-Rodent hearts can regenerate myocardium lost to apical resection or myocardial infarction for up to 7 days after birth, but whether a similar window for myocardial regeneration also exists in large mammals is unknown. -Acute myocardial infarction (AMI) was surgically induced in neonatal pigs on postnatal days 1, 2, 3, 7, and 14 (i.e., the P1, P2, P3, P7, and P14 groups, respectively). Cardiac systolic function was evaluated before AMI and at 30 days post AMI via transthoracic echocardiography. Cardiomyocyte cell cycle activity was assessed via immunostaining for proliferation and mitosis markers, infarct size was evaluated histologically, and telomerase activity was measured by quantitative PCR. -Systolic function at day 30 post- AMI was largely restored in P1 animals and partially restored in P2 animals, but significantly impaired when AMI was induced on postnatal day 3 or later. Hearts of P1 animals showed little evidence of scar formation or wall thinning on day 30 after AMI, with increased measures of cell-cycle activity seen six days after AMI (i.e. postnatal day 7) compared to postnatal day 7 in noninfarcted hearts. -The neonatal porcine heart is capable of regeneration following AMI during the first 2 days of life. This phenomenon is associated with induction of cardiomyocyte proliferation, and is lost when cardiomyocytes exit cell cycle shortly after birth.
Cardiovascular physiology exhibits time-of-day-dependent oscillations, which are mediated by both extrinsic (e.g., environment/behavior) and intrinsic (e.g., circadian clock) factors. Disruption of circadian rhythms negatively affects multiple cardiometabolic parameters. Recent studies suggest that the cardiomyocyte circadian clock directly modulates responsiveness of the heart to metabolic stimuli (e.g., fatty acids) and stresses (e.g., ischemia/reperfusion). The aim of this study was to determine whether genetic disruption of the cardiomyocyte circadian clock impacts insulin-regulated pathways in the heart. Genetic disruption of the circadian clock in cardiomyocyte-specific Bmal1 knockout (CBK) and cardiomyocyte-specific Clock mutant (CCM) mice altered expression (gene and protein) of multiple insulin signaling components in the heart, including p85α and Akt. Both baseline and insulin-mediated Akt activation was augmented in CBK and CCM hearts (relative to littermate controls). However, insulin-mediated glucose utilization (both oxidative and non-oxidative) and AS160 phosphorylation were attenuated in CBK hearts, potentially secondary to decreased Inhibitor-1. Consistent with increased Akt activation in CBK hearts, mTOR signaling was persistently increased, which was associated with attenuation of autophagy, augmented rates of protein synthesis, and hypertrophy. Importantly, pharmacological inhibition of mTOR (rapamycin; 10 days) normalized cardiac size in CBK mice. These data suggest that disruption of cardiomyocyte circadian clock differentially influences insulin-regulated processes, and provide new insights into potential pathologic mediators following circadian disruption.
Exosomes hold great potential to deliver therapeutic reagents for cancer treatment due to its inherent low antigenicity. However, several technical barriers need be overcome before widely clinical applications, such as low productivity and ineffective cancer targeting. The present study aimed at creating a new biomanufacturing platform of cancer-targeted exosomes for drugs delivery.Specifically, we created a scalable, robust, high-yield, cell line-based exosomes production process in stirred-tank bioreactor, and developed an efficient surface tagging technique to generate mAbexosomes. The in vitro characterization using transmission electron microscopy, NanoSight nanoparticle tracking analysis and Western blotting confirmed the high quality of exosomes. Flow cytometry and confocal laser scanning microscopy imaging demonstrated mAb-exosomes had strong surface binding to cancer cells. Furthermore, to validate the targeted drug delivery efficiency, we loaded Romidepsin, a histone deacetylase inhibitor, into mAb-exosomes. The in vitro anti-cancer toxicity study showed high cytotoxicity of mAb-exosome-Romidepsin to cancer cells. Finally, the in vivo study using tumor xenograft animal model validated the cancer targeting specificity, anti-cancer efficacy, and drug delivery capability of the targeted exosomes. In summary, we developed new techniques enabling targeted exosomes for drug delivery to support large-scale animal studies and facilitate the translation from research to clinics.
