Melatonin alleviates myocardial dysfunction through inhibition of endothelial-to-mesenchymal transition via the NF-κB pathway.

Endothelial-to-mesenchymal transition (EndMT) is a complex biological process of cellular transdifferentiation by which endothelial cells (ECs) lose their characteristics and acquire mesenchymal properties, leading to cardiovascular remodeling and complications in the adult cardiovascular diseases environment. Melatonin is involved in numerous physiological and pathological processes, including aging, and has anti-inflammatory and antioxidant activities. This molecule is an effective therapeutic candidate for preventing oxidative stress, regulating endothelial function, and maintaining the EndMT balance to provide cardiovascular protection. Although recent studies have documented improved cardiac function by melatonin, the mechanism of action of melatonin on EndMT remains unclear. The present study investigated the effects of melatonin on induced EndMT by transforming growth factor-ß2/interleukin-1ß in both in vivo and in vitro models. The results revealed that melatonin reduced the migratory ability and reactive oxygen species levels of the cells and ameliorated mitochondrial dysfunction in vitro. Our findings indicate that melatonin prevents endothelial dysfunction and inhibits EndMT by activating related pathways, including nuclear factor kappa B and Smad. We also demonstrated that this molecule plays a crucial role in restoring cardiac function by regulating the EndMT process in the ischemic myocardial condition, both in vessel organoids and myocardial infarction (MI) animal models. In conclusion, melatonin is a promising agent that attenuates EC dysfunction and ameliorates cardiac damage compromising the EndMT process after MI.

Epigallocatechin-3-Gallate Attenuates Myocardial Dysfunction via Inhibition of Endothelial-to-Mesenchymal Transition.

Cardiac tissue damage following ischemia leads to cardiomyocyte apoptosis and myocardial fibrosis. Epigallocatechin-3-gallate (EGCG), an active polyphenol flavonoid or catechin, exerts bioactivity in tissues with various diseases and protects ischemic myocardium; however, its association with the endothelial-to-mesenchymal transition (EndMT) is unknown. Human umbilical vein endothelial cells (HUVECs) pretreated with transforming growth factor ß2 (TGF-ß2) and interleukin 1ß (IL-1ß) were treated with EGCG to verify cellular function. In addition, EGCG is involved in RhoA GTPase transmission, resulting in reduced cell mobility, oxidative stress, and inflammation-related factors. A mouse myocardial infarction (MI) model was used to confirm the association between EGCG and EndMT in vivo. In the EGCG-treated group, ischemic tissue was regenerated by regulating proteins involved in the EndMT process, and cardioprotection was induced by positively regulating apoptosis and fibrosis of cardiomyocytes. Furthermore, EGCG can reactivate myocardial function due to EndMT inhibition. In summary, our findings confirm that EGCG is an impact activator controlling the cardiac EndMT process derived from ischemic conditions and suggest that supplementation with EGCG may be beneficial in the prevention of cardiovascular disease.

Regulation of Inflammation-Mediated Endothelial to Mesenchymal Transition with Echinochrome a for Improving Myocardial Dysfunction.

Endothelial-mesenchymal transition (EndMT) is a process by which endothelial cells (ECs) transition into mesenchymal cells (e.g., myofibroblasts and smooth muscle cells) and induce fibrosis of cells/tissues, due to ischemic conditions in the heart. Previously, we reported that echinochrome A (EchA) derived from sea urchin shells can modulate cardiovascular disease by promoting anti-inflammatory and antioxidant activity; however, the mechanism underlying these effects was unclear. We investigated the role of EchA in the EndMT process by treating human umbilical vein ECs (HUVECs) with TGF-ß2 and IL-1ß, and confirmed the regulation of cell migration, inflammatory, oxidative responses and mitochondrial dysfunction. Moreover, we developed an EndMT-induced myocardial infarction (MI) model to investigate the effect of EchA in vivo. After EchA was administered once a day for a total of 3 days, the histological and functional improvement of the myocardium was investigated to confirm the control of the EndMT. We concluded that EchA negatively regulates early or inflammation-related EndMT and reduces the myofibroblast proportion and fibrosis area, meaning that it may be a potential therapy for cardiac regeneration or cardioprotection from scar formation and cardiac fibrosis due to tissue granulation. Our findings encourage the study of marine bioactive compounds for the discovery of new therapeutics for recovering ischemic cardiac injuries.

