Isolation and characterization of human apatite-inducing aortic proteolipid

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

51,

149-158 (1989)

Isolation and Characterization of Human Apatite-Inducing Aortic Proteolipid’ R. ROMEO, J.M.

AUGUSTYN,ANDG.MANDEL

Department of Pathology, Albany Medical College, Albany, New York 12208; Veterans Administration Medical Center, Albany, New York 12208; and National Veterans Administration Crystal Identification Center, Milwaukee, Wisconsin 53201 Received December 19, 1988, revised June 15, 1989 A proteolipid, capable of inducing hydroxyapatite in vitro, can be isolated from human aortic calcified lesions, noncalcified lesions, and nonlesion tissue obtained at autopsy. Analysis of the amino acid composition after acid hydrolysis showed all nucleators to be high in the hydrophobic amino acids glycine and alanine. Estimation of the molecular weight of the nucleator, extracted from calcified lesions, by gel filtration and lipid content showed a minimum molecular weight of 469,000 Da. All nucleators were characterized by the presence of acidic phospholipids which may have a role in the mechanism of calcification. 8 1989 Academic Press, Inc.

INTRODUCTION Studies on the development and regression of atheromatous lesions in experimental animal models and man have shown (i) that continued exposure to atherogenie stimuli (e.g., diet and/or injury) can lead to the formation of complicated lesions that have undergone one or more of the following changes, ulceration, hemorrhage, aneurysmal dilation, and calcification (12, 14, 16, 18-21, 30-32); and (ii) that the removal of atherogenic stimuli (e.g., diet modification) can induce regression with subsequent healing of the lesion (1, 6-9, 13, 25, 29, 35). The one aspect of experimental atherosclerosis that does not appear to regress is calcification. In the swine model, removal of atherogenic stimuli in animals with calcified lesions has been shown to increase the calcium content of the affected vessel (13). This suggests that the process of calcification may be induced by stimuli other than lipid accumulation and smooth muscle cell death. One such factor is an aortic proteolipid first described by Ennever and co-workers (10). This proteolipid has the ability to induce the formation of hydroxyapatite in vitro. The purpose of this investigation was to isolate and begin to characterize the apatiteinducing proteolipid (AIP) from human aortic calcified lesions, noncalcified lesions, and nonlesion tissue in an effort to elucidate its role in vivo in the initiation of calcification. METHODS Adult human aortas (35 to 65 years) were removed and frozen (- 20°C) within 3 hr of death at the morgue at Albany Medical Center Hospital or the Veterans Administration Medical Center (Albany, NY). The aortas were kept at -20°C until a sufficient quantity was obtained to begin the isolation protocol. Aortas were defrosted overnight at 4°C and then stripped of adventitia and outer media. Lesion areas were dissected from nonlesion areas to prevent dilution or contami This research was supported by the Veterans Administration

and by Grant 210038 NIHL.

