by

عون عبد سارة نوري فاضل سجاد موشنة سالم حسين اياد ندى خميس حسن

formation of new blood vesselsThere are two ways to form the blood vessels in the body ?

1-Vasculogenesis2-Angiogenesis

Vasculogenesis is the generation of blood vessels from endothelial cell progenitors (angioblasts). It is responsible for the formation of the primary vasculature of the body during early embryonic development development of the cardiovascular system. this is followed by formation of a vascular tree and finally the cardiovascular system.

after which angiogenesis is responsible for most, if not all, blood vessel growth during development and in disease like cardiovascular disease

What is Angiogenesis? angiogenesis is the formation of new blood vessels from pre-existing vesselsand is a normal process in growth and development, as well as in wound healing.o angiogenesis is defined as the growth of blood vessels and is an

important natural process used by the body for reproduction and for healing injured tissues

o blood vessels bring oxygen and nutrients via the circulation to nourish all tissues in the body

o the cells comprising blood vessels are called endothelial cells o the endothelial cells of a blood vessel also produce molecules

that support the growth of tissues o cancer cells take over the body's control of angiogenesis in

order to recruit their own private blood supply And the angiogenesis have important role in the tumor to give nutrient an oxygen for growth the tumor and for proliferation and

important in metastasis the tumor

Intussusceptive angiogenesis

Sproutingangiogenesis

TYPE

Sprouting angiogenesis

Sprouting angiogenesis is the basic mechanism seen in the growth of new blood vessels. It was the first identified form of angiogenesis. It occurs in several well-characterized stages .

There are sequential steps that are finely regulated by chemical mediators in the body .

Significant role is played by factors such as VEGFFirst, biological signals known as angiogenic growth

factors VEGF and FGF-Β produced by fibroblasts, macrophages, endothelial cells and keratinocytes

are principal factors which regulate angiogenesis activate receptors present on endothelial cells

Sprouting angiogenesis

Second step : activated endothelial cells begin to release enzymes like proteasesMatrix metalloprotease Plasminogen activator …that activate plasminogen into plasmid ;which degrades several components of ECM

.These proteases can break down proteins and cells of the basement membrane. This creates opening in the existing blood vessel that allows the escape of the activated endothelial cells from the existing orginal blood vessel.

The endothelial cells then proliferate into the surrounding matrix and form solid sprouts connecting neighboring vessels. As sprouts extend toward the source of the angiogenic stimulus, new endothelial cells using adhesion molecules, called integrins that help them bind to each other to form chains. These sprouts then form loops to become tubular blood vessels. Sprouting occurs at a rate of several millimeters per day, and enables new vessels to grow across gaps in the vasculature. It is markedly different from splitting angiogenesis, however, because it forms entirely new vessels as opposed to splitting existing vessels.

Sprouting angiogenesis

FINAL STEP Maturation of neovasculature When sufficient neovascularization has occurred the angiogenic factors are down regulated Balance equilibrium

http://www.angio.org/img/cascadehi.jpg

Intussusceptive Angiogenesis Intussusception, also known as splitting angiogenesis, was first observed in neonatal rats. In this type of vessel formation, the capillary wall extends into the lumen to split a single vessel in two. There are four phases of intussusceptive angiogenesis.

First, the two opposing capillary walls establish a zone of contact.

Second, the endothelial cell junctions are reorganized and the vessel bilayer is perforated to allow growth factors and cells to penetrate into the lumen.

Third, a core is formed between the two new vessels at the zone of contact that is filled with pericytes and myofibroblasts. These cells begin laying collagen fibers into the core to provide an extracellular matrix for growth of the vessel lumen.

Finally, the core is fleshed out with no alterations to the basic structure.

Intussusceptive Angiogenesis

Intussusception is important because it is a reorganization of existing cells. It allows a vast increase in the number of capillaries without a corresponding increase in the number of endothelial cells. This is especially important in embryonic development as there are not enough resources to create a rich microvasculature with new cells every time a new vessel develops.

