The relationship between markers of oxidative stress, inflammation and arterial compliance in a bi-ethnic population : the SABPA study

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The relationship between markers of

oxidative stress, inflammation and arterial

compliance in a bi-ethnic population: The

SABPA study

MC Mokhaneli

21275203

Dissertation submitted in fulfillment of the requirements for the

degree Master of Science in Physiology at the Potchefstroom of

the North-West University

Supervisor:

Prof CMC Mels

Co-supervisor:

Prof CMT Fourie

Co-supervisor:

Dr S Botha

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ACKNOWLEDGEMENTS

Thanks to my God for making the completion of this dissertation possible, thank you for strength, courage, provision, wisdom and spirit of excellence. (Deuteronomy 8:18)

I would like to sincerely acknowledge the following people for the roles they played in making this project a success and for their constant support:

 Carina Mels, for her willingness to be my supervisor, for the continuous guidance, support and valuable insights throughout this study. Thank you for being positive always and for believing in me.

 Carla Fourie and Shani Botha, my sincere gratitude is extended towards their excellent technical advice, constant motivation and inspiration.

 Close friends and family, your support and encouragement is highly appreciated.  To fellow postgrad student, Moliehi Mothae, Gontse Mokwatsi and Mellissa Maritz,

thank you for your support, help during my studies.

 My dad and my sister, thank you for your unconditional love, support and encouragement during my studies.

 Bonang and Blessing, thank you for giving me strength to do well in life.

 To the National Research Foundation (Innovation) for the financial support for this study.

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PREFACE

The article-format has been chosen for this dissertation. This is the format approved and recommended by the North-West University for postgraduate studies. The layout of the dissertation is as follows:

Chapter 1: Background and motivation

Chapter 2: Literature study

Chapter 3 Methodology

Chapter 4: Research article consisting of an abstract, introduction, methods, results, discussion, conclusion and acknowledgements.

Chapter 5: Summary of main findings, conclusion and recommendations

References are provided at the end of each chapter according to the referencing style of the journal Free Radical Research.

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AFFIRMATIONS OF AUTHORS

The contribution of each of the authors to the research was the following:

MC Mokhaneli: Responsible for conducting the literature search. The candidate performed all statistical analyses, designed, wrote and compiled the dissertation.

Prof CMC Mels: Supervisor: Supervised all stages of compiling the dissertation, was responsible for collection of data, initial design of the dissertation, acquirement of funding and gave professional input.

Prof CMT Fourie: Co-supervisor: Supervised the writing of the manuscript. Responsible for collection of data, reviewing the dissertation and gave recommendations.

Dr S Botha: Co-supervisor: Provided recommendations and responsible for collection of data and reviewing the dissertation.

STATEMENT BY THE AUTHORS

The following is a statement of the co-authors to verify their individual contributions and involvement in this study and to grant their permission that the relevant research article may form part of this thesis:

Prof CMC Mels

Prof CMT Fourie

Dr S Botha

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SUMMARY

Motivation

Cardiovascular disease (CVD) has become the major cause of mortality among the black population in South Africa. This might be due to rapid increase in urbanization seen in the last decade. It was further indicated that black Africans are more frequently diagnosed with heart failure than any of the other ethnic groups.

Unhealthy lifestyle behaviors, such as excessive alcohol consumption and tobacco use, are known to augment oxidative stress and inflammation, and as a result these two factors may lead to increased arterial stiffness, either directly by oxidative stress, or indirectly by activating inflammatory processes. Oxidative stress is an imbalance between oxidants and antioxidants. Oxidative damage is characterized by an increase in the levels of oxidation products of macromolecules, such as thiobarbituric acid reactive substances (TBARS) and protein carbonyls. TBARS are an end-product of lipid peroxidation and levels of TBARS were shown to independently predict cardiovascular events such as coronary heart disease. The antioxidant defense system, such as the enzymes superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase acts to combat oxidative stress. However, lower antioxidant enzyme activity such as GPx activity allows accumulation of peroxide molecules and is associated with increased risk of cardiovascular events.

Increased arterial stiffness is independently associated with cardiovascular morbidity and mortality in various population groups. Arterial stiffness is characterized by structural and functional alterations of the intrinsic elastic properties of arteries, leading to decreased arterial compliance (Cwk) and increased total peripheral resistance (TPR). Differences between black and white Africans have been established by numerous investigators, finding that blacks are more prone to increased oxidative stress, inflammation (CRP, IL-6) and arterial stiffness; however, studies investigating the link between these factors in a population with high prevalence of CVD are scant.

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Aim

The aim of the study was to compare markers of oxidative stress (TBARS), antioxidant capacity (GPx), inflammation (CRP, IL-6), arterial compliance (Cwk) and vascular resistance (TPR) between black and white South Africans. A further aim was to determine whether oxidative stress, antioxidant capacity and inflammation are related to Cwk and TPR in this study population.

Methodology

This sub-study, which is embedded in the Sympathetic Activity and Ambulatory Blood Pressure in Africans (SABPA) study, included a total of 359 participants, 173 black and 186 white men and women between the age of 28 and 68. The participants of this study were from North-West Province of South Africa and data of the follow up phase of this study was used in this sub-study. The follow up phase was conducted between February and May 2011/2012.

