The Effect of Six Different Statins on the Proliferation, Migration, and Invasion of Human Smooth Muscle Cells

doi:10.1016/j.jss.2005.05.016

Journal of Surgical Research

Volume 129, Issue 1, November 2005, Pages 52-56

https://doi.org/10.1016/j.jss.2005.05.016 Get rights and content

Intimal hyperplasia (IH) can occur after any vascular injury and results from smooth muscle cells (SMC) proliferation, migration, and invasion into the subintimal space. The purpose of this study was to investigate the effect of six different statins on the proliferation, migration, and invasion of human venous SMC. The statins were all used at their Cmax concentrations. SMCs were used to construct growth curves in the presence of 10% fetal calf serum or 10% fetal calf serum supplemented with the six statins. Migration and invasion experiments were performed using modified Boyden chambers. The invasion experiments were performed using Matrigel coated plates. We found that all of the statins significantly inhibited SMC proliferation compared to the platelet-derived growth factor control (ranging from fluvastatin 33% of control to pravastatin 72% of control, P = 0.03). SMC migration through uncoated polycarbonate membranes in presence of the six statins was significantly reduced (ranging from lovastatin 43% to pravastatin 57% of control, P = 0.006). All six statins also significantly reduced SMC invasion (ranging from fluvastatin 65% to simvastatin 87% of control, P = 0.002). We conclude that the inhibitory effect of statins on SMC proliferation, migration, and invasion is a class, rather than drug specific effect.

Introduction

The treatment of peripheral vascular disease, although greatly improved over recent decades by surgical and endovascular procedures, remains limited by intimal hyperplasia (IH). IH leads to focal stenoses that threaten the patency of up to 35% of lower limb vein bypass grafts [1]. IH is thought to result from abnormal activation of smooth muscles cells after vessel manipulation. Normally, smooth muscle cells (SMCs) reside in the vessel wall media in a quiescent differentiated and contractile state. However, SMCs seem to be easily activated after injury and this activation of SMCs is associated with a shift from a contractile to a synthetic phenotype, itself leading to SMC proliferation, migration, and synthesis of extracellular matrix [2].

Although several pharmacological approaches have been shown to reduce IH in animal models, these results have not been reproduced in human clinical trials. Statins are a class of drugs used to reduce plasma cholesterol levels in patients with hypercholesterolemia [3]. Large clinical trials have shown that the beneficial effects of statins in patients with atherosclerotic disease are attributable to the inhibition of HMG-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis [4, 5, 6]. Laufs and colleagues [7] have shown that some of the effects of HMG-CoA reductase inhibitors on SMC are independent of their effects on plasma cholesterol levels, probably through inhibition of isoprenoid synthesis that is a precursor of prenylated proteins. Corsini and colleagues [8] have shown that statins can decrease rat aortic SMC migration and proliferation by interfering with signaling pathways requiring prenylated proteins. The effect of simvastastin on IH development in human saphenous vein organ culture has previously been reported by Porter et al. [9] who found that simvastatin reduced IH and that simvastatin inhibited isolated human SMC proliferation and migration. The purpose of the present study was to investigate whether the beneficial effects of HMG-CoA reductase inhibitors on human SMC proliferation is a class effect or is confined to specific statins.

Section snippets

SMC Isolation and Culture

Human saphenous SMCs were prepared using an explant technique. Vein segments were chopped into small fragments (approximately 1 mm³) within a 2-ml volume of RPMI medium supplemented with 10% fetal calf serum (FCS). The vein fragments together with the medium were transferred to 25 cm² tissue culture flasks and maintained at 37°C in a humidified incubator in 5% CO₂ in air. After 1 to 2 weeks SMCs were seen to migrate out from the explants, and at 4 to 5 weeks cells were passaged into 80 cm²

Effects of Statins on SMC Proliferation

The results of the proliferation studies are shown in Fig. 1. To calculate the effect of the statins on proliferation the area under the curve (AUC) for each experiment was calculated and the AUCs for each statin expressed as a percentage of the 10% FCS control. The median AUC for atorvastatin was 61% of control, cerivastatin 58%, fluvastatin 33%, lovastatin 60%, pravastatin 72%, and simvastatin 57%. These percentage AUCs were all significantly lower than control (P = 0.03). The percent

Discussion

Our results show that all six statins significantly reduce human vascular SMC proliferation, migration, and invasion. Although all of the statins were equally effective at reducing migration and invasion, fluvastatin appeared particularly effective at reducing SMC proliferation. We have not been able to find any other comparisons of the effects of different statins on human venous SMC proliferation. However, Nègre-Aminou et al. [10] did compare the effect of the same six statins on human

Acknowledgments

We are grateful to Dawn Croston for technical assistance and expertise.

