Exosomal MALAT1 Derived from High Glucose-Treated Macrophages Up-Regulates Resistin Expression via miR-150-5p Downregulation.

Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a crucial role in the pathophysiological process associated with diabetes-related complications. The effect of high glucose levels on macrophage-derived exosomal MALAT1 is unknown. Therefore, we investigated the molecular regulatory mechanisms controlling exosomal MALAT1 in macrophages under high glucose treatment and the therapeutic target of macrophage-derived exosomal MALAT1 using a balloon injury model of vascular disease in diabetic rats. High glucose (25 mM) significantly increased MALAT1 expression in macrophage-derived exosomes. MALAT1 suppressed miR-150-5p expression in macrophage-derived exosomes under high-glucose conditions. Silencing MALAT1 using MALAT1 siRNA significantly reversed miR-150-5p expression induced by macrophage-derived exosomes. Macrophage-derived exosomes under high-glucose treatment significantly increased resistin expression in macrophages. Silencing MALAT1 and overexpression of miR-150-5p significantly decreased resistin expression induced by macrophage-derived exosomes. Overexpression of miR-150-5p significantly decreased resistin luciferase activity induced by macrophage-derived exosomes. Macrophage-derived exosome significantly decreased glucose uptake in macrophages and silencing MALAT1, resistin or overexpression of miR-150-5p significantly reversed glucose uptake. Balloon injury to the carotid artery significantly increased MALAT1 and resistin expression and significantly decreased miR-150-5p expression in arterial tissue. Silencing MALAT1 significantly reversed miR-150-5p expression in arterial tissue after balloon injury. Silencing MALAT1 or overexpression of miR-150-5p significantly reduced resistin expression after balloon injury. In conclusion, high glucose up-regulates MALAT1 to suppress miR-150-5p expression and counteracts the inhibitory effect of miR-150-5p on resistin expression in macrophages to promote vascular disease. Macrophage-derived exosomes containing MALAT1 may serve as a novel cell-free approach for the treatment of vascular disease in diabetes mellitus.

Cyclic stretching boosts microRNA-499 to regulate Bcl-2 via microRNA-208a in atrial fibroblasts.

MicroRNAs that modulate transcription can regulate other microRNAs and are also up-regulated under pathological stress. MicroRNA-499 (miR-499), microRNA-208a (miR-208a) and B-cell lymphoma 2 (Bcl-2) play roles in cardiovascular diseases, such as direct reprogramming of cardiac fibroblast into cardiomyocyte and cardiomyocyte apoptosis. Whether miR208a, miR499 and Bcl-2 were critical regulators in cardiac fibroblast apoptosis under mechanical stretching conditions in human cardiac fibroblasts-adult atrial (HCF-aa) was investigated. Using negative pressure, HCF-aa grown on a flexible membrane base were cyclically stretched to 20% of their maximum elongation. In adult rats, an aortocaval shunt was used to create an in vivo model of volume overload. MiR208a was up-regulated early by stretching and returned to normal levels with longer stretching cycles, whereas the expression of miR499 and Bcl-2 was up-regulated by longer stretching times. Pre-treatment with antagomir-499 reversed the miR-208a down-regulation, whereas Bcl-2 expression could be suppressed by miR-208a overexpression. In the HCF-aa under stretching for 1 h, miR-499 overexpression decreased pri-miR-208a luciferase activity; this inhibition of pri-miR-208a luciferase activity with stretching was reversed when the miR-499-5p binding site in pri-miR-208a was mutated. The addition of antagomir-208a reversed the Bcl-2-3'UTR suppression from stretching for 1 h. Flow cytometric analysis revealed that pre-treatment with miR-499 or antagomir-208a inhibited cellular apoptosis in stretched HCF-aa. In hearts with volume overload, miR-499 overexpression inhibited myocardial miR-208a expression, whereas Bcl-2 expression could be suppressed by the addition of miR-208a. In conclusion, miR-208a mediated the regulation of miR-499 on Bcl-2 expression in stretched HCF-aa and hearts with volume overload.

Chrysin boosts KLF2 expression through suppression of endothelial cell-derived exosomal microRNA-92a in the model of atheroprotection.

