Differential effects of imatinib on PDGF-induced proliferation and PDGF receptor signaling in human arterial and venous smooth muscle cells

Platelet-derived growth factor (PDGF) has been implicated in smooth muscle cell (SMC) proliferation, a key event in the development of myointimal hyperplasia in vascular grafts. Recent evidence suggests that the PDGF receptor (PDGFR) tyrosine kinase inhibitor, imatinib, can prevent arterial proliferative diseases. Because hyperplasia is far more common at the venous anastomosis than the arterial anastomosis in vascular grafts, we investigated whether imatinib also inhibited venous SMC (VSMC) proliferation, and examined possible differences in its mechanism of action between VSMC and arterial SMC (ASMC). Human ASMC and VSMC were stimulated with PDGF-AB, in the presence or absence of imatinib (0.1-10 microM). Proliferation was assayed using the 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, while PDGFR, Akt and ERK1/2-mitogen activated protein kinase (MAPK) signaling pathways were investigated by immunoblotting. The proliferative response to PDGF at 50 and 100 ng/ml was 32 and 43% greater, respectively, in VSMC than in ASMC. Similarly, PDGF-stimulated proliferation was more sensitive to inhibition by imatinib in VSMC than ASMC (IC(50) = 0.05 microM vs. 0.4 microM; P < 0.01). Imatinib also more effectively inhibited PDGF-induced phosphorylation of PDGFRbeta and Akt in VSMC, compared to ASMC. These data highlight inherent pharmacodynamic differences between VSMC and ASMC in receptor and cell signaling functions and suggest that imatinib therapy may be useful for the prevention of venous stenosis in vascular grafts.     

PDGF induces osteoprotegerin expression in vascular smooth muscle cells by multiple signal pathways

Acetohydroxyacid synthase (AHAS; EC 4.1.3.18) contains catalytic and regulatory subunits, the latter being required for sensitivity to feedback regulation by leucine, valine and isoleucine. The regulatory subunit of Arabidopsis thaliana AHAS possesses a sequence repeat and we have suggested previously that one repeat binds leucine while the second binds valine or isoleucine, with synergy between the two sites. We have mutated four residues in each repeat, based on a model of the regulatory subunit. The data confirm that there are separate leucine and valine/isoleucine sites, and suggest a complex pathway for regulatory signal transmission to the catalytic subunit.     

Additive effects of PDGF receptor beta signaling pathways in vascular smooth muscle cell development

The platelet-derived growth factor beta receptor (PDGFRbeta) is known to activate many molecules involved in signal transduction and has been a paradigm for receptor tyrosine kinase signaling for many years. We have sought to determine the role of individual signaling components downstream of this receptor in vivo by analyzing an allelic series of tyrosine-phenylalanine mutations that prevent binding of specific signal transduction components. Here we show that the incidence of vascular smooth muscle cells/pericytes (v/p), a PDGFRbeta-dependent cell type, can be correlated to the amount of receptor expressed and the number of activated signal transduction pathways. A decrease in either receptor expression levels or disruption of multiple downstream signaling pathways lead to a significant reduction in v/p. Conversely, loss of RasGAP binding leads to an increase in this same cell population, implicating a potential role for this effector in attenuating the PDGFRbeta signal. The combined in vivo and biochemical data suggest that the summation of pathways associated with the PDGFRbeta signal transduction determines the expansion of developing v/p cells.     

Mechanical strain increases PDGF-B and PDGF beta receptor expression in vascular smooth muscle cells

Cyclic mechanical strain causes proliferation of vascular smooth muscle cells, mediated in part by platelet-derived growth factor (PDGF). We examined the effect of cyclic strain on expression of PDGF-B and the PDGF beta receptor. Neonatal rat vascular smooth muscle cells were exposed to 1 hertz cyclic strain on silicone elastomer plates. PDGF-B mRNA increased after 6 h of strain. In cells transfected with a PDGF-B promoter chloramphenicol acetyl transferase construct (psisCAT 6A), activity increased by 12-fold following 12 h of strain. Two neutralizing antibodies to the PDGF beta receptor both reduced strain-induced [(3)H]thymidine incorporation by 50%. Expression of the PDGF beta receptor protein increased 1.8-fold following 24 h of strain. During strain, PDGF beta receptor expression was not significantly altered by neutralizing antibodies to PDGF-B. Thus, both PDGF-B and the PDGF beta receptor are induced by cyclic mechanical strain and both contribute to cell proliferation in response to strain.     

