Postangioplasty Restenosis Research Articles

The Crataegus extract WS ® 1442 inhibits balloon catheter-induced intimal hyperplasia in the rat carotid artery by directly influencing PDGFR-β

Objective Effective systemic drugs against restenosis upon percutaneous transluminal coronary angioplasty (PTCA) are largely lacking. Polyphenols have been suggested to ameliorate post-angioplasty restenosis. Hawthorn ( Crataegus spp.) extracts, which are among the most frequently used herbal medicinal products against mild forms of congestive heart failure, contain polyphenols, but have not been investigated in this context. We aimed to assess the potential of the hawthorn extract WS ® 1442 to prevent balloon catheter-induced intimal hyperplasia and to elucidate the underlying mechanisms. Methods We analyzed the effects of WS ® 1442 on serum-induced vascular smooth muscle cell (VSMC) and endothelial cell (EC) growth and migration, growth factor-induced proliferation, growth factor receptor activity, and neointima formation in the rat carotid artery model. Results WS ® 1442 (100 μg/ml) decreased VSMC migration by 38% and proliferation by 44%, whereas EC migration and proliferation were unaltered. The extract inhibited VSMC DNA synthesis induced by platelet-derived growth factor (PDGF) (IC 50: 47 μg/ml), but not that of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Along this line, WS ® 1442 blocked recombinant human PDGF receptor (PDGFR)-β kinase activity (IC 50: 1.4 μg/ml) and decreased PDGFR-β activation and extracellular signal-regulated kinase (ERK) activation in VSMCs. In rats, orally administered WS ® 1442 significantly reduced neointima formation after balloon catheter dilatation of the carotid artery. Conclusion WS ® 1442 inhibits migration and proliferation of VSMCs, but not of ECs, and reduces balloon catheter-evoked neointima formation probably through inhibition of PDGFR-β. Thus, the present study suggests a novel adjunct pharmacological strategy to prevent angioplasty-related restenosis.

A20 inhibits post-angioplasty restenosis by blocking macrophage trafficking and decreasing adventitial neovascularization

Objective Neointimal hyperplasia is an inflammatory and proliferative process that occurs as a result of injury to the vessel wall. We have shown that the homeostatic protein A20 prevents neointimal hyperplasia by affecting endothelial cell (EC) and smooth muscle cell (SMC) responses to injury. In this work, we questioned whether A20 impacts other pathogenic effectors of neointimal hyperplasia including homing of monocyte/macrophages and EC/SMC precursors to the site of vascular injury, vascular endothelial growth factor (VEGF) secretion, and adventitial neovascularization. Methods and results Carotid balloon angioplasty was performed on rat recipients of a bone marrow transplant from green fluorescent rats. Adenoviral delivery of A20 prevented neointimal hyperplasia and decreased macrophage infiltration. This was associated with decreased ICAM-1 and MCP-1 expression in vitro. Additionally, A20 reduced neovascularization in the adventitia of balloon injured carotid arteries, which correlated with fewer VEGF positive cells. Conclusions A20 downregulates adhesion markers, chemokine production, and adventitial angiogenesis, all of which are required for macrophage trafficking to sites of vascular injury. This, in turn, diminishes the inflammatory milieu to prevent neointimal hyperplasia.

AS-128: Pattern of Percutaneous Transluminal Coronary Angioplasty and Stenting in NICVD in 2008

Percutaneous transluminal coronary angioplasty (PTCA) is an established mode of treatment for significant coronary artery disease (CAD). Postangioplasty restenosis is a major limitation of coronary interventions, and it has been significantly reduced with the widespread application of coronary stents. Stent length, diameter, and type of stent are 3 important recognized predictors of recurrent restenosis.

