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Testini, Chiara
Publications (10 of 10) Show all publications
Testini, C., Smith, R. O., Jin, Y., Martinsson, P., Sun, Y., Hedlund, M., . . . Claesson-Welsh, L. (2019). Myc-dependent endothelial proliferation is controlled by phosphotyrosine 1212 in VEGF receptor-2. EMBO Reports, 20(11), Article ID e47845.
Open this publication in new window or tab >>Myc-dependent endothelial proliferation is controlled by phosphotyrosine 1212 in VEGF receptor-2
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2019 (English)In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 20, no 11, article id e47845Article in journal (Refereed) Published
Abstract [en]

Exaggerated signaling by vascular endothelial growth factor (VEGF)-A and its receptor, VEGFR2, in pathologies results in poor vessel function. Still, pharmacological suppression of VEGFA/VEGFR2 may aggravate disease. Delineating VEGFR2 signaling in vivo provides strategies for suppression of specific VEGFR2-induced pathways. Three VEGFR2 tyrosine residues (Y949, Y1212, and Y1173) induce downstream signaling. Here, we show that knock-in of phenylalanine to create VEGFR2 Y1212F in C57Bl/6 and FVB mouse strains leads to loss of growth factor receptor-bound protein 2- and phosphoinositide 3 '-kinase (PI3K)p85 signaling. C57Bl/6 Vegfr2(Y1212F/Y1212F) show reduced embryonic endothelial cell (EC) proliferation and partial lethality. FVB Vegfr2(Y1212F/Y1212F) show reduced postnatal EC proliferation. Reduced EC proliferation in Vegfr2(Y1212F/Y1212F) explants is rescued by c-Myc overexpression. We conclude that VEGFR2 Y1212 signaling induces activation of extracellular-signal-regulated kinase (ERK)1/2 and Akt pathways required for c-Myc-dependent gene regulation, endothelial proliferation, and vessel stability.

Keywords
angiogenesis, GRB2, Nck, PI3Kp85, proliferation
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-397954 (URN)10.15252/embr.201947845 (DOI)000496229500001 ()31545012 (PubMedID)
Funder
Swedish Research Council, 2015-02375Swedish Cancer Society, CAN2016/578Knut and Alice Wallenberg Foundation, KAW 2015.0030Knut and Alice Wallenberg Foundation, 2015.0275
Available from: 2020-01-02 Created: 2020-01-02 Last updated: 2020-01-02Bibliographically approved
Morin, E., Sjöberg, E., Tjomsland, V., Testini, C., Lindskog, C., Franklin, O., . . . Claesson-Welsh, L. (2018). VEGF receptor-2/neuropilin 1 trans-complex formation between endothelial and tumor cells is an independent predictor of pancreatic cancer survival. Journal of Pathology, 246(3), 311-322
Open this publication in new window or tab >>VEGF receptor-2/neuropilin 1 trans-complex formation between endothelial and tumor cells is an independent predictor of pancreatic cancer survival
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2018 (English)In: Journal of Pathology, ISSN 0022-3417, E-ISSN 1096-9896, Vol. 246, no 3, p. 311-322Article in journal (Refereed) Published
Abstract [en]

Unstable and dysfunctional tumor vasculature promotes cancer progression and spread. Signal transduction by the pro-angiogenic vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) is modulated by VEGFA-dependent complex formation with neuropilin 1 (NRP1). NRP1 expressed on tumor cells can form VEGFR2/NRP1 trans-complexes between tumor cells and endothelial cells which arrests VEGFR2 on the endothelial surface, thus interfering with productive VEGFR2 signaling. In mouse fibrosarcoma, VEGFR2/NRP1 trans-complexes correlated with reduced tumor vessel branching and reduced tumor cell proliferation. Pancreatic ductal adenocarcinoma (PDAC) strongly expressed NRP1 on both tumor cells and endothelial cells, in contrast to other common cancer forms. Using proximity ligation assay, VEGFR2/NRP1 trans-complexes were identified in human PDAC tumor tissue, and its presence was associated with reduced tumor vessel branching, reduced tumor cell proliferation, and improved patient survival after adjusting for other known survival predictors. We conclude that VEGFR2/NRP1 trans-complex formation is an independent predictor of PDAC patient survival. 

