Hwan Myung Lee

3-Morpholinosydnonimine participates in the attenuation of neointima formation via inhibition of annexin A2-mediated vascular smooth muscle cell migration

3‐Morpholinosydnonimine (SIN‐1) affects vascular smooth muscle cell migration and proliferation, processes essential for atherosclerosis. However, the mechanism by which SIN‐1 exerts these effects has not been elucidated. We used 2‐DE followed by MALDI‐TOF/TOF MS to identify responses in protein expression to SIN‐1 in rat aortic smooth muscle. Platelet‐derived growth factor‐BB increased cell migration and proliferation in rat aortic smooth muscle cells, and subsequent SIN‐1 treatment inhibited it. Administration of SIN‐1 in vivo attenuated neointima formation in balloon‐injured rat carotid arteries. Proteomic analysis showed that glutathione peroxidase and 40S ribosomal protein S12 were differentially expressed in aortic strips exposed to SIN‐1. Expression of annexin A2 was decreased by SIN‐1. Platelet‐derived growth factor‐BB‐induced cell migration was increased and inhibited in rat aortic smooth muscle cells with overexpression and knockdown of annexin A2 gene, respectively. The expression of annexin A2 was increased in vascular neointima compared with the intact control, which was inhibited by SIN‐1 treatment. These results demonstrate that SIN‐1 may attenuate vascular neointima formation by inhibiting annexin A2‐mediated migration. Therefore, annexin A2 may be a potential target for therapeutic strategies for atherosclerosis.

Carvacrol inhibits atherosclerotic neointima formation by downregulating reactive oxygen species production in vascular smooth muscle cells

OBJECTIVE Carvacrol (2-methyl-5-(1-methylethyl) phenol), a cyclic monoterpene, exerts protective activities in a variety of pathological states including tumor growth, inflammation, and oxidative stress. However, it is unknown whether carvacrol affects events in vascular cells during the development of atherosclerotic neointima. We investigated the effects of carvacrol on the migration and proliferation of rat aortic smooth muscle cells (RASMCs) and on vascular neointima formation. METHODS AND RESULTS Carvacrol significantly inhibited platelet-derived growth factor (PDGF)-BB-stimulated RASMC migration and proliferation in a concentration-dependent manner. Cell viability was not affected by treatment with carvacrol. Carvacrol attenuated the expression of NADPH oxidase (NOX) 1 and the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1/2 in response to PDGF-BB. Moreover, carvacrol suppressed the PDGF-BB-stimulated generation of H2O2 and inhibited the activity of NOX in RASMCs. Treatment with carvacrol inhibited PDGF-BB-induced aortic sprout outgrowth, balloon injury-evoked vascular neointima formation, and expression of proliferating cell nuclear antigen in the neointima. CONCLUSION These findings indicate that carvacrol inhibits migration and proliferation of RASMCs by suppressing the reactive oxygen species-mediated MAPK signaling pathway in these cells, thereby attenuating vascular neointimal formation. Carvacrol may be a promising agent for preventing vascular restenosis or atherosclerosis.

DJ-1/park7 modulates vasorelaxation and blood pressure via epigenetic modification of endothelial nitric oxide synthase

