The microRNA-dependent cell fate of multipotent stromal cells differentiating to endothelial cells.

In the endothelial recovery process, bone marrow-derived MSCs are a potential source of cells for both research and therapy, and their capacities to self-renew and to differentiate into all the cell types in the human body make them a promising therapeutic agent for remodeling cellular differentiation and a valuable resource for the treatment of many diseases. Based on the results provided in a miRNA database, we selected miRNAs with unique targets in cell fate-related signaling pathways. The tested miRNAs targeting GSK-3ß (miR-26a), platelet-derived growth factor receptor, and CD133 (miR-26a and miR-29b) induced MSC differentiation into functional ECs, whereas miRNAs targeting VEGF receptor (miR-15, miR-144, miR-145, and miR-329) inhibited MSC differentiation into ECs through VEGF stimulation. In addition, the expression levels of these miRNAs were correlated with in vivo physiological endothelial recovery processes. These findings indicate that the miRNA expression profile is distinct for cells in different stages of differentiation from MSCs to ECs and that specific miRNAs can function as regulators of endothelialization.

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

BACKGROUND: Pathologic 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. RESULTS: Among 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. CONCLUSION: The 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.

Alterations in Cardiomyocyte Differentiation-Related Proteins in Rat Mesenchymal Stem Cells Exposed to Hypoxia.

BACKGROUND/AIMS: It is known that mesenchymal stem cells (MSCs) can have variable responses to hypoxic conditions and that hypoxia may specifically stimulate differentiation into osteogenic, chondrogenic, or adipogenic cells. Based on our previous study, we hypothesized that hypoxia may also induce MSC differentiation into cardiomyocytes and/or cells with comparable phenotypes. METHODS: The differences in the proteomes were specifically investigated in bone marrow-derived rat MSCs (BM-rMSCs) under normoxic and hypoxic conditions using 2-DE combined with a MALDI-TOF-MS analysis and western blot analysis. In addition, genetic and/or proteomic interactions were assessed using a String network analysis. RESULTS: Among the 35 markedly changed spots from a total of 393 matched spots, 24 were highly up-regulated and 11 were significantly down-regulated in hypoxic rMSCs based on a proteomic analysis. Although hypoxia failed to induce the direct differentiation of rMSCs into cardiomyocytes, several cardiomyocyte differentiation-related genes and proteins were significantly increased by hypoxic stress. CONCLUSION: We found that BM-rMSCs alter their expression of several cardiomyocyte differentiation-related genes and proteins under hypoxic conditions, and we examined the interactions between these genes and/or proteins, providing new insights for the applicability of MSCs preconditioned by hypoxic stimulation for use in cardiac diseases.

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.

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 (H2 O2 )-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 H2 O2 . 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.

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.

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.

Na(+)-Ca(2+) exchanger targeting miR-132 prevents apoptosis of cardiomyocytes under hypoxic condition by suppressing Ca(2+) overload.

During ischemia-reperfusion (IR) injury of the heart, Ca(2+) overload occurs, leading to cardiomyocyte dysfunction and eventual cell death by apoptosis. Since preventing Ca(2+) overload during IR injury has been reported to protect cardiomyocytes, interrupting Ca(2+) signaling cascades leading to Ca(2+) overload may exert protective effect on cardiomyocytes under hypoxic condition. One of the key regulators of the intracellular Ca(2+) level during IR injury is Na(+)-Ca(2+) exchanger 1 (NCX1), whose down-regulation during IR injury conferred protection of heart. In the present study, we examined whether down-regulation of NCX1 using exogenous microRNA ameliorates apoptosis of cardiomyocytes under hypoxic condition. Here, we identified miR-132 as a novel microRNA targeting the NCX1, whose expression increased during hypoxia. Delivery of miR-132 suppressed the increase of intracellular Ca(2+) in cardiomyocytes under hypoxia, and the expressions of apoptotic molecules, such as Bax, cytochrome C, and caspase 3, and the number of apoptotic cells were also decreased by exogenous miR-132 treatment. These results suggest the potential of miR-132 as an effective therapeutic agent against IR damage to heart by preventing Ca(2+) overload during hypoxic condition and warrant further studies to validate its anti-apoptotic effect in vivo.

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.

Suppression of miR-181a attenuates H2O2-induced death of mesenchymal stem cells by maintaining hexokinase II expression.

