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.

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.

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.

Abstract 374: Inhibition of Mitochondrial CaMKII Reduces Vascular Smooth Muscle Migration and Neointima Formation and Halts Mitochondrial Mobility

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Emily Nguyen ◽  
Olha Koval ◽  
Isabella Grumbach
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Mitochondrial Fission ◽  
Leading Edge ◽  
Neointima Formation ◽  
Vsmc Proliferation ◽  
Mitochondrial Motility ◽  
And Migration

Background: Restenosis after angioplasty for coronary vascular disease remains a critical problem in cardiovascular medicine. Vascular smooth muscle cell (VSMC) migration and proliferation cause restenosis through neointima formation. Mitochondrial motility is likely necessary for cell proliferation and migration, and is inhibited in microdomains with increased Ca 2+ . The Ca 2+ /calmodulin-dependent kinase II (CaMKII) in mitochondria (mtCaMKII) is proposed to control mitochondrial matrix Ca 2+ uptake through mitochondrial Ca 2+ uniporter (MCU). Thus, we hypothesized that blocking mtCaMKII decreases VSMC migration and neointima formation by decreasing mitochondrial motility. Methods: mtCaMKII was inhibited by expression of the mitochondria-targeted CaMKII inhibitor peptide (CaMKIIN) in a novel transgenic mouse model in smooth muscle only (SM-mtCaMKIIN) or delivered by adenoviral transduction (Ad-mtCaMKIIN). Results: In our models, mtCaMKIIN was detected selectively in mitochondria of VSMC. mtCaMKIIN significantly reduced mitochondrial Ca 2+ current and Ca 2+ content compared to WT in vivo and in vitro. SM-mtCaMKIIN mice showed significantly reduced neointimal area 28 days after endothelial injury (n=8, p<0.05) and fewer proliferating neointimal cells by PCNA staining. In vitro, Ad-mtCaMKIIN mildly reduced VSMC proliferation and mitochondrial ROS production without altering maximal respiration after PDGF treatment. Ad-mtCaMKIIN abolished VSMC migration, as did mitoTEMPO and MCU inhibitor Ru360. Ad-mtCaMKIIN blocked mitochondrial mobility towards the leading edge, while relocation of mitochondria was seen in WT cells 6 h after PDGF treatment. Mitochondrial redistribution was also inhibited by Ru360, but not by mitoTEMPO or cytoplasmic CaMKII inhibition. Mitochondrial fission promotes cell migration. Accordingly, PDGF increased mitochondrial particles in WT VSMC, while mitochondria in Ad-mtCaMKIIN cells were fragmented and unresponsive to PDGF treatment. Conclusions: mtCaMKIIN prevents mitochondrial distribution to the leading edge and reduces VSMC migration and neointima formation. These data suggest mitochondrial Ca 2+ regulation plays an important role in VSMC migration by altering mitochondrial location.

Inhibition of vascular smooth muscle cell proliferation by chronic hemin treatment

2008 ◽  
Vol 295 (3) ◽  
pp. H999-H1007 ◽  
Author(s):  
Tuanjie Chang ◽  
Lingyun Wu ◽  
Rui Wang
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Cell Cycle Progression ◽  
Control Level ◽  
Inhibitory Effect ◽  
Cycle Progression ◽  
Vsmc Proliferation ◽  
Long Term Treatment

Hemin, an oxidized form of heme, is an essential regulator of gene expression and cell cycle progression. Our laboratory previously reported ( 34 ) that chronic hemin treatment of spontaneously hypertensive rats reversed the eutrophic inward remodeling of small peripheral arteries. Whether long-term treatment of cultured vascular smooth muscle cells (VSMCs) with hemin alters the proliferation status of these cells has been unknown. In the present study, hemin treatment at 5 μM for 4, 7, 14, and 21 days significantly inhibited the proliferation of cultured rat aortic VSMCs (A-10 cells) by arresting cells at G0/G1 phases so as to decelerate cell cycle progression. Heme oxygenase (HO) activity and inducible HO-1 protein expression were significantly increased by hemin treatment. HO inhibitor tin protoporphyrin IX (SnPP) abolished the effects of hemin on cell proliferation and HO activity. Interestingly, hemin-induced HO-1 expression was further increased in the presence of SnPP. Hemin treatment had no significant effect on the expression of constitutive HO-2. Expression of p21 protein and the level of reactive oxygen species (ROS) were decreased by hemin treatment, which was reversed by application of SnPP. After removal of hemin from culture medium, inhibited cell proliferation and increased HO-1 expression in VSMCs were returned to control level within 1 wk. Transfection with HO-1 small interfering RNA significantly knocked down HO-1 expression and decreased HO activity, but had no effect on HO-2 expression, in cells treated with or without hemin for 7 days. The inhibitory effect of hemin on cell proliferation was abolished in HO-1 silenced cells. It is concluded that induction of HO-1 and, consequently, increased HO activity are responsible for the chronic inhibitory effect of hemin on VSMC proliferation. Changes in the levels of p21 and ROS might also participate in the cellular effects of hemin.

