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  • 1.
    Ahl, David
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Eriksson, Olle
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Sedin, John
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Seignez, Cedric
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Schwan, Emil
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Christoffersson, Gustaf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Phillipson, Mia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Turning Up the Heat: Local Temperature Control During in vivo Imaging of Immune Cells2019In: Frontiers in Immunology, ISSN 1664-3224, E-ISSN 1664-3224, Vol. 10, article id 2036Article in journal (Refereed)
    Abstract [en]

    Intravital imaging is an invaluable tool for studying the expanding range of immune cell functions. Only in vivo can the complex and dynamic behavior of leukocytes and their interactions with their natural microenvironment be observed and quantified. While the capabilities of high-speed, high-resolution confocal and multiphoton microscopes are well-documented and steadily improving, other crucial hardware required for intravital imaging is often developed in-house and less commonly published in detail. In this report, we describe a low-cost, multipurpose, and tissue-stabilizing in vivo imaging platform that enables sensing and regulation of local tissue temperature. The effect of tissue temperature on local blood flow and leukocyte migration is demonstrated in muscle and skin. Two different models of vacuum windows are described in this report, however, the design of the vacuum window can easily be adapted to fit different organs and tissues.

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  • 2.
    Barkefors, Irmeli
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fredlund Fuchs, Peder
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Heldin, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bergström, Tobias
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Forsberg-Nilsson, Karin
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Exocyst Complex Component 3-like 2 (EXOC3L2) Associates with the Exocyst Complex and Mediates Directional Migration of Endothelial Cells2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 27, p. 24189-24199Article in journal (Refereed)
    Abstract [en]

    The exocyst is a protein complex that ensures spatial targeting of exocytotic vesicles to the plasma membrane. We present microarray data obtained from differentiating mouse embryonic stem cell cultures that identify an up-regulation of exocyst complex component 3-like 2 (exoc3l2) mRNA in sprouting blood vessels. Vascular expression of exoc3l2 is confirmed by qPCR analysis of different mouse tissues and immunofluorescence analyses of mouse brain sections. We detect an up-regulation of exoc3l2 mRNA synthesis in primary human endothelial cells in response to VEGFA, and this response is enhanced when the cells are grown on a three-dimensional collagen I matrix. Myc-tagged EXOC3L2 co-precipitates with the exocyst protein EXOC4, and immunofluorescence detection of EXOC3L2 shows partial subcellular colocalization with EXOC4 and EXOC7. Finally, we show that exoc3l2 silencing inhibits VEGF receptor 2 phosphorylation and VEGFA-directed migration of cultured endothelial cells.

  • 3.
    Barkefors, Irmeli
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Le Jan, Sébastien
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jakobsson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Hejll, Eduar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Carlson, Gustav
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Johansson, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jarvius, Jonas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Park, Jeong Won
    Li Jeon, Noo
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Endothelial cell migration in stable gradients of vascular endothelial growth factor A and fibroblast growth factor 2: effects on chemotaxis and chemokinesis2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 283, no 20, p. 13905-13912Article in journal (Refereed)
    Abstract [en]

    Gradients of secreted signaling proteins guide growing blood vessels during both normal and pathological angiogenesis. However, the mechanisms by which endothelial cells integrate and respond to graded distributions of chemotactic factors are still poorly understood. We have in this study investigated endothelial cell migration in response to hill-shaped gradients of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2) using a novel microfluidic chemotaxis chamber (MCC). Cell migration was scored at the level of individual cells using time-lapse microscopy. A stable gradient of VEGFA165 ranging from 0 to 50 ng/ml over a distance of 400 microm was shown to strongly induce chemotaxis of endothelial cells of different vascular origin. VEGFA121, unable to bind proteoglycan and neuropilin coreceptors, was also shown to induce chemotaxis in this setup. Furthermore, a gradient of FGF2 was able to attract venular but not arterial endothelial cells, albeit less efficiently than VEGFA165. Notably, constant levels of VEGFA165, but not of FGF2, were shown to efficiently reduce chemokinesis. Systematic exploration of different gradient shapes led to the identification of a minimal gradient steepness required for efficient cell guidance. Finally, analysis of cell migration in different regions of the applied gradients showed that chemotaxis is reduced when cells reach the high end of the gradient. Our findings suggest that chemotactic growth factor gradients may instruct endothelial cells to shift toward a nonmigratory phenotype when approaching the growth factor source.

  • 4.
    Barkefors, Irmeli
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Thorslund, Sara
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Nikolajeff, Fredrik
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A fluidic device to study directional angiogenesis in complex tissue and organ culture models2009In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 9, no 4, p. 529-535Article in journal (Refereed)
    Abstract [en]

    Many signals that induce angiogenesis have been identified; however, it is still not clear how these signals interact to shape the vascular system. We have developed a fluidic device for generation of molecular gradients in 3-dimensional cultures of complex tissues and organs in order to create an assay for precise induction and guidance of growing blood vessels. The device features a centrally placed culture chamber, flanked by channels attached to a perfusion system used to generate gradients. A separate network of vacuum channels permits reversible attachment of the device to a flat surface. We show that the fluidic device can be used to create growth factor gradients that induce directional angiogenesis in embryonic mouse kidneys and in clusters of differentiating stem cells. These results demonstrate that the device can be used to accurately manipulate complex morphogenetic processes with a high degree of experimental control.

  • 5.
    Blom, Magdalena
    et al.
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet.
    Reis, Katarina
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet.
    Heldin, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Aspenström, Pontus
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet.
    The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration2017In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 352, no 2, p. 255-264Article in journal (Refereed)
    Abstract [en]

    RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as cortical actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration.

  • 6.
    Christoffersson, Gustaf
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Lomei, Jalal
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Engblom, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computational Science.
    Phillipson, Mia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Vascular sprouts induce local attraction of proangiogenic neutrophils2017In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 102, p. 741-751Article in journal (Refereed)
  • 7.
    Fatsis-Kavalopoulos, Nikos
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Gradientech AB, Uppsala, Sweden.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Xie, Beichen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Hernández Vera, Rodrigo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Idevall Hagren, Olof
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution2019In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 19, no 6, p. 1071-1081Article in journal (Refereed)
    Abstract [en]

