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Purpose: To determine whether homocyst(e)ine (H(e)) is related to hemostatic factors in a population-based sample without evidence of cardiovascular disease., Methods: A subsample of 660 participants--67 African-American women, 53 African-American men, 201 white women, and 339 white men--was selected from the Atherosclerosis Risk in Communities Study baseline cohort. This was based on carotid intimal-medial wall thickness above the 90th percentile or below the 75th percentile of the population distribution, assessed by B-mode ultrasonography. Unadjusted and multivariable-adjusted associations between fasting plasma H(e) and the hemostatic factors fibrinogen, factor VII:c, factor VIII:c, protein C antigen, hematocrit, platelet count, beta-thromboglobulin (beta-TG), tissue plasminogen activator (tPA), PAI-1, D-dimer, and lipoprotein[a] were examined., Results: Mean age-adjusted H(e) was positively, albeit weakly, correlated with beta-TG, tPA, hematocrit, D-dimer and PAI-1; inversely correlated with protein C; and was higher in smokers, men and African-Americans. In multivariable regression, beta-TG, tPA, and factor VII:c were positively associated with H(e), as well as age, black race, male sex, and current cigarette smoking., Conclusions: These cross-sectional data for a biracial group of middle-aged individuals suggest that H(e) levels falling below values consistent with homocyst(e)inemia are associated with several prothrombotic factors after adjustment for sociodemographic factors. If H(e) change is antecedent to altered hemostasis, FDA-mandated fortification of grain products with folic acid for prevention of fetal neural tube defects may lead to both reduced plasma H(e) levels and improved hemostatic profiles.

To examine the effects of atherosclerosis on the protein C anticoagulant pathway in vivo, we measured anticoagulant responses to intravenous administration of human alpha-thrombin or activated protein C (APC) in cynomolgus monkeys. Two groups of monkeys were fed either a control diet (n=18) or an atherogenic diet (n=12) that produces both hypercholesterolemia and moderate hyperhomocyst(e)inemia. A third group (n=8) was fed an atherogenic diet for 15 months, and then fed the atherogenic diet supplemented with B vitamins for 6 months to correct the hyperhomocyst(e)inemia. The plasma homocyst(e)ine level was higher in monkeys fed the atherogenic diet (9.6+/-1.0 micromol/L) than in monkeys fed the control diet (3.7+/-0.2 micromol/L) or the atherogenic diet with B vitamins (3.6+/-0.2 micromol/L) (P<0.001). Infusion of thrombin produced a much greater prolongation of the activated partial thromboplastin time in monkeys fed the control diet (52+/-10 seconds) than in monkeys fed the atherogenic diet either with (24+/-4 seconds) or without (27+/-5 seconds) supplemental B vitamins (P<0.02). Thrombin-dependent generation of circulating APC was higher in control (294+/-17 U/mL) than in atherosclerotic (240+/-14 U/mL) monkeys (P<0.05), although levels of fibrinogen, plasminogen, D-dimer, and thrombin-antithrombin complexes were similar in each group. Injection of human APC produced a similar prolongation of the activated partial thromboplastin time in control (31+/-3 seconds) and atherosclerotic (29+/-2 seconds) monkeys. These findings provide evidence for impaired anticoagulation, due partly to decreased formation of APC, in atherosclerosis. The blunted anticoagulant response to thrombin in hypercholesterolemic monkeys was not corrected by supplementation with B vitamins.

