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?
© Naik et al.; licensee BioMed Central Ltd. 2014
Received: 23 December 2013
Accepted: 7 April 2014
Published: 23 April 2014
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.
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.
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.
Tobacco smoke (TS) is a major public health hazard, accounting for more than 5.4 million premature deaths worldwide and over 440,000 deaths each year in the United States alone . In addition to the onset of various forms of cancer , smoking has been associated with the pathogenesis and/or progression of a number of major neurological disorders. These include, but are not limited to, silent cerebral infarction (SCI) , stroke  due to the pro-coagulant and atherogenic effects of smoking [5, 6] and cerebral aneurysms . There is also a strong correlation between smoking and an increased risk for multiple sclerosis [8, 9], Alzheimer’s disease, small vessel ischemic disease (SVID) and neurodevelopmental damage during pregnancy . Although it is possible to explain some of the neuropathological effects of TS with nicotine specific pathways , the precise harmful mechanisms activated by tobacco smoke remain unclear. Thus the neuropathology of cigarette smoking and underlying pathogenic pathways remain largely unknown, although TS-dependent impairment of blood–brain barrier (BBB) function is certainly a critical prodromal factor.
A burgeoning yet incomplete body of evidence suggests that cerebrovascular inflammation and impairment of endothelial physiology are primarily responsible for a large number of neurological disorders associated with BBB dysfunction . This provides a solid link to TS-dependent impairment of BBB function whereas cigarette smoke extracts have been shown to act as a powerful activator of immune/inflammatory response pathways altering the integrity/function of the BBB [13, 14].
Mainstream TS contains over 4000 chemical compounds including a harmful cloud of free radicals and other reactive oxygen (ROS) and nitrogen species (RNS) contained in both the gaseous phase and the tar . At the vascular level free radicals can lead to oxidative damage of endothelial cells  involving DNA strand breakage and inflammation [17–19]. Active and passive tobacco smoking can spawn these highly reactive oxygen species (hydrogen peroxide, epoxides, nitric oxide (NO), nitrogen dioxide, peroxynitrite (ONOO) ) beyond the levels which the human body can eliminate effectively. In fact, several studies have shown that: 1) chronic smokers suffer from antioxidant shortage caused by increased anti-oxidative mobilization in response to systemic oxidative stress evoked by ROS-enriched TS [21, 22]; 2) antioxidant supplementation reduces the oxidation and inflammation induced by TS in animals and cells [14, 23]; 3) TS contributes to a pro-atherosclerotic environment by triggering a complex pro-inflammatory response and mediates the recruitment of leukocytes  through cytokine signaling.
The tobacco industry has developed “reduced exposure” and “light” products containing lower levels of nicotine, nitrosamines or other chemicals deemed to be potentially toxic. However, experimental and clinical data supporting the claim that these products reduce the health hazard of tobacco smoking are lacking. To date, only a handful of studies have investigated the effect of TS on BBB function and integrity, thus limiting our understanding of mechanisms involved in TS-related toxicity at BBB and associated risks for neuropathological disorders.
Therefore, in our study we investigated the effects of various tobacco products (including ultralow nicotine and tobacco-free cigarettes) on BBB endothelium in vitro, using a well characterized human BBB endothelial cell line (hCMEC/D3; [25, 26]. Data from this study indicates that smoking-related dysfunction of BBB endothelial physiology (e.g., increased oxidative stress, impaired tight junction expression/distribution, etc.) positively correlate with the total content of tar of various tobacco products and associated oxidative stress (ROS and NO output) rather than nicotine content.
Exposure to nicotine concentrations equivalent to that observed in plasma in chronic human smoker does not affect endothelial cell viability
Nitrate and nitrite levels in CSE correlates with corresponding cigarette’s tar and nicotine content
Release of hydrogen peroxide (H2O2) in CSE increases with the tar content of cigarettes and leads to progressive oxidative damage in BBB ECs
Exposure to CSE from 3R4F, NF and ultralow nicotine cigarettes negatively impacts ZO-1 and occludin expression/distribution as well as BBB integrity
Exposure to CSE from 3R4F, NF and ultralow nicotine cigarettes promotes the pro-inflammatory activation of BBB endothelial cells
Importantly, analysis of the culture conditioned media by ELISA revealed a significant increase of interleukin-6 (IL-6) release from the endothelial cells exposed to either NF (p < 0.01) or ultralow nicotine cigarette extracts (p < 0.005), compared to controls (Figure 6C). A modest, yet significant increase in the release of matrix metalloproteinase-2 (MMP-2) was also observed in cultures treated with either 3R4F or NF smoke extracts, but not ultralow nicotine (Figure 6C). However, MMP-9, IL-1β and TNF-α levels in the conditioned media from all treatment conditions were below the reading sensitivity (data not shown).
