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.     

Serotonin-induced deoxyribonucleic acid synthesis in vascular smooth muscle cells involves a novel, pertussis toxin-sensitive pathway

Serotonin-induced DNA synthesis in bovine aortic smooth muscle cells was totally abolished by pretreatment of cultures with 5 ng/ml pertussis toxin. The half maximally effective concentration of toxin was approximately 10 pg/ml. Pertussis toxin did not affect platelet-derived growth factor (PDGF)-stimulated DNA synthesis and actually enhanced the mitogenic effect of the phorbol ester, phorbol 12-myristate 13-acetate. Pertussis toxin did not inhibit serotonin-stimulated inositol phosphate accumulation or increases in intracellular calcium or cAMP concentrations under conditions sufficient to completely inhibit serotonin-induced (3H)thymidine incorporation. These results demonstrate that a novel, pertussis-sensitive pathway is required for serotonin-, but not platelet-derived growth factor-induced DNA synthesis in vascular smooth muscle cells. The pertussis-sensitive step does not involve cAMP, phosphoinositide hydrolysis, mobilization of intracellular calcium, or phorbol ester-sensitive protein kinase C activity.     

Role of adiponectin in preventing vascular stenosis. The missing link of adipo-vascular axis

Obesity is more linked to vascular disease, including atherosclerosis and restenotic change, after balloon angioplasty. The precise mechanism linking obesity and vascular disease is still unclear. Previously we have demonstrated that the plasma levels of adiponectin, an adipose-derived hormone, decreases in obese subjects, and that hypoadiponectinemia is associated to ischemic heart disease. In current the study, we investigated the in vivo role of adiponectin on the neointimal thickening after artery injury using adiponectin-deficient mice and adiponectin-producing adenovirus. Adiponectin-deficient mice showed severe neointimal thickening and increased proliferation of vascular smooth muscle cells in mechanically injured arteries. Adenovirus-mediated supplement of adiponectin attenuated neointimal proliferation. In cultured smooth muscle cells, adiponectin attenuated DNA synthesis induced by growth factors including platelet-derived growth factor, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), basic fibroblast growth factor, and EGF and cell proliferation and migration induced by HB-EGF. In cultured endothelial cells, adiponectin attenuated HB-EGF expression stimulated by tumor necrosis factor alpha. The current study suggests an adipo-vascular axis, a direct link between fat and artery. A therapeutic strategy to increase plasma adiponectin should be useful in preventing vascular restenosis after angioplasty.     

Phospholipase D mimics platelet-derived growth factor as a competence factor in vascular smooth muscle cells.

Recent studies have suggested the importance of phosphatidylcholine (PC) metabolism in growth factor-stimulated cells. In these cells, PC is hydrolyzed not only by PC-specific phospholipase C but also by phospholipase D (PLD). In the present investigation, we show that the simple addition of PC-hydrolyzing PLD from Streptomyces chromofuscus to the culture medium of vascular smooth muscle cells elicits choline release into the medium accompanied by the formation of phosphatidic acid. In the presence of ethanol, this treatment elicits a formation of phosphatidylethanol (PEt) at the expense of phosphatidic acid. Furthermore, we show here that exogenous addition of S. chromofuscus PLD induces a marked DNA synthesis in quiescent vascular smooth muscle cells. This DNA synthesis induced by S. chromofuscus PLD is, like platelet-derived growth factor (PDGF)-elicited DNA synthesis, largely dependent on the presence of insulin. In addition, S. chromofuscus PLD-induced PEt formation and DNA synthesis were not affected by protein kinase C down-regulation, whereas PDGF-induced PEt formation and DNA synthesis were significantly inhibited. These observations strongly suggest that protein kinase-dependent activation of PLD is involved in mitogenic signal in PDGF-stimulated cells and that exogenously added PLD acts as a competence factor in the same way as PDGF.     

