Background
Cigarette smoking accounts for 434,000 casualties/year in US and is the leading cause of preventable death. Even though there has been a marginal decline in smoking in recent years, the fact that ≈18% of the US adult population are current smokers is alarming [
1]. In 2007 diabetes was the 7th leading cause of death in the US and it is increasing at an alarming rate. One in every three US adults are projected to suffer from diabetes by 2050 [
2]. Smoking is a major risk factor for diabetes [
3], with 12% of type-2 diabetes mellitus (2DM) cases being attributed to tobacco smoke [
4]. Both active and passive smoking not only causes glucose intolerance [
5], but significantly increases the risk of diabetes by 45 or 74% in men and women, respectively [
6]. Major pathological changes in diabetic patients such as insulin resistance and high levels of glycated hemoglobin (HbA1c) have also been reported in smokers [
7]. Both 2DM and smoking have been reported independently to enhance the risk of cerebrovascular and neurological disorders like stroke [
1,
8], Alzheimer’s [
9,
10], depression [
11,
12], cognitive impairment, and vascular dementia [
13,
14]; largely due to an increase in reactive oxygen species (ROS) generation [
15‐
17], proinflammatory activity [
18,
19], and BBB impairment [
20,
21]. However, 2DM and CS—dependent pathophysiological mechanisms underlying these cerebrovascular disorders remain elusive. CS contains over 4,000 chemicals including nicotine and various ROS (e.g., H
2O
2, epoxides, nitrogen dioxide, peroxynitrite—ONOO
−, etc. [
22,
23] ) which pass through the lung alveolar wall and raise systemic ROS [
24]. At the cerebrovascular level this promotes oxidative damage and BBB breakdown via tight junction (TJ) modification and activation of proinflammatory pathways [
25,
26]. Chronic hyperglycemia, a pathogenic alteration characteristic of 2DM, also causes endogenous ROS increase by inhibiting glycolysis and promoting the formation of harmful intermediates such as advanced glycation end products (AGEs) and protein kinase-C pathway (PKC) isoforms, which have DNA and protein damaging effects [
27,
28]. At the BBB level, chronic hyperglycemia causes endothelial dysfunction leading to BBB impairment and loss of barrier integrity [
28].
Similarly, chronic hyperglycemia has also been reported to alter the expression of a number of BBB functional transporters including facilitative sodium independent glucose transporter-1 (GLUT-1), sodium dependent glucose co-transporter-1 (SGLT-1) and P-glycoprotein (P-gp) [
28]. However, the reports are controversial with certain studies reporting a decrease [
29,
30] or no change [
31‐
33] in cerebral GLUT-1 and SGLT-1 protein expression, and an increase [
34] or unaltered [
33] local cerebral glucose utilization. In addition, P-gp expression levels have been reported to decrease [
35,
36], increase [
37] or remain unaffected [
38] in animal diabetic models. Expression and activity changes of P-gp at BBB on smoke exposure have not yet been investigated. Further, despite the epidemiological and translational studies strongly suggesting activation of similar pathophysiological pathways by 2DM and CS, determination and characterization of shared key modulators in BBB impairment lies unexplored. Identification and then targeting of these putative key modulators could help in preventing the initiation of metabolic/cerebrovascular complications in smokers.
Therefore, the objective of our study was to investigate the individual and combinatorial effects of tobacco products and hyperglycemia on BBB endothelium. The experiments were conducted in vitro using a well-characterized human BBB endothelial cell line hCMEC/D3 [
39]. Data from this study indicates that hyperglycemia and tobacco smoke (TS) exposure cause dysfunction of BBB endothelium (e.g. impaired tight junction protein expression/distribution, increase in permeability, increase in proinflammatory activity, etc.). Further, our results suggest the existence of similar patterns of endothelial dysfunction in response to TS and hyperglycemia which shows additive or synergistic effects in the majority of our experimental scenarios. Together our data suggest the involvement of shared pathological pathways in TS and hyperglycemia which impact the BBB.
Methods
Antibody sources
The antibodies used in this study were obtained from the following sources: rabbit anti-ZO-1 (#D7D12), rabbit anti-ICAM-1 (#4915S) and mouse anti-PECAM-1 (#89C2) from Cell Signaling Technology (Danvers, MA, USA); mouse anti-occludin (#331500) from Life Technologies (Grand Island, NY, USA); mouse anti-glut-1 (#MABS132) and mouse anti-P-gp (517301) from EMD Millipore (Billerica, MA, USA); rabbit anti-SGLT-1 (#ab-14686) from Abcam (Cambridge, MA, USA); rabbit anti-Nrf2 (#sc-722) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); 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); goat anti-rabbit (#A11008, A21428) conjugated to Alexa Fluor® 488 and 555, respectively and anti-mouse (#A11001) conjugated to Alexa Fluor® 488 from Invitrogen (Camarillo, CA, USA).
