点击显示 收起
【摘要】
Objective- Atherosclerosis is a chronic inflammatory disease with immune cell infiltration. Various cytokines and chemokines have been characterized as pro- or antiatherogenic factors. Interleukin-20 (IL-20) belongs to the IL-10 family and is a proinflammatory cytokine involved in the pathogenesis of psoriasis. However, the association between IL-20 and atherosclerosis is undetermined. Therefore, we sought to investigate whether IL-20 is associated with atherosclerosis.
Methods and Results- We examined the expression of IL-20 and its receptor complex IL-20R1/IL-20R2 in atherosclerotic lesions of humans and mice using immunohistochemical staining. IL-20 was expressed in macrophage-rich areas. Both IL-20 and IL-20R1/IL-20R2 were expressed by endothelial cells lining the intimal microvessels, vasa vasorum, but rarely in nonatherosclerotic arteries. We used reverse-transcription polymerase chain reaction to analyze gene expression. IL-20 transcripts increased in hypoxic monocytes and monocytes treated with oxidized low-density lipoprotein. The expression of IL-20R1 and IL-20R2 was also upregulated by human umbilical vein endothelial cells in response to hypoxic treatment. Incubating IL-20 with human umbilical vein endothelial cells upregulated CXCL9 and CXCL11 transcripts. Furthermore, in vivo administration of IL-20 expression vector using intramuscular electroporation promoted atherosclerosis in apolipoprotein E-deficient mice.
Conclusions- Our data suggest that IL-20 is a proatherogenic cytokine that contributes to the progression of atherosclerosis.
We investigated the association between IL-20 and atherosclerosis. IL-20 and its receptors are expressed in the atherosclerosis plaques of human and mice. In vitro, oxidized LDL and hypoxia induced IL-20. In vivo, IL-20 promoted atherosclerosis in apoE -/- mice. Thus, we postulate that IL-20 is a proatherogenic cytokine.
【关键词】 atherosclerosis cytokines hypoxia IL oxidized lipids
Introduction
Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by the progressive accumulation of lipids, extracellular matrix, and cells, including macrophages, T lymphocytes, and smooth muscle cells. 1 Inflammation plays a major role in atherosclerotic plaque disruption and thrombosis; therefore, it greatly influences the occurrence of coronary syndromes and mortality. 2-4 Foam cell macrophages are generally thought to play a major role in the pathology of the disease. 2 Activated macrophages secrete cytokines and chemokines that direct and amplify the local immune response. Inflammatory stimuli include lipoproteins trapped within lesions in which protein and lipid moieties have been chemically modified. Lipid-engorged macrophages, or foam cells, comprise the major volume of the early lesions and are enriched in late-stage lesions as well. 3,5 In situ studies of human and animal atherosclerotic lesions have identified the increased expression of several chemokines, cytokines, and tissue-remodeling and lipid metabolism genes. 6 Antibodies to oxidized low-density lipoprotein (OxLDL) epitopes and increases in circulating proinflammatory cytokines correlate with vascular disease. 7 In addition, exposure to OxLDL alters the transcription response of macrophages to inflammatory stimuli. 8 These studies implicate activated macrophages and other inflammatory cells in the development and progression of atherosclerosis.
