Levamisole Impairs Vascular Function by Blocking α-Adrenergic Receptors and Reducing NO Bioavailability in Rabbit Renal Artery.

Levamisole is an anthelmintic drug restricted to veterinary use but is currently detected as the most widely used cocaine cutting agent in European countries. Levamisole-adulterated cocaine has been linked to acute kidney injury, marked by a decrease in glomerular filtration rate, which involves reduced renal blood flow, but data on the alteration of renovascular response produced by levamisole are scarce. Renal arteries were isolated from healthy rabbits and used for isometric tension recording in organ baths and protein analysis. We provide evidence that depending on its concentration, levamisole modulates renovascular tone by acting as a non-selective α-adrenergic receptor blocker and down-regulates α1-adrenoceptor expression. Furthermore, levamisole impairs the endothelium-dependent relaxation induced by acetylcholine without modifying endothelial nitric oxide synthase (eNOS) expression. However, exposure to superoxide dismutase (SOD) partially prevents the impairment of ACh-induced relaxation by levamisole. This response is consistent with a down-regulation of SOD1 and an up-regulation of NADPH oxidase 4 (Nox4), suggesting that endothelial NO loss is due to increased local oxidative stress. Our findings demonstrate that levamisole can interfere with renal blood flow and the coordinated response to a vasodilator stimulus, which could worsen the deleterious consequences of cocaine use.

Sodium valproate treatment reverses endothelial dysfunction in aorta from rabbits with acute myocardial infarction.

Sodium valproate (VPA), a histone deacetylase (HDAC) inhibitor, could be a promising candidate to treat acute myocardial infarction (AMI). In this study, AMI was induced in New Zealand White rabbits by occluding the left circumflex coronary artery for 1 h, followed by reperfusion. The animals were distributed into three experimental groups: the sham-operated group (SHAM), the AMI group and the AMI + VPA group (AMI treated with VPA 500 mg/kg/day). After 5 weeks, abdominal aorta was removed and used for isometric recording of tension in organ baths or protein expression by Western blot, and plasma for the determination of nitrate/nitrite (NOx) levels by colorimetric assay. Our results indicated that AMI induced a reduction of the endothelium-dependent response to acetylcholine without modifying the endothelium-independent response to sodium nitroprusside, leading to endothelial dysfunction. VPA treatment reversed AMI-induced endothelial dysfunction and even increased NO sensitivity in vascular smooth muscle. This response was consistent with an antioxidant effect of VPA, as it was able to reverse the superoxide dismutase 1 (SOD 1) down-regulation induced by AMI. Our experiments also ruled out that the VPA mechanism was related to eNOS, iNOS, sGC and arginase expression or changes in NOx plasma levels. Therefore, we conclude that VPA improves vasodilation by increasing NO bioavailability, likely due to its antioxidant effect. Since endothelial dysfunction was closely related to AMI, VPA treatment could increase aortic blood flow, making it a potential agent in reperfusion therapy that can prevent the vascular damage.

The Role of Aryl Hydrocarbon Receptor in the Endothelium: A Systematic Review.

Activation of the aryl hydrocarbon receptor (AhR) has been shown to be important in physiological processes other than detoxification, including vascular homeostasis. Although AhR is highly expressed in the endothelium, its function has been poorly studied. This systematic review aims to summarise current knowledge on the AhR role in the endothelium and its cardiovascular implications. We focus on endogenous AhR agonists, such as some uremic toxins and other agonists unrelated to environmental pollutants, as well as studies using AhR knockout models. We conclude that AhR activation leads to vascular oxidative stress and endothelial dysfunction and that blocking AhR signalling could provide a new target for the treatment of vascular disorders such as cardiovascular complications in patients with chronic kidney disease or pulmonary arterial hypertension.

Improvement of Vascular Insulin Sensitivity by Ranolazine.