BACKGROUND: Low survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated. RESULTS: H2O2 (600 µM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2. CONCLUSIONS: These findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.

Looking for Pyroptosis-Modulating miRNAs as a Therapeutic Target for Improving Myocardium Survival.

Pyroptosis is the most recently identified type of regulated cell death with inflammatory response and has characteristics distinct from those of apoptosis or necrosis. Recently, independent studies have reported that small noncoding RNAs termed microRNAs (miRNAs) are involved in the regulation of pyroptosis. Nevertheless, only a handful of empirical data regarding miRNA-dependent regulation of pyroptosis is currently available. This review is aimed to provide a current update on the role of miRNAs in pyroptosis and to offer suggestions for future studies probing miRNAs as a linker connecting pyroptosis to various cardiovascular diseases (CVDs) and their potential as a therapeutic target for preventing excessive cell death of myocardium during CVDs.

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.

Looking into a conceptual framework of ROS-miRNA-atrial fibrillation.

Atrial fibrillation (AF) has been recognized as a major cause of cardiovascular-related morbidity and mortality. MicroRNAs (miRNAs) represent recent additions to the collection of biomolecules involved in arrhythmogenesis. Reactive oxygen species (ROS) have been independently linked to both AF and miRNA regulation. However, no attempts have been made to investigate the possibility of a framework composed of ROS-miRNA-AF that is related to arrhythmia development. Therefore, this review was designed as an attempt to offer a new approach to understanding AF pathogenesis. The aim of this review was to find and to summarize possible connections that exist among AF, miRNAs and ROS to understand the interactions among the molecular entities underlying arrhythmia development in the hopes of finding unappreciated mechanisms of AF. These findings may lead us to innovative therapies for AF, which can be a life-threatening heart condition. A systemic literature review indicated that miRNAs associated with AF might be regulated by ROS, suggesting the possibility that miRNAs translate cellular stressors, such as ROS, into AF pathogenesis. Further studies with a more appropriate experimental design to either prove or disprove the existence of an ROS-miRNA-AF framework are strongly encouraged.

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.

Phorbol myristate acetate differentiates human adipose-derived mesenchymal stem cells into functional cardiogenic cells.

To achieve effective regeneration of injured myocardium, it is important to find physiological way of improving the cardiogenic differentiation of stem cells. Previous studies demonstrated that cardiomyocytes from bone marrow-derived mesenchymal stem cells (BMSCs) activated with phorbolmyristate acetate (PMA), a protein kinase C (PKC) activator, restore electromechanical function in infarcted rat hearts. In this study, we investigated the effect of PMA on cardiogenic differentiation of adipose-derived MSCs (ASCs) for clinical applications. To confirm the effect of PMA, ASCs treated with 1µM PMA were grown for nine days. The expression of cardiac-specific markers (cardiac troponin T, myosin light chain, myosin heavy chain) in PMA-treated MSCs was demonstrated by immunocytochemistry. Alhough few α(1A) receptors exist in ASCs, α(1)-adrenergic receptor subtypes were preferentially expressed in PMA-treated ASCs. Moreover, expression of the ß-adrenergic and muscarinic receptors increased in PMA-treated ASCs compared to normal cells. The mRNA levels of Ca(2+)-related factors (SERCA 2a; sarcoplasmic reticulum Ca(2+)-ATPase, LTCC; L-type Ca(2+) channel) in treated ASCs were similar to the levels in cardiomyocytes. Following the transplantation of chemically activated cardiogenic ASCs into infarcted myocardium, histological analysis showed that infarct size, interstitial fibrosis, and apoptotic index were markedly decreased and cardiac function was restored. In conclusion, PMA might induce the cardiogenic differentiation of human ASCs as well as BMSCs. This result suggests successful use of human ASCs in cardiac regeneration therapy.

Structure and property based design, synthesis and biological evaluation of γ-lactam based HDAC inhibitors: part II.