Angiogenesis is also crucial in the progression of atherosclerosis plaque formation. The clinical importance of the neovascularization of plaque has been demonstrated by a higher prevalence of neovascularization in lesions with plaque rupture, mural hemorrhage, or unstable angina. 9 Therefore, factors that stimulate plaque angiogenesis also contribute to plaque disruption, which is responsible for myocardial infarction and ischemic stroke. Angiogenesis inhibitors such as endostatin reduce intimal neovascularization and plaque growth in apolipoprotein E-deficient (apoE -/- ) mice. 10 This evidence further supports the importance of angiogenesis in the pathogenesis of atherosclerosis. See page 1929
An imbalance between the demand and supply of oxygen is another key factor for the development of atherosclerotic lesions. In vivo, zones of hypoxia occur within the atherosclerotic plaque through the mechanism of impaired oxygen diffusion capacity caused by the thickness of the lesion, together with high oxygen consumption by foam cells. 11 This observation supports the anoxemia theory of atherosclerosis. 12
IL-10, important in atherosclerotic lesion formation and stability, is a protective factor against the effect of environmental pathogens on atherosclerosis. 13 IL-10 was originally described as a cytokine-synthesis inhibitory factor because of its inhibitory effect on cytokine production. 14 Several new members of the IL-10 family, including IL-19, IL-20, IL-22, MDA-7 (IL-24), and AK155 (IL-26), have recently been discovered. 15 Overexpression of IL-20 in transgenic mice causes neonatal death as well as skin abnormalities, including aberrant epidermal differentiation. 16 IL-20 selectively enhances multipotential hematopoietic progenitors in vitro and in vivo. 17 IL-20 is preferentially expressed in monocytes 18 and induces STAT 3 activation through binding to 2 types of IL-20 receptor (R) complexes, either IL-20R1/IL-20R2 or IL-22R1/IL-20R2. 19 Our recent study 20 demonstrated that IL-20 induced endothelial cell proliferation, migration, and vascular tube formation in vitro and tumor angiogenesis in vivo. IL-20 stimulates angiogenic activity either directly or indirectly through inducing vascular endothelial growth factor (VEGF), bFGF, and IL-8 production. Angiogenesis is one of the characteristics of atherosclerosis. We therefore wanted to investigate the molecular mechanism of IL-20 to determine whether it, like IL-10, had any association with atherosclerosis. ApoE -/- mice reveal the phenotype of atherosclerosis. 21 Thus, we investigated the expression pattern of IL-20 and its receptors on atherosclerotic lesions of humans and apoE -/- mice using immunohistochemical staining. We also examined the regulation of IL-20 and its receptors under hypoxia or OxLDL stimulation and the effect of IL-20 on chemokine regulation in endothelial cells. To study the in vivo activity of IL-20, using intramuscular electroporation, we injected IL-20 expression vector into apoE -/- mice and monitored the progression of atherosclerosis plaques.
Materials and Methods
Please see http://atvb.ahajournals.org for detailed Materials and Methods
Results
Upregulation of IL-20 and Its Receptors in Atherosclerotic Lesions of apoE -/- Mice
Chronic inflammation plays a pivotal role in the progression of atherosclerosis. IL-10 exerts important protective effects against the development of atherosclerotic lesions in experimental animals. IL-20 promotes angiogenesis in vitro and in vivo. Therefore, we wanted to see whether IL-20 was also associated with atherosclerosis. ApoE -/- mice demonstrate the atherosclerosis phenotype. 21 Thus, we performed immunostaining to analyze the expression of IL-20 and its receptors in the atherosclerotic lesions in apoE -/- mice and normal C57BL/6 mice. We found that IL-20 was upregulated in the atherosclerosis plaque of apoE -/- mice compared with normal C57BL/6 mice (supplemental Figure I Ai to Aj, available online at http://atvb.ahajournals.org). Strong immunoreactivity was detected for both IL-20R1 (supplemental Figure I Aa) and IL-20R2 (supplemental Figure I Ae) in atherosclerosis plaque and the endothelium of the aortic arches of apoE -/- mice. By contrast, low levels of IL-20R1 and IL-20R2 were detected in a portion of the endothelial cells in the aortic arches of normal C57BL/6 mice (supplemental Figure IAb, f). Immunoreactivity was detected in the adventitia of the aortic arches of C57BL/6 and apoE -/- mice. In the aortic root lesions, IL-20 was also detected in Mac-3-rich (foam cells/macrophages) area (supplemental Figure I Ba-b). These results indicated that both IL-20 and IL-20 receptors were induced in atherosclerosis plaque and were markedly upregulated in the endothelium of atherosclerotic aortas. It is likely that foam cells/macrophages are the major producers of IL-20 in the plaques.
Expression of IL-20, IL-20R1, and IL-20R2 in Human Atherosclerotic Artery
The expression of IL-20, IL-20R1, and IL-20R2 was also examined in paraffined sections of surgical femoral arterial samples from peripheral arterial occlusion patients. In these sections, pronounced thickening occurred in the intima and media, both of which exhibited extensive angiogenesis. IL-20 was detected in endothelial cells lining the microvessels and macrophage-derived foam cells that exhibited positive staining for CD68 ( Figure 1 Aa, f). Both IL-20R1 and IL-20R2 were detected primarily in endothelial cells of the vasa vasorum ( Figure 1 Ab, d). By contrast, IL-20R1 and IL-20R2 were expressed at lower levels in the endothelium of normal aorta sections whereas IL-20 was not detected in normal aorta sections ( Figure 1 Ag, h, j). In addition, none of these proteins was significantly expressed in the smooth muscle cells of atherosclerotic lesions. A high-power field (200 x ) magnification of an atherosclerotic lesion is shown in Figure 1 B.
