Fong To

Lack of the Cysteine Protease Inhibitor Cystatin C Promotes Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—Degradation of extracellular matrix plays an important role in growth and destabilization of atherosclerotic plaques. Cystatin C, inhibitor of the collagen- and elastin-degrading cysteine proteases of the cathepsin family, is produced by virtually all cell types. It is present in the normal artery wall but severely reduced in human atherosclerotic lesions. Methods and Results—To determine the functional role of cystatin C in atherosclerosis, we crossed cystatin C–deficient (cysC−/−) mice with apolipoprotein E–deficient (apoE−/−) mice. After 25 weeks of atherogenic diet, mice lacking apoE and cystatin C (cysC−/− apoE−/−) had larger subvalvular plaques compared with cysC+/+ apoE−/− mice (766 000±20 000 &mgr;m2 per section versus 662 000±19 000 &mgr;m2 per section; P=0.001), suggesting an atheroprotective role of cystatin C. The plaques from cysC−/− apoE−/− mice were characterized by increased total macrophage content. To determine which cellular source is important for the antiatherosclerotic effect of cystatin C, we performed bone marrow transplantations. ApoE−/− mice were transplanted with either cysC−/− apoE−/− or cysC+/+ apoE−/− bone marrow. No significant differences in plaque area, macrophage, collagen, or lipid content of subvalvular lesions between the 2 groups were detected. Conclusions—The result suggests that the protective role of cystatin C in atherosclerosis is dependent primarily on its expression in nonhematopoietic cell types.

Immunization of apoE-/- mice with aldehyde-modified fibronectin inhibits the development of atherosclerosis

AIMS Oxidation of low-density lipoprotein in the extracellular matrix of the arterial wall results in the formation of malondialdehyde (MDA) that modifies surrounding matrix proteins. This is associated with the activation of an immune response against modified extracellular matrix proteins present in atherosclerotic plaques. Clinical studies have revealed an inverse association between antibodies to MDA-modified fibronectin and risk for development of cardiovascular events. To determine the functional role of these immune responses in atherosclerosis, we performed studies in which apoE-deficient mice were immunized with MDA-modified fibronectin. METHODS AND RESULTS Immunization of apoE-deficient mice with MDA-modified fibronectin resulted in a 70% decrease in plaque area and a less inflammatory phenotype of remaining plaques. Immunization shifted a weak naturally occurring Th1 antibody response against MDA-fibronectin into a Th2 antibody response. Cytokine expression and flow cytometry analyses of spleen cells from immunized mice showed an activation of regulatory T cells. Immunization with MDA-fibronectin was also found to reduce plasma fibronectin levels. CONCLUSION Immunization with MDA-fibronectin significantly reduces the development of atherosclerosis in apoE-deficient mice suggesting that the immune response observed in humans may have a protective effect. MDA-fibronectin represents a possible novel target for immunomodulatory therapy in atherosclerosis.

Inhibition of Nuclear Factor of Activated T-Cells (NFAT) Suppresses Accelerated Atherosclerosis in Diabetic Mice

Objective of the Study Diabetic patients have a much more widespread and aggressive form of atherosclerosis and therefore, higher risk for myocardial infarction, peripheral vascular disease and stroke, but the molecular mechanisms leading to accelerated damage are still unclear. Recently, we showed that hyperglycemia activates the transcription factor NFAT in the arterial wall, inducing the expression of the pro-atherosclerotic protein osteopontin. Here we investigate whether NFAT activation may be a link between diabetes and atherogenesis. Methodology and Principal Findings Streptozotocin (STZ)-induced diabetes in apolipoprotein E−/− mice resulted in 2.2 fold increased aortic atherosclerosis and enhanced pro-inflammatory burden, as evidenced by elevated blood monocytes, endothelial activation- and inflammatory markers in aorta, and pro-inflammatory cytokines in plasma. In vivo treatment with the NFAT blocker A-285222 for 4 weeks completely inhibited the diabetes-induced aggravation of atherosclerosis, having no effect in non-diabetic mice. STZ-treated mice exhibited hyperglycemia and higher plasma cholesterol and triglycerides, but these were unaffected by A-285222. NFAT-dependent transcriptional activity was examined in aorta, spleen, thymus, brain, heart, liver and kidney, but only augmented in the aorta of diabetic mice. A-285222 completely blocked this diabetes-driven NFAT activation, but had no impact on the other organs or on splenocyte proliferation or cytokine secretion, ruling out systemic immunosuppression as the mechanism behind reduced atherosclerosis. Instead, NFAT inhibition effectively reduced IL-6, osteopontin, monocyte chemotactic protein 1, intercellular adhesion molecule 1, CD68 and tissue factor expression in the arterial wall and lowered plasma IL-6 in diabetic mice. Conclusions Targeting NFAT signaling may be a novel and attractive approach for the treatment of diabetic macrovascular complications.

Immune responses against aldehyde-modified laminin accelerate atherosclerosis in Apoe(-/-) mice.

