**6.4. Matrix vesicles**

pathway [202]. It has also been found that calcium deposits colocalize with inflammatory cells both *in vitro* [230, 231] and *in vivo* [232]. Mineral crystals may therefore be pro‐inflammatory

Hormones have pleiotropic effects on calcific vasculopathy. For example, the adipose‐derived factor, leptin, promotes vascular cells *in vitro* [235] and *in vivo* [236], while adiponectin‐defi‐ cient mice have increased levels of vascular calcification [237]. The influence of parathyroid hormone (PTH), which is involved in the bone turnover process, is also well known. PTH has a crucial role in calcium homeostasis, and so does PTH‐related peptide (PTHrP), and the two may function as pathological calcification mediators. Both PTH and PTHrP prevent VSMC calcification in a dose‐dependent manner by inhibiting ALP activity [238]. In addition, PTHrP is secreted from VSMCs, an action that is impaired by calcitriol (1,25‐dihydroxyvitamin D, the active form of vitamin D) [239]. PTH not only promotes the release of calcium from bone but also mobilizes salts, including bicarbonate and phosphate and impairs renal phosphate excretion, leading, for example, to advanced nephron loss in CKD patients, and thus result‐ ing in severe hyperphosphatemia [240]. Accrued high levels of serum phosphate then further stimulate the secretion of PTH, forming a vicious cycle [241]. Hyperphosphatemia increases FGF23 (a protein released by bone), which—together with its co‐receptor Klotho (a trans‐ membrane protein expressed by the kidney and blood vessels)—may also be a pathogenic factor in vascular calcification [242, 243]. Klotho maintains the balance of circulating calcium and phosphate [244]. Activation of the vitamin D receptor increases the expression of Klotho and FGF23 to promote renal phosphate excretion by downregulating the sodium phosphate transporters Slc34A1/NaPi‐2a and Slc34A3/NaPi‐2c. Intriguingly, Klotho inhibits vascular calcification by preventing VSMC differentiation while disrupting Klotho‐FGF23 signaling

Calcitriol may also exacerbate dystrophic calcification. Vitamin D toxicity is a common ani‐ mal model used to study vascular calcification [246]. Calcitriol dose‐dependently increases both calcification and ALP activity in VSMCs [239]. In response to interferon‐γ, macrophages express 25‐hydroxyvitamin D 1α‐hydroxylase, the enzyme needed to convert 25‐hydroxyvi‐ tamin D into calcitriol [239]. Once calcitriol binds to its receptor, signaling through this path‐ way has pleiotropic effects. The vitamin D receptor influences many genes in the vessel wall, including vascular endothelial growth factor (VEGF), matrix metalloproteinase 9, myosin, and structural proteins (including elastin and type I collagen [247–250], and this explains some of

Glucocorticoids, a class of steroid hormones with anti‐inflammatory properties, have also been shown to mediate osteoblastic differentiation and thereby promote ectopic calcification. Long‐term glucocorticoid use has been associated with osteoporosis, however, and these compounds have been shown to initiate differentiation to an osteochondrogenic phenotype in vascular cells [251, 252]. Similarly, pericytes exposed to dexamethasone exhibit a weaker

expression of MGP and OPN, and an increased ALP activity and calcium deposition.

per se, prompting and exacerbating the inflammation and calcification [233, 234].

28 Updates and Advances in Nephrolithiasis - Pathophysiology, Genetics, and Treatment Modalities

results in hyperphosphatemia with ectopic calcification [244, 245].

the effects of calcitriol on vascular calcification.

**6.3. Hormones**

Bone formation involves hydroxyapatite [Ca10(PO4 ) 6 (OH)<sup>2</sup> ] crystals, which begin to develop matrix vesicles that grow out of osteoblasts. VSMC that have undergone osteoblastic differ‐ entiation are able to release similar mineralization‐competent matrix vesicle‐like structures in the extracellular matrix too [176, 180, 215, 226, 253–256]. These matrix vesicles serve as mineral nucleation sites and are responsible for the initial deposition of calcium and phosphate in blood vessels (**Figure 7**). Matrix vesicles contain proteins related to calcification, extracellular matrix and extracellular matrix‐modifying enzymes, calcium channels, trafficking and cytoskeletal proteins, oxidant and endoplasmic stress‐related proteins, and other serum proteins [226]. All these proteins are involved in the disruption of the normal vessel architecture and thus serve as the nidus for calcification. Matrix vesicles also have an increased expression and activity of transglutaminase 2, a calcium‐dependent enzyme that promotes extracellular matrix crosslink‐ ing, and matrix metalloproteinase‐2 [226, 257]. Matrix vesicles are secreted from multivesicular bodies and are enriched with exosomes found to contain amorphous calcium‐phosphate crys‐ tals under calcifying conditions, and detected at the site of calcification [258].

Prolonged cellular stress may activate homeostatic repair processes, or cells may undergo apoptosis when overwhelmed by the stress. Apoptosis regulates VSMC calcification *in vitro* and inhibiting apoptosis reduces VSMC calcification [171, 176, 259–261]. In advanced carotid atherosclerotic plaques, the matrix vesicles contain high levels of BAX (a pro‐apoptotic mem‐ ber of the BCL2 family), indicating that they may be remnants of apoptotic cells [171, 176, 260]. Apoptotic VSMC‐derived matrix vesicle‐like structures are also able to concentrate and crys‐ tallize calcium, triggering calcification [176, 183, 189, 193, 222]. It has likewise been reported that chondrocyte‐derived apoptotic bodies might contribute to the calcification of articular cartilage [262]. All these data support the idea that the formation of apoptotic bodies may be another factor initiating ectopic calcification in cells under certain conditions.

Autophagy—a catabolic process that may be an adaptive response to cell stress—has been found to limit SMC calcification by inhibiting matrix vesicle release. When phosphate levels are high, inhibiting autophagy resulted in an increased VSMC calcium deposition. Downregulating autophagy was also associated with a loss of VSMC contractile proteins, but not with any VSMC differentiation to an osteogenic phenotype. On the other hand, inhibiting autophagy did increase the release of procalcific matrix vesicles with high levels of ALP activity [263]. In short, factors that interfere with autophagy are likely to increase VSMC and vascular calcification.

#### **6.5. MicroRNAs**

MicroRNAs (miRs) have emerged as key regulators of cell differentiation to osteoblast‐ like cells, regulating gene expression under pro‐calcifying conditions. Some studies have described a stronger expression of miRs targeting smooth muscle contractile proteins and a weaker expression of miRs targeting osteoblast differentiation markers under these conditions [264]. For example, the miR‐143/145 complex, which regulates the expression of VSMC dif‐ ferentiation markers and Kruppel‐like factor4 (KLF4), is downregulated; and KLF4 is known to control bone homeostasis by negatively regulating both osteoclast and osteoblast differen‐ tiation [265]. Other studies showed that downregulation of miR‐204, miR‐205, miR‐133a, or miR‐30b/c in VSMCs occurs prior to calcification and upregulates Runx2 expression [266, 267]. Micro‐RNA‐125b, which targets Ets1 and osterix, was found downregulated 21 days after exposing VSMCs to osteogenic medium [268]. Another set of miRs, miR‐135a(*n*), miR‐762, miR‐714, miR‐712(*n*), that target the calcium flux proteins NCX1, PMCA1, and NCKX4, have also been implicated in VSMC calcification [266]. It is still not clear, however, whether these miRs are really important in VSMC differentiation to an osteoblast‐like phenotype, or whether this process is associated with changes in the expression of a panel of miRs targeting several proteins important for calcification.
