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γ-Aminbuturic Acid A Receptor Mitigates Homocysteine-Induced
Endothelial Cell Permeability
Neetu Tyagi, Karni S. Moshal, Suresh C. Tyagi, and David Lominadze
Department of Physiology and Biophysics, University of Louisville, Louisville, Kentucky, USA
Abstract
Many cerebrovascular disorders are accompanied by an increased homocysteine (Hcy) levels. We
have previously shown that acute hyperhomocysteinemia (HHcy) leads to an increased microvascular
permeability in the mouse brain. Hcy competitively binds to γ -aminbuturic acid (GABA) receptors
and may increase vascular permeability by acting as an excitatory neurotransmitter. However, the
role of GABA-A (GABAA) receptor in Hcy-induced endothelial cell (EC) permeability remains
unclear. In the present study we attempted to determine the role of GABAA receptor and the possible
mechanisms involved in Hcy-induced EC layer permeability. Mouse aortic and brain ECs were grown
in Transwells and treated with 50 μM Hcy in the presence or absence of GABAA-specific agonist
muscimol. Role of matrix metalloproteinase-9 (MMP-9) was determined using its activity inhibitor
GM-6001. Involvement of extracellular signal-regulated kinase (ERK) signaling was assessed using
its kinase activity inhibitors PD98059 or U0126. EC permeability to the known content of bovine
serum albumin (BSA)-conjugated with Alexa Flour-488 was assessed by measuring fluorescence
intensity of the solutes in the Transwell's lower chambers. It was found that Hcy induced the formation
of filamentous actin (F-actin). Hcy-induced EC permeability to BSA was significantly decreased by
GABA and muscimol treatments. Presence of MMP-9 or ERK kinase activity inhibitors restored the
Hcy-induced EC permeability to its baseline level. The mediation BSA leakage through the ECs was
further confirmed in the experiments where Hcy-induced alterations in transendothelial electrical
resistance of confluent ECs were assessed. The data suggest that Hcy increases EC layer permeability
through inhibition of GABAA receptor and F-actin formation, in part, by transducing ERK and
MMP-9 activation.
Keywords
Albumin Leakage; Endothelial Gap Formation; ERK; F-Actin; Muscimol
Homocysteine (Hcy) is a homologue of the naturally occurring amino acid, cysteine. Normal
concentration of Hcy in plasma is in the range of 5 to 10 μM. Increased blood level of Hcy is
known as hyperhomocysteinemia (HHcy). There are three ranges of HHcy: moderate (10 to
30 μM), intermediate (31 to 100 μM), and severe (>100 μM). Increased blood levels of Hcy
Copyright © Informa Healthcare USA, Inc.
Address correspondence to David Lominadze, PhD, Department of Physiology and Biophysics, Health Sciences Center, A-1115,
University of Louisville, Louisville, KY 40292, USA. dglomi01@louisville.edu.
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NIH Public Access
Author Manuscript
Endothelium. Author manuscript; available in PMC 2010 February 10.
Published in final edited form as:
Endothelium. 2007 ; 14(6): 315. doi:10.1080/10623320701746164.
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have been linked to increased risk of premature coronary artery disease, stroke, and
thromboembolism (Lentz 2005; Lee et al. 2006). Elevation of blood Hcy content coincides
with an increased blood concentration of well-accepted inflammatory markers such as C-
reactive protein (Hackam and Anand 2003), interleukin-6 (Araki et al. 2005), and fibrinogen
(Zoccali et al. 2002). Because of this association, it was postulated that an increase in Hcy
content may promote vascular inflammation (Lentz 2005). In addition, it was shown that
increased level of Hcy may cause interleukin-6 accumulation in the monocytes (van Aken et
al. 2000), suggesting an association of HHcy and an enhanced formation of interleukin-6, the
latter being involved in synthesis of another inflammatory agent fibrinogen (Vasse et al.
