Angiogenesis

Angiogenesis - a review

© by Florence Folmer, University of Bangor, Wales, December 2000

Capillaries extend into virtually all the tissues of the body. They replenish the tissues with nutrients, and they carry off waste products. Under most conditions, capillaries do not increase in size or number, because the endothelial cells that line these narrow tubes do not divide. But occasionally, for example during menstruation or when tissue is damaged, these vessels begin to grow rapidly. This proliferation of new capillaries, called angiogenesis, or neovascularisation, is highly regulated, and it is typically short-lived (it usually lasts for less than one week). Nevertheless, neovascularisation can also occur under abnormal conditions. Tumour cells, for example, can turn-on angiogenesis. As new blood vessels bring in fresh nutrients and growth factors, the tumour mass can expand. In fact, angiogenesis appears to be one of the crucial steps in a tumour's transition from a small, harmless cluster of mutated cells to a large, malignant cancer, capable of spreading (metastasising) to other organs throughout the body. Neovascularisation is also involved in numerous other diseases termed angiogenic diseases. These include benign tumours, rheumatoidal arthritis, fibrosis of the liver and of the kidney, and serious eye-diseases in which abnormal vessels proliferate and destroy vision, such as diabetic retinopathy, macular degeneration, neovascular glaucoma, and retrolental fibroplasia. Ocular neovascularisation is, indeed, the most common cause of blindness.

The process of angiogenesis, in the context of cancer growth and metastasis in particular, and the methods currently used to cure angiogenic diseases are discussed in the present paper.

Whether healthy or cancerous, cells need to be in the proximity of capillaries in order to live and to proliferate. If a healthy cell becomes cancerous and begins dividing rapidly, the resulting daughter cells accumulate farther and farther away from the nearest capillary. After a few million such cells have accumulated, the small tumour, often called an in situ carcinoma, stops expanding and reaches a steady state in which the number of dying cells counterbalances the number of proliferating cells. This steady state results from the lack of readily available nutrients, growth factors, and oxygen. After many months or even years in steady state, an in situ tumour may abruptly induce new capillary growth and start to invade surrounding tissue. To do so, the tumour calls into service the naturally occurring angiogenesis-stimulators, such as fibroblast growth factor (FGF), vascular endothelial cell growth factor (VEGF), and vascular permeability factor (VPF) present in its neighbourhood, to increase the production of proteolytic enzymes such as matrix metallo-proteinases (MMPs) and tissue plasminogen activators (TPAs). MMPs and TPAs are protease families which facilitate endothelial cell (EC) release from the parent venule via the disruption of cell-to-cell and cell-to-matrix adhesion, and via the degradation of the venule's basement membrane.

Proteolysis is also required for the migration of ECs into and through the collagen-rich perivascular stroma. These events are followed by sprout extension of the epithelium, and by subsequent capillary-lumen formation. Once neovascularisation has occurred, hundreds of new capillaries converge on the tiny tumour, and each vessel soon becomes coated with rapidly dividing tumour cells. At this advanced stage, the tumour can expand very rapidly and reach the size of two cubic centimetres within a couple of months. In addition, the expanding tumour often releases metastasis-stimulating proteins, such as interleukin-6, which allow it to spread through the body. Advanced angiogenesis causes readily identifiable symptoms such as blood appearing in the urine, stool or sputum, and indicating that angiogenesis has taken place in the bladder, colon, or lung, respectively.

A schematic overview of the process of angiogenesis is presented in figure 1.

Figure 1. Schematic overview of the process of angiogenesis

Figure 1.1.a.: 1. The tumour releases angiogenic molecules, which act on the epithelial cells and on the basement membrane surrounding the blood vessels, and then on the endothelial cells of the blood vessel (2). 3. Tumour-derived molecules act as chemotactic factors on macrophages and on mast cells. 4. The macrophages and the mast cells release angiogenic factors and angiogenic amplifiers, which act on the epithelial cells and on the basement membrane surrounding the blood vessels. 5. The tumour also releases plasminogen activators, collagenases, and heparanases, which interact with bFGF stored in the basement membrane (6). 7. The basement membrane becomes hyperpermeable. 8. The endothelial cells start migrating and proliferating.

Figure 1.1.b.: Neovascularization provides the tumour with nutrients, and it allows tumor cells to enter the blood stream and to metastasise.

Figure 1.1.c.: The reverse process of intravasion is used at the target destination by the tumour cells to exit the blood stream, and to maintain a good nutrient supply.

(after: Folkman (1993), Moses (1997), and Klagsbrun & Moses (1999)

Matrix metallo-proteinases, which are the main actors in angiogenesis, occur naturally in humans, as well as in many other organisms. Twenty-three different matrix metallo-proteinases have been identified to date, and the MMP family can be divided into three categories on the basis of substrate specificity: the interstitial collagenases, the Type IV collagenases (gelatinases), and the stromelysins. The regulation of MMP activity occurs at several levels including gene transcriptional control, proenzyme activation, and inhibition of activated MMPs by endogenous inhibitors. All matrix metallo-proteinases are synthesised as prepro-enzymes and secreted as inactive pro-MMPs in most cases. The pro-enzymes are activated by an external proteolytic enzyme system, such as the urokinase-plasminogen system, which cuts off the propeptide domain at the N-terminal end. The pro-peptide domain (about 80 amino acids) has a conserved unique PRCG(V/N)PD sequence. The cysteine within this sequence ligates the catalytic zinc to maintain the latency of pro-MMPs. The catalytic domain (about 170 amino acids) contains a zinc binding motif and a conserved methionine, which forms a unique "Met-turn" structure.

