Calcification of α-elastin coacervates: A bulk property of elastin

Scanning electron microscopy and electron probe microanalysis studies are reported on thin sections of calcified coacervates of α-elastin. It is found that the capacity of elastin coacervates to initiate calcification is a bulk property of the coacervate and not limited to the serum-coacervate interface, that the calcium phosphate deposits act to bind the protein units together and slow the dissolution and spreading of the coacervate as it floats on an air-water interface, and that, within the limits of detectability, there is no involvement of sulfur. As the charged groups of α-elastin had been blocked, the initiation of deposition is due to neutral sites in the protein which are tightly bound to the calcium phosphate deposits.     

Development of anin vitro system for the calcification of tropoelastin and α-elastin coacervates in serum

The coacervate of the N-formyl-O-methyl ester derivative of α-elastin was incubated in a calcifying medium containing bovine serum. The lag period observed before calcium phosphate formation was shown to be influenced by the conditions under which the protein matrix was formed. Tropoelastin coacervates, both blocked and unblocked, provide a matrix for calcification. A cross-linked structure is not an obligatory prerequisite for elastin calcification, nor is the pyridinium moiety of desmosine or isodesmosine. A significant advantage of thisin vitro calcifying system for coacervates is that it may be used to study the effect of various factors, such as glycoproteins, mucopolysaccharides and lipids, on the initiation of calcification.     

Scanning electron microscopy and electron probe microanalysis of calcified α-elastin coacervates

The uptake of calcium and phosphorus from a serum calcification medium by coacervated α-elastin was studied by electron probe microanalysis and scanning electron microscopy. Calcium and phosphorus were bound by the coacervate in ratios similar to that of hydroxyapatite. A significant difference was observed in the secondary electron image of the coacervate films before and after calcification. Elemental mapping, i.e., formation of an image composed of a selected energy range, showed the calcium to be deposited evenly over the coacervate rather than at discrete foci. There appeared to be no deposition of sulfur-containing molecules from the serum during calcification of the coacervate.     

Elastin Degradation Accelerates Phosphate-Induced Mineralization of Vascular Smooth Muscle Cells

Medial layer vascular calcification is common in patients with end-stage kidney disease. Inorganic phosphate has been shown to accelerate the transformation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells, which is thought to be a major process of medial layer calcification. Although elastin degradation is associated with medial layer calcification, the linkage between elastin degradation and the transformation of VSMCs remains to be clarified. We investigated the involvement of elastin degradation in the transformation of VSMCs. Rat VSMCs were isolated and cultured with a normal- (NP, 1.0 mM) or high- (HP, 2.5 mM) phosphate medium. An elastin-derived peptide, α-elastin (500 μg/ml), was also added to the normal- (NP + E) or high- (HP + E) phosphate medium. After a culture period of 2 weeks, von Kossa staining revealed mineralization in the HP group, which was accelerated by α-elastin, whereas α-elastin did not affect the mineralization at a normal phosphate concentration. The gene expression of osteoblastic differentiation factors, i.e., Runx2 or osteocalcin (OC), in VSMCs was significantly increased in the HP (Runx2 P < 0.05, OC P < 0.05) and HP + E (OC P < 0.05) groups compared with the NP and NP + E groups. Both gene and protein expressions of tissue-nonspecific alkaline phosphatase (TNAP) were significantly increased in the HP group compared with the NP and NP + E groups (P < 0.01, respectively). This increment was augmented in the HP + E group (P < 0.01). These results suggest that elastin degradation would accelerate or stabilize the process of VSMC transformation, which is induced by high phosphate through the upregulation of TNAP.     

Identification of proteins extracted from calcium oxalate and calcium phosphate crystals induced in the urine of healthy and stone forming subjects

The purpose of our study was to identify the proteins and investigate the differences, if any, between protein components of the matrices of calcium oxalate (CaOx) and calcium phosphate (CaP) crystals induced in␣vitro in whole human urine of healthy individuals and kidney stone patients. In addition, preliminary studies were performed to understand the effect of centrifugation and filtration of urine on its protein contents. Crystallization in urine was induced by addition of an oxalate or phosphate load. Crystals were collected, washed, and analyzed by scanning electron microscopy, X-ray diffraction, and energy dispersive X-ray microanalysis. Matrix proteins were obtained by demineralization with ethylene diamine tetraacetic acid (EDTA), analyzed by polyacrylamide gel electrophoresis, and identified by western blotting technique. No significant differences were detected between protein components of the matrices of CaOx and CaP crystals and between the crystal matrices obtained from the urine of normal and stone forming subjects. Albumin (AB), inter-α-inhibitor (IαI) related proteins, α-1 microglobulin (α-1 m), osteopontin (OPN), prothrombin (PT)-related proteins and Tamm-Horsfall protein (THP) were identified in matrices of both CaOx and CaP crystals induced in urine from both the normal subjects and stone formers. AB, PT-related proteins and OPN were the main constituents. The other proteins were present in smaller but detectable amounts. However, CaP crystal matrix, contained a large amount of THP. In addition CaP crystals contained significantly more proteins than CaOx crystals. Centrifugation and/or filtration of the urine resulted in reduction of many high molecular weight proteins including THP, AB and OPN in the urine. Received: 24 July 1997 / Accepted: 2 January 1998     