1996). However, according to the recent Glasgow Myocardial Infarction Study, correlation of
Hcy with the inflammatory markers such as fibrinogen, C-reactive protein, and interleukin-6
was not significant (Woodward et al. 2006), suggesting that, within the population range,
increasing Hcy contents have no effect on the systemic inflammatory response. Therefore, it
can be stated that Hcy is an independent risk factor for cardiovascular diseases and stroke
(Seshadri et al. 2002; Tyagi 1999).
Many cardiovascular diseases, e.g., hypertension and diabetes, are considered inflammatory
diseases. Increased microvascular permeability, also typical to these diseases, is one of the
manifestations of inflammation. A net loss of blood plasma components into the interstitium
causes edema. Albumin, the most abundant plasma protein, helps maintain the oncotic pressure
and protects endothelial barrier integrity by its interaction with glycocalyx (Huxley and Curry
1985, 1987). Thus, changes in albumin transport through the endothelial cell (EC) layer may
cause significant damage to tissue and induce inflammatory responses (Mehta and Malik
2006).
It has been shown that HHcy induced by folate depletion caused increased arterial permeability
and stiffness in rats (Symons et al. 2002, 2006). The results of our previous study suggest that
an acute increase in blood Hcy content enhances leakage of pial microvessels in mice
(Lominadze et al. 2006). Although the role of Hcy-induced matrix metalloproteinase-9
(MMP-9) activation in vascular remodeling is well known, the mechanism of Hcy-induced
increased microvascular permeability was not clear. Along with extracellular matrix (ECM)
degradation, tissue plasminogen activator causes proteolytic shedding of γ-aminobutyric acid
(GABA) receptors (Mataga et al. 2002; Frey et al. 1996). GABA receptors play a significant
role in brain microvascular permeability, which if inhibited, alters the neuronal environment
and leads to ECM degradation and edema (Lee et al. 1995; Limmroth et al. 1996; Fruscella et
al. 2001; Lazzarini et al. 2001). Hcy competes with GABA receptors and acts as an excitatory
neurotransmitter. Therefore, an increase in blood Hcy level may attenuate the function of
GABA receptor, leading to the disruption of EC layer integrity. In the present study, we tested
the hypothesis that high levels of Hcy may increase EC leakage to albumin through extracellular
signal regulated kinase (ERK) signaling and filamentous actin (F-actin) formation. The role of
GABA-A (GABAA) receptor in Hcy-induced EC permeability is shown.
METHODS
Materials and Reagents
Bovine serum albumin (BSA) 1-palmitoyl-sn-glycero-3-phosphocholine (LPC), Hcy,
fibronectin, and muscimol (GABAA receptor–specific agonist) were purchased from Sigma
(St. Louis, MO). Specific inhibitors of the mitogen-activated protein kinase known as
extracellular signal-regulated kinase (ERK), kinase, PD98059 (2′-amino-3′-methoxyflavone),
and U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), and GM-6001
(MMP activity blocker) were purchased from Calbiochem (La Jolla, CA). Monoclonal
antibody to phosphorylated ERK1/2 (p44/42) was purchased from Cell Signaling Technology
(Beverly, MA) and the antibody to ERK2 was purchased from Santa Cruz Biotechnology
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(Santa Cruz, CA). Horseradish peroxidase (HRP)-conjugated antibody was obtained from
Cappel Laboratories (Durham, NC). BSA conjugated with Alexa Flour-488 dye (BSA-488),
Alexa Flour-594 Phalloidin (300 U), and a secondary antibody conjugated with Texas Red dye
were purchased from Molecular Probes (Eugene, OR).
Endothelial Cell Culture
Mouse aortic endothelial cells (MAECs) were a generous gift from Kathleen Bove, Stratton
VA Medical Center, Albany, NY. The endothelial nature of the cells was verified by uptake
of acylated low-density lipoprotein and positive staining for CD-31 (Lincoln et al. 2003).