PPARγ gene C161T substitution alters lipid profile in Chinese patients with coronary artery disease and type 2 diabetes mellitus

BackgroundPeroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated transcription factor, which regulates gene expression of the key proteins involved in lipid metabolism, vascular inflammation, and proliferation. PPARγ may contribute to attenuating atherogenesis and postangioplasty restenosis. PPARγ C161→T substitution is associated with a reduced risk of coronary artery disease (CAD). Whether or not the gene substitution alters the risk of CAD in type 2 diabetes mellitus (T2DM) patients remains unclear.MethodsA total of 556 unrelated subjects from a Chinese Han population, including 89 healthy subjects, 78 CAD patients, 86 T2DM patients, and 303 CAD combined with T2DM patients, were recruited to enroll in this study. PPARγC161→T gene polymorphism was determined by polymerase chain reaction and restriction fragment length polymorphisms. Plasma levels of lipoproteins, apolipoproteins, glucose, and insulin were measured by ELISA or radioimmunoassay (RIA). The coronary artery lesions were evaluated by coronary angiography.ResultsThe frequency of the 161T allele in CAD, T2DM, and CAD combined with T2DM patients was similar to that observed in the healthy control group. However, in CAD combined with T2DM patients, the group with angiographically documented moderate stenoses had a higher frequency of the 161T allele in comparison to the group with severe stenoses (P < 0.05). Moreover, in CAD with T2DM patients, the triglyceride levels and apoB in CC homozygote carriers were significantly higher than those in "T" allele carriers.ConclusionsPPARγC161→T genotypes weren't significantly associated with the risk of CAD, but were markedly correlated with severity of disease vessels in patients with CAD and T2DM. Furthermore, PPARγC161→T substitution was associated with an altered adipose, but not glucose metabolism. These results indicate that the PPARγ C161→T polymorphism may reduce the risk of severe atherogenesis by modulation of adipose metabolism, especially triglycerides and apoB, in Chinese patients with CAD and T2DM.

S-phase kinase-associated protein-2 (Skp2) promotes vascular smooth muscle cell proliferation and neointima formation in vivo

ObjectiveVascular smooth muscle cell (VSMC) proliferation plays an important role in the development of postangioplasty or in-stent restenosis, venous graft failure, and atherosclerosis. Our previous work has demonstrated S-phase kinase-associated protein-2 (Skp2), an F-box subunit of SCFSkp2 ubiquitin ligase, as an important mediator and common final pathway for growth factors, extracellular matrices, and cyclic-nucleotides to regulate VSMC proliferation in vitro. However, whether alteration of Skp2 function also regulates VSMC proliferation in vivo and neointimal thickening postvascular injury remains unclear. We investigated the effect of Skp2 on VSMC proliferation and neointimal formation in vivo.Methods and ResultsFirstly, we demonstrated that Skp2-null mice developed significantly smaller neointimal areas than wild-type mice after carotid ligation. Secondly, to further identify a local rather than a systemic effect of Skp2 alteration, we demonstrated that adenovirus-mediated expression of dominant-negative Skp2 in the balloon-injured rat carotid artery significantly increased medial p27Kip1 levels, inhibited VSMC proliferation, and the subsequent neointimal thickening. Lastly, to determine if Skp2 alone is sufficient to drive VSMC proliferation and lesion development in vivo, we demonstrated that adenovirus-delivery of wild-type Skp2 to the minimally-injured rat carotids is sufficient to downregulate p27Kip1 protein levels, enhanced medial VSMC proliferation, and the neointimal thickening.ConclusionThis data provides, we believe for the first time, a more comprehensive understanding of Skp2 in the regulation of VSMC proliferation and neointimal formation and suggests that Skp2 is a promising target in the treatment of vasculoproliferative diseases.

MicroRNAs and Ischemic Heart Disease: Towards a Better Comprehension of Pathogenesis, New Diagnostic Tools and New Therapeutic Targets

MicroRNAs are key, recently discovered, regulators of gene expression. They are involved in many physiological cellular pathways so it is not surprising that an altered microRNA expression pattern can be involved in the pathogenesis of many disease states. The possibility to manipulate microRNAs to obtain a therapeutical effect is very attractive since they represent specific targets in a particular cellular pathway and because it is quite easy to synthesize short oligonucleotides with the ability to interfere with microRNA mechanism of action. The main problem for microRNA-based therapy is represented by delivery. In the last two years many studies have underlined the involvement of microRNAs in many aspects of ischemic heart disease, the leading cause of morbidity and mortality in the Western World. MiR-29 is involved in fibrotic reaction after myocardial infarction while miR-21 may exert a fundamental role in post-angioplasty restenosis. MiR-208 is involved in the shift toward a fetal gene expression pattern in contractile proteins in heart failure. MiR-1 influences susceptibility to cardiac arrhythmias after myocardial infarction. This review will focus on microRNAs involvement in multiple aspects of ischemic heart disease and on their promising novel therapeutic applications including some recent patents.

