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Cell Biology International (2008) 32, 198209 (Printed in Great Britain)
Effect of nicotine and polyaromtic hydrocarbons on cerebral endothelial cells
Pilaiwanwadee Hutamekalinab, Attila E. Farkasa, Anna Orbóka, Imola Wilhelma, Péter Nagyőszia, Szilvia Veszelkaa, Mária A. Delia, Krisztina Buzásc, Éva Hunyadi‑Gulyásc, Katalin F. Medzihradszkycd, Duangdeun Meksuriyenb and István A. Krizbaia*
aInstitute of Biophysics, Biological Research Centre, Temesvári krt. 62, 6726 Szeged, Hungary
bDepartment of Biochemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Phayathai Road, Pathumwan, Bangkok 10330, Thailand
cProteomics Research Group, Biological Research Centre, Temesvári krt. 62, 6726 Szeged, Hungary
dDepartment of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94143-0446, USA
The present study was designed to investigate the effect of nicotine and polyaromatic hydrocarbon compounds on cerebral endothelial cells (CECs). Nicotine treatments from 15
Keywords: Nicotine, Blood–brain barrier, Cerebral endothelial cells, Polyaromatic hydrocarbons, Tight junction, Proteomics.
*Corresponding author. Tel.: +36 62 599 602; fax: +36 62 433 133.
Tobacco-related diseases are among the world's leading preventable causes of death affecting not only active but passive smokers as well. Cigarette smoke is a complex mixture of different chemicals including nicotine and polyaromatic hydrocarbons (PAHs). The high number of patients affected by neurological disorders related to tobacco use suggests that one of the main targets of tobacco smoke components is the central nervous system (CNS) including the blood–brain barrier (BBB). Despite the high incidence of stroke among smokers and the role of BBB in stroke, little is known about the direct effects of smoking on the BBB. One of the most controversial issues is to which extent nicotine per se is involved in the development of cerebrovascular diseases and what the role of other components of cigarette smoke is.
The cerebral endothelium is a single-cell layer lining the blood vessels of the brain and constitutes the principal component of the BBB. By forming an active interface between blood and neuronal tissue it plays a key role in the maintenance of the homeostasis of CNS. The morphological basis of the endothelial barrier forming function is constituted by the interendothelial junctions: tight junctions (TJ) and adherens junctions (AJ). In this respect the transmembrane proteins of the junctional complexes may have a special importance. These include occludin (Furuse et al., 1993), the claudin family (Furuse et al., 1998) and the junctional adhesion molecules (Martin-Padura et al., 1998). Occludin binds to the members of the zonula occludens protein family (ZO-1, ZO-2, ZO-3) and cingulin which connect the junctional complex to the cytoskeleton. The transmembrane proteins of the adherens junctions are the cadherins, in the case of vascular endothelial cells VE-cadherin which is linked through the catenins (α, β, γ) to the cytoskeleton. A proper function of the adherens junction is needed for tight junction formation, blockade of cadherin with peptides directed against the extracellular region of cadherin causes an increase in BBB permeability (Pal et al., 1997).
The direct effect of nicotine on endothelial cells has been investigated in several studies (Abbruscato et al., 2002; Conklin et al., 2002; Cucina et al., 2000). The majority of these studies have been performed on endothelial cells of non-cerebral origin, showing that endothelial cells play a crucial role in the development of vascular diseases associated with cigarette smoking. Respecting endothelial barrier function it is of special importance that nicotine is able to reorganize the cytoskeleton of bovine aortic endothelial cells (Cucina et al., 2000) and that nicotine induced upregulation of vascular endothelial growth factor expression in porcine aortic endothelial cells (Conklin et al., 2002). It has also been shown that nicotine increases plasminogen activator inhibitor-1 production by cerebral endothelial cells via a PKC dependent pathway (Zidovetzki et al., 1999) and that nicotine is able to downregulate alpha 2 isoform of Na,K-ATPase at the BBB in rats, which is an important mediator of ion homeostasis in the brain (Wang et al., 1994). Furthermore, an increase in permeability and decrease in ZO-1 expression has been recently demonstrated (Abbruscato et al., 2002; Hawkins et al., 2004).
