Vascular Smooth Muscle Cell Motility Research Articles

A major role of TWEAK/Fn14 axis as a therapeutic target for post-angioplasty restenosis

BackgroundTumor necrosis factor-like weak inducer of apoptosis (Tnfsf12; TWEAK) and its receptor Fibroblast growth factor-inducible 14 (Tnfrsf12a; Fn14) participate in the inflammatory response associated with vascular remodeling. However, the functional effect of TWEAK on vascular smooth muscle cells (VSMCs) is not completely elucidated. MethodsNext generation sequencing-based methods were performed to identify genes and pathways regulated by TWEAK in VSMCs. Flow-citometry, wound-healing scratch experiments and transwell migration assays were used to analyze VSMCs proliferation and migration. Mouse wire injury model was done to evaluate the role of TWEAK/Fn14 during neointimal hyperplasia. FindingsTWEAK up-regulated 1611 and down-regulated 1091 genes in VSMCs. Using a gene-set enrichment method, we found a functional module involved in cell proliferation defined as the minimal network connecting top TWEAK up-regulated genes. In vitro experiments in wild-type or Tnfrsf12a deficient VSMCs demonstrated that TWEAK increased cell proliferation, VSMCs motility and migration. Mechanistically, TWEAK increased cyclins (cyclinD1), cyclin-dependent kinases (CDK4, CDK6) and decreased cyclin-dependent kinase inhibitors (p15lNK4B) mRNA and protein expression. Downregulation of p15INK4B induced by TWEAK was mediated by mitogen-activated protein kinase ERK and Akt activation.Tnfrsf12a or Tnfsf12 genetic depletion and pharmacological intervention with TWEAK blocking antibody reduced neointimal formation, decreasing cell proliferation, cyclin D1 and CDK4/6 expression, and increasing p15INK4B expression compared with wild type or IgG-treated mice in wire-injured femoral arteries. Finally, immunohistochemistry in human coronary arteries with stenosis or in-stent restenosis revealed high levels of Fn14, TWEAK and PCNA in VSMCs enriched areas of the neointima as compared with healthy coronary arteries. InterpretationOur data define a major role of TWEAK/Fn14 in the control of VSMCs proliferation and migration during neointimal hyperplasia after wire injury in mice, and identify TWEAK/Fn14 as a potential target for treating in-stent restenosis. FundISCiii-FEDER, CIBERCV and CIBERDEM.

Long non-coding RNA in regulation of plasticity of vascular smooth muscle cells

Phenotypic plasticity of vascular smooth muscle cells (VSMCs) is a functional property that is essential for vascular remodeling in vessel injury healing. During phenotypic switch, quiescent VSMCs loss contractile capacity but acquire ability to proliferate and migrate to the injured site where they differentiate again to the quiescent state. However, in pathological conditions such as endothelial dysfunction or atherosclerosis, phenotypic changes in arterial VSMCs become deregulated leading to elevated VSMC dedifferentiation, proliferation, excessive extracellular matrix deposits, and intimal thickening. VSMC hyperplasia is a complex mechanism that is coordinated by a network of various regulatory factors. Long non-coding (lnc)RNAs represent an important part of this regulatory network. Some of lncRNAs are involved in VSMC differentiation, apoptosis, and maintenance of VSMC quiescence, while other lncRNAs promote VSMC dedifferentiation, proliferation, and motility. In this review, we characterize these RNAs and their function in the context of possible involvement to atherosclerosis. Normal 0 false false false EN-GB X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Table Normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:Calibri,sans-serif; mso-bidi-font-family:Times New Roman; mso-ansi-language:EN-GB; mso-fareast-language:EN-GB;}

Formyl peptide receptor-1 activation exerts a critical role for the dynamic plasticity of arteries via actin polymerization

