JOURNAL OF ULTRASTRUC RESEARCH 98, 199--211 (1988)
Proteoglycan in Fast-Frozen, Freeze-Dried, Plastic-Embedded Rabbit Arteries
M. RICHARDSON,* L. J. M C G U F F E E , AND M. W. C. HAITON Department of Patholvgy, Room 31V26, McMaster University Health Science Centre, 1200 Main Street West,
Hamilton, Ontario. Canada l..8N 3Z5, and *Department of Pharmacology, University of Ne~t, Mexico, Albuquerque, New Mexico 87106
Received December 4, 1986, and in ret,ised form July 27, 1987
The connective tissue of arteries includes a nonfibrous extracellular matrix contain- ing proteoglycans (PG). PG are large, com- plex molecules consisting of PG monomers attached to hyaluronic acid via a link pro- tein to form PG aggregates. The PG mono- mers contain sulfated glycosaminoglycans (GAG) which are unbranched polymers of repeating disaccharide units, covalently linked to a protein core (Camejo, 1982; Comper and Laurent, 1978). From bio- chemical analyses, the GAG content of the arterial wall includes hyaluronic acid, heparan sulfate, chondroitin sulfate, and dermatan sulfate (Camejo, 1982; Comper and Laurent, 1978). It is generally consid- ered that PG are present in arterial wall as aggregates and this is supported by the iso- lation of PG-hyaluronate complexes from bovine aorta (McMurtrey et al. , 1979).
The morphology of isolated arterial PG monomers, extracted from the extracellular matrix secreted by monkey aortic smooth muscle cells in culture, was shown to be a "bottlebrush" structure (Chert and Wight, 1984; Wight and Hascall, 1983), similar to
199
that observed in extracts of cartilage (Has- call, 1980); but the structure of arterial PG aggregates has not yet been fully deter- mined. Based on existing data (Chen and Wight, 1984; Hascall, 1980; Wight and Has- call, 1983), a possible structure of an arte- rial PG aggregate is represented diagram- matically in Fig. I.
PG/n situ in the arterial wall cannot be reliably or reproducibly visualized by trans- mission electron microscopy of conven- tionally prepared tissue, i.e., fixation in glu- taraldehyde, followed by postfixation in OsO4, en b loc staining in uranyl acetate, solvent dehydration, and embedding in ep- oxy resin (Hunziker and Schenk, 1984). The visualization of PG was reported im- proved by the use of low temperature em- bedded, glutaraldehyde-fixed rabbit aorta, and the PG was described as rod-like gran- ules with connecting filaments. However, fixation carried out in aqueous media, and the subsequent dehydration and embed- ding, have been shown to induce consider- able distortion of the matrix components (Rostgaard and Trenum-Jensen, 1980) and,
0889-1605/88 $3.00 Copyright ~ 1988 by Academic Press. Inc. All rights of reproduction in any ~rm reserved.
200 RICHARDSON, MC GUFFEE, AND HATTON
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FtG. 1. Diagrammatic representation of the possi- ble structure of arterial PG aggregate. The dimensions of the PG monomer are based on the observations of Wight and Hascall (1983). The spacing for the PG monomers on the hyaluronic acid is based on the ob- servations made in cartilage PG (Hascall, 1980). For clarity the GAG side chains have been omitted from this part of the diagram.
moreover, up to 37% of 35S-labeled PG was lost during these procedures (Chen and Wight, 1984). The use of cationic dyes such as ruthenium red reduces the loss of 35S- labeled PG during processing and permits visualization of the PG. However, the PG is condensed or collapsed by this staining pro- cedure, and it appears as granules (Chert and Wight, 1984; Contri et al. , 1985; Has- call, 1980; Iozzo et al. , 1982; Richardson et al., 1980, 1982; Wight and Hascall, 1983). The granules, which have been shown to contain GAG due to their degradation by GAG-specific enzymes (Richardson e t a / . , 1980, 1982; Chen and Wight, 1984) are as- sociated with fine fibrils. The GAG contain- ing granules are generally considered to represent the condensed PG monomers, and the fibrils, the hyaluronic acid back- bone of the aggregate (Hascall, 1980).