Background and Purpose: Elevated plasma homocyst(e)ine [H(e)] concentration is associated with premature atherosclerosis. A common cause of elevated plasma H(e) concentration is a thermolabile mutation (677T) in the gene encoding methylenetetrahydrofolate reductase (MTHFR). We sought to determine whether plasma H(e) concentration or MTHFR genotype would be more strongly associated with carotid plaque area (CPA), a potential intermediate phenotype of atherosclerosis., Methods: In 307 subjects who were ascertained through a premature atherosclerosis clinic, we measured CPA with 2-dimensional ultrasound and also determined traditional atherosclerosis risk factors, in addition to plasma H(e) concentration and MTHFR genotypes., Results: We found that the frequency of the MTHFR 677T allele was 0.363 in this sample. Mean plasma H(e) concentration was significantly higher in 677T/T homozygotes than in 677T/C heterozygotes and 677C/C homozygotes (17. 1+/-13.7 versus 13.5+/-6.1 versus 12.6+/-5.9 micromol/L, respectively, P<0.001). Analysis of variance showed that CPA was significantly associated with age, sex, smoking, diabetes, hypertension, and hyperlipidemia (each P<0.05). When plasma H(e) concentration was included in the model, it was significantly associated with CPA (P<0.05). However, when the MTHFR genotype was included in the model, it was not associated with CPA (P=0.50). Furthermore, there was a significant correlation of CPA with plasma H(e) (r=0.23, P<0.0001). However, the mean CPA did not differ between subjects according to genotype. CONCLUISONS: Thus, plasma H(e), but not MTHFR genotype, is significantly associated with carotid atherosclerosis, suggesting that the biochemical test may be sufficient to identify patients who may be at increased risk of atherosclerosis through this mechanism.

Blood homocyst(e)ine levels are an important, independent and frequent risk factor for clinical atherosclerosis and venous thrombosis. Folic acid, vitamins B6 and B12, renal and thyroid functions, certain medications and certain genotypes are known to modulate plasma homocyst(e)ine levels. Intake of B vitamins through diet, supplementation and fortified foods effectively reduces homocyst(e)ine concentration and thus may reduce the risk of cardiovascular disease. This is true even in individuals who are genetically predisposed to hyperhomocyst(e)inemia. Randomized clinical trials are needed to investigate these effects further.

Background: Elevated plasma total homocysteine (tHcy), low B-vitamin intake, and genetic polymorphisms related to tHcy metabolism may play roles in coronary heart disease (CHD). More prospective studies are needed., Methods and Results: We used a prospective case-cohort design to determine whether tHcy-related factors are associated with incidence of CHD over an average of 3.3 years of follow-up in a biracial sample of middle-aged men and women. Age-, race-, and field center-adjusted CHD incidence was associated positively (P<0.05) with tHcy in women but not men, and CHD was associated negatively (P<0.05) with plasma folate (women only), plasma pyridoxal 5'-phosphate (both sexes), and vitamin supplementation (women only). However, after accounting for other risk factors, only plasma pyridoxal 5'-phosphate was associated with CHD incidence; the relative risk for the highest versus lowest quintile of pyridoxal 5'-phosphate was 0.28 (95% CI=0.1 to 0.7). There was no association of CHD with the C677T mutation of the methylenetetrahydrofolate reductase gene or with 3 mutations of the cystathionine beta-synthase gene., Conclusions: Our prospective findings add uncertainty to conclusions derived mostly from cross-sectional studies that tHcy is a major, independent, causative risk factor for CHD. Our findings point more strongly to the possibility that vitamin B6 offers independent protection. Randomized trials, some of which are under way, are needed to better clarify the interrelationships of tHcy, B vitamins, and cardiovascular disease.

Moderate elevation of plasma homocyst(e)ine is associated with increased risk for atherosclerotic vascular disease. In a previous study, we observed impaired vascular function in nonatherosclerotic monkeys with moderate hyperhomocyst(e)inemia. In this study, we tested the hypothesis that dietary intervention to lower plasma homocyst(e)ine corrects vascular dysfunction in atherosclerotic monkeys. Cynomolgus monkeys were fed an atherogenic diet that produces both hypercholesterolemia and moderate hyperhomocyst(e)inemia. After 17 months, the atherogenic diet was supplemented with B vitamins (5 mg folic acid, 400 micrograms vitamin B-12, and 20 mg vitamin B-6 daily) for 6 months. Total plasma homocyst(e)ine decreased from 12.8 +/- 2.8 to 3.5 +/- 0.3 mumol/L (n = 9; mean +/- SE; P < .01) after vitamins were added to the diet, but plasma cholesterol remained elevated (522 +/- 63 versus 514 +/- 41 mg/dL; P > .05). In response to intra-arterial infusion of collagen, blood flow to the leg decreased by 30 +/- 3% and 38 +/- 5%, respectively, before and after vitamin supplementation (P > .05). In vivo responses of resistance vessels to endothelium-dependent vasodilators (acetylcholine or ADP) were impaired at baseline and did not improve after vitamin supplementation. In carotid artery studied ex vivo, relaxation to low doses of acetylcholine improved after vitamin supplementation, but maximal relaxation remained impaired. Ex vivo thrombomodulin anticoagulant activity was threefold higher in monkeys fed the atherogenic diet (with or without B vitamins) than in normal monkeys (P < .05). We conclude that normalization of plasma homocyst(e)ine is insufficient to restore normal vascular function in atherosclerotic monkeys with persistent hypercholesterolemia and that atherosclerosis, with or without hyperhomocyst(e)inemia, is associated with elevated thrombomodulin activity.

