(Circulation. 2000;101:352.)
© 2000 American Heart Association, Inc.
Brief Rapid Communications |
Laminar Shear Stress Upregulates the Complement-Inhibitory Protein Clusterin
A Novel Potent Defense Mechanism Against Complement-Induced Endothelial Cell Activation
From Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Germany.
Correspondence to Andreas M. Zeiher, MD, Department of Internal Medicine IV, Division of Cardiology, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany. E-mail zeiher{at}em.uni-frankfurt.de
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Abstract |
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Background—The complement system is implicated in the pathogenesis of atherosclerosis. Complement has been shown to activate endothelial cells (ECs) by inducing a proinflammatory response. Physiological levels of shear stress exert potent antiatherosclerotic effects. Therefore, we investigated whether shear stress antagonizes the effects of complement on ECs.
Methods and Results—Incubation of ECs with nonlytic concentrations of complement serum (CS: 0.2 U/mL for 6 hours) resulted in an upregulation of interleukin-8 (IL-8) (165±12%) and monocyte chemoattractant protein-1 (MCP-1) mRNA expression (267±34%). Preexposure of ECs for 18 hours with laminar shear stress (15 dyne/cm2) abrogated CS-induced IL-8 release to 106±10% (P<0.001) and reduced CS-induced MCP-1 expression (170±31%; P<0.05). To examine the mechanism of the protective effect of shear stress, expression of the complement-inhibitory protein clusterin was analyzed under shear exposure. Shear stress increased clusterin mRNA (225±76%, 6 hours) and protein expression (164±22%, 18 hours). Specific inhibition of clusterin by transfection with antisense oligonucleotides reversed the protective effect of shear stress on CS-induced MCP-1 and IL-8 upregulation (P<0.05 versus sense-transfected cells). Moreover, clusterin overexpression inhibited CS-induced EC activation.
Conclusions—Shear stress abrogates the complement-induced proinflammatory response of ECs by upregulation of the complement-inhibitory protein clusterin. Upregulation of clusterin may contribute to the potent antiatherosclerotic effects of shear stress by preventing endothelial activation through the complement cascade.
Key Words: atherosclerosis • shear stress • endothelium • cells • complement
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Activation of the complement system plays a key role in the pathogenesis of atherosclerosis.1 Not only can complement complexes be detected in atherosclerotic lesions,2 but more importantly, complement deficiency prevents the formation of atherosclerotic lesions.3 Although the exact mechanisms by which complement exerts its proatherosclerotic effects are not yet clear, several studies suggest that the complement complex activates the endothelial cells (ECs), thereby inducing a proinflammatory response. Exposure of ECs to complement components triggers the induction of proinflammatory cytokines, such as interleukin (IL)-6 or IL-8.4 Moreover, monocyte chemoattractant protein-1 (MCP-1), which contributes causally to the formation of atherosclerotic lesions,5 is released by ECs in response to complement.4
A major endogenous atheroprotective signal is provided by the intraluminal flow (shear stress) acting on the endothelial monolayer, as evidenced by the preferential development of atherosclerotic lesions in areas with low or unsteady shear stress.6 Exposure of ECs to fluid shear stress modulates gene expression and thereby regulates endothelial structure and function.6 7 8 9 Therefore, we investigated the effect of shear stress on complement-induced upregulation of IL-8 and MCP-1 as indicators of EC activation.
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Methods |
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Human umbilical vein ECs (HUVECs; CellSystems/Clonetics) were cultured (third passage) and exposed to shear stress in a cone-and-plate apparatus as previously described.10 HUVECs were incubated in endothelial basal medium complete medium containing 1% BSA. Complement serum (CS) was purchased from Sigma Chemical Co. IL-8 was detected in cell culture supernatants by Quantikine Immunoassay (R&D Systems).
Expression of mRNA
RNA was isolated, separated by agarose gel electrophoresis, and
transferred to nylon membranes. The blots were hybridized with a
radioactively labeled full-length human MCP-1, clusterin, or CD59
probe.
Western Blot Analysis
HUVECs (4.0x105 cells) were lysed and
protein homogenates were prepared as
described.10 Proteins (50 µg/lane) were loaded onto 10%
SDS-polyacrylamide gels, blotted onto PVDF membranes, and
incubated with clusterin antibody (Santa Cruz Biotechnology; 1:200/3%
BSA) or MCP-1 antibody (R&D Systems; 1:1000/3% BSA) and detected by
enhanced chemiluminescence.
Plasmid Construction and Transfection
Human full-length clusterin was cloned by polymerase chain
reaction (primers:
5'-CGGGATCCGGCGCCATGTCAGAACCGGCT-GGG-3'/5'-GCGATATCGGGCTTCCTCTTGGAGAAGATCAG-3')
into the BamHI/EcoRV sites of the
pcDNA3.1-Myc-His vector. HUVECs were transiently transfected as
described previously.10 Medium (150 µL) was mixed
with 3 µg plasmid and 30 µL Superfect (Qiagen) and incubated for 10
minutes, 1 mL medium was added, and HUVECs were incubated with this
mixture for 3 hours at 37°C.
