Redox Homeostasis Alteration Is Restored through Melatonin Treatment in COVID-19 Patients: A Preliminary Study.

Type II pneumocytes are the target of the SARS-CoV-2 virus, which alters their redox homeostasis to increase reactive oxygen species (ROS). Melatonin (MT) has antioxidant proprieties and protects mitochondrial function. In this study, we evaluated whether treatment with MT compensated for the redox homeostasis alteration in serum from COVID-19 patients. We determined oxidative stress (OS) markers such as carbonyls, glutathione (GSH), total antioxidant capacity (TAC), thiols, nitrites (NO2-), lipid peroxidation (LPO), and thiol groups in serum. We also studied the enzymatic activities of glutathione peroxidase (GPx), glutathione-S-transferase (GST), reductase (GR), thioredoxin reductase (TrxR), extracellular superoxide dismutase (ecSOD) and peroxidases. There were significant increases in LPO and carbonyl quantities (p ≤ 0.03) and decreases in TAC and the quantities of NO2-, thiols, and GSH (p < 0.001) in COVID-19 patients. The activities of the antioxidant enzymes such as ecSOD, TrxR, GPx, GST, GR, and peroxidases were decreased (p ≤ 0.04) after the MT treatment. The treatment with MT favored the activity of the antioxidant enzymes that contributed to an increase in TAC and restored the lost redox homeostasis. MT also modulated glucose homeostasis, functioning as a glycolytic agent, and inhibited the Warburg effect. Thus, MT restores the redox homeostasis that is altered in COVID-19 patients and can be used as adjuvant therapy in SARS-CoV-2 infection.

Reduced Levels of Selenium and Thioredoxin Reductase in the Thoracic Aorta Could Contribute to Aneurysm Formation in Patients with Marfan Syndrome.

Marfan syndrome (MFS) is an autosomal dominant disorder caused by a heterozygous mutation of the FBN1 gene. MFS patients present oxidative stress that disturbs redox homeostasis. Redox homeostasis depends in part on the enzymatic antioxidant system, which includes thioredoxin reductase (TrxR) and glutathione peroxidases (GPx), both of which require an adequate concentration of selenium (Se). Therefore, the aim of this study was to determine if Se levels are decreased in the TAA of patients with MFS since this could contribute to the formation of an aneurysm in these patients. The results show that interleukins IL-1ß, IL-6 TGF-ß1, and TNF-α (p ≤ 0.03), and carbonylation (p ≤ 0.03) were increased in the TAA of patients with MFS in comparison with control subjects, while Se, thiols (p = 0.02), TrxR, and GPx (p ≤ 0.001) were decreased. TLR4 and NOX1 (p ≤ 0.03), MMP9 and MMP2 (p = 0.04) and NOS2 (p < 0.001) were also increased. Therefore, Se concentrations are decreased in the TAA of MFS, which can contribute to a decrease in the activities of TrxR and GPx, and thiol groups. A decrease in the activities of these enzymes can lead to the loss of redox homeostasis, which can, in turn, lead to an increase in the pro-inflammatory interleukins associated with the overexpression of MMP9 and MMP2.

PPAR Alpha Activation by Clofibrate Alleviates Ischemia/Reperfusion Injury in Metabolic Syndrome Rats by Decreasing Cardiac Inflammation and Remodeling and by Regulating the Atrial Natriuretic Peptide Compensatory Response.

Metabolic syndrome (MetS) is a cluster of factors that increase the risk of developing diabetes, stroke, and heart failure. The pathophysiology of injury by ischemia/reperfusion (I/R) is highly complex and the inflammatory condition plays an important role by increasing matrix remodeling and cardiac apoptosis. Natriuretic peptides (NPs) are cardiac hormones with numerous beneficial effects mainly mediated by a cell surface receptor named atrial natriuretic peptide receptor (ANPr). Although NPs are powerful clinical markers of cardiac failure, their role in I/R is still controversial. Peroxisome proliferator-activated receptor α agonists exert cardiovascular therapeutic actions; however, their effect on the NPs' signaling pathway has not been extensively studied. Our study provides important insight into the regulation of both ANP and ANPr in the hearts of MetS rats and their association with the inflammatory conditions caused by damage from I/R. Moreover, we show that pre-treatment with clofibrate was able to decrease the inflammatory response that, in turn, decreases myocardial fibrosis, the expression of metalloprotease 2 and apoptosis. Treatment with clofibrate is also associated with a decrease in ANP and ANPr expression.

