© 1998 American Heart Association, Inc.
Original Contributions |
Cyclic Stretch Upregulates Production of Interleukin-8 and Monocyte Chemotactic and Activating Factor/Monocyte Chemoattractant Protein-1 in Human Endothelial Cells
From the Department of Cardiovascular Medicine, Kyoto University, Kyoto (M.O., A.M., Y.F., T.S., A.I., S.S.); the Department of Pharmacology, Cancer Research Institution, Kanazawa University, Kanazawa (K.M.); and the Department of Molecular Preventive Medicine, School of Medicine, University of Tokyo, Tokyo (K.M.), Japan.
Correspondence to Akira Matsumori, MD, PhD, Department of Cardiovascular Medicine, Kyoto University, 54 Kawaracho Shogoin, Sakyo-ku, Kyoto 606, Japan. E-mail amat{at}kuhp.kyoto-u.ac.jp
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Abstract |
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Abstract—In vivo, vascular walls are exposed to mechanical stretch, which may promote atherogenesis. This study was designed to investigate the effect of mechanical stretch on the production and gene expression of cytokines in endothelial cells (ECs) of human umbilical veins. ECs were cultured on flexible silicone membranes and exposed to cyclic mechanical stretch. Although the secretion levels of interleukin (IL)-1ß, tumor necrosis factor-
, IL-6, granulocyte (G) -colony
stimulating factor(CSF), G and macrophage (M) -CSF, and M-CSF
were not affected by cyclic stretch over 24 hours, the levels of IL-8
and monocyte chemotactic and activating factor (MCAF)/monocyte
chemoattractant protein-1 (MCP-1) were significantly increased by
cyclic stretch. Northern blot analysis indicated that the mRNA
levels of IL-8 and MCAF/MCP-1 were upregulated by cyclic stretch as a
function of its intensity. Cytochalasin D, which disrupts the actin
cytoskeleton, abolished the stretch-induced gene expression of IL-8 and
MCAF/MCP-1. In contrast, neither inhibition of
stretch-activated ion channels nor disruption of microtubules
affected the induction of these chemokines by cyclic stretch. Northern
blot analysis using enzyme inhibitors showed that
phospholipase C, protein kinase C, and tyrosine kinase were involved in
the stretch-induced gene expression of IL-8 and MCAF/MCP-1, whereas
cAMP- or cGMP-dependent protein kinase was not. In conclusion, cyclic
stretch enhanced the secretion and gene expression of IL-8 and
MCAF/MCP-1 in a stretch-dependent fashion, and the integrity of the
actin cytoskeleton and activities of phospholipase C, protein kinase C,
and tyrosine kinase may be essential in the process of stretch-induced
gene induction of IL-8 and MCAF/MCP-1.
Key Words: atherosclerosis • interleukin-8 • macrophage chemotactic and activating factor • monocyte chemoattractant protein-1 • hemodynamic forces
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Introduction |
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In vivo, vascular walls are exposed to three main hemodynamic forces: (1) shear stress, a tangential frictional force due to blood flow; (2) transmural pressure, a perpendicular cyclic force due to blood pressure; and (3) mechanical stretch, a cyclic tensile stress caused by blood pressure. Physiologically, vascular ECs sense such forces and secrete various vasoactive substances to regulate vascular tone and maintain normal homeostasis. Among these hemodynamic forces, shear stress has been widely and intensely investigated. Changes in shear stress regulate the secretion of several factors, including vasodilators such as NO1 and prostacyclin,2 vasoconstrictors such as endothelin-1,3 and growth factors such as PDGF.4 5 The role of shear stress on the pathogenesis of vascular disease, especially atherogenesis, has been well characterized.6 7 8 On the other hand, although a growing number of recent reports have clarified the effects of mechanical stretch on vascular wall cells,9 10 11 12 13 its role in atherogenesis has been less precisely defined.
