# Lowering Caveolin-1 Expression in Human Vascular Endothelial Cells Inhibits Signal Transduction in Response to Shear Stress

Authors: A. D. van der Meer; M. M. J. Kamphuis; A. A. Poot; J. Feijen; I. Vermes
Journal: International Journal of Cell Biology
 (2009)

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## Abstract

Vascular endothelial cells have an extensive response to physiological levels of shear stress. There is evidence that the protein caveolin-1 is involved in the early phase of this response. In this study, caveolin-1 was downregulated in human endothelial cells by RNAi. When these cells were subjected to a shear stress of 15 dyn/cm2 for 10 minutes, activation of Akt and ERK1/2 was significantly lower than in control cells. Moreover, activation of Akt and ERK1/2 in response to vascular endothelial growth factor was significantly lower in cells with low levels of caveolin-1. However, activation of integrin-mediated signaling during cell adhesion onto fibronectin was not hampered by lowered caveolin-1 levels. In conclusion, caveolin-1 is an essential component in the response of endothelial cells to shear stress. Furthermore, the results suggest that the role of caveolin-1 in this process lies in facilitating efficient VEGFR2-mediated signaling.


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## Body

## 1. Introduction

Vascular endothelial cells (ECs) are
constantly subjected to shear stress caused by the flow of blood. ECs are
highly responsive to changes in this shear stress. They are able to convert these
mechanical stimuli into relevant biological signals by a process that is known
as mechanotransduction [1]. The best-known elements in the
early stages of this response are the cell-anchoring integrins [2] and certain membrane-associated
receptors, such as the vascular endothelial growth factor receptor 2 (VEGFR2) [3, 4] and G-protein coupled receptors [5]. After the initial activation of
these molecules, the biological signal is transmitted into the cell by activation
of major signal transduction pathways, such as mitogen-activated protein kinase
(MAPK) pathways, the protein kinase B (PKB/Akt) pathway, and the endothelial
nitric oxide synthase (eNOS) signaling route. These events lead to a functional
response of the cell, influencing rate of apoptosis and proliferation [1, 6], sensitivity to inflammation [7], and cytoskeletal remodeling [8].Studies have shown that 50-nanometer,
omega-shaped membrane invaginations, known as caveolae, are linked to
mechanotransduction. The majority of membrane-associated proteins that are
phosphorylated in response to shear stress localize to these domains [9, 10]. Also, as ECs are subjected to shear
stress, the density of caveolae in the cell membrane increases, modulating the
activation of signaling pathways [11, 12]. Moreover, mice that lack a
structural protein of the caveolar domain, caveolin-1, have an abnormal
vascular response when shear stress is altered [13].In order to gain more mechanistic insight
into the exact role of caveolae in the EC response to shear stress, in vitro studies were carried out to interfere directly with caveolar
function. In some studies, caveolae were disrupted by cholesterol extraction [9, 14, 15], and in one study caveolar
functioning was inhibited by introducing blocking antibodies to caveolin-1 [16]. These studies have shown that interfering
with caveolar function causes an impaired response to shear stress, as
characterized by a lowered activation of MAPKs [9, 14, 16], Akt [15], and eNOS [10].An important molecular biological
tool to study the role of proteins in cellular processes is RNA interference
(RNAi). By transfecting cells with short interfering RNA (siRNA) molecules with
a sequence that is complementary to the mRNA of the protein of interest, a dramatic
lowering of the expression of this protein can be achieved. The tool is very
specific and using it to lower expression levels of caveolin-1 could be useful
in confirming the results obtained by the more crude method of cholesterol
extraction.In this study, we use RNAi in human
umbilical vein endothelial cells (HUVECs) to confirm the essential role of
caveolin-1 in EC mechanotransduction. We also show that lowering caveolin-1 levels leads to
impaired VEGFR2 signaling, but not integrin signaling, in these cells. This
suggests that the role of caveolin-1
in mechanotransduction lies in coupling VEGFR2
activation to downstream signaling.


## 2. Materials and Methods

### 2.1. Antibodies and Reagents

Caveolin-1 siRNA was bought from Qiagen (West Sussex, Ireland)
(cat. no. SI00299635); negative control siRNA was obtained from Invitrogen (Calif, USA) (Stealth RNAi negative control with medium GC content). Antibodies for immunodetection were from the following companies: caveolin-1 (Sigma, Mo, USA; C4490), Akt (Abcam (Cambridge, UK); ab28422), phospho-Akt (Abcam, ab27773), ERK1 (Abcam, ab9363), phospho-ERK (Santa Cruz, Calif, USA; sc-7976), GAPDH (Abcam, ab9485), goat anti-rabbit IgG-Alexa 633 (Molecular Probes, Ore, USA; A21071), goat anti-rabbit IgG-HRP (Sigma, A0545), and mouse anti-goat IgG-HRP (Zymed, Calif, USA; 81-1620). All products for cell culturing were from Lonza (Breda, The Netherlands) except for the partially purified fibronectin, which was obtained as a coproduct during purification of human factor VIII at Sanquin, Amsterdam, The Netherlands, and which was used at 2 mg/mL in phosphate buffered saline (PBS) to coat surfaces for cell culture. All other reagents were from Sigma, except when specified differently.


