EPC-Derived Exosomal miR-1246 and miR-1290 Regulate Phenotypic Changes of Fibroblasts to Endothelial Cells to Exert Protective Effects on Myocardial Infarction by Targeting ELF5 and SP1

Myocardial infarction (MI) remains a leading cause of morbidity and mortality worldwide. Endothelial progenitor cell (EPC)-derived exosomes have been found to be effective in alleviating MI, while the detailed mechanisms remain unclear. The present study aimed to determine the protective effects of EPC-derived exosomal miR-1246 and miR-1290 on MI-induced injury and to explore the underlying molecular mechanisms. The exosomes were extracted from EPCs; gene expression levels were determined by quantitative real-time PCR, and protein expression levels were determined by western blot and immunofluorescence staining, respectively. The angiogenesis and proliferation of human cardiac fibroblasts (HCFs) were determined by tube formation assay and immunofluorescence staining of PKH67, respectively. Luciferase reporter, CHIP, and EMSA assays determined the interaction between miR-1246/1290 and the targeted genes (EFL5 and SP1). The protective effects of miR-1246/1290 on MI were evaluated in a rat model of MI. EPC-derived exosomes significantly upregulated miR-1246 and miR-1290 expression and promoted phenotypic changes of fibroblasts to endothelial cells, angiogenesis, and proliferation in HCFs. Exosomes from EPCs with miR-1246 or miR-1290 mimics transfection promoted phenotypic changes of fibroblasts to endothelial cells and angiogenesis in HCFs, while exosomes from EPCs with miR-1246 or miR-1290 knockdown showed opposite effects in HCFs. Mechanistically, miR-1246 and miR-1290 from EPC-derived exosomes induced upregulation of ELF5 and SP1, respectively, by targeting the promoter regions of corresponding genes. Overexpression of both ELF5 and SP1 enhanced phenotypic changes of fibroblasts to endothelial cells and angiogenesis in HCFs pretreated with exosomes from EPCs with miR-1246 or miR-1290 mimics transfection, while knockdown of both EFL5 and SP1 exerted the opposite effects in HCFs. Both ELF5 and SP1 can bind to the promoter of CD31, leading to the upregulation of CD31 in HCFs. Furthermore, in vivo animal studies showed that exosomes from EPCs with miR-1246 or miR-1290 overexpression attenuated the MI-induced cardiac injury in the rats and caused an increase in ELF5, SP1, and CD31 expression, respectively, but suppressed α-SMA expression in the cardiac tissues. In conclusion, our study revealed that miR-1246 and miR-1290 in EPC-derived exosomes enhanced in vitro and in vivo angiogenesis in MI, and these improvements may be associated with amelioration of cardiac injury and cardiac fibrosis after MI.

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Exosomal miR-1290 promotes angiogenesis in hepatocellular carcinoma via targeting SMEK1

10.21203/rs.2.13182/v2 ◽

2020 ◽

Author(s):

Qiong Wang ◽

Guanwen Wang ◽

Lianjie Niu ◽

Shaorong Zhao ◽

Jianjun Li ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Endothelial Cells ◽

