PDGF-induced Migration Research Articles

MiR-503 inhibits platelet-derived growth factor-induced human aortic vascular smooth muscle cell proliferation and migration through targeting the insulin receptor.

Abnormal proliferation and migration of vascular smooth muscle cells (VSMC) is a common feature of disease progression in atherosclerosis. Here, we investigated the potential role of miR-503 in platelet-derived growth factor (PDGF)-induced proliferation and migration of human aortic smooth muscle cells and the underlying mechanisms of action. miR-503 expression was significantly downregulated in a dose- and time-dependent manner following PDGF treatment. Introduction of miR-503 mimics into cultured SMCs significantly attenuated cell proliferation and migration induced by PDGF. Bioinformatics analyses revealed that the insulin receptor (INSR) is a target candidate of miR-503. miR-503 suppressed luciferase activity driven by a vector containing the 3'-untranslated region of INSR in a sequence-specific manner. Downregulation of INSR appeared critical for miR-503-mediated inhibitory effects on PDGF-induced cell proliferation and migration in human aortic SMCs. Based on the collective data, we suggest a novel role of miR-503 as a regulator of VSMC proliferation and migration through modulating INSR.

408 - Cinnamic Aldehyde Inhibits PDGF-Induced Migration and Proliferation of Smooth Muscle Cells, and Neointimal Hyperplasia in Diabetic Rats

Introduction Patients with Diabetes Mellitus have a higher risk of developing peripheral arterial and cardiovascular disease, which is partially attributed to inflammation, increased reactive oxygen species, and endothelial cell dysfunction. Nrf2 (NF-E2-related factor 2) plays a key role in protecting against environmental stress by inducing expression of detoxification and antioxidant enzymes. Cinnamic aldehyde (CA) is a small molecule Nrf2 activator. Our goal is to assess the protective effect of CA against diabetes-induced vascular damage. We hypothesize that CA will activate the Nrf2 pathway, inhibit proliferation and migration of diabetic vascular smooth muscle cells in vitro, as well as protect against restenosis in vivo after carotid balloon injury in Zucker Diabetic Fatty (ZDF) rats. Methods Arterial smooth muscle cells from ZDF rats (D-SMC) were isolated and cultured in high glucose medium. The effect of CA at different concentrations (0.1, 1, 10, 50, and 100 μM) on platelet-derived growth factor (PDGF)-induced cell proliferation was assessed by the MTT assay and by counting viable cells. Cell migration was evaluated following CA treatment via the scratch assay. A Muse-based assay was used to determine the effect of 100 μM CA on the cell cycle. Nrf2 translocation was studied by Western blotting of the nuclear fraction, and immunofluorescence after CA treatment (100 μM). Sulforaphane (4 μM) was used as a positive control. 10 week-old ZDF rats were fed a high fat diet for one month to induce diabetes. The balloon carotid injury model was performed with or without periadventitial application of CA (100 μM in 150 μl of Pluronic gel). Two weeks after surgery the carotids were harvested for morphometric analysis. Results CA (100 μM) inhibited PDGF-induced cell proliferation of D-SMC by 50%, assessed by both MTT (P Conclusion These data show that CA has a protective effect, inhibiting proliferation and migration of diabetic arterial smooth muscle cells. CA also has a protective effect in vivo inhibiting the development of neointimal hyperplasia. This inhibition suggests that CA could afford protection against vascular restenosis in diabetic arteries after vascular interventions.

Abstract 553: The Matricellular Protein Ccn1 is a Key Mediator of Smooth Muscle Cell Migration and Neointimal Formation