### 2.2. Huvec Isolation and Culturing

HUVECs were isolated from umbilical
cords by the method of Jaffe et al. [17], using trypsin solution (0.05% (w/v)
trypsin, 0.02% (w/v) EDTA in PBS). The obtained endothelial cells were cultured
in fibronectin-coated culture flasks in endothelial growth medium-2 (EGM-2),
containing 2% fetal bovine serum, until they reached confluency. When
confluent, cells were detached from the surface using trypsin solution and
diluted 1 : 3 in a fresh fibronectin-coated culture flask. Cells were kept in a
humidified incubator (37°C, 5%CO2) up to passage 8, after which they were discarded.


### 2.3. Sirna Transfection

The day before transfection, HUVECs were seeded in a 6-well plate,300⋅103 cells per well in EGM-2. The
following day, cells were washed once with OptiMEM medium (Gibco, Okla, USA) and then overlaid with
800 μL OptiMEM. To transfect one well with siRNA, the following protocol was
used. 15 μL of 20 μM siRNA solution were mixed with 145 μL of 37°C OptiMEM medium and left at
room temperature for 15 minutes. 8 μL of Oligofectamine (Invitrogen) were mixed
with 32 μL OptiMEM, and after 5 minutes the Oligofectamine mixture was added to
the tube with siRNA. Complexes were allowed to form for 15 minutes, and they
were added to the cells. After 4 hours in the incubator, 500 μL EGM-2 with
three times the normal amount of bovine serum were added to the wells. After
overnight incubation, the cells were washed with PBS and incubated in normal
EGM-2 for 6 hours.


### 2.4. Cell Treatments

For shear stress experiments, four
wells of siRNA-transfected cells were trypsinized and replated on a
fibronectin-coated glass plate of 40cm2. The cells were left to
adhere for two hours in EGM-2, after which the medium was replaced by endothelial
basal medium-2 (EBM-2) to starve the cells overnight. The next day, 48 hours
after transfection, cells were subjected to shear stress in a custom-built
parallel plate flow chamber. All parts of the setup were sterilized before use.
The chamber consisted of two parallel glass plates, spaced 0.6 mm apart by
glass spacers, held together by a stainless steel housing. The chamber was
connected to a glass reservoir containing 200 mL shear medium (medium 199 with
100 units/mL penicillin and 100 μg/mL streptomycin) and to a peristaltic pump
(Watson-Marlow, Rotterdam, The Netherlands). The different parts of the setup
were connected to each other in a closed circuit using silicone tubing (Versitec
silicone, 5 mm inner diameter, Rubber BV, The Netherlands), and the entire
setup was put in an incubator to maintain proper culturing conditions. Medium
was then pumped through the flow chamber for 10 or 30 minutes at a rate of 200 mL
per minute. This yields a theoretical estimate for the shear stress of approximately
15 dyne/cm2. After the treatment, the flow chamber was disassembled; the cells were washed once with PBS and scraped in 100 μL ice-cold lysis buffer (1% (w/v) Triton X-100/PBS with added protease inhibitors (4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin) and phosphatase inhibitors (microcystin LR, cantharidin, (−)-p-bromotetramisole, sodium vanadate, sodium molybdate, sodium tartrate, and imidazole)). Then, the lysate was incubated on ice for 15 minutes and centrifuged at 12000×g for 10
minutes. The supernatant was transferred to a fresh tube, and the protein
concentration was determined using a bicinchonic acid assay (Pierce, Etten-Leur, The Netherlands). An equivolume of 2× Laemmli sample buffer was
added to the lysate. The resulting sample was boiled for 5 minutes and then
stored at −20°C until use.For VEGF stimulation experiments,
siRNA transfected cells were incubated overnight in growth medium and then
starved for 6 hours with EBM-2. Subsequently, 48 hours after transfection, the
cells were washed once with PBS, followed by addition of EBM-2 with 200 ng/mLVEGF165.
Cells were put in the incubator for 10 minutes, after which a lysate was
prepared as described above.The integrin activation experiments
were performed as follows. After siRNA transfection, cells were starved
overnight in EBM-2. The following day, 48 hours after transfection, cells were
trypsinized, spun down, and resuspended in 2 mL EBM-2 with 2% (w/v) bovine
serum albumin (BSA). The cells were kept in suspension in the incubator under
constant, light agitation for 30 minutes. Then, 1 mL of the cell suspension was
plated on a fibronectin-coated surface, while the remaining cells were spun
down, washed once with PBS, and then resuspended in lysis buffer. The plated
cells were left to adhere for 20 minutes, after which a lysate was prepared as
described above.


### 2.5. Western Blot and Immunodetection

Samples were subjected to sodium
dodecyl sulphate (SDS) poly(acrylamide) gel electrophoresis, Western blotting,
and immunodetection according to common protocols. Shortly, samples were
separated by size on a 10% (w/v) poly(acrylamide) gel, and the protein band
pattern was transferred to a poly(vinylidene difluoride) membrane, using a
Bio-Rad Mini-Protean 3 system. The membrane was blocked with 1% (w/v) nonfat
dry milk in 25 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween-20, pH 8.3 (TBS-T).
The primary antibodies were applied to the membranes in blocking buffer,
overnight at4°C. After washing the membrane with TBS-T four times for 20
seconds and one time for 15 minutes, the secondary antibody was applied to the
membrane in blocking buffer for one hour at room temperature. The same washing
regime was repeated, and the membrane was overlayed with SuperSignal West Femto
substrate (Pierce). After three minutes of incubation, the chemiluminescent
signal was detected with a Kodak Image station with CCD camera.