149 0014-4800/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ination with nonlesion tissue during extraction. Lesion tissue was designated as either noncalcified or calcified, pooled, and treated as separate samples. Each tissue sample was cut into approximately l-cm sections and weighed wet. The calcified lesion, noncalcified lesion, and nonlesion tissue pools were then brought through the initial steps of the purification protocol of Ennever et al. (10). Briefly, the individual tissue pools were washed with constant stirring at 4°C in (i) saline to remove soluble protein, the completion of which was determined by absorbance at 280 nm; (ii) 2 M formic acid to bring about decalcification as determined by atomic absorption spectroscopy; and (iii) distilled deionized water to remove formic acid as determined by conductivity. Tissues were lyophilized and approximately 10 g (dry wt) of each tissue type was extracted twice in 1000 ml of chloroform-methanol-HCl(2: l:O.Ol, v:v:v) at room temperature. The extracts for each tissue type were pooled and reduced to a volume of 50 ml under a stream of nitrogen. Cold acetone (one-half of the original volume of extract) was then added resulting in the precipitation of crude AIP. The precipitate, collected by centrifugation, was resolubilized in chloroform:methanol(2: 1, v:v) and then analyzed for protein according to a modified Lowry method (22). Cholesterol was determined by the method of LeMer (23) triglyceride by the method of Van Handel and Zilversmit (33), and phospholipid by Bartlett’s method (3). AIP was further purified by a departure from the method of Ennever et al. (10) by using high-pressure liquid chromatography and a Bio-Sil TSK-250 column (Bio-Rad). Samples were prepared for chromatography by resolubilizing an aliquot of precipitate in chloroform:methanol (2: 1, v:v) followed by low speed centrifugation. Aliquots of clear supernatant were injected into the column and eluted isocratically with chloroform:methanol (2: 1). Protein-containing fractions (absorbance at 280 nm) were pooled as applicable, evaporated under a stream of nitrogen, and stored at -20°C. Each peak was then assessed for the ability to form apatite in vitro using the nucleation protocol described by Ennever et al. (11). The presence of apatite, as determined by X-ray diffraction analysis, was made by Dr. Gretchen Mandel, VA Crystal Identification Center (Milwaukee, WI). Samples were characterized by high resolution X-ray powder diffraction, using a germanium crystal monochromated Guiner camera (Huber Diffracktionstechnik, West Germany) and a Rigaku high brilliance rotating anode X-ray generator equipped with a copper target (Rigaku USA, Danvers, MA). Diffraction films were taken using CL& 1 radiation at room temperature for 2 hr at 50 kV and 150 mA. Diffraction patterns were compared with known standards. Aliquots of purified nucleating and nonnucleating proteolipids were also analyzed for protein and lipid as described previously. Amino acid analysis was carried out in the laboratory of Dr. Theordore Peters, The Mary Imogene Basset Hospital, (Cooperstown, NY) by the method of Peters and Reed (26). The morphological integrity of the samples was confirmed at several steps in the purification protocol by light and/or electron microscopic examination. Representative samples were examined (a) after dissection, (b) following formic acid decalcification, and (c) following delipidation with acidic chloroform-methanol. Samples for light microscopy were fixed in buffered formaldehyde solution and embedded in paraffin. Sections were stained with hematoxylin-eosin or VerhoeffVan Gieson stains. Tissue for electron microscopy was fixed in glutaraldehyde, postfixed in osmium tetroxide, and embedded in epoxy resin. Thick plastic sec-

HUMAN

APATITE-INDUCING

AORTIC

PROTEOLIPID

151

tions were stained with toluidine blue and examined by light microscopy. Finer grids were prepared from all three types of tissue and examined with a Phillips 300 transmission electron microscope. The molecular weight of the apoprotein portion of AIP from calcified lesion tissue was determined on Sephadex G-200. AIP was delipidated by the procedure of Boyan-Myers and Boskey (5). Delipidated AIP was resolubilized in 0.1% sodium dodecyl sulfate (SDS) applied to Sephadex G-200 and eluted isocratically. Elution volume of delipidated AIP was compared with the elution volumes of molecular weight standards purchased from Sigma Chemical Co. Delipidated AIP was also assessed for its ability to form apatite in vitro by the nucleation protocol of Ennever et al. (11). Qualitative analysis of phospholipids present in purified AIP was by twodimensional thin-layer chromatography on silica gel G (28). SDS-polyacrylamide gel electrophoresis (PAGE) of purified AIP was by the method of Gaal et al. (15). RESULTS Light and electron microscopic examination of the three tissue preparations confirmed that the tissue pools were homogeneous and accurately classified. Light microscopic examination of paraffin and plastic sections of tissue following formic acid treatment showed no calcium deposits in any of the tissue preparations including those from calcified lesions. However, a small focus of nonmineralized osteoid tissue was noted in one of the toluidine blue sections. The sections from both types of lesion showed typical atheromata with the necrotic core and fibrous cap. Poorly stained cells were seen in both the toluidine blue- and the hematoxylin-eosin-stained sections. The sections from the tissue subjected to acidic chloroform-methanol extraction showed collapse of the tissue architecture with loss of definition of the necrotic core. Only a few ill-defined cells could be observed. Electron microscopic examination of the decalcified tissue showed no hydroxyapatite crystals in or outside the matrix vesicles. The latter were, in general, collapsed and their limiting membrane appeared thick and smudgy. Smooth muscle cells and foam cells were easily identifiable. The intracellular lipid inclusions were also well preserved; however, the myofilaments were not. The ultrastructural appearance of the tissue following acidic chloroformmethanol extraction was extensively altered and none of the normal or pathological features were recognizable with certainty. Characteristic chromatograms on TSK-250 are shown in Fig. 1. Calcified and noncalcified lesion tissue extracts as well as nonlesion tissue extracts were characterized by three major peaks. Peaks from each tissue type were pooled, evaporated under nitrogen at 37°C and analyzed for (a) protein and lipid content, (b) the ability to induce calcium hydroxyapatite in vitro, (c) homogeneity on SDSPAGE, (d) amino acid composition, and (e) molecular weight. Table I summarizes the protein content and recovery at various steps in the purification protocol. Lipid content of peaks eluted from TSK-250 for the three tissue types is shown in Table II. The results of nucleation studies were as follows: The first peak from each tissue preparation failed to induce hydroxyapatite, while the second peak consis-

