Carotid Specimens Research Articles

Epigallocatechin-3-gallate is a potent phytochemical inhibitor of intimal hyperplasia in the wire-injured carotid artery

Epigallocatechin-3-gallate (EGCG), a catechin gallate ester, is the major component of green tea and has been demonstrated to inhibit tumor growth as well as inhibit smooth muscle cell migration. We evaluated the effect of the phytochemicals resveratrol, allicin, sulforaphane (SFN), and EGCG on intimal hyperplasia in the carotid artery injury model. Intimal hyperplasia was induced in carotid arteries of adult Sprague-Dawley rats with a wire injury. Experimental animals received intraperitoneal injections of one of the four phytochemicals daily beginning 1 day prior to surgery and continued for up to 4 weeks. Control animals were administered saline. Carotid specimens were harvested at 2 weeks and subjected to quantitative image analysis. In addition, EGCG specimens were analyzed for cell proliferation, immunohistochemistry, and Western blot analysis. Quantitative image analysis showed significant phytochemical suppression of intimal hyperplasia at 2 and 4 weeks postoperatively with EGCG (62% decrease in intimal area). Significant decreases were also noted at 2 weeks for SFN (56%) and resveratrol (44%), whereas the decrease with allicin (24%) was not significant. Quantification of intimal hyperplasia by intima:media ratio showed similar results. Cell proliferation assay of specimens demonstrated suppression by EGCG. Immunohistochemical staining of EGCG-treated specimens showed extracellular signal-regulated kinase (ERK) suppression but not of the c-jun N-terminal kinase or p38 pathways. Western blot analysis confirmed reduced ERK activation in arteries treated with EGCG. Intraperitoneal injection of the phytochemicals EGCG, SFN, resveratrol, and allicin have suppressive effects on the development of intimal hyperplasia in the carotid artery injury model, with maximal effect due to EGCG. The mechanism of EGCG action may be due to inhibition of ERK activation. EGCG may affect a common pathway underlying either neoplastic cellular growth or vascular smooth muscle cellular proliferation.

Carotid baroreceptors are mainly localized in the medial portions of the proximal internal carotid artery

To visualize baroreceptors in the human carotid bifurcation by light microscopy. Baroreceptor location is investigated in order to provide recommendations for the extent of adventitial stripping in the treatment for carotid sinus syndrome (CSS). Human carotid specimens were transversely cut in 20 μm sections. After immunohistochemical staining using antibodies to vesicular glutamate transporter 2 (VGLUT2) and protein gene product 9.5 (PGP 9.5), the presence of baroreceptor tissue was studied using light microscopic techniques. Visual assessment indicated that VGLUT2 and PGP 9.5 immunoreactivity was present in the adventitia of the carotid arteries and that nerve density was highest in the medial wall of the proximal first cm of the internal carotid artery (ICA). Human carotid baroreceptors, as reflected in immunoreactivity for VGLUT2 and PGP 9.5, are mainly localized in the medial portions of the proximal ICA. If surgical carotid denervation is indicated in patients suffering from carotid sinus syndrome, adventitial stripping of the proximal portion of the ICA should be sufficient.

Abstract TP228: Fluorescent Pegylated Nanoparticles are Localized in Macrophage-rich but Relatively Stable Human Carotid Atheromata

