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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:523-532.)
© 1996 American Heart Association, Inc.

Articles

Apolipoproteins B, (a), and E Accumulate in the Morphologically Early Lesion of `Degenerative' Valvular Aortic Stenosis

Presented in part at the Western Section, American Federation for Clinical Research, Carmel, Calif, February 11, 1994.

Kevin D. O'Brien; Dennis D. Reichenbach; Santica M. Marcovina; Johanna Kuusisto; Charles E. Alpers; Catherine M. Otto

From the Division of Cardiology (K.D.O'B., J.K., C.M.O.) and Northwest Lipid Research Laboratories (S.M.M.), Departments of Medicine and Pathology (D.D.R., C.E.A.), University of Washington, Seattle. Dr Kuusisto is now with Kuopio (Finland) University.

Correspondence to Kevin D. O'Brien, MD, Division of Cardiology, Box 356422, University of Washington, Seattle, WA 98195-6422.

	Abstract

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Abstract Nonrheumatic aortic stenosis of trileaflet aortic valves has been considered to be a "degenerative" process, but the early lesion of aortic stenosis contains the chronic inflammatory cells, macrophages and T lymphocytes. Because lipoprotein deposition is prominent in atherosclerosis, another chronic inflammatory process, this study examined whether lipoproteins accumulate in aortic valve lesions. Immunohistochemical studies were performed to detect apolipoprotein (apo) B, apo(a), apoE, macrophages, and -actin–expressing cells on 18 trileaflet aortic valves that ranged from normal to stenotic. All three apolipoproteins were detected in early through end-stage lesions of aortic stenosis but not in histologically normal regions. Comparison with oil red O staining suggested that most of the extracellular neutral lipid in these valves was associated with either plasma-derived or locally produced apolipoproteins. Thus, in early through end-stage aortic valve lesions, apolipoproteins accumulate and are associated with the majority of extracellular valve lipid. These results are consistent with the hypothesis that lipoprotein accumulation in the aortic valve contributes to pathogenesis of aortic stenosis.

Key Words: lipids • macrophages • inflammation • foam cells

	Introduction

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A prominent feature of "degenerative" valvular aortic stenosis is the accumulation of lipid, particularly in the fibrosa, the anatomic layer of the valve located immediately below the endothelium on the aortic side of the valve.^{1 2 3 4 5 6 7 8 9 10} Most authors have suggested that this lipid deposition is a consequence of degenerative changes in the valve^{1 2 3 4 5 6} rather than a result of an active inflammatory process, despite the fact that the earliest description of the disease noted the presence of an inflammatory infiltrate in stenotic valve leaflets.¹ Since the prevalence of clinical aortic stenosis in the elderly is only 2.9%,¹¹ it has been suggested that factors in addition to age-related degenerative changes must play a role in the development of this disease.^{10 12 13 14} Several authors have noted that aortic valvular lesions have some similarities to atherosclerosis,^{7 8 9 10} and recent immunohistochemical studies have demonstrated evidence of chronic inflammation in aortic valve leaflets, including the presence of macrophages^{10 12} and T lymphocytes^{10 12 13} and the expression of HLA-DR antigen on valve fibroblasts.¹² Molecules implicated in calcification, including osteopontin^{15 16 17 18 19} and BMP-2a,²⁰ have been detected recently in atherosclerotic lesions, and osteopontin also has been found recently in aortic valve lesions.¹⁴ These observations have raised the possibility that aortic valve lesion development might be actively regulated, and thus potentially modifiable, rather than being an inevitable consequence of aging.^{10 14}

Despite the widespread recognition that lipid accumulates in aortic valve lesions, little is known about its origin and thus whether its accumulation might be abrogated. One previous study has suggested that some aortic valve lipid is derived from plasma lipoproteins, on the basis of the demonstration of colocalization of a histological stain for neutral lipid with immunohistochemical staining for LDL.⁹ However, this finding had not been confirmed, and no studies to date had characterized whether other lipoproteins implicated in atherogenesis, such as lipoprotein(a) [Lp(a)]^{21 22 23 24} or apoE-containing lipoproteins,^{25 26 27 28} might be present in aortic valvular lesions or whether apolipoproteins might be present in all areas containing extracellular neutral lipid.

Therefore, this study was undertaken to address the following questions: (1) Are the protein components of the plasma lipoproteins LDL and Lp(a), specifically, apoB and apo(a), associated with human aortic valve lesions? (2) Is apoE, a prominent component of atherosclerotic lesions, present in aortic valve lesions? and (3) Does the extent of neutral lipid deposition correlate with the extent of apolipoprotein deposition?

	Methods

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Human Aortic Valvular Tissue
Human aortic valvular tissue was obtained at autopsy (n=15) or surgery (n=3) from 18 patients. Tissue from these patients was used in previous studies characterizing the cellular components¹⁰ and osteopontin content¹⁴ of aortic valvular lesions. Macroscopically, the leaflets were normal or only mildly thickened (morphologically early disease) in 15 patients. In 3 patients, the leaflets were severely thickened and there was clinical evidence for significant aortic valve obstruction. Sections of the valve leaflets were fixed in methanol–Carnoy's solution and paraffin-embedded. Because all of these leaflets had been paraffin-embedded, they could not be stained for neutral lipid. To evaluate the colocalization of apolipoproteins and neutral lipid, an additional five valves were obtained from the excised native hearts of 5 consecutive patients undergoing cardiac transplantation. None of these 5 patients had clinically evident or echocardiographically detectable aortic stenosis. These five leaflets, which macroscopically were normal to mildly thickened, were placed in OCT embedding solution (Miles Laboratories, Inc) and snap-frozen in liquid nitrogen within 2 hours of organ removal. Frozen sections were postfixed in acetone at 4°C for 10 minutes before immunohistochemical staining.

Histological Stains
Paraffin-embedded specimens were examined for morphology with hematoxylin and eosin staining, for the pattern of elastin fibers and protein deposition with Verhoeff–van Gieson's (VVG) stain, for mineralization with von Kossa's stain, and for calcification with McGee-Russell alizarin red stain. Stains for mineralization and calcification were not performed on specimens that had been decalcified by formic acid treatment. However, because the methanol Carnoy's solution used to fix all specimens contains a small amount of acetic acid, the possibility of slight decalcification by the acetic acid cannot be excluded. Frozen specimens were stained for neutral lipid with oil red O.

