Statins (HMG-CoA reductase inhibitors) exhibit beneficial effects on the vasculature independently of their cholesterol-lowering properties. These pleiotropic effects underlie the ability of statins to reduce intimal hyperplasia in saphenous vein (SV) bypass grafts by attenuating smooth muscle cell (SMC) invasion and proliferation. Although all statins can effectively lower cholesterol, the pleiotropic effects of individual statins may well differ. We therefore compared the concentration-dependent effects of 4 lipophilic statins (simvastatin, atorvastatin, fluvastatin, and lovastatin) and 1 hydrophilic statin (pravastatin) on the proliferation and invasion of SMC cultured from SV of 9 different patients undergoing coronary artery bypass grafting (CABG). The lipophilic statins inhibited SV-SMC proliferation over a 4-day period with an order of potency of fluvastatin > atorvastatin > simvastatin > lovastatin (IC50 range = 0.07 to 1.77 μM). Similarly, these statins also inhibited SV-SMC invasion through an artificial basement membrane barrier (fluvastatin > atorvastatin > simvastatin ≫ lovastatin; IC50 range = 0.92 to 26.9 μM). In contrast, the hydrophilic pravastatin had no significant effect on SV-SMC proliferation at concentrations up to 10 μM, nor did it attenuate SV-SMC invasion (up to 30 μM). Our data provide strong evidence that individual statins possess differential pleiotropic effects on SV-SMC function. This may be of clinical relevance in the selection of individual statins for the treatment of CABG patients.
From the *Academic Unit of Cardiovascular Medicine, Leeds Institute of Genetics, Health and Therapeutics (LIGHT), University of Leeds, Leeds, UK; and †Department of Cardiac Surgery, The Yorkshire Heart Centre, Leeds General Infirmary, Leeds, UK.
Received for publication April 20, 2007; accepted May 18, 2007.
This work was supported by the British Heart Foundation, Research Councils UK. NAT is in receipt of a Research Councils UK Academic Fellowship.
The authors state that they have no financial interest in the products mentioned within this article.
Reprints: Dr. Karen E. Porter, PhD, Institute for Cardiovascular Research, Worsley Building, Clarendon Way, University of Leeds, Leeds LS2 9JT, UK (e-mail: firstname.lastname@example.org).
Statins (HMG-CoA reductase inhibitors) are a class of drugs prescribed to individuals at risk of coronary heart disease. In addition to their primary function of lowering plasma cholesterol levels, statins also have beneficial effects on the cardiovascular system through lipid-independent mechanisms.1 These are referred to collectively as pleiotropic effects and arise independently of effects on cholesterol synthesis, predominantly via inhibition of small GTPases (eg, Ras and Rho). These G-proteins are extensively implicated in the mechanism of a diversity of cardiovascular pathologies2,3 and inhibition of Ras/Rho underlies many of the cholesterol-independent effects of statins on the vascular wall.4-6
There is increasing clinical evidence that statins can reduce the incidence of coronary artery bypass graft (CABG) occlusion in saphenous vein (SV) conduits, effects that are independent of cholesterol levels.7-9 Indeed, statin therapy can retard the progression of vein graft disease and reduce the need for repeat revascularization procedures.10 The underlying pathology of SV graft occlusion is intimal hyperplasia (IH), a process initiated by endothelial damage during vein harvesting and implantation. The resultant increase in expression of inflammatory mediators such as platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) induces matrix metalloproteinase (MMP)-2 and -9 activity, which permits intimally directed smooth muscle cell (SMC) invasion and proliferation.11,12 We5,13,14 and others4,15-18 have demonstrated using organ-cultured human SV or SV-derived SMC cultures that statins reduce neointima formation, MMP-9 secretion, and SV-SMC invasion and proliferation.
Although all statins effectively lower cholesterol, the pleiotropic effects of individual statins may well differ.19,20 Given the potential clinical implications of statin pleiotropy, it is therefore important to compare the direct cellular effects of these compounds in addition to their lipid-lowering properties. In the present study, we investigated the concentration-dependent effects of 4 lipophilic statins (simvastatin, atorvastatin, fluvastatin, and lovastatin) and 1 hydrophilic statin (pravastatin) on modulation of human SV-SMC proliferation and invasion.
