Human Aortic Intima Research Articles

HAEMOSTATIC FACTORS IN HUMAN AORTIC INTIMA

Accumulation of lipid in developing fibrous atherosclerotic plaques is associated with high concentrations of fibrin and accumulation of a tightly bound lipoprotein fraction that can be released by incubation with fibrinolytic enzymes, suggesting that fibrin may play a key role in lesion development. It is not known whether this fibrin represents incorporated mural thrombus or is produced in situ by clotting of the fibrinogen that is present in intima in high concentration. Immunoelectro-phoresis was used to measure the concentrations of fibrinogen and components of the clotting system in human aortic intima and lesions, and in mural thrombi. In the lipid and fibrin rich plaque centres prothrombin concentration was almost twice that in normal intima, but concentrations of the thrombin inhibitors antithrombin III and α2-macro-globulin fell, so that the molar ratios of inhibitor/prothrombin fell from 3:1 in normal intima to 1:1 in the plaque centre. In mural thrombi there was preferential sequestration of fibrinogen-like antigen and prothrombin. Distribution of factor-VIII-related antigen was highly unpredictable; in both normal intima and all types of intimal lesion substantial amounts were recovered from some samples, whereas none was recovered from others. The highest concentrations were found in samples free of endothelium from the deep layers of lesions. The results are compatible with the idea that fibrinogen may be converted to fibrin within lesions; once some fibrin has accumulated within the intima it seems to bind low-density lipoprotein and sequester fibrinogen and clotting factors, thereby producing a self-amplifying system.

Characterization of free and tightly bound lipoprotein in intima by thin layer isoelectric focusing

Thin layer isoelectric focusing followed by second dimension quantitative immunoelectrophoresis has been used to characterize the free and tightly bound lipoprotein (LP) fractions in human aortic intima. In 3.3% acrylamide gels plasma low density lipoprotein (LDL) focuses rapidly, but very low density lipoprotein (VLDL) fails to enter the gel and remains at the origin (point of application). Samples of normal intima and lesions were placed directly on the gel and focused for 10–12 h; in normal intima and gelatinous lesions which had not accumulated cholesterol 60–70% of the free LP focused with plasma LDL, but with increasing accumulation of cholesterol the proportion focusing with LDL decreased and the component remaining at the origin increased. In 9 of the 39 samples examined one or more components with pI acid to LDL were present, and in one sample the whole peak focused at a more acid pI. In 4 of 5 lipid-rich centres of gelatinous plaques all the free LP remained at the origin, and the bound LP released by incubation with plasmin remained at the origin in all samples in 3.3% gels although it would focus in 2.6% gels. With antiserum to apo-C this large LP did not behave like VLDL, and it appears to be an aggregated LDL. An increased proportion of aggregated LDL was associated with (a) accumulation of cholesterol, (b) topographical location towards the centre of plaques, and (c) partial destruction of endogenous LP during incubation of fresh samples of tissue.

Elastin-lipid interaction action in the arterial wall: Part 1. Extraction of elastin from human aortic intima

Elastin was extracted from human aortic plaque and adjacent grossly normal intima by the following methods: (1) 0.1 N NaOH at 100°C, (2) hot NaOH and 0.2 M EDTA, (3) 5 M guanidine-HCI and collagenase, (4) guanidine-collagenase and dithioerythritol-urea-sodium dodecyl sulfate, (5) guanidinecollagenase and EDTA, (6) 10% NaCl and collagenase, and (7) NaCl-collagenase and EDTA. All elastin samples contained small amounts of carbohydrate and hydroxyproline. The lipid content of non-plaque intimal elastin samples was small (2–3%), whereas it increased to 4–6% in plaque intima. The lipid composition of elastin preparations varied significantly with the extraction procedure. Elastin from plaque intima contained significantly more cholesterol (50–60%) and less triglyceride and phospholipid than elastin of non-plaque intima (30–50% cholesterol). The contents of free and esterified cholesterol were comparable in all preparations. The main phospholipid in all samples was sphingomyelin, which comprised between 50 and 80% of the total phospholipid. Compared with NaOH-purified elastin, the other elastin samples were characterized by an increased phosphatidyl-choline content, while they all contained an almost equal amount of phosphatidylethanolamine. In elastin samples from plaque intima, the polar amino acids were increased, whereas cross-linking amino acids were decreased. The polarity and hydroxy-proline content of elastin samples were slightly decreased after treatment with EDTA or diethioerythritol-urea-sodium dodecyl sulfate.