ROMEO, AUGUSTYN,

152

AND MANDEL

.6463

.56--

.63

.46--

.-,x E a,E nc

.36

EN 8 .o h 0

27

1

0.06~-

A

5

I6 Volume

B

1%

5

1015

Volume

(ml)

C

5 IO 15 Volume (ml I

(ml)

FIG. 1. Elution profiles of tissue extracts on TSK-250. Aliquots of the acetone precipitate from each tissue type were reconstituted with chloroform:methanol (2:1, v:v). After centrifugation, ahquots of clear supematant were injected into the column, 300 X 7.5 cm, and eluted as described under Methods. Aliquots (0.5 ml) were collected at a flow rate of 1 ml/min and monitored at 280 nm. Protein-absorbing fractions were pooled as peaks, evaporated under nitrogen, and stored at - 20°C. (A) Calcified lesion tissue extract; (B) noncalcified lesion tissue extract; (C) nonlesion tissue extract.

TABLE PROTEIN

CONTENT

I

AND RECOVERY ACCORDING TO TISSUE TYPE AT EACH PURIFICATION PROTOCOL

Tissue type

Wet weight (g)

Dry weight” (d

Total proteinb (Kid

Calcified lesion

207.1

17.17

3657

Noncalcitied lesion

213.6

33.98

8801

Nonlesion

186.2

28.32

6174

Peak 1 2 3 1 2 3 1 2 3

I% protein/ peak’ 33 53 45 363 152 1184 loo0 333 1244

STEW IN THE

Total proteind

Percentage recovered

131

3.6

1699

19.3

2577

41.7

Note. Data shown are from a representative purification at each step of the protocol. Protein was determined as described under Methods. u Dry tissue weight was recorded after lyophilization. b Total protein is the content after acetone precipitation. ’ The protein determination is that of individual peaks eluted from TSK-250. d The sum of the peaks for each tissue type.

HUMAN

APATITE-INDUCING

AORTIC

153

PROTEOLIPID

tently formed apatite in vitro, regardless of the tissue type. The third peak induced apatite only when it originated from noncalcified lesion tissue. Homogeneity of the delipidated nucleating fraction (Peak 2) from all three tissue preparations was assessedby SDS-PAGE. The results of the migration of the apoprotein portion in 0.1 M phosphate buffer, pH 7.1, are shown in Fig. 2. All three nucleating fractions showed a single area of protein. SDS-PAGE with a citrate+phosphate buffer (pH 5.0) or a Tris-HCl buffer (pH 9.0) showed similar protein patterns. Homogeneity of individual peaks was also verified by a second chromatography on Bio-Sil TSK-250. The results of the amino acid analysis of nucleating preparations from all three tissues are shown in Table III. All nucleating preparations were high in glycine and alanine. The molecular weight of the apoprotein moiety of the nucleating AIP from calcified lesions was determined to be 117,000Da on Sephadex G-200 (Fig. 3). The apoprotein moiety eluted as a single symmetrical peak after the void volume. TABLE LIPIDCONTENTOFPEAKSELUTEDFROM

Tissue type Calcified lesion Noncalcitied lesion Nonlesion

Calcified lesion Noncalcified lesion Nonlesion

Calcified lesion Noncalcified lesion Nonlesion

Peak

II

TSK-250 FORTHETHREETISSUETYPES n

Mean

A. Cholesterol content (pg/pg protein) 1 1 3.83 2 1 2.15 3 2 236.90 1 4 1.91 2 4 7.81 3 4 0.81 1 2 1.97 2 3 1.30 3 2 0.40 B. Triglyceride content (p&g protein) 1 2 1.89 2 2 0.67 3 2 7.05 1 2 2.93 2 4 0.94 3 3 0.60 1 1 2.09 2 1 0.29 3 1 0.77 C. Phospholipid content (kmole P/w protein) 1 1 0.006 2 1 0.002 3 1 0.003 1 5 0.004 2 3 0.019 3 3 0.010 1 3 0.002 2 3 0.031 3 3 0.003

