Mechanisms of malignant and non-malignant angiogenesis using zebrafish models

Pathological angiogenesis significantly contribute to the onset, development and progression of most common and severe human diseases including cancer, metastatic disease, cardiovascular disease, age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity. Under these pathological conditions, tissue hypoxia often acts as a trigger to switch on angiogenesis. However, there has been lacking non-invasive and clinically relevant animal models that allow us to study mechanisms of human diseases. Zebrafish, as a complementary animal model to mice, is a highly genetically and pharmacologically tractable vertebrate which is easily visualized during development. Zebrafish offers a unique opportunity to study angiogenesis under hypoxia.

This thesis describes development and characterization of four novel zebrafish models in relation to hypoxia-induced angiogenesis, vascular and tumor pathology. Using these models, we demonstrate that hypoxia plays a causal role in development of retinopathy and cancer cell metastasis and thus provide important insights needed for the development of therapeutic approaches aimed at interfering with these processes.

In paper I, we showed that hypoxia could induce neovascular retinopathy in zebrafish and this model is highly relevant to clinical retinopathy caused by diabetes. This zebrafish retinopathy model also allows us study the therapeutic potential of various antiangiogenic agents. In paper II, we demonstrate a novel principle that regulates blood perfusion in lymphatics as an effective defense against tissue hypoxia in zebrafish and kryptopterus bicirrhis. The arterial-lymphatic shunt is controlled by nitric oxide and the implication of this work is that NO-induced lymphatic perfusion might facilitate tumor cell spread from the blood stream into the lymphatic system. In paper III, we take advantage of the transparent nature of zebrafish embryos and availability of the transgenic strain fli1:EGFP to develop a zebrafish metastasis model. Using this model, we are the first to study the role of hypoxia in relation to angiogenesis in facilitating tumor cell dissemination, invasion and metastasis. To the best of our knowledge, this is the first animal model that allows scientists to study the early events of metastasis at a single cell level. In paper IV, We show that PI3 kinase is a key signaling component that mediates angiogenesis in the developing embryonic retina and in the regenerating adult fins.

In conclusion, development of these zebrafish disease models have paved new avenues for studying mechanisms of pathological angiogenesis in malignant and non malignant diseases and offers unique opportunities for assessment of therapeutic potentials of known and novel drugs against these most common and lethal diseases.

List of scientific papers

I. Cao R, Jensen LD, Söll I, Hauptmann G, Cao Y (2008). "Hypoxia-induced retinal angiogenesis in zebrafish as a model to study retinopathy." PLoS One 3: e2748
https://pubmed.ncbi.nlm.nih.gov/18648503

II. Dahl Ejby Jensen L, Cao R, Hedlund EM, Söll I, Lundberg JO, Hauptmann G, Steffensen JF, Cao Y (2009). "Nitric oxide permits hypoxia-induced lymphatic perfusion by controlling arterial-lymphatic conduits in zebrafish and glass catfish." Proc Natl Acad Sci U S A 106: 18408-18413
https://pubmed.ncbi.nlm.nih.gov/19822749

III. Lee SL, Rouhi P, Dahl Jensen L, Zhang D, Ji H, Hauptmann G, Ingham P, Cao Y (2009). "Hypoxia-induced pathological angiogenesis mediates tumor cell dissemination, invasion, and metastasis in a zebrafish tumor model." Proc Natl Acad Sci U S A 106: 19485-19490
https://pubmed.ncbi.nlm.nih.gov/19887629

IV. Alvarez Y, Astudillo O, Jensen L, Reynolds AL, Waghorne N, Brazil DP, Cao Y, O´Connor JJ, Kennedy BN (2009). "Selective inhibition of retinal angiogenesis by targeting PI3 kinase." PLoS One 4: e7867
https://pubmed.ncbi.nlm.nih.gov/19924235