1. |
- Andersson, Linda, 1973, et al.
(author)
-
Glucosylceramide synthase deficiency in the heart compromises β1-adrenergic receptor trafficking
- 2021
-
In: European Heart Journal. - : Oxford University Press. - 0195-668X .- 1522-9645. ; 42:43, s. 4481-4492
-
Journal article (peer-reviewed)abstract
- AIMS: Cardiac injury and remodelling are associated with the rearrangement of cardiac lipids. Glycosphingolipids are membrane lipids that are important for cellular structure and function, and cardiac dysfunction is a characteristic of rare monogenic diseases with defects in glycosphingolipid synthesis and turnover. However, it is not known how cardiac glycosphingolipids regulate cellular processes in the heart. The aim of this study is to determine the role of cardiac glycosphingolipids in heart function.METHODS AND RESULTS: Using human myocardial biopsies, we showed that the glycosphingolipids glucosylceramide and lactosylceramide are present at very low levels in non-ischaemic human heart with normal function and are elevated during remodelling. Similar results were observed in mouse models of cardiac remodelling. We also generated mice with cardiomyocyte-specific deficiency in Ugcg, the gene encoding glucosylceramide synthase (hUgcg-/- mice). In 9- to 10-week-old hUgcg-/- mice, contractile capacity in response to dobutamine stress was reduced. Older hUgcg-/- mice developed severe heart failure and left ventricular dilatation even under baseline conditions and died prematurely. Using RNA-seq and cell culture models, we showed defective endolysosomal retrograde trafficking and autophagy in Ugcg-deficient cardiomyocytes. We also showed that responsiveness to β-adrenergic stimulation was reduced in cardiomyocytes from hUgcg-/- mice and that Ugcg knockdown suppressed the internalization and trafficking of β1-adrenergic receptors.CONCLUSIONS: Our findings suggest that cardiac glycosphingolipids are required to maintain β-adrenergic signalling and contractile capacity in cardiomyocytes and to preserve normal heart function.
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2. |
- Kijani, Siavash, et al.
(author)
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Filter-Dense Multicolor Microscopy
- 2015
-
In: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 10:3
-
Journal article (peer-reviewed)abstract
- Immunofluorescence microscopy is a unique method to reveal the spatial location of proteins in tissues and cells. By combining antibodies that are labeled with different fluorochromes, the location of several proteins can simultaneously be visualized in one sample. However, because of the risk of bleed-through signals between fluorochromes, standard multicolor microscopy is restricted to a maximum of four fluorescence channels, including one for nuclei staining. This is not always enough to address common scientific questions. In particular, the use of a rapidly increasing number of marker proteins to classify functionally distinct cell populations and diseased tissues emphasizes the need for more complex multistainings. Hence, multicolor microscopy should ideally offer more channels to meet the current needs in biomedical science. Here we present an enhanced multi-fluorescence setup, which we call Filter-Dense Multicolor Microscopy (FDMM). FDMM is based on condensed filter sets that are more specific for each fluorochrome and allow a more economic use of the light spectrum. FDMM allows at least six independent fluorescence channels and can be applied to any standard fluorescence microscope without changing any operative procedures for the user. In the present study, we demonstrate an FDMM setup of six channels that includes the most commonly used fluorochromes for histology. We show that the FDMM setup is specific and robust, and we apply the technique on typical biological questions that require more than four fluorescence microscope channels.
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3. |
- Krishnaswamy, Jayendra Kumar, et al.
(author)
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Migratory CD11b+ conventional dendritic cells induce T follicular helper cell-dependent antibody responses.
- 2017
-
In: Science immunology. - : American Association for the Advancement of Science (AAAS). - 2470-9468. ; 2:18
-
Journal article (peer-reviewed)abstract
- T follicular helper (Tfh) cells are a subset of CD4+ T cells that promote antibody production during vaccination. Conventional dendritic cells (cDCs) efficiently prime Tfh cells; however, conclusions regarding which cDC instructs Tfh cell differentiation have differed between recent studies. We found that these discrepancies might exist because of the unusual sites used for immunization in murine models, which differentially bias which DC subsets access antigen. We used intranasal immunization as a physiologically relevant route of exposure that delivers antigen to all tissue DC subsets. Using a combination of mice in which the function of individual DC subsets is impaired and different antigen formulations, we determined that CD11b+ migratory type 2 cDCs (cDC2s) are necessary and sufficient for Tfh induction. DC-specific deletion of the guanine nucleotide exchange factor DOCK8 resulted in an isolated loss of CD11b+ cDC2, but not CD103+ cDC1, migration to lung-draining lymph nodes. Impaired cDC2 migration or development in DC-specific Dock8 or Irf4 knockout mice, respectively, led to reduced Tfh cell priming, whereas loss of CD103+ cDC1s in Batf3-/- mice did not. Loss of cDC2-dependent Tfh cell priming impaired antibody-mediated protection from live influenza virus challenge. We show that migratory cDC2s uniquely carry antigen into the subanatomic regions of the lymph node where Tfh cell priming occurs-the T-B border. This work identifies the DC subset responsible for Tfh cell-dependent antibody responses, particularly when antigen dose is limiting or is encountered at a mucosal site, which could ultimately inform the formulation and delivery of vaccines.
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4. |
- Levin, Malin, 1973, et al.
(author)
-
Rip2 deficiency leads to increased atherosclerosis despite decreased inflammation.
- 2011
-
In: Circulation research. - 1524-4571. ; 109:11, s. 1210-8
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Journal article (peer-reviewed)abstract
- The innate immune system and in particular the pattern-recognition receptors Toll-like receptors have recently been linked to atherosclerosis. Consequently, inhibition of various signaling molecules downstream of the Toll-like receptors has been tested as a strategy to prevent progression of atherosclerosis. Receptor-interacting protein 2 (Rip2) is a serine/threonine kinase that is involved in multiple nuclear factor-κB (NFκB) activation pathways, including Toll-like receptors, and is therefore an interesting potential target for pharmaceutical intervention.
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5. |
- Nilton, Anna, et al.
(author)
-
Targeting Zfp148 activates p53 and reduces tumor initiation in the gut
- 2016
-
In: OncoTarget. - : Impact Journals, LLC. - 1949-2553. ; 7:35, s. 56183-56192
-
Journal article (peer-reviewed)abstract
- The transcription factor Zinc finger protein 148 (Zfp148, ZBP-89, BFCOL, BERF1, htβ) interacts physically with the tumor suppressor p53, but the significance of this interaction is not known. We recently showed that knockout of Zfp148 in mice leads to ectopic activation of p53 in some tissues and cultured fibroblasts, suggesting that Zfp148 represses p53 activity. Here we hypothesize that targeting Zfp148 would unleash p53 activity and protect against cancer development, and test this idea in the APCMin/+ mouse model of intestinal adenomas. Loss of one copy of Zfp148 markedly reduced tumor numbers and tumor-associated intestinal bleedings, and improved survival. Furthermore, after activation of β-catenin-the initiating event in colorectal cancer-Zfp148 deficiency activated p53 and induced apoptosis in intestinal explants of APCMin/+ mice. The anti-tumor effect of targeting Zfp148 depended on p53, as Zfp148 deficiency did not affect tumor numbers in APCMin/+ mice lacking one or both copies of Trp53. The results suggest that Zfp148 controls the fate of newly transformed intestinal tumor cells by repressing p53 and that targeting Zfp148 might be useful in the treatment of colorectal cancer.
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6. |
- Pandita, Ankur, et al.
(author)
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Intussusceptive angiogenesis in human metastatic malignant melanoma. : Intussusception in human melanoma
- 2021
-
In: The American Journal of Pathology. - : Elsevier BV. - 1525-2191 .- 0002-9440. ; 191:11, s. 2023-2038
-
Journal article (peer-reviewed)abstract
- Angiogenesis supplies oxygen and nutrients to growing tumors. Inhibiting angiogenesis may stop tumor growth, but vascular endothelial growth factor inhibitors have limited effect in most tumors. The limited effect may be explained by an additional, less vascular endothelial growth factor-driven, form of angiogenesis known as intussusceptive angiogenesis. The importance of intussusceptive angiogenesis in human tumors is not known. Epifluorescence and confocal microscopy was used to visualize intravascular pillars, the hallmark structure of intussusceptive angiogenesis, in tumors. Human malignant melanoma metastases, patient-derived melanoma xenografts in mice (PDX), and genetically engineered BRAF-induced, PTEN-deficient (BPT) mice (BrafCA/+Ptenf/fTyr-Cre+/0-mice) were analyzed for pillars. Gene expression in human melanoma metastases and PDXs was analyzed by RNA sequencing. Matrix metalloproteinase 9 (MMP9) protein expression and T-cell and macrophage infiltration in tumor sections were determined with multiplex immunostaining. Intravascular pillars were detected in human metastases but rarely in PDXs and not in BPT mice. The expression of MMP9 mRNA was higher in human metastases compared with PDXs. High expression of MMP9 protein as well as infiltration of macrophages and T-cell infiltration were detected in proximity to intravascular pillars. MMP inhibition blocked formation of pillars, but not tubes or tip cells, invitro. In conclusion, intussusceptive angiogenesis may contribute to the growth of human melanoma metastases. MMP inhibition blocked pillar formation invitro and should be further investigated as a potential anti-angiogenic drug target in metastatic melanoma.