Tumor Angiogenesis ◽

Molecular Mechanisms ◽

Target Genes ◽

Primary Liver Cancer ◽

Tumor Model ◽

Tube Formation ◽

Luciferase Reporter

Abstract Background: Hepatocellular carcinoma (HCC), the most common primary liver cancer, rely on the formation of new blood vessel for growth and frequent intrahepatic and extrahepatic metastasis. Therefore, it is important to explore the underlying molecular mechanisms of tumor angiogenesis of HCC. Recently, microRNAs have been shown to modulate angiogenic processes by modulating the expression of critical angiogenic factors. However, the potential roles of tumor-derived exosomal microRNAs in regulating tumor angiogenesis remain to be elucidated. Methods: MiRNome sequencing was performed to uncover the miRNAs that are dysregulated in HCC patient serum-derived exosomes. Expression levels of miR-1290 in tissues and cells were determined by quantitative real-time PCR. The effect of mir-1290 on proliferation was evaluated by CCK-8 assay. The angiogenic ability of cells were determined by transwell, wound-healing, tube formation and matrigel plug assays. SMMC-7721 xenograft tumor model was established in NOD-SCID nude mice using miR-1290 and NC antagomirs to determin the angiogenic effect of mir-1290 in vivo. Target protein expression was determined by western blotting. Dual luciferase reporter assay was performed to confirm the action of miR-1290 on downstream target genes including SMEK1. Results are reported as means ± S.D. and differences were tested for significance using 2-sided Student’s t-test.Results: In this study, our miRNome sequencing demonstrated that miR-1290 was overexpressed in HCC patient serum-derived exosomes, and we found that delivery of miR-1290 into human endothelial cells enhanced their angiogenic ability. Our results further revealed that SMEK1 is a direct target of miR-1290 in endothelial cells. MiR-1290 exerted its pro-angiogenic function, at least in part, by alleviating the inhibition of VEGFR2 phosphorylation done by SMEK1. Conclusions: Collectively, our findings provide evidence that miR-1290 is overexpressed in HCC and promotes tumor angiogenesis via exosomal secretion, implicating its potential role as a therapeutic target for HCC.

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Exosomal miR-1290 promotes angiogenesis in hepatocellular carcinoma via targeting SMEK1

10.21203/rs.2.13182/v1 ◽

2019 ◽

Author(s):

Qiong Wang ◽

Guanwen Wang ◽

Lianjie Niu ◽

Shaorong Zhao ◽

Jianjun Li ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Endothelial Cells ◽

Tumor Angiogenesis ◽

Molecular Mechanisms ◽

Target Genes ◽

Primary Liver Cancer ◽

Tumor Model ◽

Luciferase Reporter ◽

Dependent Manner

Abstract Abstract Background: Hepatocellular carcinoma (HCC), the most common primary liver cancer, rely on the formation of new blood vessel for growth and frequent intrahepatic and extrahepatic metastasis. Therefore, it is important to explore the underlying molecular mechanisms of tumor angiogenesis of HCC. Recently, microRNAs have been shown to modulate angiogenic processes by modulating the expression of critical angiogenic factors. However, the potential roles of tumor-derived exosomal microRNAs in regulating tumor angiogenesis remain to be elucidated. Methods: MiRNome sequencing was performed to uncover the miRNAs that are dysregulated in HCC patient serum-derived exosomes. Expression levels of miR-1290 in tissues and cells were determined by quantitative real-time PCR. The effect of mir-1290 on proliferation was evaluated by CCK-8 assay. The angiogenic ability of cells were determined by transwell, wound-healing, tube formation and matrigel plug assays. SMMC-7721 xenograft tumor model was established in NOD-SCID nude mice using miR-1290 and NC antagomirs to determin the angiogenic effect of mir-1290 in vivo. Target protein expression was determined by western blotting. Dual luciferase reporter assay was performed to confirm the action of miR-1290 on downstream target genes including SMEK1. Results are reported as means ± S.D. and differences were tested for significance using 2-sided Student’s t-test. Results: In this study, our miRNome sequencing demonstrated that miR-1290 was overexpressed in HCC patient serum-derived exosomes, and we found that delivery of miR-1290 into human endothelial cells enhanced their angiogenic ability. Our results further revealed that SMEK1 is a direct target of miR-1290 in endothelial cells. MiR-1290 exerted its pro-angiogenic function, at least in part, by inhibiting the VEGFR2 signaling pathway in a SMEK1-dependent manner. Conclusions: Collectively, our findings provide evidence that miR-1290 is overexpressed in HCC and promotes tumor angiogenesis via exosomal secretion, implicating its potential role as a therapeutic target for HCC.

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MicroRNA-132, Delivered by Mesenchymal Stem Cell-Derived Exosomes, Promote Angiogenesis in Myocardial Infarction

Stem Cells International ◽

10.1155/2018/3290372 ◽

2018 ◽

Vol 2018 ◽

pp. 1-11 ◽

Cited By ~ 40

Author(s):

Teng Ma ◽

Yueqiu Chen ◽

Yihuan Chen ◽

Qingyou Meng ◽

Jiacheng Sun ◽

...