Tormentic acid reduces vascular smooth muscle cell proliferation and survival

The triterpene tormentic acid (TA) has been reported to exhibit anticancer, anti-inflammatory and anti-atherogenic properties, and minimal toxicity has been detected in in vivo. Vascular smooth muscle cell (VSMC) proliferation and apoptosis resistance are hallmarks of vasculoproliferative diseases, such as post-angioplasty restenosis. The present study was designed to assess the effects of TA on the phenotype of cultured VSMC. The exposure of VSMC to TA (30 μM) significantly increased apoptosis of serum-deprived A7r5 cells, whereas cell survival in the presence of 10% fetal bovine serum was less affected by the drug. On the other hand, even in the presence of serum, A7r5 cell proliferation was significantly inhibited by TA, an effect that persisted for at least 8 days of daily administration of TA. As preservation of endothelial integrity is critical to normal vascular function, we also evaluated the effects of TA on human umbilical cord endothelial cells (HUVEC). Interestingly, TA did not produce significant changes in the levels of apoptosis and proliferation of HUVEC. Our data indicate that TA is a VSMC apoptosis inducer and proliferation inhibitor. The anti-growth action in VSMC in the presence of serum, and the absence of significant effects in endothelial cells suggest that TA may control VSMC abnormal proliferation and cell death resistance without affecting the normal vasculature. We conclude that TA should be investigated further as a potential tool for the prevention and treatment of proliferative vascular diseases, particularly in the setting of post-angioplasty restenosis.

Vascular smooth muscle cell expression of ectonucleotidase CD39 (ENTPD1) is required for neointimal formation in mice

Vascular smooth muscle cell (VSMC) migration and proliferation are critical steps in the pathogenesis of atherosclerosis, post-angioplasty restenosis, neointimal hyperplasia, and chronic allograft rejection. Extracellular nucleotides are known to influence both migration and proliferation of VSMC. Although it is well established that vascular endothelial Cd39/ENTPD1 regulates blood nucleotide concentrations, whether Cd39 associated with VSMC also impacts vascular wall pathology has not been investigated. The objective of this paper is to determine levels of expression of Cd39 on VSMC and functional consequences of gene deletion in vitro and in vivo. Cd39 is the major ectonucleotidase in VSMC, as shown by substantive decreases in ecto-ATPase and -ADPase activity in Cd39-null cells compared to wild type. Significant decreases in neointimal lesion formation are observed in Cd39-null mice at 21 days post arterial balloon injury. Stimulated Cd39-null VSMC have pronounced proliferative responses in vitro. However, using Transwell systems, we show that Cd39-null VSMC fail to migrate in response to ATP, UTP, and PDGF. Cd39 is the dominant ectonucleotidase expressed by VSMC. Deletion of Cd39 in mice results in decreased neointimal formation after vascular injury and is associated with impaired VSMC migration responses in vitro.

E013 Resveratrol inhibits the mTOR mitogenic signaling evoked by oxidized LDL in smooth muscle cells

Smooth muscle cell (SMC) proliferation is a major feature in atherosclerosis, since it contributes to the formation of the fibrous cap, thus to plaque stability, but also to arterial stenosis and post-angioplasty restenosis. Among the various mitogenic signaling pathways involved in SMC proliferation, the mTOR pathway regulates both the cell cycle and cell growth. Resveratrol, a polyphenolic compound from grapes and red wine, has potential anti-atherogenic and anti-cancer properties. This work was designed to investigate the activation of the mTOR pathway by the proatherogenic oxidized LDL (oxLDL) in SMC, and the potential inhibitory effect of resveratrol. mTOR and its downstream target p70S6 kinase are phosphorylated and activated by mitogenic concentrations of oxLDL (50mug/ml), and are involved in SMC proliferation, as assessed by the inhibitory effect of the mTOR inhibitor rapamycin. The activation of mTOR signaling by oxLDL, requires the upstream activation of PI3K and Akt, as assessed by the inhibitory effect of the PI3K inhibitor Ly294002 on mTOR activation and DNA synthesis. Resveratrol blocked the oxLDL-induced phosphorylation and activation of the PI3K/Akt/mTOR/p70S6K pathway and strongly inhibited both the DNA synthesis and proliferation of SMC. This activity is independent of the anti-oxidant effect and of AMPK activation by resveratrol. These data indicate that the mTOR pathway is activated by oxLDL via PI3K/PDK1/Akt, and is required for SMC proliferation. Resveratrol blocks specifically this pathway, thereby inhibiting oxLDL-induced SMC proliferation. These data highlight a new property for resveratrol that could contribute to the general anti-atherogenic properties of this polyphenol.