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7. |
- Sjögren, Lovisa, et al.
(author)
-
Postprandial effects of the phosphodiesterase-5 inhibitor tadalafil in people with well-controlled Type 2 diabetes mellitus: a randomized controlled trial.
- 2016
-
In: Diabetic medicine : a journal of the British Diabetic Association. - : Wiley. - 1464-5491. ; 33:9, s. 1299-1301
-
Journal article (peer-reviewed)abstract
- Type 2 diabetes mellitus is a serious global health problem that is hard to treat in the long term, prompting great efforts to find new drug targets. Little attention has been paid, however, to the metabolic significance of the microcirculation in insulin-sensitive tissues, despite the fact that microvascular insulin resistance and endothelial dysfunction are closely associated and have been shown to precede Type 2 diabetes [1]. Endothelial nitric oxide plays a major role in mediating the beneficial effects of insulin on capillary recruitment and muscle glucose uptake [2] and in suppression of inflammatory pathways, including those activated by a high-fat diet [3]. This article is protected by copyright. All rights reserved.
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8. |
- Wilhelmson, Anna S K, et al.
(author)
-
Increased Intimal Hyperplasia After Vascular Injury in Male Androgen Receptor-Deficient Mice
- 2016
-
In: Endocrinology. - : The Endocrine Society. - 1945-7170 .- 0013-7227. ; 157:10, s. 3915-3923
-
Journal article (peer-reviewed)abstract
- Intimal hyperplasia is a vascular pathological process involved in the pathogenesis of atherosclerosis. Data suggest that T, the most important sex steroid hormone in males, protects men from atherosclerotic cardiovascular disease. T mainly acts via the androgen receptor (AR), and in this study we evaluated formation of intimal hyperplasia in male AR knockout (ARKO) mice using a vascular injury model. Two weeks after ligation of the carotid artery, male ARKO mice showed increased intimal area and intimal thickness compared with controls. After endothelial denudation by an in vivo scraping injury, there was no difference in the reendothelialization in ARKO compared with control mice. Ex vivo, we observed increased outgrowth of vascular smooth muscle cells from ARKO compared with control aortic tissue explants; the number of outgrown cells was almost doubled in ARKO. In vitro, stimulation of human aortic vascular smooth muscle cells with a physiological T concentration inhibited both migration and proliferation of the cells. Analyzing the expression of central regulators of cell proliferation and migration, we found that mRNA and protein levels of p27 were lower in uninjured arteries from ARKO mice and that T replacement to castrated male mice increased p27 mRNA in an AR-dependent manner. In conclusion, AR deficiency in male mice increases intimal hyperplasia in response to vascular injury, potentially related to the effects of androgens/AR to inhibit proliferation and migration of smooth muscle cells.
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9. |
- Yrlid, Ulf, 1971, et al.
(author)
-
Endothelial repair is dependent on CD11c(+) leukocytes to establish regrowing endothelial sheets with high cellular density
- 2019
-
In: Journal of Leukocyte Biology. - : Oxford University Press (OUP). - 0741-5400 .- 1938-3673. ; 105:1, s. 195-202
-
Journal article (peer-reviewed)abstract
- Endothelial injury makes the vessel wall vulnerable to cardiovascular diseases. Injured endothelium regenerates by collective sheet migration, that is, the endothelial cells coordinate their motion and regrow as a sheet of cells with retained cell-cell contacts into the wounded area. Leukocytes appear to be involved in endothelial repair in vivo; however, little is known about their identity and role in the reparative sheet migration process. To address these questions, we developed a high-quality en face technique that enables visualizing of leukocytes and endothelial cells simultaneously following an endoluminal scratch wound injury of the mouse carotid artery. We discovered that regrowing endothelium forms a broad proliferative front accompanied by CD11c(+) leukocytes. Functionally, the leukocytes were dispensable for the initial migratory response of the regrowing endothelial sheet, but critical for the subsequent formation and maintenance of a front zone with high cellular density. Marker expression analyses, genetic fate mapping, phagocyte targeting experiments, and mouse knock-out experiments indicate that the CD11c(+) leukocytes were mononuclear phagocytes with an origin from both Ly6C(high) and Ly6C(low) monocytes. In conclusion, CD11c(+) mononuclear phagocytes are essential for a proper endothelial regrowth following arterial endoluminal scratch injury. Promoting the endothelial-preserving function of CD11c(+) leukocytes may be a strategy to enhance endothelial repair following surgical and endovascular procedures.
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10. |
- Andersson, Linda, 1973, et al.
(author)
-
Deficiency in perilipin 5 reduces mitochondrial function and membrane depolarization in mouse hearts.
- 2017
-
In: The international journal of biochemistry & cell biology. - : Elsevier BV. - 1878-5875 .- 1357-2725. ; 91:Pt A, s. 9-13
-
Journal article (peer-reviewed)abstract
- Myocardial triglycerides stored in lipid droplets are important in regulating the intracellular delivery of fatty acids for energy generation in mitochondria, for membrane biosynthesis, and as agonists for intracellular signaling. Previously, we showed that deficiency in the lipid droplet protein perilipin 5 (Plin5) markedly reduces triglyceride storage in cardiomyocytes and increases the flux of fatty acids into phospholipids. Here, we investigated whether Plin5 deficiency in cardiomyocytes alters mitochondrial function. We found that Plin5 deficiency reduced mitochondrial oxidative capacity. Furthermore, in mitochondria from Plin5((-/)(-)) hearts, the fatty acyl composition of phospholipids in mitochondrial membranes was altered and mitochondrial membrane depolarization was markedly compromised. These findings suggest that mitochondria isolated from hearts deficient in Plin5, have specific functional defects.
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11. |
- Andersson, Linda, 1973, et al.
(author)
-
Rip2 modifies VEGF-induced signalling and vascular permeability in myocardial ischemia
- 2015
-
In: Cardiovascular Research. - : Oxford University Press (OUP). - 0008-6363 .- 1755-3245. ; 107:4, s. 478-486
-
Journal article (peer-reviewed)abstract
- In myocardial ischemia, vascular endothelial growth factor (VEGF) induces permeability by activating a signalling pathway that includes VEGF receptor 2 (VEGFR2), resulting in increased oedema and inflammation and thereby expanding the area of tissue damage. In this study, we investigated the role of receptor-interacting protein 2 (Rip2) in VEGF signalling and myocardial ischemia/reperfusion injury.
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12. |
- Breuer, Silke, et al.
(author)
-
Introduction of embryonic stem cells into vein grafts reduces intimal hyperplasia in mice.