Besides nicotine, polyaromatic hydrocarbons may play an important role in endothelial damage. PAHs are present at high concentrations in cigarette smoke (1-methylanthracene: 1500
Previous experimental data suggested that smoking may influence BBB function. The present study was designed to clarify the structural and functional alterations at interendothelial junction (TJ and AJ) sites elicited by nicotine and PAHs and to identify proteins which may be involved in the mediation of the effect of cigarette smoke components on CECs.
2 Materials and methods
2.1 Chemicals and antibodies
All chemicals, unless otherwise stated, were purchased from Sigma (Budapest, Hungary). 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) was from Alexis, protein G sepharose was from Amersham-Pharmacia Biotech. The following antibodies were used: anti-occludin, claudin-5, ZO-1, ZO-2 (Zymed, San Francisco, USA), anti-pan-cadherin, α-catenin, β-catenin (Sigma), Cy3 labelled anti-rabbit (Jackson). Fine chemicals for 2D electrophoresis were purchased from BioRad.
2.2 Cell culture
Rat brain endothelial cells were isolated from 2-week old rats. Brains were cut into small pieces and digested in two steps with collagenase type II (Sigma) and collagenase/dispase (Roche), followed by centrifugation on percoll gradient. The microvessel fragments were plated on collagen type IV and fibronectin coated dishes (Orange), glass coverslips or corneal extracellular matrix coated cell culture inserts (Transwell clear, 1
For co-culture primary cultures of glial cells were prepared from two-day old Wistar rats. After removing the meninges, cortices were mechanically dissociated in DMEM (Sigma) containing 10% fetal bovine serum. The cells were plated on poly-
2.3 Preparation of cell extracts and western-blot
Confluent monolayers of brain endothelial cells were treated with different concentrations of nicotine, phenanthrene and methylanthracene. The highest concentrations used (nicotine: 10−5
For the preparation of Triton X-100 soluble fraction, cells were scraped into lysis buffer (20
For immunoprecipitation, cells were homogenized as described above in 500
For immunofluorescent studies cells were cultured on collagen/fibronectin coated coverslips. Cells were fixed using a mixture of ice cold ethanol:acetic acid (95:5) for 10
2.6 Transendothelial electric resistance (TEER)
In order to obtain a better BBB model, endothelial cells were co-cultured with astrocytes which is a widely accepted model of the BBB (Dehouck et al., 1990). Brain endothelial cells were cultured on cell culture inserts in plates containing astroglia at the bottom of the wells with endothelial culture medium in both compartments. TEER was measured using a chopstick electrode and an EVOM epithelial voltohmmeter (World Precision Instruments). The resistance of the coated filter was substracted from the measured value to obtain the resistance of the endothelial monolayer itself.
2.7 Statistical analysis
All TEER data presented are means
2.8 2D electrophoresis
Primary cultures of rat brain endothelial cells were treated with 10−5
2.9 Protein identification by mass spectrometry
Proteins were in-gel digested by side-chain protected porcine trypsin according to a published protocol (http://donatello.ucsf.edu/ingel.html). The unfractionated digests were analyzed on a Bruker Reflex III MALDI-TOF mass spectrometer, using 2,5-dihydroxy-benzoic acid as the matrix. Database searches were performed with the monoisotopic MH+ lists against the NCBI protein database using the MS-Fit program of ProteinProspector. (http://prospector.ucsf.edu). Protein IDs were confirmed by MS/MS (Post-source decay) analysis.
2.10 Induction of oxidative stress
To investigate the additional effects of oxidative stress, nicotine treatment was combined with DMNQ (10−5
3.1 Expression of junctional proteins
Treatment of cerebral endothelial cells with nicotine (10−8, 10−7, 10−6 and 10−5
Expression of occludin in response to nicotine. Cells were treated with 10−8–10−4
Expressional changes of ZO-1 and cadherin in response to nicotine. Cells were treated with 10−6–10−5
Treatment with 3
Expression of junctional proteins in response to 3
Effect of phenanthrene (Ph) on the interaction of β-catenin with α-catenin and cadherin. CECs were treated with 3
3.2 Localization of junctional proteins by immunofluorescence
Immunofluorescent staining of primary RBEC cultures confirmed the results of western-blot analysis. The most pronounced decrease in response to 24
Immunofluorescent localization of junctional proteins in response to nicotine treatment. Cells were treated with 10−5
Treatment with 3
Distribution of junctional proteins in response to phenanthrene. Cells were treated with 3
Localization of junctional proteins after a combined nicotine and phenanthrene treatment was comparable to that seen after nicotine treatment alone. After the combined treatment we observed a marked decrease in ZO-1 staining intensity and continuity, while ZO-2 and occludin staining showed a slight disruption of continuity (Fig. 7).
