The apolipoprotein A‐I mimetic peptide,D‐4F,restrains neointimal formation through heme oxygenase‐1 up‐regulation

D‐4F, an apolipoprotein A‐I (apoA‐I) mimetic peptide, possesses distinctly anti‐atherogenic effects. However, the biological functions and mechanisms of D‐4F on the hyperplasia of vascular smooth muscle cells (VSMCs) remain unclear. This study aimed to determine its roles in the proliferation and migration of VSMCs. In vitro, D‐4F inhibited VSMC proliferation and migration induced by ox‐LDL in a dose‐dependent manner. D‐4F up‐regulated heme oxygenase‐1 (HO‐1) expression in VSMCs, and the PI3K/Akt/AMP‐activated protein kinase (AMPK) pathway was involved in these processes. HO‐1 down‐regulation with siRNA or inhibition with zinc protoporphyrin (Znpp) impaired the protective effects of D‐4F on the oxidative stress and the proliferation and migration of VSMCs. Moreover, down‐regulation of ATP‐binding cassette transporter A1 (ABCA1) abolished the activation of Akt and AMPK, the up‐regulation of HO‐1 and the anti‐oxidative effects of D‐4F. In vivo, D‐4F restrained neointimal formation and oxidative stress of carotid arteries in balloon‐injured Sprague Dawley rats. And inhibition of HO‐1 with Znpp decreased the inhibitory effects of D‐4F on neointimal formation and ROS production in arteries. In conclusion, D‐4F inhibited VSMC proliferation and migration in vitro and neointimal formation in vivo through HO‐1 up‐regulation, which provided a novel prophylactic and therapeutic strategy for anti‐restenosis of arteries.     

Crucial Role of Hyaluronan in Neointimal Formation after Vascular Injury

Background

Hyaluronan (HA) is a primary component of the extracellular matrix of cells, and it is involved in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the role of HA in neointimal formation after vascular injury and determine its tissue-specific role in vascular smooth muscle cells (VSMCs) by using a cre-lox conditional transgenic (cTg) strategy.

Methods and Results

HA was found to be expressed in neointimal lesions in humans with atherosclerosis and after wire-mediated vascular injury in mice. Inhibition of HA synthesis using 4-methylumbelliferone markedly inhibited neointimal formation after injury. In vitro experiments revealed that low-molecular-weight HA (LMW-HA) induced VSMC activation, including migration, proliferation, and production of inflammatory cytokines, and reactive oxygen species (ROS). The migration and proliferation of VSMCs were mediated by the CD44/RhoA and CD44/ERK1/2 pathways, respectively. Because HA synthase 2 (HAS2) is predominantly expressed in injured arteries, we generated cTg mice that overexpress the murine HAS2 gene specifically in VSMCs (cHAS2/CreSM22α mice) and showed that HA overexpression markedly enhanced neointimal formation after cuff-mediated vascular injury. Further, HA-overexpressing VSMCs isolated from cHAS2/CreSM22α mice showed augmented migration, proliferation, and production of inflammatory cytokines and ROS.

Conclusion

VSMC-derived HA promotes neointimal formation after vascular injury, and HA may be a potential therapeutic target for cardiovascular disease.     

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.     

Effect of sinomenine on vascular smooth muscle cell dedifferentiation and neointima formation after vascular injury in mice