Human embryonic stem cell-specific role of YAP in maintenance of self-renewal and survival.

Human embryonic stem cells (hESCs) have unique characteristics, such as self-renewal and pluripotency, which are distinct from those of other cell types. These characteristics of hESCs are tightly regulated by complex signaling mechanisms. In this study, we demonstrate that yes-associated protein (YAP) functions in an hESC-specific manner to maintain self-renewal and survival in hESCs. hESCs were highly sensitive to YAP downregulation to promote cell survival. Interestingly, hESCs displayed dynamic changes in YAP expression in response to YAP downregulation. YAP was critical for the maintenance of self-renewal. Additionally, the function of YAP in maintenance of self-renewal and cell survival was hESC-specific. Doxycycline upregulated YAP in hESCs and attenuated the decreased cell survival induced by YAP downregulation. However, decreased expression of self-renewal markers triggered by YAP downregulation and neural/cardiac differentiation were affected by doxycycline treatment. Collectively, the results reveal the mechanism underlying the role of YAP and the novel function of doxycycline in hESCs.

MicroRNA-dependent inhibition of WEE1 controls cancer stem-like characteristics and malignant behavior in ovarian cancer.

Cancer stem-like cells (CSCs) have been suggested to be responsible for chemoresistance and tumor recurrence owing to their self-renewal capacity and differentiation potential. Although WEE1 is a strong candidate target for anticancer therapies, its role in ovarian CSCs is yet to be elucidated. Here, we show that WEE1 plays a key role in regulating CSC properties and tumor resistance to carboplatin via a microRNA-dependent mechanism. We found that WEE1 expression is upregulated in ovarian cancer spheroids because of the decreased expression of miR-424 and miR-503, which directly target WEE1. The overexpression of miR-424/503 suppressed CSC activity by inhibiting WEE1 expression, but this effect was reversed on the restoration of WEE1 expression. Furthermore, we demonstrated that NANOG modulates the miR-424/503-WEE1 axis that regulates the properties of CSCs. We also demonstrated the pharmacological restoration of the NANOG-miR-424/503-WEE1 axis and attenuation of ovarian CSC characteristics in response to atorvastatin treatment. Lastly, miR-424/503-mediated WEE1 inhibition re-sensitized chemoresistant ovarian cancer cells to carboplatin. Additionally, combined treatment with atorvastatin and carboplatin synergistically reduced tumor growth, chemoresistance, and peritoneal seeding in the intraperitoneal mouse models of ovarian cancer. We identified a novel NANOG-miR-424/503-WEE1 pathway for regulating ovarian CSCs, which has potential therapeutic utility in ovarian cancer treatment.

A bioisosteric approach to the discovery of novel N-aryl-N'-[4-(aryloxy)cyclohexyl]squaramide-based activators of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation.

Inhibition of translation initiation has emerging implications for the development of mechanism-based anticancer therapeutics. Phosphorylation of eIF2α is recognized as a key target that regulates the translation initiation cascade. Based on the bioisosteric replacement of urea-derived eIF2α phosphorylation activator 1, a novel series of N-aryl-N'-[4-(aryloxy)cyclohexyl]squaramide derivatives was designed and synthesized; their effects on the activation of eIF2α phosphorylation was assessed systematically. A brief structure-activity relationship analysis was established by stepwise structural optimization of the squaramide series. Subsequently, the antiproliferative activities of the selected analogues were determined in human leukemia K562 cells. We then identified 10 potent eIF2α phosphorylation activators with considerable anticancer activity. The most promising analogues 19 and 40 possessed higher cancer cell selectivity (SI = 6.16 and 4.83, respectively) than parent 1 (SI = 2.20). Finally, protein expression analysis revealed that compounds 19 and 40 induced eIF2α phosphorylation and its downstream effectors ATF4 and CHOP.

Hypermethylation of PDX1, EN2, and MSX1 predicts the prognosis of colorectal cancer.