Regulation of SM22 alpha expression by arginine vasopressin and PDGF-BB in vascular smooth muscle cells

Vascular smooth muscle (SM) cells (VSMC) undergo phenotypic modulation in vivo and in vitro. This process involves coordinated changes in expression of multiple SM-specific genes. In cultured VSMC, arginine vasopressin (AVP) increases and PDGF decreases expression of SM alpha-actin (SMA), the earliest marker of SM cells (SMC). However, it is unknown whether these agents regulate other SM genes in a similar fashion. SM22 alpha appears secondary to SMA during development and is also a marker for SMC. This study examined the regulation of SM22 alpha expression by AVP and PDGF in cultured VSMC. Levels of SM22 alpha mRNA and protein were increased by AVP and suppressed by PDGF. Consistent with these changes, AVP increased SM22 alpha promoter activity, whereas PDGF inhibited basal promoter activity and blocked AVP-induced increase. Activation of both JNK and p38 MAPK pathways was necessary for AVP-mediated induction of SM22 alpha promoter. Expression of constitutively active Ras produced similar suppressions on SM22 alpha promoter activity as PDGF. Signaling relayed from PDGF/Ras activation involved Raf, or a protein that competes for this site, Ral-GDS, and phosphatidylinositol 3-kinase activation. Truncational analysis showed that the proximal location of three CArG boxes in the promoter was sufficient for AVP stimulation. Mutations in this CArG box reduced basal and AVP-stimulated promoter activity without effecting PDGF suppression. Overexpression of serum response factor enhanced basal and AVP-stimulated promoter activity but had no effect on PDGF-BB-induced suppression. These data indicate that AVP and PDGF initiate specific signaling pathways that control expression of multiple SM genes leading to phenotypic modulation.     

Angiotensin II and noradrenaline increase PDGF-BB receptors and potentiate PDGF-BB stimulated DNA synthesis in vascular smooth muscle

The effects of angiotensin II and noradrenaline were examined on PDGF-BB and PDGF-AB induced mitogenesis in primary cultures of rat aortic smooth muscle. Incubation of the smooth muscle with either angiotensin II or noradrenaline potentiated the submaximal but not maximal mitogenic effects of PDGF-BB but not PDGF-AB. These effects on PDGF-BB stimulated mitogenesis correlated with an increase in receptor number specific for this homodimer when the smooth muscle was incubated with either angiotensin II or noradrenaline. Mitogenic concentrations of PDGF-AB did not interact with this PDGF receptor subtype. These results indicate that the mitogenic effects of PDGF-AB and -BB are elicited via different PDGF receptor subtypes. Angiotensin II and noradrenaline potentiate the mitogenic effects of PDGF-BB by increasing the steady state concentrations of membrane receptors for this homodimer.     