Ruthenium red-stained PG granules are of two sizes, large proteoglycan granules
(LPG), 20-50 nm diameter, which are present in the extracellular space, and small proteoglycan granules (SPG) <20 nm diam- eter, associated with the basement mem- brane of endothelial and smooth muscle cells. Much of the presently available infor- mation relating to PG distribution and ac- cumulatio,n in the arterial wall in s i tu has been obtained by stereological analysis of ruthenium red-stained tissue (Richardson et al . , 1980, 1982). Stereological analysis of ruthenium red-stained tissue was shown to reflect biochemical changes in the sulfated GAG content of human colon carcinoma (Iozzo et al., 1982). Ruthenium red staining has also been used to examine the interac- tion of PG with collagen (Myers et al. , 1973; Vidal and Mello, 1984) and elastin (Contri et al. , 1985; Goldfischer et al. , 1983) in the arterial wall and in other tissues. However, it has been impossible to determine with precision the structural organization of the PG aggregates and the relationship that these macromolecules have to the fibrous connective tissue components and the cells of the vessel wall using such techniques, because of the alterations in the morpholo- gy of the PG induced by the chemical fixa- tion.
Hunziker (1984) reported that the PG in cartilage was preserved as a reticulum of fine filamentous threads in tissue prepared without aqueous fixation, by high-pressure freezing followed by freeze substitution in methanol, and low temperature embedding. The reticulum was described as one which closely resembled the native morphology of PG. Two morphologically distinct compo- nents were identified and, based on their size and their selective affinity for uranyl acetate stain, the thicker, more intensely
FIG. 2. The extracellular space of the intima of rabbit thoracic aorta prepared by conventional methods without cationic dye stain. The matrix contains faintly staining amorphous material (C, collagen; E, elastin). Scale bar = 100 rim.
FIG. 3. The extracellular matrix of fast-frozen rabbit abdominal aorta adjacent to the internal elastic lamina (IEL). The matrix contains collagen (C) and a meshwork of PG filaments. The heavily staining long filaments (LF) are indicated by arrowheads; the short thick filaments (TF) are indicated by arrows; the fine filaments (FF) are indicated by open arrowheads. Scale bar = 100 nm.
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202 RICHARDSON, M( GUFFEE, AND HATrON
staining strands were considered to be the protein and the thinner strands, the carbo- hydrate of the PG.
In the present study we have explored the possibility of fast-freezing, freeze- drying, and plastic-embedding arterial tis- sue as a method of visualizing this connec- tive tissue without the use of cationic dyes, chemical fixatives and solvent dehydration. We have examined the extracellular matrix of rabbit aorta and renal artery, and com- pared the freeze-dried material to adjacent samples of the same vessels after glutaral- dehyde fixation and staining with the cat- ionic dyes ruthenium red. To explore fur- ther the nature of the extracellular matrix, the tissue was also examined before and af- ter treatment with GAG-specific enzymes.
METHODS
Heallhy New Zealand White rabbits of both sexes, weighing 3--4 kg, were killed by an overdose of anes- thetic (sodium pentobarbital, 50 mg/kg body wt); the renal arteries and the abdominal aorta, or the thoracic aorta and the aortic arch, were removed within 3 rain of death. The excised arterial segments were immedi- ately immersed in oxygenated Krebs-Henzleit solu- tion at room temperature. The adventitial tissue was carefully dissected away from the vessels, and the re- maining vessel was diced into i-ram square portions of the full thickness vessel wall. During the dissection, the vessels were immersed in Krebs-Henzleit solu- tion. The l-mm samples were fast-frozen, freeze- dried, and embedded by the technique described by McGuffee and co-workers (1981; Chiovetti et al., 1986). The arterial samples were frozen from the inti- real surface by bringing them rapidly into contact with a highly polished copper bar cooled with liquid nitro- gen to approximately -196°C. Frozen samples were immediately transferred to a liquid nitrogen holding bath until they were loaded into the specimen contain- er of the freeze-drying apparatus. The freeze-drying
apparatus is based on liquid nitrogen-cooled 5 A mo- lecular seive material which acts as a cryogenic pump. The design and operation is detailed in earlier reports (McGuffee etal., 1981; Chiovetti ef at., 1986). All the samples were exposed to vapors of osmium tetroxide ia vacuo. The vacuum in the freeze-dryer was broken with dry nitrogen gas, and the samples were immedi- ately transferred to Spurr's resin ($purr, 1969). For embedding, the samples were diced into four pieces and transferred to t/at embedding moulds containing fresh resin. Samples from the same vessels which were frozen were also fixed in 2.5% glutaraldehyde for 18 hr at 4°C, were rinsed in wash buffer alone and postfixed in aqueous i% OsO, for 1 hr or rinsed in wash buffer containing ruthenium red (0.75 mg/ml) for 18 hr, and postfixed in aqueous 1% OsO, containing ruthenium red (0.75 mglml) for i hr, All glutaralde- hyde-fixed samples were stained en bloc with 10% aqueous uranyl acetate for I hr, dehydrated through graded ethanol, and embedded in Spurr's resin (Spurr, 1969).
