BACKGROUND
tobacco smoke  is a major public health hazard, accounting for more than  <dig>  million premature deaths worldwide and over  <dig>  deaths each year in the united states alone  <cit> .
in addition to the onset of various forms of cancer  <cit> , 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   <cit> , stroke  <cit>  due to the pro-coagulant and atherogenic effects of smoking  <cit>  and cerebral aneurysms  <cit> .
there is also a strong correlation between smoking and an increased risk for multiple sclerosis  <cit> , alzheimer’s disease, small vessel ischemic disease  and neurodevelopmental damage during pregnancy  <cit> .
although it is possible to explain some of the neuropathological effects of ts with nicotine specific pathways  <cit> , 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  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  <cit> .
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  <cit> .
mainstream ts contains over  <dig> chemical compounds including a harmful cloud of free radicals and other reactive oxygen  and nitrogen species  contained in both the gaseous phase and the tar  <cit> .
at the vascular level free radicals can lead to oxidative damage of endothelial cells  <cit>  involving dna strand breakage and inflammation  <cit> .
active and passive tobacco smoking can spawn these highly reactive oxygen species , nitrogen dioxide, peroxynitrite   <cit> ) 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  <cit> ; 2) antioxidant supplementation reduces the oxidation and inflammation induced by ts in animals and cells  <cit> ; 3) ts contributes to a pro-atherosclerotic environment by triggering a complex pro-inflammatory response and mediates the recruitment of leukocytes  <cit>  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  on bbb endothelium in vitro, using a well characterized human bbb endothelial cell line  positively correlate with the total content of tar of various tobacco products and associated oxidative stress  rather than nicotine content.
RESULTS
exposure to nicotine concentrations equivalent to that observed in plasma in chronic human smoker does not affect endothelial cell viability
hplc studies were performed to determine the dilution factor for freshly prepared 3r4f cigarette-derived cse stock solution necessary to achieve cse exposure yielding 100 ng/ml of nicotine .
this nicotine concentration was chosen to model the plasma levels seen in human smokers  <cit> .
3r4f cigarette was used as a reference to calculate the dilution factor for the cse stock which was then uniformly applied to all the test cigarettes.
as shown in figure 1b, 100 ng/ml of nicotine did not affect the cell viability at  <dig> and 48 h exposure.
cytotoxic effects of nicotine exposure were observed at higher concentrations .
note also that 24 h exposure to 5% diluted cse from test cigarettes did not affect endothelial viability with the exception of nf-derived extracts .
a small yet significant decrease in cell viability was observed in response to nf-derived cse exposure, as compared to controls .
nitrate and nitrite levels in cse correlates with corresponding cigarette’s tar and nicotine content
a generation of highly carcinogenic tobacco-specific nitrosamines   <cit>  has been suggested to arise from the reaction of amines with nitrite derived from nitrate in the tobacco  <cit> .
we measured nitrate, no3−/nitrite andno2− content of cse derived from 1r5f , 3r4f , ultralow nicotine and nf  cigarettes .
in addition, commercially available marlboro light, medium and full cigarettes were also analyzed for comparison.
no3−/no2− content in cse from ultralow nicotine cigarettes was significantly higher than any other brand tested including 3r4f , as observed with marlboro full or medium cigarettes .
in contrast, no3−/no2− content of nf cigarette was significantly lower  when compared to “light” cigarettes .
a positive correlation between no3−/no2− content of cse and tar for the corresponding cigarette brands was also observed as demonstrated by the regression analysis shown in figure 2b.
importantly, the ultralow nicotine brand stands out in terms of no3−/no2− content when compared to other brands containing a similar amount of tar.
no3−/no2− positively correlate with the nicotine content of corresponding cigarette types with an exception of the ultralow nicotine brand .
results from the regression analyses  suggests that an alteration of the tobacco product to reduce the nicotine content, such as in ultralow nicotine and nf brands, could be responsible for the higher output of no3−/no2− during cigarette combustion.
release of hydrogen peroxide  in cse increases with the tar content of cigarettes and leads to progressive oxidative damage in bbb ecs
ts is a major exogenous source of free radicals contained in both gaseous phase and tar, which can spawn sustained high levels of ros  that may directly affect the bbb integrity.
