University of Pennsylvania
Departmental Papers (CBE)
Department of Chemical & Biomolecular
Hemodynamic Regulation of Inflammation at the
Julie Y. Ji
University of Pennsylvania
University of Pennsylvania
Scott L. Diamond
University of Pennsylvania, firstname.lastname@example.org
Postprint version. Published inAnnals of Biomedical Engineering, Volume 36, Issue 4, April 2008, pages 586-595.
Publisher URL: http://dx.doi.org/10.1007/s10439-008-9465-4
This paper is posted at ScholarlyCommons.http://repository.upenn.edu/cbe_papers/113
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Ji J.Y., Jing H. and Diamond S.L. - 1
Hemodynamic Regulation of Inflammation
at the Endothelial-Neutrophil Interface
Julie Y. Ji1, Huiyan Jing2, and Scott L. Diamond1,2*
Institute for Medicine and Engineering
Departments of Bioengineering1 and Chemical and Biomolecular Engineering2
University of Pennsylvania
1024 Vagelos Research Laboratory
3340 Smith Walk
Philadelphia PA 19104
Tel: (215) 573-5702
Fax: (215) 573-6815
* Corresponding author
Shear Stress is anti-inflammatory
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Ji J.Y., Jing H, and Diamond S.L. - Page 2
Arterial shear stress can regulate endothelial phenotype. The potential for anti-
inflammatory effects of shear stress on TNFα-activated endothelium was tested in assays of
cytokine expression and neutrophil adhesion. In cultured human aortic endothelial cells
(HAEC), arterial shear stress of 10 dyne/cm2 blocked by > 80% the induction by 5 ng/ml TNFα
of interleukin-8 (IL-8) and IL-6 secretion (50% and 90% reduction, respectively, in the presence
of nitric oxide synthase antagonism with 200 μM nitro-L-arginine methylester, L-NAME).
Exposure of TNFα-stimulated HAEC to arterial shear stress for 5 hr also reduced by 60% (P <
0.001) the conversion of neutrophil rolling to firm arrest in a venous flow assay conducted at 1
dyne/cm2. Also, neutrophil rolling lengths at 1 dyne/cm2 were longer when TNFα-stimulated
HAEC were presheared for 5 hr at arterial stresses. In experiments with a synthetic promoter
that provides luciferase induction to detect cis interactions of glucocorticoid receptor (GR) and
NFκB, shear stress caused a marked 40-fold induction of luciferase in TNFα-treated cells,
suggesting a role for GR pathways in the anti-inflammatory actions of fluid shear stress.
Hemodynamic force exerts anti-inflammatory effects on cytokine activated endothelium by
attenuation of cytokine expression and neutrophil firm arrest.
Ji J.Y., Jing H, and Diamond S.L. - Page 3
shear stress, inflammation, glucucorticoid, neutrophils
Ji J.Y., Jing H, and Diamond S.L. - Page 4
Atherosclerosis is a chronic inflammatory disease that is often hemodynamically
localized at sites of low and reversing shear stress.1, 2 Inflammatory markers are distinct
indicators in the development and the progression of atherosclerotic lesions, which are heavily
infiltrated with macrophages, T-lymphocytes and other cellular components of inflammation.3
During inflammation, the initial rolling interaction of neutrophils on activated endothelium is
mediated by selectins.4 Firm adhesion occurs when β2 integrins on neutrophils are up regulated
by chemokines such as IL-8 to mediate binding to adhesion molecules (ICAM-1) on the
activated endothelium.5 Proinflammatory cytokines such as TNFα or IL-1 induce the expression
of several cytokines and cell adhesion molecules by endothelial cells.3
With respect to steroidal anti-inflammatory drugs, there are two main mechanisms by
which glucocorticoids are thought to exert their therapeutic actions in suppressing inflammatory
and immune responses. Glucocorticoids diffuse into the cytoplasm and bind to glucocorticoid
