calpain and Atherosclerosis

Protein catabolism in macrophages, which is accomplished mainly through autophagy- lysosomal degradation, ubiquitin-proteasome system, and calpains, is disturbed in atheroprone vessels. Moreover, growing evidence suggests that defects in protein catabolism interfere with cholesterol handling in macrophages. Indeed, decreases in autophagy facilitate the deposition of cholesterol in atheroprone macrophages and the subsequent development of vulnerable atherosclerotic plaques due to impaired catabolism of lipid droplets and limited efferocytic clearance of dead cells. The proteasome is responsible for the degradation of ATP-binding cassette transporters, which leads to impaired cholesterol efflux from macrophages. Overactivation of conventional calpains contributes to excessive processing of functional proteins, thereby accelerating receptor-mediated uptake of oxidized low-density lipoproteins (LDLs) and slowing cholesterol efflux. Furthermore, calpain-6, an unconventional nonproteolytic calpain in macrophages, potentiates pinocytotic uptake of native LDL and attenuates the efferocytic clearance of dead cells. Herein, we focus on recent progress in understanding how defective protein catabolism is associated with macrophage cholesterol handling and subsequent atherogenesis.

Topics: Animals; Atherosclerosis; ATP-Binding Cassette Transporters; Calpain; Cholesterol; Humans; Lipoproteins, LDL; Macrophages; Proteasome Endopeptidase Complex

Topics: Aneurysm; Animals; Aorta; Atherosclerosis; Calpain; Catalysis; Cell Communication; Cell Proliferation; Cholesterol, LDL; Diabetic Angiopathies; Diabetic Retinopathy; Endothelial Cells; Extracellular Matrix; Humans; Hypertension, Pulmonary; Inflammation; Isoenzymes; Janus Kinase 1; Lipoproteins, LDL; Macrophages; Mice; Mice, Transgenic; Neoplasms; Neovascularization, Pathologic; Nitric Oxide Synthase; Phenotype; Proteolysis; Signal Transduction; Vascular Diseases

Calpains are Ca

Topics: Animals; Atherosclerosis; ATP-Binding Cassette Transporters; Calpain; CD36 Antigens; Cholesterol; Humans; Lipoproteins, LDL; Macrophages; Phagocytosis; Pinocytosis; Plaque, Atherosclerotic; Proteolysis; Receptors, Scavenger

Arterial aging is the major contributing factor to increases in the incidence and prevalence of cardiovascular disease, due mainly to the presence of chronic, low-grade, 'sterile' arterial inflammation. Inflammatory signaling driven by the angiotensin II cascade perpetrates adverse age-associated arterial structural and functional remodeling. The aged artery is characterized by endothelial disruption, enhanced vascular smooth muscle cell (VMSC) migration and proliferation, extracellular matrix (ECM) deposition, elastin fracture, and matrix calcification/amyloidosis/glycation. Importantly, the molecular mechanisms of arterial aging are also relevant to the pathogenesis of hypertension and atherosclerosis. Age-associated arterial proinflammation is to some extent mutable, and interventions to suppress or delay it may have the potential to ameliorate or retard age-associated arterial diseases.

Topics: Aged; Aging; Angiotensin II; Animals; Antigens, Surface; Arteries; Arteritis; Atherosclerosis; Calpain; Chemokine CCL2; Endothelin-1; Humans; Hypertension; Inflammation; Inflammation Mediators; Matrix Metalloproteinase 2; Middle Aged; Milk Proteins; Muscle, Smooth, Vascular; Nitric Oxide; Reactive Oxygen Species; Receptors, CCR2; Transforming Growth Factor beta1

This review highlights the pro-atherogenic roles of Ca(2+)-sensitive intracellular protease calpains. Among more than ten species of calpain isozymes, µ- and m-calpains have been characterized most extensively. These two isozymes are ubiquitously expressed in mammalian tissues, including blood vessels, and tightly regulate functional molecules in the vascular component cells through limited proteolytic cleavage. Indeed, previous cell-based experiments showed that calpains play significant roles in nitric oxide production in vascular endothelial cells (ECs), maintenance of EC barrier function and angiogenesis for maintaining vascular homeostasis. Recently, we demonstrated that modified-low density lipoprotein (LDL)-induced m-calpain causes hyperpermeability in ECs, leading to the infiltration of monocytes/macrophages and plasma lipids into the intimal spaces (Miyazaki T. et al., Circulation. 2011; 124: 2522-2532). Calpains also mediate oxidized LDL-induced apoptotic death in ECs. In monocytes/macrophages, calpains induce proteolytic degradation of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1), which results in impaired cholesterol efflux and subsequent macrophage foam cell formation. In vascular smooth muscle cells, calpains may be involved in the conversion from contractile phenotype to proliferative phenotype. In hepatocytes, calpains disrupt the biogenesis of high-density lipoprotein via proteolytic degradation of ABCA1. Thus, calpains may serve as novel candidate molecular targets for control of atherosclerosis.