Characterization of low density lipoprotein-like particle in the human aorta from grossly normal and atherosclerotic regions

Physical and chemical criteria of lipoproteins containing apolipoprotein B, extracted from human aortic intima, were compared with those of plasma low density lipoproteins (LDL). Homogenates of grossly normal intima and advanced atherosclerotic lesions were subjected to differential ultracentrifugation to isolate a d = 1.006–1.063 g/ ml density fraction which was extensively characterized. By electroimmunoassay, over 90% of the recovered apolipoprotein B immunological reactivity was found in isolates from both plaques and normal intima. In isolates of plaque and normal intima, particles of the same size as LDL were found, although a small population of very large structures was also present in plaque fractions. Apolipoprotein composition was similar to that of plasma LDL except for the presence of human serum albumin in aortic isolates. Fractions from aorta demonstrated greater electrophoretic mobilities than LDL. The lipid composition of isolates from normal intima was similar to that of LDL. The lipid composition of plaque fractions showed a significant decrease in the cholesteryl ester to free cholesterol ratio and in the triglyceride content in comparison to LDL and to fractions from normal intima. The fatty acid pattern of the cholesteryl ester fraction from isolates of both normal and plaque aortic homogenates demonstrated a significant decrease in the linoleate to oleate ratio as compared to LDL. Our initial studies suggest that although aortic fractions are similar to LDL by certain criteria, some differences observed are more pronounced in fractions from lesions than from normal intima.

Lipoproteins in human atherosclerotic vessels: I. Biochemical properties of arterial low density lipoproteins, very low density lipoproteins, and high density lipoproteins

Lipoproteins extracted from the human aortic intima into 1.65 M NaCl were quantitated and characterized biochemically and by electron microscopy following separation in the preparative ultracentrifuge. The arterial lipoproteins, although separated and designated according to the density classes used for the serum lipoproteins, were distinctly different from their serum counterparts. The amount of lipoproteins in the low density range of d 1.063 to 1.006 (arterial LDL) and in the very low density range of d < 1.006 (arterial VLDL) extracted from arterial intima increased with increasing intimal lipid content. In contrast, the concentration of lipoproteins in the high density range of d 1.210 to 1.063 (arterial HDL) was small and did not change with the severity of atherosclerosis. Arterial VLDL, LDL and its subfractions, LDL 1 (d 1.006 to 1.019) and LDL 2 (d 1.019 to 1.063), were markedly heterogenous and contained unusually large particles, which were isolated by Bio-Gel A-150. These particles showed a pitted and cratered appearance by scanning electron microscopy and were immunochemically unreactive and had no electrophoretic mobility. The lipid and amino acid composition of the arterial VLDL and LDL fractions as well as their electrophoretic, chromatographic and analytical flotation behavior was distinctly different from that of their serum lipoprotein counterparts. Arterial VLDL, in sharp contrast to serum VLDL, was rich in cholesteryl ester and poor in triglycerides. Arterial VLDL also showed no electrophoretic mobility and only half of the preparations reacted to LDL antisera. Acid mucopolysaccharides were detected in the arterial VLDL and LDL fractions in association with the large size particles which lacked electrophoretic mobility and immunochemical reactivity and showed only a “saw tooth” pattern in the analytical ultracentrifuge. Arterial LDL and LDL 2 contained a smaller sized population of particles as separated by Bio-Gel A-150. These particles exhibited a reaction of complete identity with serum LDL when reacted against LDL antiserum. However these particles had a greater electrophoretic mobility and different amino acid composition than did serum LDL and LDL 2. An asymmetrical peak with a mean SF of 7.3 was demonstrated by these particles in the analytical ultracentrifuge. The over-all studies suggest that lipid deposition in atherosclerotic plaques is associated with the accumulation of lipoproteins with biochemical and ultrastructural properties unlike those of serum lipoproteins. The presence of these lipoproteins in the arteries may be a result of the interaction of serum and arterial lipoproteins with acid mucopolysaccharides and of lipoprotein synthesis and degradation in the arteries.

