Historia de la calidad

Vascular calcification is characterised by the extracellular deposition of calcium in the vascular wall. It is a complex biological process that is based on a

continuous balance between promoting and inhibiting factors (519;520). Cell types potentially involved in calcium deposition with the vessel wall include VSMCs (521), interstitial valve cells (522), circulating osteoprogenitor cells (523) and mesenchymal pluripotent cells (524).

1.7.8.1 Types of vascular calcification

Two main types of extracellular vascular calcification are recognized, intimal and medial (122). Intimal calcification is primarily associated with

atherosclerosis and appears as punctate and disorganized mineral deposition in the intima. Intimal calcification forms an important part of atherosclerotic plaques, which mainly constitute VSMCs, lipids, macrophages, connective tissue, and necrotic debris (525).

Coronary artery calcification is very important in the development of CVD and predominantly affects the intima (526). A major complication of atherosclerosis is plaque rupture followed by serious sequelae including myocardial infarction (MI) and stroke. Although the role of plaque rupture is certain, the direct contribution of calcification to plaque rupture is still unclear as recent studies suggest that the distribution of calcification, rather than its mere presence, may predispose to plaque rupture. It has been found that diffuse and speckled micro calcium deposits (spotty calcification) are associated with greater risk of plaque rupture (527;528). Hypertension is an independent risk factor for the

development of atherosclerosis and is also associated with acute plaque rupture; by increasing the pulsatile mechanical stress on plaques (529).

Medial calcification is predominantly associated with ageing, diabetes mellitus, hypertension and uraemia, and morphologically appears as organized mineral deposition along the elastic lamellae of media. Medial calcification and

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as BP stress on vascular walls promotes calcification (530). Medial calcification decreases vascular elasticity and increases stiffness which accelerates pulse wave velocity and contributes to increases in BP (531;532). Patients with

resistant hypertension also have exaggerated arterial stiffness and calcification (533). Apart from other factors arterial calcification is independently associated with arterial stiffness. This association between arterial calcification and

arterial stiffness is mainly due to medial calcification as intimal calcification (atherosclerosis) has only a modest association with arterial stiffness (534).

Vascular calcification is quantified by non-contrast computed tomography (CT). It is a sensitive method of measuring total vessel calcium content but is not specific as it does not distinguish between intimal and medial mineralization (529).

1.7.8.2 Calcification and VSMCs

Vascular calcification is now recognized as an active and regulated process and has similarities with developmental osteogenesis. VSMCs are the chief

modulators and orchestrator of vascular calcification. In response to stress stimuli or any damage signals (such as hyperphosphatemia, oxidative stress and inflammation) VSMCs change their phenotype (as a repair mechanism) and transforms to an osteogenic/calcifying phenotype and are then called calcifying vascular cells (CVCs) (535;536). This VSMC modification is also accompanied by increase in inflammatory cytokines and oxidized lipids along with mineral

imbalance (530). Moreover, VSMC phenotypic transformation is also accompanied by the secretion of micro vesicles (MVs) which are integral to calcification

process (537). Osteogenic VSMCs have the capacity to secrete an osteoid-like matrix which can calcify (529). The release of MVs by VSMC is one of the earliest events in calcification. Researchers have identified two populations of MVs: 1) relatively large apoptotic bodies (200–800 nm) derived from dying cells, and 2) smaller MVs (50–150 nm) released by living VSMCs, particularly in response to calcium stress; both furnishing nucleation site for crystals (537).

Potent inhibitors of calcification, such as matrix GLA protein (MGP) and inorganic pyrophosphate (PPi), are locally produced (by vascular cells) and expressed in the arterial wall and prevent mineralization of elastic lamellae

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(538) and also prevent differentiation of VSMC into chondrogenic cells. It is plausible that the differentiation of vascular cells into a chondro/osteoblast-like phenotype is accompanied by reduced production of calcification inhibitors by these modified cells themselves or the ‘adjacent’ vascular elements; as

calcification inhibitors are expressed differentially in calcified arterial walls as compared to healthy artery walls (539). In the absence of calcification

inhibitors, MVs form the nidus for nucleation of mineral calcium, by providing a micro-environment which raises the calcium:phosphate product above the threshold for precipitation (537;540). In non-stress state, these inhibitors are loaded into MVs and inhibit calcification, but are differentially expressed in calcified MVs; favouring calcification (537). Under conditions of acute stress, VSMC release MVs as an initial adaptive response to prevent cell death by

removing excess calcium, which is bound by the inhibitors in the MVs. These are then deposited in the ECM. However, in the presence of prolonged stress (e.g. mineral imbalance) this adaptive mechanism becomes overwhelmed and calcification ensues (537).