Figure 1. Immunohistochemical staining of IL-20 and receptor subunits in the human atherosclerotic lesions. Paraffin sections of human femoral artery were obtained from patients with peripheral artery occlusion disease after bypass surgery. A, IL-20 was detected by an anti-IL-20 monoclonal antibody 7E (a,g). IL-20R1 expression was detected by the mouse anti-hIL-20R1 monoclonal antibody (b,h). IL-20R2 expression was detected with a rabbit anti-hIL-20R2 polyclonal antibody (d,j). Staining with mouse IgG 1 isotype was used as the negative control for IL-20R1 and IL-20(c,i). Staining with rabbit preimmune serum alone was used as the negative control for IL-20R2 (e,k). Monocytes/macrophages were detected using an anti-CD68 monoclonal antibody (f,l). Reactions were detected by AEC (red, a-e, g-k) or DAB (brown, f,l), and nuclei were counterstained with hematoxylin (blue). B, 200-fold magnification. Macrophages ( ) and endothelial cells ( ) were indicated. Bar represents 100 µm. Sections represent similar patterns in 3 individual specimens.
OxLDL Induced the Expression of IL-20 in Monocytes
IL-20 and its receptors were upregulated both in human and mice atherosclerotic lesions (supplemental Figure I and Figure 1 ). We speculated that IL-20 was induced by existing inflammatory stimuli in the atherosclerotic lesions. OxLDL has been shown to promote foam cell formation and compromise endothelial function by triggering the secretion of chemokines and increasing the expression of leukocyte adhesion molecules. 5 To examine whether OxLDL stimulated IL-20 expression, we used reverse-transcription polymerase chain reaction (RT-PCR) to determine IL-20 mRNA level in OxLDL-treated human peripheral monocytes. Our results revealed that OxLDL, like the positive control lipopolysaccharide (LPS), induced IL-20 expression in human peripheral monocytes ( Figure 2 A) and mouse RAW264.7 macrophages ( Figure 2 B). We further confirmed the result using real-time PCR ( Figure 2 C). These results could explain the co-localization of foam cells and IL-20 in immunohistochemical staining ( Figure 1 ).
Figure 2. OxLDL induced IL-20 in monocytes. Human peripheral monocytes (A) and mouse RAW264.7 macrophages (B) were exposed to PBS, native LDL (100 µg/mL), OxLDL (1, 20, 100 µg/mL), or LPS (10 ng/mL) for 6 hours. Total RNA was then isolated for RT-PCR analyses, and ß-actin was the internal control. C, Total RNA underwent real-time PCR analysis to confirm the result of OxLDL-treated human monocytes. Fold increases of the transcripts are represented as 2 - Ct Ct: retention time.
IL-20 Did Not Affect OxLDL Uptake by Macrophages
We further analyzed whether IL-20 affected OxLDL uptake by macrophages in vitro. Mouse peritoneal macrophages (supplemental Figure II A-B) or human THP-1 macrophages (supplemental Figure II C-D) were incubated with IL-20, phorbol 12-myristate 13-acetate (PMA), or IL-10 together with OxLDL. Unlike IL-10-enhanced OxLDL uptake, 22 IL-20 had no effect on OxLDL uptake by macrophages. Thus, OxLDL induced IL-20 production by monocytes, but it did not enhance OxLDL-induced foam cells formation.
Hypoxia Induced the Expression of IL-20 and Its Receptors
Hypoxia induces various angiogenesis factors and results in neovascularization in ischemic tissues. Neovascularization in atherosclerotic plaque was associated with its hypoxic zones. IL-20 and its receptors were upregulated in atherosclerotic lesions in a mouse model and human samples. Therefore, we speculated that IL-20 and its receptors might be induced under hypoxic conditions. CoCl 2 is known to elicit hypoxia-like responses by activating hypoxia-inducible factor-1 (HIF-1 ) in vitro. 23,24 To evaluate whether IL-20 is upregulated under hypoxic conditions, we used RT-PCR to determine IL-20 mRNA level in CoCl 2 -treated human peripheral monocytes. Our results demonstrated that CoCl 2 induced IL-20 in monocytes ( Figure 3A, 3 B). We further examined the effect of hypoxia on the expression of IL-20 and its receptors in endothelial cells. CoCl 2 treatment upregulated the transcripts of IL-20, IL-20R1, and IL-20R2, but it did not significantly upregulate the transcripts of IL-22R1, in human umbilical vein endothelial cells (HUVECs) ( Figure 3 C). Furthermore, we incubated HUVECs in the chamber supplied with only 1% O 2 to mimic a hypoxia condition for 12 hours and analyzed the transcripts of IL-20 and its receptors. The similar results of upregulation of IL-20 and its receptors were observed ( Figure 3 D). Thus, hypoxia stimulated the expression of IL-20 on monocytes and HUVECs and its receptors on HUVECs. These data are consistent with observations in immunohistochemical staining of atherosclerotic lesions.