Despite numerous observations regarding the relationship between DNA methylation changes and cancer progression, only a few genes have been verified as diagnostic biomarkers of colorectal cancer (CRC). To more practically detect methylation changes, we performed targeted bisulfite sequencing. Through co-analysis of RNA-seq, we identified cohort-specific DNA methylation markers: CpG islands of the intragenic regions of PDX1, EN2, and MSX1. We validated that these genes have oncogenic features in CRC and that their expression levels are increased in correlation with the hypermethylation of intragenic regions. The reliable depth of the targeted bisulfite sequencing data enabled us to design highly optimized quantitative methylation-specific PCR primer sets that can successfully detect subtle changes in the methylation levels of candidate regions. Furthermore, these methylation levels can divide CRC patients into two groups denoting good and poor prognoses. In this study, we present a streamlined workflow for screening clinically significant differentially methylated regions. Our discovery of methylation markers in the PDX1, EN2, and MSX1 genes suggests their promising performance as prognostic markers and their clinical application in CRC patients.

Multiplexed targeting of microRNA in stem cell-derived extracellular vesicles for regenerative medicine.

Regenerative medicine is a research field that develops methods to restore damaged cell or tissue function by regeneration, repair or replacement. Stem cells are the raw material of the body that is ultimately used from the point of view of regenerative medicine, and stem cell therapy uses cells themselves or their derivatives to promote responses to diseases and dysfunctions, the ultimate goal of regenerative medicine. Stem cell-derived extracellular vesicles (EVs) are recognized as an attractive source because they can enrich exogenous microRNAs (miRNAs) by targeting pathological recipient cells for disease therapy and can overcome the obstacles faced by current cell therapy agents. However, there are some limitations that need to be addressed before using miRNA-enriched EVs derived from stem cells for multiplexed therapeutic targeting in many diseases. Here, we review various roles on miRNA-based stem cell EVs that can induce effective and stable functional improvement of stem cell-derived EVs. In addition, we introduce and review the implications of several miRNA-enriched EV therapies improved by multiplexed targeting in diseases involving the circulatory system and nervous system. This systemic review may offer potential roles for stem cell-derived therapeutics with multiplexed targeting. [BMB Reports 2022;55(2): 65-71].

Multiplexed targeting of miRNA-210 in stem cell-derived extracellular vesicles promotes selective regeneration in ischemic hearts.

Extracellular vesicles (EVs) are cell derivatives containing diverse cellular molecules, have various physiological properties and are also present in stem cells used for regenerative therapy. We selected a "multiplexed target" that demonstrates multiple effects on various cardiovascular cells, while functioning as a cargo of EVs. We screened various microRNAs (miRs) and identified miR-210 as a candidate target for survival and angiogenic function. We confirmed the cellular and biological functions of EV-210 (EVs derived from ASCmiR-210) secreted from adipose-derived stem cells (ASCs) transfected with miR-210 (ASCmiR-210). Under hypoxic conditions, we observed that ASCmiR-210 inhibits apoptosis by modulating protein tyrosine phosphatase 1B (PTP1B) and death-associated protein kinase 1 (DAPK1). In hypoxic endothelial cells, EV-210 exerted its angiogenic capacity by inhibiting Ephrin A (EFNA3). Furthermore, EV-210 enhanced cell survival under the control of PTP1B and induced antiapoptotic effects in hypoxic H9c2 cells. In cardiac fibroblasts, the fibrotic ratio was reduced after exposure to EV-210, but EVs derived from ASCmiR-210 did not communicate with fibroblasts. Finally, we observed the functional restoration of the ischemia/reperfusion-injured heart by maintaining the intercommunication of EVs and cardiovascular cells derived from ASCmiR-210. These results suggest that the multiplexed target with ASCmiR-210 is a useful tool for cardiovascular regeneration.

MicroRNA 34a-AXL Axis Regulates Vasculogenic Mimicry Formation in Breast Cancer Cells.

