Cargo proteins in extracellular vesicles: potential for novel therapeutics in non-alcoholic steatohepatitis

Background Extracellular vesicles (EVs) are recognized as novel cell-free therapeutics. Non-alcoholic steatohepatitis (NASH) remains a critical health problem. Herein, we show that EVs from pan peroxisome proliferator-activated receptor agonist-primed induced mesenchymal stem cell (pan PPAR-iMSC-EVs) has unique cargo protein signatures, and demonstrate its therapeutic function in NASH. Results A unique protein signatures were identified in pan PPAR-iMSC-EVs against those from non-stimulated iMSC-EVs. NASH mice receiving pan PPAR-iMSC-EVs showed reduced steatotic changes and ameliorated ER stress and mitochondiral oxidative stress induced by inflammation. Moreover, pan PPAR-iMSC-EVs promoted liver regeneration via inhibiting apoptosis and enhancing proliferation. Conclusions We conclude that our strategy for enriching unique cargo proteins in EVs may facilitate the development of novel therapeutic option for NASH. Graphical AbstractThis work was supported by the Technology Development Program (S2823001) from the Ministry of SMEs and Startups (MSS, Korea). This work was also supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2018R1D1A1A02085481)

Epigenetic analyses and modulation of nuclear reprogramming

This dissertation is composed of three lines of research regarding nuclear reprogramming. At first, expression profiles in deceased and live cloned pigs were compared to controls using microarray. Differentially expressed (DE) genes in deceased clones were potentially causing pathological anomalies such as dysregulated surfactant homeostasis in the lung. A large extent of DE genes in live clones demonstrates that live clones may be not merely the least affected animals but harbor magnified abnormalities, for example, delayed alveologenesis in the lung. Global DNA methylation analysis revealed hypomethylation in deceased clones whereas hypermethylation in live clones. Together, incomplete reprogramming by somatic cell nuclear transfer appears to contribute to dysregulated gene expression, resulting in pathogenesis in clones. ^ To determine the compatibility of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in cloned pigs, maternal lineages of clones were traced by mtDNA sequencing followed by expression profiling of nDNA- and mtDNA-encoded genes. While expression profiles of nDNA-encoded genes in clones were distinct from controls, mtDNA-encoded genes particularly in deceased clones showed an intermixed clustering. These results demonstrate that incomplete reprogramming adversely affects the gene expression of mtDNA. ^ To evaluate oxidative guanine as an alternative epigenetic modification, degree of guanine oxidation was measured in oxidative stress-induced mouse embryonic stem cells (ESC) Qualitative and quantitative analyses of oxidative guanine revealed that oxidative stress-induced ESCs harbored significantly increased level of 8oxoG Furthermore, the incidence of beating embryoid bodies differentiated from oxidative stress-induced ESCs was significantly increased, suggesting oxidative guanine has transmittable effects on cardiac-lineage specification after initiation of ESC differentiation. ^ In subsequent study, the reprogramming potential of carcinogens was evaluated. A putative reprogramming protocol was applied to mouse embryonic fibroblasts with 16 chemical candidates identified through function-oriented in silico high-throughput screening. From recovery day 2, alkaline phosphatase and surface specific embryonic antigen-1 positive colonies appeared at an efficiency of 0.02%. Expression profiling revealed that the colonies gained neuromuscular characteristics by partial reprogramming in addition to chemically induced adverse effects. ^ In conclusion, knowledge on epigenetic regulation of genes accumulated through these studies can possibly lead to precise regulation of nuclear reprogramming in the future.

Mesenchymal Stem Cell-derived Extracellular Vesicles for Skin Wound Healing

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Drug Signature-based Finding of Additional Clinical Use of LC28-0126 for Neutrophilic Bronchial Asthma

In recent decades, global pharmaceutical companies have suffered from an R&D innovation gap between the increased cost of a new drug's development and the decreased number of approvals. Drug repositioning offers another opportunity to fill the gap because the approved drugs have a known safety profile for human use, allowing for a reduction of the overall cost of drug development by eliminating rigorous safety assessment. In this study, we compared the transcriptional profile of LC28-0126, an investigational drug for acute myocardial infarction (MI) at clinical trial, obtained from healthy male subjects with molecular activity profiles in the Connectivity Map. We identified dyphilline, an FDA-approved drug for bronchial asthma, as a top ranked connection with LC28-0126. Subsequently, we demonstrated that LC28-0126 effectively ameliorates the pathophysiology of neutrophilic bronchial asthma in OVA(LPS)-OVA mice accompanied with a reduction of inflammatory cell counts in the bronchoalveolar lavage fluid (BALF), inhibition of the release of proinflammatory cytokines, relief of airway hyperactivity, and improvement of histopathological changes in the lung. Taken together, we suggest that LC28-0126 could be a potential therapeutic for bronchial asthma. In addition, this study demonstrated the potential general utility of computational drug repositioning using clinical profiles of the investigational drug.Y