Sinomenine, a pure alkaloid extract from Sinomenium acutum, has anti-inflammatory and immunoregulatory functions. This study investigated the efficiency and the signalling pathways involved in the effect of sinomenine on vascular smooth muscle cell (VSMC) dedifferentiation in response to platelet-derived growth factor (PDGF)-BB stimulation and vascular injury. VSMCs were isolated from rat aorta and preincubated with sinomenine before being stimulated with PDGF-BB. WST and BrdU incorporation assays were used to evaluate VSMC proliferation. Flow cytometric analysis was performed for testing the cell cycle progression. The cell migration of VSMCs were analysed using a Transwell system. The expression of VSMC specific genes and signalling proteins were tested by Western blot. For the animal study, C57/BL6 mice were fed either normal rodent chow diets or sinomenine chow diets that supplemented with 0.09 % sinomenine (w/w) in the normal chows for 14 days before carotid artery wire injury. PDGF-BB activated the dedifferentiation of VSMCs characterised by decreased expression of SMA, Smoothelin and SM22α. However, sinomenine treatment preserved the dedifferentiation in response to PDGF-BB. The activations of mitogen-activated protein kinase extracellular signal-regulated kinases, Akt, GSK3β and STAT3 induced by PDGF-BB were also inhibited in sinomenine-treated VSMCs. In vivo evidence with wire-injured mice exhibited a reduction in neointimal area and an increase in smooth muscle-specific gene expression in the sinomenine-treated group. In this study, we found that sinomenine-suppressed VSMC phenotype switching induced by PDGF-BB in vitro and neointimal formation in vivo. Therefore, sinomenine is a potential candidate to be used in the treatment of vascular proliferative disease.     

Ceramide 1-phosphate induces neointimal formation via cell proliferation and cell cycle progression upstream of ERK1/2 in vascular smooth muscle cells

Ceramide 1-phosphate (C1P) is a novel bioactive sphingolipid formed by ceramide kinase (CERK)-catalyzed phosphorylation of ceramide. It has been implicated in the regulation of such vital pathophysiological functions as phagocytosis and inflammation, but there have been no reports ascribing a biological function to CERK in vascular disorders. Here the potential role of CERK/C1P in neointimal formation was investigated using rat aortic vascular smooth muscle cells (VSMCs) in primary culture and a rat carotid injury model. Exogenous C8-C1P stimulated cell proliferation, DNA synthesis, and cell cycle progression of rat aortic VSMCs in primary culture. In addition, wild-type CERK-transfected rat aortic VSMCs induced a marked increase in rat aortic VSMC proliferation and [³H]-thymidine incorporation when compared to empty vector transfectant. C8-C1P markedly activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) within 5 min, and the activation could be prevented by U0126, a MEK inhibitor. Also, K1, a CERK inhibitor, decreased the ERK1/2 phosphorylation and cell proliferation on platelet-derived growth factor (PDGF)-stimulated rat aortic VSMCs. CERK expression and C1P levels were found to be potently increased during neointimal formation using a rat carotid injury model. However, ceramide levels decreased during the neointimal formation process. These findings suggest that C1P can induce neointimal formation via cell proliferation through the regulation of the ERK1/2 protein in rat aortic VSMCs and that CERK/C1P may regulate VSMC proliferation as an important pathogenic marker in the development of cardiovascular disorders.     

Studies on microRNAs that are correlated with the cancer stem cells in chronic myeloid leukemia

Vascular remodeling is characterized by the aggregation of vascular smooth muscle cells (VSMCs) in intima. Previous studies have demonstrated that dehydroepiandrosterone (DHEA), a steroid hormone, can reverse vascular remodeling. However, it is still far clear that whether and how DHEA participates in the modulation of VSMCs activation and vascular remodeling. VSMCs were obtained from the thoracic aorta of SD rats. Cell proliferation was evaluated by CCK-8 assay and BrdU assay. To measure VSMCs migration activity, a transwell chamber assay was performed. Quantitative real-time RT-PCR and western blot were used to explore the molecular mechanisms. ROS generation by VSMCs was measured by DCF fluorescence. NADPH oxidase activity and SOD activity were measured by the corresponding kits. NF-κB activity was detected by NF-κB luciferase reporter gene assay. A rat carotid artery balloon injury model was built to evaluate the neointimal formation, and plasma PGF2 was measured by ELISA. Our results showed that DHEA significantly inhibited VSMCs proliferation after angiotensin (Ang II) stimulation by down-regulation of NADPH oxidase activity and ERK1/2 phosphorylation. Ang II can increase IL-6 and MCP-1 expression, but DHEA reverses these changes via inhibiting p38-MAPK/NF-κB (p65) signaling pathway. DHEA has no significant effects on VSMCs phenotype transition, but can reduce the neointimal to media area ratio after balloon injury. DHEA can alleviate oxidative stress and inflammation in VSMCs via ERK1/2 and NF-κB signaling pathway, but has no effect on VSMCs phenotype transition. Furthermore, DHEA attenuates VSMCs activation and neointimal formation after carotid injury in vivo. Taken together, DHEA might be a promising treatment for vascular injury under pathological condition.     

