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ATVB In Focus Endothelium: Signaling, , and Gene Expression Series Editor: Frank M. Faraci

Endothelium-Dependent Effects of

Sebastian Wolfrum, Kristin S. Jensen, James K. Liao

Abstract—The vascular endothelium is a dynamic endocrine organ that regulates contractile, secretory, and mitogenic activities in the vessel wall and hemostatic processes within the vascular lumen. Risk factors for , such as cigarette smoking, hypertension, and elevated serum lipid levels, impair endothelial function and lead to the development of atherosclerotic vessels. Recent studies suggest that statins reduce cardiovascular events in part by improving endothelial function. Statins reduce plasma cholesterol levels, thereby decreasing the uptake of modified lipoproteins by vascular wall cells. There is increasing evidence, however, that statins may also exert effects beyond cholesterol lowering. Indeed, many of these cholesterol-independent or “pleiotropic” vascular effects of statins appear to involve restoring or improving endothelial function through increasing the of , promoting re-endothelialization, reducing oxidative stress, and inhibiting inflammatory responses. Thus, the endothelium- dependent effects of statins are thought to contribute to many of the beneficial effects of therapy in cardiovascular disease. (Arterioscler Thromb Vasc Biol. 2003;23:729-736.) Key Words: 3-hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitor Ⅲ endothelium Ⅲ atherosclerosis Ⅲ cholesterol Ⅲ protein kinase Akt

he vascular endothelium is the inner lining of tion.8 The activated endothelium expresses cell–surface ad- Tvessels and serves as an important autocrine/paracrine hesion molecules, such as vascular cell adhesion molecule-1, organ that regulates vascular wall contractile state and cellu- intercellular adhesion molecule-1, and endothelial-leukocyte lar composition. Because of its strategic location between the adhesion molecule, which facilitate the attachment of circu- circulation and the vascular wall, the endothelium interacts lating leukocytes to the endothelium.9 Monocyte adhesion to with both cellular and hormonal mediators from these two the vessel wall and its subsequent differentiation into macro- compartments. There is growing evidence that endothelial phages are crucial events leading to the development of dysfunction, which is often defined as the decreased synthe- macrophage-derived foam cells in atherosclerotic plaques. sis, release, and/or activity of endothelial-derived nitric oxide Cytokines, oxidized LDLs (ox-LDLs), and infectious agents, (NO), is an important factor leading to atherosclerosis and such as cytomegalovirus and Chlamydia pneumonia, promote acute coronary syndromes.1,2 This realization, in part, is based vascular oxidation and inflammation, which lead to endothe- on the fact that the lack of NO in atherosclerotic vessels lial cell activation.10,11 Thus, endothelial dysfunction and contributes to impaired vascular relaxation,3 aggre- activation caused by coronary risk factors and vascular gation,4 increased vascular smooth muscle proliferation,5 and inflammation are the basis for the development of atheroscle- enhanced leukocyte adhesion to the endothelium.6 Indeed, , platelet activation, and thrombosis caused rotic lesions. by the rupture of atherosclerotic plaques are primary features of acute coronary syndromes and other cardiovascular Endothelial Dysfunction and Atherosclerosis events.7 The endothelium produces vasoactive substances in response In addition to endothelial dysfunction, another important to environmental factors, such as blood flow, oxygen tension, feature of atherosclerotic vessels is endothelial cell activa- and receptor-mediated .12–14 Endothelium-

Received December 10, 2002; revision accepted February 11, 2003. From the Vascular Medicine Research, Brigham & Women’s Hospital and Harvard Medical School, Cambridge, Mass. Correspondence to James K. Liao, MD, Brigham & Women’s Hospital, 65 Landsdowne Street, Room 275, Cambridge, MA 02139. E-mail [email protected] © 2003 American Association, Inc. Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org DOI: 10.1161/01.ATV.0000063385.12476.A7 729 730 Arterioscler Thromb Vasc Biol. May 2003