Sense (5'-ATGATGAAGACTCTGCTGCTG-3'; bases 53 to 73) or antisense (5'-CAGCAGCAGAGTCTTCATCAT-3') oligonucleotides corresponding to the human clusterin sequence were incubated in 100 µL RPMI medium in the presence of 5 µL lipofectamine (Gibco) for 30 minutes at room temperature. HUVECs (4.0x105 cells) were washed with RPMI and incubated with 2 mL RPMI before the lipofectamine/oligonucleotide mixture was added. After incubation for 5 hours, 3 mL complete endothelial basal medium medium was added.
Statistical Analysis
Data are expressed as mean±SEM. Statistical analysis
was performed by t test or ANOVA for multiple
comparisons.
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Results |
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Shear Stress Inhibits Complement-Induced Upregulation of IL-8 and MCP-1
Nonlytic concentrations of CS (0.2 U/mL) stimulated HUVECs to release IL-8 into the medium (Figure 1A
).
Similarly, MCP-1 mRNA expression was time- and dose-dependently
upregulated by complement, with a maximal response after 6 hours
(Figure 1B, data not shown). Control experiments demonstrated
that concentrations up to 0.5 U/mL CS did not affect EC viability (data
not shown). To test the effect of shear stress, HUVECs were preexposed
to laminar shear stress (15 dynes/cm2) for 18
hours and then stimulated with 0.2 U/mL CS for 6 hours under prolonged
application of laminar shear stress. As shown in Figure 1A, shear stress prevented the CS-induced IL-8 release. Furthermore,
CS-stimulated MCP-1 mRNA expression was significantly reduced by
exposure to shear stress (Figure 1B). These data were confirmed
by Western blot analysis of MCP-1 protein expression (data not
shown).
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Shear Stress Upregulates the Complement-Inhibitory
Protein Clusterin
To elucidate the mechanism of the
complement-inhibitory effect of shear stress, we examined
whether shear stress upregulates the complement-inhibitory
proteins clusterin and CD59. Shear stress exposure time-dependently
increased the expression of clusterin mRNA (6 hours: 225±76%) and
clusterin protein levels (164±22% increase after 18 hours; Figure 1C
). In contrast, expression of the complement
inhibitor CD59 was not affected by shear stress (data not
shown).
To assess a causal role for shear stress–stimulated upregulation of
clusterin to mediate the complement-inhibitory effect of
shear stress, clusterin expression was specifically inhibited by
transfection with antisense oligonucleotides (Figure 2A). Clusterin antisense
oligonucleotides significantly reduced the inhibition
of CS-induced MCP-1 expression by shear stress, compared with HUVECs
transfected with sense or scrambled oligonucleotides
(Figure 2B; data not shown). Similar data were obtained for IL-8
(data not shown). Most importantly, overexpression of clusterin
significantly reduced CS-induced expression of MCP-1 and IL-8 compared
with mock-transfected cells (Figure 2C and 2D, data not shown).
Thus, clusterin appears to be causally involved in the suppression of
complement-induced activation of ECs.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The results of the present study demonstrate that exposure of ECs to laminar shear stress prevents complement-induced activation of ECs by upregulation of the complement-inhibi-tory protein clusterin. Complement components stimulate the activation of ECs, resulting in an enhanced expression of MCP-1 and other cytokines,4 which trigger the firm adhesion of monocytes to the endothelium, a key event in initiation of the pathogenesis of atherosclerosis.5 11 Clusterin is a heterodimeric multifunctional glycoprotein capable of interacting with complement components C7, C8, and C9, thereby preventing the formation of the membrane attack complex.12 The results of the present study demonstrate that overexpression of clusterin is sufficient to prevent complement-induced activation of ECs. More importantly, clusterin upregulation is necessary for the complement-inhibitory effects of shear stress.
Abrogation of the complement-induced proinflammatory response of ECs
might importantly contribute to the potent antiatherosclerotic effects
of shear stress.6 Previous studies have demonstrated that
prolonged exposure to physiological levels of shear
stress reduces the expression of adhesion molecules and MCP-1 in part
via increased expression of the endothelial NO synthase
(eNOS),8 13 which alters endothelial redox
state and inhibits nuclear factor-
B–mediated gene
transcription.13 Shear stress–induced upregulation
of clusterin, however, occurs independently of enhanced NO synthesis,
because NOS inhibitors did not affect shear
stress–stimulated clusterin expression (data not shown). Thus,
inhibition of complement-induced EC activation does not require eNOS
activation. It has been reported that transforming growth factor
(TGF)-ß stimulates the upregulation of clusterin.14
Because shear stress induces gene expression of TGF-ß,15
one may speculate that TGF-ß mediates the enhanced expression of
clusterin in response to shear stress.
