scholarly journals Inhibitory Effects of Genistein on Vascular Smooth Muscle Cell Proliferation Induced by Ox-LDL: Role of BKCa Channels

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Bing Bai ◽  
Nanjuan Lu ◽  
Wei Zhang ◽  
Jinghan Lin ◽  
Tingting Zhao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Protein Expression ◽  
Molecular Mechanism ◽  
Vascular Diseases ◽  
Nuclear Antigen ◽  
Dependent Manner ◽  
Bkca Channels ◽  
Vsmc Proliferation

Background. Oxidized low-density lipoprotein (Ox-LDL) is a crucial pathogenic factor for vascular diseases, which can induce the proliferation of vascular smooth muscle cells (VSMCs). Genistein is the main component of soybean isoflavone. Genistein has a variety of pharmacological properties in the treatment of vascular diseases and a promising clinical application. Large-conductance calcium-activated potassium (BKCa) channels are the primary type of potassium channels in VSMCs, which regulate various biological functions of VSMCs. However, whether genistein exerts an antiproliferation effect on Ox-LDL-stimulated VSMCs remains unclear. The current study is aimed at elucidating the effect of genistein on the Ox-LDL-stimulated proliferation of VSMCs and its possible molecular mechanism, especially the electrophysiological mechanism related to BKCa channels. Methods. Monoculture VSMC was obtained by an acute enzyme-dispersing method. The proliferation of cells was measured by CCK-8, cell cycle, and proliferating cell nuclear antigen (PCNA) expression. The BKCa whole-cell currents were measured by patch-clamp. Results. Ox-LDL treatment induced the proliferation of VSMCs, upregulated the BKCa protein expression, and increased the density of BKCa currents, while genistein significantly inhibited these effects caused by Ox-LDL. BKCa channels exerted a regulatory role in the proliferation of VSMCs in response to Ox-LDL. The inhibition of BKCa channels suppressed Ox-LDL-stimulated VSMC proliferation, while the activation of BKCa channels showed the opposite effect. Moreover, genistein suppressed the activity of BKCa, including protein expression and current density in a protein tyrosine kinase- (PTK-) dependent manner. Conclusion. This study demonstrated that genistein inhibited the Ox-LDL-mediated proliferation of VSMCs by blocking the cell cycle progression; the possible molecular mechanism may be related to PTK-dependent suppression of BKCa channels. Our results provided novel ideas for the application of genistein in the treatment of vascular diseases and proposed a unique insight into the antiproliferative molecular mechanism of genistein.

scholarly journals Aldose Reductase Regulates Vascular Smooth Muscle Cell Proliferation by Modulating G1/S Phase Transition of Cell Cycle

Endocrinology ◽  
2010 ◽  
Vol 151 (5) ◽  
pp. 2140-2150 ◽  
Author(s):  
Ravinder Tammali ◽  
Ashish Saxena ◽  
Satish K. Srivastava ◽  
Kota V. Ramana
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Aldose Reductase ◽  
S Phase ◽  
Nuclear Antigen ◽  
Proliferative Cell Nuclear Antigen ◽  
Vsmc Proliferation ◽  
Tnf Α ◽  
Proliferative Cell

Abnormal proliferation of vascular smooth muscle cells (VSMC) is a key feature of development of cardiovascular complications, atherosclerosis, and restenosis. Patients with diabetes have higher risk for restenosis after coronary angioplasty than nondiabetic patients due to hyperglycemia-induced release of cytokines such as TNF-α. However, the molecular mechanisms regulating VSMC proliferation remain unclear. Herein, we report that inhibition of the polyol pathway enzyme aldose reductase (AR) prevents high glucose (HG)- and/or TNF-α-induced VSMC proliferation by accumulating cells at the G1 phase of the cell cycle. Treatment of VSMC with AR inhibitor sorbinil prevented HG- as well as TNF-α-induced phosphorylation of retinoblastoma protein and activation of E2F-1. Inhibition of AR also prevented HG- and TNF-α-induced phosphorylation of cyclin-dependent kinase (cdk)-2 and expression of G1/S transition regulatory proteins such as cyclin D1, cyclin E, cdk-4, c-myc, and proliferative cell nuclear antigen. More importantly, inhibition of AR prevented the increased expression of E2F-1 and proliferative cell nuclear antigen in diabetic rat aorta. Treatment of VSMC with the most abundant and toxic lipid aldehyde 4-hydroxy-trans-2-nonenal (HNE) or its glutathione conjugate [glutathionyl (GS)-HNE] or AR-catalyzed product of GS-HNE, GS-1,4-dihydroxynonane, resulted in increased E2F-1 expression. Inhibition of AR prevented HNE- or GS-HNE-induced but not GS-1,4-dihydroxynonane-induced up-regulation of E2F-1. Collectively, these results show that AR could regulate HG- and TNF-α-induced VSMC proliferation by altering the activation of G1/S-phase proteins such as E2F-1, cdks, and cyclins. Thus, inhibition of AR may be a useful therapeutic approach in preventing vascular complications.