Standardized methods were used to capture all data and included anthropometric measurements, cardiovascular measurements namely, Windkessel compliance (Cwk), total peripheral resistance (TPR), mean arterial pressure (MAP), pulse pressure (PP), diastolic blood pressure (DBP) and systolic blood pressure (SBP). Biochemical analyses included the antioxidant markers, superoxide dismutase (SOD), glutathione reductase (GR), glutathione peroxidase (GPx), total glutathione (tGSH) and the lipid peroxidation marker thiobarbituric acid reactive substances (TBARS), as well as inflammatory makers C-reactive protein (CRP) and interleukin-6 (IL-6). The groups were stratified by ethnicity in line with the aims but not by sex, since no interaction of sex existed for the associations between cardiovascular and oxidative stress markers. T-tests and Chi-square tests were used to compare means and proportions, respectively. Pearson and partial regression analyses were used to determine correlations between cardiovascular markers and markers of oxidative stress and inflammation. This was followed by multiple regression analyses to investigate whether

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independent associations exist between the cardiovascular and oxidative stress variables in both ethnic groups. P-values ≤0.05 were deemed significant.

Results and conclusion

In this study black participants had worse cardiovascular (as indicated by higher SBP, DBP, TPR, MAP and lower Cwk all p≤0.013), inflammatory (as indicated by higher CRP and IL-6 levels p<0.001) and lipid profiles (as indicated by higher total cholesterol, low density cholesterol and, triglyceride levels p≤0.01) when compared to the white participants. Black participants also had higher levels of TBARS (p=0.018) despite their higher antioxidant enzyme activities GPx (p=0.005) and GR (p=0.019) compared to their white counterparts.

Unadjusted regression analyses showed a negative relationship of Cwk with TBARS (r=-0.25; p=0.002) and a positive relationship of TPR with TBARS (r=0.21; p=0.031) in black participants. In white participants a negative correlation existed between Cwk and GPx (r=-0.16; p=0.025), whereas TPR correlated positively with GPx (r=0.17; p=0.026). After adjustments were made for age, sex and BMI, the above mentioned correlations remained and an association between MAP and TBARS (r=0.17; p=0.049) became significant in the blacks, while a positive correlation of DBP (r=0.17; p=0.028) and MAP (r=0.17; p=0.032) with GPx became significant in the white group.

In multiple regression analyses, a positive independent association of both MAP (β=0.21;

p=0.032) and TPR (β=0.18; p=0.018) with TBARS was found, as well as a negative

independent association between Cwk and TBARS in the black group. These associations

re mained after excluding hypertensive participants. In the white group a positive association of

both MAP (β=0.17; p=0.011) and TPR (β=0.13; p=0.047) with GPx and a negative association between Cwk and GPx (β=-0.10; p=0.019) were also indicated to be independent of various covariates. In conclusion, decreased arterial compliance and increased vascular resistance associated with increased oxidative damage independent of

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hypertensive status in black participants. These results suggest that oxidative stress plays a role in early vascular changes in a black population prone to the development of cardiovascular disease.

Key words: Arterial elasticity, Glutathione peroxidase Thiobarbituric acid reactive

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LIST OF ABBREVIATIONS

BMI Body mass index

CAD Coronary artery disease

CAT Catalase

CIMT Carotid intima-media thickness

CO Cardiac output

CVD Cardiovascular diseases

Cwk Windkessel compliance

cm centimeters

CRP C-reactive protein

DBP Diastolic blood pressure

EDTA Ethylene diaminetetraacetic acid

eNOS Endothelial nitric oxide synthase

GGT Gamma glutamyl transferase

GPx Glutathione peroxidase

GR Glutathione reductase

GSSG Oxidized glutathione

g/l gram per liter

HbA1c Glycosylated hemoglobin

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xii HIV Human immunodeficiency virus

H2O2 Hydrogen peroxide

H2O Water

IL-6 Interleukin-6

kg/m2 _{Kilogram per meter squared}

kg kilogram

LDL-C Low density lipoprotein cholesterol

LPO Lipid peroxidation

MAD Malondialdehyde

MAP Mean arterial pressure

mg/L milligram per liter

ml/min milliliter per minute

mm millimeter

mmHg millimeter mercury

mmol/l millimole per liter

MMP Matrix metalloproteinases

MR Multiple regression

NADPH Nicotinamide-adenine dinucleotide oxidase

ng/ml nanograms per milliliter

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xiii oxLDL oxidized low density lipoprotein

PWV Pulse wave velocity

ROS Reactive oxygen species

RNS Reactive nitrogen species

SBP Systolic blood pressure

SD Standard deviation

SMCs Smooth muscle cells

TBA Thiobarbituric acid

TBARS Thiobarbituric acid reactive substances

TC Total cholesterol

TC:HDL Total cholesterol-to-high-density lipoprotein ratio

tGSH Total glutathione

TEE Total energy expenditure

TNF-α Tumor necrosis factor alpha

TPR Total peripheral resistance

TG Triglycerides

U/l Units per liter

VSMCs Vascular smooth muscle cells

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LIST OF TABLES

Chapter 2

Table 2.1 Different types of ROS and RNS 13

Chapter 4

Table 4.1: Characteristics of the study population. 60

Table 4.2: Partial regression analyses of cardiovascular markers with oxidative stress and inflammatory markers. 63

Table 4.3: Independent associations of cardiovascular, inflammatory and oxidative stress measures. 65

Table 4.4: Independent associations between cardiovascular, inflammatory and

oxidative stress measures in normotensive black and white Africans.

