- Email: [email protected]
Lysyl oxidase resolves inflammation by reducing monocyte chemoattractant protein-1 in abdominal aortic aneurysm
Atherosclerosis 208 (2010) 366–369
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Short communication
Lysyl oxidase resolves inflammation by reducing monocyte chemoattractant protein-1 in abdominal aortic aneurysm Masahiko Onoda a , Koichi Yoshimura b,∗ , Hiroki Aoki b , Yasuhiro Ikeda b , Noriyasu Morikage a , Akira Furutani a , Masunori Matsuzaki b , Kimikazu Hamano a a b
Department of Surgery and Clinical Science, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami Kogushi, Ube, Japan Department of Molecular Cardiovascular Biology, Yamaguchi University School of Medicine, 1-1-1 Minami Kogushi, Ube, Japan
a r t i c l e
i n f o
Article history: Received 21 April 2009 Received in revised form 14 July 2009 Accepted 15 July 2009 Available online 23 July 2009 Keywords: Lysyl oxidase Monocyte chemoattractant protein -1 Abdominal aortic aneurysm Inflammation
a b s t r a c t Lysyl oxidase (LOX) is an enzyme critical for the stability of extracellular matrix and also known to have diverse biological functions. Little is known, however, about the role of LOX in regulating inflammation. Here we demonstrate that LOX suppresses secretion of monocyte chemoattractant protein-1 (MCP-1) in cultured vascular smooth muscle cells. Furthermore, enhancement of LOX activity reduces MCP-1 in a mouse model of abdominal aortic aneurysm (AAA), thereby preventing macrophage infiltration and AAA progression. These findings suggest that LOX has a novel function in resolving inflammation by reducing MCP-1 in AAA. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Lysyl oxidase (LOX) is a copper-dependent amine oxidase that catalyzes the crosslinking of collagens and elastin, and is thus critical for the stability of the extracellular matrix (ECM). Many lines of evidence have shown that LOX has diverse biological functions, including regulation of gene transcription, cell migration, adhesion and transformation [1–3]. Little is known, however, regarding the role of LOX in regulating inflammation. Previously we found that inflammatory signalling through c-Jun N-terminal kinase (JNK) inhibits LOX activity in vascular smooth muscle cells (VSMCs), and demonstrated that JNK inhibition induced regression of experimental abdominal aortic aneurysms (AAA) [4] that are characterized by chronic inflammation and ECM degradation [5]. We further showed that overexpression of LOX prevents AAA expansion in mice [4]. In addition, inhibition of LOX by -aminopropionitrile fumarate (BAPN) in rat elastase-induced AAA frequently results in aortic dissection, which is another manifestation of ECM degradation [6]. Together these findings indicate that LOX plays an important role in stabilizing the ECM in AAA. In the present study, we investigated another possibility that LOX might regulate not only ECM stability but also inflammatory responses in cultured VSMCs and in the mouse model of AAA.
2.1. Adenoviral vectors Adenoviral vectors encoding LOX with a C-terminal HA epitope tag (Ad-LOX), -galactosidase (lacZ) gene with a nuclear localizing signal (Ad-LacZ), constitutively active MKK7 (Ad-caMKK7), and wild type JNK1 (Ad-JNK1) were prepared as previously described [4,7,8]. 2.2. Cell culture experiments VSMCs were obtained from the aortae of 8-week-old male Wistar rats as described previously [9]. All experiments were performed using cells at passage 3. Confluent VSMCs were serum-starved for 24 h and then treated with 200 M -aminopropionitrile fumarate (BAPN, Sigma) for inhibition of LOX activity. For overexpression of LOX, VSMCs were transfected with Ad-LOX for 24 h prior to serum starvation. Ad-LacZ was used at the same multiplicity of infection as a control. For specific activation of the JNK pathway, VSMCs were cotransfected with Ad-caMKK7 and Ad-JNK1. 2.3. Cytokine array
∗ Corresponding author. Tel.: +81 836 22 2361; fax: +81 836 22 2362. E-mail address: [email protected] (K. Yoshimura). 0021-9150/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2009.07.036
The conditioned media was analyzed with the cytokine array membrane (Rat Cytokine Antibody Array, Ray Biotech), according to the manufacturer’s instructions.
M. Onoda et al. / Atherosclerosis 208 (2010) 366–369
2.4. ELISA analysis
3. Results
The concentration of monocyte chemoattractant protein-1 (MCP-1) in the conditioned media was quantified by a sandwich enzyme immunoassay technique using the rat MCP-1 ELISA Kit (Pierce), according to the manufacturer’s instructions.
3.1. Array analysis of cytokines after overexpression of LOX in VSMCs
2.5. LOX activity assay The cell lysates were subjected to the fluorometric assay for LOX enzyme activity as previously described [4,10].
2.6. Quantitative reverse transcription–polymerase chain reaction Total RNA was isolated from VSMCs using RNeasy (Qiagen). mRNA was quantified by the LightCycler Instrument (Roche Applied Science) using the QuantiTect SYBR Green RT-PCR Kit (Qiagen) and QuantiTect Primer Assay (Qiagen). MCP-1 transcripts were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control.