Endogenous testosterone attenuates neointima formation after moderate coronary balloon injury in male swine.

Previous studies from our laboratory have demonstrated that testosterone increases coronary smooth muscle protein kinase C delta (PKC delta) both in vivo and in vitro and inhibits coronary smooth muscle proliferation by inducing G(0)/G(1) cell cycle arrest in a PKC delta-dependent manner. The purpose of the present study was to determine whether endogenous testosterone limits coronary neointima (NI) formation in a porcine model of post-angioplasty restenosis. Sexually mature, male Yucatan miniature swine were either left intact (IM), castrated (CM), or castrated with testosterone replacement (CMT; Androgel, 10 mg/day). Angioplasty was performed in both the left anterior descending and left circumflex coronary arteries with balloon catheter overinflation to induce either moderate (1.25-1.3 x diameter; 3 x 30 s) or severe (1.4x diameter; 3 x 30 s) injury, and animals were allowed to recover for either 10 or 28 days. Injured coronary sections were dissected, fixed, stained (Verheoff-Van Gieson, Ki67, PKC delta, p27), and analysed. Vessels without internal elastic laminal rupture were excluded. Following moderate injury, intimal area, intima-to-media ratio (I/M), and I/M normalized to rupture index (RI) were increased in CM compared with IM and CMT. RI, medial area, and intimal/medial thickness (IMT) were not different between groups. NI formation was inversely related to serum testosterone concentration. Conversely, following severe injury, there were no significant differences between the groups. Testosterone inhibited proliferation and stimulated PKC delta and p27(kip1) expression during NI formation (10 days post-injury). These findings demonstrate that endogenous testosterone limits coronary NI formation in male swine and provides support for a protective role for testosterone in coronary vasculoproliferative diseases, such as restenosis and atherosclerosis.

ADAMTS-7 Mediates Vascular Smooth Muscle Cell Migration and Neointima Formation in Balloon-Injured Rat Arteries

The migration of vascular smooth muscle cells (VSMCs) plays an essential role during the development of atherosclerosis and restenosis. Extensive studies have implicated the importance of extracellular matrix (ECM)-degrading proteinases in VSMC migration. A recently described family of proteinases, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTs), is capable of degrading vascular ECM proteins. Here, we sought to determine whether ADAMTS-7 is involved in VSMC migration and neointima formation in response to vascular injury. ADAMTS-7 protein accumulated preferentially in neointima of the carotid artery wall after balloon injury. In primary VSMCs, ADAMTS-7 level was enhanced by the proinflammatory cytokine tumor necrosis factor alpha and growth factor platelet-derived growth factor-BB. ADAMTS-7 overexpression greatly accelerated and small interfering RNA knockdown markedly retarded VSMC migration/invasion in vitro. In addition, luminal delivery of ADAMTS-7 adenovirus to carotid arteries exacerbated intimal thickening nearly sixfold 7 days after injury. Conversely, perivascular administration of ADAMTS-7 small interfering RNA but not scramble small interfering RNA to injured arteries attenuated intimal thickening by 50% at 14 days after injury. Furthermore, ADAMTS-7 mediated degradation of the vascular ECM cartilage oligomeric matrix protein (COMP) in injured vessels. Replenishing COMP circumvented the promigratory effect of ADAMTS-7 on VSMCs. Enforced expression of COMP significantly suppressed VSMC migration and neointima formation postinjury, which indicates that ADAMTS-7 facilitated intimal hyperplasia through degradation of inhibitory matrix protein COMP. ADAMTS-7 may therefore serve as a novel therapeutic target for atherosclerosis and postangioplasty restenosis.