    The function and behaviour of any given cell in a healthy tissue, or in a tumor, is affected by interactions with its neighboring cells. It is therefore important to create methods that allow for reconstruction of tissue niches in vitro for studies of cell-cell signaling and associated cell behaviour. To this end we created the cell assembly generator (CAGE), a microfluidic device which enables the organization of different cell types into precise cell clusters in a flow chamber compatible with high-resolution microscopy. In proof-of-concept paracrine signalling experiments, 4-cell clusters consisting of one pancreatic -cell and three breast cancer cells were formed. It has previously been established that extracellular ATP induces calcium (Ca2+) release from the endoplasmic reticulum (ER) to the cytosol before it is cleared back into the ER via sarcoplasmic/ER Ca2+ ATPase (SERCA) pumps. Here, ATP release from the -cell was stimulated by depolarization, and dynamic changes in Ca2+ levels in the adjacent cancer cells measured using imaging of the calcium indicator Fluo-4. We established that changes in the concentration of cytosolic Ca2+ in the cancer cells were proportional to the distance from the ATP-releasing -cell. Additionally, we established that the relationship between distance and cytosolic calcium changes were dependent on Ca2+-release from the ER using 5-cell clusters composed of one -cell, two untreated cancer cells and two cancer cells pretreated with Thapsigargin (to deplete the ER of Ca2+). These experiments show that the CAGE can be used to create exact cell clusters, which affords precise control for reductionist studies of cell-cell signalling and permits the formation of heterogenous cell models of specific tissue niches.

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  • 8.
    Filipek-Gorniok, Beata
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Holmborn, Katarina
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology.
    Haitina, Tatjana
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Habicher, Judith
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Oliveira, Marta Bastos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hellgren, Charlotte
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ledin, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Expression of chondroitin/dermatan sulfate glycosyltransferases during early zebrafish development2013In: Developmental Dynamics, ISSN 1058-8388, E-ISSN 1097-0177, Vol. 242, no 8, p. 964-975Article in journal (Refereed)
    Abstract [en]

    Background: Chondroitin/dermatan sulfate (CS/DS) proteoglycans present in the extracellular matrix have important structural and regulatory functions. Results: Six human genes have previously been shown to catalyze CS/DS polymerization. Here we show that one of these genes, chpf, is represented by two copies in the zebrafish genome, chpfa and chpfb, while the other five human CS/DS glycosyltransferases csgalnact1, csgalnact2, chpf2, chsy1, and chsy3 all have single zebrafish orthologues. The putative zebrafish CS/DS glycosyltransferases are spatially and temporally expressed. Interestingly, overlapping expression of multiple glycosyltransferases coincides with high CS/DS deposition. Finally, whereas the relative levels of the related polysaccharide HS reach steady-state at around 2 days post fertilization, there is a continued relative increase of the CS amounts per larvae during the first 6 days of development, matching the increased cartilage formation. Conclusions: There are 7 CS/DS glycosyltransferases in zebrafish, which, based on homology, can be divided into the CSGALNACT, CHSY, and CHPF families. The overlap between intense CS/DS production and the expression of multiple CS/DS glycosyltransferases suggests that efficient CS/DS biosynthesis requires a combination of several glycosyltransferases.

  • 9.
    Heindryckx, Femke
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Binet, F.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology.
    ER Stress Enhances Liver Fibrosis Through MIR-150 Degradation and XBP-1 Splicing2015In: Journal of Hepatology, ISSN 0168-8278, E-ISSN 1600-0641, Vol. 62, no S2, p. S336-S336Article in journal (Other academic)
  • 10.
    Heldin, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fredlund Fuchs *, Peder
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    O’Callaghan *, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kasza, Zsolt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Le Jan, Sebastien
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kampf, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Aspenström, Pontus
    Dept. of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    FGD5 promotes VEGF-A signaling via inhibition of VEGF-receptor ubiquitination and degradationManuscript (preprint) (Other academic)
  • 11.
    Heldin, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Hernández Vera, Rodrigo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Fredlund Fuchs, Peder
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    FGD5 sustains vascular endothelial growth factor A (VEGFA) signaling through inhibition of proteasome-mediated VEGF receptor 2 degradation2017In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 40, p. 125-132Article in journal (Refereed)
    Abstract [en]

    The complete repertoire of endothelial functions elicited by FGD5, a guanine nucleotide exchange factor activating the Rho GTPase Cdc42, has yet to be elucidated. Here we explore FGD5's importance during vascular endothelial growth factor A (VEGFA) signaling via VEGF receptor 2 (VEGFR2) in human endothelial cells. In microvascular endothelial cells, FGD5 is located at the inner surface of the cell membrane as well as at the outer surface of EEAl-positive endosomes carrying VEGFR2. The latter finding prompted us to explore if FGD5 regulates VEGFR2 dynamics. We found that depletion of FGD5 in microvascular cells inhibited their migration towards a stable VEGFA gradient. Furthermore, depletion of FGD5 resulted in accelerated VEGFR2 degradation, which was reverted by lactacystin-mediated proteasomal inhibition. Our results thus suggest a mechanism whereby FGD5 sustains VEGFA signaling and endothelial cell chemotaxis via inhibition of proteasome-dependent VEGFR2 degradation.

  • 12.
    Hernández Vera, Rodrigo
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Fatsis-Kavalopoulos, Nikos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Gradientech AB, Uppsala Science Park, Uppsala, Sweden.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Modular microfluidic systems cast from 3D-printed molds for imaging leukocyte adherence to differentially treated endothelial cultures2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 11321Article in journal (Refereed)
    Abstract [en]

    Microfluidic systems are very useful for in vitro studies of interactions between blood cells and vascular endothelial cells under flow, and several commercial solutions exist. However, the availability of customizable, user-designed devices is largely restricted to researchers with expertise in photolithography and access to clean room facilities. Here we describe a strategy for producing tailor-made modular microfluidic systems, cast in PDMS from 3D-printed molds, to facilitate studies of leukocyte adherence to endothelial cells. A dual-chamber barrier module was optimized for culturing two endothelial cell populations, separated by a 250 μm wide dividing wall, on a glass slide. In proof-of-principle experiments one endothelial population was activated by TNFα, while the other served as an internal control. The barrier module was thereafter replaced with a microfluidic flow module, enclosing both endothelial populations in a common channel. A suspension of fluorescently-labeled leukocytes was then perfused through the flow module and leukocyte interactions with control and tnfα-treated endothelial populations were monitored in the same field of view. Time-lapse microscopy analysis confirmed the preferential attachment of leukocytes to the TNFα-activated endothelial cells. We conclude that the functionality of these modular microfluidic systems makes it possible to seed and differentially activate adherent cell types, and conduct controlled side-by-side analysis of their capacity to interact with cells in suspension under flow. Furthermore, we outline a number of practical considerations and solutions associated with connecting and switching between the microfluidic modules, and the advantages of simultaneously and symmetrically analyzing control and experimental conditions in such a microfluidic system.