Several human diseases, include cancer and stroke are characterized by changes in immune system activation and vascular contractility. However, the mechanistic foundation of a vascular immuno-physiology network is still largely unknown. Formyl peptide receptor-1 (FPR-1), which plays a vital role in the function of the innate immune system, is widely expressed in arteries, but its role in vascular plasticity is unclear. We questioned why a receptor that is crucial for immune defense, and cell motility in leukocytes, would be expressed in vascular smooth muscle cells (VSMCs). We hypothesized that activation of FPR-1 in arteries is important for the temporal reorganization of actin filaments, and consequently, changes in vascular function, similar to what is observed in neutrophils. To address our hypothesis, we used FPR-1 knockout and VSMCs lacking FPR-1. We observed that FPR-1 activation induces actin polymerization in wild type VSMCs. Absence of FPR-1 in the vasculature significantly decreased vascular contraction and induced loss of myogenic tone to elevated intraluminal pressures via disruption of actin polymerization. Actin polymerization activator ameliorated these responses. In conclusion, we have established a novel role for FPR-1 in VSMC contractility and motility, similar to the one observed in sentinel cells of the innate immune system. This discovery is fundamental for vascular immuno-pathophysiology, given that FPR-1 in VSMCs not only functions as an immune system receptor, but it also has an important role for the dynamic plasticity of arteries.

BMP-2 enhances the migration and proliferation of hypoxia-induced VSMCs via actin cytoskeleton, CD44 and matrix metalloproteinase linkage

The persistent proliferation of hypoxia-induced vascular smooth muscle cells (VSMCs) in the arterial wall underlie the development of atherosclerosis. However, the mechanism that regulates the behavior of VSMCs, which involve in actin aggregation, and impedes their migration is still elusive. Here, we report that bone morphogenetic protein 2 (BMP-2) leads to enrichment of CD44 and F-actin stress fiber and secretion of matrix metalloproteinases-2 (MMP-2) during hypoxia in vitro and following artificial hypoxia-induced atherosclerosis exacerbation in vivo. To test this hypothesis, fluorescence immunostaining, immune-hybridization and flow cytometry analyses were performed to understand the relationship among BMP-2, CD44 and MMP-2 linkage. The cellular actin cytoskeleton was reduced, and smaller adhesion plaques were formed in hypoxia-induced T/G HA-VSMC cell line, but BMP-2 against disruption of F-actin and increase the motility and migration behaviors of VSMC during hypoxic cultured. Aggregation of F-actin dependents on the interaction between the cell surface integral membrane protein CD44 and Vinculin which enhanced by rBMP-2. This activity of Actin/CD44/ linkage was inhibited by competing with the active site of the CD44 using recombined the hemopexin-like C-terminal domain (PEX) of MMP-2. These results lead to the proliferation and migration of VSMCs were inhibited in response to MMP-2 activity when the cell is in a hypoxic environment. Collectively, our discovery indicates that BMP-2 could enhance migration and proliferation of hypoxia-induced VSMCs via the Actin/CD44/MMP-2 molecular pathway.

Formyl Peptide Receptor Exerts a Sentinel Role and is Important for the Dynamic Plasticity of the Vasculature

