Stem cell-derived extracellular vesicles reduce the expression of molecules involved in cardiac hypertrophy-In a model of human-induced pluripotent stem cell-derived cardiomyocytes.

Cardiac pathological hypertrophy is the major risk factor that usually progresses to heart failure. We hypothesized that extracellular vesicles (EVs), known to act as important mediators in regulating physiological and pathological functions, could have the potential to reduce the cardiac hypertrophy and the ensuing cardiovascular diseases. Herein, the effects of mesenchymal stem cell-derived extracellular vesicles (EV-MSCs) on cardiac hypertrophy were investigated. EVs were isolated from the secretome of human adipose tissue-derived stem cells (EV-ADSCs) or bone marrow-derived stem cells (EV-BMMSCs). Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were stimulated with AngII and TGF-ß1, in absence or presence of EVs. The results showed that exposure of hiPSC-CMs to AngII and TGF-ß1 generated in vitro model of hypertrophic cardiomyocytes characterized by increases in surface area, reactive oxygen species production, protein expression of cardiac-specific biomarkers atrial natriuretic factor, migration inhibitory factor, cTnI, COL1A1, Cx43, α-SMA and signalling molecules SMAD2 and NF-kBp50. The presence of EV-ADSCs or EV-BMMSCs in the hiPSC-CM culture along with hypertrophic stimuli reduced the protein expressions of hypertrophic specific markers (ANF, MIF, cTnI, COL1A1) and the gene expressions of IL-6 molecule involved in inflammatory process associated with cardiac hypertrophy and transcription factors SMAD2, SMAD3, cJUN, cFOS with role in cardiomyocyte hypertrophic response induced by AngII and TGF-ß1. The EV-ADSCs were more effective in reducing the protein expressions of hypertrophic and inflammatory markers, while EV-BMMSCs in reducing the gene expressions of transcription factors. Notably, neither EV-ADSCs nor EV-BMMSCs induced significant changes in cardiac biomarkers Cx43, α-SMA and fibronectin. These different effects of stem cell-derived EVs could be attributed to their miRNA content: some miRNAs (miR-126-3p, miR-222-3p, miR-30e-5p, miR-181b-5p, miR-124-3p, miR-155-5p, miR-210-3p hsa-miR-221-3p) were expressed in both types of EVs and others only in EV-ADSCs (miR-181a-5p, miR-185-5p, miR-21-5p) or in EV-BMMSCs (miR-143-3p, miR-146a-5p, miR-93-5p), some of these attenuating the cardiac hypertrophy while others enhance it. In conclusion, in hiPSC-CMs the stem cell-derived EVs through their cargo reduced the expression of hypertrophic specific markers and molecules involved in inflammatory process associated with cardiac hypertrophy. The data suggest the EV potential to act as therapeutic mediators to reduce cardiac hypertrophy and possibly the subsequent cardiovascular events.

VLA4-Enhanced Allogeneic Endothelial Progenitor Cell-Based Therapy Preserves the Aortic Valve Function in a Mouse Model of Dyslipidemia and Diabetes.

The number and function of endothelial progenitor cells (EPCs) are reduced in diabetes, contributing to deteriorated vascular repair and the occurrence of cardiovascular complications. Here, we present the results of treating early diabetic dyslipidemic mice or dyslipidemic with disease-matched EPCs modified to overexpress VLA4 (VLA4-EPCs) as compared with the treatment of EPCs transfected with GFP (GFP-EPCs) as well as EPCs from healthy animals. Organ imaging of injected PKH26-stained cells showed little pulmonary first-pass effects and distribution in highly vascularized organs, with splenic removal from circulation, mostly in non-diabetic animals. Plasma measurements showed pronounced dyslipidemia in all animals and glycaemia indicative of diabetes in streptozotocin-injected animals. Echocardiographic measurements performed 3 days after the treatment showed significantly improved aortic valve function in animals treated with VLA4-overexpressing EPCs compared with GFP-EPCs, and similar results in the groups treated with healthy EPCs and VLA4-EPCs. Immunohistochemical analyses revealed active inflammation and remodelling in all groups but different profiles, with higher MMP9 and lower P-selectin levels in GFP-EPCs, treated animals. In conclusion, our experiments show that genetically modified allogeneic EPCs might be a safe treatment option, with bioavailability in the desired target compartments and the ability to preserve aortic valve function in dyslipidemia and diabetes.

Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles on Atherosclerosis-Induced Vascular Dysfunction and Its Key Molecular Players.

Atherosclerosis is a progressive, chronic inflammatory disease of the large arteries caused by the constant accumulation of cholesterol, followed by endothelial dysfunction and vascular inflammation. We hypothesized that delivery of extracellular vesicles (EVs), recognized for their potential as therapeutic targets and tools, could restore vascular function in atherosclerosis. We explored by comparison the potential beneficial effects of EVs from subcutaneous adipose tissue stem cells (EVs (ADSCs)) or bone marrow mesenchymal stem cells (EVs (MSCs)) on the consequences of atherogenic diet on vascular health. Also, the influences of siRNA-targeting Smad2/3 (Smad2/3siRNA) on endothelial dysfunction and its key molecular players were analyzed. For this study, an animal model of atherosclerosis (HH) was transplanted with EVs (ADSCs) or EVs (MSCs) transfected or not with Smad2/3siRNA. For controls, healthy or HH animals were used. The results indicated that by comparison with the HH group, the treatment with EVs(ADSCs) or EVs(MSCs) alone or in combination with Smad2/3siRNA of HH animals induced a significant decrease in the main plasma parameters and a noticeable improvement in the structure and function of the thoracic aorta and carotid artery along with a decrease in the selected molecular and cellular targets mediating their changes in atherosclerosis: 1) a decrease in expression of structural and inflammatory markers COL1A1, α-SMA, Cx43, VCAM-1, and MMP-2; 2) a slight infiltration of total/M1 macrophages and T-cells; 3) a reduced level of cytosolic ROS production; 4) a significant diminution in plasma concentrations of TGF-ß1 and Ang II proteins; 5) significant structural and functional improvements (thinning of the arterial wall, increase of the inner diameter, enhanced distensibility, diminished VTI and Vel, and augmented contractile and relaxation responses); 6) a reduced protein expression profile of Smad2/3, ATF-2, and NF-kBp50/p65 and a significant decrease in the expression levels of miR-21, miR-29a, miR-192, miR-200b, miR-210, and miR-146a. We can conclude that 1) stem cell-derived EV therapies, especially the EVs (ADSCs) led to regression of structural and functional changes in the vascular wall and of key orchestrator expression in the atherosclerosis-induced endothelial dysfunction; 2) transfection of EVs with Smad2/3siRNA amplified the ability of EVs(ADSCs) or EVs(MSCs) to regress the inflammation-mediated atherosclerotic process.

Extracellular vesicles-incorporated microRNA signature as biomarker and diagnosis of prediabetes state and its complications.

Extracellular vesicles (EVs) are small anuclear vesicles, delimited by a lipid bilayer, released by almost all cell types, carrying functionally active biological molecules that can be transferred to the neighbouring or distant cells, inducing phenotypical and functional changes, relevant in various physio-pathological conditions. The microRNAs are the most significant active components transported by EVs, with crucial role in intercellular communication and significant effects on recipient cells. They may also server as novel valuable biomarkers for the diagnosis of metabolic disorders. Moreover, EVs are supposed to mediate type 2 diabetes mellitus (T2DM) risk and its progress. The T2DM development is preceded by prediabetes, a state that is associated with early forms of nephropathy and neuropathy, chronic kidney disease, diabetic retinopathy, and increased risk of macrovascular disease. Although the interest of scientists was focused not only on the pathogenesis of diabetes, but also on the early diagnosis, little is known about EVs-incorporated microRNA involvement in prediabetes state and its microvascular and macrovascular complications. Here, we survey the biogenesis, classification, content, biological functions and the most popular primary isolation methods of EVs, review the EVs-associated microRNA profiling connexion with early stages of diabetes and discuss the role of EVs containing specific microRNAs in prediabetes complications.

Extracellular Vesicles from Adipose Tissue Stem Cells in Diabetes and Associated Cardiovascular Disease; Pathobiological Impact and Therapeutic Potential.