- 2014
-
In: The Journal of cardiovascular surgery. - 0021-9509. ; 55:2, s. 235-46
-
Journal article (peer-reviewed)abstract
- Aim: Atherosclerosis with its cardiovascular events including cardiac and peripheral ischemia represents the main cause of death in the developed countries. Although interventional treatments like percutaneous transluminal angioplasty (PTA) or stents are increasingly applied for the treatment of peripheral arterial disease, they are not always technically applicable or durable and bypass surgery is needed. Compared to synthetic grafts, vein grafts show a better patency especially when used for the lower leg as well as a lower risk for infection compared to synthetic grafts. Still the long-term patency rates are unsatisfactory due to accelerated intimal hyperplasia, a thickening of the vessel wall. The aim of this study was to elucidate, if the implantation of embryonic stem cells into vein grafts can reduce the development of intimal hyperplasia in a mouse in vivo model. Methods: In this study we implanted LacZ-tagged (ROSA26) murine embryonic stem cells into decellularized vein grafts. Control groups were: 1) untreated veins; 2) decellularized veins; 3) decellularized veins with gel and plastic film; and 4) decellularized veins with smooth muscle cells in gel surrounded by plastic film. Six weeks after insertion into the carotid artery of mice, the grafts were excised and analyzed immunohistochemically, morphologically, and by x-gal staining and compared to the control groups. The Mann-Whitney-U test was used to compare groups. Statistical significance was indicated by a value of P<0.05. Results: Decellularized veins with implanted stem cells showed significantly less intimal thickening compared to all control groups (intimal hyperplasia vs. luminal circumference mean±SD 7.3±3.5 µm, median 8 µm). The control groups: 1) untreated veins (60.3±25.5 µm, median 58.5 µm); 2) decellularized veins (53.9±22.4 µm, median 48.4 µm); 3) decellularized veins with gel and plastic film (70.6±22.4 µm, median 72.6 µm); and 4) decellularized veins with smooth muscle cells in gel surrounded by plastic film (73.5±18.1 µm, median 73.6 µm) all showed the same high degree of intimal hyperplasia. Conclusion: This study demonstrates that embryonic stem cells have a therapeutic competence to favourably modulate intimal hyperplasia in vivo.
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13. |
- Chaudhari, Aditi, et al.
(author)
-
ARAP2 promotes GLUT1-mediated basal glucose uptake through regulation of sphingolipid metabolism
- 2016
-
In: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - : Elsevier BV. - 1388-1981. ; 1861:11, s. 1643-1651
-
Journal article (peer-reviewed)abstract
- Lipid droplet formation, which is driven by triglyceride synthesis, requires several droplet-associated proteins. We identified ARAP2 (an ADP-ribosylation factor 6 GTPase-activating protein) in the lipid droplet proteome of NIH-3T3 cells and showed that knockdown of ARAP2 resulted in decreased lipid droplet formation and triglyceride synthesis. We also showed that ARAP2 knockdown did not affect fatty acid uptake but reduced basal glucose uptake, total levels of the glucose transporter GLUT1, and GLUT1 levels in the plasma membrane and the lipid micro-domain fraction (a specialized plasma membrane domain enriched in sphingolipids). Microarray analysis showed that ARAP2 knockdown altered expression of genes involved in sphingolipid metabolism. Because sphingolipids are known to play a key role in cell signaling, we performed lipidomics to further investigate the relationship between ARAP2 and sphingolipids and potentially identify a link with glucose uptake. We found that ARAP2 knockdown increased glucosylceramide and lactosylceramide levels without affecting ceramide levels, and thus speculated that the rate-limiting enzyme in glycosphingolipid synthesis, namely glucosylceramide synthase (GCS), could be modified by ARAP2. In agreement with our hypothesis, we showed that the activity of GCS was increased by ARAP2 knockdown and reduced by ARAP2 overexpression. Furthermore, pharmacological inhibition of GCS resulted in increases in basal glucose uptake, total GLUT1 levels, triglyceride biosynthesis from glucose, and lipid droplet formation, indicating that the effects of GCS inhibition are the opposite to those resulting from ARAP2 knockdown. Taken together, our data suggest that ARAP2 promotes lipid droplet formation by modifying sphingolipid metabolism through GCS.
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14. |
- Drevinge, Christina, 1983, et al.
(author)
-
Perilipin 5 is protective in the ischemic heart
- 2016
-
In: International Journal of Cardiology. - : Elsevier BV. - 0167-5273 .- 1874-1754. ; 219, s. 446-454
-
Journal article (peer-reviewed)abstract
- Background: Myocardial ischemia is associated with alterations in cardiac metabolism, resulting in decreased fatty acid oxidation and increased lipid accumulation. Here we investigate how myocardial lipid content and dynamics affect the function of the ischemic heart, and focus on the role of the lipid droplet protein perilipin 5 (Plin5) in the pathophysiology of myocardial ischemia. Methods and results: We generated Plin5(-/-) mice and found that Plin5 deficiency dramatically reduced the triglyceride content in the heart. Under normal conditions, Plin5(-/-) mice maintained a close to normal heart function by decreasing fatty acid uptake and increasing glucose uptake, thus preserving the energy balance. However, during stress or myocardial ischemia, Plin5 deficiency resulted in myocardial reduced substrate availability, severely reduced heart function and increased mortality. Importantly, analysis of a human cohort with suspected coronary artery disease showed that a common noncoding polymorphism, rs884164, decreases the cardiac expression of PLIN5 and is associated with reduced heart function following myocardial ischemia, indicating a role for Plin5 in cardiac dysfunction. Conclusion: Our findings indicate that Plin5 deficiency alters cardiac lipid metabolism and associates with reduced survival following myocardial ischemia, suggesting that Plin5 plays a beneficial role in the heart following ischemia. (C) 2016 The Authors. Published by Elsevier Ireland Ltd.
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15. |
- Dunér, Pontus, et al.
(author)
-
Antibodies against apoB100 peptide 210 inhibit atherosclerosis in apoE(-/-) mice
- 2021
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
-
Journal article (peer-reviewed)abstract
- Atherosclerotic plaques are characterized by an accumulation and subsequent oxidation of LDL, resulting in adaptive immune responses against formed or exposed neoepitopes of the LDL particle. Autoantibodies against native p210, the 3136-3155 amino acid sequence of the LDL protein apolipoprotein B-100 (apoB100) are common in humans and have been associated with less severe atherosclerosis and decreased risk for cardiovascular events in clinical studies. However, whether apoB100 native p210 autoantibodies play a functional role in atherosclerosis is not known. In the present study we immunized apoE(-/-) mice with p210-PADRE peptide to induce an antibody response against native p210. We also injected mice with murine monoclonal IgG against native p210. Control groups were immunized with PADRE peptide alone or with control murine monoclonal IgG. Immunization with p210-PADRE induced an IgG1 antibody response against p210 that was associated with reduced atherosclerotic plaque formation in the aorta and reduced MDA-LDL content in the lesions. Treatment with monoclonal p210 IgG produced a similar reduction in atherosclerosis as immunization with p210-PADRE. Our findings support an atheroprotective role of antibodies against the apoB100 native p210 and suggest that vaccines that induce the expression of native p210 IgG represent a potential therapeutic strategy for lowering cardiovascular risk.
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16. |
- Ekstrand, Matias, et al.
(author)
-
Depletion of ATP and glucose in advanced human atherosclerotic plaques
- 2017
-
In: PLoS One. - : Public Library of Science (PLoS). - 1932-6203. ; 12:6
-
Journal article (peer-reviewed)abstract
- Severe hypoxia develops close to the necrotic core of advanced human atherosclerotic plaques, but the energy metabolic consequences of this hypoxia are not known. In animal models, plaque hypoxia is also associated with depletion of glucose and ATP. ATP depletion may impair healing of plaques and promote necrotic core expansion. To investigate if ATP depletion is present in human plaques, we analyzed the distribution of energy metabolites (ATP, glucose, glycogen and lactate) in intermediate and advanced human plaques.Snap frozen carotid endarterectomies from 6 symptomatic patients were analyzed. Each endarterectomy included a large plaque ranging from the common carotid artery (CCA) to the internal carotid artery (ICA). ATP, glucose, and glycogen concentrations were lower in advanced (ICA) compared to intermediate plaques (CCA), whereas lactate concentrations were higher. The lowest concentrations of ATP, glucose and glycogen were detected in the perinecrotic zone of advanced plaques.Our study demonstrates severe ATP depletion and glucose deficiency in the perinecrotic zone of human advanced atherosclerotic plaques. ATP depletion may impair healing of plaques and promote disease progression.
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17. |
- Ekstrand, Matias, et al.