Anti-atherosclerotic effects of sirolimus on human vascular smooth muscle cells

Sirolimus is a potent immunosuppressive agent and has an anti-atherosclerotic effect through its anti-proliferative property. The present study was undertaken to investigate the effect of sirolimus on intracellular cholesterol homeostasis in human vascular smooth muscle cells (VSMCs) in the presence of inflammatory cytokine IL-1 beta. We explored the effect of sirolimus on the lipid accumulation of VSMCs in the presence of IL-1 beta, using Oil Red O staining and quantitative measurement of intracellular cholesterol. The effect of sirolimus on the gene and protein expression of lipoprotein receptors and ATP binding cassettes (ABCA1 and ABCG1) was examined by real-time PCR and Western blotting, respectively. Furthermore, the effect of sirolimus on cholesterol efflux from VSMCs in the presence or absence of IL-1 beta was also investigated using [(3)H] cholesterol efflux. Finally, we examined the effect of sirolimus on the production of inflammatory cytokines in VSMCs using ELISA. Sirolimus reduced intracellular lipid accumulation in VSMCs mediated by IL-1 beta possibly due to the reduction of expression of low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) receptors. Sirolimus increased cholesterol efflux from VSMCs and overrode the suppression of cholesterol efflux induced by IL-1 beta. Sirolimus also increased ABCA1 and ABCG1 genes expression, even in the presence of IL-1 beta. We further confirmed that sirolimus inhibited mRNA and protein expression of inflammatory cytokines IL-6, tumor necrosis factor-alpha, IL-8, and monocyte chemoattractant protein-1. Inhibition of lipid uptake together with increasing cholesterol efflux and the inhibition of inflammatory cytokines are all important aspects of the anti-atherosclerotic effects of sirolimus on VSMCs.     

Mechanical strain induces growth of vascular smooth muscle cells via autocrine action of PDGF

The effect of cyclic mechanical strain on growth of neonatal rat vascular smooth muscle (VSM) cells were examined. Cells were grown on silicone elastomer plates subjected to cyclic strain (60 cycle/min) by application of a vacuum under the plates. A 48 h exposure to mechanical strain increased the basal rate of thymidine incorporation by threefold and increased cell number by 40% compared with cells grown on stationary rubber plates. Strain also increased the rate of thymidine incorporation in response to alpha-thrombin (from 15- to 33-fold), but not to PDGF. As determined by thymidine autoradiography, strain alone induced a fourfold increase in labeled nuclei at the periphery of dishes, where strain is maximal, and a 2-3-fold increase at the center of dishes. Strain appeared to induce the production of an autocrine growth factor(s), since conditioned medium from cells subjected to strain induced a fourfold increase in DNA synthesis in control cells. Western blots of medium conditioned on the cells subjected to strain indicate that the cells secrete both AA and BB forms of PDGF in response to strain. Northern blots of total cell RNA from cells exposed to strain for 24 h show increased steady-state level of mRNA for PDGF- A. Lastly, polyclonal antibodies to the AA form of PDGF reduced by 75% the mitogenic effect of strain and polyclonal antibodies to AB-PDGF reduced mitogenicity by 50%. Antibodies to bFGF did not significantly reduce the strain-induced thymidine incorporation. Thus, the mechanism of strain-induced growth appears to involve the intermediary action of secreted PDGF.     

17Beta-estradiol attenuates PDGF signaling in vascular smooth muscle cells at the postreceptor level

Estrogens are known to display significant vasoprotective effects in premenopausal women. PDGF is an important mediator of vascular smooth muscle cell (VSMC) migration and proliferation, and thus atherogenesis. We analyzed the effects of 17beta-estradiol (E2) on beta-PDGF receptor (beta-PDGFR) expression/activation and PDGF-dependent VSMC proliferation, migration, and downstream signaling events. Pretreatment of VSMCs with E2 (0.3 microM-0.1 mM) for 24 h concentration-dependently inhibited PDGF-induced proliferation and migration up to 85.5 +/- 15.8% and 79.4 +/- 9.8%, respectively (both P < 0.05). These effects were prevented by coincubation with the ER antagonist ICI-182780. E2 did not alter beta-PDGFR expression, nor did it impair the ligand-induced tyrosine phosphorylation of the beta-PDGFR and consecutive binding of the receptor-associated signaling molecules Src homology region 2-containing phosphatase-2, PLC-gamma, phosphatidylinositol 3-kinase, and RasGAP. Thus estrogens inhibited PDGF-induced cellular responses at the postreceptor level. Although stimulation of VSMCs with PDGF-BB led to a transient increase of rac-1 activity, pretreatment with E2 for 24 h concentration-dependently inhibited PDGF-induced rac-1 activation. Furthermore, inhibition of rac-1 by Clostridium sordellii lethal toxin or overexpression of dominant-negative rac-1 (rac-N17) significantly inhibited PDGF-induced VSMC migration, indicating that rac-1 activity is essential for PDGF-dependent cellular responses. E2 did not further reduce PDGF-induced migration in rac-N17-overexpressing cells, suggesting that it diminishes VSMC migration by altering rac-1 activity. We conclude that E2 attenuates PDGF-dependent cellular functions of VSMCs downstream of the beta-PDGFR via inhibition of rac-1. These observations offer a molecular explanation for the vasoprotective effects of estrogens.     

