In atherosclerosis, and other major cardiovascular disorders, nuclear factor kB (NF-kB) signalling represents a critical regulating mechanism involved in disease onset and progression, including inflammation, cell proliferation, migration, differentiation and apoptosis. Several line of evidence indicate that NF-kB is a redox-sensitive transcription factor which is robustly activated by ROS possible generated by activated Nox. Interestingly, a positive feed-back loop of Nox activation by NF-kB has been proposed in several studies. Thus, a new integrative concept has emergedthe "vicious cycle", to describe the interconnection between metabolic dysfunction, inflammation, and oxidative stress that converges to vascular disorders (Manea, 2010). In murine monocytes, the expression of the Nox2 is induced by NF-kB. Moreover, the up-regulation of p47phox and p22phox expression by LPS/IFNγ was blunted in IkBα-overexpressing cells suggesting the involvement of the NF-kB signaling in the regulation of the Nox components (Anrather et al. 2006). Similar finding were reported in human monocytes/macrophages exposed to TNFα (Gauss et al., 2007). Moreover, in previous studies we have shown that, NF-kB is an important transcriptional regulator of the genes coding for p22phox, Nox1, and Nox4, and has a profound impact in the up-regulation of Nox activity in TNFα-treated human aortic

The molecular mechanisms that facilitate hypoxia sensing and related signalling events are critical for the maintenance of vascular cell homeostasis. Compelling data depicts that hypoxic conditions up-regulate the expression and activity various Nox subtypes (Goyal et al., 2004). It has been demonstrated that persistent hypoxia induces Nox4 gene and protein expression levels in pulmonary artery SMCs and in pulmonary vessels in mice exposed to hypoxic conditions (Diebold et al., 2010). Mechanistically, the response is dependent on hypoxia inducible factor-1α (HIF-1α), which interacts with the corresponding elements in the Nox4 promoter. As a result, the HIF-1α dependent upregulation of Nox4 by may be an essential mechanism to preserve ROS level after hypoxia and the hypoxia-induced proliferation of pulmonary artery SMCs. Furthermore,

•- production.

CCAAT/enhancer-binding protein (C/EBP)β and C/EBPδ (Maitra et al., 2009).

(Jak)/STAT-dependent mechanisms are implicated in the ensuing O2

Nox4 transcriptional program (Zhang et al., 2008).

SMCs (Manea et al., 2007; Manea et al., 2010b).

a significant increase in H2O2 generation rather that O2•- and a significant augmentation of the pro-oxidative status. Despite of these aspects, the blood pressure of the animals was lower under basal conditions and after angiotensin II treatment. Interestingly, endotheliumdependent relaxation was significantly improved compared with wild-type animals. Notably, these effects were sensitive to the *ex vivo* addition of catalase and *in vivo* administration of *N*-acetylcysteine, indicating that they were mediated by peroxide-type, namely H2O2, mechanisms. Mechanistically, it seems that an H2O2 action on potassium channels may be responsible for the elaboration of endothelium-derived hyperpolarizing factor (EDHF) type. Thus, one has to ponder that the type of ROS released in the vascular system determines their biological function.

As mentioned above, less is know about the role of Nox5 in atherogenesis, and this is due mainly to the absence of *Nox5* gene in the rodents' genome and therefore to the lack of a reliable animal model., Still, it has been reported a significant correlation between Nox5 expression and atherosclerotic lesion progression. Interestingly, a specific expression pattern was reported; with Nox5 being expressed mainly by the endothelium in the early stages of the disease while its expression is significantly increased in SMCs underlying fibro-lipid atherosclerotic lesions (Guzik et al., 2008).

#### **8. Mechanisms of Nox regulation**

#### **8.1 Phosphorylation pathways and transcription factors**

Nox activity and expression is highly regulated at multiple levels by various physiologic and pathological factors which, by this means, dictate the enzyme complex function. The activities of both Nox1 and Nox2 isoforms are primarily regulated by complex networking of phosphorylation cascades involving regulatory components (i.e., p40phox, p47phox, p67phox, Noxo1, Noxa1) which induce the assembly of the enzyme complex. Nox4 is constitutively active and does not necessitate phosphorylation of regulatory proteins, whereas Nox5 has been demonstrated to be Ca2+-responsivenes. Yet, activation mechanisms involving protein kinase C and the proto-oncogenic tyrosine kinase c-Abl phosphorylation of Nox5 have been reported (El Jamali et al., 2008; Serrander et al., 2007).

The phosphorylation mechanisms responsible for Nox1 and Nox2 activation consist of a large spectrum of signalling molecules such as protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), GTP-binding proteins (Ras, Rac1/2), members of the mitogen-activated protein kinase (MAPK) family (p38MAPK, ERK1/2), phospholipases (PLCβ/γ, PLD), arachidonic acid metabolites, and non-receptor protein tyrosine kinases (Kilpatrick et al., 2010; Yamamori et al., 2004). Besides the aforementioned kinases, chaperone proteins (e.g., protein disulfide isomerase) have been proved to be important regulators on Nox function (Janiszewski et al., 2005).

In addition to the phosphorylation of cytosolic regulatory subunits, alterations of the Nox isoforms expression have been shown to be critical for their activity. Multiple transcription factors are coordinately implicated in the modulation of Nox expression and function. PU.1, Elf-1, IRF-1 (interferon regulatory factor-1), and ICSBP (interferon consensus sequence binding protein) are important transcriptional regulators of Nox2 in the myelomonocytic cell lineage (Kakar et al., 2005). In human colon epithelial Caco-2 cells, GATA-binding factors are critical for Nox1 transcriptional activity (Brewer et al., 2006), whereas in murine macrophages, the up-regulation of Nox1 in response to lipopolysaccharide (LPS)

a significant increase in H2O2 generation rather that O2•- and a significant augmentation of the pro-oxidative status. Despite of these aspects, the blood pressure of the animals was lower under basal conditions and after angiotensin II treatment. Interestingly, endotheliumdependent relaxation was significantly improved compared with wild-type animals. Notably, these effects were sensitive to the *ex vivo* addition of catalase and *in vivo* administration of *N*-acetylcysteine, indicating that they were mediated by peroxide-type, namely H2O2, mechanisms. Mechanistically, it seems that an H2O2 action on potassium channels may be responsible for the elaboration of endothelium-derived hyperpolarizing factor (EDHF) type. Thus, one has to ponder that the type of ROS released in the vascular

As mentioned above, less is know about the role of Nox5 in atherogenesis, and this is due mainly to the absence of *Nox5* gene in the rodents' genome and therefore to the lack of a reliable animal model., Still, it has been reported a significant correlation between Nox5 expression and atherosclerotic lesion progression. Interestingly, a specific expression pattern was reported; with Nox5 being expressed mainly by the endothelium in the early stages of the disease while its expression is significantly increased in SMCs underlying fibro-lipid

Nox activity and expression is highly regulated at multiple levels by various physiologic and pathological factors which, by this means, dictate the enzyme complex function. The activities of both Nox1 and Nox2 isoforms are primarily regulated by complex networking of phosphorylation cascades involving regulatory components (i.e., p40phox, p47phox, p67phox, Noxo1, Noxa1) which induce the assembly of the enzyme complex. Nox4 is constitutively active and does not necessitate phosphorylation of regulatory proteins, whereas Nox5 has been demonstrated to be Ca2+-responsivenes. Yet, activation mechanisms involving protein kinase C and the proto-oncogenic tyrosine kinase c-Abl phosphorylation of Nox5 have been reported (El Jamali et al., 2008; Serrander et al.,

The phosphorylation mechanisms responsible for Nox1 and Nox2 activation consist of a large spectrum of signalling molecules such as protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K), GTP-binding proteins (Ras, Rac1/2), members of the mitogen-activated protein kinase (MAPK) family (p38MAPK, ERK1/2), phospholipases (PLCβ/γ, PLD), arachidonic acid metabolites, and non-receptor protein tyrosine kinases (Kilpatrick et al., 2010; Yamamori et al., 2004). Besides the aforementioned kinases, chaperone proteins (e.g., protein disulfide isomerase) have been proved to be important regulators on Nox function

In addition to the phosphorylation of cytosolic regulatory subunits, alterations of the Nox isoforms expression have been shown to be critical for their activity. Multiple transcription factors are coordinately implicated in the modulation of Nox expression and function. PU.1, Elf-1, IRF-1 (interferon regulatory factor-1), and ICSBP (interferon consensus sequence binding protein) are important transcriptional regulators of Nox2 in the myelomonocytic cell lineage (Kakar et al., 2005). In human colon epithelial Caco-2 cells, GATA-binding factors are critical for Nox1 transcriptional activity (Brewer et al., 2006), whereas in murine macrophages, the up-regulation of Nox1 in response to lipopolysaccharide (LPS)

system determines their biological function.

atherosclerotic lesions (Guzik et al., 2008).

**8. Mechanisms of Nox regulation**

2007).

(Janiszewski et al., 2005).

**8.1 Phosphorylation pathways and transcription factors**

stimulation is mediated at least in part, by pro-inflammatory transcription factors CCAAT/enhancer-binding protein (C/EBP)β and C/EBPδ (Maitra et al., 2009).

In previous studies, we have shown that in human aortic SMCs exposed angiotensin II or tumor necrosis factor-α, the pro-inflammatory transcription factor AP-1 is an essential regulator of the genes coding for p22phox, Nox1, and Nox4 components (Manea et al., 2008; Raicu & Manea, 2010). Other than, activator protein -1 (AP-1), the vascular inflammationrelated and growth-promoting transcription factors signal transducer and activator of transcription (STAT1 and STAT3) proteins physically interact with the promoters of human Nox1 and Nox4 genes in SMCs exposed to interferon (IFN) γ and a Jauns kinase (Jak)/STAT-dependent mechanisms are implicated in the ensuing O2 •- production. Moreover, the promoter activities of the genes coding for p22phox, p47phox, and p67phox, have been demonstrated to be considerably augmented in SMCs overexpressing STAT1/STAT3, a result that suggests the existence of functionally gamma activated sequence (GAS)/interferon-stimulated response element (ISRE) consensus sequences (Manea et al., 2010a). In human aortic SMCs, Ets1, a critical mediator of vascular inflammation and remodelling, regulates p47phox expression in response to AngII (Ni et al., 2007). Similar observations were made in A7r5 cells and primary mouse aortic SMCs, in which the growth-promoting transcription factor E2F actually interacts and controls the Nox4 transcriptional program (Zhang et al., 2008).

In atherosclerosis, and other major cardiovascular disorders, nuclear factor kB (NF-kB) signalling represents a critical regulating mechanism involved in disease onset and progression, including inflammation, cell proliferation, migration, differentiation and apoptosis. Several line of evidence indicate that NF-kB is a redox-sensitive transcription factor which is robustly activated by ROS possible generated by activated Nox. Interestingly, a positive feed-back loop of Nox activation by NF-kB has been proposed in several studies. Thus, a new integrative concept has emergedthe "vicious cycle", to describe the interconnection between metabolic dysfunction, inflammation, and oxidative stress that converges to vascular disorders (Manea, 2010). In murine monocytes, the expression of the Nox2 is induced by NF-kB. Moreover, the up-regulation of p47phox and p22phox expression by LPS/IFNγ was blunted in IkBα-overexpressing cells suggesting the involvement of the NF-kB signaling in the regulation of the Nox components (Anrather et al. 2006). Similar finding were reported in human monocytes/macrophages exposed to TNFα (Gauss et al., 2007). Moreover, in previous studies we have shown that, NF-kB is an important transcriptional regulator of the genes coding for p22phox, Nox1, and Nox4, and has a profound impact in the up-regulation of Nox activity in TNFα-treated human aortic SMCs (Manea et al., 2007; Manea et al., 2010b).

The molecular mechanisms that facilitate hypoxia sensing and related signalling events are critical for the maintenance of vascular cell homeostasis. Compelling data depicts that hypoxic conditions up-regulate the expression and activity various Nox subtypes (Goyal et al., 2004). It has been demonstrated that persistent hypoxia induces Nox4 gene and protein expression levels in pulmonary artery SMCs and in pulmonary vessels in mice exposed to hypoxic conditions (Diebold et al., 2010). Mechanistically, the response is dependent on hypoxia inducible factor-1α (HIF-1α), which interacts with the corresponding elements in the Nox4 promoter. As a result, the HIF-1α dependent upregulation of Nox4 by may be an essential mechanism to preserve ROS level after hypoxia and the hypoxia-induced proliferation of pulmonary artery SMCs. Furthermore,

Vascular Biology of Reactive Oxygen Species and NADPH Oxidases: Role in Atherogenesis 437

Fig. 1. Schematic depiction of the major mechanisms responsible for the up-regulation of Nox enzymes and installation of oxidative stress in atherosclerosis. In response to cardiovascular risk factors, vascular cells through their receptors activate a range of signalling pathways that up-regulate Nox expression, activity, and the ensuing ROS

production. This triggers a chain of critical events that generally amplify the initial response to vascular insults (i.e., activation of other cellular sources of ROS and redox-sensitive signalling effectors). Persistent Nox activation leads to oxidative stress that is a major contributor to the initiation and the development of atherosclerotic lesions. The diagram highlights that genetic, epigenetic, as well as genetic/non-epigenetic-independent

mechanisms linked to Nox up-regulation and hyperactivity in atherosclerosis may be used to target and control pharmacologically the Nox-derived oxidative stress (green text).

The Nox-derived ROS may have both beneficial and deleterious effects. Thus, we can safely assume that these effects are function of the expression pattern and regulation of various Nox isoforms, their subcellular compartmentalization, and the rate of ROS generation. Despite of the numerous existing data, the precise mechanisms of Nox regulation in atherosclerosis and the stream of signalling molecules (up-, or down-regulated) responsible for the increased oxidative stress that is associated with the onset and development of cardiovascular dysfunction, is poorly understood. Thus, a complex interplay of genetic, epigenetic and non-epigenetic factors, transcription factors, co-activators, and/or corepressors may be coordinately involved in the up-regulation of Nox activity in

**9. Conclusion**

activating transcription factor-1 (ATF-1), a transcription factor of the CREB (CRE-binding protein)/ATF family, proved to play a key role in the induction of Nox1 in rat vascular SMCs (Katsuyama et al., 2005).

Nuclear factor (erythroid-derived 2)-like 2, also known as Nrf2 represents a master modulator of the antioxidant responses by inducing genes (e.g., *Sod* genes*)* with important function in combating oxidative stress. Interestingly, it has been demonstrated that Nrf2, also controls Nox4 expression in mouse lung and human lung endothelium in response to hyperoxia (Pendyala & Natarajan, 2010).

#### **8.2 Genetic and epigenetic mechanisms of Nox regulation**

Genetic studies highlight that several Nox-related polymorphisms are closely associated with an increased susceptibility for cardiovascular disorders. One of the most investigated genes from the Nox complex is *CYBA* which encodes the p22phox essential subunit. The p22phox is ubiquitously expressed in cardiovascular cells and forms stable and functional heterodimers with Nox1, Nox2 or Nox4, a critical structure for enzyme activity as shown by studies employing siRNA technology to knock-down p22phox expression (Kawahara et al., 2005). Moreover, it has been demonstrated that p22phox is more abundant in advanced atherosclerotic plaques than in nonatherosclerotic arteries, suggesting a correlation between p22phox expression, O2•- production, and the severity of atherosclerosis (Azumi et al., 1999).

