VNS improves VSMC metabolism and arteriogenesis in infarcted hearts through m/n-AChR-Akt-SDF-1α in adult male rats.

Vagal nerve stimulation (VNS) provides a novel therapeutic strategy for injured hearts by activating cholinergic anti-inflammatory pathways. However, little information is available on the metabolic pattern and arteriogenesis of VSMCs after MI. VNS has been shown to stimulate the expression of CPT1α, CPT1ß, Glut1, Glut4 and SDF-1α in coronary VSMCs, decreasing the number of CD68-positive macrophages while increasing CD206-positive macrophages in the infarcted hearts, leading to a decrease in TNF-α and IL-1ß accompanied by a reduced ratio of CD68- and CD206-positive cells, which were dramatically abolished by atropine and mecamylamine in vivo. Knockdown of SDF-1α substantially abrogated the effect of VNS on macrophagecell alteration and inflammatory factors in infarcted hearts. Mechanistically, ACh induced SDF-1α expression in VSMCs in a dose-dependent manner. Conversely, atropine, mecamylamine, and a PI3K/Akt inhibitor completely eliminated the effect of ACh on SDF-1α expression. Functionally, VNS promoted arteriogenesis and improved left ventricular performance, which could be abolished by Ad-shSDF-1α. Thus, VNS altered the VSMC metabolism pattern and arteriogenesis to repair the infarcted heart by inducing SDF-1α expression, which was associated with the m/nAChR-Akt signaling pathway.

A Dihydroartemisinin-Loaded Nanoreactor Motivates Anti-Cancer Immunotherapy by Synergy-Induced Ferroptosis to Activate Cgas/STING for Reprogramming of Macrophage.

Infiltration of tumor-associated macrophages (TAM) characterized by an M2 phenotype is an overriding feature in malignant tumors. Reprogramming TAM is the most cutting-edge strategy for cancer therapy. In the present study, an iron-based metal-organic framework (MOF) nanoreactor loaded with dihydroartemisinin (DHA) is developed, which provides high uptake by TAM and retains their viability, thus effectively addressing the inefficiency of the DHA at low concentrations. Impressively, DHA@MIL-101 can selectively accumulate in tumor tissues and remodel TAM to the M1 phenotype. The results of RNA sequencing further suggest that this nanoreactor may regulate ferroptosis, a DNA damage signaling pathway in TAM. Indeed, the outcomes confirm that DHA@MIL-101 triggers ferroptosis in TAM. In addition, the findings reveal that DNA damage induced by DHA nanoreactors activates the intracellular cGAS sensor, resulting in the binding of STING to IRF3 and thereby up-regulating the immunogenicity. In contrast, blocking ferroptosis impairs DHA@MIL-101-induced activation of STING signaling and phenotypic remodeling. Finally, it is shown that DHA nanoreactors deploy anti-tumor immunotherapy through ferroptosis-mediated TAM reprogramming. Taken together, immune efficacy is achieved through TAM's remodeling by delivering DHA and iron ions into TAM using nanoreactors, providing a novel approach for combining phytopharmaceuticals with nanocarriers to regulate the immune microenvironment.

Vagus nerve stimulation-induced stromal cell-derived factor-l alpha participates in angiogenesis and repair of infarcted hearts.

AIMS: We aim to explore the role and mechanism of vagus nerve stimulation (VNS) in coronary endothelial cells and angiogenesis in infarcted hearts. METHODS AND RESULTS: Seven days after rat myocardial infarction (MI) was prepared by ligation of the left anterior descending coronary artery, the left cervical vagus nerve was treated with electrical stimulation 1 h after intraperitoneal administration of the α7-nicotinic acetylcholine inhibitor mecamylamine or the mAChR inhibitor atropine or 3 days after local injection of Ad-shSDF-1α into the infarcted heart. Cardiac tissue acetylcholine (ACh) and serum ACh, tumour necrosis factor α (TNF-α), interleukin 1ß (IL-1ß) and interleukin 6 (IL-6) levels were detected by ELISA to determine whether VNS was successful. An inflammatory injury model in human coronary artery endothelial cells (HCAECs) was established by lipopolysaccharide and identified by evaluating TNF-α, IL-1ß and IL-6 levels and tube formation. Immunohistochemistry staining was performed to evaluate CD31-positive vessel density and stromal cell-derived factor-l alpha (SDF-1α) expression in the MI heart in vivo and the expression and distribution of SDF-1α, C-X-C motif chemokine receptor 4 and CXCR7 in HCAECs in vitro. Western blotting was used to detect the levels of SDF-1α, V-akt murine thymoma viral oncogene homolog (AKT), phosphorylated AKT (pAKT), specificity protein 1 (Sp1) and phosphorylation of Sp1 in HCAECs. Left ventricular performance, including left ventricular systolic pressure, left ventricular end-diastolic pressure and rate of the rise and fall of ventricular pressure, should be evaluated 28 days after VNS treatment. VNS was successfully established for MI therapy with decreases in serum TNF-α, IL-1ß and IL-6 levels and increases in cardiac tissue and serum ACh levels, leading to increased SDF-1α expression in coronary endothelial cells of MI hearts, triggering angiogenesis of MI hearts with increased CD31-positive vessel density, which was abolished by the m/nAChR inhibitors mecamylamine and atropine or knockdown of SDF-1α by shRNA. ACh promoted SDF-1α expression and its distribution along with the branch of the formed tube in HCAECs, resulting in an increase in the number of tubes formed in HCAECs. ACh increased the levels of pAKT and phosphorylation of Sp1 in HCAECs, resulting in inducing SDF-1α expression, and the specific effects could be abolished by mecamylamine, atropine, the PI3K/AKT blocker wortmannin or the Sp1 blocker mithramycin. Functionally, VNS improved left ventricular performance, which could be abolished by Ad-shSDF-1α. CONCLUSIONS: VNS promoted angiogenesis to repair the infarcted heart by inducing SDF-1α expression and redistribution along new branches during angiogenesis, which was associated with the m/nAChR-AKT-Sp1 signalling pathway.

