Kuzuya, Masafumi MD, PhD; Cheng, Xian Wu MD; Sasaki, Takeshi PhD; Tamaya-Mori, Norika PhD; Iguchi, Akihisa MD, PhD
It has been suggested that constrictive arterial remodeling plays a major role in lumen narrowing following angioplasty. 1–4 An injured artery wall resembles a healing wound in its sequence of remodeling events. Wound repair occurs through integrated cellular and molecular events leading to healing of the wound defect by a combination of epithelization, contraction, and extracellular matrix biosynthesis. 5 The reorganization of extracellular matrix proteins by smooth muscle cells (SMCs) appears to play an important role in vascular remodeling following angioplasty. 1–4
Fibroblast cultures embedded in hydrated collagen lattice have been used as in vitro models of skin wound healing. 5,6 The ability of the fibroblast culture to reorganize and contract three-dimensional collagen gels is considered an in vitro model for wound contraction. Since remodeling of the arterial wall may be analogous to wound healing, collagen gel contraction by SMCs, which are cultured in collagen gel, is also used as a model of vascular remodeling. In this system, it has been demonstrated that α2β1 integrin mediates collagen reorganization 7 and that glycosaminoglycan yaluronan enhances collagen gel contraction by SMCs through CD44 receptors. 8
Recent clinical data have demonstrated that cholesterol-lowering drugs, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors (statins), can induce regression of vascular atherosclerosis as well as reduction of cardiovascular-related morbidity and mortality in patients with and without coronary artery disease. Recent studies have suggested that the clinical benefit of statins may result from multiple effects on the vasculature beyond the lowering of serum cholesterol levels. 9 It has been shown that statins up-regulate the expression of endothelial nitric synthase and tissue plasminogen activator in endothelial cells. 10,11 In addition, statins suppress SMCs proliferation and migration. 12 However, no attempts to evaluate the effect of statins on constrictive arterial remodeling have been reported.
The aim of the present study was to test the hypothesis that statins play a beneficial role in constrictive vascular remodeling. We investigated the effect of pitavastatin, a newly synthesized statin, 13 on SMC-populated collagen lattice contraction, an in vitro model of constrictive arterial remodeling.
Smooth muscle cells were isolated from calf aortic media by means of the explant method and cultured with Dulbecco modified Eagle medium (DMEM, Nissui Pharmaceutical; Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS) and antibiotics as previously described. 14 For the experiments, SMCs in a subconfluent state at the 7th to the12th passage were used.
Collagen Gel Contraction Assay
Native type I collagen gels were prepared by mixing collagen, 1N NaOH, 10× DMEM on ice such that the final mixture contained 1.0 mg/ml collagen, 1× DMEM, at pH 7.4. SMCs were trypsinized and suspended in the collagen solutions at 3 × 105 cells/ml. The collagen/cell mixtures (500 μL) were added to 24-well plates precoated with 1% agarose. The mixtures were allowed to polymerize for 1 hour at 37°C, and then incubated overnight in 1 mL of DMEM in the presence or absence of various chemicals. To initiate collagen gel contraction, the collagen gels were reacted with 10% FBS or platelet-derived growth factor-BB (PDGF, 10 ng/ml), and the diameters of the gels were recorded at the indicated intervals. Contraction was measured relative to the initial gel diameter of 10 mm. Before the addition of FBS or PDGF, no contraction of gels was observed even after overnight incubation in DMEM.
Treatment of Smooth Muscle Cells with C3 Exoenzyme
Smooth muscle cells growing on 60-mm dishes in serum-containing medium were washed in phosphate buffered saline (PBS) and scraping buffer (114 mM KCl, 15 mM NaCl, 5.5 mM MgCl2, 10 mM Tris-HCl). Cells were then gently scraped in 0.25 ml of scraping buffer in the presence or absence of C3 exoenzyme, and resuspended in 10% FBS + DMEM as described previously. 15 Following overnight incubation, the cells were used for the experiments.
Preparation of Liposomes Containing Isoprenoids
To make liposomes containing isoprenoids, an aliquot of a mixture of dipalmitoylphosphatidylcholine (5 μM) and 200 μg of geranylgeranyl pyrophosphate (GGPP) or farnesyl pyrophosphate (FPP) was added to a pear-shaped flask, and the solvent was removed by means of rotary evaporation and a vacuum pump. The dried lipid film was then dispersed in 0.5 mL of PBS. Warming the flask to 50°C facilitates smooth dispersion. The liposomes were sonicated and stored at 4°C.