Apolipoprotein B retention in the grossly normal and atherosclerotic human aorta.

Apoliporotein B (apoB) was measured in buffer-extracted homogenates of grossly normal and artherosclerotic human aortic intima by means of an electroimmunoassay procedure. The apoB values which were expressed as microgram per mg tissue dry weight, varied widely, ranging from 0.34 to 18.45 in normal intima and from 0.8 to 12.5 in fatty fibrous plaques. No consistent differences in apoB content were found between normal intimas from thoracic and abdominal aortic regions. There was a statistically significant positive correlation between the quantity of buffer-extractable apoB in normal regions and the plasma cholesterol and triglyceride concentration. Buffer-extractable apoB values were significantly higher in fatty fibrous plaques than in ulcerated lesions from the same vessel. However, fatty fibrous plaque apoB values were significantly lower than those from grossly normal regions from the same aorta, although the topographical distribution of apoB was more widespread in plaques than in normal regions, as shown by immunofluorescence studies. This apparent discrepancy reflected the incomplete extraction of apoB from plaques as contrasted to normal regions. The relatively loosely bound apoB, extractable by standard buffers, may represent intact low density lipoprotein (LDL) and/or very low density lipoprotein (VLDL), while the tightly bound fraction may represent insoluble complexes of intact lipoproteins within the plaque or delipidated apoB.

The release of an immobilized lipoprotein fraction from atherosclerotic lesions by incubation with plasmin

A large amount of plasma low density lipoprotein is present in human aortic intima, and this can be removed and measured by electrophoresis directly from the minced tissue into an antibody-containing gel. We now find that, in addition to this electrophoretically mobile lipoprotein, there is an immobilized lipoprotein fraction that can be released from lesions by incubation of the tissue sample with plasmin or other proteolytic enzymes after the mobile lipoprotein has been removed. The concentration of immobilized lipoprotein is highly correlated with the concentration of the residual cholesterol (not mobile on electrophoresis) that has accumulated in the tissue ( r = 0.702; P ⪡ 0.001). Thus, in normal intima and early gelatinous lesions it is about 15% of the concentration of mobile lipoprotein, whereas in the atheroma lipid layers of fibrous or gelatinous plaques it may be 2 or 3 times greater than the concentration of mobile lipoprotein. This suggests that immobilization of plasma lipoprotein is an intermediate step in the irreversible deposition of extracellular cholesterol in atherosclerotic lesions. Incubation with plasmin allowed maximum release of lipoprotein: plasmin = crude collagenase > trypsin > “pure” collagenase > chondroitinase ABC in order of their relative effectiveness. The concentration of immobilized lipoprotein was significantly correlated ( r = 0.793; P ⪡ 0.001) with the concentration in the tissue of fibrin or other insoluble derivatives of fibrinogen (“fibrin”). In aliquots of lesions incubated with varying amounts of plasmin for varying times there was a constant relation between release of lipoprotein and release of fibrin-degradation products. Together, these findings suggest that the lipoprotein is associated with insoluble “fibrin”. This appears to be of considerable clinical interest, suggesting a synergism between lipoprotein and fibrinogen in the accumulation of lipid in lesions.

Purification and properties of cholesterol ester hydrolase from human aortic intima and media

1. 1. Cholesterol ester hydrolase of human aortic intima and media was isolated and purified about 650-fold with 10–15% recovery of the original activity by sequential precipitation with 35% acetone, gel filtration on Sephadex G-75 and DEAE-cellulose column chromatography. 2. 2. Two pH optima of 4.5–5.0 and 7.0–7.5 were consistently observed for the partially purified cholesterol ester hydrolase of human aortic intima and media 3. 3. In the system used in the present study, the increasing concentration of emulsifiers, sodium taurocholate and phosphatidylcholine, inhibited the activity of the neutral enzymes but not on the acid enzymes. On the contrary, reaction products, cholesterol and oleic acid, were much more inhibitory on the acid enzymes than on the neutral ones. 4. 4. Results of studies on the effect of presentation of substrate on the enzyme activity and on the difference between acid and neutral enzymes are also discussed.