2.7. Animal experiments AAA was induced in mice by periaortic application of 0.5 M CaCl2 as previously described [4,11]. We performed adenoviral gene transfer with 20 l of Ad-LOX (n = 8) or Ad-LacZ (n = 5) at the same titer (1.5 × 109 PFU/ml) 3 weeks after CaCl2 treatment as previously described [4]. After an additional 3 weeks, mice were sacrificed and perfusion-fixed with 3.7% formaldehyde/PBS for histological analyses. For immunostaining of MCP-1, mice were sacrificed 3 days after adenoviral gene transfer and excised aortae were frozen with Tissue-Tek OCT compound (Sakura Finetek). The animal experimental protocols were approved by the Animal Review Board of Yamaguchi University School of Medicine.
367
To explore the possibility that LOX directly regulates inflammatory molecules, we transfected rat aortic VSMCs with an adenovirus encoding full-length LOX (Ad-LOX) and used the conditioned media to perform a comprehensive analysis of 19 cytokines and chemokines using the cytokine antibody array. The results showed that secretion of MCP-1 was decreased in the VSMCs transfected with Ad-LOX compared to those with the control Ad-LacZ (Fig. 1). The protein levels of other cytokines remained unchanged after enhancement of LOX activity in VSMCs. Therefore, we focused on MCP-1 as a candidate molecule that links LOX activity with inflammation. 3.2. Role of LOX activity in regulation of MCP-1 secretion in vitro To demonstrate that LOX regulates MCP-1 secretion in VSMCs, LOX activity was enhanced by transfection with Ad-LOX or inhibited by treatment with BAPN (200 M), a chemical inhibitor of LOX. Treatment of VSMCs with BAPN reduced LOX activity (48 ± 12% reduction) and significantly increased MCP-1 secretion (1.8 ± 0.3 fold, p < 0.01 compared to Control, Fig. 2A). Enhancement of LOX activity by Ad-LOX markedly reduced secretion of MCP-1 (63 ± 9% reduction, p < 0.01 compared to Ad-LacZ, Fig. 2A) as well as mRNA level of MCP-1 (40 ± 16% reduction, p < 0.01 compared to Ad-LacZ). These data clearly indicate that LOX activity downregulates MCP-1 in VSMCs. We next examined whether activation of the JNK pathway, an inflammatory signalling pathway, affects the role of LOX activity in regulation of MCP-1 secretion. To specifically activate the JNK
2.8. Histological analysis Paraffin-embedded sections were stained with hematoxylin–eosin (HE) and elastica van-Gieson (EVG) stains for histologic analysis and antibodies to appropriate antigens were used for immunohistochemistry as previously described [4,12]. Phosphorylated JNK (P-JNK) was detected with anti-phosphospecific JNK antibody (Promega). MCP-1 was detected with an anti-mouse MCP-1 antibody (R&D Systems) using frozen sections. These proteins were visualized by an avidin–biotin complex technique. Macrophages were detected with an anti-mouse Mac-3 antibody (BD Biosciences) and visualized by indirect immunofluorescence staining with the Alexa Fluor 488 conjugated antibody (Molecular Probes). TO-PRO-3 (Molecular Probes) was used for nuclear staining. The number of macrophages was determined by counting Mac-3-positive cells in four high-power fields (HPFs, 143 m × 143 m) per mouse.
2.9. Statistical analysis All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using student’s unpaired t-test or analysis of variance (ANOVA), in which the post-test comparison was performed by a method of Bonferroni.
Fig. 1. Array analysis of cytokines after overexpression of LOX in VSMCs. Cultured VSMCs were transfected with Ad-LOX or Ad-LacZ. The conditioned media was analyzed with the cytokine array. (A) Representative images of array membranes. Arrowheads indicate the signals for MCP-1. (B) The position of cytokines and controls on the membrane. Pos: positive control, Neg: negative control, CINC: cytokineinduced neutrophil chemoattractants, CNTF: ciliary neutrophil factor, GM-CSF: granulocyte macrophage-colony stimulating factor, IFN: interferon, IL: interleukin, LIX: lipopolysaccharide-induced CXC chemokine, MIP: macrophage inflammatory protein, NGF: nerve growth factor, TIMP: tissue inhibitor of metalloproteinase, TNF: tumor necrosis factor, VEGF: vascular endothelial growth factor.
368
M. Onoda et al. / Atherosclerosis 208 (2010) 366–369
almost completely suppressed by additional transfection of Ad-LOX (Fig. 2B). We previously reported that inhibition of JNK prevented a decrease of LOX expression due to serum starvation [4]. In addition, JNK inhibition also blocked MCP-1 expression induced by tumor necrosis factor (TNF)-␣ [13]. These indicate that activation of JNK pathway causes an increase in MCP-1 secretion through reduction of LOX activity. Taken together, our findings suggest that LOX plays a critical role in inhibiting the secretion of MCP-1 in VSMCs under both basal and inflammatory conditions. 3.3. Role of LOX activity in inflammatory responses during AAA progression in vivo
Fig. 2. Secretion of MCP-1 after inhibition or enhancement of LOX activity in VSMCs. (A) Cultured VSMCs were treated with BAPN (200 M) to inhibit LOX activity, transfected with Ad-LOX or transfected with the Ad-LacZ. Amount of MCP-1 in the conditioned media was determined by ELISA. Data are means ± SD of 8 independent observations. ** and §§ indicate p < 0.01 compared with control and Ad-LacZ, respectively. (B) Cultured VSMCs were transfected with Ad-LOX or Ad-LacZ, and cotransfected with Ad-caMKK7 and Ad-JNK1 for specific activation of JNK pathway as indicated. Data are means ± SD of 4 independent observations. ** and §§ indicate p < 0.01 compared with Ad-LacZ and with Ad-caMKK7 + Ad-JNK1 + Ad-LacZ, respectively.
