Emanuel Dragan

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.

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.

Circulating Endothelial Progenitor Cell and Platelet Microparticle Impact on Platelet Activation in Hypertension Associated with Hypercholesterolemia

Aim The purpose of this project was to evaluate the influence of circulating endothelial progenitor cells (EPCs) and platelet microparticles (PMPs) on blood platelet function in experimental hypertension associated with hypercholesterolemia. Methods Golden Syrian hamsters were divided in six groups: (i) control, C; (ii) hypertensive-hypercholesterolemic, HH; (iii) ‘prevention’, HHin-EPCs, HH animals fed a HH diet and treated with EPCs; (iv) ‘regression’, HHfin-EPCs, HH treated with EPCs after HH feeding; (v) HH treated with PMPs, HH-PMPs, and (vi) HH treated with EPCs and PMPs, HH-EPCs-PMPs. Results Compared to HH group, the platelets from HHin-EPCs and HHfin-EPCs groups showed a reduction of: (i) activation, reflected by decreased integrin 3β, FAK, PI3K, src protein expression; (ii) secreted molecules as: SDF-1, MCP-1, RANTES, VEGF, PF4, PDGF and (iii) expression of pro-inflammatory molecules as: SDF-1, MCP-1, RANTES, IL-6, IL-1β; TFPI secretion was increased. Compared to HH group, platelets of HH-PMPs group showed increased activation, molecules release and proteins expression. Compared to HH-PMPs group the combination EPCs with PMPs treatment induced a decrease of all investigated platelet molecules, however not comparable with that recorded when EPC individual treatment was applied. Conclusion EPCs have the ability to reduce platelet activation and to modulate their pro-inflammatory and anti-thrombogenic properties in hypertension associated with hypercholesterolemia. Although, PMPs have several beneficial effects in combination with EPCs, these did not improve the EPC effects. These findings reveal a new biological role of circulating EPCs in platelet function regulation, and may contribute to understand their cross talk, and the mechanisms of atherosclerosis.

Conjugation of curcumin-loaded lipid nanoemulsions with cell-penetrating peptides increases their cellular uptake and enhances the anti-inflammatory effects in endothelial cells.

To prepare and characterize in vitro and in vivo lipid nanoemulsions (LN) loaded with curcumin (Cm) and functionalized with a cell‐penetrating peptide.

Interaction of platelets with endothelial progenitor cells in the experimental atherosclerosis: Role of transplanted endothelial progenitor cells and platelet microparticles.

Recent studies suggest that endothelial progenitor cells (EPCs) and platelets have an important role in repair following vascular injury. Although evidence suggest that platelets are essential in EPC attracting, homing and differentiation to the injury site; however, the platelet effects on EPC function in atherosclerosis have received less attention. In this context, we followed the consequences of circulating EPCs and platelet microparticles (PMPs) administration on platelet–EPC interaction in atherosclerosis and the involved mechanisms. The experiments were performed on Golden Syrian hamsters divided in five equal groups: control (C), hypertensive–hypercholesterolemic (HH), HH treated with EPCs (HH–EPCs) or PMPs (HH–PMPs) and HH treated with EPCs and PMPs (HH–EPCs–PMPs).

Cardiomyocyte apoptosis in ischaemia‐reperfusion due to the exogenous oxidants at the time of reperfusion

Various studies performed on different models have demonstrated that apoptosis occurs in ischaemic‐reperfused myocardium in vivo; however, the individual contribution of ischaemia and reperfusion to CMC (cardiomyocyte) apoptosis remains uncertain. We have determined the main inducer of CMC apoptosis in ischaemia‐reperfusion by exposing CMCs to either 30 min ischaemia followed by reperfusion or to 25‐OH‐cholesterol (25‐hydroxycholesterol) for 1–3 days. Both ischaemia‐reperfusion and exogenous oxidants increased the Bax/Bcl‐2 ratio, a favourable effect for the apoptotic process. However, apoptosis was not observed in ischaemic CMCs in the absence of reperfusion. Moreover, reperfusion after 30 min ischaemia did not make an important contribution to CMC apoptosis in culture in terms of caspase 3 activation. In contrast, 25‐OH‐cholesterol promoted CMC apoptosis by a caspase 3‐dependent mechanism that involved the transcriptional activation of the pro‐apoptotic protein, Bax and post‐translational degradation of the anti‐apoptotic protein, Bcl‐2. From these results, we conclude that CMC apoptosis is not induced by ischaemia per se, but by the oxidants from the surrounding environment at the time of reperfusion. These exogenous oxidants exacerbate the alterations induced by ischaemia and complete the apoptotic process at the time when ATP and glucose levels are restored.

