Jeffrey M. Hoeg

Active Serum Vitamin D Levels Are Inversely Correlated With Coronary Calcification

BACKGROUND Arterial calcification is a common feature of atherosclerosis, occurring in >90% of angiographically significant lesions. Recent evidence from this and other studies suggests that development of atherosclerotic calcification is similar to osteogenesis; thus, we undertook the current investigation on the potential role of osteoregulatory factors in arterial calcification. METHODS AND RESULTS We studied two human populations (173 subjects) at high and moderate risk for coronary heart disease and assessed them for associations between vascular calcification and serum levels of the osteoregulatory molecules osteocalcin, parathyroid hormone, and 1alpha,25-dihydroxyvitamin D3 (1,25-vitamin D). Our results revealed that 1,25-vitamin D levels are inversely correlated with the extent of vascular calcification in both groups. No correlations were found between extent of calcification and levels of osteocalcin or parathyroid hormone. CONCLUSIONS These data suggest a possible role for vitamin D in the development of vascular calcification. Vitamin D is also known to be important in bone mineralization; thus, 1,25-vitamin D may be one factor to explain the long observed association between osteoporosis and vascular calcification.

The role of nitric oxide in endothelium-dependent vasodilation of hypercholesterolemic patients.

BackgroundPatients with hypercholesterolemia have a reduced response to endothelium-dependent vasodilators. However, the regulatory function of the endothelium on vascular tone is mediated through the release of several vasoactive substances; therefore, a reduced response to endothelium-dependent agents does not identify which of the factors released by the endothelium is involved in this abnormality. Methods and Result. To investigate the role of nitric oxide in the endothelium-dependent vasodilation in hypercholesterolemia, we studied the effect of NG-monomethylRL-arginine (L-NMMA), an inhibitor of endothelial nitric oxide synthesis, on basal vascular tone and on the responses to acetylcholine, an endothelium-dependent vasodilator, and to sodium nitroprusside, a direct smooth muscle dilator. The study included 33 hypercholesterolemic patients (17 men; 51±8 years; plasma cholesterol, .240 mg/dL) and 23 normal controls (12 men; 48±7 years; plasma cholesterol, <210 mgldL). Drugs were infused into the brachial artery, and the response of the forearm vasculature was measured by strain-gauge plethysmography. Basal blood flow and vascular resistance were similar in hypercholesterolemic patients and normal controls (3.1±1 versus 2.6±0.8 mL/min per 100 mL and 32.1±13 versus 36.1±12 mm Hg/mL-1 min−1. 100 mL-1, respectively). The reduction in basal blood flow and increase in vascular resistance produced by L-NMMA were not significantly different between the two groups. L-NMMA markedly blunted the response to acetylcholine in normals (maximum flow decreased from 16.4±8 to 7.0±3; P<.005); however, the arginine analogue did not significantly modify the response to acetylcholine in the hypercholesterolemic patients (maximum flow, 11.1±8 versus 10.0±8). L-NMMA did not modify the vasodilator response to sodium nitroprusside in either controls or patients. ConclusionsThese findings indicate that hypercholesterolemic patients have a defect in the bioactivity of nitric oxide that may explain their impaired endothelium-dependent vascular relaxation.

CUMULATIVE EFFECTS OF HIGH CHOLESTEROL LEVELS, HIGH BLOOD PRESSURE, AND CIGARETTE SMOKING ON CAROTID STENOSIS

BACKGROUND Single measurements of cardiovascular risk factors may not accurately reflect a persons past exposure to those risk factors. We therefore studied the long-term associations of cardiovascular risk factors such as high serum cholesterol levels, high blood pressure, and cigarette smoking with the prevalence of carotid stenosis. METHODS We studied cross-sectional and longitudinal information from a sample of 429 men and 661 women in the Framingham Heart Study who underwent B-mode ultrasound measurements of the carotid artery. Their mean age was 75 years, and each had attended most of the biennial clinic examinations over the 34 years before the carotid ultrasound study. We used time-integrated measurements to assess the associations between various cardiovascular risk factors and the degree of carotid stenosis. RESULTS Moderate carotid stenosis (> or =25 percent) was present in 189 men and 226 women. We assessed the odds ratios for this degree of stenosis as compared with minimal stenosis (<25 percent) according to increases in risk factors. In the men, the odds ratio for moderate carotid stenosis associated with an increase of 20 mm Hg in systolic blood pressure was 2.11 (95 percent confidence interval, 1.51 to 2.97). The odds ratio for an increase of 10 mg per deciliter (0.26 mmol per liter) in the cholesterol level was 1.10 (95 percent confidence interval, 1.03 to 1.16), and for an increase of five pack-years of smoking it was 1.08 (95 percent confidence interval, 1.03 to 1.13). The results were similar in the women. Time-integrated measurements of diastolic blood pressure showed significant associations with carotid stenosis in men and insignificant associations in women. CONCLUSIONS Over the long term, high systolic blood pressure, high cholesterol levels, and smoking were associated with an increased risk of carotid stenosis in this elderly population.

