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Vascular Biology

Cristina Sanina1, Olga L. Bockeria2, Karlo A. Wiley3 and Jonathan E. Feig4

1Department of Internal Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY, USA2Department of Cardiovascular Surgery, Bakoulev Center for Cardiovascular Surgery, Moscow, Russia3Cornell University, College of Agriculture and Life Sciences, Ithaca, NY, USA4Johns Hopkins Heart and Vascular Institute, The Johns Hopkins Hospital, Baltimore, MD, USA

IntroductionLike many contemporary sciences, vascular biology hasbeen progressively developing at the junction of manydisciplines. New knowledge has been obtained in regardto vessel growth biology, physiology, and genetics as wellas physiological and pathophysiological mechanismsunderlying endothelial dysfunction and atherogenesis.Based on studies that extend back to the 1920s, regres-sion and stabilization of atherosclerosis in humans havegone from just a dream to something that is achievable.Review of the literature indicates that successful attemptsat regression applied robust measures to improve plasmalipoprotein profiles. Examples include extensive loweringof plasma concentrations of atherogenic apolipoproteinB and enhancement of reverse cholesterol transport fromatheromata to the liver. Possible mechanisms respons-ible for lesion shrinkage include decreased retention ofatherogenic apolipoprotein B within the arterial wall,efflux of cholesterol and other toxic lipids from plaques,emigration of lesional foam cells out of the arterial wall,and an influx of healthy phagocytes that remove necroticdebris as well as other components of the plaque. Untilvery recently, with the approval of the PCSK9 inhibitors,the available clinical agents caused less dramatic changesin plasma lipoprotein levels, and thereby failed to stopmost cardiovascular events. In addition, although the useof angioplasty and stenting has undoubtedly been bene-ficial, it does not offer a cure or address the underlyingmechanisms of vascular disease.

Endovascular Interventions, First Edition. Edited by Jose M. Wiley, Cristina Sanina, Peter Faries, Ian Del Conde, George D. Dangas and Prakash Krishnan.© 2019 John Wiley & Sons Ltd. Published 2019 by John Wiley & Sons Ltd.

Vascular AnatomyBlood vessels are composed of three layers: the innerlining of intima (a monolayer of endothelial cells), themiddle layer, the media (a layer or layers of vascularsmooth muscle cells), and the outer layer, the adventitia(contains collagen type 1, elastic fibers, myofibroblasts,mesenchymal stem cells, vasa vasorum, and nerves).These three layers are separated with internal and exter-nal elastic laminas, a thin layer of connective tissue. Largearteries contain more layers of smooth muscle cells andmore elastin, and medium-sized arteries contain morecollagen. The smallest vessels (capillaries) are built froma single layer of endothelial cells with surrounding basallamina and pericytes. A number of pericytes and theirfunctions differ in respect to the organs in which theyare found. Vascular smooth muscle cells and pericytesregulate peripheral vascular resistance, vascular diameter,and direction of blood flow [1].

Endothelium, the Largest BodyOrganThe endothelium is a large and complex organ withendocrine, autocrine, and paracrine proprieties that pro-duces nitric oxide (NO), endothelin-1, prostacyclin-2,interleukin-6, vascular endothelial growth factor (VEGF),von Willebrand factor, plasminogen activator, plasmino-gen activator inhibitor-1, angiopoietin-2, adhesion mole-cules such as P-selectin, E-selectin, integrins, and other

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bioactive molecules. Endothelium controls the recruit-ment of inflammatory cells and thrombocytes, regulatesthe coagulation process, extravasation, and vascular tone,and is involved in wound healing through angiogen-esis. Endothelial cells cover the entire vasculature invertebrates with the largest estimated surface amount-ing to 3000–6000 m2. The total weight of endotheliumin an adult person is approximately 720 g, of which600 g is capillaries [2]. Interestingly, endothelial cellsnot only from arteries and veins but also from dif-ferent tissues possess diverse tissue-specific proteinexpression [3]. NO is a major vasodilator moleculethat was discovered by Dr. Furchott in 1980 andnamed endothelium-derived relaxing factor. In 1992NO was identified and in 1998 three US scientists,Robert F. Furchott, Louis J. Ignarro, and Ferid Murad,were awarded the Nobel Prize for NO discovery [4].NO plays an essential role in vascular smooth musclecell relaxation, thrombocyte aggregation, endothelialcell turnover, and immune/anti-inflammatory processes.Endogenous NO is generated from L-arginine by afamily of three calmodulin-dependent NO synthase(NOS) enzymes that are primarily expressed by threecell types: endothelial cells (eNOS), neurons (nNOS),and immune cells (iNOS) [5]. However, NO can alsobe released non-enzymatically from S-nitrosothiols ornitrate/nitrate. Decreased production or bioavailabil-ity of NO and increased expression of endothelin-1,an endothelium-derived potent vasoconstrictor, sug-gest endothelial dysfunction and are associated withhypertension, inflammation, prothrombogenesis, athero-genesis, and cardiovascular events [1]. Inflammation oran increase in proinflammatory circulating moleculessuch as interleukin-1 and interleukin-6, tumor necro-sis factor-𝛼, C-reactive protein, and neutrophils andmacrophages boost C-reactive protein production bythe liver which, in turn, causes eNOS downregulationand increases endothelin-1 bioavailability, leading todecreased vasodilation, increased shear stress, and vascu-lar atherogenesis. In particular, inflammation upregulatesthe expression of endothelial cell adhesion moleculesthat facilitate low-density lipids (LDLs) and macrophagemigration across the vascular endothelium via monocytechemoattractant protein 1 [6]. Inflammatory cytokinesalso induce tissue factor and von Willebrand factorsynthesis by endothelial cells, initiating coagulationcascade and platelet aggregation. MetalloproteinaseADAMTS-13, also produced by endothelial cells, stellarliver cells, platelets, and kidney podocytes, cleaves largemolecules of von Willebrand factor, but inflammatoryconditions decrease ADAMTS-13 activity, promoting

the prothrombotic state. Endothelial cells also providea rescue mechanism for thrombogenesis by continuallyproducing tissue plasminogen activator, which is clearedby the liver unless fibrin binds to it. Furthermore, inflam-matory cytokines promote endothelial cells to produceanother tissue plasminogen activator–urokinase-type tocleave substantial fibrin deposition. Thrombin, a pro-coagulation protease that converts soluble fibrinogen intoinsoluble fibrin, in turn activates eNOS leading to NO andprostacyclin-2 production, causing vasodilatation andplatelet aggregation inhibition. In this way endotheliumregulates thrombogenesis and thrombolysis [2].