Chapter 4: Data supplement

Table S1: Single regression analyses of cardiovascular markers with oxidative stress and inflammatory markers. 77

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LIST OF FIGURES

Chapter 2

Figure 2.1: Imbalance between oxidants and antioxidants 14

Figure 2.2: A schematic representation of the enzymatic antioxidant system 15

Figure 2.3: Lipid peroxidation pathway 17

Figure 2.4: Structure of the arterial wall 19

Figure 2.5: The Windkessel model 20

Figure 2.6: Summary 23

Chapter 3

Figure 3.1: Areas involved in this study from the North West Province, South Africa 43

Figure 3.2: The study population 44

Figure 3.3: Finometer device 47

Chapter 4

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CHAPTER 1

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2 1.1 Background and motivation

It is well established that hypertension is one of the common risk factors in populations worldwide, which may lead to the development of cardiovascular disease [1]. The development of hypertension could be the result of many factors including increasing age, race, environmental factors, unhealthy lifestyle and diet [2]. In addition, structural changes of the arterial wall as a result of inflammation and oxidative stress [3], may also lead to cardiovascular disease [4,5].

Oxidative stress occurs as a result of an imbalance between oxidants and antioxidants [6]. The effects of oxidative stress can be indicated by damage to macro-molecules such as DNA [7], lipids and proteins [8], and as a consequence of the accumulation of markers such as s 8-hydroxy-2 deoxyguanosine (8OHdG) and thiobarbituric acid reactive substances (TBARS) [6] may ensue. TBARS are formed as a by-product of lipid peroxidation [9] and it was indicated that elevated TBARS can predict carotid atherosclerotic plaque progression [10].

Physiologically, reactive oxygen species (ROS) are generated in a controlled manner to act as signaling molecules [11] and to defend against infections [12]. ROS is further involved in the maintenance of vascular function by regulating endothelial function and vascular contraction-relaxation [13]. However, under pathological conditions, excessive ROS may lead to endothelial dysfunction [13,14] through reduction of nitric oxide (NO) synthesis or inactivation of NO via the chemical reaction of superoxide with NO, which results in the formation of peroxynitrate [13,15]. Furthermore, peroxynitrate formation may decrease NO bioavailability and increase oxidative stress through the oxidation of tetrahydrobiopterin (BH4) [12], the co-factor of nitric oxide synthase (NOS). Consequently, this may lead to the uncoupling of endothelial nitric oxide synthase (eNOS), where eNOS produces superoxide instead of NO [16], which lead to impaired vascular compliance [17]. Antioxidant defense systems, such as the enzymes superoxide

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dismutase (SOD), glutathione peroxidase (GPx) and catalase can scavenge ROS in the vasculature, resulting in increased NO bioavailability [18]. Oxidative stress also plays a role in arterial stiffness, as injury due to increased oxidative stress may result in vascular inflammation, and increased cellular proliferation may subsequently lead to increased arterial stiffness [19].

Inflammation is a protective response to injury or infection [20] and it has been implicated in the etiology of atherosclerosis [21]. Low grade inflammation is often a critical step in the progression of vascular disease [22,23], and various inflammatory markers such as tumor necrosis factor α (TNF-α), interleukin-6 (IL-6) and C-reactive protein (CRP) are associated with various forms of cardiovascular disease (CVD) [21,24,25].

During infection or injury leukocytes are recruited into the inflammatory tissue to eliminate the offending agent and repair the site of injury [12]. However, sustained inflammation may not only lead to an overproduction of ROS by the innate immune cells, macrophage and neutrophils [26] but may also lead to endothelial dysfunction due to altered NO synthesis and degradation [27]. Endothelial dysfunction may in turn result in increased expression of pro-inflammatory cytokines and cell-surface adhesion molecules [27]. Furthermore, vascular inflammation can increase vascular fibrosis and smooth muscle cell proliferation as well as impair endothelial vasodilation, which may subsequently lead to increased arterial stiffness, impaired arterial distensibility [19,28,29] and a decrease in vascular compliance [30].

Increased arterial stiffness is also indicated by decreased arterial compliance [31-34] and is characterized by the thickening of the intima-media, VSMC hyperplasia, increased collagen synthesis and elastin degradation [27]. Arterial compliance is defined as the change in volume of the artery per unit pressure (DV/DP) [30]. In a stiff vessel the volume change, and therefore compliance, is reduced for any given pressure change [35]. The consequence of reduced arterial compliance is an increased propagation velocity of the pressure pulse along the arterial tree, called pulse wave velocity, which indicates increased arterial stiffness [35]. Furthermore, a

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decrease in the levels of vasodilators, such as NO, could favor vascular constriction and could result in greater vascular resistance [17].

Thus, oxidative stress plays a potent role in the development of cardiovascular disease, such as hypertension and atherosclerosis [36,37]. Previous studies have indicated that blacks are more prone to increased oxidative stress [38], inflammation (CRP) [39,40] and arterial stiffness [41,42] than whites. However, knowledge on how markers of oxidative stress and inflammation associate with arterial compliance and vascular resistance in South Africans is limited.

A previous study on oxidative stress and arterial stiffness in South African blacks focused on limited markers of oxidative stress (serum peroxides) and stiffness markers such as pulse pressure (PP) and ambulatory arterial stiffness index [41]. The above study indicated that serum peroxides is positively associated with PP (indicative of arterial stiffness) in black men only [41]. In this study the added role of inflammation (TNF-α, IL-6 & CRP) to oxidative stress and its association with arterial compliance and vascular resistance, which was not previously investigated in black and white South Africans, will be further investigated.

