Virchows Archives of Pathological Anatomy, 1990, Number 417, Pages 105-111
Lipoprotein(a) [Lp(a)] is a lipoprotein that closely resembles low-density lipoprotein (LDL). Both lipoproteins have a similar lipid composition and both contain apoprotein B (apoB). In addition, Lp(a) consists of a protein called apoprotein (a) [apo(a)], which is attached to the apoB by disulphide bonds (Utermann et al. 1969, 1987; Simons et al. 1970; Ehnholm et al. 1972). Lp(a) was first described by Blumberg et al. (1962) and Berg (1963). The molecular weight of apo(a) is higher than 500kDa (Utermann et al. 1987). Lp(a) floats in a density range of 1.05-1.12 g/ml. It binds to the LDL receptor with lower affinity than LDL (Krempler et al. 1984; Maartmann and Berg 1981; Armstrong et al. 1985). Its clearance from the plasma has not yet been fully evaluated. Lp(a) is synthesized in the liver and is probably secreted by hepatocytes, associated with apoB.
The pathophysiological relevance of Lp(a) is evident in the positive correlation between elevated serum levels of Lp(a) and coronary heart disease shown in a number of epidemiological studies (Wottawa et al. 1984; Schriewer et al. 1984; Dahlen et al. 1986; Armstrong et al. 1986). Approximately 30% of a normal population express atherogenic serum levels of Lp(a) that are above 25 mg/dl; 40% of patients suffering from coronary heart disease express elevated Lp(a) serum levels.
In previous morphological studies apoB has been detected in the arterial wall by means of immunohistochemistry (Hoff et al. 1977, 1978; Hoff and Bond 1983; Yomantas et al. 1984; Yamauchi and Hoff 1984; Carter et al. 1987). From these investigations it has been deduced that either LDL and/ or very low-density lipoprotein (VLDL) are present. Only one study (Walton et al. 1974) has described apo(a) in the arterial wall. Interestingly, in that report it was concluded that Lp(a) does not participate in atherogenesis.
Against a background of increasing epidemiological evidence about the atherogenic properties of Lp(a) and with the availability of an apo(a)-specific monoclonal antibody, the present study investigates whether or not there exists a correlation between immunoreactivity of apo(a) and apoB in the arterial wall. A positive finding indicates that the localization of apoB has to be related, at least in part, to the presence of Lp(a). Autopsy tissue from the thoracic aorta and the left coronary artery has been examined by means of immunohistochemistry (Hsu et al. 1981; Niendorf et al. 1987) in combination with morphometric analysis in order to determine whether Lp(a) can be detected preferentially in atheromatous lesions of the arterial wall.
Autopsy tissue (n = 74) from patients who died at the age of 0-98 years (39 females, 35 males, approximately 10 samples representing each decade) was obtained from the Institutes of Pathology and Forensic Medicine at the University of Hamburg and was taken between 24 and 47 h post mortem. The slices were cut from the thoracic aorta in the region of the first intercostal arteries and from the first 2 cm of the left coronary artery. From atherosclerotic vessels two samples were taken: one from an atheromatous area and a second, if possible, from an adjacent area which was grossly normal. The tissue was formalin-fixed [3.7% buffered in phosphate-buffered saline (PBS)] and paraffin-embedded by standard procedures. Haematoxylin and eosin and elastic-van-Gieson staining were performed by routine methods.
Polyclonal goat anti-apoB (Immuno, Vienna) was applied to the tissue sections in a 1 : 1000 dilution. Monoclonal mouse-anti(a) has been produced in the laboratory of one of the authors (U. Beisiegel) and will be described and characterised elsewhere (paper in preparation). This antibody has been checked for a possible cross-reactivity with plasminogen, which was excluded.
