Joseph H. Rapp

Triglyceride-rich lipoproteins isolated by selected-affinity anti-apolipoprotein B immunosorption from human atherosclerotic plaque.

We isolated and characterized immunoreactive apolipoprotein B (apoB)-containing lipoproteins from human atherosclerotic plaque and plasma to determine whether very-low-density lipoprotein (VLDL) can enter and become incorporated into the atherosclerotic lesion and how plaque apoB-containing lipoproteins differ from apoB-containing lipoproteins isolated from plasma. Atherosclerotic plaques were obtained during aortic surgery and processed immediately. Lipoproteins were extracted from minced plaque in a buffered saline solution (extract A). In selected cases a second extraction was done after plaque was incubated with collagenase (extract B). Lipoproteins were then isolated from the extracts by anti-apoB immunosorption and separated into VLDL + intermediate-density lipoprotein (IDL) (d < 1.019 g/mL) and low-density lipoprotein (LDL) (1.019 < d < 1.070 g/mL) fractions by ultracentrifugation. The VLDL + IDL fractions from plaque contained more than one third of the total apoB-associated lipoprotein cholesterol in both extracts A and B. The lipid composition of VLDL + IDL in both extracts was related to that of plasma VLDL + IDL. By electron microscopy mean particle diameters of VLDL + IDL from extracts A and B were 9% and 23%, respectively, greater than VLDL + IDL diameters from plasma. Mean diameters of LDL from extracts A and B were 11% and 31% greater than LDL diameters from plasma. The apoE-apoB ratio of extract A VLDL + IDL was nearly twice that of plasma VLDL + IDL and severalfold higher than that of extract A LDL. Immunoblots of both VLDL + IDL and LDL from extract A demonstrated minimal fragmentation of apoB.(ABSTRACT TRUNCATED AT 250 WORDS)

Human very low density lipoproteins and chylomicrons can protect against endotoxin-induced death in mice.

Endotoxemia stimulates many physiologic responses including disturbances in lipid metabolism. We hypothesized that this lipemia may be part of a defensive mechanism by which the body combats the toxic effects of circulating endotoxin. We tested the effects of mixtures of endotoxin, lipoproteins, and lipoprotein-free plasma and determined the ability of varying concentrations of human very low density lipoproteins (VLDL) and chylomicrons, as well as low density lipoproteins (LDL) and high density lipoproteins (HDL), and of the synthetic lipid emulsion SOYACAL to prevent endotoxin-induced death in mice. This study demonstrates that the triglyceride-rich VLDL and chylomicrons, as well as cholesterol-rich LDL and HDL, and cholesterol-free SOYACAL can protect against endotoxin-induced death. Protection required small amounts of lipoprotein-free plasma, and depended on the incubation time and the concentration of lipoprotein lipid. Despite stringent techniques to prevent exogenous endotoxin contamination eight of ten duplicate VLDL preparations contained endotoxin (5,755 +/- 3,514 ng endotoxin/mg triglyceride, mean +/- SEM) making the isolation of endotoxin-free VLDL difficult. In contrast, simultaneous preparations of LDL and HDL were relatively free of endotoxin contamination (3 +/- 3 and 320 +/- 319 ng/mg total cholesterol, respectively), suggesting that the contamination of VLDL occurs in vivo and not during the isolation procedure. These observations suggest a possible role for increased triglyceride-rich lipoproteins in the hosts defense against endotoxemia and infection.

Rupture rate of large abdominal aortic aneurysms in patients refusing or unfit for elective repair

CONTEXT Among patients with abdominal aortic aneurysm (AAA) who have high operative risk, repair is usually deferred until the AAA reaches a diameter at which rupture risk is thought to outweigh operative risk, but few data exist on rupture risk of large AAA. OBJECTIVE To determine the incidence of rupture in patients with large AAA. DESIGN AND SETTING Prospective cohort study in 47 Veterans Affairs medical centers. PATIENTS Veterans (n = 198) with AAA of at least 5.5 cm for whom elective AAA repair was not planned because of medical contraindication or patient refusal. Patients were enrolled between April 1995 and April 2000 and followed up through July 2000 (mean, 1.52 years). MAIN OUTCOME MEASURE Incidence of AAA rupture by strata of initial and attained diameter. RESULTS Outcome ascertainment was complete for all patients. There were 112 deaths (57%) and the autopsy rate was 46%. Forty-five patients had probable AAA rupture. The 1-year incidence of probable rupture by initial AAA diameter was 9.4% for AAA of 5.5 to 5.9 cm, 10.2% for AAA of 6.0 to 6.9 cm (19.1% for the subgroup of 6.5-6.9 cm), and 32.5% for AAA of 7.0 cm or more. Much of the increased risk of rupture associated with initial AAA diameters of 6.5-7.9 cm was related to the likelihood that the AAA diameter would reach 8.0 cm during follow-up, after which 25.7% ruptured within 6 months. CONCLUSION The rupture rate is substantial in high-operative-risk patients with AAA of at least 5.5 cm in diameter and increases with larger diameter.

