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.

(-)-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.

Hyperbaric oxygen activates visfatin expression and angiogenesis via angiotensin II and JNK pathway in hypoxic human coronary artery endothelial cells.

Visfatin is an adipocytokine with important roles in endothelial angiogenesis. Hyperbaric oxygen (HBO) has been widely used to treat various medical illness with enhanced angiogenesis. The molecular effects of HBO on visfatin under hypoxia are poorly understood. This study aimed to investigate the effect of HBO on visfatin in hypoxic human coronary arterial endothelial cells (HCAECs). HCAECs under chemical hypoxia (antimycin A, 0.01 mmol/L) were exposed to HBO (2.5 atmosphere absolute; ATA) for 2-4 hours. Western blot, real-time polymerase chain reaction, electrophoretic mobility shift assay, luciferase promoter activity, migration and tube formation assay, and in vitro glucose uptake were measured. Visfatin protein expression increased in hypoxic HCAECs with earlier angiotensin II (AngII) secretion and c-Jun N-terminal kinase (JNK) phosphorylation, which could be effectively suppressed by the JNK inhibitor (SP600125), AngII antibody or AngII receptor blocker (losartan). In hypoxic HCAECs, HBO further induced earlier expression of visfatin and AngII. Hypoxia significantly increased DNA-protein binding activity of hypoxia-inducible factor-1α (HIF-1α) and visfatin. Hypoxia, hypoxia with HBO and exogenous addition of AngII also increased promoter transcription to visfatin; SP600125 and losartan blocked this activity. In HCAECs, glucose uptake, migration and tube formation were increased in the presence of hypoxia with HBO, but were inhibited by visfatin small interfering RNA, SP600125 and losartan. In conclusion, HBO activates visfatin expression and angiogenesis in hypoxic HCAECs, an effect mediated by AngII, mainly through the JNK pathway.

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.

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.

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.

Hyperbaric oxygen activates discoidin domain receptor 2 via tumour necrosis factor-alpha and the p38 MAPK pathway to increase vascular smooth muscle cell migration through matrix metalloproteinase 2.

DDR2 (discoidin domain receptor 2) regulates collagen turnover mediated by SMCs (smooth muscle cells) in atherosclerosis. HBO (hyperbaric oxygen) has been used in medical practice; however, the molecular mechanism of the beneficial effects of HBO is poorly understood. Furthermore, the effect of HBO on DDR2 has not been reported previously. In the present study, we investigated the cellular and molecular mechanisms of DDR2 regulation by HBO in VSMCs (vascular SMCs). Cells were exposed to 2.5 ATA (atmosphere absolute) of oxygen in a hyperbaric chamber. DDR2 protein (3.63-fold) and mRNA (2.34-fold) expression were significantly increased after exposure to 2.5 ATA HBO for 1 h. Addition of SB203580 and p38 MAPK (mitogen-activated protein kinase) siRNA (small interfering RNA) 30 min before HBO inhibited the induction of DDR2 protein. HBO also significantly increased DNA-protein binding activity of Myc/Max. Addition of SB203580 and an anti-TNF-alpha (tumour necrosis factor-alpha) monoclonal antibody 30 min before HBO abolished the DNA-protein binding activity induced by HBO. HBO significantly increased the secretion of TNF-alpha from cultured VSMCs. Exogenous addition of TNF-alpha significantly increased DDR2 protein expression, whereas anti-TNF-alpha and anti-(TNF-alpha receptor) antibodies blocked the induction of DDR2 protein expression. HBO significantly increased VSMC migration and proliferation, whereas DDR2 siRNA inhibited the migration induced by HBO. HBO increased activated MMP2 (matrix metalloproteinase 2) protein expression, and DDR2 siRNA abolished the induction of activated MMP2 expression induced by HBO. In conclusion, HBO activates DDR2 expression in cultured rat VSMCs. HBO-induced DDR2 is mediated by TNF-alpha and at least in part through the p38 MAPK and Myc pathways.

Evodiamine inhibits in vitro angiogenesis: Implication for antitumorgenicity.