Smooth muscle cells on glass cover slips coated with type I collagen were pretreated overnight with DMEM without serum, and then incubated with 10% FBS or PDGF (10 ng/ml) for 1 hour. The cells were fixed with 3% paraformaldehyde followed by permeabilization for 10 minutes with 0.1% Triton X-100. To stain for F-actin, the samples were incubated with rhodamine-conjugated phalloidin (8 U/ml) for 30 minutes followed by washes with PBS. For the detection of focal adhesions, the samples were immunostained with monoclonal anti-vinculin antibody for 30 minutes, washed in PBS followed by 30 minutes incubation with Alexa-Flour-488 anti-mouse IgG. After additional washes, samples were mounted on glass slides. Micrographs were obtained with a fluorescence microscope (Orympus, Japan).
Values were expressed as means ± SD. Significant differences were analyzed using a Student t test or ANOVA followed by a Dunnett multiple-comparison post hoc test. A value of P < 0.05 is considered to be statistically significant.
Recombinant human PDGF was obtained from Pepro Tech. (Rocky Hill, NJ). Pitavastatin was a kind gift from Kowa Company Ltd. (Tokyo, Japan). Dipalmitoylphosphatidylcholine, lovastatin, mevalonate, FPP, GGPP, rhodamine-conjugated phalloidin, anti-vinculin antibody, and secondary antibody were purchased from Sigma-Aldrich. FTI-277, GGTI-298, and Y-27632 were obtained from Calbiochem and Biomedical Technologies Inc. FTI-277 and GGTI-298 were dissolved in DMSO. Clostridum botulinim C3 exoenzyme was obtained from Biomol Research Laboratories Inc. The inactive lactone of lovastatin was converted to the active form as described by Kita et al. 16
Pitavastatin Inhibits Smooth Muscle Cell-Populated Collagen Lattice Contraction
FBS at a concentration of 10% induced the SMC-populated collagen lattice contraction in a time-dependent manner over a 24-hour period (Fig. 1 and Fig. 2A). PDGF also had a similar effect on SMC-mediated collagen gel contraction, although the effect was weaker than that of FBS (Fig. 1 and Fig. 2B). These effects were dramatic until 6 hours after addition of these stimuli. The pretreatment of the SMC-populated collagen lattice with pitavastatin prevented the FBS-induced as well as PDGF-induced gel contraction in a concentration-dependent manner (Figs. 2A and 2B).
Mevalonate Metabolites Reverse the Effect of Pitavastain
As shown in Figure 3, the effect of pitavastatin on collagen gel contraction by SMCs was prevented by the addition of mevalonate, but not by squalene, a precursor of cholesterol. We also observed that lovastatin, another HMG-CoA reductase inhibitor, had a similar effect on SMC-mediated collagen gel contraction, on which the effect was also prevented by the addition of mevalonate (Fig. 3), indicating that this effect is not restricted to pitavastatin but is common to other statins.
Mevalonate is a donor for the synthesis of several critical metabolites involved in the posttranslational modification of proteins. It acts as an isoprenyl precursor for farnesyl and geranylgeranyl molecules, which have an important signaling function. 17 The direct addition of FPP or GGPP had no effect on pitavastatin's effect on SMC-mediated collagen gel contraction. However, when liposomes containing these isoprenoids were used to increase their membrane permeability, GGPP prevented the effect of pitavastatin, although neither liposomes containing FPP nor containing vehicle affected the pitavastatin-induced effect (Fig. 4). Isoprenyldiphosphate intermediates, FPP and GGPP are substrates for prenyl:protein transferase (FTase and geranylgeranyl:protein transferase [GGTases] I and II), which catalyze the post-translational modification of the small guanosine triphosphate (GTP)-binding proteins such as Ras, Rho, Rac, and Rab. 18 As shown in Figures 5A and B, GGTI-298, a specific cell-permeable GGTase inhibitor, 19 blocked FBS- and PDGF-induced collagen gel contractions by SMCs in a concentration-dependent manner, but FTI-277, a farnesylation inhibitor, 20 had no effect.
Rho and Rho Kinase Inhibitors Block Smooth Muscle Cells Populated Collagen Lattice Contraction
It has been demonstrated that a small GTPase protein, Rho, is a target for geranylgeranylation and that statins suppress Rho activation in various cell types. To examine whether FBS- and PDGF-induced collagen gel contraction by SMCs is mediated by the Rho pathway, we used the C3 exoenzyme and Y-27632, a specific inhibitor of Rho and Rho kinase, respectively. 21,22 Rho kinase is a major target protein of Rho and regulates stress fiber formation. 23 Treatment of SMCs with C3 exoenzyme as well as with Y-27632 suppressed FBS- as well as PDGF-induced collagen gel contractions by SMCs (Figs. 5C and 5D).
