LIPOPROTEIN ASSOCIATED PHOSPHOLIPASE A2 AS A MARKER OF VULNERABLE ATHEROSCLEROTIC PLAQUE IN PATIENTS WITH INTERNAL CAROTID ARTERY STENOSIS.

The aim of this study was to compare the concentration of inflammatory vascular markers and morphological structure of atherosclerotic plaque in symptomatic and asymptomatic patients with the stenosis of internal carotid artery (ICA). The research was carried out in 70 patients with hemodynamically significant stenosis of ICA out of which 40 (57%) were asymptomatic patients and 30 (43%) were symptomatic patients, of which 20 patients (66%) have had a stroke, or transient ischemic attack (TIA), 10 patients (33%). All the patients were indicated to carotid endarterectomy as a surgical prevention of stroke. All the patients were taken their blood for biochemical testing (T-Chol, LDL, HDL, TG, Fibrinogen, CRP and specific markers IL-4 and Lp-PLA2) early morning prior to surgery. The highest concentrations of T-Chol, LDL, HDL, CRP and Fibrinogen were measured in symptomatic patients, however, these did not feature a significant difference compared with the group of asymptomatic patients (P>0.05). Significant difference was found in IL-4 (P<0.001) and in Lp-PLA2 (P<0.001). When evaluating concentration of tracked parameters in patients with soft atherosclerotic plaque and patients with calcified atherosclerotic plaque, significant differences were found in these markers: TG (P<0.05), CRP (P<0.01), IL-4 (P<0.001) and Lp-PLA2 (P<0.001). The paper deals with higher concentrations of Lp-PLA2 in patients with a soft atherosclerotic plaque. Higher concentration of Lp-PLA2 and systemic inflammatory markers (CRP, IL-4) could be used along with ultrasonography to detect mainly asymptomatic patients who are in urgent need of surgical or endovascular treatment as a prevention of stroke.

Hydrocortisone supresses inflammatory activity of metalloproteinase - 8 in carotid plaque / Hidrocortisona suprime a atividade inflamatória da metaloproteinase - 8 presente na placa carotídea

AbstractObjective:Matrix metalloproteinases are inflammatory biomarkers involved in carotid plaque instability. Our objective was to analyze the inflammatory activity of plasma and carotid plaque MMP-8 and MMP-9 after intravenous administration of hydrocortisone.Methods:The study included 22 patients with stenosis ≥ 70% in the carotid artery (11 symptomatic and 11 asymptomatic) who underwent carotid endarterectomy. The patients were divided into two groups: Control Group - hydrocortisone was not administered, and Group 1 - 500 mg intravenous hydrocortisone was administered during anesthetic induction. Plasma levels of MMP-8 and MMP-9 were measured preoperatively (24 hours before carotid endarterectomy) and at 1 hour, 6 hours and 24 hours after carotid endarterectomy. In carotid plaque, tissue levels of MMP-8 and MMP-9 were measured.Results:Group 1 showed increased serum levels of MMP- 8 (994.28 pg/ml and 408.54 pg/ml, respectively; P=0.045) and MMP-9 (106,656.34 and 42,807.69 respectively; P=0.014) at 1 hour after carotid endarterectomy compared to the control group. Symptomatic patients in Group 1 exhibited lower tissue concentration of MMP-8 in comparison to the control group (143.89 pg/ml and 1317.36 respectively; P=0.003). There was a correlation between preoperative MMP-9 levels and tissue concentrations of MMP-8 (P=0.042) and MMP-9 (P=0.019) between symptomatic patients in the control group.Conclusion:Hydrocortisone reduces the concentration of MMP- 8 in carotid plaque, especially in symptomatic patients. There was an association between systemic and tissue inflammation.