PDGF-BB enhances expression of,and reduces adhesion to,laminin-5 in vascular smooth muscle cells

The laminin family of extracellular matrix (ECM) proteins plays crucial roles in regulating cellular growth, migration, and differentiation. We report here that laminin-5 is expressed in the tunica media of the rat aorta and pulmonary arteries. Using indirect immunofluorescence microscopy, Western blots, and RT-PCR analysis, we found that primary cultures of rat arterial smooth muscle cells express laminin-5 and deposit it into their insoluble ECM. These cells also attach strongly to laminin-5 via beta1 integrin receptors in 30 min adhesion assays. Laminin-5 expression in these cells is upregulated by growth factors in vitro and platelet-derived growth factor (PDGF-BB) stimulation reduces adhesion to laminin-5. As laminin-5 promotes enhanced migration of other cell types, the production of and adhesion to laminin-5 by vascular smooth muscle cells may play a role in the pathological growth and migration of these cells associated with restenosis following vascular injury.     

Rapid effects of aldosterone on clonal human vascular smooth muscle cells

It has been increasingly appreciated that aldosterone elicits acute vascular effects through nongenomic signaling pathways. Our previous studies demonstrated that aldosterone attenuated phenylephrine-mediated constriction in intact vessels [via phosphatidylinositol 3-kinase-dependent nitric oxide synthase activation] but enhanced vasoconstrictor responses in endothelium-denuded arteries. To determine the mechanism of this vasoconstrictor response, we assessed the effect of aldosterone on myosin light-chain phosphorylation and contraction in clonal adult human vascular smooth muscle cells. Acute aldosterone exposure mediated dose-dependent myosin light-chain phosphorylation, inhibited by spironolactone and phosphatidylinositol 3-kinase inhibition. These rapid effects of aldosterone were mimicked by estradiol and hydrocortisone and were also inhibitable by both spironolactone and eplerenone. In parallel to its effects on myosin light-chain phosphorylation, aldosterone mediated dose-dependent contraction responses that were inhibited by spironolactone. Comparable contractile responses were seen with both 17-estradiol and hydrocortisone. In total, these data are consistent with a mechanism of acute aldosterone-mediated contraction common to both glucocorticoids and estrogen. Steroid-mediated vasoconstriction may represent an important pathobiological mechanism of vascular disease, especially in the setting of preexisting endothelial dysfunction. steroid hormones; contraction; nongenomic     

Differential effects of fatty acids on glycolysis and glycogen metabolism in vascular smooth muscle

The effects of fatty acids of different chain lengths on aerobic glycolysis, lactic acid production, glycogen metabolism and contractile function of vascular smooth muscle were investigated. Porcine carotid artery segments were treated with 50 microM iodoacetate and perchloric acid tissue extracts were then analyzed by 31P-NMR spectroscopy to observe the accumulation of phosphorylated glycolytic intermediates so that the activity of the Embden-Myerhof pathway could be tracked under various experimental paradigms. Aerobic glycolysis and lactate production in resting arteries were almost completely inhibited with 0.5 mM octanoate, partially inhibited with 0.5 mM acetate and unaffected by 0.5 mM palmitate. Inhibition of glycolysis by octanoate was not attributable to inhibition of glucose uptake or glucose phosphorylation. Basal glycogen synthesis was unchanged with palmitate and acetate, but was inhibited by 52% with octanoate incubation. The characteristic glycogenolysis which occurs upon isometric contraction with 80 mM KCl in the absence of fatty acid in the medium was not demonstrable in the presence of any of the fatty acids tested. Glycogen sparing was also demonstrable in norepinephrine contractions with octanoate and acetate, but not with palmitate. Additionally, norepinephrine-stimulated isometric contraction was associated with enhanced synthesis of glycogen amounting to 6-times the basal rate in medium containing octanoate. Contractile responses to norepinephrine were attenuated by 20% in media containing fatty acids. Thus, fatty acids significantly alter metabolism and contractility of vascular smooth muscle. Fatty acids of different chain lengths affect smooth muscle differentially; the pattern of substrate utilization during contraction depends on the contractile agonist and the fatty acid present in the medium.     

