Domenico Palombo

Eversion versus conventional carotid endarterectomy: Late results of a prospective multicenter randomized trial

OBJECTIVE The durability of carotid endarterectomy (CEA) may be affected by carotid restenosis. The data from randomized trials show that the highest incidence of restenosis after CEA occurs from 12 to 18 months after surgery. The optimal CEA technique to reduce perioperative complications and restenosis rates is still undefined. This study examines the long-term clinical outcome and incidence of recurrent stenosis in patients who undergo eversion CEA. Previously published perioperative results of this study did not show statistically significant differences in study endpoints between the eversion and standard techniques. METHODS From October 1994 to March 1997, 1353 patients with surgical indications for carotid stenosis were randomly assigned to undergo eversion (n = 678) or standard CEA (n = 675; primary closure, 419; patch, 256). Withdrawal from the assigned treatment occurred in 1.6% of the patients (in 13 assigned to eversion CEA, and in nine assigned to standard CEA). The clinical and duplex scan follow-up examination was 99% complete, and the mean follow-up interval was 33 months (range, 12 to 55 months). The primary outcomes were perioperative and late major stroke and death, carotid restenosis (stenosis >/= 50% of the lumen diameter detected at duplex scanning), and carotid occlusion. The primary evaluation of study outcomes was conducted on the basis of an intention-to-treat analysis. RESULTS Restenosis was found at duplex scanning in 56 patients (19 in the eversion group, and 37 in the standard group). Within the standard group, the restenosis rates were 7.9% in the primary closure population and 1.5% in the patched population. Of the patients with restenosis, 36% underwent cerebral angiography that confirmed restenosis in all cases. The cumulative restenosis risk at 4 years was significantly lower in the group that underwent treatment with eversion CEA as compared with the standard group (3.6% vs 9.2%; P =.01), with an absolute risk reduction of 5. 6% and a relative risk reduction of 62%. Eighteen patients would have had to undergo treatment with eversion CEA to prevent one restenosis during the 4-year period. The incidence rate of ipsilateral stroke was 3.3% in the eversion population and 2.2% in the standard group. There were no significant differences in the cumulative risks of ipsilateral stroke (3.9% for eversion, and 2.2% for standard; P =.2) and death (13.1% for eversion, and 12.7% for standard; P =.7)) in the two groups. Of the 18 variables that were examined for their influence on restenosis, eversion CEA (hazard ratio, 0.3; 95% confidence interval, 0.2 to 0.6; P =.0004) and patch CEA (hazard ratio, 0.2; 95% confidence interval, 0.07 to 0.6; P =. 002) were negative independent predictors of restenosis with multivariate Cox proportional hazards regression analysis. CONCLUSION The EVEREST (EVERsion carotid Endarterectomy versus Standard Trial) showed that eversion CEA is safe, effective, and durable. No statistically significant differences were found in late outcome between the eversion and standard techniques at the available follow-up examination.

Anti-Apolipoprotein A-1 auto-antibodies are active mediators of atherosclerotic plaque vulnerability

AIMS Anti-Apolipoprotein A-1 auto-antibodies (anti-ApoA-1 IgG) represent an emerging prognostic cardiovascular marker in patients with myocardial infarction or autoimmune diseases associated with high cardiovascular risk. The potential relationship between anti-ApoA-1 IgG and plaque vulnerability remains elusive. Thus, we aimed to investigate the role of anti-ApoA-1 IgG in plaque vulnerability. METHODS AND RESULTS Potential relationship between anti-ApoA-1 IgG and features of cardiovascular vulnerability was explored both in vivo and in vitro. In vivo, we investigated anti-ApoA-1 IgG in patients with severe carotid stenosis (n = 102) and in ApoE-/- mice infused with polyclonal anti-ApoA-1 IgG. In vitro, anti-ApoA-1 IgG effects were assessed on human primary macrophages, monocytes, and neutrophils. Intraplaque collagen was decreased, while neutrophil and matrix metalloprotease (MMP)-9 content were increased in anti-ApoA-1 IgG-positive patients and anti-ApoA-1 IgG-treated mice when compared with corresponding controls. In mouse aortic roots (but not in abdominal aortas), treatment with anti-ApoA-1 IgG was associated with increased lesion size when compared with controls. In humans, serum anti-ApoA-1 IgG levels positively correlated with intraplaque macrophage, neutrophil, and MMP-9 content, and inversely with collagen. In vitro, anti-ApoA-1 IgG increased macrophage release of CCL2, CXCL8, and MMP-9, as well as neutrophil migration towards TNF-α or CXCL8. CONCLUSION These results suggest that anti-ApoA-1 IgG might be associated with increased atherosclerotic plaque vulnerability in humans and mice.

