Magnolol inhibits migration of vascular smooth muscle cells via cytoskeletal remodeling pathway to attenuate neointima formation

BackgroundIncreased proliferation and migration of vascular smooth muscle cells (VSMCs) contribute importantly to the formation of both atherosclerotic and restenotic lesions. The objective of this study was to investigate the effect of magnolol on VSMC migration.MethodsThe proteolytic activity of matrix metalloproteinases (MMPs) in tumor necrosis factor alpha (TNF-α) stimulated VSMCs was performed by gelatin zymography. VSMC migration was assessed by wound healing and Boyden chamber methods. Collagen induced VSMC adhesion was determined by spectrofluorimeter and stress fibers formation was evaluated by fluorescence microscope. The expression of signaling molecules involved in stress fibers formation was determined by western blot. The phosphorylation of myosin light chain (MLC20) was determined by urea-glycerol polyacrylamide gel electrophoresis. Immunohistochemistry was performed to determine the expression of β1-integrin and collagen type I in the injured carotid arteries of rats on day 35 after vascular injury.ResultsVSMC migration was strongly inhibited by magnolol without affecting MMPs expression. Also, magnolol inhibited β1-integrin expression, FAK phosphorylation and RhoA and Cdc42 activation to inhibit the collagen induced stress fibers formation. Moreover, magnolol inhibited the phosphorylation of MLC20. Our in vivo results showed that magnolol inhibited β1-integrin expression, collagen type I deposition and FAK phosphorylation in injured carotid arteries without affecting MMP-2 activity.ConclusionsMagnolol inhibited VSMC migration via inhibition of cytoskeletal remodeling pathway to attenuate neointima formation.General significanceThis study provides a rationale for further evaluation of magnolol for the management of atherosclerosis and restenosis.     

Genistein suppresses leptin‐induced proliferation and migration of vascular smooth muscle cells and neointima formation

Obesity is a strong risk factor for the development of cardiovascular diseases and is associated with a marked increase in circulating leptin concentration. Leptin is a peptide hormone mainly produced by adipose tissue and is regulated by energy level, hormones and various inflammatory mediators. Genistein is an isoflavone that exhibits diverse health‐promoting effects. Here, we investigated whether genistein suppressed the atherogenic effect induced by leptin. The A10 cells were treated with leptin and/or genistein, and then the cell proliferation and migration were analysed. The reactive oxygen species (ROS) and proteins levels were also measured, such as p44/42MAPK, cell cycle‐related protein (cyclin D1 and p21) and matrix metalloproteinase‐2 (MMP‐2). Immunohistochemistry and morphometric analysis were used for the neointima formation in a rat carotid artery injury model. Genistein (5 μM) significantly inhibited both the proliferation and migration of leptin (10 ng/ml)‐stimulated A10 cells. In accordance with these finding, genistein decreased the leptin‐stimulated ROS production and phosphorylation of the p44/42MAPK signal transduction pathway. Meanwhile, genistein reversed the leptin‐induced expression of cyclin D1, and cyclin‐dependent kinase inhibitor, p21. Genistein attenuated leptin‐induced A10 cell migration by inhibiting MMP‐2 activity. Furthermore, the leptin (0.25 mg/kg)‐augmented neointima formation in a rat carotid artery injury model was attenuated in the genistein (5 mg/kg body weight)‐treated group when compared with the balloon injury plus leptin group. Genistein was capable of suppressing the atherogenic effects of leptin in vitro and in vivo, and may be a promising candidate drug in the clinical setting.     

Protocatechuic aldehyde inhibits migration and proliferation of vascular smooth muscle cells and intravascular thrombosis

TAR DNA-binding protein of 43 kDa (TDP-43) is the major component of the intracellular inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here, we show that both monoclonal (60019-2-Ig) and polyclonal (10782-2-AP) anti-TDP-43 antibodies recognize amino acids 203-209 of human TDP-43. The monoclonal antibody labeled human TDP-43 by recognizing Glu204, Asp205 and Arg208, but failed to react with mouse TDP-43. The antibodies stained the abnormally phosphorylated C-terminal fragments of 24-26 kDa in addition to normal TDP-43 in ALS and FTLD brains. Immunoblot analysis after protease treatment demonstrated that the epitope of the antibodies (residues 203-209) constitutes part of the protease-resistant domain of TDP-43 aggregates which determine a common characteristic of the pathological TDP-43 in both ALS and FTLD-TDP. The antibodies and methods used in this study will be useful for the characterization of abnormal TDP-43 in human materials, as well as in vitro and animal models for TDP-43 proteinopathies.     

