Increased Vascularization in the Vulnerable Upstream Regions of Both Early and Advanced Human Carotid Atherosclerosis.

BACKGROUND: Vascularization of atherosclerotic plaques has been linked to plaque vulnerability. The aim of this study was to test if the vascularization was increased in upstream regions of early atherosclerotic carotid plaques and also to test if the same pattern of vascularization was seen in complicated, symptomatic plaques. METHODS: We enrolled 45 subjects with early atherosclerotic lesions for contrast enhanced ultrasound and evaluated the percentage of plaque area in a longitudinal ultrasound section which contained contrast agent. Contrast-agent uptake was evaluated in both the upstream and downstream regions of the plaque. We also collected carotid endarterectomy specimens from 56 subjects and upstream and downstream regions were localized using magnetic resonance angiography and analyzed using histopathology and immunohistochemistry. RESULTS: Vascularization was increased in the upstream regions of early carotid plaques compared with downstream regions (30% vs. 23%, p = 0.033). Vascularization was also increased in the upstream regions of advanced atherosclerotic lesions compared with downstream regions (4.6 vs. 1.4 vessels/mm2, p = 0.001) and was associated with intra-plaque hemorrhage and inflammation. CONCLUSIONS: Vascularization is increased in the upstream regions of both early and advanced plaques and is in advanced lesions mainly driven by inflammation.

Cadmium exposure as measured in blood in relation to macrophage density in symptomatic atherosclerotic plaques from human carotid artery.

BACKGROUND AND AIMS: The general population is exposed to cadmium through diet and smoking. Cadmium is pro-atherogenic and pro-inflammatory in experimental and observational studies. Cadmium levels in blood and carotid plaque endarterectomies correlate. Cadmium concentrations are much higher in plaque-areas that most frequently rupture. Here we investigated if blood cadmium concentrations are associated with macrophage density and the accumulation of CD14 as indicator of macrophage activation by lipopolysaccharide (LPS) in endarterectomies from patients with symptomatic carotid plaques. METHODS: Endarterectomies from ninety nine patients were fixed in formalin, embedded in paraffin, serially sectioned and stained for assessment of morphology. As predefined, the two section levels with most prevalent plaque rupture were used for further analyses. Macrophages were assessed as area of staining for CD68 (%). Blood cadmium was measured with ICP-MS. RESULTS: The CD68 median [25,75 percentiles] from the average of both sections were higher in cadmium tertile 3 than in tertile 1 (9.8 [4.9,16.1] % and 3.8 (0.6,12.4) %, p = 0.017). This difference remained in a multiple linear regression analysis with (10)log meanCD68 as dependent variable and adjustment for sex, age, smoking, statin treatment, index event, time between event and surgery (beta coefficient 0.44 [95% CI 0.05-0.87]. CD14 was not associated with blood cadmium. CONCLUSIONS: The results showed that blood cadmium was associated with proinflammatory macrophage density in the sections of carotid plaques with most frequent rupture, previously shown to contain most cadmium. No association between cadmium and LPS-mediated macrophage-activation was found. Cadmium exposure may promote plaque inflammation.

Increased vascularization of shoulder regions of carotid atherosclerotic plaques from patients with diabetes.

