Simvastatin inhibits the migration and adhesion of monocytic cells and disorganizes the cytoskeleton of activated endothelial cells
Introduction
Statins are competitive inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, a key microsomal enzyme that catalyzes the rate limiting step in the biosynthesis of cholesterol, which converts by deacylation of HMG-CoA to mevalonate and CoA. They are considered the most potent and effective therapeutic agents currently available for lowering plasma levels of cholesterol. Several large, and controlled clinical trials have convincingly shown that statin therapy significantly reduces the risks of coronary heart disease morbidity and mortality in patients with hypercholesterolemia, and slows the progression of new cardiovascular events (Blumenthal, 2000, Maron et al., 2000, Weitz-Schmidt, 2002, Pedersen and Tobert, 2004, Balk et al., 2004, Elrod and Lefer, 2005).
Several recent studies demonstrate that the effectiveness of statins therapy cannot be fully explained on the basis of their potential in the reduction of circulating lipoproteins and cholesterol levels. Statins appear to have, beyond normalization of hypercholesterolemia, a variety of pleiotropic effects that may contribute to the overall clinical improvement, including their antiinflammatory action (Ross, 1999, Blumenthal, 2000, Maron et al., 2000, Kwak et al., 2000, Weitz-Schmidt, 2002, Hernandez-Presa et al., 2002, Elrod and Lefer, 2005), reduction of endothelial barrier dysfunction and permeability (van Nieuw Amerongen et al., 2000, Stalker et al., 2001, Balk et al., 2004, Jacobson et al., 2003), enhanced fibrinolysis, antithrombotic properties (Undas et al., 2001), induction of apoptosis (Guijarro et al., 1998), prevention of development of cardiac hypertrophy (Takemoto et al., 2001), and regulation of immune responses (Kwak et al., 2000, Hillyard et al., 2004). Moreover, these beneficial properties of statins occur earlier and are more rapidly induced than what would be expected from regression of coronary events or stabilization of unstable atheroma, besides the lack of correlation with the decrease in serum cholesterol concentration.
Several authors attribute this multifunctional mode of action of statins to their ability to suppress the biosynthesis of important isoprenoid intermediates, such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), rather than their ability to inhibit cholesterol synthesis (Ross, 1999, Takemoto and Liao, 2001, Blumenthal, 2000, Pedersen and Tobert, 2004). These isoprenoids are important lipid attachments for post-translational modifications of a variety of proteins, such as Rho and Ras small guanosine triphosphate (GTP)-binding proteins. It is well known that isoprenylation of proteins plays key regulatory roles in intracellular trafficking, cytoskeleton architecture, cell migration, proliferation, and cellular adhesion (Fenton et al., 1992, Miquel et al., 1996, Koch et al., 1997, Evers et al., 2000). In other words, statins are important in altering the processing of signal transduction proteins that require lipidation.
It has been recognized that cellular interactions in atherogenesis are similar to those in other inflammatory diseases. Leukocyte migration, expression of adhesion molecules, and cell accumulation after adhesion to endothelium and extravasation play a crucial role in the perpetuation of a chronic inflammatory reaction, lipid deposition, and formation of atheromas (Bevilacqua et al., 1994, Ross, 1999, Libby, 2000, Hansson, 2001). Several authors have demonstrated the antiproliferative, antimigratory, and antiadherence properties of statins (Hidaka et al., 1992, Corsini et al., 1995, Yang et al., 2000, Sadeghi et al., 2000, Teupser et al., 2001, Bernot et al., 2003, Zapolska-Downar et al., 2004), but there are some controversial data about the specific actions of statins and the role of isoprenylated proteins in those inflammatory responses. Moreover, the available “in vitro” studies on statin actions have been focused on long-term (overnight or longer) incubation periods. As far as we know, examination of these inflammatory events that may be affected by short-term exposure (brief cell pretreatment of about one to 4 h) to statins has not been reported. Hence, the purpose of the present work is to perform comparative studies between short-term and overnight treatments to determine the role of simvastatin, and the isoprenoid intermediates, in cell migration, adhesion, and cytoskeletal organization of cytokine endothelial cell activation.
Section snippets
Culture of endothelial cells and monocytic cell line
Human endothelial cells (HUVEC) were obtained from umbilical cords, and cultured as described elsewhere (Jaffe et al., 1973). Briefly, umbilical veins were cannulated and perfused with M199 culture medium to remove blood, and then incubated with 0.1% collagenase type P (Roche Diagnostics) for 20 min at 37 °C. Cells were harvested and cultured in M199 medium (BioWittaker) with 20% heat-inactivated fetal calf serum (FCS), and antibiotics. Routinely, we used confluent cells between the first and
Simvastatin is a potent inhibitor of chemotaxis of THP-1 monocytic cell line after a short-term period of treatment
The effect of simvastatin on MCP-1-directed monocytic cell migration was examined by performing Transwell assays on porous polycarbonate filters. These studies demonstrate that pretreatment of THP-1 monocytes with 40 μM and 20 μM for 24 h, almost completely abolished the migratory response of these cells toward MCP-1, being this chemotactic effect concentration dependent (Fig. 1A). In order to assess the effects of exogenous isoprenoids, THP-1 cells were also treated for 24 h simultaneously
Discussion
Directed leukocyte migration and their adhesion to endothelial cells are critical cellular responses of immune and inflammatory processes. Thus, cell migration involves actin cytoskeleton reorganization, polarization, and communication with adjacent cells and extracellular matrix proteins; and cell adhesion to endothelium is accomplished, at least in part, by up-regulation of cell surface adhesion molecules in response to cytokines.
We have specifically studied the ability of simvastatin to
Acknowledgments
This work was supported by grants from the Ministerio de Educación y Cultura (PM97-0089) and Ministerio de Ciencia y Tecnología (BFI2002-03892) to Jesús González Cabrero, and the Ministerio de Sanidad y Consumo (FIS, PI021047), Ministerio de Educación y Cultura (SAF 2004/0619), Comunidad de Madrid (GR/SAL/0411/2004) and Fundación Mutua Mdrileña to Jesús Egido. This study was partially supported by Merck, Sharp and Dohme. Mayte Pozo was a recipient of a predoctoral fellowship of Fundación
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