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    fulltext
  • 13.
    Hernández Vera, Rodrigo
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Schwan, Emil
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Fatsis-Kavalopoulos, Nikos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Gradientech AB, Uppsala Sci Pk, Uppsala, Sweden..
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D Printed Parts, a Smartphone, and Off-The-Shelf Electronics2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 12, article id e0167583Article in journal (Refereed)
    Abstract [en]

    Time-lapse imaging is a powerful tool for studying cellular dynamics and cell behavior over long periods of time to acquire detailed functional information. However, commercially available time-lapse imaging systems are expensive and this has limited a broader implementation of this technique in low-resource environments. Further, the availability of time-lapse imaging systems often present workflow bottlenecks in well-funded institutions. To address these limitations we have designed a modular and affordable time-lapse imaging and incubation system (ATLIS). The ATLIS enables the transformation of simple inverted microscopes into live cell imaging systems using custom-designed 3D-printed parts, a smartphone, and off-the-shelf electronic components. We demonstrate that the ATLIS provides stable environmental conditions to support normal cell behavior during live imaging experiments in both traditional and evaporation-sensitive microfluidic cell culture systems. Thus, the system presented here has the potential to increase the accessibility of time-lapse microscopy of living cells for the wider research community.

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  • 14.
    Holmborn, Katarina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Habicher, Judith
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kasza, Zsolt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eriksson, Anna S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gorniok, Beata Filipek
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gopal, Sandeep
    Couchman, John R.
    Ahlberg, Per Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wiweger, Malgorzata
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ledin, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    On the Roles and Regulation of Chondroitin Sulfate and Heparan Sulfate in Zebrafish Pharyngeal Cartilage Morphogenesis2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 40, p. 33905-33916Article in journal (Refereed)
    Abstract [en]

    The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes.

  • 15.
    Huang, Hua
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Langenkamp, Elise
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Georganaki, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Loskog, Angelica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical Immunology.
    Fuchs, Peder Fredlund
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Dieterich, Lothar C
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Kreuger, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Dimberg, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    VEGF suppresses T-lymphocyte infiltration in the tumor microenvironment through inhibition of NF-κB-induced endothelial activation2015In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 29, no 1, p. 227-238Article in journal (Refereed)
    Abstract [en]

    Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) signaling pathway is in clinical use, but its effect on vascular function and the tumor microenvironment is poorly understood. Here, we investigate cross-talk between VEGF and proinflammatory TNF-α signaling in endothelial cells and its impact on leukocyte recruitment. We found that cotreatment with VEGF decreased TNF-α-induced Jurkat cell adhesion to human microvascular endothelial cells by 40%. This was associated with inhibition of TNF-α-mediated regulation of 86 genes, including 2 T-lymphocyte-attracting chemokines, CXCL10 and CXCL11 [TNF-α concentration 1 ng/ml; 50% inhibition/inhibitory concentration (IC50) VEGF, 3 ng/ml]. Notably, VEGF directly suppressed TNF-α-induced gene expression through negative cross-talk with the NF-κB-signaling pathway, leading to an early decrease in IFN regulatory factor 1 (IRF-1) expression and reduced phosphorylation of signal transducer and activator of transcription 1 (p-Stat1) at later times. Inhibition of VEGF signaling in B16 melanoma tumor-bearing mice by sunitinib treatment resulted in up-regulation of CXCL10 and CXCL11 in tumor vessels, accompanied by up to 18-fold increased infiltration of CD3(+) T-lymphocytes in B16 tumors. Our results demonstrate a novel role of VEGF in negative regulation of NF-κB signaling and endothelial activation in the tumor microenvironment and provide evidence that pharmacological inhibition of VEGF signaling enhances T-lymphocyte recruitment through up-regulation of chemokines CXCL10 and CXCL11.-Huang, H., Langenkamp, E., Georganaki, M., Loskog, A., Fuchs, P. F., Dieterich, L. C., Kreuger, J., Dimberg, A. VEGF suppresses T-lymphocyte infiltration in the tumor microenvironment through inhibition of NF-κB-induced endothelial activation.

  • 16.
    Hufnagel, Lars
    et al.
    University of California.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Cohen, Stephen M.
    European Molecular Biology Laboratory.
    Shraiman, Boris I.
    University of California.
    On the role of glypicans in the process of morphogen gradient formation2006In: Developmental Biology, ISSN 0012-1606, E-ISSN 1095-564X, Vol. 300, no 2, p. 512-522Article in journal (Refereed)
    Abstract [en]

    Glypicans are cell surface molecules that influence signaling and gradient formation of secreted morphogens and growth factors. Several distinct functions have been ascribed to glypicans including acting as co-receptors for signaling proteins. Recent data show that glypicans are also necessary for morphogen propagation in the tissue. In the present study, a model describing the interaction of a morphogen with glypicans is formulated, analyzed and compared with measurements of the effect of glypican Dally-like (Dlp) overexpression on Wingless (Wg) morphogen signaling in Drosophila melanogaster wing imaginal discs. The model explains the opposing effect that Dlp overexpression has on Wg signaling in the distal and proximal regions of the disc and makes a number of quantitative predictions for further experiments. In particular, our model suggests that Dlp acts by allowing Wg to diffuse on cell surface while protecting it from loss and degradation, and that Dlp rather than acting as Wg co-receptor competes with receptors for morphogen binding.

  • 17.
    Jakobsson, Lars
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Building blood vessels- Stem cell models in vascular biology2007In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 177, no 5, p. 751-755Article, review/survey (Refereed)
    Abstract [en]

    Spheroids of differentiating embryonic stem cells, denoted embryoid bodies, constitute a high-quality model for vascular development, particularly well suited for loss-of-function analysis of genes required for early embryogenesis. This review examines vasculogenesis and angiogenesis in murine embryoid bodies and discusses the promise of stem cell–based models for the study of human vascular development.

  • 18.
    Jakobsson, Lars
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Holmborn, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lundin, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Heparan sulfate in trans potentiates VEGFR-mediated angiogenesis2006In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 10, no 5, p. 625-634Article in journal (Refereed)
    Abstract [en]

    Several receptor tyrosine kinases require heparan sulfate proteoglycans (HSPGs) as coreceptors for efficient signal transduction. We have studied the role of HSPGs in the development of blood capillary structures from embryonic stem cells, a process strictly dependent on signaling via vascular endothelial growth factor receptor-2 (VEGFR-2). We show, by using chimeric cultures of embryonic stem cells defective in either HS production or VEGFR-2 synthesis, that VEGF signaling in endothelial cells is fully supported by HS expressed in trans by adjacent perivascular smooth muscle cells. Transactivation of VEGFR-2 leads to prolonged and enhanced signal transduction due to HS-dependent trapping of the active VEGFR-2 signaling complex. Our data imply that direct signaling via HSPG core proteins is dispensable for a functional VEGF response in endothelial cells. We propose that transactivation of tyrosine kinase receptors by HSPGs constitutes a mechanism for crosstalk between adjacent cells.