The occurrence of particular polymorphisms of the *CYBA* gene has been shown to predispose to oxidative stress and to be independently correlated with cardiovascular risk factors and disease occurrence namely hypertension, coronary artery disease, myocardial infarction, cerebrovascular disease, diabetic and non-diabetic nephropathy) (San José et al., 2008). Various *CYBA* allelic variants were detected in both exonic sequences such as C242T, A640G, C549T (Dinauer et al., 1990; Guzik et al., 2000; Inoue et al., 1998), and promoter regions namely -930A*/*G, -675A*/*T, -852C*/*G, -536C*/*T (Lim et al., 2006; Moreno et al., 2007), which potentially affect the p22phox expression and consequently the Nox activity. Thus far, data indicating the existence of functional Nox1-5 polymorphisms with a relevant impact on vascular pathology are not available yet.

Emerging evidence demonstrates that epigenetic events such as DNA methylation and modifications of histone tails are important processes of oxidative stress onset. DNA methylation mechanisms of the promoter CpG islands has been shown to be involved in the up-regulation of 15-lipoxygenase, a pro-oxidative enzyme with implications in plaque formation and vulnerability, and down-regulation of superoxide dismutase 3, endothelial NO synthase, and various anti-proliferative genes (estrogen receptor-α), a condition that leads to oxidative stress, impaired vascular relaxation, and aberrant SMC hyperplasia (Fernandez et al., 2010). Hitherto, data about the role of epigenetics in the regulation of Nox subtypes are missing. Nevertheless, using both *in vitro* (e,g., human aortic SMCs exposed to pro-inflammatory conditions) and *in vivo* (ApoE-/- mice fed a high fat, cholesterol rich diet) models, we have found recently that an aberrant methylation of the Nox1 promoter may be responsible for the up-regulated expression and activity of this enzyme (Manea et al., unpublished data).

A schematic representation of the key molecular pathways implicated in the up-regulation of Nox enzymes as well as the potential pharmacological targets intended to counteract oxidative stress are presented in the figure below.

Fig. 1. Schematic depiction of the major mechanisms responsible for the up-regulation of Nox enzymes and installation of oxidative stress in atherosclerosis. In response to cardiovascular risk factors, vascular cells through their receptors activate a range of signalling pathways that up-regulate Nox expression, activity, and the ensuing ROS production. This triggers a chain of critical events that generally amplify the initial response to vascular insults (i.e., activation of other cellular sources of ROS and redox-sensitive signalling effectors). Persistent Nox activation leads to oxidative stress that is a major contributor to the initiation and the development of atherosclerotic lesions. The diagram highlights that genetic, epigenetic, as well as genetic/non-epigenetic-independent mechanisms linked to Nox up-regulation and hyperactivity in atherosclerosis may be used to target and control pharmacologically the Nox-derived oxidative stress (green text).

#### **9. Conclusion**

436 Atherogenesis

activating transcription factor-1 (ATF-1), a transcription factor of the CREB (CRE-binding protein)/ATF family, proved to play a key role in the induction of Nox1 in rat vascular

Nuclear factor (erythroid-derived 2)-like 2, also known as Nrf2 represents a master modulator of the antioxidant responses by inducing genes (e.g., *Sod* genes*)* with important function in combating oxidative stress. Interestingly, it has been demonstrated that Nrf2, also controls Nox4 expression in mouse lung and human lung endothelium in response to

Genetic studies highlight that several Nox-related polymorphisms are closely associated with an increased susceptibility for cardiovascular disorders. One of the most investigated genes from the Nox complex is *CYBA* which encodes the p22phox essential subunit. The p22phox is ubiquitously expressed in cardiovascular cells and forms stable and functional heterodimers with Nox1, Nox2 or Nox4, a critical structure for enzyme activity as shown by studies employing siRNA technology to knock-down p22phox expression (Kawahara et al., 2005). Moreover, it has been demonstrated that p22phox is more abundant in advanced atherosclerotic plaques than in nonatherosclerotic arteries, suggesting a correlation between p22phox expression, O2•- production, and the severity of

The occurrence of particular polymorphisms of the *CYBA* gene has been shown to predispose to oxidative stress and to be independently correlated with cardiovascular risk factors and disease occurrence namely hypertension, coronary artery disease, myocardial infarction, cerebrovascular disease, diabetic and non-diabetic nephropathy) (San José et al., 2008). Various *CYBA* allelic variants were detected in both exonic sequences such as C242T, A640G, C549T (Dinauer et al., 1990; Guzik et al., 2000; Inoue et al., 1998), and promoter regions namely -930A*/*G, -675A*/*T, -852C*/*G, -536C*/*T (Lim et al., 2006; Moreno et al., 2007), which potentially affect the p22phox expression and consequently the Nox activity. Thus far, data indicating the existence of functional Nox1-5 polymorphisms with a relevant

Emerging evidence demonstrates that epigenetic events such as DNA methylation and modifications of histone tails are important processes of oxidative stress onset. DNA methylation mechanisms of the promoter CpG islands has been shown to be involved in the up-regulation of 15-lipoxygenase, a pro-oxidative enzyme with implications in plaque formation and vulnerability, and down-regulation of superoxide dismutase 3, endothelial NO synthase, and various anti-proliferative genes (estrogen receptor-α), a condition that leads to oxidative stress, impaired vascular relaxation, and aberrant SMC hyperplasia (Fernandez et al., 2010). Hitherto, data about the role of epigenetics in the regulation of Nox subtypes are missing. Nevertheless, using both *in vitro* (e,g., human aortic SMCs exposed to pro-inflammatory conditions) and *in vivo* (ApoE-/- mice fed a high fat, cholesterol rich diet) models, we have found recently that an aberrant methylation of the Nox1 promoter may be responsible for the up-regulated expression and activity of this enzyme (Manea et al.,

A schematic representation of the key molecular pathways implicated in the up-regulation of Nox enzymes as well as the potential pharmacological targets intended to counteract

SMCs (Katsuyama et al., 2005).

hyperoxia (Pendyala & Natarajan, 2010).

atherosclerosis (Azumi et al., 1999).

unpublished data).

impact on vascular pathology are not available yet.

oxidative stress are presented in the figure below.

**8.2 Genetic and epigenetic mechanisms of Nox regulation**

The Nox-derived ROS may have both beneficial and deleterious effects. Thus, we can safely assume that these effects are function of the expression pattern and regulation of various Nox isoforms, their subcellular compartmentalization, and the rate of ROS generation. Despite of the numerous existing data, the precise mechanisms of Nox regulation in atherosclerosis and the stream of signalling molecules (up-, or down-regulated) responsible for the increased oxidative stress that is associated with the onset and development of cardiovascular dysfunction, is poorly understood. Thus, a complex interplay of genetic, epigenetic and non-epigenetic factors, transcription factors, co-activators, and/or corepressors may be coordinately involved in the up-regulation of Nox activity in

Vascular Biology of Reactive Oxygen Species and NADPH Oxidases: Role in Atherogenesis 439

Chung, S.W., Park, J.W., Lee, S.A., Eo, S.K. & Kim, K. (2010). Thrombin promotes

Dejana, E., Simionescu, M. & Wolburg, H. (2009). Endothelial cell biology and pathology.

Diebold, I., Petry, A., Hess, J. & Görlach, A. (2010). The NADPH oxidase subunit NOX4 is a

Dikalova, A., Clempus, R., Lassègue, B., Cheng, G., McCoy, J., Dikalov, S., San Martin, A.,

Dikalova, A.E., Góngora, M.C., Harrison, D.G., Lambeth, J.D., Dikalov, S. & Griendling, K.K.

*Physiology Heart and Circulatory Physiology,* 299, 673-679, ISSN 0363-6135 Dinauer, M.C., Pierce, E.A., Bruns, G.A., Curnutte, J.T. & Orkin, S.H. (1990). Human

Ding, H., Hashem, M. & Triggle, C. (2007). Increased oxidative stress in the streptozotocin-

kinase c-Abl. *Free Radical Biology and Medicine,* 44, 868-881, ISSN 0891-5849 El-Benna, J., Dang, P.M. & Gougerot-Pocidalo, M.A. (2007). Role of the NADPH oxidase

Fearon, I.M. & Faux, S.P. (2009). Oxidative stress and cardiovascular disease: novel tools

Fenyo, I.M., Florea, I.C., Raicu, M. & Manea, A. (2011). Tyrphostin AG490 reduces NAPDH

Fernandez, Z., Siebel, A.L. & El-Osta, A. (2010). Atherogenic factors and their epigenetic relationships. *International Journal of Vascular Medicine,* doi:10.1155/2010/437809 Forman, H.J., Maiorino, M. & Ursini, F. (2010). Signaling functions of reactive oxygen

E-deficient mice. *Vascular Pharmacology,* 54, 100-106, ISSN 1537-1891

*Immunology.* 3, 111-115, ISSN 1744-666X

species. *Biochemistry,* 49, 835–842, ISSN 0006-2960

*Biophysical Research Communications,* 396, 748-754, ISSN 0006-291X

transgenic mice. *Circulation,* 112, 2668-2676, ISSN 0009-7322

*Cell and Tissue Research,* 335, 1-3, ISSN 0302-766X

2087-2096, ISSN 1059-1524

8424

9738

ISSN 0022-2828

*Proceedings of the National Academy of Sciences USA,* 101, 13014-13019, ISSN 0027-

proinflammatory phenotype in human vascular smooth muscle cell. *Biochemical and*

new target gene of the hypoxia-inducible factor-1. *Molecular Biology of the Cell,* 21,

Lyle, A., Weber, D.S., Weiss, D., Taylor, W.R., Schmidt, H.H., Owens, G.K., Lambeth, J.D. & Griendling, K.K. (2005). Nox1 overexpression potentiates angiotensin II-induced hypertension and vascular smooth muscle hypertrophy in

(2010). Upregulation of Nox1 in vascular smooth muscle leads to impaired endothelium-dependent relaxation via eNOS uncoupling. *American Journal of*

neutrophil cytochrome b light chain (p22-phox). Gene structure, chromosomal location, and mutations in cytochrome-negative autosomal recessive chronic granulomatous disease. *Journal of Clinical Investigation,* 86, 1729–1737, ISSN 0021-

induced diabetic apoE-deficient mouse: changes in expression of NADPH oxidase subunits and eNOS. *European Journal of Pharmacology,* 561, 121-128, ISSN 0014-2999 El Jamali, A., Valente, A.J., Lechleiter, J.D., Gamez, M.J., Pearson, D.W., Nauseef, W.M. &

Clark, R.A. (2008). Novel redox-dependent regulation of NOX5 by the tyrosine

systems Nox and Duox in host defense and inflammation. *Expert Review of Clinical*

give (free) radical insight. *Journal of Molecular and Cellular Cardiology,* 47, 372-381,

oxidase activity and expression in the aorta of hypercholesterolemic apolipoprotein

atherogenesis. Therefore, elucidation of the complex interactions among different mechanisms that control Nox subtypes expression/activity and subcellular compartmentalization of ROS production and its subsequent biological meaning, may lead to a more focused and effective antioxidant therapies. The expected impact of these pharmacological strategies goes well beyond the atherosclerosis field. Oxidative stress is a common occurrence in most pathologies i.e. diabetes, neurodegenerative diseases, cancer, etc. The knowledge gained will be applicable to all these pathologies since controlling oxidative stress ought to have a beneficial knock-on effect on these diseases. Identifying the basic molecular mechanism regulating the oxidative stress will be used to find ways to manage its occurrence and correct its adverse effects.

#### **10. Acknowledgement**

This work was supported by grants from the Romanian Ministry of Education, and Research (CNCSIS-UEFISCSU project numbers PNII-IDEI 1005/2009 and PNII-TE 65/2010), and from the European Foundation for the Study of Diabetes - New Horizons. The financial support of European Social Fund – "Cristofor I. Simionescu" Postdoctoral Fellowship Programme (ID POSDRU/89/1.5/S/55216), Sectoral Operational Programme Human Resources Development 2007 – 2013 is acknowledged.

#### **11. References**

atherogenesis. Therefore, elucidation of the complex interactions among different mechanisms that control Nox subtypes expression/activity and subcellular compartmentalization of ROS production and its subsequent biological meaning, may lead to a more focused and effective antioxidant therapies. The expected impact of these pharmacological strategies goes well beyond the atherosclerosis field. Oxidative stress is a common occurrence in most pathologies i.e. diabetes, neurodegenerative diseases, cancer, etc. The knowledge gained will be applicable to all these pathologies since controlling oxidative stress ought to have a beneficial knock-on effect on these diseases. Identifying the basic molecular mechanism regulating the oxidative stress will be used to find ways to

This work was supported by grants from the Romanian Ministry of Education, and Research (CNCSIS-UEFISCSU project numbers PNII-IDEI 1005/2009 and PNII-TE 65/2010), and from the European Foundation for the Study of Diabetes - New Horizons. The financial support of European Social Fund – "Cristofor I. Simionescu" Postdoctoral Fellowship Programme (ID POSDRU/89/1.5/S/55216), Sectoral Operational Programme Human Resources

Ago, T., Kuroda, J., Pain, J., Fu, C., Li, H. & Sadoshima, J. (2010). Upregulation of Nox4 by

Anrather, J., Racchumi, G. & Iadecola, C. (2006). NF-kappaB regulates phagocytic NADPH

Azumi, H., Inoue, N., Takeshita, S., Rikitake, Y., Kawashima, S., Hayashi, Y., Itoh, H. &

BelAiba, R.S., Djordjevic, T., Petry, A., Diemer, K., Bonello, S., Banfi, B., Hess, J., Pogrebniak,

Brewer, A.C., Sparks, E.C. & Shah, A.M. (2006). Transcriptional regulation of the NADPH

Briones, A.M. & Touyz, R.M. (2010). Oxidative stress and hypertension: current concepts.

Chen, Z., Keaney, J.F. Jr., Schulz, E., Levison, B., Shan, L., Sakuma, M., Zhang, X., Shi, C.,

myocytes. *Circulation Research,* 106, 1253–1264, ISSN 0009-7330

coronary arteries. *Circulation,* 100, 1494-1498, ISSN 0009-7322

*Radical Biology and Medicine,* 40, 260-274, ISSN 0891-5849

*Current Hypertension Reports,* 12, 135-142, ISSN 1522-6417

hypertrophic stimuli promotes apoptosis and mitochondrial dysfunction in cardiac

oxidase by inducing the expression of gp91phox. *Journal of Biological Chemistry,* 281,

Yokoyama, M. (1999). Expression of NADH/NADPH oxidase p22phox in human

A., Bickel, C. & Görlach, A. (2007). NOX5 variants are functionally active in endothelial cells. *Free Radical Biology and Medicine,* 42, 446–459, ISSN 0891-5849 Belia, S., Santilli, F., Beccafico, S., De Feudis, L., Morabito, C., Davi, G., Fanò, G. & Mariggiò,

M.A. (2009). Oxidative-induced membrane damage in diabetes lymphocytes: effects on intracellular Ca(2+) homeostasis. *Free Radical Biology and Medicine,* 43, 138–148,

oxidase isoform, Nox1, in colon epithelial cells: role of GATA-binding factor(s). *Free*

Hazen, S.L. & Simon, D.I. (2004). Decreased neointimal formation in Nox2-deficient mice reveals a direct role for NADPH oxidase in the response to arterial injury.

manage its occurrence and correct its adverse effects.

Development 2007 – 2013 is acknowledged.

