Fetuin-B regulates vascular plaque rupture via TGF-ß receptor-mediated Smad pathway in vascular smooth muscle cells.

Fetuin-B is a serum protein linked to the regulation of physiological or pathophysiological events such as fertility, energy metabolism, and liver disease. Recently, fetuin-B has been reported to be involved in the modulation of the rupture of atherosclerotic plaques associated with acute myocardial infarction. However, the exact mechanism involved in the modulation of atherosclerotic plaque rupture event by fetuin-B is not fully elucidated yet. In the present study, we investigated whether fetuin-B could influence atherosclerotic plaque rupture through vascular smooth muscle cells (VSMCs). Immunoprecipitation assay using membrane proteins from VSMCs revealed that fetuin-B tightly bound to transforming growth factor-ß receptor (TGF-ßR). Fetuin-B treatment elevated TGF-ßR signals (e.g., phosphorylation of Smad2 and Smad3) in VSMCs. Fetuin-B also stimulated nuclear translocation of phosphorylated Smads. Phosphorylation of Smad and its nuclear translocation by treatment with fetuin-B were inhibited in VSMCs by treatment with SB431542, a selective inhibitor of TGF-ßR. Fetuin-B enhanced expression levels of plasminogen activator inhibitor-1 (PAI-1) and matrix metalloproteinase-2 (MMP-2) in VSMCs through its epigenetic modification including recruitments of both histone deacetylase 1 and RNA polymerase II. These epigenetic alterations in VSMCs were also inhibited by treatment with SB431542. In vivo administration of fetuin-B protein increased expression levels of PAI-1 and MMP-2 in the vascular plaque. However, these increases in expression were inhibited by the administration of SB43154. These results indicate that fetuin-B may modulate vascular plaque rupture by promoting expression of PAI-1 and MMP-2 in VSMCs via TGF-ßR-mediated Smad pathway.

A monoclonal antibody derived by inoculation of human umbilical vein endothelial cells is a potential inhibitor of acetylcholine receptor-linked vasorelaxation.

In a previous study, we produced antibodies from rats immunized with human umbilical vein endothelial cells (HUVECs) and determined the vascular function of the monoclonal antibodies. However, unanswered question remains still about their role in vascular function. The current study explored vasoreactivity, in particular, focusing on the vascular contractility of a functional antibody against proteins expressed on the plasma membrane of HUVECs developed in a previous study. Among the antibodies developed, A-7 significantly attenuated endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to sodium nitroprusside or histamine. In addition, the A-7 antibody did not affect norepinephrine-stimulated contraction in both endothelium-intact and -denuded aorta. Immunocytochemical and immunoblotting analyses showed that A-7 attenuated ACh-increased expression of ACh receptor on the plasma membrane of HUVECs. These findings suggest that the monoclonal A-7 antibody may act as an inhibitor of endothelium-dependent vasorelaxation, probably in part via downregulation of ACh receptor expression.

Sabinene Prevents Skeletal Muscle Atrophy by Inhibiting the MAPK-MuRF-1 Pathway in Rats.

Chrysanthemum boreale Makino essential oil (CBMEO) has diverse biological activities including a skin regenerating effect. However, its role in muscle atrophy remains unknown. This study explored the effects of CBMEO and its active ingredients on skeletal muscle atrophy using in vitro and in vivo models of muscle atrophy. CBMEO reversed the size decrease of L6 myoblasts under starvation. Among the eight monoterpene compounds of CBMEO without cytotoxicity for L6 cells, sabinene induced predominant recovery of reductions of myotube diameters under starvation. Sabinene diminished the elevated E3 ubiquitin ligase muscle ring-finger protein-1 (MuRF-1) expression and p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase1/2 (ERK1/2) phosphorylations in starved myotubes. Moreover, sabinene decreased the increased level of reactive oxygen species (ROS) in myotubes under starvation. The ROS inhibitor antagonized expression of MuRF-1 and phosphorylation of MAPKs, which were elevated in starved myotubes. In addition, levels of muscle fiber atrophy and MuRF-1 expression in gastrocnemius from fasted rats were reduced after administration of sabinene. These findings demonstrate that sabinene, a bioactive component from CBMEO, may attenuate skeletal muscle atrophy by regulating the activation mechanism of ROS-mediated MAPK/MuRF-1 pathways in starved myotubes, probably leading to the reverse of reduced muscle fiber size in fasted rats.

