Oxidized LDL and NO synthesis—Biomarkers of endothelial dysfunction and ageing

Daniela Gradinaru; Claudia Borsa; Cristina Ionescu; Gabriel Ioan Prada

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Mechanisms of Ageing

Mechanisms of Ageing and Development xxx (2015) xxx-xxx

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ELSEVIER

Mechanisms of Ageing and Development

journal homepage www.elsevier.com/locate/mechagedev

1 Oxidized LDL and NO synthesis—Biomarkers of endothelial

2 dysfunction and ageing

3 Q2 Daniela Gradinaru ab*, Claudia Borsaa, Cristina Ionescua, Gabriel loan Pradaac

4 a Ana Aslan National Institute of Gerontology and Geriatrics, 9 Caldarusani Street, Sector 1, P.O. Box 2-4, 011241 Bucharest, Romania

5 b Carol Davila University of Medicine and Pharmacy, Faculty of Pharmacy, Department of Biochemistry, 6 Taian Vuia Street,

6 Sector2, 020956 Bucharest, Romania

7 c Carol Davila University of Medicine and Pharmacy, Faculty of Medicine, Department of Geriatrics and Gerontology, 37 Dionisie Lupu Street,

8 Sector2, 020021 Bucharest, Romania

ARTICLE INFO

ABSTRACT

Article history: Available online xxx

Keywords: Oxidized LDL Nitric oxide Ageing Biomarker

Endothelial dysfunction

Oxidized LDL (oxLDL) and nitric oxide (NO) exert contradictory actions within the vascular endothelium microenvironment influencing key events in atherogenesis. OxLDL and NO are so far regarded as representative parameters of oxidative stress and endothelial dysfunction, new targets in prevention, diagnosis and therapy of cardiovascular diseases, and also as candidate biomarkers in evaluating the human biological age. The aim of this review is to explore recent literature on molecular mechanisms and pathophysiological relationships between LDL oxidation, NO synthesis and vascular endothelium function/dysfunction in ageing, focusing on the following aspects: (1) the impact of metabolic status on both LDL oxidation and NO synthesis in relation with oxidative stress, (2) the use of oxidized LDL and NO activity as biomarkers in human studies reporting on cardiovascular outcomes, and (3) evidences supporting the importance of oxidized LDL and NO activity as relevant biomarkers in vascular ageing and age-related diseases.

22 "longevity is a vascular question. A man is as old as his arteries"

23 William Osler, 1892

24 "It has been said that one is as old as one's arteries. In view of

25 the supreme importance of endothelium in arterial function, I

26 should like to modify... this statement by saying that

27 one is as old as one's endothelium."

28 Rudolf Altschul, 1954

Abbreviation: LDL, low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; LOX-1, lectin-like oxidized LDL receptor-1; FFA, free fatty acids; ApoB-100, apolipoprotein B-100; H2O2, hydrogen peroxide; SOD, superoxide dismutase; ROS, reactive oxygen species; O2^-, superoxide anion; HO% hydroxyl radical; LO% alkoxyl radical; LOO% peroxyl radical; ONOO-, peroxynitrite; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; NF-kB, nuclear factor kB; p66Shc, 66-kDa isoform of Shc adaptor protein; BH4, tetrahydrobiopterin; Dyslip, dyslipidemia; Hypergly, hyperglycemia; IR, insulin resistance.

* Corresponding author at: Ana Aslan National Institute of Gerontology and Geriatrics, 9 Caldarusani Street, Sector 1, P.O. Box 2-4, 011241 Bucharest, Romania. Tel.: +40 744339630; fax: +40 212231480.

E-mail address: daniela.gradinaru@umf.ro (D. Gradinaru).

1. Introduction 29

The vascular endothelium, with its broad spectrum of paracrine 30

and autocrine functions, can be regarded as a multifunctional organ 31

and "chief governor" of body homeostasis. Occupying a strategic 32

location between the blood and tissues, the endothelial cells exist in 33

a "high-risk position" and react progressively to aggressive factors, 34

at first by modulation of the constitutive functions: permeabil- 35

ity (i.e., increased transcytosis of lipoproteins) and biosynthesis 36

(i.e., enhanced synthesis of the basement membrane and extra- 37

cellular matrix) (Simionescu and Antohe, 2006; Sima et al., 2009). 38

Even though the endothelial cells are resourceful cells that have 39

the functional-structural attributes to adapt to the ever-changing 40

surrounding milieu, to use innate mechanisms to confront and 41

defend against insults, the ageing process induces a progressive 42

failure of protective mechanisms, leading to vascular alterations 43

(Dantas et al., 2012). It is becoming evident that ageing results in 44

well-defined phenotypic changes and, as a consequence, a height- 45

ened susceptibility of the cardiovascular system to diseases, even 46

in absence of traditional risk factors (e.g., hypertension, hyper- 47

cholesterolemia, diabetes, and smoking). Moreover, age-related 48

alterations in cellular homeostatic mechanisms also impact the 49

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2 D. Gradinaru et al. / Mechanisms of Ageing and Development xxx (2015)xxx-xxx

50 aged vasculature making it more liable to the damaging effects of prevents abnormal constriction (vasospasm) of the coronary arter- 113

51 the traditional pathophysiological conditions (Ungvari et al., 2010). ies, which favors intraluminal clot formation and inhibits platelets 114

52 Endothelial dysfunction, a systemic pathological state defined aggregation and adhesion to endothelium surface (anti-thrombotic 115

53 as imbalance between vasodilating and vasoconstricting com- effect); (2) inhibits the release and action of the vasoconstrictor 116

54 pounds produced by and acting on the endothelium, precedes and mitogenic peptide endothelin-1, decreases endothelial perme- 117

55 development of atherosclerosis, leading to reduced vasodilation, ability and reduces vessel tone, reducing lipoproteins flux into the 118

56 pro-inflammatory and pro-thrombotic states (Deanfield et al., vessel wall, vascular smooth muscle cell proliferation and migra- 119

57 2007). tion (cell growth inhibition and anti-atherogenic effect) (Verma 120

58 In the last three decades, oxidation of low density lipoproteins et al., 2003); (3) inhibits the NF-kB activation and determines 121

59 (LDL) and nitric oxide (NO) synthesis have been discovered in paral- a disruption of pro-inflammatory cytokine - induced signaling 122

60 lel, studied extensively and considered as important mechanisms pathways; NO reduces the endothelial expression of intracellular 123

61 contributing to endothelial dysfunction, vascular ageing and dis- adhesion molecule-1 (1CAM-1), vascular cell adhesion molecule-1 124

62 ease. Oxidized LDL and NO exert contradictory actions within the (VCAM-1), and endothelial leukocyte adhesion molecule-1(ELAM) 125

63 vascular endothelium microenvironment influencing key events (anti-inflammatory effect) (Rubio and Morales-Segura, 2004); (4) 126

64 in endothelial dysfunction and atherogenesis such as: leukocyte suppresses endothelial cell apoptosis and preserves the endothe- 127

65 adhesion, platelet aggregation and vascular smooth-muscle cell lial progenitor cell (EPC) function and regulation of tissue energy 128

66 proliferation and migration. While oxidized LDL (oxLDL)- an oxida- metabolism (anti-apoptotic, vasoprotective and cardioprotective 129

67 tive stress biomarker has been identified as a non-traditional, effect) (Ungvari et al., 2010); (5) controls mitochondrial oxygen 130

68 pro-atherogenic emerging risk factor for coronary heart disease, consumption and maintains cellular redox state. At physiological 131

