Formation of Dysfunctional High-Density Lipoprotein by Myeloperoxidase

Formation of Dysfunctional High-Density Lipoprotein by Myeloperoxidase Stephen J. Nicholls, Lemin Zheng, and Stanley L. Hazen*

Recent studies identify the presence of high-density lipoprotein (HDL) particles in patients with cardiovascular disease, which are bdysfunctional,Q lacking in typical atheroprotective properties, and promoting proinflammatory effects. The mechanisms for generating dysfunctional HDL have been unclear. New evidence points to a role for myeloperoxidase (MPO)-generated oxidants as participants in rendering HDL dysfunctional within human atherosclerotic plaque. Myeloperoxidase was recently shown to bind to HDL within human atherosclerotic lesions, and biophysical studies reveal MPO binding occurs via specific interactions with apolipoprotein (apo) A-I, the predominant protein of HDL. This likely facilitates the observed selective targeting of apoA-I for site-specific chlorination and nitration by MPO-generated reactive oxidants in vivo. One apparent consequence of MPO-catalyzed apoA-I oxidation includes the functional impairment of the ability of HDL to promote cellular cholesterol efflux via the adenosine triphosphate binding cassette-1 transport system. Myeloperoxidase-mediated loss of the atheroprotective functional properties of HDL may thus provide a novel mechanism linking inflammation and oxidative stress to the pathogenesis of atherosclerosis. (Trends Cardiovasc Med 2005;15:212–219) D 2005, Elsevier Inc. Abbreviations: ABCA-1, adenosine triphosphate binding cassette-1; apo, apolipoprotein; ClTyr, chlorotyrosine; CVD, cardiovascular disease; H2O2, hydrogen peroxide; HDL, high density lipoprotein; HOCl, hypochlorous acid; LDL, low-density lipoprotein; MPO, myeloperoxidase; NO, nitric oxide (nitrogen monoxide); NO2
, nitrite; NO2Tyr, nitrotyrosine; OCl
, hypochlorite; oxLDL, oxidized lowdensity lipoprotein; SR-BI, scavenger receptor type B, class I; VCAM-1, vascular cell adhesion molecule-1. Stephen J. Nicholls, Lemin Zheng and Stanley L. Hazen are at Center for Cardiovascular Diagnostics and Prevention, Cleveland Clinic Foundation, Cleveland, Ohio. * Address correspondence to: Stanley L. Hazen, MD, PhD, Cleveland Clinic Foundation, Lerner Research Institute, Center for Cardiovascular Diagnostics and Prevention, 9500 Euclid Avenue, NE-10, Cleveland, OH 44195, USA. Tel.: (+1) 216-445-9763; fax: (+1) 216-636-0392; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter

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Since the seminal observation in the Framingham study that levels of plasma high-density lipoprotein (HDL) cholesterol serve as the strongest lipoprotein predictor of clinical risk (Gordon et al. 1977), numerous population (Gordon et al. 1989) and animal (Badimon et al. 1989, Plump et al. 1994) studies have established the atheroprotective properties of HDL. In addition to the wellcharacterized ability to promote reverse cholesterol transport (Fielding et al. 1995), HDL has reported antioxidant, antiinflammatory, and antithrombotic activities (Nofer et al. 2002, Bielicki and Oda 2002, Barter et al. 2004). Direct infusion of apolipoprotein (apo) A-I in animal models inhibits progression of atherosclerotic plaque (Miyazaki et al. 1995), and in recent studies, reconstituted forms of HDL in humans demonstrate both dramatic benefit on

