Maria Luisa Balestrieri

High glucose downregulates endothelial progenitor cell number via SIRT1

Increasing evidence indicates that mammalian SIRT1 mediates calorie restriction and influences lifespan regulating a number of biological molecules such as FoxO1. SIRT1 controls the angiogenic activity of endothelial cells via deacetylation of FoxO1. Endothelial dysfunction and reduced new blood vessel growth in diabetes involve a decreased bioactivity of endothelial progenitor cells (EPCs) via repression of FoxO1 transcriptional activity. The relative contribution of SIRT1 with respect to the direct effects of high glucose on EPC number is poorly understood. We report that treatment of EPCs with high glucose for 3 days determined a consistent downregulation of EPC positive to DiLDL/lectin staining and, interestingly, this was associated with reduced SIRT1 expression levels and enzyme activity, and increased acetyl-FoxO1 expression levels. Moreover, EPCs responded to high glucose with major changes in the expression levels of cell metabolism-, cell cycle-, and oxidative stress-related genes or proteins. Proteomic analysis shows increased expression of nicotinamide phosphoribosyl transferase and mitochondrial superoxide dismutase whereas a glucose-related heat shock protein is reduced. These findings show that SIRT1 is a critical modulator of EPCs dysfunction during alteration of glucose metabolism.

Bioavailability of encapsulated resveratrol into nanoemulsion-based delivery systems.

Different O/W nanoemulsion-based delivery systems were developed in order to optimize the bioavailability of encapsulated resveratrol for potential oral administration. Blank formulations without resveratrol had no negative effect on cell viability or the cytoskeleton structure of Caco-2 cells (XTT viability assay and confocal microscopy). All nanoemulsions were then evaluated based on permeability tests on Caco-2 cells. As a result, the most efficient formulations were lecithin-based nanoemulsions which were able to transport resveratrol through cell monolayers in characteristically shorter times (1-6h) than those required for their metabolization (3-12h), allowing for better preservation of the integrity of the emulsion droplets, thus better protecting the resveratrol molecule. Fluorescence spectroscopy studies confirmed that resveratrol was encapsulated in the inner core of the nanoemulsions, which provides protection against chemical degradation. Furthermore, the developed systems also demonstrated the capability of nanoemulsions in sustained release of resveratrol from dialysis bags compared to the unencapsulated compound.

Rethinking Primary Prevention of Atherosclerosis-Related Diseases

Crucial advances in our understanding of basic pathogenic mechanisms involved in atherogenesis have been achieved during the past 2 decades. The historical hypothesis of pathogenesis (“lipid accumulation”) has evolved to integrate several causal events contributing to the initiation and evolution of atherosclerosis. Vascular inflammation and apoptosis may play a joint pivotal role in its progression and onset. Hypercholesterolemia and hypertension have synergistic deleterious effects on coronary endothelial function.1 Impaired fasting glucose, triglycerides and triglyceride-rich lipoprotein remnants, lipoprotein(a), homocysteine, and high-sensitivity C-reactive protein (hsCRP) might contribute to an increased risk of atherosclerosis.2 The disease also has been related to infiltration of immune cells, which are involved in both systemic and local, innate as well as adaptive, immune responses.3 Distinct pathways of atherothrombosis seem to develop at different sites of the vascular system (brain, heart, and peripheral circulation). Endothelial dysfunction induced by cardiovascular risk factors is considered to be 1 of the earliest stages in vascular damage and is associated independently with cardiovascular events.4 There is a synergic action between genetic, ambient, local, and systemic factors, and ultimately the progression of atherosclerosis is responsible for coronary heart disease (CHD) and its complications (such as unstable “in crescendo” angina, myocardial infarction, and sudden death), peripheral arterial disease, and ischemic stroke. The evolution of atherosclerosis, however, is characterized by a long lag time between onset and clinical manifestation, thereby providing an opportunity for implementation of early detection, prevention, and intervention strategies. Because the development of atherosclerosis commences early in humans, we need to rethink the timing of what is currently considered to be “primary” prevention of atherosclerosis-related diseases. It is likely that we need to start administering effective treatments much earlier than previously assumed. Indeed, much attention would be important when subjects are in a state of wellness before the …

Sirtuin 6 Expression and Inflammatory Activity in Diabetic Atherosclerotic Plaques: Effects of Incretin Treatment

