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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 20, 2003 as doi:10.1096/fj.02-0814fje. |
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* Department of Internal Medicine II, University of Ulm, Germany;
Institute of Molecular Animal Breeding, Gene Center, Ludwig-Maximilians-University München, Germany; and
Creatogen AG, Augsburg, Germany
3Correspondence: University of Ulm, Department of Internal Medicine II-Cardiology, Robert Koch-Str. 8, 89081 Ulm, Germany. E-mail: jan.torzewski{at}medizin.uni-ulm.de
SPECIFIC AIMS
Cell proliferation and apoptosis determine accumulation of human vascular smooth muscle cells (SMCs) in atherosclerotic lesions. As the terminal complement complex C5b-9 interacts with SMCs in human atherogenesis, we investigated whether C5b-9 activates the insulin-like growth factor-1 (IGF-1) system in SMCs, resulting in the inhibition of SMC apoptosis.
PRINCIPAL FINDINGS
1. C5b-9 inhibits CD95-mediated induction of apoptosis
To determine differences in the amount of apoptosis, we performed annexin V binding experiments. Stimulation of SMCs was accomplished by preincubation with sublytic C5b-9 for 4 h, followed by incubation with an anti-CD95 monoclonal antibody CH11 for 0, 1, 3, 6, 12, and 24 h. Flow cytometric analysis revealed a time-dependent enhancement of annexin V binding (Fig. 1
). Preincubation of SMCs with sublytic C5b-9 inhibited this shift in annexin V binding. Control cells pretreated with C5b6, C8, and C9 lacking C7 underwent apoptosis at a similar rate as SMCs without complement, thus indicating that the antiapoptotic effects of C5b-9 require complete complex formation.
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Kinetic studies of caspase 3 activity after C5b-9 and CH11 stimulation at 0, 24, 48, and 72 h (Fig. 2
A) confirmed the antiapoptotic effect of C5b-9 on CD95-mediated programmed cell death. Stimulation of SMCs with CH11 alone led to a significant increase in caspase 3 activity whereas coincubation with CH11 and C5b-9 significantly (P<0.05) decreased caspase 3 activity even below the activity level of unstimulated cells (Fig. 2A
). The number of C5b-9 complexes formed on the membrane of SMCs affected the antiapoptotic properties of C5b-9. 10 MHD of C5b-9 had no antiapoptotic effect whereas 500 MHD C5b-9 inhibited apoptosis at the same rate as 100 MHD C5b-9 (P<0.05) (Fig. 2A
).
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2. C5b-9 inhibits UV-mediated induction of apoptosis
To test whether the observed antiapoptotic effect was specific to CD95-mediated apoptosis, we induced apoptosis in SMCs by an alternative method using UV light (Fig. 2B
). Pretreatment of the SMCs with sublytic C5b-9 also significantly reduced caspase 3 activity after UV light treatment (P<0.05).
3. C5b-9 increases IGF-1 secretion
To measure the direct effect of C5b-9 on IGF-1 secretion, we stimulated SMCs with sublytic C5b-9 and measured the IGF-1 accumulation in the supernatant. The supernatant was analyzed 0, 6, 12, 24, and 48 h after stimulation with C5b-9. Preincubation with C5b-9 significantly increases IGF-1 secretion from SMCs in a time-dependent manner, with maximum effect achieved at 24 h. The release of IGF-2 was markedly higher than that of IGF-1 but was not significantly influenced by C5b-9. Western ligand blot analysis of conditioned media revealed the presence of different IGFBPs. IGF binding activities were found as a band doublet between 36 and 45 kDa and weak singular bands at 32 and 24 kDa, respectively. However, no regulative function of C5b-9 could be attributed to any of these binding activities. To investigate whether IGF-1 secretion is due to an increase in RNA, we performed semiquantitative RT-PCR of IGF-1. A comparison of the IGF-1 mRNA amount in cells preincubated with sublytic C5b-9 and control cells revealed no difference in mRNA-amount, indicating that no de novo synthesis of IGF-1 mRNA is induced by C5b-9 stimulation.
4. C5b-9 increases the number of IGF-1 receptors and IGF-1 binding sites on SMCs
We used flow cytometry and IGF-1 binding assays to address whether C5b-9 affects the number of IGF-1 receptors and binding sites on SMCs. Flow cytometric analysis of IGF-1 receptors revealed a time-dependent shift of gated cells from 5.1% (0 h) to 20.9% (24 h). Analysis of control cells (24 h) without C5b-9 stimulation revealed no significant up-regulation of IGF-1 receptors. Incubation with sublytic C5b-9 for 18 h induced a significant increase in the amount of specifically bound IGF-1 (P<0.01 vs. control) as shown by 125I-IGF-1 binding assays. Linear Scatchard plots indicate that this was due to an increase in IGF-1 receptor number (101,300±18,300, control vs. 203,000±46,500, C5b-9; P<0.02) without changes in binding affinity (Kd values: 0.63±0.33 nM, control vs. 0.85±0.4 nM, C5b-9).