High-Resolution CT Imaging of Carotid Artery Atherosclerotic Plaques

BACKGROUND AND PURPOSE: Plaque morphologic features have been suggested as a complement to luminal narrowing measurements for assessing the risk of stroke associated with carotid atherosclerotic disease, giving rise to the concept of “vulnerable plaque.” The purpose of this study was to evaluate the ability of multidetector-row CT angiography (CTA) to assess the composition and characteristics of carotid artery atherosclerotic plaques with use of histologic examination as the gold standard. MATERIALS AND METHODS: Eight patients with transient ischemic attacks who underwent carotid CTA and “en bloc” endarterectomy were enrolled in a prospective study. An ex vivo micro-CT study of each endarterectomy specimen was obtained, followed by histologic examination. A systematic comparison of CTA images with histologic sections and micro-CT images was performed to determine the CT attenuation associated with each component of the atherosclerotic plaques. A computer algorithm was subsequently developed that automatically identifies the components of the carotid atherosclerotic plaques, based on the density of each pixel. A neuroradiologists reading of this computer analysis was compared with the interpretation of the histologic slides by a pathologist with respect to the types and characteristics of the carotid plaques. RESULTS: There was a 72.6% agreement between CTA and histologic examination in carotid plaque characterization. CTA showed perfect concordance for calcifications. A significant overlap between densities associated with lipid-rich necrotic core, connective tissue, and hemorrhage limited the reliability of individual pixel readings to identify these components. However, CTA showed good correlation with histologic examination for large lipid cores (κ = 0.796; P < .001) and large hemorrhages (κ = 0.712; P = .102). CTA performed well in detecting ulcerations (κ = 0.855) and in measuring the fibrous cap thickness (R2 = 0.77; P < .001). CONCLUSION: The composition of carotid atherosclerotic plaques determined by CTA reflects plaque composition defined by histologic examination.

Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins.

Aggregation and retention of LDL in the arterial wall are key events in atherogenesis, but the mechanisms in vivo are not known. Previous work from our laboratories has shown that exposure of LDL to bacterial sphingomyelinase (SMase) in vitro leads to the formation of LDL aggregates that can be retained by extracellular matrix and that are able to stimulate macrophage foam cell formation. We now provide evidence that retained LDL is hydrolyzed by an arterial-wall SMase activity. First, we demonstrated that SMase-induced aggregation is caused by an increase in particle ceramide content, even in the presence of excess sphingomyelin (SM). This finding is compatible with previous data showing that lesional LDL is enriched in SM, though its ceramide content has not previously been reported. To address this critical compositional issue, the ceramide content of lesional LDL was assayed and, remarkably, found to be 10-50-fold enriched compared with plasma LDL ceramide. Furthermore, the ceramide was found exclusively in lesional LDL that was aggregated; unaggregated lesional LDL, which accounted for 20-25% of the lesional material, remained ceramide poor. When [3H]SM-LDL was incubated with strips of rabbit aorta ex vivo, a portion of the LDL was retained, and the [3H]SM of this portion, but not that of unretained LDL, was hydrolyzed to [3H]ceramide by a nonlysosomal arterial hydrolase. In summary, LDL retained in atherosclerotic lesions is acted upon by an arterial-wall SMase, which may participate in LDL aggregation and possibly other SMase-mediated processes during atherogenesis.

Chylomicrons alter the fate of endotoxin, decreasing tumor necrosis factor release and preventing death.