Insoluble “fibrin” in human aortic intima: Quantitative studies on the relationship between insoluble “fibrin”, soluble fibrinogen and low density lipoprotein

A quantitative assay for fibrin or other insoluble fibrin-like antigens (“fibrin”) in small samples of intima is described. Tissue samples were subjected to electrophoresis directly from the intima into an antibody-containing gel to remove and measure fibrinogen and other soluble fibrin reactive antigens (FRA). The residual tissue was then exhaustively incubated with plasmin, and the soluble fragments generated from the insoluble “fibrin” were measured by quantitative immunoelectrophoresis. “Fibrin” accounted for about 2% of the tissue dry weight in normal intima and the ratio fibrinogen/“fibrin” was 1–1.5. In the gelatinous lesions, which seem to be the precursors of fibrous plaques, there was a small increase in “fibrin” but a substantial increase in fibrinogen and low density (LD)-lipoprotein, and the ratio fibrinogen/“fibrin” rose to about 3, which suggests that the increase in “fibrin” is secondary to increased permeation of fibrinogen. At the edges of larger plaques there was also a threefold increase in fibrinogen, but “fibrin” increased fivefold, and accounted for 10% of the tissue dry weight. The same high concentration was found in the centres of large fibrous plaques with advanced atheroma lipid. Raised levels of “fibrin” were accompanied by raised levels of fibrinogen in most tissue samples. About 80% of the total soluble FRA could be clotted with thrombin; there was no significant difference between normal intima and lesions, and the proportion clotted was not related to “fibrin” content.

大動脈壁中の脂質分解酵素について

In order to clarify a part of lipid metabolism in aortic wall, the properties of lipolytic enzymes in the human aortic intima and media have been studied. Human intima and media were homogenized in ice-cold 0.01M phosphate buffer, pH7.0, and the supernatant of homogenate was used as the enzyme source.With cholesterol ester hydrolase, phospholipase and lipase in aortic intima and media, two pH optima of acid and alkaline ranges were consistently observed. In the cases of cholesterol ester hydrolase and lipase, the activities of intimal enzymes were significantly higher than those of medial ones at all pH values. On the contrary, the activity of intimal phospholipase was slightly lower than that of medial one.Comparing with the phospholipases of pancreatic juice, snake venom and starfish, the enzyme of aortic wall was unstable for heat.Alkaline lipases of intima and media were activated by sodium deoxycholate and were slightly inhibited by albumin. On the other hand, both acid lipases were not influenced by these compounds. 1M sodium chloride and protamine sulphate significantly inhibited the activity of the intimal alkaline lipase, but had no effect on the medial alkaline lipase and both the acid lipases. Heparin in low concentration slightly increased the activities of both alkaline lipases, but that of the intimal lipase was inhibited by higher concentration of heparin. These observations lead us to speculate that the main part of the intimal lipase might be lipoprotein lipase, and the medial alkaline lipase might be true lipase. The intimal alkaline lipase had the absolute positional specificity for the primary ester bond of triglyceride. On the contrary, the other lipases had the relative specificities for the primary ester bonds.

Quantitative studies on fibrinogen and low-density lipoprotein in human aortic intima

The amounts of soluble, fibrinogen/fibrin related antigens (FRA) and of intact low-density (LD) lipoprotein in human aortic intima have been measured by an immunoelectrophoretic technique. Substantial amounts of FRA and LD lipoprotein were found in normal intima: in early fibrous lesions the concentrations of both antigens showed two- to four-fold increases compared with normal intima from the same aorta. In spite of the increase in concentration the ratio LD lipoprotein cholesterol/FRA did not differ significantly between normal intima and lesions. There was a significant correlation between lipoprotein and FRA ( r = 0.722, P = 0.015), which suggests that fibrinogen may be entering the intima together with lipoprotein and other plasma constituents. When tissue samples were treated with thrombin about 50% of the antigen was “clotted”; the “clottable” material was presumably fibrinogen since “clottable” fragments are not derived from lysis of a stabilized fibrin clot. The results suggest that substantial amounts of plasma fibrinogen enter the intima; if this is converted to fibrin within the intimal tissue it could be a potent factor in atherogenesis.