ROS, despite being essential for biological systems  have the potential to cause extensive oxidative damage to cells and tissues if their levels become excessive [33, 34]. At the vascular level ROS can cause oxidative damage of endothelial cells  including DNA strand breakage and inflammation . In addition to ROS, nicotine can equally elicit oxidative stress and tissue injury [35, 36] and has been shown to exacerbate brain edema following focal ischemia [37, 38]. Oxidants in the gaseous phase of cigarette smoke, including nicotine and various ROS species, ([15, 20] can pass through the lung alveolar wall and raise systemic oxidative stress . This can lead to oxidative damage to cells and tissues, including the brain vascular system and the BBB, over a period of sustained exposure to TS (e.g., chronic smokers) and facilitate the pathogenesis and progression of neurological disorders [40–42]. Thus, existing evidence strongly suggests a role for TS-dependent oxidative and inflammatory stress in the development of CNS pathologies. In fact, the cerebrovascular endothelium is highly vulnerable to oxidative stress resulting in loss of BBB function and integrity via altered expression and distribution of intercellular TJ complexes [43, 44].
In this study we assessed and compared the effects of various tobacco products on human BBB endothelial cells in relation to their corresponding oxidative potential. Specifically, several studies have demonstrated that cigarette smoke contains high concentrations of NO which may directly affect the integrity of the BBB. For this purpose we measured ROS as well as NO3−/NO2− content (Figures 2 & 3) of tobacco smoke from 1R5F (ultralight), 3R4F (full flavor), NF (tobacco free) and ultralow nicotine cigarettes. The NO3−/NO2− analysis revealed a direct correlation with the content of tar of the respective cigarettes. However, this did not hold true for NF products, whose tar content (comparable to medium strength cigarettes) produced the least amount of nitrate and nitrite. When we compared the NO3−/NO2− output with corresponding nicotine content, a significant correlation was not found, unless ultralow nicotine brand were removed from the pool (Figure 2C - insets). Together these results suggest that NO3−/NO2− is relatively independent of nicotine content while holding a strong correlation with that of tar.
Tar being a byproduct derived from combustion of tobacco or analogous products, alteration of tobacco (e.g., ultralow nicotine products) or replacement with alternative products (NF cigarette) to reduce nicotine content in a bid to decrease addiction potential, may result in an unwanted increase of nitrate/nitrite output, and risk for health hazard. In fact, tobacco nitrate levels have been previously reported to correlate with the formation of non-specific volatile nitrosamines (e.g., N-nitrosodimethylamine, N-nitroso-diethylamine, N-nitrosoethylmethyl- amine, etc.), and non-volatile Tobacco-Specific Nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, nicotine-derived nitrosamine ketone) which have been associated with carcinogenicity of tobacco smoke [30, 31].
Interestingly, H2O2 content measured in ultralow nicotine and tobacco free (NF) cigarettes, considered “reduced-exposure” products, was significantly higher than any other brand including medium and full flavor (see Figure 3). Regression analysis of H2O2 also revealed a strong correlation with the tar content of the respective cigarettes but not with that of nicotine unless both products were to be removed from the pool. These results strongly correlate with the increased oxidative stress generated in BBB endothelial cultures (see Figure 3A2) and revealing that the highest level of oxidation is in endothelial cells that are exposed to ultralow and NF cigarette smoke extracts. Interestingly, the oxidative stress potential of 3R4F cigarettes was comparable to that of ultralow and NF cigarettes despite releasing lower amounts of H2O2. This can be attributed to the higher content of nicotine in 3R4F cigarettes since nicotine equally contributes to oxidative stress (Das et al. 2012). Taken together, these results suggest that alteration and/or substitution of tobacco with alternative products in order to reduce nicotine content was responsible for the increased H2O2 output measured in these “denicotinized” cigarette products.