Epac1 is upregulated during neointima formation and promotes vascular smooth muscle cell migration

Vascular remodeling after mechanoinjury largely depends on the migration of smooth muscle cells, an initial key step to wound healing. However, the role of the second messenger system, in particular, the cAMP signal, in regulating such remodeling remains controversial. Exchange protein activated by cAMP (Epac) has been identified as a new target molecule of the cAMP signal, which is independent from PKA. We thus examined whether Epac plays a distinct role from PKA in vascular remodeling. To examine the role of Epac and PKA in migration, we used primary culture smooth muscle cells from both the fetal and adult rat aorta. A cAMP analog selective to PKA, 8-(4-parachlorophenylthio)-cAMP (pCPT-cAMP), decreased cell migration, whereas an Epac-selective analog, 8-pCPT-2'-O-Me-cAMP, enhanced migration. Adenovirus-mediated gene transfer of PKA decreased cell migration, whereas that of Epac1 significantly enhanced cell migration. Striking morphological differences were observed between pCPT-cAMP- and 8-pCPT-2'-O-Me-cAMP-treated aortic smooth muscle cells. Furthermore, overexpression of Epac1 enhanced the development of neointimal formation in fetal rat aortic tissues in organ culture. When the mouse femoral artery was injured mechanically in vivo, we found that the expression of Epac1 was upregulated in vascular smooth muscle cells, whereas that of PKA was downregulated with the progress of neointimal thickening. Our findings suggest that Epac1, in opposition to PKA, increases vascular smooth muscle cell migration. Epac may thus play an important role in advancing vascular remodeling and restenosis upon vascular injury.     

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.     

Dexamethasone inhibits leukocyte migration through endothelial cells towards smooth muscle cells

Leukocyte interactions with endothelial cell monolayers (ECM) and smooth muscle cells (SMC) play an important role during inflammatory processes. Several studies describe an inhibitory effect of dexamethasone on polymorphonuclear leukocytes (PMNL), endothelial cell function, and interleukin-1 (IL-1) release. Aim of the current study was to investigate the influence of dexamethasone on leukocyte migration through an endothelial cell monolayer towards SMC-layers stimulated by tumor necrosis factor-alpha (TNF-alpha). Using a recently developed triple chamber migration system, SMC-layers were cultured on the bottom of a 24-well plate. On the upper surface of the first filter, ECM were cultured, the second filter was a collecting filter. The amount of leukocyte migration through ECM towards TNF-alpha-stimulated smooth muscle cell layers with and without dexamethasone-pretreatment was measured using a fluorescence technique. The pretreatment of SMC-layers with dexamethasone reduced the amount of leukocyte migration down to 92 +/- 8.8% (0.001 mM, p=n.s.), to 67 +/- 5.7% (0.01 mM, p<0.05), to 53 +/- 4.6% (0.1 mM, p<0.05), and to 41 +/- 5.0% (1 mM, p<0.05). In conclusion, dexamethasone treatment of smooth muscle cell layers inhibits leukocyte migration through ECM towards smooth muscle cell layers. The inhibition seems to be due to a decrease in IL-1 release. Treatment of all cell types, PMNL, endothelial cells, as well as smooth muscle cell layers, simulating an in-vivo situation, seems to have an additive effect.     

miR‐145 inhibits the proliferation and migration of vascular smooth muscle cells by regulating autophagy

miR‐145, the most abundant miRNA in the vascular smooth muscle cells (VSMCs), regulates VSMC function in intimal hyperplasia. It has been reported that autophagy participates in the regulation of proliferation and migration of VSMCs. However, the effect of miR‐145 on autophagy and related mechanism in the proliferation and migration of VSMCs remains unclear. Therefore, we aimed to determine the effect of miR‐145 on autophagy and the mechanism in VSMCs. Cell autophagy was determined by transmission electron microscope, mRFP‐GFP‐LC3 assay and Western blotting. A recombinant lentivirus containing miR‐145 was used to construct VSMCs with miR‐145 overexpression. We found that miR‐145 expression was decreased, and autophagy was increased in the carotid arteries of C57BL/6J mice with intimal hyperplasia and TGF‐β1‐stimulated VSMCs. Furthermore, miR‐145 overexpression inhibited cell autophagy, whereas miR‐145 inhibition promoted autophagy in TGF‐β1‐stimulated VSMCs. Meanwhile, miR‐145 inhibited the proliferation and migration of VSMCs. More importantly, our study showed that autophagy inhibition augmented the inhibitory effect of miR‐145 on the proliferation and migration of VSMCs. In addition, we found that the sirtuins are not direct targets of miR‐145 in the proliferation and migration of VSMCs. These results suggest that miR‐145 inhibits the proliferation and migration of VSMCs by suppressing the activation of autophagy.     

