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Johan Thyberg

Publications and source records attributed to Johan Thyberg.

28 records · Page 2Linked to original sources
PubMed2003

Small GTP-binding protein Rac is an essential mediator of vascular endothelial growth factor-induced endothelial fenestrations and vascular permeability.

BACKGROUND: Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induces both angiogenesis and vascular permeability. Although its angiogenic activity has been well characterized, the signaling pathways of VEGF-induced permeability remain poorly understood. METHODS AND RESULTS: Using the mouse corneal micropocket assay, Miles assay, and a combination of cytochemical, electron microscopic, and biochemical assays, we demonstrate that VEGF-induced vascular leakage partly can be separated from its angiogenic activity. VEGF but not FGF-2 induced capillaries with a highly fenestrated endothelium, a feature linked with increased vascular permeability. A cell-permeable Rac antagonist (TAT-RacN17) converted VEGF-induced, leaky vascular plexuses into well-defined vascular networks. In addition, this Rac mutant blocked formation of VEGF-induced endothelial fenestrations and vascular permeability but only partially inhibited angiogenesis. Studies on endothelial cell cultures further revealed that VEGF stimulated phosphorylation of VEGF receptor-2 (VEGFR-2), leading to activation of Rac as well as increased phosphorylation of phospholipase Cgamma (PLCgamma), protein kinase B (Akt), endothelial nitric oxide synthase (eNOS), and extracellular regulated kinase (Erk1/2). We further found that phosphatidylinositol-3-OH kinase (PI3K) acted upstream of Rac and Akt-eNOS in VEGF/VEGFR-2 signaling. CONCLUSIONS: Our findings indicate that the small GTP-binding protein Rac is a key component in mediation of VEGF-induced vascular permeability but less so in neovascularization. This may have conceptual implications for applying Rac antagonists in treatment and prevention of VEGF-induced vascular leakage and edema in connection with ischemic disorders.

Animals↗
PubMed2003

Influence of intraluminal thrombus on structural and cellular composition of abdominal aortic aneurysm wall.

INTRODUCTION: It has been suggested that the intraluminal thrombus of abdominal aortic aneurysm (AAA) affects the underlying vessel wall. Aneurysm enlargement has been associated with growth of thrombus, and rupture has been proposed to occur after bleeding into the thrombus. To examine how thrombus affects the vessel wall, we compared the morphology of aneurysm wall covered with thrombus with wall segments exposed to flowing blood. Material and methods Sixteen patients (14 men, 2 women; age range, 56-79 years) undergoing elective repair of AAA, where computed tomography scans showed thrombus and segments of the aneurysm wall exposed to flowing blood, were included in the study. Specimens from the aneurysm were taken for light and electron microscopy. Masson trichrome staining was performed for wall thickness determination and demonstration of collagen, and Weigert-van Gieson staining for elastin. The cellular composition was analyzed by immunohistochemistry with antibodies against CD3 for T cells, CD4 for T helper cells, CD8 for T cytotoxic cells, CD20 for B cells, CD68 for macrophages, and smooth muscle alpha-actin for smooth muscle cells (SMCs). Caspase-3 staining and TUNEL analysis were performed to evaluate apoptosis. RESULTS: The aneurysm wall covered with thrombus was thinner and contained fewer elastin fibers, and the few that were found were often fragmented. This part of the wall also contained fewer SMCs and more apoptotic nuclei than the wall exposed to flowing blood. Clusters of inflammatory cells were detected in the media of the aneurysm wall and in higher numbers in the parts covered with thrombus. Electron microscopy showed that the aneurysm wall without thrombus contained a dense collagenous matrix with differentiated SMCs. In the segment covered with thrombus, SMCs were more dedifferentiated (synthetic) and apoptotic or necrotic. There were also an increased number of inflammatory cells located in close contact with SMCs in various stages of apoptosis. CONCLUSION: The aneurysm wall covered with thrombus is thinner and shows more frequent signs of inflammation, apoptosis of SMCs, and degraded extracellular matrix. These findings suggest that thrombus formation and accumulation of inflammatory cells may perturb the structural integrity and stability of the vessel wall and thereby increase the risk for aneurysm rupture.

Aged↗
PubMed2003

Shortened life span, bradycardia, and hypotension in mice with targeted expression of an Igf2 transgene in smooth muscle cells.