The spatiotemporal matching pattern of Ezrin/Periaxin involved in myoblast differentiation and fusion and Charcot-Marie-Tooth disease-associated muscle atrophy.

BACKGROUND: Clinically, Charcot-Marie-Tooth disease (CMT)-associated muscle atrophy still lacks effective treatment. Deletion and mutation of L-periaxin can be involved in CMT type 4F (CMT4F) by destroying the myelin sheath form, which may be related to the inhibitory role of Ezrin in the self-association of L-periaxin. However, it is still unknown whether L-periaxin and Ezrin are independently or interactively involved in the process of muscle atrophy by affecting the function of muscle satellite cells. METHOD: A gastrocnemius muscle atrophy model was prepared to mimic CMT4F and its associated muscle atrophy by mechanical clamping of the peroneal nerve. Differentiating C2C12 myoblast cells were treated with adenovirus-mediated overexpression or knockdown of Ezrin. Then, overexpression of L-periaxin and NFATc1/c2 or knockdown of L-periaxin and NFATc3/c4 mediated by adenovirus vectors were used to confirm their role in Ezrin-mediated myoblast differentiation, myotube formation and gastrocnemius muscle repair in a peroneal nerve injury model. RNA-seq, real-time PCR, immunofluorescence staining and Western blot were used in the above observation. RESULTS: For the first time, instantaneous L-periaxin expression was highest on the 6th day, while Ezrin expression peaked on the 4th day during myoblast differentiation/fusion in vitro. In vivo transduction of adenovirus vectors carrying Ezrin, but not Periaxin, into the gastrocnemius muscle in a peroneal nerve injury model increased the numbers of muscle myosin heavy chain (MyHC) I and II type myofibers, reducing muscle atrophy and fibrosis. Local muscle injection of overexpressed Ezrin combined with incubation of knockdown L-periaxin within the injured peroneal nerve or injection of knockdown L-periaxin into peroneal nerve-injured gastrocnemius muscle not only increased the number of muscle fibers but also recovered their size to a relatively normal level in vivo. Overexpression of Ezrin promoted myoblast differentiation/fusion, inducing increased MyHC-I+ and MyHC-II + muscle fiber specialization, and the specific effects could be enhanced by the addition of adenovirus vectors for knockdown of L-periaxin by shRNA. Overexpression of L-periaxin did not alter the inhibitory effects on myoblast differentiation and fusion mediated by knockdown of Ezrin by shRNA in vitro but decreased myotube length and size. Mechanistically, overexpressing Ezrin did not alter protein kinase A gamma catalytic subunit (PKA-γ cat), protein kinase A I alpha regulatory subunit (PKA reg Iα) or PKA reg Iß levels but increased PKA-α cat and PKA reg II α levels, leading to a decreased ratio of PKA reg I/II. The PKA inhibitor H-89 remarkably abolished the effects of overexpressing-Ezrin on increased myoblast differentiation/fusion. In contrast, knockdown of Ezrin by shRNA significantly delayed myoblast differentiation/fusion accompanied by an increased PKA reg I/II ratio, and the inhibitory effects could be eliminated by the PKA reg activator N6-Bz-cAMP. Meanwhile, overexpressing Ezrin enhanced type I muscle fiber specialization, accompanied by an increase in NFATc2/c3 levels and a decrease in NFATc1 levels. Furthermore, overexpressing NFATc2 or knocking down NFATc3 reversed the inhibitory effects of Ezrin knockdown on myoblast differentiation/fusion. CONCLUSIONS: The spatiotemporal pattern of Ezrin/Periaxin expression was involved in the control of myoblast differentiation/fusion, myotube length and size, and myofiber specialization, which was related to the activated PKA-NFAT-MEF2C signaling pathway, providing a novel L-Periaxin/Ezrin joint strategy for the treatment of muscle atrophy induced by nerve injury, especially in CMT4F.