Platelet-derived growth factor (PDGF), a potent chemoattractant, induces smooth muscle cell (SMC) migration via the MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified the matricellular protein CCN1, which is de novo synthesized in response to PDGF stimulation, as the key downstream mediator of the ERK and JNK pathways, independent of the p38 MAPK and AKT pathways, and, thereby, it mediates PDGF-induced SMC migration. Our results revealed that, when CCN1 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with the plasma membrane integrins α6β1 and αvβ3. We further demonstrate that CCN1 and integrins are integral components of the PDGF signaling pathway via an “outside-in” signaling route to activate intracellular focal adhesion kinase (FAK), leading to SMC migration. Therefore, our study provides new evidence that the PDGF-induced endogenous extracellular matrix component CCN1 is a key mediator in modulating SMC migration by connecting intracellular PDGF-ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular CCN1 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor-induced cell migration. We further identified that CCN1 is a key mediator of lysophosphatidic acid (LPA)-mediated SMC migration. Our recent data reveal that CCN1 mediates LPA-induced neointimal formation. The discovered novel signaling pathway may represent an important therapeutic target in growth factor/lipid-mediated cell migration related vascular diseases.

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.

Nox4 and Duox1/2 Mediate Redox Activation of Mesenchymal Cell Migration by PDGF.

Platelet derived growth factor (PDGF) orchestrates wound healing and tissue regeneration by regulating recruitment of the precursor mesenchymal stromal cells (MSC) and fibroblasts. PDGF stimulates generation of hydrogen peroxide that is required for cell migration, but the sources and intracellular targets of H2O2 remain obscure. Here we demonstrate sustained live responses of H2O2 to PDGF and identify PKB/Akt, but not Erk1/2, as the target for redox regulation in cultured 3T3 fibroblasts and MSC. Apocynin, cell-permeable catalase and LY294002 inhibited PDGF-induced migration and mitotic activity of these cells indicating involvement of PI3-kinase pathway and H2O2. Real-time PCR revealed Nox4 and Duox1/2 as the potential sources of H2O2. Silencing of Duox1/2 in fibroblasts or Nox4 in MSC reduced PDGF-stimulated intracellular H2O2, PKB/Akt phosphorylation and migration, but had no such effect on Erk1/2. In contrast to PDGF, EGF failed to increase cytoplasmic H2O2, phosphorylation of PKB/Akt and migration of fibroblasts and MSC, confirming the critical impact of redox signaling. We conclude that PDGF-induced migration of mesenchymal cells requires Nox4 and Duox1/2 enzymes, which mediate redox-sensitive activation of PI3-kinase pathway and PKB/Akt.

Dock8 interacts with Nck1 in mediating Schwann cell precursor migration

During embryonic development of the peripheral nervous system (PNS), Schwann cell precursors migrate along neuronal axons to their final destinations, where they will myelinate the axons after birth. While the intercellular signals controlling Schwann cell precursor migration are well studied, the intracellular signals controlling Schwann cell precursor migration remain elusive. Here, using a rat primary cell culture system, we show that Dock8, an atypical Dock180-related guanine-nucleotide exchange factor (GEF) for small GTPases of the Rho family, specifically interacts with Nck1, an adaptor protein composed only of Src homology (SH) domains, to promote Schwann cell precursor migration induced by platelet-derived growth factor (PDGF). Knockdown of Dock8 or Nck1 with its respective siRNA markedly decreases PDGF-induced cell migration, as well as Rho GTPase activation, in precursors. Dock8, through its unique N-terminal proline-rich motif, interacts with the SH3 domain of Nck1, but not with other adaptor proteins composed only of SH domains, e.g. Grb2 and CrkII, and not with the adaptor protein Elmo1. Reintroduction of the proline-rich motif mutant of Dock8 in Dock8 siRNA-transfected Schwann cell precursors fails to restore their migratory abilities, whereas that of wild-type Dock8 does restore these abilities. These results suggest that Nck1 interaction with Dock8 mediates PDGF-induced Schwann cell precursor migration, demonstrating not only that Nck1 and Dock8 are previously unanticipated intracellular signaling molecules involved in the regulation of Schwann cell precursor migration but also that Dock8 is among the genetically-conservative common interaction subset of Dock family proteins consisting only of SH domain adaptor proteins.

Exogenous S100A8 protein inhibits PDGF-induced migration of airway smooth muscle cells in a RAGE-dependent manner.

