PDGF-induced Vascular Smooth Muscle Cell Migration Research Articles

PS-B01-8: MALAYSIAN TUALANG HONEY INHIBITS PDGF-INDUCED HUMAN VASCULAR SMOOTH MUSCLE CELLS PROLIFERATION AND MIGRATION

Introduction and Objective: Cardiovascular diseases (CVD) are the leading cause of death globally. Atherosclerosis is a common CVD which can lead to the deadliest heart failure. Increasing evidence showed that abnormal vascular smooth muscle cells (VSMC) proliferation and migration constitute the main pathological basis of atherosclerosis. These events are potently stimulated by platelet-derived growth factor (PDGF), causing phenotypic switching of VSMC from a mature, quiescent phenotype to a proliferative, migratory phenotype which is commonly observed in the development of atherosclerosis. Furthermore, study have revealed that PDGF elevates the concentration of reactive oxygen species (ROS) which stimulates VSMC proliferation and migration. Pharmacological treatment for atherosclerosis is often an ongoing, long-term regimen that may have adverse effect to an individual. Thus, alternative therapy based on natural products can potentially be the frontier in atherosclerosis treatment. Interestingly, previous study revealed that Malaysian Tualang Honey (MTH) is capable of protecting vascular endothelial cells from inflammation. However, little is known on the effect of MTH on the PDGF-induced VSMC proliferation and migration. Therefore, the present study aimed to determine the antioxidant, anti-proliferative and anti-migratory effects of MTH in PDGF-induced VSMC. Methodology: VSMC were treated with increasing concentrations of MTH (0.001–10%) for 24 hours and the cell viability were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were then be treated with non-cytotoxic concentrations of MTH and induced with 20ng/mL PDGF for 72 hours to determine the proliferation rate via MTT assay. The intracellular ROS were measured in these cultured cells by dichlorodihydrofluorescein diacetate (DCFH-DA) assay. Wound healing assay were used to determine the anti-migration activities on PDGF-induced VSMC treated with MTH. Results: Our data showed that MTH concentrations ranging from 0.01% to 1.00% showed no cytotoxic effect to VSMC. Co-culture of the cells with MTH significantly inhibits the proliferation and migration of PDGF-stimulated VSMC in a concentration-dependent manner. Pre-treatment of cells with MTH prior to culturing in 20ng/mL PDGF showed a significant decrease in the production of ROS. Conclusion: This study demonstrates that MTH inhibits VSMC proliferation and migration by suppressing the production of ROS which constitute the pharmacological basis of anti-atherosclerotic action. Therefore, MTH potentially can be a promising therapeutic agent in the treatment of atherosclerosis.

Abstract MP11: Histone Methyl Transferase (suv39h1): Function In Smooth Muscle Cell Phenotypic Plasticity

Introduction: The phenotypic plasticity of vascular smooth muscle cells (VSMCs) is central to growth and remodeling processes, but also contributes to cardiovascular disease. This unique ability of VSMCs to reversibly differentiate and de-differentiate is incompletely understood. SUV39H1 is a histone methyltransferase that specifically trimethylates Lysine-9 of histone H3 (H3K9me3), resulting in transcriptional repression through epigenetic gene silencing. Hypothesis: We hypothesized that SUV39H1 may play a role in SMC phenotypic switch. Methods: Using in vitro and in vivo approaches including knockdown, qPCR, western, and chromatin immunoprecipitation (ChIP) assays to determine role of SUV39H1 in SMC plasticity. Results: A qPCR array screen of epigenetic regulators in VSMCs identified SUV39H1 mRNA as upregulated with PDGF induced dedifferentiation and downregulated with rapamycin induced differentiation. This was confirmed at the protein level. SUV39H1 knockdown significantly increased VSMC contractile protein mRNA, protein levels and decreased dedifferentiation associated gene expression, and also decreased PDGF-induced VSMC migration (n=4). Interestingly, we found that expression of KLF4, the master transcriptional regulator of dedifferentiation in SMCs, was dramatically decreased after SUV39H1 knockdown (n=5). We further noted that SUV39H1 knockdown decreased KLF4 mRNA stability (n=3). Mechanistically, SUV39H1 knockdown increased miRNA143, a well-known repressor of KLF4. ChIP assays at contractile gene promoters showed a significant decrease in H3K9me3 mark and an increase in H3K27 acetylation, an activation mark (n=4). Carotid artery ligation induced intimal hyperplastic lesions in wild type C57BL/6 mice, showed a significant increase in SUV39H1 and H3K9me3 expression compared to uninjured vessels. Conclusion: We identify SUV39H1 an epigenetic regulator of VSMC phenotype whose expression and activity increase with dedifferentiation in vitro and in vivo . Mechanistically, SUV39H1 influences VSMC chromatin marks and KLF4 expression to repress contractile phenotype. Understanding the role of SUV39H1 and its targets may have implications for developing new therapeutic strategies for treating vascular diseases.

