© 2000 American Heart Association, Inc.
Vascular Biology |
Fluid Shear Stress Induces Heat Shock Protein 60 Expression in Endothelial Cells In Vitro and In Vivo
From the Institute of Biomedical Aging Research (B.-W.H., A.A., Q.X., G.W.), Austrian Academy of Sciences, Innsbruck; the D. Swarovski Laboratory of Transplant-Surgery (B.-W.H., T.E., R.M.), University of Innsbruck; the Institute of Chemistry and Biochemistry (E.-O.H.), University of Salzburg; and the Institute of Pathology (P.O.), University of Innsbruck, Austria.
Correspondence to Boris-Wolfgang Hochleitner, University Hospital of Innsbruck, Surgical Department, Anichstrasse 35, A-6020 Innsbruck, Austria. E-mail Boris.Hochleitner{at}uibk.ac.at
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Abstract |
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Abstract—Recent investigations indicate that the initial event in the pathogenesis of atherosclerosis involves an (auto)immunologic injury to the vessel wall. Heat shock proteins (hsps), which are expressed on the endothelial cell surface, constitute possible autoantigens. After being exposed to shear stress of 30 dyne/cm2 in vitro by means of a rotational viscometer, human umbilical vein endothelial cells were immunohistochemically stained for hsp 60 by the monoclonal antibody ML-30; static control cells were negative. Maximal hsp 60 induction was observed after 12 hours of hemodynamic stress. In Northern blots, the level of hsp 60 mRNA was markedly increased after only 1 hour of shear stress in human umbilical vein endothelial cells compared with static control cells. In vivo investigations in Lewis rats confirmed these in vitro findings: the intima and media of frozen sections of the right common carotid artery exposed to increased wall shear stress (after ligation of the left common carotid artery) were stained for hsp 60. The vessel wall of the left low-shear-stress–exposed side was negative. These findings demonstrate that shear stress results in hsp 60 induction in endothelial cells in vivo and in vitro, providing the prerequisite for humoral and cellular reactions to endothelial hsp in the earliest stages of atherosclerosis.
Key Words: atherosclerosis • endothelial cells • endothelium • shear stress • heat shock protein
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Introduction |
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It is well known that cells respond to a variety of environmental stresses (eg, elevated temperature, heavy metals, toxins, and free radicals) by rapid production of a family of proteins called the heat shock proteins (hsps).1 2 3 This response is markedly conserved throughout evolution, from bacteria to humans. Hsps are divided into 4 major families according to their molecular mass (30, 60, 70, and 90 kDa). Some of them (60-kDa and 70-kDa families) function as "molecular chaperones." They bind to immature or partially denatured proteins, helping them to fold into their native conformation and regain their functional activity.
Some microbial hsps are important target antigens, eliciting protective immune responses against many infectious organisms. As a side effect, hsps evoke interest in pathology as autoantigens by "antigenic mimicry." The have been associated with diseases like rheumatoid arthritis,4 diabetes mellitus,5 systemic sclerosis,6 rejection of transplanted organs,7 and atherosclerosis.8 9 10 A previous epidemiological study11 from our laboratory had demonstrated that serum antibodies to mycobacterial hsp 65, which is a homologue of human hsp 60, were significantly increased in clinically healthy subjects with sonographically demonstrable carotid atherosclerosis compared with those without lesions. This increased antibody level was independent of other established risk factors, such as hyperlipidemia, smoking, hypertension, diabetes mellitus, and obesity. Serum concentrations of hsp 65 antibodies were also elevated in patients with coronary heart disease compared with healthy controls.12 Furthermore, in atherosclerotic lesions of rabbits and humans, endothelial cells showed higher levels of hsp 60 than those in other parts of the normal arterial intima.13 14 In fact, immunization of rabbits with recombinant mycobacterial hsp 65 induced the development of atherosclerotic lesions at characteristic predilection sites, such as the curve, bifurcation, branch, or attachment of arteries.15