receptor (GR), which then translocate into the nucleus and bind as dimers to the glucocorticoid
response elements (GRE) present in various promoters.6 Examples of genes regulated through
GRE in their promoters include IL-8 and IL-2 receptor α.7 Activated GR can also suppress
inflammation by directly interacting with activated transcription factors, such as nuclear factor-
kappa B (NFκB) and activator protein-1 (AP-1), thus altering NFκB or AP-1 participation in
inflammatory gene expression.8, 9 In most cases, the active form of NFκB is a heterodimer of
RelA (p65) and NFκB1 (p50) released from its inhibitor IκB. Steroid-liganded GR can directly
interact with the p65 subunit of NFκB10 as well as interfering with transcriptional cofactors
CREB binding protein (CBP) and steroid receptor coactivator-1 (SRC-1).11 In the endothelium,
TNFα inducible genes that are down regulated by the presence of dexamethasone include:
Ji J.Y., Jing H, and Diamond S.L. - Page 5
interferon β, platelet-derived growth factor (PDGF) B subunit, transforming growth factor (TGF)
β2, vascular endothelial growth factor receptor 3 (VEGFR3), IL-1, 2, 7, and 8.12 Glucocorticoids
also suppress the expression of inflammatory markers such as adhesion molecule expression
(ICAM-1, E-selectin, VCAM-1)13-15 and various interleukins. GR suppresses NFκB induction of
the IL-6 gene in vascular endothelial cells,16 indicating that the precise mechanisms of NFκB
down-regulation by nuclear steroid receptors can be gene-specific since IL-6 lacks any apparent
GRE in its promoter.
The endothelium also acts as a dynamic interface between biochemical triggers and
mechanical factors (hemodynamics) and inflammatory cell adhesion. Various kinases activated
during mechanotransduction can alter the activity of transcription factors such as NFκB, AP-1,
erg-1, and GR.17-20 A recent study by Chiu et al. showed that shear stress on endothelial cells
alters TNFα-stimulated expression of ICAM-1, VCAM-1 and E-selectin while decreasing TNFα-
stimulated NFκB -DNA binding activity in mobility shift assay,21 although no tests of cytokine
secretion or endothelial adhesiveness were conducted in that study. Glucocorticoid receptors are
present in endothelium and smooth muscle cells.22, 23 In our prior study, we demonstrated that
shear stress caused endothelial GR nuclear localization and activated transcription from a GRE
promoter through pathways sensitive to inhibitors of the shear-activated kinases, MEK1/2
kinases and PI-3 kinase.20 This finding suggests certain parallels between the atheroprotective
role of unidirectional shear stress and the anti-inflammatory actions of the GR. We now test the
hypothesis that shear stress is anti-inflammatory, specifically in suppressing TNFα-induced
endothelial activation with respect to the expression of secreted cytokines and neutrophil
Ji J.Y., Jing H, and Diamond S.L. - Page 6
Materials and Methods
Cell Culture and Reagents
Human aortic endothelial cells (HAEC) were maintained in EGM-2 endothelial media
system (Clonetics). Glass slides were coated with type I collagen (BD Biosciences). For flow
chamber experiments, cells were seeded on collagen coated 38 x 75 mm glass slides at a density
of 1 to 2 x 106 cells per slide and cultured to confluency. TNFα was obtained from Sigma.
Incubated cell culture media from HAEC was collected and measured for cytokine content using
human IL-8 and IL-6 ELISA immunoassays (R & D Systems) according to the manufacturer’s
instructions. IL-8 and IL-6 concentrations were used to calculate the total amount of cytokine
produced after accounting for volume changes, and normalized with respect to the total number
of cells in each experimental group.
