Their importance

The vasculature is the first organ to develop during development. Blood vessels run through virtually every organ in the body, supplying oxygen and nutrients. Not surprisingly therefore, vessels are critical for organ growth in the embryo and for repair of wounded tissue in the adult.

Excess angiogenesis promotes the progression of numerous diseases, including cancer, ocular neovascularization (age related macular degeneration), inflammation, infection etc. Inhibition of angiogenesis has thus emerged as an attractive therapeutic strategy to combat various diseases.

Insufficient angiogenesis also plays a major role in the pathogenesis of many disorders, including ischemic heart and brain disease, peripheral arterial occlusive disease and even, as we recently showed, neurodegeneration. Novel approaches to revascularize ischemic tissues or promote post-infarction repair using angiogenic factors or progenitor cells offer great promise.

The lymphatic vascular system is crucial for reabsorption of extravasated protein-rich fluid and lymphocytes into the blood circulation. When the lymphatic system is dysfunctional, the immune response is compromised and debilitating lymphedema develops, with chronic swelling, tissue fibrosis and poor wound healing, often leading to amputation. Importantly, malignant cells often escape along lymphatics to lymph nodes and, via entry into the circulation, metastasize to distant organs. Because of an insufficient understanding of the molecular mechanisms how lymph vessels grow (a process termed lymphangiogenesis), there is still no clinically approved treatment to inhibit the excessive growth of these lymphatics in cancer, or to stimulate lymphangiogenesis in lymphedema.

Vascular development in zebrafish and tadpoles

Our research is focused on unraveling the key angiogenic and lymphangiogenic mechanisms in health and disease, and, at the same time, has a strong interest in translating these genetic insights into the clinic by developing novel therapeutic strategies. To study vascular development in the embryo, the VRC has set-up extensive small animal facilities. State-of-the-art genetic manipulation and physiology of the mouse are being routinely used to study the role and therapeutic potential of angiogenic and lymphangiogenic candidates (see below).
Compared to the mouse model, smaller animal models such as zebrafish embryos or tadpoles can be more easily and rapidly genetically manipulated. In addition, chemico-genetics offer conditional, dose-dependent knockdown of target genes. Moreover, superior imaging techniques offer unprecedented opportunities to visualize the growing vasculature in living embryos. They are therefore attractive tools to characterize the role of novel gene candidates or the therapeutic potential of novel drug candidates at a much larger scale than in the mouse. These models can thus be used to pre-screen gene candidates identified in genomic or proteomic profiling, thereby allowing one to rapidly narrow down the dozens of putative candidates to the few truly interesting (disease) candidate genes, which can then be studied by gene targeting in the mouse.

Both small aquatic animal models (zebrafish embryos and Xenopus tadpoles link naar aquatic core facility) and mouse models are being used in a complementary manner to unravel the genetic basis of lymphangiogenesis. Our lab was the first to develop the Xenopus tadpole as a powerful model to study the molecular basis of lymphangiogenesis (Ny et al, NMED 2005). This tadpole model was recently applied to dissect the importance of the lymphendothelial receptor VEGFR-3 and its ligands in developmental lymphangiogenesis, revealing a modifier role of VEGF-D in concert with VEGF-C, and a critical requirement of their receptor VEGFR-3 for the formation of functional lymph vessels (Ny et al, Blood 2008).
Of note, unlike in mouse embryos, survival of the early-stage, small-sized Xenopus (or zebrafish) embryos is supported by passive diffusion of oxygen, even when the vasculature is dysfunctional. This enables the investigation of lymph/angiogenic genes that in mice are indispensable for embryonic development. Novel angiogenic and lymphangiogenic candidates are currently being studied in these aquatic models. Interesting phenotypes are further investigated in mice during adult physiology and pathology.

Another exciting new research field is the role of classical axon guidance signals in vessel navigation and formation. The discovery of key axon guidance molecules over the past decade has shown that axons are guided to their targets by finely tuned codes of attractive an repulsive cues. Recent studies reveal that these cues also help blood vessels to navigate to their targets. In some cases, vessels produce signals that attract axons to track alongside the pioneer vessel. The ability of axons to guide vessels, and vice versa, extends the well know facts that axons can guide other axons. The same cues and receptors - that control axon guidance - also function to pattern blood vessels. This provides a distinct mechanism to explain how axons and vessels align – by responding to common cues.

Recently, we demonstrated that morpholino knock-down of the zebrafish orthologue of Unc5b or its ligand Netrin-1a led to aberrant pathfinding of intersegmental vessels (ISVs; see figure). This phenotype indicates that netrin-1 and Unc5b provide critical repulsive cues for navigating vessels. They also open novel avenues for therapeutic strategies aimed not only at stimulating the formation of new blood vessels in ischemic diseases (like heart and limb ischemic diseases), but also at guiding them into a coordinated and functional network.

 
Peter Carmeliet / Mieke Dewerchin

Angiogenesis and lymphangiogenesis in disease

By applying gene targeting, silencing or overexpression in mice (link naar mouse gene targeting facility) complemented with various in vitro cell culture experimentations, we are analyzing the role of family-members of the vascular endothelial growth factor system (VEGF system) and its homologue PlGF as wekk as components of the coagulation system (e.g. Gas6; eventueel link to Stichting tegen kanker projects website, see below). We investigate their role in angiogenesis in development, health and disease, with a strong focus on tumor angiogenesis and metastasis.

In addition, the involvement of the transmembrane protein Notch3 in arterial integrity and arteriopathy is studied with respect to the pathobiology of CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), an adult onset autosomal-dominant neurovascular disorder caused by stereotyped mutations in the extracellular domain of the transmembrane protein Notch3. Mice with targeted mutation of Notch3 analogous to a prevailing human Cadasil mutation were generated.

These genetic studies have yielded promising therapeutic targets. For instance, we developed anti-PlGF antibodies, which not only efficiently block tumor angiogenesis and growth, but also lack the side effects of typical VEGF (receptor) inhibitors (Cell, Nov 2007; collaboration with ThromboGenics).

In the future, we will further explore the therapeutic potential of anti-PlGF therapy for cancer (additional tumor models), ocular disease (age-related macular degeneration), inflammatory disease (rheumatoid arthritis), etc, and delineate the molecular and cellular mechanisms of anti-PlGF therapy.

We will also focus on the role of lymphatic candidates in pathological diseases. Finding compounds or strategies to stimulate or inhibit the growth of lymph vessels would offer treatment opportunities for lymphedema and cancer, respectively. These findings will elucidate possible targets for development of drugs for lymphatic disorders important in human diseases.

Using various techniques (delivery of protein, gene transfer, cell transplantation, etc), we are exploring the therapeutic potential of novel pro-angiogenic factors, such as for instance of PlGF and VEGF-B, another homologue of VEGF.