Basement membrane proteins in various arterial beds from individuals with and without type 2 diabetes mellitus: a proteome study.

BACKGROUND: Basement membrane (BM) accumulation is a hallmark of micro-vessel disease in diabetes mellitus (DM). We previously reported marked upregulation of BM components in internal thoracic arteries (ITAs) from type 2 DM (T2DM) patients by mass spectrometry. Here, we first sought to determine if BM accumulation is a common feature of different arteries in T2DM, and second, to identify other effects of T2DM on the arterial proteome. METHODS: Human arterial samples collected during heart and vascular surgery from well-characterized patients and stored in the Odense Artery Biobank were analysed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We included ascending thoracic aortas (ATA) (n = 10 (type 2 DM, T2DM) and n = 10 (non-DM)); laser capture micro-dissected plaque- and media compartments from carotid plaques (n = 10 (T2DM) and n = 9 (non-DM)); and media- and adventitia compartments from ITAs (n = 9 (T2DM) and n = 7 (non-DM)). RESULTS: We first extended our previous finding of BM accumulation in arteries from T2DM patients, as 7 of 12 pre-defined BM proteins were significantly upregulated in bulk ATAs consisting of > 90% media. Although less pronounced, BM components tended to be upregulated in the media of ITAs from T2DM patients, but not in the neighbouring adventitia. Overall, we did not detect effects on BM proteins in carotid plaques or in the plaque-associated media. Instead, complement factors, an RNA-binding protein and fibrinogens appeared to be regulated in these tissues from T2DM patients. CONCLUSION: Our results suggest that accumulation of BM proteins is a general phenomenon in the medial layer of non-atherosclerotic arteries in patients with T2DM. Moreover, we identify additional T2DM-associated effects on the arterial proteome, which requires validation in future studies.

Cholesterol Crystals are Associated with Carotid Plaque Vulnerability: An Optical Coherence Tomography Study.

OBJECTIVE: Vulnerable carotid plaque is associated with cerebrovascular events. Cholesterol crystals are often seen in the atherosclerotic plaques. However, the potential role of cholesterol crystals in carotid plaques destabilization is unknown. We aimed to identify the association between cholesterol crystals and carotid plaque vulnerability. METHODS: Optical coherence tomography assessment of carotid plaque was performed in 95 patients. Clinical characteristics and plaque morphology were examined. The differences in plaque characteristics (thrombus, calcification, neovascularization, and macrophage accumulations) and clinical parameters (age, symptom, coronary heart disease, total cholesterol, triglycerides, and C-reactive protein) between patients with or without cholesterol crystals were analyzed with multivariate logistic regression. RESULTS: Among 66 patients with acceptable carotid atherosclerotic optical coherence tomography images, 16 were with and 50 were without cholesterol crystals. 56.3% patients (9 of 16) with cholesterol crystals had cerebrovascular ischemic symptom related to ipsilateral internal carotid artery, whereas only 26.0% patients (13 of 50) without cholesterol crystals had symptom (OR, 3.66; 95% CI, 1.13-11.82; P = .025). 75.0% of the plaques with cholesterol crystals had concomitant macrophage accumulation (OR, 4.14; 95% CI, 1.17-14.65; P = .04). In segments with cholesterol crystals, a higher presence of calcification could be demonstrated compared to those without cholesterol crystals (62.5% versus 32.0%, P = .03). 70.0% plaques with cholesterol crystals and calcification were classified as symptomatic plaques (OR, 6.38; 95% CI, 1.46-27.91; P = .01). No association between plaque rupture and cholesterol crystals was identified. Multivariate logistic regression showed that age and macrophage accumulation were independently associated with cholesterol crystals. CONCLUSIONS: Carotid atherosclerotic plaques with cholesterol crystals were more likely to have concomitant macrophage and calcification accumulations. Patients with cholesterol crystals plaque experienced more cerebrovascular symptoms. Thus, cholesterol crystals, especially together with macrophage and calcification, may serve as an important component of venerable carotid plaques.

The Relevance of Vascular Endothelial Growth Factor, Hypoxia Inducible Factor-1 Alpha, and Clusterin in Carotid Plaque Instability.

