Kikuo Isoda

Osteopontin Plays an Important Role in the Development of Medial Thickening and Neointimal Formation

Osteopontin (OPN) is a soluble secreted phosphoprotein that binds with high affinity to several integrins and it has been found at the site of atherosclerotic lesions. However, the role of OPN expression in vivo is still poorly understood. To investigate the physiological role of OPN in detail, we generated transgenic mice (Tg) overexpressing the OPN gene under control of the cytomegalovirus enhancer/chicken &bgr;-actin promoter. We detected OPN mRNAs in almost all tissues of 3 lines of Tg mice by Northern blotting. The serum levels of OPN were significantly higher in Tg than in non-Tg mice (782±107 versus 182±44 ng/mL;P <0.001). Compared with non-Tg mice, a 73% (88±6 versus 51±7 &mgr;m;P <0.001) and 94% (126±15 versus 73±11 &mgr;m;P <0.0001) increase in the medial thickness of the aorta was determined in Tg mice at 16 and 32 weeks after birth. However, we found no evidence of inflammatory cells adhering to endothelial cells, intimal hyperplasia, or calcification in any region of Tg mice without artery injury. We then investigated the effect of cuff-induced injury to the femoral artery. The intimal thickening in Tg mice increased 2.9-fold more than that in non-Tg mice (4.9±1.9 versus 1.7±0.4 &mgr;m;P =0.022). The expression of OPN induces both medial thickening without injury and neointimal formation after injury, thus suggesting that OPN plays a role in the development of atherosclerosis, vascular remodeling, and restenosis after angioplasty in vivo.

Lack of Interleukin-1 Receptor Antagonist Modulates Plaque Composition in Apolipoprotein E–Deficient Mice

Objective—Interleukin (IL)-1 plays an important role in atherosclerosis. IL-1 receptor antagonist (IL-1Ra) is an endogenous inhibitor of IL-1. However, the role of IL-1Ra in the development of atherosclerosis is poorly understood. Methods and Results—Mice that lacked IL-1Ra (IL-1Ra−/−) were crossed with apolipoprotein E-deficient (E−/−) mice and formation of atherosclerotic lesions was analyzed after 16 weeks or 32 weeks consumption of a normal chow diet. This study focused on the comparison of atherosclerotic lesion between IL-1Ra+/+/apoE−/− (n=12) and IL-1Ra+/−/apoE−/− mice (n=12), because of the significantly leaner phenotype in IL-1Ra−/−/apoE−/− mice compared with the others. Interestingly, atherosclerotic lesion size in IL-1Ra+/−/apoE−/− mice at age 16 weeks was significantly increased (30%) compared with IL-1Ra+/+/apoE−/− mice (P< 0.05). At 32 weeks, the differences of lesion size between these mice failed to achieve statistical significance. However, immunostaining demonstrated an 86% (P< 0.0001) increase in the MOMA-2–stained lesion area of IL-1Ra+/−/apoE−/− mice. In addition, α-actin staining in these lesions was significantly decreased (−15%) compared with those in IL-1Ra+/+/apoE−/− mice (P< 0.05). Conclusions—These results suggest an important role of IL-1Ra in the suppression of lesion development during early atherogenesis and furthermore indicate its role in the modulation of plaque composition.

Deficiency of Interleukin-1 Receptor Antagonist Promotes Neointimal Formation After Injury

Background—The cytokine interleukin (IL)-1 is an important mediator of inflammation and cardiovascular disease. Activity of this cytokine is modulated endogenously via the IL-1 receptor antagonist (IL-1Ra). The role of IL-1Ra in neointima formation after injury, however, is poorly understood. Methods and Results—Using IL-1Ra–deficient (IL-1Ra−/−; backcrossed 8 generations into the C57BL/6J background) and wild-type (IL-1Ra+/+) mice, we investigated neointimal formation 3 weeks after femoral artery injury induced by an external vascular cuff model. Intima and media thicknesses were measured, and the intima/media ratio was calculated. The mean intimal thickness and the intima/media ratio of IL-1Ra−/− mice increased by 249% (31.8±2.9 &mgr;m [n=10] versus 9.1±0.7 &mgr;m [n=10]; P <0.0001) and 257% (2.5±0.2 versus 0.7±0.1; P <0.0001), respectively, compared with IL-1Ra+/+ mice. No significant differences were observed in the medial thickness. Control immunostaining for IL-1Ra in injured vessels localized IL-1&bgr; and the endogenous inhibitor in the endothelium and inflammatory cells of the adventitia in IL-1Ra+/+ but not IL-1Ra−/− mice. Conclusions—The absence of IL-1Ra promotes neointimal formation in mice after injury. These results suggest that endogenous IL-1Ra may suppress other occlusive vascular responses to injury, such as atherosclerosis and restenosis after angioplasty.

