- Email: [email protected]
Statins and ischemic stroke
Atherosclerosis Supplements 3 (2002) 21 – 25 www.elsevier.com/locate/atherosclerosis
Statins and ischemic stroke James K. Liao * Vascular Medicine Research, Brighan & Women’s Hospital Ha6ard Medical School, 65 Landsdowne Street, Room 275, Cambridge, Massachusetts, 02139, USA
Abstract The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors or statins are potent inhibitors of cholesterol synthesis. Several large clinical trials have demonstrated that these agents reduce serum cholesterol levels and the incidence of cardiovascular diseases. However, overlap and meta-analyses of these clinical trials suggest that the beneficial effects of statins may extend beyond their effects on serum cholesterol levels. Because statins also inhibit the synthesis of isoprenoid intermediates in the cholesterol biosynthetic pathway, they may have pleiotropic effects on the vascular wall. In particular, the ability of statins to decrease the incidence of ischemic stroke highlights some of their non-cholesterol effects since serum cholesterol levels are poorly correlated with the risk for ischemic stroke. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Endothelium; Nitric oxide; Isoprenoids; Cholesterol; G-proteins; Gene regulation; Stroke
1. Introduction Recent large clinical trials have demonstrated that a class of cholesterol-lowering agents called statins decrease the incidence of myocardial infarctions and ischemic strokes in hypercholesterolemic and atherosclerotic individuals [1 – 4]. These agents inhibit an early step in cholesterol biosynthesis by blocking the conversion of 3-hydroxy-3-methylglutaryl (HMG)-CoA to mevalonate. Because serum cholesterol level is strongly associated with coronary atherosclerotic disease [5], it has been generally assumed that cholesterol reduction by statins is the predominant, if not the only mechanism, underlying their beneficial effects in cardiovascular diseases. However, subgroup analyses of large clinical trials have challenged this notion and suggest that the beneficial effects of statins may extend to mechanisms beyond cholesterol reduction [6,7], possibly involving direct effects on the vascular wall. Subgroup analyses of the WOSCOP, CARE, and LIPID trials indicate that despite comparable serum cholesterol levels, statin-treated individuals have significantly lower risks for coronary heart disease compared to age-matched placebo-controlled individuals [2,4,7,8]. Furthermore, meta-analyses of past clinical trials sug* Tel.: + 1-617-768-8424; fax: + 1-617-768-8425. E-mail address: [email protected] (J.K. Liao).
gest that the risk of myocardial infarctions in individuals treated with statins is significantly lower compared to individuals treated with other cholesterol-lowering agents or modalities despite comparable reduction in serum cholesterol levels in both groups [9,10]. Taken together, these findings suggest that some of the beneficial effects of statins may be due to their cholesterol-independent effects on the vascular wall.
2. Statins and endothelial function Hypercholesterolemia impairs endothelial function and endothelial dysfunction is one of the earliest markers of atherosclerosis, occurring even in the absence of angiographic evidence of disease [11,12]. The vascular endothelium serves as an important autocrine/paracrine organ that regulates vascular wall contractile state and cellular composition. An important characteristic of endothelial dysfunction is the impaired synthesis, release and activity of endothelial-derived nitric oxide (NO). Endothelial NO has been shown to inhibit several components of the atherogenic process. For example, endothelium-derived NO mediates vascular relaxation and inhibits platelet aggregation, vascular smooth muscle proliferation and endothelial-leukocyte interactions. In addition, factors which limit the bioavailability of NO such as superoxide anion lead to
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nitrate tolerance, vasoconstriction, and hypertension [13,14]. Improvement in endothelial function by statins can be attributed to the lowering of serum cholesterol levels since hypercholesterolemia impairs endothelial function [13]. Indeed, acute lowering of serum cholesterol by plasma LDL apheresis rapidly improves endotheliumdependent vasodilatation [15]. However, in some studies, restoration of endothelial function by statins occurs before significant reduction in serum cholesterol levels [16–18] suggesting that there are additional effects on endothelial function beyond that of cholesterol reduction. Indeed, statins upregulate endothelial nitric oxide synthase (eNOS) expression and activity and reverse the downregulation of eNOS expression by hypoxia and oxidized low-density lipoprotein (ox-LDL) under cholesterol-clamped conditions [19,20]. Therefore, it is possible that statins may have other beneficial effects, irrespective of serum cholesterol levels, in conditions associated with endothelial dysfunction such as atherosclerosis, pulmonary hypertension, and congestive heart failure.