BACKGROUND: Stroke is one of the leading causes of morbidity and mortality. Thromboembolism, as a major cause of carotid artery-related stroke, can be caused by plaque rupture which is associated with neoangiogenesis within the carotid plaque. AIM: We sought to investigate a possible correlation between angiogenesis-related factors and preoperative neurological manifestations in patients with internal carotid artery stenosis, for a better understanding of thromboembolism in internal carotid artery stenosis-related stroke. METHODS: This study included 54 patients (asymptomatic, n = 20 and symptomatic, n = 34) undergoing carotid endarterectomy for high-grade internal carotid artery stenosis. In the retrieved carotid plaques, angiogenesis-related factors (vascular endothelial growth factor [VEGF], hypoxia inducible factor-1 alpha [HIF-1α], and Clusterin) were measured by immunohistochemistry and quantified by real-time polymerase chain reaction. RESULTS: We demonstrated the expression of VEGF, HIF-1α, and Clusterin by endothelial cells and smooth muscle cells in the carotid plaques. Noteworthy, mRNA VEGF levels were .7-fold higher in symptomatic patients (P = .017) compared to asymptomatic patients. In contrast, mRNA Clusterin levels were 1.8-fold lower (P = .021). Levels of mRNA HIF-1α were 1.5-fold higher in asymptomatic patients, but no statistical significance was reached between the 2 groups. CONCLUSIONS: Our results show an association between VEGF and Clusterin and neurological symptoms of patients with high-grade carotid artery stenosis.

A Microscopic and Biomarker Evaluation of Embolic Filter Debris Collected During Carotid Artery Stenting.

PURPOSE: To evaluate and characterize debris retrieved from the cerebral embolic protection devices (EPDs) used during carotid artery stenting (CAS) and compare debris size, volume, tissue types, cellular composition, and protein biomarker expression in symptomatic and asymptomatic patients. METHODS: Distal protection filters were retrieved from 22 consecutive patients (mean age 71.6 years, range 52-85; 16 men) undergoing elective CAS between July 2012 and February 2014 for >70% internal carotid artery stenosis (mean 85.4% ± 10.3%). Six patients were symptomatic. The debris within each EPD was visually characterized using stereomicroscopy and then processed for histology and immunohistochemistry. Biomarkers were immunohistochemically measured to evaluate plaque stability [matrix metalloproteinase-9 (MMP-9)], inflammation [glycoprotein CD68 and interleukin-6 (IL-6)], or phenotype [smooth muscle (SM)-actin and type IV collagen]. The immunohistochemical results were measured using semiquantitative grading criteria based on both staining intensity and distribution in the samples. RESULTS: Macroscopic debris was visible in 5/22 EPDs; 3 of the 5 filters came from symptomatic patients. Microscopic debris was detected in all filters and ranged in size from 0.01 to 8.57 mm(2). Debris consisted of calcified, fibrous, and necrotic tissue, as well as fibrin and foam cells with no significant difference between the symptomatic and asymptomatic groups. There was no association between the degree or type of embolic material and stenosis severity, carotid tortuosity, calcium grade, soft plaque, or arch type. Symptomatic patients had a larger volume of debris (8.24 vs 0.58 mm(3), p<0.01), mean particle size (1.30 vs 0.32 mm(2), p<0.001), and expression of biomarkers IL-6 (2.17 vs 0.81, p<0.05), CD68 (2.00 vs 0.38, p<0.01), SM-actin (1.00 vs 0.25, p=0.055), type IV collagen (1.17 vs 0.25,p=0.082), and MMP-9 (1.00 vs 0.06, p<0.05). CONCLUSION: Histological analysis revealed particulate embolization in all EPDs used during CAS. Symptomatic patients had a larger volume of embolic debris, mean particle size, and the biomarkers associated with inflammation, necrotic core, and diminished fibrous cap.

Near-infrared spectroscopy combined with intravascular ultrasound in carotid arteries.