For immunohistochemistry deparaffinised 2-µm sections were mounted on Zementit-coated slides. They were allowed to dry at 60¡C overnight. Endogenous peroxidase was inhibited by a 10-min treatment with methanol-H2O2 (174 ml methanol + 6 ml 30% H2O2). Thereafter tissue sections were washed for 10 min in PBS and pre-incubated with normal horse serum [apo(a)] or normal-rabbit serum (apoB) for 30 min at room temperature. The serum was decanted and the specific first antibody [monoclonal anti-apo(a) or polyclonalanti-apoB] was applied for 24 h at 6¡ C. Incubation with the primary antibody was followed by three washes in PBS for 10 min each. After that the sections were incubated with biotinylated secondary antibodies [horse anti-mouse for Lp(a) detection and rabbit anti-goat for apoB] was applied for 24 h at 6¡ C. Incubation with the primary antibody was followed by three washes in PBS for 10 min each. After that the sections were incubated with biotinylated secondary antibodies [horse anti-mouse for Lp(a) detection and rabbit anti-goat for apoB] which were both diluted 1 : 200 in PBS. This was again followed by three washes of 10 min each. Thereafter the sections were incubated for 30 min with the avidin-biotin reagents (Vector Laboratories, Burlingame, Calif.) and washed again three times for 10 min. Sections were then incubated with diaminobenzidine as a chromogenic substrate for 6 min. They were rinsed for 5 min in plain water and counter-stained with haemalaum for 5 min, dehydrated in alcohol and mounted in Eukit.
In control incubations non-immune serum of the same species in which the specific first antibody had been raised (normal mouse serum or normal goat) was applied instead of the first anybody.
Sections of the arterial wall were classified as “normal” (no lesion and intimal thickening), “fatty streak” (accumulation of foam cells), “fibrous plaque” (smooth muscle cell and collagen accumulation) and “complicated lesion” (any fibrous lesion including in addition necrosis, haemorrhage, thrombosis or calcification). Immunoreactivity within the arterial wall was estimated by semiquantitative methods and a staining score was deduced from the following formula: SC = (%SALA/100) x (ISA) x (%LATSA/100), (where SC = staining score; SALA=stained area with regard to lesional area; ISA = intensity of stained area; LATSA =lesion area with regard to total section area).
This included the area and intensity of staining in a given tissue section. A value ranging from 0 to 12 thus resulted from the product of intensity (0-12) and the percentage of a stained area (divided by 100) after consideration of the percentage of lesional area within the given total section through the arterial wall. Thus the highest possible staining score was 12. The morphometric analysis was performed by two independent investigators.
The distribution of staining for apo(a) and apoB was found to be located almost exclusively in the intima of the arterial wall in both thoracic aorta and left coronary artery. The media expressed staining for either antigen in general. The media expressed staining for either antigen in general. In 5% of the total sample number a restricted immunoreactivity at the cellular level was observed in smooth muscle cells of the media. The lumina of adventitial vessles (vasa vasora) showed staining for both antigens. The adventitia itself expressed a low level of diffuse staining in some cases.
The deposition pattern of both antigens in the normal intima is summarized schematically in Fig. 1 A-G. In non-lesional areas there was either no staining at all (Fig. 2), or fine spot-like or a fine striped staining. Both patterns were found in the near luminal part and the near media part (Fig. 3) or were distributed over the whole area of the intima. In no case was a marked aggregation of staining observed in non-lesional areas. In contrast, in fibrous plaques and complicated lesions dense bundle-like staining pattern was seen, for both antigens. Predominant localization of both antigens within fibrous caps was seen this either reached to the luminal border of the intima (Fig. 1; CL and FP type A) or covered the necrotic core of a complicated lesion or was found at the edges of necrotic cores (Fig. 5). The distribution patterns of apo(a) and apoB in lesional areas are summarized in Fig. 1 H – L.
At the cellular level predominant localization of apo(a) and apoB in the extracellular matrix has been observed.
Exceptionally, some foam cells within fatty streaks or complicated lesions (Fig. 4) and a few smooth muscle cells of the media contain certain amounts of both antigens. In summary apo(a) and apoB are located extracellularly almost exclusively.
Comparison of the distribution pattern of both antigens with regard to localization and intensity exhibits a high degree of congruency for apo (a)- and apoB staining in all lesion areas. Localization of apo(a) and apoB is shown to be present in atheromatous lesions of the aorta as well as in the coronary artery as shown in Figs. 2 Ð 5 [apo(a) localization]. The co-localization of both proteins is demonstrated in Figs. 6 and 7.
