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BACKGROUND: Apolipoprotein E ε4 allele (APOE-ε4) is the main genetic risk factor for late-onset Alzheimers disease (AD) and may impact cognitive function also via other neuropathological lesions. However, there is limited evidence available from diverse populations, as APOE associations with dementia seem to differ by race. Therefore, we aimed to evaluate the pathways linking APOE-ε4 to cognitive abilities through AD and non-AD neuropathology in an autopsy study with an admixed sample. METHODS: Neuropathological lesions were evaluated following international criteria using immunohistochemistry. Participants were classified into APOE-ε4 carriers (at least one ε4 allele) and non-carriers. Cognitive abilities were evaluated by the Clinical Dementia Rating Scale sum of boxes. Mediation analyses were conducted to assess the indirect association of APOE-ε4 with cognition through AD-pathology, lacunar infarcts, hyaline arteriosclerosis, cerebral amyloid angiopathy (CAA), Lewy body disease (LBD), and TAR DNA-binding protein 43 (TDP-43). RESULTS: We included 648 participants (mean age 75 ± 12 years old, mean education 4.4 ± 3.7 years, 52% women, 69% White, and 28% APOE-ε4 carriers). The association between APOE-ε4 and cognitive abilities was mediated by neurofibrillary tangles (β = 0.88, 95% CI = 0.45; 1.38, p

Intracerebral hemorrhage (ICH) is a significant cause of morbidity and mortality worldwide. Hypertension and cerebral amyloid angiopathy (CAA) are the most common causes of primary ICH, but the mechanism of hemorrhage in both conditions is unclear. Although fibrinoid necrosis and Charcot-Bouchard aneurysms (CBAs) have been postulated to underlie vessel rupture in ICH, the role and significance of CBAs in ICH has been controversial. First described as the source of bleeding in hypertensive hemorrhage, they are also one of the CAA-associated microangiopathies along with fibrinoid necrosis, fibrosis and "lumen within a lumen appearance." We describe clinicopathologic findings of CBAs found in 12 patients out of over 2700 routine autopsies at a tertiary academic medical center. CBAs were rare and predominantly seen in elderly individuals, many of whom had multiple systemic and cerebrovascular comorbidities including hypertension, myocardial and cerebral infarcts, and CAA. Only one of the 12 subjects with CBAs had a large ICH, and the etiology underlying the hemorrhage was likely multifactorial. Two CBAs in the basal ganglia demonstrated associated microhemorrhages, while three demonstrated infarcts in the vicinity. CBAs may not be a significant cause of ICH but are a manifestation of severe cerebral small vessel disease including both hypertensive arteriopathy and CAA.

Cerebral microbleeds (CMB) are a common MRI finding, representing underlying cerebral microhemorrhages (CMH). The etiology of CMB and microhemorrhages is obscure. We conducted a pathological investigation of CMH, combining standard and immunohistological analyses of postmortem human brains. We analyzed 5 brain regions (middle frontal gyrus, occipital pole, rostral cingulate cortex, caudal cingulate cortex, and basal ganglia) of 76 brain bank subjects (mean age ± SE 90 ± 1.4 years). Prussian blue positivity, used as an index of CMH, was subjected to quantitative analysis for all 5 brain regions. Brains from the top and bottom quartiles (n = 19 each) were compared for quantitative immunohistological findings of smooth muscle actin, claudin-5, and fibrinogen, and for Sclerosis Index (SI) (a measure of arteriolar remodeling). Brains in the top quartile (i.e. with most extensive CMH) had significantly higher SI in the 5 brain regions combined (0.379 ± 0.007 vs 0.355 ± 0.008; p

PurposeSubclinical coronary artery calcification is an established predictor of cardiovascular events. While a history of kidney stones has been linked to subclinical carotid atherosclerosis, to our knowledge no study has examined its relationship with coronary artery calcification. We studied the association between kidney stone history and prevalent coronary artery calcification in MESA (Multi-Ethnic Study of Atherosclerosis).Materials and methodsMESA is a multisite cohort study of participants 45 to 84 years old without known cardiovascular disease at baseline from 2000 to 2002. Computerized tomography was done in 3,282 participants at followup in 2010 to 2012 to determine coronary artery calcification and kidney stone history was assessed by self-report. Coronary artery calcification scores were categorized as none-0, mild-1 to 99, moderate-100 to 399 or severe-400 or greater. Cross-sectional analysis was performed adjusting for demographic and dietary factors related to kidney stones.ResultsThe prevalence of kidney stone disease history was approximately 9%, mean ± SD participant age was 69.5 ± 9.3 years, 39% of participants were Caucasian, 47% were men and 69% had detectable coronary artery calcification (score greater than 0). No difference in the score was seen between single stone formers and nonstone formers. Recurrent kidney stone formation was associated with moderate or severe calcification on multivariable logistic regression vs none or mild calcification (OR 1.80, 95% CI 1.22-2.67). When coronary artery calcification scores were separated into none, mild, moderate and severe calcification, recurrent stone formation was associated with a higher score category on multivariable ordinal logistic regression (OR 1.44 per category, 95% CI 1.04-2.01).ConclusionsRecurrent kidney stone formation is associated with subclinical coronary atherosclerosis. This association appeared stronger with coronary artery calcification severity than with coronary artery calcification presence.

Whether inclusion of the coronary artery calcium score improves cardiovascular risk prediction in individuals with CKD, a population with unique calcium-phosphate homeostasis, is unknown. Among 6553 participants ages 45-84 years without prior cardiovascular disease in the Multi-Ethnic Study of Atherosclerosis, coronary artery calcium score was assessed for cardiovascular risk prediction beyond the Framingham predictors in those with (n=1284) and without CKD and contrasted with carotid intima-media thickness and ankle-brachial index (two other measures of subclinical atherosclerosis). During a median follow-up of 8.4 years, 650 cardiovascular events (coronary heart disease, stroke, heart failure, and peripheral artery disease) occurred (236 events in subjects with CKD). In Cox proportional hazards models adjusted for Framingham predictors, each subclinical measure was independently associated with cardiovascular outcomes, with larger adjusted hazard ratios (HRs; per 1 SD) for coronary artery calcium score than carotid intima-media thickness or ankle-brachial index in subjects without and with CKD (HR, 1.69; 95% confidence interval [95% CI], 1.45 to 1.97 versus HR, 1.12; 95% CI, 1.00 to 1.25 and HR, 1.20; 95% CI, 1.08 to 1.32, respectively). Compared with inclusion of carotid intima-media thickness or ankle-brachial index, inclusion of the coronary artery calcium score led to greater increases in C statistic for predicting cardiovascular disease and net reclassification improvement. Coronary artery calcium score performed best for the prediction of coronary heart disease and heart failure, regardless of CKD status. In conclusion, each measure improved cardiovascular risk prediction in subjects with CKD, with the greatest improvement observed with coronary artery calcium score.