### 2.6. Confocal Microscopy

Cells were seeded at a density of150⋅103 cells per well on
fibronectin-coated glass coverslips in a 12-well plate. They were transfected with siRNA as
described earlier, but with half the reagents per well. After transfection, the
cells were incubated overnight in EGM-2, then washed with PBS, and fixed with
4% (w/v) formaldehyde/PBS for 15 minutes at room temperature. The coverslips
were then covered with permeabilization buffer (PBS with 1 mg/mL BSA and 0,1%
(w/v) Triton X-100) for 10 minutes. Primary antibodies were diluted in
permeabilization buffer and then incubated on the coverslips for 1 hour at
37°C. The coverslips were washed three times for 5 minutes with PBS and were
subsequently covered with the secondary antibody in a mild permeabilization
buffer (PBS with 1 mg/mL BSA and 0.05% (w/v) Triton X-100). After incubating for 1 hour at 37°C, coverslips were washed and a 100 ng/mL solution of 4′,6-Diamidino-2-phenylindole
(DAPI) was applied for 5 minutes to stain nuclei. After three more washes with
PBS, coverslips were mounted on microscope slides using Mowiol (CalBiochem, Darmstadt, Germany) and stored at 4°C in the dark until
they were imaged with a Zeiss LSM 510 confocal microscope. An estimate of the
average staining intensity was performed by dividing the total signal in a
field by the number of nuclei, as determined by using ImageJ image analysis
software [18].


### 2.7. Statistical Analysis

Signal intensities of immunodetection
on Western blots were quantified using ImageJ. The intensity of the specific
signal was normalized to total protein content, as assessed by the loading
control in the same lane. In order to compare these signal intensities between
different experiments, the ratios between the intensity level of a sample and
the total intensity of all samples in that experiment were determined. The
averages of these normalized ratios were plotted, with the error bars
representing standard deviation. Differences between these means were tested
for statistical significance by performing an unpaired, two-tailed Student’st-test. Differences were considered to be statistically significant at P-values
smaller than .05.


## 2.1. Antibodies and Reagents

Caveolin-1 siRNA was bought from Qiagen (West Sussex, Ireland)
(cat. no. SI00299635); negative control siRNA was obtained from Invitrogen (Calif, USA) (Stealth RNAi negative control with medium GC content). Antibodies for immunodetection were from the following companies: caveolin-1 (Sigma, Mo, USA; C4490), Akt (Abcam (Cambridge, UK); ab28422), phospho-Akt (Abcam, ab27773), ERK1 (Abcam, ab9363), phospho-ERK (Santa Cruz, Calif, USA; sc-7976), GAPDH (Abcam, ab9485), goat anti-rabbit IgG-Alexa 633 (Molecular Probes, Ore, USA; A21071), goat anti-rabbit IgG-HRP (Sigma, A0545), and mouse anti-goat IgG-HRP (Zymed, Calif, USA; 81-1620). All products for cell culturing were from Lonza (Breda, The Netherlands) except for the partially purified fibronectin, which was obtained as a coproduct during purification of human factor VIII at Sanquin, Amsterdam, The Netherlands, and which was used at 2 mg/mL in phosphate buffered saline (PBS) to coat surfaces for cell culture. All other reagents were from Sigma, except when specified differently.


## 2.2. Huvec Isolation and Culturing

HUVECs were isolated from umbilical
cords by the method of Jaffe et al. [17], using trypsin solution (0.05% (w/v)
trypsin, 0.02% (w/v) EDTA in PBS). The obtained endothelial cells were cultured
in fibronectin-coated culture flasks in endothelial growth medium-2 (EGM-2),
containing 2% fetal bovine serum, until they reached confluency. When
confluent, cells were detached from the surface using trypsin solution and
diluted 1 : 3 in a fresh fibronectin-coated culture flask. Cells were kept in a
humidified incubator (37°C, 5%CO2) up to passage 8, after which they were discarded.


## 2.3. Sirna Transfection

The day before transfection, HUVECs were seeded in a 6-well plate,300⋅103 cells per well in EGM-2. The
following day, cells were washed once with OptiMEM medium (Gibco, Okla, USA) and then overlaid with
800 μL OptiMEM. To transfect one well with siRNA, the following protocol was
used. 15 μL of 20 μM siRNA solution were mixed with 145 μL of 37°C OptiMEM medium and left at
room temperature for 15 minutes. 8 μL of Oligofectamine (Invitrogen) were mixed
with 32 μL OptiMEM, and after 5 minutes the Oligofectamine mixture was added to
the tube with siRNA. Complexes were allowed to form for 15 minutes, and they
were added to the cells. After 4 hours in the incubator, 500 μL EGM-2 with
three times the normal amount of bovine serum were added to the wells. After
overnight incubation, the cells were washed with PBS and incubated in normal
EGM-2 for 6 hours.


