Hyang-Hee Seo

MicroRNA-145 suppresses ROS-induced Ca2+ overload of cardiomyocytes by targeting CaMKIIδ.

A change in intracellular free calcium (Ca(2+)) is a common signaling mechanism of reperfusion-induced cardiomyocyte death. Calcium/calmodulin dependent protein kinase II (CaMKII) is a critical regulator of Ca(2+) signaling and mediates signaling pathways responsible for functions in the heart including hypertrophy, apoptosis, arrhythmia, and heart disease. MicroRNAs (miRNA) are involved in the regulation of cell response, including survival, proliferation, apoptosis, and development. However, the roles of miRNAs in Ca(2+)-mediated apoptosis of cardiomyocytes are uncertain. Here, we determined the potential role of miRNA in the regulation of CaMKII dependent apoptosis and explored its underlying mechanism. To determine the potential roles of miRNAs in H2O2-mediated Ca(2+) overload, we selected and tested 6 putative miRNAs that targeted CaMKIIδ, and showed that miR-145 represses CaMKIIδ protein expression and Ca(2+) overload. We confirmed CaMKIIδ as a direct downstream target of miR-145. Furthermore, miR-145 regulates Ca(2+)-related signals and ameliorates apoptosis. This study demonstrates that miR-145 regulates reactive oxygen species (ROS)-induced Ca(2+) overload in cardiomyocytes. Thus, miR-145 affects ROS-mediated gene regulation and cellular injury responses.

Cyclin G1 overcomes radiation-induced G2 arrest and increases cell death through transcriptional activation of cyclin B1

Although cyclin G1 has been implicated in certain p53-related biological phenomena, other aspects of its function remain unclear. Here we report hitherto unknown mechanism by which cyclin G1 increases radiation sensitivity by regulating the level of cyclin B1. Overexpression of cyclin G1 was observable in lung carcinoma tissues. Irradiation of human lung cells with cyclin G1 overexpression resulted in increased cell death and γ-H2AX foci suggesting that cyclin G1 rendered the cells more susceptible to DNA damage. Enhanced radiosensitivity by cyclin G1 was correlated with increased cyclin B1, CDC2/cyclin B1 complex, and MPM2. Cell cycle synchronization clearly showed coexpression of cyclin G1 and cyclin B1 in G2/M phase. Depletion of cyclin G1 by interference RNA revealed that cyclin G1 regulated transcription of cyclin B1 in a p53-independent manner, and confirmed that the increased mitotic cells and cell death by cyclin G1 were dependent upon cyclin B1. Therefore, our data suggest that cyclin G1 enhanced radiation sensitivity by overriding radiation-induced G2 arrest through transcriptional upregulation of cyclin B1.

MicroRNA‐365 Inhibits the Proliferation of Vascular Smooth Muscle Cells by Targeting Cyclin D1

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a common feature of disease progression in atherosclerosis. Cell proliferation is regulated by cell cycle regulatory proteins. MicroRNAs (miR) have been reported to act as important gene regulators and play essential roles in the proliferation and migration of VSMCs in a cardiovascular disease. However, the roles and mechanisms of miRs in VSMCs and neointimal formation are far from being fully understood. In this study, cell cycle‐specific cyclin D1 was found to be a potential target of miR‐365 by direct binding. Through an in vitro experiment, we showed that exogenous miR‐365 overexpression reduced VSMC proliferation and proliferating cell nuclear antigen (PCNA) expression, while miR‐365 was observed to block G1/S transition in platelet‐derived growth factor‐bb (PDGF‐bb)‐induced VSMCs. In addition, the proliferation of VSMCs by various stimuli, including PDGF‐bb, angiotensin II (Ang II), and serum, led to the downregulation of miR‐365 expression levels. The expression of miR‐365 was confirmed in balloon‐injured carotid arteries. Taken together, our results suggest an anti‐proliferative role for miR‐365 in VSMC proliferation, at least partly via modulating the expression of cyclin D1. Therefore, miR‐365 may influence neointimal formation in atherosclerosis patients. J. Cell. Biochem. 115: 1752–1761, 2014.

let-7b suppresses apoptosis and autophagy of human mesenchymal stem cells transplanted into ischemia/reperfusion injured heart 7by targeting caspase-3

