Angiogenesis
Cell Proliferation

ANGIOGENESIS AND TUMOUR GROWTH

VASCULOGENESIS, ANGIOGENESIS, ARTERIOGENESIS

The development of the cardiovascular system is regulated by vasculogenesis and by angiogenesis wich regulates the growth of vessels both in the embryo and in the adult.
In the adult, angiogenesis characterises some physiological situationes, such as the vascularisation of ovary and uterus during the mestrual cycle, of mammary glands during lactation, and of granulation tissue during wound healing. Not less significant is the role of angiogenesis in pathological settings, such as tumours, chronic inflammatory disease and vasculopathies
Five biological phases of angiogenesis have been established:

1. initiation
2. progression
3. differentiation
4. maturation
5. remodelling and guidance

INITIATION :

This phase is characterised by changes in the EC ( endothelial cell ) shape and by increased permeability.

PROGRESSION:

Begin the degradation of extracellular matrix, and migration and proliferation of ECs ( external endothelial cells )

DIFFERENTIATION:
During this phase, ECs stop the growth, survive in suboptimal conditions, and differentiate into primitive blood vessels
MATURATION:
This phase implies formation of new extracellular matrix, recruitment of pericytes and smooth muscle cells ( SMCs ), and remodelling of the primitive vascular network.
GUIDANCE:
The aim of the last part is delineate the architecture of the mature vasculature tree.

All these steps are in part regulated by vascular endothelium-specific growth factors that now include members of the VEGF ( vascular endothelial growth factor ), Angiopoietin ( Ang ), ephrin, and semaphorin families

ANGIOGENESIS and CANCER PROGRESSION

At the beginning of its history, the cancer is not vascularised, and it does not grow beyond 2 mm in size unless vascularisation has occurred.
The interaction between tumour cells and blood vessels may be supported by:
1- the ability of tumour cells to directly modify the homeostasis of inducers and inhibitors of angiogenesis
2- the ability of tumour cells to disturb this balance through stromal and inflammatory cells
3- the possibility of tumour cells to grow around pre-existing vessels
4- the colonisation of nascent vessels by CD 34+ cells wich differentiate in ECs
5- the formation of capillaries by tumour cells themselves through a trandifferentiation
6- the transient and dynamic participation of tumour cells to vessel walls
The first model implies a change in the local equilibrium between positive and negative regulators of microvessel growth.
Usually the up-regulation of an angiogenic inducer is not sufficient by itself for a tumour cell to become angiogenic and therefore certain inhibitors have to be down regulated. Similarly, tumour cells may stimulate stromal and infiltrating leukocytes to produce angiogenic factors.
The third model represent a new and provocative concept in cancer biology that has raised some criticism. Such a model would suggest that tumour cells metamorphose into vessels that either carry blood or connect to the host’s blood supply. This model has been named vasculogenic mimicry, because it is reminiscent of the differentiation process occurring during vasculogenesis.
Recently it has been noted the existence of angioblast-like circulating endothelial precursor cell in adult human blood. These precursors may be endowed with the phenotype of embryonic angioblasts, which are migratory EC precursor capable of circulating, proliferating, and differentiating into mature ECs.
The last model may explain the observation that metastasising tumour cells transiting into the vascular lumen may reside temporarily on the microvessel wall and occupy up to 4% of the total vascular surface area.

THE TUMOR VASCULATURE

The tumor vasculature shows structural and functional anomalies:
suboptimal intake of O2, drugs, nutrients…
The use of inhibitors of VEGF or VEGF-R with the intention to
block tumor angiogenesis presents the clinical effect more
relevant of normalization / maturation of vessels.
The anti-VEGF therapy is not due tumor remission, however:
-Optimizes the distribution of chemotherapeutic into the tumor.
-The metastasis is less likely.
The combination therapies to improve the effect of normalization of vessels
to enhance the contribution of the cytotoxic drug is the best application of the therapy

Drugs inhibitors of angiogenesis
Bevacizumab (Avastin ®)
The first inhibitor of angiogenesis on the market, approved in 2004 by the FDA for the treatment of
colorectal cancer.
Humanized monoclonal antibody
VEGF ligand.
Cetuximab (Erbitux ®)
Humanized monoclonal antibody-binding extracellular domain
EGF-R (Epidermal Growth Factor Receptor).