The canonical view accepts that the vascular smooth muscle cells (VSMCs) cytoskeleton is not plastic and remains stationary during a contractile event. However, it is now recognized that VSMCs display a highly dynamic process that undergoes polymerization and depolymerization based upon cellular demand.One of the most powerful signaling pathways that induces actin polymerization, Ca2+ influx and movement in neutrophils is due to formyl peptide receptor‐1 (FPR) activation. These receptors were originally identified by their ability to bind bacterial N‐formyl peptides. Interestingly, mitochondria carry hallmarks of their bacterial ancestry and one of these hallmarks is that this organelle uses an N‐formyl‐methionyl‐tRNA as an initiator of protein synthesis. Consequently, both mitochondrial and bacterial N‐formyl peptides are recognized by FPR‐1 as ligands and are known to play a role in the activation of the innate immune system.We have observed that FPR‐1 is not only expressed in sentinel cells such as leukocytes, but also in endothelial cells and VSMCs. To better understand the role of FPR‐1 in the regulation of the vascular plasticity, we studied the hypothesis that F‐MITs (formylated peptide corresponding to the NH2‐terminus of mitochondria ND6) binds FPR‐1 and leads to actin polymerization in VSMCs similar to the observed in neutrophils when activated with bacterial fragments. On the other hand, FPR‐1 absence would disrupt VSMCs actin polymerization and decrease contraction. For this, VSMCs isolated from aorta [14 weeks male C57BL6 (WT) mice] were treated with F‐MIT (20 min, 10 μM) or nonformylated peptide (control) in the presence or absence of a cocktail of FPR antagonists (FPR‐1 antagonist, cyclosporine H: CsH, 1 μmol/L and FPR‐2 antagonist, WRW4, 10 μmol/L). We observed that F‐MIT induced VSMCs actin polymerization via a shift in the filamentous:globular actin equilibrium in favor of F‐actin (1.8 fold vs. control, p<0.05). Using confocal microscopy, it was observed that VSMCs from aorta or intrarenal arteries (resistance arteries) that do not express the FPR‐1 present disrupted F‐actin and cell migration as measured by scratch assay. Also, measurements of isometric tension in aorta, demonstrated that the absence of the FPR‐1 decreased both the first ‐(fast, Ca2+ influx) and second‐phase (slow, actin polymerization) (Figure 1) of KCl (120 mmol/L)‐induced contraction and the concentration response curve to phenylephrine (Emax: WT: 8.4±1.2 vs. FPR‐1 KO: 0.6±0.2 mN, p<0.05). Jasplakinolide, an actin polymerization inducer (0.1 μmol/L), but not RhoA activator (2 μg/mL, 3 hours) restored contraction. L‐type calcium channels (Cav1.2) and AHNAK (anchors Cav 1.2 to the actin) protein expression are decreased in VSMCs from FPR‐1 KO, and Cav 1.2 activator (BayK8644, 0.1 nmol/L −1 μmol/L) was able to restore contraction. This suggests a link between actin polymerization via FPR‐1 activation and Cav 1.2 integrity and function.We established a remarkable role for an innate immune receptor FPR‐1 in VSMCs contractility and motility. This discovery is fundamental for vascular immuno‐pathophysiology, given that FPR‐1 in VSMCs not only functions as an immune regulator and danger signal sensor, but also has an important role for the dynamic plasticity of the vasculature.Support or Funding InformationNational Institutes of Health (NIH:1K99GM118885‐01)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelin-1 promotes vascular smooth muscle cell migration across the artery wall: a mechanism contributing to vascular remodelling and intimal hyperplasia in giant-cell arteritis

BackgroundGiant-cell arteritis (GCA) is an inflammatory disease of large/medium-sized arteries, frequently involving the temporal arteries (TA). Inflammation-induced vascular remodelling leads to vaso-occlusive events. Circulating endothelin-1 (ET-1) is increased in patients...

System Study of Vascular Smooth Muscle Cell (VSMC) Activation-Related Signaling Pathways by Monocyte and Macrophage Cells

Background: The vessel stenosis and re-stenosis processes are mainly related to cellular and molecular events. The vascular smooth muscle cell (VSMC) activation is the basic element in these lesions. Based on monocyte and macrophage inflammatory responses and also VSMC motility, a complex protein network is dynamically proposed between these cells. The aim of this study was to highlight the protein communications involved in VSMC proliferation and migration signaling pathways. Keywords: Atherosclerosis, network, cluster, pathway, VSMC, macrophage, monocyte.

P2Y12 Promotes Migration of Vascular Smooth Muscle Cells Through Cofilin Dephosphorylation During Atherogenesis.