Topics: Atherosclerosis; Calpain; Humans; Macrophages; Monocytes

The Ca

Topics: Animals; Atherosclerosis; Autophagy; Calpain; Foam Cells; Laminaria; Lipoproteins, LDL; Macrophages; Mice; Polysaccharides; Signal Transduction

We previously reported that a calpain inhibitor (CAI) prevents the development of atherosclerosis in rats. This study aimed to investigate the effects of CAI (1 mg/kg) on atherosclerosis in apolipoprotein E knockout (ApoE KO) mice that were fed a high-fat diet (HFD) and explore the underlying mechanism by analyzing the expression of genes related to the uptake and efflux of cholesterol.. Atherosclerotic plaques were evaluated. The activity of calpain in the aorta and that of superoxide dismutase (SOD) in the serum were assessed. Lipid profiles in the serum and liver were examined. Serum oxidized low-density lipoprotein (oxLDL), malondialdehyde (MDA), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6) levels were measured. The mRNA expressions of CD68, TNF-α, IL-6, CD36, scavenger receptor (SR-A), peroxisome proliferator-activated receptor gamma (PPAR-γ), liver-x-receptor alpha (LXR-α), and ATP-binding cassette transporter class A1 (ABCA1) in the aorta and peritoneal macrophages were also evaluated.. CAI reduced calpain activity in the aorta. CAI also impeded atherosclerotic lesion formation and mRNA expression of CD68 in the aorta and peritoneal macrophages of ApoE KO mice compared with those of mice receiving HFD. However, CAI had no effect on body weight and lipid levels in both the serum and liver. CAI significantly decreased MDA, oxLDL, TNF-α, and IL-6 levels and increased SOD activity in the serum. Moreover, CAI significantly inhibited the mRNA expression of TNF-α and IL-6 genes in the aorta and peritoneal macrophages. In addition, CAI significantly downregulated the mRNA expression of scavenger receptors CD36 and SR-A and upregulated the expression of genes involved in the cholesterol efflux pathway, i.e., PPAR-γ, LXR-α, and ABCA1 in the aorta and peritoneal macrophages.. CAI inhibited the development of atherosclerotic lesions in ApoE KO mice, and this effect might be related to the reduction of oxidative stress and inflammation and the improvement of cholesterol intake and efflux pathways.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Aorta; Aortic Diseases; Atherosclerosis; ATP Binding Cassette Transporter 1; Calpain; Cholesterol; Cysteine Proteinase Inhibitors; Disease Models, Animal; Gene Expression Regulation; Leupeptins; Lipid Metabolism; Liver X Receptors; Macrophages, Peritoneal; Male; Mice, Inbred C57BL; Mice, Knockout, ApoE; Plaque, Atherosclerotic; PPAR gamma; RNA, Messenger; Scavenger Receptors, Class A

ATP-binding cassette transporter A1 (ABCA1) is an essential regulator of intracellular cholesterol efflux. Secreted cholesterol binds to lipid-free apolipoprotein A-I (apoA-I) in peripheral blood to constitute high-density lipoprotein cholesterol (HDL) complexes. ABCA1 protein on the surface of macrophages acts as a crucial controller in preventing cholesterol accumulation. Importantly, ABCA1 is unstable and easily degraded via a series of biochemical activities, including but not limited to calpain-mediated and ubiquitin-proteasome system-mediated processes. How accelerated ABCA1 degradation impacts disordered lipid metabolism in macrophages and foam cell formation is unclear. N-methyl d-aspartate receptors (NMDARs) are ionotropic glutamate receptors with high calcium permeability. Calcium influx via NMDARs activates downstream signaling pathways. Over-activation of NMDARs stimulated by NMDA contributes to dysfunctional lipid metabolism in macrophages and foam cell formation via promotion of calpain-mediated ABCA1 proteolysis. However, increased NMDAR activity does not affect liver X receptor expression or ABCA1 mRNA levels. Following NMDA receptor silencing or calpain inhibition, NMDA treatment did not reduce ABCA1 protein levels, nor caused lipid accumulation in macrophages. In addition, NMDAR over-activation activates NF-κB signaling to promote IL-1β and IL-6 macrophage marker expression. However, NMDAR silencing and calpain inhibition reduce inflammatory macrophage responses. In summary, our study suggests that NMDAR activation reduces surface ABCA1 protein, promotes lipid accumulation, and induces the production and secretion of many inflammatory mediators in macrophages, possibly through enhanced calpain-mediated ABCA1 protein degradation. Thus, the NMDAR receptor may be a novel pharmacologic target for atherosclerosis therapy.