OBJECTIVE: Increased vascularization is considered an important contributing factor for plaque vulnerability. Microvascular proliferative disease in patients with diabetes results in renal damage and visual loss. We assessed the hypothesis that vascularization in carotid atherosclerotic tissue is increased in diabetic patients, especially in the critical shoulder regions of the plaque. METHODS: Carotid endarterectomy specimens, clinical data, and blood samples were collected from patients with symptomatic carotid artery stenosis (median 85 days after clinical event) and pharmacologic treatment for diabetes (n = 26) or no diabetes (n = 85). Plaques were fixed in formalin and transverse tissue sections prepared. Histopathology and immunohistochemistry were performed for detection of endothelial cells (anti-CD34), macrophages (anti-CD68), vascular endothelial growth factor (VEGF), and its receptor (VEGFR-2). Neovascularization was assessed as CD34(+) neovessel density in the entire section area and by the presence or absence of CD34(+) vessels in the shoulder and cap regions of the plaques. RESULTS: The patient groups did not differ significantly in neovascularization in the entire transverse sections (2.0 vs 2.1 vessels/mm(2); P = .61) or in the fibrous cap (52% of the patients in both groups; P = .95). Neovascularization of the plaque shoulder regions was observed in 52% of the diabetic patients and in 26% of the nondiabetic patients (P = .028). VEGF-stained areas were similar in the two patient groups (0.4% and 0.2% of shoulder area; P = .61). Patients with diabetes had more VEGFR-2 (1.0% vs 0.2% of shoulder area; P < .016) and less CD68 staining (0.4% vs 3.6% of shoulder area; P < .008). Time from clinical event to surgery was positively associated with neovascularization of the plaque shoulder regions (≤90 days, 18% of patients; >90 days, 50% of patients; P = .002), independently of diabetes status. CONCLUSIONS: Diabetes was associated with increased vascularization of the shoulder regions in patients with symptomatic carotid atherosclerotic plaques. This was accompanied by increased expression of VEGFR-2. The increased vascularization of the plaque shoulder regions may help explain why patients with diabetes are at increased risk of atherosclerotic complications.

Differences in lesion severity and cellular composition between in vivo assessed upstream and downstream sides of human symptomatic carotid atherosclerotic plaques.

BACKGROUND: The heterogeneous structure of carotid atherosclerotic plaques may be better understood if it is related to blood flow variations, influencing gene expression and cellular functions. Upstream of the maximum stenosis there is laminar blood flow and high shear stress, downstream there is turbulence and low shear stress. We studied if these variations were associated with differences in plaque morphology and composition between sites located up- and downstream of the maximum stenosis in symptomatic carotid plaques. METHODS: Patients with symptomatic carotid stenosis were examined with magnetic resonance angiography to localize the maximum stenosis in-vivo, prior to endarterectomy. In 41 endarterectomized specimens, transverse tissue sections prepared up- and downstream of the maximum stenosis were compared using histopathology and immunohistochemistry. RESULTS: The location of maximum stenosis relative the carotid bifurcation varied considerably between plaques. Compared with the downstream side, the upstream side of the stenosis had higher incidence of severe lesions with cap rupture and intraplaque hemorrhage, more macrophages, less smooth muscle cells and more collagen. CONCLUSIONS: The up- and downstream sides of symptomatic carotid plaques differed in plaque morphology and composition. This implies that the intraplaque location of sampling sites may be a confounding factor in studies of atherosclerotic plaques.

Expression of chemokine (C-C motif) ligand 18 in human macrophages and atherosclerotic plaques.

OBJECTIVE: Using gene expression profiling, we aimed to identify genes that are predominantly expressed in human carotid atherosclerotic plaques. Such genes may be important in atherogenesis and pathophysiology of the plaque, and genes that encode for secreted proteins may be potential biomarkers for atherosclerosis and cardiovascular disease. METHODS: DNA microarray generated expression profiles of human carotid atherosclerotic plaques were compared to expression profiles of 80 different human tissues and cell types, to identify plaque-specific genes. RESULTS: We identified the chemokine (C-C motif) ligand 18 (CCL18) as predominantly expressed in human carotid plaque. Immunohistochemistry showed that CCL18 protein was localized to a subset of macrophages in carotid plaques. Monocyte-derived macrophages from subjects with atherosclerosis had threefold higher expression of CCL18 than macrophages from control subjects (p=0.012). Subjects with A/G genotype of the rs2015086 SNP in the promoter region of the CCL18 gene had threefold higher macrophage expression of CCL18 than subjects with A/A genotype (p=0.049), but we found no association of this SNP with an increased risk of coronary heart disease. We also compared serum levels of CCL18 from subjects with symptomatic carotid artery disease with control subjects. There were no differences in serum levels of CCL18 between the two groups, however CCL18 correlated with measurements of adiposity. CONCLUSION: CCL18 is predominantly expressed in human atherosclerotic plaques and may participate in the atherosclerotic plaque formation.