Limited insights into the pathophysiology of the atherosclerotic carotid stenosis are available in vivo. We conducted a prospective study to assess safety and feasibility of intravascular ultrasound (IVUS) combined with near-infrared spectroscopy (NIRS) in carotid arteries. In addition, we described the size and the distribution of lipid rich plaques in significant atherosclerotic carotid stenoses. In a prospective single centre study 45 consecutive patients (mean age 66 ± 8 years) with symptomatic (≥50 %) or asymptomatic (≥70 %) stenosis of internal carotid artery (ICA) amendable to carotid stenting were enrolled. A 40 mm long NIRS-IVUS pullback through the stenosis was performed. IVUS and NIRS data were analyzed to assess minimal luminal area (MLA), plaque burden (PB), remodeling index (RI), calcifications, lipid core burden index (LCBI), maximal LCBI in any 4 mm segment of the artery (LCBImx) and LCBI in the 4 mm segment at the site of minimal luminal area (LCBImxMLA). NIRS-IVUS pullbacks were safely performed without overt clinical events. LCBImx was significantly higher than LCBImxMLA (369.1 ± 221.1 vs. 215.7 ± 2589; p = 0.004). Conversely, PB was significantly larger at the site of MLA (87.4 ± 4.8 % vs. 58.3 ± 18.2 %; p < 0001). Distance of the NIRS-IVUS frame with the highest LCBI from the site of MLA was 6.5 ± 7.7 mm. Eighty percent of frames with maximal LCBI were localized within 10 mm from the site of MLA and 67 % proximally to or at the site of MLA. This study suggested safety and feasibility of the NIRS-IVUS imaging of the carotid stenosis and provided insights on the distribution of lipids in the carotid stenosis. Lipid rich plaques were more often located in the sites with a milder stenosis and smaller plaque burden than at the site of MLA.

MicroRNA deregulation in symptomatic carotid plaque.

OBJECTIVE: Embolization of carotid stenotic plaques is the direct cause of stroke in nearly 20% of cases. Genetic mechanisms and especially the roles played by microRNAs in the regulation of plaque destabilization and rupture are mostly unknown. The aim of this pilot study was to compare the expression of seven microRNAs allegedly involved in plaque growth and instability (miR-100, 125a, 127, 133a, 145, 155, and 221), between symptomatic and asymptomatic human carotid plaques. METHODS: Thirty patients undergoing carotid endarterectomy in our department were prospectively included. Carotid plaques were subdivided into symptomatic (n = 15) and asymptomatic (n = 15) according to the presence or absence of stroke. After isolation of total RNA from atherosclerotic plaques, microRNAs were quantified by real-time polymerase chain reaction. RESULTS: The two groups of patients were comparable in terms of age, gender, risk factors for cerebral ischemia, medication, and stenosis severity. All seven microRNAs were quantified in extracted carotid plaques. miR-100, miR-125a, miR-127, miR-133a, miR-145, and miR-221 were significantly overexpressed in symptomatic vs asymptomatic plaques. miR-125a expression was significantly inversely correlated with the circulating level of low-density lipoprotein cholesterol in the symptomatic group. CONCLUSIONS: This pilot study evaluated the expression of seven selected miRNAs in human carotid plaques from a small group of patients and suggested a potential regulatory role for these miRNAs in evolution of the plaque towards growth, instability and rupture. Studies based on larger sample sizes are required to determine the potential use of miR-100, miR-125a, miR-127, miR-133a, miR-145, and miR-221 as biomarkers or therapeutic targets for stroke.

Oxidized LDL in human carotid plaques is related to symptomatic carotid disease and lesion instability.

BACKGROUND: Oxidative stress is an important determinant in atherosclerosis development. Various markers of oxidative stress, such as oxidation of low-density lipoprotein (LDL), nitrosative stress, lipid peroxidation, and protein oxidation, have been implicated in the initiation and/or progression of atherosclerosis, but their association with plaque erosion and symptomatic carotid disease has not been fully defined. In addition, certain oxidative markers have been shown in various models to promote plaque remodeling through matrix metalloproteinase (MMP) activation. OBJECTIVE: To perform a global investigation of various oxidative stress markers and assess for potential relationships with destabilization and symptomatic development in human carotid plaques. METHODS: Thirty-six patients undergoing endarterectomy were evaluated and compared with 20 control specimens obtained at the time of autopsy. Differences between stable and unstable plaques, symptomatic and asymptomatic patients, and >or=90% and <90% stenosis were evaluated. Oxidized LDL (ox-LDL), nitrotyrosine (NT), malondialdehyde (MDA), and protein carbonyls (PCs) levels were determined in atheromatic plaques homogenates by corresponding biochemical assays. Immunohistochemical (IHC) analysis was also employed to determine the percentage and topological distribution of cells expressing NT and metalloproteinase-9 (MMP-9) in serial sections from corresponding atheromatic plaques. MMP-9 expression was further verified using Western blot analysis. RESULTS: Ox-LDL was increased in symptomatic patients (P < .05). Also, ox-LDL and NT levels were significantly higher in unstable versus stable carotid plaques (P < .05, respectively). Furthermore, IHC serial section analysis, corroborated by statistical analysis, showed a topological and expressional correlation between NT and MMP-9 (P < .05). MDA and PCs levels, although increased in carotid plaques, did not distinguish stable from unstable carotid plaques as well as symptomatic from asymptomatic patients with various degrees of stenosis. CONCLUSION: All types of investigated oxidative stress markers were significantly increased in human carotid plaques, but only ox-LDL levels were associated with clinical symptoms, while peroxynitrite products and MMP-9 were specifically related to plaque instability.