Cinnamon and its Components Suppress Vascular Smooth Muscle Cell Proliferation by Up-Regulating Cyclin-Dependent Kinase Inhibitors

2015 ◽  
Vol 43 (04) ◽  
pp. 621-636 ◽  
Author(s):  
Hyeeun Kwon ◽  
Jung-Jin Lee ◽  
Ji-Hye Lee ◽  
Won-Kyung Cho ◽  
Min Jung Gu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Signal Transduction ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Regulatory Proteins ◽  
Nuclear Antigen ◽  
Active Components ◽  
Vsmc Proliferation ◽  
Cinnamon Extract ◽  
Early Signal

Cinnamomum cassia bark has been used in traditional herbal medicine to treat a variety of cardiovascular diseases. However, the antiproliferative effect of cinnamon extract on vascular smooth muscle cells (VSMCs) and the corresponding restenosis has not been explored. Hence, after examining the effect of cinnamon extract on VSMC proliferation, we investigated the possible involvement of signal transduction pathways associated with early signal and cell cycle analysis, including regulatory proteins. Besides, to identify the active components, we investigated the components of cinnamon extract on VSMC proliferation. Cinnamon extract inhibited platelet-derived growth factor (PDGF)-BB-induced VSMC proliferation and suppressed the PDGF-stimulated early signal transduction. In addition, cinnamon extract arrested the cell cycle and inhibited positive regulatory proteins. Correspondingly, the protein levels of p21 and p27 not only were increased in the presence of cinnamon extract, also the expression of proliferating cell nuclear antigen (PCNA) was inhibited by cinnamon extract. Besides, among the components of cinnamon extract, cinnamic acid (CA), eugenol (EG) and cinnamyl alcohol significantly inhibited the VSMC proliferation. Overall, the present study demonstrates that cinnamon extract inhibited the PDGF-BB-induced proliferation of VSMCs through a G0/G1 arrest, which down-regulated the expression of cell cycle positive regulatory proteins by up-regulating p21 and p27 expression.

scholarly journals Hsa_circ_0054633 regulates PDGF-BB-induced proliferation, migration and oxidative stress of vascular smooth muscle cells through miR-107/TXNIP axis

2021 ◽  
Author(s):  
Xiaochun Lei ◽  
Yi Ding ◽  
Ye Tian ◽  
Peng Ding
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Cell Counting ◽  
Nuclear Antigen ◽  
Luciferase Reporter ◽  
Human Artery ◽  
Interacting Protein ◽  
Vsmc Proliferation ◽  
And Migration