SD

206.70-267.10“ 0.56 0.93 0.21 1.80-2.14” 0.26 0.35-0.45” 1.78-2.00” 0.61-0.73” 6.80-7.30” 2.73-3.13” 0.25 0.29 -

0.002 0.002 0.002 0.008 0.011 0.001

Note. Fractions showing absorbance at 280 run after elution from TSK-250 were pooled into peaks and analyzed for their cholesterol, triglyceride, and phospholipid contents as cited under Methods. n, number of determinations; SD, standard deviation. “Rangewhenn = 2.

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FIG. 2. Electrophoretograms of nucleating peaks. Peak 2 from each tissue type was delipidated as described under Methods. The resulting apoprotein was resolubilized in 0.1% SDS. Samples were saturated with sucrose and layered on 5% polyacrylamide gels. Phosphate buffer 0.1 M, pH 7.1, containing 0.1% SDS was layered on top of the gels. The gels were preelectrophoresed at 2 mA/tube for 15 min and electrophoresed at 3 mA/tube for an additional 80 min in the same phosphate-SDS buffer. Protein was visualized by staining with Coomassie blue. (A) Calcified lesion apoprotein; (B) noncalcified lesion apoprotein; (C) nonlesion apoprotein.

Calculation of grams of lipid per 117,000 g of protein using data in Table II resulted in an estimate of a minimum molecular weight of 469,000 Da for the nucleating AIP extracted from calcified human lesions. Qualitative determination of phospholipids present in nucleating peaks from all tissue types on thin-layer chromatography showed the presence of phosphatidylserine. Phosphatidylinositol was present only in nucleating peaks from noncalcified lesions and nonlesion tissues. Phosphatidylserine. phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin were detected only in Peak 2 of noncalcified lesion tissue. DISCUSSION A proteolipid(s), capable of inducing hydroxyapatite in vitro, has been isolated from human aortic calcified lesions, noncalcified lesions, and nonlesion tissue obtained at autospy. The presence of AIP in calcified tissue confirms data first reported by Ennever ef al. (IO). The presence of AIP in tissue extracts from both noncalcified lesions and nonlesion tissue lends weight to the suggestion of Anderson (2) that atherosclerotic calcification is a dynamic, ongoing process, and not an end stage of the disease process.

HUMAN

APATITE-INDUCING

AORTIC

155

PROTEOLIPID

The nucleation assay of Ennever et al. (11) demonstrates the ability to induce hydroxyapatite in vitro but does not necessarily reflect the ability to do so in viva. The apatite formed in vitro does, however, have the same X-ray diffraction pattern as biologic apatite. Furthermore, AIP is the only aortic constituent to date which can induce apatite in vitro (4, 27). The amino acid composition of the nucleating AIPs proved to be distinct from the composition of atherocalcin, a y-carboxyglutamic acid-containing protein known to occur in atheroma (24). Differences were also observed when comparing our data with those reported by Ennever et al. (10). This difference is likely to be a reflection of the modified purification protocol described under Methods, i.e., purification on Bio-Sil TSK-250. The amino acid composition of extracellular matrix constituents, such as collagen and elastin (both of which have been implicated in the mechanism of soft tissue mineralization) also showed differences when compared with the amino acid composition of the nucleating AIPs isolated in this study (17, 36). One of the proposed mechanisms for hydroxyapatite formation requires the presence of acidic phospholipids (34). This “lipid-dependent” mechanism was substantiated in this study as follows: (i) Aliquots of nucleating AIP were delipidated (5) and subjected to the nucleation protocol of Ennever et al. (11). Delip-

AMINO

ACID

TABLE COMPOSITION

Calcified

Peak n Asp Thr Ser Glu pro

GUY Ala CYS Val Met Be Leu ‘Or Phe His LYS Ml

III OF NUCLEATING

PEAKS

Noncalcified

residues/1000 amino acids bead

residues/1000 amino acids (me@

(mean)

2 2 66 38 62 89 ? 224 93 0 93 14 47 87 ? 45 12 44 41

2 3 52 35 74 66 92 165 140 0 93 14 4s 82 14 57 lo” 29 36

3 2 64 47 90 90 61 147 122 0 52 13 46 86 19 34 13 50 64

Nonlesion residues/l000 ammo acids (mead 2 3 38 33 66 45 122 223 177 0 103 0 44 51 16

