Seung Hyo Jung

DJ-1/park7 protects against neointimal formation via the inhibition of vascular smooth muscle cell growth

AIMS DJ-1/park7 is a ubiquitously expressed multifunctional protein that plays essential roles in a variety of cells. However, its function in the vascular system has not been determined. We investigated the protective roles of DJ-1/park7 in vascular disorders, especially in neointimal hyperplasia. METHODS AND RESULTS DJ-1/park7 was strongly expressed in the neointimal layer, in which its oxidized form was predominant. Treatment of vascular smooth muscle cells (VSMCs) from the mouse aorta with H(2)O(2) increased the oxidation of DJ-1/park7 visualized on two-dimensional electrophoresis gels. The growth of VSMCs in FBS-containing media and the release of H(2)O(2) were significantly increased in DJ-1/park7(-/-) knockout mice compared with DJ-1/park7(+/+) wild-type mice. The expression of cyclin D1 and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 were greater in VSMCs from the DJ-1/park7(-/-) aorta than from the DJ-1/park7(+/+) aorta. Both of these measures were inhibited by treatment with an ERK1/2 inhibitor or antioxidants and in DJ-1/park7-overexpressing cells. VSMC proliferation, cyclin D1 expression, and ERK1/2 phosphorylation in response to platelet-derived growth factor-BB were upregulated in DJ-1/park7(-/-) compared with DJ-1/park7(+/+) mice. VSMCs of DJ-1/park7(-/-) mice exhibited higher levels of sprout outgrowth of aortic strips and neointimal plaque formation elicited by carotid artery ligation compared with those of DJ-1/park7(+/+) mice. CONCLUSION These results indicate that DJ-1/park7 is involved in the growth of VSMCs, thereby inhibiting neointimal hyperplasia, and suggest that it might play protective roles in vascular remodelling.

Compound K, an intestinal metabolite of ginsenosides, inhibits PDGF-BB-induced VSMC proliferation and migration through G1 arrest and attenuates neointimal hyperplasia after arterial injury.

OBJECTIVE Compound K (CK), an intestinal metabolite of ginsenosides, has pharmacological properties such as anti-angiogenesis, anti-inflammation, anti-platelet and anti-cancer activities. In the present study, we investigated the inhibitory effect of CK on vascular smooth muscle cell (VSMC) proliferation and migration in vitro and neointima formation in a rat carotid artery injury model. RESULTS CK significantly inhibited both the proliferation and migration of PDGF-BB-stimulated VSMCs in a concentration-dependent manner. In accordance with these findings, CK blocked the PDGF-BB-induced progression of synchronized cells through the G0/G1 phase of the cell cycle. CK also decreased the expressions of cell cycle-related proteins, including cyclin-dependent kinase (CDK) 2, cyclin E, CDK4, cyclin D1, and proliferative cell nuclear antigen (PCNA) in response to PDGF. However, CK did not affect early signal transduction through PDGF-Rβ, Akt, ERK1/2 and PLC-γ1 phosphorylation. CK attenuated PDGF-BB-induced VSMC migration by inhibiting MMP-2 and MMP-9 expression. Furthermore, the CK-treated groups showed a significant reduction in neointima formation vs. the control group. Immunohistochemical staining demonstrated decreased expression of PCNA in the neointima of the CK-treated group. CONCLUSION Our findings demonstrated that CK was capable of suppressing the abnormal VSMC proliferation and migration. It suggested that CK can be a therapeutic agent to control pathologic cardiovascular conditions such as restenosis and atherosclerosis.

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.

DJ-1 protein regulates CD3+ T cell migration via overexpression of CXCR4 receptor

OBJECTIVE DJ-1-a multifunctional protein responding to oxidative stress-is a possible regulator of the inflammatory response that plays an important role in atherosclerosis. Stromal cell-derived factor (SDF)-1 and its receptor, chemokine receptor type 4 (CXCR4), have been implicated in the recruitment of inflammatory cells during atherosclerosis. Here we investigated the hypothesis that DJ-1 protein might participate in CD3+ T cell functions in response to SDF-1 and contribute to the pathogenesis of atherosclerosis. METHODS AND RESULTS SDF-1 stimulated migration in mouse CD3+ T cells in a dose-dependent manner. SDF-1 also elevated the phosphorylation level of extracellular-regulated kinase (ERK) 1/2 in CD3+ T cells. These SDF-1-induced responses were greater in CD3+ T cells from DJ-1 gene knockout (DJ-1(-/-)) mice than in those from wild type (DJ-1(+/+)) mice and were abolished by treatment with WZ811 and PD98059, inhibitors of CXCR4 and ERK1/2, respectively. Flow cytometry revealed that expression of the CXCR4 receptor was greater in CD3+ T cells from DJ-1(-/-) mice than in those from the controls. Moreover, expression of the CD3 protein was observed in the neointimal plaque from carotid artery-ligated mice and was stronger in DJ-1(-/-) mice compared with controls. The CD3+ T cell subsets, Th1 and Th17, showed increased production of interferon-γ and interleukin-17 in DJ-1(-/-) compared with DJ-1(+/+) mice. CONCLUSION DJ-1 protein is involved in the SDF-1-induced CD3+ T cell migration via overexpression of the CXCR4 receptor, and that DJ-1 acts as an inhibitory regulator in vascular remodeling such as neointima formation.

DJ-1 protects against undernutrition-induced atrophy through inhibition of the MAPK-ubiquitin ligase pathway in myoblasts.

AIMS The purpose of this study is to explore whether antioxidant DJ-1 protein affects the atrophy of skeletal muscle cell induced by undernutrition. MAIN METHODS To determine cell atrophic responses, L6 cell line and skeletal primary cells from mouse hind limbs were cultivated under condition of FBS-free and low glucose. Changes of protein expression were analyzed using Western blot. Overexpression and knockdown of DJ-1 was performed in cells to assess its influence on cell atrophic responses. KEY FINDINGS Undernutrition decreased cell size and increased the abundance of oxidized form and total form of DJ-1 protein in L6 myoblasts. The undernourished cells revealed an elevation in the expression of muscle-specific RING finger-1 (MuRF-1) and atrogin-1, and in the phosphorylations of p38 mitogen-activated protein kinase (MAPK) and stress-activated protein kinase/c-Jun N-terminal kinase compared with control groups. Moreover, DJ-1-knockout mice showed a decrease in cell size and an enhancement in the expression of MuRF-1 and atrogin-1, as well as in the phosphorylation of MAPKs in gastrocnemius muscles; these changes were also observed in L6 cells transfected with siRNA of DJ-1. On the other hand, L6 cells overexpressing full-length DJ-1 did not exhibit the alterations in cell size and ubiquitin ligases seen after undernourished states of control cells. Myotubes differentiated from L6 cells also showed elevated expression of MuRF-1 and atrogin-1 in response to undernutrition. SIGNIFICANCE These results suggest that DJ-1 protein may contribute to undernutrition-induced atrophy via MAPKs/ubiquitin ligase pathway in skeletal muscle cells.

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.