Publications by authors named "Byeong Hwa Jeon"

111 Publications

Downregulation of CR6-interacting factor 1 suppresses keloid fibroblast growth via the TGF-β/Smad signaling pathway.

Sci Rep 2021 Jan 12;11(1):500. Epub 2021 Jan 12.

Department of Medical Science, Chungnam National University, Daejeon, Republic of Korea.

Keloids are a type of aberrant skin scarring characterized by excessive accumulation of collagen and extracellular matrix (ECM), arising from uncontrolled wound healing responses. While typically non-pathogenic, keloids are occasionally regarded as a form of benign tumor. CR6-interacting factor 1 (CRIF1) is a well-known CR6/GADD45-interacting protein, that has both nuclear and mitochondrial functions, and also exerts regulatory effects on cell growth and apoptosis. In this study, cell proliferation, cell migration, collagen production and TGF-β signaling was compared between normal fibroblasts (NFs) and keloid fibroblasts (KFs). Subsequently, the effects of CRIF1 deficiency were investigated in both NFs and KFs. Cell proliferation, cell migration, collagen production and protein expressions of TGF-β, phosphorylation of Smad2 and Smad3 were all found to be higher in KFs compared to NFs. CRIF1 deficiency in NFs and KFs inhibited cell proliferation, migration, and collagen production. In addition, phosphorylation of Smad2 and Smad3, which are transcription factors of collagen, was decreased. In contrast, mRNA expression levels of Smad7 and SMURF2, two important inhibitory proteins of Smad2/3, were increased, suggesting that CRIF1 may regulate collagen production. CRIF1 deficiency decreases the proliferation and migration of KFs, thereby inhibiting their overgrowth via the transforming growth factor-β (TGF-β)/Smad pathway. CRIF1 may therefore represent a potential therapeutic target in keloid pathogenesis.
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http://dx.doi.org/10.1038/s41598-020-79785-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7804403PMC
January 2021

SIRT1 Activation Attenuates the Cardiac Dysfunction Induced by Endothelial Cell-Specific Deletion of CRIF1.

Biomedicines 2021 Jan 8;9(1). Epub 2021 Jan 8.

Department of Physiology and Medical Science, College of Medicine, Chungnam National University, Daejeon 301-747, Korea.

The CR6-interacting factor1 (CRIF1) mitochondrial protein is indispensable for peptide synthesis and oxidative phosphorylation. Cardiomyocyte-specific deletion of CRIF1 showed impaired mitochondrial function and cardiomyopathy. We developed an endothelial cell-specific CRIF1 deletion mouse to ascertain whether dysfunctional endothelial CRIF1 influences cardiac function and is mediated by the antioxidant protein sirtuin 1 (SIRT1). We also examined the effect of the potent SIRT1 activator SRT1720 on cardiac dysfunction. Mice with endothelial cell-specific CRIF1 deletion showed an increased heart-to-body weight ratio, increased lethality, and markedly reduced fractional shortening of the left ventricle, resulting in severe cardiac dysfunction. Moreover, endothelial cell-specific CRIF1 deletion resulted in mitochondrial dysfunction, reduced ATP levels, inflammation, and excessive oxidative stress in heart tissues, associated with decreased SIRT1 expression. Intraperitoneal injection of SRT1720 ameliorated cardiac dysfunction by activating endothelial nitric oxide synthase, reducing oxidative stress, and inhibiting inflammation. Furthermore, the decreased endothelial junction-associated protein zonula occludens-1 in CRIF1-deleted mice was significantly recovered after SRT1720 treatment. Our results suggest that endothelial CRIF1 plays an important role in maintaining cardiac function, and that SIRT1 induction could be a therapeutic strategy for endothelial dysfunction-induced cardiac dysfunction.
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http://dx.doi.org/10.3390/biomedicines9010052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7827654PMC
January 2021

Ref-1 protects against FeCl-induced thrombosis and tissue factor expression the GSK3β-NF-κB pathway.

Korean J Physiol Pharmacol 2021 Jan;25(1):59-68

Department of Physiology and Medical Science, Chungnam National University School of Medicine, Daejeon 35015, Korea.

Arterial thrombosis and its associated diseases are considered to constitute a major healthcare problem. Arterial thrombosis, defined as blood clot formation in an artery that interrupts blood circulation, is associated with many cardiovascular diseases. Oxidative stress is one of many important factors that aggravates the pathophysiological process of arterial thrombosis. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ref-1) has a multifunctional role in cells that includes the regulation of oxidative stress and anti-inflammatory function. The aim of this study was to investigate the therapeutic effect of adenovirus-mediated Ref-1 overexpression on arterial thrombosis induced by 60% FeCl solution in rats. Blood flow was measured to detect the time to occlusion, thrombus formation was detected by hematoxylin and eosin staining, reactive oxygen species (ROS) levels were detected by high-performance liquid chromatography, and the expression of tissue factor and other proteins was detected by Western blot. FeCl aggravated thrombus formation in carotid arteries and reduced the time to artery occlusion. Ref-1 significantly delayed arterial obstruction the inhibition of thrombus formation, especially by downregulating tissue factor expression through the Akt-GSK3β-NF-κB signaling pathway. Ref1 also reduced the expression of vascular inflammation markers ICAM-1 and VCAM1, and reduced the level of ROS that contributed to thrombus formation. The results showed that adenovirus-mediated Ref-1 overexpression reduced thrombus formation in the rat carotid artery. In summary, Ref-1 overexpression had anti-thrombotic effects in a carotid artery thrombosis model and could be a target for the treatment of arterial thrombosis.
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http://dx.doi.org/10.4196/kjpp.2021.25.1.59DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756532PMC
January 2021

High Salt Intake Recruits Tonic Activation of NR2D Subunit-Containing Extrasynaptic NMDARs in Vasopressin Neurons.

J Neurosci 2021 Feb 10;41(6):1145-1156. Epub 2020 Dec 10.

Department of Physiology, Chungnam National University, Daejeon 35015, Republic of Korea

