William Hollander

Unified concept on the role of acid mucopolysaccharides and connective tissue proteins in the accumulation of lipids, lipoproteins, and calcium in the atherosclerotic plaque

Abstract The biochemical and morphological data reviewed supported the concept that the interaction of lipoproteins with acid mucopolysaccharides and calcium, together with the binding of these complexes to the connective tissue proteins, may play an important role in the formation of the fibrous plaque. The interactions between these macromolecules could provide an explanation for the accumulation of lipids, lipoproteins, and calcium in the plaque. The possibility also was considered that the interaction of the arterial lipoproteins, particularly very low density lipoproteins, with acid mucopolysaccharides might be an important mechanism involved in the formation of the fatty streak.

Soluble proteins in the human atheroschlerotic plaque

A number of soluble proteins contained in human aortic intimal tissue was extracted into buffered saline (pH 7.4) and identified and quantitated by immunoelectrophoresis and immunodiffusion. The proteins included IgA, IgG, IgM, B1C (C3), α1-antitrypsin, α2-macroglobulin, fibrinogen, albumin, LDL, HDL, α1-acid glycoprotein, β2-glyprotein, transferrin and ceruloplasmin. The concentration of soluble proteins was significantly higher in the atherosclerotic intima than in the normal intima. The diseased intima also contained a small amount of tissue-bound IgG, IgA and B1C which was extractable with citrate buffer at pH 3.2. The vascular band IgG, and B1C were shown by enzymatic and immunohistochemical studies to be closely associated with the collagenous tissue of the plaque. The Ig contained in the atherosclerotic plaque may be derived in part from the biosynthesis of Ig by the artery, since the incorporation of 14C-labeled leucine into IgG by the atheromatous plaque was demonstrable by radioimmunoelectrophoresis. In contrast to the diseased artery, the normal artery did not synthesize IgG and did not contain vascular bound IgG or complement. However, the normal artery was capable of fixing IgG and B1C eluted from the diseased artery. The present studies suggested that the IgG contained and synthesized by the plaque might represent an immune response to an endogenous or exogenous antigen closely associated with plaque collagen. IgG and B1C either alone or in the form of an immune complex also may play an important role in phagocytosis in the plaque and thereby influence the course of atherosclerosis. The proteolytic inhibitors, α1-antitrypsin and α2-macroglobulin, found in relatively high concentrations in the plaque, could enhance fibrosis of the lesion because of their known inhibitory effects on collagenase and elastase.Abstract A number of soluble proteins contained in human aortic intimal tissue was extracted into buffered saline (pH 7.4) and identified and quantitated by immunoelectrophoresis and immunodiffusion. The proteins included IgA, IgG, IgM, B 1 C (C 3 ), α 1 -antitrypsin, α 2 -macroglobulin, fibrinogen, albumin, LDL, HDL, α 1 -acid glycoprotein, β 2 -glyprotein, transferrin and ceruloplasmin. The concentration of soluble proteins was significantly higher in the atherosclerotic intima than in the normal intima. The diseased intima also contained a small amount of tissue-bound IgG, IgA and B 1 C which was extractable with citrate buffer at pH 3.2. The vascular band IgG, and B 1 C were shown by enzymatic and immunohistochemical studies to be closely associated with the collagenous tissue of the plaque. The Ig contained in the atherosclerotic plaque may be derived in part from the biosynthesis of Ig by the artery, since the incorporation of 14 C-labeled leucine into IgG by the atheromatous plaque was demonstrable by radioimmunoelectrophoresis. In contrast to the diseased artery, the normal artery did not synthesize IgG and did not contain vascular bound IgG or complement. However, the normal artery was capable of fixing IgG and B 1 C eluted from the diseased artery. The present studies suggested that the IgG contained and synthesized by the plaque might represent an immune response to an endogenous or exogenous antigen closely associated with plaque collagen. IgG and B 1 C either alone or in the form of an immune complex also may play an important role in phagocytosis in the plaque and thereby influence the course of atherosclerosis. The proteolytic inhibitors, α 1 -antitrypsin and α 2 -macroglobulin, found in relatively high concentrations in the plaque, could enhance fibrosis of the lesion because of their known inhibitory effects on collagenase and elastase.

Role of hypertension in atherosclerosis and cardiovascular disease.