Reagents
Sterile culture ware was obtained from Fisher Scientific (Pittsburgh, PA, USA), reagents and chemicals were purchased from Sigma-Aldrich or Bio-rad laboratories (Hercules, CA, USA), while Mini-Protean® TGX™ gels 4–15% (#456-1084) from Bio-rad laboratories was used for gel electrophoresis. Dextran-Cascade Blue® (10,000 MW; #D-1976) was obtained from Life Technologies, while Fluorescein isothiocyanate (FITC)-dextran (3,000–5,000 MW; #FD4) and Rhodamine B isothiocyanate (RITC)—dextran (70,000 MW; #R9379) were purchased from Sigma-Aldrich.
Tobacco smoke preparation
Cigarettes (3R4F) equivalent to full flavor commercial brands with 9.4 mg tar and 0.726 mg nicotine per cigarette were obtained from the University of Kentucky. Cigarette smoke extracts (CSE) was prepared by bubbling eight puffs per cigarette directly into phosphate buffered saline (PBS). This was done in accordance to the ISO/FTC standard smoking protocol (draw of 35 ml, puff duration of 2 s, 1 puff per 60 s), using a single cigarette smoking machine (SCSM, CH Technologies Inc., Westwood, NJ, USA). A 3× concentration (or 300%) of stock solution was first prepared by using three cigarettes. It was then diluted to 5% concentrations in low serum media depending upon the treatment conditions as described later.
Cell culture
The immortalized hCMEC/D3 endothelial cell line was obtained from Dr. Couraud (INSERM, Paris). These hCMEC/D3 cells (passages no. 28–30) were seeded on collagen-coated cell culture flasks or glass chamber slides (seeding density of 2.5 × 104/cm2) and maintained at 37°C with 5% CO2 exposure. Cell culture medium consisted of EBM-2 basal medium (Lonza, Walkersville, MD, USA), which was supplemented with 5% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), Fibroblast Growth Factor (Sigma Aldrich), chemically defined lipid concentrate (Life Technologies, Carlsbad, CA, USA), antibiotic/antimycotic (1:1, Atlanta Biologicals, GA, USA) and HEPES (10 mM). The culture medium was changed every other day until the cells reached confluency. Phase contrast microscopy and the expression of characteristic phenotypic markers confirmed the monolayer integrity of the hCMEC/D3 cells at confluency.
Transwell cell culture setup
Clear polyester transwell inserts (0.4 µm pore size membranes) were seeded with hCMEC/D3 cells (passage no. 28–29) on the luminal side and grown in the culture medium containing EBM-2 basal media and supplements as mentioned above. The wells were coated with collagen prior to seeding. Trans-endothelial electrical resistance (TEER) measurement and phase contrast microscopy was employed to confirm cell layer confluency and integrity.
Treatment
HCMEC/D3 cells in culture flasks, chamber slides and transwell setup were maintained overnight in media containing 1% FBS with no growth factors (referred to as low serum media). Next day the cell monolayer was exposed to fresh low serum medium of different treatment conditions containing 5.5 mM (normal/control), 35.0 mM d-glucose (HG), normal media containing 5% CSE, HG media containing 5% CSE. These concentrations are based on our previously-published reports.
Cell viability
Extracellular lactate dehydrogenase (LDH) in the media increases with plasma membrane damage. The LDH levels in the culture medium were measured after 12 and 24 h of experimental exposures to various media conditions—5.5 mM control with or without (w/wo) 5% CSE, 35.0 mM HG w/wo 5% CSE, by a colorimetric enzymatic reaction (Pierce LDH cytotoxicity assay kit, Thermo Scientific, Rockford, IL, USA) in accordance with the manufacturer guidelines.