Down Regulation of Catsper1 Expression by Calmodulin Inhibitor (Calmidazolium): Possible Implications for Fertility.

The CatSper channel localizes exclusively in the flagella of sperm cells. The Catsper1 protein, together with three pore units, is essential for the CatSper Channel formation, which produces flagellum hyperactivation and confers sperm fertility. Catsper1 expression is dependent on Sox transcription factors, which can recognize in vitro at least three Sox binding sites on the promoter. Sox transcription factors have calmodulin-binding domains for nuclear importation. Calmodulin (CaM) is affected by the specific inhibitor calmidazolium (CMZ), which prevents the nuclear transport of Sox factors. In this work, we assess the regulation of the Catsper1 promoter in vivo by Sox factors in the murine testis and evaluate the effects of the inhibitor calmidazolium on the expression of the Casper genes, and the motility and fertility of the sperm. Catsper1 promoter has significant transcriptional activity in vivo; on the contrary, three Sox site mutants in the Catsper1 promoter reduced transcriptional activity in the testis. CaM inhibition affects Sox factor nuclear transport and has notable implications in the expression and production of Catsper1, as well as in the motility and fertility capability of sperm. The molecular mechanism described here might conform to the basis of a male contraceptive strategy acting at the transcriptional level by affecting the production of the CatSper channel, a fundamental piece of male fertility.

Rich fatty acids diet of fish and olive oils modifies membrane properties in striatal rat synaptosomes.

Background: Essential fatty acids (EFAs) and non-essential fatty acids (nEFAs) exert experimental and clinical neuroprotection in neurodegenerative diseases. The main EFAs, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), nEFAs, and oleic acid (OA) contained in olive and fish oils are inserted into the cell membranes, but the exact mechanism through which they exert neuroprotection is still unknown. Objectives and Methods: In this study, we assessed the fatty acids content and membrane fluidity in striatal rat synaptosomes after fatty acid-rich diets (olive- or a fish-oil diet, 15% w/w). Then, we evaluated the effect of enriching striatum synaptosomes with fatty acids on the oxidative damage produced by the prooxidants ferrous sulfate (FeSO4) or quinolinic acid (QUIN). Results and Discussion: Lipid profile analysis in striatal synaptosomes showed that EPA content increased in the fish oil group in comparison with control and olive groups. Furthermore, we found that synaptosomes enriched with fatty acids and incubated with QUIN or FeSO4 showed a significant oxidative damage reduction. Results suggest that EFAs, particularly EPA, improve membrane fluidity and confer antioxidant effect.

Modulation of Renal Function in a Metabolic Syndrome Rat Model by Antioxidants in Hibiscus sabdariffa L.

Metabolic syndrome (MS) is the association of three or more pathologies among which obesity, hypertension, insulin resistance, dyslipidemia, and diabetes are included. It causes oxidative stress (OS) and renal dysfunction. Hibiscus sabdariffa L. (HSL) is a source of natural antioxidants that may control the renal damage caused by the MS. The objective of this work was to evaluate the effect of a 2% HSL infusion on renal function in a MS rat model induced by the administration of 30% sucrose in drinking water. 24 male Wistar rats were divided into 3 groups: Control rats, MS rats and MS + HSL rats. MS rats had increased body weight, systolic blood pressure, triglycerides, insulin, HOMA index, and leptin (p ≤ 0.04). Renal function was impaired by an increase in perfusion pressure in the isolated and perfused kidney, albuminuria (p ≤ 0.03), and by a decrease in clearance of creatinine (p ≤ 0.04). The activity of some antioxidant enzymes including the superoxide dismutase isoforms, peroxidases, glutathione peroxidase, glutathione-S-transferase was decreased (p ≤ 0.05). Lipoperoxidation and carbonylation were increased (p ≤ 0.001). The nitrates/nitrites ratio, total antioxidant capacity, glutathione levels and vitamin C were decreased (p ≤ 0.03). The treatment with 2% HSL reversed these alterations. The results suggest that the treatment with 2% HSL infusion protects renal function through its natural antioxidants which favor an improved renal vascular response. The infusion contributes to the increase in the glomerular filtration rate, by promoting an increase in the enzymatic and non-enzymatic antioxidant systems leading to a decrease in OS and reestablishing the normal renal function.