PURPOSE: Atherosclerosis and its related clinical complications are the leading cause of death. MicroRNA (miR)-92a in the inflammatory endothelial dysfunction leads to atherosclerosis. Krüppel-like factor 2 (KLF2) is required for vascular integrity and endothelial function maintenance. Flavonoids possess many biological properties. This study investigated the vascular protective effects of chrysin in balloon-injured carotid arteries. MATERIALS AND METHODS: Exosomes were extracted from human coronary artery endothelial cell (HCAEC) culture media. Herb flavonoids and chrysin were the treatments in these atheroprotective models. Western blotting and real-time PCRs were performed. In situ hybridization, immunohistochemistry, and immunofluorescence analyses were employed. RESULTS: MiR-92a increased after balloon injury and was present in HCAEC culture media. Chrysin was treated, and significantly attenuated the miR-92a levels after balloon injury, and similar results were obtained in HCAEC cultures in vitro. Balloon injury-induced miR-92a expression, and attenuated KLF2 expression. Chrysin increased the KLF2 but reduced exosomal miR-92a secretion. The addition of chrysin and antagomir-92a, neointimal formation was reduced by 44.8 and 49.0% compared with balloon injury after 14 days, respectively. CONCLUSION: Chrysin upregulated KLF2 expression in atheroprotection and attenuated endothelial cell-derived miR-92a-containing exosomes. The suppressive effect of miR-92a suggests that chrysin plays an atheroprotective role. Proposed pathway for human coronary artery endothelial cell (HCAEC)-derived exosomes induced by chrysin to suppress microRNA (miR)-92a expression and counteract the inhibitory effect of miR-92a on KLF2 expression in HCAECs. This provides an outline of the critical role of the herbal flavonoid chrysin, which may serve as a valuable therapeutic supplement for atheroprotection.

Hyperbaric oxygen-induced long non-coding RNA MALAT1 exosomes suppress MicroRNA-92a expression in a rat model of acute myocardial infarction.

Hyperbaric oxygen (HBO) improves angiogenesis. The effect of HBO on metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a pro-angiogenic long non-coding RNA, in cardiac myocyte-derived exosomes and acute myocardial infarction (AMI) is unknown. We aimed to investigate whether MALAT1 is altered in cardiac myocyte-derived exosomes in response to HBO as well as the molecular regulatory mechanisms of MALAT1 in cardiac myocytes treated with HBO. Cardiac myocytes were cultured, and HBO was applied at 2.5 atmosphere absolute in a hyperbaric chamber. Exosomes were extracted from the culture media. A rat model of AMI generated by the ligation of the left anterior descending artery was used. HBO significantly increased MALAT1 expression in cardiac myocytes and HBO-induced MALAT1 and exosomes attenuated miR-92a expression after myocardial infarction. Expression of krüppel-like factor 2 (KLF2) and CD31 was significantly decreased after infarction and HBO-induced exosomes significantly reversed the expression. Silencing of MALAT1 using MALAT1-locked nucleic acid GapmeR significantly attenuated KLF2 and CD31 protein expression after infarction induced by HBO-induced exosomes. HBO-induced exosomes also decreased infarct size significantly. HBO-induced exosomes from cardiac myocytes up-regulate MALAT1 to suppress miR-92a expression and counteract the inhibitory effect of miR-92a on KLF2 and CD31 expression in left ventricular myocardium after myocardial infarction to enhance neovascularization.

Catalpol Ameliorates Neointimal Hyperplasia in Diabetic Rats.

Catalpol, an iridoid glycoside, is an isolated natural product of Rehmannia glutinosa, which has been reported to have antidiabetic properties. This study investigated the vascular protective effects of catalpol in hyperglycemic rats with balloon-injured carotid arteries. Balloon injury stress led to the upregulation of monocyte chemoattractant protein-1 expression in rats with streptozotocin-induced diabetes. Western blotting and real-time PCR were performed. In situ hybridization, immunohistochemistry, and confocal analyses were employed. Monocyte chemoattractant protein-1 levels were increased through streptozotocin induction or balloon injury. After treatment with catalpol, the neointimal hyperplasia area was reduced 2 weeks after balloon injury in hyperglycemic rats. Real-time PCR and immunohistochemical analysis demonstrated reduced levels of monocyte chemoattractant protein-1 2 weeks after the balloon injury. Monocyte chemoattractant protein-1 expression was significantly increased in balloon-injured rats compared with the control groups. Thus, treatment with catalpol affected monocyte chemoattractant protein-1 expression. This study demonstrated that catalpol downregulated monocyte chemoattractant protein-1 expression in carotid arteries and ameliorated neointimal hyperplasia in hyperglycemic rats. The suppressive effect of monocyte chemoattractant protein-1 suggests that it plays a key role in neointimal hyperplasia. The results imply that catalpol is potentially effective for preventing hyperglycemia-related ischemic cardiac diseases.