PDGF stimulates DNA synthesis in human vascular smooth muscle cells via a novel wortmannin-insensitive phosphatidylinositol 3-kinase

The class 1(A) phosphatidylinositol 3-kinase enzymes consist of a number of heterodimeric complexes of regulatory and catalytic subunits and have been implicated in a number of cellular responses. While platelet-derived growth factor (PDGF)-induced chemotaxis of human vascular smooth muscle cells (HVSMC) is inhibited by both wortmannin and LY294002, DNA synthesis is only inhibited by LY294002. Serum-induced DNA synthesis however is inhibited by LY294002, wortmannin and rapamycin. Similarly PDGF-induced protein kinase B (PKB) activation is inhibited by LY294002 but not by wortmannin or rapamycin. In conclusion PDGF-induced DNA synthesis appears to occur through a phosphatidylinositol 3-kinase (PI3-K)-dependent, but wortmannin-insensitive, PKB/Akt pathway.     

Ligand-independent trans-activation of the platelet-derived growth factor receptor by reactive oxygen species requires protein kinase C-delta and c-Src

Reactive oxygen species are involved in the mitogenic signal transduction cascades initiated by several growth factors and play a critical role in mediating cardiovascular diseases. Interestingly, H(2)O(2) induces tyrosine phosphorylation and trans-activation of the platelet-derived growth factor receptor and the epidermal growth factor receptor in many cell lines including vascular smooth muscle cells. To investigate the molecular mechanism by which reactive oxygen species contribute to vascular diseases, we have examined a signal transduction cascade involved in H(2)O(2)-induced platelet-derived growth factor receptor activation in vascular smooth muscle cells. We found that H(2)O(2) induced a ligand-independent phosphorylation of the platelet-derived growth factor-beta receptor at Tyr(1021), a phospholipase C-gamma binding site, involving the requirement of protein kinase C-delta and c-Src that is distinct from a ligand-dependent autophosphorylation. Also, H(2)O(2) induced the association of protein kinase C-delta with the platelet-derived growth factor-beta receptor and c-Src in vascular smooth muscle cells. These findings will provide new mechanistic insights by which enhanced reactive oxygen species production in vascular smooth muscle cells induces unique alleys of signal transduction distinct from those induced by endogenous ligands leading to an abnormal vascular remodeling process.     

Interleukin-1 promotes proliferation of vascular smooth muscle cells in coordination with PDGF or a monocyte derived growth factor

We report that interleukin-1 (IL-1) potentiates the proliferation of vascular smooth muscle cells. Growth of early passage smooth muscle cells was not significantly affected by IL-1 alone. Treatment with IL-1 together with the platelet derived growth factor (PDGF) or another polypeptide growth factor derived from mitogen activated human monocytes (MDGF) resulted in a significant enhancement of cell growth over either PDGF or MDGF alone. DNA synthesis was enhanced only marginally (30-40%) in quiescent cultures treated with an optimal concentration of IL-1 alone. In the presence of 5 units/ml of PDGF or MDGF, IL-1 produced about six- to eightfold higher DNA synthesis than the untreated cultures. Induction of DNA synthesis was linear between 0.1 and 1.0 pM IL-1, dependent on PDGF concentration, and was effectively neutralized by monoclonal antibodies against IL-1 beta. The growth promoting activity of IL-1 was extremely potent producing half-maximum stimulation at a concentration of 0.5 pM. These results suggest that IL-1 may play an important role in the modulation of growth and other activities of vascular smooth muscle cells. These observations are especially important with regard to defining the potential macrophage derived mediators contributing to vascular cell proliferation during inflammation and the pathogenesis of atherosclerosis. It is shown here that elicitation of IL-1 induced growth response requires a coordinated action with another priming growth factor such as PDGF. In this regard, IL-1 mediated proliferation of smooth muscle cells may have analogy with the IL-1 mediated T-cell activation and IL-2 production where concerted actions of antigen/mitogen and IL-1 are required.     