Effect of Pitavastatin on Smooth Muscle Cells Actin Cytoskeletal Reorganization
To examine the effect of pitavastatin on actin's cytoskeletal reorganization, stress fibers and focal adhesion complexes were visualized by palloidin and vinculin staining, respectively. In quiescent SMCs pretreated overnight with serum-free medium, weak levels of stress fibers were observed. FBS or PDGF treatment enhanced the assembly of stress fibers and focal adhesion complexes. Pitavastatin pretreatment suppressed these formations (Fig. 6). Mevalonate completely prevented these pitavastatin effects. FBS or PDGF also induced SMCs lamelliopodia extension, which was not inhibited by pitavastatin (Fig. 6).
Although SMC appears to be a key cell in constrictive arterial remodeling, its exact mechanisms remain unknown. 1–4 We observed that pitavastatin inhibited serum- and PDGF-induced collagen gel contraction by SMCs. This inhibitory effect of pitavastatin was prevented in the presence of excess of HMG-CoA reductase products, mevalonate, but not in the presence of squalene, suggesting that the observed effect of pitavastatin on collagen gel contraction by SMCs was attributable to the decrease of mevalonate production occurring as a consequence of HMG-CoA reductase inhibition, and that metabolites of mevalonate other than cholesterol might play a critical role in this effect of pitavastatin. We also observed that GGPP but not FPP prevented the effect of pitavastatin. These results indicated that pitavastatin's effect appeared to be related to the inhibition of geranylgeranylation of small G-proteins such as Rho GTPase. This is consistent with our finding that that the specific inhibitor of GGTase suppressed serum- and PDGF-induced SMC-populated collagen gel contraction.
Recent studies have shown that activation of Rho protein modulates the organization of actin filaments in cells, including formation of stress fibers and focal adhesions. 23,24 Rho protein regulates signal transduction from receptors in the membrane to a variety of cellular events related to cell morphology, motility, and cytoskeletal dynamics. Rho-kinase (Rho-associated protein kinase, p160ROCK), one of the target proteins of Rho, is implicated in many downstream processes of Rho; this includes stress fiber and focal adhesion formation. 23 Rho kinase regulates myosin light chain (MLC) phosphorylation by the direct phosphorylation of MLC and by the inactivation of myosin phosphatase. Indeed, a large number of studies have demonstrated the central role of MLC phosphorylation and the Rho–Rho kinase pathway in SMC migration, contraction, and relaxation. 25–27 In the present study, we demonstrated that the Rho–Rho kinase pathway is required to induce FBS- and PDGF-dependent collagen gel contraction by SMCs based on the observation of the C3 exoenzyme and Y-27632, specific inhibitors of Rho and Rho kinase, respectively. The activity of the Rho protein is regulated by a critical step in posttranslational processing, namely, the addition of isoprenoid lipid geranylgeranyl, mediated by GGTase I. 28 Therefore, this conclusion is consistent with our observation that a geranylgeranyl lipid from the cholesterol intermediates is essential for the mechanisms of collagen contraction by SMCs.
We demonstrated that serum or PDGF increased the stress fiber organization in SMCs, which was blocked by pitavastatin pretreatment. Again this effect of pitavastatin was prevented by the addition of mevalonate, suggesting that the products synthesized from mevalonate play a prominent role in serum- and PDGF-induced actin's cytoskeletal reorganization. However, pitavastatin had no effect on serum- or PDGF-induced lamelliopodia extension. These results may suggest that enhanced assembly of stress fibers is essential for collagen gel contraction. Lamelliopodia formations are not required for the serum- and PDGF-induced SMC-populated collagen gel contraction.
It has been known that stress fiber formation is also regulated by the Ca2+-dependent calmodulin/MLC kinase (MLCK) system. 29 In fact, we observed that chelation of intracellular Ca2+ as well as the treatment of SMCs with a specific MLCK inhibitor blocked collagen gel contraction (unpublished observations), providing supporting evidence that the Ca2+ calmodulin/MLCK system is also essential for SMC-mediated collagen gel contraction. Although the exact roles of Rho–Rho kinase and MLCK pathway-induced MLC phosphorylation in SMC contraction remain unknown, it has been demonstrated that the Rho–Rho kinase pathway is involved in the assembly of stress fibers in the center of cells and that MLCK is at the cell periphery. 29 Therefore, actin assembly at both the center and at the periphery appears necessary for SMC-induced collagen gel contraction.
In the present study, we demonstrated that pitavastatin, an HMG-COA reductase inhibitor, blocked the serum- and PDGF-induced SMC-populated type I collagen lattice contractions. These results may reproduce the possible action of statin on vascular contractive remodeling after vascular injury. During the preparation of this manuscript, Li et al 30 and Pearce et al 31 have reported that Rho–Rho kinase pathway is involved in SMC-populated collagen gel contraction.