Background: Foam cell macrophages to produce matrix-disorganizing proteolytic enzymes may render atherosclerotic plaques prone to rupture, which causes thromboembolic stroke. Macrophages within atherosclerotic lesions were reported to take up low density lipoprotein (LDL)-sized nanoparticles by fluid-phase pinocytosis, indicating that fluid-phase pinocytosis of LDL is a mechanism for macrophage foam cell formation in vivo. Objective: To invesigate if fluorescent pegylated nanoparticles that are taken up by macrophages via pinocytosis could visualize vulnerable areas of human atheromata. Methods: Fresh carotid specimens from 36 patients undergoing carotid endarterectomy (after 3T carotid MRI) were tissue-cultured in DMEM (4 mL) with the fluorescent molecular imaging probe in 37°C CO 2 incubator. Before and 4 hours after the incubation, molecular optical imaging was performed using a Cy5.5 near-infrared fluorescent (NIRF) imaging machine with a charge-coupled device camera. The atherosclerotic plaques were classified according to the American Heart Association Report using 35 microsections (5μm thickness) from various pre-determined regions of selected carotid tissues (n=11). Immunohistochemical staining for macrophages (CD68) was performed using the avidin-biotin-peroxidase method. Results: High risk lesions (AHA Type IV ~ VIII) tended to show more frequent plaque regions with strong CD68 immunoreactivity (22 / 27) than did low risk lesion (2 / 8, p = 0.003). Unexpectedly however, low risk lesions (Type I: intial lesion with foam cells; Type II: fatty steak with multiple foam cell layers) tended to show more frequent in plaque regions with both strong CD68 immunoreactivity and high Cy5.5 NIRF signal (11 / 14) than the others (10 / 21, p = 0.07), suggesting that the nanoparticles could be taken up by relatively fresh foam cells in early atherosclerotic lesions rather than end-stage apoptonecrotic macrophages in advanced atheromata. Conclusion: Preliminary analysis of this on-going prospective study shows that fluorescent pegylated nanoparticles may localize macrophage-rich but relatively stable regions of human atheromata.

Sex-related differences in carotid plaque features and inflammation

Severe carotid stenosis is a frequent cause of stroke in both men and women. Whereas several sex-related comparisons are available on coronary atherosclerosis, there are few data appraising gender-specific features of carotid plaques. We aimed to systematically compare the pathology and inflammatory features of carotid plaques in men vs women. Carotid plaque specimens were collected from patients undergoing surgical endarterectomy for asymptomatic or symptomatic carotid stenosis. Histologic analysis was performed, as well as measurements of plaque composition and inflammation. A total of 457 patients were included (132 women, 325 men). Baseline analyses showed a greater prevalence of hypercholesterolemia, hypertension, and former smoking status in women, despite a higher Framingham Heart Score in men (all P < .05). Women had a lower prevalence of thrombotic plaques, smaller percentage area of necrotic core, and hemorrhage extension (all P < .05). Plaque inflammation analysis showed a lower concentration of inflammatory and, in particular, of macrophage foam cells in the plaque cap of women (both P < .05). These differences were, however, no longer significant at multivariable analysis, including several baseline features, such as symptom status and stenosis severity. Carotid plaques seem significantly different in women and men, but the main drivers of such pathologic differences are baseline features, including stenosis severity and symptom status.

MONITORING THE PROGRESSION OF ATHEROSCLEROSIS IN RABBITS MODEL WITH TARGETED ULTRASOUND DETECTION OF VASCULAR CELL ADHESION MOLECULE-1

ObjectivesThe aim of our study was to investigate the use of targeted contrast-enhanced high-frequency ultrasonography for molecular imaging of vascular cell adhesion molecule-1 (VCAM-1) expression on carotid artery in a...

Kidney Stone Volume Estimation from Computerized Tomography Images Using a Model Based Method of Correcting for the Point Spread Function

We propose a method to improve the accuracy of volume estimation of kidney stones from computerized tomography images. The proposed method consisted of 2 steps. A threshold equal to the average of the computerized tomography number of the object and the background was first applied to determine full width at half maximum volume. Correction factors were then applied, which were precalculated based on a model of a sphere and a 3-dimensional gaussian point spread function. The point spread function was measured in a computerized tomography scanner to represent the response of the scanner to a point-like object. Method accuracy was validated using 6 small cylindrical phantoms with 2 volumes of 21.87 and 99.9 mm(3), and 3 attenuations, respectively, and 76 kidney stones with a volume range of 6.3 to 317.4 mm(3). Volumes estimated by the proposed method were compared with full width at half maximum volumes. The proposed method was significantly more accurate than full width at half maximum volume (p <0.0001). The magnitude of improvement depended on stone volume with smaller stones benefiting more from the method. For kidney stones 10 to 20 mm(3) in volume the average improvement in accuracy was the greatest at 19.6%. The proposed method achieved significantly improved accuracy compared with threshold methods. This may lead to more accurate stone management.