Adipose tissue-derived stem cells (ADSCs) are pluripotent mesenchymal stem cells found in relatively high percentages in the adipose tissue and able to self-renew and differentiate into many different types of cells. "Extracellular vesicles (EVs), small membrane vesicular structures released during cell activation, senescence, or apoptosis, act as mediators for long distance communication between cells, transferring their specific bioactive molecules into host target cells". There is a general consensus on how to define and isolate ADSCs, however, multiple separation and characterization protocols are being used in the present which complicate the results' integration in a single theory on ADSCs' and their derived factors' way of action. Metabolic syndrome and type 2 diabetes mellitus (T2DM) are mainly caused by abnormal adipose tissue size, distribution and metabolism and so ADSCs and their secretory factors such as EVs are currently investigated as therapeutics in these diseases. Moreover, due to their relatively easy isolation and propagation in culture and their differentiation ability, ADSCs are being employed in preclinical studies of implantable devices or prosthetics. This review aims to provide a comprehensive summary of the current knowledge on EVs secreted from ADSCs both as diagnostic biomarkers and therapeutics in diabetes and associated cardiovascular disease, the molecular mechanisms involved, as well as on the use of ADSC differentiation potential in cardiovascular tissue repair and prostheses.

Integrins α4ß1 and αVß3 are Reduced in Endothelial Progenitor Cells from Diabetic Dyslipidemic Mice and May Represent New Targets for Therapy in Aortic Valve Disease.

Diabetes reduces the number and induces dysfunction in circulating endothelial progenitor cells (EPCs) by mechanisms that are still uncovered. This study aims to evaluate the number, viability, phenotype, and function of EPCs in dyslipidemic mice with early diabetes mellitus and EPC infiltration in the aortic valve in order to identify possible therapeutic targets in diabetes-associated cardiovascular disease. A streptozotocin-induced diabetic apolipoprotein E knock-out (ApoE-/-) mouse model was used to identify the early and progressive changes, at 4 or 7 days on atherogenic diet after the last streptozotocin or citrate buffer injection. Blood and aortic valves from diabetic or nondiabetic ApoE-/- animals were collected.EPCs were identified as CD34 and vascular endothelial growth factor receptor 2 positive monocytes, and the expression levels of α4ß1, αVß3, αVß5, ß1, αLß2, α5 integrins, and C-X-C chemokine receptor type 4 chemokine receptor on EPC surface were assessed by flow cytometry. The number of CD34 positive cells in the aortic valve, previously found to be recruited progenitor cells, was measured by fluorescence microscopy. Our results show that aortic valves from mice fed 7 days with atherogenic diet presented a significantly higher number of CD34 positive cells compared with mice fed only 4 days with the same diet, and diabetes reversed this finding. We also show a reduction of circulatory EPC numbers in diabetic mice caused by cell senescence and lower mobilization. Dyslipidemia induced EPC death through apoptosis regardless of the presence of diabetes, as shown by the higher percent of propidium iodide positive cells and higher cleaved caspase-3 levels. EPCs from diabetic mice expressed α4ß1 and αVß3 integrins at a lower level, while the rest of the integrins tested were unaffected by diabetes or diet. In conclusion, reduced EPC number and expression of α4ß1 and αVß3 integrins on EPCs at 4 and 7 days after diabetes induction in atherosclerosis-prone mice have resulted in lower recruitment of EPCs in the aortic valve.

Intravenous Administration of Allogenic Cell-Derived Microvesicles of Healthy Origins Defend Against Atherosclerotic Cardiovascular Disease Development by a Direct Action on Endothelial Progenitor Cells.

Atherosclerosis and cardiovascular disease development is the outcome of intermediate processes where endothelial dysfunction and vascular inflammation are main protagonists. Cell-derived microvesicles (MVs), endothelial progenitor cells (EPCs), and circulating microRNAs (miRNAs) are known as biomarkers and potential regulators for atherosclerotic vascular disease, but their role in the complexity of the inflammatory process and in the mechanism of vascular restoration is far from clear. We aimed to evaluate the biological activity and functional role of MVs, in particular of the EPCs-derived MVs (MVEs), of healthy origins in reducing atherosclerotic vascular disease development. The experiments were performed on hamsters divided into the following groups: simultaneously hypertensive-hyperlipidemic (HH group) by combining two feeding conditions for 4 months; HH with retro-orbital sinus injection containing 1 × 105 MVs or MVEs from control hamsters, one dose per month for 4 months of HH diet, to prevent atherosclerosis (HH-MVs or HH-MVEs group); and controls (C group), age-matched normal healthy animals. We found that circulating MV and MVE transplantation of healthy origins significantly reduces atherosclerosis development via (1) the mitigation of dyslipidemia, hypertension, and circulating EPC/cytokine/chemokine levels and (2) the structural and functional remodeling of arterial and left ventricular walls. We also demonstrated that (1) circulating MVs contain miRNAs; this was demonstrated by validating MVs and MVEs as transporters of Ago2-miRNA, Stau1-miRNA, and Stau2-miRNA complexes and (2) MV and MVE administration significantly protect against atherosclerotic cardiovascular disease via transfer of miR-223, miR-21, miR-126, and miR-146a to circulating late EPCs. It should be mentioned that the favorable effects of MVEs were greater than those of MVs. Our findings suggest that allogenic MV and MVE administration of healthy origins could counteract HH diet-induced detrimental effects by biologically active miR-10a, miR-21, miR-126, and miR-146a transfer to circulating EPCs, mediating their vascular repair function in atherosclerosis processes.