endothelial function (Spieker et al. 2002, Bisoendial et al. 2003) and regression of atherosclerotic burden (Nissen et al. 2003). Despite the numerous demonstrated atheroprotective effects of HDL, high levels of the lipoprotein are not always protective in subjects, suggesting that not all HDLs function to prevent atherosclerosis. There remains a lack of consensus on how to manage patients with low levels of HDL. Raising HDL is a secondary goal in the current lipid management guidelines of the National Cholesterol Education Program (Grundy et al. 2004). Moreover, clinically available therapeutic agents have only limited ability to substantially elevate plasma HDL. As a result, there is no consensus supporting a target level for HDL elevation. This discrepancy results, in part, from the recognition that the quality of HDL may be as important as its quantity. A growing body of evidence supports the notion that some HDL is bdysfunctionalQ or bproinflammatoryQ, facilitating leukocyte recruitment and cellular activation phenotypes. Oxidative processes have been linked to the presence of proinflammatory HDL, though the precise pathways responsible for its formation are not established (Barter et al. 2004). Navab et al. (2000, 2001a, 2001b, 2001c) have shown that detection of proinflammatory HDL may serve as a useful marker for gauging susceptibility for atherosclerosis in subjects (Ansell et al. 2003). These investigators found that HDL isolated from patients with coronary artery disease and plasma HDL levels in the normal or high range promoted rather than inhibited monocyte chemotaxis in response to oxidized low-density lipoprotein (LDL), suggesting a possible mechanism for why normal levels of HDL are not always protective. This paper focuses on studies characterizing dysfunctional or proinflammatory forms of HDL, with particular emphasis on recent insights into pathways contributing to the oxidative modification of HDL in vivo, and their apparent influence on the functional properties of the lipoprotein. A series of recent reports identify novel in vivo modifications of apoA-I within human atherosclerotic plaque that are catalyzed by the enzyme myeloperoxidase TCM Vol. 15, No. 6, 2005

(MPO). These modifications appear to have a detrimental impact on the beneficial properties of the lipoprotein, inhibiting both lipid binding and adenosine triphosphate binding cassette-1 (ABCA-1)-dependent cholesterol efflux activities. The relationships between MPO-generated reactive oxidant species and impaired atheroprotection of HDL provide additional mechanistic links between inflammation, oxidative stress and atherogenesis.



Dysfunctional Forms of High-Density Lipoprotein

Despite the consistent demonstration that a low plasma HDL is a strong predictor of clinical risk, it is apparent that many patients with bnormalQ or even belevatedQ plasma HDL experience clinical events. In fact, nearly half of the clinical events in the Framingham cohort occurred in subjects with plasma HDL concentrations z40 mg/dL (Kwiterovich 1998). It has been proposed that HDL with impaired functional properties within subjects of this cohort may lead to either a loss of protective benefit or even an actual promotion of atherogenic events (Ansell et al. 2003). It is therefore of interest that HDL recovered from different subjects often demonstrates marked heterogeneity in its in vitro functional properties. For example, Ashby et al. (2001) demonstrated that HDL isolated from distinct subjects differed markedly in their ability to inhibit cytokine-induced expression of the adhesion molecule vascular cell adhesion molecule-1 by endothelial cells. Several investigators have similarly reported that HDL isolated from diabetic subjects has impaired ability both to promote cellular cholesterol efflux and to prevent the oxidation of LDL (Gowri et al. 1999, Syvanne et al. 1996). Another intriguing observation is that the antiinflammatory properties of HDL reportedly decline in the setting of the acute phase response. Van Lenten et al. (1995) compared the functional properties of HDL isolated from subjects before versus after elective surgery. High-density lipoprotein isolated preoperatively demonstrated antiinflammatory properties, such as the ability to inhibit LDL oxidation and subsequent monoctye TCM Vol. 15, No. 6, 2005

chemotaxis. In contrast, HDL isolated postoperatively promoted both LDL oxidation and monocyte chemotaxis. Functional properties of HDL during acute phase responses have been further studied in humans, rabbits, and mice (Ashby et al. 2001, Van Lenten et al. 1995, 2001). The transition to a proinflammatory form of HDL is reportedly associated with alterations in the composition of circulating HDL-associated proteins. Both reductions in the HDL contents of paraoxonase and platelet-activating factor acetylhydrolase, as well as parallel elevations in HDL content of serum amyloid A and ceruloplasmin, are reported (Van Lenten et al. 1995). These alterations in composition may underscore the observed effects of such proinflammatory HDL on monocyte chemotaxis (Ansell et al. 2003). Of interest, 6 weeks of treatment with simvastatin reportedly reduced the extent of monocyte chemotaxis induced by HDL preparations isolated from subjects with prior proinflammatory HDL (Ansell et al. 2003). These results further support the notion that the quality, rather than the

quantity, of circulating HDL may serve as the more important determinant of overall cardiovascular risk. Whereas HDL with apparent proinflammatory properties has been widely reported, the underlying mechanism(s) responsible for generating these functionally heterogeneous HDLs, and the chemical components responsible, remain largely unexplored. A leading hypothesis suggests that the nature and degree of oxidative modification of HDL may be responsible for the functional and biologic heterogeneity. 