Mouse brain endothelial cells (MBECs) were purchased from American Type Culture
Collection (ATCC, Manassas, VA) at 21th passage. The endothelial nature of these cells was
confirmed by the observed expression of von Willebrand factor and uptake of fluorescently
labeled low-density lipoprotein (Montesano et al. 1990). The MAECs were grown in
Dulbecco's modified Eagle's medium/Ham's F-12 50/50 mix (DMEM/F12–50/50; Cellgrow,
Mediatech, Herndon, VA), whereas MBEC were grown in DMEM (ATCC). Both culture
media were supplemented with 10% fetal bovine serum (HyClone, Logan, UT) 1% L-glutamine
and 1% penicillin-streptomycin (Gibco, Grand Island, NY). Cells were grown at 37°C at 5%
CO2 in a humidified environment. For the experimentation, MAECs and MBECs were used
at the 10th and 22nd passages, respectively.
Endothelial Cell Permeability Assays
Albumin leakage through endothelial cell monolayer was assessed according to the method
described earlier (Tyagi et al. 2007b). Transwell permeable supports (Corning, Corning, NJ)
with polycarbonate membranes (Nuclepore Track-Etch; 6.5 mm in diameter, 0.4-μm pore size,
and pore density of 108/cm2) were coated with fibronectin for 1 h. The membranes were seeded
with MAECs or MBECs and the cells were grown to confluence. To confirm the cell confluence
and the presence of an intact monolayer on the membranes, cells in a separate test well (not
membrane) were monitored by a light microscope. Cells in the test membranes were labeled
with fluorescence (2′ ,7′-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl
ester [BCECF, AM]; Molecular Probes, Eugene, OR) and observed under a microscope (Carl
Zeiss Axiovert-100, objective 10×) with a fluorescence filter (488-nm excitation and 516-nm
emission) for absence of visible gaps in the cell monolayer. The permeability assays were done
after confirming that the cells in the test wells and Transwell membranes were fully confluent
and formed an intact monolayer.
Unlabeled BSA was added to each well to maintain its concentration similar to a normal plasma
concentration of albumin (440 μM) and maintain its activity coefficient close to that in blood
(Rivas et al. 1999). The surface levels of solutions in the luminal (200 μL) and abluminal (600
μL) compartments of the Transwells were the same. The experimental set-up is similar to that
used by Cooper et al. (1987), but the presence of albumin-mediated osmotic pressure in the
present study makes it closer to in vivo conditions (Tyagi et al. 2007b).
For the permeability assay, cells were washed with phosphate-buffered saline (PBS) and
incubated with various doses of Hcy (5, 25, or 50 μM) for 24 h, or with 50 μM of Hcy for 1,
12, or 24 h. To determine a possible role of GABAA receptor, MMPs, and the mitogen-activated
protein kinase kinase (MEK) signaling in Hcy-induced EC permeability, the following
substances were added to the Transwells: Hcy (50 μM) with muscimol (50 μM), Hcy (50 μM)
with MEK inhibitors (PD98059 or U0126; 50 μM each), Hcy (50 μM) with GABA (50 μM),
or Hcy (50 μM) with GM6001 (1 μM).
To determine the effect of GABA, muscimol, GM6001, and MEK inhibitors on EC
permeability to albumin, the following substances were added to other Transwells without Hcy:
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GABA (50 μM), muscimol (50 μM), GM6001 (1 μM), PD98059 (50 μM), or U0126 (50 μM).
Cells incubated with medium alone were used as a control group. BSA-488 (3 mg/mL) was
added to each of the wells described above and experiments were performed five times in
duplicate for each treatment.
The cells were incubated in humidified conditions at 37°C. After incubation, media samples
were collected from lower and upper wells. Fluorescence intensity of the samples was measured
by a microplate reader (SpectraMax M2; Molecular Devices, Sunnyvale, CA) with excitation
at 494 nm and emission at 518 nm. Results are expressed as fluorescence intensity units (FIU)
and presented as a percent of the control values.