(author)
-
Imaging of Intracellular and Extracellular ROS Levels in Atherosclerotic Mouse Aortas Ex Vivo: Effects of Lipid Lowering by Diet or Atorvastatin
- 2015
-
In: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 10:6
-
Journal article (peer-reviewed)abstract
- Objective The first objective was to investigate if intracellular and extracellular levels of reactive oxygen species (ROS) within the mouse aorta increase before or after diet-induced lesion formation. The second objective was to investigate if intracellular and extracellular ROS correlates to cell composition in atherosclerotic lesions. The third objective was to investigate if intracellular and extracellular ROS levels within established atherosclerotic lesions can be reduced by lipid lowering by diet or atorvastatin. To address our objectives, we established a new imaging technique to visualize and quantify intracellular and extracellular ROS levels within intact mouse aortas ex vivo. Using this technique, we found that intracellular, but not extracellular, ROS levels increased prior to lesion formation in mouse aortas. Both intracellular and extracellular ROS levels were increased in advanced lesions. Intracellular ROS correlated with lesion content of macrophages. Extracellular ROS correlated with lesion content of smooth muscle cells. The high levels of ROS in advanced lesions were reduced by 5 days high dose atorvastatin treatment but not by lipid lowering by diet. Atorvastatin treatment did not affect lesion inflammation (aortic arch mRNA levels of CXCL 1, ICAM-1, MCP-1, TNF-alpha, VCAM, IL-6, and IL-1 beta) or cellular composition (smooth muscle cell, macrophage, and T-cell content). Aortic levels of intracellular ROS increase prior to lesion formation and may be important in initiation of atherosclerosis. Our results suggest that within lesions, macrophages produce mainly intracellular ROS whereas smooth muscle cells produce extracellular ROS. Short term atorvastatin treatment, but not lipid lowering by diet, decreases ROS levels within established advanced lesions; this may help explain the lesion stabilizing and anti-inflammatory effects of long term statin treatment.
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18. |
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19. |
- Fogelstrand, Per, 1971, et al.
(author)
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Dependence of proliferative vascular smooth muscle cells on CD98hc (4F2hc, SLC3A2).
- 2009
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In: The Journal of experimental medicine. - : Rockefeller University Press. - 1540-9538 .- 0022-1007. ; 206:11, s. 2397-406
-
Journal article (peer-reviewed)abstract
- Activation of vascular smooth muscle cells (VSMCs) to migrate and proliferate is essential for the formation of intimal hyperplasia. Hence, selectively targeting activated VSMCs is a potential strategy against vaso-occlusive disorders such as in-stent restenosis, vein-graft stenosis, and transplant vasculopathy. We show that CD98 heavy chain (CD98hc) is markedly up-regulated in neointimal and cultured VSMCs, and that activated but not quiescent VSMCs require CD98hc for survival. CD98hc mediates integrin signaling and localizes amino acid transporters to the plasma membrane. SMC-specific deletion of CD98hc did not affect normal vessel morphology, indicating that CD98hc was not required for the maintenance of resident quiescent VSMCs; however, CD98hc deletion reduced intimal hyperplasia after arterial injury. Ex vivo and in vitro, loss of CD98hc suppressed proliferation and induced apoptosis in VSMCs. Furthermore, reconstitution with CD98hc mutants showed that CD98hc interaction with integrins was necessary for the survival of VSMCs. These studies establish the importance of CD98hc in VSMC proliferation and survival. Furthermore, loss of CD98hc was selectively deleterious to activated VSMCs while sparing resident quiescent VSMCs, suggesting that activated VSMCs are physiologically dependent on CD98hc, and hence, CD98hc is a potential therapeutic target in vaso-occlusive disorders.
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20. |
- Fogelstrand, Per, 1971, et al.
(author)
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External collar inhibits balloon-induced intimal hyperplasia in rabbits.
- 2002
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In: Journal of vascular research. - 1018-1172. ; 39:4, s. 361-7
-
Journal article (peer-reviewed)abstract
- Intimal hyperplasia is a common complication following vascular interventions. To understand the underlying pathophysiology, the focus has mainly been on the intima and media. The adventitia has been less investigated, although adventitial hyperplasia is seen together with intimal hyperplasia. If the adventitial response is an important part of the process, the adventitia might be a target to inhibit intimal hyperplasia. In the present study we investigated whether an external collar attenuating the adventitial thickness could inhibit a balloon-induced intimal hyperplasia. The common carotid artery was injured in rabbits (n = 6) with a 3-french balloon catheter. The mid portion of the injured artery was encircled with a silicone collar (diameter = 2.0 mm). After 14 days the balloon-induced neointima was reduced by 54 +/- 6.3% underneath the collar. The adventitial and medial thickenings were also attenuated (36 +/- 8.7 and 44 +/- 4.3%, respectively). This study demonstrates that intimal hyperplasia following balloon injury can be inhibited with an external collar. This supports the idea of the adventitia as a potential target to inhibit intimal hyperplasia.
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21. |
- Fogelstrand, Per, 1971, et al.
(author)
-
Increased Vascular Injury Reduces the Degree of Intimal Hyperplasia following Angioplasty in Rabbits.
- 2011
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In: Journal of vascular research. - : S. Karger AG. - 1423-0135 .- 1018-1172. ; 48:4, s. 307-15
-
Journal article (peer-reviewed)abstract
- Background/Aims: Formation of intimal hyperplasia following angioplastic procedures can lead to complications, including restenosis and accelerated atherosclerosis. The vessel wall media is a main source of neointimal cells. However, evidence suggests that there are additional cell sources, such as the adventitia. Here we investigate whether an extensive loss of vascular smooth muscle cells (VSMCs) in the media results in less intimal hyperplasia or if there is compensatory cell recruitment from the adventitia. Methods:A balloon catheter was pulled through the rabbit carotid artery 4 times (major injury) or 2 times (minor injury). Adventitial cells were labeled with 5-bromo-2-deoxyuridine or PKH26. Results:The major injury, but not the minor injury, resulted in a complete loss of VSMCs in large parts of the media and significant leukocyte infiltration. The major injury resulted in less neointima compared with the minor injury. The thinnest neointima was seen at the most injured parts of the media in the major injury group. Cell-tracking experiments showed that the media, but not the adventitia, served as a source of neointimal cells. Conclusion: An augmented angioplastic injury with extensive VSMC loss in rabbits reduced the degree of intimal hyperplasia. No compensatory recruitment of neointimal cells from the adventitia occurred.
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22. |
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23. |
- Fogelstrand, Per, 1971, et al.
(author)
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Retention of atherogenic lipoproteins in the artery wall and its role in atherogenesis.
- 2012
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In: Nutrition, Metabolism and Cardiovascular Diseases: NMCD. - : Elsevier BV. - 1590-3729 .- 0939-4753. ; 22:1, s. 1-7
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Research review (peer-reviewed)abstract
- In this review, we discuss the mechanisms behind the binding of low-density lipoproteins (LDL) to the arterial wall and how this interaction might be targeted to prevent atherosclerosis.
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24. |
- Fogelstrand, Per, 1971
(author)
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The role of the adventitia and perivascular tissue for the formation of intimal hyperplasia
- 2004
-
Doctoral thesis (other academic/artistic)abstract
- Symptomatic atherosclerosis is commonly treated with balloon angioplasty or bypass surgery. About 30% of the interventions will re-narrow within a year due to restenosis and graft-stenosis. Thickening of the luminal layer, the intima, is an important component in these side-effects. Smooth muscle cells (SMCs) from the media have been regarded as responsible for the formation of the intimal hyperplasia. During the last decade the outermost vessel wall layer, the adventitia, and the perivascular tissue have been recognized as additional sources for intimal SMCs. The aim of this thesis was to investigate the role of the adventitia and the perivascular tissue in the formation of intimal hyperplasia. The specific aims were to investigate 1) if intimal hyperplasia can be inhibited from the adventitial side of a vessel, 2) if the adventitia and perivascular tissue can contribute with neointimal cells and 3) if the adventitia and perivascular tissue have a dynamic relation to other cellular sources in the formation of intimal hyperplasia. We used three different animal models for intimal hyperplasia: balloon injury to the rabbit carotid artery, implantation of vein grafts in mice and artificial grafts in pigs. Intimal hyperplasia following balloon injury could be inhibited from the adventitial side by placing a silicone collar around the treated artery. An external inhibition was also achieved in mice with a plastic shielding that isolated acellular vein grafts from the perivascular tissue.We could not find any cell migration from the adventitia / the perivascular tissue into the neointima in the rabbit carotid artery. However, in vascular grafts the perivascular tissue contributed with neointimal cells in both mouse and pig. A contribution from the blood was seen in rabbit and suggested in mouse and pig. In mouse, there was a dynamic relationship between different cellular sources. The donor vein and the perivascular tissue could fully compensate each other in the neointimal formation. In rabbit, we could not find this phenomenon. The media was the main cellular source and could not be fully compensated for by other sources.The results in this thesis show that different cellular sources, with a varying degree of compensatory abilities, contribute with neointimal cells. The cellular sources and their dynamic potential must be known and considered when inhibiting intimal hyperplasia following balloon injury and implantation of vascular grafts.