Hypertension Associated With Hyperlipidemia Induced Different MicroRNA Expression Profiles in Plasma, Platelets, and Platelet-Derived Microvesicles; Effects of Endothelial Progenitor Cell Therapy.

Aim: The aim of this study was to analyze the expressed profiles of miRNAs in plasma, platelets, and platelet-derived microvesicles (PMVs) obtained from experimental induced atherosclerosis animal model and to investigate the effect of EPC transplantation on these profiles. Methods: Seventeen selected circulating miRNAs (miR-19a,-21,-126,-146a,-223,-26b,-92a,-222,-210,-221,-143,-10a,-145,-155,-34a,-204, and miR-214) were individually analyzed in plasma, platelets, and PMVs isolated from peripheral blood of hypertensive-hyperlipidemic hamsters treated or not with endothelial progenitor cells (EPCs), and of healthy hamsters taken as control group. Results: Comparative with control group, in hypertension associated with hyperlipidemia the investigated miRNA expression profiles were different: (i) in plasma, the levels of all investigated miRNAs were significantly increased, the highest enhances being noticed for miR-21,-146a,-221,-143,-34a, and miR-204; (ii) in platelets, the expressions of almost all miRNAs were significantly elevated, remarkable for miR-126,-146a,-223,-222, and miR-214, while levels of miR-143, miR-10a, and miR-145 were significantly reduced; (iii) in PMVs, numerous miRNAs were found to have significantly increased levels, especially miR-222,-221,-210, and miR-34a, whereas expressions of various miRNAs as miR-223,-214,-146a,-143,-10a, and miR-145 were significantly decreased. The treatment with EPCs had the following reverse effects: (i) in plasma, significantly reduced the expression of miR-26b,-143,-34a,-204, and miR-214; (ii) in platelets, significantly decreased the levels of almost investigated miRNAs, with remarkably diminishing for miR-126 and miR-221; and (iii) in PMVs, significantly lowered the expression of some miRNAs, with considerably reductions for miR-222,-221,-210, and miR-19a, while the level of miR-214 was found elevated. Conclusions: The present study revealed that miRNAs have differential expression profiles in plasma, platelets, and PMVs under hypertension associated with hyperlipidemia conditions. The different miRNA profile in PMVs compared with platelets indicated an active mechanism of selective packing of miRNAs into PMVs from maternal cells; various miRNAs such as miR-19a,-21,-126,-26b,-92a,-155,-204,-210,-221,-222, and-34a delivered by PMVs may contribute to enrichment of circulating plasma miRNA expression. In addition, our study showed that the EPC-based therapy can regulate the expressions of investigated miRNAs into the three sources. These results provide novel information that could help in finding potential targets for the development of new therapeutic strategies in the cardiovascular disease.

Diabetes-induced early molecular and functional changes in aortic heart valves in a murine model of atherosclerosis.

Diabetes contributes directly to the development of cardiovascular aortic valve disease. There is currently no drug therapy available for a dysfunctional valve and this urges the need for additional research to identify distinctive mechanisms of cardiovascular aortic valve disease evolution. The aim of this study was to evaluate changes of valvular aortic lesions induced in a hyperlipemic ApoE-/- mouse model by early type 1 diabetes onset (at 4 and 7 days after streptozotocin induction). The haemodynamic valve parameters were evaluated by echography and blood samples and aortic valves were collected. Plasma parameters were measured, and inflammatory, remodelling and osteogenic markers were evaluated in the aortic valves. Next, correlations between all parameters were determined. The results showed early aortic valve dysfunction detected by echography after 1 week of diabetes; lesions were found in the aortic root. Moreover, increased expression of cell adhesion molecules, extracellular matrix remodelling and osteogenic markers were detected in hyperlipemic ApoE-/- diabetic mice. Significant correlations were found between tissue valve biomarkers and plasmatic and haemodynamic parameters. Our study may help to understand the mechanisms of aortic valve disease in the diabetic milieu in order to discover and validate new biomarkers of cardiovascular aortic valve disease in diabetes and reveal new possible targets for nanobiotherapies.

