Cardiovascular Research 60 (2003) 488 – 496
www.elsevier.com/locate/cardiores
Shear stress-induced up-regulation of the intermediate-conductance
Ca2+-activated K+ channel in human endothelium
Susanne Brakemeier a, Anne Kersten a, Ines Eichler a, Ivica Grgic a, Andreas Zakrzewicz b,
Hartmut Hopp c, Ralf Köhler a,*, Joachim Hoyer a
a
Department of Nephrology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany
b
Department of Physiology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Arnimallee 22, D-14195 Berlin, Germany
c
Department of Gynecology, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, D-12200 Berlin, Germany
Received 8 April 2003; received in revised form 27 August 2003; accepted 9 September 2003
Time for primary review 29 days
Abstract
Objective: Wall shear stress associated with blood flow is a major stimuli for generation of endothelial vasodilating and antithrombotic
factors and it also regulates endothelial gene expression. Activation of endothelial intermediate-conductance Ca2 +-activated K+ channels
(IKCa) is important for the control of endothelial function by inducing cell hyperpolarization and thus generation of the endothelium-derived
hyperpolarizing factor. In the present study we tested whether the IKCa encoding IKCa1 gene is regulated by laminar shear stress (LSS).
Methods: Human umbilical vein endothelial cells (HUVEC) were subjected to LSS with a magnitude of 0.5 – 15 dyn/cm2 and time intervals
of 2 – 24 h in a flow cone apparatus. Expression of the IKCa1 gene and IKCa-functions were determined by using real time RT-PCR and
patch-clamp techniques. Results: A short 2 – 4 h—or long 24 h—exposure to a LSS with a low (venous) magnitude of 0.5 dyn/cm2 had no
effect on IKCa1 expression levels. An exposure for 2 and 4 h to LSS with an intermediate magnitude of 5 dyn/cm2 was also ineffective,
whereas an exposure for 24 h induced a significant threefold up-regulation of IKCa1 expression levels. An exposure to LSS with a higher
(arterial) magnitude of 15 dyn/cm2, resulted in an eightfold up-regulation of IKCa1 expression levels after a 4 h—exposure and a fourfold
increase of IKCa1 expression levels at 24 h. The increased IKCa1 expression levels following exposure to high levels of LSS resulted in
enhanced IKCa whole-cell currents and in an increased hyperpolarization of the endothelium in response to ATP and the IKCa opener 1-EBIO.
Inhibition of the mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK) kinase 1/2 (MEK/ERK) pathway by PD98059
prevented the LSS-induced up-regulation of IKCa1 expression levels and IKCa whole-cell currents indicating that augmentation of IKCa1
expression levels is mediated by the LSS-induced activation of the MEK/ERK pathway. Conclusion: Long term exposure to LSS upregulates expression and function of endothelial IKCa. This increase might represent a new important mechanism in endothelial adaptation to
altered hemodynamics.
D 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Blood flow; Endothelial function; Signal transduction; Gene expression; IKCa1; K-channel
This article is referred to in the Editorial by E. van
Bavel (pages 457 – 459) in this issue.
1. Introduction
In vivo, the vascular endothelium is constantly exposed
to wall shear stress generated by the streaming blood.
* Corresponding author. Tel.: +49-30-84452398; fax: +49-3084452398.
E-mail address: koe@zedat.fu-berlin.de (R. Köhler).
Alterations in shear stress levels lead to complex changes
in endothelial functions [1,2]. Early responses to elevated
shear stress include activation of ion channels, cell hyperpolarization, elevation of the intracellular Ca2 + concentration, and protein phosphorylation stimulating the synthesis
of vasodilatory factors such as NO, EDHF, and prostacyclin
[2,3]. Later responses include endothelial remodeling and
modulation of gene expression levels such as of endothelial
nitric oxide synthase (eNOS), prostacyclin, cyclooxygenase,
connexins, and adhesion molecules [1,4 – 6].
This shear stress-dependent modulation of gene expression was related to long term vessel adaptation and vaso-
0008-6363/$ - see front matter D 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2003.09.010
S. Brakemeier et al. / Cardiovascular Research 60 (2003) 488–496
regulation especially in disease states like hypertension [1].
Moreover, shear stress induced modulation of gene expression levels is regarded as an atheroprotective mechanism by
increasing the generation of antithrombotic and antimitogenic factors [8].
With respect to mechanisms by which the endothelium
senses alterations in shear stress levels, opening of mechanosensitive cation channels and Ca2 +-activated or inwardly
rectifying K+-channels have been proposed to play an
important role by inducing mechanosensitive Ca2 +-entry
and membrane hyperpolarization [9– 13], respectively, as
part of the very rapid responses to elevated shear stress.
In particular, intermediate-conductance Ca2 +-activated
+
K channels (IKCa) encoded by the IKCa1 gene [14 – 19]
have been reported to induce endothelial hyperpolarization
within seconds following shear stress stimulation [11,12].
However, whether also a modulation of IKCa1 expression
occurs, maybe as part of the adaptive response of the
endothelium to altered hemodynamics, is still elusive.
Therefore, we tested whether shear stress regulates
endothelial IKCa1 gene expression in human endothelium.