References (20)

M.G. Davies et al.
Pathophysiology of vein graft failurea review
Eur. J. Vasc. Endovasc. Surg.
(1995)
J.A. Herd et al.
Effects of fluvastatin on coronary atherosclerosis in patients with mild to moderate cholesterol elevations. (Lipoprotein and Coronary Arteriosclerosis Study [LCAS])
Am. J. Cardiol.
(1997)
G. Riegger et al.
The effect of fluvastatin on cardiac events in patients with symptomatic coronary artery disease during one year of treatment
Atherosclerosis
(1999)
U. Laufs et al.
3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate smooth muscle cell proliferation by preventing rho GTPase-induced down-regulation of p27 (Kip1)
J. Biol. Chem.
(1999)
A. Corsini et al.
New insights into the pharmacodynamic and pharmacokinetic properties of statins
Pharmacol. Ther.
(1999)
K.E. Porter et al.
Simvastatin inhibits human saphenous vein neointima formation via inhibition of smooth muscle cell proliferation and migration
J. Vasc. Surg.
(2002)
P. Nègre-Aminou et al.
Inhibition of proliferation of human smooth muscle cells by various HMG-CoA reductase inhibitorsComparison with other human cell types
Biochim. Biophys. Acta
(1997)
H. Kouchi et al.
inhibitor of ras farnesyltransferase, inhibits proliferation and migration of rat vascular smooth muscle cells
Biochem. Biophys. Res. Comm.
(1999)
S. Ai et al.
Rho-Rho kinase is involved in smooth muscle cell migration through myosin light chain phosphorylation-dependent and independent pathways
Atherosclerosis
(2001)
P. Nègre-Aminou et al.
Differential effect of simvastatin on various signal transduction intermediates in cultured human smooth muscle cells
Biochem. Pharmacol.
(2001)

There are more references available in the full text version of this article.

Cited by (46)

