hESC-Derived Epicardial Cells Promote Repair of Infarcted Hearts in Mouse and Swine.

Myocardial infarction (MI) causes excessive damage to the myocardium, including the epicardium. However, whether pluripotent stem cell-derived epicardial cells (EPs) can be a therapeutic approach for infarcted hearts remains unclear. Here, the authors report that intramyocardial injection of human embryonic stem cell-derived EPs (hEPs) at the acute phase of MI ameliorates functional worsening and scar formation in mouse hearts, concomitantly with enhanced cardiomyocyte survival, angiogenesis, and lymphangiogenesis. Mechanistically, hEPs suppress MI-induced infiltration and cytokine-release of inflammatory cells and promote reparative macrophage polarization. These effects are blocked by a type I interferon (IFN-I) receptor agonist RO8191. Moreover, intelectin 1 (ITLN1), abundantly secreted by hEPs, interacts with IFN-ß and mimics the effects of hEP-conditioned medium in suppression of IFN-ß-stimulated responses in macrophages and promotion of reparative macrophage polarization, whereas ITLN1 downregulation in hEPs cancels beneficial effects of hEPs in anti-inflammation, IFN-I response inhibition, and cardiac repair. Further, similar beneficial effects of hEPs are observed in a clinically relevant porcine model of reperfused MI, with no increases in the risk of hepatic, renal, and cardiac toxicity. Collectively, this study reveals hEPs as an inflammatory modulator in promoting infarct healing via a paracrine mechanism and provides a new therapeutic approach for infarcted hearts.

Therapeutic silencing of lncRNA RMST alleviates cardiac fibrosis and improves heart function after myocardial infarction in mice and swine.

Rationale: Cardiac fibrosis is an adverse consequence of aberrant fibroblast activation and extracellular matrix (ECM) deposition following myocardial infarction (MI). Recently, long noncoding RNAs (lncRNAs) have been reported to participate in multiple cardiac diseases. However, the biological functions of lncRNA rhabdomyosarcoma 2-associated transcript (RMST) in cardiac fibrosis remain largely unknown. Methods: The role of RMST in regulating cardiac fibroblast (CF) proliferation, fibroblast-to-myofibroblast transition (FMT), and ECM production, which were induced by transforming growth factor-ß1, was evaluated through immunofluorescence staining, cell contraction assay, cell migration assay, qRT-PCR, and western blot. The therapeutic effect of RMST silencing was assessed in murine and porcine MI models. Results: The present study showed that RMST expression was upregulated and associated with cardiac fibrosis in murine and porcine MI models. Further loss-of-function studies demonstrated that RMST silencing in vitro significantly inhibited CF proliferation, FMT, and ECM production. Accordingly, RMST knockdown in vivo alleviated cardiac fibrosis and improved cardiac contractile function in MI mice. Moreover, RMST acted as a competitive endogenous RNA of miR-24-3p. miR-24-3p inhibition abolished, while miR-24-3p agomir reproduced, the RMST knockdown-mediated effects on CF fibrosis by regulating the lysyl oxidase signaling pathway. Finally, the therapeutic potential of RMST knockdown was evaluated in a porcine MI model, and local RMST knockdown significantly inhibited cardiac fibrosis and improved myocardial contractile function in pigs after MI. Conclusion: Our findings identified RMST as a crucial regulator of cardiac fibrosis, and targeting RMST may develop a novel and efficient therapeutic strategy for treating fibrosis-related cardiac diseases.

Therapeutic delivery of microRNA-125a-5p oligonucleotides improves recovery from myocardial ischemia/reperfusion injury in mice and swine.

Rationale: Clinical application of mesenchymal stem cells (MSCs) and MSC-derived exosomes (MSC-Exos) to alleviate myocardial ischemia/reperfusion (I/R) injury is compromised by the low cell engraftment rate and uncontrolled exosomal content. As one of their active ingredients, single-component microRNA therapy may have more inherent advantages. We sought to find an ideal microRNA candidate and determine whether it could reproduce the cardioprotective effects of MSCs and MSC-Exos. Methods: Cardiac function and myocardial remodeling in MSC, MSC-Exo, or microRNA oligonucleotide-treated mouse hearts were investigated after I/R injury. The effects of microRNA oligonucleotides on cardiac cells (macrophages, cardiomyocytes, fibroblasts, and endothelial cells) and their downstream mechanisms were confirmed. Large animals were also employed to investigate the safety of microRNA therapy. Results: The results showed that microRNA-125a-5p (miR-125a-5p) is enriched in MSC-Exos, and intramyocardial delivery of their modified oligonucleotides (agomir) in mouse I/R myocardium, as well as MSCs or MSC-Exos, exerted obvious cardioprotection by increasing cardiac function and limiting adverse remodeling. In addition, miR-125a-5p agomir treatment increased M2 macrophage polarization, promoted angiogenesis, and attenuated fibroblast proliferation and activation, which subsequently contributed to the improvements in cardiomyocyte apoptosis and inflammation. Mechanistically, Klf13, Tgfbr1, and Daam1 are considered the targets of miR-125a-5p for regulating the function of macrophages, fibroblasts, and endothelial cells, respectively. Similar results were observed following miR-125a-5p agomir treatment in a porcine model, with no increase in the risk of arrhythmia or hepatic, renal, or cardiac toxicity. Conclusions: This targeted microRNA delivery presents an effective and safe strategy as a stem cell and exosomal therapy in I/R cardiac repair.

