**2. The biology of vascular calcification**

The calcification of the vasculature refers to the pathological deposition of phospho-calcium minerals in the arteries, leading to its stiffness [19] and thickening, as a result of a complex interaction of factors [4, 23]. Regardless of that, phospho-calcium can spontaneously precipitate [31]. Oversaturation increases of those ions concentrations and acidic environment favors their precipitation with a formation of various intermediates minerals, gradually bigger, until the least soluble, **hydroxyapatite** (HA) which will then crystalize [32]. The deposition of this crystal, or **nucleation process**, occurs chiefly in the apoptotic cells [9, 17, 33] or in mineral containing vesicles [34], which will then release more calcium to extracellular matrix (ECM), promoting more apoptosis when entering the neighboring cells, in a vicious cycle [32].

#### **2.1 Calcification types**

The histological location of that deposition and the pathobiology has made a distinction between types of calcifications [4, 35]. This phenomenon can arise in: i) the inner layer of endothelial and connective subendothelial tissue of arteries, ii) the medial muscular layer, iii) in the cardiac valves (resulting in valve sclerosis and stenosis) and iv) calcific uremic arteriolopathy, previously known as calciphylaxis [3]. These are the calcifications referring to the vascular system, of which we will focus on the first two. Besides VC, other extra-skeletal calcifications may happen [3, 36].

**Atherosclerosis**, an accumulation of calcium in the intimal layer of large and medium sized arteries, is observed following a long inflammatory process and as *Vascular Calcification and Cardiovascular Risk in Chronic Kidney Disease: A Problem That Is… DOI: http://dx.doi.org/10.5772/intechopen.99886*

a last stage of that series of events [23, 37, 38]. It involves the infiltration of macrophages in the epithelial and subepithelial connective tissue and the formation of a lipid plaque [3, 23]. Hence, it is related to dyslipidaemia, arterial hypertension and tabagism, but also with age [8].

In **medial calcific sclerosis** (MCS) or **Mönckeberg's medial arteriosclerosis**, the target of calcification is the muscular medial layer of elastic or muscular arteries, and posteriorly, fibrosis, stiffness and thickening ensues [36, 37, 39]. This form is widely related to mineral imbalance, and it is seen in diabetes, some rare genetic diseases [40] and strongly related to CKD [17, 37]. This is the form to which we will be referring to.

It is important to point out that atherosclerosis is due to inflammation and lipid deposition and for that depends on the so-called traditional CV factors (dyslipidaemia, arterial hypertension and tabagism) while medial VC does not [23, 41, 42]. In atherosclerosis, plaque calcification creates areas of different compliance [23, 39, 43, 44]. It ultimately leads to plaque rupture and acute vessel obstruction and is more closely associated with an increase in mortality [23, 43, 45, 46].

The association of MCS and mortality was brought up by many studies but not yet proven to be a cause-effect [28, 29] and some opinions still diverge, as we will discuss ahead. Research efforts have improved our knowledge on the molecular mechanisms involved, showing a passive process of physicochemical reaction resulting in vesicles [17, 47]. As stated earlier, apoptotic debris are good mineral nucleation sites but extracellular vesicles (EV) are regarded as having much higher mineralization potential [48].

Cells capable of osteo-transdifferentiation include not only vascular smooth muscle cells (VSMC) in the media, but also miofibroblasts in the adventitia, pericytes under endothelial cells (related to atherosclerosis), multipotent vascular mesenchymal cells and cardiac valve interstitial cells [42, 49, 50]. EVs, originally termed matrix vesicles, are nanoparticles of cellular origin, heterogeneous in size, shape and content, with two origins: membrane budding or in an endosomal pathway where multivesicular bodies fuse with the plasma membrane and then releases to the ECM by the regulation of sphingomyelin 3 [1, 17, 47, 51]. These EVs are designated exosomes [1, 52]. These vesicles are known for many decades to be related to bone mineralization but only recently were identified in VC. Indeed, EV's membrane have affinity to matrix proteins and, contrary to non-calcifying vesicles, contain phosphatidilserin (acidic phospholipid) and calcium-channel annexin family molecules in the membrane, responsible for the vesicle's release. Annexin-6 has been described as a regulator of VC in vivo [1, 37, 47, 53].

EV's release is promoted by high levels of calcium and their calcifying potential depends on its altered content compared to normal vesicles, with lower calcification modulating proteins – which we will analyze in detail later –, higher calcium and phosphate content, lipids, microRNAs, matrix metalloproteinases (MMP) for matrix digestion and alkaline phosphatase (ALP), which normally is not present in vascular tissue. ALP releases free inorganic phosphate (Pi) – enhancing more crystal formation [47].

The nucleation of HA begins inside the vesicles, with molding crystals' size and shape, and continues with the of in the ECM. Even tough origin and release of EVs are still poorly understood, vesicles are regarded as nucleating foci for VC [52, 54].

#### **2.2 Transdifferentiation of VSMC**

The unregulated osteogenesis is owed to the influence of external and internal factors (age, inflammation, toxins, CKD or diabetes) that induce transformation of VSMC to osteogenic cells responsible for a pathological mineralization [23, 37, 54, 55].

Gene silencing lead to the loss of contractile properties by the underexpression of α-smooth muscle actin (SMA) and smooth muscle protein 22 (SM2) – the de-differentiation of VSMCs – whereas the upregulation of several genes coding for bone-like cells, give VSMCs the **osteogenic characteristics** [48, 49, 56]:

