Promoting Mechanisms of Vascular Health Circulating Progenitor Cells, Angiogenesis, and Reverse Cholesterol Transport

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provided by Elsevier - Publisher Connector Journal of the American College of Cardiology Vol. 53, No. 25, 2009 © 2009 by the American College of Cardiology Foundation ISSN 0735-1097/09/$36.00 Published by Elsevier Inc. doi:10.1016/j.jacc.2009.02.057

STATE-OF-THE-ART PAPER

Pedro R. Moreno, MD,* Javier Sanz, MD,* Valentin Fuster, MD, PHD,*† New York, New York; and Madrid, Spain

To understand and promote vascular health, we must reduce the aggression to the vessel wall and enhance the physiologic mechanisms leading to restoration of vessel wall function. Three main defense mechanisms are responsible for maintaining cardiovascular homeostasis: the regenerative production of endothelial progenitor cells, vessel wall angiogenesis, and macrophage-mediated reverse cholesterol transport. Endothelial progenitor cells can restore vessel wall function and reduce atherosclerosis. In patients with risk factors, high levels of circulating progenitor cells increase event-free survival from cardiovascular events. Mobilization of progenitor cells includes physical and pharmacological approaches, of which exercise and statin therapy have great potential. Angiogenesis is a pivotal defense mechanism to counteract hypoxia and is needed for plaque regression. How- ever, neovessels are susceptible for intraplaque hemorrhage, particularly in diabetes mellitus. In these patients, the haptoglobin 2-2 genotype is the more affected, and may beneﬁt from an antioxidant approach. Finally, the reverse cholesterol transport system is the main mechanism for plaque regression. In addition to high-density lipoprotein cholesterol, apolipoprotein A-I therapies and the promotion of cholesterol efﬂux from macrophages by the ABCA1 and ABCG1 transporter systems hold great promise and may be available for therapeutic application in the near future. (J Am Coll Cardiol 2009;53:2315–23) © 2009 by the American College of Cardiology Foundation

Independent of sex, race, or income, cardiovascular disease sion, triggers an inflammatory response, a defense mechanism (CVD) is by far the leading cause of death in the world, to restore arterial wall integrity. However, persistence of risk with an unsustainable economic burden for our society (1). factor–mediated arterial wall injury leads to endothelial dys- As a result, our health system needs to be revised to reduce function, atheromatous plaque formation (9), plaque rupture, cost and promote health. This implies a major shift of and thrombotic complications, the overall process of athero- mentality: from disease treatment to promotion of health. thrombosis (10,11). Simultaneously, inflammation also orches- At the vascular level, this can be achieved by reducing vessel trates a process of repair through 3 main defense mechanisms, wall injury and promoting physiologic repair. Vessel wall illustrated in Figure 1. These include: 1) endothelial repair by homeostasis is a fine balance between injury and the defense progenitor cells; 2) plaque neovascularization; and 3) reverse mechanisms of repair. This review will address these defense cholesterol transport. mechanisms to promote health, with special focus on the coronary arteries. Endothelial Repair by Progenitor Cells Atherosclerosis evolves from subendothelial retention of lipoproteins through the leaky and defective endothelium, Endothelial dysfunction finally leads to endothelial cell where these plasma molecules are modified (e.g., oxidized) and death, lipid entry, invasion of inflammatory cells, and become cytotoxic, proinflammatory, chemotaxic, and vascular smooth muscle cell proliferation. An initial func- proatherogenic (2). The response-to-injury hypothesis consid- tional impairment of the endothelial monolayer with a high ers inflammation as a central mechanism responsible for early chance of reversibility now turns into structural damage atherogenesis (3–8). Arterial wall injury, mostly mediated by (12). However, regeneration can occur. In 1997, Asahara aging, diabetes, smoking, hypercholesterolemia, and hyperten- et al. (13) identified bone marrow–derived circulating endothelial progenitor cells (EPCs) from the bloodstream and established their regenerative potential. EPCs are a sub-