Cell culture reagents were purchased from Invitrogen (Paisley, UK) with the exception of fetal calf serum (FCS), which was from Biosera Ltd (Ringmer, UK). PDGF-BB and IL-1α were from Sigma (Poole, UK), and statin sodium salts were purchased from Calbiochem (Nottingham, UK).
Samples of SV were obtained from a total of 9 separate patients undergoing elective CABG surgery at the Leeds General Infirmary. Local ethical committee approval and informed patient consent were obtained. SV-SMC were cultured from explants of SV tissue and characterized as described previously.13 Cells were maintained at 37°C in full growth medium (Dulbecco Modified Eagle Medium containing 10% FCS) in a humidified atmosphere of 5% CO2 in air. All experiments were performed using SV-SMC of passage number 3-4.
Cell proliferation assays were performed essentially as described previously.13 Briefly, SV-SMC from 5 different patients were seeded into 24-well cell culture plates at a density of 1 × 104 cells per well in full growth medium. Cells were incubated overnight and then quiesced in serum free medium for 3 days before transfer to full growth medium (10% FCS) containing 5 different statins at a range of concentrations. All statins were tested on cells from each individual patient. Medium and drugs were replaced after 2 days, and viable cell numbers were determined in triplicate wells after 4 days using Trypan Blue and a hemocytometer. The increase in cell number was calculated by subtracting the starting cell number (day 0) from the final cell number (day 4). Data were then normalized to control values (no statin) to correct for differences in proliferation rates between cells from different patients.
SV-SMC invasion was assessed using a modified Boyden chamber technique with polycarbonate membrane pores occluded with Matrigel basement membrane matrix (BD Biosciences, Oxford, UK), as we have described previously.13 Cells (1 × 105) from 4 different patients were loaded in the upper chamber in medium supplemented with 0.4% FCS with or without statins at the appropriate concentrations. All 5 statins at 3 concentrations were tested in parallel on cells from each different patient. The lower chamber contained 0.4% FCS supplemented with chemoattractant (10 ng/mL PDGF-BB plus 10 ng/mL IL-1α). After incubation for 24 h at 37°C in a tissue culture incubator, duplicate membranes were processed and evaluated by counting cells in 10 random fields under high power (×400) light microscopy.13 Data were normalized to the number of invaded cells in control wells (ie, in the absence of statin).
Results are expressed as mean ± SEM with n representing the number of experiments on cells from different patients. Curves were fitted to proliferation and invasion data using nonlinear regression analysis (GraphPad Prism software, www.graphpad.com), and IC50 values were calculated as the concentration of statin causing a 50% reduction in proliferation/invasion.
Effect of Statins on SV-SMC Proliferation
SV-SMC were plated at equal densities, quiesced, and then exposed to 10% FCS (maximal growth stimulus) alone or supplemented with simvastatin (0.3 to 3 μM), atorvastatin (0.03 to 1 μM), fluvastatin (0.03 to 0.3 μM), lovastatin (1 to 10 μM), or pravastatin (1 to 10 μM) for 4 days before determining changes in cell number by counting viable cells. The 4 lipophilic statins inhibited FCS-induced proliferation with an order of potency of fluvastatin > atorvastatin > simvastatin > lovastatin (Fig. 1A). The concentration of statin that elicited a 50% reduction in cell number (IC50 values) ranged from 0.07 μM for fluvastatin to 1.77 μM for lovastatin (Table 1). In contrast to the lipophilic statins, the hydrophilic pravastatin had no significant effect on SV-SMC proliferation at concentrations up to 10 μM (Fig. 1A).
Effect of Statins on SV-SMC Invasion
The effects of the same 5 statins on PDGF/IL-1-induced invasion through a Matrigel basement membrane barrier were then investigated (Fig. 1B). The lipophilic statins inhibited SV-SMC invasion with the same order of potency observed in the proliferation assays (fluvastatin > atorvastatin > simvastatin > lovastatin); IC50 values ranged from 0.92 μM for fluvastatin to 26.9 μM for lovastatin (Table 1). Pravastatin had no effect on SV-SMC invasion, even at concentrations up to 30 μM (Fig. 1B).