High-Density Lipoprotein as a Selective Target of Oxidation in the Artery Wall

Low-density lipoprotein oxidation is widely believed to play an important role in the development of atherosclerotic plaque. Native LDL has little effect on cells of the arterial wall, whereas oxidatively modified forms of LDL induce numerous proatherosclerotic effects, including promotion of cholesterol deposition and foam cell formation (Partha-

Table 1. Preferential nitration and chlorination of apoA-I in serum of patients with established cardiovascular disease and in atherosclerotic lesions NO2Tyr Median (IQR) (mmol oxTyr/molTyr)

ClTyr P value

Median (IQR) (mmol oxTyr/molTyr)

P value

Serum Total protein Control CVD apoA-I Control CVD

6.1 (3.9–7.8) 9.0 (5.7–12.9)

b.001

1.6 (0.6–2.4) 1.9 (1.3–3.1)

.07

438 (335–598) 629 (431–876)

.005

186 (114–339) 500 (335–650)

b.001

Normal artery Total protein apoA-I

55 (24–143) 401 (185–637)

b.001

63 (25–128) 678 (299–1311)

b.001

Atheroma Total protein apoA-I

108 (51–346) 2340 (1665–5050)

b.001

232 (111– 431) 3930 (1679–7005)

b.01

Results are shown for median and interquartile (IQR) ranges of NO2Tyr and ClTyr contents of (A) total protein and apoA-I circulating in serum of patients with and without cardiovascular disease (CVD) and (B) total protein and apoA-I isolated from a normal arterial wall and atherosclerotic lesions, expressed as the mole ratio of oxidized to parent amino acid tyrosine. The P values shown are for comparisons of NO2Tyr or ClTyr content (A) in serum between control and CVD groups and (B) between normal arterial wall and atherosclerotic lesions. Adapted with permission from J Clin Invest 2004;114:529.

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Table 2. Relationship between serum nitrotyrosine and chlorotyrosine content in total protein versus isolated apoA-I and the prevalence of cardiovascular disease Odds ratio (95% CI) of CVD per tertile

Total protein NO2Tyr apoA-I NO2Tyr Total protein ClTyr apoA-I ClTyr

1

2

1.0 1.0 1.0 1.0

1.1 1.7 1.5 3.7

3 (0.4–3.6) (0.5–5.3) (0.5–4.6) (1.1–13.0)

5.1 5.5 2.1 16.0

(1.6–16.4) (1.6–18.4) (0.7–6.6) (4.0–64.0)

Results are presented as the odds ratio and 95% confidence intervals (CIs) of the prevalence of CVD for the second and third tertiles compared with the lowest tertile. Adapted with permission from J Clin Invest 2004;114:529.

sarathy et al. 1989, Podrez et al. 1999, 2000, Glass and Witztum 2001, Chisholm and Steinberg 2000). Whereas considerable interest has focused upon LDL as a target for oxidation in vivo, little research by comparison has examined HDL modification in vivo and the corresponding alterations in lipoprotein function. This is remarkable given the fact that HDL has long been appreciated as the major carrier of lipid hydroperoxides in the plasma (Bowry et al. 1992). Acting as a sink, HDL efficiently delivers these oxidative products to the liver, where they are preferentially taken up and catabolized. Zheng et al. (2004) first demonstrated that apoA-I serves as a selective target of oxidation both within the systemic circulation and in human atherosclerotic plaque (Table 1). Remarkably, the nitrotyrosine (NO2Tyr) and chlorotyrosine (ClTyr) contents of apoA-I recovered from serum of subjects were noted to be approximately 100-fold higher than that of other typical proteins within the systemic circulation (Table 1). Similarly, apoA-I recovered from normal human arterial tissues, and even more so, from atherosclerotic plaques, demonstrated large and incremental enrichment in both NO2Tyr and ClTyr content over that observed in apoA-I recovered from the circulation. Overall, a remarkable 1000-fold selective preference for oxidative modification of apoA-I recovered from human atherosclerotic plaque relative to a typical protein within the systemic circulation was noted (Table 1). These results are consistent with apoA-I serving as a preferred and selective target for oxidative modification in the protected environment of the subendothelial compartment of the artery wall.

