Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. However, its pathogenesis remains unknown. To explore key regulatory connections and crucial pathways underlying the TOF, gene or microRNA expression profile datasets of human TOF were obtained from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. The differentially expressed mRNAs (DEmRNAs) and microRNAs (DEmiRs) between TOF and healthy groups were identified after data preprocessing, followed by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Then, we further constructed protein-protein interaction (PPI) network and subnetwork of modules. Ultimately, to investigate the regulatory network underlying TOF, a global triple network including miRNAs, mRNAs, and transcription factors (TFs) was constructed based on the integrated data. In the present study, a total of 529 DEmRNAs, including 115 downregulated and 414 upregulated DEmRNAs, and 7 significantly upregulated DemiRs, including miR-499, miR-23b, miR-222, miR-1275, miR-93, miR-155, and miR-187, were found between TOF and control groups. Furthermore, 22 hub genes ranked by top 5% genes with high connectivity and six TFs, including SRF, CNOT4, SIX6, SRRM3, NELFA, and ONECUT3, were identified and might play crucial roles in the molecular pathogenesis of TOF. Additionally, an miRNA-mRNA-TF co-regulatory network was established and indicated ubiquitin-mediated proteolysis, energy metabolism associated pathways, neurodevelopmental disorder associated pathways, and ribosomes might be involved in the pathogenesis of TOF. The current research provides a comprehensive perspective of regulatory mechanism networks underlying TOF and also identifies potential molecule targets of genetic counseling and prenatal diagnosis for TOF.

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Identification of miRNA-mRNA-TFs Regulatory Network and Crucial Pathways Involved in Tetralogy of Fallot.

microRNAs signatures as potential biomarkers of structural cardiotoxicity in human-induced pluripotent stem-cell derived cardiomyocytes

The therapeutic effect of mesenchymal stem cells (MSCs) on sepsis has been well-known. However, a comprehensive understanding of the relationship between MSCs and macrophages remains elusive. Superparamagnetic iron oxide (SPIO) is one of the most commonly used tracers for MSCs. Our previous study has shown that SPIO enhanced the therapeutic effect of MSCs in a macrophage-dependent manner. However, the fate of SPIO-labeled MSCs (MSCSPIO) after infusion remains unknown and the direct interaction between MSCSPIO and macrophages remains unclear. Mice were injected intravenously with MSCSPIO at 2 h after Escherichia coli infection and sacrificed at different times to investigate their distribution and therapeutic effect. We found that MSCSPIO homed to lungs rapidly after infusion and then trapped in livers for more than 10 days. Only a few MSCSPIO homed to the spleen and there was no MSCSPIO detectable in the brain, heart, kidney, colon, and uterus. MSCSPIO tended to stay longer in injured organs compared with healthy organs and played a long-term protective role in sepsis. The mRNA expression profiles between MSCs and MSCSPIO were rather different, genes related to lipid metabolism, inflammation, and oxidative stress were changed. The levels of ROS and lipid peroxide were elevated in MSCSPIO, which confirmed that SPIO-induced ferroptosis in MSCSPIO. Ferroptosis of MSCSPIO induced by SPIO enhanced the efferocytosis of macrophages and thus enhanced the protective effect on septic mice, while the benefits were impaired after MSCSPIO were treated with Ferrostatin-1 (Fer-1) or Liproxtatin-1 (Lip-1), the inhibitors of ferroptosis. SPIO-induced ferroptosis in MSCs contributes to better therapeutic effects in sepsis by enhancing the efferocytosis of macrophages. Our data showed the efficacy and advantage of MSCSPIO as a therapeutic tool and the cell states exert different curative effects on sepsis. 

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Ferroptotic MSCs protect mice against sepsis via promoting macrophage efferocytosis

New and creative approaches are required to treat chronic infections caused by increasingly drug-resistant strains of bacteria. One strategy is the use of cellular therapy employing mesenchymal stromal cells (MSC) to kill bacteria directly and to also activate effective host immunity to infection. We demonstrated previously that activated MSC delivered systemically could be used effectively together with antibiotic therapy to clear chronic biofilm infections in rodent models. Therefore, we sought in the current studies to gain new insights into the antimicrobial properties of activated canine MSC and to evaluate their effectiveness as a novel cellular therapy for treatment of naturally-occurring drug resistant infections in dogs. These studies revealed that canine MSC produce and secrete antimicrobial peptides that synergize with most classes of common antibiotics to trigger rapid bactericidal activity. In addition, activated canine MSC migrated more efficiently to inflammatory stimuli, and secreted factors associated with wound healing and fibroblast proliferation and recruitment of activated neutrophils. Macrophages incubated with conditioned medium from activated MSC developed significantly enhanced bactericidal activity. Clinical studies in dogs with chronic multidrug resistant infections treated by repeated i.v. delivery of activated, allogeneic MSC demonstrated significant clinical benefit, including infection clearance and healing of infected tissues. Taken together, the results of these studies provide new insights into antimicrobial activity of canine MSC, and their potential clinical utility for management of chronic, drug-resistant infections.