AIMS DJ-1/park7, a multifunctional protein, may play essential roles in the vascular system. However, the function of DJ-1/park7 in vascular contractility has remained unclear. The present study was designed to investigate whether the DJ-1/park7 is involved in the regulation of vascular contractility and systolic blood pressure (SBP). METHODS AND RESULTS Norepinephrine (NE) elevated contraction in endothelium-intact vessels in a dose-dependent manner, to a greater extent in DJ-1/park7 knockout (DJ-1/park7(-/-)) mice than in wild-type (DJ-1/park7(+/+)) mice. Acetylcholine inhibited NE-evoked contraction in endothelium-intact vessels, and this was markedly impaired in DJ-1/park7(-/-) mice compared with DJ-1/park7(+/+). Nitric oxide (NO) production (82.1 ± 2.8% of control) and endothelial NO synthase (eNOS) expression (61.7 ± 8.9%) were lower, but H2O2 production (126.4 ± 8.6%) was higher, in endothelial cells from DJ-1/park7(-/-) mice than in those from DJ-1/park7(+/+) controls; these effects were reversed by DJ-1/park7-overexpressing endothelial cells from DJ-1/park7(-/-) mice. Histone deacetylase (HDAC)-1 recruitment and H3 histone acetylation at the eNOS promoter were elevated and diminished, respectively, in DJ-1/park7(-/-) mice compared with DJ-1/park7(+/+) controls. Moreover, SBP was significantly elevated in DJ-1/park7(-/-) mice compared with DJ-1/park7(+/+) controls, but this elevation was inhibited in mice treated with valproic acid, an inhibitor of Class I HDACs including HDAC-1. CONCLUSION These results demonstrate that DJ-1/park7 protein may be implicated in the regulation of vascular contractility and blood pressure, probably by the impairment of NO production through H2O2-mediated epigenetic inhibition of eNOS expression.

Effects of gintonin on the proliferation, migration, and tube formation of human umbilical-vein endothelial cells: involvement of lysophosphatidic-acid receptors and vascular-endothelial-growth-factor signaling

Background Ginseng extracts are known to have angiogenic effects. However, to date, only limited information is available on the molecular mechanism underlying the angiogenic effects and the main components of ginseng that exert these effects. Human umbilical-vein endothelial cells (HUVECs) are used as an in vitro model for screening therapeutic agents that promote angiogenesis and wound healing. We recently isolated gintonin, a novel ginseng-derived lysophosphatidic acid (LPA) receptor ligand, from ginseng. LPA plays a key role in angiogenesis and wound healing. Methods In the present study, we investigated the in vitro effects of gintonin on proliferation, migration, and tube formation of HUVECs, which express endogenous LPA1/3 receptors. Results Gintonin stimulated proliferation and migration of HUVECs. The LPA1/3 receptor antagonist, Ki16425, short interfering RNA against LPA1 or LPA3 receptor, and the Rho kinase inhibitor, Y-27632, significantly decreased the gintonin-induced proliferation, migration, and tube formation of HUVECs, which indicates the involvement of LPA receptors and Rho kinase activation. Further, gintonin increased the release of vascular endothelial growth factors from HUVECs. The cyclooxygenase-2 inhibitor NS-398, nuclear factor kappa B inhibitor BAY11-7085, and c-Jun N-terminal kinase inhibitor SP600125 blocked the gintonin-induced migration, which shows the involvement of cyclooxygenase-2, nuclear factor kappa B, and c-Jun N-terminal kinase signaling. Conclusion The gintonin-mediated proliferation, migration, and vascular-endothelial-growth-factor release in HUVECs via LPA-receptor activation may be one of in vitro mechanisms underlying ginseng-induced angiogenic and wound-healing effects.

Olibanum Extract Inhibits Vascular Smooth Muscle Cell Migration and Proliferation in Response to Platelet-Derived Growth Factor

Olibanum (Boswellia serrata) has been shown to have anti-inflammatory, anti-arthritic and anti-cancer effects. This study determined the role of a water extract of olibanum in platelet-derived growth factor (PDGF)-stimulated proliferation and migration of rat aortic smooth muscle cells (RASMCs). PDGF-BB induced the migration and proliferation of RASMCs that were inhibited by olibanum extract in a dose-dependent manner. The PDGF-BB-increased phosphorylation of p38 mitogen-activated protein kinase (MAPK); the heat shock protein (Hsp) 27 was significantly inhibited by the olibanum extract. The effects of PDGF-BB-induced extracellular signal-regulated kinase1/2 was not altered by the olibanum extract. Treatment with olibanum extract inhibited PDGF-BB-stimulated sprout out growth of aortic rings. These results suggest that the water extract of olibanum inhibits PDGF-BB-stimulated migration and proliferation in RASMCs as well as sprout out growth, which may be mediated by the inhibition of the p38 MAPK and Hsp27 pathways.