S100A8 is an important member of the S100 protein family, which is involved in intracellular and extracellular regulatory activities. We previously reported that the S100A8 protein was differentially expressed in the asthmatic respiratory tracts. To understand the potential role of S100A8 in asthma, we investigated the effect of recombinant S100A8 protein on the platelet-derived growth factor (PDGF)-induced migration of airway smooth muscle cells (ASMCs) and the underlying molecular mechanism by using multiple methods, such as impedance-based xCELLigence migration assay, transwell migration assays and wound-healing assays. We found that exogenous S100A8 protein significantly inhibited PDGF-induced ASMC migration. Furthermore, the migration inhibition effect of S100A8 was blocked by neutralizing antibody against the receptor for advanced glycation end-products (RAGE), a potential receptor for the S100A8 protein. These findings provide direct evidence that exogenous S100A8 protein inhibits the PDGF-induced migration of ASMCs through the membrane receptor RAGE. Our study highlights a novel role of S100A8 as a potential means of counteracting airway remodeling in chronic airway diseases.

Abstract 14743: Intracellular Adenosine Suppresses Vsmc Phenotypic Switch Through Klf4 Gene Methylation

Background and objective: It is well known that adenosine affects the growth of vascular smooth muscle cells via adenosine receptors. Adenosine kinase (ADK) is an intracellular enzyme regulating the levels of adenosine in intracellular and further extracellular compartments. The effect of ADK on VSMC phenotypic switch is unknown. In this study, we evaluate phenotypic changes of VSCMs both in vitro and in vivo following deletion of ADK in VSMCs. Methods and results: The expression of ADK in vascular wall was significant upregulated in mouse carotid artery ligation model. Heterogeneous deletion of ADK or specific ADK deletion in VSMCs led to remarkably decreased neointima in ligated mouse carotid arteries (Fig. A&B). This was accompanied with increased expression of VSMC differentiation markers and transcriptional factor KLF4 (Fig. D). In an in vitro setup, treatment of VSMCs with ADK shRNA or ADK inhibitors suppressed serum or PDGF-induced SMC proliferation and migration (Fig. C). This effect remained intact in the presence of adenosine receptor antagonists to A1, A2A, A2B and A3. Deletion of ADK in VSMCs increased the levels of intracellular adenosine, decreased the methylation levels of genes and histones, and augmented KLF4 expression. It is being investigated that upregulation of KLF4 was resulted from its gene hypomethylation in response to elevated intracellular adenosine following ADK deletion/knockdown or inhibition. Conclusions: Our data demonstrate that ADK deletion/inhibition suppresses neointima formation in injured arteries through a novel epigenetic pathway but not classic adenosine receptors, and ADK is a promising therapeutic target in the treatment of arterial diseases such as atherosclerosis and arterial neointima formation following arterial angioplasty.

Urokinase-type plasminogen activator receptor interaction with β1 integrin is required for platelet-derived growth factor-AB-induced human mesenchymal stem/stromal cell migration.

IntroductionMesenchymal stem cells (MSC) are well described for their role in tissue regeneration following injury. Migratory properties of endogenous or administrated MSC are critical for tissue repair processes. Platelet-derived growth factor (PDGF) is a chemotactic growth factor that elicits mesenchymal cell migration. However, it is yet to be elucidated if signaling pathways other than direct activation of PDGF receptor (PDGF-R) are involved in PDGF-induced cell migration.MethodsKnocking down and co-immunoprecipitation approaches were used to evaluate urokinase-type plasminogen activator receptor (uPAR) requirement and its interactions with proteins involved in migration mechanisms, in human MSC induced to migrate under PDGF-AB effect.ResultsWe demonstrated that uPAR activation and its association with β1-integrin are required for PDGF-AB-induced migration. This phenomenon takes place in MSC derived from bone marrow and from adipose tissue.ConclusionsWe showed that PDGF-AB downstream signaling requires other effector molecules in MSC such as the uPA/uPAR system and β1 integrin signaling pathway known for their role in migration. These findings provide new insights in molecular mechanisms of PDGF-AB-induced migration of human MSC that may be relevant to control MSC function and tissue remodeling after injury.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0163-5) contains supplementary material, which is available to authorized users.

