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.