5657-5667, ISSN 0021-9258

ISSN 0891-5849

**10. Acknowledgement**

**11. References**

*Proceedings of the National Academy of Sciences USA,* 101, 13014-13019, ISSN 0027- 8424

Vascular Biology of Reactive Oxygen Species and NADPH Oxidases: Role in Atherogenesis 441

Heistad, D.D., Wakisaka, Y., Miller, J., Chu, Y. & Pena-Silva, R. (2009). Novel aspects of

Hoyal, C.R., Gutierrez, A., Young, B.M., Catz, S.D., Lin, J.H., Tsichlis, P.N. & Babior, B.M.

Hwang, J., Saha, A., Boo, Y.C., Sorescu, G.P., McNally, J.S., Holland, S.M., Dikalov, S.,

Inoue, N., Kawashima, S., Kanazawa, K., Yamada, S., Akita, H. & Yokoyama, M. (1998).

Janiszewski, M., Lopes, L.R., Carmo, A.O., Pedro, M.A., Brandes, R.P., Santos, C.X. &

Kakar, R., Kautz, B. & Eklund, E.A. (2005). JAK2 is necessary and sufficient for interferon-

Katsuyama, M., Fan, C., Arakawa, N., Nishinaka, T., Miyagishi, M., Taira, K. & Yabe-

Kawahara, T., Ritsick, D., Cheng, G. & Lambeth, J.D. (2005). Point mutations in the proline-

Kilpatrick, L. E., Sun S., Li, H., Vary, T.C. & Korchak, H.M. (2010). Regulation of TNF-

Kondo, T., Hirose, M. & Kageyama, K. (2009). Roles of oxidative stress and redox regulation in atherosclerosis. *Journal of Atherosclerosis and Thrombosis,* 16, 532-538 Kuroda, J. & Sadoshima, J. (2010). NADPH oxidase and cardiac failure. *Journal of*

Lambeth J. D. (2004). NOX enzymes and the biology of reactive oxygen. *Nature Reviews*

Lambeth, J.D. (2007). Nox enzymes, ROS, and chronic disease: an example of antagonistic pleiotropy. *Free Radical Biology and Medicine,* 43, 332–347, ISSN 0891-5849 Lassègue, B. & Griendling, K.K. (2002). Out phoxing the endothelium: what's left without

coronary artery disease. *Circulation,* 97, 135–137, ISSN 0009-7322

*Sciences USA,* 100, 5130–5135, ISSN 0027-8424

1346-9843

0021-9258

47298, ISSN 0021-9258

280, 40813-40819, ISSN 0021-9258

*Biology,* 77, 120-127, ISSN 0741-5400

*Biochemical Journal,* 386, 255-261, ISSN 0264-6021

*Journal of Leukocyte Biology,* 87, 153-164, ISSN 0741-5400

p47? *Circulation Research,* 90, 123–124, ISSN 0009-7330

*Cardiovascular Translational Research,* 3, 314–320

*Immunology,* 4, 181-189, ISSN 1474-1733

oxidative stress in cardiovascular diseases. *Circulation Journal* 73, 201-207, ISSN

(2003). Modulation of p47PHOX activity by sitespecific phosphorylation: Aktdependent activation of the NADPH oxidase. *Proceedings of the National Academy of*

Giddens, D.P., Griendling, K.K., Harrison, D.G. & Jo, H. (2003). Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion. *Journal of Biological Chemistry,* 278, 47291-

Polymorphism of the NADH/NADPH oxidase p22phox gene in patients with

Laurindo, F.R. (2005). Regulation of NAD(P)H oxidase by associated protein disulfide isomerase in vascular smooth muscle cells. *Journal of Biological Chemistry,*

gamma-induced transcription of the gene encoding gp91PHOX. *Journal of Leukocyte*

Nishimura, C. (2005). Essential role of ATF-1 in induction of NOX1, a catalytic subunit of NADPH oxidase: involvement of mitochondrial respiratory chain.

rich region of p22phox are dominant inhibitors of Nox1- and Nox2-dependent reactive oxygen generation. *Journal of Biological Chemistry,* 280, 31859-31869, ISSN

induced oxygen radical production in human neutrophils: role of delta-PKC.

Förstermann, U. (2008). Oxidative stress in vascular disease: causes, defense mechanisms

Fulton, D.J. (2009). Nox5 and the regulation of cellular function. *Antioxidants and Redox*

Gao, L. & Mann, G.E. (2009). Vascular NAD(P)H oxidase activation in diabetes: a doubleedged sword in redox signalling. *Cardiovascular Research,* 82, 9–20, ISSN 0008-6363 Gauss, K.A., Nelson-Overton, L.K., Siemsen, D.W., Gao, Y., DeLeo, F.R. & Quinn, M.T.

Gavazzi, G., Deffert, C., Trocme, C., Schäppi, M., Herrmann, F.R. & Krause, K.H. (2007).

Go, Y.M. & Jones, D.P. (2010) Redox control systems in the nucleus: mechanisms and

Goyal, P., Weissmann, N., Grimminger, F., Hegel, C., Bader, L., Rose, F., Fink, L., Ghofrani,

Gu, J.L., Pei, H., Thomas, L., Nadler, J.L., Rossi, J.J., Lanting, L. & Natarajan R. (2001).

Guzik, T.J., Chen, W., Gongora, M.C., Guzik, B., Lob, H.E., Mangalat, D., Hoch, N., Dikalov,

Guzik, T.J., West, N.E., Black, E., McDonald, D., Ratnatunga, C., Pillai, R. & Channon, K.M.

Harrison, D., Griendling, K.K., Landmesser, U., Hornig, B. & Drexler H. (2003). Role of

Hazen, S.L., Gaut, J.P., Hsu, F.F., Crowley, J.R., d'Avignon, A. & Heinecke, J.W. (1997). p-

*the American College of Cardiology,* 52, 1803-1809, ISSN 0735-1097

ISSN 1743-4297

ISSN 0741-5400

0891-5849

1452, ISSN 0009-7330

102, 1744-1747, ISSN 0009-7322

ISSN 0002-9149

16998, ISSN 0021-9258

*Signaling,* 11, 2443–2452

*Hypertension,* 50, 189-196, ISSN 0194-911X

functions. *Antioxidants and Redox Signaling,* 13, 489–509

and potential therapies. *Nature Clinical Practice Cardiovascular Medicine,* 5, 338-349,

(2007). Role of NF-kappaB in transcriptional regulation of the phagocyte NADPH oxidase by tumor necrosis factor-alpha. *Journal of Leukocyte Biology,* 82, 729-741,

NOX1 deficiency protects from aortic dissection in response to angiotensin II.

H.A., Schermuly, R.T., Schmidt, H.H., Seeger, W. & Hänze, J. (2004). Upregulation of NAD(P)H oxidase 1 in hypoxia activates hypoxia-inducible factor 1 via increase in reactive oxygen species. *Free Radical Biology and Medicine,* 36, 1279-1288, ISSN

Ribozyme-mediated inhibition of rat leukocyte-type 12-lipoxygenase prevents intimal hyperplasia in balloon-injured rat carotid arteries. *Circulation,* 103, 1446 –

S., Rudzinski, P., Kapelak, B., Sadowski, J. & Harrison, D.G. (2008). Calciumdependent NOX5 nicotinamide adenine dinucleotide phosphate oxidase contributes to vascular oxidative stress in human coronary artery disease. *Journal of*

(2000). Functional effect of the C242T polymorphism in the NAD(P)H oxidase p22phox gene on vascular superoxide production in atherosclerosis. *Circulation,*

oxidative stress in atherosclerosis. *American Journal of Cardiology,* 91, 7A–11A, ISSN

Hydroxyphenylacetaldehyde, the major product of L-tyrosine oxidation by the myeloperoxidase-H2O2-chloride system of phagocytes, covalently modifies epsilonamino groups of protein lysine residues. *Journal of Biological Chemistry*, 272, 16990–

Vascular Biology of Reactive Oxygen Species and NADPH Oxidases: Role in Atherogenesis 443

Manea, A., Raicu, M. & Simionescu, M. (2005). Expression of functionally phagocyte-type

Manea, A., Tanase, L.I., Raicu, M. & Simionescu, M. (2010b). Transcriptional regulation of

Manea, A., Tanase, L.I., Raicu, M. & Simionescu, M. (2010a). Jak/STAT signaling pathway

dietary lipid lowering. *Circulation Research,* 88, 733–739, ISSN 0009-7330 Martinez-Hervas, S., Real, J.T., Ivorra, C., Priego, A., Chaves, F.J., Pallardo, F.V., Viña, J.R.,

Matsuno, K., Yamada, H., Iwata, K., Jin, D., Katsuyama, M., Matsuki, M., Takai, S.,

Miller, F.J. Jr., Filali, M., Huss, G.J., Stanic, B., Chamseddine, A., Barna, T.J. & Lamb, F.S.

Moreno, M.U., San José, G., Fortuño, A., Beloqui, O., Redón, J., Chaves, F.J., Corella, D.,

Negre-Salvayre, A., Auge, N., Ayala, V., Basaga, H., Boada, J., Brenke, R., Chapple, S.,

*Arteriosclerosis, Thrombosis, and Vascular Biology,* 14, 133-140, ISSN

ISSN 1079-5642

*the Cell,* 97, 723–734, ISSN 1059-1524

*Diseases,* 20, 734-739, ISSN 0939-4753

*Circulation,* 112, 2677-2685, ISSN

101, 663–671, ISSN 0009-7330

901-907, ISSN 0006-291X

role of p22phox subunit. *Arteriosclerosis Thrombosis and Vascular Biology,* 28, 878-885,

NAD(P)H oxidase in pericytes: effect of angiotensin II and high glucose. *Biology of*

NADPH oxidase isoforms, Nox1 and Nox4, by nuclear factor-kappaB in human aortic smooth muscle cells. *Biochemical and Biophysical Research Communications,* 396,

regulates Nox1 and Nox4-based NADPH oxidase in human aortic smooth muscle cells. *Arteriosclerosis Thrombosis and Vascular Biology,* 30, 105-112, ISSN 1079-5642 Martinet, W., Knaapen, M.W., De Meyer, G.R., Herman, A.G. & Kockx, M.M. (2001).

Oxidative DNA damage and repair in experimental atherosclerosis are reversed by

Redon, J., Carmena, R. & Ascaso J.F. (2010). Increased plasma xanthine oxidase activity is related to nuclear factor kappa beta activation and inflammatory markers in familial combined hyperlipidemia. *Nutrition, Metabolism & Cardiovascular*

Yamanishi, K., Miyazaki, M., Matsubara, H. & Yabe-Nishimura, C. (2005). Nox1 is involved in angiotensin II-mediated hypertension: a study in Nox1-deficient mice.

(2007). Cytokine activation of nuclear factor kappa B in vascular smooth muscle cells requires signaling endosomes containing Nox1 and ClC-3. *Circulation Research,*

Díez, J. & Zalba, G. (2007). A novel CYBA variant, the -675A/T polymorphism, is associated with essential hypertension. *Journal of Hypertension,* 25, 1620-1626, ISSN Nakashima, Y., Plump, A.S., Raines, E.W., Breslow, J.L. & Ross, R. (1994). ApoE-deficient

mice develop lesions of all phases of atherosclerosis throughout the arterial tree.

Cohen, G., Feher, J., Grune, T., Lengyel, G., Mann, G.E., Pamplona, R., Poli, G., Portero-Otin, M., Riahi, Y., Salvayre, R., Sasson, S., Serrano, J., Shamni, O., Siems, W., Siow, R.C., Wiswedel, I., Zarkovic K. & Zarkovic, N. (2010). Pathological aspects of lipid peroxidation. *Free Radical Research,* 44, 1125-1171, ISSN 1071-5762 Ni, W., Zhan, Y., He, H., Maynard, E., Balschi, J.A. & Oettgen, P. (2007). Ets-1 is a critical

transcriptional regulator of reactive oxygen species and p47(phox) gene expression in response to angiotensin II. *Circulation Research,* 101, 985-994, ISSN 0009-7330 Olukman, M., Orhan, C.E., Celenk, F.G. & Ulker, S. (2010). Apocynin restores endothelial

dysfunction in streptozotocin diabetic rats through regulation of nitric oxide

Lee, M.Y. & Griendling, K.K. (2008). Redox signaling, vascular function, and hypertension.

Lee, M.Y., San Martin, A., Mehta, P.K., Dikalova, A.E., Garrido, A.M., Datla, S.R., Lyons, E.,

Leeuwenburgh, C., Rasmussen, J.E., Hsu, F.F., Mueller, D.M., Pennathur, S. & Heinecke,

Li, J.M. & Shah, A.M. (2003). Mechanism of endothelial cell NADPH oxidase activation by

Lim, S.C., Goh, S.K., Lai, Y.R., Tee, W.W., Koh, A., Xu, X.H., Wu, Y.S., Yap, E.,

Liu, H., Colavitti, R., Rovira, I.I. & Finkel, T. (2005). Redox-dependent transcriptional

Lyle, A.N., Deshpande, N.N., Taniyama, Y., Seidel-Rogol, B., Pounkova, L., Du, P.,

Madamanchi N.R., Vendrov, A. & Runge M.S. (2005). Oxidative stress and vascular disease. *Arteriosclerosis Thrombosis and Vascular Biology*, 25, 29-38, ISSN 1079-5642 Maitra, U., Singh, N., Gan, L., Ringwood, L. & Li, L. (2009) IRAK-1 contributes to

Manea, A. (2010). NADPH oxidase-derived reactive oxygen species: involvement in vascular physiology and pathology. *Cell and Tissue Research,* 342, 325-339, ISSN 0302-766X Manea, A., Manea, S.A., Gafencu, A.V. & Raicu, M. (2007). Regulation of NADPH oxidase

Manea, A., Manea, S.A., Gafencu, A.V., Raicu, M. & Simionescu, M. (2008). AP-1-dependent

Manea, A., Manea, S.A., Gafencu, A.V., Raicu, M. & Simionescu, M. (2008). AP-1-dependent

regulation. *Circulation Research,* 97, 967–974, ISSN 0009-7330

*Circulation Research,* 105, 249-259, ISSN 0009-7330

*Physiology and Biochemistry,* 113, 163-172, ISSN

Krause, K.H., Banfi, B., Lambeth, J.D., Lassègue, B. & Griendling, K.K. (2009). Mechanisms of vascular smooth muscle NADPH oxidase 1 (Nox1) contribution to injury-induced neointimal formation. *Arteriosclerosis Thrombosis and Vascular*

J.W. (1997). Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques. *Journal of Biological Chemistry*, 272, 3520–3526,

angiotensin II. Role of the p47phox subunit. *Journal of Biological Chemistry,* 278,

Subramaniam, T. & Sum, C.F. (2006). Relationship between common functional polymorphisms of the p22phox gene (-930A > G and +242C > T) and nephropathy as a result of Type 2 diabetes in a Chinese population. *Diabetic Medicine,* 23, 1037-

Papaharalambus, C., Lassègue, B. & Griendling, K.K. (2009). Poldip2, a novel regulator of Nox4 and cytoskeletal integrity in vascular smooth muscle cells.

lipopolysaccharide-induced reactive oxygen species generation in macrophages by inducing NOX-1 transcription and Rac1 activation and suppressing the expression of antioxidative enzymes. *Journal of Biological Chemistry,* 284, 35403-35411, ISSN

subunit p22(phox) by NF-kB in human aortic smooth muscle cells. *Archives of*

transcriptional regulation of NADPH oxidase in human aortic smooth muscle cells: role of p22phox subunit. *Arteriosclerosis Thrombosis and Vascular Biology*, 28, 878-885,

transcriptional regulation of NADPH oxidase in human aortic smooth muscle cells:

*Antioxidant and Redox Signaling*, 10, 1045–1059

*Biology,* 29, 480-487, ISSN 1079-5642

12094–12100, ISSN 0021-9258

1041, ISSN 0742-3071

ISSN 0021-9258

ISSN 1079-5642

ISSN 0021-9258

role of p22phox subunit. *Arteriosclerosis Thrombosis and Vascular Biology,* 28, 878-885, ISSN 1079-5642

Vascular Biology of Reactive Oxygen Species and NADPH Oxidases: Role in Atherogenesis 445

Tonks, N.K. (2006). Protein tyrosine phosphatases: from genes, to function, to disease.