## 2.4. Cell Treatments

For shear stress experiments, four
wells of siRNA-transfected cells were trypsinized and replated on a
fibronectin-coated glass plate of 40cm2. The cells were left to
adhere for two hours in EGM-2, after which the medium was replaced by endothelial
basal medium-2 (EBM-2) to starve the cells overnight. The next day, 48 hours
after transfection, cells were subjected to shear stress in a custom-built
parallel plate flow chamber. All parts of the setup were sterilized before use.
The chamber consisted of two parallel glass plates, spaced 0.6 mm apart by
glass spacers, held together by a stainless steel housing. The chamber was
connected to a glass reservoir containing 200 mL shear medium (medium 199 with
100 units/mL penicillin and 100 μg/mL streptomycin) and to a peristaltic pump
(Watson-Marlow, Rotterdam, The Netherlands). The different parts of the setup
were connected to each other in a closed circuit using silicone tubing (Versitec
silicone, 5 mm inner diameter, Rubber BV, The Netherlands), and the entire
setup was put in an incubator to maintain proper culturing conditions. Medium
was then pumped through the flow chamber for 10 or 30 minutes at a rate of 200 mL
per minute. This yields a theoretical estimate for the shear stress of approximately
15 dyne/cm2. After the treatment, the flow chamber was disassembled; the cells were washed once with PBS and scraped in 100 μL ice-cold lysis buffer (1% (w/v) Triton X-100/PBS with added protease inhibitors (4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin A, E-64, bestatin, leupeptin, and aprotinin) and phosphatase inhibitors (microcystin LR, cantharidin, (−)-p-bromotetramisole, sodium vanadate, sodium molybdate, sodium tartrate, and imidazole)). Then, the lysate was incubated on ice for 15 minutes and centrifuged at 12000×g for 10
minutes. The supernatant was transferred to a fresh tube, and the protein
concentration was determined using a bicinchonic acid assay (Pierce, Etten-Leur, The Netherlands). An equivolume of 2× Laemmli sample buffer was
added to the lysate. The resulting sample was boiled for 5 minutes and then
stored at −20°C until use.For VEGF stimulation experiments,
siRNA transfected cells were incubated overnight in growth medium and then
starved for 6 hours with EBM-2. Subsequently, 48 hours after transfection, the
cells were washed once with PBS, followed by addition of EBM-2 with 200 ng/mLVEGF165.
Cells were put in the incubator for 10 minutes, after which a lysate was
prepared as described above.The integrin activation experiments
were performed as follows. After siRNA transfection, cells were starved
overnight in EBM-2. The following day, 48 hours after transfection, cells were
trypsinized, spun down, and resuspended in 2 mL EBM-2 with 2% (w/v) bovine
serum albumin (BSA). The cells were kept in suspension in the incubator under
constant, light agitation for 30 minutes. Then, 1 mL of the cell suspension was
plated on a fibronectin-coated surface, while the remaining cells were spun
down, washed once with PBS, and then resuspended in lysis buffer. The plated
cells were left to adhere for 20 minutes, after which a lysate was prepared as
described above.


## 2.5. Western Blot and Immunodetection

Samples were subjected to sodium
dodecyl sulphate (SDS) poly(acrylamide) gel electrophoresis, Western blotting,
and immunodetection according to common protocols. Shortly, samples were
separated by size on a 10% (w/v) poly(acrylamide) gel, and the protein band
pattern was transferred to a poly(vinylidene difluoride) membrane, using a
Bio-Rad Mini-Protean 3 system. The membrane was blocked with 1% (w/v) nonfat
dry milk in 25 mM Tris, 150 mM NaCl, 0.05% (v/v) Tween-20, pH 8.3 (TBS-T).
The primary antibodies were applied to the membranes in blocking buffer,
overnight at4°C. After washing the membrane with TBS-T four times for 20
seconds and one time for 15 minutes, the secondary antibody was applied to the
membrane in blocking buffer for one hour at room temperature. The same washing
regime was repeated, and the membrane was overlayed with SuperSignal West Femto
substrate (Pierce). After three minutes of incubation, the chemiluminescent
signal was detected with a Kodak Image station with CCD camera.


## 2.6. Confocal Microscopy

Cells were seeded at a density of150⋅103 cells per well on
fibronectin-coated glass coverslips in a 12-well plate. They were transfected with siRNA as
described earlier, but with half the reagents per well. After transfection, the
cells were incubated overnight in EGM-2, then washed with PBS, and fixed with
4% (w/v) formaldehyde/PBS for 15 minutes at room temperature. The coverslips
were then covered with permeabilization buffer (PBS with 1 mg/mL BSA and 0,1%
(w/v) Triton X-100) for 10 minutes. Primary antibodies were diluted in
permeabilization buffer and then incubated on the coverslips for 1 hour at
37°C. The coverslips were washed three times for 5 minutes with PBS and were
subsequently covered with the secondary antibody in a mild permeabilization
buffer (PBS with 1 mg/mL BSA and 0.05% (w/v) Triton X-100). After incubating for 1 hour at 37°C, coverslips were washed and a 100 ng/mL solution of 4′,6-Diamidino-2-phenylindole
(DAPI) was applied for 5 minutes to stain nuclei. After three more washes with
PBS, coverslips were mounted on microscope slides using Mowiol (CalBiochem, Darmstadt, Germany) and stored at 4°C in the dark until
they were imaged with a Zeiss LSM 510 confocal microscope. An estimate of the
average staining intensity was performed by dividing the total signal in a
field by the number of nuclei, as determined by using ImageJ image analysis
software [18].


