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Am J Physiol Heart Circ Physiol 292: H3190-H3197, 2007. First published February 16, 2007; doi:10.1152/ajpheart.01177.2006
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Regulation of bovine brain microvascular endothelial tight junction assembly and barrier function by laminar shear stress

Olga C. Colgan,1 Gail Ferguson,1 Nora T. Collins,1 Ronan P. Murphy,1 Gerardeane Meade,2 Paul A. Cahill,1 and Philip M. Cummins

1Vascular Health Research Centre, Dublin City University, Glasnevin, and 2Department of Molecular and Cellular Therapeutics (Bio-Imaging Facility), Royal College of Surgeons in Ireland, Dublin, Ireland

Submitted 26 October 2006 ; accepted in final form 13 February 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Blood-brain barrier (BBB) controls paracellular solute diffusion into the brain microenvironment and is maintained primarily by tight junctions between adjacent microvascular endothelial cells. Studies implicate blood flow-associated shear stress as a pathophysiological mediator of BBB function, although detailed biochemical data are scarce. We hypothesize that shear stress upregulates BBB function via direct modulation of expression and properties of pivotal tight-junction proteins occludin and zonula occludens-1 (ZO-1). Bovine brain microvascular endothelial cells (BBMvECs) were exposed to either steady or pulsatile shear stress (10 and 14 dyn/cm2, respectively) for 24 h. Sheared BBMvECs were monitored for occludin-ZO-1 expression, association, and subcellular localization, and transendothelial permeability of BBMvECs to FITC-dextran and 14[C]sucrose was assessed. Actin reorganization and BBMvEC realignment were observed following steady shear stress for 24 h. Substantial increases in occludin mRNA and protein expression (2.73 ± 0.26- and 1.83 ± 0.03-fold) and in occludin-ZO-1 association (2.12 ± 0.15-fold) were also observed. Steady shear stress also induced clear relocalization of both proteins to the cell-cell border in parallel with reduced transendothelial permeability to FITC-dextran (but not sucrose). Following pulsatile shear stress, increased protein levels for both occludin and ZO-1 (2.15 ± 0.02- and 1.67 ± 0.21-fold) and increased occludin-ZO-1 association (2.91 ± 0.14-fold) were observed in parallel with a reduction in transendothelial permeability to 14[C]sucrose. Shear stress upregulates BBMvEC barrier function at the molecular level via modulation of expression, association, and localization of occludin and ZO-1. The pulsatile shear model appeared to give the most profound biochemical responses.

blood-brain barrier; occludin; zonula occludens-1

THE BLOOD-BRAIN BARRIER (BBB) maintains homeostasis of the central nervous system (CNS) by restricting paracellular diffusion of water-soluble substances from blood to the brain microenvironment. It is comprised of microvascular endothelial cells, which exhibit many specialized properties, including extremely low permeability, high transendothelial electrical resistance, and low occurrence of pinocytotic vesicles (6, 10). Barrier function is maintained primarily by the regulated apposition of apical tight-junction complexes between adjacent cells and comprise occludin, claudins, junctional adhesion molecules 1–3, cingulin, 7H6, spectrin, and zonula occludens-1, -2, and -3 (ZO-1, -2, and -3), the latter linking tight-junction proteins to each other and the actin cytoskeleton (21).

In recent years, investigations have highlighted the potential importance of basolateral conditions (e.g., presence of astrocytes) to BBB function (19, 23, 24, 34), although the mechanisms involved are poorly understood. Of the physiological stimuli that have an impact on the vascular endothelium, however, blood flow-associated hemodynamic forces such as shear stress and cyclic strain are also of crucial importance. These forces profoundly affect endothelial gene expression, morphology, and cell fate (18, 36, 44). In this regard, recent work in our laboratory has demonstrated how cyclic strain regulates the expression, phosphorylation, association, and subcellular localization of occludin and ZO-1, two pivotal tight-junction proteins, with direct consequences for barrier function in bovine aortic endothelial cells (BAECs) (8). Moreover, a few isolated studies exist that implicate shear stress in regulating occludin expression and phosphorylation, again in endothelial cells of macrovascular origin (9, 13, 35).

Within the brain microvasculature, levels of shear stress range from 4 to 20 dyn/cm2 (14). Recent studies by Krizanac-Bengez and co-workers (27) have suggested that shear stress plays a significant role in homeostasis and pathophysiology of the cerebral microvasculature. These researchers have shown that a combination of factors, including anoxia, aglycemia, and, most importantly, loss of shear stress (leading to leukocyte-mediated cytokine release and matrix metalloproteinase/tissue inhibitor of matrix metalloproteinase imbalance) causes the BBB failure and changes in endothelial/glial signaling associated with ischemia (28–30). Moreover, an earlier study by Stannes et al. (42) has also indicated that shear stress is required for induction and maintenance of the BBB. These findings implicate shear stress as a putative physiological and pathological mediator of BBB function, although detailed biochemical data on tight-junction assembly is lacking. Consequently, we hypothesize that shear stress upregulates BBB function via direct modulation of the expression and properties of tight-junction proteins.

In this study, we investigated this hypothesis via a detailed examination of the effects of shear stress on the expression, association, and subcellular localization of occludin and ZO-1 in bovine brain microvascular endothelial cells (BBMvECs), in parallel with a functional index of barrier integrity (i.e., transendothelial permeability). Moreover, the effects of both steady (nonpulsatile) and pulsatile shear stress in this context were compared.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All reagents used in this study were of the highest purity and unless otherwise stated were obtained from Sigma-Aldrich (Dorset, UK).