Figure 3. Hypoxia regulated the expression of IL-20 and its receptors. Human peripheral monocytes (A, B) and HUVECs (C) were exposed to normoxia (PBS), 100 µmol/L CoCl 2, 500 µmol/L CoCl 2, or 100 ng/mL LPS for 6 hours. In another experiment, HUVECs were incubated in chambers under normoxia (21% O 2, 5% CO 2 ) or hypoxia (1% O 2, 5% CO 2 ) condition for 12 hours (D). RNA was then isolated for RT-PCR analysis, and HPRT or ß-actin was used as an internal control. Equal amounts of cDNA and primers specific for IL-20, IL-20R1, IL-20R2, and IL-22R1 were used in PCR to amplify the transcripts. HUVECs (E) or HMECs (F) were incubated with PBS, hIL-20 (200 ng/mL), or IFN- (10 ng/mL) for 6 hours. Total RNA was then isolated for RT-PCR analysis as described. The relative quantity of PCR products was analyzed using the BIO-PROFIL program and expressed as a fold-increase relative to untreated control cells. The experiment was repeated three times with similar results.
IL-20 Induced Chemokines, Mig/CXCL9, and I-TAC/CXCL11, in Endothelial Cells
Recruitment of T lymphocyte into the atherosclerotic lesions is a prominent process during progression of atherosclerosis. 2 Three CXCR3 ligands, chemokines induced by IFN-, Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11, mediate the process of T lymphocyte recruitment and are present in atheroma-associated cells. 25 To investigate whether IL-20 induced chemokine production and thus, recruited T lymphocyte, we analyzed the effect of IL-20 on chemokines production in endothelial cells. HUVECs were incubated with IL-20 and analyzed using RT-PCR. The transcripts of CXCL9 and CXCL11, but not CXCL10, were upregulated by IL-20 in HUVECs ( Figure 3 E) as well as in human microvessel endothelial cells (HMECs) ( Figure 3 F).
Mouse IL-20 Promoted Angiogenesis In Vivo
We have shown human IL-20 promotes HUVECs proliferation, migration in vitro, and tumor angiogenesis in vivo. 20 To further examination whether mouse (m) IL-20 also enhanced angiogenesis in vivo, mouse hepatoma cells, ML-1, were dorsally co-injected with Matrigel-containing saline, mIL-20, mIL-20 plus anti-mIL-20R1 antibody, or VEGF into Balb/c mice (Laboratory Animal Center, NCKU; Tainan, Taiwan). After 7 days, tumors were excised and immunohistochemical stained with CD31 for microvessel density analysis (supplemental Figure III). mIL-20, similar to VEGF, enhanced vascularization around the solid tumors compared with saline-treated control (supplemental Figure IIIA). CD31 staining also showed higher microvessel density in mIL-20 treated tumors than that in saline-treated tumors (supplemental Figure IIIB-C). In addition, antibody against mouse IL-20R1 inhibited mIL-20-induced angiogenesis, indicating IL-20R1 was involved in the signaling of IL-20-induced angiogenesis. These results provided evidences of the angiogenic activity of mIL-20 in vivo.
IL-20 Promoted Atherosclerosis in ApoE -/- Mice
To further investigate the role of IL-20 in atherosclerosis in vivo, we performed intramuscular electroporation to deliver the mIL-20 expression construct into apoE -/- mice. The serum levels of mIL-20 after electroporation were measured using enzyme-linked immunosorbent assay ( Figure 4 A). The mIL-20 protein level peaked at the first week after electroporation and gradually leveled off in the mice treated with mIL-20 plasmid DNA. Administration of mIL-20 led to enhanced en face lesion areas of aortas ( Figure 4B, 4 C), lesion area of aortic sinus, and an increased area of Mac-3-positive (macrophages) in the aortic sinus. However, no significant difference in the percentage of Mac-3-positive area (Mac-3-positive area/lesion area) was observed between the groups (supplemental Table II). Additionally, in consistent with the in vitro result, the expression of Mig and I-TAC were also higher in aortas form pcDNA3.1-mIL-20-treated mice than that from control mice. Furthermore, proinflammatory cytokines tumor necrosis factor- and IL-6 were also upregulated in the aortas ( Figure 4 D). These results suggest that IL-20 is a proatherogenic factor and may contribute to the pathogenesis of atherosclerosis.