Effects of calcium-antagonists and calmodulin inhibitors on DNA synthesis in cultured rat vascular smooth muscle cells

To investigate the role of intracellular Ca2+ in the mechanism of cellular proliferation of vascular smooth muscle cells (VSMC), the effects of Ca2+-antagonists and calmodulin (CaM) inhibitors on DNA synthesis stimulated by serum-derived growth factors were studied in cultured VSMCs derived from rat aorta. DNA synthesis assessed by incorporation of [3H]thymidine into the cells was significantly stimulated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF) or fetal bovine serum (FBS), of which the effects were dose-dependently inhibited by a variety of Ca2+-antagonists, such as verapamil, diltiazem and nicardipine. Trifluoperazine and W-7, both specific CaM inhibitors, similarly inhibited DNA synthesis stimulated by EGF, PDGF or FBS in a dose-dependent manner, whereas W-5, a less specific CaM inhibitor, was minimally effective. These data suggest that the Ca2+-CaM system plays an important role in the mechanism of growth factor-induced DNA synthesis in VSMCs.     

Factor Xa induces mitogenesis of vascular smooth muscle cells via autocrine production of epiregulin

Factor Xa has been reported to elicit smooth muscle cell proliferation via autocrine release of platelet-derived growth factor. However, this study has shown that factor Xa-induced mitogenesis of rat aortic smooth muscle cell is independent of platelet-derived growth factor. We also could not observe any platelet-derived growth factor isoforms in the cultured medium of factor Xa-stimulated cells. Our finding that the cultured medium of factor Xa-stimulated cells strongly induces rat aortic smooth muscle cell mitogenesis in the absence of factor Xa activity led us to explore the existence of a novel autocrine pathway. The autocrine growth factor was purified from the cultured medium and was identified to be epiregulin. Recombinant epiregulin was also able to induce the mitogenesis. The secretion of epiregulin from factor Xa-stimulated rat aortic smooth muscle cell required mRNA expression and protein synthesis of the growth factor. The mitogenic effect of factor Xa on rat aortic smooth muscle cell was significantly reduced by anti-epiregulin antibody or by antisense oligodeoxynucleotide to epiregulin. Several lines of experimental evidence clearly indicate that the autocrine production of epiregulin, an epidermal growth factor-related ligand, is induced in the factor Xa-stimulated mitogenic process of rat aortic smooth muscle cell.     

Stimulation of platelet-derived growth factor-induced DNA synthesis by angiotensin II in rabbit vascular smooth muscle cells

In cultured rabbit vascular smooth muscle cells (VSMCs), angiotensin II by itself had little mitogenic effect even in the presence of cell-free plasma-derived serum (PDS), but markedly stimulated the platelet-derived growth factor (PDGF)-induced DNA synthesis in the presence of PDS. The maximal extent of DNA synthesis induced by PDGF plus angiotensin II was about twice that induced by PDGF alone. The stimulatory effect of angiotensin II was dose-dependent with the maximal response seen at 1 microM and was inhibited by the specific angiotensin II receptor antagonist, [Sar1, Ile8]angiotensin II. In VSMCs, both PDGF and angiotensin II induced expression of the c-fos gene in dose-dependent manners. In contrast to the synergistic effect of angiotensin II and PDGF on DNA synthesis, they induced expression of the c-fos gene in an additive manner. These results suggest that angiotensin II may act as a growth regulator for VSMCs in addition to acting as a vasoconstrictor.     

Heparin-binding growth factor type one and platelet-derived growth factor are required for the optimal expression of cell surface low density lipoprotein receptor binding activity in human adult arterial smooth muscle cells

Summary Purified heparin-binding growth factor-1 (HBGF-1) stimulated low density lipoprotein binding, internalization, and degradation in isolated human adult arterial smooth muscle cells. Exposure of quiescent cells to HBGF-1 in serum-free, defined medium increased both low density lipoprotein (LDL) receptor activity and de novo cholesterol biosynthesis. Both events preceded the onset of DNA synthesis by 6 to 9 h. HBGF-1 acted additively with platelet-derived growth factor (PDGF) to maximally stimulate cell surface LDL receptor binding activity and DNA synthesis in the smooth muscle cells. The presence of LDL was required for maximal mitogenic activity of HBGF-1 and PDGF. In the presence of LDL, growth factor-stimulated, proliferating human smooth muscle cells accumulated cholesterol ester and triglycerides. The results suggest that HBGF-1, PDGF, and LDL act together to promote the maximal proliferation of smooth muscle cells in culture. Chronic exposure to the three growth promoters may contribute to the smooth muscle cell hyperplasia and lipid accumulation observed in atherosclerotic lesions. This work was supported by the National Cancer Institute grants CA 37589 and HD 03275, National Council for Tobacco Research grant 1718, and a grant from RJR Nabisco, Inc.     