Ranolazine (RN) is a drug used in the treatment of chronic coronary ischemia. Different clinical trials have shown that RN behaves as an anti-diabetic drug by lowering blood glucose and glycosylated hemoglobin (HbA1c) levels. However, RN has not been shown to improve insulin (IN) sensitivity. Our study investigates the possible facilitating effects of RN on the actions of IN in the rabbit aorta. IN induced vasodilation of the abdominal aorta in a concentration-dependent manner, and this dilatory effect was due to the phosphorylation of endothelial nitric oxide synthase (eNOS) and the formation of nitric oxide (NO). On the other hand, IN facilitated the vasodilator effects of acetylcholine but not the vasodilation induced by sodium nitroprusside. RN facilitated all the vasodilatory effects of IN. In addition, IN decreased the vasoconstrictor effects of adrenergic nerve stimulation and exogenous noradrenaline. Both effects were in turn facilitated by RN. The joint effect of RN with IN induced a significant increase in the ratio of p-eNOS/eNOS and pAKT/AKT. In conclusion, RN facilitated the vasodilator effects of IN, both direct and induced, on the adrenergic system. Therefore, RN increases vascular sensitivity to IN, thus decreasing tissue resistance to the hormone, a key mechanism in the development of type II diabetes.

Glucose 6-P Dehydrogenase Overexpression Improves Aging-Induced Endothelial Dysfunction in Aorta from Mice: Role of Arginase II.

The increase of vascular arginase activity during aging causes endothelial dysfunction. This enzyme competes with the endothelial nitric oxide synthase (eNOS) for L-arginine substrate. Our hypothesis is that glucose 6-P dehydrogenase (G6PD) overexpression could improve the endothelial function modulating the arginase pathway in aorta from mice. For this study, three groups of male mice were used: young wild type (WT) (6-9 months), old WT (21-22 months) and old G6PD-Tg (21-22 months) mice. Vascular reactivity results showed a reduced acetylcholine-dependent relaxation in the old WT but not old G6PD-Tg group. Endothelial dysfunction was reverted by nor-NOHA, an arginase inhibitor. Mice overexpressing G6PD underexpressed arginase II and also displayed a lower activity of this enzyme. Moreover, histological analyses demonstrated that age causes a thickness of aortic walls, but this did not occur in G6PD-Tg mice. We conclude that the overexpressing G6PD mouse is a model to improve vascular health via the arginase pathway.

Cerium dioxide nanoparticles modulate antioxidant defences and change vascular response in the human saphenous vein.

Nanoparticles have a promising future in biomedical applications and knowing whether they affect ex vivo vascular reactivity is a necessary step before their use in patients. In this study, we have evaluated the vascular effect of cerium dioxide nanoparticles (CeO2NPs) on the human saphenous vein in response to relaxing and contractile agonists. In addition, we have measured the protein expression of key enzymes related to vascular homeostasis and oxidative stress. We found that CeO2NPs increased expression of both SOD isoforms, and the consequent reduction of superoxide anion would enhance the bioavailability of NO explaining the increased vascular sensitivity to sodium nitroprusside in the presence of CeO2NPs. The NOX4 reduction induced by CeO2NPs may lead to lower H2O2 synthesis associated with vasodilation through potassium channels explaining the lower vasodilation to bradykinin. In addition, we showed for the first time, that CeO2NPs increase the expression of ACE2 in human saphenous vein, and it may be the cause of the reduced contraction to angiotensin II. Moreover, we ruled out that CeO2NPs have effect on the protein expression of eNOS, sGC, BKca channels and angiotensin II receptors or modify the vascular response to noradrenaline, endothelin-1 and TXA2 analogue. In conclusion, CeO2NPs show antioxidant properties, and together with their vascular effect, they could be postulated as adjuvants for the treatment of cardiovascular diseases.

Deleterious effects of levamisole, a cocaine adulterant, in rabbit aorta.