Atherosclerosis is commonly limited to well-defined segments rather than the entire course of the artery. In humans, atherosclerotic lesions occur preferentially at bifurcations and curvatures,14 whereas recent observations have shown that in the arterial tree, mechanical stretch is significantly higher at bifurcations and curvatures than in straight segments.15 Furthermore, high blood pressure, which increases vascular wall stretch,16 17 is a well-recognized risk factor in atherogenesis. These considerations suggest that mechanical stretch may play an important role in atherogenesis.
There is also growing evidence to suggest that cytokines play a
pivotal role in the formation and development of atherosclerotic
lesions. In the process of atherogenesis, they are thought to
contribute to cell recruitment and migration, cell proliferation, and
the control of lipid and protein synthesis.18
This contribution of various cytokines has indeed been shown in
human atherosclerotic lesions and in experimental
animals.19 20 21 22 23 24 25 26 Therefore, we hypothesized that
high mechanical stretch exerted cyclically on vascular walls would
enhance the production of cytokines from ECs and
participate in the formation of atherosclerotic lesions. The
cytokines specifically examined consisted of IL-1ß, TNF-,
IL-6, IL-8, MCAF/MCP-1, G-CSF, GM-CSF, and M-CSF. These
cytokines are secreted by ECs in various pathological
conditions and are thought to play important roles in
atherogenesis.19 20 21 22 23 24 25 26
The mechanism by which ECs "sense" cyclic stretch and convert it into the gene induction of cytokines was also investigated. We examined the involvement of the actin cytoskeleton, microtubule network, and stretch-activated ion channels. The mechanical stress applied to the cells is transmitted, directly or indirectly, to the cytoskeleton, and stretch-activated ion channels on ECs are activated by mechanical stretch and cause the influx of cations, particularly Ca2+.27 Therefore, these cellular components may act as mechanotransducers and link cyclic stretch with intracellular responses. Furthermore, using enzyme inhibitors, we examined the intracellular signal transduction responsible for stretch-induced gene expression.
In this study, we demonstrate that cyclic stretch enhances the production and gene expression of IL-8 and MCAF/MCP-1 in a stretch-dependent manner and that the actin cytoskeleton and activities of phospholipase C (PLC), protein kinase C (PKC), and tyrosine kinase (TK) are involved in the stretch-induced gene expression of these chemokines. This observation may be one of the explanations for the link between hypertension and atherosclerosis, as well as for the preferential involvement of bifurcations and curvatures in atherosclerosis.
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Methods |
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Biochemicals
Cytochalasin D, 2-nitro-4-carboxyphenyl-N, N-diphenylcarbamate (NCDC), medium 199, and heparin sodium salt (grade I-A from porcine intestinal mucosa) were obtained from Sigma Chemical Co. IL-1ß was obtained from Genzyme. Gadolinium chloride hexahydrate, calphostin C, KT-5720, KT-5823, genistein, and human fibronectin were obtained from Wako Pure Chemical Co. Fluorescein phalloidin was obtained from Molecular Probes Inc. Fetal calf serum was obtained from GIBCO BRL.
Cell Culture
ECs were isolated from human umbilical veins as previously
described28 and cultured in medium 199
supplemented with 20% heat-inactivated fetal calf serum,
90 µg/mL heparin, and antibiotics (50 U/mL penicillin, 50 µg/mL
streptomycin, and 125 ng/mL amphotericin B). Cells at passage level 3
or 4 were seeded on silicone elastomer–bottomed six-well culture
plates (Flex1 dishes, Flexcell International Corp) coated with human
fibronectin and incubated at 37°C in a humidified atmosphere of 5%
CO2/95% air. After the monolayer had become
confluent, the culture medium was changed to medium 199 with 5%
heat-inactivated fetal calf serum, and the cells were
incubated overnight and exposed to cyclic stretch. The cells were
characterized by the typical "cobblestone" appearance and staining
for factor VIII antigen by immunofluorescence.
