NRF2 and NF-қB interplay in cerebrovascular and neurodegenerative disorders: Molecular mechanisms and possible therapeutic approaches.

Electrophiles and reactive oxygen species (ROS) play a major role in modulating cellular defense mechanisms as well as physiological functions, and intracellular signaling. However, excessive ROS generation (endogenous and exogenous) can create a state of redox imbalance leading to cellular and tissue damage (Ma and He, 2012) [1]. A growing body of research data strongly suggests that imbalanced ROS and electrophile overproduction are among the major prodromal factors in the onset and progression of several cerebrovascular and neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), stroke, Alzheimer's disease (AD), Parkinson's disease (PD), and aging (Ma and He, 2012; Ramsey et al., 2017; Salminen et al., 2012; Sandberg et al., 2014; Sarlette et al., 2008; Tanji et al., 2013) [1-6]. Cells offset oxidative stress by the action of housekeeping antioxidative enzymes (such as superoxide dismutase, catalase, glutathione peroxidase) as well direct and indirect antioxidants (Dinkova-Kostova and Talalay, 2010) [7]. The DNA sequence responsible for modulating the antioxidative and cytoprotective responses of the cells has been identified as the antioxidant response element (ARE), while the nuclear factor erythroid 2-related factor (NRF2) is the major regulator of the xenobiotic-activated receptor (XAR) responsible for activating the ARE-pathway, thus defined as the NRF2-ARE system (Ma and He, 2012) [1]. In addition, the interplay between the NRF2-ARE system and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB, a protein complex that controls cytokine production and cell survival), has been further investigated in relation to neurodegenerative and neuroinflammatory disorders. On these premises, we provide a review analysis of current understanding of the NRF2-NF-ĸB interplay, their specific role in major CNS disorders, and consequent therapeutic implication for the treatment of neurodegenerative and cerebrovascular diseases.

In Vitro Modulation of Redox and Metabolism Interplay at the Brain Vascular Endothelium: Genomic and Proteomic Profiles of Sulforaphane Activity.

Sulforaphane (SFN) has been shown to protect the brain vascular system and effectively reduce ischemic injuries and cognitive deficits. Given the robust cerebrovascular protection afforded by SFN, the objective of this study was to profile these effects in vitro using primary mouse brain microvascular endothelial cells and focusing on cellular redox, metabolism and detoxification functions. We used a mouse MitoChip array developed and validated at the FDA National Center for Toxicological Research (NCTR) to profile a host of genes encoded by nuclear and mt-DNA following SFN treatment (0-5 µM). Corresponding protein expression levels were assessed (ad hoc) by qRT-PCR, immunoblots and immunocytochemistry (ICC). Gene ontology clustering revealed that SFN treatment (24 h) significantly up-regulated ~50 key genes (>1.5 fold, adjusted p < 0.0001) and repressed 20 genes (<0.7 fold, adjusted p < 0.0001) belonging to oxidative stress, phase 1 & 2 drug metabolism enzymes (glutathione system), iron transporters, glycolysis, oxidative phosphorylation (OXPHOS), amino acid metabolism, lipid metabolism and mitochondrial biogenesis. Our results show that SFN stimulated the production of ATP by promoting the expression and activity of glucose transporter-1, and glycolysis. In addition, SFN upregulated anti-oxidative stress responses, redox signaling and phase 2 drug metabolism/detoxification functions, thus elucidating further the previously observed neurovascular protective effects of this compound.

Unhealthy smokers: scopes for prophylactic intervention and clinical treatment.