Angiogenesis in cancer

During tumor growth, angiogenesis is required for proper nourishment and removal of metabolic wastes from tumor sites .In physiologic conditions, cells are located within

100 and 200 micro meter. from blood vessels, their source of oxygen. When a multicellular organism is growing, cells induce angiogenesis and vasculogenesis in

order to recruit newblood supply. In a pathological condition such as cancer, angiogenesis is required for

tumor survival and proliferation. The microenvironment of solid humantumors is characterized by heterogeneity in oxygenation

Effect of hypoxia on tumer and angiogenisis Hypoxia in tumors is primarily a pathophysiologic consequence of structurally and

functionally disturbed microcirculation and the deterioration of diffusion conditionsTumor hypoxia appears to be strongly associated with tumor propagation, malignant progression, and resistance to therapy, and it has thus become a central issue in tumor physiology and cancer treatmentHypoxia arises early in the process of tumor development because rapidly proliferating tumor cells outgrow the capacity of the host vasculatureTumor cells located more than 100 micro m away from blood vessels become hypoxicSome clones will survive by activating an angiogenic pathway. If new blood vessels do not form, tumor clones will be confined within 1–1.5 mm diameterSuch clones remain dormant from months to years before they switch to an angiogenic phenotype Vascular cooption is confined only in the tumor periphery and gradual tumor expansion causes a progressive central hypoxia. Hypoxia induces the expression of proangiogenic factors through hypoxia-inducible factor-a, and if proangiogenic factors are inexcess of antiangiogenic factors, it may lead to the switch to an angiogenic phenotype

Carmeliet and Jain. Nature. 2000= Proangiogenic factor, eg.

VEGF= Angiogenic inhibitor

Angiogenesis

Mutation

Hypoxia

HIF-1

VEGF

dormant

Secretion of angiogenic factors Rapid

growth of cancer

Regression of cancer

THE ANGIOGENIC SWITCHThe transition from a pre-vascular to a vascularized tumor phenotype is

called the angiogenic switch. This switch is controlled by a balance between pro- and anti-angiogenic factors, which are secreted by the tumor cells

themselves or by cells of the tumor microenvironment (in particular stromal cells and immune cells). The most prominent pro-angiogenic factors are

vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Conversely, proteolytic fragments of the extracellular matrix (ECM) can act

as potent angiogenesis inhibitors (e.g., endostatin). Other anti-angiogenic factors include cleaved derivatives of plasminogen (angiostatin) or antithrombin III (C-terminal antithrombin-fragment). The expression of pro and anti-angiogenic factors by cancer cells is controlled directly by oncogenes, tumor suppressor genes and transcription factors, but also indirectly by environmental factors (such as oxygen or glucose supply).. Genetic and epigenetic changes can modulate the response of the endothelial cells to VEGFand FGF and thus influence the angiogenic balance.. In patients, the angiogenic switch has been shown to occur in a number of cancer types, most prominently in breast and cervical cancer

Structure of tumor vasculature

In tumors, the normal configuration of blood vessels is spreading without any organization, following tortuous paths and changing in diameter without any organization. Largecaliber tumor

vessels may have thin walls usually belonging to capillaries or an incomplete basement membrane and an unusual pericyte coat

Pericytes of normal capillaries have skeletal shapes and are closely attached to endothelial cells. In contrast, pericytes in a tumor model show irregular shapes and are loosely attached to endothelial cells. Many projections are observed from the pericyte into the interstitial space. Arrow:pericytes of normal capillary. Arrowhead: pericytes of tumor capillary.