pathway, we cotransfected VSMCs with Ad-caMKK7 and Ad-JNK1. Cotransfection markedly reduced LOX activity in VSMCs (69 ± 14% reduction), which was completely restored by additional transfection of Ad-LOX (data not shown). Interestingly, MCP-1 secretion was significantly increased after cotransfection of Ad-caMKK7 and Ad-JNK1 (2.3 ± 0.2 fold, p < 0.01 compared to Ad-LacZ), which was
We next investigated the effects of LOX activity on inflammatory responses during progression of AAA in mice. For this purpose, we created CaCl2 -induced AAA in mice and performed local gene transfer with Ad-LOX or Ad-LacZ 3 weeks after CaCl2 treatment. Interestingly, expression of MCP-1 protein was reduced in the aortae transfected with Ad-LOX compared to those with Ad-LacZ at 3 days after transfection (Fig. 3A). At 6 weeks after CaCl2 treatment, the aortae from Ad-LOX transfected mice showed a significantly smaller diameter compared with those of mice transfected with Ad-LacZ (Ad-LOX, 1.1 ± 0.2 mm; Ad-LacZ, 1.5 ± 0.1 mm, p < 0.01), consistent with our previous results [4]. Importantly, the control aortae transfected with Ad-LacZ showed marked cellular infiltration with a substantial number of macrophages. Immunostaining for P-JNK revealed that JNK was activated in infiltrating cells as well as in interstitial cells (Fig. 3B). In contrast, the aortae from Ad-LOX transfected mice had much less inflammatory cells. Immunostaining showed significantly fewer macrophages in the aortae from Ad-LOX transfected mice than in those from Ad-LacZ transfected mice (1.1 ± 0.9 versus 10.6 ± 5.2 cells/HPF, respectively, p < 0.05) (Fig. 3C). Less P-JNK positive cells were observed, and morphology of elastic lamella was well preserved in the aortae in Ad-LOX transfected mice (Fig. 3B). These findings indicate that the enhancement of LOX activity not only stabilized the ECM but also reduced inflammatory responses, including MCP-1 secretion, macrophage infiltration and JNK activation, thereby preventing the AAA progression.
Fig. 3. Effects of LOX overexpression on inflammatory responses in the mouse model of AAA. AAA was induced in mice by application of CaCl2 . Local gene transfer with Ad-LOX or the Ad-LacZ was performed 3 weeks after CaCl2 treatment. (A) MCP-1 was detected by immunostaining of the mice aortae 3 days after transfection. (B) Mice aortae excised 3 weeks after transfection were examined by histologic analysis with HE and EVG staining, immunofluorescence for detection of macrophages and immunostaining for P-JNK. Green and red signals in the immunofluorescence image indicate staining for macrophages and cell nuclei, respectively. Scale bars indicate 50 m (A and B). (C) Quantitative analyses of the number of macrophages per HPF. Data are means ± SD of 4 independent observations. * indicates p < 0.05 compared with Ad-LacZ (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).
M. Onoda et al. / Atherosclerosis 208 (2010) 366–369
369
4. Discussion
Acknowledgments
We demonstrated for the first time that LOX negatively regulates MCP-1 secretion and subsequent inflammatory responses in cultured VSMCs and in the CaCl2 -induced mouse model of AAA. As we previously reported [4], LOX activity is increased early after CaCl2 treatment in this AAA model. These findings indicate a potential role of LOX in suppressing inflammation by reducing MCP-1 secretion after exposure to proinflammatory stimuli. Recently, resolution of inflammation is regarded as an active and coordinated program that initiates to return to homeostasis early after an inflammatory response begins [14]. Therefore, our results strongly suggest that LOX is involved in the physiological program of inflammation resolution. In AAA, however, JNK is highly activated by various stimuli and suppresses LOX activity [4], resulting in persistent inflammation. MCP-1 was identified as a target of LOX-mediated suppression by our comprehensive analysis. In fact, MCP-1 is one of the highly upregulated chemokines in human AAA walls [15], and is increased in the aortic wall before the onset of the chronic inflammatory responses and development of AAA in mice [16]. Moreover, deletion of the MCP-1 receptor CCR2 prevents inflammatory responses and development of AAA in mice [17]. Because these reports indicate a critical role for MCP-1 in AAA, the role of LOX in regulating MCP-1 is also likely to be important in pathogenesis of AAA. While LOX cross-links ECM proteins, recent reports showed that LOX oxidizes cell surface proteins, such as PDGFR-, and enhances the chemotactic response of VSMCs [18]. Moreover, mature LOX translocates from the extracellular space into cytosolic and nuclear compartments [1–3], indicating that LOX can act in both ECM-dependent and ECM-independent manners. Thus, whether LOX regulates MCP-1 secretion in an ECM-dependent or ECM-independent manner remains to be clarified. Interestingly, pentagalloyl glucose was reported to prevent the progression of CaCl2 -induced AAA in mice by stabilization of aortic elastin without interfering with inflammatory cell infiltration [19], indicating that ECM stabilization is not enough to suppress inflammatory responses. Therefore, we propose that LOX inhibits MCP-1 secretion in VSMCs and the subsequent inflammatory responses in AAA, possibly in an ECM-independent manner. According to our results, treatments that recover LOX activity might be beneficial to patients with AAA. A very recent report has demonstrated that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, abrogated the reduction of vascular LOX expression caused by TNF-␣ [20]. Thus, it is possible that statins restore LOX activity and consequently cause the resolution of inflammation as well as the restoration of ECM stability. In conclusion, we demonstrated that LOX has a novel function in resolving inflammation by reducing MCP-1 secretion in VSMCs and in AAA. Our findings also suggest that reduction of LOX activity contributes to persistence of inflammation in the pathogenesis of AAA, and that enhancement of LOX activity may represent a new therapeutic target for chronic inflammatory diseases, including AAA and atherosclerosis.