Vasculogenesis, Angiogenesis,and ArteriogenesisEndothelial cells originate from mesoderm (heman-gioblasts), which gives rise to hematopoietic stem cellsand endothelial progenitor cells (angioblasts). The vas-cular network is formed due to three primary processes:vasculogenesis, angiogenesis, and arteriogenesis. Theterm “vasculogenesis” was defined by Risau in 1997 as thede novo formation of vessels from endothelial progenitorcells, i.e. angioblasts [7]. During vasculogenesis stem cellsform primitive primary vascular plexus, i.e. capillaries.Initially, it was considered that vasculogenesis occursonly during the early stages of embryogenesis; however,further studies suggested that vasculogenesis occurs invarious diseases, tumorogenesis, and regenerative pro-cesses. Prenatal vasculogenesis begins after initiation ofgastrulation with the formation of blood islets in the yolksac and angioblast precursors in the head mesenchymeand posterior lateral plate mesoderm. Blood islets aremostly composed of hemangioblast, the precursor ofendothelial and hematopoietic cells. Angioblasts, futureendothelial cells, and the peripheral cells of blood isletsjoin together to construct primary vascular plexus. Mul-tiple molecules and growth factors, including FGF-2,VEGF, Tie-1, Tie-2, angiopoietin, TGF-β, neuropilins,hedgehog, fibronectin, β1 integrin, etc., are involved atdifferent times in fetal vasculogenesis [7–9]. In 2010Ricci-Vitiani et al. showed that tumor vasculogenesisexists and a variable number (range from 20% to 90%,mean 60.7%) of endothelial cells in glioblastoma carriedthe same genetic alteration as tumor cells, indicatingthat tumor stem-like cells partially give rise to tumorvasculature [10].

Angiogenesis is the growth of blood vessels fromanlage (preexisting blood vessels) that provides a mas-sive proliferation of the vascular plexus. Angiogenesisoccurs in utero and in adults. Angiogenesis is the most

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extensively studied area in vascular biology. The term“angiogenesis” was introduced in 1935 by Arthur GeorgeTansley, who studied the formation of new vessels in theplacenta. The modern history of angiogenesis began withJudah Folkman, who in 1971 described tumor growthas angiogenesis-dependent [11]. There are two types ofangiogenesis: sprouting angiogenesis and intussuscep-tive or splitting angiogenesis. Sprouting angiogenesis orhypoxia-induced angiogenesis mostly is initiated in thehypoxic environment by parenchymal cells that secreteVEGF in response to hypoxia. Sprouting angiogenesisstarts when an endothelial tip cell guides the developingcapillary sprout through the extracellular matrix. Furtherendothelial cell migration and proliferation, tubulo-genesis, vessel fusion, vessel pruning, and pericytesstabilization occur. The delta-notch signaling pathwayis a key component of sprouting angiogenesis [12, 13].Intussusceptive or splitting angiogenesis occurs when theexisting vessel wall protrudes into the lumen, causing asingle vessel to split in two. This type of angiogenesis isfast and more efficient, but it mainly exists in utero wherethe growth is rapid. Splitting angiogenesis is less studied,yet it is known that it is VEGF dependent [14].

Arteriogenesis is a process of development of maturearteries. Unlike angiogenesis, arteriogenesis is a bloodflow-mediated process, and it is defined by enlarge-ment of existing vessels and collateral formation. A keymechanism of arteriogenesis is the mechanical stress ofvessels that often occurs in arterial obstruction/occlusion(i.e. occlusive peripheral vascular disease, obstructivecoronary artery disease, ischemic stroke). Thus a com-plex vascular rescue system is formed which providesnecessary tissue oxygenation, immunity, thrombogene-sis, thrombolysis, removal of decomposition products,temperature regulation, and maintenance of blood pres-sure. During arteriogenesis migration and proliferationof endothelial cells, smooth muscle cells and pericytesoccur. Mechanical stress of the vessels upregulates mul-tiple genes/proteins, including MCP-1, VEGF, FGF-2,Abra, nitric oxide, thymosin β4, cofilin, Erg-1, MMP2,and MMP9, and the vessels gain vasomotor propertiesand elasticity and undergo the remodeling required toadjust to the needs of tissue blood supply [15].

AtherogenesisAtherosclerosis, a chronic inflammatory disease thatoccurs within the artery wall, is one of the underlyingcauses of vascular complications such as myocardialinfarction, stroke, and peripheral vascular disease.Atherogenesis is a process that occurs over many years,