The results show that shear stress stimulation leads to an
up-regulation of IKCa1 expression levels and an increase
of IKCa functions in human umbilical vein endothelium.
489
single cells (22 F 2 pF, n = 29) were recorded with an
EPC-9 patch-clamp amplifier (HEKA) using voltage
ramps (duration 1000 ms) from
120 to 100 mV and
were low-pass filtered ( 3 dB, 1000 Hz) at a sampling
time of 0.5 ms [17]. In another set of current-clamp
experiments, cells were not electrically uncoupled and
membrane hyperpolarization in response to ATP (1 AM),
and 1-ethyl-2-benzimidazolinone (1-EBIO, 100 AM) were
measured.
For activation of IKCa currents in whole-cell patchclamp experiments, cells were dialyzed with a pipette
solution containing (mM): KCl 135; MgCl2 1, EGTA 1,
CaCl2 0.955 ([Ca2 +]free = 3 AM), and HEPES 5 (pH 7.2).
The NaCl bath solution contained (mM): NaCl 137,
Na2HPO4 4.5, KCl 3, KH2PO4 1.5, MgCl2 0.4, and
CaCl2 0.7 (pH 7.4). For current-clamp experiments, the
pipette solution was prepared without CaCl2 to avoid
activation of IKCa by Ca2 + dialysis in the patch-clamped
cell. All experiments were performed at room temperature. Data analysis was performed as described previously
[17]. Series resistance (4– 10 MV) was not compensated
and symmetric background leak currents were subtracted
manually.
2.3. RNA isolation and quantitative realtime RT-PCR
2. Materials and methods
2.1. Cell culture and shear stress experiments
Human umbilical vein endothelial cells (HUVEC) were
isolated and cultured as described previously [20]. The
investigation conformed with the principles outlined in the
Declaration of Helsinki (Cardiovasc. Res. 1997;35:2 – 3).
For shear stress (LSS) experiments, HUVEC of second
passage were used to avoid senescence. After reaching
confluence in fibronectin-coated petri dishes, HUVEC were
subjected to defined LSS levels of 0.5, 5 and 15 dyn/cm2
for 2, 4 and 24 h in a cone-plate viscosimeter as described
previously [20]. For controls, each LSS experiment was
accompanied by HUVEC from the same cell preparation
and kept for equal time intervals under static conditions. In
additional subsets of experiments, cells were pretreated for
30 min with either the p38-inhibitor SB203580 (10 AM) or
the MEK1/2 inhibitor PD98059 (25 AM) before LSS
exposure.
2.2. Patch-clamp experiments
Patch-clamp experiments were carried out as described
previously [14]. Patch pipettes were pulled from borosilicate glass capillaries with 0.3 mm wall thickness and had
a tip resistance of 4 MV in symmetric KCl solution. To
induce complete electrical uncoupling, cells were pretreated for 5 min with 1 mM heptanol, a gap-junction
inhibitor. Membrane currents in the electrically uncoupled
Cells were harvested by cell scraping and RNA was
isolated and purified using TRIzol (Life Technologies,
Eggenstein, Germany), following the manufacturer’s
instructions. RNA (2 Ag) was reverse transcribed using
random hexamers (Boehringer, Mannheim, Germany) and
M-MLV reverse transcriptase (Life Technologies) in a 50Al reaction. Expression was quantified with an ABI Prism
7700 Sequence detection system (Perkin-Elmer Applied
Biosystems). Primer pairs and internal oligos for IKCa1,
eNOS, intercellular adhesion molecule 1 (ICAM1), von
Willebrand factor (vWF), and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (Table 1) were positioned in the
coding region and primer pairs spanned intronic sequences. Internal oligonucleotides (Biotez, Berlin, Germany)
were labeled with 6-carboxy-fluorescein (FAM) on the
5Vend and 6-carboxytetramethylrhodamine (TAMRA) on
the 3Vend. Identity of PCR products was verified by
sequencing and linearity of each PCR assay were confirmed by serial dilutions of cDNA.
Each 25 Al PCR reaction consisted of 500 nM forward
primer, 500 nM reverse primer, 150 nM probe, 3
Al cDNA, and 1 (final concentration) TaqMan Universal Master Mix (Perkin-Elmer Applied Biosystems). PCR
parameters were 50 jC 2 min, 95 jC 10 min, and 50
cycles at 95 jC 15 s, 60 jC 1 min.
The TaqManR software was employed to calculate a
threshold cycle (Ct) which is defined as the cycle at
which the reporter fluorescence is distinguishable from the
background in the extension phase of the PCR reaction
(ABI User Bulletin #2). Real-time RT-PCR signals for
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S. Brakemeier et al. / Cardiovascular Research 60 (2003) 488–496
Table 1
Primer pairs and internal oligos
Gene
Accession no.
