PDGF-stimulated Smooth Muscle Cell Research Articles

A novel PDGF receptor inhibitor-eluting stent attenuates in-stent neointima formation in a rabbit carotid model.

A novel drug-eluting stent (DES) is required to target vascular smooth muscle cells (SMCs) without harming endothelial cells (ECs). Platelet-derived growth factor (PDGF) is critical for the proliferation and migration of SMCs. Sunitinib [a PDGF receptor (PDGFR) tyrosine kinase inhibitor]-eluting stents may therefore inhibit neointimal formation. The aim of the present study was to examine the stent-based delivery of sunitinib in a rabbit carotid model; in addition, the effects of sunitinib were evaluated in vitro. Local administration of sunitinib markedly reduced neointimal formation without delaying re-endothelialization in the carotid artery model. In vitro, sunitinib inhibited SMC proliferation; however, no effects were observed on ECs. Sunitinib caused necrosis of SMCs. In addition, sunitinib attenuated PDGF-stimulated SMC migration in a scratch wound assay and inhibited α-SMA cytoskeleton polymerization. Furthermore, sunitinib inhibited PDGF-induced phosphorylation of extracellular signal-regulated kinase in vitro and in vivo. Therefore, this novel DES may be a potential strategy for the treatment of vascular disorders.

Thrombin Stimulated Platelet-Derived Exosomes Inhibit Platelet-Derived Growth Factor Receptor-Beta Expression in Vascular Smooth Muscle Cells

Background/Aims: It is difficult to predict acute thrombotic cardiovascular events in the clinic. Few studies have reported the presence of plasma exosomes containing microRNAs (miRNAs) in cardiovascular events. Therefore, we aimed to investigate the levels of miR-223, miR-339 and miR-21 in plasma exosomes before thrombosis in mouse models of carotid tandem stenosis, as well as the mechanisms underlying the origin and function of these exosomal miRNAs. Methods: Plasma samples were collected from the carotid tandem stenosis and sham control groups of our successfully developed atherothrombosis mouse models before thrombosis. Platelets from healthy volunteers and mice were purified to obtain thrombin stimulated platelet-derived exosomes. Exosomes were isolated via differential ultracentrifugation, and western blotting and transmission electron microscopy were used for their identification. The total RNA was extracted, and quantitative real-time PCR was performed to determine the expression levels of miR-223, miR-339 and miR-21. DAVID Tools were used to analyze the pathways that were enriched among the miRNA target genes. Immuno-fluorescence staining was performed to identify the protein expression levels of platelet-derived exosome target genes in vascular smooth muscle cells (SMCs) in vitro and in vivo. Results: The levels of miR-223, miR-339 and miR-21, which are associated with platelet activation, were elevated in pooled mouse plasma exosomes before thrombosis and enriched in thrombin-stimulated platelet-derived exosomes in vitro. Platelet-derived growth factor receptor-beta (PDGFRβ) was a target of these miRNAs, and PDGFRβ expression in vascular smooth muscle cells (SMCs) was inhibited following incubation with platelet-derived exosomes. Platelet-derived exosomes could also inhibit PDGF-stimulated SMC proliferation. Furthermore, a decrease in PDGFRβ expression was observed in vascular SMCs around thrombotic areas in vivo. Conclusions: Our data indicate that activated platelet-derived exosomes containing miR-223, miR-339 and miR-21 could be transferred into SMCs and inhibit PDGFRβ expression; these exosomal miRNAs may be a biomarker for predicting atherothrombosis.

Integration of proteomic and transcriptomic profiles identifies a novel PDGF-MYC network in human smooth muscle cells.

BackgroundPlatelet-derived growth factor-BB (PDGF-BB) has been implicated in the proliferation, migration and synthetic activities of smooth muscle cells that characterize physiologic and pathologic tissue remodeling in hollow organs. However, neither the molecular basis of PDGFR-regulated signaling webs, nor the extent to which specific components within these networks could be exploited for therapeutic benefit has been fully elucidated.ResultsExpression profiling and quantitative proteomics analysis of PDGF-treated primary human bladder smooth muscle cells identified 1,695 genes and 241 proteins as differentially expressed versus non-treated cells. Analysis of gene expression data revealed MYC, JUN, EGR1, MYB, RUNX1, as the transcription factors most significantly networked with up-regulated genes. Forty targets were significantly altered at both the mRNA and protein levels. Proliferation, migration and angiogenesis were the biological processes most significantly associated with this signature, and MYC was the most highly networked master regulator. Alterations in master regulators and gene targets were validated in PDGF-stimulated smooth muscle cells in vitro and in a model of bladder injury in vivo. Pharmacologic inhibition of MYC and JUN confirmed their role in SMC proliferation and migration. Network analysis identified the diaphanous-related formin 3 as a novel PDGF target regulated by MYC and JUN, which was necessary for PDGF-stimulated lamellipodium formation.ConclusionsThese findings provide the first systems-level analysis of the PDGF-regulated transcriptome and proteome in normal smooth muscle cells. The analyses revealed an extensive cohort of PDGF-dependent biological processes and connected key transcriptional effectors to their regulation, significantly expanding current knowledge of PDGF-stimulated signaling cascades. These observations also implicate MYC as a novel target for pharmacological intervention in fibroproliferative expansion of smooth muscle, and potentially in cancers in which PDGFR-dependent signaling or MYC activation promote tumor progression.