Topics: Apolipoprotein A-I; Atherosclerosis; ATP Binding Cassette Transporter 1; Biological Transport; Calcium; Calpain; Cholesterol, HDL; Foam Cells; Gene Expression Regulation; Humans; Lipid Metabolism; Liver X Receptors; Macrophages; N-Methylaspartate; NF-kappa B; Proteolysis; Receptors, N-Methyl-D-Aspartate

Accumulation of foam cells in the neointima represents an early stage of atherosclerosis. 1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl) urea (TPPU), a novel soluble epoxide hydrolase inhibitor (sEHi), effectively elevates epoxyeicosatrienoic acid (EET) levels. The effects of EETs on macrophages foam cells formation are poorly understood.Methods and Results:Incubation of foam cells with TPPU markedly ameliorate cholesterol deposition in oxidized low-density lipoprotein (oxLDL)-loaded macrophages by increasing the levels of EETs. Notably, TPPU treatment significantly inhibits oxLDL internalization and promotes cholesterol efflux. The elevation of EETs results in a decrease of class A scavenger receptor (SR-A) expression via downregulation of activator protein 1 (AP-1) expression. Additionally, TPPU selectively increases protein but not the mRNA level of ATP-binding cassette transporter A1 (ABCA1) through the reduction of calpain activity that stabilizes the protein. Moreover, TPPU treatment reduces the cholesterol content of macrophages and inhibits atherosclerotic plaque formation in apolipoprotein E-deficient mice. These changes induced by TPPU are dependent on heme oxygenase-1 (HO-1) activation.. The present study findings elucidate a precise mechanism of regulating cholesterol uptake and efflux in macrophages, which involves the prevention of atherogenesis by increasing the levels of EETs with TPPU.

Topics: Animals; Arachidonic Acids; Atherosclerosis; ATP Binding Cassette Transporter 1; Calpain; Cholesterol; Disease Models, Animal; Enzyme Inhibitors; Epoxide Hydrolases; Foam Cells; Heme Oxygenase-1; Humans; Lipoproteins, LDL; Macrophages; Male; Membrane Proteins; Mice, Knockout, ApoE; Phenylurea Compounds; Piperidines; Plaque, Atherosclerotic; Scavenger Receptors, Class A; Signal Transduction; THP-1 Cells

In the previous in vitro study, we found that simvastatin decreased the protein expression of CD36, the scavenger receptor, and calpain-1, the Ca2+-sensitive cysteine protease, in oxidized low-density lipoprotein (oxLDL)-treated macrophages. In this in vivo study, we investigated whether simvastatin downregulates the expression of CD36 and calpain-1 and inhibits the inflammation and atherosclerosis in apolipoprotein E knockout (ApoE KO) mice.. Twenty male 6-week-old ApoE KO mice were divided into 2 groups: the ApoE KO group and the ApoE KO + simvastatin (ApoE KO + Sim) group. Atherosclerotic lesions were evaluated and the expressions of CD68, CD36, and calpain-1 in aorta were examined.. Simvastatin inhibited the atherosclerotic lesion in ApoE KO mice. In addition, simvastatin reduced the contents of oxLDL, thiobarbituric acid reactive substances, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in serum, decreased the mRNA and protein expressions of CD36 and reduced the mRNA expression of TNF-α and IL-6 in the aortas. Furthermore, simvastatin reduced the calpain activity and the protein expression of calpain-1 in the aorta.. The results suggested that the attenuation of atherosclerotic lesions in ApoE KO mice by simvastatin might be associated with the downregulations of CD36 and calpain-1 and with inflammation.

Topics: Animals; Anti-Inflammatory Agents; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Aorta; Aortic Diseases; Apolipoproteins E; Atherosclerosis; Calpain; CD36 Antigens; Disease Models, Animal; Down-Regulation; Genetic Predisposition to Disease; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Inflammation; Inflammation Mediators; Interleukin-6; Lipoproteins, LDL; Male; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Plaque, Atherosclerotic; Simvastatin; Thiobarbituric Acid Reactive Substances; Tumor Necrosis Factor-alpha

Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice.. To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor(-/-) mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII.. The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Topics: Angiotensin II; Animals; Aortic Aneurysm, Abdominal; Atherosclerosis; Bone Marrow Transplantation; Calcium-Binding Proteins; Calpain; Cell Adhesion; Cell Movement; Cells, Cultured; Coculture Techniques; Cysteine Proteinase Inhibitors; Diet, High-Fat; Disease Models, Animal; Endothelial Cells; Genetic Predisposition to Disease; Inflammation; Leukocytes; Macrophages; Male; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Phenotype; Receptors, LDL; Whole-Body Irradiation