Experimental Cyclic Stretch
Cyclic mechanical stretch was applied to the ECs with the
Flexercell unit (Flexcell International Corp) as described
previously.29 Cells plated on silicone
elastomer–bottomed culture plates could be deformed by application of
a vacuum. A vacuum line is connected to regulator solenoid valves that
in turn are controlled by a computer with a timer program.
Consequently, the intensity, frequency, and duration of stretch can be
changed freely. In these experiments, cyclic deformations (-5 to -20
kPa) were applied at a frequency of 60 cycles per minute (0.5 second of
deformation alternating with 0.5 second of relaxation). In this model,
the stretch pattern across the membrane is inhomogeneous.
Cells seeded on the periphery of the membrane are exposed to the
greatest and cells in the center to the least stretch. Therefore, to
apply a more homogeneous stretch, a cylindrical glassy
"fence" was placed in the center of the well, and cells were seeded
at the periphery of the well only. In this modified model, -20, -12,
and -5 kPa of vacuum produce an average stretch of 15.1%, 11.6%, and
6.1%, respectively. After the stretch was applied for various periods
of time, supernatant and total RNAs were collected from the cells.
Supernatant and total RNAs from cells kept static for identical periods
of time were collected as unstretched, static controls. After
completion of all experiments, the viability of cells was ascertained
by a trypan blue exclusion study.
Experimental Protocols
Effect of Cyclic Stretch on Cytokine Secretion
The effects of cyclic stretch at a peak level of -20 kPa at 60
cycles/min for 24 hours on the protein levels of IL-1ß, TNF-,
IL-6, IL-8, MCAF/MCP-1, G-CSF, GM-CSF, and M-CSF were measured and
compared with those from control cells kept static for the same length
of time. The levels of IL-1ß, TNF-, IL-6, G-CSF, and GM-CSF in the
supernatants were measured by a specific ELISA kit (Otsuka
Pharmaceutical Co), and those of IL-8 and MCAF/MCP-1 by a separate
specific ELISA kit (Toray Industries Inc). The level of M-CSF was
measured by a specific radioimmunoassay kit (Otsuka Pharmaceutical Co).
With respect to the sensitivity and technical quality of the assay,
standard curves were generated for each set of samples assayed by using
human recombinant cytokines.
RNA Isolation and Northern Blot Hybridization
To examine whether the rises in IL-8 and MCAF/MCP-1
secretion depended on the induction of gene expression, total cellular
RNAs were isolated from cells stretched for 0, 3, 6, 12, or 24 hours,
and Northern blot analysis was performed. Total RNA was
isolated by the guanidinium
thiocyanate–phenol-chloroform-isoamylalcohol
procedure30 and quantified by measuring the
absorbance at 260 nm. Equal amounts of RNA were electrophoresed through
a 1.2% agarose/formaldehyde gel and transferred to a nylon membrane
(Hybond-N+, Amersham Corp) by standard
procedures.31 The blots were sequentially
hybridized with cDNA probes for human IL-8 and MCAF/MCP-1, which had
been labeled with [32P]dCTP by the
random-primer method. After overnight hybridization, the membranes were
washed with 2x SSPE/0.1% SDS at room temperature, 1x SSPE/0.1% SDS
at 65°C, and 0.1x SSPE/0.1% SDS at 65°C. The blots were
analyzed with a FUJIX bioimaging analyzer BAS 2000
(Fujix) with normalization to the corresponding 18S rRNA level.
Effect of Stretch Intensity on Gene Expression of IL-8 and
MCAF/MCP-1
To determine whether the increases in IL-8 and MCAF/MCP-1 gene
expression depended on the intensity of stretch, we exposed ECs to
various levels of stretch (6.1%, 11.6%, or 15.1% average stretch)
for 6 hours and compared the mRNA levels induced at each step.