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25. |
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26. |
- Glise, Lars, 1988, et al.
(author)
-
Disturbed Laminar Blood Flow Causes Impaired Fibrinolysis and Endothelial Fibrin Deposition In Vivo
- 2019
-
In: Thrombosis and Haemostasis. - : Georg Thieme Verlag KG. - 0340-6245 .- 2567-689X. ; 119:2, s. 223-233
-
Journal article (peer-reviewed)abstract
- Endothelial expression of tissue-type plasminogen activator (t-PA) is crucial for maintaining an adequate endogenous fibrinolysis. It is unknown how endothelial t-PA expression and fibrinolysis are affected by blood flow in vivo. In this study, we investigated the impact of different blood flow profiles on endothelial t-PA expression and fibrinolysis in the arterial vasculature. Induction of disturbed laminar blood flow (D-flow) in the mouse carotid artery potently reduced endothelial t-PA messenger ribonucleic acid and protein expression, and caused fibrin deposition. En face immunohistochemistry demonstrated that arterial areas naturally exposed to D-flow had markedly lower endothelial t-PA levels than areas with sustained laminar blood flow (S-flow), and displayed pronounced fibrin deposition despite an intact endothelium. In t-PA and plasminogen-deficient mice, fibrin deposition did not extend into S-flow areas, indicating that areas of D-flow and S-flow differ, not only in fibrinolytic capacity, but also in coagulation. Furthermore, plasminogen accumulation was found at D-flow areas, and infusion of recombinant t-PA activated fibrinolysis and significantly reduced the fibrin deposits. In conclusion, D-flow potently impairs the fibrinolytic capacity and causes endothelial fibrin deposition in vivo. Our data also indicate that t-PA is the limiting factor for efficient fibrinolysis at the thrombosis-prone D-flow areas in the arterial vasculature.
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27. |
- Glise, Lars, 1988, et al.
(author)
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pH-Dependent Protonation of Histidine Residues Is Critical for Electrostatic Binding of Low-Density Lipoproteins to Human Coronary Arteries
- 2022
-
In: Arteriosclerosis Thrombosis and Vascular Biology. - : Ovid Technologies (Wolters Kluwer Health). - 1079-5642 .- 1524-4636. ; 42:8, s. 1037-1047
-
Journal article (peer-reviewed)abstract
- Background: The initiating step in atherogenesis is the electrostatic binding of LDL (low-density lipoprotein) to proteoglycan glycosaminoglycans in the arterial intima. However, although proteoglycans are widespread throughout the intima of most coronary artery segments, LDL is not evenly distributed, indicating that LDL retention is not merely dependent on the presence of proteoglycans. We aim to identify factors that promote the interaction between LDL and the vessel wall of human coronary arteries. Methods: We developed an ex vivo model to investigate binding of labeled human LDL to human coronary artery sections without the interference of cellular processes. Results: By staining consecutive sections of human coronary arteries, we found strong staining of sulfated glycosaminoglycans throughout the arterial intima, whereas endogenous LDL deposits were focally distributed. Ex vivo binding of LDL was uniform at all intimal areas with sulfated glycosaminoglycans. However, lowering the pH from 7.4 to 6.5 triggered a 35-fold increase in LDL binding. The pH-dependent binding was abolished by pretreating LDL with diethyl-pyrocarbonate, which blocks the protonation of histidine residues, or cyclohexanedione, which inhibits the positive charge of site B on LDL. Thus, both histidine protonation and site B are required for strong electrostatic LDL binding to the intima. Conclusions: This study identifies histidine protonation as an important component for electrostatic LDL binding to human coronary arteries. Our findings show that the local pH will have a profound impact on LDL's affinity for sulfated glycosaminoglycans, which may influence the retention and accumulation pattern of LDL in the arterial vasculature.
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28. |
- Heckenkamp, J, et al.
(author)
-
Photodynamic therapy reduces intimal hyperplasia in prosthetic vascular bypass grafts in a pig model.
- 2007
-
In: European journal of vascular and endovascular surgery. - : Elsevier BV. - 1078-5884. ; 34:3, s. 333-9
-
Journal article (peer-reviewed)abstract
- BACKGROUND: Bypass surgery has a failing frequency of 30% during the first year, mainly due to intimal hyperplasia (IH). This negative effect is most pronounced in artificial grafts. Photodynamic therapy (PDT) is a technique in which light activates photosensitizer dyes to produce free-radicals resulting in an eradication of cells in the vascular wall. The aim of this study was to determine the effectiveness of PDT to reduce IH in a preclinical porcine PTFE bypass model. MATERIAL AND METHODS: Ten pigs were used. After a pilot PDT dosimetry study (n=3) PTFE grafts were bilaterally placed into the circulation as bypasses from the common to the external iliac arteries (n=7). The right sides served as controls (C). Before implantation of the left grafts, the arterial connecting sites of the left distal anastomoses were PDT-treated. The arteries were pressurized at 180 mmHg for 5 minutes with the photosensitizer Methylene Blue (330 microg/ml), and thereafter endoluminally irradiated with laser light (lambda = 660 nm, 100 mW/cm(2), 150 J/cm(2)). After 4 weeks the specimens were retrieved and formalin fixed. Cross sections through the midportions of the distal anastomoses and the grafts were used for histology, immunohistochemistry to identify inflammatory cells and morphometric evaluation (n=7). RESULTS: No systemic side effects and no graft occlusions were noted. PDT-treated anastomoses showed reduced IH in the mid-portions of the anastomoses (Area of IH: microm(2)/microm graft: C: 6970+/-1536, PDT: 2734+/-2560; P<0.005) as well as in the grafts (C: 5391+/-4031, PDT: 777+/-1331; P<0.02). The number of inflammatory cells per microscopic field was increased after PDT (C: 24+/-16, PDT: 37+/-15; P<0.009). CONCLUSIONS: Adjuvant PDT, performed in an endovascular fashion, was a safe method to reduce prosthetic graftstenosis in a preclinical setting. This study underscores the clinical potential of PDT to inhibit the development of clinical bypass graftstenosis.
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29. |
- Håversen, Liliana, 1963, et al.
(author)
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Vimentin deficiency in macrophages induces increased oxidative stress and vascular inflammation but attenuates atherosclerosis in mice
- 2018
-
In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 8
-
Journal article (peer-reviewed)abstract
- The aim was to clarify the role of vimentin, an intermediate filament protein abundantly expressed in activated macrophages and foam cells, in macrophages during atherogenesis. Global gene expression, lipid uptake, ROS, and inflammation were analyzed in bone-marrow derived macrophages from vimentin-deficient (Vim(-/-)) and wild-type (Vim(-/-)) mice. Atherosclerosis was induced in Ldlr(-/-) mice transplanted with a PCSK9 gain-of-function virus. The mice were fed an atherogenic diet for 12-15 weeks. We observed impaired uptake of native LDL but increased uptake of oxLDL in Vim(-/-) macrophages. FACS analysis revealed increased surface expression of the scavenger receptor CD36 on Vim(-/-) macrophages. Vim(-/-) macrophages also displayed increased markers of oxidative stress, activity of the transcription factor NF-kappa B, secretion of proinflammatory cytokines and GLUT1-mediated glucose uptake. Vim(-/- )mice displayed decreased atherogenesis despite increased vascular inflammation and increased CD36 expression on macrophages in two mouse models of atherosclerosis. We demonstrate that vimentin has a strong suppressive effect on oxidative stress and that Vim(-/-) mice display increased vascular inflammation with increased CD36 expression on macrophages despite decreased subendothelial lipid accumulation. Thus, vimentin has a key role in regulating inflammation in macrophages during atherogenesis.
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30. |
- Jacobsen, M. T., et al.
(author)
-
Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction
- 2017
-
In: Cell Chemical Biology. - : Elsevier BV. - 2451-9448 .- 2451-9456. ; 24:8, s. 1040-47
-
Journal article (peer-reviewed)abstract
- Chemical modification of proteins provides great opportunities to control and visualize living systems. The most common way to modify proteins is reaction of their abundant amines with N-hydroxysuccinimide (NHS) esters. Here we explore the impact of amine number and positioning on protein-conjugate behavior using streptavidin-biotin, a central research tool. Dye-NHS modification of streptavidin severely damaged ligand binding, necessitating development of a new streptavidin-retaining ultrastable binding after labeling. Exploring the ideal level of dye modification, we engineered a panel bearing 1-6 amines per subunit: "amine landscaping.'' Surprisingly, brightness increased as amine number decreased, revealing extensive quenching following conventional labeling. We ultimately selected Flavidin (fluorophore- friendly streptavidin), combining ultrastable ligand binding with increased brightness after conjugation. Flavidin enhanced fluorescent imaging, allowing more sensitive and specific cell labeling in tissues. Flavidin should have wide application in molecular detection, providing a general insight into how to optimize simultaneously the behavior of the biomolecule and the chemical probe.
