As You Like It, Part 3: A Critique and Historical Review of Calcification as Seen with the Electron Microscope

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Reduced serum levels of the calcification inhibitor fetuin-A associate with increased cardiovascular mortality in dialysis patients. Fetuin-A-deficient mice display calcification of various tissues but notably not of the vasculature. This absence of vascular calcification may result from the protection of an intact endothelium, which becomes severely compromised in the setting of atherosclerosis. To test this hypothesis, we generated fetuin-A/apolipoprotein E (ApoE)-deficient mice and compared them with ApoE-deficient and wild-type mice with regard to atheroma formation and extraosseous calcification. We assigned mice to three treatment groups for 9 wk: (1) Standard diet, (2) high-phosphate diet, or (3) unilateral nephrectomy (causing chronic kidney disease [CKD]) plus high-phosphate diet. Serum urea, phosphate, and parathyroid hormone levels were similar in all genotypes after the interventions. Fetuin-A deficiency did not affect the extent of aortic lipid deposition, neointima formation, and coronary sclerosis observed with ApoE deficiency, but the combination of fetuin-A deficiency, hyperphosphatemia, and CKD led to a 15-fold increase in vascular calcification in this model of atherosclerosis. Fetuin-A deficiency almost exclusively promoted intimal rather than medial calcification of atheromatous lesions. High-phosphate diet and CKD also led to an increase in valvular calcification and aortaassociated apoptosis, with wild-type mice having the least, ApoE-deficient mice intermediate, and fetuin-A/ApoE-deficient mice the most. In addition, the combination of fetuin-A deficiency, high-phosphate diet, and CKD in ApoE-deficient mice greatly enhanced myocardial calcification, whereas the absence of fetuin-A did not affect the incidence of renal calcification. In conclusion, fetuin-A inhibits pathologic calcification in both the soft tissue and vasculature, even in the setting of atherosclerosis. 20: 126420: -127420: , 200920: . doi: 10.1681 Hemodialysis (HD) patients experience a cardiovascular mortality of up to 20% per year, and vascular calcification is a strong independent risk factor of cardiovascular death. 1,2 Pathologic calcification is driven both by an elevated serum calcium phosphate product and by differentiation of vascular or mesenchymal cells into osteoblastlike cells, becoming mineralization competent. J Am Soc NephrolSerum is a metastable solution with respect to calcium phosphate precipitation. Once started, calcification proceeds rapidly in the presence of calcifiable templates such as collagen, elastin, and cell debris. [3][4][5] Fetuin-A accounts for approximately 50% of the capacity of serum to inhibit the spontaneous apatite formation from solutions supersaturated in

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Fetuin-A Protects against Atherosclerotic Calcification in CKD

Westenfeld

Schäfer

Krüger

et al. 2009

159

101

“…24 A weakness of our method is that, as a serum test, it does not by nature take into account the established contribution of cells, including vascular smooth muscle cells 25 and calcifying myeloid cells, 26 in promoting vascular calcifications in vivo. [27][28][29][30] Because serum represents the endogenous milieu surrounding all cells in the body, however, investigating possible CPP-cell interactions will likely also broaden our understanding of calcification processes in vivo.…”

Section: Discussionmentioning

confidence: 99%

Nanoparticle-Based Test Measures Overall Propensity for Calcification in Serum

Pasch

Farese

Gräber

et al. 2012

278

372

Vascular and soft tissue calcification contributes to cardiovascular morbidity and mortality in both the general population and CKD. Because calcium and phosphate serum concentrations are near supersaturation, the balance of inhibitors and promoters critically influences the development of calcification. An assay that measures the overall propensity for calcification to occur in serum may have clinical use. Here, we describe a nanoparticle-based assay that detects, in the presence of artificially elevated calcium and phosphate concentrations, the spontaneous transformation of spherical colloidal primary calciprotein particles (CPPs) to elongate crystalline secondary CPPs. We used characteristics of this transition to describe the intrinsic capacity of serum to inhibit the precipitation of calcium and phosphate. Using this assay, we found that both the sera of mice deficient in fetuin-A, a serum protein that inhibits calcification, and the sera of patients on hemodialysis have reduced intrinsic properties to inhibit calcification. In summary, we developed a nanoparticle-based test that measures the overall propensity for calcification in serum. The clinical use of the test requires evaluation in a prospective study.

“…Whether the mitochondrial Ca overload observed in normal vessels, which is likely to be a prelude to necrotic cell death, also occurs in vivo remains unclear 19,20 ; the inability of VSMCs in normal vessel rings to adapt in vitro may represent a tissue culture phenomenon, as a result of their acute and prolonged exposure to high Ca and P in suboptimal, serum-free culture conditions.…”

Section: Ca Acts To Enhance Phenotypic Modulation Of Vsmcsmentioning

confidence: 99%

Chronic Mineral Dysregulation Promotes Vascular Smooth Muscle Cell Adaptation and Extracellular Matrix Calcification

Shroff¹,

McNair²,

Skepper³

et al. 2010

285

229

In chronic kidney disease (CKD) vascular calcification occurs in response to deranged calcium and phosphate metabolism and is characterized by vascular smooth muscle cell (VSMC) damage and attrition. To gain mechanistic insights into how calcium and phosphate mediate calcification, we used an ex vivo model of human vessel culture. Vessel rings from healthy control subjects did not accumulate calcium with long-term exposure to elevated calcium and/or phosphate. In contrast, vessel rings from patients with CKD accumulated calcium; calcium induced calcification more potently than phosphate (at equivalent calcium-phosphate product). Elevated phosphate increased alkaline phosphatase activity in CKD vessels, but inhibition of alkaline phosphatase with levamisole did not block calcification. Instead, calcification in CKD vessels most strongly associated with VSMC death resulting from calcium-and phosphate-induced apoptosis; treatment with a pan-caspase inhibitor ZVAD ameliorated calcification. Calcification in CKD vessels was also associated with increased deposition of VSMC-derived vesicles. Electron microscopy confirmed increased deposition of vesicles containing crystalline calcium and phosphate in the extracellular matrix of dialysis vessel rings. In contrast, vesicle deposition and calcification did not occur in normal vessel rings, but we observed extensive intracellular mitochondrial damage. Taken together, these data provide evidence that VSMCs undergo adaptive changes, including vesicle release, in response to dysregulated mineral metabolism. These adaptations may initially promote survival but ultimately culminate in VSMC apoptosis and overt calcification, especially with continued exposure to elevated calcium.