We thank M. Oishi, S. Nishino and T. Hozawa for their technical assistance. This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (to K.Y.), Japan Foundation of Cardiovascular Research (to K.Y.), Takeda Medical Research Foundation (to K.Y.) and a grant from Daiichi Sankyo Company to the Department of Molecular Cardiovascular Biology, Yamaguchi University School of Medicine. References [1] Kagan HM, Li W. Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell. J Cell Biochem 2003;88:660–72. [2] Payne SL, Hendrix MJC, Kirschmann DA. Paradoxical roles for lysyl oxidases in cancer - A prospect. J Cell Biochem 2007;101:1338–54. [3] Rodriguez C, Martinez-Gonzalez J, Raposo B, et al. Regulation of lysyl oxidase in vascular cells: lysyl oxidase as a new player in cardiovascular diseases. Cardiovasc Res 2008;79:7–13. [4] Yoshimura K, Aoki H, Ikeda Y, et al. Regression of abdominal aortic aneurysm by inhibition of c-Jun N-terminal kinase. Nat Med 2005;11:1330–8. [5] Thompson RW, Geraghty PJ, Lee JK. Abdominal aortic aneurysms: basic mechanisms and clinical implications. Curr Probl Surg 2002;39:110–230. [6] Huffman MD, Curci JA, Moore G, et al. Functional importance of connective tissue repair during the development of experimental abdominal aortic aneurysms. Surgery 2000;128:429–38. [7] Yamada M, Ikeda Y, Yano M, et al. Inhibition of protein phosphatase 1 by inhibitor-2 gene delivery ameliorates heart failure progression in genetic cardiomyopathy. FASEB J 2006;20:1197–9. [8] Aoki H, Kang PM, Hampe J, et al. Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes. J Biol Chem 2002;277:10244–50. [9] Adachi M, Katsumura KR, Fujii K, et al. Proteasome-dependent decrease in Akt by growth factors in vascular smooth muscle cells. FEBS Lett 2003;554:77–80. [10] Palamakumbura AH, Trackman PC. A fluorometric assay for detection of lysyl oxidase enzyme activity in biological samples. Anal Biochem 2002;300:245–51. [11] Longo GM, Xiong W, Greiner TC, et al. Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. J Clin Invest 2002;110:625–32. [12] Nomura S, Yoshimura K, Akiyama N, et al. HMG-CoA reductase inhibitors reduce matrix metalloproteinase-9 activity in human varicose veins. Eur Surg Res 2005;37:370–8. [13] Chen YM, Chiang WC, Lin SL, et al. Dual regulation of tumor necrosis factor-␣-induced CCL2/monocyte chemoattractant protein-1 expression in vascular smooth muscle cells by nuclear factor-B and activator protein-1: Modulation by type III phosphodiesterase inhibition. J Pharmacol Exp Ther 2004;309:978–86. [14] Serhan CN, Savill J. Resolution of inflammation: the beginning programs the end. Nat Immunol 2005;6:1191–7. [15] Kudo J, Yoshimura K, Hamano K. Simvastatin reduces secretion of monocyte chemoattractant proteins and matrix metalloproteinase-9 in human abdominal aortic aneurysms. Bull Yamaguchi Med School 2007;54:47–56. [16] Colonnello JS, Hance KA, Shames ML, et al. Transient exposure to elastase induces mouse aortic wall smooth muscle cell production of MCP-1 and RANTES during development of experimental aortic aneurysm. J Vasc Surg 2003;38:138–46. [17] Ishibashi M, Egashira K, Zhao Q, et al. Bone marrow-derived monocyte chemoattractant protein-1 receptor CCR2 is critical in angiotensin II-induced acceleration of atherosclerosis and aneurysm formation in hypercholesterolemic mice. Arterioscler Thromb Vasc Biol 2004;24:e174–178. [18] Lucero HA, Ravid K, Grimsby JL, et al. Lysyl oxidase oxidizes cell membrane proteins and enhances the chemotactic response of vascular smooth muscle cells. J Biol Chem 2008;283:24103–17. [19] Isenburg JC, Simionescu DT, Starcher BC, Vyavahare NR. Elastin stabilization for treatment of abdominal aortic aneurysms. Circulation 2007;115:1729–37. [20] Rodriguez C, Alcudia JF, Martinez-Gonzalez J, et al. Statins normalize vascular lysyl oxidase down-regulation induced by proatherogenic risk factors. Cardiovasc Res 2009;83:595–603.