Clinical, experimental and pathologic studies strongly indicate that hypertension is a major factor in coronary heart disease, sudden death, stroke congestive heart failure and renal insufficiency. The deleterious effect of the elevated blood pressure on the cardiovascular system appears to be due mainly to the mechanical stress placed on the heart and blood vessels. Humoral factors and vasoactive hormones such as angiotensin, catecholamines and prostaglandins may play a role in the pathogenesis of hypertensive cardiovascular disease but this role has not yet been defined and is probably secondary. Hypertension and the resulting increase in tangential tension on the myocardial and arterial walls, leads to the development of hypertensive heart disease and congestive heart failure as well as hypertensive vascular disease that affects not only the kidneys but also the heart and brain. Hypertensive vascular disease involves both large and small arteries as well as arterioles and is characterized by fibromuscular thickening of the intima and media with luminal narrowing of the small arteries and arterioles. The physical stress of hypertension on the arterial wall also results in the aggravation and acceleration of atherosclerosis, particularly of the coronary and cerebral vessels. Moreover, hypertension appears to increase the susceptibility of the small and large arteries to atherosclerosis. Thus the patient with hypertension is a candidate for both hypertensive and atherosclerotic vascular disease of the coronary and cerebral vessels leading to occlusive disease of both the large and small arteries and resulting in myocardial infarction and stroke. Other major complications of hypertensive vascular disease include rupture and thrombotic occlusion of blood vessels, especially in the brain. Disease of the arterial media, which begins in childhood with the deposition of calcium in the vessels, may be an important cause of arterial hypertension. This form of hypertension may manifest itself in adults as arteriosclerotic hypertension and lead to cardiovascular complications very similar to those of essential hypertension. The relation of arteriosclerotic hypertension to nutritional factors, including dietary salt intake, deserves study.

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, LDL1 (d 1.006 to 1.019) and LDL2 (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 LDL2 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 LDL2. 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.

Studies on the progression and regression of coronary and peripheral atherosclerosis in the cynomolgus monkey. I. Effects of dipyridamole and aspirin.

Abstract The morphological and biochemical changes in the arteries of the cynomolgus monkey were investigated during the induction and regression of atherosclerosis. During the feeding of an atherogenic diet containing 2% cholesterol and 10% butter for 5 months, the animals developed fibro-fatty plaques which involved the coronary and peripheral arteries and caused significant luminal narrowing of these vessels. The induced aortic lesions contained increased amounts of free and esterified cholesterol, collagen, elastin, and calcium. These changes were associated with an elevation of plasma cholesterol and an increased net influx of plasma cholesterol and LDL into the aorta. Dipyridamole (10 mg/kg) and aspirin (50 mg/kg) had no effect on the arterial uptake of plasma LDL and cholesterol and did not protect against atherosclerosis in any of the vessels examined. During the regression period (low cholesterol diet) of 12 months duration, the induced lesions became more fibrotic and calcified while the cellularity and lipid content of the same lesions decreased. As a result of these changes there were no significant decreases in the atherosclerotic narrowing of the coronary and peripheral vessels. The net influx of plasma LDL and cholesterol into the aorta returned to normal during the regression period. This finding together with the slow rate of aortic cholesterol equilibration suggests that the retention of cholesterol in the regressed aortic lesions is due to a defect in cholesterol transport rather than to an abnormality in intimal permeability. The addition of dipyridamole and aspirin to the regression diet did not alter the course of the atherosclerosis.

Comparative effects of cetaben (PHB) and dichlormethylene diphosphonate (Cl2MDP) on the development of atherosclerosis in the cynomolgus monkey

Abstract The comparative effects of cetaben [sodium p-hexadecylaminobenzoate, (PHB)] and dichloromethylene diphosphate (Cl 2 MDP) on the development of atherosclerosis was investigated in the cynomolgus monkey. The atherosclerosis was induced by feeding a 2.5% cholesterol and 10% butter diet for 6 months and was assessed by morphological and biochemical examination of the arteries at necropsy. The experimental disease was also evaluated clinically by the electrocardiographic responses to isoproterenol and by measurements of regional myocardial blood flow using microspheres. Both the clinical and necropsy findings indicated that PHB was an effective antiatherosclerotic agent which reduced the extent and severity of the disease and protected against obstructive atherosclerotic disease of the coronary and peripheral arteries. On the other hand, (CL 2 MDP) was found to have no antiatherosclerotic effect except for preventing calcium deposition in the induced lesions. The atherosclerotic narrowing of the major coronary arteries in the PHB-treated group averaged about 27% as compared to 55% in the untreated animals. PHB also reduced the luminal narrowing of the carotid, subclavian, iliac and tibial arteries by 50% or more. The drug had actions on both the circulating blood and arteries that could account for its antiatherosclerotic effects. These actions included a lowering of plasma cholesterol and an effect on the plasma lipoproteins resulting in a decrease in LDL and an increase in HDL. Other important actions of PHB included the inhibition of calcium, cholesterol and cholesteryl ester deposition in the induced lesions as well as reduction in the fibrous protein content of the lesions.

Lipoproteins in human atherosclerotic vessels. II. Biochemical properties of the major apolipoproteins of arterial low density and very low density lipoproteins.