Primer pairs
TaqMan probe
IKCa1
AF022797
5V-FAM-TGG TGA CGT GGT GCC GGG C-TAMRA-3V
eNOS
L26914
vWF
K03028
GAPDH
BC 013310
ICAM1
XM049516
F 5V-CATCACATTCCTGACCATCG-3V
R 5V-ACGTGCTTCTCTGCCTTGTT-3V
F 5V-CGGCATCACCAGGAAGAAGA-3V
R 5V-CATGAGCGAGGCGGAGAT-3V
F 5V-TGGGAGAAGAGGGTCACAGG-3V
R 5V-CATAATTTTACCTCCCTCAGCCA-3V
F 5V-CACCGTCAAGGCTGAGAACG-3V
R 5V-GCCCCACTTGATTTTGGAGG-3V
F 5V-CAAGAACCAGACCCGGGAG-3V
R 5V-TTTCCCGGACAATCCCTCTC-3V
IKCa1, eNOS, ICAM1, and vWF were standardized to
GAPDH by using the equation: CtX CtGAPDH = DCt,
where CtX is the value for the IKCa1, eNOS, ICAM1,
or vWF probe, and CtGAPDH is the value calculated for
GAPDH. The equation, DCtstatic DCtshear stress = DDCt,
was used to determine changes in expression levels of
the respective gene following LSS stimulation. Fold
increases in expression were calculated by the equation,
2DDCt = fold change in expression (ABI User Bulletin #2).
2.4. Reagents
5V-FAM-TTTAAAGAAGTGGCCAACGCCGTGAA-TAMRA-3V
5V-FAM-TGCCCACCCTTTGATGAACACAAGTG-TAMRA-3V
5V-FAM-CCCATCACCATCTTCCAGGAGCGA-TAMRA-3V
5V-FAM-CGTGTCCTGTATGGCCCCCGACT-TAMRA-3V
3. Results
3.1. Up-regulation of endothelial IKCa1 expression levels
by LSS
To evaluate the regulation of endothelial IKCa1 expression by LSS, we quantified IKCa1 expression in HUVECs
after different time intervals and magnitude of LSS exposure and in an equal number of static controls accompanying each LSS experiment. A LSS exposure for 2, 4 or
24 h with a magnitude of 0.5 dyn/cm2 (n = 3) as reported
PD98059 and SB203580 were obtained from TOCRIS
(Ballwin, MO). TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) was a kind gift of Dr. H. Wulff
(UCI, CA). All other chemicals and toxins were obtained
from Sigma (Deisenhofen, Germany).
2.5. Statistical analysis
Data are given as mean F SEM. Student’s t-test was used
to assess differences between groups. P-values of < 0.05
were considered significant.
Fig. 1. Shear stress-induced up-regulation of endothelial IKCa1 gene
expression levels. Quantitative real time RT-PCR analysis of IKCa1 gene
expression levels in human umbilical vein endothelial cells after exposure
to LSS of 0.5, 5 or 15 dyn/cm2 for the indicated time intervals.
DDCt = DCtstatic DCtshear stress (left axis). Data are given as mean F SEM;
*P < 0.05, n = 3 – 8.
Fig. 2. LSS increases IKCa functions in human umbilical vein endothelial
cells. (A) Representative whole-cell recordings of HUVEC exposed for 24
h to LSS with a magnitude of 15 dyn/cm2 and of HUVEC kept under static
conditions. IKCa currents were activated by dialysis of the cells with 3
Amol/l Ca2 + (B) Quantitative analysis of KCa current densities (pA/pF) at a
holding potential of 0 mV in HUVEC subjected to LSS with a magnitude of
15 dyn/cm2 for 4 h (cells, n = 9), 6 h (cells, n = 12), and 24 h (cells, n = 13)
compared to controls kept in stationary culture (cells, n = 21). Data are
given as mean F SEM. *P < 0.01 vs. static controls.
S. Brakemeier et al. / Cardiovascular Research 60 (2003) 488–496
to be present in veins [21] had no effects on IKCa1
expression levels (Fig. 1). Also an exposure for 2 and 4
h to a LSS with an intermediate magnitude of 5 dyn/cm2
(n = 5 each) did not induce considerable changes of IKCa1
expression levels. However, at this higher magnitude of
LSS, an exposure for 24 h (n = 6) resulted in a threefold
increase in IKCa1 expression levels (Fig. 1).
At a LSS with a higher magnitude of 15 dyn/cm2 as
reported to be present in arteries [21], the time course of
modulation of IKCa1 expression levels was different (Fig.
1). A steep eightfold increase in IKCa1 expression was
already observed after an exposure interval of 4 h (n = 5),
but with no significant increase at 2 h (n = 7). A longer
491
exposure for 24 h induced a fourfold increase in IKCa1
expression (n = 8), similar to the increase in channel expression levels detected after an exposure for 24 h to a LSS with
a magnitude of 5 dyn/cm2.