Platelets of Healthy Origins Promote Functional Improvement of Atherosclerotic Endothelial Progenitor Cells.

The purpose was to evaluate the effect of platelets on functional properties of late endothelial progenitor cells (EPCs), in the direct co-culture conditions, and to investigate the involved mediators, in experimental induced atherosclerosis. The late EPCs obtained from two animal groups, hypertensive-hyperlipidemic (HH) and control (C) hamsters, named late EPCs-HH and late EPCs-C, were co-incubated with or without platelets isolated from both groups. Our results have showed that exposure to platelets from control animals: (i) promoted the late EPCs-C capacity to form colonies and capillary-like structures, and also to proliferate and migrate; (ii) improved the functional properties of late EPCs-HH; (iii) strengthened the direct binding EPCs-platelets; (iv) increased SDF-1α,VEGF, PDGF, and reduced CD40L, IL-1ß,-6,-8 levels; and (v) enhanced miR-223 and IGF-1R expressions. Platelets from HH group diminished functional abilities for both EPC types and had opposite effects on these pro-angiogenic and pro-inflammatory molecules. Furthermore, testing the direct effect of miR-223 and IGF-1R on late EPCs disclosed that these molecular factors improve late EPC functional properties in atherosclerosis in terms of stimulation of the proliferation and migration abilities. In conclusion, in vitro exposure to platelets of healthy origins had a positive effect on functional properties of atherosclerotic late EPCs. The most likely candidates mediating EPC-platelet interaction can be SDF-1α, VEGF, CD40L, PDGF, IL-1ß,-6,-8, miR-223, and IGF-1R. The current study brings evidences that the presence of healthy origin platelets is of utmost importance on functional improvement of EPCs in atherosclerosis.

Sera of Obese Type 2 Diabetic Patients Undergoing Metabolic Surgery Instead of Conventional Treatment Exert Beneficial Effects on Beta Cell Survival and Function: Results of a Randomized Clinical Study.

BACKGROUND: Pancreatic beta cells are highly sensitive to oxidative and endoplasmic reticulum (ER) stress, commonly occurring in type 2 diabetes (T2D) and obesity. OBJECTIVE: We aimed at investigating cellular responses of human beta cells exposed to sera from obese T2D patients treated differently, namely by conventional therapy or laparoscopic sleeve gastrectomy (LSG). METHODS: Serum samples from obese T2D men randomized to conventional treatment or LSG were taken at baseline and 6 months later. After exposing 1.1B4 cells to study patients' sera, the following were assessed: cellular viability and proliferation (by MTT and xCELLigence assays), reactive oxygen species (ROS) production (with DCFH-DA), and expression of ER stress markers, oxidative- or autophagy-related proteins and insulin (by real-time PCR and Western blot). RESULTS: At 6-month follow-up, patients undergoing LSG achieved an adequate glycemic control, whereas conventionally treated patients did not. As compared to 1.1B4 cells incubated with baseline sera (control), cells exposed to sera from LSG-treated participants exhibited (i) increased viability and proliferation (p < 0.05); (ii) diminished levels of ROS and p53 (p < 0.05); (iii) enhanced protein expression of autophagy-related SIRT1 and p62/SQSTM1 (p < 0.05); (iv) significantly decreased transcript levels of ER stress markers (p < 0.05); and (v) augmented insulin expression (p < 0.05). Conversely, the 6-month conventional therapy appeared not to impact on circulating redox status. Moreover, 1.1B4 cells exposed to sera from conventionally treated patients experienced mild ER stress. CONCLUSION: Circulating factors in patients with improved diabetes after metabolic surgery exerted favorable effects on beta cell function and survival.

The Distinct Effects of Palmitic and Oleic Acid on Pancreatic Beta Cell Function: The Elucidation of Associated Mechanisms and Effector Molecules.