Previous reports by Hossain and co-workers  have shown a dose dependent loss of BBB integrity directly correlating to TS-derived oxidative stress. Furthermore, loss of BBB function and integrity caused by TS exposure was prevented or at least reduced by antioxidant vitamins. These findings by others clearly support our results which outlined a strong correlation between the impairment of tight junction protein expression/distribution and BBB integrity with the oxidative stress generated by the TS extracts. As clearly shown in the results (see Figure 4) BBB endothelial ZO-1 expression and distribution is completely deregulated upon exposure to TS extract from 3R4F, NF and Ultralow Nicotine cigarettes. This is also reflected in the increased BBB permeability to dextran paracellular markers observed under the same conditions.
ZO-1 is a cytoplasmic accessory protein which plays a crucial role in BBB integrity by connecting transmembrane proteins (such as occludin, claudins and JAM) to cytoskeletal proteins and is actively involved in signal transduction and transcriptional modulation [45, 46]. Interestingly, the effect of CSE on ZO-1 expression/distribution reflects the overall oxidative potential of the corresponding cigarettes (see Figure 3), thus suggesting a correlation between TS-dependent oxidative potential and dysregulation of TJs and BBB integrity. ZO-1 TJ protein closely associates with the actin cytoskeletal network. When we observed the actin structure with respect to ZO-1, it appeared intact. In addition, membrane expression of occludin was significantly down-regulated as evidenced by the WB analysis of the corresponding membrane fractions. Similar to ZO-1, membrane distribution of occludin was also altered deteriorating from a homogenous pattern at cell-cell junctions in controls to a patchy distribution in cultures exposed to 3RF4, NF and ultralow nicotine cigarettes. This can be a reflection of the parallel loss of ZO-1 which provides a positioning system and anchoring scaffold for the transmembrane TJ proteins.
In contrast to ZO-1 and occludin, the expression of VE-cadherin and claudin-5 was proportionally increased with respect to the oxidative potential of the corresponding CSE treatment. In fact, as shown in Figure 5, VE-cadherin membrane expression was progressively up-regulated by exposure to 3RF4, NF and ultralow nicotine cigarettes, although statistical significance was proven only for the last two cigarette products. In parallel, claudin-5 membrane expression was similarly up-regulated (see Figure 5B). This is in agreement with emerging evidences suggesting that VE-cadherin controls claudin-5 expression by preventing the nuclear accumulation of FoxO1 and beta-catenin which repress the claudin-5 promoter  thus reducing its expression. Although, these results were surprising, they actually seem to be in agreement with the above mentioned observations. In fact, recent in vitro studies have shown a direct positive correlation between VE-cadherin expression and oxidative stress  suggesting this being part of a cytoprotective response mechanism. In fact, VE-cadherin acts as a master regulator of various endothelial functions including modulation of cell-cell adhesion, angiogenesis, and vascular permeability to leukocytes in response to VCAM-1 activation , whose expression level was also increased (see Figure 6). Note also that an up-regulation of claudin-5 (in this case mediated by VE-cadherin) does not necessarily translate into an improved BBB integrity. Although this is true from a biological standpoint under normal circumstances we have to take into consideration that the mere expression of TJ proteins is not sufficient as a standalone determinant for BBB integrity. Other important factors play a significant role here such as the link between TJ proteins with the cytoskeleton. An important interaction mediated by first order regulatory proteins such as ZO-1 is of critical importance for the positioning and interaction of TJ proteins with their homologues on adjacent endothelial cells. Moreover, although claudin-5 was up-regulated (see Figure 5) the pattern of expression presented as an homogenous distribution throughout the cells and lacked a demarcated membrane localization which does not suggest improvements of cell-cell adhesion. This hypothesis well copes with the evident loss of barrier functions outlined by the increased permeability to dextran markers.
In addition, a similar increase in PECAM-1 expression was observed as well as an increased endothelial release of IL-6 and MMP-2 (see Figure 6). Regarding MMP-2, previous reports by others have shown how ROS regulate the activity of vascular matrix metalloproteinases in vitro including MMP-2 and MMP-9  which have an implication in atherosclerotic plaque stability. Expression and activation of MMP-2 has been demonstrated as a key event in oxidative stress injury to heart  and hyperglycaemia promoted BBB dysfunction . Together these results strengthen the link between tobacco smoke, it’s corresponding oxidative and inflammatory stress, and potential risk for BBB dysfunction. Although outside the scope of the present work, more studies will be necessary to dissect the molecular mechanisms involved in the generation of cellular oxidative stress at the brain microvascular endothelium by CSE and its impact on BBB function and integrity.
