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VEGF-mediated vascular functions in health and disease
Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Health Sciences.
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Angiogenesis is essential for physiological processes including embryonic development, tissue regeneration, and reproduction. Under various pathological conditions the same angiogenic process contribute to the onset, development, and progression of many human diseases including cancer, diabetic complications, ocular disease, chronic inflammation and cardiovascular disease. Vascular endothelial growth factor (VEGF) is a key angiogenic factor for physiological and pathological angiogenesis. In addition to its strong angiogenic activity, VEGF also potently induces vascular permeability, often causing tissue edema in various pathological tissues. VEGF transduces its vascular signal through two tyrosine kinase receptors-VEGFR1 and VEGFR2, the latter being a functional receptor that mediates both angiogenic and vascular permeability effects. To study physiological and pathological functions of VEGF, we developed novel zebrafish disease models that permit us to study hypoxia-induced retinopathy and cancer metastasis processes. We have also administered anti-VEGF and anti-VEGFR specific antibodies to healthy mice to study the homeostatic role of VEGF in the maintenance of vascular integrity and its functions in various tissues and organs.

Finally, using a zebrafish model, we evaluated if VEGF expression is regulated by circadian clock genes. In paper I, we developed protocols that create hypoxia-induced retinopathy in adult zebrafish. Adult fli1:EGFP zebrafish were placed in hypoxic water for 3-10 days with retinal neovascularization being analyzed using confocal microscopy. This model provides a unique opportunity to kinetically study the development of retinopathy in adult animals using non-invasive protocols and to assess the therapeutic efficacy of orally administered anti-angiogenic drugs. In paper II, we developed a zebrafish metastasis model to dissect the complex events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent DiI-labeled human or mouse tumor cells were implanted into the perivitelline cavity of 48-hour-old zebrafish embryos, which were subsequently placed in hypoxic water for 3 days. Tumor cell invasion, metastasis and pathological angiogenesis were analyzed using fluorescent microscopy in the living fish. The average experimental time for this model is 7 days. Our protocol offers an opportunity to study molecular mechanisms of hypoxia-induced cancer metastasis. In paper III, we show that systemic delivery of an anti-VEGF or an anti-VEGF receptor (VEGFR)-2 neutralizing antibody cause global vascular regression in mice. Among all examined tissues, the vasculature in endocrine glands, intestinal villi, and the uterus are most affected in response to VEGF or VEGFR-2 blockades. Pro-longed anti-VEGF treatment resulted in a significant decrease in the circulating levels of the predominant thyroid hormone, free thyroxine, but not the minimal isoform of triiodothyronine, suggesting that chronic anti-VEGF treatment impairs thyroid function. These findings provide structural and functional bases of anti-VEGF-specific druginduced side effects in relation to vascular changes in healthy tissues. In paper IV, we show that disruption of the circadian clock by constant exposure to light coupled with genetic manipulation of key genes in the zebrafish led to impaired developmental angiogenesis. A bmal1-specific morpholino inhibited developmental angiogenesis in zebrafish embryos without causing obvious nonvascular phenotypes. Conversely, a period2 morpholino accelerated angiogenic vessel growth, suggesting that Bmal1 and Period2 display opposing angiogenic effects. These results offer mechanistic insights into the role of the circadian clock in regulation of developmental angiogenesis, and our findings may be reasonably extended to other types of physiological or pathological angiogenesis. Overall, the results in this thesis provide further insight to angiogenic mechanistic properties in tissues and suggest possible novel therapeutic targets for the treatment of various angiogenesis-dependent diseases.

Abstract [sv]

Blodkärlsnybildning, så kallad angiogenes, är viktigt för fysiologiska processer vid embryonal utveckling, vävnadsregenerering och reproduktion. Samma angiogena process kan också under olika sjukdomstillstånd bidra till uppkomst, utveckling och progress av många sjukdomar, såsom cancer, diabeteskomplikationer, ögonsjukdomar, kronisk inflammation samt hjärtkärlsjukdom. Vascular endothelial growth factor (VEGF) är mycket viktig för fysiologisk och patologisk angiogenes. Utöver sin starka angiogena effekt inducerar VEGF även ökad kärlpermeabilitet, som ofta orsakar ödem. VEGF utövar sin effekt på kärlen via två tyrosinkinasreceptorer: VEGFR1 och VEGFR2, där den senare är en funktionell receptor som förmedlar både angiogena signaler och har effekter på vaskulär permeabilitet. För att öka möjlgheterna att studera fysiologiska och patologiska funktioner av VEGF, har vi utvecklat sjukdomsmodeller i zebrafisk - hypoxi-inducerad retinopati och metastasering av cancer. Vi har också givit anti-VEGF och anti-VEGFR-specifika antikroppar till friska möss för att utvärdera VEGFs roll vid stabiliseringen av kärlfunktionen i olika vävnader och organ.