In additional experiments, permeability of the Nuclepore membranes alone to albumin was
compared to albumin leakage through the EC layer in Tranwells. The results showed that
albumin leakage through the membrane alone (583 ± 34 FIU) was about nine times greater
than that (66 ± 12 FIU) in the presence of the cells.
Transendothelial electrical resistance measurement was preformed using an electrical cell-
substrate impedance system (ECIS; Applied Biophysics, Troy, NY). ECs were seeded onto
evaporated gold microplates of the ECIS. The cells were grown to a confluent monolayer that
covered the microelectrodes of the system's 8-well chamber (Tiruppathi et al.1992). Effect of
Hcy (50 μM) was determined in the presence or absence of muscimol (50 μM), MEK inhibitors
(PD98059 or U0126; 50 μM each), GABA (50 μM), or with GM6001 (1 μM). To determine
the effect of GABA, muscimol, GM6001, and MEK inhibitors on transendothelial electrical
resistance of the cells, the following substances were added to wells without Hcy: GABA (50
μM), muscimol (50 μM), GM6001 (1 μM), PD98059 (50 μM), or U0126 (50 μM). Cells
incubated with medium alone were used as a control group. Resistance values from each
microelectrode were collected and plotted as relative resistance versus time.
F-Actin Formation Assay
Formation of F-actin in cultured ECs was studied according to the method described earlier
(Lominadze et al. 2004, 2006; Tyagi et al. 2007). Briefly, MAECs and MBECs were grown
until confluent in 8-well coverglass plates coated with fibronectin. The cells were washed and
incubated with or without PD98059 or U0126 (50 μM each), muscimol (50 μM), or GABA
(50 μM), followed by Hcy (50 μM) treatment for 24 h. To determine the effect of GABA,
muscimol, PD98059, or U0126 on F-actin formation, the separate groups of cells were
incubated with following substances without Hcy: GABA (50 μM), muscimol (50 μM),
PD98059 (50 μM), or U0126 (50 μM). Cells incubated with medium alone were used as a
control. After the treatments the cells were washed twice with PBS and incubated with Alexa
Flour-594 Phalloidin (10 U) and LPC (100 μg/mL, dissolved in 3.7% formaldehyde) for 30
min at 4°C in the dark (Lominadze et al. 2006). After incubation, the cells were washed with
PBS and digital images of intracellular F-actin were taken with the confocal microscope
(Olympus FV1000, objective 100×) using a HeNe-G laser (596 nm) to excite the dye, whereas
emission was observed above 620 nm. The images were compared for the formation of
individual stress fibers and presence of actin foci (Lominadze et al. 2006).
In separate experiments, Hcy-induced formation of F-actin in ECs were compared to F-actin
formation induced by thrombin. The cells were treated with or without 50 μMHcy for 24 h and
0.5 U/mL thrombin for 15 min (Qiao et al. 1995;Trepat et al. 2005; Tyagi et al. 2007) and
analyzed for intracellular F-actin formation using confocal microscopy.
Hcy-induced formation of F-actin fibers was assessed for each well by analyzing the total
fluorescence intensity in four random fields with image analysis software (Image-Pro Plus;
Media Cybernetics). Four experiments were done to study Hcy-induced F-actin formation and
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three experiments were done to study Hcy- and thrombin-induced F-actin formation. All the
experiments were done in duplicate (two wells per experimental group). Results are expressed
as a percent of the control values.
Western Blot Analysis for ERK Phosphorylation
Hcy-induced phosphorylation of ERK was studied using a method described previously
(Moshal et al. 2006). Briefly, MAECs were grown in 6-well plates (Corning, Corning, NY)
until they become 80% to 90% confluent. Before experimentation, cells were serum starved
by depriving fetal bovine serum to 0.01% in the medium for 16 h. Then cells were incubated
with one of the following: Hcy (5 or 50 μM), Hcy (50 μM) with muscimol (50 μM), Hcy (50
μM) with MEK inhibitors (PD98059 or U0126; 50 μM each), or Hcy (50 μM) with GABA (50
μM) at 37°C for 24 h. Other cells were incubated with one of the following: muscimol (50
μM), PD98059 or U0126 (50 μM each), or GABA (50 μM) at 37°C for 24 h. Cells incubated
with low-serum medium were used as a control.