To relate this up-regulation of IKCa1 expression levels to
previously reported shear stress-sensitive or -insensitive
endothelial cell-specific gene regulation, we determined
expression of eNOS and ICAM1 as shear stress-sensitive
genes [5,7] and of vWF as a shear stress-insensitive gene
[22]. Similar to earlier reports on the regulation of eNOS
expression by LSS in HUVECs [7,23], a twofold upregulation of eNOS expression levels in HUVECs was only
present after a 24-h exposure to an arterial LSS of 15 dyn/
Fig. 3. Properties and pharmacology of IKCa in human umbilical vein endothelial cells. (A) IKCa currents after substitution of the 4 mM K+ bath solution (a) by
a 140 mM K+ solution (b). Inhibition of IKCa current in HUVEC by (B) TRAM-34 (100 nM), by (C) clotrimazole (CLT; 100 nM), and by (D) charybtotoxin
(ChTX; 100 nM). Apamin (APA; 100 nM), a selective blocker of SKCa had no blocking effects. (E) Concentration-dependent blockade of IKCa currents by
clotrimazole (o) KD 23 F 2 nM, TRAM-34 (.) KD 13 F 3 nM, or charybdotoxin (5) KD 5 F 1 nM, n = 3 – 4, for calculation of KD values, data points were
fitted to the Boltzmann equation. (F) Increase of IKCa currents by 1-EBIO (100 AM).
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Table 2
Endothelial hyperpolarizing response following exposure to LSS
Treatment
n
Static
LSS
LSS + PD98059
LSS + SB203580
8
5
4
4
RMP
(mV)
17 F 3
18 F 2
13 F 4
19 F 1
ATP
(1 AM)
(mV)
21 F 4
39 F 5*
19 F 5**
33 F 2
1-EBIO
(100 AM)
(mV)
30 F 5
49 F 6*
25 F 6**
42 F 7
RMP = resting membrane potential; n = number of experiments; LSS = laminar shear stress; values are given as mean F SEM.
* P < 0.05 vs. static controls.
** P < 0.05 vs. LSS in the absence of MAPK inhibitors.
cm2 (DDCt 1.1 F 0.4, P < 0.05, n = 7) but not at earlier time
points (at 2 h: DDCt
0.39 F 0.5, n = 7; at 4 h: DDCt
0.7 F 0.4, n = 4). Exposure intervals for 2, 4 and 24 h with
an intermediate LSS of 5 dyn/cm2 had no effect on eNOS
expression (at 2 h: DDCt 0.6 F 0.4, n = 6; at 4 h: DDCt
0.05 F 0.3, n = 5; at 24 h: DDCt 0.2 F 0.6, n = 7).
Regarding ICAM1 expression, a transient twofold increase in expression levels was detected after a 2-h exposure to 5 dyn/cm2 (DDCt 1.0 F 0.4, P < 0.05, n = 5) which
returned to control levels already after a 4 hrs-exposure
(DDCt
0.9 F 1.8, n = 4). After a 24-h exposure there
was no further change (DDCt 0.0 F 0.5, n = 5). A LSS
exposure of 15 dyn/cm2 induced a steeper fourfold
increase in ICAM1 expression levels after 2 h (DDCt
2.0 F 1.1, P < 0.05, n = 5) which returned towards controls
levels after 4 h (DDCt 1.1 F 0.6, n = 7). After 24-h exposure to 15 dyn/cm2 expression levels completely returned
to control levels (DDCt 0.2 F 0.5, n = 7). Expression
levels of vWF were not altered by LSS with magnitudes
of up to 15 dyn/cm2 and at exposure intervals of up to
24 h (data not shown). These results indicate that in
contrast to ICAM1 expression but similar to eNOS ex-
pression, up-regulation of IKCa1 expression levels arises
after prolonged LSS exposure ( z 4 h) and persists at
least for 24 h.
3.2. LSS-induced up-regulation of IKCa1 gene expression
leads to increased IKCa functions and endothelial
hyperpolarization
To test whether the up-regulation of IKCa1 expression
levels results in enhanced IKCa functions, we determined
mean IKCa current densities in individual cells as well as
membrane hyperpolarization in the confluent monolayer
after stimulation with ATP and the IKCa opener 1-EBIO [24].
After LSS exposure with a magnitude of 15 dyn/cm2,
an increase in IKCa current densities in individual cells
and thus increased surface expression of the IKCa channel
was first observed after 6 h of LSS exposure ( p = 0.0002
vs. controls kept under static conditions), but not after 4
h, although upregulation of IKCa1mRNA expression was
already present at this time point. After 24 h of LSS
exposure IKCacurrent densities were still up-regulated
( p = 0.001, Fig. 2A,B), but did not reach the level
observed at 6 h after LSS exposure. Thus, in keeping
with the bell shape like up-regulation of IKCa1 expression, LSS exposure resulted in a similar bell shape like
up-regulation of IKCa functions.
This IKCa current reversed near the K+ equilibrium
potential of
89 mV at a low extracellular K+ concentration of 4 mM and at near 0 mV at a high extracellular
K+ concentration of 140 mM thus demonstrating K+
selectivity (Fig. 3A). IKCa currents in HUVECs were
blocked by the selective IKCa-blockers TRAM-34, CLT,
and ChTX [25], with potencies (KD 13 F 3 nM for
TRAM-34, KD 23 F 2 nM for CLT, and KD 5 F 1 nM
for ChTX; Fig. 3B –E) similar to cloned IKCa1 channels,
Fig. 4. Representative current-clamp recordings of ATP (1 AM)- and 1-EBIO (100 AM)-induced hyperpolarizations in HUVEC exposed to LSS of 15 dyn/cm2
for 24 h and in controls kept in stationary culture.