IntroductionHsa_circ_0054633 has been found to be elevated in the blood of coronary artery disease (CAD) patients. However, the molecular mechanism and the role of hsa_circ_0054633 in the pathogenesis of CAD have not been reported in detail.Material and methodsThe expression of hsa_circ_0054633, microRNA (miR)-107 and thioredoxin-interacting protein (TXNIP) mRNA was measured using quantitative real-time polymerase chain reaction. Human artery vascular smooth muscle cell (HA-VSMC) proliferation, cell cycle, and migration were detected by cell counting kit-8 assay, flow cytometry and transwell assay, respectively. The generation of reactive oxygen species (ROS) was analyzed by dichlorofluorescein diacetate (DCFH-DA) assay. Western blot was utilized to determine the levels of proliferating cell nuclear antigen (PCNA), cyclin D1, matrix metallopeptidase 9 (MMP-9), Mn-superoxide dismutase (SOD2) and TXNIP protein. The interaction between miR-107 and hsa_circ_0054633 or TXNIP was confirmed by dual-luciferase reporter, RNA immunoprecipitation assay or pull-down assay.ResultsHsa_circ_0054633 was elevated in the plasma of CAD patients, and might be a potential blood biomarker for CAD prediction. Hsa_circ_0054633 silencing reversed PDGF-BB-induced promotion on HA-VSMC proliferation, cell cycle, migration and ROS production. MiR-107 directly interacted with hsa_circ_0054633 and TXNIP, and hsa_circ_0054633 regulated TXNIP expression by sponging miR-107. Besides, rescue assay indicated that the action of hsa_circ_0054633 silencing on PDGF-BB-treated HA-VSMCs could be attenuated by miR-107 inhibition or TXNIP overexpression, respectively.ConclusionsHsa_circ_0054633 knockdown protected HA-VSMCs against PDGF-BB-induced dysfunction through regulating miR-107/TXNIP axis, suggesting a potential therapeutic target for coronary atherosclerosis.

Inhibitory Effects of Brazilin on the Vascular Smooth Muscle Cell Proliferation and Migration Induced by PDGF-BB

2013 ◽  
Vol 41 (06) ◽  
pp. 1283-1296 ◽  
Author(s):  
Jing Guo ◽  
Li Li ◽  
Yu-Jie Wu ◽  
Yu Yan ◽  
Xiao-Na Xu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Vascular Diseases ◽  
Inhibitory Effects ◽  
Vsmc Proliferation ◽  
And Migration ◽  
Proliferation And Migration

Abnormal vascular smooth muscle cell (VSMC) proliferation and migration contribute to the pathogenesis of vascular diseases including atherosclerosis and restenosis. Brazilin isolated from the heartwood of Caesalpinia sappan L. has been reported to exhibit various biological activities, such as anti-platelet aggregation, anti-inflammation, vasorelaxation and pro-apoptosis. However, the functional effects of Brazilin on VSMCs remain unexplored. The present study investigated the potential effects of Brazilin on platelet-derived growth factor (PDGF)-BB induced VSMC proliferation and migration as well as the underlying mechanism of action. VSMC proliferation and migration were measured by Crystal Violet Staining, wound-healing and Boyden chamber assays, respectively. Cell cycle was analyzed by flow cytometry. Enzymatic action of matrix metalloproteinase-9 (MMP-9) was carried out by gelatin zymography. Expression of adhesion molecules, cell cycle regulatory proteins, the phosphorylated levels of PDGF receptor β (PDGF-Rβ), Src, extracellular signal regulated kinase (ERK) and Akt were tested by immunoblotting. The present study demonstrated that pretreatment with Brazilin dose-dependently inhibited PDGF-BB stimulated VSMC proliferation and migration, which were associated with a cell-cycle arrest at G0/G1 phase, a reduction in the adhesion molecule expression and MMP-9 activation in VSMCs. Furthermore, the increase in PDGF-Rβ, Src, ERK1/2 and Akt phosphorylation induced by PDGF-BB were suppressed by Brazilin. These findings indicate that Brazilin inhibits PDGF-BB induced VSMC proliferation and migration, and the inhibitory effects of Brazilin may be associated with the blockade of PDGF-Rβ - ERK1/2 and Akt signaling pathways. In conclusion, the present study implicates that Brazilin may be useful as an anti-proliferative agent for the treatment of vascular diseases.

Abstract 173: MicroRNA-365 Inhibits the Proliferation of Vascular Smooth Muscle Cells by Targeting Cyclin D1

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Myung-Hyun Kim ◽  
Onju Ham ◽  
Se-Yeon Lee ◽  
Eunmi Choi ◽  
Chang Yeon Lee ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cyclin D1 ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Nuclear Antigen ◽  
Neointimal Formation ◽  
Vsmc Proliferation

