| 1. |
- Englund, Mikael C. O., 1971
(författare)
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Regulation of human macrophage gene expression by oxidized low density lipoprotein. Studies on tumor necrosis factor-a and global gene expression profiles
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Atherosclerosis is a multifactorial and progressive disease that is characterized by a focal thickening of the arteries caused by accumulation of lipoproteins, extracellular matrix, and the migration and proliferation of various cell types. Among the cells found in atherosclerotic lesions are macrophages, which accumulate lipids and are transformed into foam cells. The atherosclerotic lesion is also characterized as a site of inflammation. In vivo and in vitro data support the role of macrophages in the development of an inflammatory response in the vessel wall. Several pro-inflammatory cytokines have been detected in the atherosclerotic plaque, among those are tumor necrosis factor-a (TNF-a). Major sources of TNF-a are activated macrophages. Macrophages have been connected to expression of several cytokines in the atherosclerotic plaque that can modulate plaque development.Oxidative modification of lipoproteins is regarded as a key event in the development of atherosclerosis. The oxidation of the low density lipoprotein (LDL) particle produces several biologically active molecules. Among the products formed are oxysterols, which are found in atherosclerotic plaques. Oxysterols have been implicated in the development of atherosclerosis. The main aim of this thesis was to investigate the impact of oxidized LDL (oxLDL) and oxysterols on gene expression in human macrophages.The results obtained in this thesis demonstrate that oxLDL and oxysterols, in particular 25-hydroxyxcholesterol (25-OH), modulates expression of TNF-a. OxLDL decreased the lipopolysaccharide (LPS)-induced DNA binding of the transcription factor nuclear factor kappaB (NF-kB) to the TNF-a promoter. This suggests that oxLDL can decrease the inflammatory response in macrophages. Further, it was demonstrated that 25-OH could decrease an LPS-induced TNF-a secretion, but also that 25-OH together with interferon-gamma (IFN-g) could contribute to an increased expression of TNF-a. 25-OH affects both transcriptional and post-transcriptional events including kinase activity, and also intracellular levels of H2O2 that could influence gene expression. Microarray experiments suggest that oxLDL induces a coordinated stress response, activates detoxification enzymes, as well as the more expected changes in lipid and cholesterol metabolism in macrophages.In conclusion, this thesis shows that components of oxLDL can influence expression of genes in macrophages, which could contribute to the development of atherosclerosis.
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| 2. |
- Englund, Mikael C. O., 1971, et al.
(författare)
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The establishment of 20 different human embryonic stem cell lines and subclones; a report on derivation, culture, characterisation and banking.
- 2010
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Ingår i: In vitro cellular & developmental biology. Animal. - : Springer Science and Business Media LLC. - 1543-706X .- 1071-2690. ; 46:3-4, s. 217-30
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Tidskriftsartikel (refereegranskat)abstract
- This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means.
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| 3. |
- Hansson, Mattias, et al.
(författare)
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Artifactual insulin release from differentiated embryonic stem cells.
- 2004
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Ingår i: Diabetes. - 0012-1797. ; 53:10, s. 2603-9
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Tidskriftsartikel (refereegranskat)abstract
- Several recent reports claim the generation of insulin-producing cells from embryonic stem cells via the differentiation of progenitors that express nestin. Here, we investigate further the properties of these insulin-containing cells. We find that although differentiated cells contain immunoreactive insulin, they do not contain proinsulin-derived C-peptide. Furthermore, we find variable insulin release from these cells upon glucose addition, but C-peptide release is never detected. In addition, many of the insulin-immunoreactive cells are undergoing apoptosis or necrosis. We further show that cells cultured in the presence of a phosphoinositide 3-kinase inhibitor, which previously was reported to facilitate the differentiation of insulin(+) cells, are not C-peptide immunoreactive but take up fluorescein isothiocyanate-labeled insulin from the culture medium. Together, these data suggest that nestin(+) progenitor cells give rise to a population of cells that contain insulin, not as a result of biosynthesis but from the uptake of exogenous insulin. We conclude that C-peptide biosynthesis and secretion should be demonstrated to claim insulin production from embryonic stem cell progeny.
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| 4. |
- Heins, Nico, et al.
(författare)
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Derivation, characterization, and differentiation of human embryonic stem cells.
- 2004
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Ingår i: Stem cells (Dayton, Ohio). - 1066-5099. ; 22:3, s. 367-76
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Tidskriftsartikel (refereegranskat)abstract
- The derivation of human embryonic stem (hES) cells establishes a new avenue to approach many issues in human biology and medicine for the first time. To meet the increased demand for characterized hES cell lines, we present the derivation and characterization of six hES cell lines. In addition to the previously described immunosurgery procedure, we were able to propagate the inner cell mass and establish hES cell lines from pronase-treated and hatched blastocysts. The cell lines were extensively characterized by expression analysis of markers characteristic for undifferentiated and differentiated hES cells, karyotyping, telomerase activity measurement, and pluripotency assays in vitro and in vivo. Whereas three of the cell lines expressed all the characteristics of undifferentiated pluripotent hES cells, one cell line carried a chromosome 13 trisomy while maintaining an undifferentiated pluripotent state, and two cell lines, one of which carried a triploid karyotype, exhibited limited pluripotency in vivo. Furthermore, we clonally derived one cell line, which could be propagated in an undifferentiated pluripotent state.