Osteopontin Transgenic Mice Fed a High-Cholesterol Diet Develop Early Fatty-Streak Lesions

Background—Osteopontin (OPN) is a noncollagenous adhesion protein found at the site of atherosclerotic lesions. However, it has not yet been clarified whether or not OPN can promote atherosclerotic lesions. Methods and Results—We investigated the contribution of OPN to atherosclerosis by evaluating aortic sinus lesions of both OPN transgenic (Tg) and non-Tg mice fed an atherogenic diet (1.25% cholesterol) for 16 weeks. The atherosclerotic lesions were found to be significantly larger in OPN-Tg compared with those in non-Tg (17 859±2010 versus 6469±485 &mgr;m2, P <0.01). The lesions in both mice were fatty-streak lesions with an accumulation of mononuclear cells and lipids. We next investigated the production of interleukin (IL)-10 by macrophages from both mice. Compared with the non-Tg mice, a 42% (P <0.01) and 73% (P <0.001) decrease in the IL-10 production was identified in the OPN-Tg mice either without or with lipopolysaccharide. Conclusions—The expression of OPN induces fatty-streak lesion formation in mice fed an atherogenic diet and inhibits IL-10 production by macrophages, thus suggesting that OPN plays an important role in the development of fatty-streak lesions in vivo.

Fluorescence Analysis of Biochemical Constituents Identifies Atherosclerotic Plaque With a Thin Fibrous Cap

Vulnerable plaque generally contains a thin fibrous cap, lipid pools, and reduced internal plaque collagen. Arterial fluorescence analysis can differentiate atherosclerotic lesions from normal arteries; however, the contribution of the lipid core to atherosclerotic arterial fluorescence remains controversial. This study aimed to identify lipid core fluorophores and to differentiate the lipid core from normal artery and atheroma. The helium-cadmium laser–induced fluorescence spectra of cadaveric arteries and known chemical constituents were recorded. Lipid core fluorescence spectra exhibited marked red shifts and broadening compared with the fluorescence spectra of normal tissue and atheroma. Similar fluorescence spectra were obtained for lipid core and oxidized low density lipoprotein, for atheroma and collagen, and for normal artery and elastin. A classification based on collagen, elastin, and oxidized low density lipoprotein spectral decomposition could discriminate the lipid core (n=29), normal artery (n=74), atheroma (n=73), and preatheroma (n=10) with 86% accuracy. Fibrous cap thickness was correlated with the spectral collagen content index (r =0.65, P <0.0001), especially at a thickness of <200 &mgr;m. We conclude that a classification algorithm based on chemical spectral decomposition can accurately classify the fluorescence spectra of normal artery, atheroma, and lipid core and may be useful in identifying vulnerable atheroma in vivo.

Deficiency of Interleukin-1 Receptor Antagonist Induces Aortic Valve Disease in BALB/c Mice

Objective—Interleukin-1 receptor antagonist (IL-1Ra), one of the most important antiinflammatory cytokines, is crucial for homeostasis of the immune system. However, the role of IL-1Ra in aortic valve stenosis (AS) remains poorly understood. Methods and Results—IL-1Ra–deficient (IL-1Ra−/−) mice on the BALB/c background showed increased aortic valve leaflet thickness compared to wild-type mice at the age of 12 weeks (P<0.001). We used peripheral T-cell transplantation to examine the role of T cells in the development of AS. T cells from IL-1Ra−/− but not from wild-type mice induced increased aortic valve thickness in nu/nu mice. Moreover, IL-1Ra−/− T cells produced much higher levels of tumor necrosis factor (TNF)-&agr; in culture supernatants after anti-CD3 antibody stimulation compared to wild-type mice (P<0.001). Finally, we studied the role of TNF-&agr; in the development of AS in IL-1Ra−/− mice by generating double-gene-deficient (TNF-&agr;−/−/IL-1Ra−/−) mice. Interestingly, TNF-&agr;−/−/IL-1Ra−/− mice did not have AS. Conclusion—IL-1Ra deficiency in inflammatory cells induced aortic valve inflammation and TNF-&agr; participates importantly in the development of AS in IL-1Ra−/− mice.