Targeted Folate Receptor β Fluorescence Imaging as a Measure of Inflammation to Estimate Vulnerability Within Human Atherosclerotic Carotid Plaque

The probability of atherosclerotic plaque rupture and its thrombotic sequelae are thought to be increased at sites of macrophage accumulation. Folate receptor β (FR-β) is present on activated macrophages but not on quiescent macrophages or other immune cells. By conjugating the ligand folate with a fluorescent contrast agent, fluorescein isothiocyanate (FITC), we aimed to explore the potential role of FR-β fluorescence imaging in the distinction of vulnerable sites from more stable regions. Carotid specimens were taken from 20 patients and incubated with folate-FITC for 30 min. Ex vivo fluorescence imaging was performed to determine the exact location of folate-FITC uptake. Sections displaying regions of high uptake (determined as hot spots) were compared with sections showing low uptake (cold spots) through immunohistochemistry and real-time quantitative reverse-transcription polymerase chain reaction for FR-β. Hot spots showed significantly higher folate-FITC uptake than cold spots (P < 0.001). Hot spots tended to contain more macrophages and areas of hypoxia than cold spots. A positive correlation between messenger RNA levels of CD68 (marker for macrophages), FR-β (r = 0.53, P = 0.045), and hypoxia-inducible factor-1α expression (marker for intraplaque hypoxia; r = 0.55, P = 0.034) was found. Compared with areas with low folate-FITC uptake, areas of high folate-FITC uptake within human atherosclerotic plaques had an increased number of activated macrophages and higher areas of hypoxia. These characteristics of vulnerability imply that molecular imaging of FR-β through folate conjugates might be a good indicator for plaque vulnerability in future noninvasive imaging studies.

Abstract 122: Circulating CD28- T Cells Are Associated with Histological Features of Carotid Plaque Instability

Background: Circulating CD28- T-cells have been found to be higher in patients with acute coronary syndromes, suggesting a role for these cells in plaque instability. However, the association between circulating CD28- T-cells and carotid plaque instability as assessed by histology/immunohistochemistry has not been investigated. Methods/Results: 40 patients referred for carotid endarterectomy were recruited pre-operatively at the Royal Victoria Hospital in Montreal, Canada. A blood sample was obtained pre-operatively. Flow cytometry was performed on whole blood using a CD3, CD4, CD28 antibody cocktail (eBioscience, San Diego, United States) and a BD Bioscience (Mississauga, Canada) flow cytometer. Data was acquired as percentage of CD4+CD28- T-cells. The carotid plaque specimen was obtained from the operating room, fixed, decalcified, embedded in paraffin, and sections of 4μm were obtained from the site of maximum stenosis. Sections were stained with hematoxylin and eosin and with anti-CD3 (lymphocytes), anti-CD68 (macrophages/foam cells), and von Willebrand Factor (vWF, neovessels, all antibodies from Dako, Burlington, Canada). A vascular pathologist classified the plaques according to American Heart Association (AHA) classifications and graded CD3, CD68, and vWF staining on a semi-quantitative scale. Patients with more advanced lesions (AHA type 5 or 6) had a greater percentage of circulating CD4+CD28- T-cells [2.9% (2.7-5.7)] compared to patients with lower classifications [1.6% (1.0-4.2), P&lt;0.05]. Furthermore, patients with plaques that had ≥10 new vessels per section (feature of plaque instability) had a median of 4.5% [2.8-8.0] CD4+CD28- T-cells whereas patients with &lt;10 new vessels per section had only 2.3% [0.8-5.0] (P&lt;0.05). There were no significant differences when patients were separated according to high/low plaque lymphocytes/macrophages. Conclusion: We have shown that circulating CD28- T-cells are found in greater amounts in patients who have more advanced/unstable carotid plaques as assessed by histology and immunohistochemistry. This may point to a role for CD28- T-cells in the pathogenesis of atherosclerosis. Further studies are currently being carried out to confirm these results.