Integration of proteomic and transcriptomic profiles identifies a novel PDGF-MYC network in human smooth muscle cells

Platelet-derived growth factor-BB (PDGF-BB) has been implicated in the proliferation, migration and synthetic activities of smooth muscle cells that characterize physiologic and pathologic tissue remodeling in hollow organs. However, neither the molecular basis of PDGFR-regulated signaling webs, nor the extent to which specific components within these networks could be exploited for therapeutic benefit has been fully elucidated. Expression profiling and quantitative proteomics analysis of PDGF-treated primary human bladder smooth muscle cells identified 1,695 genes and 241 proteins as differentially expressed versus non-treated cells. Analysis of gene expression data revealed MYC, JUN, EGR1, MYB, RUNX1, as the transcription factors most significantly networked with up-regulated genes. Forty targets were significantly altered at both the mRNA and protein levels. Proliferation, migration and angiogenesis were the biological processes most significantly associated with this signature, and MYC was the most highly networked master regulator. Alterations in master regulators and gene targets were validated in PDGF-stimulated smooth muscle cells in vitro and in a model of bladder injury in vivo. Pharmacologic inhibition of MYC and JUN confirmed their role in SMC proliferation and migration. Network analysis identified the diaphanous-related formin 3 as a novel PDGF target regulated by MYC and JUN, which was necessary for PDGF-stimulated lamellipodium formation. These findings provide the first systems-level analysis of the PDGF-regulated transcriptome and proteome in normal smooth muscle cells. The analyses revealed an extensive cohort of PDGF-dependent biological processes and connected key transcriptional effectors to their regulation, significantly expanding current knowledge of PDGF-stimulated signaling cascades. These observations also implicate MYC as a novel target for pharmacological intervention in fibroproliferative expansion of smooth muscle, and potentially in cancers in which PDGFR-dependent signaling or MYC activation promote tumor progression.

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.

An Akt- and Fra-1-Dependent Pathway Mediates Platelet-Derived Growth Factor-Induced Expression of Thrombomodulin, a Novel Regulator of Smooth Muscle Cell Migration

Overdistension of hollow organs evokes pathological changes characterized by smooth muscle remodeling. Mechanical stimuli induce smooth muscle cell (SMC) growth through acute activation of signaling cascades and by increased expression of soluble mitogens. Physical forces have also been implicated in ligand-independent activation of receptor tyrosine kinases, including the platelet-derived growth factor (PDGF) receptor, although the extent to which this occurs in intact tissue is unknown. Previously, we implicated Akt and activator protein-1 (AP-1) as mediators of growth and gene expression in SMC exposed to cyclic stretch or PDGF. Here we show that bladder wall distension leads to PDGFR activation and identify thrombomodulin (TM) as an Akt and AP-1 target in SMC. We demonstrate that TM, also induced by bladder stretch injury, is regulated at the transcriptional level by the AP-1 components c-jun and Fra1. Mutation of an AP-1 motif at -2010/-2004 abolished both AP-1 binding and PDGF responsiveness of the TM promoter. Fra1 silencing diminished PDGF-induced TM expression and SMC cell cycle transit. In contrast, TM knockdown did not affect cell growth but attenuated PDGF-stimulated SMC migration. Taken together, these results reveal new facets of TM regulation in SMC and provide the first demonstration of a role for endogenous TM in PDGF-induced cell migration. Moreover, TM induction on bladder injury suggests that it may be a biomarker for pathological smooth muscle remodeling.

