Vascular Smooth Muscle Cells Cell Cycle Research Articles

Greater TIMP-1 protein levels and neointimal formation represent sex-dependent cellular events limiting aortic vessel expansion in female rats.

Fragmentation/loss of the structural protein elastin represents the precipitating event translating to aortic expansion and subsequent aneurysm formation. The present study tested the hypothesis that greater protein expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and neointimal growth secondary to a reduction of medial elastin content represent sex-dependent events limiting aortic vessel expansion in females. TIMP-1 protein levels were higher in the ascending aorta of female versus male patients diagnosed with a bicuspid aortic valve (BAV). The latter paradigm was recapitulated in the aorta of adult male and female rats complemented by greater TIMP-2 expression in females. CaCl2 (0.5 M) treatment of the infrarenal aorta of adult male and female rats increased the in situ vessel diameter and expansion was significantly smaller in females despite a comparable reduction of medial elastin content. The preferential appearance of a neointimal region of the CaCl2-treated infrarenal aorta of female rats may explain in part the smaller in situ expansion and neointimal growth correlated positively with the % change of the in situ diameter. Neointimal formation was secondary to a significant increase in the density of medial/neointimal vascular smooth muscle cells (VSMCs) that re-entered the G2-M phase whereas VSMC cell cycle re-entry was attenuated in the CaCl2-treated infrarenal aorta of male rats. Thus, greater TIMP-1 expression in the aorta of female BAV patients may prevent excessive elastin fragmentation and preferential neointimal growth following CaCl2-treatment of the infrarenal aorta of female rats represents a sex-dependent biological event limiting vessel expansion secondary to a significant loss of the structural protein.

Abstract P3020: O-GlcNAc Transferase Regulates Vascular Smooth Muscle Cell Cycle Progression In Diabetes

Diabetic patients show increased proclivity for vascular smooth muscle cell (VSMC) migration and proliferation. Hyperglycemia, hallmark of diabetes, increases intracellular glucose signaling via O-linked N-acetylglucosamine transferase (OGT), a key regulator of protein O-GlcNAcylation. We previously showed that smooth muscle OGT (smOGT) deletion prevents hyperglycemia-induced atherosclerosis. Goal of this work was to interrogate the role of smOGT in VSMC cell cycle progression in diabetes. For this, we used conditional SMC-specific OGT knockout (smOGT KO ) and wild-type (smOGT WT ) mice generated via tamoxifen (tmx)-inducible VSMC-restricted Cre driver mice ( Itga8-CreER T2 ). Briefly, following tmx injection, 8-wks-old smOGT KO and smOGT WT mice were placed on Western diet feeding regimen for study duration. At 14-wks-age, mice were injected with 50mg/Kg/day streptozotocin i.p. once daily for 5 consecutive days. Mice were harvested at 16-18-wks-age and plasma and aortic tissues were collected for biochemical and molecular studies. Immunoblotting of aortic lysates revealed a significant decrease (59%) in OGT and O-GlcNAc expression, validating the smOGT KO mice. Loss of smOGT reduced (40%) CDK6 expression (regulator of G1-S phase cell cycle progression) concomitant to enhanced (62%) p21 Cip1 expression (cyclin-dependent kinase inhibitor) in aortic vessels from diabetic smOGT KO vs. smOGT WT mice. This was further accompanied with 69% attenuation in SRF expression (transcriptional regulator of SM proliferation) in diabetic smOGT KO vs. smOGT WT aortic vessels. Similar results were obtained with OGT loss-of-function studies in human coronary artery SMC (HCASMC) primary cultures under diabetic conditions in vitro . Briefly, control (siCtrl) and OGT siRNA (siOGT)-transfected HCASMC were treated with or without 25mM glucose+100μM PUGNAc for 48 hours. Immunoblotting revealed that reduced OGT-mediated O-GlcNAcylation in siOGT cells in a diabetic milieu significantly attenuated SRF expression coupled with augmented p21 Cip1 expression compared to diabetic siCtrl cells. Together, our results suggest a direct regulatory role of smooth muscle OGT in VSMC cell cycle progression in diabetes.