Endoleak Detection after Endovascular Aortic Aneurysm Repair Using Ultrasound Based on Nanoscale Bubble Contrast Agents and Their Effects on Vascular Smooth Muscle Cell Proliferation and Migration

Abdominal aortic aneurysm (AAA) is the most common vascular disease that causes disability and death. Its morbidity is relatively subtle, and the mortality rate is high. Clinically, endovascular aortic aneurysm repair (EVAR) has gradually become the primary treatment of AAA due to its unique advantages such as low trauma and low incidence of short-term complications. However, the outcome of EVAR is greatly compromised by the possible occurrence of endoleaks. Contrast-enhanced ultrasound (CEUS) is a promising alternative technique to detect endoleaks following EVAR due to lack of exposure to ionizing radiation. Traditional ultrasound contrast agents with an overlarge size (microscale) leading to reluctant accumulation in target organs and instability trigger the requirement of nanoscale contrast agents that enter tumor tissues through the enhanced permeability and retention effect. In this study, we used ultrasound based on nanoscale bubble contrast agents to evaluate endoleak detection after endovascular aortic aneurysm repair and analyzed the effects of nanoscale bubble contrast agents on vascular smooth muscle cell (VSMC) proliferation and migration. Among 52 AAA patients a month following EVAR, there were 16 cases of endoleaks after EVAR detected by nanobubble contrast-enhanced ultrasound, including 6 cases of type I endoleak (1 case of type Ia endoleak and 5 cases of type Ib endoleak), 7 cases of type II endoleak, and 3 cases of type III endoleak; there were 12 cases of endoleak after EVAR detected by computed tomography angiography (CTA), including 6 cases of type I endoleak (1 case of type Ia and 5 cases of type Ib), 5 cases of type II endoleak, and 1 case of type III endoleak. Six months after EVAR, 3 cases of type III endoleak were detected by both nanobubble contrast-enhanced ultrasound and CTA. Vascular smooth muscle cells (VSMCs) used for in vitro experiments were subjected to ultrasound irradiation and platelet-derived growth factor (PDGF) treatment with or without the addition of nanobubble contrast agents. After high-intensity and long-term irradiation (0.75 W/cm2 and 1 W/cm2 irradiation for 120 s and 150 s, respectively) by ultrasound with or without the addition of nanobubble contrast agents, PDGF-induced VSMC migration was inhibited ( P < 0.01 ). Low-intensity and short-term ultrasound irradiation did not differ PDGF-induced VSMC migration ( P > 0.05 ), but 0.5 W/cm2 and 90 s ultrasound irradiation could significantly inhibit PDGF-induced VSMC migration without the addition of nanobubble contrast agents ( P < 0.05 ). When VSMCs were irradiated at 1, 0.75, 0.5, and 0.35 W/cm2 for 30 s to 150 s, ultrasound irradiation with or without the addition of nanobubble contrast agents remarkably reduced PDGF-induced VSMC proliferation, as evidenced by reduced OD values ( P < 0.05 ). In conclusion, ultrasound based on nanoscale bubble contrast agents is an effective alternative detection method for the occurrence of AAA patients who are not suitable for CTA.