3. Statins, mevalonate pathway, and eNOS By inhibiting L-mevalonate synthesis, statins also prevent the synthesis of other important isoprenoid intermediates of the cholesterol biosynthetic pathway, such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) [21]. These intermediates serve as important lipid attachments for the post-translational modification of a variety of proteins, including the g subunit of heterotrimeric G-proteins, Heme-a, nuclear lamins, and small GTP-binding protein Ras, and Ras-like proteins, such as Rho, Rab, Rac, Ral or Rap [22]. Thus, protein isoprenylation permits the covalent attachment, subcellular localization, and intracellular trafficking of membrane-associated proteins such as Rho. Members of the Ras and Rho GTPase family are major substrates for post-translational modification using isoprenoids [22– 24]. In endothelial cells, Ras translocation from the cytoplasm to the plasma membrane is dependent upon farnesylation while Rho translocation is dependent upon geranylgeranylation [25,26]. Statins inhibit both Ras and Rho isoprenylation and lead to accumulation of inactive Ras and Rho in the cytoplasm. While the effects of statins on Ras and Rho isoprenylation are reversed in the presence of FPP and GGPP, respectively, the effects of statins on eNOS expression are only reversed with GGPP and not by FPP or LDL-cholesterol [25]. These findings are consistent with a non-cholesterol-lowering effect of statins and suggest that inhibition of Rho by statins upregulates eNOS expression. Indeed, statins upregulate eNOS expression
by prolonging eNOS mRNA half-life but not eNOS gene transcription [20]. Since hypoxia, oxidized LDL, and cytokines such as TNF-a decrease eNOS expression by reducing eNOS mRNA stability, the ability of statins to prolong eNOS half-life may make them effective agents in counteracting conditions which downregulate eNOS expression. Indeed, statins prevent the downregulation of eNOS by oxidized LDL and TNF-a and under hypoxic conditions [19,20]. Because Rho is a major target of geranylgeranylation, inhibition of Rho and its downstream target, the actin cytoskeleton, is a likely mechanism by which statins upregulate eNOS expression. The evidence that Rho-mediated actin cytoskeletal changes negatively regulate eNOS expression comes from three sets of experiments. First, direct inhibition of Rho by Clostridium botulinum C3 transferase increases eNOS expression independent of isoprenylation. The C3 transferase ADP-ribosylates asparagine-41 of Rho and renders it biologically inactive in the GDP-bound state [23]. Second, inhibition of Rho by overexpression of a dominant-negative RhoA mutant, N19RhoA, also increases eNOS expression [25]. Finally, disruption of the actin cytoskeleton leads to increases in eNOS expression in vitro and in vivo [27]. These results, therefore, identify Rho and the actin cytoskeleton as a negative regulator of eNOS expression (Fig. 1).
4. Statins and ischemic stroke Ischemic stroke is the third leading cause of death in the United States and is a major cause of long-term disability, especially in the elderly population and in patients undergoing cardiovascular surgery. Currently, few therapeutic options are available for the treatment
Fig. 1. Upregulation of eNOS by statins. Statins block the synthesis of isoprenoids and cholesterol. The isoprenoid, geranylgeranyl (GG) pyrophosphate (PP), is important for the membrane translocation and function of Rho. Rho, in turn, negatively regulates the expression of eNOS via effects on the actin cytoskeleton.
J.K. Liao / Atherosclerosis 3 (2002) 21–25
of ischemic strokes with prophylactic treatment strategies limited mainly to agents which block platelet aggregation or the coagulation cascade [28]. Although clinical trials have demonstrated the effectiveness of antiplatelet agents in reducing the incidence of ischemic strokes, these agents, however, do not address the larger issue of reducing cerebral infarct size. In fact, only therapies directed at restoring cerebral blood flow such as tissue-type plasminogen activator (t-PA) have been shown to be effective in the acute treatment of ischemic stroke [29,30]. These studies, therefore, suggest that neuroprotectants are relatively ineffective if cerebral blood flow is not restored and that cerebral blood flow is probably the single most important determinant of stroke severity. An intriguing result of large clinical trials with statins is the reduction in ischemic stroke [31]. Although myocardial infarction is closely associated with serum cholesterol levels, neither the Framingham Study nor the Multiple Risk Factor Intervention Trial (MRFIT) demonstrated significant correlation between ischemic stroke and serum cholesterol levels [32,33]. Thus, the findings of these large statin trials raise the interesting question of how a class of cholesterol-lowering agents can reduce ischemic stroke when ischemic stroke is not related to cholesterol levels. It appears likely that there are pleiotropic effects of statins, which are beneficial for ischemic stroke. Some of these beneficial effects may be attributed to the effects of statins on endothelial function and the vascular wall. Cerebral vascular tone and blood flow are regulated by endothelium-derived NO [34]. Mutant mice lacking eNOS (eNOS − /−) are relatively hypertensive and develop greater proliferative and inflammatory response to vascular injury [35]. Indeed, eNOS−/ − mice develop larger cerebral infarcts following cerebrovascular occlusion [36]. Thus, the beneficial effects of statins in ischemic stroke may be due, in part, to their ability to upregulate eNOS expression and activity. Indeed, mice which were prophylactically treated with statins for up to 2 weeks, have 25–30% higher cerebral blood flow and 50% smaller cerebral infarct sizes following cerebrovascular occlusion [37]. Furthermore, no increase in cerebral blood flow or neuroprotection was observed in eNOS − /− mice treated with statins indicating that the upregulation of eNOS accounts for most, if not all, of the neuroprotective effects of these agents. A recent study showed that statins can also activate eNOS via the phosphatidylinositol 3-kinase/protein kinase Akt pathway [38]. However, direct inhibition of Rho or the actin cytoskeleton mimics the effect of statins on eNOS upregulation and stroke protection [27], suggesting that inhibition of isoprenoid synthesis and Rho is the predominant mechanism by which statins exert their neuroprotective effects (Fig. 2). Interestingly, treatment with statins did not affect blood
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Fig. 2. Neuroprotective effect of statins. Statins upregulate eNOS by inhibiting Rho geranylgeranylation. This leads to vasodilatation, increase in cerebral blood flow, and protection against ischemic stroke.