Touyz, R.M. (2003) Reactive oxygen species in vascular biology: role in arterial

Turrens, J.F. (2003). Mitochondrial formation of reactive oxygen species. *Journal of Physiology*,

Valdivia, A., Pérez-Alvarez, S., Aroca-Aguilar, J.D., Ikuta, I. & Jordán, J. (2009). Superoxide

Vendrov, A.E., Hakim, Z.S., Madamanchi, N.R., Rojas, M., Madamanchi, C. & Runge, M.S.

Vendrov, A.E., Hakim, Z.S., Madamanchi, N.R., Rojas, M., Madamanchi, C. & Runge, M.S.

Woo, H.A., Yim, S.H., Shin, D.H., Kang, D., Yu, D.Y. & Rhee, S.G. (2010). Inactivation of

Wu, W.S., Wu, J.R. & Hu, C.T. (2008). Signal cross talks for sustained MAPK activation and

Xu, X., Gao, X., Potter, B.J., Cao, J.M. & Zhang, C. (2007). Anti-LOX-1 rescues endothelial

*Arteriosclerosis Thrombosis and Vascular Biology,* 27, 871-877, ISSN 1079-5642 Yamamori, T., Inanami, O., Nagahata, H. & Kuwabara, M. (2004). Phosphoinositide 3-kinase

Yusuf, S., Dagenais, G., Pogue, J., Bosch, J., & Sleight, P. (2000) Vitamin E supplementation

Zadák, Z., Hyspler, R., Tichá, A., Hronek, M., Fikrová, P., Rathouská, J., Hrnciariková, D. &

Zalba, G., Fortuño, A., San José, G., Moreno, M.U., Beloqui, O. & Díez, J. (2007). Oxidative

dismutases: a physiopharmacological update. *Journal of Physiology and Biochemistry*,

(2007). Atherosclerosis is attenuated by limiting superoxide generation in both macrophages and vessel wall cells. *Arteriosclerosis Thrombosis and Vascular Biology,*

(2007). Atherosclerosis is attenuated by limiting superoxide generation in both macrophages and vessel wall cells. *Arteriosclerosis Thrombosis and Vascular Biology*,

peroxiredoxin I by phosphorylation allows localized H(2)O(2) accumulation for cell

cell migration: the potential role of reactive oxygen species. *Cancer Metastasis Rev,*

function in coronary arterioles in atherosclerotic ApoE knockout mice.

regulates the phosphorylation of NADPH oxidase component p47(phox) by controlling cPKC/PKCdelta but not Akt. *Biochemical and Biophysical Research*

and cardiovascular events in high-risk patients. The Heart Outcomes Prevention Evaluation Study Investigators. *New England Journal of Medicine,* 342, 154-160, ISSN

Stetina, R. (2009). Antioxidants and vitamins in clinical conditions. *Physiological*

stress, endothelial dysfunction and cerebrovascular disease. *Cerebrovascular Diseases*

*Nature Reviews Molecular Cell Biology,* 7, 833-846, ISSN 1471-0072

hypertension. Expert Review *of Cardiovascular Therapy,* 1, 91-106

103, 149–158, ISSN 0009-7330

552, 335-344, ISSN 0022-3751

65, 195–208, ISSN 1138-7548

27, 2714-2721, ISSN 1079-5642

27, 2714-2721, ISSN 1079-5642

27, 303–314

0028-4793

signaling. *Cell,* 140, 517–528, ISSN 0092-8674

*Communications,* 316, 720-730, ISSN 0006-291X

*Research*, 58, 13–17, ISSN 0862-8408

24, 24-29, ISSN 1015-9770

phosphatase SHP-2 in vascular smooth muscle cells from SHR. *Circulation Research,*

synthase and NADPH oxidase expressions. *Journal of Diabetes and its Complications,* 24, 415-423, ISSN 1056-8727

Pendyala, S. & Natarajan, V. (2010). Redox regulation of Nox proteins. Respiratory

Raicu, M. & Manea, A. (2010). Activator protein-1 regulates Nox1 and Nox4-containing

Ray, R., Murdoch, C.E., Wang, M., Santos, C.X., Zhang, M., Alom-Ruiz, S., Anilkumar, N.,

Riahi, Y., Cohen, G., Shamni, O. & Sasson, S. (2010). Signaling and cytotoxic functions of 4-

San José, G., Fortuño, A., Beloqui, O., Díez, J. & Zalba, G. (2008). NADPH oxidase CYBA

Savenkova, M.L., Mueller, D.M. & Heinecke, J.W. (1994). Tyrosyl radical generated by

Sedeek, M., Hébert, R.L., Kennedy, C.R., Burns, K.D. & Touyz, R.M. (2009). Molecular

Serrander, L., Jaquet, V., Bedard, K., Plastre, O., Hartley, O., Arnaudeau, S., Demaurex, N.,

Shao, B. & Heinecke, J.W. (2009). HDL, lipid peroxidation, and atherosclerosis. *Journal of*

Shao, B. & Heinecke, J.W. (2009). HDL, lipid peroxidation, and atherosclerosis. *Journal of*

Sima, A.V., Stancu, C.S. & Simionescu, M. (2009). Vascular endothelium in atherosclerosis.

Simionescu, M. (2007). Implications of early structural-functional changes in the

Simionescu, M., Popov, D. & Sima, A. (2009). Endothelial transcytosis in health and disease.

Tabet, F., Schiffrin, E.L., Callera, G.E., He, Y., Yao, G., Ostman, A., Kappert, K., Tonks, N.K.

endothelium for vascular disease. *Arteriosclerosis Thrombosis and Vascular Biology*, 27,

& Touyz, R.M. (2008). Redox-sensitive signaling by angiotensin II involves oxidative inactivation and blunted phosphorylation of protein tyrosine

*in Nephrology and Hypertension,* 18, 122–127, ISSN 0194-911X

*Physiology &* Neurobiology*,*174, 265-271, ISSN 1569-9048

*Vascular Biology,* 31, 1368-1376, ISSN 1079-5642

24, 415-423, ISSN 1056-8727

*RSCB*, XV, 11-17

879-886, ISSN 0193-1849

1159-1167, ISSN 0300-9084

266-274, ISSN 1079-5642

*Lipid Research,* 50, 599-601, ISSN 0022-2275

*Lipid Research* 50, 599-601, ISSN 0022-2275

*Cell and Tissue Research,* 335, 191-203, ISSN 0302-766X

*Cell and Tissue Research,* 335, 27-40, ISSN 0302-766X

0021-9258

*(Lond),* 114, 173-182, ISSN 0143-5221

synthase and NADPH oxidase expressions. *Journal of Diabetes and its Complications,*

NADPH oxidase transcription in human vascular smooth muscle cells. *Annals of*

Ouattara, A., Cave, A.C., Walker, S.J., Grieve, D.J., Charles, R.L., Eaton, P., Brewer, A.C. & Shah, A.M. (2011). Endothelial Nox4 NADPH oxidase enhances vasodilatation and reduces blood pressure in vivo. *Arteriosclerosis Thrombosis and*

hydroxyalkenals. *American Journal of Physiology Endocrinology and Metabolism,* 299,

polymorphisms, oxidative stress and cardiovascular diseases. *Clinical Science*

myeloperoxidase is a physiological catalyst for the initiation of lipid peroxidation in low density lipoprotein. *Journal of Biological Chemistry*, 269, 20394–20400, ISSN

mechanisms of hypertension: role of Nox family NADPH oxidases. *Current Opinion*

Schlegel, W. & Krause, K.H. (2007). NOX5 is expressed at the plasma membrane and generates superoxide in response to protein kinase C activation. *Biochimie,* 89, phosphatase SHP-2 in vascular smooth muscle cells from SHR. *Circulation Research,* 103, 149–158, ISSN 0009-7330

**21**

*Spain*

**Modified Forms of LDL in Plasma**

José Luis Sánchez-Quesada1 and Sandra Villegas2 *1Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona*

*2Biochemistry and Molecular Biology Department, Universitat Autonoma de Barcelona*

High blood concentration of low-density lipoprotein (LDL) cholesterol is a major risk factor for early development of atherosclerosis. Massive accumulation of cholesterol in the arterial wall and formation of lipid-laden cells (foam cells) typical of the atheromatous plaque occur after LDL entrapment in the subendothelial space. However, there is a general agreement that native non-modified LDL does not present any of the typical features of an atherogenic lipoprotein; native LDL does not promote foam cell formation and has no inflammatory, proliferative or apoptotic capacity. It is therefore assumed that when LDL is trapped in the arterial wall it is modified by several mechanisms such as lipoperoxidation, non-enzymatic glycosylation, enzymatic lipolysis and/or proteolysis. As a consequence, modified LDL particles acquire inflammatory and apoptotic capacity and are recognizable by scavenger receptors to promote foam cell formation. Although most studies on LDL modification have focused on mechanisms that could occur in the vessel wall, modified LDL particles have been reported in blood. The concentration of these particles is increased in subjects with high cardiovascular risk. The present chapter reviews the possible mechanisms leading to LDL modification. It then focuses on a subfraction of modified LDL particles detected in

Pioneering studies by Goldstein and Brown demonstrated the involvement of LDL receptor (LDLr) in the plasma clearance of LDL and its major role in the development of atherosclerosis (Goldstein & Brown, 1985). However, these authors' outstanding observations gave rise to what was called the "cholesterol paradox" (Brown & Goldstein, 1983). Patients with homozygous familial hypercholesterolemia lack functional LDLr. Moreover, LDLr expression is negatively regulated by the intracellular cholesterol content, which results in a tight control of the concentration of esterified cholesterol in the cytoplasm (Brown & Goldstein, 1986). However, atherosclerosis is characterized by an abundance of foam cells loaded with esterified cholesterol droplets. Hence, there must be an alternative pathway leading to the massive accumulation of cholesterol in foam cells of the atherosclerotic lesion. This alternative pathway was discovered through the study of the scavenger receptors (SR) expressed by monocyte-derived macrophages (Krieger, 1992). These receptors have the ability to bind an unusual variety of ligands with the common

**1. Introduction**

plasma, named electronegative LDL.

**2. Modification of LDL as a key event for atherosclerosis**

### **Modified Forms of LDL in Plasma**

José Luis Sánchez-Quesada1 and Sandra Villegas2 *1Biomedical Research Institute Sant Pau (IIB Sant Pau), Barcelona 2Biochemistry and Molecular Biology Department, Universitat Autonoma de Barcelona Spain*

#### **1. Introduction**

446 Atherogenesis

Zhang, L., Sheppard, O.R., Shah, A.M. & Brewer, A.C. (2008). Positive regulation of the

Zhang, M., Brewer, A.C., Schröder, K., Santos, C.X., Grieve, D.J., Wang, M., Anilkumar, N.,

*Radical Biology and Medicine,* 45, 679-685, ISSN 0891-5849

18121-18126, ISSN 0027-8424

NADPH oxidase NOX4 promoter in vascular smooth muscle cells by E2F. *Free*

Yu, B., Dong, X., Walker, S.J., Brandes, R.P. & Shah, A.M. (2010). NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis. *Proceedings of the National Academy of Sciences USA,* 107,

> High blood concentration of low-density lipoprotein (LDL) cholesterol is a major risk factor for early development of atherosclerosis. Massive accumulation of cholesterol in the arterial wall and formation of lipid-laden cells (foam cells) typical of the atheromatous plaque occur after LDL entrapment in the subendothelial space. However, there is a general agreement that native non-modified LDL does not present any of the typical features of an atherogenic lipoprotein; native LDL does not promote foam cell formation and has no inflammatory, proliferative or apoptotic capacity. It is therefore assumed that when LDL is trapped in the arterial wall it is modified by several mechanisms such as lipoperoxidation, non-enzymatic glycosylation, enzymatic lipolysis and/or proteolysis. As a consequence, modified LDL particles acquire inflammatory and apoptotic capacity and are recognizable by scavenger receptors to promote foam cell formation. Although most studies on LDL modification have focused on mechanisms that could occur in the vessel wall, modified LDL particles have been reported in blood. The concentration of these particles is increased in subjects with high cardiovascular risk. The present chapter reviews the possible mechanisms leading to LDL modification. It then focuses on a subfraction of modified LDL particles detected in plasma, named electronegative LDL.

#### **2. Modification of LDL as a key event for atherosclerosis**

Pioneering studies by Goldstein and Brown demonstrated the involvement of LDL receptor (LDLr) in the plasma clearance of LDL and its major role in the development of atherosclerosis (Goldstein & Brown, 1985). However, these authors' outstanding observations gave rise to what was called the "cholesterol paradox" (Brown & Goldstein, 1983). Patients with homozygous familial hypercholesterolemia lack functional LDLr. Moreover, LDLr expression is negatively regulated by the intracellular cholesterol content, which results in a tight control of the concentration of esterified cholesterol in the cytoplasm (Brown & Goldstein, 1986). However, atherosclerosis is characterized by an abundance of foam cells loaded with esterified cholesterol droplets. Hence, there must be an alternative pathway leading to the massive accumulation of cholesterol in foam cells of the atherosclerotic lesion. This alternative pathway was discovered through the study of the scavenger receptors (SR) expressed by monocyte-derived macrophages (Krieger, 1992). These receptors have the ability to bind an unusual variety of ligands with the common

Modified Forms of LDL in Plasma 449

been described in the last two decades (SRBI, CD36, LOX-1, CD68, LRP1, TLR4) (Adachi & Tsujimoto, 2006; Llorente-Cortes & Badimon, 2005; Miller, 2003; Van Berkel, 2000). These receptors play a role in innate immunity, scavenging a number of ligands and extracellular debris. Some of these are also expressed in endothelial cells or in smooth muscle cells (Adachi & Tsujimoto, 2006). The derivatization of lysines has an additional effect on LDL; since a lysine-rich cluster is the LDLr binding site of apoB (residues 3359- 3369), the loss of their positive charge abolishes the interaction between oxLDL and LDLr

Besides lipid accumulation, other phenomena occur during the evolution of the disease. Atherosclerosis is a chronic inflammatory process that begins with leukocyte recruitment to the lesion area (Ross, 1999). A number of inflammation mediators, such as cytokines (IL6), chemokines (IL8, MCP1) or vascular adhesion molecules (VCAM, ICAM1, eselectin) are hyperexpressed in response to oxLDL by all cells involved in atherogenesis, including endothelial cells, monocytes/macrophages, lymphocytes and smooth muscle cells (Berliner, 1997; Tedgui & Mallat, 2006). Related to the inflammatory activity of oxLDL is its ability to promote the expression of growth factors and colony stimulating factors. This proliferative activity is mediated by oxidation-derived lipids, and in smooth muscle cells it induces the change of the normal contractile phenotype to a proliferative phenotype (Auge, 2002). The proliferative phenotype of smooth muscle cells is characterized by the high production of collagen that will contribute decisively to the

Another hallmark of the atheromatous plaque in its advanced stages is the presence of a necrotic core formed by debris from dead cells (Stary, 2000). OxLDL also contributes to the formation of this necrotic core, because when the content of esterified cholesterol exceeds the storage capacity of foam cells apoptotic processes are induced (Hessler, 1983). Another mechanism of cytotoxicity is the high content of lysophosphatidylcholine in oxLDL that disrupts the integrity of cytoplasmatic membrane and promotes cell death

OxLDL is immunogenic, and autoantibodies against oxidation-specific epitopes are detected in normal and hyperlipemic subjects (Lopes-Virella & Virella, 2010). The role of these autoantibodies is not well defined and published results are divergent; positive or negative associations between autoantibodies and atherosclerosis have been reported. In general, antibodies of IgG class are presumed to be pro-atherogenic, whereas IgM antibodies would play a protective role (Frostegard, 2010). Further deleterious properties of oxLDL are its capacity to increase the tissue factor activity (Petit, 1999), favouring thrombosis, or the

The lipoperoxidative process is sequential and lipids degrade to produce new products which in turn form other molecules (Quehenberger, 1988). As a consequence, oxLDL comprises an extremely heterogeneous group of particles that have different atherogenic characteristics depending on the relative content of each molecule (Esterbauer, 1993). For instance, minimally oxidized LDL (mmLDL) has a high inflammatory activity due to its high content in oxidized phospholipids formed during the early phases of oxidation. However, mmLDL has normal affinity to the LDLr and is not recognized by SR. In contrast, extensively oxidized LDL has relatively low inflammatory activity but it is a major inductor of cytotoxicity due to its high content of oxysterols. Indeed, the more oxidized the LDL the

inhibition of the release of nitric oxide (Minuz, 1995), promoting vasoconstriction.