Targeting the tumor vasculature is an attractive strategy for cancer treatment. However, the tumor vasculature is heterogeneous, and the mechanisms involved in the neovascularization of tumors are highly complex. Vasculogenic mimicry (VM) refers to the formation of vessel-like structures by tumor cells, which can contribute to tumor neovascularization, and is closely related to metastasis and a poor prognosis. Here, we report a novel function of AXL receptor tyrosine kinase (AXL) in the regulation of VM formation in breast cancer cells. MDA-MB-231 cells exhibited VM formation on Matrigel cultures, whereas MCF-7 cells did not. Moreover, AXL expression was positively correlated with VM formation. Pharmacological inhibition or AXL knockdown strongly suppressed VM formation in MDA-MB-231 cells, whereas the overexpression of AXL in MCF-7 cells promoted VM formation. In addition, AXL knockdown regulated epithelial-mesenchymal transition (EMT) features, increasing cell invasion and migration in MDA-MB-231 cells. Finally, the overexpression of microRNA-34a (miR-34a), which is a well-described EMT-inhibiting miRNA and targets AXL, inhibited VM formation, migration, and invasion in MDA-MB 231 cells. These results identify a miR-34a-AXL axis that is critical for the regulation of VM formation and may serve as a therapeutic target to inhibit tumor neovascularization.

A simple metastatic brain cancer model using human embryonic stem cell-derived cerebral organoids.

Every year, hundreds of thousands of people die because of metastatic brain cancer. Most metastatic cancer research uses 2D cell culture or animal models, but they have a few limitations, such as difficulty reproducing human tissue structures. This study developed a simple 3D in vitro model to better replicate brain metastasis using human cancer cells and human embryonic stem cell-derived cerebral organoids (metastatic brain cancer cerebral organoid [MBCCO]). The MBCCO model successfully reproduced metastatic cancer processes, including cell adhesion, proliferation, and migration, in addition to cell-cell interactions. Using the MBCCO model, we demonstrated that lung-specific X protein (LUNX) plays an important role in cell proliferation and migration or invasion. We also observed astrocyte accumulation around and their interaction with cancer cells through connexin 43 in the MBCCO model. We analyzed whether the MBCCO model can be used to screen drugs by measuring the effects of gefitinib, a well-known anticancer agent. We also examined the toxicity of gefitinib using normal cerebral organoids (COs). Therefore, the MBCCO model is a powerful tool for modeling human metastatic brain cancer in vitro and can also be used to screen drugs.

Regulation of alternative macrophage activation by MSCs derived hypoxic conditioned medium, via the TGF-ß1/Smad3 pathway.

Macrophages are re-educated and polarized in response to myocardial infarction (MI). The M2 anti-inflammatory phenotype is a known dominator of late stage MI. Mesenchymal stem cells (MSCs) represent a promising tool for cell therapy, particularly heart related diseases. In general, MSCs induce alteration of the macrophage subtype from M1 to M2, both in vitro and in vivo. We conjectured that hypoxic conditions can promote secretome productivity of MSCs. Hypoxia induces TGF-ß1 expression, and TGF-ß1 mediates M2 macrophage polarization for anti-inflammation and angiogenesis in infarcted areas. We hypothesized that macrophages undergo advanced M2 polarization after exposure to MSCs in hypoxia. Treatment of MSCs derived hypoxic conditioned medium (hypo-CM) promoted M2 phenotype and neovascularization through the TGF-ß1/Smad3 pathway. In addition, hypo-CM derived from MSCs improved restoration of ischemic heart, such as attenuating cell apoptosis and fibrosis, and ameliorating microvessel density. Based on our results, we propose a new therapeutic method for effective MI treatment using regulation of macrophage polarization. [BMB Reports 2020; 53(11): 600-604].

Angiogenesis and vasculogenic mimicry as therapeutic targets in ovarian cancer.

Tumor angiogenesis is an essential process for growth and metastasis of cancer cells as it supplies tumors with oxygen and nutrients. During tumor angiogenesis, many pro-angiogenic factors are secreted by tumor cells to induce their own vascularization via activation of pre-existing host endothelium. However, accumulating evidence suggests that vasculogenic mimicry (VM) is a key alternative mechanism for tumor vascularization when tumors are faced with insufficient supply of oxygen and nutrients. VM is a tumor vascularization mechanism in which tumors create a blood supply system, in contrast to tumor angiogenesis mechanisms that depend on pre-existing host endothelium. VM is closely associated with tumor progression and poor prognosis in many cancers. Therefore, inhibition of VM may be a promising therapeutic strategy and may overcome the limitations of anti-angiogenesis therapy for cancer patients. In this review, we provide an overview of the current anti-angiogenic therapies for ovarian cancer and the current state of knowledge regarding the links between microRNAs and the VM process, with a focus on the mechanism that regulates associated signaling pathways in ovarian cancer. Moreover, we discuss the potential for VM as a therapeutic strategy against ovarian cancer. [BMB Reports 2020; 53(6): 291-298].