We determined the concentration-dependent effects of 5 different statins on human SV-SMC proliferation and invasion; important events in the development of neointimal lesions in SV bypass grafts. The order of potency of the lipophilic statins was identical for both aspects of SMC function with fluvastatin > atorvastatin > simvastatin > lovastatin, whereas the hydrophilic pravastatin had no effect. For both assays, the difference in efficacy between fluvastatin and lovastatin was approximately 25-fold to 30-fold. In contrast to the lipophilic statins, which can enter cells by passive diffusion, hydrophilic statins such as pravastatin (and rosuvastatin) are unable to penetrate the plasma membrane of most cell types, with the exception of hepatic cells, whose membranes contain specific transporters for uptake of such hydrophilic substances.21 One would therefore predict that although hydrophilic statins would reduce cholesterol synthesis in the liver, they would exhibit reduced pleiotropic effects on the vasculature. Indeed, fluvastatin, simvastatin, and lovastatin all reduced neointimal formation in normocholesterolaemic rabbits after carotid artery restriction, whereas pravastatin was without significant effect.22 Similar findings were reported in a rabbit atheroma model.23 However, some more recent in vivo6,24 and clinical studies25,26 have reported beneficial effects of hydrophilic statins (pravastatin and rosuvastatin) on IH; therefore hydrophilicity alone may not fully predict the pleiotropic effects of statins.
Our proliferation data are in keeping with previous reports that have demonstrated that lipophilic, but not hydrophilic, statins inhibit vascular SMC proliferation.17,27 The IC50 values calculated from our proliferation studies compared favorably with the manufacturers' Cmax values for different statins quoted in the study of Corpateaux and colleagues (1 μM for simvastatin, atorvastatin, lovastatin, pravastatin; 0.1 μM for fluvastatin).16 However, in contrast to our findings, these authors reported that pravastatin (10 μM) could reduce neointima formation in organ-cultured SV15 and inhibit SV-SMC proliferation and invasion16 to the same extent as lipophilic statins, although the reasons for these differences are not clear.
In our invasion assay, cells must first degrade a Matrigel basement membrane barrier before undergoing chemotaxis towards a PDGF/IL-1 stimulus. Statins can potentially modulate this process in at least 2 distinct ways: (1) by inhibiting MMP-9 secretion; and (2) by reducing chemotaxis. It has been previously reported that several statins can reduce human SV-SMC migration in the absence of a Matrigel barrier (chemotaxis) by approximately 50%.16 Additionally, we have demonstrated that simvastatin and atorvastatin, but not lovastatin or pravastatin, can inhibit phorbol ester-induced MMP-9 secretion in human SV-SMC.5 Thus, the relatively low efficacy of lovastatin in inhibiting SV-SMC invasion compared with other lipophilic statins may be due to its inability to effectively reduce MMP-9 secretion. In our experiments, pravastatin had no effect on SV-SMC invasion, consistent with our findings on cell proliferation.
A particular strength of our study was the use of human SV-SMC, the exact cell type that causes the clinical problem of SV graft stenosis. Moreover, by examining concentration-response profiles of 5 different statins on both SV-SMC proliferation and invasion, we have gained important insights into the differential pleiotropic effects of members of this class of drugs. The IC50 values for the lipophilic statins were consistently lower for cell proliferation than invasion. This may be due to the nature of our proliferation assay protocol, in which statins were added on 2 separate occasions over a 4-day period, compared with a single addition for the 24-hour invasion assay. Another explanation is that cell proliferation may be more sensitive to changes in small GTPase activity than cellular invasion.
Lipophilic statins inhibited both proliferation and invasion of human SV-SMC with the same order of potency (fluvastatin > atorvastatin > simvastatin > lovastatin), whereas hydrophilic pravastatin had no significant effect. These pleiotropic effects of statins on SV-SMC function likely underlie their ability to reduce SV graft occlusion. Given the widespread prescribing of statins, it may be necessary to consider not only the lipid-lowering but also the pleiotropic properties of individual statins for optimal treatment of CABG patients.
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Keywords:© 2007 Lippincott Williams & Wilkins, Inc.
smooth muscle cells; human; statin; proliferation; invasion; intimal hyperplasia