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5 1.2 References

[1] Twagirumukiza M, De Bacquer D, Kips JG, de Backer G, Vander Stichele R, Van Bortel LM. Current and projected prevalence of arterial hypertension in sub-Saharan Africa by sex, age and habitat: an estimate from population studies. Journal of Hypertension 2011;29(7):1243-1252.

[2] Mancia G, Fagard R, Narkiewicz K, Redon J, Zanchetti A, Böhm M, Christiaens T, Cifkova R, De Backer G, Dominiczak A. 2013 ESH/ESC guidelines for the management of arterial hypertension: the Task Force for the Management of Arterial Hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC). Blood pressure 2013;22(4):193-278.

[3] Zalba G, San José G, Moreno MU, Fortuño MA, Fortuño A, Beaumont FJ, Díez J. Oxidative stress in arterial hypertension role of NAD (P) H oxidase. Hypertension 2001;38(6):1395-1399.

[4] Wu J, Xia S, Kalionis B, Wan W, Sun T. The role of oxidative stress and inflammation in cardiovascular aging. BioMed Research International 2014;2014:1-13.

[5] Reverri EJ, Morrissey BM, Cross CE, Steinberg FM. Inflammation, oxidative stress, and cardiovascular disease risk factors in adults with cystic fibrosis. Free Radical Biology and Medicine 2014;76:261-277.

[6] Strobel NA, Fassett RG, Marsh SA, Coombes JS. Oxidative stress biomarkers as predictors of cardiovascular disease. International Journal of Cardiology 2011;147(2):191-201.

[7] Marnett LJ. Lipid peroxidation—DNA damage by malondialdehyde. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 1999;424(1):83-95.

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[8] Cabiscol E, Tamarit J, Ros J. Oxidative stress in bacteria and protein damage by reactive oxygen species. International Microbiology 2010;3(1):3-8.

[9] Repetto M, Boveris A, Semprine J. Lipid peroxidation: chemical mechanism, biological implications and analytical determination. INTECH Open Access Publisher; 2012:1-30.

[10] Salonen JT, Nyysso K, Salonen R, Porkkala-Sarataho E, Tuomainen T-P, Diczfalusy U, Bjo I. Lipoprotein oxidation and progression of carotid atherosclerosis. Circulation 1997;95(4):840-845.

[11] Touyz R. Reactive oxygen species and angiotensin II signaling in vascular cells: implications in cardiovascular disease. Brazilian Journal of Medical and Biological Research 2004;37(8):1263-1273.

[12] Dinh QN, Drummond GR, Sobey CG, Chrissobolis S. Roles of inflammation, oxidative stress, and vascular dysfunction in hypertension. BioMed Research International 2014;2014:1-11.

[13] Touyz RM. Reactive oxygen species, vascular oxidative stress, and redox signaling in hypertension what is the clinical significance? Hypertension 2004;44(3):248-252.

[14] Ceconi C, Boraso A, Cargnoni A, Ferrari R. Oxidative stress in cardiovascular disease: myth or fact? Archives of Biochemistry and Biophysics 2003;420(2):217-221.

[15] Reiter RJ, Tan D-x, Manchester LC, Qi W. Biochemical reactivity of melatonin with reactive oxygen and nitrogen species. Cell Biochemistry and Biophysics 2001;34(2):237-256.

[16] Vásquez-Vivar J, Kalyanaraman B, Martásek P, Hogg N, Masters BSS, Karoui H, Tordo P, Pritchard KA. Superoxide generation by endothelial nitric oxide synthase: the

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influence of cofactors. Proceedings of the National Academy of Sciences 1998;95(16):9220-9225.

[17] Higashi Y, Yoshizumi M. Exercise and endothelial function: role of endothelium-derived nitric oxide and oxidative stress in healthy subjects and hypertensive patients. Pharmacology & Therapeutics 2004;102(1):87-96.

[18] Faraci FM, Didion SP. Vascular protection superoxide dismutase isoforms in the vessel wall. Arteriosclerosis, Thrombosis, and Vascular Biology 2004;24(8):1367-1373.

[19] Patel RS, Al Mheid I, Morris AA, Ahmed Y, Kavtaradze N, Ali S, Dabhadkar K, Brigham K, Hooper WC, Alexander RW. Oxidative stress is associated with impaired arterial elasticity. Atherosclerosis 2011;218(1):90-95.

[20] Medzhitov R. Origin and physiological roles of inflammation. Nature 2008;454(7203):428-435.

[21] Bruunsgaard H, Skinhøj P, Pedersen AN, Schroll M, Pedersen B. Ageing, tumour necrosis factor‐ alpha (TNF‐ α) and atherosclerosis. Clinical & Experimental Immunology 2000;121(2):255-260.

[22] Csiszar A, Ungvari Z, Koller A, Edwards JG, Kaley G. Aging-induced proinflammatory shift in cytokine expression profile in coronary arteries. The FASEB Journal 2003;17(9):1183-1185.

[23] Belmin J, Bernard C, Corman B, Merval R, Esposito B, Tedgui A. Increased production of tumor necrosis factor and interleukin-6 by arterial wall of aged rats. American Journal of Physiology-Heart and Circulatory Physiology 1995;268(6):H2288-H2293.

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[24] Mendall M, Patel P, Ballam L, Strachan D, Northfield T. C reactive protein and its relation to cardiovascular risk factors: a population based cross sectional study. BMJ 1996;312(7038):1061-1065.