The cAMP-producing agonist beraprost inhibits human vascular smooth muscle cell migration via exchange protein directly activated by cAMP.

AimsDuring restenosis, vascular smooth muscle cells (VSMCs) migrate from the vascular media to the developing neointima. Preventing VSMC migration is therefore a therapeutic target for restenosis. Drugs, such as prostacyclin analogues, that increase the intracellular concentration of cyclic adenosine monophosphate (cAMP) can inhibit VSMC migration, but the mechanisms via which this occurs are unknown. Two main downstream mediators of cAMP are protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). This study has examined the effects of the prostacyclin analogue beraprost on VSMC migration and investigated the intracellular pathways involved.Methods and resultsIn a chemotaxis chamber, human saphenous vein VSMC migrated towards a platelet-derived growth-factor-BB (PDGF) chemogradient. Incubation with therapeutically relevant concentrations of cAMP-producing agonist beraprost significantly decreased PDGF-induced migration. Direct activation of either PKA or Epac inhibited migration whereas inhibition of PKA did not prevent the anti-migratory effect of beraprost. Direct activation of Epac also prevented hyperplasia in ex vivo serum-treated human veins. Using fluorescence resonance energy transfer, we demonstrated that beraprost activated Epac but not PKA. The mechanisms of this Epac-mediated effect involved activation of Rap1 with subsequent inhibition of RhoA. Cytoskeletal rearrangement at the leading edge of the cell was consequently inhibited. Interestingly, Epac1 was localized to the leading edge of migrating VSMC.ConclusionsThese results indicate that therapeutically relevant concentrations of beraprost can inhibit VSMC migration via a previously unknown mechanism involving the cAMP mediator Epac. This may provide a novel target that could blunt neointimal formation.

Abstract 719: PFKFB3 Mediated Glycolysis in Vascular Smooth Muscle Cells Is Essential for Arterial Neointima Formation

Background: Mature vascular smooth muscle cells (VSMCs) exhibit limited proliferation and migration ability. However, in response to stresses, VSMCs undergo phenotypic modulation characterized by increased proliferation and motility. This phenotypic modulation is associated with several vascular diseases, including atherosclerosis, arterial neointima formation. Elevated glycolysis has been described in proliferative VSMCs. The isoform 3 of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase (PFKFB3) is a regulatory factor for VSMC glycolysis. PFKFB3 catalyze the synthesis of fructose 2, 6-bishosphate (F2, 6 BP). The latter is a powerful activator of 6-phosphofructo-1-kinase, the rate-limiting enzyme in glycolytic flux. It is unclear whether PFKFB3 mediated glycolysis play a role in VSMC phenotypic modulation andarterial neointima formation. Methods and results: PDGF treatment increased PFKFB3 expression in human aortic smooth muscle cells (HASMCs). This was accompanied with enhanced proliferation and migration of HASMCs. Knockdown of PFKFB3 with adenovirus carrying PFKFB3 short hairpin RNA suppressed PDGF-induced HASMC proliferation and migration. Examination of intracellular signaling indicated PI3K/Akt participated into PFKFB3-mediated glycolysis, and further cooperated with PFKFB3 to modulate phenotype of HASMCs. In a model of the left common carotid artery ligation, both PFKFB3 heterozygotes and mice with deficiency of PFKFB3 in VSMCs showed the decreased arterial neointima compared with controls mice. Conclusions: PFKFB3 mediates glycolysis and cooperates with PI3K/AKT, thereafter promoting proliferation and migration of VSMCs and leading to the formation of an increased arterial neointima. Thus, PFKFB3 is a promising therapeutic target for the treatment of atherosclerosis, arterial neointima and other peripheral arterial diseases.