Xeroderma Pigmentosum Group D (XPD) Inhibits the Proliferation Cycle of Vascular Smooth Muscle Cell (VSMC) by Activating Glycogen Synthase Kinase 3β (GSK3β).

BackgroundVSMC proliferation plays a key role in atherosclerosis, but the role of XPD in VSMC proliferation remains unknown. We investigated the expression of XPD, which is involved in cell cycle regulation, and its role in VSMC proliferation response to atherogenic stimuli.Material/MethodsHuman umbilical vein VSMCs were transfected with recombinant plasmid pEGFP-N2/XPD and pEGFP-N2 and incubated with PDGF-BB in vitro. Cell viability was determined by MTT assay. The expressions of XPD, GSK3β, p-GSK3β, CDK4, and cyclin D1 protein were detected by Western blot analysis. Cell cycle was examined by flow cytometry.ResultsPDGF inhibited the expression of XPD in VSMCs and promoted VSMC proliferation. Overexpression of XPD significantly augmented cell cycle arrest, and attenuated protein expression levels of CDK4 and cyclin D1 in VSMCs. XPD overexpression suppressed the effects of PDGF-BB in promoting G1/S transition and accelerating protein expression levels of CDK4 and cyclin D1. XPD diminished the phosphorylation of GSK3β, and SB216763 inhibited the reduction effect of XPD on CDK4 and cyclin D1.ConclusionsXPD induces VSMC cell cycle arrest, and the activation of GSK3β plays a crucial role in inhibitory effect of XPD on VSMC proliferation.

Vasculoprotective Effects of 3-Hydroxybenzaldehyde against VSMCs Proliferation and ECs Inflammation.

3-hydroxybenzaldehyde (3-HBA) is a precursor compound for phenolic compounds like Protocatechuic aldehyde (PCA). From recent reports, PCA has shown vasculoprotective potency, but the effects of 3-HBA remain unclear. The aim of this study is to investigate the vasculoprotective effects of 3-HBA in endothelial cells, vascular smooth muscle cells and various animal models. We tested effects of 3-HBA in both vitro and vivo. 3-HBA showed that it prevents PDGF-induced vascular smooth muscle cells (VSMCs) migration and proliferation from MTS, BrdU assays and inhibition of AKT phosphorylation. It arrested S and G0/G1 phase of VSMC cell cycle in PI staining and it also showed inhibited expression levels of Rb1 and CD1. In human umbilical vein endothelial cells (HUVECs), 3-HBA inhibited inflammatory markers and signaling molecules (VCAM-1, ICAM-1, p-NF-κB and p-p38). For ex vivo, 3-HBA has shown dramatic effects in suppressing the sprouting from aortic ring of Spargue Dawley (SD) rats. In vivo data supported the vasculoprotective effects of 3-HBA as it inhibited angiogenesis from Matrigel Plug assay in C57BL6 mouse, prevented ADP-induced thrombus generation, increased blood circulation after formation of thrombus, and attenuated neointima formation induced by common carotid artery balloon injury of SD rats. 3-HBA, a novel therapeutic agent, has shown vasculoprotective potency in both in vitro and in vivo.

MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism.

BackgroundOverexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo.Methods and ResultssiRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G0/G1 to S. Protein expression of the cyclin-dependent kinase inhibitor p27kip1, but not p21cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27kip1-/- mice indicating that the effect of MARCKS is p27kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27kip1 at serine 10 by KIS is required for nuclear export and degradation of p27kip1. MARCKS knockdown caused nuclear trapping of p27kip1. Both p27kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization.ConclusionsMARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated reestablishment of an intact endothelium. MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.

Salusin-β induces smooth muscle cell proliferation by regulating cyclins D1 and E expression through MAPKs signaling pathways.