5. IGF-1 is involved in antiapoptotic properties of C5b-9
We further tested the role of IGF-1 in C5b-9-mediated inhibition of apoptosis. SMCs were stimulated with sublytic C5b-9 and incubated with CH11 for 0, 6, 12, and 24 h. To assess whether IGF-1 may be involved in C5b-9-mediated inhibition of apoptosis, SMCs were preincubated with a monoclonal IGF-1 antibody to immuno-neutralize autocrine released IGF-1. The IGF-1 antibody abolished the antiapoptotic effects of C5b-9 as assayed by annexin V binding. In control experiments, no apoptotic effect of the IGF-1 antibody was seen when SMCs were incubated with the antibody alone. No additional apoptotic effect was observed in combination with CH11.
6. IGF-1 is antiapoptotic for SMCs
To evaluate the extend of the antiapoptotic effect of C5b-9 in comparison to IFG-1, we performed further annexin V assays. Substitution of C5b-9 with recombinant IGF-1 inhibited enhancement of annexin V binding after CH11-stimulation of SMCs in a similar manner as C5b-9 preincubation.
CONCLUSIONS
In this paper we demonstrate by annexin V labeling and TUNEL stain of apoptotic cells that generation of sublytic C5b-9 on SMCs in vitro inhibits CD95-mediated apoptosis in these cells (Fig. 3
). We demonstrate that the observed antiapoptotic effect is not limited to CD95-mediated apoptosis. The antiapoptic effect is dependent on formation of the complete transmembrane pore as omission of C7 abolishes the regulative function of C5b-9 for SMC apoptosis. We demonstrate that C5b-9 induces both IGF-1 release and up-regulation of IGF-1 binding sites in SMCs, whereas secretion of IGF-2 and IGFBPs is not influenced. Induction of IGF-1-release did not involve IGF-1 de novo-synthesis, as shown by RT-PCR.
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Immunoneutralization of endogenously released IGF-1 by a blocking antibody abolished the inhibitory effects of C5b-9 on SMC apoptosis. Substitution of C5b-9 with recombinant IGF-1 completely inhibited apoptosis in our cell system. Thus, the endogenously released IGF-1 is a major mediator of the antiapoptotic action of C5b-9. Our findings are in line with former reports on C5b-9-mediated growth factor, cytokine release, and inhibition of apoptosis in other nucleated cells. However, this study describes for the first time that an autocrine IGF-1 loop causes the antiapoptotic effect of C5b-9 on SMCs. The IGF system is essential in the pathogenesis of atherosclerosis. The fact that IGF-1 is involved in C5b-9-mediated inhibition of apoptosis links inflammation to growth factor activation in atherogenesis.
Complement activation is a pathogenic feature in both human and experimental atherogenesis and in other cardiovascular diseases. C-reactive protein (CRP), an important cardiovascular risk factor, has been identified as a major complement activating molecule in the arterial wall. The interaction of complement activation products with vascular cells may be important in the understanding of the inflammatory mechanisms underlying atherosclerosis. As C5b-9 colocalizes with SMCs during early atherogenesis, an investigation of direct effects of C5b-9 on SMC proliferation and apoptosis might contribute to understanding SMC accumulation in the arterial wall. Sublytic C5b-9 has been shown to stimulate SMC proliferation. As it is demonstrated in this paper that C5b-9 also inhibits SMC apoptosis, complement activation may be significantly involved in the disbalance between cell death and proliferation observed during atherogenesis. Apoptosis in atherosclerotic lesions is related to plaque stage, with early lesions showing little apoptosis and advanced lesions showing apoptotic levels of 2%. Since C5b-9 in advanced lesions is mainly confined to macrophages, SMCs in early but not in advanced plaques may be protected against ongoing cell death by the antiapoptotic effects of C5b-9.
Apoptosis in atherosclerotic lesions can be induced by other factors intimately involved in atherogenesis, such as oxidized LDL. Future research may determine whether C5b-9 also inhibits apoptosis mediated by these molecules.
Inhibition of the complement system may be a potential new therapeutic option for the treatment of atherosclerosis. Further studies must be undertaken to investigate the mechanisms by which C5b-9 mediates IGF-1 release and up-regulation of IGF-1 binding sites in SMCs.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0814fje; doi: 10.1096/fj.02-0814fje 
2 Present address: National Primate Research Center, University of Wisconsin, Madison, Wisconsin, USA.
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  C. Cudrici, F. Niculescu, T. Jensen, E. Zafranskaia, M. Fosbrink, V. Rus, M. L. Shin, and H. Rus C5b-9 Terminal Complex Protects Oligodendrocytes from Apoptotic Cell Death by Inhibiting Caspase-8 Processing and Up-Regulating FLIP. J. Immunol., March 1, 2006; 176(5): 3173 - 3180. [Abstract] [Full Text] [PDF]
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