Keyword(s):

Myocardial Infarction ◽

Endothelial Cells ◽

Real Time ◽

Real Time Pcr ◽

Target Gene ◽

Tube Formation ◽

Luciferase Reporter ◽

Reporter Assay ◽

Ischemic Diseases

Background. To cure ischemic diseases, angiogenesis needs to be improved by various strategies in ischemic area. Considering that microRNA-132 (miR-132) regulates endothelial cell behavior during angiogenesis and the safe and efficacious delivery of microRNAs in vivo is rarely achieved, an ideal vehicle for miR-132 delivery could bring the promise for ischemic diseases. As a natural carrier of biological molecules, exosomes are more and more developed as an ideal vehicle for miRNA transfer. Meanwhile, mesenchymal stem cells could release large amounts of exosomes. Thus, this study aimed to investigate whether MSC-derived exosomes can be used for miR-132 delivery in the treatment of myocardial ischemia. Methods. MSC-derived exosomes were electroporated with miR-132 mimics and inhibitors. After electroporation, miR-132 exosomes were labelled with DiI and added to HUVECs. Internalization of DiI-labelled exosomes was examined by fluorescent microscopy. Expression levels of miR-132 in exosomes and HUVECs were quantified by real-time PCR. The mRNA levels of miR-132 target gene RASA1 in HUVECs were quantified by real-time PCR. Luciferase reporter assay was performed to examine the targeting relationship between miR-132 and RASA1. The effects of miR-132 exosomes on the angiogenic ability of endothelial cells were evaluated by tube formation assay. Matrigel plug assay and myocardial infarction model were used to determine whether miR-132 exosomes can promote angiogenesis in vivo. Results. miR-132 mimics were effectively electroporated and highly detected in MSC-derived exosomes. The expression level of miR-132 was high in HUVECs preincubated with miR-132 mimic-electroporated exosomes and low in HUVECs preincubated with miR-132 inhibitor-electroporated exosomes. The expression level of RASA1, miR-132 target gene, was reversely correlated with miR-132 expression in HUVECs pretreated with exosomes. Luciferase reporter assay further confirmed that RASA1 was a direct target of miR-132. Exosomes loaded with miR-132, as a vehicle for miRNA transfer, significantly increased tube formation of endothelial cells. Moreover, subcutaneous injection of HUVECs pretreated with miR-132 exosomes in nude mice significantly increased their angiogenesis capacity in vivo. In addition, transplantation of miR-132 exosomes in the ischemic hearts of mice markedly enhanced the neovascularization in the peri-infarct zone and preserved heart functions. Conclusions. The findings suggest that the export of miR-132 via MSC-derived exosomes represents a novel strategy to enhance angiogenesis in ischemic diseases.

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MicroRNA-142-3p Induces Atherosclerosis-Associated Endothelial Cell Apoptosis by Directly Targeting Rictor

Cellular Physiology and Biochemistry ◽

10.1159/000490932 ◽

2018 ◽

Vol 47 (4) ◽

pp. 1589-1603 ◽

Cited By ~ 18

Author(s):

Bing Qin ◽

Yaqing Shu ◽

Ling Long ◽

Haiyan Li ◽

Xuejiao Men ◽

...

Keyword(s):