pressure or heart rate before, during, or after cerebrovascular ischemia and did not alter serum cholesterol levels in mice, consistent with the previous finding that rodents are relatively resistant to changes in steady-state cholesterol levels by statins [37]. In addition to increases in cerebral blood flow, other beneficial effects of statins are likely to occur. For example, Lefer et al. reported that statins attenuate P-selectin expression and leukocyte adhesion via increases in NO production in a model of cardiac ischemia and reperfusion [39]. Statins have also been shown to decrease the expression of the potent vasoconstrictor/mitogen, endothelin-1, by inhibiting preproendothelin-1 gene transcription [40]. Others have reported that statins upregulate tissue-type plasminogen activator (t-PA) and downregulate plasminogen activator inhibitor (PAI)-1 expression through a similar mechanism involving inhibition of Rho geranylgeranylation [41]. Thus, the absence of neuroprotection in eNOS-deficient mice emphasizes the importance of endothelium-derived NO in not only augmenting cerebral blood flow, but also, potentially, in limiting the impact of endothelin-1-induced vasoconstriction and platelet and white blood cell accumulation on tissue viability following ischemia. Thus, it is highly likely that statins may decrease in the incidence of ischemic strokes in clinical trials, in part, by reducing cerebral infarct size to levels that are clinically unappreciated. Since most ischemic strokes occur in individuals with average cholesterol levels who may not otherwise be taking statin therapy, the upregulation of eNOS by statins may be an effective prophylactic treatment strategy for limiting the severity of ischemic strokes in normocholesterolemic high risk individuals (i.e. such as patients with history of stroke or transient ischemic attacks) or for patients undergoing elective cardiovascular procedures which have high peri-operative risk for cerebral ischemia (such as coronary artery bypass graft surgery or carotid endarterectomy). Because statins increase cerebral blood flow, these agents may serve as a useful adjunctive therapeutic modality for increasing the delivery of other co-administered drugs to the CNS. Finally, since eNOS expression is upregulated in the systemic vasculature, HMG-CoA reductase inhibitors
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J.K. Liao / Atherosclerosis 3 (2002) 21–25
may also have beneficial effects in other cardiovascular conditions where endothelial dysfunction may cause or worsen the pathological process such as atherosclerosis, pulmonary hypertension, and congestive heart failure.
5. Conclusions Statins exert many pleiotropic effects in addition to the lowering of serum cholesterol levels. Most of these effects, especially on the upregulation of eNOS, are mediated by statin’s inhibitory effect on isoprenoid synthesis. These effects of statins, therefore, may explain their ability to reduce the incidence of ischemic stroke whose risk is poorly tied to serum cholesterol levels.
Acknowledgements The work described in this paper was supported in part by the National Institutes of Health (HL-52233, HL-34836, and NS-10828) and the American Heart Association. Dr Liao is an Established Investigator and a Bugher Foundation Awardee of the American Heart Association.
References [1] Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 1994;344:1383 –9. [2] The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group. Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. N Engl J Med 1998;339:1349 –57. [3] Packard CJ. Influence of pravastatin and plasma lipids on clinical events in the West of Scotland Coronary Prevention Study (WOSCOPS). Circulation 1998;97:1440 – 5. [4] Sacks FM, Pfeffer MA, Moye LA, Rouleau JL, Rutherford JD, Cole, TG, Brown L, Warnica JW, Arnold JM, Wun CC, Davis BR, Braunwald E. Cholesterol and Recurrent Events Trial Investigators. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med 1996;335:1001 –9. [5] Klag MJ, Ford DE, Mead LA, He J, Whelton PK, Liang KY, Levine DM. Serum cholesterol in young men and subsequent cardiovascular disease. N Engl J Med 1993;328:313 – 8. [6] Blum CB. Comparison of properties of four inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Am J Cardiol 1994;73:3D – 11D. [7] Massy ZA, Keane WF, Kasiske BL. Inhibition of the mevalonate pathway: benefits beyond cholesterol reduction? Lancet 1996;347:102 – 3. [8] Shepherd J, Cobbe SM, Ford, I, Isles CG, Lorimer AR, MacFarlane PW, McKillop JH, Packard CJ. West of Scotland Coronary Prevention Study Group. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med 1995;333:1301 –7.