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Short communication
Lysyl oxidase resolves inflammation by reducing monocyte chemoattractant protein-1 in abdominal aortic aneurysm Masahiko Onoda a , Koichi Yoshimura b,∗ , Hiroki Aoki b , Yasuhiro Ikeda b , Noriyasu Morikage a , Akira Furutani a , Masunori Matsuzaki b , Kimikazu Hamano a a b
Department of Surgery and Clinical Science, Yamaguchi University Graduate School of Medicine, 1-1-1 Minami Kogushi, Ube, Japan Department of Molecular Cardiovascular Biology, Yamaguchi University School of Medicine, 1-1-1 Minami Kogushi, Ube, Japan
a r t i c l e
i n f o
Article history: Received 21 April 2009 Received in revised form 14 July 2009 Accepted 15 July 2009 Available online 23 July 2009 Keywords: Lysyl oxidase Monocyte chemoattractant protein -1 Abdominal aortic aneurysm Inflammation
a b s t r a c t Lysyl oxidase (LOX) is an enzyme critical for the stability of extracellular matrix and also known to have diverse biological functions. Little is known, however, about the role of LOX in regulating inflammation. Here we demonstrate that LOX suppresses secretion of monocyte chemoattractant protein-1 (MCP-1) in cultured vascular smooth muscle cells. Furthermore, enhancement of LOX activity reduces MCP-1 in a mouse model of abdominal aortic aneurysm (AAA), thereby preventing macrophage infiltration and AAA progression. These findings suggest that LOX has a novel function in resolving inflammation by reducing MCP-1 in AAA. © 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
2. Materials and methods
Lysyl oxidase (LOX) is a copper-dependent amine oxidase that catalyzes the crosslinking of collagens and elastin, and is thus critical for the stability of the extracellular matrix (ECM). Many lines of evidence have shown that LOX has diverse biological functions, including regulation of gene transcription, cell migration, adhesion and transformation [1–3]. Little is known, however, regarding the role of LOX in regulating inflammation. Previously we found that inflammatory signalling through c-Jun N-terminal kinase (JNK) inhibits LOX activity in vascular smooth muscle cells (VSMCs), and demonstrated that JNK inhibition induced regression of experimental abdominal aortic aneurysms (AAA) [4] that are characterized by chronic inflammation and ECM degradation [5]. We further showed that overexpression of LOX prevents AAA expansion in mice [4]. In addition, inhibition of LOX by -aminopropionitrile fumarate (BAPN) in rat elastase-induced AAA frequently results in aortic dissection, which is another manifestation of ECM degradation [6]. Together these findings indicate that LOX plays an important role in stabilizing the ECM in AAA. In the present study, we investigated another possibility that LOX might regulate not only ECM stability but also inflammatory responses in cultured VSMCs and in the mouse model of AAA.
2.1. Adenoviral vectors Adenoviral vectors encoding LOX with a C-terminal HA epitope tag (Ad-LOX), -galactosidase (lacZ) gene with a nuclear localizing signal (Ad-LacZ), constitutively active MKK7 (Ad-caMKK7), and wild type JNK1 (Ad-JNK1) were prepared as previously described [4,7,8]. 2.2. Cell culture experiments VSMCs were obtained from the aortae of 8-week-old male Wistar rats as described previously [9]. All experiments were performed using cells at passage 3. Confluent VSMCs were serum-starved for 24 h and then treated with 200 M -aminopropionitrile fumarate (BAPN, Sigma) for inhibition of LOX activity. For overexpression of LOX, VSMCs were transfected with Ad-LOX for 24 h prior to serum starvation. Ad-LacZ was used at the same multiplicity of infection as a control. For specific activation of the JNK pathway, VSMCs were cotransfected with Ad-caMKK7 and Ad-JNK1. 2.3. Cytokine array
∗ Corresponding author. Tel.: +81 836 22 2361; fax: +81 836 22 2362. E-mail address: [email protected] (K. Yoshimura). 0021-9150/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2009.07.036
The conditioned media was analyzed with the cytokine array membrane (Rat Cytokine Antibody Array, Ray Biotech), according to the manufacturer’s instructions.
M. Onoda et al. / Atherosclerosis 208 (2010) 366–369
2.4. ELISA analysis
3. Results
The concentration of monocyte chemoattractant protein-1 (MCP-1) in the conditioned media was quantified by a sandwich enzyme immunoassay technique using the rat MCP-1 ELISA Kit (Pierce), according to the manufacturer’s instructions.
3.1. Array analysis of cytokines after overexpression of LOX in VSMCs
2.5. LOX activity assay The cell lysates were subjected to the fluorometric assay for LOX enzyme activity as previously described [4,10].
2.6. Quantitative reverse transcription–polymerase chain reaction Total RNA was isolated from VSMCs using RNeasy (Qiagen). mRNA was quantified by the LightCycler Instrument (Roche Applied Science) using the QuantiTect SYBR Green RT-PCR Kit (Qiagen) and QuantiTect Primer Assay (Qiagen). MCP-1 transcripts were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control.