Role of the OPG/RANK/RANKL triad in calcifications of the atheromatous plaques: Comparison between carotid and femoral beds

Recent works demonstrated the difference of calcification genesis between carotid and femoral plaques, femoral plaques being more calcified. It has been clearly demonstrated that the molecular triad osteoprotegerin (OPG)/Receptor Activator of NFkB (RANK)/RANK Ligand (RANKL) exerts its activities in the osteoimmunology and vascular system. The aim of this study was to determine their expression and their potential role in calcifications of the atheromatous plaques located in two different peripheral arterial beds, carotid and femoral. The expression of OPG, RANK and RANKL was analyzed by immunochemistry in 40 carotid and femoral samples. Blood OPG and RANKL were quantified using specific ELISA assays. OPG staining was more frequently observed in carotid than in femoral plaques, especially in lipid core. Its expression correlated with macrophage infiltration more abundantly observed in carotid specimens. Surprisingly, serum OPG concentration was significantly lower in carotid population compared to femoral population while RANK and RANKL were equally expressed in both arterial beds. Carotid plaques that are less rich in calcium than femoral specimens, express more frequently OPG, this expression being correlated with the abundance of macrophages in the lesions. These data strengthen the key role played by OPG in the differential calcification in carotid and femoral plaques.

Planar biaxial characterization of diseased human coronary and carotid arteries for computational modeling

Computational models have the potential to provide precise estimates of stresses and strains associated with sites of coronary plaque rupture. However, lack of adequate mathematical description of diseased human vessel wall mechanical properties is hindering computational accuracy. The goal of this study is to characterize the behavior of diseased human coronary and carotid arteries using planar biaxial testing. Diseased coronary specimens exhibit relatively high stiffness (50–210kPa) and low extensibility (1–10%) at maximum equibiaxial stress (250kPa) compared to human carotid specimens and values commonly reported for porcine coronary arteries. A thick neointimal layer observed histologically appears to be associated with heightened stiffness and the direction of anisotropy of the specimens. Fung, Choi–Vito and modified Mooney–Rivlin constitutive equations fit the multiaxial data from multiple stress protocols well, and parameters from representative coronary specimens were utilized in a finite element model with fluid–solid interactions. Computed locations of maximal stress and strain are substantially altered, and magnitudes of maximum principal stress (48–65kPa) and strain (6.5–8%) in the vessel wall are lower than previously predicted using parameters from uniaxial tests. Taken together, the results demonstrate the importance of utilizing disease-matched multiaxial constitutive relationships within patient-specific computational models to accurately predict stress and strain within diseased coronary arteries.

Early atherosclerotic plaques show evidence of infection by Chlamydia pneumoniae

Chlamydia pneumoniae (Cpn) could play an important role in the development of atherosclerosis. Cpn interferes with HIF-1alpha regulation in infected host cells during intracellular replication in hypoxia. We obtained carotid artery specimens with low (n=38), high (n=25) levels of stenosis and 10 middle cerebral arteries. Fifty eight percent of the carotids with low levels of stenosis showed evidence of the viable organism. Ninety one percent of the positive results were derived from pre-atheromatous lesions. Only 12 percent of plaques removed at endarterectomy showed the presence of Cpn DNA. All middle cerebral arteries failed to show evidence of live Chlamydia. Ninety one percent of sera from 22 endarterectomy patients failed to show the presence of Cpn antibodies. Immunohistology of carotid arteries with low levels of stenosis was used to confirm the presence of HIF-1alpha in infected specimens and showed a correlation between the over-expression of HIF-1alpha and Cpn in the plaque (p less than 0.05). Cpn might play an important role in activation and development of the initial stages of atherosclerotic lesions.