with the initiation phase being the subendothelialaccumulation of apolipoprotein B-containing lipopro-teins (apoB). These particles undergo modifications,including oxidation and hydrolysis, leading to theactivation of endothelial cells. These cells secretechemoattractants called chemokines that interact withspecific receptors expressed on monocytes essentially“recruiting” the cells into the lesion. The monocytes thenroll along the endothelial cells via interactions of specificselectins (i.e. P-selectin glycoprotein ligand-1 [PSGL-1]),with attachment being mediated by monocyte integrinssuch as very late antigen-4 (VLA-4) and lymphocytefunction-associated antigen-1 (LFA-1) to the respectiveendothelial ligands vascular cell adhesion molecule-1(VCAM-1) and intercellular adhesion molecule-1(ICAM-1). Once attached, a process called diapedesisoccurs by which monocytes enter the subendothe-lial space. Having accessed the subendothelial space,recruited monocytes differentiate into macrophages,a process driven by interactions with the extracellularmatrix (ECM) and cytokines, including macrophagecolony-stimulating factor and members of the tumornecrosis factor family. The uptake of oxidized LDL bythe macrophages occurs via scavenger receptors, notablythe type A scavenger receptor (SRA) and CD36, a mem-ber of the type B family. The cholesteryl esters of theapoB particles that are ingested are hydrolyzed intofree cholesterol, which occurs in late endosomes. Thefree cholesterol is then delivered to the endoplasmicreticulum (ER) where it is re-esterified by acyl-CoA:cholesterol ester transferase (ACAT). It is this processthat leads to the macrophages having a “foamy” appear-ance. It is well-known that macrophages contribute to theformation of the necrotic core and fibrous cap thinningthat characterizes the vulnerable plaque. How do thesemacrophages ultimately contribute to the vulnerableplaque? Macrophage-derived matrix metalloproteinases(MMPs) are a family of proteins that can degrade varioustypes of ECM and hence promote rupture. Moreover,once activated, certain MMPs can activate other ones.Studies have shown a temporal and spatial correlationbetween the presence of macrophages in rupture-proneshoulder regions of plaques, thinning of the fibrous cap inthese regions, and local accumulation of activated MMPs.Another potential mechanism of how macrophages maypromote plaque thinning and increase vulnerability is viasmooth muscle cell (SMC) apoptosis. Vulnerable plaquesshow evidence of SMC death and decreased numbersof SMCs. Even after plaque rupture, the macrophagecontinues to play a role as it secretes prothrombotic tissuefactor, thereby accelerating thrombus formation [16–18].

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The idea that human atheromata can regress at all has metconsiderable resistance over the decades [16–18]. Resis-tance to the concept of lesion regression has been due tothe fact that advanced atheromata in humans and animalmodels contains components that give an impression ofpermanence, such as necrosis, calcification, and fibrosis.Furthermore, numerous theories have been proposed toexplain atherogenesis that included processes thoughtto be difficult, if not impossible, to reverse, includinginjury [19, 20], oxidation [21], and cellular transforma-tions resembling carcinogenesis [22]. In this review, datawill be presented that demonstrate that changes in theplaque environment can indeed stabilize and regress evenadvanced lesions.

Plaque Regression: Evidence fromAnimal StudiesIn the 1920s, Anichkov and colleagues reported thatswitching cholesterol-fed rabbits to low-fat chow overtwo to three years resulted in arterial lesions becomingmore fibrous with a reduced lipid content [23], whichfrom a modern perspective suggests plaque stabilization[20, 24]. To our knowledge, however, the first prospec-tive, interventional study demonstrating substantialshrinkage of atherosclerotic lesions was performed incholesterol-fed rabbits and reported in 1957 [25]. Thedietary regimen raised total plasma cholesterol to around26 mmol l−1 (∼1 000 mg dl−1) and induced widespreadlesions involving about 90% of the aorta. To mobilizetissue stores of cholesterol, animals received intravenousbolus injections of phosphatidylcholine (PC). After lessthan a week and a half of treatment, the remainingplaques were scattered and far less severe than initially,and three-quarters of arterial cholesterol stores had beenremoved.

Over the next 20 years, similar arterial benefits frominjections of dispersed phospholipids were reported bya number of groups using a variety of atheroscleroticanimal models, including primates [26]. Given the heavyreliance of atherosclerosis research on animal models, itis surprising that these impressive, reproducible resultswere largely ignored, even in numerous historical reviewsof regression [16, 18, 23, 27, 28].

The concept of regression gained support with ashort-term study in squirrel monkeys by Maruffo andPortman [29], and more extensive work by Armstrongand colleagues. The latter reported that advanced arteriallesions in cholesterol-fed Rhesus monkeys underwentshrinkage and remodeling during long-term follow-upwhen their diet was switched to low-fat or linoleate-rich

diets [27, 30]. The cholesterol-feeding induction periodlasted 17 months, producing widespread coronarylesions, with fibrosis, cellular breakdown, intracellularand extracellular lipid accumulation, and 60% luminalnarrowing. The subsequent regression period lasted40 months, bringing total plasma cholesterol values downto approximately 3.6 mmol l−1 (∼140 mg dl−1) and result-ing in the loss of approximately two-thirds of coronaryartery cholesterol, substantial reduction in necrosis, someimprovement in extracellular lipid levels and fibrosis, andsubstantial lesion shrinkage so that only 20% luminalnarrowing remained [27, 30]. Further work by Wisslerand Vesselinovich as well as Malinow confirmed andextended these findings [23, 28]. Three decades ago, in anoverview of this work, Armstrong concluded that “In theprimate the answer is clear: all grades of induced lesionsstudied to date improve… the primate lesion showsamazing metabolic responsiveness: some extracellular, aswell as intracellular lipid, is depleted, there is resolutionof necrotic lesions, crystalline lipid tends to diminishslowly, and fibroplasia is eventually contained.” [27]

Regression of advanced lesions in cholesterol-fedswine after reversion to a chow diet demonstrated animportant sequence of events. Histologic examinationof atheromata from these animals immediately after thehigh-cholesterol induction phase showed the hallmarksof complex plaques, including necrosis and calcification.The regression regimen reduced total plasma cholesterolto approximately 1.8 mmol l−1 (∼70 mg dl−1), implyingan even lower LDL-cholesterol level. Interestingly, theearly phase of regression showed loss of foam cells fromthe lesions and an increase in non-foam-cell macrophagesaround areas of necrosis. Long term, the necrotic areasvirtually disappeared, indicating removal of the materialby a flux of functioning, healthy phagocytes [31].