The role of sirtuin 6 (SIRT6) in atherosclerotic progression of diabetic patients is unknown. We evaluated SIRT6 expression and the effect of incretin-based therapies in carotid plaques of asymptomatic diabetic and nondiabetic patients. Plaques were obtained from 52 type 2 diabetic and 30 nondiabetic patients undergoing carotid endarterectomy. Twenty-two diabetic patients were treated with drugs that work on the incretin system, GLP-1 receptor agonists, and dipeptidyl peptidase-4 inhibitors for 26 ± 8 months before undergoing the endarterectomy. Compared with nondiabetic plaques, diabetic plaques had more inflammation and oxidative stress, along with a lesser SIRT6 expression and collagen content. Compared with non-GLP-1 therapy–treated plaques, GLP-1 therapy–treated plaques presented greater SIRT6 expression and collagen content, and less inflammation and oxidative stress, indicating a more stable plaque phenotype. These results were supported by in vitro observations on endothelial progenitor cells (EPCs) and endothelial cells (ECs). Indeed, both EPCs and ECs treated with high glucose (25 mmol/L) in the presence of GLP-1 (100 nmol/L liraglutide) presented a greater SIRT6 and lower nuclear factor-κB expression compared with cells treated only with high glucose. These findings establish the involvement of SIRT6 in the inflammatory pathways of diabetic atherosclerotic lesions and suggest its possible positive modulation by incretin, the effect of which is associated with morphological and compositional characteristics of a potential stable plaque phenotype.

Effect of gestational hypercholesterolaemia on omental vasoreactivity, placental enzyme activity and transplacental passage of normal and oxidised fatty acids

Objective  Maternal hypercholesterolaemia during pregnancy increases lipid peroxidation in mothers and fetuses and programs increased susceptibility to atherosclerosis later in life. The objective of this study was to elucidate the role of the placenta in mediating oxidative stress from mother to offspring.

Beneficial effects of autologous bone marrow cell infusion and antioxidants/L-arginine in patients with chronic critical limb ischemia.

Background Short-term (within 6 weeks follow-up) clinical studies indicate that implantation of bone marrow cells (BMCs) into ischemic limbs may improve peripheral ischemia. Here, the long-term safety and feasibility of intraarterial autologous BMCs with oral treatment with antioxidants and L-arginine were investigated in patients with critical ischemia on account of advanced atherosclerotic peripheral arterial disease (PAD). Methods Eighteen patients with PAD (advanced III/IV Fontaine stages) were enrolled in this study (NCT00306085). An additional group of 18 patients taking maximal drug therapy that refused BMC therapy served as control. The BMC-treated group received two doses of BMCs in the leg arteries (time 0 and 45 days). After 30 days from the first BMC dose, patients received daily antioxidants, and l-arginine. Therapeutic neoangiogenesis was estimated by angiography and laser Doppler capillaroscopy. Results Ankle brachial index improvement (ΔABI: >0.1) was seen in 10 patients at 3 months and in 12 patients at 12–18 months. Ischemic ulcers improved in 13 patients (after 6–12 months). Although two patients underwent amputation, the mean maximum walking distance significantly increased at 3 months and was sustained up to 18 months. Among conservative patients, 10 underwent amputation in comparison with two BMC-treated patients (55.6 vs. 13.3%; P = 0.014). Conclusion This small study shows that intraarterial autologous BMC and antioxidants and l-arginine therapy is safe and effective in patients with advanced atherosclerotic PAD with positive effects until 18 months. Eur J Cardiovasc Prev Rehabil 15:709–718

Understanding the immunoangiostatic CXC chemokine network.

Chemokines, originally discovered as mediators of directional migration of immune cells to sites of inflammation and injury, have a function beyond their role in leukocyte chemotaxis. Indeed, they participate in organ development, angiogenesis, tumourigenesis and, more importantly, in the immune response. The chemokine family characterized by four highly conserved cysteine amino acid residues, with two cysteine residues (C) and a non-cysteine amino acid (X) between them (CXC), is known for its ability to promote trafficking of various leukocytes and to regulate angiogenesis and vascular remodelling. Intriguingly, the presence or absence of a structural-functional domain constituted by glutamic acid-leucine-arginine motif that precedes the first cysteine amino acid residue accounts for their unique property to induce or inhibit angiogenesis (angiogenic or angiostatic activity). The ability of CXC chemokine receptor 3 to promote Th1-dependent immunity and, at the same time, inhibit angiogenesis (immunoangiostasis) is of critical importance for inducing tumour regression. Agents that are able to inhibit angiogenic activities or promote angiostatic activities of CXC chemokines are future targets for research on cancer treatment. Here, we review insights on CXC chemokines in the context of immunoangiostasis and vascular damage.

Therapeutic effects of autologous bone marrow cells and metabolic intervention in the ischemic hindlimb of spontaneously hypertensive rats involve reduced cell senescence and CXCR4/Akt/eNOS pathways.