Antibodies and Antiserum
Single-label immunohistochemistry was performed with monoclonal antibody anti-CD68 (Dako Corporation) to identify macrophages²⁹ and a monoclonal antibody anti–-actin (Boehringer-Mannheim Biochemica) to identify smooth muscle cells or myofibroblasts.³⁰ Both antibodies were used at a 1:1000 dilution. ApoB and apo(a) were identified with the use of monoclonal antibodies 9A and a-5 produced and immunochemically characterized as reported previously.^{31 32} Briefly, 9A is an IgG1 antibody with a high affinity constant (2.1x10⁹ L/mol) recognizing an epitope located within residues 2657-3248 of the apoB molecule. Additionally, this antibody has been demonstrated to inhibit cellular uptake and degradation of LDL in a dose-dependent manner.³¹ Monoclonal antibody a-5 has an affinity constant of 2.1x10¹⁰ L/mol and is directed to an epitope located in the carboxyl-terminal part of kringle 4 types 1 and 2 of apo(a). The binding of a-5 is not affected by oxidation of Lp(a) or by carbohydrate removal.³² Both antibodies were used at a concentration of 1 µg/mL. ApoE was detected with the use of a goat anti-human apoE polyclonal antiserum, the immunohistochemical specificity of which has been described previously.²⁶

Single-Label Immunohistochemistry
Single-label immunohistochemistry was performed as described previously.^{10 26} Briefly, tissue sections were deparaffinized with xylene and then rehydrated with graded alcohols. The slides were blocked with 3% H₂O₂, washed with PBS, incubated for 60 minutes with the primary antibody or antiserum, and then washed again with PBS. A biotin-labeled secondary antibody (anti-mouse or anti-goat, as appropriate) then was applied for 30 minutes, followed by an avidin-biotin-peroxidase conjugate (ABC Elite, Vector Laboratories) for 30 minutes. Standard peroxidase enzyme substrate, 3,3'-diaminobenzidine with nickel chloride, then was added to yield a black reaction product. The slides were counterstained with methyl green.

Negative controls included substitution of primary antisera/antibody with either PBS or, as appropriate, isotype-matched, irrelevant monoclonal antibodies or normal goat serum to abolish specific immunohistochemical staining.

Data Collection and Statistical Analysis
Each set of histological and immunohistochemical stains was evaluated by two experienced observers with side-by-side comparisons of the different stains on sequential tissue sections to allow correlation of the location of abnormalities. Each slide was examined systematically, with evaluation for each characteristic on a semiquantitative scale in which 0=none, 1=mild, 2=moderate, and 3=severe. Mantel-Haenszel tests for linear association were used for comparisons of semiquantitative assessments of characteristics and were performed with the SPSS for MS Windows, Release 6, software, running on an 80486 microprocessor. These statistical semiquantitative methods have been validated previously.^{10 14}

	Results

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Plasma-Derived ApoB and Apo(a) Are Associated With Mild Aortic Valvular Lesions and Aortic Valvular Mineralization
The normal aortic valve leaflet (Fig 1a through 1e, left-hand portions) is composed of three anatomic layers: the fibrosa, a collagen-rich layer on the aortic side of the valve (top layer); the ventricularis, a layer with prominent elastin fibers located on the ventricular side of the valve (bottom layer); and the spongiosa, a layer of variable thickness lying between the fibrosa and ventricularis, with a less dense matrix and often containing adipose cells. Isolated macrophages often can be found in the ventricularis and spongiosa of normal adult aortic valve leaflets but are not present in the normal fibrosa. In contrast, the morphologically early lesion of aortic stenosis (Fig 1a through 1e, right-hand portions) is characterized by displacement and fragmentation of an elastic lamina that normally lies immediately beneath the endothelium, the accumulation of protein and chronic inflammatory cells, and thickening of the adjacent fibrosa.

View larger version (186K): [in this window] [in a new window]   Figure 1. Apolipoproteins in morphologically early aortic valve lesions but not in histologically normal regions of the valve. Fig 1a through 1e are low-power photomicrographs of sections obtained at the junction of the middle third of an aortic valve leaflet (left-hand portion) and the leaflet base (right-hand portion) and are oriented with the aortic surface of the leaflet at the top. The left-hand portion in each photomicrograph (delimited by the arrow shown in Fig 1a) is a normal region of valve, while the area to the right of the arrow is a morphologically early lesion of aortic stenosis. VVG stain demonstrates that the histologically normal region of the valve (Fig 1a, left) consists of the collagen-rich fibrosa, lying underneath the endothelium on the aortic surface of the valve; the elastin-rich ventricularis, lying on the ventricular surface of the valve; and a third layer, the spongiosa, sandwiched between the fibrosa and the ventricularis. Scattered macrophages (black immunoreaction product, Fig 1b) are present in the ventricularis and spongiosa but not in the fibrosa of the normal region(Fig 1b, left). ApoB (Fig 1c) and apo(a) (Fig 1d) also are absent from the normal region (lack of black immunoreaction product, Fig 1c and 1d, left), and no calcium mineral is detected in the normal region by von Kossa's stain (Fig 1e, left). In contrast, the early lesion present in the leaflet base (Fig 1a through 1e, right) is characterized on VVG stain by downward displacement and fragmentation of the black elastic membrane that normally lies between the fibrosa and the aortic surface endothelium, accumulation of protein (Fig 1a, right), and the subendothelial accumulation of macrophages (Fig 1b, right). Both apoB (Fig 1c, right) and apo(a) (Fig 1d, right) accumulate in the lesion as well as in the adjacent fibrosa. Von Kossa's stain reveals accumulation of a black product identifying calcium mineral (Fig 1e, right) in the deeper regions of apolipoprotein accumulation (Fig 1c and 1d, right). (Original magnification x100 [Fig 1a through 1e]; VVG stain [Fig 1a], methyl green counterstain [Fig 1b through 1d], and von Kossa's stain [Fig 1e].)

Normal regions of the leaflets that did not contain lesions typically were devoid of staining for apoB and apo(a). Fig 1a shows a section of a valve leaflet stained with the VVG stain and is obtained at the junction of the midportion of the leaflet, located to the left of the arrow, and the leaflet base, located to the right of the arrow. ApoB (Fig 1c) was not present in the normal region of the valve identifiable by histological criteria defined above and by the absence of macrophages (Fig 1b, left-hand portion). Similarly, apo(a) (Fig 1d, left-hand portion) was not detected in the normal region of the valve. Note that the ventricularis of the normal valve typically contains scattered, resident macrophages (Fig 1b, bottom).