2.7. Animal experiments AAA was induced in mice by periaortic application of 0.5 M CaCl2 as previously described [4,11]. We performed adenoviral gene transfer with 20 l of Ad-LOX (n = 8) or Ad-LacZ (n = 5) at the same titer (1.5 × 109 PFU/ml) 3 weeks after CaCl2 treatment as previously described [4]. After an additional 3 weeks, mice were sacrificed and perfusion-fixed with 3.7% formaldehyde/PBS for histological analyses. For immunostaining of MCP-1, mice were sacrificed 3 days after adenoviral gene transfer and excised aortae were frozen with Tissue-Tek OCT compound (Sakura Finetek). The animal experimental protocols were approved by the Animal Review Board of Yamaguchi University School of Medicine.
367
To explore the possibility that LOX directly regulates inflammatory molecules, we transfected rat aortic VSMCs with an adenovirus encoding full-length LOX (Ad-LOX) and used the conditioned media to perform a comprehensive analysis of 19 cytokines and chemokines using the cytokine antibody array. The results showed that secretion of MCP-1 was decreased in the VSMCs transfected with Ad-LOX compared to those with the control Ad-LacZ (Fig. 1). The protein levels of other cytokines remained unchanged after enhancement of LOX activity in VSMCs. Therefore, we focused on MCP-1 as a candidate molecule that links LOX activity with inflammation. 3.2. Role of LOX activity in regulation of MCP-1 secretion in vitro To demonstrate that LOX regulates MCP-1 secretion in VSMCs, LOX activity was enhanced by transfection with Ad-LOX or inhibited by treatment with BAPN (200 M), a chemical inhibitor of LOX. Treatment of VSMCs with BAPN reduced LOX activity (48 ± 12% reduction) and significantly increased MCP-1 secretion (1.8 ± 0.3 fold, p < 0.01 compared to Control, Fig. 2A). Enhancement of LOX activity by Ad-LOX markedly reduced secretion of MCP-1 (63 ± 9% reduction, p < 0.01 compared to Ad-LacZ, Fig. 2A) as well as mRNA level of MCP-1 (40 ± 16% reduction, p < 0.01 compared to Ad-LacZ). These data clearly indicate that LOX activity downregulates MCP-1 in VSMCs. We next examined whether activation of the JNK pathway, an inflammatory signalling pathway, affects the role of LOX activity in regulation of MCP-1 secretion. To specifically activate the JNK
2.8. Histological analysis Paraffin-embedded sections were stained with hematoxylin–eosin (HE) and elastica van-Gieson (EVG) stains for histologic analysis and antibodies to appropriate antigens were used for immunohistochemistry as previously described [4,12]. Phosphorylated JNK (P-JNK) was detected with anti-phosphospecific JNK antibody (Promega). MCP-1 was detected with an anti-mouse MCP-1 antibody (R&D Systems) using frozen sections. These proteins were visualized by an avidin–biotin complex technique. Macrophages were detected with an anti-mouse Mac-3 antibody (BD Biosciences) and visualized by indirect immunofluorescence staining with the Alexa Fluor 488 conjugated antibody (Molecular Probes). TO-PRO-3 (Molecular Probes) was used for nuclear staining. The number of macrophages was determined by counting Mac-3-positive cells in four high-power fields (HPFs, 143 m × 143 m) per mouse.
2.9. Statistical analysis All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using student’s unpaired t-test or analysis of variance (ANOVA), in which the post-test comparison was performed by a method of Bonferroni.
Fig. 1. Array analysis of cytokines after overexpression of LOX in VSMCs. Cultured VSMCs were transfected with Ad-LOX or Ad-LacZ. The conditioned media was analyzed with the cytokine array. (A) Representative images of array membranes. Arrowheads indicate the signals for MCP-1. (B) The position of cytokines and controls on the membrane. Pos: positive control, Neg: negative control, CINC: cytokineinduced neutrophil chemoattractants, CNTF: ciliary neutrophil factor, GM-CSF: granulocyte macrophage-colony stimulating factor, IFN: interferon, IL: interleukin, LIX: lipopolysaccharide-induced CXC chemokine, MIP: macrophage inflammatory protein, NGF: nerve growth factor, TIMP: tissue inhibitor of metalloproteinase, TNF: tumor necrosis factor, VEGF: vascular endothelial growth factor.
368
M. Onoda et al. / Atherosclerosis 208 (2010) 366–369
almost completely suppressed by additional transfection of Ad-LOX (Fig. 2B). We previously reported that inhibition of JNK prevented a decrease of LOX expression due to serum starvation [4]. In addition, JNK inhibition also blocked MCP-1 expression induced by tumor necrosis factor (TNF)-␣ [13]. These indicate that activation of JNK pathway causes an increase in MCP-1 secretion through reduction of LOX activity. Taken together, our findings suggest that LOX plays a critical role in inhibiting the secretion of MCP-1 in VSMCs under both basal and inflammatory conditions. 3.3. Role of LOX activity in inflammatory responses during AAA progression in vivo
Fig. 2. Secretion of MCP-1 after inhibition or enhancement of LOX activity in VSMCs. (A) Cultured VSMCs were treated with BAPN (200 M) to inhibit LOX activity, transfected with Ad-LOX or transfected with the Ad-LacZ. Amount of MCP-1 in the conditioned media was determined by ELISA. Data are means ± SD of 8 independent observations. ** and §§ indicate p < 0.01 compared with control and Ad-LacZ, respectively. (B) Cultured VSMCs were transfected with Ad-LOX or Ad-LacZ, and cotransfected with Ad-caMKK7 and Ad-JNK1 for specific activation of JNK pathway as indicated. Data are means ± SD of 4 independent observations. ** and §§ indicate p < 0.01 compared with Ad-LacZ and with Ad-caMKK7 + Ad-JNK1 + Ad-LacZ, respectively.