thus, we determined the amount of h2o <dig> release in the cse and assessed its oxidative effect on bbb endothelial cell cultures.
as shown in figure 3a <dig>  the highest levels of h2o <dig> were found in cse from ultralow nicotine and nf  cigarettes when compared to light products .
h2o <dig> content in cse from 3r4f  and marlboro medium/full cigarettes, although statistically higher that light cigarettes, was considerably less compared with ultralow nicotine and nf products.
this was in accordance with measurements of cellular oxidative stress  and revealed the highest level of cellular oxidation in endothelial cultures chronically exposed  to ultralow nicotine and nf smoke extracts.
interestingly, 3r4f cigarettes released lower amounts of h2o <dig> than ultralow nicotine and nf cigarettes which also demonstrated a comparable oxidative stress potential in endothelial cells.
furthermore, regression analysis of h2o <dig> content revealed a direct relationship between tar content of the corresponding cigarette brand  and h2o <dig>  a positive correlation between h2o <dig> and nicotine content was also observed but only with the exclusion of nf and ultralow cigarettes from the analytical pool .
exposure to cse from 3r4f, nf and ultralow nicotine cigarettes negatively impacts zo- <dig> and occludin expression/distribution as well as bbb integrity
immunofluorescence analysis of bbb endothelial confluent monolayers revealed a significant loss of zo- <dig> at cell-cell junctions following exposure to cse from 3r4f, ultralow and nf cigarettes and to a lesser extent in 1r5f treated cultures .
results were further confirmed by western blot  analysis of the corresponding membrane fractions  in which exposure to ultralow nicotine cse caused the most significant reduction of zo- <dig> expression at the membrane level .
however, actin distribution and expression were not altered by cse exposure when compared to controls.
parallel immunofluorescence analyses also revealed a similar down-regulation and altered pattern of distribution at cell-cell junction for occludin .
results were also confirmed by wb analysis of occludin expression in the corresponding membrane fractions .
expression levels of occludin were severely impaired in endothelial cultures exposed to cse from 3r4f cigarette.
the effect was even more significant in culture exposed to nf and ultralow nicotine smoke extracts .
note also that a modest  alteration in the distribution and expression level of occludin was observed in cell cultures treated with 1r5f-derived cse when compared to controls.
as expected, alteration in tj expression/distribution impacted bbb integrity as demonstrated by permeability measurements to dextran molecules .
increased permeability to 70 kda dextran was noted in transwells exposed to ts extracts from nf, ultralow and 3r4f although results were deemed significant  only for the last two conditions.
on the other hand, permeability to lower molecular weight dextrans  was significantly increased for all the three conditions mentioned above.
further, immunofluorescence analysis revealed a marked up-regulation of ve-cadherin at cell-cell contacts in response to cse exposure from 3r4f, nf and ultralow nicotine .
results were confirmed by parallel wb analysis of corresponding membrane fractions  demonstrating a statistically significant  increase in ve-cadherin expression following exposure to nf and ultralow nicotine derived cse, compared to controls and cultures exposed to 1r5f.
a noticeable  increase in ve-cadherin expression was also observed in endothelial cultures treated with 3r4f-derived cse.
similarly, claudin- <dig> expression was up-regulated by exposure to cse derived from 3r4f, ultralow nicotine and nf cigarettes, as demonstrated by immunofluorescence and wb analyses of corresponding membrane fractions .
interestingly, the patterns/level of expression of these junction proteins remarkably reflects the oxidative stress/h2o <dig> content of the cse extracts of the respective brands .
exposure to cse from 3r4f, nf and ultralow nicotine cigarettes promotes the pro-inflammatory activation of bbb endothelial cells
as shown in figure 6a, endothelial cell expression of vascular endothelial adhesion molecule- <dig>  was up-regulated following exposure to 3r4f, nf and ultralow nicotine cses, as compared to controls and 1r5f cigarette treated cultures.
however, no expression changes were observed with respect to e-selectin.
in addition, immunofluorescence analysis indicated an up-regulation of endothelial platelet endothelial cell adhesion molecule- <dig>  expression following exposure to 3r4f, nf and ultralow nicotine cigarettes .