Leukotriene-C4 Synthase,a Critical Enzyme in the Activation of Store-independent Orai1/Orai3 Channels,Is Required for Neointimal Hyperplasia

Leukotriene-C₄ synthase (LTC₄S) generates LTC₄ from arachidonic acid metabolism. LTC₄ is a proinflammatory factor that acts on plasma membrane cysteinyl leukotriene receptors. Recently, however, we showed that LTC₄ was also a cytosolic second messenger that activated store-independent LTC₄-regulated Ca²⁺ (LRC) channels encoded by Orai1/Orai3 heteromultimers in vascular smooth muscle cells (VSMCs). We showed that Orai3 and LRC currents were up-regulated in medial and neointimal VSMCs after vascular injury and that Orai3 knockdown inhibited LRC currents and neointimal hyperplasia. However, the role of LTC₄S in neointima formation remains unknown. Here we show that LTC₄S knockdown inhibited LRC currents in VSMCs. We performed in vivo experiments where rat left carotid arteries were injured using balloon angioplasty to cause neointimal hyperplasia. Neointima formation was associated with up-regulation of LTC₄S protein expression in VSMCs. Inhibition of LTC₄S expression in injured carotids by lentiviral particles encoding shRNA inhibited neointima formation and inward and outward vessel remodeling. LRC current activation did not cause nuclear factor for activated T cells (NFAT) nuclear translocation in VSMCs. Surprisingly, knockdown of either LTC₄S or Orai3 yielded more robust and sustained Akt1 and Akt2 phosphorylation on Ser-473/Ser-474 upon serum stimulation. LTC₄S and Orai3 knockdown inhibited VSMC migration in vitro with no effect on proliferation. Akt activity was suppressed in neointimal and medial VSMCs from injured vessels at 2 weeks postinjury but was restored when the up-regulation of either LTC₄S or Orai3 was prevented by shRNA. We conclude that LTC₄S and Orai3 altered Akt signaling to promote VSMC migration and neointima formation.     

黄芩苷通过降低活性氧生成抑制血管平滑肌细胞伪足形成和迁移

已知黄芩苷（baicalin）通过削弱肌动蛋白相关蛋白（actin-related protein, Arp）2/3复合物的活性抑制血管平滑肌细胞（vascular smooth muscle cell, VSMC）伪足形成和迁移,然而,其抑制该信号途径的机制尚不明确。本研究证明,黄芩苷通过抑制VSMC活性氧（reactive oxygen species,ROS）生成降低Arp2/3活性,发挥阻止细胞伪足形成和迁移的功能。分别利用TRITC 鬼笔环肽和ROS荧光探针标记VSMCs,结果显示,黄芩苷能显著抑制血小板源性生长因子（platelet derived growth factor, PDGF）-BB诱导的VSMC伪足形成和迁移,伴有ROS生成减少。用超氧物歧化酶（superoxide dismutase, SOD）清除胞内过氧化物后,PDGF-BB引发的VSMC伪足形成被逆转,且该过程与降低皮层肌动蛋白微丝（F-actin）成核蛋白Arp2/3活性有关。免疫沉淀分析结果进一步表明,黄芩苷降低p47phox磷酸化水平,与ROS生成减少相一致。体内的实验也表明,黄芩苷（70 mg/kg/d）能有效抑制球囊损伤诱导的大鼠颈总动脉ROS生成。以上结果表明,黄芩苷通过抑制NADPH氧化酶介导的ROS生成,降低细胞皮质区F-actin成核活性,阻止细胞伪足形成、迁移,进而发挥血管保护作用。     

