Endothelial cells (EC) are the simple squamous epithelium that lines blood and lymphatic vessels. The endothelium partitions the blood from the intima and media of the arteries and veins, and from the interstitium of tissues throughout the body. The endothelium is responsible for determining the movement of macromolecules, and also directed migration of circulating cells from the blood into extra vascular tissues.
The major endocrine and paracrine functions of the endothelium are to regulate the vascular tone and this is done through the production of vasoconstrictive and vasodilatory molecules such as endothelin [ET]-1 (La and Reid, 1995) and nitric oxide respectively (Moncada et al., 1987; Palmer et al., 1987); to regulate platelet function; maintain blood fluidity and finally to control inflammation. Other functions include a number of cellular activities such as maintaining homeostasis of solutes, hormones and macromolecules,` and preventing the formation of thrombus (Cines et al., 1998; van Hinsbergh, 1997).
Endothelial dysfunction can be due to cell injury which can be induced by a number of different mechanisms which include bacterial (Schouten et al., 2008), oxidative stress through abnormal regulation of reactive oxygen species (ROS) (Kojda and Harrison, 1999), environmental irritants such as smoking (Oida et al., 2003) and hyperlipidaemia (Toma et al., 2009; Zeman et al., 2006).
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Therefore EC dysfunction can lead to compromise of vasoregulation (Furchgott and Zawadzki, 1980; Motz et al., 1991), increased blood coagulation (Cerinic et al., 2003), promote infiltration of inflammatory cells and lipids into the intima (O'Brien et al., 1996), dysregulation of the production of nitric oxide and increase smooth muscle cell migration and proliferation (Dubey et al., 1995). The endothelium is responsive to stimuli from circulating blood, neighbouring cells and tissues. Loss of normal function, both biochemical and physiological, is associated with disease.
Injury to endothelial cells can be caused by a variety of different sources such as viral infection (Patel et al., 1995), homocysteine (Harker et al., 1976; Zhang et al., 2001), reactive oxygen species (Kojda and Harrison, 1999), oxidised lipids (Rubbo et al., 2002) and hypoxia (Santilli et al., 1991). The responses generated as a result of these stimuli are leukocyte adhesion, altered permeability, procoagulation activity, production of vasoactive substances, and growth factor/cytokine release.
1.2 Nitric Oxide Functions
Under normal conditions the production of Nitric oxide (NO) by the vascular endothelium is important in the regulation of blood flow. Vascular action of NO includes direct vasodilation (Ignarro et al., 1987), indirect vasodilation (Steinberg et al., 1994), anti-thrombotic effects, anti-inflammatory effects, anti-proliferative effects and modulates neutrophil adhesion to exposed arterial media and the vascular endothelium (Provost et al., 1994; Radomski et al., 1987). NO also prevents the oxidative modification of low density lipoprotein cholesterol (Rubbo et al., 2002).
NO is a signalling molecule that is produced by the endothelial isoform of nitric oxide synthase (eNOS) (Lowenstein and Michel, 2006; Palmer et al., 1988). In the vascular system NO activates a cascade of events that lead to the relaxation of smooth muscle which in turn leads to a decrease in blood pressure (Palmer et al., 1987). NO serves as a potent neurotransmitter at neuronal synapses (Garthwaite et al., 1988).
In normal physiology NO is known to have an anti-inflammatory effect but can be a pro-inflammatory mediator in irregular conditions such as inflammatory lung disease (Grisham et al., 1999) so therefore can be implicated in the immune response (Beckman and Koppenol, 1996). NO is a contributing factor to the non-adhesive properties of the vascular endothelium meaning that it inhibits platelet adhesion to vascular endothelium (Provost et al., 1994; Radomski et al., 1987).
1.3 Inflammation & Reactive Oxygen Species
Inflammation can be defined as a protective localised response of tissue that eradicates the agents, debris or injury that cause the inflammation and can be linked intimately to repair. I.e. the recruitment and activation of leukocytes (Kelly et al., 2007). During a normal inflammatory response highly unstable superoxide radicals are generated by leukocytes, which is known as a respiratory burst.
Production of superoxide in phagocytes is mediated by the NADPH-oxidase complex (Babior, 1999; Takeya and Sumimoto, 2003). In quiescent cells this system exists in a disassembled state but following activation of the phagocyte the multi protein system comes together to produce superoxide (Babior, 1999; Robinson, 2008). The NADPH-oxidase complex also exists in the endothelium, therefore superoxide and ROS are also produced by the endothelium (Al-Mehdi et al., 1998). Superoxide dismutase is the enzyme that clears superoxide radicals from general circulation by converting it to oxygen and hydrogen peroxide. Dysfunction of superoxide dismutase can be attributed to development of high volume hypertension (Jung et al., 2007).