Reactive sulfur species inhibit the migration of PDGF-treated vascular smooth muscle cells by blocking the reactive oxygen species-regulated Akt signaling pathway

Vascular smooth muscle cell (VSMC) migration contributes to vascular remodeling after injury, whereas oxidative stress generated through dysfunctional redox homeostasis induces hypermigration, leading to arteriosclerosis. Platelet-derived growth factor (PDGF)-induced reactive oxygen species (ROS) serve as intracellular signaling molecules in VSMCs. Reactive sulfur species (RSS) may serve as a biological defense system because of the antioxidative properties of highly nucleophilic sulfane sulfur. However, insufficient information is available on its function in PDGF-induced VSMC migration. Here we show that PDGF significantly increased the levels of intracellular sulfane sulfur and that intracellular sulfane sulfur donors, donor 5a and Na2S4, inhibited the increase in ROS levels in PDGF-treated VSMCs and inhibited their migration. Consistent with the migration results, sulfane sulfur donors inhibited Akt phosphorylation, a downstream signaling molecule in the PDGF cascade, without affecting the autophosphorylation of PDGF receptor-β. Further, sulfane sulfur donors inhibited vinculin and paxillin recruitment to the leading edge of VSMCs in response to PDGF to decrease focal adhesion formation. These findings suggest that RSS are required for PDGF-stimulated VSMC migration through the regulation of the ROS-regulated Akt pathway, which may contribute to focal adhesion formation. Our findings provide insight into RSS as novel regulators of vascular redox homeostasis.

Abstract 16659: Nogo-B Receptor is Required for the Recruitment of Vascular Smooth Muscle Cells to Developing Blood Vessels

The blood vessel maturation involves the coordination of two cell types - endothelial cells (EC) and vascular smooth muscle cells (VSMC). Hence, understanding of EC and VSMC migration is an essential step in developing new therapeutic approaches for diseases-associated with blood vessel maturation defects. PDGF plays a critical role in blood vessel maturation by promoting VSMC proliferation and migration via Ras signaling pathway. Although Nogo-B receptor (NgBR) was identified as a cell surface receptor special for the ligand Nogo-B, it acts as a scaffold protein requested for Ras translocation to the plasma membrane, which is a critical step for Ras activation by PDGF receptor and other receptor tyrosine kinases. NgBR is expressed in both EC and VSMC. Previous reports demonstrated the genetic deletion of NgBR in endothelial cells causes the embryonic lethality due to cerebrovascular defects in mice. Here, we further elucidate the roles of NgBR in regulating the recruitment of VSMC in the developing blood vessels. The genetic deletion of NgBR in VSMC also results in embryonic lethality due to impaired VSMC coverage on blood vessels and hemorrhage. Mechanistically, our data demonstrated that NgBR is required for PDGF-induced Ras signaling pathway and VSMC migration. Interestingly, we also appreciate the defects of VSMC recruitment in the blood vessels of NgBR EC-specific knockout mice. Gene profile analysis reveals that NgBR knockdown decreased the expression of PDGF in EC. Collectively, these data demonstrate that NgBR is an integral part of blood vessel maturation process by facilitating VSMC recruitment. Our results suggest that NgBR may be one of the essential genes coordinating the blood vessel development.

Copper transporter ATP7A interacts with IQGAP1, a Rac1 binding scaffolding protein: role in PDGF-induced VSMC migration and vascular remodeling.

Vascular smooth muscle cell (VSMC) migration contributes to neointimal formation after vascular injury. We previously demonstrated that copper (Cu) transporter ATP7A is involved in platelet-derived growth factor (PDGF)-induced VSMC migration in a Cu- and Rac1-dependent manner. The underlying mechanism is still unknown. Here we show that ATP7A interacts with IQGAP1, a Rac1 and receptor tyrosine kinase binding scaffolding proteins, which mediates PDGF-induced VSMC migration and vascular remodeling. In cultured rat aortic SMCs, PDGF stimulation rapidly promoted ATP7A association with IQGAP1 and Rac1 and their translocation to the lipid rafts and leading edge. Cotransfection assay revealed that ATP7A directly bound to NH2-terminal domain of IQGAP1. Functionally, either ATP7A or IQGAP1 depletion using siRNA significantly inhibited PDGF-induced VSMC migration without additive effects, suggesting that IQGAP1 and ATP7A are in the same axis to promote migration. Furthermore, IQGAP1 siRNA blocked PDGF-induced ATP7A association with Rac1 as well as its translocation to leading edge, while PDGF-induced IQGAP1 translocation was not affected by ATP7A siRNA or Cu chelator. Overexpression of mutant IQGAP1 lacking a Rac1 binding site prevented PDGF-induced translocation of Rac1, but not ATP7A, to the leading edge, thereby inhibiting lamellipodia formation and VSMC migration. In vivo, ATP7A colocalized with IQGAP1 at neointimal VSMCs in a mice wire injury model, while neointimal formation and extracellular matrix deposition induced by vascular injury were inhibited in ATP7A mutant mice with reduced Cu transporter function. In summary, IQGAP1 functions as ATP7A and Rac1 binding scaffolding protein to organize PDGF-dependent ATP7A translocation to the lamellipodial leading edge, thereby promoting VSMC migration and vascular remodeling.