BACKGROUND LDL oxidation in the vascular wall is associated with aldehyde modification of surrounding extracellular matrix proteins that may target autoimmune responses against vascular tissues. Here we investigated the possible influence of immunity against a malondialdehyde (MDA)-modified form of the basement membrane protein laminin on atherosclerosis. METHODS AND RESULTS IgM and IgG autoantibodies were present in human plasma and a prospective clinical study demonstrated that individuals who later suffered from acute cardiovascular events had lower levels of MDA-laminin antibodies compared to those in the control group. Immunohistochemical analysis of atherosclerotic plaques from Apoe-/- mice demonstrated co-localization between laminin and MDA epitopes, however MDA-laminin IgG was absent in mouse plasma. To determine the effect of MDA-laminin immunity, Apoe-/- mice were immunized with MDA-laminin. Analysis of circulating leukocytes at 12 weeks demonstrated increased T-cell activation, expansion of Th17 cells and a lower fraction of regulatory T cells (Tregs) in mice immunized with MDA-laminin. At 25 weeks, aortic atherosclerosis was increased by more than 60% in mice immunized with MDA-laminin, together with increased levels of MDA-laminin IgG1 and MDA-laminin-specific T-cells expressing IL-2, IL-4 and IL-6 in the spleen. CONCLUSION The clinical observations suggest that immune responses against MDA-laminin may be involved in the development of cardiovascular disease in humans. Furthermore, observations in mice provide evidence for the presence of aldehyde-modified laminin in atherosclerotic lesions and demonstrate that induction of an immune response against these structures is associated with activation of Th17 cells, reduced fraction of Tregs and a more aggressive development of atherosclerosis.

IL-25 Inhibits Atherosclerosis Development in Apolipoprotein E Deficient Mice

Objective IL-25 has been implicated in the initiation of type 2 immunity and in the protection against autoimmune inflammatory diseases. Recent studies have identified the novel innate lymphoid type 2 cells (ILC2s) as an IL-25 target cell population. The purpose of this study was to evaluate if IL-25 has any influence on atherosclerosis development in mice. Methods and Results Administration of 1 μg IL-25 per day for one week to atherosclerosis-prone apolipoprotein (apo)E deficient mice, had limited effect on the frequency of T cell populations, but resulted in a large expansion of ILC2s in the spleen. The expansion was accompanied by increased levels of anti-phosphorylcholine (PC) natural IgM antibodies in plasma and elevated levels of IL-5 in plasma and spleen. Transfer of ILC2s to apoE deficient mice elevated the natural antibody-producing B1a cell population in the spleen. Treatment of apoE/Rag-1 deficient mice with IL-25 was also associated with extensive expansion of splenic ILC2s and increased plasma IL-5, suggesting ILC2s to be the source of IL-5. Administration of IL-25 in IL-5 deficient mice resulted in an expanded ILC2 population, but did not stimulate generation of anti-PC IgM, indicating that IL-5 is not required for ILC2 expansion but for the downstream production of natural antibodies. Additionally, administration of 1 μg IL-25 per day for 4 weeks in apoE deficient mice reduced atherosclerosis in the aorta both during initiation and progression of the disease. Conclusions The present findings demonstrate that IL-25 has a protective role in atherosclerosis mediated by innate responses, including ILC2 expansion, increased IL-5 secretion, B1a expansion and natural anti-PC IgM generation, rather than adaptive Th2 responses.

Immune responses against fibronectin modified by lipoprotein oxidation and their association with cardiovascular disease.

Objectives.  Accumulation and subsequent oxidation of LDL in the arterial wall are considered as key events in the development of atherosclerosis. We have investigated the possibility that LDL oxidation results in release of aldehydes that modify surrounding matrix proteins and that this may target immune responses against the plaque extracellular matrix and modulate the disease progression.

Increased Inflammation in Atherosclerotic Lesions of Diabetic Akita-LDLr(-/-) Mice Compared to Nondiabetic LDLr(-/-) Mice.

Background. Diabetes is associated with increased cardiovascular disease, but the underlying cellular and molecular mechanisms are poorly understood. One proposed mechanism is that diabetes aggravates atherosclerosis by enhancing plaque inflammation. The Akita mouse has recently been adopted as a relevant model for microvascular complications of diabetes. Here we investigate the development of atherosclerosis and inflammation in vessels of Akita mice on LDLr −/− background. Methods and Results. Akita-LDLr −/− and LDLr −/− mice were fed high-fat diet from 6 to 24 weeks of age. Blood glucose levels were higher in both male and female Akita-LDLr −/− mice (137% and 70%, resp.). Male Akita-LDLr −/− mice had markedly increased plasma cholesterol and triglyceride levels, a three-fold increase in atherosclerosis, and enhanced accumulation of macrophages and T-cells in plaques. In contrast, female Akita-LDLr −/− mice demonstrated a modest 29% increase in plasma cholesterol and no significant increase in triglycerides, atherosclerosis, or inflammatory cells in lesions. Male Akita-LDLr −/− mice had increased levels of plasma IL-1β compared to nondiabetic mice, whereas no such difference was seen between female diabetic and nondiabetic mice. Conclusion. Akita-LDLr −/− mice display considerable gender differences in the development of diabetic atherosclerosis. In addition, the increased atherosclerosis in male Akita-LDLr −/− mice is associated with an increase in inflammatory cells in lesions.