An immuno-electrophoretic assay of beta-lipoprotein and albumin in human aortic intima by direct electrophoresis from the tissue sample into an antibody-containing gel.

An immuno-electrophoretic assay of beta-lipoprotein and albumin in human aortic intima by direct electrophoresis from the tissue sample into an antibody-containing gel.

The chemical and immunological assay of low density lipoprotriens extracted from human aortic intima

A quantitative immunoplate technique was used to measure the amount of immunologically active plasma low density lipoprotein (immuno-β-cholesterol) in the ultracentrifugal S f 0–20 LP fraction of extracts of normal intima and early fibrous and fatty lesions. The preparation of satisfactory extracts was a major difficulty as homogenization appears both to produce non-specific lipid-protein complexes which float in the plasma LP range in the ultracentrifuge, and to destroy the immunological activity of plasma S f 0–20 LP. Maximum immunological activity was obtained from saline extracts of scissor-minced tissue. Neither immuno-β-cholesterol nor S f 0–20 cholesterol of normal intima were significantly correlated with age, but both were significantly correlated with intimal thickness ( P = 0.001). Total tissue cholesterol was more highly correlated with age than with thickness. In early fibrous lesions the immuno-β-cholesterol was significantly higher than in adjacent control samples, whereas it was significantly lower in lesions containing fat-filled cells. The levels in control samples adjacent to lesions with fat-filled cells were also reduced, and were significantly lower than in all other normals in the same age group. The proportion of samples with high levels of immuno-β-cholesterol was significantly greater in men with coronary heart disease (CHD) than in men of the same age group with other conditions.

Antithrombin activity in the human aortic intima, with special reference to its correlation to atherosclerotic changes

Since what is called thrombogenic theory was established by DUGUID, blood coagulation mechanism has been considered to take a part in pathogenesis of atherosclerosis. Later, it became clear that not only a strong thromboplastic activity but also anticoagulant activity existed in the aortic intimas. As to the anticoagulant activity of the vascular walls, KIRK et al. have pointed out that some of acid mucopolysaccharides (AMPS) in human aortic walls have a function of anticoagulant activity. Very recently OHE of our laboratory described originally the presence of antithrombin activity in aortic intimas of pigs. This is of intermedate type and has an activity derectly to inactivated thrombin. Purpose of the present experiments was to know a nature of intimal antithrombin of human aortas and correlation between the activity and variable atherosclerotic lesions of the aortas, ageing of the patient and amount of metachromatic materials in the human aortic intimas. Materials and Methods About 130 cases of fresh human aortic intimas were used as materials. Selerosing grade of human aortas was classified into 4 groups, as shown in Table I. By means of Endothelial Membrane Method (EMD), described by OHE, aortic intimal extracts were prepared from each group of the intimal lesions. Differential centrifugation of homogenates of each layers of the aortas, in particular of each of different sclerotic lesions of the intimas was made for test of antithrombin activity. Sephadex G-100 gel filtration of aortic intimal supernatants obtained by 105, 000G centrifugation was performed for the purpose of purification of antithrombin activity. Blood coagulation tests were performed in following method: plasma clotting activity (Fig. 1), thromboplastin generation test (Fig. 2), antithrombin activity test to use thrombin-fibrinogen system (Fig. 3, and Fig. 9) and TAMe system (Fig. 8) and so on. Histological examination of the aortic walls was made to use conventional staining method and OHNO's toluidin blue metachromasia method for detection of vascular AMPS. Results Plasma clotting time of aortic intimal extracts, obtained from many intact intimas by means of EMD, was found to be much longer than that of control solution. On the other hand, in cases of sclerotic intimas of grade I and II plasma clotting activity was significantly shortened with a parallelism to severity of atheroscletosis. It was also found that thromboplastin generation activity of the extracts from severe sclerotic intimas of grade II was as high as that of cephalin used routinely, intimal extracts from intact intimas, however, showed clearly inhibition of thromboplastin gene-ration activity. These facts suggested that sclerotic intimas may release substances rich in pro-coagulant activity rather than in anticoagulant activity into the blood. And in intact intimas the reverse may be also true. When antithrombin activity of the extracts from intact intimas was measured by means of two-stage method of thrombin-fibrinogen system. Characteristic developmental pattern of the activity was obtained and refered to as intermediate type of antithrombin (Fig. 3).