Levels of lipoprotein Lp(a) decline with neomycin and niacin treatment

Total and low density lipoprotein cholesterol concentration reduction in patients with markedly increased levels of these substances, leads to a decline in the incidence of myocardial infarction and death. A unique cholesterol-rich lipoprotein, lipoprotein Lp(a), has been identified which not only can be confused with low density lipoproteins, but has also been associated with premature cardiovascular disease. Using the cholesterol-lowering drugs neomycin and niacin in 14 type II hyperlipoproteinemic subjects, we determined the effect of lipid-lowering therapy on lipoprotein Lp(a) concentrations. Neomycin (2g/day) reduced low density lipoprotein cholesterol and lipoprotein Lp(a) concentrations by 23% and 24%, respectively. Combination therapy with neomycin (2 g/day) and niacin (3 g/day) induced a 48% decline in low density lipoprotein cholesterol levels and a 45% reduction in the concentration of lipoprotein Lp(a). These changes in lipoprotein Lp(a) levels were associated with a striking decline in the intensity of the slow pre-beta-lipoprotein fraction determined Lp(a) by lipoprotein electrophoresis. This slow pre-beta-lipoprotein fraction contained Lp(a) determined by immunofixation. These observations indicate that lipoprotein Lp(a) concentrations can be altered pharmacologically and that the progression of cardiovascular disease may be altered through changes in lipoprotein (a) levels.

Cardiovascular features of homozygous familial hypercholesterolemia: Analysis of 16 patients

Familial hypercholesterolemia (FH) is characterized by an autosomal codominant inheritance, an abnormality in low-density lipoprotein (LDL) receptor function, elevated plasma cholesterol levels and premature atherosclerosis. Sixteen patients with homozygous FH were studied to correlate the extent of their atherosclerotic disease with their lipid levels and receptor function. The age range at initial presentation was 3 to 38 years (mean 12), and at the last examination, 6 to 43 years (mean 20). The mean pretreatment total plasma cholesterol concentration for all patients was 729 +/- 58 mg/dl (+/- standard error of the mean), and the mean LDL cholesterol level was 672 +/- 58 mg/dl (normal 60 to 176). High-density lipoprotein cholesterol was 28 +/- 3 mg/dl (normal 30 to 74). In the 7 patients with FH who had symptoms of myocardial ischemia (Group I), the mean pretreatment LDL cholesterol value (817 +/- 62 mg/dl) was higher than that of the 9 asymptomatic patients (Group II) (560 +/- 74 mg/dl). In Group I, 5 of 7 patients had left or right coronary ostial narrowing and 3 had significant left ventricular outflow obstruction. Most coronary arterial narrowing occurred in the right coronary and left anterior descending arteries and the least amount in the left circumflex coronary artery. A femoral bruit was the physical finding that correlated best with the Group I population; brother:sister pairs revealed a milder clinical course for the female. Seven of the 16 patients have survived into their third decade without symptoms. Comparison of these persons with those in whom angina developed reveals a marked heterogeneity in their clinical course, which appears to be associated with receptor negative/defective status.

Quantitation of plasma apolipoproteins in the primary and secondary prevention of coronary artery disease.

