In Vitro Assessment of Tobacco Smoke Toxicity at the BBB: Do Antioxidant Supplements Have a Protective Role?

<p>Abstract</p> <p>Background</p> <p>Tobacco smoke (TS) contains highly reactive oxygen species (such as hydrogen peroxide, peroxynitrite, etc), which cause oxidative damage in vascular tissue and may exacerbate inflammatory events leading to the blood-brain barrier damage (BBBD) which accompanies the development of a variety of neurological disorders. Smokers often have elevated leukocyte counts (primarily neutrophils and monocytes), and significant decreases in plasma alpha-tocopherol (vitamin E) and ascorbic acid (vitamin C) levels due to increased anti-oxidative mobilization in response to oxidative stress evoked by TS. For this purpose, using static culture systems and a well-established dynamic <it>in vitro </it>BBB model (DIV-BBB) we tested the hypothesis that antioxidant vitamin supplementation (E and/or C) can protect the BBB during exposure to whole soluble TS.</p> <p>Results</p> <p>TS exacerbates inflammatory events and leads to endothelial overexpression of vascular adhesion molecules (VCAM-1, P-selectin and E-selectin), release of pro-inflammatory cytokines (TNF-α and IL-6) and nitric oxide (NO), release and activation of matrix metalloproteinases (MMP-2 and MMP-9), monocytic maturation into macrophages, and adhesion to the vascular endothelium. Furthermore, TS altered the normal glucose metabolic behaviour of <it>in vitro </it>BBB capillaries and caused a period of transient anaerobic respiration to meet the cellular bioenergetic demand. Pre-treatment with antioxidant vitamins (C and/or E) effectively reduced the pro-inflammatory activity associated with TS, protecting the viability and functions of the BBB.</p> <p>Conclusion</p> <p>Our results have shown that loss of endothelial viability as well as BBB function and integrity caused by TS exposure can be prevented or at least reduced by normal physiologic concentrations of antioxidant vitamins <it>in vitro</it>.</p

The role of shear stress in Blood-Brain Barrier endothelial physiology

<p>Abstract</p> <p>Background</p> <p>One of the most important and often neglected physiological stimuli contributing to the differentiation of vascular endothelial cells (ECs) into a blood-brain barrier (BBB) phenotype is shear stress (SS). With the use of a well established humanized dynamic <it>in vitro </it>BBB model and cDNA microarrays, we have profiled the effect of SS in the induction/suppression of ECs genes and related functions.</p> <p>Results</p> <p>Specifically, we found a significant upregulation of tight and adherens junctions proteins and genes. Trans-endothelial electrical resistance (TEER) and permeability measurements to know substances have shown that SS promoted the formation of a tight and highly selective BBB. SS also increased the RNA level of multidrug resistance transporters, ion channels, and several p450 enzymes. The RNA level of a number of specialized carrier-mediated transport systems (e.g., glucose, monocarboxylic acid, etc.) was also upregulated.</p> <p>RNA levels of modulatory enzymes of the glycolytic pathway (e.g., lactate dehydrogenase) were downregulated by SS while those involved in the Krebs cycle (e.g., lactate and other dehydrogenases) were upregulated. Measurements of glucose consumption versus lactate production showed that SS negatively modulated the glycolytic bioenergetic pathways of glucose metabolism in favor of the more efficient aerobic respiration. BBB ECs are responsive to inflammatory stimuli. Our data showed that SS increased the RNA levels of integrins and vascular adhesion molecules. SS also inhibited endothelial cell cycle via regulation of BTG family proteins encoding genes. This was paralleled by significant increase in the cytoskeletal protein content while that of membrane, cytosol, and nuclear sub-cellular fractions decreased. Furthermore, analysis of 2D gel electrophoresis (which allows identifying a large number of proteins per sample) of EC proteins extracted from membrane sub-cellular endothelial fractions showed that SS increased the expression levels of tight junction proteins. In addition, regulatory enzymes of the Krebb's cycle (aerobic glucose metabolism) were also upregulated. Furthermore, the expression pattern of key protein regulators of the cell cycle and parallel gene array data supported a cell proliferation inhibitory role for SS.</p> <p>Conclusions</p> <p>Genomic and proteomic analyses are currently used to examine BBB function in healthy and diseased brain and characterize this dynamic interface. In this study we showed that SS plays a key role in promoting the differentiation of vascular endothelial cells into a truly BBB phenotype. SS affected multiple aspect of the endothelial physiology spanning from tight junctions formation to cell division as well as the expression of multidrug resistance transporters. BBB dysfunction has been observed in many neurological diseases, but the causes are generally unknown. Our study provides essential insights to understand the role played by SS in the BBB formation and maintenance.</p