Asiatic acid alleviates ischemic myocardial injury in mice by modulating mitophagy- and glycophagy-based energy metabolism.

Myocardial infarction (MI) causes disturbances in myocardial energy metabolism, ultimately leading to a poor prognosis. Cytosolic glycogen autophagy (glycophagy) and mitochondrial autophagy (mitophagy) are upregulated in MI to optimize energy metabolism but to a limited extent. Asiatic acid (AA), a pentacyclic triterpene derived from the traditional Chinese herb Centella asiatica, displays anti-inflammatory, antioxidant, and antiapoptotic activities. AA has been found to alleviate focal cerebral and liver ischemic injury by reversing mitochondrial dysfunction. In this study, we investigated whether AA exerted cardioprotective effects against MI by activating glycophagy and mitophagy to improve the energy balance. In vitro cardioprotective effects were examined in neonatal mouse cardiomyocytes subjected to oxygen-glucose deprivation for 12 h. Treatment with AA (2-50 µM) significantly increased cell viability and improved the energy metabolism evidenced by increased ATP level and phosphocreatine/ATP ratio. In vivo cardioprotective effects were studied in a mouse model of MI. Administration of AA (5-125 mg·kg-1·d-1, ig) significantly reduced infarct size and ischemic myocardial injury, and improved cardiac function. AA treatment also promoted mitophagy and relieved mitochondrial edema evidenced by increased number of mitophagosomes in ischemic myocardium in vivo and increased mitochondria-light chain 3 (LC3)-II colocalization in ODG-treated cardiomyocytes in vitro. Mitophagy activation was accompanied by activation of the AMPK signaling pathway. Knockdown of AMPK abolished AA-activated mitophagy. Furthermore, we showed that glycophagy was upregulated in OGD cardiomyocytes evidenced by increased starch binding domain protein 1 (STBD1)-GABA type A receptor-associated protein-like 1(GABARAPL1) interaction and extracellular acidification rate, whereas AA treatment further promoted glycophagy accompanied by PI3K/Akt activation. PI3K inhibitor LY294002 or Akt inhibitor GSK690693 blocked the effects of AA on glycophagy and glycolysis. Finally, simultaneous inhibition of glycophagy and mitophagy abolished the cardioprotective effects and energy regulation of AA. These results demonstrate that AA protects ischemic cardiomyocytes by modulating glycophagy- and mitophagy-based energy metabolism through the PI3K/Akt and AMPK pathways.

NGF nanoparticles enhance the potency of transplanted human umbilical cord mesenchymal stem cells for myocardial repair.

In this study, we investigated whether human umbilical cord mesenchymal stem cell (hUCMSC) fibrin patches loaded with nerve growth factor (NGF) poly(lactic-co-glycolic acid) (PLGA) nanoparticles could enhance the therapeutic potency of hUCMSCs for myocardial infarction (MI). In vitro, NGF significantly improved the proliferation of hUCMSCs and mitigated cytotoxicity and apoptosis under hypoxic injury. NGF also promoted the paracrine effects of hUCMSCs on angiogenesis and cardiomyocyte protection. The tyrosine kinase A (TrkA) and phosphoinositide 3-kinase (PI3K)-serine/threonine protein kinase (Akt) signaling pathways in hUCMSCs were involved in the NGF-induced protection. NGF PLGA nanoparticles continued to release NGF for at least 1 mo and also exerted a protective effect on hUCMSCs, the same with free NGF. In vivo, we treated MI mice with nothing (MI group), a cell-free fibrin patch with blank PLGA nanoparticles (MI + OP group), a cell-free fibrin patch with NGF nanoparticles (MI + NGF group), and hUCMSC fibrin patches with blank PLGA nanoparticles (MI + MSC group) or NGF PLGA nanoparticles (MSC + NGF group). Among these groups, the MSC + NGF group exhibited the best cardiac contractile function, the smallest infarct size, and the thickest ventricular wall. The application of NGF PLGA nanoparticles significantly improved the retention of transplanted hUCMSCs and enhanced their ability to reduce myocardial apoptosis and promote angiogenesis in the mouse heart after MI. These findings demonstrate the promising therapeutic potential of hUCMSC fibrin cardiac patches loaded with NGF PLGA nanoparticles.NEW & NOTEWORTHY NGF PLGA nanoparticles can exert a protective effect on hUCMSCs and promote the paracrine effects of hUCMSCs on angiogenesis and cardiomyocyte protection through TrkA-PI3K/Akt signaling pathway, the same with free NGF. The application of NGF PLGA nanoparticles in the hUCMSC fibrin cardiac patches can significantly improve the retention of transplanted hUCMSCs and enhance their ability to reduce myocardial apoptosis and promote angiogenesis in the mouse heart after MI.