BackgroundFibroblast growth factor 23 (FGF23) levels are elevated in chronic kidney disease (CKD) and elevated values have been associated with both heart disease and mortality. Recent studies show that FGF23, a protein synthesized by osteocytes, is also present in calcified atherosclerotic plaques and may be induced by heart disease. Whether vascular expression of FGF23 is associated with progressive CKD, however, remains unknown. Therefore, the relationship between kidney function, vascular calcification and FGF23 expression was evaluated in patients with heart disease.MethodsImmunohistochemistry for FGF23 was performed in coronary arteries of all patients undergoing heart transplantation at UCLA between February 2008 and 2010. Immunohistochemical staining for Klotho, DMP1, FGFR1, and FGFR3; calcium deposition; and RNA expression of Klotho and DMP1 were assessed in a subset of eight samples.ResultsFGF23 was detected by immunohistochemistry in 56% of the coronary artery specimens. Vascular FGF23 expression correlated with declining kidney function, as evidenced by reduced creatinine clearance. FGFR1 and FGFR3 were detected throughout the vascular tissue and in calcified plaques. Calcium deposition, Klotho expression and DMP1 expression correlated with FGF23 immunoreactivity.ConclusionsThe findings suggest that the Klotho-FGF23-FGFR system is active in coronary arteries and its upregulation correlates with impaired renal function and matrix calcium deposition.

The diagnosis of vascular dementia (VaD) describes a group of various vessel disorders with different types of vascular lesions that finally contribute to the development of dementia. Most common forms of VaD in the elderly brain are subcortical vascular encephalopathy, strategic infarct dementia, and the multi infarct encephalopathy. Hereditary forms of VaD are rare. Most common is the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). Sporadic forms of VaD are caused by degenerative vessel disorders such as atherosclerosis, small vessel disease (SVD) including small vessel arteriosclerosis, arteriolosclerosis, and lipohyalinosis, and cerebral amyloid angiopathy (CAA). Less frequently inflammatory vessel disorders and tumor-associated vessel lesions (e.g. angiocentric T-cell or angiotropic large cell lymphoma) can cause symptoms of dementia. Here, we review and discuss the impact of vessel disorders to distinct vascular brain tissue lesions and to the development of dementia in elderly individuals. The impact of coexisting neurodegenerative pathology in the elderly brain to VaD as well as the correlation between SVD and CAA expansion in the brain parenchyma with that of Alzheimer's disease (AD)-related pathology is highlighted. We conclude that "pure" VaD is rare and most frequently caused by infarctions. However, there is a significant contribution of vascular lesions and vessel pathology to the development of dementia that may go beyond tissue damage due to vascular lesions. Insufficient blood blow and alterations of the perivascular drainage mechanisms of the brain may also lead to a reduced protein clearance from extracellular space and subsequent increase of proteins in the brain parenchyma, such as the amyloid β-protein, and foster, thereby, the development of AD-related neurodegeneration. As such, it seems to be important for clinical practice to consider treatment of potentially coexisting AD pathology in cognitively impaired patients with vascular lesions.

ObjectiveThe purpose of this study was to fine map previously identified quantitative trait loci affecting atherosclerosis in mice using association analysis.Methods and resultsWe recently showed that high-resolution association analysis using common inbred strains of mice is feasible if corrected for population structure. To use this approach for atherosclerosis, which requires a sensitizing mutation, we bred human apolipoprotein B-100 transgenic mice with 22 different inbred strains to produce F1 heterozygotes. Mice carrying the dominant transgene were tested for association with high-density single nucleotide polymorphism maps. Here, we focus on high-resolution mapping of the previously described atherosclerosis 30 locus on chromosome 1. Compared with the previous linkage analysis, association improved the resolution of the atherosclerosis 30 locus by more than an order of magnitude. Using expression quantitative trait locus analysis, we identified one of the genes in the region, desmin, as a strong candidate.ConclusionsOur high-resolution mapping approach accurately identifies and fine maps known atherosclerosis quantitative trait loci. These results suggest that high-resolution genome-wide association analysis for atherosclerosis is feasible in mice.

PPARalpha, beta/delta, and gamma regulate genes involved in the control of lipid metabolism and inflammation and are expressed in all major cell types of atherosclerotic lesions. In vitro studies have suggested that PPARs exert antiatherogenic effects by inhibiting the expression of proinflammatory genes and enhancing cholesterol efflux via activation of the liver X receptor-ABCA1 (LXR-ABCA1) pathway. To investigate the potential importance of these activities in vivo, we performed a systematic analysis of the effects of PPARalpha, beta, and gamma agonists on foam-cell formation and atherosclerosis in male LDL receptor-deficient (LDLR(-/-)) mice. Like the PPARgamma agonist, a PPARalpha-specific agonist strongly inhibited atherosclerosis, whereas a PPARbeta-specific agonist failed to inhibit lesion formation. In concert with their effects on atherosclerosis, PPARalpha and PPARgamma agonists, but not the PPARbeta agonist, inhibited the formation of macrophage foam cells in the peritoneal cavity. Unexpectedly, PPARalpha and PPARgamma agonists inhibited foam-cell formation in vivo through distinct ABCA1-independent pathways. While inhibition of foam-cell formation by PPARalpha required LXRs, activation of PPARgamma reduced cholesterol esterification, induced expression of ABCG1, and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner. In concert, these findings reveal receptor-specific mechanisms by which PPARs influence macrophage cholesterol homeostasis. In the future, these mechanisms may be exploited pharmacologically to inhibit the development of atherosclerosis.

PPARalpha, beta/delta, and gamma regulate genes involved in the control of lipid metabolism and inflammation and are expressed in all major cell types of atherosclerotic lesions. In vitro studies have suggested that PPARs exert antiatherogenic effects by inhibiting the expression of proinflammatory genes and enhancing cholesterol efflux via activation of the liver X receptor-ABCA1 (LXR-ABCA1) pathway. To investigate the potential importance of these activities in vivo, we performed a systematic analysis of the effects of PPARalpha, beta, and gamma agonists on foam-cell formation and atherosclerosis in male LDL receptor-deficient (LDLR(-/-)) mice. Like the PPARgamma agonist, a PPARalpha-specific agonist strongly inhibited atherosclerosis, whereas a PPARbeta-specific agonist failed to inhibit lesion formation. In concert with their effects on atherosclerosis, PPARalpha and PPARgamma agonists, but not the PPARbeta agonist, inhibited the formation of macrophage foam cells in the peritoneal cavity. Unexpectedly, PPARalpha and PPARgamma agonists inhibited foam-cell formation in vivo through distinct ABCA1-independent pathways. While inhibition of foam-cell formation by PPARalpha required LXRs, activation of PPARgamma reduced cholesterol esterification, induced expression of ABCG1, and stimulated HDL-dependent cholesterol efflux in an LXR-independent manner. In concert, these findings reveal receptor-specific mechanisms by which PPARs influence macrophage cholesterol homeostasis. In the future, these mechanisms may be exploited pharmacologically to inhibit the development of atherosclerosis.