Resveratrol inhibits the mTOR mitogenic signaling evoked by oxidized LDL in smooth muscle cells

ObjectivesSmooth muscle cell (SMC) proliferation is a major feature in atherosclerosis, since it contributes to the formation of the fibrous cap, thus to plaque stability, but also to arterial stenosis and post-angioplasty restenosis. Among the various mitogenic signaling pathways involved in SMC proliferation, the mTOR pathway regulates both the cell cycle and cell growth. Resveratrol, a polyphenolic compound from grapes and red wine, has potential anti-atherogenic and anti-cancer properties. This work was designed to investigate the activation of the mTOR pathway by the proatherogenic oxidized LDL (oxLDL) in SMC, and the potential inhibitory effect of resveratrol. ResultsmTOR and its downstream target p70S6 kinase are phosphorylated and activated by mitogenic concentrations of oxLDL (50μg/ml), and are involved in SMC proliferation, as assessed by the inhibitory effect of the mTOR inhibitor rapamycin. The activation of mTOR signaling by oxLDL, requires the upstream activation of PI3K and Akt, as assessed by the inhibitory effect of the PI3K inhibitor Ly294002 on mTOR activation and DNA synthesis. Resveratrol blocked the oxLDL-induced phosphorylation and activation of the PI3K/Akt/mTOR/p70S6K pathway and strongly inhibited both the DNA synthesis and proliferation of SMC. This activity is independent of the anti-oxidant effect and of AMPK activation by resveratrol. ConclusionThese data indicate that the mTOR pathway is activated by oxLDL via PI3K/PDK1/Akt, and is required for SMC proliferation. Resveratrol blocks specifically this pathway, thereby inhibiting oxLDL-induced SMC proliferation. These data highlight a new property for resveratrol that could contribute to the general anti-atherogenic properties of this polyphenol.

Multimodality Cardiovascular Molecular Imaging, Part II

In Part I of this consensus article, the imaging methodology, evolving imaging technology, and development of novel targeted molecular probes relevant to the developing field of cardiovascular molecular imaging were reviewed. Novel reporter gene and reporter probe imaging approaches for tracking of cardiac transgene expression were also discussed and have important future implications for evaluation of gene- and cell-based therapies for the failing heart. The current role of metabolic and receptor imaging was also briefly reviewed, as these represent the beginning of our clinical application of molecular imaging within the cardiovascular system. Part II will summarize the available targeted imaging probes as well as specific future applications of molecular imaging for identification and evaluation of critical pathophysiological processes of the cardiovascular system.

Chemokines in Atherosclerosis, Thrombosis, and Vascular Biology

Series Editor: Christian Weber ATVB In Focus Chemokines in Atherosclerosis, Thrombosis, and Vascular Biology The trafficking of leukocytes is essential for many aspects of vascular biology and pathology in health and disease. Sequential steps of leukocyte recruitment to the vascular wall, eg, adhesion and emigration, are governed by chemokines, a family of small chemotactic peptides, which serve both inflammatory and homeostatic functions. Beyond controlling …

Utility of Vascular Endothelial Specific Peptides for Enhancement of Adeno-Associated Virus-Mediated Gene Transfer

Vascular endothelial cells (EC) have been targeted for the treatment of pathological conditions such as atherosclerosis, hypercholesterolemia, post-angioplasty restenosis and hypertension. Non-pathogenic adeno-associated virus (AAV) has been shown as a good gene delivery tool in a variety of cell lines as well as in animal models. However, AAV has been reported to induce less endothelial cell transduction. AAV vector alone transduced HUVEC much lower than other cell lines including Hela, PAC1, and C2C12. Preincubation of AAV vector with EC membrane specific peptides markedly increased AAV transduction of HUVEC. On the contrary, those peptides did not affect AAV expression in other cell types. These EC-specific peptides may be a strategy for enhancement of AAV mediated-gene expression.