The flow of blood is associated with shear stress, the tractive force per unit area, which acts in the direction of blood flow on the surface of the inner wall of the blood vessel. In the simplifying case of laminar Poiseuille-Hagen flow, wall shear stress is proportional to flow velocity and medium viscosity and is inversely proportional to the third power of the internal radius.16 Although the effect of inflammatory mediators and cytokines on endothelial cells has been extensively studied, less is known about the effect of shear stress. Endothelial cells rapidly respond to a change of shear stress by signal transduction events, such as an increase of intracellular calcium,17 18 19 decrease of intracellular pH,20 or increase of inositol trisphosphate and diacylglycerol.21 22 The expression of many genes is altered by shear stress. Examples of such genes are c-fos,23 24 25 platelet-derived growth factor B chain,26 mitogen-activated protein kinases (ERK/JNK),27 and intercellular adhesion molecule-1.28
Atherosclerosis is restricted to arteries, and venosclerosis usually does not exist. However, if a vein is transferred to a site subjected to arterial blood pressure, as in coronary bypass surgery, sclerosis and stenosis may often be observed. The patency rate of aortocoronary saphenous vein bypass grafts is estimated at 66% in 10 years. The histopathologic examination of surgically removed saphenous vein bypass grafts showed that stenosis resulted either from intimal fibrocellular proliferation or mainly from progressive venosclerosis.29
We speculate that increased hemodynamic stress may be the decisive factor for the occurrence of venosclerosis when veins are exposed to arterial pressure. However, shear forces should not be regarded apart from other factors (eg, oxidized LDL, cytokines, and elevated blood glucose) to which vascular endothelium is exposed. Possibly, prestress by arterial blood pressure lowers the threshold for hsp 60/adhesion molecule induction by other stress factors, including classic risk factors for atherosclerosis. To establish the missing link between shear forces and the immunologic hypothesis of atherosclerosis, we addressed whether hemodynamic forces could also induce hsp 60 expression in endothelial cells in vitro and in vivo.
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Methods |
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Cell Culture
Human umbilical vein endothelial cells (HUVECs) were harvested from umbilical cords obtained from the Department of Gynecology and Obstetrics of the Innsbruck University Hospital by collagenase type II (Sigma Chemical Co) digestion for 15 minutes.30 Subsequent cultivation was maintained at 37°C in a humidified atmosphere (5% CO2 in air) by using endothelial cell growth medium (Promocell, catalogue No. C-22010). Petri dishes with a diameter of 100 mm (Falcon, item 3003, Becton Dickinson Co) were precoated with 28 µg/mL human fibronectin (HFN, Inotech AG). Cells were identified as endothelial by the typical cobblestone-like morphological appearance and positive von Willebrand factor immunohistochemical staining (mouse/anti-human von Willebrand factor antibody, Dako, code No. M0616). Only cultures of high purity with fibroblast contamination <3% were accepted for further experimentation. Trypsin/EDTA solution (0.025%/0.02%, Sigma) was applied for subcultivation. Second and third passages were used for shear stress experiments and static controls. Endothelial cells were seeded into precoated Petri dishes (Falcon, item 3003) at seeding densities of 0.8 to 1.2x106 cells per dish. Experiments were initiated after a cultivation period of 5 days, when a semiconfluent monolayer had been established.
Shear Stress Device
A semiconfluent monolayer of HUVECs was fed with growth
medium (see above) 24 hours before the onset of
hemodynamic stress. The monolayer was then exposed to
laminar shear stress by use of a purpose-built cone and plate
viscometer, specifically designed to accept 100-mm-diameter culture
plates at 37°C and a humidified atmosphere of 5%
CO2.31 Reynolds number, R, and fluid
shear stress magnitude, 
, were computed at different values of
radius, r, as shown in Equations 1
, and 2 below
according to Sdougos et al31 :
 | (1) |
 | (2) |
is kinematic viscosity,
is rotational velocity, and 
is the cone angle. Static viscosity
of the medium, obtained by measuring kinematic viscosity with a
calibrated capillary viscometer and multiplying by the density of the
medium, was 0.79 centipoise at 37°C. Average Reynolds number and
shear stress magnitudes were obtained by integrating their respective
values over the area of the tissue culture plate. Our cone with an
angle of 0.5° and spinning at 5 revolutions per second achieved an
average shear stress of 30 dyne/cm2 and Reynolds
number of 0.20. Other shear stress magnitudes were attained by
variation of rotational speed.