Atherogenesis is enhanced in arterial segments exposed to disturbed blood flow, indicating the active participation of the hemodynamic environment in lesion formation. Turbulent shear stress selectively regulates responsive genes in the endothelium and increases the damage induced by free radicals. The purpose of the present study was to evaluate the effects of intervention with antioxidants and l-arginine on endothelial NO synthase (eNOS) and oxidation-sensitive gene perturbation induced by disturbed flow in vitro and in vivo. Both human endothelial cells exposed to shear stress and high atherosclerosis-prone areas of hypercholesterolemic low-density lipoprotein receptor knockout (LDLR(-/-)) mice showed increased activities of redox-transcription factors (ELK-1, p-Jun, and p-CREB) and decreased expression of eNOS. Intervention with antioxidants and l-arginine reduced the activation of redox-transcription factors and increased eNOS expression in cells and in vivo. These results demonstrate that atherogenic effects induced by turbulent shear stress can be prevented by cotreatment with antioxidants and l-arginine. The therapeutic possibility to modulate shear stress-response genes may have important implications for the prevention of atherosclerosis and its clinical manifestations.

BackgroundMaternal hypercholesterolemia during pregnancy is associated with markedly enhanced fatty streak formation in human fetal aortas and accelerated progression of atherosclerosis in normocholesterolemic children.Methods and resultsTo establish the causal role of maternal hypercholesterolemia in a genetically homogeneous murine model and to test the hypothesis that pathogenic events during fetal development result in persistent changes in arterial gene expression, female LDL receptor-deficient (LDLR(-/-)) mice were fed regular chow or high-fat diets supplemented with 0.075% or 1.25% cholesterol during pregnancy. Lesion sizes were determined in the aortic origin of their chow-fed offspring at 3 months. Maternal hypercholesterolemia more than doubled lesion sizes in male offspring (P1.7-fold. Quantitative PCR for one of these genes using RNA from individual mice yielded similar results. Comparative immunostaining for several of the above genes also indicated increased protein content in offspring of hypercholesterolemic mothers.ConclusionsThese findings establish an atherogenic effect of maternal hypercholesterolemia in genetically uniform mice and indicate that changes in aortic gene expression persist long after fetal exposure to hypercholesterolemia. In addition to elucidating pathogenic mechanisms initiated during fetal development, this approach may identify genes in morphologically normal arteries that influence the susceptibility to classical risk factors of atherosclerosis.

Transgenic and knockout mice are widely used as models for atherogenesis studies. While developing a Helicobacter infection model in LDL receptor-negative (LDLR(-/-)) mice, we noticed that mice fed a high-fat, high-cholesterol diet often contracted gastritis independent of infection. To further investigate this finding, we studied 27 male and 18 female LDLR(-/-) mice fed high-fat, 1% or 1.25% cholesterol diets for 3 to 4 months. The extent of atherosclerosis was morphometrically analyzed in the whole aorta, and the degree of gastric inflammation was scored histologically in hematoxylin-eosin-stained stomach sections. The autoantibody titers to epitopes of oxidized LDL were also measured. Mice fed high-fat, high-cholesterol diets had a significantly higher incidence of gastritis than mice fed normal chow, 62% versus 5%, respectively (P

HypothesisPatency after primary percutaneous transluminal angioplasty (PTA) and stenting of superficial femoral artery (SFA) occlusions is better than historical experience with PTA alone.DesignConsecutive case series of primary PTA with stenting, and follow-up with duplex imaging every 6 months (mean +/- SD follow-up, 32 +/- 15 months).SettingVeterans Affairs medical center.Patients and methodsPatients were 57 previously untreated men with 71 limbs having chronic atherosclerotic SFA occlusion with suprageniculate reconstitution and patent tibial runoff. Critical ischemia (Society for Vascular Surgery [SVS] category, 4-6) was present in 7 (10%), the remainder had intermittent claudication only (SVS, 1-3).InterventionsGuidewire recanalization followed by PTA, Wallstent deployment, and adjunctive thrombolysis as necessary; 19 limbs (27%) required thrombolysis to manage periprocedural thrombosis.Main outcome measuresCumulative patency, limb salvage, and complications.ResultsLength (mean +/- SD) of occlusion was 14.4 +/- 9.9 cm. Length of stented artery was 24.3 +/- 11.1 cm. Ankle brachial index increased from 0.59 +/- 0.14 to 0.86 +/- 0.16 (P

Autoantibodies to oxidized low density lipoprotein (OxLDL) are elevated in some human populations with increased risk of atherosclerosis. To determine whether autoantibody levels to epitopes of OxLDL reflect the extent of aortic atherosclerosis and the content of OxLDL, we measured IgG and IgM autoantibody titers to malondialdehyde (MDA)-LDL and copper-oxidized LDL (Cu-OxLDL) in 43 LDL receptor-deficient mice consuming atherogenic and regression diets. Antibody titers were correlated to percent atherosclerotic surface area, aortic weight, and aortic OxLDL content, measured as the in vivo uptake of (125)I-MDA2, a monoclonal antibody to MDA-LDL. All mice were fed an atherogenic diet for 6 months, and 1 group was euthanized. The other 3 groups were fed an atherogenic diet (fat/CHOL group), normal mouse chow (chow group), or mouse chow supplemented with vitamins E and C (chow+VIT group) for an additional 6 months. After dietary intervention, compared with their own baseline, autoantibody titers to MDA-LDL and Cu-OxLDL increased significantly in the fat/CHOL group, whereas they did not change or decreased significantly in the chow and chow+VIT groups. Aortic weight and surface area showed significant progression in the fat/CHOL group, mild progression in the chow group, and no progression in the chow+VIT group (P

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that regulates fat-cell development and glucose homeostasis and is the molecular target of a class of insulin-sensitizing agents used for the management of type 2 diabetes mellitus. PPARgamma is highly expressed in macrophage foam cells of atherosclerotic lesions and has been demonstrated in cultured macrophages to both positively and negatively regulate genes implicated in the development of atherosclerosis. We report here that the PPARgamma-specific agonists rosiglitazone and GW7845 strongly inhibited the development of atherosclerosis in LDL receptor-deficient male mice, despite increased expression of the CD36 scavenger receptor in the arterial wall. The antiatherogenic effect in male mice was correlated with improved insulin sensitivity and decreased tissue expression of TNF-alpha and gelatinase B, indicating both systemic and local actions of PPARgamma. These findings suggest that PPARgamma agonists may exert antiatherogenic effects in diabetic patients and provide impetus for efforts to develop PPARgamma ligands that separate proatherogenic activities from antidiabetic and antiatherogenic activities.