MiR-145-5p Inhibits Vascular Smooth Muscle Cells (VSMCs) Proliferation and Migration by Dysregulating the Transforming Growth Factor-b Signaling Cascade.

BackgroundThere is accumulating evidence demonstrating that microRNAs (miRNA) play essential roles in proliferation, migration, and invasion of vascular smooth muscle cells (VSMCs). However, the exact function of these molecules and the mechanisms involved are not fully understood. In this study, we defined the role of miR-145-5p in VSMCs.Material/MethodsThis study used the PDGF-bb-induced VSMCs proliferation model. Expression of miR-145-5p and its target, Smad4, were detected and measured by real-time PCR and Western blot analysis. The luciferase reporter of miR-145-5p was used to elucidate miRNA-target interactions. The functions of miR-145-5p in proliferation and migration were detected by CCK-8 assay, Transwell assay, and scratch test.ResultsThis study demonstrates that miR-145-5p is downregulated in PDGF-mediated VSMCs in both time- and dose-dependent manners. The in vitro results suggest that overexpression of miR-145-5p results in a reduction in SMAD4 and an increase in SMAD2, Smad3, and TGF-β at the mRNA and protein levels. Overexpression of miR-145-5p inhibited PDGF-induced VSMCs proliferation and migration. Moreover, SMAD4 was identified as a direct target of miR-145-5p and is involved in PDGF-mediated VSMC proliferation. Downstream factors such as Smad2, Smad3, and TGF-β were also influenced by miR-145-5p.ConclusionsWe identify miR-145-5p as a novel regulator of VSMC. Moreover, miR-145-5p inhibits VSMCs proliferation and migration by directly targeting Smad4 and dysregulating the transforming growth factor-β signaling cascade, including Smad2, Smad3, and TGF-β.

Rubiarbonone C inhibits platelet-derived growth factor-induced proliferation and migration of vascular smooth muscle cells through the focal adhesion kinase, MAPK and STAT3 Tyr705 signalling pathways.

The proliferation and migration of vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor (PDGF) are important steps in cardiovascular diseases, including neointimal lesion formation, myocardial infarction and atherosclerosis. Here, we evaluated the rubiarbonone C-mediated signalling pathways that regulate PDGF-induced VSMC proliferation and migration. Cell proliferation and migration were measured in cells treated with rubiarbonone C followed by PDGF BB using the MTT assay, [3 H]-thymidine incorporation, flow cytometry and wound-healing migration assay, MMP gelatin zymography, a fluorescence assay for F-actin. Western blotting of molecules including MAPK, focal adhesion kinase (FAK) and STAT3 and an immunofluorescence assay using anti-PCNA and -STAT3 antibodies were performed to evaluate rubiarbonone C signalling pathway(s). The medial thickness of the carotid artery was evaluated using a mouse carotid ligation model. Rubiarbonone C inhibited PDGF-induced VSMC proliferation and migration and diminished the ligation-induced increase in medial thickness of the carotid artery. In PDGF-stimulated VSMCs rubiarbonone C decreased the following: (i) levels of cyclin-dependent kinases, cyclins, PCNA and hyperphosphorylated retinoblastoma protein; (ii) levels and activity of MMP2 and MMP9; (iii) activation of MAPK; (iv) F-actin reorganization, by reducing FAK activation; (v) activation of STAT3. These findings suggest that rubiarbonone C inhibits the proliferation and migration of VSMCs by inhibiting the FAK, MAPK and STAT3 signalling pathways. Therefore, rubiarbonone C could be a good candidate for the treatment of cardiovascular disease.