Quantification of immunoreactivity shows a significantly more intensive staining for both antigens in regions of the intima that show either fatty streaks, fibrous plaques or complicated lesions than in areas of normal intima. Plaque area as a total has a staining score (mean value for both lipoproteins in aorta and coronary artery) of 6.5 compared with 2.5 in non-lesional areas. The most pronounced aggregation of both proteins is observed in the coronary artery exhibiting a staining score of 5.1 for apo(a) and 6.7 for apoB in lesional areas compared with 1.2 [apo (a)] and 1.8 (apoB) in non-lesional areas. All values for the immunoreactivity are listed in Table 1.
Increasing numbers of epidemiological studies (Wottawa et al. 1984; Schriewer et al. 1984; Dahlen et al. 1986; Armstrong et al. 1986) have indicated the value of investigation the distribution of apo(a) and apoB in a larger number of cases. The intriguing immunological problem that results from a close similarity between plasminogen and apo(a) (Havekes et al. 1981; Floren et al. 1981) was resolved by the availability of a monoclonal antibody that detects the apo(a) and does not cross-react. The results from this study reveal a staining pattern for apo(a) and apoB located in the intima of the arterial wall with a preference for lesions is predominantly in the extracellular space, and is congruent in adjacent sections.
Determination of staining intensity in lesions and non-lesional areas clearly demonstrates that apo(a) and apoB are enriched in lesions. This finding supports the epidemiological observation that Lp(a) is associated with the appearance of atherosclerosis. Both antigens show a comparable tissue distribution in the thoracic aorta and the coronary artery. Since the aorta exhibits fatty streaks that are composed of foam cells more frequently than the coronary artery, with its rather fibrous or complicated lesions, it is noteworthy that the number of intracellularly stained cells is higher in the aorta than in the coronary vessel. Under the premise that intimal thickening can be considered to be a physiological age-related process it is important to note that deposition of both proteins occurs in parts of the intima not affected by morphologically detectable lesions. However, this is observed to a much lesser extent than in lesions of the arterial wall. The demonstration of protein deposition in non-lesional areas might be interpreted as the detection of earliest stages of the development of atheromatous lesions.
The mechanism of accumulation of lipoproteins in the arterial wall is still under debate (Kratzin et al. 1972; Smith et al. 1976; Hollander et al. 1979; Hoff et al. 1979). LDL is suspected to be modified and enriched in the intima that has undergone a primary injury at the site of the endothelial barrier. Concerning the Lp(a) particle two hypotheses are under discussion: Lp(a) as a lipoprotein might diffuse into the intima and either stick to the intercellular components such as glycosaminoglycans or be directly taken up by monocyte-derived macrophages or smooth muscle cells. Since Lp(a) is known to bind to glycosaminoglycans with a higher affinity than does LDL (Smith et al. 1976; Hollander et al. 1979) this particle is predisposed to be entrapped in the intercellular matrix. Subsequently it might be phagocytosed, in the sense of a repair or clearance mechanism and phagocytosis and receptor-mediated endocytosis lead to lysosomal degradation of the incorporated ligand. One would not expect ligands which have been undergone lysosomal degradation to be detectable by immunological methods and it seems unlikely that we are detecting apoproteins that have undergone phagocytosis.
We therefore favour a second hypothesis to explain Lp(a) accumulation in the arterial wall, based on the similarity of this lipoprotein with plasminogen. The apo(a) molecule is made up predominantly of kringle structures having a high degree of homology with plasminogen (Eaton et al. 1987; McLean et al. 1987). Lp(a) might therefore bind to sites where fibrinogen is polymerized. In this regard Lp(a) might play a fatal role interfering with fibrinolysis, which has to occur in a regular manner to prevent fibrotic organization of mural thrombi triggering the formation of complicated lesions. Preliminary results from our group confirm speculations about a Lp(a)-fibrin association. Morphologically a striking congruency of fibrinogen and apo(a) was observed in a limited number of cases (data not shown). The distribution patterns of both proteins with regard to the intra- and extracellular localization also favour a mechanism where Lp(a) either sticks to extracellular matrix components or is entrapped in thrombi, due to its similarity with plasminogen. Biochemical data (Rath et al. 1989) reveal a considerable amount of gradient-extracted apo(a) associated with intact lipoprotein particles. This finding corroborates the concept of a predominantly extracellular deposition. In this work we find apo(a) and apoB to be deposited mainly extracellularly, although weak intracellular staining within foam cells can be observed occasionally.