In addition to producing a classical excitatory postsynaptic current via activation of synaptic NMDA receptors (NMDARs), glutamate in the brain also induces a tonic NMDAR current () via activation of extrasynaptic NMDARs (eNMDARs). However, since Mg blocks NMDARs in nondepolarized neurons, the potential contribution of eNMDARs to the overall neuronal excitatory/inhibitory (E/I) balance remains unknown. Here, we demonstrate that chronic (7 d) salt loading (SL) recruited NR2D subunit-containing NMDARs to generate an Mg-resistant tonic in nondepolarized [ (holding potential) -70 mV] vasopressin (VP; but not oxytocin) supraoptic nucleus (SON) neurons in male rodents. Conversely, in euhydrated (EU) and 3 d SL mice, Mg-resistant tonic was not observed. Pharmacological and genetic intervention of NR2D subunits blocked the Mg-resistant tonic in VP neurons under SL conditions, while an NR2B antagonist unveiled Mg-sensitive tonic but not Mg-resistant tonic In the EU group VP neurons, an Mg-resistant tonic was not generated by increased ambient glutamate or treatment with coagonists (e.g., d-serine and glycine). Chronic SL significantly increased NR2D expression but not NR2B expression in the SON relative to the EU group or after 3 d under SL conditions. Finally, Mg-resistant tonic selectively upregulated neuronal excitability in VP neurons under SL conditions, independent of ionotropic GABAergic input. Our results indicate that the activation of NR2D-containing NMDARs constitutes a novel mechanism that generates an Mg-resistant tonic in nondepolarized VP neurons, thus causing an E/I balance shift in VP neurons to compensate for the hormonal demands imposed by a chronic osmotic challenge. The hypothalamic supraoptic nucleus (SON) consists of two different types of magnocellular neurosecretory cells (MNCs) that synthesize and release the following two peptide hormones: vasopressin (VP), which is necessary for regulation of fluid homeostasis; and oxytocin (OT), which plays a major role in lactation and parturition. NMDA receptors (NMDARs) play important roles in shaping neuronal firing patterns and hormone release from the SON MNCs in response to various physiological challenges. Our results show that prolonged (7 d) salt loading generated a Mg-resistant tonic NMDA current mediated by NR2D subunit-containing receptors, which efficiently activated nondepolarized VP (but not OT) neurons. Our findings support the hypothesis that NR2D subunit-containing NMDARs play an important adaptive role in adult brain in response to a sustained osmotic challenge.
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http://dx.doi.org/10.1523/JNEUROSCI.1742-20.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7888215PMC
February 2021

Plasma APE1/Ref-1 Correlates with Atherosclerotic Inflammation in ApoE Mice.

Biomedicines 2020 Sep 21;8(9). Epub 2020 Sep 21.

Research Institute for Medical Sciences, College of Medicine, Chungnam National University, 266 Munhwa-ro, Jung-gu, Daejeon 35015, Korea.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is involved in DNA base repair and reducing activity. However, the role of APE1/Ref-1 in atherosclerosis is unclear. Herein, we investigated the role of APE1/Ref-1 in atherosclerotic apolipoprotein E (ApoE) mice fed with a Western-type diet. We found that serologic APE1/Ref-1 was strongly correlated with vascular inflammation in these mice. Neutrophil/lymphocyte ratio (NLR), endothelial cell/macrophage activation, and atherosclerotic plaque formation, reflected by atherosclerotic inflammation, were increased in the ApoE mice fed with a Western-type diet. APE1/Ref-1 expression was upregulated in aortic tissues of these mice, and was co-localized with cells positive for cluster of differentiation 31 (CD31) and galectin-3, suggesting endothelial cell/macrophage expression of APE1/Ref-1. Interestingly, APE1/Ref-1 plasma levels of ApoE mice fed with a Western-type diet were significantly increased compared with those of the mice fed with normal diet (15.76 ± 3.19 ng/mL vs. 3.51 ± 0.50 ng/mL, < 0.05), and were suppressed by atorvastatin administration. Correlation analysis showed high correlation between plasma APE1/Ref-1 levels and NLR, a marker of systemic inflammation. The cut-off value for APE1/Ref-1 for predicting atherosclerotic inflammation at 4.903 ng/mL showed sensitivity of 100% and specificity of 91%. We conclude that APE1/Ref-1 expression is upregulated in aortic endothelial cells/macrophages of atherosclerotic mice, and that plasma APE1/Ref-1 levels could predict atherosclerotic inflammation.
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http://dx.doi.org/10.3390/biomedicines8090366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555038PMC
September 2020

Effect of a new herbal composition comprised of red clover and hop extract on human endothelial cell damage and vasorelaxant activity.

J Food Biochem 2020 08 16;44(8):e13314. Epub 2020 Jun 16.

R&D Center, Naturetech Co., Ltd, 450-86, Maebong-Ro, Dongnam-Gu, Cheonan-Si, Chungnam, Republic of Korea.

Hormone replacement therapy may cause various side effects, including enhancing the risk of cardiovascular disease (CVD) in postmenopausal women. Here, we investigated the effect of red clover and hop extract combination (RHEC) on estrogen receptor (ER) binding and endothelial function of human umbilical vein endothelial cells (HUVECs) to develop an herbal agent for reducing the risk of CVDs. In ER competitor assay, RHEC showed binding affinity toward ERα and ERβ with IC50 values of 5.92 µg/ml and 1.66 µg/ml, respectively. In HUVECs, RHEC significantly increased the cell viability and reduced the reactive oxygen species production against oxidative stress-induced damage. We also showed that RHEC increased the NO production through upregulating the endothelial nitric oxide synthase expression via ER activation in estrogen depleted condition. In particular, RHEC showed greater efficacy with increase in NO and decrease in endothelin-1 than red clover or hop treatment alone. Additionally, 0.3-0.5 mg/ml of RHEC-induced vasorelaxation of rat aortic rings precontracted by phenylephrine. PRACTICAL APPLICATIONS: Recently, a large interest has grown in the synergistic effects of phytochemicals for better therapies to treat various diseases. Red clover and hop are well-known edible plants which are widely used to help relieve postmenopausal symptoms including CVD. However, their combination has not been studied so far. For the first time, we demonstrated that RHEC, a new herbal combination comprising the extracts from red clover and hop, appeared to be effective in protection of endothelial function against oxidative stress and estrogen depletion. Therefore, RHEC could be a potent herbal agent for reducing the risk of endothelial damage.
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http://dx.doi.org/10.1111/jfbc.13314DOI Listing
August 2020

Positive allosteric modulation of GABA receptors by a novel antiepileptic drug cenobamate.

Eur J Pharmacol 2020 Jul 20;879:173117. Epub 2020 Apr 20.

Department of Physiology, College of Medicine and Brain Research Institute, Chungnam National University, 6 Munhwa-ro, Jung-gu, Daejeon, 35015, Republic of Korea. Electronic address:

Cenobamate is a novel antiepileptic drug under investigation for use in patients with focal (partial-onset) seizures. To understand its potential molecular mechanism of action, the effects of cenobamate on GABA-mediated currents and GABA receptors in rodent hippocampal neurons were examined. Cenobamate potentiated GABA-induced currents (I) in acutely isolated CA3 pyramidal cells in a concentration-dependent manner (EC, 164 μM), which was not affected by flumazenil, a benzodiazepine receptor antagonist. Cenobamate enhanced tonic GABA currents (I), which is defined as a holding current shift by the GABA receptor antagonist bicuculline (EC, 36.63 μM). At therapeutically relevant concentrations, cenobamate induced minimal changes in the frequency, amplitudes, and decay time of spontaneous inhibitory postsynaptic currents in the CA1 neurons. Flumazenil failed to affect cenobamate-potentiated I and I in CA1 neurons. Cenobamate showed positive allosteric modulation of GABA-induced I mediated by GABA receptors. This effect was similar for all tested hGABA receptors containing six different alpha subunits (αβγ or αβγ), with EC values ranging from 42 to 194 μM. Cenobamate did not displace the binding of flunitrazepam, a benzodiazepine derivative, or flumazenil to GABA receptors. The results showed that cenobamate, a novel antiepileptic drug, acts as a positive allosteric modulator of high-affinity GABA receptors, activated by GABA at a site independent of the benzodiazepine binding site and efficiently enhances I inhibition in hippocampal neurons, which could be an underlying molecular mechanism stabilizing neural circuits of the epileptic hippocampus.
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http://dx.doi.org/10.1016/j.ejphar.2020.173117DOI Listing
July 2020

The Biological Role of Apurinic/Apyrimidinic Endonuclease1/Redox Factor-1 as a Therapeutic Target for Vascular Inflammation and as a Serologic Biomarker.