Roles of Stretch-Activated Ion Channels, Actin
Cytoskeleton, and Microtubule Network on Stretch-Induced Gene
Expression of IL-8 and MCAF/MCP-1
To clarify the contribution of stretch-activated ion
channels in stretch-induced gene expression of IL-8 and MCAF/MCP-1, ECs
were pretreated with the stretch-activated ion channel blocker
gadolinium (Gd3+) for 1 hour and then exposed to
cyclic stretch (-20 kPa) for 3 hours in the presence of
Gd3+.
To elucidate whether the integrity of the actin cytoskeleton was indispensable for stretch-induced gene expression of IL-8 and MCAF/MCP-1, treatment with cytochalasin D, which induces actin depolymerization and suppresses the formation of actin fibers, was started 1 hour before exposure of ECs to cyclic stretch (-20 kPa) for 3 hours. To examine the role of the integrity of microtubules, treatment with colchicine, which binds tubulin and inhibits its assembly to form microtubules, was started 1 hour before exposure of ECs to cyclic stretch (-20 kPa) for 3 hours.
Roles of PLC-, PKC-, TK-, cAMP-, and cGMP-Dependent Protein Kinases
(A and G Kinases) on Stretch-Induced Gene Expression of IL-8 and
MCAF/MCP-1
To study the roles of PLC, PKC, TK, A kinase, and G kinase
in stretch-induced gene expression of IL-8 and MCAF/MCP-1, ECs were
pretreated with NCDC, a PLC inhibitor; calphostin C, a
specific PKC inhibitor (IC50 values
for PKC, A kinase, and G kinase are 0.05 µmol/L, >50
µmol/L, and >25 µmol/L, respectively), genistein, a TK
inhibitor; KT-5720, an A-kinase inhibitor
(Ki=0.056 µmol/L); and
KT-5823, a G-kinase inhibitor
(Ki=0.234 µmol/L), each
for 1 hour before the stretch applied for 3 hours.
Fluorescence Staining
Cells were washed three times with PBS and fixed with 3.7%
paraformaldehyde in PBS for 10 minutes at room
temperature. After three washes with PBS, the cells were
permeabilized with 0.5% Triton X-100 in PBS for 5
minutes at room temperature and washed three times with PBS.
Filamentous actin was stained with fluorescein phalloidin
for 60 minutes at room temperature, washed three times with PBS, and
viewed on a fluorescence microscope equipped with
epifluorescence using narrow bandpass excitation and emission
filters for fluorescein. Fluorescence images were
recorded on T-Max (ISO 400) films (Kodak).
Statistical Analyses
Values are presented as mean±SEM. Results were
analyzed by Student's t test for comparison of
results from two experimental groups or by ANOVA when data from more
than two groups were compared.
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Results |
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Effects of Cyclic Stretch on Cytokine Secretion
Figure 1
shows the mean levels of
cytokines measured by ELISA and radioimmunoassay in three
separate experiments. The levels of IL-1ß, TNF-, IL-6, G-CSF,
GM-CSF, and M-CSF were not affected by cyclic stretch. In contrast,
levels of IL-8 and MCAF/MCP-1 were increased significantly by the
application of cyclic stretch when compared with control cells. The
IL-8 and MCAF/MCP-1 levels from ECs stretched for 24 hours were,
respectively, 2.6±0.2-fold (P<0.001) and 2.8±0.3-fold
(P<0.001) of those from cells kept static for 24 hours.
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Effects of Cyclic Stretch on IL-8 and MCAF/MCP-1 mRNA
Levels
Figure 2 shows a
representative Northern blot indicating the time course
of mRNA levels of IL-8 and MCAF/MCP-1. The IL-8 mRNA level was elevated
at 3 hours, peaked at 6 hours, and then declined. Densitometric
analysis of the IL-8 mRNA level normalized to the 18S rRNA
level showed a 2.9±0.3-fold (n=3) increase in ECs exposed to stretch
for 6 hours when compared with static controls. The MCAF/MCP-1 mRNA
level was elevated at 3 hours and showed a sustained increase over the
entire period of observation. Densitometric analysis of the
MCAF/MCP-1 mRNA level indicated a 2.1±0.2-fold (n=3) increase in ECs
exposed to stretch for 3 hours when compared with static controls. The
IL-8 and MCAF/MCP-1 mRNA levels of the unstretched controls showed no
significant change during the observation period.