Slutligen,utvärderade vi om expressionen av VEGF regleras av dygnsrytmen genom så kallade klock-gener. I papper I utvecklade vi en modell för hypoxiinducerad retinopati hos vuxna zebrafiskar. Vuxna fli1:EGFP zebrafiskar placeras i syrefattigt vatten i 3-10 dagar, varpå retinal nybildning av kärl analyserades. Denna modell ger en unik icke-invasiv möjlighet att studera kinetiskt utveckling av retinopati och den möjliggör bedömning av terapeutiska effekter av oralt givna anti-angiogena läkemedel. I papper II utvecklade vi en zebrafiskmodell för utvärdering av cancermetastasering, som möjliggör studier av detaljerade delprocesser vid hypoxi-inducerad tumörcellsinvasion och metastasering i samband med angiogenes på encellig nivå. I denna modell användes fluorescerande Dil-märkta humana- eller mustumörceller som implanterades vid den perivitellina hålighet hos 48-h-gamla zebrafiskembryon placerade i syrefattigt vatten i 3 dagar. Tumörcellinvasion, metastasering och patologisk angiogenes analyserades med mikroskopi i levande fiskar. Vårt protokoll möjliggör studier av molekylära mekanismer bakom hypoxi-inducerad cancermetastasering. I papper III visas, att systemisk administration av anti-VEGF eller anti-VEGF-receptor (VEGFR)-2 neutraliserande antikroppar in en musmodell orsakar generell kärlregression. Bland alla undersökta vävnader påverkades endokrina körtlar, tarmslemhinna och uterus mest av VEGF eller VEGFR-2 blockad. Långvarig anti-VEGF behandling resulterade i en signifikant minskning av cirkulerande nivåer av det dominerande sköldkörtelhormonet, fritt tyroxin, men inte av trijodtyronin, vilket tyder på att kronisk anti-VEGF behandling försämrar sköldkörtelfunktionerna. Resultaten påvisar risken för biverkningar i friska vävnader av anti-VEGF behandling. I papper IV visar vi att störningar i dygnsrytm genom konstant exponering för ljus och genetisk manipulation av nyckelgener i zebrafisk ledde till nedsatt angiogenes under embryonal utveckling. En bmal1-specifik morfolino hämmade angiogenes i zebrafisk utan att orsaka andra kärl-oberoende fenotyper. Omvänt, en period2 morfolino accelererade angiogeneskärltillväxt, vilket tyder på att Bmal1 och Period2 utövar motsatta effekter påkärlstillväxt. Dessa resultat ger mekanistisk kunskap om den roll som dygnsrytmen har i regleringen av angiogenes, och resultat kan rimligen utvidgas till andra typer av fysiologisk eller patologisk angiogenes. Sammanfattningsvis ger resultaten i denna avhandling ytterligare kunskap om angiogenetiska mekanismer och pekar på möjliga nya terapeutiska mål för behandling av olika angiogenes-beroende sjukdomar.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. , 69 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1459
National Category
Clinical Medicine Surgery
Identifiers
URN: urn:nbn:se:liu:diva-117356DOI: 10.3384/diss.diva-117356ISBN: 978-91-7519-079-2 (print)OAI: oai:DiVA.org:liu-117356DiVA: diva2:807622
Public defence
2015-05-25, Nilsholger, Campus US, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2015-04-24 Created: 2015-04-24 Last updated: 2017-03-27Bibliographically approved
List of papers
1. Hypoxia-induced retinopathy model in adult zebrafish
Open this publication in new window or tab >>Hypoxia-induced retinopathy model in adult zebrafish
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2010 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 5, no 12, 1903-1910 p.Article in journal (Refereed) Published
Abstract [en]

Hypoxia-induced vascular responses, including angiogenesis, vascular remodeling and vascular leakage, significantly contribute to the onset, development and progression of retinopathy. However, until recently there were no appropriate animal disease models recapitulating adult retinopathy available. In this article, we describe protocols that create hypoxia-induced retinopathy in adult zebrafish. Adult fli1: EGFP zebrafish are placed in hypoxic water for 3-10 d and retinal neovascularization is analyzed using confocal microscopy. It usually takes 11 d to obtain conclusive results using the hypoxia-induced retinopathy model in adult zebrafish. This model provides a unique opportunity to study kinetically the development of retinopathy in adult animals using noninvasive protocols and to assess therapeutic efficacy of orally active antiangiogenic drugs.

Place, publisher, year, edition, pages
Nature Publishing Group, 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-63381 (URN)10.1038/nprot.2010.149 (DOI)000284884100003 ()
Available from: 2010-12-17 Created: 2010-12-17 Last updated: 2017-12-11Bibliographically approved
2. Hypoxia-induced metastasis model in embryonic zebrafish
Open this publication in new window or tab >>Hypoxia-induced metastasis model in embryonic zebrafish
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2010 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 5, no 12, 1911-1918 p.Article in journal (Refereed) Published
Abstract [en]

Hypoxia facilitates tumor invasion and metastasis by promoting neovascularization and co-option of tumor cells in the peritumoral vasculature, leading to dissemination of tumor cells into the circulation. However, until recently, animal models and imaging technology did not enable monitoring of the early events of tumor cell invasion and dissemination in living animals. We recently developed a zebrafish metastasis model to dissect the detailed events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent DiI-labeled human or mouse tumor cells are implanted into the perivitelline cavity of 48-h-old zebrafish embryos, which are subsequently placed in hypoxic water for 3 d. Tumor cell invasion, metastasis and pathological angiogenesis are detected under fluorescent microscopy in the living fish. The average experimental time for this model is 7 d. Our protocol offers a remarkable opportunity to study molecular mechanisms of hypoxia-induced cancer metastasis.