[9] Brown BG, Zhao XQ, Sacco DE, Albers JJ. Lipid lowering and plaque regression. New insights into prevention of plaque disruption and clinical events in coronary disease. Circulation 1993;87:1781 – 91. [10] Pekkanen J, Linn S, Heiss G, Suchindran CM, Leon A, Rifkind BM, Tyroler HA. Ten-year mortality from cardiovascular disease in relation to cholesterol level among men with and without preexisting cardiovascular disease. N Engl J Med 1990;322:1700 – 7. [11] Liao JK. Endothelium and acute coronary syndromes. Clin Chem 1998;44:1799 – 808. [12] Selwyn AP, Kinlay S, Libby P, Ganz P. Atherogenic lipids, vascular dysfunction, and clinical signs of ischemic heart disease. Circulation 1997;95:5 – 7. [13] Harrison DG. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest 1997;100:2153 – 7. [14] Munzel T, Sayegh H, Freeman BA, Tarpey MM, Harrison DG. Evidence for enhanced vascular superoxide anion production in nitrate tolerance. A novel mechanism underlying tolerance and cross-tolerance. J Clin Invest 1995;95:187 – 94. [15] Tamai O, Matsuoka H, Itabe H, Wada Y, Kohno K, Imaizumi T. Single LDL apheresis improves endothelium-dependent vasodilatation in hypercholesterolemic humans. Circulation 1997;95:76 – 82. [16] Anderson TJ, Meredith IT, Yeung AC, Frei B, Selwyn AP, Ganz P. The effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion. N Engl J Med 1995;332:488 – 93. [17] O’Driscoll G, Green D, Taylor RR. Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month. Circulation 1997;95:1126 – 31. [18] Treasure CB, Klein JL, Weintraub WS, Talley JD, Stillabower ME, Kosinski AS, Zhang J, Boccuzzi SJ, Cedarholm JC, Alexander RW. Beneficial effects of cholesterol-lowering therapy on the coronary endothelium in patients with coronary artery disease. N Engl J Med 1995;332:481 – 7. [19] Laufs U, Fata VL, Liao JK. Inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase blocks hypoxia-mediated downregulation of endothelial nitric oxide synthase. J Biol Chem 1997;272:31725 – 9. [20] Laufs U, Fata VL, Plutzky J, Liao JK. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 1998;97:1129 – 35. [21] Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature 1990;343:425 – 30. [22] Van Aelst L, D’Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev 1997;11:2295 – 322. [23] Aktories K. Bacterial toxins that target Rho proteins. J Clin Invest 1997;99:827 – 9. [24] Hall A. Rho GTPases and the actin cytoskeleton. Science 1998;279:509 – 14. [25] Laufs U, Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem 1998;273:24266 – 71. [26] Laufs U, Liao JK. Targeting rho in cardiovascular disease. Circ Res 2000;87:526 – 8. [27] Laufs U, Endres M, Stagliano N, Amin-Hanjani S, Chui DS, Yang SX, Simoncini T, Yamada M, Rabkin E, Allen PG, Huang PL, Bohm M, Schoen FJ, Moskowitz MA, Liao JK. Neuroprotection mediated by changes in the endothelial actin cytoskeleton. J Clin Invest 2000;106:15 – 24. [28] Patrono C. Aspirin as an antiplatelet drug. N Engl J Med 1994;330:1287 – 94. [29] Caplan LR. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med 1999;341:1240 – 1. [30] The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med 1995;333:1581 – 7.
J.K. Liao / Atherosclerosis 3 (2002) 21–25 [31] Crouse JR, Byington RP, Furberg CD. HMG-CoA reductase inhibitor therapy and stroke risk reduction: an analysis of clinical trials data. Atherosclerosis 1998;138:11 –24. [32] Multiple Risk Factor Intervention Trial Research Group. Multiple risk factor intervention trial. Risk factor changes and mortality results. J Am Med Assoc 1982;248:1465 –77. [33] Sytkowski PA, Kannel WB, D’Agostino RB. Changes in risk factors and the decline in mortality from cardiovascular disease. The Framingham Heart Study. N Engl J Med 1990;322:1635 – 41. [34] Dalkara T, Yoshida T, Irikura K, Moskowitz MA. Dual role of nitric oxide in focal cerebral ischemia. Neuropharmacology 1994;33:1447 – 52. [35] Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 1995;377:239 – 42. [36] Huang Z, Huang PL, Ma J, Meng W, Ayata C, Fishman MC, Moskowitz MA. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine. J Cereb Blood Flow Metab 1996;16:981 –7.
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[37] Endres M, Laufs U, Huang Z, Nakamura T, Huang P, Moskowitz MA, Liao JK. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci USA 1998;95:8880 –5. [38] Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ, Sessa WC, Walsh K. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med 2000;6:1004 – 10. [39] Lefer AM, Campbell B, Shin YK, Scalia R, Hayward R, Lefer DJ. Simvastatin preserves the ischemic-reperfused myocardium in normocholesterolemic rat hearts. Circulation 1999;100:178 – 84. [40] Hernandez-Perera O, Perez-Sala D, Soria E, Lamas S. Involvement of rho GTPases in the transcriptional inhibition of preproendothelin-1 gene expression by simvastatin in vascular endothelial cells. Circ Res 2000;87:616 – 22. [41] Essig M, Nguyen G, Prie D, Escoubet B, Sraer JD, Friedlander G. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase fibrinolytic activity in rat aortic endothelial cells. Role of geranylgeranylation and Rho proteins. Circ Res 1998;83:683 – 90.