(Boren, 1998a).

(Naito, 1994).

thickening of the arterial wall (Negishi, 2004).

more these particles promote foam cell formation.

characteristic of high electronegative charge. The first SR described was the type A SR (SRA) (Goldstein, 1979). SRA do not recognize native LDL but is able to bind modified forms of LDL; indeed, SRA expression is not regulated by the cytoplasm concentration of cholesterol. Early studies were performed using acetylated LDL (acLDL), a good ligand for SRA. However, acetylation does not occur spontaneously "in vivo". Soon, researchers looked for further modifications of LDL that would increase its negative charge and were able to occur "in vivo". The finding that oxidized LDL (oxLDL) could be internalized through SR and promote the formation of foam cells opened a vast field of research (Steinberg, 1989).

#### **2.1 Oxidative modification of LDL**

Oxidative modifications of lipid and proteins are frequent in many pathophysiological processes "in vivo" and it is now well-established that LDL undergoes oxidative modifications that confer to these modified particles a number of atherogenic properties (Witztum & Steinberg, 1991). In the early 80s several researchers independently observed that the incubation of LDL with endothelial cells in culture drastically altered these particles, transforming them into more electronegative LDL, after which they become a ligand for macrophages SR (Henriksen, 1981; Hessler, 1979). These studies concluded that the changes induced by endothelial cells on LDL were due to free radical modification. This was the origin of the oxidative modification hypothesis of atherogenesis (Steinberg, 1989). This hypothesis was gradually strengthened by subsequent findings regarding the properties of oxLDL obtained over the ensuing years. Table 1 summarizes the atherogenic properties observed in oxLDL and their contribution to atherosclerosis.

Table 1. Atherogenic characteristics of oxLDL

Oxidation of LDL primarily attacks the double bonds of unsaturated fatty acids in phospholipids and generates a plethora of lipid-derived compounds, including oxidized cholesterol (oxysterols), oxidized-fragmented phospholipids, lysophosphatidylcholine, hydroperoxids, aldehydes and ketones (Esterbauer, 1990). The protein moiety of LDL can be modified by reacting with lipid-oxidation products. Both aldehydes and ketones, for example, malondialdehyde (MDA) or 4-hydroxynonenal (HNE), have the ability to derivatize lysine and arginine residues in apoB (Fogelman, 1980). This reaction forms an adduct that eliminates the positive charge of these aminoacids and increases the negative charge of the particle, favouring its recognition by SRA (Goldstein, 1979). In addition to SRA, further SRs and other receptors which bind different forms of modified LDLs have

characteristic of high electronegative charge. The first SR described was the type A SR (SRA) (Goldstein, 1979). SRA do not recognize native LDL but is able to bind modified forms of LDL; indeed, SRA expression is not regulated by the cytoplasm concentration of cholesterol. Early studies were performed using acetylated LDL (acLDL), a good ligand for SRA. However, acetylation does not occur spontaneously "in vivo". Soon, researchers looked for further modifications of LDL that would increase its negative charge and were able to occur "in vivo". The finding that oxidized LDL (oxLDL) could be internalized through SR and

promote the formation of foam cells opened a vast field of research (Steinberg, 1989).

Oxidative modifications of lipid and proteins are frequent in many pathophysiological processes "in vivo" and it is now well-established that LDL undergoes oxidative modifications that confer to these modified particles a number of atherogenic properties (Witztum & Steinberg, 1991). In the early 80s several researchers independently observed that the incubation of LDL with endothelial cells in culture drastically altered these particles, transforming them into more electronegative LDL, after which they become a ligand for macrophages SR (Henriksen, 1981; Hessler, 1979). These studies concluded that the changes induced by endothelial cells on LDL were due to free radical modification. This was the origin of the oxidative modification hypothesis of atherogenesis (Steinberg, 1989). This hypothesis was gradually strengthened by subsequent findings regarding the properties of oxLDL obtained over the ensuing years. Table 1 summarizes the atherogenic properties

**2.1 Oxidative modification of LDL**

Increase in the expression of cytokines, chemokines and vascular adhesion

Table 1. Atherogenic characteristics of oxLDL

molecules

observed in oxLDL and their contribution to atherosclerosis.

Recognition by SR Foam cell formation

Inhibition of NO release and function Vasoconstriction Increase of tissue factor activity Thombosis

Promotion of apoptosis and cytotoxicity Formation of necrotic core

**Characteristic Consequence in atherosclerosis**  Impaired binding to the LDL receptor Decreased plasma clearance

Increase in the expression of growth factors Cell proliferation and collagen secretion

Immunogenicity Immunogenic component of atherosclerosis

Oxidation of LDL primarily attacks the double bonds of unsaturated fatty acids in phospholipids and generates a plethora of lipid-derived compounds, including oxidized cholesterol (oxysterols), oxidized-fragmented phospholipids, lysophosphatidylcholine, hydroperoxids, aldehydes and ketones (Esterbauer, 1990). The protein moiety of LDL can be modified by reacting with lipid-oxidation products. Both aldehydes and ketones, for example, malondialdehyde (MDA) or 4-hydroxynonenal (HNE), have the ability to derivatize lysine and arginine residues in apoB (Fogelman, 1980). This reaction forms an adduct that eliminates the positive charge of these aminoacids and increases the negative charge of the particle, favouring its recognition by SRA (Goldstein, 1979). In addition to SRA, further SRs and other receptors which bind different forms of modified LDLs have

Leukocyte recruitment, inflammation

been described in the last two decades (SRBI, CD36, LOX-1, CD68, LRP1, TLR4) (Adachi & Tsujimoto, 2006; Llorente-Cortes & Badimon, 2005; Miller, 2003; Van Berkel, 2000). These receptors play a role in innate immunity, scavenging a number of ligands and extracellular debris. Some of these are also expressed in endothelial cells or in smooth muscle cells (Adachi & Tsujimoto, 2006). The derivatization of lysines has an additional effect on LDL; since a lysine-rich cluster is the LDLr binding site of apoB (residues 3359- 3369), the loss of their positive charge abolishes the interaction between oxLDL and LDLr (Boren, 1998a).

Besides lipid accumulation, other phenomena occur during the evolution of the disease. Atherosclerosis is a chronic inflammatory process that begins with leukocyte recruitment to the lesion area (Ross, 1999). A number of inflammation mediators, such as cytokines (IL6), chemokines (IL8, MCP1) or vascular adhesion molecules (VCAM, ICAM1, eselectin) are hyperexpressed in response to oxLDL by all cells involved in atherogenesis, including endothelial cells, monocytes/macrophages, lymphocytes and smooth muscle cells (Berliner, 1997; Tedgui & Mallat, 2006). Related to the inflammatory activity of oxLDL is its ability to promote the expression of growth factors and colony stimulating factors. This proliferative activity is mediated by oxidation-derived lipids, and in smooth muscle cells it induces the change of the normal contractile phenotype to a proliferative phenotype (Auge, 2002). The proliferative phenotype of smooth muscle cells is characterized by the high production of collagen that will contribute decisively to the thickening of the arterial wall (Negishi, 2004).

Another hallmark of the atheromatous plaque in its advanced stages is the presence of a necrotic core formed by debris from dead cells (Stary, 2000). OxLDL also contributes to the formation of this necrotic core, because when the content of esterified cholesterol exceeds the storage capacity of foam cells apoptotic processes are induced (Hessler, 1983). Another mechanism of cytotoxicity is the high content of lysophosphatidylcholine in oxLDL that disrupts the integrity of cytoplasmatic membrane and promotes cell death (Naito, 1994).

OxLDL is immunogenic, and autoantibodies against oxidation-specific epitopes are detected in normal and hyperlipemic subjects (Lopes-Virella & Virella, 2010). The role of these autoantibodies is not well defined and published results are divergent; positive or negative associations between autoantibodies and atherosclerosis have been reported. In general, antibodies of IgG class are presumed to be pro-atherogenic, whereas IgM antibodies would play a protective role (Frostegard, 2010). Further deleterious properties of oxLDL are its capacity to increase the tissue factor activity (Petit, 1999), favouring thrombosis, or the inhibition of the release of nitric oxide (Minuz, 1995), promoting vasoconstriction.

The lipoperoxidative process is sequential and lipids degrade to produce new products which in turn form other molecules (Quehenberger, 1988). As a consequence, oxLDL comprises an extremely heterogeneous group of particles that have different atherogenic characteristics depending on the relative content of each molecule (Esterbauer, 1993). For instance, minimally oxidized LDL (mmLDL) has a high inflammatory activity due to its high content in oxidized phospholipids formed during the early phases of oxidation. However, mmLDL has normal affinity to the LDLr and is not recognized by SR. In contrast, extensively oxidized LDL has relatively low inflammatory activity but it is a major inductor of cytotoxicity due to its high content of oxysterols. Indeed, the more oxidized the LDL the more these particles promote foam cell formation.

Modified Forms of LDL in Plasma 451

Several studies have focused on the atherogenic effects of LDL modified with different types of secreted phospholipase A2 (sPLA2) (Divchev & Schieffer, 2008). Some sPLA2 have been detected in atherosclerotic lesions and their products, lysophosphatidylcholine (LPC) and NEFA, induce cytotoxicity at relatively high concentrations by disrupting membrane integrity (Dersch, 2005; Naito, 1994). Moreover, both molecules have inflammatory potential by stimulating the expression of cytokines and chemokines (Sonoki, 2003). In addition, extensively lipolyzed LDL with some (type X), but not all (type IIA or V), sPLA2 are able to induce foam cell formation (Curfs, 2008). An interesting approach was performed by Sparrow and coworkers, which combined sPLA2 and lipoxygenase to modify LDL (Sparrow, 1988). This combination of lipoperoxidation and phospholipolysis generated LDL particles with similar properties to those promoted by endothelial cell-induced oxidative modification. Another atherogenic characteristic of sPLA2-modified LDL is its increased affinity for PG binding due to the exposition of an alternative binding site in apoB that specifically recognizes PG (Boren, 1998b). As occurs with SMase-modified LDL, this would

PAF-acetylhydrolase (PAF-AH), another PLA2 with relevance in the metabolism of LDL, merits special mention. This enzyme is transported in plasma bound to lipoproteins (approximately 70% in LDL and 25% in HDL) but, in contrast to sPLA2, its substrate is not native phospholipids but fragmented phospholipids that have been generated by oxidation (Tjoelker & Stafforini, 2000). Thus, LPC content is high in oxLDL due to the action of PAF-AH. However, there is controversy regarding the pro- or anti-atherogenic role of PAF-AH (Tellis & Tselepis, 2009). On one hand, its function should be atheroprotective since it degrades highly-inflammatory fragmented phospholipids. On the other hand, however, the by-products formed are LPC and short-chain NEFA, which also have inflammatory

The hyperexpression of acid and neutral SMases in atherosclerotic lesions has been known for several years (Tabas, 1999). SMase hydrolyzes sphingomyelin (SM) yielding phosphorylcholine and ceramide. Ceramide directly exerts several biological effects, but its main action is to elicit the production of bioactive sphingolipids, such as sphingosine-1 phosphate that plays a major role in apoptosis (van Blitterswijk, 2003). Another action of SMase is to promote extensive LDL aggregation (Oorni, 2000). Aggregation of LDL confers several atherogenic properties. First, aggregated LDL (agLDL) is able to induce foam cell formation, not through SR in this case but through the LDL-receptor related protein 1 (LRP1), a receptor of aggregated lipoproteins highly expressed in smooth muscle cells (Llorente-Cortes & Badimon, 2005).  As discussed below, this binding promotes structural changes in apoB. The association of agLDL to PG precludes the exit of this lipoprotein from the subendothelial

potential, though to a lesser extent than oxidized phospholipids (MacPhee, 1999).

space, and consequently favours that agLDL could undergo further modifications.

Although non-enzymatic glycosylation of LDL occurs in all subjects, it has stronger consequences in people with diabetes mellitus. Glucose can interact with the free amino groups of lysines and arginines in apoB, forming a Schiff base that rearranges to yield an

**2.2.2 Phospholipase A2-modified LDL (PLA2-LDL)**

lead to increased subendothelial retention.

**2.2.3 Sphingomyelinase-modified LDL (SMase-LDL)**

**2.2.4 Glycated LDL (glLDL)**

#### **2.2 Further modifications of LDL occurring in the arterial wall**

Oxidative modification is not the only process affecting the native properties of lipoproteins in the arterial wall. Disappointing results from large-antioxidant trials have led to the concept that alternative mechanisms of modification could be involved (Steinberg, 2009). Current knowledge indicates that although oxidation is still relevant, other processes could contribute substantially to the development of atherosclerosis. A number of modifications have been studied over the past three decades.

Enzymes that are hyperexpressed in the microenvironment of the lesion area, such as lipases (cholesterol esterase (CEase), sphingomyelinase (SMase) or secretary phospholipase A2 (sPLA2)) or proteases (matrix metalloproteases or cathepsins) can modify LDL (Pentikainen, 2000). In the case of diabetes, non-enzymatic glycosylation could have a major role in LDL modification (Witztum, 1997). The interaction of LDL with the proteoglycans (PG) could modify apoB conformation, destabilizing its conformation and promoting aggregation (Pentikainen, 1997). Other putative physiological processes that could modify LDL are carbamylation (Basnakian, 2010) or desialylation (Tertov, 1990). Table 2 summarizes several possible physiological mechanisms leading to LDL modification.