Macrophages contribute to the development of atherosclerosis through pinocytotic deposition of native LDL-derived cholesterol in macrophages in the vascular wall. Inhibiting macrophage-mediated lipid deposition may have protective effects in atheroprone vasculature, and identifying mechanisms that potentiate this process may inform potential therapeutic interventions for atherosclerosis. Here, we report that dysregulation of exon junction complex-driven (EJC-driven) mRNA splicing confers hyperpinocytosis to macrophages during atherogenesis. Mechanistically, we determined that inflammatory cytokines induce an unconventional nonproteolytic calpain, calpain-6 (CAPN6), which associates with the essential EJC-loading factor CWC22 in the cytoplasm. This association disturbs the nuclear localization of CWC22, thereby suppressing the splicing of target genes, including those related to Rac1 signaling. CAPN6 deficiency in LDL receptor-deficient mice restored CWC22/EJC/Rac1 signaling, reduced pinocytotic deposition of native LDL in macrophages, and attenuated macrophage recruitment into the lesions, generating an atheroprotective phenotype in the aorta. In macrophages, the induction of CAPN6 in the atheroma interior limited macrophage movements, resulting in a decline in cell clearance from the lesions. Consistent with this finding, we observed that myeloid CAPN6 contributed to atherogenesis in a murine model of bone marrow transplantation. Furthermore, macrophages from advanced human atheromas exhibited increased CAPN6 induction and impaired CWC22 nuclear localization. Together, these results indicate that CAPN6 promotes atherogenicity in inflamed macrophages by disturbing CWC22/EJC systems.

Topics: Adult; Aged; Aged, 80 and over; Animals; Aorta; Atherosclerosis; Bone Marrow Transplantation; Calpain; Cell Nucleus; Cytokines; Female; Gene Expression Regulation; History, Ancient; Humans; Inflammation; Lipoproteins, LDL; Macrophages; Male; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Middle Aged; Monocytes; Neuropeptides; Phenotype; Pinocytosis; Plaque, Atherosclerotic; rac1 GTP-Binding Protein; RNA Precursors; RNA Splicing; Signal Transduction

We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, inhibits atherosclerosis in rats. The present study was designed to investigate the effect of simvastatin on mouse peritoneal macrophage foam cell formation, the early feature of atherosclerosis, and explore its mechanisms. The results showed that simvastatin decreased cholesterol content and DiI-oxLDL (1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate - oxidized low-density lipoprotein) uptake, reduced the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the medium, down-regulated the mRNA and protein expression of CD36 (a fatty acid receptor), and reduced the mRNA expressions of peroxisome proliferator-activated receptor gamma (PPARγ), TNF-α, and IL-6 in macrophages treated with oxLDL. However, PPARγ agonist troglitazone partly abolished the effects of simvastatin on foam cells. In addition, simvastatin reduced the protein expression of calpain-1, a Ca

Topics: Animals; Atherosclerosis; Calpain; CD36 Antigens; Cells, Cultured; Humans; Lipoproteins, LDL; Macrophages; Mice; PPAR gamma; Signal Transduction; Simvastatin

Paeonol, a potent antioxidant isolated from cortex moutan, possesses athero‑protective activity, yet the detailed mechanisms are not fully investigated. This study was conducted to explore the role of paeonol and its underlying mechanisms in RAW264.7 macrophages and apolipoprotein E‑deficient (ApoE(‑/‑)) mice. Paeonol treatment significantly attenuated intracellular lipid accumulation in macrophages, which may be the result of decreased oxidized low‑density lipoprotein (ox‑LDL) uptake and increased cholesterol efflux. Additionally, paeonol markedly inhibited the mRNA and protein expression of the cluster of differentiation 36 (CD36) by decreasing nuclear translocation of c‑Jun [a subunit of activator protein‑1 (AP‑1)]. Moreover, paeonol upregulated the protein stability of ATP‑binding cassette transporter A1 (ABCA1) by inhibiting calpain activity, while ABCA1 mRNA expression was not altered. Furthermore, small hairpin RNA (shRNA) targeting haem oxygenase‑1 (HO‑1) inhibited the paeonol‑mediated beneficial effects on the expression of c‑Jun, CD36, ABCA1, calpain activity and lipid accumulation in macrophages. Accordingly, paeonol retarded the progress of atherosclerosis in ApoE(‑/‑) mice and modulated the expression of CD36 and ABCA1 in aortas similarly to that observed in macrophages. These results indicate that paeonol provides protective effects on foam cell formation by a novel HO‑1‑dependent mediation of cholesterol efflux and lipid accumulation in macrophages.