these results were further supported by wb analysis of the corresponding membrane fractions .
importantly, analysis of the culture conditioned media by elisa revealed a significant increase of interleukin- <dig>  release from the endothelial cells exposed to either nf  or ultralow nicotine cigarette extracts , compared to controls .
a modest, yet significant increase in the release of matrix metalloproteinase- <dig>  was also observed in cultures treated with either 3r4f or nf smoke extracts, but not ultralow nicotine .
however, mmp- <dig>  il-1β and tnf-α levels in the conditioned media from all treatment conditions were below the reading sensitivity .
discussion
ros, despite being essential for biological systems  <cit>  have the potential to cause extensive oxidative damage to cells and tissues if their levels become excessive  <cit> .
at the vascular level ros can cause oxidative damage of endothelial cells  <cit>  including dna strand breakage and inflammation  <cit> .
in addition to ros, nicotine can equally elicit oxidative stress and tissue injury  <cit>  and has been shown to exacerbate brain edema following focal ischemia  <cit> .
oxidants in the gaseous phase of cigarette smoke, including nicotine and various ros species,  and facilitate the pathogenesis and progression of neurological disorders  <cit> .
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  <cit> .
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  of tobacco smoke from 1r5f , 3r4f , nf  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  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 .
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  or replacement with alternative products  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 , and non-volatile tobacco-specific nitrosamines  such as 4--1--1-butanone  which have been associated with carcinogenicity of tobacco smoke  <cit> .
interestingly, h2o <dig> content measured in ultralow nicotine and tobacco free  cigarettes, considered “reduced-exposure” products, was significantly higher than any other brand including medium and full flavor .
regression analysis of h2o <dig> 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  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 h2o <dig>  this can be attributed to the higher content of nicotine in 3r4f cigarettes since nicotine equally contributes to oxidative stress .
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 h2o <dig> output measured in these “denicotinized” cigarette products.
previous reports by hossain and co-workers  <cit>  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  bbb endothelial zo- <dig> 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- <dig> is a cytoplasmic accessory protein which plays a crucial role in bbb integrity by connecting transmembrane proteins  to cytoskeletal proteins and is actively involved in signal transduction and transcriptional modulation  <cit> .
interestingly, the effect of cse on zo- <dig> expression/distribution reflects the overall oxidative potential of the corresponding cigarettes , thus suggesting a correlation between ts-dependent oxidative potential and dysregulation of tjs and bbb integrity.
zo- <dig> tj protein closely associates with the actin cytoskeletal network.
when we observed the actin structure with respect to zo- <dig>  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- <dig>  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 3rf <dig>  nf and ultralow nicotine cigarettes.
this can be a reflection of the parallel loss of zo- <dig> which provides a positioning system and anchoring scaffold for the transmembrane tj proteins.
in contrast to zo- <dig> and occludin, the expression of ve-cadherin and claudin- <dig> was proportionally increased with respect to the oxidative potential of the corresponding cse treatment.
in fact, as shown in figure  <dig>  ve-cadherin membrane expression was progressively up-regulated by exposure to 3rf <dig>  nf and ultralow nicotine cigarettes, although statistical significance was proven only for the last two cigarette products.
in parallel, claudin- <dig> membrane expression was similarly up-regulated .
this is in agreement with emerging evidences suggesting that ve-cadherin controls claudin- <dig> expression by preventing the nuclear accumulation of foxo <dig> and beta-catenin which repress the claudin- <dig> promoter  <cit>  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  <cit>  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- <dig> activation  <cit> , whose expression level was also increased .
note also that an up-regulation of claudin- <dig>  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- <dig> is of critical importance for the positioning and interaction of tj proteins with their homologues on adjacent endothelial cells.
moreover, although claudin- <dig> was up-regulated  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- <dig> expression was observed as well as an increased endothelial release of il- <dig> and mmp- <dig> .
regarding mmp- <dig>  previous reports by others have shown how ros regulate the activity of vascular matrix metalloproteinases in vitro including mmp- <dig> and mmp- <dig>  <cit>  which have an implication in atherosclerotic plaque stability.
expression and activation of mmp- <dig> has been demonstrated as a key event in oxidative stress injury to heart  <cit>  and hyperglycaemia promoted bbb dysfunction  <cit> .
