Vascular effects of adiponectin: molecular …...Healthy Aging, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, People’s Republic of China ABSTRACT Adiponectin

Clinical Science (2008) 114, 361–374 (Printed in Great Britain) doi:10.1042/CS20070347 361

R E V I E W

Vascular effects of adiponectin:molecular mechanisms and potential

therapeutic intervention

Weidong ZHU∗, Kenneth K. Y. CHENG∗, Paul M. VANHOUTTE†‡, Karen S. L. LAM∗‡and Aimin XU∗†‡∗Department of Medicine, University of Hong Kong, Hong Kong, People’s Republic of China, †Department of Pharmacology,University of Hong Kong, Hong Kong, People’s Republic of China, and ‡Research Centre of Heart, Brain, Hormone andHealthy Aging, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, People’s Republic of China

A B S T R A C T

Adiponectin is a major adipocyte-secreted adipokine abundantly present in the circulation as threedistinct oligomeric complexes. In addition to its role as an insulin sensitizer, mounting evidencesuggests that adiponectin is an important player in maintaining vascular homoeostasis. Numerousepidemiological studies based on different ethnic groups have identified adiponectin deficiency(hypoadiponectinaemia) as an independent risk factor for endothelial dysfunction, hypertension,coronary heart disease, myocardial infarction and other cardiovascular complications. Conversely,elevation of circulating adiponectin concentrations by either genetic or pharmacologicalapproaches can alleviate various vascular dysfunctions in animal models. Adiponectin exerts itsvasculoprotective effects through its direct actions in the vascular system, such as increasingendothelial NO production, inhibiting endothelial cell activation and endothelium–leucocyteinteraction, enhancing phagocytosis, and suppressing macrophage activation, macrophage-to-foamcell transformation and platelet aggregation. In addition, adiponectin reduces neointima formationthrough an oligomerization-dependent inhibition of smooth muscle proliferation. The presentreview highlights recent research advances in unveiling the molecular mechanisms that underpinthe vascular actions of adiponectin and discusses the potential strategies of using adiponectin orits signalling pathways as therapeutic targets to combat obesity-related metabolic and vasculardiseases.

Key words: adipokine, adiponectin, adiponectin receptor, atherosclerosis, cardiovascular disease, diabetic complication, endothelialdysfunction.Abbreviations: ACE, angiotensin-converting enzyme; ACTA, acyl-CoA:cholesterol acyltransferase; AMPK, AMP-activatedprotein kinase; AngII, angiotensin II; apoE, apolipoprotein E; APPL1, adaptor protein containing pleckstrin homology domain,phosphotyrosine-binding domain and leucine zipper motif 1; ARB, AngII-receptor blocker; BMI, body mass index; (hs-)CRP, (high-sensitivity) C-reactive protein; eNOS, endothelial NO synthase; Erg-1, early growth response protein-1; ERK1/2, extracellular-signal-regulated kinase 1/2; GPCR, G-protein-coupled receptor; HMW, high-molecular-weight (‘mass’); HSP90, heat-shock protein90; HUVEC, human umbilical vein endothelial cell; IL, interleukin; ICAM-1, intercellular adhesion molecule-1; IMT, intima-media thickness; LDL, low-density lipoprotein; LMW, low-molecular-weight (‘mass’); LPS, lipopolysaccharide; MCP-1, monocytechemoattractant protein-1; MMP, matrix metalloproteinase; MMW, middle-molecular-weight (‘mass’); NF-κB, nuclear factor κB;ox-LDL, oxidized LDL; PDGF-BB, platelet-derived growth factor-BB; PKA, cAMP-dependent protein kinase; PPAR, peroxisome-proliferator-activated receptor; ROS, reactive oxygen species; SMC, smooth muscle cell; T2DM, Type 2 diabetes mellitus; TIMP-1,tissue inhibitor of metalloproteinases-1; TLR, Toll-like receptor; TNF-α, tumour necrosis factor-α; TZD, thiazolidinedione; VCAM-1, vascular cell adhesion molecule-1; VSMC, vascular SMC.Correspondence: Dr Aimin Xu (email [email protected]).

C© The Authors Journal compilation C© 2008 Biochemical Society

362 W. Zhu and others

INTRODUCTION

Obesity, which is the accumulation of excessive adiposetissue, is closely associated with an increased risk ofcardiovascular morbidities, including hypertension,atherosclerosis and other vascular complications. Prev-iously regarded as just a storage depot of excess energy,the adipose tissue is now recognized to be a highlyversatile endocrine gland, secreting a large number ofbioactive molecules (collectively called adipokines oradipocytokines) [1]. Adipokines are actively involved indiverse biological processes, including energy metabol-ism, insulin sensitivity, immune responses and vascularhomoeostasis. As obesity develops, adipose tissue isinfiltrated with numerous activated macrophages, leadingto the augmented production of various proinflammatoryfactors, such as TNF-α (tumour necrosis factor-α), IL(interleukin)-6, MCP-1 (monocyte chemoattractantprotein-1), resistin, leptin, serum amyloid A3, lipocalin-2and PAI-1 (plasminogen activator inhibitor-1) [2]. Theseadipose-tissue-derived factors act either in a paracrinemanner to perpetuate local inflammation in adipose tissueor in an endocrine manner to induce insulin resistance andvascular dysfunction. Aberrant production of adipokineshas recently been proposed to be a key mechanism thatlinks obesity to increased risk of vascular complications[3].