Is Antioxidant Therapy a Useful Complementary Measure for Covid-19 Treatment? An Algorithm for Its Application.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes the corona virus disease-19 which is accompanied by severe pneumonia, pulmonary alveolar collapses and which stops oxygen exchange. Viral transmissibility and pathogenesis depend on recognition by a receptor in the host, protease cleavage of the host membrane and fusion. SARS-CoV-2 binds to the angiotensin converting enzyme 2 receptor. Here, we discuss the general characteristics of the virus, its mechanism of action and the way in which the mechanism correlates with the comorbidities that increase the death rate. We also discuss the currently proposed therapeutic measures and propose the use of antioxidant drugs to help patients infected with the SARS-CoV-2. Oxidizing agents come from phagocytic leukocytes such as neutrophils, monocytes, macrophages and eosinophils that invade tissue. Free radicals promote cytotoxicity thus injuring cells. They also trigger the mechanism of inflammation by mediating the activation of NFkB and inducing the transcription of cytokine production genes. Release of cytokines enhances the inflammatory response. Oxidative stress is elevated during critical illnesses and contributes to organ failure. In corona virus disease-19 there is an intense inflammatory response known as a cytokine storm that could be mediated by oxidative stress. Although antioxidant therapy has not been tested in corona virus disease-19, the consequences of antioxidant therapy in sepsis, acute respiratory distress syndrome and acute lung injury are known. It improves oxygenation rates, glutathione levels and strengthens the immune response. It reduces mechanical ventilation time, the length of stay in the intensive care unit, multiple organ dysfunctions and the length of stay in the hospital and mortality rates in acute lung injury/acute respiratory distress syndrome and could thus help patients with corona virus disease-19.

Use of silver nanoparticles to control Vibrio fluvialis in cultured angelfish Pterophyllum scalare.

Nanoparticles have multiple applications, among which is their use as antimicrobial agents in aquaculture. The objective of this work was to determine the antibacterial effect of silver nanoparticles (AgNPs) against Vibrio fluvialis in cultured angelfish Pterophyllum scalare. AgNPs were synthetized through chemical reduction and characterized by UV-visible and infrared spectroscopy. Particle size ranged from 60 to 170.8 nm, and scanning electron microscopy revealed cubic and spherical forms. A minimal inhibitory concentration of 222.5 ppm was determined, as well as inhibition halos between 8.66 and 14.3 mm. Inhibition of V. fluvialis growth was observed upon contact with AgNPs. An 88% survival of infected fish was obtained when treated with AgNPs, in contrast to 100% mortality of fish that were not treated. No damage to internal or external organs was observed in fish exposed to AgNPs. We conclude that AgNPs exert an antimicrobial effect against V. fluvialis, and thus represent a new alternative to control diseases caused by this microorganism in P. scalare culture.

Clofibrate Treatment Decreases Inflammation and Reverses Myocardial Infarction-Induced Remodelation in a Rodent Experimental Model.

Myocardial infarction (MI) initiates an inflammatory response that promotes both beneficial and deleterious effects. The early response helps the myocardium to remove damaged tissue; however, a prolonged later response brings cardiac remodeling characterized by functional, metabolic, and structural pathological changes. Current pharmacological treatments have failed to reverse ischemic-induced cardiac damage. Therefore, our aim was to study if clofibrate treatment was capable of decreasing inflammation and apoptosis, and reverse ventricular remodeling and MI-induced functional damage. Male Wistar rats were assigned to (1) Sham coronary artery ligation (Sham) or (2) Coronary artery ligation (MI). Seven days post-MI, animals were further divided to receive vehicle (V) or clofibrate (100 mg/kg, C) for 7 days. The expression of IL-6, TNF-α, and inflammatory related molecules ICAM-1, VCAM-1, MMP-2 and -9, nuclear NF-kB, and iNOS, were elevated in MI-V. These inflammatory biomarkers decreased in MI-C. Also, apoptotic proteins (Bax and pBad) were elevated in MI-V, while clofibrate augmented anti-apoptotic proteins (Bcl-2 and 14-3-3ε). Clofibrate also protected MI-induced changes in ultra-structure. The ex vivo evaluation of myocardial functioning showed that left ventricular pressure and mechanical work decreased in infarcted rats; clofibrate treatment raised those parameters to control values. Echocardiogram showed that clofibrate partially reduced LV dilation. In conclusion, clofibrate decreases cardiac remodeling, decreases inflammatory molecules, and partly preserves myocardial diameters.