Role of Pin1 in neointima formation: Down-regulation of Nrf2-dependent heme oxygenase-1 expression by Pin1

Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intima formation after stenting and balloon angioplasty. Pin1, a peptidyl prolyl isomerase recognizing phosphorylated Ser/Thr–Pro, isomerizes the peptide bond. Because Pin1 overexpression is associated with transformation and the uncontrolled cell growth of tumors, we hypothesized that Pin1 functions as a chronic stimulator of VSMC proliferation. Pin1-positive smooth muscle cells were seen in the neointimal region of the femoral artery after guidewire injury. Exposure of VSMCS to platelet-derived growth factor (PDGF) increased Pin1 expression in a concentration-dependent manner. Basal cell growth rate and cyclin D1 expression were enhanced in Pin1-overexpressing VSMCs (Pin1-VSMCs). Moreover, PDGF-induced production of reactive oxygen species (ROS) in Pin1-VSMCs was higher than in control VSMCs. In Pin1-VSMCs, heme oxygenase-1 (HO-1) induction in response to nitric oxide donor was suppressed compared to control VSMCs. Nuclear translocation of nuclear factor E2-related factor-2 (Nrf2) was also diminished in Pin1-VSMCs. In contrast, the activity of the inducible minimal antioxidant response element (ARE) was potentiated in Pin1-null mouse embryonic fibroblasts (MEFs), compared to Pin1-wild-type MEFs. Moreover, Nrf2 ubiquitination was stimulated by Pin1 overexpression. Intraperitoneal injection of juglone (a Pin1 inhibitor) for 3 weeks (1 mg/kg, two times a week) significantly suppressed neointimal formation induced by wire injury. In conclusion, Pin1 induction during neointimal formation may be associated with ROS-mediated VSMC proliferation via down-regulation of Nrf2/ARE-dependent HO-1 expression. Pin1 may be a novel therapeutic target for several vascular diseases including atherosclerosis and stenosis.     

Puerarin attenuates high-glucose-and diabetes-induced vascular smooth muscle cell proliferation by blocking PKCβ2/Rac1-dependent signaling

Oxidative stress has been implicated in several steps leading to the development of diabetic vascular complications. The purpose of this study was to determine the efficacy and the possible mechanism of puerarin on high-glucose (HG; 25 mM)-induced proliferation of cultured rat vascular smooth muscle cells (VSMCs) and neointimal formation in a carotid arterial balloon injury model of obese Zucker rats. Our data demonstrated that puerarin significantly inhibited rat VSMC proliferation as well as reactive oxygen species (ROS) generation and NADPH oxidase activity induced by HG treatment. Further studies revealed that HG treatment resulted in phosphorylation and membrane translocation of PKCβ2 as well as Rac1, p47phox, and p67phox subunits, leading to NADPH oxidase activation. Puerarin treatment remarkably disrupted the phosphorylation and membrane translocation of PKCβ2 as well as Rac1, p47phox, and p67phox subunits. Blocking PKCβ2 by infection with AdDNPKCβ2 also abolished HG-induced phosphorylation and membrane translocation of Rac1, p47phox, and p67phox subunits as well as ROS production and NADPH oxidase activation in VSMCs. In vivo neointimal formation of obese Zucker rats evoked by balloon injury was evidently attenuated by the administration of puerarin. These results demonstrate that puerarin may exert inhibitory effects on HG-induced VSMC proliferation via interfering with PKCβ2/Rac1-dependent ROS pathways, thus resulting in the attenuation of neointimal formation in the context of hyperglycemia in diabetes mellitus.     