Although most adipokines impair insulin sensitivityand promote vascular diseases, adiponectin appears topossess antidiabetic, anti-atherogenic and anti-inflam-matory activities [4]. In recent years, adiponectin hasattracted much attention due to its pleiotrophic salutaryeffects on obesity-related cardiometabolic complications.The pathophysiological roles of adiponectin andadiponectin receptors in insulin resistance, T2DM (Type 2diabetes mellitus) and the metabolic syndrome have beenextensively reviewed elsewhere [4,5]. In this review, wewill focus on the recent research related to the vascularactions of adiponectin. In addition, the potential of usingadiponectin or its receptors as targets for therapeuticintervention of vascular diseases will also be discussed.

STRUCTURAL PROPERTIES AND DIVERSEFUNCTIONS OF ADIPONECTIN

Adiponectin, also termed Acrp30, AdipoQ, apM1 orGBP28, was identified originally by four independentgroups in both mice and humans [6–9]. The gene encodinghuman adiponectin is located on chromosome 3q27 [10],a susceptibility locus for diabetes and cardiovascular dis-ease. Indeed, a growing body of genetic studies suggeststhat the genetic deficits in adiponectin production or itsaction may contribute to the pathogenesis of obesity-related disorders [11].

Full-length adiponectin is composed of 247 aminoacid residues, including the N-terminal hypervariableregion, followed by a conserved collagenous domaincomprising 22 Gly-Xaa-Yaa repeats and a C-terminalC1q-like globular domain. In both mouse and humanplasma, adiponectin is present predominantly as threemajor oligomeric forms [12,13]. The monomeric formof adiponectin has never been detected under nativeconditions. The basic unit of the oligomeric adiponectin isthe homotrimer, also called LMW [low-molecular-weight(‘mass’)] adiponectin [14,15]. Two subunits of the adipo-nectin trimer are linked by a disulfide bond mediatedthrough a cysteine residue in the collagen-like domain toform a hexamer, also termed MMW [middle-molecular-weight (‘mass’)] adiponectin. The hexamer provides thebuilding block for the formation of a bouquet-like HMW[high-molecular-weight (‘mass’)] adiponectin comprising12–18 protomers. Post-translational modifications arerequired for the intracellular assembly of the HMWoligomeric complex in adipocytes [16]. Different oligo-meric forms of adiponectin act on different targets andpossess distinct biological functions.

Adiponectin is abundantly present in the circulation,accounting for approx. 0.01 % of the total human plasmaprotein [17]. Unlike most other adipokines, circulatinglevels of adiponectin are decreased significantly in obesityand its co-morbidities, including insulin resistance,T2DM, coronary heart disease, stroke, non-alcoholicfatty liver disease and steatohepatitis, and several types ofcancers (breast, colon and prostate) [18–20]. Prospectivestudies have demonstrated that adiponectin deficiency(hypoadiponectinaemia) is associated causally withincreased prevalence and/or poor prognosis of thesediseases, independent of other classical risk factors. Onthe other hand, elevation of circulating adiponectin byeither genetic or pharmacological approaches in animalmodels has been shown to be effective in preventingmost obesity-related medical complications, by its directactions on multiple target tissues [21–25].

ADIPONECTIN RECEPTORS

Two adiponectin receptors (AdipoR1 and AdipoR2) havebeen identified [26]. Both receptors contain seven trans-membrane domains, but are structurally and functionallydistinct from classical GPCRs (G-protein-coupledreceptors). Both AdipoR1 and AdipoR2 have an invertedmembrane topology with a cytoplasmic N-terminus and ashort extracellular C-terminus of approx. 25 amino acids.In C2C12 myotubes, AdipoR1 and AdipoR2 mediate in-creased AMPK (AMP-activated protein kinase), PPARα

(peroxisome-proliferator-activated receptor α) ligandactivities, and glucose uptake and fatty acid oxidationby adiponectin [26]. Recent studies have identifiedAPPL1, an adaptor protein containing a PH (pleckstrin


Vascular effects of adiponectin 363

homology) domain, PTB (phosphotyrosine binding)domain and leucine zipper motif, as a direct interactingpartner of AdipoR1 and AdipoR2 [27,28]. APPL1appears to play a key role in coupling the adiponectin re-ceptors to their downstream signalling cascades, althoughthe detailed molecular events remain to be elucidated.

Several groups have recently investigated the phen-otypic changes in AdipoR1- and AdipoR2-knockoutmice. Yamauchi et al. [29] reported that targeted dis-ruption of AdipoR1 resulted in the abrogation ofadiponectin-induced activation of AMPK, whereasablation of AdipoR2 diminished adiponectin-stimulatedPPARα signalling [29]. Simultaneous disruption ofboth AdipoR1 and AdipoR2 abolished the binding andactions of adiponectin, leading to insulin resistanceand marked glucose intolerance [29]. These findingssupport the physiological roles of AdipoR1 and AdipoR2as the predominant receptors for adiponectin in theregulation of glucose and lipid metabolism. AdiopR1-null mice generated by Bjursell et al. [30] had increasedadiposity with augmented glucose intolerance, whereasAdipoR2-null mice were lean and resistant to diet-induced glucose intolerance, indicating that AdipoR1and AdipoR2 might have opposing effects. On the otherhand, Liu et al. [31] have shown that ablation of AdipoR2reduced diet-induced insulin resistance, but promotedT2DM. The precise physiological roles of these tworeceptors need to be further clarified in future studies.