Calcification of osteoblastlike rat osteosarcoma cells in agarose suspension cultures

Summary Ros 17/2 clonal rat osteosarcoma cells calcify when cultured in the presence of 10 μg/ml β-glycerol phosphate in an agarose gel. Culture in 1% agarose inhibited cell division while allowing cells to remain metabolically active and viable for over 21 days. Serial photography of the same microscopic field shows a progressive deposition of calcium phosphate during the course of the experiment. The deposition of calcium around cells was confirmed by calcium-specific stains, and by energy dispersive X-ray analysis (EDX) during scanning electron microscopy. Cells with high calcium content analyzed by EDX had Ca:P ratios similar to hydroxyapatite. Total calcium progressively increased in β-glycerol phosphate-treated cultures whereas the control plates maintained a constant calcium content over 16 days. Alkaline phosphatase activity increased with time in culture whereas cells with β-glycerol phosphate maintained the alkaline phosphatase values achieved at the time of initial calcification. Alkaline phosphatase staining revealed no correlation between the presence of the enzyme activity and calcification. Radioimmunoassay for the bone-specific vitamin K-dependent protein bone Gla protein showed that β-glycerol phosphate-treated cells accumulate over sixfold greater amounts of this protein. Our studies show that ROS cells can calcify and accumulate bone-specific matrix components when cultured in a 3-dimensional agarose matrix.     

Effects of 1α-hydroxy vitamin D3 on the rachitic chick intestine: A comparison to the effects of 1,25-dihydroxyvitamin D3

Summary The timed sequence of events following the oral administration of 1α-hydroxy vitamin D₃ (1αOHD₃) to rachitic chicks was compared to that following a comparable dose of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). RNA polymerase activity was maximally increased 20% by 1αOHD₃ within 1 to 2 h and returned to control values after 8 h. Alkaline phosphatase activity was stimulated by 4 h and was maximal (3- to 5-fold increase) at 24 h. Calcium binding protein (CaBP) was detected initially within epithelial cells at the proximal end of the villus (just above the crypt) 6 to 8 h after 1αOHD₃ administration, in epithelial cells lining the proximal half of the villus by 24 h, and in epithelial cells along nearly the entire villus by 48 h. At no time did goblet cells contain CaBP. Serum calcium concentrations were significantly elevated in 2 h and maximal by 12 h (an increase of 3.6 mg/dl). Calcium accumulation by the intestinal mucosa in vitro was increased by 6 to 8 h and maximal (60% increase over controls) at 24 h. Phosphate accumulation by the intestinal mucosa in vitro was increased by 6 h and maximal (105% increase over controls) between 8 and 24 h. 1,25(OH)₂D₃ increased CaBP and calcium accumulation by 4 h, 2 h sooner than did 1αOHD₃. 1,25(OH)₂D₃ decreased serum calcium levels and increased serum phosphate levels at 2 h unlike 1αOHD₃. No difference in the effects of these compounds on alkaline phosphatase activity, RNA polymerase activity, and phosphate accumulation could be demonstrated. These results are consistent with the possibility that 1αOHD₃ may not require conversion to 1,25(OH)₂D₃ for all of its biological effects. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. In conducting the research described in this report, the investigators adhered to the Guide for Laboratory Animal Facilities and Care, as promulgated by the Committee on the Guide for Laboratory Animal Facilities and Care, of the Institute of Laboratory Animal Resources, National Academy of Sciences-National Research Council.     

Role of inter-α-inhibitor and its related proteins in urolithiasis. Purification of an inter-α-inhibitor related protein from the bovine kidney

Urine contains several macromolecules that inhibit calcium oxalate (CaOx) crystallization. Among them is bikunin, the light chain of most of the inter-α-inhibitor (IαI) family of glycoproteins. This study aimed to verify whether bikunin and other members of the IαI family are synthesized in the kidneys or derived exclusively from the plasma. Proteins extracted from homogenized bovine kidney were applied successively to three chromatographic steps on DEAE-Sephacel, Sephacryl S-300, and Mono Q column. The inhibitory activity was assayed using a CaOx crystallization system. The presence of IαI-related proteins was determined by␣electrophoresis and Western blotting. The results showed that kidney extract contained a 125-kDa protein that cross-reacted with anti-IαI antibodies. This protein inhibited CaOx crystallization efficiently. According to its molecular weight and immunoreaction with anti-IαI antibody, the 125-kDa protein could be pre-α-inhibitor. The latter is known to encompass a heavy chain and bikunin, which may explain its inhibitory activity against CaOx crystallization. Consequently, we hypothesize that kidneys may produce some IαI-related proteins that are involved in the inhibition of stone formation. Received: 18 February 1998 / Accepted: 9 July 1998     

Nephrocalcinosis and hyperlipidemia in rats fed a cholesterol- and fat-rich diet: association with hyperoxaluria, altered kidney and bone minerals, and renal tissue phospholipid–calcium interaction