The critical role of monocyte recruitment in atherogenesis has been appreciated for some time. However, until recently, there have been no sufficiently sensitive methods for measuring rates of monocyte recruitment to the arterial wall in vivo. We have developed a novel highly sensitive method, based on the polymerase chain reaction, for quantitatively tracking DNA-marked monocytes and have adapted it for use in mice. We use the uniquely male gene, SRY:, on the Y chromosome as a gene marker. We transfuse monocytes from a male donor into a congenic female mouse, euthanize the mouse after 24 to 48 hours, and then quantify the arterial uptake of monocytes by quantitative polymerase chain reaction. This study describes the techniques used and their sensitivity and reproducibility and demonstrates the approach by assessing the effects of cytokines. In control low density lipoprotein receptor-negative mice, monocyte recruitment decreased slightly but significantly as lesions progressed. Intraperitoneal injection of a combination of tumor necrosis factor-alpha and interleukin-1 beta more than doubled the rate of monocyte recruitment into developing lesions. However, the response to the cytokines was much greater in younger mice with less advanced lesions than in older animals with more advanced lesions.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that regulates fat-cell development and glucose homeostasis and is the molecular target of a class of insulin-sensitizing agents used for the management of type 2 diabetes mellitus. PPARgamma is highly expressed in macrophage foam cells of atherosclerotic lesions and has been demonstrated in cultured macrophages to both positively and negatively regulate genes implicated in the development of atherosclerosis. We report here that the PPARgamma-specific agonists rosiglitazone and GW7845 strongly inhibited the development of atherosclerosis in LDL receptor-deficient male mice, despite increased expression of the CD36 scavenger receptor in the arterial wall. The antiatherogenic effect in male mice was correlated with improved insulin sensitivity and decreased tissue expression of TNF-alpha and gelatinase B, indicating both systemic and local actions of PPARgamma. These findings suggest that PPARgamma agonists may exert antiatherogenic effects in diabetic patients and provide impetus for efforts to develop PPARgamma ligands that separate proatherogenic activities from antidiabetic and antiatherogenic activities.

Oxidatively modified LDL mimics several aspects of atherogenesis. In this disease, degradation of the matrix proteins' network also occurs. By a new morphological ex vivo approach, not requiring sample processing, we explored the relationship between the degradation of matrix protein and oxidatively modified LDL. Two-photon excitation fluorescence microscopy images of fresh cross-section rings of rat aorta, acquired while the sample was maintained in a glucose- and oxygen-supplemented buffer, showed straight, parallel, thick, long extracellular matrix proteins. Traditional microscopic examination, requiring sample fixation and staining, shows smaller and curved fibers. Instead, we observed curved and broken fibers after a 30-min incubation of aorta with either LDL containing lipid hydroperoxides, or tert-butyl-hydroperoxide. The adhesion of LDL to the endothelium and its internalization was directly visualized by using a lipid fluorophore. The damage to aorta matrix proteins induced by LDL and tert-butyl-hydroperoxide was fully prevented by antioxidants, such as ascorbate or Trolox C, or inhibitors of proteases. The image spectroscopy of the fibers' autofluorescence (polarization and lifetime) revealed an increased mobility of the fluorescent cross-link in fibers. Damaged matrix proteins were also imaged in aorta samples from apolipoprotein E knock-out mice. Our ex vivo images directly visualized the activation of a fast redox-sensitive proteolytic process in the arterial wall triggered by lipid hydroperoxides in LDL.

We previously established that probucol decreases basal expression of VCAM-1 in the aorta of WHHL rabbits and inhibits the up-regulation of VCAM-1 expression that normally accompanies atherogenesis. To determine whether this effect is shared by other antioxidants in vivo, we now investigated whether a structurally unrelated antioxidant, vitamin E, also inhibits arterial VCAM-1 expression and whether the degree of VCAM-1 inhibition correlates with the reduction of atherosclerosis or the antioxidant protection of LDL. Atherogenesis and VCAM-1 mRNA and protein were determined in four groups of NZW rabbits (n = 6;-8) fed 0.5% cholesterol alone or supplemented with 0.1% vitamin E, a low dose (0.04;-0.075%) of probucol yielding the same degree of antioxidant protection of plasma LDL as vitamin E, or a high dose (0.5%) of probucol, and in normocholesterolemic rabbits. After 81 days, extensive atherosclerosis and a greater than 4-fold up-regulation of VCAM-1 mRNA was seen in rabbits on high cholesterol diet, mostly in the intima. Treatment with vitamin E, high-dose probucol, and low-dose probucol significantly decreased VCAM-1 mRNA by 49.0, 74.9, and 57. 5%, respectively, and reduced atherosclerosis in adjacent segments of the thoracic aorta to a similar degree as reported by previous studies. Immunocytochemistry confirmed that lesions of antioxidant-treated animals also contained less VCAM-1 protein. Neither the degree of VCAM-1 inhibition nor the extent of atherosclerosis correlated with the degree of antioxidant protection of plasma LDL.In summary, treatment with structurally unrelated antioxidants conveyed different degrees of antioxidant protection to plasma LDL but significantly reduced VCAM-1 expression in vivo and inhibited atherogenesis. This is consistent with the assumption that antiatherogenic effects of antioxidants may in part be mediated by interference with oxidation-dependent intracellular signaling.

BackgroundChildren generally have low cholesterol and no clinical manifestations of atherosclerosis, but fatty-streak formation begins in fetuses and is greatly increased by maternal hypercholesterolaemia during pregnancy. In the FELIC study we assessed the evolution of such lesions during childhood.MethodsComputer-assisted imaging was used to measure the area of the largest individual lesion and the cumulative lesion area per section in serial cross-sections through the entire aortic arch and abdominal aorta of 156 normocholesterolaemic children aged 1-13 years, who died of trauma and other causes. Children were classified by whether their mother had been normocholesterolaemic (n=97) or hypercholesterolaemic (n=59) during pregnancy. Atherosclerosis was correlated with 13 established or potential risk factors. Findings The largest fatty streaks in the aortic arch of children younger than 3 years of hypercholesterolaemic mothers were 64% smaller than those previously found in corresponding fetuses (p

The role of insulin resistance (IR) in atherogenesis is poorly understood, in part because of a lack of appropriate animal models. We assumed that fructose-fed LDL receptor-deficient (LDLR-/-) mice might be a model of IR and atherosclerosis because (1) fructose feeding induces hyperinsulinemia and IR in rats; (2) a preliminary experiment showed that fructose feeding markedly increases plasma cholesterol levels in LDLR-/- mice; and (3) hypercholesterolemic LDLR-/- mice develop extensive atherosclerosis. To test whether IR could be induced in LDLR-/- mice, 3 groups of male mice were fed a fructose-rich diet (60% of total calories; n=16), a fat-enriched (Western) diet intended to yield the same plasma cholesterol levels (n=18), or regular chow (n=7) for approximately 5.5 months. The average cholesterol levels of both hypercholesterolemic groups were similar (849+/-268 versus 964+/-234 mg/dL) and much higher than in the chow-fed group (249+/-21 mg/dL). Final body weights in the Western diet group were higher (39+/-6.2 g) than in the fructose- (27.8+/-2.7 g) or chow-fed (26.7+/-3.8 g) groups. Contrary to expectation, IR was induced in mice fed the Western diet, but not in fructose-fed mice. The Western diet group had higher average glucose levels (187+/-16 versus 159+/-12 mg/dL) and 4.5-fold higher plasma insulin levels. Surprisingly, the non-insulin-resistant, fructose-fed mice had significantly more atherosclerosis than the insulin-resistant mice fed Western diet (11.8+/-2.9% versus 7.8+/-2. 5% of aortic surface; P