35 8 15 21

Note. Representative aliquots from each peak were hydrolyzed in 6 M hydrochloric acid. Each hydrolysate was applied to a Type W3H spherical crosslinked sulfonated styrene copolymer as described by Peters and Reed (26). Ammo acids were eluted with a 0.2 M sodium-citrate buffer, pH 3.25-7.25, by increasing the concentration of sodium chloride. Absorbance, following injection with ninhydrin, was monitored at 570 and 440 mn. n, number of determinations; ?, not consistently detected. “n=2.

156

ROMEO,

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AND MANDEL

idated AIP failed to induce hydroxyapatite in vitro. (ii) The nucleating AIPs isolated in this study consistently showed the presence of phosphatidylserine and/or phosphatidylinositol. The existence of an in vitro nucleator in the aortic tissue types studied suggests the possibility that an in vivo nucleator may be present in aortic tissue in normal and diseased states. There are a variety of possible mechanisms through which progressive atherosclerosis could alter the AIP and convert it to a viable inducer of hydroxyapatite in vivo. One mechanism could involve the chronic proliferation of cells within the lesion and the increase in cellular products leading to cellular necrosis and degradation. These occurrences, typical of the advanced atheroma, could lead to the physical exposure of the proteolipid (e.g., the exposure of a normally buried active site) or could serve to provide an environment with the correct pH, ionic strength, temperature, and dielectric constant what would favor apatite induction in vivo. 10,000 Thyroglobulin

(669,000)

-Amylose

(200,000)

lcohol al

Dehydrogenose Peak

2

Albumin

(150,000)

(117,000)

(66,000)

lO--

1.0

1.5

2.0

Ve /Vo FIG. 3. Molecular weight for the apoprotein portion of nucleating fraction from calcified lesion tissue on Sephadex G-200. The interface of the delipidated nucleating fraction (Peak 2) from calcified lesion tissue was washed twice with distilled deionized water to remove any contaminating buffer. The pellet was reconstituted with 400 )~l of 0.1% SDS and incubated in a 37°C water bath for at least 30 min to facilitate solubilization. The entire sample was applied to a 41 x 2.5cm Sephadex G-200 column and eluted isocratically with 0.1% SDS at a flow rate of 20 mhhr. Three-milliliter fractions were collected and monitored for absorbance at 280 nm. The elution volume was compared to that obtained when 400-u.l aliquots of the following standards were applied and eluted in a similar manner: 8 mg/ml of thyroglobulin (molecular weight = 669,000), 4 mgknl of p-amylase (molecular weight = 200,000), 4 mg/ml of alcohol dehydrogenase (molecular weight = lSO,OOO),and 10 m&nl of albumin (molecular weight = 66,000).

HUMAN

APATITE-INDUCING

AORTIC

PROTEOLIPID

157

While the data in this study do not offer a specific explanation as to why the noncalcified lesion extract contains two nucleators, the nature of the disease may again offer some clues. The existence of two nucleators in noncalcified lesion extracts may reflect the fact that the role of AIP in calcification is to initiate apatite formation in the lipid lesion. Once the process has been initiated only one AIP may be sufficient to maintain calcification. Qualitative analysis of phospholipids was carried out to verify reports that acidic phospholipids play a role in apatite formation (5, 10, 34). The presence of acidic phospholipids in all nucleators together with the observation that the delipidated apoprotein portion of each nucleator failed to induce hydroxyapatite in vitro suggests that phospholipids play a role in calcification. Anderson (2) has suggested that the ability of phospholipids to bind calcium is a possible mechanism for the sequestration of calcium and its focal accumulation in the atherosclerotic lesion. CONCLUSION The presence of a proteolipid capable of inducing hydroxyapatite in vitro in both calcified and noncalcified lesions as well as in nonlesion tissue suggests that atheromatous calcification is a dynamic, ongoing process. If this is the case, then the further characterization and cellular localization of AIP could lead to an improved understanding of the mechanism of calcification and ultimately to its inhibition in human blood vessels. ACKNOWLEDGMENTS The authors thank Leslie Davidson for her time and technical assistance and Dr. Theodore Peters for the use of equipment and laboratory space.

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