P2Y12 is a well-recognized receptor expressed on platelets and the target of thienopyridine-type antiplatelet drugs. However, recent evidence suggests that P2Y12 expressed in vessel wall plays a role in atherogenesis, but the mechanisms remain elusive. In this study, we examined the molecular mechanisms of how vessel wall P2Y12 mediates vascular smooth muscle cells (VSMCs) migration and promotes the progression of atherosclerosis. Using a high-fat diet-fed apolipoprotein E-deficient mice model, we found that the expression of P2Y12 in VSMCs increased in a time-dependent manner and had a linear relationship with the plaque area. Moreover, administration of P2Y12 receptor antagonist for 12 weeks caused significant reduction in atheroma and decreased the abundance of VSMCs in plaque. In cultured VSMCs, we found that activation of P2Y12 receptor inhibited cAMP/protein kinase A signaling pathway, which induced cofilin dephosphorylation and filamentous actin disassembly, thereby enhancing VSMCs motility and migration. In addition, the number of P2Y12-positive VSMCs was decreased in the carotid artery plaque from patients receiving clopidogrel. Vessel wall P2Y12 receptor, which promotes VSMCs migration through cofilin dephosphorylation, plays a critical role in the development of atherosclerotic lesion and may be used as a therapeutic target for atherosclerosis.

Prelamin A Accumulation Attenuates Rac1 Activity and Increases the Intrinsic Migrational Persistence of Aged Vascular Smooth Muscle Cells.

Vascular smooth muscle cell (VSMC) motility is essential during both physiological and pathological vessel remodeling. Although ageing has emerged as a major risk factor in the development of cardiovascular disease, our understanding of the impact of ageing on VSMC motility remains limited. Prelamin A accumulation is known to drive VSMC ageing and we show that presenescent VSMCs, that have accumulated prelamin A, display increased focal adhesion dynamics, augmented migrational velocity/persistence and attenuated Rac1 activity. Importantly, prelamin A accumulation in proliferative VSMCs, induced by depletion of the prelamin A processing enzyme FACE1, recapitulated the focal adhesion, migrational persistence and Rac1 phenotypes observed in presenescent VSMCs. Moreover, lamin A/C-depleted VSMCs also display reduced Rac1 activity, suggesting that prelamin A influences Rac1 activity by interfering with lamin A/C function at the nuclear envelope. Taken together, these data demonstrate that lamin A/C maintains Rac1 activity in VSMCs and prelamin A disrupts lamin A/C function to reduce Rac1 activity and induce migrational persistence during VSMC ageing.

Substance-specific importance of EGFR for vascular smooth muscle cells motility in primary culture

Besides their importance for the vascular tone, vascular smooth muscle cells (VSMC) also contribute to pathophysiological vessel alterations. Various G-protein coupled receptor ligands involved in vascular dysfunction and remodeling can transactivate the epidermal growth factor receptor (EGFR) of VSMC, yet the importance of EGFR transactivation for the VSMC phenotype is incompletely understood. The aims of this study were (i) to characterize further the importance of the VSMC-EGFR for proliferation, migration and marker gene expression for inflammation, fibrosis and reactive oxygen species (ROS) homeostasis and (ii) to test the hypothesis that vasoactive substances (endothelin-1, phenylephrine, thrombin, vasopressin and ATP) rely differentially on the EGFR with respect to the abovementioned phenotypic alterations.In primary, aortic VSMC from mice without conditional deletion of the EGFR, proliferation, migration, marker gene expression (inflammation, fibrosis and ROS homeostasis) and cell signaling (ERK 1/2, intracellular calcium) were analyzed.VSMC-EGFR loss reduced collective cell migration and single cell migration probability, while no difference between the genotypes in single cell velocity, chemotaxis or marker gene expression could be observed under control conditions. EGF promoted proliferation, collective cell migration, chemokinesis and chemotaxis and leads to a proinflammatory gene expression profile in wildtype but not in knockout VSMC. Comparing the impact of five vasoactive substances (all reported to transactivate EGFR and all leading to an EGFR dependent increase in ERK1/2 phosphorylation), we demonstrate that the importance of EGFR for their action is substance-dependent and most apparent for crowd migration but plays a minor role for gene expression regulation.