TIPE2 deficiency accelerates neointima formation by downregulating smooth muscle cell differentiation

Phenotypic switching of vascular smooth muscle cells (VSMCs) is known to play a key role in the development of atherosclerosis. However, the mechanisms that mediate VSMC phenotypic switching are unclear. We report here that TIPE2, the tumor necrosis factor (TNF) α-induced protein 8-like 2 (TNFAIP8L2), plays an atheroprotective role by regulating phenotypic switching of VSMCs in response to oxidized low-density lipoprotein (ox-LDL) stimuli. TIPE2-deficient VSMCs treated with ox-LDL expressed lower levels of contractile proteins such as SMαA, SM-MHC and calponin, whereas the proliferation, migration and the synthetic capacity for growth factors and cytokines were increased remarkably. Furthermore, TIPE2 inhibited VSMCs proliferation by preventing G₁/S phase transition. Interestingly, these effects of TIPE2 on VSMCs were dependent on P38 and ERK1/2 kinase signals. As a result, neointima formation was accelerated in the carotid arteries of TIPE2-deficient mice. These results indicate that TIPE2 is a potential inhibitor of atherosclerosis.     

Carotid artery ligation induced intimal thickening and proliferation is unaffected by ageing

Following interventions to treat atherosclerosis, such as coronary artery bypass graft surgery, restenosis occurs in approximately 40% of patients. Identification of proteins regulating intimal thickening could represent targets to prevent restenosis. Our group previously demonstrated that in a murine model of vascular occlusion, Wnt4 protein expression and β-catenin signalling was upregulated which promoted vascular smooth muscle cell (VSMC) proliferation and intimal thickening. In this study, the effect of age on VSMC proliferation, intimal hyperplasia and Wnt4 expression was investigated. In vitro proliferation of VSMCs isolated from young (2 month) or old (18–20 month) C57BL6/J mice was assessed by immunocytochemistry for EdU incorporation. As previously reported, 400 ng/mL recombinant Wnt4 protein increased proliferation of VSMCs from young mice. However, this response was absent in VSMCs from old mice. As our group previously reported reduced intimal hyperplasia in Wnt4^+/? mice compared to wildtype controls, we hypothesised that impaired Wnt4 signalling with age may result in reduced neointimal formation. To investigate this, carotid artery ligation was performed in young and old mice and neointimal area was assessed 21 days later. Surprisingly, neointimal area and percentage lumen occlusion were not significantly affected by age. Furthermore, neointimal cell density and proliferation were also unchanged. These data suggest that although Wnt4-mediated proliferation was impaired with age in primary VSMCs, carotid artery ligation induced neointimal formation and proliferation were unchanged in old mice. These results imply that Wnt4-mediated proliferation is unaffected by age in vivo, suggesting that therapeutic Wnt4 inhibition could inhibit restenosis in patients of all ages.     

Angiogenic factor AGGF1 blocks neointimal formation after vascular injury via interaction with integrin α7 on vascular smooth muscle cells

Angiogenic factor AGGF1 (AngioGenic factor with G-patch and FHA (Forkhead-Associated) domain 1) blocks neointimal formation (formation of a new or thickened layer of arterial intima) after vascular injury by regulating phenotypic switching of vascular smooth muscle cells (VSMCs). However, the AGGF1 receptor on VSMCs and the underlying molecular mechanisms of its action are unknown. In this study, we used functional analysis of serial AGGF1 deletions to reveal the critical AGGF1 domain involved in VSMC phenotypic switching. This domain was required for VSMC phenotypic switching, proliferation, cell cycle regulation, and migration, as well as the regulation of cell cycle inhibitors cyclin D, p27, and p21. This domain also contains an RDDAPAS motif via which AGGF1 interacts with integrin α7 (ITGA7), but not α8. In addition, we show that AGGF1 enhanced the expression of contractile markers MYH11, α-SMA, and SM22 and inhibited MEK1/2, ERK1/2, and ELK phosphorylation in VSMCs, and that these effects were inhibited by knockdown of ITGA7, but not by knockdown of ITGA8. In vivo, deletion of the VSMC phenotypic switching domain in mice with vascular injury inhibited the functions of AGGF1 in upregulating α-SMA and SM22, inhibiting MEK1/2, ERK1/2, and ELK phosphorylation, in VSMC proliferation, and in blocking neointimal formation. Finally, we show the inhibitory effect of AGGF1 on neointimal formation was blocked by lentivirus-delivered shRNA targeting ITGA7. Our data demonstrate that AGGF1 interacts with its receptor integrin α7 on VSMCs, and this interaction is required for AGGF1 signaling in VSMCs and for attenuation of neointimal formation after vascular injury.     