From the *Zena and Michael A. Wiener Cardiovascular Institute, and the Marie- group of peripheral blood monocytes that express stem Josee and Henry R. Kravis Cardiovascular Health Center, The Mount Sinai School cell–like antigenic determinants including CD34 and the of Medicine, New York, New York; and †The Centro Nacional de Investigaciones vascular endothelial growth factor 2 receptor (14). Other Cardiovasculares (CNIC), Madrid, Spain. ϩ ϩ ϩ progenitor markers, including CD31 , CD133 , CD117 , Manuscript received November 14, 2008; revised manuscript received January 27, ϩ ϩ ϩ ϩ 2009, accepted February 6, 2009. P1H12 , cKit , Sca1 , and CXCR4 , may also be relevant 2316 Moreno et al. JACC Vol. 53, No. 25, 2009 Promoting Biological Pathways for Vascular Health June 23, 2009:2315–23

Ͼ Abbreviations (14). The homing process, medi- are observed in patients with carotid stenosis 70% (22). and Acronyms ated by the activation of chemo- EPC levels directly correlated with the ankle-brachial index, kines and perhaps activated plate- with extremely low levels observed in patients with athero- apolipoprotein A-I ؍ ApoA-I lets, provides the signals for sclerosis obliterans (22). Furthermore, diabetic patients with cholesterol ester ؍ CETP transporter protein dynamic trafﬁcking of EPCs coronary artery disease show a further reduction in the (14,15). Several studies have in- number of circulating EPCs (23). Most importantly, in- cardiovascular ؍ CVD disease vestigated the mechanistic pro- creased concentration and function of EPCs decrease the endothelial tective role of EPCs in experi- likelihood of severe coronary disease. For every 10-colony ؍ EPC progenitor cell mental animal models (16), with forming unit increase in EPCs, a patient’s likelihood for

.(hemoglobin recent inhibition of plaque for- multivessel coronary disease declined by 20% (23 ؍ Hb mation by local administration of -high-density EPCs and cardiovascular events. The number of circulat ؍ HDL lipoprotein EPCs in a rabbit model (17). We ing EPCs was associated with a reduced risk for cardiac high-density will now review the role of EPCs death and all other coronary events, as shown in Figure 2 ؍ HDL-C lipoprotein cholesterol in predicting CVD in patients (24). This and other studies suggest that decreased levels of haptoglobin with risk factors. circulating EPCs are seen in patients with coronary risk ؍ Hp EPC consumption, cardiovascu- factors and reflect senescence, endothelial dysfunction, im- lar risk factors, and disease. The paired vascular repair, and increased cardiovascular events. number of circulating and colony-forming units of EPCs is Modulating EPCs: implications for therapy. Enhance- associated with a lower risk score in men without history of ment of EPCs is considered one of the most promising CVD (18). A competent bone marrow can translate injury therapeutic alternatives for cardiovascular disease (25). The into productive EPC consumption and recruitment, re- process of EPC mobilization leads to accelerated re- storing endothelial function. However, a high risk factor endothelialization, successfully achieved by erythropoietin profile perpetuates injury, the bone marrow becomes and other growth factors (26,27). Physical exercise increases incompetent, and so the EPCs number in the process of nitric oxide availability, improving EPC regeneration (28). repair (19). Several pharmacological pathways may mobilize and in- EPC function in subclinical CVD. Several studies have crease EPCs, and statin therapy is the most studied so far evaluated the relationship between EPCs and the presence (29–33). Although statins may have a direct stimulating of subclinical atherosclerosis. In the elderly, reduced flow- effect on EPC synthesis and release into the circulation, the mediated brachial artery reactivity correlates with dysfunc- lipid-lowering and anti-inflammatory effects will reduce tional EPCs (20). The number of circulating EPCs is 44% aggression to the vascular wall. All together will eventually lower in diabetes mellitus, correlating with high levels of mitigate inflammation and finally passivate the system (34). hemoglobin (Hb) A1c (21) and the severity of diabetic Other potential pharmacological approaches to enhance arteriopathy in different vascular territories. Thus, in dia- EPCs include the peroxisome proliferator-activated recep- betic patients with carotid disease, the lowest levels of EPCs tor agonists, and medications involved in the renin-