## 2.7. Statistical Analysis

Signal intensities of immunodetection
on Western blots were quantified using ImageJ. The intensity of the specific
signal was normalized to total protein content, as assessed by the loading
control in the same lane. In order to compare these signal intensities between
different experiments, the ratios between the intensity level of a sample and
the total intensity of all samples in that experiment were determined. The
averages of these normalized ratios were plotted, with the error bars
representing standard deviation. Differences between these means were tested
for statistical significance by performing an unpaired, two-tailed Student’st-test. Differences were considered to be statistically significant at P-values
smaller than .05.


## 3. Results

### 3.1. Mechanotransduction in Ecs

In order to investigate
mechanotransduction in our flow chamber system, HUVECs were subjected to
physiologically relevant shear stresses for 10 minutes and 30 minutes. After these
treatments, cell lysates were tested for phosphorylated Akt and ERK1/2 by
immunoblotting. As shown in Figure1, 10 minutes of shear stress led to
significantly enhanced phosphorylation of both Akt and ERK1/2. After 30 minutes
of shear stress, phosphorylation levels had dropped to values comparable to the
static situation. Therefore, we decided to focus on the early timepoint for our
studies into the role of caveolin-1 in the EC mechanotransduction.Figure 1
Activation of mechanotransduction pathways by
shear. HUVECs were subjected to shear stress for different periods.
Subsequently, the amount of phosphorylated Akt and ERK1/2 was determined by
immunoblotting. After 10 minutes, phosphorylation levels were significantly
higher (P*<.01, Student’s t-test) compared to levels in statically cultured cells. After 30 minutes, this significant increase was no
longer detected.


### 3.2. Caveolin-1 Downregulation

When HUVECs were treated with
caveolin-1 siRNA, the expression of caveolin-1 decreased significantly to less
than one-third of the caveolin-1 level in untreated cells, as shown by
immunoblotting (see Figures2(a) and 2(b)). When cells were transfected with
negative control siRNA, no statistically significant decrease in caveolin-1
expression was detected, showing the specificity of the siRNA treatment. Moreover,
expression of Akt and ERK1/2 was not affected by the transfection procedure or
the lowering of caveolin-1 levels (see Figure 2(a)). The downregulation of
caveolin-1 was confirmed by using immunocytofluorescence (see Figure 2(c)). Using the same microscope settings, staining intensity was approximately four
times higher in untreated cells than in caveolin-1 siRNA-treated cells. The
remaining caveolin-1 in the siRNA-treated cells was located mostly in the
perinuclear area, with a lack of intense membrane staining like in the
untreated and control RNA treated cells.Downregulation of caveolin-1 expression by RNAi.
HUVECs were treated with caveolin-1 siRNA, negative control siRNA, or left
untreated. Caveolin-1 levels were determined 48 hours after transfection. (a)
Immunoblot analysis of cell lysates. Equal loading of proteins of each
transfection condition was ensured by detecting the unrelated metabolic enzyme
GAPDH on the same blot. (b) Quantification of the signal intensities in the
immunoblot assay. The bars represent average intensities of the caveolin-1 band
in seven separate experiments. Error bars represent standard deviation.
Caveolin-1 siRNA treated cells had significantly lower caveolin-1 expression
than control siRNA-treated cells (P*<.0001, Student’s t-test). No significant differences were found between untreated cells and negative control siRNA-treated cells. Bars represent averages of seven separate
experiments. (c) Confocal microscopic imaging of caveolin-1. Treated cells were
fixed and labeled with caveolin-1 antibodies that were detected by Alexa 633-coupled
secondary antibodies. Nuclei were stained with DAPI. All images were taken with
the same microscope settings. Scale bar is 50 μm.
(a)(b)(c)


### 3.3. Mechanotransduction in Ecs with Lowered Caveolin-1 Levels

HUVECs with normal and lowered levels
of caveolin-1 were subjected to physiological levels of shear stress in a
parallel plate flow chamber. After 10 minutes of shear stress, phosphorylation
status of the important mechanotransducing molecules Akt and ERK1/2 was
determined by phospho-specific immunoblotting. Total expression levels of these
proteins were not affected by siRNA treatment, as determined by immunoblotting
(see Figure2(a)). After subjecting HUVECs to shear stress, phosphorylation
status of Akt and ERK1/2 increased approximately five to ten times (see Figure 3). We found that the activation of both signal transduction pathways was
significantly lower in caveolin-1 siRNA-treated cells than in cells treated
with negative control siRNA. No significant differences were found among the activation levels of control siRNA-treated cells and untreated cells.Activation of Akt and
ERK1/2 in response to shear stress. HUVECs were transfected with either
caveolin-1 siRNA, negative control siRNA, or left untreated. 48 hours after
transfection, cells were subjected to a shear stress of approximately 15 dyn/cm2 for 10 minutes. (a) After treatment, cells were lysed, and the amount of
phosphorylated Akt and ERK1/2 was determined by immunoblotting. Equal loading
of proteins of each transfection condition was ensured by detecting the
unrelated metabolic enzyme GAPDH on the same blot. The phosphorylation status
of statically cultured cells is shown as a reference. (b) Quantification of the
signal intensities in the immunoblot assay. Each bar represents the average
intensity of four separate experiments. Error bars denote standard deviation.
Akt and ERK1/2 phosphorylations increase five- to tenfold when untreated cells
are subjected to shear stress. Both Akt and ERK1/2 phosphorylations are
significantly lower (P*<.05, P**<.01, Student’s t-test)
when cells are treated with caveolin-1 siRNA instead of negative control siRNA
prior to subjecting the cells to shear stress. No significant difference was
detected between untreated cells and cells transfected with negative control
siRNA, showing that the transfection procedure itself has no influence. Graphs
represent the averages of four separate experiments.
(a)(b)


