Nicoleta Alexandru

Dysfunction of human subcutaneous fat arterioles in obesity alone or obesity associated with Type 2 diabetes

The aim of the present study was to examine the effects of obesity alone and obesity associated with Type 2 diabetes on the structure, vascular reactivity and response to insulin of isolated human subcutaneous fat arterioles; these effects were correlated with the expression of insulin signalling proteins. Periumbilical subcutaneous adipose tissue was explanted during surgery, small arterioles (internal diameter 220 ± 40 μm) were dissected out and investigated by electron microscopy, myography and immunoblotting. Compared with the subcutaneous arterioles of lean subjects, obesity activated the endothelium, enhanced the accumulation of collagen within vascular wall and increased the sensitivity of adrenergic response; obesity also diminished eNOS (endothelial NO synthase) protein expression, NO production, and endothelium-dependent and insulin-induced vasodilatation, as well as the protein expression of both IRS (insulin receptor substrates)-1 and IRS-2 and of the downstream molecules in the insulin signalling pathway, such as PI3K (phosphoinositide 3-kinase), phospho-Akt and Akt. When obesity was associated with Type 2 diabetes, these changes were significantly augmented. In conclusion, obesity alone or obesity associated with Type 2 diabetes alters human periumbilical adipose tissue arterioles in terms of structure, function and biochemsitry, including diminished eNOS expression and reduced levels of IRS-1, IRS-2, PI3K and Akt in the insulin signalling pathway.

Platelet dysfunction in vascular pathologies and how can it be treated

Cardiovascular diseases are one of the leading causes of morbidity and mortality in industrialized countries, and although many processes play a role in the development of vascular disease, thrombosis is the primary event that precipitates stroke and acute coronary syndromes. The blood platelets are of significant importance in medicine. These cells are involved in many physiological processes, particularly haemostasis through their ability to aggregate and form clots in response to activation. In addition, these dynamic cells display activities that extend beyond thrombosis, including an important role in initiating and sustaining vascular inflammation. The expansion of knowledge from basic and clinical research has highlighted the critical position of platelets in several inflammatory diseases such as arthritis and atherosclerosis. Platelets are emerging as important mediators of inflammation and provide important signals to mediate phenotype of other blood and vascular cells. The important role of platelets in arterial thrombosis and the onset of acute myocardial infarction after atherosclerotic plaque rupture make inhibition of platelet aggregation a critical step in preventing thrombotic events associated with stroke, heart attack, and peripheral arterial thrombosis. However, the use of platelet inhibitors for thrombosis prevention must seek a delicate balance between inhibiting platelet activation and an associated increased bleeding risk. The aim of this review is to up-date the knowledge on platelets physiology and dysfunction in pathologies, such as diabetes mellitus, hypercholesterolemia, and hypertension, emphasizing the link between platelets and the inflammation-related atherosclerosis. The review evaluates the opportunities offered by the novel platelet inhibitors to efficiently alleviate the thrombotic events.

Effect of homocysteine on calcium mobilization and platelet function in type 2 diabetes mellitus

Type 2 diabetes mellitus induces a characteristic platelet hyperactivity that might be due to several factors including oxidativ stress and abnormal intracellular Ca2+ homeostasis. Hyperhomocysteinaemia is considered a risk factor in the development of thrombosis although its effect on platelet function and the mechanisms involved are still poorly understood. Here we show tha homocysteine induce a concentration‐dependent increase in endogenous production of reactive oxygen species (ROS), which was significantly greater in platelets from diabetic patients than in controls. Platelet treatment with homocysteine resulted in Ca2+ release from the dense tubular system and the acidic stores. Ca2+ mobilization‐induced by homocysteine consisted in two components, an initial slow increase in intracellular free Ca + concentration ([Ca +]i) and a rapid and marked increase in [Ca2+]i, th second leading to the activation of platelet aggregation. As well as ROS generation, Ca2+ mobilization and platelet aggregation were significantly greater in platelets from diabetic donors than in controls, which indicate that platelets from diabetic donors are more sensitive to homocysteine. These findings, together with the hyperhomocysteinaemia reported in diabetic patients, strongly suggest that homocysteine might be considered a risk factor in the development of cardiovascular complications associated to type 2 diabetes mellitus.