Endothelial Cells ◽

Signaling Pathway ◽

Molecular Mechanisms ◽

Apoptotic Cell ◽

Luciferase Reporter ◽

In Vivo Model ◽

Bioinformatics Analyses ◽

Endothelial Apoptosis

Background/Aims: Atherosclerosis, a multifactorial chronic disease, is the main cause of death and impairment in the world. Endothelial cells (ECs) apoptosis plays a crucial role in the onset and development of atherosclerosis, whereas the underlying molecular mechanisms are unclear. MicroRNA-142-3p (miR-142-3p) is a well-defined tumor suppressor in several types of cancer, while the role of miR-142-3p in ECs apoptosis and the development of atherosclerosis has yet to be elucidated. Therefore, the present study aimed to investigate the role of miR-142-3p in ECs apoptosis during atherosclerosis and the underlying mechanism. Methods: Human aortic endothelial cells (HAECs) were treated with oxidized low-density lipoprotein (ox-LDL). The expression level of miR-142-3p was detected using qRT-PCR. Apoptosis was determined via flow cytometry and Caspase-3 activity assay. Prediction of the binding between miR-142-3p and 3’-UTR of Rictor mRNA was performed by bioinformatics analyses and confirmed by a dual luciferase reporter assay. The effects of miR-142-3p on endothelial apoptosis and atherosclerosis were further analyzed in an in vivo model using ApoE-/- mice fed with high-fat diet (HFD). Results: MiR-142-3p expression was substantially up-regulated during the ox-LDL-elicited apoptosis in HAECs. Forced expression of miR-142-3p exacerbated apoptosis in ECs whereas inhibition of miR-142-3p could partly alleviate apoptotic cell death mediated by ox-LDL. Further analysis identified Rictor as a direct target of miR-142-3p, and Rictor knockdown abolished the anti-apoptotic effect of miR-142-3p inhibitor. Moreover, the Akt/endothelial nitric oxide synthase (eNOS) signaling pathway was found to mediate the beneficial effect of miR-142-3p inhibitor on endothelial apoptosis. Finally, systemic treatment with miR-142-3p antagomir attenuated endothelial apoptosis and retarded the progression of atherosclerosis in the aorta of ApoE-/- mice. Conclusions: Down-regulation of miR-142-3p inhibited ECs apoptosis and atherosclerotic development by up-regulating the expression of Rictor and activating the Akt/eNOS signaling pathway. This indicates that miR-142-3p may be a potential target for the prevention and treatment of atherosclerosis.

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Endothelial Cell Autophagy in Atherosclerosis is Regulated by miR-30-Mediated Translational Control of ATG6

Cellular Physiology and Biochemistry ◽

10.1159/000430402 ◽

2015 ◽

Vol 37 (4) ◽

pp. 1369-1378 ◽

Cited By ~ 25

Author(s):

Tao Zhang ◽

Feng Tian ◽

Jing Wang ◽

Jing Jing ◽

Shan-Shan Zhou ◽

...

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Translational Control ◽

Cell Injury ◽

Protein Translation ◽

Luciferase Reporter ◽

Protective Effects ◽

Bioinformatics Analyses

Background/Aims: Endothelial cell injury and subsequent death play an essential role in the pathogenesis of atherosclerosis. Autophagy of endothelial cells has a protective role against development of atherosclerosis, whereas the molecular regulation of endothelial cell autophagy is unclear. MicroRNA-30 (miR-30) is a known autophagy suppressor in some biological processes, while it is unknown whether this regulatory axis may be similarly involved in the development of atherosclerosis. Here, we aimed to answer these questions in the current study. Methods: We examined the levels of endothelial cell autophagy in ApoE (-/-) mice suppled with high-fat diet (HFD), a mouse model for atherosclerosis (simplified as HFD mice). We analyzed the levels of autophagy-associated protein 6 (ATG6, or Beclin-1) and the levels of miR-30 in the purified CD31+ endothelial cells from mouse aorta. Prediction of the binding between miR-30 and 3'-UTR of ATG6 mRNA was performed by bioinformatics analyses and confirmed by a dual luciferase reporter assay. The effects of miR-30 were further analyzed in an in vitro model using oxidized low-density lipoprotein (ox-LDL)-treated human aortic endothelial cells (HAECs). Results: HFD mice developed atherosclerosis in 12 weeks, while the control ApoE (-/-) mice that had received normal diet (simplified as NOR mice) did not. Compared to NOR mice, HFD mice had significantly lower levels of endothelial cell autophagy, resulting from decreases in ATG6 protein, but not mRNA. The decreases in ATG6 in endothelial cells were due to HFD-induced increases in miR-30, which suppressed the translation of ATG6 mRNA via 3′-UTR binding. These in vivo findings were reproduced in vitro on ox-LDL-treated HAECs. Conclusion: Upregulation of miR-30 by HFD may impair the protective effects of endothelial cell autophagy against development of atherosclerosis through suppressing protein translation of ATG6.