Topics: Acetophenones; Animals; Aorta; Atherosclerosis; ATP Binding Cassette Transporter 1; Calpain; CD36 Antigens; Cell Line; Gene Expression Regulation, Developmental; Heme Oxygenase-1; Humans; Lipid Metabolism; Lipoproteins, LDL; Macrophages; Mice; Mice, Knockout

Activation of Na(+)/H(+) exchanger 1 (NHE1) by lipopolysaccharide (LPS) via Ca(2+)/calpain is responsible in vascular smooth muscle cell (VSMC) apoptosis and to the process of atherosclerosis. Probucol is a lipid-lowering drug which has an anti-atherosclerosis effect. The mechanism remains poorly understood. Here we hypothesized that probucol via inhibition of NHE1 in VSMCs attenuates LPS-accelerated atherosclerosis and promotes plaque stability. Our results revealed that treatment of VSMCs with LPS increased the NHE1 activity in a time-dependent manner, associated with the increased Ca(2+)i. Probucol inhibited the LPS-induced increase of NHE1 activity in a dose-dependent manner in VSMCs for 24-hour co-incubation, as well as the change of Ca(2+)i. In addition, LPS enhanced the calpain activity. Both probucol and calcium chelation of Ca(2+) abolished the LPS-induced increase of calpain activity. Treatment of VSMCs with LPS reduced the expression of Bcl-2 without altering the mRNA level. Probucol inhibited the LPS-reduced expression of Bcl-2 protein in VSMCs. Animal studies indicated administration of probucol suppressed LPS-accelerated apoptosis, atherosclerosis and plaque instability in Apoe(-/-) mice. In conclusion, probucol via inhibition of NHE1 attenuates atherosclerosis lesion growth and promotes plaque stability.

Topics: Animals; Atherosclerosis; Calcium; Calpain; Cation Transport Proteins; Humans; Lipopolysaccharides; Mice; Plaque, Atherosclerotic; Probucol; Proto-Oncogene Proteins c-bcl-2; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers

To investigate the effects of the potassium-sparing diuretic amiloride on endothelial cell apoptosis during lipopolysaccharide (LPS)-accelerated atherosclerosis.. Human umbilical vein endothelial cells (HUVECs) were exposed to LPS (100 ng/mL) in the presence of drugs tested. The activity of Na(+)/H(+) exchanger 1 (NHE1) and calpain, intracellular free Ca(2+)level ([Ca(2+)](i)), as well as the expression of apoptosis-related proteins in the cells were measured. For in vivo study, ApoE-deficient (ApoE(-/-)) mice were fed high-fat diets with 0.5% (w/w) amiloride for 4 weeks and LPS (10 μg/mouse) infusion into caudal veins. Afterwards, atherosclerotic lesions, NHE1 activity and Bcl-2 expression in the aortic tissues were evaluated.. LPS treatment increased NHE1 activity and [Ca(2+)](i) in HUVECs in a time-dependent manner, which was associated with increased activity of the Ca(2+)-dependent protease calpain. Amiloride (1-10 μmol/L) significantly suppressed LPS-induced increases in NHE1 activity, [Ca(2+)](i). and calpain activity. In the presence of the Ca(2+) chelator BAPTA (0.5 mmol/L), LPS-induced increase of calpain activity was also abolished. In LPS-treated HUVECs, the expression of Bcl-2 protein was significantly decreased without altering its mRNA level. In the presence of amiloride (10 μmol/L) or the calpain inhibitor ZLLal (50 μmol/L), the down-regulation of Bcl-2 protein by LPS was blocked. LPS treatment did not alter the expression of Bax and Bak proteins in HUVECs. In the presence of amiloride, BAPTA or ZLLal, LPS-induced HUVEC apoptosis was significantly attenuated. In ApoE(-/-) mice, administration of amiloride significantly suppressed LPS-accelerated atherosclerosis and LPS-induced increase of NHE1 activity, and reversed LPS-induced down-regulation of Bcl-2 expression.. LPS stimulates NHE1 activity, increases [Ca(2+)](i), and activates calpain, which leads to endothelial cell apoptosis related to decreased Bcl-2 expression. Amiloride inhibits NHE1 activity, thus attenuates LPS-accelerated atherosclerosis in mice.

Topics: Amiloride; Animals; Apoptosis; Atherosclerosis; Calcium; Calpain; Cation Transport Proteins; Endothelial Cells; Epithelial Sodium Channel Blockers; Human Umbilical Vein Endothelial Cells; Humans; Lipopolysaccharides; Male; Mice; Proto-Oncogene Proteins c-bcl-2; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers

Helical apolipoproteins remove cellular phospholipid and cholesterol to generate nascent HDL and this reaction is the major source of plasma HDL. ABCA1 is mandatory and rate-limiting for this reaction. Besides regulation of the gene expression by transcriptional factors including LXR, AP2 and SREBP, the ABCA1 activity is regulated post-translationally by calpain-mediated proteolytic degradation of ABCA1 protein that occurs in the early endosome after its endocytosis. When the HDL biogenesis reaction is ongoing as helical apolipoproteins interact with ABCA1, ABCA1 becomes resistant to calpain and is recycled to cell surface after endocytosis. Biogenesis of HDL is most likely to take place on cell surface. Clearance rate of ABCA1 by this mechanism is also retarded by various factors that interact with ABCA1, such as α1-syntrophin, LXRβ and calmodulin. Physiological relevance of the retardation by these factors is not entirely clear. Pharmacological inhibition of the calpain-mediated ABCA1 degradation results in the increase of the ABCA1 activity and HDL biogenesis in vitro and in vivo, and potentially suppresses atherogenesis. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Topics: alpha-Synuclein; Animals; Aorta; Atherosclerosis; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; BALB 3T3 Cells; Calmodulin; Calpain; Endocytosis; Endosomes; Lipoproteins, HDL; Liver X Receptors; Mice; Orphan Nuclear Receptors; Protein Processing, Post-Translational; Protein Transport; Proteolysis; Quinones; Rabbits

