uu.seUppsala University Publications
Change search
Link to record
Permanent link

Direct link
BETA
Publications (10 of 16) Show all publications
Fatsis-Kavalopoulos, N., O'Callaghan, P., Xie, B., Hernández Vera, R., Idevall Hagren, O. & Kreuger, J. (2019). Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution. Lab on a Chip, 19(6), 1071-1081
Open this publication in new window or tab >>Formation of precisely composed cancer cell clusters using a cell assembly generator (CAGE) for studying paracrine signaling at single-cell resolution
Show others...
2019 (English)In: Lab on a Chip, ISSN 1473-0197, E-ISSN 1473-0189, Vol. 19, no 6, p. 1071-1081Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
ROYAL SOC CHEMISTRY, 2019
National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-381586 (URN)10.1039/c8lc01153b (DOI)000462666200012 ()30783638 (PubMedID)
Funder
Swedish Cancer Society, CAN 2017/703EU, Horizon 2020, 642866Swedish Research Council, MH2015-03087Göran Gustafsson Foundation for Research in Natural Sciences and Medicine
Available from: 2019-04-12 Created: 2019-04-12 Last updated: 2019-04-24Bibliographically approved
Hernández Vera, R., O'Callaghan, P., Fatsis-Kavalopoulos, N. & Kreuger, J. (2019). Modular microfluidic systems cast from 3D-printed molds for imaging leukocyte adherence to differentially treated endothelial cultures. Scientific Reports, 9, Article ID 11321.
Open this publication in new window or tab >>Modular microfluidic systems cast from 3D-printed molds for imaging leukocyte adherence to differentially treated endothelial cultures
2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 11321Article in journal (Refereed) Published
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.

National Category
Cell Biology
Identifiers
urn:nbn:se:uu:diva-382276 (URN)10.1038/s41598-019-47475-z (DOI)000478743700033 ()
Funder
Swedish Cancer Society, CAN 2017/703EU, Horizon 2020, 642866
Note

De två första författarna delar förstaförfattarskapet.

Available from: 2019-04-24 Created: 2019-04-24 Last updated: 2019-09-27Bibliographically approved
O'Callaghan, P., Zhang, X. & Li, J.-P. (2018). Heparan Sulfate Proteoglycans as Relays of Neuroinflammation. Journal of Histochemistry and Cytochemistry, 66(4), 305-319
Open this publication in new window or tab >>Heparan Sulfate Proteoglycans as Relays of Neuroinflammation
2018 (English)In: Journal of Histochemistry and Cytochemistry, ISSN 0022-1554, E-ISSN 1551-5044, Vol. 66, no 4, p. 305-319Article, review/survey (Refereed) Published
Abstract [en]

Heparan sulfate proteoglycans (HSPGs) are implicated as inflammatory mediators in a variety of settings, including chemokine activation, which is required to recruit circulating leukocytes to infection sites. Heparan sulfate (HS) polysaccharide chains are highly interactive and serve co-receptor roles in multiple ligand:receptor interactions. HS may also serve as a storage depot, sequestering ligands such as cytokines and restricting their access to binding partners. Heparanase, through its ability to fragment HS chains, is a key regulator of HS function and has featured prominently in studies of HS's involvement in inflammatory processes. This review focuses on recent discoveries regarding the role of HSPGs, HS, and heparanase during inflammation, with particular focus on the brain. HS chains emerge as critical go-betweens in multiple aspects of the inflammatory responserelaying signals between receptors and cells. The molecular interactions proposed to occur between HSPGs and the pathogen receptor toll-like receptor 4 (TLR4) are discussed, and we summarize some of the contrasting roles that HS and heparanase have been assigned in diseases associated with chronic inflammatory states, including Alzheimer's disease (AD). We conclude by briefly discussing how current knowledge could potentially be applied to augment HS-mediated events during sustained neuroinflammation, which contributes to neurodegeneration in AD.