Statins and ischemic stroke James K. Liao * Vascular Medicine Research, Brighan & Women’s Hospital Ha6ard Medical School, 65 Landsdowne Street, Room 275, Cambridge, Massachusetts, 02139, USA
Abstract The 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors or statins are potent inhibitors of cholesterol synthesis. Several large clinical trials have demonstrated that these agents reduce serum cholesterol levels and the incidence of cardiovascular diseases. However, overlap and meta-analyses of these clinical trials suggest that the beneficial effects of statins may extend beyond their effects on serum cholesterol levels. Because statins also inhibit the synthesis of isoprenoid intermediates in the cholesterol biosynthetic pathway, they may have pleiotropic effects on the vascular wall. In particular, the ability of statins to decrease the incidence of ischemic stroke highlights some of their non-cholesterol effects since serum cholesterol levels are poorly correlated with the risk for ischemic stroke. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Endothelium; Nitric oxide; Isoprenoids; Cholesterol; G-proteins; Gene regulation; Stroke
1. Introduction Recent large clinical trials have demonstrated that a class of cholesterol-lowering agents called statins decrease the incidence of myocardial infarctions and ischemic strokes in hypercholesterolemic and atherosclerotic individuals [1 – 4]. These agents inhibit an early step in cholesterol biosynthesis by blocking the conversion of 3-hydroxy-3-methylglutaryl (HMG)-CoA to mevalonate. Because serum cholesterol level is strongly associated with coronary atherosclerotic disease [5], it has been generally assumed that cholesterol reduction by statins is the predominant, if not the only mechanism, underlying their beneficial effects in cardiovascular diseases. However, subgroup analyses of large clinical trials have challenged this notion and suggest that the beneficial effects of statins may extend to mechanisms beyond cholesterol reduction [6,7], possibly involving direct effects on the vascular wall. Subgroup analyses of the WOSCOP, CARE, and LIPID trials indicate that despite comparable serum cholesterol levels, statin-treated individuals have significantly lower risks for coronary heart disease compared to age-matched placebo-controlled individuals [2,4,7,8]. Furthermore, meta-analyses of past clinical trials sug* Tel.: + 1-617-768-8424; fax: + 1-617-768-8425. E-mail address: [email protected] (J.K. Liao).
gest that the risk of myocardial infarctions in individuals treated with statins is significantly lower compared to individuals treated with other cholesterol-lowering agents or modalities despite comparable reduction in serum cholesterol levels in both groups [9,10]. Taken together, these findings suggest that some of the beneficial effects of statins may be due to their cholesterol-independent effects on the vascular wall.
2. Statins and endothelial function Hypercholesterolemia impairs endothelial function and endothelial dysfunction is one of the earliest markers of atherosclerosis, occurring even in the absence of angiographic evidence of disease [11,12]. The vascular endothelium serves as an important autocrine/paracrine organ that regulates vascular wall contractile state and cellular composition. An important characteristic of endothelial dysfunction is the impaired synthesis, release and activity of endothelial-derived nitric oxide (NO). Endothelial NO has been shown to inhibit several components of the atherogenic process. For example, endothelium-derived NO mediates vascular relaxation and inhibits platelet aggregation, vascular smooth muscle proliferation and endothelial-leukocyte interactions. In addition, factors which limit the bioavailability of NO such as superoxide anion lead to
0021-9150/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 5 6 7 - 5 6 8 8 ( 0 1 ) 0 0 0 1 1 - 3
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J.K. Liao / Atherosclerosis 3 (2002) 21–25
nitrate tolerance, vasoconstriction, and hypertension [13,14]. Improvement in endothelial function by statins can be attributed to the lowering of serum cholesterol levels since hypercholesterolemia impairs endothelial function [13]. Indeed, acute lowering of serum cholesterol by plasma LDL apheresis rapidly improves endotheliumdependent vasodilatation [15]. However, in some studies, restoration of endothelial function by statins occurs before significant reduction in serum cholesterol levels [16–18] suggesting that there are additional effects on endothelial function beyond that of cholesterol reduction. Indeed, statins upregulate endothelial nitric oxide synthase (eNOS) expression and activity and reverse the downregulation of eNOS expression by hypoxia and oxidized low-density lipoprotein (ox-LDL) under cholesterol-clamped conditions [19,20]. Therefore, it is possible that statins may have other beneficial effects, irrespective of serum cholesterol levels, in conditions associated with endothelial dysfunction such as atherosclerosis, pulmonary hypertension, and congestive heart failure.
3. Statins, mevalonate pathway, and eNOS By inhibiting L-mevalonate synthesis, statins also prevent the synthesis of other important isoprenoid intermediates of the cholesterol biosynthetic pathway, such as farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) [21]. These intermediates serve as important lipid attachments for the post-translational modification of a variety of proteins, including the g subunit of heterotrimeric G-proteins, Heme-a, nuclear lamins, and small GTP-binding protein Ras, and Ras-like proteins, such as Rho, Rab, Rac, Ral or Rap [22]. Thus, protein isoprenylation permits the covalent attachment, subcellular localization, and intracellular trafficking of membrane-associated proteins such as Rho. Members of the Ras and Rho GTPase family are major substrates for post-translational modification using isoprenoids [22– 24]. In endothelial cells, Ras translocation from the cytoplasm to the plasma membrane is dependent upon farnesylation while Rho translocation is dependent upon geranylgeranylation [25,26]. Statins inhibit both Ras and Rho isoprenylation and lead to accumulation of inactive Ras and Rho in the cytoplasm. While the effects of statins on Ras and Rho isoprenylation are reversed in the presence of FPP and GGPP, respectively, the effects of statins on eNOS expression are only reversed with GGPP and not by FPP or LDL-cholesterol [25]. These findings are consistent with a non-cholesterol-lowering effect of statins and suggest that inhibition of Rho by statins upregulates eNOS expression. Indeed, statins upregulate eNOS expression
by prolonging eNOS mRNA half-life but not eNOS gene transcription [20]. Since hypoxia, oxidized LDL, and cytokines such as TNF-a decrease eNOS expression by reducing eNOS mRNA stability, the ability of statins to prolong eNOS half-life may make them effective agents in counteracting conditions which downregulate eNOS expression. Indeed, statins prevent the downregulation of eNOS by oxidized LDL and TNF-a and under hypoxic conditions [19,20]. Because Rho is a major target of geranylgeranylation, inhibition of Rho and its downstream target, the actin cytoskeleton, is a likely mechanism by which statins upregulate eNOS expression. The evidence that Rho-mediated actin cytoskeletal changes negatively regulate eNOS expression comes from three sets of experiments. First, direct inhibition of Rho by Clostridium botulinum C3 transferase increases eNOS expression independent of isoprenylation. The C3 transferase ADP-ribosylates asparagine-41 of Rho and renders it biologically inactive in the GDP-bound state [23]. Second, inhibition of Rho by overexpression of a dominant-negative RhoA mutant, N19RhoA, also increases eNOS expression [25]. Finally, disruption of the actin cytoskeleton leads to increases in eNOS expression in vitro and in vivo [27]. These results, therefore, identify Rho and the actin cytoskeleton as a negative regulator of eNOS expression (Fig. 1).