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Dexmedetomidine Reduces Propofol-Induced Hippocampal Neuron Injury by Modulating the miR-377-5p/Arc Pathway

10.21203/rs.3.rs-841956/v1 ◽

2021 ◽

Author(s):

Zong Chen ◽

Yong Ding ◽

Ying Zeng ◽

Xue-Ping Zhang ◽

Jian-Yan Chen

Keyword(s):

Promoter Region ◽

Hippocampal Neurons ◽

Hippocampal Neuron ◽

Molecular Mechanisms ◽

Expression Level ◽

Terminal Deoxynucleotidyl Transferase ◽

Luciferase Reporter ◽

Protective Effects ◽

Expression Levels ◽

Neuron Injury

Abstract BackgroundPropofol and dexmedetomidine (DEX) are widely used in general anesthesia, and exert toxic and protective effects on hippocampal neurons, respectively. The study sought to investigate the molecular mechanisms of DEX-mediated neuroprotection against propofol-induced hippocampal neuron injury in mouse brains.MethodsHippocampal neurons of mice were treated with propofol, DEX, and propofol+DEX in vitro and in vivo. Neuronal apoptosis was evaluated by a means of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) or Hochest 33258 staining; Arc positive expression in hippocampus tissues was detected using a microscope in immunohistochemistry assays; miRNA-377-5p expression levels were quantified by RT-PCR; the protein levels of Arc, DNMT3A, and DNMT3B were determined using western blot; CCK-8 kit was used to evaluated neuron viability; methylation analysis in miR-377-5p promoter was performed through the methylated DNA immunoprecipitation (MeDIP) assay; luciferase reporter assay was performed to confirm whether Arc was under targeted regulation of miR-377-5p.Results In the current study, both in vitro and in vivo, propofol treatment induced hippocampal neuron apoptosis and suppressed cell viability. DNMT3A and DNMT3B expression levels were decreased following propofol treatment, resulting in lowered methylation in the miR-377-5p promoter region and then enhanced expression of miR-377-5p, leading to a decrease in the expression level of downstream Arc. Conversely, the expression levels of DNMT3A and DNMT3B were increased following DEX treatment, thus methylation in miR-377-5p promoter region was improved, and miR-377-5p expression levels were decreased, leading to an increase in the expression level of downstream Arc. Finally, DEX pretreatment protected hippocampal neurons against propofol-induced neurotoxicity by recover the expression levels of DNMT3A, miR-377-5p, and Arc to the normal levels.ConclusionsDEX reduced propofol-induced hippocampal neuron injury via the miR-377-5p/Arc signaling pathway.

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Long Non-coding RNA MIAT Knockdown Prevents the Formation of Intracranial Aneurysm by Downregulating ENC1 via MYC

Frontiers in Physiology ◽

10.3389/fphys.2020.572605 ◽

2021 ◽

Vol 11 ◽

Author(s):

Xinguo Li ◽

Hang Zhao ◽

Jihui Liu ◽

Jing Tong

Keyword(s):