A model of cerebrovascular injury in rats

Although the pathophysiology of post-angioplasty restenosis has been extensively studied in extracranial arteries using transluminal vascular injury model in rodents, it is still not well known in the intracranial arteries, which have quite different structures from extracranial arteries. Here, we examined whether 1-min placement of modified intraluminal suture could induce an injury in the internal carotid artery (ICA) in rats and observed temporal profile of histological change after the injury. HE staining showed that the injured intracranial ICA was dilated, while the media was markedly thinned at 1 day after injury. The internal elastic lamina was not observed, and the media contained few cells. At 1 week after injury, a thin layer of neointimal hyperplasia was observed on the luminal side of the internal elastic lamina. Neointimal hyperplasia developed until at least 4 weeks after injury. Morphometric analysis demonstrated that the healing process of the injury was related to arterial remodeling. Immunohistochemical staining for α-smooth muscle actin and electron microscopic analysis showed that the neointima was composed of smooth muscle cells. Re-endothelialization was observed from 1 to 4 weeks after injury by immunohistochemical staining for von Willebrand’s factor and electron microscopic analysis. Vascular endothelial growth factor was expressed in neointima on days 7 and 14. Interestingly, superoxide anion was not increased in injured arteries on day 3, when the infiltration of macrophages was intensive, but increased on day 7, when infiltrating macrophages almost disappeared. These findings might shed new light on pathophysiology of post-angioplasty restenosis in intracranial arteries.

Ginsenoside Rb1 Attenuates Homocysteine-Augmented Guidewire Injury-Induced Intimal Hyperplasia in Mice

Intimal hyperplasia (IH) is the primary cause for post-angioplasty restenosis. The purpose of this study is to investigate the effects of homocysteine (Hcy) and ginsenoside Rb1 (Rb1) on IH using a guidewire injury animal model. In 12-wk-old C57BL/6J mice, the left common carotid artery (CCA) was denudated with a guidewire and the right CCA was used as the uninjured control. They were treated with saline (NS), Hcy, Rb1, or Hcy + Rb1 for 4 wk prior to sacrifice. Animals were sacrificed at 4, 6, or 8 wk. Both CCAs were harvested and intimal-medium thickness (IMT) ratios were calculated. Local macrophage distribution was also studied. Histology analyses demonstrated consistent internal elastic lamina disruption and focal IH in the injured CCA segments. The degree of IH correlated to the lengths of time following injury. Hcy treated group had significant increase in IMT compared with the NS group (P < 0.05), while Rb1 group was similar to the NS group. In addition, Hcy + Rb1 group showed significant improvement in IMT compared with Hcy group (P < 0.01). Furthermore, Hcy significantly increased local macrophage content as compared with either lesion alone or Rb1 treated animals. Our study showed that Hcy increased the degree of IH and macrophage content in the injured CCA and that Rb1 attenuated these adverse effects. These changes might be mediated through antioxidative effects of Rb1. Our data suggests a potential clinical application of ginseng in controlling Hcy-related vascular injuries.

Lentivirus-mediated RNA Interference Targeting STAT4 Inhibits the Proliferation of Vascular Smooth Muscle Cells

STAT4 is a key transcription factor regulating Th1 development. However, its presence and role in vascular smooth muscle cells (VSMCs) has not been well studied. In the current study, we have utilized lentivirus-mediated shRNA for functional gene knockdown in human umbilical artery smooth muscle cells in order to access the potential role of STAT4 in VSMC growth. Cells were isolated from the umbilical arteries of newborns and used at passage 3-5. Recombinant lentivirus producing STAT4 siRNA was prepared. Protein and mRNA expression of STAT4 and relevant genes were examined by Western blot, ELISA, and quantitative RT-PCR analysis, and the effects of the lentivirus on cell growth and apoptosis were determined using MTT assay and flow cytometry, respectively. Lentivirus-mediated RNAi effectively reduced endogenous STAT4 expression and downregulation of STAT4 in VSMCs and significantly reduced VSMC growth rate in vitro. We found that STAT4 knockdown led to impaired pSTAT4 protein expression. SOCS-3 as well as MCP-1 production were also markedly decreased, consistent with the suppression of STAT4 expression. Results from our study suggest that STAT4 may play a role in VSMC proliferation, and thus is a novel therapeutic target for neointima formation following vascular injury, e.g., post-angioplasty restenosis.