Figure 1 Mechanisms of Vascular Health

Defense mechanisms responsible for maintaining endothelial and vessel wall homeostasis, including: 1) endothelial progenitor cells; 2) angiogenesis and plaque neovascularization; and 3) reverse cholesterol transport and plaque regression. LDL ϭ low-density lipoprotein; MMP ϭ matrix metalloproteinase; TF ϭ tissue factor. Modi- ﬁed, with permission, from Fuster et al. (10). JACC Vol. 53, No. 25, 2009 Moreno et al. 2317 June 23, 2009:2315–23 Promoting Biological Pathways for Vascular Health

a result, plaque neovascularization, either from pre-existing endothelial cells (angiogenesis [45]) or from bone marrow– derived EPCs (vasculogenesis [47]), is an essential defense mechanism to compensate hypoxia and restore homeostasis in the vessel wall (48,49). Major insights from the role of neovessels in atherosclerosis originally evolved from progression and regression experimental studies in primates (50). Disease progression is associated with a 10-fold increase in vessel wall flow through vasa vasorum, a defense mechanism response for the removal of waste products (51). As soon as the stimuli for progression stopped, flow through vasa vasorum started to diminish, coming back to baseline levels. Lipid content was reduced along with very low levels of inflammatory cells, leading to plaque regression (51,52). As a result, neovessels serve as a pathway for macrophages to get out of lipid-rich plaques and stabilize atherosclerotic plaques (52). Despite all of these beneficial effects, plaque neovascularization is associated with inflammation and may paradoxi- cally contribute to plaque rupture (53). Hence, an important question needs to be addressed. How does a defense mechanism fail and trigger complex disease? The answer appears to be intraplaque hemorrhage. Plaque neovessels are fragile structures, with single-layer endothelial cells prone to Figure 2 Improved Survival in Patients With High Levels of Circulating Endothelial Progenitor Cells leakage and/or rupture, allowing for extravasation of red blood cells (RBCs) (48). Erythrocyte membranes are very Cumulative event-free survival in an analysis of death from cardiovascular rich in cholesterol, contributing to lipid expansion (54). In causes at 12 months according to levels of circulating CD34ϩ KDRϩ endothelial progenitor cells at the time of enrollment. Reprinted, with permission, from addition, RBC lysis releases free Hb, generating reactive Werner et al. (24). oxygen species (ROS) and increasing lipid peroxidation and macrophage activation within the atherosclerotic plaque. angiotensin system (35–38). A better understanding of how Intraplaque hemorrhage, haptoglobin, and macrophage EPCs generate and interact with each other (39–41), as interaction in plaque stability. The heme iron compo- nent of Hb generates ROS and activates the proinflam- well as the identification of specific functions through ␬ imaging technology (42,43), may significantly contribute to matory nuclear transcription factor- B(55), leading to the enhancement of such defense mechanisms (44). inflammation. The haptoglobin (Hp) pathway promotes clearance of free Hb through the Hp-Hb complex. This Angiogenesis and Plaque Neovascularization clearance is finally scavenged by the macrophage receptor CD163 (56). Thus, the ability of the macrophage to Neovascularization is a pivotal defense mechanism to main- eliminate the Hp-Hb complex may influence plaque tain homeostasis and restore healthy tissue in wound heal- stability. However, the Hp genotype has been shown to ing, myocardial necrosis, chronic ischemia, and regeneration play a role in disease progression (57). Two classes of of heart muscle by stem cell therapy (45). In the normal alleles (Hp-1 and Hp-2) synthesize proteins that are vessel wall, oxygen is provided to the tunica intima by direct structurally and functionally distinct. Functionally, the diffusion from the lumen, whereas the tunic media and the Hp-1 allele protein product is superior to the Hp-2 allele adventitia are nurtured by vasa vasorum, maintaining met- protein. Multiple epidemiological studies have demon- abolic homeostasis and removing waste products. When an strated that diabetic individuals with the Hp 2-2 geno- atherosclerotic