Cardioprotective kinase signaling to subsarcolemmal and interfibrillar mitochondria is mediated by caveolar structures.

The demonstration that caveolin-3 overexpression reduces myocardial ischemia/reperfusion injury and our own finding that multiprotein signaling complexes increase in mitochondria in association with caveolin-3 levels, led us to investigate the contribution of caveolae-driven extracellular signal-regulated kinases 1/2 (ERK1/2) on maintaining the function of cardiac mitochondrial subpopulations from reperfused hearts subjected to postconditioning (PostC). Rat hearts were isolated and subjected to ischemia/reperfusion and to PostC. Enhanced cardiac function, reduced infarct size and preserved ultrastructure of cardiomyocytes were associated with increased formation of caveolar structures, augmented levels of caveolin-3 and mitochondrial ERK1/2 activation in PostC hearts in both subsarcolemmal (SSM) and interfibrillar (IFM) subpopulations. Disruption of caveolae with methyl-ß-cyclodextrin abolished cardioprotection in PostC hearts and diminished pho-ERK1/2 gold-labeling in both mitochondrial subpopulations in correlation with suppression of resistance to permeability transition pore opening. Also, differences between the mitochondrial subpopulations in the setting of PostC were evaluated. Caveolae disruption with methyl-ß-cyclodextrin abolished the cardioprotective effect of postconditioning by inhibiting the interaction of ERK1/2 with mitochondria and promoted decline in mitochondrial function. SSM, which are particularly sensitive to reperfusion damage, take advantage of their location in cardiomyocyte boundary and benefit from the cardioprotective signaling driven by caveolae, avoiding injury propagation.

Extracts of Crataegus oxyacantha and Rosmarinus officinalis Attenuate Ischemic Myocardial Damage by Decreasing Oxidative Stress and Regulating the Production of Cardiac Vasoactive Agents.

Numerous studies have supported a role for oxidative stress in the development of ischemic damage and endothelial dysfunction. Crataegus oxyacantha (Co) and Rosmarinus officinalis (Ro) extracts are polyphenolic-rich compounds that have proven to be efficient in the treatment of cardiovascular diseases. We studied the effect of extracts from Co and Ro on the myocardial damage associated with the oxidative status and to the production of different vasoactive agents. Rats were assigned to the following groups: (a) sham; (b) vehicle-treated myocardial infarction (MI) (MI-V); (c) Ro extract-treated myocardial infarction (MI-Ro); (d) Co extract-treated myocardial infarction (MI-Co); or (e) Ro+Co-treated myocardial infarction (MI-Ro+Co). Ro and Co treatments increased total antioxidant capacity, the expression of superoxide dismutase (SOD)-Cu2+/Zn2+, SOD-Mn2+, and catalase, with the subsequent decline of malondialdehyde and 8-hydroxy-2'-deoxyguanosine levels. The extracts diminished vasoconstrictor peptide levels (angiotensin II and endothelin-1), increased vasodilators agents (angiotensin 1-7 and bradikinin) and improved nitric oxide metabolism. Polyphenol treatment restored the left intraventricular pressure and cardiac mechanical work. We conclude that Ro and Co treatment attenuate morphological and functional ischemic-related changes by both an oxidant load reduction and improvement of the balance between vasoconstrictors and vasodilators.

Fenofibrate Therapy Restores Antioxidant Protection and Improves Myocardial Insulin Resistance in a Rat Model of Metabolic Syndrome and Myocardial Ischemia: The Role of Angiotensin II.

