Arnaldo Ippoliti

MicroRNA 217 Modulates Endothelial Cell Senescence via Silent Information Regulator 1

Background— Aging is a major risk factor for the development of atherosclerosis and coronary artery disease. Through a microarray approach, we have identified a microRNA (miR-217) that is progressively expressed in endothelial cells with aging. miR-217 regulates the expression of silent information regulator 1 (SirT1), a major regulator of longevity and metabolic disorders that is progressively reduced in multiple tissues during aging. Methods and Results— miR-217 inhibits SirT1 expression through a miR-217–binding site within the 3′-UTR of SirT1. In young human umbilical vein endothelial cells, human aortic endothelial cells, and human coronary artery endothelial cells, miR-217 induces a premature senescence-like phenotype and leads to an impairment in angiogenesis via inhibition of SirT1 and modulation of FoxO1 (forkhead box O1) and endothelial nitric oxide synthase acetylation. Conversely, inhibition of miR-217 in old endothelial cells ultimately reduces senescence and increases angiogenic activity via an increase in SirT1. miR-217 is expressed in human atherosclerotic lesions and is negatively correlated with SirT1 expression and with FoxO1 acetylation status. Conclusions— Our data pinpoint miR-217 as an endogenous inhibitor of SirT1, which promotes endothelial senescence and is potentially amenable to therapeutic manipulation for prevention of endothelial dysfunction in metabolic disorders.

TIMP3 Is Reduced in Atherosclerotic Plaques From Subjects With Type 2 Diabetes and Increased by SirT1

OBJECTIVE Atherosclerosis is accelerated in subjects with type 2 diabetes by unknown mechanisms. We identified tissue inhibitor of metalloproteinase 3 (TIMP3), the endogenous inhibitor of A disintegrin and metalloprotease domain 17 (ADAM17) and other matrix metalloproteinases (MMPs), as a gene modifier for insulin resistance and vascular inflammation in mice. We tested its association with atherosclerosis in subjects with type 2 diabetes and identified Sirtuin 1 (SirT1) as a major regulator of TIMP3 expression. RESEARCH DESIGN AND METHODS We investigated ADAM10, ADAM17, MMP9, TIMP1, TIMP2, TIMP3, and TIMP4 expression levels in human carotid atherosclerotic plaques (n = 60) from subjects with and without diabetes. Human vascular smooth muscle cells exposed to several metabolic stimuli were used to identify regulators of TIMP3 expression. SirT1 small interference RNA, cDNA, and TIMP3 promoter gene reporter were used to study SirT1-dependent regulation of TIMP3. RESULTS Here, we show that in human carotid atherosclerotic plaques, TIMP3 was significantly reduced in subjects with type 2 diabetes, leading to ADAM17 and MMP9 overactivity. Reduced expression of TIMP3 was associated in vivo with SirT1 levels. In smooth muscle cells, inhibition of SirT1 activity and levels reduced TIMP3 expression, whereas SirT1 overexpression increased TIMP3 promoter activity. CONCLUSIONS In atherosclerotic plaques from subjects with type 2 diabetes, the deregulation of ADAM17 and MMP9 activities is related to inadequate expression of TIMP3 via SirT1. Studies in vascular cells confirmed the role of SirT1 in tuning TIMP3 expression.

Hyperfibrinogenemia Is Associated With Specific Histocytological Composition and Complications of Atherosclerotic Carotid Plaques in Patients Affected by Transient Ischemic Attacks