Biomedicines 2020 Mar 10;8(3). Epub 2020 Mar 10.

Research Institute for Medical Sciences, College of Medicine, Chungnam National University, 266 Munhwa-ro, Jung-gu, Daejeon 35015, Korea.

Endothelial dysfunction promotes vascular inflammation by inducing the production of reactive oxygen species and adhesion molecules. Vascular inflammation plays a key role in the pathogenesis of vascular diseases and atherosclerotic disorders. However, whether there is an endogenous system that can participate in circulating immune surveillance or managing a balance in homeostasis is unclear. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (henceforth referred to as APE1/Ref-1) is a multifunctional protein that can be secreted from cells. It functions as an apurinic/apyrimidinic endonuclease in the DNA base repair pathway and modulates redox status and several types of transcriptional factors, in addition to its anti-inflammatory activity. Recently, it was reported that the secretion of APE1/Ref-1 into the extracellular medium of cultured cells or its presence in the plasma can act as a serological biomarker for certain disorders. In this review, we summarize the possible biological functions of APE1/Ref-1 according to its subcellular localization or its extracellular secretions, as therapeutic targets for vascular inflammation and as a serologic biomarker.
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http://dx.doi.org/10.3390/biomedicines8030057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7148461PMC
March 2020

CR6 interacting factor 1 deficiency induces premature senescence via SIRT3 inhibition in endothelial cells.

Free Radic Biol Med 2020 04 25;150:161-171. Epub 2020 Feb 25.

Department of Physiology & Medical Science, Chungnam National University College of Medicine, Daejeon, 301-747, Republic of Korea. Electronic address:

Vascular endothelial cell senescence is an important cause of cardiac-related diseases. Mitochondrial reactive oxygen species (mtROS) have been implicated in cellular senescence and multiple cardiovascular disorders. CR6 interacting factor 1 (CRIF1) deficiency has been shown to increase mtROS via the inhibition of mitochondrial oxidative phosphorylation; however, the mechanisms by which mtROS regulates vascular endothelial senescence have not been thoroughly explored. The goal of this study was to investigate the effects of CRIF1 deficiency on endothelial senescence and to elucidate the underlying mechanisms. CRIF1 deficiency was shown to increase the activity of senescence-associated β-galactosidase along with increased expression of phosphorylated p53, p21, and p16 proteins. Cell cycle arrested in the G0/G1 phase were identified in CRIF1-deficient cells using the flow cytometry. Furthermore, CRIF1 deficiency was also shown to increase cellular senescence by reducing the expression of Sirtuin 3 (SIRT3) via ubiquitin-mediated degradation of transcription factors PGC1α and NRF2. Downregulation of CRIF1 also attenuated the function of mitochondrial antioxidant enzymes including manganese superoxide dismutase (MnSOD), Foxo3a, nicotinamide-adenine dinucleotide phosphate, and glutathione via the suppression of SIRT3. Interestingly, overexpression of SIRT3 in CRIF1-deficient endothelial cells not only reduced mtROS levels by elevating expression of the antioxidant enzyme MnSOD but also decreased the expression of cell senescence markers. Taken together, these results suggest that CRIF1 deficiency induces vascular endothelial cell senescence via ubiquitin-mediated degradation of the transcription coactivators PGC1α and NRF2, resulting in decreased expression of SIRT3.
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http://dx.doi.org/10.1016/j.freeradbiomed.2020.02.017DOI Listing
April 2020

CR6-interacting factor 1 deficiency reduces endothelial nitric oxide synthase activity by inhibiting biosynthesis of tetrahydrobiopterin.

Sci Rep 2020 01 21;10(1):842. Epub 2020 Jan 21.

Department of Physiology & Medical Science, School of Medicine, Chungnam National University, Daejeon, 301-747, Republic of Korea.

Downregulation of CR6 interacting factor 1 (CRIF1) has been reported to induce mitochondrial dysfunction, resulting in reduced activity of endothelial nitric oxide synthase (eNOS) and NO production in endothelial cells. Tetrahydrobiopterin (BH4) is an important cofactor in regulating the balance between NO (eNOS coupling) and superoxide production (eNOS uncoupling). However, whether the decreased eNOS and NO production in CRIF1-deficient cells is associated with relative BH4 deficiency-induced eNOS uncoupling remains completely unknown. Our results showed that CRIF1 deficiency increased eNOS uncoupling and depleted levels of total biopterin and BH4 by reducing the enzymes of BH4 biosynthesis (GCH-1, PTS, SPR, and DHFR) in vivo and vitro, respectively. Supplementation of CRIF1-deficient cells with BH4 significantly increased the recovery of Akt and eNOS phosphorylation and NO synthesis. In addition, scavenging ROS with MitoTEMPO treatment replenished BH4 levels by elevating levels of GCH-1, PTS, and SPR, but with no effect on the level of DHFR. Downregulation of DHFR synthesis regulators p16 or p21 in CRIF1-deficient cells partially recovered the DHFR expression. In summary, CRIF1 deficiency inhibited BH4 biosynthesis and exacerbated eNOS uncoupling. This resulted in reduced NO production and increased oxidative stress, which contributes to endothelial dysfunction and is involved in the pathogenesis of cardiovascular diseases.
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http://dx.doi.org/10.1038/s41598-020-57673-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6972730PMC
January 2020

Bestrophin1-mediated tonic GABA release from reactive astrocytes prevents the development of seizure-prone network in kainate-injected hippocampi.

Glia 2020 05 13;68(5):1065-1080. Epub 2019 Dec 13.

Department of Medical Science, College of Medicine and Brain Research Institute, Chungnam National University, Daejeon, Republic of Korea.

Tonic extrasynaptic GABA receptor (GABA R) activation is under the tight control of tonic GABA release from astrocytes to maintain the brain's excitation/inhibition (E/I) balance; any slight E/I balance disturbance can cause serious pathological conditions including epileptic seizures. However, the pathophysiological role of tonic GABA release from astrocytes has not been tested in epileptic seizures. Here, we report that pharmacological or genetic intervention of the GABA-permeable Bestrophin-1 (Best1) channel prevented the generation of tonic GABA inhibition, disinhibiting CA1 pyramidal neuronal firing and augmenting seizure susceptibility in kainic acid (KA)-induced epileptic mice. Astrocyte-specific Best1 over-expression in KA-injected Best1 knockout mice fully restored the generation of tonic GABA inhibition and effectively suppressed seizure susceptibility. We demonstrate for the first time that tonic GABA from reactive astrocytes strongly contributes to the compensatory shift of E/I balance in epileptic hippocampi, serving as a good therapeutic target against altered E/I balance in epileptic seizures.
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http://dx.doi.org/10.1002/glia.23762DOI Listing
May 2020

CRIF1 deficiency induced mitophagy via p66shc-regulated ROS in endothelial cells.