Place, publisher, year, edition, pages
Nature Publishing Group, 2010
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-63382 (URN)10.1038/nprot.2010.150 (DOI)000284884100004 ()
Available from: 2010-12-17 Created: 2010-12-17 Last updated: 2017-12-11Bibliographically approved
3. Anti-VEGF- and anti-VEGF receptor-induced vascular alteration in mouse healthy tissues
Open this publication in new window or tab >>Anti-VEGF- and anti-VEGF receptor-induced vascular alteration in mouse healthy tissues
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2013 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 29, 12018-12023 p.Article in journal (Refereed) Published
Abstract [en]

Systemic therapy with anti-VEGF drugs such as bevacizumab is widely used for treatment of human patients with various solid tumors. However, systemic impacts of such drugs in host healthy vasculatures remain poorly understood. Here, we show that, in mice, systemic delivery of an anti-VEGF or an anti-VEGF receptor (VEGFR)-2 neutralizing antibody caused global vascular regression. Among all examined tissues, vasculatures in endocrine glands, intestinal villi, and uterus are the most affected in response to VEGF or VEGFR-2 blockades. Thyroid vascular fenestrations were virtually completely blocked by VEGF blockade, leading to marked accumulation of intraendothelial caveolae vesicles. VEGF blockade markedly increased thyroid endothelial cell apoptosis, and withdrawal of anti-VEGF resulted in full recovery of vascular density and architecture after 14 d. Prolonged anti-VEGF treatment resulted in a significant decrease of the circulating level of the predominant thyroid hormone free thyroxine, but not the minimal isoform of triiodothyronine, suggesting that chronic anti-VEGF treatment impairs thyroid functions. Conversely, VEGFR-1-specific blockade produced virtually no obvious phenotypes. These findings provide structural and functional bases of anti-VEGF-specific drug-induced side effects in relation to vascular changes in healthy tissues. Understanding anti-VEGF drug-induced vascular alterations in healthy tissues is crucial to minimize and even to avoid adverse effects produced by currently used anti-VEGF-specific drugs.

Place, publisher, year, edition, pages
National Academy of Sciences, 2013
Keyword
angiogenesis, antiangiogenic therapy, off-tumor targets, vascular homeostasis, vessel regression
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-102114 (URN)10.1073/pnas.1301331110 (DOI)000322086100078 ()23818623 (PubMedID)
Available from: 2013-12-01 Created: 2013-12-01 Last updated: 2017-12-06Bibliographically approved
4. Opposing Effects of Circadian Clock Genes Bmal1 and Period2 in Regulation of VEGF-Dependent Angiogenesis in Developing Zebrafish
Open this publication in new window or tab >>Opposing Effects of Circadian Clock Genes Bmal1 and Period2 in Regulation of VEGF-Dependent Angiogenesis in Developing Zebrafish
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2012 (English)In: Cell Reports, ISSN 2211-1247, Vol. 2, no 2, 231-241 p.Article in journal (Refereed) Published
Abstract [en]

Molecular mechanisms underlying circadian-regulated physiological processes remain largely unknown. Here, we show that disruption of the circadian clock by both constant exposure to light and genetic manipulation of key genes in zebrafish led to impaired developmental angiogenesis. A bmal1-specific morpholino inhibited developmental angiogenesis in zebrafish embryos without causing obvious nonvascular phenotypes. Conversely, a period2 morpholino accelerated angiogenic vessel growth, suggesting that Bmal1 and Period2 display opposing angiogenic effects. Using a promoter-reporter system consisting of various deleted vegf-promoter mutants, we show that Bmal1 directly binds to and activates the vegf promoter via E-boxes. Additionally, we provide evidence that knockdown of Bmal1 leads to impaired Notch-inhibition-induced vascular sprouting. These results shed mechanistic insight on the role of the circadian clock in regulation of developmental angiogenesis, and our findings may be reasonably extended to other types of physiological or pathological angiogenesis.

Place, publisher, year, edition, pages
Elsevier (Cell Press), 2012
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-85304 (URN)10.1016/j.celrep.2012.07.005 (DOI)000309715100004 ()
Note

Funding Agencies|Swedish Research Council||Swedish Cancer Foundation||Karolinska Institute Foundation||Karolinska Institute||Tianjin Natural Science Foundation (CMM-Tianjin)|09ZCZDSF04400|Torsten Soderbergs Foundation||European Union|222741|European Research Council (ERC)|250021|

Available from: 2012-11-16 Created: 2012-11-15 Last updated: 2017-03-27Bibliographically approved

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