BackgroundAtherosclerotic lesions in intracranial arteries occur later and are less extensive than in extracranial arteries. To investigate potential mechanisms responsible for this difference, in particular the atherogenic response to hypercholesterolemia and LDL oxidation, we compared the extent of fatty streak formation and the composition of these very early lesions in intracranial arteries of human fetuses from normocholesterolemic and hypercholesterolemic mothers with those in extracranial arteries.Methods and resultsLesions were quantified by computer-assisted image analysis of 30 oil red O-stained sections, each from the middle cerebral, basilar, and common carotid arteries and the abdominal aorta of human fetuses (spontaneous abortions and premature newborns who died within 12 hours of birth; both of fetal age 6.2+/-1.3 months) from 43 hypercholesterolemic mothers and 34 normocholesterolemic mothers. Macrophages, apolipoprotein B, and 2 epitopes of oxidized LDL in lesions were determined immunocytochemically. Activities of superoxide dismutase, catalase, and glutathione peroxidase in the arterial wall were also determined. Lesion numbers and sizes were dramatically greater in the abdominal aorta (area of the largest lesion per section: 66.5+/-10.9 x10(3) microm2) and the carotid (11. 6+/-5.3 x10(3) microm2) than in the basilar and middle cerebral artery (0.4+/-0.1 and 0.8+/-0.2 x10(3) microm2, respectively; P

BackgroundThe purpose of this investigation was to quantify the amount of intralesional calcium detected by intravascular ultrasound (IVUS) compared with undecalcified histology in human arteries. This method preserves intralesional calcium and reduces sectioning artifacts, thereby providing an accurate measure of calcium plaque morphology.Methods and resultsTen arterial segments (5 coronary, 5 iliac) were obtained at autopsy. IVUS imaging was performed with a 4.9F catheter at an automated pullback rate of 1.0 mm/s. The undecalcified arteries were dehydrated in ascending alcohol and polymerized in glycol methylmethacrylate. The arteries were cut into 200-microm sections with an Isomet low-speed saw and stained with Goldner's trichrome. The lumen cross-sectional area, the calcium plaque cross-sectional area, the calcium plaque depth, length, and angle of arc of calcified plaque were measured from the IVUS images and histologic sections. In 24 selected cross sections, there were 38 separate calcium plaques. An independent observer correctly identified 34 of 38 calcified plaques for a sensitivity of 89% and specificity of 97%. The total mean calcified plaque cross-sectional area measured from histology was 4.6 +/- 4.1 mm2 compared with 2.8 +/- 2.3 mm2 by IVUS (P =.002). Plaque depth measured by histology was 1.2 +/- 0.4 mm versus 0.7 +/- 0.2 mm by IVUS (P =.001). The length of calcium plaques measured by histology was 3.6 +/- 1.78 mm versus 3.6 +/- 1.5 mm for IVUS (r = 0.79).ConclusionsIVUS accurately depicts circumferential calcified lesions with high sensitivity (89%) and specificity (97%). However, IVUS underestimates the total calcified plaque cross-sectional area by 39%. This is mainly because of the inability of the ultrasound to penetrate intralesional calcium, which leads to an underestimation of the depth of calcium by 45%.

We and others previously showed that immunization of rabbits with different forms of oxidized low density lipoprotein (LDL) significantly reduced atherogenesis. We now investigated the effect of continued immunization on atherosclerosis in LDL receptor-deficient (LDLR-/-) mice to determine whether a similar reduction of atherosclerosis occurred in murine models and whether this was due to humoral immune responses, ie, formation of high titers of antibodies to oxidation-specific epitopes. Three groups of LDLR-/- mice were repeatedly immunized with homologous malondialdehyde-modified LDL (MDA-LDL), native LDL, or phosphate-buffered saline (PBS) for 7 weeks. Extensive hypercholesterolemia and accelerated atherogenesis were then induced by feeding a cholesterol-rich diet for 17 weeks, during which immunizations were continued. Binding of immunoglobulin (Ig) M and IgG antibodies, as well as IgG1 and IgG2a isotypes, to several epitopes of oxidized LDL were followed throughout the study. After 24 weeks of intervention, atherosclerosis in the aortic origin was significantly reduced by 46.3% and 36.9% in mice immunized with MDA-LDL and native LDL, respectively, compared with PBS (133 558 and 157 141 versus 248 867 microm2 per section, respectively). However, the humoral immune response to oxidative neoepitopes in the MDA-LDL group was very different from that of the LDL or PBS group. IgG antibody binding to MDA-LDL and other epitopes of oxidized LDL, such as oxidized phospholipid (cardiolipin), oxidized cholesterol, or oxidized cholesteryl linoleate, but not native LDL, increased markedly in mice immunized with MDA-LDL, but not in mice immunized with native LDL or PBS. In the MDA-LDL group, both T helper cell (Th)2-dependent IgG1 antibody and Th1-dependent IgG2a antibody binding to oxidative neoepitopes increased significantly over time. The fact that mice immunized with both MDA-LDL and native LDL had a significant reduction in atherosclerosis, whereas only the MDA-LDL group developed very high titers of antibodies to oxidation-specific epitopes, suggests that the antiatherogenic effect of immunization is not primarily dependent on very high titers of antibodies to oxidation-specific epitopes but is more likely to result from the activation of cellular immune responses.

Probucol is a powerful inhibitor of atherosclerosis in a number of animal models. However, it is unknown whether this is due to the strong antioxidant protection of low density lipoprotein (LDL), to antioxidant effects in the artery wall, or to cellular effects not shared by other antioxidants. To investigate whether murine models are suitable to study the antiatherogenic mechanisms of probucol, three experiments following different protocols were carried out in 135 male and female LDL receptor-deficient (LDLR-/-) mice. Treatment groups received a high (0.5%) or low (0.025%) dose of probucol, or low-dose probucol plus a high dose (0.1%) of vitamin E for periods ranging from 6 to 26 weeks. In all experiments, probucol strongly protected LDL against ex vivo oxidation (lag times exceeding 1400 min in 0.5% probucol-treated mice). Treatment with 0.5% probucol significantly lowered both HDL-cholesterol and plasma apolipoprotein (apo)A-I concentrations. In all three experiments, treatment with 0.5% probucol consistently increased the size of lesions in the aortic origin, from 1.3-fold (n.s.) to 2.9-fold (P < 0.05) in female mice and from 3.6- to 3.7-fold in males (P < 0.001). Even treatment with 0.025% probucol increased atherosclerosis 1.6-fold in male mice (P < 0.01). Addition of the high dose of vitamin E did not attenuate the pro-atherogenic effect of 0.025% probucol. In conclusion, probucol not only failed to decrease but actively increased atherogenesis in LDLR-/- mice in a dose-dependent manner, even though it provided a very strong antioxidant protection of LDL. This suggests that the reduction of atherosclerosis observed in other animal models is due to intracellular effects of probucol not found in mice, to differences in the metabolism of probucol, and/or to an overriding atherogenic effect of the decrease in HDL in murine models.