Systemic and Intraplaque Mediators of Inflammation Are Increased in Patients Symptomatic for Ischemic Stroke

Background and Purpose— The concept of “vulnerable plaque” has been extended to the more recent definition of the “cardiovascular vulnerable patient,” in which “intraplaque” and “systemic” factors contribute to the cumulative risk of acute cardiovascular events. Thus, we investigated the possible role of systemic and intraplaque inflammation in patients asymptomatic versus symptomatic for ischemic stroke. Methods— Regions upstream and downstream the blood flow were isolated from internal carotid plaques of patients asymptomatic (n=63) or symptomatic (n=18) for ischemic stroke. Specimens were analyzed for lipid, collagen, macrophage, lymphocyte, neutrophil, mast cell and smooth muscle cell content, and chemokine and cytokine mRNA expression. Chemokine receptors and adhesion molecules were assessed on circulating leukocytes by flow cytometry. Systemic inflammatory markers and biochemical parameters were measured on total blood, plasma, and serum. Results— Tumor necrosis factor-&agr; and CCL5 serum levels as well as intercellular adhesion molecule-1 expression on circulating neutrophils were increased in symptomatic as compared with asymptomatic patients. Collagen content and smooth muscle cell infiltration were decreased in symptomatic plaques. In upstream regions of symptomatic plaques, lipid content and lymphocyte infiltration were increased. In downstream regions of symptomatic plaques, macrophage, neutrophil, and mast cell infiltration were increased. Intraplaque collagen content was positively correlated with smooth muscle cell infiltration and inversely correlated with macrophages, neutrophils, or serum tumor necrosis factor-&agr;. Collagen reduction in downstream regions and serum tumor necrosis factor-&agr; were independently associated with the likelihood of being symptomatic. Conclusions— Inflammatory mediators are increased in ischemic stroke. Despite statistically significant, the correlation between tumor necrosis factor-&agr; serum level and intraplaque vulnerability was weak and probably of limited biological importance.

Adipose tissue immune response: novel triggers and consequences for chronic inflammatory conditions.

Adipose tissue inflammation mediates the association between excessive body fat accumulation and several chronic inflammatory diseases. A high prevalence of obesity-associated adipose tissue inflammation was observed not only in patients with cardiovascular conditions but also in patients with inflammatory bowel diseases, abdominal aortic aneurysm, or cardiorenal syndrome. In addition to excessive caloric intake, other triggers promote visceral adipose tissue inflammation followed by chronic, low-grade systemic inflammation. The infiltration and accumulation of immune cells in the inflamed and hypertrophied adipose tissue promote the production of inflammatory cytokines, contributing to target organ damages. This comorbidity seems to delimit subgroups of individuals with systemic adipose tissue inflammation and more severe chronic inflammatory diseases that are refractory to conventional treatment. This review highlights the association between adipose tissue immune response and the pathophysiology of visceral adiposity-related chronic inflammatory diseases, while suggesting several new therapeutic strategies.

The activation of the cannabinoid receptor type 2 reduces neutrophilic protease-mediated vulnerability in atherosclerotic plaques

AIMS The activation of cannabinoid receptor type 2 (CB(2))-mediated pathways might represent a promising anti-atherosclerotic treatment. Here, we investigated the expression of the endocannabinoid system in human carotid plaques and the impact of CB(2) pharmacological activation on markers of plaque vulnerability in vivo and in vitro. METHODS AND RESULTS The study was conducted using all available residual human carotid tissues (upstream and downstream the blood flow) from our cohort of patients symptomatic (n = 13) or asymptomatic (n = 27) for ischaemic stroke. Intraplaque levels of 2-arachidonoylglycerol, anandamide N-arachidonoylethanolamine, N-palmitoylethanolamine, N-oleoylethanolamine, and their degrading enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) were not different in human plaque portions. In the majority of human samples, CB(1) (both mRNA and protein levels) was undetectable. In downstream symptomatic plaques, CB(2) protein expression was reduced when compared with asymptomatic patients. In these portions, CB(2) levels were inversely correlated (r = -0.4008, P = 0.0170) with matrix metalloprotease (MMP)-9 content and positively (r = 0.3997, P = 0.0174) with collagen. In mouse plaques, CB(2) co-localized with neutrophils and MMP-9. Treatment with the selective CB(2) agonist JWH-133 was associated with the reduction in MMP-9 content in aortic root and carotid plaques. In vitro, pre-incubation with JWH-133 reduced tumour necrosis factor (TNF)-α-mediated release of MMP-9. This effect was associated with the reduction in TNF-α-induced ERK1/2 phosphorylation in human neutrophils. CONCLUSION Cannabinoid receptor type 2 receptor is down-regulated in unstable human carotid plaques. Since CB(2) activation prevents neutrophil release of MMP-9 in vivo and in vitro, this treatment strategy might selectively reduce carotid vulnerability in humans.