Plasma homocyst(e)ine [H(e)] levels correlate with the prevalence of arterial occlusive diseases. Recently, transesophageal echocardiography (TEE) has been used to evaluate patients with atherosclerotic plaques in the thoracic aorta. The purpose of this study was to determine whether H(e) levels correlate with the degree of atherosclerotic plaque in the thoracic aorta (ATH) as seen on TEE. Maximum plaque areas for three locations in the thoracic aorta (arch, proximal descending, and distal descending) were measured with TEE in 156 patients. Maximum plaque areas for these locations were added to yield an estimate of ATH. ATH and H(e) levels, and levels of folic acid, vitamin B12, and pyridoxal 5'-phosphate were measured in a double-blind manner. Univariate analysis demonstrated a significant correlation of H(e) with ATH (r = 0.3, p< 0.001). On multivariate analysis, H(e) was independently predictive of ATH (r for the model including H(e) was 0.63, p < 0.0001). Plasma H(e) levels are therefore significantly and independently correlated with the degree of atherosclerosis in the thoracic aorta.

Homocysteine is an intermediate compound formed during metabolism of methionine. The results of many recent studies have indicated that elevated plasma levels of homocyst(e)ine are associated with increased risk of coronary atherosclerosis, cerebrovascular disease, peripheral vascular disease, and thrombosis. The plasma level of homocyst(e)ine is dependent on genetically regulated levels of essential enzymes and the intake of folic acid, vitamin B6 (pyridoxine), and vitamin B12 (cobalamin). Impaired renal function, increased age, and pharmacologic agents (e.g. nitrous oxide, methotrexate) can contribute to increased levels of homocyst(e)ine. Plausible mechanisms by which homocyst(e)ine might contribute to atherogenesis include promotion of platelet activation and enhanced coagulability, increased smooth muscle cell proliferation, cytotoxicity, induction of endothelial dysfunction, and stimulation of LDL oxidation. Levels of homocysteine can be reduced with pharmacologic doses of folic acid, pyridoxine, vitamin B12, or betaine, but further research is required to determine the efficacy of this intervention in reducing morbidity and mortality associated with atherosclerotic vascular disease.

Background: Plasma levels of homocyst(e)ine are elevated in certain patients with occlusive arterial diseases. We extended these findings to asymptomatic adults., Methods and Results: We determined plasma homocyst(e)ine levels in 287 pairs of asymptomatic adults. Cases and controls were defined on the basis of intimal-medial thickness of the carotid wall as measured by B-mode ultrasound. Study subjects had no history of atherosclerotic disease and were selected from a probability sample of 15,800 men and women between 45 and 64 years old. Subjects with thickened intimal-medial carotid walls (cases) had higher plasma homocyst(e)ine levels than controls (p < 0.001). The odds ratio for having a thickened carotid artery wall was 3.15 (p < 0.001) for subjects in the top quintile of plasma homocyst(e)ine levels (> 10.5 mumol/L) compared with those in the bottom quintile (< 5.88 mumol/L)., Conclusions: The present study as well as observations on the common occurrence of elevated plasma homocyst(e)ine levels in patients with occlusive arterial diseases suggest that clinical trials should be conducted to determine whether normalization of hyperhomocyst(e)inemia may prevent progression of atherosclerosis.