Immunofluorescence
HCMEC/D3 cells were seeded in two-well chamber slides and grown as mentioned earlier. Cells were fixed with 16%, methanol free formaldehyde (diluted 1 in 4 in 1X PBS; from Polysciences Inc. # 18814) after specified experimental exposure duration. This was followed by three PBS washes and cell permeabilization using 0.02% Triton 100X. After another three PBS washes, fixed cells were blocked with 5% goat serum in PBS (blocking buffer) at room temperature (RT) for 45 min and incubated overnight at 4°C with primary antibodies prepared in blocking buffer. The following day, cells were incubated for 1 h at RT with Alexa Fluor® 488 or 555 conjugated goat anti-rabbit or anti-mouse antibodies or vice versa, respectively (1:1,000) after three washes with PBS. Thereafter, cells were rinsed, dried and mounted with DAPI in prolonged gold anti-fade mounting media (invitrogen). Mounted slides were examined with EVOS digital inverted fluorescence microscope after overnight drying. Cell slides stained with only secondary antibodies served as negative controls.
Western blotting
Cells were lysed using a subcellular protein fractionation kit for cultured cells (Thermo scientific, # 78840) as per manufacturer’s guidelines, such that nuclear, cytosolic and membrane fractions were collected. Protein quantification was carried out using Pierce BCA Protein Assay Kit (Thermo Scientific, # 23225). Sample preparation and the entire process was followed as described in our previous report [
25]. In brief, denatured samples (15–30 μg) were subjected to SDS-PAGE (4–15% gradient gel) and transferred to PVDF membranes for further blotting. Western blotting was used to measure the protein expression of ZO-1, Occludin, PECAM-1, ICAM-1, GLUT-1, SGLT-1, and P-gp in cell membrane fraction. Nrf-2 protein expression in cytosol vs. nuclear fraction was evaluated. Band densities were analyzed by Image Studio Lite Ver 3.1 and calculated as fold change over control protein expression.
ELISA
Cell culture media from flasks were collected after 24 h of exposure to treatment conditions (5.5 mM control w/wo 5% CSE, 35.0 mM HG w/wo 5% CSE). These cell culture supernatant samples were then analyzed by Quantikine ELISA kits (R&D systems, Minneapolis, MN, USA) for quantitative determination of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6) and interleukin-8 (IL-8), according to the manufacturer’s protocol.
BBB integrity
In order to evaluate BBB integrity, our previously-reported method was followed [
31]. In brief, a mixture of labeled dextrans in PBS (FITC ~4 kDa, 8 mg/ml; Cascade Blue ~10 kDa, 5 mg/ml; and RITC ~70 kDa, 8 mg/ml) were added to the luminal compartment of the transwells upon treatment exposure of the cells for 24 h. The media was sampled from the abluminal compartment (50 µl) and replaced with the equal volumes of fresh media to maintain appropriate sink conditions. The concentration of each fluorescent dye in the sample was determined by fluorescent measurements at their specific excitation and emission wavelengths. Media samples without dextran and that from abluminal compartments of cell free inserts with dextran added to the luminal compartment, served as references. The permeability measurements were reported as percentage of controls. In addition, we measured TEER (Ω cm
2) using EVOM 2 (World Precision Instruments, Sarasota, FL, USA), as described earlier [
40]. Cell free inserts were also evaluated for TEER for subtraction.
P-gp efflux activity
To assess the activity of P-gp, the cellular retention of P-gp substrate rhodamine-123 was measured fluorimetrically at excitation/emission wavelengths of 485/535 nm. The cells were cultured as detailed above and were treated for 24 h with 35.0 mM d-glucose (HG), normal media containing 5% CSE, HG media containing 5% CSE, apart from 5.5 mM (normal/control) exposure. At the end of 24 h, cells were washed with cold Hank’s balanced salt solution (HBSS) without calcium and magnesium (Life Technologies) and incubated on ice with rhodamine123 (10 µg/ml) in efflux buffer for 1 h. The efflux buffer consisted of 10 mM HEPES, 1% BSA in EBM-2 basal medium. Cells were subsequently washed with pre-warmed HBSS and incubated in efflux buffer at 37°C for 30 min under gentle agitation. Finally, cells were rinsed with ice-cold PBS and lysed with 0.3% triton X-100. The fluorescence of cell lysates was measured and normalized to protein content of the sample.
Statistical analysis
Data from all experiments were expressed as mean ± standard deviation (SD) and analyzed by one-way ANOVA using GraphPad Prism 6 Software Inc. (La Jolla, CA, USA). Post hoc multiple comparison tests were performed with Tukey’s test. P value <0.05 was considered statistically significant.