### 3.4. Signal Transduction in Ecs with Lowered Caveolin-1 Levels

In order to uncover the mechanism for
the ineffective mechanotransduction in cells with low caveolin-1 levels, we induced
signal transduction by activating cell surface receptors. We chose activation
of integrins and VEGFR2, because these proteins are well known to be essential
for mechanotransduction. In order to assess integrin-mediated signal
transduction, cells were kept in suspension for 30 minutes and then plated for
20 minutes on a fibronectin-coated surface. When checking the phosphorylation
status of Akt and ERK1/2 by phospho-specific immunoblotting, no significant
differences could be detected between the activation of signal transduction
pathways in negative control siRNA-treated cells and in cells with low caveolin-1 levels (see Figures4(a) and 4(b)). Activation of VEGFR2 was accomplished by stimulating starved cells with 200 ng/mL VEGF165 for 10 minutes. In this case, signal transduction was found to be significantly lower in cells that had been treated with caveolin-1 siRNA than in cells with normal levels of caveolin-1 (see Figures 4(c) and 4(d)).Activation of Akt and
ERK1/2 in response to external stimuli. HUVECs were transfected with either
caveolin-1 siRNA, negative control siRNA, or left untreated. 48 hours after
transfection, cells were subjected to the following stimuli. (a), (b) Cells
were kept in suspension for 30 minutes and then plated on fibronectin-coated
culture plates to activate integrin-mediated signaling. Phosphorylation of both
(a) Akt and (b) ERK1/2 increased significantly when compared to cells in
suspension. No significant differences in activation levels were detected
between HUVECs with high or low caveolin-1 levels. (c), (d) Cells were starved
in basal medium and then treated with 200 ng/mL VEGF for 10 minutes. Phosphorylation
of both (c) Akt and (d) ERK1/2 in response to this stimulus was significantly
lower in cells with low levels of caveolin-1 than in cells that were treated
with negative control siRNA (P*<.01). All bars are averages of
three separate experiments.
(a)(b)(c)(d)


## 3.1. Mechanotransduction in Ecs

In order to investigate
mechanotransduction in our flow chamber system, HUVECs were subjected to
physiologically relevant shear stresses for 10 minutes and 30 minutes. After these
treatments, cell lysates were tested for phosphorylated Akt and ERK1/2 by
immunoblotting. As shown in Figure1, 10 minutes of shear stress led to
significantly enhanced phosphorylation of both Akt and ERK1/2. After 30 minutes
of shear stress, phosphorylation levels had dropped to values comparable to the
static situation. Therefore, we decided to focus on the early timepoint for our
studies into the role of caveolin-1 in the EC mechanotransduction.Figure 1
Activation of mechanotransduction pathways by
shear. HUVECs were subjected to shear stress for different periods.
Subsequently, the amount of phosphorylated Akt and ERK1/2 was determined by
immunoblotting. After 10 minutes, phosphorylation levels were significantly
higher (P*<.01, Student’s t-test) compared to levels in statically cultured cells. After 30 minutes, this significant increase was no
longer detected.


## 3.2. Caveolin-1 Downregulation

When HUVECs were treated with
caveolin-1 siRNA, the expression of caveolin-1 decreased significantly to less
than one-third of the caveolin-1 level in untreated cells, as shown by
immunoblotting (see Figures2(a) and 2(b)). When cells were transfected with
negative control siRNA, no statistically significant decrease in caveolin-1
expression was detected, showing the specificity of the siRNA treatment. Moreover,
expression of Akt and ERK1/2 was not affected by the transfection procedure or
the lowering of caveolin-1 levels (see Figure 2(a)). The downregulation of
caveolin-1 was confirmed by using immunocytofluorescence (see Figure 2(c)). Using the same microscope settings, staining intensity was approximately four
times higher in untreated cells than in caveolin-1 siRNA-treated cells. The
remaining caveolin-1 in the siRNA-treated cells was located mostly in the
perinuclear area, with a lack of intense membrane staining like in the
untreated and control RNA treated cells.Downregulation of caveolin-1 expression by RNAi.
HUVECs were treated with caveolin-1 siRNA, negative control siRNA, or left
untreated. Caveolin-1 levels were determined 48 hours after transfection. (a)
Immunoblot analysis of cell lysates. Equal loading of proteins of each
transfection condition was ensured by detecting the unrelated metabolic enzyme
GAPDH on the same blot. (b) Quantification of the signal intensities in the
immunoblot assay. The bars represent average intensities of the caveolin-1 band
in seven separate experiments. Error bars represent standard deviation.
Caveolin-1 siRNA treated cells had significantly lower caveolin-1 expression
than control siRNA-treated cells (P*<.0001, Student’s t-test). No significant differences were found between untreated cells and negative control siRNA-treated cells. Bars represent averages of seven separate
experiments. (c) Confocal microscopic imaging of caveolin-1. Treated cells were
fixed and labeled with caveolin-1 antibodies that were detected by Alexa 633-coupled
secondary antibodies. Nuclei were stained with DAPI. All images were taken with
the same microscope settings. Scale bar is 50 μm.
(a)(b)(c)