Suppressive effect of epigallocatechin-3-O-gallate on endoglin molecular regulation in myocardial fibrosis in vitro and in vivo.

Epigallocatechin-3-O-gallate (EGCG), derived from green tea, has been studied extensively because of its diverse physiological and pharmacological properties. This study evaluates the protective effect of EGCG on angiotensin II (Ang II)-induced endoglin expression in vitro and in vivo. Cardiac fibroblasts (CFs) from the thoracic aorta of adult Wistar rats were cultured and induced with Ang II. Western blotting, Northern blotting, real-time PCR and promoter activity assay were performed. Ang II increased endoglin expression significantly as compared with control cells. The specific extracellular signal-regulated kinase inhibitor SP600125 (JNK inhibitor), EGCG (100 µM) and c-Jun N-terminal kinase (JNK) siRNA attenuated endoglin proteins following Ang II induction. In addition, pre-treated Ang II-induced endoglin with EGCG diminished the binding activity of AP-1 by electrophoretic mobility shift assay. Moreover, the luciferase assay results revealed that EGCG suppressed the endoglin promoter activity in Ang II-induced CFs by AP-1 binding. Finally, EGCG and the JNK inhibitor (SP600125) were found to have attenuated endoglin expression significantly in Ang II-induced CFs, as determined through confocal microscopy. Following in vivo acute myocardial infarction (AMI)-related myocardial fibrosis study, as well as immunohistochemical and confocal analyses, after treatment with endoglin siRNA and EGCG (50 mg/kg), the area of myocardial fibrosis reduced by 53.4% and 64.5% and attenuated the left ventricular end-diastolic and systolic dimensions, and friction shortening in hemodynamic monitor. In conclusion, epigallocatechin-3-O-gallate (EGCG) attenuated the endoglin expression and myocardial fibrosis by anti-inflammatory effect in vitro and in vivo, the novel suppressive effect was mediated through JNK/AP-1 pathway.

A novel cancer therapeutic using thrombospondin 1 in dendritic cells.

Induction of thrombospondin 1 (TSP-1) is generally assumed to suppress tumor growth through inhibiting angiogenesis; however, it is less clear how TSP-1 in dendritic cells (DCs) influences tumor progression. We investigated tumor growth and immune mechanism by downregulation of TSP-1 in dendritic cells. Administration of TSP-1 small hairpin RNA (shRNA) through the skin produced anticancer therapeutic effects. Tumor-infiltrating CD4(+) and CD8(+) T cells were increased after the administration of TSP-1 shRNA. The expression of interleukin-12 and interferon-γ in the lymph nodes was enhanced by injection of TSP-1 shRNA. Lymphocytes from the mice injected with TSP-1 shRNA selectively killed the tumor cells, and the cytotoxicity of lymphocytes was abolished by depletion of CD8(+) T cells. Injection of CD11c(+) TSP-1-knockout (TSP-1-KO) bone marrow-derived DCs (BMDCs) delayed tumor growth in tumor-bearing mice. Similarly, antitumor activity induced by TSP-1-KO BMDCs was abrogated by depletion of CD8(+) T cells. In contrast, the administration of shRNAs targeting TSP-2, another TSP family member, did not extend the survival of tumor-bearing mice. Finally, TSP-1 shRNA functioned as an immunotherapeutic adjuvant to augment the therapeutic efficacy of Neu DNA vaccination. Collectively, the downregulation of TSP-1 in DCs produces an effective antitumor response that is opposite to the protumor effects by silencing of TSP-1 within tumor cells.

Exosomal MALAT1 from macrophages treated with high levels of glucose upregulates LC3B expression via miR-204-5p downregulation.