scholarly journals Fluvastatin Upregulates theα1CSubunit of CaV1.2 Channel Expression in Vascular Smooth Muscle Cells via RhoA and ERK/p38 MAPK Pathways

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Qiu-Fang Ouyang ◽  
Ying Han ◽  
Zhi-Hong Lin ◽  
Hong Xie ◽  
Chang-Sheng Xu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
P38 Mapk ◽  
Marker Gene ◽  
Flow Cytometric Analysis ◽  
Immunofluorescent Staining ◽  
Potential Candidate ◽  
Mapk Activation ◽  
Vsmc Proliferation

Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis. And this process has been related to remodeling of L-type calcium channel (LTCC). We attempted to investigate whether fluvastatin has any effect on VSMC proliferation and LTCCα1Csubunit(LTCCα1C)expression as well as the potential mechanisms involved. The VSMCs proliferation was assayed by osteopontin immunofluorescent staining and [3H]-thymidine incorporation. The cell cycle was detected by flow cytometric analysis. The activity of RhoA was determined with pull-down assay. MAPK activity andLTCCα1Cexpression were assessed by western blotting. We demonstrated fluvastatin prevented the VSMCs dedifferentiating into a proliferative phenotype and induced cell cycle arrest in the G0/G1 phase in response to PDGF-BB stimulation. Fluvastatin dose-dependently reversed the downregulation ofLTCCα1Cexpression induced by PDGF-BB. Inhibition of ROCK, ERK, or p38 MAPK activation largely enhanced the upregulation effect of fluvastatin(P<0.01). However, blockade of JNK pathway had no effect onLTCCα1Cexpression. We concludedLTCCα1Cwas a VSMC contractile phenotype marker gene. Fluvastatin upregulatedLTCCα1Cexpression, at least in part, by inhibiting ROCK, ERK1/2, and p38 MAPK activation. Fluvastatin may be a potential candidate for preventing or treating vascular diseases.

scholarly journals PYK2 signaling is required for PDGF-dependent vascular smooth muscle cell proliferation

2011 ◽  
Vol 301 (1) ◽  
pp. C242-C251 ◽  
Author(s):  
Jessica Perez ◽  
Rebecca A. Torres ◽  
Petra Rocic ◽  
Mary J. Cismowski ◽  
David S. Weber ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Protein Kinase ◽  
Vascular Smooth Muscle ◽  
Dna Synthesis ◽  
Smooth Muscle Cell ◽  
Protein Kinase B ◽  
Signaling Cascades ◽  
Vsmc Proliferation ◽  
Dependent Protein

Aberrant vascular smooth muscle cell (VSMC) growth is associated with many vascular diseases including atherosclerosis, hypertension, and restenosis. Platelet-derived growth factor-BB (PDGF) induces VSMC proliferation through control of cell cycle progression and protein and DNA synthesis. Multiple signaling cascades control VSMC growth, including members of the mitogen-activated protein kinase (MAPK) family as well as phosphatidylinositol 3-kinase (PI3K) and its downstream effector AKT/protein kinase B (PKB). Little is known about how these signals are integrated by mitogens and whether there are common receptor-proximal signaling control points that synchronize the execution of physiological growth functions. The nonreceptor proline-rich tyrosine kinase 2 (PYK2) is activated by a variety of growth factors and G protein receptor agonists in VSMC and lies upstream of both PI3K and MAPK cascades. The present study investigated the role of PYK2 in PDGF signaling in cultured rat aortic VSMC. PYK2 downregulation attenuated PDGF-dependent protein and DNA synthesis, which correlated with inhibition of AKT and extracellular signal-regulated kinases 1 and 2 (ERK1/2) but not p38 MAPK activation. Inhibition of PDGF-dependent protein kinase B (AKT) and ERK1/2 signaling by inhibitors of upstream kinases PI3K and MEK, respectively, as well as downregulation of PYK2 resulted in modulation of the G1/S phase of the cell cycle through inhibition of retinoblastoma protein (Rb) phosphorylation and cyclin D1 expression, as well as p27Kip upregulation. Cell division kinase 2 (cdc2) phosphorylation at G2/M was also contingent on PDGF-dependent PI3K-AKT and ERK1/2 signaling. These data suggest that PYK2 is an important upstream mediator in PDGF-dependent signaling cascades that regulate VSMC proliferation.