Despite the clear mitogenic effect of salusin-β on vascular smooth muscle cells (VSMCs), which contributes to its proatherosclerotic effects, additional studies are needed to explore its underlying mechanisms. The aim of this study was to investigate the mechanism of salusin-β's effects on VSMCs cell cycle regulation and the possible signal pathways. Salusin-β accelerated the G1/S phase transition in VSMCs and increased the expression levels of cyclins D1 and E. Silencing either cyclin D1 or cyclin E gene inhibited salusin-β-induced VSMCs proliferation, cell cycle progression, phosphorylation of the Rb protein, and dissociation of the E2F-Rb complex. Importantly, expression of cyclin E was also induced by cyclin D1. Next, we found that salusin-β increased the protein expressions of activator protein-1 (AP-1) subunits c-Jun and c-Fos, and enhanced binding of AP-1 to the promoter region of cyclin D1. In addition, inhibition of AP-1 activity could lead to significant suppression of salusin-β-induced cyclin D1 expression. Furthermore, MPAKs pathways were found to mediate salusin-β-induced VSMCs proliferation, cyclin D1, cyclin E, c-Jun, and c-Fos expression. These results suggest that salusin-β promotes cell cycle progression of VSMCs by upregulating the cyclin D1 and cyclin E, in an AP-1-dependent manner through mitogen-activated protein kinases signaling pathways.

THE ROLE OF GEMININ GENE ON PROLIFERATION OF VASCULAR SMOOTH MUSCLE CELL

ObjectivesThe proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of vascular diseases. Geminin is a 25 kDa nuclear protein, which regulate the proliferation of...

Protocatechuic aldehyde inhibits migration and proliferation of vascular smooth muscle cells and intravascular thrombosis

The migration and proliferation of vascular smooth muscle cells (VSMCs) and formation of intravascular thrombosis play crucial roles in the development of atherosclerotic lesions. This study examined the effects of protocatechuic aldehyde (PCA), a compound isolated from the aqueous extract of the root of Salvia miltiorrhiza, an herb used in traditional Chinese medicine to treat a variety of vascular diseases, on the migration and proliferation of VSMCs and platelets due to platelet-derived growth factor (PDGF). DNA 5-bromo-2′-deoxy-uridine (BrdU) incorporation and wound-healing assays indicated that PCA significantly attenuated PDGF-induced proliferation and migration of VSMCs at a pharmacologically relevant concentration (100μM). On a molecular level, we observed down-regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathways, both of which regulate key enzymes associated with migration and proliferation. We also found that PCA induced S-phase arrest of the VSMC cell cycle and suppressed cyclin D2 expression. In addition, PCA inhibited PDGF-BB-stimulated reactive oxygen species production in VSMCs, indicating that PCA’s antioxidant properties may contribute to its suppression of PDGF-induced migration and proliferation in VSMCs. Finally, PCA exhibited an anti-thrombotic effect related to its inhibition of platelet aggregation, confirmed with an aggregometer. Together, these findings suggest a potential therapeutic role of PCA in the treatment of atherosclerosis and angioplasty-induced vascular restenosis.

Potent Inhibition of Platelet-Derived Growth Factor-Stimulated Rat Aortic Vascular Smooth Muscle Cell Cycle and Proliferation by (2E)-3-(4-hydroxy-3-methoxyphenyl)phenylpro-2-en-1-one, a Newly Synthesized Benzylideneacetophenone Derivative

One of the principal regulators of mitogenesis in vascular smooth muscle cells (VSMCs) is platelet-derived growth factor-BB (PDGF-BB). An increase of PDGF-BB expression has been observed in atherosclerotic lesions. The aim of this study was to elucidate the effects and molecular mechanism of (2E)-3-(4-hydroxy-3-methoxyphenyl) phenylpro-2-en-1-one (KTJ2242), a newly synthesized benzylideneacetophenone derivative, on PDGF-BB-stimulated rat aortic VSMCs. KTJ2242 induced accumulation of cells in the G1 phase of the cell cycle of VSMCs. We observed that KTJ2242 inhibited PDGF-BB-stimulated [3H]-thymidine incorporation into the DNA of VSMCs, and the cell number was significantly reduced in a concentration-dependent manner. Also, we observed that KTJ2242 decreased PDGF-BB-stimulated extracellular-regulated kinase 1 and 2 (ERK1/2) and Akt phosphorylation. These results suggest the possibility that KTJ2242 may be a potential agent with which to control vascular disorders and its antiproliferative mechanism may be mediated through partial Akt and ERK1/2-dependent signaling pathways.