After incubation, the cells were washed twice with ice-cold 1× PBS and lysed with ice-cold
lysis buffer (composition: Tris·HCl [50 mM], NaCl [150 mM], Triton X-100 [1%], and EGTA
[1 mM], pH 7.4) supplemented with phenylmethylsulfonyl fluoride (1 mM), leupeptin (1 μg/
mL), sodium orthovan-date (200 μM), and aprotinin (1 μg/mL). Cell lysates were assayed for
protein concentration using Bradford assay (Bradford 1976). Equal amount of protein was
resolved on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
and blotted onto a polyvinylidene difluoride (PVDF) membrane. After being transferred, blots
were washed with Tris-buffered saline with 0.1% Tween-20 (TBST) for 5 min at room
temperature and blocked with 5% BSA in TBST for 1 h at room temperature. The blots were
then incubated with polyclonal anti-rabbit phospho-ERK1/2 antibody (1:1000 dilution)
overnight at 4°C with gentle agitation. The blots were washed three times (8-min wash each
time) with TBST and incubated with appropriate secondary antibody (1:3000 dilution).
Proteins of interest were immuno detected using an enhanced chemiluminescence (ECL) plus
kit (Amersham Biosciences, Piscataway, NJ). The membranes were stripped and reprobed for
ERK2 as loading control. The blots were analyzed with Gel-Pro Analyzer software (Media
Cybernetics, Silver Spring, MD) as described earlier (Lominadze et al. 2002). The protein
expression intensity was assessed by integrated optical density (IOD), i.e., the area of the band
in the lane profile. To account for possible differences in the protein load, the results of the
measurements are presented as a ratio of IOD of each band under the study (phosphorylated
ERK1/2) to the IOD of the respective total protein (total ERK2).
Statistics
All data are expressed as mean ± SEM. The experimental groups were compared by one-way
analysis of variance (ANOVA). If ANOVA indicated a significant difference (p < .05), Tukey's
multiple comparison test was used to compare group means. Differences were considered
statistically signifi-cant if p < .05.
RESULTS
Hcy induced a dose-dependant increase in the permeability of the EC layer to albumin as
determined by the fluorescence intensity of albumin that leaked though MAECs into the lower
wells of the Transwells (Figure 1). The permeability of the MAECs to albumin, induced by
the highest dose of Hcy (50 μM), was increased (128% ± 9%, 186% ± 25%, 303% ± 34 % of
control) with treatment time (1, 12, and 24 h, respectively). The permeability of MAEC layer
to albumin induced by Hcy (50 μM) during 24 h was decreased significantly by GABA,
muscimol, GM6001, and MEK inhibitors (PD98059 or U0126) (Figure 1). In the absence of
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Hcy, treatment of MAECs with GABA, muscimol, GM-6001, or MEK inhibitors (PD98059
or U0126) had no effect on albumin leakage through MAEC layer (Table 1).
The enhanced permeability of the MBECs to albumin, induced by Hcy (50 μM), was decreased
significantly by GABA, muscimol, MEK inhibitors (PD98059 or U0126), and GM6001
(Figure 2). In the absence of Hcy, treatment of MBECs with GABA, muscimol, MEK inhibitors
(PD98059 or U0126), or GM-6001 had no effect on MBEC albumin leakage (Table 2).
In separate experiments, Hcy treatment significantly decreased transendothelial electrical
resistance compared to untreated control MBECs (Figure 3). This decrease in resistance was
significantly less in the presence of muscimol and GABA (Figure 3). Treatment of the MBECs
with GABA and muscimol had no effect on transendothelial electrical resistance (Figure 3).