The abdominal aorta is injured infrequently in blunt trauma to the abdomen. When injury does occur, aortic rupture, dissection, and traumatic aneurysm most commonly result. An aneurysmal abdominal aorta would appear to be at increased risk for injury from blunt abdominal trauma. This case report appears to be the first description of iliac and femoral artery occlusion by thromboemboli dislodged from an atherosclerotic abdominal aortic aneurysm at the time of a motor vehicle accident. We believe that this peripheral embolization was caused by direct compression of the abdominal aortic aneurysm by a seatbelt.

Investigations into the mechanisms by which diabetes accelerates atherosclerosis have been hampered by the lack of suitable animal models. We hypothesized that streptozotocin-treated LDL receptor-deficient mice would be a good model of diabetic atherosclerosis because streptozotocin causes diabetes in the parent C57BL/6 strain and because in these mice diet-induced hypercholesterolemia leads to the formation of advanced atherosclerotic lesions throughout the aorta. Diabetes was induced in 18 mice by intraperitoneal injection of streptozotocin. Low-dose insulin was given subcutaneously to prevent excessive mortality and extreme elevations in triglyceride levels. The control group was subjected to sham injections. Both groups were fed a diet containing .075% cholesterol for six months. Average blood glucose was higher in the diabetic group than in the control group (257 +/- 67 mg/dL versus 111 +/- 7 mg/dL, P < 0.05). Although plasma cholesterol was similar (966 +/- 399 versus 1002 +/- 180 mg/dL) in both groups, VLDL cholesterol was higher whereas LDL cholesterol was lower in the diabetic group. Immunocytochemical analysis demonstrated significantly more advanced glycation end-product (AGE) epitopes in the artery wall of the diabetic group, whereas staining for oxidation-specific epitopes was similar in both groups. Sera of diabetic mice also contained significantly more IgG autoantibodies that bound to several AGE epitopes than did sera from control mice. Despite the presence of hyperglycemia, diabetic dyslipidemia, and enhanced AGE formation in the diabetic mice, both groups had a similar extent of atherosclerosis (diabetic, 17.3 +/- 5.2; control, 16.5 +/- 6.6% of the aortic surface). These data suggest that, at least under conditions of marked hypercholesterolemia; hyperglycemia and enhanced AGE formation do not contribute significantly to atherogenesis in LDL-/- mice.

Different intravascular ultrasound (IVUS) systems vary in their image presentation. The purpose of this study was to compare four IVUS systems in vitro to determine the accuracy of tissue characterization of atherosclerotic plaque compared with histology. Ninety-eight plaque segments from 23 formalin-fixed human iliac arteries were imaged in saline at room temperature with four different IVUS systems. To assess the accuracy of IVUS in describing plaque, three types of analysis were performed: (1) the ability to identify the presence and extent of lumen or plaque boundary; (2) sensitivity, specificity, and interobserver variability of IVUS in qualitatively identifying plaque components compared with histology; and (3) quantification of calcification. The synthetic aperture device had a lower sensitivity in identifying lumen and plaque boundaries (87%, 38% respectively) compared with other machines (96%-100%, 95%-100%). All three mechanically rotating systems had fair to good sensitivities for identifying calcification (57%-73%) or lipid filled areas (50%-83%). The sensitivity of discriminating fibrous tissue from fatty areas was low (39%-52%). The synthetic aperture system had a significantly lower sensitivity for identifying all three tissue types (4%-21%). There was significant interobserver variability (kappa value = 0.47-0.68) as well as machine to machine variability (kappa value = 0.52) for tissue characterization. Calcified areas were underestimated by System 1 (p < .05) and System 4 (p < .01) because of weaker echo reflections or poor image quality. There are significant differences in image representation among these four IVUS systems in the diagnosis of tissue components of complex atherosclerotic plaque. These variabilities should be considered when interpreting studies performed with different machines.

The purpose of this investigation was to compare the accuracy of intravascular ultrasound (IVUS) and electron-beam computed tomography (EBCT) in quantitating human atherosclerotic plaque and calcium. In experiment 1, 12 human atherosclerotic arterial segments were obtained at autopsy and imaged by using IVUS and EBCT. The plaque from each arterial segment was dissected and a volume measurement of the dissected plaque was obtained by water displacement. The plaque from each arterial segment was ashed at 700 degrees F, and the weight of the remaining ashes was used as an estimate of the calcium mass. In experiment II, 11 calcified arterial segments were obtained at autopsy and imaged by using IVUS at one site along the artery. A corresponding histologic cross section stained with Masson's trichrome was prepared. In experiment I, the mean plaque volume measured by water displacement was 165.3 +/- 118.4 microliters. The mean plaque volume calculated by IVUS was 166.1 +/- 114.4 microliters and correlated closely with that by water displacement (r = 0.98, p < 0.0001). The mean calcium mass measured by ashing was 19.4 +/- 15.8 mg. The mean calculated calcium mass by EBCT was 19.9 mg and correlated closely with that by ashing (r=0.98, p

Murine strains susceptible to atherosclerosis provide valuable models to study factors involved in atherogenesis. In some murine models, limited hypercholesterolemia can be achieved and lesions develop primarily in the aortic origin, in the vicinity of the aortic valve. In other models such as LDL receptor-deficient and apoE-deficient mice, diet-induced or spontaneous hypercholesterolemia and atherogenesis are much greater. To determine whether lesion formation in the aortic origin, where particular pathogenic conditions may exist, correlates with lesion formation throughout the entire aorta, we measured the extent of atherosclerosis in both areas in 8 apoE- and 11 LDL receptor-deficient mice fed cholesterol-rich diets for 3-6 months, as well as in 9 C57BL/6 mice fed an atherogenic diet for a year, using two different morphometric methods. Both apoE-deficient and LDL receptor-deficient mice developed extensive lesions throughout the aorta, and in these models a significant correlation was observed between the extent of lesions in the entire aorta (measured as percent of surface area) and that at the aortic origin (measured as averaged lesion area per cross-section) (r = 0.77, P < 0.0001). In contrast, the plasma cholesterol levels achieved in C57BL/6 mice were much lower, and atherosclerotic lesions were found almost exclusively in the aortic origin. These results demonstrate that in murine models developing extensive aortic lesions, both morphometric methods provide valid and complementary information on the degree and distribution of atherosclerosis, and suggest that under severe atherogenic conditions lesion formation throughout the aorta is determined by the same pathological factors, in each model. Comparison of the extent of atherosclerosis in the entire aorta between genders also showed that male LDL receptor-deficient mice had significantly more lesions than females (29.2 vs. 14.8%, P < 0.005, n = 16). A similar trend was also seen in apoE-deficient mice.