Table 2. Atherogenic properties of several modified LDLs. 1 The characteristics of oxLDL, and also those of the other modifications, depend on the extent of modification

#### **2.2.1 Enzymatically-modified LDL (E-LDL)**

In an attempt to find a modified LDL alternative to oxLDL, Bhakdi and coworkers performed a series of studies using LDL that was modified by means of one protease and CEase. They found that this "enzymatically-modified LDL" (E-LDL) acquired a number of atherogenic properties (Bhakdi, 1995). As a result of the enzymatic treatment, E-LDL presents mild apoB fragmentation, and it has high free cholesterol and non-esterified fatty acids (NEFA) content (Klouche, 1998). E-LDL induces inflammation, proliferation and apoptosis (Dersch, 2005; Klouche, 1999; Klouche, 2000). The high content of NEFA seems to be major factor responsible for these atherogenic properties (Suriyaphol, 2002). E-LDL adds further atherogenic characteristics because it binds to C-reactive protein and activates the classical complement pathway (Bhakdi, 2004). The activation of complement by E-LDL concurs with the emerging concept of the innate immune response as a potentially important factor in atherosclerosis (Hartvigsen, 2009).

Oxidative modification is not the only process affecting the native properties of lipoproteins in the arterial wall. Disappointing results from large-antioxidant trials have led to the concept that alternative mechanisms of modification could be involved (Steinberg, 2009). Current knowledge indicates that although oxidation is still relevant, other processes could contribute substantially to the development of atherosclerosis. A number of modifications

Enzymes that are hyperexpressed in the microenvironment of the lesion area, such as lipases (cholesterol esterase (CEase), sphingomyelinase (SMase) or secretary phospholipase A2 (sPLA2)) or proteases (matrix metalloproteases or cathepsins) can modify LDL (Pentikainen, 2000). In the case of diabetes, non-enzymatic glycosylation could have a major role in LDL modification (Witztum, 1997). The interaction of LDL with the proteoglycans (PG) could modify apoB conformation, destabilizing its conformation and promoting aggregation (Pentikainen, 1997). Other putative physiological processes that could modify LDL are carbamylation (Basnakian, 2010) or desialylation (Tertov, 1990). Table 2 summarizes several

**formation**

Oxidation1 +++ +++ +++

protease -/+ ++ ++ Lipolysis by SMase + - -/+ Lipolysis by PLA2 -/+ ++ + Non-enzymatic glycosylation -/+ -/+ -/+ Binding to PG ++ - - Carbamylation ++ ++ ++ Desialylation ++ + ++ Table 2. Atherogenic properties of several modified LDLs. 1 The characteristics of oxLDL,

and also those of the other modifications, depend on the extent of modification

In an attempt to find a modified LDL alternative to oxLDL, Bhakdi and coworkers performed a series of studies using LDL that was modified by means of one protease and CEase. They found that this "enzymatically-modified LDL" (E-LDL) acquired a number of atherogenic properties (Bhakdi, 1995). As a result of the enzymatic treatment, E-LDL presents mild apoB fragmentation, and it has high free cholesterol and non-esterified fatty acids (NEFA) content (Klouche, 1998). E-LDL induces inflammation, proliferation and apoptosis (Dersch, 2005; Klouche, 1999; Klouche, 2000). The high content of NEFA seems to be major factor responsible for these atherogenic properties (Suriyaphol, 2002). E-LDL adds further atherogenic characteristics because it binds to C-reactive protein and activates the classical complement pathway (Bhakdi, 2004). The activation of complement by E-LDL concurs with the emerging concept of the innate immune response as a potentially

**Inflammation/ proliferation**

**Cytotoxicity/ apoptosis**

**2.2 Further modifications of LDL occurring in the arterial wall**

possible physiological mechanisms leading to LDL modification.

**Mechanisms of modification Foam cell**

**2.2.1 Enzymatically-modified LDL (E-LDL)**

important factor in atherosclerosis (Hartvigsen, 2009).

Modification by CEase and

have been studied over the past three decades.

#### **2.2.2 Phospholipase A2-modified LDL (PLA2-LDL)**

Several studies have focused on the atherogenic effects of LDL modified with different types of secreted phospholipase A2 (sPLA2) (Divchev & Schieffer, 2008). Some sPLA2 have been detected in atherosclerotic lesions and their products, lysophosphatidylcholine (LPC) and NEFA, induce cytotoxicity at relatively high concentrations by disrupting membrane integrity (Dersch, 2005; Naito, 1994). Moreover, both molecules have inflammatory potential by stimulating the expression of cytokines and chemokines (Sonoki, 2003). In addition, extensively lipolyzed LDL with some (type X), but not all (type IIA or V), sPLA2 are able to induce foam cell formation (Curfs, 2008). An interesting approach was performed by Sparrow and coworkers, which combined sPLA2 and lipoxygenase to modify LDL (Sparrow, 1988). This combination of lipoperoxidation and phospholipolysis generated LDL particles with similar properties to those promoted by endothelial cell-induced oxidative modification. Another atherogenic characteristic of sPLA2-modified LDL is its increased affinity for PG binding due to the exposition of an alternative binding site in apoB that specifically recognizes PG (Boren, 1998b). As occurs with SMase-modified LDL, this would lead to increased subendothelial retention.

PAF-acetylhydrolase (PAF-AH), another PLA2 with relevance in the metabolism of LDL, merits special mention. This enzyme is transported in plasma bound to lipoproteins (approximately 70% in LDL and 25% in HDL) but, in contrast to sPLA2, its substrate is not native phospholipids but fragmented phospholipids that have been generated by oxidation (Tjoelker & Stafforini, 2000). Thus, LPC content is high in oxLDL due to the action of PAF-AH. However, there is controversy regarding the pro- or anti-atherogenic role of PAF-AH (Tellis & Tselepis, 2009). On one hand, its function should be atheroprotective since it degrades highly-inflammatory fragmented phospholipids. On the other hand, however, the by-products formed are LPC and short-chain NEFA, which also have inflammatory potential, though to a lesser extent than oxidized phospholipids (MacPhee, 1999).

#### **2.2.3 Sphingomyelinase-modified LDL (SMase-LDL)**

The hyperexpression of acid and neutral SMases in atherosclerotic lesions has been known for several years (Tabas, 1999). SMase hydrolyzes sphingomyelin (SM) yielding phosphorylcholine and ceramide. Ceramide directly exerts several biological effects, but its main action is to elicit the production of bioactive sphingolipids, such as sphingosine-1 phosphate that plays a major role in apoptosis (van Blitterswijk, 2003). Another action of SMase is to promote extensive LDL aggregation (Oorni, 2000). Aggregation of LDL confers several atherogenic properties. First, aggregated LDL (agLDL) is able to induce foam cell formation, not through SR in this case but through the LDL-receptor related protein 1 (LRP1), a receptor of aggregated lipoproteins highly expressed in smooth muscle cells (Llorente-Cortes & Badimon, 2005).  As discussed below, this binding promotes structural changes in apoB. The association of agLDL to PG precludes the exit of this lipoprotein from the subendothelial space, and consequently favours that agLDL could undergo further modifications.

#### **2.2.4 Glycated LDL (glLDL)**

Although non-enzymatic glycosylation of LDL occurs in all subjects, it has stronger consequences in people with diabetes mellitus. Glucose can interact with the free amino groups of lysines and arginines in apoB, forming a Schiff base that rearranges to yield an

Modified Forms of LDL in Plasma 453

atherosclerosis development but also a reflection of the presence of unstable and ruptured atherosclerotic plaques (Fraley & Tsimikas, 2006). The fact that oxLDL increases temporarily during the acute phase of myocardial infarction or stroke supports this notion (Fraley & Tsimikas, 2006; Nishi, 2002; Uno, 2003) and some studies have raised the possibility that plasma oxLDL could predict future cardiovascular events (Meisinger, 2005). The concentration of oxLDL in plasma is very low, and data reported by several authors are disperse, ranging from 0.01% to 0.5% of total LDL. The heterogeneous nature of oxLDL is one reason to explain variation in the reports of oxLDL concentration, but another could be the use of several antibodies that recognize different epitopes (Ishigaki, 2009). The fact that in vitro oxidation does not reproduce the same epitopes generated during in vivo

Several studies have shown that besides its utility as a biomarker, oxLDL in plasma acts as a pathogenic factor. It has been reported that oxLDL contributes to increase the systemic inflammatory status by stimulating the activity of the transcription factor NF-kB in peripheral blood mononuclear cells (Cominacini, 2005). More direct demonstration of the implication of oxLDL in blood has been obtained by increasing the expression of SR, such as SRA1 (Whitman, 2002), LOX-1 (Ishigaki, 2008), or the chimerical fusion protein SRA1 growth hormone (Laukkanen, 2000), which favours oxLDL removal from blood. These studies showed an inhibition of atherosclerosis development. It was recently reported that repeated administration of the chimerical fusion protein Fc-CD68 decreases the extent of atherosclerosis in hyperlipemic mice (Zeibig, 2011). However, these studies have been tested to date in animal models only. Regarding humans, several trials have reported that lipidlowering therapy in atherosclerotic patients lowers oxLDL, although this decrease is parallel to that of LDL cholesterol (Ky, 2008). However, no trial with a therapy specifically focused on oxLDL is yet available. The development of novel therapies for lowering oxLDL itself is a

The concentration of glLDL in plasma from patients with diabetes is increased, reflecting the hyperglycemia in these patients. This concentration varies depending on the method used for its quantification (ELISA, affinity chromatography), but it is generally higher than that of oxLDL (Cohen, 1993; Reaven, 1995). The proportion of glLDL in diabetics can reach up to 7- 8% of total LDL and approximately half in normoglycemic subjects. It is important to note that glLDL is present in all individuals, and even in normoglycemic subjects the concentration in plasma is higher than that of oxLDL. This suggests glLDL plays a role in the development of atherosclerosis even in absence of hyperglycemia. Interestingly, glLDL is more abundant in the subfractions of LDL that are smallest and have the highest density, probably because these small-dense LDL particles are prone to non-enzymatic glycosylation (Younis, 2009). These particles are also more susceptible to oxidation and have a relatively low affinity to the LDL receptor. These properties are related to the strong association

Alternative pathways can also result in LDL glycosylation without a direct involvement of glucose. Metabolites of glucose such as glyoxal, methylglyoxal or glycaldehyde have a higher reducing capacity as glycating agents (Rabbani & Thornalley, 2011). Minimal modification by methylglyoxal renders LDL particles with atherogenic properties, including binding to PG and susceptibility to aggregation (Rabbani, 2011). LDL particles with this low

between small-dense LDL and high cardiovascular risk (Krauss, 1995).

level of modification are increased in diabetic patients (Rabbani, 2010).

modification makes it difficult to develop a golden standard.

promising strategy for atherosclerosis treatment.

**3.2 Glycated LDL in plasma**

Amadori product (Brownlee, 1992). This modification leads to a loss of electropositive charges in glycated LDL (glLDL), decreasing its affinity to the LDLr and consequently prolonging its mean lifetime in plasma (Witztum, 1982). The increase in lifetime can result in further modification of LDL forming advanced glycation end-products (AGE) and producing a form of LDL named AGE-LDL (Menzel, 1997). AGE-LDL can also be internalized by specific receptors (RAGE) (Bucala, 1996). AGE-LDL and glLDL increase chemotactic activity in monocytes, stimulate cell proliferation and enhance platelet aggregation, although the relative contribution that the coexistence of oxidation could have is not well established. The formation of AGE involves oxidative reactions and it has been demonstrated that lipoperoxidation and non-enzymatic glycosylation are mutually potentiated processes (Sobal, 2000). Thus, glycosylation of LDL is not only noxious per se but also because it promotes LDL oxidation.

#### **2.2.5 Proteoglycan-bound LDL (PG-LDL)**

PG are the main constituents of the arterial intima. It has been hypothesized that subendothelial retention of lipoproteins due to the binding of LDL to PG is the initial event in atherogenesis, even prior to endothelial dysfunction or inflammation (Williams & Tabas, 2005). The retention itself increases the time of LDL in the subendothelial space and, therefore, the possibility that further modifications occur. Furthermore, the binding LDL-PG also has a direct effect on LDL modification because this binding promotes changes in the structure of apoB that facilitate processes such as oxidation or lipolysis mediated by SMase or sPLA2 (Hevonoja, 2000). On the other hand, LDL-PG complexes are taken up by cells through different types of SR, promoting foam cell formation.

#### **3. Modified LDL in human plasma**

For many years it was considered that LDL modification was a phenomenon occurring mainly in the intima layer of the arterial wall. Oxidative modification was the most studied process, and most researchers accepted that the abundance in plasma of soluble molecules with antioxidant capacity (albumin, uric acid, bilirubin, glutathione, ascorbic acid) would inhibit oxidation of lipoproteins. Moreover, the binding of oxLDL by SR expressed in circulating monocytes is known to promote a rapid clearance of extensively oxidized LDL. However, progress in enzyme immunoassay procedures has provided direct evidence of oxLDL in circulating plasma. Besides oxLDL, similar methods have been used to detect other forms of modified LDL in blood. These include glLDL, AGE-LDL, carbamylated LDL (ca-LDL), desialylated LDL and electronegative LDL (LDL(-)).

#### **3.1 Oxidized LDL in plasma**

Despite the abundance of antioxidant defences in blood, increased oxidative stress has been described in plasma from patients with atherosclerosis. Early studies reported that increased levels of oxidized lipids in plasma were associated with atherosclerosis development (Avogaro, 1986). In agreement with that, the existence of oxLDL in blood is increased in subjects with high cardiovascular risk (Holvoet, 1999; Holvoet, 1998) or in diseases, such as diabetes, obesity, metabolic syndrome and hyperlipemia (Ishigaki, 2009). Although it cannot be totally ruled out that a part of oxLDL could be formed in blood, it is generally accepted that oxLDL originates primarily in the arterial wall and that the molecules reach the blood from the subendothelial space. For this reason, oxLDL is considered not only a biomarker of

Amadori product (Brownlee, 1992). This modification leads to a loss of electropositive charges in glycated LDL (glLDL), decreasing its affinity to the LDLr and consequently prolonging its mean lifetime in plasma (Witztum, 1982). The increase in lifetime can result in further modification of LDL forming advanced glycation end-products (AGE) and producing a form of LDL named AGE-LDL (Menzel, 1997). AGE-LDL can also be internalized by specific receptors (RAGE) (Bucala, 1996). AGE-LDL and glLDL increase chemotactic activity in monocytes, stimulate cell proliferation and enhance platelet aggregation, although the relative contribution that the coexistence of oxidation could have is not well established. The formation of AGE involves oxidative reactions and it has been demonstrated that lipoperoxidation and non-enzymatic glycosylation are mutually potentiated processes (Sobal, 2000). Thus, glycosylation of LDL is not only noxious per se but also because it promotes LDL oxidation.