In contrast, apoB and apo(a) could be detected in areas of the aortic valve that contained early valvular lesions (Fig 1a, right-hand portion), characterized by displacement and fragmentation of the black elastic lamina and by the presence of macrophages (Fig 1b, right-hand portion). The apolipoproteins were detected within both the lesion and the adjacent fibrosa. ApoB and apo(a) typically were detected in similar distributions (Fig 1c and 1d, right-hand portion). The apparent colocalization of apoB and apo(a) has been reported both in atherosclerosis^{33 34 35} and in tendinous xanthomas³¹ and is consistent with either of two possibilities: first, that both LDL and Lp(a) have been deposited; or second, since Lp(a) contains both apoB and apo(a), that only Lp(a) is present. Expression of -actin, which would denote the presence of smooth muscle cells or myofibroblasts, was not detected in these regions (data not shown).

Von Kossa's stain (Fig 1e), which detects inorganic phosphate and thus mineral deposition, identified fine deposits of mineral in the region of apolipoprotein staining. However, mineralization was not present throughout the entire region of apolipoprotein staining but instead was localized to the deeper portions of the region most distant from the aortic lumen. It should be noted that the fixative used, methanol Carnoy's solution, contains a 1:10 dilution of glacial acetic acid. Therefore, the possibility that this dilute concentration of acetic acid may have partially decalcified these specimens cannot be excluded.

The McGee-Russell alizarin red S stain also was used to demonstrate that calcium was present in areas with mineralization as detected by von Kossa's stain. As shown in Fig 2, staining of adjacent sections with the McGee-Russell alizarin red S stain (Fig 2a and 2c) and with von Kossa's stain (Fig 2b and 2d) demonstrates colocalization of calcium and phosphate in the regions with mineralization. The colocalization of calcium and phosphate in these regions is consistent with, though not conclusive evidence for, the presence of hydroxyapatite in these specimens.

View larger version (124K): [in this window] [in a new window]   Figure 2. Colocalization of staining for calcium and phosphate in an early aortic valvular lesion. Low-power (Fig 2a and 2b) and higher-power (Fig 2c and 2d) photomicrographs demonstrate through histological staining of adjacent tissue sections with the McGee-Russell alizarin red S stain for calcium (Fig 2a and 2c) and von Kossa's stain for inorganic phosphate (Fig 2b and 2d) the presence of both calcium and phosphate in this region (Fig 2a vs 2b and Fig 2c vs 2d). Colocalization of calcium and phosphate in this region would be consistent with, although not conclusive evidence for, the presence of hydroxyapatite, the form in which calcium is actively deposited in bone. (Original magnification x200 [Fig 2a and 2b] or x400 [Fig 2c and 2d].)

Presence of ApoB and Apo(a) in Moderate and Severe Lesions of Aortic Stenosis
ApoB and apo(a) also could be detected in moderate lesions of aortic stenosis (Fig 3), which are characterized both by larger accumulations of protein in the lesion itself and by more marked thickening of the fibrosa. VVG stain of a moderate lesion (Fig 3a) demonstrates pale regions in the fibrosa, suggesting accumulation of substances other than collagen, and von Kossa's stain (Fig 3b) localizes mineralization to these areas of pale staining. Immunohistochemical staining shows that apoB (Fig 3c and 3d) and apo(a) (Fig 3e and 3f) are present in the portion of the fibrosa adjacent to the lesion in regions with more pale staining with the VVG stain (Fig 3a). Similar to the results in early lesions, von Kossa's stain (Fig 3b) localized calcium mineral to the region of apolipoprotein staining most distant from the aortic lumen. The lesion contains an infiltrate of macrophages (Fig 3g and 3h).

View larger version (123K): [in this window] [in a new window]   Figure 3. Accumulation of apolipoproteins in the fibrosa adjacent to a moderate aortic valvular lesion. VVG stain (Fig 3a) demonstrates a moderate aortic valvular lesion, with substantial displacement of the elastic membrane and prominent thickening of the fibrosa. The fibrosa contains pale regions, consistent with accumulation of substances other than collagen. Substantial calcium mineral is detected with von Kossa's stain (Fig 3b) in the deeper portion of the region of the fibrosa, with a pale appearance on VVG stain (Fig 3a). There is substantial accumulation of apoB (Fig 3c and 3d) and apo(a) (Fig 3e and 3f) in both the lesion and adjacent fibrosa, and macrophage accumulation in the lesion (Fig 3g and 3h). (Original magnification x100 [Fig 3a through 3c, 3e, and 3g] or x200 [Fig 3d, 3f, and 3h]; VVG stain [Fig 3a], von Kossa's stain [Fig 3b], or methyl green counterstain [Fig 3c through 3h].)

Apolipoproteins also could be detected in leaflets removed from patients with clinical aortic stenosis (Fig 4). VVG staining of clinically stenotic leaflets (Fig 4a) revealed marked thickening of the fibrosa, displacement and loss of the elastic lamina, and the presence of macrophages (Fig 4b). ApoB (Fig 4c) was widely distributed in the fibrosa, and apo(a) also was present. However, in the example shown in Fig 4, the distribution of apo(a) (Fig 4d) was much more restricted than that of apoB, being found only in the central portion of the fibrosa. The demonstration that some areas had apoB without colocalized apo(a) suggests that LDL is deposited in aortic valve lesions in addition to Lp(a).

View larger version (65K): [in this window] [in a new window]   Figure 4. Accumulation of apolipoproteins in a clinically stenotic valve leaflet. VVG stain (Fig 4a) demonstrates absence of the black elastic membrane, obliterating the distinction between a large lesion and the thickened fibrosa, and the presence of a large, round calcium deposit (central portion, Fig 4a). Macrophages have accumulated around the circumference of the calcium deposit (Fig 4b), and there is substantial apoB (Fig 4c) and apo(a) (Fig 4d) accumulation. In contrast to Figs 1 and 3, there are large areas with apoB without corresponding apo(a), suggesting that LDL has been deposited, in addition to Lp(a). (Original magnification x100 [Fig 4a through 4d]; VVG stain [Fig 4a] and methyl green counterstain [Fig 4b through 4d].)

