| 1. |
- Ares, Isabella, et al.
(författare)
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Oxidized low-density lipoprotein induces calpain-dependent cell death and ubiquitination of caspase 3 in HMEC-1 endothelial cells.
- 2003
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Ingår i: Biochemical Journal. - 1470-8728. ; 374:Pt 2, s. 403-411
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Tidskriftsartikel (refereegranskat)abstract
- Oxidized low-density lipoprotein (oxLDL) is known to induce apoptosis in endothelial cells, and this is believed to contribute to the progression of atherosclerosis. In the present study we made the novel observation that oxLDL-induced death of HMEC-1 cells is accompanied by activation of calpain. The mu-calpain inhibitor PD 151746 decreased oxLDL-induced cytotoxicity, whereas the general caspase inhibitor BAF (t-butoxycarboryl-Asp-methoxyfluoromethylketone) had no effect. Also, oxLDL provoked calpain-dependent proteolysis of cytoskeletal a-fodrin in the HMEC-1 cells. Our observation of an autoproteolytic cleavage of the 80 kDa subunit of mu-calpain provided further evidence for an oxLDL-indunced stimulation of calpain activity. The Bcl-2 protein Bid was also cleaved during oxLDL-elicited cell death, and this was prevented by calpain inhibitors, but not by inhibitors of cathepsin B and caspases. Treating the HMEC-1 cells with oxLDL did not result in detectable activation of procaspase 3 or cleavage of PARP [poly(ADP-ribose) polymerase], but it did cause polyubiquitination of caspase 3, indicating inactivation and possible degradation of this protease. Despite the lack of caspase 3 activation, oxLDL treatment led to the formation of nucleosomal DNA fragments characteristic of apoptosis. These novel results show that oxLDL initiates a calpain-mediated death-signalling pathway in endothelial cells.
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| 2. |
- Goncalves, Isabel, et al.
(författare)
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Activation of calpain-1 in human carotid artery atherosclerotic lesions
- 2009
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Ingår i: BMC Cardiovascular Disorders. - : Springer Science and Business Media LLC. - 1471-2261. ; 9:26
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Tidskriftsartikel (refereegranskat)abstract
- Background: In a previous study, we observed that oxidized low-density lipoprotein-induced death of endothelial cells was calpain-1-dependent. The purpose of the present paper was to study the possible activation of calpain in human carotid plaques, and to compare calpain activity in the plaques from symptomatic patients with those obtained from patients without symptoms. Methods: Human atherosclerotic carotid plaques (n = 29, 12 associated with symptoms) were removed by endarterectomy. Calpain activity and apoptosis were detected by performing immunohistochemical analysis and TUNEL assay on human carotid plaque sections. An antibody specific for calpain-proteolyzed alpha-fodrin was used on western blots. Results: We found that calpain was activated in all the plaques and calpain activity colocalized with apoptotic cell death. Our observation of autoproteolytic cleavage of the 80 kDa subunit of calpain-1 provided further evidence for enzyme activity in the plaque samples. When calpain activity was quantified, we found that plaques from symptomatic patients displayed significantly lower calpain activity compared with asymptomatic plaques. Conclusion: These novel results suggest that calpain-1 is commonly active in carotid artery atherosclerotic plaques, and that calpain activity is colocalized with cell death and inversely associated with symptoms.
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| 3. |
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| 4. |
- Reeve, Janice L. V., et al.
(författare)
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OxLDL-induced gene expression patterns in CASMC are mimicked in apoE(-/-) mice aortas
- 2007
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Ingår i: Biochemical and Biophysical Research Communications. - : Elsevier BV. - 1090-2104 .- 0006-291X. ; 356:3, s. 681-686
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Tidskriftsartikel (refereegranskat)abstract
- Oxidized low density lipoprotein (oxLDL) contributes to the pathophysiology of atherosclerosis, partly by altering gene expression in vascular cells. Here, we show 221 genes differentially regulated by oxLDL in coronary artery smooth muscle cells (CASMC), using oligonucleotide microarrays. These genes were classified into 14 functional groups. A comparable gene expression pattern was detected in apoE(-/-) mice. OxLDL induced an oxidative stress response in CASMC, but not the unfolded protein response. OxLDL also caused CASMC death which was accompanied by increased expression of FasL, Bax, and p53 but was caspase-independent. This approach provides further insight into disease pathology and prognosis. (c) 2007 Elsevier Inc. All rights reserved.
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| 5. |
- Stollenwerk, Maria M, 1959-, et al.