The Influence of Caldesmon Suppression on Proliferation and Motilities of Vascular Smooth Muscle Cells

Migration of vascular smooth muscle cells (VSMCs) from the media to intima constitutes a critical step in the development of proliferative vascular diseases. To elucidate the regulatory mechanism of VSMCs motility, the roles of caldesmon (CaD) were investigated previously. CaD is an actin-binding protein dynamically regulating cytoskeleton structure. In this study, the potential role of CaD in mediating proliferation and motility of VSMCs were discussed. First, structural effect of CaD on cytoskeleton integrity was analyzed with CaD knock-down; second, the proliferation of VSMCs was measured in CaD knock-down and control cells; third, the specific role of CaD on VSMCs motilities was evaluated with in vitro migration and invasion assays. We found that CaD is an integral component to maintain cytoskeleton integrity of VSMCs. Our data indicated that CaD suppression does not show significant influence on VSMCs proliferation, but negatively modulates the motilities of VSMCs, and CaD depletion would significantly facilitate migration and invasion of VSMCs.

TonEBP/NFAT5 regulates ACTBL2 expression in biomechanically activated vascular smooth muscle cells.

Cytoskeletal reorganization and migration are critical responses which enable vascular smooth muscle cells (VSMCs) cells to evade, compensate, or adapt to alterations in biomechanical stress. An increase in wall stress or biomechanical stretch as it is elicited by arterial hypertension promotes their reorganization in the vessel wall which may lead to arterial stiffening and contractile dysfunction. This adaptive remodeling process is dependent on and driven by subtle phenotype changes including those controlling the cytoskeletal architecture and motility of VSMCs. Recently, it has been reported that the transcription factor nuclear factor of activated T-cells 5 (TonEBP/NFAT5) controls critical aspects of the VSMC phenotype and is activated by biomechanical stretch. We therefore hypothesized that NFAT5 controls the expression of gene products orchestrating cytoskeletal reorganization in stretch-stimulated VSMCs. Automated immunofluorescence and Western blot analyses revealed that biomechanical stretch enhances the expression and nuclear translocation of NFAT5 in VSMCs. Subsequent in silico analyses suggested that this transcription factor binds to the promotor region of ACTBL2 encoding kappa-actin which was shown to be abundantly expressed in VSMCs upon exposure to biomechanical stretch. Furthermore, ACTBL2 expression was inhibited in these cells upon siRNA-mediated knockdown of NFAT5. Kappa-actin appeared to be aligned with stress fibers under static culture conditions, dispersed in lamellipodia and supported VSMC migration as its knockdown diminishes lateral migration of these cells. In summary, our findings delineated biomechanical stretch as a determinant of NFAT5 expression and nuclear translocation controlling the expression of the cytoskeletal protein ACTBL2. This response may orchestrate the migratory activity of VSMCs and thus promote maladaptive rearrangement of the arterial vessel wall during hypertension.

Endocrine Disruptors Differently Influence Estrogen Receptor β and Androgen Receptor in Male and Female Rat VSMC

Sex steroid hormones differently control the major physiological processes in male and female organisms. In particular, their effects on vascular smooth muscle cells (VSMCs) migration are at the root of sex/gender-related differences reported in the cardiovascular system. Several exogenous substances, defined endocrine disruptor chemicals (EDCs), could interfere with these androgen and estrogen effects; however, the sex/gender-related susceptibility of VSMC motility to EDCs is completely unknown. Here, the effect of naturally occurring (naringenin, Nar) and synthetic (bisphenol A, BPA) EDCs on male and female VSMC motility has been evaluated. 17β-estradiol (E2, 0.1 nM-1 µM) induced a dose-dependent inhibition of motility in female-derived VSMC. In contrast, neither dihydrotestosterone (DHT, 0.01-100 nM) nor the common precursor of sex steroid hormones, testosterone (Tes, 0.01-100 nM) modified male-derived VSMC motility. Estrogen receptor (ER) β subtype-dependent activation of p38 was necessary for the E2 effect on cell motility. High BPA concentration prevented E2 effects in female-derived cells being without any effect in male-derived cells. Nar mimicked E2 effects on female-derived cells even in the presence of E2 or BPA. Intriguingly, Nar also inhibited the male-derived VSMC mobility. This latter effect was prevented by ERβ inhibitor, but not by the androgen receptor (AR) inhibitor. As a whole, ERβ-dependent signals in VSMC results more susceptible to the impact of EDCs than AR signals suggesting a possible high and overall susceptibility of female to EDCs. However, several male-derived cells, including VSMC, express ERβ, which could also serve as target of EDC disruption in male organisms.