In summary, this study is one of the first attempts to assess and compare the potential toxic impact of various cigarette products on BBB endothelial cells using whole smoke extracts. We further correlated the oxidative and inflammatory potential of these cigarette products with respect to their tar, nicotine, H2O2 and nitric oxide content. We also clearly showed that the alteration of tobacco in an attempt to reduce cigarette nicotine content to attenuate addiction can result in an increased toxicity and endothelial inflammatory response. This can ultimately impair the BBB function and increase the risk for the pathogenesis of a number of CNS disorders.
Materials and reagents
The antibodies used in this study were obtained from the following sources: Rabbit anti-ZO-1 (#8193), rabbit anti-claudin-3 (#341700), rabbit anti-VE-cadherin (#D87F2), rabbit anti-VCAM-1 (#12367), mouse PECAM-1 (#89C2) from Cell Signaling Technology (Danvers, MA, USA); mouse anti-E-selectin (#S 9555), β-actin (#A5441) from Sigma-Aldrich (St. Louis, MO, USA); donkey anti-rabbit (#NA934) and sheep anti-mouse (#NA931) HRP-linked secondary antibodies from GE Healthcare (Piscataway, NJ, USA); mouse anti-claudin 5 (#35-2500), goat anti-rabbit (#A11008) and anti-mouse (#A21422) conjugated to Alexa Fluor® 488 and 555 from Invitrogen (Camarillo, CA, USA). Sterile cultureware was obtained from Fisher Scientific (Pittsburgh, PA, USA), while other reagents and chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Bio-Rad laboratories (Hercules, CA, USA).
The immortalized hCMEC/D3 cell line was donated by Dr. Couraud (INSERM, Paris). The hCMEC/D3 cells (passage 28–32) were seeded on collagen coated culture flasks (2.5-3 × 104/cm2) or glass slides (4 × 104/cm2) and maintained at 37°C with 5% CO2 exposure in EBM-2 basal medium (Lonza, Walkersville, MD, USA) supplemented with 5% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), chemically defined lipid concentrate (Life Technologies, Carlsbad, CA, USA), growth factors, antibiotic/antimycotic (1:1, Atlanta Biologicals, GA, USA and HEPES (10 mM). Medium was changed every 2 days until the cells reached confluence. Monolayer integrity of hCMEC/D3 cells at confluence was confirmed by phase contrast microscopy and the expression of endothelial cell-specific phenotypic markers at cell-cell junctions, as previously described . For treatment, cell monolayers were exposed to CSE concentration (5-20%) diluted from freshly prepared smoke extracts as described above. Cultures exposed to CSE-free vehicle (PBS) served as controls.
Cell viability assay
The effects of CSE exposure on cell viability were determined by MTT (3 (4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay. Briefly, HCMEC/D3 cells were passaged in a 96-well plate and allowed to attach for a period of 48 h. Following exposure to CSE, cells were incubated with 10 μM MTT for 5 h at 37°C. MTT was removed and DMSO was added to solubilize the formazan crystals for 20 min. Color development corresponding to viable cells was quantitated by measuring absorbance at 520 nm.
Nitric oxide (NO) content analysis
Cigarette smoke was bubbled through an impinger into PBS using the CSM-SCCM smoking machine as described above. NO content of the different types of cigarettes was determined indirectly through the estimation of nitrate/nitrite content using Griess reagent reaction based NO kit from R&D Systems, according to manufacturer’s guidelines.
Hydrogen peroxide (H2O2) content analysis
H2O2 content in smoke extracts of various types of cigarettes was determined by TBR4100 free radical analyzer with 100 μm HPO sensor. Briefly, aliquots from cigarette preparation were titrated in PBS to obtain a sensor reading which was extrapolated against a hydrogen peroxide standard curve to quantitate the amount per cigarette.
HPLC analysis of CSE preparation
For sample preparation, CSE obtained from CSM was subjected to liquid/liquid extraction using dichloromethane. Briefly, 500 μl aliquot of CSE was mixed with 5 μl of 1 M NaOH followed by the addition of 2 ml DCM. After centrifugation of the mixture at 1500 g for 10 min, the upper aqueous layer was discarded. The lower organic layer was evaporated under nitrogen gas, and the precipitate was resuspended in mobile phase, filtered and then injected on to the column. Nicotine (Sigma Aldrich, St. Louis, MA, USA #36733) dissolved in mobile phase was used to prepare the standard curve. Isocratic separation was performed on Agilent A1220 HPLC System (Agilent Technologies, Santa Clara, CA, USA) coupled to a UV detector, using Zorbax Rx-C18 column (4.6x150mm, 5 μm) with an inline guard column filter. The mobile phase consisted of 50 mM KH2PO4 buffer with 10 mM sodium heptane 1-sulfonate, pH adjusted to 3.0 using orthophosphoric acid and methanol (70:30 v/v). The flow rate was set to 1 ml/min with column temperature at 30°C and injection volume was 50 μl. Wavelength corresponding to maximum absorption of nicotine (259 nm) was used.