Involvement of Tumor Necrosis Factor-α in the Development of T Cell–Dependent Aortitis in Interleukin-1 Receptor Antagonist–Deficient Mice

Background—Interleukin-1 receptor antagonist–deficient (IL-1Ra−/−) mice on the BALB/c background spontaneously develop inflammatory arthropathy that resembles rheumatoid arthritis in humans. These mice also frequently develop aortitis at the root of the aorta, but the mechanism underlying the development of this disease has not been completely elucidated. Methods and Results—Using IL-1Ra−/− mice (backcrossed 8 generations to the BALB/c background) and wild-type mice, we studied the histopathology and examined the immunologic mechanisms involved in the development of aortic inflammation by cell transplantation experiments. Half of the IL-1Ra−/− mice developed aortitis at the root of the aorta, with massive infiltration of macrophages and monocytes and loss of elastic lamellae in the aortic media. Left ventricular hypertrophy and mild aortic stenosis were also shown by transthoracic echocardiography. Transplantation of T cells from IL-1Ra−/− mice induced aortitis in recipient nu/nu mice. Bone marrow cell transplants from IL-1Ra−/− mice also induced aortitis in irradiated wild-type recipient mice. Furthermore, tumor necrosis factor (TNF)-&agr; deficiency completely suppressed the development of aortitis in IL-1Ra−/− mice, whereas IL-6 deficiency did not affect pathology. Conclusions—These observations suggest that IL-1Ra deficiency in T cells activates them excessively, resulting in the development of aortitis in IL-1Ra−/− mice in a TNF-&agr;–dependent manner.

Emergent Z stent placement for treatment of cor pulmonale due to pulmonary emboli after failed lytic treatment: Technical considerations

A patient with a pulmonary embolism due to deep vein thrombosis of a lower extremity developed hypotension and cor pulmonale despite prior placement of an inferior vena caval filter and treatment with a thrombolytic agent. After failure of percutaneous guidewire fragmentation and thrombosuction, self-expandable bilateral Z stents were positioned into the lower branches through the pulmonary arterial trunks. The patient experienced immediate relief of her cor pulmonale and successful recovery from hypotesnion.

Interleukin-1 and Occlusive Arterial Diseases

Interleukin (IL)-1 is a pro-inflammatory cytokine and a central mediator in the cytokine network, and is known to control important functions both in the immune system and inflammation. The activity of IL-1 is counter-regulated by its endogenous inhibitor, IL-1 receptor antagonist (IL-1Ra). IL-1 and IL-1Ra are produced and secreted by a variety of cells including those responsible for controlling immunity. A recent study indicated that IL-1 and IL-1Ra transcripts were expressed in the vessel wall, suggesting that these cytokines contribute to the development and progression of vascular diseases. In this review, we will discuss the recent advances in our understanding of the mechanism of action of IL-1 in occlusive arterial diseases such as neointimal hyperplasia and atherosclerosis, specifically in a mouse model.

Dipeptidyl‐Peptidase‐4 Inhibitor, Alogliptin, Attenuates Arterial Inflammation and Neointimal Formation After Injury in Low‐Density Lipoprotein (LDL) Receptor‐Deficient Mice

Background The results of recent studies suggest that dipeptidyl‐peptidase‐4 inhibitors have antiatherogenic effects. However, whether or not dipeptidyl‐peptidase‐4 inhibitors could suppress arterial inflammation and intimal hyperplasia after injury remains undetermined. The present study aims to clarify the anti‐inflammatory effects of the dipeptidyl‐peptidase‐4 inhibitor, alogliptin (AGP), on the arteries of atherogenic low‐density lipoprotein receptor‐deficient (LKO) mice. Methods and Results We compared intimal hyperplasia in LKO mice 2 weeks after femoral artery injury using an external vascular cuff model. All mice received oral injection of AGP (20 mg/kg per day) or normal saline (control) once daily for 14 days. Fasting blood sugar levels, serum cholesterol levels, or blood pressure did not significantly differ between the 2 groups. Plasma levels of active glucagon‐like peptide‐1 were higher in the AGP than in the control LKO mice (22.2±1.9 versus 15.6±0.9 pg/mL; P<0.05). Compared with saline, AGP significantly reduced intimal hyperplasia (1087±127 versus 1896±140 μm2; P<0.001) as well as the intima/media ratio (0.08±0.01 versus 0.16±0.02; P<0.001). Immunostaining showed that AGP reduced proliferating cells (proliferating cell nuclear antigen–positive nuclei; P<0.001), percent smooth‐muscle cell area (α‐SMA‐positive cells; P<0.001), inflammatory cells infiltration (lymphocyte antigen 6 complex–positive cells; P<0.05), tumor necrosis factor‐α expression (P<0.05), and percent phospho‐NF‐κB‐positive cell compared with saline. Levels of tumor necrosis factor ‐α (0.5‐fold P<0.05), monocyte chemoattractant protein 1 (0.3‐fold P<0.01), and interleukin‐1β (0.2‐fold P<0.05) mRNA were lower in the injured arteries of the AGP than in the control group. Conclusions AGP appeared to suppress neointimal formation by inhibiting inflammation, independently of its effects on glucose or cholesterol metabolism in atherogenic LKO mice.