Biochem Biophys Res Commun 2020 02 2;522(4):869-875. Epub 2019 Dec 2.

Department of Physiology & Medical Science, College of Medicine, Chungnam National University, Daejeon, 301-747, Republic of Korea. Electronic address:

Inhibition of mitochondrial protein CR6 interacting factor 1 (CRIF1) disturbs mitochondrial function, depolarizes membrane potential, and increases reactive oxygen species (ROS) levels in endothelial cells. Impaired mitochondrial function accompanied by oxidative damage is a major contributor to the initiation of mitophagy. We hypothesized that CRIF1 deficiency-induced harmful effects may promote mitophagy, and explored the mechanism underlying this effect in human umbilical vein endothelial cells (HUVECs). Our results showed that CRIF1 downregulation not only induced the mitophagy-related markers LC3 (LC3-II/Ⅰ), PTEN-induced putative kinase 1 (PINK1) and parkin, but also stimulated redox enzyme p66shc expression. Scavenging mitochondrial ROS markedly blunted the CRIF1 deficiency-induced increase in p66shc expression. In addition, knockdown of p66shc inhibited the CRIF1 deletion-triggered mitochondrial ROS increase, membrane potential depolarization, and mitochondrial fusion. The restoration of mitochondrial dysfunction by p66shc downregulation also decreased CRIF1 deficiency-induced mitophagy, by elevating the levels of LC3-II/Ⅰ, PINK1 and parkin. These findings suggest that CRIF1 deficiency induces mitophagy via p66shc-regulated ROS in endothelial cells.
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http://dx.doi.org/10.1016/j.bbrc.2019.11.109DOI Listing
February 2020

Far-Infrared-Emitting Sericite Board Upregulates Endothelial Nitric Oxide Synthase Activity through Increasing Biosynthesis of Tetrahydrobiopterin in Endothelial Cells.

Evid Based Complement Alternat Med 2019 31;2019:1813282. Epub 2019 Oct 31.

Department of Physiology & Medical Science, School of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea.

Far-infrared ray (FIR) therapy has been reported to exert beneficial effects on cardiovascular function by elevating endothelial nitric oxide synthesis (eNOS) activity and nitric oxide (NO) production. Tetrahydrobiopterin (BH) is a key determinant of eNOS-dependent NO synthesis in vascular endothelial cells. However, whether BH synthesis is associated with the effects of FIR on eNOS/NO production has not yet been investigated. In this study, we investigated the effects of FIR on BH-dependent eNOS/NO production and vascular function. We used FIR-emitting sericite boards as an experimental material and placed human umbilical vein endothelial cells (HUVECs) and Sprague-Dawley rats on the boards with or without FIR irradiation and then evaluated vascular relaxation by detecting NO generation, BH synthesis, and Akt/eNOS activation. Our results showed that FIR radiation significantly enhanced Akt/eNOS phosphorylation and NO production in human endothelial cells and aorta tissues. FIR can also induce BH storage by elevating levels of enzymes (e.g., guanosine triphosphate cyclohydrolase-1, 6-pyruvoyl tetrahydrobiopterin synthase, sepiapterin reductase, and dihydrofolate reductase), which ultimately results in NO production. These results indicate that FIR upregulated eNOS-dependent NO generation via BH synthesis and Akt phosphorylation, which contributes to the regulation of vascular function. This might develop potential clinical application of FIR to treat vascular diseases by augmenting the BH/NO pathway.
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http://dx.doi.org/10.1155/2019/1813282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6875339PMC
October 2019

ATP Binding Cassette Transporter A1 is Involved in Extracellular Secretion of Acetylated APE1/Ref-1.

Int J Mol Sci 2019 Jun 28;20(13). Epub 2019 Jun 28.

Research Institute of Medical Sciences, Department of Physiology, College of Medicine, Chungnam National University, 266 Munhwa-ro, Jung-gu, Daejeon 35015, Korea.

Acetylation of nuclear apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is associated with its extracellular secretion, despite the lack of an N-terminal protein secretion signal. In this study, we investigated plasma membrane targeting and translocation of APE1/Ref-1 in HEK293T cells with enhanced acetylation. While APE1/Ref-1 targeting was not affected by inhibition of the endoplasmic reticulum/Golgi-dependent secretion, its secretion was reduced by inhibitors of ATP-binding cassette (ABC) transporters, and siRNA-mediated down-regulation of ABC transporter A1. The association between APE1/Ref-1 and ABCA1 transporter was confirmed by proximal ligation assay and immunoprecipitation experiments. An APE1/Ref-1 construct with mutated acetylation sites (K6/K7R) showed reduced co-localization with ABC transporter A1. Exposure of trichostatin A (TSA) induced the acetylation of APE1/Ref-1, which translocated into membrane fraction. Taken together, acetylation of APE1/Ref-1 is considered to be necessary for its extracellular targeting via non-classical secretory pathway using the ABCA1 transporter.
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http://dx.doi.org/10.3390/ijms20133178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6651529PMC
June 2019

The extracellular role of Ref-1 as anti-inflammatory function in lipopolysaccharide-induced septic mice.

Free Radic Biol Med 2019 08 15;139:16-23. Epub 2019 May 15.

Research Institute of Medical Sciences, Department of Physiology, School of Medicine, Chungnam National University, Daejeon, South Korea. Electronic address:

Apurinic/apyrimidinic endonuclease/redox factor-1 (Ref-1), a multifunctional protein secreted from stimulated cells, has been identified as a new serological biomarker. Despite recent reports on the role of Ref-1 in inflammation, the biological function of secreted Ref-1 remains unknown, especially in vivo. This study aimed to evaluate the possible roles of secreted Ref-1 in lipopolysaccharide-induced systemic inflammation in vivo. We generated a secretory Ref-1 adenoviral vector system, AdPPT-LS-Ref-1, by conjugation of preprotrypsin leading sequence (PPT-LS) with full-length Ref-1 sequences. Expression of tumor necrosis factor-α (TNF-α)-induced vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells and lipopolysaccharide (LPS)-induced cyclooxygenase-2 in Raw264.7 cells was inhibited by secretory Ref-1, and this inhibitory effect was abrogated following neutralization of Ref-1 with anti-Ref-1 antibody. Plasma Ref-1 levels following administration of AdPPT-LS-Ref-1 (2 × 10 ifu, i.p.) for 24 h were substantially higher than those recorded following administration of Adβgal (84.6 ± 7.2 ng/ml vs. 4.4 ± 1.5 ng/ml). Treatment with LPS (10 mg/kg, i.v. for 6 h) markedly increased VCAM-1 expression, cathepsin or myeloperoxidase activity, which were significantly suppressed by treatment with AdPPT-LS-Ref-1. Furthermore, LPS-induced cytokines, such as TNF-α, interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein 1, were significantly inhibited in AdPPT-LS-Ref-1-treated mice. However, LPS-induced myeloperoxidase activities were not suppressed by treatment with the redox mutant of secretory Ref-1, AdPPT-LS-Ref-1(C65A/C93A), or wild-type AdRef-1. Collectively, these results suggest that secreted Ref-1 has anti-inflammatory properties and that its redox cysteine residue is associated with the anti-inflammatory activity in vivo. Furthermore, our findings indicate that secretory Ref-1 may be useful as a therapeutic biomolecule against systemic inflammation.
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http://dx.doi.org/10.1016/j.freeradbiomed.2019.05.013DOI Listing
August 2019

Arginase II Contributes to the Ca/CaMKII/eNOS Axis by Regulating Ca Concentration Between the Cytosol and Mitochondria in a p32-Dependent Manner.