Immunohistochemistry
Sections were fixed in absolute ethanol for 10 minutes at room
temperature. Subsequently, the sections were placed in a humidified
chamber and overlaid with the monoclonal antibody ML-30 (gift from Dr
J. Ivanyi, London, UK) recognizing a cross-reactive epitope of
mycobacterial hsp 65 and human hsp 60.32 After they were
washed with Tris buffer (0.005 mol/L, pH 7.6), the sections were
incubated with rabbit anti-mouse immunoglobulin antibodies (Dako Corp)
for 30 minutes. After 3 further washes in Tris buffer, the sections
were incubated with alkaline phosphatase mouse anti–alkaline
phosphatase complex (Dako) for 30 minutes, washed in Tris buffer 3
times, and developed for 10 minutes at room temperature with use of a
substrate solution containing 9.8 mL Tris buffer (0.1 mol/L, pH 7.8),
0.2 mL dimethylformamide, 8 mg naphthol AS-MX phosphate, 3 mg
levamisole, and 10 mg Fast Red TR salt (Sigma).
RNA Isolation and Hybridization
The acid guanidinium thiocyanate–phenol–chloroform
method33 was used to isolate total cellular RNA. After
determination of RNA purity and concentration, 10 µg of total RNA was
fractionated with the use of 1% agarose gels containing 8%
formaldehyde and 0.04 mol/L Bicin (pH 8,4, Serva). RNA was then
transferred overnight by capillary action in 10x SSC (3 mol/L sodium
chloride and 300 mmol/L sodium citrate, pH 7) on a blotting
membrane (Zeta-Probe, Bio-Rad) and immobilized by UV
irradiation. Prehybridization and hybridization of membranes were
accomplished in Church buffer (1% BSA, 7% SDS, 0.075% EDTA, and 0.5
mol/L sodium phosphate). Northern blot hybridization was performed with
a random-primer 32P-labeled fragment of hsp
60.15 After incubation, the blots were washed in 2x
SSC/1% SDS for 15 minutes at room temperature, followed by 2 washes of
20 minutes in 05x SSC/1% SDS at 65°C and then exposed to x-ray
film.
In Vivo Experiments
Adult female Lewis rats (Harlan-Winkelmann, Borchen, Germany),
weighing 200 to 300 g, were anesthetized with an
intraperitoneal injection of 5 mg Vetanarcol
(Veterinaria AG). A cervical midline incision was made caudal to the
thyroid cartilage, and then the left common carotid artery was exposed
and ligated just rostral to the origin of the superior thyroid artery
branch with 2.0 silk. The incision was closed in layers, and the animal
was allowed to recover. According to Walpola and
colleagues,34 35 this procedure results in an increase of
shear stress in the right common carotid artery of 
170% and a
decrease in the downstream left carotid artery of 70%.
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Results |
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In Vitro Data: Hsp 60 Staining
At least 4 different HUVEC isolates were used for the in vitro studies. Semiconfluent monolayers of HUVECs underwent laminar shear stress of 30 dyne/cm2 in a cone-plate viscometer for 3, 6, 12, and 24 hours. Fluid shear stress induced a characteristic cell-shape change and cell alignment, as previously described.36 Cells appeared longer and narrower and displayed a protrusion of the nuclear area. After 24 hours of constant shear stress, there was complete and uniform alignment in the direction of the flow.
HUVECs were immunohistochemically stained with the monoclonal antibody
anti–hsp 65/60 ML-30. Static controls showed very weak, if any, hsp 60
staining, whereas mechanically stressed HUVECs revealed distinctive
positive staining (Figure 1).