Hcy caused a dose-dependant increase of F-actin formation in MAECs (Figure 4). The
formation of F-actin, induced by the highest dose of Hcy (50 μM), was significantly decreased
by GABA, muscimol, or by MEK inhibitors (PD98059 or U0126) (Figure 4). Formation of F-
actin in the MAECs treated with GABA, muscimol, or with MEK inhibitors (PD98059 or
U0126) alone was not different from that in control group (Table 3). The treatment of ECs with
Hcy (50 μM) for 24 h caused the formation of F-actin (160% ± 6% of control), which was
similar to that (141% ± 4% of control) induced by thrombin (0.5 U/mL for 15 min) (Figure
4A). In the parallel series of experiments, treatment of MBECs with Hcy (50 μM) for 24 h
caused the formation of F-actin, which was not different from that induced by thrombin (0.5
U/ml for 15 min) (Figure 5).
Hcy induced a dose-dependant increase of ERK phosphorylation in MAECs that was
significantly decreased in the presence of muscimol (Figure 6). Treatment of MAECs with
muscimol alone did not alter ERK phosphorylation in the ECs compared to that in the control
group (Figure 6).
DISCUSSION
The present study shows that pathological concentrations of Hcy (<12 μM) can enhance
albumin leakage through an EC monolayer. These results coincide with our data showing that
Hcy increases albumin leakage of pial vessels in mice (Lominadze et al. 2006). It is known
that thrombin-induced intercellular gaps are accompanied by formation of F-actin (Qiao et al.
1995). In the present study, the treatment of the ECs with higher than normal dose of Hcy (50
μM) for 24 h caused F-actin formation to the level that was caused by thrombin (0.5 U/mL 15
min; Figure 4), which is known to increase F-actin formation (Trepat et al. 2005). These data
suggest that an increased concentration of Hcy in the blood stream has a role in increasing EC
permeability similar to thrombin.
Increased content of Hcy may enhance albumin leakage by inducing the formation of F-actin
(Figures 4 and 5), which may cause stiffening of the cells and opening of interendothelial
junctions (IEJs). Role of Hcy on mouse brain IEJs was clearly shown using ECIS (Figure 3),
where, the effect of Hcy was significantly diminished by GABA or muscimol. The results of
the present study also coincide with our previous results, where we showed that Hcy may affect
EC by activating MMPs (Lominadze et al. 2006; Moshal et al. 2006). The present results show
a restoration of Hcy-induced albumin leakage through EC layer in the presence of MMP
inhibitor (GM6001), which confirms the role of MMPs in Hcy-induced microvascular leakage
(Lominadze et al. 2006). Changes in MMPs may cause subendothelial matrix remodeling by
altering collagen/elastin ratio (Shastry et al. 2006) and target focal adhesion molecules and cell
junction molecules at the basal side of the ECs, such as cadherins and/or connexins (Mehta
and Malik 2006).
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Significant decrease in Hcy-induced albumin leakage and F-actin formation by GABA or its
agonist, muscimol, suggests that Hcy may directly affect EC properties through binding to
GABAA receptor. This binding suppressed the activity of GABAA receptor (Shastry et al.
2006), making Hcy an excitatory neurotransmitter. Our studies show that mouse EC also
express GABAA receptor, and its activity can be suppressed by Hcy (Tyagi et al. 2007a). The
results of the present study suggest that the activation of GABAA receptor may prevent Hcy-
induced EC leakage and therefore highlight the functional role of GABAA receptor in
endothelium and its possible involvement in Hcy-induced EC layer permeability.
Mitogen-activated protein kinase kinase (MEK) is a protein kinase that is phosphorylated and
activated by Raf. Once activated, MEK phosphorylates and activates ERK (Kolch 2000).