In this study, we aimed to identify the mechanisms underlying the different effects of palmitic acid and oleic acid on human pancreatic beta cell function. To address this problem, the oxidative stress, endoplasmic reticulum stress, inflammation, apoptosis and their mediator molecules have been investigated in the insulin releasing beta cells exposed to palmitic and/or oleic acid. Herein, we have demonstrated that in cultured 1.1B4 beta cells oleic acid promotes neutral lipid accumulation and insulin secretion, whereas palmitic acid is poorly incorporated into triglyceride and it does not stimulate insulin secretion from human pancreatic islets at physiologically glucose concentrations. In addition, palmitic acid caused: (1) oxidative stress through a mechanism involving increases in ROS production and MMP-2 protein expression/gelatinolytic activity associated with down-regulation of SOD2 protein; (2) endoplasmic reticulum stress by up-regulation of chaperone BiP protein and unfolded protein response (UPR) transcription factors (eIF2α, ATF6, XBP1u proteins) and by PTP-1B down-regulation in both mRNA and protein levels; (3) inflammation through enhanced synthesis of proinflammatory cytokines (IL6, IL8 proteins); and (4) apoptosis by enforced proteic expression of CHOP multifunctional transcription factor. Oleic acid alone had opposite effects due to its different capacity of controlling these metabolic pathways, in particular by reduction of the ROS levels and MMP-2 activity, down-regulation of BiP, eIF2α, ATF6, XBP1u, CHOP, IL6, IL8 and by SOD2 and PTP-1B overexpression. The supplementation of saturated palmitic acid with the monounsaturated oleic acid reversed the negative effects of palmitic acid alone regulating insulin secretion from pancreatic beta cells through ROS, MMP-2, ATF6, XBP1u, IL8 reduction and SOD2, PTP-1B activation. Our findings have shown the protective action of oleic acid against palmitic acid on beta cell lipotoxicity through promotion of triglyceride accumulation and insulin secretion and regulation of some effector molecules involved in oxidative stress, endoplasmic reticulum stress, inflammation and apoptosis.

Microparticles: From Biogenesis to Biomarkers and Diagnostic Tools in Cardiovascular Disease.

Cardiovascular diseases (CVD) are the main object of morbidity and death in the world. There are different triggers of CVD, but atherosclerosis and/or hypertension are the most usual. CVD risk factors are used as prognosticators of disease process, while the biomarkers that envisage cardiovascular risk have an important biological basis. The integration of CVD risk factors and new biomarkers can improve CVD risk stratification and disease management. The identification of microparticles (MPs) in body fluids has opened new perspectives in diagnosis, prognosis and therapy of CVD pathogenesis. MPs are submicron vesicles released from a diversity of cell types with pleiotropic biological effects on signaling among cells. They are correlated with disease state and play a major role in atherosclerosis associated with inflammation, thrombosis as well as in CVD development and progression. This review reveals many sides of MPs as risk factors, biomarkers, novel forms of intercellular communication, non-invasive potential diagnostic tools and discusses their positive and negative impact on CVD.

Role of MicroRNA in Endothelial Dysfunction and Hypertension.

PURPOSE OF REVIEW: Hypertension is either a cause or a consequence of the endothelial dysfunction and a major risk factor for cardiovascular disease (CVD). In vitro and in vivo studies established that microRNAs (miRNAs) are decisive for endothelial cell gene expression and function in various pathological conditions associated with CVD. This review provides an overview of the miRNA role in controlling the key connections between endothelial dysfunction and hypertension. RECENT FINDINGS: Herein we summarize the present understanding of mechanisms underlying hypertension and its associated endothelial dysfunction as well as the miRNA role in endothelial cells with accent on the modulation of renin-angiotensin-aldosterone-system, nitric oxide, oxidative stress and on the control of vascular inflammation and angiogenesis in relation to endothelial dysfunction in hypertension. In particular, latest insights in the identification of endothelial-specific microRNAs and their targets are added to the understanding of miRNA significance in hypertension. This comprehensive knowledge of the role of miRNAs in endothelial dysfunction and hypertension and of molecular mechanisms proposed for miRNA actions may offer novel diagnostic biomarkers and therapeutic targets for controlling hypertension-associated endothelial dysfunction and other cardiovascular complications.