Inhibition of cell proliferation by alpha-tocopherol. Role of protein kinase C

The effect of alpha-tocopherol (vitamin E) on the proliferation of vascular smooth muscle cells (A7r5), human osteosarcoma cells (Saos-2), fibroblasts (Balb/3T3), and neuroblastoma cells (NB2A) has been studied. The proliferation of vascular smooth muscle cells was inhibited by physiologically relevant concentrations of alpha-tocopherol, neuroblastoma cells were only sensitive to higher alpha-tocopherol concentrations, and proliferation of the other cell lines was not inhibited. The inhibition of smooth muscle cell proliferation was specific for alpha-tocopherol. Trolox, phytol, and alpha-tocopherol esters had no effect. Proliferation of smooth muscle cells stimulated by platelet-derived growth factor or endothelin was completely sensitive to alpha-tocopherol. If smooth muscle cells were stimulated by fetal calf serum, proliferation was 50% inhibited by alpha-tocopherol. No effect of alpha-tocopherol was observed when proliferation of smooth muscle cells was stimulated by bombesin and lysophosphatidic acid. The possibility of an involvement of protein kinase C in the cell response to alpha-tocopherol was suggested by experiments with the isolated enzyme and supported by the 2- to 3-fold stimulation of phorbol ester binding induced by alpha-tocopherol in sensitive cells. Moreover, alpha-tocopherol also caused inhibition of protein kinase C translocation induced by phorbol esters and inhibition of the phosphorylation of its 80-kDa protein substrate in smooth muscle cells. A model is discussed by which alpha-tocopherol inhibits cell proliferation by interacting with the cytosolic protein kinase C, thus preventing its membrane translocation and activation.     

Epidermal growth factor controls smooth muscle alpha-isoactin expression in BC3H1 cells

We have examined the effects of epidermal growth factor (EGF), platelet-derived growth factor, and insulin on the differentiation of a mouse vascular smooth muscle-like cell line, the BC3H1 cells. On the basis of cell morphology and smooth muscle alpha-isoactin synthesis, we demonstrate that EGF at physiological concentrations prevents the differentiation of these cells, whereas platelet-derived growth factor has no apparent effect. The induction of alpha-isoactin synthesis by serum deprivation is inhibited by EGF in a dose-dependent manner with a half-maximal effect at 3-5 ng/ml and a maximal inhibition at approximately 30 ng/ml. Northern analysis also shows that EGF blocks the accumulation of alpha-isoactin mRNA normally observed during cell differentiation. Addition of EGF to differentiated cells results in a repression of alpha-isoactin synthesis, a stimulation of beta- and gamma-isoactin synthesis, and the stabilization of the nonmuscle isoactins. The synthesis of creatine phosphokinase, a muscle-specific noncontractile protein, is also regulated by EGF in a similar fashion. Modulation by EGF of alpha-isoactin expression is not affected by aphidicolin and is therefore independent of its mitogenic effect on these cells. Insulin is not required for observation of the EGF-dependent effects but instead seems to promote differentiation. Our results show that EGF can replace serum in controlling the differentiation of BC3H1 cells.     