To test for colocalization of apoB with apo(a), Mantel-Haenszel tests for linear association were performed for each of the three anatomic regions of the valve leaflets for the comparisons of the semiquantitative assessments of the degree of accumulation of apoB versus apo(a). Highly statistically significant associations were found for apoB and apo(a) for all three anatomic regions, the leaflet base (P=.00001), the midleaflet (P=.00002), and the leaflet tip (P=.00005), confirming the impression from visual analysis of sections (and as illustrated in Figs 1 and 3) that apoB and apo(a) were colocalized in the majority of leaflet regions.

Localization of ApoE in Aortic Valve Lesions
ApoE often was present in regions with apoB and apo(a) (Fig 5) but also could be detected in regions without apoB (Fig 6).

View larger version (103K): [in this window] [in a new window]   Figure 5. Comparison of apoB and apoE localization in an early aortic valve lesion. VVG stain demonstrates the presence of early lesions of aortic stenosis, characterized by mild elastic lamina displacement and mild fibrosa thickening, and the extreme right-hand portion of the photomicrograph (right of arrow) shows a histologically normal region (Fig 5a). Macrophages have accumulated in the region between the aortic surface endothelium and the elastic lamina in lesion areas (Fig 5b, left-hand portion and center) but not in the normal region (Fig 5b, extreme right-hand portion). Both apoB (Fig 5c) and apoE (Fig 5d) have accumulated in the thickened fibrosa adjacent to the elastic lamina of lesion areas but are absent from the histologically normal region. (Original magnification x100 [Fig 5a through 5d]; VVG stain [Fig 5a] or methyl green counterstain [Fig 5b through 5d].)

View larger version (138K): [in this window] [in a new window]   Figure 6. ApoE intracellularly and extracellularly in areas without apoB. High-power photomicrographs of the early aortic valve lesion shown in Fig 1 demonstrate that substantial apoB (Fig 6a) and apoE (Fig 6b) are present extracellularly. However, comparison of Fig 6a and 6b demonstrates that there are regions in which only apoE is detected extracellularly (Fig 6a and 6b, upper right). (Original magnification x400; methyl green counterstain.)

There were statistically significant associations between the areas stained for apoB and apoE, but in only two of the three anatomic regions, ie, at the leaflet base (P=.02) and in the midleaflet (P=.004); no significant association was found at the leaflet tip (P=.20). These findings confirm the visual impression that although apoB and apoE are both present in lesion areas, their distribution is somewhat different, as characterized both by intracellular staining, which was found for apoE but not for apoB, and by the presence of similarities but also differences in the extracellular localization of apoB and apoE.

Association of Neutral Lipid With Apolipoproteins
Because the 18 valves described previously had been embedded in paraffin, they could not be stained for neutral lipid, since it is extracted from the valves during the deparaffinization process. Therefore, to determine whether all regions containing extracellular lipid in aortic valves contained apolipoproteins, serial frozen sections of aortic valve leaflets from 5 patients were stained with oil red O (to detect neutral lipid), apoB, apo(a), and apoE. As shown in Fig 7, the total distribution of extracellular neutral lipid could be accounted for by staining with one or more of the apolipoproteins. In addition, apoE could be detected intracellularly in subsets of lesion non–foam cell and foam cell macrophages (Fig 7). In contrast, detectable apoB and apo(a) were exclusively extracellular.

View larger version (167K): [in this window] [in a new window]   Figure 7. Colocalization of extracellular neutral lipid and apoB and apoE. A low-power photomicrograph of a hematoxylin-eosin–stained frozen section (Fig 7a) demonstrates the presence of two aortic valve lesions. Higher-power views of adjacent sections focus on the left-hand lesion of Fig 7a and demonstrate patterns of oil red O staining for neutral lipid (Fig 7b) and immunohistochemical staining of apoB (Fig 7c) and apoE (Fig 7d). Intracellular oil red O staining is present in subendothelial cells (Fig 7b, arrows). Still higher-power views demonstrate that these foam cells, which contain apoE (Fig 7e), are macrophages (Fig 7f). However, all regions of extracellular oil red O staining in the fibrosa (Fig 7b) have associated staining for apoB (Fig 7c) and/or apoE (Fig 7d). (Original magnification x100 [Fig 7a], x200 [Fig 7b through 7d], and x400 [Fig 7e and 7f]; hematoxylin and eosin counterstain [Fig 7a] or hematoxylin only [Fig 7b through 7f].)

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Stenosis of trileaflet aortic valves is a significant cause of morbidity and mortality among the elderly, but it does not uniformly afflict individuals in this age group.¹¹ Thus, it does not seem reasonable to assume that this disease is simply a degenerative condition. The incidence of atherosclerosis, which clearly is not simply a degenerative process, also increases with age,³⁶ and aortic stenosis and atherosclerosis have many common risk factors in addition to age. These include male sex,³⁷ hypertension,³⁸ dyslipidemia,^{38 39} and diabetes.^{38 39} The two diseases also have several common histological features, including the presence of a chronic inflammatory infiltrate,^{1 7 8 10 12 13 14 26 27 28} dystrophic calcification,^{1 2 3 4 5 6 7 8 9 10 14 15 16 17 20} and the accumulation of lipid.^{1 2 3 4 5 6 7 8 9 26 27 28 33 34 35} Although the presence of lipid as a prominent feature of aortic valve lesions long has been appreciated, only one previous human study provided primary evidence that some lipid may be derived from circulating lipoproteins,⁹ and this finding has neither been confirmed subsequently nor appreciated widely. The present study demonstrates that apoB and apo(a), the apolipoproteins of the "atherogenic" lipoproteins LDL and Lp(a),^{21 22 23 24 33 34 35} accumulate in developing lesions of aortic stenosis. Further, apoE, a prominent apolipoprotein in lesions of atherosclerosis,^{26 27 28} also is a significant component of lesions of aortic stenosis.

