these studies are limited by complex inflammatory conditions. In this study, we investigated whether pro-autophagic γ-aminobutyric acid receptor-associated protein (GABARAP) promotes BMSCs proliferation and osteogenic differentiation by modulating autophagy in the presence or absence of interleukin-1 beta (IL-1ß) in vitro. The expression levels of all relevant factors were evaluated by qRT-PCR or western blotting where appropriate. BMSCs differentiation were assessed by Alizarin Red, alkaline phosphatase, safranin O, and Oil Red O staining. Furthermore, the interactions between autophagy and osteogenic differentiation were investigated by co-treatment with the autophagy inhibitor 3-methyladenine (3-MA). As the results, we found that treatment with recombinant human His6-GABARAP protein promoted cell proliferation, inhibited apoptosis, and reduced ROS generation by increasing autophagic activity, particularly when co-cultured with IL-1ß. Moreover, His6-GABARAP could effectively increase the osteogenic differentiation of BMSCs. The expression levels of inflammatory factors were significantly decreased by His6-GABARAP treatment, whereas its protective effects were attenuated by 3-MA. This study demonstrates that GABARAP maintains BMSCs survival and strengthens their osteogenic differentiation in an inflammatory environment by upregulating mediators of the autophagy pathway.

RP1, a RAGE antagonist peptide, can improve memory impairment and reduce Aß plaque load in the APP/PS1 mouse model of Alzheimer's disease.

Amyloid-ß (Aß) accumulation is a pathological hallmark of Alzheimer's disease (AD). The receptor for advanced glycation end products (RAGE) is involved in the production and accumulation of Aß. RP1, a peptide antagonist of RAGE, was screened by phage display technology in our previous studies, and its neuroprotective effects on an AD cell model have been confirmed. However, its efficacy in vivo remains unclear. Here, the intranasal delivery of RP1 to APPSwe/PS1dE9 (APP/PS1) mice significantly improved memory impairment and relieved the Aß burden by decreasing the expression of amyloid precursor protein and ß-secretase. RNA-sequencing (RNA-seq) was utilized to identify differentially expressed genes (DEGs) in APP/PS1 mice after RP1 administration. Several DEGs in RAGE downstream signalling pathways were downregulated. Some transcription factors (such as Fos) and the pathways enriched in the remarkable modules may also be related to the efficacy of RP1. In conclusion, RP1 significantly improves the AD symptoms of APP/PS1 mice, and the RNA-seq results provide new ideas for elucidating the possible mechanisms of RP1 treatment.

Flux-Based Assay for the Identification of Autophagy Modulators for Osteoarthritis.

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify regulators of autophagy could be used for the identification of novel therapeutics. This article describes a cell-based imaging screening workflow developed to monitor autophagic flux using LC3 as a reporter of autophagic flux (mCherry-EGFP-LC3B) in human chondrocytes. Data acquisition is performed using an automated High Content Imaging Screening System microscope. An algorithm-based automated image analysis protocol was developed and validated to identify molecules activating autophagic flux. Critical steps, explanatory notes, and improvements over current autophagy monitoring protocols are reported. Physiologically relevant phenotypic screening approaches to target hallmarks of aging can facilitate more effective drug discovery strategies for age-related musculoskeletal diseases.

Hepatitis B virus X protein (HBx) enhances centrosomal P4.1-associated protein (CPAP) expression to promote hepatocarcinogenesis.

BACKGROUND: Our previous report suggested that centrosomal P4.1-associated protein (CPAP) is required for Hepatitis B virus (HBV) encoded non-structure protein X (HBx)-mediated nuclear factor kappa light chain enhancer of activated B cells (NF-κB) activation. CPAP is overexpressed in HBV-associated hepatocellular carcinoma (HCC); however, the interaction between CPAP and HBx in HBV-HCC remains unclear. METHODS: The mRNA expression of CPAP and HBx was analyzed by quantitative-PCR (Q-PCR). NF-κB transcriptional activity and CPAP promoter activity were determined using a reporter assay in Huh7 and Hep3B cells. Immunoprecipitation (IP) and in situ proximal ligation assay (PLA) were performed to detect the interaction between CPAP and HBx. Chromatin-IP was used to detect the association of cAMP response element binding protein (CREB) and HBx with the CPAP promoter. Cell proliferation was measured using cell counting kit CCK-8, Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) incorporation, and clonogenic assays. The tumorigenic effects of CPAP were determined using xenograft animal models. RESULTS: HBx can transcriptionally up-regulate CPAP via interacting with CREB. Overexpressed CPAP directly interacted with HBx to promote HBx-mediated cell proliferation and migration; SUMO modification of CPAP was involved in interacting with HBx. Knocked-down expression of CPAP decreased the HBx-mediated tumorigenic effects, including cytokines secretion. Interestingly, overexpressed CPAP maintained the HBx protein stability in an NF-κB-dependent manner; and the expression levels of CPAP and HBx were positively correlated with the activation status of NF-κB in HCC. Increased expression of CPAP and CREB mRNAs existed in the high-risk group with a lower survival rate in HBV-HCC. CONCLUSION: The interaction between CPAP and HBx can provide a microenvironment to facilitate HCC development via enhancing NF-κB activation, inflammatory cytokine production, and cancer malignancies. This study not only sheds light on the role of CPAP in HBV-associated HCC, but also provides CPAP as a potential target for blocking the hyper-activated NF-κB in HCC.