Continuous exposure to isoprenaline reduced myotube size by delaying myoblast differentiation and fusion through the NFAT-MEF2C signaling pathway.

We aimed to explore whether superfluous sympathetic activity affects myoblast differentiation, fusion, and myofiber types using a continuous single-dose isoprenaline exposure model in vitro and to further confirm the role of distinct NFATs in ISO-mediated effects. Compared with delivery of single and interval single, continuous single-dose ISO most obviously diminished myotube size while postponing myoblast differentiation/fusion in a time- and dose-dependent pattern, accompanied by an apparent decrease in nuclear NFATc1/c2 levels and a slight increase in nuclear NFATc3/c4 levels. Overexpression of NFATc1 or NFATc2, particularly NFATc1, markedly abolished the inhibitory effects of ISO on myoblast differentiation/fusion, myotube size and Myh7 expression, which was attributed to a remarkable increase in the nuclear NFATc1/c2 levels and a reduction in the nuclear NFATc4 levels and the associated increase in the numbers of MyoG and MEF2C positive nuclei within more than 3 nuclei myotubes, especially in MEF2C. Moreover, knockdown of NFATc3 by shRNA did not alter the inhibitory effect of ISO on myoblast differentiation/fusion or myotube size but partially recovered the expression of Myh7, which was related to the slightly increased nuclear levels of NFATc1/c2, MyoG and MEF2C. Knockdown of NFATc4 by shRNA prominently increased the number of MyHC +, MyoG or MEF2C + myoblast cells with 1 ~ 2 nuclei, causing fewer numbers and smaller myotube sizes. However, NFATc4 knockdown further deteriorated the effects of ISO on myoblast fusion and myotube size, with more than 5 nuclei and Myh1/2/4 expression, which was associated with a decrease in nuclear NFATc2/c3 levels. Therefore, ISO inhibited myoblast differentiation/fusion and myotube size through the NFAT-MyoG-MEF2C signaling pathway.

Platelets are highly efficient and efficacious carriers for tumor-targeted nano-drug delivery.

The present work aims to prove the concept of tumor-targeted drug delivery mediated by platelets. Doxorubicin (DOX) attached to nanodiamonds (ND-DOX) was investigated as the model payload drug of platelets. In vitro experiments first showed that ND-DOX could be loaded in mouse platelets in a dose-dependent manner with a markedly higher efficiency and capacity than free DOX. ND-DOX-loaded platelets (Plt@ND-DOX) maintained viability and ND-DOX could be stably held in the platelets for at least 4 hr. Next, mouse Lewis lung cancer cells were found to activate Plt@ND-DOX and thereby stimulate cargo unloading of Plt@ND-DOX. The unloaded ND-DOX was taken up by co-cultured cancer cells which consequently exhibited loss of viability, proliferation suppression and apoptosis. In vivo, Plt@ND-DOX displayed significantly prolonged blood circulation time over ND-DOX and DOX in mice, and Lewis tumor grafts demonstrated infiltration, activation and cargo unloading of Plt@ND-DOX in the tumor tissue. Consequently, Plt@ND-DOX effectively reversed the growth of Lewis tumor grafts which exhibited significant inhibition of cell proliferation and apoptosis. Importantly, Plt@ND-DOX displayed a markedly higher therapeutic potency than free DOX but without the severe systemic toxicity associated with DOX. Our findings are concrete proof of platelets as efficient and efficacious carriers for tumor-targeted nano-drug delivery with the following features: 1) large loading capacity and high loading efficiency, 2) good tolerance of cargo drug, 3) stable cargo retention and no cargo unloading in the absence of stimulation, 4) prolonged blood circulation time, and 5) excellent tumor distribution and tumor-activated drug unloading leading to high therapeutic potency and few adverse effects. Platelets hold great potential as efficient and efficacious carriers for tumor-targeted nano-drug delivery.

ATG7-mediated autophagy facilitates embryonic stem cell exit from naive pluripotency and marks commitment to differentiation.