Cell Culture and Shear Stress

Cell culture. BBMvECs between passages 5 and 15 (Cell Applications, San Diego, CA) were cultured in high-glucose DMEM supplemented with 10% FCS (GIBCO, Paisley, UK), 3 µg/ml heparin, 3 ng/ml human recombinant basic fibroblast growth factor (Chemicon International, Temecula, CA), and antibiotics (50 U/ml penicillin, 50 µg/ml streptomycin). Cells were grown in a humidified atmosphere of 5% CO2-95% air at 37°C.

Steady shear stress. BBMvECs were seeded at 1 x 104 cells/cm2 in regular six-well plates and were allowed to come to confluency, typically in 5–7 days. Medium was then removed and replaced with 4 ml of fresh growth medium, and cells were exposed to either 0 or 10 dyn/cm of steady (laminar, nonpulsatile) shear stress for 24 h on an orbital rotator using the equation {tau}w = {alpha}{surd}{rho}{eta}(2{pi}f)3, where {tau}w is shear stress, {alpha} is radius of rotation (cm), {rho} is density of liquid (g/l), {eta} is fluid viscosity (0.0075 dyn/cm2 at 37°C), and f is rotations per second (22).

Pulsatile shear stress. The CellMax artificial capillary system (Spectrum Laboratories, Rancho Dominguez, CA) is an automated closed perfusion system comprising a bundle of 50 semipermeable, ProNectin-coated polypropylene capillaries (capillary length, 13 cm; internal diameter, 330 µm; wall thickness, 150 µm; pore size, 0.5 µm; internal surface area, 70 cm2) (38). This system operates within a humidified CO2 incubator where medium is pumped through gas-permeable silicone tubing from a media reservoir at a chosen flow rate. As the gear pump rotates, the motor shaft forces the pump pins to depress the silicone tubing on the capillary module, thereby forcing media to flow in a pulsatile fashion through the capillary bundle. Flow rate (pulse frequency and height) can be regulated by using an external control unit. With the use of this system, shear stress is calculated according to the equation {tau} = 4{eta}Q/{pi}R3, where {tau} is shear stress, {eta} is fluid viscosity (0.0075 dyn·s–1·cm–2 at 37°C), Q is fluid flow rate per fiber (ml/s), and R is internal fiber radius (cm). Following capillary preequilibration in growth medium for 3 days, BBMvECs grown to confluency in a T75 culture flask were trypsinized, suspended in 10 ml of growth medium, and seeded into the capillary bundle lumen. Cells were allowed to adhere for 3 h, after which the pump was set to a low flow rate (0.2 dyn/cm, 0.2 Hz). When cells had formed a confluent monolayer (7–10 days), control cells were maintained at low flow, whereas test cells were gradually "ramped up" to high flow (14 dyn/cm, 3 Hz) over 5 h and maintained thus for a further 24 h. Following shear stress, cells were harvested first by washing both the luminal and extracapillary spaces with HBSS and subsequently by trypsinization.

Transendothelial Permeability Studies

Permeability studies were performed by using the CellMax artificial capillary system described above. Following 24 h of either low or high shearing in perfused capillaries, flow was momentarily stopped and 2 µCi of 14[C]sucrose (2) was added to each medium reservoir. Following restoration of flow, diffusion of 14[C]sucrose from the intraluminal to the extracapillary space was allowed to proceed for 3 h. Media samples (100 µl) were collected every 15 min by syringe from the extracapillary space port. All samples were subsequently monitored in triplicate for 14[C]sucrose by scintillation counting. Permeability data is presented as percent transendothelial exchange, which can be derived from the "flux" variable within the permeability coefficient (5).

Protein Assay

Protein was routinely quantified by using the BCA protein microassay procedure (40) with BSA as standard (Pierce, Cramlington, UK).

Western Immunoblotting

Following shearing experiments, BBMvECs were harvested and total lysate samples (or, as in the case of occludin, immunoprecipitates) were resolved by SDS-PAGE under reducing conditions (occludin, 12% polyacrylamide; ZO-1, 6% polyacrylamide) according to the method of Laemmli (31). Gels were electroblotted and immunostained as previously described (8). Primary antisera were 1:1,000 mouse anti-occludin monoclonal IgG and 1:2,500 mouse anti-ZO-1 monoclonal IgG (Zymed Laboratories, South San Francisco, CA). Secondary antisera were 1:4,000 horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham Biosciences, Little Chalfont, UK). For quantitative comparisons between bands, scanning densitometry was performed by using NIH Image v. 1.61 software. Normalization of densitometric data to untreated control (assigned a value of 1.0) was routinely used for data presentation to factor out run-to-run variability between absolute densitometry values assigned to individual bands. Samples were typically analyzed in triplicate on a given blot, whereas blots from at least three experiments were used for statistical comparisons. All nitrocellulose membranes were routinely stained with Ponceau S to normalize for protein loading/transfer.

Immunocytochemistry

Following shearing experiments (in six-well dishes only), BBMvECs were prepared for immunocytochemical analysis according to the method of Groarke et al. (20) with modifications. Primary antibody was 10 µg/ml mouse anti-occludin monoclonal IgG or 0.25 µg/ml mouse anti-ZO-1 monoclonal IgG (Zymed Laboratories). Secondary antibody was 1:400 Alexa 488-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR). Nuclear (DAPI, 500 ng/ml, 3 min) and F-actin staining (rhodamine-phalloidin, 1:200, 1 h) were also routinely conducted. Visualization was by standard fluorescent microscopy (Olympus BX50). Suitable antibody controls were included.