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Activated Mesenchymal Stromal Cell Therapy for Treatment of Multi-Drug Resistant Bacterial Infections in Dogs

Background Myocardial dysfunction caused by sepsis (SIMD) leads to high mortality in critically ill patients. We investigated the function and mechanism of long non-coding RNA MAPKAPK5-AS1 (lncRNA MAPKAPK-AS1) on lipopolysaccharide (LPS)-induced inflammation response in vivo and in vitro. Method Male SD rats were utilized for in vivo experiments. Rat cardiomyocytes (H9C2) were employed for in vitro experiments. Western blotting was employed to measure protein expression, and RT-PCR was performed to measure mRNA expression of inflammation factors. TUNEL and flow cytometry were carried out to evulate cell apoptosis. Result The results showed that the expression of MAPKAPK5-AS1 was increased, while the expression of miR-124-3p was decreased in the inflammatory damage induced by LPS in vivo and in vitro. Knockdown of MAPKAPK5-AS1 reduced LPS-induced cell apoptosis and inflammation response, while overexpression of miR-124-3p weakened the effects of MAPKAPK5-AS1 knockdown on LPS-induced cell apoptosis and inflammation response. Moreover, miR-124-3p was identified as a downstream miRNA of MAPKAPK5-AS1, and E2F3 was a target of miR-214-3p. MAPKAPK5-AS1 knockdown increased the expression of miR-124-3p, while miR-124-3p overexpression reduced the expression of MAPKAPK5-AS1. In addition, miR-124-3p was found to downregulate E2F3 expression in H9C2 cells. Conclusion MAPKAPK5-AS1/miR-124-3p/E2F3 axis regulates LPS-related H9C2 cell apoptosis and inflammatory response.

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Long noncoding RNA MAPKAPK5-AS1 promoted lipopolysaccharide-induced inflammatory damage in the myocardium by sponging microRNA-124-3p/E2F3.

Sepsis-induced myocardial dysfunction (MD) is an important pathophysiological feature of multiorgan failure caused by a dysregulated host response to infection. Patients with MD continue to be managed in intensive care units with limited understanding of the molecular mechanisms controlling disease pathogenesis. Emerging evidences support the use of mesenchymal stem/stromal cell (MSC) therapy for treating critically ill septic patients. Combining this with the known role that microRNAs (miRNAs) play in reversing sepsis-induced myocardial-dysfunction, this study sought to investigate how MSC administration alters miRNA expression in the heart. Mice were randomized to experimental polymicrobial sepsis induced by cecal ligation and puncture (CLP) or sham surgery, treated with either MSCs (2.5 × 105) or placebo (saline). Twenty-eight hours post-intervention, RNA was collected from whole hearts for transcriptomic and microRNA profiling. The top microRNAs differentially regulated in hearts by CLP and MSC administration were used to generate a putative mRNA-miRNA interaction network. Key genes, termed hub genes, within the network were then identified and further validated in vivo. Network analysis and RT-qPCR revealed that septic hearts treated with MSCs resulted in upregulation of five miRNAs, including miR-187, and decrease in three top hit putative hub genes (Itpkc, Lrrc59, and Tbl1xr1). Functionally, MSC administration decreased inflammatory and apoptotic pathways, while increasing cardiac-specific structural and functional, gene expression. Taken together, our data suggest that MSC administration regulates host-derived miRNAs production to protect cardiomyocytes from sepsis-induced MD.

Mesenchymal Stem/Stromal Cells Increase Cardiac MIR-187-3P Expression in Polymicrobial Animal Model of Sepsis.

Regulation of mir-187b in endotoxemic primary cardiomyocytes and septic murine hearts treated with mesenchymal stromal/stem cells

Despite diminution of myocardial dysfunction, organ injury, and animal mortality in polymicrobial models of sepsis upon systemic administration of bone marrow-derived mesenchymal stem cell (MSC), t...

Keisuke Mori

Papers

Mesenchymal Stem/Stromal Cells Increase Cardiac MIR-187-3P Expression in Polymicrobial Animal Model of Sepsis.

Regulation of mir-187b in endotoxemic primary cardiomyocytes and septic murine hearts treated with mesenchymal stromal/stem cells

Abstract 203: Identification of Immunoregulatory microRNAs in Cardiac Tissue of Septic Mice Treated with Mesenchymal Stem Cells