A comparative study of carotid atherosclerotic plaque microvessel density and angiogenic growth factor expression in symptomatic versus asymptomatic patients.

OBJECTIVE: A challenge facing clinicians is identifying patients with asymptomatic carotid disease at risk of plaque instability. We hypothesise that locally released angiogenic growth factors contribute to plaque instability. METHODS: Carotid endarterectomy specimens from eight symptomatic and eight asymptomatic patients were interrogated for microvessel density and angiogenic growth factor expression histologically using immunofluorescence, and biochemically using quantitative real-time polymerase chain reaction (q-RT-PCR). Bio-Plex suspension array was used to assess circulating biomarkers in venous blood from the same patients and six healthy age-matched controls. RESULTS: Immunofluorescence demonstrated significantly greater neovessel density in symptomatic plaques (P=0.010) with elevated expression of hepatocyte growth factor (HGF) (P=0.001) and its receptor MET (P=0.011) than in asymptomatic plaques. The q-RT-PCR demonstrated up-regulation of Endoglin (CD105), HGF (P=0.001) and MET (P=0.011) in the plaques of symptomatic versus asymptomatic patients. Bio-Plex suspension array demonstrated elevated HGF (P=0.002) serum levels in symptomatic versus asymptomatic patients and healthy controls, and decreased platelet-derived growth factor (PDGF) (P=0.036) serum levels in symptomatic versus asymptomatic patients. CONCLUSION: Plaque instability may be mediated by HGF-induced formation of new microvessels, and decreased vessel stability resulting from decreased PDGF. Suspension array technology has the potential to identify circulating biomarkers that correlate with plaque rupture risk.

Carotid plaque composition in chronic kidney disease: a retrospective analysis of patients undergoing carotid endarterectomy.

OBJECTIVES: Calcified plaques are suggested to represent atherosclerotic lesions with stabilising properties. However, patients with chronic kidney disease (CKD) frequently have calcified plaques but significant higher prevalence of cardiovascular complications. The aim of our study was therefore to analyse the effect of CKD in patients with advanced carotid stenosis (>70%) on plaque composition, lesion stability and risk of rupture. METHODS: We investigated retrospectively, by histology, carotid plaques of patients with high-grade internal carotid artery stenosis undergoing carotid endarterectomy. Comparison of plaque morphology was performed on 41 patients with CKD with estimated glomerular filtration rate (eGFR) <60 ml min(-1) (according to the Modification of Diet in Renal Disease formula, MDRD-eGFR) and 56 patients with normal renal function. RESULTS: Patients with CKD had significantly higher percentage of total calcification (17% vs. 7%, p<0.001), unstable and ruptured plaques (83% vs. 52%, p=0.001 and 59% vs. 36%, p=0.039, respectively) compared with patients with normal renal function. By contrast, the content of collagenous fibres was significantly reduced in CKD patients (40% vs. 57%, p=0.011). No significant differences were found for neurological symptoms and soft plaque content. CONCLUSION: Our results demonstrate that CKD significantly affects plaque composition in patients with advanced carotid artery stenosis. Enhanced calcification and reduced collagenous plaque may lead to plaque instability and rupture.

Up-regulation of a hydrogen peroxide-responsive pre-mRNA binding protein in atherosclerosis and intimal hyperplasia.

