Phosphatidylinositol 3-kinase but not tuberin is required for PDGF-induced cell migration

The loss of function of the tumor suppressor gene TSC2 and its protein product tuberin promotes the development of benign lesions by stimulating cell growth, although the role of tuberin in regulating cell migration and metastasis has not been characterized. In addition, the role of phosphatidylinositol 3-kinase (PI 3-kinase), an important signaling event regulating cell migration, in modulating tuberin-deficient cell motility remains unknown. Using a tuberin-deficient rat smooth muscle cell line, ELT3, we demonstrate that platelet-derived growth factor (PDGF) stimulates cell migration by 3.2-fold, whereas vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-alpha, and basic fibroblast growth factor (bFGF) increase migration by 2.1-, 2.1-, and 2.6-fold, respectively. Basal and PDGF-induced migration in tuberin-deficient ELT3, ELT4, and ERC15 cells was not significantly different from that of tuberin-positive transformed rat kidney epithelial 2, airway smooth muscle, and pulmonary arterial vascular smooth muscle cells. Expression of tuberin in tuberin-deficient ELT3 cells also had little effect on cell migration. In parallel experiments, the role of PI 3-kinase activation in ELT3 cell migration was investigated. LY-294002, a PI 3-kinase inhibitor, decreased PDGF-induced migration in a concentration-dependent manner with an IC(50) of approximately 5 microM. LY-294002 also abrogated ELT3 cell migration stimulated by bFGF and TGF-alpha but not by VEGF and phorbol 12-myristate 13-acetate. Furthermore, transient expression of constitutively active PI 3-kinase (p110*) was sufficient to induce ELT3 cell migration. However, the migration induced by p110* was less than that induced by growth factors, suggesting other signaling pathways are also critically important in modulating growth factor-induced cell migration. These data suggest that PI 3-kinase is required for growth factor-induced cell migration and loss of tuberin appears to have little effect on cell migration.     

PDGF-BB induces vascular smooth muscle cell expression of high molecular weight FGF-2, which accumulates in the nucleus

Basic fibroblast growth factor (FGF-2) and platelet-derived growth factor (PDGF) are implicated in vascular remodeling secondary to injury. Both growth factors control vascular endothelial and smooth muscle cell proliferation, migration, and survival through overlapping intracellular signaling pathways. In vascular smooth muscle cells PDGF-BB induces FGF-2 expression. However, the effect of PDGF on the different forms of FGF-2 has not been elucidated. Here, we report that treatment of vascular aortic smooth muscle cells with PDGF-BB rapidly induces expression of 20.5 and 21 kDa, high molecular weight (HMW) FGF-2 that accumulates in the nucleus and nucleolus. Conversely, PDGF treatment has little or no effect on 18 kDa, low-molecular weight FGF-2 expression. PDGF-BB-induced upregulation of HMW FGF-2 expression is controlled by sustained activation of extracellular signal-regulated kinase (ERK)-1/2 and is abolished by actinomycin D. These data describe a novel interaction between PDGF-BB and FGF-2, and indicate that the nuclear forms of FGF-2 may mediate the effect of PDGF activity on vascular smooth muscle cells.     

Basic fibroblast growth factor modulates the mitogenic potency of the platelet-derived growth factor (PDGF) isoforms by specific upregulation of the PDGF alpha receptor in vascular smooth muscle cells.

Platelet-derived growth factor AA (PDGF AA), in contrast to PDGF AB and BB, is a poor mitogen for smooth muscle cells (SMC). However, together with basic fibroblast growth factor (bFGF) it acts synergistically on DNA synthesis of these cells. Northern blot analysis revealed that bFGF selectively increases the PDGF-receptor alpha subtype (PDGF-R alpha) mRNA level without a significant effect on the PDGF-R beta mRNA level. The amount of PDGF-R alpha protein is also selectively increased after stimulating SMC with bFGF as shown by immunoprecipitation of lysates from SMC with anti-PDGF-R alpha antibodies. The number of binding sites for 125I-PDGF AA is more than doubled after bFGF-treatment, whereas the specific binding for PDGF AB and BB increased only by approximately 30 and 20%, respectively. The increase in the number of PDGF-R alpha renders the SMC responsive for PDGF AA as demonstrated by the induction of the proto-oncogene c-fos as well as by an increased cell proliferation. The enhanced PDGF binding after bFGF treatment may in fact explain the observed synergistic behavior. These data are discussed with regard to a possible role of growth factor-induced transmodulation of receptor expression during atherogenesis.     