Immunoprecipitation

Following shearing experiments, total BBMvEC lysate samples were monitored by immunoprecipitation (IP) for changes in occludin-ZO-1 coassociation. IP was performed according to the method of Ferguson et al. (15) with minor modifications. Briefly, lysate containing 60 µg protein was incubated with 1.5 µg of mouse anti-ZO-1 monoclonal IgG, 6 µl of 10% BSA, and 7.5 µl of protein G-Sepharose beads (Amersham Biosciences) to give a final reaction volume of 500 µl. Incubation proceeded overnight at 4°C with continuous Eppendorf rotation. Beads were then washed extensively, resuspended in 15 µl of SDS-PAGE sample solubilization buffer, and heated for 10 min at 90°C. Beads were subsequently pelleted, and supernatant (containing solubilized proteins) was removed to a fresh tube for Western immunoblotting as described. Immunoblots were subsequently probed and visualized in the normal manner with mouse anti-occludin monoclonal IgG.

Real-Time PCR

Following shearing experiments, extraction of total BBMvEC RNA and performance of real-time PCR were employed as previously described (48) to monitor changes in occludin and ZO-1 mRNA levels. Gene-specific primers for occludin, ZO-1, and GAPDH (included for normalization purposes) have been published previously (8, 48). All primer pairs used were routinely screened for nonspecific primer-dimer products by melt-curve analysis and by standard PCR in conjunction with agarose gel electrophoresis.

Statistical Analysis

Results are expressed as means ± SE. Experimental points were performed in triplicate with a minimum of three independent experiments (n = 3). Means were compared by Student's unpaired t-test. For permeability assays, two-factor ANOVA was used for comparisons, the two factors being time and treatment (control vs. shear). For either test, a value of *P ≤ 0.05 was significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of Steady Shear Stress on BBMvEC Morphology

Following exposure of BBMvECs to 0 or 10 dyn/cm2 shear stress, cellular realignment was monitored by phase-contrast microscopy and F-actin staining. In the unsheared control cells, cellular alignment was random and multidirectional. Following shear, cells realigned in the direction of flow (Fig. 1, i and iii). In parallel, F-actin staining with rhodamine-phalloidin revealed significant redistribution of actin bundles in the direction of shear (Fig. 1, ii and iv).


Figure 1
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  		Fig. 1. Effect of steady shear stress on bovine brain microvascular endothelial cell (BBMvEC) morphology. Following shear stress (10 dyn/cm2, 24 h), BBMvEC realignment was monitored by phase-contrast microscopy (i and iii) and standard fluorescent microscopy (rhodamine-phalloidin staining for F-actin; ii and iv). Dotted arrows highlight alignment in direction of flow. Images are representative of 3 independent experiments.

 
Regulation of Occludin and ZO-1 Levels in BBMvECs by Steady Shear Stress

Following exposure of BBMvECs to 0 or 10 dyn/cm shear stress, mRNA and protein levels of occludin and ZO-1 were monitored by real-time PCR and Western blotting, respectively. In response to shear, mRNA levels for occludin increased by 2.73 ± 0.26-fold (Fig. 2A) compared with 1.25 ± 0.003-fold for ZO-1 (Fig. 2B). Additionally, protein levels for occludin increased by 1.83 ± 0.03-fold compared with 1.32 ± 0.03-fold for ZO-1. With respect to both proteins, shear-dependent increases in mRNA and protein were significant (P ≤ 0.05). Moreover, the increases for occludin were statistically higher than those for ZO-1 (P ≤ 0.05).


Figure 2
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  		Fig. 2. Effect of steady shear stress on occludin and zonula occludens-1 (ZO-1) levels in BBMvECs. Following shear stress (10 dyn/cm2, 24 h), BBMvECs were monitored for occludin (A) and ZO-1 (B) mRNA and protein levels by real-time PCR and Western blotting, respectively. Histograms represent fold change in mRNA level and are averaged from 3 independent experiments ± SE. *P ≤ 0.05 vs. unsheared control. Representative protein blots are also shown.

 
Regulation of Occludin-ZO-1 Association and Subcellular Localization in BBMvECs by Steady Shear Stress

Following exposure of BBMvECs to 0 or 10 dyn/cm shear stress, occludin-ZO-1 association was monitored in cell lysates by IP/Western blotting. Postshear, the level of occludin detected in anti-ZO-1 immunoprecipitates increased by 2.12 ± 0.15-fold (Fig. 3A). Moreover, inclusion of cyclohexamide (1 µg/ml) reduced this increase by ~55% (data not shown). Subcellular localization of occludin and ZO-1 was also monitored by immunocytochemistry. Occludin immunoreactivity became appreciably more concentrated along the cell border in response to shear stress (Fig. 3B, i and iii). Moreover, ZO-1 immunoreactivity at the cell-cell border, which exhibited a jagged localization pattern in unsheared cells, became appreciably more continuous and well-defined following shear (Fig. 3B, ii and iv).


Figure 3
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  		Fig. 3. Effect of steady shear stress on occludin and ZO-1 association and localization in BBMvECs. Following shear stress (10 dyn/cm2, 24 h), BBMvECs were monitored for occludin and ZO-1 association (A) and subcellular localization (B) by immunoprecipitation (IP)/Western blotting and immunocytochemistry (IB), respectively. A: fold change in band intensity averaged from 3 independent experiments ± SE; *P ≤ 0.05 vs. unsheared control. Protein blot shown is representative. B: shear-dependent membrane localization of occludin and ZO-1. White arrows highlight plasma membrane localization. Images are representative of 3 independent experiments.