BACKGROUND: Multiple lines of investigation have implicated hydrogen peroxide (H(2)O(2)) as an important endogenous mediator of cell proliferation in the vessel wall. Heterogeneous nuclear ribonucleoprotein C (hnRNP-C), a nuclear pre-mRNA binding protein that plays roles in vertebrate cell proliferation and differentiation, has been identified as a component of a vascular cell signaling pathway activated by low physiologic levels of H(2)O(2). The expression of hnRNP-C in human arteries has not previously been assessed. METHODS: Segments of human proximal internal carotid arteries were evaluated for the expression of hnRNP-C by immunohistochemistry. RESULTS: In normal proximal internal carotid arteries, hnRNP-C is expressed predominantly by the endothelium, with significantly lower expression by medial smooth muscle. In preatherosclerotic intimal hyperplasia, hnRNP-C is up-regulated in the artery wall, due to the robust expression by the intimal smooth muscle cells, without up-regulation in the medial smooth muscle cells. In arteries with atherosclerotic lesions, there is strong expression of hnRNP-C not only by intimal cells but also by medial smooth muscle cells. CONCLUSIONS: The H(2)O(2) responsive pre-mRNA binding protein hnRNP-C is up-regulated in atherosclerosis and in preatherosclerotic intimal hyperplasia in humans, supporting the hypothesis that H(2)O(2) is a regulator of vascular cell proliferation in these conditions. These data also suggest that hnRNP-C may be useful as a marker of vascular cell activation.

Quantitative evaluation of carotid plaque echogenicity by integrated backscatter analysis: correlation with symptomatic history and histologic findings.

BACKGROUND AND PURPOSE: Echogenicity of carotid plaque well reflects the risk of ischemic stroke and may be predictive of the histologic content of the plaque. However, objective evaluation of plaque echogenicity has been hampered by a lack of established quantitative measures. This study examined the relation between echogenicity assessed by integrated backscatter (IBS) analysis and (1) symptomatic history and (2) histologic features of carotid plaques. METHODS: We used acoustic densitometry to quantify by IBS analysis the echogenicity of 31 carotid plaques of 26 patients undergoing carotid endarterectomy or stenting. IBS was subsequently compared with histologic findings of the respective tissue in 10 patients who underwent endarterectomy. The IBS value was calibrated with 2 reference structures (vessel lumen and adventitia) as the IBS index. RESULTS: The IBS index of symptomatic plaques was lower than that of asymptomatic plaques (23.1 +/- 12.5 vs. 36.5 +/- 18.2, p < 0.05). The IBS index in fatty/necrotic atheromatous sites (n = 20, 16.6 +/- 10.7) was lower than that in fibrous (n = 26, 42.4 +/- 13.6, p < 0.01) or calcified (n = 11, 87.7 +/- 17.4, p < 0.01) sites and the same as that in intraplaque hemorrhagic sites (n = 50, 23.6 +/- 16.9). CONCLUSIONS: Carotid plaque echogenicity, as quantitatively assessed by IBS analysis, correlates well with the presence or absence of prior symptoms and histologic contents of the plaques. IBS analysis may aid in the assessment of carotid plaque-related risk of stroke.

Contrast-enhanced ultrasound imaging of atherosclerotic carotid plaque neovascularization: a new surrogate marker of atherosclerosis?

An atherosclerotic plaque requires a nutrient blood supply, which is predominantly derived from arterial vasa vasorum. A variety of factors (environmental and genetic) contribute to the initiation and growth of atherosclerosis within vessel walls. Chemotactic factors, such as tissue ischemic and hypoxic factors, stimulate the release of vascular endothelial growth factor (VEGF) proteins, resulting in vessel wall angiogenesis. These developments often precede the formation of the luminal plaque. In this report, we describe the use of contrast-enhanced carotid ultrasound (CECU) imaging for the detection and quantification of intra-plaque neovascularization. The efficacy of CECU was measured against the neovascular density observed within the tissue specimens obtained at the time of carotid endarterectomy surgery. The objective of this study was to provide a histologic correlation between CECU and carotid artery atherosclerotic plaque neovascularization. Fifteen patients with significant atherosclerotic carotid artery disease received a CECU examination prior to undergoing a carotid endarterectomy (CEA). Two patients received bilateral endarterectomies, resulting in a total of 17 cases. At the time of surgery, carotid plaque samples were surgically removed and stained with specific vascular markers (CD31, CD34, von Willebrand factor, and hemosiderin) designed to identify the presence and degree of neovascularization. The intra-plaque neovascularization recorded on preoperative CECU was correlated with the degree of neovascularization noted in the tissue specimens. The CECU neovascularization was correlated to CD31-stained tissue specimens. This correlation value was 0.68 using Spearman's rank method. When CECU results were correlated with the other histologic markers (CD34, von Willebrand factor, and hemosiderin), a correlation of 0.50 was obtained. In conclusion, contrast-enhanced carotid ultrasound correlated to the presence and degree of intra-plaque neovascularization as determined from histology specimens.