S. Brakemeier et al. / Cardiovascular Research 60 (2003) 488–496
493
native IKCa in rat arterial ECs [14] and T-lymphocytes
[16]. Iberiotoxin, a selective blocker of large-conductance
KCa (not shown), or apamin, a selective blocker of smallconductance KCa (SKCa), had no blocking effect on the
KCa current (Fig. 3D). The SKCa/IKCa opener 1-EBIO
enhanced IKCa currents (Fig. 3F). This pharmacological
profile indicates that KCa currents in these HUVEC were
indeed mediated by IKCa.
In keeping with the observed increased IKCa-current
densities in individual cells, we tested whether LSS exposure also increases the hyperpolarisation response induced
by ATP-mediated intracellular Ca2 +-mobilization and subsequent IKCa activation.
In confluent and electrically coupled cells (membrane
capacity>500 pF) resting membrane potentials (RMP)
ranged between
15 and
26 mV (Table 2), similar to
values reported by others [12] and human mesenteric
endothelium in situ [17]. Following LSS exposure for 24
h with a magnitude of 15 dyn/cm2, RMP was not altered
while ATP-induced hyperpolarisation was significantly increased (Fig. 4, Table 2). In addition, the potentiation of this
hyperpolarization response by the SKCa/IKCa opener 1EBIO was more pronounced following LSS exposure (Fig.
4, Table 2).
3.3. Up-regulation of IKCa1 expression requires activation
of the Ras/Raf/MEK/ERK signal transduction cascade
Activation of the Ras/Raf/MEK/ERK-signaling system
has been shown to up-regulate functional IKCa expression in
rat fibroblast in vitro [26]. The activation of this signaling
cascade has also been considered important for modulation
of endothelial gene expression in response to LSS stimulation [27,28]. We therefore tested whether the Ras/Raf/MEK/
ERK-signaling cascade is involved in LSS-induced upregulation of IKCa1 expression levels.
A pretreatment of the cells with the MEK1/2 inhibitor
PD98059 (25 AM, n = 4) prevented the LSS-induced upregulation of IKCa1 expression levels and also abolished the
LSS-induced up-regulation of IKCa current densities (Fig.
5A,B). Moreover, the hyperpolarization responses to ATP
and 1-EBIO were significantly reduced (Table 2). In contrast to inhibition of MEK/ERK-signaling, inhibition of p38
signaling cascade by SB203580 (10 AM, n = 3) was ineffective in preventing LSS-induced up-regulation of IKCa1
expression levels as well as IKCa current densities (Fig.
5A,C). Accordingly, hyperpolarization responses to ATP
and 1-EBIO were similar to those observed in absence of
SB203580 (Table 2).
To rule out that PD98059 or SB203580 may exert direct
effects on IKCa activity, PD98059 or SB203580 were
directly applied to cells exhibiting IKCa activity. However,
both substances, PD98059 or SB203580, had no detectable
effects on channel activity (data not shown).
Taken together, these results suggest that LSS-induced
up-regulation of IKCa1 expression levels and IKCa function
Fig. 5. Inhibition of the MEK/ERK signaling cascade prevents LSS-induced
up-regulation of IKCa1 expression levels. (A) Real time RT-PCR analysis
of IKCa1 expression levels after exposure for 24 h to a LSS with a
magnitude of 15 dyn/cm2 with or without pretreatment with the MEK
inhibitor PD98059 (25 AM) or the p38 inhibitor SB203580 (10 AM).
DDCt = DCtshear stress + PD or SB DCtshear stress (left axis). Data are given as
mean F SEM. **P < 0.01, n = 3 – 4 each. (B) Representative IKCa currents
in HUVEC after LSS exposure following pretreatment with PD98059 or
SB203580. (C) Quantitative analysis of KCa-current densities (pA/pF) at a
holding potential of 0 mV in HUVEC following LSS exposure with or
without pretreatment with PD98059 or SB203580. Data are given as
mean F SEM. *P < 0.05, n = 11 – 20 each.
was mediated through activation of the MEK/ERK signaling
cascade, similar to the increased IKCa functions in mitogenactivated fibroblasts [26].
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4. Discussion
Shear stress-dependent gene expression has been shown
for several genes such as the eNOS, cyclooxygenases, and
the adhesion molecule ICAM1 [1,4 – 6]. However, there are
only few reports regarding shear stress-dependent modulation of endothelial ion channel expression. For instance,
recent in vitro studies revealed a shear stress-induced upregulation of stretch-activated channels in HUVEC [20] and
of KATP channels in pulmonary endothelium of the rat [29].
In the present study using the HUVEC/cone-and-plate
apparatus system as a standard in vitro model system for
investigating shear stress-modulated gene regulation in
human EC [3], we demonstrated that LSS up-regulates
IKCa1 expression levels and IKCa functions in human
umbilical vein endothelium in vitro. This up-regulation
arose in a time- and shear stress magnitude-dependent
manner. Since IKCa activation plays a major role in endothelium-dependent vasodilation [12,30], it is likely that the
shear stress-dependent up-regulation of IKCa1 expression
levels and subsequently increased IKCa functions might
represent a novel mechanism to enhance endothelial vasodilatory and atheroprotective functions.