In previous morphological studies the distribution pattern of apoB has been determined (Hoff et al. 1977, 1978; Hoff and Bond 1983; Yomantas et al. 1984; Yamauchi and Hoff 1984; Carter et al. 1987). The results of these investigators are confirmed with regard to tissue distribution. The presence of apoB indicates that VLDL or LDL must be present, and from the findings of this study the presence of LDL cannot be excluded. The major new conclusions is that immunoreactivity for apoB has to be related to the presence of Lp(a) at least in part, since apo(a) and apoB are detected in congruency. One should therefore keep in mind the possibility that Lp(a) is present when apoB is detected in atheromatous lesions.
Walton (1974) first reported the observation of similar distribution patterns for apo(a) and apoB. However, he found it to be “rather unlikely that the finding of apo (a)-immunoreactivity in patients with clinical evidence of coronary artery disease has any special sinister significance”. On the basis of our data we can clearly contradict this statement.
In conclusion, this work describes Lp(a) for the first time as an atherogenic particle that can be detected immunologically by the demonstration of apo (a). It has a preferential deposition in atheromatous lesions and should be considered as a lipoprotein that deserves close attention with regard to its diagnostic and therapeutic implications.
1.Armstrong VW, Walli AK, Seidel D (1985) Isolation, characterization, and uptake in human fibroblasts of an Apo(a)-free lipoprotein obtained on reduction of lipoprotein(a). Journal of Lipid Research 26: 1314-1323
2. Armstrong VW, Cremer P, Eberle E, Manke A, Schulze F, Wieland H, Kreuzer H, Seidel D (1986) The association between serum Lp(a) concentrations and angiographically assessed coronary atherosclerosis: dependance on serum LDL levels. Atherosclerosis 62: 249-257
3. Berg KA (1963) New serum type system in man Ð the Lp system. Acta Pathologica 59: 369-382
4. Blumberg BS, Bernanke D, Allison AC (1962) A human lipoprotein polymorphism. Journal of Clinical Investigation 41: 1936-1944
5. Carter RS, Siegel RJ, Chai AU, Fishbein MC (1987) Immunohistochemical localization of apolipoproteins A-I and B in human carotid arteries. Journal of Pathology 153: 31-36
6. Dahlen G, Guyton JR, Attar M, Farmer JA, Kautz JA, Gotto AM jr. (1986) Association of levels of lipoprotein(a) (Lp(a)), plasma lipids and other lipoproteins with coronary artery disease documented by angiography. Circulation 74: 758-765
7. Eaton DL, Fless GM, Kohr WJ, Mc Lean JW, Xu QT, Miller CB, Lawn RM, Scann AM (1987) Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to plasminogen. Proceedings of the National Academy of Sciences USA 84: 3224-3228
8. Ehnholm C, Groff H, Renkonen O, Simons K (1972) Protein and carbohydrate composition of Lp(a) lipoprotein from human plasma. Biochemistry 11: 3220-3232
9. Floren CH, Albers JJ, Biermann EL (1981) Uptake of Lp(a) by cultured fibroblasts. Biochem Biophys Research Communication 102: 636-639
10. Havekes L, Vermeer BJ, Brugmann T, Emeis J (1981) Binding of Lp(a) to the low density lipoprotein receptor of human fibroblasts. FEBS Letters 132: 169-173
11. Hoff HF, Bond MG (1983) Apolipoprotein B localization in coronary atherosclerotic plaques from cynomolgus monkeys. Artery 12: 104-116
12. Hoff HF, Heidemann CL, Gotto AM, Gaubatz JW (1977) Apolipoprotein B retention in the grossly normal and atherosclerotic human aorta. Circulation Research 41: 684-690
13. Hoff HF, Heidemann CI, Gaubatz JW, Scott DW, Titus JL, Gotto AM (1978) Correlation of apolipoprotein B retention with the structure of atherosclerotic placques. Laboratory Investigation 38: 560