An imbalance in angiogenic factors, like vascular endothelial growth factor (VEGF) and angiopoietins, is the main cause of this chaotic structure in a tumor vessels from which new

vessels originate are characterized by degradation of the basement membrane and decreased number of perycites and Tumor vessels are hyperpermeable, mostly described as

‘leaky’, because of loss of adherence between endothelial junctions as well as a discontinuous basement membrane. Vascular permeability allows the extravasation of

plasma proteins that constitute a momentary scaffold for migrating endothelial cells.Another very common feature in tumor blood vessels is the presence of focal hemorrhages that occurspontaneously mainly if the tumor cells express VEGF121 or VEGF165 The structural aberrations described so far in tumor vessels are also coupled to molecular and functional disorders such as the overexpression of growth factors, integrins, and the uptake of cationic liposomes

ENDOGENOUS ANGIOGENIC FACTORS More than a dozen different proteins have been identified as angiogenic activators, including

• Vascular endothelial growth factor (VEGF)

• Basic fibroblast growth factor (bFGF)

• Angiogenin , transforming growth factor (TGF)-α, TGF-β

• Tumor necrosis factor (TNF)-α

• Platelet-derived endothelial growth factor

• Granulocyte colony-stimulating factor

• Placental growth factor

• Interleukin-8

• Hepatocyte growth factor

• Epidermal growth factor

SOURCE OF ANGIOGENIC FACTORS

• Angiogenic activity arises from the tumor cells itself in the form of the release of angiogenic molecules such as basic fibroblast growth factor;

• angiogenic activity arises from host cells recruited by the tumor (ex. Macrophages), or is mobilized from the extracellular matrix, or requires concomitant loss of physiological inhibition of endothelial cell proliferation.

ENDOGENOUS ANGIOGENIC FACTORS

• VEGF is a powerful angiogenic agent in neoplastic tissues, as well as in normal tissues. Under the influence of certain cytokines and other growth factors

• Some angiogenic phenotypes can be triggered by hypoxia resulting from the increasing distance between the growing tumor cells and the capillaries or from the inefficiency of new vessels. Hypoxia induces the expression of VEGF and its receptor via hypoxia-inducible factor-1α (HIF-1α).

• Tumor cells feed on the new blood vessels by producing VEGF and then secreting it into the surrounding tissue. When the tumor cells encounter endothelial cells, they bind to receptors on the outer surface of the endothelial cell.

ENDOGENOUS ANGIOGENIC FACTORS • The binding of VEGF to its receptor activates relay proteins that transmit a signal into the nucleus of

the endothelial cell.

• The nuclear signal prompts a group of genes to make products needed for new endothelial cell growth.

• Endothelial cells activated by VEGF produce matrix metalloproteinases (MMPs).

• The MMPs break down the extracellular matrix which fills the spaces between cells and is made of protein and polysaccharides.

• This matrix permits the migration of endothelial cells. The endothelial cells begin to divide as they migrate into the surrounding tissues. Soon they organize into hollow tubes that evolve gradually into a mature network of blood vessels with the help of an adhesion factor, such as integrin α or β.

• Newly formed blood vessels need to stabilize or mature. Angiotensin-1, -2, and their receptor Tie-2 can stabilize and govern vascular growth.

The binding of VEGF to its receptor

activates relay

proteins

VEGF produce matrix metalloproteinases

(MMPs).

MMPs break down the extracellular

matrix

migration of endothelial

cells

dividing of endothelial cell

stabilize or mature by Angiotensin-1, -2

ENDOGENOUS ANGIOGENIC FACTORS

VEGF Family

• VEGFA• VEGFB• VEGFC• VEGFD• VEGFE

ENDOGENOUS ANGIOGENIC FACTORS • Among the VEGF family, VEGF-A, VEGF-B, VEGFC and VEGF-E acting on their

respective receptors cause proliferation of blood vessels, while VEGF-C and VEGFD are involved in lymphangiogenesis.

• Vascular endothelial growth factor-A is also known as VEGF/vascular permeability factor (VPF). Vascular endothelial growth factor-A is a heparin binding glycoprotein that occurs in at least six molecular isoforms, which consist of 121, 145, 165, 183, 189, and 206 amino acids and are the result of alternative splicing of the mRNA.

• VEGF-A is a potent and very specific mitogen for vascular endothelial cells and stimulates the full cascade of events required for angiogenesis, and is overexpressed in a variety of tumors.