Ultrathin sections were mounted on uncoated high- transmission copper grids. Sections of froze~ tissue and tissue not stained by cationic dye were stained with lead citrate (2 rain) and uranyl acetate (2 rain); the tissue stained by ca/ionic dye was viewed with no fur- ther staining. The sections were examined in a Philips TEM 301 at 60 KV.
Enzyme Treatment
Unfixed thoracic aorta or renal artery segments were diced into l~mm square samples as above and exposed to chondroitinase ABC (5 units/ml) or hepar- itinase (5 units/ml) (Miles Laboratories, Elkhart, IN) in oxygenated Krebs-Henzleit solution for 20 rain at 370C. The tissue samples were divided into two groups, one group was fast-frozen, freeze-dried, and embedded, the other was fixed in glutaraldehyde and stained with ruthenium red as above.
Mmphometric Evaluation
For morphometric analyses, all micrographs were enlarged to ×80 000. Measurements were made using an image analysis system (VIAS, Pella, Inc., CA). The measurements obtained were based on comparison to micrographs of a calibrated grid (Ladd Research In-
FIG. 4. The extracellular matrix of fast-frozen rabbit renal artery. Filaments are in contact with collagen fibrils (arrows) and elastin (E). A long filament appears to be wrapped around one collagen fibril (open arrow- head). The pericellular matrix appears to contain a continuous fibril (large arrows) which is attached to the cell surface and the rest of the extracellular matrix by fine filaments (SMC, smooth muscle cell; C, collagen;). Scale bar = 1{30 nm.
FiG. 5. The extraceilular matrix of the media of rabbit renal artery, stained with ruthenium red. The medial smooth muscle basement membrane contains SPG (arrowhead); LPG (arrow) are present in the extracellular space and associated with collagen (C) and elastin (E) (SMC, smooth muscle cell). Scale bar = 100 nm.
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dustries, Inc., VT), printed at the same final magnifi- cation. The data are presented as the means "- SE for a minimum of 50 measurements.
Representative micrographs of fast-frozen, tissue and the corresponding ruthenium red-stained tissue from control and enzyme-treated material are pre- sented.
RESULTS
Frozen Tissue
The ultrastructural appearance of the ex- tracellular matrix, of normal arterial.tissue, processed by fast-freezing, contrasted sharply from that of glutaraldehyde-fixed tissue, with or without ruthenium red. In glutaraldehyde-fixed tissue, with no ruthe- nium red, the extracellular matrix con- tained collagen and elastin, and the remain- der of the space contained faintly staining amorphous material (Fig. 2).
In contrast, in frozen tissue, as well as collagen and elastin, the extracellular ma- trix contained a reticulum of filaments (Figs. 3, 4). Three morphologically differ- ent sizes of filaments were distinguished; long heavily staining filaments (LF), which were up to 1000 nm long, shorter thick fil- aments (TF), which were 50-70 nm long and appeared to branch from the LF, and less intensely staining fine filaments (FF), 30-50 nm long, which appeared to branch from the TF.
Collagen and elastin were closely associ- ated with the filaments of the'extracellular matrix. TF were present at regular intervals along the collagen fibers, corresponding to the periodicity of the collagen banding. In freeze-dried tissue the collagen periodicity was 56 -+ 3 nm. FF were also seen associ- ated with collagen fibers, but at less regular intervals, and sometimes arising from the same site as the TF. The LF ran parallel to the collagen, and sometimes appeared to wrap around the collagen fibers (Fig. 4). TF
RICHARDSON, MC GUFFEE, AND HATTON
and FF were observed at the surface of elastin and associated with elastin mi- crofibrils. The TF frequently appeared to link adjacent collagen fibers and also formed bridges between the fibrous con- nective tissues and the smooth muscle cells.