CeReS-18, a cell regulatory sialoglycopeptide, inhibits proliferation and migration of rat vascular smooth muscle cells

CeReS-18, a cell regulatory sialoglycopeptide, has been shown to inhibit proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on vascular smooth muscle cell (SMC) proliferation was characterized in cultured rat aorta SMCs (A7r5). More extensively, the effect of CeReS-18 on platelet-derived growth factor (PDGF)-induced SMC migration was examined using a modified Boyden's chamber assay. CeReS-18 inhibits both SMC proliferation and migration in a concentration-dependent, calcium-sensitive, and reversible manner. Furthermore, cells preincubated with the inhibitor had an increased sensitivity to CeReS-18-mediated inhibition of SMC migration. Immunoprecipitation and in vitro phosphorylation assays demonstrated that MAP kinase activity was inhibited in the CeReS-18-treated cells and pretreatment with CeReS-18 suppressed the activation of MAP kinase stimulated by PDGF. However, it is not likely that the suppression of the MAP kinase pathway was directly responsible for the ability of CeReS-18 to inhibit migration of the rat aorta smooth muscle cells since a MEK-specific inhibitor, PD98059, did not influence A7r5 cell migration.     

Malabaricone C inhibits PDGF-induced proliferation and migration of aortic smooth muscle cells through induction of heme oxygenase-1

Malabaricone C (Mal-C), isolated from nutmeg, is known to exert a variety of pharmacological activities. However, the effect of Mal-C on vascular smooth muscle cells (VSMCs) is unknown. This study examined the effect of Mal-C on proliferation and migration of primary rat aortic smooth muscle cells (RASMCs) as well as its underlying mechanisms. Treatment of RASMCs with Mal-C induced both protein and mRNA expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Mal-C-mediated HO-1 induction was inhibited by treatment with actinomycin D or by cycloheximide. SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (a MEK inhibitor), and N-acetylcysteine (NAC, an antioxidant) did not suppress Mal-C-induced HO-1 expression. In contrast, LY294002 (a PI3K inhibitor) blocked Mal-C-induced HO-1 expression. Moreover, RASMCs treated with Mal-C exhibited activation of AKT in a dose- and time-dependent manner. Treatment of RASMCs with Mal-C increased nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2), which is a key regulator of HO-1 expression, and this translocation was also inhibited by LY294002. Consistent with the notion that HO-1 has protective effects against VSMCs, Mal-C remarkably inhibited platelet-derived growth factor (PDGF)-induced proliferation and migration of RASMCs. However, inhibition of HO-1 significantly attenuated the inhibitory effects of Mal-C on PDGF-induced proliferation and migration of RASMCs. Taken together, these findings suggest that Mal-C could suppress PDGF-induced proliferation and migration of RASMCs through Nrf2 activation and subsequent HO-1 induction via the PI3K/AKT pathway, and may be a potential HO-1 inducer for preventing or treating vascular diseases.     

Manumycin A, inhibitor of ras farnesyltransferase, inhibits proliferation and migration of rat vascular smooth muscle cells.

Restenosis after angioplasty is thought to be caused by proliferation and migration of vascular smooth muscle cells (VSMCs), and it is a most serious problem in medical treatment. A low dose (50 ng/ml) of manumycin A, an inhibitor of p21(ras) (ras) farnesylation, significantly inhibited proliferation of rat VSMCs stimulated by the platelet-derived growth factor (PDGF). The mitoinhibitory effect of manumycin A was dose- and time-dependent but was independent of cell density. Western blot analysis showed that manumycin A reduced the amount of functional ras localized at the cytoplasmic membrane and inhibited the phosphorylation of p42/44 mitogen-activated protein kinase (MAPK). Manumycin A also inhibited VSMC migration and disorganized alpha actin fibers, as shown by immnofluorecence staining. These results indicate that the interruption of the ras/MAPK signal transduction pathway and the disorganization of alpha actin fibers are the main cause of manumycin A inhibition of VSMC proliferation and migration induced by PDGF.     