Differentiation of cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) is critically dependent upon the regulation of the Wnt signaling pathway. The mechanisms remain unclear with regard to the dose and timing of each differentiation inducer, and the interaction of the inducers that regulate the Wnt in mesendoderm specification to cardiac mesoderm. Consequently, it remains far from optimal in differentiation efficiency and consistency from hiPSC lines to CMs. Here, we have carefully deciphered the role of Wnt signaling pathway manipulation on mesoderm specification in a dosage and time dependent manner. To examine the hypothesis of that fate specification of hiPSC-CMs differentiation is dictated by temporal and spatial factors that regulate Wnt, we evaluate hiPSC-CM differentiation with: (1) two-phase modulation of Wnt, (2) dosage variant of GSK3β inhibitors, (3) treatment with insulin, and (4) 3-dimentional suspension culture environment on iPSC-CM differentiation. The results highlight the importance of mesendoderm specification to cardiac mesoderm, which needs precisely regulation of Wnt in a dosage dependent and temporal on/off manner. This temporal regulation dictates the final efficiency and purity of derived cardiomyocytes. After the initial activation of Wnt signaling pathway to generate mesendoderm, the maintenance of Wnt signaling at an appropriate dose is critical to direct the cell fate into cardiac mesoderm. Otherwise, lower Wnt signals lead to definitive endoderm and higher Wnt signals induce presomitic mesoderm differentiation. The precisely specification of cardiac mesoderm results in not only greater than 90% of cTnT+ cardiomyocytes but also high cardiomyocytes yield under both monolayer and suspension culture conditions. Thus, the current findings provide critical insights to decipher the temporal mechanism of Wnt activation in regulation of hiPSC-CMs differentiation, and more importantly provide the guidelines for the consistent and high-yield and high-quality hiPSC-CMs production in cardiovascular research.
Background-We have shown that genetic overexpression of cell cycle proteins can increase the proliferation of transplanted cardiomyocytes derived from human induced-pluripotent stem cells (hiPSC-CMs) in animal models of myocardial infarction (MI). Here, we introduce a new, nongenetic approach to promote hiPSC-CM cell cycle activity and proliferation in transplanted human cardiomyocyte patches (hCMPs).Methods-Mice were randomly distributed into 5 experimental groups (n = 10 per group). One group underwent Sham surgery, and the other 4 groups underwent MI induction surgery followed by treatment with hCMPs composed of hiPSC-CMs and nanoparticles that contained CHIR99021 and FGF1 (the NP CF -hCMP group), with hCMPs composed of hiPSC-CMs and empty nanoparticles (the NP E -hCMP group); with patches containing the CHIR99021/FGF-loaded nanoparticles but lacking hiPSC-CMs (the NP CF -Patch group), or patches lacking both the nanoparticles and cells (the E-Patch group). Cell cycle activity was evaluated via Ki67 and Aurora B expression, bromodeoxyuridine incorporation, and phosphorylated histone 3 levels (immunofluorescence); engraftment via human cardiac troponin T or human nuclear antigen expression (immunofluorescence) and bioluminescence imaging; cardiac function via echocardiography; infarct size and wall thickness via histology; angiogenesis via isolectin B4 expression (immunofluorescence); and apoptosis via TUNEL and caspace 3 expression (immunofluorescence).
Preconditioning with the ROCK inhibitor Y-27632 increased the engraftment of transplanted hiPSC-CM in a murine MI model, while reversibly impairing hiPSC-CM contractility and promoting adhesion.
Analysis of transcriptomic data demonstrates extensive epigenetic gene silencing of the transcription factor PRDM16 in renal cancer. We show that restoration of PRDM16 in RCC cells suppresses in vivo tumor growth. RNaseq analysis reveals that PRDM16 imparts a predominantly repressive effect on the RCC transcriptome including suppression of the gene encoding semaphorin 5B (SEMA5B). SEMA5B is a HIF target gene highly expressed in RCC that promotes in vivo tumor growth. Functional studies demonstrate that PRDM16’s repressive properties, mediated by physical interaction with the transcriptional corepressors C-terminal binding proteins (CtBP1/2), are required for suppression of both SEMA5B expression and in vivo tumor growth. Finally, we show that reconstitution of RCC cells with a PRDM16 mutant unable to bind CtBPs nullifies PRDM16’s effects on both SEMA5B repression and tumor growth suppression. Collectively, our data uncover a novel epigenetic basis by which HIF target gene expression is amplified in kidney cancer and a new mechanism by which PRDM16 exerts its tumor suppressive effects.
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