 
Regulation of Occludin and ZO-1 Levels in BBMvECs by Pulsatile Shear Stress

Following exposure of BBMvECs to low or high shear stress, protein expression of occludin and ZO-1 were monitored by Western blotting. Protein levels for occludin and ZO-1 increased by 2.15 ± 0.02 and 1.67 ± 0.21, respectively (Fig. 4, A and b). With respect to both proteins, shear-dependent increases in mRNA and protein were significant (P ≤ 0.05). Moreover, the increases for occludin were statistically higher than those for ZO-1 (P ≤ 0.05). Furthermore, when compared with increases in protein levels following steady shear stress, increases following pulsatile shear stress were statistically higher (Students two-sample t-test assuming unequal variances, P ≤ 0.0001).


Figure 4
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  		Fig. 4. Effect of pulsatile shear stress on occludin and ZO-1 levels and occludin-ZO-1 association in BBMvECs. Following shear stress (0.2 or 14 dyn/cm2, 24 h), cells were monitored for occludin (A) and ZO-1 (B) protein levels by Western blotting. C: association of occludin and ZO-1 was also monitored by IP/Western blotting. Histogram represents fold change in band intensity and is averaged from 3 independent experiments ± SE; *P ≤ 0.05 vs. low shear control (0.2 dyn/cm). Protein blots shown are representative.

 
Regulation of Occludin-ZO-1 Association in BBMvECs by Pulsatile Shear Stress

Following exposure of BBMvECs to low or high shear stress, occludin-ZO-1 association was monitored in cell lysates by IP/Western blotting. Postshear, the level of occludin detected in anti-ZO-1 immunoprecipitates increased by 3.18 ± 0.14-fold (Fig. 4C). When compared with increases in occludin-ZO-1 association following steady shear stress, increases following pulsatile shear stress were statistically higher (Students two-sample t-test assuming unequal variances, P ≤ 0.0001).

Regulation of BBMvEC Permeability to 14[C]Sucrose by Pulsatile Shear Stress

Following exposure of BBMvECs to low and high shear stress, permeability to 14[C]sucrose was assessed as described. High shear stress resulted in a 19.7 ± 7.74% decrease in 14[C]sucrose crossing from the luminal space to the extracapillary space over the 60-min monitoring period (Fig. 5).


Figure 5
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  		Fig. 5. Effect of pulsatile shear stress on BBMvEC transendothelial permeability. Following shear stress (0.2 or 14 dyn/cm2, 24 h), BBMvECs were monitored for permeability to 14[C]sucrose. Points represent total extraluminal radioactivity at a given time point (from 0–60 min) as a percentage of total luminal radioactivity at t = 0 min. %TEE, percent transendothelial exchange of 14[C]sucrose. Results are averaged from 3 independent experiments ± SE, with statistical analysis by two-factor ANOVA (P = 0.014, F = 17.76, Fcrit = 7.71). No significant difference in permeability was found in capillaries without cells under either low- or high-flow conditions.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Homeostatic maintenance of the brain microenvironment is essential for normal brain function and neuronal activity and is regulated by the BBB, a continuum of intercellular tight junctions between adjacent microvascular endothelial cells. This barrier forms an effective seal to prevent paracellular diffusion of charged particles, proteins, ions, hydrophilic molecules, and hormones, thus protecting the CNS from changes in blood composition (6, 10, 21). Despite its protective actions on the CNS, it presents a major obstacle to drug delivery. Conversely, disruption of BBB integrity is associated with a number of pathophysiological conditions, including multiple sclerosis, progressive multifocal leukoencephalopathy, and stroke (27, 45, 49). Thus development of novel strategies to selectively modulate BBB permeability necessitates elucidating the biochemistry and pathophysiology of microvascular endothelial tight-junction assembly. In this context, shear stress is highly relevant. Recent studies by Krizanac-Bengez et al. (27), for example, indicate that shear stress plays a significant role in homeostasis and pathophysiology of the cerebral microvasculature. These workers have shown that loss of shear stress leads to the BBB failure associated with ischemia (28–30). Moreover, Stannes et al. (42) have also indicated that shear stress is required for BBB induction and maintenance. These findings implicate shear stress as a putative physiological and pathological mediator of BBB function, although detailed biochemical data on tight-junction protein assembly is lacking. Consequently, our objective was to show that shear stress regulates BBB function via direct modulation of the expression and properties of the pivotal tight-junction proteins occludin and ZO-1.

Our initial investigations clearly demonstrate that exposure of BBMvECs to steady (nonpulsatile) shear stress (10 dyn/cm2, 24 h) causes actin cytoskeletal reorganization and cell morphological realignment in the direction of the shear vector (18). Concomitantly, mRNA and protein levels for occludin were significantly increased, whereas ZO-1 exhibited comparatively minor changes. Increased occludin levels may be due to increased gene expression and/or reduced mRNA/protein turnover. Consistent with these findings, earlier studies by Conklin et al. (9) and DeMaio et al. (13), albeit in aortic endothelial cells, also report shear-dependent upregulation of occludin levels in parallel with negligible effects on ZO-1 levels, respectively.