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Effects of Stretch Intensity on Gene Expression of IL-8 and
MCAF/MCP-1
Densitometric analysis of Northern blots revealed
1.2±0.2-fold, 1.6±0.2-fold, and 2.8±0.3-fold increases in IL-8 mRNA
level after exposure to 6.1%, 11.6%, and 15.1% average stretch,
respectively, when compared with static controls (Figure 3, hatched bars). Likewise, the
MCAF/MCP-1 mRNA level increased 1.3±0.2-fold, 1.7±0.3-fold, and
2.2±0.3-fold after exposure to 6.1%, 11.6%, and 15.1% average
stretch, respectively (Figure 3, solid bars). These data indicate that
gene expression of IL-8 and MCAF/MCP-1 is upregulated as a function of
the amplitude of stretch applied to ECs.
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Roles of Stretch-Activated Ion Channels, Actin
Cytoskeleton, and Microtubule Network on Stretch-Induced Gene
Expression of IL-8 and MCAF/MCP-1
In preliminary experiments, Gd3+ >60
µmol/L caused cell detachment; therefore, the effect of
Gd3+ 
30 µmol/L was investigated.
Stretch-induced gene expression of both IL-8 and MCAF/MCP-1 was not
blocked by Gd3+ 30 µmol/L (Figure 4A).
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Application of cyclic stretch (-20 kPa) to ECs altered their shape
from the typical cobblestone pattern to fusiform and uniformly aligned
in the direction perpendicular to the force vector. The actin-staining
pattern of cells exposed to cyclic stretch showed that the filamentous
actin cytoskeleton had reorganized into parallel arrays of elongated
stress fibers that were also aligned in the direction perpendicular to
the force vector (compare Figure 5A and 5B). Treatment with >100 nmol/L cytochalasin D caused marked shrinkage
and detachment of cells. Treatment of the cells with this drug at 50
nmol/L disrupted the actin cytoskeleton without affecting cell adhesion
and viability under static condition (Figure 5C) and abolished
reorganization of the actin cytoskeleton into actin stress fibers
induced by cyclic stretch (Figure 5D). Northern blot analysis
revealed that disruption of the actin cytoskeleton by 50 nmol/L
cytochalasin D (Figure 4B
) inhibited the stretch-induced gene
expression of IL-8 by 42±16% and of MCAF/MCP-1 by 39±21%
(P<0.01 and P<0.05 versus stretched,
untreated controls, respectively). IL-1ß (10 ng/mL, stimulation for 3
hours) –induced gene expression of IL-8 and MCAF/MCP-1 was not
affected by 50 nmol/L cytochalasin D (Figure 4C), suggesting that
inhibition of stretch-induced gene expression of these molecules by
cytochalasin D was not due to a nonspecific effect.
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In contrast, disruption of microtubules by 1 µmol/L colchicine,
which was the maximal concentration with minimal cytotoxic effect, did
not inhibit the stretch-induced increases in IL-8 and MCAF/MCP-1 mRNA
levels (Figure 4D). These results suggest that the integrity of the
actin cytoskeleton is essential for stretch-induced gene expression of
IL-8 and MCAF/MCP-1, whereas stretch-activated ion channels or
the integrity of microtubules is not.