Levamisole, a veterinary anthelmintic drug, is one of the most widely used and dangerous cocaine adulterants. Like cocaine, levamisole acutely blocks noradrenaline reuptake but with much less potency, although its vascular effects are not well known. In this study, we evaluated the vascular effects of levamisole and cocaine in rabbit aortic rings used for isometric recording of tension in organ baths and protein expression by western blot. Our results indicated that levamisole (10-5-10-3 M) induced a concentration-dependent relaxation in rings precontracted with noradrenaline (10-7-3 × 10-7 M). Furthermore, it reduced the contractile response to phenylephrine (10-9-3 × 10-5 M) that was not modified by cocaine (10-5-10-4 M), and reduced α1-adrenergic receptor expression. Levamisole (10-6-10-4 M) produced a potentiation of the electrical field stimulation that was not further enhanced by the combination of both drugs. However, high concentrations of levamisole (10-3 M) abolished adrenergic neurotransmission whether administered alone or with cocaine (10-4 M). In addition, levamisole (10-5-10-3 M) also decreased endothelium-dependent relaxation to acetylcholine that was not further impaired by cocaine (10-4 M), and that was partially reversed by superoxide dismutase (SOD, 200 U/ml). These results demonstrate that levamisole has a dual effect on the adrenergic system, and its effects are independent of the presence of cocaine. At lower concentrations, it enhances the contractile sympathetic response by blocking presynaptic α2-adrenergic receptors, while at high concentrations, the effect of the antagonism of α1-adrenergic receptor prevails. In addition, levamisole induces endothelial dysfunction by reducing NO bioavailability, and this effect could be in part mediated by oxidative stress.

PPARγ as an indicator of vascular function in an experimental model of metabolic syndrome in rabbits.

BACKGROUND AND AIMS: Underlying mechanisms associated with vascular dysfunction in metabolic syndrome (MetS) remain unclear and can even vary from one vascular bed to another. METHODS: In this study, MetS was induced by a high-fat, high-sucrose diet, and after 28 weeks, aorta and renal arteries were removed and used for isometric recording of tension in organ baths, protein expression by Western blot, and histological analysis to assess the presence of atherosclerosis. RESULTS: MetS induced a mild hypertension, pre-diabetes, central obesity and dyslipidaemia. Our results indicated that MetS did not change the contractile response in either the aorta or renal artery. Conversely, vasodilation was affected in both arteries in a different way. The aorta from MetS showed vascular dysfunction, including lower response to acetylcholine and sodium nitroprusside, while the renal artery from MetS presented a preserved relaxation to acetylcholine and an increased sensitivity to sodium nitroprusside. We did not find vascular oxidative stress in the aorta from MetS, but we found a significant decrease in PPARγ, phospho-Akt (p-Akt) and phospho-eNOS (p-eNOS) protein expression. On the other hand, we found oxidative stress in the renal artery from MetS, and PPARγ, Akt and p-Akt were overexpressed. No evidence of atherosclerosis was found in arteries from MetS. CONCLUSIONS: MetS affects vascular function differently depending on the vessel. In the aorta, it decreases both the vasodilation and the expression of the PPARγ/Akt/eNOS pathway, while in the renal artery, it increases the expression of PPARγ/Akt signalling pathway without decreasing the vasodilation.

Action of low doses of Aspirin in Inflammation and Oxidative Stress induced by aß1-42 on Astrocytes in primary culture.

Aspirin has been used as anti-inflammatory and anti-aggregate for decades but the precise mechanism(s) of action after the presence of the toxic peptide Aß1-42 in cultured astrocytes remains poorly resolved. Here we use low-doses of aspirin (10-7 M) in astrocytes in primary culture in presence or absence of Aß1-42 toxic peptide. We noted an increase of cell viability and proliferation with or without Aß1-42 peptide presence in aspirin treated cells. In addition, a decrease in apoptosis, determined by Caspase 3 activity and the expression of Cyt c and Smac/Diablo, were detected. Also, aspirin diminished necrosis process (LDH levels), pro-inflammatory mediators (IL-ß and TNF-α) and NF-ᴋB protein expression, increasing anti-inflammatory PPAR-γ protein expression, preventing Aß1-42 toxic effects. Aspirin inhibited COX-2 and iNOS without changes in COX-1 expression, increasing anti-oxidant protein (Cu/Zn-SOD and Mn-SOD) expression in presence or absence of Aß1-42. Taken together, our results show that aspirin, at low doses increases cell viability by decreasing inflammation and oxidative stress, preventing the deleterious effects of the Aß1-42 peptide on astrocytes in primary culture. The use of low doses of aspirin may be more suitable for Alzheimer's disease.