In addition to AdipoR1/AdipoR2, T-cadherin, a GPI(glycosylphosphatidylinol)-linked cell-surface molecule,has also been suggested as a potential receptor for adipo-nectin [32]. T-cadherin is highly expressed in the heart,smooth muscle and endothelium, which are also thetargets of adiponectin; however, the functional relevanceof adiponectin binding to T-cadherin remains elusive.

PROTECTION BY ADIPONECTIN AGAINSTVASCULAR DISEASES

Clinical dataOver the past several years, numerous epidemiologicalinvestigations based on different ethnic groups haverepeatedly documented a close association of adiponectindeficiency with the development of almost every stageof vascular disease [18,19]. Hypoadiponectinaemiawas found to be a significant predictor of endothelialdysfunction in both periphery and coronary arteries,independent of the insulin resistance index, BMI (bodymass index) and dyslipidaemia [33,34]. In addition, resultsobtained from both cross-sectional and prospectiveinvestigations have demonstrated low levels of adipon-ectin as an independent risk factor for hypertension[35,36]. Subjects with essential hypertension hadsignificantly lower concentrations of plasma adiponectincompared with normotensive healthy subjects, even

after adjustment for confounding factors by multipleregression analysis [37]. In a recent 5-year follow-upstudy including 577 non-diabetic Chinese subjects [36],we found that normotensive subjects with baseline serumadiponectin levels in the lowest sex-specific tertile hada significantly increased risk of becoming hypertensive,suggesting that hypoadiponectinaemia may contributeto the pathogenesis of hypertension in humans.

The inverse correlation between serum adiponectinconcentrations and carotid IMT (intima-media thick-ness), a well-established measure of subclinical athero-sclerosis, has been reported in a number of studies inboth healthy subjects and patients with T2DM [38,39].In a study of 140 obese juveniles (mean age, 13.5 years),serum levels of adiponectin were correlated negativelywith carotid IMT, even after controlling for BMI, theinsulin resistance index, cholesterol, triacylglycerols(triglycerides), blood pressure, hs-CRP (high-sensitivityC-reactive protein), gender and age [40]. On the otherhand, in another recent study involving 887 middle-agedindividuals, a significant inverse age-adjusted associationof serum adiponectin with carotid IMT was onlyobserved in men, but not in women [41]. This associationwas attenuated after adjustments for other risk factors.

Numerous epidemiological studies have identifiedhypoadiponectinaemia as a predictor for coronary arterydisease, acute coronary syndrome, myocardial infarctionand ischaemic cerebrovascular disease, independent ofclassical cardiovascular risk factors [42]. A prospectivestudy of patients with renal failure demonstrated thatthe risk of adverse cardiovascular events decreased by3 % for each 1 mg/ml increase in serum adiponectinlevels, and the relative risk of adverse cardiovascularevents was 1.56 times higher among patients in the firstadiponectin tertile compared with those in the thirdtertile [43]. In another prospective nested case-controlstudy, high plasma levels of adiponectin were found to beassociated with a significantly decreased risk of myocar-dial infarction over a follow-up period of 6 years among18 225 male participants without a previous historyof cardiovascular disease [44]. This association wasindependent of hypertension, diabetes or inflammation,and was only partly explained by changes in lipid profiles.

Plasma adiponectin levels have been shown to beclosely associated with several surrogate markers ofvascular diseases. A significant inverse correlation wasobserved between adiponectin and hs-CRP, a well-estab-lished inflammatory marker closely associated with ather-osclerosis [45,46]. Recent studies have also demonstrateda negative association of plasma adiponectin with othermarkers of inflammation and atherosclerosis, includingA-FABP (adipocyte fatty-acid-binding protein) [47] andlipocalin-2 [48]. On the other hand, plasma adiponectincorrelated positively with the number of endothelialprogenitor cells, which have recently been identified assignificant contributors to vascular repair [49]. These



Figure 1 Adiponectin exerts its vasculoprotective activities through multiple mechanismsSM cells, SMCs.

clinical findings collectively support an aetiological roleof adiponectin deficiency in the development of variousvascular complications in humans.

Animal-based investigationsConsistent with the clinical observations describedabove, adiponectin-deficient mice have been shownto be more susceptible to develop vascular disorders.Adiponectin-knockout mice had a significantly increasedneointimal hyperplasia after acute vascular injury [50,51],an impaired endothelium-dependent vasodilation on anatherogenic diet [52] and an elevated blood pressure com-pared with their wild-type littermates [53]. On the otherhand, both adenovirus-mediated overexpression of full-length adiponectin [54] and transgenic overexpression ofglobular adiponectin [50] resulted in a marked alleviationof atherosclerotic lesions in apoE (apolipoprotein E)-deficient mice, and also caused a significant improvementin endothelial dysfunction and hypertension in severalmouse models of obesity [52,53].