Renin-angiotensin system (RAS) activation promotes oxidative stress which increases the risk of cardiac dysfunction in metabolic syndrome (MetS) and favors local insulin resistance. Fibrates regulate RAS improving MetS, type-2 diabetes and cardiovascular diseases. We studied the effect of fenofibrate treatment on the myocardic signaling pathway of Angiotensin II (Ang II)/Angiotensin II type 1 receptor (AT1) and its relationship with oxidative stress and myocardial insulin resistance in MetS rats under heart ischemia. Control and MetS rats were assigned to the following groups: (a) sham; (b) vehicle-treated myocardial infarction (MI) (MI-V); and (c) fenofibrate-treated myocardial infarction (MI-F). Treatment with fenofibrate significantly reduced triglycerides, non-high density lipoprotein cholesterol (non-HDL-C), insulin levels and insulin resistance index (HOMA-IR) in MetS animals. MetS and MI increased Ang II concentration and AT1 expression, favored myocardial oxidative stress (high levels of malondialdehyde, overexpression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4), decreased total antioxidant capacity and diminished expression of superoxide dismutase (SOD)1, SOD2 and catalase) and inhibited expression of the insulin signaling cascade: phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PkB, also known as Akt)/Glut-4/endothelial nitric oxide synthase (eNOS). In conclusion, fenofibrate treatment favors an antioxidant environment as a consequence of a reduction of the Ang II/AT1/NOX4 signaling pathway, reestablishing the cardiac insulin signaling pathway. This might optimize cardiac metabolism and improve the vasodilator function during myocardial ischemia.

PPARα Stimulation Modulates Myocardial Ischemia-induced Activation of Renin-Angiotensin System.

We have recently demonstrated that peroxisome proliferator-activated receptor alpha (PPARα) stimulation lowers the production of angiotensin II while increasing the production of Ang-(1-7), both in cardiac and plasmatic level. This stimulation improves nitric oxide bioavailability, preserving cardiac histologic features and functioning. Based on these results, we decided to study the effect of PPARα stimulation on renin-angiotensin system components of ischemic myocardium. Male Wistar rats (weighing 300-350 g) were assigned to the following groups: (1) sham, (2) myocardial ischemia vehicle-treated (MI-V), and (3) myocardial ischemia clofibrate-treated. Expression of the angiotensin-converting enzyme increased during ischemia, whereas clofibrate-treated group remained comparable to control. Activation of the PPARα receptor stimulated the expression of angiotensin-converting enzyme-2; while the activity of this enzyme was increased in MI-V, clofibrate inhibited any change. The concentration of bradykinin and phospho-Akt(SER473) in homogenate increased in the animals treated with the drug. Mas receptor expression increased in MI-V rats. In conclusion, stimulation of PPARα by clofibrate prevents an increase in the activity of renin-angiotensin system and promotes the production of vasodilator substances.

Effect of High Sodium Intake on Gut Tight Junctions' Structure and Permeability to Bacterial Toxins in a Rat Model of Chronic Kidney Disease.

INTRODUCTION: Uremic toxicity changes the gut structure and permeability, allowing bacterial toxins to translocate from the lumen to the blood during chronic kidney failure (CKD). Clinical fluid overload and tissue edema without uremia have similar effects but have not been adequately demonstrated and analyzed in CKD. AIMS: To investigate the effect of sodium intake on the plasma concentration of gut-derived uremic toxins, indoxyl sulfate (IS), and p-cresyl sulfate (pCS) and the expression of genes and proteins of epithelial gut tight junctions in a rat model of CKD. METHODS: Sham-operated (control group, CG) and five-sixths nephrectomized (5/6Nx) Sprague-Dawley rats were randomly assigned to low (LNa), normal (NNa), or high sodium (HNa) diets., Animals were then sacrificed at 8 and 12 weeks and analyzed for IS and pCS plasma concentrations, as well as for gene and protein expression of thigh junction proteins, and transmission electron microscopy (TEM) in colon fragments. RESULTS: The HNa 5/6Nx groups had higher concentrations of IS and pCS than CG, NNa, and LNa at eight and twelve weeks. Furthermore, HNa 5/6Nx groups had reduced expression of the claudin-4 gene and protein than CG, NNa, and LNa. HNa had reduced occludin gene expression compared to CG. Occludin protein expression was more reduced in HNa than in CG, NNa, and LNa. The gut epithelial tight junctions appear dilated in HNa compared to NNa and LNa in TEM. CONCLUSION: Dietary sodium intake and fluid overload have a significant role in gut epithelial permeability in the CKD model.

