Howard H.T. Hsu

Isolation of calcifiable vesicles from human atherosclerotic aortas

Advanced mineralization can cause brittleness of aortic walls with decreased elasticity thereby causing the wall to rupture. Although the precise mechanisms of dystrophic calcification remain unknown, morphological evidence reveals the presence of mineral-associated vesicles in the lesions and defective bioprosthetic valves. In an attempt to demonstrate the calcifiability of the vesicles, small segments of human atherosclerotic aortas with calcified lesions were removed at autopsy and then digested in a crude collagenase solution to release vesicles. A differential centrifugation was then used to isolate calcifiable vesicles, which was precipitated at 300,000 x g for 20 min. An exposure of the vesicles to a calcifying medium containing physiologic levels of Ca2+, Pi, and 1 mM ATP caused Ca deposition in a vesicle protein-concentration dependent manner. The calcifiability of the vesicles was further demonstrated by electron microscopy. Fourier transform spectroscopic analysis of the deposited mineral revealed the presence of a hydroxyapatite phase, closely resembling the native form of mineral in atherosclerotic plaques. In addition, calcifiable vesicles were enriched in ATP-hydrolyzing enzymes including Mg2+ or Ca2+-ATPase and NTP pyrophosphohydrolase that may be involved in normal and pathological calcification. Triton X-100 at 0.01% abolished 80% of both ATPase activity and ATP-initiated calcification. A comparison of vesicles isolated from non-atherosclerotic and atherosclerotic aortas indicated that atherosclerotic vesicles tended to have higher calcifiability. These observations suggest that the calcifiable vesicles play a part in dystrophic calcification of aortas in atherosclerosis.

Isolation of calcifiable vesicles from aortas of rabbits fed with high cholesterol diets

Advanced arterial wall calcification in atherosclerosis imposes a serious rupturing effect on the aorta. However, the mechanism of dystrophic calcification linked to hyperlipidemia, that causes atherosclerosis remains unknown. Emerging morphological and biochemical evidence reveals that calcifiable vesicles may have a role in plaque calcification. To determine whether a high cholesterol diet can induce arterial calcification and produce or activate calcifiable vesicles in aortas, a rabbit model was used. After 2 months of daily high lipid feeding (supplemented with 2% cholesterol and 6% peanut oil), typical atherosclerotic lesions developed. However, the mineral, if present in aortas, was insufficient to be detected by Fourier transform-infrared spectroscopy (FT-IR) or alizarin red staining, indicative of a non-calcifying stage of atherosclerosis. Small segments of thoracic aortas were digested in a crude collagenase solution to release calcifiable vesicles. Vesicles were also isolated from normal aortas as control to consider the possibility that membrane vesicles may be produced by crude collagenase digestion, which could cause the degradation of some cells. Calcifiable vesicles were precipitated at 300,000 x g after subcellular particles were removed by centrifugation at 30,000 x g. Calcifiability of isolated vesicles was then tested using calcifying media containing physiological levels of Ca2+ and Pi and 1 mM ATP. Electron microscopic observations showed that the isolated vesicles were heterogeneous in size and shape and capable of depositing electron dense particles. Fourier transform infrared spectroscopic analysis of the deposited particles revealed the presence of an amorphous mineral phase. The spectroscopic mineral to matrix ratios, related to the amount of mineralization, indicated that vesicles from cholesterol-fed rabbits produced more minerals than control vesicles obtained from the normal aortas. Alizarin red staining for mineral further demonstrated substantially higher calcifiability of the experimental vesicles. A 3-5 h exposure of the vesicles to calcifying media caused significant deposition of 45Ca and 32Pi in a vesicle protein-concentration dependent manner. Similar to previously reported observations with human atherosclerotic aorta vesicles, rabbit vesicles were enriched in ATP-hydrolyzing enzymes including Mg2+- or Ca2+-ATPase and NTP pyrophosphohydrolase that are implicated in normal and pathological calcification. Altogether, these observations suggest that accumulation of the released calcifiable vesicles, as a result of high cholesterol diets, may have a role in dystrophic calcification in hyperlipidemia-related atherosclerosis.