[25] Harris TB, Ferrucci L, Tracy RP, Corti MC, Wacholder S, Ettinger WH, Heimovitz H, Cohen HJ, Wallace R. Associations of elevated interleukin-6 and C-reactive protein levels with mortality in the elderly. The American Journal of Medicine 1999;106(5):506-512.

[26] Crowley SD. The cooperative roles of inflammation and oxidative stress in the pathogenesis of hypertension. Antioxidants & Redox Signaling 2014;20(1):102-120.

[27] Park S, Lakatta EG. Role of inflammation in the pathogenesis of arterial stiffness. Yonsei Medical Journal 2012;53(2):258-261.

[28] Pasceri V, Willerson JT, Yeh ET. Direct proinflammatory effect of C-reactive protein on human endothelial cells. Circulation 2000;102(18):2165-2168.

[29] Pasceri V, Chang J, Willerson JT, Yeh ET. Modulation of C-reactive protein–mediated monocyte chemoattractant protein-1 induction in human endothelial cells by anti-atherosclerosis drugs. Circulation 2001;103(21):2531-2534.

[30] Van Bortel L, Spek J. Influence of aging on arterial compliance. Journal of Human Hypertension 1998;12(9):583-586.

[31] Hall JE. Guyton and Hall textbook of medical physiology. 11th_{Ed Elsevier Health}

Sciences; 2015.

[32] Mackenzie I, Wilkinson I, Cockcroft J. Assessment of arterial stiffness in clinical practice. QJM 2002;95(2):67-74.

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[33] Laurent S, Cockcroft J, Van Bortel L, Boutouyrie P, Giannattasio C, Hayoz D, Pannier B, Vlachopoulos C, Wilkinson I, Struijker-Boudier H. Expert consensus document on arterial stiffness: methodological issues and clinical applications. European Heart Journal 2006;27(21):2588-2605.

[34] Aggoun Y, Szezepanski I, Bonnet D. Noninvasive assessment of arterial stiffness and risk of atherosclerotic events in children. Pediatric Research 2005;58(2):173-178.

[35] Cecelja M, Chowienczyk P. Role of arterial stiffness in cardiovascular disease. JRSM Cardiovascular Disease 2012;1(4):11.

[36] Heitzer T, Schlinzig T, Krohn K, Meinertz T, Münzel T. Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Circulation 2001;104(22):2673-2678.

[37] Gokce N, Keaney JF, Hunter LM, Watkins MT, Menzoian JO, Vita JA. Risk stratification for postoperative cardiovascular events via noninvasive assessment of endothelial function A prospective study. Circulation 2002;105(13):1567-1572.

[38] Morris AA, Zhao L, Patel RS, Jones DP, Ahmed Y, Stoyanova N, Gibbons GH, Vaccarino V, Din-Dzietham R, Quyyumi AA. Differences in systemic oxidative stress based on race and the metabolic syndrome: the Morehouse and Emory Team up to Eliminate Health Disparities (META-Health) study. Metabolic Syndrome and Related Disorders 2012;10(4):252-259.

[39] Schutte A, Van Vuuren D, Van Rooyen J, Huisman H, Schutte R, Malan L, Malan N. Inflammation, obesity and cardiovascular function in African and Caucasian women from South Africa: the POWIRS study. Journal of Human Hypertension 2006;20(11):850-859.

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[40] Kruger R, Schutte R, Huisman HW, Hindersson P, Olsen MH, Eugen-Olsen J, Schutte AE. NT-proBNP, C-reactive protein and soluble uPAR in a bi-ethnic male population: the SAfrEIC study. PloS one 2013;8(3):e58506.

[41] Kruger R, Schutte R, Huisman H, Van Rooyen J, Malan N, Fourie C, Louw R, Van der Westhuizen F, Van Deventer C, Malan L. Associations between reactive oxygen species, blood pressure and arterial stiffness in black South Africans: the SABPA study. Journal of Human Hypertension 2012;26(2):91-97.

[42] Morris AA, Patel RS, Binongo JNG, Poole J, al Mheid I, Ahmed Y, Stoyanova N, Vaccarino V, Din‐ Dzietham R, Gibbons GH. Racial differences in arterial stiffness and microcirculatory function between Black and White Americans. Journal of the American Heart Association 2013;2(2):e002154.

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CHAPTER 2

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12 2.1 General introduction

The World Health Organization estimates that about 63% of global deaths are due to non-communicable diseases of which 30% are caused by cardiovascular disease (CVD) [1]. In sub-Saharan Africa, non-communicable diseases are also increasing at an alarming rate due to accelerated urbanization accompanied by lifestyle changes such as excess alcohol intake and smoking [2,3]. The South African population also suffers under the increased burden of CVD, as noted by the increased prevalence of cardiovascular risk factors such as hypertension [4,5], obesity [6] and diabetes mellitus [7].

Age dependent development of CVD is accompanied by stiffening of the large arteries which in turn is associated with hypertension [8], heart failure [9], stroke, coronary artery diseases [10] and arterial fibrillation [11]. These factors are known to be some of the main causes of mortality in developing countries. Black South Africans tend to have increased arterial stiffness at a younger age compared to their white South African counterparts [12,13]. This underlines the urgent need to identify markers for the early detection of arterial stiffness and CVD, to aid with the reduction of the high rate of cardiovascular mortality.