Abstract 445: Beraprost, a Prostacyclin Analogue, Inhibits Human Vascular Smooth Muscle Cell Migration via Activation of Exchange Protein Activated by cAMP (Epac)

Following vein grafting, endothelial damage initiates vascular smooth muscle cell (VSMC) migration to the intima leading to neointimal hyperplasia and restenosis. Inhibition of VSMC migration is therefore a therapeutic target to prevent restenosis. Previous studies have shown that cyclic adenosine monophosphate (cAMP) inhibits VSMC migration although the downstream mediators are unknown. This suggests that cyclic AMP-producing agonists, such as the prostacyclin analogue beraprost which binds to prostanoid receptors, could be used as a potential drug against neointimal formation. We hypothesised that prostacyclin analogues inhibit VSMC migration by activating specific cAMP downstream mediators. The aims of this project were to examine the role of cAMP mediators protein kinase A (PKA) and Exchange protein activated by cAMP (Epac) in this inhibitory effect. Saphenous veins were obtained from patients undergoing coronary artery bypass. VSMCs were primary cultured and migration measured using a chemotaxis chamber. Cell migration stimulated by PDGF was significantly inhibited by 68 ± 5% (n=6) in the presence of a therapeutically relevant concentration of beraprost (1 nM). To examine potential mechanisms of this inhibition, selective activators of PKA and Epac were used. Both Epac and PKA activators significantly inhibited migration towards PDGF. However, fluorescence resonance energy transfer using cells transfected with specific probes revealed that 1 nM beraprost only activates Epac and not PKA in VSMCs (n=4). The downstream mechanisms were further investigated. Pulldown assays demonstrated that beraprost-mediated stimulation of Epac produces subsequent activation of the monomeric G-protein Rap1 and ultimately inhibits RhoA-induced cytoskeleton rearrangement required for migration (n=3). Using siRNA to knockdown Rap1, the inhibition of PDGF-induced migration by beraprost in VSMC cells was shown to be dependent on the Rap1 pathway (n=3). In conclusion, the prostacyclin analogue beraprost inhibits VSMC migration via activation of Epac/Rap1. This may provide a new therapeutic target to prevent neointimal hyperplasia.

Dihydroaustrasulfone alcohol inhibits PDGF-induced proliferation and migration of human aortic smooth muscle cells through inhibition of the cell cycle.

Dihydroaustrasulfone alcohol is the synthetic precursor of austrasulfone, which is a marine natural product, isolated from the Taiwanese soft coral Cladiella australis. Dihydroaustrasulfone alcohol has anti-inflammatory, neuroprotective, antitumor and anti-atherogenic properties. Although dihydroaustrasulfone alcohol has been shown to inhibit neointima formation, its effect on human vascular smooth muscle cells (VSMCs) has not been elucidated. We examined the effects and the mechanisms of action of dihydroaustrasulfone alcohol on proliferation, migration and phenotypic modulation of human aortic smooth muscle cells (HASMCs). Dihydroaustrasulfone alcohol significantly inhibited proliferation, DNA synthesis and migration of HASMCs, without inducing cell death. Dihydroaustrasulfone alcohol also inhibited platelet-derived growth factor (PDGF)-induced expression of cyclin-dependent kinases (CDK) 2, CDK4, cyclin D1 and cyclin E. In addition, dihydroaustrasulfone alcohol inhibited PDGF-induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whereas it had no effect on the phosphorylation of phosphatidylinositol 3-kinase (PI3K)/(Akt). Moreover, treatment with PD98059, a highly selective ERK inhibitor, blocked PDGF-induced upregulation of cyclin D1 and cyclin E and downregulation of p27kip1. Furthermore, dihydroaustrasulfone alcohol also inhibits VSMC synthetic phenotype formation induced by PDGF. For in vivo studies, dihydroaustrasulfone alcohol decreased smooth muscle cell proliferation in a rat model of restenosis induced by balloon injury. Immunohistochemical staining showed that dihydroaustrasulfone alcohol noticeably decreased the expression of proliferating cell nuclear antigen (PCNA) and altered VSMC phenotype from a synthetic to contractile state. Our findings provide important insights into the mechanisms underlying the vasoprotective actions of dihydroaustrasulfone alcohol and suggest that it may be a useful therapeutic agent for the treatment of vascular occlusive disease.