BACKGROUND: Globally, tobacco use causes approximately 6 million deaths per year, and predictions report that with current trends; more than 8 million deaths are expected annually by 2030. Cigarette smokings is currently accountable for more than 480,000 deaths each year in United States (US) and is the leading cause of preventable death in the US. On average, smokers die 10 years earlier than nonsmokers and if smoking continues at its current proportion among adolescents, one in every 13 Americans aged 17 years or younger is expected to die prematurely from a smoking-related illness. Even though there has been a marginal smoking decline of around 5% in recent years (2005 vs 2015), smokers still account for 15% of the US adult population. What is also concerning is that 41,000 out of 480,000 deaths results from secondhand smoke (SHS) exposure. Herein, we provide a detailed review of health complications and major pathological mechanisms including mutation, inflammation, oxidative stress, and hemodynamic and plasma protein changes associated with chronic smoking. Further, we discuss prophylactic interventions and associated benefits and provide a rationale for the scope of clinical treatment. CONCLUSIONS: Considering these premises, it is evident that much detailed translational and clinical studies are needed. Factors such as the length of smoking cessation for ex-smokers, the level of smoke exposure in case of SHS, pre-established health conditions, genetics (and epigenetics modification caused by chronic smoking) are few of the criteria that need to be evaluated to begin assessing the prophylactic and/or therapeutic impact of treatments aimed at chronic and former smokers (especially early stage ex-smokers) including those frequently subjected to second hand tobacco smoke exposure. Herein, we provide a detailed review of health complications and major pathological mechanisms including mutation, inflammation, oxidative stress, and hemodynamic and plasma protein changes associated with chronic smoking. Further, we discuss about prophylactic interventions and associated benefits and provide a rationale and scope for clinical treatment.

Offsetting the impact of smoking and e-cigarette vaping on the cerebrovascular system and stroke injury: Is Metformin a viable countermeasure?

Recently published in vitro and in vivo findings strongly suggest that BBB impairment and increased risk for stroke by tobacco smoke (TS) closely resemble that of type-2 diabetes (2DM) and develop largely in response to common key modulators such oxidative stress (OS), inflammation and alterations of the endogenous antioxidative response system (ARE) regulated by the nuclear factor erythroid 2-related factor (Nrf2). Preclinical studies have also shown that nicotine (the principal e-liquid's ingredient used in e-cigarettes) can also cause OS, exacerbation of cerebral ischemia and secondary brain injury. Herein we provide evidence that likewise to TS, chronic e-Cigarette (e-Cig) vaping can be prodromal to the loss of blood-brain barrier (BBB) integrity and vascular inflammation as well as act as a promoting factor for the onset of stroke and worsening of post-ischemic brain injury. In addition, recent reports have shown that Metformin (MF) treatment before and after ischemic injury reduces stress and inhibits inflammatory responses. Recent published data by our group revealead that MF promotes the activation of counteractive mechanisms mediated by the activation of Nrf2 which drastically reduce TS toxicity at the brain and cerebrovascular levels and protect BBB integrity. In this study we provide additional in vivo evidence showing that MF can effectively reduce the oxidative and inflammatory risk for stroke and attenuate post-ischemic brain injury promoted by TS and e-Cig vaping. Our data also suggest that MF administration could be extended as prophylactic care during the time window required for the renormalization of the risk levels of stroke following smoking cessation thus further studies in that direction are warrated.

Role of Nrf2 and protective effects of Metformin against tobacco smoke-induced cerebrovascular toxicity.

Cigarette smoking (CS) is associated with vascular endothelial dysfunction in a causative way primarily related to the TS content of reactive oxygen species (ROS), nicotine, and inflammation. TS promotes glucose intolerance and increases the risk of developing type-2 diabetes mellitus (2DM) with which it shares other pathogenic traits including the high risk of cerebrovascular and neurological disorders like stroke via ROS generation, inflammation, and blood-brain barrier (BBB) impairment. Herein we provide evidence of the role played by nuclear factor erythroid 2-related factor (Nrf2) in CS-induced cerebrobvascular/BBB impairments and how these cerebrovascular harmful effects can be circumvented by the use of metformin (MF; a widely prescribed, firstline anti-diabetic drug) treatment. Our data in fact revealed that MF activates counteractive mechanisms primarily associated with the Nrf2 pathway which drastically reduce CS toxicity at the cerebrovascular level. These include the suppression of tight junction (TJ) protein downregulation and loss of BBB integrity induced by CS, reduction of inflammation and oxidative stress, renormalization of the expression levels of the major BBB glucose transporter Glut-1 and that of the anticoagulant factor thrombomodulin. Further, we provide additional insights on the controversial interplay between Nrf2 and AMPK.