Taken together, shear stress–induced upregulation of clusterin might interfere with endothelial activation in response to infectious agents, which activate the complement cascade and are currently receiving increasing attention in relation to atherogenesis.16 Furthermore, additional biological effects of the multifunctional protein clusterin, including the transport of lipoproteins12 and its chaperone-like activity17 in combination with the promotion of cell survival,18 may be involved in preserving the integrity and the biological functions of the endothelial monolayer.
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft (Di600/2-3). We would like to thank Susanne Ficus, Christiane Mildner-Rihm, and Iris Henkel for expert technical assistance.
Received November 5, 1999; accepted November 29, 1999.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Bhakdi S. Complement and atherogenesis: the unknown connection. Ann Med. 1998;30:503–507.[Medline] [Order article via Infotrieve]
2.
Torzewski M, Klouche M, Hock J, Messner M, Dorweiler
B, Torzewski J, Gabbert HE, Bhakdi S. Immunohistochemical demonstration
of enzymatically modified human LDL and its colocalization with the
terminal complement complex in the early atherosclerotic lesion.
Arterioscler Thromb Vasc Biol. 1998;18:369–378.
3.
Schmiedt W, Kinscherf R, Deigner HP, Kamencic H, Nauen
O, Kilo J, Oelert H, Metz J, Bhakdi S. Complement C6 deficiency
protects against diet-induced atherosclerosis in
rabbits. Arterioscler Thromb Vasc Biol. 1998;18:1790–1795.
4. Kilgore KS, Schmid E, Shanley TP, Flory CM, Maheswari V, Tramontini NL, Cohen H, Ward PA, Friedl HP, Warren JS. Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-kappa B activation. Am J Pathol. 1997;150:2019–2031.[Abstract]
5. Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK, Libby P, Rollins BJ. Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol Cell. 1998;2:275–281.[Medline] [Order article via Infotrieve]
6. Gimbrone MA, Nagel T, Topper JN. Biomechanical activation: an emerging paradigma in endothelial adhesion biology. J Clin Invest. 1997;99:1809–1813.[Medline] [Order article via Infotrieve]
7.
Traub O, Berk BC. Laminar shear stress: mechanisms by
which endothelial cells transduce an atheroprotective
force. Arterioscler Thromb Vasc Biol. 1998;18:677–685.
8.
Chien S, Li S, Shyy YJ. Effects of mechanical forces
on signal transduction and gene expression in
endothelial cells. Hypertension. 1998;31:162–169.
9. Dimmeler S, Fisslthaler B, Fleming I, Hermann C, Busse R, Zeiher AM. Activation of nitric oxide synthase in endothelial cells via Akt-dependent phosphorylation. Nature. 1999;399:601–605.[Medline] [Order article via Infotrieve]
10.
Dimmeler S, Assmus B, Hermann C, Haendeler J, Zeiher
AM. Fluid shear stress stimulates phosphorylation of
Akt in human endothelial cells: involvement in
suppression of apoptosis. Circ Res. 1998;83:334–342.
11. Gerszten RE, Garcia-Zepeda EA, Lim YC, Yoshida M, Ding HA, Gimbrone MA Jr, Luster AD, Luscinskas FW, Rosenzweig A. MCP-1 and IL-8 trigger firm adhesion of monocytes to vascular endothelium under flow conditions. Nature. 1999;398:718–723.[Medline] [Order article via Infotrieve]
12. Jenne DE, Tschopp J. Clusterin: the intriguing guises of a widely expressed glycoprotein. Trends Biochem Sci. 1992;17:154–159.[Medline] [Order article via Infotrieve]
13.
Zeiher AM, Fisslthaler B, Schray-Utz B, Busse R. Nitric
oxide modulates the expression of monocyte chemoattractant protein 1 in
cultured human endothelial cells. Circ Res. 1995;76:980–986.
14.
Jin G, Howe PH. Regulation of clusterin gene expression
by transforming growth factor beta. J Biol Chem. 1997;272:26620–26626.
15. Ohno M, Cooke JP, Dzau VJ, Gibbons GH. Fluid shear stress induces endothelial transforming growth factor beta-1 transcription and production: modulation by potassium channel blockade. J Clin Invest. 1995;95:1363–1369.
16.
Libby P, Egan D, Skarlatos S. Roles of infectious
agents in atherosclerosis and restenosis.
Circulation. 1997;96:4095–4103.
17.
Humphreys DT, Carver JA, Easterbrook-Smith SB, Wilson
MR. Clusterin has chaperone-like activity similar to that of small heat
shock proteins. J Biol Chem. 1999;274:6875–6881.
18. Koch-Brandt C, Morgans C. Clusterin: a role in cell survival in the face of apoptosis? Prog Mol Subcell Biol. 1996;16:130–149.[Medline] [Order article via Infotrieve]
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