ResumoObjetivo:As metaloproteinases são biomarcadores inflamatórios envolvidos na instabilidade da placa carotídea. O objetivo deste estudo foi analisar a atividade inflamatória da MMP-8 e MMP-9 plasmática e presente na placa carotídea, após administração intravenosa de hidrocortisona.Métodos:Participaram do estudo 22 pacientes portadores de estenose ≥ 70% em artéria carótida (11 sintomáticos e 11 assintomáticos), submetidos à endarterectomia de artéria carótida. Os pacientes foram divididos em dois grupos: Grupo Controle - não foi administrado hidrocortisona e Grupo 1 - foi administrado 500 mg intravenoso de hidrocortisona durante a indução anestésica. As dosagens plasmáticas de MMP-8 e MMP-9 foram efetuadas no pré-operatório (24 horas antes da endarterectomia de artéria carótida) e em 1 hora, 6 horas e 24 horas após endarterectomia de artéria carótida. Na placa carotídea foram mensurados os níveis teciduais de MMP-8 e MMP-9.Resultados:O grupo 1 exibiu elevação dos níveis séricos da MMP-8 (994,28 pg/ml e 408,54 pg/ml, respectivamente; P=0.045) e MMP-9 (106.656,34 e 42.807,69, respectivamente; P=0.014) em 1 hora após a endarterectomia de artéria carótida, em relação ao grupo controle. Os pacientes sintomáticos do grupo 1 exibiram menor concentração tecidual de MMP-8, em relação ao grupo controle (143,89 pg/ml e 1317,36, respectivamente; P=0.003). Houve correlação entre os níveis pré-operatórios de MMP-9 e as concentrações teciduais de MMP-8 (P=0.042) e MMP-9 (P=0.019) entre os pacientes sintomáticos do grupo controle.Conclusão:A hidrocortisona reduz a concentração de MMP-8 na placa carotídea, em especial nos pacientes sintomáticos. Houve associação entre a inflamação sistêmica e a tecidual.

Hydrocortisone supresses inflammatory activity of metalloproteinase-8 in carotid plaque.

OBJECTIVE: Matrix metalloproteinases are inflammatory biomarkers involved in carotid plaque instability. Our objective was to analyze the inflammatory activity of plasma and carotid plaque MMP-8 and MMP-9 after intravenous administration of hydrocortisone. METHODS: The study included 22 patients with stenosis ≥ 70% in the carotid artery (11 symptomatic and 11 asymptomatic) who underwent carotid endarterectomy. The patients were divided into two groups: Control Group - hydrocortisone was not administered, and Group 1 - 500 mg intravenous hydrocortisone was administered during anesthetic induction. Plasma levels of MMP-8 and MMP-9 were measured preoperatively (24 hours before carotid endarterectomy) and at 1 hour, 6 hours and 24 hours after carotid endarterectomy. In carotid plaque, tissue levels of MMP-8 and MMP-9 were measured. RESULTS: Group 1 showed increased serum levels of MMP- 8 (994.28 pg/ml and 408.54 pg/ml, respectively; P=0.045) and MMP-9 (106,656.34 and 42,807.69 respectively; P=0.014) at 1 hour after carotid endarterectomy compared to the control group. Symptomatic patients in Group 1 exhibited lower tissue concentration of MMP-8 in comparison to the control group (143.89 pg/ml and 1317.36 respectively; P=0.003). There was a correlation between preoperative MMP-9 levels and tissue concentrations of MMP-8 (P=0.042) and MMP-9 (P=0.019) between symptomatic patients in the control group. CONCLUSION: Hydrocortisone reduces the concentration of MMP- 8 in carotid plaque, especially in symptomatic patients. There was an association between systemic and tissue inflammation.

Association of the endogenous nitric oxide synthase inhibitor ADMA with carotid artery intimal media thickness in the Framingham Heart Study offspring cohort.