Elastin- and Collagen-Rich Human Carotid Plaques Have Increased Levels of the Cysteine Protease Inhibitor Cystatin C

Background: Cystatin C is a major inhibitor of the elastin- and collagen-degrading cysteine proteases and may therefore have an important role in preserving atherosclerotic plaque stability. In this study we analyzed the associations between human carotid plaque cystatin C expression and the plaque content of collagen and elastin. Methods: Thirty-one plaques were removed by endarterectomy and homogenized. Cystatin C levels were analyzed by densitometry of Western blots and elastin and collagen levels were determined colorimetrically. Results: The plaque content of cystatin C correlated with total elastin (r = 0.58, p = 0.001) and collagen (r = 0.50, p = 0.004), as well as with cross-linked forms of elastin (r = 0.42, p = 0.022) and collagen (r = 0.52, p = 0.003). Immunohistochemical analysis demonstrated that cystatin C colocalized with elastin and collagen. No correlation was seen between cystatin C and the amount of degraded elastin or collagen in plaques. Conclusion: The positive correlation between cystatin C levels and collagen and elastin levels in plaques supports the notion that cystatin C plays an important role in maintaining atherosclerotic plaque stability.

Increased aldehyde-modification of collagen type IV in symptomatic plaques - A possible cause of endothelial dysfunction.

OBJECTIVE Subendothelial LDL-adhesion and its subsequent oxidation are considered as key events in the development of atherosclerotic lesions. During oxidation of LDL, reactive aldehydes such as malondialdehyde (MDA) are formed, which modify apolipoprotein B100. However, the possibility that these reactive aldehydes could leak out of the LDL-particle and modify surrounding extracellular matrix proteins has been largely unexplored. We have investigated if aldehyde-modification of collagen type IV, one of the major basement membrane components, in plaques is associated with cardiovascular events. METHODS The amount of MDA-modified collagen type IV and native collagen type IV were determined in homogenates from 155 carotid artery lesions, removed by endarterectomy from patients with or without previous cerebrovascular events. RESULTS Plaque MDA-collagen type IV, but not native collagen type IV, correlated with oxidized LDL (r=0.31, P<0.001) and lipoprotein-associated phospholipase A2 (r=0.44, P<0.001). MDA-collagen type IV was increased in lesions from symptomatic patients compared to lesions from asymptomatic patients. Auto-antibodies against MDA-collagen type IV in plasma correlated with the amount of MDA-collagen type IV in lesions. MDA-modification of collagen type IV decreased endothelial cell attachment. In addition, culture of endothelial cells with MDA-modified collagen type IV increased vascular cell adhesion molecule expression and reduced the anti-coagulant proteins thrombomodulin and endothelial protein C receptor. In the lesions native collagen type IV, but not MDA-collagen type IV, was positively associated with thrombomodulin. CONCLUSION The present observations imply that aldehyde-modification of collagen type IV, associated with LDL oxidation, in atherosclerotic plaques may cause endothelial dysfunction and increase the risk of clinical events.

Interleukin 25 (IL-25) has a protective role in atherosclerosis development in the aortic arch in mice

Atherosclerosis is a chronic inflammatory disease characterized by the entrapment of apolipoprotein B–containing lipoproteins in the arterial intima, leading to local inflammation. T helper (Th) cell 1–mediated immune responses have been associated with atherosclerosis, and the cytokine interleukin-25 (IL-25 or IL-17E) has been reported to potentially regulate Th1 cell– and Th17 cell–related immune responses. In this study, we evaluated the effects of complete IL-25 deficiency or of a temporal IL-25 blockade on atherosclerosis development in apolipoprotein E–deficient (Apoe−/−) mice. Mice deficient in both apolipoprotein E and IL-25 (Apoe−/−/IL-25−/−) had more Th1 cells in the spleen, along with elevated plasma levels of IL-17 and an increased release of splenic interferon-γ (INF-γ). In support of this observation, a 4-week-long treatment of young Apoe−/− mice (at 10–14 weeks of age) with an IL-25–blocking antibody increased the release of Th1/Th17-associated cytokines in the spleen. In both mouse models, these findings were associated with increased atherosclerotic plaque formation in the aortic arch. We conclude that complete IL-25 deficiency and a temporal IL-25 blockade during early plaque development aggravate atherosclerosis development in the aortic arch of Apoe−/− mice, accompanied by an increase in Th1/Th17-mediated immune responses. Our finding that endogenous IL-25 has an atheroprotective role in the murine aortic arch has potential implications for atherosclerosis development and management in humans.