Seahyoung Lee

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.

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.

ROS-mediated bidirectional regulation of miRNA results in distinct pathologic heart conditions.

Under distinct pathological heart conditions, the expression of a single miRNA can display completely opposite patterns. However, the mechanism underlying the bidirectional regulation of a single miRNA and the clinical implications of this regulation remain largely unknown. To address this issue, we examined the regulation of miR-1, one of the most abundant miRNAs in the heart, during cardiac hypertrophy and ischemia/reperfusion (I/R). Our data indicated that different magnitudes and chronicities of ROS levels in cardiomyocytes resulted in differential expression of miR-1, subsequently altering the expression of myocardin. In animal models, the administration of a miR-1 mimic attenuated cardiac hypertrophy by suppressing the transverse aortic constriction-induced increase in myocardin expression, whereas the administration of anti-miR-1 ameliorated I/R-induced cardiac apoptosis and deterioration of heart function. Our findings indicated that a pathologic stimulus such as ROS can bidirectionally alter the expression of miRNA to contribute to the development of pathological conditions exhibiting distinct phenotypes and that the meticulous adjustment of the pathological miRNA levels is required to improve clinical outcomes.

Interaction of small G protein signaling modulator 3 with connexin 43 contributes to myocardial infarction in rat hearts

Connexin 43 (Cx43), a ubiquitous connexin expressed in the heart and skin, is associated with a variety of hereditary conditions. Therefore, the characterization of Cx43-interacting proteins and their dynamics is important to understand not only the molecular mechanisms underlying pathological malfunction of gap junction-mediated intercellular communication but also to identify novel and unanticipated biological functions of Cx43. In the present study, we observed potential targets of Cx43 to determine new molecular functions in cardio-protection. MALDI-TOF mass spectrometry analysis of Cx43 co-immunoprecipitated proteins showed that Cx43 interacts with several proteins related to metabolism. In GeneMANIA network analysis, SGSM3, which has not been previously associated with Cx43, was highly correlated with Cx43 in heart functions, and high levels of SGSM3 appeared to induce the turnover of Cx43 through lysosomal degradation in myocardial infarcted rat hearts. Moreover, we confirmed that lysosomal degradation of Cx43 is dependent upon the interaction between SGSM3 and Cx43 in H9c2 cardiomyocytes. The functional importance of the interaction between SGSM3 and Cx43 was confirmed by results showing that Cx43 expression was enhanced by SGSM3 siRNA knockdown in H9c2 cells. In summary, the results of this study elucidate the molecular mechanisms in which Cx43 with SGSM3 is degraded in myocardial infarcted rat hearts, which may contribute to the establishment of new therapeutic targets to modulate cardiac function in physiological and pathological conditions.

Potential therapeutic application of small molecule with sulfonamide for chondrogenic differentiation and articular cartilage repair

The restoration of damaged articular cartilage is a long-pursued goal in regenerative medicine. Chondrocyte-specific differentiation of mesenchymal stem cells (MSCs) may be an effective means of repairing damaged cartilage. We identified small molecule 6 with sulfonamide as an agent that promotes specific chondrogenic differentiation of human adipose-derived MSCs (hASCs). Unlike other chondrogenic differentiation media composed of various defined components, simply adding compound 6 into culture medium was sufficient to induce chondrogenesis in this study. In an animal osteoarthritis model, both the small molecule 6 and the 6-treated hASCs exhibited enhanced recovery of injured articular cartilage. This work provides new insight into MSC differentiation induced by small molecules and potential new therapeutic approaches for articular cartilage injury.

7-cyclopentyl-5-(4-phenoxyphenyl)−7H-pyrrolo[2,3-d] pyrimidin-4-ylamine inhibits the proliferation and migration of vascular smooth muscle cells by suppressing ERK and Akt pathways

ABSTRACT Excessive vascular smooth muscle cell (VSMC) proliferation and migration after vascular injury significantly contributes to the development of occlusive vascular disease. Therefore, inhibiting the proliferation and migration of VSMCs is a validated therapeutic modality for occlusive vascular disease such as atherosclerosis and restenosis. In the present study, we screened chemical compounds for their anti‐proliferative effects on VSMCs using multiple approaches, such as MTT assays, wound healing assays, and trans‐well migration assays. Our data indicate that 7‐cyclopentyl‐5‐(4‐phenoxyphenyl)−7H‐pyrrolo[2,3‐d] pyrimidin‐4‐ylamine, a lymphocyte‐specific protein tyrosine kinase (Lck) inhibitor, significantly inhibited both VSMC proliferation and migration. 7‐cyclopentyl‐5‐(4‐phenoxyphenyl)−7H‐pyrrolo[2,3‐d]pyrimidin‐4‐ylamine suppresses VSMC proliferation and migration via down‐regulating the protein kinase B (Akt) and extracellular signal regulated kinase (ERK) pathways, and it significantly decreased the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 and, the phosphorylation of retinoblastoma protein (pRb). Additionally, 7‐cyclopentyl‐5‐(4‐phenoxyphenyl)−7H‐pyrrolo[2,3‐d] pyrimidin‐4‐ylamine suppressed the migration of VSMCs from endothelium‐removed aortic rings, as well as neointima formation following rat carotid balloon injury. The present study identified 7‐cyclopentyl‐5‐(4‐phenoxyphenyl)−7H‐pyrrolo[2,3‐d]pyrimidin‐4‐ylamine as a potent VSMC proliferation and migration inhibitor and warrants further studies to elucidate its more detailed molecular mechanisms, such as its primary target, and to further validate its in vivo efficacy as a therapeutic agent for pathologic vascular conditions, such as restenosis and atherosclerosis.