ZIPK is critical for the motility and contractility of VSMCs through the regulation of nonmuscle myosin II isoforms

Migration of medial vascular smooth muscle cells (VSMCs) into the intimal layer contributes to pathological remodeling of the blood vessel in arterial hypertension and atherosclerosis. It is well established that reorganization of cytoskeletal networks is an essential component of cellular motile events. However, there is currently a lack of insight into the cellular characteristics of VSMC migration under three-dimensional environments. Here, we investigated the mechanisms of VSMC migration and remodeling using two different collagen matrix assays as in vitro models: migration of VSMCs within a collagen matrix for VSMC invasion and contraction of a collagen gel by VSMCs for VSMC remodeling and contraction. We found that nonmuscle myosin IIA (NMIIA) and nonmuscle myosin IIB (NMIIB) differentially contribute to the migratory capacity of VSMCs via NMII isoform-dependent cytoskeletal reorganization. Depletion of NMIIA by short hairpin RNA revealed a unique interplay between actomyosin and microtubules during VSMC migration. On the other hand, NMIIB was required for the structural maintenance of migrating VSMC. Interestingly, there was a significant difference between NMIIA and NMIIB knockdown in the VSMC migration but not in the VSMC-mediated collagen gel contraction. Furthermore, depletion of zipper-interacting protein kinase by short hairpin RNA resulted in an impairment of VSMC migration and a substantial decrease of VSMC-mediated collagen gel contraction. These results suggest that NMIIA and NMIIB uniquely control VSMC migration and may contribute to vascular remodeling, which are both regulated by zipper-interacting protein kinase.

BMP-2 Overexpression Augments Vascular Smooth Muscle Cell Motility by Upregulating Myosin Va via Erk Signaling

Background. The disruption of physiologic vascular smooth muscle cell (VSMC) migration initiates atherosclerosis development. The biochemical mechanisms leading to dysfunctional VSMC motility remain unknown. Recently, cytokine BMP-2 has been implicated in various vascular physiologic and pathologic processes. However, whether BMP-2 has any effect upon VSMC motility, or by what manner, has never been investigated. Methods. VSMCs were adenovirally transfected to genetically overexpress BMP-2. VSMC motility was detected by modified Boyden chamber assay, confocal time-lapse video assay, and a colony wounding assay. Gene chip array and RT-PCR were employed to identify genes potentially regulated by BMP-2. Western blot and real-time PCR detected the expression of myosin Va and the phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2). Immunofluorescence analysis revealed myosin Va expression locale. Intracellular Ca2+ oscillations were recorded. Results. VSMC migration was augmented in VSMCs overexpressing BMP-2 in a dose-dependent manner. siRNA-mediated knockdown of myosin Va inhibited VSMC motility. Both myosin Va mRNA and protein expression significantly increased after BMP-2 administration and were inhibited by Erk1/2 inhibitor U0126. BMP-2 induced Ca2+ oscillations, generated largely by a “cytosolic oscillator”. Conclusion. BMP-2 significantly increased VSMCs migration and myosin Va expression, via the Erk signaling pathway and intracellular Ca2+ oscillations. We provide additional insight into the pathophysiology of atherosclerosis, and inhibition of BMP-2-induced myosin Va expression may represent a potential therapeutic strategy.