A decade of e-cigarettes: Limited research & unresolved safety concerns.

It is well known that tobacco consumption is a leading cause of preventable deaths worldwide and has been linked to major diseases ranging from cancer to chronic obstructive pulmonary disease, atherosclerosis, stroke and a host of neurological/neurodegenerative disorders. In the past decade a number of alternative vaping products have hit the market, rapidly gaining consumers especially among the younger population. Electronic nicotine delivery systems or e-cigarettes have become the sought-after product due to the belief that they are much safer than traditional cigarettes. However, inadequate research and lack of regulatory guidelines for both the manufacturing process and the content of the vaping solution of the e-cigarette has become a major concern. Highly debated and unresolved questions such as whether e-cigarettes may help smokers quit and whether e-cigarettes will promote the use of nicotine among non-smokers add to the confusion of the safety of e-cigarettes. In this review article, we summarize the current understanding (and lack thereof) of the potential health impacts of e-cigarettes. We will also highlight the most recent studies (in vivo/in vitro) which seem to conflict with the broad safety claims put forward by the manufacturers. Finally, we provide potential solutions to overcome the research gap of the short and long-term health impact of e-cigarettes.

Hyperglycemia exacerbates antiretroviral drug combination induced blood-brain barrier endothelial toxicity.

In this study, we sought to investigate how concomitant hyperglycemia influences the impact of combination antiretroviral therapy on blood-brain barrier (BBB) endothelial function. Immortalized human brain microvascular endothelial cell line (hCMEC/D3) was exposed to azidothymidine (AZT; a nucleoside reverse transcriptase inhibitor) and/or indinavir (IND; protease inhibitor) in normal glycemic (5.5mM) or hyperglycemic (HG; 25mM) media containing D-glucose for 24-72h. Cellular reactive oxygen species (ROS) and mitochondria-specific superoxide levels were assayed in addition to membrane potential to determine the extent of mitochondrial dysfunction. Nrf2 expression was analyzed by immunofluorescence. Our results indicated a significant increase in BBB endothelial toxicity (decreased ATP) by HG and AZT+IND with progression of time (24-72h). Concurrent HG and antiviral drug combination synergistically elevated BBB endothelial ROS induced by either condition alone. Further, HG and AZT+IND mutually interact to elicit a pronounced increase in mitochondrial superoxide levels post 24h (vs. either condition alone or controls). In addition, HG and AZT+IND complemented each other to induce potential loss of mitochondrial membrane potential. While HG or AZT+IND alone for 24h increased Nrf2 nuclear distribution, co-exposure conditions induced a potential loss of Nrf2 expression/nuclear translocation in BBB endothelium. In summary, our data strongly suggest that antiretroviral drug combination potentially interacts with concomitant HG and triggers exacerbated mitochondrial dysfunction and BBB endothelial toxicity, possibly through dysregulation of Nrf2 signaling. Thus, this study warrants the critical need for safety evaluation and monitoring of neurovascular complications of HAART regimens in HIV-infected diabetic patient cohort.

Protecting the BBB endothelium against cigarette smoke-induced oxidative stress using popular antioxidants: Are they really beneficial?