4. Statins and ischemic stroke Ischemic stroke is the third leading cause of death in the United States and is a major cause of long-term disability, especially in the elderly population and in patients undergoing cardiovascular surgery. Currently, few therapeutic options are available for the treatment
Fig. 1. Upregulation of eNOS by statins. Statins block the synthesis of isoprenoids and cholesterol. The isoprenoid, geranylgeranyl (GG) pyrophosphate (PP), is important for the membrane translocation and function of Rho. Rho, in turn, negatively regulates the expression of eNOS via effects on the actin cytoskeleton.
J.K. Liao / Atherosclerosis 3 (2002) 21–25
of ischemic strokes with prophylactic treatment strategies limited mainly to agents which block platelet aggregation or the coagulation cascade [28]. Although clinical trials have demonstrated the effectiveness of antiplatelet agents in reducing the incidence of ischemic strokes, these agents, however, do not address the larger issue of reducing cerebral infarct size. In fact, only therapies directed at restoring cerebral blood flow such as tissue-type plasminogen activator (t-PA) have been shown to be effective in the acute treatment of ischemic stroke [29,30]. These studies, therefore, suggest that neuroprotectants are relatively ineffective if cerebral blood flow is not restored and that cerebral blood flow is probably the single most important determinant of stroke severity. An intriguing result of large clinical trials with statins is the reduction in ischemic stroke [31]. Although myocardial infarction is closely associated with serum cholesterol levels, neither the Framingham Study nor the Multiple Risk Factor Intervention Trial (MRFIT) demonstrated significant correlation between ischemic stroke and serum cholesterol levels [32,33]. Thus, the findings of these large statin trials raise the interesting question of how a class of cholesterol-lowering agents can reduce ischemic stroke when ischemic stroke is not related to cholesterol levels. It appears likely that there are pleiotropic effects of statins, which are beneficial for ischemic stroke. Some of these beneficial effects may be attributed to the effects of statins on endothelial function and the vascular wall. Cerebral vascular tone and blood flow are regulated by endothelium-derived NO [34]. Mutant mice lacking eNOS (eNOS − /−) are relatively hypertensive and develop greater proliferative and inflammatory response to vascular injury [35]. Indeed, eNOS−/ − mice develop larger cerebral infarcts following cerebrovascular occlusion [36]. Thus, the beneficial effects of statins in ischemic stroke may be due, in part, to their ability to upregulate eNOS expression and activity. Indeed, mice which were prophylactically treated with statins for up to 2 weeks, have 25–30% higher cerebral blood flow and 50% smaller cerebral infarct sizes following cerebrovascular occlusion [37]. Furthermore, no increase in cerebral blood flow or neuroprotection was observed in eNOS − /− mice treated with statins indicating that the upregulation of eNOS accounts for most, if not all, of the neuroprotective effects of these agents. A recent study showed that statins can also activate eNOS via the phosphatidylinositol 3-kinase/protein kinase Akt pathway [38]. However, direct inhibition of Rho or the actin cytoskeleton mimics the effect of statins on eNOS upregulation and stroke protection [27], suggesting that inhibition of isoprenoid synthesis and Rho is the predominant mechanism by which statins exert their neuroprotective effects (Fig. 2). Interestingly, treatment with statins did not affect blood
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Fig. 2. Neuroprotective effect of statins. Statins upregulate eNOS by inhibiting Rho geranylgeranylation. This leads to vasodilatation, increase in cerebral blood flow, and protection against ischemic stroke.