Platelet activation in hypertension associated with hypercholesterolemia: effects of irbesartan

Summary.  Aim: The aim of this study was to determine the effect of simultaneous hypertension and hypercholesterolemia on platelet activation, nitric oxide (NO) production and oxidative stress, and to evaluate the role of irbesartan, an angiotensin II type 1 receptor antagonist. Methods: Golden Syrian hamsters were divided into three groups: controls, C (fed a standard diet); hypertensive‐hypercholesterolemic, HH (fed a diet enriched in 3% cholesterol, 15% butter and 8% NaCl, for 4 months); and hypertensive‐hypercholesterolemic treated with irbesartan, HHI (fed as HH group, plus irbesartan 10 mg kg−1 per day, for 4 months). Results: Compared with the C group, platelets isolated from the HH group showed: morphological modifications; increased integrin β3 exposure and protein expression of P‐selectin, FAK, PI3K, Akt and Src; reduced eNOS protein expression and NO production; higher generation of ROS, mostly produced by NADPH‐oxidase, cyclooxygenase‐1 (COX‐1) and 12‐lipoxygenase; and enhanced NAD(P)H oxidase activity and protein expression of gp91phox and p22phox subunits, 12‐lipoxygenase, COX‐1, cPLA2 and PKC. Compared with the HH group, the treatment with irbesartan (HHI group) significantly attenuates the changes in all the molecules tested, reduces platelet aggregation, and improves intraplatelet redox balance. Conclusions: Experimental hypertension associated with hypercholesterolemia produces major changes in morphology, signaling mechanisms and oxidative stress in blood platelets. These changes were significantly diminished by irbesartan administration, which functions as an antioxidant on platelets.

Vascular complications in diabetes: Microparticles and microparticle associated microRNAs as active players

The recognition of the importance of diabetes in vascular disease has greatly increased lately. Common risk factors for diabetes-related vascular disease include hyperglycemia, insulin resistance, dyslipidemia, inflammation, hypercoagulability, hypertension, and atherosclerosis. All of these factors contribute to the endothelial dysfunction which generates the diabetic complications, both macro and microvascular. Knowledge of diabetes-related vascular complications and of associated mechanisms it is becoming increasingly important for therapists. The discovery of microparticles (MPs) and their associated microRNAs (miRNAs) have opened new perspectives capturing the attention of basic and clinical scientists for their potential to become new therapeutic targets and clinical biomarkers. MPs known as submicron vesicles generated from membranes of apoptotic or activated cells into circulation have the ability to act as autocrine and paracrine effectors in cell-to-cell communication. They operate as biological vectors modulating the endothelial dysfunction, inflammation, coagulation, angiogenesis, thrombosis, subsequently contributing to the progression of macro and microvascular complications in diabetes. More recently, miRNAs have started to be actively investigated, leading to first exciting reports, which suggest their significant role in vascular physiology and disease. The contribution of MPs and also of their associated miRNAs to the development of vascular complications in diabetes was largely unexplored and undiscussed. In essence, with this review we bring light upon the understanding of impact diabetes has on vascular biology, and the significant role of MPs and MPs associated miRNAs as novel mediators, potential biomarkers and therapeutic targets in vascular complications in diabetes.

Circulating microparticles and endothelial progenitor cells in atherosclerosis: pharmacological effects of irbesartan

Summary.  Aims: This study aimed to (i) employ our newly designed model, the hypertensive–hypercholesterolemic hamster (HH), in order to find out whether a correlation exists between circulating microparticles (MPs), endothelial progenitor cells (EPCs) and their contribution to vascular dysfunction and (ii) to assess the effect of irbesartan treatment on HH animals (HHI).Methods and Results: The results showed that compared with the control (C) group, HH displayed: (i) a significant increase in plasma cholesterol and triglyceride concentration, and an augmentation of systolic and diastolic arterial blood pressure, and of heart rate; (ii) a marked elevation of MPs and a significant decrease in EPCs; (iii) structural modifications of the arterial wall correlated with altered protein expression of MMP2, MMP9, MMP12, TIMP1, TIMP2 and collagen type I and III; (iv) a considerably altered reactivity of the arterial wall closely correlated with MPs and EPC adherence; and (v) an inflammatory process characterized by augmented expression of P‐Selectin, E‐Selectin, von Willebrand factor, tissue factor, IL‐6, MCP‐1 and RANTES. Additionally, the experiments showed the potential of irbesartan to correct all altered parameters in HH and to mobilize EPCs by NO, chemokines and adhesion molecule‐dependent mechanisms.Conclusions: Hypertension associated with hypercholesterolemia is accompanied by structural modifications and expression of pro‐inflammatory molecules by the vessel wall, the alteration of vascular tone, enhanced release of MPs and reduced EPCs; the ratio between the latter two may be considered as a marker of vascular dysfunction. Irbesartan, which exhibits a pharmacological control on the levels of MPs and EPCs, has the potential to restore homeostasis of the arterial wall.

Carbonylation of platelet proteins occurs as consequence of oxidative stress and thrombin activation, and is stimulated by ageing and type 2 diabetes.