BACKGROUND: Metastasis-associated lung adenocarcinoma transcript 1 ( MALAT1 ) plays a critical role in the pathophysiology of diabetes-related complications. However, whether macrophage-derived MALAT1 affects autophagic activity under hyperglycemic conditions is unclear. Therefore, we investigated the molecular regulatory mechanisms of macrophage-derived MALAT1 and autophagy under hyperglycemic conditions. METHODS: Hyperglycemia was induced by culturing macrophages in 25 mM glucose for 1 hour. Exosomes were extracted from the culture media. A rat model of carotid artery balloon injury was established to assess the effect of MALAT1 on vascular injury. Reverse transcription, real-time quantitative polymerase chain reaction, western blotting, immunohistochemical staining, and luciferase activity assays were performed. RESULTS: Stimulation with high levels of glucose significantly enhanced MALAT1 expression in macrophage-derived exosomes. MALAT1 inhibited miR-204-5p expression in macrophage-derived exosomes under hyperglycemic conditions. siRNA-induced silencing of MALAT1 significantly reversed macrophage-derived exosome-induced miR-204-5p expression. Hyperglycemic treatment caused a significant, exosome-induced increase in the expression of the autophagy marker LC3B in macrophages. Silencing MALAT1 and overexpression of miR-204-5p significantly decreased LC3B expression induced by macrophage-derived exosomes. Overexpression of miR-204-5p significantly reduced LC3B luciferase activity induced by macrophage-derived exosomes. Balloon injury to the carotid artery in rats significantly enhanced MALAT1 and LC3B expression, and significantly reduced miR-204-5p expression in carotid artery tissue. Silencing MALAT1 significantly reversed miR-204-5p expression in carotid artery tissue after balloon injury. MALAT1 silencing or miR-204-5p overexpression significantly reduced LC3B expression after balloon injury. CONCLUSION: This study demonstrated that hyperglycemia upregulates MALAT1 . MALAT1 suppresses miR-204-5p expression and counteracts the inhibitory effect of miR-204-5p on LC3B expression in macrophages to promote vascular disease.

Angiotensin II mediates urotensin II expression by hypoxia in cultured cardiac fibroblast.

BACKGROUND: Urotensin II plays a role in myocardial remodelling. Cardiac fibroblasts play a critical role in the development of cardiac fibrosis. The effect of hypoxia on urotensin II expression in cardiac fibroblasts is poorly understood. We sought to investigate the regulation of urotensin II by hypoxia in cardiac fibroblasts and the effect of angiotensin II in the interaction with urotensin II. METHODS AND RESULTS: Rat cardiac fibroblasts were cultured in hypoxic chamber. Hypoxia significantly increased urotensin II expression and reactive oxygen species (ROS) production in cultured cardiac fibroblasts. Hypoxia-induced increase in urotensin II protein and ROS was significantly attenuated after the addition of SP600125, JNK siRNA or N-acetylcysteine before hypoxia treatment. The phosphorylated JNK protein was induced by hypoxia and was abolished by pretreatment with SP600125, losartan (an angiotensin II receptor antagonist) or N-acetylcysteine. The increased urotensin II expression by exogenous addition of angiotensin II was similar to that by hypoxia. Addition of losartan and angiotensin II antibody before hypoxia almost completely inhibited the increase in urotensin II induced by hypoxia. Hypoxia significantly increased the secretion of angiotensin II from cardiac fibroblasts and increased the collagen I protein expression. Hypoxia significantly increased the urotensin II promoter activity by 4·3-fold as compared to normoxic control. Urotensin II siRNA almost completely attenuated the collagen I protein expression induced by hypoxia. CONCLUSIONS: Hypoxia-induced urotensin II expression in cardiac fibroblast is mediated by angiotensin II and through ROS and JNK pathway. Urotensin II is a mediator of angiotensin II-induced cardiac fibrosis under hypoxia.

Skin delivery of short hairpin RNA of indoleamine 2,3 dioxygenase induces antitumor immunity against orthotopic and metastatic liver cancer.