GABARAP promotes bone marrow mesenchymal stem cells-based the osteoarthritis cartilage regeneration through the inhibition of PI3K/AKT/mTOR signaling pathway.

Osteoarthritis (OA) is a degenerative disease of the cartilage prevalent in the middle-aged and elderly demographic. Direct transplantation of bone marrow mesenchymal stem cells (BMSCs) or stem cell-derived chondrocytes into the damaged cartilage is a promising therapeutic strategy for OA, but is limited by the poor survival and in situ stability of the chondrocytes. Autophagy is a unique catabolic pathway conserved across eukaryotes that maintains cellular homeostasis, recycles damaged proteins and organelles, and promotes survival. The aim of this study was to determine the role of the proautophagic γ-aminobutyric acid receptor-associated protein (GABARAP) on the therapeutic effects of BMSCs-derived chondrocytes in a rat model of OA, and elucidate the underlying mechanisms. Anterior cruciate ligament transection (ACLT) was performed in Sprague-Dawley rats to simulate OA, and the animals were injected weekly with recombinant human His6-GABARAP protein, BMSCs-derived differentiated chondrocytes (DCs) or their combination directly into the knee cartilage. The regenerative effects of GABARAP and/or DCs were determined in term of International Cartilage Repair Society scores and cartilage thickness. The combination treatment of DCs and GABARAP significantly increased the levels of the ECM proteins Col II and SOX9, indicating formation of hyaline-like cartilage, and decreased chondrocyte apoptosis and inflammation. DCs + GABARAP treatment also upregulated the mediators of the autophagy pathway and suppressed the PI3K/AKT/mTOR pathway, indicating a mechanistic basis of its therapeutic action.

Exogenous CLASP2 protein treatment enhances wound healing in vitro and in vivo.

Proliferative and migratory abilities of fibroblasts are essential for wound healing at the skin surface. Cytoplasmic linker-associated protein-2 (CLASP2) was originally found to interact with cytoplasmic linker protein (CLIP)-170. CLASP2 plays an important role in microtubule stabilization and the microtubule-stabilizing activity of CLASP2 depends on its interactions with end binding (EB)-1 and CLIP-170. Although the microtubule-stabilizing role of CLASP2 is well established, the effects of CLASP2 on the migration and proliferation of fibroblasts remain unclear in the context of wound healing. Therefore, we tested the utilization of CLASP2 as a directly applied protein drug to improve wound healing by promoting the migration of effector cells, including skin fibroblasts, to the site of repair or injury using an in vivo excisional wound mouse model and in vitro Hs27 skin fibroblast model. Epidermal growth factor, which is a recognized contributor to cell proliferation and migration, was used as positive control. In vitro and in vivo, CLASP2 treatment significantly enhanced cell migration and accelerated wound closure. Furthermore, in vivo, the CLASP2-treated animal group displayed enhanced epidermal repair and collagen deposition. Next, we studied the mechanism of CLASP2 for wound healing. Increasing the abundance of intracellular free CLASP2 in skin fibroblasts by supplying exogenous CLASP2 seemed to stabilize microtubules through an interaction between CLASP2 and CLIP-170, as well as EB1. Exogenous CLASP2 also showed direct binding with IQGAP1, increasing both cyclic adenosine monophosphate activity and phosphorylation of glycogen synthase kinase 3ß, which in turn reinstated the binding between free CLASP2 and IQGAP1. In summary, exogenous CLASP2 increased Hs27 skin fibroblast migration by interacting with IQGAP1 and other cytoskeletal linker proteins, such as CLIP-170 and EB1. Our results strongly suggest that CLASP2 can be developed in wound healing drugs for skin repair and/or regenerating cosmetic products.