## 3.3. Mechanotransduction in Ecs with Lowered Caveolin-1 Levels

HUVECs with normal and lowered levels
of caveolin-1 were subjected to physiological levels of shear stress in a
parallel plate flow chamber. After 10 minutes of shear stress, phosphorylation
status of the important mechanotransducing molecules Akt and ERK1/2 was
determined by phospho-specific immunoblotting. Total expression levels of these
proteins were not affected by siRNA treatment, as determined by immunoblotting
(see Figure2(a)). After subjecting HUVECs to shear stress, phosphorylation
status of Akt and ERK1/2 increased approximately five to ten times (see Figure 3). We found that the activation of both signal transduction pathways was
significantly lower in caveolin-1 siRNA-treated cells than in cells treated
with negative control siRNA. No significant differences were found among the activation levels of control siRNA-treated cells and untreated cells.Activation of Akt and
ERK1/2 in response to shear stress. HUVECs were transfected with either
caveolin-1 siRNA, negative control siRNA, or left untreated. 48 hours after
transfection, cells were subjected to a shear stress of approximately 15 dyn/cm2 for 10 minutes. (a) After treatment, cells were lysed, and the amount of
phosphorylated Akt and ERK1/2 was determined by immunoblotting. Equal loading
of proteins of each transfection condition was ensured by detecting the
unrelated metabolic enzyme GAPDH on the same blot. The phosphorylation status
of statically cultured cells is shown as a reference. (b) Quantification of the
signal intensities in the immunoblot assay. Each bar represents the average
intensity of four separate experiments. Error bars denote standard deviation.
Akt and ERK1/2 phosphorylations increase five- to tenfold when untreated cells
are subjected to shear stress. Both Akt and ERK1/2 phosphorylations are
significantly lower (P*<.05, P**<.01, Student’s t-test)
when cells are treated with caveolin-1 siRNA instead of negative control siRNA
prior to subjecting the cells to shear stress. No significant difference was
detected between untreated cells and cells transfected with negative control
siRNA, showing that the transfection procedure itself has no influence. Graphs
represent the averages of four separate experiments.
(a)(b)


## 3.4. Signal Transduction in Ecs with Lowered Caveolin-1 Levels

In order to uncover the mechanism for
the ineffective mechanotransduction in cells with low caveolin-1 levels, we induced
signal transduction by activating cell surface receptors. We chose activation
of integrins and VEGFR2, because these proteins are well known to be essential
for mechanotransduction. In order to assess integrin-mediated signal
transduction, cells were kept in suspension for 30 minutes and then plated for
20 minutes on a fibronectin-coated surface. When checking the phosphorylation
status of Akt and ERK1/2 by phospho-specific immunoblotting, no significant
differences could be detected between the activation of signal transduction
pathways in negative control siRNA-treated cells and in cells with low caveolin-1 levels (see Figures4(a) and 4(b)). Activation of VEGFR2 was accomplished by stimulating starved cells with 200 ng/mL VEGF165 for 10 minutes. In this case, signal transduction was found to be significantly lower in cells that had been treated with caveolin-1 siRNA than in cells with normal levels of caveolin-1 (see Figures 4(c) and 4(d)).Activation of Akt and
ERK1/2 in response to external stimuli. HUVECs were transfected with either
caveolin-1 siRNA, negative control siRNA, or left untreated. 48 hours after
transfection, cells were subjected to the following stimuli. (a), (b) Cells
were kept in suspension for 30 minutes and then plated on fibronectin-coated
culture plates to activate integrin-mediated signaling. Phosphorylation of both
(a) Akt and (b) ERK1/2 increased significantly when compared to cells in
suspension. No significant differences in activation levels were detected
between HUVECs with high or low caveolin-1 levels. (c), (d) Cells were starved
in basal medium and then treated with 200 ng/mL VEGF for 10 minutes. Phosphorylation
of both (c) Akt and (d) ERK1/2 in response to this stimulus was significantly
lower in cells with low levels of caveolin-1 than in cells that were treated
with negative control siRNA (P*<.01). All bars are averages of
three separate experiments.
(a)(b)(c)(d)