To revive the long-neglected finding of rapid athero-sclerosis regression after injections of dispersed phospho-lipids, Williams and colleagues sought to determine theunderlying mechanism of action [26, 32]. Aqueous dis-persions of PC spontaneously form vesicular structurescalled liposomes. Initially, cholesterol-free PC liposomesremain intact in the circulation [33] and can mobilizecholesterol from tissues in vivo [33–36] by acting ashigh-capacity sinks into which endogenous HDL choles-terol shuttles lipid [26, 35, 37]. Bolus injections of PCliposomes rapidly restore normal macrovascular andmicrovascular endothelial function in hyperlipidemicanimals [36], remove lipid from advanced plaques in rab-bits in vivo [38], and rapidly mobilize tissue cholesterol invivo in humans [39]. Importantly, the optimum liposo-mal size (∼120 nm) has been achieved in animal model

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studies, which allows these particles to gradually delivertheir cholesterol to the liver without suppressing hepaticLDL receptor expression or raising plasma concentrationsof LDL cholesterol [35, 37]. Eventually, in 1976 successin atherosclerosis regression was also achieved in rabbitsfollowing reversion to normal-chow diet in combinationwith hypolipidemic and other agents [23]. Decades later,a series of studies achieved shrinkage of atheromata inrabbits with injections of HDL or HDL-like apolipopro-tein A-I (apoA-I) and PC disks [40, 41]. Interestingly, alipid-lowering regimen in rabbits was found to diminishlocal proteolytic and prothrombotic factors in the arterywall, again consistent with the remodeling of atheromatainto a more stable phenotype [42].

Unlike humans, mice have a naturally high plasmaHDL:LDL ratio, providing a strong intrinsic resistanceto atherosclerosis. Drastic manipulations of plasmalipoproteins are required, therefore, to induce arteriallipoprotein accumulation and sequelae. A revolutionin murine atherosclerosis research began in the 1980swhen Breslow and colleagues began applying trans-genic techniques to create mice that were models ofhuman lipoprotein metabolism [43]. With the emergingtechnique of gene inactivation through homologousrecombination (“knock out”), came the ability to recreateimportant aspects of human lipid metabolism in mice.Most mouse models of atherosclerosis are derived fromtwo basic models: the apolipoprotein E (apoE)-null(apoE−/−) mouse [44, 45] and the LDL receptor-null(LDLR−/−) mouse [46]. In these models, the normallylow plasma apoB levels are increased to atherogeniclevels by eliminating either a ligand (apoE−/−) or areceptor (LDLR−/−) for lipoprotein clearance. Feedingthese modified mice with a cholesterol-enriched andfat-enriched diet (Western diet (WD)) increased plasmaapoB levels to an even greater degree, resulting in acceler-ated plaque formation in the major arteries. Gene transferwas the first strategy used to achieve plaque regression inmice. For example, injection of LDLR−/− mice that haddeveloped fatty streak lesions after a five-week WD withan adenoviral vector containing cDNA encoding humanapoA-I caused a significant increase in HDL-cholesterollevel and, importantly, regression of fatty streak lesionsat a sampling point four weeks later [47]. The ability ofHDL-like particles to rapidly remodel plaques in micewas shown by infusion of apoA-IMilano/PC complexes, avariant of apolipoprotein A-I identified in individualswho exhibit very low HDL-cholesterol levels. Infusion ofthis complex reduced foam cell content in arterial lesionsin apoE−/− mice within 48 hours [48]. This finding wascorroborated by a specific transplantation model that we

reported in 2001 [49], described later. Although anotherHDL protein, apolipoprotein M, has been overexpressedin mice to retard plaque progression [50], evaluation ofits role in regression has not yet been reported.

Another major target of gene transfer to achieveregression in mice is hepatic overexpression of apoE,which increases the clearance of plasma atherogeniclipoproteins through receptors in the liver for LDL [46]and for postprandial lipoprotein remnants [32, 51–53].Following successful transient reduction of atheroscle-rosis progression in apoE−/− mice with short-termadenoviral-mediated expression of apoE [54], a numberof laboratories capitalized on the greater duration ofapoE expression afforded by “second-generation” viralvectors [55]. For example, in LDLR−/− mice fed a WDfor 14 weeks to develop plaques abundant in foam cells(∼50% macrophage content), increased expression ofapoE resulted in significant plaque regression, despitehaving no discernible effect on fasting plasma lipoproteinlevels [56]. These findings were attributed in part to theentry of expressed apoE into the vessel wall, consistentwith other studies [36, 57]; however, another plausi-ble mechanism is that expressed apoE might have alsoimproved clearance of atherogenic lipoproteins in thepostprandial state.

Transplantation Modelof Atherosclerosis RegressionTo further explore cellular and molecular mechanismsof atherosclerosis regression in murine models, we andothers have developed new approaches to rapidly inducerobust improvements in the plaque environment and trig-ger lesion remodeling and regression. Our study groupdeveloped the technique of transplanting a segment ofthe plaque-containing aorta from a (WD-fed) hyperlipi-demic apoE−/−mouse (i.e. an extremely pro-atherogenicmilieu consisting of high plasma apoB levels and lowHDL-cholesterol levels) into a wild-type recipient (i.e.rapidly normalizing the lipoprotein environment, whichis sustainable indefinitely). This approach allows analysisof plaques of any degree of complexity. We found thattransplanting early lesions [58, 59] or advanced, com-plicated plaques into wild-type recipients substantiallyreduced foam cell content and increased the number ofsmooth muscle cells, particularly in the cap, which is con-sistent with plaque stabilization and regression [60, 61].The loss of foam cells from early lesions was surprisinglyrapid, with large decreases evident as early as three dayspost-transplantation (Figure 1.1) [58, 59]. With advanced

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Baseline WT Rec.

3 days post-transplantation

EKO Rec.

Figure 1.1 Regression of plaques. ApoE−/− mice were fed a West-ern diet for 16 weeks to develop advanced atherosclerosis. Aorticarches from these mice were either harvested and analyzed by his-tochemical methods, or were transplanted into apoE−/− (“progres-sion”) or wild-type (“regression”) recipient mice. Three or seven dayslater, the same analyses were performed. Shown are the histochemi-cal results for the foam-cell marker CD68 (red). The pictures show theimmunostaining of representative aortic lesions in cross section. Thevirtual absence of foam cells can be seen in the “regression” group. Incontrast with the “regression” results, the “progression” group showedpersistence of foam cells.

lesions, all features regressed after nine weeks, includingnecrosis, cholesterol clefts, and fibrosis [60, 61].