Resveratrol counteracts systemic and local inflammation involved in early abdominal aortic aneurysm development

BACKGROUND Monocyte activation, macrophage infiltration, vascular oxidative stress and matrix proteolysis are inflammatory key steps contributing to abdominal aortic aneurysm (AAA) development. A phenotypical and functional heterogeneity is recognizable in monocytes by the differential expression of surface molecules: CD62L- subset corresponds to activated monocytes, while CD143/ACE surface expression increases during their differentiation into macrophages. In this work, Resveratrol, which is an antioxidant polyphenol with vasoprotective properties, has been evaluated for its potential to limit aneurysm development and monocyte-dependent inflammatory response in a model of elastase-induced AAA. METHODS Male Sprague-Dawley rats received Resveratrol (10 mg/kg/die) (Rsv group, n=15) or vehicle (ethanol) alone (Et-OH group, n=15) continuously from 7 d before until 14 d after the AAA induction with elastase; five littermates were used as untreated control group (Ctr group, n=5). At the end of treatment, CD143 and CD62L monocyte expression was analyzed by flow cytometry, serum antioxidant capacity was evaluated using the TRAP method and circulating TNFα, and MMP-9 were measured with ELISA and gel zymography, respectively. Aortas were subjected to histology and immunohistochemistry for morphological analysis, macrophage infiltration, and MMP-9, TNFα, and VEGF expression. RESULTS Resveratrol counteracted the CD62L-monocyte subset expansion, CD143 monocyte expression, and circulating levels of MMP-9 activity and TNFα associated to AAA induction. Similarly, treatment with Resveratrol significantly attenuated AAA expansion, vessel wall macrophage infiltration and MMP-9, VEGF, and TNFα expression, compared with AAA from Et-OH group. CONCLUSIONS Resveratrol limited the monocyte-dependent inflammatory response, macrophage differentiation and aortic lumen enlargement in elastase-induced AAA. These data suggest that Resveratrol might be tested in selected patients with small AAA to modulate the early systemic and local inflammatory response associated to AAA progression.

Endothelial and smooth muscle cells from abdominal aortic aneurysm have increased oxidative stress and telomere attrition.

Background Abdominal aortic aneurysm (AAA) is a complex multi-factorial disease with life-threatening complications. AAA is typically asymptomatic and its rupture is associated with high mortality rate. Both environmental and genetic risk factors are involved in AAA pathogenesis. Aim of this study was to investigate telomere length (TL) and oxidative DNA damage in paired blood lymphocytes, aortic endothelial cells (EC), vascular smooth muscle cells (VSMC), and epidermal cells from patients with AAA in comparison with matched controls. Methods TL was assessed using a modification of quantitative (Q)-FISH in combination with immunofluorescence for CD31 or α-smooth muscle actin to detect EC and VSMC, respectively. Oxidative DNA damage was investigated by immunofluorescence staining for 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG). Results and Conclusions Telomeres were found to be significantly shortened in EC, VSMC, keratinocytes and blood lymphocytes from AAA patients compared to matched controls. 8-oxo-dG immunoreactivity, indicative of oxidative DNA damage, was detected at higher levels in all of the above cell types from AAA patients compared to matched controls. Increased DNA double strand breaks were detected in AAA patients vs controls by nuclear staining for γ-H2AX histone. There was statistically significant inverse correlation between TL and accumulation of oxidative DNA damage in blood lymphocytes from AAA patients. This study shows for the first time that EC and VSMC from AAA have shortened telomeres and oxidative DNA damage. Similar findings were obtained with circulating lymphocytes and keratinocytes, indicating the systemic nature of the disease. Potential translational implications of these findings are discussed.

Treatment with Angiotensin-(1-7) reduces inflammation in carotid atherosclerotic plaques

Angiotensin (Ang)-(1-7), acting through the receptor Mas, has atheroprotective effects; however, its role on plaque vulnerability has been poorly studied. Here, we investigated the expression of the renin-angiotensin system (RAS) components in stable and unstable human carotid plaques. In addition, we evaluated the effects of the chronic treatment with an oral formulation of Ang-(1-7) in a mouse model of shear stress-determined carotid atherosclerotic plaque. Upstream and downstream regions of internal carotid plaques were obtained from a recently published cohort of patients asymptomatic or symptomatic for ischaemic stroke. Angiotensinogen and renin genes were strongly expressed in the entire cohort, indicating an intense intraplaque modulation of the RAS. Intraplaque expression of the Mas receptor mRNA was increased in the downstream portion of asymptomatic patients as compared to corresponding region in symptomatic patients. Conversely, AT1 receptor gene expression was not modified between asymptomatic and symptomatic patients. Treatment with Ang-(1-7) in ApoE-/- mice was associated with increased intraplaque collagen content in the aortic root and low shear stress-induced carotid plaques, and a decreased MMP-9 content and neutrophil and macrophage infiltration. These beneficial effects were not observed in the oscillatory shear stress-induced plaque. In vitro incubation with Ang-(1-7) did not affect ICAM-1 expression and apoptosis on cultured endothelial cells. In conclusion, Mas receptor is up regulated in the downstream portions of human stable carotid plaques as compared to unstable lesions. Treatment with the oral formulation of Ang-(1-7) enhances a more stable phenotype in atherosclerotic plaques, depending on the local pattern of shear stress forces.