69 NO is a free radical signal-transducing molecule that maintains levels, NO is a highly reactive free radical that can also attenuate the 132

70 the vasodilating tone, modulates in vitro lipid peroxidation reac- metal/peroxide oxidative chemistry, as well as lipid peroxidation, 133 71Q4 tions and alters pro-inflammatrory gene expression (Holvoet et al., and may limit oxidative injury to mammalian cells (antioxidative 134

72 2008a,b; Borsa et al., 2012). effect) (Wink et al., 2001; Müller et al., 2004). 135

73 The LDL oxidation and NO activity are so far regarded as Oxidative stress is one of the causative factors involved in ageing 136

74 representative parameters of oxidative stress and endothelial dys- and pathogenesis of cardiovascular disease. While the chrono- 137

75 function, new targets in prevention, diagnosis and therapy of logical age is classified as major nonmodifiable risk factor for 138

76 cardiovascular diseases, and also as candidate biomarkers in eval- cardiovascular disease, the majority of modifiable atherosclerotic 139

77 uating the human biological age (Rodriguez-Manas et al., 2009; risk factors like hypertension, dyslipidemia, chronic hyperglycemia 140

78 Verhoye and Langlois, 2009; Maiolino et al., 2013a,b; Zuliani et al., and cigarette smoking are real harmful stimuli that accelerate dis- 141

79 2012; Paiket al., 2013; Burkle et al., 2015; Moreno-Villanueva et al., ease progression by augmenting the production of reactive oxygen 142

80 2015a,b; Capri et al., 2015; Baur et al., 2015). species (ROS) (Nilsson, 2008). 143

81 The aim of this review is to explore recent literature on molec- The damaging effects of oxidative stress on cardiovascular sys- 144

82 ular mechanisms and pathophysiological relationships between tem determine endothelial dysfunction through reduction in nitric 145

83 LDL oxidation, NO synthesis and vascular endothelium func- oxide (NO) synthesis and bioavailability, inflammatory response, 146

84 tion/dysfunction in ageing, focusing on the following aspects: (1) and lipid peroxidation. The endothelium is continuously exposed 147

85 the impact of metabolic status on both LDL oxidation and NO to various physiological molecules that may have a direct impact 148

86 synthesis in relation with vascular oxidative stress, (2) the use on nitric oxide actions (Chikani et al., 2004). Plasma lipoproteins, 149

87 of oxidized LDL and NO activity as representative biomarkers in by virtue of their close interactions with endothelial cells in the 150

88 human studies reporting on cardiovascular outcomes, and (3) evi- vasculature and the susceptibility of their surface lipids to oxida- 151

89 dences supporting the importance of oxidized LDL and NO activity tive modification, are perfect biological "sensors" of oxidative stress 152

90 as relevant biomarkers in vascular ageing and age-related diseases. in the arterial wall (Le, 2015). LDLs as main blood cholesterol car- 153

riers, containing relevant amount of polyunsaturated fatty acids 154

(PUFAs) - major substrate for lipid peroxidation, are among various 155

91 2. Relationships between NO synthesis and LDL oxidation molecular targets the most affected by the oxidative stress asso- 156

92 in endothelial dysfunction ciated with metabolic imbalance (hyperlipidemia, hyperglycemia, 157

insulin resistance). Therefore, the oxidative modification hypoth- 158

93 In the endothelial microenvironment, concurrently, a variety of esis of atherosclerosis recognizes the crucial role of oxLDL as a 159

94 substances that influence endothelial function have been recog- byproduct of LDLs exposure to ROS (Steinberg and Witzum, 2010). 160

95 nized, but among them NO and oxLDL are the best characterized key OxLDL promotes endothelial dysfunction and contributes to the 161

96 players sharing significant antagonistic roles, and being involved in atherosclerotic plaque formation, progression and destabilization, 162

97 all phases of atherogenesis (Borsa et al., 2012). by several mechanisms described in numerous recent review arti- 163

98 Nitric oxide, a non-eicosanoid component of endothelial- cles (Maiolino et al., 2013a,b; Pirillo et al., 2013; Xu et al., 2012; 164

99 derived relaxation factor (EDRF) is the most important vasodilating Le, 2015): (1) chemotactic recruitment, activation, and prolifera- 165

100 molecule being continuously synthesized bythe endothelial consti- tion of monocytes/macrophages in the arterial wall, through the 166

101 tutive isoform of nitric oxide synthase (eNOS and NOS111) under the induction of the expression of intercellular adhesion molecule-1 167

102 action of different neurohumoral mediators such as acetylcholine (1CAM-1) and vascular-cell adhesion molecule-1 (VCAM-1), thus 168

103 and circulating hormones (catecholamines, vasopressin and aldos- stimulating their binding to endothelial cells; (2) its identifica- 169

104 terone), plasma constituents (thrombin, sphingosine 1-phosphate), tion and rapid uptake by macrophages, followed by foam cells 170

105 platelet products (serotonin, adenosine diphosphate), and auta- formation; (3) stimulation of smooth muscle cells (SMCs) migra- 171

106 coids (histamine, bradykinin and prostaglandin E4) (Michel and tion and proliferation in the tunica intima, following the increase 172

107 Vanhoutte, 2010). of the expression of growth factors, such as platelet-derived growth 173

108 1n addition to maintenance of normal organ blood flow, factor (PDGF) and basic fibroblast growth factor (FGF) by endothe- 174

109 endothelium derived NO has the following pleiotropic vasoprotec- lial cells and macrophages. Subsequently, oxLDL stimulate collagen 175

110 tive, cardioprotective and anti-atherogenic effects, summarized in production by SMCs and increase secretion of matrix metallopro- 176

111 numerous review articles (Michel and Vanhoutte, 2012; Ungvari teinases 1 and 9 (MPP-1 and MPP-9) inducing SMCs apoptosis; 177

112 et al., 2010; Jin and Loscalzo, 2010; Bermudez et al., 2008): (1) (4) cytotoxicity exerted mainly on the endothelial cells, which 178

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Fig. 1. Key significant anti-atherogenic effects of nitric oxide (NO) vs. pro-atherogenic actions of oxidized LDL(oxLDL) as biomarkers involved in the endothelial dysfunction.

179 promote their apoptosis and the release in the subendothelial space

180 of lipids and lysosomal enzymes; (5) stimulation of platelet adhe-

181 sion and aggregation by decreasing endothelial production of nitric

182 oxide, increasing prostacyclin production; (6) blockage of coro-

183 nary artery relaxation (vasoconstriction) by downregulating eNOS

184 expression, by inhibiting NO and increasing endothelin production.

185 The above evidences explain the pro-inflammatory, pro-oxidant,

186 pro-thrombotic, and vasoconstrictor actions of oxLDL, accounting

187 for its global pro-atherogenic effect on vascular endothelium.

188 Consequently, NO and oxLDL are important biological mediators

189 that promote protective vs. pathogenic effects in the vasculature,

190 simultaneously and concurrently. By reducing NO synthesis and

191 bioavailability, oxLDL breaks the balance of the vessel wall and

192 results in defective endothelial-dependent vasodilation (Fig. 1).

193 Endothelial dysfunction refers to inability of the endothe-

194 lium to regulate vascular homeostasis, and essentially describes

195 "tipping" of the physiological balance in favor of vasoconstric-

196 tive, pro-inflammatory and pro-thrombotic effects that promote

197 atherosclerosis. These endothelial function abnormalities strongly

198 related to both biomarkers - NO and oxLDL, could be detected early

199 in the development of CVD, often before symptoms are clinically

200 evident (Roberts and Porter, 2013).