siRNAs target sites selection of ezrin and the influence of RNA interference on ezrin expression and biological characters of osteosarcoma cells

Tissue kallikrein 1 cleaves kininogen substrate to produce vasoactive kinin peptides that have been implicated in inhibiting neointimal hyperplasia in rat carotid arteries after balloon injury. However, its effects on the proliferation, cell cycle and its mechanisms, for example, cyclin-dependent kinase inhibitors, p27(Kip1) and p2l(Cip1) in vascular biology are poorly understood. The objective of this study was to explore the effects of human tissue kallikrein 1 (hTK1) mediated by recombinant adenovirus (Ad-hTK1) on proliferation and cell cycle of vascular smooth muscle cells (VSMCs) derived from spontaneously hypertensive rats induced by platelet-derived growth factor-BB (PDGF-BB) in vitro. The results showed that, within a given multiplicity of infection (MOI) and time, the hTK1 gene delivery inhibited PDGF-BB-stimulating VSMCs growth in a concentration-dependent (20-100 MOI) and time-dependent (2-5 days) manner by cell counting, with a peak inhibition rate at 36.3% at 72 h (P < 0.01). In addition, hTK1 gene delivery significantly suppressed PDGF-BB-induced proliferation of VSMCs by methyl thiazolyl tetrazoliuin assay, and decreased the percentage of cells in the S phase and in DNA synthesis by flow cytometry, with a peak inhibition rate at 30.2 and 36.4%, respectively (P < 0.01). Western blot assay showed that the protein levels of p27(Kip1) and p2l(Cip1) in cells infected with Ad-hTK1 were much more abundant than those in cells only induced by PDGF-BB, with up-modulating rates at 51.8 and 58.7%, respectively (P < 0.001). We also observed that the effects of hTK1 gene delivery in inhibiting VSMCs proliferation, arresting cell cycling in G(0)/G(1) phase and up-regulating the expression of p27(Kip1) and p2l(Cip1) could be blocked by icatibant (Hoe 140), a specific bradykinin B(2) receptor antagonist. Taken together, these results demonstrated that hTK1 overexpressed by recombinant adenovirus potently inhibits VSMCs proliferation that is required for neointimal hyperplasia and restenosis, and may activate p27(Kip1) and p2l(Cip1) signaling pathways via bradykinin B(2) receptor.     

Role of platelet-derived growth factor-BB (PDGF-BB) in human pulmonary artery smooth muscle cell proliferation

Abstract

Pulmonary arterial hypertension (PAH) is a vascular remodeling disease characterized by enhanced proliferation of pulmonary artery smooth muscle cells (PASMCs) and suppressed apoptosis. Platelet-derived growth factor (PDGF) is a potent mitogen involved in cell proliferation and migration. PDGF-BB induces the proliferation and migration of PASMCs and has been proposed to be a key mediator in the progression of PAH. Previous studies have shown that PDGF and its receptor are substantially elevated in lung tissues and PASMCs isolated from patients and animals with PAH, but the underlying mechanisms are still poorly manifested. MAP kinases, including extracellular signal-regulated kinase1/2 (ERK1/2), c-Jun NH2-terminal kinase1/2 (JNK1/2), and p38 are the key intracellular signals for stimuli-induced cell proliferation, survival, and apoptosis. Therefore, the purpose of this study is to determine whether PDGF-BB on cell proliferation process is mediated through the MAP kinases pathway in human PASMCs (HPASMCs). Our results showed PDGF-BB-induced proliferating cell nuclear antigen (PCNA), Cyclin A and Cyclin E expression in a concentration-dependent manner. The expression levels of phosphorylated JNK (p-JNK) was upregulated with 20?ng/ml PDGF-BB treatment, while PDGF-BB could not increase phosphorylated ERK1/2 (p-ERK1/2) and p-38 (p-p38) expression. The effects of PDGF-BB on cell proliferation and survival were weakened after the administration of antagonist of the JNK pathway or si-JNK. In addition, PDGF-BB protected against the loss of mitochondrial membrane potentials evoked by serum deprivation (SD) in a JNK-dependent manner. These results suggest that PDGF-BB promotes HPASMCs proliferation and survival, which is likely to be mediated via the JNK pathway.     