J Am Heart Assoc 2018 09;7(18):e009579

1 Department of Biology School of medicine Kangwon National University Chuncheon Korea.

Background Arginase II activity contributes to reciprocal regulation of endothelial nitric oxide synthase ( eNOS ). We tested the hypotheses that arginase II activity participates in the regulation of Ca/Ca/calmodulin-dependent kinase II / eNOS activation, and this process is dependent on mitochondrial p32. Methods and Results Downregulation of arginase II increased the concentration of cytosolic Ca ([Ca]c) and decreased mitochondrial Ca ([Ca]m) in microscopic and fluorescence-activated cell sorting analyses, resulting in augmented eNOS Ser1177 phosphorylation and decreased eNOS Thr495 phosphorylation through Ca/Ca/calmodulin-dependent kinase II . These changes were observed in human umbilical vein endothelial cells treated with small interfering RNA against p32 (sip32). Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, fluorescence immunoassay, and ion chromatography, inhibition of arginase II reduced the amount of spermine, a binding molecule, and the release of Ca from p32. In addition, arginase II gene knockdown using small interfering RNA and knockout arginase II -null mice resulted in reduced p32 protein level. In the aortas of wild-type mice, small interfering RNA against p32 induced eNOS Ser1177 phosphorylation and enhanced NO -dependent vasorelaxation. Arginase activity, p32 protein expression, spermine amount, and [Ca]m were increased in the aortas from apolipoprotein E (ApoE) mice fed a high-cholesterol diet, and intravenous administration of small interfering RNA against p32 restored Ca/Ca/calmodulin-dependent kinase II -dependent eNOS Ser1177 phosphorylation and improved endothelial dysfunction. The effects of arginase II downregulation were not associated with elevated NO production when tested in aortic endothelia from eNOS knockout mice. Conclusions These data demonstrate a novel function of arginase II in regulation of Ca-dependent eNOS phosphorylation. This novel mechanism drives arginase activation, mitochondrial dysfunction, endothelial dysfunction, and atherogenesis.
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http://dx.doi.org/10.1161/JAHA.118.009579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222941PMC
September 2018

Isocitrate dehydrogenase 2 deficiency induces endothelial inflammation via p66sh-mediated mitochondrial oxidative stress.

Biochem Biophys Res Commun 2018 09 30;503(3):1805-1811. Epub 2018 Jul 30.

Department of Physiology & Medical Science, School of Medicine, Chungnam National University, Daejeon, 301-747, Republic of Korea. Electronic address:

Isocitrate dehydrogenase 2 (IDH2) is an essential enzyme in the mitochondrial antioxidant system, which produces nicotinamide adenine dinucleotide phosphate, and thereby defends against oxidative stress. We have shown that IDH2 downregulation results in mitochondrial dysfunction and reactive oxygen species (ROS) generation in mouse endothelial cells. The redox enzyme p66shc is a key factor in regulating the level of ROS in endothelial cells. In this study, we hypothesized that IDH2 knockdown-induced mitochondrial dysfunction stimulates endothelial inflammation, which might be regulated by p66shc-mediated oxidative stress. Our results showed that IDH2 downregulation led to mitochondrial dysfunction by decreasing the expression of mitochondrial oxidative phosphorylation complexes I, II, and IV, reducing oxygen consumption, and depolarizing mitochondrial membrane potential in human umbilical vein endothelial cells (HUVECs). The dysfunction not only increased mitochondrial ROS levels but also activated p66shc expression in HUVECs and IDH2 knockout mice. IDH2 deficiency increased intercellular adhesion molecule (ICAM)-1 expression and mRNA levels of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, and interleukin [IL]-1β) in HUVECs. The mRNA expression of ICAM-1 in endothelial cells and plasma levels of TNF-α and IL-1β were also markedly elevated in IDH2 knockout mice. However, p66shc knockdown rescued IDH2 deficiency-induced mitochondrial ROS levels, monocyte adhesion, ICAM-1, TNF-α, and IL-1β expression in HUVECs. These findings suggest that IDH2 deficiency induced endothelial inflammation via p66shc-mediated mitochondrial oxidative stress.
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http://dx.doi.org/10.1016/j.bbrc.2018.07.117DOI Listing
September 2018

Therapeutic positioning of secretory acetylated APE1/Ref-1 requirement for suppression of tumor growth in triple-negative breast cancer in vivo.

Sci Rep 2018 06 7;8(1):8701. Epub 2018 Jun 7.

Research Institute of Medical Sciences, Department of Physiology, School of Medicine, Chungnam National University, Daejeon, 35015, South Korea.

Triple-negative breast cancer (TNBC) represents a relatively small proportion of all BCs but a relatively large proportion of BC-related death. Thus, more effective therapeutic strategies are needed for the management of TNBC. We demonstrated that the stimulation of apoptosis by the binding of secreted acetylated-apurinic apyrimidinic endonuclease 1/redox factor-1 (Ac-APE1/Ref-1) to the receptor for advanced glycation end products (RAGE) was essential for TNBC cell death in response to hyperacetylation. The aim of the present study was to assess the potential therapeutic efficacy of secretory Ac-APE1/Ref-1 in orthotopic TNBC xenografts in vivo. We found that hyperacetylation in xenografts caused secretion of Ac-APE1/Ref-1 into the blood, where the factor bound directly to RAGE in hyperacetylated tumor tissues. Hyperacetylation in the TNBC xenografts induced strong inhibition of tumor growth and development, leading to apoptotic cell death, accompanied by increased RAGE expression and generation of reactive oxygen species. Tissues exhibited markedly higher counts of apoptotic bodies, a reduced proliferation index, and reduced neovascularization compared with control tumors. Ac-APE1/Ref-1-stimulated apoptosis was markedly reduced in RAGE-knockdown tumors compared with RAGE-overexpressing tumors, even in the presence of hyperacetylation. The function of secreted Ac-APE1/Ref-1 was confirmed in other hyperacetylated TNBCs xenografts using BT-549 and MDA-MB-468 cells, demonstrating its relevance as an anti-cancer molecule.
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http://dx.doi.org/10.1038/s41598-018-27025-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5992149PMC
June 2018

Angiotensin II facilitates neointimal formation by increasing vascular smooth muscle cell migration: Involvement of APE/Ref-1-mediated overexpression of sphingosine-1-phosphate receptor 1.

Toxicol Appl Pharmacol 2018 05 30;347:45-53. Epub 2018 Mar 30.