A spleen tyrosine kinase inhibitor attenuates the proliferation and migration of vascular smooth muscle cells

BackgroundPathologic vascular smooth muscle cell (VSMC) proliferation and migration after vascular injury promotes the development of occlusive vascular disease. Therefore, an effective chemical agent to suppress aberrant proliferation and migration of VSMCs can be a potential therapeutic modality for occlusive vascular disease such as atherosclerosis and restenosis. To find an anti-proliferative chemical agent for VSMCs, we screened an in-house small molecule library, and the selected small molecule was further validated for its anti-proliferative effect on VSMCs using multiple approaches, such as cell proliferation assays, wound healing assays, transwell migration assays, and ex vivo aortic ring assay.ResultsAmong 43 initially screened small molecule inhibitors of kinases that have no known anti-proliferative effect on VSMCs, a spleen tyrosine kinase (Syk) inhibitor (BAY61-3606) showed significant anti-proliferative effect on VSMCs. Further experiments indicated that BAY61 attenuated the VSMC proliferation in both concentration- and time-dependent manner, and it also significantly suppressed the migration of VSMCs as assessed by both wound healing assays and transwell assays. Additionally, BAY61 suppressed the sprouting of VSMCs from endothelium-removed aortic rings.ConclusionThe present study identified a Syk kinase inhibitor as a potent VSMC proliferation and migration inhibitor and warrants further studies to elucidate its underlying molecular mechanisms, such as its primary target, and to validate its in vivo efficacy as a therapeutic agent for restenosis and atherosclerosis.

1H-pyrrole-2,5-dione-based small molecule-induced generation of mesenchymal stem cell-derived functional endothelial cells that facilitate rapid endothelialization after vascular injury

IntroductionDespite the success of interventional processes such as drug-eluting stents, complete prevention of restenosis is still hindered by impaired or delayed endothelialization or both. Here, we report that 1H-pyrrole-2,5-dione-based small molecule-generated mesenchymal stem cell-derived functional endothelial cells (MDFECs) facilitated rapid transmural coverage of injured blood vessels.MethodsSmall molecules that induced CD31 expression were screened by principal component analysis (PCA). Rat mesenchymal stem cells (MSCs) were treated with selected small molecules for up to 16 days, and the expression levels of CD90 and CD31 were examined by immunocytochemistry. In vitro functional assays of MDFECs, including tube formation assays and nitric oxide production assays, were performed. After MDFECs (intravenous, 3×106 cells per animal) were injected into balloon-injured rats, neointima formation was monitored for up to 21 days. The endothelial coverage of denuded blood vessels was evaluated by Evans Blue staining. The functionality of repaired blood vessels was evaluated by measuring vasorelaxation and hemodynamic changes. Additionally, derivatives of the selected small molecules were examined for their ability to induce endothelial markers.ResultsPCA indicated that 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione effectively induced MDFECs. MDFECs inhibited the neointima formation of denuded blood vessels by facilitating more rapid endothelialization. Further examination indicated that derivatives with a 1H-pyrrole-2,5-dione moiety are important for initiating the endothelial cell differentiation of MSCs.ConclusionsSmall molecules with 1H-pyrrole-2,5-dione as a core structure have great potential to improve the efficacy of MSC-based cell therapy for vascular diseases, such as atherosclerosis and restenosis.

microRNA-133a attenuates cardiomyocyte hypertrophy by targeting PKCδ and Gq

During the past decade, microRNAs have continuously been suggested as a promising therapeutic tool due to their beneficial effects, such as their multi-targets and multi-functions in pathologic conditions. As a pathologic phenotype is generally regulated by multiple signaling pathways, in this study we identified a microRNA regulating multiple target genes within cardiac hypertrophic signaling pathways. microRNA-133a is known to play a crucial role in cardiac hypertrophy. However, the role of microRNA-133a, which may regulate several signaling pathways in norepinephrine-induced cardiac hypertrophy via multi-targeting, has not been investigated. In the current study, we showed that microRNA-133a can protect cardiomyocyte hypertrophy against norepinephrine stimulation in neonatal rat ventricular cardiomyocytes via new targets, PKCδ and Gq, all of which are related to downstream signaling pathways of the α1-adrenergic receptor. Taken together, these results suggest the advantages of the therapeutic use of microRNAs as an effective potential drug regulating multiple signaling pathways under pathologic conditions.

Effects of donor age on human adipose-derived adherent stromal cells under oxidative stress conditions

Objective Adipose-derived stromal vascular fractions (SVFs) are heterogeneous complex populations of cells with therapeutic efficacy for tissue generation and vascular stabilization. SVFs have cardiomyogenic potential, and many researchers have examined the possibility of SVF transplantation for heart disease. In cell-based therapies, donor age affects the regenerative capability, cell yield, and differentiation potential of adult tissues; however, opposing or controversial results have been found in humans. We examined whether SVF transplantation into impaired heart tissue shows differential effects according to donor age. Methods We investigated differences in protein expression in human umbilical vein endothelial cells (HUVECs) co-cultured with adipose-derived adherent stromal cells (ADASs) from donors of different ages [>40-year-olds (40s group) and >60-year-olds (60s group)] under oxidative stress conditions. Results Although co-culturing HUVECs with ADASs ameliorated inflammation due to increased oxidative stress conditions, few differences were observed between the ADASs from the 40s and 60s groups. Moreover, the Database for Annotation, Visualization, and Integrated Discovery classification tool revealed differentially expressed genes in the Kyoto Encyclopedia of Genes and Genomes pathway associated with cytokine–cytokine receptor interaction in response to ADASs. Conclusion Protein expression profiles were unchanged in HUVECs induced by isolated ADASs from donors of different ages under oxidative stress conditions.