Liver cancer is one of the most malignant cancers in the world and has a high rate of metastasis. Therefore, development of a novel therapy for liver cancer is a critical issue. Indoleamine 2,3-dioxygenase (IDO) is known as a negative immune regulator in dendritic cells. Our previous study demonstrated that skin delivery of IDO short hairpin RNA (shRNA) induced antitumor immunity in subcutaneous bladder and colon tumor models. Because the immunological environment is quite different between skin and liver, it is essential to evaluate whether skin delivery of IDO shRNA is an effective treatment in metastatic and orthotopic animal tumor models. In the present study, IDO shRNA inhibited tumor growth in subcutaneous, metastatic and orthotopic liver tumor models. The cytotoxicity of splenocytes was significantly elevated in mice treated with IDO shRNA in the orthotopic and metastatic tumor models. Interleukin (IL)-12 and interferon (IFN)-gamma mRNA expression were upregulated while IL-10 was downregulated in the inguinal lymph nodes, which were collected from IDO shRNA-treated mice. Similar results were observed in the spleens of mice inoculated with IDO shRNA by gene gun. The results indicate that skin administration of IDO shRNA is an effective therapy in orthotopic and metastatic liver cancer animal models. Indoleamine 2,3-dioxygenase shRNA might be a potential new treatment for liver cancer in the future.

Tumor necrosis factor-α enhances hyperbaric oxygen-induced visfatin expression via JNK pathway in human coronary arterial endothelial cells.

BACKGROUND: Visfatin, a adipocytokine with insulin-mimetic effect, plays a role in endothelial angiogenesis. Hyperbaric oxygen (HBO) has been used in medical practice. However, the molecular mechanism of beneficial effects of HBO is poorly understood. We sought to investigate the cellular and molecular mechanisms of regulation of visfatin by HBO in human coronary arterial endothelial cells (CAECs). METHODS: Human CAECs were exposed to 2.5 atmosphere absolute (ATA) of oxygen in a hyperbaric chamber. Western blot, real-time polymerase chain reaction, and promoter activity assay were performed. In vitro glucose uptake and tube formation was detected. RESULTS: Visfatin protein (2.55-fold) and mRNA (2.53-fold) expression were significantly increased after exposure to 2.5 ATA HBO for 4 to 6 h. Addition of SP600125 and JNK siRNA 30 min before HBO inhibited the induction of visfatin protein. HBO also significantly increased DNA-protein binding activity of AP-1 and visfatin promoter activity. Addition of SP600125 and TNF-α monoclonal antibody 30 min before HBO abolished the DNA-protein binding activity and visfatin promoter activity induced by HBO. HBO significantly increased secretion of TNF-α from cultured human CAECs. Exogenous addition of TNF-α significantly increased visfatin protein expression while TNF-α antibody and TNF-α receptor antibody blocked the induction of visfatin protein expression induced by HBO. HBO increased glucose uptake in human CAECs as HBO and visfatin siRNA and TNF-α antibody attenuated the glucose uptake induced by HBO. HBO significantly increased the tube formation of human CAECs while visfatin siRNA, TNF-α antibody inhibited the tube formation induced by HBO. CONCLUSIONS: HBO activates visfatin expression in cultured human CAECs. HBO-induced visfatin is mediated by TNF-α and at least in part through JNK pathway.

Promoting effect of Antrodia camphorata as an immunomodulating adjuvant on the antitumor efficacy of HER-2/neu DNA vaccine.

It is well known that DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. Antrodia camphorata (AC) is a unique basidiomycete fungus of the Polyporaceae family that only grows on the aromatic tree Cinnamomum kanehirai Hayata (Lauraceae) endemic to Taiwan. Importantly, AC has been shown to be highly beneficial in the treatment and prevention of cancer. The goal of this study was to investigate whether AC is able to augment the antitumor immune properties of a HER-2/neu DNA vaccine in a mouse model in which p185neu is overexpressed in MBT-2 tumor cells. Compared with the mice that received the HER-2/neu DNA vaccine alone, co-treatment with AC suppressed tumor growth and extended the survival rate. This increase in the antitumor efficacy was attributed to the enhancement of the Th1-like cellular immune response by the HER-2/neu DNA vaccine-AC combination. Evidence for this came from the marked increase in the IFN-gamma mRNA expression in CD4+ T cells in the draining inguinal lymph nodes, an increase in the number of functional HER-2/neu-specific CTLs, and the increased tumor infiltration of both CD4+ and CD8+ T cells, depletion of which abolishes the antitumor effect of the HER-2/neu DNA vaccine-AC therapy. Our results further indicate that the treatment of mice with AC enhanced DC activation and production of Th1-activating cytokines (e.g. IL-12, and IFN-alpha) in the draining lymph nodes, which were sufficient to directly stimulate T cell proliferation and higher IFN-gamma production in response to ErbB2. Overall, these results clearly demonstrate that AC represents a promising immunomodulatory adjuvant that could enhance the therapeutic potency of HER-2/neu DNA vaccines in cancer therapy.