Low levels of Nogo-B in human carotid atherosclerotic plaques are associated with an atheromatous phenotype, restenosis, and stenosis severity.

OBJECTIVE: Reticulon-4/Nogo (Nogo-B) protects mouse arteries from lumen loss by reducing smooth muscle cell (SMC) migration and intimal thickening. Our goal was to determine plaque and circulating levels of Nogo-B in atherosclerotic and control subjects. Therefore, we studied the relationships between local Nogo-B, plaque characteristics, and clinical data in patients undergoing carotid endarterectomy. METHODS AND RESULTS: Western blot analysis showed that endarterectomy specimens from the femoral (n=19) and carotid arteries (n=145) contained significantly less Nogo-B than nonatherosclerotic mammary arteries (n=8; P<0.003) and aortas (n=15; P=0.03). Immunohistochemistry revealed that in atherosclerotic lesions, Nogo-B was expressed by macrophage/foam cells, SMC rich, and neo-vascularized areas. Atheromatous plaques (>40% fat content) showed a significant reduction in Nogo-B expression (P=0.002). Nogo-B expression levels were significantly lower in patients with more than 90% of carotid stenosis (P=0.04) or restenotic lesions after prior carotid intervention (duplex; P=0.01). In contrast, plasmatic levels of Nogo-B (soluble Nogo-B) did not differ between atherosclerotic subjects (n=68) and risk-factor matched controls (n=63; P=0.5). CONCLUSION: Our findings suggest that local reduction of Nogo-B in atherosclerotic tissue might contribute to plaque formation and/or instability triggering luminal narrowing. In contrast, plasma Nogo-B levels are not associated with clinically manifested atherosclerotic disease.

Quantification and characterization of carotid calcium with multi-detector CT-angiography.

OBJECTIVE: The aim of this study was to assess the accuracy of CT-angiography for identification and measurement of calcification of carotid atherosclerotic plaques and to characterise the content and distribution pattern of mineral calcium (hydroxyapatite, Ca) in carotid bifurcations and investigate its relationship with neurological symptoms. METHODS: Twenty-six patients with ICA stenosis > 60% (13 symptomatic, 13 asymptomatic) were selected for study. Ca was estimated from the weight of the ashed remnants of carotid endarterectomy (CEA) specimens in 11 patients. Calcium content (calcification volume (mm3),CV), and average calcium density (Hounsfield units (HU),CD), were determined by CT-angiography. The distribution pattern of calcium within the lesion (base (posterior), shoulder or luminal surface) was assessed in all cases. RESULTS: CT-derived estimation of CV and Ca mass (modified Agatston Score, (mAS) = CV x CD) showed a good correlation with its direct measurement in CEA specimens (r = 0.911 and 0.993 respectively, p < 0,005). Asymptomatic patients with ICA stenosis > 60% showed statistically significant higher content of Ca than those who were symptomatic (mAS: 122.6 +/- 138.0 HU mm3 vs 42.8 +/- 59.1 HU mm3, p = 0.04). Calcification on the surface of the plaque was observed more commonly in asymptomatic patients (9/12 vs 3/15, p = 0.006). Non-calcified or plaques with posterior calcification were 12 times more likely to be symptomatic (OR: 12, 95%CI 1.5-91.1, p = 0.021). CONCLUSIONS: CT-angiography permits the reliable quantification of calcification of carotid plaques. A lower content of calcium in carotid plaques, as well as its distribution in the base of the lesion, was associated with a greater prevalence of neurological symptoms. These parameters may be useful to identify those patients at higher risk of stroke.