Erythromycin and clarithromycin modulation of growth factor-induced expression of heparanase mRNA on human lung cancer cells in vitro.

Heparanase activity is correlated with the metastatic potential of several cancer cells and is a key enzyme in the breakdown of tissue barriers. It is also involved in the regulation of growth factor and cytokine activity. However, little is known about the factors that induce heparanase in cancer cells. We investigated the effect of three growth factors, platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF), on heparanase mRNA induction in lung cancer cells in vitro. In addition, we examined the effect of erythromycin (EM) and clarithromycin (CAM), which are 14-membered ring macrolide antibiotics that act as biological response modifiers, on the expression of heparanase mRNA induced by growth factors. PDGF, HGF and bFGF stimulated cell migration activity and enhanced the expression of heparanase mRNA in the human lung adenocarcinoma cell line A549. Via different mechanisms, EM and CAM modulate the induction by these factors of heparanase mRNA expression on A549 cells. EM also significantly suppressed A549 cell migration induced by PDGF and HGF, and CAM significantly suppressed A549cell migration induced by bFGF. The results suggest that the growth factors PDGF, HGF and bFGF are important inducers of heparanase in potentially invasive and metastatic cancer cells. The suppressive effect of heparanase mRNA expression by EM and CAM may have interestingtherapeutic applications in the prevention of metastasis.     

非磷酸化肌球蛋白在血小板衍生生长因子诱导的血管平滑肌细胞迁移中的作用

为了阐明非磷酸化肌球蛋白在平滑肌细胞迁移中的作用,研究探讨了非磷酸化肌球蛋白是否介导了血小板衍生生长因子(PDGF)诱导豚鼠脑基底动脉平滑肌细胞(GbaSM-4)的迁移。研究结果显示,20ng/ml以下剂量的PDGF可诱导GbaSM-4细胞发生迁移,此时肌球蛋白轻链(MLC20)磷酸化水平无变化。该迁移作用可被肌球蛋白特异性抑制剂blebbistatin所拮抗。应用RNA干扰技术抑制肌球蛋白轻链激酶表达,经免疫印迹检测经果显示,MLC20的磷酸化水平发生了显著下降;但对PDGF诱导的迁移作用无影响;在RNA干扰后blebbistatin也可抑制其迁移作用。体外ATP酶活性测定结果显示,blebbistatin对从平滑肌中提取的非磷酸化肌球蛋白的ATP酶活性有明显的抑制作用,其主要作用位点位于肌球蛋白头的头部S1。上述结果提示,非磷酸化的肌球蛋白参与了PDGF诱导的平滑肌细胞迁移。     