CD64-directed microtubule associated protein tau kills leukemic blasts ex vivo.

Fc gamma receptor I (FcγRI, CD64) is a well-known target antigen for passive immunotherapy against acute myeloid leukemia and chronic myelomonocytic leukemia. We recently reported the preclinical immunotherapeutic potential of microtubule associated protein tau (MAP) against a variety of cancer types including breast carcinoma and Hodgkin's lymphoma. Here we demonstrate that the CD64-directed human cytolytic fusion protein H22(scFv)-MAP kills ex vivo 15-50% of CD64+ leukemic blasts derived from seven myeloid leukemia patients. Furthermore, in contrast to the nonspecific cytostatic agent paclitaxel, H22(scFv)-MAP showed no cytotoxicity towards healthy CD64+ PBMC-derived cells and macrophages. The targeted delivery of this microtubule stabilizing agent therefore offers a promising new strategy for specific treatment of CD64+ leukemia.

Genetic Demonstration of a Role for Stathmin in Adult Hippocampal Neurogenesis, Spinogenesis, and NMDA Receptor-Dependent Memory.

Neurogenesis and memory formation are essential features of the dentate gyrus (DG) area of the hippocampus, but to what extent the mechanisms responsible for both processes overlap remains poorly understood. Stathmin protein, whose tubulin-binding and microtubule-destabilizing activity is negatively regulated by its phosphorylation, is prominently expressed in the DG. We show here that stathmin is involved in neurogenesis, spinogenesis, and memory formation in the DG. tTA/tetO-regulated bitransgenic mice, expressing the unphosphorylatable constitutively active Stathmin4A mutant (Stat4A), exhibit impaired adult hippocampal neurogenesis and reduced spine density in the DG granule neurons. Although Stat4A mice display deficient NMDA receptor-dependent memory in contextual discrimination learning, which is dependent on hippocampal neurogenesis, their NMDA receptor-independent memory is normal. Confirming NMDA receptor involvement in the memory deficits, Stat4A mutant mice have a decrease in the level of synaptic NMDA receptors and a reduction in learning-dependent CREB-mediated gene transcription. The deficits in neurogenesis, spinogenesis, and memory in Stat4A mice are not present in mice in which tTA/tetO-dependent transgene transcription is blocked by doxycycline through their life. The memory deficits are also rescued within 3 d by intrahippocampal infusion of doxycycline, further indicating a role for stathmin expressed in the DG in contextual memory. Our findings therefore point to stathmin and microtubules as a mechanistic link between neurogenesis, spinogenesis, and NMDA receptor-dependent memory formation in the DG. SIGNIFICANCE STATEMENT: In the present study, we aimed to clarify the role of stathmin in neuronal and behavioral functions. We characterized the neurogenic, behavioral, and molecular consequences of the gain-of-function stathmin mutation using a bitransgenic mouse expressing a constitutively active form of stathmin. We found that stathmin plays an important role in adult hippocampal neurogenesis and spinogenesis. In addition, stathmin mutation led to impaired NMDA receptor-dependent and neurogenesis-associated memory and did not affect NMDA receptor-independent memory. Moreover, biochemical analysis suggested that stathmin regulates the synaptic transport of NMDA receptors, which in turn influence CREB-mediated gene transcription machinery. Overall, these data suggest that stathmin is an important molecule for neurogenesis, spinogenesis, and NMDA receptor-dependent learning and memory.

[Endothelial monocyte-activating polypeptide-II: properties, functions, and pathogenetic significance].

Endothelial dysfunction is implicated in the pathogenesis of diabetes and atherosclerosis. Endothelial monocyte-activating polypeptide-II (EMAP-II) is a multifunctional polypeptide with proinflammatory and antiangiogenic activity. EMAP-II induces procoagulant activity on the surface of endothelial cells, increases expression of E- and P-selectins and tumor necrosis factor-1, directs migration of monocytes and neutrophils, induces apoptosis in endothelial cells. The mechanisms of effects on endothelial cells, inflammatory action, anti-tumor properties, pathogenic role in diseases of the central nervous system involved in the development of the lungs during embryogenesis and pathogenic role in diseases of the lungs, in the development of cardiovascular disease.