Abstract The biochemical behavior of the major apoproteins of low density lipoproteins (LDL) and very low density lipoproteins (VLDL) isolated from human atheroslerotic arteries was examined. On Sephadex G-150, delipidated arterial LDL behaved like delipidated serum LDL and eluted as a single peak (apoLDL) at the void volume. The apoLDL fractions migrated as a single band when examined by electrophoresis on cellulose acetate or acrylamide gel and had a mean sedimentation coefficient of 3.2. Upon double immunodiffusion arterial apoLDL formed immunopreciptin lines of complete identity with serum apoLDL, when reacted against antisera to either human serum apoLDL or LDL. However the amino acid composition of arterial and serum apoLDL was significantly different. When delipidated arterial VLDL was examined on Sephadex G-150, it separated into two major apoprotein fractions, SF-1 and SF-3, and into a minor SF-2 fraction. These fractions appeared to have similar elution volumes and immunochemical reactivity as the corresponding Sephadex fractions of delipidated serum VLDL. Arterial and serum SF-1, SF-2, and SF-3, when reacted respectively against apoLDL antisera, HDL antisera and apo C-III antisera, formed immunoprecipitin lines of complete identity. The sedimentation coefficients of arterial and serum SF-1 appeared similar, as were the coefficients of arterial and serum of SF-3. However the amino acid composition of arterial SF-1 and SF-3 was clearly distinguishable from that of their corresponding serum apoprotein fractions. When examined by acrylamide electrophoresis arterial and serum SF-3 showed a similar band pattern but with different electrophoretic mobility. SF-1 of arterial apoVLDL behaved much like apoLDL from arterial LDL as indicated by electrophoretic and immunochemical behavior and amino acid composition. Although the mean sedimentation coefficients of these apoprotein fractions were similar, the analytical pattern suggested SF-1 of arterial apoVLDL to be more polydisperse in its composition. The present studies indicate that major differences exist between the arterial and serum apoproteins especially in regard to their amino acid composition. They also suggest that arterial LDL and VLDL contain apoproteins which have some characteristics that are similar to the apoB and apoC proteins contained in serum LDL and VLDL.

Studies on the role of arterial microsomes in the contractile function of the arteries

Abstract Microsomal vesicles were isolated from canine arteries by differential ultracentrifugation between 12,000 and 40,000g. The arterial microsomes were capable of accumulating calcium in the presence of Mg2+ and ATP. Calcium binding and uptake appeared to be relatively slow and dependent on Mg2+ activated ATPase activity. The capacity of the microsomes to accumulate Ca2+ appeared to be maximal at about 40 μM Ca2+ and averaged 0.020 μmoles/mg without K-oxalate and 0.034 μmoles/mg with K-oxalate. About half of the total calcium accumulated by the microsomes occurred in 5–15 min. The arterial microsomes also were able to release Ca2+ after being preloaded with calcium. The partial replacement of K+ by Na+ in the test system augmented Ca2+ release as well as reduced calcium uptake by the microsomes. The superprecipitation of arterial actomyosin was partially inhibited by the arterial microsomes. However this inhibition of superprecipitation was demonstrable only after preincubation of the microsomes with Ca2+ and adjustment of the ATP concentrations in the test system. The substitution of Na+ for K+ in the reaction mixture decreased calcium uptake and reduced the inhibiting effects of the microsomes on superprecipitation. The present findings are consistent with the concept that the calcium uptake and ATPase activities of arterial microsomes may play a role in regulating the contractility of arterial smooth muscle by influencing the intracellular concentrations of Ca2+ and ATP.

Biochemical and morphological characteristics of arterial actomyosin

Abstract An active preparation of natural actomyosin was extracted from eanine arteries in solutions of low ionic strength containing ATP. In this solution, arterial actomyosin appeared by electron microscopy to dissociate into thick filaments corresponding to myosin and thin filaments corresponding to F-actin. In a high ionic strength solution without ATP, electronmicrographs of arterial actomyosin showed an “arrow-head” filamentous structure which also has been described for skeletal actomyosin (myosin B). The actomyosin preparation also had many of the biochemical characteristics reported for skeletal actomyosin including the ability to hydrolyze ATP, undergo superprecipitation (contraction), and respond to the changes in the concentration of free Ca 2+ . The concentration of ATP had a noticeable effect on the superprecipitation (contraction) and clearing (relaxation) of arterial actomyosin with relatively low concentrations promoting superprecipitation and high concentrations promoting clearing. Varying concentrations of Na + and K + also influenced the superprecipitation with the partial replacement of K + by equimolar concentrations of Na + resulting in an enhanced superprecipitation of arterial actomyosin. The present study could provide an explanation for the manner in which changes in intracellular concentrations of different cations and ATP alter the contractile function of the arteries.