lesion evolves in the intima, the intima type (homozygous for the Hp-2 allele) are at 4 to 5 times becomes thicker, and the distance between the deep layers greater risk of cardiovascular events (58–61). This is of the intima and the luminal surface increases and ulti- related to a reduced clearance of macrophage-Hp-Hb mately exceeds the oxygen diffusion threshold (250 to 500 complex, favoring iron deposition, oxidative stress, and ␮m), resulting in local hypoxia and induction of neovascu- active macrophage accumulation (62–64). Furthermore, larization. Indeed, in rabbit aortic plaques thicker than 500 diabetic patients with the Hp 2-2 genotype exhibit a ␮m, the majority of viable macrophages present in the lipid severe down-regulation of the macrophage scavenger core were severely hypoxic, glucose-depleted, and adenosine receptor CD163 (65). It has been suggested that such triphosphate–depleted, a condition likely leading to macro- overall accumulation of activated macrophages failing to phage death and formation of a necrotic lipid core (46). As target Hb clearance may contribute, by matrix metallo- 2318 Moreno et al. JACC Vol. 53, No. 25, 2009 Promoting Biological Pathways for Vascular Health June 23, 2009:2315–23 proteinase expression, to the digestion of the internal ulating neovessel growth is subject to controversy. Proan- elastic lamina, and so to plaque disruption, as seen in giogenic therapy to promote the above mentioned de- Figure 3 (66). fense mechanism might lead to neovessel growth in Modulating deleterious effects of Hb genotype and oncogenic regions. On the other hand, antiangiogenic plaque neovascularization: implications for protective therapy to prevent erythrocyte-macrophage active accu- therapy. Increased oxidative stress in diabetic individuals mulation may have clinical detrimental effects (76–78). with the Hp 2-2 genotype may be antagonized by the use As a result, pharmacological or immunological modula- of antioxidant therapy. Although vitamin E does not tion of angiogenesis still faces difficult obstacles before prevent cardiovascular events in the overall population, a application for clinical therapy (79). subgroup analysis of diabetic individuals with the Hp 2-2 genotype in the Heart Outcomes Prevention Evaluation Reverse Cholesterol Transport study suggested a reduction in the primary composite events (67). Recent prospective studies are encouraging The third presumed defense mechanism to preserve vessel (68–70), but clearly require more extensive documenta- wall integrity is known as reverse cholesterol transport. tion before being considered for clinical application. Of Introduced by Glomset (80) in 1968, the term describes a interest, the Hp-Hb complex binds to apolipoprotein process by which extrahepatic (peripheral) cholesterol re- (Apo) A-I, inducing oxidation and leading to dysfunction turns to the liver for excretion in the bile and feces. of high-density lipoprotein (HDL) (71), as seen in Figure Unesterified (free) cholesterol is toxic to cells, and on the 4. Vitamin E decreased oxidative modification of HDL, basis of this concept, Ross and Glomset (81) proposed that improving HDL function in diabetic patients with the atherosclerosis is the result of an imbalance between depo- Hp 2-2 genotype (71). sition and removal of arterial cholesterol after endothelial Regarding potential therapies for modulation of plaque injury. This theory was further strengthened by the inverse neovascularization, statin therapy has shown plaque re- relationship between high-density lipoprotein cholesterol gression in experimental animals (72,73) and most re- (HDL-C) and cardiovascular disease. Increasing the cently in humans by means of magnetic resonance imag- HDL-C level by 1 mg/dl may reduce the risk of cardiovas- ing (74,75). Presumably, statins establish a concentration cular disease by 2% to 3%. This is consistent with the gradient tissue/blood of low-density lipoprotein choles- concept of a beneficial role of HDL-C, which is based on terol (LDL-C), thus favoring its removal from tissue and free cholesterol efflux from macrophages out of the vessel subsequent regression of the neovessels. However, mod- wall. This efflux occurs either by passive diffusion (82)orby

