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Identification and characterization of VEGF-A-responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans
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 Medical Cell Biology.
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 Medical Cell Biology.
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2015 (English)In: Blood, ISSN 0006-4971, E-ISSN 1528-0020, Vol. 126, no 17, 2016-2026 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
2015. Vol. 126, no 17, 2016-2026 p.
National Category
Physiology
Identifiers
URN: urn:nbn:se:uu:diva-265201DOI: 10.1182/blood-2015-03-631572ISI: 000366389200012PubMedID: 26286848OAI: oai:DiVA.org:uu-265201DiVA: diva2:864083
Funder
Swedish Research CouncilThe Royal Swedish Academy of SciencesMagnus Bergvall FoundationSwedish Diabetes AssociationÅke Wiberg FoundationRagnar Söderbergs stiftelseKnut and Alice Wallenberg Foundation
Available from: 2015-10-25 Created: 2015-10-25 Last updated: 2017-12-01Bibliographically approved
In thesis
1. A close-up on neutrophils: Visualizing the mechanisms of their in vivo recruitment and function
Open this publication in new window or tab >>A close-up on neutrophils: Visualizing the mechanisms of their in vivo recruitment and function
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

A successful immune response depends on prompt and sufficient recruitment of leukocytes from the circulation to infected or injured sites. Mobilization of leukocytes to hypoxic tissues is vital for angiogenesis, i.e. the formation of new blood vessels from preexisting vasculature, and thus crucial for tissue growth and regeneration. Deviations from normal leukocyte recruitment drive a variety of pathologies, including chronic inflammation, autoimmune diseases and cancer, for which therapeutic options are limited or unspecific. Understanding the mechanisms by which the body controls leukocyte recruitment is therefore critical for the development of novel therapeutic strategies.

The present investigations focused on delineating the mechanisms behind leukocyte mobilization from the bloodstream to afflicted sites, by means of in vivo imaging techniques and in vitro assays. We demonstrate that, in response to inflammation, increased vascular permeability enhances transendothelial transport of tissue-released chemokines. Within the vasculature, chemokines form a chemotactic gradient sequestered on heparan sulfate, which directs crawling neutrophils and expedites their extravasation to the inflamed tissue. Consequently, gradient formation grants efficient bacterial clearance. Citrullination of chemokines by leukocyte-derived PAD enzymes in the inflamed tissue prevents chemokine transport into blood vessels, which dampens further neutrophil recruitment and thereby controls the amplitude of the inflammatory response. Moreover, the mechanisms of neutrophil recruitment in response to proangiogenic factors released during hypoxia are revealed to differ from those observed during classical inflammation. Particularly, VLA-4 integrin and VEGFR1 expressed on a defined subset of neutrophils, along with endothelial VEGFR2, are required for efficient neutrophil recruitment to hypoxia. Rather than stimulus-induced phenotypic changes on neutrophils, specific neutrophil subtypes with innate proinflammatory or proangiogenic functions (respectively, CD49d-VEGFR1lowCXCR4low and CD49d+VEGFR1highCXCR4high) coexist in the circulation of humans and mice.

In summary, this dissertation provides relevant information on specific steps of neutrophil recruitment to inflamed or hypoxic tissues, which may represent future means to down-regulate aberrant immune responses during chronic inflammation and autoimmune diseases; to increase angiogenesis during ischemia; or to limit pathological angiogenesis, a characteristic of tumor growth and of several chronic inflammatory disorders.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2015. 56 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1159
Keyword
angiogenesis, chemokine, chemotactic gradient, citrullination, hypoxia, inflammation, intraluminal crawling, intravital imaging, PAD enzymes, permeability, proangiogenic, proinflammatory
National Category
Physiology
Identifiers
urn:nbn:se:uu:diva-265203 (URN)978-91-554-9401-8 (ISBN)
Public defence
2015-12-18, A1:107a, Biomedicinskt Centrum, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2015-11-26 Created: 2015-10-25 Last updated: 2016-01-13
2. Exploring immune cell functions and ways to make use of them
Open this publication in new window or tab >>Exploring immune cell functions and ways to make use of them
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In addition to host defense, alternative functions of immune cells are emerging. Immune cells are crucial during healing of injured tissue, in formation of new blood vessels, angiogenesis, and also in maintaining the balance in inflammation having immune regulating functions. Over the last decade a higher degree of heterogeneity and plasticity of immune cells have been reported and immune cells develop different characteristics in different situations in vivo.

This thesis investigates roles for immune cells in situations of muscle hypoxia and reduced blood perfusion, wound healing in skin and at sites of transplantation of allogeneic islets of Langerhans and on top of this, ways to steer immune cell function for future therapeutic purposes. A specific neutrophil subset (CD49d+VEGFR1+CXCR4high) was found to be recruited to VEGF-A released at hypoxia and these neutrophils were crucial for functional angiogenesis. In muscle with restricted blood flow macrophages were detected in perivascular positions and started to express aSMA and PDGFR1b and were found to directly assist in blood flow regulation by iNOS-dependent NO production. This essential function in muscle regain of function could be boosted by plasmid overexpression of CXCL12 where the effect of these macrophages chaperoning the vasculature was amplified improving limb blood flow regulation. The effect on macrophages accelerating tissue regeneration being amplified by CXCL12 was tested in a model of cutaneous wound healing where the administration of CXCL12 was optimized for high bioavailability. In the skin, CXCL12-treatment induced accumulation of TGFb-expressing macrophages close to the wound driving the healing process, and subsequently the wounds healed with an efficiency never reported before. In the last study means to circumvent systemic immune suppressive therapy required in allogeneic transplantation was investigated. Allogeneic islets of Langerhans transplanted to muscle were immediately destroyed by the host immune system. Co-transplanting islets and CCL22-encoding plasmids we could curb this fast rejection for 10 days by accumulating CD4+CD25+FoxP3+ regulatory T lymphocytes at the site for transplantation preventing islet grafts from being attacked by the host cytotoxic T lymphocytes.

In summary this thesis outlines distinct immune cell subsets being essential for regain of tissue function in hypoxia, ischemia and post injury and ways to amplify specific immune cell functions in these situations that are feasible for clinical use.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 53 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1242
Keyword
leukocytes, neutrophils, macrophages, regulatory T cells, chemokines, VEGF-A, hypoxia, ischemia, muscle, Islets of Langerhans, diabetes, transplantation, wound healing
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-299683 (URN)978-91-554-9641-8 (ISBN)
External cooperation:
Public defence
2016-09-23, A1:107a, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-09-02 Created: 2016-07-25 Last updated: 2016-09-05

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Massena, SaraChristoffersson, GustafVågesjö, EvelinaSeignez, CédricGustafsson, KarinHerrera Hidalgo, CarmenGiraud, AntoineLomei, JalalWeström, SimoneClaesson-Welsh, LenaGerwins, PärWelsh, MichaelKreuger, JohanPhillipson, Mia

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Massena, SaraChristoffersson, GustafVågesjö, EvelinaSeignez, CédricGustafsson, KarinHerrera Hidalgo, CarmenGiraud, AntoineLomei, JalalWeström, SimoneClaesson-Welsh, LenaGerwins, PärWelsh, MichaelKreuger, JohanPhillipson, Mia
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