Discussion
Cerebrovascular pathological conditions such as stroke, Alzheimer’s, and multiple sclerosis involving a change in brain microenvironment are characteristically accompanied by BBB dysfunction [
20], thereby emphasizing the necessity of maintaining a proper barrier function and integrity to conserve the brain tissue microenvironment. As mentioned earlier, both 2DM and TS have been considered to increase the chances of developing and progressing the above-mentioned cerebrovascular pathological conditions [
1,
8‐
10]. Recent findings from our laboratory demonstrated that CSE exposure, comparable to physiological nicotine plasma concentrations of 100 ng/ml as observed in an average chronic smoker, induces BBB endothelial dysfunction and a strong inflammatory response. This included a down-regulation and redistribution of TJ protein expression, an up-regulation in expression of adhesion molecules and an increase in release of proinflammatory cytokines such as matrix metalloproteinase-2 (MMP-2) and IL-6 [
25]. On similar lines, reports from our lab and others have demonstrated an altered expression and distribution of TJ and glucose transporter proteins in BBB endothelial cell cultures following exposure to DM like altered hyperglycemic conditions (35.0 mM
d-glucose levels) [
31].
As a TJ scaffolding protein anchored to the actin cytoskeleton, ZO-1 is crucial for the regulation of inter-endothelial TJ complexes and BBB structural integrity [
42,
43]. Results from this study demonstrate a progressive down-regulation and disruption of ZO-1 expression/continuity at cell–cell contacts following 24 h exposure to HG, CSE or both, although an initial increase in expression of ZO-1 in membrane fractions was observed at 12 h under CSE conditions (Figure
2). These data further corroborate our previous studies indicating a 50% reduction in ZO-1 expression by exposure to CSE alone [
25] and a marked disruption of ZO-1 bands at cell–cell contacts by HG [
31]. In addition, diabetes-related hyperglycemia significantly suppressed ZO-1 expression in rodent BBB [
44]. Based on these findings, it is likely that HG down-regulates ZO-1 expression with an apparent increase in its cytosolic redistribution [
31]. However, we did not observe additive effects on ZO-1 reduction by HG + CSE exposure compared to either treatment alone, suggesting a saturated common pathway (Figure
2). Additionally, reduction of ZO-1 expression was accompanied by a decreased TEER across endothelial monolayers and significant increase in paracellular permeability to labeled dextrans in a size-selective manner, especially by CSE (Figure
3). This is in agreement with previous findings from our laboratory and others showing a proportionate increase in BBB permeability due to loss of TJ proteins [
25,
31,
45]. Importantly, HG aggravated the CSE-induced BBB permeability of all labeled dextrans, thus implicating an exacerbated BBB damage by smoking co-morbid with diabetes. Moreover, as shown in Figure
3, loss of BBB integrity by CSE could also be plausibly explained by an increased endothelial release of VEGF, a potent mediator of BBB disruption that was previously shown to be involved in hyperglycemia–induced BBB disruption [
31,
46]. Interestingly, our results correlate with previous findings [
31] by Sajja et al. which have also shown only a modest increase of VEGF release from hCMEC/D3 monocultures whereas a significant higher output of this growth factor was observed in hCMEC/D3 co-cultures with human astrocytes. This is an additional experimental condition we are planning to exploit in future along with direct animal experimentation to validate our findings. Further, VEGF blockade restored the BBB integrity by preventing the loss of ZO-1 and other TJ proteins [
47]. Thus, concomitant exposure to HG exacerbates CSE-induced BBB damage by potentiating VEGF release (Figure
3).
Glucose flux across the BBB is a dynamic phenomenon and is majorly mediated by facilitative and insulin-independent GLUT-1 (~55 kDa isoform) in response to the circulating glucose levels and cerebral metabolic demand [
28]. In this study, we have shown for the first time that CSE exposure alone or concomitant with HG significantly increases the membrane expression of GLUT-1 in hCMEC/D3 cultures and the increase is sustained up to 24 h treatment (Figure
4). However, we observed a time-dependent effect (delayed onset) of HG on GLUT1 expression with an overall increase at 24 h but not 12 h exposure. Previously, conflicting lines of evidence were reported for the effects of hyperglycemia on BBB GLUT1 and glucose transport [
28]. For example, experimental models of diabetes revealed a significant repression [
48] or no change [
49] in GLUT1 density and glucose transport at BBB. In this line, our recent findings have demonstrated unaltered total cellular GLUT1 (unfractionated) levels following 24 h exposure to hyperglycemic conditions in hCMEC/D3 cells [
31]. In this study, we examined the changes in GLUT1 expression in membrane fractions isolated from cells exposed to HG and/or CSE. Given the increased membrane GLUT1 and unchanged total GLUT1 expression [
31], we speculate that HG promotes the translocation and membrane deposition of cytosolic GLUT1. Additional functional studies are required to validate these findings. Nevertheless, our data shows a significant additive increase in membrane GLUT1 expression following combined exposure to HG and CSE, which would contribute to elevated levels of oxidative stress in BBB endothelium.