Selective synthesis of bone morphogenetic proteins-1, -3, -4 and bone sialoprotein may be important for osteoinduction by Saos-2 cells

Abstract. An ability to induce new bone formation at a required site would represent a considerable advance in bone repair and tissue engineering. It has been shown that the healing of critical-size bone defects in rats can be augmented by extracts of Saos-2 cells. These human osteosarcoma cells uniquely contain a bone-inducing activity, whereas other human osteosarcoma cells, e.g., U-2 OS cells, cannot replicate the osteoinductive capacity. To understand the necessary components of the Saos-2 bone-inducing activity, this study compared osteoinductive Saos-2 cells with non-osteoinductive U-2 OS cells with respect to the synthesis of bone morphogenetic proteins (BMPs)-1, -2, -3, -4, -5, -6, and -7 and the non-collagenous matrix proteins bone sialoprotein (BSP), osteonectin (ON), osteopontin (OPN), and osteocalcin (OC). The main differences were abundant synthesis of BMP-1/tolloid, BMP-3, -4, and BSP by Saos-2 cells, but absence or reduced synthesis in U-2 OS cells. BMP-2 and -7 were present in low amounts in both cell types, while BMP-5 and -6 were more abundant in U-2 OS cells, suggesting that these BMPs were of lesser importance for the osteoinductivity of Saos-2 cells. However, a relatively high expression of BMP-3 and -4, together with BMP-1/tolloid, may be important for the osteoinductive capacity of Saos-2 cells. The inability of U2-OS cells to induce bone, despite expressing most of the BMPs, may be due to an insufficiency of tolloid, BMP-3 or -4, BSP, and/or other unknown factors. A better understanding of the necessary components of the Saos-2 cell bone-inducing agent may, in future, lead to clinically useful Saos-2 cell products for bone repair and tissue engineering.

Bone-inducing agent (BIA) from cultured human Saos-2 osteosarcoma cells

The Saos-2 line of human osteosarcoma cells was established in culture in 1975. These cells produce a large amount of alkaline phosphatase but little or no matrix in vitro, and are unable to grow when transplanted into athymic mice. We decided to test our local strain of Saos-2 cells for bone-inducing ability in the skeletal muscle of athymic mice by implanting freeze-dried, acetone-defatted cells, with and without a collagen carrier. A bone-inducing activity (BIA) thus was demonstrated in 88% of 90 implants of devitalized Saos-2 cells. In further studies, we have used guanidinium hydrochloride (Gu-HCl) to extract, solubilize, and remove the Saos bone-inducing agent(s) in an active state which when reprecipitated by aqueous dialysis was able to induce ultrastructurally typical endochondrial bone formation in nude mouse muscle in 92% of 48 implants. This preliminary report is offered to alert investigators to the presence of an extractable BIA in Saos-2 cells.

Induction of calcification in rabbit aortas by high cholesterol diets: roles of calcifiable vesicles in dystrophic calcification

Atherosclerotic calcification may weaken the aorta wall and thereby lead to rupture of the vessel. The mechanism whereby aortas undergo calcification remains unclear. Previous reports in this laboratory showed that, after 2 months of cholesterol-supplemental feeding, an increase in calcifiability of membrane vesicles isolated from rabbit aortas precedes substantial arterial calcification. Further, the mineral was deposited by isolated calcifiable vesicles as an amorphous phase similar to minerals in human aortas at an early stage of atherosclerosis. In the current study, atherosclerotic calcification was induced by exposing rabbits to a 1% cholesterol-rich diet for 3 or 6 months. After 3 months of dietary interventions, atherosclerotic lesions were fully developed. Fatty streaks were evident in areas proximal to the heart and became less frequent in the distal areas. However, calcification was not yet identifiable histologically or by using Fourier transform spectroscopy (FT-IR). After 6 months of high cholesterol treatment, aortas were partially calcified. Histochemical staining for mineral revealed that calcification appeared to occur predominantly in the intimal areas immediately adjacent to the media. Fourier Transform Imaging analysis demonstrated that the mineral deposited in atherosclerotic rabbit aortas was a hydroxyapatite-like phase. To determine whether aorta vesicles play a role in mineral formation in aortas, vesicles were isolated from calcified aortas and then their calcifiability was compared to that in normal vesicles. Interestingly, during the course of vesicle isolation, we found that calcifiable vesicles with much higher calcifiability than normal vesicles could be readily isolated from atherosclerotic aortas simply by suspending minced tissues in PBS. The characteristics of the calcification process and the enzymatic contents of isolated vesicles were similar to those obtained using collagenase digestion. Correlatively, mineral deposited by calcifiable vesicles isolated from the calcified aortas was also of hydroxyapatite-like phases. Altogether, these observations indicate that (1) aortic calcification is a later event during atherogenesis, (2) calcifiable vesicles are loosely bound to the matrices of the lesions as the result of the disease process and (3) similarities in the mineral phases between those in aortas and by vesicles during atherogenesis further support the role of calcifiable vesicles in dystrophic calcification.