BACKGROUND Epidemiological studies have demonstrated that hyperfibrinogenemia is an independent risk factor for cerebrovascular atherosclerosis. However, the underlying mechanisms are poorly understood. We studied whether hyperfibrinogenemia could modify the histological composition of atherosclerotic plaque and precipitate carotid thrombosis resulting from rupture of the plaque. METHODS AND RESULTS We studied the histological composition of 71 carotid atherosclerotic plaques from patients who had undergone surgical endarterectomy after a first episode of transient ischemic attack. Patients were divided into 3 groups corresponding to the tertiles of plasma fibrinogen values. Hypercholesterolemia, hypertriglyceridemia, hypertension, diabetes, and smoking habit were also assessed. At the histological analysis, plaques of patients in the highest tertile of fibrinogen (>407 mg/dL) were characterized by a high incidence of thrombosis (66.7% of cases) compared with plaques of subjects in the lower (21.7%) (P=0.002) and middle (29. 2%) (P=0.009) tertiles. Plaque rupture was significantly associated with high fibrinogen levels (54.2%, P=0.003). Multivariate logistic regression indicated that hyperfibrinogenemia was an independent risk factor for a decrease in cap thickness (P=0.0005), macrophage foam cell infiltration of the cap (P=0.003), and thrombosis (P=0. 003). When the presence of other risk factors was accounted for, hyperfibrinogenemia remained an independent predictor of carotid thrombosis with an odds ratio of 5.83, compared with other risk factors. CONCLUSIONS The results of the present study add to the evidence that hyperfibrinogenemia, independently of other risk factors, is associated with a specific histological composition of carotid atherosclerotic plaques that predisposes them to rupture and thrombosis.

MiR-216a: a link between endothelial dysfunction and autophagy

Endothelial dysfunction and impaired autophagic activity have a crucial role in aging-related diseases such as cardiovascular dysfunction and atherosclerosis. We have identified miR-216a as a microRNA that is induced during endothelial aging and, according to the computational analysis, among its targets includes two autophagy-related genes, Beclin1 (BECN1) and ATG5. Therefore, we have evaluated the role of miR-216a as a molecular component involved in the loss of autophagic function during endothelial aging. The inverse correlation between miR-216a and autophagic genes was conserved during human umbilical vein endothelial cells (HUVECs) aging and in vivo models of human atherosclerosis and heart failure. Luciferase experiments indicated BECN1, but not ATG5 as a direct target of miR-216a. HUVECs were transfected in order to modulate miR-216a expression and stimulated with 100 μg/ml oxidized low-density lipoprotein (ox-LDL) to induce a stress repairing autophagic process. We found that in young HUVECs, miR-216a overexpression repressed BECN1 and ATG5 expression and the ox-LDL induced autophagy, as evaluated by microtubule-associated protein 1 light chain 3 (LC3B) analysis and cytofluorimetric assay. Moreover, miR-216a stimulated ox-LDL accumulation and monocyte adhesion in HUVECs. Conversely, inhibition of miR-216a in old HUVECs rescued the ability to induce a protective autophagy in response to ox-LDL stimulus. In conclusion, mir-216a controls ox-LDL induced autophagy in HUVECs by regulating intracellular levels of BECN1 and may have a relevant role in the pathogenesis of cardiovascular disorders and atherosclerosis.

A pathobiologic link between risk factors profile and morphological markers of carotid instability

OBJECTIVE Although cardiovascular risk factors have been strongly linked to carotid intimal-media thickness, their association with plaque progression towards instability is poorly understood. We evaluated a large database of endarterectomy specimens removed from symptomatic and asymptomatic patients to determine the correlation between major cardiovascular risk factors and carotid plaque morphology. METHODS Incidence of thrombotic, vulnerable and stable plaques together with the degree of plaque inflammatory infiltration was evaluated in 457 carotid atherosclerotic lesions. Clinical records were reviewed in all cases for risk factors profile. RESULTS Thrombotic plaques were more frequently observed in patients affected by stroke (66.9%) as compared to TIA (36.1%) and asymptomatic patients (26.8%, p<0.001). Out of 457 carotid plaques removed during carotid endarterectomy, 181 (39.6%) were represented by thrombotic plaques, 72 (15.8%) by vulnerable plaques (thin cap fibroateroma) and 204 (44.6%) by stable plaques. At the multivariate analysis, a strong association was observed between hypertension, low HDL-cholesterol (HDL-C) and ratio of total to HDL-C >5 with vulnerable and thrombotic carotid plaques. Hypertension (p=0.001), hypercholesterolemia (p=0.05) and low HDL-C (p=0.001) significantly also correlated with the presence of high inflammatory infiltrate of the plaque. When multivariate analysis was restricted to asymptomatic patients, hypertension (p=0.009, OR 2.29), low HDL-cholesterol (p=0.01 OR 2.21) and the ratio of total to HDL-C >5 (p=0.03, OR 2.07) were confirmed to be the risk factors most significantly associated to unstable plaques. The relative risk to carry an unstable plaque for asymptomatic patients with high Framingham Risk Score as compared with those with low risk score was 2.06 (95% C.I., 1.26-3.36). CONCLUSIONS The present histopathological study identifies risk factors predictive of increased risk of carotid plaque rupture and thrombosis. Asymptomatic patients with high risk factors profile may constitute a specific target to reduce the likelihood of cerebrovascular accidents even in the presence of non-flow-limiting plaque.