PD9805 and U0126 used in the present study are chemically unrelated MEK/ERK inhibitors
with different mechanisms of action (Alessi et al. 1995; Favata et al. 1998). Whereas PD98059
binds to the inactive forms of MEK and prevents its activation by upstream activators such as
Raf (Alessi et al. 1995), U0126 binds to the activated form of MEK (Favata et al. 1998). It is
known that formation of F-actin can occur through ERK signaling (Bourguignon et al. 2005;
Chandrasekar et al. 2003). Furthermore, ERK signaling pathway is involved in expression of
GABAA receptor (Bulleit and Hsieh 2000; Kalluri and Ticku 2002). Our data show that Hcy-
induced albumin leakage may be regulated by ERK signaling (Figure 6). Furthermore, these
results, in combination with the data showing decreased albumin leakage through EC layer in
the presence of GABA or muscimol, suggest that competitive binding of Hcy to GABAA
receptor on EC surface involves the activation of MEK/ERK signal. In our previous study, we
showed that increased levels of Hcy enhance phosphorylation of ERK and activate MMP-9
(Moshal et al. 2006). The present study indicates the role of MMPs in Hcy-induced albumin
leakage through the EC layer. Increased content of Hcy enhances EC layer permeability, which
involves inhibition of GABAA receptor activity and formation of F-actin through ERK
signaling. Our data related to the muscimol-induced increase ERK phosphorylation confirm
the result by others showing that muscimol activates ERK signaling (Obrietan et al. 2002).
However, muscimol-induced ERK phosphorylation was not as robust as the one induced by
50 μM Hcy. The treatment of the cells with muscimol prior to addition of Hcy (50 μM) did not
increase ERK phosphorylation to the extent that was caused by the same dose of Hcy. This
suggests that msucimol competes with Hcy for GABAA receptor binding sites and induces
ERK phosphorylation.
Increased blood Hcy concentration may increase binding of Hcy to endothelial GABAA
receptors. Cytoskeletal ezrinradixin-moesin (ERM) family protein radixin has been identified
as an essential clustering factor that anchors GABAA receptor with actin cytoskeleton of a cell
(Tsukita and Yonemura 1999; Loebrich et al. 2006). A role of radixin in ERK signaling pathway
has been suggested (Jung et al. 2005). In the present study the role of radixin was not evaluated;
however, the role of GABAA receptor in radixin-mediated ERK signaling pathway can't be
ruled out during Hcy-induced endothelial monolayer leakage, which may cause the formation
of F-actin in ECs. F-actin formation may increase the rigidity of the cells, widen the IEJ, and
may even increase gaps between the cells (Ehringer et al. 1999; Gordon et al. 2005; Lominadze
et al. 2004, 2006). The enlarged IEJ gaps may be enough to allow albumin (diameter ~5 nm)
to pass though the EC monolayer.
Increased blood Hcy concentration is typical for many cardiovascular and cerebrovascular
diseases (Seshadri et al. 2002; Tyagi et al. 1999). Almost all of these diseases are accompanied
with increased ECs permeability. We provide the evidence that elevated levels of Hcy have a
role in increasing EC layer permeability through its binding to endothelial GABAA receptor
and suppressing its activity.
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Limitations of the Present Study
The results of the present study show that elevated content of Hcy increases EC layer
permeability to albumin through IEJs. However, the design of our experiments did not permit
us to rule out transcellular migration of albumin (Mehta and Malik 2006). In addition to
affecting IEJs, Hcy may alter transcytosis affecting caveolae-involved an absorptive or fluid-
phase pathways, or formation of transendothelia channels. The experiments to address the role
of Hcy in transendothelial albumin transport are in progress.
Acknowledgments
This work was supported in part by the American Heart Association (AHA), National, SDG (grant 0235317N), NIH
(grant HL-080394 to DL, grants HL-71010 and NS-051568 to SCT), and the AHA, postdoctoral training grant (award
0625579B) to KSM.
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FIG. 1.
Hcy-induced albumin leakage through the mouse aortic endothelial cell monolayer.
Fluorescence intensity of bovine serum albumin conjugated with Alexa Flour-488 detected in
lower chambers of Transwells. *p < .05 versus 5 μM of Hcy. †p < .05 versus the lower dose
of Hcy. #p < .05 versus 50 μMof Hcy. Number of experiments n = 5 for all groups.