201 However, taking into account data derived from studies based

202 on molecular and clinical approaches these two biomarkers do not

203 seem to exert their antagonistic influence on vascular endothelium

204 with the same significance. Impairment in NO, a common feature

205 in patients with endothelial dysfunction, is considered to pre-

206 dict atherosclerosis and cardiovascular events (Ignarro and Napoli,

207 2004), whereas upregulation of oxLDL levels is a hallmark feature

208 of atherosclerosis development (Steinberg and Witzum, 2010). Ele-

209 vated levels of oxidized LDL, mainly formed within the arterial wall,

210 are commonly related to the pro-atherogenic profile (Steinberg,

211 2009).

212 Numerous reviews summarized the mechanisms of oxidative

213 stress in association with endothelial dysfunction, LDL oxidation

214 and NO degradation, with ROS being the common mechanism by

215 which different CVD risk factors trigger atherosclerosis (Cai and

216 Harrison, 2000; El Assar et al., 2012; Pirillo et al., 2013; Le, 2015).

217 On the whole, the decline in NO bioavailability is caused

218 by the cumulative effects of many factors and processes: the

219 decreased expression of the endothelial NO synthase, a reduction

220 of substrate or cofactors for eNOS, alterations of cellular signaling,

221 eNOS inhibition by asymmetric dimethyl arginine, reduced NO

production and accelerated NO degradation by hyperlipidemia, 222

chronic 223

hyperglycemia, oxidative stress and obviously, LDL oxidation. 224

Endothelial dysfunction occurs in response to vascular risk, hence 225

metabolic status may influence both ageing-related vascular 226

oxidative stress and inflammation (Borsa et al., 2012). 227

3. OxLDL and NO synthesis - oxidative stress and 228

cardiovascular risk biomarkers 229

Given the central role of the endothelium in the development 230

and clinical course of atherosclerosis, endothelial biomarkers test- 231

ing may serve as useful tools in cardiovascular risk and outcomes 232

assessments. Current evidence suggests that endothelial function 233

is an integrative marker of the net effects of damage from tradi- 234

tional and emerging risk factors on the arterial wall and its intrinsic 235

capacity for repair. These endothelial-dependent biomarkers are 236

important in the atherosclerosis initiation/progression, the disease 237

state of stable/unstable transitions, as well as clinical research out- 238

come (Deanfield et al., 2007). 239

An important step forward in clinical studies was to explore the 240

metabolic determinants of oxLDL and the relation between oxLDL 241

and endothelial function measured as endothelium NO release 242

and action. Hence, in this review we focused mainly on the inter- 243

relations of metabolic atherogenic risk factors with lipoxidative 244

stress and endothelial dysfunction biomarkers. Numerous oxida- 245

tive stress biomarkers show strong associations with the onset and 246

progression of coronary artery disease (CAD) and predict cardiovas- 247

cular events, being surrogate biomarkers and likely complementing 248

diagnostic investigations (Meisinger et al., 2005; Tsimikas et al., 249

2012; Wang et al., 2013). Particularly, the LDL particles' susceptib- 250

lity to oxidation is included among the "downstream markers" of 251

oxidative stress (Borsa et al., 2012). 252

An indirect marker of in vivo oxidation of plasma LDL is the 253

increase of the titer of autoantibodies against neo-epitopes in 254

oxLDL (Tsimikas et al., 2007) but its relevance in coronary heart 255

disease (CHD) remains controversial. By contrast, a direct, widely 256

applied sensitive immunoassay quantifying the circulating levels of 257

oxLDL uses the monoclonal antibody 4E6, directed against oxidized 258

apolipoprotein B-100 moiety of LDL. However, all the methods 259

using different antibodies to oxLDL most probably detect and mea- 260

sure the circulating minimally oxidized LDL which represents only 261 a minor fraction of LDL ranging from 0.001%, in healthy controls, to

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262 approximately 5%, in patients with acute coronary events (Holvoet

263 et al., 2008a,b). Moreover, circulating oxLDLs are strongly corre-

264 lated to LDL-cholesterol and apoB100, making it difficult to unravel

265 their independent contribution to cardiovascular risk (van der

266 Zwan et al., 2009). For this reason, it has been suggested that the

267 oxLDL/LDL-cholesterol ratio (i.e., the relative amount of oxLDLs)

268 could be the best indicator of the risk associated with oxLDLs levels.

269 Most cohort studies underline the associations between oxLDL

270 and cardiovascular events or mortality, in particular those includ-

271 ing a very high-risk population, specifically with age-related

272 chronic metabolic diseases and their complications. For an

273 overview on the association of oxLDL with cardiovascular events

274 the reader is referred to a recent review by Maiolino et al. (2013a,b).

275 In middle-aged and elderly subjects, obesity and dyslipidemia

276 are the strongest predictors of levels of oxLDL (Kopprasch et al.,

277 2002; Holvoet et al., 2004). Recently, the association between dys-

278 lipidemia and oxidation of LDL has been demonstrated in elderly

279 individuals, even in the pre-diabetic state (Gradinaru et al., 2013).

280 In this regard, an increase in immunologically detected epitopes of

281 oxLDL in subjects with abdominal obesity was reported by several

282 authors (Couillard et al., 2005; Weinbrenner et al., 2006; Njajou

283 et al., 2009; Babakr et al., 2014). Thus, higher levels of oxLDL

284 were associated with increased incidence of metabolic syndrome

285 (MetS) overall, as well as its components of abdominal obesity,

286 hyperglycemia and hypertriglyceridemia in the population-based,

287 prospective, observational study CARDIA (The Coronary Artery Risk

288 Development in Young Adults) (Holvoet et al., 2008a,b). More-

289 over, oxLDL was suggestively associated with a greater prevalence

290 of internal carotid intimal-medial thickness (IMT) and detectable

291 coronary artery calcium (CAC) in a 997 participants (aged 45-84

292 years) of MESA Study (Multi-Ethnic Study of Atherosclerosis) with-

293 out clinical CAD. However, this study pointed out that metabolic

294 abnormalities and oxidative endothelial damage may lead to

295 atherosclerotic disease through distinct mechanisms (Vaidya et al.,

296 2011). Increased circulating oxLDL levels have been related to

297 CVD in some studies, although not always independently after

298 adjustment of classical lipid markers. In this sense, Wu et al.

299 (2006) suggested in a prospective cohort study that oxLDL as an

300 individual parameter, measured with antibody 4E6, was not an

301 independent overall predictor of CHD, being less predictive than

302 apoB and total cholesterol/HDL-cholesterol ratio. Also, in 2524

303 healthy middle-aged subjects (Asklepios study) oxLDL was affected

304 by many biological and lifestyle factors, as well as subclinical

305 atherosclerosis (Verhoye and Langlois, 2009). Autoantibodies to

306 oxLDLs (anti-oxLDLAbs) were detectable in the serum of subjects

307 with and without atherosclerosis, but it is unclear if they play a

308 pathogenic or a protective role in atherogenesis or if they are sim-

309 ply a marker of atherosclerosis. In a prospective cohort study (748

310 patients) of the GENICA study who underwent coronary angiogra-

311 phy and assessment of incident CV events at follow-up, high titer of

312 anti-oxLDLAbs is a marker which predicts long term cardiovascular

313 mortality in high risk patients (Maiolino et al., 2013a,b). Conversely,

314 also in a recent study, Zuliani et al. (2012) using data for the

315 InCHlANTI dataset (1025 older community dwelling individuals), a

316 9-year follow-up population-based study, no association emerged

317 between higher oxLDLs levels (measured with antibody 4E6) and

318 CVD/cardiac mortality, suggesting that in advanced age the prog-

319 nostic information added by oxLDLs might be negligible. This study

320 underlined also that LDL-cholesterol (LDL-C), triglycerides, and

321 HDL-cholesterol (HDL-C) are the most important determinants of

322 oxLDLs levels, indirectly suggesting an association between small

323 dense LDLs and LDLs oxidation. Interestingly, a negative association

324 between oxLDLs levels and age was found in this study popula-

325 tion, perfectly mirroring the relationship between LDL-C and age.