IGF2 is known to affect the normal development and pathology of the cardiovascular system. We previously created mutant mice with targeted expression of an Igf2 transgene in the smooth muscle cells and showed that these mice spontaneously develop aortic intimal cushions. In the present work, we provide a general description of the phenotype of two independent lines of heterozygous transgenics. These mice showed organomegaly and a shortened life span. The latter trait was stronger in the line with a relatively more marked organomegaly and more pronounced in males than females in both lines. Postmortem histology revealed gross abnormalities of the cardiac architecture, suggesting that transgenic mice may accumulate lethal cardiovascular defects. Accordingly, apparently normal transgenic mice had mild cardiomegaly, an enlarged left ventricle, bradycardia, and hypotension. These observations are discussed in the light of the proposed therapeutic use of IGF2 in human cardiac diseases.

Animals↗
PubMed2003

Arteriosclerosis in rat aortic allografts: dynamics of cell growth, apoptosis and expression of extracellular matrix proteins.

Transplant vasculopathy is a key factor behind the late loss of transplanted organs. Since effective treatment is still lacking, a further understanding of the pathology of this process is important. Here, a rat model of aortic allografts was used and analyzed by immunohistochemistry and biochemical tests. Infrarenal aortic segments were transplanted from F344 to Lewis rats and analysed after 1-12 weeks using isografts as controls. After 1 week, endothelial cells gradually disappeared at the graft lumen as shown by von Willebrand factor staining and cellular activation was detected in the adventitia and intima using cellular retinol-binding protein-1 as a marker. Subsequently, proliferating smooth muscle cells, lymphocytes and macrophages accumulated in the intima as indicated by the appearance of staining for cell- and proliferation-specific antigens (smooth muscle alpha-actin, CD45RC, ED1, cyclin D1 and proliferating cell nuclear antigen). After 4-8 weeks, TUNEL- and Fas-positive cells were observed in the media, denoting progressive apoptosis. In parallel, the developing neointima contained increased immunoreactivity for fibronectin and osteopontin. At the end of the observation period, an accumulation of macrophages and calcification was observed in the media and endothelial cells reappeared at the graft surface. The findings demonstrate major cellular and structural changes in the transplanted artery, including activation, proliferation and apoptosis of SMCs, and an altered composition of the extracellular matrix. Possibly, the observed changes in SMC phenotype, cell cycle and apoptosis during development of transplant arteriosclerosis are related to the expression of extracellular matrix proteins.

Animals↗
PubMed2002

Smooth-muscle progenitor cells of bone marrow origin contribute to the development of neointimal thickenings in rat aortic allografts and injured rat carotid arteries.

This study indicates that circulating progenitors of bone marrow origin give rise to cells with smooth muscle-like properties during formation of neointimal thickenings in the arterial wall after allotransplantation and after balloon injury. A segment of abdominal aorta was transplanted from female F344 to male LEW rats, and the grafts were analyzed for male cells by using the gene as a marker. Immunostaining demonstrated that CD45-positive leukocytes made up 35-45% of the neointimal cells during the 8-week period examined. Concurrently, up to 70% of the neointimal cells were of host origin, as shown by real-time polymerase chain reaction for the gene (Y chromosome). This suggests that the neointima contained host cells also of noninflammatory character. Accordingly, many cells positive for smooth-muscle alpha-actin were detected in this layer. To explore the possible bone marrow origin of allograft cells, female LEW rats were irradiated and substituted with bone marrow from male LEW rats. Subsequently, the animals received an aortic transplant from female F344 rats or were exposed to a balloon injury of the carotid artery. Immunostaining and real-time polymerase chain reaction confirmed the above findings, but the fractions of leukocytes and -positive cells were lower in the carotids than in the allografts. Combined primed in situ labeling and immunostaining verified that not only inflammatory but also smooth muscle-like cells of male origin appeared in the vessel wall in both situations. These observations suggest that the smooth-muscle cells that participate in the development of neointimal lesions during vascular disease may, in part, originate from circulating progenitors.

Animals↗
PubMed2002

Charge attraction and beta propensity are necessary for amyloid fibril formation from tetrapeptides.

Amyloid fibrils in which specific proteins have polymerized into a cross-beta-sheet structure are found in about 20 diseases. In contrast to the close structural similarity of fibrils formed in different amyloid diseases, the structures of the corresponding native proteins differ widely. We show here that peptides as short as 4 residues with the sequences KFFE or KVVE can form amyloid fibrils that are practically identical to fibrils formed in association with disease, as judged by electron microscopy and Congo red staining. In contrast, KLLE or KAAE do not form fibrils. The fibril-forming KFFE and KVVE show partial beta-strand conformation in solution, whereas the non-fibril-forming KLLE and KAAE show random structure only, suggesting that inherent propensity for beta-strand conformation promotes fibril formation. The peptides KFFK or EFFE do not form fibrils on their own but do so in an equimolar mixture. Thus, intermolecular electrostatic interactions, either between charged dipolar peptides or between complementary charges of co-fibrillating peptides favor fibril formation.