Department of Medicine, School of Medicine, Konkuk University, Seoul 05029, Republic of Korea. Electronic address:

Angiotensin II (Ang II) is implicated in the development of cardiovascular disorders including hypertension and atherosclerosis. However, the role of Ang II in the interaction between apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) and sphingosine-1-phosphate (S1P) signals in relation to vascular disorders remains to be clarified. This study aimed to determine whether APE/Ref-1 plays a role in epigenetic regulation of the S1P receptor (S1PR) in response to Ang II in vascular smooth muscle cell (VSMC) migration and vascular neointima formation. Ang II augmented the expression of S1PR1 in aortic smooth muscle cells of Sprague Dawley rats (RASMCs), which was attenuated by Ang II receptor (AT) 1 inhibitors, antioxidants, and APE/Ref-1 knockdown with small interference RNA. Ang II stimulation produced HO, and exogenous HO elevated S1PR1 expression in RASMCs. Moreover, Ang II caused translocation of cytoplasmic APE/Ref-1 into the nucleus in RASMCs. H3 histone acetylation and APE/Ref-1 binding at the S1PR1 promoter were increased in RASMCs treated with Ang II. In addition, Ang II induced migration in RASMCs, which was suppressed by AT1 and S1PR1 inhibitors. The expression of S1PR1, and colocalization of APE/Ref-1 and acetylated histone H3 in vascular neointima, were greater in Ang II-infused rats compared with a control group. These findings demonstrate that Ang II stimulates the epigenetic regulation of S1PR1 expression via HO-mediated APE/Ref-1 translocation, which may consequently be involved in Ang II-induced VSMC migration and vascular neointima formation. Therefore, APE/Ref-1-mediated overexpression of S1PR1 may be implicated in the vascular dysfunction evoked by Ang II.
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http://dx.doi.org/10.1016/j.taap.2018.03.032DOI Listing
May 2018

Anthocyanin-Rich Extract from Red Chinese Cabbage Alleviates Vascular Inflammation in Endothelial Cells and Apo E Mice.

Int J Mol Sci 2018 Mar 12;19(3). Epub 2018 Mar 12.

Research Institute of Medical Sciences, Department of Physiology, School of Medicine, Chungnam National University, 266 Munhwa-ro, Jung-gu, Daejeon 35015, Korea.

Anthocyanins, the most prevalent flavonoids in red/purple fruits and vegetables, are known to improve immune responses and reduce chronic disease risks. In this study, the anti-inflammatory activities of an anthocyanin-rich extract from red Chinese cabbage (ArCC) were shown based on its inhibitory effects in cultured endothelial cells and hyperlipidemic apolipoprotein -deficient mice. ArCC treatment suppressed monocyte adhesion to tumor necrosis factor-α-stimulated endothelial cells. This was validated by ArCC's ability to downregulate the expression and transcription of endothelial adhesion molecules, determined by immunoblot and luciferase promoter assays, respectively. The regulation of adhesion molecules was accompanied by transcriptional inhibition of nuclear factor-κB, which restricted cytoplasmic localization as shown by immunocytochemistry. Administration of ArCC (150 or 300 mg/kg/day) inhibited aortic inflammation in hyperlipidemic apolipoprotein E-deficient mice, as shown by in vivo imaging. Immunohistochemistry and plasma analysis showed that the aortas from these mice exhibited markedly lower leukocyte infiltration, reduced plaque formation, and lower concentrations of blood inflammatory cytokines than those observed in the control mice. The results suggest that the consumption of anthocyanin-rich red Chinese cabbage is closely correlated with lowering the risk of vascular inflammatory diseases.
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http://dx.doi.org/10.3390/ijms19030816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877677PMC
March 2018

CR6 interacting factor 1 deficiency promotes endothelial inflammation by SIRT1 downregulation.

PLoS One 2018 23;13(2):e0192693. Epub 2018 Feb 23.

Department of physiology & Medical Science, School of Medicine, Chungnam National University, Daejeon, Republic of Korea.

Aims: CR6 interacting factor 1 (CRIF1) deficiency impairs mitochondrial oxidative phosphorylation complexes, contributing to increased mitochondrial and cellular reactive oxygen species (ROS) production. CRIF1 downregulation has also been revealed to decrease sirtuin 1 (SIRT1) expression and impair vascular function. Inhibition of SIRT1 disturbs oxidative energy metabolism and stimulates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-induced inflammation. Therefore, we hypothesized that both CRIF1 deficiency-induced mitochondrial ROS production and SIRT1 reduction play stimulatory roles in vascular inflammation.

Methods And Results: Plasma levels and mRNA expression of proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) were markedly elevated in endothelium-specific CRIF1-knockout mice and CRIF1-silenced endothelial cells, respectively. Moreover, CRIF1 deficiency-induced vascular adhesion molecule-1 (VCAM-1) expression was consistently attenuated by the antioxidant N-acetyl-cysteine and NF-κB inhibitor (BAY11). We next showed that siRNA-mediated CRIF1 downregulation markedly activated NF-κB. SIRT1 overexpression not only rescued CRIF1 deficiency-induced NF-κB activation but also decreased inflammatory cytokines (TNF-α, IL-1β, and IL-6) and VCAM-1 expression levels in endothelial cells.

Conclusions: These results strongly suggest that CRIF1 deficiency promotes endothelial cell inflammation by increasing VCAM-1 expression, elevating inflammatory cytokines levels, and activating the transcription factor NF-κB, all of which were inhibited by SIRT1 overexpression.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0192693PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5825004PMC
April 2018

Elevation of Serum APE1/Ref-1 in Experimental Murine Myocarditis.

Int J Mol Sci 2017 Dec 8;18(12). Epub 2017 Dec 8.

Division of Cardiology, Department of Internal Medicine, Chungnam National University Hospital, Chungnam National University School of Medicine, 282 Munhwa-ro, Jung-gu, Daejeon 35015, Korea.

Myocarditis is an inflammatory disease of the myocardium that causes cardiogenic shock and death. However, endomyocardial biopsy that is, the gold standard for a diagnosis is limited. Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1) is a multifunctional protein, which is involved in DNA-based excision repair pathway, and in redox signaling, its changes are observed in various cardiovascular diseases including hypertension and coronary artery disease. We analyzed serum APE1/Ref-1 in experimental murine myocarditis. To induce myocarditis, coxsackievirus B3 was injected intraperitoneally to BALB/c mice. The serum APE1/Ref-1, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and troponin I were measured. The histology and virus titers measurements were performed. The troponin I and inflammation were significantly elevated at day 3, peaked to day 7 and decreased at day 10. The NT-proBNP and virus titers were significantly peaked at day 3, and dropped at day 7 and 10. The serum APE1/Ref-1 was gradually raised and its elevation is still maintained until a later time, namely day 10. Also, its level was positively correlated with myocardial inflammation, reflecting severity of myocardial injury. We suggest that serum APE1/Ref-1 can be used to assess for myocardial injury in viral myocarditis without endomyocardial biopsy.
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http://dx.doi.org/10.3390/ijms18122664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751266PMC
December 2017

Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions.

J Biol Chem 2018 01 30;293(3):847-862. Epub 2017 Nov 30.