Figure 4. IL-20 promoted atherosclerosis in apoE -/- mice. Fourteen-week-old apoE -/- mice were fed by the atherogenic diet. 50 µg per mouse of pcDNA3.1 or pcDNA3.1-mIL-20 were delivered to the muscles of hind leg using intramuscular electroporation (n=7 in each group). Electroporation was performed once each week for a total of ten times per mouse. A, Serum levels of mIL-20 in apoE -/- mice were analyzed using enzyme-linked immunosorbent assay. B, The aortas were oil red O-stained and showed red lipid-rich atherosclerotic lesions. Figure represents 7 mice from each group. C, Quantitative analysis showed that the en face lesion area of aortas were higher in pcDNA3.1-mIL-20 treated mice than that in pcDNA3.1 treated mice. Bar represents 1 mm (A). * P <0.05. D, RT-PCR analyses of the expression of Mig and I-TAC in the aortas from mice treated with pcDNA3.1-mIL-20 or control vector.
Discussion
More characteristics of the biological activities of IL-20 were demonstrated by the expression profiles of IL-20 and its receptors. 16,18,26,27 Our current work provides evidence to support the notion that expression of IL-20 and its receptors is associated with the development of atherosclerosis, though much remains to be elucidated about the physiological and pathogenic roles of IL-20 and its receptors. Results from immunohistochemical staining demonstrated that IL-20 and its receptor complex IL-20R1/IL-20R2 were indeed expressed in atherosclerotic lesions from human patients as well as apoE -/- mice (supplemental Figure I and Figure 1 ). Because macrophages are known to secrete IL-20, co-distribution of IL-20 and the macrophage marker Mac-3 (mouse) and CD68 (human) in atherosclerotic lesions suggests that macrophages are the major source of IL-20 in the atherosclerotic lesions (supplemental Figure I and Figure 1 ). IL-20 and its receptor complex IL-20R1/IL-20R2 were expressed at minimum to undetectable level in the aorta of C57BL/6 normal mice and normal human aorta, whereas their expression increased in endothelial cells lining the intimal microvessel, vasa vasorum. These results indicate that IL-20 may involve in the progression of atherosclerosis plaque by regulating the intimal neovascularization.
The formation of new microvessels in human atherosclerotic plaque is associated with high cellular proliferation activity in the plaque. 28 Neovascularization is required for atherosclerotic plaque development. 12 Administration of the antiangiogenic drugs endostatin and TNP-470 to apoE -/- mice reduces both plaque growth and intimal neovascularization. 10 Recently, our study showed IL-20 induced the proliferation and migration of endothelial cells in vitro, and promoted tumor angiogenesis in vivo. In addition, IL-20 also induced matrix metalloproteinase-2, VEGF, and IL-8 expression by HUVECs. 20 In the present study, we also showed mouse IL-20 promoted angiogenesis in vivo through IL-20R1 in a mouse model (supplemental Figure III). Therefore, it can be postulated that an increased secretion of IL-20 by macrophages and endothelial cells may serve as paracrine or autocrine factors for endothelial cells. The increased IL-20 secretion in combination with upregulation of IL-20 receptors on endothelial cells will promote the migration and proliferation of endothelial cells, thereby stimulating angiogenesis in atherosclerotic lesions. In another report, 29 IL-20 inhibited PMA-induced angiogenesis by inhibiting COX-2. It is possible that IL-20 regulates angiogenesis via proangiogenic and antiangiogenic activities in different tissue types according to the microenvironment and different receptor complex used.
In the animal model, we did not detect significant CD31-positive staining on the intimal vasa vasorum in the aortic arches sections (data not shown). This may be attributed to the angiogenesis-mediated neovascularization occurred at the late stage of plaque development. 30 Thus, it was not clear whether IL-20 regulated angiogenesis during atherosclerotic plaque development in the apoE -/- model. Nevertheless, we did not exclude the possibility that IL-20 regulates angiogenesis in human atherosclerotic lesions. There may be some difference in the molecular mechanism of atherosclerotic plaque development between animal model and human. The effect of IL-20 on the regulation of angiogenesis during atherosclerotic plaque development awaits further investigation.