  • 19.
    Jemth, Per
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kusche-Gullberg, Marion
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sturiale, Luisa
    Giménez-Gallego, Guillermo
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Biosynthetic oligosaccharide libraries for identification of protein-binding heparan sulfate motifs: exploring the structural diversity by screening for fibroblast growth factor (FGF)1 and FGF2 binding.2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 34, p. 30567-30573Article in journal (Refereed)
    Abstract [en]

    Heparan sulfate is crucial for vital reactions in the body because of its ability to bind various proteins. The identification of protein-binding heparan sulfate sequences is essential to our understanding of heparan sulfate biology and raises the possibility to develop drugs against diseases such as cancer and inflammatory conditions. We present proof-of-principle that in vitro generated heparan sulfate oligosaccharide libraries can be used to explore interactions between heparan sulfate and proteins, and that the libraries expand the available heparan sulfate sequence space. Oligosaccharide libraries mimicking highly 6-O-sulfated domains of heparan sulfate were constructed by enzymatic O-sulfation of O-desulfated, end-group (3)H-labeled heparin octasaccharides. Acceptor oligosaccharides that were 6-O-desulfated but only partially 2-O-desulfated yielded oligosaccharide arrays with increased ratio of iduronyl 2-O-sulfate/glucosaminyl 6-O-sulfate. The products were probed by affinity chromatography on immobilized growth factors, fibroblast growth factor-1 (FGF1) and FGF2, followed by sequence analysis of trapped oligosaccharides. An N-sulfated octasaccharide, devoid of 2-O-sulfate but with three 6-O-sulfate groups, was unexpectedly found to bind FGF1 as well as FGF2 at physiological ionic strength. However, a single 2-O-sulfate group in the absence of 6-O-sulfation gave higher affinity for FGF2. FGF1 binding was also augmented by 2-O-sulfation, preferentially in combination with an adjacent upstream 6-O-sulfate group. These results demonstrate the potential of the enzymatically generated oligosaccharide libraries.

  • 20. Kasza, Zsolt
    et al.
    Fredlund Fuchs, Peder
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tamm, Christoffer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eriksson, Anna S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heindryckx, Femke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Erik
    Le Jan, Sebastien
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Radiology, Oncology and Radiation Science, Radiology.
    Kjellen, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    MicroRNA-24 Suppression of N-Deacetylase/N-Sulfotransferase-1 (NDST1) Reduces Endothelial Cell Responsiveness to Vascular Endothelial Growth Factor A (VEGFA)2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 36, p. 25956-25963Article in journal (Refereed)
    Abstract [en]

    Heparan sulfate (HS) proteoglycans, present at the plasma membrane of vascular endothelial cells, bind to the angiogenic growth factor VEGFA to modulate its signaling through VEGFR2. The interactions between VEGFA and proteoglycan co-receptors require sulfated domains in the HS chains. To date, it is essentially unknown how the formation of sulfated protein-binding domains in HS can be regulated by microRNAs. In the present study, we show that microRNA-24 (miR-24) targets NDST1 to reduce HS sulfation and thereby the binding affinity of HS for VEGFA. Elevated levels of miR-24 also resulted in reduced levels of VEGFR2 and blunted VEGFA signaling. Similarly, suppression of NDST1 using siRNA led to a reduction in VEGFR2 expression. Consequently, not only VEGFA binding, but also VEGFR2 protein expression is dependent on NDST1 function. Furthermore, overexpression of miR-24, or siRNA-mediated reduction of NDST1, reduced endothelial cell chemotaxis in response to VEGFA. These findings establish NDST1 as a target of miR-24 and demonstrate how such NDST1 suppression in endothelial cells results in reduced responsiveness to VEGFA.

  • 21.
    Kreuger, Johan
    et al.
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Genetics and Pathology.
    Jemth, Per
    Department of Medical Biochemistry and Microbiology.
    Sanders-Lindberg, Emil
    Eliahu, Liat
    Ron, Dina
    Basilico, Claudio
    Salmivirta, Markku
    Lindahl, Ulf
    Department of Medical Biochemistry and Microbiology.
    Fibroblast growth factors share binding sites in heparan sulphate.2005In: Biochem J, ISSN 1470-8728, Vol. 389, no Pt 1, p. 145-50Article in journal (Refereed)
    Abstract [en]

    HS (heparan sulphate) proteoglycans bind secreted signalling proteins, including FGFs (fibroblast growth factors) through their HS side chains. Such chains contain a wealth of differentially sulphated saccharide epitopes. Whereas specific HS structures are commonly believed to modulate FGF-binding and activity, selective binding of defined HS epitopes to FGFs has generally not been demonstrated. In the present paper, we have identified a series of sulphated HS octasaccharide epitopes, derived from authentic HS or from biosynthetic libraries that bind with graded affinities to FGF4, FGF7 and FGF8b. These HS species, along with previously identified oligosaccharides that interact with FGF1 and FGF2, constitute the first comprehensive survey of FGF-binding HS epitopes based on carbohydrate sequence analysis. Unexpectedly, our results demonstrate that selective modulation of FGF activity cannot be explained in terms of binding of individual FGFs to specific HS target epitopes. Instead, different FGFs bind to identical HS epitopes with similar relative affinities and low selectivity, such that the strength of these interactions increases with increasing saccharide charge density. We conclude that FGFs show extensive sharing of binding sites in HS. This conclusion challenges the current notion of specificity in HS-FGF interactions, and instead suggests that a set of common HS motifs mediates cellular targeting of different FGFs.