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31. |
- Jenndahl, L., et al.
(author)
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Personalized tissue-engineered arteries as vascular graft transplants : A safety study in sheep
- 2022
-
In: Regenerative Therapy. - : Japanese Society of Regenerative Medicine. - 2352-3204. ; 21, s. 331-341
-
Journal article (peer-reviewed)abstract
- Patients with cardiovascular disease often need replacement or bypass of a diseased blood vessel. With disadvantages of both autologous blood vessels and synthetic grafts, tissue engineering is emerging as a promising alternative of advanced therapy medicinal products for individualized blood vessels. By reconditioning of a decellularized blood vessel with the recipient's own peripheral blood, we have been able to prevent rejection without using immunosuppressants and prime grafts for efficient recellularization in vivo. Recently, decellularized veins reconditioned with autologous peripheral blood were shown to be safe and functional in a porcine in vivo study as a potential alternative for vein grafting. In this study, personalized tissue engineered arteries (P-TEA) were developed using the same methodology and evaluated for safety in a sheep in vivo model of carotid artery transplantation. Five personalized arteries were transplanted to carotid arteries and analyzed for safety and patency as well as with histology after four months in vivo. All grafts were fully patent without any occlusion or stenosis. The tissue was well cellularized with a continuous endothelial cell layer covering the luminal surface, revascularized adventitia with capillaries and no sign of rejection or infection. In summary, the results indicate that P-TEA is safe to use and has potential as clinical grafts.
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32. |
- Kijani, Siavash, et al.
(author)
-
Intimal hyperplasia induced by vascular intervention causes lipoprotein retention and accelerated atherosclerosis
- 2017
-
In: Physiological Reports. - : Wiley. - 2051-817X. ; 5:14
-
Journal article (peer-reviewed)abstract
- Accelerated atherosclerosis diminishes the long term patency of vascular interventions, such as percutaneous coronary intervention and implantation of saphenous vein grafts. However, the cause of this accelerated atherosclerosis is unclear. In this study, we tested the hypothesis that intimal hyperplasia formed following vascular intervention promotes retention of atherogenic lipoproteins. Intimal hyperplasia was surgically induced in the mouse common carotid artery. The surgery was combined with different mouse models of hypercholesterolemia to obtain different cholesterol levels and to control the onsets of hypercholesterolemia. Three weeks after surgery, samples were immunostained for apoB lipoproteins, smooth muscle cells and leukocytes. Already at mild hypercholesterolemia (193mg/dL), pronounced apoB lipoprotein retention was found in the extracellular matrix in both intimal hyperplasia and the injured underlying media. In contrast, minimal retention was detected in the uninjured proximal region of the same vessel, or in vessels from mice with normal cholesterol levels (81mg/dL). Induction of aggravated hypercholesterolemia 3weeks after surgery, when a mature intimal hyperplasia had been formed, caused a very rapid development of atherosclerotic lesions. Mechanistically, we show that lipoprotein retention was almost exclusively dependent on electrostatic interactions to proteoglycan glycosaminoglycans, and the lipoprotein retention to intimal hyperplasia could be inhibited invivo using glycosaminoglycan-binding antibodies. Thus, formation of intimal hyperplasia following vascular intervention makes the vessel wall highly susceptible for lipoprotein retention and accelerated atherosclerosis. The increased lipoprotein retention in intimal hyperplasia can be targeted by blocking the interaction between apoB lipoproteins and glycosaminoglycans in the extracellular matrix.
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33. |
- Klevstig, Martina, et al.
(author)
-
Targeting acid sphingomyelinase reduces cardiac ceramide accumulation in the post-ischemic heart
- 2016
-
In: Journal of Molecular and Cellular Cardiology. - : Elsevier BV. - 0022-2828 .- 1095-8584. ; 93, s. 69-72
-
Journal article (peer-reviewed)abstract
- Ceramide accumulation is known to accompany acute myocardial ischemia, but its role in the pathogenesis of ischemic heart disease is unclear. In this study, we aimed to determine how ceramides accumulate in the ischemic heart and to determine if cardiac function following ischemia can be improved by reducing ceramide accumulation. To investigate the association between ceramide accumulation and heart function, we analyzed myocardial left ventricle biopsies from subjects with chronic ischemia and found that ceramide levels were higher in biopsies from subjects with reduced heart function. Ceramides are produced by either de novo synthesis or hydrolysis of sphingomyelin catalyzed by acid and/or neutral sphingomyelinase. We used cultured HL-1 cardiomyocytes to investigate these pathways and showed that acid sphingomyelinase activity rather than neutral sphingomyelinase activity or de novo sphingolipid synthesis was important for hypoxia-induced ceramide accumulation. We also used mice with a partial deficiency in acid sphingomyelinase (Smpd1(+/-) mice) to investigate if limiting ceramide accumulation under ischemic conditions would have a beneficial effect on heart function and survival. Although we showed that cardiac ceramide accumulation was reduced in Smpd1(+/-) mice 24 h after an induced myocardial infarction, this reduction was not accompanied by an improvement in heart function or survival. Our findings show that accumulation of cardiac ceramides in the post-ischemic heart is mediated by acid sphingomyelinase. However, targeting ceramide accumulation in the ischemic heart may not be a beneficial treatment strategy.
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34. |
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35. |
- Li, Lu, 1964, et al.
(author)
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ARF6 Regulates Neuron Differentiation through Glucosylceramide Synthase
- 2013
-
In: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 8:3
-
Journal article (peer-reviewed)abstract
- The small GTPase ADP ribosylation factor 6 (ARF6) mediates endocytosis and has in addition been shown to regulate neuron differentiation. Here we investigated whether ARF6 promotes differentiation of Neuro-2a neuronal cells by modifying the cellular lipid composition. We showed that knockdown of ARF6 by siRNA in Neuro-2a cells increased neuronal outgrowth as expected. ARF6 knockdown also resulted in increased glucosylceramide levels and decreased sphingomyelin levels, but did not affect the levels of ceramide or phospholipids. We speculated that the ARF6 knockdown-induced increase in glucosylceramide was caused by an effect on glucosylceramide synthase and, in agreement, showed that ARF6 knockdown increased the mRNA levels and activity of glucosylceramide synthase. Finally, we showed that incubation of Neuro-2a cells with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) normalized the increased neuronal outgrowth induced by ARF6 knockdown. Our results thus show that ARF6 regulates neuronal differentiation through an effect on glucosylceramide synthase and glucosylceramide levels.
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36. |
- Mardani, Ismena, et al.
(author)
-
Plin2-deficiency reduces lipophagy and results in increased lipid accumulation in the heart.
- 2019
-
In: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1
-
Journal article (peer-reviewed)abstract
- Myocardial dysfunction is commonly associated with accumulation of cardiac lipid droplets (LDs). Perilipin 2 (Plin2) is a LD protein that is involved in LD formation, stability and trafficking events within the cell. Even though Plin2 is highly expressed in the heart, little is known about its role in myocardial lipid storage. A recent report shows that cardiac overexpression of Plin2 result in massive myocardial steatosis suggesting that Plin2 stabilizes LDs. In this study, we hypothesized that deficiency in Plin2 would result in reduced myocardial lipid storage. In contrast to our hypothesis, we found increased accumulation of triglycerides in hearts, and specifically in cardiomyocytes, from Plin2-/- mice. Although Plin2-/- mice had markedly enhanced lipid levels in the heart, they had normal heart function under baseline conditions and under mild stress. However, after an induced myocardial infarction, stroke volume and cardiac output were reduced in Plin2-/- mice compared with Plin2+/+ mice. We further demonstrated that the increased triglyceride accumulation in Plin2-deficient hearts was caused by altered lipophagy. Together, our data show that Plin2 is important for proper hydrolysis of LDs.
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37. |
- Mellander, Stefan, 1960, et al.
(author)
-
Healing of PTFE grafts in a pig model recruit neointimal cells from different sources and do not endothelialize.