Zona occludens-2 protects against podocyte dysfunction induced by ADR in mice.

Zona occludens-2 (ZO-2) is a protein present at the tight junction and nucleus of epithelial cells. ZO-2 represses the transcription of genes regulated by the Wnt/ß-catenin pathway. This pathway plays a critical role in podocyte injury and proteinuria. Here, we analyze whether the overexpression of ZO-2 in the glomerulus, by hydrodynamics transfection, prevents podocyte injury mediated by the Wnt/ß-catenin pathway in the mouse model of adriamycin (ADR) nephrosis. By immunofluorescence and immunogold electron microscopy, we show that ZO-2 is present in mice glomerulus, not at the slit diaphragms where nephrin concentrates, but in the cytoplasm and at processes of podocytes. Our results indicate that in the glomeruli of mice treated with ADR, ZO-2 overexpression increases the amount of phosphorylated ß-catenin, inhibits the expression of the transcription factor snail, prevents nephrin and podocalyxin loss, reduces podocyte effacement and massive fusions, restrains proteinuria, and supports urea and creatinine clearance. These results suggest that ZO-2 could be a new target for the regulation of hyperactive Wnt/ß-catenin signaling in proteinuric kidney diseases.

Sulforaphane modifies mitochondrial-endoplasmic reticulum associations through reductive stress in cardiomyocytes.

Mitochondria-endoplasmic reticulum (ER) communication relies on platforms formed at the ER membrane with the mitochondrial outer membrane contact sites (MERCs). MERCs are involved in several processes including the unfolded protein response (UPR) and calcium (Ca2+) signaling. Therefore, as alterations in MERCs greatly impact cellular metabolism, pharmacological interventions to preserve productive mitochondrial-ER communication have been explored to maintain cellular homeostasis. In this regard, extensive information has documented the beneficial and potential effects of sulforaphane (SFN) in different pathological conditions; however, controversy has arisen regarding the effect of this compound on mitochondria-ER interaction. Therefore, in this study, we investigated whether SFN could induce changes in MERCs under normal culture conditions without damaging stimuli. Our results indicate that non-cytotoxic concentration of 2.5 µM SFN increased ER stress in cardiomyocytes in conjunction with a reductive stress environment, that diminishes ER-mitochondria association. Additionally, reductive stress promotes Ca2+ accumulation in the ER of cardiomyocytes. These data show an unexpected effect of SFN on cardiomyocytes grown under standard culture conditions, promoted by the cellular redox unbalance. Therefore, it is necessary to rationalize the use of compounds with antioxidant properties to avoid triggering cellular side effects.

Actin-Cytoskeleton Drives Caveolae Signaling to Mitochondria during Postconditioning.

Caveolae-associated signaling toward mitochondria contributes to the cardioprotective mechanisms against ischemia-reperfusion (I/R) injury induced by ischemic postconditioning. In this work, we evaluated the role that the actin-cytoskeleton network exerts on caveolae-mitochondria communication during postconditioning. Isolated rat hearts subjected to I/R and to postconditioning were treated with latrunculin A, a cytoskeleton disruptor. Cardiac function was compared between these hearts and those exposed only to I/R and to the cardioprotective maneuver. Caveolae and mitochondria structures were determined by electron microscopy and maintenance of the actin-cytoskeleton was evaluated by phalloidin staining. Caveolin-3 and other putative caveolae-conforming proteins were detected by immunoblot analysis. Co-expression of caveolin-3 and actin was evaluated both in lipid raft fractions and in heart tissue from the different groups. Mitochondrial function was assessed by respirometry and correlated with cholesterol levels. Treatment with latrunculin A abolishes the cardioprotective postconditioning effect, inducing morphological and structural changes in cardiac tissue, reducing F-actin staining and diminishing caveolae formation. Latrunculin A administration to post-conditioned hearts decreases the interaction between caveolae-forming proteins, the co-localization of caveolin with actin and inhibits oxygen consumption rates in both subsarcolemmal and interfibrillar mitochondria. We conclude that actin-cytoskeleton drives caveolae signaling to mitochondria during postconditioning, supporting their functional integrity and contributing to cardiac adaption against reperfusion injury.