Human blood was collected from healthy adult donors by venipuncture and
anticoagulated with Na-citrate (9 parts blood to 1 part Na-citrate) and neutrophils were isolated
over neutrophil isolation medium (Robbins Scientific) as previously described.24 After isolation,
neutrophils were resuspended in Hank’s balanced salt solution (HBSS, Gibco Laboratories)
supplemented with 2% HBS, counted, and diluted to a final concentration of 0.75 x 106 cells/ml.
Shear Stress Exposure and Neutrophil Adhesion Studies
Cells were exposed to laminar shear stress in parallel plate flow chambers attached to
flow loops for media recirculation (15 ml) in a 37°C incubator as previously described.20 Wall
shear stress was calculated as:τwall = 6μQ/bh2 for viscosity, μ = 0.01 dynes-sec/cm2; Q,
volumetric flow rate (cm3/s); b, flow chamber width (2.5 cm); h, the total plate separation (0.025
Ji J.Y., Jing H, and Diamond S.L. - Page 7
cm). For neutrophil adhesion studies, following 5 hr arterial shear stress exposure at 10
dyne/cm2 (+ 5 ng/ml TNFα), the flow chambers were reconnected to a Harvard syringe pump for
infusion of a neutrophil suspension at wall shear stress of 1 dyne/cm2. During the neutrophil
adhesion studies at venous flow conditions, flow chambers were imaged by phase contrast
microscopy (Zeiss Axiovert 135, 20X Plan Apochromat) and recorded on videotape for
subsequent digital image analysis. Neutrophils were perfused over HAEC for 5 min before the
start of image acquisition. Each field of view (FOV; 0.1 mm2) of neutrophils flowing over
HAEC was recorded in 10-sec video segments from which total and firmly adherent neutrophil
counts were determined. “Firm adhesion” refers to neutrophils that remained stationary during
10 seconds, and “total” refers to average number of neutrophils that interacted with the
endothelial monolayer in the FOV over the 10-sec interval. Rolling distance was generated
using the multi-tracking function of ImageJ (NIH).
The pGRED was kindly provided by Dr. Alexander Whitehead (U. Penn.) 25. The
pGRED contains the SAA2 promoter with a deletion of a 9-basepair interruption of the GRE
consensus sequence, thus providing an active GRE and an active NFkB site in the promoter
upstream of firefly luciferase. Renilla luciferase transfection control plasmid was from Promega.
For dual luciferase assays, endothelial cultures were washed in PBS and lysed in Passive Lysis
Buffer (Promega). Lysates were assayed for luciferase and Renilla activity using the LAR II and
Stop and Glo Reagents (Promega) in a dual-injection luminometer.
Ji J.Y., Jing H, and Diamond S.L. - Page 8
Shear stress attenuates TNFα induced IL-8 and IL-6 secretion
Addition of 5 ng/ml TNFα under static conditions induced a marked 45-fold increase (P
< 0.001, n = 3) in the total amount of IL-8 secreted by HAEC in 8 hr compared to static control
(Figure 1A). However, shearing the cells during TNFα exposure blocked this IL-8 secretion by
80% (P < 0.001, n = 3). Shear stress alone caused a small increase relative to static culture of
IL-8 secretion from 1 to 3 ng/106cells at 8 hr. This small up-regulation of IL-8 by flow alone
was not seen in the presence of LNAME (Figure 2A) since LNAME-treated cells maintained in
static culture produced about 4 ng of IL-8 per 106 cells at 8 hr.