Macroautophagy/autophagy is a conserved cellular mechanism to degrade unneeded cytoplasmic proteins and organelles to recycle their components, and it is critical for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Whereas autophagy is essential for early development of embryos, no information exists regarding its functions during the transition from naive-to-primed pluripotency. Here, by using an in vitro transition model of ESCs to epiblast-like cells (EpiLCs), we find that dynamic changes in ATG7-dependent autophagy are critical for the naive-to-primed transition, and are also necessary for germline specification. RNA-seq and ATAC-seq profiling reveal that NANOG acts as a barrier to prevent pluripotency transition, and autophagy-dependent NANOG degradation is important for dismantling the naive pluripotency expression program through decommissioning of naive-associated active enhancers. Mechanistically, we found that autophagy receptor protein SQSTM1/p62 translocated into the nucleus during the pluripotency transition period and is preferentially associated with K63 ubiquitinated NANOG for selective protein degradation. In vivo, loss of autophagy by ATG7 depletion disrupts peri-implantation development and causes increased chromatin association of NANOG, which affects neuronal differentiation by competitively binding to OTX2-specific neuroectodermal development-associated regions. Taken together, our findings reveal that autophagy-dependent degradation of NANOG plays a critical role in regulating exit from the naive state and marks distinct cell fate allocation during lineage specification.Abbreviations: 3-MA: 3-methyladenine; EpiLC: epiblast-like cell; ESC: embryonic stem cell; PGC: primordial germ cell.

Patient-specific iPSC-derived cardiomyocytes reveal abnormal regulation of FGF16 in a familial atrial septal defect.

AIMS: Congenital heart disease (CHD) frequently occurs in newborns due to abnormal formation of the heart or major blood vessels. Mutations in the GATA4 gene, which encodes GATA binding protein 4, are responsible for atrial septal defect (ASD), a common CHD. This study aims to gain insights into the molecular mechanisms of CHD using human-induced pluripotent stem cells (iPSCs) from a family cohort with ASD. METHODS AND RESULTS: Patient-specific iPSCs possess the same genetic information as the donor and can differentiate into various cell types from all three germ layers in vitro, thus presenting a promising approach for disease modelling and molecular mechanism research. Here, we generated a patient-specific iPSC line (iPSC-G4T280M) from a family cohort carrying a hereditary ASD mutation in GATA4 gene (T280M), as well as a human embryonic stem cell line (ESC-G4T280M) carrying the isogenic T280M mutation using the CRISPR/Cas9 genome editing method. The GATA4-mutant iPSCs and ESCs were then differentiated into cardiomyocytes (CMs) to model GATA4 mutation-associated ASD. We observed an obvious defect in cell proliferation in cardiomyocytes derived from both GATA4T280M-mutant iPSCs (iPSC-G4T280M-CMs) and ESCs (ESC-G4T280M-CMs), while the impaired proliferation ability of iPSC-G4T280M-CMs could be restored by gene correction. Integrated analysis of RNA-Seq and ChIP-Seq data indicated that FGF16 is a direct target of wild-type GATA4. However, the T280M mutation obstructed GATA4 occupancy at the FGF16 promoter region, leading to impaired activation of FGF16 transcription. Overexpression of FGF16 in GATA4-mutant cardiomyocytes rescued the cell proliferation defect. The direct relationship between GATA4T280M and ASD was demonstrated in a human iPSC model for the first time. CONCLUSIONS: In summary, our study revealed the molecular mechanism of the GATA4T280M mutation in ASD. Understanding the roles of the GATA4-FGF16 axis in iPSC-CMs will shed light on heart development and provide novel insights for the treatment of ASD and other CHD disorders.

Histone Lysine Methylation and Long Non-Coding RNA: The New Target Players in Skeletal Muscle Cell Regeneration.

Satellite stem cell availability and high regenerative capacity have made them an ideal therapeutic approach for muscular dystrophies and neuromuscular diseases. Adult satellite stem cells remain in a quiescent state and become activated upon muscular injury. A series of molecular mechanisms succeed under the control of epigenetic regulation and various myogenic regulatory transcription factors myogenic regulatory factors, leading to their differentiation into skeletal muscles. The regulation of MRFs via various epigenetic factors, including DNA methylation, histone modification, and non-coding RNA, determine the fate of myogenesis. Furthermore, the development of histone deacetylation inhibitors (HDACi) has shown promising benefits in their use in clinical trials of muscular diseases. However, the complete application of using satellite stem cells in the clinic is still not achieved. While therapeutic advancements in the use of HDACi in clinical trials have emerged, histone methylation modulations and the long non-coding RNA (lncRNA) are still under study. A comprehensive understanding of these other significant epigenetic modulations is still incomplete. This review aims to discuss some of the current studies on these two significant epigenetic modulations, histone methylation and lncRNA, as potential epigenetic targets in skeletal muscle regeneration. Understanding the mechanisms that initiate myoblast differentiation from its proliferative state to generate new muscle fibres will provide valuable information to advance the field of regenerative medicine and stem cell transplant.

Common mtDNA variations at C5178a and A249d/T6392C/G10310A decrease the risk of severe COVID-19 in a Han Chinese population from Central China.