- 2005
-
In: European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery. - : Elsevier BV. - 1078-5884. ; 30:1, s. 63-70
-
Journal article (peer-reviewed)abstract
- OBJECTIVES: The aim of this study was to analyze the cellular sources for the neointima and the cell type that is lining the lumen in artificial grafts implanted in pigs. MATERIALS AND METHODS: We used polytetrafluoroethylene grafts as bypasses from the common to the external iliac arteries. The animals were sacrificed after 1, 4, 7, 14, 21, 30, 60 and 90 days. Morphological, immunohistochemical and electron microscope assessments were made. RESULTS: After 7 days a circumferential neoadventitia was formed. At day 14 isolated cellular islets of proliferating cells were observed on the luminal side of the graft without connection to the neoadventitia or the adjacent arteries. In the anastomotic regions at day 14 we observed an isolated neointima in contact with the adjacent artery. The cells lining the lumen had characteristics of both smooth muscle cells and endothelial cells. CONCLUSIONS: Our study suggests that in artificial porcine grafts, the perivascular tissue, the blood and the adjacent artery contribute to the formation of the neointima. The luminal surface is covered by a hybrid cell with both smooth muscle cell and endothelial cell properties.
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38. |
- Petrich, Brian G, et al.
(author)
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The antithrombotic potential of selective blockade of talin-dependent integrin alpha IIb beta 3 (platelet GPIIb-IIIa) activation.
- 2007
-
In: The Journal of clinical investigation. - 0021-9738. ; 117:8, s. 2250-9
-
Journal article (peer-reviewed)abstract
- In vitro studies indicate that binding of talin to the beta(3) integrin cytoplasmic domain (tail) results in integrin alpha(IIb)beta(3) (GPIIb-IIIa) activation. Here we tested the importance of talin binding for integrin activation in vivo and its biological significance by generating mice harboring point mutations in the beta(3) tail. We introduced a beta(3)(Y747A) substitution that disrupts the binding of talin, filamin, and other cytoplasmic proteins and a beta(3)(L746A) substitution that selectively disrupts interactions only with talin. Platelets from animals homozygous for each mutation showed impaired agonist-induced fibrinogen binding and platelet aggregation, providing proof that inside-out signals that activate alpha(IIb)beta(3) require binding of talin to the beta(3) tail. beta(3)(L746A) mice were resistant to both pulmonary thromboembolism and to ferric chloride-induced thrombosis of the carotid artery. Pathological bleeding, measured by the presence of fecal blood and development of anemia, occurred in 53% of beta(3)(Y747A) and virtually all beta(3)-null animals examined. Remarkably, less than 5% of beta(3)(L746A) animals exhibited this form of bleeding. These results establish that alpha(IIb)beta(3) activation in vivo is dependent on the interaction of talin with the beta(3) integrin cytoplasmic domain. Furthermore, they suggest that modulation of beta(3) integrin-talin interactions may provide an attractive target for antithrombotics and result in a reduced risk of pathological bleeding.
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39. |
- Simsa, Robin, et al.
(author)
-
Brain organoid formation on decellularized porcine brain ECM hydrogels
- 2021
-
In: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 16:1
-
Journal article (peer-reviewed)abstract
- Human brain tissue models such as cerebral organoids are essential tools for developmental and biomedical research. Current methods to generate cerebral organoids often utilize Matrigel as an external scaffold to provide structure and biologically relevant signals. Matrigel however is a nonspecific hydrogel of mouse tumor origin and does not represent the complexity of the brain protein environment. In this study, we investigated the application of a decellularized adult porcine brain extracellular matrix (B-ECM) which could be processed into a hydrogel (B-ECM hydrogel) to be used as a scaffold for human embryonic stem cell (hESC)-derived brain organoids. We decellularized pig brains with a novel detergent- and enzyme-based method and analyzed the biomaterial properties, including protein composition and content, DNA content, mechanical characteristics, surface structure, and antigen presence. Then, we compared the growth of human brain organoid models with the B-ECM hydrogel or Matrigel controls in vitro. We found that the native brain source material was successfully decellularized with little remaining DNA content, while Mass Spectrometry (MS) showed the loss of several brain-specific proteins, while mainly different collagen types remained in the B-ECM. Rheological results revealed stable hydrogel formation, starting from B-ECM hydrogel concentrations of 5 mg/mL. hESCs cultured in B-ECM hydrogels showed gene expression and differentiation outcomes similar to those grown in Matrigel. These results indicate that B-ECM hydrogels can be used as an alternative scaffold for human cerebral organoid formation, and may be further optimized for improved organoid growth by further improving protein retention other than collagen after decellularization.
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40. |
- Simsa, Robin, et al.
(author)
-
Effect of fluid dynamics on decellularization efficacy and mechanical properties of blood vessels
- 2019
-
In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 14:8
-
Journal article (peer-reviewed)abstract
- Decellularization of blood vessels is a promising approach to generate native biomaterials for replacement of diseased vessels. The decellularization process affects the mechanical properties of the vascular graft and thus can have a negative impact for in vivo functionality. The aim of this study was to determine how detergents under different fluid dynamics affects decellularization efficacy and mechanical properties of the vascular graft. We applied a protocol utilizing 1% TritonX, 1% Tributyl phosphate (TnBP) and DNase on porcine vena cava. The detergents were applied to the vessels under different conditions; static, agitation and perfusion with 3 different perfusion rates (25, 100 and 400 mL/min). The decellularized grafts were analyzed with histological, immunohistochemical and mechanical tests. We found that decellularization efficacy was equal in all groups, however the luminal ultrastructure of the static group showed remnant cell debris and the 400 mL/min perfusion group showed local damage and tearing of the luminal surface. The mechanical stiffness and maximum tensile strength were not influenced by the detergent application method. In conclusion, our results indicate that agitation or low-velocity perfusion with detergents are preferable methods for blood vessel decellularization.
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41. |
- Simsa, Robin, et al.
(author)
-
Extracellular Heme Proteins Influence Bovine Myosatellite Cell Proliferation and the Color of Cell-Based Meat
- 2019
-
In: Foods. - : MDPI AG. - 2304-8158. ; 8:10
-
Journal article (peer-reviewed)abstract
- Skeletal muscle-tissue engineering can be applied to produce cell-based meat for human consumption, but growth parameters need to be optimized for efficient production and similarity to traditional meat. The addition of heme proteins to plant-based meat alternatives was recently shown to increase meat-like flavor and natural color. To evaluate whether heme proteins also have a positive effect on cell-based meat production, bovine muscle satellite cells (BSCs) were grown in the presence of hemoglobin (Hb) or myoglobin (Mb) for up to nine days in a fibrin hydrogel along 3D-printed anchor-point constructs to generate bioartificial muscles (BAMs). The influence of heme proteins on cell proliferation, tissue development, and tissue color was analyzed. We found that the proliferation and metabolic activity of BSCs was significantly increased when Mb was added, while Hb had no, or a slightly negative, effect. Hb and, in particular, Mb application led to a very similar color of BAMs compared to cooked beef, which was not noticeable in groups without added heme proteins. Taken together, these results indicate a potential benefit of adding Mb to cell culture media for increased proliferation and adding Mb or Hb for the coloration of cell-based meat.
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42. |
- Simsa, Robin, et al.
(author)
-
Systematic in vitro comparison of decellularization protocols for blood vessels.
- 2018
-
In: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 13:12
-
Journal article (peer-reviewed)abstract
- Decellularization of native blood vessels is a promising technology to generate 3D biological scaffolds for vascular grafting. Blood vessel decellularization has been performed in previous studies under various experimental conditions, that complicates comparison and optimization of suitable protocols. The goal of this work was to systematically compare the decellularization and recellularization efficacy of 5 different protocols utilizing the detergents sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), CHAPS and TritonX-100 together with DNA-removing enzymes on porcine vena cava in a perfusion bioreactor setup. Additionally, we tested the effect of DNase on the extracellular matrix (ECM) properties. We found that all protocols could efficiently decellularize blood vessels. Mechanical strength, collagen preservation and ECM integrity were similar among all tested detergents, yet TritonX protocols required long-term DNase application for complete decellularization. However, TritonX-based protocols showed the greatest recellularization efficacy with HUVECs in vitro. Furthermore, we developed a novel protocol for TritonX which improved recellularization and reduced total process time and ECM stiffness compared to previous protocols. SDS, SDC and CHAPS based protocols had a lower recellularization potential. In conclusion, decellularization of blood vessels can be achieved with all tested reagents, but TritonX treated ECM can be most efficiently recellularized with endothelial cells.