ENDOGENOUS ANGIOGENIC FACTORS

• VEGF-B exists as two protein isoforms, VEGF-B167 and VEGF-B186, resulting from different spliced mRNA and binds specifically to VEGFR-1. However, VEGF-B forms a heterodimer with VEGF-A, which may alter its interaction with its biological receptors and modify its normal physiological effects. While VEGF-B is widely expressed in heart, skeletal muscle, and vascular cells, its biological function remains unclear. It has also been reported that VEGF-B levels increase both throughout development and after birth, closely correlating with the progression of cardiac angiogenesis.

ENDOGENOUS ANGIOGENIC FACTORS • VEGF-C has a mature form that consists of a VEGF homology domain, Unlike VEGF-A, the

expression of VEGF-C does not appear to be regulated by hypoxia.

• The expression of VEGF-C appears to be restricted to early development and certain pathological settings such as tumor angiogenesis and lymphangiogenesis.

• VEGF-D is known as induced growth factor (FIGF), these growth factors bind to the same receptors on human endothelial cells, namely VEGFR-2 and -3. VEGF-C and VEGF-D bind and activate VEGFR-3, regulating lymphangiogenesis as well as angiogenesis in the mid-stages of embryogenesis.

• VEGF-E the interaction of VEGF-E with its receptor seems to promote endothelial cell growth There is a significant overlap between the binding pocket of VEGF-A and VEGF-E on VEGFR-2 which may suggest alliance of the two molecules in final response or alternatively an antagonistic relationship between these two factors.

THE FIBROBLAST GROWTH FACTOR (FGF)• The fibroblast growth factor (FGF) family with its prototype members FGF-1 (acidic FGF) and

FGF-2 (basic FGF) consists to date of at least 22 known members.

• FGF-1 stimulates the proliferation and differentiation of all cell types necessary for building an arterial vessel, including endothelial cells and smooth muscle cells; this fact distinguishes FGF-1 from other pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF), which primarily drives the formation of new capillaries.

• One of the most important functions of fibroblast growth factor-2 (FGF-2 or bFGF) is the promotion of endothelial cell proliferation and the physical organization of endothelial cells into tube-like structures, thus promoting angiogenesis. FGF-2 is a more potent angiogenic factor than VEGF or PDGF (platelet-derived growth factor); however, it is less potent than FGF-1.

Balance of angiogenesis in cancer

Angiogenesis is stimulated when tumor tissues require nutrients and oxygen. Angiogenesis is regulated by both activator and inhibitor molecules. However, up-regulation of the activity of

angiogenic factors is itself not sufficient for angiogenesis of the neoplasm. Negative regulators or inhibitors of vessel growth

need to also be down-regulated

An angiogenesis inhibitor

An angiogenesis inhibitor is a substance that inhibits the growth of new blood vessels (

angiogenesis). Some angiogenesis inhibitors are endogenous and a normal part of the body's

control, angiogenesis inhibitors are often derived from circulating extracellular matrix proteins, and

others are obtained exogenously through pharmaceutical drugs or diet

VEGF pathway inhibition

Inhibiting angiogenesis requires--- treatment with anti-angiogenic factors, or drugs

Which reduce the production of pro-angiogenic factors, prevent them binding to their receptors or block their actions

Inhibition of the VEGF pathway has become the focus of angiogenesis research as approximately 60% of malignant tumors express high

concentrations of VEGF

Inhibitors of angiogenesis

. There are many naturally occurring proteins that can inhibit angiogenesis, including

angiostatin, Endostatin Tumstatin Interferon Platelet factor 4 Thorombospondin prolactin 16 kd fragment Tissue inhibitor of metalloproteinase-1, -2, and -3

Inhibitors of angiogenesis

Angiostatin is composed of one or more fragments of plasminogen. It induces apoptosis in endothelial cells and tumor cells, and inhibits migration and the formation of tubules in endothelial cells. Immunohistochemical examination of angiostatin-treated tumors indicated a decrease in the expression of mRNA for VEGF and bFGF.