326 This finding suggests that after 65 years of age, although the oxida-

327 tive stress might increase with ageing, circulating oxLDLs tend to

decrease as a consequence of the progressive reduction of the sub- 328

strate. 329

It is noteworthy that circulating levels of oxLDL reflect both the 330

quantity and the "quality" of LDL particles, as cholesterol, phos- 331

pholipids, polyunsaturated fatty acids and apolipoprotein B-100 332

are the LDL substrates for oxidation, being significantly influ- 333

enced by the presence of endogenous lipophilic antioxidants: 334

a- and 7-tocopherol, P-carotene, ubiquinol-10 (Esterbauer et al., 335

1992). Therefore, the estimation of in vitro LDL susceptibility to 336

oxidation includes evaluation of specific products of the lipid 337

peroxidation chain reaction, after the exposure of isolated LDL 338

particles to a standard oxidative stress. The extent in forma- 339

tion of thiobarbituric reactive substances (TBARS), conjugated 340

dienes, lipid hydroperoxides, and aldehydes, could indicate its 341

oxidizability. 342

As indicator of NO activity, the measurement of endothelium- 343

dependent vasodilation (flow-mediated dilation method, FMD) by 344

inducing reactive hyperemia in the brachial artery, is the golden 345

standard in clinical practice (Hirata et al., 2010). NO present in the 346

circulation is resulting from endothelial and smooth muscle cells, 347

thrombocytes, leukocytes and cardiomyocytes (Pacher et al., 2007). 348

Systemic NO activity is the net result of a balance between its pro- 349

duction and its inactivation by oxygen free radicals. NO released 350

in vivo rapidly autooxidizes to yield nitrite (NO2-), which interacts 351

with oxyhemoglobin yielding nitrate (NO3 -). Because nitrite plus 352

nitrate are relatively stable compounds in blood, their levels may 353

be a biochemical index of systemic NO production being assessed 354

as NO metabolic-pathway products, NOx (NO2 - + NO3 -) (Lundberg 355

and Weitzberg, 2005). 356

Given the vast range of vasoprotective effects of NO, the term 357

endothelial dysfunction generally refers to a reduced NO bioavail- 358

ability, through decreased eNOS expression. lndeed, using these 359

procedures, it has been demonstrated that patients with con- 360

firmed CVD, and importantly, even those who carry risk factors for 361

future CVD events, had impaired endothelial-dependent vasodila- 362

tion (Roberts and Porter, 2013; van der Zwan et al., 2009). 363

Among the twenty-five recent human studies reporting on asso- 364

ciations between oxidized LDL, vascular endothelial function and 365

different cardiovascular outcomes, fifteen reported the clinical 366

assessment of the brachial artery flow-mediated dilation (FMD) 367

as a measure of endothelium (NO)-dependent response, and ten 368

studies reported the biochemical evaluation of plasma NO stable 369

metabolites, NOx (nitrite + nitrate). Their application in the assess- 370

ment of endothelial dysfunction in adult and elderly subjects, is 371

summarized in the Tables 1 and 2. 372

With respect to the contradictory actions and interrelations of 373

oxLDL and NO within the vascular endothelium, we recently pro- 374

posed the use of a new marker of endothelial dysfunction, namely 375

the ratio of oxLDL to NOx (oxLDL/NOx), which could be a more 376

accurate estimation of the in vivo cumulative implications of oxLDL 377

and NO in atherogenesis (Borsa et al., 2012; Gradinaru et al., 2012, 378

2013). Thus, in 170 elderly hyperlipidemic subjects we pointed out 379

the strong positive associations of this ratio with the atherogenic 380

index and the atherogenic risk markers: total cholesterol/HDL-C 381

and oxLDL/HDL-C ratios. These significant relationships were in 382

support to propose the ratio oxLDL/NOx as a potential marker of 383

endothelial dysfunction. The future in depth studies, should take 384

into consideration its association with clinical parameters ofvascu- 385

lar endothelial functions, to further test this new candidate marker 386

as biomarker of vascular ageing. 387

For these reasons, we used the susceptibility of LDL to oxidation 388

and NO metabolic pathway products in MARK-AGE European Study 389

to Establish Biomarkers of Human ageing, as biomarkers of oxida- 390

tive stress and endothelial dysfunction in evaluation of biological 391

age (Burkle et al., 2015; Moreno-Villanueva et al., 2015a,b; Capri 392 et al., 2015; Baur et al., 2015).

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Table 1

Human studies reporting the relationship between oxidized LDL (oxLDL) and vascular endothelial function (VEF) assessed by clinical methods, in the investigation of different cardiovascular (CV) outcomes in adult and elderly subjects.

Population under study

OxLDL tests

VEF tests

Relevant findings

References

Healthy middle-aged and older adults, 127 men and women, aged 48-77 years Hypertensive patients (N=88),

overweight (OW) and obese (OB) Randomized, double-blind, placebo-controlled study (N=30) of smoking cessation and ^-tocopherol (t-T) supplementation Double control sandwich model intervention study, 21 hypercholesterolemic subjects 47 overweight and obese postmenopausal women completed a 4-month program of 1 h low-intensity physical activity (PA) 22 healthy sedentary subjects aged 50-77 years, treated with fenofibrate (N =12) Healthy, nonsmoking adults, 187 men and 127 women, aged 18-79 years Random population sample, 89 smokers and 261 non-smokers

Hypertensive subjects (N=94), naive of antihypertensive medication Cross-sectional study, N= 70 anuric hemodialysis patients

Prospective study, 19 subjects with

primary biliary cirrhosis (PBC) Randomized, controlled, parallel feeding trial, 50 adults with metabolic syndrome (MS) received diet supplemented with mixed nuts Multicenter study, 25 patients with hereditary gp91(phox) deficiency, 25 healthy, 25 obese Population-based cohort study: 624 men and women (age range 50-87 years)

25 men with a previous hospital-diagnosed myocardial infarction

Relationship between dietary niacin intake, VEF and oxLDL

Relationship between BMI, immune and VEF

Effect of improvements in 7-T status on VEF and oxidative stress

Effects of hazelnut consumption on CV risk markers

Impact of a lower-than-advised level of PA on small artery VEF and oxidative stress

Influence of short-term treatment (7 days) on endothelial function

Relationship between, plasma norepinephrine (PNE) and VEF

Association between WBC telomere length, oxLDL and VEF

Effect of antihypertensive therapy on EVF

Relationship between malnutrition-inflammation score (MIS) and the VEF

Effect of low-dose atorvastatin treatment on dyslipidemia and VEF Effect of nuts consumption on markers of oxidation and endothelial function

Relationship between NADPH-oxidase and FMD

Metabolic determinants of oxLDL and the relation between oxLDL and FMD

Relationship between oxLDL and C-reactive protein (CRP) with endothelial function, in CHD subjects

OxLDL-Ab-4E6 - ELISA

Anti-oxLDL Abs OxLDL-Ab-4E6 - ELISA

OxLDL-Ab-4E6 - ELISA OxLDL-Ab-4E6 - ELISA

OxLDL-Ab-4E6 - ELISA OxLDL-Ab-4E6 - ELISA OxLDL-Ab-4E6 - ELISA Anti-oxLDL Abs OxLDL-Ab-4E6 - ELISA

