Cardiovascular disease (CVD)

Cardiovascular disease (CVD) is a common term that is employed to describe diseases of the heart and blood vessels. In Europe, CVD is categorized as the main cause of mortality accounting for about four million deaths per year, nearly half of all deaths (48%) each year in Europe (Allender et al., 2008; Chong et al., 2010). Many environmental and genetic factors can cause this chronic and multi-factorial disease.

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Well-known environmental factors which cause and/or enhance the CVD risk are high saturated fat consumption, hypertension, diabetes mellitus (DM), hyperlipidemia, physical inactivity and smoking (Huang, 2009, Vauzour et al., 2010). Some of these risk factors can be controlled or modified, for example lifestyle modification; changing dietary habit and physical activity behaviour both led to decreasing CVD risk (Chong et al., 2010), or if appropriate by pharmacological treatment (Kahn et al., 2005, Huang 2009). Many studies have studied the relationship between diets rich in fruit and vegetables and decreasing CVD incidence. Fruits and vegetables contain many compounds that may have cardio-protective effects such as, folate, vitamin and non-nutrient phytochemicals, such as flavonoids and carotenoids (Chong et al., 2010).

1.5.1 Endothelial cell function

Endothelial cells are arranged in a single layer known as the endothelium, separating the vascular lumen from the smooth muscle cells of the vessels. The endothelium is a semi-permeable barrier which helps in water and nutrient exchange between the blood and the vessel wall (Celermajer, 1997). Endothelial cells play a fundamental role in the regulation of vascular tone and homeostasis (Bonetti et al., 2003). Responding to different hemodynamic and hormonal factors, the endothelium releases various vasoactive mediators to maintain vascular tone (Celermajer, 1997, Hinderliter and Caughey, 2003), namely nitric oxide (NO) as a vasodilator and endothelin-1 (ET-1) as a vasoconstrictor (Widlansky et al., 2003, Shenouda and Vita 2007).

NO derived from endothelial cells is a strong vasodilator, and plays a crucial role in modulating both vascular tone and BP (Vallance and Chan, 2001, Moncada and Higgs, 2006). NO may play a crucial role in regulatory of atherosclerosis development, including restricting the activation of platelet aggregation, adhesion of leukocytes to the endothelial surface, synthesis of pro-inflammatory cytokines and vascular smooth muscle cell growth (Widlansky et al., 2003, Rush et al., 2005, Esper et al., 2006).

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NO is produced after the amino acid from arginine is oxidised (also producing L-citrulline), catalysed by endothelial nitric oxide synthase (eNOS) which can be found in caveolae (invaginations in cell membranes), in the presence of many cofactors including oxygen, tetrahydrobiopterin (BH4) and nicotinamide adenine dinucleotide phosphate (NADPH), flavan adenine dinucleotide (FAD), flavan mono nucleotide (FMN), and one heam group. The enzyme eNOS is activated to produce NO either by increase in blood flow (hyperaemia), which results in the increase of shear stress subsequently increasing intracellular Ca²⁺ (Davignon and Ganz, 2004), or by alteration in a number of chemical factors, such as histamine, bradykinin, acetylcholine (Moncada and Higgs, 1993, Kone et al. 2003, Fonseca et al., 2004, Kawashima and Yokoyama, 2004, Stuehr and Griffith, 2006).

From the endothelial cells, NO will diffuse across the cell membrane into the smooth muscle cells in the vascular wall, where it activates guanylate cyclase resulting in an increase in cyclic guanosine 3,5-monophosphate (cGMP) level. This leads to smooth muscle cell relaxation (Vallance and Chan, 2001), as shown in figure 1.4.

Figure 1.4: Vasoactive mediator effects on endothelial cells. Taken from Tousoulis et al., (2005).

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Low levels of NO bioavailability are recognised as an important factor in dysfunction of endothelial cells (Malek et al., 1999, Vita and Keaney 2002).

Alterations in normal function of endothelial cells play a fundamental role in pathogenesis of CVD. Thus atherosclerotic plaque formation, thrombosis, inflammation and vasoconstriction are all known as dysfunction of endothelial cells (Widlansky et al., 2003, Bonetti et al., 2003, Huang and Vita, 2006; Shenouda and Vita, 2007). Cardiovascular risk factors which raise vascular production of reactive oxygen species (ROS), and decrease the endothelial nitric oxide availability are shown in Figure 1.5. These include: reduced eNOS activity due to increased ADMA levels (an endogenous eNOS inhibitor), resulting from redox-sensitive inhibition of DDAH; eNOS “uncoupling” due to increased oxidation of the cofactor BH4; and inactivation of NO by reaction with superoxide resulting in peroxynitrite (ONOO‾) (Landmesser et al., 2004). In fact NO bioavailability can be effected by many reasons such as decreased expression of eNOS, by altered activation of eNOS, or reduced level of eNOS cofactors (Venugopal et al., 2002, Förstermann and Münzel, 2006), as well as increased synthesis of ROS from oxidative stress (Barua et al., 2003, Mudau et al., 2012).

Figure 1.5: The major cardiovascular risk factors increase endothelial dysfunction through decreasing the endothelial NO availability, reproduced from (Landmesser et al., 2004).

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When there is deficiency in the enzyme cofactors or L-arginine, eNOS can raise the superoxide anion (O₂^-) and oxygen radical production. The O₂^- in the absence of antioxidant defence mechanisms will react with NO to form peroxynitrite (ONOO^-) or hydrogen peroxide (H2O2) decreasing NO bioavailability (Vanhoutte, 2003, Spieker et al., 2006).

Dysfunction of endothelial cells is an early contributor to atherosclerosis (Bonetti et al., 2003). It can therefore be an indicator for probable vascular complications (Anderson, 2004, Esper et al., 2006,). Endothelial cell dysfunction is considered as an independent vascular risk factor that can be treated by medication, abstinent smoking, and increased physical activity. In addition to improving food habits and the healthy dietary food consumption rich in flavonoid compounds tends to reverse endothelial cell dysfunction, and thus reduce the CVD risk.

1.5.2 Atherosclerosis

Atherosclerosis is a potentially serious condition whereby arteries become clogged up by fatty substances known as plaques or atheroma (Martini et al., 2012).

Atherosclerosis is a chronic vascular disease which entails thickening and toughening of arterial walls and narrowing of arterial lumen. As a result, blood flow and thus oxygen and nutrient supply to the body’s tissues is diminished.

Atherosclerosis is mainly observed in large and medium-sized arteries.

Atherosclerosis pathogenesis requires several factors, such as vascular inflammation, endothelial dysfunction, and vascular cell and immune system interaction (Napoli et al., 2006).

Atherosclerosis pathogenesis (theroma formation) comprises many steps as shown in figure 1.6. Firstly, low-density lipoprotein (LDL) accumulates within the intima layer where it is oxidized and modified. This gives rise to the release of leukocyte adhesion molecules by endothelial cells, which results in macrophages in the intima engulfing the modified LDL (De Winther and Hofker, 2000) eventually forming lipid-containing macrophages foam cells. It is the accumulation of foam cells which leads to fatty streaks. The fatty streak is covered by a cap of both smooth muscle cells and collagen matrix which forms the plaque. As the plaque grows, the blood vessel lumen narrows restricting the flow. The plaque might also rupture

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which is of particular importance since it plays a crucial role in causing of ischaemic heart disease.

1.6 Dietary flavonoids and cardiovascular disease