With respect to IL-6 secretion, adding TNFα to cell culture media induced a striking 33-
fold increase (P < 0.01, n = 3) over static control that was reduced significantly by 90% (P <
0.01, n = 3) by flow (Figure 1B). Shear stress alone caused an increase of IL-6 secretion at 8 hr
compared to static control from 0.05 to 0.25 ng/106cells that was not seen in the presence of
LNAME (Figure 2B). These data demonstrated that shear stressed endothelium, when compared
to stationary cultures, were considerably less responsive to TNFα with respect to IL-8 and IL-6
Either TNFα receptor mediated signaling proximal of NFκB activation was disrupted in
sheared cells and/or shear stress triggered factors, e.g. nitric oxide (NO), that antagonized NFκB
function. To test the role of shear induced NO production26 on TNFα stimulation of HAEC, we
used an eNOS inhibitor nitro-L-arginine methylester (L-NAME) in conjunction with TNFα and
shear stress (Figure 2). HAEC were pretreated with the L-NAME (200 μM, 1 hr), a
concentration known to block shear induced NO release.27 HAEC preconditioned in static
culture or shear condition (10 dynes/cm2, 1 hr) in the presence of L-NAME, were then
Ji J.Y., Jing H, and Diamond S.L. - Page 9
maintained further in the presence or absence of TNFα (5 ng/ml). In comparing the static control
groups from Figure 1 and Figure 2, there was a small increase of basal IL-8 secretion and a
marked increase in IL-6 secretion by HAEC cells pretreated with L-NAME, indicating that the
basal production of NO by static cells limited IL-8 and IL-6 expression. In static HAEC
cultures, pretreatment with L-NAME did not prevent the marked increase of IL-8 and IL-6
secretion by TNFα, and again shearing in media with TNFα yielded a significant reduction of
cytokine expression (50% for IL-8 and 90% for IL-6). With L-NAME present, shear stress
reduced IL-6 production in TNFα-stimulated cells to levels below the matched static control
cultures. These data indicate that shear stress interfered with TNFα-induced increase of IL-8 and
IL-6 without a strict requirement for flow-induced NO.
Shear stress attenuates neutrophil firm arrest on TNFα-activated HAEC
HAEC monolayers were treated with 5 ng/ml TNFα in the presence or absence of arterial
shear stress for 5 hr before a neutrophil adhesion assay at 1 dyne/cm2. Time averaged images
(Figure 3) allowed detection of rolling and arrested neutrophils. On control cultures without
TNFα, neutrophils passed over the endothelial surfaces with essentially no rolling or arrest.
HAEC exposed to shear stress for 5 hr alone (no TNFα) did not promote neutrophil adhesion,
indicating that shear stress alone was not pro-adhesive. HAEC cells treated with TNFα, on the
other hand, were strongly activated with about 38.61 ± 6.92% (n = 15 FOV) of interacting
neutrophils becoming firmly arrested (Figure 4A). Cells maintained under arterial shear stress
for 5 hr during the TNFα induction, however, had 60% (P < 0.001) fewer neutrophils converting
to firm arrest (11.7 ± 4.59% of all interacting neutrophils, n = 15 FOV). The total number of
neutrophils (rolling and arrested) that came to interact with shear stressed HAEC plus TNFα
Ji J.Y., Jing H, and Diamond S.L. - Page 10
versus static HAEC treated with TNFα was not significantly different (56.1 ± 18.8 versus 46.3 ±
17.9 per FOV).
To further quantify the neutrophil interactions with endothelial surfaces at 1 dyne/cm2,
the rolling length in microns over a 10-sec interval was determined. The results are presented in
Figure 4B, in the form of a histogram, generated on the tracked movement of 188 and 212
neutrophils over static and sheared HAEC, respectively. On TNFα-stimulated HAEC in the
absence of arterial shear stress exposure, most neutrophils had short rolling lengths (90% < 21
μm) with the longest being 37 μm and with a median length of 8.83 μm. On sheared HAEC
treated with TNFα, however, the rolling length increased up to 113 μm with the median length of
24 μm. The mean rolling length for both cases was also significantly different: 10.6 μm (no
shear) versus 30.1 μm (10 dyne/cm2) (P < 0.001).