The extracellular matrix of ruthenium red-s ta ined t i ssue showed the well- established appearance of dense granules (LPG) connected by projections (Fig. 5). The granules were associated with colla- gen, coinciding with the periodicity of the collagen banding, which was 64 - 3 nm, and were also associated with elastin.
For comparison of the appearance of the extracellular matrix visualized by fast- freezing and by staining with ruthenium red, Figs. 6 and 7 are from similar areas of the vessel wall taken from the same seg- ment of aortic arch, and are printed at the same final magnification. In this tissue when prepared by fast-freezing (Fig. 6) the spacing between the TF branches from the LF was 51 -+ 4 nm; when stained with ru- thenium red (Fig. 7) the spacing between adjacent LPG was 69 --- 5 nm.
The plasma membrane of the smooth muscle cells in fast-frozen tissue was close- ly associated with a dense meshwork of fil- aments. In regions where the trilamellar structure of the plasma membrane was clearly visible, the pericellular coat fre- quently showed a continuous filament, of- ten parallel to the plasma membrane, and connected to the cell by much finer fila- ments (Fig. 4). In areas where the extracel- lular matrix was dense, the continuous fil- ament could not be distinguished from the reticulum of the extraceilular matrix.
In ruthenium red-stained tissue, the base- ment membrane of the smooth muscle cells was visualized as an electron semi-dense
FIG. 6. Fast.frozen rabbit aortic arch. The branch points on the LF are shown by arrows (C. collagen; E, elastin). Scale bar = 100 nm.
FiG. 7. Rabbit aortic arch adjacent to the tissue illustrated in Fig. 6, stained with ruthenium fred. The spacing between adjacent LPG is shown by arrows (C, collagen; E, elastin). Scale bar = 100 rim.
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206 RICHARDSON. MC GU
layer containing SPG mainly at the outer margin (Fig. 5). Some LPG were closely associated with the SPG.
Enzyme 7)'eatment
The effect of GAG-specific enzymes on the extracellular matrix of the media of the renal artery is shown in Figs. 8--11. Un- treated renal artery media is shown in Figs. 4 and 5.
Chondroitinase ABC
C h o n d r o i t i n a s e ABC se lec t ive ly re- moved the FF from the extracellular matrix of fast-frozen, freeze-dried tissue. TF asso- ciated with collagen and elastin were still p resent . The per ice l lu lar f i laments ap- peared largely unchanged except that the continuous filament was interrupted (Fig. 8). In comparison, chondroitinase ABC re- moved the LPG from the extracellular ma- trix, and from collagen and elastin, in tissue subsequently stained with ruthenium red (Fig. 9).
Heparitinase
Incubation of tissue with heparitinase, followed by fast-freezing, removed the con- t inuous f i lament from the per iphery of smooth muscle cells. However, some of the filaments associated with the cell surface were not removed (Fig. 10). In hepariti- nase-treated tissue, which was then ruthe- nium red-stained, the SPG were removed f rom the b a s e m e n t m e m b r a n e of the smooth muscle cells, but heparitinase did not destroy or prevent the formation of the electron semi-dense layer (Fig. I 1).
DISCUSSION
The ultrastructure of the arterial nonfi- brous extracellular matrix in fast-frozen, freeze-dried material is significantly differ-
FFEE, AND HA'Iq'ON
ent from that of conventionally prepared, i.e., glutaraldehyde-fixed tissue. In the lat- ter, there is amorphous material in the ex- tracellular space, but the PG is not clearly visible. In contrast, in fast-frozen, fi'eeze- dried arteries there is a reticulum of fine filaments. Some of these filaments interact with collagen, elastin, and the cell surface. This general appearance of the extracellular matrix is similar to that described as PG in cartilage prepared by high-pressure freez- ing and freeze-substitution (Hunziker and Schenk, 1984). Further characterization of the reticulum shows that some of the fila- ments are degraded by GAG-specific en- zymes, confirming that the reticulum con- tains PG.