Redistribution of junctional proteins in response to combined nicotine and phenantrene treatment. Cells were treated with 3
3.3 Assessment of the barrier function by the measurement of the transendothelial electrical resistance
For TEER measurements, CECs were co-cultured with astrocytes on Transwell filters coated with collagen IV/fibronectin and supplemented with physiological concentrations of hydrocortisone and CPT-cAMP. With this protocol we could achieve average TEER values of 200–300
The CECs were treated with 10−5
Effect of nicotine (A, 10−5
3.4 Cumulative effect of nicotine treatment and oxidative stress
Cigarette smoke contains oxidants that remain stable in aqueous solution and can penetrate into the blood through the lung alveolar wall (Yamaguchi et al., 2007). Thus the effect of nicotine is often combined with oxidative stress in smokers. Since oxidative stress is known to impair BBB function (Krizbai et al., 2005), a study of the combined effect of nicotine and oxidative stress could lead to a better understanding of the damaging effect of smoking on the BBB. Combination of 24
Effect of nicotine (10−5
Effect of nicotine and oxidative stress on the localization of ZO-1. Cells were treated with 10−5
3.5 Proteomic analysis
Primary cultures of rat brain endothelial cells treated with 10−5
Differentially expressed proteins in cerebral endothelial cells following nicotine treatment
Differentially expressed proteins in cerebral endothelial cells following phenanthrene treatment
Both agents caused changes in the levels of different shock induced proteins: after nicotine treatment we observed a slight realignment of chaperonin containing TCP subunits, and an increase in the Hsp70/Hsp90-organizing protein (Hop), while phenanthrene caused a rise in the Hsp90 level of the cells.
We also noted changes in the levels of certain proteins involved in intracellular signaling. Nicotine increased the calcium binding protein 1 level, while phenanthrene treatment greatly diminished it. Nicotine also increased the abundance of the PICOT protein, which possibly plays a role in regulating stress induced signalization through the inhibition of PKC theta. Phenanthrene induced an increase in the expression of zyxin, which is a component of focal adhesion plaques and it has been shown to shuttle to the nucleus where it interacts with a variety of proteins.
Both nicotine and phenanthrene treatment increased the levels of particular proteins involved in replication, transcription and translation: Elf4A and–to a lesser extent–a protein similar to Tardbp in nicotine treatment, minichromosome maintenance protein 7 and transcription elongation factor elongin A in phenanthrene-treated cells.
Phenanthrene decreased the level of the proteasome subunit PSMC2 which is one of the six putative ATPases in the regulatory complex of the proteasome. Interestingly, the expression of GMP synthase–the key enzyme of guanine nucleotide synthesis, and a stimulator of histone deubiquitylation–was also increased by phenanthrene.
Furthermore, several enzymes have also been identified among the proteins which showed expressional changes after nicotine or phenanthrene treatment. Nicotine decreased the level of dihydrolipoamide dehydrogenase, slightly increased that of glucose-6-phosphate dehydrogenase and lactate dehydrogenase B, while markedly increasing that of transketolase. On the other hand, phenanthrene increased the expression of dihydrolipoamide acetyltransferase.
Other proteins affected by nicotine treatment were: GDP dissociation inhibitor 3, similar to dihydropyrimidinase related protein-2 and–to a greater extent–metavinculin. Phenanthrene decreased the expression of annexin I which is a member of the annexins implicated in several cellular processes, including modulation of phospholipase A2 (PLA2) activity and inflammation, immune response, proliferation, blood coagulation, differentiation, exocytosis, membrane skeletal linkage and intracellular signal transduction.