In HUVECs, IKCa1 expression and IKCa functions were
not altered after exposure to venous levels of shear stress,
while both, intermediate and higher arterial levels resulted in
comparable increases in IKCa1 expression after a 24-h exposure. However, the time course of up-regulation of IKCa1
expression was markedly different at these two different
LSS levels. While up-regulation of IKCa1 expression was
first detectable after a 24-h exposure to a LSS of intermediate magnitude, up-regulation of IKCa1 expression by the
higher LSS of arterial magnitude occurred in a more bell
shape like fashion with the highest increase of IKCa1
expression already present after 4 hrs and of IKCa functions
after 6 h, which was followed by a less strong increase of
IKCa1 expression and IKCa functions after 24 h. This may
indicate that an exposure to higher LSS levels activates
more potently the signal transduction pathway or even
additional signal transduction pathways resulting in stimulation of IKCa1 expression at earlier time points. Time and
magnitude dependent effects of LSS on gene expression in
endothelial cells have also been decribed for, i.e. the
monocyte chemotactic protein 1 expression [31] and for
ICAM1 as shown in the present study and by others [5].
However, the up-regulation of expression of these genes
was transient and expression levels returned to basal levels
within 4 h. In contrast, IKCa1 expression was still upregulated after a 24-h exposure to intermediate and arterial
LSS levels. Such persisting alterations in gene expression
have also been shown for, e.g. eNOS expression as shown
here and previously by others [7,23] and have been interpreted as an adaptive response to altered hemodynamics [1].
In keeping with the magnitude dependent effects on the time
course of LSS-induced IKCa1 expression, it is tempting to
speculate that the more rapid and persisting up-regulation of
IKCa1 gene expression levels by high LSS could represent a
rapid adaptation to increased hemodynamic forces. On the
other hand, the slowly occurring up-regulation in IKCa1
gene expression levels induced by prolonged exposure to
intermediate LSS levels may be related to a LSS-mediated
control of basal endothelial gene expression for establishing
or maintaining adequate endothelial function. With respect
to the mechanisms of shear stress-dependent gene regulation, alterations of gene expression have been shown to be
mediated by LSS response element containing promoters
[32] as found in several shear stress-regulated genes such as
the ICAM1 gene [32] and as a result of activation of MAPK
signaling cascades [33]. The promoter of the IKCa1 gene
lacks shear stress response elements [34]. Regarding the
control of IKCa1 transcription, up-regulation of IKCa1
expression levels have been reported to occur in mitogenstimulated fibroblast [29] and human T-lymphocytes [16].
This mitogen-induced up-regulation of IKCa1 expression
levels is mediated via the MEK/ERK signaling cascade in
fibroblast, and in T-lymphocytes augmentation of IKCa1
levels occurs as a result of AP1-dependent transcription. In
the present study, the shear stress-induced up-regulation of
IKCa1 gene expression levels in HUVECs was prevented
by inhibition of the MEK/ERK signaling cascade but not of
the p38 signaling cascade. This indicates that in shear stressstimulated HUVECs similar to mitogen-stimulated fibroblasts [26], augmentation of IKCa1 expression levels and
IKCa functions requires the activation of MEK/ERK signaling cascade.
With respect to the role of IKCa channels in endothelial
function in general, previous studies revealed so far, that
endothelial IKCa are rapidly activated after humoral or shear
stress stimulation and induce membrane hyperpolarization
[10,17 – 19,35]. This IKCa-mediated hyperpolarization has
been proposed to be essential for initial endothelial mechanoreception and short term regulation of vascular tone
[10,11,30] by stimulating synthesis of vasodilating factors
either indirectly by stimulating membrane potential-driven
Ca2 + influx and subsequent stimulation of Ca2 + dependent
NO and prostacyclin synthesis [36] or directly, given that
endothelial hyperpolarization is propagated via gap-junctions to the smooth muscle cells [34]. It has also been
suggested that K+ efflux from the endothelium through IKCa
could stimulate inwardly rectifying K+ channel and Na/K
ATPases leading to vasodilation [37]. As such EDHFtermed responses can be eliminated by blocking these
endothelial IKCa [18,38] it is widely accepted that besides
other presumed EDHFs such as cytochrome P450 generated
metabolites of arachidonic acid [38] activation of endothelial IKCa channels is essential for the generation of the
EDHF response [14,18,19,35,39].
The findings of the present study show that arterial levels
of LSS induced an augmentation of endothelial IKCa1
transcription and thereby enhances IKCa function in our in
vitro model system. As a consequence of increased IKCa1
expression and function, the ability of the endothelium to
S. Brakemeier et al. / Cardiovascular Research 60 (2003) 488–496
hyperpolarize is increased as shown in the present study.
This increased ability to hyperpolarize in turn enhances
membrane potential-driven Ca2 + influx and subsequently
Ca2 +-dependent synthesis of endothelial vasodilators and in
a more direct fashion the EDHF signaling. Such an upregulation of the EDHF signaling may be especially important in compensating the diminished availability or loss of
other endothelium-derived vasodilator such as NO as
reported in salt-sensitive hypertension as well as in eNOS
knock-out mice [40,41]. Furthermore, an increased capacity
of the endothelium to hyperpolarize may be artheroprotective presumably by increasing the synthesis of antithrombotic factors [8].