Role of heat shock protein 27 phosphorylation in migration of vascular smooth muscle cells

Objective The aim of the present study was to investigate the role of heat shock protein 27 (HSP27) phosphorylation in the migration of vascular smooth muscle cells (VSMCs) induced by angiotensin II (AngII) and platelet derived growth factor-BB (PDGF-BB). Methods The activity of HSP27 was evaluated by Western blot with specific phospho-HSP27 antibody. F-actin polymerization was detected by FITC-Phalloidine staining using confocal microscopy. Modified Boyden chamber technique was employed for VSMCs migration assessment. Results The phosphorylation of HSP27 was induced by AngII and PDGF-BB in a time- and concentration-dependent manner in VSMCs, which was significantly blocked by the HSP inhibitor Quercetin in a concentration-dependent manner. Reorganization of actin stimulated by AngII and PDGF-BB was markedly inhibited by pretreatment with 100 μmol/l Quercetin. The migration of VSMCs induced by AngII and PDGF-BB was partially inhibited by Quercetin with peak inhibition concentration at 100 μmol/l. Conclusions HSP27 phosphorylation plays an important role in mediating the rearrangement of F-actin and migration of VSMCs induced by AngII and PDGF-BB. HSP27 may be a potential target for the interventional treatment of pathological process related to cell migration.     

Myricetin suppresses the proliferation and migration of vascular smooth muscle cells and inhibits neointimal hyperplasia via suppressing TGFBR1 signaling pathways

BackgroundNeointimal formation, mediated by the proliferation and migration of vascular smooth muscle cells (VSMCs), is a common pathological basis for atherosclerosis and restenosis. Myricetin, a natural flavonoid, reportedly exerts anti-atherosclerotic effects. However, the effect and mechanism of myricetin on VSMCs proliferation and migration and neointimal hyperplasia (NIH) remain unknown.PurposeWe investigated myricetin's effect on NIH, as well as the potential involvement of transforming growth factor-beta receptor 1 (TGFBR1) signaling in mediating myricetin's anti-atherosclerotic and anti-restenotic actions.MethodsMyricetin's effects on the proliferation and migration of HASMCs and A7R5 cells were determined by CCK-8, EdU assays, wound healing, Transwell assays, and western blotting (WB).Molecular docking, molecular dynamics (MD) simulation, surface plasmon resonance (SPR) and TGFBR1 kinase activity assays were employed to investigate the interaction between myricetin and TGFBR1. An adenovirus vector encoding TGFBR1 was used to verify the effects of myricetin. In vivo, the left common carotid artery (LCCA) ligation mouse model was adopted to determine the impacts of myricetin on neointimal formation and TGFBR1 activation.ResultsMyricetin dose-dependently inhibited the migration and proliferation in VSMCs, suppressed the expression of CDK4, cyclin D3, MMP2, and MMP9. Molecular docking revealed that myricetin binds to key regions for TGFBR1 antagonist binding, and the binding energy was -9.61 kcal/mol. MD simulation indicated stable binding between TGFBR1 and myricetin. Additionally, SPR revealed an equilibrium dissociation constant of 4.35 × 10⁻⁵ M between myricetin and TGFBR1. According to the TGFBR1 kinase activity assay, myricetin directly inhibited TGFBR1 kinase activity (IC₅₀ = 8.551 μM). Furthermore, myricetin suppressed the phosphorylation level of TGFBR1, Smad2, and Smad3 in a dose-dependent pattern, which was partially inhibited by TGFBR1 overexpression. Consistently, TGFBR1 overexpression partially rescued the suppressive roles of myricetin on VSMCs migration and proliferation. Moreover, myricetin dramatically inhibited NIH and reduced TGFBR1, Smad2, and Smad3 phosphorylation in the LCCA.ConclusionThis is the first study to demonstrate that myricetin suppresses NIH and VSMC proliferation and migration via inhibiting TGFBR1 signaling. Myricetin can be developed as a potential therapeutic candidate for treating atherosclerosis and vascular restenosis.     