Figure 1. ox-LDL impairs endothelial func- tion and leads to endothelial cell activation. Statins counteract these effects by reduc- ing circulating LDL levels and in part by direct actions on the endothelial cells, lead- ing to an increased activity of eNOS, a reduced expression of vasoconstricting agents, and a decreased production of reactive oxygen species.

dependent vascular relaxation is predominantly mediated by mechanisms by which LDL inhibits endothelial-derived NO NO and to lesser degrees by prostacyclin and activators of activity include downregulation of endothelial NOS expres- ATP- and -sensitive potassium channels (ie, hyperpo- sion,22 decreased receptor-mediated NO release,23 and NO larizing factor). Endothelium-derived vasoconstrictive sub- inactivation via increases in superoxide anion production.24 15 stances include (ET)-1 and thromboxane A2. The Furthermore, LDL facilitates the development of atheroscle- balance between these opposing endothelium-derived vaso- rosis by enhancing monocyte adhesion to endothelial cells in active substances under normal and pathological conditions vitro,25 a process that might be mediated by an increased ultimately determines the contractile and perhaps the mito- expression of adhesion molecules, such as intercellular adhe- genic state of the underlying vascular smooth muscle. Endo- sion molecule-1 (Figure 1).26 thelium-derived NO plays an important physiological role in the regulation of , and blood flow and has been Statins and Endothelial Function widely used as a clinical marker of endothelial function. Hypercholesterolemia impairs endothelial function and by Indeed, endothelial dysfunction or decreased NO bioavail- blocking the conversion of 3-hydroxy-3-methylglutaryl coen- ability is often associated with cardiovascular diseases, such zyme A (HMG-CoA) to mevalonate, statins inhibit an early as atherosclerosis.3 rate-limiting step in cholesterol biosynthesis. This leads to Endothelial dysfunction may also be attributed to abnormal increased hepatic LDL receptors and enhanced uptake of or excessive release of vasoconstricting substances, such as cholesterol by the liver. Indeed, therapeutic doses of statins ET-1. Circulating levels and tissue immunoreactivity of ET-1 potently reduce serum cholesterol levels in humans,27 and are elevated in patients with advanced atherosclerosis and several large clinical trials have demonstrated that inhibition acute coronary syndromes.16,17 Whereas NO is vasodilatory of HMG-CoA reductase by statins markedly decreases the and antiproliferative in its effects on the underlying vascular incidence of cardiovascular events in hypercholesterolemic smooth muscle, ET-1 is vasoconstrictive and mitogenic. individuals.27–30 Because of the strong association between Exposure to cardiovascular risk factors, such as ox-LDL, elevated serum cholesterol levels and coronary atheroscle- enhances the production and release of ET-1.18 Increased rotic disease, reduction of serum cholesterol levels by statins ET-1 levels in combination with platelet-derived growth has been proposed to be the predominant mechanism under- factors promote vascular smooth muscle proliferation in the lying the beneficial effects of statins. Indeed, acute plasma neointima of atherosclerotic lesions.19 Although definitive LDL apheresis improves endothelium-dependent vasodilata- proof that ET-1 is a primary inducer of atherosclerosis is still tion,31 suggesting that statins could restore endothelial func- elusive, it is highly likely that ET-1 is at least an important tion, in part, by lowering serum cholesterol levels. contributor to the atherogenic process. In some studies with statins, however, restoration of Numerous studies demonstrate that endothelial dysfunction endothelial function occurs before significant reduction in is one of the earliest manifestations of atherosclerosis, even in serum cholesterol levels,32,33 suggesting that there are addi- the absence of angiographic evidence of disease.20 Con- tional effects on endothelial function beyond that of choles- versely, improved endothelial function is one of the earliest terol reduction. In a recent study in normocholesteremic clinical markers after atherogenic risk factor modification. patients, improvement of endothelial function was observed There is a strong association between atherogenic risk factors within 3 hours after a single bolus of cerivastatin.34 Indeed, in and endothelial dysfunction. For example, LDL, especially patients, statin therapy has been found to rapidly improve ox-LDL, is a potent inhibitor of endothelial function.21 The vasomotor response to endothelium-dependent agonists,35 Wolfrum et al Statin Pleiotropism and Endothelial Function 731