OxLDL-Ab-4E6 - ELISA

OxLDL- Ab-4E6 - ELISA LDL oxidation susceptibility

OxLDL-Ab-4E6 - ELISA

OxLDL-Ab-4E6 - ELISA LDL oxidation susceptibility

IgG and IgM antibodies (Ab) against oxLDL

Brachial artery FMD

Brachial artery FMD Brachial artery FMD

Brachial artery FMD

Small artery reactive hyperemia index (saRHl)

Brachial artery FMD

Distensibility of the carotid artery (CA)

Brachial artery FMD

Peripheral artery tonometry (PAT)

Brachial artery FMD

FMD in isolated resistance arteries from subcutaneous fat biopsies

OxLDL was inversely related to niacin intake; FMD was positively related to dietary niacin intake OB had lower levels of anti-oxLDL Abs and impaired FMD OxLDL levels were unaffected by smoking cessation or 7-T-rich supplementation, whereas FMD was significantly improved

Antiatherogenic effect of hazelnut-enriched diets by improving endothelial function and oxLDL levels PA improves antioxidant capacity, resting heart rate (RHR), and saRHl in postmenopausal women

Fenofibrate improved FMD after 2 and 7 days, reduced oxLDL and increased eNOS expression in vascular EC VEF was inversely related to sympathetic activity

Higher level of oxLDL is associated with shorter WBC telomeres and increased stiffness of the CA Anti-oxLDL Abs titer increased and FMD was improved after antihypertensive therapy MlS and oxLDL were independent significant predictors of FMD in a multivariate analysis Statin treatment improved CV risk markers and VEF

No significant differences in oxLDL or endothelial function during the intervention

NOx significantly correlated with FMD; gp91(phox) is involved in the modulation of arterial tone oxLDL/apoB100 ratio was negatively related to FMD after adjustment for age, sex, glucose tolerance status, and Framingham risk score Maximum vessel dilatation was inversely related to IgG-Ab levels; correlation between FMD, CRP and plasma levels of IgG-Ab

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Demiret al. (2010)

Stojakovic et al. (2010) López-Uriarte et al. (2010)

Violi et al. (2009)

van der Zwan et al. (2009)

Crisby et al. (2009)

Plasma oxLDL were assessed using the competitive ELISA method with monoclonal antibody 4E6 (Ab-4E6), or the anti-oxidized LDL antibodies (anti-oxLDL Abs). The endothelial function was assessed by flow-mediated dilation (FMD) of the brachial artery, FMD in isolated resistance arteries from subcutaneous fat biopsies, as small artery reactive hyperemia index (saRHl), as distensibility of the carotid artery (CA) or by peripheral artery tonometry (PAT).

3 ста

ГD О) t

ста _

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Table 2

Human studies reporting the relationship between oxidized LDL (oxLDL) and vascular endothelial function (VEF) assessed as plasma NO metabolites, in the investigation of different cardiovascular (CV) outcomes in adult and elderly subjects.

Population under study

OxLDL tests

VEF tests

Relevant findings

References

6i j. i maze

3 D. О

35 subjects with normolipidemia and 30 subjects with hyperlipidemia

273 subjects, aged 60-75 years, with pre-diabetes (N=90, IFG), and type 2 diabetes mellitus (N=95,T2DM), vs. control

Cross-sectional study: 90 subjects aged 65-78 years, with pre-diabetes (N=30, IFG), and type 2 diabetes mellitus (N=35) 170 elderly, aged 60-70 years: hypercholesterolemia (N =125) vs. control (N =45) 84 patients with metabolic syndrome (MS) and 42 healthy controls

24 patients with rheumatoid arthritis (RA), 25 with psoriatic arthritis (PsA), vs. control Double-blind, randomized, crossover dietary-intervention study, 24 women with high-normal blood pressure (BP) or stage 1 essential hypertension 50 stable coronary artery disease (CAD) patients, 50 unstable CAD and 50 control

Randomized crossover study, 20 participants

62 patients diagnosed with essential arterial hypertension and 45 healthy controls

Relationship between VEF, asymmetric

dimethylarginine (ADMA) and oxLDL

Relationship between advanced oxidation and advanced glycation of proteins (AOPP and AGEs), oxLDL and NOx Relationship between vitamin D status, systemic oxidative stress and VEF

Metabolic determinants of oxLDL and NOx in hyperlipidemia Relationship between plasma lipoprotein (a) (Lp[a]) levels and atherosclerotic burden Association oxLDL, NOx, with subclinical atherosclerosis Influence of a polyphenol-rich olive oil diet on BP, oxLDL and endothelial function

Relationships between myeloperoxidase (MPO), oxLDL and NOx levels Effect of Mediterranean diet supplemented with coenzyme Q10 (Med+CoQ diet) on postprandial oxidative stress Diagnostic value of endothelial dysfunction and oxidative stress markers

OxLDL-Ab-4E6 - ELISA

LDL oxidation susceptibility

OxLDL-Ab-4E6 - ELISA

OxLDL-Ab-4E6 - ELISA Anti-oxLDLAbs

OxLDL-Ab-4E6 - ELISA

IgG antibodies against oxLDL

Plasma NOx

Plasma NOx brachial artery FMD

Plasma NOx

Plasma NOx brachial artery FMD

Plasma NOx

OxLDL values were significantly higher and NOx decreased in dyslipidemic subjects vs. normo-lipidemic subjects

Higher levels of plasma AOPPs, AGEs, oxLDL, NOx, atherosclerosis risk markers, and insulin resistance were pointed out in IFG and T2DM

Serum 25-hydroxyvitamin was inversely associated with oxidative stress and endothelial dysfunction, in subjects with hypovitaminosis D

OxLDL/NOx ratio correlated significantly with traditional cardiovascular risk markers

NOx levels were significantly lower in patients with high Lp(a) as compared with those with normal Lp(a)

Ox-LDLs and NOx may be markers of accelerated atherosclerosis in RA and PsA

Polyphenol-rich olive oil diet led to a significant decrease in BP, oxLDL, and increase in plasma NOx and FMD

Plasma MPO levels were significantly positively correlated with oxLDL and negatively correlated with NOx levels Med and Med+CoQdiets produced a lower postprandial decrease in total nitrite, a higher postprandial increase in FMD, and a lower postprandial oxLDL

Prostacyclin and oxLDL had the best diagnostic value

Ercan et al. (2014)

Gradinaru et al. (2013)

Gradinaru et al. (2012)

Borsa et al. (2012)

Muñoz-Torrero et al. (2012)

Profumoetal. (2012) Moreno-Luna et al. (2012)

Samsamshariat et al. (2011)

Yubero-Serrano et al. (2011)

Kuklinska et al. (2009)

Plasma oxidized LDL were assessed using the competitive ELISA method with monoclonal antibody 4E6 (Ab-4E6), the anti-oxidized LDL antibodies (anti-oxLDL Abs), or the LDL susceptibility to in vitro oxidation. The endothelial function was evaluated as plasma NO metabolic pathway products, NOx (NO2- + NO3-).