Increased expression of hsp 60 was found at 6 hours, and there was a
peak at 12 hours and a subsequent slight decline at 24 hours. At 12
hours, the percentage of responsive cells was determined as 94%
(average of cell counts from 3 subsequent experiments). Positive
controls were obtained by means of a heat shock of 42°C for 30
minutes. Figure 2 demonstrates a positive
"dose"-response correlation between the level of shear stress and
immunohistochemical staining for hsp 60. Semiconfluent HUVECs underwent
6 hours of laminar shear stress of varied intensities (5, 20, and 50
dyne/cm2). Shear forces corresponding to venous
flow (5 dyne/cm2) only had a minimal effect.
However, simulating arterial conditions in vitro (20
dyne/cm2) produced more distinctive, but
moderate, staining. Excessive shear forces >50
dyne/cm2, as found
physiologically at the branching of
arterial vessels, induced strongly positive staining.
Figure 3 illustrates how long an episode
of elevated shear stress must persist to induce a significantly
increased expression of hsp 60. HUVECs underwent 5, 10, or 30 minutes
of shear stress (30 dyne/cm2) and remained in the
incubator under static conditions until 6 hours after the onset of
shear forces. Five or 10 minutes of increased
hemodynamic stress did not significantly influence hsp
60 protein levels, as determined by immunohistochemistry, whereas 30
minutes of shear stress was enough to cause a translational
upregulation of hsp 60.
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Hsp 60 mRNA
Semiconfluent monolayers of HUVECs were subjected to laminar shear
stress of 30 dyne/cm2 in a cone-plate viscometer
for 30 minutes (plus 30-minute static), 1 hour, 3 hours (plus 3-hour
static), and 8 hours. mRNA was isolated from these cells at the above
time points after onset of shear stress. The cells expressed hsp 60
transcripts after 30 minutes of shear stress and 30 minutes of
relaxation at significantly higher levels than did static controls
(Figure 4A). However, as expected and
known from previous studies, static controls were not completely
negative for hsp 60 mRNA but showed a baseline expression of hsp 60
mRNA, which was possibly due to the stress of cell culture
itself.37 38 Hsp 60 mRNA levels increased between 1 hour
and 8 hours of constant shear stress. Cells in lane C underwent 3 hours
of shear stress and were allowed to rest for a further 3 hours before
harvesting of the cells. During the 3 hours of relaxation, hsp 60 mRNA
significantly decreased. Figure 4B demonstrates a correlation
between dose of shear stress and hsp 60 mRNA formation, as also shown
above on the protein level.
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In Vivo Data: Hsp 60 Staining
Three animals per time point were euthanized 3, 6, 12, and 24
hours after ligation of the left common carotid artery. Left and right
common carotid artery samples were put into slices of liver tissue for
processing purposes and frozen in liquid nitrogen. Serial cross
sections were immunohistochemically stained with monoclonal antibodies
against mycobacterial hsp 65 (ML-30). Sham-operated rats and rats that
were anesthetized only (without subsequent operation) showed no
staining on either side. Intimal endothelium, media,
and adventitia of left carotid arteries, experiencing low shear stress,
showed weak, if any, hsp 60 staining at all time points. Carotid
endothelium and media from the right side exposed to
high shear stress revealed distinctive positive staining. The time
course is shown in Figure 5: hsp 60
staining of intima and media started between 3 and 6 hours after the
operation and steadily increased from 12 to 24 hours.