scholarly journals Regulation of Vascular Smooth Muscle Proliferation by Heparin

2005 ◽  
Vol 280 (16) ◽  
pp. 15682-15689 ◽  
Author(s):  
Stephen Fasciano ◽  
Rekha C. Patel ◽  
Indhira Handy ◽  
Chandrashekhar V. Patel
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
S Phase ◽  
Mrna Levels ◽  
Cyclin Dependent Kinase ◽  
Cellular Targets ◽  
Vsmc Proliferation ◽  
Protein Levels ◽  
Smooth Muscle Proliferation ◽  
Antiproliferative Agent

Uncontrolled proliferation of vascular smooth muscle cells (VSMCs) contribute to intimal hyperplasia during atherosclerosis and restenosis. Heparin is an antiproliferative agent for VSMCs and has been shown to block VSMC proliferation both in tissue culture systems and in animals. Despite the well documented antiproliferative actions of heparin, its cellular targets largely remain unknown. In an effort to characterize the mechanism of the antiproliferative property of heparin, we have analyzed the effect of heparin on cell cycle in VSMC. Our results indicate that the heparin-induced block in G1to S phase transition is imposed by p27kip1-mediated inhibition of cyclin-dependent kinase 2 activity. Further analysis of p27kip1mRNA levels showed that the increase in p27kip1protein levels in heparin-treated VSMC occurs at posttranscriptional levels. We present evidence that heparin causes stabilization of p27kip1protein during G1phase and thereby prevents activation of cyclin-dependent kinase 2.

scholarly journals c-myc and skp2 Coordinate p27 Degradation, Vascular Smooth Muscle Proliferation, and Neointima Formation Induced by the Parathyroid Hormone-Related Protein

Endocrinology ◽  
2012 ◽  
Vol 153 (2) ◽  
pp. 861-872 ◽  
Author(s):  
Brian M. Sicari ◽  
Ronnie Troxell ◽  
Fatimah Salim ◽  
Mansoor Tanwir ◽  
Karen K. Takane ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Smooth Muscle ◽  
Parathyroid Hormone ◽  
Vascular Smooth Muscle ◽  
Molecular Mechanisms ◽  
Neointima Formation ◽  
Related Protein ◽  
Down Regulation ◽  
Parathyroid Hormone Related Protein ◽  
Arterial Restenosis

Parathyroid hormone-related protein (PTHrP) contains a classical bipartite nuclear localization signal. Nuclear PTHrP induces proliferation of arterial vascular smooth muscle cells (VSMC). In the arterial wall, PTHrP is markedly up-regulated in response to angioplasty and promotes arterial restenosis. PTHrP overexpression exacerbates arterial restenosis, and knockout of the PTHrP gene results in decreased VSMC proliferation in vivo. In arterial VSMC, expression of the cell cycle inhibitor, p27, rapidly decreases after angioplasty, and replacement of p27 markedly reduces neointima development. We have shown that PTHrP overexpression in VSMC leads to p27 down-regulation, mostly through increased proteosomal degradation. Here, we determined the molecular mechanisms through which PTHrP targets p27 for degradation. S-phase kinase-associated protein 2 (skp2) and c-myc, two critical regulators of p27 expression and stability, and neointima formation were up-regulated in PTHrP overexpression in VSMC. Normalization of skp2 or c-myc using small interfering RNA restores normal cell cycle and p27 expression in PTHrP overexpression in VSMC. These data indicate that skp2 and c-myc mediate p27 loss and proliferation induced by PTHrP. c-myc promoter activity was increased, and c-myc target genes involved in p27 stability were up-regulated in PTHrP overexpression in VSMC. In primary VSMC, PTHrP overexpression led to increased c-myc and decreased p27. Conversely, knockdown of PTHrP in primary VSMC from PTHrPflox/flox mice led to cell cycle arrest, p27 up-regulation, with c-myc and skp2 down-regulation. Collectively, these data describe for the first time the role of PTHrP in the regulation of skp2 and c-myc in VSMC. This novel PTHrP-c-myc-skp2 pathway is a potential target for therapeutic manipulation of the arterial response to injury.

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.

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