The hypertriglyceridemia of infection was traditionally thought to represent the mobilization of substrate to fuel the bodys response to the infectious challenge. However, we have previously shown that triglyceride-rich lipoproteins can protect against endotoxin-induced lethality. The current studies examine the mechanism by which this protection occurs. Rats infused with a lethal dose of endotoxin preincubated with chylomicrons had a reduced mortality compared with rats infused with endotoxin alone (15 vs. 76%, P < 0.001). Preincubation with chylomicrons increased the rate of clearance of endotoxin from plasma and doubled the amount of endotoxin cleared by the liver (30 +/- 1 vs. 14 +/- 2% of the total infused radiolabel, P < 0.001). In addition, autoradiographic studies showed that chylomicrons directed more of the endotoxin to hepatocytes and away from hepatic macrophages. Rats infused with endotoxin plus chylomicrons also showed reduced peak serum levels of tumor necrosis factor as compared with controls (14.2 +/- 3.3 vs. 44.9 +/- 9.5 ng/ml, mean +/- SEM, P = 0.014). In separate experiments, chylomicrons (1,000 mg triglyceride/kg) or saline were infused 10 min before the infusion of endotoxin. Chylomicron pretreatment resulted in a reduced mortality compared with rats infused with endotoxin alone (22 vs. 78%, P < 0.005). Therefore, chylomicrons can protect against endotoxin-induced lethality with and without preincubation with endotoxin. The mechanism by which chylomicrons protect against endotoxin appears to involve the shunting of endotoxin to hepatocytes and away from macrophages, thereby decreasing macrophage activation and the secretion of cytokines.

Relationship of age, gender, race, and body size to infrarenal aortic diameter

PURPOSE To assess the effects of age, gender, race, and body size on infrarenal aortic diameter (IAD) and to determine expected values for IAD on the basis of these factors. METHODS Veterans aged 50 to 79 years at 15 Department of Veterans Affairs medical centers were invited to undergo ultrasound measurement of IAD and complete a pre-screening questionnaire. We report here on 69,905 subjects who had no previous history of abdominal aortic aneurysm (AAA) and no ultrasound evidence of AAA (defined as IAD > or = 3.0 cm). RESULTS Although age, gender, black race, height, weight, body mass index, and body surface area were associated with IAD by multivariate linear regression (all p < 0.001), the effects were small. Female sex was associated with a 0.14 cm reduction in IAD and black race with a 0.01 cm increase in IAD. A 0.1 cm change in IAD was associated with large changes in the independent variables: 29 years in age, 19 cm or 40 cm in height, 35 kg in weight, 11 kg/m2 in body mass index, and 0.35 m2 in body surface area. Nearly all height-weight groups were within 0.1 cm of the gender means, and the unadjusted gender means differed by only 0.23 cm. The variation among medical centers had more influence on IAD than did the combination of age, gender, race, and body size. CONCLUSIONS Age, gender, race, and body size have statistically significant but small effects on IAD. Use of these parameters to define AAA may not offer sufficient advantage over simpler definitions (such as an IAD > or = 3.0 cm) to be warranted.

Assessment of carotid artery stenosis by ultrasonography, conventional angiography, and magnetic resonance angiography: Correlation with ex vivo measurement of plaque stenosis

PURPOSE Several studies have investigated the correlation between Doppler ultrasonography (DUS), angiography (CA), and magnetic resonance angiography (MRA) in the evaluation of stenosis of the carotid bifurcation. However, these studies suffer from the lack of a true control-the lesion itself-and therefore conclusions about the diagnostic accuracy of each method remain relative. To determine the absolute accuracy of these modalities, we have prospectively studied lesion size with DUS, MRA, and CA in 28 patients undergoing 31 elective carotid endarterectomies and compared the percent of carotid stenosis determined by each technique to the carotid atheroma resected en bloc. METHODS All patients were evaluated by each modality within 1 month before the thromboendarterectomy. With DUS, stenosis size was determined by standard flow criteria. For angiography and MRA, stenosis was defined as residual lumenal diameter/estimated normal arterial diameter (European Carotid Surgery Trial criteria). At surgery the carotid atheroma was removed en bloc in all patients. Patients in whom the lesion could not be removed successfully without damage were excluded from the study. Stenosis of the atheroma was determined ex vivo with high-resolution (0.03 mm3) magnetic resonance and confirmed by acrylic injection of the specimen under pressure and measurement of the atheroma wall and lumen. RESULTS The measurements of the ex vivo stenosis by high-resolution magnetic resonance imaging correlated closely with the size of stenosis determined by the acrylic specimen casts (r = 0.92). By ex vivo measurement, the lesions were placed in the following size categories: 40% to 59% stenosis (n = 2), 60% to 79% stenosis (n = 6), 80% to 89% stenosis (n = 7), and 90% to 99% stenosis (n = 16). CONCLUSIONS In general, the correlation of measurements of ex vivo stenosis with all modalities was good in these severely diseased arteries, although it was better for DUS (r = 0.80; p < 0.001) and MRA (r = 0.76; p < 0.001) than for CA (r = 0.56; p < 0.05).

