Growth factors controlling angiogenesis and lymphangiogenesis

The underlying causes of cancer are lesions in critical genes regulating cell growth and differentiation and maintenance of the genome integrity. Mutations activating oncogenes or inactivating tumor suppressor genes can be found in most types of human cancer. Mutations in gene products controlling DNA damage repair result in predisposition to cancer because of increased genomic instability. Damaged normal cells may activate the programmed cell death pathway, presumably when cellular damage is beyond repair. In cancer cells these control points are lost, and the cells continue unrestricted proliferation. They also induce the cellular proteolytic machinery for invasion and growth factor gene expression for stimulation of tumor vascularization or angiogenesis (Fig. 1 ).

Mechanisms of angiogenesis

Our main interest is to understand the mechanisms of angiogenesis and to learn analogous mechanisms of lymphatic vessel growth, lymphangiogenesis. Growth factors appear to mediate vascular endothelial cell (EC) reprogramming and invasion in tumor angiogenesis. We have discovered a number of new vascular endothelial growth factors (VEGFs) and their receptors, as well as another endothelial growth factor receptor family (Tie family) (Fig. 2 ). Growth factors binding to these receptors regulate the proteolytic activity of ECs. In these studies we are attempting to combine our knowledge on one side of the receptors and their signalling, and on the other side on cell cycle regulation and apoptosis. Bridging the gap between the angiogenic growth factor receptors and nuclear reprogramming of gene expression in growth factor stimulated cells is a challenging task, but we already have several novel observations regarding these events. As part of the larger Molecular Cancer Biology Program our laboratory wants to elucidate mechanisms of cross-talk and interaction between the ECs, periendothelial cells and cancer cells, to identify novel growth regulatory genes involved and to develop new molecular tools for studies of these interactions. These should then result in innovative new treatments of not only cancer, but also of cardiovascular diseases.

Growth factors and receptors involved in angiogenesis and lymphangiogenesis

To grow beyond the microscopic stage tumors need to generate a microvasculature. This is achieved in part in a paracrine fashion with the help of tumor cell secreted VEGF, also known as the vascular permeability factor. VEGF is an EC specific mitogenic and chemotactic factor. Although encoded by a single gene, VEGF has several isoforms generated by alternative splicing. It is essential for embryonic vasculogenesis, especially in the early differentiation of mesodermal cells into hemangioblasts. Mice with disrupted VEGF or VEGFR genes have impaired vasculogenesis/angiogenesis and die in utero. VEGF acts via its two known receptors VEGFR-1 and VEGFR-2 (see Fig. 2). We have cloned the related VEGFR-3, which does not bind VEGF; its expression becomes restricted mainly to lymphatic endothelia during development. Homozygous VEGFR-3 gene-deleted embryos die at midgestation due to failure of cardiovascular development. We have also purified and cloned the VEGFR-3 ligand, VEGF-C, which is proteolytically processed, and binds and activates VEGFR-3. Transgenic mice expressing VEGF-C in the skin developed a hyperplastic lymphatic vessel network, showing evidence of lymphangiogenesis. However, proteolytically processed VEGF-C was also capable of stimulating VEGFR-2 and was weakly angiogenic. VEGF-C also induced vascular permeability, but its point mutant, which retained lymphangiogenic properties and activated only VEGFR-3 did not. VEGF-D was found to be closely related to VEGF-C, similarly processed and to bind to the same receptors. Thus, VEGF-C and VEGF-D appear to be angiogenic and lymphangiogenic growth factors. Another related novel growth factor, VEGF-B was cloned in collaboration with Dr. Ulf Eriksson's group and found to be especially abundantly expressed in the heart. VEGF-B bound VEGFR-1 and neuropilin-1 and formed cell surface-associated, disulfide-linked homodimers and heterodimers with VEGF when coexpressed.

Another step in the angiogenic pathway involves the angiopoietins (Ang:s). Ang-1 activates the Tie-2 receptor and is involved in the inhibition of permeability changes induced by VEGF. Ang-2 is involved in the destabilization of blood vessels. Together, the Angs and VEGFs constitute an important regulatory system for the development and maintenance of functional blood and lymphatic vessels. Tie-1, one of the receptor tyrosine kinases we have cloned, is expressed in mouse hematopoietic progenitor cells and in ECs. In transgenic mice the Tie-1 gene promoter directed endothelial specific expression of heterologous genes (such as the GFP gene), allowing the isolation of primary cultures of ECs, which can also be immortalized. Tie-1 was required during embryonic development for the sprouting and survival of new vessels, particularly in the regions undergoing angiogenic growth of capillaries. Our results thus demonstrate an increased complexity of signalling for EC proliferation, migration, differentiation and survival. Knowledge of these signals would be essential for the control of angiogenesis in a variety of diseases including cancer, where a direct correlation has been shown between increased vascularization and poor prognosis.

Angiogenesis and lymphangiogenesis are involved in major human diseases

In the EU and USA over 10 million patients suffer from cancers dependent on angiogenesis. Because most forms of cancer as well as their metastatic spread, are directly or indirectly related to angiogenesis and lymphangiogenesis, this is a very large patient population for therapy development. On the other hand, cardiovascular diseases form another common group of serious illnesses in the Western world, responsible for over 40 % of deaths in the USA and Europe. The most common underlying problem is atherosclerosis, a condition of the arteries in which abnormal accumulation of cells and lipids narrow the vessel wall and act as a focal point for the formation of blood clots. In the heart this results in myocardial infarction and in peripheral muscles this leads to ischaemia, sometimes necessitating leg amputation. In addition to cholesterol-lowering drugs, the only treatment is to reopen the blocked blood vessels with angioplasty or bypass grafting. While inhibition of angiogenesis is a desirable goal for cancer, promotion of angiogenesis in atherosclerotic patients could alleviate tissue ischaemia (Fig. 3 ).