Lipids are transported in the circulation by lipoproteins, which consist of lipids (cholesterol, triglycerides, and phospholipids) and proteins (called apolipoproteins). Apolipoproteins have many physiologic functions in lipoprotein metabolism, acting as structural proteins for lipoprotein particles, cofactors for enzymes, and ligands for cell-surface receptors. Table 1 summarizes the major apolipoproteins and their known functions. Table 1. Major Apolipoproteins and Their Functions* Traditionally, lipoproteins have been separated on the basis of their hydrated densities; the major density classes of lipoprotein particles include chylomicrons, very low-density lipoproteins (VLDL), intermediate-density lipoproteins, low-density lipoproteins (LDL), and high-density lipoproteins (HDL) [1]. Figure 1 depicts the metabolism of these lipoproteins. Chylomicrons are intestinal lipoproteins that transport dietary lipids to peripheral tissues and the liver. They are triglyceride rich and contain one form of apolipoprotein B (apo B), apo B-48. The triglycerides in chylomicrons are hydrolyzed by the endothelial enzyme lipoprotein lipase, which requires apolipoprotein C-II (apo C-II) as a cofactor [2]. The resulting chylomicron remnants are removed from the circulation by the liver through a process that involves the binding of apolipoprotein E (apo E) on the chylomicron remnants to a putative hepatic remnant receptor (or apo E receptor) [3]. Very low-density lipoproteins are triglyceride-rich lipoproteins secreted by the liver and contain another form of apo B, apo B-100. These triglycerides are also hydrolyzed by lipoprotein lipase, with conversion to the more dense VLDL remnants, or intermediate-density lipoprotein. Some VLDL remnants are removed from the circulation by the liver through an apo E-mediated process, but others are further hydrolyzed by the endothelial enzyme hepatic lipase, ultimately resulting in conversion to LDL. Low-density lipoprotein transports cholesterol ester to various peripheral tissues, but a substantial amount of LDL is eventually removed from the circulation by the liver when apo B-100 is bound to the hepatic LDL receptor [4]. Low-density lipoprotein can undergo oxidative modification, producing a form of oxidized LDL that can cause cholesterol loading in cells [5]. Figure 1. Schematic diagram of lipoprotein metabolism. High-density lipoproteins are synthesized and secreted by the intestine and the liver and also are generated by hydrolysis of triglyceride-rich lipoproteins [6, 7]. The major apolipoproteins in HDL are apolipoprotein A-I (apo A-I) and apolipoprotein A-II (apo A-II) (Table 1). High-density lipoprotein stimulates the efflux from cells of unesterified cholesterol, which is then converted to the esterified form by lecithin-cholesterol acyltransferase Figure 1, a plasma enzyme activated primarily by apo A-I [8]. As small, dense HDL3 accumulates cholesteryl ester, it is transformed into larger, less dense HDL2. High-density lipoprotein cholesteryl ester can be transferred to apo B-containing lipoproteins by the cholesteryl ester transfer protein [9]. This may be one important route of human reverse cholesterol transport [10]. High-density lipoprotein is a substrate for hepatic lipase, which hydrolyzes HDL phospholipids and triglycerides, creating smaller HDL particles [7] (Figure 1). Plasma Lipoproteins and Coronary Artery Disease The risk for premature atherosclerotic coronary artery disease is directly correlated with plasma concentrations of LDL cholesterol [11, 12] and inversely correlated with levels of HDL cholesterol (reviewed in reference 13). The independent association of plasma triglycerides with coronary artery disease risk is less certain [14, 15]. Interventions designed to decrease plasma LDL concentrations are effective in the primary prevention of coronary artery disease [16-18]. Lowering plasma LDL is also very effective in secondary prevention of coronary artery disease (reviewed in [19]), decreasing overall mortality rate [20], decreasing cardiovascular events [21-25], and producing an objective regression of atherosclerotic disease [22-26]. Although one primary prevention trial suggested an independent benefit of increasing plasma HDL cholesterol concentrations [18], no clinical trials have been done specifically to evaluate the effect of selectively increasing HDL in primary or secondary prevention of coronary artery disease. Despite the association of plasma lipid levels with coronary artery disease risk, many patients with premature coronary artery disease do not have very high levels of LDL cholesterol or very depressed HDL cholesterol concentrations. Therefore, investigators continue to search for other clinical markers that will allow better prediction of coronary artery disease risk and can be used to guide therapeutic decisions to prevent or treat coronary artery disease. Quantitation of plasma apolipoproteins was proposed as one such clinical tool. In