  • 22.
    Kreuger, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Heparan Sulfate Biosynthesis: Regulation and Variability2012In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 60, no 12, p. 898-907Article in journal (Refereed)
    Abstract [en]

    Nearly all vertebrate cells have been shown to express heparan sulfate proteoglycans (HSPGs) at the cell surface. The HSPGs bind to many secreted signaling proteins, including numerous growth factors, cytokines, and morphogens, to affect their tissue distribution and signaling. The heparan sulfate (HS) chains may have variable length and may differ with regard to both degree and pattern of sulfation. As the sulfation pattern of HS chains in most cases will determine if an interaction with a potential ligand will take place, as well as the affinity of the interaction, a key to understanding the function of HSPGs is to clarify how HS biosynthesis is regulated in different biological contexts. This review provides an introduction to the current understanding of HS biosynthesis and its regulation, and identifies research areas where more knowledge is needed to better understand how the HS biosynthetic machinery works. (J Histochem Cytochem 60:898-907, 2012)

  • 23.
    Kreuger, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Nitrocellulose filter binding to assess binding of glycosaminoglycans to proteins2003In: Recognition of carbohydrates in biological systems, Part B: Specific applications / [ed] Yuan C. Lee, Reiko T. Lee, Elsevier , 2003, Vol. 363, p. 327-339Chapter in book (Refereed)
  • 24. Kreuger, Johan
    et al.
    Matsumoto, Taro
    Vanwildemeersch, Maarten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sasaki, Takako
    Timpl, Rupert
    Claesson-Welsh, Lena
    Spillmann, Dorothe
    Lindahl, Ulf
    Role of heparan sulfate domain organization in endostatin inhibition of endothelial cell function2002In: EMBO Journal, Vol. 21, no 23, p. 6303-6311Article in journal (Refereed)
  • 25.
    Kreuger, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Nilsson, Ingrid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kerjaschki, Dontscho
    Petrova, Tatiana
    Alitalo, Kari
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Early lymph vessel development from embryonic stem cells2006In: Arteriosclerosis, Thrombosis and Vascular Biology, ISSN 1079-5642, E-ISSN 1524-4636, Vol. 26, no 5, p. 1073-1078Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE:

    The purpose of this study was to establish a model system for lymph vessel development based on directed differentiation of murine embryonic stem cells.

    METHODS AND RESULTS:

    Stem cells were aggregated to form embryoid bodies, and subsequently cultured in 3-dimensional collagen matrix for up to 18 days. Treatment with vascular endothelial growth factor (VEGF)-C and VEGF-A individually enhanced formation of lymphatic vessel structures, although combined treatment with VEGF-C and VEGF-A was most potent and gave rise to a network of LYVE-1, podoplanin, Prox1, and VEGF receptor-3 positive lymphatic vessel structures running parallel to and apparently emanating from, capillaries. In contrast, fibroblast growth factor-2, hepatocyte growth factor, or hypoxia had little or no effect on the development of the early lymphatics. Further, cells of hematopoietic origin were shown to express lymphatic markers. In summary, different subpopulations of lymphatic endothelial cells were identified on the basis of differential expression of several lymphatic and blood vessel markers, indicating vascular heterogeneity.

    CONCLUSIONS:

    We conclude that the present model closely mimics the early steps of lymph vessel development in mouse embryos.

  • 26.
    Kreuger, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    O'Callaghan, Paul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Failure to Genotype: A Cautionary Note on an Elusive loxP Sequence2016In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 10, article id e0165012Article in journal (Refereed)
    Abstract [en]

    Here we report on a technical difficulty we encountered while optimizing genotyping strategies to identify mice derived from Exoc3l2(tm1a( KOMP)Wtsi) embryonic stem cells obtained from the Knockout Mouse Project Repository. The Exoc3l2(tm1a(KOMP)Wtsi) construct encodes a "knockout-first" design with loxP sites that confer conditional potential (KO1st). We designed primers that targeted wild-type sequences flanking the most downstream element of the construct, an 80 base pair synthetic loxP region, which BLAST alignment analysis reveals is an element common to over 10,000 conditional gene-targeting mouse models. As PCR products amplified from KO1st and wild-type templates would have different lengths (and different mobility in an agarose gel) this strategy was designed to determine the zygosity of individual mice from a single PCR. In parallel we performed PCR with a primer specifically targeting the synthetic loxP sequence. Unexpectedly, while the latter strategy detected the synthetic loxP region and correctly genotyped KO1st chimeric mice, the same individuals were genotyped as wild-type when using the primers that flanked the synthetic loxP region. We discuss the possibility that secondary DNA structures, formed due to the palindromic nature of the synthetic loxP region, may have caused the KO1st template to elude the PCR when using primers that flanked this region. This brief report aims to raise awareness regarding this potential source of false-negative genotype results, particularly for those who are devising genotyping strategies for similarly engineered animal models.

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  • 27.
    Kreuger, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Perez, Lidia
    Giraldez, Antonio J
    Cohen, Stephen M
    Opposing activities of Dally-like glypican at high and low levels of Wingless morphogen activity.2004In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 7, no 4, p. 503-512Article in journal (Refereed)
    Abstract [en]

    The glypican family of heparan sulfate proteoglycans has been implicated in formation of morphogen gradients. Here, we examine the role of the glypican Dally-like protein (Dlp) in shaping the Wingless gradient in the Drosophila wing disc. Surprisingly, we find that Dlp has opposite effects at high and low levels of Wingless. Dlp promotes low-level Wingless activity but reduces high-level Wingless activity. We present evidence that the Wg antagonist Notum acts to induce cleavage of the Dlp glypican at the level of its GPI anchor, which leads to shedding of Dlp. Thus, spatially regulated modification of Dlp by Notum employs the ligand binding activity of Dlp to promote or inhibit signaling in a context-dependent manner. Notum-induced shedding of Dlp could convert Dlp from a membrane-tethered coreceptor to a secreted antagonist.

  • 28.
    Kreuger, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Phillipson, Mia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology, Integrative Physiology.
    Targeting vascular and leukocyte communication in angiogenesis, inflammation and fibrosis2016In: Nature reviews. Drug discovery, ISSN 1474-1776, E-ISSN 1474-1784, Vol. 15, no 2, p. 125-142Article, review/survey (Refereed)
    Abstract [en]

    Regulation of vascular permeability, recruitment of leukocytes from blood to tissue and angiogenesis are all processes that occur at the level of the microvasculature during both physiological and pathological conditions. The interplay between microvascular cells and leukocytes during inflammation, together with the emerging roles of leukocytes in the modulation of the angiogenic process, make leukocyte-vascular interactions prime targets for therapeutics to potentially treat a wide range of diseases, including pathological and dysfunctional vessel growth, chronic inflammation and fibrosis. In this Review, we discuss how the different cell types that are present in and around microvessels interact, cooperate and instruct each other, and in this context we highlight drug targets as well as emerging druggable processes that can be exploited to restore tissue homeostasis.