BACKGROUND AND PURPOSE: Higher plasma concentrations of the endogenous nitric oxides synthase inhibitor asymmetrical dimethylarginine (ADMA) are associated with increased risk of cardiovascular and cerebrovascular events and death, presumably by promoting endothelial dysfunction and subclinical atherosclerosis. We hypothesized that plasma ADMA concentrations are positively related to common carotid artery intimal-media thickness (CCA-IMT) and to internal carotid (ICA)/bulb IMT. METHODS: We investigated the cross-sectional relations of plasma ADMA with CCA-IMT and ICA/bulb IMT in 2958 Framingham Heart Study participants (mean age, 58 years; 55% women). RESULTS: In unadjusted analyses, ADMA was positively related to both CCA-IMT (beta per SD increment, 0.012; P<0.001) and ICA/bulb IMT (beta per SD increment, 0.059; P<0.001). In multivariable analyses (adjusting for age, sex, systolic blood pressure, antihypertensive treatment, smoking status, diabetes, BMI, total-to-HDL cholesterol ratio, log C-reactive protein, and serum creatinine), plasma ADMA was not associated with CCA-IMT (P=0.991), but remained significantly and positively related to ICA/bulb IMT (beta per SD increment, 0.0246; P=0.002). CONCLUSIONS: In our large community-based sample, we observed that higher plasma ADMA concentrations were associated with greater ICA/bulb IMT, but not with CCA-IMT. These data are consistent with the notion that ADMA promotes subclinical atherosclerosis in a site-specific manner, with a greater proatherogenic influence at known vulnerable sites in the arterial tree.

Markers of instability in high-risk carotid plaques are reduced by statins.

BACKGROUND: Macrophage infiltration and expression of matrix metalloproteinase-9 (MMP-9) are markers of high-risk atherosclerotic carotid plaques and strong indicators of plaque instability. Use of statins is associated with a decreased risk of stroke and reportedly improves stability of atherosclerotic plaques, but available data addressing the mechanism of this effect are conflicting. METHODS: We retrospectively analyzed data from 94 consecutive patients with internal carotid artery stenosis who underwent carotid endarterectomy. Excised plaques underwent systematic quantitative immunohistochemical analysis to determine the percentage of macrophage area and the percentage of MMP-9 area. Associations between percentage of macrophage area and percentage of MMP-9 area and use of statins and cerebrovascular disease were examined by univariate and multivariate analysis. RESULTS: We found significantly higher values of percentage of macrophage area and of MMP-9 area in recently symptomatic (n = 26) compared with asymptomatic (n = 68) internal carotid artery stenoses: median (IQR) percentage of macrophage area was 2.29 (1.53-4.129) vs 0.53 (0.27-0.96) and percentage of MMP-9 area was 0.61 (0.36-0.89) vs 0.08 (0.02-0.27; both P < .0005). Patients treated with statins (n = 49) showed lower percentage values of macrophage area and MMP-9 area than untreated patients: the percentage of macrophage area was 0.54 (0.31-1.18) vs 1.03 (0.57-2.08; P = .01) and percentage of MMP-9 area was 0.06 (0.02-0.22) vs 0.36 (0.16-0.62; P < .0005). These associations between statin treatment and percentages of macrophage area and MMP-9 area did not change after controlling for symptomatic cerebrovascular disease and the effects of other potential confounders in multivariable analysis. CONCLUSIONS: Our results confirm the value of percentage of macrophage area and percentage of MMP-9 area as markers of plaque instability and provide further evidence to support the hypothesis that statins reduce inflammatory responses and thereby stabilize carotid atherosclerotic plaques.

Lysozyme, a mediator of sepsis that produces vasodilation by hydrogen peroxide signaling in an arterial preparation.