In the present study the extracellular ma- trix contained three morphologically dis- tinct filaments; the FF, which were selec- tively removed by chondroi t inase ABC, and consist presumably of the GAGs, chon- droitin sulfate, and/or dermatan sulfate; the TF, which were resistant to the GAG- specific enzymes and may represent the protein core; and the LF which, because of their relative length, may represent hyal- uronic acid. The pericellular matrix, al- though morphologically similar to the retic- ulum in the extracellular space, reacted dif- ferently to GAG-specific enzymes.
The morphology of PG aggregates, ex- tracted from cartilage (Hascall, 1980) has been described and correlated with ruthe- nium red staining characteristics, but the morphological appearance of arterial PG aggregates has not, to our knowledge, been reported. PG monomers extracted from monkey arterial smooth muscle cells in cul- ture were shown to contain a central core averaging 140 nm in length, with 8 to 10 side arms, each 70 to 75 nm in length (Wight and Hascall, 1983). In comparison, in rabbit ar-
FI6. 8. The effect of incubation in chondroitinase ABC on the PG of the media of renal artery prior to fast-freezing. The FF have been removed from the PG meshwork; the pericellular filaments (open arrowhead) are largely unchanged (C, collagen; SMC, smooth ,muscle cell). Scale bar = 100 nm.
Fic. 9. The effect of incubation in chrondrotinase ABC on the PG of the media of the renal artery prior to staining with ruthenium red. The LPG have been removed, but the SPG (open arrowhead) are still present (C, collagen; E, elastin; SMC, smooth muscle cell). Scale bar -- 100 nm.
PROTEOGLYCAN IN FAST-FROZEN ARTERIES 207
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terial PG preserved ht situ by fast-freezing, the TF were 50 to 70 nm long, and the FF were 30 to 70 nm. These observations are consistent with these componen~.s of retie- ulum in the extracellular matrix represent- ing PG monomers. The discrepancies in the sizes may reflect differences due to species or tissue preparation. Furthermore, the pe- r iodicity of the collagen banding was smaller in fast-frozen material than in glu- taraldehyde-fixed tissue. Based on a peri- odicity of 67 nm (Myers et al., 1973) for glutaraldehyde-fixed tissue the dimensions of the PG observed in the present study are consistent with those described for isolated PG monomers by Wight and Hascall (1983).
PG has been demonstrated by transmis- sion electron microscopy of sections of ar- terial tissue following staining with a vari- ety of cationic dyes, as well as ruthenium red, including aician blue, acridine orange, safranin O (Chen and Wight, I984), tolu- idine blue O, osmium-ferrocyanide (Colt- off-Schiller and Goldfischer, 1981), and cu- prolinic blue (Volker et al., 1986).
The morphology of the PG demonstrated by these stains included granules as shown by ruthenium red and osmium-ferro- cyanide, or rods as shown by alcian blue, acridine orange, or cuprolinic blue. In con- trast, safranin O stain resulted in a more mesh-like appearance, and was described as more closely resembling the morphology of isolated PG molecules (Chert and Wight, 1984).
The appearance of the extraceUular ma- trix in fast-frozen arteries was compared to that in similar tissue stained by ruthenium red, in order to support the interpretation of the appearance of PG in situ as demonstrat- ed by fast-freezing. The density of the re-
ticulum in fast-frozen arteries was compa- rable to that of the PG stained by ruthenium red. There is good correlation of the spac- ing between adjacent ruthenium red-stained LPG with the spacing of TF branching points from LF, if the measurements are compared to the collagen banding in the same preparation. The LPG were removed by chondroitinase-ABC, as previously de- scribed (Chen and Wight, 1984; Richardson et al., I980). These observations are con- sistent with the interpretation that, in ruthe- nium red-stained tissue, the LPG contain GAG and represent the collapsed PG monomer (Chen and Wight, 1984), whereas in the fast-frozen tissue, the TF represent the protein core of the PG monomer; the FF, the GAG chains and the LF represent hyaluronic acid. Ruthenium red-stained LPG were associated with collagen with the same distribution as the TF seen in frozen tissue.