Pharmacologic Management of Isolated Low High-Density Lipoprotein Syndrome

High-density lipoprotein (HDL) cholesterol is a heterogeneous group of lipoproteins exhibiting a variety of properties like prostacyclin production stimulation, decrease in platelet aggregation, endothelial cell apoptosis inhibition, and low-density lipoprotein oxidation blockade. Epidemiologic studies have shown an inverse relation between HDL cholesterol levels and cardiovascular risk. Low HDL cholesterol is associated with increased risk for myocardial infarction, stroke, sudden death, peripheral artery disease, and postangioplasty restenosis. In contrast, high HDL levels are associated with longevity and protection against atherosclerotic disease development. Given the evolving epidemic of obesity, diabetes mellitus, and metabolic syndrome, the prevalence of low HDL will continue to rise. In the United States, low HDL is present in 35% of men, 15% of women, and approximately 63% of patients with coronary artery disease. Data extracted from the Framingham study highlight that 1-mg increase in HDL levels decreases by 2% to 3% the risk of cardiovascular disease. There is no doubt regarding clinical importance about isolated low HDL, but relatively few clinicians consider a direct therapeutic intervention of this dyslipidemia. In this sense, lifestyle measures should be the first-line strategy to manage low HDL levels. On the other hand, pharmacologic options include niacin, fibrates, and statins. Fibrates appear to reduce risk preferentially in patients with low HDL with metabolic syndrome, whereas statins reduce risk across all levels of HDL. Torcetrapib, a cholesteryl esters transfer protein inhibitor, represented a hope to raise this lipoprotein; however, all clinical trials on this drug had ceased after ILLUMINATE, RADIANCE and ERASE trials had recorded an increase in mortality, rates of myocardial infarction, angina, and heart failure. In the near future, drugs as beta-glucans, Apo-A1 mimetic peptides, and ACAT inhibitors, are the new promises to treat this condition.

RNA interference targeting ORC1 gene suppresses the proliferation of vascular smooth muscle cells in rats

Background The proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of vascular diseases such as atherosclerosis and postangioplasty restenosis. The largest subunit of the origin recognition complex (ORC), ORC1, plays a critical role during the initiation of DNA replication in eukaryotes. However, the involvement of ORC1 in the initiation of DNA replication in VSMCs has not been studied yet. Objective The aim of this study was to silence ORC1 gene selectively by using RNA interference and analyze the effects of ORC1 gene on the proliferation and apoptosis of rat VSMCs. Methods Freshly isolated rat VSMCs were transfected with siRNA targeting ORC1 gene capsulated in liposome. ORC1 protein expression was determined by Western blotting and ORC1 mRNA level by RT-PCR. DNA synthesis was analyzed by 3H thymidine ( 3H-TdR) incorporation and cell proliferative activity and cell cycle distribution by flow cytometry. Two apoptosis-related proteins, Bax and Bcl-2, were examined immunohistochemically. Results Down-regulation of ORC1 mRNA and protein expression was observed in rat VSMCs at 24 h after transfection with the three pairs of siRNA targeting ORC1 gene and this reduction persisted at least 7 days post-transfection. Down-regulation of ORC1 mRNA (60%) and protein (80%) expression was observed at 72 h post-transfection in the cells transfected with B-ORC1 siRNA. A significant decrease in 3H thymidine incorporation was observed in rat VSMCs with ORC1 gene silencing after serum challenge, but not in the non-silenced control. A significant increase in the proliferation index and a significant decrease in the percentage of cells at G 0/G 1 phase after serum challenge were observed in the non-silenced control, but not in ORC1 gene silenced cells. A significant increase in the ratio of Bcl-2/Bax was observed after serum challenge in the non-silenced control, but only a slight increase was found in the ORC1 gene silenced cells. ORC1 gene silencing disappeared 7 days after transfection. Continuous serum challenge stimulated VSMCs to synchronously reenter the cell cycle as evidenced by increases in [ 3H] thymidine incorporation, the proliferation index, and the ratio of Bcl-2/Bax, as non-silenced cells were induced to resume cell cycle progression by the addition of 15% fetal bovine serum to the culture medium. Conclusion ORC1 gene silencing causes rat VSMCs to enter a reversible G 0 quiescent, growth arrested state; thus, ORC1 gene may be an important new target for suppressing VSMCs proliferation.