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NRP1 Presented in trans to the Endothelium Arrests VEGFR2 Endocytosis, Preventing Angiogenic Signaling and Tumor Initiation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Cancer and Vascular Biology.
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2014 (English)In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 28, no 6, p. 633-646Article in journal (Refereed) Published
Abstract [en]

Neuropilin 1 (NRP1) modulates angiogenesis by binding vascular endothelial growth factor (VEGF) and its receptor, VEGFR2. We examined the consequences when VEGFR2 and NRP1 were expressed on the same cell (cis) or on different cells (trans). In cis, VEGF induced rapid VEGFR2/NRP1 complex formation and internalization. In trans, complex formation was delayed and phosphorylation of phospholipase C gamma (PLC gamma) and extracellular regulated kinase 2 (ERK2) was prolonged, whereas ERK1 phosphorylation was reduced. Trans complex formation suppressed initiation and vascularization of NRP1-expressing mouse fibrosarcoma and melanoma. Suppression in trans required high-affinity, steady-state binding of VEGF to NRP1, which was dependent on the NRP1 C-terminal domain. Compatible with a trans effect of NRP1, quiescent vasculature in the developing retina showed continuous high NRP1 expression, whereas angiogenic sprouting occurred where NRP1 levels fluctuated between adjacent endothelial cells. Therefore, through communication in trans, NRP1 can modulate VEGFR2 signaling and suppress angiogenesis.

Place, publisher, year, edition, pages
2014. Vol. 28, no 6, p. 633-646
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-224353DOI: 10.1016/j.devcel.2014.02.010ISI: 000333765900005OAI: oai:DiVA.org:uu-224353DiVA, id: diva2:716988
Available from: 2014-05-13 Created: 2014-05-09 Last updated: 2018-10-26
In thesis
1. Regulation of VEGFR2 signaling in angiogenesis and vascular permeability
Open this publication in new window or tab >>Regulation of VEGFR2 signaling in angiogenesis and vascular permeability
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Angiogenesis and vascular permeability occur in physiological and pathological conditions. Angiogenesis denotes the process of blood vessel formation from preexisting quiescent vessels. Angiogenesis is initiated by proangiogenic factors, inducing endothelial cell sprouting, migration and anastomosis, followed by regression of the new vessels or maturation into a quiescent status. Vascular permeability is the process where blood vessels exchange nutrients, solutes and inflammatory cells with the surrounding tissue. Small molecules freely cross the endothelial wall, however macromolecules and cells leak out from the vasculature only after stimulation by certain factors, including VEGF. Angiogenesis and vascular permeability are tightly regulated physiological processes, but uncontrolled angiogenesis and excessive leakage lead to pathological conditions and the progression of several diseases.

VEGF and its receptor VEGFR2 are critical players in angiogenesis and in vascular permeability. The binding of the ligand to the receptor is not the only event involved in the activation and regulation of the signaling cascade. Coreceptors, kinases, phosphatases, and other proteins involved in the trafficking of the complex modulate the signal amplitude and duration.

VEGF/VEGFR2 complex combined with the coreceptor NRP1 has a strong pro-angiogenic action and a critical role in angiogenesis. Both VEGFR2 and NRP1 bind VEGF and can present VEGF in cis, when both VEGFR2 and NRP1 are expressed on the same endothelial cell or in trans, when NRP1 is expressed on an adjacent endothelial cell or another type of cell.

Y949 and Y1212 are two of the main phosphorylation sites of VEGFR2 induced by VEGFA. The binding of phosphorylated Y949 to the SH2 domain of TSAd regulates vascular permeability leading to Src activation and adherens junction opening in vitro. Phospho-Y1212 is implicated in actin stress fiber remodeling via the adapter Nck, affecting the actin cytoskeleton and endothelial cell migration in vitro.

Paladin is a vascular-enriched phosphatase-domain containing protein without reported phosphatase activity and is a negative regulator of insulin receptor and Toll-like receptor 9 signaling.