Myocardial Infarction ◽

Endothelial Cells ◽

Intracranial Aneurysm ◽

Cell Viability ◽

Quantitative Polymerase Chain Reaction ◽

Cell Counting ◽

Luciferase Reporter ◽

Immunoprecipitation Assay

Intracranial aneurysm (IA) is vascular enlargement occurred on the wall of cerebral vessels and can result in fatal subarachnoid hemorrhage when ruptured. Recent studies have supported the important role of long non-coding RNAs (lncRNAs) in IA treatment. This study identified functional significance of lncRNA myocardial infarction associated transcript (MIAT) in IA. Myocardial infarction associated transcript and ectodermal-neural cortex 1 (ENC1) expression was detected by reverse transcription quantitative polymerase chain reaction. Cell counting kit 8 assay flow cytometry were conducted to detect cell viability and apoptosis of endothelial cells in IA. The interaction among MIAT, ENC1, and myelocytomatosis oncogene (MYC) was analyzed by RNA pull down, RNA immunoprecipitation assay, chromatin immunoprecipitation assay, and dual luciferase reporter assay. Intracranial aneurysm was induced by ligating the left carotid artery and the bilateral posterior branch of the renal artery in rats for studying the role of MIAT and ENC1 in vivo. Myocardial infarction associated transcript and ENC1 were upregulated in IA. Endothelial cells in IA presented a decreased cell viability and an increased apoptotic rate. Myocardial infarction associated transcript could regulate the expression of ENC1, and MYC could bind to the promoter region of ENC1. High expression of MIAT increased endothelial cell apoptosis and vascular endothelial injury, while MIAT knockdown was identified to reduce the risk of IA both in vitro and in vivo through regulating ENC1. To sum up, MIAT silencing is preventive for IA occurrence by decreasing the MYC-mediated ENC1 expression, which represents a novel therapeutic target for IA.

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YAP/TEAD1 Complex Is a Default Repressor of Cardiac Toll-Like Receptor Genes

International Journal of Molecular Sciences ◽

10.3390/ijms22136649 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6649

Author(s):

Yunan Gao ◽

Yan Sun ◽

Adife Gulhan Ercan-Sencicek ◽

Justin S. King ◽

Brynn N. Akerberg ◽

...

Keyword(s):

Heart Development ◽

Molecular Mechanisms ◽

Developmental Stages ◽

Heart Diseases ◽

Luciferase Reporter ◽

Binding Motif ◽

Expression Levels ◽

Genomic Regions

Toll-like receptors (TLRs) are a family of pattern recognition receptors (PRRs) that modulate innate immune responses and play essential roles in the pathogenesis of heart diseases. Although important, the molecular mechanisms controlling cardiac TLR genes expression have not been clearly addressed. This study examined the expression pattern of Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, Tlr8, and Tlr9 in normal and disease-stressed mouse hearts. Our results demonstrated that the expression levels of cardiac Tlr3, Tlr7, Tlr8, and Tlr9 increased with age between neonatal and adult developmental stages, whereas the expression of Tlr5 decreased with age. Furthermore, pathological stress increased the expression levels of Tlr2, Tlr4, Tlr5, Tlr7, Tlr8, and Tlr9. Hippo-YAP signaling is essential for heart development and homeostasis maintenance, and YAP/TEAD1 complex is the terminal effector of this pathway. Here we found that TEAD1 directly bound genomic regions adjacent to Tlr1, Tlr2, Tlr3, Tlr4, Tlr5, Tlr6, Tlr7, and Tlr9. In vitro, luciferase reporter data suggest that YAP/TEAD1 repression of Tlr4 depends on a conserved TEAD1 binding motif near Tlr4 transcription start site. In vivo, cardiomyocyte-specific YAP depletion increased the expression of most examined TLR genes, activated the synthesis of pro-inflammatory cytokines, and predisposed the heart to lipopolysaccharide stress. In conclusion, our data indicate that the expression of cardiac TLR genes is associated with age and activated by pathological stress and suggest that YAP/TEAD1 complex is a default repressor of cardiac TLR genes.

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Selenium Attenuates Chronic Heat Stress-Induced Apoptosis via the Inhibition of Endoplasmic Reticulum Stress in Mouse Granulosa Cells

Molecules ◽

10.3390/molecules25030557 ◽

2020 ◽

Vol 25 (3) ◽

pp. 557 ◽

Cited By ~ 3

Author(s):

Yongjie Xiong ◽

Qirun Yin ◽

Erhui Jin ◽

Huatao Chen ◽

Shaojun He

Keyword(s):