Loss of PIKFyve Kinase Function Driven by Platelet Factor 4 Promoter Results in Platelet Lysosomal Storage Defects and Infiltration of Multiple Organs with Vacuolated Macrophages

Abstract 697Phosphatidylinositol kinases are crucial in the generation of diverse membrane phosphoinositides that regulate the intracellular signaling for essential cellular processes including membrane trafficking. PIKFyve is a kinase that generates phosphatidylinostol 3,5 bisphosphate (PtdIns(3,5)P2) from PtdIns(3)P. Yeast mutants lacking the PIKFyve homologue were unable to synthesize PtdIns(3,5)P2 and exhibited enlarged vacuoles, defective membrane recycling and abnormal protein sorting to multivesicular bodies. In human platelets, PtdIns(3,5)P2 synthesis increased following thrombin stimulation, and our data indicated that murine platelets express PIKFyve mRNA. We hypothesized that PIKFyve and its phospholipid product, PtdIns(3,5)P2 would be critical in the membrane trafficking during the biogenesis of platelet granules. To test this hypothesis, we engineered mice to lack PIKFyve specifically within their platelets and megakaryocytes. The resulting PIKFyvefl/fl mice contained a conditional targeting mutation within two exons encoding for a critical component of its kinase domain. The conditionally targeted mice were crossed with transgenic mice expressing Cre recombinase under the control of platelet factor 4 (PF4) promoter. As expected, RT-PCR analysis confirmed a complete loss of normal PIKFyve mRNA in platelets derived from PIKFyvefl/fl PF4 Cre+ mice. Surprisingly, by 3 weeks of age, PIKFyvefl/fl PF4 Cre+ mice started to display a robust pleomorphic phenotype characterized by dorsal hair loss and progressive weight gain due to generalized body swelling. By 5–6 months of age, the body weight of the PIKFyvefl/fl PF4 Cre+ mice was about 30% higher than that of the control littermate mice (PIKFyvefl/fl PF4 Cre- or PIKFyvefl/+ PF4 Cre+) and appeared acutely ill with decreased ambulation and feeding. A DEXA scan revealed that PIKFyvefl/fl PF4 Cre+ mice had approximately 50% lower body fat content compared to their control littermates. Necropsy of PIKFyvefl/fl PF4 Cre+ mice at about 6 months of age demonstrated that their weight gain was due to massive infiltration of largely vacuolated F4/80 expressing macrophages in multiple organs including skin, muscle, lung, heart, liver, pancreas, small and large intestines, kidney, genital organs, thymus and bone marrow. The vacuoles within these macrophages occupied the entire cytoplasm and were reminiscent of PIKFyve null yeast mutant cells. Unexpectedly, PCR analysis of genomic DNA from tissues infiltrated by these vacuolated macrophages revealed that PIKFyve deletion had occurred within these cells. This indicated that the PF4 Cre was expressed in these macrophages and led to targeting of PIKFyve and ultimately to the development of vacuolated macrophages that infiltrated multiple organs. Despite the pleomorphic phenotype, the platelet counts of the PIKFyvefl/fl PF4 Cre+ mice were normal. Furthermore, platelets lacking PIKFyve normally exposed P-selectin from alpha granules and released ATP from dense granules in response to agonist stimulation. Nevertheless, the PIKFyvefl/fl PF4 Cre+ platelets exhibited a three-fold increase of basal b-hexosaminidase, a lysosomal hydrolase. This data suggests a role for PIKFyve in the biogenesis of platelet lysosomes. Furthermore, in vivo platelet thrombus formation analysis using the ferric chloride induced carotid artery injury model revealed that PIKFyvefl/fl PF4 Cre+ mice made an occluding thrombus significantly faster than the control littermate mice demonstrating that they are prothrombotic. Histological analysis of the carotid artery specimen at the site of thrombus demonstrated that the intravascular side did not contain vacuolated macrophages. This suggests that the enhanced thrombosis formation was due to the increased lysosomal content within the platelets, and not due to infiltration by abnormal macrophages. We conclude that PF4 promoter driven selective deletion of PIKFyve kinase is associated with defective biogenesis of platelet lysosomes and a prothrombotic effect in vivo. Furthermore, PIKFyvefl/fl PF4 Cre+ mice display a pleomorphic phenotype associated with vacuolated macrophage accumulation in multiple organs and suggest that PF4 promoter driven Cre expression is not restricted to megakaryocytes and platelets. This study also highlights a potential problem in previous and current work using the PF4 Cre transgenic model. Disclosures:No relevant conflicts of interest to declare.