In this thesis work, I have investigated the spatial dynamics of NRP1/VEGFR2 complex formation (in cis and in trans) for coordinating VEGF-mediated angiogenesis in physiological and in pathological conditions (Paper I). I have studied, in vivo, the role of VEGFR2 Y949 in vascular permeability and metastatic spread (Paper II) and the role of VEGFR2 Y1212 in angiogenic remodeling and vessel stability (Paper III). Furthermore, I have examined paladin’s role in regulating VEGF/VEGFR2 signaling and VE-cadherin junction stability, in angiogenic sprouting and vascular permeability (Paper IV).

In conclusion, VEGF/VEGFR2 signaling is regulated by a multifactor system and each individual regulatory mechanism leads to a specific outcome in angiogenesis, vascular permeability and vessel stability.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. p. 56
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1240
Keywords
VEGFR2, angiogenesis, permeability, NRP1, Y949, Y1212, paladin
National Category
Cell and Molecular Biology Cardiac and Cardiovascular Systems Cancer and Oncology
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-300084 (URN)978-91-554-9637-1 (ISBN)
External cooperation:
Public defence
2016-09-29, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2016-09-02 Created: 2016-08-02 Last updated: 2018-01-10
2. Neuropilin-1 regulation of tumor vascularization and growth
Open this publication in new window or tab >>Neuropilin-1 regulation of tumor vascularization and growth
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Angiogenesis, the formation of new blood vessels from existing ones, is dysregulated during tumor progression as a result of chronic hypoxia and inflammation. Such alterations lead to a lack of vessel hierarchy, and the formation of poorly perfused, leaky and blunt-ended vessels, contributing to disease progression. This thesis explores the impact of neuropilin-1 (NRP1) presentation of vascular endothelial growth factor-A (VEGF-A) to its cognate receptor, VEGFR2. NRP1 presentation of VEGF-A occurs in cis (when NRP1 and VEGFR2 are present on the same cell) or in trans (when molecules are present on adjacent cells). As shown in this thesis, the different modes of NRP1 presentation influence endothelial cell signaling and tumor angiogenesis. The overall aim with the studies has been to identify new biomarkers for cancer survival and potential therapeutic targets.

In paper I, we explored if signaling downstream of VEGFR2 was affected by NRP1 presentation in cis compared to trans. Complex formation in trans was readily identified, however, the kinetics were delayed and prolonged, inhibiting VEGFR2 internalization and downstream signaling. Additionally, in vivo tumor studies in mice demonstrated that trans presentation of NRP1 led to early inhibition of angiogenesis and suppressed tumor initiation.

In paper II, the presence and clinical impact of trans VEGFR2/NRP1 complexes in human cancer was investigated. We first identified gastric and pancreatic adenocarcinomas (PDAC) as candidates for further investigation. VEGFR2/NRP1 complexes were identified in both tumor types but were more prevalent in PDAC. Trans presentation of NRP1 in PDAC correlated with a reduction in several vessel parameters and tumor cell proliferation. Importantly, this study identified the presence of trans complexes as an independent marker of longer overall survival for PDAC patients.

In paper III, we explored the impact of NRP1 presentation modes on renal cell carcinoma (RCC) patient survival. We performed in situ proximity ligation assay (PLA) and immunofluorescence staining on a RCC cohort. Tumor cell NRP1, either trans-complexed with endothelial cell-expressed VEGFR2 as detected by in situ PLA, or alternatively, detected by immunofluorescent staining, was identified as an independent predictor of increased overall survival. These data reinforce the importance of the cell type-specific expression of cancer biomarkers.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 69
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1512
Keywords
Angiogenesis, Neuropilin-1, VEGFR2, tumor biology, pancreatic ductal adenocarcinoma, renal cell carcinoma
National Category
Cell and Molecular Biology Cancer and Oncology
Research subject
Medical Cell Biology
Identifiers
urn:nbn:se:uu:diva-364415 (URN)978-91-513-0495-3 (ISBN)
Public defence
2018-12-14, Rudbecksalen, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2018-11-22 Created: 2018-10-26 Last updated: 2018-11-30

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Morin, EricTestini, ChiaraWeström, SimoneClaesson-Welsh, Lena

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