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43. |
- Sommer, Felix, et al.
(author)
-
Site-specific programming of the host epithelial transcriptome by the gut microbiota
- 2015
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In: Genome Biology. - : Springer Science and Business Media LLC. - 1465-6906 .- 1474-760X. ; 16
-
Journal article (peer-reviewed)abstract
- Background: The intestinal epithelium separates us from the microbiota but also interacts with it and thus affects host immune status and physiology. Previous studies investigated microbiota-induced responses in the gut using intact tissues or unfractionated epithelial cells, thereby limiting conclusions about regional differences in the epithelium. Here, we sought to investigate microbiota-induced transcriptional responses in specific fractions of intestinal epithelial cells. To this end, we used microarray analysis of laser capture microdissection (LCM)-harvested ileal and colonic tip and crypt epithelial fractions from germ-free and conventionally raised mice and from mice during the time course of colonization. Results: We found that about 10% of the host's transcriptome was microbially regulated, mainly including genes annotated with functions in immunity, cell proliferation, and metabolism. The microbial impact on host gene expression was highly site specific, as epithelial responses to the microbiota differed between cell fractions. Specific transcriptional regulators were enriched in each fraction. In general, the gut microbiota induced a more rapid response in the colon than in the ileum. Conclusions: Our study indicates that the microbiota engage different regulatory networks to alter host gene expression in a particular niche. Understanding host-microbiota interactions on a cellular level may facilitate signaling pathways that contribute to health and disease and thus provide new therapeutic strategies. RAMS GD, 1963, LABORATORY INVESTIGATION, V12, P355
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44. |
- Tkachenko, Eugene, et al.
(author)
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The nucleus of endothelial cell as a sensor of blood flow direction.
- 2013
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In: Biology open. - : The Company of Biologists. - 2046-6390. ; 2:10, s. 1007-12
-
Journal article (peer-reviewed)abstract
- Hemodynamic shear stresses cause endothelial cells (ECs) to polarize in the plane of the flow. Paradoxically, under strong shear flows, ECs disassemble their primary cilia, common sensors of shear, and thus must use an alternative mechanism of sensing the strength and direction of flow. In our experiments in microfluidic perfusion chambers, confluent ECs developed planar cell polarity at a rate proportional to the shear stress. The location of Golgi apparatus and microtubule organizing center was biased to the upstream side of the nucleus, i.e. the ECs polarized against the flow. These in vitro results agreed with observations in murine blood vessels, where EC polarization against the flow was stronger in high flow arteries than in veins. Once established, flow-induced polarization persisted over long time intervals without external shear. Transient destabilization of acto-myosin cytoskeleton by inhibition of myosin II or depolymerization of actin promoted polarization of EC against the flow, indicating that an intact acto-myosin cytoskeleton resists flow-induced polarization. These results suggested that polarization was induced by mechanical displacement of EC nuclei downstream under the hydrodynamic drag. This hypothesis was confirmed by the observation that acute application of a large hydrodynamic force to ECs resulted in an immediate downstream displacement of nuclei and was sufficient to induce persistent polarization. Taken together, our data indicate that ECs can sense the direction and strength of blood flow through the hydrodynamic drag applied to their nuclei.
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45. |
- Tsiantoulas, D., et al.
(author)
-
APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans
- 2021
-
In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 597, s. 92-96
-
Journal article (peer-reviewed)abstract
- Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide(1). The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs)(2) and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall(2). A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs(3), but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits the retention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis.
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46. |
- Wilhelmson, Anna S K, et al.
(author)
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Catechol-O-methyltransferase is dispensable for vascular protection by estradiol in mouse models of atherosclerosis and neointima formation.
- 2011
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In: Endocrinology. - : The Endocrine Society. - 1945-7170 .- 0013-7227. ; 152:12, s. 4683-90
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Journal article (peer-reviewed)abstract
- Estradiol is converted to the biologically active metabolite 2-methoxyestradiol via the activity of the enzyme catechol-O-methyltransferase (COMT). Exogenous administration of both estradiol and 2-methoxyestradiol reduces experimental atherosclerosis and neointima formation, and COMT-dependent formation of 2-methoxyestradiol likely mediates the antimitogenic effect of estradiol on smooth muscle cells in vitro. This study evaluated whether 2-methoxyestradiol mediates the vasculoprotective actions of estradiol in vivo. Wild-type (WT) and COMT knockout (COMTKO) mice on an apolipoprotein E-deficient background were gonadectomized and treated with estradiol or placebo. Exogenous estradiol reduced atherosclerotic lesion formation in both females (WT, -78%; COMTKO, -82%) and males (WT, -48%; COMTKO, -53%) and was equally effective in both genotypes. We further evaluated how exogenous estradiol affected neointima formation after ligation of the carotid artery in ovariectomized female mice; estradiol reduced intimal hyperplasia to a similar extent in both WT (-80%) and COMTKO (-77%) mice. In ovarian-intact female COMTKO mice, atherosclerosis was decreased (-25%) compared with WT controls. In conclusion, the COMT enzyme is dispensable for vascular protection by exogenous estradiol in experimental atherosclerosis and neointima formation in vivo. Instead, COMT deficiency in virgin female mice with intact endogenous production of estradiol results in relative protection against atherosclerosis.
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47. |
- Wilhelmson, Anna S K, et al.
(author)
-
Testosterone is an endogenous regulator of BAFF and splenic B cell number
- 2018
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 9:1
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Journal article (peer-reviewed)abstract
- Testosterone deficiency in men is associated with increased risk for autoimmunity and increased B cell numbers through unknown mechanisms. Here we show that testosterone regulates the cytokine BAFF, an essential survival factor for B cells. Male mice lacking the androgen receptor have increased splenic B cell numbers, serum BAFF levels and splenic Baff mRNA. Testosterone deficiency by castration causes expansion of BAFF-producing fibro-blastic reticular cells (FRCs) in spleen, which may be coupled to lower splenic noradrenaline levels in castrated males, as an alpha-adrenergic agonist decreases splenic FRC number in vitro. Antibody-mediated blockade of the BAFF receptor or treatment with the neurotoxin 6-hydroxydopamine revert the increased splenic B cell numbers induced by castration. Among healthy men, serum BAFF levels are higher in men with low testosterone. Our study uncovers a previously unrecognized regulation of BAFF by testosterone and raises important questions about BAFF in testosterone-mediated protection against autoimmunity.
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48. |
- Wilhelmson, Anna S K, et al.
(author)
-
Testosterone Protects Against Atherosclerosis in Male Mice by Targeting Thymic Epithelial Cells.
- 2018
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In: Arteriosclerosis, thrombosis, and vascular biology. - 1524-4636. ; 38:7, s. 1519-1527
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Journal article (peer-reviewed)abstract
- Androgen deprivation therapy has been associated with increased cardiovascular risk in men. Experimental studies support that testosterone protects against atherosclerosis, but the target cell remains unclear. T cells are important modulators of atherosclerosis, and deficiency of testosterone or its receptor, the AR (androgen receptor), induces a prominent increase in thymus size. Here, we tested the hypothesis that atherosclerosis induced by testosterone deficiency in male mice is T-cell dependent. Further, given the important role of the thymic epithelium for T-cell homeostasis and development, we hypothesized that depletion of the AR in thymic epithelial cells will result in increased atherosclerosis.Prepubertal castration of male atherosclerosis-prone apoE-/- mice increased atherosclerotic lesion area. Depletion of T cells using an anti-CD (cluster of differentiation) 3 antibody abolished castration-induced atherogenesis, demonstrating a role of T cells. Male mice with depletion of the AR specifically in epithelial cells (E-ARKO [epithelial cell-specific AR knockout] mice) showed increased thymus weight, comparable with that of castrated mice. E-ARKO mice on an apoE-/- background displayed significantly increased atherosclerosis and increased infiltration of T cells in the vascular adventitia, supporting a T-cell-driven mechanism. Consistent with a role of the thymus, E-ARKO apoE-/- males subjected to prepubertal thymectomy showed no atherosclerosis phenotype.We show that atherogenesis induced by testosterone/AR deficiency is thymus- and T-cell dependent in male mice and that the thymic epithelial cell is a likely target cell for the antiatherogenic actions of testosterone. These insights may pave the way for new therapeutic strategies for safer endocrine treatment of prostate cancer.
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