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.     

Bone marrow-derived mesenchymal stem cells inhibit vascular smooth muscle cell proliferation and neointimal hyperplasia after arterial injury in rats

We investigated whether mesenchymal stem cell (MSC)-based treatment could inhibit neointimal hyperplasia in a rat model of carotid arterial injury and explored potential mechanisms underlying the positive effects of MSC therapy on vascular remodeling/repair. Sprague-Dawley rats underwent balloon injury to their right carotid arteries. After 2 days, we administered cultured MSCs from bone marrow of GFP-transgenic rats (0.8 × 10⁶ cells, n = 10) or vehicle (controls, n = 10) to adventitial sites of the injured arteries. As an additional control, some rats received a higher dose of MSCs by systemic infusion (3 × 10⁶ cells, tail vein; n = 4). Local vascular MSC administration significantly prevented neointimal hyperplasia (intima/media ratio) and reduced the percentage of Ki67 + proliferating cells in arterial walls by 14 days after treatment, despite little evidence of long-term MSC engraftment. Notably, systemic MSC infusion did not alter neointimal formation. By immunohistochemistry, compared with neointimal cells of controls, cells in MSC-treated arteries expressed reduced levels of embryonic myosin heavy chain and RM-4, an inflammatory cell marker. In the presence of platelet-derived growth factor (PDGF-BB), conditioned medium from MSCs increased p27 protein levels and significantly attenuated VSMC proliferation in culture. Furthermore, MSC-conditioned medium suppressed the expression of inflammatory cytokines and RM-4 in PDGF-BB-treated VSMCs. Thus, perivascular administration of MSCs may improve restenosis after vascular injury through paracrine effects that modulate VSMC inflammatory phenotype.     

The signal transduction pathways of heat shock protein 27 phosphorylation in vascular smooth muscle cells

The objective of this study is to investigate the signal transduction pathways that regulate heat shock protein 27 (HSP27) phosphorylation and migration of vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) induced by angiotensin II (AngII) and platelet derived growth factor-BB (PDGF-BB). The activity of HSP27 was evaluated by Western blot with specific phospho-HSP27 antibody. F-actin polymerization was detected by FITC-Phalloidine staining using confocal microscopy. Modified Boyden chamber technique was employed for VSMCs migration assessment. Within a given concentration, the phosphorylation of HSP27 induced by AngII and PDGF-BB was blocked by the specific P38MAPK inhibitor SB202190, the specific PI3K inhibitor LY294002 and the specific ERK1/2 inhibitor U0126 in a concentration-dependent manner, with a peak inhibition rate at 87.2%, 78.4% and 37.3%, respectively, induced by AngII (P < 0.01), with a peak inhibition rate at 85.0%, 55.3% and 41.0%, respectively, induced by PDGF-BB (P < 0.01).The migration of VSMCs induced by AngII and PDGF-BB was inhibited by 100 μmol/l SB202190, 30 μmol/l LY294002, and 30 μmol/l U0126, with a inhibition rate at 60.1%, 71.7% and 47.3%, respectively, provoked by AngII (P < 0.01), with a inhibition rate at 55.3%, 55.6% and 38.1%, respectively, provoked by PDGF-BB (P < 0.01). P38MAPK and PI3 K/Akt are important pathways that contribute to the phosphorylation of HSP27 and migration of VSMCs in response to AngII and PDGF-BB. ERK1/2 might be involved in HSP27 phosphorylation and migration of VSMCs provoked by AngII and PDGF-BB.