The anti-tubercular activity of simvastatin is mediated by cholesterol-driven autophagy via the AMPK-mTORC1-TFEB axis
2020, Journal of Lipid Research
Citation Excerpt :
Our conclusions are strongly supported by the findings that the anti-mycobacterial activity of statins is phenocopied only by cholesterol-branch but not by prenylation-branch inhibitors and the biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis are reversed by adding exogenous cholesterol to the cells. We propose that the different conclusions reached by the previous authors most likely depend on their employment of murine cells and very high simvastatin doses (500-fold higher than the dose we used), which cannot distinguish between anti-cholesterol and anti-prenylation effects of the statin, are toxic for human (THP1) cells, and far exceed the plasma levels attained during therapeutic administration of simvastatin (72). Our work shows that the cholesterol-lowering activity of statins can affect autophagy in multiple interconnected ways.
The rise of drug-resistant tuberculosis poses a major risk to public health. Statins, which inhibit both cholesterol biosynthesis and protein prenylation branches of the mevalonate pathway, increase anti-tubercular antibiotic efficacy in animal models. However, the underlying molecular mechanisms are unknown. In this study, we used an in vitro macrophage infection model to investigate simvastatin's anti-tubercular activity by systematically inhibiting each branch of the mevalonate pathway and evaluating the effects of the branch-specific inhibitors on mycobacterial growth. The anti-tubercular activity of simvastatin used at clinically relevant doses specifically targeted the cholesterol biosynthetic branch rather than the prenylation branches of the mevalonate pathway. Using Western blot analysis and AMP/ATP measurements, we found that simvastatin treatment blocked activation of mechanistic target of rapamycin complex 1 (mTORC1), activated AMP-activated protein kinase (AMPK) through increased intracellular AMP:ATP ratios, and favored nuclear translocation of transcription factor EB (TFEB). These mechanisms all induce autophagy, which is anti-mycobacterial. The biological effects of simvastatin on the AMPK-mTORC1-TFEB-autophagy axis were reversed by adding exogenous cholesterol to the cells. Our data demonstrate that the anti-tubercular activity of simvastatin requires inhibiting cholesterol biosynthesis, reveal novel links between cholesterol homeostasis, the AMPK-mTORC1-TFEB axis, and Mycobacterium tuberculosis infection control, and uncover new anti-tubercular therapy targets.
Harnessing the pleiotropic effects of atorvastatin-fenofibrate combination for cardiovascular stents
2018, Materials Science and Engineering C
Atorvastatin and fenofibrate have been conventionally employed as lipid-lowering agents. They also exhibit beneficial effects in the treatment of endothelial dysfunction, oxidative stress and vascular inflammation due to their pleiotropic effects that include vasodilatory and anti-inflammatory effects. These pleiotropic effects may serve to overcome the drawbacks of late stent thrombosis and delayed endothelialization that plague conventional drug eluting stents. However, the combination has not been explored yet as therapeutic coatings in drug eluting stents. The present study aims to investigate the potential of atorvastatin-fenofibrate combination loaded in a biodegradable poly(l-lactide-co-caprolactone) polymer film to inhibit thrombus formation and macrophage activation apart from exploring their effect on the proliferation of smooth muscle cells and endothelial cells. The dual drug-loaded polymer films were characterized by spectroscopy and calorimetry. In vitro studies revealed that the combination effectively retarded the proliferation of only smooth muscle cells but not the endothelial cells which augers well for stent applications where rapid re-endothelialization is preferred. Further, the dual drug-loaded films exhibited a marked decrease in the adhesion and activation of platelets and macrophages revealing the potent anti-thrombogenic and anti-inflammatory effects of the combination. The pleiotropic effects of the combination may be attributed to their ability to activate nitric oxide synthase in endothelial cells while mTOR levels remained unaltered by the combination.
Design and characterization of a perivascular PLGA coated PET mesh sustaining the release of atorvastatin for the prevention of intimal hyperplasia
2018, International Journal of Pharmaceutics
Citation Excerpt :
A local application is therefore relevant as it will deliver the active compound close to the graft without systemic distribution related to oral administration with the risk of adverse effects. As for the drug, we have previously validated in vitro the efficacy of atorvastatin (ATV) for inhibiting human vascular smooth muscle cell proliferation and migration (Corpataux et al., 2005; Dubuis et al., 2013). The relatively high ATV concentration required to trigger this effect (EC50 of 10 μg/mL) is orders of magnitude higher than the ATV plasma concentration achieved upon oral administration (0.01 μg/mL), showing the advantage of a local ATV release to inhibit hyperplasia (Mylonaki et al., 2016).
Following vascular bypass interventions, autologous saphenous vein grafts are prone to fail due to intimal hyperplasia development. An atorvastatin (ATV)-eluting tubular mesh coated with poly(d,l-lactide-co-glycolisde) acid (PLGA) was designed for perivascular application, in order to prevent the development of this pathology. Formulation parameters such as PLGA molecular weight, concentration of ATV and PLGA in the coating solution and number of coatings were investigated to optimise the mesh in terms of drug loading efficacy, drug release kinetics and mechanical properties. Using the dip-coating technique, 1.6 mg of ATV was loaded on a tubular 5 cm long mesh. The most important parameter influencing ATV loading was the concentration of the drug in the coating solution. In vitro an ATV release profile combining an initial fast release over 3 days and a sustained release over 40 days was obtained; consistent with the timeframe of hyperplasia development. The amount of PLGA polymer coating as well as the molecular weight of the polymer were optimized to achieve these kinetics. A poly(d,l-lactide-co-caprolactone) (PLCL) layer was sprayed on the external side of the PLGA coated tubular mesh to restrict the ATV release to the vascular tissue. Scanning electron microscopy observation showed that the macroporosity of the mesh was preserved after coating, while texture analysis demonstrated that its elasticity decreased slightly.
Perivascular sustained release of atorvastatin from a hydrogel-microparticle delivery system decreases intimal hyperplasia