Intercellular cleft size and degree of permeability are regulated by actin cytoskeletal tension and junctional protein cointeractions (1). As such, elevated occludin-ZO-1 association, coupled to subcellular redistribution of both proteins along the cell-cell border where tight junctions form, should accompany an increase in endothelial barrier function, as evidenced in previous studies (8, 17, 32). We therefore decided to build on our initial investigations by examining the impact of steady shear stress in this context. Our results clearly indicate a significant shear-dependent increase in occludin-ZO-1 association, likely driven by a combination of new protein synthesis (i.e., 55% reduction in association following cyclohexamide treatment) and posttranslational modification of existing occludin/ZO-1 cellular pools (e.g., phosphorylation), observations consistent with the findings of Collins et al. (8) in BAECs. Furthermore, occludin, observed within the nucleus and cytosol of unsheared BBMvECs, exhibited more appreciable immunoreactivity along the cell-cell border in response to shear stress, whereas ZO-1 exhibited an even more dramatic linear redistribution along the plasma membrane. These observations are also consistent with our previous findings in BAECs (8). It should be noted that in other BBB models, occludin was seen to localize only at the endothelial cell border (16, 41), in contrast to the cytosolic and nuclear pools of occludin seen in this study. Because we observed this in both unstimulated and stimulated cells, phenotypic dedifferentiation of BBMvECs under unstimulated conditions was ruled out as a likely cause. Other possible explanations may be nonspecific antisera binding or a partial dedifferentiation of cell phenotype with passage number (in both studies, commercial cell lines between passages 5 and 15 were used). The latter possibility is more likely, because published models showing occludin localization exclusively to the microvascular endothelial cell border appear to use unmodified (16, 41) or immortalized (12, 25, 33) primary microvascular endothelial cells and whole tissues (3), thus potentially highlighting a limitation of commercially available vascular endothelial cells for in vitro BBB modeling studies.

The use of orbital rotation was, unfortunately, problematic when attempting to monitor the effects of steady shear on BBMvEC barrier function. BBMvECs could not be sheared by orbital rotation in Transwell-Clear plates (six-well format, 0.4-µm pore) due to the possibility of turbulence effects and basolateral shearing. This necessitated that BBMvECs be sheared by orbital rotation in regular six-well plates, after which cells were trypsinized, replated into Transwell-Clear plates, and monitored for transendothelial permeability to either 40-kDa FITC-dextran or 14[C]sucrose. Using this approach, we observed that steady shear stress reduced BBMvEC transendothelial permeability to FITC-dextran (but not 14[C]sucrose) by almost two-thirds (data not shown). It should also be noted that we monitored a number of occludin/ZO-1 properties and confirmed that shear-induced changes in these molecules (e.g., localization changes) persisted 24 h after passage into Transwell-Clear plates (data not shown). Although these collective data lead us to conclude that tight-junction expression, assembly, and barrier integrity are upregulated by blood flow within the brain microvasculature, the orbital rotation model is clearly limited with respect to the latter barrier integrity study. For example, BBMvEC permeability to 14[C]sucrose is apparently unaffected, suggesting that despite shear-induced barrier upregulation, interendothelial gaps are still sufficiently large to allow transendothelial movement of very small molecules. This may reflect nonspecific effects on endothelial cells of "orbitally applied" shear stress, as recently implied in the findings of Dardik et al. (11). Alternately (and more likely), the necessity for replating sheared BBMvECs into Transwell-Clear plates for permeability measurements may lead to either a temporal reduction in barrier function (i.e., measuring permeability 24 h after cessation of stimulus) or "proteolytic weakening" of the tight junction due to trypsinization. Moreover, blood flow in vivo is pulsatile, a condition not reflected in the aforementioned orbital rotation model. Endothelial characteristics (i.e., remodeling rate, actin cytoskeletal reorganization, endothelial nitric oxide synthase expression, membrane potential, etc.) are uniquely responsive to flow type (4, 37).

To overcome these drawbacks, it was decided to revisit some of our initial experiments using a CellMax Artificial Capillary System. The usefulness of this model can be appreciated at a number of levels. In addition to mimicking the unique three-dimensional environment of the blood-brain microvasculature, an additional feature of the CellMax model is pulsatile flow. Due to extensive branching, cerebral capillaries experience little or no pulse pressure (and thus cyclic strain). However, opening and closing of precapillary sphincters (present at arteriole/capillary branch points), in conjunction with the dilation/contraction of capillaries, likely exerts a degree of "pulsatility" to blood flow within the brain microvasculature (36a). With the use of this system, therefore, BBMvECs could be exposed to pulsatile flow (0.2 or 14 dyn/cm2, 24 h) and monitored for occludin-ZO-1 expression and association. Additionally, transendothelial permeability to 14[C]sucrose could be monitored during shear "without" the need to trypsinize and replate cells into Transwell-Clear dishes. It should also be mentioned that an additional feature of the CellMax model is hydrostatic pressure exerted on endothelial cells. Although the precise contribution of hydrostatic pressure to vascular endothelial properties in vivo is not well understood, it is a potentially important consideration when interpreting results from this system. Relative to steady shear stress, pulsatile shear stress caused a more statistically significant upregulation of both occludin and ZO-1 protein levels in parallel with levels of protein-protein association, although a possible dose-dependent shear effect (i.e., 10 vs. 14 dyn/cm2) cannot be completely discounted here. Furthermore, we observed ~20% reduction in BBMvEC permeability to 14[C]sucrose in response to pulsatile shear stress.