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Overproduction of ROS leads to oxidative stress. NADPH oxidases (Takeya and Sumimoto, 2003), xanthine oxidase (Godber et al., 2000; McNally et al., 2003), uncoupled eNOS (dysfunctional eNOS) (Verhaar et al., 2004) and the leakage of "activated" oxygen from the mitochondria during oxidative respiration (Ballinger et al., 2000) are all important sources of superoxide in the cardiovascular system. Cellular sources of these enzymes mentioned
1.4 Peroxynitrite Production and Its Contribution to Disease
Inflammation after tissue injury can be associated with the increased generation of ROS such as the anion superoxide (O2-) and nitric oxide (NO). Peroxynitrite (PN) is generated by the reaction between nitric oxide (NO) and anion superoxide (O2-) (Pryor and Squadrito, 1995; Wang and Zweier, 1996). It is a powerful oxidant that can readily react with many cellular components (Hurst and Lymar, 1997). PN can easily penetrate the phospholipid bilayer and produce substrate nitration, which leads to inactivating receptors and enzymes such as free radical scavengers (Harrison, 1997; van der Loo et al., 2000).
PN can interact with target molecules directly through one or two electron oxidation processes (thiols and iron sulphur centres in proteins) or by the generation of radicals. PN breaks down into highly reactive products, nitrogen dioxide (Â·NO2), hydroxyl radical (Â·OH) and carbonate radical (CO3Â·âˆ’). These PN derived radicals can oxidise proteins and nitrate tyrosine residues (Alvarez and Radi, 2003), and also induce cell membrane lipid peroxidation (Hall et al., 2004).
Melatonins, Flavonoids, Mercaptoethylguanidine are all known scavenger of peroxynitrite (Gilad et al., 1997; Haenen et al., 1997; Zingarelli et al., 1998). Direct scavengers react with the PN anion and increase the rate at which the anion is decomposed at. On the other hand indirect scavengers scavenge the secondary reactive species produced by PN.
1.5 Atherosclerosis Formation
Atherosclerosis is formation of lipid deposits in the tunica media which is associated with damage to the endothelial lining. The cells involved in atherosclerosis are the endothelial cells, monocytes, macrophages and platelets. The mediators involved are oxidised LDL, growth factors and ROS. Atherosclerosis is a chronic inflammatory fibroproliferative disease and can be dangerous since a restricted blood flow can lead to vital organs being deprived of oxygen and nutrients which could lead to necrosis of tissue. Both early and advanced formation of atherosclerosis can be associated with endothelial cell dysfunction (Pettersson et al., 1993; Ross, 1999).
The process of atheroma formation is called atherogenesis and contains a number of steps. The first step is injury to the endothelium occurs, this leads to the up regulation of adhesion molecules (Price and Loscalzo, 1999). Monocytes then infiltrate the area which leads to macrophages forming foam cells (Bobryshev, 2006; Gerrity, 1981). Smooth muscle cells then migrate and form extra cellular matrix. This leads to the formation of a fibro fatty lesion. Arterial calcification then occurs (Doherty et al., 2003).
PN has been attributed to a wide variety of inflammatory diseases. Reduction in normal detoxification mechanisms of PN and endothelial dysfunction lead to a cycle of chronic inflammation which triggers further generation PN. The overload of PN on both the endothelial cells and the vascular smooth muscle leads to the formation of atheroma (Pacher et al., 2007).
In unregulated chronic inflammatory conditions such as atherosclerosis there is an excessive production of peroxynitrite and also accumulation of free radicals (White et al., 1994). Peroxynitrite releases zinc from the zinc thiolate cluster of endothelial eNOS; disruption of eNOS leads to decreased synthesis of NO and increases superoxide anion production by the enzyme contributing to increased cellular stress (Zou et al., 2002). Superoxide dismutase was found to be inactivated by peroxynitrite (Grzelak et al., 2000). Superoxide dismutase converts superoxide to oxygen and hydrogen peroxide; therefore if this enzyme is inactivated further inflammation can occur as superoxide is not broken down.
Since the levels of NO which normally stop oxidation of LDL, are decreased because of eNOS dysfunction, there will be an increase in oxidation of LDL which has been implicated as a major mechanism in the atherosclerotic process. (Rubbo et al., 2002; Steinberg, 2009; Steinberg and Witztum, 2002).
An additional risk factor for the development of atherosclerosis is the reduction in bioavailability of NO which can be caused by numerous factors. Firstly due to NO combining with O2- to form PN. Secondly also known to reduce bioavailability of NO is homocysteine by conversion to hydrogen peroxide (Upchurch et al., 1997) and finally decreased amounts of expression of eNOS (Wilcox et al., 1997).
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1.6 Map Kinases
Mitogen activated protein kinases (MAPK) are a family of Ser/Thr protein kinases which are widely conserved amongst eukaryotes (Zheng and Guan, 1994). MAPK are involved in essential cellular processes such as cell proliferation, differentiation, movement and death. The system is organised into a three tier cascade signalling system (Junttila et al., 2008). MAPKKK (MAP-kinase-kinase-kinase) are activated by phosphorylation by interaction with a small family of GTPases which connect the MAPKKK to the cells surface receptor which has been stimulated by an external stimuli (Zhang et al., 1995). The MAPKK is then phosphorylated by the MAPKKK which in turn phosphorylates the MAPK. Figure 1 shows this in detail for the p38 pathway and the transcription factors that are activated as a result.