Vascular Smooth Muscle Cell Motility Is Mediated by a Physical and Functional Interaction of Ca2+/Calmodulin-dependent Protein Kinase IIδ2 and Fyn

In vascular smooth muscle (VSM) cells, Ca(2+)/calmodulin-dependent protein kinase IIδ2 (CaMKIIδ2) activates non-receptor tyrosine kinases and EGF receptor, with a Src family kinase as a required intermediate. siRNA-mediated suppression of Fyn, a Src family kinase, inhibited VSM cell motility. Simultaneous suppression of both Fyn and CaMKIIδ2 was non-additive, suggesting coordinated regulation of cell motility. Confocal immunofluorescence microscopy indicated that CaMKIIδ2 and Fyn selectively (compared with Src) co-localized with the Golgi in quiescent cultured VSM cells. Stimulation with PDGF resulted in a rapid (<5 min) partial redistribution and co-localization of both kinases in peripheral membrane regions. Furthermore, CaMKIIδ2 and Fyn selectively (compared with Src) co-immunoprecipitated, suggesting a physical interaction in a signaling complex. Stimulation of VSM cells with ionomycin, a calcium ionophore, resulted in activation of CaMKIIδ2 and Fyn and disruption of the complex. Pretreatment with KN-93, a pharmacological inhibitor of CaMKII, prevented activation-dependent disruption of CaMKIIδ2 and Fyn, implicating CaMKIIδ2 as an upstream mediator of Fyn. Overexpression of constitutively active CaMKII resulted in the dephosphorylation of Fyn at Tyr-527, which is required for Fyn activation. Taken together, these data demonstrate a dynamic interaction between CaMKIIδ2 and Fyn in VSM cells and indicate a mechanism by which CaMKIIδ2 and Fyn may coordinately regulate VSM cell motility.

Bisdemethoxycurcumin inhibits PDGF‐induced vascular smooth muscle cell motility and proliferation

A key event in the development of plaque in the arteries is the migration and proliferation of smooth muscle cells (SMCs) from the media to the intima of the blood vessel. This study was conducted to evaluate the effects of bisdemethoxycurcumin (BC), a naturally occurring structural analog of curcumin (CC), on platelet-derived growth factor (PDGF)-stimulated migration and proliferation of SMCs. CC and BC were synthesized by condensing acetyl acetone with vanillin and 4-hydroxybenzaldehyde, respectively. SMCs isolated from adult rat aorta were stimulated with PDGF in the presence or absence of CC or BC following which, cell migration and proliferation were assessed by monolayer wound healing assay and [(3) H]-thymidine incorporation respectively. PDGF-stimulated phosphorylation of PDGF receptor-β and its downstream effectors Akt and ERK were assessed by Western blotting. Intracellular reactive oxygen species was assessed using the fluorescent dye 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. BC elicited a concentration-dependent inhibition of PDGF-stimulated phosphorylation of PDGF receptor-β, Akt and Erk as well as the PDGF-stimulated SMC migration and proliferation. BC was more potent than CC in inhibiting migration and proliferation and suppressing PDGF-signaling in SMCs. Both compounds were equipotent in inhibiting PDGF-stimulated generation of intracellular reactive oxygen species. BC may be of potential use in the prevention or treatment of vascular disease.

Proprotein convertase furin enhances survival and migration of vascular smooth muscle cells via processing of pro-nerve growth factor

Maturation of nerve growth factor (NGF) in neuronal cells requires endoproteolytic processing of the precursor protein proNGF to β-NGF by the proprotein convertase furin. Pro- and β-NGF elicit opposite biological functions by differential neurotrophin-receptor binding, leading to apoptosis via sortilin or survival via neurotrophic tyrosine kinase receptor type-1 (TrkA), respectively. The present study was done to investigate the impact of furin-dependent proNGF processing on vascular smooth muscle cell (VSMC) function. We found that β-NGF mRNA and protein expression was upregulated in platelet-derived growth factor-BB/transforming growth factor-β1-stimulated, proliferating rat aortic VSMCs. Although β-NGF itself did not affect VSMC proliferation, it promoted VSMC motility in an autocrine fashion via TrkA/Akt-dependent integrin inside-out signalling. The β-NGF-induced migration of VSMCs required proNGF processing by furin, which was co-regulated with NGF. Furin-inhibition increased proNGF and reduced β-NGF secretion, leading to apoptosis rather than migration. In line with our in vitro demonstration, we found co- and upregulation of NGF, its convertase furin and its high-affinity receptor TrkA in the neointima of balloon-injured rodent arteries. These results indicate that furin determines the balance between proNGF and β-NGF in proliferating VSMCs, thus impacting on VSMC survival and migration and is also important in neointima formation.