Interactions between glucocorticoid receptor and NFkB in sheared endothelium
While disturbed hemodynamics may enhance endothelium susceptibility to
atherosclerosis, we have detected the anti-inflammatory effects of unidirectional arterial shear
stress in attenuating TNFα-activated endothelial cytokine production (Figures 1 and 2) and
neutrophil adhesion (Figures 3 and 4). The net effect of shear stress, however, encompasses a
variety of transcriptional factors such as AP-1, SP-1, and GR 17-20 to potentially regulate NF-κB
function in a promoter specific manner. We sought to investigate possible interactions between
shear stress activated GR functions, independent of dexamethasone 20, on NFκB function. To
detect cis interactions between GR and NFκB on a promoter, we employed an artificial promoter
construct that is induced when GR and NFκB bind the promoter. The wildtype SAA2 promoter
contains binding sites for NFκB, AP-1, and NF-IL6, along with a disrupted GRE site 28. The
inactive GRE sequence is interrupted in the middle by a function-blocking 9-bp insertion.
Ji J.Y., Jing H, and Diamond S.L. - Page 11
Removal of this 9-bp insertion renders the promoter responsive to dexamethasone potentiation
(Figure 5A) in the presence of cytokine stimulation 25. This artificial promoter construct based on
the SAA2 deletion (pGRED) is unique in that, when induced by a cytokine, its transcriptional
activity is enhanced, not repressed, by dexamethasone. This allows "light-up" detection of
NFκB-GR cross talk. While dexamethasone typically down regulates NFκB function, this may
occur via GR binding to NFκB, either on or off the promoter. Because GRED involves
dexamethasone potentiation of NFκB, the GRED construct allows detection of GR modulation of
activated NFκB on the promoter.
TNFα (5 ng/ml) caused a 15-fold induction of GRED which was further enhanced by
dexamethasone (10 pM) (Figure 5B). The GRED promoter was not responsive to
dexamethasone alone, consistent with the enhancer function of GR when in the presence of
NFκB on the promoter. Applying shear stress alone activated GRED, thus detecting activation
of both GR and NFκB by shear stress. The combination of TNFα with shear stress caused a
striking 40-fold elevation of transcriptional activity from GRED. Taken together, these data
suggest that having the intact GRE sequence present renders the GRED promoter highly
responsive to shear stress, particularly in the presence of strong NFκB functionality in TNFα-
Ji J.Y., Jing H, and Diamond S.L. - Page 12
Shear stress activation of GR receptor and GRE transcriptional regulation20 provides a
mechanism for potential cross talk between mechanotransduction and anti-inflammatory actions.
In this study, we demonstrated that shear stress at 10 dynes/cm2 attenuated TNFα-stimulated IL-
6 and IL-8 expression in cultured human endothelial cells (Figures 1 and 2). Shear induced
inhibition of TNFα-stimulated IL-6 and IL-8 expression did not strictly require the presence of
NO (Figure 2), which suggests that the anti-inflammatory actions of shear stress is independent
of its vasodilatory effects through stimulated NO release. We have previously shown that
pretreating endothelial cells with L-NAME had no effect on shear induced GRE-SEAP promoter
construct activation at 6 hr, as a metric of shear activation of endogenous GR function. The anti-
inflammatory effect of shear stress was also apparent in neutrophil-endothelial interactions.
Endothelial monolayers exposed to arterial shear and static endothelium respond differently to
TNFα stimulation, as evidenced by more sustained neutrophil rolling (longer rolling length) and
less conversion to firm arrest on cells pre-exposed to 10 dyne/cm2 (Figure 3 and Figure 4). A
likely explanation for the increased rolling length could be the decreased expression of IL-8
and/or reduced presentation of ICAM-1 or VCAM-1 by sheared endothelial cells. The GRED
promoter construct was designed to provide a "light-up" signal to detect GR-NFkB interactions
at the level of a promoter. Shear stress proved to be a particularly strong inducer of the GRED
promoter in TNFα-stimulated endothelium.