Roles of PLC, PKC, TK, A, and G Kinases on Stretch-Induced Gene
Expression of IL-8 and MCAF/MCP-1
Stretch-induced increases in IL-8 and MCAF/MCP-1 mRNA levels were
inhibited (Figure 6A) by both NCDC
(300 µmol/L) (56±11%, P<0.01 and 65±3%,
P<0.01, versus stretched, untreated controls,
respectively) and calphostin C (2 µmol/L) (33±4%,
P<0.001 and 39±6%, P<0.001, versus
stretched, untreated controls, respectively). Stretch-induced increases
in IL-8 and MCAF/MCP-1 mRNA levels were also inhibited (Figure 6B) by
genistein (20 µmol/L) (32±4%, P<0.001 and
39±4%, P<0.001, versus stretched, untreated
controls, respectively). In contrast, 0.5 µmol/L KT-5720 and
1 µmol/L KT-5823 had no significant effect on stretch-induced
gene expression of IL-8 and MCAF/MCP-1 (Figure 6C). All drugs were used
at optimal concentration, as determined in preliminary experiments, and
the concentrations used were not toxic to ECs and did not affect cell
adhesion under static and stretched conditions. These results suggest
that PLC, PKC, and TK are involved in stretch-induced gene expression
of IL-8 and MCAF/MCP-1.
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Discussion |
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The role of hemodynamic forces in the pathogenesis of vascular disease, especially atherogenesis, is receiving increased attention. The participation of shear stress in atherogenesis has been widely and intensely investigated. The nonrandom distribution of early atherosclerotic lesions observed in the natural disease process in humans14 has been explained by local disturbances in flow (flow separation, flow reversal, low amplitude flow, and fluctuating wall shear stresses).6 7 8 However, a recent report has revealed that the area of pressure-induced high strain is related to the sites of atherosclerotic plaques.15 Furthermore, although atherosclerotic lesions do not develop in veins in their normal environment of low pressure, such lesions do indeed develop when the veins are used as arterial bypass grafts and are subsequently subjected to high pressure and cyclic stretch. These considerations suggest that mechanical stretch, as well as shear stress, may play an important role in atherogenesis.
The earliest recognizable lesion of atherosclerosis is the fatty streak, an aggregation of lipid-laden macrophages and T lymphocytes within the subendothelial space.18 In the formation of atherosclerotic lesions, migration of monocytes and T lymphocytes is thought to be the earliest and most significant event. Our experiments have demonstrated that among the several cytokines examined, mechanical stretch enhances human EC production of IL-8, a chemoattractant for neutrophils,32 33 T lymphocytes,34 and others, and of MCAF/MCP-1, a chemoattractant for monocytes.35 36 37 These observations may be one of the explanations for an early link between mechanical stretch to vascular walls and atherogenesis. Furthermore, an enhanced production of IL-8 and MCAF/MCP-1 has recently been reported in human abdominal aortic aneurysms,38 which develop in the late stage of atherosclerosis, particularly in hypertensive patients. Our results are consistent with the finding of such chemokines' being involved in the late stage of atherosclerosis, such as aortic aneurysms.