MAPKs are activated by dual phosphorylation specificity kinases by phosphorylation of threonine and tyrosine in a Thr-Xaa-Tyr motif in a loop near the active site. In p38 the intervening amino acid is Gly and this kinase is activated by cellular stress (Raingeaud et al., 1995).
1.7 P38 MAP Kinase
Five members of the p38 group of MAP kinases have been characterised and clones; they are p38, also known as p38Î± (Han et al., 1994), p38Î²1 (Jiang et al., 1996), p38Î²2, (Enslen et al., 1998), p38Î³ (or ERK6, SAPK3), (Lechner et al., 1996; Li et al., 1996) and p38Î´ (or SAPK4) (Jiang et al., 1997), which are expressed differentially between different cell types. The p38Î± and p38Î² isoforms are expressed ubiquitously in adult tissue. The expression of p38 Î³ is mainly restricted to skeletal muscle whereas p38 Î´ can be predominantly found in lung, kidney, pancreas, placenta and testis.
Figure 1 The p38 activation pathway. From the pathway it can be seen that p38 not only activates other kinases but also leads to gene expression of transcription factors in the nucleus. Activation of the various transcription factors leads to a range of responses listed in the figure.
MLK3, TAK, DLK, MKK1-4
Î±, Î², Î³, Î´
P38 protein has a wide spectrum of downstream targets which allows it to mediate a range of responses. (See figure 1). The MAPK signalling pathway plays an important role in inflammation and other physiological processes. Inhibitors of p38 Î± and Î² MAPK block production of the major inflammatory cytokines i.e. Tumour Necrosis Factor (TNF) Î± and IL-1 (Xu et al., 2001) but TNF induced p38 MAPK, mediates the phosphorylation of Bcl-x(L) in endothelial cells which leads to degradation of Bcl-x(L) in proteasomes and successive induction of apoptosis (Grethe et al., 2004).
The major function of the pathway is post transcriptional control of inflammatory gene expression. Many of the mRNAs are unstable because of AU-rich elements in the 3' untranslated region. Signalling in the p38 pathway counteracts these and therefore stabilises the mRNAs by prevent their otherwise rapid degeneration (Winzen et al., 1999). Inhibiting the p38 MAPK pathway leads to suppression of the production of the key mediators in inflammation it is seen as an obvious target for therapy of chronic inflammatory diseases.
During oxidative stress induced cellular injury, p38 has been shown an important role in a pro-apoptotic signalling system (Zhou et al., 2006).
Hypoxia induces Discoidin domain receptor-2 (DDR2) expression in vascular smooth muscle cells (VSMCs) at the transcriptional level, which is mediated by the p38 MAPK pathway and contributes to VSMC migration. Hypoxia-induced VSMC migration may contribute to the pathophysiological effects of hypoxia on the vasculature and, thus, may play a role in the development of atherosclerosis (Chen et al., 2008).
PN is known to activate MAPK pathways. PN can target EPGFR (Epidermal Growth Factor Receptor) Raf-1 and MEK independently of each other (Zhang et al., 2000).
Enhanced p38 MAPK signalling in the cardiomyocytes has shown to lead to dysfunction, promoting the growth of individual cardiomyocytes which is a contributing factor in the development of ischemia. The inhibition of this pathway has shown to have a protective effect in the cardiovascular system (Barancik et al., 2000). During ischemia p38 MAPK is activated by the scaffold protein TAB1, not by the MKK signalling system (Ge et al., 2002; Tanno et al., 2003). Inhibition of angiogenesis and induction of apoptosis requires activation of CD36 by p38 MAPK (Jimenez et al., 2000). Further finding show that the CD-36 dependent signalling pathway is initiated by oxLDL is necessary for foam cell formation (Rahaman et al., 2006).
PN is known to induce detrimental effects in a range of tissues using various signalling pathways. In ischemic heart muscle, PN is known to stimulate p38 MAPK activation and in vitro this has shown to lead to necrosis of the tissue (Tanno et al., 2003). Homocysteine mediated apoptosis in trigeminal sensory neurones can be induced by addition of peroxynitrite and nitric oxide (Williams et al., 2008).
PN is also known to generate a concentration and time dependent activation of ERK in cardiomyocytes which is a secondary to the upstream activation of MEK 1 (Pesse et al., 2005).
Current studies on peroxynitrite and endothelial function both show that they are key mediators in events leading to the development of atherosclerosis. Studies on p38 show that its activation leads to apoptosis in different tissues. A definitive link between p38 MAPK signalling and activation in endothelial cells has yet to be identified.