Initiation and progression of atherosclerosis are dependent on an inflammatory environment. Proinflammatory cytokines play a pivotal role in perpetuating this environment. However, mechanisms regulating the expression of IL-20 and its receptors remain poorly understood. Our results demonstrated that LPS, OxLDL, and hypoxic condition could contribute to increase of IL-20 production by monocytes. Furthermore, hypoxic condition also increased the expression of IL-20, IL-20R1, and IL-20R2 by HUVECs ( Figure 3 ).
A hypoxic zone occurs at an atherosclerotic lesion 11 and is associated with the angiogenesis process and inflammation. 31 In response to hypoxia, HIF-1 induces gene expression by binding to the hypoxia response element on the promoter of hypoxia-inducible genes. Treatment with CoCl 2 induced IL-20 transcription by human monocytes. IL-20 and the receptors, IL-20R1 and IL-20R2, were expressed on HUVECs under normoxia. Induction of hypoxia by CoCl 2 or 1% O 2 treatment significantly increased the expression of IL-20 and its receptors in HUVECs ( Figure 3 ). We have found some potential hypoxia response elements upstream of IL-20 gene. These HIF-1 binding motifs may be responsible for upregulation of IL-20 under hypoxia condition (unpublished data). This provides a mechanism for upregulation of IL-20 in atherosclerotic lesions by macrophages and endothelial cells, and upregulation of the IL-20 receptors by endothelial cells.
The 3 CXCR3 ligands (Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11) were differentially expressed by human atheroma-associated cells and mediate the recruitment of T lymphocytes into the inflammation sites. 25,32 Our result demonstrated that IL-20 induced Mig/CXCL9 and I-TAC/CXCL11 but not IP-10/CXCL10 in endothelial cells ( Figure 3 E, F). In consistent with the in vitro data, the mRNA levels of Mig and I-TAC were also elevated in the aortas from IL-20 plasmid DNA treated mice ( Figure 4 D). Thus, IL-20 induced Mig and I-TAC in vitro and in vivo, indicating IL-20 may play an important role to recruit T lymphocytes to accumulate in the atheroma during the plaque formation.
Systemic delivery of IL-20 by intramuscular electroporation resulted in increased atherosclerotic lesion areas in apoE -/- mice ( Figure 4 and supplemental Table II). These results suggest that IL-20 acts as a proatherogenic cytokine. We found that IL-20, unlike IL-10, did not enhance OxLDL uptake and foam cells formation in vitro (supplemental Figure II). The proatherogenic effect of IL-20 might be mediated through persistent higher serum levels of IL-20 by acting on vascular endothelial cells directly or indirectly through other mediators (Mig, I-TAC, VEGF) in inflamed lesions. The upregulation of IL-6 and tumor necrosis factor- also revealed the role of IL-20 in enhancing intraplaque inflammation ( Figure 4 D). Thus, neutralization of IL-20 may constitute an attractive new strategy for the treatment of atherosclerosis.
Transgenic mice expressing IL-20 controlled by various tissue promoters reveal similar neonatal death effect. 16 However, injection of IL-20 protein into normal Balb/c mice has no effect on skin phenotype or any lethal effect. 17 In this study, we used the intramuscular electroporation to systemically deliver the IL-20 gene construct to the apoE -/- mice without any lethal effect or significant pathological change. Therefore this approach could be useful for further exploring the biological function of IL-20 in vivo. However, repeated electroporation may induce muscle necrosis or tolerance that could explain mIL-20 protein gradually leveled off after the first week.
In summary, we found that IL-20 is associated with atherosclerosis because both IL-20 and its receptor subunits are expressed in atherosclerotic lesions of apoE -/- mice and in human atherosclerotic arteries. In vitro, the expression of IL-20 was upregulated in monocytes with OxLDL and hypoxic stimulation. IL-20 also induced chemokines expression in HUVECs and systemic delivery of IL-20 in apoE -/- mice led to increased atherosclerotic lesion areas, suggesting that IL-20 is a novel proatherogenic cytokine. Because the biological functions and clinical implications of IL-20 remain to be explored, our findings provide evidence of the association between IL-20 and atherosclerosis.