Figure 3 Hp Genotype, Inﬂammation, and Plaque Destabilization

The haptoglobin (Hp)-1 and -2 genotypes play opposite roles in macrophage function after plaque hemorrhage. In individuals with the Hp-1 genotype, a redox-inactive, hemoglobin (Hb)–Hp-1 complex is generated that binds to the macrophage CD163 receptor to induce the secretion of anti-inflammatory cytokines such as interleukin-10 (IL-10). Conversely, after plaque hemorrhage in individuals with the Hp-2 genotype, a redox-active Hb–Hp-2 complex is generated that produces reactive oxygen species (ROS) and induces macrophages to secrete proinflammatory cytokines by both CD163-dependent and -independent pathways, as shown. NF ϭ nuclear transcription factor. Reprinted, with permission, from Moreno et al. (48). JACC Vol. 53, No. 25, 2009 Moreno et al. 2319 June 23, 2009:2315–23 Promoting Biological Pathways for Vascular Health interaction with the SR-BI receptor (83) or the ABCA1 protein, ApoA-I (89–92). A second group of studies sup- transporter (84), as shown in Figure 5. Of these, the porting the concept that increasing HDL-C reduces ath- ABCA1 transporter system is the most efficient, responsible erosclerosis relates to the CETP enzyme pathway. From an for over 50% of cholesterol efflux from macrophages to experimental point of view, the data on CETP expression poorly lipidated ApoA-I. This pivotal protein is then have been controversial. In some studies, overexpression of converted to mature alpha-HDL after esterification. Mature CETP reduced atherosclerosis (93), whereas in others, HDL-C also transfers esterified cholesterol to other li- atherosclerosis is actually increased (94,95). Studies in poproteins by the enzyme cholesterol ester transporter humans with CETP deficiency are also controversial. In protein (CETP), increasing the efficiency of the system some studies, CETP deficiency has been atherogenic (85). There is also evidence that HDL-C also reduces (96,97), whereas in others, it has not (98,99). The hypoth- LDL-C oxidation, and endothelial cell adhesion expression esis that HDL-C could be increased via CETP inhibition (86). In addition, HDL-C improves re-endothelialization was tested in 15,000 patients randomized to torcetrapib plus through EPC activation and proliferation (87). Further- atorvastatin versus atorvastatin alone. Results prove to be more, the antiatherosclerotic role of anti-inflammatory cy- detrimental, with increases in death, heart failure, angina, tokines such as interleukin-10 may be associated with and revascularization procedures in the combined torce- HDL-C reverse cholesterol transport. As a result, increas- trapib plus atorvastatin group when compared to the ator- ing HDL-C may participate in several pathways to prevent vastatin group (100). The question is whether other agents CVD. with different interactions with CETP will provide favor- Experimental and clinical studies on HDL-C protective able results. Torcetrapib was associated with activation in therapy. The first study demonstrating atherosclerotic the renin-angiotensin-aldosterone system, perhaps in part plaque regression with exogenous administration of HDL explaining the increased risk of death in the trial. Intravas- was performed by Badimon et al. (88) in hypercholester- cular ultrasound studies showed no changes in atheroma olemic rabbits with significant reductions in both esterified volume (101). and free cholesterol in aortic plaques. Several other studies Clinical studies on niacin and on CETP deficiency or confirmed these beneficial effects of HDL-C, mostly with inhibition. Niacin has been the most consistent medication the direct infusion and/or overexpression of HDL’s major to increase HDL-C, leading to significant reductions in

Figure 4 Haptoglobin Genotype and HDL Function

Hemoglobin (Hb) released intravascularly from red blood cells (RBCs) is rapidly bound by haptoglobin (Hp) protein to form an Hp-Hb complex. In Hp 2-2 diabetic individuals, the complex is cleared by the scavenger receptor CD163 more slowly than in Hp 1-1 diabetic individuals. The Hp-Hb complex can bind to apolipoprotein (Apo)A-I in high-density lipoprotein (HDL), with increased binding of Hp 2-2-Hb occurring due to its increased avidity for HDL and its increased plasma concentration. The Hp 2-2–Hb, but not the Hp 1-1–Hb complex, when bound to HDL can produce reactive oxygen species, which can oxidize protein (i.e., ApoA-I; GPx-glutatione peroxidase; lecithin cholesterol acyltransferase [LCAT]) and lipid components (cholesterol [chol]) of HDL and render the HDL dysfunctional (due to decreased reversed cholesterol transport [RCT] and antioxidant activity), proatherogenic, and prothrombotic. DM ϭ diabetes mellitus. Reprinted, with permission, from Asleh et al. (71). 2320 Moreno et al. JACC Vol. 53, No. 25, 2009 Promoting Biological Pathways for Vascular Health June 23, 2009:2315–23