Background: Abnormal proliferation of vascular smooth muscle cells (VSMCs) is a common feature of disease progression in atherosclerosis. Cell proliferation is regulated by cell cycle regulatory proteins. MicroRNAs (miRNAs) have been reported to act as important gene regulators and play essential roles in the proliferation and migration of VSMCs in cardiovascular disease. However, the roles and mechanisms of miRNAs in VSMCs and neointimal formation are far from being fully understood. Methods & Results: In this study, cell cycle specific cyclin D1 was found to be a potential target of miR-365 by direct binding. Through an in vitro experiment, we showed that exogenous miR-365 overexpression reduced VSMC proliferation and proliferating cell nuclear antigen (PCNA) expression, while miR-365 was observed to block G1/S transition in platelet-derived growth factor (PDGF)-induced VSMCs. In addition, the proliferation of VSMCs by various stimuli, including PDGF, angiotensin II (Ang II), and serum, led to the downregulation of miR-365 expression levels. The expression of miR-365 was confirmed in balloon injured carotid arteries. Taken together, our results suggest an anti-proliferative role for miR-365 in VSMC proliferation, at least partly via modulating the expression of cyclin D1. Conclusions: Therefore, miR-365 may influence neointimal formation in atherosclerosis patients.

Abstract P110: EBP50 Regulates Vascular Smooth Muscle Cell Growth by Skp2-Mediated Degradation of p21/cip1

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Gyun Jee Song ◽  
Stacey Barrick ◽  
Kristen L Leslie ◽  
Nathalie M Fiaschi-Taesch ◽  
Alessandro Bisello
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Pdz Domain ◽  
Scaffolding Protein ◽  
Mrna Levels ◽  
Neointima Formation ◽  
Vsmc Proliferation ◽  
Genetic Ablation ◽  
Skp2 Expression

The PDZ domain-containing scaffolding protein, Ezrin-Radixin-Moesin-binding phosphoprotein 50 (EBP50) regulates vascular stenosis following endoluminal vessel injury. Its expression in vascular smooth muscle cells (VSMC) increases after wire injury, and neointima formation is significantly reduced in EBP50 knockout (KO) mice. The molecular mechanisms underlying EBP50 actions in VSMC are unknown. Genetic ablation of EBP50 reduced VSMC proliferation and was associated with increased (5-fold) expression of the cell cycle inhibitor p21cip1 both in vessels and in primary cells. No differences in mRNA levels of p21cip1 were observed in WT and KO cells. However, the half-life of p21cip1 in KO VSMC was significantly longer than in WT VSMC (80 min vs. 45 min) and p21cip1 levels were similar in WT and KO VSMC treated with the proteasome inhibitor MG132. These observations suggest that EBP50 regulates post-translational degradation of p21cip1. The S-phase kinase-associated protein 2 (skp2) is a component of the E3 ligase complex that degrades p21cip1. The C-terminal four amino acids of skp2 (ProSerCysLeu) are a canonical PDZ-binding sequence. Indeed, co-immunoprecipitation and in-gel overlay assays demonstrated the direct interaction between EBP50 and skp2. Mutation of the C-terminal Leu to Ala (L424A-skp2) abrogated the interaction with EBP50. Skp2 expression was significantly lower in KO than in WT cells and inhibition of EBP50 expression by an shRNA lentivirus decreased skp2 expression in WT cells. Moreover, expression of skp2, but not of the mutant L424A-skp2, in WT cells reduced p21cip1 levels. Therefore, EBP50 regulates both expression and activity of skp2 with attendant effects on p21cip1 and VSMC proliferation. Collectively, these experiments show that EBP50, by regulating skp2 and p21cip1 expression, controls VSMC proliferation and the progression of neointima formation. These studies identify a novel function for EBP50 in the direct regulation of the cell cycle and provide a mechanistic basis for the remarkable effect of this scaffolding protein on vascular remodeling.

scholarly journals An essential role of PDCD4 in vascular smooth muscle cell apoptosis and proliferation: implications for vascular disease

2010 ◽  
Vol 298 (6) ◽  
pp. C1481-C1488 ◽  
Author(s):  
Xiaojun Liu ◽  
Yunhui Cheng ◽  
Jian Yang ◽  
Thomas J. Krall ◽  
Yuqing Huo ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cell ◽  
Muscle Cell ◽  
Vascular Smooth Muscle Cell ◽  
Carotid Arteries ◽  
Vascular Diseases ◽  
Vsmc Proliferation ◽  
Vascular Walls