Inhibitory effect of PMC, a potent hydrophilic α-tocopherol derivative, on vascular smooth muscle cell proliferation: The pivotal role of PKC-α translocation

Content: Vascular smooth muscle cells (VSMCs) play a major role in the pathogenesis of atherosclerosis and restenosis, and thus the excessive proliferation of VSMCs contributes to neointimal thickening during atherosclerosis and restenosis. PMC (2,2,5,7,8-pentamethyl-6-hydroxychromane) is the most potent hydrophilic derivative of the α-tocopherols; it acts as a potent anti-inflammatory and free-radical scavenger.Objective: The present study was designed to examine the inhibitory mechanisms of PMC in VSMC proliferation.Materials and methods: VSMC proliferation and cytotoxicity were measured by MTT and LDH assays, respectively. The cell cycle and translocation of PKC-α in VSMCs were used by flow cytometry and confocal microscope, respectively. To detect PKC-α translocation and activation in VSMCs, immunoblotting was performed in the present study.Results: In this study, we demonstrate an anti-proliferative effect of PMC in VSMCs. Concentration-dependent inhibition of serum-induced VSMC proliferation was observed in PMC (20 and 50 μM)-treated cells. PMC pretreatment also arrested VSMC cell cycle progression at the G2/M phase. Furthermore, PMC exhibited obvious inhibitory effects on phorbol 12-myristate 13-acetate (PMA)-induced protein kinase C (PKC)-α translocation and phospho-(Ser/Thr) substrate phosphorylation.Discussion and conclusion: The inhibitory mechanisms of PMC on VSMC proliferation is mediated, at least in part, by inhibition of PKC-α translocation and causes cell cycle arrest in the G2/M phase. PMC treatment may represent a novel approach for lowering the risk of or improving function in abnormal VSMC proliferation-related vascular diseases.

Effect of endothelial progenitor cells on the proliferation of co-cultured vascular smooth muscle cells

To explore the effect of endothelial progenitor cells (EPCs) on the proliferation of co-cultured rat vascular smooth muscle cells (VSMCs). Mononuclear cells were isolated from fresh cord blood by 6% hydroxyethyl starch (HES) and density gradient centrifugation. Isolated mononuclear cells were cultured in EGM-2 medium supplemented with 20% fetal bovine serum (FBS), VEGF,bFGF and other growth factors. Biological features of EPCs were observed at different time points, and EPCs were identified by morphology, fluorescence double-staining and flow cytometry. Indirect immunofluorescence was performed to analyze the expression of alpha-SM-actin, calponin of VSMCs special antigen. Co-culture system of EPCs and VSMCs was established by transwell permeable support. FBS (20%) was used to stimulate the proliferation of VSMCs. In a VSMCs/EPCs co-culture system, the DNA synthesis ability, total protein level and cell cycle of VSMCs were determined by BrdU marking method,protein quantitation and flow cytometry after co-culture for 6, 12, 24,48 and 72 h. After co-culture for 12, 24, 48, and 72 h, the DNA synthesis ability and total protein level of VSMCs significantly decreased compared with the control group (P<0.05). Flow cytometry showed that the percentage of S phase of VSMCs in VSMCs/EPCs co-cultured group significantly decreased and the percentage of G1 phase increased markedly compared with the control group (P<0.05). The maximal inhibitory effect was observed at 48 h. Early EPCs could inhibit the proliferation of VSMCs.