BACKGROUND: Mitochondria have been shown to play vital roles during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) development. Currently, it is unclear whether mitochondrial DNA (mtDNA) variants, which define mtDNA haplogroups and determine oxidative phosphorylation performance and reactive oxygen species production, are associated with COVID-19 risk. METHODS: A population-based case-control study was conducted to compare the distribution of mtDNA variations defining mtDNA haplogroups between healthy controls (n = 615) and COVID-19 patients (n = 536). COVID-19 patients were diagnosed based on molecular diagnostics of the viral genome by qPCR and chest X-ray or computed tomography scanning. The exclusion criteria for the healthy controls were any history of disease in the month preceding the study assessment. MtDNA variants defining mtDNA haplogroups were identified by PCR-RFLPs and HVS-I sequencing and determined based on mtDNA phylogenetic analysis using Mitomap Phylogeny. Student's t-test was used for continuous variables, and Pearson's chi-squared test or Fisher's exact test was used for categorical variables. To assess the independent effect of each mtDNA variant defining mtDNA haplogroups, multivariate logistic regression analyses were performed to calculate the odds ratios (ORs) and 95% confidence intervals (CIs) with adjustments for possible confounding factors of age, sex, smoking and diseases (including cardiopulmonary diseases, diabetes, obesity and hypertension) as determined through clinical and radiographic examinations. RESULTS: Multivariate logistic regression analyses revealed that the most common investigated mtDNA variations (> 10% in the control population) at C5178a (in NADH dehydrogenase subunit 2 gene, ND2) and A249d (in the displacement loop region, D-loop)/T6392C (in cytochrome c oxidase I gene, CO1)/G10310A (in ND3) were associated with a reduced risk of severe COVID-19 (OR = 0.590, 95% CI 0.428-0.814, P = 0.001; and OR = 0.654, 95% CI 0.457-0.936, P = 0.020, respectively), while A4833G (ND2), A4715G (ND2), T3394C (ND1) and G5417A (ND2)/C16257a (D-loop)/C16261T (D-loop) were related to an increased risk of severe COVID-19 (OR = 2.336, 95% CI 1.179-4.608, P = 0.015; OR = 2.033, 95% CI 1.242-3.322, P = 0.005; OR = 3.040, 95% CI 1.522-6.061, P = 0.002; and OR = 2.890, 95% CI 1.199-6.993, P = 0.018, respectively). CONCLUSIONS: This is the first study to explore the association of mtDNA variants with individual's risk of developing severe COVID-19. Based on the case-control study, we concluded that the common mtDNA variants at C5178a and A249d/T6392C/G10310A might contribute to an individual's resistance to developing severe COVID-19, whereas A4833G, A4715G, T3394C and G5417A/C16257a/C16261T might increase an individual's risk of developing severe COVID-19.

Efficient Delivery of Chlorin e6 by Polyglycerol-Coated Iron Oxide Nanoparticles with Conjugated Doxorubicin for Enhanced Photodynamic Therapy of Melanoma.

Chlorin e6 (Ce6) is a promising photosensitizer for tumor photodynamic therapy (PDT). However, the efficacy of Ce6 PDT is limited by Ce6's poor water solubility, rapid blood clearance, and inadequate accumulation in the tumor tissue. This problem is tackled in this work, wherein functionalized superparamagnetic iron oxide nanoparticles (IO-NPs) were used as carriers to deliver Ce6 to melanoma. The IO-NPs were coated with polyglycerol (PG) to afford good aqueous solubility. The chemotherapeutic agent doxorubicin (DOX) was attached to the PG coating via the hydrazone bond to afford affinity to the cell membrane and thereby promote the cell uptake. The hydrophobic nature of DOX also induced the aggregation of IO-NPs to form nanoclusters. Ce6 was then loaded onto the IO nanoclusters through physical adsorption and coordination with surface iron atoms, yielding the final composites IO-PG-DOX-Ce6. In vitro experiments showed that IO-PG-DOX-Ce6 markedly increased Ce6 uptake in mouse melanoma cells, leading to much-enhanced photocytotoxicity characterized by intensified reactive oxygen species production, loss of viability, DNA damage, and stimulation of tumor cell immunogenicity. In vivo experiments corroborated the in vitro findings and demonstrated prolonged blood clearance of IO-PG-DOX-Ce6. Importantly, IO-PG-DOX-Ce6 markedly increased the Ce6 distribution and retention in mouse subcutaneous melanoma grafts and significantly improved the efficacy of Ce6-mediated PDT. No apparent vital organ damage was observed at the same time. In conclusion, the IO-PG-DOX NPs provide a simple and safe delivery platform for efficient tumor enrichment of Ce6, thereby enhancing antimelanoma PDT.

Establishment and characterization of a human embryonic stem cell line carrying a heterozygous GATA4T280M mutation.

Heterozygous T280M mutation in the GATA4 gene, encoding GATA binding protein 4, was recently identified in patients with congenital heart disease (CHD). Here, a human embryonic stem cell line carrying a heterozygous GATA4T280M mutation was generated by using the CRISPR/Cas9 and piggyBac technologies. This GATA4T280M cell line remains normal morphology, pluripotency and karyotype, and could differentiate into all three germ layers both in vivo and in vitro. Thus, the GATA4T280M cell line will be a useful platform to model GATA4-associated CHD through the induction of cardiomyocyte differentiation in vitro.