Autocrine expression of osteopontin contributes to PDGF-mediated arterial smooth muscle cell migration

Migration of smooth muscle cells (SMCs) from the media to the intima of arteries is involved in intimal thickening. The platelet-derived growth factor (PDGF) BB is recognized as a major migratory factor for arterial SMCs both in vitro and during neointima formation. Since PDGF acts in synergy with the matrix protein osteopontin (OPN) and also induces its expression, the present study was conceived to explore the role of the OPN produced in an autocrine fashion by PDGF-stimulated SMCs in the migration process and to define regulatory mechanisms of OPN expression. PDGF stimulation of quiescent rat aortic SMCs induced their migration (transfilter assays) and the increase of OPN expression (mRNA and protein assays). Blockade of either OPN expression by a specific short interference RNA (siRNA) or of its function by a blocking antibody decreased the PDGF-stimulated migration by about 70%, demonstrating that autocrine production and excretion of OPN are integral to the PDGF-induced SMC migration. In parallel, SMC stimulation by PDGF also activated the transcription factor CREB essentially through mitogen-activated protein kinase (MAPK) 1/2 and protein kinase A (PKA) pathways. Inhibition of either CREB expression (via siRNA) or function (via dominant-negative CREB) decreased both PDGF-induced SMC migration and OPN expression. SMC transfection with OPN promoter reporter constructs demonstrated that PDGF-induced OPN transcription is mediated by CREB binding to two functional sites of the OPN promoter: a CRE site located at -1403 and an AP-1 site located at -76. The present study demonstrates that the autocrine expression of OPN plays a major role in PDGF-induced SMC migration. It further shows that the transcription factor CREB, activated in PDGF-stimulated SMCs, plays a key role in PDGF-induced SMC migration, probably by regulating OPN expression.

Decorin Inhibition of PDGF-Stimulated Vascular Smooth Muscle Cell Function: Potential Mechanism for Inhibition of Intimal Hyperplasia after Balloon Angioplasty

Decorin is a small proteoglycan that binds to transforming growth factor-beta (TGF-beta) and inhibits its activity. However, its interaction with platelet-derived growth factor (PDGF), involved in arterial repair after injury, is not well characterized. The objectives of this study were to assess decorin-PDGF and decorin-PDGF receptor (PDGFR) interactions, the in vitro effects of decorin on PDGF-stimulated smooth muscle cell (SMC) functions and the in vivo effects of decorin overexpression on arterial repair in a rabbit carotid balloon-injury model. Decorin binding to PDGF was demonstrated by solid-phase binding and affinity cross-linking assays. Decorin potently inhibited PDGF-stimulated PDGFR phosphorylation. Pretreatment of rabbit aortic SMC with decorin significantly inhibited PDGF-stimulated cell migration, proliferation, and collagen synthesis. Decorin overexpression by adenoviral-mediated gene transfection in balloon-injured carotid arteries significantly decreased intimal cross-sectional area and collagen content by approximately 50% at 10 weeks compared to beta-galactosidase-transfected or balloon-injured, non-transfected controls. This study shows that decorin binds to PDGF and inhibits its stimulatory activity on SMCs by preventing PDGFR phosphorylation. Decorin overexpression reduces intimal hyperplasia and collagen content after arterial injury. Decorin may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.

Inhibition of vascular smooth muscle cell migration by cytochrome p450 epoxygenase-derived eicosanoids.

Vascular smooth muscle cell (SMC) migration and proliferation contribute to neointimal hyperplasia and restenosis after vascular injury. The epoxyeicosatrienoic acids (EETs), which are products of cytochrome P450 (CYP) epoxygenases, possess vasodilatory, antiinflammatory, and fibrinolytic properties. To determine whether these compounds also possess antimigratory and antiproliferative properties, we stimulated rat aortic SMCs with either 20% serum or platelet-derived growth factor (PDGF-BB, 20 ng/mL). In a concentration-dependent manner, treatment with EETs, particularly 11,12-EET, inhibited SMC migration through a modified transwell filter by 53% to 60%. EETs, however, have no inhibitory effects on PDGF-stimulated SMC proliferation. Adenoviral-mediated overexpression of the CYP isoform, CYP2J2, in SMCs also inhibited serum- and PDGF-induced SMC migration by 32% and 26%, respectively; both effects of which were reversed by the CYP inhibitors SKF525A or clotrimazole, but not by the K(Ca) channel blocker, charybdotoxin, or the cyclooxygenase inhibitor, diclofenac. The effect of EETs correlated with increases in intracellular cAMP levels. Indeed, forskolin and 8-bromo-cAMP exert similar inhibitory effects on SMC migration as 11,12-EET and the effects of 11,12-EET were blocked by cAMP and protein kinase A (PKA) inhibitors. These findings indicate that EETs possess antimigratory effects on SMCs through the cAMP-PKA pathway and suggest that CYP epoxygenase-derived eicosanoids may play important roles in vascular disease and remodeling.