Extracellular space calcium is elevated in people with chronic renal failure and in atherosclerosis plaques (at sites of cell death and necrosis), however, in hypertension there is intracellular calcium overload (541). Mechanistically calcium promotes the loss of inhibitors such as MGP from MVs, and also exposes the calcium binding protein annexin A6 together with phosphatidylserine (PSer) on the surface of MVs; forming a complex. This complex is highly efficient at nucleating hydroxyapatite, thus enabling MVs to seed extracellular matrix calcification in the vessel wall (541;542). In vitro studies have suggested that calcium channel blockers may prevent calcification by preventing MVs from mineralizing (543). Similarly animal models of medial calcification have demonstrated that various anti-hypertensive therapies (diuretics, calcium channel blockers, ARBs and endothelin receptor antagonists) can reduce pulse wave velocity and slow or prevent medial calcification (544-547) but their efficacy in humans still needs further work. Both calcification and hypertension are related to each other and may influence each other as shown in Figure 1.8 and explained below.

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Figure 1.8 The cyclical association of vascular calcification, arterial stiffness, atherosclerosis and hypertension.

Redrawn and modified with permission from Jeffcoate et al. 2009 (548)

Red coloured compounds are calcification inhibitors. MSC= mesenchymal stem cells, VSMC= vascular smooth muscle cells, CVC= calcifying vascular cells, MV= micro-vesicles, OPG= osteoprotegerin, NO=Nitric oxide , BMPs= Bone morphogenetic protein, Cbfa1= Transcription factor core-binding protein, ROS= reactive oxygen species, RANKL/RANK= Receptor activator for nuclear factor κB/ Receptor activator for nuclear factor κB ligand, PTH= parathyroid hormone, CaPO4= calcium phosphate.

1.7.8.3 Hypertensive remodelling causing calcification

Hypertension is associated with remodelling of the arterial wall mainly characterised by changes in composition and quantity of ECM, along with proliferation/differentiation of VSMC (321;549;550). Elastic fibres are an important constituent of aorta and large and medium sized arteries. They are composed of an elastin core which is surrounded by fibrillin rich microfibrils (551). The elastic properties of large conduit arteries are determined by the presence as well as special arrangement of elastic fibres; organized in

concentric rings of fenestrated lamellae intercalated with aligned VSMCs (116). However, increased pressure on elastic fibres (e.g. hypertension) can induce quantitative and qualitative changes in the elastic fibre organization. Increased BP increases elastin production and deposition in vascular wall (552). Increased

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pressure causes fatigue and damage to elastic fibres, leading to fragmentation and generation of elastin degradation products (EDPs) (116). EDPs are endowed with chemotactic activity and also have proliferative and migratory effects on VSMCs (553). Hypertension is also associated with increased deposition of several ECM components, including collagen, fibronectin and proteoglycans by vascular cells especially VSMC (116;552). Adijiang et al showed the effect of high BP in calcification by exposing Dahl salt-sensitive hypertensive rats to a uremic (indoxyl sulfate) toxin; and this accelerated vascular calcification (554). In contrast exposure of normotensive rats to the same uremic toxin did not induce calcium deposition (554). It is suggested that hypertension-associated changes in ECM and vascular cells might create an environment which is prone to calcium deposition, and calcification is accelerated in the presence of noxious or pro- calcification mediators.

EDPs are represented as a preferential site for hydroxyapatite crystal nucleation (555); they also induce phenotypic transition of VSMC toward an osteoblast-like profile and amplify phosphate driven calcium deposition (556-558). EDPs also promote the release of metalloproteinases (MMPs), through interaction with specific receptors (556;557). During early hypertension MMP activation helps to limit the pulse pressure rise by increasing vascular compliance (559), however, MMP accumulation amplifies ECM damage, including elastin degradation and elastin calcification. Moreover, blockade of MMPs prevents the calcification of elastin (560-562).

Elastic fibres are composed of several microfibrillar molecules including the latent transforming growth factor-β (TGFβ) binding proteins and these

contribute to formation of TGFβ large latent complex (LLC) (563). LLC interacts with ECM components and in response to any insult (including MMP), converts from latent TGFβ to active TGFβ (563). During hypertensive remodelling active TGFβ is known to drive the synthetic and proliferative response of vascular cells (564). TGFB has also been shown to be involved in the osteogenic differentiation of VSMC in synergy with other procalcific mediators, including EDPs (556;565).

VSMCs also produce type I collagen during hypertensive remodelling (552). Type 1 collagen represents an ideal matrix for apatite crystal nucleation and

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and on exogenous administration it facilitates matrix calcification along with collagen and fibronectin (566;567). Increased type I collagen production has been also documented in senescent VSMCs (568). Moreover, it provides the matrix for MV driven calcification (569) and also acts as modulator of VSMC differentiation into osteoblast-like cells (567;570). Type 1 collagen gene deletion leads to significant reduction in calcium deposition (568). Similarly its receptor gene [discoidin domain receptor-1 (DDR1)] deletion was also associated with significant reduction in vascular calcification (570). VSMCs deficient for DDR1 show reduced ability of osteogenic differentiation and also express higher level of the calcification inhibitors such as ENPP1 (570). Arterial proteoglycan content (including chondroitin sulfate, biglycan and decorin) increases during

hypertension (571) and they are also associated with increased vascular calcification (565;572).