The comparability of the two shearing models employed clearly warrants some discussion. With respect to shear-dependent changes in occludin-ZO-1 expression and association, both models are basically comparable because cells are sheared, immediately harvested by trypsinization in either instance, and subsequently lysed for analysis. With respect to functional assays of barrier integrity, however, comparing cells from nonpulsatile/trypsinized (orbital shaker) and pulsatile/nontrypsinized (CellMax) models is not ideal, with the latter model clearly being the more physiologically (and technically) acceptable. As such, this prevents us from drawing any conclusions as to the precise role of "pulsatility" on BBMvEC barrier function proper. A more ideal study model in which cells are exposed to pulsatile and nonpulsatile shear stresses under identical experimental conditions (e.g., a cone-plate viscometer adapted for nonpulsatile and pulsatile shearing in Transwell-Clear plates, accompanied by real-time high transendothelial electrical resistance measurement) therefore, remains a central objective of future studies in this area.

The mechanotransduction pathway(s) regulating shear-dependent changes in endothelial barrier integrity remain elusive. Vascular endothelial cells use multiple sensing mechanisms to detect changes in mechanical force, leading to activation of signaling networks. For example, shear-dependent integrin-Shc association, leading to JNK activation and AP-1/TRE-mediated gene transcription, has been reported in BAECs by Chen et al. (7). A role for Flk-1 in these events has also been reported by the same authors (7). The cytoskeleton is also critically important in this context as it provides a structural framework for endothelial cells to transmit hemodynamic forces such as shear between its apical, basolateral and junctional surfaces and the cell interior (which includes the cytoplasm, nucleus and focal adhesion sites where integrins are clustered). In this regard, numerous studies have confirmed the contribution of cytoskeletal dynamics to endothelial barrier regulation. For example, the well-established antagonistic relationship between the cytoskeletal regulators RhoA and Rac-1 is pivotal to this process (50). Because shear-dependent integrin activation has been shown to inactivate RhoA and activate Rac-1 in BAECs (46, 47), events jointly consistent with barrier upregulation, the integrin:Rho-GTPase signaling axis therefore represents a highly plausible signaling route for flow-mediated permeability changes within the BBB. Also worthy of mention, studies by Kolosova et al. (26) indicate that ATP, released from vascular endothelial cells in response to hemodynamic force and vascular injury, can lead to barrier enhancement in a Gi{alpha}-dependent, ERK-independent manner.

With respect to mechanotransduction, our own studies have already confirmed that posttranslational modifications to the phosphorylation state of both occludin and ZO-1 are directly causal of the upregulation of tight-junction assembly and barrier integrity in BAECs in response to cyclic strain (8), an important hemodynamic component of blood flow (particularly in the aorta). Moreover, these events proceed via a Gi{alpha}-/p38-dependent, ERK-independent pathway (unpublished observations), consistent with a putative role for ATP as suggested by Kolosova (26). Furthermore, a role for Rac-1 activation in these events has also been confirmed by our group (unpublished observations). Delineating the involvement of these signaling events with respect to shear-dependent regulation of BBMvEC tight junctions will undoubtedly be a focus of our future investigations in this field.

In summary, this study shows the effects of both steady and pulsatile shear stress on tight-junction assembly and function in brain microvascular endothelial cells in vitro. Our findings demonstrate that shear stress modulates the expression, association, and membrane localization of occludin and ZO-1, pivotal components of intercellular tight junctions, with consequences for BBMvEC barrier integrity. To our knowledge, this is one of the first attempts to define these physiologically important events at the tight-junction protein level with respect to shearing of the brain microvasculature. Because this work clearly complements another recent capillary flow study (39), we anticipate that it will contribute much to the future development of more advanced, multivariable in vitro models for examining, and ultimately exploiting, BBB integrity at the molecular and functional levels.