Connective tissue proteinases and inhibitors in abdominal aortic aneurysms. Involvement of the vasa vasorum in the pathogenesis of aortic aneurysms.

Recent studies have shown that increases in proteolytic activity are associated with abdominal aortic aneurysms (AAAs). We have studied samples of the dilated aortic wall, taken during corrective surgery for AAAs, in terms of the number, type, and tissue location of connective tissue proteinases and their inhibitors. Five distinct caseinolytic serine proteinases and six gelatinolytic metalloproteinases were resolved by molecular weight by use of sodium dodecyl sulfate-substrate gel electrophoresis. Isoforms of the Mr 92,000 neutrophil gelatinase were identified by immunoprecipitation of biosynthetically labeled organ culture media. About 50% of the total radiolabeled protein secreted by AAA organ cultures was identified as the Mr 30,000 glycoprotein, tissue inhibitor of metalloproteinase (TIMP), by immunoprecipitation. Both TIMP and gelatinase were localized to the vasa vasorum by immunoperoxidase staining. However, interstitial collagenase could not be detected by any method. These results suggest the involvement of the vasa vasorum in the maintenance and possibly the genesis of AAAs.

The “Natural History” of Segmental Wall Motion Abnormalities in Patients Undergoing Noncardiac Surgery

Intraoperative segmental wall motion abnormalities (SWMA) detected by transesophageal echocardiography (TEE) are sensitive, but not always specific, markers of myocardial ischemia. To determine their incidence, characteristics, and relation to postoperative cardiac morbidity, we continuously recorded the left ventricular short-axis view and 12-lead ECG in 156 high-risk patients undergoing non-cardiac surgery. Monitoring was clinically blinded. Wall motion was scored at predefined clinical, hemodynamic, and ECG events and at periodic intervals (26 +/- 11 samples per patient). We detected 44 episodes of new or worsened SWMA in 32 patients (20%). The severity of most episodes was limited to severe hypokinesis (24/44, 55%) followed by akinesis (16/44, 36%) and dyskinesis (4/44, 9%). The remaining 124 patients had normal wall motion or only mild hypokinesis (56/156, 36%) or chronic SWMA (68/156, 44%). The incidence of new SWMA did not differ for patients with known coronary artery disease (CAD) and those with cardiac risk factors only (22% vs. 19%, P = not significant), although CAD patients had a significantly greater incidence of chronic SWMA (62% vs. 41%, P = 0.02). The incidence of new or worsened SWMA was significantly greater during aortic vascular surgery (38% vs. 17%, P = 0.05). Approximately 40% of all new TEE changes occurred in the absence of either an apparent clinical event or a significant change in systolic blood pressure or heart rate. Ten patients had new or worsened SWMA persisting until the end of surgery, 8 with new akinesis, only 1 developing myocardial infarction. The distribution of new or worsened SWMA and significant intraoperative ST-T changes (n = 19) in this cohort was discordant: temporal overlap between modalities was present in only 5 patients. Major cardiac complications occurred in 5 patients (3.2%), all of whom underwent peripheral vascularization. All patients with cardiac complications and new or worsened SWMA also had intraoperative or early postoperative ST-T changes. We conclude that: 1) continuous TEE recording with offline analysis in this high-risk group of patients revealed a relatively low incidence of new or worsened SWMA (20%), most episodes of which were characterized by severe hypokinesis (55%); 2) episodes were more common in patients undergoing aortic vascular surgery; 3) approximately 40% of episodes were unaccompanied by clinical events or significant hemodynamic changes; 4) episodes were poorly correlated with postoperative cardiac complications; and 5) the discordant relation between TEE and ECG changes observed here necessitates careful monitoring of the ECG when TEE is used clinically.