Inhibitory effects of scoparone from chestnut inner shell on platelet-derived growth factor-BB-induced vascular smooth muscle cell migration and vascular neointima hyperplasia.

BACKGROUND: Compounds of the inner shell of chestnut (Castanea crenata) have diverse biological activities, including anti-cancer and anti-oxidant activities. Here we explored the effects of an extract of chestnut inner shells and of its bioactive component scoparone on vascular smooth muscle cell migration and vessel damage. RESULTS: The ethanol extract of chestnut inner shells, containing 11 major compounds, inhibited platelet-derived growth factor (PDGF)-BB-induced migration of rat aortic smooth muscle cells (RASMCs). Among these compounds, scoparone (6,7-dimethoxycoumarin) suppressed RASMC migration and wound healing in response to PDGF-BB but did not affect RASMC proliferation. In RASMCs, scoparone inhibited the PDGF-BB-induced rat aortic sprout outgrowth and attenuated the PDGF-BB-mediated increase in phosphorylation of mitogen-activated protein kinases (MAPKs), p38 MAPK and extracellular signal-regulated kinase 1/2. The in vivo administration of scoparone resulted in the attenuation of neointima formation in balloon-injured carotid arteries of rats. CONCLUSION: These findings demonstrate that scoparone, found in chestnut inner shells, may inhibit cell migration through suppression of the phosphorylation of MAPKs in PDGF-BB-treated RASMCs, probably contributing to the reduction of neointimal hyperplasia induced after vascular injury. Therefore, scoparone and chestnut inner shell may be a potential agent or functional food, respectively, for the prevention of vascular disorders such as vascular restenosis or atherosclerosis. © 2019 Society of Chemical Industry.

Corrigendum to: Dual mechanisms for the regulation of brain-derived neurotrophic factor by valproic acid in neural progenitor cells.

[This corrects the article on p. 679 in vol. 22, PMID: 30402028.].

Glucose-regulated protein 78 in lipid rafts elevates vascular smooth muscle cell proliferation of spontaneously hypertensive rats by controlling platelet-derived growth factor receptor signaling.

The multifunctional glucose-regulated protein 78 (GRP78) is known to be differentially expressed in the lipid rafts of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) and normal Wistar-Kyoto (WKY) rats. However, its role in VSMCs from SHRs remains to be elucidated. This work was conducted to investigate the contribution made by GRP78 in VSMCs. GRP78 expression in VSMC lipid rafts decreased in WKY rats with age, but not in SHRs. Transfection with GRP78-siRNA attenuated not only platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation and aortic sprout outgrowth but also the phosphorylation of PDGF receptor (PDGFR)-ß, Akt, and extracellular signal-regulated kinase (Erk) 1/2 in VSMCs in response to PDGF-BB. Moreover, GRP78 knockdown also reduced the PDGF-BB-induced dimerization of PDGFR-ß and GRP78 in SHR VSMCs. The phosphorylation of GRP78 and PDGFR-ß was elevated in VSMCs treated with PDGF-BB and was completely abolished by AG1296 (a PDGFR inhibitor). Moreover, the binding of PDGFR-ß to GRP78 and the co-localization of GRP78 to PDGFR-ß in VSMCs were stronger in SHRs than in WKY rat controls. This study demonstrates that the PDGF-BB-induced proliferation of SHR VSMCs is mediated by the expressional upregulation of GRP78 on VSMC lipid rafts in SHRs, probably via the regulation of PDGFR-ß-GRP78 binding and their cross-activation. These observations indicate that GRP78 may play important roles in the pathological progression of SHR VSMCs.

DJ-1 is involved in epigenetic control of sphingosine-1-phosphate receptor expression in vascular neointima formation.

