Eugenio Martelli

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.

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.

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.

Carotid Bypass: A Safe and Durable Solution for Recurrent Carotid Stenosis

BACKGROUND The long-term results of carotid artery stenting (CAS) for post-carotid endarterectomy (CEA) restenosis are disappointing (4-year patency rates: ∼75%). Since 1988, our group has offered carotid bypass (CB) as an alternative to redo CEA and later also to CAS in this setting. The aim of this retrospective study was to investigate early and late outcomes associated with CB in this population. METHODS Data were collected from patients treated with CB in the year 2000-2012 for significant/symptomatic post-CEA restenosis (or intra-stent restenosis [ISR] after CAS for post-CEA restenosis). All patients had good life expectancy. CB was performed under loco-regional anesthesia. With the aid of sequential vessel clamping, the graft (great saphenous vein [GSV] or polytetrafluoroethylene) was anastomosed with the common carotid artery (side-to-end) and the distal internal carotid artery (end-to-side). Patients were followed with clinical and duplex scan assessments at 1, 3, and 6 months and yearly thereafter. RESULTS The study population comprised 21 patients (mean age 67.3 years; 17 men). CB was performed for post-CEA restenosis (or ISR after CAS for post-CEA restenosis, n=3) 51.2 months (mean) after the previous operation. GSV grafts were used in half of the cases (n=11; 52.4%); temporary shunting was used in 4 (19%) patients. Intraoperative complications (none fatal) occurred in 4 (19%) patients (3 transient peripheral nerve injuries, 1 cervical hematoma). During follow-up (mean 64.8 months), there were no neurologic complications or restenoses. Overall mortality was 33.3% (6 deaths from acute myocardial infarctions, 1 from a ruptured abdominal aortic aneurysm). CONCLUSIONS For post-CEA restenosis (or ISR after CAS for post-CEA restenosis), CB offers superior long-term patency rates than CAS (or redo angioplasty) and an acceptable risk of cranial nerve damage.

Insulin Resistance Affects Gene Expression in Endothelium

To the editor: We and others have found that either genetic insulin resistance or hyperinsulinemia may dampen insulin ability to activate the PI3-kinase/Akt pathway, leading to perturbation of endothelial nitric oxide synthase (eNOS) activation.1,2 However, little is known about insulin effects on transcription of antiatherogenic and proatherogenic genes in insulin resistant conditions. Therefore, we used a genetic model of vascular insulin resistance, primary human umbilical vein endothelial cells (HUVECs) naturally carrying the G972R Insulin Receptor Substrate-1 (IRS-1) variant,2 to investigate the effect of both genetic insulin resistance and hyperinsulinemia on transcription of atherosclerosis related genes. G972R IRS-1 variant reduces IRS-1 activation of the PI3-K/Akt pathway,3 and it has been associated to coronary artery disease and obesity.3,4 HUVECs carrying the wild-type (WT) or G972R IRS-1 variant were obtained as previously described.3 For the experiments, 3rd through 5th passage HUVEC-WT and 972 were incubated in serum free EGM-2 medium for 16 hours in the presence or absence of insulin 5×10−7 mol/L, to obtain a full effect of insulin on nitric oxide production.5 Total RNA was extracted from the HUVECs with Trizol reagent according to the manufacturer’s protocol. To profile gene expression pattern we made use of U133A Affymetrix DNA microarray containing a total number of 22283 probe sets corresponding to about 15 000 genes, using previously described methods.6 Gene expression profiling was obtained by …