   ACKNOWLEDGMENTS
 
This research was supported by grants from and the Wellcome Trust, Enterprise, Ireland, and ELAN Corporation, Athlone, Ireland.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. M. Cummins, Vascular Health Research Centre, Dublin City University, Glasnevin, Dublin 9, Ireland (e-mail: phil.cummins{at}dcu.ie)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Balda MS, Matter K. Tight Junctions. J Cell Sci 111: 541–547, 1998.[Abstract]
  2. Berezowski V, Landry C, Dehouck MP, Cecchelli R, Fenart L. Contribution of glial cells and pericytes to the mRNA profiles of P-glycoprotein and multidrug resistance-associated proteins in an in vitro model of the blood-brain barrier. Brain Res 1018: 1–9, 2004.[CrossRef][ISI][Medline]
  3. Bertossi M, Virgintino D, Maiorano E, Occhiogrosso M, Roncali L. Ultrastructural and morphometric investigation of human brain capillaries in normal and peritumoral tissues. Ultrastruct Pathol 21: 41–49, 1997.[ISI][Medline]
  4. Blackman BR, Garcia-Cardena G, Gimbrone MA Jr. A new in vitro model to evaluate differential responses of endothelial cells to simulated arterial shear stress waveforms. J Biomech Eng 124: 397–407, 2002.[CrossRef][ISI][Medline]
  5. Brown RC, Mark KS, Egleton RD, Huber JD, Burroughs AR, Davis TP. Protection against hypoxia-induced increase in blood-brain barrier permeability: role of tight junction proteins and NfkappaB. J Cell Sci 116: 693–700, 2003.[Abstract/Free Full Text]
  6. Butt AM, Jones HC, Abbot NJ. Electrical resistance across the blood-brain barrier in anaesthetized rats: a developmental study. J Physiol 49: 47–62, 1990.
  7. Chen KD, Li YS, Kim M, Li S, Yuan S, Chien S, Shyy JY. Mechanotransduction in response to shear stress: roles of receptor tyrosine kinases, integrins and Shc. J Biol Chem 274: 18393–18400, 1999.[Abstract/Free Full Text]
  8. Collins NT, Cummins PM, Colgan OC, Ferguson G, Birney YA, Murphy RP, Meade G, Cahill PA. Cyclic strain-mediated regulation of vascular endothelial occludin and ZO-1: influence on intercellular tight junction assembly and function. Arterioscler Thromb Vasc Biol 26: 62–68, 2006.[CrossRef][ISI][Medline]
  9. Conklin BS, Zhong DS, Zhao W, Lin PH, Chen C. Shear stress regulates occludin and VEGF expression in porcine arterial endothelial cells. J Surg Res 102: 13–21, 2002.[CrossRef][ISI][Medline]
  10. Crone C, Olesen SP. Electrical resistance of brain microvascular endothelium. Brain Res 241: 49–55, 1982.[CrossRef][ISI][Medline]
  11. Dardik A, Chen L, Frattini J, Asada H, Aziz F, Kudo FA, Sumpio B. Differential effects of orbital and laminar shear stress on endothelial cells. J Vasc Surg 41: 869–880, 2005.[CrossRef][ISI][Medline]
  12. Deli MA, Abraham CS, Kataoka Y, Niwa M. Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology. Cell Mol Neurobiol 25: 59–127, 2005.[CrossRef][ISI][Medline]
  13. DeMaio L, Chang YS, Gardner TW, Tarbell JM, Antonetti DA. Shear stress regulates occludin content and phosphorylation. Am J Physiol Heart Circ Physiol 281: H105–H113, 2001.[Abstract/Free Full Text]
  14. Desai SY, Marroni M, Cucullo L, Krizanac-Bengez L, Mayberg MR, Hossain MT, Grant GG, Janigro D. Mechanisms of endothelial survival under shear stress. Endothelium 9: 89–102, 2002.[CrossRef][ISI][Medline]
  15. Ferguson G, Watterson KR, Palmer TM. Subtype-specific kinetics of inhibitory adenosine receptor internalization are determined by sensitivity to phosphorylation by G protein-coupled receptor kinases. Mol Pharmacol 57: 546–552, 2000.[Abstract/Free Full Text]
  16. Forster C, Silwedel C, Golenhofen N, Burek M, Kietz S, Mankertz J, Drenckhahn D. Occludin as direct target for glucocorticoid-induced improvement of blood brain barrier properties in a murine in vitro system. J Physiol 565: 475–486, 2005.[Abstract/Free Full Text]
  17. Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, Tsukita S, Tsukita S. Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol 127: 1617–1626, 1994.[Abstract/Free Full Text]
  18. Galbraith CG, Skalak R, Chien S. Shear stress induces spatial reorganization of the endothelial cell cytoskeleton. Cell Motil Cytoskeleton 40: 317–330. 1998.[CrossRef][ISI][Medline]
  19. Gee JR, Keller JN. Astrocytes: regulation of brain homeostasis via apolipoprotein E. Int J Biochem Cell Biol 37: 1145–1150, 2005.[CrossRef][ISI][Medline]
  20. Groarke DA, Drmota T, Bahia DS, Evans NA, Wilson S, Milligan G. Analysis of the C-terminal tail of the rat thyrotropin-releasing hormone receptor-1 in interactions and co-internalization with beta-arrestin 1 green fluorescent protein. Mol Pharmacol 59: 375–385, 2001.[Abstract/Free Full Text]
  21. Hawkins BT, Davis TP. The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev 57: 173–185, 2005.[Abstract/Free Full Text]
  22. Hendrickson RJ, Cahill PA, Sitzmann JV, Redmond EM. Ethanol enhances basal and flow-stimulated nitric oxide synthase activity in vitro by activating an inhibitory guanine nucleotide binding protein. J Pharmacol Exp Ther 289: 1293–1300, 1999.[Abstract/Free Full Text]
  23. Hoheisel D, Nitz T, Franke H, Wegener J, Hakvoort A, Tilling T, Galla HJ. Hydrocortisone reinforces the blood-brain barrier properties in a serum-free cell culture system. Biochem Biophys Res Commun 244: 312–316, 1998.[CrossRef][ISI][Medline]
  24. Jeliazkova-Mecheva VV, Bobilya DJ. A porcine astrocyte/endothelial cell co-culture model of the blood-brain barrier. Brain Res Brain Res Protoc 12: 91–98, 2003.[CrossRef][Medline]