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| 5. |
- Hägg, Daniel, 1974, et al.
(författare)
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Oxidized LDL induces a coordinated up-regulation of the glutathione and thioredoxin systems in human macrophages.
- 2006
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Ingår i: Atherosclerosis. - : Elsevier BV. - 0021-9150 .- 1879-1484. ; 185:2, s. 282-9
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Tidskriftsartikel (refereegranskat)abstract
- Using DNA microarray analysis, we found that human macrophages respond to oxidized low-density lipoprotein (oxLDL) by activating the antioxidative glutathione and thioredoxin systems. Several genes of the glutathione and thioredoxin systems were expressed at high levels in macrophages when compared to 80 other human tissues and cell types, indicating that these systems may be of particular importance in macrophages. The up-regulation of three genes in these systems, thioredoxin (P < 0.005), thioredoxin reductase 1 (P < 0.001) and glutathione reductase (P < 0.001) was verified with real-time RT-PCR, using human macrophages from 10 healthy donors. To investigate the possible role of these antioxidative systems in the development of atherosclerosis, expression levels in macrophages from 15 subjects with atherosclerosis (12 men, 3 women) and 15 matched controls (12 men, 3 women) were analyzed using DNA microarrays. Two genes in the glutathione system Mn superoxide dismutase (P < 0.05) and catalase (P < 0.05) differed in expression between the groups. We conclude that macrophage uptake of oxidized LDL induces a coordinated up-regulation of genes of the glutathione and thioredoxin systems, suggesting that these systems may participate in the cellular defense against oxidized LDL and possibly modulate the development of atherosclerosis.
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| 6. |
- Svensson, Per-Arne, 1969, et al.
(författare)
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Copper induces the expression of cholesterogenic genes in human macrophages.
- 2003
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Ingår i: Atherosclerosis. - 0021-9150. ; 169:1, s. 71-6
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Tidskriftsartikel (refereegranskat)abstract
- Accumulation of lipids and cholesterol by macrophages and subsequent transformation into foam cells are key features in development of atherosclerosis. Serum copper concentrations have been shown to be associated with cardiovascular disease. However, the mechanism behind the proatherogenic effect of copper is not clear. We used DNA microarrays to define the changes in gene expression profile in response to copper exposure of human macrophages. Expression monitoring by DNA microarray revealed 91 genes that were regulated. Copper increased the expression of seven cholesterogenic genes (3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase, IPP isomerase, squalene synthase, squalene epoxidase, methyl sterol oxidase, H105e3 mRNA and sterol-C5-desaturase) and low-density lipoprotein receptor (LDL-R), and decreased the expression of CD36 and lipid binding proteins. The expression of LDL-R and HMG CoA reductase was also investigated using real time PCR. The expression of both of these genes was increased after copper treatment of macrophages (P<0.01 and P<0.01, respectively). We conclude that copper activates cholesterogenic genes in macrophages, which may provide a mechanism for the association between copper and atherosclerosis. The effect of copper on cholesterogenic genes may also have implications for liver steatosis in early stages of Wilson's disease.
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| 7. |
- Svensson, Per Anders, 1959, et al.
(författare)
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Identification of genes predominantly expressed in human macrophages
- 2004
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Ingår i: Atherosclerosis. - : Elsevier BV. ; 177, s. 287-290
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Tidskriftsartikel (refereegranskat)abstract
- Identification of cell and tissue specific genes may provide novel insights to signaling systems and functions. Macrophages play a key role in many diseases including atherosclerosis. Using DNA microarrays we compared the expression of approximately 10,000 genes in 56 human tissues and identified 23 genes with predominant expression in macrophages. The identified genes include both genes known to be macrophage specific and genes previously not well described in this cell type. Tissue distribution of two genes, liver X receptor (LXR) alpha and interleukin-1 receptor antagonist (IL1RN), was verified by real-time RT-PCR. We conclude that comparison of expression profiles from a large number of tissues can be used to identify genes that are predominantly expressed in certain tissues. Identification of novel macrophage specific genes may increase our understanding of the role of this cell in different diseases.
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| 8. |
- Svensson, Per-Arne, 1969, et al.
(författare)
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Major role of HSP70 as a paracrine inducer of cytokine production in human oxidized LDL treated macrophages.