Effect of substrate stiffness and PDGF on the behavior of vascular smooth muscle cells: Implications for atherosclerosis

Vascular disease, such as atherosclerosis, is accompanied by changes in the mechanical properties of the vessel wall. Although altered mechanics is thought to contribute to disease progression, the molecular mechanisms whereby vessel wall stiffening could promote vascular occlusive disease remain unclear. It is well known that platelet‐derived growth factor (PDGF) is a major stimulus for the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs) and contributes critically to vascular disease. Here we used engineered substrates with tunable mechanical properties to explore the effect of tissue stiffness on PDGF signaling in VSMCs as a potential mechanism whereby vessel wall stiffening could promote vascular disease. We found that substrate stiffness significantly enhanced PDGFR activity and VSMC proliferation. After ligand binding, PDGFR followed distinct routes of activation in cells cultured on stiff versus soft substrates, as demonstrated by differences in its intensity and duration of activation, sensitivity to cholesterol extracting agent, and plasma membrane localization. Our results suggest that stiffening of the vessel wall could actively promote pathogenesis of vascular disease by enhancing PDGFR signaling to drive VSMC growth and survival. J. Cell. Physiol. 225: 115–122, 2010. © 2010 Wiley‐Liss, Inc.     

AngⅡ对血管平滑肌细胞PDGF受体表达的影响

目的:研究血管紧张素Ⅱ(AngⅡ)对血管平滑肌细胞血小板源生长因子(PDGF)受体表达的影响.方法:采用大鼠主动脉球囊内皮剥脱术制备主动脉再狭窄模型,观察形态学变化;放免法测定主动脉AngⅡ含量;免疫印迹法测定主动脉PDGF-β受体含量,并与假手术组相比较.培养大鼠主动脉血管平滑肌细胞(VSMC),AngⅡ刺激正常培养的与洛沙坦预处理过的VSMC 6 h,测定PDGF-β受体含量.结果:球囊内皮剥脱术后14 d,主动脉中层VSMC大量增殖,内膜显著增厚,AngⅡ含量显著升高(P＜0.05),PDGF-β受体表达显著增强(P＜0.05).AngⅡ诱导VSMC PDGF-β受体表达显著增强(P＜0.01),AngⅡ受体拮抗剂洛沙坦完全抑制AngⅡ对PDGF-β受体上调的诱导作用.结论:AngⅡ可通过其Ⅰ型受体诱导血管平滑肌细胞PDGF受体上调,这可能是AngⅡ促VSMC发生增殖的一个重要机制.     

Differential effect of atorvastatin and tacrolimus on proliferation of vascular smooth muscle and endothelial cells