(författare)
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Very low-density lipoprotein induces interleukin-1beta expression in macrophages
- 2005
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier. - 0006-291X .- 1090-2104. ; 335:2, s. 603-608
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Tidskriftsartikel (refereegranskat)abstract
- Elevated plasma level of very low-density lipoprotein (VLDL) is a risk factor for coronary heart disease. We investigated the effect of VLDL on expression of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) in human peripheral blood monocyte-derived macrophages. IL-1beta mRNA and protein expression was analysed by PCR and ELISA, respectively. Caspase activation was assessed by immunoblotting. Apart from potentiating lipopolysaccharide-induced secretion of IL-1beta, VLDL alone induced secretion of IL-1beta from human monocyte-derived macrophages. This effect was suppressed by an inhibitor of caspase-1, the protease which cleaves pro-IL-1beta. VLDL treatment activated caspase-1, as indicated by increased levels of the caspase-1 p20 subunit. Furthermore, VLDL increased IL-1beta mRNA expression, which was associated with activation of transcription factor AP-1. Inhibition of caspase-1 did not influence IL-1beta mRNA expression. In conclusion, VLDL induces IL-1beta mRNA expression, caspase-1 activation, and IL-1beta release from macrophages, suggesting that VLDL can promote inflammation in atherosclerotic lesions.
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| 6. |
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| 7. |
- Ares, Mikko, et al.
(författare)
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Decreased inducibility of TNF expression in lipid-loaded macrophages.
- 2002
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Ingår i: BMC Immunology. - 1471-2172. ; 3:1, s. 13-13
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Inflammation and immune responses are considered to be very important in the pathogenesis of atherosclerosis. Lipid accumulation in macrophages of the arterial intima is a characteristic feature of atherosclerosis which can influence the inflammatory potential of macrophages. We studied the effects of lipid loading on the regulation of TNF expression in human monocyte-derived macrophages. RESULTS: In macrophages incubated with acetylated low density lipoprotein (ac-LDL) for 2 days, mRNA expression of TNF in cells stimulated with TNF decreased by 75%. In cell cultures stimulated over night with IL-1beta, lipid loading decreased secretion of TNF into culture medium by 48%. These results suggest that lipid accumulation in macrophages makes them less responsive to inflammatory stimuli. Decreased basal activity and inducibility of transcription factor AP-1 was observed in lipid-loaded cells, suggesting a mechanism for the suppression of cytokine expression. NF-kappaB binding activity and inducibility were only marginally affected by ac-LDL. LDL and ac-LDL did not activate PPARgamma. In contrast, oxidized LDL stimulated AP-1 and PPARgamma but inhibited NF-kappaB, indicating that the effects of lipid loading with ac-LDL were not due to oxidation of lipids. CONCLUSIONS: Accumulation of lipid, mainly cholesterol, results in down-regulation of TNF expression in macrophages. Since monocytes are known to be activated by cell adhesion, these results suggest that foam cells in atherosclerotic plaques may contribute less potently to an inflammatory reaction than newly arrived monocytes/macrophages.
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| 8. |
- Ares, Mikko
(författare)
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Effects of lipid oxidation on transcriptional regulation and cell death
- 1998
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Elevated levels of low density lipoprotein (LDL) in plasma are associated with an increased risk for atherosclerosis. Excessive accumulation of lipoproteins and lipids in the vascular wall is thought to lead to the development of advanced atherosclerotic lesions prone to rupture. In the present investigation, we have studied the cellular effects of oxidized lipids and lipoproteins, as well as lipid accumulation, on the regulation of gene expression. We have also characterized cell death induced by oxysterols, oxidized derivatives of cholesterol, in smooth muscle cells. Oxidized LDL (ox-LDL) inhibited activation of NF-KB, a transcription factor which induces the expression of a large number of genes encoding proteins involved in immune and inflammatory responses. While this effect would seem to be anti-inflammatory, ox-LDL may have other, pro-inflammatory effects, such as activation of the transcription factor AP- 1, which was for the first time demonstrated in the present study. The biologically active component of ox-LDL responsible for AP-1 induction appears to be Iysophosphatidylcholine, while inhibition of NF-KB may be due to other lipid oxidation products such as aldehydes and oxysterols. The effects of ox-LDL on AP-I and NF-KB were similar in smooth muscle cells, endothelial cells and macrophages stimulated with lipopolysaccharide or TNF, whether LDL was oxidized with copper or UV light. Activation of AP-I by ox-LDL was associated with increased TNF mRNA expression and protein secretion in adherent monocytes. Conditioned medium from monocytes treated with ox-LDL stimulated proliferation of smooth muscle cells. Antibodies against TNF abolished 45% of the ox-LDL-dependent mitogenicity of conditioned medium, indicating that TNF is a major smooth muscle cell mitogen secreted by monocytes in response to ox-LDL. Cytotoxic oxysterols are formed during oxidation of LDL. We have shown that several oxysterols can induce apoptosis in smooth muscle cells. Side-chain hydroxylated oxysterols were most potent in inducing apoptosis, but the reasons for this remain to be explained. Both 7ß-hydroxycholesterol and 25-hydroxycholesterol induced intracellular Ca2+ oscillations and activated extracellular signal-regulated protein kinases within a few minutes after addition. Within a few hours, oxysterol treatment caused disorganization, but not swelling, of the ER and Golgi membranes, suggesting that Ca2+ is a critical mediator of oxysterol toxicity. When macrophages were loaded with lipid by incubation in the presence of acetylated LDL, inducibility of AP-I and TNF expression were decreased, suggesting that lipid accumulation and foam cell formation decrease the inflammatory potential of macrophages. NF-KB was unaffected by lipid loading, but ox-LDL suppressed NF-KB activity, demonstrating that the effects of lipid loading on AP-I were not due to lipoprotein oxidation. Taken together, our results support a model for the pathogenesis of atherosclerosis where atherogenic lipoproteins initially stimulate but subsequently suppress cell proliferation and inflammation.