Gelatinases [matrix metalloproteinase-2 (MMP-2) and MMP-9] induce carotid plaque instability but their systemic levels are not predictive of local events.

Matrix metalloproteinases (MMPs) appear to play a central role in atherosclerotic plaque remodeling; however, the relationship of increased MMP levels in inducing carotid plaque instability remains controversial. We investigated whether gelatinases (MMP-2 and MMP-9) are implicated in carotid intraplaque hemorrhage and whether their serum levels may predict local carotid events. Nineteen carotid specimens obtained by endarterectomy of 18 patients were studied. The presence of gross intraplaque hemorrhage was recorded before plaque removal and quantification of MMP-2, MMP-9, and tissue inhibitor of metalloproteinase-1 (TIMP-1) in extracts from (1) the more stenotic area of the plaque, (2) the periphery of the plaque, and (3) serum was performed by enzyme-linked immunosorbent assay. MMP-9 levels measured in extracts from the most stenotic area were significantly higher in patients with intraplaque hemorrhage (p = 0.007); however, serum levels showed no difference, while those taken from the periphery of the lesion were also increased but did not reach a statistically significant level (p = 0.06). An increase in MMP-2 values was observed in the periphery of the lesion (p = 0.04) in patients with intraplaque hemorrhage. TIMP-1 levels showed no difference between the two groups regardless of the presence or absence of intraplaque hemorrhage. No significant differences in MMP levels were observed between symptomatic and asymptomatic patients. Increased levels of MMPs, particularly MMP-9, have been implicated in carotid intraplaque hemorrhage without their serum levels being predictive of local events.

Regulation of thrombomodulin expression in human vascular smooth muscle cells by COX-2-derived prostaglandins.

There is concern that cyclooxygenase (COX)-2 inhibitors may promote atherothrombosis by inhibiting vascular formation of prostacyclin (PGI2) and an increased thrombotic risk of COX-2 inhibitors has been reported. It is widely accepted that the prothrombotic effects of COX-2 inhibitors can be explained by the removal of platelet-inhibitory PGI2. Using microarray chip technology, we have previously demonstrated that thrombomodulin (TM) mRNA is upregulated in cultured human coronary artery smooth muscle cells by the stable prostacyclin mimetic iloprost. This study is the first to demonstrate a stimulation of the expression of functionally active thrombomodulin in human smooth muscle cells by prostaglandins, endogenously formed via the COX-2 pathway. Because TM is an important inhibitor of blood coagulation, these findings provide a novel platelet-independent mechanism to explain the prothrombotic effects of COX-2 inhibitors. The full text of this article is available online at http://circres.ahajournals.org.

Multi-sequence in vivo MRI can quantify fibrous cap and lipid core components in human carotid atherosclerotic plaques.

OBJECTIVES: Risk of thrombo-embolic stroke is thought to be better reflected by carotid plaque composition than by luminal stenosis. We set out to determine whether high resolution MRI was a valid method of quantifying plaque components in vivo. DESIGN: A prospective cohort study validating in vivo MRI against histological analysis of excised carotid plaques. MATERIALS: Twenty-five recently symptomatic patients with severe internal carotid artery stenosis underwent pre-operative in vivo multi-sequence MRI of the carotid artery using a 1.5 T system. METHODS: Individual plaque constituents were characterized on axial MR images according to net signal intensities. Analysis of fibrous cap and lipid core content was quantified proportional to overall plaque area. Bland-Altman plots were generated, and intra-class coefficients computed to determine the level of agreement between the two methods and inter-observer variability. RESULTS: The intra-class correlation coefficients between two MR readers were 0.94 and 0.88 for quantifying fibrous cap and lipid core components, respectively. There was good agreement between MR and histology derived quantification of both fibrous cap and lipid core content; the mean % difference for fibrous cap was 0.75% (+/-2.86%) and for lipid core was 0.86% (+/-1.76%). CONCLUSION: High resolution carotid MRI can be used to quantify plaque components and may prove useful in risk stratification.

Carotid artery calcium: accuracy of a calcium score by computed tomography-an in vitro study with comparison to sonography and histology.