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.
CONCLUSIONS
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, h2o <dig> 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.

 tobacco smoke is a major public health hazard, accounting for more than million premature deaths worldwide and over 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, stroke due to the pro-coagulant and atherogenic effects of smoking and cerebral aneurysms. there is also a strong correlation between smoking and an increased risk for multiple sclerosis, alzheimer’s disease, small vessel ischemic disease 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 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. mainstream ts contains over chemical compounds including a harmful cloud of free radicals and other reactive oxygen and nitrogen species 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. active and passive tobacco smoking can spawn these highly reactive oxygen species, nitrogen dioxide, peroxynitrite ) 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; 2) antioxidant supplementation reduces the oxidation and inflammation induced by ts in animals and cells; 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 on bbb endothelium in vitro, using a well characterized human bbb endothelial cell line positively correlate with the total content of tar of various tobacco products and associated oxidative stress rather than nicotine content.  exposure to nicotine concentrations equivalent to that observed in plasma in chronic human smoker does not affect endothelial cell viability hplc studies were performed to determine the dilution factor for freshly prepared 3r4f cigarette-derived cse stock solution necessary to achieve cse exposure yielding 100 ng/ml of nicotine. this nicotine concentration was chosen to model the plasma levels seen in human smokers. 3r4f cigarette was used as a reference to calculate the dilution factor for the cse stock which was then uniformly applied to all the test cigarettes. as shown in figure 1b, 100 ng/ml of nicotine did not affect the cell viability at and 48 h exposure. cytotoxic effects of nicotine exposure were observed at higher concentrations. note also that 24 h exposure to 5% diluted cse from test cigarettes did not affect endothelial viability with the exception of nf-derived extracts. a small yet significant decrease in cell viability was observed in response to nf-derived cse exposure, as compared to controls. nitrate and nitrite levels in cse correlates with corresponding cigarette’s tar and nicotine content a generation of highly carcinogenic tobacco-specific nitrosamines has been suggested to arise from the reaction of amines with nitrite derived from nitrate in the tobacco. we measured nitrate, no3−/nitrite andno2− content of cse derived from 1r5f, 3r4f, ultralow nicotine and nf cigarettes. in addition, commercially available marlboro light, medium and full cigarettes were also analyzed for comparison. no3−/no2− content in cse from ultralow nicotine cigarettes was significantly higher than any other brand tested including 3r4f, as observed with marlboro full or medium cigarettes. in contrast, no3−/no2− content of nf cigarette was significantly lower when compared to “light” cigarettes. a positive correlation between no3−/no2− content of cse and tar for the corresponding cigarette brands was also observed as demonstrated by the regression analysis shown in figure 2b. importantly, the ultralow nicotine brand stands out in terms of no3−/no2− content when compared to other brands containing a similar amount of tar. no3−/no2− positively correlate with the nicotine content of corresponding cigarette types with an exception of the ultralow nicotine brand. results from the regression analyses suggests that an alteration of the tobacco product to reduce the nicotine content, such as in ultralow nicotine and nf brands, could be responsible for the higher output of no3−/no2− during cigarette combustion. release of hydrogen peroxide in cse increases with the tar content of cigarettes and leads to progressive oxidative damage in bbb ecs ts is a major exogenous source of free radicals contained in both gaseous phase and tar, which can spawn sustained high levels of ros that may directly affect the bbb integrity. thus, we determined the amount of h2o release in the cse and assessed its oxidative effect on bbb endothelial cell cultures. as shown in figure 3a the highest levels of h2o were found in cse from ultralow nicotine and nf cigarettes when compared to light products. h2o content in cse from 3r4f and marlboro medium/full cigarettes, although statistically higher that light cigarettes, was considerably less compared with ultralow nicotine and nf products. this was in accordance with measurements of cellular oxidative stress and revealed the highest level of cellular oxidation in endothelial cultures chronically exposed to ultralow nicotine and nf smoke extracts. interestingly, 3r4f cigarettes released lower amounts of h2o than ultralow nicotine and nf cigarettes which also demonstrated a comparable oxidative stress potential in endothelial cells. furthermore, regression analysis of h2o content revealed a direct relationship between tar content of the corresponding cigarette brand and h2o a positive correlation between h2o and nicotine content was