Significance of MDR1 and multiple drug resistance in refractory human epileptic brain

BACKGROUND: The multiple drug resistance protein (MDR1/P-glycoprotein) is overexpressed in glia and blood-brain barrier (BBB) endothelium in drug refractory human epileptic tissue. Since various antiepileptic drugs (AEDs) can act as substrates for MDR1, the enhanced expression/function of this protein may increase their active extrusion from the brain, resulting in decreased responsiveness to AEDs. METHODS: Human drug resistant epileptic brain tissues were collected after surgical resection. Astrocyte cell cultures were established from these tissues, and commercially available normal human astrocytes were used as controls. Uptake of fluorescent doxorubicin and radioactive-labeled Phenytoin was measured in the two cell populations, and the effect of MDR1 blockers was evaluated. Frozen human epileptic brain tissue slices were double immunostained to locate MDR1 in neurons and glia. Other slices were exposed to toxic concentrations of Phenytoin to study cell viability in the presence or absence of a specific MDR1 blocker. RESULTS: MDR1 was overexpressed in blood vessels, astrocytes and neurons in human epileptic drug-resistant brain. In addition, MDR1-mediated cellular drug extrusion was increased in human 'epileptic' astrocytes compared to 'normal' ones. Concomitantly, cell viability in the presence of cytotoxic compounds was increased. CONCLUSIONS: Overexpression of MDR1 in different cell types in drug-resistant epileptic human brain leads to functional alterations, not all of which are linked to drug pharmacokinetics. In particular, the modulation of glioneuronal MDR1 function in epileptic brain in the presence of toxic concentrations of xenobiotics may constitute a novel cytoprotective mechanism

Oxidative and pro-inflammatory impact of regular and denicotinized cigarettes on blood brain barrier endothelial cells: is smoking reduced or nicotine-free products really safe?

Background: Both active and passive tobacco smoke (TS) potentially impair the vascular endothelial function in a causative and dose-dependent manner, largely related to the content of reactive oxygen species (ROS), nicotine, and pro-inflammatory activity. Together these factors can compromise the restrictive properties of the blood–brain barrier (BBB) and trigger the pathogenesis/progression of several neurological disorders including silent cerebral infarction, stroke, multiple sclerosis and Alzheimer’s disease. Based on these premises, we analyzed and assessed the toxic impact of smoke extract from a range of tobacco products (with varying levels of nicotine) on brain microvascular endothelial cell line (hCMEC/D3), a well characterized human BBB model. Results: Initial profiling of TS showed a significant release of reactive oxygen (ROS) and reactive nitrogen species (RNS) in full flavor, nicotine-free (NF, “reduced-exposure” brand) and ultralow nicotine products. This release correlated with increased oxidative cell damage. In parallel, membrane expression of endothelial tight junction proteins ZO-1 and occludin were significantly down-regulated suggesting the impairment of barrier function. Expression of VE-cadherin and claudin-5 were also increased by the ultralow or nicotine free tobacco smoke extract. TS extract from these cigarettes also induced an inflammatory response in BBB ECs as demonstrated by increased IL-6 and MMP-2 levels and up-regulation of vascular adhesion molecules, such as VCAM-1 and PECAM-1. Conclusions: In summary, our results indicate that NF and ultralow nicotine cigarettes are potentially more harmful to the BBB endothelium than regular tobacco products. In addition, this study demonstrates that the TS-induced toxicity at BBB ECs is strongly correlated to the TAR and NO levels in the cigarettes rather than the nicotine conten