Vagus nerve stimulation optimized cardiomyocyte phenotype, sarcomere organization and energy metabolism in infarcted heart through FoxO3A-VEGF signaling.

Vagus nerve stimulation (VNS) restores autonomic balance, suppresses inflammation action and minimizes cardiomyocyte injury. However, little knowledge is known about the VNS' role in cardiomyocyte phenotype, sarcomere organization, and energy metabolism of infarcted hearts. VNS in vivo and acetylcholine (ACh) in vitro optimized the levels of α/ß-MHC and α-Actinin positive sarcomere organization in cardiomyocytes while reducing F-actin assembly of cardiomyocytes. Consistently, ACh improved glucose uptake while decreasing lipid deposition in myocytes, correlating both with the increase of Glut4 and CPT1α and the decrease of PDK4 in infarcted hearts in vivo and myocytes in vitro, attributing to improvement in both glycolysis by VEGF-A and lipid uptake by VEGF-B in response to Ach. This led to increased ATP levels accompanied by the repaired mitochondrial function and the decreased oxygen consumption. Functionally, VNS improved the left ventricular performance. In contrast, ACh-m/nAChR inhibitor or knockdown of VEGF-A/B by shRNA powerfully abrogated these effects mediated by VNS. On mechanism, ACh decreased the levels of nuclear translocation of FoxO3A in myocytes due to phosphorylation of FoxO3A by activating AKT. FoxO3A overexpression or knockdown could reverse the specific effects of ACh on the expression of VEGF-A/B, α/ß-MHC, Glut4, and CPT1α, sarcomere organization, glucose uptake and ATP production. Taken together, VNS optimized cardiomyocytes sarcomere organization and energy metabolism to improve heart function of the infarcted heart during the process of delaying and/or blocking the switch from compensated hypertrophy to decompensated heart failure, which were associated with activation of both P13K/AKT-FoxO3A-VEGF-A/B signaling cascade.

A Selective TRPC3 Inhibitor Pyr3 Attenuates Myocardial Ischemia/Reperfusion Injury in Mice.

An emerging body of evidence indicates that transient receptor potential TRP channels act as important mediators for a wide variety of physiological functions and are potential targets for drug discovery. Our previous study has identified transient receptor potential channel 3 (TRPC3) and TRPC6 as cation channels through which most of the damaging calcium enters, aggravates pathological changes in vivo and increases ischemia/reperfusion (I/R) injury in mice. This study aimed to verify the effects of TRPC3 inhibitor Pyr3 on myocardial I/R injury in mice. C57BL/6J wild-type male mice (8 to 12 weeks old) were anesthetized with 3.3% chloral hydrate. A murine I (30 min)/R (24 h) injury model was established by temporary occlusion of the left anterior descending (LAD) coronary artery. Pyr3 was administered at concentrations of 0, 2.5, 5, or 10 mg/kg via the right jugular vein 5 min before reperfusion. We observed that the selective TRPC3 inhibitor, 10 mg/kg Pyr3, significantly decreased the infarct size of left ventricle, and reduced the myocardial cell apoptosis rate and inflammatory response in mice. In a conclusion, TRPC3 can function as a candidate target for I/R injury prevention, and Pyr3 may directly bind to TRPC3 channel protein, inhibit TRPC3 channel activity, and improve TRPC3-related myocardial I/R injury. Pyr3 may be used for clarification of TRPC3 functions and for treatments of TRPC3-mediated diseases.

S100B is required for maintaining an intermediate state with double-positive Sca-1+ progenitor and vascular smooth muscle cells during neointimal formation.

