Cyclophilin A induces macrophage apoptosis and enhances atherosclerotic lesions in high-fat diet-fed hyperglycemic rabbits.

Macrophage apoptosis is a key contributor to the progression of atherosclerosis. Cyclophilin A, a monocyte secretory protein associated with the initiation of atherosclerosis has an inherent nuclease activity. This study reports the mechanism by which cyclophilin A causes apoptosis of macrophages and accelerates the progression of atherosclerosis. Aortic lesion formation and apoptosis were studied in New Zealand White rabbits (NZW) which were fed high-fat diet (HFD) for 12 weeks. Using monocytes and HFD-fed rabbits we demonstrate that cyclophilin A induces mitochondrial membrane potential loss and mitochondrial pore transition protein opening through caspase 3 activation. En face staining revealed a significant increase in the lesion area in HFD-fed rabbits. Levels of glucose, cholesterol and proinflammatory cytokines were higher in these animals compared to rabbits fed with a normal diet. In the aorta of HFD-fed rabbits, medial vascular smooth muscle cells were disorganized and there was a loss of integrity of the endothelium. An 8-fold increase was seen in the number of apoptotic cells in the lesion area of HFD-fed NZW rabbits which were associated with an elevation in plasma cyclophilin A levels. siRNA knockdown of cyclophilin A gene reduced activation of caspase 3 in macrophages. Treatment with cyclosporine A, an inhibitor of cyclophilin A, significantly attenuated apoptosis in macrophages. Our study indicates that inhibitors of proinflammatory cytokines such as cyclophilin A may arrest macrophage apoptosis and result in a regression of advanced atherosclerotic lesions.

Metformin attenuates effects of cyclophilin A on macrophages, reduces lipid uptake and secretion of cytokines by repressing decreased AMPK activity.

Growing evidence implicates cyclophilin A secreted by vascular wall cells and monocytes as a key mediator in atherosclerosis. Cyclophilin A in addition to its proliferative effects, during hyperglycemic conditions, increases lipid uptake in macrophages by increasing scavenger receptors on the cell's surface. It also promotes macrophage migration across endothelial cells and conversion of macrophages into foam cells. Given the known effects of metformin in reducing vascular complications of diabetes, we investigated the effect of metformin on cyclophilin A action in macrophages. Using an ex vivo model of cultured macrophages isolated from patients with type 2 diabetes with and without coronary artery disease (CAD), we measured the effect of metformin on cyclophilin A expression, lipid accumulation, expression of scavenger receptors, plasma cytokine levels and AMP-activated protein kinase (AMPK) activity in macrophages. In addition, the effects of metformin on migration of monocytes, reactive oxygen species (ROS) formation, lipid uptake in the presence of cyclophilin A inhibitors and comparison with pioglitazone were studied using THP-1 monocytes. Metformin reduced cyclophilin A expression in human monocyte-derived macrophages. Metformin also decreased the effects of cyclophilin A on macrophages such as oxidized low-density lipoprotein (oxLDL) uptake, scavenger receptor expression, ROS formation and secretion of inflammatory cytokines in high-glucose conditions. Metformin reversed cyclophilin A-induced decrease in AMPK-1α activity in macrophages. These effects of metformin were similar to those of cyclophilin A inhibitors. Metformin can thus function as a suppressor of pro-inflammatory effects of cyclophilin A in high-glucose conditions by attenuating its expression and repressing cyclophilin A-induced decrease in AMPK-1α activity in macrophages.

Cyclophilin A enhances macrophage differentiation and lipid uptake in high glucose conditions: a cellular mechanism for accelerated macro vascular disease in diabetes mellitus.

BACKGROUND: Vascular disease in diabetes is initiated by monocyte adhesion to vascular endothelium, transmigration and formation of foam cells. Increasing clinical evidence supports a role for the secretory protein, cyclophilin A in diabetic vascular disease. The means by which cyclophilin A contributes to vascular lesion development in diabetes is however largely unknown. METHODS: In this study we investigated using THP1 cells and human monocytes whether cyclophilin A under hyperglycemic conditions, functions in the inflammatory cascade as a chemoattractant and increases lipid uptake by formation of foam cells invitro. We developed an invitro model of monocytes cultured in 20 mm glucose (high glucose) equivalent to 360 mg/dL of plasma glucose levels. These monocytes were then differentiated into macrophages using PMA and subsequently transformed to lipid laden foam cells using oxidized low density lipoproteins in the presence and absence of cyclophilin A. This cellular model was used to study monocyte to macrophage differentiation, transmigration and foam cell formation. A similar cellular model using siRNA mediated transient elimination of the cyclophilin A gene as well as chemical inhibitors were used to further confirm the role of cyclophilin A in the differentiation and foam cell formation process. RESULTS: Cyclophilin A effectively increased migration of high glucose treated monocytes to the endothelial cell monolayer (p < 0.0001). In the presence of cyclophilin A, differentiated macrophages, when treated with oxLDL had a 36 percent increase in intracellular lipid accumulation (p = 0.01) when compared to cells treated with oxLDL alone. An increased flux of reactive oxygen species was also observed (p = 0.01). Inflammatory cytokines such as TNF-α, MCP-1 and cyclophilin A were significantly increased. Silencing cyclophilin A in THP-1 cells and human monocytes using siRNA or chemical inhibitor, TMN355 resulted in decrease in lipid uptake by 65-75% even after exposure to oxidized LDL. The expression of scavenger receptors expressed during differentiation process, CD36 and LOX-1 were decreased (p < 0.0001). Levels of extracellular cyclophilin A and other inflammatory cytokines such as TNF-α and MCP-1also significantly reduced. CONCLUSIONS: Taken together, we describe here a possible cellular basis by which cyclophilin A may accelerate atherogenesis in diabetes mellitus.

Treatment with TNF-alpha or bacterial lipopolysaccharide attenuates endocardial endothelial cell-mediated stimulation of cardiac fibroblasts.

BACKGROUND: The endocardial endothelium that lines the inner cavity of the heart is distinct from the microvascular endothelial cells and modulates cardiac muscle performance in a manner similar to the vascular endothelial modulation of vascular structure and vasomotor tone. Although the modulatory effects of endocardial endothelium (EE) on cardiomyocytes are firmly established, the regulatory effects of endocardial endothelium on the cardiac interstitium and its cellular components remain ill defined. METHODS AND RESULTS: We investigated whether the stimulatory effect of EE on cardiac fibroblasts would be altered when EECs are activated by the cytokine tumor necrosis factor-alpha (TNF-alpha) or the endotoxin bacterial lipopolysaccharide (LPS). Both TNF-alpha and LPS were found to independently attenuate the stimulatory effect of EE on cardiac fibroblasts. These agents lowered the synthesis or release of ET-1 and increased the secretion of TGF-beta and NO. CONCLUSION: The findings of this study using endocardial endothelial cells (EECs) and neonatal cardiac fibroblasts demonstrate that pro-inflammatory cytokines cause altered secretion of paracrine factors by EECs and inhibit proliferation and lower collagen synthesis in fibroblasts. These changes may influence fibroblast response and extra cellular matrix remodeling in pathological conditions of the heart.