Although considered promising for use in drug-eluting stents (DES), tacrolimus failed clinically. Tacrolimus inhibits growth factor production but can also act as a growth factor on vascular smooth muscle cells (VSMC). This unexpected proliferative stimulus could reverse the beneficial effects of the drug on restenosis. We hypothesized that tacrolimus' association with statins, which lower cholesterol and impair cell proliferation, could restore tacrolimus' beneficial effect by abrogating the aberrant proliferative stimulus. Additionally, since maintenance of endothelial function represents a challenge for new-generation DES, we investigated the combined effect of tacrolimus and atorvastatin on endothelial cells. Human VSMC and umbilical vein endothelial cells (HUVEC) were incubated with 100 nM tacrolimus and increasing doses of atorvastatin (0-3.0 μM). Atorvastatin plus tacrolimus dose-dependently inhibited VSMC proliferation. The percentage of cells incorporating 5-bromo-2'-deoxyuridine (BrdU) in their DNA was 49 ± 14% under basal conditions, 62 ± 15% (P = 0.01) with tacrolimus, 40 ± 22% with 3 μM atorvastatin, and 30 ± 7% (P < 0.05) with 3 μM atorvastatin plus tacrolimus. Atorvastatin downregulated β-catenin, Erk1 and Erk2, and cyclin B in tacrolimus-stimulated VSMC. In contrast, atorvastatin plus tacrolimus did not affect proliferation of endothelial cells. The percentage of HUVEC incorporating BrdU in their DNA was 47 ± 8% under basal conditions, 58 ± 6% (P = 0.01) with tacrolimus, 45 ± 4% with 3 μM atorvastatin, and 49 ± 1% with 3 μM atorvastatin plus tacrolimus. Both agents stimulated endoglin production by HUVEC. Taken together, these results suggest that, when combined with tacrolimus, atorvastatin exerts a dose-dependent antiproliferative effect on VSMC. In contrast, atorvastatin acts in concert with tacrolimus in HUVEC to stimulate production of endoglin, a factor that has an important role in endothelial repair. Our study supports the conclusion that prevention of postcoronary in-stent restenosis and late thrombosis may benefit of concomitant association of tacrolimus and high doses of atorvastatin.     

Mechanisms of inhibition by heparin of PDGF stimulated MAP kinase activation in vascular smooth muscle cells

Heparin and heparan are potent inhibitors of vascular smooth muscle cell (VSMC) proliferation. To investigate the mechanisms by which heparin suppresses growth factor stimulated mitogenesis, the present experiments investigated the effects of heparin on platelet-derived growth factor (PDGF) stimulated signal transduction pathways. Heparin treatment substantially inhibited PDGF-BB stimulated rat VSMC growth. Western analysis showed a 30 min PDGF-BB treatment of VSMC induced the tyrosine phosphorylation of multiple protein bands; cotreatment with heparin inhibited mitogen-activated protein (MAP) kinase tyrosine phosphorylation but had little effect on PDGF receptor tyrosine phosphorylation. In-gel kinase assays demonstrated that heparin inhibited PDGF-BB stimulated MAP kinase activity at late (25 min) but not early (10 min) time points. These data indicate that heparin does not inhibit the initial signalling events after PDGF-BB binding but instead acts through an alternate mechanism to inhibit MAP kinase. To investigate if heparin directly stimulates tyrosine phosphatase-mediated suppression of MAP kinase, we treated VSMC with orthovanadate, a tyrosine phosphatase inhibitor. Heparin inhibited MAP kinase tyrosine phosphorylation after orthovanadate treatment, indicating that heparin does not suppress MAP kinase by enlistment of a tyrosine phosphatase. Experiments were performed to investigate signalling pathways upstream of MAP kinase. To determine if protein kinase C (PKC) mediates PDGF-BB, serum, and EGF stimulation of MAP kinase, we treated VSMC overnight with phorbol ester (PMA) to downregulate PKC. Abolition of conventional and novel PKC activity significantly suppressed both serum and PDGF-BB induced MAP kinase activation, indicating protein kinase C is an important mediator for these mitogens. In contrast, downregulation of these PKC isoforms had little effect on EGF stimulation of MAP kinase. As heparin inhibits PDGF and serum but not EGF stimulation of MAP kinase, these data precisely correlate heparin inhibition of MAP kinase with activation through PKC-dependent pathways. Immunoprecipitation analysis found that heparin inhibited serum, PMA, and PDGF but not EGF induced raf-1 phosphorylation. These studies demonstrate that heparin did not block PDGF-BB receptor activation, which initiates the mitogenic signalling cascade. Heparin did inhibit specific postreceptor second messenger signals, such as the late phase activation of MAP kinase, which may be mediated by suppression of PKC-dependent pathways. J. Cell. Physiol. 172:69–78, 1997. © 1997 Wiley-Liss, Inc.