The protective effect of adiponectin against vasculardisorders has also been observed in a recent studyin a rabbit model with spontaneous atherosclerosis[55]. Intravascular ultrasonography analysis revealeda significantly reduced atherosclerotic plaque area inabdominal aortas of rabbits after local treatment withadiponectin through injection of recombinant aden-

oviruses compared with those treated with adenovirusexpressing β-galactosidase as a control. Adiponectin-mediated attenuation of atherosclerosis in this modelwas associated with the decreased expression of adhesionmolecules such as VCAM-1 (vascular cell adhesion mole-cule-1) and ICAM-1 (intercellular adhesion molecule-1).

PLEIOTROPIC EFFECTS OF ADIPONECTIN ONTHE VASCULAR SYSTEM

In addition to its beneficial effects on insulin sensitivityand lipid metabolism, adiponectin exerts its multiplevasculoprotective effects through its direct actions on thevascular system, including endothelial cells, monocytesand macrophages, leucocytes, platelets and SMCs(smooth muscle cells) (Figure 1). Adiponectin is effectivein preventing almost every pathogenic event involved inatherosclerotic plaque formation, as summarized below.

Augmentation of endothelial NOproductionEndothelium-derived NO protects the vascular systemby enhancing vasodilation and inhibiting plateletaggregation, monocyte adhesion and SMC proliferation[56]. Decreased bioavailability and/or impaired pro-duction of NO cause endothelial dysfunction, whichis now recognized as one of the earliest changes in



Figure 2 Intracellular signalling events that mediate theactions of adiponectin in endothelial cellsEC, endothelial cell; gAd, globular adiponectin.

atherosclerosis. Both the full-length and globular domainof adiponectin have been shown to increase endothelialNO production in several types of endothelial cells,through activation of eNOS (endothelial NO synthase)[57–60]. Adiponectin stimulates eNOS phosphorylationat Ser1177 and also enhances the interaction betweeneNOS and HSP90 (heat-shock protein 90), a complexrequired for the maximal activation of eNOS (Figure 2).

A more recent study from our laboratory has shownthat both AdipoR1 and AdipoR2 are required for mediat-ing adiponectin-induced NO production [27]. Adipon-ectin stimulation facilitates the interaction between itstwo receptors with APPL1, a novel adaptor proteininvolved in signal transduction of multiple extracellularstimuli. Suppression of APPL1 expression by RNAi(RNA interference) significantly attenuated adiponectin-induced phosphorylation of AMPK at Thr172 and eNOSat Ser1177, as well as the complex formation betweeneNOS and HSP90, resulting in a marked reduction of NOproduction. In db/db diabetic mice, APPL1 expression insmall mesenteric arteries was decreased markedly com-pared with their lean controls. Notably, decreased APPL1expression was associated with impaired vasodilation in

response to adiponectin [27]. These findings support thekey role of APPL1 as a signalling relay that mediates theadiponectin-induced cellular signalling cascade leading toNO production. On the other hand, overexpression of aconstitutively active form of AMPK alone was sufficientto stimulate eNOS activation and NO production, evenwhen APPL1 expression was suppressed [27], suggestingthat AMPK acts downstream of APPL1 and is directlyresponsible for both eNOS phosphorylation at Ser1177

and its interaction with HSP90 (Figure 2). However, thedetailed signalling events that link APPL1 with AMPKactivation remain to be elucidated. There is also evidencesuggesting the involvement of PI3K (phosphoinos-itide 3-kinase) in adiponectin-induced endothelial NOproduction, possibly through activation of AMPK[59–61].

Antioxidant activities of adiponectinin the endotheliumIncreased oxidative stress underlies the pathogenesis ofvascular dysfunction in obesity and diabetes [62]. Thekey feature of oxidative stress is the increased productionof vascular ROS (reactive oxygen species), resulting in thequenching of NO and activation of pro-inflammatorysignalling pathways, such as PKC (protein kinase C) andNF-κB (nuclear factor κB) [62]. Globular adiponectin in-hibits both basal and ox-LDL [oxidized LDL (low-den-sity lipoprotein)]-induced ROS release in bovine endo-thelial cells, possibly through suppression of NADPHoxidase [63]. A more recent study has demonstrated thatboth full-length and globular adiponectin inhibited high-glucose-induced ROS generation in cultured HUVECs(human umbilical vein endothelial cells) [64]. In addition,the cardioprotection of adiponectin against myocardialischaemia/reperfusion might also be attributed, at least inpart, to the reduction of oxidative stress by adiponectin[65]. Consistent with these in vitro findings, clinicalstudies have observed a negative association betweenplasma adiponectin and markers of oxidative stress (suchas urinary 8-epi-prostaglandin-F2α) [66,67].