In septic shock, systemic vasodilation and myocardial depression contribute to the systemic hypotension observed. Both components can be attributed to the effects of mediators that are released as part of the inflammatory response. We previously found that lysozyme (Lzm-S), released from leukocytes, contributed to the myocardial depression that develops in a canine model of septic shock. Lzm-S binds to the endocardial endothelium, resulting in the production of nitric oxide (NO), which, in turn, activates the myocardial soluble guanylate cyclase (sGC) pathway. In the present study, we determined whether Lzm-S might also play a role in the systemic vasodilation that occurs in septic shock. In a phenylephrine-contracted canine carotid artery ring preparation, we found that both canine and human Lzm-S, at concentrations similar to those found in sepsis, produced vasorelaxation. This decrease in force could not be prevented by inhibitors of NO synthase, prostaglandin synthesis, or potassium channel inhibitors and was not dependent on the presence of the vascular endothelium. However, inhibitors of the sGC pathway prevented the vasodilatory activity of Lzm-S. In addition, Aspergillus niger catalase, which breaks down H(2)O(2), as well as hydroxyl radical scavengers, which included hydroquinone and mannitol, prevented the effect of Lzm-S. Electrochemical sensors corroborated that Lzm-S caused H(2)O(2) release from the carotid artery preparation. In conclusion, these results support the notion that when Lzm-S interacts with the arterial vasculature, this interaction results in the formation of H(2)O(2), which, in turn, activates the sGC pathway to cause relaxation. Lzm-S may contribute to the vasodilation that occurs in septic shock.

Endothelial apoptosis does not determine symptom status in carotid artery disease.

BACKGROUND: We examined the hypothesis that endothelial denudation in advanced carotid plaques (CPs) occurs by increased apoptosis of endothelial cells (ECs) using scanning electron microscopy (SEM) as well as markers of cellular proliferation and apoptosis in advanced symptomatic CPs (SCPs) and asymptomatic CPs (ACPs). METHODS: 93 consecutive patients underwent carotid endarterectomy. Five additional specimens were studied by SEM. We performed TUNEL assays, and immunostaining against Fas receptor (FasR), Fas ligand (FasL), activated caspase 3 (ACA3) and Ki-67. RESULTS: SEM revealed morphological changes consistent with EC detachment. Surprisingly, ACA3 positivity was more pronounced on the endothelium of ACPs (4.6 +/- 0.7% of total EC count) than on SCPs (3.3 +/- 0.7%, p = 0.049), and was found to correlate positively with nuclear Ki-67 expression (r(s) = 0.275, p = 0.040). FasL expression was significantly increased on the endothelium of SCPs compared with ACPs (66.4 +/- 4.4 vs. 53.9 +/- 4.5%, p = 0.047). CONCLUSIONS: Absence of increased positivity of apoptotic markers dismisses apoptosis as a dominant mechanism underlying endothelial detachment of SCPs. Rather, increased ACA3 with co-expression of Ki-67 in ACPs might suggest that renewal of endothelium by active cell turnover may contribute to clinically silent evolution of plaques with preserved EC integrity. These observations may assist in designing novel therapies to prevent endothelial decay and symptom generation in advanced carotid artery disease.

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.

Relationship of matrix metalloproteinases and macrophages to embolization during endoluminal carotid interventions.

PURPOSE: To investigate if relationships exist among macrophage infiltration, plasma matrix metalloproteinase (MMP) levels, and the number of emboli generated during endoluminal carotid interventions. METHODS: Carotid endarterectomy specimens excised as intact cylinders (n=27) were subjected to a standardized angioplasty procedure under radiological guidance in an ex vivo pulsatile flow model. Emboli collected in distal filters were counted and sized using microscopy. Preoperative plasma gelatinase activity was determined by gelatin zymography and quantified using image analysis software. Levels of tissue inhibitors of metalloproteinases (TIMP) 1 and 2 were determined by ELISA. Macrophages within postangioplasty plaques were analyzed using immunohistochemical staining for CD68 antigen and graded by a blinded examiner. Statistical analysis was performed using Spearman's rank correlation. RESULTS: The median number of emboli recorded during angioplasty was 104 (interquartile range 33.75-242.5, absolute range 13-1090). Plasma MMP-9 and MMP-2 levels correlated with emboli number (r=0.544 [p=0.003] and r=0.412, [p=0.033], respectively), while TIMP-1 and TIMP-2 levels did not. Macrophage infiltration within the plaques correlated with emboli number (r=0.722, p<0.001) and the plasma MMP-9 level (r=0.489, p=0.010). CONCLUSIONS: These data indicate that plaque macrophage infiltration may play a role in the generation of emboli during endoluminal carotid intervention, possibly via modulation of protease activity.