  • 29.
    Kreuger, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Prydz, K.
    Petersson, R. F.
    Lindahl, Ulf
    Salmivirta, M.
    Characterization of fibroblast growth factor 1 binding heparan sulfate domain1999In: Glycobiology, Vol. 9, no 7, p. 723-729Article in journal (Refereed)
  • 30.
    Kreuger, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Salmivirta, M.
    Sturiale, L.
    Giménez-Gallego, G.
    Lindahl, Ulf
    Sequence analysis of heparan sulfate epitopes with graded affinities for FGF-1 amd FGF22001In: J Biol Chem, Vol. 276, no 33, p. 30744-30752Article in journal (Refereed)
  • 31.
    Kreuger, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sasaki, T.
    Timpl, R.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Endostatin is recognized by heparan sulfate epitopes containing both sulfated and acetylated N-substituentsManuscript (Other academic)
  • 32.
    Kreuger, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Li, Jin-ping
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Interactions between heparan sulfate and proteins: the concept of specificity2006In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 174, no 3, p. 323-327Article, review/survey (Refereed)
    Abstract [en]

    Proteoglycan (PG) coreceptors carry heparan sulfate (HS) chains that mediate interactions with growth factors, morphogens, and receptors. Thus, PGs modulate fundamental processes such as cell survival, division, adhesion, migration, and differentiation. This review summarizes recent biochemical and genetic information that sheds new light on the nature of HS-protein binding. Unexpectedly, many interactions appear to depend more on the overall organization of HS domains than on their fine structure.

  • 33. Larsson, Erik
    et al.
    Fredlund Fuchs, Peder
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Heldin, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Barkefors, Irmeli
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Bondjers, Cecilia
    Genové, Guillem
    Arrondel, Christelle
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kurschat, Christine
    Schermer, Bernhard
    Benzing, Thomas
    Harvey, Scott J
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lindahl, Per
    Discovery of microvascular miRNAs using public gene expression data: miR-145 is expressed in pericytes and is a regulator of Fli12009In: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 1, no 11, p. 108-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND

    A function for the microRNA (miRNA) pathway in vascular development and angiogenesis has been firmly established. miRNAs with selective expression in the vasculature are attractive as possible targets in miRNA-based therapies. However, little is known about the expression of miRNAs in microvessels in vivo. Here, we identified candidate microvascular-selective miRNAs by screening public miRNA expression datasets.

    METHODS

    Bioinformatics predictions of microvascular-selective expression were validated with real-time quantitative reverse transcription PCR on purified microvascular fragments from mouse. Pericyte expression was shown with in situ hybridization on tissue sections. Target sites were identified with 3' UTR luciferase assays, and migration was tested in a microfluid chemotaxis chamber.

    RESULTS

    miR-145, miR-126, miR-24, and miR-23a were selectively expressed in microvascular fragments isolated from a range of tissues. In situ hybridization and analysis of Pdgfb retention motif mutant mice demonstrated predominant expression of miR-145 in pericytes. We identified the Ets transcription factor Friend leukemia virus integration 1 (Fli1) as a miR-145 target, and showed that elevated levels of miR-145 reduced migration of microvascular cells in response to growth factor gradients in vitro.

    CONCLUSIONS

    miR-126, miR-24 and miR-23a are selectively expressed in microvascular endothelial cells in vivo, whereas miR-145 is expressed in pericytes. miR-145 targets the hematopoietic transcription factor Fli1 and blocks migration in response to growth factor gradients. Our findings have implications for vascular disease and provide necessary information for future drug design against miRNAs with selective expression in the microvasculature.

  • 34.
    Le Jan, Sébastien
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hayashi, Makoto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Kasza, Zsolt
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eriksson, Inger
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bishop, Joseph R
    Weibrecht, Irene
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Heldin, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Holmborn, Katarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Jakobsson, Lars
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools.
    Spillmann, Dorothe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Esko, Jeffrey D
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
    Kjellén, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Functional Overlap Between Chondroitin and Heparan Sulfate Proteoglycans During VEGF-Induced Sprouting Angiogenesis2012In: Arteriosclerosis, Thrombosis and Vascular Biology, ISSN 1079-5642, E-ISSN 1524-4636, Vol. 32, no 5, p. 1255-1263Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: Heparan sulfate proteoglycans regulate key steps of blood vessel formation. The present study was undertaken to investigate if there is a functional overlap between heparan sulfate proteoglycans and chondroitin sulfate proteoglycans during sprouting angiogenesis.

    METHODS AND RESULTS: Using cultures of genetically engineered mouse embryonic stem cells, we show that angiogenic sprouting occurs also in the absence of heparan sulfate biosynthesis. Cells unable to produce heparan sulfate instead increase their production of chondroitin sulfate that binds key angiogenic growth factors such as vascular endothelial growth factor A, TGFβ, and platelet-derived growth factor B. Lack of heparan sulfate proteoglycan production however leads to increased pericyte numbers and reduced adhesion of pericytes to nascent sprouts, likely due to dysregulation of TGFβ and platelet-derived growth factor B signal transduction.

    CONCLUSIONS: The present study provides direct evidence for a previously undefined functional overlap between chondroitin sulfate proteoglycans and heparan sulfate proteoglycans during sprouting angiogenesis. Our findings provide information relevant for potential future drug design efforts that involve targeting of proteoglycans in the vasculature.

  • 35. Loo, B. M.
    et al.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jalkanen, M.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Salmivirta, M.
    Binding of heparin/heparan sulfate to fibroblast growth factor receptor 42001In: J Biol Chem, Vol. 276, no 20, p. 16868-16876Article in journal (Refereed)
  • 36.
    Lundin, Lars
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Larsson, Helena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kanda, S
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Salmivirta, Markku
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Selectively desulfated heparin inhibits fibroblast growth factor-induced mitogenicity and angiogenesis.2000In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 275, no 32, p. 24653-24660Article in journal (Refereed)
    Abstract [en]

    Fibroblast growth factors (FGFs) are known to induce formation of new blood vessels, angiogenesis. We show that FGF-induced angiogenesis can be modulated using selectively desulfated heparin. Chinese hamster ovary cells (CHO677) deficient in heparan sulfate biosynthesis were employed to assess the function of heparin/heparan sulfate in FGF receptor-1 (FGFR-1) signal transduction and biological responses. In the presence of FGF-2, FGFR-1 kinase and subsequent mitogen-activated protein kinase Erk2 activities were augmented in a dose-dependent manner, whereas high concentrations of heparin resulted in decreased activity. The length of the heparin oligomer, minimally an 8/10-mer, was critical for the ability to enhance FGFR-1 kinase activity. The N- and 2-O-sulfate groups of heparin were essential for binding to FGF-2, whereas stimulation of FGFR-1 and Erk2 kinases by FGF-2 also required the presence of 6-O-sulfate groups. Sulfation at 2-O- and 6-O-positions was moreover a prerequisite for binding of heparin to a lysine-rich peptide corresponding to amino acids 160-177 in the extracellular domain of FGFR-1. Selectively 6-O-desulfated heparin, which binds to FGF-2 but fails to bind the receptor, decreased FGF-2-induced proliferation of CHO677 cells, presumably by displacing intact heparin. Furthermore, FGF-2-induced angiogenesis in chick embryos was inhibited by 6-O-desulfated heparin. Thus, formation of a ternary complex of FGF-2, heparin, and FGFR-1 appears critical for the activation of FGFR-1 kinase and downstream signal transduction. Preventing complex formation by modified heparin preparations may allow regulation of FGF-2 functions, such as induction of angiogenesis.