Chronic infusion of angiotensin II (AngII) augments atherosclerosis and abdominal aortic aneurysm (AAA) formation in hypercholesterolemic mice. AngII-induced AAAs are associated with medial macrophage accumulation and matrix metalloproteinase (MMP) activation. Inhibition of calpain, a calcium-activated neutral cysteine protease, by overexpression of its endogenous inhibitor, calpastatin, attenuates AngII-induced leukocyte infiltration, perivascular inflammation, and MMP activation in mice. The purpose of this study was to define whether pharmacological inhibition of calpain influences AngII-induced AAAs in hypercholesterolemic mice. Male low-density lipoprotein receptor-/- mice were fed a fat-enriched diet and administered with either vehicle or a calpain-specific inhibitor, BDA-410 (30 mg/kg per day) for 5 weeks. After 1 week of feeding, mice were infused with AngII (1000 ng/kg per minute) for 4 weeks. AngII-infusion profoundly increased aortic calpain protein and activity. BDA-410 administration had no effect on plasma cholesterol concentrations or AngII-increased systolic blood pressure. Calpain inhibition significantly attenuated AngII-induced AAA formation and atherosclerosis development. BDA-410 administration attenuated activation of MMP12, proinflammatory cytokines (IL-6, monocyte chemoattractant protein-1), and macrophage infiltration into the aorta. BDA-410 administration significantly attenuated thioglycolate-elicited macrophage accumulation in the peritoneal cavity. We conclude that calpain inhibition using BDA-410 attenuated AngII-induced AAA formation and atherosclerosis development in low-density lipoprotein receptor-/- mice.

Topics: Angiotensin II; Animals; Aortic Aneurysm, Abdominal; Atherosclerosis; Blotting, Western; Calpain; Cysteine Proteinase Inhibitors; Diet, High-Fat; Disease Models, Animal; Hypercholesterolemia; Male; Mice; Mice, Knockout; Receptors, LDL; Sulfonamides

Curcumin, a potent antioxidant extracted from Curcuma longa, confers protection against atherosclerosis, yet the detailed mechanisms are not fully understood. In this study, we examined the effect of curcumin on lipid accumulation and the underlying molecular mechanisms in macrophages and apolipoprotein E-deficient (apoE⁻/⁻) mice.. Treatment with curcumin markedly ameliorated oxidized low-density lipoprotein (oxLDL)-induced cholesterol accumulation in macrophages, which was due to decreased oxLDL uptake and increased cholesterol efflux. In addition, curcumin decreased the protein expression of scavenger receptor class A (SR-A) but increased that of ATP-binding cassette transporter (ABC) A1 and had no effect on the protein expression of CD36, class B receptor type I (SR-BI), or ATP-binding cassette transporter G1 (ABCG1). The downregulation of SR-A by curcumin was via ubiquitin-proteasome-calpain-mediated proteolysis. Furthermore, the curcumin-induced upregulation of ABCA1 was mainly through calmodulin-liver X receptor α (LXRα)-dependent transcriptional regulation. Curcumin administration modulated the expression of SR-A, ABCA1, ABCG1, and SR-BI in aortas and retarded atherosclerosis in apoE⁻/⁻ mice.. Our findings suggest that inhibition of SR-A-mediated oxLDL uptake and promotion of ABCA1-dependent cholesterol efflux are two crucial events in suppression of cholesterol accumulation by curcumin in the transformation of macrophage foam cells.

Topics: Animals; Anticholesteremic Agents; Apolipoprotein A-I; Apolipoproteins E; Atherosclerosis; ATP Binding Cassette Transporter 1; ATP Binding Cassette Transporter, Subfamily G, Member 1; ATP-Binding Cassette Transporters; Calmodulin; Calpain; CD36 Antigens; Cholesterol; Curcumin; Foam Cells; Lipid Metabolism; Lipoproteins; Lipoproteins, LDL; Liver X Receptors; Mice; Mice, Mutant Strains; Orphan Nuclear Receptors; Proteasome Endopeptidase Complex; Scavenger Receptors, Class A; Scavenger Receptors, Class B