Emergency Endograft Placement for Recurrent Aortocaval Fistula after Conventional AAA Repair

Purpose: To report a novel case in which a stent-graft was used to emergently treat an aortocaval fistula that recurred after conventional abdominal aortic aneurysm (AAA) repair. Case Report: A 67-year-old man was treated urgently for ruptured AAA with surgical placement of a 16-mm Dacron interposition graft. During the procedure, an aortocaval fistula was repaired primarily. The patient was discharged in satisfactory condition but returned 20 days later with dyspnea, bilateral perimalleolar edema, and a bruit in the mesogastric region. The high flow fistula was again present just above the aortic bifurcation at the distal anastomosis of the existing graft. The patients condition deteriorated rapidly, so a bifurcated Vanguard stent-graft was deployed in an emergency procedure. Subsequent imaging confirmed satisfactory closure of the fistula. The patient was discharged 8 days after endograft placement, and he continues to be without signs of fistula recurrence at 2 years. Conclusions: Endograft treatment of vascular lesions in the acute setting is becoming more common as our experience with the devices grows. Endovascular repair of primary aortocaval fistulas appears to be an efficacious and minimally invasive means of dealing with these lesions in AAA patients.

Sex-related differences in carotid plaque features and inflammation

OBJECTIVE Severe carotid stenosis is a frequent cause of stroke in both men and women. Whereas several sex-related comparisons are available on coronary atherosclerosis, there are few data appraising gender-specific features of carotid plaques. We aimed to systematically compare the pathology and inflammatory features of carotid plaques in men vs women. METHODS Carotid plaque specimens were collected from patients undergoing surgical endarterectomy for asymptomatic or symptomatic carotid stenosis. Histologic analysis was performed, as well as measurements of plaque composition and inflammation. RESULTS A total of 457 patients were included (132 women, 325 men). Baseline analyses showed a greater prevalence of hypercholesterolemia, hypertension, and former smoking status in women, despite a higher Framingham Heart Score in men (all P < .05). Women had a lower prevalence of thrombotic plaques, smaller percentage area of necrotic core, and hemorrhage extension (all P < .05). Plaque inflammation analysis showed a lower concentration of inflammatory and, in particular, of macrophage foam cells in the plaque cap of women (both P < .05). These differences were, however, no longer significant at multivariable analysis, including several baseline features, such as symptom status and stenosis severity. CONCLUSIONS Carotid plaques seem significantly different in women and men, but the main drivers of such pathologic differences are baseline features, including stenosis severity and symptom status.