人主动脉硫酸乙酰肝素蛋白聚糖对培养的人血管壁细胞生长的作用

通过培养的人主动脉平滑肌细胞（ｈＡＳＭＣ）及脐静脉内皮细胞（ｈＵＶＥＣ）,应用３Ｈ－ＴｄＲ参入、Ｎｏｒｔｈｅｒｎｂｌｏｔ分析、逆转录多聚酶链反应（ＲＴ－ＰＣＲ）、放射免疫分析（ＲＩＡ）、和紫外比色法等技术观察了人主动脉中硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对ｈＡＳＭＣ和ｈＵＶＥＣＤＮＡ合成的作用及对血小板源生长因子（ＰＤＧＦ）、ＰＤＧＦ受体、转化生长因子β（ＴＧＦ－β）、内皮素－１（ＥＴ－１）或碱性成纤维细胞生长因子（ｂＦＧＦ）基因表达和肾素－血管紧张系统（ＲＡＳ）的影响,结果显示,ＨＳＰＧ明显抑制培养的ｈＡＳＭＣ基础的ＤＮＡ合成（ｃｐｍ值为：１０３８５±３２６３ｖｓ,２５５４１±６４２１,Ｐ＜０．０１）及外源性ＰＤＧＦ诱导的ＤＮＡ合成（ｃｐｍ值为：９８７８±１９４７ｖｓ．１３４８１±４４ｌ０,Ｐ＜０．０５）;抑制ＰＤＧＦＡ链、ＴＧＦ－Ｂｐ和ＥＴ－１ｍＲＮＡ表达,提高ＰＤＧＦａ和β受体ｍＲＮＡ的表达;显著降低ｈＡＳＭＣ培养液中血管紧张素Ⅱ（ＡｎｇⅡ）的浓度和血管紧张素转换酶（ＡＣＥ）的活性,推测ＨＳＰＧ抑制ＰＤＧＦＡ链、ＴＧＦ－β及ＥＴ－１ｍＲＮＡ表达,降低ＡＣＥ活性及ＡｎｇⅡ浓度是其抑制ｈＡＳＭＣ增殖的重要机     

Both platelet-derived growth factor receptor (PDGFR)-alpha and PDGFR-beta promote murine fibroblast cell migration

Cell motility plays a critical role for many physiological and pathological processes including wound healing, fibrosis, angiogenesis, and tumor metastasis. Platelet-derived growth factor (PDGF) is among the most potent stimuli for mesenchymal cell migration. The PDGF B-chain homodimer PDGF BB activates both alpha- and beta-receptor subunits (alpha-PDGFR and beta-PDGFR), and promotes cell migration in many cell types including fibroblasts and smooth muscle cells. PDGF-A chain homodimer PDGF AA activates alpha-PDGFR only, and its role for cell migration is still debatable. PDGF BB, but not PDGF AA, induces smooth muscle cell migration. Interestingly, alpha-PDGFR was shown to antagonize beta-PDGFR-induced smooth muscle cell migration. In the present study, we investigated the role of alpha-PDGFR and beta-PDGFR in PDGF-mediated cell migration of murine fibroblasts (NIH 3T3). Unlike smooth muscle cells, both PDGF AA and PDGF BB promoted NIH 3T3 cell migration. The effect of PDGF BB activation of beta-PDGFR alone for cell migration was examined using previously established NIH 3T3 clones in which alpha-PDGFR signaling is inhibited by a dominant-negative alpha-PDGFR, or an antisense construct of alpha-PDGFR. PDGF BB activation of beta-PDGFR alone was sufficient to induce cell migration, but the efficiency was significantly lower compared to PDGF activation of both receptors. These results showed that both alpha- and beta-PDGFRs promote fibroblast cell migration and their effects are additive. Taken together, we propose that cell-type specific alpha-PDGFR signaling is critical for regulation of mesenchymal cell migration in response to PDGF isoform, whereas beta-PDGFR mainly promotes cell migration.     

Apatinib attenuates phenotypic switching of arterial smooth muscle cells in vascular remodelling by targeting the PDGF Receptor‐β

Apatinib (YN968D1) is a small‐molecule tyrosine kinase inhibitor（TKI）which can inhibit the activity of vascular endothelial growth factor receptor‐2 (VEGFR‐2). It has been reported that apatinib has anti‐tumour effect of inhibiting proliferation and inducing apoptosis of a variety of solid tumour cells, whereas its effect on vascular smooth muscle cells (VSMC) remains unclear. This study investigated the effect of apatinib on phenotypic switching of arterial smooth muscle cells in vascular remodelling. Compared to the vehicle groups, mice that were performed carotid artery ligation injury and treated with apatinib produced a reduction in abnormal neointimal area. For in vitro experiment, apatinib administration inhibited VSMC proliferation, migration and reversed VSMC dedifferentiation with the stimulation of platelet‐derived growth factor type BB (PDGF‐BB).In terms of mechanism, with the preincubation of apatinib, the activations of PDGF receptor‐β (PDGFR‐β) and phosphoinositide‐specific phospholipase C‐γ1 (PLC‐γ1) induced by PDGF‐BB were inhibited in VSMCs. With the preincubation of apatinib, the phosphorylation of PDGFR‐β, extracellular signal‐related kinases (ERK1/2) and Jun amino‐terminal kinases (JNK) induced by PDGF‐BB were also inhibited in rat vascular smooth muscle cell line A7r5. Herein, we found that apatinib attenuates phenotypic switching of arterial smooth muscle cells induced by PDGF‐BB in vitro and vascular remodelling in vivo. Therefore, apatinib is a potential candidate to treat vascular proliferative diseases.     