Carotid Plaque Vulnerability

Rupture of atherosclerotic plaques is one of the main causes of ischemic strokes. The aim of this study was to investigate carotid plaque vulnerability markers in relation to blood flow direction and the mechanisms leading to plaque rupture at the upstream side of carotid stenoses. Frequency and location of rupture, endothelial erosion, neovascularization, and hemorrhage were determined in longitudinal sections of 80 human carotid specimens. Plaques were immunohistochemically analyzed for markers of vulnerability. Plaque geometry was measured to reconstruct shape profiles of ruptured versus stable plaques and to perform computational fluid dynamics analyses. In 86% of ruptured plaques, rupture was observed upstream. In this region, neovascularization and hemorrhage were increased, along with increased immunoreactivity of vascular endothelial and connective tissue growth factor, whereas endothelial erosion was more frequent downstream. Proteolytic enzymes, mast cell chymase and cathepsin L, and the proapoptotic protein Bax showed significantly higher expression upstream as compared with the downstream shoulder of atherosclerotic lesions. Comparison of geometric profiles for ruptured and stable plaques showed increased longitudinal asymmetry of fibrous cap and lipid core thickness in ruptured plaques. The specific geometry of plaques ruptured upstream induced increased levels of shear stress and increased pressure drop between the upstream and the downstream plaque shoulders. Vulnerability of the upstream plaque region is associated with enhanced neovascularization, hemorrhage, and cap thinning induced by proteolytic and proapoptotic mechanisms. These processes are reflected in structural plaque characteristics, analyses of which could improve the efficacy of vascular diagnostics and prevention.

Statin treatment is associated with reduced toll-like receptor 4 immunohistochemical expression on carotid atherosclerotic plaques: a novel effect of statins

Toll-like receptor 4 (TLR4) has been recently implicated in inflammatory pathways involved in carotid plaque destabilization. Given that statins have plaque stabilization and inflammation reduction effects, we investigated whether TLR4 expression on carotid atherosclerotic plaques correlates with statin intake. Carotid atherosclerotic plaques were obtained on 140 patients (preoperative statin intake, n = 70). TLR4 immunohistochemical expression was investigated in endothelial cells (ECs), macrophages (MACs) and smooth muscle cells (SMCs) of carotid atheroma. TLR4 positivity, over-expression and intensity of immunostaining were compared in statin versus no-statin users. The results of this study showed that statin users had a significantly lower expression of TLR4 in ECs (P = 0.02, 0.001, 0.006 for TLR4 positivity, increased intensity and over-expression, respectively). Similarly, TLR4 positivity was less pronounced in carotid plaque MACs of statin users (P = 0.03). No carotid specimen with increased EC TLR4 intensity or over-expression was observed among statin users. The prevalence of any cerebrovascular accident was 61.4% in the 'no statin' versus 18.6% in the 'statin' group (odds ratio for statin use: 0.14, 95% CI: 0.07-0.31, P < 0.001). In conclusion, statin treatment is associated with attenuated TLR4 expression on human carotid atherosclerotic plaques and a reduced risk of carotid-related cerebrovascular events. TLR4 may potentially mediate statins' plaque stabilization effects. Further investigation is necessary.

Multispectral optoacoustic tomography of matrix metalloproteinase activity in vulnerable human carotid plaques.