It is well established that vascular smooth muscle cell (VSMC) apoptosis and proliferation are critical cellular events in a variety of human vascular diseases. However, the molecular mechanisms involved in controlling VSMC apoptosis and proliferation are still unclear. In the current study, we have found that programmed cell death 4 (PDCD4) is significantly downregulated in balloon-injured rat carotid arteries in vivo and in platelet-derived growth factor-stimulated VSMCs in vitro. Overexpression of PDCD4 via adenovirus (Ad-PDCD4) increases VSMC apoptosis in an apoptotic model induced by serum deprivation. In contrast, VSMC apoptosis is significantly decreased by knockdown of PDCD4 via its small interfering RNA. In the rat carotid arteries in vivo, VSMC apoptosis is increased by Ad-PDCD4. We have further identified that activator protein 1 is a downstream signaling molecule of PDCD4 that is associated with PDCD4-mediated effects on VSMC apoptosis. In addition, VSMC proliferation was inhibited by overexpression of PDCD4. The current study has identified, for the first time, that PDCD4 is an essential regulator of VSMC apoptosis and proliferation. The downregulation of PDCD4 expression in diseased vascular walls may be responsible for the imbalance of VSMC proliferation and apoptosis. The results indicate that PDCD4 may be a new therapeutic target in proliferative vascular diseases.

scholarly journals PDGF-induced vascular smooth muscle cell proliferation is associated with dysregulation of insulin receptor substrates

2011 ◽  
Vol 300 (6) ◽  
pp. C1375-C1385 ◽  
Author(s):  
Yan Zhao ◽  
Swarajit K. Biswas ◽  
Patrick H. McNulty ◽  
Mark Kozak ◽  
John Y. Jun ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Insulin Receptor ◽  
Serine Phosphorylation ◽  
Phenotypic Switching ◽  
Dependent Manner ◽  
Receptor Signaling ◽  
Vsmc Proliferation ◽  
Insulin Receptor Substrates ◽  
Insulin Receptor Signaling

In vascular smooth muscle cells (VSMCs), platelet-derived growth factor (PDGF) plays a major role in inducing phenotypic switching from contractile to proliferative state. Importantly, VSMC phenotypic switching is also determined by the phosphorylation state/expression levels of insulin receptor substrate (IRS), an intermediary signaling component that is shared by insulin and IGF-I. To date, the roles of PDGF-induced key proliferative signaling components including Akt, p70S6kinase, and ERK1/2 on the serine phosphorylation/expression of IRS-1 and IRS-2 isoforms remain unclear in VSMCs. We hypothesize that PDGF-induced VSMC proliferation is associated with dysregulation of insulin receptor substrates. Using human aortic VSMCs, we demonstrate that prolonged PDGF treatment led to sustained increases in the phosphorylation of protein kinases such as Akt, p70S6kinase, and ERK1/2, which mediate VSMC proliferation. In addition, PDGF enhanced IRS-1/IRS-2 serine phosphorylation and downregulated IRS-2 expression in a time- and concentration-dependent manner. Notably, phosphoinositide 3-kinase (PI 3-kinase) inhibitor (PI-103) and mammalian target of rapamycin inhibitor (rapamycin), which abolished PDGF-induced Akt and p70S6kinase phosphorylation, respectively, blocked PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. In contrast, MEK1/ERK inhibitor (U0126) failed to block PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. PDGF-induced IRS-2 downregulation was prevented by lactacystin, an inhibitor of proteasomal degradation. Functionally, PDGF-mediated IRS-1/IRS-2 dysregulation resulted in the attenuation of insulin-induced IRS-1/IRS-2-associated PI 3-kinase activity. Pharmacological inhibition of PDGF receptor tyrosine kinase with imatinib prevented IRS-1/IRS-2 dysregulation and restored insulin receptor signaling. In conclusion, strategies to inhibit PDGF receptors would not only inhibit neointimal growth but may provide new therapeutic options to prevent dysregulated insulin receptor signaling in VSMCs in nondiabetic and diabetic states.

scholarly journals Cytoglobin overexpression facilitates proliferation and migration of vascular smooth muscle cells