RLIP76, a non-ABC transporter, and drug resistance in epilepsy

BACKGROUND: Permeability of the blood-brain barrier is one of the factors determining the bioavailability of therapeutic drugs and resistance to chemically different antiepileptic drugs is a consequence of decreased intracerebral accumulation. The ABC transporters, particularly P-glycoprotein, are known to play a role in antiepileptic drug extrusion, but are not by themselves sufficient to fully explain the phenomenon of drug-resistant epilepsy. Proteomic analyses of membrane protein differentially expressed in epileptic foci brain tissue revealed the frequently increased expression of RLIP76/RALBP1, a recently described non-ABC multi-specific transporter. Because of a significant overlap in substrates between P-glycoprotein and RLIP76, present studies were carried out to determine the potential role of RLIP76 in AED transport in the brain. RESULTS: RLIP76 was expressed in brain tissue, preferentially in the lumenal surface of endothelial cell membranes. The expression was most prominent in blood brain barrier tissue from excised epileptic foci. Saturable, energy-dependent, anti-gradient transport of both phenytoin and carbamazepine were demonstrated using recombinant RLIP76 reconstituted into artificial membrane liposomes. Immunotitration studies of transport activity in crude membrane vesicles prepared from whole-brain tissue endothelium showed that RLIP76 represented the dominant transport mechanism for both drugs. RLIP76(-/- )knockout mice exhibited dramatic toxicity upon phenytoin administration due to decreased drug extrusion mechanisms at the blood-brain barrier. CONCLUSION: We conclude that RLIP76 is the predominant transporter of AED in the blood brain barrier, and that it may be a transporter involved in mechanisms of drug-resistant epilepsy

Impact of in-utero electronic cigarette exposure on neonatal neuroinflammation, oxidative stress and mitochondrial function

Introduction: Despite the prevalence of the perception that electronic cigarettes (e-cig) are a safer alternative to tobacco smoke, growing concern about their potential toxic impact warrants adequate investigation focusing on special populations like maternal and pediatric groups. This study evaluated the consequences of maternal e-cig use on neonatal neuroinflammation, oxidative stress, and mitochondrial function in primary cultured neurons and postnatal day (PD) 7 and 90 brain.Methodology: Pregnant CD1 mice were exposed to e‐cig vapor (2.4% nicotine) from gestational day 5 (E5) till PD7, and the primary neurons were isolated from pups at E16/17. Cellular total reactive oxygen species (ROS) and mitochondrial superoxide were measured in primary neurons using CM-H2DCFDA and Mitosox red, respectively. Mitochondrial function was assessed by Seahorse XF Cell Mitostress analysis. The level of pro-inflammatory cytokines was measured in primary neurons and PD7 and PD90 brains by RT-PCR and immunobead assay. Western blot analysis evaluated the expression of antioxidative markers (SOD-2, HO-1, NRF2, NQO1) and that of the proinflammatory modulator NF-κB.Results: Significantly higher level of total cellular ROS (p &lt; 0.05) and mitochondrial superoxide (p &lt; 0.01) was observed in prenatally e-cig-exposed primary neurons. We also observed significantly reduced antioxidative marker expression and increased proinflammatory modulator and cytokines expression in primary neurons and PD7 (p &lt; 0.05) but not in PD90 postnatal brain.Conclusion: Our findings suggest that prenatal e-cig exposure induces postnatal neuroinflammation by promoting oxidative stress (OS), increasing cytokines’ levels, and disrupting mitochondrial function. These damaging events can alter the fetal brain’s immune functions, making such offspring more vulnerable to brain insults