Potential Relevance of Lipoprotein Accumulation to Aortic Valve Lesion Pathogenesis
The presence of atherogenic apolipoproteins in aortic valvular lesions may provide some clues as to the pathogenesis of this disorder. First, their presence helps to explain the predilection for lesion development on the aortic side of the valve, which is an area of high mechanical and low shear stress, where endothelial injury could lead to the infiltration of plasma lipoproteins, similar to atherosclerosis-prone sites in arteries.⁴⁰ These abnormal mechanical forces would be accentuated in bicuspid aortic valves, perhaps predisposing them to even greater lipoprotein deposition. Subsequent minimal oxidative modification of the insudated lipoproteins then could lead to elaboration of a number of factors, including leukocyte adhesion molecules,⁴¹ monocyte chemoattractants,⁴² and leukocyte growth factors,⁴³ thus accounting for the chronic inflammatory cell infiltrate seen in aortic valvular lesions.^{10 12 13} More extensive oxidative modification of the lipoproteins then could lead to their recognition and uptake by macrophage scavenger receptors, resulting in formation of foam cells,^{44 45} the presence of which has been documented in aortic valve lesions.¹⁰ Extensively oxidized LDL has been shown to have a number of prothrombotic effects, including induction of endothelial cell tissue factor and plasminogen activator inhibitor-1 expression, as well as endothelial cell cytotoxicity. However, this sequence of events is speculative, since the present study provides no direct evidence for the presence of oxidatively modified lipoproteins in aortic valve lesions. Finally, fibrinogen also accumulates in aortic valve lesions,⁹ and fibrinogen accumulation may in part explain the accumulation of Lp(a) in lesions, since partially degraded fibrin has a high affinity for Lp(a).²¹

Over the long term, substantial apolipoprotein accumulation in lesions likely is mediated by elaboration of matrix proteins with affinity for apolipoproteins. This theory is supported by the observation of a previous study⁹ that, while LDL is present in aortic valve lesions, a number of proteins either similar in size to or smaller than LDL do not accumulate in these areas. The specific molecules that mediate LDL accumulation in aortic valvular lesions, especially in the adjacent fibrosa, are not known. Proteoglycans are likely candidates, on the basis of data from older histological studies demonstrating colocalization of aortic valve extracellular lipid with acidic mucopolysaccharides.⁹ Several in vitro studies have documented increased affinity of apolipoproteins for specific proteoglycans,^{46 47 48 49} and in vivo studies have colocalized specific apolipoproteins with particular proteoglycans.⁵⁰

Finally, three features of early aortic valve lesion apolipoprotein deposition are comparable with what is seen in early lesions of atherosclerosis. These are (1) the predilection of aortic valve lipoprotein deposition in areas of low shear stress, (2) the accumulation of lipoproteins in regions in which histological staining characteristics are consistent with the accumulation of proteoglycans, and (3) the associated macrophage and T-lymphocyte accumulation in regions of lipoprotein deposition. These similarities between early aortic valvular lesions and early lesions of atherosclerosis raise the possibility that, like early lesions of atherosclerosis, early lesions of the aortic valve might be controllable through identification and treatment of factors that promote their development.

Lipid Accumulation and Calcification
The association of calcification and lipid has been well described in atherosclerosis, and this study confirms the observations of others that they also colocalize in aortic valve lesions. The exact mechanisms by which lipids might participate in calcification are not known; however, extracellular matrix vesicles, composed in part by phospholipids and probably derived primarily from cell membranes, are believed to be important in lipid accumulation and calcification.⁵¹ These vesicles are abundant in atherosclerotic lesions as well as in older aortic valves.^{52 53} Also, through hydrolysis of their phospholipids or cholesteryl esters, lipoproteins in lesions might provide fatty acids to the calcification process. Further, oxidized lipids, which have been shown to cause cell membrane "blebbing" might induce formation of matrix vesicles from cells as a manifestation of cytotoxicity.

ApoE Accumulation and Lesion Pathogenesis
The role of apoE expression by lesion macrophages is not known but represents another similarity between aortic valvular lesions and atherosclerosis. Atherosclerotic lesions contain large amounts of apoE,^{26 27 28} some of which may be derived from circulating remnant lipoproteins that contain both apoE and apoB.²⁵ However, the findings that plaques and aortic valve lesions have regions with extracellular apoE but not apoB and that a subset of macrophages contains intracellular apoE suggest that much of the apoE in lesions may be produced locally. Macrophages increase their secretion of apoE in response to intracellular cholesterol loading,^{54 55 56} and in vitro studies with an apoE-deficient macrophage cell line have shown that, after cholesterol loading, apoE-transfected cells were much more effective at decreasing cell cholesterol content than were untransfected, apoE-deficient cells.⁵⁷ Thus, cells may secrete apoE/lipid vesicles as a means of removing excess cholesterol. Similar to this study's immunohistochemical findings for apoE in aortic valves, in situ hybridization^{26 28} and immunohistochemical studies^{26 27 28} of human atherosclerotic lesions have shown that foam cell macrophages contain the most abundant apoE mRNA and protein. Finally, it has been shown recently in human atherosclerotic lesions that apoE colocalizes to a striking degree with biglycan,⁵⁰ a small dermatan sulfate proteoglycan that accumulates in atherosclerotic plaques⁵⁸ and also is secreted by fibroblasts in a bleomycin-induced lung injury model.⁵⁹ Thus, it is tempting to speculate that a specific proteoglycan expressed in a variety of tissues in response to injury might contribute to apoE accumulation in aortic valves.

This constellation of observations has led us to develop a schema, shown in Fig 8, for how lipoproteins might be involved, at several levels, in the pathogenesis of aortic valvular lesions. If aortic stenosis develops through a cascade of biological events, then it becomes easier to understand how a number of risk factors, including dyslipidemia, diabetes, hypertension, and age, as well as alterations in valve morphology that increase hemodynamic stress,^{60 61 62 63} could contribute to the pathogenesis of the disease. Similarly, a number of the potential risk factors for aortic stenosis contribute to the pathogenesis of another multifactorial disease, namely, atherosclerosis. However, given the lack of concordance between the degree of coronary atherosclerosis and aortic valve stenosis, there must be other factors that contribute uniquely to the pathogenesis of clinically evident aortic stenosis. Possible differentiating factors include variations in the response of fibroblasts to inflammatory and hemodynamic injury as well as differences in calcium metabolism⁶⁴ that predispose to the massive calcium deposition in this tissue. The net effect of these differences is that one group of patients develops rigid, calcified aortic valves in response to injury, while another group develops coronary artery plaques that are structurally weak and prone to rupture. Finally, the presence of fibrinogen in aortic valve lesions⁹ suggests that rupture and thrombosis might contribute to aortic valve lesion development; however, while a relatively small thrombus may partially or completely occlude a coronary artery, formation of thrombi of the same size or greater on aortic valves easily could be clinically silent.