MCAF, also known as MCP-1, a 14-kD glycoprotein with potent monocyte chemotactic activity,35 36 37 is believed to be one of the important molecules involved in atherogenesis. This involvement was particularly illustrated in hypercholesterolemic human and animals.23 24 39 IL-8, which like MCAF/MCP-1, belongs to the chemokine superfamily, has received relatively little attention in atherogenesis, probably because IL-8 was originally identified as a chemotactic factor for neutrophils,32 33 which are uncommon in human atherosclerotic lesions. However, a recent study has shown that IL-8 is a potent chemotactic factor for T lymphocytes,34 which are found in human atherosclerotic lesions and are recognized to participate in atherogenesis.40 41 42 Another study has found that IL-8 also has mitogenic and chemotactic activities toward vascular smooth muscle cells.43 Migration and proliferation of vascular smooth muscle cells are other important phenomena in atherogenesis.18 Therefore, IL-8 may play important roles in atherogenesis as well as does MCAF/MCP-1 and in fact, the presence of IL-8 protein and mRNA in the human atherosclerotic arterial wall has been shown in previous reports.22 38
The mechanisms by which ECs sense cyclic stretch and convert this hemodynamic force into gene induction of IL-8 and MCAF/MCP-1 are still poorly understood. Our experiments have shown that the actin cytoskeleton and activities of PLC, PKC, and TK play important roles in stretch-induced gene expression of these chemokines. The involvement of phosphatidylinositol turnover and PKC in stretch-induced signal transduction was previously demonstrated in experiments using the same stretch apparatus and the same types of cells,44 45 and several stimuli were reported to upregulate gene expression of IL-8 and MCAF/MCP-1 through a PKC-dependent pathway in ECs.46 47 48 However, the correlation of these enzymes with the actin cytoskeleton remains to be clarified. The effect of mechanical forces on cell behavior involving gene expression has recently received increased attention, and both integrins and the actin cytoskeleton have been recognized to be strong candidates as mechanotransducers.49 50 51 Integrins are the main family of cell surface receptors that mediate attachment to the extracellular matrix and the cell-cell adhesive interactions, and integrin receptor engagement and clustering lead to the formation of focal adhesion, in which integrins link to intracellular cytoskeletal complexes and bundles of actin filaments.52 53 54 The integrin-mediated interactions transmit extracellular stimuli into the cell and mediate a multitude of cellular responses, including the response to mechanical stress.49 50 51 55 56 57 58 Wilson et al59 60 have demonstrated that mechanical strain of rat vascular smooth muscle cells is sensed by specific extracellular matrix/integrin interactions and induces growth via an autocrine action of PDGF. Yano et al61 62 63 have clearly demonstrated that in ECs, integrins and focal adhesion proteins play important roles in transducing mechanical stimuli into intracellular signals.9 In addition, recent observations have clarified that through integrins, many signal transduction molecules are activated, such as Ras, Grb2, MAP kinase, Rho, and PI-3 kinase, as well as PLC and PKC.57 64 The actin cytoskeleton, which is intensified and leads to the formation of actin stress fibers by cyclic stretch, is believed to sustain the integrin-mediated activation of these signal transduction molecules.64 65 Miyamoto et al65 have reported that disruption of the actin cytoskeleton by cytochalasin D blocks the accumulation and activation of these signaling molecules in fibroblasts. Furthermore, the importance of integrins in the endothelial response to stretch is supported by our finding that inhibition of TK by genistein, which is reported to disrupt signals from integrins to intracellular molecules,64 65 abolished stretch-induced gene expression. In our experiments, disruption of the actin cytoskeleton by cytochalasin D and inhibition of TK by genistein may have blocked the integrin-mediated activation of PLC and PKC induced by cyclic stretch. Another report, however, has suggested that PKC activity itself regulates integrin-mediated responses,66 such that further investigations will be necessary to clarify these signal transduction pathways.
Recent work by Wung et al67 has shown that cyclic stretch increases MCP-1 secretion in human ECs not dependent on the actin cytoskeleton but dependent on stretch-activated ion channels.67 We, however, have demonstrated here that the stretch-induced gene expression of not only IL-8 but also MCAF/MCP-1 is dependent not on stretch-activated ion channels but on the actin cytoskeleton. Although this discrepancy cannot be easily explained, it is possible that our choice of fibronectin in plate coating, as opposed to type I collagen by Wung et al, may be the source of these different results.
In summary, our study shows that cyclic stretch upregulates secretion and gene expression of IL-8 and MCAF/MCP-1 from human ECs, which may play a role in the pathogenesis of atherosclerosis. The integrity of the actin cytoskeleton and the activities of PLC, PKC, and TK could be involved in the stretch-induced gene expression of these chemokines.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by a research grant from the Japanese Ministry of Health and Welfare and a grant-in-aid for General Scientific Research from the Japanese Ministry of Education, Science and Culture (A.M. and S.S.). We wish to thank Y. Ohmoto for assistance in the assays of cytokines.
Received October 10, 1997; accepted December 8, 1997.
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