There is a known inverse association between type 2 diabetes (T2D) and prostate cancer (PrCa) that is poorly understood. Genetic studies of the T2D-PrCa association may provide insight into the underlying mechanisms of this association. We evaluated associations in the Atherosclerosis Risk in Communities study between PrCa and nine T2D single nucleotide polymorphisms from genome-wide association studies of T2D (in CDKAL1, CDKN2A/B, FTO, HHEX, IGF2BP2, KCNJ11, PPARG, SLC30A8, and TCF7L2) and four T2D single nucleotide polymorphisms from pre-genome-wide association studies (in ADRB2, CAPN10, SLC2A2, and UCP2). From 1987 to 2000, there were 397 incident PrCa cases among 6,642 men ages 45 to 64 years at baseline. We used race-adjusted Cox proportional hazards models to estimate associations between PrCa and increasing number of T2D risk-raising alleles. PrCa was positively associated with the CAPN10 rs3792267 G allele [hazard ratio (HR) 1.20; 95% confidence interval (CI), 1.00-1.44] and inversely associated with the SLC2A2 rs5400 Thr110 allele (HR, 0.85; 95% CI, 0.72, 1.00), the UCP2 rs660339 Val55 allele (HR, 0.84; 95% CI, 0.73, 0.97) and the IGF2BP2 rs4402960 T allele (HR, 0.79; 95% CI, 0.61-1.02; blacks only). The TCF7L2 rs7903146 T allele was inversely associated with PrCa using a dominant genetic model (HR, 0.79; 95% CI, 0.65-0.97). Further knowledge of T2D gene-PrCa mechanisms may improve understanding of PrCa etiology.

Topics: Atherosclerosis; Calpain; Cohort Studies; Diabetes Mellitus, Type 2; Genetic Predisposition to Disease; Genome-Wide Association Study; Glucose Transporter Type 2; Humans; Ion Channels; Male; Middle Aged; Mitochondrial Proteins; Polymorphism, Single Nucleotide; Proportional Hazards Models; Prostatic Neoplasms; RNA-Binding Proteins; TCF Transcription Factors; Transcription Factor 7-Like 2 Protein; Uncoupling Protein 2

Apoptosis is a key feature of advanced atherosclerotic plaques. Attraction signals such as p43 released from apoptotic cells play a crucial role in the timely removal of the apoptotic remnants by recruiting fresh phagocytes. Here, we sought to determine whether p43 may link apoptosis to inflammation and plaque progression.. RT-PCR and immunohistochemistry showed that p43 was abundantly expressed in human plaques compared with nonatherosclerotic mammary arteries and colocalized with splicing factor SC-35. Cell culture experiments indicated that p43 expression was associated with enhanced protein translation. On initiation of apoptosis or necrosis, p43 was cleaved by calpains and released as truncated protein p43(apoptosis-released factor [ARF]). Processing of p43 into endothelial monocyte activating polypeptide II was not observed. Full-length p43, but not p43(ARF) or endothelial monocyte activating polypeptide II, activated THP1 monocytes (upregulation of tumor necrosis factor alpha, interleukin 1 beta, interleukin 8, macrophage inflammatory protein (MIP)-1 alpha, MIP1 beta, MIP2 alpha) and endothelial cells (enhanced synthesis of E-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, tissue factor). The chemotactic activity of p43 or fragments thereof was poor compared with ATP. Treatment of smooth muscle cells with p43 did not induce cell death.. p43 is cleaved during apoptosis by calpains and released as a truncated protein that is harmless for the structure of the plaque.

Topics: Animals; Apolipoproteins E; Apoptosis; Atherosclerosis; Calpain; Carotid Artery Diseases; Case-Control Studies; Cells, Cultured; Coronary Artery Disease; Cytokines; Disease Models, Animal; Disease Progression; Endothelial Cells; Female; Humans; Immunohistochemistry; Inflammation; Inflammation Mediators; Male; Mice; Mice, Knockout; Monocytes; Myocytes, Smooth Muscle; Neoplasm Proteins; Nuclear Proteins; Protein Biosynthesis; Protein Processing, Post-Translational; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleoproteins; RNA-Binding Proteins; RNA, Messenger; Serine-Arginine Splicing Factors; Time Factors; Transfection; Up-Regulation

Accumulation of foam cells in the intima is a hallmark of early-stage atherosclerotic lesions. Ginkgo biloba extract (EGb761) has been reported to exert anti-oxidative and anti-inflammatory properties in atherosclerosis, yet the significance and the molecular mechanisms of action of EGb761 in the formation of macrophage foam cells are not fully understood.. Treatment with EGb761 resulted in a dose-dependent decrease in oxidized low-density lipoprotein (oxLDL)-mediated cholesterol accumulation in macrophages, a consequence that was due to a decrease in cholesterol uptake and an increase in cholesterol efflux. Additionally, EGb761 significantly down-regulated the mRNA and protein expression of class A scavenger receptor (SR-A) by decreasing expression of activator protein 1 (AP-1); however, EGb761 increased the protein stability of ATP-binding cassette transporter A1 (ABCA1) by reducing calpain activity without affecting ABCA1 mRNA expression. Small interfering RNA (siRNA) targeting haem oxygenase-1 (HO-1) abolished the EGb761-induced protective effects on the expression of AP-1, SR-A, ABCA1, and calpain activity. Accordingly, EGb761-mediated suppression of lipid accumulation in foam cells was also abrogated by HO-1 siRNA. Moreover, the lesion size of atherosclerosis was smaller in EGb761-treated, apolipoprotein E-deficient mice compared with the vehicle-treated mice, and the expression of HO-1, SR-A, and ABCA1 in aortas was modulated similar to that observed in macrophages.. These findings suggest that EGb761 confers a protection from the formation of foam cells by a novel HO-1-dependent regulation of cholesterol homeostasis in macrophages.