View larger version (124K): [in this window] [in a new window]   Figure 8. Schematic diagram of the potential roles of lipoproteins in the pathogenesis of aortic valvular lesions. Endothelial injury allows insudation of circulating lipoproteins into the subendothelial space. Sequestered in a microenvironment in which actively metabolizing cells (ie, resident fibroblasts and endothelial cells) consume antioxidants, these lipoproteins become minimally oxidized and possibly stimulate leukocyte adhesion. After more extensive oxidative modification, lipoproteins could be recognized and endocytosed by macrophage scavenger receptors, resulting in foam cell formation. Studies showing that HLA antigens and interleukin-2 receptors are present in lesions suggest the possibility of induction of cytokine and growth factor release by macrophages and fibroblasts. Lipid accumulation in macrophages leads to expression of apoE. The cytokines released by macrophages and fibroblasts stimulate fibroblast production of proteoglycans. LDL and apoE then may be trapped by proteoglycans and Lp(a) by both proteoglycans and fibrinogen, leading to lipoprotein accumulation and repetition of the cycle of events described above. Several factors in this cascade of events, including lipoproteins, release of calcification-mediating molecules such as osteopontin by inflammatory cells and possibly by fibroblasts, and participation of proteoglycans in matrix vesicle formation could facilitate calcium deposition. Those features whose presence is inferred but not proved are indicated with question marks.

In summary, this study has demonstrated the accumulation in aortic valvular lesions of three apolipoproteins, apoB, apo(a), and apoE, that have been implicated previously in the pathogenesis of atherosclerosis. Similar to what happens in atherosclerosis, apoB and apo(a) typically are colocalized in lesions, suggesting deposition of both LDL and Lp(a). While apoE often is detected in areas with apoB, it often is present in regions without apoB, consistent with the hypothesis that some apoE in valvular lesions is produced locally by macrophages. Furthermore, there is prominent accumulation of all three apolipoproteins in the thickened valvular fibrosa adjacent to lesions, in regions in which, based on histological appearance, proteoglycans may be present. Finally, there is prominent deposition of calcium in many regions with apolipoprotein accumulation. These findings suggest that a number of potentially modifiable factors may participate in the development of aortic stenosis.

	Acknowledgments

 
This study was supported in part by Grants-in-Aid 93-WA-505 (Dr O'Brien) from the American Heart Association, Washington Affiliate, Seattle, and 91-007520 (Dr Otto) from the American Heart Association, Dallas, Tex, and by grants HL-02788 (Dr O'Brien), HL-30086 (Dr Marcovina), and HL-42270 and HL-47151 (Dr Alpers) from the National Institutes of Health. The authors gratefully acknowledge Lisa Anne Billings for assistance in manuscript preparation and the expert technical assistance of Winnie Chiu, Marina Ferguson, Susan Rozell, and Randy Small.

Received April 24, 1995; accepted January 4, 1996.

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				R. O. Bonow, B. A. Carabello, K. Chatterjee, A. C. de Leon Jr, D. P. Faxon, M. D. Freed, W. H. Gaasch, B. W. Lytle, R. A. Nishimura, P. T. O'Gara, et al. ACC/AHA 2006 Guidelines for the Management of Patients With Valvular Heart Disease: A Report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Revise the 1998 Guidelines for the Management of Patients With Valvular Heart Disease) Developed in Collaboration With the Society of Cardiovascular Anesthesiologists Endorsed by the Society for Cardiovascular Angiography and Interventions and the Society of Thoracic Surgeons J. Am. Coll. Cardiol., August 1, 2006; 48(3): e1 - e148. [Full Text] [PDF]

				K. D. O'Brien Pathogenesis of Calcific Aortic Valve Disease: A Disease Process Comes of Age (and a Good Deal More) Arterioscler Thromb Vasc Biol, August 1, 2006; 26(8): 1721 - 1728. [Abstract] [Full Text] [PDF]

				S. Helske, S. Syvaranta, K. A. Lindstedt, J. Lappalainen, K. Oorni, M. I. Mayranpaa, J. Lommi, H. Turto, K. Werkkala, M. Kupari, et al. Increased Expression of Elastolytic Cathepsins S, K, and V and Their Inhibitor Cystatin C in Stenotic Aortic Valves Arterioscler Thromb Vasc Biol, August 1, 2006; 26(8): 1791 - 1798. [Abstract] [Full Text] [PDF]

				M. Briand, I. Lemieux, J. G. Dumesnil, P. Mathieu, A. Cartier, J.-P. Despres, M. Arsenault, J. Couet, and P. Pibarot Metabolic Syndrome Negatively Influences Disease Progression and Prognosis in Aortic Stenosis J. Am. Coll. Cardiol., June 6, 2006; 47(11): 2229 - 2236. [Abstract] [Full Text] [PDF]

				L. A. Cuniberti, P. G. Stutzbach, E. Guevara, G. G. Yannarelli, R. P. Laguens, and R. R. Favaloro Development of Mild Aortic Valve Stenosis in a Rabbit Model of Hypertension J. Am. Coll. Cardiol., June 6, 2006; 47(11): 2303 - 2309. [Abstract] [Full Text] [PDF]

				D E Newby, S J Cowell, and N A Boon Emerging medical treatments for aortic stenosis: statins, angiotensin converting enzyme inhibitors, or both? Heart, June 1, 2006; 92(6): 729 - 734. [Abstract] [Full Text] [PDF]

				R. Katz, N. D. Wong, R. Kronmal, J. Takasu, D. M. Shavelle, J. L. Probstfield, A. G. Bertoni, M. J. Budoff, and K. D. O'Brien Features of the Metabolic Syndrome and Diabetes Mellitus as Predictors of Aortic Valve Calcification in the Multi-Ethnic Study of Atherosclerosis Circulation, May 2, 2006; 113(17): 2113 - 2119. [Abstract] [Full Text] [PDF]

				M.-C. Drolet, E. Roussel, Y. Deshaies, J. Couet, and M. Arsenault A High Fat/High Carbohydrate Diet Induces Aortic Valve Disease in C57BL/6J Mice J. Am. Coll. Cardiol., February 21, 2006; 47(4): 850 - 855. [Abstract] [Full Text] [PDF]

				D M Shavelle Are angiotensin converting enzyme inhibitors beneficial in patients with aortic stenosis? Heart, October 1, 2005; 91(10): 1257 - 1259. [Abstract] [Full Text] [PDF]

				N. M. Rajamannan, M. Subramaniam, F. Caira, S. R. Stock, and T. C. Spelsberg Atorvastatin Inhibits Hypercholesterolemia-Induced Calcification in the Aortic Valves via the Lrp5 Receptor Pathway Circulation, August 30, 2005; 112(9_suppl): I-229 - I-234. [Abstract] [Full Text] [PDF]