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indicates lumen; x, intima; 
, media; and *,
adventitia. |
Discussion |
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Previous work8 9 10 11 13 14 15 defined a possible role of hsp 60 as an (auto)antigen in atherogenesis. Because hsp 60 is imperceptibly expressed constitutively on the cell surface, a certain prestress is a prerequisite for making endothelial cells immunologic targets of anti–hsp 60 antibodies and/or effective T cells. Using a combination of immunohistochemistry and Northern analysis, we were able to demonstrate that fluctuations in blood flow can exert such a prestress on endothelial cells, upregulating hsp 60 expression in vitro and in vivo. Although the in vitro fluorescent staining of hsp 60 is clearly intracellular in Figures 1 to 3
, there is
ample evidence from previous work that hsp 60 is also expressed on the
surface of stressed endothelial cells.13
As a matter of fact, it has also been shown that anti–hsp 60/65
antibodies are able to lyse stressed but not unstressed
endothelial cells.39
The in vivo studies (Figure 5) demonstrate that staining
of hsp 60 is not restricted to the intima but also includes the media
and adventitia. This finding complies with the previous observation
that hypertensive stress in vivo lead to the expression of hsp 70 by
vascular smooth muscle cells,40 and we deem it likely that
similar mechanisms may underlie the expression of hsp 60 in our
experiments at the mentioned sites. As a matter of fact, it has been
demonstrated previously that smooth muscle cells in atherosclerotic
lesions express hsp 60.14
Static controls did not stain for hsp 60 protein, but we constantly
observed a basal hsp 60 mRNA transcription of static HUVECs in vitro.
Although culture medium was exchanged every 48 hours and pH was kept
stable, the basal hsp 60 transcription is possibly due to the stress of
cell culture itself. Another discrepancy between Northern
analysis and fluorescent staining is the fact that in
Figure 4B a lack of hsp 60 mRNA induction with 20
dyne/cm2 is noted, whereas in Figure 2, positive immunohistochemical staining for hsp 60 is seen with
only 5 dyne/cm2. However, we do not feel that
this is an experimental error, because both phenomena were very
reproducible.
To our knowledge, the only other stress protein that has been studied regarding the application of shear stress on endothelial cells is the small molecular mass hsp of 27 kDa (hsp 27).41 Interestingly, after an application of shear stress of 16 dyne/cm2, hsp 27 became more highly phosphorylated, whereas hsp 27 antigen levels did not change. Thus, the modulation of hsp 27 by shear stress is merely posttranslational.
Shear stress is a less ideal parameter for predilection
sites of atherosclerosis. Gibson et al42
used quantitative angiography to calculate coronary
arterial diameter during a 3-year period in 20
arterial coronary segments. Low shear stress was
correlated with an increased rate of atherosclerosis
progression. However, the emphasis on low shear stress as a dominant
factor in the predilection for localization of atherosclerotic lesions
is not justified in view of the development of
atherosclerosis at sites of high shear stress. In the
present study, we selected higher shear stress compared with
physiological arterial flow. For in
vitro tests, 30 dyne/cm2 was used, and the wall
shear stress in the right common carotid artery increased from 12
dyne/cm2 to 30 dyne/cm2
after ligating the left common carotid artery in vivo.
Texon 43 identified the sites of predilection for atherosclerosis as precisely those locations characterized by a relative reduction in lateral pressure. The lateral pressure is predictable in a nonviscid fluid on the basis of Bernoulli’s theorem, which states that the sum of pressure and the square of the velocity times density divided by 2 is constant for any 2 points of flow on the same stream line. For example, fluid in a vessel with converging boundaries causes the lateral pressure to be reduced at the narrow portion, where the velocity is increased.
The transductional events and transcriptional factors for shear stress–mediated events are currently under investigation in many laboratories. Resnick and colleagues44 45 defined a "shear stress response element" in the promoter of the platelet-derived growth factor B chain gene that interacts with DNA binding proteins in the nuclei of shear-stressed endothelial cells to upregulate transcriptional activity.
The transductional events after the onset of shear stress, which finally lead to hsp 60 expression, are not yet clear. One possible mechanism may involve the endothelial nitric oxide synthase (eNOS), which is expressed constitutively in endothelial cells. Shear stress is a powerful modulator of eNOS expression, increasing eNOS mRNA significantly.46 Early data from our laboratory show that intracellular nitric oxide is able to upregulate hsp 60 mRNA formation.47 Another early event leading to upregulation of hsp 60 mRNA is the generation of heat shock factor, which also has been shown to be shear stress inducible.47
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Acknowledgments |
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This study was supported by the Austrian Science Fund (grant No. P-12213 to Dr Wick).
Received May 11, 1999; accepted September 3, 1999.
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