From the World Class Institute, Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology, 30 Yeongudanji-ro, Ochang, Cheongwon, Chungbuk 28116, Korea,

Elevated expression of human enhancer filamentation 1 (HEF1; also known as NEDD9 or Cas-L) is an essential stimulus for the metastatic process of various solid tumors. This process requires HEF1 localization to focal adhesions (FAs). Although the association of HEF1 with FAs is considered to play a role in cancer cell migration, the mechanism targeting HEF1 to FAs remains unclear. Moreover, up-regulation of Polo-like kinase 1 (Plk1) positively correlates with human cancer metastasis, yet how Plk1 deregulation promotes metastasis remains elusive. Here, we report that casein kinase 1δ (CK1δ) phosphorylates HEF1 at Ser-780 and Thr-804 and that these phosphorylation events promote a physical interaction between Plk1 and HEF1. We found that this interaction is critical for HEF1 translocation to FAs and for inducing migration of HeLa cells. Plk1-docking phosphoepitopes were mapped/confirmed in HEF1 by various methods, including X-ray crystallography, and mutated for functional analysis in HeLa cells. In summary, our results reveal the role of a phosphorylation-dependent HEF1-Plk1 complex in HEF1 translocation to FAs to induce cell migration. Our findings provide critical mechanistic insights into the HEF1-Plk1 complex-dependent localization of HEF1 to FAs underlying the metastatic process and may therefore contribute to the development of new cancer therapies.
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http://dx.doi.org/10.1074/jbc.M117.802587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777258PMC
January 2018

APE1/Ref-1 Inhibits Phosphate-Induced Calcification and Osteoblastic Phenotype Changes in Vascular Smooth Muscle Cells.

Int J Mol Sci 2017 Sep 25;18(10). Epub 2017 Sep 25.

Research Institute of Medical Sciences, Department of Physiology, School of Medicine, Chungnam National University, 266 Munhwa-ro, Jung-gu, Daejeon 35015, Korea.

Vascular calcification plays a role in the pathogenesis of atherosclerosis, diabetes, and chronic kidney disease; however, the role of apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) in inorganic phosphate (Pi)-induced vascular smooth muscle cell (VSMC) calcification remains unknown. In this study, we investigated the possible role of APE1/Ref-1 in Pi-induced VSMC calcification. We observed that Pi decreased endogenous APE1/Ref-1 expression and promoter activity in VSMCs, and that adenoviral overexpression of APE1/Ref-1 inhibited Pi-induced calcification in VSMCs and in an ex vivo organ culture of a rat aorta. However, a redox mutant of APE1/Ref-1(C65A/C93A) did not reduce Pi-induced calcification in VSMCs, suggesting APE1/Ref-1-mediated redox function against vascular calcification. Additionally, APE1/Ref-1 overexpression inhibited Pi-induced intracellular and mitochondrial reactive oxygen species production, and APE1/Ref-1 overexpression resulted in decreased Pi-induced lactate dehydrogenase activity, pro-apoptotic Bax levels, and increased anti-apoptotic Bcl-2 protein levels. Furthermore, APE1/Ref-1 inhibited Pi-induced osteoblastic differentiation associated with alkaline phosphatase activity and inhibited Pi-exposure-induced loss of the smooth muscle phenotype. Our findings provided valuable insights into the redox function of APE1/Ref-1 in preventing Pi-induced VSMC calcification by inhibiting oxidative stress and osteoblastic differentiation, resulting in prevention of altered osteoblastic phenotypes in VSMCs.
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http://dx.doi.org/10.3390/ijms18102053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5666735PMC
September 2017

Enhanced peripheral nerve regeneration through asymmetrically porous nerve guide conduit with nerve growth factor gradient.

J Biomed Mater Res A 2018 Jan 23;106(1):52-64. Epub 2017 Sep 23.

Department of Advanced Materials and Chemical Engineering, Hannam University, Daejeon, 34054, Republic of Korea.

In this study, we fabricated a nerve guide conduit (NGC) with nerve growth factor (NGF) gradient along the longitudinal direction by rolling a porous polycaprolactone membrane with NGF concentration gradient. The NGF immobilized on the membrane was continuously released for up to 35 days, and the released amount of the NGF from the membrane gradually increased from the proximal to distal NGF ends, which may allow a neurotrophic factor gradient in the tubular NGC for a sufficient period. From the in vitro cell culture experiment, it was observed that the PC12 cells sense the NGF concentration gradient on the membrane for the cell proliferation and differentiation. From the in vivo animal experiment using a long gap (20 mm) sciatic nerve defect model of rats, the NGC with NGF concentration gradient allowed more rapid nerve regeneration through the NGC than the NGC itself and NGC immobilized with uniformly distributed NGF. The NGC with NGF concentration gradient seems to be a promising strategy for the peripheral nerve regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 52-64, 2018.
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http://dx.doi.org/10.1002/jbm.a.36216DOI Listing
January 2018

Protein kinase C beta II upregulates intercellular adhesion molecule-1 via mitochondrial activation in cultured endothelial cells.

Korean J Physiol Pharmacol 2017 Jul 26;21(4):377-384. Epub 2017 Jun 26.

Research Institute for Medical Sciences, Department of Physiology, School of Medicine, Chungnam National University, Daejeon 35015, Korea.

Activation of protein kinase C (PKC) is closely linked with endothelial dysfunction. However, the effect of PKCβII on endothelial dysfunction has not been characterized in cultured endothelial cells. Here, using adenoviral PKCβII gene transfer and pharmacological inhibitors, the role of PKCβII on endothelial dysfucntion was investigated in cultured endothelial cells. Phorbol 12-myristate 13-acetate (PMA) increased reactive oxygen species (ROS), p66shc phosphorylation, intracellular adhesion molecule-1, and monocyte adhesion, which were inhibited by PKCβi (10 nM), a selective inhibitor of PKCβII. PMA increased the phosphorylation of CREB and manganese superoxide dismutase (MnSOD), which were also inhibited by PKCβi. Gene silencing of CREB inhibited PMA-induced MnSOD expression, suggesting that CREB plays a key role in MnSOD expression. Gene silencing of PKCβII inhibited PMA-induced mitochondrial ROS, MnSOD, and ICAM-1 expression. In contrast, overexpression of PKCβII using adenoviral PKCβII increased mitochondrial ROS, MnSOD, ICAM-1, and p66shc phosphorylation in cultured endothelial cells. Finally, PKCβII-induced ICAM-1 expression was inhibited by Mito-TEMPO, a mitochondrial ROS scavenger, suggesting the involvement of mitochondrial ROS in PKC-induced vascular inflammation. Taken together, the results suggest that PKCβII plays an important role in PMA-induced endothelial dysfunction, and that the inhibition of PKCβII-dependent p66shc signaling acts as a therapeutic target for vascular inflammatory diseases.
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http://dx.doi.org/10.4196/kjpp.2017.21.4.377DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5507776PMC
July 2017

Ethanol Extract of Brassica rapa ssp. pekinensis Suppresses Tumor Necrosis Factor-α-Induced Inflammatory Response in Human Umbilical Vein Endothelial Cells.

J Med Food 2017 May;20(5):511-518

1 Department of Physiology, BK21plus CNU Integrative Biomedical Education Initiative, School of Medicine, Chungnam National University , Daejeon, Korea.