ω-3 Polyunsaturated fatty acids inhibit migration of human vascular smooth muscle cells in vitro

Arterial smooth muscle cell migration from the media to the intima is a crucial process in the pathogenesis of atherosclerosis. Platelet-derived growth factor (PDGF) has been proposed to play a key role in the development of advanced atherosclerotic lesions by stimulating the migration and proliferation of vascular smooth muscle cells. Polyunsaturated fatty acids (PUFA) of the ω-3 series, extracted from fish oil has been shown to have beneficial effects on atherosclerosis. In this study, we evaluated the effects of ω-3 PUFA on the migration of human aortic smooth muscle cell (hASMC) in vitro. The migration assay was performed according to the Capsoni's method using transwell culture plates. PDGF, fibrinogen or 10%FCS significantly stimulated hASMC migration, however, ω-3 PUFA significantly inhibited PDGF-induced migration of hASMC. These results suggest that the inhibitory effect of ω-3 PUFA on cell migration may be an important aspect by which ω-3 PUFA exerts its antiatherosclerotic influence.     

Basic FGF regulates interstitial collagenase gene expression in human smooth muscle cells

Basic fibroblast growth factor (bFGF) is a mitogenic factor that is implicated in smooth muscle cell growth in atherosclerosis and vascular restenosis. In this study, we examined the effect of bFGF on the expression of the interstitial collagenase gene in human vascular smooth muscle cells. Results from Northern transfer analysis showed that bFGF increased collagenase mRNA levels greater than threefold as early as 24 h. Collagenase pre-mRNA levels were elevated approximately threefold by bFGF, according to RT-PCR analysis. Transient transfections of the smooth muscle cells with a 4.4-kb human collagenase promoter-CAT reporter gene, however, failed to show upregulation of the promoter activity by bFGF. Interestingly, transfections with deleted fragments containing promoter sequences from -1047 to -2271 resulted in modest stimulation of the collagenase-CAT promoter activity by bFGF. bFGF did not alter the stability of the collagenase mRNA, as demonstrated by degradation studies. The enhanced collagenase mRNA levels elicited by bFGF were reflected in increased amounts of collagenase protein that were detected by Western blot analysis. In summary, bFGF upregulates the interstitial collagenase expression, resulting in turnover of the extracellular matrix, an event that could facilitate smooth muscle cell migration and proliferation during the early stages of atherosclerosis and restenosis. J. Cell. Biochem. 65:32–41. © 1997 Wiley-Liss, Inc.     

Platelet-derived growth factor is a chemoattractant for vascular smooth muscle cells

In previous experiments (Grotendorst et al, 1981), we showed that platelet-derived growth factor promotes the migration of smooth muscle cells in vitro. Using a "checkerboard" analysis, we now establish that platelet-derived growth factor (PDGF) acts as a true chemoattractant for cultured aortic smooth muscle cells. Other growth factors such as epidermal growth factor, fibroblast growth factor, and insulin are not chemoattractants. The chemotactic response occurs before the initiation of DNA synthesis and is not affected by inhibition of DNA synthesis. Chemotaxis occurs at levels of PDGF lower than required for mitogenesis. RNA and protein synthesis are required for the chemotactic response. As found previously in bacteria and leucocytes, we find that methylation reactions are required for the chemotactic response. The possibility is discussed that PDGF acts in vivo at sites of vascular injury to attract smooth muscle cells from the medial layer to the luminal surface, and is involved in the early stages of the formation of atherosclerotic plaques.     