To determine whether an “atherogenic” diet (excess of cholesterol and neutral fat) induces pathological calcification in various organs, including the kidney, and abnormal oxalate metabolism, 24 male Sprague-Dawley rats were fed either normal lab chow (controls, n=12) or the cholesterol- and fat-rich experimental diet (CH-F, n=12) for 111 ± 3 days. CH-F rats developed dyslipidemia [high blood levels of triglycerides, total, low-density lipoprotein (LDL)-, very low-density lipoprotein (VLDL)-, high-density lipoprotein (HDL)-bound cholesterol, total phospholipids], elevated serum total alkaline phosphatase and lactate dehydrogenase (LDH) levels, in the absence of changes in overall renal function, extracellular mineral homeostasis [serum protein-corrected total calcium, magnesium, parathyroid hormone (PTH), 1,25-dihydroxyvitamin D (1,25(OH)₂D)], plasma glycolate and oxalate levels. There was a redistribution of bone calcium and enhanced exchange of this within the extraosseous space, which was accompanied by significant bone calcium loss, but normal bone histomorphometry. Liver oxalate levels, if expressed per unit of defatted (DF) dry liver, were three times higher than in the controls. Urinary glycolate, oxalate, calcium and total protein excretion levels were elevated, the latter showing an excess of proteins >100 kD and a deficit of proteins >30–50 kD. Urinary calcium oxalate supersaturation was increased, and calcium phosphate supersaturation was unchanged. There were dramatically increased (by number, circumference, and area) renal calcium phosphate calcifications in the cortico-medullary region, but calcium oxalate deposits were not detectable. Electron microscopy (EM) and elemental analysis revealed intratubular calcium phosphate, apparently needle-like hydroxyapatite. Immunohistochemistry of renal tissue calcifications revealed co-localization of phospholipids and calcium phosphate. It is concluded that rats fed the CH-F diet exhibited: (1) a spectrum of metabolic abnormalities, the more prominent being dyslipidemia, hyperoxaluria, hypercalciuria, dysproteinuria, loss of bone calcium, and calcium phosphate nephrocalcinosis (NC); and (2) an interaction between calcium phosphate and phospholipids at the kidney level. The biological significance of these findings for the etiology of idiopathic calcium urolithiasis in humans is uncertain, but the presented animal model may be helpful when designing clinical studies. Received: 12 April 2000 / Accepted: 3 August 2000     

1α-Hydroxyvitamin D2 Partially Dissociates Between Preservation of Cancellous Bone Mass and Effects on Calcium Homeostasis in Ovariectomized Rats

Vitamin D metabolites can prevent estrogen depletion-induced bone loss in ovariectomized (OVX) rats. Our aim was to compare the bone-protective effects of 1α,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), 1α,25-dihydroxyvitamin D₂ (1,25(OH)₂D₂), 1α-hydroxyvitamin D₃ (1α(OH)D₃), and 1α-hydroxyvitamin D₂ (1α(OH)D₂) in OVX rats. 1α(OH)D₃ and 1α(OH)D₂ are thought to be activated in the liver to form 1,25(OH)₂D₃ and 1,25(OH)₂D₂, respectively. Forty-four 12-week-old female Fischer-344 rats were either OVX or sham-operated (SHAM). Groups of OVX rats (n = 7 each) received vehicle alone, 1,25(OH)₂D₃, 1,25(OH)₂D₂, 1α(OH)D₃, or 1α(OH)D₂, starting 2 weeks after surgery. All vitamin D metabolites were administered orally at a dose of 15 ng/day/rat. Urine and blood samples were collected 6, 9, 12, and 16 weeks after surgery. Serum samples were analyzed for total calcium and phosphate. Calcium, phosphate, creatinine, and free collagen cross-links (ELISA) were determined in urine. After tetracycline double labeling, the rats were sacrificed 16 weeks postsurgery, and the proximal tibiae and the first lumbar vertebrae were processed undecalcified for static and dynamic bone histomorphometry. 1,25(OH)₂D₃ and, to a slightly lesser extent, 1,25(OH)₂D₂ elevated vertebral cancellous bone mass in OVX rats to a level beyond that observed in SHAM animals, and both compounds increased serum calcium and urinary calcium excretion to similar extents. 1α(OH)D₃ and 1α(OH)D₂ resulted in a 64% and 84%, respectively, inhibition of ovariectomy-induced vertebral cancellous bone loss. In the proximal tibial metaphysis, all vitamin D metabolites tested could only partially prevent post-OVX trabecular bone loss, with a tendency for 1α(OH)D₃ to be the least active compound. The effects of 1α(OH)D₃ and 1α(OH)D₂ on calcium homeostasis differed markedly, however. The mean increase in urinary calcium excretion over the whole experiment was fivefold for 1α(OH)D₃, whereas the corresponding increase for 1α(OH)D₂ was only twofold. We conclude that, compared with 1α(OH)D₃, 1α(OH)D₂ combined at least equal or higher bone-protective activity in OVX rats with distinctly less pronounced effects on calcium homeostasis. This effect was not due to a differential action of the corresponding main activation products, 1,25(OH)₂D₃ and 1,25(OH)₂D₂. Received: 2 May 1996 / Accepted: 18 October 1996