DJ-1 and sphingosine-1-phosphate (S1P) receptors (S1PRs) are implicated in the control of physiology and pathophysiology of cardiovascular systems such as blood pressure, atherosclerosis, and restenosis. Here, we investigated whether DJ-1 with antioxidant function participates in the regulation of S1PR1 and S1PR2 expression in vascular smooth muscle cells (VSMCs) and whether this response is related to vascular neointima formation. In vitro studies used cellular migration assay, western blot, reverse transcriptase and real-time PCR analysis, and immunocytochemistry. In vivo studies were performed using the carotid artery ligation model together with immunohistochemistry in DJ-1 knockout (DJKO) and corresponding wild-type (DJWT) mice. S1P stimulated migration of VSMCs from DJKO and DJWT mice. VSMC migration was suppressed by S1PR1 inhibitor but was elevated by S1PR2 inhibitor. Compared with DJWT mice, S1PR1 expression was higher in VSMCs and neointimal plaque from DJKO mice, but S1PR2 expression was lower. Overexpression of DJ-1 in DJKO VSMCs reduced S1PR1 expression and elevated S1PR2 expression. Compared with DJWT mice, histone deacetylase-1 recruitment and histone H3 acetylation at the S1PR1 promoter region were lower and higher, respectively, but this pattern was reversed at the S1PR2 promoter region in DJKO VSMCs. S1PR expressions and epigenetic changes at S1PR promoter regions in DJWT VSMCs treated with H2O2 showed similar patterns to those in DJKO VSMCs. Our findings suggest that DJ-1 may be involved in the regulation of S1PR1 and S1PR2 expression via H2O2-mediated histone modification in VSMCs. Consequently, this modification may affect S1P-induced VSMC migration and be related to vascular neointima formation.

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.

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 H2O2, and exogenous H2O2 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 H2O2-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.

Dual mechanisms for the regulation of brain-derived neurotrophic factor by valproic acid in neural progenitor cells.

Autism spectrum disorders (ASDs) are neurodevelopmental disorders that share behavioral features, the results of numerous studies have suggested that the underlying causes of ASDs are multifactorial. Behavioral and/or neurobiological analyses of ASDs have been performed extensively using a valid model of prenatal exposure to valproic acid (VPA). Abnormal synapse formation resulting from altered neurite outgrowth in neural progenitor cells (NPCs) during embryonic brain development has been observed in both the VPA model and ASD subjects. Although several mechanisms have been suggested, the actual mechanism underlying enhanced neurite outgrowth remains unclear. In this study, we found that VPA enhanced the expression of brain-derived neurotrophic factor (BDNF), particularly mature BDNF (mBDNF), through dual mechanisms. VPA increased the mRNA and protein expression of BDNF by suppressing the nuclear expression of methyl-CpG-binding protein 2 (MeCP2), which is a transcriptional repressor of BDNF. In addition, VPA promoted the expression and activity of the tissue plasminogen activator (tPA), which induces BDNF maturation through proteolytic cleavage. Trichostatin A and sodium butyrate also enhanced tPA activity, but tPA activity was not induced by valpromide, which is a VPA analog that does not induce histone acetylation, indicating that histone acetylation activity was required for tPA regulation. VPA-mediated regulation of BDNF, MeCP2, and tPA was not observed in astrocytes or neurons. Therefore, these results suggested that VPA-induced mBDNF upregulation was associated with the dysregulation of MeCP2 and tPA in developing cortical NPCs.

DJ-1 Regulates Differentiation of Human Mesenchymal Stem Cells into Smooth Muscle-like Cells in Response to Sphingosylphosphorylcholine.

Although multiple factors contribute to the differentiation of human mesenchymal stem cells (hMSCs) into various types of cells, the differentiation of hMSCs into smooth muscle cells (SMCs), one of central events in vascular remodeling, remains to be clarified. ROS participate in the differentiation of hMSCs into several cell types and were regulated by redox-sensitive molecules including a multifunctional protein DJ-1. Here, we investigated the correlation between altered proteins, especially those related to ROS, and SMC differentiation in sphingosylphosphorylcholine (SPC)-stimulated hMSCs. Treatment with SPC resulted in an increased expression of SMC markers, namely α-smooth muscle actin (SMA) and calponin, and an increased production of ROS in hMSCs. A proteomic analysis of SPC-stimulated hMSCs revealed a distinctive alteration of the ratio between the oxidized and reduced forms of DJ-1 in hMSCs in response to SPC. The increased abundance of oxidized DJ-1 in SPC-stimulated hMSCs was validated by immunoblot analysis. The SPC-induced increase in the expression of α-SMA was stronger in DJ-1-knockdown hMSCs than in control cells. Moreover, the expression of α-SMA, and the calponin and generation of ROS in response to SPC were weaker in normal hMSCs than in DJ-1-overexpressing hMSCs. Exogenous H2 O2 mimicked the responses induced by SPC treatment. These results indicate that the ROS-related DJ-1 pathway regulates the differentiation of hMSCs into SMCs in response to SPC.

Low-power laser irradiation inhibits PDGF-BB-induced migration and proliferation via apoptotic cell death in vascular smooth muscle cells.