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393 4. Oxidized LDL and NO synthesis in human vascular ageing

394 Ageing of the cardiovascular system has represented a real chal-

395 lenge for the human health, ever since we start to age at the

396 first heartbeat (Thorin andThorin-Trescases, 2009). Atherosclerosis

397 is associated with premature biological ageing, as atherosclerotic

398 plaques show evidence of cellular senescence characterized by

399 reduced cell proliferation, irreversible growth arrest and apopto-

400 sis, elevated DNA damage, epigenetic modifications, and telomere

401 shortening and dysfunction (Wang and Bennett, 2011). Diseases of

402 the vascular system such as hypertension, chronic coronary disease

403 and diabetes have long been considered to be age-related in terms

404 of their onset and progression. ln endothelial cells these changes

405 result in a phenotype that is pro-inflammatory, pro-atherosclerotic,

406 and pro-thrombotic. Endothelial cell (EC) senescence can be

407 induced by a number of factors implicated in vascular patholo-

408 gies, particularly by sustained cell replication and oxidative stress

409 (Erusalimski, 2009).

410 Ageing is associated with an increase in arterial stiffness and an

411 impairment of endothelial function - early important event leading

412 to CVD (El Assar et al., 2012). Many studies attempting to elucidate

413 the mechanisms behind this decline have used animal models (Wu

414 et al., 2014). The key role of endothelium derived NO in protecting

415 the cardiovascular system during ageing is underscored by the find-

416 ings that eNOS knockout mice exhibit a premature cardiac ageing

417 phenotype associated with early mortality (Li et al., 2004)

418 Among mechanisms that are proposed to contribute to

419 age-dependent endothelial dysfunction, the most acknowledged

420 evidenced the following aspects: (1) reduction of NO bioavail-

421 ability, caused by diminished NO synthesis and/or by augmented

422 NO scavenging due to oxidative stress; (2) increased oxidative

423 stress in the endothelial microenvironment; (3) increased oxida-

424 tion of LDL; (4) development of a low-grade pro-inflammatory

425 environment; (5) increased activities of vasoconstrictor factors; (5)

426 impaired endothelial cell function and maintenance repair systems

427 by endothelial progenitor cells (EPC) (El Assar et al., 2012; Wadley

428 et al., 2013; Ungvari et al., 2010).

429 Higher levels of oxidative stress and inflammation are major

430 determinants of reduced vascular function in human ageing, being

431 included in the vascular health triad concept (Wadley et al., 2013).

432 Elevated circulating levels of high sensitivity CRP (hsCRP) and

433 oxidized LDL were associated with CVD (Obradovic et al., 2015);

434 and other inflammatory markers (e.g., tumor necrosis factor-alpha

435 [TNFa], sVCAM-1, sE-selectin, interleukin [lL]-6, lL-18, and MCP-1)

436 were correlated with age, independently of other cardiovascular

437 risk factors (Ungvari et al., 2010). In particular, elderly people

438 often present a low-grade, chronic, systemic inflammation with

439 age, termed "inflammaging" (Franceschi et al., 2007). Ageing and

440 increased levels of systemic pro-inflammatory markers and oxLDL

441 were associated with arterial stiffness (Donato et al., 2007; Kampus

442 et al., 2007; Brinkley et al., 2009; Kim et al., 2013). Reduced brachial

443 artery flow-mediated dilation (FMD) and elevated nitrotyrosine

444 expression in brachial artery ECs were pointed out in older com-

445 pared to young subjects (Kim et al., 2013). Additionally, in older

446 men increased NAD(P)H oxidase-p47(phox) enzyme expression,

447 together with nuclear factor-kappa B p65, a component of the

448 redox-sensitive NF-kB, were pointed out (Donato et al., 2007). Of

449 particular interest is the impact of oxidative stress on plasma LDL,

450 but very low-density lipoprotein (VLDL), beta-VLDL and even HDL

451 undergo oxidative modification that must be taken into consid-

452 eration in the complex process of atherosclerosis (Parthasarathy

453 et al., 2008). Age-related evaluations of arteries markers of func-

454 tional and structural changes in healthy subjects (175 subjects,

455 aged 40-70 years), showed a significant association between oxLDL

456 and carotid intima-media thickness (lMT) (Kampus et al., 2007). ln

457 a cross-sectional study in 2295 elderly persons from the health,

ageing, and body composition study, elevated plasma oxLDL lev- 458 els were associated with higher arterial stiffness, independent of 459

other traditional CVD risk factors (Brinkley et al., 2009). In a recent 460

3-year longitudinal study in 57 nonobese men (aged 34-55 years) 461

changes in arterial stiffness measured as brachial-ankle pulse wave 462

velocities (ba-PWV) were positively correlated with the changes in 463

waist-hip ratio (WHR), oxLDLs, plasma malondialdehyde (MDA), 464

hs-CRP, and lL-6 levels suggesting that changes in oxidative stress, 465

pro-inflammation or abdominal obesity could play important roles 466 in accelerating arterial stiffness (Kim et al., 2013). The effect of 467

age on atherogenicity of LDL and inflammatory markers studied 468

in 2944 healthy women (aged 30-79 years), underlined increases 469

in plasma oxLDL levels after 50 years and higher levels of inflam- 470

matory markers: hCRP, TNF-a) and lL-6 after 60 years of age (Paik 471

et al., 2013). Since total cholesterol levels decrease after 60 years 472

of age, the oxLDL increases with age may be due to enhancement 473

in LDL atherogenicity under preponderantly pro-oxidant and pro- 474

inflammatory environment. 475

Circulating oxLDL is associated with the development of not only 476

atherosclerosis but also numerous other degenerative and age- 477

related diseases, such as rheumatoid arthritis, multiple sclerosis, 478

osteoporosis, macular degeneration and Alzheimer's and Parkin- 479

son's diseases (Profumo et al., 2012; Besler and Comoglu, 2003; 480

Dildar et al., 2010; Robman et al., 2004; Maziere et al., 2010; 481

Kankaanpaa et al., 2009). 482

Recent studies underscore the association between white blood 483

cell (WBC) telomere length, as index of systemic ageing, oxidized 484

LDL, and human vascular ageing expressed by the distensibility 485

of the carotid artery. Higher levels of oxidized LDL were associ- 486

ated with shorter WBC telomeres and increased stiffness of the 487

carotid artery (Nawrot and Staessen, 2008; Nawrot et al., 2010). 488

Also, emerging evidence suggests that increasing NO bioavailabil- 489

ity or eNOS expression activates telomerase and delays endothelial 490

cell senescence (Hayashi et al., 2008). lnterestingly, NO can play a 491

dual role in atherosclerosis, some pathological vascular conditions 492

being linked with excessive rather than reduced NO production. 493

The inducible nitric oxide synthase isoform (iNOS), involved in 494

immune response is synthesized by many cell types in response to 495

pro-inflammatory cytokines (lL-6, TNF-a, and gamma-interferon) 496

and produces quantities of NO far exceeding those produced by 497

the eNOS isoform. These elevated levels of NO are associated with 498

the general cytotoxic effects of NO, as NO is a free radical with an 499 unpaired electron (Armitage et al., 2009). Therefore, regulation of 500

nitric oxide synthases and bioavailability of their product become 501

critical for the development and progression of vascular diseases, 502

such as atherosclerosis (Napoli et al., 2006). High levels of NO pro- 503

duced from iNOS in endothelial cells and macrophages can induce 504

injury to the endothelium. Peroxynitrite (ONOO-), the harmful 505

product of NO interaction with superoxide (O2is also pro- 506

duced in significant amounts in atherosclerotic lesions (Chatterjee 507

et al., 2009). Thus, while the low concentrations of NO generated 508

by eNOS protect against atherosclerosis by promoting vasodilata- 509

tion, inhibiting leucocyte and platelet adhesion and/or aggregation 510 and smooth muscle cell proliferation; higher concentrations of 511