pathway, we cotransfected VSMCs with Ad-caMKK7 and Ad-JNK1. Cotransfection markedly reduced LOX activity in VSMCs (69 ± 14% reduction), which was completely restored by additional transfection of Ad-LOX (data not shown). Interestingly, MCP-1 secretion was significantly increased after cotransfection of Ad-caMKK7 and Ad-JNK1 (2.3 ± 0.2 fold, p < 0.01 compared to Ad-LacZ), which was
We next investigated the effects of LOX activity on inflammatory responses during progression of AAA in mice. For this purpose, we created CaCl2 -induced AAA in mice and performed local gene transfer with Ad-LOX or Ad-LacZ 3 weeks after CaCl2 treatment. Interestingly, expression of MCP-1 protein was reduced in the aortae transfected with Ad-LOX compared to those with Ad-LacZ at 3 days after transfection (Fig. 3A). At 6 weeks after CaCl2 treatment, the aortae from Ad-LOX transfected mice showed a significantly smaller diameter compared with those of mice transfected with Ad-LacZ (Ad-LOX, 1.1 ± 0.2 mm; Ad-LacZ, 1.5 ± 0.1 mm, p < 0.01), consistent with our previous results [4]. Importantly, the control aortae transfected with Ad-LacZ showed marked cellular infiltration with a substantial number of macrophages. Immunostaining for P-JNK revealed that JNK was activated in infiltrating cells as well as in interstitial cells (Fig. 3B). In contrast, the aortae from Ad-LOX transfected mice had much less inflammatory cells. Immunostaining showed significantly fewer macrophages in the aortae from Ad-LOX transfected mice than in those from Ad-LacZ transfected mice (1.1 ± 0.9 versus 10.6 ± 5.2 cells/HPF, respectively, p < 0.05) (Fig. 3C). Less P-JNK positive cells were observed, and morphology of elastic lamella was well preserved in the aortae in Ad-LOX transfected mice (Fig. 3B). These findings indicate that the enhancement of LOX activity not only stabilized the ECM but also reduced inflammatory responses, including MCP-1 secretion, macrophage infiltration and JNK activation, thereby preventing the AAA progression.
Fig. 3. Effects of LOX overexpression on inflammatory responses in the mouse model of AAA. AAA was induced in mice by application of CaCl2 . Local gene transfer with Ad-LOX or the Ad-LacZ was performed 3 weeks after CaCl2 treatment. (A) MCP-1 was detected by immunostaining of the mice aortae 3 days after transfection. (B) Mice aortae excised 3 weeks after transfection were examined by histologic analysis with HE and EVG staining, immunofluorescence for detection of macrophages and immunostaining for P-JNK. Green and red signals in the immunofluorescence image indicate staining for macrophages and cell nuclei, respectively. Scale bars indicate 50 m (A and B). (C) Quantitative analyses of the number of macrophages per HPF. Data are means ± SD of 4 independent observations. * indicates p < 0.05 compared with Ad-LacZ (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).
M. Onoda et al. / Atherosclerosis 208 (2010) 366–369
369
4. Discussion
Acknowledgments
We demonstrated for the first time that LOX negatively regulates MCP-1 secretion and subsequent inflammatory responses in cultured VSMCs and in the CaCl2 -induced mouse model of AAA. As we previously reported [4], LOX activity is increased early after CaCl2 treatment in this AAA model. These findings indicate a potential role of LOX in suppressing inflammation by reducing MCP-1 secretion after exposure to proinflammatory stimuli. Recently, resolution of inflammation is regarded as an active and coordinated program that initiates to return to homeostasis early after an inflammatory response begins [14]. Therefore, our results strongly suggest that LOX is involved in the physiological program of inflammation resolution. In AAA, however, JNK is highly activated by various stimuli and suppresses LOX activity [4], resulting in persistent inflammation. MCP-1 was identified as a target of LOX-mediated suppression by our comprehensive analysis. In fact, MCP-1 is one of the highly upregulated chemokines in human AAA walls [15], and is increased in the aortic wall before the onset of the chronic inflammatory responses and development of AAA in mice [16]. Moreover, deletion of the MCP-1 receptor CCR2 prevents inflammatory responses and development of AAA in mice [17]. Because these reports indicate a critical role for MCP-1 in AAA, the role of LOX in regulating MCP-1 is also likely to be important in pathogenesis of AAA. While LOX cross-links ECM proteins, recent reports showed that LOX oxidizes cell surface proteins, such as PDGFR-, and enhances the chemotactic response of VSMCs [18]. Moreover, mature LOX translocates from the extracellular space into cytosolic and nuclear compartments [1–3], indicating that LOX can act in both ECM-dependent and ECM-independent manners. Thus, whether LOX regulates MCP-1 secretion in an ECM-dependent or ECM-independent manner remains to be clarified. Interestingly, pentagalloyl glucose was reported to prevent the progression of CaCl2 -induced AAA in mice by stabilization of aortic elastin without interfering with inflammatory cell infiltration [19], indicating that ECM stabilization is not enough to suppress inflammatory responses. Therefore, we propose that LOX inhibits MCP-1 secretion in VSMCs and the subsequent inflammatory responses in AAA, possibly in an ECM-independent manner. According to our results, treatments that recover LOX activity might be beneficial to patients with AAA. A very recent report has demonstrated that statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, abrogated the reduction of vascular LOX expression caused by TNF-␣ [20]. Thus, it is possible that statins restore LOX activity and consequently cause the resolution of inflammation as well as the restoration of ECM stability. In conclusion, we demonstrated that LOX has a novel function in resolving inflammation by reducing MCP-1 secretion in VSMCs and in AAA. Our findings also suggest that reduction of LOX activity contributes to persistence of inflammation in the pathogenesis of AAA, and that enhancement of LOX activity may represent a new therapeutic target for chronic inflammatory diseases, including AAA and atherosclerosis.