By using the transplantation model, we characterizedcellular and molecular features of the regressing plaque.An early question we sought to answer concerned the fateof the disappearing foam cells – was their disappearancedue to apoptosis and phagocytosis by newly recruitedmacrophages or emigration? Interestingly, we found thatthe rapid loss of foam cells was largely accounted forby their emigration into regional and systemic lymphnodes. Furthermore, we found that the wild-type milieuprovoked foam cells to display markers characteristicof both macrophages and, surprisingly, dendritic cells,which enabled emigration [58, 59, 62]

Using laser microdissection to remove foam cellsfrom regressing and non-regressing plaques [63, 64],analyses revealed the presence of mRNA for CCR7 [59],chemokine (C—C motif) receptor 7, which is requiredfor dendritic cell emigration [65]. Interestingly, injectionof wild-type recipient animals with antibodies againstthe two CCR7 ligands, CCL19 and CCL21, inhibited themajority of foam cells from emigrating from the aortictransplant lesions, establishing a functional role for CCR7in regression [59].

In addition, mRNA concentrations of several well-known proteins implicated in atherothrombosis, such asvascular cell adhesion protein-1 (VCAM-1), monocytechemotactic protein 1 (MCP-1), and tissue factor, are

decreased in foam cells during regression. Also, thelevel of mRNA for the nuclear oxysterol liver X recep-tor [alpha] (LXRα) – known to be induced in vitroby oxidized sterols [66, 67] – significantly increasedin vivo, as did its anti-atherogenic target ATP-bindingcassette 1 (ABCA-1) [59]. Intriguingly, systemic admin-istration of an LXR agonist caused lesion regressionin LDLR−/− mice [68], although the concomitantdevelopment of fatty liver has dampened enthusi-asm for this approach in humans [69]. Interestingly,we discovered that LXR activation in macrophagespromoted regression in vivo and was dependent onCCR7 expression [70]. It is unlikely that regressionof atherosclerosis occurs only through one mecha-nism. A recent report showed that netrin-1, a neuro-immune guidance cue, was secreted by macrophages inhuman and mouse atheroma, where it inactivated themigration of macrophages toward chemokines (such asCCL19, a ligand for CCR7) linked to their egress fromplaques [70]. These findings suggest that inhibition ofnetrin-1 may be one method of inducing regressionof atherosclerosis. Overall, these findings indicate thatregression does not simply comprise the events leadingto lesion progression in reverse order; instead, it involvesspecific cellular and molecular pathways that eventuallymobilize all pathologic components of the plaque.

HDL and Plaque RegressionAt least three plasma parameters are changed in thetransplantation model when the regression is observed:(i) non-HDL levels decreased, (ii) HDL levels wererestored from ∼33% of normal to wild-type levels, and(iii) apoE was now present. For this review, we willfocus on the HDL change. To selectively test this as aregression factor, we adopted the transplant approach byusing as recipients human apoAI transgenic/apoE−/−mice (hAI/EKO) or apoAI−/− mice [70]. Briefly,plaque-bearing aortic arches from apoE−/− mice (lowHDL-C, high non-HDL-C) were transplanted into recipi-ent mice with differing levels of HDL-C and non-HDL-C:C57BL/6 mice (normal HDL-C, low non-HDL-C),apoAI−/− mice (low HDL-C, low non-HDL-C), orhAI/EKO mice (normal HDL-C, high non-HDL-C).Remarkably, despite persistently elevated non-HDL-Cin hAI/EKO recipients, plaque CD68(+) cell contentdecreased by >50% by one week after transplantation,whereas there was little change in apoAI−/− recipientmice despite hypolipidemia. Interestingly, the reducedcontent of plaque CD68+ cells was associated with theiremigration and induction of their chemokine receptor

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CCR7 [70]. These data are consistent with a recentmeta-analysis of clinical studies in which it was shownthat atherosclerosis regression (assessed by intravascularultrasonography (IVUS)) after LDL lowering was mostlikely to be achieved when HDL was also significantlyincreased [71].

The induction of CCR7 is also likely related to changesin the sterol content of foam cells when they are placedin a regression environment, given that its promoter hasa putative sterol regulatory element (SRE). This idea is inagreement with a report that demonstrated that loadingTHP-1 human monocytes with oxidized LDL suppressesthe expression of this gene [72]. Notably, we have foundthat statins, potent regulators of SRE-dependent tran-scription, can induce CCR7 expression in vivo andpromote regression via emigration of CD68+ cells in aCCR7-dependent manner [73]. Recently, it was reportedthat both atorvastatin and rosuvastatin can promoteregression of atherosclerosis as assessed by IVUS [74].Our data, therefore, suggest that activation of the CCR7pathway may be one contributing mechanism.

Another aspect of interest has been the effect of HDLon the inflammatory state of CD68+ cells in plaques. Anumber of benefits from this can be envisioned, such asreduced production of monocyte-attracting chemokinesand plaque “healing” by macrophages prodded to becometissue re-modelers (M2 macrophages). There are multi-ple reasons for HDL to have anti-inflammatory effectson plaques, including the antioxidant properties of itsenzymatic and non-enzymatic components, the abilityto remove normal and toxic lipid species from cells, andthe dampening of TLR signaling by regulating plasmamembrane cholesterol content [75]. It is important tonote that in CD68+ cells laser-captured from the plaques,normalization of HDL-C led to decreased expressionof inflammatory factors and enrichment of markers ofthe M2 macrophage state [76, 77]. Macrophage het-erogeneity in human atherosclerotic plaques is widelyrecognized, with both M1 (activated) and M2 markersbeing detectable in lesions [78] but little is known aboutthe factors that regulate M2 marker expression in plaquesin vivo.