Chrysanthemum boreale Makino essential oil induces keratinocyte proliferation and skin regeneration

We investigated the effect of essential oil from the flower of Chrysanthemum boreale Makino (CBMEO) on growth of human keratinocytes (HaCaTs) and explored a possible mechanism for this response. CBMEO was extracted using the steam distillation method. CBMEO contained a total of 33 compounds. CBMEO stimulated HaCaT proliferation (EC50, 0.028 μg/mL) and also induced phosphorylation of Akt and ERK1/2 in HaCaTs (EC50, 0.007 and 0.005 μg/mL, for phosphorylated Akt and ERK1/2, respectively). Moreover, CBMEO promoted wound closure in the dorsal side skin of rat tail. This study demonstrated that CBMEO can stimulate growth of human skin keratinocytes, probably through the Akt and ERK1/2 pathways. Therefore, CBMEO may be helpful in skin regeneration and wound healing in human skin, and may also be a possible cosmetic material for skin beauty.

The serum protein fetuin-B is involved in the development of acute myocardial infarction.

The rupture of an atherosclerotic plaque is one of the main causes of coronary artery thrombotic occlusion, leading to myocardial infarction. However, the exact mechanism and causal risk factors for plaque rupture remain unclear. To identify a potential molecule that can influence atherosclerotic plaque rupture, we investigated protein expression in serum from patients with acute myocardial infarction (AMI) and stable angina (SA), using proteomic analysis. The expression of six proteins, including fibrinogen, fetuin-B, keratin 9, proapolipoprotein and fibrinogen, were altered in serum from patients with AMI compared with serum from those with SA. Of these, fetuin-B, proapolipoprotein, fibrinogen γ-B-chain precursors and fibrinogen expression were greater in serum from patients with AMI than from patients with SA. Increased fetuin-B expression in serum from AMI patients was also confirmed by Western blot analysis. Treatment with recombinant human fetuin-B increased the migration in monocytes and macrophages in a concentration-dependent manner. Fetuin-B also affected vascular plaque-stabilizing factors, including lipid deposition and cytokine production in macrophages, the activation of matrix metalloproteinase (MMP)-2 in monocytes, and the activation of apoptosis and MMP-2 in vascular smooth muscle cells. Moreover, in vivo administration of fetuin-B decreased the collagen accumulation and smooth muscle cell content and showed an increased number of macrophages in the vascular plaque. From these results, we suggest that fetuin-B may act as a modulator in the development of AMI. This study may provide a therapeutic advantage for patients at high risk of AMI.

DJ-1 regulates the expression of renal (pro)renin receptor via reactive oxygen species-mediated epigenetic modification

BACKGROUND DJ-1 protein plays multifunctional roles including transcriptional regulation and scavenging oxidative stress; thus, it may be associated with the development of renal disorders. We investigated whether DJ-1 protein regulates the expression of (pro)renin receptor (PRR), a newly identified member of renin-angiotensin system. METHODS The levels of mRNA and protein were determined by real-time PCR and western blot, respectively. H2O2 production was tested by using fluorescence probe. Histone modification was determined by chromatin immunoprecipitation. RESULTS The expression of PRR was significantly higher in the kidney from DJ-1 knockout mice (DJ-1-/-) compared with wild-type mice (DJ-1+/+). Histone deacetylase 1 recruitment at the PRR promoter was lower, and histone H3 acetylation and RNA polymerase II recruitment were higher in DJ-1-/- than in DJ-1+/+. Knockdown or inhibition of histone deacetylase 1 restored PRR expression in mesangial cells from DJ-1+/+. H2O2 production was greater in DJ-1-/- cells compared with DJ-1+/+ cells. These changes in PRR expression and epigenetic modification in DJ-1-/- cells were induced by H2O2 treatment and reversed completely by addition of an antioxidant reagent. Prorenin-stimulated ERK1/2 phosphorylation was greater in DJ-1-/- than in DJ-1+/+ cells and this was inhibited by a PRR-inhibitory peptide, and by AT1 and AT2 receptor inhibitors. The expression of renal fibrotic genes was higher in DJ-1-/- than in DJ-1+/+ cells and decreased in PRR-knockdown DJ-1-/- cells. CONCLUSIONS We conclude that DJ-1 protein regulates the expression of renal PRR through H2O2-mediated epigenetic modification. GENERAL SIGNIFICANCE We suggest that renal DJ-1 protein may be an important molecule in the acceleration of renal pathogenesis through PRR regulation.