Previous research has shown that IL-8 in solution rapidly induces rolling neutrophils to
arrest. Also, increasing immobilized IL-8 decreases neutrophil rolling distance and promotes
firm adhesion.29 IL-8 in the fluid phase or bound to endothelium glycoaminoglycan may
increase β2-integrin avidity, leading to neutrophil firm arrest through ICAM-1/β2 integrin
Ji J.Y., Jing H, and Diamond S.L. - Page 13
interactions.5 Sheared endothelial cells express less IL-8 upon cytokine stimulation (Figure 1A),
which may lead to less neutrophil firm adhesion and longer rolling lengths. Chiu et al showed
decreased DNA binding activity of NFκB in cells that were exposed to shear stress in addition to
TNFα.21 This finding of NFκB down regulation is quite consistent with the reduced IL-6 and IL-
8 expression in sheared HAEC stimulated with TNFα that we demonstrated. In fact, this
decreased IL-6 and IL-8 presentation may contribute significantly to altered interactions between
neutrophils and sheared endothelium since its mechanism is independent of NO inhibitor. On
the other hand, NO inhibitor has been shown to abolish the attenuating effect of shearing on
elevated endothelial VCAM-1 expression induced by TNFα and lipopolysaccharide.30 Finally,
we also saw that shearing endothelial cells alone, in the absence of cytokine stimulation, does not
promote neutrophil adhesiveness, possible because shear stress alone does not substantially
induce expression of VCAM-1 or ICAM-1 on HAEC.31
While increased P-selectin or E-selectin facilitates neutrophil rolling and ICAM-1 or
VCAM-1 expression aids in neutrophil firm adhesion, our findings in Figure 4A suggest that
shear stress influenced only the conversion to firm arrest since total interacting neutrophils
(rolling and arrested) were the same regardless of preshearing. Prior studies have investigated
various aspects of endothelial response to stimulation by TNFα in the presence of shear stress,
although none have previously measured endothelial adhesiveness to human neutrophils.
Yamawaki et al 32 showed in an ex vivo model of rabbit aorta that shear stress inhibited TNFα
stimulated VCAM-1 expression. Chiu et al21 also demonstrated in human umbilical endothelial
cells that that stress decreases TNFα-induced VCAM-1and E-selectin expression, while
enhancing TNFα-induced ICAM-1 mRNA and protein expression. It is difficult to predict the
net effect on neutrophil adhesion from these two prior studies since E-selectins, ICAM-1 and
Ji J.Y., Jing H, and Diamond S.L. - Page 14
VCAM-1 are being altered in differing ways. Additionally, the membrane-cytoskeletal structure
function is likely altered in endothelial cells during shear stress exposure and this may have
subsequent effect on bond life, independent of receptor number due to changes in membrane
extension and tethering. The VCAM-1 data from these studies correlates well with the reduced
neutrophil interactions that we saw. No change in net rolling, a selectin-mediated process, was
observed in our measurements due to preshearing of the TNFα-stimulated endothelium. On the
other hand, ICAM-1 levels under shear has been consistently observed to be different from
VCAM-1 or E-selectin,21, 33-35 and these studies suggested that both NFκB transcriptional
activation and oxidative stress (reactive oxygen species) differentially influence TNFα induced
secretion of cytokines and adhesion molecules. The ICAM-1 promoter region contains binding
sites for AP-1, SP-1 and NFκB, rendering its expression sensitive to regulation by a number of
transcriptional factors under both shear stress and cytokine stimulations.36 The effect of arterial
shear stress on TNFα activation of endothelium has been addressed before in previous studies.32
However, this is the first functional assay of direct neutrophil-endothelial interactions under both
TNFα and shear stress stimulation. Though previous studies have presented data on expression
levels of adhesion molecules, there were no direct measurements of altered endothelial
adhesiveness toward neutrophils. This is the first study to measured alteration of neutrophil
rolling on TNFα-stimulated endothelium due to pre-exposure to arterial shear stress.