Effects of polyphenol extract from olive pomace on anoxia-induced endothelial dysfunction.

Anoxia modulates the expression of molecules associated with endothelial dysfunction and vascular diseases. Polyphenols have potent antioxidant properties due to their ability to modulate genes involved in oxidative tissue damage. In this study, we investigated the effect of polyphenol extract from olive pomace (PEOP) and its main constituents, Tyrosol and Oleuropein, on endothelial cells subjected to anoxia by evaluating the expression of molecules critical for endothelial function, proliferation and migration, and the signaling pathway involved. EAhy926 human endothelial cells were exposed to anoxic stress in the presence or absence of PEOP. Anoxia increased the nitric oxide (NO) level and the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and tumor necrosis factor-α (TNFα). These effects were prevented by PEOP treatment in a dose-dependent manner. Moreover, PEOP prevented the proliferation and migration associated with anoxia in EAhy926 cells, down-regulated the levels of matrix metalloproteinase (MMP)-2, MMP-9 and membrane type-1 MMP (MT1-MMP) and increased tissue MMP inhibitor-1 (TIMP-1) expression. Purified Oleuropein or Tyrosol restored to a basal level the anoxia-induced expression of MMP-9 and partially of MMP-2. The expression of TNFα was reduced by both polyphenols in a dose-dependent manner, but more efficiently by Tyrosol. Conversely, Oleuropein and Tyrosol had no significant effects on iNOS, COX-2 and TIMP-1 expression when used at the concentration found in PEOP. PEOP induced a time-dependent phosphorylation of p38 MAPK and ERK1/2 and inhibited anoxia-induced NF-κB activation. PEOP treatment restores the endothelial functions that are impaired by anoxia by regulating the expression of genes involved in proteolysis, angiogenesis and inflammation more efficiently than the single purified components. Therefore, the combined use of polyphenols, as in PEOP, could represent a powerful tool for the treatment and chemoprevention of endothelial dysfunction-associated vascular diseases.

Serum levels of anti-apolipoprotein A-1 auto-antibodies and myeloperoxidase as predictors of major adverse cardiovascular events after carotid endarterectomy

We aimed at challenging the prognostic accuracies of myeloperoxidase (MPO) and antibodies anti-apolipoprotein A-1 (anti-apoA-1 IgG), alone or in combination, for major adverse cardiovascular events (MACE) prediction, one year after carotid endarterectomy (CEA). In this prospective single centre study, 178 patients undergoing elective CEA were included. Serum anti-apoA-1 IgG and MPO were assessed by enzyme-linked immunosorbent assay prior to the surgery. Post-hoc determination of the MPO cut-off was performed by receiver operating characteristics (ROC) analyses. MACE was defined by the occurrence of fatal or non-fatal acute coronary syndromes or stroke during one year follow-up. Prognostic accuracy of anti-apoA-1 IgG was assessed by ROC curve analyses, survival analyses and reclassification statistics. During follow-up, 5% (9/178) of patients presented a MACE, and 29% (52/178) were positive for anti-apoA-1 IgG. Patients with MACE had higher median MPO and anti-apoA-1 IgG levels at admission (p=0.01), but no difference for the 10-year global Framingham risk score (FRS) was observed (p=0.22). ROC analyses indicated that both MPO and anti-apoA-1 IgG were significant predictors of subsequent MACE (area under the curve [AUC]: 0.75, 95% confidence interval [95%CI]: 0.61-0.89, p=0.01; and 0.74, 95%CI: 0.59-90; p=0.01), but combining anti-apoA-1 IgG positivity and MPO>857 ng/ml displayed the best predictive accuracy (AUC: 0.78, 95%CI: 0.65-0.91; p=0.007). It was associated with a poorer MACE-free survival (98.2% vs. 57.1%; p<0.001, LogRank), with a positive likelihood ratio of 13.67, and provided incremental predictive ability over FRS. In conclusion, combining the assessment of anti-apoA-1 IgG and MPO appears as a promising risk stratification tool in patients with severe carotid stenosis.