				D. Weisenberg, Y. Sahar, G. Sahar, Y. Shapira, Z. Iakobishvili, B. A. Vidne, and A. Sagie Atherosclerosis of the aorta is common in patients with severe aortic stenosis: An intraoperative transesophageal echocardiographic study J. Thorac. Cardiovasc. Surg., July 1, 2005; 130(1): 29 - 32. [Abstract] [Full Text] [PDF]

				R. V. Freeman and C. M. Otto Spectrum of Calcific Aortic Valve Disease: Pathogenesis, Disease Progression, and Treatment Strategies Circulation, June 21, 2005; 111(24): 3316 - 3326. [Full Text] [PDF]

				N. M. Rajamannan, T. B. Nealis, M. Subramaniam, S. Pandya, S. R. Stock, C. I. Ignatiev, T. J. Sebo, T. K. Rosengart, W. D. Edwards, P. M. McCarthy, et al. Calcified Rheumatic Valve Neoangiogenesis Is Associated With Vascular Endothelial Growth Factor Expression and Osteoblast-Like Bone Formation Circulation, June 21, 2005; 111(24): 3296 - 3301. [Abstract] [Full Text] [PDF]

				S. J. Cowell, D. E. Newby, R. J. Prescott, P. Bloomfield, J. Reid, D. B. Northridge, N. A. Boon, and the Scottish Aortic Stenosis and Lipid Lowering Tr A Randomized Trial of Intensive Lipid-Lowering Therapy in Calcific Aortic Stenosis N. Engl. J. Med., June 9, 2005; 352(23): 2389 - 2397. [Abstract] [Full Text] [PDF]

				R. Rosenhek Statins for Aortic Stenosis N. Engl. J. Med., June 9, 2005; 352(23): 2441 - 2443. [Full Text] [PDF]

				F. W.H. Sutherland, T. E. Perry, Y. Yu, M. C. Sherwood, E. Rabkin, Y. Masuda, G. A. Garcia, D. L. McLellan, G. C. Engelmayr Jr, M. S. Sacks, et al. From Stem Cells to Viable Autologous Semilunar Heart Valve Circulation, May 31, 2005; 111(21): 2783 - 2791. [Abstract] [Full Text] [PDF]

				D. W. Quinn Jr. and S. A. Spinler Efficacy of statins in preventing progression of aortic stenosis Am. J. Health Syst. Pharm., May 1, 2005; 62(9): 979 - 981. [Full Text] [PDF]

				J Kuusisto, K Rasanen, T Sarkioja, E Alarakkola, and V-M Kosma Atherosclerosis-like lesions of the aortic valve are common in adults of all ages: a necropsy study Heart, May 1, 2005; 91(5): 576 - 582. [Abstract] [Full Text] [PDF]

				K. D. O'Brien, J. L. Probstfield, M. T. Caulfield, K. Nasir, J. Takasu, D. M. Shavelle, A. H. Wu, X.-Q. Zhao, and M. J. Budoff Angiotensin-Converting Enzyme Inhibitors and Change in Aortic Valve Calcium Arch Intern Med, April 25, 2005; 165(8): 858 - 862. [Abstract] [Full Text] [PDF]

				C. A. Simmons, G. R. Grant, E. Manduchi, and P. F. Davies Spatial Heterogeneity of Endothelial Phenotypes Correlates With Side-Specific Vulnerability to Calcification in Normal Porcine Aortic Valves Circ. Res., April 15, 2005; 96(7): 792 - 799. [Abstract] [Full Text] [PDF]

				G. M. Novaro Electron Beam Computed Tomography: The Latest "Stethoscope" for Calcific Aortic Valve Disease Mayo Clin. Proc., October 1, 2004; 79(10): 1239 - 1241. [PDF]

				N. M. Rajamannan and C. M. Otto Targeted Therapy to Prevent Progression of Calcific Aortic Stenosis Circulation, September 7, 2004; 110(10): 1180 - 1182. [Full Text] [PDF]

				R. Rosenhek, F. Rader, N. Loho, H. Gabriel, M. Heger, U. Klaar, M. Schemper, T. Binder, G. Maurer, and H. Baumgartner Statins but Not Angiotensin-Converting Enzyme Inhibitors Delay Progression of Aortic Stenosis Circulation, September 7, 2004; 110(10): 1291 - 1295. [Abstract] [Full Text] [PDF]

				J. R Ortlepp, F. Schmitz, V. Mevissen, S. Weiss, J. Huster, R. Dronskowski, G. Langebartels, R. Autschbach, K. Zerres, C. Weber, et al. The amount of calcium-deficient hexagonal hydroxyapatite in aortic valves is influenced by gender and associated with genetic polymorphisms in patients with severe calcific aortic stenosis Eur. Heart J., March 2, 2004; 25(6): 514 - 522. [Abstract] [Full Text] [PDF]

				C. M. Otto Why is aortic sclerosis associated with adverse clinical outcomes? J. Am. Coll. Cardiol., January 21, 2004; 43(2): 176 - 178. [Full Text] [PDF]

				G. M. Novaro, R. Sachar, G. L. Pearce, D. L. Sprecher, and B. P. Griffin Association Between Apolipoprotein E Alleles and Calcific Valvular Heart Disease Circulation, October 14, 2003; 108(15): 1804 - 1808. [Abstract] [Full Text] [PDF]

				T. E. David and J. Ivanov Is degenerative calcification of the native aortic valve similar to calcification of bioprosthetic heart valves? J. Thorac. Cardiovasc. Surg., October 1, 2003; 126(4): 939 - 941. [Full Text] [PDF]

				J R Ortlepp, F Schmitz, T Bozoglu, P Hanrath, and R Hoffmann Cardiovascular risk factors in patients with aortic stenosis predict prevalence of coronary artery disease but not of aortic stenosis: an angiographic pair matched case-control study Heart, September 1, 2003; 89(9): 1019 - 1022. [Abstract] [Full Text] [PDF]

				K.-L. Chan Is aortic stenosis a preventable disease? J. Am. Coll. Cardiol., August 20, 2003; 42(4): 593 - 599. [Abstract] [Full Text] [PDF]

				N. M Rajamannan, B. Gersh, and R. O Bonow CALCIFIC AORTIC STENOSIS: FROM BENCH TO THE BEDSIDE--EMERGING CLINICAL AND CELLULAR CONCEPTS Heart, July 1, 2003; 89(7): 801 - 805. [Full Text] [PDF]