  • 37.
    Massena, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Christoffersson, Gustaf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Vågesjö, Evelina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Seignez, Cédric
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Gustafsson, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Binet, François
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Herrera Hidalgo, Carmen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Giraud, Antoine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Lomei, Jalal
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Weström, Simone
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Dermatology and Venereology.
    Shibuya, Masabumi
    Jobu Univ, Gakubunkan Inst Physiol & Med, Gunma, Japan.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Gerwins, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Radiology.
    Welsh, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Phillipson, Mia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Identification and characterization of VEGF-A-responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans2015In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 126, no 17, p. 2016-2026Article in journal (Refereed)
    Abstract [en]

    Vascular endothelial growth factor A (VEGF-A) is upregulated during hypoxia and is the major regulator of angiogenesis. VEGF-A expression has also been found to recruit myeloid cells to ischemic tissues where they contribute to angiogenesis. This study investigates the mechanisms underlying neutrophil recruitment to VEGF-A as well as the characteristics of these neutrophils. A previously undefined circulating subset of neutrophils shown to be CD49d(+)VEGFR1(high)CXCR4(high) was identified in mice and humans. By using chimeric mice with impaired VEGF receptor 1 (VEGFR1) or VEGFR2 signaling (Flt-1tk(-/-), tsad(-/-)), we found that parallel activation of VEGFR1 on neutrophils and VEGFR2 on endothelial cells was required for VEGF-A-induced recruitment of circulating neutrophils to tissue. Intravital microscopy of mouse microcirculation revealed that neutrophil recruitment by VEGF-A versus by the chemokine macrophage inflammatory protein 2 (MIP-2 [CXCL2]) involved the same steps of the recruitment cascade but that an additional neutrophil integrin (eg, VLA-4 [CD49d/CD29]) played a crucial role in neutrophil crawling and emigration to VEGF-A. Isolated CD49d(+) neutrophils featured increased chemokinesis but not chemotaxis compared with CD49d(-) neutrophils in the presence of VEGF-A. Finally, by targeting the integrin α4 subunit (CD49d) in a transplantation-based angiogenesis model that used avascular pancreatic islets transplanted to striated muscle, we demonstrated that inhibiting the recruitment of circulating proangiogenic neutrophils to hypoxic tissue impairs vessel neoformation. Thus, angiogenesis can be modulated by targeting cell-surface receptors specifically involved in VEGF-A-dependent recruitment of proangiogenic neutrophils without compromising recruitment of the neutrophil population involved in the immune response to pathogens.

  • 38.
    Nilsson, Ingrid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Bahram, Fuad
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Li, Xiujuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Gualandi, Laura
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Koch, Sina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Jarvius, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Anisimov, Andrey
    Kholová, Ivana
    Pytowski, Bronislaw
    Baldwin, Megan
    Ylä-Herttuala, Seppo
    Alitalo, Kari
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    VEGF receptor 2/-3 heterodimers detected in situ by proximity ligation on angiogenic sprouts2010In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 29, no 8, p. 1377-1388Article in journal (Refereed)
    Abstract [en]

    The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/-3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell filopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/-3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting.

  • 39.
    Nunes de Almeida *, Francisca
    et al.
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
    Heldin *, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Reis, Katarina
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Aspenström, Pontus
    Dept. of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
    A RhoD-regulated pathway is needed for PDGF-BB-induced chemotaxisManuscript (preprint) (Other academic)
  • 40.
    O'Callaghan, Paul
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Zarb, Yvette
    Zurich Univ, Zurich Univ Hosp, Clin Neurosci Ctr, Dept Neurosurg, Zurich, Switzerland.
    Noborn, Fredrik
    Univ Gothenburg, Inst Biomed, Dept Clin Chem & Transfus Med, Sahlgrenska Acad, Gothenburg, Sweden.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Modeling the structural implications of an alternatively spliced Exoc3l2, a paralog of the tunneling nanotube-forming M-Sec2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 8, article id e0201557Article in journal (Refereed)
    Abstract [en]

    The exocyst is a molecular tether that retains secretory vesicles at the plasma membrane prior to SNARE-mediated docking and fusion. However, individual exocyst complex components (EXOCs) may also function independently of exocyst assembly. Alternative splice variants of EXOC mRNA and paralogs of EXOC genes have been described and several have been attributed functions that may be independent of the exocyst complex. Here we describe a novel splice variant of murine Exoc3l2, which we term Exoc3l2a. We discuss possible functional implications of the resulting domain excision from this isoform of EXOC3L2 based on structural similarities with its paralog M-Sec (EXOC3L3), which is implicated in tunneling nanotube formation. The identification of this Exoc3l2 splice variant expands the potential for subunit diversity within the exocyst and for alternative functionality of this component independently of the exocyst.

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  • 41.
    Olsson, Anna-Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Dimberg, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Claesson-Welsh, Lena
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    VEGF receptor signalling: in control of vascular function2006In: Nature reviews. Molecular cell biology, ISSN 1471-0072, E-ISSN 1471-0080, Vol. 7, no 5, p. 359-371Article in journal (Refereed)
    Abstract [en]

    Vascular endothelial growth-factor receptors (VEGFRs) regulate the cardiovascular system. VEGFR1 is required for the recruitment of haematopoietic precursors and migration of monocytes and macrophages, whereas VEGFR2 and VEGFR3 are essential for the functions of vascular endothelial and lymphendothelial cells, respectively. Recent insights have shed light onto VEGFR signal transduction and the interplay between different VEGFRs and VEGF co-receptors in development, adult physiology and disease.