Figure 2. Inhibition of HMG-CoA reductase by statins decreases the synthesis of isoprenoids and cholesterol. The isoprenoid geranylgeranyl (GG) is an important lipid attachment for Rho, which per- mits the subsequent membrane translocation and activation of Rho and Rho kinase. Inhibition of Rho/Rho-kinase activity by statins leads to increase in eNOS expression and activity. Further- more, statins activate eNOS by stimulating the PI3 kinase/Akt pathway by a yet-unidentified mechanism.

enhance coronary blood flow,36 and reduce the levels of attachment, subcellular localization, and intracellular traffick- adhesion molecules.37 The mechanism is caused, in part, by ing of several membrane-associated proteins. Although the statin’s ability to increase endothelial NO production by effects of statins on Ras and Rho isoprenylation are reversed stimulating and upregulating endothelial in the presence of FPP and GGPP, respectively, the effect of (eNOS).38,39 Furthermore, statins have been shown to restore statins on eNOS expression is only reversed with GGPP and eNOS activity in the presence of hypoxia40 and ox-LDL,39 not by FPP or LDL–cholesterol.49 Indeed, direct inhibition of conditions that lead to endothelial dysfunction. Statins also geranylgeranyltransferase or Rho leads to increases in eNOS increase the expression of tissue-type plasminogen activator expression.49,50 These findings are consistent with a noncho- (t-PA)41 and inhibit the expression of endothelin-1, a potent lesterol-lowering effect of statins and suggest that inhibition vasoconstrictor and mitogen.42 Statins, therefore, exert many of Rho by statins mediates the increase in eNOS expression. favorable effects on the endothelium and attenuate endothe- Indeed, statins upregulate eNOS expression by prolonging lial dysfunction in the presence of atherosclerotic risk factors eNOS mRNA half-life but not eNOS gene transcription.49 (Figure 1). Because hypoxia, ox-LDL, and cytokines, such as tumor necrosis factor-␣, decrease eNOS expression by reducing Statin Pleiotropism and Endothelial Function eNOS mRNA stability, the ability of statins to prolong eNOS A number of recent studies have focused on the pleiotropic half-life may make them effective agents in counteracting effects of statins. It has been reported that statins decrease the conditions which downregulate eNOS expression. extent of cerebral and myocardial /reperfusion injury Furthermore, it has been recently reported by Kureishi et in rodents without changes in serum cholesterol levels.43–46 al38 that statins can activate protein kinase Akt. The serine– The vascular protective effects of statins were associated with threonine kinase Akt is an important regulator of various increased blood flow, attenuated P-selectin expression, and cellular processes, including cell and apoptosis.51 leukocyte adherance.43,44 By using genetic and pharmacolog- Stimulation of receptor tyrosine kinases and G-protein– ical approaches, the role of eNOS in cholesterol independent coupled receptors lead to activation phosphatidylinositol protection by statins was also delineated in in vivo models. 