				C.A Glader, L.S Birgander, S Soderberg, H.P Ildgruben, P Saikku, A Waldenstrom, and G.H Dahlen Lipoprotein(a), Chlamydia pneumoniae, leptin and tissue plasminogen activator as risk markers for valvular aortic stenosis Eur. Heart J., January 2, 2003; 24(2): 198 - 208. [Abstract] [Full Text] [PDF]

				D. Proudfoot, J.D. Davies, J.N. Skepper, P.L. Weissberg, and C.M. Shanahan Acetylated Low-Density Lipoprotein Stimulates Human Vascular Smooth Muscle Cell Calcification by Promoting Osteoblastic Differentiation and Inhibiting Phagocytosis Circulation, December 10, 2002; 106(24): 3044 - 3050. [Abstract] [Full Text] [PDF]

				M. F. Bellamy, P. A. Pellikka, K. W. Klarich, A. J. Tajik, and M. Enriquez-Sarano Association of cholesterol levels, hydroxymethylglutaryl coenzyme-a reductase inhibitor treatment, and progression of aortic stenosis in the community J. Am. Coll. Cardiol., November 20, 2002; 40(10): 1723 - 1730. [Abstract] [Full Text] [PDF]

				A. S. Pearlman Medical treatment ofaortic stenosis: Promising, or wishful thinking? J. Am. Coll. Cardiol., November 20, 2002; 40(10): 1731 - 1734. [Full Text] [PDF]

				K. D. O'Brien, D. M. Shavelle, M. T. Caulfield, T. O. McDonald, K. Olin-Lewis, C. M. Otto, and J. L. Probstfield Association of Angiotensin-Converting Enzyme With Low-Density Lipoprotein in Aortic Valvular Lesions and in Human Plasma Circulation, October 22, 2002; 106(17): 2224 - 2230. [Abstract] [Full Text] [PDF]

				C M Otto Calcification of bicuspid aortic valves Heart, October 1, 2002; 88(4): 321 - 322. [Full Text] [PDF]

				L Wallby, B Janerot-Sjoberg, T Steffensen, and M Broqvist T lymphocyte infiltration in non-rheumatic aortic stenosis: a comparative descriptive study between tricuspid and bicuspid aortic valves Heart, October 1, 2002; 88(4): 348 - 351. [Abstract] [Full Text] [PDF]

				B. Iung, C. Gohlke-Barwolf, P. Tornos, C. Tribouilloy, R. Hall, E. Butchart, and A. Vahanian Recommendations on the management of the asymptomatic patient with valvular heart disease Eur. Heart J., August 2, 2002; 23(16): 1253 - 1266. [PDF]

				L. L Demer Vascular calcification and osteoporosis: inflammatory responses to oxidized lipids Int. J. Epidemiol., August 1, 2002; 31(4): 737 - 741. [Full Text] [PDF]

				N. M. Rajamannan, M. Subramaniam, M. Springett, T. C. Sebo, M. Niekrasz, J. P. McConnell, R. J. Singh, N. J. Stone, R. O. Bonow, and T. C. Spelsberg Atorvastatin Inhibits Hypercholesterolemia-Induced Cellular Proliferation and Bone Matrix Production in the Rabbit Aortic Valve Circulation, June 4, 2002; 105(22): 2660 - 2665. [Abstract] [Full Text] [PDF]

				F. Robicsek, M. J. Thubrikar, and A. A. Fokin Cause of degenerative disease of the trileaflet aortic valve: review of subject and presentation of a new theory Ann. Thorac. Surg., April 1, 2002; 73(4): 1346 - 1354. [Abstract] [Full Text] [PDF]

				I. Hisar, M. Ileri, E. Yetkin, I. Tandogan, S. Cehreli, R. Atak, K. Senen, and D. Demirkan Aortic Valve Calcification: Its Significance and Limitation as a Marker for Coronary Artery Disease Angiology, March 1, 2002; 53(2): 165 - 169. [Abstract] [PDF]

				C. M OTTO and K. D O'BRIEN Why is there discordance between calcific aortic stenosis and coronary artery disease? Heart, June 1, 2001; 85(6): 601 - 602. [Full Text]

				C. M. Otto Aortic Stenosis -- Listen to the Patient, Look at the Valve N. Engl. J. Med., August 31, 2000; 343(9): 652 - 654. [Full Text]

				G. M. LONDON, B. PANNIER, S. J. MARCHAIS, and A. P. GUERIN Calcification of the Aortic Valve in the Dialyzed Patient J. Am. Soc. Nephrol., April 1, 2000; 11(4): 778 - 783. [Full Text] [PDF]

				A. Wierzbicki Lipids, cardiovascular disease and atherosclerosis in systemic lupus erythematosus Lupus, March 1, 2000; 9(3): 194 - 201. [Abstract] [PDF]

				C. M. Otto, B. K. Lind, D. W. Kitzman, B. J. Gersh, D. S. Siscovick, and The Cardiovascular Health Study Association of Aortic-Valve Sclerosis with Cardiovascular Mortality and Morbidity in the Elderly N. Engl. J. Med., July 15, 1999; 341(3): 142 - 147. [Abstract] [Full Text] [PDF]

				M. Olsson, J. Thyberg, and J. Nilsson Presence of Oxidized Low Density Lipoprotein in Nonrheumatic Stenotic Aortic Valves Arterioscler Thromb Vasc Biol, May 1, 1999; 19(5): 1218 - 1222. [Abstract] [Full Text] [PDF]

				K. D. O'Brien, K. L. Olin, C. E. Alpers, W. Chiu, M. Ferguson, K. Hudkins, T. N. Wight, and A. Chait Comparison of Apolipoprotein and Proteoglycan Deposits in Human Coronary Atherosclerotic Plaques : Colocalization of Biglycan With Apolipoproteins Circulation, August 11, 1998; 98(6): 519 - 527. [Abstract] [Full Text] [PDF]

				C. M. Otto, I. G. Burwash, M. E. Legget, B. I. Munt, M. Fujioka, N. L. Healy, C. D. Kraft, C. Y. Miyake-Hull, and R. G. Schwaegler Prospective Study of Asymptomatic Valvular Aortic Stenosis : Clinical, Echocardiographic, and Exercise Predictors of Outcome Circulation, May 6, 1997; 95(9): 2262 - 2270. [Abstract] [Full Text]

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