In conclusion, shear stress-induced modulation in endothelial IKCa1 gene expression and function might therefore
be an important mechanism in long term vessel adaptation
and vasoregulation in response to increased hemodynamic
forces as present in hypertension [1]. In addition to other
genes such as eNOS and cyclooxygenase [7,23] the endothelial IKCa1 can now be regarded as new class of shear
stress-regulated gene.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (FOR 341/1/5/7, HO 1103/2-4, GRK 276/2).
The authors wish to thank Heike Wulff for the gift of
TRAM-34. We are grateful to Klaus Schlotter for excellent
technical assistance.
References
[1] Chien S, Li S, Shyy YJ. Effects of mechanical forces on signal transduction and gene expression in endothelial cells. Hypertension 1998;
31:162 – 9.
[2] Pohl U, Holtz J, Busse R, Bassenge E. Crucial role of endothelium in
the vasodilator response to increased flow in vivo. Hypertension
1986;8:37 – 44.
[3] Frangos JA, Eskin SG, McIntire LV, Ives CL. Flow effects on prostacyclin production by cultured human endothelial cells. Science
1985;227:1477 – 9.
[4] Malek AM, Izumo S. Mechanism of endothelial cell shape change
and cytoskeletal remodeling in response to fluid shear stress. J Cell
Sci 1996;109:713 – 26.
[5] Nagel T, Resnick N, Atkinson WJ, Dewey Jr CF, Gimbrone Jr MA.
Shear stress selectively upregulates intercellular adhesion molecule-1
expression in cultured human vascular endothelial cells. J Clin Invest
1994;94:885 – 91.
[6] DePaola N, Davies PF, Pritchard Jr WF, Florez L, Harbeck N, Polacek
DC. Spatial and temporal regulation of gap junction connexin43 in
vascular endothelial cells exposed to controlled disturbed flows in
vitro. Proc Natl Acad Sci U S A 1999;96:3154 – 9.
[7] Ranjan V, Xiao Z, Diamond SL. Constitutive NOS expression in
cultured endothelial cells is elevated by fluid shear stress. Am J
Physiol 1995;269:H550 – 5.
[8] Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vasc Biol 1998;18:677 – 85.
495
[9] Lansman JB, Hallam TJ, Rink TJ. Single stretch-activated ion channels in vascular endothelial cells as mechanotransducers? Nature
1987;325:811 – 3.
[10] Olesen SP, Clapham DE, Davies PF. Haemodynamic shear stress
activates a K+ current in vascular endothelial cells. Nature 1988;
331:168 – 70.
[11] Hoyer J, Köhler R, Distler A. Mechanosensitive Ca2 + oscillations and
STOC activation in endothelial cells. FASEB J 1998;12:359 – 66.
[12] Nilius B, Droogmans G. Ion channels and their functional role in
vascular endothelium. Physiol Rev 2001;81:1415 – 59.
[13] Hoger JH, Ilyin VI, Forsyth S, Hoger A. Shear stress regulates the
endothelial Kir2.1 ion channel. Proc Natl Acad Sci U S A 2002;99:
7780 – 5.
[14] Köhler R, Brakemeier S, Kühn M, et al. Impaired hyperpolarization in
regenerated endothelium after balloon catheter injury. Circ Res
2001;89:174 – 9.
[15] Ishii TM, Silvia C, Hirschberg B, Bond CT, Adelman JP, Maylie J. A
human intermediate conductance calcium-activated potassium channel. Proc Natl Acad Sci U S A 1997;94:11651 – 6.
[16] Ghanshani S, Wulff H, Miller MJ, et al. Up-regulation of the IKCa1
potassium channel during T-cell activation. Molecular mechanism and
functional consequences. J Biol Chem 2000;275:37137 – 49.
[17] Köhler R, Degenhardt C, Kühn M, Runkel N, Paul M, Hoyer J.
Expression and function of endothelial Ca2 +-activated K+ channels
in human mesenteric artery: a single-cell reverse transcriptase-polymerase chain reaction in situ. Circ Res 2000;87:496 – 503.
[18] Eichler I, Wibawa J, Grgic I, et al. Selective blockade of endothelial
Ca2 +-activated small- and intermediate-conductance K+-channels
suppresses EDHF-mediated vasodilation. Br J Pharmacol 2003;138:
594 – 601.
[19] Bychkov R, Burnham MP, Richards GR, Edwards G, Weston AH,
Feletou M, et al. Characterization of a charybdotoxin-sensitive intermediate conductance Ca2 +-activated K+ channel in porcine coronary endothelium: relevance to EDHF. Br J Pharmacol 2002;137:
1346 – 54.
[20] Brakemeier S, Eichler I, Hopp H, Köhler R, Hoyer J. Up-regulation of
endothelial stretch-activated cation channels by fluid shear stress.
Cardiovasc Res 2002;53:209 – 18.
[21] Pries AR, Secomb TW, Gaehtgens P. Design principles of vascular
beds. Circ Res 1995;77:1017 – 23.
