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.

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.

Nicotinamide Riboside and Pterostilbene Cooperatively Delay Motor Neuron Failure in ALS SOD1G93A Mice.

Oxidative stress-induced damage is a major mechanism in the pathophysiology of amyotrophic lateral sclerosis (ALS). A recent human clinical trial showed that the combination of nicotinamide riboside (NR) and pterostilbene (PT), molecules with potential to interfere in that mechanism, was efficacious in ALS patients. We examined the effect of these molecules in SOD1G93A transgenic mice, a well-stablished model of ALS. Assessment of neuromotor activity and coordination was correlated with histopathology, and measurement of proinflammatory cytokines in the cerebrospinal fluid. Cell death, Nrf2- and redox-dependent enzymes and metabolites, and sirtuin activities were studied in isolated motor neurons. NR and PT increased survival and ameliorated ALS-associated loss of neuromotor functions in SOD1G93A transgenic mice. NR and PT also decreased the microgliosis and astrogliosis associated with ALS progression. Increased levels of proinflammatory cytokines were observed in the cerebrospinal fluid of mice and humans with ALS. NR and PT ameliorated TNFα-induced oxidative stress and motor neuron death in vitro. Our results support the involvement of oxidative stress, specific Nrf2-dependent antioxidant defenses, and sirtuins in the pathophysiology of ALS. NR and PT interfere with the mechanisms leading to the release of proapoptotic molecular signals by mitochondria, and also promote mitophagy.

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.

Efficacy and tolerability of EH301 for amyotrophic lateral sclerosis: a randomized, double-blind, placebo-controlled human pilot study.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, characterized by progressive loss of spinal and cortical motor neurons, leading to muscular atrophy, respiratory failure, and ultimately death. There is no known cure, and the clinical benefit of the two drugs approved to treat ALS remains unclear. Novel disease-modifying therapeutics that are able to modulate the disease course are desperately needed. Our objective was to evaluate the efficacy and tolerability of Elysium Health's candidate drug EH301 in people with ALS (PALS). METHODS: This was a single-center, prospective, double-blind, randomized, placebo-controlled pilot study. Thirty-two PALS were recruited thanks to the collaboration of the Spanish Foundation for ALS Research (FUNDELA). Study participants were randomized to receive either EH301 or placebo and underwent evaluation for 4 months. Differences between EH301 and placebo-treated participants were evaluated based on standard clinical endpoints, including the revised ALS functional rating scale (ALSFRS-R), forced vital capacity (FVC), and the Medical Research Council (MRC) grading scale. RESULTS: Compared to placebo, participants treated with EH301 demonstrated significant improvements in the ALSFRS-R score, pulmonary function, muscular strength, and in skeletal muscle/fat weight ratio. EH301 was shown to significantly slow the progression of ALS relative to placebo, and even showed improvements in several key outcome measures compared with baseline. CONCLUSIONS: This study provides evidence in support of the disease-modifying effects of EH301 for the treatment of ALS.

Glucocorticoid receptor antagonism overcomes resistance to BRAF inhibition in BRAFV600E-mutated metastatic melanoma.

Clinical applications of glucocorticoids (GC) in Oncology are dependent on their pro-apoptotic action to treat lymphoproliferative cancers, and to alleviate side effects induced by chemotherapy and/or radiotherapy. However, the mechanism(s) by which GC may also promote tumor progression remains unclear. GC receptor (GR) knockdown decreases the antioxidant protection of highly metastatic B16-F10 melanoma cells. We hypothesize that a GR antagonist (RU486, mifepristone) could increase the efficacy of BRAF-related therapy in BRAFV600E-mutated metastatic melanoma. In vivo formed spontaneous skin tumors were reinoculated into nude mice to expand the metastases of different human BRAFV600E melanoma cells. The GR content of melanoma cell lines was measured by [3H]-labeled ligand binding assay. Nuclear Nrf2 and its transcription activity was investigated by RT-PCR, western blotting, and by measuring Nrf2- and redox state-related enzyme activities and metabolites. GR knockdown was achieved using lentivirus, and GR overexpression by transfection with the NR3C1 plasmid. shRNA-induced selective Bcl-xL, Mcl-1, AKT1 or NF-κB/p65 depletion was used to test the efficacy of vemurafenib (VMF) and RU486 against BRAFV600E-mutated metastatic melanoma. During early progression of skin melanoma metastases, RU486 and VMF induced a drastic metastases regression. However, treatment at an advanced stage of growth demonstrated the development of resistance to RU486 and VMF. This resistance was mechanistically linked to overexpression of specific proteins of the Bcl-2 family (Bcl-xL and Mcl-1 in our experimental models). We found that melanoma resistance is decreased if AKT and NF-κB signaling pathways are blocked. Our results highlight mechanisms by which metastatic melanoma cells adapt to survive.

Chronic exercise impairs nitric oxide pathway in rabbit carotid and femoral arteries.

KEY POINTS: Some of the beneficial effects of exercise in preventing vascular related diseases are mediated by the enhancement of endothelial function where the role of nitric oxide (NO) is well documented, although the relevance of calcium activated potassium channels is not fully understood. The impact of oxidative stress induced by training on endothelial function remains to be clarified. By evaluating different endothelial vasodilator pathways on two vascular beds in a rabbit model of chronic exercise, we found a decreased NO bioavailability and endothelial nitric oxide synthase expression in both carotid and femoral arteries. Physical training induced carotid endothelial dysfunction as a result of an increase in oxidative stress and a reduction in superoxide dismutase expression. In the femoral artery, the lower production of NO was counteracted by an increased participation of large conductance calcium activated potassium channels, preventing endothelial dysfunction. ABSTRACT: The present study aimed to evaluate the effects of chronic exercise on vasodilator response in two different arteries. Rings of carotid and femoral arteries from control and trained rabbits were suspended in organ baths for isometric recording of tension. Endothelial nitric oxide synthase (eNOS), Cu/Zn and Mn-superoxide dismutase (SOD), and large conductance calcium activated potassium (BKCa) channel protein expression were measured by western blotting. In the carotid artery, training reduced the relaxation to ACh (10-9 to 3 × 10-6  m) that was reversed by N-acetylcysteine (10-3  m). l-NAME (10-4  m) reduced the relaxation to ACh in both groups, although the effect was lower in the trained group (in mean ± SEM, 39 ± 2% vs. 28 ± 3%). Physical training did not modify the relaxation to ACh in femoral arteries, although the response to l-NAME was lower in the trained group (in mean ± SEM, 41 ± 5% vs. 17 ± 2%). Charybdotoxin (10-7  m) plus apamin (10-6  m) further reduced the maximal relaxation to ACh only in the trained group. The remaining relaxation in both carotid and femoral arteries was abolished by KCl (2 × 10-2  m) and BaCl2 (3 × 10-6  m) plus ouabain (10-4  m) in both groups. Physical training decreased eNOS expression in both carotid and femoral arteries and Cu/Zn and Mn-SOD expression only in the carotid artery. BKCa channels were overexpressed in the trained group in the femoral artery. In conclusion, chronic exercise induces endothelial dysfunction in the carotid artery as a result of oxidative stress. In the femoral artery, it modifies the vasodilator pathways, enhancing the participation of BKCa channels, thus compensating for the impairment of NO-mediated vasodilatation.

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.