Dominant negative insulin-like growth factor-1 receptor inhibits neointimal formation through suppression of vascular smooth muscle cell migration and proliferation, and induction of apoptosis

Blocking of the IGF-1 signaling pathway targeting the IGF-1 receptor (IGF-1R) provides a potential treatment strategy for restenosis. In this study, we have examined the effects of a dominant negative IGF-1R (IGF-1Rt) on primary rat VSMCs in vitro and on injured rat carotid artery in vivo. Ad/IGF-1Rt infection inhibited VSMC migration and proliferation, and it also induced apoptosis by inhibiting phosphorylation of Akt and phosphorylation of ERK1/2. Consistent with the anti-proliferative and apoptotic effects in vitro, the Ad/IGF-1Rt infection markedly reduced neointimal formation in carotid injury model. Ad/IGF-1Rt treated carotid arteries exhibited a suppressed proliferation index, PCNA expression, and also were stained positive for TUNEL assay. These results indicate that a dominant negative IGF-1R has the potential to reduce neointimal formation of injured rats' carotid arteries. The delivery of dominant negative IGF-1R by adenoviral or other vectors may provide a useful strategy for inhibiting restenosis after angioplasty.     

人SOD1基因在大鼠血管平滑肌细胞的表达及对抗氧自由基损伤的作用

本实验构建含人铜锌超氧化物歧化酶（ｈＳＯＤ１）基因的逆转录病毒载体,将其导入离体培养的鼠血管平滑肌细胞,观察ｈＳＯＤ１基因表达及其抗氧自由基损害作用,结果表明：（１）载体构建策略和方法正确,ｈＳＯＤ１基因可在靶细胞中高效稳定表达;（２）转化ｈＳＯＤ１的ＶＳＭＣｓ可对抗大剂量氧自由基对细胞的直接损伤作用;（３）小剂量氧自由基刺激ＶＳＭＣｓ增殖,而转化ｈＳＯＤ１的ＶＳＭＣｓ增殖反应受到抑制,本研究结果     

USP10 exacerbates neointima formation by stabilizing Skp2 protein in vascular smooth muscle cells

The underlying mechanism of neointima formation remains unclear. Ubiquitin-specific peptidase 10 (USP10) is a deubiquitinase that plays a major role in cancer development and progression. However, the function of USP10 in arterial restenosis is unknown. Herein, USP10 expression was detected in mouse arteries and increased after carotid ligation. The inhibition of USP10 exhibited thinner neointima in the model of mouse carotid ligation. In vitro data showed that USP10 deficiency reduced proliferation and migration of rat thoracic aorta smooth muscle cells (A7r5) and human aortic smooth muscle cells (HASMCs). Mechanically, USP10 can bind to Skp2 and stabilize its protein level by removing polyubiquitin on Skp2 in the cytoplasm. The overexpression of Skp2 abrogated cell cycle arrest induced by USP10 inhibition. Overall, the current study demonstrated that USP10 is involved in vascular remodeling by directly promoting VSMC proliferation and migration via stabilization of Skp2 protein expression.     

PLD1 promotes reactive oxygen species production in vascular smooth muscle cells and injury-induced neointima formation

Phospholipase D (PLD) generates the signaling lipid phosphatidic acid (PA) and has been known to mediate proliferation signal in vascular smooth muscle cells (VSMCs). However, it remains unclear how PLD contributes to vascular diseases. VSMC proliferation directly contributes to the development and progression of cardiovascular disease, such as atherosclerosis and restenosis after angioplasty. Using the mouse carotid artery ligation model, we find that deletion of Pld1 gene inhibits neointima formation of the injuried blood vessels. PLD1 deficiency reduces the proliferation of VSMCs in both injured artery and primary cultures through the inhibition of ERK1/2 and AKT signals. Immunohistochemical staining of injured artery and flow cytometry analysis of VSMCs shows a reduction of the levels of reactive oxygen species (ROS) in Pld1^?/? VSMCs. An increase of intracellular ROS by hydrogen peroxide stimulation restored the reduced activities of ERK and AKT in Pld1^?/? VSMCs, whereas a reduction of ROS by N-acetyl-l-cysteine (NAC) scavenger lowered their activity in wild-type VSMCs. These results indicate that PLD1 plays a critical role in neointima, and that PLD1 mediates VSMC proliferation signal through promoting the production of ROS. Therefore, inhibition of PLD1 may be used as a therapeutic approach to suppress neointimal formation in atherosclerosis and restenosis after angioplasty.