Cholesterol homeostasis has also recently beeninvestigated with microRNAs (miRNA), which aresmall endogenous non-protein-coding RNAs that arepost-transcriptional regulators of genes involved in phys-iological processes. MiR-33, an intronic miRNA locatedwithin the gene-encoding sterol-regulatory elementbinding protein-2, inhibits hepatic expression of bothABCA-1 and ABCG-1, reducing HDL-C concentrations,as well as ABCA-1 expression in macrophages, thus

resulting in decreased cholesterol efflux. Interestingly,enrichment of M2 markers in plaque CD68+ cells wasobserved in LDLR−/− mice treated with an antagomirof miR-33 [79]. The treated mice also exhibited plaqueregression (fewer macrophages). The therapeutic poten-tial of miR-33 antagomirs to cause similar benefits inpeople was suggested by plasma levels of HDL beingraised in treated non-human primates [80]. Thus, antag-onism of miR-33 may represent a novel approach toenhancing macrophage cholesterol efflux and raisingHDL-C levels in the future.

Recently, Voight and colleagues [81] reported, usingMendelian randomization, that some genetic mecha-nisms (i.e. endothelial lipase polymorphisms) that raiseplasma HDL cholesterol do not seem to lower the risk ofmyocardial infarction. These data potentially challengethe concept that raising of plasma HDL cholesterol willuniformly translate into reductions in the risk of myocar-dial infarction. However, it is important to note thatthese results should not lead one to abandon the conceptthat HDL is beneficial but rather may indicate that it istime to alter the HDL hypothesis – it is not the quantityof HDL but rather the quality or functionality that iscritical. We need clinical trials that have HDL function asan endpoint rather than simply the level.

Plaque Regression: Evidence fromClinical StudiesStatins, Niacin, HDL, and CETP InhibitorsThe first prospective, interventional study to demonstrateplaque regression in humans was in the mid-1960s, inwhich approximately 10% of patients (n = 31) treatedwith niacin showed improved femoral angiograms [82].Larger trials of lipid-lowering have since demonstratedangiographic evidence of regression; however, thoughstatistically significant, the effects were surprisingly small,particularly in light of large reductions in clinical events[16–18, 83]. This “angiographic paradox” was resolvedwith the realization that lipid-rich, vulnerable plaqueshave a central role in acute coronary syndromes. A vul-nerable plaque is characterized by being small, causingless than 50% occlusion, and being full of intracellularand extracellular lipid, rich in macrophages and tissuefactor, with low concentrations of smooth muscle cells,and with only a thin fibrous cap under an intact endothe-lial layer [20, 24, 83, 84]. Rupture of a vulnerable plaqueprovokes the formation of a robust local clot, and hencevessel occlusion and acute infarction [85]. Lipid lowering,which promoted measurable shrinkage of angiographi-cally prominent but presumably stable lesions, probably

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8 Endovascular Interventions

had a greater impact on risk reduction by the remodelingand stabilization of small, rupture-prone lesions [83, 84].Regression studies in animal models strongly support thisinterpretation, given that macrophage content, a key hall-mark of instability, can be rapidly corrected with robustimprovements in the plaque lipoprotein environment.

In order to track potentially more important changesin plaque composition, to avoid the confounding effectsof lesion remodeling on lumen size, arterial wall imagingis required. Recent human trials have switched fromquantitative angiography, which images only the vascu-lar lumen, to techniques that image plaque calcium (e.g.electron-beam CT) and plaque volume (e.g. IVUS). A ret-rospective analysis found that aggressive LDL-cholesterollowering with statins correlated significantly with reduc-tion in coronary calcium volume score by electron-beamCT, indicating that coronary artery calcifications canshrink [86]. In the Reversal of Atherosclerosis withAggressive Lipid Lowering (REVERSAL) study [87]and A Study to Evaluate the Effect of Rosuvastatin onIntravascular Ultrasound-Derived Coronary AtheromaBurden (ASTEROID) [88], patients with acute coronarysyndromes were treated for over a year with high-dosestatins and evaluated by IVUS. The REVERSAL trialcompared the high-dose statin therapy with a conven-tional, less-potent statin regimen. During 18 monthsof treatment, patients treated with the conventionalregimen exhibited statistically significant progressionof atheroma volume (+2.7%), despite achieving averageLDL-cholesterol levels of 2.8 mmol l−1 (110 mg dl−1)and, therefore, meeting the then-current Adult Treat-ment Panel III goal [89]. By contrast, the high-dosestatin group experienced no significant progressionof atheroma volume (average LDL-cholesterol level,2 mmol l−1 [79 mg dl−1]). Importantly, analysis across thetreatment groups found that LDL reduction exceedingapproximately 50% was associated with a decrease inatheroma volume. In ASTEROID, all patients receivedthe same high-dose therapy for 24 months, and IVUSfindings pretreatment and post-treatment were com-pared. During treatment, LDL cholesterol dropped to1.6 mmol l−1 (60.8 mg dl−1), and atheroma volume shrankby a median of 6.8%. Thus, in both of these studies,extensive LDL-cholesterol lowering for extended periodscaused established plaques to shrink. The greater effi-cacy seen in ASTEROID could be explained by the lowermedian LDL-cholesterol level, but also by the longer treat-ment period and higher HDL-cholesterol levels achievedthan those in REVERSAL. As in earlier angiographicstudies, we believe that these reductions in plaque vol-ume are accompanied by favorable alterations in plaque

biology, a theory which is further supported by evidencethat robust plasma LDL lowering to 1.0–1.6 mmol l−1

or below (≤40–60 mg dl−1) is associated with furtherreductions in cardiovascular events [90].