3-(PI3) kinase, the products of which, namely 3Ј phospholip- Pretreatment with statins increased eNOS mRNA expression ids, provoke the phosphorylation and activation of Akt.52 as well as eNOS activity, and protection by statins was Indeed, inhibitors of PI3 kinase, such as wortmannin, block completely abolished in eNOS knockout mice or after inhi- the effects of statins on Akt activation.38 Akt has shown to bition of eNOS with L-NAME.43,44,46 modulate several targets, such as caspase-9 and eNOS, by The realization that inhibition of HMG-CoA reductase by phosphorylation.53–55 Consequently, activation of Akt by statins not only reduces cholesterol production but also statins inhibits apoptosis and increases NO production in prevents the formation of various isoprenoid intermediates cultured endothelial cells.38 Therefore, in addition to stabiliz- has given rise to statin pleiotropism on the vascular wall, in ing eNOS mRNA by inhibition of Rho, there is increasing particular, the endothelium47 (Figure 2). Farnesylpyrophos- evidence that activation of the PI3 kinase/Akt pathway may phate (FPP) and geranylgeranylpyrophosphate (GGPP), for also contribute to the endothelium-dependent effects of st- example, serve as important lipid attachments for the post- atins although the precise mechanisms how PI3 kinase is translational modification of a variety of proteins, including activated by statins are not yet identified (Figure 2). the subunit of heterotrimeric G-proteins and small GTP- Because several vasoconstricting agents counteract the binding protein Ras, and Ras-like proteins, such as Rho, Rab, vasodilating effect of NO, endothelial dysfunction and devel- Rac, Ral, or Rap.48 Protein isoprenylation allows the covalent opment of atherosclerosis may also be attributed to the 732 Arterioscler Thromb Vasc Biol. May 2003 release of potent vasoconstrictors like ET-1 or II whereas another cholesterol lowering agent, cholestyramine, (Ang II). Indeed, circulating concentrations and tissue immu- had no effect, despite similar reduction in cholesterol level as noreactivity of ET-1 are increased in patients with severe that of statins.70 Finally, patients receiving statin therapy in atherosclerosis.16,17 ET-1 acts as a vasoconstrictive and mi- addition to antihypertensive drugs have a more pronounced togenic agent. Exposure to ox-LDL leads to an increased reduction in blood pressure, an effect that was independent production and release of ET-1,18 which promotes neointima from cholesterol lowering.71 It is interesting to speculate that proliferation of atherosclerotic lesions.19 Statins have been a reduction in blood pressure, and not cholesterol, by statins shown to inhibit preproET-1 mRNA expression in a may explain some of their protective effects in ischemic concentration-dependent manner and to reduce immunoreac- stroke, a disease that is not generally associated with elevated tive ET-1 in bovine endothelial cells, a phenomenon that has cholesterol levels. been suggested to be mediated by Rho proteins (Figure 1).42,56 Furthermore, statins attenuate the increased expression Statins and Endothelial Inflammatory Response of endothelin receptors achieved by basic fibroblastic growth Atherosclerosis is a complex inflammatory process that is factor.57 Ichiki et al58 recently reported that statins also characterized by the presence of monocytes or macrophages modulate the renin–angiotensin system. In the study, statins and T lymphocytes in the atheroma.72 Inflammatory cyto- downregulated the expression of angiotensin receptor subtype kines secreted by these macrophages and T lymphocytes can