Vascular restenosis after injury of blood vessel has been implicated in various responses including apoptosis, migration, and proliferation in vascular smooth muscle cells (VSMCs) stimulated by diverse growth factors underlying platelet-derived growth factor (PDGF). Previous studies evaluated the effects of low-power laser (LPL) irradiation over various wavelength ranges on VSMC events in normal and pathologic states. However, whether VSMC responses are affected by LPL irradiation remains unclear. The purpose of this study is to explore the effects of LPL (green diode laser 532-nm pulsed wave of 300 mW at a spot diameter of 1 mm) irradiation on the responses, apoptosis, migration, and proliferation of VSMCs. The effect of LPL irradiation was tested on VSMCs through cytotoxicity, proliferation, migration, and apoptotic assays. Aortic ring assay was used to assess the effect of LPL irradiation on aortic sprout outgrowth. Protein expression levels were determined by western blotting. LPL irradiation did not affect VSMC viability but slightly attenuated PDGF-BB-induced proliferation in VSMCs. In addition, LPL irradiation inhibited PDGF-BB-evoked migration of VSMCs. Aortic sprout outgrowth in response to PDGF-BB was diminished in cells treated with LPL. In contrast, LPL irradiation evoked apoptosis in VSMCs in the presence of PDGF-BB. Similarly, activation of caspase-3 and Bax, as well as p38 mitogen-activated protein kinase (MAPK), in VSMCs treated with PDGF-BB was enhanced by exposure to LPL. These findings indicate that LPL irradiation induces vascular apoptosis via p38 MAPK activation and simultaneously inhibits VSMC proliferation and migration in response to PDGF-BB.

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.

Diminished Lipid Raft SNAP23 Increases Blood Pressure by Inhibiting the Membrane Fluidity of Vascular Smooth-Muscle Cells.

Synaptosomal-associated protein 23 (SNAP23) is involved in microvesicle trafficking and exocytosis in various cell types, but its functional role in blood pressure (BP) regulation has not yet been defined. Here, we found that lipid raft SNAP23 expression was much lower in vascular smooth-muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) than in those from normotensive Wistar-Kyoto (WKY) rats. This led us to investigate the hypothesis that this lower expression may be linked to the spontaneous hypertension found in SHR. The expression level of lipid raft SNAP23 and the fluidity in the plasma membrane of VSMCs were lower in SHR than in WKY rats. Cholesterol content in the VSMC membrane was higher, but the secreted cholesterols found in VSMC-conditioned medium and in the blood serum were lower in SHR than in WKY rats. SNAP23 knockdown in WKY rat VSMCs reduced the membrane fluidity and increased the membrane cholesterol level. Systemic overexpression of SNAP23 in SHR resulted in an increase of cholesterol content in their serum, a decrease in cholesterol in their aorta and the reduction of their BP. Our findings suggest that the low expression of the lipid raft SNAP23 in VSMCs might be a potential cause for the characteristic hypertension of SHR.

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.

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.

Monoclonal antibody against α-actinin 4 from human umbilical vein endothelial cells inhibits endothelium-dependent vasorelaxation.

BACKGROUND: This study was attempted to identify new molecules expressed on the plasma membrane of human umbilical vein endothelial cells (HUVECs) using monoclonal antibody-based proteomics technology and to determine the effect of the identified antibody on vascular reactivity. METHODS: Twenty-two antibodies were developed from rats inoculated with HUVECs, and their effects were determined by observing vascular reactivity. RESULTS: Among the 22 antibodies, the C-7 antibody significantly inhibited endothelium-dependent vasorelaxation in response to acetylcholine (ACh) but not to histamine. Moreover, the C-7 antibody did not affect norepinephrine-induced contraction in either the endothelium-intact or -denuded aorta. A proteomics study involving immunoprecipitation of the C-7 antibody with biotinylated HUVECs showed that this antibody binds to plasma membrane proteins corresponding to immunoglobulin heavy chain (VHDJ region), chaperonin-containing T-complex polypeptide 1 and α-actinin 4. The muscarinic M3 ACh receptor and α-actinin 4 were colocalized on the plasma membrane of HUVECs, and the colocalization was found to increase in response to ACh and was inhibited by pretreatment with the C-7 antibody. CONCLUSIONS: These results demonstrate that monoclonal C-7 antibody exerts an inhibitory effect on endothelium-dependent vasorelaxation induced by ACh and that this response may at least partially result from the inhibition of α-actinin 4.