A CRAC-like motif in BAX sequence: relationship with protein insertion and pore activity in liposomes.

Several proteins that interact with cholesterol have a highly conserved sequence, corresponding to the cholesterol recognition/interaction amino acid consensus. Since cholesterol has been proposed to modulate both oligomerization and insertion of the pro-apoptotic protein BAX, we investigated the existence of such a motif in the BAX sequence. Residues 113 to 119 of the recombinant BAX α5-helix, LFYFASK, correspond with the sequence motif described for the consensus pattern, -L/V-(X)(1-5)-Y-(X)(1-5)-R/K. Functional characterization of the point mutations, K119A, Y115F, and L113A in BAX, was performed in liposomes supplemented with cholesterol, comparing binding, integration, and pore forming activities. Our results show that the mutations Y115F and L113A changed the cholesterol-dependent insertion observed in the wild type protein. In addition, substitutions in the BAX sequence modified the concentration dependency of carboxyfluorescein release in liposomes, although neither pore activity of the wild type or of any of the mutants significantly increased in cholesterol-enriched liposomes. Thus, while it is likely that the putative CRAC motif in BAX accounts for its enhanced insertion in cholesterol-enriched liposomes; the pore forming properties of BAX did not depend on cholesterol content in the membranes, albeit those mutations changed the pore channeling activity of the protein.

TiO2 nanoparticles induce dysfunction and activation of human endothelial cells.

Nanoparticles can reach the blood and cause inflammation, suggesting that nanoparticles-endothelial cells interactions may be pathogenically relevant. We evaluated the effect of titanium dioxide nanoparticles (TiO2) on proliferation, death, and responses related with inflammatory processes such as monocytic adhesion and expression of adhesion molecules (E- and P-selectins, ICAM-1, VCAM-1, and PECAM-1) and with inflammatory molecules (tissue factor, angiotensin-II, VEGF, and oxidized LDL receptor-1) on human umbilical vein endothelial cells (HUVEC). We also evaluated the production of reactive oxygen species, nitric oxide production, and NF-κB pathway activation. Aggregates of TiO2 of 300 nm or smaller and individual nanoparticles internalized into HUVEC inhibited proliferation strongly and induced apoptotic and necrotic death starting at 5 µg/cm². Besides, TiO2 induced activation of HUVEC through an increase in adhesion and in expression of adhesion molecules and other molecules involved with the inflammatory process. These effects were associated with oxidative stress and NF-κB pathway activation. In conclusion, TiO2 induced HUVEC activation, inhibition of cell proliferation with increased cell death, and oxidative stress.

Clofibrate PPARα activation reduces oxidative stress and improves ultrastructure and ventricular hemodynamics in no-flow myocardial ischemia.

Peroxisome proliferator-activated receptors (PPAR) play a critical physiological role in energy homeostasis, in inflammation, and a protective role in cardiovascular function. We assessed the antioxidant effect of clofibrate-induced Peroxisome proliferator-activated receptor alpha (PPARα) stimulation on ischemic myocardium on myocardial morphology and hemodynamics. Male Wistar rats (300 g) were distributed into the following groups: (1) Sham, (2) myocardial ischemia vehicle treated (MI-V), and (3) myocardial ischemia clofibrate [100 mg/kg/ intraperitoneally) treated (MI-C). Reactive oxygen species (ROS) and lipid peroxidation increased in MI-V, whereas clofibrate prevented this effect. Superoxide dismutase (SOD)-1 and SOD-2 expression increased 4 times upon PPARα stimulation. SOD-1, SOD-2, and catalase activity also increased in response to clofibrate. eNOS mRNA and tetrahydrobiopterin increased in the MI-C group. Clofibrate was able to decrease Angiotensin II (AngII), AngII AT1-receptor, whereas Ang-(1-7) and AngII AT2-receptor expression increased. Assessment of myocardial morphology and cardiac function show that clofibrate improved histological features and hemodynamic parameters. Our results suggest that PPARα stimulation by clofibrate increases the antioxidant defense, leading to improved cardiac function.