Differential Response of Mesoderm- and Neural Crest-Derived Smooth Muscle to TGF-β1: Regulation of c-myb and α1 (I) Procollagen Genes

Previously, we demonstrated that avian vascular smooth muscle cells (VSMC) derived from embryonic abdominal and thoracic aorta grow differently in the presence of transforming growth factor beta (TGF-β1) and platelet-derived growth factor (PDGF-BB) (Wrennet al., In Vitro Cell. Dev. Biol.29, 73–78, 1992). The thoracic VSMC (N-VSMC) are derived from neural crest, and therefore differentiate from ectoderm; the abdominal VSMC (M-VSMC) are derived from mesoderm. The present study was designed to identify factors that mediate the differential responses of the VSMC to TGF-β1. We found that TGF-β1 increased DNA synthesis by approximately sevenfold in N-VSMC. Levels of both α1 (I) procollagen and c-myb mRNAs were markedly induced in N-VSMC treated with TGF-β1. Chimeric plasmids containing up to 3.5 kb of α1 (I) procollagen 5′ flanking DNA were induced to equivalent levels as procollagen mRNA in N-VSMC. However, TGF-β1 increased DNA synthesis by threefold in M-VSMC; there was no effect on α1 (I) procollagen expression, and c-myb was not expressed, as demonstrated by immunohistochemistry staining and RNA analyses. Antisense c-myb oligodeoxynucleotides blocked the TGF-β1 induction of α1 (I) procollagen and the growth of N-VSMC. The increase in DNA synthesis by M- and N-VSMC was correlated with the secretion of PDGF-AA, and staurosporine and antibodies directed against PDGF-AA suppressed DNA synthesis. Our results demonstrate that TGF-β1 activity and c-myb expression modulate the expression of α1 (I) collagen and cell proliferation in neural crest-derived smooth muscle. The regulation of these events by TGF-β1 may be important during morphogenesis of blood vessels and vascular diseases.     

Platelet-derived growth factor BB-dimer suppresses the expression of macrophage colony-stimulating factor in human vascular smooth muscle cells.

Vascular smooth muscle cell is a major cell component involved in the process of atherosclerosis. In the present study, we investigated the effects of platelet-derived growth factor (PDGF)-BB dimer on the expression of macrophage-colony stimulating factor (M-CSF) in vascular smooth muscle cells isolated from human umbilical artery. On Northern blot analysis of total RNAs isolated from smooth muscle cells, with human cDNA for M-CSF, a marked dose-dependent reduction of mRNA level was found in PDGF-BB-treated smooth muscle cells. Cellular production of M-CSF was estimated by immunoblot analysis of cell lysate with specific polyclonal antibody against recombinant human M-CSF. A concentration of 10 ng/ml PDGF-BB significantly reduced M-CSF mass in smooth muscle cells compared with that in the absence of PDGF-BB. These results suggest that PDGF-BB plays an important role in the cellular metabolism of vascular wall by regulating the rate of M-CSF production in vascular smooth muscle cells.     

Apolipoprotein E binding to low density lipoprotein receptor-related protein-1 inhibits cell migration via activation of cAMP-dependent protein kinase A

Smooth muscle cell migration and proliferation contribute to neointimal hyperplasia and vascular stenosis after endothelial denudation. Previous studies revealed that apolipoprotein E (apoE) is an effective inhibitor of platelet-derived growth factor-directed smooth muscle cell migration and proliferation and that the anti-migratory function is mediated via apoE binding to low density lipoprotein receptor-related protein-1 (LRP-1). This study was undertaken to identify the intracellular pathway by which apoE binding to LRP-1 results in inhibition of smooth muscle cell migration. The results showed that apoE increased intracellular cAMP levels 3-fold after 5 min, and the increase was sustained for more than 1 h. As a consequence, apoE also increased protein kinase A (PKA) activity in smooth muscle cells. Importantly, suppression of PKA activity with a cell-permeable peptide inhibitor of PKA abolished the inhibitory effect of apoE on smooth muscle cell migration. These results indicated that apoE inhibition of smooth muscle cell migration is mediated via the activation of cAMP-dependent PKA. Additional experiments revealed that apoE also inhibited fibroblasts migration toward platelet-derived growth factor by a similar mechanism of cAMP-dependent PKA activation. It is noteworthy that apoE failed to increase cAMP levels or inhibit migration of LRP-1-negative mouse embryonic fibroblasts and LRP-1-deficient smooth muscle cells. Taken together, these findings established the mechanism by which apoE inhibits cell migration, i.e. via cAMP-dependent protein kinase A activation as a consequence of its binding to LRP-1.     