Evodiamine, the major bioactive compound isolated from Chinese herbal drug named Wu-Chu-Yu, has been reported to exhibit anti-tumor growth and metastasis. However, the effect of evodiamine on angiogenesis remains to be investigated. We used the fresh medium containing evodiamine or human lung adenocarcinoma cell (CL1 cells) derived conditioned media free of evodiamine to test their capability to induce in vitro angiogenesis, i.e., human umbilical vein endothelial cells (HUVECs) tube formation and invasion. We demonstrated that evodiamine could directly inhibit in vitro HUVECs tube formation and invasion. Locally administered evodiamine also inhibited the in vivo angiogenesis in the chick embryo chorioallantoic membrane (CAM) assay. The gene expression of vascular endothelial growth factor (VEGF) and the p44/p42 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells angiogenesis were inhibited by evodiamine. We found that the evodiamine-treated CL1 cells derived conditioned medium showed decreased VEGF release and reduced ability of inducing in vitro tube formation. After the collection of conditioned media, the VEGF expression of remaining CL1 cells were determined by Western analyses and revealed that evodiamine decreased VEGF expression. Moreover, administration of recombinant human VEGF(165) (rhVEGF(165)) induced tube formation and ERK phosphorylation by HUVECs, and partially attenuated inhibitory effect of evodiamine. From these results, we suggested that evodiamine is a potent inhibitor of angiogenesis. The mechanism might involve at least the inhibition of VEGF expression, probably through repression of ERK phosphorylation.

Hyperbaric oxygen induces VEGF expression through ERK, JNK and c-Jun/AP-1 activation in human umbilical vein endothelial cells.

Hyperbaric oxygen (HBO) is increasingly used in a number of areas of medical practice, such as selected problem infections and wounds. The beneficial effects of HBO in treating ischemia-related wounds may be mediated by stimulating angiogenesis. We sought to investigate VEGF, the main angiogenic regulator, regulated by HBO in human umbilical vein endothelial cells (HUVECs). In this study, we found that VEGF was up regulated both at mRNA and protein levels in HUVECs treated with HBO dose- and time-dependently. Since there are several AP-1 sites in the VEGF promoter, and the c-Jun/AP-1 is activated through stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and extracellular signal regulated kinase (ERK), we further examined the c-Jun, JNK and ERK that might be involved in the VEGF induced by HBO. The VEGF mRNA induced by HBO was blocked by both PD98059 and SP600125, the ERK and JNK inhibitors respectively. HBO induced phospho-ERK and phospho-JNK expressions within 15 min. We further demonstrated that c-Jun phosphorylation was induced within 60 min of HBO treatment. HBO also induced the nuclear AP-1 binding ability within 30-60 min, but the AP-1 induction was blocked by treatment with either the ERK or JNK inhibitor. To verify that the VEGF expression induced by HBO is through the AP-1 trans-activation and VEGF promoter, both the VEGF promoter and AP-1 driving luciferase activity were found increased by the cells treated with HBO. The c-Jun mRNA, which is also driven by AP-1, was also induced by HBO, and the induction of c-Jun was blocked by ERK and JNK inhibitors. We suggest that VEGF induced by HBO is through c-Jun/AP-1 activation, and through simultaneous activation of ERK and JNK pathways.

Saikosaponin C induces endothelial cells growth, migration and capillary tube formation.

Saikosaponin C is one of the saikosaponins that are consisted in a Chinese herb, Radix Bupleuri. Recently, saikosaponins have been reported to have properties of cell growth inhibition, inducing cancer cells differentiation and apoptosis. However, saikosaponin C had no correlation with cell growth inhibition. In this study, we investigated the role of saikosaponin C on the growth of endothelial cells and angiogenesis. We found that saikosaponin C yielded a potent effect on inducing human umbilical vein endothelial cells (HUVECs) viability and growth. In addition to inducing endothelial cells growth, saikosaponin C also induced endothelial cells migration and capillary tube formation. The gene expression or activation of matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF) and the p42/p44 mitogen-activated protein kinase (MAPK, ERK) that correlated with endothelial cells growth, migration and angiogenesis were also induced by saikosaponin C. From these results, we suggest that saikosaponin C may have the potential for therapeutic angiogenesis but is not suitable for cancer therapy.

Hyperbaric oxygen selectively induces angiopoietin-2 in human umbilical vein endothelial cells.

One of the biological effects of hyperbaric oxygen (HBO) therapy in enhancing ischemia-related wound healing is the induction of angiogenesis. To elucidate the mechanism(s) underlying the HBO-induced angiogenesis, we studied the expression of several angiogenesis-related genes in human umbilical vein endothelial cells exposed to HBO. Western blot analyses showed that HBO enhanced the expression of angiopoietin-2 (Ang2) with no effect on the expression of Tie2, angiopoietin-1, and VEGF. The induction of Ang2 was further confirmed by immunohistochemistry, quantitative PCR, and Northern blot analyses. Inhibition of endothelial nitric oxide synthase blocked the HBO-induced Ang2 expression, but failed to block hypoxia-induced Ang2 expression. These data indicated that HBO-induced Ang2 expression may be through transcriptional stimulation, and requires the nitric oxide signaling pathway, which may play an important role in HBO-induced angiogenesis.