Dysregulation of the Acrosome Formation Network by 8-oxoguanine (8-oxoG) in Infertile Sperm: A Case Report with Advanced Techniques

8-Hydroxyguanine (8-oxoG) is the most common oxidative DNA lesion and unrepaired 8-oxoG is associated with DNA fragmentation in sperm. However, the molecular effects of 8-oxoG on spermatogenesis are not entirely understood. Here, we identified one infertile bull (C14) due to asthenoteratozoospermia. We compared the global concentration of 8-oxoG by reverse-phase liquid chromatography/mass spectrometry (RP-LC/MS), the genomic distribution of 8-oxoG by next-generation sequencing (OG-seq), and the expression of sperm proteins by 2-dimensional polyacrylamide gel electrophoresis followed by peptide mass fingerprinting (2D-PAGE/PMF) in the sperm of C14 with those of a fertile bull (C13). We found that the average levels of 8-oxoG in C13 and C14 sperm were 0.027% and 0.044% of the total dG and it was significantly greater in infertile sperm DNA (p = 0.0028). Over 81% of the 8-oxoG loci were distributed around the transcription start site (TSS) and 165 genes harboring 8-oxoG were exclusive to infertile sperm. Functional enrichment and network analysis revealed that the Golgi apparatus was significantly enriched with the products from 8-oxoG genes of infertile sperm (q = 2.2 × 10−7). Proteomic analysis verified that acrosome-related proteins, including acrosin-binding protein (ACRBP), were downregulated in infertile sperm. These preliminary results suggest that 8-oxoG formation during spermatogenesis dysregulated the acrosome-related gene network, causing structural and functional defects of sperm and leading to infertility

Disruption of Mitochondrion-To-Nucleus Interaction in Deceased Cloned Piglets.

Most animals produced by somatic cell nuclear transfer (SCNT) are heteroplasmic for mitochondrial DNA (mtDNA). Oxidative phosphorylation (OXPHOS) in clones therefore requires the coordinated expression of genes encoded by the nuclear DNA and the two sources of mitochondria. Such interaction is rarely studied because most clones are generated using slaughterhouse oocytes of unrecorded origin. Here we traced the maternal lineages of seven diseased and five one-month-old live cloned piglets by sequencing their mtDNA. Additionally by using a 13K oligonucleotide microarray, we compared the expression profiles of nuclear and mtDNA-encoded genes that are involved in mitochondrial functions and regulation between the cloned groups and their age-matched controls (n=5 per group). We found that the oocytes used to generate the cloned piglets were of either the Large White or Duroc background, and oocyte genetic background was not related to the clones' survival. Expression profiles of mtDNA-encoded genes in clones and controls showed intermixed clustering patterns without treatment or maternal lineage-dependency. In contrast, clones and controls clustered separately for their global and nuclear DNA-encoded mitochondrial genes in the lungs for both the deceased and live groups. Functional annotation of differentially expressed genes encoded by both nuclear and mtDNA revealed abnormal gene expression in the mitochondrial OXPHOS pathway in deceased clones. Among the nine differentially expressed genes of the OXPHOS pathway, seven were down-regulated in deceased clones compared to controls, suggesting deficiencies in mitochondrial functions. Together, these data demonstrate that the coordination of expression of mitochondrial genes encoded by nuclear and mtDNA is disrupted in the lung of diseased clones

Differential expression levels of 8-oxoG repair genes <i>Ogg1</i> and <i>PolB</i> in ESCs induced by oxidative stress.

<p>Relative mRNA levels of (A) <i>Ogg1</i> mRNA and (B) <i>PolB</i>. Gapdh was used as an internal control. Mean ± SD, **<i>p</i> < 0.01, ***<i>p</i> < 0.001, one-sided t-test. (C) Levels of Ogg1 and PolB proteins by Western blotting. α-tubulin was used as an internal control.</p

G∙C to C∙G transversions at exon 1 of the <i>Tbx5</i> gene in R1 mouse ESCs subjected to oxidative stress.

<p>G∙C to C∙G transversions at exon 1 of the <i>Tbx5</i> gene in R1 mouse ESCs subjected to oxidative stress.</p