PG are the main constituents of the arterial intima. It has been hypothesized that subendothelial retention of lipoproteins due to the binding of LDL to PG is the initial event in atherogenesis, even prior to endothelial dysfunction or inflammation (Williams & Tabas, 2005). The retention itself increases the time of LDL in the subendothelial space and, therefore, the possibility that further modifications occur. Furthermore, the binding LDL-PG also has a direct effect on LDL modification because this binding promotes changes in the structure of apoB that facilitate processes such as oxidation or lipolysis mediated by SMase or sPLA2 (Hevonoja, 2000). On the other hand, LDL-PG complexes are taken up by cells

For many years it was considered that LDL modification was a phenomenon occurring mainly in the intima layer of the arterial wall. Oxidative modification was the most studied process, and most researchers accepted that the abundance in plasma of soluble molecules with antioxidant capacity (albumin, uric acid, bilirubin, glutathione, ascorbic acid) would inhibit oxidation of lipoproteins. Moreover, the binding of oxLDL by SR expressed in circulating monocytes is known to promote a rapid clearance of extensively oxidized LDL. However, progress in enzyme immunoassay procedures has provided direct evidence of oxLDL in circulating plasma. Besides oxLDL, similar methods have been used to detect other forms of modified LDL in blood. These include glLDL, AGE-LDL, carbamylated LDL

Despite the abundance of antioxidant defences in blood, increased oxidative stress has been described in plasma from patients with atherosclerosis. Early studies reported that increased levels of oxidized lipids in plasma were associated with atherosclerosis development (Avogaro, 1986). In agreement with that, the existence of oxLDL in blood is increased in subjects with high cardiovascular risk (Holvoet, 1999; Holvoet, 1998) or in diseases, such as diabetes, obesity, metabolic syndrome and hyperlipemia (Ishigaki, 2009). Although it cannot be totally ruled out that a part of oxLDL could be formed in blood, it is generally accepted that oxLDL originates primarily in the arterial wall and that the molecules reach the blood from the subendothelial space. For this reason, oxLDL is considered not only a biomarker of

**2.2.5 Proteoglycan-bound LDL (PG-LDL)**

**3. Modified LDL in human plasma**

**3.1 Oxidized LDL in plasma**

through different types of SR, promoting foam cell formation.

(ca-LDL), desialylated LDL and electronegative LDL (LDL(-)).

atherosclerosis development but also a reflection of the presence of unstable and ruptured atherosclerotic plaques (Fraley & Tsimikas, 2006). The fact that oxLDL increases temporarily during the acute phase of myocardial infarction or stroke supports this notion (Fraley & Tsimikas, 2006; Nishi, 2002; Uno, 2003) and some studies have raised the possibility that plasma oxLDL could predict future cardiovascular events (Meisinger, 2005). The concentration of oxLDL in plasma is very low, and data reported by several authors are disperse, ranging from 0.01% to 0.5% of total LDL. The heterogeneous nature of oxLDL is one reason to explain variation in the reports of oxLDL concentration, but another could be the use of several antibodies that recognize different epitopes (Ishigaki, 2009). The fact that in vitro oxidation does not reproduce the same epitopes generated during in vivo modification makes it difficult to develop a golden standard.

Several studies have shown that besides its utility as a biomarker, oxLDL in plasma acts as a pathogenic factor. It has been reported that oxLDL contributes to increase the systemic inflammatory status by stimulating the activity of the transcription factor NF-kB in peripheral blood mononuclear cells (Cominacini, 2005). More direct demonstration of the implication of oxLDL in blood has been obtained by increasing the expression of SR, such as SRA1 (Whitman, 2002), LOX-1 (Ishigaki, 2008), or the chimerical fusion protein SRA1 growth hormone (Laukkanen, 2000), which favours oxLDL removal from blood. These studies showed an inhibition of atherosclerosis development. It was recently reported that repeated administration of the chimerical fusion protein Fc-CD68 decreases the extent of atherosclerosis in hyperlipemic mice (Zeibig, 2011). However, these studies have been tested to date in animal models only. Regarding humans, several trials have reported that lipidlowering therapy in atherosclerotic patients lowers oxLDL, although this decrease is parallel to that of LDL cholesterol (Ky, 2008). However, no trial with a therapy specifically focused on oxLDL is yet available. The development of novel therapies for lowering oxLDL itself is a promising strategy for atherosclerosis treatment.

#### **3.2 Glycated LDL in plasma**

The concentration of glLDL in plasma from patients with diabetes is increased, reflecting the hyperglycemia in these patients. This concentration varies depending on the method used for its quantification (ELISA, affinity chromatography), but it is generally higher than that of oxLDL (Cohen, 1993; Reaven, 1995). The proportion of glLDL in diabetics can reach up to 7- 8% of total LDL and approximately half in normoglycemic subjects. It is important to note that glLDL is present in all individuals, and even in normoglycemic subjects the concentration in plasma is higher than that of oxLDL. This suggests glLDL plays a role in the development of atherosclerosis even in absence of hyperglycemia. Interestingly, glLDL is more abundant in the subfractions of LDL that are smallest and have the highest density, probably because these small-dense LDL particles are prone to non-enzymatic glycosylation (Younis, 2009). These particles are also more susceptible to oxidation and have a relatively low affinity to the LDL receptor. These properties are related to the strong association between small-dense LDL and high cardiovascular risk (Krauss, 1995).

Alternative pathways can also result in LDL glycosylation without a direct involvement of glucose. Metabolites of glucose such as glyoxal, methylglyoxal or glycaldehyde have a higher reducing capacity as glycating agents (Rabbani & Thornalley, 2011). Minimal modification by methylglyoxal renders LDL particles with atherogenic properties, including binding to PG and susceptibility to aggregation (Rabbani, 2011). LDL particles with this low level of modification are increased in diabetic patients (Rabbani, 2010).

(Fig. 1).

Modified Forms of LDL in Plasma 455

the content in lipoperoxides decreased (De Castellarnau, 2000; Demuth, 1996; Sevanian, 1997). Discrepancies regarding the increased content in oxidized lipids in LDL(-), continue; some authors do not find differences compared to native LDL (Benitez, 2007b; Demuth, 1996; Sanchez-Quesada, 2003), and others report increased amounts of oxidized lipids in LDL(-) (Asatryan, 2003; Sevanian, 1997; Ziouzenkova, 2002). But in any case, this level of lipoperoxidation is closer to minimally oxidized LDL than to extensively oxidized LDL suggesting that alternative mechanisms should be involved in LDL(-) formation

Fig. 1. Formation of LDL(-). The modification of native LDL by one or several mechanisms

inflammatory, proliferative and apoptotic lipids and non-apoB proteins in LDL(-), as well as

LDL is a heterogeneous mixture of particles that differ in size (24-28 nm of diameter) and density (1.019-1.063 g/ml). Small-dense particles have fewer lipid molecules while largebuoyant particles have more. It is known that LDL particles at both extremes of the density range have increased electronegative charge compared with mid-density particles (Lund-Katz, 1998). It has been proposed that the origin of LDL(-) could be related with the impairment of the catabolic cascade that transforms VLDL-to-IDL-to-LDL in blood (Sanchez-Quesada, 2004). Such impairment leads to the formation of small-dense or largebuoyant LDL particles. In agreement with this, LDL(-) is most abundant (>80% of LDL(-)) in small-dense particles in normolipemic subjects. Interestingly, in hypercholesterolemic and hypertriglyceridemic patients, LDL(-) are also abundant large-buoyant particles (Sanchez-Quesada, 2002). Another consequence of impaired LDL catabolism is the presence of non-apoB proteins in LDL. Theoretically, the protein moiety of LDL consists of a single copy of a very large protein, apoB. However, it is known that some particles of LDL also contain other proteins. In native LDL, the content of non-apoB proteins is less than 1%. In contrast, LDL(-) contains up to 5% of non-apoB proteins (Bancells, 2010a;

alters the composition of LDL surface. These alterations include the increase of

structural abnormalities in apoB.

**4.1.1 Impaired catabolism of LDL**

Yang, 2003).

A more advanced form of glLDL is AGE-LDL, in which AGE are formed due to autooxidation of Amadori adducts yielding a number of products, such as carboxymethyl lysine or pentosyl lysine (Brownlee, 2000). AGE-LDL has atherogenic characteristics that are similar to oxLDL, probably because their oxidized lipid content is similar. Although it was generally considered that AGE-LDL was generated in the arterial wall this modified form of LDL has also been detected in blood (Lopes-Virella & Virella, 2010). AGE-LDL, like other AGE-containing proteins, is recognized in circulation by RAGE. RAGE activation stimulates cytokyne and growth factors release (Ramasamy, 2009). An excess of stimulation (i.e. an excess of AGE-LDL) plays an essential role in atherogenic alterations.

#### **3.3 Carbamylated LDL (ca-LDL)**

Carbamylation of proteins is a post-translational modification in which amine-containing residues react with cyanate, a compound that derives from urea or from thiocyanate. This modification is relatively frequent in patients with chronic uremia or in heavy smokers. Both situations are closely related to increased cardiovascular risk and carbamylated LDL (ca-LDL) is increased in the plasma in both groups of subjects (Basnakian, 2010). Ca-LDL is recognized by SR, promotes monocyte adhesion to endothelium, stimulates cell proliferation and causes cell injury (Apostolov, 2007; Carracedo, 2011).

#### **3.4 Desialylated LDL (ds-LDL)**

Native LDL has high content of sialic acid in the carbohydrate chains attached to apoB. Tertov and colleagues isolated a fraction of desialylated LDL from plasma (Tertov, 1990). This fraction, which was increased in patients with advanced atherosclerosis, induced foam cell formation in cultured smooth muscle cells and presented inflammatory properties (Orekhov, 1991). They suggested that low sialic acid in LDL was a cardiovascular risk factor (Ruelland, 1993) but this idea was not supported by other authors (Cerne, 2002). Later studies revealed that desialylated LDL was oxidized and that the loss of sialic acid was a consequence of oxidative modification (Tertov, 1995).

#### **4. Electronegative LDL, a pool of modified LDL in blood**

A common characteristic of most of the previously described modifications is an increase of the negative electric charge. Taking advantage of this property, Avogaro and co-workers fractionated total LDL from human plasma by anion-exchange chromatography, into two subfractions, a major subfraction of native LDL and an electronegatively-charged fraction of LDL (LDL(-)) (Avogaro, 1988). In this first report, LDL(-) accounted for 5-20% of total LDL in normolipemic subjects and presented a number of atherogenic characteristics, including impaired binding to LDLr, high aggregation level, capacity to induce cholesterol accumulation in macrophages and higher conjugated diene (a by-product of lipid peroxidation) content. Since then, a number of studies have tried to elucidate the physicochemical and biological characteristics of LDL(-) and its relationship with atherosclerosis.

#### **4.1 Origin of LDL(-) – Discrepancies regarding the oxidative origin**

Early studies focused on the physico-chemical characteristics LDL(-) concluded, in accordance with the high content of oxidized lipids, that LDL(-) was the in vivo counterpart of in vitro oxidized LDL (Cazzolato, 1991). However, as isolation procedures improved and measures to prevent modification increased, the proportion of LDL(-) and

A more advanced form of glLDL is AGE-LDL, in which AGE are formed due to autooxidation of Amadori adducts yielding a number of products, such as carboxymethyl lysine or pentosyl lysine (Brownlee, 2000). AGE-LDL has atherogenic characteristics that are similar to oxLDL, probably because their oxidized lipid content is similar. Although it was generally considered that AGE-LDL was generated in the arterial wall this modified form of LDL has also been detected in blood (Lopes-Virella & Virella, 2010). AGE-LDL, like other AGE-containing proteins, is recognized in circulation by RAGE. RAGE activation stimulates cytokyne and growth factors release (Ramasamy, 2009). An excess of stimulation (i.e. an

Carbamylation of proteins is a post-translational modification in which amine-containing residues react with cyanate, a compound that derives from urea or from thiocyanate. This modification is relatively frequent in patients with chronic uremia or in heavy smokers. Both situations are closely related to increased cardiovascular risk and carbamylated LDL (ca-LDL) is increased in the plasma in both groups of subjects (Basnakian, 2010). Ca-LDL is recognized by SR, promotes monocyte adhesion to endothelium, stimulates cell proliferation

Native LDL has high content of sialic acid in the carbohydrate chains attached to apoB. Tertov and colleagues isolated a fraction of desialylated LDL from plasma (Tertov, 1990). This fraction, which was increased in patients with advanced atherosclerosis, induced foam cell formation in cultured smooth muscle cells and presented inflammatory properties (Orekhov, 1991). They suggested that low sialic acid in LDL was a cardiovascular risk factor (Ruelland, 1993) but this idea was not supported by other authors (Cerne, 2002). Later studies revealed that desialylated LDL was oxidized and that the loss of sialic acid was a

A common characteristic of most of the previously described modifications is an increase of the negative electric charge. Taking advantage of this property, Avogaro and co-workers fractionated total LDL from human plasma by anion-exchange chromatography, into two subfractions, a major subfraction of native LDL and an electronegatively-charged fraction of LDL (LDL(-)) (Avogaro, 1988). In this first report, LDL(-) accounted for 5-20% of total LDL in normolipemic subjects and presented a number of atherogenic characteristics, including impaired binding to LDLr, high aggregation level, capacity to induce cholesterol accumulation in macrophages and higher conjugated diene (a by-product of lipid peroxidation) content. Since then, a number of studies have tried to elucidate the physicochemical and biological characteristics of LDL(-) and its relationship with atherosclerosis.

Early studies focused on the physico-chemical characteristics LDL(-) concluded, in accordance with the high content of oxidized lipids, that LDL(-) was the in vivo counterpart of in vitro oxidized LDL (Cazzolato, 1991). However, as isolation procedures improved and measures to prevent modification increased, the proportion of LDL(-) and

excess of AGE-LDL) plays an essential role in atherogenic alterations.

and causes cell injury (Apostolov, 2007; Carracedo, 2011).

consequence of oxidative modification (Tertov, 1995).

**4. Electronegative LDL, a pool of modified LDL in blood**

**4.1 Origin of LDL(-) – Discrepancies regarding the oxidative origin**

**3.3 Carbamylated LDL (ca-LDL)**

**3.4 Desialylated LDL (ds-LDL)**

the content in lipoperoxides decreased (De Castellarnau, 2000; Demuth, 1996; Sevanian, 1997). Discrepancies regarding the increased content in oxidized lipids in LDL(-), continue; some authors do not find differences compared to native LDL (Benitez, 2007b; Demuth, 1996; Sanchez-Quesada, 2003), and others report increased amounts of oxidized lipids in LDL(-) (Asatryan, 2003; Sevanian, 1997; Ziouzenkova, 2002). But in any case, this level of lipoperoxidation is closer to minimally oxidized LDL than to extensively oxidized LDL suggesting that alternative mechanisms should be involved in LDL(-) formation (Fig. 1).

Fig. 1. Formation of LDL(-). The modification of native LDL by one or several mechanisms alters the composition of LDL surface. These alterations include the increase of inflammatory, proliferative and apoptotic lipids and non-apoB proteins in LDL(-), as well as structural abnormalities in apoB.

#### **4.1.1 Impaired catabolism of LDL**

LDL is a heterogeneous mixture of particles that differ in size (24-28 nm of diameter) and density (1.019-1.063 g/ml). Small-dense particles have fewer lipid molecules while largebuoyant particles have more. It is known that LDL particles at both extremes of the density range have increased electronegative charge compared with mid-density particles (Lund-Katz, 1998). It has been proposed that the origin of LDL(-) could be related with the impairment of the catabolic cascade that transforms VLDL-to-IDL-to-LDL in blood (Sanchez-Quesada, 2004). Such impairment leads to the formation of small-dense or largebuoyant LDL particles. In agreement with this, LDL(-) is most abundant (>80% of LDL(-)) in small-dense particles in normolipemic subjects. Interestingly, in hypercholesterolemic and hypertriglyceridemic patients, LDL(-) are also abundant large-buoyant particles (Sanchez-Quesada, 2002). Another consequence of impaired LDL catabolism is the presence of non-apoB proteins in LDL. Theoretically, the protein moiety of LDL consists of a single copy of a very large protein, apoB. However, it is known that some particles of LDL also contain other proteins. In native LDL, the content of non-apoB proteins is less than 1%. In contrast, LDL(-) contains up to 5% of non-apoB proteins (Bancells, 2010a; Yang, 2003).

