DOCUMENTACIÓN DEL SISTEMA DE GESTIÓN DE CALIDAD

diabetes, atherosclerosis and coronary heart disease (245).

1.6.1 Inflammation related vascular changes in ageing and CV

risk

Chronic inflammation is strongly associated with arterial ageing and occurs in the absence of any microorganisms and with little or no white blood cell

infiltration (246). Phenotypic shifts in arterial ECs and VSMCs during the ageing process, promote pathogenic inflammation (247-250). Most of the changes in the vessel wall during ageing are also associated with inflammation including;

endothelial disruption, enhanced VSMC migration and proliferation and matrix calcification/amyloidosis/glycation (246). Vascular inflammation is also related to the pathogenesis of hypertension and atherosclerosis. Age-associated arterial pro-inflammation is to some extent modifiable and may have the potential to ameliorate or retard age-associated arterial diseases. The transcription and activity of ACE1 and chymase (both increasing Ang II production) increases with age (251). This leads to increased Ang II in older arteries (30 months old rats) (252) and is also associated with up regulation of AT1 receptor expression in old coronary arteries (246;252).

Ageing is also associated with increased aldosterone/mineralocorticoid receptor (MR) signalling and increased sensitivity of MR to aldosterone thus increasing MR activity (252;253), promoting a pro-inflammatory phenotype via an extracellular signal-regulated kinase 1/2/mitogen-activated protein kinase/epidermal growth factor receptor (ERK/MAPK/EGFR)-dependent pathway (253). Moreover

aldosterone mediated increase in the expression of EGFR in VSMCs also reinforces the inflammatory effects (253).

In contrast, the key defence system of antioxidant enzymes protecting against the cytotoxic effects of oxidative stress is downregulated with age. This is due to the inactivation of transcription factors of detoxifying and antioxidant genes by ROS in the vasculature of older animals (254). Similarly levels of the

antioxidant enzymes like glutathione are reduced in old age as compared with young animals (254).

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The relationship between inflammation and endothelial dysfunction explained above is biologically plausible but does not confirm causality. Chronic low grade inflammation promotes cellular and biochemical changes in vessel wall which favour endothelial dysfunction (246). Low grade inflammation also decreases basal NO production promoting increased expression of cell-surface adhesion molecules for leucocytes and platelets, promoting interaction between these cells and the vascular endothelium, and inducing pro-coagulant activity (255). In addition TNF-α (a pro-inflammatory factor) has been shown to reduce the half- life of the mRNA encoding endothelial NO synthase (256). Cumulatively these changes may cause endothelial dysfunction and increase the likelihood of vasospasm, thrombosis and vessel occlusion (255).

1.6.1.1 Inflammation and phenotypic shift of vascular cells in ageing

Low grade chronic inflammation has been suggested as the key to most of the age-related alterations in arterial structure and function such as diffuse intima- medial thickening, increased stiffening and VSMC migration, proliferation and senescence (246). Many characteristics of vascular ageing like endothelial dysfunction, oxidative stress and increased apoptosis can be reproduced by recombinant TNF-α and chronic infusion of Ang II (246;252). These pro-

inflammatory molecules increases activity of pro-inflammatory molecules, for example, MMP-2, MCP-1, TGF- β1, NADPH oxidase and calpain-1, affecting the arterial wall cells and matrix and leading to adverse arterial restructuring (246;252). Continuous ACE inhibition, AT1 blockade and/or inhibition of MMPs from an early age delays the progression of age-associated aortic remodelling in animal models, by markedly inhibiting the pro-inflammatory molecules (246).

Age-related vascular changes involve inflammation as an intermediary step. The phenotypic shift in different vascular cells will be discussed in relation to both ageing and inflammation in the following section.

Endothelial cells

Cellular senescence is a condition in which the cell is metabolically active but loses the ability to proliferate. With each cell division the telomere length is shortened until a critical length is exceeded, at which cell signalling is triggered

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for the arrest of cell proliferation and start of senescence and apoptosis (257). Telomere dysfunction and endothelial senescence are related to increased ROS, decreased NO, and increased production of pro-inflammatory molecules (258). The senescent endothelial cells impact negatively on neighbouring cells; further enhancing endothelial dysfunction (258).

In vitro, the number of cellular replications is correlated with a decrease in NO synthase and an increase in the number of monocytes adhering to the ECs (257). Ageing also increases the sensitivity of the endothelium to apoptotic stimuli. Oxidized LDL also increase the inflammatory activity more than three times in old cells as compared to young cells (259).

ECs are in direct contact with the blood and carry the components of the pro- inflammatory burden that originates within the circulation. The Ang II, MCP-1, and MFG-E8 inflammatory load is increased in ECs isolated from the vessels of older animals (246;258). This pro-inflammatory state enhances ROS generation, which damages endothelial mitochondrial DNA and also interferes with the mitochondrial life cycle (246-250). All of these mechanisms initiate, and also promote EC senescence and apoptosis (247-250). MMPs break down the ECM ultimately damaging basement membrane and old enlarged ECs are likely to detach from the damaged basement membrane (247-250). The disrupted basement membrane is more likely to recruit and also concentrate the

inflammatory factors such as Ang II and MFGE8, which form a local inflammatory focus that disturbs EC (247;248). The pro-inflammation and associated cellular and micro environmental changes lead to endothelial dysfunction and are also the perpetrators of enhanced permeability, infiltration, pro-thrombosis or coagulation within the vessel wall (247;248).

