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.

Hypertension induces compensatory left ventricular hypertrophy by a mechanism involving gap junction lateralization and overexpression of CD36, PKC and MMP-2.

Hypertension-induced left ventricular hypertrophy evolves initially as an adaptive response meant to minimize ventricular wall stress. The mechanisms involved in the preservation of the cardiac function during the "compensatory" phase of the left ventricular hypertrophy are still unclear. Therefore, we aimed at uncovering fine changes that aid the heart to cope with the increased stress in hypertension. Male golden Syrian hamsters were given NG-nitro-L-arginine methyl ester (L-NAME) for 16 weeks, and they became hypertensive (HT), developing left ventricular hypertrophy with no impaired contractility or fibrosis. As compared to age-matched control hamsters, the hypertrophied left ventricles in L-NAME-induced HT hamsters exhibited the following structural and molecular changes: (i) accumulation of lipid droplets (LDs) within cardiomyocytes and relocation of gap junctions to the lateral membrane of cardiomyocytes or close to mitochondria (revealed by electron microscopy); (ii) overexpression of the cluster of differentiation 36 (CD36) fatty acid transporter, protein kinase C (PKC), and matrix metalloproteinase-2 (MMP-2), enhanced activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway, and unchanged expression of the connexin 43 (Cx43) and N-cadherin junctional proteins (assessed by Western blot); (iii) increased protein carbonyl content, assessed with a 2,4-Dinitrophenylhydrazine (DNPH)-based spectrophotometric assay, indicative of an enhanced reactive oxygen species (ROS) production; and (iv) augmented MMP-2 activity (determined by gelatin zymography). These changes may participate in an orchestrated adaptive hypertrophic growth response that helps to maintain cardiac performance, in HT hamsters. Together, these findings could provide support for designing future strategies meant to prevent the transition from compensatory left ventricular hypertrophy to decompensated heart failure.

Proteomics of regenerated tissue in response to a titanium implant with a bioactive surface in a rat tibial defect model.

Due to their excellent mechanical and biocompatibility properties, titanium-based implants are successfully used as biomedical devices. However, when new bone formation fails for different reasons, impaired fracture healing becomes a clinical problem and affects the patient's quality of life. We aimed to design a new bioactive surface of titanium implants with a synergetic PEG biopolymer-based composition for gradual delivery of growth factors (FGF2, VEGF, and BMP4) during bone healing. The optimal architecture of non-cytotoxic polymeric coatings deposited by dip coating under controlled parameters was assessed both in cultured cells and in a rat tibial defect model (100% viability). Notably, the titanium adsorbed polymer matrix induced an improved healing process when compared with the individual action of each biomolecules. High-performance mass spectrometry analysis demonstrated that recovery after a traumatic event is governed by specific differentially regulated proteins, acting in a coordinated response to the external stimulus. Predicted protein interactions shown by STRING analysis were well organized in hub-based networks related with response to chemical, wound healing and response to stress pathways. The proposed functional polymer coatings of the titanium implants demonstrated the significant improvement of bone healing process after injury.

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.

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.

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.

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.

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

OBJECTIVES: To prepare and characterize in vitro and in vivo lipid nanoemulsions (LN) loaded with curcumin (Cm) and functionalized with a cell-penetrating peptide. METHODS: Curcumin-loaded lipid nanoemulsions (CmLN) functionalized with a nona-arginine peptide (R9-CmLN) have been obtained, characterized and optimized for size, entrapment efficiency and in vitro Cm release. The interaction of R9-CmLN with human endothelial cells (HEC) was investigated using cultured EA.hy926 cells, and in vivo biodistribution studies were performed using C57BL6 mice. KEY FINDINGS: When used in therapeutically relevant concentration, R9-CmLN have low haemolytic activity, low cytotoxicity on HEC, and show anti-inflammatory effects by reducing the monocytes adhesion to TNF-α activated HEC. Moreover, HEC uptake and internalization of R9-CmLN was significantly higher compared to the non-functionalized CmLN. In vivo biodistribution studies in mice revealed a higher accumulation of R9-CmLN in the liver and the lungs compared to CmLN and the body clearance of the both nanoformulations after 72 h. CONCLUSIONS: Cell-penetrating peptides-functionalized CmLN have superior characteristics compared to their non-functionalized counterparts: are more efficiently internalized by the cells, produces anti-inflammatory effects in HEC and when administrated intravenously in mice exhibit increased accumulation in the liver and the lungs, suggesting their potential therapeutic applications in different inflammatory pathologies localized in the liver or the lungs.

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.

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.

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.

Effect of depletion of monocytes/macrophages on early aortic valve lesion in experimental hyperlipidemia.

Monocytes/macrophages are key players throughout atheroma development. The aim of this study was to determine the role of macrophages in lesion formation in heart valves in hyperlipidemia. We examined whether systemic depletion of monocytes/macrophages had a beneficial or adverse effect on the development of lesions in hyperlipemic hamsters injected twice weekly (for 2 months) with clodronate-encapsulated liposomes (H+Lclod), a treatment that selectively induces significant monocyte apoptosis. Hyperlipemic hamsters were employed as controls, as were hyperlipemic hamsters treated with plain liposomes. We assayed serum cholesterol (CH) and triglycerides (TG), the lipid and collagen contents and the size of the valve lesions, the matrix metalloproteinases (MMPs) in the serum and vessel wall, apolipoprotein E (ApoE), interleukin-1beta (IL-1beta), and superoxide anion production. In comparison with controls, H+Lclod hamsters exhibited: (1) increased lipid and collagen accumulation within the lesions, (2) decreased activity of MMP-9 and MMP-2 in sera and aortic homogenates, (3) decreased serum CH and TG and decreased expression of ApoE in sera and liver, (4) reduced expression of IL-1beta in aorta and liver homogenates, and (5) no change in the level of superoxide anion in the aorta. Thus, initially, the presence of the macrophages is beneficial in valvular lesion formation. Depletion of monocytes/macrophages is a two-edged sword having a beneficial effect by decreasing the expression of IL-1beta and MMP activities but an adverse effect by inducing a significant increase in the lipid and collagen content and expansion of valvular lesions.