also observed but only with the exclusion of nf and ultralow cigarettes from the analytical pool. exposure to cse from 3r4f, nf and ultralow nicotine cigarettes negatively impacts zo- and occludin expression/distribution as well as bbb integrity immunofluorescence analysis of bbb endothelial confluent monolayers revealed a significant loss of zo- at cell-cell junctions following exposure to cse from 3r4f, ultralow and nf cigarettes and to a lesser extent in 1r5f treated cultures. results were further confirmed by western blot analysis of the corresponding membrane fractions in which exposure to ultralow nicotine cse caused the most significant reduction of zo- expression at the membrane level. however, actin distribution and expression were not altered by cse exposure when compared to controls. parallel immunofluorescence analyses also revealed a similar down-regulation and altered pattern of distribution at cell-cell junction for occludin. results were also confirmed by wb analysis of occludin expression in the corresponding membrane fractions. expression levels of occludin were severely impaired in endothelial cultures exposed to cse from 3r4f cigarette. the effect was even more significant in culture exposed to nf and ultralow nicotine smoke extracts. note also that a modest alteration in the distribution and expression level of occludin was observed in cell cultures treated with 1r5f-derived cse when compared to controls. as expected, alteration in tj expression/distribution impacted bbb integrity as demonstrated by permeability measurements to dextran molecules. increased permeability to 70 kda dextran was noted in transwells exposed to ts extracts from nf, ultralow and 3r4f although results were deemed significant only for the last two conditions. on the other hand, permeability to lower molecular weight dextrans was significantly increased for all the three conditions mentioned above. further, immunofluorescence analysis revealed a marked up-regulation of ve-cadherin at cell-cell contacts in response to cse exposure from 3r4f, nf and ultralow nicotine. results were confirmed by parallel wb analysis of corresponding membrane fractions demonstrating a statistically significant increase in ve-cadherin expression following exposure to nf and ultralow nicotine derived cse, compared to controls and cultures exposed to 1r5f. a noticeable increase in ve-cadherin expression was also observed in endothelial cultures treated with 3r4f-derived cse. similarly, claudin- expression was up-regulated by exposure to cse derived from 3r4f, ultralow nicotine and nf cigarettes, as demonstrated by immunofluorescence and wb analyses of corresponding membrane fractions. interestingly, the patterns/level of expression of these junction proteins remarkably reflects the oxidative stress/h2o content of the cse extracts of the respective brands. exposure to cse from 3r4f, nf and ultralow nicotine cigarettes promotes the pro-inflammatory activation of bbb endothelial cells as shown in figure 6a, endothelial cell expression of vascular endothelial adhesion molecule- was up-regulated following exposure to 3r4f, nf and ultralow nicotine cses, as compared to controls and 1r5f cigarette treated cultures. however, no expression changes were observed with respect to e-selectin. in addition, immunofluorescence analysis indicated an up-regulation of endothelial platelet endothelial cell adhesion molecule- expression following exposure to 3r4f, nf and ultralow nicotine cigarettes. these results were further supported by wb analysis of the corresponding membrane fractions. importantly, analysis of the culture conditioned media by elisa revealed a significant increase of interleukin- release from the endothelial cells exposed to either nf or ultralow nicotine cigarette extracts, compared to controls. a modest, yet significant increase in the release of matrix metalloproteinase- was also observed in cultures treated with either 3r4f or nf smoke extracts, but not ultralow nicotine. however, mmp- il-1β and tnf-α levels in the conditioned media from all treatment conditions were below the reading sensitivity. discussion ros, despite being essential for biological systems have the potential to cause extensive oxidative damage to cells and tissues if their levels become excessive. 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 and has been shown to exacerbate brain edema following focal ischemia. oxidants in the gaseous phase of cigarette smoke, including nicotine and various ros species, and facilitate the pathogenesis and progression of neurological disorders. 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. 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 of tobacco smoke from 1r5f, 3r4f, nf 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 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. 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 or replacement with alternative products 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, and non-volatile tobacco-specific nitrosamines such as 4--1--1-butanone which have been associated with carcinogenicity of tobacco smoke. interestingly, h2o content measured in ultralow nicotine and tobacco free cigarettes, considered “reduced-exposure” products, was significantly higher than any other brand including medium and full flavor. regression analysis of h2o 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 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 h2o this can be attributed to the higher content of nicotine in 3r4f cigarettes since nicotine equally contributes to oxidative stress. 