The effect of adiponectin on the suppression ofexcessive ROS production under hyperglycaemicconditions was abolished by pretreatment of cells withthe PKA (cAMP-dependent protein kinase) inhibitorH-89, but not by the AMPK inhibitor compoundC [64]. Although the two adiponectin receptors arestructurally distinct from classical GPCRs, both full-length and globular adiponectin have been shown toelevate intracellular cAMP levels in endothelial cells.Furthermore, activation of cAMP signalling by treatmentwith forskolin or dibutyryl-cAMP mimicked the effectsof adiponectin in decreasing glucose-induced ROSgeneration, whereas activation of AMPK by AICAR(5-amino-4-imidazolecarboxamide riboside) only hada modest effect. Together, these results suggest that the



antioxidant activities of adiponectin are mediated by thecAMP/PKA pathway (Figure 2). Nevertheless, whetheror not AdipoR1 and/or AdipoR2 are involved in theadiponectin-induced increase in cAMP and suppressionof ROS production remains to be clarified.

Effects of adiponectin on endothelial cellactivationEndothelial cell activation, characterized by increasedexpression of adhesion molecules (such as ICAM-1,VCAM-1 and E-selectin), is an early event in athero-genesis. Adiponectin exerts its anti-inflammatory effectson the endothelium by suppressing both TNF-α- andresistin-induced expression of adhesion moleculesand IL-8, which, in turn, results in the attenuationof monocyte attachment to endothelial cells [68]. Inaddition, adenovirus-mediated expression of adiponectindecreases the expression of adhesion molecules in theaortic tissue of apoE-deficient mice [54] and a rabbitmodel with atherosclerosis [60]. The anti-inflammatoryeffects of adiponectin in endothelial cells appear to bemediated by PKA-dependent suppression of NF-κBactivation [69] (Figure 2).

By contrast, a more recent study has demonstratedthat acute treatment with globular adiponectin activatesNF-κB and enhances the expression of adhesionmolecules and MCP-1 in endothelial cells, throughactivation of the sphingosine kinase signalling pathway[70]. The discrepancy between these reports can beattributed to different forms of adiponectin or differentincubation times in each study. Indeed, a previous studyhas shown that different oligomeric forms of adiponectinmay have opposite functions in modulating NF-κBactivity in C2C12 myotubes [71].

Suppression of the leucocyte–endotheliuminteractionsRecruitment of circulating leucocytes into the endothe-lium is now recognized as an important step in thepathophysiology of both macrovascular and microvascu-lar diseases [72]. The pathological leucocyte–endotheliuminteraction triggers the exposure of the vascular wall andsurrounding tissues to the damaging action of activatedleucocytes. A recent study in adiponectin-knockout micefound that adiponectin deficiency caused a 2-fold increasein leucocyte rolling and a 5-fold increase in leucocyteadhesion in the microcirculation [73]. These changeswere associated with a significantly decreased NO level,but an increased expression of E-selectin and VCAM-1 in the vascular endothelium. On the other hand,systemic administration of recombinant adiponectinto adiponectin-deficient mice significantly restoredendothelial NO to a physiological level and suppressedthe expression of adhesion molecules, and attenuated theleucocyte–endothelium interactions. In addition, pre-

treatment with adiponectin also protected wild-type miceagainst TNF-α-induced leucocyte–endothelium interac-tions [73]. The inhibitory effects of adiponectin on leu-cocyte adhesion and adhesion molecule expression wereabolished by the eNOS inhibitor l-NAME (NG-nitro-l-arginine methyl ester), suggesting that eNOS/NOsignalling is required for the anti-inflammatory activitiesof adiponectin in endothelial cells (Figure 2).

Protection of the endothelium fromapoptosisEndothelial cell injury is considered a critical eventin the pathogenesis of atherosclerosis, plaque erosionand thrombus formation [74]. In atherosclerotic lesions,the turnover rate of endothelial cells is acceleratedand local apoptosis of these cells is implicated in thisprocess. Endothelial cell injury can be induced by highglucose, AngII (angiotensin II) and ox-LDL. The HMWoligomeric form of adiponectin, but not its trimericor hexameric complexes, inhibits apoptosis and caspase3 activity in HUVECs, through the activation of theAMPK signalling pathway [75]. On the other hand,the globular domain of adiponectin dose-dependentlyinhibited AngII-induced apoptosis in HUVECs, possiblythrough restoring the eNOS–HSP90 interaction andeNOS activation [76]. Whether or not the twoadiponectin receptors and/or APPL1 is obligatory forthe anti-apoptotic activity of this adipokine in endothelialcells needs to be clarified further in future studies.

Inhibition of macrophage activation andfoam cell formationA critical step in the development of atheroscleroticplaques is the infiltration of monocytes into thesubendothelial space of arteries where they differentiateinto macrophages [77]. Activated macrophages expressscavenger receptors and internalize modified lipopro-teins, thereby transforming themselves into foam cells.The pro-inflammatory factors produced from activatedmacrophages are the major contributors to endothelialcell activation and atherosclerotic lesion formation.Recent studies have demonstrated that adiponectinsuppresses both macrophage activation and foam cellformation.