Arterial stiffness is defined as the reduced ability of the artery to expand for a given blood pressure [14] and is caused by structural changes in the arterial wall as well as functional alterations in the elastic properties and vascular smooth muscle tone [15,16]. These changes may be the result of increased oxidative stress and inflammation [17-19]. Oxidative stress and inflammation are interrelated [20,21] since oxidative injury may induce vascular inflammation, which in turn may result in endothelial dysfunction [22,23]. Endothelial dysfunction is furthermore related to increased arterial stiffness and the development of CVD [24].

This study will focus on several relevant aspects that may be important when investigating oxidative stress, inflammation and arterial stiffness.

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2.2 Reactive oxygen species, oxidative stress and the antioxidant system

2.2.1 Reactive species

Reactive species are molecules that can be produced enzymatically or through chemical reactions [25]. Reactive species play an important role as mediators of cell signaling [26,27]. During normal cellular metabolism these species are present at low concentrations [26,28] and are divided into three groups, namely reactive nitrogen species (RNS), reactive chloride species and reactive oxygen species (ROS) [29]. ROS is a term that describes radical and non-radical derivatives of oxygen (O2), whereas RNS include species that are derived from nitrogen [30].

The different types of ROS and RNS are summarized in Table 2.1 [27].

The cellular sources of ROS production are the endothelium [32,33], vascular smooth muscle cells [34] and fibroblasts within the adventitia [30], whereas vascular ROS are derived from different enzyme reactions, including nicotinamide adenine dinucleotide phosphate oxidase (NOX) [32], xanthine oxidase [35] and uncoupled endothelial nitric oxide synthase (eNOS) [36]. ROS plays an important role in regulation of cell physiology and function as well as other biological processes [37]. Under normal conditions, the rate of ROS production is balanced by the rate of elimination [38]. However, under pathological conditions an imbalance between ROS production and antioxidant activity leads to the accumulation of ROS and RNS resulting in a state of oxidative stress or nitrosative stress (Figure 2.1) [38]. ROS and RNS have been

Table 2.1 Different types of ROS and RNS (modified from Halliwell & Gutteridge et al)

[27,29,31].

ROS RNS

Name Symbol Name Symbol

Radicals Hydroxyl Peroxyl Superoxide OH• RO2• O2 •-Nitric oxide Nitrogen dioxide NO• NO2•

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identified as oxidative damage molecules, which might reflect the cardiovascular disease process, and is therefore an indication of the important role of oxidative stress in cardiovascular pathophysiology [37,39].

2.2.2 Oxidative stress and the antioxidant system

Oxidative stress is a state in which the presence of oxidants (free radicals) overwhelms the antioxidant system [40] (Figure 2.1). This imbalance may arise as a result of decreased antioxidant capacity, or excess production of reactive oxygen species (ROS) or a combination of these factors [27,41]. The effects of oxidative stress can be seen by the accumulation of products formed as a result of oxidative damage to macro-molecules such as DNA, lipids and proteins, including 8-hydroxy-2’-deoxyguanosine (8OHdG), thiobarbituric acid reactive substances (TBARS) and 3-nitrotyrosine (3-NT) [41,42]. Due to the short half live of ROS, most ROS cannot be measured directly, but the products formed as a result of oxidative damage are frequently used as indication of oxidative stress [43].

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The antioxidant system plays a potent role to combat oxidative stress and to maintain homeostasis [26]. This system consists of endogenous and exogenous antioxidants [26,29]. The exogenous antioxidants include dietary antioxidants such as vitamin C, vitamin E, ubiquinol, thiols, flavonoids and carotenoids [45]. Endogenous antioxidants consist of an enzyme system which includes superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT) and glutathione reductase (GR), whereas non-enzymatic endogenous antioxidants include reduced glutathione (GSH) (Figure 2.2) [27]. The enzyme SOD is involved in the dismutation of superoxide to hydrogen peroxide (H2O2) [46]. Glutathione peroxidase further reduces H2O2 to

water (H2O). During this reaction GSH is oxidized to gluthathione disulfide (GSSG) [27]. In order

to maintain the redox environment [47], GSSG is then reduced back to GSH by the enzyme GR at the expense of NADPH [27,48].

Figure 2.2: A schematic representation of the enzymatic antioxidant system (adapted from Li S et al.) [49]

2.2.2.1 Glutathione peroxidase activity and cardiovascular diseases

Lower GPx activity is associated with increased risk for the development of CVD in patients with coronary artery diseases [50]. The attenuation in GPx levels affects the level of peroxides. In other words, a decrease in GPx activity allows for increased intracellular levels of peroxides and

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vice versa [51]. Glutathione peroxidase deficiency has been shown to promote endothelial

dysfunction and abnormal structural changes in the vasculature [52]. Furthermore, Sorokin and colleagues have indicated that a polymorphism that decreases GPx4 activity is associated with arterial stiffness in young Russians [53].

2.2.3 Lipid peroxidation

Lipid peroxidation refers to the oxidative breakdown of lipids [54] and is considered the main mechanism involved in the oxidative damage to cell structures [55]. Lipid peroxidation occurs particular in polyunsaturated fatty acids, since they contain a large number of hydrogen chains and are more susceptible to oxidation [40,41,56]. Lipid peroxidation is a chain reaction mediated by superoxide (Figure 2.3) [55,57]. This chain reaction occurs in three phases known as the initiation phase, the propagation phase and the termination phase [54,55,57]. The initiation phase involves the abstraction of a hydrogen atom from the methyl group of the unsaturated fatty acid, producing a fatty acid radical [55,58]. The propagation phase involves the stabilizing of the fatty acid radical by rearranging the double bonds to form a conjugated diene, which then reacts with O2 to form an unstable peroxyl radical [55,57,58]. The unstable lipid peroxyl radical

further reacts with another free fatty acid to yield lipid peroxide [59]. In the termination phase two lipid peroxyl radicals conjugate with each other to form a non-radical product, thereby terminating the chain reaction [56].