Marimastat
Inhibitor of matrix metalloproteases.
Thalidomide (Thalomid ®)
Known teratogenic but has anti-angiogentics.
Administered to patients with multiple myeloma. Mechanism of Action
not clear.
Small Molecules Tyrosine Kinase Inhibitors
The integrins are found only on the surface of endothelial cells

In this attachment " VASCULOGENESIS"we use these angiogenesis factors to develope a " angiogenesis mathematical model "

ANGIOGENIC AND ANTIANGIOGENIC FACTORS

Angiogenesys is a complex process that usually takes place under close control of factors that can stimulate or inhibit it.

ANGIOGENIC FACTORS

Angiogenic factors are:
• VEGF
• ANGIOPOIETINE
• bFGF-2 (basic Fibroblast Growth Factor)
• TNF; TGF
• INTERLEUCHIN
• PD-ECGF (Platelet-Derived Growth Factor/ Thymidine Phosphorylase)
• COX-2

ANTIANGIOGENIC FACTORS

The most studied angiogenic inhibitors mediators are:
• INTERFERON alpha (IFNa)
• INTERLEUCHIN 12
• RETINOIC ACID
• TNP-470
• ANGIOSTATINE
• ENDOSTATINE
• TETRAHYDROCORTISOL

TETRAHYDROCORTISOL
Tetrahydrocortisol was the most potent inhibitor of angiogenesis between occurring corticosteroids. Tetrahydrocortisol lacks the 4,5 double bond in its A ring, and, for this reason, lacks all of the known functions of cortisone. In fact, it is one of the major metabolites of cortisone previously believed to be without biological activity. Steroids are mainly inactivated in the liver by the enzymatic reduction of the 4,5 double bond in the A ring forming the dihydro derivative.
The dihydro derivative is converted to a tetrahydro derivative by the enzymatic reduction of the 3 oxo-group to a 3 hydroxyl group. This compound is a lipid soluble. It is then conjugated with glucuronic acid to become a water soluble product that can be excreted by the kidney.
Tetrahydrocortisol represent a steroid with pure antiangiogenic activity independent of either glucocorticoid or mineralcorticoid activity. It defines a new class of steroid named “angiostatic steroids”. Three rules predict antiangiogenic activity:
1) The 11 and 21-hydroxyl groups can be removed without significant loss of angiostatic activity;
2) The 17-hydroxyl group is essential, because its removal reduces antiangiogenic activity by approximately 76% of the activity of hydrocortisone. Thus, maintenance of antiangiogenic activity is governed mainly by structural components on the D ring of the pregnane nucleus;
3)Reduction of the A ring increases antiangiogenic activity by almost twice that of hydrocortisone;