Effects of dietary calcium on atherosclerosis, aortic calcification, and icterus in rabbits fed a supplemental cholesterol diet

BackgroundVascular calcification is implicated in myocardial infarction, instability and rigidity of the aortic wall, and bioprosthetic failures. Although an increase in the calcium (Ca) content in atherogenic diets has been shown to decrease atherosclerosis in rabbits, whether Ca supplementation and deficiency can affect atherosclerosis-related aortic calcification remains unknown.ResultsNew Zealand White male rabbit littermates were fed an atherogenic diet containing 0.5% cholesterol and 2% peanut oil. The Ca content of the diet, which normally contains 1%, was adjusted to 0.5 or 3%. Segments of thoracic aortas were dissected from rabbits for histological evaluations and Ca and Pi determinations. Rabbits with calcium supplementation were maintained for 4 months, whereas those with calcium deficiency were maintained for 2 1/2 months due to severe icterus beyond this stage. The ratios of intimal to medial areas and calcified to intimal areas were used to semi-quantify lesion accumulation and calcification, respectively. Icterus was estimated from the extent of yellowing of the skin, sclera, and mucous membranes along with gross evidence of hepatic lipidosis and/or biliary obstructions. Statistical analysis of 16 matched littermates shows that Ca supplementation significantly decreased the lesions by 41% (p < 0.05) and markedly inhibited calcification by 62% (p < 0.05). Statistical analysis of 11 matched littermates shows that Ca deficiency significantly increased the lesions by 2.7-fold (p < 0.05) and that the diet caused a small but significant calcification not seen in the sibling groups with normal dietary Ca. Ca supplementation caused a significant 30% decrease in serum cholesterol (p < 0.05). Calcium deficiency increased serum cholesterol by 57% (p < 0.001). Serum cholesterol and LDL-cholesterol levels in Ca deficient rabbits were 2-fold higher than those with high Ca diets. Ca supplementation decreased soluble Ca and Pi content in aortas, suggesting that this effect may underlie the effects of Ca supplementation on calcification. Calcium deficiency increased icterus by 33% (p < 0.05), which may affect hepatic clearance of cholesterol, while calcium supplementation decreased it by 43% (p < 0.001).ConclusionCa supplementation to an atherogenic diet inhibits atherosclerosis, aortic calcification, and icterus, whereas a Ca deficient-diet promotes them.

Further characterization of ATP-initiated calcification by matrix vesicles isolated from rachitic rat cartilage. Membrane perturbation by detergents and deposition of calcium pyrophosphate by rachitic matrix vesicles.

Although membrane associated enzymes such as ATPase, alkaline phosphatase, and NTP pyrophosphohydrolase in matrix vesicles (MVs) may underlie the mechanisms of ATP-promoted calcification, prior to the current investigation, the role of the MV membrane in calcification had not been addressed. In this study, various perturbations were introduced to the MV membrane in in vitro calcification systems to determine ideal conditions for ATP-initiated calcification by MVs isolated from rachitic rat epiphyseal cartilage. Membrane integrity appears to be required, since the rupture of the vesicular membrane by vigorously mixing with 10% butanol abolished calcification. In contrast, a mild treatment of MVs with low concentrations (e.g., 0.01%, which is much below the critical concentration for micelle formation) of either neutral Triton X-100 or anionic deoxycholate stimulated calcification by >2-fold, without inducing obvious changes in vesicular appearance. Fourier transform infrared spectroscopic studies were done to identify the mineral phase formed in these experiments. For the first time, rachitic MVs were shown to induce the formation of a calcium pyrophosphate dihydrate-like phase after their exposure to calcifying medium with 1 mM ATP. The integration of spectral areas indicated that calcification was enhanced by Triton X-100. The detergent effect was reversible and appeared to be not mediated through activation of ATPase, alkaline phosphatase, or ATP pyrophosphohydrolase. In contrast to neutral Triton X-100 and anionic deoxycholate, cationic cetyltrimethylammonium bromide inhibited both ATPase activity (I50=10 microM) and ATP-initiated calcification. These observations suggest that membrane perturbations can affect calcification and that the presence of NTP-pyrophosphohydrolase in MVs may play a role in the deposition of CaPPi in rachitic cartilage.