Malondialdehyde (MDA), as seen in Figure 2.3, is a product of oxidized unsaturated fatty acids [60] which can react with thiobarbituric acid to form a thiobarbituric acid-reactive substance (TBARS) [61-63]. This reaction has been used as a sensitive assay for lipid peroxidation [62]. Furthermore, the process of lipid peroxide results in structural damage to membranes (endothelial lesions), loss of fluidity, decline in membrane function and increased vascular permeability [26,55,64]. In addition, lipid peroxidation inhibits the synthesis of prostacyclin, an antiplatelet-aggregation substance which may result in platelet adherence and adhesion [65].

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Figure 2.3: Steps in lipid peroxidation (adapted from Mimic-Dukic et al.) [66]

2.2.3.1 TBARS and cardiovascular diseases

Previous studies have demonstrated that elevated TBARS can predict carotid atherosclerotic plaque progression [67] and cardiovascular events in patients with stable coronary artery disease (CAD), independent of traditional factors and inflammatory markers [68]. It was also indicated that TBARS are elevated in conditions associated with CVD such as cigarette smoking [69,70], hypertension [71], hyperlipidemia [72] and diabetes [67,73].

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18 2.3 Inflammation

The immune system plays a role in protecting the body against injury or infection [74]. An imbalance in the immune system may lead to an adverse pro-inflammatory state. Low-grade inflammation is often a critical step in the progression of vascular disease [75-77] and is characterized by increased levels of pro-inflammatory cytokines and immune activation markers, as well as a raised white blood cell count [78]. Inflammatory markers such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and C-reactive protein (CRP) are associated with various forms of cardiovascular disease (CVD) [79-81]. These inflammatory markers levels were reported to be higher in black than in white Africans [82,83].

2.3.1 Inflammatory markers and cardiovascular diseases

TNF-α is released in response to T cell activation [84]. TNF-α further induce the production of IL-6 by a wide range of cells, including immune cells, endothelial cells, smooth muscle cells and ischemic heart cells [85]. Higher IL-6 levels correlate with increased arterial stiffness [86,87] and IL-6 may further lead to the release of CRP from the liver [88]. CRP plays an important role in inflammation and is currently deemed the golden standard for the measurement of low-grade inflammation [78]. CRP is also a good marker of metabolic inflammation and is associated with body mass index (BMI) and lipids such as low-density lipoprotein and triglycerides [78,89]. CRP levels of less than 1 μg/mL are considered as normal for healthy subjects, while an increase in CRP levels above 3 μg/mL is associated with greater arterial stiffness of elastic [86,90-94] and muscular arteries [95].

The mechanisms by which inflammation play a role in loss of elasticity will be discussed later in this chapter.

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19 2.4 Vascular function and stiffening

The arterial system acts as a conduit to deliver nutrients and oxygenated blood to the organs and to act as a cushion to soften pulsation of blood flow from the heart [96,97].

The arterial wall consists of three layers, the intima, media and adventitia [98] (Figure 2.4). The intima is the innermost layer and it consists of a small amount of connective tissue below the endothelium [100]. The second layer, the media, is composed of smooth muscle cells and elastin [97,98]. Lastly, the external layer, the adventitia, comprises of a large amount of collagen fibers and fewer elastin fibers [96]. The composition of the central and peripheral arteries differs, as central arteries are more compliant and contain more elastin, while the peripheral muscular arteries contain more collagen and are therefore stiffer [97]. This difference in composition is important as it aids the central vessels to maintain the Windkessel function [99] and it requires a high degree of aortic compliance [100].

Figure 2.4: Structure of the arterial wall (adapted from McPhee et al.) [101].

Arterial compliance is defined as the change in arterial blood volume (or change in diameter ∆D) per unit pressure (∆P) [102,103]. Decreased compliance is used as an indication of loss of elasticity or increased arterial stiffness [74]. Arterial stiffness is described as a reduced ability of the artery to contract and dilate at any given pressure [14] and may be influenced by the

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diameter of vessels. In other words, smaller vessels are stiffer than larger vessels because of their smaller radius [104]. Not only does it describe decreased compliance but it also relates to endothelial dysfunction [24]. The arterial wall structure may change as a consequence of age, loss of muscle attachment, fractures of elastic lamina, increase in collagen fibers, inflammation, infiltration of vascular smooth muscle, fibrosis, macrophages and calcification [105]. Arterial stiffness is characterized by structural and functional alteration of the intrinsic elastic properties of the artery and it has been indicated to be independently associated with cardiovascular morbidity and mortality in different population groups [15].

2.4.1 Vascular stiffness measurements

Various techniques are available to measure arterial stiffness along the arterial tree [106]. The non-invasive methods for the measurement of arterial stiffness include systemic, regional and local determination [107]. Systemic arterial stiffness determination includes arterial compliance (Windkessel compliance) and reflects the opposition of large arteries to the beating effects of ventricular ejection [107].