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Moreover, these findings support a direct role for nitric oxide (NO)-derived oxidants and MPO-catalyzed halogenation

as oxidation pathways relevant to apoA-I oxidation in vivo, because NO 2 Tyr and ClTyr serve as selective molecular markers of posttranslational protein modification by these respective pathways (Ischiropoulos and Beckman 2003, Brennan et al. 2002, Hazen and Heinecke 1997). Independent confirmation of apoA-I as a preferred target for both NO-derived oxidants and MPO-catalyzed oxidation pathways in vivo has recently been reported (Bergt et al. 2004, Pennathur et al. 2004). Complementary immunohistochemical studies demonstrate the colocalization of apoA-I, NO2Tyr, HOClmodified proteins, and MPO-containing macrophages within human atheroma. Interestingly, several studies now show

Figure 1. Myeloperoxidase-catalyzed modification of apoA-I inhibits the ability of HDL to promote ABCA-1-dependent cholesterol efflux. High-density lipoprotein (A) and apoA-I (B) were modified with either ONOO
, MPO/H2O2/NO2
or MPO/H2O2/Cl
and subsequently incubated with cholesterol-loaded macrophages in the presence or absence of 8-Br-cAMP pretreatment. Modification by chlorination and nitration both resulted in selective inhibition of ABCA-1dependent efflux. In contrast, exposure to ONOO
had no influence on this activity. Results are expressed as meanFSD. Reprinted with permission from J Clin Invest 2004;114:529.

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Figure 2. Correlation between the apoA-I content of NO2Tyr (A) and ClTyr (B) of HDL isolated from subjects attending a preventive cardiology clinic and the functional impairment of ABCA-1-dependent cholesterol efflux. Reprinted with permission from J Clin Invest 2004;114:529.

mammals capable of generating chlorinating oxidants (Hazen and Heinecke 1997), and multiple studies with the use of MPO knockout mice in various inflammatory models confirm the essential role of MPO in generation of ClTyr in vivo (Brennan et al. 2001, 2002, Askari et al. 2003, Zhang et al. 2002). The selective enrichment of ClTyr within apoA-I recovered from atherosclerotic plaque, thus, places MPO and its reactive halogenating species near apoA-I. The marked chemical reactivity of MPO-generated chlorinating oxidants against a wealth of common moieties in biologic matrices (e.g. amines, thiols, thioether, imidazole, indole, phenolic ring, double bonds) strongly argues that the observed selective targeting of apoAI amongst alternative proteins in plasma and atherosclerotic plaque would only occur if a short diffusional distance exists between MPO and apoA-I. Consistent with this notion, MPO was demonstrated to directly bind with apoA-I in cross immunoprecipitation studies within human plasma (Zheng et al. 2004). Further support for a direct physiologic interaction between MPO and apoA-I in vivo is the observation

from two independent laboratories that MPO copurifies with HDL isolated from human atherosclerotic lesions (Zheng et al. 2004, Bergt et al. 2004). Indeed, a specific binding site for MPO on HDL within the helix 8 domain of apoA-I has been identified through biophysical studies employing hydrogen deuterium exchange coupled to tandem mass spectrometry (Zheng et al. 2004). A major functional consequence of MPO-catalyzed oxidative modification of HDL is inhibition in the cholesterol efflux activity of the modified lipoprotein. Incubation of both HDL and apoA-I with physiologic levels of MPO-generated nitrating and chlorinating systems markedly reduced the ABCA-1-dependent cholesterol efflux activity of the lipoprotein from cholesterol-loaded macrophages (Figure 1). Importantly, the physiologic relevance of these findings is supported by clinical studies of isolated HDL recovered from multiple subjects, revealing a strong inverse correlation between the degree of apoA-I nitration and chlorination versus the intrinsic capacity of the isolated lipoprotein to promote ABCA-1-dependent cholesterol efflux (Figure 2).

that apoA-I recovered from patients with coronary artery disease possesses a greater content of both NO2Tyr and ClTyr than apoA-I from healthy controls (Zheng et al. 2004, Bergt et al. 2004, Pennathur et al. 2004). In the largest study, where sequential subjects presenting to a preventive cardiology clinic were examined, the degree of modification of apoA-I correlated with the prevalence of cardiovascular disease in the cohort (Zheng et al. 2004). Compared with the lowest tertile, subjects with apoA-I NO2Tyr and ClTyr contents in the highest tertiles were 5.5-fold and 16-fold, respectively, more likely to have cardiovascular disease (Table 2). 