CTRP3/cartducin is induced by transforming growth factor‐β1 and promotes vascular smooth muscle cell proliferation

CTRP3 (C1q and tumour necrosis factor‐related protein 3)/cartducin, a novel serum protein, is a member of the CTRP superfamily. Although the CTRP3/cartducin gene is markedly up‐regulated in rat carotid arteries after balloon injury, little is known about its biological roles in arterial remodelling and neointima formation in injured blood vessels. We have investigated the mechanisms underlying CTRP3/cartducin up‐regulation and the in vitro effects of CTRP3/cartducin on vascular smooth muscle cells. CTRP3/cartducin expression in cultured p53LMAC01 vascular smooth muscle cells was induced by TGF‐β1 (transforming growth factor‐β1), but not by bFGF (basic fibroblast growth factor) or PDGF‐BB (platelet‐derived growth factor‐BB). Exogenous CTRP3/cartducin promoted the proliferation of p53LMAC01 cells in a dose‐dependent manner via ERK1/2 (extracellular signal‐regulated kinase 1/2)‐ and MAPK (p38 mitogen‐activated protein kinase)‐signalling pathways. In contrast, CTRP3/cartducin exhibited no effect on the migration of p53LMAC01 cells. Taken together, the results of the present study demonstrate a novel biological role of CTRP3/cartducin in promoting vascular smooth muscle cell proliferation in blood vessel walls after injury.     

A naturally occurring carotenoid,lutein, reduces PDGF and H<Subscript>2</Subscript>O<Subscript>2</Subscript> signaling and compromises migration in cultured vascular smooth muscle cells

Background  

Platelet-derived growth factor (PDGF) is a potent stimulator of growth and motility of vascular smooth muscle cells (VSMCs). Abnormalities of PDGF/PDGF receptor (PDGFR) are thought to contribute to vascular diseases and malignancy. We previously showed that a carotenoid, lycopene, can directly bind to PDGF and affect its related functions in VSMCs. In this study we examined the effect of the other naturally occurring carotenoid, lutein, on PDGF signaling and migration in VSMCs.     

Bifunctional effects of transforming growth factor-beta on migration of cultured rat aortic smooth muscle cells

Migration of smooth muscle cells (SMC) in the arterial wall is important in the pathogenesis of atherosclerosis and is presumably regulated in both normal and atherosclerotic tissues. In this study, the effect of transforming growth factor-beta (TGF-beta) on the migration of rat aortic SMC was examined. TGF-beta alone enhanced the migration of SMC at concentrations of 10 to 50 pg/ml and its maximal effect was similar to that of platelet-derived growth factor (PDGF). Checker board analysis showed that TGF-beta had a chemotactic, but not a chemokinetic effect. PDGF also enhanced the migration in a dose-dependent manner and TGF-beta inhibited the PDGF-induced migration dose-dependently at 1.0 pg/ml to 1.0 ng/ml. These data suggest that TGF-beta is a bifunctional regulator of the migration of aortic SMC.     

CeReS-18, a cell regulatory sialoglycopeptide, inhibits proliferation and migration of rat vascular smooth muscle cells

CeReS-18, a cell regulatory sialoglycopeptide, has been shown to inhibit proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on vascular smooth muscle cell (SMC) proliferation was characterized in cultured rat aorta SMCs (A7r5). More extensively, the effect of CeReS-18 on platelet-derived growth factor (PDGF)-induced SMC migration was examined using a modified Boyden's chamber assay. CeReS-18 inhibits both SMC proliferation and migration in a concentration-dependent, calcium-sensitive, and reversible manner. Furthermore, cells preincubated with the inhibitor had an increased sensitivity to CeReS-18-mediated inhibition of SMC migration. Immunoprecipitation and in vitro phosphorylation assays demonstrated that MAP kinase activity was inhibited in the CeReS-18-treated cells and pretreatment with CeReS-18 suppressed the activation of MAP kinase stimulated by PDGF. However, it is not likely that the suppression of the MAP kinase pathway was directly responsible for the ability of CeReS-18 to inhibit migration of the rat aorta smooth muscle cells since a MEK-specific inhibitor, PD98059, did not influence A7r5 cell migration.     