IntroductionMesenchymal stem cells (MSCs) have therapeutic potential for the repair of myocardial injury. The efficacy of MSC therapy for myocardial regeneration mainly depends on the survival of cells after transplantation into the infarcted heart. In the transplanted regions, reactive oxygen species (ROS) can cause cell death, and this process depends on caspase activation and autophagosome formation.MethodsA Software TargetScan was utilized to search for microRNAs (miRNAs) that target caspase-3 mRNA. Six candidate miRNAs including let-7b were selected and transfected into human MSCs in vitro. Expression of MEK-EKR signal pathways and autophagy-related genes were detected. Using ischemia/reperfusion model (I/R), the effect of MSCs enriched with let-7b was determined after transplantation into infarcted heart area. Miller catheter was used to evaluate cardiac function.ResultsHere, we report that let-7b targets caspase-3 to regulate apoptosis and autophagy in MSCs exposed to ROS. Let-7b-transfected MSCs (let-7b-MSCs) showed high expression of survival-related proteins, including p-MEK, p-ERK and Bcl-2, leading to a decrease in Annexin V/PI- and TUNEL-positive cells under ROS-rich conditions. Moreover, autophagy-related genes, including Atg5, Atg7, Atg12 and beclin-1, were significantly downregulated in let-7b-MSCs. Using a rat model of acute myocardial infarction, we found that intramyocardial injection of let-7b-MSCs markedly enhanced left ventricular (LV) function and microvessel density, in accordance with a reduced infarct size and the expression of caspase-3.ConclusionsTaken together, these data indicate that let-7b may protect MSCs implanted into infarcted myocardium from apoptosis and autophagy by directly targeting caspase-3 signaling.

Upregulation of miR-23b enhances the autologous therapeutic potential for degenerative arthritis by targeting PRKACB in synovial fluid-derived mesenchymal stem cells from patients.

The use of synovial fluid-derived mesenchymal stem cells (SFMSCs) obtained from patients with degenerative arthropathy may serve as an alternative therapeutic strategy in osteoarthritis (OA) and rheumatoid arthritis (RA). For treatment of OA and RA patients, autologous transplantation of differentiated MSCs has several beneficial effects for cartilage regeneration including immunomodulatory activity. In this study, we induced chondrogenic differentiation of SFMSCs by inhibiting protein kinase A (PKA) with a small molecule and microRNA (miRNA). Chondrogenic differentiation was confirmed by PCR and immunocytochemistry using probes specific for aggrecan, the major cartilaginous proteoglycan gene. Absorbance of alcian blue stain to detect chondrogenic differentiation was increased in H-89 and/or miRNA-23btransfected cells. Furthermore, expression of matrix metalloproteinase (MMP)-9 and MMP-2 was decreased in treated cells. Therefore, differentiation of SFMSCs into chondrocytes through inhibition of PKA signaling may be a therapeutic option for OA or RA patients.

Regulation of Mitochondrial Morphology by Positive Feedback Interaction Between PKCδ and Drp1 in Vascular Smooth Muscle Cell

Dynamin‐related protein‐1 (Drp1) plays a critical role in mitochondrial fission which allows cell proliferation and Mdivi‐1, a specific small molecule Drp1 inhibitor, is revealed to attenuate proliferation. However, few molecular mechanisms‐related to Drp1 under stimulus for restenosis or atherosclerosis have been investigated in vascular smooth muscle cells (vSMCs). Therefore, we hypothesized that Drp1 inhibition can prevent vascular restenosis and investigated its regulatory mechanism. Angiotensin II (Ang II) or hydrogen peroxide (H2O2)‐induced proliferation and migration in SMCs were attenuated by down‐regulation of Drp1 Ser 616 phosphorylation, which was demonstrated by in vitro assays for migration and proliferation. Excessive amounts of ROS production and changes in mitochondrial membrane potential were prevented by Drp1 inhibition under Ang II and H2O2. Under the Ang II stimulation, activated Drp1 interacted with PKCδ and then activated MEK1/2‐ERK1/2 signaling cascade and MMP2, but not MMP9. Furthermore, in ex vivo aortic ring assay, inhibition of the Drp1 had significant anti‐proliferative and ‐migration effects for vSMCs. A formation of vascular neointima in response to a rat carotid artery balloon injury was prevented by Drp1 inhibition, which shows a beneficial effect of Drp1 regulation in the pathologic vascular condition. Drp1‐mediated SMC proliferation and migration can be prevented by mitochondrial division inhibitor (Mdivi‐1) in in vitro, ex vivo and in vivo, and these results suggest the possibility that Drp1 can be a new therapeutic target for restenosis or atherosclerosis. J. Cell. Biochem. 116: 648–660, 2015.

The promotion of cardiogenic differentiation of hMSCs by targeting epidermal growth factor receptor using microRNA-133a.

Human bone marrow-derived mesenchymal stem cells (hMSCs) are an attractive candidate for cell therapy in heart disease. Low survival and incomplete electromechanical integration between resident cardiomyocytes and transplanted hMSCs remain unsolved. In order for an infarcted heart to tolerate transplantation, differentiation capacity in stem cells must be reinforced. In this study, we found that compound 56, an epidermal growth factor receptor (EGFR) inhibitor, promotes cardiogenic differentiation of hMSCs and the transplantation of hMSCs treated with compound 56 resulted in enhancement of heart functions. Furthermore, hMSCs transfected with microRNA-133a (miR-133a), which targets EGFR, were observed to express cardiac-specific markers. We also discovered that luciferase activity is exclusively decreased by targeting EGFR in hMSCs transfected with miR-133a mimic. These results suggest that EGFR plays a key role in the regulation of cardiogenic differentiation in hMSCs.