The observations so far conducted in cynomologus monkeys on semipurified diets containing butter and cholesterol suggest that this nonhuman primate is an excellent model for studying the therapy of established coronary atherosclerosis. (1) This species is available at a reasonable cost and can be kept in captivity in good health for prolonged periods of time. (2) It readily accepts semipurified diets with a percentage composition similar to that of human diets in the U.S. (3) Ingestion of these diets leads quite rapidly (around 6 months) to moderate coronary atherosclerosis. More prolonged feeding leads to lesions which are histologically very similar to those in man. (4) The distribution of lesions in the main coronary arteries is similar to that in man. (5) Methods to quantify the coronary lesions are available. (6) The diets can be so modified that cholesterol levels closely resemble those in hypercholesterolemic man. (7) The monkeys are amenable to several therapeutic regimens which show promise of arresting the progress or inducing the regression of the coronary lesions.

We studied the ultrastructure of the left anterior descending coronary artery in 6 female cynomolgus macaques fed atherogenic food containing 0.5% cholesterol for 6 months, and in 2 female cynomolgus macaques that had received food low in cholesterol. Animals given the atherogenic food had coronary artery lesions of a characteristic two-level architecture consisting of a lipid-poor upper cap, and of a lipid-rich lower core. The cap consisted of layers of smooth muscle cells that were rich in rough endoplasmic reticulum and contained few myofilaments and relatively few lipid droplet inclusions. The core consisted mainly of macrophages overloaded with droplet inclusions (macrophage foam cells), droplet-laden smooth muscle cells, and extracellular lipid and cell debris. Some macrophage foam cells were in mitosis. The largest lipid cores contained multinucleate giant cells with large intracellular crystal clefts. Dead macrophage foam cells were the source of the extracellular lipid and debris particles. Intimal cores often extended into the adjacent media. The adventitia was involved in the intima-media lesions in 3 of the animals. Compared with the coronary artery lesions of rhesus macaques and patas monkeys given similar atherogenic diets, cynomolgus monkey lesions were morphologically closer to human atherosclerotic plaques with respect to their stratification into a cap and a core.

It has been observed that atherosclerotic lesions (AL) concentrate certain porphyrins. We evaluated the usefulness of 111In-labelled Photofrin II (PFII), a porphyrin derived from haematoporphyrin derivative, for detection of experimental AL in cholesterol fed rabbits. Three groups of rabbits were studied: non-atherosclerotic (n = 3), 6 and 12 week cholesterol fed (n = 4, 3). 111In-PFII was injected intravenously and gamma camera images were obtained at 24 and 48 h. At 48 h, explanted aortas were also imaged. Aortic arch (AA) to background (BKG) count ratios were calculated from images of the whole body and explanted aortas. AA/BKG ratios were significantly higher in the 12 week cholesterol fed rabbits (3.9 +/- 0.72 at 24 h) and (4.0 +/- 0.67 at 48 h) than in the non-atherosclerotic rabbits (2.2 +/- 0.07 at 24 h) and (2.3 +/- 0.18 at 48 h) (p less than 0.05). The AA/BKG ratio for the explanted aortas showed similar results. Additionally, in two of three 12 week cholesterol fed rabbits, focal count deposition was visible on the whole animal images at the site of aortic arch atherosclerosis. We conclude that 111In-PFII concentrates in AL as early as 24 h after injection and has the potential to be used as an imaging agent for experimental atherosclerosis.

Regression of atherosclerosis has been demonstrated in several species of animals, including rabbits, chickens, rats, dogs, pigeons, pigs, and nonhuman primates. Regression has been associated with withdrawal of cholesterol from the diet or with the ingestion of cholestyramine, alfalfa meal, or alfalfa saponins in monkeys fed high-cholesterol diets. Although regression of atherosclerosis has been documented by sequential contrast arteriography in humans, the correspondence of regression in animals and in humans needs to be established.