In an experiment of this type, LNAME may have regulatory effects on baseline properties of
endothelium, effects on TNFα-stimulated properties, and effects on mechanobiological responses via NO
in the presence or absence of TNFα. For example, LNAME is known to have unexpected additional
actions on endothelium beyond the inhibition of NO production37 since NO is active as an autocrine
agent. We note that LNAME reduced IL-8 production by TNFα-stimulated endothelium under no-flow
conditions. In contrast, LNAME on its own (without TNFα or flow) enhanced IL-6 production indicating
Ji J.Y., Jing H, and Diamond S.L. - Page 15
an additional role of LNAME on IL-6 regulation not seen for IL-8. Complex autocrine loops regulating
the IL-6 and IL-8 genes may become unmasked with the use of chemical inhibitors such as LNAME and
this is seen with IL-6 which was up-regulated by the use of LNAME on its own. Still, shear stress
markedly reduced TNFα-stimulated IL-8 and IL-6 production as seen in Fig. 1 and 2.
The anti-inflammatory effect of shear stress on TNFα activation is further supported by
microarray studies of endothelial gene expression.38 As microarray studies of gene expression
became more sophisticated, a recent study analyzed differential changes in endothelial
transcription profiles of disturbed versus undisturbed laminar flow regions of the same pig
aorta.39 Proinflammatory adhesion molecules such as VCAM-1, ICAM-1, E-selectin, P-selectin
were not differentially expressed in these regions, while IL-6 and IL-8 receptor β are up-
regulated in disturbed regions and IL-8 is slightly down-regulated in disturbed regions. These
data are in good agreement with our data on the attenuating effect of elevated shear stress on IL-
6 and IL-8 expression. Taken together, these data suggest that interleukins (IL-6, IL-8) display
increased sensitivity toward varying flow conditions, and changes in their expression may be a
precursor to altered presentation of inflammatory adhesion molecules.
As an initial step toward studying shear activated GR and NFκB function, we studied the
interaction between shear activated GR and NFκB at their corresponding promoter sites, utilizing
a modified SAA2 promoter constructs that presents binding sites for AP-1, SP-1, and a
functional GRE sequence resulted in overall increased activation of reporter gene. It should also
be noted that binding of shear induces and activates fos/jun (the AP-1 complex) which can also
antagonize NFκB function. Shear stress activated GR pathway, independent of dexamethasone,
may interfere with cytokine enhanced NFκB functions in inflammation. However, the overall
effect of shear stress encompasses a variety of transcriptional factors, including AP-1, SP-1, and
NF-κB that may interfere with GR transcriptional functions as well.
Ji J.Y., Jing H, and Diamond S.L. - Page 16
TNFα is a strong activator of NFκB,40, 41 a key transcription factor in the up-regulated
expression of inflammatory markers including IL-6 and IL-8.42, 43 Previous studies have shown
that NFκB mediated interleukin expression can be repressed by ligand activated GR,9, 44
suggesting that shear stress may also exert its inhibitory effects against TNFα through the
activation of GR and GRE pathway. The atheroprotective effects of shear stress on the
endothelium may be attributed to various anti-inflammatory processes, along with its ability to
modulate the release of vasoactive factors such as NO, prostacyclin, endothelin-1, MCP-1, and
vascular epidermal growth factor (VEGF).45-48 Suppressing inflammation could be a key
mechanism by which shear stress exerts its atheroprotective functions in the endothelium.
Recent analysis of gene expression profile in normal pig aorta revealed that the endothelium in
disturbed flow region is primed for inflammation39 where genes for several general pro-
inflammatory cytokines and receptors such as interleukin 1α, IL-1 receptor 1, IL-6, IL-8 receptor
β, and monocyte chemotactic protein 1 (MCP-1) are up regulated compared to laminar flow area.