Keywords
VEGF, neuropilin 1, pancreatic adenocarcinoma, trans-complex, branching
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-363966 (URN)10.1002/path.5141 (DOI)000447161600007 ()30027561 (PubMedID)
Funder
Swedish Research Council, 2015-02375Swedish Cancer Society, CAN2016/578Knut and Alice Wallenberg Foundation, KAW 2015.0030Knut and Alice Wallenberg Foundation, KAW 2015.0275
Available from: 2018-10-22 Created: 2018-10-22 Last updated: 2019-06-26Bibliographically approved
Testini, C. (2016). Regulation of VEGFR2 signaling in angiogenesis and vascular permeability. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
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
Li, X., Padhan, N., Sjöström, E. O., Roche, F. P., Testini, C., Honkura, N., . . . Claesson-Welsh, L. (2016). VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread. Nature Communications, 7, Article ID 11017.
Open this publication in new window or tab >>VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread
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2016 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 7, article id 11017Article in journal (Refereed) Published
Abstract [en]

The specific role of VEGFA-induced permeability and vascular leakage in physiology and pathology has remained unclear. Here we show that VEGFA-induced vascular leakage depends on signalling initiated via the VEGFR2 phosphosite Y949, regulating dynamic c-Src and VE-cadherin phosphorylation. Abolished Y949 signalling in the mouse mutant Vegfr2(Y949F/Y949F) leads to VEGFA-resistant endothelial adherens junctions and a block in molecular extravasation. Vessels in Vegfr2(Y949F/Y949F) mice remain sensitive to inflammatory cytokines, and vascular morphology, blood pressure and flow parameters are normal. Tumour-bearing Vegfr2(Y949F/Y949F) mice display reduced vascular leakage and oedema, improved response to chemotherapy and, importantly, reduced metastatic spread. The inflammatory infiltration in the tumour micro-environment is unaffected. Blocking VEGFA-induced disassembly of endothelial junctions, thereby suppressing tumour oedema and metastatic spread, may be preferable to full vascular suppression in the treatment of certain cancer forms.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-288617 (URN)10.1038/ncomms11017 (DOI)000372721400001 ()27005951 (PubMedID)
Funder
Swedish Cancer SocietySwedish Research CouncilKnut and Alice Wallenberg FoundationEU, European Research Council, 294556 BBBARRIERWenner-Gren Foundations
Available from: 2016-05-11 Created: 2016-04-28 Last updated: 2017-11-30Bibliographically approved
Koch, S., van Meeteren, L. A., Morin, E., Testini, C., Weström, S., Björkelund, H., . . . Claesson-Welsh, L. (2014). NRP1 Presented in trans to the Endothelium Arrests VEGFR2 Endocytosis, Preventing Angiogenic Signaling and Tumor Initiation. Developmental Cell, 28(6), 633-646
Open this publication in new window or tab >>NRP1 Presented in trans to the Endothelium Arrests VEGFR2 Endocytosis, Preventing Angiogenic Signaling and Tumor Initiation
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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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-224353 (URN)10.1016/j.devcel.2014.02.010 (DOI)000333765900005 ()
Available from: 2014-05-13 Created: 2014-05-09 Last updated: 2018-10-26
Yao, L.-C., Testini, C., Tvorogov, D., Anisimov, A., Vargas, S. O., Baluk, P., . . . McDonald, D. M. (2014). Pulmonary Lymphangiectasia Resulting From Vascular Endothelial Growth Factor-C Overexpression During a Critical Period. Circulation Research, 114(5), 806-822
Open this publication in new window or tab >>Pulmonary Lymphangiectasia Resulting From Vascular Endothelial Growth Factor-C Overexpression During a Critical Period
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2014 (English)In: Circulation Research, ISSN 0009-7330, E-ISSN 1524-4571, Vol. 114, no 5, p. 806-822Article in journal (Refereed) Published
Abstract [en]