Recently, it has been found that glucuronic acid can be synthetically conjugated to the 3-hydroxyl position of tetrahydrocortisol and that it retains very high antiangiogenic activity in the presence of heparin.The conjugated compound is water soluble. This is the first demonstration of a biological activity for this urinary metabolite of hydrocortisone. Tetrahydrocortisol, when administered at pharmacologic doses with exogenous heparin, can induce capillary regression in the growing vessels of the chick embryo,and at the doses used, can inhibit but not regress capillary growth induced by intracorneal tumor. It is unclear whether circulating tetrahydrocortisol has a physiologic
role. It may act synergistically with the heparin-like molecules that are known to be on the surface of the endothelial cells and embedded within its basement membrane, as well as within nearby
mast cells, to prevent capillary growth under normal conditions. Recent experiments of Falkman e coll. demonstrate that the administration of hydrocortisone with a sulfatase inhibitor
but without exogenous heparin is just as effective in causing capillary regression in the chick embryo as is the steroid with heparin. The steroid alone has no effect. We found that sulfatase activity is abundant in the chorioallantoic membrane. The added sulfatase inhibitor appears to retard degradation of heparin and allows endogenous heparin to accumulate locally. Thus, it is possible that endogenous heparin together with circulating angiostatic steroids may act at low concentrations over long periods to defend against abnormal capillary growth at the wrong place or time.
In this experiment, removal of an angiogenic stimulus (ethylene vinyl acetate copolymer pellet containing tumor extract) led to platelet aggregation and the formation of microthrombi at the
tips of the new capillaries within 24-48 hours. Endothelial cells desquamated and macrophages migrated into the lumen of the vessels to phagocytize platelets and dying endothelial cells. In contrast, when capillaries regressed under the influence of angiostatic steroids and heparin, histologic sections showed no platelet accumulation,macrophage infiltration, or microthrombi. This suggested that a novel mechanism of capillary involution was taking place. A clue to such a mechanism came from an examination of what is known about other tissues that undergo physiologic involution, e.g., the mammary gland and the Mullerian duct. Involution of epithelium in these tissues is associated with degradation of basement membrane. Furthermore, the modulation of basement membrane integrity in these epithelial tissues appears to be regulated through a balance of steroids and specific polypeptide growth factors. By analogy, it seemed possible that angiostatic steroids could regulate the growth and viability of capillary endothelium through their effects on the capillary basement membrane. Degradation of basement membrane could lead to gradual rounding and eventual detachment of endothelial cells, as predicted by experimental models of epithelial cells. Normal endothelial cells progressively lose their ability to grow as they take on increasingly rounded forms, even when supplied with optimal growth factors. They die after only a few hours of detachment from a substratum.
The normal vascular basement membrane contains collagen type IV, laminin, fibronectin, and glycosaminoglycans.
We used immunofluorescence microscopy to study the distribution of two basement membrane components, fibronectin and laminin Chorioallantoic membranes treated with steroid-heparin
mixtures revealed avascular zones with complete loss of capillary structure in the center, and partial involution of capillary blood vessels at the periphery. At the periphery of the avascular zones, the distance between capillaries was more than 10-fold greater than normal intercapillary distance in untreated parts of the chorioallantoic membrane). As the avascular zones developed over 48 hours, there was fragmentation of capillary basement membrane and progressive loss of fibronectin and laminin within the regions of capillary involution. Eventually these basement membrane components disappeared completely as the capillary itself underwent total involution. The rapid breakdown of capillary basement membrane in the steroid-heparintreated embryos correlated with endothelial cell rounding and desquamation. Capillary retraction presented a pattern that was the reverse of capillary growth. In the treated embryos, the basement membranes of surrounding large vessels, nongrowing capillaries, and neighboring epithelium were not affected.
In contrast, in untreated embryos, or in those treated with heparin or steroid alone, fibronectin and laminin appeared in continuous linear patterns within the basement membrane surrounding growing capillaries. Fibronectin outlined both the large vessels and fine capillaries throughout the entire vascular network of the chorioallantoic membrane.
The pathway by which only basement membrane associated with growing capillaries breaks down, leaving basement membrane of neighboring epithelium and large vessels intact, is not obvious. Nevertheless, the specificity of angiostatic steroids for growing capillaries is unparalleled. A possible explanation for this specificity is that normal capillary growth may require high turnover rates of basement membrane. Adjacent tissue and larger vessels may have much slower basement membrane turnover. Basement membranes of large vessels are more heavily stained with fibronectin antibodies than are fine capillaries. Older capillaries that are not growing are surrounded by a slightly more dense basement membrane that. contains increased amounts of laminin and type IV collagen.
This biological system has also made it possible to demonstrate that the relation between matrix dissolution and tissue involution clearly correlates with changes in cell shape. For example, endothelial cell rounding was generally associated with progressive degradation of the vascular basement membrane. This supports a concept that we previously proposed, that cell shape and especially matrix-dependent alterations of cell shape, may serve as physiologic regulators of tissue development in vivo. In support of this hypothesis, we have recently been able to select successful pharmacologic inhibitors of angiogenesis based simply on their ability to modulate capillary endothelial cell form in vitro.

REFERENCES

ANGIOSTATIC STEROIDS: METHOD OF DISCOVERY AND MECHANISM OF ACTION Judah Folkman M.D and Donald E. Ingber M.D Ph.D. Department of Surgery and Pathology, Harvard Medical School, Boston, Massuchussetts;

STEROIDI, CALEIDOSCOPIO ITALIANO Ivan Manduchi, Laboratorio Analisi Ospedale “Infermi” Rimini, 1995;

DALL’IPOTESI DI FOLKMAN ALLE TERAPIE ANTI-VEGF: ANGIOGENESI, TARGET E NUOVI TRATTAMENTI, Aiom Associazione Italiana Oncologia Medica 2008;
ANGIOGENESYS INHIBITOR THERAPY, U.S National Cancer Institute 2008;
S.Liekens, E.De clercq, J.Neyts. Angiogenesis: regulators and clinical applications. Biochemical Pharmacology 61 (2001) 253-270
Angiogenesi: Regolazione e controllo farmacologico nello sviluppo delle neoplasie. Salesi N., Tribuna Biologica e Medica Anno 11- Vol. 11 n° 3-4
National Cancer Institute Fact Sheet 7.49, Targeted Cancer Therapies: Questions and Answers
(http://www.cancer.gov/cancertopics/factsheet/Therapy/targeted)