Endostatin is a naturally-occurring fragment derived from type XVIII collagen. It is reported to serve as an anti-angiogenic agent, similar to angiostatin and thrombospondin. Endostatin inhibits the growth factor

Endostatin is a broad-spectrum angiogenesis inhibitor and may interfere with the pro-angiogenic action of growth factors such as basic fibroblast growth factor (bFGF/FGF-2) and vascular endothelial growth factor (VEGF)

Tumstatin is a protein fragment cleaved from collagen that serves as both an antiangiogenic and proapoptotic agent.[1]

  tumstatin has been shown to reduce angiogenesis in tumors, there is great potential to use this

knowledge as treatment for cancer. Tumstatin binds to the endothelium of the tumor and is thus able to affect tumor growth.[9] By affecting the apoptotic pathway, tumstatin inhibits the

proliferation of endothelial cells.[6][1] It has been shown that the efficacy of tumstatin in reducing angiogenesis in tumors increases with tumor size.

 One study showed that mice with a genetic αvβ3 integrin showed accelerated tumor growth

(integrins consisting of two noncovalently bound transmembrane α and β subunits, are an important molecular family involved in tumor angiogenesis. Integrin αvβ3 is highly expressed on

activated endothelial cells, new-born vessels as well as some tumor cells, but is not present in resting endothelial cells and most normal organ systems, making it a suitable target for anti-

angiogenic therapy.) When tumstatin was replaced into the system, the tumor growth was disrupted and the tumor shrunk.[11]

Prolactin 16-kD FragmentA 16 kD N-terminal fragment of prolactin formed

endogenously by cleavage of intact prolactin. Possessing anti-angiogenic properties, prolactin 16-kD

fragment inhibits the pro-angiogenic effects of basal fibroblast growth factor (bFGF) and vascular

endothelial growth factor (VEGF) on endothelial cell proliferation, migration, and capillary organization

Vascular Disrupting Agents in Cancer Therapy Vascular disrupting agents (VDAs) are

distinguished from anti-angiogenic agents by their ability to cause a catastrophic vascular collapse in tumour tissue within minutes to hours of drug administration, leading to extensive tumour cell necrosis.

. Tumors cannot grow larger than 2mm without angiogenesis

Flavonoids

DMXAA (5,6-dimethylxanthenone-4-acetic acid; and FAA (fl avone acetic acid) are members of a series of drugs, structurally distinct from the combretastatins, whose primary site of action is unknown but which have multiple antivascular actions including the induction of cytokines. Initial work found that FAA induced extensive haemorrhagic necrosis and reduced blood fl ow in animal tumour models, leading to the development of derivatives, one of which, DMXAA, is 16 times more potent than FAA and active in human tumours (Baguley 2003).

Other Biological VDAs

Various peptides, antibodies, antibody fragments or growth factors are being designed to selectively bind to tumour blood vessels and induce coagulation and/or endothelial cell death. As for the low-molecular-weight VDAs, these compounds are designed to induce rapid tumour vascular shut-down, leading to extensive tumour cell necrosis.

Antiangiogenic treatment of cancer

Inhibitors Mechanismbevacizumab (Avastin) VEGF

carboxyamidotriazole inhibit cell proliferation and cell migration of endothelial cells

IL-12 stimulate angiogenesis inhibitor formation

thrombospondin inhibits binding of angiogenesis stimulators inhibit basement membrane degradation

matrix metalloproteinase inhibitorsangiostatin inhibit cell proliferation and induce apoptosis of

endothelial cells

Antiangiogenic treatment of cancerInhibitors Mechanism

endostatin inhibit cell migration, cell proliferation and survival of endothelial cells

thalidomide inhibit cell proliferation of endothelial cells

thrombospondin inhibit cell migration, cell proliferation, cell adhesion and survival of endothelial cells

prolactin VEGF

anti-angiogenesis factors One of the most recent methods that is being developed for the delivery of