Like many other enzyme families, matrix metallo-proteinases are a key component of a system of "balanced proteolysis" wherein a fine-tuned equilibrium exists between the amount of active enzyme and its endogenous proteinase inhibitor(s). In the early 1980's, Folkman discovered that many of the natural angiogenesis inhibitors are constantly present in the body, as a fragment of a larger, inactive protein, and hence they are readily available for immediate use when the body needs to stop normal angiogenesis, i.e. after wound healing. Cartilage, which is a highly avascular tissue, appears to be particularly rich in angiogenesis inhibitors, which protect it from becoming vascularised. In the mid 1990's, Bouck and his co-workers demonstrated that even certain tumour cells do not only produce angiogenesis-stimulating proteins, but also the latter's inhibitors, and that the balance between them determines whether the tumour can switch on angiogenesis. It has also been shown that some of the naturally occurring angiogenesis inhibitors are normally under the control of p53, which has been implicated in various cancers.

The native MMP inhibitors comprise a family of proteins generally referred to by the acronym TIMPs (Tissue inhibitors of matrix metallo-proteinases). Sixteen of these inhibitors, grouped into four TIMP families (TIMP-(1-4)) have been identified to date, and some have been cloned and expressed in E. coli. The TIMPs are specific inhibitors of MMPs. The complex formed between TIMPs and MMPs is a tight noncovalently bound complex. But amongst the MMPs, TIMPs show little differences in their binding specificity. Each TIMP is able to inhibit all members of the MMP family. TIMPs are in many cases structurally similar and in most cases functionally similar. The molecular weight of TIMPs is ~ 21-45 kDa. The amino acid sequence similarity between the TIMP family members is about 45% (25% identity), and includes total conservation of 12 Cysteine residues known to form the characteristic six disulphide bonds. These Cysteine residues play, in fact, a critical role in the inhibition of MMPs. The major structural and functional differences known to date between the TIMP family members lie in their glycosylation and in their specificity of binding the latent pro-enzyme form of MMPs.

Progress in angiogenesis research has been rising very rapidly since the early 1970's, when Judah Folkman from the Harvard Medical School first developed the concept of angiogenic disease, and purified the first angiogenic molecule and the first angiogenesis inhibitor. A lot of research effort has been put into the elucidation of the process of angiogenesis, and a couple of anti-angiogenesis drugs are already under late-stage clinical trial. Yet, there are still a lot of questions that need to be answered, and a lot of hurdles (i.e. financial problems, unforeseen inefficiency in humans or other uncertainties from clinical trials,...) that need to be overcome before angiogenesis inhibitors can be used on a regular base in the clinic. Other processes involved in metastasis, such as the loss in E-cadherin-mediated cell-to-cell adhesion, also need to be further investigated. But it is now believed that anti-angiogenic drugs will become very promising in cancer treatment in the near future, either in conjunction with radio-, chemo-, or immuno-therapy, or as a long-term treatment after surgery or instead of surgery, if the tumour has spread out to too many different parts of the body. At present, patients diagnosed with any type of cancer typically rely on surgery or radiation to remove the original tumour, followed by further radiation or chemotherapy to try to eliminate any remaining cancerous cells in the body. Anti-angiogenic therapy, in contrast to many other therapeutic approaches, does not aim to destroy tumours. Instead, by limiting the blood supply, it attempts to shrink tumours and prevent them from growing. As a matter of fact, it has been shown that tumour cells, unless they become vascularised, do not become larger than about the size of a pea. The specificity of angiogenesis inhibitors has powerful benefits in the way that they cause very little toxic side-effects or drug resistance. Indeed, since angiogenesis inhibitors stop new capillary growth, but do not attack healthy vessels, they only do little, if any, harm to blood vessels serving normal tissues.

The fact that metastasis is the deadliest aspect of cancer urges researchers to thoroughly understand and prevent the whole process of angiogenesis, which is capable to turn a cancer from a benign locally growing tumour into a malign metastatic killer. Furthermore, once the anti-angiogenic agents will be on the market, it will also be possible to use them in the treatment of other angiogenic diseases.

Abbreviations

a.a.:

Amino acid

bFGF:

Basic fibroblast growth factor

BME:

-mercapto ethanol

cDNA:

Complimentary DNA

DNA:

Deoxyribonucleic acid

EC:

Epithelial cell

ECM:

Extra-cellular matrix

FGF:

Fibroblast growth factor

MMP:

Matrix metallo-proteinase

MMPI:

Synthetic MMP inhibitor

MW:

Molecular weight

TPA:

Tissue plasminogen inhibitor

TIMP:

Tissue inhibitor of matrix metallo-proteinases

VEGF:

Vascular endothelial cell growth factor

VPF:

Vascular permeability factor

Selected References

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