We thank M. Oishi, S. Nishino and T. Hozawa for their technical assistance. This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (to K.Y.), Japan Foundation of Cardiovascular Research (to K.Y.), Takeda Medical Research Foundation (to K.Y.) and a grant from Daiichi Sankyo Company to the Department of Molecular Cardiovascular Biology, Yamaguchi University School of Medicine. References [1] Kagan HM, Li W. Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell. J Cell Biochem 2003;88:660–72. [2] Payne SL, Hendrix MJC, Kirschmann DA. Paradoxical roles for lysyl oxidases in cancer - A prospect. J Cell Biochem 2007;101:1338–54. [3] Rodriguez C, Martinez-Gonzalez J, Raposo B, et al. Regulation of lysyl oxidase in vascular cells: lysyl oxidase as a new player in cardiovascular diseases. Cardiovasc Res 2008;79:7–13. [4] Yoshimura K, Aoki H, Ikeda Y, et al. Regression of abdominal aortic aneurysm by inhibition of c-Jun N-terminal kinase. Nat Med 2005;11:1330–8. [5] Thompson RW, Geraghty PJ, Lee JK. Abdominal aortic aneurysms: basic mechanisms and clinical implications. Curr Probl Surg 2002;39:110–230. [6] Huffman MD, Curci JA, Moore G, et al. Functional importance of connective tissue repair during the development of experimental abdominal aortic aneurysms. Surgery 2000;128:429–38. [7] Yamada M, Ikeda Y, Yano M, et al. Inhibition of protein phosphatase 1 by inhibitor-2 gene delivery ameliorates heart failure progression in genetic cardiomyopathy. FASEB J 2006;20:1197–9. [8] Aoki H, Kang PM, Hampe J, et al. Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes. J Biol Chem 2002;277:10244–50. [9] Adachi M, Katsumura KR, Fujii K, et al. Proteasome-dependent decrease in Akt by growth factors in vascular smooth muscle cells. FEBS Lett 2003;554:77–80. [10] Palamakumbura AH, Trackman PC. A fluorometric assay for detection of lysyl oxidase enzyme activity in biological samples. Anal Biochem 2002;300:245–51. [11] Longo GM, Xiong W, Greiner TC, et al. Matrix metalloproteinases 2 and 9 work in concert to produce aortic aneurysms. J Clin Invest 2002;110:625–32. [12] Nomura S, Yoshimura K, Akiyama N, et al. HMG-CoA reductase inhibitors reduce matrix metalloproteinase-9 activity in human varicose veins. Eur Surg Res 2005;37:370–8. [13] Chen YM, Chiang WC, Lin SL, et al. Dual regulation of tumor necrosis factor-␣-induced CCL2/monocyte chemoattractant protein-1 expression in vascular smooth muscle cells by nuclear factor-B and activator protein-1: Modulation by type III phosphodiesterase inhibition. J Pharmacol Exp Ther 2004;309:978–86. [14] Serhan CN, Savill J. Resolution of inflammation: the beginning programs the end. Nat Immunol 2005;6:1191–7. [15] Kudo J, Yoshimura K, Hamano K. Simvastatin reduces secretion of monocyte chemoattractant proteins and matrix metalloproteinase-9 in human abdominal aortic aneurysms. Bull Yamaguchi Med School 2007;54:47–56. [16] Colonnello JS, Hance KA, Shames ML, et al. Transient exposure to elastase induces mouse aortic wall smooth muscle cell production of MCP-1 and RANTES during development of experimental aortic aneurysm. J Vasc Surg 2003;38:138–46. [17] Ishibashi M, Egashira K, Zhao Q, et al. Bone marrow-derived monocyte chemoattractant protein-1 receptor CCR2 is critical in angiotensin II-induced acceleration of atherosclerosis and aneurysm formation in hypercholesterolemic mice. Arterioscler Thromb Vasc Biol 2004;24:e174–178. [18] Lucero HA, Ravid K, Grimsby JL, et al. Lysyl oxidase oxidizes cell membrane proteins and enhances the chemotactic response of vascular smooth muscle cells. J Biol Chem 2008;283:24103–17. [19] Isenburg JC, Simionescu DT, Starcher BC, Vyavahare NR. Elastin stabilization for treatment of abdominal aortic aneurysms. Circulation 2007;115:1729–37. [20] Rodriguez C, Alcudia JF, Martinez-Gonzalez J, et al. Statins normalize vascular lysyl oxidase down-regulation induced by proatherogenic risk factors. Cardiovasc Res 2009;83:595–603.