  25. Kang YS, Lee KE, Terasaki T. Donepezil, tacrine and alpha-phenyl-n-tert-butyl nitrone (PBN) inhibit choline transport by conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB). Arch Pharmacol Res (Seoul) 28: 443–450, 2005.
  26. Kolosova IA, Mirzapoiazova T, Adyshev D, Usatvuk P, Romer LH, Jacobson JR, Natarajan V, Pearse DB, Garcia JG, Verin AD. Signaling pathways involved in adenosine triphosphate-induced endothelial barrier enhancement. Circ Res 97: 115–124, 2005.[Abstract/Free Full Text]
  27. Krizanac-Bengez L, Mayberg MR, Janigro D. The cerebral vasculature as a therapeutic target for neurological disorders and the role of shear stress in vascular homeostatis and pathophysiology. Neurol Res 26: 846–853, 2004.[CrossRef][ISI][Medline]
  28. Krizanac-Bengez L, Kapural M, Parkinson F, Cucullo L, Hossain M, Mayberg MR, Janigro D. Effects of transient loss of shear stress on blood-brain barrier endothelium: role of nitric oxide and IL-6. Brain Res 977: 239–246, 2003.[CrossRef][ISI][Medline]
  29. Krizanac-Bengez L, Hossain M, Fazio V, Mayberg M, Janigro D. Loss of flow induces leukocyte-mediated MMP/TIMP imbalance in dynamic in vitro blood-brain barrier model: role of pro-inflammatory cytokines. Am J Physiol Cell Physiol 291: C740–C749, 2006.[Abstract/Free Full Text]
  30. Krizanac-Bengez L, Mayberg MR, Cunningham E, Hossain M, Ponnampalam S, Parkinson FE, Janigro D. Loss of shear stress induces leukocyte-mediated cytokine release and blood-brain barrier failure in dynamic in vitro blood-brain barrier model. J Cell Physiol 206: 68–77, 2006.[CrossRef][ISI][Medline]
  31. Laemmli U. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970.[CrossRef][Medline]
  32. Lee HS, Namkoong K, Kim DH, Kim KJ, Cheong YH, Kim SS, Lee WB, Kim KY. Hydrogen peroxide-induced alterations of tight junction proteins in bovine brain microvascular endothelial cells. Microvasc Res 68: 231–238, 2004.[CrossRef][ISI][Medline]
  33. Murugandam A, Herx LM, Monette R, Durkin JP, Stanimirovic DB. Development of an immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood brain barrier. FASEB J 11: 1187–1197, 1997.[Abstract]
  34. Nitz T, Eisenblatter T, Psathaki K, Galla HJ. Serum-derived factors weaken the barrier properties of cultured porcine brain capillary endothelial cells in vitro. Brain Res 981: 30–40, 2003.[CrossRef][ISI][Medline]
  35. Pang Z, Antonetti DA, Tarbell JM. Shear stress regulates HUVEC hydraulic conductivity by occludin phosphorylation. Ann Biomed Eng 33: 1536–1545, 2005.[CrossRef][ISI][Medline]
  36. Patrick CW Jr, McIntire LV. Shear stress and cyclic strain modulation of gene expression in vascular endothelial cells. Blood Purif 13: 112–124, 1995.[ISI][Medline]
  37. Peppiatt CM, Howarth C, Mobbs P, Attwell D. Bidirectional control of CNS capillary diameter by pericytes. Nature 443: 642–643, 2006.[Medline]
  38. Qiu WP, Hu O, Paalocci N, Ziegelstein RC, Kass DA. Differential effects of pulsatile versus steady flow on coronary endothelial membrane potential. Am J Physiol Heart Circ Physiol 285: H341–H346, 2003.[Abstract/Free Full Text]
  39. Redmond EM, Cahill PA, Sitzmann JV. Perfused transcapillary smooth muscle and endothelial cell co-culture: a novel in vitro model. In Vitro Cell Dev Biol Anim 31: 601–609, 1995.[CrossRef]
  40. Santaguida S, Janigro D, Hossain M, Oby E, Rapp E, Cucullo L. Side-by-side comparison between dynamic versus static models of blood-brain-barrier in vitro: a permeability study. Brain Res 1109: 1–13, 2006.[CrossRef][ISI][Medline]
  41. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76–85, 1985.[CrossRef][ISI][Medline]
  42. Song L, Ge S, Pachter JS. Caveolin-1 regulates expression of junction-associated proteins in brain microvascular endothelial cells. Blood 109: 1515–1523, 2006.[ISI][Medline]
  43. Stanness KA, Westrum LE, Fornaciari E, Mascagni P, Nelson JA, Stenglein SG, Myers T, Janigro D. Morphological and functional characterization of an in vitro blood-brain barrier model. Brain Res 771: 329–342, 1997.[CrossRef][ISI][Medline]
  44. Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vasc Biol 18: 677–685, 1998.[Abstract/Free Full Text]
  45. Tunkel AR, Scheld WM. Pathogenesis and pathophysiology of bacterial meningitis. Annu Rev Med 44: 103–120, 1993.[CrossRef][ISI][Medline]
  46. Tzima E, del Pozo MA, Shattil SJ, Chien S, Schwartz MA. Activation of integrins in endothelial cells by fluid shear stress mediates Rho-dependent cytoskeletal alignment. EMBO J 20: 4639–4647, 2001.[CrossRef][ISI][Medline]
  47. Tzima E, del Pozo MA, Kiosses WB, Mohamed SA, Li S, Chien S, Schwartz MA. Activation of Rac-1 by shear stress in endothelial cells mediates both cytoskeletal reorganization and effects on gene expression. EMBO J 21: 6791–6800, 2002.[CrossRef][ISI][Medline]
  48. von Offenberg Sweeney N, Cummins PM, Cotter EJ, Fitzpatrick PA, Birney YA, Redmond EM, Cahill PA. Cyclic strain-mediated regulation of vascular endothelial cell migration and tube formation. Biochem Biophys Res Commun 329: 573–582, 2005.[CrossRef][ISI][Medline]
  49. Williams KC, Ulvestad E, Hickey WF. Immunology of multiple sclerosis. Clin Neurosci 2: 229–245, 1994.[ISI][Medline]
  50. Wojciak-Stothard B, Ridley AJ. Rho GTPases and the regulation of endothelial permeability. Vascul Pharmacol 39: 187–199, 2002.[CrossRef][ISI][Medline]




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