2016, Journal of Controlled Release
Citation Excerpt :
For this reason, we selected a non-antiproliferative pharmacological approach, atorvastatin (ATV). Although statins are traditionally thought to exert their beneficial effects on saphenous vein graft patency by reducing the blood's lipid content, it was also demonstrated that they inhibit IH in arterialized vein grafts by limiting macrophage infiltration and VSMC migration and proliferation [24,31–34]. We previously showed that when ATV is released from the clHA gel, it maintains its activity and inhibits cell proliferation and migration of hVSMC in vitro [35].
Intimal hyperplasia (IH) is the major cause of grafted vessel occlusion and occurs frequently after bypass intervention. No pharmaceutical formulation is currently available to prevent this pathology. Local perivascular delivery of an appropriate active compound released in a time-dependent manner (from day one up to 4 weeks) is necessary for an efficient single-administration preventive therapy. To this aim, we propose the combination of gel and microparticles delivery system containing atorvastatin (ATV). The incorporation of ATV in a cross-linked hyaluronic acid gel, provided in vitro a fast release over 3 days, while ATV-loaded poly-lactic-co-glycolic acid (PLGA) microparticles dispersed in the gel gave a sustained release over 4 weeks. In vivo, ATV formulations were applied perivascularly in mice undergoing carotid artery ligation. IH was significantly reduced (‐ 68%) in presence of ATV incorporated in hyaluronic acid gel and encapsulated in microparticles compared to control. No significant IH alteration was observed when ATV was incorporated only in the gel (fast release) or only in the microparticles (slow release) demonstrating that a biphasic release of ATV is essential to interfere with the development of IH.
ATV was detected in adjacent tissues 28 days after the intervention, showing the sustained presence of the drug in vivo. After four weeks ATV was not detected in remote tissues, except at a very low concentration (0.044 ng/mg) in the liver, suggesting a very low risk of systemic toxicity of locally delivered ATV. Additionally, the ex vivo data showed that ATV in solution permeates through isolated human saphenous veins and thus is a good candidate for perivascular delivery.
Our data demonstrate that a local biphasic ATV release on the mice ligated carotid efficiently prevents the development of IH without apparent toxicity.
Expression of OATP2B1 as determinant of drug effects in the microcompartment of the coronary artery
2015, Vascular Pharmacology
Clinical success of coronary drug-eluting stents (DES) is hampered by simultaneous reduction of smooth muscle cell (HCASMC) and endothelial cell proliferation due to unspecific cytotoxicity of currently used compounds. Previous in vitro data showing SMC-specific inhibition of proliferation suggested that statins may be suitable candidates for DES. It was aim of this study to further investigate statins as DES drug candidates to identify mechanisms contributing to their cell-selectivity. In vitro proliferation assays comparing the influence of various statins on HCASMC and endothelial cells confirmed that atorvastatin exhibits HCASMC-specificity. Due to similar expression levels of the drug target HMG-CoA reductase in both cell types, cellular accumulation of atorvastatin was assessed, revealing enhanced uptake in HCASMC most likely driven by significant expression of OATP2B1, a known uptake transporter for atorvastatin. In accordance with the finding that endogenous OATP2B1 influenced cellular accumulation in HCASMC we used this transporter as a tool to identify teniposide as new DES candidate drug with HCASMC-specific effects. We describe OATP2B1 as a determinant of pharmacokinetics in the coronary artery. Indeed, endogenously expressed OATP2B1 significantly influences the uptake of substrate drugs, thereby governing cell specificity. Screening of candidate drugs for interaction with OATP2B1 may be used to promote SMC-specificity.
Apolipoprotein(a) acts as a chemorepellent to human vascular smooth muscle cells via integrin αvβ<inf>3</inf>and RhoA/ROCK-mediate dmechanisms
2013, International Journal of Biochemistry and Cell Biology
Citation Excerpt :
Statins exhibit multiple pleiotropic effects, one of which is inhibition of RhoA prenylation and activity, as a downstream consequence of HMG CoA reductase inhibition (Porter and Turner, 2011). As SV-SMC migration in response to PDGF is known to be modulated by statins (Corpataux et al., 2005), and we had consistently demonstrated that apo(a) exhibited chemorepellent activity independently of PDGF (Fig. 1C), we explored the effects of statins on SV-SMC migration in the absence of PDGF. SV-SMC were pre-treated with 1 μM fluvastatin for 2 h prior to use in Boyden chamber assays.
Lipoprotein(a) (Lp(a)) is an independent risk factor for the development of cardiovascular disease. Vascular smooth muscle cell (SMC) motility and plasticity, functions that are influenced by environmental cues, are vital to adaptation and remodelling in vascular physiology and pathophysiology. Lp(a) is reportedly damaging to SMC function via unknown molecular mechanisms. Apolipoprotein(a) (apo(a)), a unique glycoprotein moiety of Lp(a), has been demonstrated as its active component. The aims of this study were to determine functional effects of recombinant apo(a) on human vascular SMC motility and explore the underlying mechanism(s). Exposure of SMC to apo(a) in migration assays induced a potent, concentration-dependent chemorepulsion that was RhoA and integrin α_Vβ₃-dependent, but transforming growth factor β-independent. SMC manipulation through RhoA gene silencing, Rho kinase inhibition, statin pre-treatment, α_Vβ₃ neutralising antibody and tyrosine kinase inhibition all markedly inhibited apo(a)-mediated SMC migration. Our data reveal unique and potent activities of apo(a) that may negatively influence SMC remodelling in cardiovascular disease. Circulating levels of Lp(a) are resistant to lipid-lowering strategies and hence a greater understanding of the mechanisms underlying its functional effects on SMC may provide alternative therapeutic targets.

View all citing articles on Scopus

View full text

VascularThe Effect of Six Different Statins on the Proliferation, Migration, and Invasion of Human Smooth Muscle Cells

Introduction

Section snippets

SMC Isolation and Culture

Effects of Statins on SMC Proliferation

Discussion

Acknowledgments

Eur. J. Vasc. Endovasc. Surg.

Am. J. Cardiol.

Atherosclerosis

J. Biol. Chem.

Pharmacol. Ther.

J. Vasc. Surg.

Biochim. Biophys. Acta

Biochem. Biophys. Res. Comm.

Atherosclerosis

Biochem. Pharmacol.

Vascular
The Effect of Six Different Statins on the Proliferation, Migration, and Invasion of Human Smooth Muscle Cells