MYBPH inhibits vascular smooth muscle cell migration and attenuates neointimal hyperplasia in a rat carotid balloon-injury model

Vascular smooth muscle cell (VSMC) migration is implicated in restenosis. Myosin binding protein H (MYBPH) is capable of reducing cell motility and metastasis. In this study, we sought to determine whether MYBPH is involved in VSMC migration and neointima formation in response to vascular injury. To determine the expression of MYBPH in injured artery, we used a standard rat carotid artery balloon-injury model. In vivo studies have demonstrated that MYBPH is upregulated after vascular injury. VSMCs treated with platelet-derived growth factor (PDGF)-BB displayed increased MYBPH mRNA and protein levels. PDGF-induced VSMC migration was inhibited by adenovirus-mediated expression of MYBPH whereas it was enhanced by small interfering RNA knockdown of MYBPH. The activation of ROCK1 was repressed by MYBPH. Luminal delivery of MYBPH adenovirus to carotid arteries decreased neointimal hyperplasia in vivo. MYBPH may, therefore, serve as a novel therapeutic target for postangioplasty restenosis.

Aspirin-triggered resolvin D1 attenuates PDGF-induced vascular smooth muscle cell migration via the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway.

Background and objectivesResolvin D1 (RvD1) is a specialized pro-resolving lipid mediator that has been previously shown to attenuate vascular smooth muscle cell (VSMC) migration, a key process in the development of intimal hyperplasia. We sought to investigate the role of the cAMP/PKA pathway in mediating the effects of the aspirin-triggered epimer 17R-RvD1 (AT-RvD1) on VSMC migration.MethodsVSMCs were harvested from human saphenous veins. VSMCs were analyzed for intracellular cAMP levels and PKA activity after exposure to AT-RvD1. Platelet-derived growth factor (PDGF)-induced migration and cytoskeletal changes in VSMCs were observed through scratch, Transwell, and cell shape assays in the presence or absence of a PKA inhibitor (Rp-8-Br-cAMP). Further investigation of the pathways involved in AT-RvD1 signaling was performed by measuring Rac1 activity, vasodilator stimulated phosphoprotein (VASP) phosphorylation and paxillin translocation. Finally, we examined the role of RvD1 receptors (GPR32 and ALX/FPR2) in AT-RvD1 induced effects on VSMC migration and PKA activity.ResultsTreatment with AT-RvD1 induced a significant increase in cAMP levels and PKA activity in VSMCs at 5 minutes and 30 minutes, respectively. AT-RvD1 attenuated PDGF-induced VSMC migration and cytoskeletal rearrangements. These effects were attenuated by the PKA inhibitor Rp-8-Br-cAMP, suggesting cAMP/PKA involvement. Treatment of VSMC with AT-RvD1 inhibited PDGF-stimulated Rac1 activity, increased VASP phosphorylation, and attenuated paxillin localization to focal adhesions; these effects were negated by the addition of Rp-8-Br-cAMP. The effects of AT-RvD1 on VSMC migration and PKA activity were attenuated by blocking ALX/FPR2, suggesting an important role of this G-protein coupled receptor.ConclusionsOur results suggest that AT-RvD1 attenuates PDGF-induced VSMC migration via ALX/FPR2 and cAMP/PKA. Interference with Rac1, VASP and paxillin function appear to mediate the downstream effects of AT-RvD1 on VSMC migration.

Abstract 31: Resolvin D1 Attenuates PDGF-induced Vascular Smooth Muscle Cell Migration via the cAMP Pathway

Introduction: Resolvin D1 (RvD1), a specialized pro-resolving lipid mediator (SPM), attenuates migration in vascular smooth muscle cells (VSMC), which is critical to the development of neointimal hyperplasia. SPM are known to interact with G-protein coupled receptors (GPCR). We sought to investigate the pathways by which RvD1 influences VSMC migration. Methods: VSMC were harvested from human saphenous veins. cAMP levels were measured via ELISA in the absence or presence of RvD1 receptor (ALX, GPR32) blockers. NF449, a G s -protein inhibitor, was also used. PDGF-BB (10ng/ml) was used as an agonist in a VSMC scratch assay as well as the receptor blockers. PDGF-BB-induced cytoskeletal changes were measured as aspect ratio after actin staining, and a scratch assay was used to assess migration. PDGF-induced Rac1 activity was measured via ELISA; VASP phosphorylation was assessed by Western blot and Paxillin translocation by Immunofluorescence. PKA inhibitor Rp-8-Br-cAMP (10μM) was used in the phenotypic and downstream signaling studies. Results: RvD1 treatment (10nM) of VSMC induced a significant acute flux in cAMP levels at 5 minutes (Fig. 1A; n≥3); this increase was abolished by an ALX antagonist (WRW4), the anti-GPR32 blocking Ab, and NF449. RvD1 (10nM) attenuated PDGF-induced VSMC migration (Fig. 1B-C; n≥3), cytoskeletal rearrangements (n=4), Rac1 activation (n≥3) and increased phosphorylation of VASP (n=3). These effects were negated by the addition of Rp-8-Br-cAMP, suggesting cAMP involvement. RvD1’s anti-migratory effect was reversed by blocking ALX or GPR32 (Fig. 1C; n≥8). Conclusion: Our results suggest that RvD1 attenuates VSMC migration by increasing levels of cAMP through ALX and GPR32 via a G s -protein-mediated action. Interference with Rac1, VASP and Paxillin function appear to be important for the anti-migratory activity of RvD1.