Relaxant and antiadrenergic effects of ranolazine in human saphenous vein.

OBJECTIVES: Ranolazine improves vascular function in animal models. We evaluate the effects of ranolazine on vascular function and adrenergic response in human saphenous vein. METHODS: Rings from 53 patients undergoing coronary artery bypass grafting were mounted in organ baths. Concentration-response curves to ranolazine were constructed in rings precontracted with phenylephrine, endothelin-1, vasopressin, KCl and the thromboxane A2 analogue U-46619. In rings precontracted with phenylephrine, relaxation to ranolazine was tested in the absence and presence of endothelial factors inhibitors, K+ channel blockers and verapamil. The effects of ranolazine on frequency-response and concentration-response curves to phenylephrine were performed in the absence and presence of endothelial factors inhibitors and K+ channel blockers. Endothelial nitric oxide synthase, α1 adrenergic receptor and large conductance Ca2+-activated K+ channel protein expressions were measured by Western blotting. RESULTS: Ranolazine (10-9-10-4 M) produced a concentration-dependent relaxation only in rings precontracted with phenylephrine that was reduced by endothelial denudation, NG-nitro-l-arginine methyl ester (10-4 M), charybdotoxin (10-7 M) and verapamil (10-6 M). Ranolazine diminished adrenergic contractions induced by electrical field stimulation (2-4 Hz) and phenylephrine (10-9-10-5 M) that were prevented by tetraethylammonium (10-3 M) and charybdotoxin (10-7 M). Ranolazine significantly decreased α1 adrenergic receptor and increased large conductance Ca2+-activated K+ channel protein expression in the saphenous vein. CONCLUSIONS: Ranolazine diminishes the adrenergic vasoconstriction, acting as α1 antagonist, and by increasing large conductance Ca2+-activated K+ channel involvement. The relaxant effects of ranolazine are partially mediated by endothelial nitric oxide, large conductance Ca2+-activated K+ channels and the blockade of voltage-dependent Ca2+ channels.

Targeting Early Atherosclerosis: A Focus on Oxidative Stress and Inflammation.

Atherosclerosis is a chronic vascular inflammatory disease associated to oxidative stress and endothelial dysfunction. Oxidation of low-density lipoprotein (LDL) cholesterol is one of the key factors for the development of atherosclerosis. Nonoxidized LDL have a low affinity for macrophages, so they are not themselves a risk factor. However, lowering LDL levels is a common clinical practice to reduce oxidation and the risk of major events in patients with cardiovascular diseases (CVD). Atherosclerosis starts with dysfunctional changes in the endothelium induced by disturbed shear stress which can lead to endothelial and platelet activation, adhesion of monocytes on the activated endothelium, and differentiation into proinflammatory macrophages, which increase the uptake of oxidized LDL (oxLDL) and turn into foam cells, exacerbating the inflammatory signalling. The atherosclerotic process is accelerated by a myriad of factors, such as the release of inflammatory chemokines and cytokines, the generation of reactive oxygen species (ROS), growth factors, and the proliferation of vascular smooth muscle cells. Inflammation and immunity are key factors for the development and complications of atherosclerosis, and therefore, the whole atherosclerotic process is a target for diagnosis and treatment. In this review, we focus on early stages of the disease and we address both biomarkers and therapeutic approaches currently available and under research.

Changes in Chemokines and Chemokine Receptors Expression in a Mouse Model of Alzheimer's Disease.