Both AdipoR1 and AdipoR2 are expressed inmonocytes and macrophages [78]. Adiponectin has beenshown to dampen the early phases of macrophage inflam-matory responses [79], acting to inhibit the growth ofmyelomonocytic progenitor cells and decrease the abilityof mature macrophages to respond to various activations[79,80]. Both full-length and globular forms of adipon-ectin inhibited leptin- and LPS (lipopolysaccharide)-induced macrophage production of pro-inflammatorycytokines, such as TNF-α, IL-1, IL-6 and IL-8 [81–85]. Inaddition, adiponectin binds to LPS and other chemokines,



Figure 3 Molecular mechanisms underlying the multiple actions of adiponectin in macrophagesgAd, globular adiponectin; SRA, scavenger receptor A.

such as MIP-1α (macrophage-inflammatory protein-1α)and MCP-1, in vitro, which may lead to the decreasedbioavailability of these pro-inflammatory factors [86,87].

Prolonged treatment of macrophages with adiponectin(6–24 h) caused desensitization of LPS-stimulated NF-κB and ERK1/2 (extracellular-signal-regulated kinase1/2) activation [79,80,88]. On the other hand, short-term treatment with adiponectin (<30 min) activated theNF-κB and ERK1/2 pathways, and induced the pro-duction of TNF-α and IL-6 in various types ofmacrophages [81,85]. These apparently paradoxicalobservations can be explained by the ability ofadiponectin to induce ‘macrophage tolerance’ (Figure 3).This hypothesis was initially proposed by Tsatsanis andco-workers [85], who demonstrated that pre-exposureof macrophages with 10 µg/ml adiponectin renderedthe cells tolerant to further adiponectin exposure or toother pro-inflammatory stimuli, such as the TLR3 (Toll-like receptor 3) ligand polyI:C (polyinosine:polycytidylicacid) and the TLR4 ligand LPS. A more recent report by

Park et al. [81] provided further a detailed molecular basisto support this hypothesis. This study showed that tran-sient activation of NF-κB and ERK1/2 by adiponectinincreased the expression of Erg-1 (early growth responseprotein-1), which consequently transactivated TNF-αgene transcription and protein production. Transientelevation of TNF-α by adiponectin was obligatory forthe subsequent induction of IL-10, an anti-inflammatorycytokine which renders macrophages tolerant to LPS andother pro-inflammatory stimuli [81]. Indeed, adiponectinhas been shown to induce IL-10 production inboth macrophages and human leucocytes [89,90]. Co-culture of macrophages with a neutralizing antibodyagainst IL-10 during 18 h of exposure to adiponectinprevented the adiponectin-mediated desensitization ofLPS-stimulated TNF-α mRNA accumulation [81]. Theseresults collectively suggest that induction of IL-10 is akey step in establishing adiponectin-induced macrophagetolerance to activation by various pro-inflammatorystimuli. Furthermore, induction of IL-10 by adiponectin



also resulted in the augmented expression of TIMP-1(tissue inhibitor of metalloproteinases-1), a physiologicalinhibitor of MMPs (matrix metalloproteinases) that isinvolved in the rupture of atherosclerotic plaques [91].Therefore adiponectin-mediated induction of TIMP-1 expression through IL-10 might contribute to itsvasculoprotective activity by the stabilization of ather-osclerotic lesions. Consistent with these in vitro findings,a positive correlation between plasma adiponectin andIL-10 expression was observed in a clinical study [93].

In addition to macrophages, it is interesting to notethat the rapid activation of NF-κB by adiponectin hasrecently been reported in human endothelial cells [94].Although this study did not investigate the long-termeffects of adiponectin, previous findings published fromother groups have demonstrated that chronic treatmentwith this adipokine inhibited TNF-α or high-glucose-mediated NF-κB activation in endothelial cells [69,95].Therefore further studies are warranted to investigatewhether or not the rapid and transient activation ofNF-κB is also an obligatory step in adiponectin-mediatedlong-term tolerance of endothelial cells to the activationinduced by pro-inflammatory factors. Although thehypothesis that the short-term activation of NF-κB byadiponectin renders the long-term tolerance to inflam-mation is attractive, the pathophysiological relevance ofthese in vitro observations remains unclear at this stage.Plasma adiponectin concentrations in both humans androdents are relatively stable and do not vary substantiallyin response to acute stimuli such as transient nutritionalchanges. Therefore adiponectin-induced short-term andrapid activation of NF-κB observed in these in vitrostudies might not occur under physiological conditions.

The inhibitory effects of adiponectin on macrophage-to-foam cell transformation might be mediated by itsability to suppress class A scavenger receptor expression,resulting in reduced uptake of acetylated LDL particles[96]. In addition, adiponectin decreased ACTA (acyl-CoA:cholesterol acyltransferase) activity, the enzymethat catalyses cholesteryl ester formation and enhancesmacrophage-to-foam cell transformation [66].