Low density lipoprotein receptor-related protein mediates apolipoprotein E inhibition of smooth muscle cell migration.

This research was undertaken to identify the cell surface receptor responsible for mediating apolipoprotein E (apoE) inhibition of platelet-derived growth factor (PDGF)-directed smooth muscle cell migration. Initial studies revealed the expression of the low density lipoprotein receptor (LDLR), the LDL receptor-related protein (LRP), the very low density lipoprotein receptor (VLDL), and apoE receptor-2 in mouse aortic smooth muscle cells. Smooth muscle cells isolated from LDLR-null, VLDL-null, and apoE receptor-2-null mice were responsive to apoE inhibition of PDGF-directed smooth muscle cell migration, suggesting that these receptors were not involved. An antisense RNA expression knockdown strategy, utilizing morpholino antisense RNA against LRP, was used to reduce LRP expression in smooth muscle cells to assess the role of this receptor in apoE inhibition of cell migration. Results showed that apoE was unable to inhibit PDGF-directed migration of LRP-deficient smooth muscle cells. The role of LRP in mediating apoE inhibition of PDGF-directed smooth muscle cell migration was confirmed by experiments showing that antibodies against LRP effectively suppressed apoE inhibition of PDGF-directed smooth muscle cell migration. Taken together, these results document that apoE binding to LRP is required for its inhibition of PDGF-directed smooth muscle cell migration.

Diltiazem, a calcium antagonist, inhibits matrix metalloproteinase-1 (tissue collagenase) production and collagenolytic activity in human vascular smooth muscle cells.

Calcium antagonists (CAs) are widely prescribed for patients with cardiovascular diseases. CAs have been reported to inhibit smooth muscle cell (SMC) proliferation in addition to their effects on vascular tone. To determine whether CAs potentially affect vascular remodeling, we measured the expression of matrix-degrading enzymes in growth factor-stimulated SMC. Human cultured SMC were stimulated with 10 ng/ml of platelet-derived growth factor (PDGF)-BB with or without a calcium antagonist, diltiazem. In the cell counting assay, diltiazem (10-5 M) alone had no effect on the proliferation of quiescent SMC, however 10-6-10-5 M of diltiazem dose-dependently inhibited PDGF-stimulated SMC proliferation. The inhibitory effects of diltiazem on SMC proliferation were further confirmed by a 5-bromo-2'-deoxyuridine (BrdU) incorporation assay and flow cytometry. In Western blotting, matrix metalloproteinase (MMP)-1 (tissue collagenase) but not MMP-2 (72-kDa gelatinase) expression was upregulated by PDGF and phorbol ester (TPA), which were reduced by diltiazem in a dose-dependent manner. The downregulation of MMP-1 expression was consistent with the reduction of collagenolytic activity measured by a FITC-conjugated type I collagen breakdown assay. PDGF-stimulated c-Jun/AP-1 expression, a major transcriptional factor for MMP-1, was not affected by diltiazem. In contrast, intracellular calcium ions measured with a fluorometric assay of Fluo-3AM-loaded cells revealed that the PDGF-stimulated increase in the intracellular calcium content was dose-dependently reduced by diltiazem. Our data suggest that diltiazem inhibits not only proliferation but also MMP-1 expression and collagenolytic activity in PDGF-stimulated SMC. The administration of CAs potentially influences the process of vascular remodeling, and this possibility should be further verified in vivo.

Apolipoprotein E Receptor Binding VersusHeparan Sulfate Proteoglycan Binding in Its Regulation of Smooth Muscle Cell Migration and Proliferation