[22] Galbusera M, Zoja C, Donadelli R, et al. Fluid shear stress modulates
von Willebrand factor release from human vascular endothelium.
Blood 1997;90:1558 – 64.
[23] Topper JN, Cai J, Falb D, Gimbrone Jr MA. Identification of vascular endothelial genes differentially responsive to fluid mechanical
stimuli: cyclooxygenase-2, manganese uperoxide dismutase, and endothelial cell nitric oxide synthase are selectively up-regulated by
steady laminar shear stress. Proc Natl Acad Sci U S A 1996;93:
10417 – 22.
[24] Walker SD, Dora KA, Ings NT, Crane GJ, Garland CJ. Activation of
endothelial cell IK(Ca) with 1-ethyl-2-benzimidazolinone evokes
smooth muscle hyperpolarization in rat isolated mesenteric artery.
Br J Pharmacol 2001;134:1548 – 54.
[25] Wulff H, Miller MJ, Hansel W, Grissmer S, Cahalan MD, Chandy
KG. Design of a potent and selective inhibitor of the intermediateconductance Ca2 +-activated K+ channel, IKCa1: a potential immunosuppressant. Proc Natl Acad Sci U S A 2000;97:8151 – 6.
[26] Pena TL, Chen SH, Konieczny SF, Rane SG. Ras/MEK/ERK Upregulation of the fibroblast KCa channel FIK is a common mechanism
for basic fibroblast growth factor and transforming growth factor-beta
suppression of myogenesis. J Biol Chem 2000;275:13677 – 82.
[27] Traub O, Monia BP, Dean NM, Berk BC. PKC-epsilon is required for
mechano-sensitive activation of ERK1/2 in endothelial cells. J Biol
Chem 1997;272:31251 – 7.
[28] Tseng H, Peterson TE, Berk BC. Fluid shear stress stimulates mitogen-activated protein kinase in endothelial cells. Circ Res 1995;77:
869 – 78.
496
S. Brakemeier et al. / Cardiovascular Research 60 (2003) 488–496
[29] Chatterjee S, Al-Mehdi AB, Levitan I, Stevens T, Fisher AB. Shear
stress increases expression of a KATP channel in rat and bovine pulmonary vascular endothelial cells. Am J Physiol Cell Physiol 2003
(Jun 25) [Epub ahead of print].
[30] Sun D, Huang A, Koller A, Kaley G. Endothelial KCa channels
mediate flow-dependent dilation of arterioles of skeletal muscle and
mesentery. Microvasc Res 2001;61:179 – 86.
[31] Shyy YJ, Hsieh HJ, Usami S, Chien S. Fluid shear stress induces a
biphasic response of human monocyte chemotactic protein 1 gene
expression in vascular endothelium. Proc Natl Acad Sci U S A
1994;91:4678 – 82.
[32] Khachigian LM, Resnick N, Gimbrone Jr MA, Collins T. Nuclear
factor-kappa B interacts functionally with the platelet-derived growth
factor B-chain shear-stress response element in vascular endothelial
cells exposed to fluid shear stress. J Clin Invest 1995;96:1169 – 75.
[33] Surapisitchat J, Hoefen RJ, Pi X, Yoshizumi M, Yan C, Berk BC.
Fluid shear stress inhibits TNF-alpha activation of JNK but not
ERK1/2 or p38 in human umbilical vein endothelial cells: inhibitory
crosstalk among MAPK family members. Proc Natl Acad Sci U S A
2001;98:6476 – 81.
[34] Edwards G, Feletou M, Gardener MJ, Thollon C, Vanhoutte PM,
[35]
[36]
[37]
[38]
[39]
[40]
[41]
Weston AH. Role of gap junctions in the responses to EDHF in rat
and guinea-pig small arteries. Br J Pharmacol 1999;128:1788 – 94.
Garland CJ, Plane F. Endothelium-derived hyperpolarizing factor.
London: Harwood Academic Publishers; 1996. p. 173 – 9.
Luckhoff A, Busse R. Calcium influx into endothelial cells and formation of endothelium-derived relaxing factor is controlled by the
membrane potential. Pflugers Arch 1990;416:305 – 11.
Edwards G, Dora KA, Gardener MJ, Garland CJ, Weston AH. K+ is
an endothelium-derived hyperpolarizing factor in rat arteries. Nature
1998;396:269 – 72.
Fisslthaler B, Popp R, Kiss L, et al. Cytochrome P450 2C is an EDHF
synthase in coronary arteries. Nature 1999;401:493 – 7.
Busse R, Edwards G, Feletou M, Fleming I, Vanhoutte PM, Weston
AH. EDHF: bringing the concepts together. Trends Pharmacol Sci
2002;23:374 – 80.
Sofola OA, Knill A, Hainsworth R, Drinkhill M. Change in endothelial function in mesenteric arteries of Sprague – Dawley rats fed a high
salt diet. J Physiol 2002;543:255 – 60.
Waldron GJ, Ding H, Lovren F, Kubes P, Triggle CR. Acetylcholineinduced relaxation of peripheral arteries isolated from mice lacking
endothelial nitric oxide synthase. Br J Pharmacol 1999;128:653 – 8.