The amyloid precursor protein plus presenilin-1 (APP/PS1) mice are a frequently-used model for Alzheimer's disease studies (AD). However, the data relevant to which proteins are involved in inflammatory mechanism are not sufficiently well-studied using the AD mouse model. Using behavioral studies, quantitative RT-PCR and Western-blot techniques, significant findings were determined by the expression of proteins involved in inflammation comparing APP/PS1 and Wild type mice. Increased GFAP expression could be associated with the elevation in number of reactive astrocytes. IL-3 is involved in inflammation and ABDF1 intervenes normally in the transport across cell membranes and both were found up-regulated in APP/PS1 mice compared to Wild type mice. Furthermore, CCR5 expression was decreased and both CCL3 and CCL4 chemokines were highly expressed indicating a possible gliosis and probably an increase in chemotaxis from lymphocytes and T cell generation. We also noted for the first time, a CCR8 increase expression with diminution of its CCL1 chemokine, both normally involved in protection from bacterial infection and demyelination. Control of inflammatory proteins will be the next step in understanding the progression of AD and also in determining the mechanisms that can develop in this disease.

Effects of photodynamic therapy in periodontal treatment: A randomized, controlled clinical trial.

AIM: To evaluate the effects of photodynamic therapy (PDT) in the nonsurgical treatment of chronic periodontitis. MATERIALS AND METHODS: A randomized, single-blind, controlled, parallel-group clinical trial was performed. Sixty patients were enrolled: 20 healthy controls and 40 patients with periodontitis. The 40 patients were randomized for scaling and root planing (SRP) or SRP + PDT. Periodontal (plaque index, probing depth, clinical recession, clinical attachment level, bleeding on probing and gingival crevicular fluid volume, corresponding to 381 versus 428 critical sites), microbiological (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia and Campylobacter rectus presence, 18 versus 19 samples) and biochemical (interleukin (IL)-1ß, IL-6 and tumour necrosis factor (TNF)-α, receptor activator of nuclear factor-kappaB ligand (RANK-L) and osteoprotegerin (OPG) levels, 18 versus 19 samples) parameters were recorded. RESULTS: Within each group, significant improvements were found for clinical parameters, though without significant differences between groups. RANK-L was significantly decreased at week 13 in the SRP + PDT group compared with the SRP group. SRP + PDT, but not SRP alone, significantly reduced the abundance of A. actinomycetemcomitans. CONCLUSIONS: Except for a significant decrease in the pathogenic burden of A. actinomycetemcomitans, coadjuvant PDT resulted in no additional improvement compared with SRP alone in patients diagnosed with moderate-to-advanced chronic periodontitis.

Neuronal Effects of Sugammadex in combination with Rocuronium or Vecuronium.

Rocuronium (ROC) and Vecuronium (VEC) are the most currently used steroidal non-depolarizing neuromuscular blocking (MNB) agents. Sugammadex (SUG) rapidly reverses steroidal NMB agents after anaesthesia. The present study was conducted in order to evaluate neuronal effects of SUG alone and in combination with both ROC and VEC. Using MTT, CASP-3 activity and Western-blot we determined the toxicity of SUG, ROC or VEC in neurons in primary culture. SUG induces apoptosis/necrosis in neurons in primary culture and increases cytochrome C (CytC), apoptosis-inducing factor (AIF), Smac/Diablo and Caspase 3 (CASP-3) protein expression. Our results also demonstrated that both ROC and VEC prevent these SUG effects. The protective role of both ROC and VEC could be explained by the fact that SUG encapsulates NMB drugs. In BBB impaired conditions it would be desirable to control SUG doses to prevent the excess of free SUG in plasma that may induce neuronal damage. A balance between SUG, ROC or VEC would be necessary to prevent the risk of cell damage.

Effects of Ranolazine on Astrocytes and Neurons in Primary Culture.