Acknowledgments
We thank Dr Chauying J. Jen and S.L. Kao (Department of Physiology, NCKU) for their help in preparing the OxLDL, and Dr Pin Ling (Department of Microbiology & Immunology, NCKU) for his assistance in preparing the manuscript.
Sources of Funding
This study was supported by grants CMFHT-9303 from the Chi-Mei Medical Center and 94-2320-B-006-093 from the National Science Council, Taiwan.
Disclosures
None.
【参考文献】
Ross R. Atherosclerosis-an inflammatory disease. N Engl J Med. 1999; 340: 115-126.
Glass CK, Witztum JL. Atherosclerosis. the road ahead. Cell. 2001; 104: 503-516.
Lusis AJ. Atherosclerosis. Nature. 2000; 407: 233-241.
Lee RT, Libby P. The unstable atheroma. Arterioscler Thromb Vasc Biol. 1997; 17: 1859-1867.
Steinberg D. Atherogenesis in perspective: hypercholesterolemia and inflammation as partners in crime. Nat Med. 2002; 8: 1211-1217.
Lawn RM, Wade DP, Couse TL, Wilcox JN. Localization of human ATP-binding cassette transporter 1 (ABC1) in normal and atherosclerotic tissues. Arterioscler Thromb Vasc Biol. 2001; 21: 378-385.
Hansson GK. Cell-mediated immunity in atherosclerosis. Curr Opin Lipidol. 1997; 8: 301-311.
Mikita T, Porter G, Lawn RM, Shiffman D. Oxidized low density lipoprotein exposure alters the transcriptional response of macrophages to inflammatory stimulus. J Biol Chem. 2001; 276: 45729-45739.
Tenaglia AN, Peters KG, Sketch MH, Jr., Annex BH. Neovascularization in atherectomy specimens from patients with unstable angina: implications for pathogenesis of unstable angina. Am Heart J. 1998; 135: 10-14.
Moulton KS, Heller E, Konerding MA, Flynn E, Palinski W, Folkman J. Angiogenesis inhibitors endostatin or TNP-470 reduce intimal neovascularization and plaque growth in apolipoprotein E-deficient mice. Circulation. 1999; 99: 1726-1732.
Bjornheden T, Levin M, Evaldsson M, Wiklund O. Evidence of hypoxic areas within the arterial wall in vivo. Arterioscler Thromb Vasc Biol. 1999; 19: 870-876.
Barger AC, Beeuwkes R, 3rd, Lainey LL, Silverman KJ. Hypothesis: vasa vasorum and neovascularization of human coronary arteries. A possible role in the pathophysiology of atherosclerosis. N Engl J Med. 1984; 310: 175-177.
Mallat Z, Besnard S, Duriez M, Deleuze V, Emmanuel F, Bureau MF, Soubrier F, Esposito B, Duez H, Fievet C, Staels B, Duverger N, Scherman D, Tedgui A. Protective role of interleukin-10 in atherosclerosis. Circ Res. 1999; 85: e17-24.
Gesser B, Leffers H, Jinquan T, Vestergaard C, Kirstein N, Sindet-Pedersen S, Jensen SL, Thestrup-Pedersen K, Larsen CG. Identification of functional domains on human interleukin 10. Proc Natl Acad Sci U S A. 1997; 94: 14620-14625.
Pestka S, Krause CD, Sarkar D, Walter MR, Shi Y, Fisher PB. Interleukin-10 and related cytokines and receptors. Annu Rev Immunol. 2004; 22: 929-979.
Blumberg H, Conklin D, Xu WF, Grossmann A, Brender T, Carollo S, Eagan M, Foster D, Haldeman BA, Hammond A, Haugen H, Jelinek L, Kelly JD, Madden K, Maurer MF, Parrish-Novak J, Prunkard D, Sexson S, Sprecher C, Waggie K, West J, Whitmore TE, Yao L, Kuechle MK, Dale BA, Chandrasekher YA. Interleukin 20: discovery, receptor identification, and role in epidermal function. Cell. 2001; 104: 9-19.
Liu L, Ding C, Zeng W, Heuer JG, Tetreault JW, Noblitt TW, Hangoc G, Cooper S, Brune KA, Sharma G, Fox N, Rowlinson SW, Rogers DP, Witcher DR, Lambooy PK, Wroblewski VJ, Miller JR, Broxmeyer HE. Selective enhancement of multipotential hematopoietic progenitors in vitro and in vivo by IL-20. Blood. 2003; 102: 3206-3209.