Blood Brain Barrier (BBB) exposed to realistic concentrations (comparable to a chronic heavy smoker) of Cigarette Smoke Extract (CSE) triggers a strong endothelial inflammatory response which can lead to the onset of neurological disorders. The involvement of Reactive Oxygen Species (ROS) in this inflammatory cascade is evident from the up-regulation of nuclear factor erythroid 2 related factor 2 (Nrf-2), a transcription factor involved in anti-oxidant response signaling in CSE exposed endothelial cells. We have shown that pre-treatment with α-tocopherol and/or ascorbic acid is highly protective for the BBB, thus suggesting that, prophylactic administration of antioxidants can reduce CSE and/or inflammatory-dependent BBB damage. We have assessed and ranked the protective effects of 5 popular OTC antioxidants (Coenzyme Q10, melatonin, glutathione, lipoic acid and resveratrol) against CSE-induced BBB endothelial damage using hCMEC/D3 cells. The analysis of pro-inflammatory cytokines release by ELISA revealed that resveratrol, lipoic acid melatonin and Co-Q10 inhibited the BBB endothelial release of pro-inflammatory cytokines IL-6 and IL-8, reduced (not Co-Q10) CSE-induced up-regulation of Platelet Cell Adhesion Molecule-1 (PECAM-1), Vascular Cell Adhesion Molecule-1 (VCAM-1) & E-selectin and inhibited monocytes-endothelial cell adhesion. The anti-inflammatory effects correlated with the anti-oxidative protection endowed by these compounds as evidenced by upregulation of NADPH: Quinone Oxidoreductase 1 (NQO1) and reduced cellular oxidative stress. CSE-induced release of Vascular Endothelial Growth Factor (VEGF) was inhibited by all tested compounds although the effect was not strictly dose-dependent. Further in vivo studies are required to validate our results and expand our current study to include combinatorial treatments.

Impact of cigarette smoke extract and hyperglycemic conditions on blood-brain barrier endothelial cells.

BACKGROUND: Diabetes and tobacco smoking are significant public health concerns which have been shown to independently impact the blood-brain barrier (BBB). Since smoking is a risk factor for diabetes and shares some of the common pathological pathways leading to metabolic abnormalities, it is hypothesized that their combination would produce additive or synergistic BBB dysfunction. Therefore, the objective of this study was to assess this hypothesis and evaluate the magnitude of these effects in vitro using hCMEC/D3 cells; a well-established human BBB endothelial cell line. METHODS: Monolayers of hCMEC/D3 cells were exposed to hyperglycemic conditions (HG; 35 mM) or 5% soluble cigarette smoke extracts (CSE, model of mainstream smoke exposure) for 12-24 h. Cells were then harvested for subsequent biochemical analyses. Transendothelial electrical resistance (TEER) and paracellular permeability to florescent dextrans were used to assess monolayer integrity. Analysis of released factors and cytokines was carried out by ELISA. Western blot (WB) analysis/immunofluorescence of relevant molecular targets was carried out. P-gp efflux activity was measured using rhodamine 123. RESULTS: Immunofluorescence and WB data showed a significant ZO-1 down-regulation by HG and/or CSE over 24 h exposure. CSE in presence of HG produced a synergistic increase in release of vascular endothelial growth factor that was accompanied by decreased TEER and augmented permeability to labeled dextrans in a size-dependent manner. Moreover, CSE increased the expression of GLUT-1 and SGLT-1 in isolated membrane fractions of hCMEC/D3 cells. The effect was amplified by HG. Both, HG and CSE elicited the membrane upregulation of P-glycoprotein (P-gp) expression which however, was not paralleled by a comparable efflux activity. Interestingly, concomitant exposure to HG and CSE evoked a marked upregulation of PECAM-1 and other pro-inflammatory markers including IL-6 and -8, when compared to each condition alone. Moreover, exposure to all tested conditions amplified (to a different degree) cellular oxidative stress response denoted by increased Nrf2 nuclear translocation. CONCLUSION: Overall, our results have clearly shown an additive pattern in the release of angiogenic and inflammatory factors following concomitant exposure to HG and CSE. This suggests the involvement of common key modulators in BBB impairment by both CS and HG possibly through the activation of oxidative stress responses.

Effect of full flavor and denicotinized cigarettes exposure on the brain microvascular endothelium: a microarray-based gene expression study using a human immortalized BBB endothelial cell line.