Cerebral haemodynamics during carotid cross-clamping

Carotid artery cross-clamping ischaemia during carotid endarterectomy (CEA) causes 5-30% of perioperative neurological deficits. This study was performed to identify possible clinical situations at higher risk for carotid cross-clamping ischaemia. 606 consecutive patients underwent CEA and were retrospectively studied; they were grouped according to risk factors, presence of associated vascular diseases, clinical pattern, angiographic and CT scan findings. Stump pressure measurement was provided in all patients, perioperative monitoring during CEA was performed by electroencephalogram (EEG) in 469 (77%) and somatosensorial evoked potentials (SEP) in 137 (23%). Local anaesthesia was used in 88 (14.5%) patients. Ischaemic changes during carotid cross-clamping were registered in 118 patients (19.5%). The incidence of cross-clamping ischaemia was then related to different factors; it affected 5.6% of asymptomatics, 25.4% of patients with fixed stroke and 38.5% of those with stenosis and contralateral occlusion. Angiographic and clinical correlation showed that patients with more severe lesions are mostly affected by clamping ischaemia (up to 55% in those with stroke and stenosis with contralateral occlusion). Age, hypertension and diabetes do not significantly affect incidence of ischaemic changes. Positive CT scan increased this risk; statistical relevance was found in regard to patients with unilateral or bilateral stenosis and in those with transient ischaemic attacks. A higher risk can be expected for subjects with more severe clinical and instrumental findings, even if no patients can be considered completely at risk or risk free. Perioperative monitoring is always mandatory and is of great importance in detecting ischaemic changes and preventing cerebral damage using a temporary intraluminal shunt.

Can knitting structure affect dilation of polyester bifurcated prostheses? A randomized study with the use of helical computed tomography scanning

PURPOSE The aim of this study was to prospectively evaluate the postoperative dilation of two types of knitted polyester arterial prostheses with the use of helical computed tomographic scanning. METHODS Thirty-four patients who underwent aortoiliac or aortofemoral bifurcation grafting were randomized to receive a collagen-sealed warp-knitted polyester graft (n = 16 patients) or a gelatin-sealed Köper-knitted polyester graft (n = 18 patients). Alterations in size of all parts of the grafts were evaluated by helical computed tomographic scanning at postoperative day 8, at 3 months, and at 6 months. RESULTS On postoperative day 8, the mean dilation of the Köper-knitted grafts was 18% +/- 8% for the stem and 15% +/- 12% for the limbs. At the same time period, the mean dilation of warp-knitted grafts was 27% +/- 13% for the stem and 33% +/- 18% for the limbs. No increase in graft dilation was observed at 3 and 6 months. Despite the wide range of values among patients with the same graft type, at each time interval, the Köper-knitted grafts dilated significantly less than the warp-knitted grafts (P <. 05). CONCLUSION In this randomized study, helical computed tomographic scanning was an accurate technique with which to assess graft dilation. For a 6-month follow-up interval, the Köper-knitted polyester structure dilated less than the warp-knitted structure. Longer-term serial scans should allow a better understanding of the clinical significance of graft dilation.

Occult impaired glucose regulation in patients with atherosclerosis is associated to the number of affected vascular districts and inflammation

OBJECTIVE The role of inflammatory adipokines has clear mechanistic effects in the promotion of both DM2 and cardiovascular diseases (CVDs), but it is unknown to what extent atherosclerosis-related inflammation might promote defects of glucose metabolism. The purpose of this study was to test the hypothesis that in subjects with atherosclerotic vascular disease and no previous medical record of type 2 diabetes mellitus (DM2), the diagnosis of occult impaired glucose regulation (IGR) is related to the severity of atherosclerosis, measured as the single or combined presence of an history of coronary artery disease (CAD), carotid atherosclerosis (Car-ATS) and peripheral artery disease (PAD). METHODS In a population of 551 subjects (440 men and 111 women) with a previous history of atherosclerosis, we investigated the presence of IGR (including both impaired glucose tolerance and DM2). To test the correlation between conventional and non-conventional risk factors for cardiovascular disease and diabetes we used logistic and regression analysis models. RESULTS IGR was more prevalent in patients with a documented vascular disease in two or three vessel districts compared with patients with only one symptomatic district (p=0.016). Among classic risk factors we found that waist circumference was correlated neither to IGR nor to symptomatic vascular disease extension. By contrast, adiponectin level was independently associated to vascular and glucose regulation status (p=0.012 and p<0.001, respectively). CONCLUSION In subjects affected by atherosclerotic vascular diseases, the presence of impaired glucose regulation is associated to the number of vascular districts affected and to a reduced adiponectin level.