Decreasing mitochondrial fission diminishes vascular smooth muscle cell migration and ameliorates intimal hyperplasia

Vascular smooth muscle cell (VSMC) migration in response to arterial wall injury is a critical process in the development of intimal hyperplasia. Cell migration is an energy-demanding process that is predicted to require mitochondrial function. Mitochondria are morphologically dynamic, undergoing continuous shape change through fission and fusion. However, the role of mitochondrial morphology in VSMC migration is not well understood. The aim of the study is to understand how mitochondrial fission contributes to VSMC migration and provides its in vivo relevance in the mouse model of intimal hyperplasia. In primary mouse VSMCs, the chemoattractant PDGF induced mitochondrial shortening through the mitochondrial fission protein dynamin-like protein 1 (DLP1)/Drp1. Perturbation of mitochondrial fission by expressing the dominant-negative mutant DLP1-K38A or by DLP1 silencing greatly decreased PDGF-induced lamellipodia formation and VSMC migration, indicating that mitochondrial fission is an important process in VSMC migration. PDGF induced an augmentation of mitochondrial energetics as well as ROS production, both of which were found to be necessary for VSMC migration. Mechanistically, the inhibition of mitochondrial fission induced an increase of mitochondrial inner membrane proton leak in VSMCs, abrogating the PDGF-induced energetic enhancement and an ROS increase. In an in vivo model of intimal hyperplasia, transgenic mice expressing DLP1-K38A displayed markedly reduced ROS levels and neointima formation in response to femoral artery wire injury. Mitochondrial fission is an integral process in cell migration, and controlling mitochondrial fission can limit VSMC migration and the pathological intimal hyperplasia by altering mitochondrial energetics and ROS levels.

Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells

Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca2+ signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.

Antirestenosis Effect of Butein in the Neointima Formation Progression

The development of restenosis involves migration and hyperproliferation of vascular smooth muscle cells (VSMCs). Platelet-derived growth factor (PDGF) is one of the major factors. Butein modulates inflammatory pathways and affects the proliferation and invasion of the tumor. We investigated the hypothesis that butein might prevent the restenosis process via a similar pathway. Our results demonstrated that butein inhibited PDGF-induced VSMC proliferation and migration as determined by BrdU proliferation and two-dimensional migration scratch assay. Butein also concentration-dependently repressed PDGF-induced phosphorylation of PDGF-receptor β, mitogen-activated protein kinases, phosphoinositide 3-kinase/Akt, and phopholipase Cγ/c-Src in VSMCs. In addition, in vivo results showed that butein attenuated neointima formation in balloon-injured rat carotid arteries. These results indicate that butein may inhibit PDGF-induced VSMC proliferation and migration, resulting in attenuation of neointima formation after percutaneous transluminal coronary angioplasty. Our study demonstrates for the first time that systemic administration of butein is able to reduce neointima formation after vascular injury.