A role for ATPase in the mechanisms of ATP-dependent Ca and phosphate deposition by isolated rachitic matrix vesicles

Physiological levels of ATP support mammalian matrix vesicle-mediated calcium deposition although the released Pi from ATP did not cause the [Ca] × [PO 4 ] ion product to exceed the threshold for spontaneous precipitation of calcium phosphate (Hsu, Biochem. Biophys. Acta 1116, 227–233, 1992). The aim of this report was to see if a specific ATPase is involved in ATP-stimulated Ca 2+ and phosphate deposition. Since ATP can be non-specifically cleaved by alkaline phosphatase (ALP), thus obscuring a specific ATPase activity, phosphatidylinositol specific phospholipase C (PI-PLC) which selectively removes ALP from isolated matrix vesicle membranes and l -tetramisole (LT), a specific ALP inhibitor were used to allow studies on specific membrane associated ATPase. The specific ATPase activity and ATP-dependent Ca and P deposition were then tested for their correlation. [ γ - 32 P]ATP hydrolytic activity of PI-PLC-released alkaline phosphatase (ALP) was not stimulated by Ca 2+ whereas membrane-associated ATPase was stimulated dose-dependently by addition of Ca 2+ or Mg 2+ . LT at 1 mM completely inhibited solubilized ALP activity whereas it only partially inhibited membrane ATPase activity. The apparent affinity of membrane ATPase, [K 0.5 ], for Ca 2+ , Mg 2+ , and ATP was 10, 20, and 49 μ M, respectively. Membrane perturbing agents such as 1% Triton X-100 or 5% butanol abolished either Ca 2+ or Mg 2+ stimulation of membrane associated ATPase. The ALP-depleted matrix vesicles with ATPase activity was able to deposit both Ca and Pi when ATP was present in calcifying media. In contrast, PI-PLC released ALP failed to deposit Ca or Pi. ATP-dependent Ca and P depositing activity by matrix vesicles was completely inhibited when the membrane was ruptured by 10% butanol. The high affinity of ATPase for Ca 2+ and ATP, insensitivity of ATPase and ATP-dependent Ca and P depositing activities to LT or PI-PLC treatments, and membrane dependence of stimulation of ATPase by Ca 2+ suggest the presence of a specific ATPase responsible for a part of [ γ - 32 P]ATP hydrolysis and involved in ATP-dependent deposition of Ca and phosphate by matrix vesicles.

Further studies on ATP-mediated Ca deposition by isolated matrix vesicles.

Summary ATP at serum levels can support matrix vesicle-mediated Ca deposition even though the Ca × P ion product is held at physiological levels

A new method to measure 45calcium accumulation by matrix vesicles in slices of rachitic growth plate cartilage

Abstract Slices of rachitic growth plate cartilage have proven a difficult tissue in which to study 45Ca uptake because, even in rickets, there exists an area of preformed mineral at the base of the growth plate. This extraneous mineral accumulates 45Ca through exchange and apatite crystal proliferation when exposed to a calcifying solution, thus creating a background of radioactivity which obscures 45Ca uptake into vesicle-initiated mineral. We have succeeded in removing the preformed mineral, and hence much background radioactivity by exposing slices of rachitic rat epiphyseal plate to 30 mM EGTA for 30 minutes prior to incubation in a calcifying solution. After EGTA, an alizarin-red stainable extraneous mineral line is removed, and then following incubation all newly formed mineral is deposited primarily in vesicle-containing areas. Thus, it was possible to enhance significantly the 45Ca uptake into living versus heat inactivated growth plates. Preliminary studies with the EGTA-treated rachitic cartilage also indicate that supplying a matrix vesicle phosphatase substrate such as beta-glycerophosphate (BGP) rather than supplying orthophosphate (Pi) in the incubation mixture leads to more active 45 Ca deposition. We conclude that BGP can be hydrolyzed by vesicle phosphatases to supply Pi for nascent mineral formation, and that our new system promises to be useful in analyzing agents which may stimulate matrix vesicle calcification.