Additionally, our results indicate a significant and transient increase of SGLT-1 in hCMEC/D3 membranes following CSE alone or in combination with HG (Figure
5). As previously reported [
31], prolonged exposure to HG (24 h) did not elicit significant changes in SGLT-1 expression. This is also in line with current findings showing that membrane expression of SGLT-1 return to baseline levels at 24 h following a transient increased at 12 h. Although the relative contribution of SGLT-1 to glucose transport at the BBB is currently unknown, it was previously shown that BBB SGLT-1 expression could be induced under pathophysiological conditions such as stroke [
50] and hypoglycemia [
31]. Therefore, it is likely that CSE-induced increase in SGLT-1 expression (12 h) in presence of HG could be mediated by an acute stressful response in hCMEC/D3 cells.
Diabetes-related hyperglycemia was previously shown to attenuate the expression of functional P-gp in rodent brain, thereby altering the CNS distribution of its substrates [
51‐
53]. By contrast, other studies demonstrated a lack of effect [
38] or a dramatic increase in P-gp expression [
37] following hyperglycemia in mouse brain endothelial cells. Recently, we have shown that acute and chronic hyperglycemia differentially influences functional expression of BBB efflux transporters, including P-gp where repeated hyperglycemic stimuli over 3 days caused a significant increase in its efflux activity [
54]. However, the effects of CSE alone and in the presence of HG on transporter activity of P-gp, remain unknown. Our data indicated a sustained up-regulation of membrane-bound P-gp expression by HG or CSE exposure following 24 h acute exposure although, no additive effects were observed under combined exposure conditions (Figure
5). Interestingly, P-gp activity (as measured by rhodamine123 efflux) was increased only by HG + CSE co-exposure at 24 h (as opposed to repetitive hits of HG exposure over several days as reported by Sajja et al.), suggesting a possible synergistic effect and an increased potential for altered CNS drug disposition of P-gp substrates in diabetic smokers [
54]. It is also possible that repetitive stimuli [
54] will further enhance this effect which we plan to test in future experiments. Given the extensive characterization of hCMEC/D3 cell line for its applicability to human BBB drug transport studies and its broad utility for understanding the molecular regulation of BBB efflux transporters in vivo [
39,
55,
56], the findings of our study may hold clinical significance. However, in vivo validation studies will be necessary to confirm our results.
Existing evidence associates the role of inflammation and oxidative stress with the development of various CNS disorders in diabetes as well as chronic smoking. Previously we have shown that hyperglycemia induces transient up-regulation of inflammatory cell adhesion molecules such as VCAM-1 [
31]. Similarly in a separate study we observed a moderate increase in the expression level of PECAM-1 and IL-8 following exposure to CSE from 3R4F cigarettes [
25]. Herein we observed a significant increase of PECAM-1 in the co-exposure conditions suggesting an additive effect of HG and CSE. By contrast ICAM-1 was upregulated in all the conditions tested when compared to controls but no additive effect was observed. These results suggest the possibility for leukocyte-endothelial interactions eventually facilitating transmigration of circulating white blood cells (WBC) across the BBB [
57]. This warrants further study in vivo to validate this hypothesis. In addition to vascular endothelial molecules, HG potentiated the release of IL-8 by CSE also strengthening the hypothesis for an additive inflammatory effect between HG and CSE prodromal to a possible neuroinflammatory disorder [
58].
Nrf2 is master regulator and redox-sensitive transcription factor that has a pleiotropic role in mediating cellular antioxidant responses [
59]. In fact we have recently shown the neuroprotective role of Nrf2 in regulating and maintaining BBB integrity [
60]. As previously reported by our group hyperglycemia does not overall affect the total expression level of Nrf2 [
31], however, HG modulates its distribution in the cells when we analyzed and compared the nuclear vs. cytosolic content. As shown in Figure
6d CSE exposure markedly elevated the nuclear/cytoplasmic ratio of Nrf2 suggesting activation of the antioxidant pathways. This effect was further enhanced by HG despite eliciting nuclear translocation of Nrf2 to a lesser extent when compared to CSE or HG + CSE. This latter implies the existence of a cooperative effect in term of cellular oxidative response activation. This in the long term can be more detrimental to BBB integrity if the oxidative stimuli overcome the protective antioxidant mechanisms of the BBB endothelial cells.