1. Pasterkamp G, de Kleijn DP, Borst C. Arterial remodeling in atherosclerosis, restenosis and after alteration of blood flow: potential mechanisms and clinical implications. Cardiovasc Res
2. Geary RL, Nikkari ST, Wagner WD, et al. Wound healing: a paradigm for lumen narrowing after arterial reconstruction. J Vasc Surg
3. Mondy JS, Williams JK, Adams MR, et al. Structural determinants of lumen narrowing after angioplasty in atherosclerotic nonhuman primates. J Vasc Surg
4. Schoenhagen P, Ziada KM, Vince DG, et al. Arterial remodeling and coronary artery disease: the concept of “dilated” versus “obstructive” coronary atherosclerosis. J Am Coll Cardiol
5. Eckes B, Zigrino P, Kessler D, et al. Fibroblast-matrix interactions in wound healing and fibrosis. Matrix Biol
6. Grinnell F. Fibroblast biology in three-dimensional collagen matrices. Trends Cell Biol
7. Lee RT, Berditchevski F, Cheng GC, et al. Integrin-mediated collagen matrix reorganization by cultured human vascular smooth muscle cells. Circ Res
8. Travis JA, Hughes MG, Wong JM, et al. Hyaluronan enhances contraction of collagen by smooth muscle cells and adventitial fibroblasts: Role of CD44 and implications for constrictive remodeling. Circ Res
9. Takemoto M, Liao JK. Pleiotropic effects of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors. Arterioscler Thromb Vasc Biol
10. Laufs U, La Fata V, Plutzky J, et al. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation
11. Essig M, Nguyen G, Prie D, et al. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase fibrinolytic activity in rat aortic endothelial cells. Role of geranylgeranylation and Rho proteins. Circ Res
12. Laufs U, Marra D, Node K, et al. 3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPase-induced down-regulation of p27(Kip1). J Biol Chem
13. Noji Y, Higashikata T, Inazu A, et al. Hokuriku NK-104 Study Group. Long-term treatment with pitavastatin (NK-104), a new HMG-CoA reductase inhibitor, of patients with heterozygous familial hypercholesterolemia. Atherosclerosis
14. Maeda K, Kuzuya M, Cheng XW, et al. Green tea catechins inhibit the cultured smooth muscle cell invasion through the basement barrier. Atherosclerosis
15. Koike T, Kuzuya M, Asai T, et al. Activation of MMP-2 by Clostridium difficile toxin B in bovine smooth muscle cells. Biochem Biophys Res Commun
16. Kita T, Brown MS, Goldstein JL. Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in livers of mice treated with mevinolin, a competitive inhibitor of the reductase. J Clin Invest
17. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature
18. Clarke S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem
19. McGuire TF, Qian Y, Vogt A, et al. Platelet-derived growth factor receptor tyrosine phosphorylation requires protein geranylgeranylation but not farnesylation. J Biol Chem
20. Lerner EC, Qian Y, Blaskovich MA, et al. Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes. J Biol Chem
21. Mohr C, Just I, Hall A, et al. Morphological alterations of Xenopus oocytes induced by valine-14 p21rho depend on isoprenylation and are inhibited by Clostridium botulinum C3 ADP-ribosyltransferase. FEBS Lett
22. Uehata M, Ishizaki T, Satoh H, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature
23. Fukata Y, Amano M, Kaibuchi K. Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends Pharmacol Sci
24. Etienne-Manneville S, Hall A. Rho GTPases in cell biology. Nature
25. Ai S, Kuzuya M, Koike T, et al. Rho-Rho kinase is involved in smooth muscle cell migration through myosin light chain phosphorylation-dependent and independent pathways. Atherosclerosis
26. Kureishi Y, Kobayashi S, Amano M, et al. Rho-associated kinase directly induces smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem
27. Shimokawa H. Rho-kinase as a novel therapeutic target in treatment of cardiovascular diseases. J Cardiovasc Pharmacol
28. Zhang FL, Casey PJ. Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem
29. Katoh K, Kano Y, Amano M, et al. Stress fiber organization regulated by MLCK and Rho-kinase in cultured human fibroblasts. Am J Physiol Cell Physiol
30. Li S, Moon JJ, Miao H, et al. Signal transduction in matrix contraction and the migration of vascular smooth muscle cells in three-dimensional matrix. J Vasc Res
31. Pearce JD, Li J, Edwards MS, et al. Differential effects of Rhokinase inhibition on artery wall mass and remodeling. J Vasc Surg
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