3-Deazaadenosine Prevents Smooth Muscle Cell Proliferation and Neointima Formation by Interfering With Ras Signaling

3-Deazaadenosine (c3Ado) is a potent inhibitor of S-adenosylhomocysteine hydrolase, which regulates cellular methyltransferase activity. In the present study, we sought to determine the effect of c3Ado on vascular smooth muscle cell (VSMC) function and neointima formation in vivo. c3Ado dose-dependently prevented the proliferation and migration of human coronary VSMCs in vitro. This was accompanied by an increased expression of the cyclin-dependent kinase inhibitors p21(WAF1/Cip1), p27(Kip1), a decreased expression of G(1)/S phase cyclins, and a lack of retinoblastoma protein hyperphosphorylation. In accordance with these findings, fluorescence-activated cell-sorting analysis of propidium iodide-stained cells indicated a cell cycle arrest in the G(0)/G(1) phase. Importantly, c3Ado did not affect the number of viable (trypan blue exclusion) or apoptotic cells (TUNEL). Mechanistically, c3Ado prevented FCS-induced Ras carboxyl methylation and membrane translocation and activity by inhibiting isoprenylcysteine carboxyl methyltransferase and reduced FCS-induced extracellular signal-regulated kinase (ERK)1/2 and Akt phosphorylation in a dose-dependent manner. Conversely, rescuing signal transduction by overexpression of a constitutive active Ras mutant abrogated c3Ado's effect on proliferation. For in vivo studies, the femoral artery of C57BL/6 mice was dilated and mice were fed a diet containing 150 microg of c3Ado per day. c3Ado prevented dilation-induced Ras activation, as well as ERK1/2 and Akt phosphorylation in vivo. At day 21, VSMC proliferation (proliferating-cell nuclear antigen [PCNA]-positive cells), as well as the neointima/media ratio (0.7+/-0.2 versus 1.6+/-0.4; P<0.05) were significantly reduced, without any changes in the number of apoptotic cells. Our data indicate that c3Ado interferes with Ras methylation and function and thereby with mitogenic activation of ERK1/2 and Akt, preventing VSMC cell cycle entry and proliferation and neointima formation in vivo. Thus, therapeutic inhibition of S-adenosylhomocysteine hydrolase by c3Ado may represent a save and effective novel approach to prevent vascular proliferative disease.

Morelloflavone blocks injury-induced neointimal formation by inhibiting vascular smooth muscle cell migration

Background In-stent restenosis, or renarrowing within a coronary stent, is the most ominous complication of percutaneous coronary intervention, caused by vascular smooth muscle cell (VSMC) migration into and proliferation in the intima. Although drug-eluting stents reduce restenosis, they delay the tissue healing of the injured arteries. No promising alternative anti-restenosis treatments are currently on the horizon. Methods In endothelium-denudated mouse carotid arteries, oral morelloflavone—an active ingredient of the Thai medicinal plant Garcinia dulcis—significantly decreased the degree of neointimal hyperplasia, without affecting neointimal cell cycle progression or apoptosis as evaluated by Ki-67 and TUNEL staining, respectively. At the cellular level, morelloflavone robustly inhibited VSMC migration as shown by both scratch wound and invasion assays. In addition, morelloflavone prevented VSMCs from forming lamellipodia, a VSMC migration apparatus. Mechanistically, the inhibition by morelloflavone of VSMC migration was through its negative regulatory effects on several migration-related kinases, including FAK, Src, ERK, and RhoA. Consistently with the animal data, morelloflavone did not affect VSMC cell cycle progression or induce apoptosis. Results These data suggest that morelloflavone blocks injury-induced neointimal hyperplasia via the inhibition of VSMC migration, without inducing apoptosis or cell cycle arrest. General significance We propose morelloflavone to be a viable oral agent for the prevention of restenosis, without compromising effects on the integrity and healing of the injured arteries.