Vitamin E has been postulated to be antiatherogenic because of its antioxidative potency. However, intervention studies published to date have yielded conflicting results. To assess the antiatherogenic effect of vitamin E, two groups of 10 Watanabe heritable hyperlipidemic (WHHL) rabbits each were fed chow pellets containing D-alpha-tocopherol-acetate at either 40 mg/kg (control group) or 1000 mg/kg (vitamin E group) for 28 weeks. Plasma vitamin E levels in the vitamin E group increased five-fold over those controls (475.5 mumol/l vs. 95.9 mumol/l). The average total plasma cholesterol during the treatment period was not significantly affected by vitamin E (control, 950 +/- 113 mg/dl; vitamin E, 884 +/- 90 mg/dl). Vitamin E treatment had no significant effects on body weights, lipoprotein profiles, or HDL levels. The protection of plasma LDL against oxidation was determined by ex vivo by measuring the lag time in the formation of conjugated dienes in a standardized Cu22+(-)containing system. Lag time in the vitamin E-treated group increased four-fold over that in controls (404 vs. 123 min). The extent of atherosclerosis determined at the end of the study was not significantly different in the two groups (control group, 59.2 +/- 6.0%; vitamin E group, 50.6 +/- 6.2%, P = 0.33). Analysis of the correlation between vitamin E levels and extent of lesions also failed to indicate an antiatherosclerotic effect of vitamin E treatment. We previously reported that an analogue of probucol that provided antioxidative protection similar to that provided by vitamin E failed to prevent atherogenesis in WHHL-rabbits. In contrast probucol conveyed a much greater degree of antioxidant protection and effectively reduced atherosclerosis in rabbits. The results of the present study therefore support the hypothesis that a threshold level of antioxidative protection of LDL may be required to inhibit atherosclerosis.

ObjectivesThe purpose of this study was to test the hypothesis that in occlusions of the superficial femoral artery, removal of atherosclerotic plaque would result in a higher long-term patency rate compared to balloon dilatation alone. A secondary hypothesis was that long term patency would be proportional to the amount of plaque removed.MethodsA randomized controlled study of patients with occluded superficial femoral arteries was performed comparing balloon dilatation alone versus a 2.7 mm or a larger (4.0 mm or 4.7 mm) transcutaneous extraction catheter (TEC) atherectomy device followed by balloon dilatation. The effect of these devices on plaque area was assessed directly by intravascular ultrasound imaging.ResultsThe mean occlusion length was 19.4 cm +/- 11.7 cm. The mean lumen area increased from 4.7 mm2 to 15.1 mm2, primarily due to balloon dilatation, but the mean atheroma area of 19.8 mm2 did not change with either size of TEC device. Although the initial procedure success rate was high (79%), the 6 month patency was only 45%. There was no difference in 6 month patency between the 3 groups.ConclusionsThe data indicate that the TEC atherectomy devices do not remove a significant amount of atherosclerotic plaque in occluded superficial femoral arteries. The 6 month patency is no different with these atherectomy devices than with balloon dilatation alone. The larger (4.0 mm or 4.7 mm) TEC device does not remove any more tissue than the smaller (2.7 mm) device. The use of intravascular ultrasound to quantitate the effects of this atherectomy device provides important insights into the mechanism of action and lack of efficacy of the TEC atherectomy catheter.

The engineering of mice that express a human apoB transgene has resulted in animals with high levels of human-like LDL particles and through crosses with human apo(a) transgenics, high levels of human-like lipoprotein (a) (Lp[a]) particles. In this study, these animals have been used to compare the atherogenic properties of apo(a), LDL, and Lp(a). The presence of the high expressing apoB (apoBH) transgene was associated with a 2.5-fold increase in VLDL-LDL cholesterol (primarily in the LDL fraction) and a 15-fold increase in proximal lesions compared with non-transgenic mice (P < or = 0.0001), while the presence of the low expressing human apoB (apoBL) transgene was not associated with major changes in lipoprotein profiles or increases in aortic lesion size. Examination of aortas of apoBH mice demonstrated lesions along the entire length of the aorta and immunochemical analysis of the lesions revealed features characteristically seen in human lesions including the presence of oxidized lipoproteins, macrophages, and immunoglobulins. Unlike animals with the apoBL transgene, animals with the apo(a) transgene had significant increases in proximal aortic fatty streak lesions compared to nontransgenic control animals (threefold; P < 0.02), while animals with both transgenes, the apo(a)/apo BL double transgenics, had lesions 2.5 times greater than animals expressing the apo(a) transgene alone and eightfold (P < 0.0006) greater than nontransgenic animals. These murine studies demonstrate that marked increases in apoB and LDL resulted in atherosclerotic lesions extending down the aorta which resemble human lesions immunochemically and suggest that apo(a) associated with apoB and lipid may result in a more pro-atherogenic state than when apo(a) is free in plasma.

The rate of restenosis after directional coronary atherectomy (DCA) is higher than expected. To elucidate why, the current study used intravascular ultrasound (IVUS) imaging to investigate the mechanism of DCA. An in vitro validation study was performed to determine the accuracy of the measurement of plaque removal by IVUS. DCA was performed in eight human atherosclerotic artery segments. The volume of removed plaque was measured by water displacement and was compared with the volume calculated from IVUS images. A clinical study of DCA was performed in 32 lesions. IVUS was performed in 28 lesions after successful DCA. Measurements of lumen dimensions from digital angiograms before and after DCA were compared with observations of lumen and plaque size from the cross-sectional IVUS images. In the in vitro study, the mean plaque volume removed by DCA was 19.9 +/- 8.5 microliters. The calculated estimate of removed plaque volume by IVUS was 18.6 +/- 7.9 microliters and correlated closely with the volume by water displacement (r = 0.92). The calculated volume of plaque removed from histologic sections was 14.3 +/- 6.0 microliters and was linearly correlated with plaque volume by water displacement (r = 0.81). In the clinical study, the angiographic mean minimum lumen diameter increased from 1.0 +/- 0.4 to 2.7 +/- 0.5 mm and the percentage stenosis decreased from 70% to 19% (p < 0.0001). The IVUS images before and after DCA showed that the lumen DCA improved from 2.9 +/- 1.5 to 7.0 +/- 1.5 mm2 (p < 0.0001). In addition the vessel cross-sectional area (CSA) increased from 17.1 +/- 5.9 to 18.7 +/- 5.5 mm2. The atheroma CSA was reduced from 14.2 +/- 5.0 to 11.7 +/- 4.8 mm2. This combined effect of reduction in atheroma CSA and stretching of the outer vessel diameter resulted in an improvement in percentage plaque area stenosis from 83% +/- 7% to 61% +/- 9%. It is concluded that despite a successful angiographic appearance, DCA removed an average of 2.5 mm2 from the atheroma, which corresponds to only 18% of the atheroma CSA. The total lumen CSA increased 4.1 mm2; 61% of the new lumen was created by cutting and removal of plaque, whereas 39% of the new lumen was made by stretching the external wall of the artery. Despite an excellent angiographic result, IVUS imaging reveals that after DCA a significant amount of residual atheroma remains. As in balloon dilatation, a stretching effect is a significant component of DCA.

