Mohammad A. Kaisar

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.

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.

New experimental models of the blood-brain barrier for CNS drug discovery

ABSTRACT Introduction: The blood-brain barrier (BBB) is a dynamic biological interface which actively controls the passage of substances between the blood and the central nervous system (CNS). From a biological and functional standpoint, the BBB plays a crucial role in maintaining brain homeostasis inasmuch that deterioration of BBB functions are prodromal to many CNS disorders. Conversely, the BBB hinders the delivery of drugs targeting the brain to treat a variety of neurological diseases. Area covered: This article reviews recent technological improvements and innovation in the field of BBB modeling including static and dynamic cell-based platforms, microfluidic systems and the use of stem cells and 3D printing technologies. Additionally, the authors laid out a roadmap for the integration of microfluidics and stem cell biology as a holistic approach for the development of novel in vitro BBB platforms. Expert opinion: Development of effective CNS drugs has been hindered by the lack of reliable strategies to mimic the BBB and cerebrovascular impairments in vitro. Technological advancements in BBB modeling have fostered the development of highly integrative and quasi- physiological in vitro platforms to support the process of drug discovery. These advanced in vitro tools are likely to further current understanding of the cerebrovascular modulatory mechanisms.

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.

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-liquids 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.

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.

OTC Antioxidant Products for the Treatment of Cardiovascular and other Disorders: Popular Myth or Fact?

Cardiovascular disease (CVD) is the number one cause of mortality worldwide as reported by World Health Organization (WHO), Centers for Disease Control (CDC) and American Heart Association (AHA). The role of micronutrients has been studied extensively as CVD risk minimizing intervention. Among these, dietary supplements antioxidants available over the counter are highly commercialized but scientific evidences and clinical trials supporting their use is not conclusive yet [1]. The beneficial effects of these antioxidants are focused mainly against pathogenesis of atherosclerosis, as the primary contributor to coronary artery disease and resulting cardiovascular complications. Atherosclerosis is chronic inflammatory process of deposition of fatty cholesterol filled plaque in arterial wall which narrows the diameter and obstructs blood flow down the length of coronary artery and throughout its branches. Hyperlipidemia (high HDL: LDL ratio), high blood pressure, toxins from tobacco are assumed to be the major risk factor of atherosclerosis. If plasma LDL exceeds the regulatory capacity of endothelial cells, LDL crosses the endothelial barrier and trapped in sub-endothelial space where they are susceptible for oxidation by reactive oxygen species (ROS) - superoxide anion (·O2−), hydroxyl radical (OH·), hydrogen peroxide (H2O2), reactive nitrogen species (RNS), nitric oxide (NO) and peroxynitrite (ONOO·) released by endothelial cells and macrophages [2]. These modified LDL stimulates endothelial cells to express various cell adhesion molecules (VCAM-I, P-selectin etc.) and chemotactic factors (monocyte chemotactic factor-1, MCP-1) for the recruitment of monocytes. Monocyte attachment is initiated by P-selectin on endothelial cell upon binding with P-selectin glycoprotein ligand-1 (PSGL-1) on the monocytes leading to rolling and prolonging the contact of monocyte on the arterial wall and initiate diapedesis across endothelial layers. Monocytes differentiate into macrophages under the influence of monocyte colony stimulating factor (M-CSF). Oxidized LDL is then engulfed in an unregulated fashion by macrophage scavenger receptor resulting in intracellular accumulation of cholesterol and forms foam cell formation [3,4]. Macrophages are also involved in activation of T cells to amplify the inflammatory response by secreting TNF-α and INF-γ. Finally, smooth muscle cells proliferate and migrate from tunica media and shield the fatty plaque and synthesize collagen to form a fibrous cap [5–7].

Blood-brain barrier disruption in diabetic mice is linked to Nrf2 signaling deficits: Role of ABCB10?

Blood-brain barrier (BBB) damage is a critical neurovascular complication of diabetes mellitus that adversely affects the CNS health and function. Previously, we showed the protective role of NF-E2 related factor-2 (Nrf2), a redox sensitive transcription factor, in regulation of BBB integrity. Given the pathogenic role of mitochondrial oxidative stress in diabetes-related microvascular complications, we focused on assessing: 1) the impact of diabetes on brain Nrf2 in correlation with BBB permeability and 2) Nrf2-dependent regulation of the mitochondrial transporter ABCB10, an essential player in mitochondrial function and redox balance at BBB endothelium. Using live animal fluorescence imaging, we demonstrated a strong increase in BBB permeability to 70kDa dextran in db/db diabetic mice that correlated with significant down-regulation of brain Nrf2 protein. Further, Nrf2 gene silencing in human BBB endothelial cells markedly suppressed ABCB10 protein, while Nrf2 activation by sulforaphane up-regulated ABCB10 expression. Interestingly, ABCB10 knockdown resulted in a strong-induction of Nrf2 driven anti-oxidant responses as evidenced by increased expression of Nrf2 and its downstream targets. Nrf2 or ABCB10 silencing elevated endothelial-monocyte adhesion suggesting an activated inflammatory cascade. Thus, our results demonstrate a novel mechanism of ABCB10 regulation driven by Nrf2. In summary, Nrf2 dysregulation and ABCB10 suppression could likely mediate endothelial oxidative/inflammatory stress and BBB disruption in diabetic subjects.

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.

Conventional and electronic cigarettes dysregulate the expression of iron transporters and detoxifying enzymes at the brain vascular endothelium: In vivo evidence of a gender-specific cellular response to chronic cigarette smoke exposure

It is well established that tobacco smoking is associated with vascular endothelial dysfunction in a causative and dose dependent manner primarily related to the tobacco smoke (TS) content of reactive oxygen species (ROS), nicotine, and oxidative stress (OS) -driven inflammation. Preclinical studies have also shown that nicotine (the principal e-liquids ingredient used in e-cigarettes (e-Cigs) can also cause OS, exacerbation of cerebral ischemia and secondary brain injury. Likewise, chronic e-Cig vaping could be prodromal to vascular endothelial dysfunctions. Herein, we provide direct evidence that similarly to TS, e-Cig promotes mitochondrial depolarization in primary brain vascular endothelial cells as well as the vascular endothelial cell line bEnd3. In addition, both TS and e-Cig exposure upregulated the transmembrane iron exporter Slc40a1 (crucial to maintain cellular iron and redox homeostasis) and that of porphyrin importer Abcb6 (linked to accelerated atherosclerosis). We then investigated in vivo whether gender plays a role in how chronic TS affect vascular endothelial functions. Our results clearly show chronic TS exposure differentially impacts the expression levels of Phase-II enzymes as well as the iron transporters previously investigated in vitro. Although the physiological implications of the gender-specific differential responses to TS are not fully clear, they do demonstrate that gender is a risk factor that needs to be investigated when assessing the potential impact of chronic smoking and perhaps e-Cig vaping.