Epothilone B Inhibits Neointimal Formation after Rat Carotid Injury through the Regulation of Cell Cycle-Related Proteins

The abnormal proliferation of vascular smooth muscle cells (VSMCs) in arterial walls is an important pathogenetic factor of vascular disorders such as atherosclerosis and restenosis after angioplasty. Epothilone B, a novel potential antitumor compound, has a potent effect on preventing postangioplasty restenosis. Therefore, we established an in vivo rat carotid injury model and examined the potential effects of epothilone B on cardiovascular disease. We found that epothilone B potently prevented neointimal formation and in vivo VSMCs proliferation. In addition, we also showed that epothilone B significantly inhibited 5% fetal bovine serum (FBS)- and 50 ng/ml platelet-derived growth factor (PDGF)-BB-induced proliferation and cell cycle progression in rat aortic VSMCs. Furthermore, FBS and PDGF-BB induced the activations of extracellular signal-regulated kinases 1 and 2, Akt, phospholipase C gamma 1, and PDGF-receptor beta chain tyrosine kinase were not changed by epothilone B. However, epothilone B treatment caused a significant decrease in the level of cyclin-dependent protein kinase (CDK) 2, whereas it caused no change in the levels of cyclin E and down-regulated the phosphorylation of retinoblastoma, which plays a critical role in cell cycle regulation. Furthermore, levels of p27, an inhibitor of cyclin E/CDK2 complex, were significantly increased in VSMCs treated with epothilone B, indicating that this might be a major molecular mechanism for the inhibitory effects of epothilone B on the proliferation and cell cycle of VSMCs. These findings suggest that epothilone B can inhibit neointimal formation via the cell cycle arrest by the regulation of the cell cycle-related proteins in VSMCs.

The Effect of Alpha-lipoic Acid on the Cell Cycle Arrest and Apoptosis in Rat Vascular Smooth Muscle Cells

Background: The proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of atheroscelrosis and post-angioplasty restenosis. We previously showed that alpha-lipoic acid (ALA) inhibited neointimal hyperplasia and has potential anti-atherosclerosis effect in rat carotid artery balloon injured model. Here, we investigated whether alpha-lipoic acid inhibited proliferation of cells and induced apoptosis in rat vascular smooth muscle cells. Methods: VSMCs were treated with ALA under each condition, harvested and protein was extracted. Same amount of protein was loaded into SDS-PAGE and western blot analysis was performed with various cell cycle regulation protein. To examine ALA induce apoptosis in VSMCs, FACS and DNA fragmentation assay were performed. Antioxidant effect of ALA was determined by DCF-DA staining. Results: ALA induced VSMCs cell cycle arrest and induced p21, p27 and p53 proteins. Also ALA induced PTEN expression and AMPK phosphorylation. Increased AMPK phosphorylation reduced Erk-2 phosphorylation and finally arrested cell cycle promotion. The apoptotic effect was also shown by ALA treatment. Also we confirmed that ALA reduced ROS generation in VSMCs. Conclusion: The present data suggest that ALA has anti-proliferative effect and arrests cell proliferation. Therefore, ALA may provide new strategies for the prevention of neointimal hyperplasia after angioplasty. (J Kor Diabetes Assoc 31:200~207, 2007)

Potential role of Chinese medicinal herbs in the prevention of coronary artery restenosis

Potential role of Chinese medicinal herbs in the prevention of coronary artery restenosis

Double blockade of cell cycle progression by coptisine in vascular smooth muscle cells

Coptisine, an isoquinoline alkaloid isolated from rhizome of Coptis japonica, inhibits proliferation of vascular smooth muscle cells (VSMCs). The aim of this study was to evaluate the action of coptisine, along with berberine (a structurally similar isoquinoline alkaloid), on progression of the cell cycle in VSMCs. Coptisine displayed antiproliferative action against VSMCs by blocking the cell cycle at G 1 and G 2/M phases. The G 1 block was shown by inhibition of [ 3H]thymidine incorporation into VSMCs at coptisine concentrations higher than 15 μM. The mechanism underlying the G 1 arrest involved a decrease in cyclin D1 protein, although cyclin E, A, and B were not affected by coptisine treatment. The selective reduction in cyclin D1 protein was mainly attributable to accelerated proteolysis via proteasome-dependent pathway, since it was inhibited by a proteasome inhibitor, N-carbobenzoxy- l-leucinyl- l-leucinyl- l-norleucinal (MG132) and further the mRNA level of cyclin D1, protein synthesis, and mitogen-activated protein kinase (MAPK) activity remained unaltered. The mechanism underlying the G 2/M arrest involved partial inhibition of tubulin polymerization, which was apparent at coptisine concentration of 3 μM. Berberine arrested the cell cycle at G 1 phase via a mechanism identical with coptisine, but did not cause block at G 2/M phase. The results demonstrate that a small difference in the structure between isoquinoline alkaloids produces a big difference in activity, and that coptisine has a unique double action in arresting the cell cycle of VSMCs.