Myeloperoxidase Binding to High-Density Lipoprotein in Vivo Facilitates Oxidative Modification and Functional Inactivation of the Lipoprotein

Multiple lines of evidence support a role for MPO as a major enzymatic catalyst for apoA-I oxidation in vivo. Foremost, MPO is the only known enzyme in TCM Vol. 15, No. 6, 2005

Figure 3. Schematic depiction of amino acid sequence of apoA-I and identified sites for both association with MPO and oxidation (chlorination and nitration) in HDL. Tyrosine residues Y192 and Y166 are selective targets for MPO modification. An alternative residue, Y18, appears to be the major target for modification by ONOO
within intact HDL. The degree of modification of parent peptides containing these residues correlates with the impairment of cholesterol efflux. Adapted with permission from J Biol Chem 2005;280:38 and J Clin Invest 2004;114:529.

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Figure 4. Myeloperoxidase catalyzed modification of apoA-I inhibits the ability of HDL to bind lipid, as monitored via inhibition in LDL aggregation. (A) Binding activity was determined by the ability of apoA-I to inhibit phospholipase C-induced LDL aggregation. apoA-I and H2O2-modified apoA-I inhibited LDL aggregation to a similar extent. Modification of apoA-I with either MPO/H2O2/Cl
or, to a lesser extent, MPO/H 2 O 2 /NO 2
, resulted in an impaired ability of apoA-I to bind LDL and inhibit its aggregation. (B) Coordinate reduction in ability of increasing degrees of apoA-I modification to impair lipoprotein lipid binding activity (A) and promotion of ABCA-1-dependent cholesterol efflux (B) when normalized for unmodified apoA-I. Reprinted with permission from J Biol Chem 2005;280:38.



sines (Zheng et al. 2005). Interestingly, this hierarchy was not observed when native (lipid-free) apoA-I was used as a target (Zheng et al., unpublished data). Further, exposure of HDL to sequential doses of peroxynitrite resulted in preferential targeting of an alternative tyrosine residue for nitration, Y18, with little to no observable nitration noted at Y192 (with the use of intact HDL as the target) (Figure 3). Remarkably, tandem mass spectrometry studies of apoA-I recovered from human atheroma identified oxidative modifications to Y192 and Y166, corroborating these sites as preferred targets for oxidative modification in vivo. Finally, a dose-dependent relationship was found between the degree of modification and impairment of the ability of either HDL or apoA-I to promote both ABCA-1-dependent cholesterol efflux and apoA-I lipid binding activity (Figure 4). The exact nature of the link between the tyrosine-specific nitration and chlorination sites and the alteration in

function of HDL and apoA-I remain to be elucidated. Whereas modification of specific tyrosines was found to correlate with loss of critical HDL functional activities (Zheng et al. 2005), such relationships do not establish a causal role for tyrosine oxidation in these effects. Indeed, NO2Tyr and ClTyr are merely serving as molecular fingerprints to define oxidative chemistry and exposures in specific regions of the lipoprotein, with alternative oxidation events not monitored possibly contributing to the functional alterations in lipoprotein activities. Reports of dose-dependent losses of specific parent tryptic peptides and corresponding ABCA-1-dependent efflux activity (Zheng et al. 2005) could occur from oxidative modification of alternative residues within the tryptic peptides containing Y192 and Y166. Moreover, it remains to be determined whether there are alternate pathways, independent of MPO, contributing to the nitrative modification of HDL within the artery wall.

Identification of Sites of Nitration and Chlorination on Apolipoprotein A-I Recovered from Human Atherosclerotic Plaque

Specific sites on apoA-I that serve as both targets for modification within atheroma, as well as preferred sites for MPO catalyzed modifications, have now been reported (Zheng et al. 2005) (Figure 3). Tandem mass spectrometry analyses of MPO reaction mixtures with intact HDL identify two tyrosine residues within helix 8 and 7, respectively, of apoA-I that serve as preferred targets for both nitration and chlorination, Y192 and Y166. Dose response studies with HDL as a target demonstrates a clear hierarchy for selective modification of Y192, and then Y166, compared with alternative tyro-

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Figure 5. Schematic depiction of the influence of MPO on lipoproteins promoting atheroma formation. Myeloperoxidase promotes lipid peroxidation and the formation of oxidized LDL (oxLDL). Oxidized LDL in the subendothelial space is taken up by macrophages via the scavenger receptor CD36. In addition, MPO-catalyzed modification of apoA-I leads to a reduction in the ability of HDL to promote cellular cholesterol efflux via the ABCA-1 transporter. Myeloperoxidase catalyzed oxidation of both LDL and HDL may thus contribute to foam cell formation and plaque progression in the arterial wall.