Many common cancers metastasize to the regional lymph nodes. Regional lymph node metastasis in general correlates with poor prognosis of the disease. This has lead to a hypothesis that tumor cell invasion occurs via the lymphatic vessels and raised a question of the role of lymphangiogenic factors in metastatic processes. Accordingly, VEGF-C is upregulated in many solid tumors and its expression seems to correlate with lymph node metastasis. On the other hand, the absence or dysf unction of the lymphatic vessels, which usually results from an infection, surgery, radiotherapy or from a genetic defect, causes lymphedema that is characterised by a chronic accumulation of protein-rich fluid in tissues. The importance of VEGFR-3 signalling for lymphangiogenesis is evident in the genetics of familial lymphedema, a disease characterised by a hypoplasia of lymphatic vessels mainly in the skin, which leads to a disfiguring and disabling swelling of the extremities. Some lymphedema families carry heterozygous missense mutations of the VEGFR3 exons encoding the tyrosine kinase domain, resulting in an inactive receptor protein. Interestingly, despite the genetic defect in VEGFR-3 signalling, new lymphatic vessels can be generated in a mouse model for hereditary lymphedema by using VEGF-C gene therapy.

Lymphatic vasculature, a newly emerging area of molecular and cell biology

Lymphatic vessels are present in most tissues where they collect protein-rich fluid and white blood cells from the interstitial space and transport these as a whitish opaque fluid, the lymph, via the lymph nodes into the blood circulation (Fig 3). The lymphatic vessels form a circulatory system in conjunction with and paralleling the blood vessels. The ECs of the lymphatic capillaries have frequent large inter-endothelial gaps and lack continuous basement membrane and pericytes. The lymphatic system performs many medically important functions in every tissue. The lymphatic system is critical in fat absorption from the gut and in immune responses. Foreign material such as bacteria and viruses are taken up by antigen presenting cells and transported via the lymphatic vessels to the lymph nodes. Many diseases are connected to the lymphatic system. The lymph nodes are the main sites where immune response against foreign microbial invaders is mounted. Cancer commonly spreads through the lymphatic system while the loss of lymphatic vessels results in swelling of the affected tissues and accumulation of fluid in body cavities. We have significantly promoted the understanding of the lymphatic vessels during the past few years. Indeed, it now seems that the lymphatic vessels could provide a unique opportunity for the development of new and innovative medical technology.

Until recently, in vitro studies of EC biology have been carried out with cultured ECs from various organs, considered to represent the blood vascular endothelium (BEC). With the identification of VEGF-C and VEGF-D stimulating the lymphatic endothelium and cell surface markers, such as the VEGFR-3 receptor allowing their isolation, this view needs to be altered. Lymphatic ECs (LECs) may become the next focus of angiogenesis and metastasis research. Recent studies by Mäkinen et al and Kriehuber et al showed that primary cultures of human dermal microvascular ECs consist of distinct populations of BECs and LECs. These cells were isolated and grown in culture as two separate stable and specialized cell lineages and showed no interconversion of distinct phenotypic properties.

Tumor cells and inflammatory cells exploit the lymphatic vessels

The major areas of interest regarding the LECs are their functions in lymphatic vessels in the regulation of tissue fluid and their interactions with cells migrating through the lymphatic endothelium, such as leukocytes and tumor cells. Migratory antigen presenting cells, such as dendritic cells in various epithelia monitor the extracellular environment, and when activated, traverse from peripheral tissues to regional lymph nodes via the lymphatic capillaries. Tumor-specific immune responses may depend on sufficient tumor cells reaching secondary lymphatic organs. However, in metastatic dissemination to the regional lymph nodes, it is not known whether tumor cells are passively transported to the lymphatic capillaries with the interstitial fluid, or whether they undergo active intravasation requiring cell migration, expression of adhesion receptors and digestion of pericellular matrix components. In transgenic and xenotransplanted mice, VEGF-C or VEGF-D overexpression resulted in the induction of tumor lymphangiogenesis, intralymphatic tumor growth and the formation of lymph node metastasis. On the other hand, these effects of the lymphangiogenic growth factors were inhibited by a soluble receptor. Although the soluble form of VEGFR-3 did not interfere with the normal lymphatic function in the tumor-bearing mice, it inhibited lymphatic development in embryos, suggesting that the newly formed lymphatic vessels could be more efficiently destroyed. It is known that metastasis via the blood vessels is an inefficient process as cancer patients can have millions of tumor cells circulating in their peripheral blood, yet they only rarely develop distant metastases. Whether such inefficiency applies also to lymphatic metastases is not clear.

The development of therapeutics for lymphedema and tumor metastasis

The intense interest in angiogenesis has allowed the identification of several anti-angiogenic agents, particularly for use in anti-cancer therapy. Several safe and potentially effective molecules have entered into clinical trials. The recent identification of lymphangiogenesis has revived the interest in the lymphatic vessels after many decades of dormancy. The elucida-tion of the genetic and functional programs of LECs will probably facilitate the development of growth factor therapies for lymphedemas of various origins as well as to define the contribution of lymphatic vessels to the molecular pathogenesis of cancer metastasis and inflammatory conditions. In vivo lymphangiogenesis assays should allow the testing of anti-lymphangiogenic compounds for clinical use. Thus this research should provide unique opportunities for the development of new and innovative therapies for many common diseases.

E-mail: Kari.alitalo@helsinki.fi

This article was first published in the Lundbeckfonden's Annual Report, 2001.

Forfatteren har modtaget Lundbeckfondens Nordiske Forskerpris 2002. En omtale heraf bringes i dette nummer af Ugeskrift for Læger, side 3218-9.