Regulation of Vascular Smooth Muscle Proliferation by Heparin: INHIBITION OF CYCLIN-DEPENDENT KINASE 2 ACTIVITY BY p27kip1

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 G(1) to S phase transition is imposed by p27(kip1)-mediated inhibition of cyclin-dependent kinase 2 activity. Further analysis of p27(kip1) mRNA levels showed that the increase in p27(kip1) protein levels in heparin-treated VSMC occurs at posttranscriptional levels. We present evidence that heparin causes stabilization of p27(kip1) protein during G(1) phase and thereby prevents activation of cyclin-dependent kinase 2.

Tanshinone: An inhibitor of proliferation of vascular smooth muscle cells

Tanshinone (Tan) is one of the active components of Radix Salvia miltiorrhiza (Lamiaceae), which is clinically used to treat cardiovascular diseases in China. The aim of this study was to estimate the effect of Tan on the proliferation of cultured vascular smooth muscle cells (VSMCs) induced by fatal bovine serum (FBS). It was shown that various concentrations of Tan inhibited the VSMCs proliferation in a dose-dependent manner. Tan significantly blocked VSMCs cell cycle in G 0/G 1 phase. The anti-proliferative effect of Tan was associated with the inhibition of the extracellular signal-regulated kinase1/2 (ERK1/2). On the other hand, the decrement of Tan on the cyclin D1 protein may be related to the high expression of p21 waf/cip1. The data suggest that the anti-proliferative effect of Tan on VSMCs proliferation was associated with ERK1/2 signaling pathway.

Inhibition of cell cycle progression and migration of vascular smooth muscle cells by prostaglandin D2 synthase: resistance in diabetic Goto-Kakizaki rats.

The regulation of vascular smooth muscle cell (VSMC) proliferation, migration, and apoptosis plays a clear role in the atherosclerotic process. Recently, we reported on the inhibition of the exaggerated growth phenotype of VSMCs isolated from hypertensive rats by lipocalin-type prostaglandin D2 synthase (L-PGDS). In the present study, we report the differential effects of L-PGDS on VSMC cell cycle progression, migration, and apoptosis in wild-type VSMCs vs. those from a type 2 diabetic model. In wild-type VSMCs, exogenously added L-PGDS delayed serum-induced cell cycle progression from the G1 to S phase, as determined by gene array analysis and the decreased protein expressions of cyclin-dependent kinase-2, p21(Cip1), and cyclin D1. Cyclin D3 protein expression was unaffected by L-PGDS, although its gene expression was stimulated by L-PGDS in wild-type cells. In addition, platelet-derived growth factor-induced VSMC migration was inhibited by L-PGDS in wild-type cells. Type 2 diabetic VSMCs, however, were resistant to the L-PGDS effects on cell cycle progression and migration. L-PGDS did suppress the hyperproliferation of diabetic cells, albeit through a different mechanism, presumably involving the 2.5-fold increase in apoptosis and the concomitant 10-fold increase of L-PGDS uptake we observed in these cells. We propose that in wild-type VSMCs, L-PGDS retards cell cycle progression and migration, precluding hyperplasia of the tunica media, and that diabetic cells appear resistant to the inhibitory effects of L-PGDS, which consequently may help explain the increased atherosclerosis observed in diabetes.