Cyclic stretch enhances the expression of toll-like receptor 4 gene in cultured cardiomyocytes via p38 MAP kinase and NF-kappaB pathway.

BACKGROUND: Toll-like receptor 4 (TLR4) plays an important role in innate immunity. The role of TLR4 in stretched cardiomyocytes is not known. We sought to investigate whether mechanical stretch could regulate TLR4 expression, as well as the possible molecular mechanisms and signal pathways mediating the expression of TLR4 by cyclic mechanical stretch in cardiomyocytes. METHODS: Neonatal Wistar rat cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation at 60 cycles/min. Western blot, real-time polymerase chain reaction, and promoter activity assay were performed. In vitro monocyte adhesion to stretched myocyte was detected. RESULTS: Cyclic stretch significantly increased TLR4 protein and mRNA expression after 2 h to 24 h of stretch. Addition of SB203580, TNF-alpha antibody, and p38alpha MAP kinase siRNA 30 min before stretch inhibited the induction of TLR4 protein. Cyclic stretch increased, while SB203580 abolished the phosphorylated p38 protein. Gel shifting assay showed significant increase of DNA-protein binding activity of NF-kappaB after stretch and SB203580 abolished the DNA-protein binding activity induced by cyclic stretch. DNA-binding complexes induced by cyclic stretch could be supershifted by p65 monoclonal antibody. Cyclic stretch increased TLR4 promoter activity while SB203580 and NF-kappaB siRNA decreased TLR4 promoter activity. Cyclic stretch increased adhesion of monocyte to cardiomyocytes while SB203580, TNF-alpha antibody, and TLR4 siRNA attenuated the adherence of monocyte. TNF-alpha and Ang II significantly increased TLR4 protein expression. Addition of losartan, TNF-alpha antibody, or p38alpha siRNA 30 min before Ang II and TNF-alpha stimulation significantly blocked the increase of TLR4 protein by AngII and TNF-alpha. CONCLUSIONS: Cyclic mechanical stretch enhances TLR4 expression in cultured rat neonatal cardiomyocytes. The stretch-induced TLR4 is mediated through activation of p38 MAP kinase and NF-kappaB pathways. TLR4 up-regulation by cyclic stretch increases monocyte adherence.

Zingiber officinale (ginger) compounds have tetracycline-resistance modifying effects against clinical extensively drug-resistant Acinetobacter baumannii.

Extensively drug-resistant Acinetobacter baumannii (XDRAB) is a growing and serious nosocomial infection worldwide, such that developing new agents against it is critical. The antimicrobial activities of the rhizomes from Zingiber officinale, known as ginger, have not been proven in clinical bacterial isolates with extensive drug-resistance. This study aimed to investigate the effects of four known components of ginger, [6]-dehydrogingerdione, [10]-gingerol, [6]-shogaol and [6]-gingerol, against clinical XDRAB. All these compounds showed antibacterial effects against XDRAB. Combined with tetracycline, they showed good resistance modifying effects to modulate tetracycline resistance. Using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging method, these four ginger compounds demonstrated antioxidant properties, which were inhibited by MnO2, an oxidant without antibacterial effects. After the antioxidant property was blocked, their antimicrobial effects were abolished significantly. These results indicate that ginger compounds have antioxidant effects that partially contribute to their antimicrobial activity and are candidates for use in the treatment of infections with XDRAB.

(-)-Epigallocatechin Gallate Promotes MicroRNA 145 Expression against Myocardial Hypoxic Injury through Dab2/Wnt3a/ß-catenin.