Telomerase transfection (introducing nucleic acids into cells) which stabilises the expression of telomerase in EC, induces a younger EC phenotype with an increase of NO synthase and higher NO activity (257). Ageing and endothelial dysfunction are also associated with a reduction of vascular expression of Sirtuin (SIRT), with lower SIRT1 lead to a reduction in the capacity for vascular repair in the elderly. SIRT1 is a key sensor system for regulating EC survival, proliferation and senescence and may possess beneficial effects against ageing-related

43 Vascular smooth muscle cells (VSMC)

Old VSMCs (i.e. isolated from older animals) lose their contractile function and instead become stiffened and develop heterogeneous phenotypes within the arterial wall. In addition to change in phenotype VSMC also have changes in other characteristics such as pro-inflammatory secretion, senescence, proliferation, migration, and ECM deposition (247-250).

Senescence and secretion

In arteries from older animals, both proliferative and senescent VSMC subsets coexist. When old VSMC enter an irreversible growth arrest, it is known as cellular senescence (246). Ang II is known to play a role in VSMC senescence through induction of stress induced premature senescence (SIPS) or telomere shortening (261;262). Both SIPS and progressive telomere shortening

consequently leads to activation of the DNA damage machinery and p53 enzymes (261-263). Ageing changes the VSMC phenotype from contractile to secretory and VSMC derived from arteries of old non-human primates show increased

expression of the age-associated arterial secretory phenotype (AAASP) (247;264). The AAASP in old cells is associated with increased secretion of IL-1β, IL-6, MCP- 1, and TNF-α (264). Similar to the AAASP of old untreated cells, young VSMCs when treated with Ang II, also secrete a large amount of pro-inflammatory factors, including MFG-E8 (247;265). The AAASP likely delivers signals to the neighbouring VSMC (in a paracrine/juxtacrine manner), enhancing the phenotypic shift with ageing (246).

Proliferation

VSMC proliferation increases with age and has been proposed to be due to

imbalance of calcium homeostasis or platelet derived growth factor (PDGF) gene over-expression (266). Moreover, old cultured VSMCs have an increased

replication rate compared to young cells (267). Old cultured VSMCs have a

greater percentage of cells in the S and G2/M phases, and a lower percentage in the G0/G1 phase of the cellular life cycle, compared to young cells (267). MFG- E8 increases in vessels with age and by Ang II, and triggers phosphorylation of ERK1/2 which enhances proliferation signalling in young cultured VSMCs. In

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contrast MFG-E8 silencing, or the blockade of ERK1/2 phosphorylation in young cells reduces inflammation and decelerates the cell cycle S phase, conferring a reduction in proliferative capacity (267). Oxidative stress and sympathetic

activity increases with age and both might play an important role in modification and proliferation of the muscle (258;268).

Migration/invasion

The migration/invasion of medial VSMC, into the arterial intima is a key cellular event in age-associated diffuse intimal thickening. Old VSMC cease to interact normally with the ECM due to changes in ECM composition or due to change in the expression of integrins (269). In addition, the capacity of invasion of VSMC increases many fold with ageing and has also been demonstrated in cultured old VSMC, via increased activation of MMP-2/-9 (247-250;270). Similarly exposure of cultured young VSMCs to Ang II, MFG-E8, calpain-1, PDGF-bb, or MCP-1 enhances invasive capacity to levels observed in untreated old cells (246;271;272). MFG-E8 silencing RNA considerably reduces the expression of MCP-1, PDGF, and the PDGF receptor and also reduces VSMC invasion capacity (265;267). Collectively MFGE8 inhibition can be used in future as a target to reduce proliferation and invasion of VSMC.

1.6.1.2 Changes in the vascular extracellular matrix (ECM) with ageing

The ECM is a complex mixture of structural proteins and glycoproteins, including collagens, elastins, fibronectins, and proteoglycans. Its main function is to provide and maintain the structural framework which is essential for the

functional properties of the vessel wall. The maintenance of three dimensional organization of the ECM contents especially elastin, collagens, proteoglycans and structural glycoproteins are essential for optimal vascular functions (273). In healthy (uninjured) vessels some proteases are constitutively expressed but their activity is controlled by inhibitors and balance is maintained. This balance is lost due to ageing and other vascular pathologies and there is induction of matrix metalloproteinase gene expression, activation of zymogens and secretion of enzymes by inflammatory cells (273). VSMCs have the ability to respond to these injurious stimuli and synthesize ECM (including collagen types I, II and III) and protease inhibitors but the three dimensional organization of the newly

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synthesized ECM is never functionally optimal (273-275). In old VSMC, MMP-2- activated TGF-b1 signalling is involved in the increased collagen I, II, and III production (274;275). In contrast some pathological conditions overcome the VSMC response and the quantity of ECM decreases (273).