K Ca 3.1 inhibition decreases size and alters composition of atherosclerotic lesions induced by low, oscillatory flow

Low, oscillatory flow/shear patterns are associated with atherosclerotic lesion development. Increased expression of KCa3.1 has been found in vascular smooth muscle (VSM), macrophages and T cells in lesions from humans and mice. Proliferating VSM increase expression of KCa3.1, such that it becomes a dominant K+ channel. KCa3.1 is also required for PDGF-induced VSM cell migration and we showed that the specific KCa3.1 inhibitor, TRAM-34, could inhibit coronary lesion development following balloon injury in swine. To determine the role of KCa3.1 in atherosclerotic lesion composition and development, we used the partial carotid ligation (PCL) model in high-fat fed, Apoe-/- mice which produces a low, oscillatory (i.e. atheroprone) flow pattern in the left carotid artery. PCL was performed on 6-8 week old male Apoe-/- mice. Mice were subsequently placed on a Western diet (TD.88137, Teklad) for 4 weeks and received daily s.c. injections of TRAM-34 (120 mg/kg; n=6) or equal volumes of vehicle (peanut oil, n=6). TRAM-34 treatment reduced lesion size 50% (p<0.05). In addition, lesions from TRAM-34 treated mice vs. controls contained significantly less collagen (6 ± 1% v. 15 ± 2%; p<0.05) and fibronectin (14±3% v. 32±3%; p<0.05). Consistent with the role of intimal smooth muscle in matrix-production, smooth muscle content of the lesions was also significantly reduced by TRAM-34 (19±2% v. 29±3%; p<0.05). Conversely, TRAM-34 had no effect on total cholesterol (1455 vs. 1334 mg/dl, PO and TRAM, resp.) or body weight (29.1 vs. 28.8 g, PO and TRAM, resp.). Together, these data support a major role for KCa3.1 in determining smooth muscle and matrix content of atherosclerotic lesions.

The Matricellular Protein Cyr61 Is a Key Mediator of Platelet-derived Growth Factor-induced Cell Migration

Platelet-derived growth factor (PDGF), a potent chemoattractant, induces cell migration via the MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified the matricellular protein Cyr61, which is de novo synthesized in response to PDGF stimulation, as the key downstream mediator of the ERK and JNK pathways, independent of the p38 MAPK and AKT pathways, and, thereby, it mediates PDGF-induced smooth muscle cell migration but not proliferation. Our results revealed that, when Cyr61 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with the plasma membrane integrins α6β1 and αvβ3. We further demonstrate that Cyr61 and integrins are integral components of the PDGF signaling pathway via an "outside-in" signaling route to activate intracellular focal adhesion kinase (FAK), leading to cell migration. Therefore, this study provides the first evidence that the PDGF-induced endogenous extracellular matrix component Cyr61 is a key mediator in modulating cell migration by connecting intracellular PDGF-ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular Cyr61 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor-induced cell migration. The discovered signaling pathway may represent an important therapeutic target in growth factor-mediated cell migration/invasion-related vascular diseases and tumorigenesis.

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.

Chronic kidney disease alters vascular smooth muscle cell phenotype.

Vascular access dysfunction associated with arteriovenous grafts and fistulas contributes to the morbidity and mortality of chronic kidney disease (CKD) patients receiving hemodialysis. We hypothesized that the uremic conditions associated with CKD promote a pathophysiological vascular smooth muscle cell (VSMC) phenotype that contributes to neointimal hyperplasia. We analyzed the effect of culturing human VSMC with uremic serum. Expression of VSMC contractile marker genes was reduced 50-80% in cells exposed to uremic serum and the decreased expression was accompanied by changes in histone marks. There was an increase in proliferation in cells exposed to uremic conditions, with no change in the levels of apoptosis. Interestingly, we found that uremic serum inhibited PDGF-induced migration of VSMC. Histomorphometric analysis revealed venous neointimal hyperplasia in veins from chronic kidney disease (CKD) patients prior to any surgical manipulation as compared to veins from patients with no kidney disease. We conclude that uremia associated with CKD alters VSMC phenotype in vitro and contributes to neointimal hyperplasia formation in vivo contributing to the pathogenesis of vascular access dysfunction in CKD patients.