Effects of transplanted circulating endothelial progenitor cells and platelet microparticles in atherosclerosis development.

Atherosclerosis is an inflammatory disease, in which risk factors such as hyperlipidemia and hypertension affect the arterial endothelium, resulting in dysfunction, cell damage or both. The number of circulating endothelial progenitor cells and microparticles provides invaluable outcome prediction for atherosclerosis disease. However, evidence for the therapeutic potential of endothelial progenitor cells and microparticles in atherosclerosis development is limited. Our study was designed to investigate the possible protective role of a cell therapy‐based approach, using endothelial progenitor cells and the dual behaviour of circulating platelet microparticles, on atherosclerosis development in hypertensive‐hypercholesterolemic hamster model. Consequently, control hamsters received four intravenous inoculations of: (1) 1×105 endothelial progenitor cells of healthy origins in one dose per month, during four months of diet‐induced atherosclerosis, and after hypertensive‐hypercholesterolemic diet for further four months; (2) in a second set of experiments, 1×105 endothelial progenitor cells of healthy origins or/and 1×105 platelet microparticles of atherosclerotic origins were inoculated every other month during hypertensive‐hypercholesterolemic diet.

Microparticles of healthy origins improve endothelial progenitor cell dysfunction via microRNA transfer in an atherosclerotic hamster model

In this study, we aimed: (i) to obtain and functionally characterize the cultures of late endothelial progenitor cells (EPCs) from the animal blood; (ii) to investigate the potential beneficial effects of circulating microparticles (MPs) of healthy origins on EPC dysfunctionality in atherosclerosis as well as involved mechanisms.

Inhibition of miR-486 and miR-92a decreases liver and plasma cholesterol levels by modulating lipid-related genes in hyperlipidemic hamsters

In the present study we aimed to evaluate the potential of in vivo inhibition of miR-486 and miR-92a to reverse hyperlipidemia, then to identify and validate their lipid metabolism-related target genes. Male Golden-Syrian hamsters fed a hyperlipidemic (HL) diet (standard chow plus 3% cholesterol and 15% butter, 10 weeks) were injected subcutaneously with lock-nucleic acid inhibitors for either miR-486 or miR-92a. Lipids and miRNAs levels in liver and plasma, and hepatic expression of miRNAs target genes were assessed in all HL hamsters. MiR-486 and miR-92a target genes were identified by miRWalk analysis and validated by 3′UTR cloning in pmirGLO vectors. HL hamsters had increased liver (2.8-fold) and plasma (twofold) miR-486 levels, and increased miR-92a (2.8-fold and 1.8-fold, respectively) compared to normolipidemic hamsters. After 2 weeks treatment, liver and plasma cholesterol levels decreased (23 and 17.5% for anti-miR-486, 16 and 22% for miR-92a inhibition). Hepatic triglycerides and non-esterified fatty acids content decreased also significantly. Bioinformatics analysis and 3′UTR cloning in pmirGLO vector showed that sterol O-acyltransferase-2 (SOAT2) and sterol-regulatory element binding transcription factor-1 (SREBF1) are targeted by miR-486, while ATP-binding cassette G4 (ABCG4) and Niemann-Pick C1 (NPC1) by miR-92a. In HL livers and in cultured HepG2 cells, miR-486 inhibition restored the levels of SOAT2 and SREBF1 expression, while anti-miR-92a restored ABCG4, NPC1 and SOAT2 expression compared to scrambled-treated HL hamsters or cultured cells. In vivo inhibition of miR-486 and miR-92a could be a useful and valuable new approach to correct lipid metabolism dysregulation.