AimsElevated expression of cathepsins, integrins and matrix metalloproteinases (MMPs) is typically associated with atherosclerotic plaque instability. While fluorescent tagging of such molecules has been amply demonstrated, no imaging method was so far shown capable of resolving these inflammation-associated tags with high fidelity and resolution beyond microscopic depths. This study is aimed at demonstrating a new method with high potential for noninvasive clinical cardiovascular diagnostics of vulnerable plaques using high-resolution deep-tissue multispectral optoacoustic tomography (MSOT) technology.Methods and resultsMMP-sensitive activatable fluorescent probe (MMPSense™ 680) was applied to human carotid plaques from symptomatic patients. Atherosclerotic activity was detected by tuning MSOT wavelengths to activation-dependent absorption changes of the molecules, structurally modified in the presence of enzymes. MSOT analysis simultaneously provided morphology along with heterogeneous MMP activity with better than 200 micron resolution throughout the intact plaque tissue. The results corresponded well with epi-fluorescence images made from thin cryosections. Elevated MMP activity was further confirmed by in situ zymography, accompanied by increased macrophage influx.ConclusionsWe demonstrated, for the first time to our knowledge, the ability of MSOT to provide volumetric images of activatable molecular probe distribution deep within optically diffuse tissues. High-resolution mapping of MMP activity was achieved deep in the vulnerable plaque of intact human carotid specimens. This performance directly relates to pre-clinical screening applications in animal models and to clinical decision potential as it might eventually allow for highly specific visualization and staging of plaque vulnerability thus impacting therapeutic clinical decision making.

Carotid and femoral atherosclerotic plaques show different morphology

ObjectiveResults of endovascular repair vary according to the arterial bed. We hypothesized that these differences may be related to the plaque features. To explore this hypothesis, we designed a prospective study that compared carotid and femoral atheroma. Methods and resultsPatients that underwent femoral or carotid endarterectomy were included in our study. Demographic data and blood sampling were obtained prior to surgery. Plaques were evaluated for AHA grading, calcification and lipid content. Eighty-eight plaques were harvested during this study (45 carotid specimens and 43 femoral specimens). No differences were noted between carotid and femoral groups regarding demographic and biological data. Histological data more frequently showed fibrous cap atheroma in carotid arteries (75%) and fibrocalcific plaques in femoral arteries (93%), p<0.001. Morphological analyses showed a high prevalence of osteoid metaplasia in femoral arteries (63%) compared to carotid arteries (20%, p<0.001). Biochemical analyses were consistent with histological data, showing higher calcium and lesser cholesterol concentrations in femoral than in carotid plaques (p<0.01). ConclusionsFemoral and carotid plaques showed different morphology in comparable groups of patients.

Detection of Human Herpesvirus 6 DNA but not Human Herpesvirus 7 or 8 DNA in Atherosclerotic and Nonatherosclerotic Vascular Tissues

Various viral infections are thought to play a role in the development of atherosclerosis. A number of studies suggest that certain viruses from the Herpesviridae family in particular may lead to atherosclerosis. We investigated the presence of human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), and human herpesvirus 8 (HHV-8) DNA in carotid, iliac, and coronary artery specimens obtained from a group of adult autopsy cases by means of polymerase chain reaction (PCR) analysis and nested PCR techniques. A 28-subject study group with at least type IV atherosclerosis and a 25-subject control group with no visible atherosclerosis were enrolled. HHV-6 DNA was found in the carotid artery specimen of 1 subject with atherosclerosis, in an iliac artery specimen of another subject, and in the iliac artery specimen of one of the control subjects. HHV-7 or HHV-8 DNA was not found in either the atherosclerosis or control cases. This study is the first to demonstrate the presence of HHV-6 in atherosclerotic vascular tissues. HHV-7 and HHV-8 were not found in atherosclerotic tissues; however, further research on broader study groups and with different protocols is needed to determine whether these viruses play a role in the formation of atherosclerosis.