Rationale: Lymphatic vessels in the respiratory tract normally mature into a functional network during the neonatal period, but under some pathological conditions they can grow as enlarged, dilated sacs that result in the potentially lethal condition of pulmonary lymphangiectasia. Objective: We sought to determine whether overexpression of the lymphangiogenic growth factor (vascular endothelial growth factor-C [VEGF-C]) can promote lymphatic growth and maturation in the respiratory tract. Unexpectedly, perinatal overexpression of VEGF-C in the respiratory epithelium led to a condition resembling human pulmonary lymphangiectasia, a life-threatening disorder of the newborn characterized by respiratory distress and the presence of widely dilated lymphatics. Methods and Results: Administration of doxycycline to Clara cell secretory protein-reverse tetracycline-controlled transactivator/tetracycline operator-VEGF-C double-transgenic mice during a critical period from embryonic day 15.5 to postnatal day 14 was accompanied by respiratory distress, chylothorax, pulmonary lymphangiectasia, and high mortality. Enlarged sac-like lymphatics were abundant near major airways, pulmonary vessels, and visceral pleura. Side-by-side comparison revealed morphological features similar to pulmonary lymphangiectasia in humans. The condition was milder in mice given doxycycline after age postnatal day 14 and did not develop after postnatal day 35. Mechanistic studies revealed that VEGF recptor (VEGFR)-3 alone drove lymphatic growth in adult mice, but both VEGFR-2 and VEGFR-3 were required for the development of lymphangiectasia in neonates. VEGFR-2/VEGFR-3 heterodimers were more abundant in the dilated lymphatics, consistent with the involvement of both receptors. Despite the dependence of lymphangiectasia on VEGFR-2 and VEGFR-3, the condition was not reversed by blocking both receptors together or by withdrawing VEGF-C. Conclusions: The findings indicate that VEGF-C overexpression can induce pulmonary lymphangiectasia during a critical period in perinatal development.

Keywords
chylothorax, lung, lymphatic vessels, lymphangiogenesis, lymphangiomatosis, pulmonary, pulmonary edema, VEGFR-2, VEGFR-3
National Category
Cardiac and Cardiovascular Systems Hematology
Identifiers
urn:nbn:se:uu:diva-227943 (URN)10.1161/CIRCRESAHA.114.303119 (DOI)000335586900013 ()
Available from: 2014-07-07 Created: 2014-07-02 Last updated: 2017-12-05Bibliographically approved
Yao, L.-C., Testini, C., Tvorogov, D., Anisimov, A., Vargas, S. O., Baluk, P., . . . McDonald, D. M. (2013). Pulmunary Lymphangiectasia Resulting From Vascular Endotelial Growth Factor-C Overexpression During a Critical Period. Circulation Research
Open this publication in new window or tab >>Pulmunary Lymphangiectasia Resulting From Vascular Endotelial Growth Factor-C Overexpression During a Critical Period
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2013 (English)In: Circulation Research, ISSN 0009-7330, E-ISSN 1524-4571Article in journal (Refereed) Published
Keywords
chylothorax, lung, lymphatic vessels, lymphangiogenesis, pulmunary edema, VEGFR-2, VEGFR-3
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-220644 (URN)
Available from: 2014-03-18 Created: 2014-03-18 Last updated: 2018-01-11Bibliographically approved
Coltrini, D., Di Salle, E., Ronca, R., Belleri, M., Testini, C. & Presta, M. (2012). Matrigel plug assay: evaluation of the angiogenic response by reverse transcription-quantitative PCR. Angiogenesis
Open this publication in new window or tab >>Matrigel plug assay: evaluation of the angiogenic response by reverse transcription-quantitative PCR
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2012 (English)In: Angiogenesis, ISSN 0969-6970, E-ISSN 1573-7209Article in journal (Refereed) Published
Keywords
Angiogenesis, FGF-2, Matrigel, PCR, Quantification
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-220647 (URN)
Available from: 2014-03-18 Created: 2014-03-18 Last updated: 2017-12-05Bibliographically approved
Nitzsche, A., Testini, C., Ekvärn, E., Larsson, J., Bentley, K., Philippides, A., . . . Hellström, M.Paladin (Pald1) regulates endothelial sprouting, VE-cadherin junction stability and vascular permeability.
Open this publication in new window or tab >>Paladin (Pald1) regulates endothelial sprouting, VE-cadherin junction stability and vascular permeability
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(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology Cardiac and Cardiovascular Systems Cancer and Oncology
Identifiers
urn:nbn:se:uu:diva-300083 (URN)
Available from: 2016-08-02 Created: 2016-08-02 Last updated: 2018-01-10
Testini, C., Smith, R., Li, X., He, L., Shibuya, M., Hellström, M. & Claesson-Welsh, L.VEGFR2 pY1212 signaling regulates endothelial cell proliferation and vessel stability.
Open this publication in new window or tab >>VEGFR2 pY1212 signaling regulates endothelial cell proliferation and vessel stability
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(English)Manuscript (preprint) (Other academic)
National Category
Cancer and Oncology Cell and Molecular Biology Cardiac and Cardiovascular Systems
Identifiers
urn:nbn:se:uu:diva-300081 (URN)
Available from: 2016-08-02 Created: 2016-08-02 Last updated: 2018-01-10
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