INTRODUCTION: Accumulation of vascular smooth muscle cells (VSMCs) within the neointimal region is a hallmark of atherosclerosis and vessel injury. Evidence has shown that Sca-1-positive (Sca-1+) progenitor cells residing in the vascular adventitia play a crucial role in VSMC assemblages and intimal lesions. However, the underlying mechanisms, especially in the circumstances of vascular injury, remain unknown. METHODS AND RESULTS: The neointimal formation model in rats was established by carotid artery balloon injury using a 2F-Forgaty catheter. Most Sca-1+ cells first appeared at the adventitia of the vascular wall. S100B expressions were highest within the adventitia on the first day after vessel injury. Along with the sequentially increasing trend of S100B expression in the intima, media, and adventitia, respectively, the numbers of Sca-1+ cells were prominently increased at the media or neointima during the time course of neointimal formation. Furthermore, the Sca-1+ cells were markedly increased in the tunica media on the third day of vessel injury, SDF-1α expressions were obviously increased, and SDF-1α levels and Sca-1+ cells were almost synchronously increased within the neointima on the seventh day of vessel injury. These effects could effectually be reversed by knockdown of S100B by shRNA, RAGE inhibitor (SPF-ZM1), or CXCR4 blocker (AMD3100), indicating that migration of Sca-1+ cells from the adventitia into the neointima was associated with S100B/RAGE and SDF-1α/CXCR4. More importantly, the intermediate state of double-positive Sca-1+ and α-SMA cells was first found in the neointima of injured arteries, which could be substantially abrogated by using shRNA for S100B or blockade of CXCR4. S100B dose-dependently regulated SDF-1α expressions in VSMCs by activating PI3K/AKT and NF-κB, which were markedly abolished by PI3K/AKT inhibitor wortmannin and enhanced by p65 blocker PDTC. Furthermore, S100B was involved in human umbilical cord-derived Sca-1+ progenitor cells' differentiation into VSMCs, especially in maintaining the intermediate state of double-positive Sca-1+ and α-SMA. CONCLUSIONS: S100B triggered neointimal formation in rat injured arteries by maintaining the intermediate state of double-positive Sca-1+ progenitor and VSMCs, which were associated with direct activation of RAGE by S100B and indirect induction of SDF-1α by activating PI3K/AKT and NF-κB.

Nuclear FAM289-Galectin-1 interaction controls FAM289-mediated tumor promotion in malignant glioma.

BACKGROUND: FAM92A1-289(abbreviated FAM289) is recognized as one of the newly-discovered putative oncogenes. However, its role and molecular mechanisms in promoting cancer progression has not yet been elucidated. This study was performed to reveal its oncogenic functions and molecular mechanisms in human glioblastoma multiforme (GBM) cell models with knockdown or overexpression of FAM289 in vitro and in vivo. METHODS: To elucidate the molecular mechanisms underlying FAM289-mediated tumor progression, the protein-protein interaction between FAM289 and Galectin-1 was verified by co-immunoprecipitation, followed by an analysis of the expression and activity of Galectin-1-associated signaling molecules. Knockdown and overexpression of FAM289 in glioma cells were applied for investigating the effects of FAM289 on cell growth, migration and invasion. The determination of FAM289 expression was performed in specimens from various stages of human gliomas. RESULTS: FAM289-galectin-1 interaction and concomitant activation of the extracellular signal-regulated kinase (ERK) pathway participated in FAM289-mediated tumor-promoting function. Since the expression of DNA methyl transferase 1 (DNMT1) and DNA methyl transferase 3B (DNMT3B) was regulated by FAM289 in U251 and U87-MG glioma cells, Galectin-1 interaction with FAM289 may promote FAM289 protein into the cell nucleus and activate the ERK pathway, thereby upregulating DNMTs expression. Drug resistance tests indicated that FAM289-mediated TMZ resistance was through stem-like property acquisition by activating the ERK pathway. The correlation between FAM289, Galectin-1 expression and the clinical stage of gliomas was also verified in tissue samples from glioblastoma patients. CONCLUSIONS: Our results suggest that high expression of FAM289 in GBM tissues correlated with poor prognosis. FAM289 contributes to tumor progression in malignant glioma by interacting with Galectin-1 thereby promoting FAM289 protein translocation into the cell nucleus. FAM289 in the nucleus activated the ERK pathway, up regulated DNMTs expression and induced stem-like property gene expression which affects drug resistance of glioma cells to TMZ. This study provided functional evidence for FAM289 to be developed as a therapeutic target for cancer treatment.

KPC1 alleviates hypoxia/reoxygenation-induced apoptosis in rat cardiomyocyte cells though BAX degradation.

Bax triggers cell apoptosis by permeabilizing the outer mitochondrial membrane, leading to membrane potential loss and cytochrome c release. However, it is unclear if proteasomal degradation of Bax is involved in the apoptotic process, especially in heart ischemia-reperfusion (I/R)-induced injury. In the present study, KPC1 expression was heightened in left ventricular cardiomyocytes of patients with coronary heart disease (CHD), in I/R-myocardium in vivo and in hypoxia and reoxygenation (H/R)-induced cardiomyocytes in vitro. Overexpression of KPC1 reduced infarction size and cell apoptosis in I/R rat hearts. Similarly, the forced expression of KPC1 restored mitochondrial membrane potential (MMP) and cytochrome c release driven by H/R in H9c2 cells, whereas reducing cell apoptosis, and knockdown of KPC1 by short-hairpin RNA (shRNA) deteriorated cell apoptosis induced by H/R. Mechanistically, forced expression of KPC1 promoted Bax protein degradation, which was abolished by proteasome inhibitor MG132, suggesting that KPC1 promoted proteasomal degradation of Bax. Furthermore, KPC1 prevented basal and apoptotic stress-induced Bax translocation to mitochondria. Bax can be a novel target for the antiapoptotic effects of KPC1 on I/R-induced cardiomyocyte apoptosis and render mechanistic penetration into at least a subset of the mitochondrial effects of KPC1.

