Deficiency of interleukin-1 receptor antagonist causes spontaneous femoral artery aneurysms

Interleukin (IL) -1, a proinflammatory cytokine, increases in aneurysm, and the IL-1 receptor antagonist (IL-1Ra) modulates IL-1 activity endogenously. We show here that IL-1Ra-deficiency provokes spontaneous femoral artery aneurysm in mice lacking intervention such as surgery or drugs, providing easily attained observation at different ages as well as diminished mortality from operative procedures. Therefore, IL-1Ra-deficient mice likely offer numerous opportunities to study the pathogenesis and therapy of inflammatory aneurysm. Arterial aneurysms represent a complex degenerative disorder involving chronic arterial wall inflammation and destructive remodeling of structural connective tissue. The relative balance of proand anti-inflammatory cytokines likely controls inflammation in the aneurysm wall . Secreted mainly by macrophages and to a lesser extent by lymphocytes, prominent pro-inflammatory cytokines include IL-1β, IL-6, IL-8, IL-18, IFN-γ, and TNF-α. Previous studies demonstrated abundant expression of many cytokines, including IL-1β, in the aneurysm wall. The IL-1 receptor antagonist (IL-1Ra) negatively regulates IL-1 signaling by binding and blocking the functional receptor without activation. Thus, IL-1Ra plays an anti-inflammatory role in acute and chronic inflammation. Although an earlier study investigating expression of IL-1 and IL-1Ra transcripts in the vascular wall following mechanistic insult suggested that IL-1Ra attenuates the biological function of IL-1β, direct evidence implicating endogenous IL-1Ra in aneurysm formation remained undetermined. Our study used IL-1Ra-deficient (IL-1Ra-/-) mice 11 to determine whether IL-1Ra-deficiency promotes aneurysm formation. We backcrossed IL-1Ra-/mice to the C57BL/6J background for eight generations. Although phenotypic analysis revealed that the body weight of 7-month-old IL-1Ra-/male mice was lower compared with wild-type (WT) mice (Fig. 1a, P<0.05, n=5), we observed no significant differences in systolic blood pressure, plasma total cholesterol levels, or HDL cholesterol levels (Fig. 1b, c, d). In addition diameter of the femoral artery was similar in IL-1Ra-/and WT mice. However, IL-1Ra-/mice on the C57BL/6J background had spontaneously developed fusiform femoral artery aneurysms (Fig. 1e) after 7-month-old, and 47% (15/32) were affected by nine months of age. We observed numerous inflammatory cells around the fusiform femoral artery aneurysm in IL-1Ra-/mice (Fig. 1e), and the lumen was larger in IL-1Ra-/mice compared with WT mice (Fig. 1e). We followed fusiform femoral artery aneurysms detected in 8-month-old IL-1Ra-/mice. Interestingly, aneurysm size increased in a time-dependent manner in IL-1Ra-/but not WT mice (Fig. 1f). Moreover, the intimal + medial area of aneurysms in IL-1Ra-/mice was significantly larger compared with WT mice and also increased in a time-dependent manner (Fig. 1f). We analyzed mRNA expression of genes involved in cytokines, chemokine, and the regulation genes of extracellular matrix in femoral arteries of 7and 9-month-old WT and IL-1Ra-/mice. Notably, we used the sites of femoral artery aneurysms to analyze the arteries of 9-month-old IL-1Ra-/mice. Prior to aneurysm formation (age seven months), the mRNA levels did not differ significantly between WT and IL-1Ra-/mice (Fig. 1g). In contrast, real-time PCR of 9-month-old IL-1Ra-/mice revealed significantly increased mRNA levels of IL-1β and TNF-α (6.6-fold, P<0.05 and 12.4-fold, P<0.05, respectively) compared with WT mice (Fig. 1g). Furthermore, MCP-1 mRNA increased significantly in IL-1Ra-/mice (8.2-fold, P<0.05) compared