Place, publisher, year, edition, pages
SAGE PUBLICATIONS LTD, 2018
Keywords
Alzheimer's disease (AD), extracellular matrix (ECM), heparan sulfate (HS), heparan sulfate proteoglycans (HSPGs), heparanase, inflammation, innate immunity, microglia, neuroinflammation, toll-like receptor 4 (TLR4)
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-357172 (URN)10.1369/0022155417742147 (DOI)000429872800008 ()29290138 (PubMedID)
Funder
Swedish Research Council, 2015-02595
Available from: 2018-08-14 Created: 2018-08-14 Last updated: 2018-08-14Bibliographically approved
O'Callaghan, P., Zarb, Y., Noborn, F. & Kreuger, J. (2018). Modeling the structural implications of an alternatively spliced Exoc3l2, a paralog of the tunneling nanotube-forming M-Sec. PLoS ONE, 13(8), Article ID e0201557.
Open this publication in new window or tab >>Modeling the structural implications of an alternatively spliced Exoc3l2, a paralog of the tunneling nanotube-forming M-Sec
2018 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 8, article id e0201557Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
San Francisco, California, US: , 2018
National Category
Cell and Molecular Biology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-360991 (URN)10.1371/journal.pone.0201557 (DOI)000441129300022 ()30086153 (PubMedID)
Funder
The Dementia Association - The National Association for the Rights of the DementedSwedish Cancer Society, CAN 2014/820
Available from: 2018-09-20 Created: 2018-09-20 Last updated: 2018-11-28Bibliographically approved
Heldin, J., O'Callaghan, P., Hernández Vera, R., Fredlund Fuchs, P., Gerwins, P. & Kreuger, J. (2017). FGD5 sustains vascular endothelial growth factor A (VEGFA) signaling through inhibition of proteasome-mediated VEGF receptor 2 degradation. Cellular Signalling, 40, 125-132
Open this publication in new window or tab >>FGD5 sustains vascular endothelial growth factor A (VEGFA) signaling through inhibition of proteasome-mediated VEGF receptor 2 degradation
Show others...
2017 (English)In: Cellular Signalling, ISSN 0898-6568, E-ISSN 1873-3913, Vol. 40, p. 125-132Article in journal (Refereed) Published
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.

Keywords
Angiogenesis, Cdc42, FGD5, Vascular biology, VEGFR2, Degradation
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-341984 (URN)10.1016/j.cellsig.2017.09.009 (DOI)000414620900013 ()28927665 (PubMedID)
Available from: 2018-02-19 Created: 2018-02-19 Last updated: 2018-02-19Bibliographically approved
Christoffersson, G., Lomei, J., O'Callaghan, P., Kreuger, J., Engblom, S. & Phillipson, M. (2017). Vascular sprouts induce local attraction of proangiogenic neutrophils. Journal of Leukocyte Biology, 102, 741-751
Open this publication in new window or tab >>Vascular sprouts induce local attraction of proangiogenic neutrophils
Show others...
2017 (English)In: Journal of Leukocyte Biology, ISSN 0741-5400, E-ISSN 1938-3673, Vol. 102, p. 741-751Article in journal (Refereed) Published
National Category
Physiology Bioinformatics (Computational Biology)
Identifiers
urn:nbn:se:uu:diva-196483 (URN)10.1189/jlb.1MA0117-018R (DOI)000413395700019 ()
Projects
eSSENCE
Available from: 2017-06-05 Created: 2013-03-10 Last updated: 2018-11-12Bibliographically approved
Kreuger, J. & O'Callaghan, P. (2016). Failure to Genotype: A Cautionary Note on an Elusive loxP Sequence. PLoS ONE, 11(10), Article ID e0165012.
Open this publication in new window or tab >>Failure to Genotype: A Cautionary Note on an Elusive loxP Sequence
2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 10, article id e0165012Article in journal (Refereed) Published
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.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-308918 (URN)10.1371/journal.pone.0165012 (DOI)000386205400044 ()27768725 (PubMedID)
Funder
Swedish Cancer Society, CAN 2014/820Swedish Research Council, 2010-3968NIH (National Institute of Health), U01HG004085
Available from: 2016-12-01 Created: 2016-12-01 Last updated: 2017-11-29Bibliographically approved
O'Callaghan, P., Li, J.-P., Lannfelt, L., Lindahl, U. & Zhang, X. (2015). Microglial Heparan Sulfate Proteoglycans Facilitate the Cluster-of-Differentiation 14 (CD14)/Toll-like Receptor 4 (TLR4)-Dependent Inflammatory Response. Journal of Biological Chemistry, 290(24), 14904-14914
Open this publication in new window or tab >>Microglial Heparan Sulfate Proteoglycans Facilitate the Cluster-of-Differentiation 14 (CD14)/Toll-like Receptor 4 (TLR4)-Dependent Inflammatory Response
Show others...
2015 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, no 24, p. 14904-14914Article in journal (Refereed) Published
Abstract [en]