MicroRNA 145 (miR-145) is a critical modulator of cardiovascular diseases. The downregulation of myocardial miR-145 is followed by an increase in disabled-2 (Dab2) expression in cardiomyocytes. (-)-epigallocatechin gallate (EGCG) is a flavonoid that has been evaluated extensively due to its diverse pharmacological properties including anti-inflammatory effects. The aim of this study was to investigate the cardioprotective effects of EGCG under hypoxia-induced stress in vitro and in vivo. The hypoxic insult led to the suppression of miR-145 expression in cultured rat cardiomyocytes in a concentration-dependent manner. Western blotting and real-time PCR were performed. In rat myocardial infarction study, in situ hybridization, and immunofluorescent analyses were adopted. The western blot and real-time PCR data revealed that hypoxic stress with 2.5% O2 suppressed the expression of miR-145 and Wnt3a/ß-catenin in cultured rat cardiomyocytes but augmented Dab2. Treatment with EGCG attenuated Dab2 expression, but increased Wnt3a and ß-catenin in hypoxic cultured cardiomyocytes. Following in vivo myocardial infarction (MI) study, the data revealed the myocardial infarct area reduced by 48.5%, 44.6%, and 48.5% in EGCG (50mg/kg) or miR-145 dominant or Dab2 siRNA groups after myocardial infarction for 28 days, respectively. This study demonstrated that EGCG increased miR-145, Wnt3a, and ß-catenin expression but attenuated Dab2 expression. Moreover, EGCG ameliorated myocardial ischemia in vivo. The novel suppressive effect was mediated through the miR-145 and Dab2/Wnt3a/ß-catenin pathways.

Antithrombotic Strategies in Patients with Atrial Fibrillation Following Percutaneous Coronary Intervention: A Systemic Review and Network Meta-Analysis of Randomized Controlled Trials.

Up to 10% of patients with atrial fibrillation (AF) undergo percutaneous coronary intervention (PCI). A systematic review and network meta-analysis were conducted by searching PubMed, Embase, and the Cochrane database of systematic reviews for randomized control trials that studied the safety and efficacy of different antithrombotic strategies in these patients. Six studies, including 12,158 patients were included. Compared to that in the triple antithrombotic therapy group (vitamin K antagonist (VKA) plus P2Y12 inhibitor and aspirin), thrombolysis in myocardial infarction (TIMI) major bleeding was significantly reduced in the dual antithrombotic therapy (non-vitamin K oral anticoagulants (NOACs) plus P2Y12 inhibitor) group by 47% (Odds ratio (OR), 0.53; 95% credible interval [CrI], 0.35-0.78; I2 = 0%). Besides, NOAC plus a P2Y12 inhibitor was associated with less intracranial hemorrhage compared to VKA plus single antiplatelet therapy (OR: 0.20, 95% CrI: 0.05-0.77). There was no significant difference in the trial-defined major adverse cardiac events or the individual outcomes of all-cause mortality, cardiovascular death, myocardial infarction, stroke or stent thrombosis among all antithrombotic strategies. In conclusion, antithrombotic strategy of NOACs plus P2Y12 inhibitor is safer than, and as effective as, the strategies including aspirin when used in AF patients undergoing PCI.

The effects of DNA formulation and administration route on cancer therapeutic efficacy with xenogenic EGFR DNA vaccine in a lung cancer animal model.

BACKGROUND: Tyrosine kinase inhibitor gefitinib is effective against lung cancer cells carrying mutant epidermal growth factor receptor (EGFR); however, it is not effective against lung cancer carrying normal EGFR. The breaking of immune tolerance against self epidermal growth factor receptor with active immunization may be a useful approach for the treatment of EGFR-positive lung tumors. Xenogeneic EGFR gene was demonstrated to induce antigen-specific immune response against EGFR-expressing tumor with intramuscular administration. METHODS: In order to enhance the therapeutic effect of xenogeneic EGFR DNA vaccine, the efficacy of altering routes of administration and formulation of plasmid DNA was evaluated on the mouse lung tumor (LL2) naturally overexpressing endogenous EGFR in C57B6 mice. Three different combination forms were studied, including (1) intramuscular administration of non-coating DNA vaccine, (2) gene gun administration of DNA vaccine coated on gold particles, and (3) gene gun administration of non-coating DNA vaccine. LL2-tumor bearing C57B6 mice were immunized four times at weekly intervals with EGFR DNA vaccine. RESULTS: The results indicated that gene gun administration of non-coating xenogenic EGFR DNA vaccine generated the strongest cytotoxicity T lymphocyte activity and best antitumor effects. CD8(+) T cells were essential for anti-tumor immunity as indicated by depletion of lymphocytes in vivo. CONCLUSION: Thus, our data demonstrate that administration of non-coating xenogenic EGFR DNA vaccine by gene gun may be the preferred method for treating EGFR-positive lung tumor in the future.