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 h2o 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 bbb endothelial zo- 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- is a cytoplasmic accessory protein which plays a crucial role in bbb integrity by connecting transmembrane proteins to cytoskeletal proteins and is actively involved in signal transduction and transcriptional modulation. interestingly, the effect of cse on zo- expression/distribution reflects the overall oxidative potential of the corresponding cigarettes, thus suggesting a correlation between ts-dependent oxidative potential and dysregulation of tjs and bbb integrity. zo- tj protein closely associates with the actin cytoskeletal network. when we observed the actin structure with respect to zo- 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- 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 3rf nf and ultralow nicotine cigarettes. this can be a reflection of the parallel loss of zo- which provides a positioning system and anchoring scaffold for the transmembrane tj proteins. in contrast to zo- and occludin, the expression of ve-cadherin and claudin- was proportionally increased with respect to the oxidative potential of the corresponding cse treatment. in fact, as shown in figure  ve-cadherin membrane expression was progressively up-regulated by exposure to 3rf nf and ultralow nicotine cigarettes, although statistical significance was proven only for the last two cigarette products. in parallel, claudin- membrane expression was similarly up-regulated. this is in agreement with emerging evidences suggesting that ve-cadherin controls claudin- expression by preventing the nuclear accumulation of foxo and beta-catenin which repress the claudin- 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- activation, whose expression level was also increased. note also that an up-regulation of claudin- 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- is of critical importance for the positioning and interaction of tj proteins with their homologues on adjacent endothelial cells. moreover, although claudin- was up-regulated 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- expression was observed as well as an increased endothelial release of il- and mmp-. regarding mmp- previous reports by others have shown how ros regulate the activity of vascular matrix metalloproteinases in vitro including mmp- and mmp- which have an implication in atherosclerotic plaque stability. expression and activation of mmp- 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, h2o 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. 
in parallel, membrane expression of endothelial tight junction proteins zo- and occludin were significantly down-regulated suggesting the impairment of barrier function. in summary, our results indicate that nf and ultralow nicotine cigarettes are potentially more harmful to the bbb endothelium than regular tobacco products. initial profiling of ts showed a significant release of reactive oxygen and reactive nitrogen species in full flavor, nicotine-free and ultralow nicotine 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. both active and passive tobacco smoke potentially impair the vascular endothelial function in a causative and dose-dependent manner, largely related to the content of reactive oxygen species, nicotine, and pro-inflammatory activity. ts extract from these cigarettes also induced an inflammatory response in bbb ecs as demonstrated by increased il- and mmp- levels and up-regulation of vascular adhesion molecules, such as vcam- and pecam- together these factors can compromise the restrictive properties of the blood–brain barrier and trigger the pathogenesis/progression of several neurological disorders including silent cerebral infarction, stroke, multiple sclerosis and alzheimer’s disease.
 both active and passive tobacco smoke potentially impair the vascular endothelial function in a causative and dose-dependent manner, largely related to the content of reactive oxygen species, nicotine, and pro-inflammatory activity. together these factors can compromise the restrictive properties of the blood–brain barrier 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 on brain microvascular endothelial cell line, a well characterized human bbb model.  initial profiling of ts showed a significant release of reactive oxygen and reactive nitrogen species in full flavor, nicotine-free and ultralow nicotine products. this release correlated with increased oxidative cell damage. in parallel, membrane expression of endothelial tight junction proteins zo- and occludin were significantly down-regulated suggesting the impairment of barrier function. expression of ve-cadherin and claudin- 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- and mmp- levels and up-regulation of vascular adhesion molecules, such as vcam- and pecam-  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. tobaccoin vitrosmokingoxidative stressblood–brain barrierinflammationnicotinepermeabilitynicotine freeultralow nicotine, alternative 