NO generated by iNOS promote atherosclerosis either directly or 512

via the formation of NO adducts, such as peroxynitrite (Moncada 513

and Higgs, 2006). By combining two experimental approaches in 514

a large sample of subjects with a wide age range (18-91 years), 515

Rodriguez-Manas et al. (2009) determined the existence of age- 516

dependent endothelial dysfunction, both in vitro and in vivo, in 517

subjects with no clinical cardiovascular diseases and no classical 518

risk factors. ln isolated mesenteric microvessels from these sub- 519

jects, an age-dependent impairment of the endothelium dependent 520

relaxations to bradykinin was observed. Moreover, aged microves- 521

sels showed superoxide anions (O2 ) and peroxynitrite (ONOO ) 522

formation, as well as enhancement of NADPH oxidase and iNOS 523

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synthase expression. The expression of mRNA for eNOS did not change in mesenteric microvessels from aged subjects, whereas it was markedly enhanced for the iNOS isoform. Hence, a paradox appears whereby in ageing humans there is elevated NO production within the vasculature, associated with reduced NO bioavailability, due to free radical scavenging. The induction of the inflammatory iNOS isoform can be also related to the endothe-lial dysfunction associated with ageing and age-related diseases. Higher levels of plasma NO metabolic pathway products (NOx) were also pointed out in adults and elderly subjects with metabolic disorders: impaired fasting glucose (IFG) and type 2 diabetes mellitus (T2DM) (Ghasemi et al., 2011; Gradinaru et al., 2012,2013), and also in stimulated blood cells (PBMNC) from T2DM subjects (Volpe et al., 2014). This suggests that in chronic hyperglycemia the role of eNOS changes from an anti-atherogenic effect to a pro-atherogenic effect and exacerbates in vitro inflammatory responses and iNOS expression.

In a prospective 4 years cohort study (204 subjects, aged over 55 years), Sverdlov et al. (2014) evaluated the effects of ageing on platelet aggregability and responsiveness to NO, in correlation with plasma asymmetric dimethylarginine (ADMA), an endogenous eNOS inhibitor. Thus, ageing was associated with marked deterioration of responsiveness of platelets to NO and increases in plasma ADMA concentrations to a proportional extent with adverse impact on CV outcomes.

5. OxLDL and NO - mechanisms in endothelial dysfunction and ageing

The NO-signaling system has a number of potential points of vulnerability—biochemical and cellular processes, which may result in impairment of the entire NO cascade. Enhanced ROS generation and overproduction of peroxynitrite in the presence of risk factors also facilitates activation of redox-dependent transcriptional factors such as NF-kB and increase iNOS (Sverdlov et al., 2014; Kolluruet al., 2012).

Numerous experimental studies have supported the harmful effects of hyperlipidemia, hyperglycemia and insulin resistance, at multiple steps in atherogenesis, including direct contributions to endothelial dysfunction, through several underlying mechanisms which involve together with NO and oxLDL, new causative factors: lectin-like oxidized LDL receptor-1 (LOX-1), p66Shc adaptor protein, NF-kB and tetyrahydrobiopterin (BH4) (Xu et al., 2012; Pirillo et al., 2013; El Assar et al., 2012; Nilsson, 2008; Ungvari et al., 2012; Wadley et al., 2013).

5.1. Lectin-like oxidized LDL receptor-1

Lectin-like oxidized LDL receptor-1 (LOX-1), the main oxLDL receptor in endothelial cells, macrophages and smooth muscle cells is implicated in pathogenesis of atherosclerosis. Soluble form of LOX-1 (sLOX-1) is associated with early stages of acute coronary syndrome (Hayashida et al., 2005), coronary plaque vulnerability (Zhao et al., 2011) and plaque rupture (Kobayashi et al., 2013), being considered a biochemical marker of atherosclerosis-related diseases (Pirillo and Catapano, 2013). Also, sLOX-1 is associated with metabolic disorders (obesity, T2DM, metabolic syndrome) (Tan et al., 2008). LOX-1 mediates the uptake of oxLDL by vascular cells being involved in endothelial dysfunction, monocyte adhesion, proliferation, migration, and apoptosis ofSMC, foam cell formation, platelet activation, and plaque instability. These cellular events may be inhibited by anti-LOX-1 antibodies, and vascular LOX-1 expression and activity could be regulated by vasculo-protective drugs (Xu et al., 2012). LOX-1 mediates the oxLDL uptake in endothelial cells by activation of protein kinase C (PKC) ß2 and

c-Jun N-terminal kinases (JNK), and by phosphorylation of p66Shc adaptor proteins (Shi et al., 2011). Also, LOX-1 is implicated in NO-dependent endothelial impairment of coronary arterioles (Xu et al., 2007) by activation the signaling cascade involving NADPH oxidase or NF-kB-NADH oxidase - ROS (Cominacini et al., 2000; Xu et al., 2012).Thus, due to the important role in regulating oxLDL-NO - mediated vascular reactivity, LOX-1 could represent a potential therapeutic target in endothelial dysfunction and cardiovascular diseases.

5.2. p66Shc adaptor protein

The p66Shc adaptor protein has a dual role in vascular dysfunction being a mediator of oxidative stress-induced vascular dysfunction and a modulator of endothelial NO production (Shi et al., 2014; Yamamori et al., 2005; Franzeck et al., 2012). The p66Shc may mediate endothelial dysfunction by redox-enzyme action in mitochondrial ROS generation, translation of oxidative signals into apoptosis and increased ROS production by oxidized LDL. The mechanisms of oxLDL - dependent ROS generation include: phos-phorylation of the p66Shc protein at ser36 through the lectin-like oxLDL receptor-1 (LOX-1), activation of protein kinase C beta-2, and c-Jun N terminal kinase (Shi et al., 2011). The deletion of ageing gene p66Shc increases endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) bioavailability via protein kinase B and its overexpression inhibits eNOS-dependent NO production (Yamamori et al., 2005). Recent studies on cultured human endothelial cells underline the dual role of eNOS for p66Shc protein activation and ROS generation. The eNOS uncoupling is a crucial player in oxLDL-induced and p66Shc-mediated intracellular ROS generation (Shi et al., 2014). Thus, eNOS uncoupling has been considered as a putative antioxidant therapeutical target in endothelial dysfunction and CVD.

5.3. Nuclear factor-kappa B

The transcription factor NF-kB, a key regulator of inflammation and oxidative stress provides an effective "transducer" for feeding forward activation of these processes. By stimulating inflammation and oxidative stress, NF-kB has a key role in mediating vascular endothelial dysfunction in humans (Xu et al., 2013; Ungvari et al., 2010). One of these roles includes the activation of the nuclear enzyme poly(ADP-ribose), polymerase (PARP-1), which leads to the production of inflammatory mediators such as iNOS, lCAM-1, and major histocompatibility complex class ll. Lack of NO leads to vasodilator dysfunction and promotes endothelial apo-ptosis, whereas nitrative stress and increased H2O2 levels lead to poly(ADP-ribose) polymerase (PARP)-1 activation, which contributes to NF-kB -dependent gene transcription (Ungvari et al., 2010; Mangerich and Burkle, 2012). NF-kB, an integral factor in vascular health triad (Donato et al., 2009), is involved in transcriptional activation of pro-inflammatory and pro-oxidative genes leading to impaired vascular function. Activation of NF-kB correlates with reduced endothelium-dependent dilation (EDD) with advancing age (Pierce et al., 2009; Donato et al., 2007, 2009), contributing to vascular endothelial dysfunction via oxidative stress. Inactiva-tion of NF-kB increased endothelial function and reduced NADPH oxidase activity (Pierce et al., 2009). The newly proposed model of vascular health, named vascular health triad (Wadley et al., 2013) integrate the associations and interactions ofoxidative stress and inflammation with vascular dysfunction in ageing, primarily mediated by the transcriptional factor NF-kB and targeting downstream NO bioavailability. The NF-kB signaling inhibition might limit the vicious cycles of inflammation and oxidative stress. Thus,

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D. Gradinaru et al. / Mechanisms of Ageing and Development xxx (2015)xxx-xxx

CARDIOVASCULAR DISEASES

Fig. 2. Simplified scheme ofthe interrelations between oxidative stress, LDLoxidation and NO in ageing and age-related metabolic disorders leading to endothelial dysfunction and cardiovascular diseases.