Amino Acid Sequence↗
PubMed2002

Assembling amyloid fibrils from designed structures containing a significant amyloid beta-peptide fragment.

The amyloid plaque, consisting of amyloid beta-peptide (Abeta) fibrils surrounded by dystrophic neurites, is an invariable feature of Alzheimer's disease. The determination of the molecular structure of Abeta fibrils is a significant goal that may lead to the structure-based design of effective therapeutics for Alzheimer's disease. Technical challenges have thus far rendered this goal impossible. In the present study, we develop an alternative methodology. Rather than determining the structure directly, we design conformationally constrained peptides and demonstrate that only certain 'bricks' can aggregate into fibrils morphologically identical to Abeta fibrils. The designed peptides include variants of a decapeptide fragment of Abeta, previously shown to be one of the smallest peptides that (1) includes a pentapeptide sequence necessary for Abeta-Abeta binding and aggregation and (2) can form fibrils indistinguishable from those formed by full-length Abeta. The secondary structure of these bricks is monitored by CD spectroscopy, and electron microscopy is used to study the morphology of the aggregates formed. We then made various residue deletions and substitutions to determine which structural features are essential for fibril formation. From the constraints, statistical analysis of side-chain pair correlations in beta-sheets and experimental data, we deduce a detailed model of the peptide strand alignment in fibrils formed by these bricks. Our results show that the constrained decapeptide dimers rapidly form an intramolecular, antiparallel beta-sheet and polymerize into amyloid fibrils at low concentrations. We suggest that the formation of an exposed beta-sheet (e.g. an Abeta dimer formed by interaction in the decapeptide region) could be a rate-limiting step in fibril formation. A theoretical framework that explains the results is presented in parallel with the data.

Alzheimer Disease↗
PubMed2002

Caveolae and cholesterol distribution in vascular smooth muscle cells of different phenotypes.

Vascular smooth muscle cells (SMCs) grown in primary culture are converted from a contractile to a synthetic phenotype. This includes a marked morphological reorganization, with loss of myofilaments and formation of a large ER-Golgi complex. In addition, the number of cell surface caveolae is distinctly reduced and the handling of lipoprotein-derived cholesterol changed. Here we used filipin as a marker to study the distribution of cholesterol in SMCs by electron microscopy. In contractile cells, filipin-sterol complexes were preferentially found in caveolae and adjacent ER cisternae (present in both leaflets of the membranes). After exposure to LDL or cholesterol, labeling with filipin was increased both in membrane organelles and in the cytoplasm. In contrast, treatment with mevinolin (a cholesterol synthesis inhibitor) or beta-cyclodextrin (a molecule that extracts cholesterol from cells) decreased the reaction with filipin but did not affect the close relation between the ER and the cell surface. In synthetic cells, filipin-sterol complexes were diffusely spread in the plasma membrane and the strongest cytoplasmic reaction was noted in endosomes/lysosomes, both under normal conditions and after incubation with LDL or cholesterol. On the basis of the present findings, we propose a mechanism for direct exchange of cholesterol between the plasma membrane and the ER and more active in contractile than in synthetic SMCs.

Animals↗
PubMed2001

Insulin-like growth factor II plays a central role in atherosclerosis in a mouse model.

Insulin-like growth factor II is a fetal promoter of cell proliferation that is involved in some forms of cancer and overgrowth syndromes in humans. Here, we provide two sources of genetic evidence for a novel, pivotal role of locally produced insulin-like growth factor II in the development of atherosclerosis. First, we show that homozygosity for a disrupted insulin-like growth factor II allele in mice lacking apolipoprotein E, a widely used animal model of atherosclerosis, results in aortic lesions that are approximately 80% smaller and contain approximately 50% less proliferating cells compared with mice lacking only apolipoprotein E. Second, targeted expression of an insulin-like growth factor II transgene in smooth muscle cells, but not the mere elevation of circulating levels of the peptide, causes per se aortic focal intimal thickenings. The insulin-like growth factor II transgenics presented here are the first viable mutant mice spontaneously developing intimal masses. These observations provide the first direct evidence for an atherogenic activity of insulin-like growth factor II in vivo.

Animals↗