- 2006
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Ingår i: Atherosclerosis. - : Elsevier BV. - 0021-9150. ; 185:1, s. 32-8
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Tidskriftsartikel (refereegranskat)abstract
- Lipid accumulation and inflammation are key hallmarks of the atherosclerotic plaque and macrophage uptake of oxidized low-density lipoprotein (oxLDL) is believed to drive these processes. Initial experiments show that supernatants from oxLDL treated macrophages could induce IL-1beta production in naïve macrophages. To search for potential paracrine mediators that could mediate this effect a DNA microarray scan of oxLDL treated human macrophages was performed. This analysis revealed that oxLDL induced activation of heat shock protein (HSP) expression. HSPs have been implicated in the development of atherosclerosis, but the exact mechanisms for this is unclear. Extracellular heat shock protein 70 (HSP70) has been shown to elicit a pro-inflammatory cytokine response in monocytes and could therefore be a potential paracrine pro-inflammatory mediator. After 24 h of oxLDL treatment there was a significant increase of HSP70 concentrations in supernatants from oxLDL treated macrophages (oxLDLsup) compared to untreated controls (P<0.05). OxLDLsup could induce both interleukin (IL)-1beta and IL-12 secretion in naïve macrophages. We also demonstrate that the effect of oxLDLsup on cytokine production and release could be blocked by inhibition of HSP70 transcription or secretion or by the use of HSP70 neutralizing antibodies. This suggests that extracellular HSP70 can mediate pro-inflammatory changes in macrophages in response to oxLDL.
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| 9. |
- Svensson, Per-Arne, 1969, et al.
(författare)
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Regulation and splicing of scavenger receptor class B type I in human macrophages and atherosclerotic plaques
- 2005
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Ingår i: BMC Cardiovasc Disord. - : Springer Science and Business Media LLC. - 1471-2261. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The protective role of high-density lipoprotein (HDL) in the cardiovascular system is related to its role in the reverse transport of cholesterol from the arterial wall to the liver for subsequent excretion via the bile. Scavenger receptor class B type I (SR-BI) binds HDL and mediates selective uptake of cholesterol ester and cellular efflux of cholesterol to HDL. The role of SR-BI in atherosclerosis has been well established in murine models but it remains unclear whether SR-BI plays an equally important role in atherosclerosis in humans. The aim of this study was to investigate the expression of SR-BI and its isoforms in human macrophages and atherosclerotic plaques. METHODS: The effect of hypoxia and minimally modified low-density lipoprotein (mmLDL), two proatherogenic stimuli, on SR-BI expression was studied in human monocyte-derived macrophages from healthy subjects using real-time PCR. In addition, SR-BI expression was determined in macrophages obtained from subjects with atherosclerosis (n = 15) and healthy controls (n = 15). Expression of SR-BI isoforms was characterized in human atherosclerotic plaques and macrophages using RT-PCR and DNA sequencing. RESULTS: SR-BI expression was decreased in macrophages after hypoxia (p < 0.005). In contrast, SR-BI expression was increased by exposure to mmLDL (p < 0.05). There was no difference in SR-BI expression in macrophages from patients with atherosclerosis compared to controls. In both groups, SR-BI expression was increased by exposure to mmLDL (p < 0.05). Transcripts corresponding to SR-BI and SR-BII were detected in macrophages. In addition, a third isoform, referred to as SR-BIII, was discovered. All three isoforms were also expressed in human atherosclerotic plaque. Compared to the other isoforms, the novel SR-BIII isoform was predicted to have a unique intracellular C-terminal domain containing 53 amino acids. CONCLUSION: We conclude that SR-BI is regulated by proatherogenic stimuli in humans. However, we found no differences between subjects with atherosclerosis and healthy controls. This indicates that altered SR-BI expression is not a common cause of atherosclerosis. In addition, we identified SR-BIII as a novel isoform expressed in human macrophages and in human atherosclerotic plaques.
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| 10. |
- Synnergren, Jane, 1967, et al.
(författare)
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Cardiomyogenic gene expression profiling of differentiating human embryonic stem cells.
- 2008
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Ingår i: Journal of biotechnology. - : Elsevier BV. - 0168-1656 .- 1873-4863. ; 134:1-2, s. 162-70
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem cells (hESCs) can differentiate into a variety of specialized cell types. Thus, they provide a model system for embryonic development to investigate the molecular processes of cell differentiation and lineage commitment. The development of the cardiac lineage is easily detected in mixed cultures by the appearance of spontaneously contracting areas of cells. We performed gene expression profiling of undifferentiated and differentiating hESCs and monitored 468 genes expressed during cardiac development and/or in cardiac tissue. Their transcription during early differentiation of hESCs through embryoid bodies (EBs) was investigated and compared with spontaneously differentiating hESCs maintained on feeders in culture without passaging (high-density (HD) protocol). We observed a larger variation in the gene expression between cells from a single cell line that were differentiated using two different protocols than in cells from different cell lines that were cultured according to the same protocol. Notably, the EB protocol resulted in more reproducible transcription profiles than the HD protocol. The results presented here provide new information about gene regulation during early differentiation of hESCs with emphasis on the cardiomyogenic program. In addition, we also identified regulatory elements that could prove critical for the development of the cardiomyocyte lineage.
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