Role of Coronin 1B in PDGF-Induced Migration of Vascular Smooth Muscle Cells

The type I subclass of coronins, a family of actin-binding proteins, regulates various actin-dependent cellular processes, including migration. However, the existence and role of coronins in vascular smooth muscle cell (VSMC) migration has yet to be determined. The goal of the present study was to define the mechanism by which coronins regulate platelet-derived growth factor (PDGF)-induced VSMC migration. Coronin 1B (Coro1B) and 1C (Coro1C) were both found to be expressed in VSMCs at the mRNA and protein levels. Downregulation of Coro1B by siRNA increases PDGF-induced migration, while downregulation of Coro1C has no effect. We confirmed through kymograph analysis that the Coro1B-downregulation-mediated increase in migration is directly linked to increased lamellipodial protraction rate and protrusion distance in VSMC. In other cell types, coronins exert their effects on lamellipodia dynamics by an inhibitory interaction with the ARP2/3 complex, which is disrupted by the phosphorylation of Coro1B. We found that PDGF induces phosphorylation of Coro1B on serine-2 via PKCε, leading to a decrease in the interaction of Coro1B with the ARP2/3 complex. VSMCs transfected with a phosphodeficient S2A Coro1B mutant showed decreased migration in response to PDGF, suggesting that the phosphorylation of Coro1B is required for the promotion of migration by PDGF. In both the rat and mouse, Coro1B phosphorylation was increased in response to vessel injury in vivo. Our data suggest that phosphorylation of Coro1B and the subsequent reduced interaction with ARP2/3 complex participate in PDGF-induced VSMC migration, an important step in vascular lesion formation.

A naturally occurring carotenoid, lutein, reduces PDGF and H2O2 signaling and compromises migration in cultured vascular smooth muscle cells

BackgroundPlatelet-derived growth factor (PDGF) is a potent stimulator of growth and motility of vascular smooth muscle cells (VSMCs). Abnormalities of PDGF/PDGF receptor (PDGFR) are thought to contribute to vascular diseases and malignancy. We previously showed that a carotenoid, lycopene, can directly bind to PDGF and affect its related functions in VSMCs. In this study we examined the effect of the other naturally occurring carotenoid, lutein, on PDGF signaling and migration in VSMCs.MethodsWestern blotting was performed to examine PDGF and H2O2 signaling. Flowcytometry was used to determine PDGF binding to VSMCs. Fluorescence microscopy was performed to examine intracellular ROS production. Modified Boyden chamber system (Transwell apparatus) was used for migration assay.ResultsLutein reduced PDGF signaling, including phosphorylation of PDGFR-β and its downstream protein kinases/enzymes such as phospholipase C-γ, Akt, and mitogen-activated protein kinases (MAPKs). Although lutein possesses a similar structure to lycopene, it was striking that lutein inhibited PDGF signaling through a different way from lycopene in VSMCs. Unlike lycopene, lutein not only interacted with (bound to) PDGF but also interfered with cellular components. This was evidenced that preincubation of PDGF with lutein and treatment of VSMCs with lutein followed by removing of lutein compromised PDGF-induced signaling. Lutein reduced PDGF-induced intracellular reactive oxygen species (ROS) production and attenuated ROS- (H2O2-) induced ERK1/2 and p38 MAPK activation. A further analysis indicated lutein could inhibit a higher concentration of H2O2-induced PDGFR signaling, which is known to act through an oxidative inhibition of protein tyrosine phosphatase. Finally, we showed that lutein functionally inhibited PDGF-induced VSMC migration, whereas its stereo-isomer zeaxanthin did not, revealing a special action of lutein on VSMCs.ConclusionsOur study reveals a differential action mechanism of lutein from other reported caroteinoids and suggests a possible beneficial effect of lutein but not zeaxanthin on prevention of vascular diseases.

Slingshot Isoform-Specific Regulation of Cofilin-Mediated Vascular Smooth Muscle Cell Migration and Neointima Formation

We hypothesized that cofilin activation by members of the slingshot (SSH) phosphatase family is a key mechanism regulating vascular smooth muscle cell (VSMC) migration and neoinitima formation following vascular injury. Scratch wound and modified Boyden chamber assays were used to assess VSMC migration following downregulation of the expression of cofilin and each SSH phosphatase isoform (SSH1, SSH2, and SSH3) by small interfering RNA (siRNA), respectively. Cofilin siRNA greatly attenuated the ability of VSMC migration into the "wound," and platelet-derived growth factor (PDGF)-induced migration was virtually eliminated versus a 3.5-fold increase in nontreated VSMCs, establishing a critical role for cofilin in VSMC migration. Cofilin activation (dephosphorylation) was increased in PDGF-stimulated VSMCs. Thus, we assessed the role of the SSH family of phosphatases on cofilin activation and VSMC migration. Treatment with either SSH1 or SSH2 siRNA attenuated cofilin activation, whereas SSH3 siRNA had no effect. Only SSH1 siRNA significantly reduced wound healing and PDGF-induced VSMC migration. Both SSH1 expression (4.7-fold) and cofilin expression (3.9-fold) were increased in balloon injured versus noninjured carotid arteries, and expression was prevalent in the neointima. These studies demonstrate that the regulation of VSMC migration by cofilin is SSH1 dependent and that this mechanism potentially contributes to neointima formation following vascular injury in vivo.