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In addition to inhibition in cholesterol efflux activity, modification of HDL by chlorinating oxidants is reported to promote additional functional consequences. Panzenboeck et al. (1997) demonstrated in vitro that apoA-I is modified preferentially compared with fatty acids by an MPO-generated chlorinating system. The modified lipoprotein, HOCl-HDL, is reportedly more avidly internalized and degraded by macrophages, presumably via ligand interactions with the scavenger receptor type B, class I (Marsche et al. 2002). Recent studies also suggest that HOClmodified HDL impairs the in vitro expression and activity of endothelial NOS by endothelial cells (Marsche et al. 2004). This effect was reportedly mediated by the formation of 2-chlorohexadecanal, an aldehydic lipid cleavage product generated in response to HOCl modification of HDL-associated plasmalogens (Albert et al. 2001). It is thus relevant to note that 2-chlorohexadecanal and sn-2 lysophosphatidylcholine molecular species, the coproduct of plasmenyl choline oxidation by HOCl, were both recently shown to be markedly enriched within human atherosclerotic plaques (Thukkani et al. 2003). 

Oxidized Cross-Linking of High-Density Lipoprotein

Not all oxidant modifications of HDL may result in detrimental functional consequences. Wang et al. (1998) reported that HDL exposed to a tyrosyl radical generating system in vitro produces a lipoprotein that is more effective than native HDL in promoting ABCA-1-dependent cholesterol efflux from lipid-laden fibroblasts and macrophages. Formation of cross-linked heterodimers composed of apoA-I and apoA-II reportedly account for the facilitated efflux activity in the oxidized HDL preparation. MacDonald et al. (2003) further demonstrated that intraperitoneal administration of tyrosylated HDL (i.e., HDL exposed to a tyrosyl radicalgenerating system) was more potent than native HDL at inhibiting the formation of atherosclerotic plaque in an animal model. Thus, not all oxidative modifications of lipoproteins appear to be deleterious, and the overall effect of HDL oxidation in vivo is certain to be complex. TCM Vol. 15, No. 6, 2005

Implications and Future Directions

References

Substantial evidence exists supporting a role for both MPO- and NO-derived oxidant pathways as contributors to cardiovascular disease in humans (Shishehbor et al. 2003a, 2003b, Zhang et al. 2001, Brennan et al. 2003, Baldus et al. 2003, Vita et al. 2004, Eiserich et al. 2002, Baldus et al. 2004, Asselbergs et al. 2004, Loscalzo 2001, Harrison et al. 2003). It is thus remarkable that within the past year, a constellation of studies collectively provided compelling evidence for both MPO and NO-derived oxidant pathways as key participants in HDL modification within human atheromas (Figure 5). The corresponding functional alterations in apoA-I cholesterol efflux activity observed in model systems, combined with clinical observations linking chemical signatures of apoA-I nitration and chlorination with increased risk for cardiovascular disease, strongly support the concept that oxidation of HDL generates a dysfunctional lipoprotein capable of contributing to the atherosclerotic process. Indeed, the strong inverse relationship observed in clinical studies between the NO2Tyr and ClTyr content of isolated HDL from subjects and the intrinsic ABCA-1-dependent efflux activity of the lipoprotein particle lends credence to this hypothesis. From a purely prognostic standpoint, it is also of interest that independent groups have now demonstrated that systemic levels of apoA-I modified by nitration or chlorination are correlated with cardiovascular disease prevalence (Zheng et al. 2004, Bergt et al. 2004, Pennathur et al. 2004), suggesting the potential of these modified forms of lipoprotein to serve as markers of cardiovascular risk prediction. In addition, as systemic levels of protein-bound NO2Tyr and ClTyr have been shown to decline in response to statin therapy (Shishehbor et al. 2003a, 2003b), it is tempting to speculate that enzymatic processes generating potentially detrimental modifications to HDL within the vasculature may be downregulated. As a result, the potential exists to identify individuals at increased cardiovascular risk due to enhanced levels of dysfunctional forms of HDL who might otherwise not have been identified, enabling more aggressive targeting of therapeutic efforts.