Ranolazine (Rn) is an antianginal agent used for the treatment of chronic angina pectoris when angina is not adequately controlled by other drugs. Rn also acts in the central nervous system and it has been proposed for the treatment of pain and epileptic disorders. Under the hypothesis that ranolazine could act as a neuroprotective drug, we studied its effects on astrocytes and neurons in primary culture. We incubated rat astrocytes and neurons in primary cultures for 24 hours with Rn (10-7, 10-6 and 10-5 M). Cell viability and proliferation were measured using trypan blue exclusion assay, MTT conversion assay and LDH release assay. Apoptosis was determined by Caspase 3 activity assay. The effects of Rn on pro-inflammatory mediators IL-ß and TNF-α was determined by ELISA technique, and protein expression levels of Smac/Diablo, PPAR-γ, Mn-SOD and Cu/Zn-SOD by western blot technique. In cultured astrocytes, Rn significantly increased cell viability and proliferation at any concentration tested, and decreased LDH leakage, Smac/Diablo expression and Caspase 3 activity indicating less cell death. Rn also increased anti-inflammatory PPAR-γ protein expression and reduced pro-inflammatory proteins IL-1 ß and TNFα levels. Furthermore, antioxidant proteins Cu/Zn-SOD and Mn-SOD significantly increased after Rn addition in cultured astrocytes. Conversely, Rn did not exert any effect on cultured neurons. In conclusion, Rn could act as a neuroprotective drug in the central nervous system by promoting astrocyte viability, preventing necrosis and apoptosis, inhibiting inflammatory phenomena and inducing anti-inflammatory and antioxidant agents.

WIN 55,212-2, agonist of cannabinoid receptors, prevents amyloid ß1-42 effects on astrocytes in primary culture.

Alzheimer's disease (AD), a neurodegenerative illness involving synaptic dysfunction with extracellular accumulation of Aß1-42 toxic peptide, glial activation, inflammatory response and oxidative stress, can lead to neuronal death. Endogenous cannabinoid system is implicated in physiological and physiopathological events in central nervous system (CNS), and changes in this system are related to many human diseases, including AD. However, studies on the effects of cannabinoids on astrocytes functions are scarce. In primary cultured astrocytes we studied cellular viability using MTT assay. Inflammatory and oxidative stress mediators were determined by ELISA and Western-blot techniques both in the presence and absence of Aß1-42 peptide. Effects of WIN 55,212-2 (a synthetic cannabinoid) on cell viability, inflammatory mediators and oxidative stress were also determined. Aß1-42 diminished astrocytes viability, increased TNF-α and IL-1ß levels and p-65, COX-2 and iNOS protein expression while decreased PPAR-γ and antioxidant enzyme Cu/Zn SOD. WIN 55,212-2 pretreatment prevents all effects elicited by Aß1-42. Furthermore, cannabinoid WIN 55,212-2 also increased cell viability and PPAR-γ expression in control astrocytes. In conclusion cannabinoid WIN 55,212-2 increases cell viability and anti-inflammatory response in cultured astrocytes. Moreover, WIN 55,212-2 increases expression of anti-oxidant Cu/Zn SOD and is able to prevent inflammation induced by Aß1-42 in cultured astrocytes. Further studies would be needed to assess the possible beneficial effects of cannabinoids in Alzheimer's disease patients.

Astrocytes protect neurons from Aß1-42 peptide-induced neurotoxicity increasing TFAM and PGC-1 and decreasing PPAR-γ and SIRT-1.

One of the earliest neuropathological events in Alzheimer's disease is accumulation of astrocytes at sites of Aß1-42 depositions. Our results indicate that Aß1-42 toxic peptide increases lipid peroxidation, apoptosis and cell death in neurons but not in astrocytes in primary culture. Aß1-42-induced deleterious neuronal effects are not present when neurons and astrocytes are mixed cultured. Stimulation of astrocytes with toxic Aß1-42 peptide increased p-65 and decreased IκB resulting in inflammatory process. In astrocytes Aß1-42 decreases protein expressions of sirtuin 1 (SIRT-1) and peroxisome proliferator-activated receptor γ (PPAR-γ) and over-expresses peroxisome proliferator-activated receptor γ coactivator 1 (PGC-1) and mitochondrial transcription factor A (TFAM), protecting mitochondria against Aß1-42-induced damage and promoting mitochondrial biogenesis. In summary our data suggest that astrocytes may have a key role in protecting neurons, increasing neural viability and mitochondrial biogenesis, acquiring better oxidative stress protection and perhaps modulating inflammatory processes against Aß1-42 toxic peptide. This might be a sign of a complex epigenetic process in Alzheimer's disease development.