this review, we assess evidence regarding the clinical utility of apolipoprotein quantitation and review the use of plasma apolipoprotein concentrations in the primary and secondary prevention of coronary artery disease. We focus primarily on the apolipoproteins for which the most data and the most clinical evidence exist that are relevant to coronary artery disease: apo A-I, apo B, and lipoprotein(a) (Lp[a]). For each of these apolipoproteins, we address the question of whether quantitation of the apolipoprotein enhances the ability to predict coronary artery disease risk in healthy persons or recurrent events in patients with established coronary artery disease, and we suggest how knowledge of the plasma apolipoprotein concentration might influence clinical management. We retrieved 82 articles from the English-language literature for the years 1975 to 1993 using MEDLINE (key words: apolipoproteins, quantitation, and coronary artery disease) and review of article bibliographies. We examined all retrospective and prospective studies of apolipoprotein quantitation that used some measure of coronary artery disease as a criterion for patient selection, including acute myocardial infarction, classic angina pectoris, and angiographic evidence of severe coronary artery disease [22, 23]. Many of the studies were designed to address the predictive value of the test for the development of coronary artery disease; relatively few studies assessed the predictive value of the test for recurrent events in patients with established coronary artery disease. We found 71 retrospective cross-sectional studies, including 7 in children and adolescents, and 11 prospective studies. For each apolipoprotein, we discuss the retrospective studies as a group and specific studies where appropriate; each of the studies in children and each prospective study are discussed individually. More than 90% of studies were done in men, and therefore we cannot generalize results to women. In addition, because assays for apolipoprotein quantitation have not been standardized, we included a section addressing some of the methodologic issues in apolipoprotein quantitation. Issues regarding Assay Methods and Standardization The lack of standardization and reference methods for apolipoprotein assays is a limitation to the general application of apolipoprotein quantitation in clinical practice [27-31]. Variation in apolipoprotein measurements among laboratories can be substantial. A collaborative study initiated by the International Federation of Clinical Chemistry evaluated differences in apo A-I and apo B quantitation among 28 laboratories, 25 of which were company laboratories [29]. The overall interlaboratory coefficient of variation was 7% for apo A-I and 19% for apo B. After uniform calibration of assay standards, the coefficients of variation decreased to 5% and 6%, respectively. Among the factors resulting in this variation are preanalytical factors such as differences in sampling and storage conditions [32]. In addition, matrix effects (additives, stabilization processes) on the immunoreactivity of standards also can be a source of bias in apolipoprotein quantitation [33]. Assays for apo A-I and apo B are widely available and are frequently done by commercial laboratories. These laboratories use methods that often result in good intralaboratory reproducibility, with coefficients of variation within laboratories that are generally less than 4% [29]. However, some of these assays may be subject to interference by high plasma triglyceride levels [27]. Therefore, apo A-I and apo B measurements in patients with very high hypertriglyceride levels (>4.5 mmol/L [400 mg/dL]) should be interpreted with caution. Several commercially available Lp(a) assay kits are used by research laboratories for Lp(a) quantitation. However, an Lp(a) assay has yet to be approved by the Food and Drug Administration for clinical use. As in the case of apo A-I and apo B, there has been no standardization of Lp(a) assays [34, 35]. Lipoprotein(a) is an acute-phase reactant [36] and should not be quantitated within several weeks after an acute illness or surgical procedure. Apolipoprotein A-I Background Apolipoprotein A-I is the major apolipoprotein in HDL and serves various structural and functional roles in HDL metabolism (reviewed in reference 6). It is probably important in protecting against premature atherosclerosis. Genetic defects that cause the inability to synthesize apo A-I cause very low plasma concentrations of HDL cholesterol and premature coronary artery disease in the fourth and fifth decades [37-40]. Conversely, an increased rate of apo A-I production causes high plasma levels of HDL cholesterol and may be associated with protection from premature coronary artery disease based on familial longevity [41]. Furthermore, overexpression of human apo A-I in transgenic mice inhibits the development of atherosclerosis [42]. Cross-Sectional Studies Many retrospective