Regenerative Stem Cell Therapy for Neurodegenerative Diseases: An Overview

Neurodegenerative diseases resulting from the progressive loss of structure and/or function of neurons contribute to different paralysis degrees and loss of cognition and sensation. The lack of successful curative therapies for neurodegenerative disorders leads to a considerable burden on society and a high economic impact. Over the past 20 years, regenerative cell therapy, also known as stem cell therapy, has provided an excellent opportunity to investigate potentially powerful innovative strategies for treating neurodegenerative diseases. This is due to stem cells’ capability to repair injured neuronal tissue by replacing the damaged or lost cells with differentiated cells, providing a conducive environment that is in favor of regeneration, or protecting the existing healthy neurons and glial cells from further damage. Thus, in this review, the various types of stem cells, the current knowledge of stem-cell-based therapies in neurodegenerative diseases, and the recent advances in this field are summarized. Indeed, a better understanding and further studies of stem cell technologies cause progress into realistic and efficacious treatments of neurodegenerative disorders

Assessing the protective effect of rosiglitazone against electronic cigarette/tobacco smoke-induced blood–brain barrier impairment

Abstract Background Smoking (TS) and recently e-cigarettes (EC) vaping, have been associated with vascular endothelial dysfunction primarily relevant to oxidative stress, exposure to nicotine, and smoking-induced inflammation. It is accepted that both EC and TS enhance glucose intolerance and the risk of developing type-2 diabetes mellitus which is also one of the causes of blood–brain barrier (BBB) damage and the higher risk of cerebrovascular diseases. Recent studies have shown how Metformin, the first common antidiabetic drug, can protect the BBB integrity through enhancement of nuclear factor erythroid 2-related factor (Nrf2) activity. Herein, we investigated the role of rosiglitazone (RSG; family of thiazolidinedione class used oral anti-diabetic drug) in TS/EC-induced BBB impairment. Results Although the exact mechanism of RSG is not fully understood, previous studies have revealed that RSG can promote counteractive protective mechanisms primarily associated with the enhancement of Nrf2 activity through activation of the peroxisome proliferator-activated receptor gamma. In line with these findings, our results show an increased expression of PPARy by RSG, enhancement of Nrf2 activity and BBB protection against TS/EC exposure including reduced inflammation, oxidative stress, tight junction downregulation and loss of BBB integrity. Conclusions RSG could be considered as a promising therapeutic potential to prevent TS/EC induced cerebrovascular dysfunction and possibly other xenobiotic substances which may impact the BBB via oxidative stress-mediated effects. However, additional in vivo studies and clinical setting will be needed to validate our results and assess the full extent of RSG protective effects

Cerebrovascular and Neurological Dysfunction under the Threat of COVID-19: Is There a Comorbid Role for Smoking and Vaping?

The recently discovered novel coronavirus, SARS-CoV-2 (COVID-19 virus), has brought the whole world to standstill with critical challenges, affecting both health and economic sectors worldwide. Although initially, this pandemic was associated with causing severe pulmonary and respiratory disorders, recent case studies reported the association of cerebrovascular-neurological dysfunction in COVID-19 patients, which is also life-threatening. Several SARS-CoV-2 positive case studies have been reported where there are mild or no symptoms of this virus. However, a selection of patients are suffering from large artery ischemic strokes. Although the pathophysiology of the SARS-CoV-2 virus affecting the cerebrovascular system has not been elucidated yet, researchers have identified several pathogenic mechanisms, including a role for the ACE2 receptor. Therefore, it is extremely crucial to identify the risk factors related to the progression and adverse outcome of cerebrovascular-neurological dysfunction in COVID-19 patients. Since many articles have reported the effect of smoking (tobacco and cannabis) and vaping in cerebrovascular and neurological systems, and considering that smokers are more prone to viral and bacterial infection compared to non-smokers, it is high time to explore the probable correlation of smoking in COVID-19 patients. Herein, we have reviewed the possible role of smoking and vaping on cerebrovascular and neurological dysfunction in COVID-19 patients, along with potential pathogenic mechanisms associated with it