Wolk K, Kunz S, Asadullah K, Sabat R. Cutting edge: immune cells as sources and targets of the IL-10 family members? J Immunol. 2002; 168: 5397-5402.
Dumoutier L, Leemans C, Lejeune D, Kotenko SV, Renauld JC. Cutting edge: STAT activation by IL-19, IL-20 and mda-7 through IL-20 receptor complexes of two types. J Immunol. 2001; 167: 3545-3549.
Hsieh MY, Chen WY, Jiang MJ, Cheng BC, Huang TY, Chang MS. Interleukin-20 promotes angiogenesis in a direct and indirect manner. Genes Immun. 2006; 7: 234-242.
Zhang SH, Reddick RL, Piedrahita JA, Maeda N. Spontaneous hypercholesterolemia and arterial lesions in mice lacking apolipoprotein E. Science. 1992; 258: 468-471.
Halvorsen B, Waehre T, Scholz H, Clausen OP, von der Thusen JH, Muller F, Heimli H, Tonstad S, Hall C, Froland SS, Biessen EA, Damas JK, Aukrust P. Interleukin-10 enhances the oxidized LDL-induced foam cell formation of macrophages by antiapoptotic mechanisms. J Lipid Res. 2005; 46: 211-219.
Yuan Y, Hilliard G, Ferguson T, Millhorn DE. Cobalt inhibits the interaction between hypoxia-inducible factor-alpha and von Hippel-Lindau protein by direct binding to hypoxia-inducible factor-alpha. J Biol Chem. 2003; 278: 15911-15916.
Semenza GL. Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol. 1999; 15: 551-578.
Mach F, Sauty A, Iarossi AS, Sukhova GK, Neote K, Libby P, Luster AD. Differential expression of three T lymphocyte-activating CXC chemokines by human atheroma-associated cells. J Clin Invest. 1999; 104: 1041-1050.
Parrish-Novak J, Xu W, Brender T, Yao L, Jones C, West J, Brandt C, Jelinek L, Madden K, McKernan PA, Foster DC, Jaspers S, Chandrasekher YA. Interleukins 19, 20, and 24 signal through two distinct receptor complexes. Differences in receptor-ligand interactions mediate unique biological functions. J Biol Chem. 2002; 277: 47517-47523.
Nagalakshmi ML, Murphy E, McClanahan T, de Waal Malefyt R. Expression patterns of IL-10 ligand and receptor gene families provide leads for biological characterization. Int Immunopharmacol. 2004; 4: 577-592.
O?Brien ER, Garvin MR, Dev R, Stewart DK, Hinohara T, Simpson JB, Schwartz SM. Angiogenesis in human coronary atherosclerotic plaques. Am J Pathol. 1994; 145: 883-894.
Heuze-Vourc?h N, Liu M, Dalwadi H, Baratelli FE, Zhu L, Goodglick L, Pold M, Sharma S, Ramirez RD, Shay JW, Minna JD, Strieter RM, Dubinett SM. IL-20, an anti-angiogenic cytokine that inhibits COX-2 expression. Biochem Biophys Res Commun. 2005; 333: 470-475.
Khurana R, Simons M, Martin JF, Zachary IC. Role of angiogenesis in cardiovascular disease: a critical appraisal. Circulation. 2005; 112: 1813-1824.
Murdoch C, Giannoudis A, Lewis CE. Mechanisms regulating the recruitment of macrophages into hypoxic areas of tumors and other ischemic tissues. Blood. 2004; 104: 2224-2234.
Piali L, Weber C, LaRosa G, Mackay CR, Springer TA, Clark-Lewis I, Moser B. The chemokine receptor CXCR3 mediates rapid and shear-resistant adhesion-induction of effector T lymphocytes by the chemokines IP10 and Mig. Eur J Immunol. 1998; 28: 961-972. <a href="/cgi/external_ref?access_num=10.1002/(SICI)1521-4141(199803)28:03
作者单位：Institute of Basic Medical Sciences (W.Y.C., B.C.C., M.S.C.), Department of Biochemistry and Molecular Biology (W.Y.C., M.Y.H., M.S.C.), Department of Cell Biology and Anatomy (M.J.J.), College of Medicine, National Cheng Kung University, and Chi-Mei Medical Center (W.Y.C., B.C.C., M.S.C.), Tainan,