Does phosphorylation of cofilin affect the progression of human bladder cancer?

BACKGROUND: We determined the differently expressed protein profiles and their functions in bladder cancer tissues with the aim of identifying possible target proteins and underlying molecular mechanisms for taking part in their progression. METHODS: We examined the expression of proteins by proteomic analysis and western blot in normal urothelium, non-muscle-invasive bladder cancers (NMIBCs), and muscle-invasive bladder cancers (MIBCs). The function of cofilin was analyzed using T24 human bladder cancer cells. RESULTS: The expression levels of 12 proteins were altered between bladder cancers and normal bladder tissues. Of these proteins, 14-3-3σ was upregulated in both NMIBCs and MIBCs compared with controls. On the other hand, myosin regulatory light chain 2, galectin-1, lipid-binding AI, annexin V, transthyretin, CARD-inhibitor of NF-κB-activating ligand, and actin prepeptide were downregulated in cancer samples. Cofilin, an actin-depolymerizing factor, was prominent in both NMIBCs and MIBCs compared with normal bladder tissues. Furthermore, we confirmed that cofilin phosphorylation was more prominent in MIBCs than in NMIBCs using immunoblotting and immunohistochemcal analyses. Epidermal growth factor (EGF) increased the phosphorylation of cofilin and elevated the migration in T24 cells. Knockdown of cofilin expression with small interfering RNA attenuated the T24 cell migration in response to EGF. CONCLUSIONS: These results demonstrate that the increased expression and phosphorylation of cofilin might play a role in the occurrence and invasiveness of bladder cancer. We suspected that changes in cofilin expression may participate in the progression of the bladder cancer.

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.

Bioluminescence Imaging of Matrix Metalloproteinases-2 and -9 Activities in Ethanol-injured Cornea of Mice.

BACKGROUND/AIM: This study aimed to investigate the usefulness of in vivo bioluminescence imaging (BLI) to examine the role of matrix metalloproteinases (MMP)-2 and MMP-9 activation in the development and healing of ethanol-induced damage in the cornea of mice. MATERIALS AND METHODS: Mouse corneal injury was induced by topical treatment with 20% ethanol. BLI was obtained from the ocular region of mice intravenously injected with an active-MMP-2/9 probe. In vivo results were validated in primary corneal epithelial cells. RESULTS: BLI indicated that treatment of the eye with 20% ethanol elevated MMP-2/9 activity, which was inhibited by the application of eye drops (hyaluronic acid and serum). Treatment of corneal epithelial cells with 20% ethanol-increased the activities of MMP-2 and MMP-9, which were also inhibited by eye drops. CONCLUSION: BLI can be applied in vivo in mice with corneal injury to examine the activity of MMPs and clarify the efficacy of eye drops.

Anti­inflammatory role of Prunus persica L. Batsch methanol extract on lipopolysaccharide­stimulated glial cells.

Glial cells are the resident immune cells of the central nervous system. Reactive glial cells release inflammatory mediators that induce neurotoxicity or aggravate neurodegeneration. Regulation of glial activation is crucial for the initiation and progression of neuropathological conditions. Constituents of the peach tree (Prunus persica L. Batsch), which has a global distribution, have been found to exert therapeutic effects in pathological conditions, such as rashes, eczema and allergies. However, the therapeutic potential of its aerial parts (leaves, fruits and twigs) remains to be elucidated. The present study aimed to evaluate the anti­inflammatory role of P. persica methanol extract (PPB) on lipopolysaccharide (LPS)­stimulated glial cells. High­performance liquid chromatography coupled with tandem mass spectrometry analysis showed that PPB contained chlorogenic acid and catechin, which have antioxidant properties. Western blot and reverse transcription polymerase chain reaction results indicated that PPB reduced the transcription of various proinflammatory enzymes (nitric oxide synthase and cyclooxygenase­2) and cytokines [tumor necrosis factor­α, interleukin (IL)­1ß and IL­6] in LPS­stimulated BV2 cells. In addition, PPB inhibited the activation of NF­κB and various mitogen­activated protein kinases required for proinflammatory mediator transcription. Finally, nitrite measurement and immunocytochemistry results indicated that PPB also suppressed nitrite production and NF­κB translocation in LPS­stimulated primary astrocytes. Thus, PPB may be used as a potential therapeutic agent for neurodegenerative diseases and neurotoxicity via the suppression of glial cell activation.