Nitric oxide inhibition accelerates hypertension and induces perivascular inflammation in rats.

1. Inflammatory changes in peripheral arteries have been reported in animal models of hypertension. Whether they occur in cerebral arteries (CA) with hypertension induced by deprivation of endogenous nitric oxide (NO) remains unknown. 2. In the present study, we compared the arteriolar injury score (AIS) and perivascular inflammation in CA between hypertensive and normotensive rats following NO deprivation with the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). Five-week-old male spontaneously hypertensive rats (SHR) and Wistar -Kyoto (WKY) rats were fed with L-NAME (1 mg/mL) for 4 weeks. 3. Nitric oxide deprivation resulted in time-dependent elevations in tail-cuff pressure (representing systolic blood pressure (SBP)) in both SHR and WKY rats. The magnitude of increase in SBP was larger in SHR (+81.0 +/- 3.2 vs+25.0 +/- 2.2 mmHg; P < 0.01). Arteriolar hyalinosis and AIS in various segments of the CA were assessed with periodic acid-Schiff staining and inflammatory cells were immunostained with the antibody against macrophage/monocyte marker (ED1). The ED1+ cells appeared in the middle CA of L-NAME-treated SHR as early as 2 weeks after treatment. These cells were not observed in L-NAME-treated WKY rats and untreated SHR. More ED1+ cells were found in L-NAME-treated SHR than L-NAME-treated WKY rats after 4 weeks treatment. 4. The AIS and number of ED1+ cells around the perivascular area of the internal carotid artery were significantly higher in L-NAME-treated compared with untreated rats (AIS: 137 +/- 28 vs 46 +/- 10 for WKY rats, respectively; 169 +/- 18 vs 53 +/- 6 for SHR, respectively (P < 0.01); ED1+ cells: 7.9 +/- 0.6 vs 1.3 +/- 0.9 for WKY rats, respectively; 13.6 +/- 2.7 vs 2.1 +/- 0.9 for SHR, respectively (P < 0.01)), although SBP was higher in untreated SHR than in L-NAME-treated WKY rats (170 +/- 4 vs 137 +/- 4 mmHg, respectively; P < 0.05). 5. These findings suggest that ED1+ cells appeared in the middle CA of L-NAME-SHR as early as 2 weeks after treatment. Chronic inhibition of NO accelerates hypertension and induces perivascular inflammation.

Activation of matrix-degrading metalloproteinases by mast cell proteases in atherosclerotic plaques.

Recent studies suggest that mast cell-derived neutral proteases can activate matrix-degrading metalloproteinases (MMPs). We have investigated the role of the mast cell proteases tryptase and chymase in the activation of MMPs in human carotid endarterectomy specimens (atherosclerotic, n=32) and postmortem carotid arteries (control, n=17). In vitro degranulation of mast cells in atherosclerotic carotid arteries by compound 48/80 caused a significant increase in MMP activity. Addition of the nonselective tryptase inhibitor antipain, the specific trypsinlike protease inhibitor 4-amidinophenylmethanesulfonyl fluoride, and the chymase inhibitor chymostatin reduced this increase in MMP activity by 30+/-6%, 23+/-6%, and 9+/-2%, respectively. Immunocytochemistry identified significantly higher numbers of tryptase-containing cells (mast cells) and cells expressing MMP-1 and MMP-3 in the "shoulder" regions of atherosclerotic artery lesions compared with the tunica media of control arteries. Dual immunocytochemistry showed collocation of MMP-1 and MMP-3 with mast cells in the shoulder regions. Degranulation was observed in 78+/-5% (mean+/-SEM) of mast cells in this area, whereas nonactivated mast cells were observed in all other areas. In situ zymography revealed caseinolytic and gelatinolytic activity in these areas. In conclusion, in vitro mast cell degranulation, which releases mast cell proteases, in carotid arteries increases MMP activity. Furthermore, elevated MMP-1 and MMP-3 expression is collocated with increased numbers of degranulated mast cells and with greater MMP activity in the shoulder regions of atherosclerotic plaques. Activation of MMPs by mast cell-derived proteases may be an important mechanism in atherosclerotic plaque destabilization.