Heat Stress ◽

Er Stress ◽

Granulosa Cells ◽

Molecular Mechanisms ◽

Caspase 3 ◽

Protective Action ◽

Protective Effects ◽

Expression Levels ◽

Induced Apoptosis

Heat stress induces apoptosis in various cells. Selenium, an essential micronutrient, has beneficial effects in maintaining the cellular physiological functions. However, its potential protective action against chronic heat stress (CHS)-induced apoptosis in granulosa cells and the related molecular mechanisms are not fully elucidated. In this study, we investigated the roles of selenium in CHS-induced apoptosis in mouse granulosa cells and explored its underlying mechanism. The heat treatment for 6–48 h induced apoptosis, potentiated caspase 3 activity, increased the expression levels of apoptosis-related gene BAX and ER stress markers, glucose-regulated protein 78 (GRP78), and CCAAT/enhancer binding protein homologous protein (CHOP) in mouse granulosa cells. The treatment with ER stress inhibitor 4-PBA significantly attenuated the adverse effects caused by CHS. Selenium treatment significantly attenuated the CHS- or thapsigargin (Tg, an ER stress activator)-induced apoptosis, potentiation of caspase 3 activity, and the increased protein expression levels of BAX, GRP78, and CHOP. Additionally, treatment of the cells with 5 ng/mL selenium significantly ameliorated the levels of estradiol, which were decreased in response to heat exposure. Consistently, administering selenium supplement alleviated the hyperthermia-caused reduction in the serum estradiol levels in vivo. Together, our findings indicate that selenium has protective effects on CHS-induced apoptosis via inhibition of the ER stress pathway. The current study provides new insights in understanding the role of selenium during the process of heat-induced cell apoptosis.

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Novel Mechanisms Related to DHA's Atheroprotective Effects in Endothelial Cells: Modulation of Histone H3 Modification via Activation of p38MAPK Signaling

Current Developments in Nutrition ◽

10.1093/cdn/nzaa058_016 ◽

2020 ◽

Vol 4 (Supplement_2) ◽

pp. 1258-1258

Author(s):

Shiqi Huang ◽

Carla Taylor ◽

Peter Zahradka

Keyword(s):

Endothelial Cells ◽

Time Course ◽

Molecular Mechanisms ◽

Histone H3 ◽

Specific Inhibitor ◽

Mitogen Activated Protein Kinase ◽

Protective Effects ◽

Downstream Targets ◽

Ea.Hy926 Cells

Abstract Objectives Docosahexaenoic acid (DHA) is known for its protective effects against cardiovascular disease, which has been the leading cause of death worldwide for 2 decades. The molecular mechanisms responsible for DHA's atheroprotective effects, however, remain largely unknown. DHA has been found to activate p38 mitogen-activated protein kinase (MAPK) differently in growing and quiescent human endothelial cells, which represent dysfunctional and healthy states in vivo, respectively. This study was designed to characterize the activation pattern of p38MAPK in endothelial cells in response to DHA treatment and to identify possible downstream targets by which DHA exerts its protective effects. Methods EA.hy926 cells were cultured on Matrigel-coated plates to sub-confluent, confluent, and quiescent states. The cells were treated with DHA to establish concentration (10 μM to 150 μM) and time course (10 min to 24 h) curves, with or without SB202190, a p38MAPK-specific inhibitor. The activation of p38MAPK and its downstream targets was quantified by Western blotting. Histone H3 modifications upon DHA treatment were tested with an ELISA-based kit. Results The activation of p38MAPK by DHA in EA.hy926 cells was concentration-, time-, and growth state-dependent. Upon p38MAPK inhibition, activation of mitogen and stress activated kinase 1 (MSK1), a downstream target of p38MAPK, declined. This reduction was attenuated by low concentrations of DHA in quiescent endothelial cells but not confluent or sub-confluent cells. Since MSK1 can act on histone H3 and other chromatin binding proteins like cAMP response element-binding protein (CREB), H3 modifications were monitored. In the confluent state, DHA caused a 6-fold increase in total H3, with a concomitant decrease in most methylation, acetylation, and phosphorylation marks, including H3K9me1/3, H3K27me2, H3K36me1/3, H3K9ac, H3K18ac, H3S10ph, and H3S28ph. Conclusions This study showed that DHA may exert its effects in endothelial cells via the p38MAPK signalling pathway, and MSK1 may be a downstream effector possibly leading to epigenetic changes. The results provide novel insights regarding DHA's atheroprotective actions and identify new therapeutic targets with potential for treating atherosclerosis. Funding Sources Research Manitoba, St Boniface Hospital Foundation-Research Without Borders.

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