pressure or heart rate before, during, or after cerebrovascular ischemia and did not alter serum cholesterol levels in mice, consistent with the previous finding that rodents are relatively resistant to changes in steady-state cholesterol levels by statins [37]. In addition to increases in cerebral blood flow, other beneficial effects of statins are likely to occur. For example, Lefer et al. reported that statins attenuate P-selectin expression and leukocyte adhesion via increases in NO production in a model of cardiac ischemia and reperfusion [39]. Statins have also been shown to decrease the expression of the potent vasoconstrictor/mitogen, endothelin-1, by inhibiting preproendothelin-1 gene transcription [40]. Others have reported that statins upregulate tissue-type plasminogen activator (t-PA) and downregulate plasminogen activator inhibitor (PAI)-1 expression through a similar mechanism involving inhibition of Rho geranylgeranylation [41]. Thus, the absence of neuroprotection in eNOS-deficient mice emphasizes the importance of endothelium-derived NO in not only augmenting cerebral blood flow, but also, potentially, in limiting the impact of endothelin-1-induced vasoconstriction and platelet and white blood cell accumulation on tissue viability following ischemia. Thus, it is highly likely that statins may decrease in the incidence of ischemic strokes in clinical trials, in part, by reducing cerebral infarct size to levels that are clinically unappreciated. Since most ischemic strokes occur in individuals with average cholesterol levels who may not otherwise be taking statin therapy, the upregulation of eNOS by statins may be an effective prophylactic treatment strategy for limiting the severity of ischemic strokes in normocholesterolemic high risk individuals (i.e. such as patients with history of stroke or transient ischemic attacks) or for patients undergoing elective cardiovascular procedures which have high peri-operative risk for cerebral ischemia (such as coronary artery bypass graft surgery or carotid endarterectomy). Because statins increase cerebral blood flow, these agents may serve as a useful adjunctive therapeutic modality for increasing the delivery of other co-administered drugs to the CNS. Finally, since eNOS expression is upregulated in the systemic vasculature, HMG-CoA reductase inhibitors
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J.K. Liao / Atherosclerosis 3 (2002) 21–25
may also have beneficial effects in other cardiovascular conditions where endothelial dysfunction may cause or worsen the pathological process such as atherosclerosis, pulmonary hypertension, and congestive heart failure.
5. Conclusions Statins exert many pleiotropic effects in addition to the lowering of serum cholesterol levels. Most of these effects, especially on the upregulation of eNOS, are mediated by statin’s inhibitory effect on isoprenoid synthesis. These effects of statins, therefore, may explain their ability to reduce the incidence of ischemic stroke whose risk is poorly tied to serum cholesterol levels.
Acknowledgements The work described in this paper was supported in part by the National Institutes of Health (HL-52233, HL-34836, and NS-10828) and the American Heart Association. Dr Liao is an Established Investigator and a Bugher Foundation Awardee of the American Heart Association.
References [1] Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 1994;344:1383 –9. [2] The Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID) Study Group. Prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. N Engl J Med 1998;339:1349 –57. [3] Packard CJ. Influence of pravastatin and plasma lipids on clinical events in the West of Scotland Coronary Prevention Study (WOSCOPS). Circulation 1998;97:1440 – 5. [4] Sacks FM, Pfeffer MA, Moye LA, Rouleau JL, Rutherford JD, Cole, TG, Brown L, Warnica JW, Arnold JM, Wun CC, Davis BR, Braunwald E. Cholesterol and Recurrent Events Trial Investigators. The effect of pravastatin on coronary events after myocardial infarction in patients with average cholesterol levels. N Engl J Med 1996;335:1001 –9. [5] Klag MJ, Ford DE, Mead LA, He J, Whelton PK, Liang KY, Levine DM. Serum cholesterol in young men and subsequent cardiovascular disease. N Engl J Med 1993;328:313 – 8. [6] Blum CB. Comparison of properties of four inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Am J Cardiol 1994;73:3D – 11D. [7] Massy ZA, Keane WF, Kasiske BL. Inhibition of the mevalonate pathway: benefits beyond cholesterol reduction? Lancet 1996;347:102 – 3. [8] Shepherd J, Cobbe SM, Ford, I, Isles CG, Lorimer AR, MacFarlane PW, McKillop JH, Packard CJ. West of Scotland Coronary Prevention Study Group. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. N Engl J Med 1995;333:1301 –7.