1 (AT1) in a Rho A–dependent manner and attenuated the modify endothelial function, smooth muscle cell prolifera- biological function of Ang II. tion, collagen degradation, and thrombosis.73 An early step in Another potential mechanism by which statins may im- atherogenesis involves monocyte adhesion to the endotheli- prove endothelial function is through their antioxidant effects. um and penetration into the subendothelial space.72 Statins For example, statins attenuate Ang II–induced free radical have been shown to reduce the number of inflammatory cells production in vascular smooth muscle cells by inhibiting in atherosclerotic plaques and therefore possess anti- Rac1-mediated NAD(P)H oxidase activity and downregulat- inflammatory properties. The mechanisms have yet to be 59 ing angiotensin AT1-receptor expression. More recently, fully elucidated but may involve inhibition of adhesion Wassmann et al60 reported, that atorvastatin reduced vascular molecules such as intercellular adhesion molecule-1 and mRNA expression of essential NAD(P)H oxidase subunits cytokines as interleukins 6 and 8, which are involved in the p22phox and nox1 by a mechanism that might involve the recruitment of inflammatory cells.74,75 In addition, a recent translocation of Rac1 from the cytosol to the cell membrane. study has shown that statins can suppress the inflammatory Because NO is scavenged by ROS, these findings indicate response independent of HMG-CoA reductase inhibition by ␤ that the antioxidant properties of statins may also contribute binding directly to a novel regulatory site of the 2 integrin, to their ability to improve endothelial function (Figure 1). leukocyte function antigen-1. This regulatory site serves as a Furthermore, withdrawal of statin treatment in mice has been major counterreceptor for intercellular adhesion molecule-1 shown to impair endothelium-dependent relaxation by in- on leukocytes.76 The mechanism of the anti-inflammatory creasing vascular superoxide anion generation via a pathway properties of statins was further elucidated by Yoshida et al,77 involving the Rac-dependent activation of the gp91phox- who recently demonstrated that cerivastatin reduced mono- containing vascular NAD(P)H oxidase.61 ROS directly affects cyte adhesion to vascular endothelium by decreasing expres- the endothelial function, and the endothelium itself has also sion of integrins and actin polymerization through the inac- been shown to generate ROS.62 Because the amount of ROS tivation of RhoA. generated in the endothelium is relatively low, most studies A clinical marker of inflammation is high-sensitivity focus on other sources of ROS, such as smooth muscle cells C-reactive protein (hs-CRP).78 hs-CRP is an acute phase and leukocytes.60,63 reactant that is produced by the liver in response to proin- flammatory cytokines, such as interleukin 6, and reflects Statins and Blood Pressure Control low-grade systemic inflammation.79 Elevated levels of hs- Because statins can increase eNOS activity and inhibit the CRP have been shown to be predictive of increased risk for expression of vasoconstrictive substances, such as ET-1, it is coronary disease in apparently healthy men and wom- likely that statins, either alone or given with another agent, en.80,81 hs-CRP is elevated in patients with coronary artery will have some effect on systemic blood pressure. Indeed, in disease, coronary ischemia, and com- several animal models of hypertension, such as Dahl salt- pared with normal subjects.82–84 It has been suggested that sensitive rats and spontaneously hypertensive rats, statins CRP could contribute to the development of atherosclerosis reduce blood pressure and prevent hypertension-induced by binding to modified LDL within atherosclerotic plaques.85 glomerular injury.64–67 In contrast, Yamashita et al68 did not Once CRP becomes bound, it activates complement, which find a decrease in blood pressure in stroke-prone spontane- has been shown to play a role in promoting atherosclerotic ously hypertensive rats despite a marked reduction in pro- lesion progression.86 In two recent studies CRP has also been teinuria and renal fibrosis. Thus, it is not entirely clear shown to impair endothelial function by decreasing eNOS whether statins alone can decrease systemic blood pressure. expression in cultured endothelial cells.87,88 However, further Nevertheless, a reduction in cholesterol levels is correlated studies are needed to fully elucidate the role CRP plays in with a lower diastolic blood pressure.69 Indeed, a small atherosclerosis. crossover study with 26 hypertensive and diabetic patients Statin therapy lowers hs-CRP levels in hypercholesterol- revealed that statin therapy reduced diastolic blood pressure, emic patients.78,89 In the CARE trial, statins significantly Wolfrum et al Statin Pleiotropism and Endothelial Function 733