Modulatory role of ERK MAPK-caldesmon pathway in PDGF-stimulated migration of cultured pulmonary artery SMCs

Extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) phosphorylate caldesmon in vivo, but the function of caldesmon phosphorylation in smooth muscle physiology is controversial. We hypothesized that ERK MAPKs and caldesmon modulate chemotactic migration of cultured canine pulmonary artery smooth muscle cells (PASMCs). Platelet-derived growth factor (PDGF; 10 ng/ml) and endothelin-1 (ET-1; 100 nM) transiently activated ERK MAPKs: PDGF produced higher maximal and more potent activation of ERK MAPKs over 5 h. While both PDGF and ET-1 increased caldesmon phosphorylation, only PDGF stimulated migration of cultured cells (13 times over basal migration). At concentrations from 0.01 to 10 nM, ET-1 failed to enhance migration; 100 nM ET-1 produced only a slight increase (1.31 +/- 0.18 times basal migration). ET-1 (100 nM) did not potentiate migration triggered by 0.5 or 3 ng/ml PDGF. The MEK1 inhibitor PD-98059 (50 microM) abolished the PDGF-stimulated phosphorylation of ERK MAPKs and caldesmon and reduced cell migration by 50%. We conclude that while ERK MAPK activity is not required to initiate migration, an ERK MAPK-caldesmon pathway may modulate later events necessary for PDGF-stimulated migration of cultured PASMCs.     

Aortic smooth muscle cell migration caused by platelet-derived growth factor is mediated by lipoxygenase product(s) of arachidonic acid

The relation between platelet-derived growth factor (PDGF)-induced smooth muscle cell migration, measured in Boyden chambers, and cellular arachidonic acid cascade was studied by using rat aortic smooth muscle cells. Partially purified PDGF stimulated cell migration significantly at a concentration of 1.33-133.0 micrograms/ml. Treatment of the cells with 10(-4)M of 5,8,11,14-eicosatetraynoic acid, an inhibitor of lipoxygenase and cyclooxygenase, and 10(-4)M of caffeic acid, a specific inhibitor of lipoxygenase, caused a significant suppression of PDGF-induced cell migration. Treatment with indomethacin, an inhibitor of cyclooxygenase, did not affect cell migration. These data indicate the involvement of a lipoxygenase product(s) of arachidonic acid in PDGF-associated smooth muscle cell migration.     

Human platelet-derived growth factor: structure and function

Human platelet-derived growth factor (PDGF) is a heat-stable, cationic polypeptide transported in blood in the alpha granules of platelets. It is released from platelets during blood clotting. PDGF has been resolved into at least two closely related active polypeptides, PDGF-I and PDGF-II, each consisting of two inactive chains linked together by disulfide bonds. PDGF stimulates the growth of normal cells in culture, including fibroblasts, arterial smooth muscle cells, and glial cells. In addition, PDGF has been shown to stimulate cell migration and many diverse metabolic functions such as amino acid transport, protein synthesis, cholesterol ester synthesis, phospholipid turnover, and prostacyclin synthesis. It modulates receptor binding of other active components such as epidermal growth factor, luteinizing hormone, low-density lipoprotein, and somatomedin C. Specific cell membrane receptors for PDGF have been demonstrated in arterial smooth muscle cells and fibroblasts.     

Integrin alpha(v)beta(3) is involved in stimulated migration of vascular adventitial fibroblasts by basic fibroblast growth factor but not platelet-derived growth factor

We examined the effects of basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) on the migration of vascular adventitial fibroblasts (VAFs) isolated from rat aortic adventitiae. Both bFGF and PDGF significantly stimulated VAF migration in vitro. An antibody to rat beta(3) integrin reduced bFGF-stimulated migration in a dose dependent manner. Moreover, VAF migration was inhibited in the presence of cyclic RGD (cRGD) peptide. However, PDGF-directed migration was blocked only by equivalent cRGD peptide but not by antibody to beta(3) integrin. These data suggest that alpha(v)beta(3) integrin mediates VAF migration stimulated by bFGF and that chemoattractant directed migration may be through distinct integrins.