2020 ◽  
Vol 72 (2) ◽  
pp. 165-172 ◽  
Author(s):  
Lei Li ◽  
Yilin Xie ◽  
Shen Li ◽  
Juanjuan Tan ◽  
Yingchun Qin ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Feedback Regulation ◽  
Muscle Cells ◽  
Nuclear Antigen ◽  
Matrix Remodeling ◽  
Negative Feedback Regulation ◽  
Vsmc Proliferation

Cytoglobin, a recently discovered globin, is expressed in vascular smooth muscle cells (VSMCs). Loss of cytoglobin provides a protective effect on vascular reconstruction but the effect of its overexpression is unclear. The aim of the study was to investigate the effect of cytoglobin overexpression on the migration and proliferation of VSMCs and possible mechanisms. We detected the expression of cytoglobin in hypertensive and normotensive rat aortas, with negative feedback regulation between cytoglobin and hypertension observed. The expression of cytoglobin was significantly decreased in hypertensive rats compared to normotensive rats, but VSMCs overexpressing cytoglobin displayed increased cell migration and proliferation, which led to a phenotypic switch. The increased expression of matrix metalloproteinase 9 and collagen Ia suggests a role for cytoglobin in extracellular matrix remodeling. Increased expression of proliferating cell nuclear antigen and decreased expression of p27 implies that cytoglobin is involved in modulating VSMC proliferation. Our findings indicate that cytoglobin may play an important role in vascular wall remodeling.

Abstract 1701: Deletion of β PDGF Receptor Dependent PI-3 Kinase and PLCγ Signaling Attenuates Vascular Smooth Muscle Cell Proliferation and Inhibits Neointima Formation in Mice

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Evren Caglayan ◽  
Marius Vantler ◽  
Olli Leppanen ◽  
Lenard Mustafov ◽  
Felix Gerhardt ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Balloon Angioplasty ◽  
Cell Cycle Progression ◽  
Signaling Molecules ◽  
Brdu Incorporation ◽  
Neointima Formation ◽  
Cycle Progression ◽  
Vsmc Proliferation

Platelet-derived growth factor β-receptor (βPDGFR)-mediated proliferation of vascular smooth muscle cells (VSMC) plays a pivotal role in the development of restenosis. The βPDGFR binds and activates cytoplasmic signaling molecules such as Src, PI 3-kinase (PI3K), RasGAP, SHP-2 and phospholipase C-gamma (PLCγ). However, the βPDGFR-induced signaling pathways leading to cell cycle progression are largely unknown. In order to characterize the signaling molecules, which are important for βPDGFR-induced VSMC proliferation, we generated several stable VSMC lines with mutated βPDGFRs which were unable to bind/activate each individual signaling molecule. BrdU incorporation assays revealed PI3K and PLCγ as the main mediators of βPDGFR-mediated VSMC proliferation. Deletion of the binding sites for PI3K or PLCγ diminished DNA-synthesis about 47 ± 6% and 54 ± 5%, respectively, whereas the deletion of Src, RasGAP, or SHP-2 binding to the βPDGFR had no influence on the mitogenic response. Mutant cell lines which were only able to bind/activate PI3K or PLCγ induced 43 ± 4% and 52 ± 5% of the WT response, respectively. We further investigated the influence of PI3K and PLCγ on the distinct expression of the key players of cell cycle progression. Western blot analysis revealed that βPDGFR-activated PI3K mainly mediated the up-regulation of cyclin D1, whereas activation of PLCγ mediated the down-regulation of the cyclin-dependent kinase-inhibitor p27 kip1 . Consistently, βPDGFR-dependent phosphorylation of the retinoblastoma protein which is important for the G1/S transition was attenuated when either the binding of PI3K or PLCγ to the βPDGFR was abolished. Moreover, we confirmed our in vitro results in an in vivo model of balloon-induced carotid artery neointima formation. Mice lacking binding of PI3K and PLCγ to the βPDGFR (F3 mice) developed ~50% less neointima formation after balloon angioplasty compared to control WT mice after 3 weeks (p < 0.05). These results indicate that the mitogenic signal of the β PDGFR is mediated by PI3K and PLCγ affecting distinct targets of cell cycle progression. Targeting specific downstream molecules of the βPDGFR in VSMCs might provide a novel approach to reduce neointima formation in patients undergoing balloon angioplasty.

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