Microglia rapidly mount an inflammatory response to pathogens in the central nervous system (CNS). Heparan sulfate proteoglycans (HSPGs) have been attributed various roles in inflammation. To elucidate the relevance of microglial HSPGs in a pro-inflammatory response we isolated microglia from mice overexpressing heparanase (Hpa-tg), the HS-degrading endoglucuronidase, and challenged them with lipopolysaccharide (LPS), a bacterial endotoxin. Prior to LPS-stimulation, the LPS-receptor cluster-of-differentiation 14 (CD14) and Toll-like receptor 4 (TLR4; essential for the LPS response) were similarly expressed in Ctrl and Hpa-tg microglia. However, compared with Ctrl microglia, Hpa-tg cells released significantly less tumor necrosis factor-α (TNFα), essentially failed to up-regulate interleukin-1β (IL1β) and did not initiate synthesis of proCD14. Isolated primary astroyctes expressed TLR4, but notably lacked CD14 and in contrast to microglia, LPS challenge induced a similar TNFα response in Ctrl and Hpa-tg astrocytes, while neither released IL1β. The astrocyte TNFα-induction was thus attributed to CD14-independent TLR4 activation and was unaffected by the cells HS status. Equally, the suppressed LPS-response in Hpa-tg microglia indicated a loss of CD14-dependent TLR4 activation, suggesting that microglial HSPGs facilitate this process. Indeed, confocal microscopy confirmed interactions between microglial HS and CD14 in LPS-stimulated microglia and a potential HS-binding motif in CD14 was identified. We conclude that microglial HSPGs facilitate CD14-dependent TLR4 activation and that heparanase can modulate this mechanism.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:uu:diva-257815 (URN)10.1074/jbc.M114.634337 (DOI)000356177300010 ()25869127 (PubMedID)
Funder
Swedish Research Council, 2003-5546
Available from: 2015-07-09 Created: 2015-07-09 Last updated: 2017-12-04Bibliographically approved
O'Callaghan, P., Noborn, F., Sehlin, D., Li, J.-p., Lannfelt, L., Lindahl, U. & Zhang, X. (2014). Apolipoprotein E increases cell association of amyloid-β 40 through heparan sulfate and LRP1 dependent pathways. Amyloid: Journal of Protein Folding Disorders, 21(2), 76-87
Open this publication in new window or tab >>Apolipoprotein E increases cell association of amyloid-β 40 through heparan sulfate and LRP1 dependent pathways
Show others...
2014 (English)In: Amyloid: Journal of Protein Folding Disorders, ISSN 1350-6129, E-ISSN 1744-2818, Vol. 21, no 2, p. 76-87Article in journal (Refereed) Published
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-168315 (URN)10.3109/13506129.2013.879643 (DOI)000336146700002 ()
Available from: 2012-02-08 Created: 2012-02-08 Last updated: 2017-12-08
Kasza, Z., Fredlund Fuchs, P., Tamm, C., Eriksson, A. S., O'Callaghan, P., Heindryckx, F., . . . Kreuger, J. (2013). MicroRNA-24 Suppression of N-Deacetylase/N-Sulfotransferase-1 (NDST1) Reduces Endothelial Cell Responsiveness to Vascular Endothelial Growth Factor A (VEGFA). Journal of Biological Chemistry, 288(36), 25956-25963
Open this publication in new window or tab >>MicroRNA-24 Suppression of N-Deacetylase/N-Sulfotransferase-1 (NDST1) Reduces Endothelial Cell Responsiveness to Vascular Endothelial Growth Factor A (VEGFA)
Show others...
2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 36, p. 25956-25963Article in journal (Refereed) Published
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-219873 (URN)10.1074/jbc.M113.484360 (DOI)000330623300026 ()
Available from: 2014-03-12 Created: 2014-03-06 Last updated: 2017-12-05
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0003-3117-5367

Search in DiVA

Show all publications