A semipurified diet containing 43% of the calories as fat and 1.2 mg of cholesterol/cal was fed to cynomolgus monkeys (Macaca fascicularis) for 6 months; the cholesterol content was reduced to 0.34 mg/cal for the next 18 months. During the latter period, the monkeys were assigned to 4 groups of 18 animals each and received the following dietary additions: A, none (controls); B, cholestyramine (5%, w/w); C, dextrothyroxine (0.003%); and D, Wy-14,643 (0.45%). Cholestyramine normalized plasma lipid levels and reduced the size of aortic and coronary atherosclerotic lesions in spite of the high-fat, high-cholesterol intake. Dextrothyroxine reduced cholesterolemia but did not modify the extent of arterial lesions. Wy-14,643 changed neither plasma cholesterol levels nor the extent of atherosclerosis.

plasma and aortic tissue lipid changes in atherosclerotic Macaca fascicularis monkeys were studied following various regression regimens for 18 months. Atherosclerosis was induced in groups of 18 Macaca fascicularis monkeys by feeding a semipurified diet containing 43% of the calories as fat and 1.2 mg/kcal cholesterol for 6 months. Five groups of animals were continued on the same diet containing 0.34 mg/kcal cholesterol during the regression period, and were given the following hypocholesterolemic regimens: none (positive controls); D-thyroxine; pyrimidine derivative; cholestyramine; and alfalfa. Another group of animals was maintained on regular monkey chow alone during the regression period (negative control). Among the hypocholesterolemic agents, cholestyramine very effectively reduced the plasma cholesterol, and aorta free- and esterified cholesterol and phospholipids. D-Thyroxine significantly lowered the plasma cholesterol levels but, surprisingly, tissue lipid levels were the highest among the groups studied. Alfalfa, although not as effective as cholestyramine, tended to reduce the plasma and tissue lipids more than other drugs. Pyrimidine derivative actually increased the mean levels of plasma and tissue cholesterol. Plasma cholesterol correlated positively with aortic tissue cholesterol, free and esterified cholesterol. Both the plasma percentage distribution of alpha-lipoprotein and the phospholipid/cholesterol ratio were inversely related to all tissues lipids. Plasma cholesterol accounted for 43% and 30% respectively of the variability in tissue total and esterified cholesterol. Thus the effectiveness of any hypocholesterolemic regimen is probably dependent on achieving a favorable lipoprotein distribution in plasma without any adverse effect on the arterial wall metabolism.

Previously reported findings on a large series of patients are compatible with the hypothesis that terminal cancer is associated with the arrest of progression and the regression of atherosclerosis.

A semipurified diet containing 1.2 mg of cholesterol/Cal was fed to cynomolgus monkeys (Macaca fascicularis). At the end of 6 months, a group of 18 animals was killed for evaluation of atherosclerosis in the aorta and the coronary arteries. The remaining monkeys were assigned to three groups of 18 animals each and fed, during the following 18 months, semipurified diets containing 0.34 mg of cholesterol/Cal with or without alfalfa meal, or a diet consisting entirely of Monkey Chow. a decrease in cholesterolemia and plasma phospholipid levels, normalization in the distribution of plasma lipoproteins, and reduction in the extent of aortic and coronary atherosclerosis were observed in monkeys fed the semipurified diet containing alfalfa, although the intake of cholesterol remained as high as in the usual American diet. These changes, also observed in monkeys fed a chow diet almost devoid of cholesterol, suggest that alfalfa counteracts the atherogenic effect of dietary cholesterol.

Changes of arterial wall lipid and connective tissue components were studied under various regression regimes on experimental high fat-cholesterol diet induced atherosclerosis in non-human primates. Levels of plasma lipids and aorta tissue lipids were closely related, while an inverse relationship between plasma cholesterol levels was noted with the ratio of aorta tissue free/ester cholesterol. Although the total content of glycosaminoglycan increased slightly with increasing disease, changes of individual glycosaminoglycans were most closely related to the severity of aorta atherosclerosis associated with both induction and regression of disease. With increasing atherosclerosis hyaluronic acid tended to decrease, while chondroitin sulfates decreased. Heparan sulfate decreased considerably with increasing severity of atherosclerosis. Consequently, regression regimens reducing the severity reversed the glycosominoglycan changes. Withdrawal of the dietary stimulus and cholestryamine produced the greatest reversal of atherosclerosis. In contrast, D-thyroxine treatment resulted in severe aorta atherosclerosis comparable to controls receiving no treatment. The effectiveness of regression depends on favorably altering plasma lipids but without adversely affecting the arterial wall structural elements.