Promoting effect of 5-azacytidine on the myogenic differentiation of bone marrow stromal cells.

Bone marrow stromal cells (BMSC) can differentiate into various cell types including myocytes, which may be valuable in cellular therapy of myocardial infarction. In an attempt to increase the myogenic commitment of BMSC, we investigated the extent of conversion induced by the demethylation agent 5-azacytidine. BMSC isolated from the adult rat tibia were exposed in culture to 5microM 5-azacytidine for 24h, 1 day after seeding. The treatment was repeated at weekly intervals and the expression of muscle-specific proteins and genes was assessed. The results revealed that cultured cells lost the native expression of osteocalcin and alkaline phosphatase as a function of time and began to express connexin 43. Exposure to 5-azacytidine of BMSC induced, at 14 days, a myocyte-resembling phenotype that included the expression of muscle-specific proteins (sarcomeric alpha-actin, troponin T, desmin, alpha-actinin, and GATA-4) and genes (GATA-4, myoD, desmin, and alpha-actinin), numerous mitochondria and myofilaments; however, the latter did not form sarcomeres. Although some of these myogenic markers also appeared in untreated cells, exposure to 5-azacytidine induced an enhanced response of calcium channels, as well as a threefold increase in desmin and myoD gene expression and a twofold increase in alpha-actinin gene and protein expression above the control values. In conclusion, the results demonstrate a promoting effect of 5-azacytidine on the expression of muscle-specific proteins and genes in BMSC in culture. Notably, the myogenic differentiation takes place over a short period of time. Priming of mesenchymal cells to cardiomyogenic differentiation may have significant applications in cellular approaches to ameliorate muscle loss after myocardial ischemia.

Protective effects of nebivolol and reversal of endothelial dysfunction in diabetes associated with hypertension.

This study aims to decipher the potential effects of nebivolol in prevention and/or regression of renal artery dysfunction in diabetes associated with hypertension. Renal arteries were isolated from 80 male mice divided into four experimental groups: (i) group D: diabetics, at 2 months since streptozotocin injection; (ii) group Din: mice that at the initiation of streptozotocin diabetes were treated with 10 mg/kg b.w./day nebivolol for 2 months, to test for the potential prevention of vascular dysfunction; (iii) group Dfin: mice that after 2 months of diabetes were treated daily with 10 mg/kg b.w./day nebivolol for additional 2 months, in order to follow the possible regression of the dysfunction, and (iv) controls (C), age-matched healthy animals. The following measurements were performed: arterial blood pressure, plasma glucose concentration, and the vascular reactivity of the renal arteries in response to noradrenaline (10(-4) M), acetylcholine (10(-4) M) and sodium nitroprusside (10(-4) M). To assess the molecular mechanisms involved in the reactivity of the renal artery, the contribution of mitogen-activated protein kinase (MAP kinase) pathway and of L-type voltage gated Ca(2+) channels (in the contractile response to noradrenaline), of nitric oxide (NO) and Ca(2+) activated K(+) channels (in the endothelium-dependent vasodilator response), and of cGMP (in the endothelium-independent vasodilator response) was examined by exposing the arteries to corresponding inhibitors, and by using myograph and patch-clamp techniques, immunoblotting and NO assays. Results showed that, group D was characterized by hyperglycemia (blood glucose concentration: 136.66 +/- 4.96 mg/dl, a value approximately 65% increased compared to group C) and hypertension (systolic blood pressure: 145.66 +/- 5.96 mm Hg, a value approximately 34% increased compared to group C). Compared to group D, group Din was characterized by diminished blood glucose concentration ( approximately 1.6 fold), reduced systolic and diastolic blood pressure ( approximately 1.3 fold) and heart rate ( approximately 1.6 fold), as well as by increased contractile response of the renal artery to noradrenaline ( approximately 1.84 fold) and of the impeded vasodilator response to acetylcholine ( approximately 1.81 fold) and sodium nitroprusside ( approximately 1.42 fold). Together, these effects demonstrate that administration of 10 mg/kg b.w./day nebivolol at the moment of diabetes induction has preventive effects, ameliorating diabetes dysfunctions. Compared to group D, group Dfin was characterized by diminished glucose concentration ( approximately 1.3 fold), reduced systolic and diastolic blood pressure and heart rate (both approximately 1.2 fold), and by augmentation of contractile response of the renal artery to noradrenaline ( approximately 1.62 fold) and of vasodilator response to acetylcholine ( approximately 1.13 fold) and sodium nitroprusside ( approximately 1.19 fold). These effects assess that administration of 10 mg/kg b.w./day nebivolol after 2 months of diabetes contributes to regression of diabetes-associated dysfunctionalies. Nebivolol influenced the molecular mechanisms involved in renal artery reactivity in diabetic and hypertensive mice: it increased the NO production and endothelial NO synthase (eNOS) protein expression, decreased the expression of proportional, variant protein in L-type calcium channels and Ca(2+) activated K(+) channels, and diminished the MAP kinase activity. The reported data suggest that nebivolol may offer additional vascular protection for treating diabetes associated with hypertension.