Abstract Background: In normal ageing, as well as in diabetes mellitus, blood platelets are exposed to increasing amounts of reactive oxygen species. Because occurrence of carbonyl groups is the first step in oxidative damage of proteins, we questioned the formation of carbonylated proteins (i) in vitro: platelets exposed to oxidant hydrogen peroxide (H2O2) and agonist thrombin (in the presence/absence of antioxidants), and (ii) in vivo: during ageing progression, and in type 2 diabetes. Methods: Platelets were isolated from the blood of experimental animals (rats and hamsters) and humans (healthy, young and elderly), as well as from type 2 diabetics (matures and elderly). 2,4-Dinitrophenyl derivatization of the amino acid side chains was used to quantify protein carbonyls (spectrophotometry) and to immunodetect carbonylated protein bands (Western blotting). Results: In animal models and humans, H2O2 produced dose-dependent increases in carbonylation of platelet proteins (vs. basal condition). Thrombin activation stimulated protein carbonyl formation in a process quenched by antioxidant catalase, suggesting that carbonylation was induced by the oxidative stress generated by activated platelets. Progression of ageing caused increased carbonylation of platelet proteins (vs. young age); enriched in carbonylated proteins, platelets of elderly subjects were less sensitive to H2O2. Type 2 diabetes additionally enhanced carbonylation of human platelet proteins (vs. the levels at young and elderly healthy subjects). In all experiments, protein carbonyl concentrations were correlated with changes in intensity of 2,4-dinitrophenyl-hydrazine-reactive protein bands on immunoblots. Conclusions: The results suggest that exogenous oxidative stress, thrombin activation, progression of ageing and type 2 diabetes lead to protein carbonyls formation in platelets, and this modification can be attenuated by antioxidant enzymes. Clin Chem Lab Med 2008;46:528–36.

The promise of EPC-based therapies on vascular dysfunction in diabetes

Diabetes mellitus is one of the most common metabolic diseases in the world and the vascular dysfunction represents a challenging clinical problem. In diabetes, endothelial cells (ECs), lining the inner wall of blood vessels, do not function properly and contribute to impaired vascular function. Circulating endothelial progenitor cells (EPCs), the precursor of mature EC, actively participate in endothelial repair, by moving to the vascular injury site to form mature EC and new blood vessels. Knowing that the therapeutic interventions can improve only a part of EC dysfunction in diabetes, this review addresses recent findings on the use of EPCs for cell therapy. The strategies proposed in review are based on in vivo and in vitro studies and, thus, their physiological relevance is confirmed. EPC therapy shows great promise for the prevention and cure of diabetes-induced vascular dysfunction.

Chronic venous insufficiency is associated with elevated level of circulating microparticles

Summary.  Background:  Chronic venous insufficiency (CVI) results when the veins in the legs no longer pump blood back to the heart effectively. Microparticles (MPs) are small membrane vesicles released by several circulating and vascular cells upon activation or apoptosis.

Irbesartan administration therapeutically influences circulating endothelial progenitor cell and microparticle mobilization by involvement of pro-inflammatory cytokines

Circulating microparticles (MPs) and endothelial progenitor cells (EPCs) correlate with endothelial dysfunction and contribute to the pathogenesis of atherosclerosis. In this context, we explored whether the angiotensin II type I receptor antagonist, irbesartan, exerts a pharmacological control in the atherosclerotic process by the improvement of EPC mobilization and inhibitory effects on MP release and VEGF and SDF-1α levels in the hypertensive-hypercholesterolemic (HH) hamster model. The HH hamsters were treated with irbesartan (50mg/kg b.w/day administered by gavage) for 4 month (HHI). We analyzed MP/EPC infiltration in vascular wall before and after irbesartan administration as well as the endothelial function and expression of VEGF/SDF-1α in plasma and tissue and of molecular pathways activated by them. The results showed that treatment with irbesartan significantly increased EPC infiltration and decreased MP infiltration. The mechanisms underlying this response include the reduction/increase of a number of specific membrane receptors exposed by MPs (TF, P-Selectin, E-Selectin, PSGL-1, Rantes), respectively, by EPCs (β2-Integrins, α4β1-integrin), the augmentation of endothelium-mediated vasodilation and the reduction of protein expression of VEGF/SDF-1α followed by: (1) the diminishment of pro-inflammatory endothelial cytokines: VEGFR1, VEGFR2, CXCR4, Tie2, PIGF with role in EPC homing to sites of damaged endothelium; and (2) the increase of protein expression of COX-2, PGI2 synthase molecules with role in the improvement of arterial wall vasodilatation. In conclusion, the study underlines that irbesartan administration therapeutically improves/reduces EPC, respectively, MP mobilization and this action may be of salutary relevance contributing to its beneficial cardiovascular effects.