BACKGROUND: Tobacco smoke (TS) toxicity to the brain microvasculature is still an understudied area till date. NF-E2 related factor (Nrf2) is a key transcription factor responsible for activating the antioxidant response element (ARE) genes following an oxidative insult. Till date, several studies targeting the blood brain barrier (BBB) have shown some protective role of Nrf2 in ischemia-reperfusion (IR) injury, however, its functional role in chronic smokers subjected to a life-long oxidative stress has never been addressed. This is of crucial importance since smokers have a much higher risk for cerebrovascular stroke and tobacco smoke exposure has been clearly shown to enhance BBB damage following an ischemia/reperfusion injury. Thus, the goal of our study was to investigate the defense pathways activated at the BBB endothelial level by TS exposure. Specifically we focused on Nrf2 and nuclear factor kappa-light-chain-enhancer of activated B signaling response (NF-κß) as the central protective mechanisms related to oxidative insult. RESULTS: With the exception of Nicotine, both full flavor (3R4F) and decotinized (ULN) cigarettes activated Nrf2 and NFκß pathways in hCMEC/D3 endothelial cells. Several detoxification and anti-oxidant genes including downstream products were also activated including NAD(P)H dehydrogenase quinone 1 (NQO-1), heme oxygenase-1 (HMOX-1), catalytic and modifier subunits of glutamate-cysteine ligase (GCL), solute carrier-SLC7A11). Gene expression levels of cytochrome P450s (CYP2S1 and CYP51A1) and efflux transporters P-glycoprotein (P-gp) and multi-drug resistance protein-4 (MRP4) were also enhanced. Increase of P-gp functional activity and depletion of GSH were also observed. Strikingly, toxicity of denicotinized ("reduced exposure") cigarettes was equivalent to 3R4F (or worse). CONCLUSIONS: This study provides a detailed analysis of Nrf2-related cytoprotective mechanisms activated in response to 3R4F and ULN-derived TS exposure correlating the results with their oxidative and inflammatory potential. Toxicants present in soluble cigarette smoke extracts (CSE) and not nicotine seem to be the primary determinant of vascular toxicity. In this respect our results from this and previous studies suggest that chronic TS exposure can overcome Nrf2 and NFκB-p65 dependent cytoprotective mechanisms of the brain microvascular endothelium possibly leading to BBB impairment and loss of BBB integrity.

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 content.

Targeting pioneering factor and hormone receptor cooperative pathways to suppress tumor progression.

Nuclear receptors and pioneer factors drive the development and progression of prostate cancer. In this disease, aggressive disease phenotypes and hormone therapy failures result from resurgent activity of androgen receptor (AR) and the upregulation of coactivator protein p300 and pioneer factors (e.g., GATA2 and FOXA1). Thus, a major emphasis in the field is to identify mechanisms by which castrate-resistant AR activity and pioneer factor function can be combinatorially suppressed. Here we show that the turmeric spice isoflavone curcumin suppresses p300 and CBP occupancy at sites of AR function. Curcumin reduced the association of histone acetylation and pioneer factors, thereby suppressing AR residence and downstream target gene expression. Histone deacetylase inhibitors reversed the effects of curcumin on AR activity, further underscoring the impact of curcumin on altering the chromatin landscape. These functions precluded pioneer factor occupancy, leading ultimately to a suppression of ligand-dependent and ligand-independent AR residence on chromatin. Moreover, these functions were conserved even in cells with heightened pioneer factor activity, thus identifying a potential strategy to manage this subclass of tumors. Biological relevance was further identified using in vivo xenograft models mimicking disease progression. Curcumin cooperated in vivo with androgen deprivation as indicated by a reduction in tumor growth and delay to the onset of castrate-resistant disease. Together, our results show the combinatorial impact of targeting AR and histone modification in prostate cancer, thus setting the stage for further development of curcumin as a novel agent to target AR signaling.