Topics: Animals; Apolipoproteins E; Atherosclerosis; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; Calpain; Cholesterol; Disease Models, Animal; Dose-Response Relationship, Drug; Foam Cells; Ginkgo biloba; Heme Oxygenase-1; Homeostasis; Lipid Metabolism; Macrophages; Mice; Mice, Knockout; Plant Extracts; Scavenger Receptors, Class A

Expression of ABCA1 is regulated by transcription of the gene and calpain-mediated proteolytic degradation, and inhibition ABCA1 degradation results in increased ABCA1 and HDL biogenesis in vitro. We examined whether this approach could be a potential antiatherogenic treatment. Although probucol inhibits both the activity and degradation of ABCA1, its oxidized products, spiroquinone and diphenoquinone, reduce degradation of ABCA1 without inhibiting its activity or altering transcription of the ABCA1 gene. Accordingly, both compounds enhanced apolipoprotein A-I/ABCA1-dependent generation of HDL in vitro, and increased hepatic ABCA1 and plasma HDL without increasing antioxidant activity in plasma when given to rabbits. Both compounds also decreased vascular lipid deposition in cholesterol-fed rabbits. We therefore conclude that stabilization of ABCA1 against calpain-mediated degradation is a novel and potentially important strategy to increase HDL formation and prevent atherosclerosis. Spiroquinone and diphenoquinone are potential seeds for development of such drugs.

Topics: Animals; Anticholesteremic Agents; Antioxidants; Apolipoprotein A-I; Atherosclerosis; ATP Binding Cassette Transporter 1; ATP-Binding Cassette Transporters; BALB 3T3 Cells; Blotting, Western; Calpain; Cell Line; Cholesterol, HDL; Green Fluorescent Proteins; Humans; Lipoproteins, HDL; Macrophages; Male; Mice; Microscopy, Confocal; Probucol; Quinones; Rabbits; Reverse Transcriptase Polymerase Chain Reaction; Transfection

The extracellular matrix (ECM) of the atherosclerotic lesion is a crucial determinant of its stability, while its degradation by matrix metalloproteinases (MMPs) has been implied in plaque rupture. As accumulation of both MMP-derived collagen fragments and apoptotic smooth muscle cells (SMC) is observed at sites of plaque rupture, we tested the effect of polymerized and degraded type I collagen on the susceptibility of SMC to apoptosis.. Human SMC were cultured on monomeric or polymerized collagen, and collagen gels were degraded by collagenase. Apoptosis was evaluated using antibodies to active caspases and their substrates. Calpain and caspase activity were measured using fluorogenic substrates.. Culture of SMC on polymerized collagen led to increased apoptosis compared to culture on monomeric collagen. In addition, we observed a distinct proteolytic degradation of the endogenous caspase inhibitor X-chromosome-linked inhibitor of apoptosis (xIAP). As MMP-1 was strongly activated in SMC on polymerized collagen, we examined the effect of degraded collagen fragments on xIAP cleavage and apoptosis. Degraded collagen induced rapid proteolytic processing of xIAP identical to that on polymerized collagen. We identified calpains as the proteolytic enzymes responsible for xIAP processing as: i) they were rapidly activated by degraded collagen; ii) recombinant calpain II processed xIAP in an identical manner, and iii) inhibition of calpains by BAPTA or calpeptin abrogated xIAP degradation in intact cells. The functional consequence of xIAP processing by calpains was a loss of its caspase-inhibitory potential. Calpain activation distinctly preceded caspase activation, and inhibition of calpains suppressed apoptosis.. Collagen fragments proteolytically released from the ECM by MMPs may propagate apoptosis of SMC by calpain-mediated inactivation of anti-apoptotic proteins such as xIAP. This may be a novel mechanism of SMC apoptosis in biological settings of enhanced collagen degradation such as vascular remodeling, neointima formation, and atherosclerotic plaque rupture.

Topics: Apoptosis; Atherosclerosis; Calpain; Caspases; Cells, Cultured; Collagen; Collagenases; Dipeptides; Egtazic Acid; Enzyme Inhibitors; Extracellular Matrix; Gels; Humans; Microscopy, Fluorescence; Muscle, Smooth, Vascular; Polymers; Recombinant Proteins; X-Linked Inhibitor of Apoptosis Protein