Although smoking is known to be involved in the pathogenesis of several neurological and vascular diseases, few data are available about the effect of cigarette smoke components on brain endothelial cells. Since cerebral endothelium is located at the blood-CNS interface, it can be the first target of different substances affecting the brain.
Nicotine and PAHs are abundant and biologically active components of cigarette smoke. We carried out a complex investigation on the effect of nicotine and PAHs on cerebral endothelial cells in order to dissect the physiopathological consequences of smoking on the cerebral endothelium. The experiments were focused on changes affecting the tight and adherens junction proteins. Moreover, a broader range of proteins was also analyzed using 2D electrophoresis.
Treatments of nicotine, phenanthrene or methylanthracene for 15
Results of the immunofluorescent analysis confirmed western-blot data showing that the most sensitive tight junction protein to nicotine was ZO-1. Similar results were previously reported on primary bovine brain microvessel endothelial cells and also cerebral microvessels isolated from nicotine-treated rats (Abbruscato et al., 2002; Hawkins et al., 2004). We observed a less pronounced sensitivity of endothelial ZO-1 and cadherin to PAHs. Combination of nicotine and phenanthrene treatment did not potentiate the alterations seen with nicotine alone in the localization of junctional proteins.
Co-immunoprecipitation studies showed that 3
The data suggest that only relatively high concentrations of CSCs are able to induce changes in the structure of the junctional complex. Only above peak plasma levels of nicotine influenced the expression of junctional proteins. In considering the damaging effect of nicotine, however, it should be noted that rats are significantly more resistant to nicotine toxicity than humans. Furthermore, the in vitro model system allowed only relatively short-term monitoring (max. 48
The observed changes did not lead to a significant decrease in the transendothelial electric resistance. This is in line with data obtained on bovine CECs (Abbruscato et al., 2002), showing that 12–24
Junctional proteins were much more affected when nicotine treatment was used in combination with DMNQ. Oxidative stress is known to alter the integrity of the junctions (Krizbai et al., 2005; Witt et al., 2003). We observed a pronounced decrease of TEER compared to DMNQ treatment alone, suggesting a cumulative effect of nicotine abuse and hypoxia. This was accompanied by morphological changes: an even more pronounced alteration in ZO-1 membrane staining compared to nicotine or DMNQ alone. This finding suggests that nicotine abuse may aggravate the clinical status of patients affected by hypoxia via an impaired BBB function. Our results support those of Abbruscato et al. (2002, 2004), who showed that there is a cumulative negative effect of nicotine and hypoxia/aglycemia on the BBB regarding permeability and potassium transport.
In order to explore the cellular proteins responsive to nicotine and PAHs in brain endothelial cells two-dimensional electrophoresis was performed. Proteomic analysis of changes induced by cigarette smoke components has already been performed in various cell types (Piubelli et al., 2005; Raveendran et al., 2005), indicating the up- and down-regulation of the expression of diverse proteins. Proteomic analysis of striatum from nicotine-treated rats revealed altered expression of a number of proteins involved in cellular signaling and apoptosis (Yeom et al., 2005). We observed different responses of the cells to both nicotine and phenanthrene treatment, resulting in altered expression of shock induced proteins, metabolic enzymes, signaling molecules. This confirms the cerebral endothelium as being a target of cigarette smoke components.
The effect of nicotine may be mediated by the presence of nicotinic acetylcholine receptors (subunits alpha 3, 5, 7, beta 2) on the surface of CECs (Abbruscato et al., 2002; Hawkins et al., 2005). However, non-receptor mediated mechanisms are also possible (Tonnessen et al., 2000).
Cerebral endothelial cells are also affected by polycyclic aromatic hydrocarbons. To our knowledge, the effect of PAHs has not so far been investigated on cerebral endothelial cells. However, human coronary artery endothelial cells exposed to PAHs showed important alterations: PLA
The results of our complex investigation on the effect of nicotine and polyaromatic hydrocarbons on cerebral endothelial cells suggest that cigarette smoke components probably do not cause acute alterations in the principal functional properties of the cerebral endothelial cells. However, 24
This work was supported by OTKA T037956 and Philip Morris Inc, USA.
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Received 12 April 2007/18 July 2007; accepted 29 August 2007doi:10.1016/j.cellbi.2007.08.026