The low density lipoprotein receptor is not required for normal catabolism of Lp(a) in humans.

Lipoprotein(a) [Lp(a)] is an atherogenic lipoprotein which is similar in structure to low density lipoproteins (LDL). The role of the LDL receptor in the catabolism of Lp(a) has been controversial. We therefore investigated the in vivo catabolism of Lp(a) and LDL in five unrelated patients with homozygous familial hypercholesterolemia (FH) who have little or no LDL receptor activity. Purified 125I-Lp(a) and 131I-LDL were simultaneously injected into the homozygous FH patients, their heterozygous FH parents when available, and control subjects. The disappearance of plasma radioactivity was followed over time. As expected, the fractional catabolic rates (FCR) of 131I-LDL were markedly decreased in the homozygous FH patients (mean LDL FCR 0.190 d-1) and somewhat decreased in the heterozygous FH parents (mean LDL FCR 0.294 d-1) compared with controls (mean LDL FCR 0.401 d-1). In contrast, the catabolism of 125I-Lp(a) was not significantly different in the homozygous FH patients (mean FCR 0.251 d-1), heterozygous FH parents (mean FCR 0.254 d-1), and control subjects (mean FCR 0.287 d-1). In summary, absence of a functional LDL receptor does not result in delayed catabolism of Lp(a), indicating that the LDL receptor is not a physiologically important route of Lp(a) catabolism in humans.

Detection and quantitation of calcific atherosclerosis by ultrafast computed tomography in children and young adults with homozygous familial hypercholesterolemia.

Ultrafast computed tomography (CT) is a new method for detecting calcific lesions in the coronary arteries. The ability of CT to detect and quantify coronary artery atherosclerosis in children and young adults at risk for malignant atherogenesis was evaluated. A total of 11 consecutive familial hypercholesterolemic (FH) homozygotes (3 to 37 years old) participated. Untreated total cholesterol concentrations were 488 to 1277 mg/dL (12.7 to 33.2 mmol/L). Angiography detected significant lesions in 7 of 11 patients. CT detected calcific atherosclerosis in all 9 of the patients older than 12 years of age, including all those with angina. CT was more sensitive in detecting aortic root and coronary ostial lesions, where atherosclerosis first appears in homozygous FH. The volume of calcification (in cubic millimeters) correlated with the severity and duration of the hypercholesterolemia (r = .62, P < .05) as well as with the presence of angina (P < .05). All patients with angina (7 of 7) had > 150 mm3 of calcified volume, whereas only 1 of 4 asymptomatic patients had a volume score > 150 mm3. We conclude that (1) coronary and aortic calcium phosphate deposits are common in young FH homozygotes; (2) these deposits are associated with the presence of angiographic stenoses, as has been seen in adults with coronary atherosclerosis; and (3) aortic calcific deposits are more common than calcific coronary lesions.

Lecithin-cholesterol acyltransferase: role in lipoprotein metabolism, reverse cholesterol transport and atherosclerosis.

In the past several years significant advances have been made in our understanding of lecithin-cholesterol acyltransferase (LCAT) function. LCAT beneficially alters the plasma concentrations of apolipoprotein B-containing lipoproteins, as well as HDL. In addition, its proposed role in facilitating reverse cholesterol transport and modulating atherosclerosis has been demonstrated in vivo. Analysis of LCAT transgenic animals has established the importance of evaluating HDL function, as well as HDL plasma levels, to predict atherogenic risk.

Characterization of a human hepatic receptor for high density lipoproteins.

Characterization of the membrane receptor for the low density lipoproteins (LDL) has led to insights Into cellular receptor physiology as well as mammalian llpid transport. Results with LDL have stimulated the search for specific receptors for other plasma lipoproteins. Receptors for high density lipoproteins (HDL) have been Identified In human flbroblasts and smooth muscle cells. Specificity for this receptor has been difficult to define since normal HDL contains several apollpoprotelns, and particles containing apollpoprotelns B and E have been shown to compete for HDL binding. In the present study, we demonstrate that HDL Isolated from a patient devoid of apollpoproteln E was bound specifically by human hepatic membranes. This binding reached saturation within 2 hours and was EDTA-resistant. Assuming a single receptor model, we found that 2.9 × 1O15 receptors/mg membrane protein bound with an affinity KD = 3.5 × 10−7 M at 0 to 4°C and KD = 1.9 × 10−7 M at37°C. The binding was effectively competed with Intact HDL3, with HDL3, that had undergone selective arginlne and lyslne residue modification, and with antibodies to apollpoproteins A-l and A-ll. However, LDL, asialofetuln, and HDL3 which had undergone tyrosine modification by nitration, and antl-apolipoprotein B did not compete with apo A-l HDL binding. In contrast to LDL binding, the human hepatoma cell line, HEPG2, increased HDL binding with cholesterol loading that was specific for HDL3. Thus, hepatic tissue can modulate Its recognition of HDL. Finally, hepatic membranes from a patient lacking normal hepatic LDL receptors bound apo A-l HDL normally. These data indicate that a saturable, specific regulatable receptor for apo E-free HDL Is present In human liver.