However, the NFκB system is primarily inactivated, consistent with the unaffected expression of
inflammatory cells adhesion molecules between two flow regions. Thus, while varying
hemodynamics may alter endothelium liability to atherosclerosis, we presented data supporting
the anti-inflammatory effects of shear stress in inhibiting TNFα activated endothelial activation
and neutrophil interactions. The net effect of shear stress encompasses a variety of
transcriptional factors such as AP-1, SP-1, and GR to regulate NFκB function in a promoter
Ji J.Y., Jing H, and Diamond S.L. - Page 17
This work was supported by National Institutes of Health Grant #HL64388 and
#HL56621. J.Y.J. is a National Science Foundation Graduate Fellow and NIH Bioengineering
Training Grant in Cardiovascular Physiology Recipient (5T32HL07954).
Ji J.Y., Jing H, and Diamond S.L. - Page 18
Figure 1: Effect of shear stress on TNFα activated expression of cytokines IL-8
(A) and IL-6 (B) in HAEC.
Cells were cultured and maintained under static condition (Cont) or treated with TNFα, 5
ng/ml, for 8 hrs (TNFα). HAEC were pretreated with shear stress at 10 dynes/cm2 for 1 hr
before TNFα was injected into flow media without interruption. Cells were sheared in either the
presence or absence of TNFα media for 8 hr. Data are presented as mean ± SE (n = 3). * P <
0.01 and **P < 0.001 refer to significant difference compared to static TNFα alone.
Figure 2: Effect of L-NAME on shear attenuation of TNFα induced IL-8 (A) and IL-6
(B) secretion in HAEC.
Cells were pretreated with the eNOS inhibitor L-NAME (200 μM) for 1 hr. Static cells
were maintained in media containing L-NAME and treated with TNFα (5 ng/ml) for 8 hr.
Sheared cells were further pre-conditioned at 10 dynes/cm2 for 1 hr in L-NAME media before
TNFα was injected and sheared for another 8 hr. Data are mean ± SE (n = 3 in each group). * P
< 0.005, **P < 0.001, refers to significant difference compared to static TNFα alone.
Figure 3: Neutrophil adhesion studies on HAEC monolayers.
Static HAEC cultured on glass slides are treated with either media or TNFα (5 ng/ml) for
5 hr before neutrophil assays. Sheared HAEC on glass slides are exposed to shear stress at 10
dynes/cm2 for 5 hr in media with or without TNFα before assaying with neutrophils. One-
second image sequences were captured from 10-second video segments, and processed to
generate the time-sequence images.
Ji J.Y., Jing H, and Diamond S.L. - Page 19
Figure 4: Total and firmly adhered neutrophil over TNFα treated sheared and
static endothelial cells are quantified in (A) for 5 different images in each
Data are presented as mean ± SE (n = 5). * P < 0.001, refers to significant difference in
firm adhesion between TNFα treatment of HAEC under static or shear stress conditions. Finally,
rolling length for 188 and 212 neutrophils over static and sheared HAEC, respectively, are
generated and presented in a histogram (B). In each case, neutrophils are collected from 5
Figure 5: cis-Interactions of GRE and NFkB in sheared endothelium.
(A) Alignment of the region of SAA2 and GRED promoters encompassing the GRE
sequence, as compared to a consensus GRE sequence. GRED carries an intact GRE sequence
following a 9-basepair deletion (Δ) from the SAA2 promoter. (B) BAEC transfected with GRED
plasmid with the Renilla control plasmid are maintained in medium only, 10 μM dexamethasone,
5 ng/ml TNFα, dexamethasone with TNFα, shear stress alone (10 dynes/cm2, dpc) or shear stress
and TNFα. Cells were harvested after 5 hr treatment and relative luciferase values were
quantified. Data are presented as mean + SE (n = 3). * P = 0.05, * * P < 0.005, refers to
significant difference between treatments with shear stress alone and shear stress with TNFα.
Ji J.Y., Jing H, and Diamond S.L. - Page 20
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