with WT mice (Fig. 1g). In the regulation genes the regulation genes of extracellular matrix, mRNA levels of TGFβ (7.3-fold, P<0.05), u-PA (7.5-fold, P<0.05), and MMP-9 (6.0, P<0.01) in IL-1Ra -/mice were significantly higher compared with WT mice (Fig. 1g). Our observations suggest that IL-1Ra-deficiency may induce chronic medial and adventitial inflammation and aneurysm development, possibly by enhancing mRNA expression of cytokines, chemokines, and the regulation genes of extracellular matrix. Although the femoral artery aneurysms of a few 9-month-old IL-1Ra-/mice (2/15) were red (fig. 2a), most were white (Fig. 1e). Following perfusion, we harvested, fixed, and sectioned the red aneurysms. Remarkably, the red aneurysms showed increased intimal and medial thickening as well as dissection between intima and media (Fig. 2a). Elastin staining showed destruction of the internal (black arrows in Fig. 2a) and external (red arrows in Fig. 2a) elastic lamina in IL-1Ra-/mice and immunostaining revealed several macrophages in the dissectional lumen and some CD4-positive T cells in the adventitia (Fig. 2a). Interestingly, we frequently (65%, 11/17) observed saccular aneurysms (Fig. 2b) with or without fusiform aneurysms in 10-month-old IL-1Ra-/mice. Medial thickness increased, as did SMC (Fig. 2b), which were disarranged and expressed low levels of SM-α actin (Fig. 2b). We also observed fissure between SMCs expressing low levels of SM-α actin (Fig. 2b). To test the hypothesis that IL-1Ra-deficiency in hematopoietic cell types participates crucially in the development of femoral artery aneurysm, we performed BM cell transplantation. After injecting BM from IL-1Ra-/or WT mice into lethally irradiated WT mice, we detected femoral artery aneurysms with the increase in intimal + medial area and many inflammatory cells in the adventitia in several (33%, 3/9) WT mice receiving BM cells from IL-1Ra-/but not (0%, 0/10) WT mice (Fig. 2c). Thus, IL-1Ra-deficiency in hematopoietic cells results in femoral artery aneurysm. This study demonstrates that IL-1Ra-deficiency in hematopoietic cells disrupts immune system homeostasis and causes spontaneous femoral artery aneurysms in mice lacking interventions such as drugs or surgery. Thus, IL-1Ra-/mice provide easy observations with age and diminish mortality that typically follows surgical procedures. Furthermore, since IL-1Ra-/mice contain the entire spectrum of lesions observed during inflammatory aneurysm, this mouse model likely will provide numerous opportunities to study the pathogenesis and therapy of inflammatory aneurysm. ACKNOWLEDGMENTS This work was supported by National Defense Medical College grants H14 (K.I.) and H18 (K.I.). We thank Dr Yoichiro Iwakura (University of Tokyo, Japan) for kindly providing IL-1Ra-/mice. We thank the members of our animal institute for assistance in animal care. AUTHOR CONTRIBUTIONS K.I. designed the study and performed most of the experiments; T.N. collected the arterial samples from mice; H.K. performed real time PCR analysis; F.O. helped in experiment design and manuscript editing. 1. Thompson, R.W. Curr. Opin. Cardiol. 11, 504-518 (1996). 2. Slifka, M.K., Whitton, J.L. J. Mol. Med. 78, 74-80 (2000). 3. Benelli, R. et al. Curr Pharm Des. 12, 101-115 (2006). 4. Xing, Z. et al. J.Clin. Invest. 101, 311-320 (1998). 5. Moriwaki, T. et al. Stroke. 37, 900-905 (2006). 6. Sadek, M. et al. Surgery. 144, 252-258 (2008). 7. Dinarello, C.A. Int.Rev.Immunol. 16, 457-499 (1998). 8. Dinarello, C.A. N.Engl.J.Med. 343, 732-734 (2000). 9. Wang, X. et al. Biochem Biophys.Res.Commun. 271, 138-143 (2000). 10. Horai, R. et al. J.Exp.Med. 187, 1463-1475 (1998). 11. Isoda, K. et al. J.Bio.Chem. 280, 7002-7009 (2005). 