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

BACKGROUND INFORMATION: 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) 1×10(5) 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, 1×10(5) endothelial progenitor cells of healthy origins or/and 1×10(5) platelet microparticles of atherosclerotic origins were inoculated every other month during hypertensive-hypercholesterolemic diet. RESULTS: Endothelial progenitor cell treatment had the following effects: (1) re-established plasmatic parameters: cholesterol and triglyceride concentrations, blood pressure, heart rate, cytokine and chemokine profiles, platelet microparticle pro-thrombotic activity and endothelial progenitor cell paracrine activity reflected by cytokine/chemokine detection; (2) reduced lipid, macrophage and microparticle accumulation in liver; (3) reduced atherosclerosis development, revealed by decreased lipid, macrophage and microparticle content of arterial wall; (4) induced the recruitment and incorporation of endothelial progenitor cells into liver and arterial wall; (5) improved arterial dysfunction by increasing contraction and relaxation; (6) reduced the protein expression of specific pro-inflammatory molecules in liver and arterial wall. Platelet microparticle transplantation aggravated the above-mentioned biomarkers and atherosclerosis process, which were partially reverted with co-inoculation of platelet microparticles and endothelial progenitor cells. CONCLUSIONS: With this study, we demonstrate in a hypertensive-hypercholesterolemic hamster model, that the endothelial progenitor cell-based therapy suppresses the development of atherosclerosis and reduces hepatic lipid and macrophage accumulation with the consequent alleviation of dyslipidaemia and hypertension. SIGNIFICANCE: Our results support the notion that increasing the number of circulating endothelial progenitor cells by different ways could be a promising therapeutic tool for atherosclerosis.

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.

Midkine proteins in cardio-vascular disease. Where do we come from and where are we heading to?

Midkine is a recently identified new growth factor/cytokine with pleiotropic functions in the human organism. First discovered in the late eighties, midkines have now become the subject of numerous studies in cardiovascular, neurologic, renal diseases and also various types of cancers. We summarize here the most important functions of midkine in cardiovascular diseases, emphasizing its role in inflammation and its antiapoptotic and proangiogenetic effects. Midkine has multiple roles in the organism, with the specific feature of being either beneficial or harmful depending on which tissue it acts on. Even though midkine has been shown to have cardiac protective effects against acute ischemia/reperfusion injury and to inhibit cardiac remodeling, it also promotes intimal hyperplasia and vascular stenosis. As such, different therapeutic strategies are currently being evaluated, consisting of administering either midkine proteins or midkine inhibitors depending on the desired outcome. More data is gathering to suggest that these novel therapies could become an adjunctive to standard cardiovascular therapy. Nonetheless, much is still to be learned about midkine. The encouraging results up till now require further studying in order to fully understand the complete profile of its mechanism of action and the clinical safety and efficacy of novel therapeutic opportunities offered by midkine molecular targeting.

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

BACKGROUND INFORMATION: 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). RESULTS: Compared with C group, EPCs isolated from HH and HH-PMPs groups presented a reduction of endothelial nitric oxide synthase and vascular endothelial growth factor expressions and an increase in thrombospondin-1 expression and inflammatory molecule secretion: interleukin 8 (IL)-8, myeloperoxidase (MPO) and plasminogen activator inhibitor-1 (PAI-1). EPC administration had beneficial effects, the obtained results being similar with those from the C group, while the combination with PMPs did not improve the EPC influences. Static coincubation of EPCs from HH and HH-PMPs with analogous platelets resulted in an increased EPC adhesion/migration, and IL-8, monocyte chemotactic protein-1, regulated on activation, normal T expressed and secreted, MPO and PAI-1 release, explained by the platelet hyperaggregability induced by pronounced distribution of vasodilator-stimulated phosphoprotein and filamentous actin, and the secretion of proinflammatory factors: IL-1ß, -6, -8, CD40 ligand. EPC therapy alone revealed an impaired platelet-EPC interaction directly correlated with the reduction of inflammatory markers and platelet aggregability. Moreover, in a dynamic flow system, EPCs and platelets from HH and HH-PMPs exhibited weakened interplay abilities, while EPC transplantation reinforces them. CONCLUSIONS: The present study demonstrates that HH animals revealed functional impairment of EPCs and platelets, which correlate with their reduced contribution to re-endothelialisation at the injury site, although in vitro exposure to immobilised platelets promotes their adhesion and migration. EPC administration alone recovers EPC/platelet functions and consolidates their interaction under dynamic flow conditions. These findings disclose new advances in understanding the platelet-EPC interaction and its role in the vascular repair.

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 (ß2-Integrins, α4ß1-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.

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 3ß, 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-1ß; 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.