Phosphatase OsPP2C27 directly dephosphorylates OsMAPK3 and OsbHLH002 to negatively regulate cold tolerance in rice.

Improving chilling tolerance is a major target of rice breeding. The OsMAPK3-OsbHLH002-OsTPP1 signalling pathway enhances chilling tolerance in rice: the kinase is activated by cold stress, and subsequently the transcription factor is phosphorylated by the activated kinase, triggering the expression of cold response genes. However, it is largely unknown how this pathway is suppressed in time to avoid it being in a continuously activated state. We found that a novel type 2C protein phosphatase, OsPP2C27, functions as a negative regulator of the OsMAPK3-OsbHLH002-OsTPP1 pathway. A dynamic change in OsMAPK3 activity was found during cold treatment. We show that OsPP2C27 interacts physically with and dephosphorylates OsMAPK3 in vitro and in vivo. Interestingly, OsPP2C27 can also directly dephosphorylate OsbHLH002, the target of OsMAPK3. After cold treatment, survival rates were higher in OsPP2C27-RNAi lines and a T-DNA insertion mutant, and lower in OsPP2C27-overexpression lines, compared to wild type. Moreover, expression of the OsTPP1 and OsDREBs were increased in OsPP2C27-RNAi lines and decreased in OsPP2C27-overexpression lines. These results indicate that cold-induced OsPP2C27 negatively regulates the OsMAPK3-OsbHLH002-OsTPP1 signalling pathway by directly dephosphorylating both phospho-OsMAPK3 and phospho-OsbHLH002, preventing the sustained activation of a positive pathway for cold stress and maintaining normal growth under chilling conditions.

Characterization of the hemolytic activity of Riemerella anatipestifer.

Riemerella anatipestifer infection causes serious economic losses in the duck industry worldwide. Acute septicemia and high blood bacterial loading in R. anatipestifer infected ducks indicate that R. anatipestifer may be able to obtain iron and other nutrients by lysing duck erythrocytes to support its rapid growth and proliferation in the blood. However, so far, little is known about the hemolytic activity of R. anatipestifer to duck erythrocytes. In this study, 29 of 52 R. anatipestifer strains showed hemolytic activity on duck blood agar, whereas all the tested dba+ (with hemolytic activity on duck blood agar) and dba- strains created pores in the duck red blood cells, with 4.35-9.03% hemolytic activity in a liquid hemolysis assay after incubation for 24 h. The concentrated culture supernatants of all the tested R. anatipestifer strains and the extracted outer membrane proteins (OMPs) from dba+R. anatipestifer strains showed hemolytic activity on duck blood agar. These results, together with the median lethal dose (LD50) of some dba+ and dba-R. anatipestifer strains in ducklings, suggested that there was no direct relationship between the hemolytic capacity of R. anatipestifer on duck blood agar and its virulence.

Evaluation of the protective immunity of Riemerella anatipestifer OmpA.

Riemerella anatipestifer is responsible for an economically important disease of commercially raised ducks. No or only few cross-protection was observed between different serotypes of R. anatipestifer strains, and so far no protective antigen in this bacterium has been identified. OmpA is a predominant immunogenic protein of R. anatipestifer, and within the 1467 bp ompA ORF (ompA1467), there is another 1164 bp ORF (ompA1164) with the same C-terminal. In this study, our results showed that the full sequence of ompA1467 from some R. anatipestifer strains with different serotypes shared the same amino acid sequence. Animal experiments showed that the soluble recombinant protein rOmpA1164, but not rOmpA1467, could provide partial protective immunity against challenge. Moreover, there was no significant difference in protective immunity between ducklings immunized with Th4△ompA bacterin and those immunized with Th4 bacterin. In addition, OmpA1467 was the main existing form of OmpA in R. anatipestifer cells by gel electrophoresis and western blot analyses. The results suggested that OmpA1467 was not a protective antigen of R. anatipestifer, and antibodies against proteins other than OmpA play a critical role in the process of anti-R. anatipestifer infection.