Brassica rapa L. ssp. pekinensis, commonly known as Chinese cabbage, is a cruciferous vegetable traditionally consumed in east Asia. Although its habitual consumption could account for the low incidence of chronic vascular inflammation, the therapeutic and protective potential of phytochemicals derived from Chinese cabbage has been poorly studied. In this study, we identified the phenolic compounds, kaempferol and quercetin, from the ethanol extract of Chinese cabbage (EtCC). We show for the first time that EtCC contains effective phytochemicals that suppress tumor necrosis factor (TNF)-α-induced inflammatory response in human umbilical vein endothelial cells. The EtCC inhibited TNF-α-induced monocyte adhesion to endothelial cells in a dose-dependent manner. The antiadhesive activity of EtCC directly correlated with downregulation of expression and transcription of vascular cell adhesion molecule-1 (VCAM-1). It was caused by an Nrf-2-dependent mechanism, leading to activation of antioxidant responsive element-driven promoter. Taken together, these results suggest that EtCC inhibits the expression of TNF-α-induced adhesion molecules through the indirect transcriptional modulation of VCAM-1 in endothelial cells. In conclusion, regular consumption of vegetables containing dietary phytochemicals might be a potential therapeutic strategy to protect against various stresses, to prevent several pathological conditions, and to treat chronic vascular inflammation, such as atherosclerosis.
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http://dx.doi.org/10.1089/jmf.2016.3869DOI Listing
May 2017

Rg3-enriched Korean Red Ginseng enhances blood pressure stability in spontaneously hypertensive rats.

Integr Med Res 2016 Sep 3;5(3):223-229. Epub 2016 Jun 3.

Department of Physiology, BK21Plus CNU Integrative Biomedical Education Initiative, School of Medicine, Chungnam National University, Daejeon, Korea.

Background: Korean Red Ginseng () has been shown to exert antihypertensive effects. In particular, ginsenoside Rg3 is thought to be a potent modulator of vascular function. The present study was performed to examine the antihypertensive efficacy of Korean Red Ginseng (KRG) extract and Rg3-enriched KRG (REKRG) extract.

Methods: Spontaneously hypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs) were divided into six groups (WKY control, WKY-KRG, WKY-REKRG, SHR control, SHR-KRG, and SHR-REKRG), and systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured at the carotid artery, followed by injection of 3 mg/kg KRG or 3 mg/kg REKRG.

Results: REKRG treatment significantly decreased SBP and DBP 3 hours post-treatment in the SHR group compared with SHR control group. However, SBP and DBP were not significantly different in KRG-treated SHRs compared with control SHRs. REKRG treatment did not significantly alter SBP or DBP 3 hours post-treatment in the WKY group compared with WKY control group. Similarly, there were no differences in SBP or DBP with KRG treatment in the WKY group and WKY control group. Both KRG and REKRG increased endothelial nitric oxide synthase phosphorylation levels in the aorta, and the increases in endothelial nitric oxide synthase phosphorylation levels by REKRG treatment were higher than those with KRG treatment. Similarly, nitric oxide production in plasma from WKYs and SHRs was also increased by both KRG and REKRG.

Conclusion: These results suggest that REKRG has a more beneficial effect on blood pressure control than KRG in SHRs.
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http://dx.doi.org/10.1016/j.imr.2016.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5390432PMC
September 2016

ethanol extract inhibits monocyte adhesion to tumor necrosis factor-alpha-stimulated endothelial cells.

Integr Med Res 2016 Jun 31;5(2):131-139. Epub 2016 Mar 31.

Research Institute for Medical Sciences, Department of Physiology, School of Medicine, Chungnam National University, Daejeon, Korea.

Background: var. Rehder (UD) has long been used in traditional folk medicine in Asia. This study is designed to investigate the antiadhesive activity of the ethanol extract of UD (UDE) and its underlying mechanisms in cultured endothelial cells.

Methods: The dried root bark of UD was extracted with 80% (v/v) ethanol. The antiadhesive activity of the UDE was investigated in cultured human umbilical vein endothelial cells and human embryonic kidney epithelial 293T (HEK 293T) cells stably transfected with pGL3-vascular cell adhesion molecule (VCAM)-1-luc. Monocyte adhesion in endothelial cells was induced by tumor necrosis factor-alpha (TNF-α), and the protective effects of UDE on monocyte-endothelial cell adhesion, VCAM-1 expression, reactive oxygen species production, and nuclear factor-κB activity were determined.

Results: Exposure to UDE at a concentration of 3-30 μg/mL for 24 hours produced no detectable cytotoxicity in human umbilical vein endothelial cells, but it significantly inhibited TNF-α-induced monocyte adhesion and VCAM-1 expression. TNF-α treatment of HEK 293T/ cells resulted in increased luciferase activity of the VCAM-1 promoter, which was inhibited by treatment with UDE. Additionally, TNF-α-induced reactive oxygen species generation, nuclear translocation of nuclear factor-κB, and IκBα degradation in human umbilical vein endothelial cells were effectively reduced by treatment with 30 μg/mL of UDE.

Conclusion: Our results indicated that UDE treatment inhibited TNF-α-induced monocyte adhesion in endothelial cells, suggesting that UD may reduce vascular endothelial inflammation.
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http://dx.doi.org/10.1016/j.imr.2016.03.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381430PMC
June 2016

CR6-Interacting Factor 1 Deficiency Impairs Vascular Function by Inhibiting the Sirt1-Endothelial Nitric Oxide Synthase Pathway.

Antioxid Redox Signal 2017 08 24;27(4):234-249. Epub 2017 May 24.

1 Department of Physiology, School of Medicine, Chungnam National University , Daejeon, Republic of Korea.

Aims: Mitochondrial dysfunction has emerged as a major contributing factor to endothelial dysfunction and vascular disease, but the key mechanisms underlying mitochondrial dysfunction-induced endothelial dysfunction remain to be elucidated. In this study, we aim at determining whether mitochondrial dysfunction in endothelial cells plays a key role in vascular disease, by examining the phenotype of endothelial-specific CR6-interacting factor 1 (CRIF1) knockout mice. We also used siRNA-mediated downregulation of CRIF1 gene in the endothelial cells to study about the in vitro pathophysiological underlying mechanisms.

Results: Downregulation of CRIF1 in endothelial cells caused disturbances of mitochondrial oxidative phosphorylation complexes and membrane potential, leading to enhanced mitochondrial reactive oxygen species production. Gene silencing of CRIF1 results in decreased SIRT1 expression along with increased endothelial nitric oxide synthase (eNOS) acetylation, leading to reduced nitric oxide production both in vitro and in vivo. Endothelium-dependent vasorelaxation of aortic rings from CRIF1 knockout (KO) mice was considerably less than in wild-type mice, and it was partially recovered by Sirt1 overexpression in CRIF1 KO mice.

Innovation: Our results show for the first time a relationship between mitochondrial dysfunction and impaired vascular function induced in CRIF1 deficiency conditions and also the possible underlying pathway involved.

Conclusion: These findings indicate that CRIF1 plays an important role in maintaining mitochondrial and endothelial function through its effects on the SIRT1-eNOS pathway. Antioxid. Redox Signal. 27, 234-249.
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http://dx.doi.org/10.1089/ars.2016.6719DOI Listing
August 2017