645 modulation of NF-kB signaling could be a potential therapeutic

646 target in vascular ageing prevention.

647 5.4. Tetyrahydrobiopterin

648 Tetyrahydrobiopterin (BH4), essential co-factor in eNOS regu-

649 lation has been recently considered as biomarker of endothelial

650 health (Tousoulis et al., 2013). Reduced synthesis or oxidative

651 inactivation of BH4 leads to reducing NO availability by uncou-

652 pled eNOS, which generates superoxide rather than NO. Increased

653 levels of BH4 enhance eNOS activity, promote vasodilation,

654 and reduce oxidative stress in experimentally induced diabetes,

655 ischemia/reperfusion, or hypertension (Faria et al., 2012; Shinozaki

656 et al., 2000; Perkins et al., 2012). In human studies, BH4 treatment

657 improves endothelial dysfunction and decreases arterial stiffness

658 in postmenopausal women, diabetes, hypercholesterolemia, and

659 coronary disease (Moreauetal., 2012; Holowatz and Kenney, 2011;

660 Heitzer et al., 2000). Recent studies in cultured human endothe-

661 lial cells confirmed the BH4 protective effect on eNOS coupling, as

662 BH4 treatment, prior to oxLDL stimulation, has prevented p66Shc-

663 mediated oxidative stress (Shi et al., 2014).

664 Based on the strong interrelationships pointed out in numer-

665 ous experimental and clinical research (Xu et al., 2013; Ungvari

666 et al., 2012; Wadley et al., 2013; Maiolino et al., 2013a,b; Borsa

667 et al., 2012; Gradinaru et al., 2012, 2013), we summarized the

668 mechanisms of oxidative stress-oxLDL-NO-induced endothelial

669 dysfunction (Fig. 2). In ageing and age-related metabolic disor-

670 ders (dyslipidemia, hyperglycemia, insulin resistance, metabolic

syndrome) enhancement of oxidative stress, superoxide anion 671

excessive generation and LDL oxidation are critically involved in 672

reduced NO bioactivity and endothelial dysfunction by direct NO 673

elimination. Superoxide radicals (O2'-), are scavenged by nitric 674

oxide to form peroxynitrite (ONOO-), which can oxidize tetrahy- 675

drobiopterin (BH4), the cofactor in NO production by eNOS enzyme, 676

leading to eNOS uncoupling. Uncoupled eNOS will generate more 677

O2'- and reduce NO production, activating the vicious cycle. 678

The binding of oxLDL to its specific receptor LOX-1 activates the 679

NADPH oxidase on the cell membrane, which increases intracel- 680

lular ROS formation. Increased ROS activates the redox-sensitive 681

NF-kB signaling pathway, generating: increases of NF-kB binding 682

to LOX-1 promoter and LOX-1 expression, and amplifies LOX- 683

1-mediated oxLDL uptake. Thus, the oxLDL binding to LOX-1 684

affects NO bioactivity by two mechanisms: (1) increased ROS pro- 685

duction, which reacts with intracellular NO generating cytotoxic 686

ONOO-, and down-regulates eNOS, decreasing NO bioavailability; 687

(2) oxLDL, through LOX-1 receptor activates arginase ll, competing 688

with eNOS for L-arginine substrate, down-regulating NO formation, 689

and contributing to vascular dysfunction. The eNOS uncoupling 690

also lead to the p66Shc activation, an important mediator of oxida- 691

tive stress-induced vascular dysfunction, which contributes to ROS 692

overproduction from mitochondria and/or via NADPH oxidase. The 693

oxidized LDL could also increase ROS production via phosphoryla- 694

tion of the p66Shc protein at ser36 through the LOX-1. 695

These complex mechanisms involved in oxLDL-NO inter- 696

actions and endothelial dysfunction, atherosclerosis and CVD, 697

could explain in part, the failure of antioxidant treatments in 698

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10 D. Gradinaru et al. / Mechanisms of Ageing and Development xxx (2015)xxx-xxx

699 improving cardiovascular outcome in long-term clinical trials

700 (Sesso et al., 2008; Parthasarathy et al., 2008).

701 Taken together, in dysmetabolic status and even ageing, the

702 oxidative stress and LDL oxidation determine reduced NO bioavail-

703 ability via combinatory effects of direct elimination and decreased 70Q5 production of NO. These NO reduced bioavailability compromises

705 all the anti-atherogenic functions of the endothelium. Hypothe-

706 sized mechanisms shown above could be a target for interventions

707 to protect against endothelial dysfunction, atherogenesis and car-

708 diovascular disease.

709 6. Conclusions

710 LDL oxidation and NO synthesis are direct contributors to

711 atherogenesis and also important biomarkers indicating the over-

712 all status of the organism as a result of the progressive damage

713 of the endothelium at cellular level under the action of pro-

714 oxidant pathogenic factors and ageing. oxLDL and NO are therefore

715 oxidative stress and endothelial dysfunction biomarkers that could

716 be modulated in the course of ageing, age-related diseases and

717 anti-ageing interventions. Vascular ageing, formerly considered an

718 immutable and inexorable risk factor, is now viewed as a tar-

719 get process for intervention in order to achieve a healthier old

720 age. Therapeutic approaches in the prevention and treatment of

721 atherosclerosis based on improving NO bioactivity and reducing

722 LDL oxidation, and their related molecular mechanisms targets,

723 may become a challenge for future research.

724 It has been acknowledged that a discrepancy exists between

725 the chronological age and the signs and "marks" of biological age,

726 as evidenced by the clinical assessment of a patient. Therefore,

727 establishing specific biomarkers of vascular endothelium function

728 is important in the complex evaluation of the biological age. In this

729 regard, oxLDL and NOx were included among the oxidative stress

730 biomarkers studied within the MARK-AGE project, European Study

731 to Establish Biomarkers of Human ageing.

732 Acknowledgements

733 The authors thank Prof. Alexander Burkle, Prof. Tilman Grune

734 and Dr. Maria-Moreno Villanueva for fruitful discussions and sup-

735 port during the MARK-AGE study, very helpful in the preparation

736 of this manuscript.

737 The authors gratefully acknowledge the European Commission

738 for the financial support provided through the FP7 large-scale inte-

739 grating project "European Study to Establish Biomarkers of Human

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Oxidized LDL and NO synthesis—Biomarkers of endothelial dysfunction and ageing Academic research paper on "Clinical medicine"

Abstract of research paper on Clinical medicine, author of scientific article — Daniela Gradinaru, Claudia Borsa, Cristina Ionescu, Gabriel Ioan Prada

Similar topics of scientific paper in Clinical medicine , author of scholarly article — Daniela Gradinaru, Claudia Borsa, Cristina Ionescu, Gabriel Ioan Prada

Academic research paper on topic "Oxidized LDL and NO synthesis—Biomarkers of endothelial dysfunction and ageing"