Vitisin B, a resveratrol tetramer, inhibits migration through inhibition of PDGF signaling and enhancement of cell adhesiveness in cultured vascular smooth muscle cells

Vascular smooth muscle cells (VSMCs) play an important role in normal vessel formation and in the development and progression of cardiovascular diseases. Grape plants contain resveratrol monomer and oligomers and drinking of wine made from grape has been linked to “French Paradox”. In this study we evaluated the effect of vitisin B, a resveratrol tetramer, on VSMC behaviors. Vitisin B inhibited basal and PDGF-induced VSMC migration. Strikingly, it did not inhibit VSMC proliferation but inversely enhanced cell cycle progression and proliferation. Among the tested resveratrol oligomers, vitisin B showed an excellent inhibitory activity and selectivity on PDGF signaling. The anti-migratory effect by vitisin B was due to direct inhibition on PDGF signaling but was independent of interference with PDGF binding to VSMCs. Moreover, the enhanced VSMC adhesiveness to matrix contributed to the anti-migratory effect by vitisin B. Fluorescence microscopy revealed an enhanced reorganization of actin cytoskeleton and redistribution of activated focal adhesion proteins from cytosol to the peripheral edge of the cell membrane. This was confirmed by the observation that enhanced adhesiveness was repressed by the Src inhibitor. Finally, among the effects elicited by vitisin B, only the inhibitory effect toward basal migration was partially through estrogen receptor activation. We have demonstrated here that a resveratrol tetramer exhibited dual but opposite actions on VSMCs, one is to inhibit VSMC migration and the other is to promote VSMC proliferation. The anti-migratory effect was through a potent inhibition on PDGF signaling and novel enhancement on cell adhesion.

Neuropilin-1 mediates PDGF stimulation of vascular smooth muscle cell migration and signalling via p130Cas

NRP1 (neuropilin-1) is a co-receptor for members of the VEGF (vascular endothelial growth factor) family in endothelial cells, but is increasingly implicated in signalling induced by other growth factors. NRP1 is expressed in VSMCs (vascular smooth muscle cells), but its function and the mechanisms involved are poorly understood. The present study aimed to determine the role of NRP1 in the migratory response of HCASMCs (human coronary artery smooth muscle cells) to PDGF (platelet-derived growth factor), and to identify the signalling mechanisms involved. NRP1 is highly expressed in HAoSMCs (human aortic smooth muscle cells) and HCASMCs, and modified in VSMCs by CS (chondroitin sulfate)-rich O-linked glycosylation at Ser612. HCASMC migration induced by PDGF-BB and PDGF-AA was inhibited by NRP1 siRNA (small interfering RNA), and by adenoviral overexpression of an NRP1 mutant lacking the intracellular domain (Ad.NRP1ΔC). NRP1 co-immunoprecipitated with PDGFRα (PDGF receptor α), and immunofluorescent staining indicated that NRP1 and PDGFRα co-localized in VSMCs. NRP1 siRNA also inhibited PDGF-induced PDGFRα activation. NRP1-specific siRNA, Ad.NRP1ΔC and removal of CS glycans using chondroitinase all inhibited PDGF-BB and -AA stimulation of tyrosine phosphorylation of the adapter protein, p130Cas (Cas is Crk-associated substrate), with little effect on other major signalling pathways, and p130Cas knockdown inhibited HCASMC migration. Chemotaxis and p130Cas phosphorylation induced by PDGF were inhibited by chondroitinase, and, additionally, adenoviral expression of a non-glycosylatable NRP1S612A mutant inhibited chemotaxis, but not p130Cas phosphorylation. These results indicate a role for NRP1 and NRP1 glycosylation in mediating PDGF-induced VSMC migration, possibly by acting as a co-receptor for PDGFRα and via selective mobilization of a novel p130Cas tyrosine phosphorylation pathway.