Ginsenoside Rg3 protects against iE-DAP-induced endothelial-to-mesenchymal transition by regulating the miR-139-5p-NF-κB axis.

BACKGROUND: Emerging evidence suggests that endothelial-to-mesenchymal transition (EndMT) in endothelial dysfunction due to persistent inflammation is a key component and emerging concept in the pathogenesis of vascular diseases. Ginsenoside Rg3 (Rg3), an active compound from red ginseng, has been known to be important for vascular homeostasis. However, the effect of Rg3 on inflammation-induced EndMT has never been reported. Here, we hypothesize that Rg3 might reverse the inflammation-induced EndMT and serve as a novel therapeutic strategy for vascular diseases. METHODS: EndMT was examined under an inflammatory condition mediated by the NOD1 agonist, γ-d-glutamyl-meso-diaminopimelic acid (iE-DAP), treatment in human umbilical vein endothelial cells. The expression of EndMT markers was determined by Western blot analysis, real-time polymerase chain reaction, and immunocytochemistry. The underlying mechanisms of Rg3-mediated EndMT regulation were investigated by modulating the microRNA expression. RESULTS: The NOD1 agonist, iE-DAP, led to a fibroblast-like morphology change with a decrease in the expression of endothelial markers and an increase in the expression of the mesenchymal marker, namely EndMT. On the other hand, Rg3 markedly attenuated the iE-DAP-induced EndMT and preserved the endothelial phenotype. Mechanically, miR-139 was downregulated in cells with iE-DAP-induced EndMT and partly reversed in response to Rg3 via the regulation of NF-κB signaling, suggesting that the Rg3-miR-139-5p-NF-κB axis is a key mediator in iE-DAP-induced EndMT. CONCLUSION: These results suggest, for the first time, that Rg3 can be used to inhibit inflammation-induced EndMT and may be a novel therapeutic option against EndMT-associated vascular diseases.

Echinochrome A Attenuates Cerebral Ischemic Injury through Regulation of Cell Survival after Middle Cerebral Artery Occlusion in Rat.

Of late, researchers have taken interest in alternative medicines for the treatment of brain ischemic stroke, where full recovery is rarely seen despite advanced medical technologies. Due to its antioxidant activity, Echinochrome A (Ech A), a natural compound found in sea urchins, has acquired attention as an alternative clinical trial source for the treatment of ischemic stroke. The current study demonstrates considerable potential of Ech A as a medication for cerebral ischemic injury. To confirm the effects of Ech A on the recovery of the injured region and behavioral decline, Ech A was administered through the external carotid artery in a rat middle cerebral artery occlusion model after reperfusion. The expression level of cell viability-related factors was also examined to confirm the mechanism of brain physiological restoration. Based on the results obtained, we propose that Ech A ameliorates the physiological deterioration by its antioxidant effect which plays a protective role against cell death, subsequent to post cerebral ischemic stroke.

Trolox-induced cardiac differentiation is mediated by the inhibition of Wnt/ß-catenin signaling in human embryonic stem cells.

Cardiac differentiation of human pluripotent stem cells may be induced under chemically defined conditions, wherein the regulation of Wnt/ß-catenin pathway is often desirable. Here, we examined the effect of trolox, a vitamin E analog, on the cardiac differentiation of human embryonic stem cells (hESCs). 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) significantly enhanced cardiac differentiation in a time- and dose-dependent manner after the mesodermal differentiation of hESCs. Trolox promoted hESC cardiac differentiation through its inhibitory activity against the Wnt/ß-catenin pathway. This study demonstrates an efficient cardiac differentiation method and reveals a novel Wnt/ß-catenin regulator.

Multiple Combination of Angelica gigas Extract and Mesenchymal Stem Cells Enhances Therapeutic Effect.