Regulation of phagocytosis by adiponectinMounting evidence suggests that defects in the phagocyticfunction of macrophages contribute to progressionof atherosclerosis [97,98]. Impaired clearance of earlyapoptotic cells by macrophages has been observed inatherosclerotic lesions of humans and rabbits [99].Although an earlier report demonstrated an inhibitoryeffect of adiponectin on phagocytosis in response tostimulation with LPS in vitro [79], more recent studieshave provided both in vitro and in vivo evidenceshowing that adiponectin enhances phagocytic activitiesof macrophages [84,100]. In comparison with wild-type mice, adiponectin-deficient mice had a markedlyimpaired ability to clear apoptotic thymocytes in

response to dexamethasone treatment and to remove earlyapoptotic cells that were injected into their intraperitonealcavities [100]. In addition, adiponectin deficiency in lprmice led to a further reduction in apoptotic cell clearance,which was accompanied by exacerbated systemic inflam-mation. Conversely, replenishment with recombinantadiponectin promoted the clearance of apoptotic cellsby macrophages in both adiponectin-deficient and wild-type mice, and also reduced features of autoimmunity inlpr mice [100]. The stimulatory effects of adiponectin onphagocytosis are attributed to the ability of this adipokineto opsonize apoptotic debris and to facilitate its bind-ing to the macrophage cell surface, through interactionwith calreticulin and CD91 (Figure 3). Taken together,these results suggest that adiponectin protects the org-anism from systemic inflammation by promoting theclearance of early apoptotic cells by macrophages througha mechanism involving calreticulin. Notably, none of theputative adiponectin receptors (AdipoR1, AdipoR2 andT-cadherin) are required for the phagocytotic activities ofadiponectin [100].

Antithrombotic activities of adiponectinPlatelet activation plays a crucial role in the progression ofatherosclerosis and plaque rupture. Inhibition of plateletaggregation has been suggested to prevent arterialthrombosis [101]. A clinical study has demonstratedthat the plasma adiponectin level was negativelyassociated with platelet activation independently ofother risk factors [102]. Adiponectin-deficient mice hadaccelerated thrombus formation following laser-inducedcarotid arterial injury, whereas adenovirus-mediatedexpression of adiponectin reversed these changes [103].In vitro, adiponectin inhibited collagen-induced plateletaggregation in platelet cells harvested from humansubjects and adiponectin-deficient mice [103]. It is well-established that endothelial-derived NO inhibits plateletactivation and, hence, suppresses platelet adhesion andaggregation [104]. The inhibitory effects of adiponectinon thrombosis may be attributed, at least in part, to itsability to stimulate endothelial NO production.

Inhibition of smooth muscle proliferationVSMC (vascular SMC) proliferation and migrationtoward the intima contribute to intimal thickening of thearteries during the development and progression of vas-cular lesions. Adiponectin inhibits both proliferation andmigration of human aortic SMCs induced by severalatherogenic growth factors, including HB-EGF [heparin-binding EGF (epidermal growth factor)-like growthfactor], PDGF-BB (platelet-derived growth factor-BB)and bFGF (basic fibroblast growth factor) [105,106]. Amore recent study has also demonstrated the suppressiveeffects of adiponectin on PDGF-BB-induced prolifera-tion of primary pulmonary arterial SMCs harvested fromapoE-deficient mice [107]. The antiproliferative effect of



Figure 4 Summary of therapeutic interventions that have been shown to increase adiponectin production, or up-regulateor activate adiponectin receptorsAR, AngII receptor.

adiponectin is attributed primarily to its oligomerization-dependent interaction with these growth factors, leadingsubsequently to the blockade of their binding to therespective cell-membrane receptors [106]. Consistentwith these in vitro findings, adiponectin-deficient mice,as compared with wild-type controls, had enhancedproliferation of VSMCs and increased neointimalthickening after mechanical injury [108]. Adenovirus-mediated expression of adiponectin in these miceattenuated the extent of neointimal proliferation [54].

ADIPONECTIN AND ITS PATHWAYS AS ATHERAPEUTIC TARGET FOR VASCULARDISEASES

According to the clinical and experimental evidence dis-cussed above, therapeutic interventions that can enhancethe actions of adiponectin, such as increasing plasmaadiponectin concentrations or up-regulating/activa-ting adiponectin receptors, could represent attractivestrategies to combat obesity-related diabetes and vasculardiseases.

Elevation of plasma adiponectinconcentrationsMany currently available therapies for cardiovasculardiseases, such as lifestyle modifications, calorierestriction, and pharmacological and dietary interven-tions, have been shown to increase plasma levels ofadiponectin in rodents and/or humans (Figure 4). Thereis also growing interest in the pharmaceutical industryto search for natural or synthetic compounds that canincrease adiponectin production [109].

Prolonged weight loss through either gastric bandingsurgery or calorie restriction increased circulating levelsof adiponectin in obese subjects [110]. A combination ofa Mediterranean-type diet with moderate physicalactivity also induced a significant increase in adiponectinin pre-menopausal obese women [111]. In addition,weight loss led to changes in the oligomeric distributionof adiponectin [75]. HMW adiponectin was significantlyincreased, whereas the hexameric and trimeric formswere decreased. Dietary fish oils and polyunsaturatedfatty acids increased adipose tissue mRNA expressionand plasma levels of adiponectin in several animal modelsof obesity [112–114]. Furthermore, Oolong tea [115],green tea extracts [116] and (−)-catechin (a type of greentea polyphenol) [117] have been shown to elevate plasmaadiponectin in humans and rodent models.