The interaction of PG with collagen can be visualized more extensively in fast- frozen material than in other preparations. In additions to TF-collagen interactions, FF were observed associated with colla- gen, and LF appeared to wrap around the fibrils. These observations are consistent with the arrangement proposed by Poole et al. (1982). In another report of the associa- tion of PG with collagen (Vidal and Mello, 1984), the PG was suggested to encircle the collagen fibril, and multiple branching sites for core protein and hyaluronic acid were proposed. This possible arrangement is also supported by our observations.
In fast-frozen tissue, the smooth muscle cell basement membrane, i.e., the pericel- lular matrix, was strikingly different from that seen in glutaraldehyde-fixed tissue. It
FIG. 10. The effect of incubation in heparitinase on the basement membrane of smooth muscle cells of the media of rabbit renal artery prior to fast-freezing. The continuous line has been removed but some of the extracellular filaments remain (C, collagen: SMC, smooth muscle cells). Scale bar = 100 nm.
Fro. I 1. The effect of incubation in hepartinase on the basement membrane of smooth muscle cells of rabbit renal artery prior to staining with ruthium red. The SPG and some of the LPG have been removed but the granules associated with collagen remain (C, collagen; E, elastin; SMC, smooth muscle cell). Scale bar = 100 rim.
210
consisted of filaments associated with the plasma membrane and a continuous fila- ment which was frequently parallel to the cell surface. The pericellular filaments were morphologically similar to those of the ex- tracellular matrix and appeared to be con- tinuous with the extracellular reticulum where it was very dense. This may indicate that in glutaraldehyde-fixed tissue there is condensation or collapse of the basement membrane components which results in the enhanced contrast in this area. The contin- uous filament was removed following expo- sure to heparitinase and was also inter- rupted by chondroitinase A B C . The fila- ments associated with the cell membrane were not degraded by either enzyme treat- ment. Therefore, the cont inuous filament may contain mainly heparan sulfate, which is closely associated With some chondroitin or dermatan sulfate. The remaining resis- tant filaments may contain other compo- nents of the basement membrane, e.g., lam- inin or fibronectin (Laurie et al., 1982). The morphology of the fast-frozen tissue corre- lated well with the observations in rutheni- um red-stained material. Heparit inase treatment removed the SPG from the base- ment membrane, but did not totally degrade the electron semi-dense layer and chon- droitinase ABC removed the LPG which were closely associated with the SPG.
In this report we have demonstrated that arterial PG can be visualized as a dense re- ticulum. This finding supports the concept, based on bichemical observations, that PG must exist as a complex meshwork in order to control water and solute transport, and for steric exclusion of macromolecules (Comper and Laurent, 1978) st~ch as low density lipoprotein (Camejo, 1982). Arterial PG also provide a structural component by their capacity to promote hydration, and to- gether with collagen and elastin give the strength and flexibility to withstand the pul- satile pressure of flowing blood. The retic- ulum fo rma t ion appears c o n t i n u o u s throughout the extraceilular space and therefore may provide the mechanism
RICHARDSON, MC GUFFEE. AND HATTON
which results in a unification of the varied components of the vessel wall.
We recognize that, in some regions of the extracellular matrix, the appearance of this reticulum may be altered by the formation of ice crystals. The size and periodicity of the banding of collagen fibrils was different in conventional fixed and fast-frozen tissue, but it has been previously shown that the dimensions of smooth muscle cells and their organelles are also different in fast- frozen compared to conventionally fixed tissue (McGuffee et al., 1981). However, the overall preservation of the extracellular matrix reported in this study represents a significant improvement over techniques previously employed to visualize extracel- lular matrix.
We conclude that the fast-frozen, freeze- dried tissue provides a means to visualize PG in arterial wall that avoids the distortion due to conventional fixation, staining by cationic dyes, and solvent dehydration. We presume that fast-freezing and freeze- drying may closely preserve the in vivo morphology of arterial extracellular matrix.
This work was supported by a grant from the Heart and Stroke Foundation of Ontario. The technical as- sistance of Mrs. M. MacLellan. Ms. S. Little, and Miss S. Moar is gratefully acknowledged. M. Richard- son holds a Scholarship from the Canadian Heart Foundation; L. J. McGuffee is an Established Inves- tigator of the American Heart Association; and M. W. C. Hatton is a Research Associate of the Heart and Stroke Foundation of Ontario.
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