## 4. Discussion

Our study shows that lowering the
expression level of caveolin-1 in human ECs by RNAi inhibits
mechanotransduction in response to shear stress. Thus, we confirm the essential
role of caveolae in the EC response to shear stress which has also been
suggested by research groups that interfered with caveolar function using
methods like cholesterol extraction [14] and antibody blocking [16].We show that the activation of both
Akt and ERK1/2 is affected by the lowering of caveolin-1. In both cases, this
lowering will have functional repercussions for the cells. Akt is well known
for its role in cell survival and resistance to apoptosis. Indeed, activation
of Akt in response to shear stress has been shown to suppress apoptosis in ECs [15, 19]. Based on the findings in this
study, one would expect that ECs with low caveolin-1 levels are more sensitive
to apoptosis when subjected to shear stress than untreated cells. This would be
an interesting and counterintuitive result, because lack of caveolin-1
generally renders cells less sensitive to apoptosis [20]. The exact mechanisms of this
decreased apoptosis sensitivity have not yet been elucidated, although the Akt
pathway is definitely involved [20]. A number of important membrane receptors
in ECs, such as activin receptor-like kinase 1 (ALK-1) [21], VEGFR2 [22], and epithelial growth factor
receptor (EGFR) [23], have been shown to associate with
caveolin-1. Moreover, all mentioned receptors have been shown to induce Akt
signaling [24–26]. Therefore, loss of the association
between these receptors and caveolin-1 by downregulation of the latter may be a
reason for changes in Akt activation and apoptosis sensitivity. Given the
importance of EC apoptosis in the onset and development of atherosclerosis [27], future studies should be performed
to provide more insight in this functional implication of the substantially
lowered Akt activation in cells with less caveolin-1.Another well-known downstream effect
of Akt activation is increased eNOS activity [28]. Based on our finding that lowered
levels of caveolin-1 inhibit activation of Akt, it can be expected that eNOS
activation and the resulting effects on blood vessels, such as vasodilatation,
are also impaired. Ex vivo studies
have indeed shown that eNOS activation and vasodilatation in response to fluid
flow are lower in arteries of caveolin-1 knockout mice than in blood vessels of
wild-type mice [13].The dramatically lowered activation
of ERK1/2 in response to shear stress in caveolin-1 siRNA-treated ECs that we
describe in this study will also have functional implications. Activation of ERK1/2
has an important role in modulating gene expression in response to shear stress.
Two well-described examples show that ERK1/2 activation is essential for the
shear stress-induced increase in the expression of matrix metalloprotease-9 [29] and eNOS [30]. The absence of ERK1/2 activation in
our system will surely have an impact on these shear stress-induced changes in
expression.When attempting to identify the
mechanism by which caveolin-1 contributes to mechanotransduction, we first
decided to investigate the signal transduction in response to integrin binding
in these cells. Integrin-mediated signaling is an essential process in
mechanotransduction [2, 31]. Moreover, there have been multiple
reports of the involvement of caveolin-1 in this signaling pathway [32–34]. Despite these reports, we were
unable to detect any changes in activation of Akt or ERK1/2 between caveolin-1 siRNA-treated
cells and untreated cells. It could be that in our system the remaining
caveolin-1 is still sufficient to properly induce these signaling events. Our
system differs from the ones used in the aforementioned reports by cell type,
caveolin-1 levels, and the chosen output parameters to determine efficient
signaling. Based on our experiments, we can conclude that it is unlikely that
the inefficient mechanotransduction as seen in our system is due to impaired
integrin signaling.We decided also to investigate signal
transduction in response to VEGF in caveolin-1 siRNA-treated ECs.
Ligand-independent activation of VEGFR2 is another essential event in
mechanotransduction [3], and stimulation with VEGF can serve
as a way to specifically activate and monitor this part of the signaling
network. The role of caveolin-1 in VEGF-induced signal transduction is still
ambiguous and may depend on cell type and culture conditions. In some cell
systems, downregulation of caveolin-1 leads to hyperactivation of signal
transduction pathways [35]. In addition, other studies that
focus on the role of caveolin-1 in VEGF-initiated signaling have shown that both an increase and a decrease of the levels of caveolin-1 in ECs have a negative impact on downstream signal transduction [22, 36]. The direct association between caveolin-1 and VEGFR2 inhibits activation of the latter, but on the other hand, the association also seems necessary for proper initiation of downstream signaling after growth factor receptor activation. Moreover, caveolin-1 knock-out mice have impaired vasculogenic
potential, due to impaired VEGF signaling [36]. Also in our system, lowering the
level of caveolin-1 has a negative impact on signal transduction when ECs are
stimulated with VEGF. This suggests that the lack of mechanotransduction in ECs
with low caveolin-1 levels is due to the important role that caveolin-1 plays
in coupling VEGFR2 activation to downstream signaling events.Based on the results presented in
this article, it can be concluded that caveolin-1 is needed for activation of
Akt and ERK1/2 in ECs that are subjected to shear stress. Our results suggest
that the impaired activation of these pathways in ECs with low levels of
caveolin-1 is due to inefficient VEGFR2 signaling. This study highlights the
importance of caveolin-1 in normal EC functioning. The role that caveolin-1 plays in the biology of ECs and
the impact it has on the physiology of the vasculature in vivo are becoming more and more clear. Because of the
importance of caveolin-1 in endothelial signaling, it is a potential target for
clinical applications, for example, in tumor angiogenesis and atherosclerosis.
Therefore, the role of caveolin-1 in the EC shear stress response is definitely
worth studying in more detail.



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*Source: 532432-2008-12-23.xml*