The PPARγ agonists TZDs (thiazolidinediones)are widely used antidiabetic drugs that also possessvasculoprotective and anti-inflammatory properties.TZDs, such as rosiglitazone and pioglitazone, increaseadiponectin production in humans and rodents in vivo,and in adipocytes in vitro [118–120]. In patients withdiabetes, TZD-mediated increases in adiponectin,especially its HMW oligomeric forms, correlated wellwith improvements in insulin sensitivity [121]. The TZD-induced increase in circulating adiponectin is probablymediated by their ability to transactivate adiponectingene expression [119] and selectively enhance thesecretion of the HMW oligomeric form of this adipokinefrom adipocytes [122]. Two recent independent studieson adiponectin-deficient mice have demonstrated thatthe insulin-sensitizing effects of TZDs were mediated,at least in part, by induction of adiponectin production[50,123].



PPARα agonists have also been shown to increaseadiponectin production. Fenofibrate therapy signific-antly increased plasma adiponectin levels and insulinsensitivity in patients with primary hypertriglyceri-daemia [121]. Significant correlations between the degreeof changes in serum adiponectin concentrations andinsulin levels, CRP levels and insulin sensitivity wereobserved after fenofibrate therapy [124]. Notably,fenofibrate therapy for 2 months elevated serumadiponectin levels without a change in body weight[125]. This raises the possibility that the therapeuticeffect of this drug may be, in part, mediated through theinduction of adiponectin.

Drugs blocking the RAS (renin–angiotensin system)blocking, including ACE (angiotensin-convertingenzyme) inhibitors and ARBs (AngII-receptor blockers),have repeatedly been shown to elevate plasma adiponectinlevels without affecting adiposity [126–129]. Losartanalone or a combined therapy with simvastatin and losa-rtan in patients with hypercholesterolaemia andhypertension significantly increased plasma adiponectinlevels and insulin sensitivity relative to baselinemeasurements [125]. In addition, several other agentswith either antidiabetic and/or vasculoprotectiveactivities, including glimepiride (a glucose-loweringdrug) [130], nebivolol (a new β-adrenergic blocker) [131]and rimonabant (a cannabinoid CB1 receptor antagonist)[132], have also been shown to increase plasma adipon-ectin concentrations in humans. However, it is currentlyunclear whether or not the beneficial effects of these drugson cardiovascular disease are mediated by adiponectin.

Up-regulation or activation of adiponectinreceptorsIn addition to hypoadiponectinaemia, decreasedexpression of the two adiponectin receptors (AdipoR1/AdipoR2) have also been reported in obese animals withinsulin resistance and endothelial dysfunction [133],and in patients with T2DM [134]. Therefore anotherlogical approach to combat obesity-related metabolicand vascular diseases is to induce AdipoR1/AdipoR2expression or to develop receptor agonists that canmimic adiponectin actions. A recent study [135] hasdemonstrated that exercise combined with a hypocaloricdiet increased both AdipoR1 and AdipoR2 expression inskeletal muscle in older obese adults (>60 years of age).In addition, intensive physical training has been shownto elevate AdipoR2 expression in adipose tissue [136]. Inhuman macrophages, both PPARα and PPARγ agonistsincreased AdipoR2 expression, whereas a synthetic LXR(liver X receptor) agonist induced the expression ofboth AdipoR1 and AdipoR2 [78]. In KKAy obese mice,a PPARα agonist reversed the decreases in AdipoR1and AdipoR2 expression in adipose tissue [137]. Thesefindings raise the possibility that up-regulation of

adiponectin and its receptors might represent one ofthe mechanisms accounting for the vasculoprotectiveactivities of PPARα and PPARγ agonists.

There are no currently available therapeutic strategiesthat have been shown to mimic the actions of adiponectinin activating its receptors. A recent in vitro study hasidentified osmotin, a member of the PR-5 (pathogenesisrelated-5) family of plant defence proteins, as apotential adiponectin receptor agonist [138]. The three-dimensional structure of osmotin is similar to globularadiponectin, both of which consist of antiparallel β-strands arranged in the shape of a β-barrel. Interestingly,osmotin activates AMPK via adiponectin receptors inmammalian C2C12 myocytes.

CONCLUDING REMARKS

Recent research provides compelling evidence supportingthe role of adiponectin as a physiological regulator ofvascular homoeostasis in both animal models andhumans. Major progress has been made in unveilingthe molecular mechanisms that underlie the multiplevasculoprotective actions of this adipokine. However,it should be noted that many mechanisms proposed arebased on in vitro studies and, thus, the physiologicalrelevance of these findings remains to be confirmed. Ourunderstanding of the adiponectin receptors, especiallywith respect to structure–function relationships andtheir pathophysiological role in vascular dysfunction, isstill at a very early stage. Further investigations in thisexciting field will facilitate the development of selectiveadiponectin agonists that can be potentially used for thetherapeutic intervention of diabetes and vascular disease.

ACKNOWLEDGMENTS

This work was supported by NSFC (Natural ScienceFoundation of China)/RGC (Research Grants Council)Joint Research Scheme (N HKU 727/05), MatchingFunding for National ‘973’ Basic Research Project, andan Outstanding Young Research Award from Universityof Hong Kong (to A. X).

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Received 2 October 2007/22 October 2007; accepted 24 October 2007Published on the Internet 1 February 2008, doi:10.1042/CS20070347


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Vascular effects of adiponectin: molecular …...Healthy Aging, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, People’s Republic of China ABSTRACT Adiponectin