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FIG. 2.
Hcy-induced albumin leakage through the mouse brain microvascular endothelial cell
monolayer. Fluorescence intensity of bovine serum albumin conjugated with Alexa Flour-488
detected in lower chambers of Transwells. Hcy, GABA, muscimol, and MEK inhibitors,
PD98059 and U0126, were used at concentration of 50 μM each, whereas GM6001 was 1
μM. * p < .05 versus Hcy. Number of experiments n = 4 for all groups.
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FIG. 3.
Changes in relative resistance of the confluent mouse brain endothelial cell layers after
treatment with Hcy (•), GABA and Hcy (○), muscimol and Hcy (▶), GABA (▽), and muscimol
(◀). Cells in the control group (*) were grown in medium alone. Hcy, GABA, and muscimol
were used at concentration of 50 μM each. Number of experiments n = 4 for all groups.
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FIG. 4.
Hcy-induced F-actin formation in mouse aortic endothelial cells. (A) Examples of images of
Hcy- and thrombin-induced F-actin formation (objective 100×). (B) Total fluorescence
intensity changes of F-actin staining induced by Hcy (5 and 50 μM) and inhibition of 50 μM
Hcy effect by GABA, muscimol, PD98059, and U0126 (50 μM each). * p < .05 versus 5 μM
of Hcy. # p < .05 versus 50 μM of Hcy. Each data point represents the results of duplicate
determinations from four separate experiments (n = 4).
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FIG. 5.
Hcy-induced F-actin formation in mouse brain endothelial cells. (A) Examples of images of
Hcy- and thrombin-induced F-actin formation (objective 100×). (B)Total fluorescence
intensity changes of F-actin staining induced by 50 μM of Hcy (24 h) and 0.5 U/ml of thrombin
(15 min). Each data point represents the results of duplicate determinations from four separate
experiments (n = 4).
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FIG. 6.
Hcy-induced ERK phosphorylation in MAECs. (A) A representative Western Blot for Hcy-
induced ERK1/2 phosphorylation and its inhibition in the presence of muscimol is shown
(top). Membranes were reprobed for total ERK2 (bottom). Phosphorylation of ERK in the cells
treated with medium alone was used as a control group. (B) ERK1/2 phosphorylation induced
by 50 μMHcy. The data are presented as ratios of integrated optical density (IOD) of each
phoshorylated ERK1/2 band to IOD of the respective total ERK2. Bars represent averages ±
SE from three different experiments (n = 3). * p < .05 versus control; #p < .05 versus 50 μM
of Hcy.
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TABLE 1
Effect of GABA, muscimol, GM6001, PD98059, or U0126 on albumin leakage through the mouse aortic
endothelial cell monolayer
GABA (50 μM) Muscimol (50 μM) GM6001 (1 μM) PD98059 (50 μM) U0126 (50 μM)
115 ± 13 121 ± 19 111 ± 12 108 ± 8 124 ± 21
Note. Data are presented as percent of control. Number of experiments n = 4.
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TABLE 2
Effect of GABA, muscimol, PD98059, U0126, or GM6001 on albumin leakage through the mouse brain
endothelial cell monolayer
GABA (50 μM) Muscimol (50 μM) PD98059 (50 μM) U0126 (50 μM) GM6001 (1 μM)
107 ± 12 101 ± 11 108 ± 8 111 ± 13 121 ± 14
Note. Data are presented as percent of control. Number of experiments n = 4.
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TABLE 3
Effect of GABA, muscimol, PD98059, U0126, or GM6001 on F-actin formation in the mouse aortic endothelial
cells
(50 μM) GABA (50 μM) Muscimol (50 μM) PD98059 (50 μM) U0126
109 ± 6 111 ± 6 98 ± 1 94 ± 3
Note. Data are presented as percent of control. Number of experiments n = 4.
Endothelium. Author manuscript; available in PMC 2010 February 10.