Alternative medicines attract attention because stroke is rarely expected to make a full recovery with the most advanced medical technology. Angelica gigas (AG) is a well-known herbal medicine as a neuroprotective agent. The present study introduced mesenchymal stem cells (MSCs) to identify for the advanced treatment of the cerebrovascular disease. The objective of this research is validation of the enhanced effects of multiple combined treatment of AG extract with MSCs on stroke through angiogenesis. Our results confirmed that AG extract with MSCs improved the neovascularization increasing expression of angiogenesis-regulated molecules. The changes of brain and the behavioral ability showed the increased effects of AG extract with MSCs. As a result, AG extract and MSCs may synergistically increase the therapeutic potential by enhancing neovascularization. This mixed approach provides a new experimental protocol of herbal medicine therapy for the treatment of a variety of diseases including stroke, trauma, and spinal cord injury.

Angelica gigas Nakai and Decursin Downregulate Myc Expression to Promote Cell Death in B-cell Lymphoma.

Angelica gigas Nakai (AGN) is an oriental traditional medicine to treat anemia, dysmenorrhea, and migraine. However, its anti-lymphoma effect is yet to be tested. Here, we demonstrated that AGN and its major component decursin target Myc to suppress lymphomagenesis in vitro and in vivo. AGN inhibited cell viability in multiple B lymphoma cells, while sparing normal splenocytes and bone marrow cells. Increased cleaved PARP level and caspase 3/7 activity and the repression of survival-promoting AKT/mTOR and MAPK pathways downstream of BCR, were responsible for the pro-apoptotic effects of AGN. We found that Myc, a prominent downstream target of these signaling pathways, contributes to AGN-induced cell death. Moreover, co-treatment with AGN and a Myc inhibitor, JQ1 or 10058-F4 yielded synergistic cytotoxic activities against cancer cells with markedly reduced Myc expression. AGN downregulated Myc expression and suppressed tumorigenesis in Eµ-myc transgenic mice. The proapoptotic activities of AGN were recapitulated by decursin, indicating that the anti-tumor effect of AGN was mainly caused by decursin. These findings suggest that AGN and decursin possess potent anti-lymphoma activity, and combination therapies with AGN/decursin and a Myc inhibitor to target Myc more efficiently could be a valuable avenue to explore in the treatment of B-cell lymphoma.

Exosomes derived from microRNA-584 transfected mesenchymal stem cells: novel alternative therapeutic vehicles for cancer therapy.

Exosomes are small membranous vesicles which contain abundant RNA molecules, and are transferred from releasing cells to uptaking cells. MicroRNA (miRNA) is one of the transferred molecules affecting the adopted cells, including glioma cells. We hypothesized that mesenchymal stem cells (MSCs) can secrete exosomes loading miRNA and have important effects on the progress of gliomas. To determine these effects by treating exosomal miRNA in culture media of miRNA mimic transfected MSCs, we assessed the in vitro cell proliferation and invasion capabilities, and the expression level of relative proteins associated with cell apoptosis, growth and migration. For animal studies, the mice injected with U87 cells were exposed to exosomes derived from miRNA-584-5p transfected MSCs, to confirm the influence of exosomal miRNA on the progress of glioma. Based on our results, we propose a new targeted cancer therapy wherein exosomes derived from miRNA transfected MSCs could be used to modulate tumor progress as the anticancer vehicles. [BMB Reports 2018; 51(8): 406-411].

Salvia miltiorrhiza enhances the survival of mesenchymal stem cells under ischemic conditions.

OBJECTIVES: To validate the enhanced therapeutic effect of Salvia miltiorrhiza Bunge (SM) for brain ischemic stroke through the anti-apoptotic and survival ability of mesenchymal stem cells (MSCs). METHODS: The viability and the expression level of cell apoptotic and survival-related proteins in MSCs by treatment of SM were assessed in vitro. In addition, the infarcted brain region and the behavioural changes after treatment of MSCs with SM were confirmed in rat middle cerebral artery occlusion (MCAo) models. KEY FINDINGS: We demonstrated that SM attenuates apoptosis and improves the cell viability of MSCs. In the rat MCAo model, the recovery of the infarcted region and positive changes of behaviour are observed after treatment of MSCs with SM. CONCLUSIONS: The therapy using SM enhances the therapeutic effect for brain ischemic stroke by promoting the survival of MSCs. This synergetic effect thereby proposes a new experimental approach of traditional Chinese medicine and stem cell-based therapies for patients suffering from a variety of diseases.