Atherosclerosis is known to be accelerated in diabetic patients, but the mechanisms of this acceleration are poorly understood. Nonenzymatic glycosylation of long-lived proteins results in the formation of advanced glycosylation end products (AGEs), which are extensively cross-linked and could contribute to atherogenesis. Oxidative modification of LDL is also an important process in atherogenesis. In vitro evidence suggests that hyperglycemia may enhance lipid peroxidation, and conversely, that increased lipid peroxidation may enhance AGE formation. If such interactions occur in vivo, we hypothesized that AGE should be found in atherosclerotic lesions of euglycemic LDL receptor-deficient rabbits in areas rich in lipids and oxidized lipoproteins. To demonstrate the presence of AGEs, we developed antisera against a specific "model" compound of AGE, 2-furoyl-4(5)-(2-furanyl)-1H-imidazole (FFI) by using FFI-hexanoic acid (FFI-HA)-protein adducts as the antigen and against AGEs in general by using AGE-albumin as the antigen. Antisera generated with FFI-HA-protein adducts recognized FFI-HA alone as well as FFI-protein adducts. Native proteins or proteins conjugated with aldehydes formed during lipid peroxidation in vitro were not recognized by these antisera. Immunocytochemistry with both FFI-specific and AGE-specific antisera revealed the presence of these epitopes in atherosclerotic lesions of euglycemic LDL receptor-deficient rabbits but not in normal aortic tissues. AGE epitopes within atherosclerotic lesions were predominantly found in similar locations as epitopes generated during modification of the lipoproteins by oxidation, consistent with the hypothesized interactions between oxidation and glycosylation. Indirect evidence in support of the in vivo presence of FFI-like structures was also obtained by the observation that both diabetic and euglycemic human subjects contained autoantibodies that recognize FFI-protein adducts. Taken together, these data provide immunological evidence for the in vivo presence of FFI-like structures and other AGE-protein adducts in atherosclerotic lesions, even in euglycemic conditions.

Apolipoprotein (apo) E-deficient transgenic mice develop marked hyperlipidemia and progressive atherosclerotic lesions. To explore whether oxidative modification of lipoproteins is involved in atherogenesis in this murine model, we performed extensive immunocytochemical studies. Atherosclerotic lesions ranging from early fatty streaks to very advanced plaques were examined from the aortic valve region and the thoracic and abdominal aorta. Using guinea pig antisera against malondialdehyde (MDA)-lysine and 4-hydroxynonenal-lysine, two epitopes generated during the oxidative modification of low-density lipoprotein (LDL), we demonstrated the presence of these "oxidation-specific epitopes" in atherosclerotic lesions. In early lesions, oxidation-specific epitopes were found predominantly in macrophage-rich areas, whereas diffuse extracellular staining predominated in necrotic areas of advanced lesions. We have previously shown that autoantibodies against MDA-lysine are present in the circulation of humans and rabbits and that the immunoglobulin fraction extracted from their lesions contains autoantibodies against several "oxidation-specific" epitopes. Sera from apoE-deficient mice also contained circulating autoantibodies to MDA-lysine, and both early and advanced lesions were rich in murine immunoglobulins. Titers of serum autoantibodies were significantly higher in apoE-deficient mice than in C57BL/6 mice. Autoantibodies in murine plasma recognized MDA-lysine epitopes in atherosclerotic lesions of rabbits, and the immunostaining was competitively inhibited by excess human MDA-LDL. Similar findings were obtained by competitive radioimmunoassay. Finally, a morphometric technique was developed and tested in these mice that allows a quantitative assessment of aortic atherosclerosis. These findings suggest that in apoE-deficient mice, lipoprotein oxidation is involved in atherogenesis and that these transgenic mice constitute an appropriate model with which to study the antiatherogenic effect of antioxidant intervention.

Previous studies have demonstrated the presence of apoprotein (apo) E protein and message in arterial lesions. To determine the source of the synthesized apoE, we performed simultaneous in situ hybridization and immunocytochemistry on human and rabbit atherosclerotic tissue. Studies of serial sections of aortic atherosclerotic lesions from humans and hypercholesterolemic New Zealand White rabbits and Watanabe heritable hyperlipidemic rabbits revealed a similar pattern of macrophage-specific apoE expression in the rabbit and human lesions. In early lesions of rabbit atherosclerotic tissue, in which many macrophages were present, there was abundant expression of apoE mRNA. Northern blot analyses of total mRNA obtained from arterial macrophage-derived foam cells, freshly isolated from ballooned, cholesterol-fed New Zealand White rabbits, demonstrated positive hybridization with an apoE-specific riboprobe. Western blot analyses of conditioned media from the isolated foam cells placed in culture for up to 24 hours demonstrated the presence of secreted apoE. These studies demonstrated that in atherosclerotic lesions, arterial wall macrophages synthesize and secrete apoE and probably account for most of the apoE synthesized in the atherosclerotic artery.

Oxidative modification of LDL renders it immunogenic and autoantibodies to epitopes of oxidised LDL, such as malondialdehyde (MDA)-lysine, are found in serum and recognise material in atheromatous tissue. However, there has been no prospective study to assess the importance of oxidised LDL among patients with vascular disease. We compared the titre of autoantibodies to MDA-modified LDL and native LDL in baseline serum samples of 30 eastern Finnish men with accelerated two-year progression of carotid atherosclerosis and 30 age-matched controls without progression. Neither group had specific antibody binding to native LDL. A titre was defined as a ratio of antibody binding to MDA-LDL/binding to native LDL. Cases had a significantly higher titre to MDA-LDL (2.67 vs 2.06, p = 0.003). Cases also had a greater proportion of smokers (37% vs 3%), higher LDL cholesterol (4.2 mmol/l vs 3.6 mmol/l), and higher serum copper concentration (1.14 mg/l vs 1.04 mg/l). Even after adjusting for these variables and the severity of baseline atherosclerosis, the difference in antibody titre remained significant in a multifactorial logistic model (p = 0.031). Thus, the titre of autoantibodies to MDA-LDL was an independent predictor of the progression of carotid atherosclerosis in these Finnish men. Our data provide further support for a role of oxidatively modified LDL in atherogenesis.

To determine the feasibility of intravascular ultrasound imaging in vivo, a miniaturized high frequency transducer catheter was introduced into human peripheral (n = 10) and coronary (n = 4) arteries. Cross-sectional ultrasound images were obtained from iliofemoral arteries in 10 patients using a 20 MHz transducer catheter (1.2 mm in diameter) and from coronary arteries in 4 patients using a 30 MHz transducer catheter 5 French size (Fr) following successful coronary angioplasty. Ultrasound images obtained from peripheral arteries showed a three-layered appearance (echo-reflective intima, echo-lucent media and echo-reflective adventitia) in the normal arteries. In diseased arteries, the location, amount and extent of atheromatous plaque were clearly documented. The arterial diameters measured by ultrasound closely correlated with the measurements by angiography (r = 0.91) in the peripheral arteries. Coronary angiograms obtained following balloon angioplasty revealed smooth edges at the dilatation sites without significant narrowing in all patients. However, a significant amount of residual atheromatous plaque was clearly observed on the ultrasound images at the previously dilated sites. Coronary dissection, which was identified as an echo-lucent area behind the plaque, was noted in 2 patients. Ultrasound images also revealed the presence of calcium in the plaque which was unrecognized on the angiograms in 3 patients. In addition, direct measurement of the lumen cross-sectional area was possible on the ultrasound images.(ABSTRACT TRUNCATED AT 250 WORDS)