14. Hoff HF, Karagas M, Heidemann CL, Baubatz JW, Gotto AM (1979) Correlation in the human aorta of Apo B fraction with tissue cholesterol and collagen content. Atherosclerosis 32: 259-268
15. Hollander W, Paddock J, Colombo M (1979) Lipoproteins in human atherosclerotic vessels. Experimental Molecular Pathology 30: 144-171
16. Hsu SM, Reine L, Fanger H (1981) Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabelled antibody (PAP) procedures. Journal of Histochemistry and Cytochemistry 29: 577-580
17. Kratzin H, Armstrong VW, Niehaus M, Hilschmann N, Seidel D (1987) Structural relationship of an apolipoprotein(a) phenotype (570 kDa) to plasminogen: homologous kringle domain are linked by carbohydrate-rich regions. Biol Chem Hoppe-Seyler 368: 1533-1544
18. Krempler F, Kostner GM, Roscher A, Bolzano K, Sandhofer F (1984) The interaction of human ApoB Ð containing cipoproteins with mouse peritoneal macrophages: a comparison of Lp(a) with LDL. Journal Lipid Research 25: 283-287
19. Maartmann-Moe K, Berg K (1981) Lp(a) lipoprotein enters cultured fibroplasts independently of the plasma membrane low density lipoprotein receptor. Clinical Genetics 20: 352-362
20. McLean JW, Tomlinson JE, Kuang WJ, Eaton DL, Chen EY, Fless GM, Scanu AM, Lawn RM (1987) cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature 300: 132-137
21. Niendorf A, Arps H, Sieck M, Dietel M (1987) Immunoreactivity of PTH-binding in intact bovine kidney tissue and cultured cortical kidney cells indicative for specific receptors. Acta Endocrinol (Copenh) 281: 207-211
22. Rath M, Niendorf A, Reblin T, Dietel M, Krebber J, Beisiegel U (1989) Detection and qunatification of lipoprotein(a) in the arterial wall of 107 coronary bypass patients. Arteriosclerosis 9: 579-592 Ê
23. Schriewer H, Assmann G, Sandkamp M (1984) The relationship of lipoprotein(a) (Lp(a)) to risk factors of coronary heart disease. Journal of Clinical Chemistry Clinical Biochemistry 22: 591-596
24. Simons K, Ehnholm C, Renkonen O, Bloth B (1970) Characterization of the Lp(a) lipoprotein in human plasma. Acta Pathol Microbiol Scand B 78: 459-466
25. Smith EB, Slater RS (1972) Relationship between low-density lipoprotein in aortic intima and serum-lipid levels. Lancet I: 463-469
26. Smith EB, Massie IB, Alexander KM (1976) The release of an immobilized lipoprotein fraction from atherosclerotic lesions by incubation with plasmin. Atheroslerosis 25: 71-84
27. Utermann G, Wiegandt H (1969) Darstellung und Charakterisierung eines Lipoproteins mit Antigenwirksamkeit mit LP-System. Humangenetik 8: 47-52
28. Utermann G, Menzel HJ, Kraft HG, Duba HC, Kemmler HG, Seitz C (1978) Lp(a) glycoproteins phenotypes. Journal of Clinical Investigation 80: 458-465
29. Walton KW, Hitchens J, Magnani AL, Khan M (1974) A study of methods of identification and estimation of Lp(a) lipoprotein and of its significance in health, hyperlipidemia and atheroslerosis. Atherosclerosis 20: 323-346
30. Wottawa A, Fromme K, Klein G (1984) Lipoprotein(a) bei coronarer Herzkrankheit und Myokardinfarkt. MŸnchener Medizinische Wochenschrift 3: 53-55
31. Yamauchi J, Hoff HF (1984) Apolipoprotein B Accumulation and development of foam cell lesions in coronary arteries of hypercholesterolemic swine. Laboratory Investigation 51: 325-332
32. Ylþ-Herttuala S, Jaakkola O, Ehnholm C, Tikkanen MJ, Solakioi T, Sþrkoja T, Nikkari T (1988) Characterization of two lipoproteins containing apolipoproteins B and E from lesionfree human aortic intima. Journal of Lipid Research 29: 563-572
33. Yomantas S, Elner VM, Schaffner T, Wissler RW (1984) Immunohistochemical localization of apolipoprotein B in human atherosclerotic lesions. Arch Pathol Lab Med 108: 374-378