Hyperbaric oxygen boosts long noncoding RNA MALAT1 exosome secretion to suppress microRNA-92a expression in therapeutic angiogenesis.

BACKGROUND: Hyperbaric oxygen (HBO) could improve wound healing by enhancement of angiogenesis. The effect of HBO on metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a proangiogenic long noncoding RNA, and on endothelial cell-derived exosome is unknown. We aim to investigate both whether MALAT1 is altered in human coronary artery endothelial cells (HCAECs)-derived exosomes in response to HBO as well as the molecular regulatory mechanisms of MALAT1 in HCAECs under HBO treatment. METHODS AND RESULTS: HCAECs were cultured and HBO was applied at 2.5 atmosphere absolute (ATA) in a hyperbaric chamber. Exosomes were extracted from culture media. A rat model of hind-limb ischemia was performed by ligation of the right femoral artery. HBO at 2.5 ATA significantly increased MALAT1 expression in HCAECs and HCAECs-derived exosomes. MALAT1 suppressed miR-92a expression in HCAEC-derived exosomes under HBO. Silencing MALAT1 by MALAT1 siRNA significantly inhibited KLF2 mRNA expression induced by HBO, as did MiR-92a. MiR-92a significantly decreased KLF2 luciferase activity in HCAECs under HBO. HBO and HBO-induced exosomes significantly increased cell proliferation and the capillary-like network formation of HCAECs. MALAT1 siRNA and miR-92a overexpression significantly attenuated the cell proliferation and tube formation caused by HBO-induced exosome. HBO and HBO-induced exosomes significantly improved neovascularization in a rat model of hind-limb ischemia. CONCLUSIONS: HBO upregulates MALAT1 to suppress miR-92a expression and counteracts the inhibitory effect of miR-92a on KLF2 expression in HCAECs to enhance neovascularization. HBO-induced derivation of exosomes from HCAECs enhances angiogenesis. Exosomes containing MALAT1 might serve as a valuable therapeutic tool for neovascularization by HBO.

Delivery of noncarrier naked DNA vaccine into the skin by supersonic flow induces a polarized T helper type 1 immune response to cancer.

BACKGROUND: DNA vaccine is a new and powerful approach to generate immunological responses against infectious disease and cancer. The T helper type (Th)1 immune response is usually required for generating effective anti-tumor responses. A microparticulate bombardment system can induce an immune response using very low amounts of DNA. Using nozzle aerodynamics, a low pressure gene gun has been developed to decrease the noise associated with high pressure gene guns. Particles are propelled by supersonic flow through this novel nozzle. To test whether this gun could inoculate a DNA vaccine that stimulates an anti-tumor Th1 immune response, we examined the effect of direct delivery of naked DNA (i.e. without any carrier) on the anti-tumor immune response of mice. METHODS: The luciferase reporter plasmid DNA was delivered using a low-pressure biolistic device and expressed in C3H/HeN, BALB/c, and C57BL/6 mice. RESULTS: Plasmid DNA expression was mainly in the epidermis. Noncarrier naked neu DNA vaccine and gold particle-coated neu DNA vaccine (at 1 microg per mouse) had similar anti-tumor effects in C3H mice. However, cytokine profile examination showed the Th1-bias of the response induced by naked DNA vaccine and the Th2-bias of the response induced by coated DNA vaccine. CONCLUSIONS: A shift in the immune response to favour enhanced tumor rejection can be achieved by skin delivery of naked DNA vaccine.

Spontaneous hemorrhagic angiomyolipoma present with massive hematuria leading to urgent nephrectomy.

Managing acute abdomen is a challenge for physicians in the emergency department. An immediate surgery is sometimes required for the hemorrhagic uropathy of renal angiomyolipoma. We present a case with left flank pain and findings of hematuria and pyuria. Abdominal and pelvic computed tomographic scans showed a 3 x 5-cm, fat-containing tumor at the left lower pole of the kidney and a bleeding and perirenal hematoma in the left perirenal space. An urgent left nephrectomy was lifesaving for the postoperative finding of renal angiomyolipoma. It is important to consider the possibility of the presence of such a tumor when spontaneous retroperitoneal hemorrhage and hypovolemic shock are encountered.