Combination of G-CSF and AMD3100 Improves the Anti-inflammatory Effect of Mesenchymal Stem Cells on Inducing M2 Polarization of Macrophages Through NF-κB-IL1RA Signaling Pathway.

Mobilized peripheral blood-derived mesenchymal stem cells (PB-MSCs) mainly derived from bone marrow-derived MSCs (BM-MSCs) exert a similar anti-inflammatory effect. However, the mechanism of anti-inflammatory effect of mobilized PB-MSCs by a combination of G-CSF and AMD3100 remains unclear. Cultured rat PB-MSCs mobilized by G-CSF/AMD3100 have shown typical surface markers and potential for multiple differentiations, similar to non-mobilized BM-MSCs. In a co-culture system, rat M0-type macrophages co-cultured with PB-MSCs have shown higher expression of M2 markers including CD206, Arg-1, IL-10, and CCL-22 than BM-MSCs, indicating that PB-MSCs induced greater M0 polarization to M2. Furthermore, compared with BM-MSCs, PB-MSCs in a co-culture system with lipopolysaccharide-induced M1-type macrophages more efficiently promoted M1 polarization to M2, accompanied by increasing expression of CD206, Arg-1, IL-10, and CCL-22 while decreasing expression of M1 markers including iNOS, TNF-α, IL-1ß and IL-6, indicating that PB-MSCs triggered greater M1 polarization to M2. Subsequently, polymerase chain reaction arrays showed higher expressions of both IL1rn and Tnfrsf11b in PB-MSCs versus BM-MSCs. In response to an inflammatory niche, such as TNF-α, PB-MSCs have shown higher expression and release of IL1RA, causing greater M2 polarization of macrophages, and the special effects may be almost entirely abolished through the neutralization antibody of IL1RA. Mechanistic studies determined that PB-MSCs showed higher levels NF-κBp65 and NF-κBp-p65 than BM-MSCs, which could be obviously enhanced by TNF-α. And the increased IL1RA expression by TNF-α in PB-MSCs could be markedly canceled by an NF-κB inhibitor PDTC. Interestingly, mimicking the mobilized PB-MSCs by a combination of G-CSF and AMD3100 in vivo, BM-MSCs were treated with G-CSF and/or AMD3100 in vitro, showing the increased expressions of NF-κBp65 and IL1RA, which could be prominently abolished by PDTC. Therefore, targeting IL1rn, gene modification or drug intervention for MSCs may provide a novel therapeutic strategy for human diseases, especially inflammatory diseases.

Continuous exposure of isoprenaline inhibits myoblast differentiation and fusion through PKA/ERK1/2-FOXO1 signaling pathway.

AIM: The objective of this study is to determine if exuberant sympathetic nerve activity is involved in muscle satellite cell differentiation and myoblast fusion. METHODS AND RESULTS: By using immunoassaying and western blot analyses, we found that ß1 and ß2-adrenergic receptors (AdR) were expressed in C2C12 cells. The differentiated satellite cells exhibited an increased expression of ß2-AdR, as compared with the proliferating cells. Continuous exposure of isoprenaline (ISO), a ß-AdR agonist, delayed C2C12 cell differentiation, and myoblast fusion in time- and dose-dependent manner. ISO also increased short myotube numbers while decreasing long myotube numbers, consistent with the greater reduction in MyHC1, MyHC2a, and MyHC2x expression. Moreover, continuous exposure of ISO gradually decreased the ratio of PKA RI/RII, and PKA RI activator efficiently reversed the ISO effect on C2C12 cell differentiation and myoblast fusion while PKA inhibitor H-89 deteriorated the effects. Continuous single-dose ISO increased ß1-AdR expression in C2C12 cells. More importantly, the cells showed enhanced phospho-ERK1/2 levels, resulting in increasing phospho-ß2-AdR levels while decreasing ß2-AdR levels, and the specific effects could be abolished by ERK1/2 inhibitor. Furthermore, continuous exposure of ISO induced FOXO1 nuclear translocation and increased the levels of FOXO1 in nuclear extracts while reducing pAKT, p-p38MAPK, and pFOXO1 levels. Conversely, blockade of ERK1/2 signaling partially abrogated ISO effects on AKT, p38MAPK, and FOXO1signaling, which partially restored C2C12 cell differentiation and myoblast fusion, leading to an increase in the numbers of medium myotube along with the increased expression of MyHC1 and MyHC2a. CONCLUSION: Continuous exposure of ISO impedes satellite cell differentiation and myoblast fusion, at least in part, through PKA-ERK1/2-FOXO1 signaling pathways, which were associated with the reduced ß2-AdR and increased ß1-AdR levels.