Histone deacetylases (HDACs) are involved in post-translational modification and epi-genetic expression, and have been the intriguing targets for treatment of cancer. In previous study, we reported synthesis and the biological preliminary results of γ-lactam based HDAC inhibitors. Based on the previous results, smaller γ-lactam core HDAC inhibitors are more active than the corresponding series of larger δ-lactam based analogues and the hydrophobic and bulky cap groups are required for better potency which decreased microsomal stability. Thus, γ-lactam analogues with methoxy, trifluoromethyl groups of ortho-, meta-, para-positions of cap group were prepared and evaluated their biological potency. Among them, trifluoromethyl analogues, which have larger lipophilicity, showed better HDAC inhibitory activity than other analogues. In overall, lipophilicity leads to increase hydrophobic interaction between surface of HDAC active site and HDAC inhibitor, improves HDAC inhibitory activity.

Structure and property based design, synthesis and biological evaluation of γ-lactam based HDAC inhibitors.

Histone deacetylases (HDACs) are involved in post-translational modification and gene expression. Cancer cells recruited amounts of HDACs for their survival by epi-genetic down regulation of tumor suppressor genes. HDACs have been the promising targets for treatment of cancer, and many HDAC inhibitors have been investigated nowadays. In previous study, we synthesized δ-lactam core HDAC inhibitors which showed potent HDAC inhibitory activities as well as cancer cell growth inhibitory activities. Through QSAR study of the δ-lactam based inhibitors, the smaller core is suggested as more active than larger one because it fits better in narrow hydrophobic tunnel of the active pocket of HDAC enzyme. The smaller γ-lactam core HDAC inhibitors were designed and synthesized for biological and property optimization. Phenyl, naphthyl and thiophenyl groups were introduced as the cap groups. Hydrophobic and bulky cap groups increase potency of HDAC inhibition because of hydrophobic interaction between HDAC and inhibitors. In overall, γ-lactam based HDAC inhibitors showed more potent than δ-lactam analogues.

Property based optimization of δ-lactam HDAC inhibitors for metabolic stability.

The novel δ-lactam based HDAC inhibitor, KBH-A118 (3) shows a good HDAC enzyme and cancer cell growth inhibitory activities but has undesirable pharmacokinetics profiles because of instability in mouse liver microsome. To improve metabolic stability, various analogues were prepared with substituents on aromatic ring of cap group and various chain lengths between the cap group and δ-lactam core. The newly prepared analogues showed moderate to potent in vitro activities. Among them six compounds (8a, 8e, 8j, 8n, 8t, and 8v) were evaluated on mouse liver microsome assay and it turned out that the microsomal stabilities were dependent on lipophilicity and the number of the rotatable bonds. Finally, the animal pharmacokinetic profiles of 8e displayed improving oral exposure and oral bioavailability.

Antithrombotic and Antiplatelet Effects of Cordyceps militaris.

Cordyceps is a genus of ascomycete fungi and is well known as one of the important medical fungi in Chinese, Korea, and other Asian countries, because of its various beneficial effects on human health. The pharmacological activities of Cordyceps extract are mainly focused on anti-cancer, anti-metastatic, and immune modulating effects. In the present study, we investigated whether the antiplatelet effect of ethanol extract of cultured Cordyceps militaris (CMEE) with FeCl3-induced arterial thrombosis model. We observed that CMEE exhibited a significant inhibitory effect against ADP and collagen-induced platelet aggregation. However, there were no significant differences in prothrombin time (PT) and activated partial thromboplastin time (aPTT). These results suggest that antithrombotic activity of CMEE is related to antiplatelet effect rather than anticoagulation effect, and CMEE may be a positive effect on improving blood circulation against vessel injury and occlusion.

Beneficial Effect of Cordyceps militaris on Exercise Performance via Promoting Cellular Energy Production.

Cordyceps militaris has been reported to the diverse pharmaceutical effects including cancer, inflammatory diseases, and bacteria or virus infection. However, the effect of C. militaris on exercise performance has not yet been elucidated. In this study, we investigated the beneficial effect of C. militaris on exercise performance. To evaluate exercise performance, we prepared C. militaris ethyl acetate extract (CMEE) and conducted grip strength tests every week after administration. Additionally, blood samples were collected at the end of the experiment for biochemical analysis. The administration of CMEE slightly increased grip strength, and this result was similar to the red ginseng treated group. According to the result of biochemical analysis, CMEE had an effect on the biomarkers related to ATP generation pathway but had little influence on the muscle fatigue related biomarkers. Therefore, C. militaris has the possibility of improving exercise performance, which could be associated with the increase in ATP production rather than the decrease in muscle fatigue during exercise.