MicroRNA‐29b Inhibits Migration and Proliferation of Vascular Smooth Muscle Cells in Neointimal Formation

The proliferation and migration of smooth muscle cells (SMCs) are considered to be key steps in the progression of atherosclerosis and restenosis. Certain stimuli, such as, interleukin‐3 (IL‐3) are known to stimulate proliferation and migration in vascular diseases. Meanwhile, microRNAs (miRs) have been revealed as critical modulators of various diseases in which miR‐29b is known to regulate cell growth by targeting Mcl‐1 and MMP2. However, roles of miR‐29b in vascular smooth muscle cells remain almost unknown. We hypothesized that miR‐29b may control the proliferation and migration processes induced by IL‐3 stimulation by inhibiting its own specific targets in SMCs. MiR‐29b significantly suppressed the proliferation and migration of SMCs through the inhibition of the signaling pathway related to Mcl‐1 and MMP2. We also found that miR‐29b expression levels significantly declined in balloon‐injured rat carotid arteries and that the overexpression of miR‐29b by local oligonucleotide delivery can inhibit neointimal formation. Consistent with the critical role of miR‐29b in vitro, we observed down‐regulated expression levels of Mcl‐1 and MMP2 from the neointimal region. These results indicate that miR‐29b suppressed the proliferation and migration of SMCs, possibly through the inhibition of Mcl‐1 and MMP2, and suggest that miR‐29b may serve as a useful therapeutic tool to treat cardiovascular diseases such as, atherosclerosis and restenosis. J. Cell. Biochem. 116: 598–608, 2015.

MicroRNA-17-mediated down-regulation of apoptotic protease activating factor 1 attenuates apoptosome formation and subsequent apoptosis of cardiomyocytes

Heart diseases such as myocardial infarction (MI) can damage individual cardiomyocytes, leading to the activation of cell death programs. The most scrutinized type of cell death in the heart is apoptosis, and one of the key events during the propagation of apoptotic signaling is the formation of apoptosomes, which relay apoptotic signals by activating caspase-9. As one of the major components of apoptosomes, apoptotic protease activating factor 1 (Apaf-1) facilitates the formation of apoptosomes containing cytochrome c (Cyto-c) and deoxyadenosine triphosphate (dATP). Thus, it may be possible to suppress the activation of the apoptotic program by down-regulating the expression of Apaf-1 using miRNAs. To validate this hypothesis, we selected a number of candidate miRNAs that were expected to target Apaf-1 based on miRNA target prediction databases. Among these candidate miRNAs, we empirically identified miR-17 as a novel Apaf-1-targeting miRNA. The delivery of exogenous miR-17 suppressed Apaf-1 expression and consequently attenuated formation of the apoptosome complex containing caspase-9, as demonstrated by co-immunoprecipitation and immunocytochemistry. Furthermore, miR-17 suppressed the cleavage of procaspase-9 and the subsequent activation of caspase-3, which is downstream of activated caspase-9. Cell viability tests also indicated that miR-17 pretreatment significantly prevented the norepinephrine-induced apoptosis of cardiomyocytes, suggesting that down-regulation of apoptosome formation may be an effective strategy to prevent cellular apoptosis. These results demonstrate the potential of miR-17 as an effective anti-apoptotic agent.

Proteomic Analysis and Identification of Paracrine Factors in Mesenchymal Stem Cell-Conditioned Media under Hypoxia

Background/Aims: We previously showed that a hypoxic environment modulates the antiarrhythmic potential of mesenchymal stem cells. Methods: To investigate the mechanism by which secreted proteins contribute to the pathogenesis of antiarrhythmic potential in mesenchymal stem cells, we used two-dimensional electrophoresis combined with MALDI-TOF-MS to perform a proteomic analysis to compare the paracrine media produced by normoxic and hypoxic cells. Results: The proteomic analysis revealed that 66 protein spots out of a total of 231 matched spots indicated differential expression between the normoxic and hypoxic conditioned media of mesenchymal stem cells. Interestingly, two tropomyosin isoforms were dramatically increased in the hypoxic conditioned medium of mesenchymal stem cells. An increase in tropomyosin was confirmed using Western blot to analyze the conditioned media between normoxic and hypoxic cells. In a network analysis based on gene ontology (GO) Molecular Function by GeneMANIA analysis, most of the identified proteins were found to be involved in the regulation of heart processes. Conclusion: Our results show that hypoxia up-regulates tropomyosin and other secreted proteins which suggests that tropomyosin may be involved in regulating proarrhythmic and antiarrhythmic functions.