OBJECTIVES: The aim of this study was to compare methods of quantifying calcification of the internal carotid artery. MATERIALS AND METHODS: We examined 92 internal carotid artery endarterectomy specimens. Grey scale median (GSM) values were calculated from optimized B-mode scans. The degree of calcification was assessed using radiographic calcification grading. Plaques were processed histologically, and classified into: (1) calcium-rich hard plaques, (2) lipid-rich soft plaques, and (3) combined plaques. The specimens were scanned in CT-scanner. The calcium score was determined as described by Agatston. RESULTS: Histopathology and GSM results concurred in 39 out of 92 cases (kappa=0.088). There was no significant correlation between the calcium score and the median GSM value (R=0.005; P=0.959). Histology and CT morphology showed a significant concordance (P<0.001). Also CT and radiomorphological classification showed close agreement (R=0.628, P<0.001). CONCLUSIONS: Calcium scores calculated using CT morphology enable precise in vitro evaluation of the calcium content of plaques in the internal carotid. In contrast, grey scale median values do not adequately reflect calcification of plaques.

Blockade of the angiotensin II type 1 receptor stabilizes atherosclerotic plaques in humans by inhibiting prostaglandin E2-dependent matrix metalloproteinase activity.

BACKGROUND: Clinical trials have demonstrated that agents that inhibit the angiotensin II pathway confer benefit beyond the reduction of blood pressure alone. However, the molecular mechanism underlying this effect has yet to be investigated. Recently, we have demonstrated enhanced expression of inducible cyclooxygenase (COX) and prostaglandin (PG)E2-dependent synthase (COX-2/mPGES-1) in human symptomatic plaques and provided evidence that it is associated with metalloproteinase (MMP)-induced plaque rupture. Thus, the aim of this study was to characterize the effect of the angiotensin II type 1 (AT1) receptor antagonist irbesartan on the inflammatory infiltration and expression of COX-2/mPGES-1 and MMPs in human carotid plaques. METHODS AND RESULTS: Seventy patients with symptomatic carotid artery stenosis were randomized to irbesartan (300 mg/d) or chlorthalidone (50 mg/d) for 4 months before endarterectomy. Plaques were subjected to analysis of COX-1, COX-2, mPGES-1, MMP-2, and MMP-9, angiotensin II, AT(1), AT2, and collagen content by immunocytochemistry, Western blot, and reverse-transcriptase polymerase chain reaction, whereas zymography was used to detect MMP activity. Immunohistochemistry was also used to identify CD68+ macrophages, CD3+ T lymphocytes, smooth muscle cells (SMCs), and HLA-DR+ inflammatory cells. Plaques from the irbesartan group had fewer (P<0.0001) macrophages, T lymphocytes, and HLA-DR+ cells; less (P<0.0001) immunoreactivity for COX-2/mPGES-1 and MMPs; reduced (P<0.0001) gelatinolytic activity; and increased (P<0.0001) collagen content. It is worth noting that COX-2/mPGES-1 inhibition was observed after incubation in vitro with irbesartan but not with the selective AT2 blockade PD123,319. CONCLUSIONS: This study demonstrates that irbesartan decreases inflammation and inhibits COX-2/mPGES-1 expression in plaque macrophages, and this effect may in turn contribute to plaque stabilization by inhibition of MMP-induced plaque rupture.

Expression of thrombomodulin in patients with spontaneous occlusion of the circle of Willis.

BACKGROUND AND PURPOSE: We examined the expression of thrombomodulin, a recently isolated anticoagulant protein, in endothelial cells from patients with spontaneous occlusion of the circle of Willis (cerebrovascular moyamoya disease) to determine whether lack of the expression of thrombomodulin might lead to the thrombogenicity in patients with this disease. METHODS: The intracranial internal carotid arteries, the external carotid arteries, and the vertebral or basilar arteries from 12 autopsied patients who had this disease and eight control autopsied patients were examined immunohistochemically by using the antiserum against human thrombomodulin. RESULTS: All of the endothelial cells from the patients with this disease and from the control patients were positive for thrombomodulin. Immunoelectron microscopy also disclosed normal localization of thrombomodulin on the luminal plasma membrane. Immunohistochemically, we could find no significant differences in the expression of thrombomodulin among the arteries examined in this study. CONCLUSIONS: We conclude that as far as we investigated immunohistochemically, the thrombogenicity in this disease is almost unlikely to depend on the abnormal expression of thrombomodulin.