Figure 5 Mechanisms of Reverse Cholesterol Transport

There are 3 major pathways by which HDL may mediate cholesterol efflux from cholesterol-loaded macrophages (left). The first pathway, passive diffusion, involves exchange of free cholesterol (FC) between mature spherical ␣-HDL and the cellular membrane. Net cholesterol efflux occurs after the conversion of FC to cholesterol ester (CE) by LCAT. In the SR-BI pathway, free cholesterol is transported to mature spherical ␣-HDL. The third pathway involves the ABCA1 transporter. In the ABCA1 transporter pathway, the preferred acceptor of cellular cholesterol is poorly lipidated ApoA-I, which binds to the ABCA1 transporter and facilitates the efflux of cellular cholesterol from the late endocytic compartment, thereby decreasing the cholesterol content of the cell. The efflux of cholesterol and phospholipids (PL) from macrophages and other peripheral tissues results in the formation of pre␤-HDL, which is ultimately converted to mature spherical ␣-HDL after the esterification of FC to CE by LCAT. (Right) Both the SR-BI and ABCA1 transporter pathways are regulated by the oxycholesterol content of the cell. Excess cellular cholesterol is converted, at least in part, to 27-hydroxycholesterol by 27-hydroxylase. The 27-hydroxycholesterol binds to the ligand-stimulated transcription factor LXR, which, after dimerization with RXR, binds to the LXRE promoter element and increases the expression of SR-BI and the ABCA1 transporter genes. Thus, both mature and pre␤-HDL facilitate the efflux of cellular cholesterol and participate in reverse cholesterol transport to the liver. Abbreviations as in Figure 4. Reprinted, with permission, from Brewer et al. (85). myocardial infarction and cardiac death (102). Niacin fa- mice (108). Both ABCA1 and ABCG1 are regulated by vorably affects apolipoprotein containing lipoproteins, in- liver X receptor (109), and synthetic agonists of this nuclear creasing HDL’s major protein, ApoA-I (103). As mono- receptor promote reverse cholesterol transport in vivo (110). therapy, niacin was tested in the Coronary Drug Project, Indeed, administration of such agonists has been associated which included 3,908 patients with previous myocardial with reduced atherosclerosis in mice (111). infarction. This study reported a 27% decrease in nonfatal The second potential pathway for clinical implementa- infarction and a 12% reduction in cardiac death at 15-year tion is the repeated infusion/overexpression of ApoA-I, follow-up (104). In addition, the Stockholm Ischaemic with documented regression of atherosclerosis in animal Heart Disease Secondary Prevention Study included 900 models and humans (112). Infusion of ApoA-I also shifts patients with recent infarction, and combined immediate- plaque activity to a more stable phenotype (113). One of the release niacin 3.0 g/day with clofibrate 2 g/day (105). This more interesting of these approaches is the concept of study reported a 35% reduction in cardiac death at 4-year ApoA-I mimetic peptides, which mimic the antiathroscle- follow-up. The question is whether neutralization of the rotic and anti-inflammatory properties of full-length niacin molecule implicated in the frequent side effects of ApoA-I (114). However, most peptides are not orally flushing can be obtained without causing other side effects. bioavailable and must be administered parenterally. At least Such an agent (Cordaptive, Merck, Whitehouse Station, 1 intravenously administered ApoA-I mimetic peptide, New Jersey) was recently reviewed by the Food and Drug known as the RLT peptide (ETC-642, Esperion Thera- Administration, which requested further studies before peutics Inc., Ann Arbor, Michigan), is being studied in consideration for approval (106). clinical trials. Another ApoA-I mimetic peptide is D-4F, an Future alternatives for reverse cholesterol transport and 18-amino acid peptide not degraded efficiently by gut plaque regression. Two potential pathways are being ac- peptidases and that can, therefore, be administered orally, tively investigated for clinical implementation. These are the albeit with low bioavailability (114). D-4F reduced athero- ABCA1 transporter and the ApoA-I system (107). Exper- sclerosis in mice (115), probably at least partly by increasing imental overexpression of ABCA1 transporter protein is the anti-inflammatory effects of HDL-C, as well as by associated with a significant regression of atherosclerosis in promoting macrophage reverse cholesterol transport (116). JACC Vol. 53, No. 25, 2009 Moreno et al. 2321 June 23, 2009:2315–23 Promoting Biological Pathways for Vascular Health

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