Peripheral arterial disease (PAD) is a major health problem, especially when associated with severe hypertension. Administration of autologous bone marrow cells (BMCs) is emerging as a novel intervention to induce neoangiogenesis in ischemic limb models and in patients with PAD. This study evaluates the neovascularization capacity of BMCs alone or in combination with metabolic cotreatment (0.8% vitamin E, 0.05% vitamin C, and 5% of L-arginine) in a rat model of ischemic hindlimbs of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Molecular mechanisms were investigated in bone marrow-derived endothelial progenitor cells (BM-EPC) derived from rats. BMC therapy increased blood flow and capillary densities and Ki67 proliferative marker, and it decreased interstitial fibrosis. These effects were amplified by metabolic cotreatment, an intervention that induces vascular protection at least partly through the nitric oxide (NO)/endothelial nitric oxide synthase (eNOS) pathway, reduction of systemic oxidative stress, and macrophage activation. In addition, BMC therapy alone and, more consistently, in combination with metabolic treatment, ameliorated BM-EPC functional activity via decreased cellular senescence and improved homing capacity by increasing CXCR4-expression levels. These data suggest potential therapeutic effects of autologous BMCs and metabolic treatment in hypertensive PAD patients.

Poor glycaemic control in type 2 diabetes patients reduces endothelial progenitor cell number by influencing SIRT1 signalling via platelet-activating factor receptor activation

Aims/hypothesisDownregulation of levels of endothelial progenitor cells (EPCs) during in-vitro short-term exposure to high glucose concentrations relates to reduced activity of silent information regulator 1 (SIRT1) and increased synthesis of platelet-activating factor (PAF). We investigated the possible relationship between PAF and SIRT1 pathways in EPCs during altered glucose homeostasis.MethodsSIRT1 and PAF receptor (PAF-R) levels were determined by western blot, RT-PCR and confocal laser-scanning microscopy. In-vivo experiments were performed on 48 type 2 diabetic patients (25 with poor glycaemic control and 23 with good glycaemic control) and 20 control individuals. In-vitro experiments with the PAF-R antagonist CV3988 were performed on EPCs isolated from leucocyte-rich buffy coat of healthy human donors.ResultsDecreased SIRT1 protein levels were observed in EPCs from type 2 diabetic patients compared with control individuals (p < 0.01). Notably, the SIRT1 level was consistently lower in patients with poor glycaemic control than in those with good glycaemic control (p < 0.01). Diabetic patients also showed an upregulation of PAF-Rs; this response occurred to a greater extent in individuals with poor glycaemic control than in those with good glycaemic control. In-vitro experiments confirmed that EPCs respond to PAF stimulation with decreased SIRT1 protein and SIRT1 mRNA levels. Moreover, reduction of SIRT1 levels and activity were abolished by CV3988.Conclusions/interpretationThese findings unveil a link between PAF and SIRT1 pathways in EPCs that contributes to the deleterious effect of hyperglycaemia on the functional properties of EPCs, crucial in diabetes and peripheral vascular complications.

Therapeutic dose of nebivolol, a nitric oxide-releasing β-blocker, reduces atherosclerosis in cholesterol-fed rabbits

Nitric oxide (NO) exerts a plethora of vascular beneficial effects. The NO-releasing beta-blocker nebivolol is a racemic mixture of D/L-enantiomers that displays negative inotropic as well as direct vasodilating activity. The in vivo antiatherogenic activity of therapeutic doses of the beta-blocker with antioxidant properties carvedilol (12.5mg/day) or nebivolol (5mg/day) was tested in cholesterol-fed rabbits. Animals received a 1% cholesterol-rich diet alone (controls) or mixed with drugs (treated animals) for 8 weeks. While it did not affect hyperlipidemia, nebivolol inhibited the development of atherosclerosis, expressed as computer-assisted imaging analysis of aortic area covered by lesions (23.3+/-4.1% in treated vs 38.2+/-6.4% in control animals, p<0.01). Differently, in our experimental condition of therapeutic drug doses, this antiatherogenic effect did not reach statistical significance in rabbits treated with carvedilol (32.5+/-5.1% aortic area covered by lesions, p=NS vs controls). Plasma nitrates increased in rabbits treated with nebivolol while both beta-blockers reduced LDL oxidation. Moreover, nebivolol induced a consistent increase of endothelial reactivity and aortic eNOS expression compared with control animals (p<0.05) and those receiving carvedilol (p<0.05). Since NO may exert beneficial effects in atherosclerosis, a NO-dependent mechanism could explain this data. These observations suggest that the NO-releasing beta-blocker, nebivolol, might represent an effective pharmacological approach for preventing atherosclerotic lesion progression.