Elevated expression of membrane-type 1 and 3 matrix metalloproteinases in rat vascular smooth muscle cells activated by arterial injury.

Matrix metalloproteinases (MMPs) play critical roles in tissue remodeling under various physiologic and pathologic conditions. We recently reported the expression of three membrane-type MMPs (MT-MMPs) by cultured vascular smooth muscle cells (SMCs) of rats (Shofuda et al, 1997). To investigate the roles of the MT-MMPs in the matrix remodeling of blood vessels, expression of MT1-MMP and MT3-MMP was examined in normal and balloon-injured rat carotid arteries by in situ hybridization and immunohistochemistry. Both MT-MMP mRNAs were detected in the intimal-dedifferentiated SMCs, but were negligible in the medial SMCs or in any of normal vascular cells. To elucidate the regulatory mechanism for the MT-MMPs expression, effects of various factors on cultured rat SMCs were also examined. MT1-MMP mRNA was constantly expressed at a high level, and its expression was weakly increased by treatment with interleukin-1beta or tumor necrosis factor-alpha. When the cells were incubated with type IV collagen, the MT1 -MMP expression was markedly decreased. On the other hand, expression of MT3-MMP mRNA was strongly increased by platelet-derived growth factor and fibronectin. These results suggest that type IV collagen may act as a negative regulator for the expression of MT1-MMP in the medial SMCs, whereas platelet-derived growth factor and fibronectin may up-regulate MT3-MMP expression under pathologic conditions. Furthermore, the elevated expression of MT1-MMP and MT3-MMP in SMCs was well associated with their dedifferentiated phenotype.

[Metalloproteinase in carotid endarterectomy plaques: increased activity in critical stenosis zones]. / Metaloproteinasas en placas de endarterectomía carotídea: actividad aumentada en áreas de estenosis crítica.

BACKGROUND: The presence of metalloproteinases in atherosclerotic plaques has been described but their role is not well understood. An increased secretion of these proteolytic enzymes could explain plaque instability and distal embolization. AIM: To measure metalloproteinase activity in atherosclerotic plaques obtained after carotid endarterectomy. MATERIAL AND METHODS: Plaques were divided in one segment with and one segment without stenosis, the latter being used as control. Both segments were incubated in culture media for 48 h or were fixed for histology. The conditioned medium was studied using gelatin zimography and digital densitometry. Metalloproteinases were identified by their molecular weight, inhibition with EDTA or Western Blot. Standard histologic study and immunohistochemistry were done. RESULTS: In stenotic areas, metalloproteinase 9 (92kD) secretion was 260% higher than in regular plaques (191 and 73 Kilopixels/microgram protein respectively p < 0.02). The histological study of stenotic areas showed macrophage infiltration and neoformation of blood vessels. CONCLUSIONS: The increased secretion of cellular matrix degrading enzyme metalloproteinase 9 in stenotic areas of atherosclerotic plaques, could explain plaque instability and subsequent embolization.

[Changes in the neural apparatus of the cerebral arteries in atherosclerosis]. / Izmeneniia nervnogo apparata arterii golovnogo mozga pri ateroskleroze.

Nervous apparatus of the brain arteries (450-60 microns in diameter) in 55-64-year-old patients with cerebral atherosclerosis was studied. Alterations in the afferent innervation were manifested in the increased sinuosity of fibers, areas of hyper- and hypoimpregnation, appearance of thickenings in the fibers. Changes in the vasomotor innervation consisted of the disturbances of the nerve network structure, an increase of nerve fibers concentration and varicosity in the cholinergic plexuses and their decrease in the adrenergic ones. Restructurings of the nerve apparatus were more pronounced in the arteries of large caliber.