Alk-1-enyl groups of glycerophosphatides from human aortic intima, plasma and erythrocytes

Alk-1-enyl groups of glycerophosphatides from human aortic intima, plasma and erythrocytes

Rabbit Autoantibodies to Aorta

Abstract Antisera of rabbits immunized with antigenic preparations of bovine and human aortic intima reacted also with extracts of rabbit aorta, and two antigens characteristic of this tissue could be detected by these antisera. Two analogous antigens could also be identified by using the antisera of rabbits immunized with pooled rabbit aorta preparations in Freund's adjuvant. One of these antigens was a thermolabile component with fast electrophoretic mobility, presumably a protein; the other was a thermostable ethanol-insoluble component closely related to previously described tissue-specific BE antigens. Reactions with antigenic preparations derived from the antibody-producing animal provided evidence for the autoantibody nature of the antibodies under investigation.

Topochemistry of acid carbohydrates in the human aortic and coronary intima

The paper reports a study of the topochemistry of the human aortic and coronary intimai acid carbohydrates. The particular “pattern” revealed by this investigation is characterized by : (i) a topographic difference between the staining peculiarities of superficial and deep intimai areas, which can be correlated with a particular reactivity of some acidic functions of the carbohydrates; (ii) a prevalent localization of heparin-like substances in the superficial intimai area and of chondroitin-sulphates and sialic acid in the deep intimai one; (iii) a prevalent histochemical reactivity of carboxyl groups in the whole intima, associated with the presence of some sulphate esters which “hinder” the neighbouring carboxyls; (iv) the constant presence of acid carbohydrates as mucoprotein and sialo-mucoprotein complexes, papain digestion being the unique histochemical procedure permitting in our study a complete elimination of the intimai chromotrope material.

Lipid in the aortic intima the correlation of morphological and chemical characteristics

Summary Correlated studies were made on the morphological and chemical characteristics of the lipid in 119 samples of human aortic intima. The cases covered an age range of 13–84 years and most samples were either macroscopically normal or showed diffuse thickening, diffuse fatty flecking or fatty infiltration. A small group of definite fatty streaks was included for comparison. Lipid was found in two distinct morphological forms: (a)Perifibrous lipid, fine extracellular droplets which were often aligned along collagen and elastic fibres. (b)Fat-filled cells, containing coarser lipid droplets. Chemically the two types of lipid were clearly differentiated. In perifibrous lipid the cholesterol esters contain about 28 % oleic acid and 40 % linoleic acid, which is comparable with serum lipid. In fat-filled cells there is much more cholesterol ester, which contain about 48 % oleic acid and 14 % linoleic acid. Perifibrous lipid increases in normal intima with increasing age; we suggest that this is infiltrated serum lipid whereas the lipid in fat-filled cells is synthesized in situ .

SEQUESTRATION OF SERUM LOW-DENSITY LIPOPROTEINS IN THE ARTERIAL INTIMA BY COMPLEX FORMATION.

SummaryExtracts of human aortic intima were made in 0.9% NaCl and in 12% NaCl. Ultracentrifugally isolated human serum beta and alpha-2 lipoproteins were placed in similar salt concentrations. These were then examined in the immunoelectrophoresis system using antiserum against serum beta lipoprotein (which is equally effective against alpha-2 lipoprotein). Extract of aorta gave 2 lipoprotein arcs with this antiserum, one in the beta and one in the pre-albumin region, when prepared with 0.9% NaCl; there was only one arc (in the alpha-2 region) in extracts with 12% NaCl. This is interpreted as suggesting that serum alpha-2 lipoprotein is bound in the aortic intima in a pair of complexes both of which can be disrupted with strong salt solution.

Histochemical identification of plasma proteins in the human aortic intima

Histochemical identification of plasma proteins in the human aortic intima