Aspirin-triggered 15-epi-lipoxin A4 signals through FPR2/ALX in vascular smooth muscle cells and protects against intimal hyperplasia after carotid ligation

Aspirin-triggered 15-epi-lipoxin A4 signals through FPR2/ALX in vascular smooth muscle cells and protects against intimal hyperplasia after carotid ligation

Poldip2 controls vascular smooth muscle cell migration by regulating focal adhesion turnover and force polarization.

Polymerase-δ-interacting protein 2 (Poldip2) interacts with NADPH oxidase 4 (Nox4) and regulates migration; however, the precise underlying mechanisms are unclear. Here, we investigated the role of Poldip2 in focal adhesion turnover, as well as traction force generation and polarization. Poldip2 overexpression (AdPoldip2) in vascular smooth muscle cells (VSMCs) impairs PDGF-induced migration and induces a characteristic phenotype of long cytoplasmic extensions. AdPoldip2 also prevents the decrease in spreading and increased aspect ratio observed in response to PDGF and slightly impairs cell contraction. Moreover, AdPoldip2 blocks focal adhesion dissolution and sustains H2O2 levels in focal adhesions, whereas Poldip2 knockdown (siPoldip2) significantly decreases the number of focal adhesions. RhoA activity is unchanged when focal adhesion dissolution is stimulated in control cells but increases in AdPoldip2-treated cells. Inhibition of RhoA blocks Poldip2-mediated attenuation of focal adhesion dissolution, and overexpression of RhoA or focal adhesion kinase (FAK) reverses the loss of focal adhesions induced by siPoldip2, indicating that RhoA and FAK mediate the effect of Poldip2 on focal adhesions. Nox4 silencing prevents focal adhesion stabilization by AdPoldip2 and induces a phenotype similar to siPoldip2, suggesting a role for Nox4 in Poldip2-induced focal adhesion stability. As a consequence of impaired focal adhesion turnover, PDGF-treated AdPoldip2 cells are unable to reduce and polarize traction forces, a necessary first step in migration. These results implicate Poldip2 in VSMC migration via regulation of focal adhesion turnover and traction force generation in a Nox4/RhoA/FAK-dependent manner.

Abstract 347: Chronic Kidney Disease Modulates Vascular Smooth Muscle Cell Phenotype and Increases de Novo Venous Intimal Hyperplasia

Background: Vascular access dysfunction associated with arteriovenous grafts and fistulas contributes significantly to the morbidity and mortality of end-stage renal disease (ESRD) patients receiving hemodialysis. The mechanism involved in the development of peri-anastomotic intimal hyperplasia, a major cause of the arteriovenous access dysfunction, is poorly elucidated. We hypothesized that the uremic condition associated with ESRD promotes a pathophysiological vascular smooth muscle cell (VSMC) phenotype that contributes to accelerated development of intimal hyperplasia in blood vessels, both de novo and following the arteriovenous anastomosis. Methods and Results: We examined the effect of culturing human VSMC with human uremic serum on its phenotype. We utilized quantitative real-time PCR to measure the expression of the VSMC-specific contractile marker genes, and found that these genes were reduced 50-80% in VSMC exposed to serum from hemodialysis patients as compared to serum from patients with normal renal function. Chromatin immunoprecipitation assays demonstrated that there were also significant changes in epigenetic markers associated with contractile gene promoters in VSMC cultured in uremic conditions. Moreover, there was a 60% increase in proliferation rate in cells exposed to uremic conditions, with no change in the levels of apoptosis as compared to cells exposed to non-uremic conditions. Interestingly, uremic serum from ESRD patients inhibited PDGF-induced migration of VSMC. We also examined the histopathology of veins from patients with normal kidney function and chronic kidney disease (CKD). Indeed, we found significant de novo venous intimal hyperplasia exists in CKD and ESRD patients prior to any surgical manipulation as compared to veins from patients with normal kidney function. Conclusion: CKD alters VSMC phenotype in vitro and contributes to intimal hyperplasia formation in vivo resulting in the pathogenesis of vascular access dysfunction in ESRD patients.