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| 9. |
- Ares, Mikko PS, et al.
(författare)
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Inflammatory effects of very low-density lipoprotein and fatty acids.
- 2006
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Ingår i: Future Cardiology. - : Future medicine. - 1744-8298 .- 1479-6678. ; 2:3, s. 315-323
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Forskningsöversikt (övrigt vetenskapligt/konstnärligt)abstract
- High plasma triacylglycerol (triglyceride, TG) levels is a risk factor for atherosclerosis. Very large lipoproteins, such as chylomicrons, alone are not considered atherogenic, but TG-rich remnant lipoproteins can penetrate into the vascular wall. Importantly, accumulating evidence suggests that all TG-rich lipoproteins stimulate cytokine expression in circulating monocytes. Very low-density lipoprotein (VLDL) stimulates monocyte adhesion to endothelial cells and expression of inflammatory genes in macrophages. Furthermore, fatty acids released from large lipoproteins can stimulate both vascular cells and circulating monocytes. It is likely that fatty acids released from TG-rich lipoproteins contribute to atherogenesis, but the role of fatty acids in ischemic heart disease is not as direct as that of cholesterol. Fatty acids influence plasma lipoprotein levels and either stimulate or suppress numerous cellular functions relevant to atherogenesis. While certain n-3 fatty acids are good for health, most other medium- to long-chain fatty acids appear to promote inflammation in cell culture studies and need to be studied further. Nevertheless, the existing evidence supports the general conclusion that TG-rich lipoproteins and fatty acids greatly accelerate the progression of atherosclerosis. This may be because of their inflammatory effects.
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| 10. |
- Dichtl, W, et al.
(författare)
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HMG-CoA reductase inhibitors regulate inflammatory transcription factors in human endothelial and vascular smooth muscle cells
- 2003
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Ingår i: Arteriosclerosis, Thrombosis and Vascular Biology. - 1524-4636. ; 23:1, s. 58-63
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Tidskriftsartikel (refereegranskat)abstract
- Objective-Pleiotropic atheroprotective effects of HMG-CoA reductase inhibitors may be mediated on the level of vascular gene transcription. The aim of this study was to characterize the effects of statins on the activation of transcription factors known to regulate inflammation and cell proliferation/differentiation. Methods and Results-Simvastatin, atorvastatin, and lovastatin (0.1 to 10 mumol/L) inhibited the binding of nuclear proteins to both the nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) DNA consensus oligonucleotides in human endothelial and vascular smooth muscle cells as assessed by electrophoretic mobility shift assay (EMSA). The inhibitory effects of statins on NF-kappaB or AP-1-dependent transcriptional activity were examined by transient transfection studies. HMG-CoA reductase inhibitors upregulated IkappaB-alpha protein levels in endothelial cells and decreased c-Jun mRNA expression in smooth muscle cells as analyzed by Western and Northern blotting, respectively. Furthermore, statins inhibited DNA binding of hypoxia-inducible factor-1alpha. Downstream effects of statins included inhibition of plasminogen activator inhibitor-1 and vascular endothelial growth factor-A mRNA levels in endothelial cells. Conclusions-HMG-CoA reductase inhibitors downregulate the activation of transcription factors NF-kappaB, AP-1, and hypoxia-inducible factor-1alpha. These, findings support the concept that statins have antiinflammatory and antiproliferative effects that are relevant in the treatment of atherosclerotic diseases.
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