Following exposure to CSE, the cell culture conditioned media was collected and stored at −20°C until analysis. Levels of pro-inflammatory cytokines such as IL-1b, TNF-alpha, IL-6 and matrix metalloproteinases (MMPs) such as MMP-2 and MMP-9 were measured by Quantikine ELISA kits from R&D systems as per manufacturer’s instructions.
Cells were grown in two-well chamber slides specifically for these studies. After treatment, cells were fixed with formaldehyde (15mins at 4°C). Following three PBS washes, cells were blocked using 5% goat serum (Sigma-Aldrich, St. Louis, MO, USA) in PBS at room temperature for 50 min and incubated with primary antibodies prepared in 5% GSA overnight at 4°C. After three rinses with PBS, cells were incubated for 1 h at RT with Alexa Fluor® 488 or 555 conjugated goat anti-rabbit or anti-mouse antibodies, respectively (1:1000). Thereafter, cells were rinsed and counterstained with DAPI in Prolonged Gold Anti-fade mounting media (Invitrogen, OR, USA). Slides were cover slipped and left for overnight drying in the dark before examination with EVOS digital inverted fluorescence microscope. Cells stained with secondary antibodies alone were used as negative controls.
Briefly, cells were lysed in ice-cold Urea-Tris buffer containing Phosphatase and Protease Inhibitors (Roche Diagnostics, Indianapolis, IN, USA), sonicated and centrifuged at 14000 rpm, 4°C for 15 min. Protein concentration was determined by Bradford assay (Bio-Rad laboratories (Hercules, CA, USA # 5000006). Denatured samples containing equal protein (40 μg) were subjected to SDS-PAGE (10% or 4-15% gradient gel) and electrotransferred to PVDF membranes (2 hr transfer at 100 V). Membranes were blocked for 2 h (RT) with 5% non-fat dry milk in Tris buffered saline (TBS) containing 0.1% Tween-20 (TTBS) and subsequently incubated with rabbit (1:1000) or mouse (1:500) primary antibodies. After 4 washes (10 min each) with TTBS, membranes were incubated with anti-rabbit or anti-mouse (1:2000) HRP-conjugated secondary antibodies (2 h, RT) and washed with TTBS. Bands were detected by enhanced chemiluminescence using Amersham ECL™ Prime with ChemiDoc™ XRS system. Membranes were subsequently stripped and probed for β-actin (1:1000) as a loading control. Band densities were analyzed by Quantity One Software.
Measurement of BBB integrity: dextran permeability
Differential effects of TS exposure on BBB integrity was assessed by measuring paracellular permeability (luminal to abluminal) to labeled dextrans (4-70 kDa) as previously described . After 24 h exposure to TS extracts, a mixture of labeled dextrans in PBS (FITC- 4 kDa, 5 mg/ml; Cascade Blue®- 10 kDa, 5 mg/ml; and Rhod. B-ITC - 70 kDa, 5 mg/ml) was added to the luminal compartment. Abluminal samples (50 μL) were collected over 30 min and replaced with equal volume of fresh media to allow sink conditions. Dextran fluxes were determined by fluorescent measurements using the appropriate excitation and emission wavelengths. Permeability measurements were reported as percentage of controls (the permeability coefficients of controls were as follow: FITC 0.198 ± 0.009 × 10−3 cm/min; Cascade Blue® 0.0953 ± 0.007 × 10−3 cm/min and Rhod. B-ITC 0.007 ± 0.0005 × 10−3 cm/min).
Data from all experiments were expressed as mean ± standard error of mean (S.E.M) and analyzed by one-way ANOVA using GraphPad Prism Software Inc. (La Jolla, CA, USA). Post hoc multiple comparisons were performed with Tukey’s test. P values ≤ than 0.05 were considered statistically significant.
These studies were supported by NIH/NIDA R01-DA029121-01A1 and in part by. A.R.D.F to Dr. Luca Cucullo.
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