In addition to the preclinical studies reviewed above,there are a limited number of human studies in whichHDL levels have been manipulated by infusion, andthe effects on plaques assessed. In the first [90],patients at high risk for cardiovascular disease wereinfused with either an artificial form of HDL (apoAImilano/phospholipid complexes) or saline (placebo)once a week for five weeks. By IVUS, there was a sig-nificant reduction in atheroma volume (−4.2%) in thecombined (high and low dose) treatment group, thoughno dose response was observed of a higher vs. lower doseof the artificial HDL. There was no significant differencein atheroma volume compared to the placebo group, butthe study was not powered for a direct comparison. Inthe second infusion study, high-risk patients receivedfour weekly infusions with reconstituted HDL (rHDL;containing wild-type apoAI) or saline (placebo) [90].Similar to the previous study, there was a significantdecrease in atheroma volume (−3.4%) (as assessed byIVUS) after treatment with rHDL compared to baseline,but not compared to placebo (which the study was notpowered for). However, the rHDL group had statisticallysignificant improvements in plaque characterizationindex and in a coronary stenosis score on quantitativecoronary angiography compared to the placebo group.In the third infusion trial [90], a single dose of reconsti-tuted human HDL was infused into patients undergoingfemoral atherectomies, with the procedure performed5–7 days later. Compared to the control group (receivingsaline solution), in the excised plaque samples in theHDL infusion group macrophage activation state (i.e.diminished VCAM-1 expression), as well as cell size (dueto diminished lipid content), were reduced.

In addition to the aforementioned meta-analysis ofstatin trials in which the relationships among LDL, HDL,and plaque regression were analyzed, there are also anumber of other drug studies in which effects on plaqueswere ascribed to the raising of HDL levels. This includesthe VA-HIT study, in which coronary events were reducedby 11% with gemfibrozil for every 5-mg dl−1 increase inHDL-C. In another series of studies (ARBITER [91–94]),high-risk patients were placed on either statins or statinsplus niacin. Over an 18–24 month observation period,carotid intimal-medial thickness (cIMT) measurementswere obtained as a surrogate for coronary artery plaqueburden. As expected, when niacin was part of the treat-ment, HDL-C levels were increased (by 18.4%), and the

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CHAPTER 1 Vascular Biology 9

authors attributed the improvement in cIMT particularlyto this change. It is important to note that niacin doesmore than just raise HDL-C levels; it also decreasesplasma triglyceride levels, makes LDL size increase, andpossesses anti-inflammatory properties, all of which havethe potential to limit plaque progression [95–97]. Thesepleiotropic effects obviously confound the interpretationof both the ARBITER and another statin-niacin clinicaltrial, the HATS study [98]. In the latter study, the addi-tion of niacin to statin treatment resulted not only in areduction in coronary artery stenosis but also in events.The encouraging results with niacin, however, wererecently called into question by the early termination ofthe AIM-HIGH study, which failed to show a benefit inthe treatment group [99]. This study has been criticized,however, as being underpowered and for the fact thatboth the treatment group and the control group in thestudy received statin therapy, making additional benefitsharder to detect, as well as for the placebo that the controlpatients received, which was a low dose of niacin [100].

Recently, cholesteryl ester transfer protein (CETP)inhibitors have been investigated as pharmacologicalagents to raise HDL levels. Surprisingly, torcetrapib, thefirst CETP inhibitor tested in a clinical trial, increasedthe all-cause mortality and cardiovascular events, whichled to the premature ending of the ILLUMINATE trial[101]. Subsequent studies indicated that the observedoff-target effects of torcetrapib (increased blood pressureand low serum potassium by stimulation of aldosteroneproduction) were rather molecule specific, unrelatedto CETP inhibition and thereby might have overshad-owed the beneficial effects of the raised HDL-C levels.Importantly, post hoc analysis of ILLUMINATE showedthat subjects with greater increases of HDL-C or apoAIlevels had a lower rate of major cardiovascular eventswithin the torcetrapib group [102]. Despite the generalfailure of torcetrapib, in the post hoc analysis of theInvestigation of Lipid Level Management Using Coro-nary Ultrasound to Assess Reduction of Atherosclerosisby CETP Inhibition and HDL Elevation (ILLUSTRATE)study, regression of coronary atherosclerosis (as assessedby IVUS) was observed in patients who achieved thehighest HDL-C levels with torcetrapib treatment. In vitrostudies showed an improved functionality of HDL-Cparticles under CETP inhibition, as HDL-C isolated frompatients treated with torcetrapib and anacetrapib exhib-ited an increased ability to promote cholesterol effluxfrom macrophages. Indeed, the CETP inhibitors anace-trapib, dalcetrapib, and evacetrapib increase HDL-Clevels between 30% and 138%, and have not shown theoff-target effects of torcetrapib in recent clinical phase II

trials, confirming the premise of non-class related toxicityof torcetrapib [103–106] Thus, raising HDL-C by CETPinhibition or modulation remains a potential therapeuticapproach for an atherosclerotic cardiovascular disease.Large clinical outcome trials were initiated for dalcetrapib(dal-OUTCOMES) and anacetrapib (REVEAL), includ-ing a total of approximately 45 000 patients. Surprisingly,in May 2012 Roche stopped the dal-HEART program fordalcetrapib after an interim analysis of dal-OUTCOMESdue to a lack of clinically meaningful efficacy. The failureof dal-OUTCOMES might have been a result of therather moderate increases in HDL-C levels (30%) andminor impact on LDL-C levels induced by dalcetrapib,a fate that does not necessarily apply for anacetrapib,which has been shown to increase HDL-C levels by138% accompanied by more robust reductions in LDL-Clevels [107]. Whether the failure of dal-OUTCOMESchallenges the benefits of raising HDL-C in general, orrather the underlying mechanisms of how HDL-C is tobe raised, will be answered by the phase III study withanacetrapib which is expected over the next few years.