Endothelial-Dependent Effects of Statins

Effect Mediator Benefit Reduction in activity of NAD(P)H oxidase Rac1 Reduction of oxidative stress Decrease in synthesis of endothelin-1 Rho Improvement of endothelial function

Decrease in the expression of AT1-receptor Rho Improvement of endothelial function Decrease in the expression of tissue-type plasminogen activator Rho Reduction in thrombosis Increase in the expression of plasminogen activator inhibitor-1 Rho Reduction in thrombosis Decrease in the expression of adhesion molecules Leukocyte function antigen-1, Rho Reduction of inflammation Increase in eNOS activity Rho, Akt Improvement of endothelial function Increase in No. and differentiation of circulating Akt Increase in neovascularization and re-endothelialization endothelial cells Inhibition of apoptosis Akt Increase in cell survival decreased plasma hs-CRP levels over a 5-year period in statin treatment is associated with an accelerated re-endothe- patients who did not experience recurrent coronary events.90 lialization after carotid balloon injury. Similarly, an analysis of baseline and 1-year follow-up from In contrast, some studies report an antiangiogenic effect of the AFCAPS/TexCAPS study demonstrated that hs-CRP statins101,102 that might be mediated by RhoA.103 It has been levels were reduced in statin-treated patients who were free of suggested that these conflicting results are dose related. Low acute major coronary events.78 Furthermore, preliminary data doses of a statin may activate endothelial Ras and promote from the PRINCE study confirm that statin therapy can Akt and eNOS phosphorylation leading to an angiogenic significantly reduce serum hs-CRP levels in primary and effect, whereas higher statin doses are antiangiogenic al- secondary prevention populations.91 After 24 weeks of ther- though they promote an increase in eNOS protein expres- apy with a statin, the hs-CRP level was reduced by approx- sion.104 This suggestion remains controversial because high imately 13% in primary and secondary prevention popula- doses of statins have also been shown to be angiogenic105 and tions, whereas placebo treatment of subjects in the primary further studies are necessary to clarify this topic. prevention arm of the study had no effect. These studies, therefore, indicate that statins are effective in decreasing Statins and Endothelial Fibrinolysis systemic and vascular inflammation. However, any potential Plasminogen activator inhibitor type-1 (PAI-1) is the major clinical benefits conferred by the lowering of hs-CRP are endogenous inhibitor of t-PA, and it also plays a pivotal role in the regulation of fibrinolysis. High PAI-1 plasma levels difficult to separate from that of the lipid-lowering effects of and decreased levels of t-PA activity have been shown to be statins without performing further clinical studies. associated with coronary heart disease. PAI-1 mRNA has also been found in human atherosclerotic lesions, underlining its Statins and Re-endothelialization role in the development of these disorders.106 There is Stimulation of re-endothelialization or neovascularization is a increasing evidence from in vitro studies that statins posi- therapeutic aim to reduce ischemia-induced tissue injury. tively affect the fibrinolytic system of cultured smooth Postnatal neovascularization was mainly attributed to angio- muscle cells as well as endothelial cells. In these studies a genesis, for example, proliferation, migration, and remodel- decrease in PAI-1 and an increase in t-PA was observed after 92 ing of preexisting endothelial cells. However, some studies co-treatment with statins in endothelial cells.41,107 Several in recently demonstrated that marrow–derived circulating vivo studies have also investigated the effect of statin therapy endothelial cells are also involved in this process.93,94 Circu- ϩ on the regulation of the fibrinolytic system. Although the lating endothelial cells can be grown out of isolated CD133 results are inconsistent, in some studies statins decrease ϩ 93,95 or CD34 cells. Transplantation of these cells leads to PAI-1 plasma levels.108,109 Therefore, statins may interfere postnatal neovascularization in the ischemic hindlimb, aug- with the progression of the atherosclerotic plaque as well as ments ischemia-induced neovascularization in vivo,96 and with thrombotic events in hyperlipidemic patients, indepen- even improves postischemic cardiac function.97 dently of their ability to reduce plasma cholesterol, but further Recent studies revealed that statins also promote vasculo- studies have to delineate its physiological significance. genesis. Llevadot et al98 demonstrated in vitro that simvasta- tin evokes proliferation, migration, and cell survival of Summary circulating endothelial cells. The signal pathway for this It is well established that the reduction of plasma cholesterol effect includes activation of protein kinase Akt, which was by statins improves endothelial function and limit atheroscle- confirmed by functional blocking with dominant-negative rosis. In addition, statins may exert other cholesterol- Akt overexpression. Dimmeler et al99 showed in vitro and in independent effects on the endothelium, which lead to further vivo that statins do not only increase the number of circulat- improvements in vascular function (Table). Most of these ing endothelial cells but also induce their differentiation. This effects of statins are mediated by inhibition of isoprenoid might be of clinical relevance because it has been recently synthesis and increase in NO release or bioavailability. reported by Walter et al100 that induction of these cells under Because statins also reduce plasma cholesterol levels in 734 Arterioscler Thromb Vasc Biol. 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