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PII S1050-1738(05)00085-X

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Gene Therapy for Cardiac Arrhythmias J. Kevin Donahue*, Kan Kikuchi, and Tetsuo Sasano

Myocardial gene transfer has become a routine tool to investigate the pathophysiology of cardiac diseases, although translation of gene transfer techniques into therapeutics has not come as quickly as many had hoped. In the field of cardiac arrhythmias, there is a great need for new therapeutic options. The current work reviews the use of gene transfer to evaluate cellular electrophysiology, the application of in vivo gene transfer to treat common arrhythmias, and the current problems in the field of cardiac gene therapy. Arrhythmia gene therapy is a field in its infancy, and future human applications are dependent on solutions to the problems discussed in this review. (Trends Cardiovasc Med 2005;15:219–224) D 2005, Elsevier Inc.

devices. Radiofrequency ablation is a proven technology for focal arrhythmias (e.g., atrioventricular [AV] nodal reentry tachycardia or atrial flutter) and an experimental approach for more diffuse arrhythmias such as AF or infarctrelated ventricular tachycardia (Calkins et al. 1999, Jais et al. 1997). For most life-threatening arrhythmias, implantable devices are the only option. Pacemakers prevent bradycardia, and implantable defibrillators convert malignant ventricular tachyarrhythmias back to sinus rhythm, but problems with implantable devices include significant expense, potential complications from the invasive procedures, and in the case of defibrillators, pain related to the treatment strategy (Knight et al. 1997, Parsonnet et al. 1989). An additional problem with implantable devices is the resistance of some cultures to accept this technology. Recently, interest has been expressed in the use of gene transfer technologies to develop new therapies for cardiac arrhythmias. In this review, we will first examine normal cardiac electrophysiology, then we will explore in vitro gene transfer to investigate the pathophysiology of arrhythmias, the preliminary reports of arrhythmia gene therapy, and the current problems in the field of myocardial gene therapy. Although stem cell therapy is being developed for certain arrhythmias, space in this review does not enable exploration of that topic. 

Cardiac arrhythmias are responsible for extensive morbidity and mortality in the developed world. Cardiac arrest accounts for more than 300,000 deaths,

J. Kevin Donahue is at the Division of Cardiology, Case Western Reserve University School of Medicine, Cleveland, Ohio. Kan Kikuchi and Tetsuo Sasano are at the Division of Cardiology, Johns Hopkins University School of Medicine, Baltimore, Maryland. * Address correspondence to: J. Kevin Donahue, Division of Cardiology, Case Western Reserve University School of Medicine, MetroHealth Campus Rammelkamp-653, 2500 MetroHealth Drive, Cleveland, OH; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter

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and atrial fibrillation (AF) burdens more than 2 million people in the United States (American Heart Association 2004). Similar numbers are found in most other industrialized nations. Compounding the problem is the lack of curative therapies for many arrhythmias. Until the early 1990s, antiarrhythmic drug therapy was considered the standard of care for treating most arrhythmias. Since that time, however, numerous studies of antiarrhythmic drugs have shown that they increased mortality (Echt et al. 1991, MacMahon et al. 1988, Waldo et al. 1996). The results of these trials left a void in available strategies for treatment of cardiac arrhythmias, filled inadequately by ablation and implantable cardiac

Normal cardiac electrophysiology

The cellular basis for cardiac electric activity is the action potential (AP). The AP is conventionally divided into five sections (phases 0 – 4, Figure 1) (Schram et al. 2002). Each phase is defined by the cellular membrane potential and the activity of ion channels that affect that potential (Tomaselli and Marban 1999). Phase 4 is the resting baseline. The dominant ionic current during this phase is a potassium current, I K1. Phase 0 is the initial depolarization fueled by a large sodium current, I Na. Phase 1 is a quick dip in the potential from the peak achieved at the end of phase 0. Activation of potassium and chloride currents (I to1 and I to2, respectively) and inactivation of the sodium channels are responsible for this portion of the AP. Phase 2 is the plateau period, where a calcium

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