Novel Imaging ModalitiesWhile IVUS has provided important coronary anatomicinformation, there is still a need for imaging modalitiesthat provide more details. Optical coherence tomogra-phy (OCT) has revolutionized intracoronary imaging.The unprecedented spatial resolution of this technique(15 μm) provides unique insights into the microstructureof the coronary wall. Currently, OCT is increasingly usedin clinical practice and also constitutes an emerging,highly robust research tool. OCT allows detailed visu-alization of atherosclerotic plaques and provides reliableinformation on plaque composition (lipid, fibrous, cal-cified). Importantly, OCT is the only technique allowingaccurate measurements of the thickness of the fibrouscap, a classical marker of plaque vulnerability, and readilydetects thin-cap fibroatheromas. In patients with acutecoronary syndromes, plaque ruptures, with associatedred or white thrombus, are nicely identified [108]. Thelipid core is an important plaque component, and itsrelationship with macrophages and the vulnerable plaquehas been established in animal models. Near-infraredspectroscopy (NIRS) is a technique that can identify thelipid core burden in the coronary arteries. It works by thelight of discrete wavelengths from a laser being directedonto the tissue sample via glass fibers. Light scatteredfrom the samples is then collected in fibers and launchedinto a spectrometer. The plot of signal intensity as afunction of wavelength is subsequently used to develop

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10 Endovascular Interventions

Lumen

Intima

Media

ABCA1/G1

1

2

34

5

6

HDL

Endothelial cell Macrophage

Lymphoidtissue

Monocyte

oxLDL

Scavenger receptor (SR-A & CD36)

Fibroblast

Foam cell

CholesterolLDL

CCL19

CCL21

CCR7

EOM

Adventitia

Ves

sel w

all

Figure 1.2 Retention, responses, and regression: 1, oxidation; 2, diapedesis; 3, foam cell formation; 4, RCT; 5, 6, macrophage egress from lesionto lumen and adventitia, respectively. HDL can inhibit processes 1–3 and promote 4–6. Macrophage egress can occur through the upregulationof CCR7 via activation of the sterol regulatory element binding protein (SREBP) pathway.

chemometric models to discriminate lipid-cores fromthe non-atherosclerotic tissue [109]. Ideally, it is the earlydetection and characterization of atherosclerotic lesionssusceptible to sudden rupture and thrombosis that needto be achieved. Plaque development has been extensivelystudied using magnetic resonance imaging (MRI) inanimal models of rapidly progressing atherosclerosis.MRI permits the accurate assessment of atheroscleroticplaque burden and the differentiation between the lipidand fibrous content of individual plaques, thus pro-viding a non-invasive approach to serially monitor theevolution of individual plaques. In addition, 18F-FDGpositron emission tomography (PET) is a relatively newnon-invasive tool for inflammation functional imag-ing. Low spatial resolution is now compensated for byco-registration with CT or MRI. One can envision havingnovel contrast agents that target specific plaque com-ponents or a diverse set of molecules within the plaquewhich would elucidate the changes at the cellular andmolecular levels during plaque progression and regres-sion. We have demonstrated the feasibility of this conceptin a study in which the detection of macrophages using ananoparticulate contrast agent was achieved. The abovehas important implications as pharmaceutical companies

are looking for early surrogate markers that could beevaluated in a small number of patients to predict thebeneficial effects of new drugs on atherosclerotic plaquesbefore moving to costly clinical trials with a large numberof patients [110–112].

ConclusionThe crucial event in atherosclerosis initiation is the reten-tion, or trapping, of apoB-containing lipoproteins withinthe arterial wall; this process leads to local responses tothis retained material, including a maladaptive infiltrateof macrophages that consume the retained lipoproteinsbut then fail to emigrate. Regression (i.e. shrinkage andhealing) of advanced, complex atherosclerotic plaqueshas been clearly documented in animals, and plau-sible evidence supports its occurrence in humans aswell. Data have shown that plaque regression requiresrobust improvements in the plaque environment, specif-ically large reductions in plasma concentrations ofapoB-lipoproteins and large increases in the reversetransport of lipids out of the plaque for disposal. Fur-thermore, it is important to note that regression is notmerely a rewinding of progression, but instead involves

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CHAPTER 1 Vascular Biology 11

a coordinated series of events such as emigration of themacrophage infiltrate, followed by the initiation of astream of healthy, normally functioning phagocytes thatmobilize necrotic debris and all other components ofadvanced plaques (Figure 1.2).

For regression of atheromata to become a realistic ther-apeutic goal, clinicians must be provided with tools thatextensively change plasma lipoprotein concentrations andplaque biology while avoiding adverse effects. To date, theanimal and human studies that achieved plaque regres-sion required large reductions in plasma levels of apoB,sometimes combined with brisk enhancements in reversecholesterol transport. Unfortunately, most patients whotake statins, for example, will not achieve and sustain thedramatically low LDL-cholesterol levels seen in chow-fednonhuman primates. Although the PCSK9 inhibitorsdramatically lower cholesterol, time will tell whether theyalso significantly reduce plaque burden, stabilize remain-ing plaque, and reduce cardiac events. Experimentalagents designed to accelerate reverse cholesterol trans-port from plaques into the liver include PC liposomes,apoA-I/PC complexes, and apoA-I mimetic peptides.Other small molecules have been investigated preclin-ically for their potential to enhance HDL-cholesterollevels and reverse lipid transport, such as agonists forLXR and peroxisome proliferator-activated receptors.On the basis of the experimental data summarizedabove, we expect that the best regression results will beobserved when plasma LDL-cholesterol concentrationsare reduced and HDL-cholesterol function in reverselipid transport is enhanced. Indeed, years of work havedemonstrated that the plaque and its components aredynamic. Most recently, by performing microarrays wehave discovered that regression of atherosclerosis is char-acterized by broad changes in the plaque macrophagetranscriptome with preferential expression of genes thatreduce cellular adhesion, enhance cellular motility, andoverall act to suppress inflammation [113]. Additionalstrategies, such as specific induction of pro-emigrantmolecules to provoke foam cells to leave the arterial wall(e.g. via CCR7), should attract pharmaceutical interest.Furthermore, there is a need for clinical trials that usethe imaging modalities described above to identify thespecific effects of novel agents on plaque componentsrather than just atheroma size. In conclusion, we provideevidence that the plaque is dynamic and depending onthe conditions macrophages, which play a crucial role inatherogenesis, can exit the lesions, proving that regressionis indeed possible. However, there is still much work tobe done, and ultimately the insights gained will lead tonew therapeutic targets against cardiovascular disease.

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