Ketoconazole induces apoptosis in rat cardiomyocytes through reactive oxygen species-mediated parkin overexpression.

Azole antifungals such as ketoconazole are generally known to induce a variety of heart function side effects, e.g., long-QT syndrome and ventricular arrhythmias. However, a clear mechanism for the action of ketoconazole in heart cells has not been reported. In the present study, we assessed the correlation between ketoconazole-induced apoptosis and the alteration of genes in response to ketoconazole in rat cardiomyocytes. Cardiomyocyte viability was significantly inhibited by treatment with ketoconazole. Ketoconazole also stimulated H2O2 generation and TUNEL-positive apoptosis in a dose-dependent manner. DNA microarray technology revealed that 10,571 genes were differentially expressed by more than threefold in ketoconazole-exposed cardiomyocytes compared with untreated controls. Among these genes, parkin, which encodes a component of the multiprotein E3 ubiquitin ligase complex, was predominantly overexpressed among those classified as apoptosis- and reactive oxygen species (ROS)-related genes. The expression of parkin was also elevated in cardiomyocytes treated with exogenous H2O2. Moreover, cell viability and apoptosis in response to ketoconazole were inhibited in cardiomyocytes treated with ROS inhibitors and transfected with parkin siRNA. From the present findings, we concluded that ketoconazole may increase the expression of parkin via the ROS-mediated pathway, which consequently results in the apoptosis and decreased viability of cardiomyocytes.

Effect of absolute from Hibiscus syriacus L. flower on wound healing in keratinocytes

Background: Proliferation and migration of keratinocytes are essential for the repair of cutaneous wounds. Hibiscus syriacus L. has been used in Asian medicine; however, research on keratinocytes is inadequate. Objective: To establish the dermatological properties of absolute from Hibiscus syriacus L. flower (HSF) and to provide fundamental research for alternative medicine. Materials and Methods: We identified the composition of HSF absolute using gas chromatography-mass spectrometry analysis. We also examined the effect of HSF absolute in HaCaT cells using the XTT assay, Boyden chamber assay, sprout-out growth assay, and western blotting. We conducted an in-vivo wound healing assay in rat tail-skin. Results: Ten major active compounds were identified from HSF absolute. As determined by the XTT assay, Boyden chamber assay, and sprout-out growth assay results, HSF absolute exhibited similar effects as that of epidermal growth factor on the proliferation and migration patterns of keratinocytes (HaCaT cells), which were significantly increased after HSF absolute treatment. The expression levels of the phosphorylated signaling proteins relevant to proliferation, including extracellular signal-regulated kinase 1/2 (Erk 1/2) and Akt, were also determined by western blot analysis. Conclusion: These results of our in-vitro and ex-vivo studies indicate that HSF absolute induced cell growth and migration of HaCaT cells by phosphorylating both Erk 1/2 and Akt. Moreover, we confirmed the wound-healing effect of HSF on injury of the rat tail-skin. Therefore, our results suggest that HSF absolute is promising for use in cosmetics and alternative medicine. Abbreviations used: HSF: Hibiscus syriacus L. flower, Erk 1/2: extracellular signal-regulated kinase 1/2, EGF: epidermal growth factor, GC/MS: gas chromatography-mass spectrometry, DMEM: dulbeccos modified eagle medium, FBS: fetal bovine serum, BSA: bovine serum albumin, p-Akt: phosphorylation of Akt, p-Erk 1/2: phosphorylation of Erk 1/2