Cell Proliferation

Author: Gianpiero Pescarmona
Date: 30/04/2009


Angiogenesis is a physiological process involving the growth of new blood vessels from pre-existing vessels

Circ Res. 2004 Mar 19;94(5):664-70. Epub 2004 Jan 22.
Comparative evaluation of FGF-2-, VEGF-A-, and VEGF-C-induced angiogenesis, lymphangiogenesis, vascular fenestrations, and permeability.

Cao R, Eriksson A, Kubo H, Alitalo K, Cao Y, Thyberg J.

Microbiology and Tumor Biology Center, Karolinska Institutet, Stockholm, Sweden.

Several endothelial growth factors induce both blood and lymphatic angiogenesis. However, a systematic comparative study of the impact of these factors on vascular morphology and function has been lacking. In this study, we report a quantitative analysis of the structure and macromolecular permeability of FGF-2-, VEGF-A-, and VEGF-C-induced blood and lymphatic vessels. Our results show that VEGF-A stimulated formation of disorganized, nascent vasculatures as a result of fusion of blood capillaries into premature plexuses with only a few lymphatic vessels. Ultrastructural analysis revealed that VEGF-A-induced blood vessels contained high numbers of endothelial fenestrations that mediated high permeability to ferritin, whereas the FGF-2-induced blood vessels lacked vascular fenestrations and showed only little leakage of ferritin. VEGF-C induced approximately equal amounts of blood and lymphatic capillaries with endothelial fenestrations present only on blood capillaries, mediating a medium level of ferritin leakage into the perivascular space. No endothelial fenestrations were found in FGF-2-, VEGF-A-, or VEGF-C-induced lymphatic vessels. These findings highlight the structural and functional differences between blood and lymphatic vessels induced by FGF-2, VEGF-A, and VEGF-C. Such information is important to consider in development of novel therapeutic strategies using these angiogenic factors.

Mol Cell Endocrinol. 2007 Apr 15;269(1-2):65-80. Epub 2007 Feb 6.
The gonadotropins: tissue-specific angiogenic factors?, 2007

Reisinger K, Baal N, McKinnon T, Münstedt K, Zygmunt M.

Department of Obstetrics and Gynecology, University of Giessen, Klinikstrasse 32, 35385 Giessen, Germany.

The gonadotropins, whose members are human chorionic gonadotropin (hCG), lutenizing hormone (LH) and follicle-stimulating hormone (FSH) are a well characterized hormone family known to regulate reproductive functions in both females and males. Recent studies indicate that they can modulate the vascular system of reproductive organs. It was shown that gonadotropins not only influence the expression of vascular endothelial growth factor (VEGF) and both its receptors VEGFR-1 and 2, but also modulate other ubiquitously expressed angiogenic factors like the angiopoietins and their receptor Tie-2, basic fibroblast growth factor or placental-derived growth factor. Some recent data indicates a possible direct action of gonadotropins on endothelial cells. Thus, the gonadotropins act as tissue-specific angiogenic factors providing an optimal vascular supply during the menstrual cycle and early pregnancy in the female reproductive tract as well as in testis. In pathological conditions (e.g. preeclampsia, intrauterine growth restriction, ovarian hyperstimulation or endometriosis), these tightly regulated interactions between the gonadotropins and the ubiquitous angiogenic factors appear to be disturbed. The intent of this short manuscript is to review the current knowledge of the regulatory role of the gonadotropins in vasculo and angiogenesis. We also review angiogenic actions of thyroid-stimulating hormone (TSH), a glycoprotein closely related to gonadotropins, which display strong gonodal actions.

HMGB1 a protein involved in oxygen (neuronal) DNA single strand stabilization. Released in hypoxia

Angiogenetic signaling through hypoxia: HMGB1: an angiogenetic switch molecule. 2005
Schlueter C, Weber H, Meyer B, Rogalla P, Röser K, Hauke S, Bullerdiek J., Am J Pathol. 2005
The initiation of angiogenesis, called the angiogenetic switch, is a crucial early step in tumor progression and propagation, ensuring an adequate oxygen supply. The rapid growth of tumors is accompanied by a reduced microvessel density, resulting in chronic hypoxia that often leads to necrotic areas within the tumor. These hypoxic and necrotic regions exhibit increased expression of angiogenetic growth factors, eg, vascular endothelial growth factor, and may also attract macrophages, which are known to produce a number of potent angiogenetic cytokines and growth factors. A group of molecules that may act as mediators of angiogenesis are the so-called high-mobility group proteins. Recent studies showed that HMGB1, known as an architectural chromatin-binding protein, can be extracellularly released by passive diffusion from necrotic cells and activated macrophages. To examine the angiogenetic effects of HMGB1 on endothelial cells an in vitro spheroid model was used. The results of the endothelial-sprouting assay clearly show that exogenous HMGB1 induced endothelial cell migration and sprouting in vitro in a dose-dependent manner. Thus, this is the first report showing strong evidence for HMGB1-induced sprouting of endothelial cells.

2012-02-25T08:52:17 - Federico Salotto



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


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


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

During this phase, ECs stop the growth, survive in suboptimal conditions, and differentiate into primitive blood vessels
This phase implies formation of new extracellular matrix, recruitment of pericytes and smooth muscle cells ( SMCs ), and remodelling of the primitive vascular network.
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


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 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).

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 "

2009-10-23T19:18:42 - Monica Cibinel


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


Angiogenic factors are:
• bFGF-2 (basic Fibroblast Growth Factor)
• PD-ECGF (Platelet-Derived Growth Factor/ Thymidine Phosphorylase)


The most studied angiogenic inhibitors mediators are:

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.


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;

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

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