anti-angiogenesis factors to tumour regions in cancer sufferers is using genetically modified bacteria that are able to colonize solid tumors in vivo. This method involves genetically engineering bacterial species such as Clostridium, Bifidobacteria and Salmonella by adding the genes for anti-angiogenic factors such as endostatin or IP10 chemokine and removing any harmful virulence genes. A target can also be added to the outside of the bacteria so that they are sent to the correct organ in the body. The bacteria can then be injected into the patient and they will locate themselves to the tumor site, where they release a continual supply of the desired drugs in the vicinity of a growing cancer mass, preventing it form being able to gain access to oxygen and ultimately starving the cancer cells] .This method has been shown to work both in vitro and in vivo in mice models, with very promising results.[. It is expected that this method will become commonplace for treatment of various cancer types in humans in the future

Side effects of angiogenesis inhibitors

Angiogenesis is important to many of the body’s normal processes. Therefore, these drugs can cause a wide range of side effects, including:

1. High blood pressure2. A rash and/or dry, itchy skin3. Hand-foot syndrome, which causes tender, thickened areas on

the skin, sometimes with blisters, on palms and soles4. Diarrhea5. Fatigue6. Low blood counts7. Problems with wound healing or cuts re-opening

Diet Some common components of human diets also act as

mild angiogenesis inhibitors and have therefore been proposed for angioprevention, the prevention of metastasis through the inhibition of angiogenesis. In particular, the following foods contain significant inhibitors and have been suggested as part of a healthy diet for this and other benefits:

Soy products such as tofu and tempeh, (which contain the

inhibitor "genistein")

Agaricus subrufescens mushrooms (contain the inhibitors 

sodium pyroglutamate and ergosterol)

Reishi mushrooms (via inhibition of VEGF and TGF-beta)

Black raspberry (Rubus occidentalis) extract

Maitake mushrooms (via inhibition of VEGF)

Phellinus linteus mushrooms(via active substance Interfungins A inhibition of glycation).

Green tea (catechins)Liquorice (glycyrrhizic acid)

References

Robbins Basic Pathology, 9E by  Vinay Kumar MBBS MD FRCPath (Author), Abul K. Abbas MBBS (Author), Jon C. Aster MD PhD

Tumor Angiogenesis Basic Mechanisms and Cancer Therapy https://en.wikipedia.org/wiki/Vascular_endothelial_growth_factor https://en.wikipedia.org/wiki/Angiogenesis#FGF https://en.m.wikipedia.org/wiki/Angiogenesis_inhibitor https://en.wikipedia.org/wiki/Tumstatin https://www.biooncology.com/pathways/vegf.html Cai, W., Chen, X. 2006. Anti-Angiogenic Cancer Therapy Based on Integrin avb3 Antagonism.

Anti-Cancer Agents in Medicinal Chhanabal, M., Jeffers, M., LaRochelle, W.J. 2005. Anti-Angiogenic Therapy as a cancer Treatment Paradigm. Anti-Cancer Agents in Medicinal Chemistry 5 (2). Patrickemistry 407-428. D, G.L. An Introduction to Medicinal Chemistry. New York: Oxford University Press, 2005

ReferenceMolecular basis of angiogenesis and cancer

Tiziana Tonini1, Francesca Rossi1,2 and Pier Paolo Claudio*,1,31Department of Biotechnology, Temple University, Philadelphia, PA

19122, USA; 2Dipartimento di Pediatria, Seconda Universita’ diNapoli, Italy; 3Dipartimento di Scienze Odontostomatologiche e Maxillo-

Facciali, Universita’ di Napoli ‘Federico II’, Italy Oncogene (2003) 22, 6549–6556

Tumor AngiogenesisBasic Mechanismsand Cancer Therapy

http://www.nature.com/onc/journal/v22/n42/full/1206816a.htmlhttps://en.m.wikipedia.org/wiki/Angiogenesis

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901818/

(https://en.wikipedia.org/wiki/Tumstatin)http://www.tititudorancea.org/z/prolactin_16_kd_fragment.htm