内皮素受体反义寡聚核苷酸对内皮素受体基因表达及血管平滑肌细胞增殖的影响

报道了内皮素Ａ型受体反义寡聚核苷酸（ＯＤＮｓ）对大鼠血管平滑肌细胞（ＶＳＭＣ）增殖及内皮素受体基因表达的影响．~３Ｈ－ＴｄＲ参入结果显示,内皮素Ａ型受体反义ＯＤＮｓ处理细胞可显著抑制内皮素诱导的ＶＳＭＣ的ＤＮＡ合成,反转录－ＰＣＲ及受体结合实验结果表明,ＯＤＮｓ的上述作用与降低ＶＳＭＣ内皮素Ａ型受体基因表达活性有关．     

A role for platelet-derived growth factor beta-receptor in a newborn rat model of endothelin-mediated pulmonary vascular remodeling

Newborn rats exposed to 60% O2 for 14 days develop endothelin (ET)-1-dependent pulmonary hypertension with vascular remodeling, characterized by increased smooth muscle cell (SMC) proliferation and medial thickening of pulmonary resistance arteries. Using immunohistochemistry and Western blot analyses, we examined the effect of exposure to 60% O2 on expression in the lung of receptors for the platelet-derived growth factors (PDGF), which are implicated in the pathogenesis of arterial smooth muscle hyperplasia. We observed a marked O2-induced upregulation of PDGF-alpha and -beta receptors (PDGF-alphaR and -betaR) on arterial smooth muscle. This led us to examine pulmonary vascular PDGF receptor expression in 60% O2-exposed rats given SB-217242, a combined ET receptor antagonist, which we found prevented the O2-induced upregulation of PDGF-betaR, but not PDGF-alphaR, on arterial smooth muscle. PDGF-BB, a major PDGF-betaR ligand, was found to be a potent in vitro inducer of hyperplasia and DNA synthesis in cultured pulmonary artery SMC from infant rats. A critical role for PDGF-betaR ligands in arterial SMC proliferation was confirmed in vivo using a truncated soluble PDGF-betaR intervention, which attenuated SMC proliferation induced by exposure to 60% O2. Collectively, these data are consistent with a major role for PDGF-betaR-mediated SMC proliferation, acting downstream of increased ET-1 in a newborn rat model of 60% O2-induced pulmonary hypertension.     

Physiological quiescence in plasma-derived serum: Influence of platelet-derived growth factor on cell growth in culture

A platelet-derived growth factor can be shown to be the principal stimulant of DNA synthesis in whole blood serum for those cells that require serum for maintenance and growth in culture. Cell free plasma-derived serum lacks such platelet-derived material. 3T3 cells and primate arterial smooth muscle cells can be maintained in a quiescent state in culture for as long as six weeks in plasma-derived serum. Such cells can grow logarithmically after exposure to 5% whole blood serum or as little as 100 ng/ml of partially purified platelet factor. The cell cycle of smooth muscle cells has been studied in the quiescent (5% plasma-derived serum) and growing state (5% whole blood serum or 5% plasma-derived serum plus platelet factor). The generation time of smooth muscle cells is 16 to 18 hours as shown by autoradiographic frequency of labelled mitoses. The generation time is the same for cells in the growth fraction in either 5% whole blood serum or 5% plasma-derived serum. Thus, platelet factor acts by recruiting cells into the growth fraction rather than effecting a change in the duration of the cell cycle. Flow microfluorimetry studies on cells growing logarithmically in 5% whole blood serum give the following phase durations: G₁ = 5.6 hours; S = 7.6 hours; and G₂ + M = 3.8 hours. Based on these studies the argument is presented that cells cultured in 5% plasma-derived serum provide a more physiological base for the study of quiescence than do cells in low concentrations of whole blood serum or confluent, density inhibited cells at high (5% or greater) concentrations of whole blood serum. Furthermore, 5% plasma-derived serum represents an appropriate state to examine the perturbation of quiescent cells.