This study showed that synthetic peptides containing either a single copy or tandem repeat of the receptor binding domain sequence of apolipoprotein (apo) E, or a peptide containing its C-terminal heparin binding domain, apoE-(211-243), were all effective inhibitors of platelet-derived growth factor (PDGF)-stimulated smooth muscle cell proliferation. In contrast, only the peptide containing a tandem repeating unit of the receptor binding domain sequence of apoE, apoE-(141-155)(2), was capable of inhibiting PDGF-directed smooth muscle cell migration. Peptide containing only a single unit of this sequence, apoE-(141-155), or the apoE-(211-243) peptide were ineffective in inhibiting PDGF-directed smooth muscle cell migration. Additional experiments showed that reductively methylated apoE, which is incapable of receptor binding yet retains its heparin binding capability, was equally effective as apoE in inhibiting PDGF-stimulated smooth muscle cell proliferation. However, reductively methylated apoE was unable to inhibit smooth muscle cell migration toward PDGF. Additionally, the receptor binding domain-specific apoE antibody 1D7 also mitigated the anti-migratory properties of apoE on smooth muscle cells. Finally, pretreatment of cells with heparinase failed to abolish apoE inhibition of smooth muscle cell migration. Taken together, these data documented that apoE inhibition of PDGF-stimulated smooth muscle cell proliferation is mediated by its binding to heparan sulfate proteoglycans, while its inhibition of cell migration is mediated through apoE binding to cell surface receptors.

PDGF Receptor-to-Nucleus Signaling of p91 (STAT1α) Transcription Factor in Rat Smooth Muscle Cells

Vascular smooth muscle cells (VSMC) are the predominant cell type in the media of a normal artery. Injury to the vessel wall leads to platelet deposition and the release of numerous factors, including PDGF, which exerts its biological effects by binding to specific surface receptors on the smooth muscle cell membrane. We demonstrate that PDGF-stimulated smooth muscle cells activate the STAT (signal transducers and activators of transcription) family of proteins in addition to other signaling pathways (e.g., RAS-RAF-MAP Kinase). We show that the transcription factor p91 (STAT1α) is rapidly activated by PDGF in VSMC and specifically binds to the regulatory elements SIE or GAS. We hypothesize that signal transduction by p91 plays an important role in VSMC, especially after injury with the release of growth factors such as PDGF.

Production of tissue collagenase (matrix metalloproteinase 1) by human aortic smooth muscle cells in response to platelet-derived growth factor

The production of the precursor of tissue collagenase/matrix metal loproteinase 1 (proMMP-l) by cultured human aortic medial smooth muscle cells (SMCs) was significantly enhanced by the treatment of the cells with platelet-derived growth factor (PDGF), interleukin 1 or 12- O-tetradecanoylphorbol-13-acetate (TPA). The response to PDGF of SMCs exhibited a tendency to be age-dependent: only SMCs obtained from older individuals (age: 54, 56, 72 and 74 years) responded to PDGF and synthesized proMMP-1, but not SMCs from young individuals (age: 10, 16 and 41 years), and weak responsiveness with a 19-year-old individual. On the other hand, induction of proMMP-1 synthesis in SMCs by TPA was not discriminated by age. The synthesis of two other related matrix metalloproteinases was also examined. Matrix metal loproteinase 2 was found to be constitutively expressed in zymogen form in SMCs and its synthesis was not affected by the treatments with PDGF, interleukin 1 or TPA. The synthesis of matrix metalloproteinase 3 (stromelysin) was not detected in SMCs from both young and old individuals even after the treatment with PDGF, interleukin-1, prostaglandin Ez or TPA. The ability of SMCs to synthesize and secrete proMMP-1 in response to PDGF suggests that this enzyme plays an important role in the migration of PDGF-stimulated SMCs from the media into the intima of aorta and the eventual formation of atherosclerotic plaques.

Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells.

Platelet-derived growth factor (PDGF), a smooth muscle cell (SMC) mitogen, and heparin-like glycosaminoglycans, known inhibitors of SMC growth and migration, were found to regulate thrombospondin synthesis and matrix deposition by cultured rat aortic SMC. The synthesis and distribution of thrombospondin was examined in growth-arrested SMCs, in PDGF-stimulated SMCs, and in heparin-treated SMCs using metabolic labeling and immunofluorescence techniques. Thrombospondin synthesis in response to purified PDGF occurred within 1 h after addition of growth factor to growth-arrested SMCs, peaked at 2 h, and returned to baseline levels by 5 h. The induction of synthesis of thrombospondin by PDGF was dose dependent, with a maximal effect observed at 2.5 ng/ml. Actinomycin D (2 micrograms/ml) inhibited thrombospondin induction by PDGF, suggesting a requirement for new RNA synthesis. In the presence of heparin and related polyanions, the incorporation of thrombospondin into the SMC extracellular matrix was markedly reduced. This effect was dose dependent with a maximal effect observed at a heparin concentration of 1 microgram/ml. Heparin did not affect the ability of SMCs to synthesize thrombospondin in response to PDGF. We interpret these data to suggest a role for thrombospondin in the SMC proliferative response to PDGF and in the regulation of SMC growth and migration by glycosaminoglycans.