In the Windkessel model, as seen in Figure 2.5, the arterial system is compared to a fire-hose system meaning the following: the Windkessel dome acts as the large elastic arteries, the wide bore hose acts as a conduit, while the fire-hose nozzle is compared to the arterioles [97,108]. This model separates the conduit function, which supply blood flow to the peripheral tissues and organs and the cushioning function, which is able to dampen the pressure that results from the ventricular ejection [97,109]. This model is used to describe the arterial system in terms of resistance and compliance [110], as well as to illustrate the changes seen in stiffer arteries and hypertension, that is an increase in vascular resistance (TPR) and a decrease in arterial compliance or elasticity [108].

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Figure 2.5: The Windkessel model (adapted from Ghio et al.) [111].

The effect of Windkessel decreases with age because the elastic properties of the arteries become less compliant with increased age [112] due to the loss of elastin fibers and the gain in collagen fibers [113,114]. This will subsequently result in increased pulse pressure and systolic blood pressure [112,115].

2.4.1.1 Total peripheral resistance (vascular resistance)

Total peripheral resistance (TPR) is the resistance of blood flow throughout the entire systemic circulation and is calculated as follows: TPR = change in pressure /cardiac output [116]. A number of factors may influence TPR, including vascular vessel diameter, blood viscosity and blood vessel length [116]. The diameter of a blood vessel is inversely proportional to blood pressure, in other words a smaller vessel would increase vascular resistance, hence increasing TPR and vice versa [116].

Increased TPR may be the result of endothelial dysfunction [11] or structural alterations [12] in small resistance arteries. Moreover, a mechanism that causes a decrease in vasodilators, such as nitric oxide (NO), may lead to an increase in TPR [116], which may further result in an increase in systolic blood pressure and a decrease in cardiac output [109].

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Regional arterial stiffness (measured by either carotid-dorsalis-pedis pulse wave velocity, carotid-femoral pulse wave velocity or ankle-brachial pulse wave velocity) is measured at the site of the aorta, where the arterial buffering function is expressed [107]. Carotid-femoral PWV is known to be the golden standard measurement of arterial stiffness [117]. A local determination of arterial stiffness includes intima-media thickness, carotid distensibility and Young’s elastin modulus and involves measurement of cross-sectional arterial distensibility [103,107].

In this study, the focus will only be placed on Windkessel arterial compliance, as a measure of arterial elasticity, and total peripheral resistance (TPR) as a measure of vascular resistance.

2.5 Oxidative stress, inflammation and arterial stiffness

Oxidative stress and inflammation plays a role in the etiology of arterial stiffness [20]. Both oxidative stress [118] and inflammation [119-122] has been associated with endothelial dysfunction, and oxidative stress induced endothelial dysfunction may be responsible for arterial stiffening [123]. Endothelial dysfunction describes the inability of the arteries to fully dilate [124,125], and it is characterized by a reduced bioavailability of vasodilators such as endothelium-derived NO and abundance of vasoconstrictors, such as endothelin-1 [126]. Sustained inflammation may not only lead to endothelial dysfunction but also to increased ROS production [127]. When ROS is in excess, superoxide reacts with NO to form peroxynitrate [128]. This leads to oxidation of tetrahydrobiopterin (BH4), the co-factor of nitric oxide synthase

(NOS) [127]. As a consequence, this may lead to the uncoupling of eNOS, where eNOS produces superoxide rather than NO [129]. Increased superoxide further impairs NO bioavailability and leads to impaired vascular relaxation (compliance) [127]. Moreover, peroxynitrite is a highly reactive intermediate that fuels lipid peroxidation, supporting pro-atherogenic modification of LDL, leading to vascular dysfunction and atherosclerosis [130].

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In addition, inflammation may also be promoted by oxidative stress by activating nuclear factor kappa B (NFκB) [19,38], which in turn stimulate further expression of pro-inflammatory cytokines, platelet aggregation and cell adhesion molecules, leading to plaque formation and fibrosis [5].

In summary (Figure 2.6), oxidative stress plays a role in inflammation [20,21]. The relationship between these two factors is interrelated, since oxidative stress may induce vascular inflammation, which may in turn further trigger oxidative stress [22]. Both factors are related or may lead to endothelial dysfunction [21,127], which in turn may lead to the expression of vasoconstrictors, pro-inflammatory, proliferative and pro-coagulation factors [131,132]. This may result in decreased arterial compliance and increased vascular resistance [133].

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Figure 2.6: Summary

2.6 Aims and hypothesis

2.6.1 Aims

The following are the aims of this study:

To compare markers of oxidative stress, antioxidant capacity, inflammation, arterial compliance and total peripheral resistance between black and white South Africans of the North-West Province; and

to investigate associations of oxidative stress, antioxidant capacity and inflammatory markers with arterial compliance and total peripheral resistance in black and white South Africans.

2.6.2 Hypotheses

Based on the literature the following hypotheses have been formulated:

 Oxidative stress (TBARS), inflammation (IL-6, CRP) and total peripheral resistance (TPR) are higher, while arterial compliance (Cwk) and antioxidant capacity (GPx) are lower in black compared to white participants.

 Cwk is negatively associated with TBARS and inflammation (IL-6, CRP) while TPR is positively associated with TBARS and inflammation (IL-6, CRP) in black and white participants.

 Cwk is positively associated with antioxidant capacity (GPx) while TPR is negatively associated with antioxidant capacity (GPx) in black and white participants.

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The relationship between markers of oxidative stress, inflammation and arterial compliance in a bi-ethnic population : the SABPA study