LDL-Induced Impairment of Human Vascular Smooth Muscle Cells Repair Function Is Reversed by HMG-CoA Reductase Inhibition

Growing human atherosclerotic plaques show a progressive loss of vascular smooth muscle cells (VSMC) becoming soft and vulnerable. Lipid loaded-VSMC show impaired vascular repair function and motility due to changes in cytoskeleton proteins involved in cell-migration. Clinical benefits of statins reducing coronary events have been related to repopulation of vulnerable plaques with VSMC. Here, we investigated whether HMG-CoA reductase inhibition with rosuvastatin can reverse the effects induced by atherogenic concentrations of LDL either in the native (nLDL) form or modified by aggregation (agLDL) on human VSMC motility. Using a model of wound repair, we showed that treatment of human coronary VSMC with rosuvastatin significantly prevented (and reversed) the inhibitory effect of nLDL and agLDL in the repair of the cell depleted areas. In addition, rosuvastatin significantly abolished the agLDL-induced dephosphorylation of myosin regulatory light chain as demonstrated by 2DE-electrophoresis and mass spectrometry. Besides, confocal microscopy showed that rosuvastatin enhances actin-cytoskeleton reorganization during lipid-loaded-VSMC attachment and spreading. The effects of rosuvastatin on actin-cytoskeleton dynamics and cell migration were dependent on ROCK-signalling. Furthermore, rosuvastatin caused a significant increase in RhoA-GTP in the cytosol of VSMC. Taken together, our study demonstrated that inhibition of HMG-CoA reductase restores the migratory capacity and repair function of VSMC that is impaired by native and aggregated LDL. This mechanism may contribute to the stabilization of lipid-rich atherosclerotic plaques afforded by statins.

Abstract 487: EBP50 Promotes Epidermal Growth Factor-Dependent Activation of Focal Adhesion Kinase and Vascular Smooth Muscle Cells Migration

The Ezrin-Radixin-Moesin-Binding Phosphoprotein 50 (EBP50) is a scaffolding protein that regulates a variety of physiological functions. Previous studies showed that EBP50 promotes vascular smooth muscle cells (VSMC) proliferation and neointima formation following arterial injury. In this study the role of EBP50 on VSMC migration was characterized. VSMC migration was determined by wound-healing assays. The motility of primary VSMC isolated from EBP50 KO mice was significantly reduced compared to WT cells (25 um/h WT vs. 19 um/h KO, p&lt;0.05) resulting in a 50% reduction in wound coverage. Conversely, EBP50 expression increased VSMC migration. EBP50-null VSMC had fewer and larger focal adhesions than EBP50-expressing cells. Both assembly and disassembly of focal adhesion in response to epidermal growth factor (EGF) (determined by real time TIRF microscopy) were significantly reduced in KO cells. Immunoprecipitation experiments showed that EBP50 interacts with both focal adhesion kinase (FAK) and EGF receptor (EGFR) and the formation of a complex containing both EGFR and FAK was increased by EBP50. Stimulation of VSMC with EGF induced FAK phosphorylation on Tyr925 in WT, but not in KO cell, whereas phosphorylation of Tyr397 was unaffected. Collectively, these observations indicate that EBP50 facilitates growth factors-dependent activation of FAK, and consequently, migration of VSMC. Therefore, in addition to increasing VSMC proliferation, EBP50 promotes neointima formation following arterial injury by increasing VSMC motility.