In summary, hypertensive remodelling of the large arteries is characterised by changes in vascular cells and ECM composition that might create a favourable environment for the initiation and propagation of calcium deposition.

1.7.8.4 Aortic calcification causing systolic hypertension

Ageing is associated with progressive increase in stiffening of aorta and other large arterial conduits. The structural modifications of the vascular wall in ageing are similar to hypertension in several ways. Ageing of the large arteries is characterized by progressive collagen accumulation along with fracture and disorganisation of elastic lamellae (111). The net effect of the imbalance of collagen and elastin in ageing is a progressive reduction in vascular elasticity and compliance. This change in vascular elasticity and compliance explains the age associated rise in SBP, the fall in DBP and the acceleration of the pulse wave velocity (PWV) as observed in the elderly. Moreover, increased stiffness may result in returning of the aortic reflected wave during the systolic period, further increasing the left ventricular load, favouring cardiac hypertrophy and susceptibility to MI (111;532).

Vascular calcification is associated with arterial rigidity which in turn is responsible for the mechanical abnormalities and cardiac consequences associated with vascular stiffening (532). As mentioned above, vascular

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calcification can be both intimal and medial, and both types increase vascular rigidity. However, the relative contribution of each in arterial stiffening is still unknown as the available CT imaging technique does not differentiate between the two types.

Through the experimental generation of medial calcification (elastocalcinosis) in thoracic and abdominal aorta in rats was associated with accelerated PWV, increased SBP and augmented PP (531;545;547;573). These changes were similar to those observed in isolated systolic hypertension (ISH), and were also

accompanied by a significant increase in left ventricular mass (573).

In human studies, vascular calcification and associated vascular stiffness has mostly been shown in patients with chronic kidney disease (CKD). Population studies clearly showed that the amount of aortic calcification in CKD patients was positively correlated with PWV (574;575). It has also been shown that both PWV and the extent of arterial calcium deposition are predictive of future CVD mortality (576;577). The Twins UK cohort of middle aged women with normal kidney functions also showed that aortic calcification was significantly

correlated with carotid femoral PWV. This positive correlation remained

significant even after adjustment for age, mean arterial pressure (MAP), glucose, heart rate and menopausal status (578). More recently, Sekikawa et al. also confirmed these findings in a multi-ethnic cohort of 906 middle-aged men without history of any CVD. They showed that carotid femoral PWV was

positively and significantly correlated with the amount of calcium deposits in the aorta observed between the aortic arch and the iliac bifurcation. This

correlation was also independent of the effect of age, BMI, MAP, smoking, diabetes and medications (579).

The association between hypertension and aortic calcification had been reported previously (580;581). Recently McEniery et al. (533) confirmed that increased aortic calcium deposition was accompanied by higher aortic PWV in a group of healthy individuals. This link was independent of age and MAP and mainly

observed between PWV and the calcification of the abdominal aortic tract. Along with aortic PWV, peripheral PP was also positively associated with aortic calcium deposition in any vascular site (abdominal, ascending and descending aorta). Considering all the factors together in a multivariate analysis, they found that

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the presence of aortic calcification was independently associated only with age, aortic PWV and calcium phosphate product. When they compared normotensive people with those with ISH, they found that hypertensive individuals exhibited higher aortic PWV along with increased calcification of abdominal and

descending thoracic aorta. Moreover, patients with resistant ISH had an even higher amount of aortic calcification (533). More recently Jensky et al. (582) investigated the association of individual BP parameters (SBP, DBP and PP) with the extent of calcification in different vascular segments. Calcification in all the large and medium sized arteries (except for the iliac and subclavian arteries) was significantly associated with SBP and PP, with the latter showing an even stronger association (582). They also found that in older people (<60 years), differences in arterial calcification between hypertensive and normotensive individuals were more pronounced (582). As the studies examining direct relationship (533;582) were cross sectional and not longitudinal, causal

relationship between aortic calcification and BP cannot be confirmed. However, findings from these studies strongly suggest a significant association between calcification and ISH, SBP and PP. Moreover, exhibition of higher aortic calcium accumulation in patients with resistant ISH (533) also underscores the possibility of a contribution in the pathophysiology.

In humans, three monogenic diseases are characterized by extensive and premature onset of arterial calcification. These conditions are 1 ) Generalized arterial calcification of infancy (GACI), associated with mutations in the ENPP1 gene (583), 2) Pseudoxanthoma elasticum (PXE), which results from mutations in the ABCC6 gene (584) and 3) arterial calcification and distal joint calcification (ACDC), which is caused by CD73 deficiency (mutations in the NT5E gene) (585). Patients affected by GACI and PXE exhibit diffuse calcific deposits in the medial layer of large arteries, whereas calcification in patients with ACDC is mainly restricted to lower limbs vessels. Both GACI and PXE are characterized by severe increase in arterial BP and development of renovascular hypertension (586).

In summary, in vitro, animal and human data supports the role of vascular calcification in the generation of arterial stiffness and subsequent increase in SBP and PP but more work is needed to confirm the independent causal association.

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