Evidence for markers of hypoxia and apoptosis in explanted human carotid atherosclerotic plaques

Apoptosis and inflammation are important features of atherosclerotic plaques. We investigated whether a common signal molecule can trigger these two apparently separate pathways. Hypoxia inducible factor (HIF-1α) is known to participate in atherosclerosis and to stimulate apoptosis signal-regulating kinase 1 (ASK-1), one of the mitogen-activated protein kinases, which is activated by various extracellular stimuli and involved in a variety of cellular function. We tested carotid artery specimens from 50 subjects who underwent angioplasty and five age-matched controls for either Western blot or histologic analysis. The hypoxic status was investigated by means of HIF-1α expression in carotid specimens. HIF-1α was significantly upregulated in carotid specimens with respect to controls (P < .05), ASK-1 was detected in plaques of any composition from lipidic to calcific, and this expression increased with the stage of the plaque and with the expression of inflammatory (p-ERK, RANK-L, OPG) and apoptotic molecules (caspase 9, p-p-38, and p-JNK). Our data suggest that hypoxia is the key regulating factor that triggers inflammation as well as apoptosis in the human atherosclerotic plaque.

Distribution of Inflammatory Mediators in Carotid and Femoral Plaques

Although atherosclerosis is a known systemic process, carotid and femoral atherosclerotic plaques are associated with distinctive complications and therapeutic outcomes. The purpose of this study was to evaluate inflammatory markers associated with carotid and femoral plaques. Carotid and femoral endarterectomy specimens were harvested from surgical patients. The carotid specimens were further sectioned into central, peripheral, and relatively normal regions. Expressions of matrix metalloproteinase (MMP)-2 and MMP-9 in carotid and femoral specimens were compared and the distributions of MMP-2, MMP-9, and CD40 within the carotid specimens were further analyzed. Messenger RNA (mRNA) levels were measured with real-time polymerase chain reaction and proteins with ELISA assay and Western blot. Despite no significant difference in the MMP-2 mRNA levels between carotid and femoral specimens, the common femoral specimens had significantly lower MMP-2 protein production and higher MMP-9 mRNA and protein expressions than those of the carotid specimens (p = 0.015, p = 0.03, and p = 0.034, respectively). Among carotid specimens, MMP-2 mRNA level was significantly lower in the central region (p < 0.01) and consistent with the distribution pattern of MMP-2 proteins. Interestingly, despite significantly lower MMP-9 mRNA expression in the central region of carotid plaques, active MMP-9 protein level was significantly higher. CD40 mRNA and protein levels were also higher in the central region. Immunohistochemistry analyses showed substantially increased macrophages and CD40 in the central region of the carotid plaques. This study emphasizes that femoral and carotid plaques are distinct entities and that distribution of inflammatory molecular markers varied within these advanced atherosclerotic plaques. Additional analyses are warranted.

Diffusion-weighted magnetic resonance imaging for the detection of lipid-rich necrotic core in carotid atheroma in vivo

Research has shown that knowing the morphology of carotid atheroma improves current risk stratification for predicting subsequent thrombo-embolic events. Previous magnetic resonance (MR) ex vivo studies have shown that diffusion-weighted imaging (DWI) can detect lipid-rich necrotic core (LR/NC) and fibrous cap. This study aims to establish if this is achievable in vivo. Twenty-six patients (mean age 73years, range 54-87years) with moderate to severe carotid stenosis confirmed on ultrasound were imaged. An echo-planar DWI sequence was performed along with standard high-resolution MR imaging. Apparent diffusion coefficient (ADC) maps were evaluated. Two independent readers reported the mean ADC values from regions of interest defining LR/NCs and fibrous caps. For subjects undergoing carotid endarterectomy (n = 19), carotid specimens were obtained and stained using Nile red. The mean ADC values were 1.0 × 10(-3) mm(2)/s (±SD 0.3 × 10(-3) mm(2)/s) and 0.7 × 10(-3) mm(2)/s (±SD 0.2 × 10(-3) mm(2)/s) for fibrous cap and LR/NC, respectively; the difference was significant (p < 0.0001). The intra-class correlation coefficients summarising the agreement between the two independent readers were 0.84 and 0.60 for fibrous cap and LR/NC, respectively. Comparison of quantitative ADC values and histology (by subjective grading of lipid content) showed a significant correlation: heavier lipid staining matched lower ADC values (r = -0.435, p = 0.005). This study indicates that DWI can be used to distinguish LR/NC and the fibrous cap. The study also suggests that the mean ADC value may be linearly related to subjective graded LR/NC content determined by histology.