Modified Forms of LDL in Plasma 457

Fig. 2. Atherogenic properties of LDL(-). Modified lipids in LDL(-) surface have

formation, but could mediate the signaling of endothelial apoptosis.

and impairs its plasma clearance.

**4.2.1 Binding to receptors**

**4.2.2 Inflammatory activity**

inflammatory, proliferative and apoptotic capacity. Aggregation, which is induced by the formation of ceramide, favours the binding to PGs and increases subendothelial retention. The alteration of apoB conformation in LDL(-) induces a partial loss of affinity to the LDLr

LDL(-) presents a partial loss of affinity to the LDLr, a property that could lengthen its halflife in blood (Benitez, 2004b). It was initially believed that this was due to the derivatization of lysines in apoB involved in receptor recognition, in a mechanism similar to oxLDL (MDA-Lys) or glLDL (glucose-Lys). However, recent studies have shown that lysines in LDL(-) are not derivatized but have an altered ionization state due to differences in the conformation of apoB (Blanco, 2010). Regarding SR, LDL(-) binds differently to distinct types of SR. The increment of electronegativity in LDL(-) is not sufficient to allow its binding to SRA in macrophages (Benitez, 2004b). However, it has been reported that LDL(-) binds to another SR, LOX-1, in endothelial cells (Lu, 2009). This binding does not promote foam cell

LDL(-) has the ability to activate the transcription factors NF-kB, AP-1 and PPAR, inducing the expression of a number of inflammatory molecules in endothelial cells (Abe, 2007; Benitez, 2006; De Castellarnau, 2000; Ziouzenkova, 2003). These molecules include cytokines (IL6) chemokines (IL8, MCP-1, GRO), vascular adhesion molecules (VCAM) and growth factors (GM-CSF, PDGF). Interestingly, LDL(-) also induces the paradoxical expression of the anti-inflammatory cytokine IL10 in lymphocytes and monocytes (Benitez, 2007a). It has been suggested that IL10 production could be a mechanism to control an excessive inflammatory response limiting the extent of injury. LDL(-) could also play a role in angiogenesis modulation since it stimulates vascular endothelial growth factor (VEGF) expression and inhibits the release of the matrix metalloproteinases MMP2 and MMP9 (Lu,

#### **4.1.2 Lipolysis**

Some characteristics of LDL(-), such as high LPC and NEFA content, suggest that a possible mechanism of formation could be mediated by phospholipases. It has been described that in vitro modification of LDL with sPLA2 renders modified particles that mimics some properties of LDL(-) (Asatryan, 2005; Benitez, 2004a; Benitez, 2004b). On the other hand, PAF-AH, which has a 5-10-fold higher activity in LDL(-) than in native LDL, could also play a role in increasing the content of LPC and NEFA and in the generation of LDL(-) (Gaubatz, 2007; Sanchez-Quesada, 2005). Some type of SMase activity could also be involved in LDL(-) generation since a minor subfraction of LDL(-) is aggregated (Bancells, 2010b). It has been reported that LDL(-) has an intrinsic phospholipase C-like (PLC-like) activity that degrades with high affinity both SM and LPC (Bancells, 2008). The origin of such activity is currently unknown, but it could be due to conformational changes in apoB. One or a combination of these phospholipolityc activities could be involved in the formation of LDL(-).

#### **4.1.3 Content of non-esterified fatty acids (NEFA)**

NEFA are transported in blood mainly associated to albumin. However, in some situations that increase NEFA or decrease albumin there is a partition of NEFA towards other proteins, including lipoproteins. In posprandial lipemia or when high energy is required (such as during heavy exercise) the increase of NEFA in blood increases LDL(-) proportion (Benitez, 2002). NEFA content in LDL(-) is three to four-fold higher than in native LDL (Benitez, 2004a; De Castellarnau, 2000; Demuth, 1996). In this context, it has been reported that the main determinant of the electronegativity of LDL(-) is NEFA (Gaubatz, 2007).

#### **4.1.4 Non-enzymatic glycosylation**

It would seem reasonable to consider that glLDL contributes in part to the pool of LDL(-), especially in diabetic patients. However, most glycated LDL particles isolable by affinity chromatography do not have a sufficient negative charge to be isolated with LDL(-) and the content of glLDL in LDL(-) is similar to that in native LDL (Benitez, 2007b; Sanchez-Quesada, 2005).

#### **4.1.5 Hemoglobin derivatization and carbamylation**

Patients with severe renal failure have a high proportion of LDL(-) (Asatryan, 2003). It was reported that LDL from these patients suffered a cross-linking with hemoglobin, rendering a particle with increased negative charge (Ziouzenkova, 2002). Recent studies on carbamylated LDL, however, suggest that carbamylation could underlie the high proportion of LDL(-) in patients with severe renal disease (Apostolov, 2010).

#### **4.2 Biological properties of LDL(-)**

Whatever the mechanism involved in its formation LDL(-), has several potentially atherogenic properties. These include abnormal binding to receptors, inflammatory and cytotoxic properties, high susceptibility to aggregation and increased affinity to PG (Fig. 2).

Some characteristics of LDL(-), such as high LPC and NEFA content, suggest that a possible mechanism of formation could be mediated by phospholipases. It has been described that in vitro modification of LDL with sPLA2 renders modified particles that mimics some properties of LDL(-) (Asatryan, 2005; Benitez, 2004a; Benitez, 2004b). On the other hand, PAF-AH, which has a 5-10-fold higher activity in LDL(-) than in native LDL, could also play a role in increasing the content of LPC and NEFA and in the generation of LDL(-) (Gaubatz, 2007; Sanchez-Quesada, 2005). Some type of SMase activity could also be involved in LDL(-) generation since a minor subfraction of LDL(-) is aggregated (Bancells, 2010b). It has been reported that LDL(-) has an intrinsic phospholipase C-like (PLC-like) activity that degrades with high affinity both SM and LPC (Bancells, 2008). The origin of such activity is currently unknown, but it could be due to conformational changes in apoB. One or a combination of these phospholipolityc activities could be

NEFA are transported in blood mainly associated to albumin. However, in some situations that increase NEFA or decrease albumin there is a partition of NEFA towards other proteins, including lipoproteins. In posprandial lipemia or when high energy is required (such as during heavy exercise) the increase of NEFA in blood increases LDL(-) proportion (Benitez, 2002). NEFA content in LDL(-) is three to four-fold higher than in native LDL (Benitez, 2004a; De Castellarnau, 2000; Demuth, 1996). In this context, it has been reported that the main determinant of the electronegativity of LDL(-) is NEFA

It would seem reasonable to consider that glLDL contributes in part to the pool of LDL(-), especially in diabetic patients. However, most glycated LDL particles isolable by affinity chromatography do not have a sufficient negative charge to be isolated with LDL(-) and the content of glLDL in LDL(-) is similar to that in native LDL (Benitez, 2007b; Sanchez-

Patients with severe renal failure have a high proportion of LDL(-) (Asatryan, 2003). It was reported that LDL from these patients suffered a cross-linking with hemoglobin, rendering a particle with increased negative charge (Ziouzenkova, 2002). Recent studies on carbamylated LDL, however, suggest that carbamylation could underlie the high proportion

Whatever the mechanism involved in its formation LDL(-), has several potentially atherogenic properties. These include abnormal binding to receptors, inflammatory and cytotoxic properties, high susceptibility to aggregation and increased affinity to PG

**4.1.2 Lipolysis**

(Gaubatz, 2007).

Quesada, 2005).

(Fig. 2).

involved in the formation of LDL(-).

**4.1.4 Non-enzymatic glycosylation**

**4.2 Biological properties of LDL(-)**

**4.1.3 Content of non-esterified fatty acids (NEFA)**

**4.1.5 Hemoglobin derivatization and carbamylation**

of LDL(-) in patients with severe renal disease (Apostolov, 2010).

Fig. 2. Atherogenic properties of LDL(-). Modified lipids in LDL(-) surface have inflammatory, proliferative and apoptotic capacity. Aggregation, which is induced by the formation of ceramide, favours the binding to PGs and increases subendothelial retention. The alteration of apoB conformation in LDL(-) induces a partial loss of affinity to the LDLr and impairs its plasma clearance.

#### **4.2.1 Binding to receptors**

LDL(-) presents a partial loss of affinity to the LDLr, a property that could lengthen its halflife in blood (Benitez, 2004b). It was initially believed that this was due to the derivatization of lysines in apoB involved in receptor recognition, in a mechanism similar to oxLDL (MDA-Lys) or glLDL (glucose-Lys). However, recent studies have shown that lysines in LDL(-) are not derivatized but have an altered ionization state due to differences in the conformation of apoB (Blanco, 2010). Regarding SR, LDL(-) binds differently to distinct types of SR. The increment of electronegativity in LDL(-) is not sufficient to allow its binding to SRA in macrophages (Benitez, 2004b). However, it has been reported that LDL(-) binds to another SR, LOX-1, in endothelial cells (Lu, 2009). This binding does not promote foam cell formation, but could mediate the signaling of endothelial apoptosis.

#### **4.2.2 Inflammatory activity**

LDL(-) has the ability to activate the transcription factors NF-kB, AP-1 and PPAR, inducing the expression of a number of inflammatory molecules in endothelial cells (Abe, 2007; Benitez, 2006; De Castellarnau, 2000; Ziouzenkova, 2003). These molecules include cytokines (IL6) chemokines (IL8, MCP-1, GRO), vascular adhesion molecules (VCAM) and growth factors (GM-CSF, PDGF). Interestingly, LDL(-) also induces the paradoxical expression of the anti-inflammatory cytokine IL10 in lymphocytes and monocytes (Benitez, 2007a). It has been suggested that IL10 production could be a mechanism to control an excessive inflammatory response limiting the extent of injury. LDL(-) could also play a role in angiogenesis modulation since it stimulates vascular endothelial growth factor (VEGF) expression and inhibits the release of the matrix metalloproteinases MMP2 and MMP9 (Lu,

Modified Forms of LDL in Plasma 459

to-LDL cascade. High NEFA could be a consequence of the increase in NEFA concentration in plasma (posprandial lipemia, intense exercise) (Benitez, 2002; Ursini, 1998), or it could come from lipolysis mediated by phospholipases (Benitez, 2004a). This latter possibility

The most abundant proteins in LDL(-) are apoA-I (0.15 molecules/particle of LDL(-)), apoE (0.22), apoC-III (0.37) and apoA-II (0.14), with a content 3-5 fold higher in LDL(-) than in native LDL (Bancells, 2010a). The role of these proteins is unclear but their relevance is probably low. However, other proteins whose absolute content in LDL(-) is lower, such as apoF (0.06 molecules/particle), apoJ (0.01) or PAF-AH (0.004) (Bancells, 2010a; Yang, 2007), but their relative content compared with native LDL is 10, 20 and 100-fold higher, respectively, could have a much more relevant role. ApoF is the physiological inhibitor of cholesteryl ester transfer protein (Morton, 2008), a protein that regulates the catabolism of the VLDL-IDL-LDL cascade. It could therefore be one cause of impaired LDL(-) maturation. ApoJ (also known as clusterin) is an extracellular chaperone that binds to hydrophobic unfolded proteins, favoring their extracellular clearance (Oda, 1995). ApoJ binds mainly to aggregated LDL(-), supporting the presence of misfolded apoB in this subfraction. The role of PAF-AH in LDL(-) would be to deactivate oxidized phospholipids, but its undesirable effect would be the formation of LPC and short-chain NEFA. It has been suggested that the PLC-like activity present in LDL(-) could act in cooperation with PAF-AH degrading LPC (Bancells, 2010b). Therefore, this would be a mechanism to limit the deleterious effects

The proportion of LDL(-) is increased in a number of pathologic situations having a cardiovascular risk. Familial hypercholesterolemia and hypertriglyceridemia present a proportion of LDL(-) 3-5 fold higher than normolipemic healthy subjects (Sanchez-Quesada, 2002; Sanchez-Quesada, 1999). These results were obtained using ultracentrifugation plus anion-exchange chromatography, both being laborious and time-consuming techniques. This limits their use for routine analysis of lipoprotein profiles. However, more reliable techniques for rapid analysis have recently been developed, including capillary electrophoresis and ELISA (Santo Faulin Tdo, 2008; Zhang, 2009; Zhang, 2008). These methods have confirmed previous data obtained by anion-exchange chromatography. Statin therapy decreases the proportion of LDL(-) but the process is not parallel to the lipidlowering effect. This is because total LDL cholesterol decreases very rapidly (in 2 weeks) whereas LDL(-) decreases more slowly (in up to six months) (Sanchez-Quesada, 1999). This suggests that LDL(-) generation not only depends on lipid metabolism but also on other

Both type 1 and type 2 diabetics have a high proportion of LDL(-) (Moro, 1998; Sanchez-Quesada, 1996; Sanchez-Quesada, 2001; Zhang, 2005). This would suggest that nonenzymatic glycosylation could be involved. However, insulin therapy decreases LDL(-) in type 1 diabetes but not in type 2 diabetes. The different response to insulin treatment has been attributed to differences in the systemic inflammation level, which is higher in type 2 patients. This agrees with the finding that pre-diabetic insulin-resistant subjects with high systemic inflammation also have increased LDL(-) proportion (Zhang, 2005). Therefore,

would be the same mechanism of increased LPC content in LDL(-).

exerted by minimal LDL oxidation on vascular cells.

**4.4 Association of LDL(-) with cardiovascular risk**

factors such as chronic inflammation.

**4.3.3 Proteins**

2008; Tai, 2006). The specific molecules that mediate these actions are not well defined; LPC and NEFA probably play a major role, but the action of oxidized lipids remains under discussion (Abe, 2007; Benitez, 2004a; Chen, 2004). If they are present in LDL(-) they could stimulate inflammatory responses, but it is also possible that high PAF-AH activity in LDL (-) degrades readily oxidized phospholipids (Benitez, 2003). Their degradation products, LPC and short-chain NEFA, would therefore be responsible for triggering inflammation.

#### **4.2.3 Cytotoxicity and apoptosis**

LDL(-) induces citotoxicity and apoptosis in endothelial cells and macrophages by different signaling pathways. The mechanisms involved are well defined, especially in endothelial cells, where LOX-1 signaling inhibits fibroblast growth factor 2 (FGF2) transcription and Akt phosphorilation (Chen, 2003; Chen, 2007; Lu, 2008; Tang, 2008; Yang, 2007). In contrast, macrophage apoptosis involves the Fas/FasL signaling pathway and the activation of the transcription factor Nrf2 (Pedrosa, 2010).

#### **4.2.4 Binding to proteoglycans**

LDL(-) and PG present a high affinity for binding (Bancells, 2009). This could favor the subendothelial retention of LDL(-) and trigger the inflammatory response. Some characteristics in LDL(-) are involved in this higher affinity. Aggregation of lipoproteins favors PG binding, and LDL(-) has a high tendency to aggregate (Bancells, 2010b). In fact, a subfraction of aggregated LDL(-) is responsible for the binding to PG (Bancells, 2009). This subfraction has an abnormal conformation that exposes an epitope in apoB, known as site Ib, that is an alternative binding site to PGs (Bancells, 2011).

#### **4.3 Physico-chemical characteristics of LDL(-)**

#### **4.3.1 Structure**