[9] Brown BG, Zhao XQ, Sacco DE, Albers JJ. Lipid lowering and plaque regression. New insights into prevention of plaque disruption and clinical events in coronary disease. Circulation 1993;87:1781 – 91. [10] Pekkanen J, Linn S, Heiss G, Suchindran CM, Leon A, Rifkind BM, Tyroler HA. Ten-year mortality from cardiovascular disease in relation to cholesterol level among men with and without preexisting cardiovascular disease. N Engl J Med 1990;322:1700 – 7. [11] Liao JK. Endothelium and acute coronary syndromes. Clin Chem 1998;44:1799 – 808. [12] Selwyn AP, Kinlay S, Libby P, Ganz P. Atherogenic lipids, vascular dysfunction, and clinical signs of ischemic heart disease. Circulation 1997;95:5 – 7. [13] Harrison DG. Cellular and molecular mechanisms of endothelial cell dysfunction. J Clin Invest 1997;100:2153 – 7. [14] Munzel T, Sayegh H, Freeman BA, Tarpey MM, Harrison DG. Evidence for enhanced vascular superoxide anion production in nitrate tolerance. A novel mechanism underlying tolerance and cross-tolerance. J Clin Invest 1995;95:187 – 94. [15] Tamai O, Matsuoka H, Itabe H, Wada Y, Kohno K, Imaizumi T. Single LDL apheresis improves endothelium-dependent vasodilatation in hypercholesterolemic humans. Circulation 1997;95:76 – 82. [16] Anderson TJ, Meredith IT, Yeung AC, Frei B, Selwyn AP, Ganz P. The effect of cholesterol-lowering and antioxidant therapy on endothelium-dependent coronary vasomotion. N Engl J Med 1995;332:488 – 93. [17] O’Driscoll G, Green D, Taylor RR. Simvastatin, an HMG-coenzyme A reductase inhibitor, improves endothelial function within 1 month. Circulation 1997;95:1126 – 31. [18] Treasure CB, Klein JL, Weintraub WS, Talley JD, Stillabower ME, Kosinski AS, Zhang J, Boccuzzi SJ, Cedarholm JC, Alexander RW. Beneficial effects of cholesterol-lowering therapy on the coronary endothelium in patients with coronary artery disease. N Engl J Med 1995;332:481 – 7. [19] Laufs U, Fata VL, Liao JK. Inhibition of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase blocks hypoxia-mediated downregulation of endothelial nitric oxide synthase. J Biol Chem 1997;272:31725 – 9. [20] Laufs U, Fata VL, Plutzky J, Liao JK. Upregulation of endothelial nitric oxide synthase by HMG CoA reductase inhibitors. Circulation 1998;97:1129 – 35. [21] Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature 1990;343:425 – 30. [22] Van Aelst L, D’Souza-Schorey C. Rho GTPases and signaling networks. Genes Dev 1997;11:2295 – 322. [23] Aktories K. Bacterial toxins that target Rho proteins. J Clin Invest 1997;99:827 – 9. [24] Hall A. Rho GTPases and the actin cytoskeleton. Science 1998;279:509 – 14. [25] Laufs U, Liao JK. Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase. J Biol Chem 1998;273:24266 – 71. [26] Laufs U, Liao JK. Targeting rho in cardiovascular disease. Circ Res 2000;87:526 – 8. [27] Laufs U, Endres M, Stagliano N, Amin-Hanjani S, Chui DS, Yang SX, Simoncini T, Yamada M, Rabkin E, Allen PG, Huang PL, Bohm M, Schoen FJ, Moskowitz MA, Liao JK. Neuroprotection mediated by changes in the endothelial actin cytoskeleton. J Clin Invest 2000;106:15 – 24. [28] Patrono C. Aspirin as an antiplatelet drug. N Engl J Med 1994;330:1287 – 94. [29] Caplan LR. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med 1999;341:1240 – 1. [30] The National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med 1995;333:1581 – 7.
J.K. Liao / Atherosclerosis 3 (2002) 21–25 [31] Crouse JR, Byington RP, Furberg CD. HMG-CoA reductase inhibitor therapy and stroke risk reduction: an analysis of clinical trials data. Atherosclerosis 1998;138:11 –24. [32] Multiple Risk Factor Intervention Trial Research Group. Multiple risk factor intervention trial. Risk factor changes and mortality results. J Am Med Assoc 1982;248:1465 –77. [33] Sytkowski PA, Kannel WB, D’Agostino RB. Changes in risk factors and the decline in mortality from cardiovascular disease. The Framingham Heart Study. N Engl J Med 1990;322:1635 – 41. [34] Dalkara T, Yoshida T, Irikura K, Moskowitz MA. Dual role of nitric oxide in focal cerebral ischemia. Neuropharmacology 1994;33:1447 – 52. [35] Huang PL, Huang Z, Mashimo H, Bloch KD, Moskowitz MA, Bevan JA, Fishman MC. Hypertension in mice lacking the gene for endothelial nitric oxide synthase. Nature 1995;377:239 – 42. [36] Huang Z, Huang PL, Ma J, Meng W, Ayata C, Fishman MC, Moskowitz MA. Enlarged infarcts in endothelial nitric oxide synthase knockout mice are attenuated by nitro-L-arginine. J Cereb Blood Flow Metab 1996;16:981 –7.
25
[37] Endres M, Laufs U, Huang Z, Nakamura T, Huang P, Moskowitz MA, Liao JK. Stroke protection by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitors mediated by endothelial nitric oxide synthase. Proc Natl Acad Sci USA 1998;95:8880 –5. [38] Kureishi Y, Luo Z, Shiojima I, Bialik A, Fulton D, Lefer DJ, Sessa WC, Walsh K. The HMG-CoA reductase inhibitor simvastatin activates the protein kinase Akt and promotes angiogenesis in normocholesterolemic animals. Nat Med 2000;6:1004 – 10. [39] Lefer AM, Campbell B, Shin YK, Scalia R, Hayward R, Lefer DJ. Simvastatin preserves the ischemic-reperfused myocardium in normocholesterolemic rat hearts. Circulation 1999;100:178 – 84. [40] Hernandez-Perera O, Perez-Sala D, Soria E, Lamas S. Involvement of rho GTPases in the transcriptional inhibition of preproendothelin-1 gene expression by simvastatin in vascular endothelial cells. Circ Res 2000;87:616 – 22. [41] Essig M, Nguyen G, Prie D, Escoubet B, Sraer JD, Friedlander G. 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors increase fibrinolytic activity in rat aortic endothelial cells. Role of geranylgeranylation and Rho proteins. Circ Res 1998;83:683 – 90.