  • 42.
    Razi, Nahid
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Lay, L
    Russo, G
    Panza, L
    Lindahl, Birgitta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical and Physiological Chemistry.
    Identification of O-sulphate substituents on D-glucuronic acid units in heparin-related glycosaminoglycans using novel synthetic disaccharide standards.1995In: Glycobiology, ISSN 0959-6658, E-ISSN 1460-2423, Vol. 5, no 8, p. 807-811Article in journal (Refereed)
    Abstract [en]

    The two disaccharides, methyl 4-O-(2-O-sulpho-beta-D-glucopyranosyl-uronic acid)-2-deoxy-2-amino-alpha-D-glucopyranoside and methyl 4-O-(3-O-sulpho-beta-D-glucopyranosyluronic acid)-2-deoxy-2-amino-alpha-D-glucopyranoside, were prepared by de novo synthesis, and converted to the corresponding 2,5-anhydro-D-[1-3H]mannitol derivatives by deamination with nitrous acid followed by reduction with NaB3H4. The resultant labelled products were used as standards in the identification, by anion-exchange high-performance liquid chromatography (HPLC), of disaccharides generated by HNO2/NaB3H4 treatment of heparan sulphate isolated from human brain. The two standards, containing 2-O- and 3-O-sulphated glucuronic acid, respectively, were clearly separated by the HPLC procedure. Comparison with the deamination products derived from heparan sulphate showed that the mono-O-sulphated disaccharide species containing a sulphated glucuronic acid unit co-eluted with the 2-O-sulphated standard. The corresponding component isolated from other heparan sulphate preparations, or from heparin, also eluted at the same position. No disaccharide derived from heparin or heparan sulphate appeared at the elution position of the 3-O-sulphated standard. It is concluded that D-glucuronic acid units in heparin-related glycosaminoglycans may be sulphated at C2, whereas no evidence has been found for sulphation at C3. By contrast, analysis of mono-O-sulphated disaccharides derived from a chemically sulphated, bacterial capsular polysaccharide (generated by Escherichia coli K5) clearly demonstrated the occurrence of O-sulphate groups at C-3 of D-glucuronic acid units.

  • 43.
    Saetre, Peter
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Animal Development and Genetics.
    Emilsson, Lina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Axelsson, Elin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Animal Development and Genetics.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Lindholm, Eva
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Animal Development and Genetics.
    Jazin, Elena
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolutionary Biology, Animal Development and Genetics.
    Inflammation-related genes up-regulated in schizophrenia brains2007In: BMC Psychiatry, ISSN 1471-244X, E-ISSN 1471-244X, Vol. 7, p. 46-Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Multiple studies have shown that brain gene expression is disturbed in subjects suffering from schizophrenia. However, disentangling disease effects from alterations caused by medication is a challenging task. The main goal of this study is to find transcriptional alterations in schizophrenia that are independent of neuroleptic treatment. METHODS: We compared the transcriptional profiles in brain autopsy samples from 55 control individuals with that from 55 schizophrenic subjects, subdivided according to the type of antipsychotic medication received. RESULTS: Using global and high-resolution mRNA quantification techniques, we show that genes involved in immune response (GO:0006955) are up regulated in all groups of patients, including those not treated at the time of death. In particular, IFITM2, IFITM3, SERPINA3, and GBP1 showed increased mRNA levels in schizophrenia (p-values from qPCR < or = 0.01). These four genes were co-expressed in both schizophrenic subjects and controls. In-vitro experiments suggest that these genes are expressed in both oligodendrocyte and endothelial cells, where transcription is inducible by the inflammatory cytokines TNF-alpha, IFN-alpha and IFN-gamma. CONCLUSION: Although the modified genes are not classical indicators of chronic or acute inflammation, our results indicate alterations of inflammation-related pathways in schizophrenia. In addition, the observation in oligodendrocyte cells suggests that alterations in inflammatory-related genes may have consequences for myelination. Our findings encourage future research to explore whether anti-inflammatory agents can be used in combination with traditional antipsychotics for a more efficient treatment of schizophrenia.

  • 44. Sasaki, T.
    et al.
    Larsson, H.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Salmivirta, M.
    Claesson-Welsh, L.
    Lindahl, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hohenester, E.
    Timpl, R.
    Structural basis and potential role of heparin/heparan sulfate binding to the angiogenesis inhibitor endostatin1999In: EMBO J, Vol. 18, no 22, p. 6240-6248Article in journal (Refereed)
  • 45.
    Thorslund, Sara
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Nguyen, Hugo
    Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
    Läräng, Thomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Barkefors, Irmeli
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    A disposable and multifunctional capsule for easy operation of microfluidic elastomer systems2011In: Journal of Micromechanics and Microengineering, ISSN 0960-1317, E-ISSN 1361-6439, Vol. 21, no 12, p. 127001-Article in journal (Refereed)
    Abstract [en]

    The global lab-on-chip and microfluidic markets for cell-based assays have been predicted to grow considerably, as novel microfluidic systems enable cell biologists to perform in vitro experiments at an unprecedented level of experimental control. Nevertheless, microfluidic assays must, in order to compete with conventional assays, be made available at easily affordable costs, and in addition be made simple to operate for users having no previous experience with microfluidics. We have to this end developed a multifunctional microfluidic capsule that can be mass-produced at low cost in thermoplastic material. The capsule enables straightforward operation of elastomer inserts of optional design, here exemplified with insert designs for molecular gradient formation in microfluidic cell culture systems. The integrated macro–micro interface of the capsule ensures reliable connection of the elastomer fluidic structures to an external perfusion system. A separate compartment in the capsule filled with superabsorbent material is used for internal waste absorption. The capsule assembly process is made easy by integrated snap-fits, and samples within the closed capsule can be analyzed using both inverted and upright microscopes. Taken together, the capsule concept presented here could help accelerate the use of microfluidic-based biological assays in the life science sector.

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  • 46.
    Wistrand-Yuen, Pikkei
    et al.
    Gradientech AB, Uppsala, Sweden.
    Malmberg, Christer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. Gradientech AB, Uppsala, Sweden.
    Fatsis-Kavalopoulos, Nikos
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Lübke, Moritz
    Gradientech AB, Uppsala, Sweden.
    Tängdén, Thomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Kreuger, Johan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    A Multiplex Fluidic Chip for Rapid Phenotypic Antibiotic Susceptibility Testing2020In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 11, article id e03109-19Article in journal (Refereed)
    Abstract [en]

    Many patients with severe infections receive inappropriate empirical treatment, and rapid detection of bacterial antibiotic susceptibility can improve clinical outcome and reduce mortality. To this end, we have developed a multiplex fluidic chip for rapid phenotypic antibiotic susceptibility testing of bacteria. A total of 21 clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus were acquired from the EUCAST Development Laboratory and tested against amikacin, ceftazidime, and meropenem (Gram-negative bacteria) or gentamicin, ofloxacin, and tetracycline (Gram-positive bacteria). The bacterial samples were mixed with agarose and loaded in an array of growth chambers in the chip where bacterial microcolony growth was monitored over time using automated image analysis. MIC values were automatically obtained by tracking the growth rates of individual microcolonies in different regions of antibiotic gradients. Stable MIC values were obtained within 2 to 4 h, and the results showed categorical agreement with reference MIC values as determined by broth microdilution in 86% of the cases.

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