12. Daugherty, A., Cassis, L.A. Arterioscler.Thromb.Vasc.Biol. 24, 429-434 (2004) Figure legend Figure 1 IL-1Ra-/mice spontaneously developed fusiform femoral artery aneurysms independent of systolic blood pressure and cholesterol profiles. (a) Body weight of IL-Ra-/(Ra-/-) male mice and wild type (WT) mice at seven months of age (*P < 0.05, n=5). (b) The systolic blood pressure of 7-month-old IL-Ra-/male mice and WT mice. Blood pressure was measured in nonanesthetized mice in the morning, using the tail-cuff method. (c-d) After fasting for seven hours, plasma total cholesterol and high-density lipoprotein (HDL) cholesterol were measured by enzymatic assays. (c) Plasma levels of total cholesterol of IL-Ra-/male mice and WT mice at seven months of age. (d) Plasma levels of HDL cholesterol in IL-Ra-/male mice and WT mice at seven months of age. (e) IL-1Ra-/mice spontaneously developed fusiform femoral artery aneurysms at nine months of age. Macroscopic appearance of femoral arteries of IL-1Ra-/and WT mice (upper panels). Femoral artery specimens were taken from IL-1Ra-/and WT mice at nine months of age. Femoral artery sections were prepared for histology and stained with hematoxylin and eosin (middle panels) or Masson’s trichrome (lower panels). Bar, 200μm. (f) Femoral artery specimens were taken from IL-1Ra-/and WT mice at 7 (n=6), 8 (n=6), or 9 (n=6) months of age. We corrected specimens of 9-month-old IL-1Ra-/mice from 8-month-old IL-1Ra-/mice showing fusiform femoral artery aneurysms. We analyzed maximal vessel area (left) and intima + medial area (right) (***P < 0.001). (g) Analyses of mRNA expression of genes involved in cytokine, chemokine, and the regulation genes of extracellular matrix were performed in femoral arteries of both WT and IL-1Ra-/mice at seven and nine months of age. The mRNA levels of the indicated genes in the femoral arteries of 7and 9-month old IL-1Ra-/and WT mice were quantified using real-time PCR with SYBR-green detection. Degree of change in gene expression is based on the 7-month baseline expression level of WT mice (*P < 0.05, **P < 0.01). Figure 2 IL-1Ra-/mice spontaneously developed both dissecting and saccular aneurysms, and IL-1Ra-deficiency in bone marrow (BM)-derived cells induced femoral artery aneurysms in WT mice. (a) IL-1Ra-/mice developed dissecting aneurysms. The macroscopic appearance of the femoral artery showed a red color (upper, left). After perfusion, the artery was harvested, embedded in OCT compound, and sectioned. All samples were stained with hematoxylin and eosin (upper, right, Bar, 200μm) or Masson’s trichrome (middle, left, black arrows show internal elastic lamina and red arrows show external elastic lamina). Immunohistochemical staining for CD11b (middle, right) revealed macrophages in the dissectional lumen. Furthermore, immunohistochemical staining for CD4 (lower panel) showed CD4-positive T cells in the adventitia. (b) IL-1Ra-/mice developed saccular aneurysms spontaneously. The macroscopic appearance of the femoral artery showed saccular aneurysm (upper, left). Samples were stained with hematoxylin and eosin (upper, middle, Bar, 500μm) or Masson’s trichrome (upper, right). Boxed area is shown in lower panels (hematoxylin and eosin (lower left), Masson’s trichrome (lower, middle), and immunohistochemical staining for SM-α actin (arrow shows fissure in the media). (c) BM of IL-1Ra-/(right) or WT (left) mice was injected in lethally irradiated recipient WT mice. We detected femoral artery aneurysms in several WT mice that received BM cells from IL-1Ra-/(upper, right (Bar, 500μm) and lower, right (Bar, 200μm)) but not WT mice (upper, left (Bar, 500μm) and lower, left (Bar, 200μm)).